EA021610B1 - Liquid antiviral formulation - Google Patents

Abstract

The invention relates to pharmacology and medicine and provides a liquid formulation comprising a covalent conjugate of interferone-alpha (INF-α) with polyethyleneglycol (PEG) as an active substance, a buffer, a stabilizer and a pharmaceutically acceptable solvent, in which the covalent conjugate is a linear or branched monopegilirated INF-α of general formula (I) or (II), the stabilizer is a mixture of sorbite, polysorbate 20 and L sodium glutamate, the buffer is citrate-phosphate buffer and water is the pharmaceutically acceptable solvent. The inventive result provides long-time stability of liquid formulation preserving substantial activity of nonmodified interferone-alpha, long-term circulation in blood stream and improved characteristics of antiviral activity and ummunogenicity during storage.

Description

The invention relates to the fields of pharmacology and medicine, more specifically to a liquid antiviral drug containing pegylated interferon a-2b, a physiologically compatible buffer, a stabilizer and a pharmaceutically acceptable solvent as an active substance. This drug is intended for the treatment of viral and oncological diseases, and can also serve as an immunomodulator.

State of the art

Pegylated interferon (PEG-INF) is interferon (INF) chemically modified by the covalent addition of polyethylene glycol to its molecule. PEG-INF, unlike unmodified INF, is much longer in the human body, maintaining its biological activity.

Currently, two types of PEG-INF are approved and registered in the Russian Federation: PEG-INF a-2a (Pegasys drug, manufactured by La Roche) and PEG-INF a-2b (PegIntron drug, manufactured by Scherring). Experts consider these drugs as the gold standard for antiviral therapy for the treatment of hepatitis C when combined with ribavirin.

It was shown that the introduction of polyethylene glycol does not have a significant effect on the course of hepatitis C, but leads to an increase in the duration of the action of INF. This is of great practical importance, consisting in reducing the drug load on the patient's body. So, to achieve a therapeutic effect, a single injection of PEG-INF per week is sufficient instead of triple administration of non-pegylated INF. Since PEG-INF remains in the human body longer and its effect is enhanced due to polyethylene glycol, better results can be expected with its use, without increasing the severity of side effects.

Pegylated interferons, both as part of monotherapy and in combination with ribavirin, have undergone numerous clinical studies, as a result of which an extensive evidence base has been collected regarding the effectiveness and tolerability of drugs. PEG-INF can achieve a stable virologic response of up to 60% in patients with the genotype 1 and up to 85% for patients with the genotypes 2 and 3.

The production of dosage forms of interferon is associated with a number of problems that are caused by the sensitivity of the active component to physical and chemical influences. Like other proteins, interferon in aqueous solutions is primarily subjected to chemical degradation, for example, proteolysis, oxidation, disulfide metabolism, oligomerization and deamidation. Physical degradation mainly includes aggregation, precipitation and adsorption. In addition, under the influence of light, elevated temperature and with a change in the pH of the medium, partial racemization of the amino acids that make up the active protein is possible, which can lead to a decrease in the pharmacological effect and an increase in undesirable side effects and toxicity.

Since interferon is very active and is present in dosage forms in small quantities, the stability of interferon preparations and ensuring a constant concentration of the active substance is of particular importance. Therefore, the dosage form of interferon should contain additives that maximally impede the occurrence of these undesirable transformations and counteract the influence of external factors.

For example, human serum albumin has often been used as a universal stabilizer for commercially available protein preparations. However, its introduction into the composition of the dosage form causes a number of problems, for example, the danger of viral infection and the formation of aggregates, which, in turn, can lead to the formation of antibodies. In connection with these shortcomings, human serum albumin, as a rule, is not included in modern injectable pharmacological compositions.

Pegylated interferon preparations can be divided into two types. The first type of PEG-INF preparation is PegIntron, which is a prodrug of interferon containing a linear PEG fragment with a molecular weight of 12 kDa. The polyethylene glycol molecule is attached to interferon by an unstable carbamate bond, which is easily hydrolyzed. For this reason, the drug is released in the form of a dry substance (lyophilisate).

