CA1295240C - Biologically stable interferon compositions - Google Patents
Biologically stable interferon compositionsInfo
- Publication number
- CA1295240C CA1295240C CA000514895A CA514895A CA1295240C CA 1295240 C CA1295240 C CA 1295240C CA 000514895 A CA000514895 A CA 000514895A CA 514895 A CA514895 A CA 514895A CA 1295240 C CA1295240 C CA 1295240C
- Authority
- CA
- Canada
- Prior art keywords
- interferon
- thimerosal
- solution
- alpha interferon
- alpha
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Abstract
ABSTRACT
Stable pharmaceutical compositions comprising interferon and thimerosal which are substantially resistant to microorganism contamination and growth are disclosed.
Stable pharmaceutical compositions comprising interferon and thimerosal which are substantially resistant to microorganism contamination and growth are disclosed.
Description
BIOLOGICALLY STA~LE
INTERFERON COMPOSITIONS
The present invention relates to pharmaceutical compositions comprising interferon and in particular to such compositions which are substantially resistant to microorganism contamination and growth during storage at room temperature.
By the present invention, thimerosal (i.e.
sodium ethylmercurithiosalicylate, also known as merthiolate) is employed in the compositions as an antimicrobial preservative.
Formulations of the present invention are use~ul in preparing stable interferon solutions suitable for injectable, ophthalmic and nasal products.
Interferons have qreat potential as drugs for the treatment of a variety disease states, e.~. various types of viral infections and certain cancers.
As used herein, the term "interferon" includes natural and recombinant alpha (leucocyte) and beta ~fibroblast) interferons, but alpha interferons are preferred. As used herein, the term "alpha interferon"
means a natural or recombinant interferon exhibiting biological properties similar to those of human leucocyte interferon. It should be noted tha~ a number of alpha interferon species are known, usually designated by a numeral after the Greek letter, and all are contemplated ;2f~
for use in this invention. Also included within the scope of this invention are the so-called alpha hybrid interferons wherein fragments of two or more native alpha interferon species are joined (see for instance, EP
51873). Preferred forms of alpha interferon for use in the formulations of the present invention are alpha-l and alpha-2 interferon. Particularly preferred for use in the formulations of the present invention is alpha-2 interferon. Alpha-2 interferon may be prepared by recombinant-DNA methods, for example those disclosed by Nagata et al., Nature, Vol. 284, pages 316-320 (1980), and by Weissmann, European Patent 32,13~.
Biologically stable interferon compositions are known. See Kwan U.S. Patent 4,496,537, which claims a method of improving biological stability of an inter-feron formulation comprising the addition of glycine or alanine to an alpha interferon formulation prior to lyophilization. While such lyophilized powders are not generally susceptible to microbiological contamination since interferon formulations are commonly prepared under aseptic conditions, reconstituted solutions of these lyophilized formulations are susceptible to microbio-logical contamination and growth. This makes reconsti-tuted solutions unsuitable for multiple dose appli-cations, e.g. nasal or ophthalmic applications, where the solution will not be used all at once.
Varlous preservatives and preservative combinations have been tested, but in general have been Eound to cause physical instability such as precipitation or turbidity in the reconstituted solution. Some preservatives are also ineffective in preventing microbial contamination and/or reduce interferon activity.
z~
We have surprisingly found that of the many preservatives tested, only thimerosal is effective in preventing microbial contamination in a reconstituted interferon solution for at least four weeks when the reconstituted solution is stored at room temperature or under refrigsration. Furthermore, such reconstituted solutions are stable with respect to antiviral activity, pH and physical appearance under those conditions.
Thimerosal can be incorporated with the interferon in the lyophilized powder or can be added in the diluent for reconstitution of lyophilized interferon powder. The concentration of thimerosal in the solution may be in the ranqe of 0.005 to 1 mg/ml, with 0.02 to 0.05 mg/ml being preferred, and 0.02 mg/ml being most preferred.
A particular advantage of the present invention is the ability to store the reconstituted solution at room temperature safely and effectively for prolonged periods. This advanta~e is evident especially for ophthalmic and nasal preparation where a non-hospitalized patient will be self-administerin~ the formulations for an extended time period, e.g. 1-3 weeks, and a portable and easily stored formulation is desirable. Another advantage is the increased comfort of the patient in being able to use such formulations as eye drops or nasal sprays at room temperature.
