WO2010010343A1

WO2010010343A1 - Stabilisation

Info

Publication number: WO2010010343A1
Application number: PCT/GB2009/001812
Authority: WO
Inventors: Bhabatosh Chaudhuri; Neill James Horley; Glen Justin Peter Mccann; Jacob Varghese Biboy
Original assignee: De Montfort University
Priority date: 2008-07-22
Filing date: 2009-07-22
Publication date: 2010-01-28
Also published as: GB0813384D0

Abstract

A dried composition comprising a membrane-associated recombinant cytochrome P450 (CYP) enzyme, wherein the CYP enzyme is stable at 21 °C for a period of at least 10 days, and preferably for at least 3 months. A method of storing a recombinant membrane-associated CYP enzyme, the method comprising providing a recombinant membrane-associated CYP enzyme, freeze-drying the CYP enzyme, and storing the dried CYP enzyme at room temperature.

Description

STABILISATION

The present application relates to cytochrome P450 enzymes, and in particular to recombinant membrane-associated cytochrome P450 enzymes that are stable at room temperature, and to methods for preparing and storing them.

The cytochrome P450 enzymes (CYPs) are a diverse family of proteins containing a single iron protoporphyrin IX prosthetic haem group. These enzymes catalyse a variety of reactions, including the hydroxylation of alkanes to alcohols, conversion of alkenes to epoxides, arenes to phenols, sulphides to sulphoxides and sulphones, and the oxidative split of C-N, C-O, C-C or C-S bonds. Many CYPs require another enzyme for catalysis which acts to donate electrons to the CYP; for example, microsomal CYPs require the presence of cytochrome P450 reductase (CPR) (McGinnity & Riley, 2000; Bruno & Njar, 2007).

At least 57 human CYPs are known (see Table 1 in Guengerich, 2006), which are encoded by 57 individual genes in eighteen distinct gene families (Frye, 2004). Human CYPs are primarily membrane-associated proteins, located either in the inner membrane of mitochondria or in the endoplasmic reticulum of cells. In the liver, CYPs metabolise xenobiotics, drugs and toxic compounds as well as metabolic products such as bilirubin. CYPs are also present in many other tissues of the body including the mucosa of the gastrointestinal tract, and play important roles in hormone synthesis and breakdown, cholesterol synthesis and vitamin D metabolism (Frye, 2004).

The majority of all marketed drugs are primarily cleared from the body by CYP- dependent metabolism, with CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A4 being responsible for around 95% of drug metabolism (Spatzenegger & Jaeger, 1995), making CYPs a major area of research for the pharmaceutical industry (Bertz, 1997). Further understanding of the unique functions and characteristics of CYPs is essential to the marketing of safer drugs with fewer side effects, predictable pharmacokinetic properties and quantifiable drug interactions, and thereby has significant health and economic benefits (McGinnity & Riley, 2001 ; Guengerich, 2006). Indeed, reaction phenotyping, primarily with regard to human drug-metabolising enzymes that exhibit genetic polymorphisms, for example CYP2D6 and CYP2C19, now is a standard component of the in vitro profiling of all drug candidates entering development (Baillie, 2008). As a result, the supply of CYPs to global pharmaceutical and research and development groups is critical for drug screening and pre-clinical submissions. CYPs may be obtained from a deceased person's liver or other tissues. However, this has inherent problems in relation to ethics, availability, cost and disease states/injury during death (healthy livers should be transplanted). Moreover, certain CYPs may be missing due to ethnic origin or may not be induced to high enough levels. The transmission of infectious materials (e.g. Hepatitis B) and batch-to-batch variation are also a concern.

Lotlikar et al (1976) Journal of Biochemistry 160, 401-404, describe the need for phospholipids for P450 function. Lotlikar et al freeze-dried hamster liver microsomes in water, optionally extracted them with butan-1-ol, and homogenised them in 0.25M sucrose with 10% glycerol. Both preparations were found to be stable for 1-2 weeks when stored at -15°C.

Aikawa et al (1976) Japanese Journal of Pharmacology 26, 227-232, describe the effect of lyophilisation on the storage of the 9,000 x g supernatant liver fraction (S9 fraction). The S9 fractions contained soluble fractions and membranes from livers of rat, mouse, guinea pig, rabbit and dog. When stored under reduced pressure at -20⁰C for at least one month, the CYP activity of the S9 fraction was stable, retaining 86%-96% of the activity of fresh samples.

Kamataki & Kitagawa (1974) Journal of Pharmacology 24, 195-203, describe the effect of lyophilisation on the storage of rat liver microsomes and S9 fractions. Preparations were stably stored at -20⁰C anaerobically for at least six months. Kamataki & Kitagawa concluded that S9 fractions were more stably stored than microsomes.

Patel et al (1983) Drug Metabolism and Disposition, Short Communication, 11(6), 620- 621 , describe the stability of liver microsomes after storage at -15°C. Patel et al stated that lyophilised microsome preparations were stable for several weeks, losing activity after 7-8 weeks of storage at -15⁰C.

Recombinant systems to produce CYPs have recently been developed in various types of host cells. Each potential host cell has different advantages and disadvantages based on the following criteria: ease of designing genetic constructs, the cost of growing the recombinant cells and producing the microsomes (the endoplasmic reticular membranes to which CYPs are naturally attached), the biomass of the recombinant cells, model organism status, post-transcriptional modification abilities of mammalian cells, ability of membrane system to allow integration of recombinant CYPs, and the non-pathogenicity of the host. Saccharomyces cerevisiae is a good host organism, and insect cells, bacterial cells, other fungi and lymphoblasts have also been used.

Chefson et al (2007) J. Biochem. 130, 436-440, describe sugar-mediated lyoprotection of CYP3A4 and CYP2D6. The CYP enzymes used by Chefson et al were isolated recombinant enzymes rather than membrane-associated CYPs. Long-term stability was not evaluated, and all storage was at -80⁰C.

WO 2008/047112 describes a method for storage of plant CYP74 enzymes. According to WO 2008/047112, CYP74 enzymes are very different from other P450 enzymes in that they have an atypical reaction mechanism that requires neither oxygen nor an NADPH-reductase. Although recombinant, the CYP74 enzymes used in WO 2008/047112 were isolated enzymes rather than membrane-associated CYPs. The freeze-dried recombinant CYP74 enzymes in WO 2008/047112 were said to be stable for at least 10 or 15 weeks, and to retain their activity their activity when reconstituted in the presence of detergent, but the storage temperature was not indicated.

Various recombinant CYPs are commercially available. For example, recombinant human cytochrome P450 enzymes (Human Cytochrome Biocatalyst) are available from Codexis as a lyophilised powder. According to the article dated May 2008 by Codexis in Biocatalysis, (http://www.codexis.com/pdf/Biocatalysis_May_2008.pdf), HCB is :

"Unlike other preparations of recombinant human cytochromes which are only offered as microsomes, the HCB is available as a lyophilised powder containing the

P450 enzyme, the reductase, NADPH, an enzymatic cofactor regeneration system, and an optimised reaction buffer. "

In other words, the Codexis HCB is not a membrane-associated CYP. Moreover, according to the Codexis Product Catalogue, 2009, HCB has a recommended storage temperature of -8O⁰C.

Membrane-associated recombinant CYPs are also commercially available. For example, recombinant CYPs prepared from a bacu Io virus-infected insect cell system are commercially available from Invitrogen™ (Baculosomes^®) and from BD Gentest™ (Supersomes™), and recombinant CYPs prepared from a bacterial system are commercially available from Cypex Limited (Bactosomes). However, the inherent instability and temperature sensitivity of these commercially available CYPs imposes strict transport and storage requirements. Thus, these CYPs are shipped on dry ice which, as well as being heavy and expensive, places restrictive conditions upon transportation.

For example, the product datasheets for Bactosomes^® from Cypex state that the CYPs should be stored at -80⁰C without frequent temperature changes, and thawed on ice when ready for use.

As another example, the product datasheets for Supersomes™ from BD Gentest™ states that the CYPs must be stored at -80⁰C, away from oxidising agents and in cool, dry conditions in sealed containers. According to the BD Gentest website, in response to the question, "How stable are Gentest microsome products?":

"To date, no microsome product has shown a significant loss in enzyme activity when stored at -80⁰C for two years... Cytochrome P450s are known to lose their activity after thawing. We strongly recommend quickly thawing GENTEST microsomes, placing them on ice, and then freezing the microsomes into smaller aliquots immediately after the initial use. Minimization of both freeze thaw cycles and amount of time the microsomes are above -80⁰C should allow the measurement of consistent P450 activity over time." (http://www.bdbiosciences.com/discoveryJabware/piOducts)

It is also appreciated that the need for -8O⁰C freezers may not be justified by smaller organisations that only require a small amount of CYPs at a time.

