WO2002045704A2 - Traitement de maladie - Google Patents

Traitement de maladie Download PDF

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Publication number
WO2002045704A2
WO2002045704A2 PCT/GB2001/005369 GB0105369W WO0245704A2 WO 2002045704 A2 WO2002045704 A2 WO 2002045704A2 GB 0105369 W GB0105369 W GB 0105369W WO 0245704 A2 WO0245704 A2 WO 0245704A2
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Prior art keywords
agent
enzyme
antagonist
individual
metabolic enzyme
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PCT/GB2001/005369
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English (en)
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WO2002045704A3 (fr
Inventor
Clair Adcocks
Claes Bavik
Michael Cork
Gordon Duff
Rachid Tazi-Ahnini
Simon Ward
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Molecular Skincare Limited
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Priority to AU2002222125A priority Critical patent/AU2002222125A1/en
Publication of WO2002045704A2 publication Critical patent/WO2002045704A2/fr
Publication of WO2002045704A3 publication Critical patent/WO2002045704A3/fr

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    • 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
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/4164—1,3-Diazoles
    • A61K31/4174—Arylalkylimidazoles, e.g. oxymetazolin, naphazoline, miconazole
    • 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
    • A61K31/496—Non-condensed piperazines containing further heterocyclic rings, e.g. rifampin, thiothixene or sparfloxacin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • This invention relates generally to the treatment of diseases with agents, drugs and other therapeutics.
  • the invention relates to prevention of attenuated response to and side effects associated with drug therapies, in particular, chronic drug therapies for the treatment of skin diseases.
  • Tachyphylaxis also known as tolerance
  • tolerance is a falling-off in the effects produced by a drug during continuous use or constantly repeated administration.
  • Tachyphylaxis is thought to arise from desensitisation, associated with conformational changes or modifications (such as phosphorylation) in the receptor (US Patent 5 , 403 , 590 ; Benovic, J.
  • a second cause of tachyphylaxis is thought to be down regulation of the receptor (or other components in the targeted signalling pathway ) for the drug in question (Lefkowitz, R. J., et al., (1980) Curr. Top. Cell. Regul. 17:205-230).
  • Down-regulation of the receptor decreases the number of receptor system molecules on a cell, thus decreasing the response to continued administration of the therapeutic agent.
  • alternating therapies may be used.
  • tachyphylaxis associated with intermittent pulse therapy over several days or weeks with topical steroids topical steroids may be prevented by alternating the steroid treatment with an arninoguanidine composition.
  • Agents such as tazarotene may be used in combination with corticosteroids in the treatment of psoriasis (Leitz et al., 2000, J Am Acad Dermatol 43 (2 Pt 3): S43-6).
  • vitamin D analogues for example, topical treatments for skin diseases, does not lead to tachyphylaxis (Kirsner R.S. and Federman D., American Family Physician 52:237-240, 1995).
  • tachyphylaxis does in fact develop in patients undergoing chronic therapies using vitamin D and its analogues. Indeed, tachyphylaxis is a major problem in vitamin D analogue therapy. Furthermore, we have discovered that an underlying cause of tachyphylaxis is the degradation of the drug in the patient, rather than de-sensitisation or receptor down-regulation. Thus, we have found that when a patient is exposed to a drug (for example, a vitamin D analogue) for extended periods, there is an increase in the expression of enzymes which are capable of metabolising that drug. Accordingly, we provide for a method of treatment of tachyphylaxis by inhibiting such induced metabolic enzymes.
  • a drug for example, a vitamin D analogue
  • a method of alleviating or preventing a tachyphylactic response to an agent in an individual comprising administering to the individual an antagonist of a metabolic enzyme which is induced as a result of exposure of the individual to the agent, in which the enzyme activity is capable of metabolising the agent.
  • an antagonist of a metabohc enzyme for use in a method of alleviating or preventing a tachyphylactic response to an agent in a individual , in which the metabolic enzyme is induced as a result of exposure of the individual to the agent.
  • the antagonist inhibits the breakdown of the agent by decreasing the amount or activity, or both, of the metabolic enzyme.
  • the antagonist is administered locally to apart of the individual which is exhibiting a tachyphylactic response, or a symptom of the disease being treated by the agent, or both.
  • the effects of the antagonist are substantially restricted to the part of the individual to which the antagonist is administered.
  • the metabolic breakdown of the agent in other parts of the individual are not substantially reduced.
  • the antagonist is administered to the skin of the individual .
  • the antagonist is not capable of crossing the basement membrane of the epidermis.
  • the antagonist is administered to a individual already exhibiting a tachyphylactic response
  • the antagonist is administered simultaneously with the agent.
  • the agent is selected from the group consisting of: a steroid, a topical steroid and a corticosteroid, together with natural or artificial analogues of any of these.
  • the agent comprises a macrolactam.
  • the agent comprises a vitamin D analogue selected from the group consisting of: Calcitriol (la,25(OH) 2 D 3 , la,25-dihydroxyvitamin D3, 1,25-dihyroxycholecalciferol), Calcipotriol (Dovanex; MC903) and Tacalcitol (la,24(R)-dihydroxyvitemin D 3 ; Curatoderm).
  • a vitamin D analogue selected from the group consisting of: Calcitriol (la,25(OH) 2 D 3 , la,25-dihydroxyvitamin D3, 1,25-dihyroxycholecalciferol), Calcipotriol (Dovanex; MC903) and Tacalcitol (la,24(R)-dihydroxyvitemin D 3 ; Curatoderm).
  • the metabolic enzyme comprises a P450 cytochrome.
  • the metabohc enzyme comprises a P450 cytochrome selected from the group consisting of: CYP1A1, CYP1A2, CYP24, CYP26, CYP27A1, CYP27B1, CYP2A6, CYP2B4, CYP2C3, CYP2C8, CYP2C9, CYP2D6, CYP2E1, CYP2E2, CYP3A4, CYP3A5, CYP3A6 and CYP3G1.
  • a P450 cytochrome selected from the group consisting of: CYP1A1, CYP1A2, CYP24, CYP26, CYP27A1, CYP27B1, CYP2A6, CYP2B4, CYP2C3, CYP2C8, CYP2C9, CYP2D6, CYP2E1, CYP2E2, CYP3A4, CYP3A5, CYP3A6 and CYP
  • a method of detecting the likelihood of a individual developing a tachyphylactic response to an agent comprising the steps of: administering the agent to a cell of the individual ; and detecting the amount and or activity of a metabohc enzyme capable of metabohsing the agent, in which an increased amount or activity indicates that the individual is likely to develop a tachyphylactic response.
  • a method of identifying an molecule which is capable of reducing a tachyphylactic response to an agent in a individual comprising the steps of: (a) providing a cell from the individual; (b) exposing the cell to an agent; (c) identifying a metabolic enzyme which is induced as a result of such exposure; and (d) identifying an inhibitor of the enzyme identified in (c).
  • a method of relieving tachyphylaxis in a individual induced by administration of an agent in which the agent induces activation of a metabolic pathway which leads to the breakdown of the agent, the method comprising administering an antagonist of an enzyme in the pathway.
  • the present invention in a sixth aspect, provides a pharmaceutical composition for the alleviation or prevention of a tachyphylactic response to an agent in a individual , the pharmaceutical composition comprising an inhibitor of a metabohc enzyme together with a pharmaceutically acceptable carrier or diluent.
  • the pharmaceutical composition further comprises the agent.
  • the agent comprises a steroid, a topical steroid, a corticosteroid, a macrolactam or a vitamin D analogue selected from the group consisting of: 1,25- dihyroxycholecalciferol (Calcitriol), Calcipotriol (Dovanex) and Tacalcitol (Curatiderm).
  • a steroid a topical steroid, a corticosteroid, a macrolactam or a vitamin D analogue selected from the group consisting of: 1,25- dihyroxycholecalciferol (Calcitriol), Calcipotriol (Dovanex) and Tacalcitol (Curatiderm).
  • the metabolic enzyme activity comprises a P450 cytochrome selected from the group consisting of: CYPIAI, CYP1A2, CYP24, CYP26, CYP27A1, CYP27B1, CYP2A6, CYP2B4, CYP2C3, CYP2C8, CYP2C9, CYP2D6, CYP2E1, CYP2E2, CYP3A4, CYP3A5, CYP3A6 and CYP3G1.
  • a P450 cytochrome selected from the group consisting of: CYPIAI, CYP1A2, CYP24, CYP26, CYP27A1, CYP27B1, CYP2A6, CYP2B4, CYP2C3, CYP2C8, CYP2C9, CYP2D6, CYP2E1, CYP2E2, CYP3A4, CYP3A5, CYP3A6 and CYP3G1.
  • an antagonist of a metabohc enzyme in the alleviation or prevention of a tachyphylactic response to an agent in a individual .
  • an antagonist of a metabohc enzyme in a method of preparation of a medicament for the alleviation or prevention of a tachyphylactic response to an agent in a individual .
  • a ninth aspect of the invention a method of alleviating or preventing a side effect associated with administration of an agent to a individual , the method comprising administering to a individual an antagonist of a metabohc enzyme which is induced as a result of exposure of the individual to the agent.
  • a method of identifying an molecule capable of alleviating or preventing a side effect associated with administration of an agent to a individual comprising identifying a metabohc enzyme which is induced by exposure of the individual to the agent, and identifying a molecule which is capable of antagonising the metabohc enzyme.
  • a metabolic enzyme antagonist for use in a method of alleviating or preventing a side effect associated with administration of an agent to a individual .
  • the side effect is caused by a metabolic product of the metabolic enzyme acting on the agent.
  • the metabohc product is a toxin.
  • the side effect is cutaneous irritation, itching, cutaneous atrophy, papulopustular reaction or striae.
  • a method for the treatment or prophylaxis of a disease comprising the steps of: adniinistering an antagonist of a metabohc enzyme to the individual , in which the metabohc enzyme is an enzyme which is induced as a result of exposure of the individual to an agent which is known or suspected to be effective in treating the disease, and in which the metabohc enzyme is capable of metabohsing the agent.
  • the disease is psoriasis and the metabolic enzyme antagonist is an antagonist of CYP24.
  • identifying an molecule suitable for treatment or prophylaxis of a disease comprising the steps of: identifying a metabohc enzyme which is induced as a result of exposure of a individual to an agent, in which the agent is known or suspected to be suitable for treating the disease, the metabohc enzyme being capable of metabohsing the agent; and identifying an antagonist of the metabohc enzyme.
  • the agent comprises a macrolactam, preferably Tacrolimus or Cyclosporin or both and the metabolic enzyme comprises CYPIAI.
  • the agent comprises a vitamin D analogue, preferably Calcipotriol, and the metabohc enzyme comprises CYP2E1.
  • the agent comprises a vitamin D analogue, preferably Calcitriol or Tacalcitol or both, and the metabohc enzyme comprises CYP24.
  • the inhibitor comprises ketoconazole.
  • the metabohc enzyme comprises CYP24
  • the inhibitor is selected from the group consisting of: N-[4-chlorobenzoyl]-2-(lH-infrdazol-l-yl)-2-(phenyl)-l-amino ethane (Compound A), N-[4-cHorobenzoyl]-2-(lH-imidazol- 1 -yl)-2,2-(di-4-chlorophenyl)- 1 - aminoethane (Compound B) and N-[4-(hex-l-yl)benzoyl]-2-(lH-imidazol-l-yl)-2-(phenyl)-l- amino ethane (Compound C).
  • the benefits of the methods and compositions according to the present invention include reduced tachyphylaxis, reduced systemic toxicity, reduced local adverse effects, reduced drug consumption, increased efficacy and increased dosing intervals (i.e., including less patient care needed).
  • Figure 1 shows gel electrophoresis of RT-PCR amplifications of skin equivalents treated with various combinations of vitamin D analogues (with and without metabohc enzyme inhibitors), and macrolactams (with and without metabohc enzyme inhibitors). Amplification with Kl specific primers is conducted.
  • Lanes 1 and 2 control I (7 days in growth media), Lanes 3 and 4: control II (5 days in growth media + 2 days RA), Lanes 5 and 6: control m (7 days in growth media + tazarotene), Lanes 7 and 8 : tacalcitol, Lanes 9 and 10: tacalcitol + ketoconazole, Lanes 11 and 12: tacalcitol + compound B, Lanes 13 and 14: calcitriol, Lanes 15 and 16: calcitriol + ketoconazole, Lanes 17 and 18: calcitriol + compound B, Lanes 19 and 20 : calcitriol + compound A, Lanes 21 and 22: calcitriol + compound C, Lanes 23 and 24: calcipotriol, Lanes 25 and 26: calcipotriol + ketoconazole, Lanes 27 and 28: calcipotriol + compound B, Lanes 29 and 30: calcipotriol + compound A, Lanes 31 and 32: calcipotriol + compound C, Lanes 33
  • Figure 2 shows RT-PCR gels of normal human keratinocytes cultured in the presence of calcitriol with or without ketoconazole and CYP24 inhibitors Compounds A, B and C.
  • Upper panel amplification with CYP24 specific primers,
  • Lane 1 KGM
  • Lane 2 calcitriol (12nM)
  • Lane 3 calcitriol + ketoconazole (lO ⁇ M)
  • Lane 425(OH)D 3 Lane 5 25(OH)D 3 + compound A (300nM).
  • Lower panel amplification with Kl specific primers
  • Lane 1 calcitriol (12nM)
  • Lane 2 calcitriol + ketoconazole (lO ⁇ M).
  • Figure 3 is a gel showing RT-PCR results using CYPl Al primers, on skin equivalent cultures in the presence of macrolactams tacrolimus and or cyclosporin (Example 6).
  • Lanes 1 and 2 control I (7 days in growth media)
  • Lanes 3 and 4 control II (5 days in growth media + 2 days RA)
  • Lanes 5 and 6 control in (7 days in growth media + tazarotene)
  • Lanes 7 and 8 tacalcitol
  • Lanes 9 and 10 tacalcitol + ketoconazole
  • Lanes 11 and 12 tacalcitol + compound B
  • Lanes 13 and 14 calcitriol
  • Lanes 15 and 16 calcitriol + ketoconazole
  • Lanes 17 and 18 calcitriol + compound B
  • Lanes 19 and 20 calcitriol + compound A
  • Lanes 21 and 22 calcitriol + compound C
  • Lanes 23 and 24 calcipotriol
  • Lanes 25 and 26 calcipotriol + ketocon
  • Figure 4 is a gel showing RT-PCR results using CYP2E1 primers, on skin equivalent cultures in the presence of the vitamin D analogue calcipotriol (Example 2).
  • Lanes 1 and 2 control I (7 days in growth media)
  • Lanes 3 and 4 control ⁇ (5 days in growth media + 2 days RA)
  • Lanes 5 and 6 control HI (7 days in growth media + tazarotene)
  • Lanes 7 and 8 tacalcitol
  • Lanes 9 and 10 tacalcitol + ketoconazole
  • Lanes 11 and 12 tacalcitol + compound B
  • Lanes 13 and 14 calcitriol
  • Lanes 15 and 16 calcitriol + ketoconazole
  • Lanes 17 and 18 calcitriol + compound B
  • Lanes 19 and 20 calcitriol + compound A
  • Lanes 21 and 22 calcitriol + compound C
  • Lanes 23 and 24 calcipotriol
  • Lanes 25 and 26 calcipotriol + ketoconazole
  • tachyphylaxis develops in response to the administration of a drug
  • this is manifested in a decrease over time of the effect (including therapeutic effect) of a drug.
  • a given amount of drug administered to a patient at a certain time will have less of a an effect than the same amount of drug administered to the patient initially.
  • the degree of therapeutic effect of an initial adirLimstration of a particular dose of a drug may be referred to as the "baseline level”.
  • baseline level The degree of therapeutic effect of an initial adirLimstration of a particular dose of a drug.
  • the patient fails to respond at all to the drug, and the therapeutic effect of the drug ceases altogether.
  • the drug to which the patient develops a tachyphylactic response is referred to here as a "drug in question”.
  • a patient exhibits a tachyphylactic response to a drug when the effect of the drug decreases below baseline levels upon continued or repeated administration of the drug over a period of time.
  • the effect of the drug includes therapeutic effects, for example, the effect of a vitamin D analogue may include inhibition of proliferation of epithehal cells.
  • Other effects which may be non-therapeutic, for example, a hypercalcaemic effect of a drug are also included.
  • tachyphylactic response is manifested in a decrease in a therapeutic effect of a drug, most preferably a decrease in the therapeutic effect of the drug which is responsible for the disease treatment etc.
  • Our methods and compositions are effective in restoring or increasing the effect of a drug, preferably a therapeutic effect, when administered to a tachyphylactic patient.
  • a tachyphylactic response may be exhibited by a patient to a drug which is administered continuously (for example, by infusion), or administered periodically (for example, occasional doses of a drug over time). What is important is that the administration takes place over a period of time. Preferably, the period of time the drug is administered is 7 or more days, for example, 10 days, 14 days or more, or even over a period of months, for example, a month, two months. In extreme cases of chronic therapies, the drug may be administered over the course of years.
  • tachyphylaxis against a therapeutic compound results from induction of a deactivating molecule (such as a metabohc enzyme) which is capable of reducing the activity of the therapeutic compound.
  • a deactivating molecule such as a metabohc enzyme
  • tachyphylaxis may be reversed, reheved, treated, or prevented by administration of one or more antagonists against one or more metabohc enzymes capable of degrading the administered drug (i.e., the drug in question).
  • our methods and compositions are such that the therapeutic effectiveness of the drug is increased to 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of the baseline level.
  • the therapeutic effectiveness of the drag is increased to 100% of the baseline level, in other words, complete relief of tachyphylaxis.
  • agent capable of degrading a molecule
  • this term should be taken to mean any deactivating molecule which is capable of reducing the activity of that molecule.
  • an agent preferably comprises a metabohc enzyme.
  • the effectiveness of the methods and compositions in the treatment, alleviation, rehef or prevention of tachyphylaxis may be assayed in a number of ways. Such assays may conveniently be based on assays of the therapeutic effectiveness of a drag in treating, alleviating, preventing, etc a disease or syndrome. Such drug assays are known in the art, and the skilled person will readily be able to choose and if necessary design an appropriate assay to use. Likewise, the effect of treatment with the metabohc enzyme antagonist (i.e., the reduction of tachyphylaxis) may be assessed by measuring the effect of treatment of a host with the drug with and without the metabohc enzyme antagonist.
