Pharmaceutical compositions of erythropoietin

 

The invention relates to aqueous pharmaceutical compositions of erythropoietin that are free from the products of human serum, stable amino acid, sorbitan mono-9-octadecenoate poly(oxy-1,2-ethandiyl) derivative. The present invention also relates to stable water containing preservatives pharmaceutical compositions of erythropoietin, which contain antibacterial amount of cresol and amino acid. Pharmaceutical compositions are stable and free from human blood products such as albumin. 2 C. and 50 C.p. f-crystals, 4 Il., 9 table.

Cross-reference to related application this application claims the priority in accordance with the provisional application U.S. serial 60/128596, filed on April 9, 1999, the description of which is incorporated herein by reference.

Field of invention the Present invention relates to aqueous pharmaceutical compositions of erythropoietin, free from products of human serum, stable amino acid, sorbitan mono-9-octadecenoate poly(oxy-1,2-ethandiyl) derivative. The present invention also relates to stable water containing preservative of additive pharmacy.

Background of the invention Erythropoietin (EPO) is a glycoprotein hormone secreted into the kidney in response to tissue hypoxia, which stimulates the production of red blood cells in the bone marrow (1). Gene EPO was cloned and expressed in cells of the ovaries of Chinese hamsters (2, 3). This recombinant human erythropoietin (epoetin Alfa, rhEPO) has an amino acid sequence identical to the sequence of human erythropoietin in urine and, although there are differences in the glycosylation of the protein, which affects the efficiency of in vivo, both are indistinguishable in functional and immunological assays (4, 5).

In clinical trials today rhEPO was evaluated in healthy subjects and in patients with various anemic conditions (6, 7). EPO stimulates rapid hematologic response in healthy volunteers, provided adequate revenues of iron to maintain the enhanced synthesis of hemoglobin (8). Most trials studied the reliability and efficiency of rhEPO for the treatment of patients with chronic renal failure who are on dialysis, and patients who dialysis has not yet been appointed. Other indications approved in the US include Storico is the ne. Worldwide EPO is used to treat anemia associated with rheumatoid arthritis, premature birth, myelofibrosis, a bone marrow transplant, burns, sickle cell anemia-talasemia, to increase the amount of blood in front of the fence autologous donor blood and is used as an aid before surgery (6, 7).

Although rhEPO is usually well tolerated, observed rare cases of skin rash and urticaria, which suggests the presence of allergic hypersensitivity to some of the components of the composition of epoetin alpha is likely to albumin human serum. Moreover, despite the screening of blood, if the pharmaceutical agent is obtained using human blood products, there is a risk of exposure to an infectious agent. Therefore, the pharmaceutical composition rhEPO, which would be stable and free from human blood products such as albumin.

In U.S. patent 4992419 described lyophilized compositions of erythropoietin containing 5-50 g/l of urea, 1-50 g/l amino acid, 0.05 to 5 g/l of surfactant and containing no albumin human serum. Opisie albumin human serum, and, thus, are not commercially acceptable. Therefore, the consumer or the doctor, or the patient may need to prepare a song just before the introduction. Lyophilized compositions are not preferred among clinical compositions of erythropoietin, as the cooking process is long, creates the risk of improper cleaning of the protein composition, or preparation may be incorrect and to maintain sufficient activity of the drug usually requires certain additives, such as stabilizers. Therefore, generally preferred are aqueous compositions of erythropoietin.

In U.S. patent 5376632 described water, lyophilized, or spray dried composition oror-cyclodextrins containing erythropoietin and do not contain additional stabilizers such as albumin human serum, bovine serum albumin, lecithin, methyl cellulose, polyethylene glycol, sulfur-containing reducing agents, urea, amino acids and surfactants (column 4, lines 51-54).

In U.S. patent 5661125 described aqueous compositions of erythropoietin containing Antibac the umina of human serum, received and investigated on the stability relative to compositions containing albumin of human serum. The results showed significant deposition of erythropoietin in the HSA-free krasilnik (0.5%) and chlorocresol (0,3%) compositions, even at 0o(Column 8, lines 1-29), making these compositions, therefore, unsuitable for clinical use. Moreover, the composition of EPO containing cresol in the region from 0.2% to 0.5%, described as having a weak increase stability and more rapid destruction of EPO (stables 6, lines 21-25). Commercially available multinazionale compositions currently contains 1% benzyl alcohol.

Commercially available compositions rhEPO get in citrate buffer with an activity of 1000 IU/0.5 ml, 2000 IU/ml, 5000 IU/ml and 10,000 IU/ml (10K) for intravenous (IV) and subcutaneous (SC) injection. These compositions are clinically confirmed as the immediate cause of discomfort in patients associated with SC introduction songs with citrate buffer (9, 10). Because the preferred use of rhEPO has shifted from centuries on SC, it soon became clear that the local introduction of this composition is not the best. No heating of the medicinal product re the Oia pain at the injection site. No human albumin, which is used as a stabilizer in the composition or reducing the osmotic pressure of the solution was not the cause of pain, a prospective candidate became citrate buffer rhEPO. Composition without preservatives with a single dose of 40,000 IU/ml is commercially useful, if it contains sodium phosphate, instead of citrate buffer. For the stability required a lower dose of the composition containing phosphate buffer instead of the less preferred citrate buffer.

Brief description of the invention the Present invention relates to pharmaceutical compositions of erythropoietin, comprising: (a) pH superyoshi agent; (b) a stabilizing amount of sorbitan mono-9-octadecenoate poly(oxy-1,2-ethandiyl) derivative; (C) a stabilizing amount of amino acid and (d) number of pharmaceutical erythropoietin, where the composition does not contain urea or product for human blood. The preferred composition as stabilizing agents include a combination of Polysorbate 80 and glycine. Particularly preferred compositions as stabilizing agents include a combination of Polysorbate 80 and glycine in the system phosphate buffer.

The present invention also relates to the government of albumin human serum; (C) a stabilizing amount of amino acids; (d) an antibacterial amount of cresol and (e) number of pharmaceutical erythropoietin. The preferred composition as stabilizing agents include combinations of human albumin and glycine. Particularly preferred compositions as stabilizing agents include combinations of albumin human serum and glycine in the system phosphate buffer with m-cresol used in a concentration of about 0.3%.

The compositions of the present invention exhibit a pharmacokinetic properties such as absorption, bioavailability, similar to the modern pharmaceutical products, and the levels of serum concentration of such commercially available at present, the pharmaceutical compositions of recombinant human erythropoietin. Moreover, the compositions of the present invention cause with the introduction of less discomfort to the patient and have a significantly shorter duration of discomfort at the injection site. Thus, the present invention relates to free from albumin human serum or preservative additives to pharmaceutical compositions of erythropoietin, which can be used instead of the G. 1 - graphs: the average concentration of serum erythropoietin - time (not adjusted with the basic levels of erythropoietin) in subjects receiving a single 150 IU/kg SC dose of rhEPO with preservative 0.3% m-cresol, or without it. The concentration of serum erythropoietin was determined by radioimmune assay (RIA).

Fig. 2 - graphics: the average concentration of serum erythropoietin - time (not adjusted with the basic levels of erythropoietin) in subjects receiving a single 150 IU/kg SC dose of rhEPO (2K) with the new stabilizer or without him. The concentration of serum erythropoietin was determined by radioimmune assay (RIA).

Fig. 3 - graphics: the average concentration of serum erythropoietin - time (not adjusted with the basic levels of erythropoietin) in subjects receiving a single 750 IU/kg SC dose of rhEPO (40K) with a new stabilizer or without him. The concentration of serum erythropoietin were determined using (RIA).

Fig. 4 - graphs: the average concentration of serum erythropoietin - time (not adjusted with the basic levels of erythropoietin) in subjects receiving a single 150 IU/kg SC dose of rhEPO with citrate and phosphate buffer. The concentration of serum erythropoietin was determined by radioimmunoassay of analyze is the way erythropoietin, which contain amino acids, sorbitan mono-9-octadecenoate poly(oxy-1,2-ethandiyl) derivatives as stabilizing agents in the buffer-free products from human blood or urea. Thus, the present invention relates to pharmaceutical compositions of erythropoietin, comprising: (a) pH superyoshi agent; (b) a stabilizing amount of sorbitan mono-9-octadecenoate poly(oxy-1,2-ethandiyl) derivative; (c) a stabilizing amount of amino acids;
(d) number of pharmaceutical erythropoietin,
and where the composition does not contain urea or the product of human blood.