Accordingly, there is a need in the art for the development of membrane-associated CYPs that are stable and can be stored and transported at room temperature.

We have developed a lyophilisation-based procedure that stabilises membrane- associated CYPs (CYPs bound to fragments of microsomes or bacterial periplasmic membranes) such that they can be stored and transported at room temperature. Specifically, we have demonstrated that membrane-bound enzymes from BD Gentest™ (insect cell-derived), Invitrogen™ (insect cell-derived), Cypex (bacterial cell-derived), and De Montfort Bioscience (DMB) (yeast and insect cell-derived), can be stabilised by this procedure. This is particularly surprising because the micelle format of membrane- bound CYPs might have been expected to be disrupted during the process of water removal and subsequent rehydration.

We have also shown that the activity of the stabilised enzymes was undiminished after 24 and 95 days storage at room temperature. In a few instances we even observed an increase in the rate of reaction using fluorometric substrates, indicating that the enzymes may be more active after stabilisation. This is particularly unexpected because the changes to the osmotic potential, pH, temperature and redox environments might have been expected to cause destruction of CYPs. Moreover, the IC₅₀ for inhibition of CYP2D6 using a known inhibitor remained unchanged after stabilisation indicating that the active site of the enzymes was unaltered by the treatment. We consider that this procedure can be used to produce enzymes that are stable at room temperature for 6 months or longer.

To place this into perspective, we have stored highly unstable CYP microsomal products for 95 days of fluctuating ambient temperature (~+21 °C), which is about 100⁰C above the commonly professed storage temperature.

The stabilisation procedure that we have developed provides several advantages including the increased convenience and lower cost of shipping without using dry-ice, the increased convenience of use and simplification of routine kinetic analyses via manual or robotic techniques due to a reduced requirement for temperature control, as well as the ability to distribute CYPs in formats other than microtitre/multiwell plates, such as Eppendorf™ tubes or HPLC vials.

Accordingly, a first aspect of the invention provides a dried composition comprising a membrane-associated recombinant cytochrome P450 (CYP) enzyme, wherein the CYP enzyme is stable at 21⁰C for a period of at least 10 days.

Typically, the dried composition has been dried by freeze-drying, i.e. by lyophilisation. Thus, by a dried composition we include the meaning that the composition is dried to the extent that is achieved by lyophilisation under routine laboratory conditions, such as those described below in the examples.

The CYP enzyme can be any known CYP enzyme. In an embodiment, it is preferred that the CYP enzyme is a mammalian, and preferably a human CYP enzyme. Suitable CYPs include the 57 known human CYP enzymes, including CYP1B1, CYP7A1 , CYP7B1, CYP8B1 , CYP11A1 , CYP11B1 , CYP11 B2, CYP17A1 , CYP19A1 , CYP21A2, CYP27A1 , CYP39A1 , CYP46A1 , CYP51A1 (which have sterols as a substrate), CYP1A1 , CYP1A2, CYP2A6, CYP2A13, CYP2B6, CYP2C8, CYP2C9, CYP2C18, CYP2C19, CYP2D6, CYP2E1 , CYP2F1 , CYP3A4, CYP4A5, CYP3A7 (which have xenobiotics as a substrate), CYP2J2, CYP4A11 , CYP4B1 , CYP4F12 (which have fatty acids as a substrate), CYP3F2, CYP4F3, CYP4F8, CYP5A1 , CYP8A1 (which have eiconasoids as a substrate), CYP2R1 , CYP24A1 , CYP26A1 , CYP26B1 , CYP26C1 , CYP27B1 (which have vitamins as a substrate), and CYP2A7, CYP2S1 , CYP2U1, CYP2W1 , CYP3A43, CYP4A22, CYP4F11 , CYP4F22, CYP4V2, CYP4X1 , CYP4Z1 , CYP20A1 , CYP27C1 (as listed in Table 1 of Guengerich, 2006).

More preferably, the CYP enzyme is selected from mammalian CYP3A4, CYP2D6, CYP2C8, CYP1A1 , CYP1 B1 , CYP2E1 and CYP1A2, especially human CYP3A4, CYP2D6, CYP2C8, CYP1A1 , CYP1B1, CYP2E1 and CYP1A2.

Some of the most relevant CYPs for the pharmaceutical industry include human CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A4, each of which are suitable CYPs for the practise of the present invention.

It is appreciated that certain of the CYPs are polymorphic, and thus by a specific CYP we include variant alleles thereof. For example, CYP2D6 is highly polymorphic, having null alleles and/or amino acid substitution(s) in most human populations (Sistonen et al, 2006). The CYP2D6*10 allele contains two amino acid substitutions, P34S and S486T, is quite common in Asian populations, and has been linked to reduced rates of metabolism of known CYP2D6 substrates in comparison to CYP2D6^*1. Other suitable CYP alleles that can be mentioned include CYP2C9^*1 (Arg144), CYP2C9^*2 (Cys144), and CYP2C9*1 (Leu359). A complete list of CYP alleles can be found at the website of the Human Cytochrome P450 (CYP) Allele Nomenclature Committee (http://www.cypalleles.ki.se).

Other suitable CYPs include plant CYPs, which are of interest in establishing biosynthetic routes to high value chemicals (both in vivo and in vitro).

In an embodiment, the dried composition may comprise more than one CYP enzyme. Preferred combinations of recombinant CYPs include any, two, three, four, five or all six of CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP3A4. In an embodiment, the CYP is not a CYP74, and preferably not a plant CYP74.

Recombinant DNA technology is now well established. Suitable techniques for cloning, manipulation, modification and expression of nucleic acids, and purification of expressed proteins, are well known in the art and are described, for example, in Sambrook et al (2001 ) "Molecular Cloning, a Laboratory Manuar, 3^rd edition, Sambrook et al (Eds), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA; Gould (1994) "Membrane Protein Expression Systems: A User's Guide", Gould (Ed), ISBN 9781855780316, Portland Press; Higgins & Hames (1999) "Protein expression: a practical approach", ISBN:0199636230, Oxford University Press; and Selinsky et al (2003) "Membrane protein protocols: Expression, purification and characterization", Selinsky (Ed), Humana Press. Thus suitable membrane-associated CYP enzymes can be made using well-known recombinant technology. Alternatively, suitable recombinant membrane-associated CYP enzymes can be obtained from BD Gentest^™, Invitrogen™, Cypex or DMB.

The recombinant membrane-associated CYP enzymes may be produced in any suitable expression system known in the art, including bacterial cell, yeast cell, insect cell or vertebrate, preferably mammalian, cell based systems.

For example, recombinant membrane-associated CYP enzymes have been produced in yeast cells such as Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pichia pastoris and Yarrowia lipolytica cells.

Alternatively, recombinant membrane-associated CYP enzymes have been produced in bacterial cells such as Escherichia coli cells.

Further alternatively, the recombinant membrane-associated CYP enzymes have been produced in insect cells such as Sf9, Sf21 , Tni, High Five and Tn368 cells.

Still further alternatively, the recombinant membrane-associated CYP enzymes may have been produced in vertebrate, preferably mammalian cells or cell lines, such as lymphoblastoid cells or cell lines, cancer cells including liver cancer cells or cell lines, and normal (i.e. non-cancerous) e.g. liver cells or cell lines.

The CYP enzyme in the dried compositions of these aspects of the invention is stable at about 21⁰C (room temperature) for a period of at least 10 days. More preferably, the CYP enzyme in the dried composition is stable at 21⁰C for a period of at least 24 days, more preferably at least 3 months or at least 6 months.

As shown in the Examples, once the CYP enzyme in the dried composition has been rehydrated after storage, the CYP enzyme retains its activity in comparison to the same CYP enzyme from the same batch stored at -8O⁰C for the same time period. Thus, by a CYP enzyme being 'stable', we mean that the CYP enzyme retains at least 10% of its activity compared to its activity after storage at -80⁰C for the same time period, typically at least 20%, and suitably at least 30% or 40%. Preferably, however, the stable CYP enzyme retains at least 50%, 60% or at least 70% or at least 80% or at least 90% of its activity compared to its activity after storage at -8O⁰C for the same time period. Still more preferably, the stable CYP enzyme retains at least 95% of its activity, or at least 99%, or about 100% or more of its activity in comparison to its activity after storage at -80⁰C for the same time period.

Although not measured directly, we expect that the once the CYP enzyme in the dried composition has been rehydrated after storage, the CYP enzyme will retain the majority of its activity (i.e., at least 50% or at least 70% or at least 90% or at least 95% or at least 99% or more) of its activity in comparison to its activity prior to drying.

