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roteins perform essential biological functions in living cells and are composed of individual amino acids con-nected by peptide bonds. With the advent of recombinant deoxyribonucleic acid technology, cost-effective produc-

tion of protein-based drugs such as hormones, cytokines, and vaccines is possible. Formulation of therapeutic proteins pro-vides different challenges compared with small molecule-based products. The main problems encountered during protein for-mulation 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, immunomodu-latory, and antiviral effects (1). IFNα-2b is active against a variety of human papillomavirus (HPV)-induced le-sions, 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 hepa-titis C, and chronic hepatitis B (2). In addition, it has thera-peutic 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 inject-ables (see Table I). To date, no successful topical dosage form has been developed for clinical delivery of IFNα-2b via the skin or mucosa.

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 stabi-lized emulsion. Biphasix technology allows the formulation

Stabilization of Interferon alpha-2b in a Topical CreamPraveen Kumar, Ravinderjit Batta, Geriene LaBine, Jinghui Shen, Kim Gaspar, John Docherty, and Marianna Foldvari

Protein-based drugs are common therapeutic options, but their administration has been limited primarily to injection because of formulation challenges. The authors describe a proprietary process for producing a stable, topical interferon alpha-2b formulation that can deliver large drug molecules into the skin or mucosa. They describe the resolution of one of the largest challenges to topical protein formulations, namely oxidation and discuss excipient selection, formulation optimization, and lead formulation selection.

Praveen Kumar,* M. Pharm. and PhD, is vice-president of topical drug product development, Ravinderjit Batta, M. Pharm., is group leader of formulation development, Geriene LaBine, M. Sc., is head of quality control, Jinghui Shen, B. Eng., is an analytical chemist, Kim Gaspar, PhD, is manager of quality assurance, and John Docherty, 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, pkumar@helixbiopharma.com. Marianna Foldvari, 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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Drug Delivery

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 mul-tiple concentric lipid bilayers. The initial therapeutic focus was for the vaginal administration of the cream in patients with cervical LSIL.

Development of Interferon alpha-2b CreamInterferon alpha-2b Cream was developed with the follow-ing objectives:

Retain the chemical and biological stability of IFNα-2b in the productEnhance the ability to transport IFNα-2b into the skin or mucosal layersImprove patient compliance through ease of application and emollient properties.

Identification of Component A in the formulationThe prevention of IFNα-2b oxidation was the most signifi-cant challenge in developing a topical IFNα-2b formulation. IFNα-2b contains five methionine residues (Met16, Met21, Met59, Met111, and Met148), which are susceptible to oxi-dation in solution (6). Giltlin et al. demonstrated that the oxidation of the Met111 residue produced a methionine-sulfoxide 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 gen-erally known as Component A. In the authors’ research, activity of IFNα-2b was measured using a cell-based anti-viral assay (AVA) that was shown to be nonspecific for the active pharmaceutical ingredient (API) versus Component A. A sensitive reversed-phase high-performance liquid chro-matography (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

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the principal IFNα-2b peak at approximately 1% of the prin-cipal 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 Compo-nent A peak. The forced degradation of IFNα-2b in solution was conducted in the presence of an oxidizing agent (0.25% hydrogen peroxide [H

2O

2]) for 5 h, and the reaction was

stopped by the addition of methionine (see Figure 1b) (6).The AVA and the enzyme-linked immunosorbent assay

detected no change in biological activity and content, re-spectively, as a result of oxidative stress. Formation of ag-gregates 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 elut-ing 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-α2b). 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 pla-cebo 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 Inter-feron 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 formula-tion. 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

2O

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perature 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).

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

2O

2 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.

As a result of this study, it was concluded that the oxidative degradation product of IFNα-2b, Component A, has a compa-rable 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

Table I: Some commercially available interferon products.Generic name Trade name

Interferon alpha-2a roferon a

Interferon alpha-2b Intron a

Interferon alpha-N3 alferon N injection

Interferon beta-1a rebif

Interferon beta-1a avonex

Interferon beta-1b Betaseron

Interferon alphacon-1 Infergen

Interferon gamma-1b actimmune

Drug Deliveryf

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Component A. Consequently, the RP-HPLC assay used in conjunc-tion with the AVA can be used to successfully monitor the stability of IFNα-2b in the cream.

Formulation developmentThe 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 formu-lation and oxidized into various degradants although the biologi-cal activity of IFNα-2b was not af-fected (Data not shown).

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

Besides the IFNα-2b, the lip-ids present in Interferon alpha-2b Cream are also susceptible to oxidation; therefore, the addition of antioxidants was essential. Various antioxidants, includ-ing ascorbic acid, alpha-tocoph-erol, benzyl alcohol, butylated hydroxyanisole, butylated hy-droxytoluene, 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 cata-lyze 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 environ-ment. Different pH conditions using several buffers, such as citrate buffer and phosphate buffers, were explored. Rheo-

logical properties were controlled by varying the amounts of viscosity-imparting agents such as white wax, glycerol monostearate, and cetyl alcohol to achieve a desirable con-sistency and maximum physical stability.

