RU2298560C2 - Conjugates of protein and polyethylene glycol derivative, pharmaceutical composition, method for control of viral infection - Google Patents

Abstract

FIELD: chemistry of proteins, virology, pharmacy.

SUBSTANCE: invention relates to novel physiologically active protein conjugates that can be used in medicine. Proposed conjugates is formed by a protein molecule and polyethylene glycol and corresponds to the formula:

wherein protein represents interferon α-2b; n and n₁ have values providing an average molecular mass of links from about 7500 Da to about 35000 Da. Conjugate shows the enhanced stability as compared with the known analogs. Also, invention relates to a pharmaceutical composition possessing an antiviral activity and containing the indicated conjugate, and to a method for control of viral infection.

EFFECT: valuable medicinal and biological properties of conjugates.

4 cl, 1 dwg, 6 ex

Description

The invention relates to new PEG-derivatives of proteins, in particular to PEG-derivatives of interferon, which can be used in medicine, for example in the treatment of viral diseases.

Treatment with native drugs of the peptide structure has a number of significant drawbacks: proteins are rapidly hydrolyzed in the gastrointestinal section of the digestive tract and are therefore used, as a rule, parenterally. The relatively short half-life of such drugs in the patient’s body prevents their maximum therapeutic effect and ensures their repeated use. Another important negative factor limiting the use of native or recombinant protein preparations is their high immunogenicity and the associated sensitive reactions. A fundamental change in the structure of their molecules either eliminates their biological properties, or entails an increase in side effects associated with their use. One of the ways to increase the effectiveness of drugs with a protein structure is by chemical modification of their molecule, which consists not in actually changing their structure, but in the physicochemical transformation achieved by combining the native molecule with polyethylene glycol (PEG). This process is called pegylation. Such a chemical modification of pharmacological preparations of the peptide structure is aimed at improving their tolerance, reducing immunogenicity, increasing their half-life, and as a result of all of the above, at a significant increase in the effectiveness of treatment and improving the quality of life during its implementation.

PEG as a potential object, a modifier of peptide structure substances, attracted the attention of researchers in the early 1970s. PEG molecules are water-soluble polymers of ethylene oxide with two terminal hydroxyl groups. The molecular weight of PEG molecules can vary between 300-4000 Daltons, and PEG macromolecules arranged in a chain can form both branched and linear stereochemistry. It is the mass of PEG and its stereochemical structure that, as a rule, determine the properties of the modified peptide substrate.

The hydrophilicity of the modified PEG molecules forms a fundamentally new physicochemical properties of the altered peptide. The high content of hydrogen atoms even in one PEG molecule allows it to bind to 2-3 water molecules. A similar effect entails the formation of a "water cloud" around the modified PEG-protein molecule. This peculiar “shield” of water around the modified molecule on the one hand significantly increases the solubility and bioavailability of the drug, on the other hand, protects the molecule from other proteins (neutralizing antibodies, complement). Thus, PEG-modified peptides are much more protected from opsonization and active phage and endocytosis of the cellular structures of the macroorganism.

Changes in the pharmacokinetic and pharmacodynamic properties of PEG-modified peptides depend on the mass of the PEG molecule, specific binding sites to the protein, and the structure of the molecule of the modifying agent.

Interferons are a group of biologically active proteins or glycoproteins synthesized by a cell in the process of a protective reaction to foreign agents - a viral infection, antigenic or mitogenic effect. IFNs do not have specificity for viruses and are inhibitory to the reproduction of various viruses. Other effects of IFN are also important, interest in them is due to the high efficiency of the use of interferon preparations in such pathological conditions as cancer, blood disease, as well as a steady increase in the number of patients suffering from the above diseases.

Pegylated interferon is a conjugate of an interferon molecule and a portion of a polyethylene glycol molecule covalently attached to it, which protects the interferon molecule from enzymatic destruction and provides the drug with a number of other advantages.

In particular, pegylated derivatives of interferon obtained by attaching a branched polyethylene glycol molecule to interferon α-2a are known (RF Patent No. 2180595, 03.20.2002). These pegylated derivatives are characterized by a longer serum half-life compared to unmodified interferon. From source Kozlowski A., et al. "Development of Pegylated interferons for the treatment of Chronic Hepatitis C", BioDrugs, 2001, 15 (7), conjugates of interferons alpha and a derivative of polyethylene glycol are known in which the molecular weight of polyethylene glycol units is 30 kDa or less.

