GB2457808A - Non-human transgenic animals with cytochrome b5 deletion - Google Patents

Abstract

Transgenic non-human animals comprising a complete or tissue specific deletion of the cytochrome b5 enzyme (Cyb5) are claimed. The tissue specific deletion may be in liver, lung, heart, kidneys, brain, ovaries, or testicles. The transgenic animal may have integrated a replacement targeting vector comprising exons 2-5 of the cytochrome b5 gene. The invention also includes methods of producing the non-human transgenic cytochrome b5 knockout animal models and uses of the animal models in methods of screening candidate therapeutics, methods of investigating metabolic processes and crosses into other animal models. Targeting vectors comprising a transgene of exons 2-5 of cytochrome b5 are also claimed. The animals may be used in modeling steatosis, methaemoglobinaemia, perturbations in fatty acid or triglyceride synthesis, adrenal insufficiency, testosterone insufficiency or infertility, changes in skin composition, ichthyosis, or nutritional weaning.

Description

I

Cytochrome b5 Knockout Animal Models The present invention relates to transgenic non-human animals comprising a deletion of the cytochrome b5 enzyme, the deletion may be a tissue specific, for example, hepatic or the deletion may be a complete null or complete knockout. The invention also includes inter alia, methods of producing the non-human transgenic animal models and products thereof, uses of the animal models particularly, but not exclusively, in methods of screening candidate therapeutics, methods of investigating metabolic processes. The invention further includes crosses of such non-human transgenic animals into other non-human transgenic animal models and also their associated uses.

BACKGROUND

Cytochrome b5 is a l7kDa membrane haemoprotein associated primarily with the endoplasmic reticulum along with its electron donor, cytochrome b5 reductase. It is a multifunctional redox enzyme and it is known to be involved as an electron transfer component in a number of diverse oxidative reactions in varied biological tissues. For example, cytochrome b5 is involved in the reactivation of methaemoglobin to haemoglobin and its deficiency in man is clinically manifest as a recessive congenital methaemoglobinaemia (Hegesh et al N.Eng J. Med 1986, 314 912) 757-761; Glordano et al Hum. Geet, 1994, 93 (5), 568-579; Yawata et al, Am. J. Haematol. 1992, 40 (4) 299-305). The known functions of cytochrome b5 include anabolic metabolism of fats (especially fatty acid desaturation) and steroids such as progesterone as well as catabolism of xenobiotics and compounds of endogenous metabolism by virtue of electron transfer into the cytochrome P450 system.

The role of cytochrome b5 in cytochrome P450 monooxygenase reactions has been controversial for nearly 40 years. Depending on the cytochrorne P450 involved, the experimental conditions and the substrate utilised, cytochrome b5 has been shown to stimulate or inhibit P450 metabolic reactions or in the case of CYP2B4 to both stimulate or inhibit depending on the concentration of cytochrome b5. These studies have been based on the measurement of enzyme activities or reduction rates in vitro using reconstituted systems and are difficult to interpret. However, the presence of cytochrome b5 has been shown to consistently increase a range of P450 reactions, this includes the metabolism of commonly-used drugs by human P450s such as CYP2C8, CYP2C9, CYP2C19, CYP2EI and CYP3A4.

Evidence to date suggests that in the NADH-cytochrome reductase enzyme system cytochrome b5 has an obligatory role in the metabolism of (I) stearoyl coenzyme A by stearoyl coenzyme A desaturase, (ii) 8, 11, 14-elcosatrienoic acid and linoleic acid by -desaturase (iii) 1-0-alkyl phosphatidyl ethanol amine by plasmalogen synthetase (iv) cholest-7-en-3f3-ol by 7-sterol 6-desaturase (v) 4,4-dimethyl -5c-cholest-7-ene-3-ol by methylsterol (v) metemyoglobin by either metmygloblin reductase or methemoglobin reductase. In the same reductase system cytochrome b5 has a role as a modifier in 17a-OH-progesterone metabolism by cytochrome P450-hAl; C17,20 lyase, 17a-hydroxylase. In the NADPH-cytochrome P450 reductase system cytochrome b5 has a major role in the metabolism of (I) methoxyflurane by cytochrome P450 284 (ii) p-nitrophenetole by cytochrome P450 2B1 (iii) prostaglandins Al, El and E2 by cytochrome P450 284 (iv) arachadonic acid by cytochrome P450 4A7 (v) p-nitroanisole by cytochrome P450 3A6 (vi) testosterone by cytochrome P450 3A4 and (v) drug-drug interactions. In this reductase system cytochrome b5 also has a role as a modifier in multiple cytochrome CYP 1, 2, 3 and 4 P450's.

It seems clear that at least in vitro, cytochrome b5 modulates the rate of a number of cytochrome P450-dependent monooxygenation reactions. However, the role of this enzyme in determining drug pharmacokinetics in vivo and the consequential effects on absorption distribution, metabolism, excretion (ADME) and toxicity has not been previously determined and is controversial There is a need for a tissue specific or a complete cytochrome Li5 null non-human transgenic animal in order to establish and to further investigate the relevance of the in vitro observations to metabolism in vivo and in pathways of chemical toxicity. Such an animal model would offer immediate benefit to the art by providing the means for examining the role of cytochrome b5 in metabolism and disposition of drugs, this is of especial importance to individuals having varying levels of cytochrome b5 or for whom cytochrome b5 is polymorphic. Such an animal model would also enable the role of cytochrome b5 in normal and metabolic process/homeostasis to be elucidated.

BRIEF SUMMARY OF THE DISCLOSURE

According to a first aspect of the invention there is provided a transgenic non-human animal comprising a deletion of cytochrome b5 or an offspring thereof or tissue or cells derived from such transgenic non-human animals or their progeny in which cytochrome b5 is deleted.

Preferably the deletion is a broad deletion or is a tissue specific deletion.

Throughout the description and claims of this specification, the words "comprise" and "contain" and variations of the words, for example "comprising" and "comprises", means "including but not limited to", and is not intended to (and does not) exclude other moieties, additives, components, integers or steps.

Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.

Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith.

Reference herein to "deletion" of cytochrome b5 is intended to include partial or complete ablation, hepatic null or complete null, "knock-our or inability to express cytochrome b5, the animals having an endogenous cytochrome b5 gene that can be deleted may involve removal or all of part of the cytochrome b5 gene. A null animal may also include an animal where cytochrome b5 expression is prevented for example by inhibition of cytochrome b5 mRNA processing.

Reference herein to a "broad deletion" is synonymous with a complete deletion or complete ablation or a null that is to say that the non-human transgenic non-human animal has no appreciable cytochrome b5 expression in any of its tissues/organs where expression is normally seen.

Preferably, the tissue specific deletion may be in, for example and without limitation, the liver, lung, heart, kidneys, brain, ovaries or testicles but preferably, the tissue specific deletion is hepatic tissue.

Preferably, the tissue specific deletion is a conditional deletion. The term conditional deletion refers to the switching "on" or "off' of a particular gene, in this instance the cytochrome b5 gene, which is conditional on a specific stimulus. The deletion maybe tissue specific or time specific and is typically conditional on Cm recombinase activation. The conditional deletion may use the albumin gene promoter to drive Gre recombinase expression and thus deletion of the cytochrome b5 in specific tissues, for example, in the liver. The albumin gene comes "on" just after birth and as such the conditional deletion may be effective at this point. Alternatively Ore recombinase expression may be driven by, for example, the Cypi al promoter and activated by a specific chemical stimulus in this way the conditional deletion may be temporally controlled. A yet further specific deletion could be achieved in embryonic stem cells.

i 0 Animals suitable for carrying out the present invention are, in general, non-human animals, such as monkeys, swine, dogs, cats, rabbits, hamsters, gerbils, guinea pigs, rats and mice. Rodent species are preferred, and the mouse is the most preferred species. Animals in every stage of development, including embryonic, neonatal, juvenile, adolescent and adult are included in this description.

The present invention provides non-human transgenic animals having a complete cytochrome b5 knockout (BCN) or a conditional deletion of cytochrome b5 in, for example the liver, to create the hepatic cytochrome b5 null (HBN) mouse. It has been most surprising, given the multifunctional role of cytochrome b5 in a variety of diverse biological processes that the HBN mice showed no overt phenotype and that BCN mice developed despite significantly elevated levels of methaemoglobin and that they bred normally exhibiting no gross physical changes except for observed changes to skin and hair. Lifter sizes from BCN crosses were smaller than wild-type crosses, albeit not significantly; however, BCN pups were significantly smaller in size during the weaning period, and thereafter remained smaller, but insignificantly so, in adulthood. As shown hereinafter, the fact that the mice were able to breed at all is most unexpected given the profound effect of the lack of cytochrome b5 on androstenedione production.

According to a yet further aspect of the invention there is provided a transgenic non-human animal whose genome has integrated therein a replacement targeting vector nucleic acid construct comprising a transgene of DNA containing exons 2-5 of the cytochrome b5 gene.

The invention also includes the nucleic acid construct itself, as depicted in either of Figurea IA or I IA and uses thereof to produce the non-human transgenic animals of the present invention.

Preferably, the nucleic acid construct is included in a cassette, flanked by same-orientated IoxP sites and optionally containing a selectable marker such as neomycin which is optionally driven by the herpes simplex thymidine kinase promoter.

According to a further aspect of the invention there is provided a transgenic knockout non-human animal having somatic and germline cells comprising a chromosomally incorporated transgene wherein at least one allele of a cytochrome b5 gene is disrupted by the transgene such that the expression of cytochrome b5 is inhibited.

Preferably, both alleles are disrupted so that the non-human transgenic animal is homozygous for the disrupted allele.

The present invention provides a non-human transgenic animal whose genome comprises a disruption in its endogenous cytochrome b5 gene, wherein said disruption reduces or eliminates the expression of a functional cytochrome bS protein. The disruption comprises either a heterozygous or homozygous allele.

According to a further aspect of the invention there is provided a method of producing a non-human transgenic animal comprising a broad or tissue specific deletion of cytochrome b5 the method comprising introducing a targeting vector as hereinbefore described and as depicted in either of Figures IA or I IA into a non-human zygote or a non-human embryonic stem cell, generating a transgenic non-human animal from said zygote or embryonic stem cell, selecting non-human transgenic animals carrying a target cytochrome b5 allele and crossing said animals to produce a transgenic non-human animal having tissue specific or complete cytochrome b5 knockout.

The animals of the present invention may preferably be produced by a method comprising the steps of transfecting embryonic stem cells with a cytochrome b5 targeting construct, selecting animals homozygous for a floxed cytochrome b5 locus (Cytb5 loX/lox) and crossing said animals with a line where Cre expression is regulated.

The genotype of the broad range or complete knockout transgenic non-human animal is homozygous for cytochrome b5 deletion, (cyt b5) that is to say it is a cytochrome b5 null. Whereas the genotype for the conditional deletion tissue specific non-human transgenic animal is cyt b5 l00)(.:: Cre ALB In order to establish the relevance of the in vitm observations to metabolism in viva, the present invention provides a non-human transgenic animal model in which cytochrome b5 is specifically deleted in the liver or is completely ablated in the broad knockout or null animal. As is demonstrated hereinafter, the rate of NADPH-driven P450 metabolism of a range of both model substrates and probe drugs is profoundly reduced -in some cases essentially abolished -in the absence of cytochrome b5. Furthermore, it is demonstrated that when probe drugs are administered to mice, significant changes in drug pharmacokinetics occur, demonstrating that cytochrome b5 can play a major role in the in viva disposition of drugs and its expression levels need to be taken into account when investigating the variability of drug response in humans.

It will be appreciated that the non-human transgenic animals of the present invention provide a powerful research tool to asses the impact of cytochrome b5 on candidate therapeutics and in drug development and represent a significant contribution to the art.

According to a yet further aspect of the invention there is provided use of the non human transgenic animals of the present invention as an in viva screen or tissues or cells derived therefrom as an in vitro screen to determine any one or more of the following parameters: (I) role of cytochrome b5 in drug/product disposition; (ii) routes of Phase Il drug/product disposition of parent compound; (iii) pathways of drug/product disposition; (iv) role of drug transporters in drug uptake; (v) pathways of chemical toxicity; (vi) role of cytochrome b5 in hormone function and regulation; (vii) role of cytochrome b5 in normal metabolic processes/homeostasis; and (viii) role of cytochrome b5 in pathogenesis of disease.

A particular advantage of examining the role of cytochrome b5 in metabolism and disposition of drugs will be for human patients having varying levels of cytochrome b5 or for whom cytochrome b5 is polymorphic.

In another embodiment of the invention the non-human transgenic animals and tissues or cells derived therefrom of the present invention may be used as an in viva screen to investigate any one or more of the following events or parameters: (i) pharmacological potency of agents subject to first pass metabolism; (ii) occurrence and rate of extrahepatic metabolism; (iii) rate of hepatic metabolic clearance as a determinant in product/drug distribution and pharmacokinetics; (iv) facilitation of selection of lead product/compound based on in vivo parameters; (v) assessment of relevance and occurrence of pharmacologically active metabolites; (vi) distinguish between toxicity due to product activation of other mechanism (vii) establish role of cytochrome b5 as a rate limiting step in drug/compound disposition and; (viii) study drug/drug interactions due either to cytochrome b5 or drug transporter effects.

In another embodiment of the invention the non-human transgenic animals and tissues or cells derived therefrom of the present invention may be used as an in vitro screen to investigate any one or more of the following events/parameters: (i) role of transporters in product/drug uptake and efflux; (ii) identification of metabolites produced by cytochrome b5; (iii) evaluate whether drugs/compounds are cytochrome b5 substrates and; (iv) assess drug/drug interactions due to cytochrome b5 effects; According to a yet further aspect of the invention there is provided a method assessing any one of the aforementioned parameters or events ascribed to uses, comprising exposing a transgenic non-human animal of the present invention or tissue or cells derived therefrom and assessing a selected parameter and comparing the outcome with that obtained from a similarly exposed wild type non-human transgenic non-human animal that has cytochrome b5 function.

In summary, one of the embodiments of the present invention provides a conditional hepatic knockout of cytochrome b5 and shows that both in vitro and in vivo the absence of this enzyme can result in significantly reduced metabolism of model substrates and probe drugs by cytochromes P450. To our knowledge, after decades of in vitro and ex vivo work, this is the first definitive in vivo demonstration of a modulatory role for cytochrome b5 in P450-mediated metabolism. Although the mechanism(s) of cytochrome b5 enhancement of P450 activity remains to be elucidated, the hepatic null mouse line should play a important role in defining how these two enzyme systems interact. a

Furthermore, the data presented hereinafter suggests that the level and/or activity of hepatic cytochrome b5 may play a significant role in the metabolism and disposition of therapeutic drugs.

According to a yet further aspect of the invention there is a transgenic non-human animal model for steatosis comprising a broad or tissue specific deletion of cytochrome b5 or an offspring thereof or tissue or cells derived from such transgenic non-human animals or their progeny in which cytochrome b5 is deleted.

Reference herein to "steatosis" is intended to include disease conditions of the liver manifest in the condition known as fatty liver which is the collection of excessive amounts of triglycerides and other fats inside liver cells.

Preferably, the steatosis model non-human transgenic animals of the present invention may be used to screen for agents that prevent the condition or may be used to identify agents that exacerbate the condition, or agents which might ameliorate the condition, the model may also be used to study the underlying mechanism of acquired or a predisposition towards the condition of fatty liver.

According to a yet further aspect of the invention there is a transgenic non-human animal model for methaemoglobinaemia comprising a broad or tissue specific deletion of cytochrome b5 or an offspring thereof or tissue or cells derived from such transgenic non-human animals or their progeny in which cytochrome b5 is deleted.

Preferably, the methaemoglobinaemia model non-human transgenic animals of the present invention may be used to screen for agents that prevent the condition or may be used to identify agents that exacerbate the condition, or agents which might ameliorate the condition the model may also be used to study the underlying mechanism of acquired or a predisposition towards the condition of methaemoglobinaemia.

According to a yet further aspect of the invention there is a transgenic non-human animal model for perturbations in fatty acid and triglyceride synthesis comprising a broad or tissue specific deletion of cytochrome b5 or an offspring thereof or tissue or cells derived from such transgenic non-human animals or their progeny in which cytochrome b5 is deleted.

Preferably, the non-human transgenic animals models perturbations in fatty acid and triglyceride synthesis of the present invention may be used to screen for agents that prevent such conditions or may be used to identify agents that exacerbate such conditions, or agents which might ameliorate the condition, the model may also be used to study the underlying mechanism of acquired or a predisposition towards perturbations in fatty acid and triglyceride synthesis.

Results indicate that the non-human transgenic animals of the present invention exhibit significant changes in fatty acid species such as mono and unsaturated fatty acids (such as omega-3 as they are trivially known) both in the liver and circulating in plasma (see Figures 28 and 29 herein after). As such they will provide useful models for investigating conditions such as and without limitation heart disease, atherosclerosis, inflammatory disease and hepatic lipid homeostasis and also as models for screening for agents which affect such conditions.