The present invention also includes aqueous compositions of erythropoietin with preservative-cresol, stable combination of albumin human serum and amino acid. Therefore, the present invention relates to pharmaceutical compositions of erythropoietin containing:
(a) pH superyoshi agent;
(b) a stabilizing amount of serum albumin human blood;
(c) a stabilizing amount of amino acids;
(d) an antibacterial amount of cresol and
(e) number of pharmaceutical erythropoietin.

One of the tasks for these compositions was to minimize described the output buffer. Therefore, all of the compositions of the present invention is especially preferred are phosphate buffer.

The number BufferedReader agent used in the pharmaceutical compositions of the present invention largely depends on the particular buffer used and the desired pH of the composition. The preferred pH of the solution is between about 5 to 8, more preferred between about 6-7,5, and most preferred is a pH value of approximately 6,9. In accordance with these pH values, the amount of buffer is typically in the range from about 10 to about 30 mm. Using the buffer system of dibasic sodium phosphate and monobasic phosphate is preferred. Other suitable buffer system to maintain the desired pH from about 5 to about 8 include, but are not limited to, sodium citrate/citric acid, sodium acetate/acetic acid and any other pharmaceutically acceptable pH superyoshi agent known in this field.

Can be added to the agent, establishing pH, such as, but not limited to, hydrochloric acid, citric acid, sodium hydroxide, or salt of either is in the range of compositions of the present invention, is any agent capable of imparting the compositions of the present invention isoosmotic with human blood. Normal osmotic agents are widely known in the field and include, but are not limited to, sodium chloride, mannitol, glycine, glucose and sorbitol. In the compositions of the present invention using sodium chloride as the osmotic agent is preferred.

Sorbitan mono-9-octadecenoate poly(oxy-1,2-ethandiyl) derivatives, including, but not limited to, Polysorbate 80 or Polysorbate 20, are examples of derivatives used as stabilizing agents to prevent the adsorption of erythropoietin on the surfaces of the containers, which are containing erythropoietin compositions. In the present invention is used with a wide variety of sorbitan mono-9-octadecenoate poly(oxy-1,2-ethandiyl) derivatives, known to specialists in this field. The amount of Polysorbate 20 or 80, used in the compositions of the present invention, is in the range from about 0.01 to about 1.0 mg per 1 ml of a composition containing 1000-100000 Units of erythropoietin per vial. In the compositions of the present invention the use of Polysorbate 80 is one the of necessaty, including, but not limited to, glycine, L-isoleucine, L-leucine, L-2-phenylalanine, L-glutamate acid and L-threonine, are used in quantities of from 0.1 to 50 g/l and preferably in quantities of from 0.25 to 20 g/l L-Alanine and L-arginine are preferred in the compositions of the present invention, because their inclusion causes conformational changes in the structure of the EPO. Glycine is the preferred amino acid and is used preferably in an amount of from 0.25 to 20 g/l, in particular in the amount of 0.5 g/l, in compositions containing Polysorbate 80 and not containing albumin human serum. Glycine is the preferred amino acid, and compositions containing the albumin human serum and a preservative cresol, it is used preferably in an amount of from 0.5 to 50 g/l, in particular 2.0 g/l In the compositions of the present invention, suitable for use is any amino acid, or L-or D-isomer form, with the exception of L-arginine and L-alanine.

The most preferred composition selected from the group listed in table a or table Century.

Unexpectedly, it was found that aqueous compositions of erythropoietin using phosphate BU the e urea or the product of human blood, represent a stable composition. In particular, compositions containing glycine and Polysorbate 80, provide a wonderful and unexpected stability compared to previous compositions described in this field.

Erythropoietin is located in the compositions in therapeutically effective amounts. Erythropoietin may include proteins that have biological activity of human erythropoietin and analogs of erythropoietin, erythropoietin isoforms, the mimetics of erythropoietin, erythropoietin fragments, hybrid erythropoietin proteins, fused protein oligomers and multinary the above proteins, homologues of the above proteins, glycosylated examples of the above-mentioned proteins and mutiny the above proteins, regardless of their biological activity and, moreover, regardless of the method of synthesis or industrial production, including, but not limited to, recombinant derived from cDNA or from genomic DNA, synthetic, transgenic, and methods of gene activation. Typical examples of erythropoietin include epoetin Alfa (EPREXThat ERYPO), a new protein, stimulirovany in European patent publication EP 640619), slit proteins analog of human erythropoietin - albumin human serum, described in international patent publication WO 9966054, erythropoietin mutants described in international patent publication WO 9938890, erythropoietin omega, which can be obtained from the ApaI restriction fragment gene human erythropoietin described in U.S. patent 5688679 changed by glycoarrays human erythropoietin described in international patent publication WO 9911781, PEG conjugated analogs of erythropoietin described in WO 9805363 or U.S. patent 5643575. Typical examples of cell lines, modified for the expression of endogenous human erythropoietin described in international patent publications WO 9905268 and WO 9412650.

Effect of erythropoietin can be traced by measuring hematocrit in relation to the target hematocrit in the 30-33%. Adjustment of dose may be conducted by monitoring the hematocrit. Vials for single use erythropoietin contain 2000, 3000, 4000, 10000 or 40000 units of EPO (1 U corresponds to approximately 8.4 ng of recombinant erythropoietin). In view of the fact that the composition in the first embodiment of the present invention with the holding replicated as many times as the number of units of erythropoietin, presented in a disposable vial. In the present invention include compositions containing 1000-100000 units or more erythropoietin on the bottle. It is generally assumed that the effective amount is from about 1 to 500 U/kg of body weight, and more preferably from 50 to 300 Units/kg of body weight, especially erythropoietin input SC Effective amount, moreover, depends on species and size of the subject undergoing treatment, individual condition or disease that is being treated, its severity, and the route of administration. In any case, your dose should be non-toxic for the patient.

Unexpectedly, it was found that erythropoietin can be represented in the form of stable conserved water compositions containing <0.5% cresol and containing albumin human serum and amino acids as stabilizing agents. Preferred levels of cresol is from 0.2 to 0.5%, most preferred is 0.3%. Glycine is the preferred amino acid stabilizer. The present invention relates to phosphatherium compositions containing cresol as a preservative, the current activity of 10,000 IU/2.5 ml, 25000 IU/2.5 ml and 40,000 IU/2.0 ml (40K). Higher Eeyore 1 according to the present invention show the use of citrate or phosphate as a buffer and the inclusion in the composition of m-cresol does not cause reduction characteristics of absorption and bioavailability, as well as the security settings of erythropoietin. Thus, we can conclude that the new multinazionale phosphatebuffered compositions of erythropoietin with preservatives comparable to currently available compositions for a single use, and multilaterally erythropoietin can be used in all indications, the currently accepted for erythropoietin.

Being pharmacokinetic compared with non-reusable EPREX songs, new song, therefore, also involves clinical use. Moreover, multinationally fosfatirovannoyj erythropoietin has a possible additional advantage compared with once used by erythropoietin in bottles; economic advantage. The use of erythropoietin to treat anemia or predetermine autologous blood requires repeated injections of the hormone once every 4 weeks once every 2 weeks, once a week, twice a week or three times a week. Designed for odnorazovoe, while using appropriate amount multinazionale conserved erythropoietin, the amount of released erythropoietin will be minimized. In addition, the antibacterial properties of cured compositions of the present invention makes them more suitable for self-injection for the treatment of a large number of patients in a hospital that brings significant savings in healthcare costs. Multinazionale vials of EPO also provide additional convenience in the administration of a medicinal product by minimizing the number of disposable bottles and vials, which the patient/health care uses at high volumes doses. Higher activity (25000 IU/2.5 ml and 40,000 IU/2.0 ml) multinazionale erythropoietin may also minimize the amount of injection, especially in cases when high doses. Multinationally erythropoietin may also be in the form of a disposable bottle, used as input dose. For example, the recommended dose for the program autologous donor blood is 600 IU/kg twice a week (7). For a patient weighing 70 kg, the recommended dosage regimen will be priblizitelen is.

The following examples illustrate the present invention but do not restrict.

Example 1.

