US20050244373A1 - Composition for treatment of and method of monitoring hepatitis C virus using interferon-tau - Google Patents

Composition for treatment of and method of monitoring hepatitis C virus using interferon-tau Download PDF

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US20050244373A1
US20050244373A1 US11/177,010 US17701005A US2005244373A1 US 20050244373 A1 US20050244373 A1 US 20050244373A1 US 17701005 A US17701005 A US 17701005A US 2005244373 A1 US2005244373 A1 US 2005244373A1
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Yoshihiro Sokawa
Chih-Ping Liu
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Pepgen Corp
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    • 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]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses

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- ⁇ ).
  • HCV hepatitis C virus
  • IFN- ⁇ Interferon- ⁇
  • 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 1 ⁇ 10 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).
  • 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 2′,5′-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 ⁇ 10 8 Units/day.
  • the dosage of ovine IFN- ⁇ is greater than 2 ⁇ 10 8 Units/day.
  • the dosage of ovine IFN- ⁇ is greater than 4 ⁇ 10 8 Units/day.
  • the dosage of ovine IFN- ⁇ is greater than 1 ⁇ 10 9 Units/day.
  • the dosage of ovine IFN- ⁇ can be greater than 4 ⁇ 10 9 Units/day.
  • the dosage of ovine IFN- ⁇ is greater than 7 ⁇ 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.
  • FIG. 1 shows OAS levels in mice whole blood following intraperitoneal (I.P.) or gastric administration (G.A.) of ovIFN- ⁇ .
  • FIG. 2 shows dose-dependent induction of blood OAS by gastric administration (G.A.) of ovIFN- ⁇ .
  • FIGS. 3-5 illustrate HCV RNA and ALT levels in three human patients following oral administration of 4.9 ⁇ 10 8 units/day ovIFN- ⁇ .
  • FIGS. 6 and 7 illustrate HCV RNA and ALT levels in two human patients following oral administration of 1.5 ⁇ 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 fines, 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 FIG. 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 FIG. 4 and the proteins with 90% sequence homology to the sequence shown in FIG. 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 (Pearson and Lipman, 1988; Pearson, 1990; program available from William R. Pearson, Department of Biological Chemistry, Box 440, Jordan Hall, Charlottesville, Va.).
  • OvIFN- ⁇ ovine IFN- ⁇
  • oTP-1 ovine trophoblast protein-one
  • 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- ⁇ II, 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- ⁇ 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 IFN- ⁇ is produced than other type I IFNs (Cross and Roberts, 1991).
  • IFN- ⁇ gene differs from the regulatory regions of the IFN- ⁇ gene. For example, transfection of the human trophoblast cell line JAR with the gene for bovine IFN- ⁇ resulted in antiviral activity while transfection with the bovine IFN- ⁇ gene did not. This implies unique transacting factors involved in IFN- ⁇ fourth root ⁇ gene expression. Consistent with this is the observation that while the proximal promoter region (from 126 to the transcriptional start site) of IFN- ⁇ is highly homologous to that of IFN- ⁇ and IFN- ⁇ ; the region from ⁇ 126 to ⁇ 450 is not homologous and enhances only IFN- ⁇ expression (Cross and Roberts, 1991). Thus, different regulatory factors appear to be involved in IFN- ⁇ expression as compared with the other type I IFNs.
  • 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, Ill.; 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, et al., 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 Wis.); pGEX (Smith, et al., 1985); pGEMEX (Promega); and pBS (Strategene, La Jolla Calif.) vectors.
  • Other bacterial expression vectors containing suitable promoters, such as the T7 RNA polymerase promoter or the tac promoter may also be used. Cloning of the OvIFN- ⁇ synthetic polynucleotide into a modified pIN 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, Calif.).
  • 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 (Hitzeman, et al., 1988; Rutter, et al., 1988; Oeda, et al., 1988).
  • 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 GAL 10 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 Calif.); plant cell expression, transgenic plant expression, and expression in mammalian cells (Clontech, Palo Alto Calif.; 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.
  • 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 be used
  • OAS 2′,5′-oligoadenylate synthetase
  • 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 FIG. 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.
  • OvIFN- ⁇ was orally administered in units of 0, 1 ⁇ 10 3 , 1 ⁇ 10 4 , 1 ⁇ 10 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 FIG. 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.
  • 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).
  • 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.
  • 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 1 ⁇ 10 5 and 1 ⁇ 10 10 units/day, preferably between about 1 ⁇ 10 8 and 1.5 ⁇ 10 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.
  • 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 StulISStl fragment (540 bp) may be cloned into a modified pIN III omp-A expression vector and transformed into a competent SB221 strain of E. coli .
  • IFN- ⁇ protein For expression of the IFN- ⁇ protein, 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 Stul and Sacl restriction sites at the 5′ and 3′ ends, respectively.
  • the amplified fragments were digested with Stul and SaclI and ligated into the SaclI and Smal sites of pBLUESCRIPT+(KS), generating pBSY-IFN ⁇ .
  • Plasmid pBSY-IFN ⁇ was digested with SaclI and EcoRV and the fragment containing the synthetic IFN- ⁇ gene was isolated.
  • the yeast expression vector pBS24Ub (Ecker, et al., 1989) was digested with SalI.
  • 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 SaclI, purified by agarose gel electrophoresis, and ligated to the SaclI-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, Calif.). 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 0.44 ⁇ 10 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 ⁇ 10 8 U/mg as measured by the anti-viral activity bioassay.
  • OvIFN- ⁇ (4.99 ⁇ 10 8 units/mg protein; Pepgen Corp., California or Biological Process Development Facility, Dept. of Food Science and Technology, University of NE-Lincoln, Lincoln, Nebr.; SEQ ID NO:4) was dissolved in 10% maltose solution to prepare ovIFN- ⁇ Solution.
  • the use of OvIFN- ⁇ (SEQ ID NO:2) is also contemplated in the present invention.
  • Two hundred microliters of ovIFN- ⁇ solution was orally administered to ICR mice (average body weight approximately 30 g, 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).
  • I.P. intraperitoneal administration
  • 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).
  • OvIFN- ⁇ was orally administered in units of 0, 10 3 , 10 4 , or 10 5 to an ICR mouse. 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 FIG. 2 , the OAS activity in whole blood increased in a dose dependent manner.
  • Ov-IFN- ⁇ SEQ ID NO:4
  • Ov-IFN- ⁇ SEQ ID NO:2
  • Ov-IFN- ⁇ SEQ ID NO:2
  • TABLE 2 Recombinant Ov-IFN- ⁇ Patient Dose Administration Number of Ov-IFN- ⁇ Volume (ml) Total Daily Dose Group Patients (mg/ml) per Dose (TID)
  • Dose (ml) I 6 1.0 0.33 1.0 II 6 1.0 1.0 3.0 III 6 1.0 3.0 9.0 IV 6 1.0 5.0 15.0
  • 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.