According to a yet further aspect of the invention there is provided a transgenic non-human animal model for adrenal insufficiency comprising a broad or tissue specific deletion of cytochrome b5 or an offspring thereof or tissue or cells derived from such transgenic non-human animals or their progeny in which cytochrome b5 is deleted.

Preferably, the adrenal insufficiency model non-human transgenic animals of the present invention may be used to screen for agents that prevent the condition or may be used to identify agents that exacerbate the condition or agents which might ameliorate the condition, the model may also be used to study the underlying mechanism of acquired or a predisposition towards the condition of adrenal insufficiency.

According to a yet further aspect of the invention there is provided a transgenic non-human animal model for testosterone insufficiency or infertility comprising a broad or tissue specific deletion of cytochrome bS or an offspring thereof or tissue or cells derived from such transgenic non-human animals or their progeny in which cytochrome b5 is deleted.

Preferably, the testosterone insufficiency or infertility model non-human transgenic animals of the present invention may be used to screen for agents that prevent the condition or may be used to identify agents that exacerbate the condition or agents which might ameliorate the condition, the model may also be used to study the underlying mechanism of acquired or a predisposition towards testosterone insufficiency or infertility.

Results have shown that the transgenic non-human animals of the present invention have dramatically compromised testosterone levels (see Figures 13 and 36 herein after) and as such they will prove as useful models to investigate the underlying mechanisms of the condition and agents which may increase fertility.

According to a yet further aspect of the invention there is provided a transgenic non-human animal model for changes in skin composition and physiology comprising a broad or tissue specific deletion of cytochrome b5 or an offspring thereof or tissue or cells derived from such transgenic non-human animals or their progeny in which cytochrome b5 is deleted.

Preferably, the skin changes and skin physiology model non-human transgenic animals of the present invention may be used to screen for agents that prevent the condition or may be used to identify agents that exacerbate the condition or agents which might ameliorate the condition, the model may also be used to study the underlying mechanism of changes in skin composition and physiology.

Results have shown that the transgenic non-human animals of the present invention have profound alterations in the physiology of the skin (see Figure 31 hereinafter) and as such they will prove as useful models to investigate the underlying mechanisms of changes in the skin and agents which may effect skin physiology and composition.

According to a yet further aspect of the invention there is provided a transgenic non-human animal model for ichthyosis comprising a broad or tissue specific deletion of cytochrome b5 or an offspring thereof or tissue or cells derived from such transgenic non-human animals or their progeny in which cytochrome b5 is deleted.

Preferably, the ichthyosis model non-human transgenic animals of the present invention may be used to screen for agents that prevent the condition or may be used to identify agents that exacerbate the condition or agents which might ameliorate the condition, the model may also be used to study the underlying mechanism of ichthyosis.

Results have shown that the transgenic non-human animals of the present invention have marked thickening of the upper skin layers such as that observed in the condition of ichthyosis (see Figure 31A and D hereinafter) and as such they will prove as useful models to investigate the underlying mechanisms of the condition and effects of agents on the condition.

According to a yet further aspect of the invention there is provided a transgenic non-human animal model for nutritional weaning comprising a broad or tissue specific deletion of cytochrome b5 or an offspring thereof or tissue or cells derived from such transgenic non-human animals or their progeny in which cytochrome b5 is deleted.

Preferably, the nutritional weaning model non-human transgenic animals of the present invention may be used to screen for agents that prevent the condition or may be used to identify agents that exacerbate the condition or agents which might ameliorate the condition, the model may also be used to study the underlying mechanism and value of maternal nurturing.

Results have shown that the transgenic non-human animals of the present invention have stunted off-spring and that when removed and weaned on wild-type mothers they regain weight and no longer are stunted. Conversely, normal pups when put to a lactating transgenic animal of the present invention to wean they lose weight and become stunted (see Figures 32 and 33 hereinafter). The nurturing phenotype of the present invention will provide a model for the investigating the constituency of milk and a useful model with which to investigate the nutritional value of maternal milk and agents which affect the constituency of milk.

According to a yet further aspect of the invention there is provided a non-human transgenic animal comprising a cross of a broad or tissue specific deletion of cytochrome b5 with another non-human transgenic animal of the same species comprising a null expression of an enzyme associated with drug metabolism.

Preferably, the cross is into Mdrl or Mdr2 or hepatic null cytochrome P450 null reductase model.

The HRNTM mouse model as described in WO 2004/007708 comprises a hepatic CPR null genotype but displays a residual (approximately 5%) residual cytochrome P450 activity. A cross of this model into the hepatic or complete cytochrome b5 animal of the present invention would improve provide a double knockout and would ablate all P450 function thereby providing an improved testing model.

It will be appreciated that each aspect of the invention includes features that may be ascribed to any one or more of the aspects of the invention mutatis mutandis.

BRIEF DESCRIPTION OF THE DRAWl NGS

Figure IA shows the construction of hepatic cytochrome b5 null mice.

Figure 2A shows characterisation of protein expression in cytochrome b5 hepatic null mice by immunoblot analysis for cytochrome b5 expression in liver, kidney, lung and small intestine microsomes. Figure 2B shows immunoblot analysis for expression of cytochrome P450-dependent monooxygenase components in liver microsomes from C$bi00x::CreL (HBN) and Cytbj0M0X (wild-type). Figure 2C shows immunoblot analysis for expression of cytochrome b5 and P450-dependent monoxygenase components in the liver, kidney, lung and small intestine of Cytb54' (wild-type) and Cytb (RON) mice (20sof protein per lane).

Figures 3 A-F show NADPH-and NADH-dependent cytochrome P450 activities in hepatic microsomes from wild type and cytochrome b5 hepatic null mice with a variety of model substrates.

Figures 4 A-E show NADPH-and NADH-dependent probe drug metabolism in hepatic microsomes from wild type and cytochrome b5 hepatic null mice with a variety of probe drugs.

Figures 4 F-J show NADPH-dependent probe drug metabolism in Cytochrome b5 complete null mice with a variety of probe drugs.

Figures 5 A-E show pharmacokinetic disposition of an orally administered P450 drug cocktail in wild-type and hepatic cytochrome b5 null mice with a variety of probe drugs.

Figures 6 A-E show pharmacokinetic disposition of an intravenously administered P450 drug cocktail in wild-type and hepatic cytochrome b5 null mice with a variety of probe drugs.

Figures 6 F-I show pharmacokinetic disposition of an orally administered P450 drug cocktail in hepatic cytochrome b5 null mice with a variety of probe drugs.

Figures 7 A-D show detailed fatty acid analysis on endoplasmic reticulum membranes from wild-type and hepatic cytochrome b5 null mice.

Figure 8 shows age related changes in P450 expression in wild-type and hepatic cytochrome b5 null mice.

Figure 9 shows densitometric analysis of the immunoreactive bands of Figure 8.

Figure 10 shows age related changes in hepatic lipids in wild-type and hepatic cytochrome b5 null mice Figure 11 shows recapitulation of NADPFI-mediated P450 activity towards (A) BEG and (B) chlorzoxazone, respectively, by supplementation of microsomes with exogenous cytochrome b5 (11.5, 23, 34.5 and 46 pmol respectively) Figure 12 shows the construction of complete cytochrome b5 null mice.

Figure 13 shows genotypic spread of live births.

Figure 14 shows growth rates of wild type, heterozygous complete cytochrome b5 knockouts and cytochrome b5 male (A) and female (B) knockout mice.

Figure 15 shows Western blotting and densitometry on microsomes prepared from 15 week old wild type and complete cytochrome b5 knockout mice.

Figure 16 shows Western blotting on microsomes prepared from 12 week old wild type and complete cytochrome b5 knockout mice.

Figure 17 shows Western blotting on microsomes prepared from male old wild type and complete cytochrome b5 knockout mice at increasing time points.

Figure 18 shows Fe24 -Co versus Fe24 difference spectra for liver microsomes prepared from male 15 week old wild type and complete cytochrome bS knockout mice, Figure 19 shows activities of microsomes prepared from wild type and complete cytochrome b5 knockout mouse tissues towards fluorescent probe 7-ethoxy-4-trifluoromethylcoumarin (EEC).

Figure 20 shows activities of liver microsomes prepared from wild type and complete cytochrome bb knockout mouse tissues towards the fluorescent probes benzylresorufin (BR), ethoxyresorufin (ER), methoxyresorufin (MR) and pentoxyresorufin (PR).

Figure 21 shows activities of microsomes prepared from wild type and complete knockout cytochrome b5 mouse tissues towards the skeletal muscle relaxant drug chlorzoxazone.

Figure 22 shows activities of microsomes prepared from wild type and complete knockout cytochrome b5 mouse tissues towards the benzodiazepine derivative midazolam.

Figure 23 shows activities of testicular microsomes prepared from 12 week old wild type and complete knockout cytochrome b5 mice towards the steroid hormones progesterone (A and C) and I 7cchydoxyprogesterone (B and C).

Figure 24 shows the time course of l7czhydoxyprogesterone metabolism (androstenediorie production) by testicular microsomes prepared from 12 week old wild type and complete cytochrome b5 knockout mice.

Figure 25A shows the effect of complete cytochrome b5 knockout on levels of methaemoglobin.

Figure 25B shows the % methaemoglobin in wild type and BCN mice.

Figure 28 shows lipid profiling of complete cytochrome b5 knockout mice.

Figure 27 shows liver and plasma lipid content of wild type and complete cytochrome b5 knockout mice.

Figures 28 A-D show fatty acid profiling of wild type and complete cytochrome b5 knockout mouse livers.

Figures 29 A-D show fatty acid profiling of wild type and complete cytochrome b5 knockout mouse plasma.

Figure 30 shows growth curves for wild type and BCN mice.

Figure 31 shows analysis of skin pathology.

Figure 32 shows time course of weanling weights for three sequential litters from a BCN female crossed with a 8GW male.

Figure 33 shows fostering of wild-type pups to a BCN mother and BCN pups to a wild-type mother indicates growth retardation is due to nutrient deficiencies.

Figure 34 shows microsomal fractions characterisation.

Figure 35 shows Western blotting analysis of testicular microsomal fractions from wild-type and BCN mice.

Figure 36A shows the effect of cytochrome b5 deletion on NADPH-clependent steroid hormone metabolism, Figure 36B shows testosterone levels in testicular tissue In wild-type and BCN mice and Figure 36C shows the pathway of NADPH-dependnet testosterone formation from progesterone.

Figure 37 shows drug cocktail compound metabolism in vitro, Figure 37A shows chlorzoxazone 6-hydroxylase activity, Figure 37B shows midazolam 1'-and 4-hydroxylase activity, Figure 37C shows metoprolol 0-demethylase and 1 -hydroxylase activity, Figure 37D shows phenacetin 0-deethylase activity and Figure 37E shows tolbutamide hydroxylase activity.

Figure 38 shows the effect of exogenous cytochrome b5 addition to microsomal cytochrome P450 activities. Activities of Cytb5' (WT) and Cytb5" (BCN) liver microsomes towards 7-benzyloxy-4-trifluoromethylcoumarirt (Figure 38A) and benzyloxy-resorufin (Figure 38B) in the absence and presence of exogenous cytochrome b5.

Samples were treated as detaDed in Experimental Procedures, where bars are mean � SD from triplicate incubations. Activities of Cytb5 (WT) and Cytb8 (BCN) lung microsomes towards 7-Benzyloxy-4-trlfluoromethylcoumarin are shown in Figure 38C and benzyloxy-resorufin (Figure 38D) in the absence and presence of exogenous cytochrome b5. Bars are mean � SD from triplicate incubations.

Figure 39 shows the pharmacokinetic disposition of an orally administered P450 drug cocktail in wild-type and microsomal cytochrome b5 null (BCN) mice. A P450 drug cocktail containing chlorzoxazone (5mg/kg), metoprolol (2mg/kg), midazolam (5mg/kg), phenacetin (5mg/kg) and tolbutamide (5mg/kg) was administered orally to wild-type (black circles) or BCN (open circles) mice and blood samples taken at timed intervals to determination the in vivo pharmacokinetic parameters of the parent compounds. * = p 0.05, ** = p 0.005, = p �= 0.001 where n = 5..

Figure 40 shows the pharmacokinetic profile for oral doses of cyclophoshamide and placlitaxel.

Figure 41 shows immunoblotting of liver microsomes (20g per lane) for Stearoyt-C0A desaturase I (SCDI) and NAD(P)H cytochrome b5 oxidoreductase (Ncb5OR).

DETAILED DESCRIPTION

Materials and Methods Generation of hepatic cytochrome b5 null (HBN) mice and Mouse breeding.

A replacement targeting vector was constructed from an 18 kb DNA fragment, produced by fusing overlapping PCR fragments generated from mouse I 29/Ola genomic DNA (Fig. IA), containing exons 2 -5 of the mouse cytochrome b5 gene. With reference to Figure 1 there is shown diagrams of the wild type, foxed and disrupted Cytb5 alleles.

LoxP sites are indicated by triangles and the selectable marker (hsvtk-neo) is indicated by a shaded box. The hatched box indicates the position of the probe used in Southern screening, and the numbered arrows indicate the position of the PCR primers used for screening. A cassette, flanked by same orientation loxP sites and containing a selectable marker (neomycin (neo)), driven by the herpes simplex thymidine kinase (hsv-tk) promoter, was inserted into a Bcll site in intron 1. A third loxP site was cloned into a Kpnl site in intron 5. The correct arrangement of the construct and orientation of all three IoxP sites was confirmed by detailed PGR and sequence analysis. The construct was transfected into GK129/1 embryonic stem (ES) cells by electroporation and the ES cells were subsequently cultured in 96-well plates under G418 selection. G418-resistant clones were screened for specific homologous recombination by Southern blot analysis, digesting genomic DNA with BglIl and using a 800bp PCR fragment generated using following 5'-GGCACAACACCAATTAmGTC-a' (SEQ ID NO:1) and 5'-GACAC3TCCTTAACACAAGCTC-3' (SEQ ID NO:2) as forward and reverse primers respectively. Two correctly targeted ES cell clones (Cytbs0X) were expanded, injected into C57BL16 blastocysts and transferred into 2.5 day post-coitum (dpc) recipient pseudopregnant mice. Male chimaeric mice were bred to C57BLJ6 mice and heterozygous offspring were screened by Southern blot analysis and multiplex PCR using the following primer set; (1): forward primer 5'-CCAATGGTCTCTCCTTGGTC-3' (SEQ ID NO:3), (2) lox/neo reverse primer 5'-CAATAGCAGCCAGTCCCTTC-3' (SEQ ID NO:4) and (3) wild type reverse primer 5'-GATGGAGTTCCCCGATGAT-S' (SEQ ID NO:5) to confirm germline transmission of the Cytb5t° genotype (Fig. I B). Figure I B shows Southern blot (i) and PCR (ii) analysis of genomic DNA from Cytbi°''4, Cytbi0Mox and Cytbj' mice. For Southern blotting, the wild type allele is represented by a band at kb and the targeted allele (CytbjOX) containing the selectable marker by a band at 11 kb. For PCR analysis, the wild type allele is represented by a band at 500 bp and the targeted allele (Cytbj°') containing the selectable marker by a band at 900 bp.

Cytb51°" mice were crossed to produce homozygous Cytb°°" mice and maintained by random breeding on a 1 29P2 X C57BU6 genetic background. Cytb"°' mice were crossed with a transgenlc mouse line expressing Cre recombinase under the control of the rat albumin promoter (Cre8) on a C57BL16 background, and Cb5::Cre offspring were backcrossed with Cytb5b0 mice to generate liver-specific cytochrome b5 conditional knockout mice (HBN, C4b5::CreM') and control (wild-type, Cytb°°'t) mice. The HBN line was thereafter maintained by random inter-crossing of these two lines. The presence of the Cre transgene was determined by PCR using the following 5'-ACCTGAAGATGTTCGCGATTATCT-3' (SEQ ID NO:6) and 5'-ACCGT CAGTACGTGAGATATCTT-3' (SEQ ID NO:7) as forward and reverse primers respectively.

Generation of complete hepatic cytochrome b5 null (HBN) mice and Mouse Breeding.

The procedure for generating the Cytb5 mice is as per above for generating the hepatic null version and the construct is as Figure IA and Figure IIA.

However, the mouse breeding programme is as follows, Cytb510 mice were crossed to produce homozygous CytbsI00X mice and maintained by random breeding on a 129P2 X C57BL16 genetic background. Cytb51°"°' mice were crossed with a transgenic mouse line expressing Cre recombinase under the control of a phosphoglycerate kinase promoter (PGK) on a C57BL16 background, and Cytb5: :PGKOe offspring were backcrossed with Cytb5 mice to generate heterozygous (het) complete cytochrome b5 knockout mice minus PGKC (Cytb5), which were subsequently crossed to generate honiozygous complete cytochrome b5 knockout mice (BCN, Cytb5 and control (wild-type, Cytb54') mice. Mice were screened by PCR (Figure 1 B(iii)) using the following primer set; (1): wild type forward primer 5'-TCCCCCTGAGAACGTAATTG-3',(SEQ ID NO:8) (2) null forward primer 5'-GGTCTCTCCTTGGTCCACAC-3' (SEQ ID NO:9) and (3) common reverse primer 5'-GAGTCTTCGTCAGTGCGTGA-3' (SEQ ID NO:1O) to confirm germiine transmission of the Cytb50x genotype. The BCN line was thereafter maintained by random inter-crossing of these two lines. The presence of the PGK transgene was determined by PCR using the following 5lACCTGMGATGTTCGCGATTATCT3' (SEQ ID NO:6) and 5'-ACCGTCAGTACGTGAGATATCTT-3P (SEQ ID NO:7) as forward and reverse primers respectively.

