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]

Definitions

• the present invention relates to the composition for treatment of conditions relating to hepatitis caused by hepatitis C virus (HCV) infection using Interferon- ⁇ (IFN- ⁇ ).
• the present invention also relates to a method of monitoring treatment of HCV by measuring the blood levels of 2', 5'-oligoadenylate synthetase.
• HCV Hepatitis C virus
• HCV is a positive-stranded, lipid-enveloped RNA virus of the Flaviviridae family, approximately ten thousand nucleotides in length (Choo, et al., 1989). HCV, unlike hepatitis B virus, has no DNA intermediate, and therefore cannot be integrated into the host genome (Berenguer, et al., 1996). Although HCV has been cloned, the virus has been difficult to culture in vitro (Trepo, 2000). HCV is extremely persistent, producing a chronic infection in 85% of infected individuals, although the mechanism of this persistence is unknown (Trepo, 2000).
• HCV cirrhosis and hepatocellular carcinoma
• therapies that are currently available for HCV are only effective for a small subpopulation of infected patients (Magrin, et al., 1994; Choo, et al., 1991; Choo, et al., 1989).
• IFN- ⁇ was introduced as therapy for chronic hepatitis C in the United States in 1991 and in Japan in 1992 (Saito, et al., 2000).
• IFN- ⁇ in sufficient dosage to yield clinical efficacy (i.e., at amounts of about 1x10 6 units/treatment and above) is usually associated with a "flu-like" syndrome characterized by fever, headache, lethargy, arthalgias and myalgias (Tyring, et al., 1992).
• other toxicities such as nausea, vomiting, diarrhea and anorexia, become more frequent.
• Neuropsychiatric symptoms have also been reported in association with IFN- ⁇ treatment (Dieperink, et al., 2000).
• IFN- ⁇ treatment is not dose dependent (Saito, et al., 2000), and that treatment with IFN- ⁇ is associated with the development or exacerbation of autoimmune disorders in patients with neoplasms or viral hepatitis (Jimenez-Saenz, et al., 2000).
• Ribavirin (1- ⁇ -D-ribofuranosyl-1 ,2,4-triazole-3-carboxamide) is a purine nucleoside analogue that has been found to interfere with viral mRNA synthesis and to inhibit in vivo and in vitro replication of a wide range of RNA and DNA viruses (Fernandez, et. al., 1986; Balzarini, et al., 1991). Ribavirin has been shown to be efficient in normalizing aminotransferase levels, but has minor activity on serum HCV RNA titres in chronic hepatitis C patients (Di Bisceglie, et. al., 1992).
• the invention includes an oral-delivery composition for use in treating HCV in a HCV-infected patient.
• the composition includes ovine Interferon-tau (OvIFN- ⁇ ), in a dosage effective to stimulate levels of 2', 5'-oligoadenylate synthetase (OAS) observed in the bloodstream 24 hours after administration of the composition.
• OAS oligoadenylate synthetase
• the composition also includes an oral-delivery vehicle containing- IFN- ⁇ and effective to release the IFN- ⁇ in active form in the stomach.
• the composition provides a preferred dose of ovine IFN- ⁇ between 10 8 - 10 10 units.
• the composition provides a preferred dose of ovine IFN- ⁇ between 10 8 - 10 10 units.
• the dosage of ovine IFN- ⁇ is greater than 1 x 10 8 Units/day.
• the dosage of ovine IFN- ⁇ is greater than 2 x 10 8 Units/day.
• the dosage of ovine IFN- ⁇ is greater than 4 x 10 8 Units/day.
• the dosage of ovine IFN- ⁇ is greater than 1 x 10 9 Units/day.
• the dosage of ovine IFN- ⁇ can be greater than 4 x 10 9 Units/day.
• the dosage of ovine IFN- ⁇ is greater than 7 x 10 9 Units/day.
