MXPA02000804A - Multi dose erythropoietin formulations - Google Patents

Abstract

The present invention is directed to multi dose erythropoietin formulations for parenteral administrations. The formulations contain one or more of the following preservatives benzethonium chloride, phenoxyethanol and phenylethyl alcohol.

Description

FORMULATIONS OF ERITROPOYETIN IN MULTIPLE DOSE ANTEC EDENTl IS OF I NVE NTION I. Field of the invention The present invention pertains to formulations of multiple doses of erythropoietin (hereinafter "EPO"), which comprises a particular preservative see tajoso or combination of preservatives. Specifically, the present invention pertains to the use of the preservatives benzethonium chloride, phenoxyethanol and ethyl alcohol, alone or in combination, in multi-dose EPO formulations. The invention described in the present invention also relates to a method for treating a patient with an EPO-containing composition comprising benzethonium chloride, phenoxyethyl-and / or ethyl ethyl alcohol. The present invention also relates to a pharmaceutical carrier composition, a bottle for containing a composition; and a method for inhibiting microbial growth in a solution; wherein all compositions or solutions comprise EPO and one or more of the preservatives benzethonium chloride, phenoxyethanol and phenylethyl alcohol.

I I. Background of the invention Sterility is one of the most important characteristics of parenteral products. For parenteral products that are sterilized and intended for injection ie a single dose, maintenance of sterility is a function of both the sterilization method and the integrity of the packaging system. For parenteral products that Intended for multiple dosing, antimicrobial agents must be added to the product formulation to protect the product from accidental microbial contamination during storage and / or use.