Abstract

A method of monitoring treatment of HCV by oral administration of ovine IFN-τ is disclosed. 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-τ until a measurable increase in blood 2′,5′-oligoadenylate synthetase level, relative to the level observed prior to administration, is observed. Also disclosed are oral-delivery compositions for use in treating HCV in an HCV-infected patient comprising ovine IFN-τ, in a dosage effective to stimulate bloodstream levels of 2′,5′-oligoadenylate synthetase.

Description

  • This application is a continuation of U.S. application Ser. No. 09/910,406 filed Jul. 19, 2001, now pending, which claims the benefit of U.S. Provisional Application No. 60/219,128 filed Jul. 19, 2000 and JP 317160 filed Oct. 17, 2000, all of which are hereby incorporated by reference in their entirety.
    • FIELD OF THE INVENTION
  • The present invention relates to the composition for treatment of conditions relating to hepatitis caused by hepatitis C virus (HCV) infection using Interferon-τ (IFN-τ). The present invention also relates to a method of monitoring treatment of HCV by measuring the blood levels of 2′,5′-oligoadenylate synthetase.
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    BACKGROUND OF THE INVENTION
  • Hepatitis C virus (HCV) is a major public health problem affecting an estimated 170 million people worldwide and more than 10% of the population in some countries (Lechner, et al., 2000). HCV is transmitted primarily by transfusion of infected blood and blood products (Cuthbert, et al., 1994; Mansell, et al., 1995). The Centers for Disease Control and Prevention estimate that HCV is responsible for 160,000 new cases of acute hepatitis in the United States each year. Therefore, an urgent medical need exists for an effective anti-HCV agent.
  • 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).
  • Treatment of HCV is aimed at reducing inflammation and liver cell damage, thus preventing cirrhosis and hepatocellular carcinoma (Horiike, et al., 1998; Benvegnu, et al., 1998). 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-α was introduced as therapy for chronic hepatitis C in the United States in 1991 and in Japan in 1992 (Saito, et al., 2000). However, use of IFN-α in sufficient dosage to yield clinical efficacy (i.e., at amounts of about 1×106 units/treatment and above) is usually associated with a “flu-like” syndrome characterized by fever, headache, lethargy, arthalgias and myalgias (Tyring, et al., 1992). At doses of 5-10×106 units/treatment and above, other toxicities, such as nausea, vomiting, diarrhea and anorexia, become more frequent. Neuropsychiatric symptoms have also been reported in association with IFN-α treatment (Dieperink, et al., 2000). In addition, some studies suggest that the efficacy of IFN-α treatment is not dose dependent (Saito, et al., 2000), and that treatment with IFN-α is associated with the development or exacerbation of autoimmune disorders in patients with neoplasms or viral hepatitis (Jimenez-Saenz, et al., 2000).
  • Ribavirin (1-β-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). Even the beneficial effects of ribavirin, however, are transient (Clarke, 2000; Koskinas, et al., 1995), and because of severe side effects, ribavirin, in combination with IFN-α, can be difficult to tolerate (Cotler, et al., 2000).
  • Because of the shortcomings associated with current HCV treatment methods, the inventors have set out to identify a new therapeutic candidate that will have more potent antiviral activity and less severe side effects.
    • SUMMARY OF THE INVENTION
  • In one aspect, the invention includes an oral-delivery composition for use in treating HCV in a HCV-infected patient. The composition includes ovine Interferon-tau (OvIFN-τ), in a dosage effective to stimulate levels of 2′,5′-oligoadenylate synthetase (OAS) observed in the bloodstream 24 hours after administration of the composition. In one embodiment the composition also includes an oral-delivery vehicle containing IFN-τ and effective to release the IFN-τ in active form in the stomach. The composition provides a preferred dose of ovine IFN-τ between 108-1010 units.
  • The composition provides a preferred dose of ovine IFN-τ between 108-1010 units. In one embodiment, the dosage of ovine IFN-τ is greater than 1×108 Units/day. In another embodiment, the dosage of ovine IFN-τ is greater than 2×108 Units/day. In yet another embodiment, the dosage of ovine IFN-τ is greater than 4×108 Units/day. In yet, still another embodiment, the dosage of ovine IFN-τ is greater than 1×109 Units/day. The dosage of ovine IFN-τ can be greater than 4×109 Units/day. Preferably, the dosage of ovine IFN-τ is greater than 7×109 Units/day.
  • In another aspect, the composition for treating HCV in a HCV-infected individual comprises ovine IFN-τ 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 108-1010 units.
  • In still another aspect, the composition of the invention includes ovine IFN-τ as an effective ingredient, where the composition avoids the absorption of ovine IFN-τ through the tunica mucosa oris.
  • In related aspects, a composition of the invention is for the treatment of hepatitis caused by HCV comprises ovine IFN-τ as an effective ingredient, and a 2′,5′-oligoadenylate synthetase activity inducer in animals other than sheep comprising ovine IFN-τ.
