Long-acting insulin composition

FIELD: medicine, pharmaceutics.

SUBSTANCE: claimed invention relates to pharmaceutical composition, which contains compound of insulin in concentration, sufficient for supplying therapeutically effective level of insulin compound in blood plasma for at least 3 days. Insulin compound relates to pro-medical compound, which represents insulin conjugate with linker, bound with hydrogel carrier. Also described is suspension, containing pharmaceutical composition of insulin conjugate, method of suspension obtaining, set, including pharmaceutical composition of insulin conjugate and container for introduction of composition.

EFFECT: pharmaceutical composition of insulin conjugate by invention is characterised by the fact that it has pharmacokinetic profile in vivo in fact without release of insulin compound.

26 cl, 9 dwg, 21 ex

 

Area of technology

The present invention relates to pharmaceutical compositions containing a compound of insulin, the use composition, method of treatment, collection, and also to the combined treatment with compound GLP-1, such as an agonist of GLP-1. The pharmaceutical composition can be administered less frequently relative to existing drugs with long-acting insulin, and it is distinguished by the release of structurally intact insulin during the entire period of time between the introduction of essentially no release of the compounds of insulin. This treatment may help patients reduce the frequency of injection, while maintaining the possibility of optimal control of insulin levels in the plasma and, accordingly, the level of glucose in the blood.

Background of the invention

Treatment with insulin is characterized in that the release of drug insulin is necessary to maintain in a very limited concentration range due to the narrow "therapeutic window", and the side effects of hyperinsulinemia potentially life-threatening. For sale is a lot of insulin preparations with different action profiles that meet specific needs of a population of people with diabetes. Fast-acting insulin analogs administered immediately before a meal to control peak concentrations of glucose in plasma after �of Renate food while analogs of long-acting insulin is usually taken once or twice a day to maintain a constant basal level of insulin.

Modern developments also include oral insulin and inhaled insulin. However, due to the fact that insulin is a protein, if oral it is easily broken down in the stomach and gastrointestinal tract. Alternatively, within a short period of time on sale was available inhaled insulin is introduced into the lungs by using a nebulizer (Exubera®, Pfizer has ceased in 2007). This composition provided insulin for a long period of time (hours), but nevertheless requiring patients basal long-acting insulin by injection. Other disadvantages of inhaled insulin include the complexity of manufacturing, leading to prohibitively expensive delivery system. As a result, all commercially available formulations of insulin, you must enter through injections, subcutaneous or intravenous.

Various commercially available insulins differ in different profiles of their concentrations in plasma. These concentration profiles in the plasma can be described as profiles with maximum and minimum plasma concentrations, which depend on the composition and type of insole�and. In order to make it possible to predict the effect of reducing the level of glucose in the plasma as a result of the action of insulin, it is very important to obtain the concentration profile in the plasma, reproducible from patient to patient and from one patient. In addition, when multiple doses of basal insulin, it is desirable that the difference between the maximum and minimum plasma concentration was as low as possible. This leads to a more constant concentration of insulin in plasma, and, therefore, to a more common effect of lowering the glucose level during the entire dosing interval.

Existing basal insulin therapy consists of daily injection or injection twice daily basal long-acting insulins, such as NPH insulin, insulin glargine or insulin detemir. Although the development of new insulin analogues goal was the reduction of variability in the insulinotropic effects, the effect of reducing the level of glucose in these compositions, the long-acting still has a strong variability as separate entities, and between that described by Heise et al. (Diabetes, 2004 (53), 1614-1620). In this study, insulin detemir showed the lowest pharmaceutical variability with a coefficient of variability (CV) of 15 compared to CV for NPH-insulin and insulin� padrina, components 26 and 34, respectively. This rather high variability is a major impediment to achieving optimal glucose control, as it is difficult to predict the availability of insulin.

In the same study studied pharmacodynamic variability, as measured by the rate of infusion of glucose (GIR). In this study it was also demonstrated that insulin detemir had a lower variability in individual subjects compared with NPH-insulin and the insulin Gladwin regarding pharmacodynamic marker - GIR. In addition, in this study it was demonstrated that the effect of insulin on the rate of infusion of glucose did not last throughout the dosing period of 24 hours, indicating a clear need for long-acting insulin that will truly reduce the glucose level throughout the time interval between doses. To overcome the problem that the existing basal insulins for daily use do not apply within 24 hours, some patients break up the dosage of basal insulin for two daily injections to control glucose levels throughout the day.

Therefore, clearly a need for new drugs long-acting insulin, which continuously release insulin throughout ne�iodine time between doses of the drug.

In addition, even if patients can control their level of glucose in the blood with the help of daily injections of basal insulin, daily injections they resist the beginning of insulin therapy. This is undesirable because the American Diabetes Association (ADA) and European Association for the Studies of Diabetes (EASD) called insulin first line drug after oral Metformin, as it provides the best result of treatment (Nathan DM et al., Diabetologica(2008) 51: 8-11).

By reducing the frequency of administration of insulin is likely to decline physiological barrier before starting insulin therapy that enables patients to begin insulin therapy at an earlier stage and greatly improves their condition.

The challenge in developing formulations of basal long-acting insulin is a narrow therapeutic range of concentrations of insulin and a high ratio between the maximum and minimum concentrations in pharmacokinetic profile of insulin, in addition, in all cases it is necessary to avoid insulin spikes.

To reduce the frequency of administration with preservation of insulin release in a narrow range of concentrations have been proposed several approaches, but they didn't increase the duration of effect of decrease of glucose level in more than a couple of days, Ho�I differed a low ratio between the maximum and minimum concentration of insulin in plasma.

In the publication WO 06003014 described are capable of releasing insulin in the hydrogel with the possibility of reducing the frequency of dosing compared with standard daily injections of basal insulin. However, the release of insulin occurs with too high speed to guarantee strict glucose control over time periods greater than 2 days. In fact, the release of insulin is approximately 30 hours, so, Pro-drug connection, you must enter at least every 30 hours to the ratio between the maximum and minimum concentrations were below 2 in equilibrium.

The concept of obtaining reversible polymer conjugate of insulin was investigated by a group Shechter and others and described in scientific articles and patent applications (for example, in European Journal of Pharmaceutics and Biopharmaceutics 2008(70), 19-28 and WO 2004/089280). Insulin is conjugated to PEG-40 kDa polymer via the GS spacer molecule 9-gidroximetil-7-(amino-3-maleimidomethyl)-fluoren-N-hydroxysuccinimide. Hydrolysis indicated the GS spacer molecules releases insulin with a half-life of approximately 30 hours, that is Pro-drug connection, you must enter at least every 30 hours, so that the ratio between the maximum and minimum concentrations were below 2 in equilibrium.

Specifically in the case of insulin was demonstrated negative impact polymer compositions (Pharm. Dev. Technol. 4 (1999) 633-642; Pharm. Dev. Technol. 5 (2000) 1-9).

In the above case, the insulin has undergone significant structural changes as a result of permanent modification of high molecular polymer as Paglierani of insulin, presumably, serves to protect the peptide from degradation in the composition of the PLGA-polymer.

Unfortunately, such high molecular weight insulin can be less efficient as a result of weakening binding to receptors, but can also give a reaction at the site of injection, such as lipodystrophy, due to a longer presence of high concentrations of high molecular weight insulin Subcommittee�Noah fabric. In addition, such Paglierani insulins have a smaller volume of distribution, which is a significant drawback in the treatment of diabetes.

In addition, the pharmacokinetic profile of the released conjugate of insulin differs by the initial ejection immediately after injection, followed by a decrease in the concentration of the conjugate of insulin in the plasma with the subsequent increase in the concentration of the conjugate of insulin in plasma during the following days. This pharmacokinetic profile typical of microencapsulated formulations of drugs and can lead to unpredictable responses to glucose in subjects receiving treatment with such a composition.

Therefore remains a challenge to develop long-acting insulin without breaking the pharmacodynamics of insulin as a result of permanent joining of high-molecular compounds.

The situation is further complicated by the fact that insulin easily enters into side reactions, due to the presence in the molecule of three disulfide bonds. For example, insulin can be split into A and b-chains resulting from cleavage of disulfide bonds, or may form dimers or oligomers due to exchange reactions between disulfide groups. This "reshuffling of disulfide bonds is particularly likely if the insulin molecule periodical�but are in close contact in a random way ("Stability of insulin: studies on the physical and chemical stability of insulin in pharmaceutical formulation", Jens Brange, Springer, 1994). Such natural lability of the insulin molecule has significantly impeded progress in the development of depot formulations of long-acting and does not allow to use other polymeric compositions in which the insulin encased in a form that is similar to amorphous precipitate, which is known to give various products of degradation resulting from intensive exchange reactions between disulfide groups.

On the rate of adverse reactions is additionally affected by the concentration of insulin - speed is increased at high concentration of insulin. Therefore, the problem is to obtain compositions with a high concentration of long-acting insulin, in which insulin does not undergo undesirable side reactions.

Therefore, there is a clear need for new drugs long-acting insulin, which constantly release of structurally intact insulin during the entire period of time between product introductions and, in addition, retain the low ratio between the maximum and minimum concentration of insulin in plasma for exclusion is too high or too low concentrations of insulin, potentially harmful for the patient.

The amount of insulin required by patients with diabetes is highly individual, and the dose depends on several physiological factors, including�th the function of pancreatic beta-cells insulin sensitivity, body weight and diet. Quite often patients require 40 IU of insulin (or more) per day. This is equal to 280 IU per week, which corresponds to 12.6 mg of human insulin per week. To minimize discomfort to the patient this should be introduced in a small volume, for example, one milliliter. Therefore, the present invention relates to the composition of insulin, in which the concentration of insulin is at least 10 mg/ml, simultaneously releasing structurally intact insulin and having a pharmacokinetic profile essentially no release of insulin. In addition, as a result of the above, the present invention is the ability of a single dose of long-acting insulin in a single injection of a composition containing at least 10 mg of the compounds of insulin.

In US2007/0207210 A1 describes a method for preparing amorphous microparticles of high molecular weight proteins and in particular antibodies. According to the description in the example mentioned insulin, introduced into the composition at concentrations up to 400 mg/ml. However, the task of the invention relates to a composition with a pharmacokinetic profile similar to the native protein, that is not relates to prolonged release. Therefore, US2007/0207210 A1 does not offer solutions to reduce the frequency of insulin administration.

Definitions

It is assumed that as used herein, the term "substantially no release" or "substantially no release" (both terms are used interchangeably in the present description means that after the introduction of the compounds of insulin, which might be a Pro-drug compound or the active compound of insulin, the ratio of the peak concentrations of the detected compounds of insulin in plasma during the first 48 hours after administration, e.g., subcutaneous or intramuscular, the minimum concentrations of the detected compounds of insulin in the blood plasma after the peak concentration during the first 48 hours after administration is less than 2 (in fact there is no detected emission), preferably less than 1.5 (no detected emission).

It is assumed that as used herein, the term "release" means that after the introduction of the compounds of insulin, which might be a Pro-drug compound or the active compound of insulin, the ratio of the peak concentrations of the detected compounds of insulin in the blood plasma within 48 hours after administration, e.g., subcutaneous or intramuscular, to the minimum concentration of the detected compounds of insulin in the blood plasma after the peak concentration�ation within 48 hours after administration is 2 or higher.

In relation to the detection of the connection of insulin in the blood plasma that a combination of insulin can be a structurally intact form entered the connection of insulin, or in the case when the connection of insulin is a Pro-drug connection, detect the connection of insulin will be intact connection of insulin released from Pro-drug compounds, for example, human insulin, insulin analogues, insulin derivatives, and fragments thereof.

It is assumed that as used herein, the term "compound GLP-1" means any compound GLP-1, such as GLP-1(7-37), GLP-1(7-36)NH2 and an analogue of GLP-1, including an agonist of GLP-1. Examples of agonists of GLP-1 used in the present invention are agonists of GLP-1, Bloomington: Indiana-3 or Bloomington: Indiana-4, including, without limitation:

(i) counterparts of Bloomington: Indiana-4 and amidarone analogues Bloomington: Indiana-4, in the sequence in which one or more amino acid residues have been replaced by other amino acid residues, including N-terminal modifications,

(ii) shortened Bloomington: Indiana-4 and amidarone shortened form,

(iii) shortened Bloomington: Indiana-4 and amidarone a shortened form, in the sequences in which one or more amino acid residues have been replaced by other amino acid residues,

(iv) GLP-1 and aminirovanie GLP-1,

(v) and�the taxes GLP-1 and amidarone analogues of GLP-1, in the sequences in which one or more amino acid residues have been replaced by other amino acid residues, including N-terminal modifications,

(vi) truncated GLP-1 and amidarone shortened form,

(vii) the truncated GLP-1 and amidarone a shortened form, in the sequences in which one or more amino acid residues have been replaced by other amino acid residues,

(viii) are known substances, AVE-0010(ZP-10) (Sanofi-Aventis Zealand Pharma), BAY-73-7977 (Bayer), TH-0318, BIM-51077 (Ipsen, Tejin, Roche), NN-2211 (Novo Nordisk), LY315902.

Agonists GLP-1 mimic the function of native GLP-1, binds to receptors that are affected by the action of GLP-1, which is useful as a glucose, and in the treatment of diabetes, or as a result of simulation of the effects of Bloomington: Indiana on the secretion of urine, slow evacuation of the stomach, increasing the feeling of satiety, increase sodium excretion with urine and/or decrease in the concentration of potassium in the urine as a result of binding to the receptor(s), through which Bloomington: Indiana performs this action.

It is assumed that as used herein, the term "the ratio of the peak concentration to a residual" means the ratio between the maximum plasma concentration minimum plasma concentration of the compounds of insulin, e.g., human insulin, in a given period of time�and between doses.

It is assumed that as used herein, the term "structurally intact insulin connection" means intact insulin, consisting of two peptides, called A and b chains connected by two disulfide bridges. In addition, the A-chain contains an intramolecular disulfide bridge. Loss of intra - or intermolecular disulfide bridges or the permutation of the two circuits, for example, A-A or b-In homodimer, can cause inactivation of insulin. The structural integrity of the measure, splitting the insulin appropriate endoprotease, for example, endoproteinase Glu-C, and analyzing the resulting fragments using mass spectrometry. The absence of signals arising from a single of the insulin chains, indicates intact insulin. It is assumed that as used herein, the term "Pro-drug connection" means the connection of insulin, which undergoes biotransformation prior to the manifestation of its pharmacological effects. Therefore, Pro-drug compounds can be considered as biologically active residues containing specialized non-toxic protective groups used temporarily to change or eliminate undesirable properties of the original molecule. For example, Pro-drug compound can be biohydrology amide and biohydrogen�th ester, but this concept also encompasses a) compounds in which biohydrology functional group in such a Pro-drug of the compound included in the compound, and b) compounds that can be oxidized biologically or reinstated by this functional group. Typical Pro-drug compounds can be associated with a bearer of the Pro-drug compound that contains a temporary linkage of a given active substance with a temporary media group, which provides improved physicochemical or pharmacokinetic properties, and which is easily removedin vivousually as a result of hydrolytic decomposition; or it can be cascaded Pro-drug compound, for which the cleavage of the carrier becomes effective only after unmasking an activating group.

To enhance physicochemical or pharmacokinetic properties of medicinal compounds, e.g., insulin, drug compound can be konjugierte with the media. If drug compound transient connected with the carrier and/or a linker, such systems are usually called associated with media Pro-drug compounds. By definition given by IUPAC (quote from http://www.chem.qmul.ac.uk/iupac.medchem as at July 22, 2009) associated with a bearer of the Pro-drug compound before�provide a Pro-drug compound, which contains a temporary linkage of a given active substance with a transient group-a carrier that provides improved physicochemical or pharmacokinetic properties, and which is easily removedin vivousually as a result of hydrolytic decomposition.

Used in such related media Pro-drug compounds linkers are transient, i.e. they are hydrolytically degraded (broken down) without the involvement of enzymes under physiological conditions (aqueous buffer at pH 7.4, 37°C) with a half-life in the range of, for example, from one hour to three months.

Suitable carriers are polymers and they can be either anywhereman with a linker directly or through unsplittable spacer. "Pro-drug compound insulin-hydrogel" refers to a Pro-drug compound, in which the transient insulin is connected with a hydrogel carrier. The terms "Pro-drug hydrogel connected" and "associated with the hydrogel Pro-drug compound" refer to Pro-drug compounds of biologically active agents, the transient associated with the hydrogel, and used as synonyms.

The terms "drug compound", "biologically active molecule", "biologically active molecule", "biologically active agent", "days�existing agent", etc. means any substance that can affect physical or biochemical properties of a biological organism, including, without limitation, viruses, bacteria, fungi, plants, animals and humans. In particular, used in the present invention, biologically active molecules include any substance intended for diagnosis, cure, mitigation, treatment, or prevention of disease in man or other animals, or otherwise to enhance physical or mental health of a person or animal.

As used herein, the expression "therapeutically effective amount" of insulin means an amount sufficient to cure, mitigate or partial suspension of clinical manifestations of this disease or its complications. The amount sufficient for this purpose, is defined as "therapeutically effective amount". Effective amounts for each purpose will depend on the severity of the disease or lesion, and the weight and General condition of the subject. It is clear that the appropriate dosage can be determined using standard experiments by creating a matrix of values and testing different elements in the matrix, which is in the competence of an experienced therapist.

"Stable" and "stability" means that within the specified time x�of anemia conjugates remain conjugated and are not hydrolyzed to a considerable extent, and also have acceptable composition of impurities relative to insulin. Stable is the composition containing less than 5% of the medicinal compounds in the free form.

As used herein, the term "biohydrology ester" is an ester compound, which is either (a) does not interfere with the biological activity of the parent substance, but gives this substance preferred propertiesin vivosuch as duration of action, onset of action, etc., or b) is biologically inactive but is easily convertedin vivosubject to the biologically active principle.

As used herein, the term "biohydrology amide" is an amide compound which is either (a) does not interfere with the biological activity of the parent substance, but gives this substance preferred propertiesin vivosuch as duration of action, onset of action, etc., or b) is biologically inactive but is easily convertedin vivosubject to the biologically active principle.

It is assumed that as used herein, the term "hydrogel" means a three-dimensional, hydrophilic or amphiphilic polymeric SEL, able to absorb large amounts of water. Networks are composed of homopolymers or copolymers, and are insoluble due to the presence of �valentich chemical or physical (ionic, hydrophobic interactions, weaves) links. Crosslinking provide the network structure and physical integrity. The hydrogels exhibit a thermodynamic compatibility with water, which allows them to swell in aqueous medium. Chains are connected into a network, forming pores, and the size of a significant portion of these pores is between 1 nm and 1000 nm.

The term "gel" refers to unsewn, the gel-like polymer solution.

It is assumed that as used herein, the term "depot-drug" means a delivery system for medicinal compounds of insulin, usually administered via subcutaneous or intramuscular injection and is capable of continuous release of active compound over an extended period of time.

It is assumed that as used herein, the term "peak concentration" means the maximum concentration obtained after administration of the compounds of insulin.

Assume that you used the term "insulin connection" means any mammalian insulin, e.g., human insulin, porcine insulin or bovine insulin with disulfide bridges between CysA7 and CysB7 and between CysA20 and CysB19, as well as intramolecular disulfide bridge between CysA6 and CysA11, recombinant insulin mammals, for example, recombinant human insulin, insulin analogues, proizvodno�e insulin and fragments thereof, typical examples are rh insulin, insulin glargine, insulin detemir, insulin glulisine, insulin aspartate, lizpro, insulin, conjugated with low molecular weight PEG, where the low molecular weight PEG has a molecular weight less than 10 kDa. The connection of insulin can be in the form of Pro-drug compounds, in which case the connection is released into the plasma, insulin is active, which is formed after the introduction of the Pro-drug compounds.

By "insulin analogue" as used in the present invention refers to a polypeptide having a molecular structure which formally can be derived from the structure of natural insulin, e.g., human insulin, as a result of removal and/or replacement of at least one amino acid residue present in the natural insulin and/or by adding at least one amino acid residue. Added and/or used to replace amino acid residues can be either encoded amino acid residues, or other natural residues or purely synthetic amino acid residues.

Insulin analogues can be proteins, in which the natural balance of Pro at position 28 in the b-chain may be changed to one of Asp, Lys, or Ile. In another aspect, the Lys at position B29 is modified to Pro. Also, Asn at position A21 may be modified to Ala, Gln, Glu, Gly, His, Ile, Leu, Met, Ser, Thr, Trp, Tyr or Val, in particular to Gly, Ala, Ser, or Thr and preferably to Gly. Furthermore, Asn at position B3 may be changed to a Lys or Asp. Further examples of insulin analogues are insulin, des(B30) human; insulin analogues des(B30) human; insulin analogs, which removed PheB1; insulin analogs with extended from N-Terminus of the A-chain and/or In-chain and insulin analogs with extended with With-end A-chain and/or In-circuit. Thus, one or two of Arg residue may be added to position B1.

"DesB30 insulin", "insulin desB30 human" is meant a natural insulin or its analog, which lacks the amino acid residue B30. Similarly, insulin desB29desB30" or "insulin desB29desB30 person" means the natural insulin or its analog, which lacks amino acid residues B29 and B30.

Under "B1", "A1", etc. refers to the amino acid residue in position 1 in the b-chain of insulin (counted from N-Terminus) and the amino acid residue in position 1 in the A-chain of insulin (counted from N-Terminus), respectively. Can also be specified amino acid residue at the specified position, as for example, PheB1, indicating that the amino acid residue at position B1 is a residue of phenylalanine.

As used herein, the term "inactive linker" means a linker which does not offer pharmacological effects lekarstvennoj� connection derived from biologically active agent.

As used herein, the term "alkyl" means a straight or branched carbon chain. Each hydrogen of carbon in the alkyl can be substituted by the Deputy.

As used herein, the term "C1-4alkyl" means an alkyl chain having 1-4 carbon atoms, for example, at the end of a molecule: methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, or e.g.-CH2-, -CH2-CH2-, -CH(CH3)-, -CH2-CH2-CH2-, -CH(C2H5)-, -C(CH3)2- when two parts of the molecule are connected by an alkyl group. Each hydrogen of carbon in the C1-4the alkyl can be substituted by the Deputy.

As used herein, the term "C1-6alkyl" means an alkyl chain having 1-6 carbon atoms, for example, at the end of the molecule: C1-4alkyl, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, n-hexyl or, for example, -CH2-, -CH2-CH2-, -CH(CH3)-, -CH2-CH2-CH2-, -CH(C2H5)-, -C(CH3)2- when two parts of the molecule are connected by an alkyl group. Each hydrogen of carbon in the C1-6the alkyl can be substituted by the Deputy.

Accordingly, "C1-18alkyl" means an alkyl chain having from 1 to 18 carbon atoms, and "C 8-18alkyl" means an alkyl chain having from 8 to 18 carbon atoms. Accordingly, "C1-50alkyl" means an alkyl chain having 1 to 50 carbon atoms.

As used herein, the term "C2-50the alkenyl" means a straight or branched alkenyl chain having 2 to 50 carbon atoms, for example, at the end of a molecule: -CH=CH2, -CH=CH-CH3, -CH2-CH=CH2, -CH=CH-CH2-CH3, -CH=CH-CH=CH2or , for example,- CH=CH-, when two parts of the molecule are connected by the alkenyl group. Each hydrogen of carbon in the C2-50alkenyl may be optionally substituted by the specified substitute. Accordingly, the term "alkenyl" refers to a carbon chain with at least one double carbon bond. Optionally, can contain one or more triple bonds.

As used herein, the term "C2-50alkynyl" means a straight or branched alkylamino chain having 2 to 50 carbon atoms, for example, at the end of a molecule: -C≡CH, -CH2-C=CH, CH2-CH2-C=CH, CH2-C≡C-CH3or , for example,- C≡C-when two parts of the molecule are connected Salcininkai group. Each hydrogen of carbon in the C2-50alkynyl may be optionally substituted by the specified substitute. Accordingly, the term "alkynyl" refers to a carbon chain with at least one t�been carbon bond. Optionally, can contain one or more double bonds.

As used herein, the term "C3-7cycloalkyl" or "C3-7cycloalkyl ring" means a cyclic alkyl chain having 3 to 7 carbon atoms, which may have a carbon double bond, wherein at least partially saturated, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclohexenyl, cycloheptyl. Each hydrogen of carbon in cycloalkyl can be substituted by the Deputy. The term "C3-7cycloalkyl" or "C3-7cycloalkyl ring" also includes bridges United Bicycle, such as Narbona or noronen. Accordingly, "C3-5cycloalkyl" means cyclic alkyl having 3 to 5 carbon atoms.

Accordingly, "C3-10cycloalkyl" means cyclic alkyl having 3 to 10 carbon atoms, for example, "C3-7cycloalkyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclohexenyl, cycloheptyl, cyclooctyl, cycloneii, cyclodecyl. The term "C3-10cycloalkyl" also includes at least partially saturated, carbamino - and-Bicycle.

As used herein, the term "halogen" means fluorine, chlorine, bromine or iodine. Usually it is preferable that the halogen was fluorine or chlorine.

Used in this document� the term "4 to 7-membered heterocyclyl" or "4 to 7-membered heterocycle" means a ring with 4, 5, 6 or 7 atoms in the ring which can contain up to the maximum number of double bonds (aromatic or non-aromatic ring that is fully saturated, partially saturated or unsaturated), wherein at least one atom in the ring (and up to 4 atoms in the ring is replaced with a heteroatom selected from the group consisting of sulfur (including-S(O)-, -S(O)2-), oxygen and nitrogen (including =N(O)-), and when the ring is attached to the rest of the molecule via a carbon atom or nitrogen. Examples 4-7-membered heterocycles are azetidin, oxetan, tieten, furan, thiophene, pyrrole, pyrrolidine, imidazole, imidazoline, pyrazole, pyrazoline, oxazole, oxazoline, isoxazole, isoxazoline, thiazole, thiazoline, isothiazole, isothiazoline, thiadiazole, thiadiazole, tetrahydrofuran, tetrahydrothiophene, pyrrolidine, imidazolidine, pyrazolidine, oxazolidine, isoxazolidine, thiazolidine, isothiazolin, thiadiazolidin, sulfolane, Piran, dihydropyran, tetrahydropyran, imidazolidine, pyridine, pyridazine, pyrazine, pyrimidine, piperazine, piperidine, morpholine, tetrazole, triazole, thiazolidine, tetrazoles, diazepan, azepin or homopiperazin.

As used herein, the term "9-11-membered heterobicycle" or "9-11-membered heterobicycle" means a heterocyclic system of two rings with 9-11 atoms in the ring, at least one ring atom of one of the two rings, and which may contain up to the maximum number of double bonds (aromatic or non-aromatic ring that is fully saturated, partially saturated or unsaturated), in which at least one atom in the ring (and up to 6 atoms in the ring) substituted with a heteroatom selected from the group consisting of sulfur (including-S(O)-, -S(O)2-), oxygen and nitrogen (including =N(O)-), and when the ring is attached to the rest of the molecule via a carbon atom or nitrogen. Examples 9-11-membered heterobicycle are indole, indoline, benzofuran, benzothiophen, benzoxazol, benzizoksazola, benzothiazol, benzisothiazol, benzimidazol, benzimidazole, quinoline, chinazoline, dihydroquinazolin, quinoline, dihydroquinoline, tetrahydroquinoline, decahydroquinoline, isoquinoline, decahydroquinoline, tetrahydroisoquinoline, dihydroisoquinoline, benzazepin, purine or pteridine. The term 9-11-membered heterobicycle also includes zerotracker of the two rings, such as 1,4-dioxa-8 azaspiro[4.5]Dean or connected by a bridge heterocycles, such as 8-Aza-bicyclo[3.2.1]octane.