• the composition for treating HCV in a HCV-infected individual comprises ovine IFN- ⁇ in a form that reaches the stomach, but not the tunica mucosa oris and at a dose effective to induce 2', 5'-oligoadenylate synthetase levels measured in the blood 24 hours after oral administration of the composition.
• a preferred dose is between about 10 8 - 10 10 units.
• the composition of the invention includes ovine IFN- ⁇ as an effective ingredient, where the composition avoids the absorption of ovine IFN- ⁇ through the tunica mucosa oris.
• composition of the invention is for the treatment of hepatitis caused by HCV comprises ovine IFN- ⁇ as an effective ingredient, and a 2', 5'- oligoadenylate synthetase activity inducer in animals other than sheep comprising ovine IFN- ⁇ .
• the invention includes a method of monitoring treatment of HCV by oral administration of ovine IFN- ⁇ .
• the method includes measuring the blood levels of 2', 5'-oligoadenylate synthetase prior to and after such oral administration, and if necessary, adjusting the dose of IFN- ⁇ until a measurable increase in blood 2', 5'- oligoadenylate synthetase level, relative to the level observed prior to administration, is observed.
• Figure 1 shows OAS levels in mice whole blood following intraperitoneal (I. P.) or gastric administration (G.A.) of ovIFN- ⁇ .
• Figure 2 shows dose-dependent induction of blood OAS by gastric administration (G.A.) of ovIFN- ⁇ .
• Figures 3-5 illustrate HCV RNA and ALT levels in three human patients following oral administration of 4.9 x 10 8 units/day ovIFN- ⁇ .
• Figures 6 and 7 illustrate HCV RNA and ALT levels in two human patients following oral administration of 1.5 x 10 9 units/day ovIFN- ⁇ .
• Hepatitis C virus or HCV refers to the viral species of which pathogenic types cause Non-A Non-B Hepatitis (NANBH), and attenuated types or defective interfering particles derived therefrom.
• the HCV genome is comprised of RNA.
• RNA containing viruses have relatively high rates of spontaneous mutation reportedly on the order of 10 "3 to 10 "4 per incorporated nucleotide. Since heterogeneity and fluidity of genotype are inherent in RNA viruses, there are multiple types/subtypes, within the HCV species which may be virulent or avirulent. The propagation, identification, detection, and isolation of various HCV types or isolates is documented in the literature.
• Treating a condition refers to administering a therapeutic substance effective to reduce the symptoms of the condition and/or lessen the severity of the condition.
• Oral refers to any route that involves administration by the mouth or direct administration into the stomach or intestines, including gastric administration.
• OAS level refers to the concentration or activity of blood 2', 5'-oligoadenylate synthetase (OAS) protein.
• Recombinant host cells, host cells, cells, cell lines, cell cultures, and other such terms denoting microorganisms or higher eukaryotic cell lines cultured as unicellular entities, are used interchangeably, and refer to cells which can be, or have been, used as recipients for recombinant vector or other transfer DNA, and include the progeny of the original cell transfected. It is understood that the progeny of a single parental cell may not necessarily be completely identical in morphology or in genomic or total DNA complement as the original parent, due to accidental or deliberate mutation.
• Progeny of the parental cell which are sufficiently similar to the parent to be characterized by the relevant property, such as the presence of a nucleotide sequence encoding a desired peptide, are included in the progeny intended yb this definition, and are covered by the above terms.
• Operably linked refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner.
• a control sequence operably linked to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences.
• An open reading frame is a region of a polynucleotide sequence which encodes for a polypeptide.
• Ovine IFN- ⁇ refers to a protein having the amino acid sequence as shown in Figure 4, and to proteins having amino acid substitutions and alterations such as neutral amino acid substitutions that do not significantly affect the activity of the protein.
• the sequence includes the ovine IFN- ⁇ sequence of Figure 4 and the proteins with 90% sequence homology to the sequence shown in Figure 4..
