EP0954304A2 - Methods for regulating nicotine metabolism - Google Patents
Methods for regulating nicotine metabolismInfo
- Publication number
- EP0954304A2 EP0954304A2 EP97930283A EP97930283A EP0954304A2 EP 0954304 A2 EP0954304 A2 EP 0954304A2 EP 97930283 A EP97930283 A EP 97930283A EP 97930283 A EP97930283 A EP 97930283A EP 0954304 A2 EP0954304 A2 EP 0954304A2
- Authority
- EP
- European Patent Office
- Prior art keywords
- cyp2a6
- nicotine
- substance
- coumarin
- individual
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
- A61K31/7032—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a polyol, i.e. compounds having two or more free or esterified hydroxy groups, including the hydroxy group involved in the glycosidic linkage, e.g. monoglucosyldiacylglycerides, lactobionic acid, gangliosides
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/12—Ketones
- A61K31/122—Ketones having the oxygen directly attached to a ring, e.g. quinones, vitamin K1, anthralin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/13—Amines
- A61K31/131—Amines acyclic
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
- A61K31/27—Esters, e.g. nitroglycerine, selenocyanates of carbamic or thiocarbamic acids, meprobamate, carbachol, neostigmine
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
- A61K31/351—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom not condensed with another ring
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/365—Lactones
- A61K31/366—Lactones having six-membered rings, e.g. delta-lactones
- A61K31/37—Coumarins, e.g. psoralen
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
- A61K31/4164—1,3-Diazoles
- A61K31/4178—1,3-Diazoles not condensed 1,3-diazoles and containing further heterocyclic rings, e.g. pilocarpine, nitrofurantoin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
- A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
- A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
- A61K31/4439—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a five-membered ring with nitrogen as a ring hetero atom, e.g. omeprazole
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/30—Drugs for disorders of the nervous system for treating abuse or dependence
- A61P25/34—Tobacco-abuse
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
Definitions
- the invention relates to methods for regulating nicotine metabolism in an individual; compositions for regulating nicotine metabolism in an individual; methods of treating conditions requiring regulation of nicotine metabolism in an individual; methods of screening for a substance that regulates nicotine metabolism in an individual; and methods of assessing nicotine metabolism in individuals.
- Nicotine is the primary alkaloid present in tobacco playing a crucial role in establishing and maintaining tobacco dependence.
- Several studies have shown that smokers adjust their smoking behaviour to try and maintain constant nicotine blood levels, and hence brain nicotine levels.
- Studies using different nicotine yield cigarettes (Finnegan et al., 1945), nicotine replacement therapy [Lucchesi et al. (intravenous infusion), 1967; Jarvik et al., 1970 (ingestion); Kaslowski et al., 1975; Russell et al., 1976; Ebert et al. , 1984 (nicotine chewing gum); Levin et al., 1994 (nicotine patches)], nicotine blockade (Stolerman et al., 1973, Nemeth-Coslett et al.
- Nicotine and its metabolites have been extensively studied over the past few decades. Nicotine is, for the most part, metabolized in the liver (80%), and to a smaller extent in the lungs and kidneys (Schielvelbein, 1982; Turner, 1975).
- Nicotine is primarily metabolized to cotinine through a two step process ( Figure 1).
- the first step in the process produces the intermediate, nicotine- ⁇ - 1 (5-) iminium ion (Peterson and Castagnoli, 1988, Williams et al, 1990), which is then further oxidized through a cytosolic aldehyde oxidase reaction in the presence of liver microsomes, O 2 and NADPH (Hill et al, 1972; Peterson et al, 1987; Brandage et al., 1979; Gorrod et al, 1982).
- cytochrome P450 The cytochrome P450 (CYP) system has been implicated in the metabolism of nicotine.
- Evidence for CYP involvement in nicotine metabolism has come from rat liver studies in which reconstituted purified CYPs, and specific antibodies were shown to inhibit nicotine metabolism.
- rat studies have shown that phenobarbital inducible CYPs (i.e., the CYPs; -2B1 , -2B2, -2C6, and -3A2) are involved in nicotine metabolism (Nakayama et al, 1982; Hibberd and Gorrod 1985; Foth et al., 1990; Seaton et al, 1991 and 1993).
- CYP2B6 showed the highest nicotine oxidase activity while CYP2E1 and CYP2C9 showed intermediate levels (Flammang et al. , 1992). McCracken et al. , (1992), have shown that human CYP2B6 and CYP2D6 displayed high rates of nicotine to cotinine metabolism, whereas the catalytic activity of CYP2E1 towards nicotine is not detectable. The results concerning CYP2E1 and CYP2D6 are in disagreement with the findings of Flammang et al., (1992). Thus, there remains some ambiguity concerning the affinities of CYPs for nicotine.
- the CYP2B proteins are expressed at low amounts in the liver (less than 5 % of the total hepatic CYP content) in all animals and humans, but their levels can be highly induced by exposure to a number of diverse chemicals, including the prototypic CYP2B inducer phenobarbital (Ryan et al. , 1990; Guengerich et al. , 1982b).
- the human CYP2B6 enzyme is expressed at variable levels among different individuals.
- CYP2B6 has poor oxidation activity towards benzopyrene, 7-ethoxycoumarin, coumarin, ethoxyresorufin, pentoxyresorufin, ethylmorphine, benphetamine, and aniline (Mimura et al. , 1993). Orphenadrine, an anti-parkinsonian agent, was found to be a specific inhibitor of CYP2B6 (Reidy et al. , 1992; Chang et al. , 1993).
- CYP2B6, CYP2C9, CYP2D6 and CYP2E1 have implicated CYP2A6 in nicotine metabolism in isolated expression systems (Flammang et al., 1992; McCracken et al. , 1992).
- CYP2A6 also displays a genetic polymorphism whereby certain individuals contain an inactive enzyme (Daly et al., 1994; Fernandez-Salguero et al., 1995).
- CYP2A6 is the predominant, if not the only, coumarin 7-hydroxylase in humans (Pearce et al., 1992).
- CYP2A6 catalyzes the hydroxylation of coumarin a naturally occurring compound in plants and essential oils (Pelkonan et al., 1985; Raunio et al., 1988; Yamano et al. , 1990; Pearce et al. , 1992). In primates, such as humans and baboons, coumarin is metabolized to 7-hydroxycoumarin ( — 80%) (Cholerton et al. , 1992; Shilling et al. , 1969; Moran et al., 1987).
- CYP2A6 protein levels in human liver microsomes varied by over 100 fold (Yun et al. , 1991).
- CYP2A6 also has been found to metabolize several procarcinogens such as NNK (Crespi et al. , 1991), aflaxtoxin Bl (Yun et al., 1991); hexamethylphosphoramide (Ding et al. , 1988), and nitrosodimethylamine (Davies et al. , 1989; Fernandez et al. , 1995).
- CYP2A6 has been shown to be the major nicotine metabolizing enzyme in human livers.
- Coumarin a specific CYP2A6 substrate, was found to specifically and selectively inhibit nicotine metabolism to cotimne by 84% ⁇ 11 % in test livers, and addition of orphenadrine (a
- Methoxsalen a.k.a. 9-Methoxy-7H- furol[3,2-g][l]benzopyran-7-one; 6-hydroxy-7-methoxy-5-benzofuranacrylic acid ⁇ - lactone; 9-methoxypsoralen; 8-methoxy-4',5':6,7-furo-coumarin; 8-methoxy[furano-
- CYP2A6 and thus of nicotine to cotinine metabolism.
- a monoclonal antibody raised against CYP2A6 also inhibited cotinine formation; however, antibodies to other CYPs did not significantly inhibit cotinine formation.
- the data indicate that variability in CYP2A6 expression results in inter-individual variation in nicotine metabolism, which in turn, can have behavioural consequences such as smoking more or less cigarettes. Therefore, selective and specific inhibitors of CYP2A6 can be used to regulate nicotine metabolism, and in particular substantially decrease nicotine metabolism, thereby affecting tobacco use.
- the terms “inhibitor” and “inhibition” in the context of the present invention, are intended to have a broad meaning and encompass substances which directly or indirectly (e.g. , via reactive intermediates, metabolites and the like) act on CYP2A6 to inhibit or otherwise regulate the ability of CYP2A6 to catalyze metabolism of nicotine.
- Other substances which act indirectly on CYP2A6 include those substances which inhibit transcription and/or translation of the gene encoding CYP2A6.
- the present invention relates to a method of regulating nicotine metabolism in an individual comprising selectively inhibiting CYP2A6.
- Inhibition of CYP2A6 may be achieved using one or more of the following: (i) substances which inhibit CYP2A6 activity; or (ii) substances which inhibit transcription and/or translation of the gene encoding CYP2A6.
- CYP2A6 may also be selectively inhibited by interfering with the transcription or translation of the gene encoding CYP2A6 using gene transfer methods.
- the present invention also provides a method of screening for a substance that regulates nicotine metabolism to cotinine in an individual comprising assaying for a substance which selectively (i) inhibits CYP2A6 activity; or (ii) inhibits transcription and/or translation of the gene encoding CYP2A6.
- the invention further provides a pharmaceutical composition for use in treating a condition requiring regulation of nicotine metabolism to cotinine comprising an effective amount of a substance which selectively inhibits CYP2A6, and/or a pharmaceutically acceptable carrier, diluent, or excipient.
- a method is also provided for treating a condition requiring regulation of nicotine metabolism to cotinine in an individual comprising administering to the individual an effective amount of a substance which selectively inhibits CYP2A6.
- the present invention also provides the use of a substance which selectively inhibits CYP2A6 for the preparation of a medicant for regulation of nicotine metabolism to cotinine in an individual.
- CYP2B6 inhibitors may also be used in combination with inhibitors of CYP2A6 to provide an enhanced inhibitory effect. Therefore, the present invention provides a method for enhancing inhibition of nicotine metabolism by a CYP2A6 inhibitor in an individual comprising administering to the individual an effective amount of a substance which selectively inhibits CYP2A6, and an effective amount of an inhibitor of CYP2B6.
- a pharmaceutical composition for use in treating a condition requiring regulation of nicotine metabolism to cotinine is also provided comprising an effective amount of a substance which selectively inhibits CYP2A6, an effective amount of an inhibitor of CYP2B6, and/or a pharmaceutically acceptable carrier, diluent, or excipient.
- a method for treating a condition requiring regulation of nicotine metabolism to cotinine in an individual comprising administering to the individual an effective amount of a substance which selectively inhibits CYP2A6, and an effective amount of an inhibitor of CYP2B6.
- compositions and methods may be used to diminish a subjects desire for nicotine and thereby can be used to alter tobacco use.
