Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K33/00—Medicinal preparations containing inorganic active ingredients
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P17/00—Drugs for dermatological disorders
• • 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
  • A61P31/02—Local antiseptics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
  • A61P31/20—Antivirals for DNA viruses
  • A61P31/22—Antivirals for DNA viruses for herpes viruses

Definitions

• the present invention relates to a complex of nitrogen oxides, arising from the interaction of nitrite and acid as an 5 antiviral composition for the treatment of viral diseases of the skin by topical application thereto.
• nitrogen oxide include particular NO which is of the importance particularly if acidified.
• WO 95/22335 we have disclosed a pharmaceutical composition comprising a pharmaceutically acceptable source of nitrites and a pharmaceutically acceptable acidifying agent, inter alia for the direct treatment of disease by topical application. These compounds have a direct effect on the organism concerned 5 but the precise mode of action is not known.
• US-A-4595591 reveals a composition comprising an aqueous solution of nitric acid and nitrous acid at a pH below 1 preferably with an organic acid and copper and cadmium ions 0 for the treatment of superficial lesions of the skin, for example tumorous growths.
• US-A-5648101 provides a vaso-active composition comprising NO adapted for delivery to a body site inter alia by means of a 5 cream or ointment.
• the NO is generated from an admixture of ferrous sulphate, an organic acid and an inorganic nitrite and caused to be reactive in the presence of moisture adjacent or at the site. Acidification is not discussed.
• WO 96/02268 reveals the inhibition of a virus by nitric oxide (N0 2 ) but the advantages of reduction of pH at the environment of use have not been appreciated. - 2 -
• WO 93/25213 reveals a composition comprising nitrous oxide contained in a der atological composition comprising as an essential feature a fatty acid or a lower alkyl ester thereof, pH values, particulary at the environment of use, are not mentioned.
• viruses In order for viruses to survive and reproduce they must evade recognition by the hosts immune responses. The mechanism by which this is achieved is largely unknown but an effective immune response eradicates the infection. Viruses are - 3 - obligate intracellular pathogens . They reproduce using the host's metabolic machinery.
• Acyclovir which is effective against herpes virus, is a deoxyguanosine analogue which competes with deoxyguanosine triphosphate as a substrate for viral thymidine kinase and when phosphorylated and incorporated in the viral DNA causes premature DNA chain termination.
• anti-viral drugs are only effective for a limited number of viral infections and viruses can mutate to overcome the effectiveness of the drugs.
• molluscum contagiosum 1 and 2 which are related to orthopox and parapox viruses and share some homology with vaccinia
• Current therapies comprise physical destruction with manual extrusion, liquid nitrogen therapy or curettage, all of which are painful and not very effective and may cause scarring. The pain of these therapies is particularly pertinent because the majority of patients are under the age of 10 years.
• warts In the case of recalcitrant warts, destructive therapies such as liquid nitrogen can be used in cases where the conventional salicylic acid paints have not resulted in the warts disappearance.
• One problem with warts is that the viral pool is in the stem cells which are found at the base of the epidermis. The aforementioned treatments often remove the virus particles and thus the infection from rhe top layer of the epidermis, but they do not penetrate deep enough to remove the stem cells and therefore the origins of the infection. This can result in the re-emergence of the warts.
• An alternative treatment for warts is by use of dinitrochlorobenzene.
• Such a treatment is intended to make the patient allergic to dinitrochlorobenzene, whereupon the the patient' s immune system mounts an immune response to the dinitrochlorobenzene at the site of the wart and the wart in some cases disappears, presumably as a result of immuno- potentiation.
• Immuno-potentiation can be an effective treatment but subjecting the patient to an allergic reaction caused by dinitrochlorobenzene can be hazardous variable and difficult to control.
• An object of this invention is to provide a treatment for viral skin infections, such as verrucae, warts and molluscum contagiosum, which works effectively and is not associated with the pain involved in the more traditional treatments.
• Another object of the invention is to provide a system for treatment of viral skin diseases which is less susceptible to mutation of viral DNA.
• the above nitrogen oxide complex comprising for example NO and/or N0 2 while it may effect replication to a degree, more importantly modifies the virally infected cells such that the immune system can recognize the viral particles.
• this is supported by the fact that the complex is markedly less effective in immunosuppressed hosts. Generally the greater the percent of nitric oxide (NO) the better the immuno potentiation. If possible up to 100% NO can be used.