Mouse Maintenance.

All mice were maintained under standard animal house conditions, with free access to food and water, and 12h light/12h dark cycle. All mouse work was carried out on male 10-week-old mice in accordance with the Animal Scientific Procedures Act (1986) and after local ethical review.

Drug treatments.

HBN and wild-type mice were administered a drug cocktail containing the following P450 substrates at the indicated doses; chlorzoxazone (5mg/kg), metoprolol (2mg/kg) midazolam (5mg/kg), phenacetin (5mg/kg) and tolbutamide (5mg/kg), dissolved in cocktail buffer (5% ethanol, 5% DMSO, 35% Polyetheylene glycol 200, 40% Phosphate buffered saline (PBS) and 20% water), by intravenous (Lv.) injection or orally by gavage.

Preparation of microsomes.

Microsomes were prepared from liver, kidney, lung, small intestine and large intestine of wild-type and HBW mice, using approximately 0.3 -0.5g of tissue, by the standard method known in the art but modified so that sonication was used instead of mechanical homogenisation. Protein concentrations of microsomal samples were determined using the Biorad Protein Assay Reagent (Bio-Rad Laboratories Ltd, Hertfordshire UK) according to the manufacturer's instructions. P450 oxidoreductase (POR) activity was estimated by NADPH-dependent cytochrome c reduction. Microsomes were stored at - 70C until required.

Immunoblotting.

Western blot analysis was carried by a standard technique using 5 p microsomal protein per lane and polyclonal antisera raised against human POR, rat cytochrome b5, rat P450s CYP2A1, CYP2B1, CYP2C6, CYP3AI and CYP4AI or human full length CYP2A4, CYP2D6 and CYP2EI. Polyclonal antiserum to NAD(P)H cytochrome b5 oxidoreductase (NCB5OR) , raised in rabbits to cytochrome b5 reductase, and a mouse monoclonal antibody raised against rat CYPIAI, were also used. Inimunoreactive proteins were detected using polyclonal goat anti-rabbit, anti-mouse or anti-sheep horseradish peroxidase (HRP) immunoglobulins as secondary antibodies (Dako, Ely, UK), and visualised using lmmobilonTM chemiluminescent HRP substrate (Millipore, Watford, UK) and a FUJIFILM LAS-3000 mini imaging system (Fujifilm UK Ltd, UK).

Densitometric analysis was performed using Multi Gauge V2.2 software (Fujifilm UK Ltd, UK).

Generic microsomal incubations.

Microsomal incubations were carried out in triplicate in 50 mM Hepes pH 7.4, 30 mM MgCI2 containing mouse liver microsomes and substrate pre-warmed to 3TC before initiation of reaction by addition of either NADPH or NADH to a final concentration of 0.5 mM or 1mM, respectively.

Fluorogenic assay incubations.

Assays were performed in a final volume of 150 p1 using white 96 well plates, using the following substrate and microsome concentrations: BFC -50 pM substrate, 20 pg mouse liver microsomes; 7-ethoxy-4-trifluoromethylcoumarin (EEC) -40 pM substrate, 15 pg mouse liver microsomes; MFC -180 pg substrate, 15 pg mouse liver microsornes; ethoxyresorufin (ER) and benzyloxyresorufin (BR) -1pM substrate, 11.25 pg mouse liver microsomes. Reactions were measured in real time for 3 mm using the recommended excitation and emission wavelengths for each probe using a Fluroskan Ascent FL plate reading fluorimeter (Labsystems, UK). Turnover rates were calculated using authentic metabolite standards (HFC for BFC, EFC and MFC assays and resorufin for ER and BR assays).

HPLC/LC MS-MS assay incubations.

Incubations were performed under the following conditions: Bufuralol -300 pM substrate, 20 pg mouse liver microsomes in a final volume of 150p1 for 6 mm; Chlorzoxazone -1 mM substrate, 20 pg mouse liver microsomes in a final volume of l5Opl for 15 mm; Midazolam -50 pM substrate, 25 pg mouse liver microsomes in a final volume of 150p1 for 9 mm; Metoprolol -800 pM substrate, 30 pg mouse liver microsomes in a final volume of l5Opl for 60 mm; Phenacetin -200 pM substrate, 20 pg mouse liver microsomes in a final volume of lOOpl for 9 mm; Tolbutamide -800 pM substrate, 30 pg mouse liver microsomes in a final volume of 150p1 for 60 mm. Assays were stopped by the addition of either 0.5 volumes (bufuralol, chlorzoxazone and tolbutamide) or I volume (midazolam and phenacetin) of ice-cold methanol and incubated on ice for 10 mm. Samples were centrifuged for 8 mm at 16000 x g to remove particulate material before HPLC (bufuralol, chlorzoxazone and tolbutarnide) or LC MS-MS (mldazolam and phenacetin) analysis.

HPLC separation for microsomal incubations.

HPLC analysis was carried out using a Hewlett Packard 1100 HPLC with IJV detection and Chemstation software following the previously described conditions. Metabolites were quantified with reference to authentic metabolite standards.

LC/MS-MS conditions for microsomal incubat ions.

Microsomal product formation from phenacetin, midazolam, metoprolol and tolbutamide was analysed by LC-MS/MS (Waters 2795 HPLC and Quattro Micro mass spectrometry system). The capillary temperature and voltage were respectively set at 250'C, 3 kV in positive and 250C, 4 kV in negative electrospray ionization mode. Multiple reaction monitoring (MRM) data wore acquired (Table 1). Substrates, phenacetin, metoprolol and tolbutamide, were diverted in the waste. The cone voltage and collision energy were optimised for each product (Table 1); a dwell time of 0.5 s was used between MRM transitions.

Table 1. Modes of detection and parameters used to quantify the different products formed during microsomal incubations with the LC-MS/MS.

Drugs Jon mode MRM Cone Collision Energy transitions voltage (kV) ___________ (V) 4-hydroxy tolbutamide -ye 285.14> 186.05 32 21 chlorzoxazone (IS) -ye 168.15> 131.94 44 25 Acetaminophen + ye 180.34> 110.34 30 30 4-hydroxy midazolam + ye 342.39 > 234.33 30 23 1-hydroxy midazolam + ye 342.42 > 324.30 30 22 N-desmethyl + ye 254.14> 177.21 35 20 254.14 > 72.04 a-hydroxy metoprolol + ye 284.07 > 73.91 35 21 284.07> 116.04 Caffeine (IS) + ye 195.00> 13797 35 25 PhenacetinfiS) +ve 180.34>110.34 30 30 Resorpine +ve 607.13>136.99 70 46 4-Hydroxy midazolam, 1-hydroxy midazolam and the internal standard phenacetin were resolved in 5 mm on a Luna C18 (2) (5.i, 100 A, 50 x 2.00 mm) column (Phenomenex, Torrance, CA). The injection volume was 20 p1. The following elution program was used at a temperature of 30'C and a flow rate of 0.5 mI/mm: Eluent A -0.1% formic acid; Eluent B -Acetonitrile; (1) a linear gradient from 3% to 50% B was run in 3 mm, (2) mobile phase was held at 50% B for 0.5 mm, (3) linear gradient was run to 60% B in 0.5 mm, (4) solvent composition was returned to 3% B for equilibration.

Acetaminophen and the internal standard caffeine were resolved with a Luna Cl 8 (5 p A, 50 x 2.00 mm) column (Phenomenex, Torrance, CA) with a run time of 5 mm.

The injection volume was 5 p1. The following elution program was used at a temperature of 20C and a flow rate of 0.2 mI/mm: 75% Eluent A -20mM ammonium formic pH 2.6 and 25% Eluent B -Acetonitrile.

N-Desmethyl metoprolol, a-hydroxy metoprolol, metoprolol acid and the internal standard caffeine were analysed with a Gemini CIB (5 p, 110 A, 50 x 2.00 mm) column (Phenomeriex, Torrance, CA). The injection volume was 20 p1. The following elution program was used at a temperature of 20CC and a flow rate of 0.3 mI/mm: Eluent A -10 mM ammonium formate pH 3.0; Eluent B -Acetonitrile wit 0.1 % formic acid;(1) mobile phase was held at 9% B for 2.5 mm, (2) a linear gradient from up to 95% B was run in 1 mm, (3) mobile phase was held at 95% B for 2.5 mm, (4) solvent composition was returned to 9% B for equilibration.

4-Hydroxy tolbutamide and the internal standard chlorzoxazone were resolved using a Hypersil C18 (3 p, 120 A, 50 x 2.00 mm) column (Phenomenex, Torrance, CA). The injection volume was 50 p1. The following elution program was used at a temperature of 20CC and a flow rate of 0.25 mI/mm: 55% Eluent A -20mM ammonium formic pH 2.6 and 45% Eluent B -Methanol.

In vivo pharmacokinetics.

For the in vivo pharmacokinetic studies, whole blood (lOpI) was taken from the tail vein at varying intervals afier drug administration and was transferred into a tube containing heparin (lOpI, 15 lU/mI). Internal standard solution (lOp1, caffeine, 500 ng and resorpine, 500 ng) was added to each tube. Protein precipitation was carried out by adding methanol (75p1) followed by 8% sulphosalicylic acid (55pQ. Samples were mixed for i mm and centrifuged at 13,000 rpm for 5 mm. The supernatant was transferred directly into HPLC vials for analysis.

The range of concentrations for the standard curves was constructed for quantifying blood levels by spiking blank blood samples with known amounts of chlorzoxazone, metoprolol, midazolam, phenacetin and tolbutamide. Extraction and protein precipitation were carried out as outlined above for the test samples.

LC/MS-MS conditions for blood samples.

Blood extracts were analysed for chlorzoxazone, metoprolol, midazolam, phenacetin, tolbutamide and the two internal standard caffeine and resorpine. LC-MS/MS was carried out using a Waters 2795 HPLC coupled to a Quattro Micro Mass spectrometry system (MicroMass, Manchester, United Kingdom) in the positive and negative electrospray ionization mode. The capillary temperature and voltage were set as outlined above for the microsomal samples. Multiple reaction monitoring (MRM) data were acquired for each substrate (Table 2). The cone voltage and collision energy was optimised for each compound (Table 2); a dwell time of 0.1 s was used between MRM transitions.

Table 2. Modes of detection and parameters used for each IndivIdual drug measured by LC-MSIMS In plasma samples.

Drug Ion mode MRM transitions Cone voltage Collision energy _______________ (V) (kV) Tolbutamide -ye 269.06>170.18 30 20 Chlorzoxazone -ye 168.15>131.94 44 25 Resorpine(IS) -ye 607.13>136.99 70 46 Phenacetin + ye 180.34> 110.34 30 30 Midazolam + ye 326.43 > 291.35 30 27 Metoprolol + ye 267.99> 132.97 30 26 Caffeine (IS) + ye 195.00> 137.97 35 25 Chromatography was carried out on a Luria C18 (2) (5p, bOA, 50 x 2.00mm) column (Phenomenex, Torrance, GA), with an injection volume of 10 p1. The following elution program was used at a temperature of 20C and a flow rate of 0.4 mI/mm: Eluent A - 20mM ammonium formic pH 2.6; Eluent B -Methanol; (1) mobile phase was held at 15% B for 1 mm, (2) linear gradient was run to 75% B in I mm, (3) linear gradient was run to 90% B in 2 mm, (4) linear gradient was run to 95% in 0.5 mm, (5) solvent composition was returned to 15% B for equilibration.

In vitro and in vivo data.

Average rates of metabolism were calculated for each triplicate incubation of mouse liver microsomes from each genotype (n = 6) and these data were then used to calculate p values using an Unpaired t-test (http://www.graphpad.com/quickcalcs/ttestl.cfm).

Pharmacokinetic variables were calculated using the WinNonLin software, version 3.1. A simple non-compartmental model was used to calculate area under the curve (AUC), ti,2, Cmax, and clearance (CL). Statistical analyses were carried out as describe above for the microsome incubations.

EXAMPLE I

Cytochrome b5 hepatic null mice (HBN) were analysed for tissue protein expression levels. Mice nulled for hepatic cytochrome b5 (HBN; Cytbi 0x::Cre1) were born at the expected Mendelian ratio, grew and developed normally and displayed no overt phenotype when compared to their wild-type littermates (Cytb51°°"). Livers from adult HBN mice looked similar to wild-type animals, with no gross changes in general morphology or pathology noted. Liver microsomes wore prepared from 10-week-old male HBN and wild-type mice (n=8), and POR activity was determined using cytochrome c as a surrogate electron acceptor. HBN demonstrated a slightly higher average rate of cytochrome c reduction than wild type mice (228 � 65 vs 180 � 26 nmol cytochrome a reduced/mm/mg), but this difference was not significant. To confirm the absence of cytochrome b5 protein in the liver and to verify that the deletion was exclusively hepatic, microsomes from lung, kidney small and large intestine were prepared and immunoblotted, with liver microsomes, for cytochrome b5 expression (Figure 2A). Figure 2A shows characterisation of protein expression in cytochrome b5 hepatic null mice.

Immunoblot analysis for cytochrome b5 expression in liver, kidney, lung and small intestine microsomes (5ig protein per lane) from Cytbi00x::Cre (HBN) and Cytb5b0Mo (wild-type). Cytochrome b5 protein was undetectable in the Uvers of HBN mice and there were no discernable differences in cytochrome b5 expression between the HBN and wild type mice in the other organs examined. No cytochrome b5 protein could be detected in the large intestine of either genotype (data not shown).

Three representative liver microsomal samples of each genotype were analysed for the expression of cytochrome P450s, POR and cytochrome b5 reductase (Figure 2B).

Figure 2B shows immunoblot analysis for expression of cytochrome P450-dependent monooxygenase components in liver microsomes (5.tg protein per lane) from Cytb5b00x::Cr? (HBN) and Cytbdox (wild-type). Although the expression of several P450s was slightly elevated in the HBN mice, these changes were not statistically significant when the immunoreactive bands were quantified by densitometry (not shown).

A slight reduction in Cyp2d was also observed. POR and cytochrome b5 reductase levels were also essentially unchanged in HBN compared to wild-type mice.

Having established that cytochrome b5 protein had been globally deleted from all tissues examined, it was necessary to determine if global deletion of cytochrome b5 resulted in compensatory changes in the expression levels of other components of the cytochrome P450-monooxygenase system. Microsomes from liver, lung, kidney and small intestine of each genotype were analysed for the expression of P450s, POR and cytochrome b5 reductase (Figure 2C). In general an increase P450 expression levels was observed in BCN livers for all isoforms except Cyp2d, however this only reached statistical significance for Cyp2a, Cyp2b and Cyp3a, with Cyp2b showing the most pronounced induction (29-fold) (Figure 2C). The associated proteins POR and cytochrome b5 reductase showed significantly increased and decreased levels respectively in the BCN livers Other changes included, decreased expression of Cyp2el in the kidney but increased in the lung, increased expression of Cyp4a and Cyp2b in the lung and finally, a 70 % decrease in Cyp2d and decrease in Cyp2e expression was observed in the small intestine (Figure 2C).

These results show that the hepatic deletion of cytochrome b5 was shown to be both specific and complete and importantly, that the deletion of cytochrome b5from the liver did not result in significantly increased expression of hepatic P450 proteins since this could have potentially complicated the interpretation of the in vitro and in vivo metabolic data. This data indicates that the reduction in P450 activity, as a consequence of cytochrome b5 deletion, does not induce any regulatory pathway aimed at compensating for this reduction by increased P450 expression.

EXAMPLE 2

In vitro cytochrome P450 activities/metabolism was investigated on model substrates and drugs. Six commonly used P450 model substrates which are metabolised by a range of individual P450 enzymes, were used to establish if there were any in vitro differences in metabolism between liver microsomes from HBN and wild type mice - (BFC (CYP3A) Figure 3A, MFC (CYP2C) Figure 3B, EFC (CYPIA/2B) Figure 3C, ER (CYPIAI/2, CYPIBI) Figure 3D, BR (CYP2B/3A) Figure 3Eand bufuralol (CYP2D) Figure SF). Figures 3 A-F show NADPH-and NADH-dependent cytochrome P450 activities in hepatic microsomes from wild type and cytochrome b5 hepatic null mice, assays were performed in triplicate, Each bar represents the mean � SD from 6 mouse liver microsome preparations. Black bars represent NADPH-and white bars NADH-mediated activities, respectively. Statistical analysis: * = p �= 0.05; ** = p �= 0.005, = p �= 0.001.