- Figure 1 shows the two step conversion of nicotine to cotinine
- Figure 2A shows the amino acid and nucleotide sequence for cytochrome CYP2A6
- Figure 2B shows the mRNA sequence for cytochrome CYP2B6
- Figure 3 shows the protein-time curves of cotinine production from 100 ⁇ M nicotine in the presence of 20 ⁇ l rat cytosol by K20 human microsomes;
- Figure 4 shows sample Michaelis-Menten curves, with inset Eddie-Hofstee plots, of nicotine to cotinine metabolism
- (A) is a graph displaying single enzyme kinetics for the L29 human liver and (B) displays multiple enzyme site kinetics for the L30 human liver
- Figure 5 is a bar graph showing apparent K m values of nicotine to cotinine metabolism by 30 human liver microsomes;
- Figure 6 is a bar graph showing apparent V ll ⁇ ax values of nicotine to cotinine metabolism by 30 human liver microsomes
- Figure 7 shows antibody activity (data from Gentest Corp.); where (A) is a graph of inhibition of coumarin oxidation by MAB-2A6 antibody and (B) is a graph of inhibition of testosterone 16 ⁇ -hydroxylation (CYP2B1), and lidocaine methyl- hydroxylation (CYP2B2) by anti-CYP2Bl antibody;
- Figure 8 shows Western blots of increasing concentrations of L64 microsomal protein (above) with respective densities plotted to show linearity of analysis (below);
- Figure 9 is a bar graph showing coumarin (150 ⁇ M) inhibition of cotinine formation by 30 human liver microsomes;
- Figure 10 is a bar graph showing percent inhibition of nicotine (100 ⁇ M) to cotinine metabolism by 150 ⁇ M coumarin by 30 human liver microsomes;
- Figure 11 is a Dixon plot of coumarin inhibition of cotinine formation in K27 liver microsomes
- Figure 12 is a bar graph showing the use of antisense oligodeoxynucleotides (ASO) for the reduction of the CYP2A6 enzyme.
- ASO antisense oligodeoxynucleotides
- Figure 13 is a graph showing the effects of MAB-2A6 on nicotine (100 ⁇ M) to cotinine metabolism by K27 human liver microsomes;
- Figure 14 shows Western blots of 30 human liver microsomes; each blot is accompanied by a 15, 30, 75 and 100 ⁇ g lanes of L64 microsomal protein such that individual blots can be compared;
- Figure 15 is a graph showing the correlation between nicotine to cotinine V max values and the amount of immunoreactive CYP2A6 by 30 human livers;
- Figure 16 is a graph showing the correlation between CYP2A6 mediated cotinine formation and the amount of immunoreactive CYP2A6 by 30 human livers;
- Figure 17 is a graph showing the correlation between nicotine to cotinine V max /K m values and the amount of immunoreactive CYP2A6 by 30 human livers;
- Figure 18 is a bar graph showing orphenadrine (150 ⁇ M) inhibition of cotinine formation by 30 human liver microsomes;
- Figure 19 is a bar graph showing the percent inhibition of cotinine formation by orphenadrine (150 ⁇ M) using 30 human liver microsomes;
- Figure 20 is a graph showing the effects of anti-rat CYP2B1 on cotinine formation by K27 human liver microsomes
- Figure 21 is a bar graph showing coumarin and orphenadrine (150 ⁇ M each) inhibition on nicotine metabolism by 30 human liver microsomes.
- Figure 22 is a bar graph showing troleandomycin (150 ⁇ M) inhibition of cotinine formation by 30 human liver microsomes;
- Figure 23 shows chemical structures of some representative CYP2A6 inhibitors;
- Figure 24 is a table showing statistical results from clinical studies using methoxsalen to inhibit the activity of CYP2A6 on nicotine to cotinine metabolism;
- Figure 25 is a graph showing the effects over time of methoxsalen on the nicotine plasma concentrations of seven subjects;
- Figure 26 is a graph showing the effects over time of methoxsalen on the cotinine plasma concentrations of seven subjects;
- Figure 27 is a bar graph summarizing significant subjective effect changes associated with methoxsalen increased plasma nicotine concentrations in seven subjects;
- Figure 28A is a graph showing a subjective rating of current nausea in seven subjects involved in the clinical study of the effect of methoxsalen on plasma nicotine concentrations
- Figure 28B is a graph showing a subjective rating of current desire for a cigarette in seven subjects involved in the clinical smdy of the effect of methoxsalen on plasma nicotine concentrations
- Figure 28C is a graph showing a subjective rating of current pleasantness of a cigarette in seven subjects involved in the clinical study of the effect of methoxsalen on plasma nicotine concentrations;
- Figure 29 is a graph showing the inhibitory effect on cotinine formation from nicotine of various antibodies including the CYP2A6 antibody;
- Figure 30A is a table showing the inhibition of the nicotine to cotinine metabolism by various chemical compounds
- Figure 30B is a table of K; values for the inhibition of the CYP2A6 substrate coumarin to 7-hydroxycoumarin metabolism by various compounds;
- Figure 30C is a table showing the percent inhibition of various compounds on cotinine formation from nicotine
- Figure 30D is a bar graph showing the percent inhibition of the nicotine to cotinine metabolism by various chemical compounds
- Figure 31 is a Dixon plot of 7-methoxycoumarin inhibition of nicotine to cotinine formation in K28 human liver microsomes
- Figure 32 is a Dixon plot of methoxsalen inhibition of nicotine to cotinine formation with 10 minute preincubation in K28 human liver microsomes;
- Figure 33 is a Cornish-Bowden plot of methoxsalen inhibition of nicotine to cotinine formation with 10 minute preincubation in K28 human liver microsomes;
- Figure 34 is a graph showing the effect of pre-incubation time of methoxsalen (100 nM) on the inhibition of nicotine (30 ⁇ M) to cotinine formation in K26 human liver microsomes;
- Figure 35 is a Dixon plot of naringenin inhibition of nicotine formation with 10 minute preincubation in K26 human liver microsomes.
- Figure 36 is a Dixon plot of diethyldithiocarbamic acid inhibition of nicotine to cotinine formation with 10 minute preincubation in K26 human liver microsomes.
- Figure 37 is a graph illustrating a correlation between fasted morning and non- fasted afternoon coumarin (C) testing sessions.
- Figure 38 is a graph showing the metabolism of nicotine over one hour in seven subjects.
- Figure 39 is a graph showing a time course of total 7-hydroxycoumarin concentration detected in the plasma of subjects given 100 mg of coumarin.
- the present invention relates to a method of regulating nicotine metabolism to cotinine in an individual comprising selectively inhibiting CYP2A6.
- Inhibition of CYP2A6 may be achieved using one or more of the following (i) substances which inhibit CYP2A6 activity; or (ii) substances which inhibit transcription and/or translation of the gene encoding CYP2A6.
- Substances which inhibit CYP2A6 activity include substances which specifically bind to CYP2A6 and thereby inhibit its activity.
- examples of such substances include antibodies which are specific for CYP2A6 including for example, the monoclonal antibody described by Pearce, R., et al, 1992., and commercially available antibodies such as MAB2A6 and monoclonal CYP2A6, sold by Gentest Corporation, Woburn, Mass., U.S.A.; XenoTech 2A6 sold by XenoTech LLC, Kansas City, KS, U.S. A and polyclonal CYP2A6 sold by Research Diagnostics, Inc, Flanders, N.J. , U.S.A.
- Substances which inhibit CYP2A6 activity also include substances having a lactone structure with a carbonyl oxygen.
- Non-limiting examples of such substances include coumarin (The Merck Index, Eleventh Edition Budavari, S. , ed. Merck & Co. Inc. , 1989, No. 2563), furanocoumarin, methoxsalen (The Merck Index, No. 5911), imperatorin (The Merck Index, No. 4839), psoralen (The Merck Index, No. 7944), ⁇ - naphthoflavone, isopimpinellin, ⁇ -naphthoflavone, bergapten (The Merck Index, No.
- SM-12502 sphondin, coumatetralyl (racumin), and ( + )-cis-3,5-dimethyl-2-(3-pyridyl)- thiazolidim-4-one (SM-12502) (Nunoya, et al. , PET 277:768-774, 1996).
- Other substances which inhibit CYP2A6 and can be used in the methods and compositions of the invention include naringenin and related flavones, diethyldithiocarbamate, nicotine (useful primarily in the screening methods of the invention), N- nitrosodialkylamine (e.g. N-nitrosodiethylamine (The Merck Index, No.
- derivatives of coumarin and methoxsalen include pharmaceutically acceptable salts, esters and complexes of coumarin and methoxsalen including potassium and sodium salts, and amino acid, carbohydrate and fatty acid complexes.
- suitable analogs of coumarin may be selected based upon their functional similarity to coumarin, including the ability to inhibit the metabolism of nicotine to cotinine by CYP2A6.
- functional analogs of coumarin include 7-methoxycoumarin, 7-methylcoumarin, and 7-ethoxycoumarin and all structures shown in Figures 23 A, 23B, 23C.
- Analogs of coumarin may also be selected based upon their three dimensional structural similarity to coumarin - i.e. , the lactone/carbonyl structure.
- Substances which inhibit transcription and/or translation of the gene encoding CYP2A6 include a nucleic acid sequence encoding the CYP2A6 gene (see Figure 2A, GenBank Accession No. HSU22027) or parts thereof (e.g. , the region which is about 20 nucleotides on either side of nucleotide 790 (ATG), and the splice sites 1237, 2115, 2499, 3207, 4257, 4873, 5577 and 6308), inverted relative to their normal orientation for transcription - i.e. , antisense CYP2A6 nucleic acid molecules.
- antisense nucleic acid molecules may be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed with CYP2A6 mRNA or the CYP2A6 gene.
- the antisense sequences may be produced biologically using an expression vector introduced into cells in the form of a recombinant plasmid, phagemid or attenuated virus in which antisense sequences are produced under the control of a high efficiency regulatory region, the activity of which may be determined by the cell type into which the vector is introduced.
- a nucleic acid molecule containing the antisense sequences may be introduced into cells in a subject using conventional techniques, such as transformation, transfection, infection, and physical techniques such as electroporation or microinjection. Chemical methods such as coprecipitation and incorporation of DNA into liposomes may also be used to deliver antisense sequences.
- the molecules may also be delivered in the form of an aerosol or by lavage.
- Suitable vectors or cloning vehicles for transferring the nucleic acid molecules are known in the art. Examples of suitable vectors include retroviral vectors, adenoviral vectors, and DNA virus vectors.
- the CYP2A6 inhibitor is at least one member selected from the group comprising coumarin, methoxsalen, derivatives thereof and analogs thereof (see Figure 23 A).
- methoxsalen is a potent inhibitor of CYP2A6.
- CYP2A6 may also be selectively inhibited in the method of the invention by interfering with the transcription of the gene encoding CYP2A6 using gene transfer methods such as targeted gene mutagenesis using allelic replacement, insertional inactivation, or deletion formation.
- allelic gene exchange using non- replicating or conditionally-replicating plasmids has been used widely for the mutagenesis of eukaryotes.
