Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
  • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
  • A61K31/19—Carboxylic acids, e.g. valproic acid
  • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/66—Phosphorus compounds
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
  • A61K31/7004—Monosaccharides having only carbon, hydrogen and oxygen atoms
• • 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]
  • A61K38/212—IFN-alpha
• • 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
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
  • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
  • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

• the present invention relates to methods of treating a broad range of viral infections in humans and animals, including birds, using pharmaceutical
• PALA is a compound which was initially developed as a transition state analogue inhibitor of aspartate transcarbamylase. Stark et al . (1974) J. Biol . Chem . 246:6599. Subsequently, PALA (NSC No. 224131) was thoroughly studied as an anti-cancer agent. See, for
• CTP as well as nucleotide intermediates, viz, UDP- GlcN, CMP-NeuNAc which are essential for elongation of the oligosaccharide chain(s) .
• PALA acts primarily by inhibiting nucleotide biosynthesis, the effect on its intermediates is also reflected in its end products: carbohydrates, proteins, as well as nucleic acids (RNA and DNA) .
• PALA exerts- its action as a competitive inhibitor of carbamyl phosphate and as a non-competitive inhibitor of aspartate. Hooengraad, N.J. (1974) Arch. Biochem . Biophys . 161:76-82. Its Ka is lOOOx more avid than that of the natural substrate, carbamyl phosphate. Moore, E.C., Friedman, J. , Valdivieso, M. , Plunkett, . Marti, J.R. et al . (1982) Biochem Pharmacol . 31:3317-3321. Because of its relative lack of toxicity and the sensitivity of several solid murine tumors lines, PALA has been used in experimental oncology studies. Recent studies have shown that PALA possesses unique modulatory activity when used in combination with halogenated pyrimidines, e .g. , 5-fluorouracil, both in vitro and in vivo .
• halogenated pyrimidines e .
• PAA phosphonoacetic acid
• acycloguanosine for HSV 2' ,3 '-dideoxythymidine analogues, e.g., AZT, ddl, and ddC for HIV
• ribavirin virazole®
• carbocyclic nucleosides, cyclobut-A which had broad spectrum antiviral activity against HIV and the herpesviruses (CMV, HSV, varicella)
• CMV herpesviruses
• HSV herpesviruses
• PMEA phosphonyl-methoxyethyladenine
• Newer drugs which do bind to the RT of HIV and f i glycosylation inhibitors (e.g., 2-dGlc) which prevent
• Cytokines e.g., interferons
• inhibitors of regulatory genes e.g., adamantidine which blocks uncoating of influenza virus and the newer compounds targeted at the receptor level
• the present invention relates to methods of treating or preventing viral infections in humans, animals and birds by administering an effective amount
• an object of the present invention to provide an antiviral compound which is effective against a broad spectrum of viruses.
• a further object of the present invention is to provide combinational therapy which prevents viruses from potentially bypassing the inhibitory effect of PALA.
• a further object of the present invention is to provide combinational therapy which allows for reduced toxicity of PALA and/or the therapeutic agent with which PALA is used.
• An object of the present invention is to provide 4 a broad spectrum antiviral compound which has a low level of toxicity, and therefore, has a higher therapeutic index.
• Yet another object of the present invention is to 10 provide a broad spectrum antiviral that has unique utility against drug resistant viral strains when used alone or in combination with other therapeutics, including but not limited to antiviral agents and/or inhibitors of viral replication. 15 Still a further object of the present invention is to provide pharmaceutically acceptable analogs of PALA which exhibit antiviral activity on oral administration.
• Figure 2 is a graph of AD 169 cell associated HCMV titers recovered from sonicated cell pellets
• Figure 3 is a graph of HCMV clinical isolate titers recovered from supernatant assay after incubation with DHPG, PALA or placebo.
• Figure 4 is a graph .of HCMV cell associated clinical isolate titers recovered from sonicated cell pellets after incubation with DHPG, PALA or placebo.
• Figure 5 is a graph of HCMV DHPG resistant isolate titers recovered from supernatant assays after incubation with DHPG, PALA or placebo.
• Figure 6 is a graph of HCMV cell associated DHPG resistant virus titers recovered from sonicated cell pellets after incubation with DHPG, PALA or placebo.
• Figure 7 is a bar graph of the vitreitis severity found in the animals of Example 5, infra.
• Figure 8 is a bar graph of the average vitreitis disease severity in single- and combination-agent therapy groups of Example 6.
• Figure 9 is a bar graph of the average vitreitis disease severity found in the therapy groups of Example 7.
• Figure 10 is a bar graph of the average optic nerve disease severity in the therapy groups of Example 7.
• Figure 13 is a plot of the log of the vaccinia viral titers for the therapy groups PALA, rifampicin and PALA + rifampicin.
• the invention is based, in part, on the discovery that PALA, a drug which has been reported to be ineffective in the treatment of cancer when used alone, is effective when used as a broad spectrum antiviral.
• PALA when used alone or in combination with other drugs, demonstrates widespread utility as an antiviral agent useful in both human and veterinary medicine.
• Viruses are obligatory intracellular parasites which take over the host cell machinery and use existing intracellular structures e.g., polysomes, endoplasmic reticulu , golgi, and specific host cell macromolecules viz, enzymes, tRNA etc. to produce a template or transcript of viral mRNA.
• Viral nucleic acids are transcribed using unique polymerases and viral proteins are translated
• viruses which are amenable to treatment with PALA encompass all types and classes of known viruses including both DNA and RNA viruses (both positive and negative stranded viruses) .
• PALA can be used against DNA and RNA viruses and virus types including but not limited to the following: Adenoviruses
• Flaviviruses yellow fever virus Japanese encephalitis virus Flaviviruses yellow fever virus Japanese encephalitis virus
• Epstein-Barr virus (EBV)
• RSV respiratory syncytial virus
• rinderpest virus veterinary
• influenza A H 2 N 2 & H 3 N 2
• influenza B certain strains
• influenza C Hepadnaviruses hepatitis B
• Hepatitis viruses (not yet fully classified) hepatitis A (HAV) hepatitis C (HCV) hepatitis E (HEV)
• Picoanaviruses polioviruses coxsackieviruses
• viral infections describes a diseased state in which a virus invades healthy cells, uses the cell's reproductive "machinery” to multiply or replicate and ultimately lyses the cell resulting in cell death, release of viral particles (virions) and the infection of other cells by the newly produced progeny viruses. Latent infection by certain viruses is also a possible result of viral infection. It is clear that one skilled in the art would understand the meaning of these terms and the disease and/or infections to which it relates.
• treating or preventing viral infections in humans, animals or birds means to inhibit the replication of the particular virus or to prevent the virus from establishing itself in its host, and to ameliorate or alleviate the symptoms of the disease caused by the viral infection.
• PALA and its pharmaceutically acceptable analogs can be used alone or in combination with other therapeutic agents when used against these viruses. It has been found that when treating herpesviruses it is preferred that PALA be used in combination with other therapeutic agents such as the antivirals acyclovir or ganciclovir.
• the use of PALA, or a pharmaceutically acceptable analog, in combination therapy against herpesviruses provides benefits over the presently available therapies; for example the reduced toxicity of the antivirals presently used to treat these viral infections.
• PALA, or a pharmaceutically acceptable analog thereof can have a unique utility against drug resistant strains of herpesviruses, such as acyclovir or ganciclovir resistant strains.
• PALA or a pharmaceutically acceptable analog thereof may be useful in prolonging or blocking the development of drug resistant viral strains e.g., DHPG or acyclovir.
• PALA or a pharmaceutically acceptable analog thereof
• other therapeutics such as the antiviral rifampicin
• the occupational hazards involved in using such vaccinia constructs would thus be minimized.
• immunocompromised individuals can be protected or treated with PALA, or a pharmaceutically acceptable analog thereof.
• PALA exerts its antiviral effect by either inhibiting early steps of pyrimidine biosynthesis, viz. ATCase, decreasing nucleotide pools or inhibition of viral DNA polymerase, yielding the activity noted in Tables 1-3, infra .
• PALA may be used in combination with another therapeutic agent(s) to enhance the antiviral effect achieved.
• additional antiviral agents include but are not limited to those which function on a different target molecule involved in viral replication; those which act at a different loci of the same molecule; those which inhibit salvage pathways (described below) in order to prevent or reduce the occurrence of viral resistance.
