Acylated insulin analogs

 

(57) Abstract:

The invention relates to medicine, in particular for the treatment of diabetes. More specifically the present invention relates to the Monomeric insulin analogue, where the chain And is a natural sequence of chain a of human insulin, and the circuit is modified in any of the provisions B28 and B or in both positions. The specified analog is manuallyand at the N - Terminus of chain a or chain, or lysine. Acylated insulin analogs have a prolonged action, which is the technical result of the invention. 4 C. and 8 C.p. f-crystals, 1 table.

The invention relates to the field of diabetes. More specifically, the present invention relates to acylated analogues of long-acting insulin.

Application insulinsensitizing therapy reduces mortality from this disease and prevent the development of acute complications of diabetes. However, complications of chronic diabetes remains a major threat to human health due to persistent metabolic disorders arising mainly as a result of poor regulation of glucose levels in the blood. Data obtained with the lc at the IX corallium with more than 35% decrease in the incidence of retinopathy.

To ensure a normal level of glucose in the blood, it is necessary to develop a therapy that it is as close as possible match the profile of secretion of endogenous insulin in normal individuals. Daily physiological need for insulin ranges and can be divided into two phases: (a) the absorptive phase, requiring release of insulin to correct caused by eating a high rise in blood glucose, and (b) postabsorptive phase, requiring constantly supported amount of insulin necessary to regulate the glucose output of the liver to establish the optimal level of blood glucose on an empty stomach.

In accordance with this effective therapy involves the integrated use of two types of exogenous insulin: rapid-acting insulin after a meal and main of slow insulin action.

At the present time to provide prolonged primary action insulin get in conditions conducive to the formation of a review of the conformation of the insoluble crystalline state. This drug prolonged action are the drugs of Ultralente, the Ribbon and the seven-Ribbon (Ultralente, Lente and semi-Lente is bathing action problems, associated with mismatch dependence "dose-effect", and with the unpredictability of time steps. In addition, one existing in today time insulin long-acting drugs, such as beef insulin Ultralente, is immunogenic. The presence of antibodies in the immunogenicity of beef insulin, Ultralente changes the pharmacokinetics of insulin fast action.

Although insoluble drug, Ultralente, due to its duration, is convenient for daily injection of basal insulin, many doctors now prefer to use insulin with an intermediate duration, insulin-proteinemia drug, commonly called insulin-NPH. Insulin - NPH is used twice a day as the main insulin, because it is relatively easy to reach the optimal dose using medicines shorter time steps. As a result, the insulin intermediate steps is 70% in the US, 64% in Japan, 45% in Europe, and only 55% of the world market of insulin.

However, as insoluble insulin-NPH and insoluble insulin Ultralente are suspensie predictable, than soluble drugs, and therefore less provide adequate regulation of blood glucose and more associated with the risk of life-threatening episodes of hypoglycemia. In line with this, the need for a soluble basal long-acting insulin for effective intensive insulinsensitizing of therapy is important. The present invention relates to acylated analogues of insulin, which can be included in the composition of medicinal products for carrying insulin therapy using soluble basal insulin.

In Japanese patent application I-S54699 Muranishi & Kiso describe the acylation of pork, beef and 1 human insulin. In particular, the following compounds: B29-N-palmitoylation ( - amino group allerban), B1-N- palmitoylation (N-terminal - amino group of the chain B allerban) and B1,B29-N,-N- dipalmitoyl ( - kinogruppa and N-terminal (amino etilirovany). Muranishi and Kiso report that acylated insulin has a biological profile similar to the profile of insulin, however, do not specify the dose, route of administration or Kirovenergo insulin.

Similarly, Hashimoto and others, in Pharmaceutical Research 6: 171-176 (1989) describe B1-N- palmitoylation (N-terminal - amino group allerban) and B1,B29-N,-N- dipalmitoyl (such as amino group, and N-terminal - aminogroup etilirovany). Hashimoto et al. studied the hypoglycemic effect of B1-N- palmitoylation and B1,B29-N,-N- dipalmitoyl in male rats at 25 u/ml, with the highest dose. In these doses, there is a very low activity when administered intravenously. In intramuscular observed only transient hypoglycemic effect B1-N- palmitoylation and minor action B1,B29-N,-N- dipalmitoyl.

