Status: approved; marketed

Organizations Involved

HemispheRx Biopharma Inc. – Manuf.; R&D; Tech.

Interferon Sciences, Inc. – R&D; Tech.; Former

Stem Cell Innovations, Inc. – Tech.; Former

Purdue Frederick Co. – Former

Armada Health Care – USA mark.

Engitech, Inc. – USA mark.

Lonza Biologics plc – Manuf. other; Tech.

Alternate Site Distributors, Inc. (ASD) – Former

Hoffmann-La Roche Inc. – Tech.; Patent dispute

Schering-Plough Corp. – Tech.; Patent dispute

Sterling Drug, Inc. – Manuf. other

Andromeco – Latin Amer. mark.

Cell Pharm – Europe mark.

Cross ref See the Interferon Products entry (#793); the entry for Multiferon (#796), another leukocyte-derived interferon; and the entry (#794) above for another natural interferon (Wellferon).

Description Interferon alfa-n3 or Alferon N Injection is an aqueous formulation of purified natural interferon alpha proteins obtained from culture of Sendai virus-induced human leukocytes, with purification including immunoaffinity chromatography using matrix-bound interferon alfa-n3 monoclonal antibody (NK2 Mab from Lonza Biologics plc). This is the only natural interferon product currently approved and marketed in the U.S. Interferon alfa-n3 is a mixture of multiple interferon alpha variant proteins (subspecies) of approximately 166 amino acids ranging in molecular weights from 16,000 to 27,000 daltons (16-27 kDa). The specific activity of Interferon alfa-n3 is approximately equal to or greater than 2 x 108 (200 million) IU/mg of protein. Interferon alfa-n3 has ha nominal molecular formala of C860H1353N227O255S9.

Alferon N Injection is manufactured from pooled units of human leukocytes which have been induced by incomplete infection with an avian virus (Sendai virus) to express interferon alfa-n3. Leukocytes are presumably Source Leukocytes from a licensed manufacturer. The Sendai virus infected leukocytes are cultured and express interferon alfa-n3. The culture medium includes the antibiotic neomycin sulfate at a concentration of 35 mg/L, but neomycin sulfate is not detectable in the final product (below 0.64 µg/ml). The manufacturing process includes immunoaffinity chromatography with an interferon alpha murine monoclonal antibody, acidification (at pH 2) for 5 days at 4˚C, and gel filtration chromatography. See the manufacture section for further information.

Alferon N Injection is packaged in 1 ml vials containing 5 million IU of interferon alfa-n3 in a pH 7.4 phosphate buffered saline solution (8.0 mg/ml sodium chloride; 1.74 mg/ml sodium phosphate dibasic; 0.20 mg/ml potassium phosphate monobasic; and 0.20 mg/ml potassium chloride), and also containing phenol (3.3 mg) as a preservative and 1 mg of Albumin (Human) as a stabilizer. The dating period is 12 months from the date of manufacture at 2-8˚C (refrigerated). The date of manufacture is the date of the last valid potency test of the final container material. Interferon Sciences may store product no longer than 6 months at -20˚C.

Nomenclature: Interferon leukocyte/HemispheRx [BIO]; ALFERON N Injection [TR]; Naturaferon [TR Argentina]; Interferon alfa-n3 (Human Leukocyte Derived) [FDA]; Interferon Alfa-n3 [USAN]; Leukocyte Interferon [former USAN]; Interferons, a- [CAS]; 74899-72-2 [CAS RN]; 9008-11-1 [CAS RN, for alpha interferons]; interferon, leukocyte-derived [SY]; alpha-n3 interferon [SY]; Cytopharm [TR in Germany]; Altemol [TR in Mexico]

Companies.: Alferon N was developed and is manufactured by Interferon Sciences, Inc. (ISI), FDA CBER est. no. 0930. In March 2004, HemispheRx Biopharma, Inc. acquired from ISI all Alferon rights, intellectual property, manufacturing and other interferon-related assets. ISI had been in dire financial condition, mostly due to its inability to obtain approval of Alferon for hepatitis C and HIV indications:, and low sales of Alferon. Besides over $7 million of Alferon N Injection in inventory, HemispheRx’s acquisition included ISI’s topical (Alferon N Gel) and oral low-dose (Alferon LDO) formulations of Alferon, and ISI’s 43,000 sq. ft. facility, including a 21,000 sq. ft. manufacturing plant.