• Amino acid homology can be determined using, for example, the ALIGN program with default parameters. This program is found in the FASTA version 1.7 suite of sequence comparison programs
• Interferon- ⁇ The first IFN- ⁇ to be identified was ovine IFN- ⁇ (OvIFN- ⁇ ), as a 18-19 kDa protein.
• OvIFN- ⁇ was originally called ovine trophoblast protein-one (oTP-1) because it was the primary secretory protein initially produced by trophectoderm of the sheep conceptus during the critical period of maternal recognition in sheep. Subsequent experiments have determined that OvIFN- ⁇ is a pregnancy recognition hormone essential for establishment of the physiological response to pregnancy in ruminants, such as sheep and cows (Bazer and Johnson, 1991).
• IFN- ⁇ cDNA obtained by probing a sheep blastocyst library with a synthetic oligonucleotide representing the N-terminal amino acid sequence (Imakawa, et al., 1987) has a predicted amino acid sequence that is 45-55% homologous with IFN- ⁇ s from human, mouse, rat and pig and 70% homologous with bovine IFN- ⁇ ll, now referred to as IFN- ⁇ .
• IFN- ⁇ Several cDNA sequences have been reported which may represent different isoforms (Stewart, et al., 1989; Klemann, et al., 1990; and Charlier, M., et al., 1991).
• IFN- ⁇ All are approximately 1 kb with a 585 base open reading frame that codes for a 23 amino acid leader sequence and a 172 amino acid mature protein.
• the predicted structure of IFN- ⁇ as a four helical bundle with the amino and carboxyl-termini in apposition further supports its classification as a type I IFN (Jarpe, et. al., 1994).
• IFN- ⁇ displays many of the activities classically associated with type I IFNs (see Table 1, above), considerable differences exist between it and the other type I IFNs. The most prominent difference is its role in pregnancy, detailed above. Also different is viral induction. All type I IFNs, except IFN- ⁇ , are induced readily by virus and dsRNA (Roberts, et al., 1992). Induced IFN- ⁇ and IFN- ⁇ expression is transient, lasting approximately a few hours. In contrast, IFN- ⁇ synthesis, once induced, is maintained over a period of days (Godkin, et al., 1982). On a per-cell basis, 300-fold more lFN- ⁇ is produced than other type I IFNs (Cross and Roberts, 1991).
• IFN- ⁇ expression may also differ between species. For example, although IFN- ⁇ expression is restricted to a particular stage (primarily days 13-21) of conceptus development in ruminants (Godkin, et al., 1982), preliminary studies suggest that the human form of IFN- ⁇ is constitutively expressed throughout pregnancy (Whaley, et al., 1994).
• OvIFN- ⁇ protein may be isolated from conceptuses collected from pregnant sheep and cultured in vitro in a modified Minimum Essential Medium (MEM) as described by Godkin, et al., (1982) and Vallet, et. al., (1987).
• MEM Minimum Essential Medium
• the IFN- ⁇ may be purified from the conceptus cultures by ion exchange chromotography and gel filtration.
• the homogeneity of isolated IFN- ⁇ may be assessed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE; Maniatis, et al., 1982; Ausubel, et al., 1988), and determination of protein concentration in purified IFN- ⁇ samples may be performed using the bicinchoninic (BCA) assay (Pierce Chemical Co., Rockford, IL; Smith, et. al., 1985).
• SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis
• BCA bicinchoninic
• Recombinant IFN- ⁇ protein may be produced from any selected IFN- ⁇ polynucleotide fragment using a suitable expression system, such as bacterial or yeast cells.
• a suitable expression system such as bacterial or yeast cells.
• the isolation of IFN- ⁇ nucleotide and polypeptide sequences is described in Bazer, et al. (1994). For example, Bazer, etal., describe the identification and isolation of a human IFN- ⁇ gene.