In addition to the in vivo experiments reported Muranishi, Kiso and Hashimoto and others , Walter and others in PCT publication WO 92/01476 describe what the half-life of proteins and peptides can be increased in vivo by chemical binding protein with a non-polar group, in particular, derivatives of fatty acids. Fatty acid bridge provides a link between protein and albumin. In addition, Walder and others indicate that non-polar group, preferably, limited only sa the main, described fatty acid derivatives of insulin. However, not specifically described or not shown in the examples of fatty acid derivatives of insulin, and there are no data indicating the preservation of biological activity of fatty acid derivatives of insulin.

It was found that the selective acylation of the free amino groups of the Monomeric insulin analogue, provides efficient activity of basal insulin. Indicates that described above neetilirovannye insulin analogs provide a rapid insulin action and faster cleaning. These analogs known in the art as Monomeric insulin analogs. The ability to modify such analogues with obtaining basal activity is highly unexpected fact.

The present invention relates to monoarylamino the insulin analogue, which when applied leads to prolonged action. These analogues can be obtained in the form of soluble drugs, which makes them more effective than the currently used therapies based on basal insulin. These analogues also have a high predictability of dependence "dose-effect", high Pres is but suitable for obtaining a mixture of drugs, containing analogue of acylated insulin or insulin analog.

The present invention relates to monoarylamino the insulin analog of the formula: SEQ ID N0:1 (sequence 1 see the end of the description),respectively stitched to the sequence SEQ ID N0:2 (see the end of the description)

or its pharmaceutically acceptable salt, where Xaa at position I of the sequence SEQ ID N0:1 (chain of insulin (A) is Gly; or acylated Gly, if Xaa in position I of the sequence SEQ ID N0:2 is Phe, Xaa at position 28 of the sequence SEQ ID N0:2 is Asp, Lys, Leu, Val or Ala, a Xaa at position 29 of the sequence SEQ ID N0:2 is Lys or Pro;

Xaa in position I of the sequence SEQ ID N0:2 (B-chain of insulin) is Phe; or acylated Phe, if Xaa in position I of the sequence SEQ ID N0: 1 is Gly, Xaa at position 28 of the sequence SEQ ID N0:2 is Asp, Lys, Leu, Val, or Ala, a Xaa at position 29 of the sequence SEQ ID N0:2 is Lys or Pro;

Xaa at position 28 of the sequence SEQ ID N0:2 is Asp, Lys, Leu, Val, Ala; or annulled Lys, if Xaa in position 1 of the sequence SEQ ID N0: 1 is Gly, Xaa at position I of the sequence SEQ ID N0:2 is Phe; Xaa at position 29 of the sequence SEQ ID N0:2 p Xaa at position 28 of the sequence SEQ ID N0: 2 is Asp, Lys, Leu, Val or Ala, Xaa in position I of the sequence SEQ ID NO:1 is Gly, a Xaa in position I of the sequence SEQ ID N0:2 is Phe.

The present invention also relates to a method for treatment of hyperglycemia by introducing a patient in need of such treatment, a pharmaceutical composition containing an effective amount of acylated insulin analog of the present invention in combination with one or more pharmaceutically acceptable excipients.

The present invention relates also to pharmaceutical preparations for parenteral administration which contain acylated insulin analog of the present invention together with one or more pharmaceutically acceptable preservatives, agents, giving isotonicity, or buffers.

All abbreviations of the amino acids used in the present description, have been accepted by the USPTO and the office for the registration of trademarks, see Art. 37 CFR 1.822 (B).

The term "cross-linking" refers to the formation of disulfide bridges between cysteine residues. Accordingly transverse cross-linked human insulin or an analogue of human insulin with eniah 6 and 11 of the circuit A. The second disulfide bridge knits cysteine residues in position 7 of the A chain and the cysteine in position 7 of the circuit B. the Third disulfide bridge knits cysteine in position 20 of the A chain and the cysteine in position 19 of the chain B.

The term "acylated Gly", "acylated Phe and acylated Lys" means Gly, Phe or Lys, acylated C6-C21-fatty acid. The term "alleluya group" means a fatty acid, a chemically related to the amino group or the amino group of the insulin analogue. Free amino groups in positions A1 and B1 are amino groups. The free amino group of Lys in position B28 and B29 is the amino group.

