A conjugate of hydroxyalkyl starch and low-molecular substance and method of obtaining it

FIELD: chemistry; medicine.

SUBSTANCE: description is given of a conjugate of hydroxyalkyl starch and a low-molecular substance, in which the binding interaction between the molecular hydroxyalkyl starch and the low-molecular substance is based on a covalent bond, which forms from a bonding reaction between (i) an end aldehyde group or carboxyl group, formed due to selective oxidation of the end aldehyde group, or activated carboxyl group, obtained from conversion of the carboxyl group of hydroxyalkyl starch molecules and (ii) a functional group of a low-molecular substance, chosen from a group, consisting of an amino group, carboxyl group, thiol group and a hydroxyl group, which is reactive, relative the given aldehyde group or carboxyl group or an activated carboxyl group of hydroxyalkyl starch molecules. The bond directly formed from the bonding reaction can be modified for further reaction until formation of the above mentioned covalent bond, if necessary.

EFFECT: use of the conjugate allows for increasing duration of the presence of the low-molecular substance in blood plasma.

35 cl, 29 ex

 

Many low molecular weight substances is of commercial interest, particularly medicinal biologically active substances and plant protection products whose use is limited or even impossible due to the poor properties of solubility in aqueous medium and/or low residence time in the body. For example, small molecule drugs due to glomerular filtration in the kidney (limit inference about 70 kDa) is often too fast again derived from the circulatory system, so you need a constant costly and unpleasant for the patient additional collateral that drug, for example, due to the often repeated injections or infusions.

To avoid this drawback, in some cases, insoluble biologically active drug substance is administered in the form of oil bolus, in which the injection often form painful deposits. Moreover, the use of such poorly soluble drugs is often associated with toxic side effects due to their deposition in organs such as the liver and/or kidney. The consequence of such unwanted side effects is the fact that greatly limited used in vivo concentration range of biologically active substances.

Observed in recent approach to overcome these problems involves the binding of these problematic substances soluble biocompatible polymers, such as polyethylene glycol and dextran. By linking, on the one hand, it is possible to increase the molecular weight is above the threshold value of 70 kDa, so that you can significantly increase the residence time in the plasma of more than small molecules, on the other hand, due to the hydrophilic polymer part can be improved solubility in aqueous medium.

Still numerous modifications are carried out using polyethylene glycol (PEG) or dextran, and, in General, the preferred PEG, as it gives a more simple products. Dextranase conjugates, however, often exhibit high allergenicity, minor metabolic stability and in many cases they have a low output when the coupling reaction. In the case of PEG-conjugates also reported side effects from unpleasant to dangerous as itching, hypersensitivity reactions and pancreatitis. Further, often the biological activity of biologically active substances after bonding with the PEG partially greatly reduced. Also still largely unknown metabolism products of decomposition of PEG-conjugates and may constitute a health threat./p>

Thus, there remains a need in a physiologically acceptable alternatives for dextranomer or PEG-conjugates, which can increase the solubility of poorly soluble low molecular weight substances and/or increase the duration of low-molecular substances in the plasma, allowing attain improved pharmacodynamic properties of the active molecule.

The objective of the invention is therefore the provision of these alternatives and the development of simple and effective ways to obtain such alternative conjugates.

Unexpectedly, it was shown that the task can be solved using conjugates hydroxyalkyloxy, which differ in that the binding interaction between the molecule hydroxyalkylated and low-molecular substance is based on the covalent bond, which is formed by the reaction of binding between the terminal aldehyde group or resulting from this aldehyde groups by chemical conversion of functional group molecules hydroxyalkylated and the functional group of the low molecular weight substances reactive with respect to the aldehyde group or generated from it functional group of the molecule hydroxyalkyloxy, the connection formed directly clause and binding assays, if necessary, can be modified by further reaction to form the above covalent bonds.

The invention also relates to pharmaceutical compositions containing these conjugates, as to the use of these conjugates and compositions for the prevention or treatment of human or animal, and also to methods of producing these conjugates and compositions.

Used according to the invention hydroxyalkylated (HAS) can be obtained in one of known methods, for example by hydroxyethylamine starch in2- and/or C6-position anhydroglucose links with accelerated or 2-chloroalkane, as, for example, 2-chloroethanol (see, for example, U.S. patent 5218108 regarding gidroksietilirovaniya starch), with a variety of desirable ranges of molecular weights and degrees of substitution. You can also use any, issued for the sale of the preparative form. The definition of the alkyl group in "hydroxyalkanoate"used in this description, includes methyl, ethyl, isopropyl and n-propyl, and especially preferred ethyl. A significant advantage of hydroxyethylamine (HES) is that he's already officially approved as a biocompatible plasma expander and is widely used in clinical settings.

The average molecular what I weight hydroxyalkylated may amount to in the range of from about 3 kDa to several million daltons, preferably from about 10 kDa to about 2000 kDa, more preferably in the range of from about 70 kDa to about 1000 kDa, particularly preferably of approximately 130 kDa. To increase the residence time of low-molecular substances in the body average molecular weight hydroxyalkylated preferably chosen so that in the case of conjugates exceeded glomerular threshold value of 70 kDa. The degree of substitution (the ratio of the number of modified anhydroglucose links among all anhydroglucose links) may also change, and often it is a value in the range from about 0.2 to 0.8, preferably from about 0.3 to 0.7, even more preferably is about 0.5. (Note: data refer to the "degree of substitution", which ranges from 0 to 1.) The value Of2-replace it With6the replacement is usually a value in the range from 4 to 16, preferably in the range from 8 to 12.

These parameters can be set by known methods. The experiments with the use of hydroxyethylamine as a blood substitute has shown that the time HES in the plasma depends on the molecular weight and the degree and type of substitution (C2-substitution or6-substitution), and higher molecular weight, the higher the degree of substitution and a higher proportion With2

These correlations are also proposed according to the invention conjugates hydroxyalkylated and low molecular weight substances, so that the time that a particular conjugate in plasma is achieved proportion of polysaccharide.

As already mentioned, is involved in binding assays functional group of the molecule hydroxyalkanoate is a terminal aldehyde group or formed from it by chemical conversion of the functional group.

An example of such a chemical transformation is the selective oxidation of this aldehyde group with a suitable oxidizing agent, such as iodine, bromine or some metal ions, or by electrochemical oxidation with the formation of carboxyl group or an activated carboxyl group, for example, a complex ester, lactone, amide, and carboxyl group, if necessary, by the second reaction is transformed into an activated derivative. This carboxyl group or an activated carboxyl group can then be linked to the primary amino group or Tilney group of low-molecular substances in the formation of amide or thioester complex communication. Another possibility is linking to a hydroxyl group of low-molecular substances in the formation of ester.

Proposed according to the invention, the conjugate, however, you can also get the fact that to introduce the desired functional group of the low molecular weight substance is injected into engagement with suitable physiologically acceptable bifunctional linker molecule. The remaining reactive group associated linker molecule according to the present invention also seen as "reactive functional group of the low molecular weight substances".

Suitable linker molecules contain at one end group that can covalently to contact with the reactive functional group of the low molecular weight substances, for example, amino group, Tilney group, a carboxyl group or a hydroxyl group, and at the other end - group that can covalently bind to the end aldehyde group or formed from it by chemical conversion of the functional group, for example, a carboxyl group, an activated carboxyl group, amino group or Tilney group. Between the two functional groups of the linker molecule is biologically compatible bridge molecule suitable length, for example, grouping, which is made from alkane, (oligo)alkylenglycol grouping or other suitable oligomeric group. predpochtitelnye groups, which can react with the amine groups are, for example, a complex N-hydroxysuccinimidyl, complex sulfo-N-hydroxysuccinimidyl, complex amidoethyl or other activated carboxylic group; preferred groups that can react with thiol groups, are, for example, maleimide and carboxyl groups; preferred groups that can react with aldehyde or carboxyl groups are, for example, an amino group or tirinya group.

Examples of linker molecules for binding of SH - and NH-groups are:

AMAS(complex N-α (maleinizatsiei)Succinimidyl);
BMPS(complex N-β (multimediafiles)Succinimidyl);
GMBS(complex N-γ (maleimidomethyl)Succinimidyl);
EMCS(complex N-ε (maleimidopropionamide)Succinimidyl);
MBS(complex m-(maleimidomethyl)-N-hydroxysuccinimidyl);
SMCCSuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate);
SMPB(Succinimidyl-4-(p-maleimidomethyl)butyrate);
SPDP(Succinimidyl-3-(pyridyl the thio)propionate);
sulfo-GMBS(complex N-γ (maleimidomethyl)sulfosuccinimidyl);
sulfo-EMCS(complex N-ε (maleimidopropionamide)sulfosuccinimidyl).