  • tachyphylaxis is reduced (i.e., the administration of the metabohc enzyme antagonist is effective) where the therapeutic effect of the drug on the disease is elevated in the presence of the metabolic enzyme antagonist, but not elevated in its absence.
  • a dose of the drug may be administered to the patient and the therapeutic response measured to establish the baseline level.
  • the metabohc enzyme antagonist is then administered to the patient.
  • a further dose is administered to the patient at a later time and the response of the patient to this dose measured.
  • the therapeutic response at this time is higher where the patient has been treated with the antagonists than otherwise.
  • the response is suitably expressed as a percentage of the baseline level; i.e., where the patient has been treated with the antagonist, the percentage is higher than without treatment.
  • a tachyphylactic response is a result of exposure of a patient to a drug inducing or up- regulating a metabohc enzyme which is capable of metabohsing or breaking down the drag.
  • the metabohc enzyme is induced in the patient generally, or in particular in the cells which are in contact with the drug.
  • induction we mean a process which leads to a higher metabohc enzyme activity than prior to exposure to the drug.
  • a metabohc enzyme may be induced by a number of ways, including upregulation of DNA replication, upregulation of transcription of a gene encoding the enzyme, upregulation of RNA processing, upregulation of RNA turnover, upregulation of translation, upregulation of transport and/or intracellular localisation of polypeptide and/or RNA within the cell, upregulation of post-transcriptional modification, upregulation of protease or other activity which activates a pro-enzyme, upregulation of enzyme cofactors, upregulation of activity of the enzyme, downregulation of breakdown of the enzyme, etc.
  • a gene encoding the metabohc enzyme for example, increased transcription, translation, etc.
  • induction may consist of processing of an inactive, or less active, form of the enzyme to an active form (such as proteolytic processing of a pro-enzyme).
  • a metabohc enzyme may further be induced by removal or processing of an inhibitor of the enzyme.
  • a co-enzyme is required for enzyme activity, anything that leads to the up-regulation, production, activation, etc of the co-enzyme may lead to induction of the metabohc enzyme.
  • the methods and compositions described here rare especially beneficial in topical use (i.e., topical application of the metabohc enzyme antagonist) where degradation in the intended targeted tissue can be inhibited while drag escaping into the general circulation may be rapidly eliminated through unimpeded catabolism. Local inhibition will thus not interfere with the important overall xenobiotic defence function of the P450 system.
  • the local inhibition of drag metabolism in the target tissue may also suitably reduce adverse effects due to build-up of toxic degradation products in the target tissue.
  • Topical administration may be contrasted with systemic application, where the drug or agent is administered to a patient (for example, by ingestion) in such a way (for example, by ingestion) that it reaches substantially throughout the patient, so that it has potential to exert its effects in substantially all parts of the patient.
  • the drug or agent in question is applied to only a portion of the body of the patient, for example, a smaU area on a tissue or organ.
  • the term “topical" or “local” adrninistration is to be understood as administration of a drag or agent to the skin or other epithelial surface of the patient.
  • a topical treatment using a drug may involve adrninistration of a drag on, say, the elbow area of a patient. More preferably, topical administration of a drag or agent is directed to a part of the body of the patient which exhibits a symptom of a disease to be treated.
  • the drug or agent is not allowed substantially to exert its effects on other parts of the body of the patient.
  • the drag or agent is not allowed to substantially exert its effects on parts of the body to which the drug/agent has not been apphed topically, but only on parts of the body to which the drag/agent has been administered.
  • the metabohc enzyme antagonist is apphed only to one part or portion of the patient's body, and is only allowed to inhibit or antagonise metabohc enzymes acting within that portion.
  • the metabohc enzyme antagonist is apphed to an area of skin which exhibits the symptoms of the disease being treated.
  • an antagonist of an enzyme responsible for breaking down vitamin D e.g., an inhibitor of the cytochrome P450 enzyme CYP24 such as ketoconazole
  • CYP24 such as ketoconazole
  • a psoriatic area of skin which is being treated has been treated, or will be treated with a vitamin D analogue.
  • such an area will also exhibit a tachyphylactic response to the drag.
  • Local adrninistration of the metabohc enzyme antagonist has particular advantages in the treatment of tachyphylaxis.
  • drags and other agents which are administered to the body may have toxic or other undesired effects (for example, hypercalcaemia in the case of vitamin D analogues), and the body has natural mechanisms for getting rid of xenobiotics such as drugs.
  • restricting the inhibition of the metabohc enzymes (which are responsible for metabolising drugs and externally administered agents) to only a portion of the body allows drag catabohsm to be inhibited locally. The rest of the body of the patient is able to continue to efficiently clear any drag molecules which escape into the general circulation.
  • Local inhibition of tachyphylaxis is therefore particularly effective in that it reduces toxicity and simplifies treatment regimes.
  • the strategy is particularly effective in relation to dermal and other "surface" conditions (for example, eye, ear, mouth & nose, pulmonary & gastro-intestinal tract, bladder, vagina, joints, cerebro-spinal fluid).
  • local or topical administration encompasses administration in such a way that overall or systemic metabohc breakdown of the drag in the patient is not substantially reduced. More preferably, inhibition of metabolic breakdown of the drug occurs in the immediate vicinity of application of the antagonist. Thus, metabolic breakdown of the relevant drag outside drag-target tissues is not substantiaUy reduced.
  • the metabolic enzyme antagonist is not capable of penetrating the basement membrane of the epidermis. In other words, the antagonist is retained substantially within the outer layers of the epidermis, above the basement membrane. Retention in such a manner stops, prevents, or retards the entry of the antagonist into the general circulation by way of the circulatory system. This is particularly useful where the disease being treated by the administered drag is a skin disease, such as a skin hype ⁇ roliferative disease being treated by a vitamin D analogue.
  • the antagonist may be dehvered in the form of a Transfersome.
  • Transfersomes (TM: IDEA, Kunststoff, Germany) are highly deformable lipid vesicles 100-200nm in diameter. They cross the skin barrier (stratum corneum) spontaneously along the same narrow paths (30-lOOnm) through which water evaporates, but in the opposite direction. It is believed that the this process is driven by a strong and inwards-directed force of hydrotaxis. This force is diminished once the stratum comeum is penetrated and the Transfersomes encounter the hyprophihc environment of the lower epidermis. In other words, Transfersomes are able to penetrate the transdermal barrier and accumulate in the viable lower part of epidermis. Transfersomes are described in further detail in a separate section below.
  • the metabohc enzyme antagonist is modified, or synthesised in such a way that it is capable of t getting specific sites in the epidermis or other portion, tissue, organ, etc where locahsed inhibition of the metabohc enzyme is desired.
  • the ionic charge, hpophihcity, etc of the metabolic enzyme antagonist may be altered in order to be compatible with retention within the epidermis.
  • the metabohc enzyme antagonist may be designed to be capable of binding to extracehular matrix molecules, preferably, extracellular matrix proteins present or abundant in the epidermis.
  • the target site may comprise an epithehal structure such as a comeodesmosome or desmosome, or a epithehal protein such as SCCE (stratum comeum chymotryptic enzyme), SKALP, corneodesmosin, etc.
  • an epithehal structure such as a comeodesmosome or desmosome
  • a epithehal protein such as SCCE (stratum comeum chymotryptic enzyme), SKALP, corneodesmosin, etc.
  • SCCE stratum comeum chymotryptic enzyme
  • SKALP corneodesmosin
  • the metabohc enzyme antagonist may be conjugated, fused, or otherwise associated with a targeting molecule such as an antibody capable of binding to the relevant structures.
  • the effects of the inhibitor or antagonist may be localised by specifically targeting metabohc enzymes which are expressed only in the relevant tissue, organ, or part of the body.
  • metabohc enzymes which are expressed only in the relevant tissue, organ, or part of the body.
  • a specific inhibitor of a metabolic enzyme which is expressed in only a certain part of the body may be apphed (even systemically).
  • the inhibitory effects on the metabohc enzyme will only be localised to the part of the body which expresses the targeted enzyme.
  • the antagonistic effects of the metabohc enzyme antagonist are thereby localised, without affecting the general metabohc breakdown of the drag in other parts of the body of the patient, for example in other tissues such as the liver.
  • CYP3 A4 The most abundantly expressed xenobiotic metabohsing liver enzyme is CYP3 A4. This enzyme has a very broad and high activity towards xenobiotics and is regarded as the main catabolic enzyme for xenobiotics. It is expressed, in order of activity in liver, intestine and lung. However, CYP3A4 is not expressed in the skin. The role of CYP3 A4 in the skin is assumed by other C YPs, for example, by CYPIAI and CYP2E1 for the catabohsm of corticosteroids in skin.
  • specific inhibitors against CYPl Al and or CYP2E1 may be used to alleviate, reduce, prevent, etc tachyphylaxis associated with administration of any xenobiotic such as a corticosteroid drug.
  • a corticosteroid drug since metabolism of the drug is inhibited specifically in the skin, tachyphylaxis against the corticosteroid drag is relieved. The drag is therefore able to exert its therapeutic effects on the part of the body which is being targeted.
  • the corticosteroid drug diffuses beyond the epithelial layer or otherwise enters the circulation, it may be carried to the liver to be catabolised in the normal way.
  • the benefits of local administration of the inhibitor to a part of the body are achieved by utilising a specific inhibitor for a metabohc enzyme expressed only in that part of the body.
  • the main catabolic enzyme for vitamin D and its analogues is the P450 enzyme CYP24. It is expressed in most tissues but not in liver.
  • a specific antagonist or inhibitor of CYP24 (such an antisense RNA, etc) may be administered to the patient to reheve tachyphylaxis associated with administration of vitamin D analogues. Tachyphylaxis is reheved in those parts of the body in which CYP24 is expressed, and the drug is able to exert its therapeutic effects at these locations. Metabolic breakdown of vitamin D analogues in other parts of the body, such as the hver, is not inhibited, and the vitamin D analogue may be cleared in the usual way.
  • Transfersomes ("carrying bodies") are complex, most often vesicular, bi- or multi- component aggregates capable of crossing barriers and of transferring material between the application and the destination sites. Transfersomes are sold by IDEA Co ⁇ oration, Kunststoff, Germany, and TRANSFERSOME is a trade mark of that company. Transfersome transdermal drug delivery technology may be used for controllable and non-invasive delivery of a wide variety of large molecules as well as for the improved delivery of small molecules, including the metabohc enzyme antagonists and/or drags provided here.
  • Transfersomes may be optimised to attain extremely flexible and self-regulating membranes. They are therefore deformable and consequently can cross microporous barriers efficiently, even when the available passages are much smaUer than the average aggregate size.
  • Transfersome formulations are typically composed of natural amphipatic compounds suspended in a water-based solution, optionally containing biocompatible surfactants.
  • Vesicular Transfersomes consist of a hpid bilayer surrounding an aqueous core and further contain at least one component, capable of softening the membrane. The bilayer of a Transfersome is therefore more flexible than a liposome membrane, even metastable. Transfersome vesicles consequently change their shape easily by adjusting locally to ambient stress.
  • Skin is one of the best biological barriers. Its outermost part, the horny layer, reaches less than 10% into the depth of the skin but contributes over 80% to the skin permeability barrier.
  • This body protecting layer consists of overlapping, flaccid corneocytes, organized in columnar clusters, sealed with multilameUar hpid sheets that are covalently attached to the cell membranes and very tightly packed.
  • the average number of and the degree of order in the intercellular hpid lamellae increases toward the skin surface. This is accompanied by a continuous, but nonlinear, decrease in local water content near the surface. Notwithstanding this, the peak skin barrier is located in the inner half of the homy layer, where the interceUular hpid seals are already formed, but not yet compromised by the skin cells detachment.
  • Transfersome vesicles Although small molecules that have crossed the homy layer of the skin (stratum comeum) are normally cleared from the skin through the blood circulation, delivery of drags by means of Transfersome vesicles allows accumulation of drag deep under the skin. Due to their large size, the vesicles are cleared slowly from the skin and associated drags can accumulate at the site. Transfersome mediated administration of weight drugs, consequently, tends to shift the drag distribution towards the deep tissue under the application site. Any method for loading Transfersomes with the metabolic enzyme antagonist and/or the drug to be targeted may be used.
  • Phosphohpid suspensions comprise a mixture of phosphatidylcholine (SPC, ex soya. 99%: Nattermann Phosphohpids, Cologne, Germany) and sodium cholate (SC, p.a.; Merck, Darmstadt, Germany).
  • SPC phosphatidylcholine
  • SC sodium cholate
  • % lOPa weight/weight ratio 89: 11
  • a suspension with 10% total hpid (by weight) is created by adding buffer (pH 6.5) and further homogenizationby sonication (titanium microtip. Heat Systems W 380, U.S.A.; 30min, 4°C). Final sterilization is achieved by filtration. The average vesicle size was measured with photon correlation spectroscopy (90°, ALV-5000 correlator; ALN-Lasermaschinegesellschaft, Langen, Germany) to be typically 150 +/- 50nm.
  • biotransformation metalabohsm/catabohsm
  • xenobiotic a foreign compound
  • biotransformation is an important aspect of its disposition in vivo.
  • One result of biotransformation is the removal of toxic compounds from the body (excretion).
  • the types of transformation are many and varied, and thus the metabohc systems are flexible and often non-specific.
  • the major factor determining the route of excretion is the structure of the xenobiotic itself. As elimination is the end point for biotransformation, metabolites are generally more polar than the parent compound, and for excretion at the kidneys or secretion into bile, polar, ionised compounds are preferred.
  • Phase 1 oxidative, reductive or hydrolytic reactions
  • Phase 2 conjugation reactions
  • the general principle is that Phase 1 reactions modify the structure of the xenobiotic to introduce a functional group suitable for conjugation with glucuronic acid, sulphate or some other highly polar moiety, making the entire product water soluble.
  • Phase 1 reactions are therefore the first step towards elimination of xenobiotics.
  • Oxidative biotransformation is a principle part of this process and the major oxidative reactions that foreign compounds undergo are catalysed by the microsomal mono-oxygenases.
  • oxidation reactions include aromatic hydroxylation, aliphatic hydroxylation, ahcychc hydroxylation, heterocyclic hydroxylation, N-, S- and O-dealkylation, N- oxidation, N-hydroxylation, S-oxidation, desulphuration and deamination.
  • the majority of these oxidative reactions are catalysed by the microsomal mono-oxygenase system based on cytochrome P-450.
  • the mono-oxygenase system is based around the enzymes cytochrome P- 450 and NADPH cytochrome P-450 reductase.
  • Cytochrome P-450 is a haemoprotein and is the terminal oxidase involved in the hydroxylation of drugs and other foreign compounds, and also of endogenous substrates such as steroids.
  • the oxidised reactants are generally more hydrophilic and can either be excreted from the body as such or serve as substrates for further oxidation or as substrates for phase II detoxifying enzymes such as glucuronosyltransferase. While generally beneficial, this defence mechanism will reduce the biological half-life of therapeutic drags and may result in toxic degradation-products, especially after prolonged drag use has up-regulated the expression of the P450 enzymes. In order to be able to catabolize the virtually unlimited number of xenobiotics the organism might be challenged with there is a large number of P450 enzymes (approximately 20) which generally have a fairly broad specificity.
  • the methods and compositions described here make use of any antagonist of a metabolic enzyme involved in the metabolism, including metabohc enzymes involved in the biotransformation, oxidation, reduction or hydrolysis, conjugation, or excretion, etc of a drag.
  • the antagonist may be an antagonist of a Phase 1 enzyme, or a Phase 2 enzyme, or both.
  • the antagonist is an antagonist of a Phase 1 enzyme.
  • the antagonist is an antagonist of a cytochrome P450 enzyme (CYP).
  • CYP cytochrome P450 enzyme
  • More than one antagonist may be used, in which case the antagonists may be against the same metabohc enzyme, or against two or more metabohc enzymes.
  • one, two or more metabohc enzyme antagonists may be targeted against members of a metabohc pathway responsible for degradation or breakdown of the drag. Some or all of the enzymes in the pathway may be induced by the administration of the drag in question.
  • Antagonists against regulatory molecules involved in the regulation of expression of the metabohc enzyme(s) may also be used.
  • Antagonists of metabohc enzymes may be identified by any means known in the art. For example, where the metabohc enzyme involved is already identified, then a known compound capable of inhibiting or antagonising its expression and/or activity may be chosen.
  • assays to identify metabohc enzyme antagonists may be designed.
  • cells for example, skin biopsies
  • genes identified whose expression is induced as a result of (or associated with) such exposure may be identified by use of probes, sequencing, size of the induced transcript or protein etc. Doing so allows metabolic enzymes whose expression is induced or activated by the exposure to the drag to be identified.
  • an antagonist may be chosen from compounds known to antagonise the metabolic enzyme.
  • the metabohc enzyme is an unknown enzyme, a bank of putative or candidate inhibitors may be tested by means known in the art for inhibitory activity.
  • such inhibitors and antagonists are specific to the metabohc enzyme induced.
  • general inhibitors of metabohc enzymes may also be used in the methods and compositions as described here.
  • P450 enzymes A general review of P450 enzymes is found in Adesnik, M. and Atchison, M. (1986) Genes for cytochrome P-450 and their regulation. CRC Crit. Rev. Biochem. 19, 247-305. Detailed information on P450 enzymes may also be found at the URL ht ⁇ ://bioinf.leeds.ac.ul ⁇ prormse/P450.html, part of the "Prosthetic groups and Metal Ions in Protein Active Sites Database" at ht ⁇ ://bioirrf.leeds.ac.uk/promi
  • CYP enzymes are known in the art, and an exhaustive list is provided at the URL http://dmelson.utmem.edu/CytocliromeP450.hti il (see also Nelson et al. (1996) Pharmacogenetics 6, 1-42).
  • a list of P450 cytochromes is also provided in Table 2. Any antagonists or inhibitors of these and other CYPs may be used in the methods and compositions described here.