In the international application \ UO 1999/048535 (publ. 30.09.1999) disclosed an aqueous composition comprising conjugates PEG-INF-α, buffer, stabilizer, cryoprotectant and solvent. A composition containing INF-α as a conjugate linked by a urethane (carbamate) bond on the histidine residue to one PEG molecule having a molecular weight of 12 kDa is intended for the preparation of a lyophilized powder, which is reconstituted into an injectable dosage form immediately before use. In a preferred embodiment, one ampoule intended to produce 0.5 ml of the preparation contains 0.1 mg of PEG-INF as an active substance, 0.75 mg of anhydrous dibasic sodium phosphate and 0.75 mg of monobasic sodium phosphate dihydrate as a buffer mixture, 40 mg of sucrose as a cryoprotectant and 0.05 mg of polysorbate 80 as a stabilizer.

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In the patent I8 7632491 (publ. 15.12.2009) a solution for the preparation of a lyophilized composition is proposed, including pegylated interferon, one or more cryoprotectants, a buffer, a stabilizer and water, in which at least 80 wt.% Of the cryoprotectant is trehalose, and the buffer provides maintenance pH in the range of 4.5 and 7.1. In one preferred embodiment, one dose of the preparation contains 0.1-0.3 mg of PEG-INF, 1.5 mg of anhydrous dibasic sodium phosphate and 1.5 mg of monobasic sodium phosphate monohydrate as a buffer, 90 mg of trehalose dihydrate as a cryoprotectant, 0.1 mg polysorbate 80 as a stabilizer and the amount of water required for dissolution.

In addition to the complexity of the production process, the main disadvantage of the known lyophilisates is the need to restore the dosage form before injection, which requires the attention of medical personnel when performing all necessary manipulations and is associated with the risk of improper handling of the pharmaceutical product.

In the patent KI 2382048 (publ. 20.02.2008) a liquid composition for injection is disclosed, comprising as an active substance a covalent conjugate of interferon-alpha with two PEG residues, in which the interferon molecule is linked via an amino alcohol or homoserine linker to the triazine fragment to which the PEG is attached . The composition retains 25-38% of the activity of the original interferon. The disadvantage of the drug is the possibility of the presence of toxic derivatives of cyanuric chloride or trifluoroethanesulfonic acid in it. In addition, the need for thorough purification of the intermediate in order to reduce the toxicity of the active substance and increase the effectiveness of the finished product reduces the yield of the final product.

In the patent I8 5762923 (published on 09.06.1998), an aqueous composition of interferon-alpha or pegylated interferon-alpha, dissolved in water with a nonionic surfactant and benzyl alcohol, does not contain human serum albumin, containing a buffer in an amount that maintains the pH in the range from 4.5 to 6.0 and isotonic agent.

The second type of pegylated interferon is the drug Pegasis, considered as the closest analogue of the proposed liquid drug. The active substance is a covalent conjugate INF-2a containing branched PEG with a molecular weight of 40 kDa. The addition of polyethylene glycol to interferon was carried out via a lysine linker connected to the interferon molecule by an amide bond, and to the PEG molecule by a carbamate bond. This drug is superior in stability to PegIntron, which allows it to be injected, for example, in the form of a solution with pH 6 containing 0.18 mg PEG-INF, 4 mg sodium chloride, 5 mg benzyl alcohol, 1.3085 mg sodium acetate trihydrate , 0.0231 mg of acetic acid, 0.025 mg of polysorbate 80, water for injection up to 0.5 ml.

The main disadvantage of this form is a small fraction of the preserved activity of the initial interferon (at the level of 1%). Another disadvantage is the presence of benzyl alcohol as a preservative, which has a particularly detrimental effect on infants. , 1982) .R. 290-291; Vgo \ yn 1., ΒτιίκΙ К.. K. M, O1kop Т.. T. S, NikYup K.K., Keppa ^ ay О.. O. Vep / y1 a1sooyo ro15oshpd: a Sayke OG SHEZHONS AS1BO515 APB BEA! W SHEOPA1A1 TGapy. Lapse! (in press)). In addition, the small capacity of the acetate buffer solution at pH 6 (Dawson R., Elliot D., Elliot W., Jones K. Handbook of biochemist: Transl. From English. - M .: Mir, 1991. 544 s) leads to a decrease in stability dosage form.