Examples of linker molecules for binding of SH and SH-groups are:

WWII(1,4-bismaleimide);
BMDB(1,4-bismaleimide-2,3-dihydroxybutyl);
UMNS(bismaleimides);
VMOE(bismaleimides);
DTME(deliberalization);
HBVS(1,6-hexanesulfonic);
VM(REO)3(1.8-vimalakirtinirdesa);
VM(REO)4(1,11-vimalakirtinirdesa).

Examples of linker molecules for binding NH - and NH-groups are:

(disuccinimidyl);
BSOCOES(bis(2-(succinimidylester)ethyl)sulfon);
BS3(bis(sulfosuccinimidyl)suberate);
DFDNB(1,5-debtor-2,4-dinitrobenzene);
DMA(.HCl);
DSG
DSS(disuccinimidyl);
EGS(etilenglikoli(succinimidylester)).

Examples of linker molecules for binding of SH - and CHO-groups are:

WMRN(g TPUK hydrazide N-β-multimediaphoto acid);
EMSA(hydrazide N-ε-multimediaphoto acid);
KMUH(hydrazide N-κ-maleimidomethyl acid);
M2With2N(4-(N-maleimidomethyl)cyclohexane-1-.HCl);
MPBH(HCl salt hydrazide of 4-(4-N-maleimidomethyl)butyric acid);
PDPH(3-(2-pyridyldithio)propenylidene).

An example of a linker molecule to bind SH and Oh groups is PMPI (N-(p-maleimidomethyl)isocyanate).

Examples of linker molecules to turn the SH-group of the COOH group are:

WMRA(N-β-maleimidomethyl acid);
AMSN(N-β-maleimidomethyl acid);
KMUA(N-κ-maleimidomethyl acid).

When the apostrophes linker molecules for the conversion of the NH-group of the COOH group are MSA (methyl-N-succinimidylester) or its homologues with longer chain or the corresponding derivatives of ethylene glycol.

Examples of linker molecules for the conversion of COOH-groups in NH-group are DAB (1,4-diaminobutane) or its homologues with longer chain or the corresponding derivatives of ethylene glycol.

An example of a linker molecule, which reacts with the amino group of the molecule and leads to the formation of protected amino group at a greater distance from the molecule to avoid spatial difficulties, is TFCS (complex N-e-(triftoratsetilatsetonom)Succinimidyl).

Other suitable linker molecules are known in the art and are commercially available or can be provided depending on the needs and depending on the available and desirable functional groups and HAS linked low molecular weight substances and can be obtained by known methods.

According to a particularly preferred way of receiving terminal aldehyde group hydroxyalkylated (HAS) selectively oxidizes in aqueous alkaline solution with a molar excess of iodine, preferably in a molar ratio of iodine to HAS from 2:1 to 20:1, particularly preferably from about 5:1 to 6:1. As described in example 1, the optimized method, first hydroxyalkylated dissolved in hot distilled water and add a little less than 1 mol-equivalent of an aqueous solution of iodine, preferably in a concentration of from PR is about of 0.05 n to 0.5 n, especially preferably about 0.1 N. Then, to the reaction solution was added slowly, dropwise, with an interval of a few minutes an aqueous solution of NaOH in a molar concentration of about 5-15-fold, preferably about 10-fold, the concentration of the solution of iodine, up until after adding the solution starts to become clear again. To the reaction solution again add less than 1 mol-equivalent of the above aqueous solution of iodine, again the addition of NaOH solution, and adding iodine and NaOH is repeated as many times until you have added about 5.5 to 6 mol equivalents of a solution of iodine and 11-12 mol-equivalents of NaOH solution in relation to hydroxyalkylated. Then the reaction terminated, the reaction solution absoluut, for example, by dialysis or ultrafiltration, is subjected to cation exchange chromatography and the reaction product obtained by lyophilization. In the case of this method, regardless of molecular weight HAS reach almost quantitative yields.

According to another particularly preferred variant implementation, the selective oxidation with alkaline stabilized solutions of metal ions, for example, Cu++or Ag+also happens with almost quantitative yield (example 2). While it is preferable to use approximately 3-10-fold molar excess of oxidant.

Then the selectively oxidized hydroxyalkylated in a suitable organic solvent is injected into the interaction with the primary amino group of the desired low molecular weight substances in the formation of amide linkages. Preferred solvents are selected from the group of polar aprotic solvents, especially preferred to use dimethyl sulfoxide (DMSO). Contrary to conventional, described in the literature methods for such reactions linking unexpectedly shown that there is no need to use otherwise binding of activators, as carbodiimide and triazoles. Binding selectively oxidized hydroxyethylamine (Oh-HES) with various model compounds (see examples) without a problem occurs in the absence of activator.

Preferably, binding assays run in the presence of carbodiimide, more preferably in the presence of DCC (dicyclohexylcarbodiimide), highly preferably in the presence of EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide).

Reactive group of the molecule hydroxyalkylated can also be an amino group or Tolna group formed by chemical transformation of terminal aldehyde groups. For example, you can conduct recovery amination of aldehyde groups is houtem reaction with ammonia in the presence of hydrogen and of a catalyst or in the presence of cyanoborohydride sodium. The resulting amino group or Tolna group can then react with the free carboxyl group or aldehyde group of low-molecular substances. In this first form amide or thioester complex communication, respectively, Schiff bases, which, if necessary, can be modified by further reaction.

Further, the terminal aldehyde group of the molecule hydroxyalkylated or formed from it by chemical conversion of the functional group may also enter into an interaction with a suitable physiologically compatible, bifunctional linker molecule. In this case, the reaction of the binding functional group, formed from the terminal aldehyde group of the molecule hydroxyalkylated due to chemical transformations, use the remaining reactive functional group of a bifunctional linker molecule, which is injected into the interaction terminal aldehyde group or generated from it functional group. Thus, the terminal aldehyde group can also be converted into the desired functional group.

Suitable linker molecules contain at one end group that can covalently bind to the end aldehyde group or formed from it by chemical transformation F. the purpose ground receiving stations group for example, a carboxyl group, an activated carboxyl group, amino group or Tilney group, and the other end is a group that can covalently to contact with the reactive functional group of the low molecular weight substances, for example, amino group, Tilney group, a carboxyl group or IT group, preferably aryl-IT-group. Between the two functional groups of the linker molecule is biologically acceptable bridging molecule suitable length, for example, grouping, which is made from alkane, (oligo)alkylenglycol grouping or other suitable oligomeric group. Preferred groups that can react with the amine groups are, for example, a complex N-hydroxysuccinimidyl, complex sulfo-N-hydroxysuccinimidyl, complex amidoethyl or other activated carboxylic group; preferred groups that can react with thiol groups, are, for example, maleimide and carboxyl groups; preferred groups that can react with aldehyde or carboxyl groups are, for example, an amino group or tirinya group.

A number of specific, non-limiting of the scope of claims of the invention examples of suitable linker molecules has already been specified to enter is in relation to the conjugation of the linker molecules with low molecular weight substances.

If an alternative method of bonding according to the present invention, the terminal aldehyde group hydroxyalkylated (HAS) directly enter into interaction with the primary amino group of low-molecular substances, respectively, associated with the substance of the linker molecule by formation of a Schiff's base. Then, or simultaneously, the resulting Schiff base reduced to amine by entering into cooperation with a suitable reducing agent, resulting in an aqueous medium, a stable connection between the low-molecular substance and HAS.

The preferred reducing agents are sodium borohydride, cyanoborohydride sodium, organic complexes of boron, for example, complex 4-(dimethylamino)pyridine and the boron complex of N-ethyldiethanolamine and boron complex N-ethylmorpholine and boron complex N-methylmorpholine and boron complex N-phenylmorpholine and boron complex lutidine and the boron complex of triethylamine and the boron complex of trimethylamine and boron; suitable stereoselective reducing agents are, for example, thiacetarsamide sodium, triethylborohydride sodium, trimethoxyborohydride sodium, three-second-butylbromide potassium (K-selected), three second-butylbromide sodium (N-selectride), three second-butylbromide lithium (L-selectride), triuniversity potassium (KS-selectride) and treamill hydrid lithium (LS-selectride).

