Kim Gaspar, PhD, is manager of quality assurance at Helix BioPharma.

Kim Gaspar

Proteins perform essential biological functions in living cells and are composed of individual amino acids connected by peptide bonds. With the advent of recombinant deoxyribonucleic acid technology, cost-effective production of protein-based drugs such as hormones, cytokines, and vaccines is possible. Formulation of therapeutic proteins provides different challenges compared with small molecule-based products. The main problems encountered during protein formulation include aggregation, thermal denaturation, oxidation, deamidation, and peptide-bond hydrolysis, all of which can lead to loss of protein potency.   

There are very few marketed topical products containing protein-derived drugs. Regranex gel (becaplermin, Johnson & Johnson, New Brunswick, NJ), which contains platelet-derived growth factor, is one example. Other protein-derived drugs could provide benefit to localized topical therapeutic targets if such topical formulations could  be developed. 

Interferon alpha-2b (IFNα-2b) is a recombinant 19 kDa protein that possesses antiproliferative, immunomodulatory, and antiviral effects (1). IFNα-2b is active against a variety of human papillomavirus (HPV)-induced lesions, particularly cutaneous lesions such as genital warts (i.e., condyloma acuminata). IFNα-2b is also used clinically to treat hairy-cell leukemia, malignant melanoma, follicular lymphoma, AIDS-related Kaposi's sarcoma, chronic hepatitis C, and chronic hepatitis B (2). In addition, it has therapeutic potential against human hyperproliferative and viral diseases such as low-grade squamous intraepithelial lesions (LSIL) (3). All currently approved interferon products such as Intron A (Schering-Plough, Kenilworth, NJ) are injectables (see Table I). To date, no successful topical dosage form has been developed for clinical delivery of IFNα-2b 

Table I: Some commercially available interferon products. 

Microencapsulation technology for interferon 

Biphasix (Helix BioPharma, Aurora, Ontario, Canada) is a microencapsulation platform technology for noninvasive delivery of drugs into or through the skin and mucosa (i.e., dermal or mucosal delivery) in a painless manner. Biphasix is a complex system in which microvesicles, consisting of  lipid bilayers, entrap the oil and aqueous phases as a stabilized emulsion. Biphasix technology allows the formulation of hydrophilic and lipophilic drugs of any size, including small molecules and macromolecules (4, 5).  

Using Biphasix technology, Helix BioPharma developed a specialized cream containing encapsulated IFNα-2b known as Interferon alpha-2b Cream (2 MIU/g). The IFNα-2b is entrapped in specialized liposomes, which are microscopic vesicles composed of a single phospholipid bilayer or multiple concentric lipid bilayers. The initial therapeutic focus was for the vaginal administration of the cream in patients with cervical LSIL.   

Interferon alpha-2b Cream was developed with the following objectives: 

 • Retain the chemical and biological stability of IFNα-2b in the product 

 • Enhance the ability to transport IFNα-2b into the skin or mucosal layers 

• Improve patient compliance through ease of application and emollient properties. 

Identification of Component A in the formulation

The prevention of IFNα-2b oxidation was the most significant challenge in developing a topical IFNα-2b formulation. IFNα-2b contains five methionine residues (Met16, Met21, Met59, Met111, and Met148), which are susceptible to oxidation in solution (6). Giltlin et al. demonstrated that the oxidation of the Met111 residue produced a methioninesulfoxide derivative that has similar biological activity to the native IFNα-2b (7). This derivative is potentially a major oxidative product in Interferon alpha-2b Cream and is generally known as Component A. In the authors' research, activity of IFNα-2b was measured using a cell-based antiviral assay (AVA) that was shown to be nonspecific for the active pharmaceutical ingredient (API) versus Component A. A sensitive reversed-phase high-performance liquid chromatography (RP-HPLC) assay using fluorescence detection was developed to monitor the formation of the oxidative degradation product (Component A) and to quantify the amount of IFNα-2b. 

When analyzing concentrated solutions containing 80µg/g IFNα-2b, Component A was identified as eluting before the principal IFNα-2b peak at approximately 1% of the principal peak area (see Figure 1a). To confirm the formation of Component A, the forced degradation of IFNα-2b in solution showed that while the principal IFNα-2b peak decreased by 16%, a corresponding increase was observed in the Component A peak. The forced degradation of IFNα-2b in solution was conducted in the presence of an oxidizing agent (0.25% hydrogen peroxide [H

]) for 5 h, and the reaction was stopped by the addition of methionine (see Figure 1b) (6). 