  • P450 enzymes also known as cytochromes P450, are members of a group of proteins known as 'r em-thiolate proteins", according to the Nomenclature Comrnittee of the International Union of Biochemistry (NC-IUB) (1991) Nomenclature of electron-transfer proteins. Recommendations 1989. Eur. J. Biochem. 200, 599-611.
  • Haem-thiolate proteins are haemoproteins in which the haem iron fifth ligand is a thiolate group (typically of a Cys residue).
  • a distinctive feature of haem-thiolate proteins is a Soret abso ⁇ tion band at around 450 nm inthe CO-difference spectrum of reduced forms.
  • cystathionine ⁇ -synthase (EC 4.2.1.22) (haemoprotein H450; serine sulphydrase; ⁇ -thionase), haem chloroperoxidase (EC 1.11.1.10), nitric oxide synthase (EC 1.14.13.39).
  • Cytochrome P450 enzymes are widely distributed in bacteria; fungi, plants and animals.
  • P450-conteining monooxygenase systems primarily fall into two major classes: bacterial/mitochondrial (I), and microsomal (II).
  • P450-containing systems can be classified according to the number of their protein components (Degtyarenko, K.N. and Archakov, A.I. (1993) Molecular evolution of P450 superfamily and P450-containing monooxygenase systems. FEBS Lett. 332, 1-8.).
  • P450 enzymes are known (Poulos, T.L. (1995) Cytochrome P450. Curr. Opin. Struct. Biol. 5, 767-774)
  • P450cam Poulos, et al, 1987, J. Mol. Biol. 195, 687-700
  • P450 domain of P450 BM - 3 Ravichandran et al., 1993, Science 261, 731-736.
  • P450 te ⁇ Hasemann et al., 1994, J. Mol. Biol. 236, 1169-1185.
  • P450eryF Cupp-Vickery et al., 1995, Nature Struct.
  • the P450 molecule is an / ⁇ protein, shaped like a triangular prism; the overall stracture has been described as being divided into -rich ('right side') and ⁇ -rich ('left side') domains (Poulos, Ravichandran, Hasemann, Cupp-Vickery, supra). However, this terminology is inappropriate, since the - and ⁇ -rich 'domains' comprise discontinuous assembhes of secondary stracture segments and do not constitute independent folding units.
  • sequence identity between any two P450s with known 3-D structure reaches only 20% or less, the overall topology of the proteins is similar, with some differences in the orientations of various helices. The most dramatic variations between P450 structures are found in regions responsible for substrate binding and access.
  • P450 CYP3A4 is involved in the oxidation of at least half the drugs used today.
  • CYP3A4 the P450's CYP1A2, CYP2C9 & CYP2D6 are known to metabolise drugs of clinical importance (Park B.K., et al., Pharmacology and therapeutics 68:385-424, 1995). In humans, five subfamilies appear to be primarily involved in drug metabohsm, namely CYPs 1 A, 2C, 2D, 2E and 3A (Stahulski AV and Lennard M.S. Journal of Chemical Education 77:349-353, 2000).
  • Preferred CYP metabohc enzymes include: CYPIAI, CYP1A2, CYP24, CYP26, CYP27A1, CYP27B1, CYP2A6, CYP2B4, CYP2C3, CYP2C8, CYP2C9, CYP2D6, CYP2E1, CYP2E2, CYP3A4, CYP3A5, CYP3A6 and CYP3G1.
  • the CYP enzymes are selected from the group consisting of: CYPl Al (GenBank Accession No: NM_000499), CYP1A2 (GenBank Accession No: NM_000761), CYP2A6 (GenBank Accession No: NM_000763), CYP2C8 (GenBank Accession No: NM_000770), CYP2C9 (GenBank Accession No: NM_000771), CYP2D6 (GenBank Accession No: NM_000106), CYP2E1 (GenBank Accession No: NM_000773), CYP3A4 (GenBank Accession No: AF280107), CYP3A5 (GenBank Accession No: NM_000777), CYP3A6 (GenBank Accession No: D00408), CYP24 (GenBank Accession No: NM_000782), CYP26 (GenBank Accession No: AC007002) and CYP27A1 (GenBank Accession No:
  • Preferred CYP enzymes further include: CYP2B4, CYP2C3, CYP2E2 and CYP3G1. Sequences of these CYPs are known (see below), and accordingly, human homologues of these and other CYP enzymes may readily be obtained by molecular biology techniques known in the art. Furthermore, inhibitors which are known, or discovered to, antagonise the activities of the CYPs in other species are likely to antagonise the corresponding homologous human CYPs. Screening assays to identify inhibitors are disclosed in further detail below. Accordingly, these antagonists (and antagonists similar to these) may be used to antagonise the relevant human metabohc enzymes.
  • rabbit CYP2B4 The sequence of rabbit CYP2B4 is known (Bo form GenBank Accession No: M 20856; Bi form, GenBank Accession No: M20857; Gasser, et. al., Mol. Pharmacol. 32: 22-30, 1988; CYP2B4 rabbit GenEMBL L10912 (2026bp) Ryan et al, (1993); GenEMBL S64259 (2028bp) PIR S35666 (491 amino acids) Ryan et al., 1993, Arch. Biochem. Biophys. 304, 454-463 (1993); Swiss P00177 PIR S31277 (491 amino acids) S31278 (491 amino acids) PIR S31279 (491 amino acids) Gasser et al., 1988, supra).
  • rabbit CYP2C3 The sequence of rabbit CYP2C3 is known (Accession Numbers: A00183, A22606,
  • the sequence of rabbit CYP2E2 is known (Accession Numbers: B27750, B27680, M18771, 1407213A, M21351, M21349, M21350, M21358, M21359, M21360, M21361, M21362, M21363, Q29508, M21364, M21365, M21366, M21367, M21368, M21369, M21370, M21371 and M21372).
  • the metabolic enzyme comprises CYP24.
  • CYP24 is also known as P450C24, P450 24> CYP24, Cyp24, 24-hydroxylase, 24-Ohase, 23-hydroxylase, 24-hydroxylase cytochrome P450, Vitamin D 24-hydroxylase, Vitamin D3-24-hydroxylase, 25- hydroxyvitamin D3 24-hydroxylase, 25-hydroxyvitamin D3-24-hydroxylase, 25-hydroxyvitamin D 3 -24-hydroxylase, 25-hydroxyvitamin D 24-hydroxylase, 25-hydroxyvitamin D-24- hydroxylase, l,25- ⁇ ycfroxyvit ⁇ minD3-24-hydroxylase, l,25-chfrydroxyvitamin D 3 -24- hydroxylase, lalpha,25-dihydroxyvitarrhnD3-24-hydroxylase, lalpha,25-dihydroxyvitamin D 3 - 24-hydroxylase, 1 alpha,25 dihydroxy-D3 24-hydroxylase, 1 alpha,25 dihydroxy-D 3 24- hydroxylase, lalpha,25(OH)(2)D(3) 24-hydroxylase, lal
  • the CYPs disclosed here preferably comprise human CYPs.
  • human CYPs include (GenBank accession numbers in brackets): CYPIAI (XM 007727; CYP1A2 (XM 007726; CYP1B1 (XM 002576; CYP2A6 (XM 012774; CYP2C8 (XM 001938; CYP2C9 (NM 000771; CYP2D6 (NM 000106; CYP2E1 (XM 051312; CYP3A4 (NM 017460; CYP3A5 (XM 011598; CYP3A7 (XM 011599; CYP24 (XM 009615; CYP26 (NM 000783; CYP27A1 (XM 002590; and CYP27B1 (XM 006670).
  • CYP24 is also known as 25-hydroxyvitamin IV24-hydroxylase, and may be antagonised by the use of 24-hydroxylase enzyme inhibitors such as Ketoconazole, Liarozole fumarate (R85,246) and Menadione.
  • Ketoconazole is a broad range CYP inhibitor. It may be used at concentrations of, for example, l ⁇ M in MCF-7 cell culture.( Zhao J. et al., J. steroid Biochem. & Mol. Biol. 57: 197- 202, 1996), 20 ⁇ M in macrophage ceU culture (Adams J.S. et al., Endocrinology 134:2567- 2573, 1994), and 0.002-1% in vivo on human skin (Kang S., et al., J. Invest. Derm. 108:513- 518, 1997.
  • Liarozole fumarate is also known as R85,246, and may be used at concentrations of, for example, l-10 ⁇ M in prostate cancer cell culture (Ly L.H., et al., Endocrinology 140:2071- 2076, 1999), l ⁇ M in MCF-7 cell culture (Zhao J. et al., J. steroid Biochem. & Mol. Biol. 57:197-202, 1996). Menadione blocks electron transfer to the P450 associated enzyme, and may in particular be used with Tacalcitol (Adams J.S. et al., Endocrinology 134:2567-2573, 1994).
  • CYP24 may also be antagonised by inhibiting the expression of CYP24, for example, by the use of inhibitors of transcription of CYP24 such as Manumycin A, H-7 and Staurosporine.
  • Manumycin A inhibits Ras farnesylation, and may be used at 50 ⁇ M in reporter-gene transfected COS-1 cell culture (Dwivedi P.P., et al., J.B.C. 275:47-55, 2000).
  • H-7 is a protein kinase C inhibitor, and has been found to be effective ex vivo in isolated mouse renal tubules (Mandla S., et al., Endocrinology 127:2639-2647, 1990), and may be used at a concentration of, for example, 20 ⁇ M in IEC-6 (rat intestinal epithehal cells) cell culture (Koyama H. et al., J. Cell. Biochem. 55:230-240, 1994).
  • Staurosporine is a protein kinase C inhibitor (Mandla S., et al., Endocrinology 127:2639-2647, 1990).
  • Forskolin may be used as an inhibitor.
  • l',9'- Dideoxyforskolin is a adenylate cyclase inactive analog of forskolin, and has been used at 1- lO ⁇ M, ex vivo in isolated mouse renal tubules and in vitro in isolated renal mitochondria (Mandla S. et al., Endocrinology 130:2145-2151, 1992).
  • 25-hydroxyvitamin D-l -hydroxylase or Vitamin Ly25-hydroxylase may be inhibited by Ketoconazole, Liarozole fumarate or Menadione, etc as for CYP24.
  • the nonspecific 24-oxidoreductase and the 22, 23-reductase involved in the metabolism of Calcipotriol may also be similarly inhibited (see Masuda et al., JBC. 269:4794-4803, 1994).
  • metabohc enzyme inhibitors include those disclosed in GB2199579 and EP0683156, as well as Schuster et al.
  • compositions described here are useful for the treatment of tachyphylaxis associated with administration of vitamin D and its analogues.
  • Vitamin D analogues have been used to treat diseases associated with cell proliferation, such as psoriasis. Vitamin D analogues have also been used to treat several other inflammatory dermatoses, including scleroderma and viteligo. Topical vitamin D analogues are also being evaluated in clinical trials for the treatment of photo ageing. Topical and oral vitamin D analogues are being evaluated for the treatment of cancer. The commonly used vitamin D analogues are described in further detail below. There are several other topical vitamin D analogues currently in clinical trial which are due for launch in the next two years. Topical vitamin D analogues are now the market leaders for the treatment of psoriasis. In the United Kingdom, sales of Calcopotriol in 1999 reached £25 million. The world market for psoriasis therapies is £650 million per annum.
  • Vitamin D analogues are known in the art, and are described in for example US Patent Nos. 6,303,315, 6,225,345, 6,184,422, 6,172,110, 6,171,845, 6,153,605, 6,051,432, 6,043,385, 6,008,209, 6,001,351, 5,986,113, 5,976,812, 5,972,642, 5,939,407 5,935,976, 5,919,986, 5,883,124, 5,872,104, 5,852,056, 5,847,165, 5,846,961, 5,843,994 5,808,029, 5,807,891, 5,786,344, 5,780,597, 5,763,651, 5,763,485, 5,753,652, 5,747,066 : 5,744,455, 5,736,129, 5,726,204, 5,712,110, 5,709,856, 5,698,222, 5,693,608, 5,686,619 5,684,045, 5,679,567, 5,668,117, 5,665,387, 5,663,200
  • tachyphylaxis Long term administration of any of these vitamin D analogues leads to tachyphylaxis developing against the drag, such tachyphylaxis may be reheved by administration of an antagonist of a metabolic enzyme capable of breaking down the vitamin D analogue, and whose expression and/or activity is up-regulated.
  • the vitamin D analogue comprises Calcipotriol and the metabolic enzyme inhibitor comprises an inhibitor of CYP2E1, such as ketoconazole.
  • the vitamin D analogue comprises Calcitriol and/or Tacalcitol and the metabohc enzyme inhibitor comprises an inhibitor of CYP24 such as ketoconazole, Compound A, B or C.
  • Other specific and non-specific inhibitors of metabohc enzymes may also be used in conjunction or in place of the ones listed above.
  • vitamin D analogues are described below:
  • Calcitriol 1 25-dihydroxyvitamin D 3 is an activated natural vitamin Ds made by Roche, Duphar.(Jones et al., Physiological Reviews IS: 1193-1231 -98. (see especially fig. 10))
  • 1,25-dihyroxycholecalciferol (Calcitriol, l,25(OH) 2 D 3 ) inhibits cell proliferation and stimulates differentiation. It also has direct anti- inflammatory actions inhibiting the production and action of pro-inflammatory cytokines, such as Interleukin l ⁇ (IL-l ⁇ ).
  • IL-l ⁇ Interleukin l ⁇
  • Calcitriol therefore appeared to be a new way to treat diseases characterised by a change in the balance between differentiation and proliferation, for example in psoriasis and cancers.
  • the agent (which may be a therapeutic drag) comprises
  • any agent capable of inhibiting CYP24 may be used to treat or alleviate tachyphylaxis associated with Calcitriol.
  • agents include ketoconazole, N-[4- cWorobenzoyl]-2-(lH-i ⁇ mdazol-l-yl)-2-(phenyl)-l-amino ethane (Compound A), N-[4- chlorobenzoyl]-2-( 1 H-imidazol- 1 -yl)-2,2-(di-4-chlorophenyl)- 1 -aminoethane (Compound B) and N-[4-(hex-l-yl)benzoyl]-2-(lH-imidazol-l-yl)-2-(phenyl)-l-amino ethane (Compound C).
  • Other inhibitors listed in Table 2 may also be used
  • the agent (which may be a therapeutic drag) comprises Calcipotriol.
  • Calcipotriol comprises Calcipotriol.
  • any agent capable of inhibiting CYP2E1 may be used to treat or alleviate tachyphylaxis associated with Calcipotriol. Examples of such agents include ketoconazole, and other inhibitors shown in Table 2.
  • Tacalcitol is l ⁇ , 24 dihydroxyvitamin D 3 , and is also known as Curatoderm (made by Teijin, hcenced by Lipha S.A., sold by Boots).
  • Curatoderm made by Teijin, hcenced by Lipha S.A., sold by Boots.
  • Tacalcitol has a similar efficacy, adverse effect profile and therapeutic ratio as Calcipotriol.
  • the agent (which may be a therapeutic drug) comprises Tacalcitol.
  • the agent comprises Tacalcitol.
  • long-term adrr ⁇ iistration of Tacalcitol is associated with expression of CYP24.
  • any agent capable of inhibiting CYP24 may be used to treat or alleviate tachyphylaxis associated with Tacalcitol.
  • ketoconazole N-[4- cMorobenzoyl]-2-(lH-imidazol-l-yI)-2-(phenyl)-l -amino ethane
  • Compound A N-[4- chlorobenzoyl]-2-( 1 H-imidazol- 1 -yl)-2,2-(di-4-chloropheny ⁇ )- 1 -aminoethane
  • Compound B N-[4-(frex-l-yl)benzoyl]-2-(lH-irmdazol-l-yl)-2- ⁇ henyl)-l-amino ethane
  • Other inhibitors listed in Table 2 may also be used.
  • the vitamin D hydroxylases constitute a family of mixed-function oxidases which contain as their specific component, a cytochrome P450 isoform.
  • cytochrome P450 isoforms have been cloned, including one putatively representing the hver vitamin D-25- hydroxylase (C YP27) and another that of the kidney 25-OH-D-24-hydroxylase (C YP24) (Usui, E., Noshiro, M., and Okuda, K. (1990) FEBSLett 262, 135-138; Ohyama, Y, Noshiro, M., and Okuda, K. (1991) FEBSLett 278, 195-198).
  • the main catabolic enzyme for inactivation of 1,25-dihydroxyvitamin Dj (Calcitrol, 1,25 (OH) 2 D 3 ) in the skin is the microsomal enzyme 25-hydroxyvitamin D-24-hydroxylase (D-24- hydroxylase, CYP24).
  • CYP24 is also the major catabolic enzyme responsible for the breakdown of vitamin D and its analogues. CYP24 is expressed in most tissues, including the skin.
  • CYP24 is a member of the cytochrome P450 protein superfamily of liaeme-containing mono-oxygenases and will sequentially oxidise 1,25 (OH) 2 D 3 to the inactive and water-soluble metabolite calcitroic acid for bilary excretion.
  • C YP27B 1 generates the active vitamin D species (25 (OH)D 3 to 1, 25 (OH) 2 D 3 ) and is probably anabolic rather than catabolic.
  • CYP27A1 is also involved in the activation of vitamin D (D 3 to 25(OH)D 3 ) but seems to serve catabolic functions as well, especially with vitamin D analogues;
  • CYP24 appears to be specific for catabohsm of l,25(OH) 2 D 3 and other active analogues but can catalyse several different reactions in their degradation.
  • the degradation of Calcitrol by P450 cytochromes is understood to involve the following pathway.
  • l,25-(OH) 2 D 3 (Calcitrol) is converted by CYP24 (24-hydroxylase) to 1,24,25- (OH) 3 D 3 (which is 1/10 as active as calcitrol).
  • l,24,25-(OH) 3 D 3 is converted by CYP24 to 24- OXO-1,25 (OH) 2 D 3 .
  • 24-0X0-1,25 (OH) 2 D 3 is converted by CYP24 to 24-0X0-1,23,25 (OH) 3 D 3 .24-0X0-1,23,25 (OH) 3 D 3 .