The binding reaction HAS or HAS oxidized low-molecular substance due to the expected poor water solubility of the substance and the lack of stability of the lactone in the aquatic environment preferably carried out in an organic solvent, more preferably in a polar aprotic solvent, which HAS and preferably also soluble low-molecular substance. Suitable solvents for HAS are, for example, DMSO, ethylene glycol, diglycol, triglycol, and N-organic. You can also use a mixture of DMSO with other solvents, if low-molecular substance insoluble in DMSO or other preferred solvent for HAS. Sometimes the reaction can be performed preferably in a heterogeneous phase.

The molar ratio HAS to low molecular weight substance in the binding assays usually ranges from about 20:1 to 1:1, preferably from about 5:1 to 1:1.

Output binding assays with respect to low molecular weight substance is, as a rule, more than 40%, often more than 60% and often more than 80% (see examples).

In the case of linking a low-molecular substance, it is preferably about drug biologically active substance, the solubility of which in an aqueous environment and/or bioavailability, stability and residence time in the body to promote the term "low molecular weight substance" should also fall peptides, includes up to about 50 amino acids. Dosage of the biologically active substance is preferably selected from the group consisting of antibiotics, anti-depressive drugs, antidiabetics, antidiuretics, anticholinergics, antiarrhythmics, antiemetics, antitussive funds, ANTIEPILEPTICS funds, antihistaminics funds, antifungal agents, protivogipertonicheskoe funds, antithrombotic agents, androgens, anti-androgens, estrogens, antiestrogen, anti-osteoporosis, anti-tumor agents, vasodilators, other, reducing the blood pressure means reducing fever agents, analgesics, suppressing inflammation funds, β-blockers, immunosuppressants and vitamins.

Some, not limiting the scope of the claims of the invention examples of biologically active drug substances with NH2group as a component binding assays with HAS are: albuterol, alendronate, amikacin, ampicillin, amoxicillin, amphotericin b, atenolol, azathioprine, cefaclor, cephalo-Smoking, Cefotaxime, ceftazidime, Ceftriaxone, cilastatin, cimetidine, ciprofloxacin, clonidine, colistin, cosyntropin, cycloserine, daunorubicin, doxorubicin, desmopressin, digidroergotamin, dobutamine, dopamine, efed is in, epinephrine, e-aminocaproic acid, ergometrine, esmolol, famotidine, flecainide, folic acid, flucytosine, furosemide, ganciclovir, gentamicin, glucagon, hydrazaline, imipenem, isoproterenol, ketamine, liotironin, localebean, perpetratin, metaraminol, hydrochlorothiazide methyldopa, metoclopramide, metoprolol, meksiletin, mitomycin, neomycin, netilmicin, nimodipine, nystatin, octreotide, oxytocin, pamidronate, pentamidine, phentolamine, phenylephrine, procainamide, procaine, propranolol, ritodrin, sotalol, teicoplanin, terbutaline, thiamine, tiludronate, tolazoline, trimethoprim, tromethamine, vancomycin, vasopressin and vinblastine.

Preferred examples of biologically active drug substances with NH2group as a component binding assays with HAS are: 6-aminopenicillanic acid, 7-aminocephalosporanic, 7-aminocephalosporanic acid and 7-aminopenicillanic acid.

Specific examples of such biologically active compounds with COOH group as a component binding assays with HAS are: acetylcysteine, azlotillin, aztreonam, benzylpenicillin, camptothecin, cefamandole, Cefazolin, cefepime, Cefotaxime, cefotetan, cefoxitin, ceftazidime, Ceftriaxone, cephalothin, cilastatin, ciprofloxacin, clavulanic acid, dicloxacillin, e-aminocaproic acid, floxacillin, formyltetrahydrofolate the I acid, furosemide, positieve acid, imipenem, indomethacin, Ketorolac, liotironin, melphalan, hydrochlorothiazide methyldopa, piperacillin, prostacyclin, prostaglandins, teicoplanin, and tikarcillin vancomycin.

Specific examples of such biologically active compounds with aryl-HE group as a component binding assays with HAS are: albuterol, allopurinol, apomorphine, Ceftriaxone, dobutamine, dopamine, doxycycline, edrophonium, isoproterenol, liotironin, metaraminol, hydrochlorothiazide methyldopa, minocycline, pentazocine, phenylephrine, phentolamine, propofol, rifamycin, ritodrin, teicoplanin, terbutaline, tetracycline and vancomycin.

Specific examples of such biologically active compounds with aliphatic HE group as a component binding assays with HAS are Taxol and portitxol.

The reaction products of the above-described chemical binding can be investigated by known methods and to determine the effectiveness of binding. For example, you can build UV calibration curve for the corresponding low-molecular substance and use it to determine the content of low-molecular substances in the sample, respectively, the share of low-molecular substances in the product obtained by binding assays. If low molecular weight substance has no UV absorption, it is possible to develop appropriate chlorimet is practical or electrochemical detection methods by analogy with known methods. The proportion of the saccharide in the conjugate can be determined, for example, by specific towards picano coloring selected reaction products. It is also possible quantification picana. The product yield of the coupling reaction with the participation of primary amines can also be set by modifying the unreacted amines using fluorescamine and determination of fluorescence.

Increased solubility in the case of sparingly soluble starting compounds can be controlled simply by experiments on dissolution. When associated with a partially water-soluble drug is biologically active substances increased hydrophilicity can be set by the OECD-method for the determination of the logP values. He correlates the retention time of the substance according to high performance liquid chromatography (HPLC) with reversed phase distribution coefficients in a mixture of n-octanol/water. All investigated by this method HES-conjugates according to the invention suiryudan in the extracted volume from the column with C18-phase, therefore, not enter into any interaction with C18-phase.

The conjugates according to the present invention, if necessary, can be used as such or in the form of a pharmaceutical composition for the prevention or treatment of human or animal.

This redcompetition include pharmaceutically effective amount proposed according to the invention conjugate as the active component, as well as pharmaceutically suitable carrier and, if necessary, other therapeutic, respectively, herbal components or excipients. Excipients may include, for example, diluents, buffers, improving the taste substances, binders, surfactants, thickeners, which imparts lubricity tablets ingredients, preservatives (including antioxidants), as well as substances that serve to give isotonicity of the composition of blood provided the recipient. Pharmaceutically effective amount is the amount sufficient for the manifestation of the desired positive action in disposable or reusable introduction in the framework of treatment to alleviate, cure or prevent disease condition. Pharmaceutically suitable carrier is a carrier that is compatible with both the dosage of the biologically active substance, and with the patient's body.

The form of the composition varies depending on the desired or suitable route of administration. Suitable routes of administration can be, for example, oral, parenteral, for example, subcutaneous, intramuscular, intravenous, intra-arterial, intra-articular, intrathecal; extradural injection, if necessary, the infusion; intranasal, intracra ealee, rectal, or local administration. Pharmaceutical compositions of more benefit can be in the form of a single dose and can be obtained by any of the well-known pharmaceutical methods.

HAS-conjugates according to the present invention can also be used in any other areas where the use of other polymer conjugates, for example, PEG-conjugates. Some specific, non-limiting of the scope of claims of the invention examples are the use of HAS-conjugate as immobilized component reaction for the reaction in heterogeneous phase or as a column material for affinity chromatography. Other possible applications for specialist obvious based on disclosed in the present description of the property proposed according to the invention HAS-conjugates.

The following examples have more to explain the invention, but without limiting its scope of claims. In particular, one can implement a similar reaction when using hydroxymethyluracil and hydroxypropylmethyl and achieve similar results.

Example 1

Selective oxidation of hydroxyethylamine (HES) using iodine

In a round bottom flask 10 g of HES with a molecular mass of 130 kDa when heated, dissolved in 12 ml of deionized water. To this solution add 2 the l of 0.1 n solution of I 2. Pipette 2 ml of 1.0 n NaOH solution associated with the flask through bilateral fitting and added dropwise a solution of NaOH at a rate of approximately 1 drop in 4 minutes. After adding approximately 0.2 ml of the NaOH solution, the solution becomes colorless at this point add the second portion of 2 ml of 0.1 n solution of iodine. After adding in total, 14 ml of a solution of iodine and 2.8 ml of NaOH solution, the reaction ends. The reaction mixture is then subjected to dialysis against deionized water.