• viruses possess their own DNA or RNA polymerases the more complex viruses viz , herpesvirus and poxvirus, to name a few, also possess individual enzymes responsible for nucleoside biosynthesis, e.g., phosphoribosyltransferases or nucleoside phosphorylase(s) which are also present as host cell enzymes. These enzymes may impart an alternative route or "salvage pathway" for pyrimidine synthesis, bypassing the inhibitory effect of antiviral agents. Thus, as with certain tumors, viral resistance to antiviral compounds could emerge.
• nucleoside analogues including but not limited to adenine arabinoside, adenine arabinoside monophosphate, idoxuridine, trifluorothymidine, acycloguanosine, bromovinyldeoxyuridine, bromovinyldeoxyarauridine (BVaraU by Bristol-Myers Squibb) fluoroiodoaracytosine, DHPA and ribavirin (virazole®) , glycosylation inhibitors (e.g., 2-dGlc) , protease inhibitors, interferons, nucleoside transport inhibitors such as dipyridamole and nitrobenzylthioinosine, DNA dependant RNA polymerase inhibitors, e.g., rifampicin (rifadin®) , chain terminators, e.g., ganciclovir (DHPG), acyclo
• PALA can also be used optionally with rifampicin (rifadin®) for vaccinia; optionally with AZT, ddl, ddC and combinations thereof for HIV-1 and 2; optionally with adamantidine for influenza; optionally with ribavirin (virazole®) for Lassa fever, Hantaan and CCHF viruses; and optionally with acyclovin ACV for varicella-zoster and optionally with interferon- ⁇ or fluorouracil for human papilloma virus.
• PALA in conjunction with another antiviral agent allows for the use of a lower dosage of one or both active agents so that the therapeutic index is increased, and toxic side effects are reduced. Because PALA does not appear to significantly alter humoral immune response - at least in tumor bearing animals (Johnson, R.K. Swyryd, E.A. and Stark, G.R. (1978) Cancer Res . 38:371-378), the use of PALA in accordance with the present invention would permit concurrent immunization for certain viruses (with appropriate vaccines, e.g., inactivated viruses or synthetic peptides as immunogens) .
• appropriate vaccines e.g., inactivated viruses or synthetic peptides as immunogens
• PALA is able to cross the blood- brain barrier and thus, relatively high concentrations can be achieved in the retina and brain; thus, PALA may also prove useful in HIV-induced encephalopathy and in CMV-induced and varicella-induced retinitis.
• PALA may be used as a prophylactic for individuals entering geographic zones where certain "exotic RNA viruses" are prevalent, yet immunization is not available (for that virus) or has not taken effect.
• polyvalent, genetically engineered vaccines may use a vaccinia construct; thus the possibility of generalized vaccinia in a patient and or a laboratory worker is real.
• the accessibility of a drug like PALA alone, or in combination with rifampicin (rifadin®) offers the treating physician a unique opportunity to intervene.
• Many of the above individuals (exposed occupationally) to such genetically engineered viruses could be treated with PALA on an outpatient basis either prophylactically or therapeutically.
• PALA may also have usage in pregnancy to prevent perinatal transmission of viruses, provided that there is no teratogenic effect.
• PALA may be useful in transplant surgery, e.g., renal and bone marrow transplant recipients undergoing chemotherapy as well as cancer patients since organ or bone marrow grafts are frequently contaminated with CMV.
• transplant surgery e.g., renal and bone marrow transplant recipients undergoing chemotherapy as well as cancer patients since organ or bone marrow grafts are frequently contaminated with CMV.
• both the recipient and the donor are treated with PALA either alone or with combinational therapy at the discretion of the treating physician, e.g., prior to donating or receiving the tissue or organ transplant.
• PALA may be useful for respiratory syncytial virus (RSV) in addition to those paramyxoviruses
• viruses of veterinary importance e.g., foot and mouth disease (FMDV) , rinderpest, Newcastle disease, pseudorabies and equine anemia and bovine rhinotracheitis viruses may be now amenable to intervention with PALA; thus PALA may prevent or control epizootics and prevent or ameliorate the severe economic loss associated with viral diseases including but not limited to livestock, birds or horses, especially race horses.
• FMDV foot and mouth disease
• rinderpest rinderpest
• Newcastle disease Newcastle disease
• pseudorabies pseudorabies
• equine anemia and bovine rhinotracheitis viruses may be now amenable to intervention with PALA; thus PALA may prevent or control epizootics and prevent or ameliorate the severe economic loss associated with viral diseases including but not limited to livestock, birds or horses, especially race horses.
• a therapeutically effective amount of PALA is administered, i.e., a dose sufficient to inhibit viral replication.
• PALA may be administered as an infusion (IV) at about 1 to about 100 mg/kilogram per day for about 1 week to about 1 month.
• IV infusion
• a preferable dose is from about 25 to about 50 mg/kg; the equivalent daily dose of PALA or a pharmaceutically acceptable analog thereof based on surface area is from about 100 to about 600 mg/m 2 .
• the most preferred dose is about 5 mg/kg to about 60 mg/kg for 1 week to about l month.
• Doses of PALA or its pharmaceutically acceptable analog should be administered in intervals of from about 1 week to about 1 month and preferably from about 7 to about 10 days.
• a preferred dose is administered to achieve peak plasma concentrations of PALA or its pharmaceutically acceptable analog from about 50 to about 100 ⁇ M. This may be achieved, for example, by the intravenous injection of a sterile about 0.05% to about 10% solution of the administered ingredients in buffered saline ( ⁇ pH 7.5) (any suitable saline solutions known to those skilled in the art of medicinal chemistry may be used) . Desirable blood levels may be maintained by a continuous infusion of PALA as ascertained by plasma levels measured by HPLC.
• Combination therapy with PALA or a pharmaceutically acceptable analog is achieved by lowering the dose of each drug about 25% to 50%
• the attending physician would know how to and when to terminate, interrupt or adjust therapy to lower dosage due to toxicity, or bone marrow, liver or kidney dysfunctions. Conversely, the attending physician would also know to adjust treatment to higher levels if the clinical response is not adequate (precluding toxicity) .
• a program comparable to that discussed above can be used in veterinary medicine.
• a daily dose range should be between about 5 to about 75 mg/kg, while most preferably a daily dose range should be between about 5 to about 60 mg/kg. Another preferred range is between about 25 to about 50 mg/kg per day.
• the therapy should be initiated at a lower dose, perhaps about 5 mg/kg to about 10 mg/kg and increased up to about 25 mg/kg or higher depending on the patient's individual response. It is further recommended that infants, children, and patients over 65 years, and those with impaired renal, or hepatic function, initially receive low doses, and that they be titrated based on individual clinical response(s) and blood level(s) .
• an amount sufficient to alleviate or prevent viral infection is meant to encompass the above described dosage amounts and dose frequency schedule.
• any suitable route of administration may be employed for providing the patient with an effective dosage of PALA.
• oral, parenteral (subcutaneous, intravenous and intramuscular) ; rectal, transdermal, vaginal and the like may be used.
• Dosage forms include tablets, troches, dispersions, suspensions, suppositories, solutions, capsules, creams, patches, minipumps (Alza Corporation) and the like.
• compositions of the invention which are useful in the treatment or prevention of viral infections in humans, animals and birds contain as an active ingredient PALA or a pharmaceutically acceptable analog thereof. These pharmaceutical compositions may also contain therapeutic agents including other antivirals, in addition to PALA or a pharmaceutically acceptable analog thereof; these novel compositions provide for combinational therapy for the treatment of viral infections. Such combinational therapy provides both additive and/or synergistic effects.
• Suitable compounds which may be used in combinational therapy with PALA within the scope of the invention include but are not limited to 2-deoxy- D-glucose(2-dGlc) , deoxynojirimycin, acycloguanosine, ribavirin (virazole®) , rifampicin (rifadin®) , adamantidine, rifabutine, ganciclovir, (DHPG) 3'- azido-3•-deoxythymidine (AZT or zidovudine®) , 2',3'- dideoxyinosine (ddl)-, 2' ,3 '-dideoxycytidine (ddC) , fluoroiodoaracytosine , idoxuridine, tri luorothy
• Novel pharmaceutical compositions encompassed by the present invention include but are not limited to PALA, or a pharmaceutically acceptable analog, and ribavirin (virazole®) ; PALA and rifampicin (rifadin®) ; PALA and AZT; PALA and ddl; PALA and ddC; PALA and adamantidine; PALA and acycloguanosine; PALA and 2- deoxy-D-glucose; PALA and deoxynojirimycin; PALA and interferon- ⁇ and PALA and ganciclovir.
• the present invention also encompasses pharmaceutical compositions which contain PALA, or a pharmaceutically acceptable analog, and, optionally more than one additional therapeutic compound to provide combinational therapy.
