Classifications

• • 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
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
  • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
• • 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
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
  • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
  • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
  • A61K31/525—Isoalloxazines, e.g. riboflavins, vitamin B2
• • 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
  • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
  • A61K31/7048—Compounds having saccharide radicals and heterocyclic rings having oxygen as a ring hetero atom, e.g. leucoglucosan, hesperidin, erythromycin, nystatin, digitoxin or digoxin
• • 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

Definitions

• the present invention relates to anti-viral agents and their use in the treatment of human and animal patients to alleviate or cure the ill-effects caused by viral infection, especially HIV.
• a detailed study of compounds according to the invention has been carried out to evaluate their efficacy against infection from several strains of HIV-1.
• the compounds have similar activity against HIV in both acutely and chronically infected cells. This is a dual property only ocassionally associated with other compounds which are in current use in the therapy of HIV infection although de nova (acute) infections of cells may be treated by compounds which act early in the replication cycle of HIV to block integration of vDNA into the host chromosome. It is this integration which signifies entry of the infection into the chronic state.
• Zidovudine for example is only active against de nova infection of HIV and has no significant activity against chronically infected cells.
• Inhibitors of gene expression of HIV (which is a positive strand RNA virus) would therefore be active in HIV chronically infected cells.
• HIV is a positive strand RNA virus which affects humans.
• the virus attaches to cell membranes by virion adsorption to CD4 surface receptor.
• the virion then passes through the cell membrane penetratively and enters the cell cytoplasm. Uncoating of the virion then takes place in the cytoplasm whereby the viral envelope and the protein coat of the genome release the viral RNA into the cytoplasm.
• Reverse transcription therein produces a double-stranded DNA transcript from host cell genetic material. This invades the host cell nucleus and integrates with the host cell chromosomal DNA. Transcription follows to produce a vRNA replicate which is translated in the cytoplasm to produce new viral proteins. The latter then assembles with vRNA at the inner cell surface to produce new virus particles which are released from the host cell.
• HIV is normally associated with an initial asymptomatic phase.
• This initial asymptomatic phase may last a number of years before the early signs of HIV disease occur.
• Apoptosis is one of these. It is a morphologically distinctive form of programmed cell death involved in many physiological and pathological processes including cellular processes which seek to maintain appropriate intracellular oxidant-antioxidant balance. Cell death in T-cells is closely associated with this balancing process. Infection with HIV is thought gradually to disturb the balance in favour of cell death. Another critical factor in determining whether cells will grow and divide in a normal fashion is intracellular ATP concentration. Low intracellular levels of ATP are associated with ischemic death. T-lymphocytes are especially vulnerable to depletion of intracellular ATP levels. HIV infection may disturb cellular oxidative phosphorylation which is the cellular process responsible for ATP levels in the cell. Cell death from whatever cause will eventually lead to cell depletion to a level that induces AIDS.
• Riboflavine is a known compound, which is also variously known as:
• Vitamin B 2
• Riboflavine is commercially available as such or as its sodium phosphate or tetrabutyrate salt, typically in the former instance as the dihydrate salt. It is also available in various mixtures with other vitamins, all essentially being for the treatment of, inter alia, vitamin B deficiency. In such mixtures the dose of riboflavin varies between 0.5 and 10 mg, with a maximum recommended daily dose being 30 mg.
• the riboflavine requirement of humans is often related to the energy intake, but it appears to be more closely related to resting metabolic requirements.
• a daily dietary intake of about 1.3 to 1.8 mg of riboflavine is recommended that is to say the basic recommended intake of riboflavine is 550 ⁇ g per 4200 kj (1000 kcal) of diet - Report of a Joint FAO/WHO Expert Group, Tech. Rep. Ser. Wld 111th Org. No. 362, 1967.
• the estimated acceptable daily intake of riboflavine is up to 500 ⁇ g per kg body weight - see Thirteenth Report of FAO/WHO Expert Committee on Food Additives, Tech. Rep. Ser. WHO. No. 445, 1971.
• Riboflavine which is a water-soluble vitamin, is essential for the utilisation of energy from food.
• the active, phosphorylated forms, flavine mono-nucleotide and flavine adenine dinucleotide, are involved as co-enzymes in oxidative/reductive metabolic reactions.
• flavouring agents Various other flavins and derivatives thereof are also known, mainly as flavouring agents.
• riboflavine as well as other flavins and derivatives thereof, at doses far higher than, previously used or recommended can be highly effective in the management and treatment of viral infections, in particular HIV.