Lactonization

Partially desalted solution is subjected to chromatography on a column of cation-exchanger (amberlite IR-120; H+-form) for turning aldonate groups in group aldonova acid. Then water is removed by lyophilization and thus receive lactoovo form.

Determination of the degree of oxidation

To, respectively, 1 ml of the sample in a nitrogen atmosphere pipette add 1 ml of alkaline copper-containing reagent (3.5 g Na2PO4, 4.0 g of K-Na-tartrate in 50 ml of water, moreover, 10 ml of 1 n NaOH solution, 8.0 ml of 10% (weight/volume) solution of CuSO4and 0,089 g of potassium Iodate in 10 ml of water, after adding 18 g of sodium sulfate complement to 100 ml). Heated for 45 minutes at a temperature of 100°C. After cooling, add 0.2 ml of 2.5%aqueous KI solution and 0.15 ml of 2M H2SO4. After 5 minutes, mixed with 1 drop of indicator solution phenol is about red (1% weight/volume) and titrated using a 5 mm solution of Na 2S2O3until the colour disappears. From the flow of funds for the titration it is possible to calculate the concentration of unreacted aldehyde groups.

Reaching nearly quantitative yield (>98%). Hydroxyethylamine with higher molecular weight (for example, 130 kDa, 250 kDa, 400 kDa), exactly the same as hydroxyethylamine with lower molecular weight (e.g., 10 kDa, 25 kDa, 40 kDa), it is possible to oxidize according to this method of obtaining such high outputs.

Example 2

Selective oxidation of hydroxyethylamine using ions of Cu2+

When heated to prepare a solution of 0.24 mmol of hydroxyethylamine (HES) with a molecular mass of 130 kDa in 10 ml of deionized water. This solution in the round bottom flask with a capacity of 100 ml are heated to a temperature of 70-80°and mixed with at 1.17 mmol stabilized Cu2+(for example, Rochelle salt as a stabilizer or other stabilizers) and water diluted solution of NaOH (final concentration of 0.1 n NaOH). Then the temperature was raised to 100°and the reaction is carried out until, until you see a reddish coloration. The reaction is stopped and the reaction mixture is cooled to a temperature of 4°C. a Reddish precipitate removed by filtration. The filtrate is subjected to dialysis against deionized water and then converted into the lactone, as described in point is the iMER 1. Oxidation proceeds quantitatively (yield >99%). According to this method, you can also oxidize low-molecular-weight HES (for example, with a molecular mass of 10 kDa, 25 kDa, 40 kDa) or more high molecular weight HES (for example, with a molecular mass of 130 kDa, 250 kDa, 400 kDa).

Example 3

Binding selectively oxidized hydroxyethylamine (ox-HES) with alendronate

In a round bottom flask with a capacity of 100 ml to 5 mg of alendronate (a bisphosphonate) and 3-5-fold molar excess of lactone ox-HES (obtained as described in example 1 or 2) is dissolved in 405 ml of dimethylsulfoxide. The suspension is heated to a temperature of 70°and under moderate stirring (magnetic stirrer) was incubated for 24-36 hours. The reaction is then stopped and the reaction mixture is cooled to room temperature. After that add 20-30 ml of water and this solution is subjected to dialysis against distilled water. Instead dialysis can also be used ultrafiltration with a suitable threshold value of the membrane in relation to molecular weight. This makes it possible not only the exchange of the solvent, but also the concentration of the solution, which is then lyophilizer. The degree of implementation of binding detect using analytical standard methods, for example, by gel chromatography and test with ninhydrin to free amino groups. The yield of binding of SOS is to place approximately 85% for binding to ox-HES with a molecular mass of 130 kDa and about 80% for the lactone binding to ox-HES with a molecular mass of 10 kDa.

Example 4

Binding selectively oxidized HES (ox-HES) with amphotericin b

12.0 g of dried lactone ox-HES with a molecular mass of 130 kDa in the atmosphere of nitrogen dissolved in 30 ml of anhydrous dimethyl sulfoxide. The solution is heated to a temperature of 70°and added 52 mg of amphotericin b C. In the absence of light, the reaction mixture was maintained under these conditions for 24 hours. Detection of binding is carried out by gel permeation chromatography with photometric detection at 385 nm (λmaxamphotericin b). After stopping the reaction by adding 80 ml of distilled water, the reaction mixture is intensively cialiswhat against water. By lyophilization receive the product binding slightly yellow in color. (Out of approximately 87%.)

In comparable conditions when linking lactone ox-HES with a molecular mass of 10 kDa with amphotericin b reach the exit approximately 75%.

Example 5

The binding of ox-HES with ampicillin

In a round bottom flask with a capacity of 100 ml to 1.3 g of dry lactone ox-HES with a molecular mass of 130 kDa dissolve 5 ml of anhydrous dimethyl sulfoxide. This solution is heated to a temperature of 45°and add 11.0 mg of ampicillin (Aldrich # 27.186-1). The reaction proceeds at moderate stirring for 20 hours and then stopped by adding 25 ml of distilled water. The reaction mixture cialiswhat against distillers the authorized water and then lyophilizer. The degree of binding is determined by analysis of the product by gel chromatography and the free amino group of ampicillin find with ninhydrin.

Example 6

The binding of ox-HES with neomycin

In a reaction vessel with a capacity of 50 ml under stirring with a magnetic stir bar and at a temperature of 60°3×10-5mol of lactone Oh-HES with a molecular mass of 25 kDa was dissolved in 5 ml of N-methylpyrrolidone. After adding 10 mg of neomycin in 2 ml of anhydrous dimethyl sulfoxide is refluxed for about 10 hours. After cooling to room temperature, the reaction is stopped by adding a further 35 ml of water. By dialysis to remove the most part of the solvent and the resulting binding product then lyophilizer. Using gel chromatography with UV-detection can be detected obtained by associating the product with a yield of about 82%.

Example 7

The binding of ox-HES with meptazinol

To dissolve completely when heated 2.5 g of the lactone Oh-HES with a molecular mass of 130 kDa and 22 mg of mepartricin (manufactured by a company Societa Prodotti Antibiotici, Mailand, Italy) is required 10 ml of ethylene glycol. The solvent before it is subjected to degassing and dried. The reaction solution is stirred for 36 hours in the absence of light and in an atmosphere of inert what about the gas, and the reaction was finally stopped by injecting 40 ml of ice water. Ethylene glycol is separated by ultrafiltration (10 kDa membrane), followed by lyophilization obtain 2.1 g of powder light yellowish color. Further purification is carried out by HPLC with reversed-phase column with phase C18 in the case of detection in the ultraviolet/visible region of the spectrum.

Example 8

The binding of ox-HES with nystatin

In a round bottom flask with a capacity of 100 ml of 2.5 g of dry lactone Oh-HES with a molecular mass of 130 kDa was dissolved in 10 ml of anhydrous dimethyl sulfoxide. After adding 9.5 mg nystatin is heated to a temperature of 60°and stirred in the atmosphere of inert gas and in the dark. The reaction proceeds at moderate stirring for 48 hours and then stopped by adding 50 ml of distilled water. The reaction mixture cialiswhat against distilled water and then lyophilizer. Binding can be detected by HPLC with reversed phase (C18 column with) and detection at 325 nm. The output is determined based on the absorption peak of the product is approximately 67%.

Example 9

The binding of ox-HES with mitomycin C

2.5 g of the lactone Oh-HES with a molecular mass of 130 kDa and 20 mg of mitomycin (Fluka # 69824) at a temperature of 60°dissolved in a mixture of dimethylsulfoxide and methanol in the ratio 9:1. The reaction solution withstand crashes-auto St is t at the boiling point under reflux for 24 hours and then to terminate the reaction, add 40 ml of water. This solution cialiswhat overnight against deionized water and then subjected to drying by freezing. Detection of binding is realized by means of HPLC with reversed phase and detection at 320 nm. The expected product link get with the release of 82%.