  • In still another aspect, the invention includes a method of monitoring treatment of HCV by oral administration of ovine IFN-τ. 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-τ until a measurable increase in blood 2′,5′-oligoadenylate synthetase level, relative to the level observed prior to administration, is observed.
  • These and other objects and features of the invention will become more fully apparent when the following detailed description is read in conjunction with the accompanying drawings.
    • BRIEF DESCRIPTION OF THE FIGURES
  • FIG. 1 shows OAS levels in mice whole blood following intraperitoneal (I.P.) or gastric administration (G.A.) of ovIFN-τ.
  • FIG. 2 shows dose-dependent induction of blood OAS by gastric administration (G.A.) of ovIFN-τ.
  • FIGS. 3-5 illustrate HCV RNA and ALT levels in three human patients following oral administration of 4.9×108 units/day ovIFN-τ.
  • FIGS. 6 and 7 illustrate HCV RNA and ALT levels in two human patients following oral administration of 1.5×109 units/day ovIFN-τ.
    • DETAILED DESCRIPTION OF THE INVENTION
  • I. Definitions
  • 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 fines, 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-τ (ovIFN-τ) refers to a protein having the amino acid sequence as shown in FIG. 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. Preferably the sequence includes the ovine IFN-τ sequence of FIG. 4 and the proteins with 90% sequence homology to the sequence shown in FIG. 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 (Pearson and Lipman, 1988; Pearson, 1990; program available from William R. Pearson, Department of Biological Chemistry, Box 440, Jordan Hall, Charlottesville, Va.).
  • II. Interferon-τ
  • The first IFN-τ to be identified was ovine IFN-τ (OvIFN-τ), as a 18-19 kDa protein. Several isoforms were identified in conceptus (the embryo and surrounding membranes) homogenates (Martal, et al., 1979). Subsequently, a low molecular weight protein released into conceptus culture medium was purified and shown to be both heat labile and susceptible to proteases (Godkin, et al., 1982). OvIFN-τ 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-τ 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).
  • An IFN-τ 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-αs from human, mouse, rat and pig and 70% homologous with bovine IFN-αII, now referred to as IFN-Ω. 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). 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-τ 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).
    TABLE 1
    Overview of the Interferons
    Aspects Type I Type I Type I Type II
    Types α & ω β τ γ
    Produced by: leukocyte fibroblast trophoblast lymphocyte
    Antiviral + + + +
    Antiproliferative + + + +
    Pregnancy Signaling − − + −
  • While IFN-τ 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-τ, are induced readily by virus and dsRNA (Roberts, et al., 1992). Induced IFN-α and IFN-β expression is transient, lasting approximately a few hours. In contrast, IFN-τ synthesis, once induced, is maintained over a period of days (Godkin, et al., 1982). On a per-cell basis, 300-fold more IFN-τ is produced than other type I IFNs (Cross and Roberts, 1991).
  • Other differences may exist in the regulatory regions of the IFN-τ gene. For example, transfection of the human trophoblast cell line JAR with the gene for bovine IFN-τ resulted in antiviral activity while transfection with the bovine IFN-Ω gene did not. This implies unique transacting factors involved in IFN-{fourth root} gene expression. Consistent with this is the observation that while the proximal promoter region (from 126 to the transcriptional start site) of IFN-τ is highly homologous to that of IFN-α and IFN-β; the region from −126 to −450 is not homologous and enhances only IFN-τ expression (Cross and Roberts, 1991). Thus, different regulatory factors appear to be involved in IFN-τ expression as compared with the other type I IFNs.
  • IFN-τ expression may also differ between species. For example, although IFN-τ 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-τ is constitutively expressed throughout pregnancy (Whaley, et al., 1994).
  • A. Isolation of IFN-τ
  • OvIFN-τ 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). The IFN-τ may be purified from the conceptus cultures by ion exchange chromotography and gel filtration. The homogeneity of isolated IFN-τ 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-τ samples may be performed using the bicinchoninic (BCA) assay (Pierce Chemical Co., Rockford, Ill.; Smith, et al., 1985).
  • B. Recombinant Production of IFN-τ
  • Recombinant IFN-τ protein may be produced from any selected IFN-τ polynucleotide fragment using a suitable expression system, such as bacterial or yeast cells. The isolation of IFN-τ nucleotide and polypeptide sequences is described in Bazer, et al. (1994). For example, Bazer, et al., describe the identification and isolation of a human IFN-τ gene.