Assays were performed at substrate concentrations approximately 5 times Km (mouse) as defined by literature or data generated with recombinant mouse P450s (data not shown).

When NADPH was used as electron donor, significantly higher turnover rates were measured in wild-type mice relative to HBN samples for all substrates tested. The fold-difference in activities ranged from 14 for ER to 9.7 fold for MFC (Figure 3B). NADH could support activity for all model substrates in the wild type samples, although the percentage of activity as compared to that with NADPH was highly substrate dependent, ranging from 4% (BR) to 51% (ER) (Figure 3D). All NADH-catalysed reactions were significantly lower in the HBN samples compared to wild type, with barely detectable activities using BFC, EFC and MFC as substrates (Figure 3 A, C, B). The metabolism of ER and bufuralol was also greatly reduced, to 52% and 28% of the wild-type rate, respectively. NADH-mediated metabolism of BR was very low in control liver microsomes, and essentially unchanged in HBN samples.

The in vitro metabolism of the probe drugs chlorzoxazone Figure 4A, metoprolol Figure 4B, midazolam Figure 4C, tolbutamide Figure 4Dand phenacetin Figure 4E were examined with liver microsomes from both HBN and wild-type mice (Figure 4). Figures 4 A-E show NADPH-and NADH-dependent probe drug metabolism in hepatic microsomes from wild type and cytochrome b5 hepatic null mice. Assays were performed in triplicate as described in the Methods. Each bar represents the mean � SD from 6 mouse liver microsome preparations. Black bars represent NADPH-and white bars NADH-mediated activities, respectively. Statistical analysis: * = p �= 0.05; ** = p �= 0.005, = p �= 0.001.

As for the model substrates, incubations were performed at substrate concentrations approximately 5 times Km (mouse), using either NADPH or NADH as co-factor. For all substrates examined, rates of metabolite production using NADPH were once again significantly higher with liver microsomes from wild-type mice compared to HBN samples (Figure 4) The difference ranged from 1.4-(midazolam, tolbutamide) to 2.3-fold (chlorzoxazone) -with only the production of 1-and 4-hydroxymidaZolam and hydroxy-tolbutamide failing to reach statistical significance. All three metabolites of metoprolol were produced at significantly lower rates in HBN microsomes with NADPH as co-factor.

NADH supported the formation of metabolites for all substrates examined in wild-type liver microsomes, with the exception of tolbutamide, the production of hydroxy-tolbutamide being undetectable. Again, the percentage of enzyme activity in wild-type liver microsomes supported by NADH as compared to the NADPH-mediated rates was significantly lower and substrate-dependent, ranging from 6% (0-desmethyl metoprolol production) to 29% (acetaminophen production) of the NADPH rate. Furthermore, NADH-driven metabolism of chlorzoxazone, metoprolol, midazolam and phenacetin was significantly higher in liver microsomes from wild-type mice as compared to those from HBN mice.

The use of a range of both model substrates and probe drugs clearly demonstrated that the absence of cytochrome b5 significantly attenuated the P450-mediated metabolism of these compounds in vitro. For all fluoro-coumarin substrates employed, there was a highly significant decrease in NADPH-mediated P450 metabolism in liver microsomes from the HBN mice (as high as 90% for EFC), whereas for the two resorufin analogues and bufuralol the change was still significant (at least 50% decrease for each compound) but less dramatic. This substrate dependency was further illustrated by the use of a series of probe drugs, where the microsomal metabolism of three such agents (chlorzoxazone, metoprolol and phenacetin) was significantly reduced in HBN mice, whereas for midazolam and tolbutamide, although NADPH-mediated P450 metabolism was lower in both cases in mice lacking hepatic cytochrome b5, the effect was not significant.

EXAMPLE 3

Microsomes from kidney, liver, lung and small intestine of wild type and BCN mice were examined for NADPH-dependent activity towards a panel of commonly used fluorescent P450 probe substrates, namely members of the alkoxy-resorufin and alkoxy-4-trifluoromethylcoumarin families (Table 3). In the liver microsome preparations, there were no differences observed in tumover of ER or MR, while the BCN samples exhibited a significantly higher rate of metabolism of BR (4.1-fold) and PR (2.4-fold) than wild type.

This increase in turnover of PR and BR, traditionally attributed to Cyp2b activity, probably reflects the 29-fold increase of Cyp2b protein observed in the liver (Figure 2C).

With respect to the alkoxy-4-trifluoromethylcoumarins, the turnover in the BCN liver microsomes was significantly lower than wild type (BFC 42-fold; EEC 5.5-fold and MFC 5.5-fold lower respectively), indicating a severely reduced metabolic capacity. P450 monooxygenase activity was completely ablated in the lung samples when challenged with the alkoxy-resorufins, and profoundly reduced with the three 4-trifluoromethylcoumarins (BFC 29.8-fold; EFC 5.5-fold and MFC 115-fold lower). Activity towards BR, PR, BFC, EFC and MFC was detected in wild type small intestine microsomes, which in the case of the alkoxy-4-trifluoromethylcoumarins and BFC was virtually or completely inhibited by the deletion of cytochrome b5. The rate of PR metabolism was however was slightly increased by cytochrome b5 deletion. No activity towards these probe substrates was detected using either wild type of BCN kidney microsomes.

To achieve a direct comparison with the in vivo pharmacokinetic data, NADPH-driven microsomal incubations were performed with the individual pharmacokinetic cocktail drugs and liver, lung, kidney and small intestine microsomes (Figures 4F-J). NADPH-dependent drug metabolism was analysed in wild-type (green bars) and BCN (red bars) mice. Assays were preformed in triplicate and values represent the mean � SD of n = 3.

* = p �= 0.05, = p �= 0.005 and *** = p 0.001. Chlorzoxazone (Figure 4F) metabolism was observed in wild type liver, lung and kidney microsomes, while in the equivalent BCN tissues activities were significantly reduced (4-, 2-fold for liver and lung respectively) or completely inhibited (kidney). Midazolam (Figure 4G) was metabolised to two metabolites: 1' hydroxy-and 4-hydroxy midazolam by all four tissues examined.

ln each case the rates of turnover achieved by the BCN samples were much lower than in the wild type preparations, reaching statistical significance in the liver and kidney for 1' hydroxy midazolam production; and in the liver for 4-hydroxy midazolam. Metoprolol (Figure 4H) was converted to 0-desmethyl metoprolol by all samples tested. Rates of turnover were significantly reduced by cytochrome b5 deletion in the liver and kidney, while no effect was observed in the lung or small intestine samples. 1-Hydroxy metoprolol was produced from liver, kidney and small intestine but not from lung incubations. Similarly to 0-desmethyl metoprolol production, formation of this metabolite was significantly decreased in the liver and kidney samples of BCN mice. Metabolism of phenacetin (Figure 41) to acetaminophen occurred only in liver and kidney, where Interestingly, rates were significantly elevated in the BCW (p<0.05) while in the kidney, ablation of cytochrome b5 had no effect on activity. Similarly, metabolism of tolbutarnide (Figure 4J) was only detected in liver and kidney microsomes, however in both cases, BCN samples showed decreased activity as compared to wild type.

Table 3, below shows the activities of mouse tissue microsomes towards seven fluorescent probe compounds For liver and small intestine samples, assays were performed in triplicate for three individual mouse samples, while for lung samples, assays were performed in triplicate on a pool of three lung preparations, precluding statistical analysis. Kidney microsomes were also tested but no activities were detected.

For BR, ER, MR and PR, metabolite formed is resorufin, and for BFC, EFO and MFC is 7-hydroxy-4-trifluoromethylcou mann.

TABLE 3 ________

Liver Lung Small intestine Activity Activity ActMty (pmol/minlmg) (pmolfminlmg) (pmol/mialmg) Compound WT BCN WT BCN WT BCN BR 3.8�1.2 15.7�1.7" 15.8�2.3 n.d. 7.5�7.6 0.6� 0.5 ER 10.1 � 1.9 9.3 � 3.7 2.4 � 0.3 n.d. n.d. n.d.

MR 53 � 14 51 � 27 0.4 � 0.2 n.d. n,d. n.d.

PR 2.2 � 0.2 5.2 � 1.2 * 0.6 � 0.4 n.d. 0.4 � 0.02 0.7 � 0.02 BFC 275�46 65�6" 45�4 13�1.7 18�20 0.05�0.09 EFC 245�59 31�5** 46�11 8.2�1.2 3.8�6.6 n.d, MFC 181�48 33�7* 90�4 0,78�1.3 20�25 n.d.

* = p 50.05, 9"! = p 5 0.005, n.d. not detected.

EXAMPLE 4

In vivo pharmacokinetics of probe drugs in wild-type and HBN mice was studied. To investigate the effect of the absence of hepatic cytochrome b5 on the in vivo pharmacokinetics of the probe drugs, wild-type and HBN mice were administered chlorzoxazone, metoprolol, midazolam, tolbutamide and phenacetin either as an intravenous or oral cocktail. Previous work with these drugs in wild-type mice, individually and as a cocktail, had demonstrated that simultaneous delivery did not result in altered pharmacokinetics as compared to single administration (not shown). Following administration of the drug cassette to wild-type and HBN mice, quantification of individual drugs and their metabolites was carried out by LC-MS/MS. The pharmacokinetic data for each drug is summarised in Tables 4 and 6, and the elimination profiles of all the drugs used are shown in Figures 5 and 6. Figures 5 A-E show pharmacokinetic disposition of an orally administered P450 drug cocktail in wild-type and hepatic cytochrome b5 null mice. A P450 drug cocktail containing chlorzoxazone (5mg/kg) (Figure 5E) , metoprolol (2mg/kg) (Figure SD) , midazolam (5mg/kg) (Figure 4A) , phenacetin (5mg/kg) (Figure 5B) and tolbutamide (5mg/kg) (Figure SC) was administered orally at the indicated doses to wild-type (black circles) or HBN (open circles) mice and blood samples taken at timed intervals to determination the in vivo pharmacokinetic parameters of the parent compounds. Statistical analysis: * = p �= 0.05; ** = p < 0.005, = p < 0.001 where n = 3. Figures 6A-E shows pharmacokinetic disposition of an intravenously administered P450 drug cocktail in wild-type and hepatic cytochrome b5 null mice. A P450 drug cocktail containing chlorzoxazone (5mg/kg) (Figure 6E) , metoprolol (2mg/kg)(Figure 4B) , midazolam (5mg/kg) (Figure 4A), phenacetin (5mg/kg) (Figure 4C) and tolbutarnide (5mg/kg) (Figure 4D) was administered by i.v. injection of the tail vein at the indicated doses to wild-type (black circles) or HBN (open circles) mice and blood samples taken at timed intervals to determination the in vivo pharmacokinetic parameters of the parent compounds. Statistical analysis: * p �= 0.05; ** = p �= 0.005, = p �= 0.001 where n = 5.

Table 4. Summary of pharmacokinetic data on drugs administered orally to HBN and wild-type controls.

Drug PK parameter WT HRN p-vaJue Midazolam AUC(min*j.xg/mI) 34.0�2.8 123.6�7.3 Cmax(p.g/ml) 0.4�0.03 1.0�0.03 Clearance (mI/mm/kg) 148.9 � 12.3 40.8 � 2.5 Tenninai Half-Life (mm) 166.5 � 57.6 69.7 � 8.0 n.s.

Phenacetin AUC(min*Lg/m1) 1.46�0.06 1.84� 0.13 n.s.

Cmax Q.ig/ml) 0.06 � 0.01 0.09 � 0.02 * Clearance (mI/mm/kg) 687.6 � 29.9 549.9 � 37.5 * Terminal Half-Life (mm) 308.0 � 43.7 278.6 � 11.2 n.s.

Metoprolol AUC (min*mg/m1) 0.20 � 0.07 0.22 � 0.04 n.s.

Cmax(ng/ml) 1.1�0.1 1.5�0.2 n.s.

Clearance (IJminJkg) 12.7 � 3.9 9.5 � 1.6 n.s.

Terminal Half-Life (mm) 167.7 � 51.0 129.6 � 8.2 n.s..

Chlorzoxazone AUC (min*.tg/mI) 16.1 � 2.4 12.9 � 2.9 n.s.

Cmax (mg/ml) 0.37 � 0.05 0.20 � 0.02 * Clearance (mI/mm/kg) 323.4 � 41.7 425.3 � 82.9 n.s.

Terminal Half-Life (mm) 194.0 � 30.6 45.9 � 3.8 ** Tolbutamide AUC (min*pg/ml) 408.9 � 57.9 314.8 � 46.9 n.s.

Cmax (jim1) 0.89 � 0.12 0.34 � 0.02 ** Clearance (mllmin/kg) 12.8 � 2.1 16.9 � 2.5 n.s Terminal Half-Life (mm) 363.8 � 33.4 639.5 � 104.6 n.s.

* = p �= 0.05, = p 0.005, = p �= 0.001, n.s.= not significant where n = 3.

Following oral administration of the P450 cocktail, significant differences in the elimination profiles of all the drugs were observed between wild-type and HBN mice. In the case of midazolam, phenacetin and metoprolol, AUG and Cmax were consistently higher in HBN mice while the clearance rate was slower (Fig. 5 and Table 3). For midazolam, AUC and Cm were significantly increased from 34.0 to 123.6 min*jmg/ml and 0.4 to 1.0.mgIml respectively, whereas clearance was decreased from 148.9 to 40.8 ml/miri/kg (Table 3). The phenacetin elimination profile also showed significant changes, with C increasing from 0.06 to 0.09 rIg/mI and clearance decreasing from 687.6 to 549.9 mI/mm/kg (Table 4). Despite changes in the parameters mentioned above for metoprolol, none of these were found to be significant (Table 4). In contrast to the differences described for midazolam, phenacetin and metoprolol, changes in the elimination profiles of chlorzoxazone and tolbutamide were reversed, with HBN mice displaying lower AUC and Cm values and higher clearance rates compare to wild-type mice (Fig. 5 and Table 3). For chlorzoxazone and tolbutamide only Cm was significantly changed, decreased from 0.37 to 0.2 flg/ml and 0.89 to 0.34 gglml respectively.

Interestingly, for chlorzoxazone, the terminal half-life was also significantly decreased in HBN mice from 194.0 to 45.9 mm (Table 4). The changes in AUC and clearance were not statistically significant.

To establish if the in vitm results obtained with the cytochrome b5 complete null would translate into a difference in in vivo pharmacokinetics; chlorzoxazone (Figure 6F) metoprolol (Figure 60), midazolam (Figure 41-1) , tolbutamide and phenacetin (Figure 61) were administered orally as cocktail to wild-type and BCN mice. Following dosing of the drug cassette to wild-type and BCN mice, quantification of individual drugs and their metabolites was carried out by LC-MS/MS. The pharmacokinetic data for each drug is summarised in Table 5 below which shows the results of adult male BCN and WT mice (n =3) were administered a drug cocktail orally; serial blood samples were collected over a 8 hour period and analysed for plasma drug concentrations by LC-MS/MS and key pharmacokinetic parameters calculated and the elimination profiles of the drugs are shown in Figure 6F-l wild-type (green line) and BCN (red line) mice and blood samples were taken at time intervals to determine the in vivo pharmacokinetic parameters of the parent compound. * p �= 0.051 ** = p S 0.005 and *** = p 50.001, where n =4.

Table 5 summarises of pharmacokinetic data on drugs administered orally to BCN and wild-type controls.

Drug PlC parameter WT BCN p-value vlidazolam AUC (min*p.g1rnl) 10.9 � 3.4 35� 14 n.s.

Cmax (ig/ml) 0.11 � 0.04 0.3 � 0.13 ns.

Clearance (mllminlkg) 281 � 47 96� 23 Terminal Half-Life (mm) 78 � 5 91 � 8 its.

henacetin AUC (mmn*p.g/ml) 3.3 � 0.48 5.8 � 0.91 * C,, (jig/mi) 0.1 � 0.01 0.14 � 0.03 * Clearance(mllmin/kg) 1333 �137 867� 103 n.s.

P120487W01.as filed Terminal Half-Life (miii) 307 d 46 172 d 24 * Vietoproloi AUC (min*pg/ml) 0.22 � 0.05 0.76 � 0.24 * Cruax (jig/mi) 0.002 � 0.001 0.008 � 0.003 * Clearance (L/min/kg) 8,4 � 0.9 2.3 � 0.5 ** Terminal Half-Life (mlii) 146 � 30 350 � 66 * Chlorzoxazone AUC (miii" jig/mi) 3.4 � 0.4 7.3 * 0.7 Cmax(j.tg/ml) 0.1 � 0.02 0.1 * 0.02 u.s.

Clearance (mI/mm/kg) 1387 � 124 627 � 65 ** Terminal Half-Life (miii) 44 � 9 92 * 34 u.s.

roibutamide AUC (mmn*j.tgfml) 213 * 18 247 � 17 u.s.

C, (jig/mi) 0.63 � 0.06 0.88 * 0.11 fl.S.