Example 10

The binding of ox-HES with daunorubicin

1.3 g of the lactone Oh-HES with a molecular mass of 130 kDa at a temperature of 70°and with stirring, dissolved in 10 ml of N-methylpyrrolidone. To the resulting solution was added dropwise a solution of 17 mg of daunorubicin (Fluka # 30450) in 3 ml of dimethylformamide. The reaction mixture is stirred for 20 hours under these conditions, cooled to room temperature and finally shaken with 40 ml of distilled water. By dialysis against water to remove the major part of the solvent and then subjected to drying by freezing. Linked daunorubicin detect by HPLC with reversed phase and detection in the ultraviolet/visible region of the spectrum.

Example 11

The binding of ox-HES from 7-aminocephalosporanic

In a round bottom flask with a capacity of 100 ml under stirring with a magnetic stirrer 3.0 g of the lactone Oh-HES with a molecular mass of 130 kDa and 20 mg of 7-aminocephalosporanic (Fluka # 07300) dissolved in 5 ml of anhydrous dimethyl sulfoxide. The temperature was raised to 50°C and maintained for 15 hours. After this time Oh what adout to a temperature of 25° C and the reaction mixture was diluted by adding 5 ml of distilled water. The sulfoxide and unreacted 7-aminocephalosporanic removed by dialysis against distilled water. The solution is then lyophilizer and the product analyzed by thin-layer chromatography (TLC) and gel chromatography.

Example 12

The binding of ox-HES 6 aminopenicillanic acid

Conduct described in example 11 reaction under the same conditions when using 16 mg 6-aminopenicillanic acid instead of 7-aminocephalosporanic and the reaction product is treated and analyzed in the same terms.

Example 13

The binding of ox-HES with LHRH

1.0 g of dried lactone Oh-HES with a molecular mass of 130 kDa with 5 mg LHRH (lutilisation; "lutenizing hormone releasing hormone") (Bachem, Switzerland) and incubated in 10 ml of dimethylsulfoxide. The reaction is performed for 15 hours at a temperature of 45°under stirring and stopped by adding 40 ml of water. Associated with HES LHRH, after extensive dialysis against water to remove the main quantity of DMSO and unreacted peptide, obtained by lyophilization. The resulting product analyzed by gel chromatography (superose 12; Amersham-Pharmacia, Sweden) and UV detection at 280 nm. To the resulting binding product stechiometry the Yu approximately 1:1 is obtained from the determination of the peptide by Trp-absorption and determine the percentage of polysaccharide by colouring the phenol/sulphuric acid.

Example 14

The binding of ox-HES with camptothecin

In a round bottom flask at a temperature of 50°20 mg camptothecin dissolved in 5 ml of anhydrous dimethyl sulfoxide. To this solution was added dropwise a solution of 36 mg of 1,4-diaminobutane in 2 ml of anhydrous dimethyl sulfoxide. The reaction mixture was kept for 24 hours under these conditions and lightly mix. The product of conjugation of purified flash chromatography. The output is approximately 83%.

For binding assays modified camptothecin with Oh-HES with a molecular mass of 130 kDa entire reaction mixture after treatment with 3.6 g of the lactone of the polysaccharide with stirring and heated to a temperature of 50°dissolved in 8 ml of anhydrous dimethyl sulfoxide. Over the course of the reaction is monitored by HPLC with reversed-phase samples from the reaction mixture. After aging for 20 hours at a temperature of 50°more is not observed any further education product, and the reaction stopped by adding 50 ml of distilled water. After dialysis against water obtained by linking the product is subjected to drying by freezing. The analysis is carried out by gel chromatography and coloring free amino groups in the modified, the unreacted camptothecin using the ninhydrin on the TLC plate.

Example 15

The binding of ox-HES what prostacyclin

a) Amidofunctional

352 mg of prostacyclin (Sigma-Aldrich) at a temperature of 0°dissolved in 5 ml of anhydrous dimethylformamide with 2% dichloromethane (volume/volume). To the resulting solution was added 1.3 g of dicyclohexylcarbodiimide (DCC) in 5 ml of anhydrous dimethylformamide. Slightly mixed, incubated for 30 minutes. Then add a 5-fold molar excess (with respect prostacyclin) simple 1,5-diaminoethylene ether and the solution slowly warmed to room temperature. Cleaning aminobenzoylamino product link perform flash chromatography on phase of silicon dioxide.

b) Linking HES

220 mg of the pure product link from the stage (a) at room temperature was dissolved in 8 ml of glycol. To the resulting solution while stirring the mixed solution of 4.0 g of the lactone Oh-HES with a molecular mass of 130 kDa in 10 ml of glycol and heated to a temperature of 45°C. in 8 hours and cooled in a bath of ice and intensively cialiswhat against water. A clear solution examined by HPLC with reversed-phase column C18. From the ratio of the area in the extracted volume of the column (product link) and the source materials you can calculate the efficiency of binding. The output is 53%.

Example 16

Linking HES with alendronate

In a round bottom flask with a capacity of 100 ml to a solution of 2.25 mg Ala is konata in 4 ml phosphate buffer (0.1 M, pH 7.5) add a tenfold molar excess HES with a molecular mass of 25 kDa. The reaction mixture was shaken to dissolve the polysaccharide and then add thirty power scope molar excess NaBH3CN. The reaction proceeds at room temperature for 48 hours, and the formation of the product binding is determined in the sample by interacting with fluorescamine that with the free amino groups gives a fluorescent product.

Example 17

Linking HES with amoxilina

In a two-neck flask contribute to 4.0 ml of 0.1 N. nutrifaster buffer (pH 7.5) and at a temperature of 60°dissolve 1.5 g HES with a molecular mass of 40 kDa. After cooling to a temperature of 25°under stirring with a magnetic stir bar add to 7.0 mg of amoxillin (Fluka # 10039). In a separate vessel, prepare a solution of NaBH3CN, which corresponds to thirty power scope molar excess, in 2 ml of the same nutrifaster buffer. Using a dropping funnel a solution of cyanoborohydride slowly added dropwise to the first solution over a period of time of 30 minutes. The reaction mixture is stirred during the next 24-36 hours and then to terminate the reaction, using 0.1 n HCl establish a pH value of 4. The solution absoluut by dialysis and lyophilizers. Detection of product binding is carried out using gel of chromatogra the AI and UV-photometer.

Example 18

Linking HES with cefaclorum

In a round bottom flask with a capacity of 100 ml, contributed 4 ml of 0.1 N. nutrifaster buffer (pH 7.0)used for solution of 110 mg of NaBH3CN. With stirring, add 6,0×10-5mol HES with a molecular mass of 130 kDa and a 2.0×10-5mol cefaclor (Fluka # 22125). The temperature of the reaction support at 25°and the reaction mixture was moderately stirred for 24 hours. Then the solution is acidified to pH 4.0 and stirred for 30 minutes; for desalting and concentration is subjected to ultrafiltration (10 kDa membrane). Product detect binding by gel chromatography high pressure (HP-GPC) at 265 nm.

Example 19

Linking HES with doxorubicin

6.0 mg of doxorubicin (Fluka # 45584) in the presence of a threefold molar excess HES with a molecular mass of 130 kDa suspended in 4 ml of 0.1 N. nutrifaster buffer (pH 7.5) at room temperature. The reaction mixture for 30 minutes intensively stirred and slowly mixed with 3 ml of 0.8 M solution of NaBH3CN. The reaction mixture was kept under stirring for 48 hours at room temperature. Then it is subjected to diafiltration when using a 10 kDa membrane to remove salts and unreacted doxorubicin. Subject diafiltration solution lyophilizer the t and the product linking examined by gel chromatography and HPLC with reversed phase.

Example 20

Linking HES with vasopressin

In a round bottom flask equipped with addition funnel, 1,25 g HES with a molecular mass of 130 kDa by heating and stirring slightly dissolved in 5 ml of 0.1 M nutrifaster buffer, pH 8.0. To this solution was added 5 mg of vasopressin (Bachem, Switzerland). In 2 ml of 0.1 M phosphate buffer (pH 7.5) dissolve 30 mg of NaBH3CN and via addition funnel slowly added dropwise to the reaction mixture. The reaction mixture was incubated for 24 hours at a temperature of 25°C. To terminate the reaction, reduce the pH value to 4.0 by addition of 0.1 n HCl. After extensive dialysis against water associated with HES product is subjected to drying by freezing. The analysis is carried out by gel chromatography, as described above, and UV detection at 220 nm.