  • To make an IFN-τ expression vector, an IFN-τ 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. Examples of suitable vectors include lambda gt11 (Promega, Madison Wis.); pGEX (Smith, et al., 1985); pGEMEX (Promega); and pBS (Strategene, La Jolla Calif.) vectors. Other bacterial expression vectors containing suitable promoters, such as the T7 RNA polymerase promoter or the tac promoter, may also be used. Cloning of the OvIFN-τ synthetic polynucleotide into a modified pIN III omp-A expression vector is described in the Materials and Methods.
  • For the experiments described herein, the OvIFN-τ 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, Calif.).
  • Other yeast vectors suitable for expressing IFN-τ 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 (Hitzeman, et al., 1988; Rutter, et al., 1988; Oeda, et al., 1988). Preferably, the vectors include an expression cassette containing an effective yeast promoter, such as the MFα1 promoter (Bayne, et al., 1988, GADPH promoter (glyceraldehyde-3-phosphate-dehydrogenase; Wu, et al., 1991) or the galactose-inducible GAL 10 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).
  • Further, a DNA encoding an IFN-τ 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 Calif.); plant cell expression, transgenic plant expression, and expression in mammalian cells (Clontech, Palo Alto Calif.; 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-τ polypeptides.
  • In addition to recombinant methods, IFN-τ proteins or polypeptides can be isolated from selected cells by affinity-based methods, such as by using appropriate antibodies. Further, IFN-τ peptides (e.g. SEQ ID NOS:2 or 4) may be chemically synthesized using methods known to those skilled in the art.
  • III. IFN-τ 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. Such criteria may not only be used to diagnose hepatitis C, but can be used to evaluate a patient's response to drug treatment.
  • Interferon causes synthesis of the enzyme 2′,5′-oligoadenylate synthetase (OAS), which in turn, results in the degradation of viral mRNA (Houglum, 1983). OAS activates an RNase that cleaves cellular and viral RNAs, thereby inactivating viral replication (Kumar et al., 1988). OAS is considered responsible, at least in part, for the antiviral state established in cells and plays a role in the elimination of HCV (Pawlotsky, et al., 1995).
  • A. IFN Administered Orally and Intraperioneally Induce OAS
  • In experiments performed in support of the present invention and detailed in Examples 1 and 3, IFN-τ, administered orally, was tested for its ability to induce OAS. OvIFN-τ was administered either orally or intraperitoneally to mice or human patients. OAS activity in whole blood in mice was determined, and is shown in FIG. 1, 24 hours after IFN-τ administration. Several human patients had 2 to 12 fold increases in their OAS enzyme activity levels as shown in Tables 3-6.
  • When OvIFN-τ was administered orally or intraperitoneally in mice, an increase in the OAS activity in whole blood was observed. When the effect of orally administered OvIFN-τ and that of intraperitoneally administered OvIFN-τ in mice were compared, both administrations provided essentially the same whole blood OAS induction activity.
  • B. Orally Administered IFN-τ Induces OAS in a Dose-Dependent Manner.
  • In experiments performed in support of the present invention and detailed in Example 2, IFN-τ, administered orally in mice, was tested for its ability to induce OAS in a dose-dependent manner. OvIFN-τ was orally administered in units of 0, 1×103, 1×104, 1×105 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 FIG. 2, the OAS activity in whole blood increased in a dose dependent manner.
  • Although it has already been established that IFN-τ is orally active (WO 96/28183), no exact determination has previously been made as to how IFN-τ was administered, or as to how IFN-τ is absorbed. In the present invention, IFN-τ 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-τ from the stomach would diminish antibody formation against IFN-τ compared to IFN-τ absorbed through the oral mucosal membrane, particularly in the case of chronic administrations of IFN-τ.
  • In addition, the present invention describes the ability of ovine IFN-τ to increase 2′,5′-oligoadenylate synthase activity in mice and humans. Prior to this work, only mouse IFN-τ had been known to be effective in mice.
  • IV. Administration of IFN-τ
  • A. Pharmaceutical Compositions
  • Therapeutic preparations or medicaments containing IFN-τ 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-τ-containing medicaments are formulated such that an effective amount of the IFN-τ is combined with a suitable carrier and/or excipient in order to facilitate effective administration of the composition. IFN-τ, 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.
  • In the case of compositions suitable for oral administration, tablets and capsules containing IFN-τ may be manufactured from IFN-τ (e.g., lyophilized IFN-τ 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.
  • Further, IFN-τ 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. By using several layers of the carrier or diluent, tablets operating with slow release can be prepared.
  • 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-τ, sugar and a mixture of ethanol, water, glycerol, propylene, glycol and possibly other additives of a conventional nature.
  • B. Dosage