• NO nitric oxide
• a topical medicament for the in vivo potentiation of the immune system during a viral skin infection resultant from virus replication in the epidermis of a topical formulation comprising a source of nitrogen oxides, and a pharmaceutically acceptable acidifying agent . - 6 -
• Nitrogen oxides for example NO and N0 2
• the nitrogen oxide complex can facilitate programmed cell death, selectively in infected cells, which may then be taken up by phagocytes and antigen presenting cells leading to immune recognition of the previously hidden viral antigens. Once recognized, specific immunity will lead to destruction of all infected cells through cellular and humoral responses.
• viruses replicating in the epidermis which cause the viral skin infection are selected from molluscum contagiosum, herpes simplex type 1 and 2, varicella zoster virus and papilloma virus.
• Treatment using the acidified nitrogen oxide source has been shown to be particularly effective in viral skin infections caused by the aforementioned viruses .
• the source of nitrogen oxides contains nitric oxide and may also contain NO " or NO" nitrosium ions or a precursor therefor, and is produced when a pharmaceutically acceptable acidifying agent and a pharmaceutically acceptable donor of nitrogen oxides, or a precursor therefor are brought into intimate contact at the site of biological action.
• the pharmaceutically acceptable acidifying agent or the pharmaceutically acceptable donor of nitrogen oxides or a precursor therefor is disposed in a pharmaceutically acceptable carrier or diluent therefor.
• the pharmaceutically acceptable acidifying agent is an organic acid and is selected from at least one of ascorbic acid, ascorbyl palmitate, salicylic acid, lactic acid, citric acid, formic acid, benzoic acid and tartaric acid.
• the choice of acidifying agent depends on the type of viral infection of the skin and the reaction of the infected areas to treatment.
• the use of reducing acids such as ascorbic acid gives a quick burst of NO and N0 2 with significantly more NO produced compared to the amount of N0 2 produced.
• the other organic acids such as salicylic acid give a sustained concentration of NO and N0 2 over a certain time period wherein the ratio of NO to N0 2 is low.
• the concentration of the inorganic nitrite for example sodium nitrite (or other alkali metal nitrites) , as the pharmaceutically acceptable donor of nitrogen oxides or a precursor therefor depends on the acid used and the concentration of the acid used.
• the reducing acid ascorbic acid is highly reactive so therefore only between 1-10% is required with stoichiometric concentrations of the pharmaceutically acceptable donor of nitrogen oxides or a precursor therefor (e.g. sodium or other alkali metal nitrite) .
• Ascorbyl palmitate is more stable but requires a higher concentration than ascorbic acid because the palmitate has a higher molecular weight. A concentration of between 3% and 25% of ascorbyl palmitate is thus required. If salicylic - 8 - acid is used, concentrations of between 0.5% and 30% are appropriate, citric acid requires a yet higher concentration of up to 45%. (All % given herein are by weight)
• the concentration of sodium nitrite required to react with the abovementioned concentrations of organic acid is between 0.5% and 30%, preferably between 5% and 20%.
• Other pharmaceutically acceptable sources of nitrogen oxides or a precursor therefor require different ranges of concentration.
• the pharmaceutically acceptable acidifying agent and the pharmaceutically acceptable donor of nitrogen oxides or a precursor therefor are in stoichiometric concentrations.
• the pharmaceutically acceptable acidifying agent and the pharmaceutically acceptable donor of nitrogen oxides or a precursor therefor are in stoichiometric concentrations, after the reaction is completed there will be no unreacted compounds left. Accordingly any compounds remaining on the infected area will not be able to contaminate healthy skin with the active medicament or anything the treated area touches such as furniture and clothing.
• the medicament is in the form of a paint, a varnish, an ointment a wax, a salve, or a cream.
• These embodiments allow the pharmaceutically acceptable acidifying agent and a pharmaceutically acceptable donor of nitrogen oxides, or a precursor therefor to be applied directly to the infected area.
• the treatment comprising the topical application of separate compositions according to this invention is preferably continued for at least one month, and more preferably two months. - 9 -
• a two-part delivery system for the topical application of a medicament for the in vivo treatment of the epidermis the said system comprising separately;
• a first waxy component comprising a pharmaceutically acceptable acidifying agent
• a second waxy component comprising a pharmaceutically acceptable source of nitrogen oxides whereby if topically applied in vivo simultaneously, or immediately sequentially, to the environment of use, active nitrogen oxides are released therefrom.
• the first and second waxy components comprise a paraffin.
• the acidifying agent is preferably a reducing organic acid or salt such as ascorbic acid or ascorbyl palmitate.
• the source of nitrogen oxides may be an alkali metal nitrite such as sodium nitrite.
• a reducing acid or salt thereof results in a product at the environment of use which comprises a major amount of NO which has significant and therapeutic and immunological effects .
• a source of oxide (s) of nitrogen in the manufacture or prophylaxis of a viral skin infection by a virus selected from herpes simplex types 1 and 2, varicella zoster, vaccinia or papilloma, and particularly from molluscum contagiosum.