For the study of example 1, was selected composition with an activity of 10,000 IU, as this activity is an acceptable dose, and the maximum recommended volume for SC injection of approximately 1 ml By reducing the amount of injection of high activity multinazionale erythropoietin potentially gives the possibility to reduce points subcutaneous injection, which undoubtedly brings benefits to improve portability of the injection the patient.

Objective: to compare the safety and pharmacokinetic properties of subcutaneously injected with recombinant human erythropoietin (epoetin Alfa, rhEPO) obtained in phosphate buffer containing 0.3% m-creosol as preservative (10000 U/ml) with commercially available citrate buffer composition rhEPO (without preservative).

Patients and methods
Patients
Eighteen healthy volunteers aged 20 to 38 years (mean age is 26.9 years) and weight of 60.8-87,4 kg (average weight of 72.3 kg) were included in the test and completed it. The average age of the subjects (patients) in group I (28.1 years) was slightly larger than that of the subjects of group II (from 25.8 years) is whether clinically significant pathological changes in laboratory tests of blood or serum biochemistry. They were negative for toxicity urine, HIV and surface antigens of hepatitis C. they didn't Have any of the following scenarios: hypertension (e.g., diastolic blood pressure95 mm RT. Art.); cases of any primary hematologic disease; significant cases of liver, kidney, cardiovascular system, gastrointestinal system, genitourinary system, metabolic, neurological diseases; cases of anemia or seizures; known sensitivity to products mammal or human serum albumin; addiction and abuse of caffeine-containing beverages; participation in any other clinical trial, or blood transfusion, or in donated blood within 30 days prior to the test; action rhEPO within three months prior to testing; disease for seven days before testing and significant cases of abnormal physical data to test or clinical laboratory data for 14 days before testing. All subjects were evaluated for safety, and all fences blood for pharmacokinetic analyses were performed in accordance with the schedule of tests. All isana included a phase I study one centre, open, randomized, crossover study with two periods in healthy male volunteers. Eighteen subjects were randomly assigned to one of two groups of sequential processing (nine subjects/group). rhEPO was administered in two independent period dose bolus SC injection in the upper thigh. Each period, the dose was divided into 14-day washout of the drug. The subjects were placed in the clinic for at least 12 hours before and was up to 72 hours after administration of the dose in each of the two periods dose, but not between periods. The dosage is given in table 1.

This test used two songs EPO. Epoetin Alfa (commercially available as EPREXcontains 10000 U/ml rhEPO in citrate buffer. The composition of rhEPO with preservative contains 25000 IU rhEPO in 2.5 ml phosphate buffer with preservative is m-cresol.

Collection of samples of blood
Serial blood samples were obtained by direct puncture of the vein before and after the introduction of the EPO. Samples of venous blood (5 ml) for determination of the concentration of erythropoietin serum was obtained for ~30, 20 and 10 minutes before a dose (3 OS is, 4, 30, 36, 48, 60 and 72 hours. Each serum sample was divided into two aliquots. All serum samples were stored at -20oC. the serum Samples were transported in dry ice. Laboratory and clinical studies on an empty stomach (blood count, serum biochemistry, and urinalysis) were performed immediately before the first dose on day 1, on the morning of the 4th day immediately before the dose on the 16th day and the morning of the 19th day.

Bioanalytical methods
For determination of the concentration of erythropoietin serum used the methodology set for radioimmune assay (RIA) (Diagnostic Systems Laboratory [DSL] , Webster TX). Commercially available RIA is a method of double antibody competitive method, which as a primary antibody using a polyclonal immune serum of rabbit to erythropoietin in urine of man and as the label uses125I-labeled human erythropoietin in urine. In a set of calibrators and quality control samples presented in DSL set erythropoietin urine was replaced with epoetin Alfa. The standard concentration used in the method was 7,8, 15,6, 31,3, 50, 62,5, 100 and 125 mIU/ml Sensitivity, defined as the average of the definition of the lower boundary of the standard, which gives acceptable accuracy, was the distribution of security
Main indicators of body condition were recorded immediately before each dose (1 and day 16) and after 6, 24, 48 and 72 hours after dose. Security definitions based on the study of deviations and changes in clinical laboratory tests from the main and nature of adverse events. In addition, before the test was estimated changes in the main indicators of the condition of the body, including blood pressure and the results of physical examination.

Data analysis
The concentration of serum after administration of doses were skorrelirovany with the main concentrations of erythropoietin to dose by subtracting from each value the concentration of serum after injection, the average primary concentration of erythropoietin determined by the average levels of erythropoietin from the three samples collected at 30, 20 and 10 minutes prior to dose. Serum concentration of erythropoietin before the introduction of the dose is not taken into account the average value, if they were below the quantitative sensitivity analysis. Pharmacokinetic parameters were determined according to the concentration of serum korregirovannye with the main concentration of erythropoietin.

Pharmacokinetic parameters of expect the Zuya program BIOAVL, the version is 8.0, (Scientific Computer Systems, RWJPRI). Identified the following pharmacokinetic parameters: peak serum concentration (Cmax); time to reach maximum serum concentration (tmax); area under the curve of concentration-time (AUC) from the start timing (zero) before the pickup time of the last blood sample (AUC0-72) was calculated using the formula of the linear trapezoid and the final half-life (t1/2) was defined as 0,693/rate constant for the decay. The rate constant of decay was determined by linear regression consecutive points in the final linear region of the logarithmic graph of the concentration-time. For each case of treatment was calculated standard deviation () and the average coefficient of variation (CI) pharmacokinetic parameters. Calculated the ratio of average parameters (with preservative composition/composition without preservative).

Results
The results of the security
The proportion of adverse events (phenomena) was equally distributed among the treated groups (treated) (39%, rhEPO without preservative; 33%, rhEPO with preservative). It was not revealed clinically significant changes in indicators or layoutwidth inspection of the physical condition and in the main indicators of the condition of the body relative to measured before the introduction of the dose. Interestingly, the average total bilirubin decreased to almost half of the main values of the 4-th day after therapy. However, the value of this reduction was similar in both the treated groups and the average levels of total bilirubin remained within normal limits. Similar changes in other studies of liver function (alkaline phosphatase, ACT, ALT, LDH) were not observed. Thus, the security settings for the two treated groups were the same.

Pharmacokinetic results
Graphs of the average concentration of serum erythropoietin - time (not skorrelirovany with the basic levels of erythropoietin) in all 18 subjects after receiving a single dose of 150 IU/kg SC rhEPO with and without preservative is m-cresol were comparable in each reference time point (Fig. 1). All subjects had major concentration of erythropoietin within the normal physiological values (<8,6-22 Miu/ml).

Pharmacokinetic parameters were determined from serum data, skorrigirovanna middle main concentrations of erythropoietin to dose (table 2). Valuesmaxlocated in a region from a low of 85 honey/ml to a high of 558 Miu/ml (mean 198123 IU the/ml) for rhEPO without preservative. It should be noted that two subjects had unusually high values Withmax(that is the subject of 111 for rhEPO with preservative [558 honey/ml] and the subject of 118 for rhEPO without preservative [601 honey/ml]) compared with 16 other entities (i.e. <400 Miu/ml). When the value Ofmaxit was calculated without these two values, the average valuemaxfor rhEPO compositions with and without preservative were more comparable (17789 honey/ml and 196102 honey/ml, respectively).

For the composition rhEPO with preservative average erythropoietin AUC(0-72h)oscillating in the field from 2973 up 11185 honeyh/ml, was 57542284 honeyh/ml similarly, the composition of rhEPO without preservative had an average value of erythropoietin AUC(0-C)65212769 honeyh/ml, ranging in area from 3096 up 14235 honeyh/ml (table 2). It should be noted that two subjects had unusually high values of AUC(0-C)(that is the subject of 111 for rhEPO with preservative [11185 honeyh/ml] and the subject of 118 for rhEPO without preservative [14235 honeyh/ml] ) compared with the other 1 for the value AUC(0-C)For rhEPO compositions with and without preservative were more comparable (54601960 and 60002100 honey/ml, respectively).

Average tmaxit was the same for rhEPO with and without preservative (125 and 135 h, respectively). The range for tmaxwas the same for both rhEPO songs (8-24 h). The final value of the half-lives were similar for rhEPO with and without preservative (20,28,1 and 19.8of 6.6 h, respectively).

The final value of the half-life observed in both parts of the test (~20 h) were similar to values obtained after SC injection, described in the literature (11, 12). These values of half-life, however, were higher than those observed previously after centuries of introduction (~5 h) (11-14). These higher values of half-life were not unexpected, if the speed of absorption at the injection site, and not the rate of elimination (excretion) was the limiting speed factor.