  • An orally active IFN-τ 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 1×105 and 1×1010 units/day, preferably between about 1×108 and 1.5×109 units/day. It will be appreciated that because of its lower toxicity, IFN-τ can be administered at higher doses than, for example, IFN-α.
  • Disorders requiring a steady elevated level of IFN-τ 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.
  • Once improvement of a patient's condition has occurred, 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.
  • C. Combination Therapies
  • It will, of course, be understood that the compositions and methods of this invention may be used in combination with other therapies. For example, the composition of ovIFN-τ for the treatment of HCV in a HCV-infected patient can be combined with an anti-viral agent such as ribavirin.
  • D. Monitoring
  • Treatment of HCV by oral administration of ovIFN-τ is monitored by measuring the blood levels of 2′,5′-oligoadenylate synthetase (OAS) prior to and following administration. The OAS levels can be monitored, for example, at 12, 24, and 48 hours after administration. If necessary, the dose of IFN-τ is adjusted until a measurable increase in blood OAS levels is observed, relative to the level observed prior to administration.
  • All patent and literature references cited in the present specification are hereby incorporated by reference in their entirety.
  • The following examples illustrate, but are not intended in any way to limit the invention.
  • Materials and Methods
  • A. Production of OvIFN-τ
  • In one embodiment, a synthetic OvIFN-τ gene was generated using standard molecular methods (Ausubel, et al., 1988) by ligating oligonucleotides containing contiguous portions of a DNA sequence encoding the OvIFN-τ 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-τ polynucleotide coding sequence may span position 16 through 531: a coding sequence of 172 amino acids.
  • In one embodiment, the full length synthetic gene StulISStl fragment (540 bp) may be cloned into a modified pIN III omp-A expression vector and transformed into a competent SB221 strain of E. coli. For expression of the IFN-τ protein, 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-τ may be liberated from the cells by sonication or osmotic fractionation.
  • For expression in yeast, the IFN-τ gene may amplified using polymerase chain reaction (PCR; Mullis, 1987; Mullis, et al., 1987) with PCR primers containing Stul and Sacl restriction sites at the 5′ and 3′ ends, respectively. The amplified fragments were digested with Stul and SaclI and ligated into the SaclI and Smal sites of pBLUESCRIPT+(KS), generating pBSY-IFNτ. Plasmid pBSY-IFNτ was digested with SaclI and EcoRV and the fragment containing the synthetic IFN-τ gene was isolated. The yeast expression vector pBS24Ub (Ecker, et al., 1989) was digested with SalI. 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 SaclI, purified by agarose gel electrophoresis, and ligated to the SaclI-EcoRV fragment isolated from pBSY-IFN-τ. The resulting recombinant plasmid was designated pBS24Ub-IFNτ.
  • The recombinant plasmid pBS24Ub-IFNτ was transformed into E. coli. Recombinant clones containing the IFN-τ insert were isolated and identified by restriction enzyme analysis. IFN-τ coding sequences were isolated from pBS24Ub-IFNτ and cloned into a Pichia pastoris vector containing the alcohol oxidase (AOX1) promoter (Invitrogen, San Diego, Calif.). 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.
  • In one embodiment, the purified IFN-τ protein has a specific activity of about 0.29 to about 0.44×108 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 4.9×108 U/mg as measured by the anti-viral activity bioassay.
    • EXAMPLE 1 Induction of OAS with Orally and Intraperitoneally Administered Ovine IFN-τ to Mice
  • OvIFN-τ (4.99×108 units/mg protein; Pepgen Corp., California or Biological Process Development Facility, Dept. of Food Science and Technology, University of NE-Lincoln, Lincoln, Nebr.; SEQ ID NO:4) was dissolved in 10% maltose solution to prepare ovIFN-τ Solution. The use of OvIFN-τ (SEQ ID NO:2) is also contemplated in the present invention. Two hundred microliters of ovIFN-τ solution was orally administered to ICR mice (average body weight approximately 30 g, 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).
  • For intraperitoneal administration (I.P.), 100 microliters of ovIFN-τ 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).
  • When the effect of orally administered 105 units of ovIFN-τ (τ GA) and that of intraperitoneally administered 105 units of OvIFN-τ (τ IP) were compared, both administrations provided essentially the same whole blood OAS induction activity. The results are shown in FIG. 1.
    • EXAMPLE 2 Dose-Dependent Induction of OAS by Oral Administration of IFN-τ in Mice
  • Using the same procedure as Example 1, OvIFN-τ was orally administered in units of 0, 103, 104, or 105 to an ICR mouse. 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 FIG. 2, the OAS activity in whole blood increased in a dose dependent manner.
    • EXAMPLE 3 Reduced ALT, Reduced HCV Viral Titer, and Induction of OAS by Oral Administration of IFN-τ in Human Patients
  • A. IFN-τ Preparation
