WO2002045704A2 - Prevention and treatment of tachyphylactic response - Google Patents

Abstract

We describe a method of alleviating or preventing a tachyphylactic response to an agent in a individual, the method 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.

Description

DISEASE TREATMENT

FIELD

This invention relates generally to the treatment of diseases with agents, drugs and other therapeutics. In particular, 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.

BACKGROUND

In the last century, the leading causes of death were infectious diseases such as tuberculosis, influenza and pneumonia and water-borne diseases such as cholera and typhoid. Today, as a result of public health measures to clean up public water supplies and also as a result of immunisation programmes, as well as improvements in medical care, such diseases have been eradicated. Instead, chronic conditions such as cardiovascular disease (which includes heart disease and stroke), cancer, chronic lung disease, and emphysema have taken their place. These diseases are all considered "chronic" because the disease process takes years to take its toll. Included are other long term conditions such as AIDS (caused by BDN infection), hepatitis, etc. Other less dangerous chronic diseases include asthma, psoriasis, hypertension and sinusitis. Treatment of such chronic diseases often requires long-term administration of drugs or other therapeutic agents.

In such chronic drug treatment regimes where drugs are administered over an extended period of time, the development of tachyphylaxis may be a problem. Tachyphylaxis (also known as tolerance) is a falling-off in the effects produced by a drug during continuous use or constantly repeated administration. Thus, for example where continuous admrnistration of a drug is taking place, it has been observed both in vivo and in vitro that with the continuous drug administration of certain drugs, the patient develops tachyphylaxis to the drug. 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. L., et al., (1988) Ann. Rev. Cell Biol. 4:405-428; Lefkowitz, R. J., et al., (1980) Curr. Top. Cell. Regul. 17:205- 230). 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.

Where tachyphylaxis is a problem, alternating therapies may be used. For example, 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 (Lebwohl et al., 2000, J Am Acad Dermatol 43 (2 Pt 3): S43-6). However, it is known that treatment with 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).

SUMMARY

We have now found that, contrary to the assumptions in the reported literature, 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. According to a first aspect of the present invention, we provide a method of alleviating or preventing a tachyphylactic response to an agent in an individual, the method 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.

There is provided, according to a second aspect of the present invention, 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.

Preferably, the antagonist inhibits the breakdown of the agent by decreasing the amount or activity, or both, of the metabolic enzyme.

Preferably, 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.

Preferably, the effects of the antagonist are substantially restricted to the part of the individual to which the antagonist is administered.

Preferably, the metabolic breakdown of the agent in other parts of the individual are not substantially reduced.

Preferably, the antagonist is administered to the skin of the individual .

Preferably, the antagonist is not capable of crossing the basement membrane of the epidermis. Preferably, the antagonist is administered to a individual already exhibiting a tachyphylactic response

Preferably, the antagonist is administered simultaneously with the agent.

Preferably, 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.

Preferably, the agent comprises a macrolactam.

Preferably, the agent comprises a vitamin D analogue selected from the group consisting of: Calcitriol (la,25(OH)₂D₃, la,25-dihydroxyvitamin D3, 1,25-dihyroxycholecalciferol), Calcipotriol (Dovanex; MC903) and Tacalcitol (la,24(R)-dihydroxyvitemin D₃; Curatoderm).

Preferably, the metabolic enzyme comprises a P450 cytochrome.

Preferably, 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.

We provide, according to a third aspect of the present invention, a method of detecting the likelihood of a individual developing a tachyphylactic response to an agent, the method 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.

As a fourth aspect of the present invention, there is provided a method of identifying an molecule which is capable of reducing a tachyphylactic response to an agent in a individual , the method 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).

We provide, according to a fifth aspect of the present invention, 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.

Preferably, the pharmaceutical composition further comprises the agent.

Preferably, 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).

Preferably, 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.

In a seventh aspect of the present invention, there is provided use of an antagonist of a metabohc enzyme in the alleviation or prevention of a tachyphylactic response to an agent in a individual . According to an eighth aspect of the present invention, we provide use of 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 .

We provide, according to 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.

There is provided, in accordance with a tenth aspect of the present invention, a method of identifying an molecule capable of alleviating or preventing a side effect associated with administration of an agent to a individual , the method 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.

As an eleventh aspect of the invention, we provide 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 .

Preferably, the side effect is caused by a metabolic product of the metabolic enzyme acting on the agent.

Preferably, the metabohc product is a toxin.

Preferably, the side effect is cutaneous irritation, itching, cutaneous atrophy, papulopustular reaction or striae.

We provide, according to a twelfth aspect of the invention, there is provided a method for the treatment or prophylaxis of a disease, the method 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.

Preferably, the disease is psoriasis and the metabolic enzyme antagonist is an antagonist of CYP24.

According to a thirteenth aspect of the present invention, we provide method of identifying an molecule 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 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.

Preferably, the agent comprises a macrolactam, preferably Tacrolimus or Cyclosporin or both and the metabolic enzyme comprises CYPIAI.

Alternatively or in addition, the agent comprises a vitamin D analogue, preferably Calcipotriol, and the metabohc enzyme comprises CYP2E1.

Alternatively or in addition, the agent comprises a vitamin D analogue, preferably Calcitriol or Tacalcitol or both, and the metabohc enzyme comprises CYP24.

There is provided, according to a fourteenth aspect of the present invention, use of an inhibitor of CYPl Al, in the treatment or alleviation of tachyphylaxis against Tacrolimus or Cyclosporin or both, or an inhibitor of CYPl Al for such use.

We provide, according to a fifteenth aspect of the present invention, use of an inhibitor of CYP2E1, in the treatment or alleviation of tachyphylaxis against Calcipotriol, or an inhibitor of CYP2Elfor such use. As a sixteenth aspect of the present invention, there is provided use of an inhibitor of CYP24, in the treatment or alleviation of tachyphylaxis against Calcitriol or Tacalcitol or both, or an inhibitor of CYP24 for such use.

Preferably, the inhibitor comprises ketoconazole.

Preferably, the metabohc enzyme comprises CYP24, and 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).

BRIEF DESCRIPTION OF THE FIGURES

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. Key: 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 and 34: tacrolimus, Lanes 35 and 36: tacrolimus + ketoconazole, Lanes 37 and 38: cyclosporin, Lanes 39 and 40: cyclosporin + ketoconazole.

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 (lOμM), Lane 425(OH)D₃, Lane 5 25(OH)D₃ + compound A (300nM). Lower panel: amplification with Kl specific primers, Lane 1: calcitriol (12nM), Lane 2: calcitriol + ketoconazole (lOμM).

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 + ketoconazole, Lanes 27 and 28: calcipotriol + compound B, Lanes 29 and 30: calcipotriol + compound A, Lanes 31 and 32: calcipotriol + compound C, Lanes 33 and 34: tacrolimus, Lanes 35 36: taCTolfmus + ketoconazole, Lanes 37 and 38: cyclosporin , Lanes 39 and 40 : cyclosporin + ketoconazole. Lanes marked - are the negative control (water) and lanes marked + are the positive control (G3PDH).

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 π (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, Lanes 27 and 28: calcipotriol + compound B, Lanes 29 and 30: calcipotriol + compound A, Lanes 31 and 32: calcipotriol + compound C, Lanes 33 and 34: tacrolimus, Lanes 35 36: tacrolimus + ketoconazole, Lanes 37 and 38: cyclosporin , Lanes 39 and 40 : cyclosporin + ketoconazole. Lanes marked - are the negative control (water) and lanes marked + are the positive control (G3PDH).

DETAILED DESCRIPTION

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of chemistry, molecular biology, microbiology, recombinant DNA and immunology, which are within the capabilities of a person of ordinary skill in the art. Such techniques are explained in the literature. See, for example, J. Sambrook, E. F. Fritsch, and T. Maniatis, 1989, Molecular Cloning: A Laboratory Manual, Second Edition, Books 1-3, Cold Spring Harbor Laboratory Press; Ausubel, F. M. et al. (1995 and periodic supplements; Current Protocols in Molecular Biology, ch. 9, 13, and 16, John Wiley & Sons, New York, N.Y.); B. Roe, J. Crabtree, and A. Kahn, 1996, DNA Isolation and Sequencing: Essential Techniques, John Wiley & Sons; J. M. Polak and James O'D. McGee, 1990, In Situ Hybridization: Principles and Practice; Oxford University Press; M. J. Gait (Editor), 1984, Oligonucleotide Synthesis: A Practical Approach, Irl Press; and, D. M. J. Lilley and J. E. Dahlberg, 1992, Methods ofEnzymology: DNA Structure Part A: Synthesis and Physical Analysis of DNA Methods in Enzymology, Academic Press. Each of these general texts is herein incoφorated by reference.

TACHYPHYLAXIS

As noted above, where 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. In other words, 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". Continued administration of the drug causes the therapeutic and other effects to decrease to below the baseline level. In extreme cases of tachyphylaxis, 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".

Accordingly, 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. Preferably, 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.

We provide for a method of improving a therapeutic regime by substantially overcoming the development of tachyphylaxis to the drug or therapeutic compound which is applied, by the use of an agent, as described here. In a preferred embodiment of the invention, 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.

According to the methods and compositions described here, 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). Preferably, 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. Most preferably, the therapeutic effectiveness of the drag is increased to 100% of the baseline level, in other words, complete relief of tachyphylaxis.

Where the term "agent capable of degrading" a molecule (such as an a<iministered drug) is used, this term should be taken to mean any deactivating molecule which is capable of reducing the activity of that molecule. Such 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. In a patient exhibiting a tachyphylactic response to a drug which has been administered over a course of time, the therapeutic response of the patient to the drug declines (i.e., the drug is not as effective as on initial administration). Accordingly, 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. Thus, in particular, 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. Preferably, the metabohc enzyme is induced in the patient generally, or in particular in the cells which are in contact with the drug. By "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.

For example, exposure of the patient's cells to the drug may lead to increased expression of a gene encoding the metabohc enzyme, for example, increased transcription, translation, etc. Furthermore, 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. Similarly, where 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. TOPICAL APPLICATION

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.

The term "topical" is to be understood in its clinical sense as being synonymous with local application of a drag or agent. The methods and compositions described here are particularly effective when applied topically, although they need not be.

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. In topical administration, 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. Preferably, 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. For example, 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.

In a preferred embodiment, the drug or agent is not allowed substantially to exert its effects on other parts of the body of the patient. Thus, preferably 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. Thus, according to this particular preferred aspect, 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. In this embodiment, the metabohc enzyme antagonist is apphed to an area of skin which exhibits the symptoms of the disease being treated. For example, an antagonist of an enzyme responsible for breaking down vitamin D (e.g., an inhibitor of the cytochrome P450 enzyme CYP24 such as ketoconazole) is only applied to a psoriatic area of skin which is being treated, has been treated, or will be treated with a vitamin D analogue. Typically, 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. As explained below, 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. Accordingly, 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).

Thus, preferably, 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. In a highly preferred embodiment, 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 hypeφroliferative disease being treated by a vitamin D analogue.

Both general metabohc enzyme antagonists (such as ketoconazole) as well as specific antagonists of particular metabohc enzymes such as particular cytochrome P450s are suitable for local administration as described above.

The restriction of the antagonistic effects of the metabohc enzyme antagonist may be achieved in a number of ways. For example, the antagonist may be dehvered in the form of a Transfersome. Transfersomes (TM: IDEA, Munich, 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.

Furthermore, 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. For example, where it is desired to retain the metabolic enzyme antagonist within the epidermis, the ionic charge, hpophihcity, etc of the metabolic enzyme antagonist may be altered in order to be compatible with retention within the epidermis. Furthermore, 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. Alternatively, 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. To achieve this, 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.

It will further be appreciated that 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. Thus, for example, 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, however, 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.

An example is provided below for the treatment of skin diseases. 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. Accordingly, 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. Thus, 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. However, once 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. Thus, the benefits of local administration of the inhibitor to a part of the body (as described above) are achieved by utilising a specific inhibitor for a metabohc enzyme expressed only in that part of the body.

Similarly, 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

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 Coφoration, Munich, 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. Generally, 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.

Passage of Transfersome aggregates across the skin is a function of vesicle membrane flexibility, hydrophihcity, and the ability to retain vesicle integrity, while the aggregate undergoes a significant change in shape. When a suspension of Transfersome vesicles is placed on the surface of the skin, water evaporates from the relatively arid skin surface and the vesicles start to dry out. Due to the strong polarity of major Transfersome ingredients, the large number of hydrophilic groups on the membrane, assisted by the softness of the membrane, the vesicles are attracted to the areas of higher water content in the narrow gaps between adjoining cells in the skin barrier, enabling skin penetration of the vehicle. This, together with the vesicle's extreme ability to deform, enables Transfersome aggregates to open, temporarily, the tiny "cracks" through which water normally evaporates out of the skin. Channels between the skin cells, two orders of magnitude wider than the original micropores, are thus created. Such newly activated passages can accommodate sufficiently deformable vesicles, which maintain their integrity but change their shape to fit the channel. Along the resulting "virtual pathways", or "virtual channels" in the homy layer, Transfersomes reach regions of high water content in the deeper skin layers. There, the vesicles (re)distribute. Since Transfersomes are too large to enter the blood vessels locally, they bypass the capillary bed and get to subcutaneous tissue, where they accumulate.