- Allelic exchange can be used to create a deletion of the CYP2A6 gene.
- Exemplary methods of making the alterations set forth above are disclosed by Sambrook et al (Molecular Cloning: A Laboratory Manual, 2d Ed. , Cold Spring Harbor Laboratory Press, 1989).
- CYP2B6 inhibitors may also be used in combination with inhibitors of CYP2A6 to provide an enhanced inhibitory effect.
- Inhibitors of CYP2B6 include one or more of the following (i) substances which inhibit CYP2B6 activity; or (ii) substances which inhibit transcription and/or translation of the gene encoding CYP2B6.
- CYP2B6 inhibitors may also be used alone to inhibit nicotine metabolism in an individual.
- Substances which inhibit CYP2B6 activity include substances which specifically bind to CYP2B6 and thereby inhibit its activity.
- examples of such substances include antibodies which are specific for CYP2B6 including for example, commercially available antibodies such as anti-CYP2B6 sold by Gentest Corporation, Woburn,
- Substances which inhibit CYP2B6 activity also include substances selected from phenylethyl amines, diphenylbarbiturates, diethyl substituted barbiturates and hydantoins.
- diphenhydramine and its derivatives including orphenadrine (The Merck Index, No. 6831), and derivatives or analogs of orphenadrine, and other antihistamines, anticholinergic substances such as cholines and analogs and derivatives thereof may be used as CYP2B6 inhibitors in various embodiments of the methods and compositions of the invention.
- Antibodies such as polyclonal CYP2B1/2, polyclonal CYP2B1 and polyclonal CYP2B6 sold by Gentest Corporation, Woburn, Mass. , U.S.A. , also bind specifically to CYP2B6 such that they also inhibit the activity of CYP2B6.
- Derivatives of orphenadrine which may be used in the methods and compositions of the invention include pharmaceutically acceptable salts, esters and complexes of orphenadrine including potassium and sodium salts, and amino acid, carbohydrate and fatty acid complexes.
- suitable analogs of orphenadrine may be selected based upon their functional similarity to orphenadrine. including the ability to inhibit CYP2B6.
- Analogs of orphenadrine may also be selected based upon their three dimensional structural similarity to orphenadrine. Substances which inhibit transcription and/or translation of the gene encoding
- CYP2B6 include a nucleic acid sequence encoding the CYP2B6 gene (see Figure 2B, GenBank Accession No. HSP452B6 for the mRNA sequence of CYP2B6), or parts thereof (e.g. , the region which is on either side of nucleotide 9 (ATG), and the sites 111 , 274, 424, 585, 762, 904, 1092, and 1234 nt), inverted relative to their normal orientation for transcription - i.e., antisense CYP2B6 nucleic acid molecules.
- antisense nucleic acid molecules may be produced and introduced into cells using conventional procedures as described herein.
- CYP2B6 may also be selectively inhibited in a method of the invention by interfering with the transcription of the gene encoding CYP2B6 using conventional gene transfer methods as discussed herein.
- the CYP2B6 inhibitor employed is orphenadrine and derivatives or analogs of orphenadrine.
- An inhibitor of CYP2A6 or CYP2B6 may be targeted to the enzyme using antibodies specific to an epitope of the enzyme.
- bispecific antibodies may be used to target an inhibitor.
- the bispecific antibodies contain a variable region of an antibody specific for at least one epitope of CYP2A6 or CYP2B6, and a variable region of a second antibody which is capable of binding to an inhibitor.
- the bispecific antibodies may be prepared by forming hybrid hybridomas, using procedures known in the art such as those disclosed in Staerz & Bevan, (1986, PNAS (USA) 83: 1453) and Staerz & Bevan, (1986, Immunology Today, 7:241).
- Bispecific antibodies may also be constructed by chemical means using conventional procedures such as those described by Staerz et al. , (1985, Nature, 314:628) and Perez et al. , (1985 Nature 316:354), or by expression of recombinant immunoglobulin gene constructs.
- the present invention provides a method of screening for a substance that regulates nicotine metabolism to cotinine in an individual comprising assaying for a substance which selectively (i) inhibits CYP2A6 activity, or (ii) inhibits transcription and/or translation of the gene encoding CYP2A6.
- the method comprises:
- Substrates of CYP2A6 which may be used in the screening method of the invention for example include nicotine, coumarin, analogs thereof and derivatives thereof.
- the corresponding reaction products for nicotine and coumarin are cotinine, and 7-hydroxycoumarin, respectively.
- CYP2A6 used in the method of the invention may be obtained from natural, recombinant, or commercial sources.
- CYP2A6 may be obtained by recombinant methods such as those described by Nesnow, S. et al., Mutation Research 1994; 324:93-102.
- Cells or liver microsomes expressing CYP2A6 may also be used in the method.
- reaction product unreacted substrate, or unreacted CYP2A6; may be isolated by conventional isolation techniques, for example, salting out, chromatography, electrophoresis, gel filtration, fractionation, absorption, polyacrylamide gel electrophoresis, agglutination, or combinations thereof.
- unreacted substrate, or unreacted CYP2A6 antibody against the reaction product or the substance, or a labelled CYP2A6 or substrate, or a labelled substance may be utilized.
- Antibodies, CYP2A6, substrate, or the substance may be labelled with a detectable marker such as a radioactive label, antigens that are recognized by a specific labelled antibody, fluorescent compounds, enzymes, antibodies specific for a labelled antigen, and chemiluminescent compounds.
- the substrate used in the method of the invention may be insolubilized.
- it may be bound to a suitable carrier.
- suitable carriers are agarose, cellulose, dextran, Sephadex, Sepharose, carboxymethyl cellulose polystyrene, filter paper, ion-exchange resin, plastic film, plastic tube, glass beads, polyamine-methyl vinyl-ether-maleic acid copolymer, amino acid copolymer, ethylene-maleic acid copolymer, nylon, silk, etc.
- the carrier may be in the shape of, for example, a tube, test plate, beads, disc, sphere etc.
- the insolubilized CYP2A6, substrate, or substance may be prepared by reacting the material with a suitable insoluble carrier using known chemical or physical methods, for example, cyanogen bromide coupling.
- a method for screening for a substance that regulates nicotine metabolism to cotinine in an individual by inhibiting transcription and/or translation of the gene encoding CYP2A6 comprising the steps of:
- a host cell for use in the method of the invention may be prepared by transfecting a suitable host with a nucleic acid molecule comprising a nucleic acid sequence encoding CYP2A6.
- a nucleic acid sequence encoding CYP2A6 may be constructed having regard to the sequence of the CYP2A6 gene ( Figure 2 A) following procedures known in the art.
- Suitable transcription and translation elements may be derived from a variety of sources, including bacterial, fungal, viral, mammalian, or insect genes. Selection of appropriate transcription and translation elements is dependent on the host cell chosen, and may be readily accomplished by one of ordinary skill in the art.
- Such elements include: a transcriptional promoter and enhancer or RNA polymerase binding sequence, a ribosomal binding sequence, including a translation initiation signal. Additionally, depending on the host cell chosen and the vector employed, other genetic elements, such as an origin of replication, additional DNA restriction sites, enhancers, and sequences conferring inducibility of transcription may be inco ⁇ orated into the expression vector. It will also be appreciated that the necessary transcription and translation elements may be supplied by the native CYP2A6 gene and/or its flanking sequences.
- reporter genes are genes encoding a protein such as ⁇ -galactosidase, chloramphenicol acetyltransferase, firefly luciferase, or an immunoglobulin or portion thereof such as the Fc portion of an immunoglobulin, preferably IgG. Transcription of the reporter gene is monitored by changes in the concentration of the reporter protein such as ⁇ -galactosidase, chloramphenicol acetyltransferase, or firefly luciferase. This makes it possible to visualize and assay for expression of CYP2A6 and in particular to determine the effect of a substance on expression of CYP2A6.
- Suitable host cells include a wide variety of prokaryotic and eukaryotic host cells, including bacterial, mammalian, yeast or other fungi, viral, plant, or insect cells.
- Nanji M. , et al., (1994) describe the expression of a cDNA encoding human CYP2A6 in a baculovirus system
- Nesnow, S. , et al. (1994) and Tiano H.F. et al, (1993) describe the expression of CYP2A6 from a retroviral vector in transformable C3H/10T1/2 mouse embryo fibroblasts
- Salonpaa, P., et al, (1993) describe the preparation of amphotropic recombinant retroviruses containing CYP2A6 using LXSN vector and PA317 packaging cells.
- Host cells which are commercially available may also be used in the method of the invention.
- the h2A3 now known as h2A6
- h2B6 cell lines available from Gentest Corporation are suitable for the screening methods of the invention.
- the above mentioned methods of me invention may be used to identity negative regulators of nicotine metabolism to cotinine in brain and liver thereby affecting conditions requiring regulation of nicotine metabolism. Identification and isolation of such regulators will permit studies of the role of the regulators in the regulation of nicotine metabolism to cotinine and permit the development of substances which affect this role, such as functional or non-functional analogs of the regulators. It will be appreciated that such substances will be useful as pharmaceuticals to modulate nicotine metabolism to cotinine as discussed herein.
- the inhibitory activity of the substances identified by the methods of the invention may be confirmed in experimental model systems, and in clinical studies, for example the studies as outlined in the Examples herein below. 3.
- compositions Substances which inhibit nicotine metabolism to cotinine described in detail herein or substances identified using die methods of the invention which selectively inhibit CYP2A6 may be incorporated into pharmaceutical compositions. Therefore the invention provides a pharmaceutical composition for use in treating a condition requiring regulation of nicotine metabolism to cotinine comprising an effective amount of one or more substances which selectively inhibit CYP2A6, and/or a pharmaceutically acceptable carrier, diluent, or excipient. A method is also provided for treating a condition requiring regulation of nicotine metabolism to cotinine in a subject comprising administering to the subject an effective amount of one or more substances which selectively inhibit CYP2A6.
- Conditions requiring regulation of nicotine metabolism to cotinine include nicotine use disorders - i.e., dependent and non-dependent tobacco use, and nicotine- induced disorders - i.e. , withdrawal.
- the conditions may develop with the use of all forms of tobacco (e.g., cigarettes, chewing tobacco, snuff, pipes, and cigars) and with prescription medications (e.g. nicotine gum, nicotine patch, spray, pulmonary inhalation or other forms).
- the pharmaceutical compositions and treatment methods of the invention may be used to diminish a subjects desire to smoke and thereby alter smoking behaviour.
- the pharmaceutical compositions and treatment methods of the invention may also be used together with other centrally active pharmaceutical compositions that modify smoking behaviour (e.g. bupropion (a.k.a. Wellbutrin * ) in its various formulations), to decrease the dose of the centrally active composition or to increase its effectiveness in the treatment of tobacco dependence.
- compositions and treatment methods of the present invention by regulating nicotine metabolism in an individual are highly effective.