• a delivery system for the topical application of a medicament for - 10 - the in vivo treatment of the epidermis comprising an adhesive layer and a support layer impregnated with at least one of the components of the medicament, characterized in that the components of the medicament are a pharmaceutically acceptable acidifying agent and a pharmaceutically acceptable donor of nitrogen oxides or a precursor therefor, and a means of combining the pharmaceutically acceptable acidifying agent with the donor of nitrogen oxides.
• the delivery system comprises two layers, which when in si tu release the oxides of nitrogen including nitric oxide.
• the activation can be by pressure or hydration from the skin.
• the delivery system is adapted for the potentiation of the immune system during a viral skin infection resultant from virus replication with the delivery system in place, such a system may, for example, resemble an adhesive plaster and it is then simple to apply physical pressure to the exterior of the plaster.
• the donor of pharmaceutically acceptable nitrogen oxides may be aqueous and encapsulated in microspheres or liposomes disposed in the support, preferably in the form of a film or a gauze.
• the film or gauze allows increased concentrations of the pharmaceutically acceptable acidifying agent to be used. If a solution of salicylic acid is used then only a concentration of 20- 26% by weight is applied, but if salicylic acid is impregnated in the film or the gauze then a concentration of 26 to 44% by weight can be applied.
• a further advantage of using an adhesive layer is that it can be used to occlude the infected area during treatment which increases the concentration of nitrogen oxides being absorbed through the epidermis . - 11 -
• the adhesive layer can be a decoratively patterned in order to appeal to children.
• the integrity of the vehicle is disrupted by pressure after application of the adhesive layer and film or gauze to a site of viral infected skin. If the pharmaceutically acceptable acidifying agent and the pharmaceutically acceptable nitrogen oxide donors or precursors therefor are not kept separate until administration at the site of biological action they will react together thus rendering the medicament less effective. Accordingly, in this embodiment it is necessary for the pharmaceutically acceptable acidifying agent and the pharmaceutically acceptable nitrites or precursors therefor to be retained separately within the film or gauze layer.
• the application to the site of biological action of pressure applied to the adhesive layer, and therefore the film or gauze layer, can result in the vehicles, such as the microspheres or liposomes, breaking and the pharmaceutically acceptable acidifying agent and the pharmaceutically acceptable nitrogen oxide donors or precursors therefor reacting, thus treating the infected area.
• the delivery system may be used in conjunction with a topically applied medicament.
• the topically applied medicament being either a pharmaceutically acceptable - 12 - acidifying agent or a pharmaceutically acceptable donor of nitrogen oxides or a precursor therefor.
• topically applied medicament without changing the compound in the delivery system.
• pharmaceutically acceptable nitrogen oxide donors or precursors therefor are impregnated in the film or gauze, then the type of pharmaceutically acceptable acidifying agent that is topically applied can be altered and the same adhesive and film or gauze layers utilized.
• the delivery system is an ideal form of treatment for the verrucae on the feet because the delivery system is hidden from view.
• Figure 3 shows a Kaplan Meier plot of the outcome of the treatment of patients with molluscum contagiosum as a function of time
• Figure 4 shows a graph of NO and N0 2 release from 0.083 g of 10% Ap wax with 0.014 g of 10% sodium nitrite wax to give 21 ⁇ moles of NaN0 2 and 25 ⁇ moles of ascorbyl palmitate.
• the curve with "squares” denotes NO values whereas the curve with "circles” denotes N0 2 values.
• the warts were prepared by scraping or abrading the skin to remove the dead skin then the sodium nitrite containing formulation was applied before applying the selected acidifying agent.
• the warts were treated every night and every three days the warts were rescrapped or abraded.
• Subjects applied 2% w/w ascorbic acid in aqueous cream to a control site and an active site. Either the low dose or the high dose nitrite cream was al ⁇ o applied to the active site. The creams were applied 3 times daily at 8 hourly intervals and both the control and the active sites were then occluded with an adhesive polythene/plastic dressing.
• the thickness of the control and active sites were measured using a Mitotoyu' spring thickness gauge and redness was measured using reflectance erythema metre.
• Two 4mm punch biopsies were taken from the active and control sites; one for - 17 - formalin fixation for histological assessment, mass cell stains, neutrophil elastase and in situ nick end labeling and the other for snap freezing and OCT embedding for the other immunohistochemical stains.
• Immunohistochemistry was performed using a streptavidin biotin method and DAB detection with the antibodies in Table 3 and using ApopTag Plus in si tu nick end labeling detection kit to identify apoptotic cells.