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. For example, the method described in Schatzlein A et al, British journal of dermatology 138:583-592, 1998 which comprises the following steps 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). To prepare a vesicle suspension, all lipophilic components are first dissolved in methanol/chloroform (1 : 1 v/v) in the weight/weight ratio 89: 11 (SPC/SC) and then condensed into a film under vacuum (% lOPa; * 12h). 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-Laser Vertriebgesellschaft, Langen, Germany) to be typically 150 +/- 50nm.

Transfersome technology is described in detail in the following documents: G. Cevc, G. Blume. (1992) Lipid vesicles penetrate into intact skin owing to the transdermal osmotic gradients and hydration force. Biochim. Biophys. Acta 1104, 226-232.; P. Gonzalez, M. E. Planas, L. Rodriguez, S. Sanchez, G. Cevc. (1992) Νoninvasive, percutaneous induction of topical analgesia by a new type of drug carriers and prolongation of the local pain-insensitivity by analgesic liposomes. Anesth. Analg. 95, 615-621.; G. Cevc. (1992) Rationale for the Production and Dermal Application of Lipid Vesicles. In: Liposome Dermatic. (O. Braun-Falco, H.C. Korting, H.I. Maibach, eds.) Springer, Berlin, pp 82-90.; G. Cevc. (1993) Dermal Insulin. In: Frontiers in Insulin Pharmacology (M. Berger, and F. A.Gries, eds.), Georg Thieme Verlag, Stuttgart, pp 161-169.; G. Cevc, A. Schatzlein, D. Gebauer, G. Blume. (1993) Ultra-high efficiency of drug and peptide transfer through the intact skin by means of novel drag-carriers, transfersomes. In: Prediction of Percutaneous Penetration. (Bain, K. R., Hadgkraft, J., W. J. James, K. A. Water, eds.), STS Publishing, Cardiff, Volume 3b, pp 226-234.; G. Cevc. (1995) Material Transport Across Permeability Barriers by Means of Lipid Vesicles. In: Handbook of Biological Physics. (Series ed. A. V. Hoff, Volume eds. R. Lipowsky, E. Sackmann), Vol. I, North-Holland, Chapter 9, pp 465-490.; A. Paul, G. Cevc. (1995) Non-Invasive Administration of Protein Antigens. Epicutaneous Immunization with the Bovine Serum Albumin. Vaccine Res. 4, 145-164.; G. Cevc, A. Schatzlein, G. Blume. (1995) Transdermal drag carriers: basic properties, optimization and transfer-efficiency in the case of epicutaneously applied peptides. J. Contr.Rel. 36, 3-16.; G. Gompper, D.M. Kroll. (1995) Driven transport of fluid vesicles through narrow pores. Phys. Rev. E 52, 4198-4208.; A. Paul, G. Cevc, B. K. Bachhawat. (1995) Transdermal Immunization With Large Proteins by Means of Ultiadeformable Drug Carriers. Eur. J. Immunol. 25, 35221-3524.; A. Schatzlein, G. Cevc. (1995) Skin penetration by phospholipid vesicles, transfersomes, as visualized by means of the confbcal laser scanning microscopy. In: Phospholipids: Characterization, Metabolism and Novel Biological Applications (G. Cevc, F. Paltauf, eds.) AOCS Press, Champain, IL, pp 191-209.; G. Cevc, G. Blume, A. Schatzlein, D. Gebauer, A. Paul. (1996) The skin: a pathway for the systemic treatment with patches and hpid-based agent carriers. Adv. Drug Del. Rev. 18, 349-378.; G. Cevc. (1996) Lipid Suspensions on the Skin. Permeation Enhancement, Vesicle Penetration and Transdermal Drag Delivery. Crit. Rev. Therap. Drag Carrier Systems. 13, 257-388.; G. Cevc, G. Blume, A. Schatzlein. (1996) Transfersomes Mediated Transepidermal Delivery Improves the Regio-Specificity and Biological Activity of Corticosteroids in vivo. J. Contr. Rel. 45, 21 , 1-226.; G. Cevc. (1997) Drug Delivery Across the Skin. Exp. Opin. Invest. Drugs, 6, 1887-1937. A. Paul, G. Cevc, B. K. Bachhawat. (1998) Transdermal Immunization With Large Membrane Bound Molecules, Gap Junction Proteins, by Means of Ultradefbrmable Drag Carriers. Vaccine, 16, 188-195.; G. Cevc, D. Gebauer, A. Schatzlein, G. Blume. (1998) Ultraflexible Vesicles, Transfersomes, Have an Extremely Low Permeation Resistance and

Transport Therapeutic Amounts of Insulin Across the Intact Mammalian Skin. Biochim. Biophys. Acta 1368, 201-215.; A. Schatzlein, G. Cevc. (1998) Non-uniform cellular packing of the stratum comeum and permeability barrier function of intact skin: a high-resolution confbcal laser scanning microscopy study using highly deformable vesicles (Transfersomes). Br. J. Dermatol. 138, 583-592.; G. Cevc. (1998) Smart Carriers for Transdermal Drag Delivery. In: Intelligent Materials: Novel Concepts for Controlled Release Technologies. (S. Dinh and J. DeNuzzio, eds.) ACS Books, Washington, DC, in the press; S. I. Simoes, H. C. Richardsen, M. B. Martins, M. E. Cruz, G. Cevc. (1998) Superoxide dismutase delivery through the narrow pores in skin models by flexible hpid aggregates. In: Proceedings of Control Release Society Satellite Meeting in Lisbon, 1998, in the press.; G. Cevc, G. Blume. (1998) New, highly efficient formulation of diclofenac for the topical, transdermal administration in ultradeformable drag carriers, Transfersomes. Under revision.

METABOLIC ENZYMES

The biotransformation (metabohsm/catabohsm) of a foreign compound (xenobiotic) 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.

For this to be achieved, biotransformation is divided into two types of reaction: Phase 1 (oxidative, reductive or hydrolytic reactions) which provides metabolites appropriate for 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.

Examples of such 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. There are a number of forms or isoenzymes of the enzyme, as shown in Table 2, the relative proportions of which are determined by such factors as species and environmental influences (e.g. chronic exposure to xenobiotics). These isoenzymes are responsible for the oxidation of different substrates or for different types of oxidation of the same substrate.

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. Preferably, the antagonist is an antagonist of a Phase 1 enzyme. More preferably, the antagonist is an antagonist of a cytochrome P450 enzyme (CYP).

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. For example, 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. Furthermore, assays to identify metabohc enzyme antagonists may be designed. Thus, cells (for example, skin biopsies) may be exposed in vitro to the drag in question, and genes identified whose expression is induced as a result of (or associated with) such exposure. Induced genes 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. Where such metabolic enzymes are known enzymes, an antagonist may be chosen from compounds known to antagonise the metabolic enzyme. Where 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. Preferably, such inhibitors and antagonists are specific to the metabohc enzyme induced. However, general inhibitors of metabohc enzymes may also be used in the methods and compositions as described here.

P450 CYTOCHROME OXIDASES

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 htφ://bioinf.leeds.ac.ul^prormse/P450.html, part of the "Prosthetic groups and Metal Ions in Protein Active Sites Database" at htφ://bioirrf.leeds.ac.uk/promi

A number of 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 (P450s), 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 absoφtion band at around 450 nm inthe CO-difference spectrum of reduced forms. Other members of the class include cystathionine β-synthase (EC 4.2.1.22) (haemoprotein H450; serine sulphydrase; β-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.

The enzymes are involved in metabolism of a plethora of both exogenous and endogenous compounds (Nebert, D.W. and Gonzalez, F.J. (1987) P450 genes: structure, evolution, and regulation. Annu. Rev. Biochem. 56, 945-993.). Usually, they act as terminal oxidases in multicomponent electron transfer chains, called P450-conteining monooxygenase systems. P450-containing monooxygenase systems primarily fall into two major classes: bacterial/mitochondrial (I), and microsomal (II). Alternatively, 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.).

The most common reaction catalysed by P450 enzymes is a monooxygenase reaction, i.e. insertion of one atom of oxygen into substrate while the other oxygen atom is reduced to water: RH + O₂ + 21^ + 2e^" ROH + H₂O

3D structures are known for a number of P450 enzymes are known (Poulos, T.L. (1995) Cytochrome P450. Curr. Opin. Struct. Biol. 5, 767-774) The 3-D structures of several P450s have been reported: P450cam (Poulos, et al, 1987, J. Mol. Biol. 195, 687-700), the P450 domain of P450_BM-₃ (Ravichandran et al., 1993, Science 261, 731-736.), P450_teφ (Hasemann et al., 1994, J. Mol. Biol. 236, 1169-1185.) and P450eryF (Cupp-Vickery et al., 1995, Nature Struct. Biol.2, 144-153). The P450 molecule is an /β protein, shaped like a triangular prism; the overall stracture has been described as being divided into -rich ('right side') and β-rich ('left side') domains (Poulos, Ravichandran, Hasemann, Cupp-Vickery, supra). However, this terminology is inappropriate, since the - and β-rich 'domains' comprise discontinuous assembhes of secondary stracture segments and do not constitute independent folding units. Although the 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.

(Guengerich F.P., et al., Drug Metabolism and Disposition 26:1175-1178, 1998). Approximately 6 P450's are responsible for the oxidation of 90% of drug & carcinogens (Guengerich FP (1995) "Human cytochrome P450 enzymes", in Cytochrome P450 (Ortiz de Montellano PR ed), pp 473-535. Plenum Press. New York). The main xenobiotic metabolizing enzymes belong to P450 famihes 1 through 4 ("The human hepatic cytochromes P450 involved in drag metabolism", Wrighton S.A. and Stevens J.C., Crit. Rev. Toxicol. 22:1-21, 1992. (16 genes)). Besides 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. More preferably, 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: NM_000784), CYP27B 1 (GenBank Accession No: NM_000785).

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.

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).

The sequence of rabbit CYP2C3 is known (Accession Numbers: A00183, A22606,

Q29525, M31245, M31246, M31247, M31248, 1002188C, A34534, M31249, M31250, M31251, M31252, 1106190A, M31253, M31254, P00182, K01523 and J02901).

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). In a highly preferred embodiment, 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 D₃-24-hydroxylase, 25-hydroxyvitamin D 24-hydroxylase, 25-hydroxyvitamin D-24- hydroxylase, l,25-α ycfroxyvitøminD3-24-hydroxylase, l,25-chfrydroxyvitamin D₃-24- hydroxylase, lalpha,25-dihydroxyvitarrhnD3-24-hydroxylase, lalpha,25-dihydroxyvitamin D₃- 24-hydroxylase, 1 alpha,25 dihydroxy-D3 24-hydroxylase, 1 alpha,25 dihydroxy-D₃ 24- hydroxylase, lalpha,25(OH)(2)D(3) 24-hydroxylase, lalpha,25(OH)(2)D(3)-24-hydroxylase, lalpha,25(OH)2D3-24-hydroxylase, and lalpha,25(OH)₂D₃-24-hydroxylase.

The CYPs disclosed here preferably comprise human CYPs. Such 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).

The methods and compositions described here therefore encompass the use of one or more antagonists against these and other metabolic enzymes.

P450 INHIBITORS

This section provides detailed information on how particular metabolic enzymes may be antagonised. 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, lμM in MCF-7 cell culture.( Zhao J. et al., J. steroid Biochem. & Mol. Biol. 57: 197- 202, 1996), 20μM 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-10μM in prostate cancer cell culture (Ly L.H., et al., Endocrinology 140:2071- 2076, 1999), lμM 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 50μM 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, 20μM 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- lOμM, 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 (CYP27) 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 (as described in further detail above) may also be similarly inhibited (see Masuda et al., JBC. 269:4794-4803, 1994). Further examples of metabohc enzyme inhibitors include those disclosed in GB2199579 and EP0683156, as well as Schuster et al. in Steroids 66:409-422, 2001 and in Steroids 66:451-462, 2001. Each of these compounds, in particular, VID400R and its derivatives as well as SDZ 89-443 and its derivatives, may be used in the methods and compositions described here.

VITAMIN D ANALOGUES

The methods and 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.