Thus, an object of the invention is the development of a drug with improved pharmacological properties and the expansion of the arsenal of liquid antiviral agents containing pegylated interferon-α.

The technical result of the invention is the long-term stability of the liquid drug while maintaining a significant proportion of the activity of unmodified interferonalf, prolonged circulation in the bloodstream and improved characteristics of antiviral activity and immunogenicity during storage.

To achieve the specified technical result, a liquid drug containing as an active substance a covalent conjugate of interferon-alpha (INF-α) with polyethylene glycol (PEG), a buffer, a stabilizer and a pharmaceutically acceptable solvent according to the invention, contains a linear mono-pegylated INF- as a covalent conjugate α of the general formula (I)

where a is an integer from 100 to 1800, b is an integer from 1 to 9, and INF indicates the remainder of interferon-α with a molecular weight of 5 to 80 kDa, or a branched monopegylated INF-α of the general formula (II)

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where η is an integer from 200 to 950, s | - an integer in the range from 1 to 5, t is an integer in the range from 1 to 5 and INF indicates the remainder of interferon-α;

as a stabilizer contains a mixture of sorbitol, polysorbate 20 and sodium b-glutamate, additionally contains citrate-phosphate buffer, and as a pharmaceutically acceptable solvent it contains water in the following ratio of components (g / 100 ml):

covalent conjugate 0.005-0.1; stabilizer: sorbitol 1.0-10.0; polysorbate 20 0.001-0.1;

B-glutamate sodium 0.1-1.0; citrate phosphate buffer 0.85-0.95; water the rest.

In addition, the drug contains the above components (g / 100 ml): covalent conjugate 0.03; stabilizer 2.0; sorbitol polysorbate 20 0.01;

Sodium b-glutamate 0.5; citrate phosphate buffer 0.92; water the rest.

SUMMARY OF THE INVENTION

As a result of extensive research, the inventors have found that these disadvantages of the solutions known from the prior art can be overcome by obtaining a liquid antiviral drug that contains as an active substance a linear or branched covalent conjugate of interferon-alpha (INF-α) with polyethylene glycol (PEG ) obtained by azide condensation of a pegylating agent (PEG agent) with INF-α. The chemistry of the applied azide condensation reaction, involving the interaction of the PEG agent of general formula (III) or (IV)

with INF-α, causes the absence in the structure of the conjugate of a hydrolytically labile carbamate bond, as well as racemization products of the substrate, which have reduced biological activity and exhibit a noticeable side effect.

Thus, the present invention provides a liquid antiviral drug containing, as an active substance, a covalent conjugate of interferonalf (INF-α) with polyethylene glycol (PEG), a buffer, a stabilizer and a pharmaceutically acceptable solvent, in which, as a covalent conjugate, it contains a linear monopegylated INF - α of the general formula (I)

where a is an integer from 100 to 1800, b is an integer from 1 to 9, and INF indicates the remainder of interferon-α with a molecular weight of 5 to 80 kDa, or a branched monopegylated INF-α of the general formula (II)

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where η is an integer from 200 to 950, s | - an integer in the range from 1 to 5, t is an integer in the range from 1 to 5 and INF indicates the remainder of interferon-α;

as a stabilizer it contains a mixture of sorbitol, polysorbate 20 and sodium b-glutamate, as a buffer it contains citrate-phosphate buffer, and as a pharmaceutically acceptable solvent it contains water in the following ratio of components (g / 100 ml):

covalent conjugate 0.005-0.1; stabilizer: sorbitol 1.0-10.0; polysorbate 20 0.001-0.1;

B-glutamate sodium 0.1-1.0; citrate phosphate buffer 0.85-0.95; water the rest.