  • CYP24 is converted by CYP24 to 24,25,26,27-tetranor-l,23 (OH) 2 D 3 (C-23 alcohol). 24,25,26,27-tetranor-l,23 (OH) 2 D 3 is converted by CYP24 to Calcitroic acid. See Jones G. et al., Physiological Reviews 78:1193-1231 -98 (especially fig. 10). Liver does not contain vitamin D receptors nor CYP24 and will not degrade calcitrol efficiently, but it will degrade Calcipotriol.
  • Calcitriol for example, any of the enzymes described above as being involved in the metabohc pathway, may be targeted for the treatment or alleviation of tachyphylaxis associated with adrninistration of Calcitriol.
  • Metabolism of Calcipotriol The metabohsm of Calcipotriol is understood to involve the following pathway.
  • a nonspecific 24-oxidoreductase acts on MC903 (Calcipotriol) to produce MCI 046 (which is 1/11 as active as MC903).
  • MC1046 is acted on by a 22, 23-reductase to produce MC1080 (which is 1/8 as active as MC903).
  • MC1080 is acted on by CYP24 to produce MC1439 (23S)/1441(23R).
  • MC1439 is converted by CYP24 to 24,25,26,27-tetranor-l,23 (OH) 2 D 3 (C-23 alcohol). This is then converted by CYP24 to produce Calcitroic acid. See Masuda et al., JBC. 269:4794-4803, 1994. (especially fig 10).
  • CYP27 is capable of 24-, 25-, or 26(27)-hydroxylation. Ref: "Transfected human hver cytochrome P-450 hydroxylates vitamin D analogs at different side-chain positions", Guo Y. et al., PNAS 90:8668-8672, 1993. CYP27 may be involved in the metabolism of l ⁇ ,24(S)(OH) 2 D 2 (requires 22,23-reduction). See Jones G. et al., Biochemical Pharmacology 52:133-140, 1996.
  • any of the enzymes involved in catalysing the various steps in the metabohsm of Calcipotriol may be targeted for the treatment or alleviation of tachyphylaxis associated with administration of Calcipotriol.
  • Tacalcitol The metabohsm of Tacalcitol is understood to involve two major mechanisms. See “Isolation and identification of l -hydroxy-24-oxovitamin ⁇ and l ⁇ ,23-dihydroxy- 24oxovitamfnD 3 ", Weinstein E. A. et al., Biochemical Pharmacology 58:1965-1973, 1999.
  • l ⁇ , 24(R) dihydroxyvitamin E (Tacalcitol) is acted on by 25-hychoxyvitemin D-l ⁇ - hydroxylase to produce l,24,25-(OH) 3 D 3 .
  • l,24,25-(OH) 3 D 3 is acted on by CYP24 to produce l ⁇ ,25(OH) 2 -24-OXO-D 3.
  • l ,25(OH) 2 -24-OXO-D 3 is acted on by CYP24 to produce l ,23,25(OH) 3 -24-OXO-D 3 .l ⁇ ,23,25(OH) 3 -24-OXO-D 3 Is acted on by CYP24 to produce 24,25,26,27-tetranor-l,23 (OH) 2 D 3 (C-23 alcohol), which is then converted by CYP24 to Calcitroic acid. This pathway appears to account for about 17% of Tacalcitol metabohsm.
  • loc, 24(R) dihydroxyvitamin E 3 (Tacalcitol) is acted on CYP24 to produce l ⁇ (OH)-24- OXO-D 3 , which is converted by CYP24 to produce l ,23(OH) 2 -24-OXO-D 3 l ⁇ ,23(OH) 2 -24-OXO-D 3 is converted by CYP24 to produce 24,25,26,27-tetranor-l,23
  • Any of the enzymes involved in catalysing the various steps in the metabohsm of Tacalcitol may be targeted for the treatment or alleviation of tachyphylaxis associated with administration of Tacalcitol.
  • the rate of metabohsm of a vitamin D analogue may for example be measured as described previously (Dilworth, F. J., Calverley, M. J., Makin, H. L. J., and Jones, G. (1994) Biochem. Pharmacol.4 ', 987-993).
  • tissue culture cells such as HPK1 A-ras cells are incubated with labelled (for example, tritiated) vitamin D analogue (23 nM) in the presence or absence of varying concentrations of analogue (0 to 23 ⁇ M) for 3 h at 37 °C.
  • Triplicate 500- ⁇ l ahquots of aqueous fraction from the cell/medium extract are mixed with aqueous scintillation mixture, and the radioactivity is measured using a scintillation counter.
  • vitamin D receptor Down-regulation of the vitamin D receptor is responsible for tachyphylaxis associated with administration of vitamin D analogues.
  • the expression of the vitamin D receptor may be assayed by any means known in the art. For example, a RT-PCR analysis may be carried out using the following primers: sense
  • tachyphylaxis is in fact caused by the induction of metabohc enzymes which break down vitamin D and its analogues as a result of adrninistration of vitamin D analogues.
  • a preferred method of assaying reduction of proliferation is by measurement of mitotic index.
  • Mitotic index as used here means the percentage of cells in a given population which are undergoing mitosis and/or cell division. Other assays are possible, for example, measurement of cell cycle period.
  • prohferation is intended to mean the division of cells resulting in growth of a tissue.
  • Prohferative cells are actively dividing, and undergo such cell cycle processes as DNA replication, mitosis, cell division etc.
  • Various methods are known by which prohferation may be assayed, for example, by radiolabelling with radioactive nucleotide triphosphates, tritiated thymidine, bromodeoxyuridine etc to detect replicating cells, by visual examination for mitotic cells etc.
  • Proliferation may also assayed by expression of markers such as Ki-67, or by determining the increase in cell numbers by direct counting of cultured cells under different conditions.
  • Tachyphylaxis associated with long term administration of vitamin D analogues may be assessed by measurement of any effect of the vitamin D analogue, preferably a therapeutic effect. Other effects may be assayed.
  • an effect which may be measured, particularly for in vitro assays, may comprise the ability of the analogue to promote differentiation and/or reduce prohferation of cells.
  • proliferation we mean the presence of cell division.
  • Differentiation refers to the process by which unspecialised cells of tissues become speciahsed for particular functions. Differentiation of a cell may be assessed in various ways, for example mo ⁇ hologically, or by assaying expression of protein markers specific for the differentiated cell type as known in the art.
  • Kl and K10 keratin are markers for commitment to terminal differentiation of epidermal keratinocytes, and expression is increased when cellular differentiation occurs.
  • other keratin subtypes may be used as markers for different differentiation stages, for example, K5, K14, K16 and K17.
  • non-keratin markers for example EGF-receptor and ⁇ -1 integrin, may also be used as markers for cellular differentiation. Differentiation and prohferation may be assayed in various ways, for example, by detection of any of the differentiation and/or prohferation markers.
  • markers include (GenBank accession numbers in brackets): Kl (NM 006121); K5 (XM 006848); K6A (XM 006847); K6 B; XM 015481); K6 E (XM 012228); K6 F (L 42611); K7 (L 42612); K10 (XM 012227); K13 (XM 008574); K14 (NM 000421); K16 (XM 008578); K17 (XM 008579); K18 (XM 029625); Cyclin Al (XM 012704); Cyclin A2 (XM 012292); involucrine (XM 001677); MDM2 (XM 017531); b3-integrin (XM 012636); c-fos (AF111167); IL-1 alpha (XM 017768); IL-6 (XM 004777); IL-8 (XM 031289); TNF alpha (NM 000594); GM- CSF
  • PCR primers comprising oligonucleotides may be designed against any one or more of the above sequences, and amplification reactions conducted on any suitable sample to detect the presence and/or absence of the differentiation and proliferation markers).
  • Corticosteroids refer to any of the steroids from the adrenal cortex (eg. hydrocortisone) excluding the sex hormones, but including synthetic or natural derivatives. They are divided in the two major groups: the glucocorticoids and the mineralocorticoids. The corticosteroids, glucocorticoids and mineralocorticoids, bind to and functions through the glucocorticoid receptor and the mineralocorticoid receptor respectively. These receptors are ligand activated transcription factors belonging to the same protein super-family as the vitamin D receptor.
  • corticosteroid should be understood to include reference to analogues of corticosteroids, mineralocorticoids and glucocorticoids, whether synthetic, artificial or natural, preferably having corticosteroid activity.
  • Preferred corticosteroid activities are set out below and include anti-mflammatory activity, ability to bind to corticosteroid receptors, etc.
  • Glucocorticoids exert both receptor-mediated and direct inhibitory effects on the inflammatory cells seen for example in biopsy specimens of psoriasis. They also inhibit mediators of inflammation such as phospholipase A2, an enzyme that initiates the arachidonic acid cascade.
  • Topical corticosteroids have been prescribed for the majority of patients with localised psoriasis for the past three decades in the United States of America. Topical steroids were prescribed to 85% of all psoriasis patients seen by dermatologists but satisfactory improvements was maintained in only 7% after 12 months. Corticosteroids are prone to tachyphylactic response. Many clinicians have had the experience where a previously beneficial topical steroid gradually loses its effectiveness and becomes "expensive Vaseline" for the patient.
  • topical corticosteroids examples include Clobetasol propionate, Amicinonide, Betamethasone dipropionate, Betamethasone-17-valerate, Aclometasone dipropionate and Dexamethasone.
  • Enzymes in the P450 system are the main metabohc enzymes for inactivation of corticosteroids.
  • Clobetasol and Dexamethasone are two corticosteroid compounds widely used in dermatology. Both of these compounds are substrates for CYP3A4 in the hver. However, CYP3 A4 is not expressed in the skin so its function there are substituted by other oxidative enzymes.
  • CYPl Al and CYP2E1 are capable of oxidising Clobetasol and Dexamethasone respectively and are expressed in skin.
  • the expression of the mineralocorticoid and glucocorticoid receptors may be assayed by any means known in the art. For example, a RT-PCR analysis may be carried out using the following primers.
  • Glucocorticoid receptor sense CCTCAACAGCAACAACAGGACC and anti-sense CTTGTGAGACTCCTGTAGTGGC.
  • Mineralocorticoid receptor sense CTTCAGCAAGACAATAATCGGC and anti-sense ACTTAGAGTGGAAGGACGATGG.
  • Macrolactams for example, Tacrolimus and Ascomycrn, are immunosupressive drags that inhibit T-lymphocyte activation and expression of cytokines. Macrolactams are administed in the treatment and prevention of diseases such as atopic eczema, irritant contact dermatitis, psoriasis, viteligo, alopecia areata, and a variety of inflammatory dermatoses.
  • Adrninistration of macrolactams for the treatment of diseases induces a tachyphylactic response in a patient against the macrolactam.
  • Catabolic enzymes such as CYP3A4, and CYPIAI, CYP1A2, CYP24, CYP26, CYP27A1, CYP27B1, CYP2A6, CYP2B4, CYP2C3, CYP2C8, CYP2C9, CYP2D6, CYP2E1, CYP2E2, CYP3A4, CYP3A5, CYP3A6 and CYP3G1 are up-regulated, resulting in increased degradation of the administered macrolactam.
  • the main enzyme responsible for breakdown of macrolactams is CYP3A4, which is known to degrade macrolactams in the liver.
  • compositions described here encompass the use of one or more antagonists against one or more metabolic enzymes responsible for breakdown of macrolactams, such as the CYP enzymes described above, in the treatment or alleviation of tachyphylaxis associated with administration of macrolactams.
  • the agent (which may be a therapeutic drug) comprises a macrolactam, preferably tacrolimus and/or cyclosporin.
  • a macrolactam such as tacrolimus and/or cyclosporin.
  • any agent capable of inhibiting CYPIAI may be used to treat or alleviate tachyphylaxis associated with a macrolactam, preferably tacrolimus and/or cyclosporin. Examples of such agents include ketoconazole and other inhibitors listed in Table 2. OTHER DRUGS
  • Drags which a patient is capable of developing tachyphylaxis to, as well as the metabolic enzymes involved in their breakdown are shown the table below.
  • the methods and compositions described here are therefore suitable for the relief and/or aUeviation and/or prevention of tachyphylaxis associated with administration of any of these drags.
  • the drags shown in Column 1 of the table below may be used generaUy or to treat the diseases in Column 2; their characteristics or mode of action are shown in Column 3.
  • CYP enzymes involved in metabohsm of the drags are shown in Column 4 of the table.
  • the relevant CYP enzyme is targeted by selecting an antagonist of the enzyme.
  • compositions and methods for treatment or aUeviation of side effects associated, or caused by, adrriinistration of a drug to a patient are provided.
  • Irritation of the skin is the principal cutaneous adverse effect of topical vitamin D analogues. Up to 15% of patients may discontinue therapy due to this irritation. In some patients, the cutaneous irritation is an immediate problem that can lead to discontinuing use of the treatment. However, in other patients, cutaneous irritation becomes more of a problem the longer they have used the products. Cutaneous irritation is also found to be a problem when other types of drugs are administered.
  • compositions for example, pharmaceutical compositions, comprising metabolic enzyme antagonists are also provided for the treatment, prevention or aUeviation of side effects caused by or associated with administration of a drug to a patient.
  • the it is the presence of the metabohc enzyme which leads to the side effect.
  • the metabohc enzyme itself may cause the side effect (i.e., the metabohc enzyme may itself be responsible for the cutaneous irritation).
  • the metabolic enzyme may be capable of metabohsing the administered drag, or any other molecule, to produce a metabolic product which is capable of causing the side effect.
  • the enzyme activity is capable of metabohsing the drag to produce the side effect.
  • the side effect is a result of the presence of a metabolic product of the metabohc enzyme acting on the drug, and the metabohc product is capable of causing the side effect.
  • the metabohc product is a toxin.
  • the presence of the metabolic enzyme may lead to the production, induction, processing, activation, etc of other molecules such as polypeptides which are capable of directly or indirectly causing the side effect.
  • the metabolic product itself, or a molecule which is produced, induced or activated, etc as a result of the metabolic product being produced may directly or indirectly lead to the side effect.
  • the side effect may be manifested immediately (i.e., on adrninistration of a drug), or it may appear after a period of time (which may be as long as days, weeks, months, or even years) after the drug is administered.
  • the side effect is associated or caused by repeated, periodic or otherwise long term administration of the drag.
  • the side effect is cutaneous irritation, itching, cutaneous atrophy, papulopustular reaction or striae.
  • a drag such as a vitamin D analogue may be used to treat psoriasis in a patient.
  • Adrninistration of an antagonist against a metabohc enzyme such as a CYP P450 inhibitor may be used to reheve or prevent any tachyphylaxis developing against the vitamin D analogue.
  • Successful treatment using the drug results in clearance of psoriatic plaques.
  • the treated patient is prevented from developing psoriasis by administration of the metabohc enzyme antagonist, without the administration of the drag in question.
  • the metabohc enzyme antagonist may itself be used as a prophylactic treatment.
  • the use of the metabohc enzyme antagonist in this aspect may be augmented by administration of a vitamin D intermediate such as 25(OH)D 3 since the patient's ceUs can efficiently activate 25(OH)D 3 to l,25(OH) 2 D 3.
  • a method for treatment or prophylaxis of a disease comprising the steps of: administering an antagonist of a metabohc enzyme to the patient, in which the metabolic enzyme is an enzyme which is induced as a result of exposure of the patient to a drug which is known or suspected to be effective in treating the disease, and in which the metabohc enzyme is capable of metabohsing the drug.
  • the metabolic enzyme antagonist alone for the treatment and/or prevention of a disease. Accordingly, we provide for the identification of a suitable metabohc enzyme antagonist for this pu ⁇ ose by identifying one or more metabohc enzymes whose expression, etc are induced by administration of a drug normaUy used, or suitable, for treating the disease.
  • a suitable antagonist is provided which is capable of antagonising the metabohc enzyme. Administration of such an antagonist is capable of preventing and/or treating that disease.
  • a method of identifying an agent suitable for treatment or prophylaxis of a disease comprising the steps of: identifying a metabohc enzyme which is induced as a result of exposure of a patient to a drag, in which the drug is known or suspected to be suitable for treating the disease, the metabohc enzyme being capable of metabohsing the drag; and identifying an antagonist of the metabohc enzyme.
  • an active ligand for example an endogenous active vitamin D species
  • an active ligand may have anti-prohferative and/or pro-differentiation effects in skin or other epithelia.
  • Inhibition of the metabohc breakdown of the active ligand by adrrrinistration of a metabohc enzyme antagonist aUows the accumulation of endogenous active species.
  • inhibition of CYP24 aUows accumulation of endogenous 1,25 (OH) 2 D 3 and inhibition of CYP26 aUows accumulation of endogenous aU- trans retinoic acid.
  • endogenously produced active ligands where inhibition of catabohsm may be of therapeutic value include: glucocorticoids, mineralocorticoids, famesoids, thyroid hormone, eicosanoids, and estrogen.
  • administration of a metabolic enzyme capable of metabohsing estrogen may be used in place of hormone replacement therapy (HRT).
  • HRT hormone replacement therapy
  • Second therapeutic agents may be chosen according to the particular disease, syndrome, or therapy in question.
  • an appropriate second therapeutic agent may be a topical steroid such as beta- methazone valerate.
  • the dosage and/or period of administration for the drag in question and the second therapeutic agent may be identical, or dissi ⁇ rUar.
  • the second therapeutic agent may be administered together with the metabohc enzyme antagomst, or subsequently, after administration of the metabohc enzyme antagonist.
  • the pharmaceutical compositions described here may suitably further comprise a second therapeutic agent.
  • the second therapeutic agent may be formulated in the composition together with the metabohc enzyme antagonist, and optionaUy together with the drag in question (i.e., the drug inducing the tachyphylactic response).
  • the second therapeutic agent preferably itself has similar therapeutic effects as the drug in question, more preferably, the second therapeutic agent acts synergisticaUy with the drug in question.
  • the second therapeutic agent may affect the same pathways as the drag in question, or it may affect related or even different pathways.
  • the chemical nature of the second therapeutic agent may be sirmlar to the drug in question, or it may be an unrelated drag.
