Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • A61K38/19—Cytokines; Lymphokines; Interferons
  • A61K38/21—Interferons [IFN]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • A61K38/19—Cytokines; Lymphokines; Interferons
  • A61K38/21—Interferons [IFN]
  • A61K38/215—IFN-beta
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals

Definitions

• Interferons are a family of proteins that have antiviral activity, inhibit cell proliferation, and modulate the natural immune response (1).
• Human interferon beta (IFN- ⁇ ) a member of this family, is a 166 amino acid glycoprotein produced by fibroblasts, as well as other cells, after induction by viral infection or by double-stranded RNA.
• IFN- ⁇ natural human IFN- ⁇ produced from human foreskin fibroblasts (n-IFN- ⁇ ), recombinant human IFN- ⁇ produced in E.Coli (IFN- ⁇ - lb, containing a genetically engineered serine substitution for cystine at position 17), and recombinant human IFN- ⁇ produced in Chinese hamster ovary cells (IFN- ⁇ -la, containing the natural human amino acid sequence).
• n-IFN- ⁇ and IFN- ⁇ -la are glycosylated with a single N-linked complex carbohydrate moiety whereas IFN- ⁇ - lb is not glycosylated.
• AVONEXTM one IFN- ⁇ -la product
• IM intramuscular
• MU 6 million units
• a second IFN- ⁇ -la product, Rebif ® is being given subcutaneously (SC) in ongoing phase HI trials in the treatment of multiple sclerosis.
• SC subcutaneously
• AVONEXTM and Rebif ® may be used interchangeably, a pharmacokinetic and pharmacodynamic intra-subject crossover comparison of the two products after IM injection in healthy male and female volunteers was conducted. The results reported here demonstrate the surprising finding that AVONEXTM and Rebif ® are not equivalent when administered intramuscularly.
• AVONEXTM is formulated in a higher concentration of albumin (15 mg mL after reconstitution versus 9 mg/mL for Rebif ® ), at a different pH (7.2 versus 5.5) and in a different buffer (phosphate versus acetate) (5,7,15,16).
• Rebif ® contains mannitol in its formulation and AVONEXTM does not. These formulation differences may contribute to the unexpected altered absorption of IFN- ⁇ after IM injection by affecting binding to the muscle extracellular matrix and or inactivation by pH-dependent proteases (17)
• Preferred embodiments of the invention for IM administration include a packaged kit for parenteral administration of the present liquid formulations.
• the package may include syringes pre-filled with the liquid formulations of the invention, several alcohol swabs, at least one needle, one or more adhesive bandages and directions for use. It will also appreciated that the present liquid formulations of the invention may be used with conventional needleless- injection systems.
• ECG electrocardiogram
• Blood was drawn for pharmacokinetic testing at 1, 3, 6, 9, 12, 15, 18, 24, and 48 hours following each SC or IM injection, and at 10, 20, and 30 minutes following the start of the IV infusion and at 5, 10, 20, 30, 45, 60, and 90 minutes, and at 2, 3, 4, 5, 6, 9, 12, 15, 18, 24, and 48 hours following the end of the IV infusion.
• Urinalysis, blood chemistry, and hematology tests were performed at 48 hours following study drug. Adverse events were monitored throughout the study period.
• AVONEXTM was supplied as a sterile lyophilized powder containing IFN- ⁇ -la, HSA, sodium phosphate, and sodium chloride; prior to injection, the vial contents were reconstituted with Sterile Water for Injection.
• Rebif ® was available as a sterile lyophilized powder containing IFN- ⁇ -la, mannitol, HSA, and sodium acetate.
• Rebif ® was reconstituted with sodium chloride solution (0.9% NaCl) for injection.
• Each product was stored as a lyophilized powder at 2-8 °C prior to reconstitution.
• AVONEXTM was packaged in 6 MU vials and Rebif ® was packaged in 3 MU vials. The activity of each was accepted as stated on the label.
• Each vial of Rebif ® was reconstituted as directed using 1 mL of the supplied vehicle. Two reconstituted vials were combined to prepare a 6 MU dose in a volume of approximately 2 mL. Each vial of AVONEXTM was also reconstituted as directed to prepare a 1 mL solution. However, in order to inject equal volumes of the two test drugs, matching placebo vials containing excipient only were also reconstituted with 1 mL of sterile water. One reconstituted vial each of AVONEXTM and placebo were then combined to prepare a 6 MU injection in a volume of 2 mL.