Although this trial was conducted in healthy male subjects, similar characteristics of absorption and graphics security can be expected in other ass is Yu, children with renal insufficiency, patients in the program predeparture autologous blood or patients scheduled surgery with electoral tactics.

In the end, subcutaneous administration of single doses of rhEPO (150 U/ml) with or without 0.3% m-cresol as a preservative was safe and well tolerated by healthy male subjects. Based on comparable cases of adverse events, laboratory and clinical indicators main indicators of body condition data and physical examination schedules security rhEPO compositions with or without preservatives were equivalent. The proposed fractional concentration multidetermined EPREX potentially provides greater clinical benefit to the patients and suppliers of medicines.

Example 2.

Comparative safety, tolerability and pharmacokinetic properties of the two compositions recombining human erythropoietin with or without the new stabilizer after a single subcutaneous administration to healthy subjects.

Objective: to compare the safety, tolerability and pharmacokinetic properties of two concentrations (2000 [the lowest at the moment] and 40,000 IU/ml [the most high at the moment]) recombinant human Erislandy with the new stabilizer (glycine and Polysorbate 80), with commercially available compositions EPREXthat the stabilizer is used albumin human serum (HSA).

Patients and methods
Patients
Twenty-four volunteers aged 18 to 35 years (mean age 21.9 years) and weighing between 57,2-92,3 kg (average 74.3 kg) participated in the first test. The average age of the two groups of sequential processing in the first test was almost the same (21,8 and 22.0 years). Twenty-three of twenty-four subjects completed the trial. One subject completed all assessments in the first period doses, but decided not to take the second period doses. Data on the safety and tolerability of this subject, which only received rhEPO without the new stabilizer, were included in the safety data. Data the concentration of EPO in the serum of the subject, however, were not included in the pharmacokinetic analysis.

The other twenty-four volunteers aged 18 to 40 years (mean age of 23.8 years) and weighing between 62,2-for 93.4 kg (average weight 73.1 kg) were recorded and fully passed the second test. The average age of the two groups of sequential processing was almost the same (24,5 and 23,the seer 16.0 g/DL; had no clinically significant abnormal values in laboratory tests of blood or serum biochemistry; had a negative indicators of toxicity urine, HIV and surface antigens of hepatitis b antibody hepatitis C. subjects were not observed cases of hypertension (for example, measured in sitting diastolic blood pressure95 mm RT. Art.); no cases of any primary hematologic disease; no significant cases of liver, kidney, cardiovascular system, gastrointestinal system, genitourinary system, metabolic, neurological diseases; no cases of anemia or seizures. The subjects had no allergies to foods mammals or HSA; not mentioned addiction and abuse of caffeine-containing beverages; were not heavy smokers (>10 cigarettes/day), did not participate in any other clinical trial, or blood transfusion, or transplantation of blood within 90 days prior to the test; not exposed to rhEPO within three months prior to testing. All subjects were evaluated for safety and all taxes blood for pharmacokinetic analyses were performed under the test
Both tests consisted of phase 1, were testing a single centre, double-blind study, a randomized, crossover study with two periods in healthy male volunteers. In each trial, 24 subject were randomly assigned to one of two groups of sequential processing (12 subjects per group). EPO was administered in two independent period dose bolus SC injection in the upper thigh. Subjects were placed in the clinic (research centre), at least 12 hours before and were at least 36 hours after the dose in each of the two periods dose. Outside of being at the centre of research subjects came to the clinic for necessary assessments and sampling of blood. Each period, the dose was divided into 28-day washout period. Subjects were not placed in the clinic in the period between doses dose (3-29 days). The dosage is given in table 3.

This test used two songs EPO. In the first test used the composition of EPO activity 2000 IU/ml (2K) with HSA as a stabilizer and a new composition rhEPO with 2K activity in phosphate buffer containing glycine and Polysorbate 80 as the new stabilizer and the new composition rhEPO with the activity of 40K in phosphate buffer, containing glycine and Polysorbate 80 as a new stabilizer.

Collection of samples of blood
Serial blood samples were obtained by direct puncture of the vein before and after the introduction of EPO; heparin tube was not used. Samples of venous blood (5 ml) for determination of the concentration of erythropoietin serum was obtained for ~30, 20 and 10 minutes before a dose (3 main sample) and after administration of the dose in about next time: 0,5, 1, 2, 5, 8, 12, 15, 18, 24, 30, 36, 48, 60 and 72 hours (all groups). The subjects in the second test additional samples were taken blood 96, 120, 144 and 168 hours. Each serum sample was divided into two aliquots. All serum samples were stored at -20oC. the serum Samples were transported in dry ice. Laboratory and clinical methods on an empty stomach (blood count, serum biochemistry, and urinalysis) were performed immediately before the first dose on day 1, on the morning of the 4th day immediately before the dose on the 30th day and the morning of the 33d day.

Bioanalytical methods
Analyses of samples for serum levels of erythropoietin were performed at LabCorp. For determination of the concentration of erythropoietin serum used set of techniques for radioimmune assay (RIA), produced Diagnostic Systems Laboratory as a primary antibody using a polyclonal immune serum of rabbit to erythropoietin in urine of man and as the label uses125I-labeled human erythropoietin in urine. DSL set was modified by substitution of urine erythropoietin epoetin Alfa in the set of calibrators and quality control samples. The standard concentration used in the method, amounted to 7,8, 15,6, 31,3, 50, 62,5, 100 and 125 mIU/ml Sensitivity, defined as the average of the definition of the lower boundary of the standard, which gives acceptable accuracy, amounted to 7.8 Miu/ml and the range of the method was expanded to 5000 Miu/ml by dilution of samples quality control.

The definition of security
Main indicators of body condition were recorded immediately before each dose (1 and 30 days) and through 6, 24, 30, 36, 48, 60 and 72 hours after administration of each dose. Security definitions based on the study of deviations and changes in clinical laboratory testing from basic (up to 72 hours after administration of the dose) and nature of adverse events. In addition, before the test was estimated changes in the main indicators of the condition of the body, including blood pressure and the results of physical examination.

The definition of portability
Tests for evaluation of tolerability at the injection sang and danced for 45 minutes after each injection (1 and 30 days). Policy two scales assessing pain: visual analogue scale (VAS), consisting of a 10 cm horizontal line with no gradations, and verbal descriptive scale (VDS), consisting of five vertically placed cells with similar descriptions. The end points on the VAS scale was in the field from "no pain" to "worst that you can imagine and the same point on the scale VDS was in the field from "no pain" to "very severe pain". The subject was then asked about the duration of pain. If the pain lasted longer than 45 minutes, testing the endurance conducted at hourly intervals before returning to normal.

Data analysis
The concentration of serum after administration of doses were skorrelirovany with the main concentrations of erythropoietin to dose by subtracting from each value the concentration of serum after injection, the average primary concentration of erythropoietin determined by the average levels of erythropoietin from the three samples collected at approximately 30, 20 and 10 minutes prior to dose. Serum concentration of erythropoietin before the introduction of the dose is not taken into account the average value, if they were below the quantitative sensitivity analysis. Pharmacokinetic parameters were determined according to the concentration of serum korregirovannye independent methods, using WinNonlin, version 1.1 (Scientific Consulting, Inc). Identified the following pharmacokinetic parameters: peak serum concentration (Cmax); time to reach maximum serum concentration (tmax); area under the curve of concentration-time (AUC) from the start timing (zero) before the pickup time of the last blood sample AUC(0-72)[group I and II] and AUC(0-168)[group III and IV] was calculated using the formula of the linear trapezoid; the final half-life (t1/2defined as 0,693/rate constant for the decay. The rate constant of decay was determined by linear regression of successive control points in the final linear region of the logarithmic graph of the concentration-time. In linear regression used a minimum of three control points. For those who have the correlation coefficient was less than 0,975, the corresponding values of t1/2were not eritropoetinovmi. For each case of treatment was calculated standard deviation () and the average coefficient of variation (CI) pharmacokinetic parameters. Calculated the ratio of average parameters (rhEPO with a new stabilizer/rhEPO without the new stabilizer).