As used herein, the term "pharmaceutically acceptable" means approved by regulatory agencies such as EMEA (Europe) and/or the FDA (USA), and/or any other national Supervisory authorities for use in animals, preferably in humans.

Used in the present�umenta the term "pharmaceutical composition" or "composition" means one or more active ingredients, and one or more inert ingredients, as well as any product obtained directly or indirectly as a result of unification, the formation of complexes or aggregation of any two or more ingredients, or as a result of dissociation of one or more of the ingredients, or from other types of reactions or interactions of one or more ingredients. Accordingly, the pharmaceutical compositions of the present invention encompass any composition made by mixing the compounds of the present invention and pharmaceutically acceptable excipients (pharmaceutically acceptable carrier).

"Free form" medicinal compounds relates to the medicinal compound in its unmodified pharmacologically active form, e.g., after release from the polymer conjugate.

"Dry composition" means that the composition of the Pro-drug compounds of insulin with the hydrogel are presented in dry form in a container. Suitable drying methods are spray drying and lyophilization (drying and freezing). Such dry composition Pro-drug compounds of insulin with the hydrogel has a residual water content of maximum 10%, preferably less than 5% and, more preferably, less than 2% (determined according to Karl Fischer). Pre�respectful method of drying is lyophilization. "Lyophilized composition" means that the composition of the Pro-drug compounds of insulin in the hydrogel of the source is frozen and then the water was removed, lowering the pressure. This terminology does not exclude additional drying step, which are present in the manufacturing process before completing the final composition of the container.

"Lyophilization (drying and freezing) is the process of removing water, wherein the freezing of the composition and then reduced pressure and optionally by heating for direct sublimation of frozen water in the composition of the solid phase to gas. Usually, water vapor is collected desublimate.

"Restore" means to add fluid to restore the original form of the composition.

"The solution is to restore" refers to a fluid used to restore dry composition Pro-drug compounds of insulin with the hydrogel before the introduction needs it the patient.

"Container" means any container that contains a composition of Pro-drug compounds of insulin in the hydrogel, and in which it can be stored until the restoration.

As used herein, the expression "therapeutically effective amount" of the compounds of insulin means an amount sufficient to cure, mitigate or partial suspension Kli�algebraic manifestations of this disease and its complications. The amount sufficient for this purpose, is defined as "therapeutically effective amount". Effective amounts for each purpose will depend on the severity of the disease or lesion, and the weight and General condition of the subject. It is clear that the appropriate dosage can be determined using standard experiments by creating a matrix of values and testing different elements in the matrix, which is in the competence of an experienced therapist or a veterinarian.

The term "buffer" or "buffering agent" refers to chemical compounds that maintain the pH within a predetermined range. Physiologically compatible buffers are, for example, sodium phosphate, succinate, histidine, bicarbonate, citrate, and acetate, sulfate, nitrate, chloride, pyruvate. You can also use antacids such as Mg(OH)2or ZnCO3. A buffer tank can fail to meet the conditions that are most sensitive to pH stability.

The term "excipients" refers to compounds injected together with therapeutic agent, for example, buffering agents, isotonicity modifiers, preservatives, stabilizatorami, anti-adsorption agents, agents that protect against oxidation, and other auxiliary agents. However, in some cases one auxiliary substance may �have double or triple functions.

"Bioprotector" is a molecule, which upon Association with target protein, significantly prevents or reduces chemical and/or physical instability of the protein during drying in General, and especially during lyophilization and subsequent storage. Examples of iprotectyou include sugars such as sucrose or trehalose; amino acids such as monolatry glutamate or histidine; methylamine, such as betaine; a lyotropic salt such as magnesium sulfate; polyhydric alcohols such as triatomic or polynuclear sugar alcohols, e.g. glycerin, aritra, glycerin, Arabic, xylitol, sorbitol and mannitol; ethylene glycol; propylene glycol; polyethylene glycol; pluronic; hydroxyalkyl starches, for example, gidroksiètilovyh starch (HES), and combinations thereof.

"Surfactant" refers to wetting agents that reduce the surface tension of the liquid.

"Isotonicity modifiers" refers to compounds that minimize the pain that can occur as a result of cell damage due to osmotic pressure difference in the location of the depot injection.

The term "stabilizers" refers to compounds used to stabilize polymeric Pro-drug compounds. Stabilization is achieved by increasing the forces stabilizing protein, destab�implementation of the denatured state, or by direct binding of excipients to the protein.

"Anti-adsorption agents" refers generally to ionic or non-ionic surfactants or other proteins or soluble polymers are used for a competitive inner surface or adsorption on the inner surface of the container containing the composition. The choice of the concentration and type of excipients depend on the effect to be avoided, but usually a monolayer of surfactant is formed on the surface of the phase slightly above the critical micelle concentrations.

"Agents that protect against oxidation" refers to antioxidants, such as ascorbic acid, ectoin, methionine, glutathione, monothioglycerol, Morin, polyethylenimine (PEI), propylgallate, vitamin E, chelating agents such as citric acid, EDTA, hexaphosphate, thioglycolic acid.

"Antimicrobial agent" refers to chemical substance that kills or inhibits the growth of microorganisms, such as bacteria, fungi, yeast, protozoa, and/or destroys viruses.

"Sealed container" means the container is closed in such a way that it is airtight, not allowing gas exchange between the external environment and the internal environment of the container, and saves the contents in a sterile condition.

It is understood that the expression "�farmacevtichesky acceptable" includes any excipient and/or additive, which do not infringe the effectiveness of the biological activity of an active ingredient and is not toxic to the master whom they are administered.

The term "reagent" refers to an intermediate connection or source material used in the method of manufacture, resulting in a Pro-drug compound of the present invention.

The term "chemical functional group" refers to a carboxylic acid and activated derivatives, amino groups, maleimide group, thiol group and derivatives, sulfonic acid and derivatives, carbonate and derivatives, carbamate and derivatives, hydroxyl, aldehyde, ketone, hydrazine, isocyanate, isothiocyanate, phosphoric acid and derivatives, phosphonic acid and derivatives, haloacetic, alkylhalides, acryloyloxy group and other alpha-beta unsaturated Michael acceptors, alleroisk agents such as aristorod, hydroxylamine, disulfides, such as pyridylsulfonyl, vinylsulfonic, vinylmation, diazoalkanes, diazoacetate compounds the oxirane and aziridine.

If a chemical functional group connected with another chemical functional group, the resulting chemical structure is referred to as a "communication". For example, reaction of amino group with a carboxyl group gives an amide bond.

"Reactive functional GRU�PY" are chemical functional groups of a frame of the molecule, which are connected to the Hyper-branched molecules.

"Functional group" is a collective term used for "reactive functional group", "degradiruem conjugate (forming an intermolecular bond) functional groups" or "conjugating functional group".

"Degradiruem conjugated functional group" is a compound containing biodegrading the link, which one side is connected with a spacer connected with the frame part of the molecule, and on the other hand are connected with cross-linking molecules. The terms "degradiruem conjugated functional group", "biodegradeableman conjugated functional group, coupled biodegradeableman functional group" and "conjugated functional group" are used as synonyms.

The term "blocking group" or "kiperousa group" are used as synonyms and refer to the parts of molecules that are irreversibly attached to reactive functional groups to block their ability to react, for example, with chemical functional groups.

The terms "protecting group" or "protective group" refer to the parts of the molecules, reversibly attached to reactive functional groups for lock�of their ability to react, for example, with chemical functional groups.

The term "capable of forming intermolecular bond functional group" refers to chemical functional groups that participate in the reaction of radical polymerization and are part of a cross-linking reagent or reagent frame.

The term "polymerizable functional group" refers to chemical functional groups that participate in the crosslinking polymerization reaction type and are part of a cross-linking reagent or reagent frame.

For conjugated functional groups, the term "hydrolytically degradiruete" refers in the context of the present invention, the compounds are hydrolytically degraded without enzymes under physiological conditions (aqueous buffer pH 7.4, 37°C) with a half-life in the range from one hour to three months, and which include, but are not limited to these compounds, aconitine, acetals, anhydrides of carboxylic acids, esters, imine, hydrazones, amides melaminovoi acid, ortepii, phosphamide, pastefire, silyl esters of phosphorus, silyl esters, sulfonic esters, aromatic carbamates, combinations thereof, etc. Preferred biodegradable compounds are carboxylic esters, carbonates, postevery and esters of sulfonic acid, and most preferred�Uo are carboxylic esters or carbonates. Obviously, in studies ofin vitrofor practical purposes, you can use accelerated conditions, e.g., pH 9, 37°C, aqueous buffer.

The skeleton of the molecule can contain GS spacer portion of the molecule, one end of which is attached to the frame of the molecule, and the other end to the binding part of the molecule.

The term "derivative" refers to chemical functional groups, respectively, substituted protecting and/or activating groups, or activated forms of the corresponding chemical functional group known to experienced specialist in this field. For example, the activated form of a carboxyl group include, but are not limited to these, reactive esters, such as Succinimidyl ether, benzotriazolyl ether, nitroanilines ether, pentafluorophenyl ether, asianstreetmeat ether, acylhomoserine, mixed and symmetrical anhydrides, allmydata.

The term "linker split without enzymes" refers to linkers, hydrolytically degradiruem under physiological conditions without enzymatic activity.

"Biologically inactive linker" means a linker which does not show the pharmacological effects of drug compound (D-H) derived from biologically active molecules.

The terms "spacer", "GS spacer group", "space�RNA molecule" and "GS spacer portion of the molecule" are used interchangeably, and when used to describe molecules present in the hydrogel carrier of the present invention, refers to any molecule that is suitable for connecting two parts of the molecule, for example, C1-50the alkyl, C2-50alkenyl or C2-50alkinyl, which optionally is interrupted by one or more groups selected from-NH-, -N(C1-4alkyl)-, -O-, -S-, -C(O)-, -C(O)NH-, -C(O)N(C1-4alkyl)-, -O-C(O)-, -S(O)-, -S(O)2-, 4-7-membered heterocycle, phenyl or naftel.

The terms "end", "end" or "far end" refers to the position of the functional group or bond in the molecule or parts of molecules, in accordance with which such a functional group may be a chemical functional group, and the communication can be degradiruem or an always-on connection, which are characterized in that they are located close to the connection or joint between the two parts of the molecule or at the end of oligomeric or polymeric chain.

The phrase "in bound form" or "part of a molecule" refer to the substructures that are part of larger molecules. The phrase "in bound form" is used to simplify references to parts of molecules by mention or enumeration of the reagents, starting materials or possible source materials, well known in this field, and according to which "in bound form" means that, �of primer, one or more hydrogen radicals (-N) or one or more activating or protective groups present in the reagent or the original substances are not present in the molecule.

It is obvious that all reagents and molecules containing polymer molecules, are macromolecular structures that are known to have variability in their molecular weight, the length of the chains or the degree of polymerization, or number of functional groups. Therefore, the structure shown reagents for frame, frame parts of the molecules, reagents for stitching or seams, are only representative examples.

The reagent or part of a molecule can be linear or branched. If the reagent or a portion of the molecules have two end groups, they are referred to as linear reagent or part of the molecule. If the reagent or a portion of the molecules have more than two end groups, they are considered branched or multifunctional reagent or part of a molecule.

The term "polymer chain on the basis of polyethylene glycol", "chain-based PEG" refers to oligomeric or polymeric molecular chain.

Preferably, such a polymer chain on the basis of polyethylene glycol has been associated with the nucleus (the branch point), and was a linear polyethylene glycol chain, which has one end attached to the core,and the other to hyperazotemia dendritic part. It is obvious that the chain on the basis of PEG can be finished (or interrupted) alkyl or aryl groups, optionally substituted by heteroatoms and chemical functional groups.

If the term "polymer chain on the basis of polyethylene glycol" is used as applied to the crosslinking reagent, it relates to cross-linking or chain containing at least 20% (by mass) of the molecules of ethylene glycol.

As used herein, the singular and similar references should be interpreted as references to the singular and the plural, unless herein otherwise expressly provided, or if this right is not contrary to the context.

Description

The present invention relates to the preparation of long-acting insulin to provide basal insulin. Basal insulins are formulations of insulin or insulin analogues long-acting, designed to mimic the basal insulin secretion by beta cells of the pancreas. Optimally, thus carried out continuous monitoring of glucose in the blood throughout the dosing interval.

The inventors have discovered that the connection of insulin can continuously released from the injection depot of the drug, (e.g., subcutaneous injection) in structurally intact form in �nterval between doses without any effect ejection. The structural integrity of the released compounds of insulin provides a highly hydrated polymeric matrix that minimizes intermolecular contacts insulin molecules. In addition, due to the lack of release of insulin reduces the risk of harmful side effects in the patient.

The present invention reduces the risk of hypoglycemia after administration of insulin due to the lack of "explosive" release of insulin, reduces the risk of hyperglycemia at the end of the dosing period, reduces the frequency of injections and provides a predictable level of insulin in the blood plasma of the patient.

An additional object of the present invention relates to a new basal insulin, requiring less frequent administration regarding existing schemes daily injection of basal insulin and provide a high level of security as a result of the release of structurally intact insulin during the entire time interval between injections and usually having a ratio of peak concentration to a residual concentration of less than 2.

Additional advantages will be apparent from the present description.

In a first aspect the present invention relates to pharmaceutical compositions containing a compound of insulin in a concentration sufficient to maintain a therapeutically effective level with�unity of insulin in blood plasma for at least 3 days normally, at least 80 hours, for example, during the week, characterized by the fact that it has a pharmacokinetic profilein vivoessentially without the release of the compounds of insulin.

This concentration will vary from subject to subject and depends on therapeutic window in individual subject, but for the duration of therapeutic effect for at least 3 days, for example, weeks (i.e., approximately 7 days), the concentration is typically at least about 10 mg/ml, for example, more than 10 mg/ml.

Accordingly, in a further aspect the present invention relates to pharmaceutical compositions containing a compound of insulin in a concentration of at least 10 mg/ml, characterized in that it has a pharmacokinetic profilein vivoessentially without the release of the compounds of insulin.

In an additional aspect, the present invention relates to pharmaceutical compositions containing a compound of insulin with a concentration of at least 11 mg/ml, for administration in a single dose of at least 10 mg of the compounds of insulin.

Used in the present description, a single dose of the compounds of insulin is given in mg, and the concentration of the compounds of insulin in the pharmaceutical composition is given in mg/ml. If the connection of insulin is a Pro-drug compound, the concentration based on the number�, a significant release of free insulin from Pro-drug compounds. Using known in the field methods an aliquot of the composition is placed in a condition for the release of insulin (aqueous buffer, pH 7.4, 37°C, or accelerated conditions at elevated pH) prior to the termination of a significant increase in insulin concentration and determine the total amount of insulin released. Obviously, in the case of instant media, the quantitative release is synonymous with quantitative hydrolysis.

In one embodiment of the present invention the concentration of the compounds of insulin is at least 11 mg/ml, for example from 11 mg/ml to 35 mg/ml, more preferably, from 15 mg/ml to 25 mg/ml, even more preferably, about 20 mg/ml, and even more preferably, the concentration is about 24 mg/ml.

The volume, which is used for the introduction of effective doses, for example, by syringe, to a subject, e.g., human, preferably less than 1.5 ml, typically 1.0 ml or less.

In an additional embodiment the single dose of the compounds of insulin is at least 5 mg, e.g., from 5 mg to 100 mg, more preferably, from 5 mg to 50 mg and, more preferably, from 5 mg to 25 mg.

In an additional embodiment, the ratio of the peak concentrations of the compounds of insulin in plasma during the first 48 hours after the introduction�Oia, for example, subcutaneous or intramuscular injection, to the minimum concentration of the compounds of insulin in the blood plasma after the peak concentration during the first 48 hours after administration is less than 2, usually less than 1.5.

The above embodiments of the invention, as well as options for implementation, described below, should be considered relative to any of the aspects described in this document, as well as any of the embodiments described herein, unless specified that the variant of implementation refers to a particular aspect or aspects of the present invention.

Typically, the pharmaceutical composition is a controlled delivery system, which includes the connection of insulin and is characterized in that the delivery connection of insulin in the blood plasma of mammals is essentially no release of insulin.

In yet another additional embodiment, the pharmacokinetic profile measured in the blood plasma of mammals, such as human blood plasma. The insulin concentration in plasma can be measured using commercially available ELISA kits for comparing the measurement results with a calibration curve obtained using an insulin standards. For statistical validity of the ex�eriment carried out with a sufficient number of biological and technical repeats and calculate the mean and median values to account for biological and technical variability.

In an additional embodiment the composition is characterized in that the ratio of peak concentration to a residual concentration of less than 2, usually less than a 1.75, preferably 1.5 or less, even more preferably less than 1.25.

In yet another additional embodiment, the composition is characterized by a constant release of compounds structurally intact insulin during the entire time interval between doses.

"Sustained release" refers to the continuous release of insulin.

In an additional embodiment, the full time interval between doses is at least about 80 hours, e.g., about 110 hours, usually at least a week, for example, 1-2 weeks or even more.

In yet another additional embodiment, the connection of insulin is a Pro-drug connection.

In an additional embodiment, the connection of insulin is associated with Pro-drug media connection.

In yet another additional embodiment, the compound of the insulin Pro-drug is a cascade connection.

Pro-drug compound can be administered in liquid form, e.g., solution or gel, or it can�t be kept in the depot of the drug, or even can be embedded in a depot of the drug, so the depot-the drug acts as a Pro-drug compounds.

In an additional embodiment, the compound of the insulin is fully contained in a depot preparation.

The term "fully-contained" refers to a depot preparation, in which less than 10% of drug compounds, that is, insulin is present in the aqueous fraction after adding 1 ml water to 1 ml of depo-preparation, mixing and separation of the depot of the drug from the water.

In yet another additional embodiment, the depot is a drug is a polymer gel, e.g., a hydrogel.

In an additional embodiment the depot drug is highly hydrated polymeric matrix.

The connection of insulin can be kept in the depot of the drug in various embodiments, for example, the connection of insulin can be ecovalence associated with depo drug or covalently linked to depot drug depot and the drug (without limitation), you can choose from a polymer gel, a hydrogel or highly hydrated polymeric matrix. Non-limiting examples of suitable polymers are polymers that can form a quasi-infinite three-dimensional highly hydrated molecular network. Such hydrogels are chemically or physical�and cross-linked or functionalized defunctionalization polyacetylene polymers, for example, polypropylene glycol or polyethylene glycol, dextran, chitosan, hyaluronic acid and derivatives, alginate, xylans, mannan, carrageenan, agarose, cellulose, starch, hydroxyethyl starch (HES) and other carbohydrate polymers, polyvinyl alcohols, polyoxazolines, polyanhydride, polyarteritis, polycarbonates, polyurethanes, polyacrylic acids, polyacrylamides, for example, polyhydroxyethylmethacrylate (HMPA), polyacrylates, polymethacrylates, for example, polyhydroxyethylmethacrylate, preorganisation, polysiloxane, polyvinylpyrrolidone, polycyanoacrylates, polyesters, for example, polylactic acid or polyglycol acid, polyaminocarboxylic, polyaminoamide, for example, polyglutamine acid or polylysine, collagen, gelatin, copolymers, grafted copolymers, cross-linked polymers, hydrogels, and block copolymers of the above polymers. These polymers can serve as the skeleton parts of crosslinking molecules or parts of molecules, and a combination of different polymers in the form of copolymers with a high degree of hydration of the molecular network. In addition to oligomeric or polymeric crosslinking parts of the molecules of these polymers, it is possible to use low molecular weight crosslinking, particularly, when using a high molecular skeleton parts of molecules to �fatality of hydrogel carriers Pro-drug compounds.

One way of minimizing contact between molecules of insulin is a homogeneous distribution of molecules of insulin in a highly hydrated polymeric matrix. Homogeneous distribution can be obtained by using covalent binding of insulin to the polymer, and as a result of the use of linkers, fissile in aqueous medium at neutral pH, it is guaranteed the slow release of structurally intact insulin.

Preferred is associated with the polymer Pro-drug compound of insulin, which essentially has no biological activity, and this property means that all insulinotropic activity relates to the released insulin. Therefore, by modeling the properties of releasing Pro-drug compounds to achieve a high degree of control of insulin levels in plasma.

Insulin may be attached via all relevant functional groups in the molecule, and such preferred functional groups of the natural amino acids are normally selected from the guanidine, imidazole, indole, carboxyl, carboxamide, primary and secondary hydroxyl, phenol, primary amine groups. For example, human insulin has the following important functional groups: carboxyl, carboxamido, primary and secondary hydroxyls�Yu, phenol, imidazole and primary amino group.

In an additional embodiment, the connection of insulin is associated with a bearer of the Pro-drug compounds either covalently with any of the lysine residues or the N-end of any of A - or b-chain of insulin connection.

In a particular embodiment, the Pro-drug compound or its pharmaceutically acceptable salt contain insulin conjugate with a linker, D-L, in which

D represents the insulin molecule; and

-L is not biologically active linker molecule-L1represented by the formula (I),

in which the dotted line indicates the accession of one of the amino groups of the insulin with the formation of the amide bond;

X is C(R3R3a); or N(R3);

R1a, R3aindependently selected from the group consisting of H, NH(R2b), N(R2b)C(O)R4and C1-4alkyl;

R1, R2, R2a, R2b, R3, R4independently selected from the group consisting of H and C1-4of alkyl,

in which L1substituted by one Deputy L2-Z and optionally further substituted, provided that the hydrogen atom marked with an asterisk in formula (I) is not substituted by the Deputy, and in which

L2is a single chemical bond or space�rum; and

Z is a hydrogel.

In the case where compounds of formula (I) contain one or more acidic or basic groups the invention also includes their corresponding pharmaceutically or toxicologically acceptable salts, in particular their pharmaceutically used salts. Therefore, the compounds of formula (I) which contain acidic groups can be used according to the invention, for example, as salts of alkali metals, salts of alkaline earth metals or ammonium salts. More specific examples of such salts include sodium salt, potassium salt, calcium salts, magnesium salts or ammonium salts or organic amines, such as, for example, ethylamine, ethanolamine, triethanolamine or amino acids. Compounds of formula (I) containing one or more basic groups, i.e. groups which can protonemata may be present, and can be used according to the invention in the form of their salts joining with inorganic or organic acids. Examples of suitable acids include hydrogen chloride, hydrogen bromide, phosphoric acid, sulfuric acid, nitric acid, methanesulfonic acid, p-toluensulfonate acid, naphthalenedisulfonic acid, oxalic acid, acetic acid, tartaric acid, lactic acid, salicylic acid, benzoic acid, formic acid, etc�peony acid, Pikalyovo acid, ditlucan acid, malonic acid, succinic acid, timelineview acid, fumaric acid, maleic acid, malic acid, sulfamic acid, phenylpropionate acid, gluconic acid, ascorbic acid, isonicotinoyl acid, citric acid, adipic acid and other acids known to those skilled in this field. If compounds of formula (I) simultaneously contain acidic and basic groups in the molecule, the invention also includes (in addition to the forms of salts) inner salt or betaine (zwitter-ions). The corresponding salt of formula (I) can be obtained by conventional methods known to the skilled person skilled in the art, for example, by contact with an organic or inorganic acid or base in a solvent or dispersing the substance or as a result of anyoneeven or cation exchange with other salts. The present invention also includes all salts of the compounds of formula (I) which, owing to low physiological compatibility, are not directly suitable for use in pharmaceuticals but which can be used, for example, as intermediates for chemical reactions or to obtain pharmaceutically acceptable salts.

Preferably, in the formula (I) R2Thames�n L 2-Z.

Preferably, in the formula (I) R1substituted L2-Z.

Preferably, in the formula (I), X is N(R3).

Preferably, in the formula (I) X is C(R3R3a), and R3ais N(R2b)C(O)R4.

Preferably, X is C(R3R3a), R3ais N(R2b)-L2-Z.

Preferred Pro-drug compounds of the present invention contain a conjugate of insulin with the linker, D-L, where L1from the formula (I) represented by formulas (Ia), (Ib), (Ic) or (Id):

in which R1, R1a, R2, R2a, R2b, R3, R4, L2, Z have the values listed in this document, and in which L1optionally further substituted, provided that the hydrogen atom marked with an asterisk in formula (Ia) to(Id), are not substituted by the Deputy.

Preferably, L1not further substituted (in addition to the mandatory Deputy L2-Z).

As shown, for example, in formulas (Ia)-(Id) one hydrogen is replaced by the group L2-Z.

In General, the L2can be attached to the L1for any position, except for substitution of the hydrogen marked with the asterisk in formula (I). Preferably, one of the hydrogen atoms of R1, R1a, R2, 2a, R2b, R3, R4directly or as hydrogen of C1-4the alkyl or additional groups was substituted L2-Z.

In addition, L1can optionally be substituted. In General, you can use any Deputy, provided that is not affected splitting principle. However, it is preferable that L1was not further substituted.

Preferably, one or more additional alternates are independently selected from the group consisting of halogen; CN; COOR9; OR9; C(O)R9; C(O)N(R9R9a); S(O)2N(R9R9a); S(O)N(R9R9a); S(O)2R9; S(O)R9; N(R9)S(O)2N(R9aR9b); SR9; N(R9R9a); NO2; OC(O)R9; N(R9)C(O)R9a; N(R9)S(O)2R9a; N(R9)S(O)R9a; N(R9)C(O)OR9a; N(R9)C(O)N(R9aR9b); OC(O)N(R9R9a); T; C1-50alkyl; C2-50alkenyl or C2-50alkynyl, where T; C1-50alkyl, C2-50of alkenyl and C2-50alkynyl optionally substituted by one or more R10that may be the same or different, and where C1-50alkyl, C2-50of alkenyl and C2-50alkynyl optionally interrupted by one or more groups selected from the group consisting of T, -C(O)O-; -O-; -C(O)-; -C(O)N(R11)-; -S(O)2N(R11)-; -S(O)N(R 11)-; -S(O)2-; -S(O)-; -N(R11S(O)2N(R11a)-; -S-; -N(R11)-; -OC(O)R11; -N(R11)C(O)-; -N(R11)S(Oh)2-; -N(R11)S(O)-; -N(R11)C(O)O-; -N(R11)C(O)N(R11a)- and-OC(O)N(R11R11a);

R9, R9a, R9bindependently selected from the group consisting of H; T; C1-50alkyl; C2-50alkenyl or C2-50alkynyl, where T; C1-50alkyl, C2-50of alkenyl and C2-50alkynyl optionally substituted by one or more R10that may be the same or different, and where C1-50alkyl, C2-50of alkenyl and C2-50alkynyl optionally interrupted by one or more groups selected from the group consisting of T, -C(O)O-; -O-; -C(O)-; -C(O)N(R11)-; -S(O)2N(R11)-; -S(O)N(R11)-; -S(O)2-; -S(O)-; -N(R11S(O)2N(R11a)-; -S-; -N(R11)-; -OC(O)R11; -N(R11)C(O)-; -N(R11)S(Oh)2-; -N(R11)S(O)-; -N(R11)C(O)O-; -N(R11)C(O)N(R11a)- and-OC(O)N(R11R11a);

T is selected from the group consisting of phenyl; naftel; indenyl; indanyl; tetralinyl;3-10cycloalkyl; 4-7-membered heterocyclyl; or 9-10-membered heterobicycle, wherein T is optionally substituted by one or more of R10that may be the same or different;

R10is halogen; CN; oxopropoxy (=O); COOR12; OR12; C(O)R12; C(O)N(R12R12a ); S(O)2N(R12R12a); S(O)N(R12R12a); S(O)2R12; S(O)R12; N(R12)S(O)2N(R12aR12b); SR12; N(R12R12a); NO2; OC(O)R12; N(R12)C(O)R12a; N(R12)S(O)2R12a; N(R12)S(O)R12a; N(R12)C(O)OR12a; N(R12)C(O)N(R12aR12b); OC(O)N(R12R12a); or1-6the alkyl, and C1-6alkyl optionally substituted by one or more Halogens (same or different);

R11, Rl1a, R12, R12a, R12bindependently selected from the group consisting of H or C1-6alkyl, and C1-6alkyl optionally substituted by one or more Halogens (same or different).