By an "activity" of a CYP we include any suitable measurable and testable activity of the CYP. Such suitable activities include metabolism of one of its known substrates. Typical and suitable methods for measuring the activity of CYP3A4, CYP2D6, CYP2C8, CYP1A1 , CYP1 B1 , CYP2E1 and CYP1A2 are provided in the Examples. Methods for determining the activity of other CYP enzymes are well known in the art and are described in the BD Gentest website (http://www.bdbiosciences.com/ discovery_labware/gentest/products/transporters/atpase_summary/HTS-FAQ.shtml) and the Invitrogen website (http://www.invitrogen.com/site/us/en/home/Products-and- Services/Applications/Drug-Discovery/Drug-Metabolism-and-Safety/Biochemical-ADME- Assays/BADMEA-misc/DME-Tech-Resource-Guide.html).

For those CYPs for which an optimal substrate has not been identified, activity can be measured by using known chemical substrates until a better one is developed. Optionally, there is a prior step of determining through CO-difference spectroscopy that the recombinant P450 exists in the holo (i.e. active) form and not in the apo (inactive) form, (the holo-form is measured as a peak at 450 nm whereas the apo-form manifests as a peak at 420 nm). The amount of CYP protein in the composition will vary depending upon its intended use after rehydration. The typical range for a single fluorescent assay is from about 1 to 4 pmols. In an HPLC/MS assay, up to 10 pmols could be used depending on the nature of the substrate. Larger amounts of CYP protein (e.g., μmols) could be used for biotransformations, and for bulk storage and transport.

In a preferred embodiment, the dried composition further comprises cytochrome P450 reductase (CPR)(also known as NADPH-hemoprotein reductase, EC 1.6.2.4). CPR transfers electrons from NADPH (NADP+) to the various isoforms of cytochrome P450. The 3-dimensional structure of CPR was determined in 1997 by Wang et al. CPR is typically present at a variety of ratios ranging from 0.5-5:1 (reductase : P450) for in vitro reconstitution assays. Mammalian CPR is preferred, such as human or rabbit. Human CPR is commercially available from a number of sources including Invitrogen™, where it is recombinantly expressed in baculovirus-infected insect cells. As for CYP, CPR is a membrane-associated enzyme, typically attached to a microsome. Typically, and preferably, the CPR in the dried composition has been expressed recombinantly. Recombinant CPR may be associated with the same membrane as the recombinant CYP enzyme through co-expression of genes encoding both enzymes. Most of the commercially available recombinant CYPs, e.g. Baculosomes^® from Invitrogen™, Supersomes™ from BD Gentest™ and Bactosomes from Cypex Ltd contain recombinantly-prepared reductase. Alternatively, but less preferred, the recombinant CPR may have been prepared separately from the CYP enzyme, and the two membrane-associated enzymes combined before drying.

In a preferred embodiment, the dried composition according to this aspect of the invention further comprises a sugar.

Without wishing to be bound by theory, the inventors consider that the presence of the sugar may reduce large and/or "sharp" water crystals, or allow a non-shrinking (i.e., physically stable) matrix for the microsomes to exist within during lyophilisation, thus aiding in the stabilisation of the dried CYP enzyme.

Typically, the sugar is selected from sorbitol, trehalose, maltose, raffinose, sucrose, mannose, glucose and galactose. Suitable sugars may include monosaccharides, including trioses, tetroses, pentoses, hexoses, heptoses, octoses, nonoses or decoses, (whether in the aldose or ketose form), such as erythrose, threose, erythrulose, arabinose, lyxose, ribose, deoxyribose, xylose, ribulose, xylulose, allose, altrose, gulose, idose, talose, fructose, psicose, sorbose, tagatose, mannoheptulose, sedoheptulose, octolose, 2-keto-3-deoxy-manno- octonate, and sialose. Alternatively, the sugar may be a disaccharide such as lactose; or a trisaccharide such as acarbose, raffinose and melezitose. Further alternatively, the sugar may be a sugar oligosaccharide, e.g. a tetrasaccharide, such as fructo- oligosaccharide (FOS), galacto-oligosaccharide (GOS) or mannan-oligosaccharides (MOS). As another alternative, the sugar may be a sugar polymer such as glycogen, starch (amylose/amylopectin), cellulose, chitin, stachyose, inulin or dextrin. As a still further alternative, the sugar may be a sugar alcohol such as glycol, erythritol, arabitol, xylitol, ribitol, mannitol, isomalt, maltitol or lactitol.

In an embodiment, disaccharides are preferred.

In an embodiment, it may also be preferred that the sugar is a non-reducing sugar such as sucrose or trehalose, rather than a reducing sugar such as mannose.

In a general embodiment, sucrose may be preferred because its presence is generally beneficial in the stabilisation of CYPs from all sources tested.

Alternatively, in a specific embodiment, when the recombinant CYP enzyme has been produced in bacterial cells, it may be preferred that the sugar is sorbitol. In another specific embodiment, when the recombinant CYP enzyme has been produced in yeast cells, it may be preferred that the sugar is trehalose. In a further particular embodiment, when the recombinant CYP enzyme has been produced in insect cells, it may be preferred that the sugar is sucrose.

Typically, the amount of sugar in the composition prior to lyophilisation is at least 2.2% w/v, more usually at least 4.5%, and preferably between about 9% and 36% w/v, or possibly more, as described below in the Examples. A suitable level is about 20% w/v. Upon lyophilisation, the mass of the sugar in the dried composition does not change, but the percentage does. When the dried composition is present in the well of a 96-well microtitre plate, it typically will comprise between 0.1 mg to 5mg of sugar. Other formats may comprise greater or lower amounts of sugar. Other components that may be present in the dried composition include those that might be left over from the microsome production methods, and so would be present at only very low concentrations, if at all. They include potassium phosphate, sorbitol, tris, EDTA, AEBSF (protease inhibitor) and dithiothreitol (DTT).

In an embodiment, it may be preferred that the dried composition does not contain glycerol.

Typically, the membrane-associated CYP is attached to a microsome. As an alternative, the membrane-associated CYP may be attached to a bacterial periplasmic membrane:

While microsomes are usually described as endoplasmic reticular (ER) membranes, it is theoretically possible that during mechanical disruption of the cells to obtain the microsomes, other cellular membranes integrate themselves within the microsomes. This may include parts (however small) of the Golgi, mitochondria, outer cell membranes and various small vesicles.

Optionally, the dried composition may also comprise cytochrome b₅. Cytochrome b₅ is a membrane-bound haemoprotein that enhances the catalytic efficiency of some P450 isoforms, including CYP2A6, CYP2B6, CYP2C10, CYP2C8, CYP2C9, CYP3A4, CYP3A5, CYP3A7, CYP4F12, CYP4F2, CYP4F3A, CYP4F3B and CYP2E1. Human cytochrome b₅ is commercially available from a number of sources including Invitrogen™, where it is recombinantly expressed in E. coli.

A second aspect of the invention provides a solid support comprising therein or thereon a dried composition according to the first aspect of the invention. The solid support may be, for example, a multiwell plate, an HPLC vial, or an Eppendorf tube.

Multi-well plates are preferred because, once rehydrated, CYP reactions could be earned out and analysed directly in the plates by HPLC or fluorometric analysis.

The multi-well plate may be a 6-well plate, in which the well area is about 960mm² per well (about 5.8ml per plate). Each well can readily accommodate up to 500 pmol per well (3000pmol per plate).

Alternatively, the multi-well plate may be a 24-well plate, in which the well area is about 175mm² per well. Each well can readily accommodate up to around 100 pmol CYP per well (is 2400 pmol per plate). Typically, the multi-well plate may be a 96-well plate, in which the well diameter is about 6mm (area of about 28mm²). A 10μl sample of microsomes + buffer mix is seen to occupy about 25% of the well surface, about 7mm². Since a 10μl volume should occupy about 10μm³ we can estimate that the microsome buffer thickness should be about 1.3μm thick.

The multi-well plate may be 386-well plate, which has a well surface area of about 9mm², so can each well accommodate 7mm² of microsomes and buffer. The 384-well plates have a well volume of about 112μl.

The use of 1536-well microplates is also possible. These plates come in two working volumes, δμl and 1.5μl.

The shape of container used, i.e., the available surface area, affects the time required for lyophilisation, as is well known to the person of skill in the art. Dried compositions of CYPs in these plate formats can be produced within a working day without significantly altering the dynamics of the lyophilisation procedure.

A third aspect of the invention provides a kit of parts comprising a dried composition according to the first aspect of the invention, or a solid support according to the second aspect of the invention, and any one, two, three, four, five or all six of: a sterile liquid for rehydrating the dried composition, a substrate for the one or more CYP enzymes in the dried composition, an inhibitor of the one or more CYP enzymes in the dried composition,

NADPH, a negative control dried membrane preparation without CYP activity, and a positive control CYP enzyme stored at -80⁰C.

