WO2001017535A2 - Enriched fraction from a porcine liver extract for treating human diseases - Google Patents

Enriched fraction from a porcine liver extract for treating human diseases Download PDF

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Publication number
WO2001017535A2
WO2001017535A2 PCT/US2000/023649 US0023649W WO0117535A2 WO 2001017535 A2 WO2001017535 A2 WO 2001017535A2 US 0023649 W US0023649 W US 0023649W WO 0117535 A2 WO0117535 A2 WO 0117535A2
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herpes
infection
extract
virus infection
herpes virus
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PCT/US2000/023649
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French (fr)
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WO2001017535A3 (en
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Carl H. Lawyer
Patricia Frank
William G. Philip
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Vigen Laboratories, Inc.
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Priority to AU77009/00A priority Critical patent/AU7700900A/en
Publication of WO2001017535A2 publication Critical patent/WO2001017535A2/en
Publication of WO2001017535A3 publication Critical patent/WO2001017535A3/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/37—Digestive system
    • A61K35/407—Liver; Hepatocytes
    • 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
    • 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
    • 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
    • A61P31/18—Antivirals for RNA viruses for HIV
    • 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
    • A61P31/20—Antivirals for DNA viruses
    • A61P31/22—Antivirals for DNA viruses for herpes viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders

Definitions

  • the present invention is directed to a method of treating numerous human diseases and to the discovery of a partially purified, i.e. an enriched fraction from porcine extract that is efficacious in treating such diseases.
  • This invention is particularly directed toward a method of treating HIV, Herpes, multiple sclerosis, and other viral diseases with this enriched fraction and/ or with specific biologically active components, alone or in combination, further purified from this partially purified fraction.
  • Mammalian liver extract has been used in the treatment of a wide range of infectious and noninfectious dermatologic conditions including acne vulgaris, Journal Invest. Dermatology, 2:205-218 (1939); first and second degree burns, Mississippi Medical Journal, 76: 199 (1954); sunburn, Clinical Medicine, 3:245 (1956); poison ivy dermatitis, Clin. Med., 3:425 (1956) and Herpes zoster, Southern Medical Journal, 50: 1524 (1957). A specific active component or a known mechanism for such treatment has not yet been defined. Crude mammalian liver extract has also been reported to have bradykinin potentiating activity, Tewksbury, et. Al., Arch. Biochem. Biophys.
  • This invention provides several bioactive fractions which have been purified or enriched from a porcine liver extract using a single reversed phase HPLC separation step.
  • VG-201 a partially purified fraction of porcine liver extract derived from porcine liver and designated herein as VG-201 if obtained from the primary separation step and VG-201 A if obtained from the secondary separation step. More specifically, the present invention provides a method of treating such diseases involving administering to humans having said diseases a therapeuticaUy effective dose of VG-201 and/or VG-201A.
  • the enriched fractions VG-201 and VG-201A may be administered independently of each other or in combination with each other.
  • fractions are characterized by being the chromatographicaUy enriched fractions from porcine liver extract or acetone powder derived from porcine liver, and having been eluted from sequential reversed phase (C18) columns in the presence of acetonitrile and low pH induced by trifluoroacetic acid (TFA) in the mobile phase.
  • the fractions originated from heat stable, acetone insoluble, water-soluble fresh porcine liver extract.
  • other forms of starting material such as acetone powdered extract may also be used.
  • the above method involving the administration of the purified fraction of porcine liver extract VG-201 and/or VG-201A provides a novel method for the treatment or prevention of a variety of human diseases characterized by neoplasia such as Hodgkin's and non-Hodgkin's lymphoma, Kaposi's sarcoma, primary effusion lymphoma, nasopharyngeal carcinoma, gastric carcinoma, leiomysarcoma, Burkitt's lymphoma, cervical cancer, prostate cancer, colon cancer, breast cancer and leukemia. It would be evident that the method will find ready application for the prevention or treatment of neoplastic disease states, particularly those specifically named, in which neoplasia is an indication.
  • neoplasia such as Hodgkin's and non-Hodgkin's lymphoma, Kaposi's sarcoma, primary effusion lymphoma, nasopharyngeal carcinoma, gastric carcinoma, leio
  • VG-201 and/or VG-201A may be utilized to treat diseases including Human Immunodeficiency Virus 1 (HIV- 1) infection, Herpes virus infections, particularly Herpes 1 (Herpes simplex 1), Herpes 2 (Herpes simplex 2), Herpes 3 (Varicella zoster virus), Herpes 4 (Epstein- Barr virus), Herpes 5 (Cytomegalovirus), Herpes 6a and 6b, Herpes 7 and Herpes 8 (Kaposi-associated herpes virus), lepromatous leprosy, multiple sclerosis and diabetes mellitus.
  • HMV- 1 Human Immunodeficiency Virus 1
  • Herpes virus infections particularly Herpes 1 (Herpes simplex 1), Herpes 2 (Herpes simplex 2), Herpes 3 (Varicella zoster virus), Herpes 4 (Epstein- Barr virus), Herpes 5 (Cytomegalovirus), Herpes 6a and 6b, Her
  • VG-201 and/or VG-201A is also effective against organ and bone marrow transplantation infection, sarcoidosis, encephalopathy syndrome, Sjogren's syndrome, and Castleman's disease, lymphoproliferative disorders, and cytomegalovirus retinitis.
  • the partially purified porcine liver extract i.e. either enriched fraction VG-201 or VG-201 A may be administered alone or in combination with other pharmaceutically acceptable agents.
  • a preferred product is an aqueous solution containing approximately 2.5% by weight of liver extract solids. Doses of from not less than about 1 ml per day to not more than about 5 ml per day, preferably about 2 ml per day, of either extract in solution are generally effective. This method has the distinct advantage that it will treat the disease as long as the appropriate dosages are used, it being understood that the dosage levels will be adjusted dependent upon the response of the subject as monitored by methods known to those skilled in the art.