The problem to which the invention is directed, is the creation of new conjugated derivatives of interferon, which have increased stability compared to known analogues.

To solve the problem in the framework of the present invention, new PEG derivatives of interferon were synthesized. During the experiments, it was unexpectedly discovered that these pegylated derivatives have a significantly longer persistence in the body than the known ones. This property of these compounds will help maintain a constant effective concentration of drugs based on them in the blood at a lower frequency of administration, which will increase the effectiveness of the therapy and reduce treatment costs.

The PEG derivatives of the proteins of the present invention are a conjugate of a protein and a polyethylene glycol derivative of the formula

where the protein is interferon α-2b, an and n ₁ have such values that the average molecular weight of polyethylene glycol units is from about 7500 Da to about 35000 Da. A preferred embodiment is a conjugate in which n = n ₁ = 120, and the molecular weight of the polyethylene glycol units is approximately 10,000 Da. The invention also relates to a pharmaceutical composition having antiviral activity comprising said conjugate and a therapeutically inert carrier; and to a method for controlling a viral infection, comprising administering to said organism a PEG conjugate.

The graph (see drawing) reflects the level of biologically active interferon in blood serum in arbitrary units of activity. The graph shows that the claimed compounds have significantly higher stability compared to other derivatives - during 14 days of observation, its elimination from the body was not observed. The following notation is used on the chart:

Compound 1 - conjugate according to the invention,

Compound 2 - conjugate of 2,4-bis- (methoxypolyethylene) triazine and interferon α-2a,

Compound 3 - conjugate of 2,4-bis- (methoxypolyethylene) triazine and interferon α-1,

Compound 4 - conjugate of 2,4-bis- (methoxypolyethylene) triazine and interferon β-1,

Compound 5 is a conjugate of 2,4-bis- (methoxypolyethylene) triazine and interferon α-4.

A well-known analogue is the conjugate of polyethylene glycol and interferon α-2a.

Example 1. Obtaining PEG derivatives

First, polyethylene oxide is produced by multistage or one-step synthesis. As the starting substance, oligoethylene oxide with a molecular weight of 250 (average degree of polymerization of 4.5) was used, obtained by reacting ethylene oxide with sodium methylate. In one-step synthesis, 2500 g of ethylene oxide was added to 100 g of the starting substance gradually over a period of 12 days with an increase in temperature from 120 to 170 ° C under a pressure of 4-5 atm. Multistage synthesis is carried out as follows:

Stage 1 To 100 g of starting material, 700 g of ethylene oxide are added. The reaction is carried out in a steel reactor with a stirrer in a nitrogen atmosphere under a pressure of 4 atm at a temperature of 120 ° C for 8 hours.

2 stage. To 100 g of the product obtained in stage 1, add 700 g of ethylene oxide. The reaction is carried out under the same conditions for 9 hours.

3 stage. To 170 g of the product obtained in stage 2, add 330 g of ethylene oxide. The reaction is carried out at a temperature of 140 ° C for 9 hours.

4 stage. To 200 g of the product obtained in stage 3, add 330 g of ethylene oxide. The reaction is carried out at a temperature of 140 ° C for 9 hours.

5 stage. To 400 g of the product obtained in stage 4, add 120 g of ethylene oxide. The reaction is carried out at a temperature of 160 ° C for 9 hours.

6th stage. To 210 g of the product obtained in step 5, 340 g of ethylene oxide are added. The reaction is carried out at a temperature of 170 ° C for 9 hours.

The synthesis of polyethylene oxides branched with cyanuric chloride is carried out in one step in a solution of 1,2-dichloroethane by reacting purified cyanuric chloride by sublimation with NaO-polyethylene oxide with different chain lengths. In this case, an interaction is carried out between a 15% solution of polyethylene oxide with cyanuric chloride in the presence of triethylamine (25% mol of the total number of end groups)

The reaction is carried out at 80 ° C for one hour. The resulting polymer is planted in a 4-fold excess of sulfuric ether, and then washed three times with it.