• Histology of all actively treated sites showed a significant increase in oedema, endothelial swelling, cloudy swelling of keratinocytes, and a mixed infiltrate of lymphocytes and neutrophils. These changes were quantified on a 0-4 ordinal scale and were similar in low dose, high dose, short exposure and long exposure.
• the number of mast cells seen in Azure A stained sections was similar in control and nitrogen oxide complex treated skin.
• Nitrogen oxide complex treated skin showed significant increases in immuno-competent cells expressing CD3, CD8, CD68 and neutrophil elastase and in the adhesion molecules which attract trafficking of the cells to the site, ICAM-1 AND VCAM- 1.
• the presence of nitrosotyrosine staining in these cells is indicative of the formation of peroxynitrite (ONOO " ) and of p53 which indicates that part of the effect of the complex is mediated through toxicity towards DNA in these cells.
• nitrogen oxide complex did cause some apoptosis but this was surprisingly small at the doses used and we postulate that the effect is likely to be in infected cells.
• the antigen processing cells of the skin were seen to lose dendricity and drop from the epidermis so there were significantly fewer in the treated skin. As these cells behave in this way when activated and functioning to process a newly recognized antigen, this would seem to offer further evidence for an immunopotentiating role for the nitrogen oxide complex. Ki-67 staining for dividing cells did not differ in control or active sites. This would suggest that in warts, for example, the action is not one of reducing cell proliferation. - 20 -
• VCAM-1 1.5 1.17 0.5 0.9 0.0357
• Ki67 was counted in the epidermis and ApopTag positive cells counted per standard section through a 3mm punch biopsy. All other counts were done by computerized image analysis on a fixed standard measuring frame and are expressed as cells per mm 2 .
• Ki67 was counted in the epidermis and ApopTag positive cells counted per standard section through a 3mm punch biopsy. All other counts were done by computerized image analysis on a fixed standard measuring frame and are expressed as cells per mm 2 .
• the nitrogen oxide complexes of the invention may be formed by a combination of ascorbic acid and nitrite on the skin, which causes the release of nitrogen oxides, inter alia nitric oxide, nitrous oxide, nitrogen dioxide and dinitrogen trioxide.
• nitrogen oxides inter alia nitric oxide, nitrous oxide, nitrogen dioxide and dinitrogen trioxide.
• the increase in T helper cells and macrophages was greater in low dose subjects and suggests that at lower doses nitrogen oxides can be pro-inflammatory but at higher doses becomes cytotoxic to the immunocompetent cells and begins to exert an inhibitory effect.
• the nitrogen oxide complex led to a marked induction of ICAM-1 and a moderate increase in VCAM-1 expression.
• the pattern of inflammation was unusual in showing a marked infiltrate of macrophages after only 24 hours, so showing that activated macrophages use nitrogen oxides to specifically attract more macrophages to kill a pathogen.
• a two part component delivery system was made up. Each component was in the form of a wax stick which can be rubbed onto an effective area at regular intervals in accordance with a physician's instructions.
• Phase B components into another vessel heat to 70°C and stir, ensure that all the sodium nitrite has dissolved.
• phase A When both phases have reached 70°C, add phase A to phase B and homogenize for 5 minutes.

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• 1998
  • 1998-03-02 GB GBGB9804469.6A patent/GB9804469D0/en not_active Ceased
• 1999
  • 1999-03-01 CN CNB998035866A patent/CN1270725C/zh not_active Expired - Fee Related
  • 1999-03-01 HU HU0101374A patent/HUP0101374A3/hu unknown
  • 1999-03-01 WO PCT/GB1999/000605 patent/WO1999044622A1/en not_active Application Discontinuation
  • 1999-03-01 CA CA002322199A patent/CA2322199A1/en not_active Abandoned
  • 1999-03-01 PL PL99342755A patent/PL342755A1/xx not_active Application Discontinuation
  • 1999-03-01 BR BR9908617-4A patent/BR9908617A/pt not_active Application Discontinuation
  • 1999-03-01 NZ NZ506678A patent/NZ506678A/xx unknown
  • 1999-03-01 MX MXPA00008583A patent/MXPA00008583A/es not_active Application Discontinuation
  • 1999-03-01 KR KR1020007009698A patent/KR20010041527A/ko not_active Application Discontinuation
  • 1999-03-01 EP EP99937878A patent/EP1059928A1/en not_active Ceased
  • 1999-03-01 JP JP2000534223A patent/JP2002505295A/ja not_active Withdrawn
  • 1999-03-01 AU AU32617/99A patent/AU758264B2/en not_active Ceased
• 2000
  • 2000-08-29 NO NO20004302A patent/NO20004302L/no not_active Application Discontinuation

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