The term "acylation" means the introduction of one acyl group covalently linked to the free amino group of a protein. The term "selective acylation" means that the acylation of the amino group (or groups) is preferred over - amino groups.

The term "fatty acid" means a saturated or unsaturated C6-C21fatty acid. Preferred fatty acids are saturated and include myristic acid (C14), pentadecylic acid (C15), palmitic acid (C16), heptadecyl acid (C17and steineke of the present invention are monosilane insulin analogs. Insulin analogs etilirovany C6-C21-fatty acid - amino group or the amino group. Preferably, these analogues monocalibre - amino group of lysine.

The term "activated ester of a fatty acid" means a fatty acid, which is activated using the General technique described in Methods of Enzymology. 25. 494-499 (1972) and Lapidot et al., in J. of Lipid Res. 8: 142-145 (1967). Activated ester of a fatty acid includes derivative commonly used alleluya agents, such as hydroxybenzotriazole (HOBT), N-hydroxysuccinimide and their derivatives. Preferred activated complex ester is N-succinimidylester.

The term "soluble" means that the acylation of insulin analogue in the liquid phase is present in a sufficient amount of ester. Preferably, in the liquid phase is 1-2 molar equivalents of activated complex ester of 1 mol of insulin analogue.

The term "Monomeric insulin analog or insulin analogue" as used in this description, means the equivalent of rapid-acting insulin, less prone to dimerization or self. Monomeric insulin analog is human insulin, where Pro at position B28 for the. in the patent application U.S. N 07/388201 (publication EP0 N 383472) and Brange and others in the publishing EP0 N 214826. It should be noted that other modifications of the Monomeric insulin analog is widely used by professionals. These modifications are: replacement of his-tag residue at position B10 on aspartic acid; replacement of the phenylalanine residue in position B1 on aspartic acid; replacement of the threonine residue in position B30 to alanine; the replacement of the serine residue at position B9 on aspartic acid; a deletion of amino acids at position B1, separately, or in combination with deletion at position B2, and the deletion of threonine from position B30.

The term "basic conditions" used in this description, means the basicity of the reaction. For the selective acylation of insulin analogue according to the amino group, the reaction must be carried out using, basically, all free deprotonated amine groups. In aqueous solvent or co-solvent basic terms, means that the reaction is carried out at a pH over 9.0 in. In an organic solvent, the reaction is carried out in the presence of a base with a basicity equivalent pKaequal in water 10,75 or higher.

SEQ ID N0:1 indicates the first serial is egovernance (see at the end of the description), where Xaa at position I of the sequence SEQ ID NO: 1 (A-chain of insulin) is Gly; or acylated Gly, if Xaa in position I of the sequence SEQ ID NO: 2 is Phe, Xaa at position 28 of the sequence SEQ ID N0:2 is Asp, Lys, Leu, Val, or Ala, a Xaa at position 29 of the sequence SEQ ID N0:2 is Lys or Pro.

SEQ ID N0: 2 indicates the second sequence specified in the list of sequences, and is the analogue of the B-chain of human insulin having the sequence (see the end of the description), where Xaa at position I of the sequence SEQ ID N0:2 (B-chain of insulin) is Phe; or acylated Phe, if Xaa in position I of the sequence SEQ ID NO: 1 is Gly, Xaa at position 28 of the sequence SEQ ID N0:2 is Asp, Lys, Leu, Val or Ala, a Xaa at position 29 of the sequence SEQ ID N0:2 is Lys or Pro;

Xaa at position 28 of the sequence SEQ ID N0:2 is Asp, Lys, Leu, Val, Ala; or acylated Lys, if Xaa in position I of the sequence SEQ ID NO: 1 (A-chain of insulin) is Gly, Xaa at position I of the sequence SEQ ID NO: 2 (B-chain of insulin) is Phe, Xaa at position 29 of the sequence SEQ ID N0:2 is Pro; and

Xaa at position 29 of the sequence SEQ ID N0:2 is Lys I sequence SEQ ID NO: 1 (A-chain of insulin) represents Gly, and Xaa in position I of the sequence SEQ ID N0:2 (B-chain of insulin) is Phe.