• an IFN- ⁇ coding sequence (e.g, SEQ ID NOS:1 or 3) is placed in an expression vector, e.g., a bacterial expression vector, and expressed according to standard methods.
• suitable vectors include lambda gt11 (Promega, Madison Wl); pGEX (Smith, et al., 1985); pGEMEX (Promega); and pBS (Strategene, La Jolla CA) vectors.
• Other bacterial expression vectors containing suitable promoters, such as the T7 RNA polymerase promoter or the tac promoter, may also be used.
• OvIFN- ⁇ synthetic polynucleotide into a modified plN III omp-A expression vector is described in the Materials and Methods.
• the OvIFN- ⁇ coding sequence present in SEQ ID NO:3 was cloned into a vector, suitable for transformation of yeast cells, containing the methanol-regulated alcohol oxidase (AOX) promoter and a Pho1 signal sequence.
• the vector was used to transform P. pastoris host cells and transformed cells were used to express the protein according to the manufacturer's instructions (Invitrogen, San Diego, CA).
• yeast vectors suitable for expressing IFN- ⁇ for use with methods of the present invention include 2 micron plasmid vectors (Ludwig, et al., 1993), yeast integrating plasmids (Yips; e.g., Shaw, et al., 1988), YEP vectors (Shen, et al., 1986), yeast centromere plasmids (YCps; e.g.), and other vectors with regulatable expression
• the vectors include an expression cassette containing an effective yeast promoter, such as the MF ⁇ 1 promoter (Bayne, et. al., 1988, GADPH promoter (glyceraldehyde-3-phosphate- dehydrogenase; Wu, et. al., 1991) or the galactose-inducible GAL10 promoter (Ludwig, et. al., 1993; Feher, et. al., 1989; Shen, et al., 1986).
• the yeast transformation host is typically Saccharomyces cerevisiae, however, as illustrated above, other yeast suitable for transformation can be used as well (e.g., Schizosaccharomyces pombe, Pichia pastoris and the like).
• a DNA encoding an IFN- ⁇ polypeptide can be cloned into any number of commercially available vectors to generate expression of the polypeptide in the appropriate host system.
• These systems include the above described bacterial and yeast expression systems as well as the following: baculovirus expression (Reilly, et al., 1992; Beames, et. al., 1991; Clontech, Palo Alto CA); plant cell expression, transgenic plant expression, and expression in mammalian cells (Clontech, Palo Alto CA; Gibco-BRL, Gaithersburg MD).
• the recombinant polypeptides can be expressed as fusion proteins or as native proteins.
• a number of features can be engineered into the expression vectors, such as leader sequences which promote the secretion of the expressed sequences into culture medium.
• the recombinantly produced polypeptides are typically isolated from lysed cells or culture media. Purification can be carried out by methods known in the art including salt fractionation, ion exchange chromatography, and affinity chromatography. Immunoaffinity chromatography can be employed, as described above, using antibodies generated based on the IFN- ⁇ polypeptides.
• IFN- ⁇ proteins or polypeptides can be isolated from selected cells by affinity-based methods, such as by using appropriate antibodies. Further, IFN- ⁇ peptides (e.g. SEQ ID NOS:2 or 4) may be chemically synthesized using methods known to those skilled in the art. III. IFN- ⁇ as a Treatment for HCV
• compositions and methods of the present invention may be used to therapeutically treat and thereby alleviate hepatitis caused by HCV.
• a person suffering from chronic hepatitis C infection may exhibit one or more of the following signs or symptoms: (a) elevated alanine aminotransferase (ALT), (b) positive test for anti-HCV antibodies, (c) presence of HCV as demonstrated by a positive test for HCV-RNA, (d) clinical stigmata of chronic liver disease, (e) hepatocellular damage, and/or (f) altered blood levels of 2', 5'- oligoadenylate synthetase.