Statistical researched the different models included the raw data AUC and log-transformed AUC data as the dependent variable, and the effects in the sequential treatment group, subjects in groups of sequential processing, and time as the independent variable. The study of the effect of sequential treatment groups was carried out at the level of 10%, using the average size in group sequential processing of the subjects as the error vector. The period effect was tested at the 5% level, using the residual error vector. The average size of the errors in the above models were used to assess the variability of the subjects in the group. Some of the least square and the variability of the subjects in the group in the above model was used to create the 90% confidence intervals for the relationship of the average EPREXwith the new stabilizer/medium HSA-containing EPREX. Based on 40% of KI obtained in the previous study, the sample size of 24 subjects should be sufficient to estimate the ratio within the20% of the true value with a confidence of 90%. Withmax, tmaxand t1/2as raw and converted paraliterary three parameters local tolerability using a crossover methods (11). Before conducting statistical studies, many VAS and values of duration of pain was transformed into normalized. Crossover study to convert the VAS and the values of the duration of pain was performed using Proc glm in SAS. The course of the study included 28-day washout period to eliminate the possibility of variation between doses. Thus, calculations were based on standard cross-model without the effect of scatter. The results of pain, assessed VDS, were analyzed using criterion rank-sum Wilcoxon signed-Mann-Whitney, according to methods a cross-sampling, as described by Koch and others (12). Briefly, this technique involves ranking periods distinguished in all patients in the experiment, and then use the test Wilcoxon signed-Mann-Whitney on the difference between the two groups of sequences.

Results
The results of the security
Proportion of all adverse events were equally distributed among all treated groups. The fraction of events for compositions with activity 2K was 63% (rhEPO without the new stabilizer) and 61% (rhEPO with a new stabilizer. the CE adverse events were transient and were held without interference, with the exception of paracetamol, which gave five subjects at a moderate headache and moderate symptoms similar to the flu (three receiving 2K rhEPO and two under 40K receiving rhEPO). The most frequent adverse event in all groups treated with EPO, was a headache.

As possibly associated with the intake of the investigational drug adverse events were classified in subjects taking composition with rhEPO activity 40K: peeling skin (n=4), headache (n= 2), myalgia (n=1) and fatigue (n=l). The subjects taking the composition of rhEPO with 2K activity, adverse events possibly associated with receiving the investigational drug include headache (n=2) and fatigue (n= 2). All four cases of skin rash caused by the EPO, occurred between the third and eighth days after administration of rhEPO with a new stabilizer; all cases were moderate and were held for two or three days. With the exception of four reports of peeling skin no clinically significant differences in the nature and proportion of adverse events were noted in any of the two tracks when both the concentration of rhEPO. It was not oath inspection of the physical condition and in the main indicators of the condition of the body, measured during the first four days after administration of either of the two compositions studied medicine at both concentrations of rhEPO. In all treated groups on day 4 there was a slight increase in reticulocytes compared to the main one. Along with this, in all groups on day 4 there was a significant decrease in the average number of total bilirubin compared to the main one. Minor changes in indicators of biochemistry serum was observed within four days of the study; however, none were considered clinically significant. Thus, the security settings for all treated groups were the same.

The results of portability
The average VAS values after a single 150 IU/kg SC dose of rhEPO (2K) with and without the new stabilizer were not significantly different (p=0,608). Similarly, the mean VAS values after a single 750 IU/kg SC dose of rhEPO (40K) with and without the new stabilizer were not significantly different (p=0,402). When evaluating VDS severity of pain through criterion's rank-sum Wilcoxon signed the difference between these two compositions were not significantly different (p=0,610) for the subjects of groups I and II and for the subjects in groups III and IV (p=0,581).

The overall average duration of pain when all the introductions. the duration of pain, it was reported that lasted up to three minutes or less for subjects in groups I or II and prior to two minutes or less for subjects in groups III and IV. Results duration of pain were consistent with results obtained for other parameters portability. There was not a statistically significant difference in the duration of pain after injection in subjects treated with EPREXwith and without the new stabilizer, in groups I and II (R=strength of 0.159) and groups III and IV (p= 0,951).

Comparison of pain at two different concentrations epoetin alpha showed that pain was observed after all the introductions investigational medicinal product (2K rhEPO) in groups I and II. In contrast, in groups III and IV subjects (40K rhEPO) there was no pain in 24-48 hours after SC injection (50%). In addition, the mean values of pain (VAS, VDS) were greater after a single 150 IU/kg dose of rhEPO (2K) than after a single 750 U/kg dose of rhEPO (40K). However, there was a higher proportion of cases of pain, as well as the high average value of pain after a single 150 IU/kg dose of rhEPO (2K) than after a single 750 U/kg dose of rhEPO (40K). This may be due to the fact that to achieve the required dose in groups I and II was necessary Bo is oppozitsii (2K) than those used in groups III and IV (40K). It should also be noted that the needle used in both introductions to the subjects of group I and II, was thinner and more flexible than the subjects of group III and IV, which made more difficult the conduct of SC injection. Finally, the average duration of pain was longer in groups III and IV (40K) than in groups I and II (2K), but it was probably due to two subjects treated with 40K rhEPO, which saw the long-term pain after both treatments, compared with all subjects in all treated groups.

Pharmacokinetic results
Graphs of the average concentration of serum erythropoietin - time all subjects who received either a single 150 IU/kg SC dose of rhEPO (groups I and II) or single 750 U/kg p. the dose of rhEPO (groups III and IV) with or without the new stabilizer shown in Fig.2 and 3, respectively (not skorrelirovany with the basic levels of erythropoietin). All subjects (n=48) before the introduction of the dose had major concentration of erythropoietin within the normal physiological values (<7,8-32 Miu/ml). 4 days after administration of a single 150 IU/kg SC dose of rhEPO (2K) with or without the new stabilizer mean levels of serum erythropoietin (3611 and 33 - 11 Miu/ml soo is 6 and 167 honey/ml, respectively). Similarly, through 7 days after administration of a single 750 IU/kg SC dose of rhEPO (40K) with or without the new stabilizer mean levels of serum erythropoietin (269 and 237, respectively) also reached average levels of endogenous erythropoietin before the introduction of the doses.

Pharmacokinetic parameters were determined from serum data, skorrigirovanna middle main concentrations of erythropoietin to dose. In groups I and II values Withmaxwas in the area from low 86,6 honey/ml to a high 681 Miu/ml (mean value 222128 Miu/ml) after administration of a single 150 IU/kg SC dose of rhEPO (2K) with the new stabilizer and from 119 honey/ml to a high of 377 Miu/ml (mean 22679 honey/ml) for rhEPO without the new stabilizer (table 4). One subject who received a single 150 IU/kg dose of rhEPO (2K) with the new stabilizer, had an unusually high valuemaxand one entity of the same group had an unusually low value ofmax. The reasons for these unusual valuesmaxnot known. In groups III and IV values Withmaxwas in the area from low 1194 honey/ml to a high 4334 Miu/ml (mean value from 1892 honey/ml to a high 3997 Miu/ml (mean 3065705 honey/ml) for rhEPO without the new stabilizer (table 5). We detected no statistically significant differences in valuesmax(p<0.05) in the compositions of rhEPO both activities and 2K 40K with or without the new stabilizer.

In groups I and II, the values of AUC(0-72)was in the area from low 3238 to high 11318 (average 66472488 honeyh/ml) after administration of a single 150 IU/kg SC dose of rhEPO (2K) with the new stabilizer. Similarly, the values of AUC(0-72)was in the area from low 4234 to high 10968 (average 69831855 honeyh/ml) after administration of a single 150 IU/kg SC dose of rhEPO (2K) without the new stabilizer (table 4). In groups III and IV AUC(0-168)was in low 48347 to high 136290 (average 10276821500 honeyh/ml) after administration of a single 750 IU/kg SC dose of rhEPO (40K) with a new stabilizer. Similarly, the values of AUC(0-168)was in the area from low 69537 to high 136689 (average 10489715781 honeyh/ml) after administration of a single 750 IU/kg SC dose of rhEPO (40K) without the new stabilizer (table 5). It should be noted that the K) without the new stabilizer.

The average value of tmaxwas similar after administration of a single 150 IU/kg SC dose of rhEPO (2K) with and without the new stabilizer (17,47,3 and 15.4of 7.5 h, respectively) (table 4). Like the average valuemaxafter the introduction of a single 750 IU/kg SC dose of rhEPO (40K) with and without the new stabilizer was 16,76,8 and 16.64,8 h, respectively (table 5). The values of tmaxwas in the field from 8-30 hours for both 2K compositions rhEPO. For concentrations of 40,000 U/ml rhEPO tmaxwas in the area 8-36 hours for songs with a new stabilizer and 12-30 hours without the new stabilizer.