• PALA PALA and a number of its analogues can be prepared according to methods described in United States Patent Nos. 4,179,464, 4,215,070, 4,267,126, 4,348,522, 4,154,759, 4,178,306 and GB 2008118 and GB 2051070, the disclosures of which are incorporated in their entirety by reference herein. Further, PALA can alternatively be prepared according to the methods disclosed by Gloede, J. et al . (1988) Pharmazie 43(6) :434; Henklein, P. et al . (1989) DD 272092 Al Sept. 27, 1989; Kafarski, P. et al .
• PALA W-(phosphonoacetyl)-L-aspartic acid
• PALA contains four highly acidic hydrogens (i.e., two carboxylic acid protons and two phosphonic acid protons) as well as a basic nitrogen substituent.
• PALA contains four highly acidic hydrogens (i.e., two carboxylic acid protons and two phosphonic acid protons) as well as a basic nitrogen substituent.
• PALA contains four highly acidic hydrogens (i.e., two carboxylic acid protons and two phosphonic acid protons) as well as a basic nitrogen substituent.
• ester, inorganic or organic salt functionalities are possible.
• Such possibilities are better appreciated with the aid of the structural representation, below, of a generic formula of PALA which encompasses the free acids,'salts, esters or compounds combining such functional groups.
• analog, analogs or analogues of PALA are meant to encompass any salts, esters or other derivatives which can be made with PALA using its available functionalities.
• PALA as used herein is meant to encompass both the protonated acid or a pharmaceutically acceptable salt of W-(phosphonoacetyl)-L-aspartic acid.
• One preferred salt is the disodium salt; another is the tetrasodium salt, infra .
• the analogs of PALA can have particular utility in the pharmaceutical compositions of the present invention, especially those formulated for oral administration.
• N- (phosphonoacetyl)-L-aspartic acid (PALA) nucleus N- (phosphonoacetyl)-L-aspartic acid (PALA) nucleus
• suitable hydrocarbon substituents include, but are not limited to, methyl ethyl, propyl, isopropyl, butyl, sec-butyl, isobutyl, tert-butyl, cyclohexyl, phenyl, benzyl, p-nitrobenzyl and the like.
• ammonium, mono-, di-, tri- and tetrasubstituted ammonium salts of PALA can be prepared by methods well-known to those skilled in the art, including, but not limited to, simple acid-base reactions between PALA and amines or passage through ion-exchange columns.
• amines can be utilized to form the amine salt, including primary, secondary or tertiary amines. Indeed, even quaternary ammonium groups can form salts of PALA, so long as the PALA is already in the salt form.
• the amine salt of PALA can be associated, depending on the stoichiometry, strength of the particular base or substituent(s) present at the other acidic portions of the molecule, with only the phosphate group, one or both carboxylic acid groups, or all the acidic portions of the PALA nucleus as depicted in the structure above.
• Organic amines are also suitable, as already mentioned.
• lower alkyl (e.g., C ⁇ C,. hydrocarbons) amine groups enjoy great utility.
• Alkanol amines in which both amino and hydroxyl groups are present also, are particularly contemplated.
• methanolamine, ethanolamine, propanolamine, isopropanolamine, butanolamine and the like make attractive amine salts or analogs of PALA.
• dialkanol, trialkanol, or tetraalkanolammonium groups are contemplated.
• Multiple amino group-containing compounds are also envisioned, such as ethylenediamine, diethylenetriamine or N-alkyl- or W-alkanol- substituted derivatives thereof.
• N- hydrocarbon substituents are defined similarly as the ester hydrocarbon groups described above, i.e., they may be cyclic, acyclic, aliphatic or aromatic and may optionally contain functional groups other than hydroxyl, such as ether groups, amino groups, thioether groups, sulfhydryl groups, fluoro groups and the like.
• compositions of the present invention comprise PALA as active ingredient, or a pharmaceutically acceptable analog thereof, and may also contain a pharmaceutically acceptable carrier, and optionally, other therapeutic ingredients.
• salts include salts, esters and other derivatives of PALA.
• the salts are prepared from pharmaceutically acceptable non-toxic acid or bases including inorganic acids or bases and organic acids or bases.
• Such salts may include alkali metal salts, such as sodium or potassium, and alkaline earth salts or ammonium salts.
• alkali metal salts such as sodium or potassium
• alkaline earth salts or ammonium salts A variety of salts of PALA can be found in the patents of Schultz et al . and Parson et al . mentioned above.
• salts may be prepared from pharmaceutically acceptable non-toxic bases or acids including inorganic and organic bases or acids as well as metals.
• suitable pharmaceutically acceptable base additions salts for the compound of the present invention include but are not limited to metallic salts made from aluminum, calcium, lithium, magnesium, potassium, sodium and zinc or organic salts made from W,W-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (n- methylglucamine) and procaine.
• a preferred salt is the tetrasodium salt of PALA and another preferred salt is the disodium salt of PALA.
• PALA can be combined as the active ingredient in intimate admixture with a pharmaceutical carrier according to conventional pharmaceutical compounding techniques.
• the carrier may take a wide variety of forms depending on the form of the preparation desired for administration, e.g., oral or parenteral.
• any of the usual pharmaceutical media may be employed.
• Rectal preparations when used can be prepared in a carbowax composition. Because of their ease of administration, tablets and capsules represent the most advantageous oral dosage unit form, in which case solid pharmaceutical carriers are employed. If desired, tablets may be further coated by standard aqueous or nonaqueous techniques.
• the compounds of the present invention may also be administered by controlled release means and/or delivery devices including Alzet® osmotic pumps which are available from Alza Corporation. Suitable delivery devices are described in U.S. Patent Nos. 3,845,770; 3,916,899; 3,536,809; 3,598,123; 3,944,064 and 4,008,719, the disclosures of which are incorporated in their entirety by reference herein.
• compositions of the present invention suitable for oral administration may be presented as discrete units (e.g., as capsules, cachets, or tablets, or aerosols sprays) each containing a predetermined amount of the active ingredient, as a powder or granules, or as a solution or a suspension in an aqueous liquid, a non-aqueous liquid, an oil-in-water emulsion, or a water-in-oil liquid emulsion, or for topical or vaginal use in an appropriate cream.
• Such compositions may be prepared by any of the well known methods employed in pharmacology, but all methods include the step of bringing into association the active ingredient with the carrier which constitutes one or more necessary ingredients.
• compositions are prepared by uniformly and intimately admixing the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product into the desired presentation.
• a tablet may be prepared by compression or molding, optionally, with one or more accessory ingredients.
• Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as powder or granules, optionally mixed with a binder, lubricant, inert diluent, surface active or dispersing agent. Molded tablets may be made by molding in a suitable machine, a mixture of the powdered compound moistened with an inert liquid diluent.
• each tablet contains from about 100 mg to about 500 mg of the active ingredient, and each cachet or capsule contains from about 100 mg to about 500 mg of the active ingredient, PALA.
• the tablet, cachet or capsule contains either one of three dosages, about 100 mg, about 200 mg or about 500 mg of the active ingredient.
• the formulation listed below is suitable for intravenous, subcutaneous, or intramuscular injection.
• Japanese encephalitis virus Groups of 10 C57B1/6 mice (VAF+, Charles River Labs.) weighing 12- 14 g are treated i.p. with phosphate-buffered saline (PBS) or drug twice daily (b.i.d.) on a 5-day schedule with the first dose administered on the day (day -1) preceding viral challenge. Five of the ten animals in each group are infected s.c. with 10-100 LD 5 ⁇ of JE virus (Beijing strain) adequate to produce 100% mortality in the diluent controls) 6 h after the first dose of compound is administered (day 0) .
• PBS phosphate-buffered saline
• b.i.d. drug twice daily
• Controls include untreated, uninfected mice; untreated, virus- infected mice, diluent-treated, virus-infected (and uninfected) mice.
• Poly (ICLC) a ribarivin®, is used as a positive treatment control.
• ICLC a ribarivin®
• Six days after viral challenge (day +6) , brains of infected mice are harvested for virus titres.
• Suspensions of brain (10% w/v) are titrated by plaque assay in Vero cell cultures. Body weights are recorded on days -l through +6. Weight change is determined as a measure of drug toxicity.
• Group #2 five animals, intravenous injection of drug daily in two divided doses on days 2, 3, 4, 5 and 6PI. Concentration of the drug is the ED90 value determined in in vitro assays.
• Group #3 five animals, intravenous injection of drug daily in two divided doses on days 2, 3, 4, 5 and 6PI. Concentration of the drug is 1 to 2 times the ED90 value determined in in vitro assays.