As indicated above, the present invention relates to monoarylamino the insulin analog of the formula SEQ ID N0:l, suitably stitched with the sequence SEQ ID N0:2, or its pharmaceutically acceptable salt. Preferred amino acid residue in position I of the sequence SEQ ID NO: 1 (A-chain of insulin) is Gly. Phenylalanine is the preferred amino acid in position I of the sequence SEQ ID N0:2 (B-chain of insulin). Preferred amino acid residue in position 28 of the sequence SEQ ID N0:2 is Asp; or acylated Lys, if the amino acid residue at position 29 of the sequence SEQ ID N0:2 is Pro. Preferred amino acid residue in position 29 of the sequence SEQ ID N0:2 is Lys; or a Pro, if amino acid residue in position 28 of the sequence SEQ ID N0:2 is acylated Lys. From standard biochemical compounds, well known in the art, the preferred analog is monocularly LysB28ProB29-human insulin. The most preferred acylated insulin analogues are analogues, monoarylamino C8-C8), nonanol (C9), decanol (C10), undecanol (C11), lauroyl (C12), tridecanol (C13), myristoyl (C14), pentadecanoic (C15), Palmitoyl (C16). Thus, preferred compounds of the present invention are B29-N- AspB28-palmitoylation person (B28is Asp; B29is acylated Lys), B28-N- Palmitoyl-LysB28ProB29-human insulin (B28 is acylated Lys; B29 is Pro), B28-N- octanoyl-LysB28ProB29-human insulin, B28-N- decanoyl-LysB28ProB29-human insulin, B28-N- myristoyl-LysB28ProB29-human insulin, B28-N- undecanoyl-LysB28ProB29-human insulin.

The acylation of the free amino groups of proteins, including insulin, are well known in the art. General methods of acylation presented in Methods of Enzymology. 25: 494-499 (1972) and include the use of activated esters, halides or anhydrides of the acids. The use of activated esters of fatty acids, in particular N-hydroxysuccinimidyl is especially preferred tool is hydroxysuccinimide and their use in obtaining N-Laurelville, N-lauryl-L-serine and N-lauroyl-L-glutamic acid.

When the selective acylation of the amino group can be used with various protective group for protecting the amino group at the time of linking. Suitable for this purpose, the protective groups are well known in the art, for example, paramethoxyamphetamine (pmZ). Preferably, if the amino group allerpet in one stage of the synthesis without using aminosidine groups. The acylation is carried out by interaction acylated complex ester of fatty acid - amino group of a protein in basic conditions in a polar solvent. The basicity of the reaction should be sufficient to deprotonation of all free amino groups of the insulin analogue. In conditions of weak bases not all amino groups are deprotonirovannymi, and the result is preferential acylation of N-terminal or amino groups. In aqueous solution or co-solvent, the term "basic conditions" means that the reaction is carried out at a pH higher than 0.9. Since the decomposition of protein occurs in the region of pH>12,0, the pH of the reaction mixture should preferably be of 10.0 to 11.5, and most preferably of 10.5. The pH of the reaction mixture in a mixed organic and aqueous solution is of amino group (s) in the nonaqueous solvent selective acylation of the amino group is carried out in the presence of a base with a basicity equivalent pKain water, equal 10,75 or higher. Thus, this base should be at least as strong as triethylamine. Preferably, the grounds are tetramethylguanidine, diisopropylethylamine or tetrabutylammonium hydroxide. The use of a weaker base leads to acylation of amino groups.

The choice of solvent is not critical and depends largely on the solubility of the insulin analogue and fatty acid ester. The solvent may be wholly organic. Usually acceptable organic solvents are DMSO, DMF, etc., are Suitable as aqueous solvent and a mixture of water and organic solvents. The choice of polar solvents is limited only by the solubility of the reagents. Preferred solvents are DMSO; DMF; acetonitrile and water, acetone and water; ethanol and water; isopropyl alcohol and water; isopropyl alcohol, ethanol and water; and ethanol, propanol and water. Preferably, the solvent is acetonitrile and water, most preferably 50% acetonitrile, to the activated ester of the fatty acid was in molar excess. Preferably, the reaction is carried out using 1-4 molar equivalents, more preferably with 1-2 molar equivalents of ester. Expert it is clear that at very high levels of activated complex ester will produce a significant amount of bis - or tri-acylated product.