Stable, protein-containing, multi-dose pharmaceutical formulations are seen by the pharmaceutical industry as particularly advantageous and commercially attractive. Multi-dose formulations are generally, but not always, contained in vials (multi-dose recipients) that allow the extraction of partial amounts of formulation at various times. This type of system is desirable, since it allows obtaining multiple doses from a single container, and allows a more controlled administration of the pharmaceutical composition, since the formulation can be withdrawn and admired in any partial quantity. The nature of the use of multi-dose formulations imposes special requirements on formulation. For example, maintaining the sterility id of the composition is particularly challenging, given the many opportunities for the introduction of microorganisms and other ingredients in the formulations. The repeated introduction of foreign elements, for example, needles, into the recession of the ductile membrane after the formulation, creates a possibility of introducing micro-organisms into the container. Additionally and alternatively, microorganisms can be introduced during the filling of the containers, or during the reconstitution of the formulations after lyophilization and before the administration. The extended periods over which the container can be stored - especially during multiple introductions of foreign elements, and / or after contaminants may have been introduced, demand that the formulation contain special additives to ensure the sterility of the contents. To ensure that multi-dose formulations maintain optimally stable properties, the United States Food and Drug Administration (FDA) and regulatory agencies in other jurisdictions require that all multi-dose formulations contain preservatives to prevent the growth of, or to kill in the affirmative, any micro-organism that can be introduced into the formulations. Given the inherent lack of ID of the proteins, and their tendency to interact adversely with conservative compounds, the development of multi-dose formulations containing protein has proved particularly difficult. Potential adverse interactions between preservatives and proteins include degradation of the protein, especially when stored for prolonged periods; inactivation of the protein; formation of protein aggregates; and other interaction is that they inactivate the formulation or make it difficult, painful or otherwise undesirable to administer the formulation to humans, particularly by infusion, injection or other parenteral administration. Additionally, the preservatives themselves are noted for causing acute adverse reactions, such as allergic reactions, in humans on parenteral administration. Ideally, the preservative contained in the pharmaceutical protein composition of multiple doses, it should be effective in low concentration against a wide variety of microorganisms, soluble in the formulation, non-toxic, compatible and non-reactive with the protein, active with long-term stability and non-reactive with compose ites of the container or system of closing. Sandeep Nema et al. We publish lists of several excipients that have been included in the formulation of injectable products marketed in the United States. The antimicrobial preservatives listed in this review article are included in Table 1: Table 1 EPO is a glycoprotein that works to stimulate the production of hemoglobin and erythrocytes in the bone marrow. It is produced in the kidney and is widely used as a treatment for anemia caused by a variety of conditions, including, for example, renal failure. The sequence of amino acids and patches of general glycosylation of EPO are known in the art. See, for example, Miyaka et al. and the US patent no. 4,703.008. The isolation and purification of EPO, from human tissues or fluids, has been described by Miyake et al. The nucleic acid sequence encoding the protein, the isolation of this sequence / the production of the protein by traditional recombination methods are also known in the art. See, for example, US Patent no. 4,703,008 to Lin, which describes the nucleic acid sequence encoding EPO; U.S. Patent No. 4,337,513 to Sugimoto et al., which describes the use of lymphoblastoid cells to produce EPO; and Sherwood et al., which describes the production of EPO by a line of human renal cell carcinoma cells. Additionally, the production, isolation and purification of the protein is also achievable by gene-activation, or homologous recombination, followed by isolation techniques. and well-known purification. The development of multi-dose formulations containing EPO has proved particularly difficult because of the particular instability of EPO, and its tendency to interact easily with common pharmaceutical ingredients. US patent no 4,806,524. Attempts have been made to develop multi-dose EPO formulations to circumvent these problems by maintaining formulations at a low pH, or by including several amino acid constructs, two approaches designed to aid in the stabilization of protein PO, or the development of freeze-dried forms in which the preservative is sublimated from the formulation prior to administration . US Patent no. 5, 503, 827 (the '827 patent) for Woog. There are few pharmaceutical formulations containing EPO of multiple doses, sterile, stable. They describe the forms described in the '827 patent. The reference '827 specifically describes and claims chloretone (chlorbutanol, 1,1,1-trichloro-2-methyl-2-propanol), benzalkonium chloride or benzyl alcohol as preservatives. Woog specifically notes the particular difficulty in providing a multi-dose EPO formulation, in which allergy ratios are reduced, and the use of specifically claimed preservatives is especially advantageous in this regard. This reference additionally points out that due to the tendency of conservatives to de-radar and proteins to be inactivated when combined, it is more desirable to mimic the contact between the preservative and the protein. The '827 patent further discloses the use of various amino acid constructs and other additives thought necessary to stabilize EPO in solution. Finally, the '827 patent discloses, in a more preferred embodiment, that any preservative used in the initial formulation is sublimated by lyophilization of the composition. Then, upon reconstitution, the preservative adds the selected g of the described group (chloretone) as defined, benzalkonium chloride and Benzyl alcohol may be introduced, but the reconstituted, injectable solution should be used within 30 days. Another example of a multi-dose formulation containing EPO, is described in the US patent no. 5,661,125 (the patent '125). This patent explicitly recognizes and affirms other references stating that EPO "is an unstable substance especially in the form of a solution" and "when used with known stabilizers, the resulting stability of E PO is varied and unpredictable". This reference continues to show then and claiming the specific use of the benzyl alcohol, a paraben and / or a phenol or a combination of these as a preservative in solutions containing EPO. Further demonstrating the difficulty of advantageous and compatible, judicious conservatives for use in multi-dose formulations containing EPO, this reference declares: "... nothing specific can be derived from the use of preservatives with other proteins that their formulation would conserve. particular for erythropoietin tubing, See, for example, Geigert, J., 'Overview of the Stability and Handling of Recombinant Protein Drugs', Journal of Parenteral Science &Technology, Vol. .43 (5): 220-224 (1989) ". Accordingly, today there is a need for a conserved, multi-dose EPO containing pharmaceutical formulation that: (1) maintains the stability of the protein component and the composition over a shelf life of the product; (2) keep the sterility of the formulation and meet the criteria of American, European and Japanese pharmacopoeia to prove a conservative challenge: (3) be safe in the concentrations used; and (4) is administrable - by any parenter-il or oral route - in a manner that is effective and minimizes pain and the likelihood of adverse reaction, eg, allergic reaction, in the patient. lll. BRIEF DESCRIPTION OF THE INVENTION The present invention provides a novel, and particularly advantageous, multidose formulation containing erythropoietin and the preservatives chloride benzethonium, phenoxyethanol and phenylethyl alcohol, either alone or in combination. The formulations of the present invention can be formulated in a variety of concentrations in various bottle sizes for various dosages of administration. For example, the formulations described in the present invention may comprise concentrations 10,000, 20,000, 40,000 or even up to or more than 100,000 Units. / ml of EPO. They may also contain any concentration between these exemplary concentrations, such as concentrations of 5,000, 15,000, 25,000 units / ml and the like. Additionally, the dosages may be formulated in a Vz, 1 or 2 ml bottle, or any other bottle size or other container preferred by the formulator. It will be clear to one of skill in the art that any combination of dosages and flasks can be used, depending on the formulator's needs. For example, one could prepare the formulations described in FIG. present as a concentration of 1000 units / ml in a 1 ml bottle, a dose of 40,000 units / ml in a 2 ml bottle, or any other combination of EPO concentration in any one bottle size . The compositions can be in the form of an aqueous solution, a suspension or they can be lyophilized. The present invention provides an alternative embodiment, a pharmaceutical carrier composition, for use as an EPO carrier, the preservatives comprising benzethonium chloride, phenoxyethanol or phenylethyl alcohol, wherein the preservatives are contained in the EPO carrier composition alone or in combination . The present invention also provides a bottle for containing multiple doses of EPO, wherein the bottle comprises EPO and an effective amount of one or a combination of the following preservatives: benzethonium chloride, fenoxietan ol and phenylethyl alcohol. Still in another mode, the present invention provides a method that inhibits microbial growth in an EPO containing solution, wherein the method comprises adding to the EPO containing solution, one or a combination of the following preservatives: chloride of benzethonium, phenoxyethanol and 1 3l phenylethyl alcohol. The additional components of the multiple dose EPO formulations of the present invention include their surfactants, buffers, osmo-ality adjusters and anti-adsorbents. Particularly advantageous additives include polysorbate-20, polysorbate-80, sodium phosphate, sodium chloride and genapo The formulations of the present invention may be in solid, semi-solid, liquid or fluid form, for example, as a tablet, aqueous solution or suspension, or may be lyophilized and reconstituted prior to administration to a patient. The formulations can be administered via any parenteral route, including intravenous, subcutaneous, intramuscular, transdermal, intraarterial, intraperitoneal, or pulmonary inhalation. They can also be administered orally.