  • the metabohc enzyme antagonist may be an antagonist of a metabohc enzyme induced by the drag in question. Alternatively, it may be an antagonist of a metabolic enzyme induced by the second therapeutic agent. Two or more metabolic enzyme antagonists may therefore be used in the methods and compositions described here. Furthermore, it may be an antagonist of both a metabohc enzyme induced by the drag, as weU as the metabolic enzyme induced by the second therapeutic agent.
  • Administration of the metabohc enzyme antagonist may therefore be supplemented by periodic discontinuation of administration of the drug in question.
  • Such a routine is referred to here as "discontinuous therapy”.
  • the period of time drag administration is discontinued should be sufficient for some degree of improvement the efficacy of the drug, preferably to 50%, 60%, 70%, 80% or more of baseline level.
  • the period is such that efficacy is restored to a level substantiaUy approaching baseline level (i.e., substantiaUy complete rehef of tachyphylaxis).
  • the administration of the drag is discontinued for a periods of 7 or more days, more preferably for a periods of up to 14 days or even more.
  • the metabohc enzyme antagonist may be administered continuously, including during the break periods when the drag is not administered. However, other administration routines are possible. For example, the metabohc enzyme antagomst may only be administered during the break periods, but not when the drag is being administered. Alternatively, the metabohc enzyme antagonist is only administered while the drug in question is administered (i.e., the break period is complete in that neither the drug nor the metabohc enzyme antagonist is being administered).
  • a preferred rotational therapy consists of administration of the drug in question, foUowed by a second therapeutic agent, foUowed by the drag and so on.
  • Our methods of relief and aUeviation of tachyphylaxis involve administration of a metabohc enzyme antagonist during administration of the drag in question, or during adrninistration of the second therapeutic agent, or both.
  • the metabohc enzyme antagonist may be administered before the course of therapy has begun (i.e., before the first administration of the drag in question), or at any time during the course of therapy, or after termination of the course of therapy.
  • the metabohc enzyme antagonist may be administered once, twice, or as many times as desired during, before or after the course of rotational therapy.
  • the dosage and or period of administration for the drag in question and the second therapeutic agent may be identical, or dissimilar.
  • the pharmaceutical compositions may suitably further comprise a second therapeutic agent.
  • the second therapeutic agent may be formulated in the composition together with the metabohc enzyme antagonist, and optionaUy together with the drug in question (i.e., the drag inducing the tachyphylactic response).
  • the second therapeutic agent preferably itself has similar therapeutic effects as the drag in question, more preferably, the second therapeutic agent acts synergisticaUy with the drug in question.
  • the second therapeutic agent may affect the same pathways as the drag in question, or it may affect related or even different pathways.
  • the chemical nature of the second therapeutic agent may be similar to the drag in question, or it may be an unrelated drag.
  • the metabolic enzyme antagonist may take the form of an adjuvant given together with the drug in question.
  • the metabohc enzyme antagonist may be an antagonist of a metabohc enzyme induced by the drag in question. Alternatively, it may be an antagonist of a metabolic enzyme induced by the second therapeutic agent. Two or more metabolic enzyme antagonists may therefore be used. Furthermore, it may be an antagonist of both a metabohc enzyme induced by the drag, as well as the metabohc enzyme induced by the second therapeutic agent.
  • combination therapy discontinuous therapy and rotational therapy have been addressed separately in the above passages
  • combinations of any or all of these may be used to treat a disease with a while avoiding or reducing tachyphylaxis.
  • rotational therapy may be combined with combination therapy, so that one of the drag and the second therapeutic agent is administered continuously.
  • the presence of the metabolic enzyme antagonist aUows the rehef and aUeviation of tachyphylaxis in this situation.
  • One or more breaks in which administration of either or both of the drag and the second therapeutic agent is discontinued may be introduced into the regime. During these breaks, the metabohc enzyme antagonist may be administered, or the adrninistration discontinued.
  • Other combinations of the above will be obvious to the person skilled in the art.
  • the metabohc enzyme antagonists may not only be used to treat tachyphylaxis to a drag, they may also be used in a preventative regime, i.e., in such a manner as to prevent tachyphylaxis from developing.
  • the metabolic enzyme antagonists may be administered to the patient before the tachyphylaxis has started to develop to the drag.
  • the metabolic enzyme antagonists may therefore be administered even before the drug in question is administered.
  • the dosage of the antagonist be varied as desired, for example, the dosage may be increased as the drag in question is administered and tolerance develops.
  • Vitamin D and its analogues have also been found to be effective in treatment of diseases such as psoriasis, scleroderma, viteligo, photoageing, cancer, etc, and the methods and compositions described here are also useful for aUeviating or preventing tachyphylaxis associated with administration over a period of vitamin D analogues for the treatment of such diseases.
  • the methods and compositions of are particularly suitable for treatment of tachyphylaxis associated with treatment of skin hype ⁇ rohferative diseases.
  • Such skin hype ⁇ roliferation diseases include psoriasis, acne vulgaris, actinic keratosis (solar keratoses - squamous carcinoma in situ), the ichthyoses, hyperkeratoses, disorders of keratinization such as Darriers disease, palmoplanter keratodermas, pityriasis rabra pUaris, epidermal naevoid syndromes, erythrokeratoderma variabihs, epidermolytic hyperkeratoses, non-buUous ichthyosiform erythroderma and hchen planus.
  • a patient exhibiting any of the symptoms associated with a skin hype ⁇ roliferative disease may be treated with a drug such as a vitamin D analogue.
  • a drug such as a vitamin D analogue.
  • Any tachyphylaxis that the patient exhibits to the drag treatment may be treated with one or more metabohc enzyme antagonist to inhibit the level and/or expression and/or activity of one or more metabohc enzymes induced by administration of the drag, and which lead to or are associated with the breakdown of the drug.
  • Such treatment leads to reduction or alleviation or prevention of the tachyphylactic response to the drag.
  • the metabolic enzyme antagonist(s) may be apphed to a patient on its own, on in the form of a pharmaceutical composition as described in more detaU below.
  • compositions and methods are suitable for the treatment or alleviation of tachyphylaxis associated with treatment of psoriasis.
  • Psoriasis manifests itself as inflamed swollen skin lesions covered with shVery white scale.
  • Characteristics of psoriasis include pus-like blisters (pustular psoriasis), severe sloughing of the skin (erythrodermic psoriasis), drop-like dots (guttate psoriasis) and smooth inflamed lesions (inverse psoriasis).
  • psoriasis The causes of psoriasis are currently unknown, although it has been established as an autoimmune skin disorder with a genetic component.
  • Whether a person actuaUy develops psoriasis may depend on "trigger factors" which include systemic infections such as strep throat, injury to the skin (the Koebner phenomenon), vaccinations, certain medications, and intramuscular injections or oral steroid medications. Once something triggers a person's genetic tendency to develop psoriasis, it is thought that in turn, the immune system triggers the excessive skin ceU reproduction.
  • Skin ceUs are programmed to follow two possible programs: normal growth or wound healing, hi a normal growth pattern, skin ceUs are created in the basal cell layer, and then move up through the epidermis to the stratum comeum, the outermost layer of the skin. This normal process takes about 28 days from cell birth to death. When skin is wounded, a wound healing program (regenerative maturation) is triggered, in which cells are produced at a much faster rate, the blood supply increases and localized inflammation occurs. Lesional psoriasis is characterized by ceU growth in the alternate growth program.
  • Skin ceUs (keratinocytes) switch from the normal growth program to regenerative maturation, ceUs are created and pushed to the surface in as little as 2-4 days, and the skin cannot shed the ceUs fast enough.
  • the excessive skin cells build up and form elevated, scaly lesions.
  • the white scale (“plaque") that usuaUy covers the lesion is composed of dead skin cells, and the redness of the lesion is caused by increased blood supply to the area of rapidly dividing skin ceUs.
  • Psoriasis is a genetically determined disease of the skin characterized by two biological hallmarks. First, there is a profound epidermal hype ⁇ roliferation related to accelerated and incomplete differentiation.
  • a common form of psoriasis psoriasis vulgaris, is characterized by well-demarcated erythematous plaques covered by thick, silvery scales.
  • a characteristic finding is the isomo ⁇ hic response (Koebner phenomenon), in which new psoriatic lesions arise at sites of cutaneous trauma.
  • guttate psoriasis a form of the disease that often erupts following streptococcal pharyngitis
  • pustular psoriasis which is characterized by numerous sterile pustules, often 2 to 5 mm in diameter, on the palms and soles or distributed over the body.
  • Objective methods which are employed for establishing the effect of treatment of psoriasis patients include the resolution of plaques by visual monitoring and with photography.
  • aU-trans-retinoic acid tretinoin
  • the methods and compositions described here rely, in some embodiments, on decreasing and/or blocking the activity of one or more metabohc enzymes involved in the breakdown of an administered drug as a means of aUeviating and/or preventing a tachyphylactic response to a drag.
  • the metabohc enzyme is one which is induced, directly or indirectly, by exposure of the patient's cells to the drug.
  • Reduction and/or inhibition of metabohc enzyme activity may take place at various levels, including regulation of DNA rephcation, regulation of transcription of a gene encoding the enzyme, regulation of RNA processing, regulation of RNA turnover, regulation of translation, regulation of transport and/or rntraceUular localisation of polypeptide and/or RNA within the cell, regulation of post-transciptional modification, regulation of protease or other activity which activates a pro-enzyme, regulation of enzyme cofactors, regulation of activity of the enzyme, regulation of breakdown of the enzyme, etc.
  • decreasing and/or blocking of the metabolic enzyme activity may be achieved at any one or more of these levels.
  • Antagonists of metabohc enzymes may achieve their effects at any one or more of the levels described above.
  • the antagonist may be an inhibitor of transcription, translation, processing, or an activator of breakdown, etc of the metabohc enzyme.
  • antagonist as used in the art, is generaUy taken to refer to a compound which binds to an enzyme and inhibits the activity of the enzyme.
  • the term as used here, however, is intended to refer broadly to any agent which inhibits the activity of a molecule, not necessarily by binding to it. Accordingly, it includes agents which affect the expression of a protein such as a protease involved in the breakdown of the metabolic enzyme, or the biosynthesis of a molecule such as a protease inhibitor (inhibitor of the protease), or the expression of modulators of the activity of the protease or protease inhibitor.
  • the specific activity which is inhibited may be any activity which is characteristic of the enzyme or molecule, for example, metabohc enzyme activity involved in or leading to the breakdown of a drag.
  • Assays for metabohc enzyme activity, in particular, any activity associated with breakdown of a drag, are known in the art.
  • the antagonist may bind to and compete for one or more sites on the relevant molecule, for example, a metabohc enzyme, preferably, the catalytic site of the metabohc enzyme. Preferably, such binding blocks the interaction between the molecule and another entity (for example, the interaction between a metabohc enzyme and its substrate).
  • the metabolic enzyme antagonist may bind to any of the P450 cytochromes in such a manner as to decrease/inhibit its interaction with a substrate (e.g., a drag) to prevent oxidation, hydroxylation, deaUcylation, etc of that substiate.
  • the antagonist need not necessarily bind directly to a catalytic site, and may bind for example to an adjacent site, another protein (for example, a protein which is complexed with the enzyme) or other entity on or in the ceU, so long as its binding reduces the activity of the enzyme or molecule.
  • an antagonist may include a substrate of the enzyme, or a fragment of this which is capable of binding to the enzyme.
  • whole or fragments of a substrate generated natively or by peptide synthesis may be used to compete with the substrate for binding sites on the enzyme.
  • a molecule which mimicks the shape or conformation of a drug molecule may be used as an inhibitor of metabohc enzyme activity.
  • an immunoglobulin for example, a monoclonal or polyclonal antibody
  • the antagonist may also include a peptide or other smaU molecule which is capable of interfering with the binding interaction.
  • Other examples of antagonists are set forth in greater detaU below, and wiU also be apparent to the skiUed person.
  • enzymatic activity of the cytochrome P450 enzyme CYP3A4 may be specificaUy inhibited by the metabohc enzyme antagonists Gestodene or Troleandomycin.
  • Other inhibitors of CYP3A4 include azole antifungals, protease inhibitors, calcium channel blockers, some macrohdes like troleandromycin and eiythromycin, and the commonly used 'SSRT antidepressants such as fluvoxamine and fluoxetine.
  • HIV protease inhibitors such as ritonavir (Norvir®), indinavir (Crixivan®), nelfinavir (Viracept®) and saquinavir (Invirase®/Fortovase®) are capable of inhibiting CYP3A4 enzyme activity.
  • Enzymatic activity of the cytochrome P450 enzyme CYPIAI may be specificaUy inhibited by the metabohc enzyme antagonists Daidzein or Genistein.
  • Enzymatic activity of the cytochrome P450 enzyme CYP2E1 may be specificaUy inhibited by the metabolic enzyme antagonists 4-Methylpyrazole or Diethyldithiocarbamate.
  • the general cytochrome P450 inhibitor ketoconazole may also be used as a metabolic enzyme antagonist.
  • Other inhibitors suitable for use as metabohc enzyme antagonists are shown in Table 2 below. Inhibition of P450 enzyme activity may be permanent or reversible.
  • Blocking the activity of a metabohc enzyme may also be achieved by reducing the level of expression of the metabohc enzyme in the cell.
  • the ceU may be treated with antisense compounds, for example oligonucleotides having sequences specific to the metabohc enzyme mRNA.
  • the level of expression of regulators of the metabohc enzyme for example, transcription factors involved in expression of the metabolic enzyme, may also be regulated this way.
  • the antagonist may regulate expression of the relevant metabohc enzyme. Regulation of expression may be achieved by regulating the transcription of a gene encoding the metabohc enzyme, or by regulating the transcription of a gene which encodes another polypeptide involved in the regulation of expression of the metabolic enzyme. For example, it is known that transcription of the gene encoding CYP24 (which is an enzyme capable of metabolic breakdown of vitamin D analogues) is dependent on the Ras signalling pathway. The drag Manumycin A is capable of inhibiting the Ras signalling pathway.
  • Manumycin A may therefore be used as an antagonist of the metabolic enzyme CYP24 (Dwivedi P.P., Omdahl, J.L., Kola I., Hume D.A., and May B.K., 2000, Regulation of rat cytochrome P450C24 (CYP24) gene expression, Journal of Biological Chemistry 275:47-55, 2000). Accordingly, we disclose methods and compositions which utilise Manumycin A in the treatment of tachyphylaxis associated with administration of vitamin D and its analogues.
  • the term "antagonist” includes but is not limited to agents such as an atom or molecule, wherein a molecule may be inorganic or organic, a biological effector molecule and/or a nucleic acid encoding an agent such as a biological effector molecule, a protein, a polypeptide, a peptide, a nucleic acid, a peptide nucleic acid (PNA), a viras, a virus- like particle, a nucleotide, a ribonucleotide, a synthetic analogue of a nucleotide, a synthetic analogue of a ribonucleotide, a modified nucleotide, a modified ribonucleotide, an amino acid, an amino acid analogue, a modified amino acid, a modified amino acid analogue, a steroid, a proteoglycan, a hpid, a fatty acid and a carbohydrate
  • antagonists are also intended to include, a protein, polypeptide or peptide including, but not limited to, a structural protein, an enzyme, a cytokine (such as an interferon and/or an interleukin) an antibiotic, a polyclonal or monoclonal antibody, or an effective part thereof, such as an Fv fragment, which antibody or part thereof may be natural, synthetic or humanised, a peptide hormone, a receptor, a signaUing molecule or other protein; a nucleic acid, as defined below, including, but not limited to, an ohgonucleotide or modified ohgonucleotide, an antisense oligonucleotide or modified antisense oligonucleotide, cDNA, genomic DNA, an artificial or natural chromosome (e.g.
  • RNA including mRNA, tRNA, rRNA or a ribozyme, or a peptide nucleic acid (PNA); a viras or virus- like particles; a nucleotide or ribonucleotide or synthetic analogue thereof, which may be modified or unmodified; an amino acid or analogue thereof, which may be modified or unmodified; a non-peptide (e.g., steroid) hormone; a proteoglycan; a lipid; or a carbohydrate.
  • PNA peptide nucleic acid
  • SmaU molecules including inorganic and organic chemicals, which bind to and occupy the active site of the polypeptide thereby making the catalytic site inaccessible to substrate such that normal biological activity is prevented, are also included.
  • smaU molecules include but are not limited to small peptides or peptide-hke molecules.
  • the antagonist or agent may itself be a protease which cleaves the metabohc enzyme.
  • proteases include aminopeptidase M, carboxypeptidase P, carboxypeptidase Y, caspase 1,4,5, caspase 2,3,7, caspase 6,8,9, chymotrypsrn, Factor Xa, pepsin, TEV, thrombin, trypsin etc.
  • cytochrome P450 cytochrome P450
  • Such metabolic enzyme antagonists are suitable for use in the methods and compositions described here for the relief or prevention of tachyphylaxis.
  • Candidate compounds may be identified from a variety of sources, for example, cells, ceU-free preparations, chemical libraries, peptide and gene libraries, and natural product mixtures.
  • Such antagonists or inhibitors so-identified may be natural or modified substrates, ligands, receptors, enzymes, etc., as the case may be, of the metabolic enzyme; or may be structural or functional mimetics thereof (see Coligan et al., Current Protocols in Immunology l(2):Chapter 5 (1991)).
  • the screening method may simply measure the binding of a candidate compound to the metabolic enzyme such as a CYP, or to ceUs or membranes bearing the metabohc enzyme, or a fusion protein thereof by means of a label directly or indirectly associated with the candidate compound.
  • the screening method may involve competition with a labeUed competitor.
  • these screening methods may test whether the candidate compound results in inhibition of the metabohc enzyme, using detection systems appropriate to the ceUs bearing the metabolic enzyme.
  • the assay system may comprise a drag, i.e., a drag known to or likely to induce tachyphylaxis, and the assay may measure the breakdown or turnover of that drag by the metabohc enzyme in the presence and absence of the putative antagonist.
  • the antagonist may affect transcription of the metabohc enzyme, and a suitable assay may measure the amount of transcription, e.g., by Northern assay, RT-PCR, etc.
  • Agents which are capable of reducing or inhibiting transcription etc are suitable for use as metabolic enzyme antagonists.