• Group #4 five animals, Placebo intravenous injections (sterile saline) on days 2, 3, 4, 5 and 6 PI.
• the clinical and histological results are evaluated together with viral recovery for all drug- treated, intravenous therapy groups and are correlated with each other and with the group receiving placebo therapy.
• the results from this study allow one to select the optimal drug concentration for use as intravenous therapy for CMV induced retinitis.
• PRIMATE MODEL FOR RSV Twenty-three young, African green monkeys, seronegative to RSV are used. All are housed individually in cages in a single room at the Primate Center. They are maintained at a temperature of 75° +/- 3°F with a relative humidity of 50-60 percent. Purina monkey chow and water are supplied ad lib and the animals are monitored daily for clinical signs and food consumption. At the termination of the experiment, all animals are killed while under ketamine anesthesia with Beuthansia D Special (euthanasia solution, Shering Corporation) and are necropsied.
• Beuthansia D Special euthanasia solution, Shering Corporation
• Two strains of RSV are used; one is the Long type strain, (ATCC VR-26) and the second is derived from an Australian human RSV isolate provided by Dr. Gail Wertz, School of Medicine, University of Alabama at Birmingham.
• the viruses are passed twice in African green monkeys and are prepared as a stock pool in BSC- 40 cells (African green monkey kidney) .
• Viral pools have a titer of ca. 10 5 TCID 50 /mL and are maintained at -70°.
• Virus inoculation consisted of a 10 " ' or 10 ⁇ 2 dilution ' of stock FSV administered by intratracheal catheter (1.0 ml) and intranasal instillation (1.0 ml). Throat swabs are taken daily and placed in 1.0 ml of tissue culture medium (minimum essential medium with 10 percent fetal bovine serum and antibiotics) . Titrations are performed on the 1.0 ml of medium after expression of fluid from the swab"(Table I) . Titrations are performed by preparation of serial ten fold dilutions of each specimen and inoculation of each dilution into duplicate wells of 24 well plates seeded with BSC-40 cells. Titers are obtained by microscopic examination of the cultures for viral induced cytopathology and the titers are expressed as TCID 50 per ml.
• a small portion of lung is taken at necropsy from each monkey, weighed and ground in glass tissue grinders to a 10 percent homogenate in pH 7.2 phosphate buffered saline of which 0.1 ml is cultured on blood agar for bacteriologic evaluation.
• a portion of the homogenate and lung lavage(s) is diluted in tenfold dilutions for titration of virus in BSC-40 cells.
• the gross and microscopic changes are evaluated together with viral titer.
• the immunoperoxidase procedures are used to define the basement membrane changes seen prominently with the A-2 (Wertz) strain of RSV.
• MTT 3-(4,5 dimethy1- thiazol-2-yl)-2,5-diphenyl tetrazol
• viruses were evaluated for antiviral efficacy against the following viruses (viral strain) : a) Japanese encephalitis virus, JE, (Nakaya a) ; b) yellow fever virus, YF, (Asibi) ; c) sandfly fever virus, SF, (Sicilian) ; d) Punta Toro virus (PT) , (Adames) ; e) Venezuelan equine encephalomyelitis virus (VEE) , (Trinidad donkey) ; f) vaccinia virus (W) , (Lederle vaccine) ; g) dengue type-4 (Caribbean) virus; h) human immunodeficiency virus type 1 or 2, HIV 1 or 2.
• viruses viral strain
• Viruses comprised the standard group against which drugs were evaluated.
• the in vitro antiviral and cytotoxic effects of the test compound were measured either: a) by observing inhibition of viral cytopathic effect using an MTT-assay [JE, YF, SF, PT, VEE, W and HIV-1 viruses], Pauwels et al . (1988) J. Virol . Methods 20:309-321, or b) by a general plaque reduction assay [all other viruses] .
• TC 50 Cellular toxicity or concentration 50%
• TC 50 is defined as the drug concentration ( ⁇ g/ml.) that reduces the cell number and their metabolic activity by 50% as compared to the viability for uninfected control cells in duplicate test wells in the MTT assay
• Viral inhibitory concentration 50%, IC 50 is defined as the drug concentration ( ⁇ g/ml) at which 50% reduction of viral cytopathic effect (CPE) is observed in triplicate test wells.
• CPE viral cytopathic effect
• the therapeutic (or antiviral) index, TI is a value proportional to the overall in vitro activity. It is calculated as a ratio of (TC 50 /IC 50 ) . It is a single drug concentration measurement of the relative anticellular and antiviral effectiveness of a compound during the same test and time period. All in vitro MTT assay results given represent an average of 2-6 individual test results.
• the assay methods described above allow for the measurement of both viral replication and toxicity levels.
• Viable cells convert MTT tetrazolium to the blue MTT formazan (using mitochondrial enzymes) ; dead cells are incapable of this conversion.
• screening is also supplemented with standard cytopathology and toxicity assays (by light microscopy) .
• Serial concentrations of PALA are assayed as previously described except the cell sheets were pretreated with PALA for 16 hours prior to viral challenge. (See, for example, the method described by Kumarasamy, R. and Blough, H.A. (1984) Virology 138:156-161.)
• Appropriate inhibitor and viral controls are used and toxicity is evaluated.
• Therapeutic indices are calculated in the usual fashion as mentioned above; the results of the screening of the flavi-, toga- and bunyaviruses (RNA viruses) are given in Table l; similarly confirmatory data for these viruses is reported in Table 2.
• TI is the Therapeutic Index, i.e., TC/IC; a) ⁇ g/ml at 50% inhibition; b) ⁇ g/ml at 50% inhibition; c) 50% inhibition; d) 95% inhibition.
• RNA positive stranded virus Coxsackie B3 required about 33 ⁇ g/ml for 50% inhibition as shown in Table 3; the therapeutic indices for most of these viruses was about 32.
• EXAMPLE 2 Using the duck hepatitis model (DHBV) , primary duck hepatocyte cells were treated with various concentrations of PALA (disodium salt) and evaluated for cytopathic effect. In addition, viral DNA replication was assessed using "slot" dot blots. At concentrations of 40-50 ⁇ M, a 50% reduction in plaques was observed (IC 50 ) ; at high concentrations, i.e., .500 ⁇ M, there was a 95% inhibition, with little or no toxicity. These experiments were performed three times with confirmatory results.
• PALA diisodium salt
• a second group of three monkeys received PALA at 50 mg/kg/day given by intravenous bolus injection into the saphenous vein.
• a third group was given a lower dose of PALA at 20 mg/kg/day administered in a similar manner.
• Acyclovir treatment was administered at a subeffective dose of 10 mg/kg/day also given as intravenous bolus injection into the saphenous vein.
• a fifth group of three monkeys received a combined treatment of PALA at 20 mg/kg/day and acyclovir at 10 mg/kg/day. The drugs were injected into opposite veins at each time of treatmen .
• Drug solutions were prepared daily prior to treatment. Treatment was begun 24 hours after virus inoculation. PALA was provided as a solution in vials containing 5 ml at 100 mg/ml. The contents of three vials were pooled and seven ml of the pooled drug diluted to 28 ml to give 25 mg/ml. Five ml of this solution was diluted to 50 ml to give 10 mg/ml. Drug was administered twice daily resulting in total daily doses of 50 or 20 mg/kg/day.
• Acyclovir received as a gift from Burroughs Wellcome was weighed out as a 200 mg quantity. This was diluted with 5 ml of PBS and pH adjusted to 11.0 with IN NaOH to effect solution. This was subsequently diluted to 40 ml to give a solution of 5 mg/ml and sterilized by filtration. Again, 1 ml per kg of body weight was administered by intravenous injection twice daily resulting in a dose of 10 mg/kg/day.
• the clinical course of simian varicella infection was followed by collection of 2 ml of blood in heparin on day 2, 5, 7, 9 and 11 post-inoculation.
• the lymphocytes in the 2 ml specimen were separated on ficol-hypaque gradients, washed twice in RPMI-1640 medium and suspended in 10 ml of this medium.
• the 10 ml volume was divided between two 25 cm 2 tissue culture flasks seeded 24 hours earlier with Vero cells. After 5-7 days incubation, the culture fluids were discarded from the flasks and the cell monolayer fixed with methanol and stained with methylene blue-basic fuchsin. When dried the number of plaques in each flask were counted and the mean number of plaques between the two paired flasks were determined and expressed as a quantitation of viremia per ml of blood.
• Rash was evaluated daily using a subjective scoring of severity from + to 4+. A + score indicates less than 10 vesicles seen on the skin of the monkey while 4+ indicates numerous vesicles covering the majority of the body surface.