Numerous 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, 5,658,949, 5,645,852, 5,643,878 5,637,589, 5,610,279, 5,610,180, 5,609,897, 5,607,975, 5,607,691, 5,605,930, 5,600,015 5,599,711, 5,597,595, 5,552,267, 5,540,917, 5,521,316, 5,447,953, 5,443,852, 5,428,006 5,425,956, 5,397,803, 5,397,771, 5,374,651, 5,356,810, 5,252,604, 5,242,901, 5,230,915, 5,153,344, 5,145,846, 5,120,722, 5,091,188, 5,091,187, 5,077,284, 5,064,655, 5,034,546, 5,024,998, 5,017,566, 4,983,586, 4,956,355, 4,906,785, 4,904,823, 4,902,818, 4,897,388, 4,851,585, 4,794,193, 4,743,696, 4,709,707, 4,686,023, 4,680,142, 4,675,421, 4,578,462, 4,559,407, 4,499,307, 4,418,087, 4,385,073, 4,355,018, 4,335,120, 4,230,701, 3,957,966, as well as US Patent Nos 6,310,226, 6,268,478, 6,207,656, 6,197,982, 6,172,110, 5,994,332, 5,932,565, 5,847,165, 5,830,885, 5,776,461, 5,726,204, 5,716,945, 5,710,142, 5,709,844, 5,679,828, 5,665,387, 5,637,589, 5,612,341, 5,612,325, 5,589,471, 5,585,368, 5,554,599, 5,545,633, 5,447,924, 5,446,034, 5,401,732, 5,401,731, 5,387,582, 5,378,695, 5,376,651, 5,374,629, 5,362,719, 5,292,727, 5,206,229, 5,190,935, 5,077,284, 4,956,355, and 4,866,048.

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.

We disclose the use of one or more inhibitors of the metabolic enzymes CYP24 and/or CYP2E1 in the treatment and/or alleviation of tachyphylaxis associated with admrmstration, preferably long-term administration, of a vitamin D analogue. Preferably, the vitamin D analogue comprises Calcipotriol and the metabolic enzyme inhibitor comprises an inhibitor of CYP2E1, such as ketoconazole. Alternatively or in addition, 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, for example those listed in Table 2, may also be used in conjunction or in place of the ones listed above.

Particularly preferred vitamin D analogues are described below:

Calcitriol 1, 25-dihydroxyvitamin D₃ is an activated natural vitamin Ds made by Roche, Duphar.(Jones et al., Physiological Reviews IS: 1193-1231 -98. (see especially fig. 10))

In addition to its calcaemic effects, 1,25-dihyroxycholecalciferol (Calcitriol, l,25(OH)₂D₃) 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 lα (IL-lα). In the 1960's, 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 initial trials of Calcitriol in psoriasis indicated that it was effective in clearing the lesions but could result in a rise in the serum calcium levels.

In a preferred embodiment, the agent (which may be a therapeutic drag) comprises

Calcitriol. We find that long-term administration of Calcitriol is associated with expression of CYP24. Accordingly, any agent capable of inhibiting CYP24 may be used to treat or alleviate tachyphylaxis associated with Calcitriol. Examples of such agents include ketoconazole, N-[4- cWorobenzoyl]-2-(lH-iιmdazol-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.

Calcipotriol (Dovanex, MC903

A search was therefore undertaken for vitamin D analogues with an improved therapeutic ratio. This would involve retaining the effects on cell proliferation and/or differentiation but reducing the hypercalcaemic effect. In 1991, the synthetic topical vitamin D analogue Calcipotriol (Dovanex) was launched for the treatment of psoriasis in Europe. This was shown in double blind clinical trials to be more effective than the previous market leaders, topical steroids and Anthalin. When used within the dose limit of lOOg per week in an adult, the use of Calcipotriol did not result in hypercalcaemia. The dose limit for Calcipotriol meant that its use was limited to mild/moderate psoriasis. This was because in severe disease, lOOg per week would not be sufficient to cover the surface area affected. However, high dose Calcipotriol therapy, c.300g per week, has been shown to be effective in cases of severe psoriasis in the short term, but was associated with a rise in serum calcium.

In a preferred embodiment, the agent (which may be a therapeutic drag) comprises Calcipotriol. We find that long-term administration of Calcipotriol is associated with expression of CYP2E1. Accordingly, 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 (Curatiderm)

In 1990s, a second vitamin D analogue Tacalcitol (Curatiderm) was launched for the treatment of psoriasis. Tacalcitol is lα, 24 dihydroxyvitamin D₃, and is also known as Curatoderm (made by Teijin, hcenced by Lipha S.A., sold by Boots). For further details on Tacalcitol, please refer to Weinstein E.A. et al., 1999, Isolation and identification of l - hydroxy-24-oxovitamin D₃ and 1 ,23-dihydroxy-24oxovitamin D₃, Biochemical Pharmacology 58:1965-1973,.

Tacalcitol has a similar efficacy, adverse effect profile and therapeutic ratio as Calcipotriol.

In a preferred embodiment, the agent (which may be a therapeutic drug) comprises Tacalcitol. We find that long-term adrrώiistration of Tacalcitol is associated with expression of CYP24. Accordingly, any agent capable of inhibiting CYP24 may be used to treat or alleviate tachyphylaxis associated with Tacalcitol. Examples of such agents include 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-chlorophenyι)- 1 -aminoethane (Compound B) and N-[4-(frex-l-yl)benzoyl]-2-(lH-irmdazol-l-yl)-2-φhenyl)-l-amino ethane (Compound C). Other inhibitors listed in Table 2 may also be used. VITAMIN D ANALOGUES AND THEIR METABOLISM

The vitamin D hydroxylases constitute a family of mixed-function oxidases which contain as their specific component, a cytochrome P450 isoform. Several such 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)₂D₃) 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)₂D₃ to the inactive and water-soluble metabolite calcitroic acid for bilary excretion.

Other enzymes are also involved in vitamin D metabohsm. C YP27B 1 generates the active vitamin D species (25 (OH)D₃ to 1, 25 (OH)₂D₃) and is probably anabolic rather than catabolic. CYP27A1 is also involved in the activation of vitamin D (D₃ to 25(OH)D₃) but seems to serve catabolic functions as well, especially with vitamin D analogues; CYP24 appears to be specific for catabohsm of l,25(OH)₂D₃ and other active analogues but can catalyse several different reactions in their degradation.

We have found that these and other metabohc enzymes capable of metabohsing vitamin D (including CYP24) are induced when vitamin D activity increases (for example, as a result of administration of vitamin D or its analogues), so that degradation of the active vitamin is up- regulated. Although this will act to reduce excessive amounts of 1,25 (OH)₂D₃, it may also result in decreasing efficacy of pharmaceutical compounds and accumulation of toxic by-products. It has been well established that most, if not all of the steps in the catabohsm of Calcitrol to calcitroic acid are mediated by CYP24. Likewise, most of the reactions in the degradation of Calcipotriol and Tacalcitol are catalysed by CYP24. Accordingly, we provide for the use of antagonists of CYP24 and other metabohc enzymes for preventing or alleviating a tachyphylactic response to administration of a vitamin D analogue in a patient.

Metabolism of Calcitrol

The degradation of Calcitrol by P450 cytochromes is understood to involve the following pathway. l,25-(OH)₂D₃ (Calcitrol) is converted by CYP24 (24-hydroxylase) to 1,24,25- (OH)₃D₃ ( which is 1/10 as active as calcitrol). l,24,25-(OH)₃D₃ is converted by CYP24 to 24- OXO-1,25 (OH)₂D₃. 24-0X0-1,25 (OH)₂D₃ is converted by CYP24 to 24-0X0-1,23,25 (OH)₃D₃.24-0X0-1,23,25 (OH)₃D₃. is converted by CYP24 to 24,25,26,27-tetranor-l,23 (OH)₂D₃ (C-23 alcohol). 24,25,26,27-tetranor-l,23 (OH)₂D₃ 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.

When the above pathway is blocked an alternative route may involve C-26 hydroxylation followed by 26,23-lactone formation. (St-Arnaud R., et al., Endocrinology 141 :2658-2666, 2000; "Isolation, identification, and metabohsm of (23S,25R)-25-hyc oxyvitamin Dj 26,23- lactol. A biosynthetic precursor of (23S,25R)-25-hydroxyvitamrn D|₃ 26,23-lactol", Yamada S., et al, J.B.C. 259:884-889, 1984).

Any of the enzymes involved in catalysing the various steps in the metabohsm of

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)₂D₃ (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 lα,24(S)(OH)₂D₂ (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, 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 administration of Calcipotriol.

Metabohsm of Tacalcitol

The metabohsm of Tacalcitol is understood to involve two major mechanisms. See "Isolation and identification of l -hydroxy-24-oxovitamin ∑ and lα,23-dihydroxy- 24oxovitamfnD₃", Weinstein E. A. et al., Biochemical Pharmacology 58:1965-1973, 1999.

lα, 24(R) dihydroxyvitamin E (Tacalcitol) is acted on by 25-hychoxyvitemin D-lα- hydroxylase to produce l,24,25-(OH)₃D₃. l,24,25-(OH)₃D₃ is acted on by CYP24 to produce lα,25(OH)₂-24-OXO-D_3. l ,25(OH)₂-24-OXO-D₃ is acted on by CYP24 to produce l ,23,25(OH)₃ -24-OXO-D₃.lα,23,25(OH)₃ -24-OXO-D₃ Is acted on by CYP24 to produce 24,25,26,27-tetranor-l,23 (OH)₂D₃ (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 lα(OH)-24- OXO-D₃, which is converted by CYP24 to produce l ,23(OH)₂-24-OXO-D₃ lα,23(OH)₂-24-OXO-D₃ is converted by CYP24 to produce 24,25,26,27-tetranor-l,23

(OH)₂D₃ (C-23 alcohol), which is converted by CYP24 to produce Calcitroic acid. This second pathway appears to account for about 83% of Tacalcitol metabohsm.

Any of the enzymes involved in catalysing the various steps in the metabohsm of Tacalcitol, 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 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). In this assay, 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 μM) for 3 h at 37 °C. Triplicate 500-μl ahquots of aqueous fraction from the cell/medium extract are mixed with aqueous scintillation mixture, and the radioactivity is measured using a scintillation counter.

It is currently thought that 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

TCAATGCTATGACCTGTGAAGG and anti-sense ATCATTCACACGAACTGGAGGC. As noted above, however, we have found that 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.

As used here, the term "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. As an Example, 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.

By "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 moφhologically, or by assaying expression of protein markers specific for the differentiated cell type as known in the art. For example, Kl and K10 keratin are markers for commitment to terminal differentiation of epidermal keratinocytes, and expression is increased when cellular differentiation occurs. In addition to Kl and K10 keratin, other keratin subtypes may be used as markers for different differentiation stages, for example, K5, K14, K16 and K17. Other non-keratin markers, for example EGF-receptor and β-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. Examples of such 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 (XM 003751); TGF-beta 1 (XM 008912); TGF-beta 2 (XM 001754); and TGF-beta 3 (XM 007417).

In particular, 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 AND THEIR METABOLISM

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.

As used in the present document, however, the term "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. Although their long-term efficacy is questionable, 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.

Examples of commonly used topical corticosteroids 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.

Accordingly, we provide for the use of antagonists of CYP24 and other metabohc enzymes for preventing or aUeviating a tachyphylactic response to administration of a corticosteroid in a patient.

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 AND THEIR METABOLISM

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, such as the diseases specified above, 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.

The methods and 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.

In a preferred embodiment, the agent (which may be a therapeutic drug) comprises a macrolactam, preferably tacrolimus and/or cyclosporin. We find that long-term aα¹ministration of a macrolactam such as tacrolimus and/or cyclosporin is associated with expression of CYPIAI. Accordingly, 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

Further examples of 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. Thus, in order to aUeviate or prevent tachyphylaxis developing against administration of a particular drug, the relevant CYP enzyme is targeted by selecting an antagonist of the enzyme.

SIDE EFFECTS OF DRUG ADMINISTRATION

We further provide for compositions and methods for treatment or aUeviation of side effects associated, or caused by, adrriinistration of a drug to a patient.

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.

Side effects are also observed during corticosteroid therapy. The most common adverse effect of topical corticosteroid use is cutaneous atrophy. This atrophy involves both the epidermis with thinning secondary to decreased mitotic activity and DNA synthesis, and the der is with decreased fibroblast synthesis of collagen and ground substance. Another common adverse effect occurring on the face is a papulopustular reaction particular in acne- or rosacea-prone individuals. The above adverse effects are reversible but another common adverse effect, striae caused by rapture of connective tissue and stietching of the epidermis, is not. Administration of macrolactams may result in the undesirable side effect of nephrotoxicity.

We have discovered that metabolic break down of an administered drug in the skin results in production of a reactive intermediate responsible for the side effect. Induction of the metabolic enzymes foUowing repeated exposure to the drag explains the increase in severity of the side effect (such as irritation) with prolonged use. Accordingly, we provide for methods and compositions which are capable of alleviating, reducing or preventing, etc undesirable side effects associated with administration of a drug to a patient.

We therefore provide a method of alleviating or preventing a side effect associated with administration of a drag to a patient, the method comprising adrninistering to a patient an antagonist of a metabohc enzyme which is induced as a result of exposure of the patient to the drug. Another aspect provides for 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 drug, and identifying an antagonist of the metabolic enzyme. 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.