• the structure of the compound indicates involvement in the process of oxidative phosphorylation within cells. It is possible that the compounds of the invention preferentially target the same target as HIV and so resist or prevent the manifestations of infection including the procreative capacity of the virus.
• the present invention in one aspect provides the use of a flavin, especially riboflavine, or a derivative thereof for the manufacture of a medicament for the management and treatment of viral infection.
• the invention in a second and broader aspect provides such certain flavins or a derivative thereof for use as anti-viral agents.
• riboflavine or other flavin may be used as such or as a derivative and the flavin derivative may be any derivative which is safe for human or animal use.
• the derivative is a riboflavine salt and more preferably the riboflavine salt is riboflavine sodium phosphate or riboflavine tetrabutyrate.
• the flavin or derivative should be of high purity and contamination with spurious ingredients should be avoided.
• the flavin or derivative for use in accordance with the invention may be defined as a compound of the formula (I), namely:
• the group X may be alkyl, or H or an aromatic or other cyclic hydrocarbon group.
• R is ribityl, alkyl, or H
• X is OH, Br, Cl, -SH, OAlk or SAlk.
• R alkyl
• R denotes the ribityl side chain for the riboflavin derivative.
• riboflavin analogs (X), 5-carba-5-deazariboflavin analogs (XI) and 1-carba-1-deazariboflavin analogs (XII), that is:
• SF2 and SF 1 can be identified as 7,8-dimethy1-10-(2,3,4-trihydroxy-4- formylbutyl) isoalloxazine and 7,8-dimethyl-10-(2,3,4-trihydroxy-4-carboxybutyl) isolloxazine, respectively.
• the flavin such as riboflavin, or derivative thereof, is preferably employed at a high dose level significantly in excess of the doses currently used or recommended.
• the riboflavin or other flavin in the clinical trial is used in the present invention at a dosage regime of at least about 1 to about 100 or more (eg 10 or above) mg/kg of body weight per day.
• use according to the invention preferably is one wherein the medicament is in orally administrable form, especially as a capsule (eg two-part).
• the invention includes a pharmaceutical or veterinary composition for use in the management and treatment of viral infections and in unit dosage form, which composition comprises a unit dose of at least about 35 mg such as 50mg or more (eg 50 to 300 mg, such as 50 to 200 or 50 to 100mg) of a flavin such as riboflavine or derivative thereof as described or defined herein, together with a pharmaceutically or veterinarily acceptable diluent, excipient or carrier.
• a pharmaceutical or veterinary composition for use in the management and treatment of viral infections and in unit dosage form, which composition comprises a unit dose of at least about 35 mg such as 50mg or more (eg 50 to 300 mg, such as 50 to 200 or 50 to 100mg) of a flavin such as riboflavine or derivative thereof as described or defined herein, together with a pharmaceutically or veterinarily acceptable diluent, excipient or carrier.
• a composition according to the invention is preferably one wherein the unit dose is from about 35 mg to about 1000 mg. More preferably, the unit dose is from about 250 to 500 mg.
• a composition according to the invention is preferably in oral or injectable form. Within that context a preferred composition is one as a solution in sterile water.
• the invention also includes a process for the manufacture of a medicament for use in the management and treatment of viral infections, which process comprises formulating a flavin such as riboflavine, or a derivative such as the tetrabutyrate salt thereof for anti-viral use.
• the invention includes a product containing a flavin such as riboflavine, or a derivative thereof, as an anti-viral agent, together with another compound(s) having anti-viral activity as a combined preparation for simultaneous, separate or sequential use in anti-viral therapy.
• a flavin such as riboflavine, or a derivative thereof
• a product according to the above definition may be one which includes one or more of the other specific features of the invention defined herein.
• the invention further includes a method for the treatment of viral infection, which method comprises orally or parenterally administering an effective amount of a flavin such riboflavine, or a derivation thereof.
• the amount administered is 1 to 100 (eg at least 10) mg/kg of patient body weight.
• a method according to the invention may include one or more of the other specific features of the invention defined herein.
• the invention is carried out with one or more of riboflavine, riboflavine sodium phosphate, flavin- adenine dinucleotide, lumiflavin, lumichrome, or especially riboflavin tetrabutyrate, whose formula is set forth below:-
• High titre virus stocks of the human immunodeficiency virus HIV-1 were grown in H9 cells with RPMI 1640 supplemented 10% fetal calf serum as growth medium. Cell debris was removed by low speed centrifugation and the supernatant stored at -70°C until required.