  • an antagonist is to be identified as being capable of affecting other activities which result in decreased metabohc enzyme function, such as regulation of DNA replication, regulation of transcription of a gene encoding the enzyme, regulation of RNA processing, regulation of RNA turnover, regulation of translation, regulation of transport and/or intraceUular localisation of polypeptide and/or RNA within the cell, regulation of post-transciptional modification, regulation of protease or other activity which activates a pro-enzyme, regulation of enzyme cofactors, regulation of activity of the enzyme, regulation of breakdown of the enzyme, etc, suitable assays as known in the art may be designed. Such assays typicaUy measure the relevant activities in the presence or absence of a putative antagonist to identify metabolic enzyme antagonists suitable for use here.
  • the screening methods may simply comprise the steps of mixing a candidate compound with a solution containing the metabohc enzyme to form a mixture, and determining whether its ability to bind a substrate (for example, a drag) is reduced.
  • Fusion proteins such as those made from Fc portion and the metabolic enzyme, may also be used for high-throughput screening assays to identify antagonists for metabolic enzyme function (see D. Bennett et al., J Mol Recognition, 8:52-58 (1995); and K. Johanson et al., J Biol Chem, 270(16): 9459-9471 (1995)).
  • a method for identifying a compound capable of inhibiting the interaction between a metabohc enzyme and a substrate of the enzyme Such a compound may be used as a metabohc enzyme antagonist.
  • the method comprises contacting a metabohc enzyme with a candidate compound in the presence of a substrate of the metabohc enzyme and determining whether the levels of metabohc enzyme binding to the substrate are reduced.
  • a fragment of metabohc enzyme capable of binding to its substrate may also be used.
  • the antagonist may comprise one or more antisense compounds, including antisense RNA and antisense DNA, which are capable of reducing the level of expression of the metabolic enzyme in the cell which is exposed to the drag.
  • the antisense compounds comprise sequences complementary to the mRNA encoding the metabohc enzyme.
  • the antisense compounds are oligomeric antisense compounds, particularly oligonucleotides.
  • the antisense compounds preferably specificaUy hybridize with one or more nucleic acids encoding the metabohc enzyme.
  • nucleic acid encoding metabohc enzyme encompasses DNA encoding the metabolic enzyme, RNA (including pre- mRNA and mRNA) transcribed from such DNA, and also cDNA derived from such RNA.
  • RNA including pre- mRNA and mRNA
  • cDNA derived from such RNA.
  • the specific hybridization of an oligomeric compound with its target nucleic acid interferes with the normal function of the nucleic acid. This modulation of function of a target nucleic acid by compounds which specificaUy hybridize to it is generaUy refe ⁇ ed to as "antisense".
  • the functions of DNA to be interfered with include rephcation and transcription.
  • RNA to be interfered with include all vital functions such as, for example, translocation of the RNA to the site of protein translation, translation of protein from the RNA, splicing of the RNA to yield one or more mRNA species, and catalytic activity which may be engaged in or facUitated by the RNA.
  • the overaU effect of such interference with target nucleic acid function is modulation of the expression of the metabohc enzyme.
  • stimulation or a decrease (inhibition) in the expression of a gene.
  • expression of a gene encoding an inhibitor of metabohc enzyme activity, or an inhibitor of expression of the metabohc enzyme may be increased.
  • inhibition of expression in particular, inhibition of metabohc enzyme expression, is the preferred form of modulation of gene expression and mRNA is a preferred target.
  • Antisense constructs are described in detail in US 6,100,090 (Monia et al), andNeckers et al., 1992, Crit Rev Oncog 3(1-2): 175-231, the teachings of which document are specifically inco ⁇ orated by reference.
  • compositions comprising one or more agents for treating or aUeviating tachyphylaxis.
  • Such pharmaceutical compositions include useful agents such as general metabohc enzyme antagonists, for example, ketoconazole or antisense RNA, as well as specific inhibitors such as Gestodene, Troleandomycin, azole antifungals, protease inhibitors, calcium channel blockers, macrohdes like troleandromycin and erythromycin, fluvoxamine and fluoxetine, ritonavir (Norvir®), indrnavir (Crixivan®), nelfinavir (Viracept®) and saquinavir (Invirase®/Fortovase®), Daidzein, Genistein, 4-Methylpyrazole, Diethyldithiocarbamate etc. Details of these and other antagonists are provided elsewhere in this document.
  • metabolic enzyme antagonists may be administered in the form of pharmaceutical compositions which may optionaUy include the drag in question (i.e., the drug against which tachyphylaxis in the patient has developed).
  • the pharmaceutical composition has as its main active ingredient the metabohc enzyme antagonist (i.e., the metabohc enzyme antagonist is not included together with the drag in question).
  • the metabohc enzyme antagonist i.e., the metabohc enzyme antagonist is not included together with the drag in question.
  • Such a pharmaceutical composition has significant advantages in that it provides a greater flexibility. For example, plaques of psoriasis are more resistant to therapy on certain parts of the body such as the knees and elbows. In these areas, the strength of formulation (i.e., the amount of metabohc enzyme antagonist in the composition), or the frequency of application may be increased. Having a separate formulation from the drag in question aUows the flexibility in dosage and administration to be achieved.
  • a formulation comprising the drug in question (for example, a vitamin D analogue for use in treating psoriasis) alone may be used as a first line therapy. If tachyphylaxis develops to this formulation, a second formulation comprising the drug and the metabohc enzyme antagonist may be used as a second line therapy. However, as noted above, a formulation comprising the metabohc enzyme antagonist alone may be used for the treatment or prevention of a disease.
  • composition comprising the agent or agents to be administered alone, it is preferable to formulate the active ingredient as a pharmaceutical formulation.
  • the composition may include the agent(s), a structuraUy related compound, or an acidic salt thereof.
  • the pharmaceutical formulations comprise an effective amount of agent together with one or more pharmaceutically-acceptable carriers.
  • An "effective amount" of an agent is the amount sufficient to aUeviate tachyphylaxis (as described above).
  • the effective amount wiU vary depending upon the particular disease or syndrome to be treated or alleviated, as well as other factors including the age and weight of the patient, the type of drag which is being administered, the metabohc enzyme involved, how advanced the disease etc state is, the general health of the patient, the severity of the symptoms, and whether the agent is being administered alone or in combination with other therapies (such as the drag in question)
  • Suitable pharmaceuticaUy acceptable carriers are weU known in the art and vary with the desired form and mode of administration of the pharmaceutical formulation.
  • they can include diluents or excipients such as fillers, binders, wetting agents, disintegrators, surface- active agents, lubricants and the like.
  • the carrier is a solid, a liquid or a vaporizable carrier, or a combination thereof.
  • Each carrier should be "acceptable” in the sense of being compatible with the other ingredients in the formulation and not injurious to the patient.
  • the carrier should be biologicaUy acceptable without eliciting an adverse reaction (e.g. immune response) when administered to the host.
  • compositions include those suitable for topical and oral administration, with topical formulations being preferred where the tissue affected is primarily the skin or epidermis (for example, psoriasis and other epidermal diseases).
  • Topical administration of the metabolic enzyme antagonist as described above in further detail, provides certain advantages.
  • the topical formulations include those pharmaceutical forms in which the composition is applied externally by direct contact with the skin surface to be treated.
  • a conventional pharmaceutical form for topical application includes a soak, an ointment, a cream, a lotion, a paste, a gel, a stick, a spray, an aerosol, a bath oil, a solution and the like.
  • Topical therapy is dehvered by various vehicles, the choice of vehicle can be important and generaUy is related to whether an acute or chronic disease is to be treated.
  • an acute skin proliferation disease generally is treated with aqueous drying preparations
  • chronic skin proliferation disease is treated with hydrating preparations. Soaks are the easiest method of drying acute moist eruptions.
  • Lotions are ideal for hairy and intertriginous areas.
  • Ointments or water-in-oil emulsions are the most effective hydrating agents, appropriate for dry scaly eruptions, but are greasy and depending upon the site of the lesion sometimes undesirable. As appropriate, they can be applied in combination with a bandage, particularly when it is desirable to increase penetration of the agent composition into a lesion.
  • Creams or oU-in-water emulsions and gels are absorbable and are the most cosmetically acceptable to the patient. (Guzzo et al, in Goodman & Oilman's Pharmacological Basis of Therapeutics, 9th Ed., p.
  • Cream formulations generaUy include components such as petroleum, lanolin, polyethylene glycols, mineral oU, glycerin, isopropyl palmitate, glyceryl stearate, cetearyl alcohol, tocopheryl acetate, isopropyl myristate, lanolin alcohol, simethicone, carbomen, methylchlorisothiazolinone, methyhsothiazolinone, cyclomethicone and hydroxypropyl methylceUulose, as weU as mixtures thereof.
  • compositions for topical application include shampoos, soaps, shake lotions, and the like, particularly those formulated to leave a residue on the underlying skin, such as the scalp (Arndt et al, in Dermatology In General Medicine 2:2838 (1993)).
  • the concentration of the agent composition in the topical formulation is in an amount of about 0.5 to 50% by weight of the composition, preferably about 1 to 30%, more preferably about 2-20%, and most preferably about 5-10%.
  • the concentration used can be in the upper portion of the range initiaUy, as treatment continues, the concentration can be lowered or the application of the formulation may be less frequent.
  • Topical applications are often apphed twice daily. However, once-daily application of a larger dose or more frequent applications of a smaUer dose may be effective.
  • the stratum comeum may act as a reservoir and aUow gradual penetration of a drug into the viable skin layers over a prolonged period of time.
  • a sufficient amount of agent must penetrate a patient's skin in order to obtain a desired pharmacological effect. It is generally understood that the abso ⁇ tion of drug into the skin is a function of the nature of the drug, the behaviour of the vehicle, and the skin. Three major variables account for differences in the rate of abso ⁇ tion or flux of different topical drugs or the same drag in different vehicles; the concentration of drug in the vehicle, the partition coefficient of drug between the stratum comeum and the vehicle and the diffusion coefficient of drag in the stratum comeum. To be effective for treatment, a drug must cross the stratum comeum which is responsible for the barrier function of the skin. In general, a topical formulation which exerts a high in vitro skin penetration is effective in vivo. Ostrenga et al (J. Pharm. Sci., 60: 1175-1179 (1971) demonstrated that in vivo efficacy of topicaUy applied steroids was proportional to the steroid penetration rate into dermatomed human skin in vitro.
  • a skin penetration enhancer which is dermatologically acceptable and compatible with the agent can be inco ⁇ orated into the formulation to increase the penetration of the active compound(s) from the skin surface into epidemal keratinocytes.
  • a skin enhancer which increases the abso ⁇ tion of the active compound(s) into the skin reduces the amount of agent needed for an effective treatment and provides for a longer lasting effect of the formulation.
  • Skin penetration enhancers are weU known in the art. For example, dimethyl sulfoxide (U.S. Pat. No. 3,711,602); oleic acid, 1,2-butanediol surfactant (Cooper, J. Pharm.
  • Te ⁇ enes such as 1,8-cineole, menthone, limonene and nerohdol (Yamane, J. Pharmacy & Pharmocology, 47:978-989 (1995)); Azone.RTM. and Transcutol (Harrison et al, Pharmaceutical Res. 13:542-546 (1996)); and oleic acid, polyethylene glycol and propylene glycol (Singh et al, Pharmazie, 51:741-744 (1996)) are known to improve skin penetration of an active ingredient. Transferosomes (as described above) may also be used.
  • Levels of penetration of an agent or composition can be determined by techniques known to those of skiU in the art. For example, radiolabeling of the active compound, foUowed by measurement of the amount of radiolabeled compound absorbed by the skin enables one of skiU in the art to determine levels of the composition absorbed using any of several methods of deterrnining skin penetration of the test compound. Publications relating to skin penetration studies include Reinfenrath, W G and G S Hawkins. The Weaning Buffalo Pig as an Animal Model for Measuring Percutaneous Penetration. ImSwine in Biomedical Research (M. E.
  • agent for some applications, it is preferable to administer a long acting form of agent or composition using formulations known in the arts, such as polymers.
  • the agent can be inco ⁇ orated into a dermal patch (Junginger, H. E., in Acta Pharmaceutica Nordica 4:117 (1992); Thacharodi et al, in Biomaterials 16:145-148 (1995); Niedner R., in Hautier 39:761- 766 (1988)) or a bandage according to methods known in the arts, to increase the efficiency of delivery of the drug to the areas to be treated.
  • the topical formulations can have additional excipients for example; preservatives such as methylparaben, benzyl alcohol, sorbic acid or quaternary ammonium compound; stabUizers such as EDTA, antioxidants such as butylated hydroxytoluene or butylated hydroxanisole, and buffers such as citrate and phosphate.
  • preservatives such as methylparaben, benzyl alcohol, sorbic acid or quaternary ammonium compound
  • stabUizers such as EDTA
  • antioxidants such as butylated hydroxytoluene or butylated hydroxanisole
  • buffers such as citrate and phosphate.
  • the pharmaceutical composition can be administered in an oral formulation in the form of tablets, capsules or solutions.
  • An effective amount of the oral formulation is administered to patients 1 to 3 times daUy until the symptoms of the proliferative disease, cancer or photoageing etc are alleviated.
  • the effective amount of agent depends on the age, weight and condition of a patient.
  • the dahy oral dose of agent is less than 1200 mg, and more than 100 mg.
  • the prefe ⁇ ed daily oral dose is about 300-600 mg.
  • Oral formulations are conveniently presented in a unit dosage form and may be prepared by any method known in the art of pharmacy.
  • the composition may be formulated together with a suitable pharmaceuticaUy acceptable carrier into any desired dosage form.
  • Typical unit dosage forms include tablets, piUs, powders, solutions, suspensions, emulsions, granules, capsules, suppositories.
  • the formulations are prepared by umformly and intimately bringing into association the agent composition with hquid carriers or finely divided solid carriers or both, and as necessary, shaping the product.
  • the active ingredient can be inco ⁇ orated into a variety of basic materials in the form of a liquid, powder, tablets or capsules to give an effective amount of active ingredient to treat skin prohferation disease.
  • Other therapeutic agents suitable for use herein are any compatible drugs that are effective for the intended purpose, or drags that are complementary to the agent formulation.
  • the treatment with an formulation can be combined with other treatments such as a topical tieatment with corticosteroids, calcipotrine, coal tar preparations, a systemic treatment with methotrexate, retinoids, cyclosporin A and photochemotherapy.
  • the combined treatment is especially important for treatment of an acute or a severe skin proliferation disease.
  • the formulation utilized in a combination therapy may be administered simultaneously, or sequentiaUy with other treatment, such that a combined effect is achieved.
  • Paragraph 1 A method of aUeviating or preventing a tachyphylactic response to a drag in a patient, the method comprising administering to the patient an antagonist of a metabohc enzyme which is induced as a result of exposure of the patient to the drag, in which the enzyme activity is capable of metabohsing the drug.
  • Paragraph 2 An antagonist of a metabohc enzyme for use in a method of aUeviating or preventing a tachyphylactic response to a drag in a patient, in which the metabohc enzyme is induced as a result of exposure of the patient to the drag.
  • Paragraph 3 A method according to Paragraph 1, or an antagonist of a metabolic enzyme according to Paragraph 2 for a use as specified therein, in which the antagonist inhibits the breakdown of the drug by decreasing the amount or activity, or both, of the metabolic enzyme.
  • Paragraph 6 A method or an antagonist according to any preceding Paragraph, in which the antagonist is administered locaUy to a part of the patient which is exhibiting a tachyphylactic response, or a symptom of the disease being treated by the drug, or both.
  • Paragraph 7 A method or an antagonist according to Paragraph 6, in which the effects of the antagonist are substantiaUy restricted to the part of the patient to which the antagonist is admimstered.
  • Paragraph 8 A method or an antagonist according to Paragraph 7, in which the metabolic breakdown of the drag in other parts of the patient are not substantiaUy reduced.
  • Paragraph 9 A method or an antagonist according to any preceding Paragraph, in which the antagonist is administered to the skin of the patient.
  • Paragraph 10 A method or an antagonist according to any preceding Paragraph, in which the antagonist is not capable of crossing the basement membrane of the epidermis.
  • Paragraph 11 A method or an antagonist according to any preceding Paragraph, in which the antagonist is administered to a patient aheady exhibiting a tachyphylactic response
  • Paragraph 12 A method or an antagonist according to any preceding Paragraph, in which the antagonist is administered simultaneously with the drug.
  • Paragraph 13 A method or an antagonist according to any preceding Paragraph, in which the drag is selected from the group consisting of: a steroid, a topical steroid and a corticosteroid, together with natural or artificial analogues of any of these.
  • Paragraph 14 A method or an antagonist according to any preceding Paragraph, in which the drag is a macrolactam.
  • Paragraph 15 A method or an antagonist according to any preceding Paragraph, in which drag is a vitamin D analogue selected from the group consisting of: Calcitriol (la,25(OH) 2 D 3 , la,25-dihydroxyvitamin D3, 1,25-dihyroxycholecalciferol), Calcipotriol (Dovanex; MC903) and Tacalcitol (la,24(R)-dihydroxyvitaminD 3 ; Curatoderm).
  • a vitamin D analogue selected from the group consisting of: Calcitriol (la,25(OH) 2 D 3 , la,25-dihydroxyvitamin D3, 1,25-dihyroxycholecalciferol), Calcipotriol (Dovanex; MC903) and Tacalcitol (la,24(R)-dihydroxyvitaminD 3 ; Curatoderm).
  • Paragraph 16 A method or an antagonist according to any preceding Paragraph, in which the metabohc enzyme is a P450 cytochrome.
  • Paragraph 17 A method or an antagonist according to any preceding Paragraph, in which the metabolic enzyme is a P450 cytochrome selected from the group consisting of: CYPIAI, CYP1A2, CYP24, CYP26, CYP27A1, CYP27B1, CYP2A6, CYP2B4, CYP2C3, CYP2C8, CYP2C9, CYP2D6, CYP2E1, CYP2E2, CYP3A4, CYP3A5, CYP3A6 and CYP3G1.