Clearance (mllminlkg) 10.8 � 2.0 12.9 � 0.6 u.s Terminal Half-Life (mm) 641 * 90 325 � 40 * * = p �= 0.05, " pS 0.005, u.s. = not significant.

Following oral administration of the drug cocktail, all the drugs show differences in their elimination profiles when BCN mice were compared to their respective controls. For midazolam a significant decrease of clearance from 281 to 96 mI/mm/kg was observed in BCN mice. In the BCN animals, this alteration of clearance has lead to a 3-fold increase of AUG and a slight increase of Cm,< and terminal half-life. As observed for midazolam, BCN mice showed a significant decrease of chlorzoxazone clearance from 1.3 to 0.6 Llmin/kg and a significant increase of AUG from 3.4 to 7.3 min*pg/ml, whereas Crnaxwas not altered. In the BCN mice, metoprolol clearance was significantly decreased as compared to wild-type (3 fold from 8.4 to 2.3 11mm/kg). Additionally, a significant increase of AUC, C and terminal half life from 0.22 to 0.76 min*pg/ml, 0.002 to 0.008 pg/mI and 146 to 250 mm were observed, respectively.

In contrast to mldazolam and metoprolol, phenacetin exhibited a less pronounced alteration of clearance from 1.3 to 0.87 11mm/kg, however AUG, Cmax and terminal half life were all significantly affected by the deletion of cytochrome b5. AUC and Cmu were increased from 3.3 to 5.8 mmn*pg/mI and 0.096 to 0.14 pg/mI, respectively, whereas terminal half life decreased from 307 to 171 mm. In the case of tolbutamide, deletion of cytochrome b5 affected only the terminal half life, which was decreased significantly from 640 to 324 mm as compared to the wild type mice.

EXAMPLE 5

Following i.v. administration of the drug cocktail, wild-type mice showed a higher concentration of parent drug in the blood at the early time points (0-60 mm) compared to the RBN mice for all drugs in the cocktail. In addition, from 2 hours, the average concentration of parent drug was much lower in the wild-type than in HBN mice, and remained so for the duration of the experiment. Pharmacokinetic analysis for each drug showed that the clearance was consistently faster, and the AUC consistently lower, in wild-type compared to HBN mice, although none of these changes reached statistical significance (Table 6). In contrast, the terminal half-life for phenacetin, tolbutaniide and midazolam were statistically different between the two lines, being significantly higher in HBN mice, increasing from 63.5 to 162.2 mm, 68.9 to 297.5 mm and 91.9 to 476.1 mm respectively (Fig. 5 and Table 6). For chlorzoxazone and metoprolol, although a 2.2-and 3.7-fold increase of the terminal half-life was found in HBN mice, respectively, this did not reach statistical significance (p = 0.056 (chlorzoxazone) and 0.06 (metoprolol)).

Table 6. Summary of pharmacokinetic data on drugs administered intravenously to HBN mice and wild-type controls.

Drug PlC parameters WT [lEN p-value Chlorzoxazone AUC (min*tgfrnl) 397.1 � 67.9 631.5 � 107.3 n.s.

Cl (mllmin/kg) 14.2 � 2.5 8.9 � 1.8 n.s.

Terminal half life (mm) 113.4� 16.8 253.8� 58.5 n.s, M:etoprolol AUC (min*pg/ml) 72.9 � 17.7 104.0 � 22.6 n.s.

Cl(mllrninlkg) 38.1 � 11.3 23.2� 5.7 n.s.

Terminal half life (mm) 110.7 � 14.7 416.5 � 138.5 n.s.

Midazolam AUC (min*g/mi) 231.3 � 56.7 247.2 � 49.8 its.

Cl (mJlmin/kg) 29.8 � 8.7 24.0 � 5.8 n.s.

Terminal half life (mm) 91.2 � 14.2 476.1 � 203.4 * Phenacetin AUC (min*g/ml) 487.4 � 101.8 580.1 � 74.7 n.s.

Cl (mllmin/kg) 13.1 � 3.4 9.2 � 1.4 n.s.

Terminal half life (mm) 63.5 � 11.4 162.2� 31.7 * Tolbutamide AUC (min*p.g/mI) 421.5 � 80.7 642.5 � 59.9 n.S.

Cl (mI/mm/kg) 13.5 � 2.4 8.0 � 0.9 its.

Tennmnalhalflifc(min) 68.9� 12.0 291.5�30.2 * = p S 0.05, ** = p S 0.005, = p 5 0.001, n.s. not significant where n = 5 The present invention represents a major advance and improvement in the ability to investigate the effect of cytochrome b5 on P450 metabolism in vivo; by deleting cytochrome b5 from the liver of HBN mice, it has been possible to administer, as a cocktail, the series of probe drugs previously employed in vitro, and determine the pharmacokinetics of these drugs in the whole animal, thus defining the role of the enzyme in regulating P450 actIvity In summary, one of the embodiments of the present invention provides a conditional hepatic knockout of cytochrome b5 and shows that both in vitro and in vivo the absence of this enzyme can result in significantly reduced metabolism of model substrates and probe drugs by cytochromes P450, as determined using NADPH as a co-factor.

EXAMPLE 6

To ensure that the observed changes in P450 activities were not a consequence of perturbation in membrane phospholipids, detailed fatty acid analysis was performed on endoplasmic reticulum (ER) membranes from HBN mice and compared to wild type mice (Figures 7A-D). The analysis comprised measuring ER levels of saturated fatty acids (Figure 7A), monounsaturated fatty acids (Figure 7B), polyunsaturated (n=6) fatty acids (Figure 7C) and polyunsaturated (n=3) fatty acids (Figure 7D). Green bars represent wild type and red bars HBN mice, respectively Each bar represents the mean � SEM of n = 3. Statistical analysis: * = p 5 0.05; ** = p �= 0.005, = p s 0.001. The data confirmed no significant changes in the consistency of the phospholipids contained in the ER membranes, confirming that the observed effects on P450 activities in HBN mice were solely due to their interaction with cytochrome b5.

EXAMPLE 7

Age related changes in P450 expression were investigated. Due to the lack of phenotypic changes at 10 weeks of age, older HBN were analysed to determine if any underlying phenotypic changes progressed with age. Liver microsomes from 6 month old females and 10 month old males were analysed for changes in P450 expression (Figure 8). Comparison with wild type mice of the same age revealed an age dependent induction of P450s belonging to the Cyp2b and Cyp3a in both sexes but also in Cyp2a proteins in males. in contrast, Cyp2e expression levels appeared to decrease with age.

POR expression levels also were found to be slightly elevated in older male HBN mice but not in females, however this maybe due to the age difference between the sexes.

These Immunoblot observations were confirmed by densitometric analysis of the immunoreactive bands (Figure 9). During the preparation of hepatic microsomes from older HBN mice, it was noticed that there appeared to be a higher hepatic lipid content.

As a result the livers were sectioned and stained with Oil Red 0 to qualitatively determine if there was an age related increase in hepatic lipid on deletion of cytochrome b5. As can be seen in Figure 10, HBN mice do indeed display an age related accumulation of hepatic lipid, however this accumulation only appears to become significant once mice reach 6 months of age (Figure 10).

EXAMPLE 8

The recapitulation of P450 activity was assessed. It has been demonstrated that uwild type" levels of in vitro activity can be regained by adding back exogenous cytochrome b5.

HBN liver microsomes (25gg) were mixed with increasing amounts of E.coli membranes containing heterologously expressed murine cytochrome b5 (0-46) pmol of cytochrome b5). NADPH dependent turnover BFC and chlorzoxazone was measured and found to increase in a cytochrome b5 dependent manner (Figure 11). The apparent kinetic parameters of the probe drugs using NAUPH as co-factor were determined in wild-type and HBN liver microsomes, and metabolism of chlorzoxazone, phenacetin, midazolam and metoprolol followed standard Michaelis Menten kinetics (Table 7).

Table 7 shows the kinetic analyses summary of metabolism of probe drugs by hepatic mlcrosomes from HBN mice and wild-type controls wild Type liver microsomes HBN liver microsomes Substrate Metabolite K, (pM) ?c,T1/minlmg) Km (pM) iwminimg) chiorroxazone 6-Hydroxy chtorzoxazone 48.9 � 8.9 1931.6 � 100.3 53.0 � 5.6 367.4 � 11.5 Midazolam 1'-HydroxyMidazolam 2.1 �0.3 389.3� 14.9 2.04�0.3 115.8 �4.3 4-Hydroxy Mldazolam 13.26 � 0.4 146.3� 1.6 7.7 � 0.9 7.3�0.3 Phenacetin Acetaminophen 56 � 1 0 133.4� 6.1 4.1 � 0,7 78.9 � 3.1 Metoprolol ct-Hydroxy Metoprolol 84.5 � 74 482.7 � 14.1 99.2 � 9.3 76.3 � 2.8 0-Desmethyl Metoproloi 86.3 � 89 1653.1 � 1.64 157.4 � 14.1 531.0 � 21.3 Tolbutamide Hydroxy tolbutamide 11256 � 3425 211.0 � 17.1 164.9 � 48.3 440 � 164.9 Assays were performed using trIplicate samples for each concentration of substrate The Michaehs constant for chlorzoxazone 6-hydroxylatian, midazolam I -hydroxylation, phenacetin oxidation and metoprolol a-hydroxylation were unchanged by the removal of microsomal cytochrome b5. Conversely, for those reactions the V was markedly reduced in the HBN samples ranging from a 2-fold (phenacetin oxidation) to a 6-fold (metoprolol a-hydroxylation) decrease. Midazolam 4-hydroxytatlon and metoprolol 0-demethylation reactions again showed decreased Vmax values for the HBN samples (20 and 3-fold respectively), however the KmS were marginally affected with the HBN samples exhibiting a 1.7-fold reduction in Km for midazolam 4-hydroxylation and a 1.8-fold increase in Km for metoprolol 0-demethylation as compared to wild-type. The data obtained for tolbutamide hydroxylation could not be fitted using the Michaelis Monten equation, but did conform to the Hill equation. Interestingly, the HBN sample exhibited 68-fold higher affinity towards tolbutamide, as well as a 5-fold reduction in Vm as compared to wild-type The Hill coefficient for both wild-type and HBN were similar (0.38 vs. 0.29) and indicative of tolbutamide binding in a negatively cooperative manner.

Pharmacokinetic studies have been performed comparing PK profiles of HBN mice with wild type, for the following substrates: paclitaxel; tamoxifen, gefitanib and cyclophosphamide as well as the previously reported cocktail drugs (midazolam, phenacetin, metoprolol, chlorzoxazone and tolbutamide). Preliminary assement of data indicate that there are differences in PK parameters for all drugs tested in the HBN model.

EXAMPLE 9

Using the construct of Figure 12A and the results of screening in Figures 12B and 12C, complete cytochrome b5 knockouts were produced. Mice were maintained on a mixed genetic background (C57BL/6Jx129). The genotypic spread of live births (Figure 13) shows that both males and females are fertile and therefore can be maintained as Het x Het (which gives reasonable Mendelian ratios); null x Het or null x null. It was also noted that pups are carried to full term, and born without gross physical defects. Other parameters of physical phenotype were investigated such as the growth rates of wild type, Hets and cytochrome b5 male (A) and female (8) knockout mice from 3 weeks to 15 weeks post partum (see Figure 13 A and B). This demonstrated that both male and female cytochrome b5 knockout pups gain weight at a similar rate to wild type and Het mice, however always maintaining a slightly lower average weight. Closer examination of the coats of the young adult complete cytochrome b5 knockouts or nulls revealed a thinner, less sleek and less glossy appearance as compared to an age-matched (8 weeks) wild-type animals (data not shown), furthermore the coat hairs of the null animals have a tendency to stand on end, as if charged with static, complete cytochrome b knockouts or nulls exhibit a "static coat appearance.

A complete cytochrome 15 knockout female (6.5 months old) had previously had two successful null lifters, however during her third pregnancy she developed poor coat condition and inflamed skin under her chin. Subsequent to the birth of this litter, she developed further hair loss with increased skin reddening, especially around the eyes and under the chin (data not shown). The female nursed pups normally, although her mammary glands were swollen. On weaning of pups, her mammary glands recovered and the coat conditioned improved, however dry skin around eyes and under chin was still present. it has been observed that repeated pregnancies resulted in a "nurturing" phenotype in complete cytochrome b knockout females as detailed above. As regards pups from a cytochrome 1,5 knockout female exhibiting a "nurturing" phenotype these appear to be underdeveloped. Pups were much slower to develop than previous complete cytochrome b5 knockout and wild type litters and suffered from poor coat cond Won, static hair and hair loss however weaned pups gained weight rapidly.

EXAMPLE 10

Western blotting and densitometry on microsomes prepared from male 15 week old wild type and complete cytochrome b knockout mice was performed for various tissues (Figure 15 A (liver), B (lung), C (kidney) and D (heart). Samples comprised approximately 0.3-0.5 g of tissue from 3 wild type and 3 cytochrome b5 knockout animals. Western blot analysis was carried out using standard conditions. Expression of cytochrome b was undetectable in all tissues examined of the complete cytochrome b knock out mouse. Cyp3a, Cyp2b and to a lesser extent Cyp2c proteins were markedly induced in the livers of the cytochrome b knockout mice while in extrahepatic tissues, Cypla proteins were highly induced. No modulation of P450 expression was observed in the small intestine, while in the brain and spleen, Cypla levels were increased in the cytochrome b knockout mice (data not shown).

Using the technique as herein before described Western blotting was performed on liver, kidney and lung derived microsomes from 12 week old wild type and complete cytochrorne b knockout male mice. Blots illustrate (Figure 16) that the induction of Cyp2b and Cyp3a proteins observed in cytochrome b knockout mice livers at 15 weeks of age is also present at 12 weeks. In contrast, there is no induction of Cypla in extrahepatic tissues at 12 weeks of age.

Western blotting on microsomes prepared from male old wild type and complete cytochrome b5 knockout mice at increasing time points was investigated (Figure 17). The Immunoblotting was performed as previously described above. Blots illustrate that the induction of Cyp2b proteins observed in cytochrome b5 knockout mice livers is maintained for at least 8 months. Equally the induction of Cypla in lung and kidney is still present at 8 months, albeit at lower levels These results demonstrate that expression of cytochrome b5 was undetectable in all tissues examined of the complete cytochrome b5 knock out mouse and that this was accompanied by a marked induction of Cypla protein in extrahepatic tissue with a marked induction of Cyp2b and Cyp3a in hepatic tissue. Thus the complete cytochrome b5 knockout animal of the present invention does indeed have ablated cytochrome b5 in all tissues and appears to have a stimulatory effect on selected forms of cytochrome P450.

EXAMPLE 11

Fe2-CO vs. Fe21' difference spectra were obtained for liver microsomes prepared from male 15 week old wild type and complete b5 knockout mice (Figure 18). Results show that levels of spectrally active P450 were significantly higher in the livers of complete cytochrome b5 knockout mice as compared to wild type (p=0.01), confirming that the increases in protein levels observed by immunoblotting correspond to functional protein not apoprotein.

EXAMPLE 12

A series of in vitro NADPH or NADH driven drug metabolism assays were performed using tissue microsomes isolated from wild type and complete cytochrome b5 knockout week old male mice. With reference to Figure 19 there is shown activities of liver and lung derived microsomes towards the fluorescent probe 7-ethoxy-4-trifiuoro-methylcoumarin (EFC). Formation of the metabolite 7-hydroxy-4-trifiuoromethylcoumarin was measured using a microplate fluorimeter. No activity was detected in brain, heart, kidney, small intestine or spleen. Rates of metabolite formation using NADPH as co-factor were significantly higher in the wild type liver (** pcO.005) and lung (*** p'cO.OOOS) as compared to the corresponding null tissue. NADH mediated activity was only detected in wild type liver microsomes, with no such corresponding activity in the cytochrome b5 knockout samples.

Figure 20 shows the NADPH driven activities of liver microsomes toward the fluorescent probes benzyloxyresorufin (BR), ethoxyresorufin (ER), methoxyresorufin (MR) and pentoxyresorufin (PR). Formation of the metabolite resorufin (product formed from all substrates) was measured using a microplate fluorimeter. Rates of ER and MR metabolism were unchanged by cytochrome b5 deletion. A marked increase was observed in BR (although this did not reach statistical significance) and PR (PC 0.02) turnover in the cytochrome b5 knockout livers.

Figure 21 shows kidney, liver and lung derived NADPH or NADH driven microsomal activity towards the skeletal muscle relaxant drug chlorzoxazone. Formation of 6-hydroxychlorzoxazone was measured by HPLC and could be detected in kidney, liver and lung microsomes using NADPH as cofactor. No metabolites were detected in brain, heart, small intestine or spleen samples, or when NADH was used as cofactor (results not shown). Rate of metabolite formation was highest in wild type liver microsomes, and in all cases, significantly higher turnover was observed in the wild type tissue as compared to corresponding cytochrome b5 knockout samples (kidney: ** pcO.05; liver ** pcO.005; lung: * pcO.0005).