The NADPH may be in the form of a regeneration system, e.g., NADP, glucose-6- phosphate, and glucose-6-phosphate dehydrogenase, which is commercially available from Sigma.

Preferred components of the kit include an NADP+ regeneration system, and specific substrates and inhibitors of the CYPs. The kit of parts may contain a sterile liquid for rehydrating the dried composition. In a preferred embodiment, the sterile liquid is water since one is simply returning what has been removed. Alternatively, the sterile liquid for rehydrating the dried composition sterile may be a phosphate or Tris buffers of a sensible range as understood by the skilled person (e.g. around 100 mM and around pH7.5) should be suitable. It is appreciated that the liquid for rehydrating the dried composition may be combined with the NADP+ regeneration system. In an embodiment, the sterile liquid for rehydrating the dried composition does not contain a detergent, such as Emulphogene or Triton X-100.

The kit of parts may comprise a substrate for the one or more CYP enzymes in the dried composition. In an embodiment, if the recombinant CYP is CYP1 B1 , CYP7A1 , CYP7B1 , CYP8B1, CYP11A1 , CYP11 B1 , CYP11 B2, CYP17A1, CYP19A1, CYP21A2, CYP27A1 , CYP39A1 , CYP46A1 or CYP51A1 , the substrate is a sterol. In another embodiment, if the recombinant CYP is CYP1A1 , CYP1A2, CYP2A6, CYP2A13, CYP2B6, CYP2C8, CYP2C9, CYP2C18, CYP2C19, CYP2D6, CYP2E1 , CYP2F1 , CYP3A4, CYP4A5 or CYP3A7, the substrate is a xenobiotic. In another embodiment, if the recombinant CYP is CYP2J2, CYP4A11 , CYP4B1 or CYP4F12, the substrate is a fatty acid. In another embodiment, if the recombinant CYP is CYP3F2, CYP4F3, CYP4F8, CYP5A1 or CYP8A1, the substrate is an eiconasoid. In another embodiment, if the recombinant CYP is CYP2R1 , CYP24A1 , CYP26A1 , CYP26B1 , CYP26C1 or CYP27B1 , the substrate is a vitamin.

According to the Kegg database (http://www.genome.jp/kegg-bin/get_htext? htext=brO8105.keg&filedir=%2ffiles&extend=B19&open=B15#B15), substrates for CYP1A1 include naphthalene, estradiol-17beta, melatonin, carbaril, benzo[a]pyrene, 4- nitroanisole, phenacetin, chlorzoxazone, ethoxycoumarin, 7-ethoxyresorufin, zoxazolamin and 1-nitronaphthalene; substrates for CYP1A2 include arachidonic acid, aniline, naphthalene, estradiol, tacrine, naproxen, warfarin, melatonin, haloperidol, 2-naphthylamine, 2- acetamidofluorene, aflatoxin B1 , acetaminophen, amitriptyline, clomipramine, clozapine, cyclobenzaprine, imipramine, theophylline, verapamil, zolmitriptan, mexiletine, olanzapine, ondansetron, propafenone, propranolol, tizanidine, caffeine, carbaril, ropivacaine, acetanillide, fluvoxamine, phenacetin, chlorzoxazone, riluzole, isosafrole, 4- aminobiphenyl, methoxychlor, 7-ethoxyresorufin, bufuralol, zoxazolamin, 1- nitronaphthalene and zileuton; substrates for CYP1 B1 include estradiol-17beta, melatonin, benzo[a]pyrene; substrates for CYP2A3 include testosterone and2,6-dichlorobenzonitrile; substrates for CYP2A5 include acetaminophen; substrates for CYP2A6 include, nicotine, coumarine, acetaminophen, phenacetin and methoxychlor; substrates for CYP2B1 include, testosterone, naphthalene, trichloroethylene, aminopyrine, bromobenzene and 1-nitronaphthalene; substrates for CYP2B2 include bromobenzene; substrates for CYP2B6 include, testosterone, nicotine, naphthalene, bupropion, ifosfamide, methadone, carbaril, efavirenz, methoxychlor, 7-ethoxycoumarin and cyclophosphamide; substrates for CYP2C6 include testosterone substrates for CYP2C8 include, arachidonic acid, tolbutamide, paclitaxel, amodiaquine, repaglinide, cerivastatin, methoxychlor and torsemide; substrates for CYP2C9 include arachidonic acid, (-)-limonene, naproxen, warfarin, ibuprofen, piroxicam, diclofenac, mefenamic acid, (+)-limonene, amitriptyline, tetrahydrocannabinol, glyburide, losartan, tamoxifen, tolbutamide, suprofen, phenytoin, irbesartan, benzo[a]pyrene, celecoxib, glimepiride, meloxicam, methoxychlor, hexobarbital, nateglinide, fluoxetine, flurbiprofen, glipizide, torasemide, seratrodast, tenoxicam and sulfaphenazole; substrates for CYP2C11 include arachidonic acid, (-)-limonene, testosterone, vitamin D2, vitamin D3, (+)-limonene; substrates for CYP2C12 include arachidonic acid; substrates for CYP2C18 include diazepam and mephenytoin; substrates for CYP2C19 include, arachidonic acid, progesterone, (-)-limonene, warfarin, indomethacin, (+)-limonene, amitriptyline, clomipramine, diazepam, imipramine, nelfinavir, omeprazole, primidone, propranolol, phenytoin, citalopram, proguanil, mephobarbital, carisoprodol, nilutamide, methoxychlor, teniposide, hexobarbital, pantoprazole, cyclophosphamide, lansoprazole and mephenytoin; substrates for CYP2C23 include arachidonic acid; substrates for CYP2D2 include dextromethorphan; substrates for CYP2D6 include, haloperidol, codeine, quinidine, amitriptyline, carvedilol, chlorpheniramine, chlorpromazine, clomipramine, desipramine, dextromethorphan, encainide, flecainide, fluphenazine, imipramine, tamoxifen, timolol, tramadol, venlafaxine, metoprolol, mexiletine, nortriptyline, ondansetron, propafenone, promethazine, propranolol, paroxetine, perphenazine, amphetamine, ethylmorphine, fluvoxamine, phenacetin, phenformin, metoclopramide, sparteine, methoxychlor, aripiprazole, mequitazine, debrisoquin, tropisetron, bufuralol, fluoxetine, lidocaine, thioridazine and risperidone; substrates for CYP2E1 include acetone, arachidonic acid, aniline, ethanol, naphthalene, p-nitrophenol, benzene, toluene, lauric acid, N,N-dimethylformamide, p- xylene, trichloroethylene, vinyl chloride, acetaminophen, theophylline, m-xylene, o- xylene, halothane, enflurane, methoxyflurane, isoflurane, sevoflurane, dapsone, chlorzoxazone, ethylene dibromide, 1 ,1-dichloroethylene, 1-nitronaphthalene; substrates for CYP2F1 include naphthalene, benzene, trichloroethylene, 3- methylindole, 1 ,1-dichloroethylene, 1-nitronaphthalene; substrates for CYP2F2 include naphthalene, benzene, trichloroethylene, 1 ,1- dichloroethylene and 1-nitronaphthalene; substrates for CYP2F4 include naphthalene, benzene, trichloroethylene, 1 ,1- dichloroethylene, 1 -nitronaphthalene; substrates for CYP2G1inlcude acetaminophen; substrates for CYP2J2 include arachidonic acid, codeine and ebastine; substrates for CYP2J3 include arachidonic acid, vitamin D2 and vitamin D3; substrates for CYP2J9 include arachidonic acid; substrates for CYP2R1 include Vitamin D2 and Vitamin D3; substrates forCYP2S1 include naphthalene; substrates for CYP2U1 include arachidonic acid; substrates for CYP3A1 include testosterone, chlorzoxazone and 1 ,8-Cineole; substrates for CYP3A4 include arachidonic acid, androstenedione, progesterone, androsterone, testosterone, Cortisol, 17beta-estradiol, sterigmatocystin, tacrolimus, cocaine, etoposide, haloperidol, erythromycin, dehydroepiandrosterone 3-sulfate, cyclosporin a, vitamin d2, vitamin d3, codeine, quinine, quinidine, parathion, aflatoxin b1 , acetaminophen, alprazolam, amiodarone, amitriptyline, amlodipine, astemizole, atorvastatin, buspirone, carbamazepin, chlorpheniramine, cisapride, clarithromycin, clomipramine, dextromethorphan, diazepam, diltiazem, disopyramide, tetrahydrocannabinol, indinavir, losartan, tamoxifen, trazodone, methadone, venlafaxine, verapamil, vincristine, Zolpidem, ritonavir, salmeterol, nelfinavir, sildenafil, nicardipine, nifedipine, nimodipine, omeprazole, propafenone, paclitaxel, propranolol, terfenadine, caffeine, zaleplon, desmethyldiazepam, carbaril, zonisamide, midazolam, 17alpha-ethinyl estradiol, benzo[a]pyrene, ethylmorphine, citalopram, phencyclidine, colchicine, proguanil, dapsone, nisoldipine, nitrendipine, sirolimus, cerivastatin, buprenorphine, amprenavir, 1 ,8-cineole, docetaxel, telithromycin, nateglinide, eplerenone, aripiprazole, cyclophosphamide, dexamethasone, felodipine, fentanyl, finasteride, ifosfamide, isradipine, ketoconazole, lidocaine, lovastatin, triazolam, miconazole, saquinavir, simvastatin, seratrodast, cilnidipine, aranidipine, domperidonel and cilostazol; substrates for CYP3A5 include testosterone, erythromycin, nifedipine, midazolam, ethylmorphine, substrates for CYP3A7 include dehydroepiandrosterone 3-sulfate and aflatoxin B1 ; substrates for CYP3A9 include testosterone; substrates for CYP3A23 include parathion and aminopyrine; substrates for CYP4A1 include fatty acid, prostaglandin F2alpha, lauric acid and prostaglandin E1 ; substrates for CYP4A2 include lauric acid; substrates for CYP4A3 include fatty acid, prostaglandin F2alpha and prostaglandin E_{1 ;} substrates for CYP4A11 include fatty acid, arachidonic acid and lauric acid; substrates for CYP4B1 include arachidonic acid and midazolam; substrates for CYP4F1 include leukotriene B4, lipoxin A4, 8(S)-HETE and 8(R)- HETE; substrates for CYP4F2 include, arachidonic acid, leukotriene B4, gamma- tocopherol, lipoxin A4, p-nitroanisole, 7-ethoxycoumarin, 8(S)-HETE, 12(S)-HETE, 12(R)-HETE and 8(R)-HETE; substrates for CYP4F3 include, Arachidonic acid, Leukotriene B4, 5- Hydroxyeicosatetraenoate, 20-OH-Leukotriene B4, Lipoxin A4, Lipoxin B4, 12(S)-HETE, 12(R)-HETE; substrates for CYP4F4 and CYP4F5 include leukotriene B4 substrates for CYP4F6 include leukotriene B4 and imipramine; substrates for CYP4F8 include arachidonic acid and prostaglandin H2; substrates for CYP4F11 include arachidonic acid, erythromycin, chlorpromazine, diazepam, ethylmorphine and benzphetamine; substrates for CYP4F12 include arachidonic acid, terfenadine and ebastine; substrates for CYP4F13 include leukotriene B4 and 5-hydroxyeicosatetraenoate; substrates for CYP4F14 include leukotriene B4, 5-hydroxyeicosatetraenoate, lipoxin A4, 8(S)-HETE and 8(R)-HETE; substrates for CYP4V2 and CYP4X1 include fatty acids; substrates for CYP5A1 include prostaglandin H2; substrates for CYP7A1 include cholesterol; substrates for CYP7B1 include 27-hydroxycholesterol and 24-hydroxycholesterof; substrates for CYP8A1 include prostaglandin H2; substrates for CYP8B1 include 7alpha-hydroxycholest-4-en-3-one; substrates for CYP11A1 include cholesterol, 20alpha-hydroxycholesterol, (22r)-