Figure 1: Reverse-phase high-performance liquid chromatography assays using fluorescence detection as follows: (a) unoxidized interferon alpha-2b (IFNÎ±-2b) in solution 80 Âµg/g; (b) oxidized IFNÎ±-2b in solution 80 Âµg/g after 5 h; (c) spiked-placebo formulation with stressed active pharmaceutical ingredient and recovery of Component A; (d) active formulation after 5 months at real-time storage; (e) forced degradation of active formulation after 5-h exposure to 0.25% hydrogen peroxide (H2O2); and (f) forced degradation of active formulation after 5-h exposure to 3% H2O2. EU is emission unit; IFN = IFNÎ±-2b. 

The AVA and the enzyme-linked immunosorbent assay detected no change in biological activity and content, respectively, as a result of oxidative stress. Formation of aggregates was not observed upon oxidative stress of IFNα-2b, as suggested by ultraviolet–visible spectroscopic data and sodium dodecyl sulfate polyacrylamide gel electrophoresis studies (Data not shown). 

In the chromatogram of stressed IFNα-2b in solution (see Figure 1b), Component A was positively identified as eluting prior to the principal IFNα-2b peak, and the peak area was used to calculate its theoretical concentration (assuming Component A was equivalent to IFN-a2b). The stressed API solution was used to spike placebo formulation at a target concentration of 125 ng/g Component A (see Figure 1c). The percent recovery of Component A from spiked placebo formulation (see Table II) confirmed that if IFNα-2b had been oxidized in the formulation, the main oxidative product would have been extracted and detected using the RP-HPLC method. 

IFNα2b is present in very small amounts in the Interferon alpha-2b Cream (i.e., about 8 µg/g, or 2MIU/g). The RP-HPLC method requires a sample size of approximately 200 mg for the extraction procedure to isolate IFNα-2b and IFNα-2b-related degradation products from the formulation. When a formulation was analyzed after five months in real-time storage, Component A was not identified (see Figure 1d). The same formulation was forced to degrade by adding 10µL of 0.25% H

 and incubating at room temperature for 1, 3, 4, or 5 h.  After 5 h, approximately 70% IFNα-2b was recovered while Component A was detected at approximately 22% peak area (see Figure 1e).  

Table II: Recovery of Component A from spiked placebo. 

To force the degradation of IFNα-2b further, 30µL of 3% H

 was added to 200 mg of cream, and the samples were left at room temperature for 1, 3, 4, or 5 h. The samples were analyzed using both the RP-HPLC and the AVA methods. Although the IFNα-2b content decreased significantly, the potency of the formulation remained unaffected (see Table III). As indicated in Figure 1f, the IFNα-2b content decreased to approximately 2% while Component A was the principal peak in the chromatogram after the sample oxidized for 5 h at room temperature. 

Table III. IFNÎ±-2b content and potency after forced degradation with 3% hydrogen peroxide. 

As a result of this study, it was concluded that the oxidative degradation product of IFNα-2b, Component A, has a comparable potency to native IFNα-2b and contributes to the overall potency of IFNα-2b in the formulation. The AVA  is therefore nonspecific and cannot differentiate between IFNα-2b and Component A. Consequently, the RP-HPLC assay used in conjunction with the AVA can be used to successfully monitor the stability of IFNα-2b in the cream. 

The initial Interferon alpha-2b Cream formulation was prepared by mixing a proliposomal gel composed of phospholipid, an oil-in-water emulsion, and an aqueous solution of IFNα-2b. The IFNα-2b was highly unstable in this formulation and oxidized into various degradants although the biological activity of IFNα-2b was not affected (Data not shown).   

The oxidation rate of IFNα-2b in the formulation was controlled by adding antioxidants, protein stabilizers, chelating agents, and buffering agents. Processing conditions, such as temperature and the use of an inert environment and storage conditions, were also optimized.  

Besides the IFNα-2b, the lipids present in Interferon alpha-2b Cream are also susceptible to oxidation; therefore, the addition of antioxidants was essential. Various antioxidants, including ascorbic acid, alpha-tocopherol, benzyl alcohol, butylated hydroxyanisole, butylated hydroxytoluene, and methionine, were screened for the ability to protect the components of the product from oxidation and also to prevent auto-oxidation of IFNα-2b .  

The presence of small amounts of metal ions is known to catalyze oxidation reactions. Because trace amounts of metal ions are present in the excipients and equipment, the chelating agent ethylenediaminetetraacetic acid was added to the product. Methionine, which acts as an antioxidant, and glycine, a protein stabilizer, was also added to further inhibit IFNα-2b oxidation.   

Oxidation is also dependent upon the pH of the environment. Different pH conditions using several buffers, such as citrate buffer and phosphate buffers, were explored. Rheological properties were controlled by varying the amounts of viscosity-imparting agents such as white wax, glycerol monostearate, and cetyl alcohol to achieve a desirable consistency and maximum physical stability. 