In mid-1995, ISI regained nearly all worldwide marketing rights for Alferon N Injection from Purdue Frederick Co. and an affiliate, Mundipharma Pharmaceutical Co. The National Patent Development Corp. (NPDC) was involved in founding ISI and was initially its majority shareholder. NPDC, Mundipharma and Purdue Frederick were all majority owned by members of the Sackler family.

In spring 1997, Alternate Site Distributors, Inc. (ASD; Dallas, TX), a subsidiary of Bergen Brunswig Corp., now AMERISOURCEBERGEN CORP., became the sole U.S. distributor for Alferon N Injection (for ISI). In Aug. 2003, ISI granted Engitech, Inc. U.S. distribution rights (apparently after expiration or withdrawal of rights from ASD). The agreement called for Engitech to deploy a sales force of at least 100 within one year, with expansion to at least 250 in the second year and after.

Fujimoto Pharmaceutical Co. holds Japanese marketing rights. The product is marketed by Cell Pharm in Germany (trade name Cytopharm), and is marketed by Andromeco in Mexico (trade name Altemol).

In Aug. 2006, HemispheRx purchased a royalty interest in the sales of its natural alpha interferon products from Stem Cell Innovations, Inc. (formerly Interferon Sciences, Inc./ISI). This interest on the interferon products was a residual from HemispheRx’s 2004 acquisition of natural interferon and other assets from ISI (see above).

In April 2007, HemispheRx formed a marketing/distribution collaboration with Armada Health Care to launch Alferon N Injection into Armada’s nationwide specialty pharmacies. This is HemispheRx’s first marketing/distribution collaboration. Hemispherx will utilize Armada’s services, including patient and physician clinical education, patient level pharmacy services, reimbursement and data reporting. Armada is the largest Specialty Pharmacy group purchasing organization in the industry; and specializes in serving unique patient populations that often utilize injectable, infusion and biopharmaceutical therapeutics.

Manufacture: The peripheral blood leukocytes (PBL) used for production are presumably Source Leukocytes obtained from licensed manufacturers. The leukocytes are suspended in an induction medium at a desired cell density. The induction culture medium contains human serum virtually free of immune globulin (agamma serum) for alpha interferon production. The medium includes the antibiotic neomycin sulfate at a concentration of 35 mg/L, but neomycin sulfate is not detectable in the final product, i.e., the concentration is below 0.64 µg/ml. Crude or purified leukocyte alpha interferon (about 10-100 IU/ml) is added as a primer to the leukocytes suspended in the induction medium. Optimal priming time for the leukocytes is about 2 to 3 hours prior to the addition of (induction with) Sendai virus at about 36˚C. After priming, Sendai virus, a well-documented inducer of interferon expression by susceptible cells, is added to the suspension of leukocytes (induction) and incubated for up to 48 hours, apparently about 15 to 22 hours at 36˚C.

The crude leukocyte cultures after priming, induction and incubation are either centrifuged (e.g., at about 2,900 g’s for 15-20 minutes) or filtered through suitable cartridge filter systems (e.g., pre-sterilized Polygard-CR high efficiency 0.1 µM pore size cartridge filters connected in series with 0.5 µ pore size Polysep-TP filters from Millipore Corp.). The resulting crude alpha interferon supernatant (crude interferon) is concentrated about 10 to 100 fold (50 fold preferred) by using a tangential flow filter system with a nominal molecular weight cut off of 10,000 Dalton (10 kDa). The concentrated interferon is centrifuged at approximately 9,000 g’s for about 30 minutes. The concentrated interferon can be stored at -70˚C. The concentrated crude interferon is clarified by centrifugation at approximately 17,700 g’s for 60 minutes and is filtered utilizing appropriate cartridge 0.22 or 0.45 micron filters prior to loading onto the monoclonal antibody column.

The crude concentrated alpha interferon is passed through an immunoaffinity chromatography column, NK2 Antibody Affinity Column, using NK2 monoclonal antibody manufactured by Lonza Biologics plc. The monoclonal antibody is coupled to cyanogen bromide (CNBr)-activated Sepharose-4B and stored at 4˚C. The NK2 monoclonal antibody binds only a sub-group of alpha interferon species, i.e., alpha.2(a/b/c), alpha8(a/b/c), alpha10a, alpha17(a/b/c/d), alpha7(a/b/c), alpha6 and alpha21(a/b). It does not bind interferon species alpha1, alpha5, alpha10b, alpha14 or alpha-omega1. The size of the affinity column is dependent on the binding capacity of the affinity gel for interferon, which is determined on each preparation. Columns are prepared by pouring an appropriate amount of Sepharose-antibody gel into a suitable glass column. About 150 million units of crude filtered interferon is loaded per mL of affinity gel.