Thus, triazine derivatives of polyethylene oxide of the formula are obtained:

Example 2. PEGylation of interferon-α-2b

The possibility of using these compounds for protein modification was demonstrated by the example of interferon. Interferon-α-2c from Hydrola Ltd. 20 mg of the drug is dissolved in 4 ml of water. Aliquots of 1 ml (5 mg) are transferred into reaction vials. To modify the pH of the solution is adjusted to 9.5 with 50 mm phosphate-carbonate buffer. Reagents are added 5 times in a 10-fold molar excess at intervals of 6-12 hours. The progress of the reaction is monitored by gel filtration HPLC. After 60 hours, the reaction was stopped by the addition of 50 mM glycine methyl ester. Physicochemical characteristics of the obtained conjugate: molecular weight 10,000 Da, pH 6.0, transparency 2.3 FTU, osmolality 375 mOsm / kg (Vogel OM 802 osmometer), bacterial endotoxins 0.15 EU / μg, purity (inverted phase HPLC) - 98% of the area.

Example 3. Antiviral activity of interferon-α-2b conjugates

Samples of 2 ml of the following calculated activity are taken:

1) standard (starting interferon) - 25 μg / ml (3.5 × 10 ⁶ )

2) the conjugate according to the invention is 24 μg / ml (3.36 × 10 ⁶ )

Antiviral activity is checked by a standard method in a culture of human lung cells L-68 against viral vesicular stomatitis.

Biological testing yielded the following results:

A drug Estimated bioactivity Bioactivity defined Bioactivity reduced Specific bioactivity % save activity Standard 3.5 × 10 ⁶ 2.7 × 10 ⁶ 3.5 × 10 ⁶ 1.4 × 10 ⁸ one hundred% The conjugate of the invention 3.36 × 10 ⁶ 2.2 × 10 ⁵ 2.9 × 10 ⁵ 0.09 × 10 ⁸ 6.4%

Reduced bioactivity - defined bioactivity, multiplied by the coefficient obtained from dividing the calculated activity of the standard by a specific activity of the standard. The reported bioactivity is calculated to determine the decrease in the specific activity of interferon during the modification process.

Thus, the modified interferon-α-2b according to this invention retains significant antiviral activity similar to the known drug, for which it is 7% with a specific bioactivity of 1.4 × 10 ⁷ .

Example 4. A study of the pharmacokinetics of drugs

Outbred rats were given the following drugs intravenously:

The conjugate of the invention is 0.56 ml = 13.5 μg / rat.

A well-known analogue is a pegylated derivative of interferon (according to RU 2180595) - 0.1 ml = 13.5 μg / rat

Each rat, 1 hour after administration, 0.5 ml of blood was taken from the tail vein. For this, 1.5-2 mm of the tail flesh was cut off with a scalpel. Blood was collected in Eppendorf tubes. The tubes were placed in a thermostat and incubated for 30 minutes at 37 ° C. A thrombus formed from the walls of the tube was separated by a glass capillary. The tubes were placed in a refrigerator and incubated for 4-6 hours at + 4 ° C. Serum was aspirated with an automatic pipette and centrifuged at 5,000 rpm for 5 minutes to remove blood cells. To sterilize serum samples, supernatants were transferred to Ultrafree-MC filtration systems (Amicon) and centrifuged at 2000 rpm for 10 minutes. Samples for testing were stored at + 4 ° C.

The results of measuring the level of biologically active interferon for a period of 14 days are shown in the graph.

Example 5. The toxicity of the obtained PEG derivatives

Acute toxicity experiments were performed on nonlinear white mice and rats. Groups of 6 animals were used to study each dose of the drug. When using a dose range of 1 to 15 IU / kg, no lethal effects were observed. Given that the maximum therapeutic dose for a person is about 0.03 IU / kg, the drugs are low toxic.

Example 6. Antiviral activity in vivo

To determine the antiviral activity, 20 mice weighing 10-12 g are injected with 0.2 ml of the drug 24 μg / ml intraperitoneally 24 and 2 hours before infection with the deadly virus. Animals are observed for 14 days. If the survival rate is more than 60%, then the test drug is considered active. For the conjugate according to the invention, this indicator was 90%, which allows it to be classified as highly active.

Claims (4)

1. Conjugates of a protein and a derivative of polyethylene glycol of the formula

where the protein is interferon α-2b, an and n ₁ have such values that the average molecular weight of polyethylene glycol units is from about 7500 Da to about 35000 Da. 2. The conjugates according to claim 1, where n = n ₁ = 120, and the molecular weight of the polyethylene glycol units is approximately 10,000 Da. 3. A pharmaceutical composition having antiviral activity, comprising the conjugate according to any one of claims 1 and 2, and a therapeutically inert carrier. 4. A method of combating a viral infection, comprising introducing into the body the conjugate according to any one of claims 1 and 2 in an effective amount.

Images