The various preservatives and preservative combinations compared to thimerosal were tested by reconstituting lyophilized alpha interferon formulations with a vehicle comprising the preservative or preservative combination. Reconstituted solutions stored at room temperature and under refriqeration were evaluated periodically for one month for physical stability and appearance; interferon activity was determined initially, after 7 days, 14 days and at the end of the study by the standard method of the inhibition ~2~5~
of cvtopathic effect (CPE) of a virus. Samples which were stable for at least 7 days at room temperature were further tested using the USP XX Antimicrobial Preservative Effectiveness (APE) Test and the British Pharmacopeia (BP) test for antimicrobial preservative effectiveness.
The followinq Table l gives the results of the testinq of physical stability of the interferon ~ormulations to which various preservatives and preservative combinations were added:
Compatibility with Preservative Concentration % alpha-Interferon*
Benzalkonium Chloride 0.005 P
0.02 P
Benzyl Alcohol 0.9 CR
1.8 P
Chlorhexidine Gluconate 0.005 P
0.01 P
Chlorohutanol 0.5 CR
Methylparaben/Propylparaben 0.12/0.012 CR
Phenylmercuric Acetate 0.004 CR
Phenylethyl Alcohol 0.5 P
Phenol 0.25 P
Benzyl Alcohol + 0.9 CR
Methylparaben ~ 0.08 Propylparaben 0.01 Benzyl Alcohol + 0-9 CR
TABLE 1 (continued) Chlorobutanol 0.3 Chlorhexidine Gluconate ~ 0.005 P
Methylparaben ~ 0.08 Propylparaben 0.01 Phenylethyl Alcohol + 0.5 P
Methylparaben + 0.08 Propylparaben 0.01 Chlorhexidine Gluconate + 0.005 P
Chlorohutanol 0.5 Chlorobutanol ~ 0-5 P
Methylparaben + 0.08 Propylparaben 0.01 Thimerosal 0.002 C
0.005 C
* C - compatible for at least 7 days at refriqeration (REF) and room temperature (RT) CR - compatible for at least 7 days at REF, but not at RT
P - turbidity and/or precipitation occurs within 7 days at REF and RT
Alpha interferon solutions prepared from thimerosal-containing lyophilized powders and from thimerosal-containin~ diluents passed physical stability, APE and CPE tests. Alpha interferon solutions prepared from thimerosal-containing diluents also passed the BP
test for antimicrobial preservative effectiveness.
Alpha interferon compositions tested comprised 1 x 107, 2.5 x 107 and 5 x 107 International Units (I.U.) interferon/ml, although it is contemplated that compositions of the present invention may contain a ran~e of 2 x 103 to 5x 108 I.U./ml, Preferably 1 x 104 to 2 x 108 I.U. alpha interferon/ml. The specific activity of the alpha interferon used in the compositions of the present invention should be at least 5 x 107 I.U./mg total protein, preferably at least 1 x 108 I.U./mq total protein. In addition to alpha interferon and thimerosal, the pharmaceutical solutions of the present invention contain 0-2 mg albumin/ml and 5-25 mg qlycine/ml in a compatible buffer to maintain the pH at 6.5 to 8Ø
Preferably the compositions comprise l mq albumin/ml and 20 mg glycine/ml in a sodium dibasic phosphate and sodium monobasic phosphate buffer system at a pH of 7.0 to 7.4.
When thimerosal is added to the alpha interferon solution before lyophilization, the diluent for reconstitution may be purified water. When thimerosal is added to alpha interferon lyophilized powder, the diluent may be water, but is preferably an aqueous solution of a compatible buffer to maintain the pH at a level which will prevent decomposition of thimerosal. The buffer system may be the same as that used in the alpha interferon lyophilized powder, e.~. pH
6.5 to 7.5 sodium phosphate buffer, but preferably is a pH 3.5 to 5.5 citric acid buffer prepared from citric acid tmonohydrate or anhydrous) and sodium citrate (dihydrate or anhydrous).
Following are examples of alpha-2 interferon formulations wherein thimerosal is present in the diluent and wherein thimerosal is present in the lyophilized powder.
EXAMPLE l Lyophilized Powder* mg/ml Alpha-2 Interferon 2 x 103 - 2 x 108 I.U.
Sodium Phosphate Dibasic 1 - 5 Sodium PhosPhate Monobasic 0.2 - 1.0 Aminoacetic Acid 5 - 25 Human Albumin 0.5 - 2 : .
.