The term "interrupted" means that between two carbon atoms or at the end of the carbon chain between carbon atoms and hydrogen inserted group.

L2represents a single chemical bond or a spacer. In the case when L2is a spacer, it is preferably described as one or more optional substituents described above, provided that L2substituted Z.

Accordingly, when L2not a single chemical bond, then L2-Z is COOR9; OR9; C(O)R9; C(O)N(R9R9a); S(O)2N(R9R9a); S(O)N(R9R9a); S(O) R9; S(O)R9; N(R9)S(O)2N(R9aR9b); SR9; N(R9R9a); OC(O)R9; N(R9)C(O)R9a; N(R9)S(O)2R9a; N(R9)S(O)R9a; N(R9)C(O)OR9a; N(R9)C(O)N(R9aR9b); OC(O)N(R9R9a); T; C1-50by alkyl; C2-50alkenyl or C2-50ukinila, and T, C1-50alkyl, C2-50of alkenyl and C2-50alkynyl optionally substituted by one or more R10that may be the same or different, and where C1-50alkyl, C2-50of alkenyl and C2-50alkynyl optionally interrupted by one or more groups selected from the group consisting of-T-, -C(O)O-; -O-; -C(O)-; -C(O)N(R11)-; -S(O)2N(R11)-; -S(O)N(R11)-; -S(O)2-; -S(O)-; -N(R11S(O)2N(R11a)-; -S-; -N(R11)-; -OC(O)R11; -N(R11)C(O)-; -N(R11)S(Oh)2-; -N(R11)S(O)-; -N(R11)C(O)O-; -N(R11)C(O)N(R11a)- and-OC(O)N(R11R11a);

R9, R9a, R9bindependently selected from the group consisting of H; Z; T; and C1-50alkyl; C2-50alkenyl or C2-50alkynyl, and T, C1-50alkyl, C2-50of alkenyl and C2-50alkynyl optionally substituted by one or more R10that may be the same or different, and where C1-50alkyl, C2-50of alkenyl and C2-50alkynyl optionally interrupted by one or several�mi groups selected from the group consisting of T, -C(O)O-; -O-; -C(O)-; -C(O)N(R11)-; -S(O)2N(R11)-; -S(O)N(R11)-; -S(O)2-; -S(O)-; -N(R11S(O)2N(R11a)-; -S-; -N(R11)-; -OC(O)R11; -N(R11)C(O)-; -N(R11)S(Oh)2-; -N(R11)S(O)-; -N(R11)C(O)O-; -N(R11)C(O)N(R11a)- and-OC(O)N(R11R11a);

T is selected from the group consisting of phenyl; naftel; indenyl; indanyl; tetralinyl;3-10cycloalkyl; 4-7-membered heterocyclyl; or 9-10-membered heterobicycle, wherein T is optionally substituted by one or more of R10that may be the same or different;

R10is Z; halogen; CN; oxopropoxy (=O); COOR12; OR12; C(O)R12; C(O)N(R12R12a); S(O)2N(R12R12a); S(O)N(R12R12a); S(O)2R12; S(O)R12; N(R12)S(O)2N(R12aR12b); SR12; N(R12R12a); NO2; OC(O)R12; N(R12)C(O)R12a; N(R12)S(O)2R12a; N(R12)S(O)R12a; N(R12)C(O)OR12a; N(R12)C(O)N(R12aR12b); OC(O)N(R12R12a); or1-6the alkyl, and C1-6alkyl optionally substituted by one or more Halogens (same or different);

R11, Rl1a, R12, R12a, R12bindependently selected from the group consisting of H; Z; or1-6alkyl, and C1-6alkyl't�certainly substituted by one or more Halogens (same or different);

provided that one of R9, R9a, R9b, R10, R11, R11a, R12, R12a, R12bis Z.

More preferably, L2was a C1-20alkyl chain that is optionally interrupted by one or more groups independently selected from-O - C(O)N(R3aa); was not necessarily substituted by one or more groups independently selected from OH; and C(O)N(R3aaR3aaa) in which R3aa, R3aaaindependently selected from the group consisting of H and C1-4of alkyl.

Preferably, the molecular weight of L2were in the range of from 14 g/mol to 750 g/mol.

Preferably, L2was attached to Z via a terminal group selected from

In the case when L2has a terminal group, more preferably, the molecular weight of L2were in the range of from 14 g/mol to 500 g/mol calculated without this limit group.

Preferably, the covalent bond formed between the linker and hydrogel-Z, was a constant.

Preferably, the hydrogel Z was a water-insoluble biodegradable hydrogel based on polyethylene glycol (PEG). In this document, the term "on the basis of PEG" means that the mass ratio of PEG chains in hydroge�e is at least 10% by weight, preferably, 25%, of the total weight of the hydrogel. The rest of the gel can be other spacers and/or oligomers or polymers, such as oligo - or polylysine.

In addition, the term "water-insoluble" refers to a swelling of three-dimensional cross-linked molecular network forming the hydrogel. Hydrogel when resuspending a large excess of water or water buffer with physiological osmolality may include significant amounts of water, for example, 10-fold weight, and, therefore, is swelling, but after removal of excess water keeps the physical stability and gel form. The gel may take any geometric shape, and it is obvious that such individual hydrogel should be considered as a single molecule, consisting of components, where each component is connected to another through chemical bonds.

According to the invention, the hydrogel may comprise frame parts, connected with each other by hydrolytically degradable bonds.

Preferably, the skeleton of the molecule had a molecular weight in the range from 1 kDa to 20 kDa, more preferably from 1 kDa to 15 kDa, and even more preferably from 1 kDa to 10 kDa. Preferably, the base frame of the molecule was PEG, and the frame portion contained one or more PEG chains.

In the hydrogel, carrier conjuga�s medicinal compound and the linker according to the invention, the skeleton of the molecule is characterized in that it carries a number of functional groups, including biodegradable conjugated functional groups, and United with the hydrogel-drug conjugates compound and the linker, and optionally - kiperousa group. This means that the skeleton of the molecule is characterized by a number United with the hydrogel-drug conjugates compound and the linker; functional groups containing conjugated biodegradable functional groups; and optionally cejrowski groups. Preferably, the amount of conjugated biodegradable functional groups, conjugates of drug compounds with the linker and kiperousa groups was 16-128, preferably 20-100, more preferably, 24 to 80 and most preferably 30-60.

Preferably, the amount of conjugated functional groups, conjugates of drug compounds with the linker and kiperousa groups in the molecule skeleton was equally divided between the number of polymer chains on the basis of the PEG projecting from the point of branching. For example, if the number of conjugated functional groups and conjugates of drug compounds with the linker and kiperousa groups is 32, then each of the four polymer chains on the basis of the PEG projecting from the branch point, we have 8 groups, PR�doctitle through parts of the dendritic molecules attached at the end of each polymer chain based on the PEG. Alternatively, each of the eight polymer chains on the basis of the PEG projecting from the nucleus, we have 4 groups, or 2 groups have on each of the sixteen polymer chains on the basis of PEG. If the number of polymer chains on the basis of the PEG projecting from the branch point, does not allow equal distribution, it is preferred that the deviation from the average of the amount of conjugated functional groups, conjugates of drug compounds with the linker and kiperousa groups on the polymer chain based on the PEG remained minimal.

In such connected to a carrier Pro-drug compounds of the invention it is desirable that the release of almost all medicinal compounds (>90%) occurred before the release of skeleton of molecules in large numbers (<10%). This can be achieved by modifying the half-life of which is connected to a carrier Pro-drug compounds on degradation kinetics of the hydrogel according to the invention.

Preferably, the skeleton of the molecule is characterized in that it has a branch point from which protrude at least three polymer chains on the basis of PEG. Accordingly, in a preferred embodiment the reagent frame includes a branch point, from which protrude by less�her least three polymer chains on the basis of PEG. Such branch point may consist of a poly - or oligospermia in bound form, preferably, of pentaerythritol, of tripentaerythritol, hexagonaria, sucrose, sorbitol, fructose, mannitol, glucose, cellulose, anilos, starches, hydroxyalkyloxy, polyvinyl alcohols, dekstranov, hyaluronate; or branch point may consist of a poly - or oligoamine, such as ornithine, diaminobutane acid, drilizen, tetralin, Pentalgin, hexalin, hepthalites, oktalyzer, nenalezen, decalin, undecaying, dodekanisos, criticality, tetragonality, pentadecanoic or oligomycin, polyethylenimine, polyvinylidine in a bound form.

Preferably, from the point of branching are from three to sixteen polymer chains on the basis of PEG, more preferably, from four to eight. Preferred branch point may consist of, pentaerythritol, ornithine, diaminoalkanes acid, Telesina, tetraline, pentamidine, hexaline, heptamethine or rigolizia, low molecular weight PEI, hexagonaria, tripentaerythritol in a bound form. Preferably, from the point of branching were from three to sixteen polymer chains on the basis of PEG, more preferably, from four to eight. Preferably, the polymer chain is based on the PEG was a linear polyethylene glycol chain�, whose one end is connected to the branch point and the other end attached to hyperazotemia dendritic part. It is obvious that the polymer chain on the basis of PEG may end or be interrupted alkyl or aryl groups, optionally substituted by heteroatoms and chemical functional groups.

Preferably, the polymer chain is based on the PEG was a correspondingly substituted derivative of polyethylene glycol (based on the PEG).

Preferred structures for the respective polymer chains on the basis of the PEG projecting from the branch point contained in the skeleton of the molecule, are multipath derivative of PEG, such as, for example described in the list of products JenKem Technology, USA (available on the website www.jenkemusa.com July 28, 2009), 4 beam PEG-derivatives (PENTAERYTHRITE core), 8-beam PEG-derivatives (exoglycosidase core) 8-beam and PEG-derivatives (tripentaerythritol the core). Most preferred are 4-beam PEG-amine (PENTAERYTHRITE core) 4 beam PEG-carboxyl (PENTAERYTHRITE core), 8-beam PEG-amine (exoglycosidase core), 8-beam PEG-carboxyl (exoglycosidase core), 8-beam PEG-amine (tripentaerythritol the core). The preferred molecular weight for such multipath PEG-derivatives in the skeleton of the molecule SOS�ulation of from 1 kDa to 20 kDa, more preferably, from 1 kDa to 15 kDa and, more preferably, from 1 kDa to 10 kDa.

It is obvious that the terminal amino group of the above-mentioned multi-beam molecules are present in a bound form in the molecule skeleton, providing additional conjugated functional groups and reactive functional groups of a frame of the molecule.

Preferably, the amount of conjugated functional groups and reactive functional groups in the molecule skeleton was equally divided between the number of polymer chains on the basis of the PEG projecting from the point of branching. If the number of polymer chains on the basis of the PEG projecting from the branch point, does not allow equal distribution, it is preferred that the deviation from the average of the sum of conjugated and reactive functional groups on the polymer chain based on the PEG remained minimal.

More preferably, the amount of conjugate and reactive functional groups in the molecule skeleton was equally divided between the number of polymer chains on the basis of the PEG projecting from the point of branching. For example, if the number of conjugated functional groups and reactive functional groups is 32, then each of the four polymer chains on the basis of the PEG projecting from the branch point, when�situated in 8 groups, preferably through dendritic molecules attached at the end of each polymer chain based on the PEG. Alternatively, each of the eight polymer chains on the basis of the PEG projecting from the nucleus, we have 4 groups, or 2 groups have on each of the sixteen polymer chains on the basis of the PEG.

Such additional functional groups can be dendritic molecules. It is preferable that each dendritic molecule had a molecular weight in the range from 0.4 kDa to 4 kDa, more preferably, from 0.4 kDa to 2 kDa. It is preferable that each dendritic molecule had at least 3 branching and at least 4 reactive functional group, and at most 63 branchings and 64 reactive functional groups, preferably at least 7 branches and at least 8 reactive functional groups and at most 31 branching and 32 reactive functional group.

Examples of such dendritic molecules include drilizen, tetralin, Pentalgin, hexalin, hepthalites, oktalyzer, nenalezen, decalin, undecaying, dodekanisos, criticality, tetragonality, pentadecanoic, hexadecanoic, heptadecanoic, octadecanoic, nonadecanoic in a bound form. Examples of such preferred� dendritic molecules include drilizen, tetralin, Pentalgin, hexalin, hepthalites in a bound form, most preferably drilizen, tantalizin or hepthalites, ornithine, diaminobutane acid in bound form.

Most preferably, the hydrogel carrier of the present invention is different in that its frame part has a quarternary carbon of formula C(A-Hyp)4in which each A independently represents a polymer chain on the basis of polyethylene glycol having one end attached to the Quaternary carbon of a permanent covalent bond and the far end of the polymer chain on the basis of PEG was covalently attached to a dendritic molecule Hyp, each dendritic molecule Hyp has at least four functional groups representing the conjugated functional groups and reactive functional groups.

Preferably, each A is independently selected from the formula -(CH2)n1(OCH2CH2)nX-, in which n1 is 1 or 2; n is an integer in the range from 5 to 50; and X is a chemical functional group covalently linking A and Hyp.

Preferably, A and Hyp were covalently connected by amide bond.

Preferably, the dendritic molecule Hyp was hyperazotemia polypeptide. Preferably, g�parasitemia polypeptide contained lysine in a bound form. It is preferable that each dendritic molecule Hyp had a molecular weight in the range from 0.4 kDa to 4 kDa. It is obvious that the skeleton of C(A-Hyp)4may consist of the same or different dendritic Hyp molecules, and each molecule Hyp can be selected independently. Each molecule Hyp consists of 5-32 of lysine, preferably at least 7 of lysine, i.e. each molecule Hyp consists of 5-32 of lysine in a bound form, preferably at least 7 of lysine in a bound form. Most preferably Hyp consisted of heptamethine.

In the reaction of the polymerizable functional groups of the reagent frame, more specifically - Hyp, with polymerizable functional groups of the crosslinking reagents based on the polyethylene glycol forms a permanent amide bond.

Preferably, the molecular weight of C(A-Hyp)4were in the range of from 1 kDa to 20 kDa, more preferably from 1 kDa to 15 kDa, and even more preferably from 1 kDa to 10 kDa.

One preferred skeleton of the molecule shown below, the dotted lines indicate paired connection with biodegradable crosslinking molecules, and n is an integer from 5 to 50:

The ability to biodegradation of the hydrogels of the present invention is achieved by introducing a hydrolytically rassal�the studied relations.

The term "hydrolytically degradiruem", "biodegradable" or "hydrolytically degradable", "autoassembly" or "camaraderie", "kumaraswamy", "transient" or "temporary" refers in the context of the present invention to relationships and connections that refermentation hydrolytically degraded or cleaved under physiological conditions (aqueous buffer pH 7.4, 37°C) with time up to three months, including but not limited to these compounds, aconitine, acetals, amides, anhydrides of carboxylic acids, esters, imine, hydrazones, amides melaminovoi acid, orthoepy, phosphamide, pastefire, silyl esters of phosphorus, silyl esters, sulfonic esters, aromatic carbamates, combinations thereof, etc.

As degradiruem intermolecular functional groups present in the hydrogel according to the invention, preferred biodegradable linkages are ester, carbonates, postevery and esters of sulfonic acid, and most preferred are esters or carbonates.

Permanent links are the links, refermentation hydrolytically degradiruete under physiological conditions (aqueous buffer at pH 7.4, 37°C), with a half-life of six months or more, for example, amides.

For the introduction of hydrolytically degradable linkages in hydrog�left the carrier according to the invention, skeleton of the molecule can directly connect to each other via biodegradable linkages.

In one embodiment the frame of the molecules of the biodegradable hydrogel carrier can be connected together directly, i.e. without the presence of crosslinks. Hyperazotemia dendritic parts of the two frame parts of the molecule such biodegrading hydrogel can be either directly connected through a conjugated functional group that connects the two hyperazotemia dendritic part. Alternatively, two hyperazotemia dendrite different parts of the two frame parts of the molecule can be connected to each other via two spacer connected to the frame part, and its other end connected with linking separated by a conjugated functional groups.

Alternatively, the frame of the molecule can be joined together via crosslinking of the molecule, with each joining the ends of at least two hydrolytically dehydroamino links. In addition to degradiruya ties on the ends of crosslinking may contain more biodegradable linkages. Thus, each end of the cross-linking of the molecule connected to the frame part of the molecule contains a hydrolytically degradable linkage, and, optionally, crosslinking supposedly part�Kula may be additional biodegradable connection.

Preferably, the biodegradable hydrogel carrier frame consisted of parts of the molecule, connected to each other hydrolytically dehydroamino connections, and frame of the molecule were connected together via crosslinking.

Biodegradable hydrogel carrier may contain one or more types of cross-links, preferably one type. Crosslinking of the molecule may be linear or branched molecule, and preferably is a linear molecule. In a preferred embodiment the crosslinking of the molecule is connected to frame parts of the molecule by at least two biodegradable bonds.

Preferably, the crosslinking of the molecule had a molecular weight in the range from 60 Da to 5 kDa, more preferably, from 0.5 kDa to 4 kDa, even more preferably from 1 kDa to 4 kDa, even more preferably from 1 kDa to 3 kDa. In one embodiment the crosslinking part is composed of a polymer.

In addition to oligomeric or polymeric crosslinking parts of the molecule, it is possible to use low molecular weight crosslinking, especially when education of the biodegradable hydrogel according to the invention are hydrophilic high molecular skeleton of the molecule.

Preferably, the linkers on the basis of polyethylene glycol were uglevodorodnye, containing ethylene glycol units, optionally containing additional chemical functional groups, wherein the crosslinking part, on the basis of polyethylene glycol contain at least m ethylene glycol units, where m is an integer in the range from 3 to 100, preferably from 10 to 70. Preferably, the crosslinking based on the polyethylene glycol had a molecular weight in the range of from 0.5 kDa to 5 kDa.

When used in connection with crosslinking or part of the polymer chain on the basis of the PEG connected to the branch point, the term "on the basis of PEG" refers to crosslinking of the molecule or polymer chain based on the PEG containing at least 20% by weight of molecules of ethylene glycol.

In one embodiment, the monomers constituting the polymer crosslinking parts, connected by biodegradable linkages. Such polymeric crosslinking parts can contain up to 100 biodegradable linkages or more, depending on the molecular weight of the crosslinking and the molecular weight of the monomer units. Examples of such crosslinking parts of the molecules are polymers based on polylactic acid or polyglycol acid. Clearly, this chain of polylactic acid or polyglycol acid may end or be interrupted alkyl or aryl groups and may (optional) be �Emesene heteroatoms and chemical functional groups.

Preferably, the crosslinking of the molecule were on the basis of PEG is preferably represented by only one molecular chain based on the PEG. Preferably, the crosslinking of the molecule on the basis of polyethylene glycol were hydrocarbon chains containing ethylene glycol units, optionally containing additional chemical functional groups, wherein the crosslinking part, on the basis of polyethylene glycol contain at least m ethylene glycol units, where m is an integer in the range from 3 to 100, preferably from 10 to 70. Preferably, the crosslinking of the molecule on the basis of polyethylene glycol had a molecular weight in the range of from 0.5 kDa to 5 kDa.

In a preferred embodiment of the present invention, crosslinking of the molecule consists of a PEG, which is symmetrically connected via ether bonds with two alpha,omega-aliphatic dicarboxylic spacers, provided that the skeleton of the molecule is connected to hyperazotemia dendritic part through the permanent amide bond.

The GS spacer dicarboxylic acid, United with a frame part of the molecule, and on the other side - stitching part, consist of 3-12 carbon atoms, most preferably 5-8 carbon atoms and may be substituted by one or more carbon atoms. Prefer�ranked substituents are alkyl groups, hydroxyl group or aminogroup, or substituted amino group. One or more aliphatic methylene groups in the dicarboxylic acids may be optionally substituted by O or NH or alkyl-substituted N. Preferred alkyl is a linear or branched alkyl with 1-6 carbon atoms.

Preferably, the constant presence of the amide bond between hyperazotemia dendritic part and a spacer connected to the frame part and the other side connected to a cross-linking molecule.

One preferred crosslinking of the molecule shown below; the dotted lines indicate paired biodegradable connection with the skeleton parts:

in which n is an integer from 5 to 50.

Preferably, the hydrogel carrier consisted of frame parts connected hydrolytically dehydroamino bonds.

More preferably, the frame contain a branch point to the following formula:

in which the dotted line indicates the connection to the rest of the frame.

More preferably, the frame portion of the molecules contain a structure with the following formula:

in which n is an integer from 5 to 50, and the dotted line indicates the connection to the other�the second part of the framework.

Preferably, the skeleton of the molecule contained hyperazotemia part of Hyp.

More preferably, the frame portion contained hyperazotemia part of Hyp to the following formula:

in which the dotted line indicates the connection to the rest of the skeleton of the molecule and carbon atoms marked with an asterisk indicate the S-configuration.

Preferably, the frame parts were attached by at least one spacer of the following formula:

in which the dotted line indicated by the asterisk indicates the bond between the hydrogel and nitrogen disuccinimidyl group,

in which the other dotted line indicates adherence to Hyp, and

in which R is an integer from 0 to 10.

Preferably, the frame parts were attached by at least one spacer of the following formula:

in which one of the dotted lines indicates the joining hyperazotemia part of the Hyp and the second dashed line indicates the connection to the rest of the molecule:

in which m is an integer from 2 to 4.

Preferably, the frame parts are connected with each other via cross-linking of the molecule with the following structure:

in which

q is an integer from 3 to 100;

on the rate of hydrolysis of the biodegradable linkages between the frame parts and parts crosslinking effect or the speed of hydrolysis determines the number and type of connected atoms adjacent to the PEG-ether carboxylic group. For example, choosing succinic, adipic or glutaric acid to form esters with PEG can be varied half time of degradation of the biodegradable hydrogel carrier according to the invention.

The total frame number of molecules can be measured in the solution after complete degradation of the hydrogel of the invention and within the fraction of soluble degradation products the degradation of the frame can be separated from the insoluble hydrogel according to the invention and explore without interference from other soluble degradation products released from the hydrogel according to the invention. The hydrogel according to the invention can be separated from excess water in the buffer with physiological osmolality by sedimentation or by centrifugation. Centrifugation can be performed so that the supernatant will be at least 10% of the volume of the swollen hydrogel according to the invention. After this stage sedimentation or centrifugation of the soluble degradation products of the hydrogel remains in the aqueous supernatant and water-soluble degradation products, aderrasi� one or more molecules of the frame detects, using aliquots of the supernatant in the respective methods of separation and/or analysis.

Preferably, the water-soluble degradation products can be separated from water-insoluble degradation products by filtering through 0.45 µm filters, after which the water-soluble degradation products can be found in the breakthrough. Water-soluble degradation products also can be separated from water-insoluble degradation products in a combination of stages of centrifugation and filtration.

For example, the skeleton of the molecule can carry groups, the UV absorption spectrum which does not coincide with the UV absorption spectrum of the other degradation products. Such groups with selective absorption in the UV spectrum can be structural components of the skeleton of the molecule, as, for example, amide bond, or they can be entered in wireframe molecule attaching to its reactive functional groups of the aromatic ring systems such as indole group.

In such related hydrogel Pro-drug compounds of insulin according to the invention it is desirable that the release of almost all medicinal compounds (>90%) occurred prior to the release of significant amounts skeleton of molecules (<10%). This can be achieved by modifying the half-life associated with the media prolec�stonoga connection with respect to the kinetics of degradation of the hydrogel according to the invention.

Associated with the hydrogel Pro-drug compound of insulin of the present invention can be obtained from the hydrogel of the present invention by standard methods known in this field. For a practitioner in this field will be obvious that there are several approaches. For example, Pro-drug linker that is listed above, to which is covalently attached biologically active molecule, i.e. insulin, can react with the reactive functional groups of the hydrogel of the present invention, already bearing the active molecule, i.e., insulin, or without it, in part or in whole.

In a preferred method of producing the hydrogel synthesized by chemical ligation. The hydrogel can be obtained from two macromolecular products with complementary functional groups that participate in the reaction, for example, the condensation reaction or accession. One source of these substances is a crosslinking reagent with at least two identical functional groups, and the other of the source substance is communitybanksonline skeleton reagent. Suitable functional groups present on the crosslinking reagent, include the terminal amino group, carboxylic acids and derivatives, maleimide and other alpha-beta unsaturated accep�ora Michael, such as vinylsulfonate, a thiol, a hydroxyl group. Suitable functional groups present on the frame reagent include, but are not limited to these, amino group, carboxylic acids and derivatives, maleimide and other alpha-beta unsaturated Michael acceptors, such as vinylsulfonate, a thiol, a hydroxyl group.

If the polymerizable group of the crosslinking reagent to use in substochiometric amounts relative to polymerizable groups of the frame, the resulting hydrogel will be reactive hydrogel with the free reactive functional groups attached to the frame structure.

Optionally, the Pro-drug linker compounds can first be konjugierte with insulin and the insulin conjugate with the Pro-drug linker compounds can then react with the reactive functional groups of the hydrogel. Alternatively, after activation of one of the functional groups of the Pro-drug linker compounds, the linker conjugate with the hydrogel may come into contact with insulin on the second stage of the reaction, and the excess insulin can be removed by filtration after conjugation of insulin with the Pro-drug linker compounds related to hydrogel.

The preferred method for the preparation�Oia Pro-drug compounds of the present invention is the following method:

The preferred starting material for the synthesis of the skeleton of the reagent is 4-beam PEG-tetraamine or 8-beam PEG-octalin, and the molecular weight of the PEG-reagent is in the range from 2000 to 10,000 daltons, most preferably in the range from 2000 to 5000 Da. Such multipath derivative of PEG lysine residues are joined sequentially, forming hyperazotemia skeleton reagent. Obviously, lysine residues can be partially or fully protected by protective groups during the stages of accession, and that is also the final frame, the reagent may contain a protective group. Preferred building block is bis-BOC-lysine. Alternatively, instead of stringing residues of lysine, you can first synthesize dendritic polylysine part of the molecules continue to attach to 4-beam PEG-tetraamine or 8-beam PEG-octomino. It is advisable to get a wireframe reagent bearing 32 amino group, respectively, seven lysine residues are attached to each shoulder 4 beam PEG or five lysine residues will be attached to each arm 8-beam PEG. In another embodiment of the invention multibeam PEG derivative is Tetra - or octacarbonyl-PEG. In this case, the dendritic part of the molecule can be synthesized from glutaric and or�paraginomai acid, and the received frame, the reagent will be 32 carboxyl group. It should be understood that all or part of the frame functional groups of the reagent may be present in free form, in the form of salts, or may be anywhereman with protective groups. It should be understood that for practical reasons the number of lysine residues skeleton of the reagent on the shoulder of the PEG will range from six to seven, more preferably, approximately, seven.