  • the porcine extract VG-201 and/ or VG- 201 A may be administered orally, topically, or parenterally, such as via intravenous or intramuscular injection, in liquid, semi-solid or solid form for use in the above treatments depending upon the extent of the disease condition, the particular disease or condition being treated, and the results desired.
  • the porcine liver extract VG-201 or VG-201 A may also be administered in combination with other pharmaceuticals, if necessary.
  • BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING Fig. 1 shows the process flow diagram for the manufacturing process used to prepare the liver extract as well as the enriched fraction VG-201 using separation A.
  • Fig. 2 shows the process flow diagram for the manufacturing process used to prepare the liver extract as well as the enriched fraction VG-201A using separation B.
  • Porcine livers were homogenated at high speed in a blender followed by the addition of three volumes of tap water to one volume of liver. For every 500kg of tapwater, add 7.9kg of concentrated sulfuric acid (H2SO4). The suspension is then agitated and heated to boiling followed by a cooling to 60°C. All solids are removed appropriately, for example, either by decanting the supernatant or filtration and the supernatant concentrated to approximately V2 volume by rotary evaporation at 80°C. The temperature must be maintained above 60°C during this evaporation. The extract may be frozen or lyophilized at this point as it is a stable hold step in the process.
  • H2SO4 concentrated sulfuric acid
  • the material is further extracted using 70% ethanol/30% water mixture (1.2L of 95% ethanol and 0.5L water to each kg of solid).
  • the organic extraction occurs at 27°C to 32°C with stirring followed by stirring for one hour at room temperature.
  • the precipitated material is allowed to settle for one to two hours and the liquid removed by decanting or filtration.
  • the liquid is then frozen at - 20°C for ten days.
  • This material is refereed to as "liver fraction 1" as well as "LFl”.
  • the LFl recipe can be found in the National Formulary XI, page 193.
  • the ethanol extract is then cold filtered and concentrated to 30% to 40% solids under vacuum at 80°C. Water may be re-added followed by additional concentration to 30% to 40% solids to drive off residual ethanol.
  • the extract is stable at this point and may be lyophilized. Dilute the extract to 16% total solids by weight and mix well. Add 90% distilled phenol to a final phenol concentration of 1% and allow the solution to sit undisturbed for seven days at room temperature. Following the seven- day incubation, filter the material through fluted filter paper to remove nay precipitated or solid material. Measure the volume of the resultant clarified solution and dilute further to 8% solids by adding an equal volume of water.
  • This material may be autoclaved at 120°C for approximately 20 minutes if it is to be held prior to chromatographic processing.
  • the chromatographic enrichment takes place in two steps. A primary reversed phase separation of the CSS was performed to obtain
  • VG-201 followed by a second reversed phase separation of active fractions to obtain VG-201A, as follows.
  • Approximately 75mg of CSS were purified by reversed phase chromatography using the following conditions:
  • Fraction collection Thirty - One minute fractions were collected, dried down under nitrogen The fractions were dried down under nitrogen and analyzed for anti-viral activity. The active fractions were compared to the elution profiles for each of the original samples. It was clear from this experiment that the active fractions do not appear to have a strong UV-absorbence at 218nm or 254nm. It is believed that the components contained within this reversed phase purified fraction may include, but is not limited to material consisting of naturally occurring, recombinant or synthetic protein, peptide, nucleic acid, glycolipid, fatty acid, small molecule or any combination thereof.
  • CEM-SS cells were passaged in T- 150 flasks for use in the assay. On the day preceding the assay, the cells were split 1:2 to assure they would be in an exponential growth phase at time of infection. On the day of assay the cells were washed twice with tissue culture medium and resuspended in fresh tissue culture medium. Total cell and viability counting were performed using hemacytometer and trypan blue dye exclusion. Cell viability was greater than 95% for the cells to be utilized in the assay. The cells were pelleted and resuspended at 2.5 x 10 4 cells/ mL in tissue culture medium. Cells were added to the drug- containing plates in a volume of 50 ⁇ L.
  • Virus Preparation A pretitered aliquot of virus was removed from the freezer (-80°C) and allowed to thaw slowly to room temperature in a biological safety cabinet. The virus was resuspended and diluted into tissue culture medium such that the amount of virus added to each well in a volume of 50 ⁇ L will be the amount determined to give complete cell killing at 6 days post- infection. In general the virus pools produced with the IIIB isolate of HIV required the addition of 5 ⁇ l of virus per well. TCID50 calculation by endpoint titration in CEM-SS cells indicated that the multiplicity of infection of these assays ranged from 0.005 to 0.01.
  • Plate format The format of the test plate has been standardized. Each plate contained cell control wells (cells only), virus control wells (cells plus virus) , drug toxicity control wells (cells plus drug only) , drug calorimetric control wells (drug only) as well as experimental wells (drug plus cells plus virus) .
  • XTT staining of screening plates After 6 days of incubation at 37°C in a 5% CO2 incubator, the test plates were analyzed by staining with the tetrazolium dye XTT. XTT-tetrazolium is metabolized by the mitochondrial enzymes of metabolically active cells to a soluble formazan product, allowing the rapid quantitative analysis of the inhibition of HIV-induced cell killing by anti-HIV test substances. On Day 6 post-infection, plates were removed from the incubator and observed. The use of round bottom microtiter plates allows rapid macroscopic analysis of the activity of a given test compound by the evaluation of pellet size. The results of the macroscopic observations were confirmed and enhanced by further microscopic analysis.
  • XTT solution was prepared daily as a stock of 1 mg/ml in PBS.
  • Phenazine methosulfate (PMS) solution was prepared at 15 mg/ml in PBS and stored in the dark at -20°C.
  • XTT/ PMS stock was prepared immediately before use by diluting the PMS 1 : 100 into PB S and adding 40 ⁇ l per ml of XTT solution.
  • HIV Reverse Transcriptase Assay This assay is performed using the rC:dG template: primer and recombinant purified HIV- 1 reverse transcriptase. Water, compound, 2X RT buffer (50 mM Tris pH 8.0, 150 mM KCl, 16 mM MgCl 2 4 mM DTT, 0.2 Units of rC:dG (template /primer) 20 ⁇ M dGTP and 4 Ci/mmol ⁇ 32P dGTP) and reverse transcriptase enzyme (pretitered concentration) are mixed in this order and incubated for 37°C for 45 min.