Formulation screening and lead-formulation selectionApplying the principles previously described led to the de-

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Figure 1: Reverse-phase high-performance liquid chromatography assays using fluorescence detection as follows: (a) unoxidized interferon alpha-2b (IFNα-2b) in solution 80 μg/g; (b) oxidized IFNα-2b in solution 80 μg/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 (H

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velopment of 39 formulations (i.e., 21Q series). The goal was to improve product stability without compromising physico-chemical properties or in vitro skin delivery of IFNα-2b.

Two trial formulations selected from the 21Q series were screened in an in vitro 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 ab-sorption 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 irritat-ing in the vaginal irritation study. Dermal irritation scores improved dramatically following a slight procedural modi-fication 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, in vitro skin absorption, and toxicology data. Formulation Q25C was selected as the lead formulation for clinical development.

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, ede-tate 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.

Samples were stored in polypropylene tubes at 2–8 °C for long-term stability studies and tested for physical appear-ance, 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.

Lead formulation improvementStability data collected for 12 months show that lead formula-tion 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

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

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at room temperature

IFNα-2b content (%) by RP-HPLC

IFNα-2b potency (%) by AVA

control 0 99.55 101.20

a 1 33.80 92.60

B 3 12.30 91.69

c 4 4.74 90.90

D 5 1.90 91.38

IfNα-2b is interferon alpha-2b. rP-hPLc is reverse-phase high-performance liquid chromatography. aVa is antiviral assay.

Table II: Recovery of Component A from spiked placebo.Sample Spiked placebo

(Concentration, ng/g)Theoretical (Component A) equivalent to IFNα-2b (μg/g)

Observed (Component A) equivalent to IFNα-2b (μg/g)

Percent recovery (%)

Mean percent recovery (%)

a 123.80 0.0449 0.044 98.00103.30 (rsD = 5.22%)

B 123.49 0.0442 0.048 108.60

c 119.58 0.0438 0.046 105.02

IfNα-2b is interferon alpha-2b. rsD is relative standard deviation.

Drug Delivery

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.

ConclusionIFNα-2b, which is used in a range of therapeutic indications, was stabilized in a specialized lipid vesicle-based top-ical Biphasix cream. L-methionine sta-bilized IFNα-2b throughout the shelf life of the Interferon alpha-2b Cream product by preventing auto-oxidation and the formation of a biologically ac-tive methioninesulfoxide derivative of IFNα-2b. A sensitive RP-HPLC method using a fluorescence detector was suc-cessfully 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 technol-ogy provides an efficient drug-delivery system for formulating macromol-ecules into a topical formulation for delivery into or across the skin.

References 1. “Interferon-α” in Human Cytokines: Handbook for Basic and

Clinical Research, B.B. Aggarwal, and J.U. Gutterman, Eds. (Blackwell Scientific Publications, Cambridge, MA, 1992), pp. 13–15.

2 C.E. Samuel, “Antiviral Actions of Interferons,” Clin. Microbiol. Rev. 14 (4), 778–809 (2001).

3. R. Cirelli and K. Tyring, “Interferons in Human Papillomavirus Infec-tions,” Antiviral Res. 24 (2–3), 191–204 (1994).

4. M.J. King et al., “Transdermal Delivery of Insulin from a Novel Bi-phasic Lipid System in Diabetic Rats,” Diabetes Technol. Ther. 4 (4), 479–488 (2002).

5. M. Foldvari et al., “Biphasic Vesicles as a Topical Delivery System for Interferon Alpha,” submitted for publication.

6. M. Cindric et a l., “Evaluation of Recombinant Human Interferon α-2b Structure and Stability by In-gel Tryptic Digestion, H/D Exchange and Mass Spectroscopy,” J. Pharm. Biomed. Anal. 40 (3), 781–787 (2006).

7. G. Giltlin et al., “Isolation and Characterization of a MonomeThi-oninesulfoxide Variant of Interferon α-2b,” Pharm. Res. 13 (5), 762–769 (1996).

8. K.H. Buchheit, A. Daas, and K.H. Jönsson, “Collaborative Study for Establishment of an HPLC Method for Batch Consistency Control of Recombinant Interferon-alfa-2,” Pharmeuropa Spec. Issue Biol. 2002 (1), 7–21 (2002).

9. K.J. Gaspar et al., “In Vitro Absorption of Interferon Alpha-2b From a Topical Biphasix Formulation,” manuscript in preparation. PT

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

1. Preparation of submicron emulsion

2. Preparation of aqueous phase with active pharmaceutical ingredient

3. Preparation of lipid phase

Preparation of water phase

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Heat and mix

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proliposomal gel

Cool

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Mix with protein stabilizer in aqueous solution

Mix IFN solution with submicron emulsion and cool

Add the mixture to proliposome gel and mix

Formation of bulk product

Package product in tubes

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

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© reprinted from Pharmaceutical technology supplement, July 2009 Printed in u.s.a.