The preferred frame of the reagent is shown below:

Synthesis of crosslinking reagent begins with a linear PEG chain with a molecular weight in the range of 0.2 to 5 kDa, more preferably, from 0.6 to 2 kDa, esterified by poluation dicarboxylic acids, mainly of adipic acid or glutaric acid. A preferred protective group for the formation of Palmyra is a benzyl group. The resulting Palmyra bis(dicarboxylic acid) and PEG turn into more reactive carboxyl compounds, such as acylchlorides or active esters, for example, pentafluorophenyl or N-hydroxysuccinimide esters, the most preferred are N-hydroxysuccinimide esters, of which preferred the chosen structure is shown below.

in which each m independently I�is an integer in the range from 2 to 4, and

q is an integer from 3 to 100.

More preferred is the following structure:

Alternatively, Palmyra bis(dicarboxylic acid) and PEG can be activated in the presence of a crosslinking reagent, such as DCC or HOBt, or PyBOP.

In an alternative embodiment the reagent frame carries a carboxyl group, and a suitable crosslinking reagent is selected from ending with the amino groups of the PEG chains containing ester groups.

Frame the reagent and crosslinking reagent can polymerize with the formation of the hydrogel according to the invention, using a polymerization in emulsion facing. After selecting the desired stoichiometric ratio between the polymerizable groups of the frame and cross-linking, frame connection and the stitching is dissolved in DMSO, and use a suitable emulsifier with an appropriately chosen value of hydrophilic-lipophilic balance, preferably, Arlacel P 135, for the formation of inverted emulsions, using a mechanical stirrer and controlling the mixing speed. The polymerization was induced by addition of the appropriate substrate, preferably, N,N,N',N'-tetramethylethylenediamine. After stirring for an appropriate period of time the reaction is stopped by adding acid, for example �kusnoy acid, and water. The bulbs are harvested, washed and fractionary in size by mechanical sieving. Not necessarily, at this stage you can remove the protective group.

In an alternative embodiment of the present invention multi-functional part of the molecule joined to the reactive functional groups of the polymerized reactive hydrogel to increase the number of functional groups, which will increase the treatment capacity of the hydrogel. Such multi-functional parts of the molecule can be suitably substituted derivatives of lysine, dilysine, Telesina, tetraline, pentamidine, hexaline, heptamethine or rigolizia, low molecular weight PEI. Preferred multi-functional part of the molecule is lysine.

In addition, such a hydrogel according to the invention can be functionalized spacer, bearing the same functional group, for example, the hydrogel can introduce amino group by joining heterobifunctional of the spacer, for example, respectively, of the activated COOH spacer-(EG)6-NH-fmoc (EG=ethylene glycol), and removing the fmoc protective group.

After the load functionalized hydrogel containing maleimide group, a conjugate of insulin and the linker, to prevent undesirable side reactions all leave�Xia functional groups kaparot appropriate blocking reagent, for example, mercaptoethanol.

In a preferred embodiment the conjugate of insulin with a linker having a free Tilney group connected to the linker part, reacts with the hydrogel bearing maleimide functional groups, at a temperature from ambient to 4°C, preferably at room temperature, in a buffered aqueous solution of pH 2-5, preferably with a pH of 2.5-4.5, more preferably pH 3.0 to 4.0. Next, the corresponding conjugate of insulin, linker and hydrogel treated with mercaptoethanol at temperatures from ambient to 4°C, preferably at room temperature, in a buffered aqueous solution of pH 2-5, preferably pH 2.5 to 4.0, more preferably pH 2.5 to 3.5.

In another preferred embodiment the insulin conjugate with a linker carrying maleimido group connected to the linker part, reacts with the hydrogel having a thiol functional group, at a temperature from ambient to 4°C, preferably at room temperature, in a buffered aqueous solution of pH 2-5, preferably with a pH of 2.5-4.5, more preferably pH 3.0 to 4.0. Further, the corresponding conjugate of insulin, linker and hydrogel treated with low-molecular-weight compound containing maleimido group, preferably, maleimide-containing with�unity with weight from 100 to 300 Yes for example, N-ethyl-maleimide, at a temperature from ambient to 4°C, preferably at room temperature, in a buffered aqueous solution of pH 2-5, preferably pH 2.5 to 4.0, more preferably pH 2.5 to 3.5.

Another aspect of the present invention is a method comprising the following steps:

(a) contact of an aqueous suspension containing hydrogel microparticles with maleimide functional groups, with a solution containing the insulin-linker reagent, bearing a thiol group, at a temperature from ambient to 4°C in aqueous buffer solution with a pH of 2-5, producing a conjugate of insulin, linker and hydrogel;

(b) optionally, processing of the insulin conjugate, the linker and hydrogel from step (a) a thiol-containing compound with a mass of from 34 to 500 kDa at a temperature from ambient to 4°C in aqueous buffer solution with a pH of 2-5.

Another aspect of the present invention is a method comprising the following steps:

(a) contact of an aqueous suspension containing hydrogel microparticles with toolname functional groups, with a solution containing the insulin-linker reagent carrying maleimide group, at a temperature from ambient to 4°C in aqueous buffer solution with a pH of 2-5, producing a conjugate of insulin, linker and hydrogel;

(b) optionally, processing of the insulin conjugate, the linker and hydrogel from the stud�and (a) maleimide-containing compound with a mass of from 100 to 300 kDa at a temperature from ambient to 4°C in aqueous buffer solution with a pH of 2-5.

Particularly preferred method of obtaining a Pro-drug compounds of insulin of the present invention comprises the steps:

(a) reaction of a compound with the formula C(A'-X1)4in which A'-X1represents A before its binding to Hyp or a precursor of Hyp and X1is an appropriate functional group, with a compound having the formula Hyp'-X2in which Hyp'-X2represents Hyp before its binding to A or predecessor Nur, and X2is a suitable functional group to participate in the reaction with X1;

(b) optionally, the participation of the compounds obtained in stage (a), in the reactions of one or more additional stages with the release of the compounds having the formula C(A-Hyp)4at least four functional groups;

(C) reaction of at least four functional groups of the compounds obtained in stage (b) with a crosslinking reagent on the basis of polyethylene glycol in which the reactive group of the crosslinking reagent is taken in substochiometric number / total number of reactive groups C(A-Hyp)4as a result of which forms a hydrogel;

(d) reaction of the remaining unreacted functional groups (representing the reactive functional groups of the frame contained in the hydrogel present�Adamu to the invention) in the frame of the hydrogel from step (C) with a covalent conjugate of insulin and transient Pro-drug linker compounds; or first the reaction of unreacted functional groups with the transient Pro-drug linker compound, and further with insulin;

(e) optionally, the phase of kupirovaniya remaining unreacted functional groups that give the Pro-drug compound of the present invention.

More specifically, hydrogels for Pro-drug compounds of insulin of the present invention are synthesized as follows:

For bulk polymerization skeleton reagent and crosslinking reagent are mixed in the ratio of amino groups to reactive ester groups is from 2:1 to 1.05:1.

Both reagents, frame and crosslinking, dissolved in DMSO, yielding a solution with a concentration of from 5 to 50 g per 100 ml, preferably from 7.5 to 20 g per 100 ml and, most preferably, from 10 to 20 g per 100 ml.

For the implementation of the polymerization solution in DMSO containing crosslinking reagent and reagent frame, add from 2 to 10% (volume) N,N,N',N'-tetramethylethylenediamine (TMEDA), and the mixture shaken for 1-20 seconds and leave still. The mixture hardens less than 1 min.

Such a hydrogel according to the invention is preferably finely chop mechanical means, such as mixing, crushing, cutting, pressing or grinding, and optionally sieving. In the case of emulsion polymerization, the reaction mixture �consists of a dispersed phase and the dispersion phase.

For dispersed phase skeleton reagent and crosslinking reagent are mixed in the ratio of amino groups to an active ester groups is from 2:1 to 1.05:1 and dissolved in DMSO, yielding a solution with a concentration of from 5 to 50 g per 100 ml, preferably from 7.5 to 20 g per 100 ml and, most preferably, from 10 to 20 g per 100 ml.

The dispersion phase is any solvent that is not miscible with DMSO, is non-core, aprotic and has a viscosity below 10 PA*sec. Preferably, the solvent that is not miscible with DMSO, was non-core, aprotic and had a viscosity below 2 PA*s, and was not toxic. More preferably, the solvent was saturated linear or branched hydrocarbon of 5-10 carbon atoms. More preferably, the solvent was n-heptane.

For the formation of the emulsion dispersed phase in continuous phase before the addition of dispersed phase to continuous phase is added an emulsifier. The amount of emulsifier is from 2 to 50 mg per ml of the dispersed phase, more preferably, from 5 to 20 mg per ml of the dispersed phase, most preferably 10 mg / ml dispersed phase.

The HLB value of the emulsifier is from 3 to 8. Preferably, the emulsifier was triavir sorbitol and fatty acids or conjugate polyhydroxystearic acid and polietilene�Kohl. More preferably, the emulsifier was conjugate polyhydroxystearic acid and of polyethylene glycol, linear polyethylene glycol with a molecular weight in the range of from 0.5 kDa to 5 kDa and links polyhydroxystearic acid with a molecular weight in the range of from 0.5 kDa to 3 kDa at the end of each circuit. The most preferred emulsifier is a polyethylene glycol-dipolyhydroxystearate, Citral DPHS (Cithrol DPHS, former name Arlacel P135, Croda International Plc).

The drops of the dispersed phase is obtained by stirring with an axial flow mixer having a geometry similar to stirrers such as Isojet, Intermig, Propeller (EKATO Ruhr - und Mischtechnik GmbH, Germany), most preferably similar to Isojet with diameter from 50 to 90% of the diameter of the reaction vessel. Preferably start mixing before adding the dispersed phase. The speed of the stirrer can be set from 0.6 to 1.7 m/sec. Dispergirovannoyj phase is added at room temperature, and the concentration of the dispersed phase ranges from 2% to 70%, preferably from 5 to 50%, more preferably from 10 to 40%, and most preferably from 20 to 35% of the total volume of the reaction. Before adding the base, initiating the polymerization, a mixture of dispersed phase, the emulsifier, and the dispersion phase was stirred for 5-60 minutes.

To a mixture of dispersed and continuous phase d�billaut from 5 to 10 equivalents of base relative to each of the formed amide bond). The base is aprotic, dinucleophiles and soluble in the dispersion phase. Preferably, the aprotic base, dinucleophiles, soluble, in the dispersion phase and in DMSO. More preferably, the aprotic base, dinucleophiles, soluble, in the dispersion phase and in DMSO, was aminoadenosine and was not toxic. The most preferred base is N,N,N',N'-tetramethylethylenediamine (TMEDA). Stirring in the presence of base is continued for 1 to 16 hours.

When mixing the droplets of dispersed phase solidifies, becoming crosslinked hydrogel beads according to the invention, which can be collected and a fractionation by size continuous vibration screening machine deck with 75 ál 32 ál, getting hydrogel microparticles according to the invention.

Hydrogel for Pro-drug compounds of insulin of the present invention can be obtained in the form of microparticles. In a preferred embodiment the reactive hydrogel is a subject of some form, as, for example, mesh or stent. Most preferably, the hydrogel was in the form of microspheres that can be entered via subcutaneous or intramuscular injection using a standard syringe. �what soft balls can have a diameter of 1-500 micrometers.

If microparticles resuspendable in an isotonic aqueous buffer, preferably, the diameter of the microparticles ranged from 10 to 100 micrometers, most preferably from 20 to 100 μm, most preferably from 25 to 80 micrometers.

Preferably, the microparticles can be entered using injection through a needle with an inner diameter less than 0.6 mm, preferably through a needle with an internal diameter of less than 0.3 mm, more preferably through a needle with an internal diameter of less 0,225 mm, even more preferably through a needle with an internal diameter of less than 0.175 mm and, most preferably, through a needle with an internal diameter of less than 0.16 mm.

It should be understood that the terms "may be administered by injection", "injection" or "ineterest" refers to a combination of factors, such as some of the force exerted on the plunger of the syringe containing biodegradable hydrogel according to the invention swollen in a liquid at a certain concentration (mass/volume) and at a certain temperature; this needle inner diameter at the end of the syringe; and the time required to displace a certain volume of the biodegradable hydrogel according to the invention from the syringe through the needle.

To ensure ineterest volume of 1 ml Pro-drug compounds of insulin according to the invention swollen in water to a concentration of p� least 5% (weight/volume) and contained in a syringe with a plunger with a diameter of 4.7 mm, can be superseded at room temperature for 10 seconds when a force of less than 50 Newton.

Preferably, ineterest achieved the Pro-drug compounds of insulin according to the invention swollen in water to a concentration of approximately 10% (weight/volume).

In an additional embodiment the composition is characterized in that it is a liquid composition, which after the injection forms a depot preparation.

In an additional embodiment the composition is characterized in that it is administered by injection, e.g., subcutaneous or intramuscular.

In an additional embodiment, the composition is intended for treating or preventing diseases or disorders associated with deficiency of insulin, treatment or prevention of which is the use of compounds of insulin is beneficial, such as hyperglycemia, prediabetes, impaired glucosetolerance, type I diabetes, type II diabetes, syndrome X. Usually, this disease or disorder is type II diabetes.

In an additional embodiment the compound selected from insulin or insulin analogues human insulin or other species, and their derivatives and conjugates. More preferred is�I insulin analogues human insulin, as, for example, insulin glargine, insulin detemir, insulin lispro., insulin aspartate, insulin glulisine. With the introduction of a maximum peak concentration of the compounds of insulin is usually achieved within the first 48 hours. In an additional embodiment, the peak concentration is achieved within the first 24 hours after administration, for example, in the first 12 hours after administration, for example, during the first 6 hours after administration.

In an additional aspect, the pharmaceutical composition of the present invention further comprises a compound GLP-1, usually, the agonist of GLP-1.

Such a compound GLP-1 is generally selected from any one of:

[Seq ID No:1] Bloomington: Indiana-4

HGEGTFTSDL SKQMEEEAVR LFIEWLKNGG PSSGAPPPS-NH2

[Seq ID No:2] Bloomington: Indiana-3

[Seq ID No. 13] GLP-1 (7-36)amide

Where the amino acid XAA is selected from P, F, Y.

Where the amino acid XAA is selected from T, α-aminobutyric acid, D-Ala, V, Gly.

Where R is selected from acetyl, pyroglutamyl, N-2-hydroxybenzoyl, N-TRANS-3-hexenol.

[Seq ID No. 20]

HXaaAEGTFTSDV SSYLEGQAAK EFIAWLVKGR-NH2

Where the amino acid XAA is 6-aminohexanoic.

In an additional aspect, the present invention relates to the use of compounds of insulin to obtain the pharmaceutical industry�tion of the composition, containing a compound of insulin in a concentration sufficient to maintain therapeutically effective levels of the compounds of insulin in blood plasma for at least 3 days, usually at least 80 hours, for example, during the week or over a longer period of time, characterized by the fact that it has a pharmacokinetic profilein vivoessentially no release of the connection of insulin, to treat or prevent diseases or disorders associated with deficiency of insulin, treatment or prevention of which is the use of compounds of insulin is useful.

This concentration will vary from subject to subject and will depend on therapeutic window in individual subject, but for the duration of therapeutic effect for at least 3 days, for example, weeks (i.e., approximately 7 days), the concentration is typically at least about 10 mg/ml, e.g., greater than 10 mg/ml.

The preferred composition of the Pro-drug compounds of insulin-hydrogel are given in the following paragraphs.

Composition Pro-drug compounds of insulin-hydrogel may be a suspension composition or a dry composition. Preferably, the pharmaceutical composition Pro-drug compounds of insulin-hydrogel before�Talala a dry composition. Suitable methods of drying include, for example, spray drying and lyophilization (drying and freezing). Preferably, the dry pharmaceutical composition of the Pro-drug compounds of insulin-hydrogel using lyophilization.

Preferably, the composition contains a dose of Pro-drug compounds of insulin-hydrogel sufficient to provide a therapeutically effective amount of insulin for at least three days, under one administration. More preferably, a single injection Pro-drug compounds of insulin-hydrogel was enough for one week.

Pharmaceutical composition Pro-drug compounds of insulin-hydrogel of the present invention contains one or more excipients.

Excipients used in parenteral compositions can be classified as buffering agents, isotonicity modifiers, preservatives, stabilizator, anti-adsorption agents, agents that protect against oxidation, thickening agents/agents that increase the viscosity, or other auxiliary agents. In some cases, these ingredients can have dual or triple functions. Composition Pro-drug compounds of insulin-hydrogel of the present invention contain one or more excipients, selected�x group, consisting of:

(i) Buffering agents: physiologically compatible buffer to maintain the pH within a predetermined range, such as sodium phosphate, bicarbonate, succinate, histidine, citrate and acetate, sulfate, nitrate, chloride, pyruvate. You can also use antacids such as Mg(OH)2or ZnCO3. A buffer tank can fail to meet the conditions that are most sensitive to pH stability.

(ii) isotonicity Modifiers: to minimize the pain that can occur as a result of cell damage due to the difference of osmotic pressure in the place of depot injections. Examples are glycerol and sodium chloride. Effective concentrations can be determined using osmometry using customary for serum osmolality - 285-315 mosmol ' /kg.

(iii) Preservatives and/or antimicrobial agents: to minimize the risk of contamination from the injection in multidose parenteral preparations need to add preservatives in sufficient concentrations, and installed by the appropriate regulatory requirements. Typical preservatives include m-cresol, phenol, methylparaben, ethylparaben,, propylparaben, butylparaben, chlorobutanol, benzyl alcohol, nitrate of finalstate, thimerosal, sorbic acid, potassium sorbate, benzoic acid, chlorocresol and benzalkonium chloride.

(iv) Stabilizat�ditch: stabilization is achieved by higher forces stabilizing protein, destabilization of the denatured state, or by direct binding of excipients to the protein. Stabilizers can be amino acids such as alanine, arginine, aspartic acid, glycine, histidine, lysine, Proline, sugars, such as glucose, sucrose, trehalose, polyols, such as glycerol, mannitol, sorbitol, salts such as potassium phosphate, sodium sulfate, chelating agents such as EDTA, hexaphosphate, ligands such as bivalent metal ions (zinc, calcium, etc.), other salts or organic molecules, such as phenolic derivatives. It is also possible to use oligomers or polymers such as cyclodextrins, dextran, dendrimers, PEG or PVP, or Protamine or HSA.

(v) Anti-adsorption agents: for a competitive inner surface or the competitive adsorption on the inner surface of the container containing the composition, mainly ionic or non-ionic surfactants or other proteins or soluble polymers. Examples are poloxamer (Pluronic F68), PEG-dodecylamine (Brij 35), Polysorbate 20 and 80, dextran, polyethylene glycol, PEG-polyhistidine, BSA and HSA, and gelatin. The choice of the concentration and type of excipients depend on the effect to be avoided, but usually the monolayer surface�but-active substances is formed on the surface of the phase slightly above the critical micelle concentrations.

(vi) Lio - and/or cryoprotectants: during lyophilization or spray drying auxiliary substances can counteract the destabilizing effects caused by the destruction of hydrogen bonds and removal of water. For this purpose you can use sugars and polyhydric alcohols, but the corresponding positive effects were observed with use of surface-active substances, amino acids, non-aqueous solvents and other peptides. Trehalose is particularly effective for reducing the aggregation caused by humidity, and it also increases thermal stability of the protein, potentially caused by exposure of hydrophobic groups of the protein in water. You can also use mannitol and sucrose, or as the only Leo/cryoprotectant or in combination with each other, and it is known that a high ratio of mannitol to sucrose increases the physical stability of lyophilized residue. Mannitol can also be combined with trehalose. Trehalose can also be combined with sorbitol or sorbitol as the sole protectants. You can also use starch or derivatives of starch.

(vii) Agents that protect against oxidation: antioxidants such as ascorbic acid, ectoin, methionine, glutathione, monothioglycerol, Morin, polyethylenimine (PEI), propylgallate, vitamin E, hela�yousie agents, such as citric acid, EDTA, hexaphosphate, thioglycolic acid.

(viii) Thickeners/agents that increase the viscosity and slow down the deposition of particles in the vial and syringe are used to facilitate mixing and resuspension of particles, and also for easier introduction of slurry by injection (i.e., for the application of less force to the syringe plunger). Suitable thickeners or amplifiers viscosity are, for example, carboneria thickeners such as Carbopol 940, Carbopol Ultrez 10, cellulose derivatives such as hydroxypropylmethyl cellulose (polymer, HPMC) or diethylaminoethylcellulose (DEAE or DEAE-C), colloidal magnesium silicate (Veegum) or sodium silicate, the gel hydroxyapatite, tricalcium phosphate gel, xantana, carrageenans, such as Satia gum 30 UTC, aliphatic polyhydroxylated, such as poly (D,L - or L-lactic acid) (PLA) and polyglycol acid (PGA), and their copolymers (PLGA), terpolymers of D,L-lactide, glycolide and caprolactone, poloxamer, hydrophilic polyoxyethylene blocks and hydrophobic polyoxypropylene blocks to create triblock polyoxyethylene-polyoxypropylene-polyoxyethylene (e.g. Pluronic®), a copolymer of simple and complex polyesters, for example, a copolymer of polyethylene-terephthalate and polybutylene-terephthalate, isobutyrate acetate sucrose (SAIB), dextran or its manufacturing�derivative, the combination of dekstranov and PEG, polydimethylsiloxane, collagen, chitosan, polyvinyl alcohol (PVA) and derivatives, polyalkylimide, poly(copolymer of acrylamide and diallyldimethylammonium (DADMA)), polyvinylpyrrolidone (PVP), glycosaminoglycans (GAG), such as dermatan sulfate, chondroitin sulfate, keratan sulfate, heparin, heparan sulfate, hyaluronan, ABA-tribloc or AB-block copolymers composed of hydrophobic A-blocks, such as polylactide (PLA) or a copolymer of lactide and glycolide (PLGA), and hydrophilic B-blocks, such as polyethylene glycol (PEG) or polyvinylpyrrolidone. Such block copolymers, as well as the above poloxamer can exhibit behaviors, the reverse thermal gelation (liquid state at room temperature to facilitate the introduction of the connection and the gel above the transition temperature of a liquid into a gel at body temperature after injection).

(ix) Agent that increases the distribution or diffusion: modifies the permeability of connective tissue as a result of hydrolysis of extracellular matrix components in the interstitial space, for example (but not limited to these), hyaluronic acid, a polysaccharide found in the intercellular matrix of connective tissue. Agent that increases the distribution, such as, but not limited to, the hyaluronidase temporarily reduces the viscosity of the extracellular Matri�sa and enhances the diffusion of the medicinal compounds administered through injection.

(x) Other auxiliary agents such as wetting agents, viscosity modifiers, antibiotics, hyaluronidase. Acids and bases, such as hydrochlorate acid and sodium hydroxide, are auxiliary agents required for leading of pH in the manufacture of the connection.

Preferably, the composition Pro-drug compounds of insulin-hydrogel contain one or more substances from the thickener and/or agent, viscosity modifier.

The term "excipient" preferably refers to a diluent, adjuvant or carrier, which is introduced therapeutic connection. Such pharmaceutical excipients can be sterile liquids, such as water and oils, including petroleum oil and oils of animal, vegetable or synthetic origin, including but not limited to this, peanut oil, soybean oil, mineral oil, sesame oil, etc. Water is the preferred auxiliary substance by oral administration of the pharmaceutical composition. Saline and aqueous dextrose solution are preferred adjuvants when administered intravenously, the pharmaceutical composition. Saline, aqueous solutions of glucose and glizer�on is preferably used as liquid excipients for injection solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, etc. Composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can be solutions, suspensions, emulsion, tablets, pills, capsules, powders, compositions with delayed release, etc., the Composition may be presented as a suppository, with traditional binders and excipients such as triglycerides. Oral composition can include standard excipients such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharinate, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical excipients are described in "Remington's Pharmaceutical Sciences" authored by E. W. Martin. Such compositions will contain a therapeutically effective amount of a therapeutic compound, preferably in purified form, together with the appropriate amount of excipients that provide the form for proper administration to the patient. The composition should �to ootvetstvovali routes of administration.

In a General embodiment the pharmaceutical composition of the present invention either in dry form or in suspension form or in different dosage forms can be a single-dose or multi-dose composition.

In one embodiment of the present invention is a dry composition Pro-drug compounds of insulin-hydrogel presented in the form of single-dose composition, meaning that the container in which it is supplied contains one pharmaceutical dose.

Therefore, in another aspect of the present invention the composition is provided as a single-dose of the composition.

Preferably, the slurry composition was multidose composition, meaning that it contains more than one therapeutic dose. Preferably, the multi-dose composition contained at least 2 doses. Such multi-dose composition of insulin-hydrogel can be used either in different patients, in need, or it can be adapted for use with a single patient, in which case after the first dose, the remaining doses are stored until you need them.

In another aspect of the present invention, the composition contained in the container. Preferably, the container was double chamber syringe. In particular, the dry compositing�tion of the present invention is in the first chamber, two-chamber syringe, and the solution to restore is located in the second chamber dual-chamber syringe.

Before the introduction of the dry composition Pro-drug compounds of insulin-hydrogel to a patient in need this, restore the dry composition. Recovery can be performed in the container in which the dry composition of the Pro-drug compounds of insulin-hydrogel, e.g., vial, syringe, dual-chamber syringe, ampoule, and cartridge. The restoration is carried out by adding a certain number of solution for the restoration to the dry composition. Solutions for recovery are sterile liquids such as water or buffer, which may contain additional additives such as preservatives and/or antimicrobial agents. If the composition of the Pro-drug compounds of insulin-hydrogel is presented in single-dose form, then the solution is to restore may contain one or more preservatives and/or antimicrobial agents. Preferably, a solution for the restoration was sterile water. If the composition of the Pro-drug compounds of insulin-hydrogel is a multi-dose composition, it is preferable that the solution to restore contain one or more preservatives and/or antimicrobial agents such as, for example, benzyl alcohol and cresol.

Additional�the DIY aspect of the present invention relates to a method of introducing the recovered composition Pro-drug compounds of insulin-hydrogel. The composition of the Pro-drug compounds of insulin-hydrogel can be entered via injection or infusion, including intradermal, subcutaneous, intramuscular, intravenous, intraosseous, and intraperitoneal.

An additional aspect is a method of obtaining a restored composition containing a therapeutically effective amount of Pro-drug compounds of insulin, and optionally one or more pharmaceutically acceptable excipients, wherein the transient insulin is connected to the hydrogel, the method includes the stage of:

• contact the compositions of the present invention with a solution to restore.

Another aspect is a composition for restoring, containing a therapeutically effective amount of Pro-drug compounds of insulin-hydrogel and optionally one or more pharmaceutically acceptable excipients, wherein the transient insulin attached to the hydrogel obtained by the above method.

Another aspect of the present invention is a method of making a dry composition Pro-drug compounds of insulin-hydrogel. In one embodiment, this suspension arrangement is made as a result of:

(i) mixing Pro-drug compounds of insulin-hydroge�I with one or more adjuvants,

(ii) transfer a quantity of one or multiple doses into a suitable container

(iii) drying the composition in the specified container, and

(iv) sealing the container.

Suitable containers are vials, syringe, dual-chamber syringes, ampoules and cartridges.