  • 2X RT buffer 50 mM Tris pH 8.0, 150 mM KCl, 16 mM MgCl 2 4 mM DTT
  • Sheared and denatured salmon sperm DNA (300 ⁇ g per reaction) is added, and samples precipitated with 10% ice cold TCA. Precipitates are harvested on glass fiber filter mats, washed twice with cold 10% TCA and dried. 3 H- ⁇ 32P dGTP incorporation is determined by scintillation counting.
  • the attachment assay is performed with the HeLa CD4 LTR ⁇ -gal cells available from the AIDS Research and Reference Reagent Repository.
  • HeLa CD4 LTR ⁇ -gal cells are routinely cultured with the required selection antibiotics. Twenty- four hours prior to initiation of the assay cells are trypsinized, counted and 1 x 10 4 cells placed in a 0.2 cm well in media without selection antibiotics. At 24h media is removed and compound in media placed on the cells and incubated for 15 to 30 min at 37°C. A known titer of virus is then added to the wells and the incubation continued for 1 h. At the end of the incubation the wells are washed 6 times with media and the culture continued for 48h.
  • ⁇ -galactosidase enzyme expression determined by chemiluminescence per manufacturers instructions (Tropix Gal- screenTM, Bedford Mass.). This chemiluminescent method uses a single solution containing cell lysis components and chemiluminescent substrates to detect activity in a single step.
  • P3HR- 1 cells are latently infected with EBV. Culturing these cells in the presence of phorbol ester induces a lytic infection cycle, resulting in the release of viral particles into the medium. Compound efficacy in this assay is determined using TaqMan PCR technology, and compound toxicity is measured by addition of the metabolic dye XTT.
  • P3HR- 1 cells are counted by the trypan blue dye exclusion method, and the cell count/ ml is determined. Cells are pelleted by centrifugation and washed once with PBS to minimize the basal level of virus released from untreated cells which spontaneously enter the lytic cycle and release virus.
  • Cells are resuspended in fresh media and adjusted to a cell density of 8 x 10 5 per ml. Cells are then plated in the interior wells of 96- well round-bottom microtiter plates in a volume of 50 ⁇ l per well (4 x 10 4 cells per well) .
  • the phorbol ester TPA is added to appropriate wells in a volume of 50 ⁇ l per well to yield a final concentration of 15 ng/ml.
  • Medium is added in place of TPA in three wells to serve as a negative control.
  • Test compounds are diluted in DMSO to a stock concentration which is 400x the desired high test concentration. These compounds are further diluted in complete medium in 1.2 ml titer tubes to yield a solution which is 2x the desired high test concentration. Serial half-log dilutions of this solution are performed in complete medium to yield a total of 6 test concentrations for each test compound. Compound dilutions are added to appropriate wells of the microtiter plate in a volume of 100 ⁇ l per well. Medium containing no test compound is added to the virus control and cell control wells. In addition, 200 ⁇ l of medium containing no test compound is added to the exterior wells of the plate.
  • Plates are incubated at 37°C in a humidified CO 2 incubator until day 4 post drug addition.
  • Efficacy plates are removed from the incubator, and cells are pelleted by centrifugation. Supernatant samples (100 ⁇ l) are removed from each well and transferred into 96-well storage plates. 2. Pronase is added to the supernatant samples to a final concentration of 0.75 mg/ml to degrade DNA which has been released by dead cells. Encapsidated DNA from intact virions is not affected by this treatment. Samples are incubated at 37°C for 30 minutes. Supernatant samples are then treated with 1 Unit of DNase per well and incubated at 37°C for 60 minutes. DNase is inactivated by heating samples to 95°C for 15 minutes.
  • PCR reaction mixtures are prepared from reagents provided in the PE Applied Biosystems TaqMan PCR Reagent Kit according to the manufacturer's directions. Total reaction volumes will be 50 ⁇ l. Master reaction mix is dispensed into optical PCR tubes in a volume of 47 ⁇ l. Samples (3 ⁇ l) are added to the reaction mix and mixed thoroughly.
  • XTT stain is prepared, and 50 ⁇ l of the stain is added to each well of the toxicity plates. Plates are incubated at 37°C in a humidified CO 2 incubator for 4 hours or until sufficient color development has occurred. Plates are read on a VMax microtiter plate reader at a wavelength of 450/650 nm. Toxicity of the test compound is determined by comparing the optical density of test wells with that of control wells.
  • VG-201 was tested at concentrations from 20 ⁇ g/ml.

Abstract

A therapeutic method for treating a range of human diseases and infectious viral disorders with particular reference to HIV, neoplasia and Herpes. The method comprises the use of a chromatographically enriched fraction from porcine liver extract or acetone-derived powdered porcine extract administered to patients in therapeutically effective doses. The extract has been shown to be stable to pH, temperature and certain organic solvents. The extract is water-soluble and can be reproducibly enriched in active form following low pH organic elution from reversed phase chromatography supports.

Description

ENRICHED FRACTION FROM A PORCINE LIVER EXTRACT FOR TREATING HUMAN DISEASES
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention is directed to a method of treating numerous human diseases and to the discovery of a partially purified, i.e. an enriched fraction from porcine extract that is efficacious in treating such diseases. This invention is particularly directed toward a method of treating HIV, Herpes, multiple sclerosis, and other viral diseases with this enriched fraction and/ or with specific biologically active components, alone or in combination, further purified from this partially purified fraction.