IV. DETAILED DESCRIPTION OF THE INVENTION The present invention provides a significant improvement over the state of the art. Novel multi-dose pharmaceutical formulations containing EPO containing preservatives are provided which individually provide stable, sterile, easily administered compositions. Furthermore, and quite unexpectedly, the present invention discloses that phenoxyethanol and benzethonium chloride, when used in combination in a multi-dose pharmaceutical composition containing EPO, have positive synergistic effects resulting in a particularly advantageous composition. Specifically, this combination of preservatives shows the following characteristics: (1) synergistic antimicrobial effect, allowing to use a lower concentration of preservatives; (2) excellent stability of EPO, at variable storage conditions, over prolonged periods; and (3) phene xethanol has a potential for a local anesthetic effect, making the composition particularly preferred for subcutaneous administration.

As used herein, the following terms have the following meanings: Erythropoietin - a protein glycogen which, when in a biologically active and glycosylated form, has the ability to induce the formation of hemoglobin and red blood cells in the blood. bone marrow . It can be obtained via isolation of human tissues or fluids, by traditional recombination methods or by activation with genes. Parenteral - by others different to the gastrointestinal tract; includes intravenous, subcutaneous or intramural and intramedullary, intra-arterial, intra-peritoneal and pulmonary inhalation. Pharmaceutically acceptable (or pharmacologically acceptable) - refers to molecular entities and compositions that do not produce an adverse, allergic or otherwise unfavorable reaction when administered to an immune or a human, as appropriate. Pharmaceutically acceptable carrier - includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, delay agents for absorption of substances and the like, which may be used as a medium for a pharmaceutically acceptable substance. . U nity - a unit of biological activity as determined by exhypoxic polyemic mouse bioassay and compared with standards of the World Health Organization. Any numerical value declared herein includes all values from the lower value grazes the higher value in increments of one unit, provided there is a separation of at least 2 units The benzethonium chloride, phenoxyethanol and phenylethyl alcohol can be used in the instant formulations herein in any effective amount. The total preservative concentration is preferably between about 0.001% and about 4.0% of the total formulation. Particularly advantageous concentrations of total preservative are those kept as low as possible to achieve the required antimicrobial effect, while minimizing the potential for adverse reactions. Although benzethonium chloride, phenoxyethanol and phenylethyl alcohol can be used individually as preservatives, doing so requires a higher concentration of preservative than if combinations of preservatives are used. In more preferred embodiments of the present invention, both benzethonium chloride and phenoxyethanol are used together. Surprisingly, when used together, these preservatives have a synergistic effect on one another. To achieve the equivalent antimicrobial effect when used alone, the concentration of benzethonium chloride or phenoxyethanol must each be greater than the total preservative concentration if they are used in combination and, in general, at least twice d 5 conservative should be used if used alone. Thus, for example, if any of benzethonium chloride or phenoxyethanol is used, only at least twice as much benzethonium chloride or phenoxyethanol will be required. It will achieve the same effect as an amount of benzene chloride in combination with phenoxyethanol.

In addition, even at these higher concentrations of benzethonium chloride and phenoxyethanol, the individual preservative formulations may not meet US, European or Japanese anti-microbial regulatory criteria. Preferred formulations include benzethonium chloride in concentrations from about 0.01 to about 0.1%, in combination with phenoxyethanol in concentrations from about 0.01 to about 1.0%. The most p -referred formulations contain benzethonium chloride in a concentration from about 0.01% to about 0.02% and phenolethanol in a concentration from about 0.25% to about 0.5%. In another embodiment, the present invention includes benzethonium chloride in combination with phenylethyl alcohol. Preferred formulations include benzethonium chloride in concentrations from about 0.001 to about 0.1% together with phenylethyl alcohol in concentrations from about 0.01 to about 1.0%. More preferred formulations contain benzethonium chloride in a concentration from about 0.15 to about 0.25% and phenylethyl alcohol in a concentration from about 0.2 to about 0.5%. A highly preferred formulation, in which benzethonium chloride and phenylethyl alcohol are used together, includes benzethonium chloride in a concentration of about 0.02% and phenylethyl alcohol in a concentration of about 0.25%.