- the methods and compositions maintain the behavioural components of smoking and modify them by reducing nicotine metabolism to cotinine.
- An individual with reduced nicotine metabolism following administration of a composition of the present invention will alter smoking behaviour and smoke exposure because of modification of nicotine requirements.
- the methods and compositions of the invention show patterns of reduction, more sustained abstinence, and lower tobacco smoke exposure than obtained with prior art methods in particular those using nicotine deprivation.
- the behavioural component of smoking is particularly important in some groups of individuals, and thus the methods and compositions of the invention in modifying and maintaining behavioural components may be particularly useful in reducing smoking in those individuals. For example, it has been found that behavioural components are significant in tobacco use by women.
- the present invention permits the development of behavioural learning on an individual/or group basis.
- compositions and treatment methods of the invention are also particularly suited to regulate nicotine metabolism in individuals or populations having high levels of CYP2A6.
- CYP2A6 For example, Caucasians in North America have high levels of CYP2A6.
- An individual or population having a high level of CYP2A6 can be identified using our methods for measuring CYP2A6.
- compositions and methods of the invention also have the advantage of individualization and flexibility in treatment duration.
- the compositions and treatment methods are particularly suitable for severely dependent individuals, previous treatment failures, individuals unable to accept the current approach of complete cessation, treatment/prevention of relapse, or concurrent treatment with other methods such as the nicotine patch. It is expected that the compositions and treatments of the invention will decrease the doses of nicotine patch and all other forms of nicotine replacement therapies that are needed and will prolong the duration of action of the therapy and/or enforce their effectiveness in the treatment of tobacco dependence.
- the methods and compositions of the invention in treating individuals with nicotine use disorders and nicotine-induced disorders are also useful in the treatment and prophylaxis of diseases or conditions, including nicotine-related disorders such as opioid related disorders; proliferative diseases; cognitive, neurological or mental disorders; and other drug dependencies in the individuals.
- nicotine-related disorders such as opioid related disorders
- proliferative diseases such as opioid related disorders
- cognitive, neurological or mental disorders such as cognitive, neurological or mental disorders
- other drug dependencies in the individuals include malignant disease, psychosis, schizophrenia, Parkinson's disease, anxiety, depression, alcoholism, and opiate dependence.
- the methods and compositions of the invention may also be used in the prophylaxis and treatment of individuals having a condition which requires a reduction in CYP2A6 or CYP2B6.
- CYP2A6 is known to metabolize several procarcinogens such as NNK (Crespi et al., 1991), aflaxtoxin Bl (Yun et al., 1991); hexamethylphosphoramide (Ding et al., 1988), and nitrosodimethylamine (Davies et al., 1989; Fernandez et al., 1995). Therefore, inhibitors of CYP2A6 may be useful in the prophylaxis and treatment of malignant diseases.
- procarcinogens such as NNK (Crespi et al., 1991), aflaxtoxin Bl (Yun et al., 1991); hexamethylphosphoramide (Ding et al., 1988), and nitrosodimethylamine (Davies et al., 1989; Fernandez et al., 1995). Therefore, inhibitors of CYP2A6 may be useful in the prophylaxis and treatment of malignant diseases.
- compositions of the invention contain substances which selectively inhibit CYP2A6 described in detail herein or substances identified using the methods of the invention.
- the active substances can be administered alone, but are generally administered with a pharmaceutical carrier etc. (see below), selected on the basis of the chosen route of administration and standard pharmaceutical practice.
- the dosage administered will vary depending on the use and known factors such as the pharmacodynamic characteristics of the particular substance, and its mode and route of administration; age, health, and weight of the individual recipient; nature and extent of symptoms, kind of concurrent treatment, frequency of treatment, and the effect desired.
- the kinetics of inhibition created by certain chemical compounds can be altered or enhanced by adding to the treatment protocol a second inhibitor to a substance (e.g. , enzyme) that is capable of inhibiting the metabolism of the CYP2A6 inhibitor.
- a second inhibitor to a substance (e.g. , enzyme) that is capable of inhibiting the metabolism of the CYP2A6 inhibitor.
- a second inhibitor By adding such a second inhibitor, the quantity of the CYP2A6 inhibitor will be maintained thus prolonging the beneficial effect of maintaining an elevated plasma concentration of nicotine.
- the use of such a second inhibitor is very beneficial since it facilitates treatment of individuals by maintaining substantially constant nicotine levels and acting locally on the kinetics of the CYP2A6 inhibitor. By using this approach, large dosages of centrally active compounds can be avoided.
- preexposure of an individual to an inhibitory substance sometimes can result in an inhibitory effect that will outlast the presence of the drug in the plasma or that will have a persistent effect in the individual despite the inhibitor's half life in the plasma.
- This phenomenon caused by preincubation or preexposure of an inhibitory substance can help increase the dose interval at which a dosage of the substance must be administered, decrease the chronic dose or enhance CYP2A6 inhibition.
- preexposure of an individual to one inhibitory substance can subsequently decrease the needed dose of a second inhibitor.
- the appropriate dosage of a substance which selectively inhibits CYP2A6 is dependent upon the amount of CYP2A6 that is present in an individual's body.
- Example 7 we confirmed that such variations can exist in the genetic material of a population. It is, therefore, an aspect of this invention to provide a method for determining the CYP2A6 activity in an individual containing two mutant alleles, one mutant allele or no mutant alleles at a gene locus for the CYP2A6 gene, the method comprising the steps of:
- step (c) correlating the results of assaying in step (a) and the amount of CYP2A6 in step (b) to determine an appropriate dosage for that individual of a substance which (i) selectively inhibits CYP2A6 activity, or (ii) selectively inhibits transcription and/or translation of the gene encoding CYP2A6.
- the individual recipient may be any type of mammal, but is preferably a human. Generally, the recipient is an individual having a CYP2A6 genotype associated with an active form of the enzyme.
- the CYP2A6 genotype of an individual and the existence of an active CYP2A6 enzyme in an individual may be determined using procedures described herein. For example, coumarin 7-hydroxylation has been used to measure CYP2A6 activity (Cholerton et al., 1992; and Rautio et al. , 1992).
- the methods and compositions of the invention may be preferably used in individuals or populations having high levels of CYP2A6, or in individuals where the behavioural components of smoking are significant. -21-
- a daily oral dosage of an active ingredient such as coumarin or methoxsalen can be about 0.1 to 80 mg/kg of body weight, preferably 0.1 to 20, more preferably 0.2 to 3 mg/kg of body weight.
- a dose of 0.5 to 50 mg/kg of coumarin or methoxsalen per day in divided doses one to multiple times a day, preferably up to four times per day, or in sustained release form is effective to obtain the desired results.
- coumarin or methoxsalen is administered twice daily for one to four days. While standard interval dose administration may be used the compositions of the invention may be administered intermittently prior to high risk smoking times, e.g. , early in the day and before the end of a working day.
- the substances for the present invention can be administered for oral, topical, rectal, parenteral, local, inhalant or intracerebral use.
- the substances are administered in intranasal form via topical use of suitable intranasal vehicles, or via transdermal routes, using forms of transdermal skin patches known to those of ordinary skill in that art.
- the dosage administration will be continuous rather than intermittent throughout the dosage regimen.
- the substances can also be administered by way of controlled or slow release capsule system and other drug delivery technologies.
- the substances described in detail herein and identified using the method of me invention form the active ingredient, and are typically administered in admixture with suitable pharmaceutical diluents, excipients, or carriers suitably selected with respect to the intended form of administration, that is, oral tablets, capsules, elixirs, syrups and the like, consistent with conventional pharmaceutical practices.
- the active substances can be combined with an oral, non-toxic, pharmaceutically acceptable, inert carrier such as lactose, starch, sucrose, glucose, methyl cellulose, magnesium stearate, dicalcium phosphate, calcium sulfate, mannitol, sorbitol and the like; for oral administration in liquid form, the oral active substances can be combined with any oral, non-toxic, pharmaceutically acceptable inert carrier such as ethanol, glycerol, water, and the like. Suitable binders, lubricants, disintegrating agents, and colouring agents can also be incorporated into the dosage form if desired or necessary.
- an oral, non-toxic, pharmaceutically acceptable, inert carrier such as lactose, starch, sucrose, glucose, methyl cellulose, magnesium stearate, dicalcium phosphate, calcium sulfate, mannitol, sorbitol and the like
- any oral, non-toxic, pharmaceutically acceptable inert carrier such as ethanol, glycerol
- Suitable binders include starch, gelatin, natural sugars such as glucose or beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, tragacanth, or sodium alginate, carboxymethylcellulose, polyethylene glycol, waxes, and the like.
- Suitable lubricants used in these dosage forms include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, and the like.
- disintegrators include starch, methyl cellulose, agar, bentonite, xanthan gum, and the like.
- Gelatin capsules may contain the active substance and powdered carriers, such as lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, and the like. Similar carriers and diluents may be used to make compressed tablets. Tablets and capsules can be manufactured as sustained release products to provide for continuous release of active ingredients over a period of time. Compressed tablets can be sugar coated or film coated to mask any unpleasant taste and protect the tablet from the atmosphere, or enteric coated for selective disintegration in the gastrointestinal tract. Liquid dosage forms for oral administration may contain colouring and flavouring agents to increase patient acceptance.
- Water a suitable oil, saline, aqueous dextrose, and related sugar solutions and glycols such as propylene glycol or polyethylene glycol s, may be used as carriers for parenteral solutions.
- Such solutions also preferably contain a water soluble salt of the active ingredient, suitable stabilizing agents, and if necessary, buffer substances.
- suitable stabilizing agents include antioxidizing agents such as sodium bisulfate, sodium sulfite. or ascorbic acid, either alone or combined, citric acid and its salts and sodium EDTA.
- Parenteral solutions may also contain preservatives, such as benzalkonium chloride, methyl- or propyl-paraben, and chlorobutanol.
- Liposome delivery systems such as small unilamellar vesicles, large unilamellar vesicles, and multilamellar vesicles.
- Liposomes can be formed from a variety of phospholipids, such as cholesterol, stearylamine, or phosphatidylcholines.
- Substances described in detail herein and identified using the methods of the invention may also be coupled with soluble polymers which are targetable drug carriers.
- soluble polymers include polyvinylpyrrolidone, pyran copolymer, polyhydroxypropy lmethacrylamide-phenol , polyhydroxyethylaspartamidephenol , or polyethyleneoxide-poly lysine substituted wid palmitoyl residues.
- the substances may also be coupled to biodegradable polymers useful in achieving controlled release of a drug.
- Suitable polymers include polylactic acid, polyglycolic acid, copolymers of polylactic and polyglycolic acid, polyepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals, polydihydropyrans, polycyanoacylates, and crosslinked or amphipathic block copolymers of hydrogels.
- the substances can also be affixed to rigid polymers and other structures such as fullerenes or Buckeyballs.