• General clinical condition was assessed daily and anorexia noted by counting the number of food biscuits consumed daily. Monkeys dying during the course of the experiment were necropsied and simian varicella was determined as the cause of death based on the typical pathology. Baseline hematology and clinical chemistry tests were performed at three days before virus inoculation and again on day 0, immediately before virus inoculation. Following inoculation of simian varicella virus blood was taken for hematology and clinical chemistry tests at 3, 7, 9 and 11 days.
• Table 4 presents data relating to the daily scoring of the rash.
• Each of the three control monkeys developed rash with one monkey developing a maximum 4+ rash on day 11. This monkey died the following day with simian varicella involving the lungs and liver. The remaining two control monkeys developed maximum rash of 2+ and 3+ persisting for two days in each monkey.
• Two of the three monkeys treated with PALA at 50 mg/kg/day developed maximum 4+ rash.
• the third monkey only showed a 1+ rash on day 9 but died on day 10 with systemic simian varicella.
• the lower dose of PALA resulted in a 1+ rash in one monkey and a 2+ rash in the second monkey.
• the third monkey showed a 3+ rash on day 10 and died later that same day.
• Acyclovir at a sub-effective dose of 10 mg/kg/day resulted in a moderately severe 3+ rash in two monkeys and mild 1+ rash in a third monkey.
• the combination at 10 mg/day appeared to moderate the rash with only + scores seen on most of the days with a maximum 1+ score in a single monkey.
• Viremia was severe in one control monkey (>1000 PFU/ml of blood) and moderate (100-300 PFU/ml of blood) in the other two control monkeys (Table 3) .
• PALA PALA
• a second had a moderately severe viremia (300-800 PFU/ml) while a third had a moderate viremia.
• Similar results were seen in the monkeys treated with 20 mg/kg/day of PALA.
• Acyclovir at 10 mg/kg/day was found to have no effect in moderating viremia.
• Two monkeys had severe viremia and one monkey presented with moderately severe viremia.
• a slight benefit of the combined treatment with PALA and acyclovir was seen.
• One monkey had a moderately severe viremia, one a moderate viremia and a third minimal viremia.
• Hematology tests showed no consistent pattern of abnormal values. Thrombocytopenia was seen on day 11 in one monkey (M636) treated with PALA at 50 mg/kg/day and in two monkeys (M642 and M639) treated with acyclovir. Chemistry values did reflect the hepatitis present as a consequence of simian varicella virus. No abnormalities were seen resulting from treatment with the drugs at the doses employed.
• Titers of serum neutralizing antibody were comparable in the monkeys in the control groups and in the monkeys treated with both doses of PALA or with acyclovir.
• the monkeys treated with the combination of PALA and acyclovir did show lower titers of antibody to simian varicella virus when compared to the titers in the other monkeys. It is likely that this reflects the effects of inhibition of virus replication by the combination therapy.
• the HCMV-inoculated drug- treated monolayers were handled as follows: The supernatant containing cell-free virus was removed from the cells and the titer of HCMV cell-free virus in the supernatant was determined by standard plaque assay. The cell monolayer was washed with HBSS to remove residual drug, and the cells harvested by scraping. The cells were sonicated and centrifuged to pellet cell debris. The titer of the cell-free HCMV released from the infected cell monolayer was determined. The efficacy of the drug was represented as reduction in HCMV PFU/ml compared to non-drug- treated HCMV PFU/ml and to DHPG HCMV inhibition.
• PALA used at a concentration of 3 ⁇ g/ml was effective in reducing HCMV titers when compared to placebo treated controls.
• the PALA was compared to DHPG in vitro therapy (19 ⁇ g/ml; ED50 for AD 169)
• the reduction in HCMV titers was similar to the DHPG treated monolayers, but, titers remained slightly higher than the DHPG titers.
• the HCMV titer reduction after therapy with the PALA was similar for the supernatant (cell free HCMV) and for the cell pellet (cell associated HCMV titer) .
• HCMV titers are presented in Table 7b and in Figures 3 and 4.
• a DHPG resistant HCMV isolate (characterized previously as a DHPG resistant isolate by in vitro decreases in DHPG sensitivity; the virus has altered thymidine kinase activity) was used in these in vitro assays.
• DHPG was not effective in reducing the titer of HCMV in either the cell associated or cell free assays.
• the titer of DHPG resistant HCMV in the DHPG treated group was the same as or higher than the placebo treated monolayers (Not statistically significant) .
• PALA was effective in reducing the DHPG resistant HCMV titer. By days 4-5 PI, the PALA treated monolayers had significantly lowered HCMV titers than DHPG treated or placebo treated monolayers.
• HCMV titers are presented in Table 8 and Figures 5 and 6.
• Table 7a Cell free (supernatant) HCMV Titers after incubation with DHPG, PALA and placebo.
• HCMV-inoculated animals were divided into 8 groups of 4 rabbits each with matched chorioretinal disease scores.
• the HCMV- infected rabbits received intravenous therapy as indicated below:
• Group #1- 4 animals intravenous injection of high dose experimental drug (50 mg/kg) daily from day 2 through 10 PI. A total of 9 IV injections.
• Group #3- 4 animals intravenous injection of low dose experimental drug (20 mg/kg) daily from day 2 through 10 PI. A total of 9 IV injections.
• Group #4- 4 animals intravenous injection of high dose experimental drug (50 mg/kg) daily from day 2 through 10 PI (a total of 9 IV injections) plus high dose DHPG 10 mg/kg/day in 2 divided doses from day 2 through 10 PI (a total of 18 IV injections) .
• Group #5- 4 animals intravenous injection of high dose experimental drug (50 mg/kg) daily from day 2 through 10 PI (a total of 9 IV injections) plus low dose DHPG 5 mg/kg/day in 2 divided doses from day 2 through 10 PI (a total of 18 IV injections) .
• Group #6- 4 animals intravenous injection of low dose experimental drug (20 mg/kg) daily from day 2 through 10 PI (a total of 9 IV injections) plus low dose DHPG 5 mg/kg/day in 2 divided doses from day 2 through 10 PI (a total of 18 IV injections) .
• Group #7- 4 animals intravenous injection of DHPG 10 mg/kg/day in 2 divided doses from day 2 through day 10 PI. A total of 18 IV injections.
• Group #8- 4 animals intravenous injection of sterile saline on days 2 through 10 PI.
• All animals received daily indirect ophthalmoscopic examinations to evaluate clinical HCMV disease progression (From days 2 through 10 PI) . The indirect ophthalmoscopic examinations were performed independently by two readers who were masked as to the therapy that the rabbits were receiving.
• Group #1 High dose PALA (50 mg/kg) IV daily single-agent therapy from day 2 through 10 PI.
• HCMV was recovered from any chorioretinal cell sonicate co-culture on day 12 PI.
• the lack of HCMV recovery may be (and probably is) directly related to the time point selected for HCMV recovery, i.e., day 12 post inoculation.
• a time course sacrifice of animals throughout the course of therapy would be necessary. (In non-treated eyes, HCMV is present usually up to day 8 or 9 PI. Recovery after day 9 or 10 PI is variable) .
• Group #2 High dose PALA (50 mg/kg) IV every other day single-agent therapy from day 2 through 10 PI.
• HCMV was recovered from any chorioretinal cell sonicate co-culture on day 12 PI.
• the lack of HCMV recovery may be (and probably is) directly related to the time point selected for HCMV recovery.
• Group #3 Low-dose PALA (20 mg/kg) IV daily single-agent therapy from day 2 through 10 PI.
• Group #7 HCMV-inoculated DHPG IV treated animals (10 mg/kg/day in 2 divided doses) from days 2 through 10 PI -
• DHPG was used in this experiment as the control therapy. Animals received DHPG therapy beginning day
• the choroid remained congested through day 10 PI.
• Vitreitis in these animals remained at moderate levels from day 4 through day 10 PI.
• the clinical impression of disease in these treated eyes was that this DHPG single agent therapy group was the most improved of all therapy groups.
• the DHPG therapy group had consistently lower vitreitis
• HCMV was recovered from any chorioretinal cell sonicate co-culture on day 12 PI.
• the lack of HCMV recovery may be (and probably is) directly related to the time point selected for HCMV recovery.
• Placebo treated animals received daily single injections of sterile saline +EDTA beginning on day 2 PI and continuing through day 10 PI.
• Placebo treated eyes had developed mild chorioretinal and vitreous disease by day 2 PI.
• the disease consisted of focal areas of retinal infiltration, optic nerve inflammation and redness and mild vitreitis.
• the vitreitis consisted of vitreous strands and peripheral cellular infiltrates and cloudiness.