Purified interferon alfa-n3 is eluted from the column at pH 2 after extensive washing. Interferon units are determined by IRMA (Celltech Ltd.). This antibody affinity chromatography purification removes most of the human serum proteins and other impurities. The purity of interferon at this stage is usually > 90; approximately 80-90% of interferon IRMA activity is recovered; and the interferon is purified about 8,000 fold.

The eluted acidic interferon solution (about pH 2) from the monoclonal affinity column is incubated at 4˚C for a minimum of 5 days. This acid incubation step is used to inactivate potential adventitious agents, such as HIV. After 5 days storage, the pH of the solution is adjusted to 7.4 with 1.0M tris.HCl (hydroxymethylaminomethane). The interferon in the solution is concentrated to ~1-3 mg/mL.

The interferon is subjected to gel filtration chromatography on preparative grade Superose 12 beads (Amersham Pharmacia Biotech). The interferon is loaded and eluted with phosphate buffered saline. All fractions constituting the main peak containing interferon are aseptically pooled. The purified interferon is filtered using a 0.2 micrometer or smaller low-binding filter and stored at -70˚C. Gel filtration removes the high and lower molecular weight impurities, such as murine IgG (antibody) leaked from the NK2 column, human IgG, interferon oligomers, and degraded interferon. The total purification after both affinity and gel filtration chromatography steps results in a recovery of 60-70% of interferon IRMA activity and a total of about 12,000 fold purification.

Purified bulk product is tested for appearance, pH, and for the presence of mouse immune globulin (IgG; residual from monoclonal antibody affinity purification) and human leukocyte DNA. Potency is measured using an interferon antiviral assay involving a cytopathic effect assay using HEp-2 human epidermoid cells infected with vesicular stomatitis virus (VSV). Protein content is measured by the Lowry method. Purity, consistency and identity of the product are confirmed using sodium dodecylsulfate (SDC)-polyacrylamine gel electrophoresis (SDS-PAGE) and Western blot techniques, amino acid sequence analysis, and reverse phase HPLC.

Bulk product manufacturing is performed at ISI’s New Brunswick, NJ, facilities. Final steps, labeling and storage are peformed Sterling Drug, Inc. in McPherson, KA (as reported at the time of original approval; no longer in operation using this name). This facility compounds the final product, sterilizes it by filtration, fills and packages the final containers, and performs some routine quality control testing, e.g., sterility, general safety, and pyrogen endotoxin. The final product is also tested for potency, protein content and identity; and for pH, physical appearance, particulates, sterility, fill volume, phenol content, identification of excipients, general safety (in vivo tests in mice and guinea pigs), and for the presence of endotoxin. The consistency of product from different manufacturing lots has been demonstrated.

Since Alferon N Injection is manufactured using Source Leukocytes, human donor screening (blood serum testing) is performed to minimize the risk that the leukocytes could be contaminated with infectious agents (e.g., HIV-1, hepatitis B virus, HTLV-1). Donor serum is also tested for alanine aminotransferase (ALT) levels (indicative of hepatitis), and donors belonging to high risk groups are eliminated.

The manufacturing process includes multiple steps which have been shown to inactivate viruses (and there has been was no evidence of viral infection among repeat recipients). In its original evaluation, the FDA concluded that the risk for transmission of infectious agents associated with Alferon N is equivalent to that of other blood- and plasma-derived products, such as immune globulin and albumin. In validation studies, Alferon N Injection was shown to be free of detectable Sendai virus (dot-blot hybridization assay), HIV-1 (ELISA plus PCR with sensitivity of 10 copies/ml), HTLV-I (ELISA), hepatitis B virus (RIA for HBsAg), herpes simplex virus type 1 (HSV-1; dot-blot hybridization), cytomegalovirus (CMV; dot-blot hybridization) and Epstein-Barr virus (EBV; dot-blot hybridization). In experiments involving spiking of source leukocytes with levels of model pathogenic viruses higher (109 infectious units/ml) than found in contaminated blood, the manufacturing process was shown to reduce viral titer by 3 x 1014 (over 14 log) for HIV-1, including 3 x 106.5 (6.5 log) reduction by the acid inactivation step and 3 x 107.9 (7.9 log) reduction by the purification processing. In validation studies, there was 108 (8 log) reduction in hepatitis B virus titer (HBsAg assay), and 109 (9 log) reduction in infectious HSV-1. [from Summary Basis of Approval and ISI patents].