  • On day one, one bottle of Ov-IFN-τ (SEQ ID NO:4) is removed from the refrigerator and the patient self-administers the proper volume of test material according to Table 2. Ov-IFN-τ (SEQ ID NO:2) may also be prepared and administered in the same manner.
    TABLE 2
    Recombinant Ov-IFN-τ Patient Dose Administration
    Number of Ov-IFN-τ Volume (ml) Total Daily
    Dose Group Patients (mg/ml) per Dose (TID) Dose (ml)
    I 6 1.0 0.33 1.0
    II 6 1.0 1.0 3.0
    III 6 1.0 3.0 9.0
    IV 6 1.0 5.0 15.0
  • B. Patient Dosing Instructions
  • 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.
  • The above steps are repeated three times per day at approximately eight-hour intervals: once in the morning, once at midday and once in the evening.
  • C. Results
  • The results of the human clinical trails in patients with HCV infections are shown in Tables 3-10 below, and graphically in FIGS. 3-7. An increase in OAS levels, and a decrease in both ALT and viral titer levels following oral ovine IFN-τ administration can be seen below.
    TABLE 3
    Human Clinical Trial Data - BB-IND9222 Dose Cohort I
    PEPGEN 2-5 (PBMC)
    OS Patient Date HCV ALT 2-5A (SERUM) pmol/5 × 106
    NUMBER Initials/# Timept. Collected RT-PCR (IU/L) pmol/dl 2-5A PBMC/ml
    180 PAB/001 Screen Nov. 17, 2000 790,000 64 12.46 —
    181 PAB/001 Day 1 Dec. 1, 2000 290,000 63 10.00 —
    337 PAB/001 day 2/24 hr. Dec. 2, 2000 — — 10.00 —
    182 PAB/001 Day 3 Dec. 3, 2000 1,700,000 57 5.00 —
    183 PAB/001 Day 8 Dec. 8, 2000 530,000 56 5.00 —
    184 PAB/001 Day 15 Dec. 15, 2000 580,000 61 0.00 —
    185 PAB/001 Day 22 Dec. 22, 2000 13,000 66 2.50 —
    186 PAB/001 Day 29 Dec. 29, 2000 230,000 40 10.00 —
    187 PAB/001 Day 43 Jan. 12, 2001 — 42 7.50 —
    188 PAB/001 Day 57 Jan. 26, 2001 640,000 37 16.67 —
    189 PAB/001 Day 71 Feb. 9, 2001 — — 12/46 —
    190 PAB/001 Day 85 Feb. 23, 2001 960,000 50 13.86 —
    191 PAB/001 Day 113 Mar. 23, 2001 160,000 53 0.00 —
    192 PAB/001 Day 169 —
    193 MSM/002 Screen Jan 27, 2000 4,600,000 258 11.05 —
    194 MSM/002 Day 1 Dec. 11, 2000 5,100,000 164 16.67 —
    337 MSM/002 Day 2/24 hr. Dec. 12, 2000 — — 10.00 —
    195 MSM/002 Day 3 Dec. 13, 2000 6,300,000 154 29.30 —
    196 MSM/002 Day 8 Dec. 18, 2000 5,100,000 133 33.08 —
    197 MSM/002 Day 15 Dec. 26, 2000 9,100,000 100 54.62 —
    198 MSM/002 Day 22 Jan. 2, 2001 — 103 51.54 —
    199 MSM/002 Day 29 Jan. 8, 2001 8,600,000 91 28.60 —
    200 MSM/002 Day 43 Jan. 23, 2001 — 86 12.46 —
    201 MSM/002 Day 57 Feb. 7, 2001 3,400,000 82 18.77 —
    202 MSM/002 Day 71 Feb. 20, 2001 — — 36.15 —
    203 MSM/002 Day 85 Mar. 2, 2001 3,700,000 49 26.14 —
    204 MSM/002 Day 113 Apr. 3, 2001 3,800,000 64 42.31 —
    205 MSM/002 Day 169
    206 DMA/003 Screen Dec. 1, 2000 780,000 115 28.60 —
    207 DMA/003 Day 1 Dec. 12, 2000 990,000 115 26.14 —
    208 DMA/003 Day 3 Dec. 14, 2000 660,000 121 30.00 —
    209 DMA/003 Day 8 Dec. 19, 2000 920,000 105 36.15 —
    210 DMA/003 Day 15 Dec. 26, 2000 580,000 107 26.14 —
    211 DMA/003 Day 22 Jan 2, 2001 — 105 24.74 —
    212 DMA/003 Day 29 Jan 9, 2001 170,000 97 27.54 —
    213 DMA/003 Day 43 Jan 22, 2001 — 85 23.33 —
    214 DMA/003 Day 57 Feb. 5, 2001 650,000 74 59.23 —
    215 DMA/003 Day 71 Feb. 20, 2001 — — 36.15 —
    216 DMA/003 Day 85 Mar. 5, 2001 11,000 49 16.00 —
    217 DMA/003 Day 107 Mar. 27, 2001 880,000 45 0.00 —
    217 DMA/003 Day 115 Apr. 4, 2000 50,000 55 20.24 —
    DMA/003 460,000 47 —
  • TABLE 4
    HEPC CLINICAL TRIALS BB-IND9222 DOSE COHORT I
    PEPGEN 2-5 (PBMC)
    OS Patient Date HCV ALT 2-5A (SERUM) pmol/5 ×106
    NUMBER Initials/# Timept. Collected RT-PCR (IU/L) pmol/dl 2-5A PBMC/ml
    219 LER/004 Screen Dec. 12, 2000 6,100,000 118 33.95 —
    220 LER/004 Day 1 Dec. 20, 2000 6,000,000 108 33.95 —
    221 LER/004 Day 3 Dec. 22, 2000 11,000,000 120 53.68 —
    222 LER/004 Day 8 Dec. 27, 2000 1,900,000 109 29.51 —
    223 LER/004 Day 15 Jan. 3, 2001 3,400,000 120 41.84 —
    224 LER/004 Day 22 Jan. 10, 2001 — 94 34.74 —
    225 LER/004 Day 29 Jan. 17, 2001 640,000 109 43.42 —
    226 LER/004 Day 43 Jan. 30, 2001 — 99 49.74 —
    227 LER/004 Day 57 Feb. 13, 2001 4,400,000 106 37.89 —
    228 LER/004 Day 71 Feb. 27, 2001 — — 81.00 —
    229 LER/004 Day 85 Mar. 14, 2001 3,900,000 67 3.20 —
    230 LER/004 Day 113 3,200,000 107 —
    231 LER/004 Day 169 —
    232 Z-I/005 Screen Dec. 20, 2000 3,400,000 151 43.42 —
    233 Z-I/005 Day 1 Jan 8, 2001 4,600,000 134 43.42 —
    338 Z-I/005 Day 2/24 hr. Jan 21, 2001 — 144 45.00 —
    234 Z-I/005 Day 3 Jan. 1, 2001 1,400,000 109 46.58 —
    235 Z-I/005 Day 8 Jan. 15, 2001 4,000,000 94 12/93 —
    236 Z-I/005 Day 15 Jan. 22, 2001 1,100,000 107 48.95 —
    237 Z-I/005 Day 22 Jan. 31, 2001 — 107 47.37 —
    238 Z-I/005 Day 29 Feb. 7, 2001 2,200,000 144 74.82 —
    239 Z-I/005 Day 43 Feb. 19, 2001 — 111 26.10 —
    240 Z-I/005 Day 57 Mar. 5, 2001 4,400,000 122 43.42 —
    241 Z-I/005 Day 71 Mar. 19, 2001 — — 10.00 —
    242 Z-I/005 Day 85 Apr. 4, 2001 1,100,000 122 17.80 —
    243 Z-I/005 Day 113 3,200,000 132 —
    244 Z-I/005 Day 169 —
    245 JRJ/006 Screen Jan. 5, 2001 21,000,000 111 52.11 —
    246 JRJ/006 Day 1 Jan. 10, 2001 8,500,000 104 21.90 —
    247 JRJ/006 Day 3 Jan. 12, 2001 6,000,000 98 26.53 —
    248 JRJ/006 Day 8 Jan. 17, 2001 950,000 124 24.21 —
    249 JRJ/006 Day 15 Jan. 24, 2001 3,700,000 118 19.09 —
    250 JRJ/006 Day 22 Jan. 30, 2001 — 109 22.07 —