The temperature of the reaction mixture is not critical. The reaction is carried out at a temperature of from 20 to 40oC and is usually completed over a period of time from 15 minutes to 24 hours.

After acylation of the product was then purified by standard methods such as treatment - phase chromatography or hydrophobic chromatography. Then the product produce by conventional methods, such as lyophilization or crystallization.

Monomeric insulin analogs of the present invention can be obtained by any of the known methods of peptide synthesis, including cusichaca (solution) methods, solid phase methods, semi-synthetic methods and more modern methods using techniques of recombinant DNA. For example, Chance et al., in the application for U.S. patent N 07/388201, EP0-publication N 383472 (Chance and others) and in EP0 214826 the Scripture by reference). Chains A and B insulin analogues according to the present invention can also be obtained through the proinsulin-like molecules predecessor using techniques of recombinant DNA. See, for example, the work of Frank et al., Peptides: Synthesis, Structure - Function, Proc. Seventh Am. Pept. Symp., Eds. D. Riche & E. Gross (1981), which is introduced in the present description by reference.

For a more visual illustration of the present invention are the following examples. It should be noted that these examples should not be construed as a limitation of the scope of the present invention.

Example 1

The acylation LysB28ProB29-human insulin using N-succinimidylester in acetonitrile and water

Crystals LysB28ProB29-human insulin (2,22 g) was dissolved in 100 ml of 50 mm boric acid solution at a pH of 2.5. the pH of the solution was brought to 2.5 with 10% HCl and the resulting solution was stirred until then, until all the crystals, by visual estimation, were not completely dissolved. The activated solution of ester (N-succinimidylester) was obtained by adding 270 mg of solid activated complex ether to 27 ml of acetonitrile, preheated to approximately the 50who areny (by visual assessment). the pH of the solution was brought to approximately 10,22 by adding 10% NaOH and the solution was stirred for 15 minutes at 4oC. To a solution with a defined pH was added acetonitrile (73 ml), and then the previously obtained solution of the activated complex ester. The reaction was carried out for 85 minutes at 4oC, and then extinguished by adding 1 N. acetic acid (600 ml), resulting in the pH of the solution was 2,35. The yield of the reaction, calculated as the number of B28-N- Palmitoyl-LysB28ProB29-human insulin in the quenched reaction relative to the original number LysB28ProB29-human insulin was 72.5%.

Example 2

C8(B28)LysB28ProB29-human insulin

Crystals Lys(B28), Pro(B29)-human insulin (EOC) (2.0 g) was dissolved in 200 ml of 50 mm borate buffer at a pH of 2.5. Then the pH of the solution was again brought to 2.5 with 10% HCl and the resulting solution was stirred until then, until all the crystals, by visual estimation, were not completely dissolved. The activated solution of ester (ether 1-octanoyl-N - hydroxysuccinimide) was obtained by adding 175 mg of solid activated complex ether to 25,62 ml of acetonitrile and vigorous mixing until until all particles complicated aviron, then the solution was stirred 5 minutes at room temperature. This pH-adjusted solution EOC) was added acetonitrile (176 ml) and then added to the previously obtained solution of the activated complex ester. The reaction was conducted for 90 minutes at room temperature, after which it was suppressed by the addition of 5.5 ml of 10% HCl (to 2.75%.about.) and three volumes (1200 ml) cold dH2O, resulting in the pH of the solution was 2,70. The yield of the reaction, calculated as the number of LysB29(C8) - EOC repaid in the reaction, relative to the original number VN, was 75.5%. This solution was divided into two 800 ml aliquots, for purification using hydrophobic chromatography (sP2Oss- After carrying out column chromatography was carried out by ultrafiltration and lyophilization.

As mentioned above, the acylated analogues of insulin of the present invention can be c used successfully for the treatment of hyperglycemia by introducing a patient in need of such treatment, an effective amount monoarylamino insulin analogue. Used in the present description, the term "effective amount" means an amount of one or more acylated analogues of the present invention, necessary for sniti from about 10 units or more per day (or from about 0.3 to about 2 mg to calculate approximately 29 u/mg). However, it should be noted that the actual number of input acylated analogue (or analogue) determined by the attending physician taking into account relevant factors, including the specific disease (i.e., disease causing hyperglycemia), specifically analog input, the selected method of parenteral administration, the age, weight, and susceptibility of the patient and the severity of the disease. So the above intervals doses do not limit the scope of the present invention.