Although it was reported only a small percentage of the subjects of groups I and II, with the final value of the half-life values were similar after administration of a single 150 IU/kg SC dose of rhEPO (2K) with and without the new stabilizer (19,712.8 and 20,110,4 h, respectively). After the introduction of a single 750 IU/kg SC dose of rhEPO (40K) with or without the new stabilizer in subjects in groups III and IV values of half-life were similar (25,714,9 and 23.811,8 h, respectively).

EPREX. Different activity of EPO (that is, 2K, 4K, 10K and 40K) with a new stabilizer can be used interchangeably with all the various activities of the HSA-containing EPO (that is, 2K, 4K, 10K and 40K).

Example 3.

The study of pharmacokinetic properties, safety and tolerability of two compositions recombining human erythropoietin after single subcutaneous administration to healthy subjects.

Objective: to compare the pharmacokinetic properties, tolerability and safety of subcutaneous administration of a single dose of two compositions of recombinant human erythropoietin (epoetin Alfa, rhEPO) in phosphate or citrate buffer and to assess the value of the buffer component in the induction of pain.

Patients and methods
Patients
Eighteen healthy volunteers in the age from 18 to 40 years (mean age 27 years), and weight between 62,6-and 82.2 kg (average weight 70.1 kg) were included in the test and completely passed this test. Included in the test subjects had no clinically Stateline indicators for toxicity urine, HIV and surface antigens of hepatitis C. The subjects were not observed cases of hypertension (ie, diastolic blood pressure > 95 mm RT.CT.); no cases of any primary hematologic disease; there were no cases of significant liver disease, kidney, cardiovascular system, gastrointestinal system, genitourinary system, metabolic, neurological diseases; no cases of anemia or seizures; known sensitivity to products mammal or human serum albumin; abuse of alcohol or drugs in the past two years; subjects did not participate in any other clinical trial, or blood transfusion, or in donated blood within 30 days prior to testing; the subjects were not exposed to rhEPO within three months prior to testing; subjects had no significant deviations of the physical data to test or clinical laboratory data for 14 days before testing. All subjects were evaluated for safety, and all fences blood for pharmacokinetic analyses were performed in accordance with the schedule of tests. All tests were performed with the consent of the Committee itoe, a randomized, crossover study with two periods in healthy male volunteers. Eighteen subjects were randomly assigned to one of two groups of sequential processing (nine subjects/group). rhEPO was administered in two independent period dose bolus SC injection in the upper thigh. Each period, the dose was divided into 14-day washout period. The subjects were placed in the clinic for at least 12 hours before and was up to 72 hours after administration of the dose in each of the two periods dose, but not between periods. The dosage is given in table 6.

This test used two songs EPO. Epoetin Alfa (commercially available as EPREX10000 U/ml) were prepared in citrate buffer and the new composition rhEPO (10000 U/ml) was prepared in phosphate buffer. Both compositions were prepared in the Ortho Biologies Division of Ortho McNeil Janssen Pharmaceuticals, Inc. (Manati, Puerto Rico).

Collection of samples of blood
Serial blood samples were obtained by direct puncture of the vein before and after the introduction of EPO; heparin tube was not used. Samples of venous blood (5 ml) for determination of the concentration of erythroped siteline the following times: 30 minutes and 1, 2, 5, 8, 12, 15, 18, 24, 48 and 72 hours. Each serum sample was divided into two aliquots. All serum samples were stored at -20oC.

Serum samples were transported in dry ice. Laboratory and clinical methods on an empty stomach (blood count, serum biochemistry, and urinalysis) were performed immediately before the first dose on day 1, on the morning of the 2nd day, immediately before the dose on the 16th day and the morning of the 17th day.

Bioanalytical methods
Analyses of samples for serum levels of erythropoietin were performed at RWJPRI. For determination of the concentration of erythropoietin serum used the methodology set for radioimmune assay (RIA) (Diagnostic Systems Laboratory [DSL], Webster, TX). Commercially available RIA is a method of double antibody competitive method, which as a primary antibody using a polyclonal immune serum of rabbit to erythropoietin in urine of man and as the label uses125I-labeled human erythropoietin in urine. DSL set was modified by substitution of urine erythropoietin epoetin Alfa in the set of calibrators and quality control samples. The standard concentration used in the method, amounted to 7,8, 15,6, 31,25, 50, 62,5, 100 and 125 mIU/ml Sensitivity, defined as the average of the expanded to 2000 Miu/ml by dilution of samples quality control.

The definition of security
Main indicators of body condition were recorded immediately before each dose (1 and day 16) and after 6, 24, 48 and 72 hours after dose. Security definitions based on the study of deviations and changes in clinical laboratory tests from the main and nature of adverse events. In addition, before the test was estimated changes in the main indicators of the condition of the body, including blood pressure and the results of physical examination.

The definition of portability
Tests for evaluation of tolerability at the injection sang and danced for 45 minutes after each injection on the day of introduction. There were two scales assessing pain: visual analogue scale (VAS) consisting of a 10 cm horizontal line without a tonal and verbal descriptive scale (VDS), consisting of five vertically placed cells with similar descriptions. The end points on the VAS scale was in the field from "no pain" to "worst that you can imagine and the same point on the school VDS was in the field from "no pain" to "very severe pain". In addition, the subject was asked about the duration of the pain.

Data analysis
The concentration of serum after administration of dose is achene concentrations of serum after injection, the average primary concentration of erythropoietin, determined by the average levels of erythropoietin from the three samples collected at 30, 20 and 10 minutes prior to dose. Serum concentration of erythropoietin before the introduction of the dose is not taken into account the average value, if they were below the quantitative sensitivity analysis. Pharmacokinetic parameters were determined according to the concentration of serum korrelirovannym with the main concentration of erythropoietin.

Pharmacokinetic parameters were calculated using independent methods for digital equipment computer operating system VAX 8600, using BIOAVL, version 8.0 a (Scientific Computer Systems, RWJPRI). Identified the following pharmacokinetic parameters: peak serum concentration (Cmax); time to reach maximum serum concentration (tmax); area under the curve of concentration-time (AUC) from the start timing (zero) before the pickup time of the last blood sample (AUC0-72) was calculated using the formula of the linear trapezoid; the final half-life (t1/2) was defined as 0,693/rate constant for the decay. The rate constant of decay was determined by linear regression of successive control points in the final linear region Logar Prov., aka regressions with coefficients in formula (r2)<0.95, and the corresponding values of t1/2was not reported. For each case of treatment was calculated standard deviation () and the average coefficient of variation (CI) pharmacokinetic parameters. Calculated the ratio of average pharmacokinetic parameters (phosphate/citrate).

Statistical analyses were performed on raw and log-transformed parameters of bioavailability. In the study of various models included data from one of the interested parameters of bioavailability (AUC, Cmaxand the raw and log-transformed) as the dependent variable and the effects in the sequential treatment group, subjects in groups of sequential processing, and time as the independent variable. The study of the effect of sequential processing group performed at the level of 10%, using the average size in group sequential processing of the subjects as the error vector. The period effect was tested at the 5% level, using the residual error vector. The average size of the errors in the above models were used to assess the variability of the subjects in the group. Some of the least square Ermolov for relations medium settings bioavailability of rhEPO with phosphate buffer to rhEPO with citrate buffer.

Statistical studies were conducted on three dimensions of sustainability. Before conducting statistical studies many VAS and values of duration of pain was transformed into normalized. All three parameters were analyzed using mean values of cross-sectional studies. Crossover study to convert the VAS and the values of the duration of pain was performed using Proc glm in SAS. The investigation included a 14-day washout period to eliminate the possibility of variation between doses. Thus, calculations were based on standard cross-model without the effect of scatter. The results of pain, assessed VDS, were analyzed using criterion rank-sum Wilcoxon signed-Mann-Whitney, according to methods a cross-sampling, as described by Koch and others. Briefly, this technique involves ranking periods distinguished in all patients in the experiment, and then use the test Wilcoxon signed-Mann-Whitney on the difference between the two groups of sequences.