In a preferred embodiment of the invention, the drug contains (g / 100 ml): covalent conjugate 0.03; stabilizer 2.0; sorbitol polysorbate 20 0.01;

Sodium b-glutamate 0.5; citrate phosphate buffer 0.92; water the rest.

The drug in accordance with the invention is prepared by mixing solutions of components that have previously undergone sterilizing filtration, followed by setting the pH in the range from 4.5 to 7.5 by adding a diluted ΝαΟΗ solution and bringing the water for injection to the required volume. The hydrated solution is again subjected to sterilizing filtration through a filter with a porosity of 0.22 μm and, under aseptic conditions, is dosed in sterile containers.

The drug in accordance with the invention may be in the form of a standard dosage form intended for parenteral administration. In this case, the sterile container is an ampoule, vial, syringe, pen, or syringe tube. The liquid drug can be administered to a patient using conventional injection devices that allow administration of 0.3 to 20 ml, preferably 0.5 to 10 ml of the composition in unit dosage form.

A drug in accordance with the invention, containing a mono-pegylated linear or branched INF-α of the general formulas (I) or (II) and having a pH of 5 to 7, is stable during storage for six months while maintaining antiviral activity at 90% of its original value . This is confirmed by the results of studies of solutions prepared in accordance with example 1 (tables. 1 and 2).

Table 1. The stability and antiviral activity of the compositions of the linear PEG-INF-a-2b formula (I) with different pH values under conditions of 6-month storage

Time, months Index The value of the indicators,% of the initial value pH 5.0 pH 6.0 pH 7.0 4’s 20 ° C 37 ° C 4C 20 ° C 37 ° C 4’s 20 ° C 37 ° C one SPI one hundred 99 97 99 99 98 one hundred 99 96 CA 0 one 3 one one 2 0 one 4 PA 102 98 94 one hundred 99 95 106 99 89 2 sleep 97 97 94 99 98 97 one hundred 99 96 CA 3 3 6 one 2 3 0 one 4 PA 98 96 85 one hundred 97 95 99 97 92 3 SPI 97 97 95 99 98 94 99 98 93 CA 3 3 5 one 2 6 one 2 7 PA one hundred 97 88 101 97 90 one hundred 94 87 4 sleep 98 95 93 98 97 95 98 98 94 CA 2 5 7 2 3 5 2 2 6 PA 98 92 81 one hundred 92 92 96 94 90 5 SPI 96 98 95 99 97 97 one hundred 95 96 CA 4 2 5 one 3 3 0 5 4 PA 93 96 92 94 94 94 98 91 90 6 SPI 97 96 95 97 95 97 98 96 96 CA 3 4 5 3 5 3 2 4 4 PA 96 89 89 97 89 93 99 91 90

SPI - the content of PEG-INF a-2; CA - content of aggregates;

PA - antiviral activity

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Table 2. Stability and antiviral activity of the branched PEG-INF-a-2b compositions of formula (II) with different pH values under 6-month storage conditions

Time, months Index The value of the indicators,% of the initial value pH 5.0 pH 6.0 pH 7.0 4 ° C 20 ° C 37 ° C 4 ° C ' 20 ° C 37 ° C 4 ° C 20 ° C 37 ° C one SPI one hundred 99 95 one hundred one hundred 95 one hundred one hundred 98 CA 0 one 5 0 0 5 0 0 2 PA 111 one hundred 89 99 97 86 102 one hundred one hundred 2 SPI one hundred 99 95 one hundred 99 98 one hundred one hundred 98 CA 0 one 5 0 one 2 0 0 2 PA 108 98 84 99 98 94 one hundred 99 98 3 SPI one hundred 99 95 one hundred 96 97 99 97 99 CA 0 one 5 0 4 3 one 3 one PA 107 94 93 106 97 90 one hundred 96 99 4 SPI 99 98 97 one hundred 96 98 one hundred 99 96 CA one 2 3 0 4 2 0 one 4 PA 97 98 93 one hundred 93 93 101 96 88 5 SPI 99 96 98 98 96 97 98 97 96 CA one 4 2 2 4 3 2 3 4 PA 102 89 87 93 90 91 98 95 91 6 SPI one hundred 97 95 99 96 96 99 98 95 CA 0 3 5 one 4 4 one 2 5 PA one hundred 94 88 98 95 87 99 98 89