In general, the it is the presence of the metabohc enzyme which leads to the side effect. Thus, the metabohc enzyme itself may cause the side effect (i.e., the metabohc enzyme may itself be responsible for the cutaneous irritation). Furthermore, 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. According to a preferred embodiment, therefore, the enzyme activity is capable of metabohsing the drag to produce the side effect. In a highly preferred embodiment, 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. Preferably, 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. Similarly, 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. Typically, the side effect is associated or caused by repeated, periodic or otherwise long term administration of the drag.

Preferably, the side effect is cutaneous irritation, itching, cutaneous atrophy, papulopustular reaction or striae.

METABOLIC ENZYME ANTAGONISTS AS PROPHYLACTICS

We have suφrisingly also found that the particular metabolic enzyme antagonists which capable of reversing, relieving, preventing, etc a tachyphylactic response in a patient to a drug may in themselves be used as therapies of treatment of the relevant disease. Thus, as noted above, administration of a drag, for example to treat a certain disease, leads to tachyphylaxis in a patient. This may be countered by administration of one or more metabohc enzyme antagonists which are capable of antagonising the metabohc breakdown of the drag.

For example, 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 (for example, an antagonist of CYP24 such as Manumycin A) 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. We have discovered that it is possible to maintain the psoriatic patient in remission by treatment with the inhibitor alone. Thus, the treated patient is prevented from developing psoriasis by administration of the metabohc enzyme antagonist, without the administration of the drag in question. In this manner, the metabohc enzyme antagonist may itself be used as a prophylactic treatment. Furthermore, the use of the metabohc enzyme antagonist in this aspect may be augmented by administration of a vitamin D intermediate such as 25(OH)D₃ since the patient's ceUs can efficiently activate 25(OH)D₃ to l,25(OH)₂D_3.

Thus, we provide for a method for 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 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.

Furthermore, it is possible to use 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 puφose 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.

Thus, we provide for 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 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.

It will be appreciated that the methods described here are not restricted to use of metabohc enzymes induced, etc, by the administration of a drag known or suitable for treating the disease. Indeed, candidate drugs may also be used as a starting point. It wUl be necessary to identify metabolic enzymes induced, etc, by these candidate drags, and also to provide agents capable of inhibiting and or antagonising them. These agents may then be tested for their efficacy in the treatment of the disease in question. Our discoveries may be based on the fact that a deficiency of vitamin D production in the skin is a cause of psoriasis. This may arise from a number of factors, for example, a geneticaUy determined enhanced and/or aberrant activity of endogenous vitamin D metabohsm. Thus, inhibition of the breakdown of the endogenous active vitamin D species by a specific metabohc enzyme inhibitor leads to accumulation of an active species and prevents eraption of the disease.

This particular aspect is particularly suitable for prophylaxis of a disease in which an administered drag exerts its therapeutic effects by mimicking the effects of an endogenously produced molecule. Thus, for example, an active ligand (for example an endogenous active vitamin D species) 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. Thus, inhibition of CYP24 aUows accumulation of endogenous 1,25 (OH)₂D₃, and inhibition of CYP26 aUows accumulation of endogenous aU- trans retinoic acid.

Other endogenously produced active ligands where inhibition of catabohsm may be of therapeutic value include: glucocorticoids, mineralocorticoids, famesoids, thyroid hormone, eicosanoids, and estrogen. For example, administration of a metabolic enzyme capable of metabohsing estrogen may be used in place of hormone replacement therapy (HRT). It will be appreciated that the use of the term "disease" here may include syndromes or conditions (such as a decrease of hormone levels associated with menopausal and post-menopausal states) which are treatable with a drag.

COMBINATION THERAPY

Within weeks of the launch of Calcipotriol in 1991, patients with psoriasis were reporting to their dermatologists that when they apphed a mixture of Calcipotriol and a topical steroid to their psoriasis, this was more effective at inducing clearance than the two products used separately at equivalent dosages. Subsequently, clinical trials demonstrated that the combination of Calcipotriol and topical steroids was synergistic. A Calcipotriol plus beta-methazone valerate combination product is currently in phase 3 clinical trials.

Accordingly, we describe the use of a second therapeutic agent or drag, in combination with the metabohc enzyme antagonist, in the prevention or aUeviation of a tachyphylactic response to a drag in a patient. The use of such second (and further) agents is referred to in this document as "combination therapy", and can lead to higher efficacy of treatment of the condition, or reduced tachyphylaxis, or preferably, both. Second therapeutic agents may be chosen according to the particular disease, syndrome, or therapy in question. For example, where tachyphylaxis arising from aclrninistration of a vitamin D analogue for treatment of psoriasis is to be avoided or treated, 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 dissiπrUar.

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.

DISCONTINUOUS THERAPY

We have found that that tachyphylaxis develops with the continuing use of any topical vitamin D analogue. However, we have also discovered that if a patient discontinues using a drag such as a topical vitamin D analogue for a period of time (for example, 2 weeks), when they resume using the same drag, its efficacy is restored to approaching baseline values. Accordingly, our methods include the use of such breaks in continuous therapy to aUeviate and/or prevent tachyphylaxis.

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. Preferably, the period is such that efficacy is restored to a level substantiaUy approaching baseline level (i.e., substantiaUy complete rehef of tachyphylaxis). Preferably, 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). ROTATIONAL THERAPY

We have discovered that, where patients have developed tolerance (tachyphylaxis) to one drug, for example, a topical vitamin D analogue such as Dovanex, when they switch to another drag (e.g., another vitamin D analogue such as Curatoderm), efficacy is restored. By continuaUy rotating the analogue, Dovanex/Curatoderm, we have discovered that the patients can maintain a much more effective therapeutic response. We refer to the alternation of a drug and a second therapeutic agent as "rotational therapy".

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.

It wiU be appreciated that although 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. For example, 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.

Furthermore, it wUl be appreciated that 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. Thus, 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. SKIN HYPERPROLIFERATTVE DISEASES

Vitamin D and its analogues (for example, Calcitriol, Calcipotriol, etc) 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 hypeφrohferative diseases.

Such skin hypeφroliferation 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.

Accordingly, a patient exhibiting any of the symptoms associated with a skin hypeφroliferative disease, for example, a disease as hsted above, may be treated with 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. The effect of treatment of a host with skin prohferation disease may be evaluated by objective criteria such as an improvement of desquamation and erythema, reduction of the size of lesions as weU as subjective criteria such as cessation of itching. In particular, the 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).

The causes of psoriasis are currently unknown, although it has been established as an autoimmune skin disorder with a genetic component. One in three people report a farmly history of psoriasis, but there is no pattern of inheritance. However, there are many cases in which children with no apparent family history of the disease will develop psoriasis. 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 hypeφroliferation related to accelerated and incomplete differentiation. Second, there is a marked inflammation of both epidermis and dermis with an increased recruitment of T lymphocytes, and in some cases, formation of neutrophU microabcesses. Many pathologic features of psoriasis can be attributed to alterations in the growth and maturation of epidermal keratinocytes, with increased prohferation of epidermal ceUs, occurring within 0.2 mm of the skin's surface. Traditional investigations into the pathogenesis of psoriasis have focused on the increased proliferation and hypeφlasia of the epidermis. In normal skin, the time for a cell to move from the basal layer through the granular layer is 4 to 5 weeks. In psoriatic lesions, the time is decreased sevenfold to tenfold because of a shortened ceU cycle time, an increase in the absolute number of cells capable of proliferating, and an increased proportion of ceUs that are actually dividing. The hypeφroliferative phenomenon is also expressed, although to a substantiaUy smaUer degree, in the clinicaUy uninvolved skin of psoriatic patients.

A common form of psoriasis, psoriasis vulgaris, is characterized by well-demarcated erythematous plaques covered by thick, silvery scales. A characteristic finding is the isomoφhic response (Koebner phenomenon), in which new psoriatic lesions arise at sites of cutaneous trauma.

Lesions are often localized to the extensor surfaces of the extremities, and the naUs and scalp are also commonly involved. Much less common forms include guttate psoriasis, a form of the disease that often erupts following streptococcal pharyngitis, and 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. The visual scoring is done using PASI (Psoriasis Area and Severity Index) score (see Fredericksson, A J, Peterssonn B C Dermatologies 157:238-244 (1978)). Our methods and compositions are also suitable for the aUeviation and/or reduction of tachyphylaxis associated with treatment of acne. Acne affects large patient populations and is a common inflammatory skin disorder which usuaUy localizes on the face. Fortunately, the disease usually disappears and in the interval of months or years between onset and resolution, therapy, although not curative, can satisfactorily suppress the disease in the majority of patients.

A small number of acne patients with severe disease show little or no response to intensive therapeutic efforts including the use of high doses of oral tetracycline, dapsone, prednisone, and, in women, estrogen. In many cases, these drugs afford only a modest degree of control while the side effects of these agents severely restrict their usefulness. Patients with nodulocystic acne suffer from large, inflammatory, suppurative nodules appearing on the face, and frequently the back and chest. In addition to their appearance, the lesions are tender and often puralently exudative and hemorrhagic. Disfiguring scars are frequently inevitable.

Therapies for acne involve local and systemic administration of retinoids. Topical application of aU-trans-retinoic acid (tretinoin) has been tried with some success, particularly against comedones or blackheads, but this condition frequently returns when the treatment is withdrawn.

ANTAGONISTS

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. Accordingly, 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. Thus, decreasing and/or blocking of the metabolic enzyme activity may be achieved at any one or more of these levels.

Agents which are capable of regulating the activity of a metabohc enzyme in a negative manner are referred to as antagonists of that activity. Antagonists of metabohc enzymes may achieve their effects at any one or more of the levels described above. Thus, the antagonist may be an inhibitor of transcription, translation, processing, or an activator of breakdown, etc of the metabohc enzyme.

The term "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). Thus, 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. However, 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.

Where antagonists of a enzyme such as a metabohc enzyme are concerned, an antagonist may include a substrate of the enzyme, or a fragment of this which is capable of binding to the enzyme. In addition, 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. Thus, a molecule which mimicks the shape or conformation of a drug molecule may be used as an inhibitor of metabohc enzyme activity. Alternatively, or in addition, an immunoglobulin (for example, a monoclonal or polyclonal antibody) capable of binding to the metabohc enzyme may be used. 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.

For example, 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. For example, 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. For example, 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.

As used herein, in general, 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. An agent may be in solution or in suspension (e.g., in crystaUine, colloidal or other particulate form). The agent may be in the form of a monomer, dirner, oligomer, etc, or otherwise in a complex.

The terms "antagonist" and "agent" 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. a yeast artificial chromosome) or a part thereof, 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. 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. Examples of 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. Examples of 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. ASSAYS

We also provide a method of screening compounds to identify antagonists to a metabohc enzyme such as a cytochrome P450 (CYP). 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. Alternatively, the screening method may involve competition with a labeUed competitor. Further, 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. For example, 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. Where 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.

Further, 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)).

We also provide 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.

ANTISENSE COMPOUNDS

As described above, 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. Preferably, the antisense compounds comprise sequences complementary to the mRNA encoding the metabohc enzyme. Preferably, 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. As used herein, the term "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. 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 refeπed to as "antisense". The functions of DNA to be interfered with include rephcation and transcription. The functions of 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.

In the context of the present document, "modulation" means either an increase

(stimulation) or a decrease (inhibition) in the expression of a gene. For example, the expression of a gene encoding an inhibitor of metabohc enzyme activity, or an inhibitor of expression of the metabohc enzyme, may be increased. However, preferably, 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 incoφorated by reference.

PHARMACEUTICAL COMPOSITIONS

We also describe pharmaceutical 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.

It should be noted that the 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).

However, and preferably, 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). 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.

Thus, whUe it is possible for the 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. For example, they can include diluents or excipients such as fillers, binders, wetting agents, disintegrators, surface- active agents, lubricants and the like. Typically, 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.

The pharmaceutical 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. As an example, an acute skin proliferation disease generally is treated with aqueous drying preparations, whereas chronic skin proliferation disease is treated with hydrating preparations. Soaks are the easiest method of drying acute moist eruptions. Lotions (powder in water suspension) and solutions (medications dissolved in a solvent) 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. 1593-15950 (1996)). 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.

Other formulations 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)).

In general, 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.

In a topical application, 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 absoφtion 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 absoφtion 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 incoφorated 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 absoφtion 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. Sci., 73:1153-1156 (1984)); a combination of ethanol and oleic acid or oleyl alcohol (EP 267,617), 2-ethyl-l,3-hexanediol (WO 87/03490); decyl methyl sulphoxide and Azone.RTM. (Hadgraft, Eur. J. Drug. Metab. Pharmacokinet, 21:165-173 (1996)); alcohols, sulphoxides, fatty acids, esters, Azone.RTM., pyrrolidones, urea and polyoles (Kalbitz et al, Pharmazie, 51:619-637 (1996));

Teφenes 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 Yorkshire Pig as an Animal Model for Measuring Percutaneous Penetration. ImSwine in Biomedical Research (M. E.