B. Erythropoietin The nucleic acid sequence, amino acid sequence, three-dimensional structure and normal glycosylation patterns of EPO are known in the art. EFO isolated and purified from several sources is also known. Accordingly, one of skill in the art can obtain EPO to be used in accordance with the present invention by isolating and purifying EPO from human tissues or fluids, through traditional recombinant techniques and through gene activation processes. All of these methods are specifically contemplated as being within the scope of this patent.

Additionally, any other EPO, obtained from any source, is contemplated for use in accordance with the present invention.

Other Active Components The optimal formulation according to the present invention may vary according to factors such as the amount of time the formulation will be stored under which conditions it will be stored and used, the particular patient population to which it can be administered, etc. Adjustments to the formulation by adjusting the constituents of the formulations and their relative relative entrances, other than the preservatives chloride of onion, phenoxyethanol and phenylethyl alcohol and EPO as described above, can be made as necessary according to the needs of the body. The formulator, administrator or patient The additional constituent elements of the multi-dose EPO formulations of the present invention may include water, a buffer, a surfactant or antiadsorbent, a wetting agent and an osmolality adjusting agent. The characteristics of the formulation that can be modified include, for example, pH and osmolality, to achieve a formulation having a pH and osmolality similar to those of human blood or tissues. The buffers are useful in the present invention for, among other purposes, manipulation of the total pH of the pharmaceutical formulation.

A variety of buffers known in the art can be used in the present formulations, such as various salts of organic or inorganic acids, bases or amino acids, and including various forms of citrate, phosphate, tartrate, succin ate, adipate, maleate, lactate ions , acetate, bicarbonate or carbonate. Particularly advantageous shorteners for use in the present invention include sodium or potassium buffers, particularly sodium phosphate. In a preferred embodiment, sodium phosphate is employed at a concentration approaching 20 ml. A particularly effective sodium phosphate buffer system comprises monohydrate monohydrate. sodium phosphate and dibasic sodium phosphate heptahydrate When this combination of monobasic and dibasic sodium phosphate is used, the advantageous concentrations of each are about 0.5 to about 1.5 mg / ml of monobasic and about 2.0 to about 4.0 mg / ml of dibasic, with referred p concentrations of approximately 0.9 mg / ml of monobasic and approximately 3.4 mg / ml of dibasic phosphate. The pH of the formulation changes according to the amount of buffer used. It is preferred to reach a pH level between 5.0 and 8.0, more preferable to have a DH of from about 6.0 to about 7.5 and most preferably to develop a formulation with a pH of about 7.0. It may also be useful to employ surfactants in the formulations described herein. The surfactants or anti-adsorbents which prove to be useful in accordance with the present invention include polyoxyethylene sorbitan, polyoxyethylene sorbitan monolaurate, polysorbate-20, such as Twee? -20 R, polysorbate-80, hydroxycellulose and genapol. In a preferred embodiment, polysorbate-20 is used. When any surfactant is employed in the present invention, it is advantageous to use it in a concentration of about 0.01 to about 0.5 mg / ml. In a particularly useful embodiment, polysorbate-20 is used in a concentration of about 0.1 mg / ml. Additional useful additives are readily determined by those skilled in the art, according to the particular needs or intended uses of the described multiple dose EPO formulations. One such particularly useful additional substance is sodium chloride, which is useful for adjusting the osmolality of the formulations to achieve the desired osmolality desired. Particularly preferred osmolalities are in the range of about 270 to about 330 Osm / kg. The optimum osmolality of the formulations described herein is about 300 mOsm / kg. Sodium chloride at concentrations of about 6.5 to about 7.5 mg / ml is effective in reaching this osmolalide d, a particularly effective one being Sodium chloride concentration of approximately 7.0 mg / ml. Or the amount of sodium chloride can be added or adjusted to achieve an osmolality of about 270 to about 330 mOsm / kg, and preferably 300 thiOm / kg. Other useful osmolality adjusting agents include manit and sorbitol.