Preserved Diluent for Reconstitution (A) mq/ml Thimerosal 0.005 - 0.1 Sodium Phosphate Dibasic 0.2 - 1.0 Sodium Phosphate Monobasic 0.4 - 2.0 Purified Water q.s. ad 1.0 ml Preserved Diluent for Reconstitution tB ? mg/ml Thimerosal 0.005 - 0.1 Sodium Citrate Dihydrate 0.1 - 0.5 Citric Acid Monohydrate 0.1 - 0.5 Purified Water q.s. ad 1.0 ml Lyophilized Powder* mg/ml Alpha-2 Interferon 2 x 103 - 2 x 108I.U.
Sodium Phosphate Dibasic 1 - 5 Sodium Phosphate Monobasic 0.2 - 1.0 Aminoacetic Acid 5 - 25 Human Albumin 0.5 - 2 Thimerosal 0.005 - 0.1 *Lyophilized powder may contain other stabilizers to improve/enhance Interferon stability.
Lyophilized powders are prepared by combining and lyophilizing the inqredients for the powder formulations using standard techniques.
Diluents are prepared by combining the in~redients of the diluents usinq standard techniques.
.... . . .~ ,
INTERFERON COMPOSITIONS
The present invention relates to pharmaceutical compositions comprising interferon and in particular to such compositions which are substantially resistant to microorganism contamination and growth during storage at room temperature.
By the present invention, thimerosal (i.e.
sodium ethylmercurithiosalicylate, also known as merthiolate) is employed in the compositions as an antimicrobial preservative.
Formulations of the present invention are use~ul in preparing stable interferon solutions suitable for injectable, ophthalmic and nasal products.
Interferons have qreat potential as drugs for the treatment of a variety disease states, e.~. various types of viral infections and certain cancers.
As used herein, the term "interferon" includes natural and recombinant alpha (leucocyte) and beta ~fibroblast) interferons, but alpha interferons are preferred. As used herein, the term "alpha interferon"
means a natural or recombinant interferon exhibiting biological properties similar to those of human leucocyte interferon. It should be noted tha~ a number of alpha interferon species are known, usually designated by a numeral after the Greek letter, and all are contemplated ;2f~
for use in this invention. Also included within the scope of this invention are the so-called alpha hybrid interferons wherein fragments of two or more native alpha interferon species are joined (see for instance, EP
51873). Preferred forms of alpha interferon for use in the formulations of the present invention are alpha-l and alpha-2 interferon. Particularly preferred for use in the formulations of the present invention is alpha-2 interferon. Alpha-2 interferon may be prepared by recombinant-DNA methods, for example those disclosed by Nagata et al., Nature, Vol. 284, pages 316-320 (1980), and by Weissmann, European Patent 32,13~.
Biologically stable interferon compositions are known. See Kwan U.S. Patent 4,496,537, which claims a method of improving biological stability of an inter-feron formulation comprising the addition of glycine or alanine to an alpha interferon formulation prior to lyophilization. While such lyophilized powders are not generally susceptible to microbiological contamination since interferon formulations are commonly prepared under aseptic conditions, reconstituted solutions of these lyophilized formulations are susceptible to microbio-logical contamination and growth. This makes reconsti-tuted solutions unsuitable for multiple dose appli-cations, e.g. nasal or ophthalmic applications, where the solution will not be used all at once.
Varlous preservatives and preservative combinations have been tested, but in general have been Eound to cause physical instability such as precipitation or turbidity in the reconstituted solution. Some preservatives are also ineffective in preventing microbial contamination and/or reduce interferon activity.
z~
We have surprisingly found that of the many preservatives tested, only thimerosal is effective in preventing microbial contamination in a reconstituted interferon solution for at least four weeks when the reconstituted solution is stored at room temperature or under refrigsration. Furthermore, such reconstituted solutions are stable with respect to antiviral activity, pH and physical appearance under those conditions.
Thimerosal can be incorporated with the interferon in the lyophilized powder or can be added in the diluent for reconstitution of lyophilized interferon powder. The concentration of thimerosal in the solution may be in the ranqe of 0.005 to 1 mg/ml, with 0.02 to 0.05 mg/ml being preferred, and 0.02 mg/ml being most preferred.
A particular advantage of the present invention is the ability to store the reconstituted solution at room temperature safely and effectively for prolonged periods. This advanta~e is evident especially for ophthalmic and nasal preparation where a non-hospitalized patient will be self-administerin~ the formulations for an extended time period, e.g. 1-3 weeks, and a portable and easily stored formulation is desirable. Another advantage is the increased comfort of the patient in being able to use such formulations as eye drops or nasal sprays at room temperature.