Acylated insulin analogs of the present invention is administered to a patient in need of such introduction (i.e., the patient suffering from hyperglycemia), in the form of pharmaceutical compositions containing an effective amount of at least one monoarylamino analogue in combination with one or more pharmaceutically acceptable excipients or carriers. For these purposes, can be manufactured in a pharmaceutical composition that contains about 100 IU/ml or equivalent to the concentration of the effective number of acylated analogue (or analogue) insulin. These compositions are intended for parenteral doing (although this is optional), can be obtained by any known method, using the. the feet, for example. Remington's Pharmaceutical Sciences. 17 th Edition, Mack Publishing Company, Easton, PA, USA (185), which is incorporated into this description by reference. For example, dosage forms for parenteral maintenance can be obtained by suspension or dissolving the desired amount of at least one monoarylamino insulin analogue in non-toxic liquid carrier suitable for injection such as aqueous medium and sterilizing the suspension or solution. Alternatively, the desired amount of coupling can be placed into the vial, and the vial and its contents sterilized and hermetically sealed. This may be accompanied by another vial or carrier for mixing prior to introduction. In pharmaceutical compositions intended for parenteral administration, use thinners, fillers and carriers, such as water and miscible with water and organic solvents such as glycerin, sesame oil, peanut oil, aqueous propylene glycol, N,N - dimethylformamide, etc. are Examples of such pharmaceutical compositions are sterile, isotonic, aqueous physiological solutions monoarylamino insulin analogue that can be buffered pharmaceutically buffet the preservatives, such as metacresol or other agents for pH correction of the final product, such as sodium hydroxide or hydrochloric acid.

Acylated insulin analogs of the present invention can also be made in the form of mixtures. Such mixed compositions may contain reallyreally insulin or analogue insulin and acylated insulin analog. The ratio of insulin or insulin analogue to acylated analogue is from 1:99 to 99:1 by weight. The preferred ratio is from 75: 25 to 25: 75; more preferred from 40:60 to 60:40, and most preferred 50: 50. The mixed composition is prepared by mixing the desired amounts of components in standard diluent for parenteral administration. Such standard diluents are an agent, giving the solution isotonicity, zinc, physiologically acceptable buffer and a preservative. Preferred physiologically acceptable buffer is a phosphate buffer, such as secondary sour nutrifaster. Other physiologically acceptable buffer is Tris or sodium acetate. The choice of buffer and its concentration can be carried out in accordance with conventional practice. Pharmaceutically acceptable preservative is carried out particularly preferred, because of the relatively fast-acting insulin and its analogue and monosilicon the insulin analogue dissolved in the composition. This allows to predict the duration profile of action of the composition.

The following is an example of a composition is provided only for purposes of illustration and should not be construed as a limitation of the scope of the invention.

Composition 1

Parenteral composition can be obtained in the following way:

Phenol, mm - 30

Glycerol, mg/ml - 16

Acylated LysB28ProB29-human insulin u - 100

Zinc, % - 0,7

Sodium acetate, mg/ml to 3.8

The solution of the above ingredients is administered by injection to a patient in need of treatment.

To illustrate the effectiveness of the compounds of the present invention, B28-N- Palmitoyl-LysB28ProB29-human insulin was studied on the model trained dogs. In the experiments performed involved adults (aged 1-2 years) short-legged hounds (females and males) weighing 8 to 15 kg, which during the night was not given food. At least 10 days before the animal studies were anestesiologi with isoflurane and did venesection in the groin area on the left or right side. Kate is the second thread 4-0. The free ends of the catheter was passed subcutaneously to the back using trocar. Then the catheter was filled with a solution of glycerol/heparin (3:1, vol/about. ; the final concentration of heparin - 250 IU/ml), and the free ends pinched and placed in a subcutaneous pocket so that they are fully covered by skin. To prevent infection was introduced Keflex before surgery (20 mg/kg, IV and 20 mg/kg, im) and after surgery (250 mg, once a day for 7 days). To relieve pain after surgery has introduced torbugesic (1.5 mg/kg, VM).