• ALT elevated alanine aminotransferase
• HCV-RNA a positive test for anti-HCV antibodies
• HCV-RNA a positive test for HCV-RNA
• clinical stigmata of chronic liver disease hepatocellular damage
• f altered blood levels of 2', 5'- oligoadenylate synthetase.
• Such criteria may not only
• OAS 2', 5'-oligoadenylate synthetase
• Examples 1 and 3 IFN- ⁇ , administered orally, was tested for its ability to induce OAS.
• OvIFN- ⁇ was administered either orally or intraperitoneally to mice or human patients. OAS activity in whole blood in mice was determined, and is shown in Figure 1, 24 hours after IFN- ⁇ administration. Several human patients had 2 to 12 fold increases in their OAS enzyme activity levels as shown in Tables 3-6.
• IFN- ⁇ Induces OAS in a Dose-dependent Manner.
• IFN- ⁇ administered orally in mice, was tested for its ability to induce OAS in a dose-dependent manner.
• OvIFN- ⁇ was orally administered in units of 0, 1x10 3 , 1x10 4 , 1x10 5 to an upper part of a mouse stomach. Twelve hours after oral administration, whole blood was taken from a mouse heart and an OAS activity of whole blood was determined. As shown in Figure 2, the OAS activity in whole blood increased in a dose dependent manner.
• IFN- ⁇ is orally active (WO 96/28183)
• no exact determination has previously been made as to how IFN- ⁇ was administered, or as to how IFN- ⁇ is absorbed.
• IFN- ⁇ was directly administered into the mouse stomach without any exposure to the tunica mucosa oris, conclusively esablishing that absorption through the stomach mucosal membrane effectively induces OAS activity.
• Direct absorption of IFN- ⁇ from the stomach would diminish antibody formation against IFN- ⁇ compared to IFN- ⁇ absorbed through the oral mucosal membrane, particularly in the case of chronic administrations of IFN- ⁇ .
• the present invention describes the ability of ovine IFN- ⁇ to increase 2', 5'-oligoadenylate synthase activity in mice and humans. Prior to this work, only mouse IFN- ⁇ had been known to be effective in mice.
• compositions Therapeutic preparations or medicaments containing IFN- ⁇ or related polypeptides or proteins can be formulated and manufactured according to known methods for preparing pharmaceutically useful compositions (medicaments). Formulations comprising interferons or interferon-like compounds have been previously described (e.g., Martin, 1976). In general, the IFN- ⁇ -containing medicaments are formulated such that an effective amount of the IFN- ⁇ is combined with a suitable carrier and/or excipient in order to facilitate effective administration of the composition. IFN- ⁇ , or related polypeptides, may be administered to a patient in any pharmaceutically acceptable dosage form, including intravenous, intramuscular, intralesional, or subcutaneous injection. Specifically, compositions and methods used for other interferon compounds can be used for the delivery of these compounds.
• tablets and capsules containing IFN- ⁇ may be manufactured from IFN- ⁇ (e.g., lyophilized IFN- ⁇ protein) and, optionally, additives such as pharmaceutically acceptable carriers (e.g., lactose, corn starch, light silicic anhydride, microcrystalline cellulose, sucrose), binders (e.g., alpha-form starch, methylcellulose, carboxymethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone), disintegrating agents (e.g., carboxymethylcellulose calcium, starch, low substituted hydroxy-propylcellulose), surfactants (e.g., Tween 80, polyoxyethylene-polyoxypropylene copolymer), antioxidants (e.g., L-cysteine, sodium sulfite, sodium ascorbate), lubricants (e.g., magnesium stearate, talc), and the like.
• pharmaceutically acceptable carriers e.g., lactose
• IFN- ⁇ polypeptides can be mixed with a solid, pulverulent or other carrier, for example lactose, saccharose, sorbitol, mannitol, starch, such as potato starch, corn starch, millopectine, cellulose derivative or gelatine, and may also include lubricants, such as magnesium or calcium stearate, or polyethylene glycol waxes compressed to the formation of tablets.