D. Preparation of the compositions The E 'O formulations described herein can be prepared in water conveniently mixed with a surfactant, such as hydroxypropylcellulose or poyoxyethylene sorbitans. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride as described arts. Prolonged absorption of the injectable compositions can be caused by the use in the compositions of agents delaying absorption, for example, aluminum monostearate or gelatin. Other agents that may be employed include, but are not limited to lecithin, urea, ethylene oxide, propylene oxide, hydroxypropylcellulose, methylcellulose or polyethylene glycol. Aqueous compositions (ínculos) as described herein, may include an effective amount of EPO dissolved or dispersed in a pharmaceutically acceptable aqueous medium. Such compositions are also referred to as inocula. The use of pharmaceutically acceptable carrier medium and agents for pharmaceutically active substances is well known in the art. Except to where any conventional means or agent is incompatible with the active ingredient, its use in therapeutic compositions is contemplated. The complementary active ingredients can also be incorporated into the compositions as described above. A proteoglycan, such as EPO can be formulated in a composition in a neural or salt form. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and those which are formed with inorganic acids, such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic , tartaric, mandélico and similar. The salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium or ferric hydroxides and such organic bases as isopropylamine, trimethylamine, his tidine, procaine and the like. The therapeutic agents of the present invention are advantageously administered in the form of injectable compositions either as liquid solutions or suspensions; Suitable solid forms for solution in, or suspension in, liquid before injection may also be prepared A normal composition for such purposes comprises a pharmaceutically acceptable carrier For example, the composition may contain 10 mg, 25 mg, 50 mg or up to about 100 mg of a human serum albumin per milliliter of phosphate-buffered saline The formulations as described herein may be contained in a bottle, canister, tube, syringe or other container for simple or multiple administrations. Such containers may be made of glass or a polymeric material, such as polypropylene, polyethylene, or polyvinyl chloride, for example. Preferred containers may include a seal or other closure systems, such as a rubber stopper that can be penetrated by a needle, in order to remove a single dose and then reseal upon removal of the needle. All such containers for injectable liquids, freeze-dried formulations, reconstituted lyophilized formulations or reconstitutable injection powders known in the art, are contemplated for use in the compositions and methods described herein.

V. EXAMPLES The following examples are illustrative only and will not be construed as limiting the scope of the invention.

A. Example 1: Conservative selection and stability test Materials and methods: Monohydrate or monobasic sodium phosphate USP, sodium phosphate dibasic heptahydrate USP, sodium chloride USP / EP and polysorbate 20 USP / NH from J.T. Baker, a division of Mallinckrodt Baker, Inc., Phillipsburg, NH 08865. Bencalzonic chloride USP / NF, 2-phenoxyethanol BP, phenylethyl alcohol USP / NF, thimerosal L SP / NF, phenol crystals USP, benzethonium chloride USP / NF, m-cresol U 3P, phenyl mercuric nitrate USP / NF, benzyl alcohol USP, chlorobutanol US, methylparaben USP / NF and propylparaben USP / NF were obtained from Spectrum Quality Products Inc., Gardena, CA 90248. Myristyl garr ma-picolinium chloride was obtained from Pharmacia & Upjohn Company, Kalamazoo, Michigan 49001 Multi-dose EPO containing solutions were formulated as sterile, non-pyrogenic, colorless water solutions for injection at concentrations of 10,000 units and 20,000 units. The pre-stopped solutions contained a 20 mM phosphate buffer (sodium phosphate monohydrate Dbasic sodium heptahydrate and sodium phosphate dibasic heptahydrate), 0.01% w / v polysorbate 20 as an antiadsorbent, 0.45-0.8% w / v chloride sodium (depending on the conservative system that was used the amount of sodium chloride was adjusted to produce an osmolality of approximately 300 mOsm / kg), and a conservative system. The prepared solutions had a pH of about 7.0 and an osmolality of about 300 mOsm / kg. The solutions were sterilized by filtration through a 0.22 micron Millipore filter, sterile. The solutions were packed in 2 ml, clear, sterile, type 1, USP glass vials and stored at room temperature.

° C and 25 ° C for chemical stability testing and in bottles of HDPP (high density polypropylene) of 250 - 500 ml, sterile, for microbial testing. Conservative studies included: benzylic alcohol 1.0% w / v, benzalkonium chloride 0.C 1% w / v, 2-phenoxyethanol 0.5% w / v, phenylethyl alcohol 0.5% w / v, thimerosal 0.005% and 0.01 % w / v, phenol crystals 0.4% p / v, benzethonium chloride 0.01% and 0.02% w / v, m-cresol 0.4% w / v, phenyl mercuric nitrate 0.002% w / v, methylparaben 0. 1 and 0. 1% w / v and propi lpraben 0. 03% and 0.035% w / v, and myristium chloride l gam ma-picol inio 0.02% w / v. In addition, the following com binations were studied: (1) benzethonium chloride 0.005% w / v with phenoxyethanol 0.25% w / v; (2) 0.005% benzethonium chloride and 0.5% p / v phenoxyethanol; (3) benzethonium chloride 0.01% w / v with phenoxyethanol 0.5% w / v; and (4) phenylethyl alcohol 0.25% w / v and benzethonium chloride 0.02% w / v.