Example 21

The binding of ox-HES with a molecular mass of 70 kDa with neomycin

In a two-neck flask in an argon atmosphere, 1.01 g of neomycin (sulfate) and 126,21 mg Oh-HES with a molecular mass of 70 kDa was dissolved in 2 ml of dimethyl sulfoxide and after adding 0,81 mg of DMAP (4-dimethylaminopyridine) is heated for 24 hours at a temperature of 70°C. Then the reaction stopped by the addition of acetone, thus precipitating the product link. The solid is dissolved in water and purified within 48 hours by dialysis against water. After freeze-drying the floor is up to 80 mg of the product binding, white (63%).

Example 22

An alternative method of binding Oh-HES with a molecular mass of 70 kDa with neomycin

The binding of neomycin with Oh-HES with a molecular mass of 70 kDa successfully can be performed at room temperature in dimethyl sulfoxide adding EDC as activator. For this 16,97 mg neomycin (sulfate), 348 mg Oh-HES with a molecular mass of 70 kDa and of 2.28 mg of DMAP are dissolved in 1 ml of dimethyl sulfoxide. After adding a 3.83 mg dicyclohexylcarbodiimide (1 equivalent) and the solution stirred for 2 hours and re-add one equivalent of dicyclohexylcarbodiimide. This method is repeated until the introduction of 10 equivalents of dicyclohexylcarbodiimide in the reaction solution. The reaction time is generally 24 hours. After addition of a solution of 20 ml of acetone product binding is deposited. The solid is dissolved in water and within 48 hours purified by dialysis against water. After freeze-drying obtain 280 mg of the product binding, white (80%).

Example 23

The binding of ox-HES with a molecular mass of 70 kDa with daunorubicin

In a two-neck flask 0.5 mg daunorubicinol, 829,0 mg Oh-HES with a molecular mass of 70 kDa and 0.108 mg DMAP in an argon atmosphere was dissolved in 2 ml of dimethylsulfoxide and heated for 24 hours at a temperature of 70°C. Then there is added 20 ml of acetone, while deposited about the SPS binding. The solution is centrifuged and the precipitate washed repeatedly with acetone and centrifuged. Get painted in a slightly pink solid, which is dissolved in water and the resulting solution cialiswhat against water. After freeze-drying receive 656 mg (80%) are painted in a slightly pink solid. The purity of the linked daunorubicin determined by HPLC with reversed phase.

Example 24

The binding of ox-HES with a molecular mass of 130 kDa with 7-aminocephalosporanic acid

In a round bottom flask with a capacity of 100 ml 383 mg Oh-HES with a molecular mass of 130 kDa and 1.22 mg 7-aminocephalosporanic acid (Fluka # 07300) under stirring with a magnetic stirrer was dissolved in 2 ml of anhydrous dimethyl sulfoxide. The temperature was raised to 70°and support within 24 hours. After this time, cooled to a temperature of 25°and the reaction product is precipitated by adding 20 ml of acetone. The solid is washed with 20 ml of acetone and dissolved in 20 ml of distilled water. Further purification of the product linking is carried out by dialysis against distilled water. The solution is then subjected to lyophilization and the product analyzed by TLC and gel chromatography. Obtain 270 mg of the product binding (70%) as a solid white color.

Example 25

The binding of ox-HES with a molecular mass is th 70 kDa with 6-aminopenicillanic acid

Described in example d reaction carried out in the same conditions with the use of 1.57 mg 6-aminopenicillanic acid instead of 7-aminocephalosporanic acid and 135,54 mg ox-HES with a molecular mass of 70 kDa. The reaction product is treated and analyzed in the same conditions. After clearing the receive and 88 mg of the product binding (65%) as a solid white color.

Example 26

Linking HES with a molecular mass of 40 kDa with amoxicillin

In a two-neck flask enter 4,0 ml of 0.1 n nutrifaster buffer (pH 7.5) and at temperatures up to 60°it dissolve 1.5 g HES with a molecular mass of 40 kDa. After cooling to a temperature of 25°under stirring with a magnetic stir bar add to 7.0 mg amoxicillin (Fluka # 10039). In a separate vessel, prepare a solution of NaBH3CN, which corresponds to thirty power scope molar excess, in 2 ml of the same nutrifaster buffer. Using a dropping funnel a solution of cyanoborohydride slowly added dropwise to the first solution over 30 minutes. The reaction mixture is stirred during the next 24-36 hours and then to terminate the reaction using 0.1 n HCl establish a pH value of 4. The solution absoluut by dialysis and lyophilizers. Detection of product binding is carried out using gel chromatography and UV-photometer.

Example 27

The binding of ox-HES from the forefront of the lar weight of 70 kDa with amoxicillin

In a round bottom flask with a capacity of 100 ml 173 mg Oh-HES with a molecular mass of 70 kDa and 0,85 mg amoxicillin under stirring with a magnetic stirrer was dissolved in 2 ml of anhydrous dimethyl sulfoxide. The temperature was raised to 70°and support within 24 hours. After this time it is cooled to a temperature of 25°and the reaction product is precipitated by adding 20 ml of acetone. The solid is washed with 20 ml of acetone and dissolved in 20 ml of distilled water. Further purification of the product linking is carried out by dialysis against distilled water. The solution is then subjected to lyophilization and the product analyzed by TLC and gel chromatography. Obtain 151 mg of product binding (87%) as a solid white color.

Example 28

The binding of ox-HES with a molecular mass of 70 kDa with cephalo-Smoking

In a round bottom flask with a capacity of 100 ml 610 mg Oh-HES with a molecular mass of 70 kDa and 2,965 mg cephalo-Smoking under stirring with a magnetic stirrer was dissolved in 2 ml of anhydrous dimethyl sulfoxide. The temperature was raised to 70°and support within 24 hours. Then cooled to a temperature of 25°and the reaction product is precipitated by adding 20 ml of acetone. The solid is washed with 20 ml of acetone and dissolved in 20 ml of distilled water. Further purification of the product binding Khujand who are by dialysis against distilled water. The solution is then subjected to lyophilization and the product analyzed by TLC and gel chromatography. Obtain 490 mg of the product binding (87%) as a solid white color.

Example 29

The binding of ox-HES with a molecular mass of 70 kDa with glucagon

In a round bottom flask 0.23 mg (66×10-9mol) of glucagon, 123 mg (6,6×10-6mol Oh-HES with a molecular mass of 70 kDa was dissolved in 1 ml of dimethyl sulfoxide. In the reaction solution with an interval of 1 hour is added in the form of 8 servings of dicyclohexylcarbodiimide (DCC) up to add to the overall results were 23.08 mg After a reaction time of 24 hours the reaction is stopped by adding 15 ml of water. Product link purified by dialysis against water. After freeze-drying obtain 79 mg of the product binding, white (65%).

1. Conjugate hydroxyalkylated and low molecular weight substances, characterized in that the binding interaction between the molecule hydroxyalkylated and low-molecular substance is based on the covalent bond, which is formed by the reaction of binding between (i) the terminal aldehyde group or a carboxyl group, which is formed by selective oxidation of the terminal aldehyde group or an activated carboxyl group, obtained by the transformation specified carboxyl groups of the molecules hydroxyethylacrylate (ii) the functional group of the low molecular weight substances, selected from the group consisting of amino group, carboxyl group, thiol group and a hydroxyl group which is reactive with respect to the aldehyde group or a carboxyl group or originating from it activated carboxyl group of the molecule hydroxyalkyloxy, the connection formed directly by the reaction of binding, if necessary, may be, if necessary, modified by further reaction to form the above covalent bonds.

2. The conjugate according to claim 1, characterized in that the carboxyl group or an activated carboxyl group of the molecule hydroxyalkylated is one of the functional groups of a bifunctional linker molecule, which interacts terminal aldehyde group or a carboxyl group is formed by selective oxidation of the terminal aldehyde group or an activated carboxyl group, obtained by the transformation specified carboxyl group.

3. The conjugate according to claim 1, characterized in that the amino group, carboxyl group, thiol group or hydroxyl group of the low molecular weight substances is one of the functional groups of a bifunctional linker molecule, which is associated with a low-molecular substance.

4. The conjugate according to claim 1, otlichalis the same time, the covalent bond is formed by the reaction of bonding between the carboxyl group formed by selective oxidation of the terminal aldehyde group of the molecule hydroxyalkyloxy, or an activated carboxyl group and a primary amino group or Tilney group of low-molecular substances.