SPI - the content of PEG-INF a-2;

CA - content of aggregates;

PA - antiviral activity

The drug in accordance with the invention is characterized by the main pharmacokinetic parameters:

circulation time (T);

area under the curve (AIS);

maximum concentration in serum (C _max );

time to reach maximum serum concentration (T _max );

clearance (Cb);

distribution volume (UD and half-life (1 _1/2 ), given in table. 3.

Table 3. The main pharmacokinetic parameters of drugs containing linear and branched PEG-INF-a-2

Active substance T, day AIS, ng / ml h Stakh, NG / ML Tscaht ⁴ Cb, ml / h \ E, ml 1 | / 2, h Linear PEG-INF a-2b 7 2,483 105 6 0.403 9.5 sixteen Branched PEG-INF a-2b 7 27,600 421 24 0,036 2,4 46

The drug according to the invention can be used to treat viral diseases such as chronic hepatitis B (HBeAd-positive or HBeAd-negative with compensated liver damage and signs of viral replication, chronic hepatitis C without cirrhosis or with compensated cirrhosis, clinically stable HIV infection and chronic hepatitis C with associated HIV infection.

Further, the invention will be illustrated by preferred examples of its implementation, confirming the achievement of the technical result.

Example 1. Preparation of a liquid drug PEG-INF-a-2b

Under sterile conditions, sterile solutions of 525 mg of citric acid monohydrate in 10 ml of water, 390 mg ΝαΗ ₂ ΡΟ ₂ · 2H ₂ O in 10 ml, 2 g of sorbitol in 10 ml of water, 500 mg of sodium glutamate in 10 ml of water and 10 mg of polysorbate 20 in 10 ml of water.

To the resulting mixture, 5% ΝαΟΗ is added dropwise with stirring until the desired pH is established and 30 ml of a solution of the corresponding PEG-INF-a-2b with a concentration of 1 are added

- 5 021610 mg / ml. Next, the pH of the resulting solution is adjusted by adding 5% ΝαΟΗ and the total volume of the mixture is adjusted to 100 ml with water for injection.

The solution is filtered through a sterilizing filter with a pore size of 0.22 μm; under aseptic conditions, 1 ml is poured into sterile vials, which are sealed and labeled with the instructions for medical use.

Example 2. The study of the stability of the liquid drug PEG-INF a-2b.

The containers are divided into 3 groups and stored at 4, 20 and 37 ° C. Next, 3 ampoules are selected from each group for research according to the following scheme: ampoules stored at 4 ° C are taken at a frequency of 1 time per month, stored at 20 ° C are taken at a frequency of 1 time in 2 weeks, and samples stored at 37 ° C, taken weekly.

Before the start of the studies, three samples that have not been incubated are combined and examined for the content of the corresponding PEG-INF-a-2B (SPI) and its aggregates (CA) by exclusion HPLC (column 8, column 6 10/300 CI, 22 ° C, eluent: 50 mmol sodium phosphate, 300 mmol No. C1, 10% ethanol, pH 6.8, 0.4 ml / min, 280 nm). In parallel, antiviral activity (PA) is examined according to the method described in Example 3. The obtained values of the parameters STI, CA and PA are taken as 100%.

Similarly, the combined samples from each group selected at the corresponding time points are examined. All test results are expressed as a percentage of the corresponding unincubated solution.

Example 3. The study of the toxicity and safety of the liquid drug PEG-INF a-2.

Male nonlinear white mice weighing 20 ± 1 g are injected with the indicated liquid drug, prepared in accordance with example 1, in a volume of 0.1 ml. In animals, clinical symptoms are recorded daily for a week, weighed, blood samples taken for general and biochemical analysis. On the last day, the animals are euthanized and necropsy with organ sampling for subsequent histological analysis. The death of experimental animals throughout the study was not noted, blood counts were normal. In a pathological morphological study of laboratory animals, no significant pathological changes were found.