20alpha,22-dihydroxycholesterol and 22beta-hydroxycholesterol; substrates for CYP1 1 B1 include 11-deoxycortisoI and 11 -deoxycorticosterone; substrates for CYP11 B2 11 -deoxycorticosterone; substrates for CYP11B3 11 -deoxycorticosterone; substrates for CYP17A1 include progesterone, 17alpha-hydroxyprogesterone, pregnenolone and 17alpha-hydroxypregnenolone; substrates for CYP19A1 include androstenedione and testosterone; substrates for CYP21A1 include progesterone and 17alpha-hydroxyprogesterone; substrates for CYP21A2 include progesterone; substrates for CYP24A1 include 25-hydroxyvitamin D3; substrates for CYP26A1 , CYP26B1 and CYP26C1 include retinoic acid; substrates for CYP27A1 include cholesterol, vitamin D2, vitamin D3, 5beta- cholestane-3alpha,7alpha-diol and 5beta-cholestane-3alpha,7alpha,12alpha-triol; substrates for CYP27B1 include 25-hydroxyvitamin D3 and 24(R),25- dihydroxyvitamin D3; substrates for CYP39A1 include 24-hydroxycholesterol; substrates for CYP46A1 include cholesterol; and substrates for CYP51A1 include lanosterol and 24,25-dihydrolanosterol.

As described below in the Examples, a suitable substrate for CYP3A4 is DBOMF (Invitrogen™); a suitable substrate for CYP2D6 is (3-[2-(N,Ndiethyl-N- methylammonium)ethyl]-7-methoxy-4-methylcoumarin) (AMMC; BD Gentest; US Patent

No. 6,130,342); a suitable substrate for CYP2C8 is DBF (Sigma); a suitable substrate for CYP1A1 and 1B1 is ethoxyresorufin (Sigma); a suitable substrate for CYP2E1 is

EOMCC (Invitrogen™); a suitable substrate for CYP1A2 is CEC (UFC Ltd, UK); a suitable substrate for CYP2A6 is Coumarin (Sigma).

An alternative CYP3A4 fluorescent substrate is 7-benzyloxy-4-(trifluoromethyl)-coumarin (BD Gentest; US Patent No. 6,207,404). The fluorescent substrate, dibenzylfluorescein (DBF; BD Gentest; US Pat 6,420,131 ) is generally used for a variety of different CYPs. The CYP3A4 fluorescent substrate benzyloxyresorufin, and the general CYP substrates CEC and CMC, are available from UFC Ltd, UK.

According to Flockhart DA. "Drug Interactions: Cytochrome P450 Drug Interaction Table", Indiana University School of Medicine (2007) http://medicine.iupui.edu/flockhart/table.htm (accessed on 15 July 2008), FDA preferred and acceptable substrates for CYP1A2 in vitro experiments include caffeine, tacrine and theophylline; for CYP2B6 they include bupropion and efavirenz; for CYP2C8 they include amodiaquine; for CYP 2C19 they include omeprazole and S-mephenytoin; for CYP2C9 they include diclofenac, tolbutamide and S-warfarin; for CYP2D6 they include bufuralol, debrisoquine and dextromethorphan; for CYP2E1 they include aniline and chlorzoxazone; and for CYP3A4, 3A5 and 3A7 they include erythromycin (not 3A5), midazolam, triazolam, terfenadine, nifedipine, testosterone and dextromethorphan, each of which are suitable in the context of the present invention.

The kit of parts may comprise an inhibitor for the one or more CYP enzymes in the dried composition. The inhibitor might be a strong inhibitor that causes a > 5-fold increase in the plasma AUC values or more than 80% decrease in clearance; a moderate inhibitor that causes a > 2-fold increase in the plasma AUC values or 50-80% decrease in clearance, or a weak inhibitor that causes a > 1.25-fold but < 2-fold increase in the plasma AUC values or 20-50% decrease in clearance.

According to Flockhart D.A. "Drug Interactions: Cytochrome P450 Drug Interaction Table", Indiana University School of Medicine (2007) http://medicine.iupui.edu/flockhart/table.htm (accessed on 15 July 2008), inhibitors for CYP1A2 include fluvoxamine (strong), ciprofloxacin (strong), cimetidine, amiodarone, fluoroquinolones, furafylline (preferred, UFC Ltd), interferon, methoxsalen, and mibefradil; inhibitors for CYP2B6 include thiotepa and ticlopidine (preferred); inhibitors for CYP2C8 include gemfibrozil, trimethoprim (moderate), glitazones, montelukast (preferred) and quercetin (preferred); inhibitors for CYP2C19 include the PPIs: lansoprazole, omeprazole (preferred), pantoprazole amd rabeprazole, chloramphenicol, cimetidine, felbamate, fluoxetine, fluvoxamine, indomethacin, ketoconazole, modafinil, oxcarbazepine, probenicid, ticlopidine (preferred) and topiramate; inhibitors for CYP2C9 include fluconazole (strong, preferred), amiodarone (moderate, preferred), fenofibrate, fluvastatin, fluvoxamine (preferred), isoniazid, lovastatin, phenylbutazone, probenicid, sertraline, sulfamethoxazole, sulfaphenazole (preferred, UFC Ltd), teniposide, voriconazole, zafirlukast; inhibitors for CYP2D6 include bupropion, fluoxetine, paroxetine, quinidine (strong, preferred), duloxetine (moderate), terbinafine (moderate), amiodarone (weak), cimetidine (weak) and sertraline (weak); inhibitors for CYP2E1 include diethyl-dithiocarbamate (preferred) and disulfiram; and inhibitors for CYP3A4, 3A5 and 3A7 include the HIV antivirals: indinavir (strong), nelfinavir (strong), ritonavir (strong), clarithromycin (strong), itraconazole (strong, preferred), ketoconazole (strong, preferred), nefazodone (strong), saquinavir (strong), telithromycin (strong), aprepitant (moderate), erythromycin (moderate), fluconazole (moderate), grapefruit juice (moderate), verapamil (moderate, preferred), diltiazem (moderate-weak), cimetidine (weak), amiodarone, chloramphenicol, delaviridine, diethyl- dithiocarbamate, fluvoxamine, gestodene, imatinib, mibefradil, mifepristone, norfloxacin, norfluoxetine, star fruit and voriconazole.