5. The conjugate according to claim 4, characterized in that the covalent bond is an amide bond, which is formed by the reaction of binding between a lactone formed by selective oxidation of the terminal aldehyde group of the molecule hydroxyalkyloxy, and the primary amino group of a low molecular weight substances.

6. The conjugate according to claim 1, characterized in that the covalent bond is an amine linkage formed by the reaction of binding between the terminal aldehyde group of the molecule hydroxyalkylated and the primary amino group of a low molecular weight substances with the formation of Schiff bases and recovery Schiff bases to obtain the amine.

7. The conjugate according to claim 1, characterized in that the molecule hydroxyalkylated has a molecular weight in the range from about 70 kDa to about 1000 kDa.

8. The conjugate according to claim 7, characterized in that the molecule hydroxyalkylated has a molecular mass of approximately 130 kDa.

9. The conjugate according to claim 1, characterized in that the Molek is and hydroxyalkylated has a degree of substitution of from about 0.3 to about 0.7.

10. The conjugate according to claim 1, characterized in that the molecule hydroxyalkylated has the value2-replace it With6-substitution of from 8 to 12.

11. The conjugate according to claim 1, characterized in that the molecule hydroxyalkanoate is a molecule hydroxyethylamine.

12. The conjugate according to claim 1, characterized in that the low molecular weight substance is a biologically active drug substance.

13. The conjugate according to clause 12, wherein the biologically active drug substance selected from the group consisting of antibiotics, anti-depressive drugs, antidiabetics, antidiuretics, anticholinergics, antiarrhythmics, antiemetics, ANTIEPILEPTICS funds, antihistaminics funds, antifungal agents, protivogipertonicheskoe funds, antithrombotic agents, androgens, anti-androgens, estrogens, antiestrogen, anti-osteoporosis, anti-tumor agents, vasodilators, and other blood pressure-lowering means for reducing fever agents, analgesics, suppressing inflammation funds, β-blockers, immunosuppressants and vitamins.

14. The conjugate according to item 12 or 13, characterized in that is involved in binding assays the functional group of the biologically active drug substance is aminor the PAP.

15. The conjugate according to 14, characterized in that the biologically active drug substance selected from the group consisting of albuterol, alendronate, amikacin, aminopenicillin, amoxicillin, atenolol, azathioprine, cefaclor, cephalo-Smoking, Cefotaxime, ceftazidime, Ceftriaxone, tsilastatina, cimetidine, ciprofloxacin, clonidine, colistin, cosyntropin, cycloserine, daunorubicin, doxorubicin, desmopressina, digidroergotamina, dobutamine, dopamine, ephedrine, epinephrine, e-aminocaproic acid, ergometrine, esmolol, famotidine, flecainide, Polevoy acid, flucytosine, furosemide, ganciclovir, gentamicin, glucagon, hydrazaline, imipenem, isoproterenol, ketamine, liotironina, localebean, perpetratin, metaraminol, hydrochlorothiazide methyldopa, metoclopramide, metoprolol, meksiletina, mitomycin, neomycin, netilmicin, nimodipina, nystatin, octreotide, oxytocin, pamidronate, pentamidine, fentolamina, phenylephrine, procainamide, procaine, propranolol, ritodrine, sotalol, teicoplanin, terbutalina, thiamine, tiludronate, tolazoline, trimethoprim, tromethamine, vancomycin, vasopressin and vinblastine.

16. The conjugate according to item 12 or 13, characterized in that is involved in binding assays functional group of medical biologically active substance is a carboxyl group and an activated carboxyl group.

17. The conjugate according to clause 16, wherein the biologically active drug substance selected from the group consisting of acetylcysteine, azlocillin, aztreonam, benzylpenicillin, camptothecin, cefamandole, Cefazolin, cefepime, Cefotaxime, cefotetan, tsefoksitina, ceftazidime, Ceftriaxone, tsefalotina, tsilastatina, ciprofloxacin, clavulanic acid, dicloxacillin, e-aminocaproic acid, floxacillin, formyltetrahydrofolate acid, furosemide, guideway acid, imipenem, indometacin, Ketorolac, liotironina, melphalan, hydrochlorothiazide methyldopa, piperazillina, prostacyclin, prostaglandin, teicoplanin, tikarcillina and vancomycin.

18. The conjugate according to item 12 or 13, characterized in that is involved in binding assays the functional group of the biologically active drug substance is an aliphatic or aryl-Oh-group.

19. Conjugate on p, characterized in that the biologically active drug substance selected from the group consisting of albuterol, allopurinol, apomorphine, Ceftriaxone, dobutamine, dopamine, doxycycline, edrophonium, isoproterenol, liotironina, metaraminol, hydrochlorothiazide methyldopa, minocycline, portitxol, pentazocine, phenylephrine, fentolamina, propofol, rifamycin, ritodrine, Taxol, teicoplanin, terbutalina, tetracycline and vancomycin.

20. Ways is getting conjugate hydroxyalkylated according to any one of claims 1 to 19, characterized in that carry out the binding reaction between the terminal aldehyde group or a carboxyl group, which is formed by selective oxidation of the terminal aldehyde group or an activated carboxyl group, obtained by the transformation specified carboxyl group of the molecule hydroxyalkylated and the functional group of the low molecular weight substance selected from the group consisting of amino group, carboxyl group, thiol group and a hydroxyl group which is reactive with respect to the aldehyde group or a carboxyl group or originating from it activated carboxyl group of the molecule hydroxyalkyloxy, and the connection formed directly by the reaction of binding, if necessary, modify due to further reaction.

21. The method according to claim 20, characterized in that the terminal aldehyde group of the molecule hydroxyalkylated by selective oxidation converted into the corresponding lactoovo group and then enter into interaction with the primary amino group of low-molecular substances, so that the molecule hydroxyalkylated through amide bond is associated with a low-molecular substance.

22. The method according to item 21, wherein the selective oxidation of aldehyde groups are using the iodine or metal ions in aqueous alkaline solution.

23. The method according to item 21, wherein the binding reaction is carried out in the presence of a carbodiimide, preferably 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC).

24. The method according to claim 20, characterized in that the binding reaction is carried out in a heterogeneous phase.

25. The method according to claim 20, characterized in that the binding reaction is carried out in a homogeneous phase in dimethyl sulfoxide or N-organic or glycol.

26. The method according to item 21, wherein the binding reaction is carried out in dimethyl sulfoxide or N-organic or glycol in the absence of activator.

27. The method according to claim 20, characterized in that the terminal aldehyde group of the molecule hydroxyalkylated associated with the primary amino group of a low molecular weight substances with the formation of Schiff bases and the resulting Schiff base reduced to amine, so that the molecule hydroxyethylamine due amine connection is associated with a low-molecular substance.

28. The method according to item 27, wherein the reducing agent is sodium borohydride, cyanoborohydride sodium or organic boron complex.

29. A method of obtaining a selectively oxidized at the terminal aldehyde group hydroxyalkyloxy, characterized in that hydroxyalkylated subjected to transformation in aqueous alkaline solution at a molar ratio of iodine to g is drexel.colombo from 2:1 to 20:1.

30. The method according to clause 29, wherein the molar ratio of iodine to hydroxyalkylated is from about 5:1 to 6:1.

31. The method according to clause 29, characterized in that

a) the number hydroxyalkylated dissolved in hot distilled water and add a little less than 1 mol-equivalent of an aqueous solution of iodine;

(b) to the reaction solution was added slowly, dropwise, with an interval of several minutes, the NaOH solution in a molar concentration which is about 5-15 times that of the solution of iodine, up until after adding the solution starts to become clear again;

c) to the reaction solution again add less than 1 mol-equivalent of an aqueous solution of iodine;

d) again, the addition of NaOH solution;

e) stage from b) to d) are repeated until then, until you have added about 5.5 to 6 mol equivalents of a solution of iodine and 11-12 mol-equivalents of NaOH solution in relation to hydroxyalkylated;

f) then the reaction is stopped and the reaction solution absoluut and subjected to cation exchange chromatography and the reaction product obtained by freeze-drying.

32. The method according to p, characterized in that in the case of an aqueous solution of iodine we are talking about approximately 0.05-0.5 n solution of iodine.

33. The method according to p or 32, characterized in that the molar concentration of the NaOH solution is in the mask, approximately 10-fold that of the solution of iodine.