• IFN- ⁇ -la (AvonexTM) was supplied as a lyophilized powder in vials containing 60 ⁇ g of IFN- ⁇ -la, as well as human serum albumin, sodium chloride and sodium phosphate. Each vial was reconstituted with 1 mL of sterile water prior to injection. The specific activity of the IFN- ⁇ -la used in this study was 200 million units (MU) of antiviral (interferon) activity per milligram of IFN- ⁇ -la protein. Thus, each vial contained 12 MU of interferon activity. Assay Methods
• CPE cytopathic effect
• the CPE assay detected the ability of interferon beta (IFN- ⁇ ) to protect human lung carcinoma cells (A549, #CCL-185, ATCC, Rockville, MD) from cytotoxicity due to encephalomyelocarditis (EMC) virus.
• IFN- ⁇ interferon beta
• the cells were prcincubated for 15 to 20 hours with serum samples to allow the induction and synthesis of interferon inducible proteins that produce an antiviral response. Following pre-incubation, EMC virus was added to each well and incubated for an additional 30 hours; cytotoxicity was determined using a crystal violet stain.
• An internal Biogen IFN- ⁇ standard was tested concurrently with samples on each assay plate. This standard has been calibrated against a natural human fibroblast interferon reference standard (WHO Second International Standard for Interferon, Human Fibroblast, Gb-23-902-531) (12).
• Serum samples and standards were tested in duplicate on each of two replicate assay plates, yielding four data points per sample. The geometric mean concentration of the four replicates was reported.
• the inter-assay variability was determined by calculating the 95% confidence interval about the mean internal IFN- ⁇ standard concentration for 323 assay plates. As defined, variabihty was less than 10% of the mean. The limit of quantitation was generally 10 U/mL. Serum neopterin concentrations were determined using a commercially available 125 I RIA kit (Immuno Biological Laboratories, Hamburg, Germany). Study personnel performing pharmacokinetic and pharmacodynamic assays were blinded to treatment assignment.
• CPE cytopathic effect
• the CPE assay used in the current study detects the ability of interferon beta (IFN- ⁇ ) to protect human lung carcinoma cells (A549, #CCL-185, ATCC, Rockville, MD) from cytotoxicity due to encephalomyelocarditis (EMC) virus.
• IFN- ⁇ interferon beta
• the cells are preincubated for 15 to 20 hours with serum samples to allow the induction and synthesis of interferon inducible proteins that then mount an antiviral response. Afterwards EMC virus is added and incubated for a further 30 hours before assessment of cytotoxicity is made using a crystal violet stain.
• An internal Biogen interferon beta standard is tested concurrently with samples on each assay plate.
• Each assay plate also includes cell growth control wells containing neither interferon beta nor EMC, and virus control wells containing cells and EMC but no interferon beta. Control plates containing the standard and samples are also prepared to determine the effect, if any, of the samples on cell growth. These plates are stained without the addition of virus.
• Serum concentrations of neopterin and ⁇ 2 -microglobulin were determined at the clinical pharmacology unit using commercially available assays. Neopterin determinations were not performed for the IV dosing group due to unavailability of the assay from the manufacturer at the time this group was enrolled.
• AUC and C ⁇ were analyzed using a two-way crossover analysis of variance (ANOVA). Terms in the analysis included sequence, subject, gender, period, and treatment (13). A term for the gender-by-treatment interaction was initially included, but subsequently removed because the interaction was not significant . AUC and C__. were logarithmically transformed prior to analysis.
• the following pharmacokinetic parameters (20) were calculated: (i) observed peak concentration, C ⁇ (U/mL); (ii) area under the curve from 0 to 48 hours, AUC (U'h/mL) using the trapezoidal rule; (iii) elimination half-life; and, additionally from IV infusion data: (iv) distribution half-life (h); (v) clearance (mL h) (vi) apparent volume of distribution, Vd (L). WinNonlin ( Version 1.0, Scientific Consulting Inc., Apex, NC) software was used to calculate the elimination half-lives after SC and IM injection.
• Figure 1 displays the mean serum interferon activity by time for each product. At each post-dose timepoint, mean serum interferon activity following AVONEXTM administration was higher than that following Rebif ® administration.
• Table I summarizes the pharmacokinetic parameters for each product and the results from the crossover analyses of variance.
• the least squares mean AUC values for AVONEXTM and Rebif ® were 824 and 403 Uxh/mL, respectively.
• the least squares mean ratio of AUC for AVONEXTM to Rebif ® was 204% with 90% confidence limits of 172 to 243% (p ⁇ 0.001).