Formulation screening and lead-formulation selection

Applying the principles previously described led to the development of 39 formulations (i.e., 21Q series). The goal was to improve product stability without compromising physicochemical properties or 

Two trial formulations selected from the 21Q series were screened in an 

 Bronaugh-type (flow-through) diffusion cell model to evaluate IFNα-2b absorption into excised human breast skin. The skin in the diffusion cells was kept at 32 °C and perfused at 37 °C with phosphate-buffered saline containing 5 mM L-methionine and 100 µg/mL bovine serum albumin. IFNα-2b absorption from infinite doses of cream was similar for formulations Q25A and Q25C as measured in the skin samples using AVA (9). 

Three types of toxicology studies were initially conducted using formulation Q25A: skin-sensitization studies in guinea pigs, repeat-dose dermal irritation studies in rabbits, and vaginal irritation studies in rabbits. Formulation Q25A was nonsensitizing in guinea pigs, transiently irritating in the dermal repeated-dose study in rabbits (approximately the first 10 days of the 30-day study), and minimally irritating in the vaginal irritation study. Dermal irritation scores improved dramatically following a slight procedural modification and the quantitative reduction of a single excipient in the formulation to create formulation Q25C. 

Formulation Q25C was found to be chemically stable with no loss of potency for at least nine months when packaged in polypropylene tubes and stored at 2–8 °C. Almost negligible amounts of Component A were formed after 15 months (see Figure 2). Based on the stability, 

 skin absorption, and toxicology data. Formulation Q25C was selected as the lead formulation for clinical development.  

Figure 2: Reverse-phase high-performance liquid chromatogram of Interferon alpha-2b (IFNÎ±-2b) Cream after 15 months at 2â8 Â°C demonstrating the absence of Component A. EU is emission unit. IFN = IFNÎ±-2b. 

Lead formulation composition, preparation, and stability 

The composition of Interferon alpha-2b Cream (Q25C) included the following excipients: benzalkonium chloride, butylated hydroxytoluene, cetyl alcohol, cholesterol, edetate disodium dihydrate, glycine, glycerol monostearate, L-methionine, methylparaben, olive oil (super-refined), polyethylene glycol (PEG 40), castor oil (hydrogenated), phospholipid, propylene glycol, propylparaben, purified water, and phosphate buffer.  

The method of preparation is summarized in Figure 3. All steps after the addition of IFNα-2b are performed under nitrogen. The product is purged with an inert gas before filling into polypropylene tubes. 

Figure 3: Interferon (IFN) alpha-2b Cream manufacturing flow chart. 

Samples were stored in polypropylene tubes at 2–8 °C for long-term stability studies and tested for physical appearance, microscopy, pH, viscosity, preservative concentration, IFNα-2b content, and total chromatographic impurities, IFNα-2b potency and microbial load. Stability was evaluated in terms of changes in IFNα-2b content using the RP-HPLC method (see Figure 4) and the AVA.  

Figure 4: Interferon alpha-2b content (%) over 12 months at 2â8 Â°C in three exhibit lots (4L8358, 4L8368, and 4L8403) of Interferon alpha-2b Cream. 

Stability data collected for 12 months show that lead formulation remains within the predefined limits for IFNα-2b (80–120% of expected) under refrigerated conditions. Studies are underway to collect stability data for up to 24 months. Either IFNα-2b in the product also degrades via pathways other than oxidation, or degradation products were formed in very small amounts that were below the detection limit of the assay. Additional studies to further improve the stability of IFNα-2b are in progress. 

IFNα-2b, which is used in a range of therapeutic indications, was stabilized in a specialized lipid vesicle-based topical Biphasix cream. L-methionine stabilized IFNα-2b throughout the shelf life of the Interferon alpha-2b Cream product by preventing auto-oxidation and the formation of a biologically active methioninesulfoxide derivative of IFNα-2b. A sensitive RP-HPLC method using a fluorescence detector was successfully developed to distinguish IFNα-2b from its oxidative variant. The precise mechanism by which L-methionine protects IFNα-2b in the topical Biphasix cream has not yet been elucidated. The Interferon alpha-2b Cream product has been used in Phase II human trials for treating LSIL, and Phase II studies are in progress to investigate its potential as a treatment for anogenital warts, another HPV-related disorder. The Biphasix technology provides an efficient drug-delivery system for formulating macromolecules into a topical formulation for delivery into or across the skin.   

,* M. Pharm. and PhD, is vice-president of topical drug product development, 

, M. Pharm., is group leader of formulation development, 

, PhD, is manager of quality assurance, and 

, M. Sc., is president and chief operating officer, all with Helix BioPharma, 301–111 Research Drive, Innovation Place, Saskatoon, Saskatchewan, Canada S7N 3R2, tel. 306.934.7471 ext 230, fax 306.934.7453, 

, D. Pharm. Sci. and PhD, is Canada research chair in bionanotechnology and nanomedicine and associate director of research and graduate studies at the School of Pharmacy, University of Waterloo, Ontario, Canada.  

*To whom all correspondence should be addressed. 

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