2. Description of prior art
Mammalian liver extract has been used in the treatment of a wide range of infectious and noninfectious dermatologic conditions including acne vulgaris, Journal Invest. Dermatology, 2:205-218 (1939); first and second degree burns, Mississippi Medical Journal, 76: 199 (1954); sunburn, Clinical Medicine, 3:245 (1956); poison ivy dermatitis, Clin. Med., 3:425 (1956) and Herpes zoster, Southern Medical Journal, 50: 1524 (1957). A specific active component or a known mechanism for such treatment has not yet been defined. Crude mammalian liver extract has also been reported to have bradykinin potentiating activity, Tewksbury, et. Al., Arch. Biochem. Biophys. (U.S.), 1 12:453 (1965); Tewksbury, Archives Intl. de Pharmacodynamie et de Therapie, 173: 426 (1964); Tewksbury, Dissertation Abstracts International-Part II, Vol 25/04, p. 2214 (1964). A once available product marketed under the tradename "Kutapressin" was shown to exert its action, according to product literature, only with respect to tissues that have been injured and when inflammation and edema are present. Further, in U.S. Pat. No. 5,055,296, the use of heat stable acetone-insoluble, water soluble mammalian liver extract was shown to be effective in the treatment of mammals infected with nondermatologic viruses, in particular, in the treatment of chronic fatigue syndrome.
In recent patents, claims have been put forth that state that a heat stable, acetone insoluble, water soluble mammalian liver extract may be used for treating Epstein-Barr viral infections (U.S. 5,334,395), hepatitis B infection (U.S. 5,316,775) and the symptoms of Alzheimer's disease (U.S. 5,284,664). Further, it has been suggested in these three patents that biologically active peptides may, in fact, be the active components of this liver extract. Sequence information for the peptides is included in these patents. These patents also state that the liver extract is free from fatty acids and vitamins, specifically Vitamin B-12, a vitamin naturally occurring in liver. While it is clear from these patents that porcine liver extract does demonstrate some effect as claimed, it is not clear that specific peptides, nucleic acids, proteoglycans, glycoproteins, hormones or other naturally occurring biomolecules demonstrate anti- viral activity. While specific peptide sequences are provided, the synthesis of these sequences and their testing in anti-viral assays was not performed. Hence, it is not clear if the activity stated in these patents is from a specific peptide or the crude mixture.
SUMMARY OF THE INVENTION
This invention provides several bioactive fractions which have been purified or enriched from a porcine liver extract using a single reversed phase HPLC separation step.
It has now been found that numerous human diseases may be effectively treated by the administration of sufficient amounts of a partially purified fraction of porcine liver extract derived from porcine liver and designated herein as VG-201 if obtained from the primary separation step and VG-201 A if obtained from the secondary separation step. More specifically, the present invention provides a method of treating such diseases involving administering to humans having said diseases a therapeuticaUy effective dose of VG-201 and/or VG-201A. The enriched fractions VG-201 and VG-201A may be administered independently of each other or in combination with each other.
These purified fractions are characterized by being the chromatographicaUy enriched fractions from porcine liver extract or acetone powder derived from porcine liver, and having been eluted from sequential reversed phase (C18) columns in the presence of acetonitrile and low pH induced by trifluoroacetic acid (TFA) in the mobile phase. The fractions originated from heat stable, acetone insoluble, water-soluble fresh porcine liver extract. However, other forms of starting material such as acetone powdered extract may also be used.
The above method, involving the administration of the purified fraction of porcine liver extract VG-201 and/or VG-201A provides a novel method for the treatment or prevention of a variety of human diseases characterized by neoplasia such as Hodgkin's and non-Hodgkin's lymphoma, Kaposi's sarcoma, primary effusion lymphoma, nasopharyngeal carcinoma, gastric carcinoma, leiomysarcoma, Burkitt's lymphoma, cervical cancer, prostate cancer, colon cancer, breast cancer and leukemia. It would be evident that the method will find ready application for the prevention or treatment of neoplastic disease states, particularly those specifically named, in which neoplasia is an indication. Additionally, it has been found that viral infection in humans may be improved dramatically by the administration of the porcine liver extract VG-201 and/or VG-201A resulting in inhibition, if not complete suppression of the viral infection of the human. Thus, it has also been discovered that VG-201 and/or VG-201A may be utilized to treat diseases including Human Immunodeficiency Virus 1 (HIV- 1) infection, Herpes virus infections, particularly Herpes 1 (Herpes simplex 1), Herpes 2 (Herpes simplex 2), Herpes 3 (Varicella zoster virus), Herpes 4 (Epstein- Barr virus), Herpes 5 (Cytomegalovirus), Herpes 6a and 6b, Herpes 7 and Herpes 8 (Kaposi-associated herpes virus), lepromatous leprosy, multiple sclerosis and diabetes mellitus.
Finally, it has been discovered that VG-201 and/or VG-201A is also effective against organ and bone marrow transplantation infection, sarcoidosis, encephalopathy syndrome, Sjogren's syndrome, and Castleman's disease, lymphoproliferative disorders, and cytomegalovirus retinitis.
The partially purified porcine liver extract, i.e. either enriched fraction VG-201 or VG-201 A may be administered alone or in combination with other pharmaceutically acceptable agents. A preferred product is an aqueous solution containing approximately 2.5% by weight of liver extract solids. Doses of from not less than about 1 ml per day to not more than about 5 ml per day, preferably about 2 ml per day, of either extract in solution are generally effective. This method has the distinct advantage that it will treat the disease as long as the appropriate dosages are used, it being understood that the dosage levels will be adjusted dependent upon the response of the subject as monitored by methods known to those skilled in the art. The porcine extract VG-201 and/ or VG- 201 A may be administered orally, topically, or parenterally, such as via intravenous or intramuscular injection, in liquid, semi-solid or solid form for use in the above treatments depending upon the extent of the disease condition, the particular disease or condition being treated, and the results desired. The porcine liver extract VG-201 or VG-201 A may also be administered in combination with other pharmaceuticals, if necessary. BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING Fig. 1 shows the process flow diagram for the manufacturing process used to prepare the liver extract as well as the enriched fraction VG-201 using separation A. Fig. 2 shows the process flow diagram for the manufacturing process used to prepare the liver extract as well as the enriched fraction VG-201A using separation B.