Results
The results of the security
In both periods dose observed the position with citrate buffer and four AEs were observed in four subjects (22%) after administration of the composition of a medicinal product with phosphate buffer. Two reported AEs case after the injection of the composition with citrate buffer was affected the system of respiratory organs (that is, pharyngitis and sinusitis). Four reported AEs case after the injection of the composition with phosphate buffer included: muscle spasms, headache, leg pain and dermatitis. All groups of AEs cases were classified as moderate and short-term nature.

The decrease in concentrations of total bilirubin was observed in all subjects after both injections of both compounds. Average total bilirubin decreased by almost half from the basic values before the introduction of the dose within two days of observation. However, the magnitude of this reduction was similar in both the treated groups and the average levels of total bilirubin remained within the normal range. This reduction seemed to be short-term, in view of the fact that two basic levels to dose (period of 16 days) were very comparable. Similar changes in other studies of liver function (alkaline phosphatase, ACT, ALT, LDH) were noted.

Except for the unexpected decrease in total serum bilirubin, General physical examination, clinical laboratory tests, blood pressure, and basic indicators of the state of the first drug. Thus, the security settings for the two treated groups were the same.

The results of portability
Two patients receiving composition with citrate buffer, were omitted from the record of pain during each period of introduction. The average VAS for all 18 phosphate injection was 21,527,5 mm, compared to 34.328,6 mm for all 16 citrate injection (p=0,0692). Taking into account that the pain after the phosphate solution is not dependent on the period of injection, pain after citrate composition was more intense during the period I than in period II (39,331,7 mm against 29.426,3 mm).

The difference between the two formulations in VDS portability was statistically significant (p= 0,0339). Thirteen subjects (72%) either did not feel pain, or felt moderate pain and five subjects (28%) had moderate/intense/very intense pain after administration of the composition of the phosphate buffer. Appropriate follow-up after introduction of the composition with citrate buffer were made in 8 (44%) and 10 (56%) subjects, respectively. In addition, VDS pain was perceived as slightly more intense when posledovatelnogo with citrate buffer (1,52.0 minutes) than after composition with phosphate buffer (0,40.5 minutes). Taking into account that the differences were already evident during the period I (the average value of 0.8 vs. 0.3 minutes), it becomes evident during period II (average of 2.2 versus 0.5 minutes), that is, when the citrate composition is administered to the subject, which was already introduced the song with a phosphate solution. It should be noted that both the composition of the medicinal product was not registered during the period I. In this test, it was noted that the standard EPREXcomposition (with citrate) causes more significant increased pain at the injection site than when the composition is used as the phosphate buffer. Painful sensation caused by citrate was more intense when the tests included the sequence of the citrate/phosphate in contrast to the phosphate/citrate sequence, demonstrating that the main event experience can change the perception of pain. Moreover, with regard to the duration of pain, the difference between the two songs was very significantly (p= 0,0057), however, in this sequence caused citrate buffer pain was perceived much on the acidity of erythropoietin serum - time (not skorrelirovany with the basic levels of erythropoietin) in all 18 subjects after receiving a single dose of 150 IU/kg SC rhEPO with citrate or phosphate buffer similar (Fig. 4). All subjects had major concentration of erythropoietin to a dose in the range of normal physiological values (<8,6-18 Miu/ml).

Pharmacokinetic parameters were determined from serum data, skorrigirovanna middle main concentrations of erythropoietin to dose (table 7). Valuesmaxlocated in a region from a low of 74 honey/ml to a high 583 Miu/ml (mean 260169 honey/ml) for rhEPO with citrate buffer and 64 honey/ml to a high 642 Miu/ml (mean 245167 honey/ml) for rhEPO with phosphate buffer. Several of the subjects in each group had an unusually high values Withmax. There was not a statistically significant difference (p>0.05) in the values Withmaxthe two compositions. For the composition of EPO with citrate buffer average erythropoietin AUC(0-72)was 7,99213,319 honeyh/ml, ranging from 3,215 to 13,077 honeyh/ml Similar to the composition of rhEPO with phosphate buffer had sreelaj from 3,445 to 17,996 honeyoh/ml (table 7). There were statistically significant differences (p>0.05) in AUC(0-72)these two compositions. Average tmaxit was the same for rhEPO with citrate phosphate buffer (145 and 1710 h, respectively). The values of tmaxwere similar for both compositions rhEPO (8-24 hours).

The average final value of the half-lives were similar for rhEPO with citrate phosphate buffer (13,410,8 and 19.714,9 h, respectively).

References
1. Koury ST, Bondurat MC. Koury MJ. (1988). Localization of erythropoietin synthesizing cells in murine kidneys by in situ hybridization. Blood 71:524-527.

2. Jacobs K, Shoemaker, Rudersdorf R, Neill SD, Kaufman RJ, A. Mufson et al. (1985). Isolation and characterization of genomic and cDNA clones of human erythropoietin. Nature 313:806-810.

3. Lin FK, Suggs S, Lin Ck, Browne JK, Smalling R, Egrie JC et al. (1985). Cloning and expression of the human erythropoietin gene. Proc Nati Acad Sci USA 82:7580-7584.

4. Jelkmann W. (1992). Erythropoietin: structure, control of production, and function. Physiol Rev 72:449-489.

5. Egric JC, Browne JK, Lai P, Lin FK (1986). Characterization and biological effects of recombinant human erythropoietin. Immunobiology 172: 213-224.

6. Faulds D, Sorkin EM (1989). Epoetin (Recombinant Human Erythropoietin): A review of its pharmacodynamic and pharmacokinetic properties and therapeutic potential in anemia and the stimulation of erythropoiesis, Drugs 38:863-899.

7. Markham A, Bryson HM (1995). Epoetin alfa: A review of its pharmacodynamic and pharmacokinetic properties and therapeutic use in nonrenal applications Drugs 49:2eers. Brit J Heamatol 92:295-301.

9. Granolleras With, Leskopf W, Shaldon S, Fourcade J (1991). Experience of pain after subcutaneous administration of different preparations of recombinant human erythropoietin: a randomized, double-blind crossover study. Clin Nephrol 36:294-298.

10. Frenken LA, Van Lier HJ, Jordans JG et al. (1993). Identification of the component part in an epoetin alfa preparation that causes pain after subcutaneous inject. Am J Kidney Dis 22:553-556.

11. Halstenson CE, Macres M, Katz SA et al. (1991). Comparative pharmacokinetics and pharmacodynamics of epoetin alfa and epoetin beta. Clin Pharmacol Ther 50:702-712.

12. Ateshkadi A, Johnson CA, Oxton LL, Hammond TG, Bohenek WS, Zimmerman SW (1993). Pharmacokinetics of intraperitoneal, intravenous, and subcutaneous recombinant human erythropoietin in patients on continuous ambulatory peritoneal dialysis. Am J Kidney Dis 21:635-642.

13. McMahon FG, Vargas R, Ryan M, et al. (1990). Pharmacokinetics and effects of recombinant human erythropoietin after intravenous and subcutaneous injections in healthy volunteers. Blood 76:1718-1722.

14. Salmonson T, Danielson BG, Wikstrom B. (1990). The pharmacokinetics of recombinant human erythropoietin after intravenous and subcutaneous administration to healthy subjects. Brit J clin Pharmacol 29:709-713.


Claims

1. The pharmaceutical composition of erythropoietin, comprising (a) a buffering agent; (b) a stabilizing amount of a derivative of sorbitan mono-9-octadecenoate poly(oxy-1,2-ethandiyl); (c) a stabilizing amount of amino acids; (d) pharmaceutical amount of erythropoietin; and does not contain urea or the product of human serum.

2. The composition according to p. 1, where the composition is water.

3. Com. the song under item 1, where the buffer agent for regulating the pH provides a range of pH from about 5 to about 8, the range of pH from about 6 to about 7.5, or pH of about 6.9.

5. The composition according to p. 1, where the buffer agent for regulating the pH selected from the group comprising monobasic sodium phosphate/dibasic sodium phosphate, sodium citrate/citric acid and sodium acetate/acetic acid.

6. The composition according to p. 1, where the derivative of sorbitan mono-9-octadecenoate poly(oxy-1,2-ethandiyl) selected from the group comprising Polysorbate 80 and Polysorbate 20.

7. The composition according to p. 1, where the amino acid is a glycine.

8. The composition according to p. 1, further comprising an osmotic agent, where the osmotic agent is selected from the group comprising sodium chloride, mannitol, glycine, glucose and sorbitol.