Another aspect is the set. If the device for introduction is just the hypodermal syringe then the kit may include a syringe, a needle and a container containing a dry composition Pro-drug compounds of insulin-hydrogel for use with a syringe, and a second container containing a solution for recovery. In more preferred embodiments, the injection device is different from ordinary hypodermal syringe, and a separate container with restored Pro-drug compound insulin-hydrogel is adapted to the injection device so that when the liquid composition in the container was connected to the outlet of the injection device with the possibility of transfer of the fluid medium. Examples of devices for administration include, but are not limited to this, hypodermal syringes and insulin pen. Particularly preferred devices for introduction are the pen, in which case the container is a cartridge, preferably one�gas cartridge.

A preferred kit includes a needle and a container containing the composition according to the present invention and optionally further comprising an solution to restore, wherein the container is adapted for use with a needle. Preferably, the container was double chamber syringe.

In another aspect, the invention relates to a cartridge containing a composition of Pro-drug compounds of insulin-hydrogel described above for use with the syringe handle. The cartridge may contain a single dose or multiple dose of insulin.

In one embodiment of the present invention, the slurry composition Pro-drug compounds of insulin-hydrogel contains not only the Pro-drug compound of insulin-hydrogel and one or more auxiliary substances, but also contains other biologically active agents, either in free form or in the form of Pro-drug compounds. Preferably, these one or more biologically active agents have been Pro-drug compounds, more preferably, Pro-drug compounds of the hydrogel. Such biologically active agents include, but are not limited to this, compounds of the following classes:

(i) Sulfonylureas, such as chlorpropamide, tolazamide, tolbutamide, glyburide, glipi�ID, glimepiride, etc.

(ii) Meglitinide, such as, for example, Repaglinide,

(iii) Glucagon-like peptide-1 (GLP-1) and its mimetics, glucoseinsulin peptide (GIP) and its mimetics, Bloomington: Indiana and its mimetics and inhibitors of dipeptidylpeptidase (DPPIV),

(iv) Biguanides, such as Metformin,

(v) a Thiazolidinedione, such as rosiglitazone, pioglitazone, troglitazone, silicate (known as MCC-555), 2-[2-[(2R)-4-hexyl-3,4-dihydro-3-oxo-2H-1,4-benzoxazin-2-yl]ethoxy]-venzolasca acid, etc.,

(vi) GW2570, etc.

(vii) the Modulators of the retinoid-X-receptors (RXR), such as, for example, targretin, 9-CIS-retinoic acid, etc.,

(viii) Other insulin sensitizing agents, such as, for example, INS-1, inhibitors of PTP-1B inhibitors, GSK3 inhibitors, glycogen-phosphorylase, inhibitors of fructose-1,6-bisphosphatase, etc.

(ix) Insulins, including regular or short-acting, average duration and long-acting, inhaled insulin and insulin analogues such as insulin molecule with minimal differences in the natural amino acid sequence,

(x) small molecule insulin mimetics, including, but not limited to these, L-783281, TE-17411, etc.

(xi) Inhibitors of Na-glucose co-Transporter, such as T-1095, T-1095 a, phlorizin, etc.

(xii) Agonists amilina that include, but are not limited to,pramlintide, etc.

(xiii) glucagon Antagonists such as AY-279955, etc.

In addition to antidiabetic agents, biologically active compounds can be agents against obesity, such as orlistat, an inhibitor of pancreatic lipase, which prevents the breakdown and absorption of fats, or sibutramine, the connection, the vast appetite and inhibiting the reuptake of serotonin, norepinephrine, and dopamine in the brain, growth factors, enhancing mobilization of fat (e.g., growth hormone, IGF-1, a releasing factor growth hormone), oxyntomodulin and modulators of ghrelin. Other potential biologically active agents against obesity include, but are not limited to these, compounds, appetite suppressants acting through adrenergic mechanisms, such as benzphetamine, phenmetrazine, phentermine, diethylpropion, mazindol, sibutramine, phenylpropanolamine or, ephedrine; agents, appetite suppressants, acting through serotonergic mechanisms, such as quipazine, fluoxetine, sertraline, fenfluramine, or deksfenfluramin; agents, appetite suppressants, acting through dopamine mechanisms, for example, apomorphine; agents, appetite suppressants, acting through histaminergic mechanisms, such as (for example, histamine mimetics, modulators of H3-receptor); enhancers of energy expenditure such as beta-3 adrenergic agonists and stimulant� functions of uncoupling proteins; leptin and latinova mimetics (for example, metreleptin); neuropeptide Y antagonists; receptor modulators of the melanocortin-1, 3, and 4; cholecystokinin agonists; mimetics and analogs of glucagon-like peptide-1 (GLP-1) (for example, Bloomington: Indiana); androgens (e.g., dehydroepiandrosterone and derivatives such as etiolation), testosterone, anabolic steroids (eg, Oxandrolone) and steroid hormones; antagonists guaninovykh receptors; cytokines, such as ciliary neurotrophic factor; amylase inhibitors; agonists/mimetics of enterostatin; antagonists of orexin/hypocretin; antagonists urocortin; bombesin agonists; modulators of protein kinase A; mimetics corticotropin-releasing factor; mimetics cocaine - and amphetamine-regulated transcript; mimetics of peptide related calcitonin gene; and inhibitors of fatty acid synthase.

In an alternative embodiment, the composition Pro-drug compounds of insulin-hydrogel of the present invention combined with a second biologically active compound in such a way that the Pro-drug compound of insulin-hydrogel is administered to the needy in the first patient, and then adding a second biologically active compound. Alternatively, the composition of insulin-hydrogel is administered to the needy patient after administration of d�ugih compounds to the same patient.

In addition to antidiabetic agents, biologically active compounds can be agents against obesity, such as orlistat, an inhibitor of pancreatic lipase, which prevents the breakdown and absorption of fats, or sibutramine, the connection, the vast appetite and inhibiting the reuptake of serotonin, norepinephrine, and dopamine in the brain, growth factors, enhancing mobilization of fat (e.g., growth hormone, IGF-1, a releasing factor growth hormone), oxyntomodulin and modulators of ghrelin. Other potential biologically active agents against obesity include, but are not limited to these, compounds, appetite suppressants acting through adrenergic mechanisms, such as benzphetamine, phenmetrazine, phentermine, diethylpropion, mazindol, sibutramine, phenylpropanolamine or, ephedrine; agents, appetite suppressants, acting through serotonergic mechanisms, such as quipazine, fluoxetine, sertraline, fenfluramine, or deksfenfluramin; agents, appetite suppressants, acting through dopamine mechanisms, for example, apomorphine; agents, appetite suppressants, acting through histaminergic mechanisms, such as (for example, histamine mimetics, modulators of H3-receptor); enhancers of energy expenditure such as beta-3 adrenergic agonists and stimulators of uncoupling functions of proteins; leptin and latinova mi�of etiki (for example, metreleptin); neuropeptide Y antagonists; receptor modulators of the melanocortin-1, 3, and 4; cholecystokinin agonists; mimetics and analogs of glucagon-like peptide-1 (GLP-1) (for example, Bloomington: Indiana); androgens (e.g., dehydroepiandrosterone and derivatives such as etiolation), testosterone, anabolic steroids (eg, Oxandrolone) and steroid hormones; antagonists guaninovykh receptors; cytokines, such as ciliary neurotrophic factor; amylase inhibitors; agonists/mimetics of enterostatin; antagonists of orexin/hypocretin; antagonists urocortin; bombesin agonists; modulators of protein kinase A; mimetics corticotropin-releasing factor; mimetics cocaine - and amphetamine-regulated transcript; mimetics of peptide related calcitonin gene; and inhibitors of fatty acid synthase.

In an alternative embodiment, the composition Pro-drug compounds of insulin-hydrogel of the present invention combined with a second biologically active compound in such a way that the Pro-drug compound of insulin-hydrogel is administered to the needy in the first patient, and then adding a second biologically active compound. Alternatively, the composition of insulin-hydrogel is administered to the needy patient after the administration of different compounds to the same patient.

To prevent and delay the development or implement a treatment selected from the above group of diseases and disorders, using a combination of the composition of long-acting insulin according to the present invention with at least one biologically active compound selected from the classes of drug compounds used to treat these conditions, including antagonists of the at1receptors; inhibitors of angiotensin-converting enzyme (ACE) inhibitors; Reina; beta-adrenergic receptor; alpha blockers-adrenergic receptors; calcium channel blockers; inhibitors aldosteronism; antagonists aldosterone receptors; inhibitors of neutral endopeptidase (NEP); dual inhibitors of angiotensin-converting enzyme/neutral endopeptidase (ACE/NEP); antagonists endothelina receptors; diuretics; statins; nitrates; anticoagulants; natriuretic peptides; digitalis compounds; modulators of PPAR.

Accordingly, in a further aspect the present invention relates to the use of compounds of insulin to obtain pharmacokinetic compositions containing the compound ince�Lina with a concentration of at least 10 mg/ml, characterized in that it has a pharmacokinetic profile essentially no release of the connection of insulin, to treat or prevent diseases or disorders associated with deficiency of insulin, in the treatment or prevention using the compounds of insulin is useful.

In one embodiment of the present invention the concentration of the compounds of insulin is at least 11 mg/ml, e.g., from 11 mg/ml to 35 mg/ml, more preferably, from 15 mg/ml to 25 mg/ml, even more preferably, about 20 mg/ml and, more preferably, about 24 mg/ml.

The volume that you enter, for example with a syringe, to a subject, such as people, preferably less than 1.5 ml, typically 1.0 ml or less.

In an additional embodiment, the implementation of a disease or disorder associated with deficiency of insulin, in the treatment or prevention using the compounds of insulin is beneficial selected from hyperglycemia, prediabetes, impaired glucosetolerance, diabetes type I, diabetes type II, syndrome X. Usually, this disease or disorder is type II diabetes.

Another variant embodiment of the invention relates to the treatment or prevention of hyperglycemia, prediabetes, impaired glucosetolerance, the distance�and type I, type II diabetes mellitus, syndrome X in a mammal, e.g., humans.

In an additional embodiment, a person diagnosed with pre-diabetes, impaired glucosetolerance, obesity, type I diabetes, type II diabetes, syndrome X.

In yet another embodiment of the present invention, the pharmacokinetic profile measured in the blood plasma of a mammal, such as human blood plasma.

In an additional embodiment of the present invention, the composition is characterized in that the ratio of peak concentration to a residual concentration of less than 2, for example, less than a 1.75, less than 1.5 or less than 1.25.

In yet another embodiment of the present invention, the composition is characterized by a constant release of compounds structurally intact insulin during the entire time interval between doses.

In an additional embodiment, the full time interval between doses is at least about 80 hours, e.g., about 110 hours, usually a week.

In yet another embodiment of the present invention, the connection of insulin is a Pro-drug compound. Such Pro-drug connection you can usually choose one of the aforementioned compounds represented by formula (D-L.

In additional Varian�e embodiment of the invention the connection of insulin is fully contained in a depot preparation typically, the polymeric gel such as a hydrogel, for example, a highly hydrated polymeric matrix. Usually, highly hydrated polymeric matrix minimizes intermolecular contacts insulin molecules.

In yet another embodiment of the present invention, the connection of insulin covalently linked to the depot, a medication, usually, polymeric gel, such as hydrogel, for example, a highly hydrated polymeric matrix.

In an additional embodiment the composition is characterized in that it is a liquid composition, which after the injection forms a depot preparation.

In yet another additional embodiment, the composition is administered by injection, e.g., subcutaneous or intramuscular.

In an additional embodiment the compound selected from insulin or insulin analogues human insulin or other species, and their derivatives and conjugates. More preferred is insulin and analogues of human insulin, such as insulin glargine, insulin detemir, insulin lispro., insulin aspartate, insulin glulisine.

In yet another additional embodiment, the peak concentration is achieved within the first 24 hours after administration, for example, in the first 12 hours after administration, for example, in the Techa�their first 6 hours after administration.

In yet another embodiment, the composition further comprises a compound of GLP-1.

Another variant implementation of the invention is the combination with the connection of GLP-1. Generally, the connection of insulin administered first, or Vice versa, and the connection of insulin and the compound of GLP-1 may be administered simultaneously or sequentially.

In an additional aspect, the present invention relates to a method of treating or preventing diseases or disorders, from lack of insulin, in the treatment or prevention using the compounds of insulin is beneficial, such as hyperglycemia, prediabetes, impaired glucosetolerance, type I diabetes, type II diabetes, syndrome X, mammal requiring such treatment or prevention, by introducing a therapeutically effective amount of the compounds of insulin in a concentration sufficient to maintain therapeutically effective levels of the compounds of insulin in blood plasma for at least 3 days, usually at least 80 hours, for example, during the week or a longer period of time, which is characterized by having a pharmacokinetic profilein vivoessentially without the release of the compounds of insulin.

In an additional aspect, the present invention relates to a method for treating or u�of eduprise disease or disorder, the insufficiency of insulin, whereby the treatment or prevention using the compounds of insulin is beneficial, such as hyperglycemia, prediabetes, impaired glucosetolerance, type I diabetes, type II diabetes, syndrome X, mammal requiring such treatment or prevention, by introducing a therapeutically effective amount of the compounds of insulin in a concentration of at least 10 mg/ml, which is characterized by having a pharmacokinetic profile essentially no release of the compounds of insulin.

Variant implementation of the invention relates to the treatment or prevention of hyperglycemia, prediabetes, impaired glucosetolerance, diabetes type I, diabetes type II, syndrome X in a mammal, e.g., humans. Usually a person diagnosed with pre-diabetes, impaired glucosetolerance, obesity, type I diabetes, type II diabetes, syndrome X.

In an additional embodiment, the concentration of the compounds of insulin is at least 11 mg/ml, e.g., from 11 mg/ml to 35 mg/ml.

In yet another embodiment of the present invention, the pharmacokinetic profile measured in the blood plasma of a mammal, such as human blood plasma.

In an additional embodiment of the present invention, the composition is characterized �eat what is the ratio of peak concentration to a residual concentration of less than 2, for example, less than a 1.75, less than 1.5 or less than 1.25.

In yet another embodiment of the present invention, the composition is characterized by constant release of compounds structurally intact insulin during the entire time interval between doses.

In an additional embodiment, the full time interval between doses is at least about 80 hours, e.g., about 110 hours, usually a week.

In yet another embodiment of the present invention, the connection of insulin is a Pro-drug compound, such as any one described herein Pro-drug compounds.

In an additional embodiment, the compound of the insulin is fully contained in a depot of the drug, typically a polymer gel such as a hydrogel, for example, a highly hydrated polymeric matrix.

In yet another embodiment of the present invention, the connection of insulin covalently linked to the depot, a medication, usually, polymeric gel, such as hydrogel, for example, a highly hydrated polymeric matrix.

In an additional embodiment the composition is characterized in that it is a liquid composition, to�area after the injection forms a depot preparation.

In yet another additional embodiment, the composition is administered by injection, e.g., subcutaneous or intramuscular.

In an additional embodiment the compound selected from insulin or insulin analogues human insulin or other species, and their derivatives and conjugates. More preferred is insulin and analogues of human insulin, such as insulin glargine, insulin detemir, insulin lispro., insulin aspartate, insulin glulisine. In yet another additional embodiment, the peak concentration is achieved within the first 24 hours after administration, for example, in the first 12 hours after administration, for example, during the first 6 hours after administration.

In an additional embodiment, the composition further comprises a compound of GLP-1.

Another variant implementation of the invention is the combination with the connection of GLP-1. Generally, the connection of insulin administered first, or Vice versa, and the connection of insulin and the compound of GLP-1 can introduce simultaneously or sequentially.

In an additional aspect, the present invention relates to a kit containing a pharmaceutical composition according to any one of the embodiments described herein, and a container for the introduction of commercial�osili. Usually, the container is a syringe.

Option kit further comprises a compound of GLP-1.

Fig.1A: UPLC-chromatogram of insulin conjugate and linker 12a.

Fig.1b: UPLC-chromatogram of insulin conjugate and linker 12b.

Figure 2 shows the average concentration of insulin in plasma in animals 1-10 after a single subcutaneous dose of the test compounds 11a containing 6 mg of insulin, healthy rats during the 2-week period. (Error bars are indicated as ± standard deviation, defined for all 10 animals, the values at t0determined 3 days prior to the introduction of the connection).

Figure 3: Average concentration of insulin in plasma in animals 1-8 after a single subcutaneous dose of the test compounds 11da containing 3 mg of insulin, healthy rats for 13 days. (Error bars are indicated as ± standard deviation, defined for all 8 animals, the values at t0determined 1 day before the introduction of the connection).

Figure 4: Concentration of insulin in plasma (gray squares) and the level of blood glucose (black circles) after a single subcutaneous dose of the test compounds 11da containing 6.4 mg of insulin, diabetic rats (n=7). (Error bars are indicated as ± standard deviation, defined for all 7 animals.�t his point in t 0was determined 4 days before the introduction of the connection).

Figure 5: Average concentration of insulin in plasma after a single subcutaneous dose of 8 mg/kg of the tested compounds 11db healthy rats during the first 24 hours after dosing (analysis of the release of insulin). 8 rats were divided into 2 groups, and blood samples for pharmacokinetic analysis were collected, alternating between both groups. None of the groups was not observed significant effect of release of insulin. (Error bars are indicated as ± standard deviation, defined for all animals in the group, the values at t0determined 1 day before the introduction of the connection).

Figure 6: Concentration of insulin in plasma (gray squares) and the level of blood glucose (black circles) during a 4-week period after 3 weekly subcutaneous doses of 8 mg/kg of the tested compounds 11da diabetic rats (n=8). (Error bars are indicated as ± standard deviation, defined for all 8 animals, the values at t0determined 3 days prior to the introduction of the connection).

Figure 7: Average concentration of insulin in plasma in animals 1-8 (1-4 animals and animals through 5-8 of 0.3, 1 hour, 2 and 4 hours, respectively) after a single subcutaneous injection of 12 mg/kg of the insulin in the composition of the tested compounds 11dc healthy rats for 13 days. (Error bars in�asana as ± standard deviation, defined for all 8 animals, the values at t0was determined 4 days before the introduction of the connection).

Figure 8: Overlay of insulin release and degradation of the hydrogel to connect the insulin-linker-hydrogel 11a. Given the dependence of the content of insulin in the insulin-linker-hydrogel (triangles) and the release of skeleton parts of a molecule (circles) during incubation insulin-linker-hydrogel at pH 7.4 and 37°C of incubation time.

Figure 9 shows a graph of the flux of force when using the 30G needle. Details: black squares = ethylene glycol; black triangles = water; black dots = Pro-drug compound of insulin and of the hydrogel.

Examples

Materials and methods:

Recombinant human insulin was obtained from Biocon Ltd., Bangalore, India.

Amino(4-radial PEG), 5 kDa, was obtained from JenKem Technology, Beijing, P. R. China.

The NHS-ester of N-(3-maleimidomethyl)-21-amino-4,7,10,13,16,19-hexaoxa-heneicosane acid (Mal-PEG6-NHS) was obtained from Celares GmbH, Berlin, Germany.

If not otherwise stated 2-chlorotriethylsilane resin, HATU, N-cyclohexyl-carbodiimide-N'-metrolotion and amino acids were obtained from Merck Biosciences GmbH, Schwalbach/Ts, Germany. Coupling Fmoc(NMe) Asp(OtBu)-OH was obtained from Bachem AG, Bubendorf, Switzerland. S-Trityl-6-mercaptohexanol acid was purchased from Polypeptide, Strasbourg, France. Unless otherwise stated, we used amino�slots were of L-configuration.

All other chemicals were from Sigma-ALDRICH Chemie GmbH, Taufkirchen, Germany.

Solid-phase synthesis was performed on 2-chlorotriethylsilane (TCP) resin with loading 1.3 mmol/g. as reaction vessels used syringes with polypropylene mitami (filter elements).

Associating a first amino acid to the resin was performed according to the manufacturer's instructions.

Removal of the Fmoc protective group:

To remove the Fmoc-protecting group the resin was washed with a mixture of piperidine/DBU/DMF in a volume ratio of 2/2/96 (two washing 10 minutes each), and washed with DMF (10 times).

The removal of the protective Fmoc group from the resin loaded with Fmoc-Aib:

Fmoc-group with immobilized Fmoc-Aib-OH was removed by stirring the resin in a mixture of DMF/piperidine in a volume ratio of 4/1 at 50°C for 20 minutes (2 times).

The Protocol for the cleavage of the synthesized product from 2-chlorotriethylsilane resin:

After completion of the synthesis the resin was washed with DCM, dried under vacuum and twice for 30 minutes was treated with a mixture of DCM/HFIP with a volumetric ratio of 6/4. Combined eluate, volatile compounds were removed under a stream of nitrogen, and purification of the obtained crude product was performed using reversed-phase HPLC. HPLC fractions containing the product were combined and liofilizirovanny.

Foods containing amines are obtained as salts of trifluoroacetic acid (TFA) was converted into suitable�sponding salt hydrochlorite acid HCl), using ion exchange resin (Discovery DSC-SAX, Supelco, USA). This stage was performed in that case, if the expected residual TFA, for example, would violate the subsequent reaction of accession.

Purification using reversed-phase HPLC:

Reversed-phase HPLC was performed on C18 column (ReproSil-Pur 300 ODS-3 5 ám (100×20 mm or 100×40 mm) (Dr. Maisch, Ammerbuch, Germany) connected to a HPLC system Waters 600 and detector Waters 2487. We used linear gradients of solution A (0.1% of TFA in H2O) and solution B (0.1% of TFA in acetonitrile). HPLC fractions containing the product, liofilizirovanny.

Flash chromatography

Purification using flash chromatography was carried out on the system Isolera One from AB biotage AB, Sweden, using silicone cartridges biotage AB KP-Sil, andn-heptane and ethyl acetate as allentow. Products were detected at 254 nm.

In the case of hydrogel beads as reaction vessels or during washing of used syringes, equipped with polypropylene ricami.

Methods of analysis

Analytical ultra-performance liquid chromatography (UPLC) was performed on the system Waters Acquity, column equipped Waters BEH300 C18 (2.1 x 50 mm, particle size 1.7 µm) coupled to a mass spectrometer LTQ Orbitrap Discovery from Thermo Scientific.

MS products PEG showed a series of links (CH2CH2O)ndue to the polydispersity of the original PEG. To facilitate an interpretation of the� in the examples cited only a single representative m/z signals. MS conjugates of insulin are shown for a representative of isotopes and relate to chetyrekhfotonnymi the adducts [M+4H]4+.

Unless otherwise stated, the gel filtration (SEC) was performed on the system, Amersham Bioscience AEKTAbasic, equipped with a column Superdex 200 5/150 GL (Amersham Bioscience/GE Healthcare) with 0.45-micron inlet filter. As the mobile phase used 20 mm sodium phosphate, 140 mm NaCl, pH 7,4.

Example 1

The synthesis of the skeleton of the reagent 1g

Frame the reagent 1g synthesized from amino(4-beam)-PEG 1A according to the following scheme:

For the synthesis of compounds 1b, 5,20 g (1.00 mmol) of the HCl salt of amino(4-beam)-PEG 1A (MW approximately 5200 g/mol) was dissolved in 20 ml anhydrous DMSO. Was added Boc-Lys(Boc)-OH (2,17 g, 6.25 mmol) in 5 ml of anhydrous DMSO, EDC•HCl (1.15 g, 6,00 mmol), HOBt•H2O (0.96 g, 6.25 mmol) and kallidin (5,20 ml, 40 mmol). The reaction mixture was stirred for 30 minutes at room temperature.

The reaction mixture was dissolved in 1200 ml of dichloromethane and 2 times washed with 600 ml of 0.1 n H2SO4once the mother liquor, 2 times with 0.1 M NaOH and 4 times with a mixture of stock solution and water in a ratio of 1/1 (volume). The aqueous phase was re-extracted with 500 ml DCM. The organic phase was dried over Na2SO4, was filtered and evaporated, yielding 6.3 g of crude product 1b as a colorless wt�and. Compound 1b was purified using reverse-phase HPLC.

The yield was 3.85 g (59%) of a colorless glassy product 1b.

MS: m/z 1294,4 = [M+5H]5+(calculated value = 1294,6).

Compound 1C was obtained, stirring 3,40 g of compound 1b (0,521 mmol) in 5 ml of methanol and 9 ml of 4n HCl in dioxane at room temperature for 15 minutes. Volatile compounds were removed under vacuum. The product was used in next step without further purification.

MS: m/z 1151,9 = [M+5H]5+(calculated value = 1152,0).

For the synthesis of compound 1d, 3,26 g of compound 1c (0,54 mmol) was dissolved in 15 ml of anhydrous DMSO. Added 2.99 g of Boc-Lys(Boc)-OH (8,64 mmol) in 15 ml of anhydrous DMSO, 1.55 g of EDC•HCl (8,1 mmol), 1,24 g of HOBt•H2O (8,1 mmol) and to 5.62 ml collidine (43 mmol). The reaction mixture was stirred for 30 minutes at room temperature.

The reaction mixture was diluted with 800 ml DCM and 2 times washed with 400 ml of 0.1 n H2SO4once the mother liquor, 2 times with 0.1 M NaOH and 4 times with a mixture of stock solution and water in a ratio of 1/1 (volume). The aqueous phase was re-extracted with 800 ml of DCM. The organic phase was dried over Na2SO4, was filtered and evaporated, receiving the vitreous product.

The product was dissolved in DCM and planted the cold (-18°C) dietilamina. This procedure was repeated twice and the precipitate was dried under vacuum.

The output was 4,01 g (89%) priceless as a whole.�ethno vitreous product 1d, which was used in next step without further purification.

MS: m/z 1405,4 = [M+6H]6+(calculated value = 1405,4).

Compound 1E was obtained by stirring a solution of compound 1d (3,96 g, 0.47 mmol) in 7 ml of methanol and 20 ml 4n HCl in dioxane at room temperature for 15 minutes. Volatile compounds were removed under vacuum. The product was used in next step without further purification.

MS: m/z 969,6 = [M+5H]7+(calculated value = 969,7).

For the synthesis of compound 1f, 3,55 g of compound 1E (0,48 mmol) was dissolved in 20 ml anhydrous DMSO. Added of 5.32 g of Boc-Lys(Boc)-OH (15,4 mmol) in 18.8 ml of anhydrous DMSO, to 2.76 g of EDC•HCl (14,4 mmol), 2,20 g of HOBt•H2O (14,4 mmol) and 10.0 ml collidine (at 76.8 mmol). The reaction mixture was stirred for 60 minutes at room temperature.

The reaction mixture was diluted with 800 ml DCM and 2 times washed with 400 ml of 0.1 n H2SO4once the mother liquor, 2 times with 0.1 M NaOH and 4 times with a mixture of stock solution and water in a ratio of 1/1 (volume). The aqueous phase was re-extracted with 800 ml of DCM. The organic phase was dried over Na2SO4, was filtered and evaporated, giving the crude product 1f as a colorless oil.

The product was dissolved in DCM and planted the cold (-18°C) dietilamina. This procedure was repeated twice and the precipitate was dried under vacuum.

The output is made 4.72 g (82%) of colorless stack�'ovidnoe product 1f, which was used in next step without further purification.

MS: m/z 1505,3 = [M+6H]8+(calculated value = 1505,4).

Frame the reagent 1g was obtained by stirring a solution of compound 1f (MW approximately 12035 g/mol, 4,72 g, 0,39 mmol) in 20 ml of methanol and 40 ml of 4n HCl in dioxane at room temperature for 30 minutes. Volatile compounds were removed under vacuum.

The yield was 3.91 g (100%) glassy frame product 1g.

MS: m/z 977,2 = [M+6H]9+(calculated value = 977,4).