Tumbleson, Ed.) Plenum, New York, 1986, and Hawkins, G. S. Methodology for the Execution of In Vitro Skin Penetration Determinations. In: Methods for Skin Absoφtion, B W Kemppainen and W G Reifenrath, Eds., CRC Press, Boca Raton, 1990, pp.67-80; and W. G. Reifenrath, Cosmetics & Toiletries, 110:3-9 (1995).

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 incoφorated 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 Hautarzt 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.

Optionally, 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.

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. In general, the dahy oral dose of agent is less than 1200 mg, and more than 100 mg. The prefeπed 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. In general, 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 incoφorated 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. As an example, 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.

FURTHER ASPECTS

Further aspects of the methods and compositions described here are set out in the following numbered paragraphs.

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)₂D₃, la,25-dihydroxyvitamin D3, 1,25-dihyroxycholecalciferol), Calcipotriol (Dovanex; MC903) and Tacalcitol (la,24(R)-dihydroxyvitaminD₃; 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.

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 (b) exposing the cell to a drug;

Paragraph (c) identifying a metabolic enzyme which is induced as a result of such exposure; and

Paragraph (d) identifying an inhibitor of the enzyme identified in (c).

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

22, in which 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.

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

Paragraphs 27 to 30, in which the metabohc product is a toxin. 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.

EXAMPLES

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.

Example 1. Characterization of Metabolic Enzyme Induction induced by Vitamin D Analogue (in vivo)

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 mm²) are taken from human psoriatic volunteers exhibiting tachyphylaxis against vitamin D analogues after three or six weeks of twice daUy treatment with 5μg g ointment Calcitriol, 50μg/g ointment Calcipotriol, or 4μg/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.

Specific PCR

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 coπesponding rabbit sequences as probes, and primers designed to amplify these three genes.

Table 1. Primers used for RT-PCR

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.

Redundant PCR

Redundant 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 coπesponding 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 coπesponding 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.

Example 2. Characterization of Metabolic Enzyme Induction induced by Vitamin D Analogue (in vitro)

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 D₅ (Calcitrol); or vehicle contiol. The drags are applied every 2 days in lOμL 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.

12 untreated SE cultures are used as controls with the first set of 3 withdrawn before tieatment (day 0). At the completion of the tieatment regimes RNA is isolated from the SE's using the RNeasy kit according to the instruction of the supplier (Qiagen Inc.).

Specific PCR

Semi-quantitative RT-PCR, primers and identification of vitamin D analogues up- regulated CYP's are identical to the method above.

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.

The results for Calcipotriol showing induction of CYP2E1 are presented in Figure 1. 0.5%) ethanol was used as the vehicle for drag dehvery and CYP2E1 is ethanol-inducible which could explain a base line expression of CYP2E1 in aU samples.

Lanes 23 and 24 show amplification of a 120bp product, the expected size, with PCR at an annealing temperature of 58oC. Thus, Calcipotriol up-regulates CYP2E1

This is in accordance with the result from the human skin biopsy trial +/- calcipotriol.

Results are also shown in Figure 4. It is clear that CYP2E1 is up-regulated by the vitamin

D analogue calcipotriol as described. This is in line with the results for the normal human skin biopsy treated with calcipotriol shown in Example 1.

Each lane of the gel equates to a skin equivalent sample described below. Lanes marked - are the negative contiol (water) and lanes marked + are the positive control (G3PDH).

Inhibition of CYP2E1 wiU therefore inhibit the degradation of Calcipotriol and hence inhibit tachyphylaxis to this compound.

Redundant PCR

Furthermore, we RT-PCR amplify DNA fragments using the pan-specific CYP primers and the RNA from the vitamin D analogue treated SE's. These fragments are cloned and sequenced, and their sequences compared to CYP sequences in the GenBank database.

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.

The above CYPs are verified to be up-regulated by the vitamin D analogue treatment, using specific primers based on the known sequences deposited in GenBank. AU vitamin D analogue responsive CYP's are fully induced by 7 days of treatment.

Changes in enzyme induction/inhibition foUowing vitamin D analogue exposure are observed and recorded.

Example 3. Characterization of Metabolic Enzyme Induction Induced by Corticosteroid (in vivo)

Examples 3 and 4 describe the identification of the catabolic enzymes responsible for catabohsm of corticosteroids in human skin (keratinocytes and lymphocytes). We use redundant P450-based RT-PCR on human volunteers with tachyphylaxis against corticosteroids, in particular Clobetasol and/or Dexamethasone.

Sin biopsies (10 mm²) 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.).

Specific PCR

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 coπesponding 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.

Redundant PCR

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. We RT-PCR amplify DNA fragments using the pan-specific CYP primers. 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.

Example 4. Characterization of Metabolic Enzyme Induction Induced by Corticosteroid (in vitro)

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 lOμL 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.

12 untreated SE cultures are used as controls with the first set of 3 withdrawn before treatment (day 0). At the completion of the treatment regimes RNA is isolated from the SE's using the RNeasy kit according to the instruction of the supplier (Qiagen Inc.). Specific PCR

Semi-quantitative RT-PCR, primers and identification of corticosteroid up-regulated CYP's are identical to the method above.

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.

Redundant PCR

Furthermore, we RT-PCR amplified 11 DNA fragments using the pan-specific CYP primers. 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.

All corticosteroid responsive CYP's are fuUy induced by 7 days of treatment.

Example 5. Characterization of Metabolic Enzyme Induction Induced by Macrolactams (in vivo)

Examples 5 and 6 describe the identification of the catabolic enzymes responsible for catabohsm of macrolactams in human skin (keratinocytes and lymphocytes). We use redundant P450-based RT-PCR on human volunteers with tachyphylaxis against macrolactams, in particular Tacrolimus, Ascomycin and Cyclosporin.

Skin biopsies (10 mm²) are taken from human psoriatic volunteers exhibiting tachyphylaxis after 4 weeks of twice daUy treatment with 0.3% ointment Tacrolimus, 0.4%> ointment Ascomycin, or 0.3%) Cyclosporin, and normal healthy volunteers. RNA is isolated from the skin biopsies using the RNeasy kit according to the instruction of the supplier (Qiagen Inc.). Specific PCR

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.

The RT-PCR shows that expression of CYPIAI is induced by Tacrolimus.

Furthermore, CYPIAI is seen to be up-regulated by exposure to Cyclosporin as compared to controls.

Redundant PCR

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. We RT-PCR amplified DNA fragments using the pan-specific CYP primers. Sequencing and comparison to the GenBank database identified the PCR products, and accordingly the metabohc enzymes which are up-regulated.

It is found that expression of CYPIAI is induced by Tacrolimus, and that CYPIAI is up-regulated by exposure to Cyclosporin as compared to controls.

The resutls are verified to be up-regulated by the macrolactam treatment as above using specific primers based on the known sequences deposited in GenBank. Example 6. Characterization of Metabolic Enzyme Induction Induced by Macrolactam (in vitro)

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 lOμL 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.

12 untreated SE cultures are used as controls with the first set of 3 withdrawn before treatment (day 0). At the completion of the treatment regimes RNA is isolated from the SE's using the RNeasy kit according to the instraction of the supplier (Qiagen Inc.). Semi-quantitative RT-PCR, primers and identification of macrolactam up-regulated CYP's are identical to the method above.

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 coπect 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. Furthermore, CYPIAI is seen to be up- regulated by exposure to Cyclosporin as compared to contiols, as shown in Lanes 37 and 38.

Results are also shown in Example 3.

We also RT-PCR amplify DNA fragments using the pan-specific CYP primers. Sequencing and comparison to the GenBank database identified these PCR products as CYPIAI. Thus, 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, in particular CYPIAI, may be used to alleviate or reheve tachyphylaxis against macrolactams, in particular tacrolimus and cyclosporin.

Example 7. In Vitro Model of Vitamin D Analogue Tachyphylaxis

In this and the foUowing Examples, 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. in Steroids 66:409-422, 2001 and in Steroids 66:451-462, 2001) and Compound C: N-[4-(hex-l-yl)benzoyl]-2-(lH-imidazol-l-yl)- 2-(phenyl)-l -amino ethane.

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). We verify that an adjunct to vitamin D analogue treatment counters a tachyphylactic response in human in vitro 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. However, the SE cultures are challenged on day 5 with hypeφroliferative stimuli by adding 1 μM retinoic acid or nothing (control) to the media. The SE cultures are maintained for 2 more day before analysis

The ceUular moφhology of all SE cultures are assessed for signs of hypeφlasia. Prohferation is assessed moφhologically by microscopic inspection of colony growth (spread) and by histology on chosen samples. On the final day of the experiments and RNA is isolated using the RNeasy kit as above.

An overview of the experiment foUows

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. Primers used are shown in the Table below.

Table. Primer sequences used to amplify differentiation and prohferation markers for RT-

PCR

We also examine if expression of vitamin D target genes are affected by the treatments. Accordingly, primers specific for vitamin D receptor (VDR), CYP24 p21^{cipl Wafl}, c-Fos and β3- integrin are included among the RT-PCR's. Skin irritation is a facet of tachyphylaxis and the expression of irritation markers such as ILl-α; IL-6; IL-8; TNF-α; GM-CSF; TGF-β is examined by RT-PCR as above.

Results

We find that various 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 hypeφroliferative 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-catøbolizing CYP's.

The response to the hypeφrohferative 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. Furthermore, continuous vitamin D analogue treatment results in upregulation of ILl-α; IL-6; IL-8; TNF-α; GM-CSF; TGF-β, markers of skin irritation. This response is diminished by co-administration of CYP inhibitors with the vitamin D analogues.

Thus, CYP24 is upregulated in the presence of calcitriol and further upregulated by a combination of calcitriol and inhibitors (ketoconazole or compound A-C).

Furthermore, 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.

FinaUy, CYP24 is not upregulated by Calcipotriol with or without inhibitors.

Results of this Experiment are shown in Figure 1.

Kl (55°c) Amplification = 645bp

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 (samples 7 - 12) shows similar effects.

Accordingly, Inhibition of CYP24 may therefore be used to inhibit the degradation of calcitriol and tacalcitol and hence inhibit tachyphylaxis to these compounds.

K14 (55°C) Amplification =104bp

Amphfication of 104bp occuπs in all samples. Reduced levels are observed in samples 13/14 (calcitriol + ketoconoazole), samples 21/22 (calcitriol + compound C) and 35 and 36 (tacrolimus + ketoconazole).

This shows that inhibition of CYP24 by compound C and ketoconazole reduces the abihty of these skin equivalents to prohferate, because the compounds tacalcitol, and calcitriol are stiU able to cause differentiation.

This experiment therefore shows that inhibition of CYP ' s by ketoconazole reducs the abUity of these skin equivalents to proliferate because tacrolimus is stiU able to cause differentiation.

CYPIAI (58°C) Amplification^ 407bp

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.

TNF (59°C). Amplification^ 429bp

The CYP24 inhibitors Compound A, B and C however show no irritancy.

Therefore, in vitro cultured human skin equivalents exposed to both vitamin D analogue and adjunct enzyme inhibitor are found to show a persistent response to vitamin D analogue treatment with lower associated irritancy and no tachyphylactic response.

Example 8. In Vitro Model of Corticosteroid Tachyphylaxis

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). We verify that an adjunct to corticosteroids treatment counters a tachyphylactic response in human in vitro 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. However, the SE cultures are challenged on day 5 with hypeφroliferative stimuh by adding 1 μM retinoic acid or nothing (contiol) to the media. The SE cultures are maintained for 2 more day before analysis.

The ceUular moφhology of all SE cultures are assessed for signs of hypeφlasia. Prohferation is assessed moφhologicaUy by microscopic inspection of colony growth (spread) and by histology on chosen samples. On the final day of the experiments and RNA is isolated using the RNeasy kit as above.

An overview of the experiment foUows

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. We also examine if expression of corticosteroid target genes are affected by the tieatments so primers specific for the glucocorticoid and mineralocorticoid receptors are included among the RT-PCR's.

Primers are designed to amplify various glucocorticoid target genes (for example, see the

URL www.bionet.nsc.ru/trrd/33/gluc.htm). For example, 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. Veπey 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-α; IL-6; IL-8; TNF-α; GM-CSF; TGF-β are examined by RT-PCR as above.

Results

We find that corticosteroid- catabolizing enzymes are up-regulated by corticosteroid treatment (see above). SE cultured in the continuos presence of corticosteroids responds to the hypeφroliferative 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 hypeφrohferative chaUenge is abohshed when CYP inhibitors) are co-aαrninistered 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. Furthermore, continuous corticosteroid treatment resulted in up-regulation of ILl-α; IL- 6; IL-8; TNF-α; GM-CSF; TGF-β, markers of skin irritation. This response is diminished by co-administration of CYP inhibitors with the corticosteroid. Thus, in vitro cultured human skin equivalent exposed to both corticosteroid and adjunct enzyme inhibitor shows a persistent response to corticosteroid treatment with lower associated irritancy and no tachyphylactic response.