  • the metabolic enzyme is a P450 cytochrome selected from the group consisting of: CYPIAI, CYP1A2, CYP24, CYP26, CYP27A1, CYP27B1, CYP2A6, CYP2B4, CYP2C3, CYP2C8, CYP2C9, CYP2D6, CYP2E1, CYP2E2, CYP3A
  • Paragraph 18 A method of detecting the likelihood of a patient developing a tachyphylactic response to a drug, the method comprising the steps of: administering the drag to a ceU of the patient; and detecting the amount and/or activity of a metabohc enzyme capable of metabohsing the drag, in which an increased amount or activity indicates that the patient is likely to develop a tachyphylactic response.
  • Paragraph 19 A method of identifying an agent which is capable of reducing a tachyphylactic response to a drag in a patient, the method comprising the steps of: Paragraph (a) providing a ceU from the patient;
  • Paragraph 20 A method of relieving tachyphylaxis in a patient induced by adrninistration of a drag, in which the drag induces activation of a metabohc pathway which leads to the breakdown of the drag, the method comprising admimstering an antagonist of an enzyme in the pathway.
  • Paragraph 21 A pharmaceutical composition for the aUeviation or prevention of a tachyphylactic response to a drag in a patient, the pharmaceutical composition comprising an inhibitor of a metabohc enzyme together with a pharmaceutically acceptable carrier or dhuent.
  • Paragraph 22 A pharmaceutical composition according to Paragraph 21, further comprising the drug.
  • Paragraph 23 A pharmaceutical composition according to Paragraph 21 or Paragraph
  • the drag is a steroid, a topical steroid, a corticosteroid, a macrolactam or a vitamin D analogue selected from the group consisting of: 1,25-dihyroxycholecalciferol (Calcitriol), Calcipotriol (Dovanex) and Tacalcitol (Curatiderm).
  • Paragraph 24 A pharmaceutical composition according to any of Paragraphs 21 to 23, in which the metabohc enzyme activity is a P450 cytochrome selected from the group consisting of: CYPIAI, CYP1A2, CYP24, CYP26, CYP27A1, CYP27B1, CYP2A6, CYP2B4, CYP2C3, CYP2C8, CYP2C9, CYP2D6, CYP2E1, CYP2E2, CYP3A4, CYP3A5, CYP3A6 and CYP3Gl.
  • a P450 cytochrome selected from the group consisting of: CYPIAI, CYP1A2, CYP24, CYP26, CYP27A1, CYP27B1, CYP2A6, CYP2B4, CYP2C3, CYP2C8, CYP2C9, CYP2D6, CYP2E1, CYP2E2, CYP3
  • Paragraph 25 Use of an antagonist of a metabohc enzyme in the aUeviation or prevention of a tachyphylactic response to a drag in a patient.
  • Paragraph 26 Use of an antagomst of a metabolic enzyme in a method of preparation of a medicament for the alleviation or prevention of a tachyphylactic response to a drug in a patient.
  • Paragraph 27 A method of aUeviating or preventing a side effect associated with adrninistration of a drag to a patient, the method comprising administering to a patient an antagonist of a metabohc enzyme which is induced as a result of exposure of the patient to the drag.
  • Paragraph 28 A method of identifying an agent capable of aUeviating or preventing a side effect associated with administration of a drag to a patient, the method comprising identifying a metabohc enzyme which is induced by exposure of the patient to the drag, and identifying an antagonist of the metabohc enzyme.
  • Paragraph 29 A metabolic enzyme antagonist for use in a method of aUeviating or preventing a side effect associated with administration of a drug to a patient.
  • Paragraph 30 A method according to Paragraph 27 or 28, or a metabolic enzyme antagonist according to Paragraph 29, in which the side effect is caused by a metabohc product of the metabohc enzyme acting on the drag.
  • Paragraph 31 A method or a metabohc enzyme antagonist according to any of
  • Paragraph 32. A method or a metabohc enzyme antagonist according to any of Paragraphs 27 to 31, in which the side effect is cutaneous irritation, itching, cutaneous atrophy, papulopustular reaction or striae.
  • Paragraph 33 A method for the treatment or prophylaxis of a disease, the method comprising the steps of: administering an antagonist of a metabohc enzyme to the patient, in which the metabolic enzyme is an enzyme which is induced as a result of exposure of the patient to a drag which is known or suspected to be effective in treating the disease, and in which the metabolic enzyme is capable of metabohsing the drag.
  • Paragraph 34 A method according to Paragraph 33, in which the disease is psoriasis and the metabolic enzyme antagomst is an antagonist of CYP24.
  • Paragraph 35 A method of identifying an agent suitable for treatment or prophylaxis of a disease, the method comprising the steps of: identifying a metabohc enzyme which is induced as a result of exposure of a patient to a drug, in which the drag is known or suspected to be suitable for treating the disease, the metabohc enzyme being capable of metabohsing the drug; and identifying an antagomst of the metabohc enzyme.
  • the Examples demonstrate in part the reduction of tachyphylaxis induced by treatment of a patient with vitamin D analogues and corticosteroids through inhibition of P450 enzymes.
  • Examples 1 and 2 describe the identification of the catabolic enzymes responsible for catabohsm of vitamin D analogues in human skin (keratinocytes and lymphocytes). We use specific and redundant P450-based RT-PCR on human volunteers with tachyphylaxis against a vitamin D analogue.
  • Skin biopsies (10 mm 2 ) are taken from human psoriatic volunteers exhibiting tachyphylaxis against vitamin D analogues after three or six weeks of twice daUy treatment with 5 ⁇ g g ointment Calcitriol, 50 ⁇ g/g ointment Calcipotriol, or 4 ⁇ g/g ointment Tacalcitol.
  • SimUar biopsies are taken from normal healthy volunteers.
  • RNA is isolated from the skin biopsies using the RNeasy kit according to the instruction of the supplier (Qiagen Inc.). Isolated RNA is subjected to semi-quantitative RT-PCR using GeneAmp Gold RNA PCR Reagent Kit (PE Biosystems, Norwalk, Conn. USA) according to manufacturer's instructions.
  • Primers used are specific for CYPIAI, CYP1A2, CYP24, CYP26, CYP27A1, CYP27B1, CYP2A6, CYP2C8, CYP2C9, CYP2D6, CYP2E1, CYP3A4, CYP3A5 and CYP3A6, and are shown in Table 1.
  • Human CYP2B4, CYP2C3 and CYP2E2 are cloned using the co ⁇ esponding rabbit sequences as probes, and primers designed to amplify these three genes.
  • the RT-PCR shows that expression of CYP24 is increased in psoriatic patients exhibiting tachyphylaxis against Calcitriol as compared to controls.
  • the RT-PCR shows that expression of CYP24 is increased in psoriatic patients exhibiting tachyphylaxis against Tacalcitol as compared to controls.
  • the RT-PCR also shows that expression of CYP2E1 is increased in psoriatic patients exhibiting tachyphylaxis against Calcipotriol as compared to controls. Tests on the biopsy of a patient treated for 3 weeks with Calcipotriol cream showed an amplification product of 120 bp, the expected size.
  • pan-specific CYP primers based on regions conserved throughout the P450 family are also used to isolate other CYPs up-regulated by the vitamin D analogue treatment.
  • the pan-specific primers are designed against human CYP sequences, positions approximately 50 - 150 downstream of ATG, and have the following sequences. Sense - CCAGTCGCVA GCBDCGGAADCC; anti-sense - AABTCCATATNNAGHHAGT.
  • the expected size of amplified PCR fragments is approximately 100 base-pairs.
  • DNA fragments are RT-PCR amplified using the pan-specific CYP primers in skin biopsies of patients treated with Calcitriol. Sequencing and comparison to the GenBank database identifies these PCR products as corresponding to CYP24.
  • DNA fragments are RT-PCR amplified using the pan-specific CYP primers in skin biopsies of patients tieated with Tacalcitol. Sequencing and comparison to the GenBank database identifies these PCR products as co ⁇ esponding to CYP24.
  • DNA fragments are RT-PCR amplified using the pan-specific CYP primers in skin biopsies of patients treated with Calcipotriol. Sequencing and comparison to the GenBank database identifies these PCR products as co ⁇ esponding to CYP2E1. The results are verified by use of specific primers based on the known sequences deposited in GenBank, as described above. The various metabolic enzymes are therefore shown to be up-regulated by the different vitamin D analogue treatments.
  • SE cultures Organotypic skin equivalent (SE) cultures are established from human keratinocytes and fibroblasts essentially as described (Matsukova E. et al., Folia Biologica 44:59-66, 1998 / Tsunenaga M. et al., Jpn. J. Cancer Res. 85:238-44, 1994). Separate SE cultures are established and then treated with 500ng Calcipotriol; 400ng Tacalcitol or 150ng l , 24(R) dihydroxyvitamin D 5 (Calcitrol); or vehicle contiol. The drags are applied every 2 days in lO ⁇ L vehicle and spread over the entire air-exposed surface. The SE cultures are exposed to each drag treatment and the SE cultures are withdrawn for analysis after 1, 4 and 14 days.
  • the RT-PCR shows that expression of CYP24 is increased in skin equivalents exposed to Calcitriol as compared to controls.
  • the RT-PCR shows that expression of CYP24 is increased in skin equivalents exposed to Tacalcitol as compared to controls.
  • the RT-PCR also shows that expression of CYP2E1 is increased in skin equivalents exposed to Calcipotriol as compared to contiols, showing an amphfication product of 120 bp, the expected size.
  • Lanes 23 and 24 show amplification of a 120bp product, the expected size, with PCR at an annealing temperature of 58oC.
  • Calcipotriol up-regulates CYP2E1
  • Lanes marked - are the negative contiol (water) and lanes marked + are the positive control (G3PDH).
  • the results using redundant PCR confirm the results obtained from specific PCR, viz, exposure of skin equivalents to Calcitriol up-regulates expression of CYP24 as compared to controls, exposure of skin equivalents to Tacalcitol up-regulates expression of CYP24 as compared to controls, and exposure of skin equivalents to Calcipotriol up-regulates expression of CYP2E1 as compared to controls.
  • Examples 3 and 4 describe the identification of the catabolic enzymes responsible for catabohsm of corticosteroids in human skin (keratinocytes and lymphocytes).
  • Sin biopsies (10 mm 2 ) are taken from human psoriatic volunteers exhibiting tachyphylaxis after 4 weeks of twice daily treatment with 0.05% ointment Clobetasol or 0.1% ointment Dexamethasone.
  • SimUar biopsies are taken from healthy volunteers.
  • RNA is isolated from the skin biopsies using the RNeasy kit according to the instruction of the supplier (Qiagen Inc.).
  • Isolated RNA is subjected to semi-quantitative RT-PCR using GeneAmp Gold RNA PCR Reagent Kit (PE Biosystems, Norwalk, Conn. USA) according to manufacturer's instructions.
  • Primers used are specific for CYPIAI, CYP1A2, CYP2A6, CYP2C8, CYP2C9, CYP2D6, CYP2E1, CYP3A4, CYP3A5 and CYP3A6 and are shown in Table 1.
  • Human CYP2B4, CYP2C3 and CYP2E2 are cloned using the co ⁇ esponding rabbit sequences as probes, and primers designed to amplify these three genes.
  • the metabolic enzymes up-regulated by exposure to the various corticosteroids are identified, by virtue of the sizes of amphfication products and confirmed by DNA sequencing.
  • Pan-specific CYP primers based on regions conserved throughout the P450 family are also used to RT-PCR amplify and isolate other CYP's up-regulated by the corticosteroid treatment.
  • Sequencing and comparison to the GenBank database identified the PCR products, and accordingly the metabolic enzymes which are up-regulated.. They are verified to be up-regulated by the corticosteroid treatment as above using specific primers based on the known sequences deposited in GenBank.
  • SE cultures Organotypic skin equivalent (SE) cultures are established from human keratinocytes and fibroblasts essentially as described (Matsukova E. et al, Foha Biologica 44:59-66, 1998 / Tsunenaga M. et al., Jpn. J. Cancer Res. 85:238-44, 1994). Separate SE cultures are established and then treated with 5ng Clobetasol; lOng Dexamethasone or vehicle control. The drags are applied every 2 days in lO ⁇ L vehicle and spread over the entire air-exposed surface. 9 SE cultures are exposed to each drag treatment and 3 SE cultures are withdrawn for analysis after 1, 4 and 14 days.
  • the metabolic enzymes up-regulated by exposure to the various corticosteroids are identified, by virtue of the sizes of amphfication products and confirmed by DNA sequencing.
  • corticosteroid responsive CYP's are fuUy induced by 7 days of treatment.
  • Examples 5 and 6 describe the identification of the catabolic enzymes responsible for catabohsm of macrolactams in human skin (keratinocytes and lymphocytes).
  • keratinocytes and lymphocytes We use redundant P450-based RT-PCR on human volunteers with tachyphylaxis against macrolactams, in particular Tacrolimus, Ascomycin and Cyclosporin.
  • Isolated RNA is subjected to semi-quantitative RT-PCR using GeneAmp Gold RNA PCR Reagent Kit (PE Biosystems, Norwalk, Conn. USA) according to manufacturer's instructions.
  • CYP2D6, CYP2E1, CYP3A4, CYP3A5 and CYP3A6 are shown in Table 1.
  • the RT-PCR shows that expression of CYPIAI is induced by Tacrolimus.
  • CYPIAI is seen to be up-regulated by exposure to Cyclosporin as compared to controls.
  • Pan-specific CYP primers based on regions conserved throughout the P450 family are also used to isolate other CYP's up-regulated by the macrolactam treatment.
  • CYPIAI is induced by Tacrolimus, and that CYPIAI is up-regulated by exposure to Cyclosporin as compared to controls.
  • SE cultures Organotypic skin equivalent (SE) cultures are established from human keratinocytes and fibroblasts essentially as described (Matsukova E. et al., Foha Biologica 44:59-66, 1998 / Tsunenaga M. et al., Jpn. J. Cancer Res. 85:238-44, 1994). Separate SE cultures are established and then tieated with 30ng Tacrolimus; 40ng Ascomycin; 15ng Cyclosporin or vehicle control. The drags are applied every 2 days in lO ⁇ L vehicle and spread over the entire air-exposed surface. 9 SE cultures are exposed to each drug treatment and 3 SE cultures are withdrawn for analysis after 1, 4 and 14 days.
  • RT-PCR assays The results of RT-PCR assays are shown in Figure 1.
  • Amphfication using CYPIAI specific primers at an annealing temperature of 58 degrees C shows co ⁇ ect amphfication of a 393 bp sample in samples cultured with tacrolimus or cyclosporin.
  • Lanes 33 and 34 show that expression of CYPIAI is induced by Tacrolimus.
  • CYPIAI is seen to be up- regulated by exposure to Cyclosporin as compared to contiols, as shown in Lanes 37 and 38.
  • CYPIAI is shown to be up-regulated by tacrolimus and cyclosporin. These enzymes are verified to be up-regulated by the macrolactam treatment as above using specific primers based on the known sequences deposited in GenBank. AU macrolactam- responsive CYP's are fully induced by 7 days of treatment.
  • Inhibitors of metabolic enzymes may be used to alleviate or reheve tachyphylaxis against macrolactams, in particular tacrolimus and cyclosporin.
  • Compounds A, B and C have the foUowing structures: Compound A: N-[4-cUorobenzoyl]-2-(lH-imidazol-l-yl)-2-(phenyl)-l-amino ethane; Compound B: N-[4-cMorobenzoyl]-2-(lH-imidazol-l-yl)-2,2-(di-4-chlorophenyl)-l- aminoethane (SDZ 89-443, described in Schuster et al.
  • This example shows the reduction/aUeviation of tachyphylaxis to vitamin D analogues by co-treatment with inhibitors of the catabolic enzyme(s) in organotypic skin equivalents (SE).
  • SE organotypic skin equivalents
  • SE cultures are tieated with vehicle with or without vitamin D analogue or an inhibitor of CYPIAI, CYP1A2, CYP24, CYP26, CYP27A1, CYP27B1, CYP2A6, CYP2B4, CYP2C3, CYP2C8, CYP2C9, CYP2D6, CYP2E1, CYP2E2, CYP3A4, CYP3A5, CYP3A6 and CYP3G1 for 7 days as in Example 2 above.
  • the SE cultures are challenged on day 5 with hype ⁇ roliferative stimuli by adding 1 ⁇ M retinoic acid or nothing (control) to the media.
  • the SE cultures are maintained for 2 more day before analysis
  • VDR vitamin D receptor
  • CYP24 p21 cipl Wafl CYP24 p21 cipl Wafl
  • c-Fos ⁇ 3- integrin
  • Skin irritation is a facet of tachyphylaxis and the expression of irritation markers such as ILl- ⁇ ; IL-6; IL-8; TNF- ⁇ ; GM-CSF; TGF- ⁇ is examined by RT-PCR as above.
  • vitamin D analogue-catabohzing enzymes are up-regulated by vitamin D analogue tieatment (see Examples above).
  • SE cultured in the continuous presence of vitamin D analogues respond to the hype ⁇ roliferative challenge by increased expression of the proliferation markers Cyclin-A, K5, K6, K14, MDM2 and decreased expression of the differentiation markers Involucrine, Kl, K10. This demonstiates that the antiprohferative effect of the vilamin D analogues is blunted by a tachyphylactic response, up-regulation of vitamin D analogue-cat ⁇ bolizing CYP's.
  • the response to the hype ⁇ rohferative chaUenge is abohshed when CYP inhibitors) are co-administered with the vitamin D analogues.
  • the expression of the vitamin D receptor is not down-regulated by the vitamin D analogue or CYP-inhibitor treatment. This demonstrates that tachyphylaxis to vitamin D analogues can be abolished by CYP inhibitors adjunct to the vitamin D analogue regime.
  • continuous vitamin D analogue treatment results in upregulation of ILl- ⁇ ; IL-6; IL-8; TNF- ⁇ ; GM-CSF; TGF- ⁇ , markers of skin irritation. This response is diminished by co-administration of CYP inhibitors with the vitamin D analogues.
  • CYP24 is upregulated in the presence of calcitriol and further upregulated by a combination of calcitriol and inhibitors (ketoconazole or compound A-C).