Discussion: Methylparaben, propylparaben, m-cresol, and phenol produced hazy solutions to nebulae when added to the EPO formulation (buffered solution). This nebulosity problem was identified as an incompatibility between the polysorbate 20 absorber and each one. of these conse; rvators (Handbook of Pharmaceutical Excipients, 1 994). Although chlorobutanol produced a clear solution when used in the formulation, its evaluation was stopped because it is not stable at pH > 3, its half life at pH 7.5 is approximately 3 months (Handbook of Pharmaceutical Excipients, 1 £ 94). In the presence of phenyl mercuric nitrate, a nebulous solution was produced. This nebulosity was identified as an incompatibility between the osmotic agent sodium chloride and the phenyl mercuric nitrate (H andbook of Pharmacuetical Excipients, 1 994).

Thimerosal produced a clear solution when used in the formulation of E PO. In addition, conservative efficacy was good.

Additionally, the EPO formulation showed good chemical stability in the presence of thimerosal. However, because it contains mercury, it is likely that it will be unacceptable by the agencies in Europe, Japan and the US. Formulations containing benzyl alcohol, benzethonium chloride, phenoxyethanol, phenylethyl alcohol, benzalkonium chloride and myristyl-gamma-picolinium chloride produced clear solutions. Based on the minimal inhibitory concentrations for these conservatives, safety and frequency of use, the conservative signs were selected for additional chemical evaluation of protein stability and anti-microbial effectiveness of the formulation. of EPO: benzyl alcohol, benzethonium chloride, phenoxyethanol, phenylethyl alcohol and various combinations of benzethonium chloride and phenoxyethanol.

B. Example 2: Proven protein stability Materials and methods: The prototype batches were made using these selected preservatives and placed in stability at 5 ° C and 25 ° C. Samples were tested by a reverse phase H PLC method. The test results (% claim on label) of the EPO containing formulations in the presence of these preservatives are shown in Table 2.

The label claim was determined by reverse phase HPLC using a C1 8 column of Waters Delta-PakD and gradient levigation using an aqueous solution containing 0.05% TFA and concentration of acetonitrile, which increases from 23 to 86%. The detection of the EPO protein was monitored at 210 nm.

Table 2 Test results in terms of concentration (% of recl &imt on label) Conservative Mese; Force Physical test (% LC) 1 5 ° C 25 ° C Benzyl alcohol 7M 1, 000 U / ml 98.6 90.0 Benzyl alcohol 7M 20, 00 U / ml 99.7 93.7 Benchiness 4M 1 0, 000 U / ml 99.9 88.9 Benchthonium 4M 20,000 U / ml 101 .5 94.4 Phenoxyethanol 4M TÓGÓOO U / m 1 00.5 96.9 Phenoxyethanol 4M 20,000 U / ml 102.6 97.5 Bencethonium & 3M '"¿b'.odo'ü / me 98.3 97.0 phenoxyethanol (0.005% and 0.5%) 1 as measured by reverse phase HPLC As can be seen from table 2, the phase HPLC data In contrast, there is no loss of EPO concentration for all formulas when stored at 5 ° C for up to 3-7 months, however, formulas containing benzyl alcohol or Benzene alone showed up to 10% loss of EPO when stored at 25 ° C for up to 3-7 months. Formulas containing phenoxyethanol or phenoxyethanol & Benchonium chloride in combination, showed no loss of EPO when stored at 25 ° C for up to 3-4 months. These results show the stabilizing effect of phenoxyethanol and phenoxyethanol & In combination with benzodiazine on EPO, this effect is surprising and unexpected, as well as extremely advantageous.

Example 3: Conservative challenge test The conservative effects tests are combinations of assays that determine the efficacy for conservative systems in multi-dose pharmaceutical preparations. In such tests, the test formulations are removed with standardized suspensions of aerobic indi cating bacteria and molds and the survival of microorganisms is monitored over a period of 28 days. Table 3 shows the results of the conservative challenge test of the US Pharmacopoeia (USP) and the European Pharmacopoeia (EP). All the formulas tested passed the USP criteria for the preservation challenge test. Formulas containing 0.01% w / v benzethonium chloride, 0.5% pv phenoxyethanol or 0.5% w / v phenylethyl alcohol failed the EP criteria for conservative challenge test. The formulas containing benzethonium chloride and phenoxyethanol in different combinations and benzethonium chloride and phenylethyl alcohol in combination, fell under the criteria of US P and EP. With Based on the data shown in Table 3, it appears that the antimicrobial activity of benzethonium chloride was surprisingly increased by the addition of phenoxyethanol, in a synergistic manner.

Table 3 Preservative challenge test Conservative Strength Conservative challenge USP EP Benzyl alcohol 20,000 U / ml Passed Not done (1.0%) Bencethonium 20,000 U / ml (0.01%) Passed Failed (0.02%) Pasó Pasó Phenoxyethanol 20,000 U / m l Passed Failed (0.5%) - Bencethonium & phenoxyethanol 20,000 U / ml Passed Passed > (0.005 &0.5%) (0.01 &0.5%) Phenylethyl alcohol 20,000 U / ml Passed Fal ló 0. 5% Bencethonium & Phenylethyl Alcohol Passed (0.02% &0.25) REFERENCES U.S. Patent No. 4,3717,573 to Sugimoto et al. U.S. Patent No. 4,70 3,008 to Lin. U.S. Patent No. 4,806,524 to Kawagachi et al. U.S. Patent No. 5,503,827 to Woog et al. U.S. Patent No. 5,661,125 to Stricklan et al. Handbook of Pharmaceutical Excipients, (Handbook of Pharmaceutical excipients), second edition 1994. L.A. Trissel, "Handbook on Injectible Drugs", ed.8, American Society of Hospital Pharmacist, Inc. 1994.