Except when present in the dried composition according to the first aspect of the invention, or on the solid support according to the second aspect of the invention, the kit of parts may further comprise cytochrome P450 reductase and/or cytochrome b₅, as described above with respect to the first aspect of the invention.

A fourth aspect of the invention provides a method of stabilising a recombinant membrane-associated CYP enzyme, the method comprising freeze-drying the CYP enzyme.

Preferences for the recombinant CYP enzyme to be stabilised by freeze-drying are discussed above with respect to the first aspect of the invention.

Usually, the membrane-associated CYP enzyme is freeze-dried in the presence of a sugar and/or a buffer. Preferences for the sugars and/or buffers to be used are also discussed above with respect to the first aspect of the invention.

The recombinant membrane-associated CYP enzyme may be freeze-dried using standard lyophilisation equipment under standard conditions, such as those described in Example 2, which are well known in the art.

In other words, this aspect of the invention includes a method of stabilising a recombinant membrane-associated CYP enzyme, the method comprising lyophilising the CYP enzyme.

In addition to the lyophilisation equipment used in the Examples, suitable equipment can be obtained from Thermo Scientific, including the Heto Power Dry LL1500, and the Micro Mod u lyo with an AC300 chamber (http://www.thermo.com/eThermo/CMA/PDFs/ Product/productPDF_27027.pdf.) Typically, the freeze-dried CYP enzyme, when reconstituted, retains at least 10% of its activity after lyophilisation, usually at least 20%, and suitably at least 30% or 40%.

Preferably, however, the freeze-dried CYP enzyme retains at least 50% of its activity after lyophilisation, more preferably at least 70% of its activity after lyophilisation, yet more preferably at least 90% of its activity after lyophilisation, still more preferably at least 95% of its activity after lyophilisation, and most preferably retains 99% or more of its activity after lyophilisation. Methods for determining the activity of a CYP enzyme are well known in the art and are described above with respect to the first aspect of the invention, and in the Examples.

In an embodiment, the CYP enzyme in the dried composition is rehydrated after storage in the absence of a detergent.

A fifth aspect of the invention provides a method of storing a recombinant membrane- associated CYP enzyme, the method comprising: providing a recombinant membrane-associated CYP enzyme; freeze-drying (i.e. lyophilising) the CYP enzyme; and storing the dried CYP enzyme at greater than 0⁰C.

The invention also includes a method of providing a membrane-associated CYP enzyme, the method comprising: storing a recombinant membrane-associated CYP enzyme according to the fifth aspect of the invention, or providing a recombinant membrane-associated CYP enzyme that has previously been stored according to the fifth aspect of the invention; and rehydrating the stored CYP enzyme.

Preferences for the recombinant CYP enzyme to be stored are discussed above with respect to the first aspect of the invention.

The recombinant membrane-associated CYP enzyme may be freeze-dried using standard lyophilisation equipment under standard conditions, such as those described in Example 2, which are well known in the art, and discussed above.

Usually and preferably, the membrane-associated CYP enzyme is freeze-dried in the presence of a sugar and/or a buffer. Preferences for the sugars and/or buffers to be used are discussed above with respect to the first aspect of the invention. The dried CYP enzyme may be stored at refrigeration temperature (about 4-8°C) or above. In a preferred embodiment, the dried CYP enzyme is stored at room temperature (about 18-22⁰C).

Typically, the dried CYP enzyme is stored for at least 10 days. More preferably, the dried CYP enzyme is stored for a period of at least 24 days, and yet more preferably for at least 3 months or for at least 6 months.

Preferably, the dried CYP enzyme, when rehydrated, retains at least 10% of its activity after storage, typically at least 20%, and suitably at least 30% or 40%. Preferably, however, the dried CYP enzyme retains at least 50% of its activity after storage, more preferably at least 70% of its activity after storage, yet more preferably at least 90% of its activity after storage, still more preferably at least 95% of its activity after storage, and most preferably retains 99% or more of its activity after storage. Methods for determining the activity of a CYP enzyme are well known in the art and are described above with respect to the first aspect of the invention, and in the Examples.

Typically, the stored CYP enzyme is rehydrated using a sterile liquid such as water. Alternatively, the sterile liquid for rehydrating the dried composition sterile may be a phosphate or Tris buffer of a sensible range as understood by the skilled person (e.g. around 100 mM and around pH7.5) should be suitable. The liquid for rehydrating the dried composition may be combined with a NADP+ regeneration system.

In an embodiment, the stored CYP enzyme is not rehydrated with a liquid containing a detergent, such as Emulphogene or Triton X-100.

The listing or discussion of an apparently prior-published document in this specification should not necessarily be taken as an acknowledgement that the document is part of the state of the art or is common general knowledge.

The invention will now be described in more detail with the aid of the following Figures and Examples.

Figure 1 is a graph demonstrating that an unlyophilised sample of DMB yeast-derived CYP3A4 microsomes shows no activity after 3 days at 3O⁰C. Figure 2 is a graph demonstrating that an unlyophilised sample of DMB yeast-derived CYP2D6 microsomes shows no activity after 3 days at 30⁰C.

Figure 3 is a graph demonstrating that an unlyophilised sample of DMB yeast-derived CYP3A4 microsomes shows no activity after 7 days at 21⁰C.

Figure 4 is a graph demonstrating that an unlyophilised sample of DMB yeast-derived CYP2C8 microsomes shows no activity after 10 days at 21⁰C.

Figure 5 is a graph demonstrating that an unlyophilised sample of DMB yeast-derived CYP1A1 microsomes shows no activity after 10 days at 21⁰C.

Figure 6 is a graph demonstrating that an unlyophilised sample of DMB yeast-derived CYP1A2 microsomes shows no activity after 10 days at 21⁰C.

Figure 7 is a picture illustrating the position of the buffer and microsomes reverse pipetted out at the 3 O'clock position.

Figure 8 is a graph depicting the activities of insect cell-derived CYP2D6 after lyophilisation treatments in buffers containing glycerol or sucrose. Controls: CYP2D6 enzyme thawed from -80⁰C and directly diluted in glycerol or sucrose containing buffer before analysis.

Figure 9 is a graph demonstrating that the lyophilisation treatment has no significant detrimental effects upon lyophilised Invitrogen CYP2D6 insect cell-derived microsomes after 20 hours storage at ~ 21⁰C.

Figure 10 is a graph demonstrating that the lyophilisation treatment has no significant detrimental effects upon DMB CYP2D6 yeast-derived microsomes after 20 hours storage at - 21⁰C.

Figure 11 is a graph demonstrating that the lyophilisation treatment has no significant detrimental effects upon Invitrogen CYP3A4 insect cell-derived microsomes after 20 hours storage at ~ 21 °C. Figure 12 is a graph demonstrating that the lyophilisation treatment has no significant detrimental effects upon BD Gentest CYP3A4 insect cell-derived microsomes after 20 hours storage at ~ 21⁰C.

Figure 13 is a graph demonstrating that the lyophilisation treatment has no significant detrimental effects upon DMB CYP3A4 yeast-derived microsomes after 20 hours storage at room temperature ~ 21⁰C.

Figure 14 is a graph demonstrating that the lyophilisation treatment has no significant detrimental effects upon BD Gentest CYP2C8 insect cell-derived microsomes after 20 hours storage at room temperature ~ 21⁰C.

Figure 15 is a graph demonstrating that the lyophilisation treatment has no significant detrimental effects upon Cypex CYP2C8 bacterial-derived microsomes after 20 hours storage at room temperature ~ 21⁰C.

Figure 16 is a graph demonstrating that the lyophilisation treatment has no significant detrimental effects upon DMB 2C8 yeast-derived microsomes after 20 hours storage at room temperature - 21 °C.

Figure 17 shows the activities of DMB yeast-derived CYP1A1 before and after lyophilisation in sucrose-containing buffer. (A) Lyophilised sample was kept at room temperature (~+21 °C, RT) for 7 days before analysis of activity. (B) Lyophilised sample was kept at room temperature (~+21⁰C, RT) for 24 days before analysis of activity.