• a solid, pulverulent or other carrier for example lactose, saccharose, sorbitol, mannitol, starch, such as potato starch, corn starch, millopectine, cellulose derivative or gelatine
• lubricants such as magnesium or calcium stearate, or polyethylene glycol waxes compressed to the formation of tablets.
• Liquid preparations for oral administration can be made in the form of elixirs, syrups or suspensions, for example solutions containing from about 0.1% to about 30% by weight of IFN- ⁇ , sugar and a mixture of ethanol, water, glycerol, propylene, glycol and possibly other additives of a conventional nature.
• An orally active IFN- ⁇ pharmaceutical composition is administered in a therapeutically effective amount to an individual in need of treatment.
• the dose may vary considerably and is dependent on factors such as the seriousness of the disorder, the age and the weight of the patient, other medications that the patient may be taking and the like. This amount or dosage is typically determined by the attending physician.
• the dosage will typically be between about 1x10 5 and 1x10 10 units/day, preferably between about 1x10 8 and 1.5x10 9 units/day. It will be appreciated that because of its lower toxicity, IFN- ⁇ can be administered at higher doses than, for example, IFN- ⁇ .
• disorders requiring a steady elevated level of IFN- ⁇ in plasma will benefit from oral administration as often as about every two to four hours or administration via injection about every 12-24 hours, while other disorders may be effectively treated by administering a therapeutically-effective dose at less frequent intervals, e.g., once every 48 hours.
• the rate of administration of individual doses is typically adjusted by an attending physician to enable administration of the lowest total dosage while alleviating the severity of the disease being treated.
• a maintenance dose is administered if necessary. Subsequently, the dosage or the frequency of administration, or both, may be reduced, as a function of the symptoms, to a level at which the improved condition is retained.
• compositions and methods of this invention may be used in combination with other therapies.
• the composition of ovIFN- ⁇ for the treatment of HCV in a HCV-infected patient can be combined with an anti-viral agent such as ribavirin.
• an anti-viral agent such as ribavirin.
• Treatment of HCV by oral administration of ovIFN- ⁇ is monitored by measuring the blood levels of 2', 5'-oligoadenylate synthetase (OAS) prior to and following administration.
• OAS 2', 5'-oligoadenylate synthetase
• the OAS levels can be monitored, for example, at 12, 24, and 48 hours after administration. If necessary, the dose of IFN- ⁇ is adjusted until a measurable increase in blood OAS levels is observed, relative to the level observed prior to administration.
• a synthetic OvIFN- ⁇ gene was generated using standard molecular methods (Ausubel, et al., 1988) by ligating oligonucleotides containing contiguous portions of a DNA sequence encoding the OvIFN- ⁇ amino acid sequence.
• the DNA sequence used may be either SEQ ID NO:1 or 3 or the sequence as shown in Imakawa, et. al., 1987.
• the resulting IFN- ⁇ polynucleotide coding sequence may span position 16 through 531: a coding sequence of 172 amino acids.
• the full length synthetic gene Stul/SStl fragment (540 bp) may be cloned into a modified plN III omp-A expression vector and transformed into a competent SB221 strain of £. coli.
• cells carrying the expression vector were grown in L-broth containing ampicillin to an OD (550 nm) of 0.1 - 1 , induced with IPTG (isopropyl-1-thio-b-D-galactoside) for 3 hours and harvested by centrifugation. Soluble recombinant IFN- ⁇ may be liberated from the cells by sonication or osmotic fractionation.
• the IFN- ⁇ gene may amplified using polymerase chain reaction (PCR; Mullis, 1987; Mullis, et al., 1987) with PCR primers containing Stu I and Sacl restriction sites at the 5' and 3' ends, respectively.
• the amplified fragments were digested with Stul and Sacll and ligated into the Sacll and Smal sites of pBLUESCRIPT+(KS), generating pBSY-IFN ⁇ .