An alternative way of synthesis of compound 1g

For the synthesis of compounds 1b, to a suspension of 4-beam-PEG - tetraamine (1a) (50.0 g, 10,0 mmol) in 250 ml of anhydrousiPrOH was added boc-Lys(boc)-OSu (26,6 g, 60,0 mmol) and DIEA (of 20.9 ml, 120 mmol) at 45°C, and the mixture was stirred for 30 minutes.

Next was added Isopropylamine (2,48 ml, 30.0 mmol). After 5 minutes the solution was diluted with 1000 ml of MTBE and left overnight at -20°C without agitation. Was decanted approximately 500 ml of the supernatant and discard. Was added 300 ml of cold MTBE, and after shaking for 1 minute product was collected by filtration through a glass filter and washed with 500 ml of cold MTBE. The product was dried under vacuum for 16 hours.

The yield was 65.6 g (74%) of compound 1b as a white lumpy sludge.

MS: m/z 937,4 = [M+7H]7+(calculated value = 937,6).

Compound 1C received�whether by stirring compound 1b from the previous stage (48,8 g, 7,44 mmol) in 156 ml of 2-propanol at 40°C. a Mixture of 196 ml of 2-propanol and to 78.3 ml of acetyl chloride was added over 1-2 minutes while stirring. Solution at a temperature of 40°C was stirred for 30 min and cooled to -30°C overnight without stirring. Added 100 ml of cold MTBE and the suspension was shaken for 1 min and cooled for 1 h at -30°C. the Product was collected by filtration through a glass filter and washed with 200 ml of cold MTBE. The product was dried under vacuum for 16 hours.

The output accounted for 38.9 g (86%) of compound 1C as a white powder.

MS: m/z 960,1 = [M+6H]6+(calculated value = 960,2).

For the synthesis of compound 1d to a suspension of compound 1C from the previous step (19.0 g, 3,14 mmol) in 80 ml 2-propanol was added Boc-Lys(boc)-OSu (16.7 g, of 37.7 mmol) and DIEA (13,1 ml, at 75.4 mmol) at 45°C, and the mixture was stirred for 30 minutes at 45°C. Next was addedn-Propylamine (1,56 ml, 18.9 mmol). After 5 minutes, the precipitate from the solution were planted 600 ml of cold MTBE and centrifuged (3000 rpm, 1 min). The precipitate was dried under vacuum for 1 hour and dissolved in 400 ml of THF. Was added 200 ml of diethylether, and the product was cooled at -30°C for 16 hours without stirring. The suspension was filtered through a glass filter and washed with 300 ml of cold MTBE. The product was dried under vacuum for 16 hours.

The yield was 21.0 g (80%) of compound 1d in in�de white precipitate.

MS: m/z 1405,4 = [M+6H]6+(calculated value = 1405,4).

Compound 1E was obtained by dissolving the compound 1d from the previous step (15.6 g, 1.86 mmol) in 3 n HCl in methanol (81 ml, 243 mmol) and stirring for 90 minutes at 40°C. was Added 200 ml of MeOH and 700 ml of iPrOH, and the mixture was left for 2 hours at -30°C. For complete crystallization was added 100 ml of MTBE and the suspension was left overnight at -30°C. was Added 250 ml of cold MTBE and the suspension was shaken for 1 minute, filtered through a glass filter and washed with 100 ml of cold MTBE. The product was dried under vacuum.

The yield was 13.2 g (96%) of compound 1E as a white powder.

MS: m/z 679,1 = [M+10H]10+(calculated value = 679,1).

For the synthesis of compound 1f to a suspension of compound 1E of the preceding stage (8,22 g, 1.12 mmol) in 165 ml of 2-propanol was added boc-Lys(boc)-OSu (11.9 g, 26.8 mmol) and DIEA (9,34 ml of 53.6 mmol) at 45°C, and the mixture was stirred for 30 minutes at 45°C. Next was addedn-Propylamine (1,47 ml, of 17.9 mmol). After 5 minutes the solution was cooled at -18°C for 2 hours, then was added 165 ml of cold MTBE and the suspension was shaken for 1 minute and filtered through a glass filter. Next, the filter cake was washed 4 times with 200 ml of a cold mixture of MTBE/iPrOH in the ratio of 4:1 and once with 200 ml of cold MTBE. The product was dried under vacuum for 16 hours.

The yield was 12.8 g(90%) of compound 1f in the form of a pale yellow lumpy sludge.

MS: m/z 1505,3 = [M+8H]8+(calculated value = 1505,4).

Frame the reagent 1g was obtained by dissolving 4-radial PEG(kDa)(-LysLys2Lys4(boc)8)4(1f) (15.5 g, 1,29 mmol) in 30 ml of MeOH and cooling to 0°C. 4n HCl in dioxane (120 ml, 480 mmol, cooled to 0°C) was added over 3 minutes and the ice bath was removed. After 20 minutes was added 3n HCl in methanol (200 ml, 600 mmol, cooled to 0°C) for 15 minutes, and the solution was stirred for 10 minutes at room temperature. The product from the solution were planted 480 ml of cold MTBE and centrifuged at 3000 rpm for 1 minute. The precipitate was dried under vacuum for 1 hour and re-dissolved in 90 ml of MeOH, precipitated with 240 ml of cold MTBE and the suspension was centrifuged at 3000 rpm for 1 minute. The 1g product was dried under vacuum.

The yield was 11.5 g (89%) as pale yellow flakes.

MS: m/z 1104,9 = [M+8H]8+(calculated value = 1104,9).

Example 2

Synthesis of crosslinking reagent 2d

Crosslinking reagent were obtained from 2d monobenzyl ester of adipic acid (English, Arthur R. et al.,Journal of Medicinal Chemistry1990, 33(1), 344-347) and PEG as follows:

A solution of PEG 2000 (2A) (11.0 g, 5.5 mmol) and Palmyra of benzimidate (4.8 g, 20.6 mmol) in dichloromethane (90,0 ml) was cooled to 0°C. was Added dicyclohexylcarbodiimide (4,47 g, and 21.7 mmol), then added kata�political amount of DMAP (5 mg), the solution was stirred, and the reaction was left overnight (12 h) at room temperature. The flask was left at +4°C for 5 hours. The precipitate was filtered, and the solvent was removed by distillation under vacuum. The precipitate was dissolved in 1000 ml of a mixture of diethylether/ethyl acetate in a ratio of 1/1 (by volume) and left at room temperature for 2 hours, during which fell a small amount of sediment in the form of flakes. The precipitate was removed by filtration through a layer of celite may®. The solution was stored in a tightly closed flask at -30°C in a freezer for 12 hours to complete the crystallization. The crystalline product was filtered through a glass filter and washed with chilled dietilamina (-30°C). The filter cake was dried under vacuum. The yield was 11.6 g (86%) of compound 2b as a colorless solid. The product was used in next step without further purification.

MS: m/z 813,1 = [M+3H]3+(calculated value = 813,3)

In a glass autoclave with a volume of 500 ml connection PEG-bis(adipic acid) bis(benzyl ether) 2b (13,3 g, 5.5 mmol) was dissolved in ethyl acetate (180 ml) was added 10% palladium on charcoal (0.4 g). The solution was gidrirovanie at a pressure of 6 bar (6×105PA) and 40°C to stop the absorption of hydrogen (5-12 hours). The catalyst was removed on a layer of celite may®, and the solvent is evaporated under vacuum. Output (quantitative) was 2.3 g of compound 2 in the form of a yellowish oil. The product was used in next step without further purification.

MS: m/z 753,1 = [M+3H]3+(calculated value = 753,2).

The solution PEG-bis(Palmyra adipic acid) with 2 (9,43 g, 4,18 mmol),N-hydroxysuccinimide (1.92 g, 16.7 mmol) and dicyclohexylcarbodiimide (3,44 g, 16.7 mmol) in 75 ml anhydrous DCM was stirred over night at room temperature. The reaction mixture was cooled to 0°C, and the precipitate was filtered. DCM was evaporated and the residue was precrystallization of THF.

The output was 8,73 g (85%) crosslinking reagent 2d in the form of a colorless solid substance.

MS: m/z 817,8 = [M+3H]3+(calculated molecular mass = 817,9 g/mol).

Example 3

Getting hydrogel beads (3) and (3A) containing free amino group

A solution of 275 mg of compound 1g and 866 mg of compound 2d in 14 ml of DMSO was added to a solution of 100 mg of Arlacel P135 (Croda International Plc) in 60 ml of heptane. The mixture was stirred at a speed of 700 rpm special metal stirrer for 10 minutes at 25°C, resulting in the obtained slurry. For the implementation of the polymerization was added 1.0 ml ofN,N,N',N'tetramethylethylenediamine. After 2 hours, the mixing speed was reduced to 400 rpm, and the mixture was stirred for additional 16 h. Added 1.5 ml of acetic acid, and after 10 minutes was added 50 ml of water. After 5 minutes, the stirrer was stopped and poured water FA�.

For fractionation of balls the size of a suspension of hydrogel and water was wet sieved on a steel city 75, 50, 40, 32 and 20 microns. The fraction of balls remaining on the sieves 32, 40 and 50 μm were combined and washed 3 times with water, 10 times with ethanol and dried for 16 hours at a pressure of 0.1 mbar, yielding compound 3 as a white powder.

3A were prepared as described for 3 except for the use of 1200 mg compound 1g, 3840 mg compounds 2d and 28.6 ml of DMSO, 425 mg Aracelia P135, 100 ml of heptane and 4.3 ml of TMEDA. To complete was added to 6.6 ml of acetic acid and after 10 minutes, 50 ml of water and 50 ml of a saturated aqueous solution of sodium chloride.

The content of amino groups in the hydrogel was determined by conjugation of fmoc-amino acids with free amino groups on the hydrogel and subsequent determination of the fmoc-group, described Gude, M., J. Ryf, et al. (2002)Letters in Peptide Science9(4): 203 to 206.

Set the content of amino groups in compounds 3 and 3A ranged from 0.11 to 0.16 mmol/g.

Example 4

Getting hydrogel beads with functionally active maleimide groups (4) and (4A) and (AA), and determination of degree of substitution maleimide groups

A solution of 600 mg of Mal-PEG6-NHS (1.0 mmol) in 4.5 ml of a mixture of acetonitrile with water at a ratio of 2/1 (volume) was added to 200 mg of dry hydrogel beads 3. Added 500 ál of sodium phosphate buffer (pH 7,4, ,5 M), and the suspension was intensively stirred for 30 minutes at room temperature. Beads were washed 4 five times: a mixture of acetonitrile/water (volume ratio 2/1), methanol and a mixture of acetonitrile/water/TFA (in a volume ratio of 1/1/0,001).

Compound 4A was synthesized as described above except that used the connection 3A instead of compound 3.

Alternatively, the hydrogel beads 3A pre-washed with a mixture of DMSO/DIEA (in a volume ratio of 99/1), washed with DMSO and incubated for 45 minutes with a solution of Mal-PEG-NHS (2.0 equivalent relative to theoretical amount of amine groups on the hydrogel) in DMSO. Balls 4aa washed two times DMSO and three times succinate buffer pH 3.0 (20 mm, 1 mm EDTA, 0,01% Tween-20). The sample is incubated in phosphate buffer with pH 6.0 (50 mm, 50 mm ethanolamine, 0.01% of Tween-20) for 1 hour at room temperature and washed five times with sodium-succinate buffer pH 3.0 (20 mm, 1 mm EDTA, 0,01% Tween-20).

For the determination of maleimide groups aliquot of hydrogel beads 4, 4a or 4aa, respectively, liofilizirovanny with excess mercaptoethanol (50 mm sodium phosphate buffer, 30 min at room temperature), and absorption of mercaptoethanol was determined using the Ellman test (Ellman, G. L. et al.,Biochem. Pharmacol.,1961, 7, 88-95). Set the content maleimide groups ranged from 0.11 to 0.13 mmol/Shogo hydrogel.

Example 5

Synthesis of linker reagent 5d

Linker reagent 5d was synthesized according to the following scheme:

Synthesis of intermediate linker reagent 5A:

4-Methoxytrityl (3 g, 9,71 mmol) was dissolved in DCM (20 ml) and added dropwise to a solution of ethylene diamine (6.5 ml, with 97.1 mmol) in (20 ml). After two hours the solution was poured into diethyl ether (300 ml) and three times washed with 50 ml of stock solution/0.1 M NaOH in a volume ratio of 30/1 and once the mother liquor (50 ml). The organic phase was dried over Na2SO4and volatile compounds were removed under reduced pressure, receiving Mmt-protected intermediate compound (3,18 g, of 9.56 mmol).

Mmt-protected intermediate compound (3,18 g, of 9.56 mmol) was dissolved in anhydrous DCM (30 ml). Added 6-(trailercaps)-hexanoic acid (4,48 g, 11,47 mmol), PyBOP (5,67 g, 11,47 mmol) and DIEA (5.0 ml, a 28.68 mmol) and the mixture was intensively stirred for 30 minutes at room temperature. The solution was diluted with diethyl ether (250 ml) and three times washed with 50 ml of stock solution/0.1 M NaOH in a volume ratio of 30/1 and once the mother liquor (50 ml). The organic phase was dried over Na2SO4and volatile compounds were removed under reduced pressure. Compound 5A was purified using flash chromatography.

The output was 5,69 g (8,09 mmol).

M�: m/z 705,4 = [M+H] +(calculated value = 705,0).

Synthesis of intermediate linker reagent 5b:

To a solution of 5a (3,19 g, 4.53 mmol) in anhydrous THF (50 ml) was added BH3•THF (1 M solution of 8.5 ml, 8.5 mmol) and the solution was stirred for 16 hours at room temperature. Added additional amount of BH3•THF (1 M solution, 14 ml, 14 mmol) and stirred for 16 hours at room temperature. The reaction was stopped by addition of methanol (8.5 ml) was added to theN,N-dimethylethylenediamine (3 ml, to 27.2 mmol) and the solution was heated to reflux and stirred for 3 hours. The mixture was diluted with ethyl acetate (300 ml) at room temperature, washed with a saturated aqueous solution of Na2CO3(2 times 100 ml) and a saturated aqueous solution of NaHCO3(2 times 100 ml). The organic phase was dried over Na2SO4and volatile compounds were removed under reduced pressure, yielding crude intermediate aminosidine (3.22 g).

Intermediate aminosidine was dissolved in DCM (5 ml) was added Boc2O (2.97 g, 13,69 mmol) dissolved in DCM (5 ml), and DIEA (3,95 ml 22,65 mmol), and the mixture was intensively stirred at room temperature for 30 minutes. The mixture was purified using flash chromatography, obtaining the crude Boc - and Mmt-protected intermediate compound (3 g).

MS: m/z 791,4 = [M+H]+, 519,3 = [M-Mmt+H]+(scheduled for�tion value = 791,1).

A 0.4 M aqueous solution of HCl (48 ml) was added to a solution of Boc - and Mmt-protected intermediate compound in acetonitrile (45 ml). The mixture was diluted with acetonitrile (10 ml) and stirred for 1 hour at room temperature. Next, the pH of the reaction mixture was let down to 5.5 by addition of 5 M NaOH solution, the acetonitrile was removed under reduced pressure, and the aqueous solution was extracted with DCM (4 times 100 ml). The combined organic phases were dried over Na2SO4and volatile compounds were removed under reduced pressure. The crude compound 5b was used without further purification.

The yield amounted to 2.52 g (3,19 mmol).

MS: m/z 519,3 = [M+H]+(calculated MW = 518,8 g/mol).

Synthesis of intermediate linker reagent 5C:

Compound 5b (780 mg, 0.98 mmol, purity ~65%) and NaCNBH3(128 mg, 1.97 mmol) was dissolved in anhydrous methanol (13 ml). Added a solution of 2,4-dimethoxybenzaldehyde (195 mg, 1,17 mmol) in DCM (2 ml) and the mixture was stirred for 2 hours at room temperature. The solvent was evaporated under reduced pressure, and the crude product was dissolved in DCM and washed with saturated solution of NaCO3. The aqueous phase three times were extracted with DCM, and the combined organic phases were washed with the mother liquor, dried over MgSO4and concentrated under reduced pressure. Compound 5C was purified using flash chromatography, and�using DCM and MeOH as allentow.

The yield amounted to 343 mg (0,512 mmol).

MS: m/z 669,37 = [M+H]+(calculated value = 669,95).

Synthesis of linker reagent 5d:

Protection with TCP-resin loaded with Fmoc-Aib (980 mg, ~0.9 mmol), was removed in DMF/piperidine, washed with DMF (5 times) and DCM (6 times) and dried under vacuum. The resin was treated with a solution ofp-nitrophenylphosphate (364 mg, is 1.81 mmol) and collidine (398 µl, 3.0 mmol) in anhydrous THF (6 ml), and rocked for 30 minutes. A solution of reagents was removed by filtration, and the resin was washed with THF (5 times) before adding a solution of amine 5c (490 mg, 0.7 mmol) and DIEA (1,23 ml, 7.1 mmol) in anhydrous THF (6 ml). After swing for 18 hours at room temperature a solution of reagents was removed by filtration, and the resin was washed with DCM (5 times). The linker reagent was tsalala from the resin and purified using reverse-phase HPLC. the pH of the fractions of the product were summed to pH 6 by the addition of saturated aqueous NaHCO3and concentrated under reduced pressure. The resulting slurry was separated between saturated aqueous NaCl solution and DCM, and the aqueous phase was extracted with DCM. The combined organic fractions were concentrated to complete dryness, which allowed to get a linker reagent 5d.

The yield was 230 mg (0,29 mmol).

MS: m/z 798,41 = [M+H]+(calculated value = 798,1).

Example 6

Synthesis of linker reagent 6s

Linker reagent 6C synthesis�were registered as follows:

Synthesis of amine 6A:

Triphenylmethanol (11,90 g, 43,08 mmol) was resuspended in DMSO (40 ml). Added DBU (7,41 ml, 49,55 mmol) and 6-Bromhexine (13,32 g, 42,94 mmol) and the mixture was allowed to undergo reaction for 15 minutes. The reaction mixture was separated in a mixture of ethyl acetate (700 ml) and 0.1 M HCl (200 ml). The aqueous phase was extracted with ethyl acetate (3 times 50 ml) and the combined organic fractions were washed with a saturated solution of NaCO3(80 ml) and the mother liquor (80 ml), dried over MgSO4, filtered and concentrated. The crude yellow oil was precrystallization fromn-heptane/ethyl acetate. The intermediate product 6-(S-trityl-)mercaptoethylamine received in the form of a white solid (13,3 g, 26.4 mmol, 62%).

6-(S-trityl-)mercaptoethylamine (14,27 g, 28.2 mmol) was resuspended in ethanol (250 ml). Was added hydrazine-hydrate (3,45 ml, at 70.5 mmol) and the mixture was heated to reflux for 2 hours. The mixture was filtered, and the filtrate concentrated in vacuo. The remaining oil was added chloroform (180 ml) and the resulting suspension was stirred at room temperature for 1.5 h. the Mixture was filtered, and the filtrate was extracted with water (60 ml) and the mother liquor (60 ml), dried over MgSO4and concentrated, obtaining the crude 6-(trailercaps)-hexylamine (10,10 g, 26,87 mmol,95%).

MS: m/z 376,22 = [M+H]+(calculated value = 376,20).

To a chilled solution (0°C) 6-(trailercaps)-hexylamine (2,44 g, of 6.49 mmol) in THF (50 ml) was added DIEA (1,41 ml, 8,11 mmol), andn-butylchloroformate (908 μl of 7.14 mmol, in 1 ml THF). After 30 minutes, was added LiAlH4(1 M in THF, to 9.74 ml, for 9.47 mmol), and the mixture was heated to reflux for 90 minutes. Add water, 3,75 M aqueous NaOH solution and water resulted in the formation of a precipitate, which was removed from the mixture by filtration. The filtrate was concentrated under vacuum to give compound 6a.

The output was 2,41 g (6,20 mmol).

MS: m/z 390,22 = [M+H]+(calculated value = 390,22).

Synthesis of intermediate linker reagent 6b:

To a solution of 6a (2.1 g, 5,31 mmol) was added 2-bromethalin (1,96 g, 7,7 mmol) and K2CO3(1,09 g, 7.9 mmol) and the mixture was heated to reflux for 6 hours. After filtration and concentration, the crude mixture was divided into ethyl acetate and a saturated aqueous solution of NaHCO3. The crude intermediate product (2-(N-methyl-N-(6-trichlorophenyl-)amino)ethyl)phthalimide was purified using flash chromatography.

The yield amounted to 1.23 g (2.18 mmol).

MS: m/z: 563,27 = [M+H]+(calculated value = 563,27).

To a solution of (2-(N-methyl-N-(6-trichlorophenyl-)amino)ethyl)phthalimide (672 mg, 1,19 mmol) in ethanol (12 ml) was added HYDR�zine monohydrate (208 μl, To 4.17 mmol), and the mixture was heated to reflux for 1 hour. The reaction mixture was filtered, concentrated, andN-(2-aminoethyl-)-N-methyl-N-(6-trichlorophenyl-)amine was purified via reverse-phase HPLC.

The yield amounted to 624 mg (0,944 mmol).

MS: m/z 433,27 = [M+H]+(calculated value = 433,26).

To a solution ofN-(2-aminoethyl-)-N-methyl-N-(6-trichlorophenyl-)amine (151 mg, 0,229 mmol) and NaCNBH3(30 mg, 0,463 mmol) in anhydrous MeOH (6 ml) was added a solution of 2,4-dimethoxybenzaldehyde in anhydrous CH2Cl2(0,6 µl). After stirring for 1 h at room temperature, the reaction mixture was concentrated, dissolved in 2 ml of a mixture water/acetonitrile in a volume ratio of 1/9 and compound 6b was purified using reverse-phase HPLC.

The yield was 177 mg (of 0.219 mmol).

MS: m/z 583,33 = [M+H]+(calculated value = 583,33).

Synthesis of linker reagent 6s

Linker reagent 6C were obtained on the resin loaded with Fmoc-Aib (704 mg, ~0.6 mmol) as described for 5d, except that the used amine 6b (in the form of a TFA-salt, 430 mg, 0.53 mmol) instead of 5c.

The output was 285 mg (0,330 mmol).

MS: m/z 712,37 = [M+H]+(calculated value = 712,37).

Example 7

Synthesis of linker reagent 7f

Linker reagent 7f was synthesized according to the following scheme:

To the cooled (0°C) solution ofN-methyl-N-boc-Ethylenediamine (0.5 ml, 2,79 mmol) and NaCNBH3(140 mg, of 2.23 mmol) in MeOH (10 ml) and acetic acid (0.5 ml) was added a solution of 2,4,6-trimethoxybenzaldehyde (0,547 mg, 2,79 mmol) in EtOH (10 ml). The mixture was stirred at room temperature for 2 hours, sekilala 2 M HCl (1 ml) and neutralized with a saturated aqueous solution of Na2CO3(50 ml). Evaporation of all volatile compounds, extraction with DCM, the resulting aqueous slurry and the concentration of the organic fractions gaveN-methyl-N-boc-N'-tmob-Ethylenediamine (7a) in the form of crude oil that was purified using reverse-phase HPLC.

The yield amounted to 593 mg (1.52 mmol).

MS: m/z 377,35 = [M+Na]+(calculated value = 377,14).

N-Fmoc-N-Me-Asp(OtBu)-OH (225 mg, 0,529 mmol) was dissolved in DMF (3 ml) was added compound 7a (300 mg, 0,847 mmol), HATU (201 mg, 0,529 mmol) and kallidin (of 0.48 ml, 3,70 mmol). The mixture was stirred at room temperature for 2 hours, obtaining a compound 7b. For removal of fmoc-protection was added piperidine (0,22 ml, 2.16 mmol) and continued stirring for 1 hour. Was added acetic acid (1 ml), and compound 7C was purified using reverse-phase HPLC.

The output was 285 mg (0.436 mmol in the form of a TFA-salt).

MS: m/z 562,54 = [M+Na]+(calculated value = 562,67).

6-Trichlorophenol acid (0,847 g, 2,17 �mol) was dissolved in anhydrous DMF (7 ml). Was added HATU (0,825 g, 2,17 mmol) and kallidin (0.8 ml, 6.1 mmol) and compound 7c (0.78 g, 1.44 mmol). The reaction mixture was stirred for 60 minutes at room temperature, sekilala AcOH (1 ml) and purified using reverse-phase HPLC. Product fractions neutralized with a saturated aqueous solution of NaHCO3and concentrated. The remaining aqueous phase was extracted with DCM, and the compound 7d was isolated after evaporation of the solvent.

The yield was 1.4 g (94%).

MS: m/z 934,7 = [M+Na]+(calculated value = 934,5).

To a solution of compound 7d (1,40 mg, 1,53 mmol) in MeOH (12 ml) and H2O (2 ml) was added LiOH (250 mg, 10.4 mmol) and the reaction mixture was stirred for 14 hours at 70°C. the Mixture sekilala AcOH (0.8 ml), and compound 7e was purified using reverse-phase HPLC. Product fractions neutralized with a saturated aqueous solution of NaHCO3and concentrated. The aqueous phase was extracted with DCM, and the compound 7E was isolated after evaporation of the solvent.

The yield amounted to 780 mg (60%).

MS: m/z 878,8 = [M+Na]+(calculated value = 878,40).

To a solution of 7e (170 mg, 0,198 mmol) in anhydrous DCM (4 ml) was added DCC (123 mg, 0,59 mmol), andN-hydroxysuccinimide (114 mg, 0,99 mmol) and the reaction mixture stirred at room temperature for 1 hour. The mixture was filtered, and the filtrate sekilala the addition of 0.5 ml of AcOH, and spent cleaning compounds 7f using the Ref�NGOs-phase HPLC. Product fractions neutralized with a saturated aqueous solution of NaHCO3and concentrated. The remaining aqueous phase was extracted with DCM, and compound 7f was isolated after evaporation of the solvent.

The output was 154 mg (0,161 mmol).

MS: m/z 953,4 = [M+H]+(calculated value = 953,43).

In an alternative embodiment, the linker reagent 7f was synthesized according to the following scheme:

An alternative reaction scheme:

To a solution ofN-methyl-N-boc-ethylene diamine (2 g, 11,48 mmol) and NaCNBH3(819 mg, 12,63 mmol) in MeOH (20 ml) was added parts of 2,4,6-trimethoxybenzaldehyde (2,08 mg, 10,61 mmol). The mixture was stirred at room temperature for 90 minutes, sekilala 3 M HCl (4 ml) and further stirred for 15 minutes. The reaction mixture was added to a saturated solution of NaHCO3(200 ml) and 5-fold was extracted with CH2Cl2. The combined organic phases were dried over Na2SO4and the solvent was evaporated in vacuum. The compound obtained inN-methyl-N-boc-N'-tmob-Ethylenediamine (7a) completely dried in high vacuum and used in the next reaction stage without further purification.

The yield was 3.76 g, (11,48 mmol, 89% purity, the ratio of compounds 7a and product with two Tmob-protective groups was 8:1).

MS: m/z 355,22 = [M+H]+(calculated value = 54,21).

To a solution of compound 7a (2 g, 5,65 mmol) in CH2Cl2(24 ml) was added COMU (4.84 g, 11.3 mmol),N-Fmoc-N-Me-Asp(OBn)-OH (2.08 g, 4,52 mmol) and kallidin (2,65 ml, 20,34 mmol). The reaction mixture was stirred for 3 hours at room temperature, diluted with CH2Cl2(250 ml) and washed with 0.1 M H2SO4(3 times 100 ml) and the mother liquor (3 times 100 ml). The aqueous phase was re-extracted with CH2Cl2(100 ml). The combined organic phases were dried over Na2SO4was filtered and the residue was concentrated to a volume of 24 ml. Compound 7g was purified using flash chromatography.