• C8166 T-lymphoblastoid cells were incubated with 10TCID 50 HIV- 1 at 37°C for 90 minutes and then washed three times with phosphate buffer saline (PBS). Aliquots of 2 ⁇ 10 5 cells were resuspended in 1.5ml of growth medium in 6ml culture tubes, and test compound at log dilutions from 0.2 to 200 ⁇ M was added immediately.
• PBS phosphate buffer saline
• test compound was dissolved in 70% ethanol and the final concentration of alcohol in the culture was ⁇ 1%. Cultures were incubated at 37°C for 72 hours in 5% CO 2 . 200 ⁇ l of supernatant was taken from each culture and assayed by optical density measurement at 450nm for
• HIV p24 core antigen (Kinchington et al 1989, Roberts et al 1990) using a commercial ELISA which recognises all the core proteins equally (Coulter Electronics Ltd, Luton, UK) .
• IC 50 values standard curves were drawn from untreated cultures containing
• cell cultures were harvested, split and fed with fresh medium approximately 18 to 24 hours before the start of assay. Addition of fresh medium stimulates the cells to enter a log phase of growth. To investigate the effect of cells reaching confluence in conditions of depleted media, cell cultures were fed and split at 72, 48 and 24 hours before being used in a standard acute assay.
• Target cells were preincubated with test compound at log dilutions of 200 to 0.2 ⁇ M for 18/24 hours before infection with HIV-1. Each sample concentration was then treated individually as in the standard acute assay. Assays for Chronically Infected Cells
• H9 cells chronically infected with HIV-1rf (H9rf) were washed three times with medium to remove extracellular virus and incubated with test compounds (200 to 0.2 ⁇ M) for three days.
• p24 antigen was then determined by optical density measurement at 450nm as described for the acute infection standard assay.
• IC 50 values standard curves were drawn from untreated cultures containing 1% alcohol.
• RO 31-8959 (Roche Proteinase inhibitor) was used as an internal control. Assays were carried out in duplicate. 2 .2 Depleted Medi um Assay
• test compound A freshly dissolved sample of test compound was split into two aliquots. They were placed either in daylight or the dark for two hours before being subjected to standard chronic assay. 3 Toxicity Assay
• F4 first antigen assay
• F5 at 450 nm versus concentration ( ⁇ M).
• the dotted line at OD 0.371 represents IC 50 (active).
• test compounds were evaluated for activity against cells both acutely and chronically infected with HIV.
• Antiviral (IC 50 ) arid toxicity (CC 50 ) data is shown below.
• compounds were tested in cell cultures in which fresh media was added at 72, 48 and 24 hours prior to infection. This experiment was carried out to investigate the effects of the compounds on cells in actively dividing and quiescent states. This data indicates that cells may be more sensitive to the test compounds when quiescent.
• the effect of light on stability, preincubation of target cells, and the activity against an African HIV-1 isolate were also investigated. Exposure to light for two hours had no effect on the activity of the compound. Preincubation with the target cells enhanced its activity and it showed significant activity against the Africa HIV-1 isolate.
• Dose level 1 1mg/kg body weight per day orally in two divided dosages
• Dose level 2 2mg/kg body weight per day orally in two divided dosages
• Dose level 3 10mg/kg body weight per day orally in two divided dosages
• Dose level 4 15mg/kg body weight per day orally in two divided dosages
• Dose level 5 20mg/kg body weight per day orally in two divided dosages
• Dose level 6 30mg/kg body weight per day orally in two to three divided dosages
• Dose level 7 40mg/kg body weight per day orally in two to three divided dosages
• Dose level 8 50mg/kg body weight per day orally in two to three divided dosages
• Dose level 9 100mg/kg body weight per day orally in two to three divided dosages
• a formulation can be prepared from the following: riboflavine-5-phosphate 10 mg
• sterile water 2 ml to provide a unit dosage of 10 mg of riboflavine for administration once per day in the treatment of viral infection.
• a formulation can be prepared from the following: riboflavine-5-phosphate 30 mg
• sterile water 2 ml to provide a unit dosage of 30 mg of riboflavine for administration once per day in the treatment of viral infection.
• Example 3 Similar formulations to those of Examples 1 and 2 can be prepared at doses of:
• Composition Compound F7 in admixture with microcrystalline cellulose Ph. Eur 166.4/156.7/118.6/108.7/50mg to give capsule weights of 191.4/206.7/218.6/ 308.7/450mg.

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