DETAILED DESCRIPTION OF THE INVENTION A method for treating a variety of human diseases has now been discovered. The material that has been shown to be active in treating these human diseases and infections is a reversed phase enriched fraction of a heat stable, acetone insoluble, water soluble porcine liver extract. There is a single reversed phase step as described below. The active fractions were collected by following the UV trace of the detector and assayed using a cell-based anti-viral assay.
1. Preparation of the Liver Extract VG-200
Porcine livers were homogenated at high speed in a blender followed by the addition of three volumes of tap water to one volume of liver. For every 500kg of tapwater, add 7.9kg of concentrated sulfuric acid (H2SO4). The suspension is then agitated and heated to boiling followed by a cooling to 60°C. All solids are removed appropriately, for example, either by decanting the supernatant or filtration and the supernatant concentrated to approximately V2 volume by rotary evaporation at 80°C. The temperature must be maintained above 60°C during this evaporation. The extract may be frozen or lyophilized at this point as it is a stable hold step in the process.
In the next step of the process, the material is further extracted using 70% ethanol/30% water mixture (1.2L of 95% ethanol and 0.5L water to each kg of solid). The organic extraction occurs at 27°C to 32°C with stirring followed by stirring for one hour at room temperature. The precipitated material is allowed to settle for one to two hours and the liquid removed by decanting or filtration. The liquid is then frozen at - 20°C for ten days. This material is refereed to as "liver fraction 1" as well as "LFl". The LFl recipe can be found in the National Formulary XI, page 193.
The ethanol extract is then cold filtered and concentrated to 30% to 40% solids under vacuum at 80°C. Water may be re-added followed by additional concentration to 30% to 40% solids to drive off residual ethanol. The extract is stable at this point and may be lyophilized. Dilute the extract to 16% total solids by weight and mix well. Add 90% distilled phenol to a final phenol concentration of 1% and allow the solution to sit undisturbed for seven days at room temperature. Following the seven- day incubation, filter the material through fluted filter paper to remove nay precipitated or solid material. Measure the volume of the resultant clarified solution and dilute further to 8% solids by adding an equal volume of water. This material is then passed over an Amberlyte column (other cation exchange supports may be substituted for the Amberlyte) that has bee sodium hydroxide activated and collect the eluent that should be the same volume as the column load. Refrigerate overnight, clarify by filtration and concentrate to 40% total solids by evaporation under vacuum at 55°C to 70°C. Add cold water (five volumes of water to 7 volumes of extract) with mixing and remove all solids by centrifugation with a continuous flow centrifuge at IL/ minute. Add phenol to a final concentration of 0.5 to 1.0%. This fraction is referred to as the "Liquid Liver Special" or "LLS".
Measure 12 L of LLS and adjust the pH to 6.0 to 7.0 using diluted HC1 USP or IN NaOH USP. Filter through a 10 micron filter and heat to 40°C for a total of twelve cycles. Add the filtered extract to a mixing volume of acetone at a ratio of IL extract to 23 liters acetone. Agitate and allow the precipitated material to settle out of solution. Remove as much acetone from the precipitate as possible and redissolve precipitate in pyrogen free water to a total volume of 30 liters. Add approximately 200 grams of boiling chips and distill off the residual acetone to a final volume of approximately 12 liters. Allow the solution to boil at 100°C for at least two hours, cool to room temperature and add pyrogen free water to a total volume of approximately 27.5 liters. Add 158ml phenol and q.s. the solution to 28 liters with pyrogen free water and mix well. This solution is known as the "Crude Stock Solution" or "CSS".
Adjust the pH to 6.0 to 7.0 using IN HC1 USP or IN NaOH USP, mix for fifteen minutes and filter through a 10 micron filter followed by filtration through 0.45 micron and 0.22 micron filters to remove any particulates. This material may be autoclaved at 120°C for approximately 20 minutes if it is to be held prior to chromatographic processing.
2. Chromatographic Separation A (VG-201)
The chromatographic enrichment takes place in two steps. A primary reversed phase separation of the CSS was performed to obtain
VG-201 followed by a second reversed phase separation of active fractions to obtain VG-201A, as follows. Approximately 75mg of CSS were purified by reversed phase chromatography using the following conditions:
Instrument: Waters 2690 HPLC with Waters 2487 detector Column: 10x150mm, reversed phase (C- 18) Platinum series 3μm, 100A Alltech Eluents: A: 0.1%TFA in water
B: 95% acetonitrile + 0.1%TFA in water Flow Rate: 3ml/ minute
Elution: 0- 10 minutes isocratic 20%B
10-25 minutes gradient 20-60%B 25-45 minutes isocratic 60%B
Fraction Collection: 2.0ml starting at approximately 1 1 minutes All fractions were dried down under vacuum and reconstituted in phosphate buffered saline. A portion of each fraction was assayed for anti-HIV activity. Fractions 14, 17 and 19 through 28 demonstrated anti- HIV activity and were combined. (VG-201)
3. Chromatographic Separation B (VG-201A)
Fractions 14, 17 and 19 were chosen to be further separated, again using reversed phase (C 18) HPLC with different column geometry and slightly different gradient conditions. The details of the secondary separation (Chromatograph Separation B) were as follows:
Instrument: Waters 2690 HPLC with Waters 2487 detector Column: 4.6x150mm, reversed phase (C-18) Platinum series 3μm, 100A Alltech PN C-6000SWB
Detection: 218nm and 254nm Eluents: A: 0.1 %TFA in water
B: 95% acetonitrile, 0.1%TFA in water
Flow Rate: lml /minute Injection volume: 50μl
Elution Conditions:
Figure imgf000010_0001
Fraction collection: Thirty - One minute fractions were collected, dried down under nitrogen The fractions were dried down under nitrogen and analyzed for anti-viral activity. The active fractions were compared to the elution profiles for each of the original samples. It was clear from this experiment that the active fractions do not appear to have a strong UV-absorbence at 218nm or 254nm. It is believed that the components contained within this reversed phase purified fraction may include, but is not limited to material consisting of naturally occurring, recombinant or synthetic protein, peptide, nucleic acid, glycolipid, fatty acid, small molecule or any combination thereof.