• Least squares mean C ⁇ values were 33.8 U/mL following AVONEXTM administration and 15.2 U/mL following Rebif ® administration.
• the least squares mean ratio of C__. for AVONEXTM to Rebif ® was 222% with 90% confidence limits of 172 to 285% (p ⁇ 0.001).
• the mean time to maximum concentration was between 12 and 16 hours for AVONEXTM and Rebif ® .
• FIG. 2 illustrates the drug-related geometric mean neopterin concentrations versus time following each treatment Following either product, neopterin concentrations rose during the initial 36 hours; concentrations plateaued from 36 through 72 hours post-dose and then gradually declined. However, neopterin induction was greater for AVONEXTM as compared to Rebif ® . Mean concentrations during the 36 to 72 hours post dose time period were approximately 12.0 nmol L for AVONEXTM and 9.3 nmol L for Rebif ® . Neopterin concentrations at 144 hours post- dose were significantly higher than pre-dose for each treatment (p ⁇ 0.001).
• E AUC pharmacodynamic parameters
• the least squares mean E AUC values for AVONEXTM and Rebif ® were 693 and 481 nmolxh/L, respectively (p ⁇ 0.001).
• the mean ratio of E AUC for AVONEXTM to Rebif ® was 144% with 90% confidence limits of 131 to 159%.
• the least squares mean E ⁇ values for AVONEXTM and Rebif 8 were 9.5 and 6.9 nmol/L, respectively (p ⁇ 0.001).
• the mean ratio of E ⁇ for AVONEXTM to Rebif ® was 138% with 90% confidence limits of 123 to 156%.
• the specific activity of the IFN- ⁇ -la in Rebif ® has been reported to be 3 x 10 8 units/mg (ie. 300 MU of antiviral activity per milligram of IFN- ⁇ -la protein) whereas AVONEXTM has a specific activity of 2 x 10 8 units/mg (14).
• Direct confirmation of this difference could not be obtained because both products are formulated with greater than a 300-fold excess of HSA which interferes with precise determination of IFN- ⁇ -la concentration.
• Table HI summarizes baseline demographics for each group. Age, height and weight were similar across the groups.
• Figure 3 shows mean interferon activity levels by time from the start of the IV infusion.
• Maximum levels, C ⁇ were detected either at 20 minutes (in one subject) or at 30 minutes, i.e., when the infusion was stopped (in all other subjects).
• C ⁇ ranged from 160 to 640 U/mL.
• the data were well described by the two-compartment model. From this model, the mean distribution and elimination half-lives were 4 minutes and 4 hours, respectively.
• the mean volume of distribution was 61.6 L, and mean total clearance was 334 mL/h/kg.
• Mean serum ⁇ 2 -microglobulin and neopterin concentrations by time are shown in Figures 5 and 6, respectively. Peak levels of both markers were observed at either 24 or 48 hours post-dosing.
• This single study has defined the single dose pharmacokinetic and pharmacodynamic response profiles of this particular IFN- ⁇ -la product, AvonexTM, after IV, SC or IM administration. After a 30 minute IV infusion, IFN- ⁇ -la is rapidly redistributed over a few minutes, then cleared more slowly over several hours. The elimination half-life seen in this study was approximately 4 hours compared to the 1.5 to 5 hours reported previously for other interferon beta products. Similarly, the minimally elevated levels of serum interferon activity after SC administration are consistent with other interferon beta products.
• both IFN- ⁇ - lb and the IFN- ⁇ -la used in the current study are formulated at physiologic pH and contain 15 mg mL of human albumin as a stabilizer.(18)
• IFN- ⁇ -lb also contains dextrose, which IFN- ⁇ -la does not.
• the second IFN- ⁇ -la product has a pH of 5-5.5 after reconstitution, and contains mannitol and 9 mg/mL of human albumin as stabilizers (15).
• Values are least squares means transformed back from the logarithmic scale, based on analysis of variance adjusting for subjects and periods. Values are arithmetic means.
• Values are least squares means transformed back from the logarithmic scale, each based on analysis of variance adjusting for subjects and periods. P- values are based on the comparison of the two products estimated from this model. Values are arithmetic means. Figures 1 and 2.
• Fig. 1 Mean Serum activities following IM administration of 6 MU EFN- ⁇ -la in 29 healthy subjects (15 male, 14 female).
• Fig. 2 Serum Neopterin Concentrations - Geometric Means versus Time. Table HI. Summary of demographics.
• Neopterin E ⁇ (nmol/L) - 16.0 ( ⁇ 2.0) 12.4 ( ⁇ 1.5)

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