• Placebo therapy did not arrest the development of chorioretinal disease and vitreitis in these animals.
• Chorioretinal disease increased and the developing vitreitis in these HCMV infected eyes developed to severe levels by day 3-4 PI interfering with comprehensive evaluation of chorioretinal disease. After day 5 PI, the vitreitis obscured comprehensive evaluation of retinal and choroidal disease.
• HCMV infection had progressed from the inner retinal areas to involve the photoreceptor layer. Histology demonstrated areas of retinal edema, mixed cellular infiltration and occasional retinal detachment. Areas of extensive retinal HCMV disease involvement were next to areas of normal retina. Histology demonstrated moderate to extensive involvement of the choroid and retina. At sacrifice, lung observation demonstrated mild to moderate opacification and hemorrhage in 2 rabbits. A mild to moderate edema (congestion) was also present.
• HCMV was recovered from any chorioretinal cell sonicate co-culture on day 12 PI.
• the lack of HCMV recovery may be (and probably is) directly related to the time point selected for HCMV recovery.
• Group #4 High dose PALA (50 mg/kg) daily IV therapy from day 2 through 10, plus IV high dose DHPG (10 mg/kg/day in 2 divided doses on days 2 through 10 PI) .
• Group #5 High dose PALA (50 mg/kg) daily IV therapy from day 2 through 10, plus IV low dose DHPG (5 mg/kg/day in 2 divided doses on days 2 through 10 PI) .
• HCMV was recovered from any chorioretinal cell sonicate co-culture on day 12 PI.
• the lack of HCMV recovery may be (and probably is) directly related to the time point selected for HCMV recovery.
• Group #6 Low dose PALA (20 mg/kg) daily IV therapy from day 2 through 10, plus low dose IV DHPG (5 mg/kg/day in 2 divided doses on days 2 through 10 Pi) .
• Combination intravenous therapy with daily low dose PALA (20 mg/kg) and daily low dose DHPG (5 mg/kg) [Group #6] was the most effective combination-agent therapy. This combination was more effective in reducing the vitreitis and optic nerve head changes than any other single-agent or combination-agent therapeutic regimen evaluated. This combination-agent therapy was superior to all other therapies throughout the course of the therapy (day 2 through 10 post inoculation) .
• the vitreitis in this combination therapy group was less severe than in any other single-agent therapy, combination-agent therapy or placebo therapy group.
• the decrease in severity of HCMV-induced disease may be interpreted as an additivity of the two compounds (additional samples will need to be evaluated before this conclusion can be supported by statistical analysis) .
• the optic nerve head Prior to vitreitis development that obscured visualization of the fundus, the optic nerve head was exhibiting moderate redness and inflammatory changes characteristic of the HCMV-induced disease. On day 10 PI, the. optic nerve head alterations had decreased in those animals where the nerve head was visible.
• PALA alone or in combination with DHPG prevented interstitial pneumonitis; thus PALA may be useful as a single agent therapy for related disorders.
• EXAMPLE 6 PALA efficacy evaluation in the rabbit: confirmation of clinical single-and combination-agent efficacy with critical analysis of reductions in HCMV titers in the chorioretina on post therapy days 3 , 4, 5, and 6. These experiments were performed to confirm the efficacy of the PALA during intravenous therapy after HCMV infection in the rabbit model by comparing clinical, and histopathological HCMV-induced disease severity. PALA intravenous therapy was evaluated by HCMV recovery during therapy. HCMV titers from chorioretinal sonicate co-cultures were compared to DHPG and placebo recovery titers. High dose PALA therapy was evaluated as a single agent and as a combination agent therapy in conjunction with DHPG.
• Group #3 6 HCMV-inoculated and 1 sham- inoculated animal, intravenous injection of mid- dose PALA (25 mg/kg) on days 2 through 10 PI (A total of 9 IV injections plus mid dose DHPG 7.5 mg/kg/day in 2 divided doses from day 2 through 10 PI (a total of 18 IV injections) .
• One animal from this therapy group was sacrificed on days 3, 4 , 5 , and 6 PI.
• the eyes were enucleated and processed for HCMV recovery by cell sonicate recovery to determine the presence of HCMV and the titer of virus in the chorioretina.
• the remaining 2 HCMV-infected and 1 sham inoculated rabbit were evaluated through day 12 PI.
• Group #4 6 HCMV-inoculated and 1 sham- inoculated animal, intravenous injection of low dose PALA (10 mg/kg) on days 2 through 10 PI (A total of 9 IV injections) plus low dose DHPG 5 mg/kg/day in 2 divided doses from day 2 through 10 PI (a total of 18 IV injections) .
• One animal from this therapy group was sacrificed on days 3, 4, 5, and 6 PI.
• the eyes were enucleated and processed for HCMV recovery by cell sonicate recovery to determine the presence of HCMV and the titer of virus in the chorioretina.
• the remaining 2 HCMV-infected and 1 sham inoculated rabbit were evaluated through day 12 PL. These remaining animals were used to confirm the clinical impressions of PALA combination efficacy as demonstrated previously in Example 5.
• Group #5 6 HCMV-inoculated animals, intravenous injection of DHPG 10 mg/kg/day in 2 divided doses from day 2 through day 10 PI. A total of 18 IV injections,
• Group #1 Rabbits #1, 2, 3, 4 and sham- inoculated rabbit Sl-50 mg/kg PALA.
• Group #4 Rabbits #18, 19, 20, 21, 22, 23 and 1 sham-inoculated animal #S4 - received daily intravenous therapy with low dose PALA (10 mg/kg) plus low dose DHPG (5 mg/kg/day) .
• Group #5 Rabbits #24, 25, 26, 27, 28, 29 and 1 sham-inoculated animal, #S5 - received daily intravenous therapy with high-dose DHPG (10 mg/kg/day) .
• Group #6 Rabbit #30, 31, 32, 33, 34, 35 and 36 received daily placebo intravenous therapy (sterile saline injections) .
• HCMV recovery from chorioretinal cell sonicate cultures The animals sacrificed at the conclusion of the efficacy evaluation were observed daily by indirect ophthalmoscopy. The clinical impressions of the HCMV disease in these animals was used to construct the vitreitis and chorioretinal disease profiles demonstrated graphically in this report.
• FIG. 6 and 7 summarizes data on the development of chorioretinal and vitreitis development in the intravenous combination and single-agent therapy groups. Chorioretinal disease development was partially obscured by the development of vitreitis on days 5-8 Pi as was demonstrated in the previous efficacy evaluation. The average scores for these days PI are based upon clinical impressions and the previous days fundus examination evaluation.
• Table 8, 9, 10, and 11 summarize clinical vitreitis, chorioretinitis and optic nerve head scorn and HCMV recovery from 2 eyes/therapy group on days 3, 4, 5, and 6 PI.
• Group #l PALA single-agent therapy 50 mg/kg/day
• HCMV recovery by cell sonicate assay in this single-agent therapy group demonstrated virus presence in the chorioretina on days 3, 4, 5, and 6 PL HCMV titer in the culture samples was highest on day 3 PI, when an average of 104 pfu HCMV was recovered from the samples.
• HCMV was recovered from both eyes of animals sacrificed in the time course evaluation.
• Optic nerve head changes in this model of HCMV infection are a reliable measurement and assessment of chorioretinal disease development and HCMV-induced pathology.
• Group #3 Combination agent PALA (25 mg/kg/day) plus DHPG (7.5 mg/kg/day) [mid-dose combination therapy] and
• Group #4 Combination agent PALA (25 mg/kg/day) plus DHPG (5 mg/kg/day) [low-dose combination agent therapy].
• Vitreitis scores in these combination agent therapy groups were not improved when compared to the high- dose combination or the single-agent DHPG therapy groups. Vitreitis remained elevated on day 10 in the mid-dose combination therapy group. Chorioretinal disease assessment demonstrated moderate levels of disease in both combination therapy groups that was clearly visible as retinal pathology on day 10 PI. The average chorioretinal and vitreous disease in these combination therapy groups was more severe than in the high dose combination agent therapy group. The disease progression in the mid-dose therapy group was not different from the disease state in the low-dose combination therapy group. Vitreitis and chorioretinal disease were evident at moderate levels in both of these combination groups.
• both the mid- dose and low-dose combination therapy groups the vitreitis and chorioretinal disease more severe than in the high-dose combination and the DHPG single-agent therapy groups.
• Optic neuritis and optic nerve head changes were present in these two mid-and low-dose therapy groups throughout the study. Both combination therapy groups demonstrated moderate levels of optic nerve head neuritis and pathology. The optic nerve head changes in these groups were not different from the single-agent DHPG therapy group or the placebo therapy group. Optic nerve head changes in these groups were worse when compared to the high dose combination agent therapy group.