4. Bioassay
• Fractions collected from both the primary (VG-201) and secondary (VG-201 A) reversed phase separation were analyzed for anti-HIV activity using the cell-based assay described elsewhere in this document. For the primary separation, the chromatographic elution profile and anti-HIV profile have been reproduced on several occasions by two separate organizations using different HPLC's. Active fraction demonstrates anti-viral activity at a level considered to be biologically relevant.
Fractions collected from the rechromatography of active fraction (Separation B) were collected, dried and submitted for bio assay. The active fractions for each of these elutions were at the approximate retention times listed below:
• Fraction 14 Approximately 14 minutes
• Fraction 17 Approximately 14.2 minutes
• Fraction 19 Approximately 14.5 minutes When corrected for the injection peak, the active fractions from each of these runs elute within 0.1 to 0.3 minutes of each other indicating a consistent elution of the active component in each of these fractions.
5. Bioassay
Fractions collected from both the primary (VG-201) and secondary (VG-201 A) reversed phase separation were analyzed for anti-HIV activity using the cell-based assay described elsewhere in this document. For the primary separation, the chromatographic elution profile and anti-HIV profile have been reproduced on several occasions by two separate organizations using different HPLC's.
Anti-HIV Activity
I. Evaluation of Anti-HIV Activity of Compounds in Established
Cell Lines
a. Cell Preparation: CEM-SS cells were passaged in T- 150 flasks for use in the assay. On the day preceding the assay, the cells were split 1:2 to assure they would be in an exponential growth phase at time of infection. On the day of assay the cells were washed twice with tissue culture medium and resuspended in fresh tissue culture medium. Total cell and viability counting were performed using hemacytometer and trypan blue dye exclusion. Cell viability was greater than 95% for the cells to be utilized in the assay. The cells were pelleted and resuspended at 2.5 x 104 cells/ mL in tissue culture medium. Cells were added to the drug- containing plates in a volume of 50 μL.
b. Virus Preparation: A pretitered aliquot of virus was removed from the freezer (-80°C) and allowed to thaw slowly to room temperature in a biological safety cabinet. The virus was resuspended and diluted into tissue culture medium such that the amount of virus added to each well in a volume of 50 μL will be the amount determined to give complete cell killing at 6 days post- infection. In general the virus pools produced with the IIIB isolate of HIV required the addition of 5μl of virus per well. TCID50 calculation by endpoint titration in CEM-SS cells indicated that the multiplicity of infection of these assays ranged from 0.005 to 0.01.
c. Plate format: The format of the test plate has been standardized. Each plate contained cell control wells (cells only), virus control wells (cells plus virus) , drug toxicity control wells (cells plus drug only) , drug calorimetric control wells (drug only) as well as experimental wells (drug plus cells plus virus) .
d. XTT staining of screening plates: After 6 days of incubation at 37°C in a 5% CO2 incubator, the test plates were analyzed by staining with the tetrazolium dye XTT. XTT-tetrazolium is metabolized by the mitochondrial enzymes of metabolically active cells to a soluble formazan product, allowing the rapid quantitative analysis of the inhibition of HIV-induced cell killing by anti-HIV test substances. On Day 6 post-infection, plates were removed from the incubator and observed. The use of round bottom microtiter plates allows rapid macroscopic analysis of the activity of a given test compound by the evaluation of pellet size. The results of the macroscopic observations were confirmed and enhanced by further microscopic analysis. XTT solution was prepared daily as a stock of 1 mg/ml in PBS. Phenazine methosulfate (PMS) solution was prepared at 15 mg/ml in PBS and stored in the dark at -20°C. XTT/ PMS stock was prepared immediately before use by diluting the PMS 1 : 100 into PB S and adding 40μl per ml of XTT solution.
Fifty micro liters of XTT/ PMS was added to each well of the plate and the plate was reincubated for 4 hours at 37°C. Adhesive plate sealers were used in place of the lids, the sealed plate was inverted several times to mix the soluble formazan product and the plate was read spectrophotometrically at 450 nm with a Molecular Devices Vmax plate reader. Using a computer program, %CPE
Reduction, %Cell Viability, IC25, 50 & 95, TC25,50 & 95 and other indices were calculated and the graphic results summary was displayed.
Efficacy of VG-201 or VG-201 A in CEM-SS Cells HIV- 1 Antiviral Activity
IC50 Activity 5.05-21.4 μg/mL
II. HIV Reverse Transcriptase Assay This assay is performed using the rC:dG template: primer and recombinant purified HIV- 1 reverse transcriptase. Water, compound, 2X RT buffer (50 mM Tris pH 8.0, 150 mM KCl, 16 mM MgCl2 4 mM DTT, 0.2 Units of rC:dG (template /primer) 20 μM dGTP and 4 Ci/mmol α32P dGTP) and reverse transcriptase enzyme (pretitered concentration) are mixed in this order and incubated for 37°C for 45 min. Sheared and denatured salmon sperm DNA (300 μg per reaction) is added, and samples precipitated with 10% ice cold TCA. Precipitates are harvested on glass fiber filter mats, washed twice with cold 10% TCA and dried. 3H-α32P dGTP incorporation is determined by scintillation counting.
HIV Reverse Transcriptase Inhibition Assay
IC50 (μg/mL)
Crude Preparation VG-201 UC38*
Activity 60.97 0.91 3.42**
* Positive Control ** μM III. Attachment assay.
The attachment assay is performed with the HeLa CD4 LTR β-gal cells available from the AIDS Research and Reference Reagent Repository. HeLa CD4 LTR β-gal cells are routinely cultured with the required selection antibiotics. Twenty- four hours prior to initiation of the assay cells are trypsinized, counted and 1 x 104 cells placed in a 0.2 cm well in media without selection antibiotics. At 24h media is removed and compound in media placed on the cells and incubated for 15 to 30 min at 37°C. A known titer of virus is then added to the wells and the incubation continued for 1 h. At the end of the incubation the wells are washed 6 times with media and the culture continued for 48h. At 48h the media is removed and β-galactosidase enzyme expression determined by chemiluminescence per manufacturers instructions (Tropix Gal- screen™, Bedford Mass.). This chemiluminescent method uses a single solution containing cell lysis components and chemiluminescent substrates to detect activity in a single step.