Figure 22 shows liver, lung, kidney and heart derived NADPI-I driven microsomal activity tissues towards the benzodiazepine derivative midazolam. Formation of 1-and 4-hydroxyl midazolam as measured by LC-MS/MS was most pronounced in mouse liver, where there was no difference in turnover observed between wild type and cytochrome b5 knockout samples. In the lung, formation of 1-hydroxymidazolam was significantly faster in the wild type tissue as compared with the cytochrome b5 knockout samples (pcO.005). In the heart, 4-hydroxymidazolam was the predominant metabolite formed in contrast to liver and lung, where cytochrome b5 knockout samples exhibited a significantly reduced turnover (pco.005). In the heart, virtually no turnover was detected in the wild type microsomes, however 4-hydroxy midazolam was formed by the cytochrome b5 knockout samples. No metabolites were detected in brain, small intestine or spleen samples (data not shown).

These series of results demonstrate that the complete cytochrome b5 knockout animals of the present invention show that absence of this enzyme results in significantly reduced metabolism of model substrates and probe drugs by cytochrome P450, as determined using NADPH or NADP as a cofactor.

EXAMPLE 13

As mentioned hereinbefore cytochrome b5 is involved in the metabolism of steroids such as progesterone to I 7cc hydroxyprogesterone by the hydroxlase pathway and I 7cc hydroxyprogesterone to androstenedione via a lyase pathway. Figure 23 shows the activities of testicular microsomes prepared from 12 week old wild type and complete cytochrome b5 knockout mice towards the steroid hormones progesterone (A & C) and 17cc hydroxyprogesterone (B & C).

Panel A illustrates a representative I-$PLC trace of the NADPH dependant metabolism of progesterone by wild type and knockout testicular microsomes mediated by the hydroxylase activity of CypI 7. Wild type microsomes show much higher rates of primary (17cc hydroxyprogesterone) and secondary (androstenedione) metabolite formation than cytochrome b5 knockout microsomes. A third unidentified metabolite was also generated in the wild type incubations. Panel B illustrates a representative HPLC trace of the NADPH dependant metabolism of 17cc progesterone to andostenedione by wild type and knockout testicular microsomes mediated by the lyase activity of CypI 7. No androstenedione was formed by the cytochrome b5 knockout microsomes, in comparison to the readily detectable levels produced by the wild type samples. A further unknown metabolite was again produced, with the same retention time as in panel A. Panel C illustrates the calculated rates of metabolite production based on triplicate assays, showing the marked difference in metabolite production between wild type and cytochrome b5 knockout testicular microsomes.

Looking at a time course of 17cc hydroxyprogesterone metabolism (androstenedione production) by testicular microsomes prepared from 12 week old wild type and complete b5 knockout mice (Figure 24), results suggested that the complete b5 knockout mice were deficient in CypI 7 lyase activity, however after an extended incubation of 80 minutes; a rate could be detected in the knockout samples, albeit at a level 21-fold slower than wild-type mice.

Taken together these results show that complete cytochrome b5 knockout male mice have severely compromised progesterone metabolism and are unable to produce a detectable amount of androstenedlone, which makes the fact that they are able to breed an even more remarkable and surprising observation.

EXAMPLE 14

The effect of complete cytochrome b5 knockout on levels of methaemoglobin was investigated (Figure 25A). Methaemoglobin was measured in four non-experimental mice to obtain a baseline percentage reading of 0.45%. Experimental wild type animals recorded similar values, while the cytochrome b5 knockout samples were significantly higher (p<0.0001). This 6-fold increase in methaemoglobin is present from at least 12 weeks of age and remains stably elevated until the animals are at least 40 weeks old.

This highly significant increased in methaemoglobin levels in neonatal and juvenile animals was surprisingly not found to be lethal or to result in any phenotypically Observable trait. Further experiments (Figure 25B) shows the highly significant difference between the % methaemoglobin in wild type compared to BCN mice.

EXAMPLE 15

A series of experiments were undertaken with a view to lipid profiling of the complete cytochrome b5 knockout animals. Total lipid content for liver and plasma samples from three wild type and three cytochrome b5 knockout mice (15 week old males) was measured (Figure 27), however no differences were observed in the lipid content of the liver or plasma from either of the two genotypes indicating that there is no difference in the lipid content of liver or plasma between wild type and cytochrome b5 knockout mice.

Liver sections from wild type and cytochrome b5 null male mice were stained with Oil Red 0 to highlight stored lipid (Figure 26). No overt changes were observed up to 16 weeks of age nor any differences between the wild type and complete cytochrome b5 knockout animals, however by 30-34 weeks, a small increase in stored lipids was noted in the null animals. It is concluded that cytochrome b5 knockout mice show an age-dependent increase in hepatic lipid content. To investigate this further, despite there being no difference in total liver and plasma lipid contents (Figure 27), closer inspection of the levels of individual fatty acid species (saturated fatty acids, monounsaturated fatty acids, polyunsaturated fatty acids where n=6 or n=3), revealed significant changes in several lipid species, both in the liver (Figure 28A-Dd) and plasma (Figure 29A-D) of complete cytochrome b5 knockout mice compared to wild type mice. Hepatic levels of C15:0, C18:3n-6 and C18:4n-3 were decreased, whereas levels of C18:0, C20:3n-6 and C20:4n-3 were increased in the complete cytochrome b5 knockout or HBN mice. In the case of plasma fatty acid levels, only C18:3n-6 was decreased, and C20:0 and C20:3n-6 were increased compared to wild type mice These results indicate that due to the age related increase in hepatic liver content in addition to the increases of hepatic fatty acids C18:0, C20:3n-6 and C20:4n-3 that the complete cytochrome b5 knockout or HBN animals of the present invention may be ideal models for studying conditions such as hepatic steatosis and candidate therapeutics for such conditions. It is believed that these complete cytochrome b5 knockout animals of the present invention offer the first such model for this disease/metabolic condition.

EXAMPLE 16

BCN mice breeding and growth were investigated. Cytb5' mice were inter-crossed to generate homozygous cytochrome b5 complete knockout mice (BCNI Cytb5) and control (wild-type, Cytb5'4) mice. Mice nulled for cytochrome b5 were born at approximately the expected Mondelian ratios, and exhibited no gross physical abnormalities. Both male and female BCN mice were fertile, and although BCN x BCN pairings gave viable BCN litters, they were very slow to breed and the females started to suffer from a "nurturing phenotype" (see below); therefore colony maintenance was routinely supplemented using heterozygous null female crossed with heterozygous null males. The average litter size of these crosses was 5.4 (n=7; range 1-11). BCN pups appeared smaller than their age-matched wild-type counterparts despite the lack of a statistical difference when growth curves were compared. However, this size difference became more pronounced in successive litters and was further underlined by comparing the growth curves of first and third litters (Figure 30; black squares and circles represent wild-type male and female mice respectively. White squares and circles represent BCN male and female mice respectively. Values represent mean � S.E.M. where n 13 for wild-type males, 19 for wild-type female, 8 for BCN males and 9 for BCN females. * p 0.05 comparing wild-type and BCN mice of the same sex).

BCN adult male mice (10 weeks old) were marginally, although not statistically smaller than aged matched wild-type mice (24.lg vs. 25.4 g (n= 19 wild-type; 22 BCN). On dissection, there were no gross anatomical differences observed, however BCN mice were found to have a significantly larger liver (1.4 g � 0.3 vs. 1.2 g � 0.3; p = 0.004 (n= 19 wild-type; 22 BCN)). From approximately 18 days old, it was noticed that the fur of the HBN pups was (by visual inspection) noticeably finer and seemingly more sparse than their wild-type counterparts, having a fluffy or a "full of static" appearance, and was often rubbed off during suckling to leave small bald patches on their heads. This "static hair" phenotype although more pronounced in the younger animals, did persist into adulthood accompanied by a thinner, less shiny coat. This thinning of the coat in the adult mice was most obvious on the underside of the animals. The epidermal and dermal thicknesses of wild-type and BCN mouse skin as well as the width of the hair shafts were measured in hematoxylin and eosin stained skin sections (Figure 31 0).

The hair shaft was significantly finer in the BCN mice as compared with wild type (12.7Dm � 0.09 vs. 14.7Dm � 0.3) while the epidermis was significantly thicker (13.2 Dm � 0.6 vs. 10.2 Dm � 0.3). There was no difference in the thickness of the dermis between the two genotypes. Furthermore, keratinization of the stratified squamous epithelium was more pronounced in the BCN skin sections (Figure 31 D). There also seemed to be proliferation of the sebaceous glands in the skin of BCN mice (Figure 31A, B, 0 & E). Oil red 0 staining of skin sections from both BCN and wild-type mice indicated that there were no significant deficiencies in lipid levels (C and F) which was further confirmed by fatty acid profile analysis (H). The tails of BCN are square ended, as if the tip has been broken off, and occasionally are dry and scaly with visible constrictions. When BCN females were bred from, some developed a marked loss of coat condition and development of dry skin, the occurrence of which did not seem to be dependent on litter number or size for example a female BCN mouse had two successful litters; however during the third pregnancy (aged 30 weeks) developed poor coat condition. After birth of this litter, the mother developed hair loss with skin reddening, particularly around the eyes and under the thin; the pups were nursed normally although swollen mammary glands were evident. These pups were under-developed and fed ravenously when weaned onto soft food. After weaning of the pups, the mother recovered coat condition and the skin inflammation healed. Analysis of the three litters showed that the weights of the pups in eath subsequent litter significantly decreased; with the pups in first litter almost double the weight of the third litter (Figure 32 which shows the time course of weanling weights for three sequential litters from a BCN female crossed with a BCN male; the first litter is denoted with open triangles, the second by open circles and the third by open squares. The "nurturing" phenotype was exhibited by this mouse after the birth of the third litter (see also Figure 310, E).

Statistical significance relates to the difference of the second or third litter from the first.

* = p �= 0.05, ** = p �= 0.005). Interestingly we have also observed a similar pattern of skin dryness and hair thinning in one third (3 out of 9) of the breeding BCN males and a single non-breeding female. Stunted size of BCN pups from BCN dams is potentially due to poor quality milk. Two pups from a WTxWT lifter and two pups from a BCNxBCN lifter were swapped and weights monitored. As can be seen from Figure 33, the wild type pups placed with the BCN dam are half the size of wild type pups, and the null pups placed with the wild type dam, grow at the same rate as the wild type pups.

EXAMPLE 17

Figure 34A shows immunoblot analysis of liver, kidney, lung and small intestine microsomes (20 jig protein per lane) from Cytb5 (WT) and Cytb5" (BCN) mice. Figure 34B shows densitometric analysis of liver immunoblot data (from A). Values are expressed as fold change normalised to wild-type expression. Bars are mean � SD from three samples of each genotype where hatched bars represent wild4ype data and white bars BCN data. Figure 34C shows Taqman analysis of hepatic gene expression. Data is normalised to lBs expression and expressed as fold change relative to wild-type expression. Bars are mean � SD from three samples of each genotype where hatched bars represent wild-type data and white bars BCN data. Figure 34D shows immunoblot analysis for expression of cytochrome P450-dependent monooxygenase components in liver microsomes (20 jig protein per lane) from one day old Cytb54" (WI) and Cytbt (BCN) pups as detailed in Experimental Procedures. Figure 34E shows representative reduced CO difference spectra of Cytht (WI) (solid line) and Cytbt (BCN) (dashed line) hepatic microsomes. Figure 34F shows cytochrome P450 oxidoreductase (POR) activities in hepatic microsomes as measured by the NADPH-dependent reduction of cytochrome c. Data is the mean � SD of three mice from each genotype run in duplicate. (* = p 0.05, ** = p �= 0.005, = p < 0.005. The complete deletion of cytochrome b5 was confirmed by western blotting of liver, lung. kidney and small intestine (Figure 34C). Densitometnc analysis or the concomitant changes in P450 levels show that deletion of cytochrome b5 caused significantly increased expression of POR, cytochrome b5 reductase, Cyp2a, Cyp2b/2b10 and Cyp3a in the liver (34B) which was transcriptional in origin (C) and present from birth (D). Extrahepatic cytochrome P450 expression was also affected but to a lesser extent. interestingly the upregulation of hepatic P450 and FOR levels translated approximately into a two fold increase in spectrally active P450 and POR activity (E and F). There was no change in testicular cytochrome P450 expression in the testes of BCN mice as shown by Western blotting analysis of testicular microsomal fractions.

EXAMPLE 18

Cytochrome P450 activity and steriod hormone synthesis was investigated. Figure 36 shows the effect of cytochrome b5 deletion on NADPH-dependent steroid hormone metabolism (Figure 36A). Assays were performed in triplicate using testicular microsomes from three mice of each genotype. Activities were determined using either progesterone (prog) or 17a hydroxy-progesterone (17a OH prog) as substrates.

F

Hatched bars (Cytb5" (wild-type)) and white bars (Cytbt (BCN)) represent the respective levels of metabolite production (andro androstenedione and test testosterone). Each bar represents mean � SD. * = p �= 0.05, ** = p �= 0.005, = p �= 0.005. The effect of cytochrome b5 deletion on in v!tm Cypi 7-hydroxylase activity was determined by measuring NADPH-dependent progesterone metabolism using testicular microsomes from wild-type and BCN mice. We observed a significant 2-fold reduction in metabolic rate in the absence of microsomal cytochrome b5 (A). When 1 7ct hydroxy-progesterone was used as a substrate to investigate the effect of cytochrome b5 deletion on Cypi 7-lyase activities, testicular HBN microsomes were found to have very low activity, exhibiting levels of androstenedione production >100-fold lower than wild-type (pc0.001) (A). Since both Cypll and 17HSD are both microsomal NADPH-dependent enzymes (C), incubations with 1 7a hydroxy-progesterone also yielded the metabolite testosterone (produced by the metabolism of androstenedione by I7I3HSD (C)). Not surprisingly, due to the difference in concentration of androstenedione present in the respective incubations, testicular microsomes from wild-type mice were 66-fold more active than HBN in the production of testosterone (A). Testicular testosterone content was also investigated, a reflection of this reduction in levels of testosterone can also be observed in vivo, as when testes from wild-type and HBN mice were analysed for testosterone content, BCN samples contained significantly less stored testosterone than wild-type (approximately 1.8 fold) (B).

EXAMPLE 19

Microsomes from kidney, liver, lung and small intestine of wild type and BCN mice were examined for NADPH-dependent activity towards a panel of commonly used fluorescent P450 probe substrates, namely members of the alkoxy-resorufin and alkoxy-4-trifluoromethylcoumarin families (below).

Table B Activities of mouse tissue microsomesa towards fluorescent probe compounds Liver Lung Small intestine Activity Activity Activity -. (pmollmln/mg)t' (pmoilmin/rng)b (pjyiol/minlmgt --compound WI BCN WT BCN WI BCN SR 38*1.2 15.7iLr 15,8�2.3 nd. 75�7.5 0.6�0.5 ER 10.1 � 1.9 9.3�3.7 2.4�03 nd. n.d. n.d.

MR 52.5�144 51.0�27.0 0.4*0.2 n.d. nd. n.d.

PR 2.2�0.2 5.2� 1.2 0.6�0.4 n.d. 0.40�0.02 0.70�0.02 SEc 275�46 65.1�6.2 447�4.4 1.5�1.7 17.5*20.0 005�0.09 EEc 245 � 59 30.5 � 5.4' 45.7 * 10.7 8.2 � 1.2 3.8 � 6.6 nd.

Mrc 181 �48 32.9 �6.9 89.7�4.0 0.8*1.3 20.1 �25.4 nd.

a assays were performed in triplicate for three individual mouse samples except for lungwhere a pool was used. No activities were detectedfor kidneyY = p �= 0.05, = p 0.005

P

In the liver microsome preparations, there were no differences observed in turnover of ER or MR between BCN and wild-type mice, while the BCN samples exhibited a significantly higher rate of metabolism of BR (4.1-fold) and PR (2.4-fold) than wild-type.

This increase in turnover of PB and BR, traditionally attributed to Cyp2b activity, probably reflects the 29-fold increase of Cyp2b protein observed in the liver (Figure 36).

With respect to the alkoxy-4-trifluorornethylcoumarins, the turnover in the BOW liver microsomes was significantly lower than wild type (BFC 4.2-fold; EFC 5.5-fold and MFO 5.5-fold lower respectively), indicating a severely reduced metabolic capacity. P450 monooxygenase activity was completely ablated in the lung samples when challenged with the alkoxy-resorufins, and profoundly reduced with the three 4-trifluoromethylcoumarins (BFC 29.8-fold; EFC 5.5-fold and MFC 115-fold lower). Activity towards BR, PR, BFC, EFC and MFC was detected in wild-type small intestine microsomes, which in the case of the alkoxy-4-trifluoromethylcoumarins and BFC was virtually or completely inhibited by the deletion of cytochrome b5. The rate of PR metabolism was, however, slightly increased by cytochrome b5 deletion. No activity towards these probe substrates was detected using either wild-type or BCN kidney microsomes. NADH-dependent metabolism of BFC, EFC and MFC was examined using liver microsomes from wild type and BCN mice. Rates in the wild type samples were approximately 25% that of the equivalent NADPH driven reactions, while no turnover was detected when the BCN microsomes were tested (data not shown).