The various preservatives and preservative combinations compared to thimerosal were tested by reconstituting lyophilized alpha interferon formulations with a vehicle comprising the preservative or preservative combination. Reconstituted solutions stored at room temperature and under refriqeration were evaluated periodically for one month for physical stability and appearance; interferon activity was determined initially, after 7 days, 14 days and at the end of the study by the standard method of the inhibition ~2~5~
of cvtopathic effect (CPE) of a virus. Samples which were stable for at least 7 days at room temperature were further tested using the USP XX Antimicrobial Preservative Effectiveness (APE) Test and the British Pharmacopeia (BP) test for antimicrobial preservative effectiveness.
The followinq Table l gives the results of the testinq of physical stability of the interferon ~ormulations to which various preservatives and preservative combinations were added:
Compatibility with Preservative Concentration % alpha-Interferon*
Benzalkonium Chloride 0.005 P
0.02 P
Benzyl Alcohol 0.9 CR
1.8 P
Chlorhexidine Gluconate 0.005 P
0.01 P
Chlorohutanol 0.5 CR
Methylparaben/Propylparaben 0.12/0.012 CR
Phenylmercuric Acetate 0.004 CR
Phenylethyl Alcohol 0.5 P
Phenol 0.25 P
Benzyl Alcohol + 0.9 CR
Methylparaben ~ 0.08 Propylparaben 0.01 Benzyl Alcohol + 0-9 CR
TABLE 1 (continued) Chlorobutanol 0.3 Chlorhexidine Gluconate ~ 0.005 P
Methylparaben ~ 0.08 Propylparaben 0.01 Phenylethyl Alcohol + 0.5 P
Methylparaben + 0.08 Propylparaben 0.01 Chlorhexidine Gluconate + 0.005 P
Chlorohutanol 0.5 Chlorobutanol ~ 0-5 P
Methylparaben + 0.08 Propylparaben 0.01 Thimerosal 0.002 C
0.005 C
* C - compatible for at least 7 days at refriqeration (REF) and room temperature (RT) CR - compatible for at least 7 days at REF, but not at RT
P - turbidity and/or precipitation occurs within 7 days at REF and RT
Alpha interferon solutions prepared from thimerosal-containing lyophilized powders and from thimerosal-containin~ diluents passed physical stability, APE and CPE tests. Alpha interferon solutions prepared from thimerosal-containing diluents also passed the BP
test for antimicrobial preservative effectiveness.
Alpha interferon compositions tested comprised 1 x 107, 2.5 x 107 and 5 x 107 International Units (I.U.) interferon/ml, although it is contemplated that compositions of the present invention may contain a ran~e of 2 x 103 to 5x 108 I.U./ml, Preferably 1 x 104 to 2 x 108 I.U. alpha interferon/ml. The specific activity of the alpha interferon used in the compositions of the present invention should be at least 5 x 107 I.U./mg total protein, preferably at least 1 x 108 I.U./mq total protein. In addition to alpha interferon and thimerosal, the pharmaceutical solutions of the present invention contain 0-2 mg albumin/ml and 5-25 mg qlycine/ml in a compatible buffer to maintain the pH at 6.5 to 8Ø
Preferably the compositions comprise l mq albumin/ml and 20 mg glycine/ml in a sodium dibasic phosphate and sodium monobasic phosphate buffer system at a pH of 7.0 to 7.4.
When thimerosal is added to the alpha interferon solution before lyophilization, the diluent for reconstitution may be purified water. When thimerosal is added to alpha interferon lyophilized powder, the diluent may be water, but is preferably an aqueous solution of a compatible buffer to maintain the pH at a level which will prevent decomposition of thimerosal. The buffer system may be the same as that used in the alpha interferon lyophilized powder, e.~. pH
6.5 to 7.5 sodium phosphate buffer, but preferably is a pH 3.5 to 5.5 citric acid buffer prepared from citric acid tmonohydrate or anhydrous) and sodium citrate (dihydrate or anhydrous).
Following are examples of alpha-2 interferon formulations wherein thimerosal is present in the diluent and wherein thimerosal is present in the lyophilized powder.
EXAMPLE l Lyophilized Powder* mg/ml Alpha-2 Interferon 2 x 103 - 2 x 108 I.U.
Sodium Phosphate Dibasic 1 - 5 Sodium PhosPhate Monobasic 0.2 - 1.0 Aminoacetic Acid 5 - 25 Human Albumin 0.5 - 2 : .
.