Example 9. In Vitro Model of Macrolactam Tachyphylaxis

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). We verify that an adjunct to macrolactam treatment counters a tachyphylactic response in human in vitro 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. However, the SE cultures are challenged on day 5 with hypeφroliferative stimuh by adding 1 μM retinoic acid or nothing (control) to the media. The SE cultures are maintained for 2 more day before analysis.

An overview of the experiment foUows:

The ceUular moφhology of all SE cultures are assessed for signs of hypeφlasia. Prohferation is assessed moφhologically by microscopic inspection of colony growth (spread) and by histology on chosen samples. On the final day of the experiments and RNA is isolated using the RNeasy kit as above.

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-α; IL-6; IL-8; TNF-α; GM- CSF; TGF- β are examined by RT-PCR as above.

Results

The macrolactam -catabolizing enzyme CYPIAI is up-regulated by macrolactam tieatment (see above). SE cultured in the continuous presence of macrolactams responded to the hypeφroliferative 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. The response to the hypeφrohferative chaUenge is abohshed when CYP inhibitors) is co-administered with the macrolactam's. This demonstrates that tachyphylaxis to macrolactam's can be abolished by CYP inhibitors adjunct to the macrolactam regime. Furthermore, continuous macrolactam treatment results in up-regulation of IL 1 - α ; IL-6; IL-8; TNF-α; GM-CSF; TGF-β, markers of skin irritation. This response is dirninishedby co- administration of CYP inhibitors with the macrolactams.

We therefore find that in vitro cultured human skin equivalent exposed to both macrolactams and adjunct enzyme inhibitor shows a persistent response to macrolactam treatment with lower associated irritancy and no tachyphylactic response.

Example 10. In Vivo Verification of Prevention of Tachyphylaxis and Testing in an Animal Model for Psoriasis (Nude Mice)

The findings of enzyme induction, differentiation/proliferation control and irritancy from the Examples above are verified in vivo. Based upon the data from the above examples, suitable enzyme inhibitors are chosen for co-administration with the drags to circumvent the tachyphylactic responses exhibited above.

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.

Results

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.

We find that the 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 response to the hypeφrohferative 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 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-α; IL-6; IL-8; TNF-α; GM-CSF; TGF-β, markers of skin irritation. This response is diminished by co-administration of CYP inhibitors with the vitamin D analogues.

Therefore, 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.

Example 11. in Vivo Verification of Prevention of Tachyphylaxis and Testing in Animal Model for Psoriasis (Mouse Tail Test)

The mouse taU model is a moφhometiy-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. 0.1% Dexamethasone ointment + 1% ketoconazole, 11. 0.3%> Tacrolimus ointment, 120.4% Ascomycin ointment, 13. 0.3%) Tacrolimus ointment + 1% ketoconazole, 14. 0.4% Ascomycin ointment + 1% ketoconazole, and 15. 1% ketoconazole treatment of mice on an 25(OH)D3 enriched diet (4 rnicrogram/kilo body weight/day).

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)D₃ enriched diet.

Example 12. Demonstration of Prophylactic Effect of Metabolic Enzyme Antagonists in vitro

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)D₃ 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)₂D₃ in the media is examined and found to be elevated in the presence of the inhibitors). Furthermore, addition of 0.5 micromolar 25(OH)D₃ is found to elevate the expression of the differentiation markers, as well as the concentration of 1 ,25(OH)₂D₃ in the media.

Figure 2 shows the results of this experiment.

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 . Thus a tachyphylactic response could be prevented by inhibition of CYP24.

Example 13. Demonstration of Prophylactic Effect of Metabolic Enzyme Antagonists in vivo

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)D₃ enriched diet.

CYP24-specific inhibitor used comprise Compounds A, B, and C.

The presence of orthokeratosis is observed in the ketoconazole tieated mice. Othokeratosis is also observed in mice treated with CYP24 specific inhibitors.

The l,25(OH)₂D₃ concentration in treated skin is examined and found to be elevated by the inhibitors). TABLE 2

Enzyme Substrates Inhibitor Inducer Expression sites

CYPIAI

Skin, Liver, Lung

TDiliberto JJ et al., Toxicol. & App. Pharm. 130:197-208, 1995

Kidney, Skin, Liver, Lung, tSantostefano et al., Tox. & App. Pharm. 151:294-310, 1998

17beta-estradiol (1 :st)(2-hydroxylation)

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD); 2,4-dichlorophenoxyacetic acid (2,4-D); dield (DED)

Liver, Kidney, Mammary tissue

TBadawi AF Carcinogenesis 21:1593-9, 2000

TCDD

Skin tReiners JJ Jr et al„ Mol. Carcinogenesis 21:135-46, 1998

Skin (mouse, induction by pyridine) TAgarwal R et al., BBRC 199:1400- 6, 1994

Fenarirnol

Kidney

TPaolini M et al.. Cancer Letters 101:171-8, 1996

Stomach (human) tTatemichi M et al.. Cancer Research 59:3893-8. 1999

3-methylcholanthrene (3MC)

Liver (esp. centriportal); Lung

(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

(renal coφuscles); Ovary (esp. meduUa); Endothelial ceUs of blood vessels (whole organism) tDey A et al., Biochem. Pharm. 58:525-37, 1999

SmaU intestine (human) tZhang QY et al.. Drag Metabolism

& Disposition 27:804-9, 1999

Colon; Rectum

TMercurio MG et al., BBRC

210:350-5, 1995

Clobetasol

Skin tLi XY et al., Carcinogenesis 16:519-24, 1995

CYP1A1/2

Phenacetin tEchizen H et al.. Drag Met. & Disposition 28:937-944, 2000

Liver, Skin t Taras- Valero D. et al.. Environmental & Molecular Mutagenesis.

35(2): 139-49, 2000

Transformed keratinocytes, oral and cervical epithehal cells TFarin FM et al, Carcinogenesis 16:1391-401, 1995

CYPIA2

Clozapine (l:st)

Caffeine, Erythromycin

Carbamazepine, Tobacco smoke TTaylorD, Brit. J. Psychiatry 171:109-112. 1997

Ethanol & Exercise

Lung (Rat)

TArdies CM et al„ Cancer Letters 103:209-18, 1996

m-Xylene

Lung tFov JW et al„ J. Tox. & Environmental Health 47:135-44. 1996

Theophylline ethylenediamine (l:st)

Fluvoxamine TRasmussenBB et al.. Therapeutic Drag Monitoring 19:56-62, 1997

Terbutaline (?, inhibits theophylline clearance) TUptonRA Clinical Pharmacokinetics 20:66-80, 1991

Tamoxifen (2:nd)

TMani C et al.. Drag Met. & Disposition 21:645-656, 1993

Methoxalen (8-Methoxypsoralen)(2:nd) tSeUers EM et al.. Clinical Pharm. & Therapeutics 68:35-43, 2000

Liver

TTaras- Valero D. et al.. Environmental & Molecular Mutagenesis.

35(2): 139-49, 2000

Liver

TSantostefano et al„ Tox. & App.

Pharm. 151:294-310. 1998

Hetercyclic amines (cooked-food mutagens)

Liver specific tOvervik E et al.. Princess Takamatsu Symposia 23:123-33, 1995

Stomach (human, pyloric gland ceUs)

tTatemicfii M et al. Cancer Research 59:3893-8. 1999

3-methylcholanthrene (3MC)

Liver (constitutive & inducible, esp centrilobular); Lung (Clara type 1 Duodenum (esp. viUous epithelia & cells suπounding glands in lamina propria); Kideney (renal coφuscles) tPey A et al., Biochem. Pharm. 58:525-37, 1999

Colon; Rectum

TMercurio MG et al.. BBRC 210:350-5. 1995

Acetaminophen (ring); Antipyrine (4,3-methyl); Bufuralol (1 and 4 others); Ondansetion (7,8); Phenacetin; Tacrine; Tamoxifen (N- demethylation); Theophyline (1,3,8); Warfarin

? Guengerich FP, Human Cytochrome P450 Enzymes, in Cytochrome P450: Stracture, Mechanism, and Biochemistry, Pg.473-535. P.R. Ortiz de MonteUano, Plenum Press, New York, 1995

CYPIBI

Cancers of the breast, colon, lung, esophagus, skin, lymph node, brai testis tMurrav et al. Cancer research 57:3026-31, 1997

17beta-estradiol (l:st)(4-hydroxylation)

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD); 2,4- dichlorophenoxyacetic acid (2,4-D); dieldrin (DED)

Liver, Kidney, Mammary tissue Badawi AF Carcinogenesis 21:1593-9, 2000

TCDD

Skin

TReiners JJ Jr et al., Mol. Carcinogenesis 21:135-46. 1998

SmaU intestine (human)

Tzhang QY et al., Drag Metabohsm

& Disposition 27:804-9. 1999

Enzyme Substrates Inhibitor Inducer Expression sites

CYP2A3

Hetercyclic amines (cooked-food mutagens)

Lung (rat & mouse)

TOvervik E et al„ Princess Takamatsu Symposia 23:123-33, 1995

Coumarin

Nasal epithelium (rat)

TBereziat JC et al., Mol. Carcinogenesis 14:130-9, 1995

CYP2A5 (Coumarin 7-hydroxylase) Coumarin

Griseofulvin, Thioacetamide, Aminotriazole TSalonpaa P et al., Naunyn-Schmiedebergs Archives of Pharmacology 351:446-452, 1995

CYP2A6

Nicotine, Coumarin

Methoxalen (8-Methoxypsoralen) TSeUers EM et al., Clinical Pharm. & Therapeutics 68:35-43, 2000

CYP2B

Ethanol & Exercise

Lung (Rat) TArdies CM et al.. Cancer Letters 103:209-18, 1996

CYP2B1

Phenobarbital

TKemper B, Prog. Nucleic acid Res. & Mol. Biol.

61:23-64, 1998

skin (mouse, induction by pyridine) TAgarwal R et al., BBRC 199:1400- 6, 1994

Fenarimol

Liver, Kidney, Lung (mouse) TPaohni M et al. Mutation res. 368:27-39, 1996

m-Xylene

Lung

TFov JW et al., J. Tox. & Environmental Health 47:135-44, 1996

CYP2B2

Phenobarbital

TKemper B, Prog. Nucleic acid Res. & Mol. Biol.

61:23-64, 1998

CYP2B6

Cyclophosphamide; Benzyloxyresorufirr, Pentoxyresorufin

Phenobarbital; Cyclophosphamide

Liver, Brain, hitestine, Kidney, Trachea TGervot L et al„ Pharmacogenetics 9:295-306, 1999

CYP2B10

Phenobarbital

TKemper B, Prog. Nucleic acid Res. & Mol. Biol.

61:23-64, 1998

CYP2B12

Arachidonic acid

Skin specific, high constitutive expression.

TKeenev PS et al. JBC 273:9279-84, 1998

CYP2C enzymes

Valproate (broad spectrum inhibitor of CYP2C's)

TAnderson GD, Annals of Pharmacothreapy 32:554-563. 1998

Liver, skin, kidney, intestine, and lung of SCID mice TRajasenan RS et a .Tox. & App. Pharm. 135(11:89-99, 1995

Uterine endometrium (human) THukkanen J. et al., Pharm. & Tox. 82:93-7, 1998

SmaU intestine (human)

Tzhang QY et al., Drag Metabohsm

& Disposition 27:804-9, 1999

CYP2C6

Tamoxifen (2:nd)

TMani C et al.. Drag Met. & Pisposition 21:645-656, 1993

CYP2C8

All- trans Retinoic acid (l:st until induction of CYP26)

Human hver, HepG2 ceUs

TMcSorlev LC et al.. Biochemical Pharm. 60:517-526, 2000

AU-trans Retinoic acid (l:st)(forms 4-OH-RA) Pachtaxel, Oiclofenac TNadinL, et al., Biochem. Pharm. 58:1201-1208, 1999

Retinoic acid, Retinol, Tienihc acid, Tolbutamide (methyl)

TGuengerich FP, Human Cytochrome P450 Enzymes, in Cytochrome P450: Stracture, Mechanism, and Biochemistry, Pg.473-535. Ed. P.R. Ortiz de Montellano, Plenum Press, New York, 1995

CYP2C9

Fluoxetine, Losartan, numerous NSAIO's, Phenytoin, Tolbutamide, Torsemide, S- Warfarin Aminodarone, Fluconazole, Phenylbutazone, Sulphinpyrazone,

Sulphaphenazole, some other Sulphonamides

Rifampicin, Carbamazepine?, Ethanol?, Phenobarbitone? TMrners et al., Brit J. Clin. Pharm. 45:525-538. 1998

HTV protease inhibitors (2:nd)(Ritonavir, hidinavir, Saquinavir) TBarrv Metal., Clinical Pharmacokinetics 32:194-209, 1997

Phenytoin (l:st)(hydroxylated to p-HPPH)

Sulfaphenazole, Phenylbutazone, Fluconazole, azapφazone, Cotrimoxazole, Propoxyphene, Miconazole, Amiodaron