  • Kl is not upregulated by calcitriol alone but is upregulated by a combination of calcitriol and inhibitors (ketoconazole or compound A-C).
  • CYP24 is upregulated by tacalcitol in the presence of ketoconazole.
  • CYP24 is not upregulated by Calcipotriol with or without inhibitors.
  • Amphfication of 645bp occurs in samples cultured in calcitriol and compound B and samples 19 and 20- cultured in calcitriol and compound A, showing that these CYP24 specific inhibitors increase the abihty of these skin equivalents to differentiate, by preventing the degradation of calcitiol. This effect of CYP24 inhibitors is also noted in keratinocyte cultures.
  • Tacalcitol +/- inhibitors shows similar effects.
  • Inhibition of CYP24 may therefore be used to inhibit the degradation of calcitriol and tacalcitol and hence inhibit tachyphylaxis to these compounds.
  • Amplification occurs in samples cultured with calcitriol and the inhibitors ketoconazole and compound A-C (particularly compound C). Inhibition of CYP IB 1 wiU therefore inhibit the degradation of calcitriol and hence inhibit tachyphylaxis to this compounds. Inhibitors of CYP IB 1 may therefore be used to reduce or aUeviate tachyphylaxis against calcitriol.
  • the CYP24 inhibitors Compound A, B and C however show no irritancy.
  • This example shows the reduction/aUeviation of tachyphylaxis to corticosteroids by co- treatment with inhibitors of the catabolic enzyme(s) in organotypic skin equivalents (SE).
  • SE organotypic skin equivalents
  • SE cultures are treated with vehicle with or without corticosteroids or an inhibitor of CYPIAI, CYP1A2, CYP24, CYP26, CYP27A1, CYP27B1, CYP2A6, CYP2B4, CYP2C3, CYP2C8, CYP2C9, CYP2D6, CYP2E1, CYP2E2, CYP3A4, CYP3A5, CYP3A6 and CYP3G1 for 7 days as in Example 2 above.
  • the SE cultures are challenged on day 5 with hype ⁇ roliferative stimuh by adding 1 ⁇ M retinoic acid or nothing (contiol) to the media. The SE cultures are maintained for 2 more day before analysis.
  • Markers of keratinocyte prohferation and differentiation are examined by semi- quantitative RT-PCR on the isolated RNA using PCR primers specific for Cyclin-A, Involucrine, Kl, K5, K6, K10, K14, MDM2 essentially as for the CYP's above.
  • PCR primers specific for Cyclin-A, Involucrine, Kl, K5, K6, K10, K14, MDM2 essentially as for the CYP's above.
  • Primers are designed to amplify various glucocorticoid target genes (for example, see the
  • primers are designed against the foUowing genes (accession numbers in brackets): Alcohol dehydrogenese 2 class I gene (A00379), Glucose-6-phosphatase gene (A00876), Pepsinogen C gene (A00878), Glycoprotein hormone a -subunit gene (A00056), Growth hormone gene-1 (A00070), Insulin receptor gene (A00170), Interleukin-6 gene (A00115), Metallothionein UA gene (A00071), Insulin-like growth factor binding protein 1 gene (A00073), Osteocalcin gene (A00364), and the Elastin gene (A00026).
  • Primers designed to amplify mineralocorticoid (aldosterone) target genes are also designed against the foUowing: Channel-inducing factor (Brennan FE. FuUer PJ. Endocrinology. 140(3): 1213-8, 1999 Mar.), Alpha 3-subunit isoform of Na(+)-K(+)- adenosinetriphosphatase (Farman N. Bonvalet JP. Seckl JR. American Journal of Physiology. 266(2 Pt l):C423-8, 1994 Feb.) Seifrie-threonine kinase SGK (Pearce D. Ve ⁇ ey F. Chen SY. Mastioberardrno L. Meijer OC. Wang J. Bhargava A. Kidney International. 57(4): 1283-9, 2000 Apr.)
  • Skin irritation is a facet of tachyphylaxis and the expression of irritation markers such as ILl- ⁇ ; IL-6; IL-8; TNF- ⁇ ; GM-CSF; TGF- ⁇ are examined by RT-PCR as above.
  • corticosteroid- catabolizing enzymes are up-regulated by corticosteroid treatment (see above).
  • SE cultured in the continuos presence of corticosteroids responds to the hype ⁇ roliferative chaUenge by increased expression of the proliferation markers Cyclin-A, K5, K6, K14, MDM2 and decreased expression of the differentiation markers Involucrine, Kl, K10 demonstrating that the antiprohferative effect of the corticosteroids is blunted by a tachyphylactic response, up-regulation of corticosteroid-catabolizing CYP's.
  • the response to the hype ⁇ rohferative chaUenge is abohshed when CYP inhibitors) are co-a ⁇ rninistered with the corticosteroids.
  • the expression of the glucocorticoid or mineralocorticoid receptors are not down-regulated by the corticosteroid or CYP-inhibitor tieatment. This demonstiates that tachyphylaxis to corticosteroids can be abolished by CYP inhibitors adjunct to the corticosteroid regime.
  • continuous corticosteroid treatment resulted in up-regulation of ILl- ⁇ ; IL- 6; IL-8; TNF- ⁇ ; GM-CSF; TGF- ⁇ , markers of skin irritation.
  • This example shows the reduction/aUeviation of tachyphylaxis to macrolactams by co- treatment with inhibitors of the catabolic enzyme(s) in organotypic skin equivalents (SE).
  • SE organotypic skin equivalents
  • SE cultures are tieated with vehicle with or without macrolactams or an inhibitor of CYPIAI, CYP1A2, CYP24, CYP26, CP27A1, CYP27B1, CYP2A6, CYP2B4, CYP2C3, CYP2C8, CYP2C9, CYP2D6, CYP2E1, CYP2E2, CYP3A4, CYP3A5, CYP3A6 and CYP3G1 for 7 days as in Example 2 above.
  • the SE cultures are challenged on day 5 with hype ⁇ roliferative stimuh by adding 1 ⁇ M retinoic acid or nothing (control) to the media.
  • the SE cultures are maintained for 2 more day before analysis.
  • Markers of keratinocyte prohferation and differentiation are examined by semi- quantitative RT-PCR on the isolated RNA using PCR primers specific for Cyclin-A, Involucrine, Kl, K5, K6, K10, K14, MDM2 essentially as for the CYP's above.
  • Skin irritation is a facet of tachyphylaxis and the expression of irritation markers such as ILl- ⁇ ; IL-6; IL-8; TNF- ⁇ ; GM- CSF; TGF- ⁇ are examined by RT-PCR as above.
  • the macrolactam -catabolizing enzyme CYPIAI is up-regulated by macrolactam tieatment (see above).
  • SE cultured in the continuous presence of macrolactams responded to the hype ⁇ roliferative challenge by increased expression of the proliferation markers Cyclin-A, K5, K6, Kl 4, MDM2 and decreased expression of the differentiation markers Involucrine, Kl , Kl 0 demonstrating that the antiprohferative effect of the macrolactam's is blunted by a tachyphylactic response, up-regulation of macrolactam-catabolizing CYP's.
  • Nude mice are topically exposed to vitamin D analogue, corticosteroid or Macrolactam for 6 weeks (42 days) aUowing suitable induction of metabohc enzymes as determined in the Examples above. Induction of enzymes and irritant response to treatment is compared with a group of mice similarly exposed but co-administered with the enzyme inhibitor determined in the relevant above examples.
  • Ajnirnals exposed to both drag and adjunct enzyme inhibitor show a persistent clinical response to drug treatment with lower associated irritancy and no tachyphylactic response as compared to animals tieated with drug alone.
  • vitamin D analogue-catabolizing enzymes CYP24 and CYP2E1 are upregulated by vitamin D analogue treatment.
  • Nude mice treated with vitamin D analogues have significantly higher expression of proliferation markers Cyclin-A, K5, K6, K14, MDM2 and decreased expression of the differentiation markers Involucrine, Kl, K10 compared to nude mice treated with both vitamin D analogs and CYP inhibitor.
  • This demonstrates that the antiprohferative effect of the vitamin D analogues is blunted by a tachyphylactic response, up-regulation of vitamin D analogue- catabolizing CYP's.
  • the expression of the vitamin D receptor is not down-regulated by the vitamin D analogue or CYP-inhibitor treatment as compared to vehicle treatment. This demonstrates that tachyphylaxis to vitamin D analogues can be abohshed by CYP inhibitors adjunct to the vitamin D analogue regime. Furthermore, continuous vitamin D analogue tieatment results in upregulation of ILl- ⁇ ; IL-6; IL-8; TNF- ⁇ ; GM-CSF; TGF- ⁇ , markers of skin irritation. This response is diminished by co-administration of CYP inhibitors with the vitamin D analogues.
  • nude mice exposed to both vitamin D analogue and adjunct enzyme inhibitor are found to show a persistent anti-proliferative and pro-differentiation response to vitamin D analogue treatment with lower associated irritancy and no tachyphylactic response.
  • the mouse taU model is a mo ⁇ hometiy-based, sensitive and reproducible method for the quantitative evaluation of the effects of drugs on epithehal differentiation and induction of orthokeratosis.
  • Orthokeratosis is determined by measuring the horizontal length of the fully developed granular layer within an individual scale in relation to its total length. (Sebok B, et al., Skin Pharmacol. Appl. Skin Physiol. 13:285-91, 2000) Mice are topically exposed to vitamin D analogue, Corticosteroids or Macrolactams for n-days with and without the CYP inhibitors.
  • mice are topically exposed to vitamin D analogue, Corticosteroids or Macrolactams for 14 days with and without the CYP inhibitors according to the foUowing protocol: 1. Vehicle alone, 2. 50mg/g ointment Calcipotriol, 3.40mg/g ointment Tacalcitol, 4. 50mg/g ointment Calcipotriol + 1% ketoconazole, 5. 40mg/g ointment Tacalcitol + 1% ketoconazole, 6. 1% ketoconazole, 7. 0.05% Clobetasol ointment, 8. 0.1% Dexamethasone ointment, 9. 0.05% Clobetasol ointment + 1% ketoconazole, 10.
  • Mouse tails exposed to both vitamin D analogs, corticosteroids or macrolactams and adjunct enzyme inhibitor are found to show an enhanced epithehal differentiation and orthokeratotic response with lower associated irritancy and no tachyphylactic response as compared to mouse taUs tieated with drug alone.
  • Mouse taUs treated with inhibitor alone show enhanced epithehal differentiation and orthokeratotic response compared to vehicle reated mouse taUs. This response is potentiated by an 25(OH)D 3 enriched diet.
  • Human normal primary keratinocyte and human psoriatic keratinocytes are cultured in defined media (retinoid and vitamin D free) in 0.1 or 1.2 mM CaCt in the presence or absence of 0.5 micromolar 25(OH)D 3 and in the presence or absence of 10 microM ketoconazole.
  • RT-PCR of differentiation markers is carried out as described above, and induction of the differentiation markers in ketocaonazole treated cells is observed.
  • the concentration of l,25(OH) 2 D 3 in the media is examined and found to be elevated in the presence of the inhibitors).
  • addition of 0.5 micromolar 25(OH)D 3 is found to elevate the expression of the differentiation markers, as well as the concentration of 1 ,25(OH) 2 D 3 in the media.
  • the Figure shows the results of RT-PCR performed on normal human keratinocytes cultured in the presence of calcitriol with or without out ketoconazole and CYP24 inhibitors (Compounds A-C).
  • PCR scans for CYP24 and Kl show that inhibition of CYP24 causes an upregulation in mRNA levels of CYP24 and an upregulation in mRNA for the differentiation marker Kl .
  • a tachyphylactic response could be prevented by inhibition of CYP24.
  • a CYP24-specific inhibitor (and or 1% ketoconazole) is tested in the mouse-tail test.
  • the inhibitor is rubbed onto the taU in a dermatologicaUy suitable inert vehicle in the presence or absence of feeding on a 25(OH)D 3 enriched diet.
  • CYP24-specific inhibitor used comprise Compounds A, B, and C.
  • Kideney (Clara type II); Brain (esp. endothelial cells in choroid plexus); Digestive tract, duodenum > jejunum > Ueu colon > esophagus > stomach (esp. vUlous epitheha & ceUs surrounding glands in lamina propria); Kideney
  • Methoxalen (8-Methoxypsoralen)(2:nd) tSeUers EM et al.. Clinical Pharm. & Therapeutics 68:35-43, 2000
  • Liver (constitutive & inducible, esp centrilobular); Lung (Clara type 1 Duodenum (esp. viUous epithelia & cells su ⁇ ounding glands in lamina propria); Kideney (renal co ⁇ uscles) tPey A et al., Biochem. Pharm. 58:525-37, 1999
  • Acetaminophen (ring); Antipyrine (4,3-methyl); Bufuralol (1 and 4 others); Ondansetion (7,8); Phenacetin; Tacrine; Tamoxifen (N- demethylation); Theophyline (1,3,8); Warfarin
  • Methoxalen (8-Methoxypsoralen) TSeUers EM et al., Clinical Pharm. & Therapeutics 68:35-43, 2000
  • Valproate (broad spectrum inhibitor of CYP2C's)
  • HTV protease inhibitors (2:nd)(Ritonavir, hidinavir, Saquinavir) TBarrv Metal., Clinical Pharmacokinetics 32:194-209, 1997
  • Piclofenac (4) Hexobarbital (3), ibuprofen (methyl, 2), Losartan (aldehyde, acid),
  • Mefanamic acid (3-methyl), Mephobarbital, Phenytoin, Piroxicam, Tenoxicam, Tienilic acid,
  • CYP2C11 (constitutively expressed, male specific)
  • HJV protease inhibitors (2:nd)(Ritonavir, Indinavir, Saquinavir) TBarry Metal. Clinical Pharmacokinetics 32:194-209. 1997
  • Clarithromycin (l:st)(macrolide antibacterial, formation of active 14-hydroxyclarithromycin), Astemidole (l:st), Cisapride (l:st), Pimozide (l: Midazolam (l:st), Triazolam (l:st)
  • CYP3A4 (the main drag metabolizing CYP in humans)
  • HJV protease inhibitors (l:st)(Ritonavir, mdinavir, Saquinavir), Azole antifungals, Macrohde antibiotics, Dapsone
  • Ketoconazole, Itiaconazole, Fluconazole (weak inhibition) TVenkatakrishnan K etal.. Clinical Pharmacokinetics 38:111-180, 2000
  • Acetaminophen quinone formation
  • AlfentanU nodeethylation
  • Bayer R5417 [-K8644] (dihydropyridine)
  • Benzphetamine N-demethylation
  • Budesonide (6 ⁇ )
  • Codeine N-demethylati Cortisol
  • Cyclophosphamide high Km
  • Cyclosporin A (AM9, AMI, AM4 ⁇ : nomenclature formerly Ml, M17, M21)
  • Cyclosporin G Papsone (N), Pehydroepiandrosterone-3-sulfate (16 ⁇ ), Pextrometho ⁇ han (N-demethylation), Piazepam (3), PUtiazem, Ebastine (alcohol), CQA 206-291, Erythromycin (N-demethylation), 17
  • Midazolam (1,4), Nifedipine (dihydropyridine), NUudipine (dihydropyridine), Nimodipine (dihydropyridine), Nisoldipine (dihydropyridine), Nitrendipine (dihydropyridine), Omeprazole, Progesterone (6 ⁇ , some 16 ), Qunindine (3, N), Rapamycin (41, others), Sertindole (N- dealkylation), Sulfamethoxazole ( ), SulfentanU, Tamoxifen (N-demethylation), Taxol (2-phenyl), Terfenadine (t-butyl, N- deaUcylation), Testosterone (6 ⁇ , trace, 15 ⁇ , 2 ⁇ ), Triazolam, Trimethadone (N-demethylation), Troleanomycin ( ⁇ ), NerapamU, Warfarin (R-10, S-dehydro Zatosetion ( ⁇ ), Zonisamide (Sites of oxid
  • Kidney human, the main CYP3A isoform
  • Liver human, the main CYP3 A isoform in embryonic -> newborn hver
  • Keratinocytes Thuman THarant H et al, J. Cell. Biochem. 78: 112-20. 2000
  • UGT Uridine diphosphate GlucuronosylTransferase

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Abstract

L'invention concerne un procédé permettant d'alléger ou de prévenir chez un individu une réponse tachyphylactique à un agent. Le procédé consiste à administrer à l'individu un antagoniste d'une enzyme métabolique induite suite à l'exposition de l'individu à l'agent, procédé selon lequel l'activité enzymatique est capable de métaboliser l'agent.
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WO2003045381A1 (fr) * 2001-11-27 2003-06-05 Molecular Skincare Limited Derives d'aminoalkylimidazole et leur utilisation dans le domaine medical
WO2003047594A1 (fr) * 2001-11-29 2003-06-12 Molecular Skincare Limited Traitement therapeutique utilisant deux composes associes, l'un de ces composes induisant une reponse de tachyphylaxie qui n'affecte pas l'autre compose
WO2006032299A1 (fr) * 2004-09-24 2006-03-30 Susanna Miettinen Utilisation d'inhibiteurs de 24-hydroxylase dans le traitement d'un cancer
US7334968B2 (en) * 2002-02-22 2008-02-26 Jennmar Corporation Yieldable prop
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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003045381A1 (fr) * 2001-11-27 2003-06-05 Molecular Skincare Limited Derives d'aminoalkylimidazole et leur utilisation dans le domaine medical
WO2003047594A1 (fr) * 2001-11-29 2003-06-12 Molecular Skincare Limited Traitement therapeutique utilisant deux composes associes, l'un de ces composes induisant une reponse de tachyphylaxie qui n'affecte pas l'autre compose
US7334968B2 (en) * 2002-02-22 2008-02-26 Jennmar Corporation Yieldable prop
WO2006032299A1 (fr) * 2004-09-24 2006-03-30 Susanna Miettinen Utilisation d'inhibiteurs de 24-hydroxylase dans le traitement d'un cancer
US20150017138A1 (en) * 2007-06-15 2015-01-15 Beth Israel Deaconess Medical Center Bacterial mediated thf alpha gene silencing

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