- compositions suitable for administration contain about 1 milligram to 1500 milligrams of active substance per unit.
- the active ingredient will ordinarily be present in an amount of about 0.5-95% by weight based on the total weight of the composition.
- Suitable pharmaceutical carriers and methods of preparing pharmaceutical dosage forms are described in Remington's Pharmaceutical Sciences. Mack Publishing Company, a standard reference text in this field.
- More than one substance described in detail herein or identified using the methods of the invention may be used to regulate metabolism of nicotine to cotinine.
- the substances can be administered by any conventional means available for the use in conjunction with pharmaceuticals, either as individual separate dosage units administered simultaneously or concurrently, or in a physical combination of each component therapeutic agent in a single or combined dosage unit.
- the active agents can be administered alone, but are generally administered with a pharmaceutical carrier selected on the basis of the chosen route of administration and standard pharmaceutical practice as described herein.
- the combination of an CYP2A6 inhibitor e.g. , coumarin, methoxsalen
- a CYP2B6 inhibitor e.g. , orphenadrine
- a preferred embodiment of the invention provides a method for treating conditions requiring regulating nicotine metabolism to cotinine comprising administering an effective amount of a CYP2A6 inhibitor and an effective amount of a CYP2B6 inhibitor to selectively inhibit nicotine metabolism to cotinine.
- the CYP2A6 inhibitor is methoxsalen or an analog or derivative thereof
- the CYP2B6 inhibitor is orphenadrine, or an analog or derivative thereof.
- the inhibitors may be administered concurrently, separately or sequentially.
- the doses of the CYP2A6 inhibitor and the CYP2B6 inhibitor are each selected so that each inhibitor alone would not show a full effect.
- the effective doses are those which are approximately the minimum doses adequate for enhanced inhibition of nicotine metabolism to cotinine.
- Pharmaceutical compositions containing combinations of CYP2A6 and CYP2B6 inhibitors may be prepared, and administered as described herein for the compositions containing CYP2A6 inhibitors.
- the pharmaceutical compositions preferably contain methoxsalen or an analog or derivative thereof, and orphenadrine, or an analog or derivative thereof, in concentrations of 1 to 1500 mg, and 25 to 400 mg, respectively.
- CYP2A6 is the major nicotine metabolizing enzyme in human livers suggests that the enzyme can be assayed in an individual to determine the individual's risk of developing tobacco dependence. Determination of CYP2A6 levels may also be used to select and monitor in an individual appropriate conventional nicotine replacement therapies such as the nicotine patch and nicotine gum. It is unlikely that conventional nicotine replacement therapies (e.g. nicotine gum, nicotine patch, spray, pulmonary inhalation or other forms) will have a high success outcome if an individual has high levels of CYP2A6. Conversely, if an individual has very low levels of CYP2A6, administering nicotine at high dosages will likely result in increased toxicity, and side effects.
- conventional nicotine replacement therapies e.g. nicotine gum, nicotine patch, spray, pulmonary inhalation or other forms
- Example 1 Materials and Methods: Biological Samples
- CYP2D6 yeast Microsomal preparations of CYP2D6 expressed in yeast (aH22/peltl cells) and control yeast (AH22/pMA91 cells) were provided by Dr. M.S. Lennard, University of Sheffield, U.K..
- Dextrorphan, methoxymorphinan, and hydroxymorphinan were provided by Hoffmann-La Roche Inc., Nutley, (N.J., USA). Budipine was obtained from Byk Gulden Pharmazeutika, Konstanz, Germany. Microsome Preparation. The partial livers ( ⁇ 2 grams) from 30 humans were thawed on ice, then minced in two volumes of cold 1.15% KC1. The samples were homogenized by applying ten strokes of a Teflon pestle powered by a Black and Decker electric drill. Each liver homogenate was then subjected to a centrifugation of 9000 g for 20 min. at 4°C in a Sorvall RC2-B.
- the supernatant which contains cytosol and microsomes, was decanted and centrifuged at 100,000 g for 60 min. at 4°C in a Sorvall Combi Plus Ultraspeed Centrifuge.
- the resulting microsomal pellet was resuspended in 1.15% KC1 and centrifuged again at 100,000 g for 60 min. at 4°C for further purification.
- the microsomal pellet was resuspended in a 2: 1 vol/wt solution of 1.15% KC1 and stored in a Forma Scientific freezer at -70°C.
- Protein Determination Protein concentration of the microsomal samples were determined with a Pierce BCA protein assay kit (Pierce Chemical Co.
- BSA bovine serum albumin standard
- Dextromethorphan to dextrorphan kinetics Dextromethorphan to dextro ⁇ han kinetics was measured as a function of protein concentration and time. Dextrometho ⁇ han at a concentration of 5 ⁇ M was incubated with 0.025, 0.05, 0.1 , 0.2, 0.3, 0.4, and 0.5 mg protein/ml, with 0.8 mM NADPH in 0.2 M phosphate buffer (pH 7.4).
- the incubation mixture was comprised of 125 ⁇ l phosphate buffer (pH 7.4), 50 ⁇ l microsomal protein, 50 ⁇ l dextrometho ⁇ han, and 25 ⁇ l NADPH for a total volume of 250 ⁇ l. Incubations were carried out at 37 °C for 30 min. in a shaking water bath, and were terminated with the addition of 10 ⁇ l perchloric acid. Budipine was used as an internal standard. Samples were then centrifuged at 3000 rpm for 5 min. , and 30 ⁇ l of the supernatant was analyzed by HPLC. Results revealed that dextro ⁇ han production was linear from 0.025 to 0.5 mg protein/ml throughout a 30 min. incubation. Apparent K.
- HPLC The HPLC system (Hewlett Packard) consists of a 1050 series pump and autosampler, connected to a HP 3396 series II integrator.
- Dextrometho ⁇ han and the various metabolites in the incubation samples were measured as described by Broley et al. (1989), except that the excitation and emission wavelengths were set at 195 nm and 280 nm, respectively for a higher sensitivity.
- Dextro ⁇ han calibration curves were linear from 0 to 120 pmoles, with the lowest detectable level of 5 pmoles for dextro ⁇ han.
- Incubation Microsomes were removed from a -70 °C freezer and thawed on ice. Incubation mixtures generally contained 100 ⁇ l (S)-nicotine, 100 ⁇ l NADPH (ImM final), 200 ⁇ l human liver microsomes (0.5 mg/ml), 20 ⁇ l Wistar rat liver cytosol, 200 ⁇ l potassium phosphate buffer (pH 7.4, 40 ⁇ M final), diluted to a 1 ml final volume with 1.15% KC1. The reaction was initiated with the addition of NADPH and placing the samples at 37 °C.
- S S-nicotine
- NADPH ImM final
- human liver microsomes 0.5 mg/ml
- 20 ⁇ l Wistar rat liver cytosol 20 ⁇ l Wistar rat liver cytosol
- 200 ⁇ l potassium phosphate buffer pH 7.4, 40 ⁇ M final
- the mixtures were incubated in a polypropylene conical 10 ml tubes then placed in a Precision Scientific Shaker Bath (Model 50) at 37 °C for 45 minutes. The reaction was stopped by adding 100 ⁇ l of 20% Na 2 CO 3 (pH 11.4). Nicotine to cotinine kinetic studies were performed by incubating 1 , 5, 10, 50, 100, and 200 ⁇ M (S)-nicotine with 0.5 mg/ml microsomal protein for 45 min, in the presence of 20 ⁇ l rat liver cytosol, 1 mM NADPH, in 40 mM phosphate buffer (pH 7.4). The reaction was started by adding NADPH.
- the incubation mixture comprised of kinetic parameters were calculated by use of computer program Enzfitter (Robin J. Leatherbarrow, 1987). The data were fit by one-site Michaelis-Menten rate equation. .Extraction: After basification with Na 2 CO 3 , 10 ⁇ l of ketamine (the internal standard) was added to each sample. Ethyl acetate (3 mis) was added for extraction pu ⁇ oses. The samples were vortexed vigorously for 5 min. , followed by centrifugation for 5 min. at 3000 ⁇ m in an GLC-2B centrifuge. The organic layer (top) was pipetted to a separate 10 ml conical mbe, which contained 400 ⁇ l of 0.01 N HCl.
- HPLC High Performance Liquid Chromatography
- the separation was performed with isocratic elution at a flow rate of 1 ml/min.
- the retention times for cotinine, nicotine and ketamine were 3.5, 4.2, and 7.0 minutes respectively.
- the HPLC system consisted of a Hewlett Packard 1090 solvent delivery system linked to a 1050 series UV detector. The UV detector was set at 210 nm to optimize for cotinine detection. Data Analysis. The UV absorbence data was transferred to a Hewlett-Packard Chemstation.
- the three peaks of interest were cotinine, nicotine, and ketamine (internal standard).
- the heights of the respective peaks were used to determine peak height ratios.
- the cotinine peak height ratio was determined by measuring the ratio between the height of the cotinine peak to the ketamine peak height.
- Peak Height Ratio (PHR) cotinine peak height ketamine peak height
- the peak height ratios were used to analyze the relative amount of cotinine production, and to determine the specific concentration of cotinine present in each sample incubation. This was achieved by producing standard curves during each session of data collection.
- Standard curves were produced by injecting various concentrations of cotinine into the HPLC. Cotinine amounts typically included 1.25, 2.5, 5.0, and 10.0 nmole concentrations. 10 ⁇ l of ketamine, at a concentration of 0.25 mg/ml, was added to each sample. The standard curve enabled any given PHR obtained from a given sample to be converted to its respective concentration of cotinine in nmoles. Within-day and Between-day variations. The within-day variation and between day variation of the assay were calculated for two concentrations of cotinine. Standard solutions contained 2.5 nmoles and 5.0 nmoles of cotinine per ml of incubation mixture. Samples contained 40 mM phosphate buffer, and 1.15% KC1.
- Protein-time assay Protein concentrations of 0.125, 0.25, 0.5, and 1 mg protein/ml from the K20 liver microsome sample were incubated at 37°C, with 20 ⁇ l rat liver cytosol, 1 mM NADPH, in 40 mM phosphate buffer (pH 7.4) across several time intervals. Results are presented in Figure 3. These results show that cotinine formation is linear at a protein concentration of 0.125 to 0.5 mg/ml for a 45 min. incubation. Cotinine formation was also dependent on NADPH concentrations. A NADPH concentration of 1 mM was determined to be optimal for the above experimental conditions. Quinidine and coumarin inhibition of cotinine formation.
- Nicotine was incubated with 0.5 mg/ml microsomal protein from K20 human liver. Incubations included 1 mM NADPH, 20 ⁇ l rat liver cytosol, 40 mM phosphate buffer (pH 7.4), and were carried out for 45 minutes at 37°C. Inhibition studies included adding 100 ⁇ M quinidine, 100 ⁇ M coumarin, 100 ⁇ M of quinidine and coumarin, with 60 ⁇ M (S)- nicotine. Incubation of nicotine in yeast expressing CYP2D6. Incubation conditions using CYP2D6 expressed in yeast supported by cumene hydroperoxide (CuOOH), were essentially those of Zanger et al., (1988) and Wu (1993).