Attachment Results
HIV Attachment Assay
Crude Chicago Sky Preparation VG-201 Blue* Activity 2.4 0.1 0.1 (μg/mL)
Cytotoxicity >200 > 100 > 10
(μg/mL)
Therapeutic Index >83.0 > 100 >100
* Positive Control Anti-Human Herpes Virus
Activity against human herpes virus is demonstrated against the specific herpes virus Epstein Barr Virus (human herpes virus 4) and human Herpes Virus 6.
EBV Assay
P3HR- 1 cells are latently infected with EBV. Culturing these cells in the presence of phorbol ester induces a lytic infection cycle, resulting in the release of viral particles into the medium. Compound efficacy in this assay is determined using TaqMan PCR technology, and compound toxicity is measured by addition of the metabolic dye XTT.
Assay Set-Up
1. P3HR- 1 cells are counted by the trypan blue dye exclusion method, and the cell count/ ml is determined. Cells are pelleted by centrifugation and washed once with PBS to minimize the basal level of virus released from untreated cells which spontaneously enter the lytic cycle and release virus.
2. Cells are resuspended in fresh media and adjusted to a cell density of 8 x 105 per ml. Cells are then plated in the interior wells of 96- well round-bottom microtiter plates in a volume of 50 μl per well (4 x 104 cells per well) .
3. The phorbol ester TPA is added to appropriate wells in a volume of 50 μl per well to yield a final concentration of 15 ng/ml. Medium is added in place of TPA in three wells to serve as a negative control.
4. Test compounds are diluted in DMSO to a stock concentration which is 400x the desired high test concentration. These compounds are further diluted in complete medium in 1.2 ml titer tubes to yield a solution which is 2x the desired high test concentration. Serial half-log dilutions of this solution are performed in complete medium to yield a total of 6 test concentrations for each test compound. Compound dilutions are added to appropriate wells of the microtiter plate in a volume of 100 μl per well. Medium containing no test compound is added to the virus control and cell control wells. In addition, 200 μl of medium containing no test compound is added to the exterior wells of the plate.
5. Plates are incubated at 37°C in a humidified CO2 incubator until day 4 post drug addition.
6. Duplicate plates are set up plating media in place of TPA for evaluation of compound toxicity.
Determination of Compound Efficacy and Toxicity 1. Efficacy plates are removed from the incubator, and cells are pelleted by centrifugation. Supernatant samples (100 μl) are removed from each well and transferred into 96-well storage plates. 2. Pronase is added to the supernatant samples to a final concentration of 0.75 mg/ml to degrade DNA which has been released by dead cells. Encapsidated DNA from intact virions is not affected by this treatment. Samples are incubated at 37°C for 30 minutes. Supernatant samples are then treated with 1 Unit of DNase per well and incubated at 37°C for 60 minutes. DNase is inactivated by heating samples to 95°C for 15 minutes.
3. PCR reaction mixtures are prepared from reagents provided in the PE Applied Biosystems TaqMan PCR Reagent Kit according to the manufacturer's directions. Total reaction volumes will be 50 μl. Master reaction mix is dispensed into optical PCR tubes in a volume of 47 μl. Samples (3 μl) are added to the reaction mix and mixed thoroughly.
4. Reaction plates are loaded into a PE Applied Biosystems 7700 Sequence Detector, and a run cycle is initiated using the manufacturer's recommended PCR conditions. 5. A standard curve prepared from known copy numbers of DNA isolated from P3HR- 1 cells and amplified is run with each plate in order to quantitate the DNA copy number in each original sample. The efficacy of the compound is determined by comparing DNA copy numbers from test wells with those of control wells.
6. XTT stain is prepared, and 50 μl of the stain is added to each well of the toxicity plates. Plates are incubated at 37°C in a humidified CO2 incubator for 4 hours or until sufficient color development has occurred. Plates are read on a VMax microtiter plate reader at a wavelength of 450/650 nm. Toxicity of the test compound is determined by comparing the optical density of test wells with that of control wells.
Activity
VG-201 Cidofovir
IC50 (μg/mL) 1.28 2.31
TC50 >50 >25
Therapeutic Index >39.1 > 10.8 * Positive control
Antiviral assay for Human Herpes virus 6 (HHV-6) VG-201 was incubated with Molt 3 cells for 0.5 hours before virus
(HHV-6B strain Z29) is added. There follows a two hour viral adsorption step during which the virus is absorbed onto the cells, cultures are incubated at 37°C for seven days at which time the cells are harvested from the cultures and stained for immunofluoresence analysis. A positive control (no anti-viral treatment) and 5 concentrations of test compound are analyzed. In a separate preliminary experiment, the cyto toxicity of the test compound is assessed. Results
VG-201 was tested at concentrations from 20 μg/ml.
Cytotoxicity: None at any concentration tested.
Antiviral Activity: Concentration Result
20 μg/mL No virus present
6.7 μg/mL No virus present
2.2 μg/mL No virus present
0.74 μg/mL < 1% of cells have virus 0.25 μg/mL < 1% of cells have virus
Although the invention has been described primarily in connection with special and preferred embodiments, it will be understood that it is capable of modification without departing from the scope of the invention. The following claims are intended to cover all variations, uses, or adaptations of the invention, following, in general, the principles thereof and including such departures from the present disclosure as come within known or customary practice in the field to which the invention pertains, or as are obvious to persons skilled in the field.

Claims

CLAIMS I claim:
1. A method for treating viral infections in a human which comprises administering to a human suffering from said infection a therapeuticaUy effective amount of a porcine liver extract, referred to herein as VG-201 sufficient to treat the infection of said human.