The exit was at 5.31 g (148%, 6,66 mmol).

MS: m/z 796,38 = [M+H]+(calculated value = 795,37).

To a solution of compound 7g [5,31 g, maximum 4,51 mmol relativelyN-Fmoc-N-Me-Asp(OBn)-OH] in THF (60 ml) was added DBU (1.8 ml, 3% by volume of the mixture). The solution was stirred for 12 minutes at room temperature, diluted with CH2Cl2(400 ml) and washed with 0.1 M H2SO4(3 times 150 ml) and the mother liquor (3 times 150 ml). The aqueous phase was re-extracted with CH2Cl2(100 ml). The combined organic phases were dried over Na2SO4and was filtered. Compound 7h was isolated after evaporation of the solvent and used in the next reaction without further purification.

MS: m/z 574,31 = [M+H]+(calculated value =573,30).

Compound 7h (5,31 g, 4,51 mmol, crude) was dissolved in acetonitrile (26 ml) was added COMU (3,87 g 9,04 mmol), 6-trichlorophenol acid (2,12 g, 5,42 mmol) and kallidin (2,35 ml 18,08 mmol). The reaction mixture was stirred for 4 hours at room temperature, diluted with CH2Cl2(400 ml) and washed with 0.1 M H2SO4(3 times 100 ml) and the mother liquor (3 times 100 ml). The aqueous phase was re-extracted with CH2Cl2(100 ml). The combined organic phases were dried over Na2SO4was filtered and the compound 7i was isolated after evaporation of the solvent. The 7i product was purified using flash chromatography.

The yield was 2.63 g (62%, 94% purity).

MS: m/z 856,41 = [M+H]+(calculated value = 855,41).

To a solution of compound 7i (2,63 g, 2,78 mmol) iniPrOH (33 ml) and H2O (11 ml) was added LiOH (267 mg, 11,12 mmol) and the reaction mixture was stirred for 70 minutes at room temperature. The mixture was diluted with CH2Cl2(200 ml) and washed with 0.1 M H2SO4(3 times 50 ml) and the mother liquor (3 times 50 ml). The aqueous phase was re-extracted with CH2Cl2(100 ml). The combined organic phases were dried over Na2SO4, was filtered and compound 7E was isolated after evaporation of the solvent. Compound 7j was purified using flash chromatography.

The yield was 1 g (88%).

MS: m/z 878,4 = [M+Na]+(calculated value = 878,40).

To a solution of compound 7e (170 mg, 0,198 mmol) in anhydrous DCM (4 ml) was added DCC (123 mg, 0,59 mmol) and catalytic amount of DMAP. After 5 minutes was added to theN-hydroxysuccinimide (114 mg, 0,99 mmol) and the reaction mixture stirred at room temperature for 1 hour. The reaction mixture was filtered, the solvent was removed in vacuo, and the residue was dissolved in 90% acetonitrile and 0.1% TFA (3.4 ml). The crude mixture was purified using reverse-phase HPLC. Product fractions were neutralized with 0.5 M phosphate buffer (pH 7.4) and concentrated. The remaining aqueous phase was extracted with DCM, and compound 7f was isolated after evaporation of the solvent.

The output was 154 mg (81%)

MS: m/z 953,4 = [M+H]+(calculated value = 953,43).

Example 8

Synthesis of NαA1-conjugates of insulin-linker, 8b and 8c

Synthesis of protected conjugate insulin-linker, 8A

Linker reagent 5d was dissolved in DCM (20 mg/ml) and activated with N-cyclohexylcarbodiimide-N'-methylpredisolone resin (1.9 mmol/g, 10 EQ.) in for 1 hour. The solution of the activated linker reagent was added to a solution of insulin (1.2 EQ.) and DIEA (3.5 EQ.) in DMSO (100 mg insulin/ml), and the mixture was shaken at room temperature for 45 minutes. RA�creative ways sekilala acetic acid, DCM was evaporated under reduced pressure, and NαA1-protected conjugated conjugate insulin-linker 8A was purified using reverse-phase HPLC.

The lyophilized product 8A was treated with a mixture of HFIP/TFA/water/triethylsilane in a volume ratio of 90/10/2/2 (2 ml per 100 mg of compound 8a) for 45 minutes at room temperature. The reaction mixture was diluted with water, and all volatiles were removed under nitrogen flow. NαA1-conjugate conjugated insulin-linker 8b was purified using reverse-phase HPLC.

8b:

The output is 139 mg, is 0.023 mmol) of 62 mg (0,078 mmol) linker 5d

MS: m/z 1524,45 = [M+4H]4+(calculated value = 1524,75).

NαA1-conjugate conjugated insulin-linker 8C was synthesized as described for 8b except that instead of compound 5d was used compound 6c (72 mg, 0,101 mmol).

8c:

The yield amounted to 237 mg (0,039 mmol)

MS: m/z 1528,23 = [M+4H]4+(calculated value = 1528,28).

Example 9

Synthesis of Nα1-insulin conjugate-linker 9

Insulin with two protective groups,Nα-boc-GlyA1-Nε-boc-LysB29-insulin, was obtained as described previously (J. Markussen, J. Halstrøm, F. C. Wiberg, L. Schaffer,J. Biol. Chem.1991,266, 18814-18818).

Linker reagent 5d (0,04 mmol) was dissolved in DCM (0.5 ml) and activated with N-cyclohex�carbodiimide-N'-methylpredisolone resin (0,205 mmol) at room temperature for 2 hours. The resulting solution of the activated linker reagent was added to a solution of insulin with two Boc protective groups (24 mg, 0,004 mmol) and DIEA (5 μl 0,0229 mmol) and was shaken at room temperature for 1 hour. The reaction mixture sekilala 100 μl of acetic acid, and protected conjugate insulin-linker was purified using reverse-phase HPLC.

The yield was 5 mg (0,00075 mmol).

MS: m/z 1660,27 = [M+4H]4+(calculated value = 1660,43).

Lyophilized protected conjugate insulin-linker was treated with a mixture of HFIP/TFA/water/TES in a volume ratio of 90/10/2/2 at room temperature for 45 minutes. The reaction mixture was diluted with 0.5 ml of water, and all volatiles were removed under nitrogen flow. Nα1-conjugate conjugated insulin-linker 9 was purified using reverse-phase HPLC.

The yield was 4 mg (0,0007 mmol).

MS: m/z 1524,46 = [M+4H]4+(calculated value = 1524,75).

Example 10

Synthesis of NεB29-insulin conjugate-linker 10

Insulin (644 mg, 0,111 mmol) was dissolved in 6.5 ml of DMSO. Was added 3 ml of chilled (4°C) of 0.5 M sodium borate buffer (pH 8.5) and compound 7f (70 mg, 0,073 mmol) in 2.5 ml of DMSO, and the mixture was stirred for 5 minutes at room temperature. Added 400 μl Acoh, and protected insulin conjugate was purified using reverse-phase HPLC.

The yield was 172 mg (0.025 mmol).

MS: m/z 1662,27 = [M+4H]4+(calculated value = 1662,48).

The protective group was removed by processing fractions lyophilized product 6 ml of a mixture of HFIP/TFA/TES/water in a volume ratio of 90/10/2/2 for 1 hour at room temperature. NεB29-conjugate conjugated insulin-linker 10 was purified using reverse-phase HPLC.

The yield was 143 mg, is 0.023 mmol).

MS: m/z 1531,46 = [M+4H]4+(calculated value = 1531,71).

Example 11

Getting the insulin-linker-hydrogel 11a, 11b, 11C, 11d, 11da, 11db 11dc and

Dry hydrogel with functional maleimide groups (compound 4, 82 mg, 10.3 μmol maleimide groups) filled the syringe, equipped Frith filter. Was added a solution of compound insulin-linker-thiol 8b (27,8 mg, 4.6 μmol) in 1.0 ml of a mixture acetonitrile/water/TFA in a volume ratio of 1/1/0,001, and the suspension was incubated 5 minutes at room temperature. Added acetate buffer (0.4 ml, pH of 4.8, 1.0 M), and the sample incubated at room temperature for 1 hour. Consumption tylnej groups was monitored using Ellman test. The hydrogel was washed 10 times with a mixture of acetonitrile/water/TFA in a volume ratio of 1/0,43/0,001 and 2 times with a mixture of 1.0 M sarcosine, pH 7.4/acetonitrile/0.5 M phosphate buffer pH 7.4/water in a ratio of 1/1/0,2/0,25. At the end of the hydrogel were resuspended� in solution sarcosine and incubated for 2 hours at room temperature.

The connection of insulin-linker-hydrogel 11a were washed 10 times with a mixture of acetonitrile/water/TFA in a ratio of 1/1/0,001 and stored at +4°C.

Insulin content was determined by complete hydrolysis of aliquots of the compounds of insulin-linker-hydrogel under reducing conditions and pH 12, and the subsequent estimate of the number of A - and b-chains of insulin was performed using reversed-phase HPLC.

The content of insulin in 11a: 175 mg insulin/g of insulin-linker-hydrogel

The amount of insulin in suspension of compound 11a in 10 mm sodium-acetate buffer, pH 5, and 135 mm sodium chloride: 12 mg insulin per 1 ml of suspension 11a.

Compounds 11b, 11C and 11d was obtained as described above except that instead of compound 8b used, respectively, compounds 8c, 9 and 10.

Connection 11da received as described above except that instead of compounds 8b and 4 were used, respectively, compounds 10 and 4A.

Connection 11db were prepared as follows: a suspension of hydrogel with functional maleimide groups 4A HCl in pH of 2.5, and 0.01% Tween-20 (5.0 ml, 119 mmol maleimide groups) filled the syringe with the filter. To the suspension was added a solution of insulin-linker-thiol (compound 10) (166 mg, up 24.4 mmol) in 8.0 ml of HCl pH 2.5, and 0.01% of Tween-20, and the suspension was incubated for 5 minutes at room temperature. Was added sodium succinate buffer (3,9 ml, pH 4.0 and 150 mm; 1 mm EDTA, 0,01% Tween-20) to obtain a pH of 3.6, and �brazes were incubated at room temperature for 90 minutes. Consumption tylnej groups was monitored using Ellman test. The hydrogel was washed with 10 times the sodium succinate buffer (pH 3.0, 50 mm; 1 mm EDTA, 0,01% Tween-20) and 3 times the sodium succinate buffer (pH 3.0, 50 mm; 1 mm EDTA, 0,01% Tween-20) containing 200 mm NAC. At the end of the hydrogel were resuspended in buffer containing acetylcysteine, and incubated for 1 hour at room temperature.

The connection of insulin-linker-hydrogel 11db were washed 10 times succinate buffer (pH 3.0, 50 mm; 1 mm EDTA, 0,01% Tween-20) and 8 times the sodium acetate buffer (pH 5.0, 10 mm; 130 mm NaCl, 0,01% Tween-20).

The content of insulin in 11db: 6,12 mg of insulin per ml suspension, the insulin-linker-hydrogel.

Connection 11dc was prepared as follows: a suspension of a hydrogel with functional maleimide groups (4A) in HCl, pH 2.5, and 0.01% of Tween-20 (of 58.3 ml, 958 mmol maleimide groups) was added to the reactor for solid phase synthesis. The compound 4A was added a solution of insulin-linker-thiol (compound 10) (117 ml, 460 mmol) in 2.5 HCl, 0.01% of Tween-20. The suspension was incubated 5 minutes at room temperature. Added succinate buffer (4.8 ml, pH 4.0 and 150 mm; 1 mm EDTA, 0,01% Tween-20) to obtain a pH of 3.6, and the sample incubated at room temperature for 90 minutes.

Consumption tylnej groups was monitored using Ellman test. The hydrogel was washed with 10 times succinate buffer (pH 3.0, 50 mm; 1 mm EDTA, 0,01% Tween-20) and 2 times the sodium succinate buffer (pH 3.0, 50 mm; 1 m� EDTA, 0,01% Tween-20) containing 10 mm mercaptoethanol. At the end of the hydrogel were resuspended in buffer containing mercaptoethanol, and incubated for 3 hours at room temperature.

The connection of insulin-linker-hydrogel (11dc) were washed 10 times succinate buffer (pH 3.0, 50 mm; 1 mm EDTA, 0,01% Tween-20) and 6 times succinate/Tris buffer (pH 5.0, 10 mm; 85 g/l trehalose, 0.01% of Tween-20).

The content of insulin in 11dc: 18,7 mg of insulin per ml suspension, the insulin-linker-hydrogel.

In an alternative embodiment, instead of compound 4A can be used microparticles maleimides derived hydrogel (a).

Example 12

The kinetics of insulin releasein vitro

Compounds of insulin-linker-hydrogel 11a, 11b, 11C and 11d (containing approximately 1 mg of insulin), respectively, were resuspended in 2 ml buffer containing 60 mm sodium phosphate, 3 mm EDTA, 0,01% Tween-20, pH of 7.4, and incubated at 37°C. the Suspension was centrifuged at certain intervals of time, and analysis of the supernatant was performed using reversed-phase HPLC with detection at 215 nm, as well as using ESI-MS. The values of the UV absorption corresponding to the released insulin were pooled and build a schedule based on time of incubation.

To determine the appropriate time provisoire insulin used software for approximation of curves.

For compounds 11a, 11b, si 11d, accordingly, it is determined following the half-life: 16 days, 10 days, 30 days and 14 days.

In an alternative embodiment, the insulin-linker-hydrogel (connection 11db) was transferred into a syringe equipped with filters were resuspended in 6 ml of 60 mm sodium phosphate, 3 mm EDTA, 0,01% Tween-20, pH of 7.4, and incubated at 37°C. At certain time intervals supernatant was exchanged, and the amount of released insulin was determined using reverse-phase HPLC at 215 nm. Built a graph of the amount of insulin released from the incubation time. Specified by software for the approximation of curves the half-life for compound 11db was 15 days.

In an alternative embodiment, the compound of the insulin-linker-hydrogel 11db filled chromatographic column and placed in thermostatically incubator (37°C). Through the column missed a sodium phosphate buffer (pH of 7.4, 60 mm; 3 mm EDTA, 0,01% Tween-20) with a constant flow rate of 0.25 ml/h (nominal) and collected a buffer outside of the incubator. At specific time points of the solution was analyzed by reversed-phase HPLC at 215 nm. Built a graph of the amount of insulin released from the incubation time. Specified by software for the approximation of curves the half-life for compound 11db amounted to 13 days.

Example 13

Synthesis of LysB29-linker conjugate and insulin (12a) and the LysB28-linker conjugate and insulin lispro. (12b):

Synthesis of LysB29-linker conjugate and insulin (12a)

1.2 g (0,206 mmol, 0.85 EQ.) insulin was dissolved in DMSO at room temperature (RT). After 30 minutes the solution was cooled to 0°C, was added borate buffer (0.5 M, pH 8.5 and 21.6 ml), 4,40 minutes. The temperature of the solution maintained 25-28°C. Compound 7f (0,239 mmol, 1 EQ.) was dissolved in 40 ml of DMSO and whatever time was added for 3 minutes. The ice bath was removed and the reaction mixture was stirred 5 minutes at room temperature. The reaction was terminated by adding 70 ml of MeCN/H2O (1:1, with 0.1% TFA) and 400 μl of AcOH. Compound 12a was purified using reverse-phase HPLC (solvent A: H2O with 0.1% TFA, solvent B: MeCN with 0.1% TFA, gradient: 30-80% B in 14 minutes, flow rate: 40 ml/min).

The selectivity UPLC-analysis (before purification using reversed-phase HPLC) was as follows: 0.70% on compound 7f was attached to GlyA1 insulin, while 76.2% of compound 7f was attached to LysB29 of insulin (see Fig.1A).

The yield amounted to 862 mg (TFA-salt, 60%).

MS: [M+H]1/4+= 1662,25 g/mol (calculated mass (MW+H)1/4= 1662,35 g/mol).

Synthesis of LysB28-linker conjugate and insulin lispro. (12b)

0,347 g (0,059 mmol, 0.85 EQ.) insulin lispro. of Sol�rely in 6 ml of DMSO at room temperature. After 30 minutes the solution was cooled to 0°C, was added borate buffer (0.5 M, pH 8.5, 5,64 ml) of 1.40 minutes. Supported the solution temperature is 25-30°C. Further, regardless of the time within 2 minutes was added a solution of 67 mg (0,070 mmol, 1 EQ.) compounds 7f, dissolved in 8 ml DMSO. The ice bath was removed and the reaction mixture was stirred 5 minutes at room temperature. The reaction was terminated by adding 20 ml of MeCN/H2O (1:1, with 0.1% TFA) and 1 ml of AcOH. Compound 12b was purified using reverse-phase HPLC (solvent A: H2O with 0.1% TFA, solvent B: MeCN with 0.1% TFA, gradient: 30-80% B in 14 minutes, flow rate: 40 ml/min).

The selectivity UPLC-analysis (before purification using reversed-phase HPLC) was as follows: 1.3% of compound 7f was attached to GlyA1 insulin lispro., and 76.7% of compound 7f was attached to LysB28 insulin lispro. (see Fig.1b).

The yield was 305 mg (TFA-salt, 72%).

MS: [M+H]1/4+= 1662,25 g/mol (calculated mass (MW+H)1/4= 1662,35 g/mol).

Example 14

Pharmacokinetic studies in rats

The pharmacokinetics of compound 11a was determined by measuring the concentration of insulin in plasma after subcutaneous administration to rats of a single dose of a compound.

One group consisting of 10 male Wistar rats (weight 200-250 g) were used for the study of insulin levels in plasma in techenie days. Each animal received a single subcutaneous injection of 500 μl of a suspension of compound 11a in acetate buffer (pH 5) containing 6 mg of insulin (12 mg insulin/ml). Each animal at specific time points were taken sublingually 200 μl of blood, yielding 100 ál of Li-heparin plasma. The samples were collected before drug administration and after 4 hours, 1, 2, 3, 4, 7, 9, 11 and 14 days after injection. Plasma samples were frozen within 15 minutes after blood collection and stored at -80°C until analysis.

The insulin concentration in plasma was measured using an ultra-sensitive ELISA kit for determining the amount of insulin (Mercodia) and following the manufacturer's Protocol. Before measurement the samples of plasma were diluted in the buffer for carrying out ELISA (1:5 and 1:10 with calibrator 0). The insulin concentration was calculated from a calibration curve, which was obtained by measurement of standards of insulin in duplicate and approximation values using linear regression. The insulin concentration was determined as the average of two independent series of dilutions, adjusted by the appropriate dilution factor, and built a dependence on time. Shown in Fig.2 average concentrations of insulin in plasma for each time point was obtained by calculating the mean for all animals in the experiment.

Observed within t�e 14 days insulin release was constant and did not have emissions.

Example 15:

Pharmacokinetic studies in rats

The pharmacokinetics of compounds 11da was determined by measuring the concentration of insulin in plasma during 13 days after subcutaneous administration to rats of a single dose of a compound.

8 Wistar rats (weight approximately 250 g) received a single subcutaneous injection of 500 μl of a suspension of the tested compounds 11da in acetate buffer (pH 5) containing 3 mg of insulin (approximately 12 mg/kg). Each animal at certain points of time took 200 μl of blood from tail vein, getting 100 ál of Li-heparin plasma. The samples were collected 1 day before drug administration and after 4 hours, 1 day, 2 days, 3 days, 6 days, 7 days, 8 days, 10 days and 13 days after administration of the tested compounds. Plasma samples were frozen and stored at -80°C until analysis. The insulin concentration in plasma samples was measured using an ultra-sensitive ELISA kit for determination of human insulin (DRG Instruments GmbH, Germany) and following the manufacturer's Protocol. Background samples (calibrator 0) included in the calibration curve and subtracted from values obtained for the samples, and the calibration curve was approximatively using polynomial equation 3rd order. Before the measurement plasma samples were processed on the vortex and was diluted in the reaction tubes (1:5 and 1:10 calibration 0). For the analysis of the measured optical density OD at 450 nm on a tablet spectrophotometer (Tecan Ultra) without correction to the reference wavelength. All the results of the content of insulin in plasma up to 13 days for all animals studied are shown in figure 3.

After a single subcutaneous injection of 500 μl of compound 11d containing 3 mg of insulin, the average level of insulin in plasma rose to a peak of approximately 500 PM in 1 day. As expected, the insulin concentration in the plasma is then gradually decreased within 2 weeks. The ratio of peak concentration to a residual concentration of insulin in plasma during the first week of the study was approximately 1.7.

Example 16:

Pharmacokinetic and pharmacodynamic studies in rats

The number and biological activity of released insulin was studied by analyzing the concentration of insulin in plasma and the effect of reducing the concentration of glucose in the blood in experimental pharmacokinetic/pharmacodynamic study using diabetic rats, Sprague-Dawley (SD).

For 8 rats caused diabetes using streptozotocin (STZ), and in this study included all animals with glucose levels above 350 mg/DL on day zero. In 7 out of 8 SD rats were formed diabetes, and they received a single subcutaneous injection of 500 µl of the tested compounds�tion 11da in acetate buffer (pH 5), containing 6.4 mg of insulin. Each animal at certain points of time took 200 μl of blood from tail vein, getting 100 ál of Li-heparin plasma. The samples were collected for 4 days before drug administration and after 2 hours, 1 day, 2 days, 3 days, 6 days, 7 days, 8 days, 10 days and 13 days after administration of the tested compounds. Plasma samples were frozen and stored at -80°C until analysis. The level of glucose in the blood was determined using the device AccuChek Comfort from the tail vein 3 times before injection connection and after 2 hours, 1 day, 2 days, 3 days, 6 days, 7 days, 8 days 10 days 13 days 15 days 17 days 20 days 22 days and 24 days after injection of the tested compounds. The content of insulin in the plasma samples was measured using an ultra-sensitive ELISA kit for determination of human insulin (DRG Instruments GmbH, Germany) and following the manufacturer's Protocol. Background samples (calibrator 0) included in the calibration curve and subtracted from values obtained for the samples, and the calibration curve was approximatively using polynomial equation 3rd order. Before the measurement plasma samples were processed on the vortex and was diluted in the reaction tubes (1:5 and 1:10 with calibrator 0). For the analysis of the measured optical density OD at 450 nm on a tablet spectrophotometer (Tecan Ultra) without correction to the reference wavelength. Insulin content in the LP�similarly controlled within 2 weeks while the content of glucose in the blood over a 3-week period, as shown in figure 4.

After a single subcutaneous injection of insulin-hydrogel 11da the level of glucose in the blood was significantly reduced within 10 days, the values were below 100 mg/ml without any symptoms of hypoglycemia. As a result a higher dose (6.4 mg) of insulin for animal maximum concentration of insulin in plasma was approximately RM 800 at day 1 and gradually decreased within 2 weeks to about 300 PM. At the same time, the level of glucose in the blood began to rise after 10 days and returned to the level before the introduction of the compound in 3 weeks.

Example 17:

24-hour pharmacokinetic study (study insulin spikes) in rats

For evidence that insulin is released from the insulin-linker-hydrogel without the release, in healthy rats was observed at the concentration of insulin in plasma within 24 hours.

8 rats Sprague-Dawley (weight 200-250 g) were divided into 2 groups, and each received a single subcutaneous injection of 2 ml of the tested compounds 11db in acetate buffer (pH 5) per kg of body weight. The tested compound has a concentration of 4 mg/ml insulin, so that each animal received 8 mg of insulin per kg of body weight. Each animal at certain points of time took 200 μl of blood from the tail�howl of Vienna, getting 100 ál of Li-heparin plasma. Samples in group a were collected before administration of the dose connection and after 5 minutes, 30 minutes, 2 hours, 4 hours and 8 hours after administration of the tested compounds, and in group before administration of the dose of the compound and after 15 minutes, 1 hour, 3 hours, 6 hours and 24 hours after administration of the tested compounds. Plasma samples were frozen and stored at -80°C until analysis. The content of insulin in the plasma samples was measured using an ultra-sensitive ELISA kit for determination of human insulin (DRG Instruments GmbH, Germany) and following the manufacturer's Protocol. Background samples (calibrator 0) included in the calibration curve and subtracted from values obtained for the samples, and the calibration curve was approximatively using polynomial equation 3rd order. Before the measurement plasma samples were processed on the vortex and was diluted in the reaction tubes (1:5 and 1:10 with calibrator 0). For the analysis of the measured optical density OD at 450 nm on a tablet spectrophotometer (Tecan Ultra) without correction to the reference wavelength. The result is shown in figure 5 and clearly shows that insulin is released without any output.

Example 18:

Pharmacokinetic and pharmacodynamic studies of repeated dosage in rats

The pharmacokinetics and pharmacodynamics after 3 weekly� doses of compounds 11da determined measuring diabetic rats the insulin concentration in plasma and the level of glucose in the blood within 4 weeks.

Used 8 rats Sprague-Dawley average weight of 239 g. Diabetes caused by using streptozotocin (STZ), and all the animals with glucose levels above 350 mg/DL on day zero were included in this study. 8 out of 8 animals treated with STZ, formed diabetes, and they received 3 weekly subcutaneous injections at 0-th, 7-th and 14-th day 2 ml of the tested compounds 11da in acetate buffer (pH 5) per kg of body weight. As the test compound has a concentration of 4 mg/ml insulin, administered dose was 8 mg of insulin per kg of body weight. Each animal at certain points of time took 200 μl of blood from tail vein, getting 100 ál of Li-heparin plasma. The samples were collected for 3 days prior to the introduction of the connection and before the 28th day after administration of the tested compounds. Plasma samples were frozen and stored at -80°C until analysis. The level of glucose in the blood is measured with the help of the device AccuChek Comfort from the tail vein 3 times before injection connection and up to 30 days after administration of the tested compounds. The content of insulin in the plasma samples was measured using an ultra-sensitive ELISA kit for determination of human insulin (DRG Instruments GmbH, Germany) and following the manufacturer's Protocol. Background samples (calibrator 0) �lucali in the calibration curve and subtracted from values obtained for the samples, and the calibration curve was approximatively using polynomial equation 3rd order. Before the measurement plasma samples were processed on the vortex and was diluted in the reaction tubes (1:5 and 1:10 with calibrator 0). For the analysis of the measured optical density OD at 450 nm on a tablet spectrophotometer (Tecan Ultra) without correction to the reference wavelength. Insulin levels in plasma and glucose content in the blood was monitored over a 4-week period, and all of these values is shown in figure 6.

The shape of the curves indicates that the released insulin was biologically active, continuously reducing the content of glucose in the blood to levels of approximately 100 mg/DL after the 3rd injection, which remained low for about a week. At the same time, the maximum insulin concentration is continuously increased, starting from RM 200 after the first dose, up to RM 300 after the second dose and RM 400 after administration of the third dose, and subsequently decreased again in 2 weeks to below 100 PM.

Example 19:

Pharmacokinetic studies in rats

The pharmacokinetics of compounds 11dc was determined by measuring the concentration of insulin in plasma during 13 days in healthy rats.