- CuOOH cumene hydroperoxide
- CuOOH 80% in cumerol, Sigma
- H 2 0 v/v
- 375 ⁇ M 0.3 M potassium phosphate buffer, pH 7.4.
- 200 ⁇ l of this solution was added to 100 ⁇ l nicotine (100 ⁇ M final) and 20 ⁇ l of rat liver cytosol, in a final volume of 1 ml (final CuOOH concentration of 75 ⁇ M).
- the incubation was initiated by the addition of 200 ⁇ l of yeast protein (0.3 mg/ml final) and was for 20 min. at room temperature. Incubations were carried out in a shaking water bath, at 37°C, for 120 min. All reactions were stopped with the addition of 100 ⁇ l 20% Na 2 CO 3 (pH 11.4).
- Dextromethorphan to dextrorphan metabolism in human liver microsomes Dextromethorphan to dextrorphan metabolism in human liver microsomes.
- the kinetics of dextro ⁇ han formation were determined using a non-linear least squares algori ⁇ im in which the data were weighted by the reciprocal of the rate of metabolism and were fit by one or two site Michaelis-Menten kinetic models.
- the Ll 1 liver sample displays a high affinity enzyme kinetics, while the L3 liver sample displays both high and low affinity enzyme kinetics.
- K m and V max values for nicotine to cotinine kinetics were calculated for all 30 human liver microsomes.
- Sample Michaelis-Menten curves for nicotine to cotinine kinetics are shown in Figure 4. These graphs show livers which display one site or multiple site enzyme kinetics.
- Figure 5 compares the respective K m values across all 30 samples. These figures were segregated into male and female liver donors so that sex differences could be examined.
- the mean K m value for all 30 livers is 66.6 ⁇ 31.8 mM (mean ⁇ SD).
- W ⁇ results revealed marked inter-individual variations in cotinine formation (Figure 6). Four human livers appeared to have very high rates of cotinine formation.
- the mean V max value for all 30 livers is 28.9 ⁇ 28.9 nmoles/mg protein/hr (mean + SD).
- Quinidine which is a specific inhibitor of the CYP2D6 enzyme, had some inhibitory effect on cotinine formation.
- Quinidine at 100 ⁇ M 1000 times greater than its K, value for inhibiting dextrometho ⁇ han to dextro ⁇ han metabolism by CYP2D6; Kerry et al., 1994
- cotinine formation was inhibited by over 80% , with little additional inhibition when quinidine was added in combination with coumarin. Nicotine metabolism with yeast expressing CYP2D6
- the possibility of inactive CYP2D6- expressing yeast was investigated; p-methoxy-amphetamine, methamphetamine, and dextrometho ⁇ han, substrates for CYP2D6, were all oxidized by the CYP2D6- expressing yeast. Nicotine incubations in yeast were performed simultaneously with para-methoxyamphetamine incubations . Discussion:
- the metabolism of dextrometho ⁇ han and nicotine was studied using a bank of 30 human liver microsomes.
- Dextrometho ⁇ han metabolism to dextro ⁇ han revealed typical Michaelis-Menten kinetics with some livers displaying a low affinity site.
- the apparent high affinity K m value was 5.79 ⁇ 2.01 (mean ⁇ SD).
- 5 ⁇ M of dextrometho ⁇ han was incubated with 30 human liver microsomes to access CYP2D6 activity in each liver. Nicotine to cotinine metabolism was also investigated in the same 30 human livers. This required the development of a novel assay which is described above.
- CYP2D6 Since the CYP2D6 enzyme appears not to be involved in nicotine to cotinine metabolism, an investigation of the role of other cytochromes P450 was undertaken. In particular the importance of CYP2A6 in contributing to inter-individual differences in nicotine metabolism was accessed in vitro. CYP2A6, heterogously expressed in human lymphoblastoid cells, has one of the highest activities in the conversion of nicotine to cotinine, second only to CYP2B6 (McCracken et al, 1992).
- Human liver microsomes The same 30 human liver samples were used in this study as were used in Example 1.
- Extensive chemical inhibition studies consisted of incubating 100 ⁇ M (S)-nicotine with 150 ⁇ M concentrations of coumarin, o ⁇ henadrine, troleandomycin, and coumarin with o ⁇ henadrine in combination, with all 30 human liver microsomes, in duplicate. To maximize inhibition, and reduce the loss due to its own metabolism, a concentration of 150 ⁇ M was chosen for each specific inhibitor. From the kinetic studies this inhibitor concentration is approximately 2 times the mean K m value for nicotine to cotinine metabolism. The nicotine concentration was set at 100 ⁇ M which was the concentration that was approaching the V max for cotinine formation.
- Immunoinhibition experiments consisted of incubating 0.5 mg/ml K27 liver microsomes with a CYP2A6 monoclonal antibody (MAB-2A6) and a CYP2B1 (anti-rat CYP2B1) polyclonal antibody.
- Antibodies and microsomes were preincubated on ice for 30 minutes followed by the addition of 100 ⁇ M nicotine, 1 mM NADPH, and 20 ⁇ l rat cytosol in 25 mM Tris-HCl buffer.
- Antibody concentrations were chosen based on immunoinhibition information provided by Gentest Co ⁇ .
- Figure 7 shows the potency and specificity of the MAB-2A6 and anti-rat CYP2B1 for their respective enzymes. Gentest Co ⁇ .
- MAB-2A6 inhibited over 95% 2A6 activity, at 0.25 mg antibody / ⁇ g microsomal protein. They used coumarin hydroxylation as a measure of 2A6 activity. They also showed that anti-rat 2B1 cross-reacts with the human CYP2B6 enzyme to inhibit its activity.
- the primary, and secondary antibodies consisted of the monoclonal CYP2A6 antibody (1/2000 dilution of 5 mg/ml stock; Gentest Co ⁇ oration), and an anti-mouse IgG horseradish peroxidase conjugate (1/2000 dilution; Amersham Co ⁇ oration, Arlington Heights, IL), respectively.
- blots were washed three times with TBST for 10 minutes each. Blots were visualized using the chemiluminescent ECL reagent (Amersham Co ⁇ oration). The densities of the visualized bands were quantified using a MCID imaging system (Imaging Co.). After determining the linearity of detection of CYP2A6 bands ( Figure 8), a concentration of 30 ⁇ g of microsomal protein was used for each liver for comparisons. Results:
- Troleandomycin a specific CYP3A inhibitor, did not show any overall inhibition of cotinine formation. The mean inhibition was 3% of control cotinine formation, with a standard deviation of 11 % ( Figure 22). Discussion:
- the rank order of potency was coumarin > 7-methoxycoumarin > 7- methylcoumarin > 7-ethoxycoumarin. It is interesting to note that 7 -ethoxy coumarin had a lesser effect of inhibiting nicotine metabolism than coumarin, since 7- ethoxycoumarin is a well known substrate for many human cytochrome P450 enzymes (i.e., CYPs 1A1, 1A2, 2A6, 2B6, 2C8, 2C9, 2E1, and 3A4) (Waxman et al. , 1991). This suggests that nicotine and coumarin metabolisms are closely related. Immunochemical inhibition studies revealed that a monoclonal antibody raised against the human CYP2A6 inhibited cotinine formation by 60% .
- Troleandomycin a substrate for several CYP3A enzymes, was used to study CYP3As role in nicotine metabolism. It was important to answer the CYP3A question since it is the most abundant CYP found in human liver (Shimada et al. , 1994). CYP3A expression is also induced by exposure to phenobarbital, hence it is potentially a great source of variability in nicotine metabolism. Thus, the use of troleandomycin in the chemical inhibition studies behaved as a negative control. The results agree with previous studies that showed no involvement of the CYP3A subfamily in nicotine metabolism.
- CYP2A6 has been shown to play an important role in nicotine metabolism, thus variations in CYP2A6 expression may be directly responsible for the high inter- individual variation seen in cotinine formation. Genetic variation in CYP2A6, and variable CYP2B6 expression may contribute to the 3-fold variation observed in nicotine metabolism in human subjects (Benowitz et al., 1982). Exposure to phenobarbital has been shown to have an inductive effect on nicotine metabolism by primarily increasing the expression of cytochrome P450 enzymes (Nakayama et al. , 1982; Hibberd et al. , 1985; Foth et al. , 1990; Seaton et al., 1991; Seaton et al., 1993).
- the CYP2A and CYP2B gene subfamilies are closely linked on chromosome 19.
- CYP2A6 is important in nicotine metabolism, and that nicotine metabolism is quite variable among individual human liver microsomes. This variability may be a consequence of previous drug use, and in the case of CYP2A6 the presence of variant CYP2A6 alleles.
- Microsomes were removed from -70 °C freezer and thawed on ice. Incubation mixtures generally contained 100 ⁇ l (S)-nicotine, 100 ⁇ l NADPH (ImM final), 200 ⁇ l human liver microsomes (0.5 mg/ml), 20 ⁇ l Wistar rat liver cytosol, 200 ⁇ l potassium phosphate buffer (pH 7.4, 40 ⁇ M final), diluted to a 1 ml final volume with 1.15 % KC1. The reaction was initiated with the addition of NADPH and placing the samples at 37°C for 45 minutes. The reaction was stopped by adding 100 ⁇ l to 20% Na 2 CO 3 (pH 11.4).
- Spherisorb-Hexyl column (15 x 0.46 cm) and a mobile phase consisting of 20% acetonitrile and 80% 20 mM potassium phosphate, pH 4.6, containing 1 mM octanesulfonic acid was used. The separation was performed with isocratic elution at a flow rate of 1 ml/min. The retention times for cotinine, nicotine and ketamine were
- Figure 31 shows a Dixon plot of 7-methoxycoumarin inhibition of nicotine to cotinine formation in K28 human liver microsomes.
- Each subject abstained from tobacco, food, beverages (other than water), and any inconsistently used drugs from midnight before each study day but continued to take any regularly scheduled drugs allowed by the protocol (e.g., oral contraceptives, daily vitamins).
- a breath CO sample (Ecolyzer) was taken to assess compliance with the smoking abstinence ( ⁇ 10 ppm expected).
- the subsequent daily schedule is summarized here. All measurements are with respect to a time zero at 8 AM, at which time the first of three hourly nicotine injections were given. Nicotine was injected at 0, +1 , and +2 hours. Baseline physiologic and subjective measures were taken at -30 minutes, +30 minutes, and hourly thereafter until +5.5 hours, approximately coinciding with the expected peak plasma nicotine concentrations after each injection. Blood samples were taken at least hourly, more often around the expected peaks after the first and third injections, as described below.
- Methoxsalen/placebo capsules were administered 30 minutes prior to the first nicotine injection.
