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Stabilization of Interferon alpha-2b in a Topical Cream
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.
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.
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 (H2O2); and
(f) forced degradation of active formulation after 5-h exposure to 3% H2O2. EU is emission unit; IFN = 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 [H2O2]) 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, 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.
Praveen Kumar, M. Pharm. and PhD, is vice-president of topical drug product development at Helix BioPharma, 301111 Research Drive, Innovation Place, Saskatoon, Saskatchewan, Canada S7N 3R2, tel. 306.934.7471 ext 230, fax 306.934.7453.
Articles by Praveen Kumar
Ravinderjit Batta
Ravinderjit Batta, M. Pharm., is group leader of formulation development at Helix BioPharma.
Articles by Ravinderjit Batta
Kim Gaspar, PhD, is manager of quality assurance at Helix BioPharma.
Articles by Kim Gaspar
John Docherty
John Docherty, M. Sc., is president and chief operating officer at Helix BioPharma.
Articles by John Docherty
Marianna Foldvari
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.
Articles by Marianna Foldvari
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