2. The method of claim 1 wherein said extract is combined with a non-toxic pharmaceutically acceptable carrier and is administered in a suitable pharmaceutical dosage form.
3. The method of claim 1 wherein said viral infection is human immunodeficiency virus- 1 infection.
4. The method of claim 1 wherein said viral infection is lepromatous leprosy.
5. The method of claim 1 wherein said viral infection is a Herpes virus infection.
6. The method of claim 5 wherein said Herpes virus infection is Herpes simplex 1.
7. The method of claim 5 wherein said Herpes virus infection is Herpes simplex 2.
8. The method of claim 5 wherein said Herpes virus infection is Herpes 3.
9. The method of claim 5 wherein said Herpes virus infection is Herpes 4.
10. The method of claim 5 wherein said Herpes virus infection if Herpes 5.
1 1. The method of claim 5 wherein said Herpes virus infection is Herpes 6a.
12. The method of claim 5 wherein said Herpes virus infection is Herpes 6b.
13. The method of claim 5 wherein said Herpes virus infection is Herpes 7.
14. The method of claim 5 wherein said Herpes virus infection is Herpes 8.
15. The method of claim 1 wherein said viral infection is multiple sclerosis.
16. The method of claim 1 wherein said viral infection is diabetes mellitus.
17. A method of treating a disease characterized by neoplasia which comprises administering to a human suffering from said disease a therapeuticaUy effective amount of a porcine liver extract, referred to herein as VG-201 , said disease selected from the group consisting of Hodgkin's lymphoma, non-Hodgkin's lymphoma, Kaposi's sarcoma, primary effusion lymphoma, nasopharyngeal carcinoma, gastric carcinoma, leiomysarcoma, Burkitt's lymphoma, cervical cancer, prostate cancer, colon cancer, breast cancer and leukemia.
18. The method of claim 17 wherein said extract is combined with a non-toxic pharmaceutically acceptable carrier and is administered in a suitable pharmaceutical dosage form.
19. A method of treating a disease selected from the group consisting of organ and bone marrow transplantation infection, sarcoidosis, encephalopathy syndrome, Sjogren's syndrome, Castleman's disease, lymphoproliferative disorders, and cytomegalovirus retinitis, which comprises administering to a human suffering from said disease a therapeuticaUy effective amount of a porcine liver extract, referred to herein as VG-201.
20. The method of claim 19 wherein said extract is combined with a non-toxic pharmaceutically acceptable carrier and is administered in a suitable pharmaceutical dosage form.
21. A method for treating viral infections in a human which comprises administering to a human suffering from said infection a therapeuticaUy effective amount of a porcine liver extract, referred to herein as VG-201 A sufficient to treat the infection of said human.
22. The method of claim 21 wherein said extract is combined with a non-toxic pharmaceutically acceptable carrier and is administered in a suitable pharmaceutical dosage form.
23. The method of claim 21 wherein said viral infection is human immunodeficiency virus- 1 infection.
24. The method of claim 21 wherein said viral infection is lepromatous leprosy.
25. The method of claim 1 wherein said viral infection is a Herpes virus infection.
26. The method of claim 25 wherein said Herpes virus infection is Herpes simplex 1.
27. The method of claim 25 wherein said Herpes virus infection is Herpes simplex 2.
28. The method of claim 25 wherein said Herpes virus infection is Herpes 3.
29. The method of claim 25 wherein said Herpes virus infection is Herpes 4.
30. The method of claim 25 wherein Herpes virus infection is Herpes 5.
31. The method of claim 25 wherein said Herpes virus infection is Herpes 6a.
32. The method of claim 25 wherein said Herpes virus infection is Herpes 6b.
33. The method of claim 25 wherein said Herpes virus infection is Herpes 7.
34. The method of claim 25 wherein said Herpes virus infection is Herpes 8.
35. The method of claim 21 wherein said viral infection is multiple sclerosis.
36. The method of claim 21 wherein said viral infection is diabetes mellitus.
37. A method of treating a disease characterized by neoplasia which comprises administering to a human suffering from said disease a therapeuticaUy effective amount of a porcine liver extract, referred to herein as VG-201 A, said disease selected from the group consisting of Hodgkin's lymphoma, non-Hodgkin's lymphoma, Kaposi's sarcoma, primary effusion lymphoma, nasopharyngeal carcinoma, gastric carcinoma, leiomysarcoma, Burkitt's lymphoma, cervical cancer, prostate cancer, colon cancer, breast cancer and leukemia.
38. The method of claim 37 wherein said extract is combined with a non-toxic pharmaceutically acceptable carrier and is administered in a suitable pharmaceutical dosage form.
39. A method of treating a disease selected from the group consisting of organ and bone marrow transplantation infection, sarcoidosis, encephalopathy syndrome, Sjogren's syndrome, Castleman's disease, lymphoproliferative disorders, and cytomegalovirus retinitis, which comprises administering to a human suffering from said disease a therapeuticaUy effective amount of a porcine liver extract, referred to herein as VG-201 A.
40. The method of claim 39 wherein said extract is combined with a non-toxic pharmaceutically acceptable carrier and is administered in a suitable pharmaceutical dosage form.
PCT/US2000/023649 1999-09-03 2000-08-29 Enriched fraction from a porcine liver extract for treating human diseases WO2001017535A2 (en)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0537722A2 (en) * 1991-10-15 1993-04-21 Kremers- Urban Co. Porcane liver polypeptides for treating viral infections
US5334395A (en) * 1988-08-04 1994-08-02 Kremers-Urban Company Method of treating an epstein-barr viral infection
US5792744A (en) * 1994-07-14 1998-08-11 Zetesis S.P.A. Proteins from mammalian liver

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5334395A (en) * 1988-08-04 1994-08-02 Kremers-Urban Company Method of treating an epstein-barr viral infection
EP0537722A2 (en) * 1991-10-15 1993-04-21 Kremers- Urban Co. Porcane liver polypeptides for treating viral infections
US5792744A (en) * 1994-07-14 1998-08-11 Zetesis S.P.A. Proteins from mammalian liver

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