As a result of the studies, it was proved that the proposed liquid drug is non-toxic, pyrogen-free, does not adversely affect the systems and organs of laboratory animals, does not have reactogenicity, and thus, low-hazard substances comply with the requirements of hazard class 4 (GOST 12.1.007-76 Harmful substances).

Example 4. Determination of the specific antiviral activity of the drug PEG-INF a-2

Antiviral activity is determined by micromethod in 96-well flat-bottom plates to suppress the cytopathic effect of the test virus of vesicular stomatitis in a diploid culture of клеток cells sensitive to interferon-alpha. To do this, use a test virus at a dose of 100 CPD ₅₀ and nutrient medium Needle ΜΕΜ with the addition of 10% serum of cattle. The international standard INF-a-2b is used as the standard.

Before starting the studies, three samples that have not been incubated are combined and their specific antiviral activity is examined, which is taken as 100%. Similarly, the combined samples from each group selected at the corresponding time points are examined. All test results are expressed as a percentage of the corresponding unincubated solution.

Example 5. The study of the level of bacterial endotoxins in the drug PEG-INF-a-2b.

The content of bacterial endotoxins in the drug PEG-INF a-2b is determined by ίη νίίτο using the LAL test according to the standard method (GFH11, part 1, p. 128). In this work, we use a set of the company Lop / a, USA. ROSOSET LAL reagent with a sensitivity of 0.03 EU / ml (catalog number: 0000184452) and an endotoxin control standard (catalog number: 0000167426). The activity of KSE 10EE / ng. LAL water (catalog number: 01022012). It was shown that the content of endotoxins in the drug PEG-INF-a-2b is less than 2.5 units. endotoxin per 1 mg of protein, which is much lower than the level of endotoxins allowed by the European Pharmacopoeia for recombinant proteins.

Example 6. Determination of the main pharmacokinetic parameters of drugs based on linear and branched PEG-INF-a-2b

Nonlinear white mice (males) are divided into two groups according to the number of studied drugs. The studied drugs containing a linear or branched conjugate are administered intraperitoneally at a dose of 1 million ΜΕ. Every 6 hours, blood samples are taken from the animals and serum is prepared from it using the standard method. The content of INF-α in serum samples (ng / ml) is determined using the immuno-enzyme assay kit (ELISA kit) Nitap ίΡΝ-α ЕЫ8А Κίί (catalog number: SK2003-1). Pharmacokinetic parameters are calculated from the data obtained using the Vogshcha 1.03 computer program. The results are presented in table. 3.

Claims (2)

CLAIM 1. Liquid antiviral drug containing as an active substance a covalent conjugate of interferon-alpha (INF-α) with polyethylene glycol (PEG), a buffer, a stabilizer and a pharmaceutically acceptable solvent, characterized in that as a covalent conjugate contains a linear monopegylated INF-α General formula (I) where a is an integer from 100 to 1800, B is an integer from 1 to 9 and INF means interferon-α residue with a molecular weight of from 5 to 80 kDa, or branched monopegylated INF-α of the general formula (II) where η is an integer from 200 to 950, s] is an integer in the range from 1 to 5, t - an integer in the range of from 1 to 5, and IFN denotes an interferon-α residue; as a stabilizer contains a mixture of sorbitol, polysorbate 20 and b-glutamate sodium, as a buffer contains citrate-phosphate buffer, and as a pharmaceutically acceptable solvent contains water in the following ratio of components, g / 100 ml: covalent conjugate of 0.005-0.1; stabilizer: sorbitol 1,0-10,0; polysorbate 20 0.001-0.1; Sodium glutamate 0.1-1.0; citrate-phosphate buffer 0.85-0.95; water the rest. 2. The drug according to claim 1, characterized in that it contains these components in the following ratio, g / 100 ml: covalent conjugate 0.03; stabilizer 2.0; sorbitol polysorbate 20 0.01; Sodium glutamate 0.5; citrate-phosphate buffer 0.92; water the rest.