P450 activities with cocktail substrates were investigated, activities NADPH-driven microsomal incubations were performed with probe drugs using liver, lung, kidney and small intestine microsomes (Figure 37A, chlorzoxazone 6-hydroxylase activity, Figure 37B midazolam 1'-and 4-hydroxylase activity, Figure 370 metoprolol 0-demethylase and I -hydroxylase activity, Figure 37D penacetin 0-deethylase activity, Figure 37E tolbutamide hydroxylase activity. Hatched bars represent Cytb5' (WT) and white bars Cytbt (BCN) samples, where each bar represents mean � SD from mouse tissue microsome preparations (Lv = liver; Ln = lung; K = kidney and SI = small intestine, * = p �= 0.05, = p �= 0.005, = p �= 0.005). Chlorzoxazone metabolism was observed in wild-type liver, lung and kidney microsomes, while in the equivalent BCN tissues activities were significantly reduced (4-, 2-fold for liver and lung respectively) or completely inhibited (kidney). Midazolam was metabolised to two metabolites (1' hydroxy-and 4-hydroxy midazolam) by all four tissues examined. In each case the rates of turnover achieved by the BON samples were much lower than in the wild-type preparations, reaching statistical significance in the liver and kidney for I' hydroxy midazolam production, and in the liver for 4-hydroxy midazolam. Metoprolol was converted to 0-desmethyl metoprolol by all samples tested. Rates of turnover were significantly reduced by cytochrome b5 deletion in the liver and kidney, while no effect was observed in the lung or small intestine samples. 1-hydroxy-metoprolol was produced from liver, kidney and small intestine but not from lung microsome incubations, Similarly to 0-desmethyl metoprolol production, formation of this metabolite was significantly decreased in the liver and kidney samples of BCN mice. Metabolism of phenacetin to acetaminophen occurred in liver, lung and kidney, where interestingly, rates were significantly elevated in the livers of BCN mice (p<0.05), probably due to the increase in CyplA and Cyp2a proteins in the liver, while in the kidney and lung, ablation of cytochrome b5 had no effect on activity. Finally, metabolism of tolbutamide was only detected in liver and kidney microsomes, however in both cases, BCN samples showed decreased activity as compared to wild-type.

The apparent kinetic parameters for the metabolism of chlorzoxazone, metoprolol, midazolam, phenacetin and tolbutamide are detailed below. For chlorzoxazone, midazolam and metoprolol, the Km was unchanged and the Vm markedly decreased in the BCN samples compared to wild-type. The Km for tolbutamide in the BCN samples was lower than wild type, and the Vmax was again reduced. This is similar to what was observed in HBN samples. BCN samples showed no change in Km towards phenacetin, however Vm was increased, again attributable to the increase levels of hepatic CyplA and Cyp2a proteins.

Table 9 Kinetic analyses5 summary of metabolism of probe drugs by hepatic microsomes from BCN mice and wild-type controls.

Wild Type liver microsomes BCN liver microsomes -Substrate Metabolito K,,, (pM) (pmo/m I(pM) (pmoI/rni9_ chlorzoxazone 6-OH Chlorzoxazone 61 4 � 7.6 527 �24 530 � 5.6 90 9 � 9.9 Midazotam 1 -OH Mldazolam 1.9 �0.2 623 � 22 2.8 � 0.3 7.3�0.3 4-OH Midazolam 21.8� 1 0 324 � 6 18.4�0.8 162 � 3 Phenacetin Acetamhiophen 5.6 � 1 0 133.4�6 1 4 1 � 0.7 76,9 � 3.1 Metoprolol a-OH Metoprolol 66.5 � 8.8 1004 � 43 73.4 � 12.4 310 * 11 0-DesM Metoprolol 59.0 � 7.7 852 � 36 78.5 � 20.3 580 � 62 Tolbutamide OH tolbutamlde 3650 � 636 60.3 � 6.4 977 � 475 17.8 � 4,3

EXAMPLE 20

Recapitulation of BCN cytochrome P450 activity by addition of exogenous cytochrome b5 was investigated by adding back exogenous cytochrome b5 (E.coIi membranes expressing murine cytochrome b5) to hepatic and lung microsomes and measuring activity towards BFC and BR (Figure 36) . In hepatic microsomes, addition of cytochrome b5 to wild-type incubations had little effect on the rate of metabolism of either substrate, indicating that in the liver, the level of cytochrome b5 is not rate-limiting for these reactions (A and B). In contrast, when exogenous cytochrome b5 was added to hepatic BCN incubations, a concentration dependent increase in actMty was observed, resulting in activities 2.3-and 7.8-fold higher than wild-type for BFC and BR respectively.

This "super induction" of turnover can be attributed to the increased levels of Cyp2b (BR and BFC activity) and Cypl a, Cyp2a and Cyp3a (BFC) present in the livers of BCN animals (A and B). In complete contrast to this, the activities of wild-type lung microsomal samples showed marked stimulation of activity when supplemented with exogenous cytochrome b5 (C and D). This was most pronounced when BR was the substrate -giving a 6.8-fold increase over basal activity. Addition of cytochrome b5 to BCN lung incubations increased the activities to levels comparable to the cytochrome b5 supplemented wild-type samples. Again this increase was most dramatic for BR 0-dealkylation. Together these data suggest that in the murine lung, for these activities mediated by (at least) the Cyp2b subfamily members, the level of cytochrome b5 is limiting. Comparison of expression of Cyp2b and cytochrome b5 protein in the liver and lung provide further evidence that this is indeed the case. Lung microsomes contain at least 10 times more Cyp2blO protein than liver; however have approximately 3-fold less cytochrome b5.

EXAMPLE 21

The pharmacokinetics of paclitaxel and cyclophosphamide were investigated. Paclitaxel (Figure 40 and Table 10) shows that BCN mice have an altered pharmacokinetic profile describing the elimination of paclitaxel as compared to wild-type, reaching a higher Cm indicating slowed metabolism. Results with cyclophosphamide (Figure 40 and Table 10) show that BCN mice exhibit an altered pharmacokinetic profile describing the elimination of cyclophosphamide as compared to wild-type, with a higher Cm and larger AUC than wild type indicating a slowed metabolism and higher exposure to parent drug.

Table 10 Summary of pharmacokinetic data on drugs administered orally to BCN and wild-type controls Drugs PK parameters --Units BCN -WT p-values cyclophosphamicie Half Life -mm 60.1 *2.52 42.5 �5.17 0016 --cmax uglrnt 26.4 �2.72 16.99 *1.62 0017 clearance mlImin/kg 82.5 �9.93 134.7 �24.53 0.084 AUc mineug/mI 2589 �366.95 1659.2 �287.33 0.081 Paciltaxel Half life mm 149.2 �24,69 147.6 �37.09 0.973 Cmax ug/mI 2.2 �0.54 1.9�0.40 0.661 AUG mllmlnllcg 292.4 �67.42 31 8.3 �8743 0.793 _Clearance min*ug4pi,_j7 �20,09 51.2�111 0.762 Looking at cocktail drugs (Figure 39 and Table 11), in order to confirm the in vitro results obtained with the cytochrome b5 complete null; chlorzoxazone, metoprolol, midazolam, tolbutamide and phenacetin were administered orally as cocktail to wild-type and BCN.

Previous work with these drugs in wild-type mice, individually and as a cocktail, had demonstrated that simultaneous delivery did not result in altered phamiacokinetics as compared to single administration (not shown).

Table 11 Summary of pharmacokinetic data on drugs administered orally to BGN and wild-type controls.

Drugs Plc Units BCN WT p-values _parameters Midazolam AUG min*uglml 35.3 � 14.4 10.9 � 3.4 0.107 cmax ug/mI 0.3� 0.13 0,11 � 0,035 0.139 Clearance mI/mm/kg 96.0 � 23.4 281.3�46.7 0.014 Terminal half mm 91.0� 6.1 78.2� 54 0.215 life ___________________________________________________ Phenacetln AUG mintug/ml 5.8 � 0.91 3.3 � 0.48 0.039 Gmax ug/mI 0.14�0.032 0.098�0.014 0.219 clearance mI/mm/kg 867.2 � 102.5 1333.0 � 136.5 0.035 Terminal half mm 171.5�23.7 307.4 � 44.5 0.041 life _____________________ _____________________ Metoprolol AUG mlnug/ml 0.76 � 0.24 0.22 � 0.05 0.043 Grnax ug/mI 0.008 � 0.003 0.002 � 0 001 0.101 Clearance L/min/kg 2.3 � 0.5 8.4 � 0.9 0.001 Terminal half mm 350.2 � 68 1 146.2 � 29.8 0.019 life __________________________ ____________________ chlorzoxazone AUG mmn*ugfml 7.3 � 0.7 3.4 � 0.4 0.001 Gmax uglml 0.096�0.019 0.103�0 015 0.723 clearance mI/mm/kg 627.2 �64.9 1387.4 � 124.1 0.002 Terminal half mm 92.3 � 33.9 44.4 � 8.9 0.171 life _______________________ Tolbutamide AUC mmn*ug/ml 247.4 � 16.5 213.2 � 18.3 0.218 Cmax ug/ml 0.88 � 0.11 0.63 � 0.06 0.067 Clearance mI/mm/kg 12.9 � 0.6 10.8 � 2.0 0.407 Terminal half mm 324.8 � 40.1 640.9 � 90.3 0.022 ___________ life _________________________ Following dosing of the drug cassette to wild-type and BGN mice, quantification of individual drugs and their metabolites was carried out by LC-MSIMS. The pharniacokinetic data for each drug is summarised in Table 9, and the elimination profiles of the drugs are shown in Figure 37. Following oral administration of the drug cocktail, all the drugs show differences in their elimination profiles when BCN mice were compared to their respective controls. For midazolam a significant decrease of clearance from 281.3 to 96.0 was observed in BCN. This alteration of clearance has lead to an increase of AUG by 3 fold and a slight increase of Cmax and terminal half life. As observed for midazolam, chlorzoxazone showed a significant decrease of clearance from 1.3 to 0.6 L/min/kg and an increase of AUC from 3.4 to 7.3 min*pg/ml, whereas Cmax was not altered. Similarly to midazolam, metoprolol showed a significant decrease of clearance by a 3 fold from 8.4 to 2.3 Llminlkg. Additionally, in this case, significant increase of AUC, Omax and terminal half life from 0.22 to 0.76 min*pg/ml, 0.002 to 0.008 pg/mI and 146 to 250 mm were respectively observed. In contrast to midazolam and metoprolol, phenacetin showed a less pronounces alteration of the clearance from 1.3 to 0.87 Liminlkg. However AUC, Cmax and terminal half life were all significantly affected by the deletion of cytochrome b5. AUC and Cmax were increased from 3.3 to 5.8 rnin*pg/ml and 0.096 to 0.14 pg/mI, whereas terminal half life decreased from 307 to 171 mm. For tolbutamide, the drug having the longer half life and one of the shorter clearances, the complete deletion of cytochrome b5 has not affected the clearance, AUG or Cmax. Only the terminal half has decreased significantly from 640 to 324 mm.

EXAMPLE 22

Plasma and hepatic fatty acid profile analysis were investigated results are shown in Tables 12, 13 and 14 which describe the fatty acid profiles of plasma, liver and liver fractions respectively, where significant increases and decreases of many fatty acid species were observed on deletion of cytochrome b5. Expression levels of SCDI and Ncb5OR, a protein that has recently been implicated in supporting electron transfer to SCDI, were not affected by the deletion of microsomal cytochrome b5 (Figure 41).

Table 12 Plasma fatty acid profile analysis, fatty acid profile analysis was carried out on the plasma of 10 and 16 week old wild-type and BCN mice. Data is presented as the mean � S.D. where n = 3. Values expressed as pg fatty acid/mI plasma.

weeks 26 Weeks -Fatty Acid Cm: -Wi/i BC? p vah Wild-i) BCN p va/i ______ Typ _______ C14:O$lyr&/call.5� 13.7�4.6 0.52 17.3�3.7 12.8�2.6 0.17 2.9 C16:OPalmlticacid 419� 516�116 0.38 739�136 629�71 0.29 C18:OStearlcacld 159�31 195�30 0.23 204�42 312�74 0.09 020:0 Arachidic acid 2.9 � 2.3 1.9 � 0.5 0.49 3.2 � 0 9 6.9 � 0.6 0.004 ** 022:0 Behenlc acid 2.9 � 2.4 2.4 � 0.8 0.78 4.9 � 2.5 9.0 � 5.3 0.28 C24:OLlgnoceric 1.6�0,6 1.9�0.4 0.47 2.3�0.4 2.1 �0.7 0.69 acid C16:1 nlPaimitoieic 54.4� 48.7�15.7 0.75 139� 29 92�10 0059 acid 24.4 C18:1 n9 Oleic acid 276 � 91 321 � 68 0.53 476 � 82 562 � 123 0.37 C18:1 nlVaccenic 39.4� 343�5.8 0.56 73.2�10.1 1042�18.3 0.06 acid 12.6 C20:1 ri9Gondolc 8.8�4.3 8.6�5.6 0.97 10.4�3.1 14.7�3.9 0.21 acid C20:1 n7Gadoleic 1.0�0.6 0.7�0.3 0.44 3.3�1.3 4.9�1.9 0.29 acid C22:1 n9 Erucic acid 1.1 � 0.7 0.9 � 1.0 0.79 n.d. 0.56 � 0.96 0.37 C24:1 n9 Nervonic 2.9 � 0.7 4.0 � 0.8 016 8.8 � 2,2 6.0 � 2.9 0.26 acid C18:2n6Linoieic 384+ 543+110 0.19 789�146 867�133 0.53 acid 134 C18:3 n6 a-Linoienlc 5 6 + 2.4 13.7 + 6.7 0.12 27.2 � 7.5 6.7 � 0.6 0.009 * acid C20:2 n6 2.0 + 0.7 1.6 + 0.5 0.45 7.9 � 1.6 10.6 � 3.6 0.31 Elcosadlenolc acid C20:3 n6 Dihomo-y -18 8 � 11.2 � 3.5 0.26 20.8 � 3.4 52.8 � 4.4 0.0006 iinoienic acid 9.3 C20:4n6 264� 338�137 0.52 499�78 420�186 0.53 Arachidonic acid 114 C22:4 n6 Adrenic 3.0 � 1.6 2.9 � 1.4 0.92 4.5 � 0.6 5.4 � 1 2 0.32 acid C22:5 n6 Osbond 4.9 � 1.9 3.6 � 2.0 0.45 7.8 � 1.0 7.3 � 0.6 0.53 acid C18:3 n3 y -Linoienic 5.4 � 3.3 5.9 � 1.6 0.80 13.4 � 4.6 14.5 � 5.9 0.81 acid C18:4 n3 Parinaric n.d. n.d -0.98 � 1.69 1.5 � 0.8 0.69 acid C20:4 n3 rtd. nd. -n.d. 0.69 � 1.20 0.37 Eicosatetraenoic acid C20:5 n3 4.4 � 2.9 6.7 � 3.0 0.39 9.5 � 2.9 9.8 � 1.6 0.86 Timnodonic acid C22:5 n3 5.0 � 3.6 6.1 � 3.1 071 &6 � 1.3 9.0 � 1.6 0.78 Clupanodonic acid C22:6 n3 83.1 � 128 � 67 0.40 160 � 28 155 � 51 0.89 Docosahexaenoic 49.2 acid Total Saturated FAa 600 + 734 + 149 0.35 976 � 185 977 � 74 0.99 Total 396+ 432+95 072 725�126 807�159 0.52 Monounsaturated 135 FAa Totain-OPUFAs 683+ 914+257 0.31 1360�235 1370�237 0.95 Total n-3 PUFAS 979 + 147 + 75 O42 192 � 36 190 � 52 0.96 Table 13 Liver fatty acid profile analysis.