8 Wistar rats (weight approx 230 g) received a single subcutaneous injection of 2 ml/kg of the tested compounds in 11dc su�synatom buffer (pH 5) (10 mm succinate/tris-buffer, 85 g/l trehalose, 0.01% of Tween-20, pH 5.0) containing 3 mg of insulin (dosage 12 mg/kg). Each animal at certain points of time took 200 μl of blood from tail vein, getting 100 ál of Li-heparin plasma. The samples were collected for 4 days before drug administration and after 0.3 hours (4 animals), 1 hour (4 animals), 2 hours (4 animals), 4 hours (4 animals), 1 day, 2 days, 3 days, 6 days, 8 days, 10 days and 13 days after administration of the tested compounds. Plasma samples were frozen and stored at -80°C until analysis. The content of insulin in the plasma samples was measured using an ultra-sensitive ELISA kit for determination of human insulin (DRG Instruments GmbH, Germany) and following the manufacturer's Protocol. Background samples (calibrator 0) included in the calibration curve and subtracted from values obtained for the samples, and the calibration curve was approximatively using polynomial equation 3rd order. Before the measurement plasma samples were processed on the vortex and was diluted in the reaction tubes (1:5 and 1:10 with calibrator 0). For the analysis of the measured optical density OD at 450 nm on a tablet spectrophotometer (Tecan Ultra) without correction to the reference wavelength. The results of the content of insulin in plasma up to 13 days for all animals studied are shown in figure 7.

After a single subcutaneous injection of 12 mg/kg of compound 11dc ur average�level of insulin in the plasma up to 1 day up to a maximum value of about 500 PM. As expected, the insulin concentration in the plasma is subsequently gradually decreased within 2 weeks. The ratio of peak concentration to a residual concentration of insulin in plasma during the first week amounted to approximately 1.4.

Example 20

Insulin release and degradation of the hydrogel at pH 7.4 in real time

Compound insulin-linker-hydrogel (11a) (730 µl, containing 3,19 mg of insulin) in acetate buffer (pH 5.0, 10 mm, 130 mm NaCl, and 0.01% (weight/volume) tween-20) was filled in a test tube for sample preparation, washed 3 times with buffer for the release of insulin from pH 7.4 (60 mm sodium phosphate, 3 mm EDTA, and 0.01% (weight/volume) Tween-20) and the volume was summed to 1.00 ml of the Aliquot of the suspension (0.5 ml, 1,59 mg of insulin) filled chromatographic column and placed in a thermostatic incubator (37°C). Through the column passed buffer for the release of insulin (pH 7.4) with a constant flow rate of 0.25 ml/h (nominal) and collected a buffer outside of the incubator. At specific time points of the solution was analyzed by reverse-phase HPLC (215 nm). Built a graph of the amount of insulin released from the incubation time, and to determine the appropriate half-time release used the software to approximate curves. It was determined that half the time visual�to study insulin is 9.4 of the day.

After 39 days of incubation at 37°C suspension of the hydrogel was transferred into a test tube for sample preparation, the remaining hydrogel was washed with column buffer to release with pH 7.4, and sample volume were summed to 1.00 ml. Two aliquots (300 μl each) were transferred to sterile tubes for samples, volume summed up to 1.5 ml, and incubated at 37°C. Samples were taken at specific intervals of time and analyzed using gel filtration. UV-absorption, the corresponding water-soluble degradation products released from the hydrogel containing one or more frame of the molecules (corresponding reactive functional groups) were combined and set aside on a graph versus time of incubation (see Fig.8).

Example 21

Ineterest Pro-drug compounds of insulin-linker-hydrogel

Used 5 ml of the Pro-drug compounds of insulin-linker-hydrogel 11dc (particle size 32-75 μm, 18 mg insulin/ml) in the buffer, succinic acid/tris with pH 5.0 (10 mm, 40 g/l mannitol, 10 g/l trehalose dihydrate, and 0.05% TWEEN-20). Suspension Pro-drug compounds of insulin-linker-hydrogel filled syringe 1 ml (length 57 mm) through a 20G needle. A 20G needle was replaced with a 30G needle and put the syringe in the holder for syringes (Aqua Computer GmbH&Co. KG), and the measurements were started at a speed of piston 172 mm/min (50 µl/s) (�the Tende strength tests: Multitest 1-d; software for data recording: EvaluatEmperor Lite, Version 1.16-015; Dynamometer: BFG 200 N (all from the company Mecmesin Ltd., UK). Experiments with increasing speed of the piston, shown in the table below, were performed with a new sample Pro-drug compounds of insulin-linker-hydrogel. Experiments with water and ethylene glycol was carried out accordingly. For all experiments used the same needle 30G. Fig.9 shows the dependence of the force relative to the flow rate when using the 30G needle.

Stream/ (sec/ml)Stream/ (μl/sec)Piston speed/ (mm/min)Force/
N (water)
Force/
N 11dc
Power/ethylene Glycol N
6167573133683
8125430102962
1010034472451
156722942235
205017231727

Abbreviations:

AcOH - acetic acid

AcOEt - ethyl acetate;

Aib - 2-aminoadamantane acid

Bn - benzyl

Boc - t-butyloxycarbonyl

COMU (1-cyano-2-ethoxy-2-oxoethylidene)dimethylamino-morpholino-carbene hexaphosphate

DBU - 1,3-diazabicyclo[5.4.0]undecen

DCC -N,N-dicyclohexylcarbodiimide

DCM - dichloro methane

DIEA - diisopropylethylamine

DMAP - dimethylaminopyridine

DMF -N,N-dimethylformamide

Dmob - 2,4-dimethoxybenzyl

DMSO-dimethyl sulfoxide

EDC - 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide

EDTA - ethylenediaminetetraacetic acid

EQ. - the stoichiometric equivalent

ESI-MS - mass spectrometry with ionization by spraying in an electromagnetic field (electrospray)

EtOH - ethanol

Fmoc - 9-fluorenylmethoxycarbonyl

HATU O-(7-asobancaria-1-yl)-N,N,N',N'-tetramethylurea hexaflurophosphate

HFIP - hexafluoroisopropanol

HPLC - high performance liquid chromatography

HOBt is N-hydroxy-benzotriazole

iPrOH - 2-�ropanol

LCMS - mass spectrometry combined with liquid chromatography

Mal - 3-maleimidomethyl

Mal-PEG6-NHS - NHS-ester of N-(3-maleimidomethyl)-21-amino-4,7,10,13,16,19-hexaoxa-heneicosane acid

Me - methyl

MeCN - acetonitrile;

MeOH - methanol;

Mmt - 4-methoxytrityl

MS - mass spectrum/mass spectrometry

MTBE - methyl tert-butyl ether

MW - molecular weight

n.d. is not defined

NHS - N-hydroxysuccinimide

OD - optical density

OBu - bucalossi

OtBu is tert-bucalossi

PEG - polyethylene glycol

Phth - ftal-

PyBOP - benzotriazole-1-yl-oxy-Tris-pyrrolidino-the phosphonium hexaflurophosphate

Reversed-phase HPLC Reversed-phase high-performance liquid chromatography

rpm - revolutions per minute

RT - room temperature

SEC gel filtration

Su - Succinimidyl

TCP - 2-chlorotriethylsilane resin

TES - triethylsilane

TFA - trifluoroacetic acid

THF - tetrahydrofuran;

TMEDA - N,N,N',N'-tetramethylethylenediamine

Tmob - 2,4,6-trimethoxybenzyl

Trt - triphenylmethyl, trityl

UPLC - ultra-performance liquid chromatography

UV - ultraviolet

VIS - visual

1. Pharmaceutical composition comprising a compound of insulin in a concentration of at least 10 mg/ml, characterized in that it has a pharmacokinetic profile in vivo essentially no emissions�and connection of insulin, thus, the connection of insulin is fully contained in a depot of the drug, and the connection of insulin covalently linked to the depot of the drug, where the connection of insulin is a Pro-drug of the compound or its pharmaceutically acceptable salt, representing a conjugate of insulin with the linker, D-L, in which
D represents the insulin molecule; and
- L is not biologically active linker molecule-L1represented by the formula (I),

in which the dotted line indicates the accession of one of the amino groups of the insulin with the formation of the amide bond;
X is C(R3R3a); or N(R3);
R1a, R3aindependently selected from the group consisting of N, NH(R2b), N(R2b)C(O)R4and C1-4alkyl;
R1, R2, R2a, R2b, R3, R4independently selected from the group consisting of N and C1-4of alkyl,
in which L1substituted by one Deputy L2-Z and optionally further substituted, provided that the hydrogen atom marked with an asterisk in formula (I) is not substituted by the Deputy, and in which
L2is a single chemical bond or L2represents C1-20alkyl chain, which optionally is interrupted by one or more groups independently selected from-O - and S(O)N(R3aa; optionally substituted by one or more groups independently selected from HE and C(O)N(R3aaR3aaa), where R3aa, R3aaaindependently selected from the group consisting of N and C1-4alkyl; and
Z is a hydrogel that contains the frame with a quarternary carbon of formula C(A-Hyp)4where each And independently selected from the formula -(CH2)n1(OCH2CH2)nX1- where n1 equals 1 or 2; n is an integer in the range from 5 to 50; and X1is a chemical functional group covalently linking A and Hyp, where Hyp consists of 5 - 32 lysine.

2. Pharmaceutical composition according to claim 1, containing a compound of insulin in a concentration of at least 11 mg/ml, for administration in a single dose of at least 10 mg of the compounds of insulin.

3. A composition according to claim 1, in which the concentration of the compounds of insulin is at least 11 mg/ml, for example from 11 mg/ml to 35 mg/ml.

4. A composition according to claim 1, characterized in that the ratio of peak concentration to a residual less than 2, for example less than 1,75, less than 1.5 or less than 1.25.

5. A composition according to claim 1, characterized by a constant release of compounds structurally intact insulin during the entire time interval between doses.

6. A composition according to claim 5, for which the complete time interval between doses �leaves at least about 80 hours, for example, at least about 110 hours, usually at least a week.

7. A composition according to claim 1, characterized by the fact that it is administered by injection, e.g., subcutaneous or intramuscular.

8. A composition according to claim 1, in which the connection of insulin is selected from human insulin, insulin glargine, insulin detemir, insulin lizpro, insulin aspart, insulin glulisine, or Pro-drug compounds.

9. A composition according to claim 1, for which the peak concentration is achieved within the first 24 hours after administration, for example in the first 12 hours after administration, for example within the first 6 hours after administration.

10. Pharmaceutical composition according to claim 1, wherein the pharmaceutical composition is dry.

11. Pharmaceutical composition according to claim 10, wherein the pharmaceutical composition is dried using lyophilization.

12. A composition according to claim 10, wherein the composition comprises a dose of Pro-drug compounds of insulin with the hydrogel sufficient to provide a therapeutically effective amount of insulin for at least three days, under one administration.

13. A composition according to claim 1, which is the single-dose composition.

14. A composition according to claim 1, wherein it is a multi-dose composition.

15. Pharmaceutical composition according to any one of claims. 1-13 in the container.

16. Pharmaceutical composition �about p. 15, wherein the container is a dual-chamber syringe.

17. Use of a compound of insulin for the manufacture of a pharmaceutical composition according to any one of claims. 1-16 for the treatment or prevention of a disease or disorder associated with deficiency of insulin, treatment or prevention of which is the use of compounds of insulin is beneficial, such as hyperglycemia, prediabetes, impaired glucosetolerance, type I diabetes, type II diabetes, and syndrome X.

18. A suspension containing a pharmaceutical composition according to any one of claims. 1-16.

19. The method of producing a slurry according to claim 18, which includes stages of restoration of a dry pharmaceutical composition according to claim 10 or 11 by adding the solution to restore.

20. A kit comprising a pharmaceutical composition according to any one of claims. 1-14 and the container for introduction of the composition.

21. A set including a needle and a container containing the solution for the restoration, and the dry composition according to claim 10 or 11 for use with a needle.

22. The kit according to claim 21, wherein the container is a dual-chamber syringe and in which one of the two chambers of the double chamber syringe contains a solution for recovery.

23. The kit according to claim 22, in which the composition can be administered by injection through a needle.

24. The kit according to claim 23, wherein the inner needle diameter less than 300 microns.

25. The kit according to claim 23, in which� inner diameter of the needle less than 225 microns.

26. The kit according to claim 23, wherein the inner diameter of the needle less than 175 microns.



 

Same patents:

FIELD: medicine.

SUBSTANCE: correcting cognitive disorders in the patients suffering arterial hypertension accompanying type 2 diabetes mellitus is ensured by combining a standard drug therapy with administering the preparation Kudesan 60 mg a day throughout two months.

EFFECT: administering Kudesan in the above dose and regimen provides the effective correction of cognitive disorders in the above group of patients in a combination with improving the cardiovascular function and metabolic processes.

2 tbl, 1 ex

FIELD: medicine.

SUBSTANCE: correcting increased levels of anxiety and depression in the patients with arterial hypertension accompanying type 2 diabetes mellitus is ensured by combining a standard drug treatment and administering Kudesan 60 mg a day for two months.

EFFECT: method provides the effective correction of anxiodepressive conditions in the given category of patients that in turn enables normalising blood pressure more effectively by reducing the negative psychosomatic effect.

1 ex, 2 tbl

FIELD: medicine, pharmaceutics.

SUBSTANCE: group of inventions refers to methods of treating type 2 diabetes, insulin resistance, insulin hyposecretion, obesity, hyperglycaemia and hyperinsulinemia, involving administering an effective amount of an anti-IL-1β antibody or its fragment into an individual, as well as to using the anti-IL-1β antibody or its fragment in preparing a composition applicable for treating the above diseases or conditions.

EFFECT: group of inventions is effective in treating type 2 diabetes mellitus, insulin resistance, insulin hyposecretion, obesity, hyperglycaemia and hyperinsulinemia.

67 cl, 13 dwg, 5 tbl, 14 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to novel N-containing heteroaryl derivatives of formula I or II or their pharmaceutically acceptable salts, which possess properties of JAK kinase, in particular JAK3, and can be applied for treating such diseases as asthma and chronic obstructive pulmonary disease (COPD). In formulae A represents carbon and B represents nitrogen or A represents nitrogen and B represents carbon; W represents CH or N; R1 and R2, independently represent hydrogen, C1-4alkyl, halogenC1-4alkyl, -CN; R3 represents C1-4alkyl, R9-C1-4alkyl, Cy1, where Cy1 is optionally substituted with one or several substituents R10; R4 represents hydrogen, C1-4alkyl, R12R7N-C0alkyl, where one of R7 and R12 represents hydrogen, and the other represents C1-4alkyl or group R13, which is selected from C1-5alkyl, Cy2-C0alkyl; R5 represents hydrogen; R6 represents hydrogen, C1-4alkyl, C1-4alkoxyC1-4alkyl, hydroxyC1-4alkyl, R12R7N-C1-4alkyl, R16CO-C0alkyl, Cy1; R7 represents hydrogen or C1-4alkyl; R9 represents halogen, -CN, -CONR7R12, -COR13, CO2R12, -OR12, -SO2R13, -SO2NR7R12, -NR7R12, -NR7COR12; R10 represents C1-4alkyl or R9-C0-4alkyl; R11 represents C1-4alkyl, halogen, -CN, -NR7R14; R12 represents hydrogen or R13; R13 represents C1-5alkyl, hydroxyC1-4alkyl, cyanoC1-4alkyl, Cy2-C0alkyl or R14R7N-C1-4alkyl; where Cy2 is optionally substituted with one or several constituents R11; R14 represents hydrogen or C1-4alkyl; R16 represents C1-4alkyl, halogenC1-4alkyl, C1-4alkoxyC1-4alkyl, hydroxyC1-4alkyl or cyanoC1-4alkyl; Cy1 represents monocyclic carbocyclic unsaturated or saturated ring, selected from C3-C6cycloalkyl, phenyl, or saturated monocyclic 4-6-membered heterocyclic ring, containing from 1 to 2 heteroatoms, selected from N and S, or partially unsaturated 10-membered bicyclic heterocyclic ring, containing oxygen atom as heteroatom, which can be substituted with group R11, where said ring is bound with the remaining part of molecule via any available C atom, and where one or several ring C or S atoms are optionally oxidised with formation of CO or SO2; and Cy2 represents monocyclic carbocyclic unsaturated ring, selected from C3-C6cycloalkyl, or aromatic monocyclic 4-6-membered heterocyclic ring, containing from 1 to 2 heteroatoms, selected from N and S, or unsaturated 10-membered bicyclic heterocyclic ring, containing oxygen atom as heteroatom, which can be substituted with group R11, where said ring is bound with the remaining part of molecule via any available atom C or N.

EFFECT: obtaining novel heteroaryl derivatives.

27 cl, 41 ex

Transdermal plaster // 2553350

FIELD: medicine.

SUBSTANCE: group of inventions relates to medicine. Described is matrix layer, suitable for application in plaster for transdermal delivery, aimed at introduction of biologically active compounds, which includes phosphate compound of tocopherol and polymer carrier. Also described is transdermal plaster and method of its production.

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48 cl, 15 tbl, 13 dwg, 12 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to pharmaceutical industry and represents clinical nutrition for prevention, treatment or relief of one or several symptoms, associated with impairment of metabolism or its disorder, which contains composition of polysaccharide high-viscosity dietary fibre, including viscous fibre mixture or its complex, consisting of from 48% to 90% in wt % of glucomannan, from 5 to 20 % in wt % of xanthan gum and from 5% to 30% in wt % of alginate, as well as, at least, one macroelement, selected from the group, consisting of protein carbohydrate and fat, where clinical nutrition is composed in order to provide dose of composition of polysaccharide high-viscosity dietary fibre from 20 g/day to 35 g/day for time period, effective for prevention, treatment and relief of one or several symptoms, associated with impairment of metabolism or its disorder.

EFFECT: invention ensures extension of arsenal of means, preventing, relieving or treating one or several symptoms, associated with impairment of metabolism or metabolic disease.

14 cl, 6 ex, 20 tbl, 48 dwg

FIELD: medicine.

SUBSTANCE: patients with diabetic microangiopathy are subjected to an examination which includes: general blood test, blood sugar, general urine analysis, ultrasonic examination of kidneys with the determination of indices of the kidney blood flow (Vmax, Vmin, S/D, PI, RI), basic ophthalmological parameters (vision acuity, examination of eye fundus vessels). Then, the intake of mildly-mineralised hydrocarbonate-chloride-sodium mineral water "Obyhovskaya" directly from the spring under sanatorium conditions is administered. Water is taken in heated to a temperature of 37°C in a dose of 3 ml per 1 kg of body weight 3 times per day 40 minutes before meal, the course constitutes 18 days.

EFFECT: application of the invention makes it possible to normalise the general blood test, blood sugar, general urine analysis, improve the condition of the visual analyser and indices of the kidney blood flow.

1 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to 2-pyridone compounds, represented by general formula [1], , where A represents benzene ring or pyridine ring, X represents structure, represented by general formula [3], V represents single bond or lower alkylene, W represents single bond, ether bond or lower alkylene, which can include ether bond, or their tautomers or stereoisomers.

EFFECT: obtaining pharmaceutically acceptable salts, which possess excellent activating activity with respect to GK and can be applied as medications.

27 cl, 23 tbl, 371 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to quinolines substituted by phosphorus-containing group of formula and applicable in medicine, wherein Z represents V1 and V2 are independently specified in hydrogen or halogen; one of R and R` represent phosphorus-containing substitute Q; the other one is specified in hydrogen or methoxyl; wherein the phosphorus-containing substitute Q represents A represents O; L represents C1-6alkyl; J represents NH or C3-6heterocycloalkyl and J is optionally substituted by G3; X is absent or represents -C(=O)-; X is absent or represents C1-6alkyl; each of R1 and R2 are independently specified in C1-6alkyl or C1-6alkoxy; G3 represents C1-6alkyl, R3S(=O)m-, R5C(=O)- or R3R4NC(=O)-; R3, R4 and R5 are independently specified in 3 or C1-6alkyl; m is equal to 0-2.

EFFECT: there are presented new protein kinase inhibitors effective for treating the diseases associated with abnormal protein kinase activity.

20 cl, 42 ex, 8 tbl, 3 dwg

FIELD: medicine.

SUBSTANCE: invention relates to medicine, namely to endocrinology, and deals with stimulation of insulin secretion. For this purpose 2 ml of concentrated nitro-glycerine solution are diluted with distilled water, cotton swab is soaked with obtained solution, stretched to 10-12 cm long and 3-4 cm wide size, applied perpendicular to spine on the left at the level of Th12, covered with cellophane and sealed with self-adhering plaster, with patient being turned onto back with preservation of said position for 1 hour.

EFFECT: method provides enhancement of insulin secretion by pancreas due to improvement of its blood supply.

2 ex, 2 tbl, 4 dwg

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to pharmaceutical industry and represents medication, possessing virucidal activity, for preventive and therapeutic treatment of viral infections, caused by virus of herpesviridae family, characterised by the fact that said medication contains pyroxican in carrier substance.

EFFECT: invention provides extension of arsenal of means, possessing virucidal activity.

7 cl

FIELD: medicine.

SUBSTANCE: ointment contains wax 13-15 wt %, glycerol 15-20 % and vegetable oil; the prepared ointment mass is exposed to ozone for 15-20 min in a yield amount of 10 mg/l.

EFFECT: invention provides the advanced healing efficacy with no side effects by reducing the time of wound cleansing from purulo-necrotic tissues.

3 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to the pharmaceutical industry and represents a pharmaceutical composition for the external application for the treatment of skin diseases in the form of a cream, which includes as an active substance methylprednisolone aceponate in a therapeutically effective amount and a lipophilic base, characterised by the fact that as the lipophilic base it contains petrolatum, liquid paraffin and oil of castor oil plant seeds and additionally white bee wax, with the components of the composition being in a specified ratio in g/100 g of the composition.

EFFECT: invention provides the creation of the stable composition, improved pharmacological properties and absence of an irritating effect.

1 tbl

FIELD: medicine.

SUBSTANCE: group of inventions refers to a pharmaceutical composition in the form of a soft dosage form for treating locomotor disorders, possessing anti-inflammatory, anaesthetic and anti-oedematous action, containing a combination of heparin sodium salt, dexpanthenol, diethylene glycol monoethyl ether and/or dimethylsulphoxide and target additives. The latter are presented by a gelation agent, a solvent/a solubiliser, a neutraliser, a flavouring agent/a moistening agent and water. The invention also discloses a method for producing the pharmaceutical composition.

EFFECT: reducing side actions, particularly allergic responses; it is characterised by high pharmacological activity, and simplified method for producing.

15 cl, 5 ex, 3 tbl

FIELD: medicine.

SUBSTANCE: pharmaceutical composition possessing a therapeutic action on various skin pathologies contains triptantrin, chitosan and distilled water, a lanoline and Vaseline mixture and protein-nucleic hydrolyzate of the salmonid fishes milt in a certain mixture ratio.

EFFECT: composition enables increasing the clinical effectiveness in the skin pathologies of various origins and extending the range of pharmaceutical compositions having the therapeutic effect on the various skin pathologies.

3 tbl, 4 dwg, 7 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to pharmaceutical industry, namely to production of medications for treating dermatosis. Medication according to invention, made in form of cream, contains mometasone furoate, preservative, hydrophilic no-aqueous solvent, emulsifying agent of 1st kind, emulsifying agent of 2nd kind, emollient, disodium edetate (trilon B), pH-regulating agent, and purified water in quantities, given in invention formula.

EFFECT: invention can be applied for treating inflammatory diseases and itching in case of dermatosis, yielding to glycocorticosteroid therapy.

9 cl, 3 tbl, 3 ex

FIELD: medicine.

SUBSTANCE: pharmaceutical composition contains drug substances and a consistency-forming base. According to the invention, it contains anaesthetics as drug substances specified in a group: anaesthesine, lidocaine, promedol and antiseptic specified in a group of: ethacridine lactate, Furacilin, dioxidine, chlorhexidine, boric acid, 0.5% silver solution in the following ratio, g in 1 ml of the mixture: anaesthetics 0.00001-0.5; antiseptics 0.00001-0.5; consistency-forming base - the rest. Besides, it contains lysozyme in an amount of 0.1-0.3 g per 1 ml of the mixture, alpha-lipoic acid as an antioxidant in an amount of 0.00001-0.5 g per 1 ml of the mixture, regenerants specified in a group of: pantothenic acid, calcium pantotenate, beta-carotene, coenzyme Q, sodium deoxyribonucleate, inosine, vitamins A, D, E, K in an amount of 0.00001-0.5 g per 1 ml of the mixture, anabolics specified in a group of: methyluracil, riboxinum, potassium orotate, orotic acid, L-carnitine in an amount of 0.00001-0.5 g per 1 ml of the mixture, glycyrrhizic acid and/or its salts in an amount of 0.00001-0.5 g per 1 ml of the mixture, recombinant interferon specified in a group of: recombinant interferon-alpha, recombinant interferon-beta, recombinant interferon-gamma in an amount of 100-1,000,000 International units, glucocorticoids specified in a group of: hydrocortisone, prenisolone, polcortolone in an amount of 0.00001-0.5 g per 1 ml of the mixture. The consistency-forming base contains the components specified in a group: hypromellose, sodium alginate, acetyl phthalyl cellulose, macrogol, polyvinylpyrrolidone.

EFFECT: improving the properties of the composition.

9 cl, 11 ex

FIELD: medicine.

SUBSTANCE: what is described is a bioactive wound coating of a hydrogel nanocomposite, which contains antimicrobial and antioxidant ingredients: silver-modified montmorillonite and fullerenol used to optimise the clinical course of the wound process, to prevent and suppress a wound infection. The wound coating can be used to treat gun-shot injuries, severe mechanical injuries, infected and uninfected wounds, including septic and persistent, granulating wounds following deep thermal, chemical and radioactive burns, in the combined therapy of trophic ulcers and bed sores at hospital, in the outpatient setting and in the field. The wound coating is elastic, not fragmented in dressing that facilitates wound care. A high sorption ability of the wound coating matrix, including of coarse-molecular ingredients of the wound effluent, provides the fast elimination of the wound bed. Using the hydrogel, i.e. possessing high degree of hydration, the wound coating meets the modern wound management in the humid medium.

EFFECT: optimum conditions for the early activation of the repair processes.

5 dwg, 2 tbl, 4 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to compositions for local application for the prevention and treatment of local eye pathologies, in particular inflammatory keratites and conjunctivitis and the dry eye syndrome, which contain as active ingredients polyunsaturated fatty acids of the omega-3 and omega-6 type, namely, EPA (eicosapentaenoic acid), DHA (docosahexaenoic acid) and GLA (γ-linolenic acid), mixed with vitamin E acetate and combined into a stable composition in a hydrogel, that is in the disperse form in a water solution, containing one or more gel-forming polymers. The claimed compositions are especially recommended for application as artificial tears.

EFFECT: invention provides an increased efficiency of the prevention and treatment of eye pathologies.

15 cl, 15 tbl, 3 dwg, 7 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to medicine, more specifically to a composition for treating dermatologic diseases, preferentially skin itching. The composition causes antiallergic action and is used in treating allergic reactions (rash, urticaria), insect bites, ultraviolet erythema and skin burns. The pharmaceutical composition contains azelastine hydrochloride and benzocaine as active substances, and a hydrophobic ingredient, a hydrophilic ingredient, an emulsifying agent and a pH corrective agent as additive agents. As the pH corrective agent, the composition contains preferentially succinic acid. The pharmaceutical composition is presented as a soft dosage form, preferentially in the form of a cream.

EFFECT: composition according to the invention is characterised by high pharmacologic activity, good package extrusion, and storage-stability.

9 cl, 1 tbl, 14 ex

FIELD: medicine.

SUBSTANCE: invention represents a dietary supplement for preventing alcoholic intoxication and relieving alcohol withdrawal syndrome presented in the form of a tablet containing silicone dioxide, taurine, succinic acid and excipients; the ingredients in the tablet are taken in certain ratio, in grams.

EFFECT: extending the range for preventing alcoholic intoxication and relieving alcohol withdrawal syndrome.

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