Miyaka et al, "Purification of Human Erythropoietin" (Purification of human erythropoietin), J. Biol. Chem.252 (15): 5558-5564, 1997 Physicians' Desk Reference, ed. 48, 1994. Physicians' Desk Reference, ed. 50, 1996. Sandeep Nema, R.J. Wahkuhn and R.J. Brendel, "Excipients and Their Use in Injectable Products" (Excipients and Their Use in Injectable Products), PDA Journal of Pharmaceutical Sciences & Technology, vol. 51 (4), July-August 1997. Shewood et al., "Erithropoietin Production by Human Renal Carcinoma Cells inCulture" (Production of erythropoietin by human renal carcinoma cells in culture), Endoc inology, vol.99 (2): 504 -510, 1976.

Sherwood et al. , "Establishment of a Human Erythropoietin-Producing Renal Carcinoma Cell Line" (Establishment of a line of human renal cell carcinoma cells, erythropoietin progenitors), Clinical Research, 31: 323A, 1983.

Claims (1)

1. REIV N DICATIONS 1 . A pharmaceutical composition comprising erythropoietin and an amount of benzene chloride Dn io effective to inhibit microbial growth in said composition 2. The composition of claim 1, wherein the composition further comprises phenoxyethanol. 3. The composition of claim 1, wherein the composition further comprises phenolic alcohol. 4. The composition of claim 1, wherein an effective amount of benzethonium chloride is a concentration from about 0.001 to about 10%. 5. The composition of claim 1, wherein an effective amount of benzethonium chloride is a concentration from about 0. 01 to approximately 0. 1%. 6. The composition of claim 1, wherein an effective amount of benzethonium chloride is a concentration of 0.005%. 7. The composition of claim 1, wherein an effective amount of benzethonium chloride is a concentration of 0.01%. 8. The com position of claim 1, wherein an effective amount of benzethonium chloride is a 0.02% co-concentration. 9. The composition of claim 2, further defined in that it comprises benzethonium chloride in a concentration from about 0.001 to about 1.0% and phenoxyethanol in a concentration from approximately 0.01 to approximately 1.0%. The composition of claim 2, further defined in that it comprises benoetonium chloride in a concentration from about 0.01 to about 0.1% and phenoxyethanol in a concentration from about 0.1 to about 0.75%. eleven . The composition of claim 2, further defined because it comprises benzethonium chloride in a concentration of 0.005% and phenoxyethanol in a concentration of 0.25%. 12. The composition of claim 2, further defined as comprising benzene chloride nio at a concentration of 0.005% and phenoxyethanol at a concentration of 0.5%. 13. The composition of claim 2, further defined in that it comprises betonium chloride at a concentration of about 0.01% and phenoxyethanol at a concentration of about 0.5%. 14. The composition of claim 3, further defined in that it comprises benzene chloride or in a concentration of 0.02% and phenylethyl alcohol in a concentration of 0.25%. 5. The composition of claim 3, further defined as comprising about 0.02% benzethonium chloride and about 0.25% phenylethyl alcohol. 1 6. The composition of claim 1, further defined in that it comprises a salt. 27. The pharmaceutical carrier of claim 20, wherein an effective amount of benzethonium chloride is a concentration of 0.02%. 28. The pharmaceutical carrier of claim 21, further defined by comprising benzethonium chloride in a concentration from about 0.001 to about 1.0% and phenoxyethanol in a concentration from about 0.01 to about 1.0%. 29. The pharmaceutical carrier of claim 21, further defined in that it comprises benzethonium chloride in a concentration from about 0.01 to about 0.1% and phenoxyethanol in a concentration from about 0. 1 to about 0.75%. 30. The pharmaceutical carrier of claim 21, further defined because it comprises benzethonium chloride at a concentration of 0.005% and phenoxyethanol at a concentration of 0.25%. 31 The pharmaceutical carrier of claim 21, further defined because it comprises benzethonium chloride in a concentration of 0.005% and phenoxyethanol in a concentration of 0.5%. 32. The pharmaceutical carrier of claim 21, further defined as comprising benzene chloride in a concentration of 0.01% and phenoxyethanol in a concentration of 0.5%. 33. The pharmaceutical carrier of claim 21, further defined as comprising benzene chloride in a concentration of 0.02% and phenylethyl alcohol in a concentration of 0.25%. 34. The pharmaceutical carrier of claim 20, further comprising one or more additives are excised from the group consisting of a buffer, a salt and an anti-adsorbent and a surfactant. 35. A bottle for containing dosages of multiple doses of erythropoietin, wherein said bottle contains a solution comprising erythropoietin and an amount of benzethonium chloride effective to inhibit microbial growth in this condition. 