    251 JRJ/006 Day 29 Feb. 7, 2001 3,300,000 93 19.75 —
    252 JRJ/006 Day 43 Feb. 22, 2001 — 122 24.88 —
    253 JRJ/006 Day 57 Mar. 7, 2001 7,000,000 78 35.62 —
    254 JRJ/006 Day 71 Mar. 21, 2001 — — 52.92 —
    255 JRJ/006 Day 85 Apr. 4, 2001 5,000,000 88 42.92 —
    256 JRJ/006 Day 113 >5,000,000 109 —
    257 JRJ/006 Day 169 —
  • TABLE 5
    HEPC CLINICAL TRIALS BB-IND9222 DOSE COHORT II
    PEPGEN 2-5 (PBMC)
    OS Patient Date HCV ALT 2-5A (SERUM) pmol/5 × 10 6
    NUMBER Initials/# Timept. Collected RT-PCR (IU/L) pmol/dl 2-5A PBMC/ml
    AMC/007 Screen Feb. 2, 2001 1,700,000 44 11.20 —
    AMC/007 Day 1 Feb. 20, 2001 1,300,000 48 18.40 —
    AMC/007 Day 3 Feb. 22, 2001 810,000 44 27.60 —
    AMC/007 Day 8 Feb. 27/2001 630,000 50 42.40 —
    AMC/007 Day 15 Mar. 6, 2001 290,000 54 50.67 —
    AMC/007 Day 22 Mar. 13, 2001 — 53 94.50 —
    AMC/007 Day 29 Mar. 20, 2001 410,000 36 120.00 —
    AMC/007 Day 43 Apr. 3, 2001 — 29 81.33 —
    AMC/007 Day 57 Apr. 17, 2001 930,000 36 55.33 —
    AMC/007 Day 71 May 1, 2001 — — 51.33 —
    AMC/007 Day 85 May 15, 2001 —
    AMC/007 Day 113 Jun. 6, 2001 —
    AMC/007 Day 169 Aug. 7, 2001 —
    ALW/008 Screen Feb. 2, 2001 30,000,000 47 53.33 —
    ALW/008 Day 1 Feb. 20, 2001 3,000,000 38 10.00 —
    ALW/008 Day 3 Feb. 22, 2001 3,200,000 42 42.00 —
    ALW/008 Day 8 Feb. 27, 2001 5,400,000 31 14.40 —
    ALW/008 Day 15 Mar. 6, 2001 17,000,000 29 10.00 —
    ALW/008 Day 22 Mar. 13, 2001 — 27 10.40 —
    ALW/008 Day 29 Mar. 20, 2001 11,000,000 25 10.00 —
    ALW/008 Day 43 Apr. 3, 2001 — 40 14.40 —
    ALW/008 Day 57 Apr. 17, 2001 18,000,000 31 12.80 —
    ALW/008 Day 71 May 1, 2001 — — 16.40 —
    ALW/008 Day 85 May 15, 2001 —
    ALW/008 Day 113 Jun. 12, 2001 —
    ALW/008 Day 169 Aug. 7, 2001 —
    DBF/012 Screen 5,300,000 84 28.80 —
    DBF/012 Day 1 9,300,000 77 26.00 —
    DBF/012 Day 3 9,400,000 71 10.00 —
    DBF/012 Day 8 7,900,000 86 53.33 —
    DBF/012 Day 15 9,100,000 67 108.00 —
    DBF/012 Day 22 — 64 42.67 —
    DBF/012 Day 29 9,900,000 58 52.00 —
    DBF/012 Day 43 — 61 58.00 —
    DBF/012 Day 57 15,000,000 70 61.33 —
    DBF/012 Day 7l — — 168.00 —
    DBF/012 Day 85 —
    DBF/012 Day 113 —
    DBF/012 Day 169 —
  • TABLE 6
    HEPC CLINICAL TRIALS BB-IND9222 DOSE COHORT II
    PEPGEN 2-5A 2-5 (PBMC)
    OS Patient Date HCV ALT (SERUM) pmol/5 × 106
    NUMBER Initials/# Timept. Collected RT-PCR (IU/L) pmol/dl 2-5A PBMC/ml
    VCC/009 Screen Feb. 2, 2001 5,100,000 113 17.20 —
    VCC/009 Day 1 Feb. 2, 2001 4,300,000 128 58.67 286.88
    VCC/009 Day Feb. 22, 2001 — — 10.00 —
    2/24 hr.
    VCC/009 Day 3 Feb. 23, 2001 3,500,000 126 18.40 218.57
    VCC/009 Day 8 Feb. 28, 2001 1,600,000 130 24.80 —
    VCC/009 Day 15 Mar. 7, 2001 2,200,000 118 25.20 624.38
    VCC/009 Day 22 Mar. 14, 2001 — 99 18.00 —
    VCC/009 Day 29 Mar. 21, 2001 1,500,000 93 30.67 1261.43
    VCC/009 Day 43 Apr. 5, 2001 — 72 15.20 —
    VCC/009 Day 57 Apr. 18, 2001 2,700,000 62 10.00 —
    VCC/009 Day 71 May 2, 2001 — — 18.40 —
    VCC/009 Day 85 May 16, 2001 — —
    VCC/009 Day 113 Jun. 13, 2001 —
    VCC/009 Day 169 Aug. 8, 2001 —
    HCM/010 Screen Feb. 2, 2001 3,00,000 60 28.84 —
    HCM/010 Day 1 Feb. 2, 2001 5,000,000 47 12.31 998.1
    HCM/010 Day Feb. 22, 2001 — — — —
    2/24 hr.
    HCM/010 Day 3 Feb. 23, 2001 5,100,000 52 22.56 1336.67
    HCM/010 Day 8 Feb. 28, 2001 5,100,000 50 18.6 —
    HCM/010 Day 15 Mar. 7, 2001 5,300,000 49 30 1336.67
    HCM/010 Day 22 Mar. 14, 2001 — 49 47.08 —
    HCM/010 Day 29 Mar. 21, 2001 3,000,000 57 50 1524.76
    HCM/010 Day 43 Apr. 4, 2001 — 45 246 —
    HCM/010 Day 57 Apr. 18, 2001 4,300,000 59 16.67 —
    HCM/010 Day 71 May 2, 2001 — — 15.26 —
    HCM/010 Day 85 May 16, 2001 —
    HCM/010 Day 113 Jun. 13, 2001 —
    HCM/010 Day 169 Aug. 8, 2001
    CLR/011 Screen Feb. 5, 2001 12,000,000 58 10.00 —
    CLR/011 Day 1 Feb. 21, 2001 19,000,000 66 30.00 960.48
    CLR/011 Day 3 Feb. 23, 2001 28,000,000 55 11.05 922.86
    CLR/011 Day 8 Feb. 28, 2001 >5,000,000 55 12.46 —
    CLR/011 Day 15 Mar. 7, 2001 23,000,000 63 12.46 1035.71
    CLR/011 Day 22 Mar. 14, 2001 — 65 19.82 —
    CLR/011 Day 29 Mar. 21, 2001 13,000,000 58 10.00 998.1
    CLR/011 Day 43 Apr. 4, 2001 — 63 36.00 —
    CLR/011 Day 57 Apr. 18, 2001 18,000,000 61 20.80 —
    CLR/011 Day 71 May 2, 2001 — — 10.00 —
    CLR/011 Day 85 May 16, 2001 —
    CLR/011 Day 113 Jun. 13, 2001 —
    CLR/011 Day 169 Aug. 8, 2001 —
  • TABLE 7
    Dose Group 1 (0.33 mg TID) - 24 Hour Serum Collection PCR Assays (HCV RNA)
    Patient ID Screen Day 1 Day 3 Day 8 Day 15 Day 22 Day 29 Day 57 Day 85 Day 113 Day 169
    001 PAB 790,000 290,000 1,700,000 530,000 580,000 13,0001 230,000 640,000 960,000   160,000   110,000
    002 MSM 4,600,000 5,100,000 6,300,000 5,100,000 9,100,000 8,600,000 3,400,000 3,700,000 3,800,000 1,900,000
    003 DMA 780,000 990,000 660,000 920,000 580,000 170,000 650,000 11,000    880,0002    340,0005
       50,0003
       460,0004
    004 LER 6,100,000 6,000,000 11,000,000 1,900,000 3,400,000 640,000 4,400,000 3,900,000 3,200,000 3,800,000
    005 Z-I 3,400,000 4,600,000 1,400,000 4,000,000 1,100,000 2,200,000 4,400,000 1,100,000 3,200,000 1,300,000
    006 JRJ 21,000,000 8,500,000 6,000,000 950,000 3,700,000 3,300,000 7,000,000 5,000,000 5,100,000 >5,000,000  