Disuhram, Metionidazole, Stiripentol TLewRH EpUepsia 36fSuppl 5 :S8-13, 1995

Phenytoin, Warfarin, Sulfamethoxazole, Losartan

Fluconazole, Miconazole, Sulfamethoxazole TVenkatakrishnan K et al.. Clinical Pharmacokinetics 38:111-180.2000

Tolbutamide

TEchizen H et al., Prug Met & Pisposition 28:937-944. 2000

AU-trans Retinoic acid (2:nd)

Human hver, HepG2 ceUs TMcSorley LC et al. Biochemical Pharm. 60:517-526, 2000

Sulfaphenazole, Tolbutamide, Torasemide

TNadin L, et al, Biochem. Pharm. 58:1201-1208, 1999

Tamoxifen (4-hydroxylation)

Liver (human)

TCrewe HK et al, Biochem. Pharm. 53:171-8, 1997

Piclofenac (4), Hexobarbital (3), ibuprofen (methyl, 2), Losartan (aldehyde, acid),

Mefanamic acid (3-methyl), Mephobarbital, Phenytoin, Piroxicam, Tenoxicam, Tienilic acid,

Tolbutamide (methyl), Trimethadone (N-demethylation), (S)- Warfarin (7)

TGuengerich FP, Human Cytochrome P450 Enzymes, in Cytochrome P450: Structure, Mechanism, and Biochemistry, Pg.473-535. Ed. P.R. Ortiz de Montellano, Plenum Press, New York, 1995

CYP2C11 (constitutively expressed, male specific)

SKF-525A TChang TK et al, J. Pharm. & Exp. Therapeutics 274:270-275, 1995

Tamoxifen (2:nd)

TMani C et al, Prag Met. & Pisposition 21:645-656, 1993

CYP2C18

Tienilic acid, Tolbutamide (methyl)

TGuengerich FP, Human Cytochrome P450 Enzymes, in Cytochrome P450: Stracture, Mechanism, and Biochemistry, Pg.473-535. Ed. P.R. Ortiz de MonteUano, Plenum Press, New York, 1995

CYP2C 19 (S-mephytoin hydroxylase) Phenytoin (2:nd)

Felbamate, Omeprazole, Cimetidine, Fluoxetine, Imiprarnine, Oiazepam TLewRH EpUepsia 36(Suppl 5):S8-13, 1995

Felbamate

T Anderson GP, Annals of Pharmacothreapy 32:554-563, 1998

Topiramate

TAnderson GP, Annals of Pharmacothreapy 32:554-563. 1998

Fluconazole

TVenkatakrishnan K et al. Clinical Pharmacokinetics 38: 111-180, 2000

Mephenytoin

TMeyer UA et al. Ann. Rev. Pharm. & Toxicol 37:269-296. 1997

S-mephenytoin

TEchizen H et al, Prag Met & Pisposition 28:937-944.2000

Piazepam (N-demethylation), (S)-Mephenytoin (4), Νirvanol, Omeprazole

TGuengerich FP, Human Cytochrome P450 Enzymes, in Cytochrome P450: Stracture, Mechanism, and Biochemistry. Pg.473-535. Ed. P.R. Ortiz de Montellano. Plenum Press. New York, 1995

CYP2P6

HJV protease inhibitors (2:nd)(Ritonavir, Indinavir, Saquinavir) TBarry Metal. Clinical Pharmacokinetics 32:194-209. 1997

Clozapine (2:nd)

TTaylorP, Brit J. Psychiatry 171:109-112, 1997

Haloperidol

Haloperidol TKudo S, etal. Clinical Pharmacokinetics 37: 435-456. 1999

Nortriptyhne, Pesipramine, Peφhenazrne, Metoprolol, Encainide, Propafenone

Terbinafine

TVenkatakrishnan K et al.. Clinical Pharmacokinetics 38:111-180, 2000

'Debrisoquine-Sparterne polymoφhism"

TMever UA et al, Ann. Rev. Pharm. & Toxicol 37:269-296. 1997

Codeine (forms moφhine)

TEichelbaum M et al.. Clin. & Exp. Pharm. & Phvsiol 23:983-985, 1996

Sparteine

TEchizen H et al. Drag Met. & Disposition 28:937-944, 2000

Transformed oral and cervical epithehal ceUs TFarinFM et al, Carcinogenesis 16:1391-401, 1995

Tamoxifen (4-hydroxylation)

Liver (human)

TCrewe HK et al. Biochem. Pharm. 53:171-8, 1997

Tricyclic SSRI antidepressants

Fluoxetine TBonin B et al, Encephale 22:221-7, 1996

SmaU intestine (human)

TZhang QY et al. Drag Metabohsm

& Disposition 27:804-9, 1999

Ajmahne, Amiflamine, Amitriptyline, Apridine, Bufuralol (1), Bupranolol, CGP 15210 G (bis[cts^,-3- hydroxy-4-(2,3-dimethly-phenoxy)piperidine]sulfate), Cloimpramine, Clozapine, Codeine,

Debrisoquine, Deprenyl, Desipramine, Dextromethoφhan (O-demthylation), Encainide, Flecainide, Guanoxan, Haloperidol, Hydrocodone, Imipramine, mdoramin, MDL 73005 (8-[[2-(2,3-ά hydro-l,4-berizo-dioxm-2-yl)me ylammo]ethyl]-8-azaspho[4,5]decane-7,9-dione) (M₂,

M₄), 4-Methoxyamphetamine, Methoxyphenamine, Metiopolol, MexUetine, Minaprine, Nortriptyhne, Paroxetine, Perhexiline, Peφhenazine, Phenformin, Propafenone, N-Propylajmaline, Propanolol, Sparteine, Thioridazine, Timolol TGuengerich FP, Human Cytochrome P450 Enzymes, in Cytochrome P450: Stracture, Mechanism and Biochemistry, Pg.473-535. Ed. P.R. Ortiz de MonteUano, Plenum Press, New York, 1995

CYP2E1

Chlorzoxazone, Trimethadione

TTanaka E. et al, J. Clrn. Pharm. & Therapeutics 25:165-175. 2000

p-nitiophenol

TEchizen H et al. Drag Met. & Disposition 28:937-944.2000

trans- 1,2-dichloroethylene (DCE)

TMathews JM. et al. Xenobiotica. 28(8):767-77, 1998

Dexamethasone, Salicylic acid Skin TSampol E. et al, BBRC 235(3 :557-61, 1997

Transformed oral and cervical epithehal ceUs

TFarin FM et al., Carcinogenesis 16:1391-401, 1995

Uterine endometrium (human)

THukkanen J. et al., Pharm. & Tox.

82:93-7, 1998

Ethanol & Exercise

Lung (Rat)

TArdies CM et al. Cancer Letters 103:209-18, 1996

Fenarimol

Lung

TPaolini M et al. Cancer Letters 101:171-8. 1996

m-Xylene

Lung

TFov JW et al. J. Tox. & Environmental Health 47:135-44. 1996

Ethanol

Gastric mucosa

TSeitz HK et al.. Alcohol & Alcoholism 32:543-9. 1997

SmaU intestine (human)

Tzhang QY et al., Drag Metabohsm

& Disposition 27:804-9, 1999

Liver (rat)

TElkahwaji J et al., Biochem

Pharm. 57:951-4, 1999

Acetaminophen (ring), Caffeine (8?), Chlorzoxazone (6), Enflurane

TGuengerich FP, Human Cytochrome P450 Enzymes, in Cytochrome P450: Stracture, Mechanism and Biochemistry, Pg.473-535. Ed. P.R. Ortiz de Montellano. Plenum Press, New York, 1995

Enzyme Substrates Inhibitor Inducer Expression sites

CYP3 A isoenzymes

Clarithromycin (l:st)(macrolide antibacterial, formation of active 14-hydroxyclarithromycin), Astemidole (l:st), Cisapride (l:st), Pimozide (l: Midazolam (l:st), Triazolam (l:st)

Omeprazole, Ritonavir

Rifampicin, Rifabutin TRodvold KA. Clinical Pharmacokinetics 37:385-389. 1999.

Cyclosporine

Ketoconazole, Ifraconazole, Selective Serotonin Reuptake Inhibitors (SSRI's)

Gasfrointestinal tract Tvon Moltke LL et al. Clinical Pharmacokinetics 29(Suppl l):33-43, 1995

EpipodophyUotoxins, Ifosphamide, Tamoxifen, Taxol, Vinca alkaloids TKivisto KT et al., Brit J Clin. Pharm.40:523-530, 1995

Cyclosporine

Liver, SmaU intestine TChristians U et al., Pharm. & Therapeutics 57:291-345. 1993

All-trans Retinoic acid

Troleandomycin (CYP3A selective), Parathion, Quinidine, Ketoconazole

TNadinL, et al, Biochem. Pharm. 58:1201-1208. 1999

No cutaneous expression

TSampol E. et al. BBRC 235(3):557-61. 1997

Liver, skin, kidney, intestine, and lung of SCID mice TRaiasenan RS etal.Tox. & App. Pharm. 135 l):89-99. 1995

Corticosteroids

TPichard et al.. Mol Pharm. 41:1047-55, 1992

skin (mouse, induction by pyridine) TAgarwal R et al. BBRC 199: 1400- 6, 1994

Fenarimol

Liver, Kidney, Lung (mouse) TPaolini M et al. Mutation res. 368:27-39, 1996

Tacrolimus

Troleandomycin; Ketoconazole

Duodenum > Jejunum > Ileum > Colon > Stomach TLampen A et al., British J. Pharm. 117:1730-4, 199

Betamethasone

TAbel SM et al. J. Steroid Biochem. & Mol Bio. 46:827-32, 1993

CYP3A1

Tamoxifen (l:st)

TMani C et al. Drag Met. & Disposition 21:645-656. 1993

CYP3A3

Colon; Rectum

TMercurio MG et al. BBRC 210:350-5, 1995

CYP3A3/4

Carbamazepine

T Ketter et al. Cellular & Molecular Neurobiology 19:511-532, 1999.

CYP3A4 (the main drag metabolizing CYP in humans)

HJV protease inhibitors (l:st)(Ritonavir, mdinavir, Saquinavir), Azole antifungals, Macrohde antibiotics, Dapsone

Carbamazepine, Phenobarbital, Phenytoin, Rifabutin, Rifampicin

TBarrv M et al. Clinical Pharmacokinetics 32:194-209, 1997 Carbamazepine

Stiripentol, Remacemide, Acetazolamide, Macrohde antibiotics, Isoniazid, Metionidazole, VerapamU, DUtiazem,

Cimetidine, Danazole, Dextiopropoxyphene

Carbamazepine, Phenytoin, Phenobarbital, Primidone

TSpin et al, Clinical Pharmacokinetics 31:198-214, 1996

Felbamate

T Anderson GD, Annals of Pharmacothreapy 32:554-563, 1998

Carbamazepine (1 :st)(formation of active CBZ-10, 11-epoxide)

Erythromycin, Troleandomycin, Clarithromycin, Josamycin, Hurythromycin, Ponsinomycin

TLewRH EpUepsia 36(Suppl 5 :S8-13, 1995

Troleandomycine, Erythromycin

Liver, Enterocyte

Tvon RosensteU NA et al.. Drag

Safety 13:105-122

Terfenadine, Astemizole, Cisapride, Pimozide, some 3-hydroxy-3methylglutaryl-coenzyme A inhibitors, SUdenafil, Midazolam, Triazolam, Alprazolam, Diazepam, Zopiclone, Buspirone, Carbamazepine, Ergotamine, Cyclosporin

Ifraconazole, Ketoconazole, Clarithromycine, Erythromycin, Nefazodone,

Ritonavir, Grapefruit juice TDresser GK Clinical Pharmacokinetics 38:41-57, 2000

Haloperidol (2:nd)

TKudo S, et al. Clinical Pharmacokinetics 37: 435-456, 1999

Zolpidem

TChournard G et al., CeUular & Molecular Neurobiology 19:533-552. 1999

Cyclosporine, Tacrohums, Alprazolam, Triazolam, Midazolam, Nifedipine, Felodipine, Simvastatin, Lovastøtin, Vincristine, Terfenadine, Astemizole

Ketoconazole, Itiaconazole, Fluconazole (weak inhibition) TVenkatakrishnan K etal.. Clinical Pharmacokinetics 38:111-180, 2000

Rifabutin

TBenedetti MS, Pharm. Res. 32:177-187, 1995

Pisopyramide

SKF-525A, Troleandomycin

TEchizen H et al. Prug Met & Pisposition 28:937-944, 2000

Ascomycine? (SPZ ASM 981)

TMollisonKW et al. Toxicology 125:169-181, 1998

Tacrolimus (FK506)

Nelfinavir TSchvarcz R et al. Transplantation 69:2194-2195, 2000

AU-trans Retinoic acid (2:nd)

Human hver, HepG2 ceUs

TMcSorley LC et al. Biochemical Pharm. 60:517-526. 2000

Salmeterol (l:st), Midazolam

Ketoconazole

Liver (human)

TManchee et al, Prag Metabolism & Pisposition 24:555-9, 1996

Tamoxifen (4-hydroxylation & N-demethylation)

Liver (human)

TCrewe HK et al, Biochem. Pharm. 53:171-8, 1997

Uterine endometrium (human) THukkanen J. et al., Pharm. & Tox. 82:93-7, 1998

Stomach (human, pyloric gland mucosa with intestinal metaplasia only) TYokose T et al, Jap. J. Cancer Res.