- Placebo and methoxsalen capsules were used.
- the following table shows the doses of methoxsalen that were administered.
- Sterile nicotine bitartrate was obtained from Sigma Chemical. The reported purity is > 99.5%. Nicotine bitartrate injections of 31 ⁇ g/kg (expressed as the base) in sterile saline were used. Each of these three daily injections delivered 2 mg to a 70 kg subject, a mass of drug comparable to that delivered by one cigarette. The solutions were passed through a viral filter to remove any remote risk of viral or bacterial contamination.
- Plasma (nicotine, cotinine) and urinary nicotine and cotinine were measured using an HPLC ion exchange column assay, using an electrochemical detector for nicotine and a UV detector for od er compounds.
- the sensitivity of the nicotine assay was ⁇ 1 ng/ml and that of the cotinine was ⁇ 5 ng/ml.
- Conjugates in urine were determined after hydrolysis with beta-glucuronidase.
- Creatinine was measured, allowing all drug concentrations to be re-expressed as a ratio of the other substance to creatinine. This provided some control for variability in urine dilution or the duration or efficiency of urine collection.
- Figure 25 shows the large changes caused by methoxsalen in nicotine concentrations.
- Figure 26 shows the effect over time of methoxsalen on the cotinine plasma concentrations of seven subjects.
- Figure 27 and Figures 28A to 28C indicate that these increases in plasma nicotine were accompanied by a significant increase in nausea, anxiousness, difficulty concentrating, systolic blood pressure and a significant decreased desire to smoke and urge to smoke and a significant decrease in the expectation that a cigarette would be pleasant.
- Immunoinhibition experiments consisted of incubating 0.5 mg/ml K12 liver microsomes with CYP2A6 (monoclonal), CYP2B1 (polyclonal), CYP2E1 (polyclonal),
- CYP2D6-peptide polyclonal
- CYP3A2 polyclonal antibodies
- BSA, rabbit and goat antisera were used as negative controls.
- Antibodies and microsomes were preincubated on ice for 30 min followed by the addition of 100 ⁇ M nicotine, 1 mM NADPH, and 20 ⁇ l rat cytosol in 0.04 M phosphate buffer (pH 7.4). Subsequent incubations were for 45 min at 37 °C.
- the CYP2A6 antibody selectively inhibits cotinine formation and produces > 80% decrease in nicotine metabolism by CYP2A6 (see Figure 29).
- ASO antisense oligodeoxynucleotides
- the h2A6 P450 cells were grown to 3xl0 6 cells, the media removed and 5 ⁇ g ASO and 20 ⁇ g/ml lipofectin was added in 1 ml of 5 % horse serum supplemented media. The cells were grown for 24 hr when a further 4 mis of complete media (10% horse serum) was added and the cells were grown for an additional 48 hr. Each cell sample was then washed 3 times with phosphate buffered saline, pelleted and frozen. Western blots were carried out on the samples and it was determined mat only the ASO #23 (e.g., 3' prime end of exon 2) was effective at removing the CYP2A6 immunoreactivity.
- ASO #23 e.g., 3' prime end of exon 2
- MSO missense oligodeoxynucleotides
- the human hepatic HepG2 cells which express CYP2A6, were grown and aliquoted at 1.0 x 10 5 cells and grown for 48 hrs in 2 mis complete media (10% fetal calf serum). After 48 hr, 2 ⁇ g ASO oligos and 15 ⁇ g/ml lipofectin was added in 1 ml 5% FCS media and grown for 24 hr. Cells then trypsinized and washed for Western blots or fixed with 4% paraformaldehyde for Immunocytochemistry. Again we found that only ASO#23 was able to decrease the CYP2A6 protein.
- Figure 12 illustrates the profound decreases in CYP2A6 protein after treatment with ASO#23, relative to control untreated and MSO#23 treated cells.
- CYP2A6 genotype was determined using nested PCR and RFLP as described by Fernandez-Salguero et al (1995).
- the first amplification which is CYP2A6 gene- specific, was used to increase the specificity for the CYP2A6 gene (versus other CYP2A genes).
- Exon 3 was utilized in the second amplification because both the CYP2A6*2 and CYP2A6*3 mutant alleles contain nucleotide changes leading to amino acid changes in this region of the CYP2A6 gene.
- the first amplification was performed using the XL-PCR kit (Parkin-Elmer Co.,
- the amplification was performed in a MJ DNA Engine (MJ Research, Inc., Watertown, Massachusetts) at 93 °C for 1 minute, 66 °C for 6 minutes and 30 seconds for 31 cycles.
- the second amplification was performed in a reaction mixture containing 0.5 ⁇ M of primers E3F and E3R, 200 ⁇ M dNTPs, 1.5 mM MgCl 2 , 2.5 U of Taq DNA polymerase (Gibco BRL, Life Technologies, Burlington, Ontario), and 2.5 ⁇ l of first amplification product, which was the template for the reaction.
- the reaction conditions were as follows: 94°C for 3 minutes, followed by 31 cycles of 94°C for 1 minute, 60°C for 1 minute and 72 °C for 1 minute.
- the second amplification yielded a PCR product 201 bp in length which was digested with Xcm I (New England Biolabs) and Dde I (New England Biolabs and Pharmacia Biotech) to detect the CYP2A6*2 and CYP2A6*3 mutations, respectively (cutting indicates the presence of the mutation).
- Concentrations of enzymes and PCR product, total volume and digestion time were determined empirically to optimize cutting efficiency with a minimal amount of time and enzyme.
- Xcm I digestion reactions were carried out at 37 °C for 2 hours in a 30 ⁇ l reaction mixture containing IX NEBuffer 3 (100 mM NaCl, 50 mM Tris-HCl, 10 mM MgCl 2 , 1 mM DTT pH 7.9 @ 25°C), dH 2 0, and 2 U of Xcm I.
- a smdy has been conducted to determine (i) if coumarin metabolic pattern (recovery and ratios of free, 7-hydroxy and conjugated 7-hydroxy coumarin) reflects CYP2A6 genotype, independent of current smoking status, and nicotine (NIC) metabolism determined from smokers' blood and urine samples; (ii) if acute smoking affects coumarin metabolism in smokers; and (iii) if mere are differences between male and females in the prediction of NIC metabolism from coumarin metabolism.
- Urine or plasma samples (0.5 ml) are hydrolyzed with 0.25 ml of ⁇ -glucuronidase acetate buffer solution (15 mg/ml acetate buffer, 0.2 M, pH 5.0) at 37 °C for 30 min. Extraction is followed with 2 ml ether by vertex for 5 min and centrifuged at 3000 ⁇ m for 10 min. Ether extract (1.2 ml) is transferred to another clean mbe and dried under nitrogen gas. The residue is reconstituted in the HPLC mobile phase (see below), and injected onto HPLC.
- HPLC Analysis The HPLC system consists of Hewlett Packard 1050 HPLC system (pump, autosampler and UV detector) and HP339611 integrator. The chromatographic separation was performed with an HP Spherisorb-ODS2 column (125 x 4 mm I.D., 5 ⁇ ). Samples were eluted with a mobile phase of acetonitrile:water:acetic acid of 150:850:2 (v/v/v) at a flow rate of l.Oml/min, and monitored by a UV detector at a wavelength of 324 nm. Samples are quantitatively determined by an external standard method. Results:
- Blank urine or plasma samples showed no interfering peak for 7- hydroxy coumarin. Sensitivity of this method is 1 ng/ml urine or plasma. Intraday and inter-day variations are less man 10% . This analysis is linear from 10 ng to 4000 ng/ml.
- Figure 37 illustrates a correlation between fasted morning and non-fasted afternoon coumarin (C) testing sessions.
- the labels on Figure 37 represent male smokers (M,S), male nonsmokers (M, NS), female smokers (F, S), female nonsmokers (F, NS).
- subjects were given 5 mg of coumarin p.o., and urine was collected for the next 4 hours.
- Figure 37 is expressed as the percent of an initial 5 mg coumarin dose excreted as 7-hydroxycoumarin within 4 hours. Morning and afternoon values were matched per subject such that the first morning value and the first afternoon value were plotted as one point and the second morning and second afternoon value were plotted as a second point per subject.
- Figure 39 is a graph showing a time course of total 7- hydroxycoumarin concentration detected in the plasma of subjects given 100 mg of coumarin. Figure 39 illustrates various time courses based on corresponding genotypes for CYP2A6.
- Benowitz has used a 30-minute deuterium-labelled NIC-d2 infusion (with COT- d4) to smdy die kinetics and fractional clearance of NIC in smokers and non-smokers.
- the kinetics of the deuterated NIC are very similar to unlabelled NIC.
- the advantage of the non-radioactive label is that the NIC-d2 and resulting COT-d2 can be used as quantitative measures of NIC metabolism in smokers while smoking. This approach will be used to obtain a quantitative estimate of NIC to COT conversion by giving a dose of NIC-d2 sufficient to detect in the urine, taking advantage of the fact that NIC and COT are found in much higher concentrations in urine than in plasma.
- Plasma and urinary NIC, COT and trans-3'-hydroxycotinine and tiieir glucuronides will be measured using a existing GC assay as modified in Dr. Jacob's lab (1988 protocol). Conjugates will be determined after alkaline hydrolysis (NIC and COT) or hydrolysis with ⁇ - glucuronidase (3'-hydroxycotinine). The quantisation limit is 1 ng/ml NIC and 10 mg/ml COT, with detection 50% lower. Coefficients of variation range from 1.1 to 7.8% for NIC (1-100 ng/ml). NIC-d2, COT-d2 will be determined by GC-MS (111).
- Most smokers' NIC metabolism can be inhibited by coumarin, the remainder by an appropriate combination of coumarin and o ⁇ henadrine; and such inhibition will reduce smoking behaviour.
- DSM-IV current tobacco dependent
- COT because of its longer half-life is less critically affected by sampling time and can be used to estimate daily NIC intake. 30 minutes after study drug, a tracer dose of Nicotine-d2 Test 1 will be given. Subjects will then collect their urine for 3 h ("Coumarin Test") and 4 h separately for determination of NIC-d23/COT-d2 ratio (7 h) and coumarin and total and free 7-OH coumarin. Additional blood samples will be collected 0.5 (coumarin/7-OH-coumarin), 3 and 7 h (NIC/COT) after the pulse tracer dose commences. Breath CO will be determined at me same times. During this period subjects will be permitted to smoke their usual brand ad lib, drink caffeinated beverages, play games, watch videos, etc. The number of cigarettes used and residual butt weight recorded.
- NIC is the addictive agent in tobacco dependence and smokers regulate their brain NIC within a fairly narrow individual concentration band
- selective inhibition of NIC conversion to COT should result in a decrease in smoke exposure (i.e. , "smoking").
- Some individuals may require different combinations of CYP2A6 and CYP2B6 inhibitors to achieve sufficient modification of NIC metabolism.