FDA class: Biologic PLA (converted to BLA)

CBER class: Biological Response Modifiers

Approvals: Date = 19891010, PLA no. 85-577

Indications: [full text of "INDICATIONS AND USAGE” section from recent product insert/labeling]:

ALFERON N Injection is indicated for the intralesional treatment of refractory or recurring external condylomata acuminata in patients 18 years of age or older (See DOSAGE AND ADMINISTRATION). The physician should select patients for treatment with ALFERON N Injection after consideration of a number of factors: the locations and sizes of the lesions, past treatment and response thereto, and the patient’s ability to comply with the treatment regimen. ALFERON N Injection is particularly useful for patients who have not responded satisfactorily to other treatment modalities, e.g., podophyllin resin, surgery, laser or cryotherapy. There have been no studies with this product in adolescents. This product is not recommended for use in patients less than 18 years of age.

Status: Although showing promise in trials for treatment of HIV, chronic hepatitis C, multiple sclerosis, cancer and other indications:, ISI has been unsuccessful at expanding the approved indications: for Alferon. Multiple failures of Alferon N Injection to show efficacy for new indications: substantially in clinical trials depleted the ISI’s financial resources, and the company divested Alferon, its primary asset, to HemispheRx Biopharma.

In mid-1999, Interferon Sciences, Inc. (ISI) announced that the FDA had advised the company that its completed Phase III trial with Alferon N Injection for treatment of chronic hepatitis C virus infection was insufficient to support approval and that an additional trial would be required. ISI had previously announced that an interim analysis of its Phase III trial with Alferon N Injection for chronic hepatitis C in previously untreated patients found that Alferon N Injection had efficacy equivalent to Intron A (recombinant interferon alfa-2b from Schering-Plough), then the standard-of-care. However, the study protocol (as approved by FDA) required demonstration of superiority of Alferon for its primary endpoint — sustained normalization of liver enzymes at the end of treatment and after six months of follow-up. With the standard-of-care having advanced since this time from alpha interferon monotherapy to the use of pegylated interferons and combinations of interferon (or peginterferon) with oral ribavirin, any new trial would likely require comparisons against these regimens with improved efficacy compared to Intron A, further raising the level of efficacy required for approval and competitive marketing of Alferon N Injection for hepatitis C.

Failure to show efficacy for hepatitis C closely followed a similar earlier conclusion by FDA that ISI’s Phase III trial with Alferon N Injection for treatment of HIV-infection was insufficient to support approval. Alferon N Gel the same active ingredient in a topical gel, was also unsuccessfully tested for treatment of vaginal genital warts.

As of March 2005, a pivotal Phase III trial for chronic fatigue syndrome (CFS) was expected to form the basis of a NDA filing for this indication, but as of 2007.

Tech. transfer: U.S. Patent 5,676,942, “Composition Containing Human alpha Interferon Species Proteins and Method of Use Thereof,” by Testa, D., et al., Jan. 14, 1997, expiring on Oct 14, 2014, assigned to Interferon Sciences; and U.S. 5,503,828, “Alpha Interferon Composition and Method for Its Production from Human Peripheral Blood Leukocytes, by Testa, D., et al., April 2, 1996, expiring Apr 2, 2013, both assigned to Interferon Sciences, describe aspects of manufacture of interferon alfa-n3 from human peripheral blood leukocytes. Example 2 of 5,503,828 apparently describes actual manufacture of interferon alfa-n3.

Interferon Sciences, then a subsidiary of National Patent Development Corp., settled a patent infringement suit and obtained a license from Hoffmann-La Roche (Nutley, NJ) in spring 1988, to market Alferon for genital warts indications:. Roche holds certain rights to patents concerning homogenous natural interferons. See the interferon alpha 2a (Roferon A)(#204) for discussion of Roche’s key natural alpha interferon patents. Roche acquired 5% of Interferon Sciences’ parent company, National Patent Development Corp., and receives unspecified royalties based on sales.

Schering-Plough Corp. filed suit against ISI in 1989 for infringement of key alpha interferon patents it had exclusively licensed from Biogen Inc. (see the interferon alpha-2b or Intron A entry (#206) for further information). In spring 1991, ISI avoided major patent difficulties with Schering-Plough (which has cross-licensed various interferon patents with Roche and others) and expanded the licensed indications: for its interferon products by concluding a licensing agreement with Hoffmann-La Roche to allow ISI to develop Alferon for indications: other than genital warts.