& Pisposition 27:804-9, 1999

Ritonavir; Indinavir (HIV protease inhibitors)

SmaU intestine (human) TKoudriakova T et al, Prag Metabohsm & Pisposition 26:552-61, 1998

Acetaminophen (quinone formation), AlfentanU (noralfentanU), Alpidem (propyl ,β), Amiodarone (N-deethylation), Bayer R4407 [+K8644 (dUiydropyridine), Bayer R5417 [-K8644] (dihydropyridine), Benzphetamine (N-demethylation), Budesonide (6β), Codeine (N-demethylati Cortisol (6β), Cyclophosphamide (high Km), Cyclosporin A (AM9, AMI, AM4Ν: nomenclature formerly Ml, M17, M21), Cyclosporin G, Papsone (N), Pehydroepiandrosterone-3-sulfate (16α), Pextromethoφhan (N-demethylation), Piazepam (3), PUtiazem, Ebastine (alcohol), CQA 206-291, Erythromycin (N-demethylation), 17β-Estiadiol (2,4), 17α-Ethynylestradiol (2), Felodipine (dmydropyridine), FK506, Gestodene, hmpramine (N-demethylation), Lidocaine (N-deethylation), Losoratn (aldehyde, acid), Lovastatin (6β, 6-e o-methylene,3,3,5- dihydrodiol), MPL 73005 (8-[[2-(2,3-d ydro-l,4-benzo-dioxm-2-yl)metfy^ (M₂, M_s),

Midazolam (1,4), Nifedipine (dihydropyridine), NUudipine (dihydropyridine), Nimodipine (dihydropyridine), Nisoldipine (dihydropyridine), Nitrendipine (dihydropyridine), Omeprazole, Progesterone (6β, 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 (6β, trace, 15β, 2β), Triazolam, Trimethadone (N-demethylation), Troleanomycin (Ν), NerapamU, Warfarin (R-10, S-dehydro Zatosetion (Ν), Zonisamide (Sites of oxidation are shown in brackets) TGuengerich FP, Human Cytochrome P450 Enzymes, in Cytochrome P450: Stracture, Mechanism, and Biochemistry, Pg.473-535. Ed. P.R. Ortiz de MonteUano, Plenum Press, New York, 1995

CYP3A5 (85% homologous to 3A4)

Uterine endometrium (human)

THukkanen J. et al., Pharm. & Tox.

82:93-7, 1998

Kidney (human, the main CYP3A isoform) TdeWUdt SN et al. Clinical Pharmacokinetics 37:485-505, 1999

SmaU intestine (human)

TZhang QY et al, Prag Metabohsm

& Pisposition 27:804-9. 1999

Ritonavir; mdinavir (HTV protease inhibitors)

SmaU intestine (human) TKoudriakova T et al. Prag Metabohsm & Pisposition 26:552-61. 1998

CYP3A7

Liver (human, the main CYP3 A isoform in embryonic -> newborn hver)

TdeWUdt SN et al. Clinical

Pharmacokinetics 37:485-505. 1999

Enzyme Substrates Inhibitor Inducer Expression sites

CYP4B1 m-Xylene

Lung

TFov JW et al, J. Tox. & Environmental Health 47: 135-44, 1996

Enzyme Substrates Inhibitor Inducer Expression sites

CYP24 (1 ,25(OH)₂O₃ 24-hydroxylaes) l ,25(OH)₂O₃ lα,25(OH)₂O₃

Keratinocytes (Thuman) THarant H et al, J. Cell. Biochem. 78: 112-20. 2000

Enzyme Substrates Inhibitor Inducer Expression sites

CYP26 (Retinoic acid 4-hydroxylase)

AU-tians Retinoic acid (not 13-cis & 9-cis)(to 4-OH-RA and 18-OH-RA) Liarozole

Retinoic acid

Breast cancer ceUs (T-47O) TSonnevald E et al, CeU Growth & Piff. 9:629-637, 1998

Retinoic acid

RI 15866

Retinoic acid

Rat hver TStoppie P et al, J. Pharm. & Exp. Therapeutics 293:304-312, 2000

AU-tians Retinoic acid (1 :st after induction)

Ketoconazole

Retinoic acid

Human hver, HepG2 ceUs

TMcSorley LC et al. Biochemical Pharm. 60:517-526, 2000

Enzyme Substrates Inhibitor Inducer Expression sites

UGT (Uridine diphosphate GlucuronosylTransferase)

Valproate (broad spectrum inhibitor of UGT enzymes) TAnderson GP, Annals of Pharmacothreapy 32:554-563, 1998

Carbamazepine

TSpina et al, Clinical Pharmacokinetics 31:198-214. 1996 Glucuronidation enzyme (unknown to CB) Haloperidol (l:st) TKudo S. et al. Clinical Pharmacokinetics 37: 435-456, 1999

Salbutamol (to sulphate conjugates), Sahnetiol (to sulphate conjugates) TSauer MJ et al, Xenobiotica 29:483-497, 1999

CYP Marker substrate Selective Inhibitors

1A2 Phenacetin O-deethylation 7,8-Benzoflavone

7-ethoxyresonrfin Fluvoxarnine

FurafyUine

2A6 Coumarin 7-hydroxylation Piethyldithiocarbamate

2C9 Tolbutamide (methyl) hydroxylation Sulfaphenazole

2C19 (S)-Mephenytoin 4-hydroxylation

2P6 Birfuralol 1 -hydroxylation Quinidine

Oebrisoquine 4-hydroxylation Ajmahcine

2E1 Chlorzoxazone 6-hydroxylation 4-Methylpyrazole

Piemyldithiocarbamate

3A4 Nifedipine oxidation Gestodene

Troleandomycin

4A11 Laurie acid 12-hydroxylation

7 Cholesterol 7α-hydroxylation

TGuengerich FP, Human Cytochrome P450 Enzymes, in Cytochrome P450: Structure, Mechanism, and Biochemistry, Pg.473-535. Ed. P.R. Ortiz de MonteUano, Plenum Press, New York, 1995.

Each of the applications and patents mentioned above, and each document cited or Each of the applications and patents mentioned in this document, and each document cited or referenced in each of the above applications and patents, including during the prosecution of each of the apphcations and patents ("application cited documents") and any manufacturer's instractions or catalogues for any products cited or mentioned in each of the apphcations and patents and in any of the application cited documents, are hereby incoφorated herein by reference. Furthermore, all documents cited in this text, and aU documents cited or referenced in documents cited in this text, and any manufacturer's instractions or catalogues for any products cited or mentioned in this text, are hereby incoφorated herein by reference.

Various modifications and variations of the described methods and system of the invention wiU be apparent to those skUled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific prefeπed embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skUled in molecular biology or related fields are intended to be within the scope of the claims.

Claims

1. A method of alleviating or preventing a tachyphylactic response to an agent in an individual, the method 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.

2. An antagonist of a metabolic 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.

3. A method according to Claim 1 , or an antagonist of a metabolic enzyme according to Claim 2 for a use as specified therein, in which the antagonist inhibits the breakdown of the agent by decreasing the amount or activity, or both, of the metabolic enzyme.

4. A method or an antagonist according to any preceding claim, in which the antagonist is administered locally to a part of the individual which is exhibiting a tachyphylactic response, or a symptom of the disease being treated by the agent, or both.

5. A method or an antagonist according to Claim 4, in which the effects of the antagonist are substantially restricted to the part of the individual to which the antagonist is administered.

6. A method or an antagonist according to Claim 5, in which the metabolic breakdown of the agent in other parts of the individual are not substantially reduced.

7. A method or an antagonist according to any preceding claim, in which the antagonist is administered to the skin of the individual .

8. A method or an antagonist according to any preceding claim, in which the antagonist is not capable of crossing the basement membrane of the epidermis.

9. A method or an antagonist according to any preceding claim, in which the antagonist is administered to a individual already exhibiting a tachyphylactic response

10. A method or an antagomst according to any preceding claim, in which the antagonist is administered simultaneously with the agent.

11. A method or an antagonist according to any preceding claim, in which 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.

12. A method or an antagonist according to any preceding claim, in which the agent comprises a macrolactam.

13. A method or an antagonist according to any preceding claim, in which agent comprises a vitamin D analogue selected from the group consisting of: Calcitriol (la,25(OH)₂D₃, la,25-dihydroxyvitamin D₃, 1,25-dihyroxycholecalciferol), Calcipotriol (Dovanex; MC903) and Tacalcitol (la,24(R)-dihydroxyvitamin D₃; Curatoderm).

14. A method or an antagonist according to any preceding claim, in which the metabolic enzyme comprises a P450 cytochrome.

15. A method or an antagonist according to any preceding claim, in which the metabolic enzyme 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 CYP3Gl.

16. A method of detecting the likelihood of a individual developing a tachyphylactic response to an agent, the method comprising the steps of: administering the agent to a cell of the individual; and detecting the amount and/or activity of a metabolic enzyme capable of metabolising the agent, in which an increased amount or activity indicates that the individual is likely to develop a tachyphylactic response.

17. A method of identifying an molecule which is capable of reducing a tachyphylactic response to an agent in a individual , the method 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).

18. 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.

19. 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 metabolic enzyme together with a pharmaceutically acceptable carrier or diluent.

20. A pharmaceutical composition according to Claim 19, further comprising the agent.

21. A pharmaceutical composition according to Claim 19 or Claim 20, in which 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).

22. A pharmaceutical composition according to any of Claims 19 to 21 , in which 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, CYP3 A5, CYP3 A6 and CYP3G1.

23. Use of an antagonist of a metabolic enzyme in the alleviation or prevention of a tachyphylactic response to an agent in a individual .

24. Use of an antagonist of a metabolic enzyme in a method of preparation of a medicament for the alleviation or prevention of a tachyphylactic response to an agent in a individual .

25. 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 metabolic enzyme which is induced as a result of exposure of the individual to the agent.

26. A method of identifying an molecule capable of alleviating or preventing a side effect associated with administration of an agent to a individual , the method comprising identifying a metabolic enzyme which is induced by exposure of the individual to the agent, and identifying a molecule which is capable of antagonising the metabolic enzyme.

27. 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 .

28. A method according to Claim 25 or 26, or a metabolic enzyme antagonist according to Claim 27, in which the side effect is caused by a metabolic product of the metabolic enzyme acting on the agent.

29. A method or a metabolic enzyme antagonist according to any of Claims 25 to 28, in which the metabolic product is a toxin.

30. A method or a metabolic enzyme antagonist according to any of Claims 25 to 29, in which the side effect is cutaneous irritation, itching, cutaneous atrophy, papulopustular reaction or striae.

31. A method for the treatment or prophylaxis of a disease, the method comprising the steps of: administering an antagonist of a metabolic enzyme to the individual , in which the metabolic 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 metabolic enzyme is capable of metabolising the agent.

32. A method according to Claim 31 , in which the disease is psoriasis and the metabolic enzyme antagonist is an antagomst of CYP24.

33. A method of identifying an molecule suitable for treatment or prophylaxis of a disease, the method comprising the steps of: identifying a metabolic 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 metabolic enzyme being capable of metabolising the agent; and identifying an antagonist of the metabolic enzyme.

34. A method, pharmaceutical composition or use according to any preceding claim, in which the agent comprises a macrolactam, preferably Tacrolimus or Cyclosporin or both, and the metabolic enzyme comprises CYPIAI .

35. A method, pharmaceutical composition or use according to any preceding claim, in which the agent comprises a vitamin D analogue, preferably Calcipotriol, and the metabolic enzyme comprises CYP2E1.

36. A method, pharmaceutical composition or use according to any preceding claim, in which the agent comprises a vitamin D analogue, preferably Calcitriol or Tacalcitol or both, and the metabolic enzyme comprises CYP24.

37. Use of an inhibitor of CYP 1 A 1 , in the treatment or alleviation of tachyphylaxis against Tacrolimus or Cyclosporin or both, or an inhibitor of CYPIAI for such use.

38. Use of an inhibitor of C YP2E 1 , in the treatment or alleviation of tachyphylaxis against Calcipotriol, or an inhibitor of CYP2Elfor such use.

39. Use of an inhibitor of CYP24, in the treatment or alleviation of tachyphylaxis against Calcitriol or Tacalcitol or both, or an inhibitor of CYP24 for such use.

40. Use according to Claim 37, 38 or 39, in which the inhibitor comprises ketoconazole.

41. Use according to Claim 39, in which the inhibitor is selected from the group consisting of: N-[4-chlorobenzoyl]-2-(lH-imidazol-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-(l H-imidazol- 1-y l)-2- (phenyl)-l -amino ethane (Compound C).