To assess the health of the animal directly on the day before the experiment, the animals took blood samples. In our experiments we used only the animals with hematocrite was above 38%, and the number of cells was less than 16000/mm3. After noon the day before the experiment the free ends of the catheters were removed from the subcutaneous pocket through a small incision made under local anesthesia (2% lidocaine), and the dog wore a set of corset with a leash and collar.

In the morning, the day of the experiment, the content of the arterial catheter was aspirated (these studies used only the arterial catheter), the catheter was rinsed with physiological restorations the cage and the catheter c of the elongated tube was attached to the hinge system, so that the dog can move freely in the cage. After a 15 minute rest (for the control of dogs - 45 minutes) took blood samples to determine the concentration of glucose in plasma. The second baseline sample was taken 15 minutes later (time 0). The test compound (phosphate buffered saline solution (10.5 mmol/kg B28-N- Palmitoyl-LysB28ProB29-human insulin; this corresponds to a molar equivalent of 1.75 IU/kg of human insulin) was injected subcutaneously into the back of the neck.

Then, at least every 30 minutes during the next 2 (control) - 6-(B28-N- Palmitoyl-LysB28ProB29-human insulin) hours took samples of arterial blood. The obtained samples were collected in a sealed tube collector for collecting blood containing disodium-EDTA and immediately placed on ice. After that, samples were centrifuged and the resulting plasma was transferred into a polypropylene laboratory test tubes, where it is kept on ice, or placed in the fridge for all the research time.

At the end of the experiment animals were anestesiologi (isoflurane), the catheter was rinsed with fresh saline and filled with a mixture of glycerol/heparin, and Svobodnyi plasma glucose was determined on the day of experiment method using glucose oxidase in glucose analyzer, Beckman. Values were expressed as mean values standard deviations from the mean (sEM).

After the injection of phosphate buffered saline concentration of glucose in plasma compared to the baseline concentration is not significantly changed during the two-hour observation period (table 1). During this same period of time after subcutaneous injection B28-N- Palmitoyl-LysB28ProB29-human insulin was observed in 15% (17 mg/DL) reduction in the concentration of glucose in plasma. The concentration of glucose in the plasma of animals, which was introduced B28-N- Palmitoyl-LysB28ProB29-human insulin, continued to gradually decline in the next 4 hours, and 6 hours after injection of the glucose level fell below the basic level and amounted to 41 mg/DL (35% reduction).

From literature it is known that the concentration of blood glucose in normal dogs is not significantly reduced even after a week of starvation. This decrease glucose levels observed in this experiment, due to the introduction of B28-N- Palmitoyl-LysB28ProB29-human insulin, indicating insulin-like activity of this compound.

1. Monocularly the insulin analogue Oh in the description, or its pharmaceutically acceptable salt.

2. Monocularly analogue insulin p. 1, where Xaa at position 28 of the sequence SEQ ID NO : 2 is acylated Lys, and Xaa at position 29 of the sequence SEQ ID NO : 2 is Pro.

3. Monocularly the insulin analogue according to p. 2, where alleluya group is C6- C17fatty acid.

4. Monocularly the insulin analogue according to p. 2, where alleluya group is C13- C17fatty acid.

5. Monocularly analogue insulin p. 1, representing B28-N-Palmitoyl-LysB28ProB29-human insulin.

6. Monocularly analogue insulin p. 1, representing B28-N-myristoyl-LysB28ProB29-human insulin.

7. Monocularly the insulin analogue according to any one of paragraphs.1 - 6, designed for use in the treatment of diabetes.

8. Pharmaceutical preparation for parenteral administration containing an effective amount monoarylamino insulin analogue according to any one of paragraphs.1 - 6 together with one or more pharmaceutically acceptable preservatives, agents, giving isotonicity, or b is on or insulin analogue, and monoarylamino insulin analogue according to any one of paragraphs.1 - 6, where the ratio by weight between the two components is about 1 to 99 : 99 to 1.

10. Pharmaceutical preparation for parenteral administration by p. 9, where the mixture contains LysB28ProB29-human insulin and B28-N-acylated LysB28ProB29-human insulin.

11. The method of treatment of a patient suffering from hyperglycemia, which includes the introduction of pharmaceutical product under item 8.

12. The way to obtain a pharmaceutical preparation for parenteral administration, which comprises mixing the compound according to any one of paragraphs.1 - 6, agent, giving isotonicity, and physiologically acceptable buffer.

 

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