U.S. 3,699,222 describes a classic method interferon production by virus induction of monolayer cell cultures.

EP0625991, "IMPROVED ALPHA INTERFERON COMPOSITION AND METHOD FOR ITS PRODUCTION FROM HUMAN PERIPHERAL BLOOD LEUKOCYTES," assigned to Interferon Sciences, expired in 2013.

Trials: A pivotal Phase III trial for treatment of chronic fatigue syndrome (CFS) was completed in Aug. 2004. This double-blinded, placebo controlled trial in >230 patients will serve as the basis for filing an NDA with the FDA.

In Feb. 2005, HemispheRx initiated a randomized, open-label, dose-ranging study of Alferon LDO, a low dose oral formulation of its leukocyte interferon, in Hong Kong, for treatment of Severe Acute Respiratory Syndrome (SARS). This protocol was designed to respond to anticipated reemergence of SARS with a prophylaxis trial at epidemic sites to evaluate the activity of Alferon LDO to prevent symptomatic infection by SARS.

In Feb. 2006, HemispheRx reported a study indicating that Alferon has potential for treatment of avian flu (influenza virus H1N5). Alferon stimulated a large bank of antiviral immunity-related genes that may control the body’s production of interferon and potentially fight a wide range of diseases, including avian flu.

HemispheRx Biopharma has attempted to develop Alferon for new indications:, e.g., multiple sclerosis (MS), following reports that recombinant interferons appear to be losing efficacy in MS patients after a year. This would likely include study of combination use of Alferon N Injection with the company’s other product Ampligen, a modified dinucleotide drug, currently in Phase III trials for treatment of chronic fatigue syndrome (CFS) and which has shown promise for diseases including HIV/AIDS, hepatitis C, MS, cancer, and human papillomavirus (HPV) infection (genital warts) (see related entry). In July 2004, HemispherRx began an open label trial with Alferon N Injection in subjects with relapsing-remitting multiple sclerosis, who have discontinued interferon-beta therapy because of clinical progression or intolerance, or who have developed neutralizing antibodies to interferon-beta. In early 2004, HemispheRx accelerated development of Alferon LDO (low-dose for oral administration) for treatment of severe acute respiratory syndrome (SARS), due to SARS coronavirus infection, including initiation of clinical trials. In Dec. 2003, the company contracted with Aplicare to package over 100,000 sachets. Alferon N has high potency against the human SARS virus in vitro.

In May 2007, clinical reported in preparation by HemispherRx for Alferon products include Alferon N Injection Phase III trials in China (PRC) for treatment of chronic hepatitis C; a U.S. Phase II Alferon N Injection trial for multiple sclerosis (MS); a U.S Alferon N Injection Phase II/III investigator-sponsored study for treatment of West Nile virus infection; and a Phase I study in Africa using Alferon LDO treatment of HIV-infection. Ongoing trials include a Phase I/II study in the U.S and Hong Kong (inititated in Spring 2007) with an oral formulation, Alferon LDO [Low Dose Oral Interferon Alfa-n3 (Human Leukocyte Derived)], that may be useful for treatment of pandemic influenza and other deadly viral infections. Alferon N Injection and Alferon LDO are in preclinical studies for treatment of avian influenza (influenza virus H5N1; pandemic influenza).

Medical Alferon N Injection is used for second (or later) line treatment of genital warts (condylomata acuminata) due to human papilloma virus (HPV) infection. Other genital warts treatments include topical podophyllin resin or podophyllotoxin, topical imiquimod, and surgical removal of warts, e.g., by laser, electrocauterization or cryosurgery. One or more of these should generally be tried before starting Alferon N therapy for genital warts.

The product in injected intralesionally, i.e., injected into base of each wart, usually with a 30 gauge needle. The recommended dose for treatment of condylomata acuminata is 0.05 ml (250,000 IU) per wart injected twice weekly for up to 8 weeks and up to a maximum of 0.5 ml (2.5 million IU) per treatment session. Injections are often very painful. For large warts, Alferon N may be injected at several points around the periphery of the wart (up to total of 0.045 mL/wart).

Market: The 2007 Average Wholesale Price (AWP) is $ $358.40/5 million IU vial (Red Book, 2007).

Total sales in 2002 were about $2 million (and likely have not increased significantly in recent years). At that time, there was no sales team in place, nor were there any promotional or marketing efforts. Alferon N Injection does not have a large market, as do recombinant interferon products approved for broader and additional indications:. Besides the lack of active marketing, competition from recombinant interferon products has kept sales low.