    1PCR Assay not scheduled for Day 22

    2 Day 1 of Retreat

    3 Day 8 of Retreat

    4 Day 29 of Retreat

    5Day 164 of Retreat
  • TABLE 8
    ALT Values (IU/L) - Dose Group 1
    Patient ID Screen Day 1 Day 3 Day 8 Day 15 Day 22 Day 29 Day 43 Day 57 Day 85 Day 113 Day 169
    001 PAB 64 63 57 56 61 66  40*  42*  37* 50 53 50
    002 MSM 258 164 154 133 100 103 91 86 82 49 64 61
    003 DMA 115 115 121 105 107 105 97 85 74 49 45 51
    004 LER 118 108 120 109 120 94 109  99 106  67 107 120
    005Z-I 151 134 144 109 94 107 107  144  111  122 132
    006 JRJ 111 104 98 124 118 109 93 122  78 88 109
    Mean 116.71 98.43 99.57 92 87.86 86.57   82.83   89.33   75.17 72.86 89 64.43
    Std

    Normal ALT (range = 1-45)

    2 Day 1 of Retreat

    3Day8 of Retreat

    4 Day 29 of Retreat

    5Day 164 of Retreat

    6Day 192 of Retreat
  • TABLE 9
    Dose Group 2 (1.0 mg TID) - 24 Hour Serum Collection PCR Assays (HCV RNA)
    Patient ID Screen Day 1 Day 3 Day 8 Day 15 Day 29 Day 57 Day 85 Day 113 Day 169
    007 AMC 1,700,000 1,300,000 810,000 630,000 290,000 410,000 930,000 900,000 310,000
    008 ALW 30,000,000 3,000,000 3,200,000 5,400,000 17,000,000 11,000,000 18,000,000 7,700,000 11,000,000
    009 VCC 5,100,000 4,300,000 3,500,000 1,600,000 2,200,000 1,500,000 2,700,000 1,700,000 670,000
    010 HMC 3,000,000 5,000,000 5,100,000 5,100,000 5,300,000 3,000,000 4,300,000 3,100,000 4,400,000
    011 CLR 12,000,000 19,000,000 28,000,000 >5,000,000 23,000,000 13,000,000 18,000,000 9,400,000 8,200,000
    012 DBF 5,300,000 9,300,000 9,400,000 7,900,000 9,100,000 9,900,000 15,000,000 9,500,000 16,000,000
  • TABLE 10
    ALT Values (IU/L) - Dose Group 2
    Patient ID Screen Day 1 Day 3 Day 8 Day 15 Day 22 Day 29 Day 43 Day 57 Day 85 Day 113 Day 169
    007 AMC  44** 48 44 50 54 53  36*  29*  36*  37* 49
    008 ALW 47  38*  42*  31*  29*  27*  25*  40*  31*  31*  25*
    009 VCC 113  128  126  130  118  99 93 72 62  38*  34*
    010 HMC 60 47 52 50 49 49 57 45 59 51 58
    011 CLR 58 66 55 55 63 65 58 63 61 60 61
    012 DBF 84 77 71 86 67 64 58 61 70 89 92

    *Normal ALT Value (range = 1-45)

    **Normal ALT Value for female 67 years of age (4-40)

Claims (15)

1. An oral-delivery composition for use in treating HCV in a HCV-infected patient comprising ovine IFN-τ, in a dosage effective to stimulate bloodstream levels of 2′,5′-oligoadenylate synthetase.
2. The oral-delivery composition of claim 1, which further comprises an oral-delivery vehicle containing IFN-τ, wherein said oral-delivery vehicle is effective to release the IFN-τ in active form in the digestive tract.
3. The composition of claim 2, wherein the vehicle is effective to release ovine IFN-τ in the stomach or intestines.
4. The composition of claim 1 wherein the dosage of ovine IFN-τ is between 108-1010 Units/day.
5. The composition of claim 1, wherein the dosage of ovine IFN-τ is greater than about 1×108 Units/day.
6. The composition of claim 1, wherein the dosage of ovine IFN-τ is greater than about 2×108 Units/day.
7. The composition of claim 1, wherein the dosage of ovine IFN-τ is greater than about 4×108 Units/day.
8. The composition of claim 1, wherein the dosage of ovine IFN-τ is greater than about 1×109 Units/day.
9. The composition of claim 1, wherein the dosage of ovine IFN-τ is greater than about 4×109 Units/day.
10. The composition of claim 1, wherein the dosage of ovine IFN-τ is greater than about 7×109 Units/day.
11. The composition of claim 1, wherein the dosage of ovine IFN-τ avoids the tunica mucosa oris.
12. The composition of claim 1, in combination with ribavirin.
13. A pharmaceutical composition for the treatment of HCV comprising:
ovine IFN-τ as an effective ingredient, wherein said composition avoids the absorption of ovine IFN-τ through the tunica mucosa oris.
14. A method of monitoring treatment of HCV by oral administration of ovine IFN-τ comprising:
measuring the blood levels of 2′,5′-oligoadenylate synthetase prior to and after such oral administration, and if necessary
adjusting the dose of IFN-τ until a measurable increase in blood 2′,5′-oligoadenylate synthetase level, relative to the level observed prior to administration, is observed.
15. The method of claim 14, wherein said adjusting includes increasing the dose above 108 units.
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