34. A method of obtaining a selectively oxidized at the terminal aldehyde group hydroxyalkyloxy, characterized in that hydroxyalkylated in aqueous alkaline solution are oxidized using a molar excess of stable ions of metals selected from Cu2+ions and Ag+-ions.



 

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21 cl, 15 tbl, 7 ex

FIELD: medicine, polymers, pharmacy.

SUBSTANCE: invention relates to a copolymer or its pharmacologically acceptable salt that comprises the following components as elemental links forming their: (a) one or some structural elemental links describes by the formula (I) given in the invention description, and (b) one or some structural links describes by the formula (II) given in the invention description. Disposition of these structural elements represented by the formulae (I) and (II) is chosen from the following sequences: (i) sequence with alternation "head-to-head"; (ii) sequence with alternation "head-to-tail"; (iii) mixed sequence with alternation "head-to-head" and "head-to-tail"; (iv) random sequence and taking into account that the ratio between structural links of the formula (I) and structural links of the formula (II) in indicated copolymer is in the range from 10:1 to 1:10. Also, the invention relates to a copolymer or its pharmacologically acceptable salt synthesized by addition of one or some links of carboxylic acid anhydride described by the formula (III) given in the invention description that comprises as elemental links: (a) one or some structural elemental links described by the formula (I), and (b) structural link comprising carboxylic acid anhydride link described by the formula (III) for one or some reactions chosen from the group consisting of: (i) hydrolysis; (ii) ammonolysis; (iii) aminolysis, and (iv) alcoholysis. Also, invention relates to a pharmaceutical composition used for prophylaxis or treatment of osseous metabolism disorder and comprising an acceptable excipient or carrier, at least one of above indicated copolymers or their pharmaceutically acceptable salts and at least one protein representing osteoclastogenesis inhibition factor (OCIF) or its analogue, or variant. Also, invention relates to a modifying agent comprising above said copolymers, to a complex between of one of above said copolymers and protein or its analogue, or variant, to a pharmaceutical composition comprising this complex. Also, invention relates to a method for time prolongation when OCIF is retained in blood stream after intake by a patient a complex between protein and at least one of above said copolymers. Also, invention relates to a method for treatment or prophylaxis of disorders of osseous metabolism involving intake by a patient the effective amount of complex comprising complex including OCIF or its analogue or variant and bound with at least one of the claimed copolymers. Also, invention relates to use of the complex comprising OCIF bound with at least one of the claimed copolymers designated for preparing a drug designated for prophylaxis or treatment of disorder of osseous metabolism and showing sensitivity to the protein effect. Modifying the protein, namely OCIF, by the claimed copolymers results to formation of complex possessing uniform properties being especially characterizing by reduced formation of disordered structure cross-linked with protein, improved retention of the protein activity and the excellent retaining protein in blood after intake of the indicated complex.

EFFECT: improved and valuable medicinal and pharmaceutical properties of agents.

110 cl, 13 tbl, 3 dwg, 40 ex

FIELD: medicine, pharmacy.

SUBSTANCE: invention relates to drugs and concerns sustained-release oral compositions. Agent comprises micelle-forming water-soluble main drug showing a positive charge at physiological pH value, polymer showing an opposite charge chosen from group consisting of polyacrylic acid, carboxymethylcellulose, xanthane gum, hellane gum, guara gum, dextran-sulfate and carragheenan, polyethylene oxide and if necessary a hydrophilic base. Also, invention proposes a method for sustained-release of micelle-forming drug.

EFFECT: improved and valuable medicinal and pharmaceutical properties of agent.

20 cl, 4 tbl, 24 dwg, 13 ex

FIELD: pharmacy, peptides.

SUBSTANCE: invention relates to a pharmaceutical composition that comprises an oligopeptide preparation and comprising oligopeptide of the formula (I): cyclo-(n-Arg-nGly-nAsp-nD-nE) and esterified β-cyclodextrin, and to a method for preparing an aqueous pharmaceutical preparation. Invention provides significant increasing solubility of oligopeptide and stability of the preparation for relatively prolonged time.

EFFECT: improved and valuable pharmaceutical properties of preparation.

13 cl, 6 tbl, 5 ex

FIELD: medicine, oncology, pharmacy.

SUBSTANCE: invention relates to drugs and concerns an antitumor composition for injection. Agent comprises derivative of anthracycline possessing the antitumor activity as an active component, and block-copolymer formed by derivative of polyethylene oxide and derivative of polyaspartic acid. The composition comprises additionally saccharide and a base represented by sodium hydrocarbonate, sodium hydrophosphate, sodium citrate and sodium hydroxide with pH value in the range 4-9. Also, invention proposes a composition based on block-copolymer micelles, solution of block-copolymer micelles with pH value in the range 4-9, and a solid composition for injection prepared by drying a micellar solution. Compositions show stability for a prolonged period and possess the improved capacity for repeated dissolving.

EFFECT: improved and valuable pharmaceutical properties of preparation.

15 cl, 2 tbl, 10 ex

FIELD: chemical-pharmaceutical industry and technology, pharmacy.

SUBSTANCE: invention relates to a composition in form of microemulsion concentrate designated for oral using, for example, in gelatin capsule. Proposed pharmaceutical composition provides high bioavailability of preparation. Pharmaceutical composition in form of microemulsion concentrate comprises ciclosporin and accessory components: (a) propanediol monocaprylate as a solvent; (b) monocaprylate glyceryl; (c) polyethylene glycol 40, hydrogenated castor oil and linoleic acid glycerides polyethylene glycol as surfactants; (d) 1,2-propylene glycol as a hydrophilic component; (e) alpha-tocoferol acetate as an antioxidant wherein in dilution microemulsion concentrate forms microemulsion to be easily dosed with particles size 15-20 nm.

EFFECT: improved and valuable properties of pharmaceutical composition.

1 tbl, 5 ex

FIELD: medicine; pharmacology.

SUBSTANCE: invention refers to medicinal agents and concerns pharmaceutical formulation for nasal absorption containing biologically active acidic polypeptide with isoelectric point 7 and lower; carrier which is water-insoluble or weak-soluble and contains particles of average size 100 mcm and less; and adjuvant for dispersion and consolidation of polypeptide on carrier surface, which is water-insoluble or weak-soluble, where average size of particle contained in specified adjuvant is lower than average size of carrier and is within the range 1 mcm to 20 mcm.

EFFECT: improved biological acceptability of biologically active polypeptide.

6 cl, 4 dwg, 11 tbl, 47 ex

FIELD: chemistry.

SUBSTANCE: invention concerns process of obtaining hydroxyl starch derivative which includes interaction of the reducing end of hydroxyalkyl starch represented by the formula (I), left unoxidised before this reaction, with a compound of formula (II) R'-NH-R", where R1 R2 and R3 independently denote hydrogen or linear or branched hydroxyalkyl group while R' or R" or R' and R" include at least one functional group X capable of interaction with at least one more compound before or after the reaction of (I) and (II). The invention also concerns hydroxyl starch derivative obtained in this process, and production process for hydroxyl starch derivative and polypeptide, a pharmaceutical composition incorporating the said hydroxyl starch derivatives.

EFFECT: improvement of compound production.

77 cl, 21 dwg, 3 tbl

FIELD: medicine; chemical and pharmaceutical industry.

SUBSTANCE: invention relates to peroral suspension containing non-dihydrated azytromicina, accipient stabilizing conversion of azytromicina, and aqueous carrier. Also a method of inhibiting conversion of non-dihydrated azytromicina in the peroral suspension has been proposed by mixing a quantity of accipient reducing the surface tension with aqueous carrier and non-dihydrated azytromicina to produce the suspension where the quantity of accipient reducing the surface tension reduced tension in a volume of water equal to the certain volume of aqueous carrier to less than 50 dine/cm (5·10-2 N/m). Besides, a method inhibiting conversion of non-dihydrated azytromicina in the peroral suspension has been proposed, where the peroral suspension contains no conversion accentuator by mixing the viscosity agent with the aqueous carrier and non-dihydrated azytromicina to make the peroral suspension with viscosity of approximately 3 cP (3 mPa·s) or more. A method has been proposed of treating bacterial and protozoal infections in patients by administering peroral suspension.

EFFECT: method inhibits conversion of a form of non-dihydrated azytromicina in peroral suspension.

12 cl, 5 ex, 14 tbl

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