Fatty acid profUe analysis was carried out on liver of 10 and 16 week old wild-type and BCN mice. Data is presented as the mean � S.D. where n 4 (wild-type 10 weeks), n = 6 (BCN 10 weeks) and n = 3 (16 weeks). Values expressed as mg fatty acidlg tissue.

weeks.16 Weeks FattyAcid Group Wik BC) p Va) Wili BCJ p val _________Tp T)p Cf4:OMyict/cack 0.26 � 0.15 � 0.16 0,38 � 0.20 � 0.09 0.14 0.09 0.11 0.08 C16:OPalmiticacid 12.6� 8,3�2.9 0.08 14.8� 11.5� 0.24 3.7 2.9 2.9 C18:OStearlcacld 3.3�0.2 3.3�0.7 0.79 2.3�0.1 3.4�0.5 0.017* C20:O Arachidic acid 0.03 � 0.03 � 0.75 0.04 � 0.15 � 0.06 0.01 0.01 0.006 0 08 C22:O Behenic acid 0.04 � 0.04 � 0.91 0.03 � 0.08 � 0.12 0.3 0.02 004 0.03 C24:O Lignoceric acid 0.03 � 0.03 � 0.78 0.02 � 0.03 � 0.80 0.01 0.004 0.02 0.006 C16:1 n7 Palmitoieic 2.2 � 11 0.73 � 0.019 * 3,7 � 0.2 1.8 � 0.7 0.08 acid 0.44 C18:1 n9 Oleic acid 13.7 � 9.3 � 5.9 0.29 13.9 � 15.4 � 0.72 6.5 2.6 6.4 C18:1 n7Vaccenicacid 1.3�0.5 0.78� 0.05* 1.6� 0.3 2.3�0.9 0.29 0.28 C20:1 n9 Gondoic acid 0.19 � 0.16 � 0.59 0.24 � 0.48 � 0.13 0.08 0.08 0.03 0.22 C20:1 n7Gadoleicacid 0.04� 0.02� 0.098 0.08� 0.16� 0.15 0.02 0.01 0.03 0.07 C22:1 n9 Erucic acid n.d n.d. n.d. n.d, 0.05 � 0.09 0.04 C24:1 n9 Nervonic acid 0.06 � 0.05 � 0.54 0 09 � 0.04 � 0.31 0.01 0.02 0.07 0.02 C18:2 n6 Linoleic acid 10.6 � 9.6 � 4.5 0.71 15.1 � 9.9 � 3.3 0.09 3.4 2.4 C18:3 n6 a-Llnolenic 0.34 � 0.19 � 0.16 0.64 � 0 10 � 0.001 *** acid 0.17 0.11 009 0.04 C20:2 n6 Eicosadlenoic 0.06 � 0.07 � 0.45 0.20 � 0.25 � 0.30 acid 0.02 0.02 0.04 0.06 C20:3 n6 Dihomo-y -0.36 � 0.22 � 0.07 0.23 � 0.68 � 0.007 linolenic acid 0.15 0.07 0.02 0.15 C20:4 n6 Arachldonic 4.4 � 0.3 3.7 � 0.5 0.05 * 3.6 � 0.2 3.3 � 0.7 0.43 acid C22:4 n6 Adrenic acid 0.19 � 0.14 � 0.20 0.14 � 0.17 � 0.37 0.09 0.04 0.04 0.04 C22:5 n6 Osbond acid 0.27 � 0.10 � 0.11 0.16 � 0 11 � 0.07 0.23 0.02 0.03 0.02 C18:3 n3 y -Llnolenic 0 35 � 0.20 � 0.19 0.55 � 0.33 � 0.25 acid 0.19 0.14 0.22 0.15 C18:4 n3 Parirtaric acid 0.06 � 0.03 � 0.09 0.20 � 0.04 � 0.03 * 0.04 0.02 0.08 0.03 C20:4 n3 nd. n.d. n.d. n.d. 0.03 � 0.008 ** Eicosatetraenolc acid 0.01 C20:5 n3 Timnodonic 0.08 � 0.09 � 0.90 0.13 � 0.09 � 0,16 acid 0.02 0.03 0.03 0.03 C22:5n3Clupanodonic 0.21 � 0.21 � 0.98 0.20� 0.19� 0.77 acid 0 06 0.09 0.03 0.05 C22:6n3 a2�o.2 2.9�06 053 2.5�0.1 t9�0.6 0.21 Docosahexaenoic acid TotalSaturatedFAs 16.2� 11.9� 0.11 17.7� 15.5� 0.46 38 3.7 3.1 3.4 Total Monounsaturated 17.5� 11.3� 0.21 19.9� 20.9� 0.87 FAa 8.2 6.8 4.2 8 6 Totaln-6PUFAs 16.2� 14.0� 0.49 20.0� 14.5� 0.11 3.7 5.1 2.2 4.2 Total n-3 PUFAs 3.9 � 0.5 3.5 � 0 8 0.45 3.6 � 0.2 2 7 � 0.9 0.16 Table 14 Uver fraction fatty acid profile analysis.

Cholesterol ester, triacylglycerol (TAG) and polar lipid fractions were prepared from the livers of 10 week old wild-type and BCN mice and subjected to fatty acid profile analysis.

Data is presented as the mean � S.D., where n = 3. Values expressed as fold change compared to wild-type mice.

Fatty Acid Gmup Choleste TAGS Polar Lip p Va esters va va C14:OMyristicacA 0.98 � 0 59 0.95 0.77 t 0.08 0.22 0.61 � 0.06 0.04 C16:O Palmltlc acId 1.3 � 0.3 0.23 0.96 � 0.06 0.37 0.94 � 0.004 0.02 * C18:OStearicacid 1.9�0.9 0.15 1.4�0.1 013 1.1�0.02 0.002 ** C20:O Arachidic acId 1.4 � 1.1 067 1 8 � 0.4 0.18 1.3 � 0.05 018 C22:O Behenic acid 1.9 � 0.9 0.29 1.2 � 1.1 0.87 0.85 � 0 08 0.44 C24:O Lignocenc acid 1.7 � 0.9 0.43 n.d. -1.0 � 0.2 1 C16:1 n7 Palmltoleic 0.57 � 0.14 0.04 0.73 � 0.05 0.02 0,70 � 0.06 0.03 * acid * * C18:1 n9Oleicacid 0.99 �0.18 0.97 1.0�0.05 0.49 1.1 � 0.03 0.01 * C18:1 nl Vaccenic acid 1.1 � 0.2 0.57 1.0 � 0.1 0.85 0.89 � 0 05 0.08 C20:1 n9Gondolcacid 1.5� 1.6 0.68 1.4�0.3 0.12 1.1 �0.03 0.003 ** C20:1 fT Gadoleic acId 2.0� 1.8 0.38 1.2 � 0.4 0.69 1.05 � 0.15 0.72 C22:1 n9 Erucic acid n.d. -2.0 � 0.6 0.20 nd. -C24:1 n9Nervonicacid n.d. n.d. -079�0.06 0.14 C18:2 n6 Linoleic acId 0.97 � 0.1 0.69 0.96 � 0.09 0.58 1.1 � 0.2 0.55 C18:3 n6 a -Linolenic 0.32 * 0.54 0.10 0.55 � 0.09 0.19 0.81 � 0.08 0.07 acid C20:2 n6 Eicosadienolc n.d. -1.6 � 0 9 0.33 0.98 � 0.11 0.91 acid C20:3 n6 Dihomo-y -1.1 � 0.6 080 1.2 � 0.2 0.09 1.2 � 0.2 0.31 linolenic acid C20:4 n6 Arachidonic 0.61 � 0.27 0.07 1.4 � 0.1 0.07 0.93 � 0.07 0.35 acid C22:4 n6 Adrenic acid nd. -1.3 � 0.1 0.20 1.4 � 0.6 0.32 C22:5 n6 Osbond acid nd. -0.77 � 0.09 0.59 0.83 � 0.23 0.49 C18:3 n3 y -Llnolenic 0.61 � 0.47 0.24 082 � 0.13 0.09 1.2 � 0.6 0.57 acid C18:4 n3 Parinaric acid n d. -0.24 � 0.41 0.17 n.d. - C20:4 n3 n.d. -0.8 � 0.2 0.35 nd. -Elcosatetraenoic acid C20:Sn3Timnodonic n.d. -1.1 �0.3 0A9 13�0.1 0.14 acid C22:5 n3 Clupanodonic nd. -0.99 � 0.29 0.96 1.1 � 0.07 0.48 acid C22:6 n3 0.49 � 0.29 007 1.1 � 0.5 0.82 0.98 � 0.04 0.76 Docosahexaenolc acid Total Saturated FAa 1.4 � 0.4 0.18 0.99 � 0.04 0.84 0.99 � 0.003 0.81 Total Monounsaturated 0.89 � 0.13 028 1.0 � 0.05 0.94 1.0 � 0.03 0.53 FAa Total n-6 PUFAs 0.88 � 0.09 0.16 1.0 � 0.09 0.92 1.0 � 0,03 0.90 Total n-3 PUFAs 0.55 � 0.36 0.11 0.97 � 0.33 0.92 0.99 � 0,04 0.94

Claims (39)

1. CLAIMS1. A non-human transgeriic animal comprising a deletion of cytochrome b5 or an offspring thereof or tissue or cells derived from such transgenic non-human animals or their progeny in which cytochrome b5 is deleted.
2. 2. A non-human transgenic animal according to claim I wherein the deletion is either a broad/complete deletion or is a tissue specific deletion.
3. 3. A non-human transgenic animal according to either claim 1 or 2 wherein the tissue specific deletion is in the liver, lung, heart, kidneys, brain, ovaries or testicles.
4. 4. A non-human transgenic animal according to any preceding claim wherein the tissue specific deletion is hepatic tissue.
5. 5. A non-human transgenic animal according to any preceding claim wherein the tissue specific deletion is a conditional deletion.
6. 6. A non-human transgenic animals according to any preceding claim wherein the animal is selected from the group comprising monkeys, swine, dogs, cats, rabbits, guinea pigs, hamsters, gerbils rats and mice.
7. 7. A non-human transgenic animal according to claim 6 wherein the animal is a mouse.
8. 8. A non-human transgenic animal according to any preceding claim in every stage of development, including embryonic, neonatal, juvenile, adolescent and adult.
9. 9. A transgenic non-human animal whose genome has integrated therein a replacement targeting vector nucleic acid construct comprising a transgene of DNA containing exons 2-5 of the cytochrome b5 gene
10. 10. A targeting vector nucleic acid construct comprising a transgene of DNA containing exons 2-5 of the cytochrome b5 gene.
11. 11. A nucleic acid construct according to claim 9 which is included in a cassette, flanked by same-orientated IoxP sites and optionally containing neomycin as a selectable marker which is optionally driven by a herpes simplex thymidine kinase promoter.
12. 12. A transgenic knockout non-human animal having somatic and gerrnline cells comprising a chromosomally incorporated transgene wherein at least one allele of a cytochrome b5 gene is disrupted by the transgene such that the expression of cytochrome b5 is inhibited.
13. 13. A transgenic knockout non-human animal according to claim 12 wherein both alleles are disrupted so that the non-human transgenic animal is homozygous for the disrupted allele.
14. 14. A method of producing a non-human transgenic animal comprising a broad or tissue specific deletion of cytochrome b5 the method comprising introducing a targeting vector according to either claim 10 or 11 into a non-human zygote or a non-human embryonic stem cell, generating a transgenic non-human animal from said zygote or embryonic stem cell, selecting non-human transgenic animals carrying a target cytochrome bS allele and crossing said animals to produce a transgenic non-human animal having tissue specific or complete cytochrome b5 knockout.
15. 15. A method according to claim 14 wherein the genotype of the broad range or complete knockout transgenic non-human animal is homozygous for cytochrome b5, (cyt b5) and wherein the genotype for the conditional deletion tissue specific non-human transgenic animal is cyt b5 loX/lOx:: Cre ALB
16. 16. Use of a non human transgenic animal or tissues or cells derived therefrom according to any one of claims 1 to 9 or 12 to 13 as an in vivo screen as an in vitro screen to determine any one or more of the following parameters: (i) role of cytochrome b5 in drug/product disposition; (ii) routes of Phase II drug/product disposition of parent compound; (iii) pathways of drug/product disposition; (iv) role of drug transporters in drug uptake; (v) pathways of chemical toxicity; (vi) role of cytochrome b5 in hormone function and regulation; (vii) role of cytochrome b5 in normal metabolic processes/homeostasis; and (viii) role of cytochrome b5 in pathogenesis of disease.
17. 17. Use of a non human transgenic animal or tissues or cells derived therefrom according to any one of claims I to 9 or 12 to 13 as an in viva screen to investigate any one or more of the following events or parameters: (I) pharmacological potency of agents subject to first pass metabolism; (ii) occurrence and rate of extrahepatic metabolism; (iii) rate of hepatic metabolic clearance as a determinant in product/drug (iv) distribution and pharmacokinetics; (v) facilitation of selection of lead product/compound based on in viva parameters; (vi) assessment of relevance and occurrence of pharmacologically active metabolites; (vii) distinguish between toxicity due to product activation of other mechanism (viii) establish role of cytochrome b5 as a rate limiting step in drug/compound disposition and; (ix) study drug/drug interactions due either to cytochrome b5 or drug transporter effects.
18. 18. Use of a non human transgenic animal or tissues or cells derived therefrom according to any one of claims I to 9 or 12 to as an in vitro screen to investigate any one or more of the following eventslparameters: (i) role of transporters in product/d rug uptake and efflux; (ii) identification of metabolites produced by cytochrome b5; (iii) evaluate whether drugs/compounds are cytochrome b5 substrates and; (iv) assess drugldrug interactions due to cytochrome b5 effects;
19. 19. A method assessing any one of the aforementioned parameters or events as defined in any one of claims 16 to 18, comprising exposing a transgenic non-human animal of the present invention or tissue or cells derived therefrom and assessing a selected parameter and comparing the outcome with that obtained from a similarly exposed wild type non-human transgenic non-human animal that has cytochrome b5 function.
20. 20. A transgenic non-human animal model for steatosis comprising a broad or tissue specific deletion of cytochrome b5 or an offspring thereof or tissue or cells derived from such transgenic non-human animals or their progeny in which cytochrome b5 is deleted.
21. 21. A model according to claim 20 for screening agents that prevent steatosis or identifying agents that exacerbate steatosis or for studying mechanisms of acquired or predisposition towards steatosis.
22. 22. A transgenic non-human animal model for methaemoglobinaemia comprising a broad or tissue specific deletion of cytochrome b5 or an offspring thereof or tissue or cells derived from such transgenic non-human animals or their progeny in which cytochrome b5 is deleted.
23. 23. A model according to claim 22 for screening agents that prevent methaemoglobinaemia or identifying agents that exacerbate methaemoglobinaemia or for studying mechanisms of acquired or predisposition towards methaemoglobinaemia.
24. 24. A transgenic non-human animal model for perturbations in fatty acid and triglyceride synthesis comprising a broad or tissue specific deletion of cytochrome b5 or an offspring thereof or tissue or cells derived from such transgenic non-human animals or their progeny in which cytochrome b5 is deleted.
25. 25. A model according to claim 24 for screening agents that prevent perturbations in fatty acid and triglyceride synthesis or identifying agents that exacerbate perturbations in fatty acid and triglyceride synthesis or for studying mechanisms of acquired or predisposition towards perturbations in fatty acid and triglyceride synthesis.
26. 26. A transgenic non-human animal model for adrenal insufficiency comprising a broad or tissue specific deletion of cytochrome b5 or an offspring thereof or tissue or cells derived from such transgenic non-human animals or their progeny in which cytochrome b5 is deleted.
27. 27. A model according to claim 26 for screening agents that prevent adrenal insufficiency or identifying agents that exacerbate adrenal insufficiency or for studying mechanisms of acquired or predisposition towards adrenal insufficiency.
28. 28. A transgenic non-human animal model for testosterone insufficiency or infertility comprising a broad or tissue specific deletion of cytochrome b5 or an offspring thereof or tissue or cells derived from such transgenic non-human animals or their progeny in which cytochrome b5 is deleted.
29. 29. A model according to claim 28 for screening for agents that prevent testosterone insufficiency or infertility or for studying mechanism of acquired or a predisposition towards testosterone insufficiency or infertility.
30. 30. A transgenic non-human animal model for changes in skin composition and physiology comprising a broad or tissue specific deletion of cytochrome b5 or an offspring thereof or tissue or cells derived from such transgenic non-human animals or their progeny in which cytochrome b5 is deleted.
31. 31. A model according to claim 30 for screening for agents that prevent skin changes and skin physiology or for studying the underlying mechanism of changes in skin composition and physiology.
32. 32. A transgenic non-human animal model for ichthyosis comprising a broad or tissue specific deletion of cytochrome b5 or an offspring thereof or tissue or cells derived from such transgenic non-human animals or their progeny in which cytochrome b5 is deleted.
33. 33. A model according to claim 32 for screening for agents that prevent ichthyosis or for studying the underlying mechanism of acquired or a predisposition towards ichthyosis.
34. 34. A transgenic non-human animal model for nutritional weaning comprising a broad or tissue specific deletion of cytochrome b5 or an offspring thereof or tissue or cells derived from such transgenic non-human animals or their progeny in which cytochrome b5 is deleted.
35. 35. A model according to claim 32 for screening for agents that affect the constituency of maternal milk during weaning or for studying the underlying mechanism of nutritional value of maternal milk during weaning.
36. 36. A model according to any one of claims 20 to 35 further including any one or more of the features of claims 2 to 9 or 12 to 13.
37. 37. A non-human transgenic animal comprising a cross of a broad or tissue specific deletion of cytochrome b5 with another non-human transgenic animal of the same species comprising a null expression of an enzyme associated with drug metabolism.
38. 38. A cross according to claim 37 wherein the null expression of an enzyme associated with drug metabolism is a Mdrl or Mdr2 or hepatic null cytochrome P450 reductase model.
39. 39. A cross according to either claim 37 or 38 further including any one or more of the features of claims 2 to 9 or 12 to 13.