- a preliminary smdy has been designed to confirm the efficacy and safety of CYP2A6 inhibitors and the need for CYP2B6 inhibition in the reduction of smoking exposure and as an aid in smoking cessation.
- susceptibility to CYP2A6 activity inhibition will be assessed by stable-labelled "Nicotine Test" after coumarin 50 or 100 mg b.i.d.
- Tobacco smoking behaviour will be monitored by daily smoking diary cards, twice weekly home carbon monoxide breath sample collected in remote CO exposure collection bags (mid-afternoon between 2:00 and 6:00 p.m.), twice weekly salivary COT.
- Subjects will be given instructions with respect to the pu ⁇ ose of treatment, plus limited supportive counseling, plus structured self-help advice. Patients will be seen weekly between 2:00 and 6:00 p.m. each day at which time plasma NIC/COT, breath CO will be done.
- Example 4 Based upon studies such as outlined in Example 4, a double-blind trial to confirm the efficacy of CYP2A6 inhibition on smoking reduction and cessation will be carried out. The drug choice and doses will be determined by the studies as outlined in Example 4. A positive treatment control (e.g., NIC patch) will be tested.
- NIC patch a positive treatment control
- CYP2A6 inhibition should decrease smoke exposure by decreasing the number of cigarettes or by altering smoking behaviour.
- Subjects will, at the end of each "active" drug/placebo 2-week phase, be told to maintain their lower smoking behaviour for 2 weeks. This 2-week period is one of behavioural change. The inhibitor-placebo cycle will be repeated. Subjects will be seen weekly at which time their self-report smoking logs will be reviewed along with their progress (minimal adjunctive care). After the trial, subjects will be contacted at 3, 6 and 12 months and provide at least a salivary COT to determine maintenance of quit rates. Breath CO, salivary, plasma NIC/COT will be determined. Established criteria for cessation will be used.
- Smokers with varying degrees of cigarette consumption will receive a dosage of coumarin (using a dose of coumarin which effectively alters nicotine levels: derived from Project 1) or placebo for three consecutive days (both conditions tested in all individuals). Smokers from both genotypes will be included, allowing us to assess safety (e.g., nausea) and effectiveness (only in wild type or works for both groups?). Cigarette consumption and behavioural indices as well as nicotine, cotinine and CO measures will be determined daily. Effectiveness of coumarin in decreasing cigarette consumption, increasing nicotine, decreasing CO (cigarette exposure) will be determined. In addition the relative effect on heavy or light smokers, and first, second and third days, will be determined. EXAMPLE 13 Acute effects of coumarin on smoking behaviour in a social setting Hypothesis: That acute administration of coumarin (inhibiting nicotine metabolism), resulting in increased nicotine, will decrease smoking behaviour in an acute social setting.
- This product may be more or less effective in an acute versus chronic dosing regime. It would also be beneficial to have a product which could be tailored to acute high risk occasions (bar nights, parties, occasions with other smokers, locations with strong smoking associations).
- Nicotine bitartrate 31 ⁇ g/kg (expressed as the base) is administered subcutaneously in the absence and die presence of pretreatment with a single dose or multiple doses of the inhibitor to be tested after at least an 8 hour smoking abstinent period and blood samples are collected at 0, 20, 30 and 60 minutes after the injection of nicotine.
- the concentration of nicotine and cotinine in the plasma is determined by a highly sensitive HPLC method.
- the method is adaptable to accommodate inhibitors with varying kinetic properties in mat longer of shorter sampling periods can be used. For example, some inhibitors may have a very long half-life and it might be desirable to obtain data over a longer period of time. The shorter screen test will nevertheless be sufficient for demonstrating the clinical inhibition and die therapeutic potential of the inhibitor. In some circumstance the ratio of nicotine/cotinine in plasma at these time points might be useful but for the pu ⁇ oses of screen for therapeutic efficacy we do not believe that cotinine concentrations are pertinent. The method described here can applied to non-smokers if the nicotine doses are decreased to 20 ⁇ g/kg s.c. Results:
- Figure 38 is a graph showing the metabolism of nicotine over one hour in seven subjects. More specifically, the graph shows the changes in the plasma nicotine concentration level over time in the presence of methoxsalen, a CYP2A6 inhibitor, versus a placebo.
- Coumarin is a selective and specific substrate for human CYP2A6 and can be used to: (1) identify individuals who are potential therapeutic exclusions for use of
- CYP2A6 CYP2A6
- risk factor assessment in identifying individuals who will not benefit from the treatment or who may be at risk to toxicity from agents which are inhibitors and substrates themselves of CYP2A6.
- the Coumarin Test exists in two forms: m Coumarin Test When Only Urine is Available
- Coumarin 5 mg formulated in a capsule or other dose form is administered orally to fasted individuals after voiding of residual bladder urine.
- Urine is collected for the first 2 hours and for the subsequent 6 hours.
- the amount of urinary excretion of e coumarin metabolite 7 hydroxy-coumarin (free and conjugated) is determined by determining the concentration of these metabolites on the urine using an HPLC assay as described in an earlier example.
- the relative activity of CYP2A6 is reflected in the total amounts of 7 hydroxy-coumarin excreted in the sampling periods separately and combined and the activity can be expressed as the ratio of the percent coumarin excretion (amount excreted in the first 2 hours/amount excreted in 8 hours) x 100.
- This percent excretion ranges from values less then 20% in individuals without CYP2A6 activity to > 80% in individuals witti high activity.
- This test can be equally effectively and reliably be applied to smokers and norii-smokers and may be used at any time of day with out apparent effect of the smoking condition or time of day on the results.
- the test demonstrates high within subject reproducibility widi a linear r of > 0.9. See Figure 37 for results of a smdy in which smokers and nonsmokers were given coumarin in the morning and afternoon on each of 2 separate days. High within subject reproducibility and reliability is demonstrated.
- a plasma-based test of CYP2A6 activity has been developed and applied to individuals of known genotype. Individuals ingest coumarin 5.0 mg orally and 45 minutes later a blood sample is drawn in a heparinized (or other anticoagulant containing tube). The sample is spun and the plasma separated. The plasma is analyzed by HPLC to quantitate 7 hydroxycoumarin (total after deconjugation with beta glucuronidase incubation). High analytical sensitivity is required in order to use 5.0 mg of coumarin. When such sensitivity is not available, the dose of coumarin may be increased up to 50 mg. HPLC analysis of 7-hydroxycoumarin in urine and plasma:
- Urine or plasma samples (0.5 ml) are hydrolyzed with 0.2 ml of ⁇ -glucuronidase acetate buffer solution (15 mg/ml acetate buffer, 0.2 M, pH 5.0) at 37°C for 30 min. Extraction is followed with 2 ml ether by vortex for 5 min and centrifuged at 3000 ⁇ m for 10 min. Ether extract (1.2 ml) is transferred to another clean mbe and dried down under nitrogen gas. The residue is reconstituted in the HPLC mobile phase (see below), and injected onto HPLC.
- HPLC analysis The HPLC system consists of Hewlett Packard 1050 HPLC system (pump, autosampler and UV detector) and HP3396II integrator. The chromatographic separation was performed with an HP Spherisorb-ODS2 column (125 x 4 mm I.D. , 5 ⁇ m). Samples were eluted with a mobile phase of acetonitrile : water : acetic acid of 150:850:2 (v/v/v) at a flow rate of 1.0 ml/min, and monitored by a UV detector at a wavelength of 324 nm for 7-hydroxycoumarin and 280 nm for coumarin. Samples are quantitatively determined by an external standard method.
- the CYP2A6 activity is expressed as d e concentration of 7 hydroxy-coumarin in the plasma at various points in time (e.g. 20, 30, 45 and 75 minutes) or as the ratio of coumarin / 7 hydroxy-coumarin in me plasma at that time.
- the preferred mode of use is a simple plasma sample at 20 or 30 minutes after the oral administration of coumarin in which both coumarin and 7-hydroxycoumarin are quantified and in which the coumarin to 7-hydroxycoumarin ration is used as the index of CYP2A6 activity.
- Blank urine or plasma samples showed no interfering peak for 7- hydroxycoumarin or coumarin.
- Sensitivity of this method is 1 ng/ml urine or plasma. Intraday and inter-day variations are less than 10% . This analysis is linear from 1 ng to 4000 ng/ml.
- Figure 39 is a graph showing a time course of total 7-hydroxycoumarin concentration detected in the plasma of subjects given coumarin.
- Figure 39 illustrates various time courses based on corresponding genotypes for CYP2A6.
- mutant alleles which decrease CYP2A6 activity in an individual can be screened in a DNA sample using the materials and screening method described in Example 7.
- Crespi C . L . , Penman B . W . , Gelboin H . V . and Gonzalez F . J . A tobacco smoke- derived nitrosamine, 4-(methylnitrosamino)-l-(3-pyridyl)-l-butanone, is activated by multiple human cytochrome P450s including the polymo ⁇ hic human cytochrome P4502D6. Carcinogenesis. 12(7): 1197-201 (1991).
- Nicotine skin patch treatment increases abstinence, decreases withdrawal symptoms, and attenuates rewarding effects of smoking. Journal of Clinical Psychopharmacology. 14(1): 41-9 (1994).
- Nemeth-Coslett R., Henningfield J.E., MK OK and Griffiths R.R. Effects of mecamylamine on human cigarette smoking and subjective ratings. Psychopharmacology. 88(4): 420-5 (1986).
- Retroviral mediated expression of human cytochrome P450 2A6 in C3H/10T1/2 cells confers transformability by 4-(methylnitrosamino)- 1 -(3-pyridyl)- 1 -butanone (NNK) .
- NNK 4-(methylnitrosamino)- 1 -(3-pyridyl)- 1 -butanone
- Waxman D.J. Lapenson D.P., Aoyama T., Gelboin H.V. , Gonzalez F.J. and Korzekwa K: Steroid hormone hydroxylase specificities of eleven cDNA- expressed human cytochrome P450s. Archives of Biochemistry & Biophysics. 290(1): 160-6 (1991).
- Yamano S. The CYP2A3 gene product catalyzes coumarin 7-hydroxylation in human liver microsomes. Biochemistry. 29: 1322-1329 (1990).
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- 1997-07-17 WO PCT/CA1997/000506 patent/WO1998003171A2/en active Application Filing
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Also Published As
Publication number | Publication date |
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BR9710728A (en) | 2000-10-31 |
CN100502860C (en) | 2009-06-24 |
AU742628B2 (en) | 2002-01-10 |
CA2227423A1 (en) | 1998-01-29 |
WO1998003171A2 (en) | 1998-01-29 |
NZ334205A (en) | 2001-04-27 |
AU3432097A (en) | 1998-02-10 |
WO1998003171A3 (en) | 1998-02-26 |
JP2000515516A (en) | 2000-11-21 |
CN1230112A (en) | 1999-09-29 |
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