Figure 18 shows the activities of DMB yeast-derived CYP1B1 before and after lyophilisation in sucrose-containing buffer. (A) Lyophilised sample was kept at room temperature (~+21 °C, RT) for 7 days before analysis of activity. (B) Lyophilised sample was kept at room temperature (~+21⁰C, RT) for 24 days before analysis of activity.

Figure 19. shows the activities of DMB yeast-derived CYP2D6 before and after lyophilisation in sucrose-containing buffer. (A) Lyophilised sample was kept at room temperature (~+21⁰C₁ RT) for 7 days before analysis of activity. (B) Lyophilised sample was kept at room temperature (~+21⁰C₁ RT) for 24 days before analysis of activity.

Figure 20 shows the activities of DMB yeast-derived CYP2C8 before and after lyophilisation in sucrose-containing buffer. (A) Lyophilised sample was kept at room temperature (~+21 °C, RT) for 7 days before analysis of activity. (B) Lyophilised sample was kept at room temperature (-+21⁰C, RT) for 24 days before analysis of activity.

Figure 21 shows the activities of DMB yeast-derived CYP1A2 before and after lyophilisation in sucrose-containing buffer. (A) Lyophilised sample was kept at room temperature (~+21 °C, RT) for 7 days before analysis of activity. (B) Lyophilised sample was kept at room temperature (-+21⁰C, RT) for 24 days before analysis of activity.

Figure 22 shows the activities of DMB yeast-derived CYP2A6 before and after lyophilisation in sucrose-containing buffer. (A) Lyophilised sample was kept at room temperature (~+21 °C, RT) for 7 days before analysis of activity. (B) Lyophilised sample was kept at room temperature (-+21⁰C, RT) for 24 days before analysis of activity.

Figure 23 shows the activities of DMB yeast-derived CYP2E1 before and after lyophilisation in sucrose-containing buffer. (A) Lyophilised sample was kept at room temperature (~+21 °C, RT) for 7 days before analysis of activity. (B) Lyophilised sample was kept at room temperature (-+21⁰C, RT) for 24 days before analysis of activity.

Figure 24 shows the activities of DMB yeast-derived CYP3A4 before and after lyophilisation in sucrose-containing buffer. (A) Lyophilised sample was kept at room temperature (-+21⁰C₁ RT) for 7 days before analysis of activity. (B) Lyophilised sample was kept at room temperature (-+21⁰C, RT) for 24 days before analysis of activity.

Figure 25 is a graph demonstrating the mean slope per minute using lyophilised DMB yeast-derived CYP1A2, in the presence of different sugars, after 12 days at room temperature (-+21⁰C).

Figure 26 is a graph demonstrating the mean slope per minute using lyophilised BD Gentest insect cell-derived CYP1A2, in the presence of different sugars, after 12 days at room temperature (-+21 °C).

Figure 27 is a graph demonstrating the mean slope per minute using lyophilised Cypex bacterial-derived CYP1A2, in the presence of different sugars, after 12 days at room temperature (-+21⁰C). Figure 28 is a graph demonstrating the conversion of dextramethorphan to dextrophan by DMB yeast-derived CYP2D6 after overnight storage at room temperature. The amount of dextramethorphan converted per pmol of CYP2D6 is graphically represented.

Figure 29 is a table showing the inhibition by quinidine of CYP2D6, obtained from Cypex, BD Gentest, and DMB expressed as IC₅₀ values.

Figure 30 is a graph demonstrating the inhibition by quinidine of CYP2D6, obtained from Cypex, BD Gentest, and DMB expressed as IC₅₀ values in μM depicted in a graphical form. The values are an average of three different experiments.

Figure 31 is an illustration of a typical plate set up for a high throughput assay.

Figure 32 is a graph demonstrating the log mean slope per min of CYP3A4 with increasing concentrations of sucrose after lyophilisation as well as fresh samples stored at -80C. 1xSD is indicated. Lyo = Lyophilised samples; M80 = -80⁰C samples.

Figure 33 is a graph demonstrating the log mean slope per min of CYP3A4 with increasing concentrations of man nose after lyophilisation as well as fresh samples stored at -80C. 1xSD is indicated. Lyo = Lyophilised samples; M80 = -80⁰C samples.

Figure 34 is a graph demonstrating the mean RFU slope per min of DMB yeast-derived CYP1A2 lyophilised in the presence of no buffer, phosphate buffer, 20% w/v sucrose + phosphate buffer, and a fresh sample stored at -80C.

Example 1 : Rapid reduction of CYP activities at 30⁰C and 21⁰C (laboratory/room temperature)

Introduction The ability of CYPs to perform their functions is graded by what is known as their activities. In simple terms this means, if one has a fixed amount of a CYP, the amount of a compound that is modified per unit of time. There are various methods of measuring CYP activities. If the CYP modification causes the molecule to become fluorescent, then a machine (e.g. a fluorometer) that counts photons may quantify the rate of this production. This method has been used most extensively in the Examples that follow. However, it is also possible to derive data from, e.g., a liquid chromatograph/mass spectrometer (LC/MS) that can purify the start compound from the modified compound and quantify the conversion based on differences in molecular charges and mass that is caused by the conversion.

The CYP enzyme system, as nature intended, works in a temperature, pH and electron (redox) controlled environment. Thus, to our test reactions we added an NADPH regenerating system that allows the reformation of hydrogen after it has been spilt into a proton and an electron. As the electron is taken away and is traded by the reductases as a powerful tool of chemical disruption for the CYPs such recycling is required to prevent electron depletion. Buffers are also added to control the pH and machines (i.e. thermostats) are used to control the temperature.

We have used a 10OmM potassium phosphate buffer pH 7.5 with a sugar at 20% w/v (as treatment buffer), followed by removal of water with a machine that operates under very low pressure, thereby greatly reducing the vapour pressure and assisting in the vaporisation of water.

To test the activities of all microsomes, 100μl reaction mixes were used which were made up as described below. The assay-specific number of picomoles (pmols) of CYP content of microsomes is diluted to 10μl in a half-molarity strength of the buffer used to make the 90μl portion of the reaction buffer (Potassium Phosphate or Tris buffers). The reaction buffer consisting of the Potassium Phosphate or Tris buffer, a NADPH regenerating system and CYP substrate is diluted to 90μl in water. The regenerating system also contains magnesium chloride and sodium citrate.

We decided that the dried microsomes should be adhered to 96-well microtitre plates using the normal ratios of buffers that are already known to work.

Due to the optimisation of the assays, between 1 and 4pmol of CYP may be required for a fluorescent assay. Due to the variable nature of the CYP expression systems, this results in a range of microsome volumes being used from less than 1 μl to more than 4μl. Since the treatment buffer tops-up the volume to 10μl, the ratio of CYPs to treatment buffer is variable.

In these experiments, we have used Ultrapure water (Millipore, MiIIi-Q; Synthesis A10 cartridge; 18.2mΩ, cm@25°C; 4ppb) to re-hydrate the microsomes from their dried state, based on the principle that only what has been taken away has been put back.

However, other buffers may be employed. Aims

In this Example, we aimed to demonstrate the reduction of microsome activity of untreated microsomes at room temperature (about 21⁰C), and at 3O⁰C.

Materials and Methods

In this Example, we used commercial samples of yeast-derived recombinant CYP3A4 and CYP2D6 from De Montfort Biopharma, UK (DBM). During their production, batches of CYP3A4 and CYP2D6 were resuspended at the final step in 1OmM Tris-HCI, 1mM EDTA and 20% v/v glycerol. These samples were thawed on ice and aliquoted into standard 1.5ml microtubes and were placed in a 3O⁰C static incubator (Sanyo, MIR-262) for 3, 7 or 10 days.

Where necessary the microsomes were diluted: the CYP3A4 assay required 1 pmol of CYP per sample in 10μl of buffer which equates to 1.1 μl of CYP3A4 in 8.9μl of 0.1 M potassium phosphate buffer, pH7.5. The CYP2D6 assay required 2.5 pmol of CYP per sample in 10μl of buffer which equates to 2.5μl of CYP2D6 in 7.5μl of 0.25M potassium phosphate buffer, pH7.5.

These assays used black, sterile, flat, clear-bottomed 96-well microtitre plates with lids (Costar, Type 3904) and the Biotek Synergy HT plate reader with KC4 (V3.9 Rev 12) software.

Unless otherwise stated, all results obtained from the following assays were obtained from experimental observations performed in triplicate.