36. The bottle of claim 35, wherein said solution further comprises phenoxyethanol. 37. The bottle of claim 35, wherein said sol- ution also comprises phenylethyl alcohol. 38. The bottle of re-excitation 35, wherein said effective amount of benzethonium chloride is a concentration from about 0.001 to about 1.0%. 39. The bottle of claim 35, wherein an effective amount of benzethonium chloride is a concentration from about 0.01 to about 0.1%. 40. The bottle of claim 35, wherein an effective amount of benzethonium chloride is a co-concentration of 0.005%. 41 The bottle of claim 35, wherein an effective amount of benzethonium chloride is a co-concentration of 0.01%. 42. The flask of claim 35, wherein an effective amount of benzethonium chloride is a concentration of 0.02%. 43. The flask of rejection 36, also defined because it comprises benzethonium chloride at a concentration of about 0.001. to about 1.0% and phenoxyethanol in a concentration from about 0.01 to about 1.0%. 44. The bottle of claim 36, further defined in that it comprises benzethonium chloride in a concentration of from about 0.01 to about 0.1% and phenoxyethanol in a concentration from about 0. 1 to about 0.75%. 45. The flask of claim 36, further defined as comprising benzethonium chloride in a concentration of 0.005% and phenoxyethanol in a concentration of 0.25%. 46. The flask of claim 36, further defined because it comprises benzethonium chloride in a concentration of 0.005% and phenoxyethanol in a concentration of 0.5%. 47. The flask of claim 36, further defined in that it comprises benzethonium chloride in a concentration of approximately 0.01% and phenoxyethanol in a concentration of approximately 0.5%. 48. The bottle of claim 37, further defined as comprising benzethonium chloride in a concentration of 0.02% and phenylethyl alcohol in a concentration of 0.25% 49. The bottle of claim 37, further defined because it comprises approximately 0.02% Benzethonium chloride and approximately 0. 25% phenylethyl alcohol. 50. The flask of claim 1, wherein said solution further comprises a salt. 51 The bottle of claim 50, wherein said salt is sodium chloride. 52. The bottle of claim 35, wherein said solution also comprises a shock absorber. 53. The bottle of claim 52, wherein said buffer is sodium phosphate. 54. A method for inhibiting microbial growth in a solution comprising erythropoietin, said method comprising adding benzethonium chloride to said solution. 55. The method of claim 54, wherein said method further comprises adding noxethanol to said solution. 56. The method of claim 54, wherein said method comprises adding phenol ethyl alcohol to said solution. 57. The method of claim 54, wherein a benzethonium chloride is added at a concentration from about 0. 001 up to about 10%. 58. The method of claim 54, wherein said benzethonium chloride is added to ur at a concentration from about 0.01 to about 0.1%. 59. The method of claim 54, wherein said benzethonium chloride is added at a concentration of 0.005%. 60. The method of claim 54, wherein the benzethonium chloride is added at a concentration of 0.01%. 61 The method of claim 54, wherein said benzethonium chloride is added at a concentration of 0.02%. 62. The method of claim 55, wherein the benzethonium chloride is added in a concentration from about 0.001 up to about 1.0% and phenoxy ethanol is added in a concentration from about 0.01 to about 1.0% 63. The method of claim 55, wherein the benzethonium chloride is added in a concentation from about 0.01 to about about 0.1% and phenoxy ethanol is added at a concentration from about 0.1 to about 0.75%. 64. The method of claim 55, wherein the benzethonium chloride is added in a concentration of 0.005% and phenoxyethanol is added in a concentration of 0.25%. 65. The method of claim 55, wherein the benzethonium chloride is added at a concentration of 0.005% and phenoxyethanol is added at a concentration of 0.5%. 66. The method of claim 55, wherein the benzethonium chloride is added in a concentration of about 0.01% and phenoxyethanol is added at a concentration of about 0.5% 67. The method of claim 56, wherein the chloride Benzethonium is added in a concentration of 0.02% and phenylethyl alcohol is added in a 0.25% concentrator. 68. The method of claim: Ion 56, wherein the benzethonium chloride is added in a concentration of about 0.02% and phenol-ethyl alcohol is added in a concentration of about 0.25%. 69. The method of claim 54, which further comprises adding a salt to said solution. 70. The method of claim 69, wherein said salt is sodium chloride. 71. The method of claim 54, further comprising adding a buffer to said solution 72. The method of claim 71, wherein said buffer is sodium phosphate.