Preserved Diluent for Reconstitution (A) mq/ml Thimerosal 0.005 - 0.1 Sodium Phosphate Dibasic 0.2 - 1.0 Sodium Phosphate Monobasic 0.4 - 2.0 Purified Water q.s. ad 1.0 ml Preserved Diluent for Reconstitution tB ? mg/ml Thimerosal 0.005 - 0.1 Sodium Citrate Dihydrate 0.1 - 0.5 Citric Acid Monohydrate 0.1 - 0.5 Purified Water q.s. ad 1.0 ml Lyophilized Powder* mg/ml Alpha-2 Interferon 2 x 103 - 2 x 108I.U.
Sodium Phosphate Dibasic 1 - 5 Sodium Phosphate Monobasic 0.2 - 1.0 Aminoacetic Acid 5 - 25 Human Albumin 0.5 - 2 Thimerosal 0.005 - 0.1 *Lyophilized powder may contain other stabilizers to improve/enhance Interferon stability.
Lyophilized powders are prepared by combining and lyophilizing the inqredients for the powder formulations using standard techniques.
Diluents are prepared by combining the in~redients of the diluents usinq standard techniques.
.... . . .~ ,
Claims (14)
1. A biologically stable pharmaceutical solution comprising per milliliter of solution 2 x 103 to 5 x 108 International Units of alpha interferon, 0-2 mg human albumin, 5-25 mg qlycine, 0.005 to 1.0 mg thimerosal, and a compatible buffer system designed to maintain the pH at 6.5 to 8Ø
2. A pharmaceutical solution as claimed in claim 1 comprising 1 x 104 to 2 x 108 International Units of alpha interferon per milliliter of solution.
3. A pharmaceutical solution as claimed in claim 2 wherein the pH is maintained at 7.0 to 7.4.
4. A pharmaceutical solution as claimed in claim 3 comprising 1 mg human albumin and 20 mg glycine per milliliter of solution.
5. A pharmaceutical solution as claimed in claim 4 comprising 0.02 to 0.05 mg thimerosal per milliliter of solution.
6. A pharmaceutical solution as claimed in claim 5 wherein the alpha interferon is alpha-2 interferon.
7. A lyophilized alpha interferon pharmaceutical composition for reconstitution with water comprising for every 2 x 103 to 5 x 108 International Units of alpha interferon, 0-2 mg human albumin, 5 - 25 mg glycine, 0.005 to 1.0 mg thimerosal and a compatible buffer system designed to maintain a pH of 605 to 8.0 after reconstitution.
8. A pharmaceutical composition as claimed on claim 7 wherein the buffer is designed to maintain a pH of 7.0 to 7.4 after reconstitution.
9. A pharmaceutical composition as claimed in claim 8 containing 1 mg human albumin and 20 mg glycine for every 1 x 104 to 2 x 108 International Units of alpha interferon.
10. A pharmaceutical composition as claimed in claim 9 containing 0.02 to 0.05 mg thimerosal for every 1 x 104 to 2 x 108 International Units of alpha interferon.
11. A pharmaceutical composition as claimed in claim 10 wherein the alpha interferon is alpha-2 interferon.
12, A process for the preparation of a lyo-philized alpha interferon composition as claimed in claim 7, which process comprises the steps of dis-solving glycine, human albumin, thimerosal a com-patible buffer system and alpha interferon in sterile water and lyophilizing the resulting solution.
13. A process for the preparation of a bio-logically stable pharmaceutical solution which process comprises reconstituting with sterile water a composition comprising for every 2 x 103 to 5 x 108 International Units of alpha interferon, 0-2 mg human albumin, 5-25 mg glycine, 0.005 to 1.0 mg thimerosal and a compatible buffer system designed to maintain a pH of 6.5 to 8.0 after reconstitution.
14. A process for the preparation of a bio-logically stable pharmaceutical solution as claimed in claim 1, which process comprises reconstituting a lyophilized powder containing the alpha interferon with a diluent containing thimerosal.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US75981785A | 1985-07-29 | 1985-07-29 | |
| US759,817 | 1985-07-29 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1295240C true CA1295240C (en) | 1992-02-04 |
Family
ID=25057067
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000514895A Expired - Fee Related CA1295240C (en) | 1985-07-29 | 1986-07-29 | Biologically stable interferon compositions |
Country Status (1)
| Country | Link |
|---|---|
| CA (1) | CA1295240C (en) |
-
1986
- 1986-07-29 CA CA000514895A patent/CA1295240C/en not_active Expired - Fee Related
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Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| MKLA | Lapsed |