• HCMV recovery from chorioretinal cell sonicate cultures in these combination therapy groups was intermediate between the placebo HCMV recovery and the single-agent DHPG HCMV recovery.
• HCMV was recovered from sonicate cultures on days 3, 4, and 6 PI. Titers decreased from an average of 104 on day 3 to and average of 101 on day 6 PI.
• the HCMV recovery was less than recovery in the placebo therapy group.
• HCMV recovery was not reduced as rapidly in the mid-dose group when compared to the high dose combination therapy group or the single-agent DHPG therapy group.
• HCMV recovery from chorioretinal cell sonicate cultures in the low-dose combination agent group was comparable to the mid-dose therapy group. Fewer chorioretinal samples were positive on days 4, 5, and 6 in this low dose combination therapy group than in the placebo group or the single agent PALA therapy groups.
• the HCMV titer and frequency of recovery in this low-dose therapy groups was similar to the mid- dose combination HCMV therapy group recovery frequency and HCMV titer.
• the pathology in this mid-dose combination therapy group was not as severe as that noted in placebo treated chorioretinal samples (Example 5) .
• the chorioretinal pathology was limited to discrete areas of immune cell infiltration separated by areas of normal retina and choroid. In areas that were involved in the HCMV reaction, the retina demonstrated edema, immune cell infiltration, necrosis and loss of the normal cellular architecture. The choroid was severely congested with marked engorgement of choroidal vessels and frequent areas of choroiditis.
• the pathology in this low-dose combination therapy group was similar to the pathology in the mid-dose therapy group.
• the pathology was more severe and geographic than the pathology in the high dose PALA plus DHPG combination therapy group and in the DHPG single agent therapy group.
• Group #5 Single-agent DHPG (10 mg/kg/day) . Animals in this single agent therapy group received DHPG therapy beginning day 2 post inoculation and continuing through day 10 post inoculation. The 10 mg/kg/day DHPG therapy in two divided doses.
• DHPG therapy did reduce the development of HCMV chorioretinal infection and disease and vitreitis. Chorioretinal disease remained focal with moderate involvement of the optic nerve head in inflammation and in immune cell infiltration of the optic nerve head. The choroid remained moderately congested.
• the clinical impression of disease in these treated eyes was similar to the high- dose PALA plus- DHPG combination therapy group.
• HCMV disease in this single-agent group was better than the mid-dose and low-dose combination therapy group and better than the placebo treatment group.
• the clinical disease in the single-agent DHPG treatment group was similar to the high-dose PALA combination treatment group.
• the high-dose combination therapy regimen may be slightly better than the single-agent DHPG treatment thus indicating an additive effect of the two intravenous therapy groups.
• HCMV recovery from cell sonicate cultures demonstrated a rapidly decreasing HCMV recovery rate and titer of HCMV recovered from the samples.
• HCMV was recovered from both chorioretinas in this therapy group.
• the titer of HCMV was determined to be 103 pfu.
• HCMV recovery had decreased to a titer of 101 and was evident in only 1 of the 2 chorioretinal samples.
• No HCMV was recovered on day 5 PI, however, a low titer (101 was recovered from 1 chorioretinal sample on day 6 PI.
• the decrease in frequency of HCMV recovery and in HCMV titer was better than the mid-dose and low-dose combination therapy group and the single agent PALA group and the placebo therapy group. The pattern of HCMV recovery was not different from that demonstrated in the high- dose combination therapy group.
• Group #6 Placebo therapy. Placebo treated animals received daily single injections of sterile saline + EDTA beginning on day 2 PI and continuing through day 10 PI. Placebo treated eyes developed mild to moderate vitreitis. The vitreitis in the placebo treated group was not as severe as in the other single-agent and combination- agent therapy groups. Chorioretinal disease in these placebo treated eyes was markedly worse than the other therapy groups.
• HCMV disease Focal retinal vein hemorrhages and intraretinal bleeding was frequent.
• the focal areas of HCMV disease were numerous and resulted in an average chorioretinal disease scores of" 1.5 to 2+.
• the disease consisted of focal to geographic areas of retinal infiltration, optic nerve inflammation and redness and mild vitreitis.
• the vitreitis consisted of vitreous strands with peripheral cellular infiltrates, cellular clumping and cloudiness. Placebo therapy did not arrest the development of chorioretinal disease.
• the average level of optic neuritis and inflammation in the placebo treated eyes was comparable to the other therapy groups.
• HCMV recovery from the placebo treatment group demonstrated HCMV recovery on days 3-6 PI in decreasing titers from 104 to 102 in the time course evaluation.
• the placebo treatment group demonstrated the highest titer recovery compared to the other therapy groups.
• Combination agent high dose PALA plus DHPG (therapy group #2) was the most effective combination agent therapy for reducing clinical disease and for reducing HCMV recovery in the chorioretinal cultures. This combination therapy was as good as single-agent DHPG therapy. This combination agent therapy demonstrated an additive antiviral effect when compared to single-agent DHPG therapy.
• Combination therapy of high dose PALA plus DHPG was the most effective at preserving retinal structure (opthalmologically) and this was confirmed by final histopathology.
• the cultures represent HCMV cell sonicate cultures during intravenous therapy. Cultures were plated onto 12 wells in a costar cluster. All negative cultures were blind passage 4 separate times for a total of 28 days in culture. The HCMV titer in positive cultures were determined by standard plaque assay after determination of HCMV presence (positive) in the cultures. Table 11
• PALA ascending dose efficacy evaluation in the rabbit clinical and HCMV recovery in a time course evaluation.
• HCMV-inoculated animals were divided into groups of 10 HCMV-inoculated .rabbits plus 1 sham-inoculated rabbit. The HCMV-infected and sham-inoculated rabbits received intravenous therapy as indicated below:
• Group #1 10 HCMV-inoculated rabbits, intravenous injection of PALA (50 mg/kg) on days
• Group #2 10 HCMV-inoculated rabbits, intravenous injection of PALA (75 mg/kg) on days 2 through 10 PI. A total of 9 IV injections per rabbit.
• Group #3 10 HCMV-inoculated rabbits, intravenous injection of PALA (100 mg/kg) on days
• Group #4 10 rabbits received 4 mg subconjunctival depo dexamethasone 1 hour prior to HCMV inoculation. Rabbits received intravenous therapy with PALA (75 mg/kg/day from days 2 through 10 PI) .
• Positive and Negative Control therapy groups Group #5: 10 HCMV-inoculated animals, intravenous injection of DHPG 10 mg/kg/day in 2 divided doses from day 2 through day 10 PI.
• Group #6 10 HCMV-inoculated animals, intravenous injection of sterile saline on days 2 through 10 PI.
• Group #1 Rabbits # 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 and sham-inoculated rabbit SI - 50 mg/kg PALA.
• Group #4 Rabbits #31, 32, 33, 34, 35, 36, 37,
• Group #5 Rabbits #41, 42, 43, 44, 45, 46, 47,
• 59, and 60 received daily placebo intravenous therapy (sterile 0.19% saline plus 1M EDTA injections) .
• Sacrifice of HCMV inoculated single-agent treated animals Day 3 post inoculation: Sacrifice and chorioretinal cell sonicate culture for recovery of HCMV -
• Group #1 Rabbit #3 and 4 Group #2: Rabbit #13 and 14 Group #3: Rabbit #23 and 24 Group #4: Rabbit #33 and 34
• Figures 9 and 10 summarize data on the development of vitrioretinal disease development in the intravenous single-agent therapy groups. Chorioretinal disease development was partially obscured by the development of vitreitis in 40% of the eyes by day 4 - 5 after inoculation. The bar graphs demonstrate trends in the vitrioretinal disease course in the ascending dose
• Tables 12, 13, 14 and 15 summarize raw data on vitreitis and optic nerve head disease severity
• Group #1 PALA single-agent therapy 50 mg/kg/day
• HCMV recovery by cell sonicate assay in this single-agent therapy group demonstrated virus presence in the choioretina on days 3, 4, 5, and 6 PI.
• HCMV titer in the culture samples was highest on day 3 PI, when an average of IO 3-5 pfu HCMV was recovered from the 4 chorioretinal cell sonicate samples.
• the frequency of recovery of HCMV from treated eyes decreased on days 4 and 5 post inoculation.
• a rebound in virus recovery (frequency of HCMV recovery) was noted on day 6 PI, when HCMV was recovered from 4/4 chorioretinal cell sonicate samples.