9. The composition according to p. 1 where the pharmaceutical amount of erythropoietin is therefore to provide a quantity per dose in the range of from about 1000 IU to about 100000 ME erythropoietin, or the number on the dose selected from the group comprising about 2000 ME, ME about 3000, about 4000 ME, ME about 10,000, about 20,000, ME, ME or about 25,000 to about 40,000 to ME.

10. The composition according to p. 9, where the derived sorbitan mono-9-octadecenoate poly(oxy-1,2-ethandiyl) is a Polysorbate 80, and AMI the Les from about 0.01 g/l to about 1.0 g/l, and the number of glycine is in the range from about 1 g/l to about 50 g/L.

12. The pharmaceutical composition of erythropoietin on p. 1, comprising (a) a buffering agent for pH regulation; (b) a stabilizing amount of a derivative of sorbitan mono-9-octadecenoate poly(oxy-1,2-ethandiyl); (c) a stabilizing amount of amino acids; (d) number of pharmaceutical erythropoietin; (e) the osmotic agent; and does not contain urea or the product of human blood.

13. The composition according to p. 12, where the composition is water.

14. The composition according to p. 12, where the amount of the buffer agent for regulating the pH is in the range from about 10 to 30 mm.

15. The composition according to p. 12, where a buffering agent for pH regulation provides a range of pH from about 5 to about 8, the range of pH from about 6 to about 7.5, or pH of about 6.9.

16. The composition according to p. 12, where the buffer agent for regulating the pH selected from the group comprising monobasic sodium phosphate/dibasic sodium phosphate, sodium citrate/citric acid and sodium acetate/acetic acid.

17. The composition according to p. 12, where the derived sorbitan mono-9-octadecenoate poly(oxy-1,2-ethandiyl) selected from the group comprising Polysorbate 80 and Polysorbate 20.

18. The composition according to p. 12, where the amino acid is own glycine, glucose and sorbitol.

20. The composition according to p. 12, where the number of pharmaceutical erythropoietin is therefore to provide a quantity per dose in the range of from about 1000 IU to about 100000 ME erythropoietin, or the number on the dose selected from the group comprising about 2000 IU ME about 3000, about 4000 ME, ME about 10,000, about 20,000, ME, ME or about 25,000 to about 40,000 to ME.

21. The composition according to p. 20, where the derived sorbitan mono-9-octadecenoate poly(oxy-1,2-ethandiyl) is a Polysorbate 80, and the amino acid is a glycine.

22. The composition according to p. 21, where the number of Polysorbate is in the range from about 0.01 g/l to about 1.0 g/l, and the number of glycine is in the range from about 1 g/l to about 50 g/L.

23. The pharmaceutical composition of erythropoietin on p. 1, selected from the group comprising (a) about 2000 ME erythropoietin, about to 4.38 mg of sodium chloride, of 1.16 mg monobasic dihydrate phosphate, 2,23 mg dihydrate dibasic phosphate, about 5.00 mg glycine, about 0.30 mg of Polysorbate 80 and brought to 1.0 ml water; (b) about 4000 ME erythropoietin, about to 4.38 mg of sodium chloride, of 1.16 mg monobasic dihydrate phosphate, 2,23 mg dihydrate dibasic phosphate, about 5.00 mg glycine, about 0.30 mg of Polysorbate 80 and brought to 1.0 is I, of 2.23 mg dihydrate dibasic phosphate, about 5.00 mg glycine, about 0.30 mg of Polysorbate 80 and brought to 1.0 ml with water; (d) about 40000 ME erythropoietin, about to 4.38 mg of sodium chloride, of 1.16 mg monobasic dihydrate phosphate, 2,23 mg dihydrate dibasic phosphate, about 5.00 mg glycine, about 0.30 mg of Polysorbate 80 and brought to 1.0 ml with water.

24. The pharmaceutical composition of erythropoietin, comprising (a) a buffering agent for pH regulation; (b) a stabilizing amount of serum albumin person; (c) a stabilizing amount of amino acids; (d) an antimicrobial amount of the cresol; (e) pharmaceutical amount of erythropoietin.

25. The composition according to p. 1, where the amount of the buffer agent for regulating the pH is in the range from about 10 to 30 mm.

26. The composition according to p. 25, where a buffering agent for pH regulation provides a range of pH from about 5 to about 8, the range of pH from about 6 to about 7.5, or pH of about 6.9.

27. The composition according to p. 24, where the buffer agent for regulating the pH selected from the group comprising monobasic sodium phosphate/dibasic sodium phosphate, sodium citrate/citric acid and sodium acetate/acetic acid.

28. The composition according to p. 24, where the preservative cresol is a met is the amount of the preservative is m-cresol is about 0.3%.

30. The composition according to p. 24, where the pharmaceutical amount of erythropoietin is therefore to provide a quantity per dose in the range of from about 1000 IU to about 100000 ME erythropoietin.

31. The composition according to p. 30 provided in mnogochasovykh bottles, with a dose selected from the group including about 10,000 IU/2.5 ml, about 25,000 IU/ml, or about 40,000 IU/2 ml

32. The composition according to p. 31, where a stabilizing amount of albumin is about 0.25 g/L.

33. The composition according to p. 24, where the amino acid is a glycine.

34. The composition according to p. 33, where the amino acid is a glycine and is in the range from about 0.25 g/l to about 20 g/L.

35. The composition according to p. 34, where the number of glycine is about 5 g/l and where a stabilizing amount of albumin is about 0.25 g/L.

36. The composition according to p. 24, where the composition is water.

37. The pharmaceutical composition of erythropoietin on p. 24, comprising (a) a buffering agent for pH regulation; (b) a stabilizing amount of serum albumin person; (c) a stabilizing amount of amino acids; (d) an antimicrobial amount of the cresol; (e) number of pharmaceutical erythropoietin; (f) the osmotic agent.

38. The composition according to p. 37, where the amount of the buffer agent regulating the pH provides a range of pH from about 5 to about 8, the range of pH from about 6 to about 7.5, or pH of about 6.9.

40. The composition according to p. 37, where the buffer agent for regulating the pH selected from the group comprising monobasic sodium phosphate/dibasic sodium phosphate, sodium citrate/citric acid and sodium acetate/acetic acid.

41. The composition according to p. 37, where the preservative cresol is a meta-cresol, and where the number of m-cresol is in the range from about 0.2% to about 0.5%.

42. The composition according to p. 41, where the amount of the preservative is m-cresol is about 0.3%.

43. The composition according to p. 41, where the pharmaceutical amount of erythropoietin is therefore to provide a quantity per dose in the range of from about 1000 IU to about 100000 ME erythropoietin.

44. The composition according to p. 43 provided in mnogochasovykh bottles, with a dose selected from the group including about 10,000 IU/2.5 ml, about 25,000 IU/ml, or about 40,000 IU/2 ml

45. The composition according to p. 37, where a stabilizing amount of albumin is about 0.25 g/L.

46. The composition according to p. 37, where the amino acid is a glycine.

47. The composition according to p. 46, where a stabilizing amount of glycine is in the range from about 5 g/l to about 50 g/L.

48. The composition according to p. 47, where the amino acid is a glycine and obespechivayushchaya sodium chloride, mannitol, glycine, glucose and sorbitol.

50. The composition according to p. 49, where the number of glycine is about 5 g/l and where a stabilizing amount of albumin is about 0.25 g/L.

51. The composition according to p. 37, where this composition is water.

52. The pharmaceutical composition of erythropoietin on p. 24, selected from the group comprising (a) about 10000 ME erythropoietin, about 6.25 mg of serum albumin human, 2,91 mg monobasic dihydrate phosphate, 11,19 mg dodecahydrate dibasic phosphate, about 50,00 mg glycine, about 7,50 mg m-cresol, brought to 2.5 ml water; (b) about 25000 ME erythropoietin, about 6.25 mg of serum albumin human, 2,91 mg monobasic dihydrate phosphate, 11,19 mg dodecahydrate dibasic phosphate, about 50,00 mg glycine, about 7,50 mg m-cresol, brought to 2.5 ml with water; (C) about 40000 ME erythropoietin, about 5.00 mg albumin human, 2,33 mg monobasic dihydrate phosphate, 8,95 mg dodecahydrate dibasic phosphate, about 40,00 mg glycine, about 6,00 mg m-cresol, brought to 2.0 ml with water.

 

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