• Plasmid pBSY-IFN ⁇ was digested with Sacll and EcoRV and the fragment containing the synthetic IFN- ⁇ gene was isolated.
• the yeast expression vector pBS24Ub (Ecker, et. al., 1989) was digested with Sail.
• Blunt ends were generated using T4 DNA polymerase.
• the vector DNA was extracted with phenol and ethanol precipitated (Sambrook, et. al., 1989).
• the recovered plasmid was digested with Sacll, purified by agarose gel electrophoresis, and ligated to the Sacll-EcoRV fragment isolated from pBSY-IFN- ⁇ .
• the resulting recombinant plasmid was designated pBS24Ub-IFN ⁇ .
• the recombinant plasmid pBS24Ub-IFN ⁇ was transformed into E. coli.
• Recombinant clones containing the IFN- ⁇ insert were isolated and identified by restriction enzyme analysis.
• IFN- ⁇ coding sequences were isolated from pBS24Ub-IFN ⁇ and cloned into a Pichia pastoris vector containing the alcohol oxidase (AOX1) promoter (Invitrogen, San Diego, CA).
• AOX1 promoter Alcohol oxidase
• the vector was then used to transform Pichia pastoris GS115 His " host cells and protein was expressed following the manufacturer's instructions.
• the protein was secreted into the medium and purified by successive DEAE-cellulose and hydroxyapatite chromatography to electrophoretic homogeneity as determined by SDS- PAGE and silver staining.
• the purified IFN- ⁇ protein has a specific activity of about 0.29 to about 0.44x10 8 U/mg as measured by anti-viral activity on Madin-Darby bovine kidney (MDBK) cells. In another embodiment, the protein has a specific activity of about 4.9 x 10 8 U/mg as measured by the anti-viral activity bioassay.
• OvIFN- ⁇ 4.99x10 8 units/mg protein; Pepgen Corp., California or Biological Process Development Facility, Dept. of Food Science and Technology, University of NE-Lincoln, Lincoln, NE; SEQ ID NO:4
• OvIFN- ⁇ 4.99x10 8 units/mg protein
• Pepgen Corp. California or Biological Process Development Facility, Dept. of Food Science and Technology, University of NE-Lincoln, Lincoln, NE; SEQ ID NO:4
• OvIFN- ⁇ SEQ ID NO:2
• ovIFN- ⁇ solution Two hundred microliters of ovIFN- ⁇ solution was orally administered to ICR mice (average body weight approximately 30g, 6 weeks of age, female) using a 20 gauge disposable oral sound (Fuchigami, Kyoto) to inject directly to an upper part of the stomach (gastric administration; GA).
• ovIFN- ⁇ solution For intra peritoneal administration (I. P.), 100 microliters of ovIFN- ⁇ solution was used. Sample injection to an upper part of a stomach was confirmed by administration of a dye. Twenty-four hours after the administration, the mouse was anesthetized with Nembutal. Blood was taken from a heart of the mouse and an OAS activity in whole blood was determined by 2-5A RIA Kit (Eiken Chemical, Tokyo; Shindo et. al., 1989).
• Ov-IFN- ⁇ SEQ ID NO:4
• Ov-IFN- ⁇ SEQ ID NO:2
• SEQ ID NO:2 may also be prepared and administered in the same manner.
• the patient keeps all vials of test material and syringes in the refrigerator maintained at 2 to 8 degrees centrigrade. Prior to the self-administration of medication, the patient removes one vial and one syringe from the refrigerator. The patient removes the cap from the tip of the syringe, places the tip of the syringe into the bottle of medication and withdraws the appropriate amount of drug into the syringe as instructed at the clinic on Day 1.
• the patient places the tip of the syringe in the mouth and empties the contents of the syringe into the mouth by depressing the plunger.
• the patient then swallows the test material.
• the patient may then drink a glass of water.
• the patient notes on his/her diary card the date and time the dose of test material was administered.

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