• HCMV titers decreased throughout the recovery course except on day 6 PI, when a slight rebound in HCMV titer to IO 2 pfu/ml was noted. HCMV recovery in this single agent therapy group was better than recovery in placebo treated eyes, and comparable to DHPG treated eyes. (DHPG treated eyes had slightly lower titers of HCMV and fewer numbers of positive chorioretinal samples in the recovery study) .
• HCMV recovery by cell sonicate assay in this single-agent therapy group demonstrated virus presence in the chorioretina on days 3, 4, 5, and 6 PI.
• HCMV titer in the culture samples was highest on days 3 and 4 PI, when an average of 10 45 pfu HCMV and IO 375 pfu HCMV were recovered from the chorioretinal cell sonicate samples at each time point.
• the HCMV titer decreased to low levels on day 5 PI.
• the frequency of recovery of HCMV from treated eyes decrease on days 4 and 5 post inoculation.
• the titer remained low.
• Group #3 PALA single-agent therapy 100 mg/kg/day
• HCMV recovery in this single agent therapy group was significantly higher than HCMV recovery in placebo treated animals. This single-agent therapy group was not effective in reducing the clinical disease progression or HCMV recovery from cell sonicate cultures.
• Group #4 PALA single-agent therapy 100 mg/kg/day plus 4 mg subconjunctival steroid injection
• HCMV recovery by cell sonicate assay in this single-agent therapy group demonstrated virus presence in the chorioretina on days 3, 4, 5, and 6 PI.
• HCMV titers in the culture samples remained elevated throughout the course of the study. The average titer of HCMV recovered was 10 3 pfu on days 3-6 post inoculation.
• the frequency of HCMV recovery (number of positive samples) was similar to the 50 mg/kg/day and 75 mg/kg/day treated groups (e.g. a gradual decrease in frequency of HCMV recovery followed by a rebound of HCMV recovery on day 6 PI) .
• the titer of virus was elevated suggesting that the steroids may have enhanced HCMV replication (or detection) .
• Group #5 Single-agent DHPG (10 mg/kg/day) .
• HCMV recovery from cell sonicate cultures demonstrated a rapidly decreasing HCMV recovery rate and titer of HCMV recovered from the samples.
• the titer of HCMV was determined to be 10 3 pfu.
• HCMV recovery had decreased to a titer of
• HCMV recovery frequency and titer continued to decrease through day 6 PI. No rebound in HCMV titer or in the number of positive HCMV tissues was demonstrated in the DHPG therapy group. The pattern of HCMV recovery and titer decreases is not different from that demonstrated previously in other single- agent DHPG therapy groups. Group #6: Placebo therapy.
• Placebo treated animals received daily single injections of sterile saline + EDTA beginning on day 2 PI and continuing through day 10 PI.
• Placebo treated eyes developed mild to moderate vitreitis.
• the vitreitis in the placebo treated group continued to progress throughout the course of the study.
• the vitreitis consisted of vitreous strands with peripheral cellular infiltrates, cellular clumping and cloudiness.
• the average level of optic neuritis and inflammation in the placebo treated eyes was comparable to the other therapy groups.
• HCMV recovery from the placebo treatment group demonstrated HCMV recovery on days 3-6 PI in decreasing titers from io 45 to 10 2 in the time course evaluation.
• the placebo treatment group demonstrated the highest titer recovery compared to the other therapy groups. There was no rebound in HCMV recovery or titer on day 6 as was demonstrated in the PALA single-agent treatment groups.
• HCMV was recovered in a time course analysis from all therapy groups. Differences in the frequency of recovery (e.g. the number of virus recovery samples that were positive HCMV) decreased with increasing time post therapy. It appears that the titer of the virus recovered from the chorioretinal sonicate samples also decreased with increasing time post inoculation. Of interest was the result that in all PALA single-agent therapy groups, there was a rebound in HCMV detection on day 6 PI and in HCMV titer on day 6 PI. This titer and frequency observation was more pronounced at higher concentrations of PALA therapy.
• DHPG therapy > PALA single-agent 50 mg/kg/day (Group #1) » PALA single-agent 75 mg/kg/day (Group #2) > or equal to placebo therapy (Group #6) » Single-agent PALA 100 mg/kg/day (Group #3) > Single-agent PALA 75 mg/kg/day plus 4 mg subconjunctival steroid injection (Group #4) .
• Scores represent sum of both eyes/rabbit.
• Cultures results are HCMV cell sonicate cultures during intravenous therap ⁇ . Cultures were plated onto 12 wells in a costar cluster. All negative cultures were lilind passaged 3 separate times for a total of 28 days in culture. The HCMV titer in positive cultures were determined by ⁇ c standard plaque assay after determination of HCMV presence (positive) in the cultures. Table 15
• Infection with vaccinia virus was a dermal infection produced by injection of 0.1 ml of a 1:100 dilution of stock virus intradermally into each of eight sites on the shaved back of each monkey. Titration of the viral inoculum showed that each injection site received 10 6 TCID 50 of virus.
• each monkey was weighed and 5 ml of blood was drawn for baseline plasma and lymphocyte samples.
• PALA and/or rifampicin Treatment with PALA and/or rifampicin was started 24 hours after virus inoculation. Both drugs were prepared fresh daily prior to treatment.
• PALA was provided in vials containing 5 ml of a solution at l 00 mg/ml. A 1:4 dilution was prepared in pH 7.2 PBS resulting in a drug concentration of 25 mg/ml.
• Monkeys in group 1 and 3 received PALA at 50 mg/kg/day which was given by intravenous injection in divided doses at 8 a.m. and 8 p.m. daily.
• the PALA solution of 25 mg/ml was given into the saphenous vein as 1 ml per kg of body weight.
• Rifampicin was weighed out in 300 mg aliquots which was dissolved in 10 ml DMSO and brought to a 60 ml volume. This dilution resulted in a concentration of 5 mg/ml.
• Groups 2 and 3 received intravenous injections of 1 ml per kg of body- eight or 5 mg/kg. Twice daily treatment, at 8 a.m. and 8 p.m. , resulted in a daily dose of 10 mg/kg.
• Monkeys in group 5 received PALA at 125 mg/kg given as single intravenous injections on Day 1 and Day 7 post-infection.
• Group 4 was an infection control group which was administered PBS by intravenous injection at 8 a.m. and 8 p.m. daily. All treatments continued for 10 days.
• Infection was evaluated by daily examination of the lesion sites and scoring them on a scale of + to 4+ in relation to increased severity.
• the total score for each monkey was determined by adding the individual lesion scores and a mean value determined by dividing the score by the number of injection sites. This provided a daily mean lesion score for each monkey.
• tissue culture medium minimum essential medium with 2 percent fetal bovine serum and antibiotics
• tissue homogenates were titrated for vaccinia virus by preparation of serial ten fold dilutions which were cultured in duplicate in 24 well culture plates containing Vero cells. After 4 days incubation, the cultures were fixed in methanol, stained with methylene blue-basic fuchsin and the number of plaques counted.
• the monkeys were weighed at 10, 14 and 21 days and bled at 14 and 21 days for determination of antibody titers to vaccinia virus.
• Antibody titers were determined by a serum neutralization assay.
• 8 mm biopsies were homogenized in 2.0 ml of tissue culture medium and titrated in duplicate wells of 24 well plates seeded with Vero cells.
• Cotton rats (outbred Sigmoden hispidu ⁇ ) , either sex, 50 to 100 g were challenged with RSV (strain Al) , using approximately 100 cotton rat median infectious doses (CRID50; 100 ⁇ l given i.n.). Therapy consisted of PALA (30 mg/kg/day) , ribavirin (30 mg/kg/day) , or combinational therapy. All test compounds were given intra-peritoneally (i.p.) for four days. Animals sacrificed on day (+)4, lungs homogenized and titered for RSV. Total number of animals used was 24. Experimental Protocol: respiratory syncytial virus (RSV) in cotton rats preliminary in vivo screens (16 animals were used) .
• RSV respiratory syncytial virus
• PROCEDURE 5 1. 50 to 100 g cotton rats of either sex inoculated i.n. with RSV A2 (pool 8-28-92) on day 0.
• Group 1 Placebo (H 2 0) i.p. Day +1 - Day +3
• Group 2 PALA 3 mg/kg/d i.p. Day +1 - Day +3
• Group 3 PALA 10 mg/kg/d i.p. Day +1 - Day +3
• Group 4 Ribavirin 40 mg/kg/d i.p. Day +1 - Day +3
• Group 5 Ribavirin 40 mg/kg/d + Day +1 - Day +3 PALA 3 mg/kg/d i.p.

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