CYP3A4 Assay

CYP3A4 reaction using DBOMF as substrate was performed in a total volume of 100 μl which contains: • 49 μl of 200 mM potassium phosphate buffer, pHδ.O,

• 34.9 μl ultra-pure water,

• 5 μl Solution A (20 mg/ml NADP⁺, 20 mg/ml glucose-6-phosphate and 13.3 mg/ml MgCI₂),

• 1 μl Solution B (5 mM sodium citrate with 40 Units per ml of glucose-6-phosphate dehydrogenase),

• 0.1 μl of 2 mM DBOMF (Invitrogen), • 10 μl of diluted microsomes containing one pmol of yeast CYP3A4 (i.e. 1.1 μl of microsomes in 8.9 μl of 100 mM potassium phosphate buffer, pHδ.O).

The total 100 μl reaction mixes were plated out in duplicate. The following plate reader parameters were used: Gain 60; Excitation 485 nm / Emission 530 nm. The mean values were plotted from the duplicates.

CYP2D6 Assay

CYP2D6 reaction using AMMC as substrate was performed in a total volume of 100 μl which contains:

• 20 μl of 0.5 M potassium phosphate buffer, pH 7.6,

• 62.37 μl water,

• 34.9 μl ultra-pure water,

• 0.63 μl Solution A (1 mg/ml NADP⁺, 20 mg/ml glucose-6-phosphate and 13.3mg/ml MgCI₂),

• 6 μl of 25 μM AMMC (BD Gentest),

• 10 μl of diluted microsomes containing 2.5 pmol of yeast CYP2D6 (i.e. 2.5 μl of microsomes in 7.5 μl of 0.25 M potassium phosphate buffer, pH7.6).

The total 100 μl reaction mixes were plated out in duplicate. The following plate reader parameters were used: Gain 100; Excitation 400 nm / Emission 460 nm.

CYP2C8 Assay

CYP2C8 reaction using DBF as substrate was performed in a total volume of 100 μl which contains:

• 10μl of 0.5M Potassium Phosphate buffer, pH7.6,

• 73μl Ultra pure water, • 5μl Solution A (20 mg/ml NADP⁺, 20 mg/ml Glucose-6-Phosphate and 13.3 mg/ml

MgCI₂),

• 1 μl Solution B (5mM Sodium Citrate with 40 Units per ml of Glucose-6-Phosphate dehydrogenase, type XV)

• 1 μl of 0.1 mM DBF (Sigma), • Lyophilised microsomes containing CYP2C8 were resuspended in 9.5μl of water. The plate was left at room temperature for 15 min. The total 100 μl reaction mixes were plated out in duplicate. The following plate reader parameters were used: Gain 75; Excitation 485 nm / Emission 530 nm.

CYP1A1 and CYP1 B1 Assay

CYP1A1/CYP1 B1 reactions using ethoxyresorufin as substrate were performed in a total volume of 100 μl which contains:

• 5μl 50OmM potassium phosphate buffer pH, 7.5

• 35μl 10OmM potassium phosphate buffer pH, 7.5 • 39μl Ultra pure Water,

• 5μl Solution A (20mg/ml NADP⁺, 20mg/ml Glucose-6-Phosphate and 13.3mg/ml MgCI₂),

• 1 μl Solution B (5mM Sodium Citrate with 40 Units per ml of Glucose-6-Phosphate dehydrogenase), • 5μl of O.1mM ethoxyresorufin (Sigma),

• 10 μl of diluted microsomes containing 1.5 pmol of CYP1A1/CYP1B1 (i.e. 1.5 μl of microsomes in 8.5 μl of 100 mM potassium phosphate buffer, pH7.6).

The total 100 μl reaction mixes were plated out in duplicate. The following plate reader parameters were used: Gain 55/65; Excitation 530 nm / Emission 590 nm.

CYP2E1 Assay

CYP2E1 reaction using EOMCC (Invitrogen) as substrate was performed in a total volume of 100 μl which contains: • 50μl of RBIII (400 mM potassium phosphate buffer pH 8.0),

• 33.5μl Ultra pure Water,

• 1 μl Solution B (5mM Sodium Citrate with 40 Units per ml of Glucose-6-Phosphate dehydrogenase),

• 0.5μl of mM EOMCC,

• 10 μl of diluted microsomes containing 2.5 pmol of CYP2E1 (i.e. 2.5 μl of microsomes in 7.5 μl of RBIII buffer).

The total 100 μl reaction mixes were plated out in duplicate. The following plate reader parameters were used: Gain 75; Excitation 400 nm / Emission 460 nm. CYP1A2 Assay

CYP1A2 reaction using CEC as substrate was performed in a total volume of 100 μl which contains: • 1 A2 Reaction buffer for 1 sample for 90μl,

• 20μl of 50OmM potassium phosphate buffer pH, 7.5

• 59μl Ultra pure Water,

• 5μl Solution A (20mg/ml NADP⁺, 20mg/ml Glucose-6-Phosphate and 13.3mg/ml MgCI₂), • 1 μl Solution B (5mM Sodium Citrate with 40 Units per ml of Glucose-6-Phosphate dehydrogenase),

• 5μl of 0.32mM CEC,

• 10 μl of diluted microsomes containing 1 pmol of CYP1A2 (i.e. 2.5 μl of microsomes in 9 μl of ^ΛA strength RBI buffer (10OmM potassium phosphate buffer, pH 8.0).

The total 100 μl reaction mixes were plated out in duplicate. The following plate reader parameters were used: Gain 82; Excitation 400 nm / Emission 460 nm.

CYP2A6 Assay

CYP2A6 reaction using Coumarin as substrate was performed in a total volume of 100 μl which contains:

• 2A6 Reaction buffer for 1 sample for 90μl,

• 20μl of 50OmM Tris buffer pH, 7.5 • 58μl Ultra pure Water,

• 5μl Solution A (1 mg/ml NADP⁺, 20mg/ml Glucose-6-Phosphate and 13.3mg/ml MgCI₂),

• 1 μl Solution B (5mM Sodium Citrate with 40 Units per ml of Glucose-6-Phosphate dehydrogenase), • 6μl of 0.32mM Coumarin (Sigma),

• 10 μl of diluted microsomes containing 1 pmol of CYP1A2 (i.e. 2.5 μl of microsomes in 9 μl of V2 strength RBI buffer (10OmM potassium phosphate buffer, pH 8.0).

The total 100 μl reaction mixes were plated out in duplicate. The following plate reader parameters were used: Gain 82; Excitation 400 nm / Emission 460 nm. Results

The loss of activity of untreated (i.e. unlyophilised) DMB yeast-derived CYP3A4 and CYP2D6 microsomes at 3O⁰C is shown in Figures 1 and 2, respectively. After 3 days storage at 30⁰C, the microsomes had lost activity.

The loss of activity of untreated (i.e. unlyophilised) DMB yeast-derived CYP3A4 microsomes at 21⁰C (laboratory/room temperature) is shown in Figure 3. After 7 days storage at 21⁰C, the microsomes had lost activity.

The loss of activity of untreated (i.e. unlyophilised) DMB yeast-derived CYP2C8,

CYP1A1 and CYP1A2 microsomes at 21⁰C (laboratory/room temperature) is shown in

Figures 4, 5 and 6, respectively. After 10 days storage at 21⁰C, the microsomes had lost activity.

Conclusion

Untreated microsomes lose CYP activities within a matter of days at 21⁰C

(laboratory/room temperature).

Example 2: Lyophilisation to prevent loss of recombinant CYP activity on storage

Aims

We aimed to prevent the reduction of microsome activity on storage at room temperature (about 21⁰C).

Materials and methods

Treatment buffers

For DMB insect cell CYP2D6 microsomes, the microsomes were diluted with treatment buffers:

(1 ) 10OmM potassium phosphate buffer pH7.5 containing 20% v/v glycerol and

(2) 10OmM potassium phosphate buffer pH7.5 containing 20% w/v sucrose to obtain 2.5 pmol of CYP2D6 in a 10μl volume (3μl of 2D6 microsomes + 7μl of 20% glycerol buffer or 3μl of 2D6 microsomes + 7μl of 20% sucrose buffer).

Other microsomes were diluted in a 10OmM potassium phosphate buffer pH7.5 containing 20% w/v sucrose. The ratios of volume (in μl) of microsomes to buffers are detailed in Table 1 , below. Due to the specific contents and the quantities of pmol required for different assays, there are differences in the ratios in the volumes of microsomes to buffer used.

Lvophilisation

Standard microsomes from a range of suppliers were treated using the following method.

Black, sterile, flat, clear-bottomed 96-well microtitre plates with lids (Costar, Type 3904) were filled with a final volume of 10μl made up of treatment buffer and microsomes. Different CYP assays require different concentrations of CYPs, and different cell type and brands have varying CYP contents per volume. Thus, for the range of enzymes tested, the microsomes occupied between 0.7μl to 4.4μl of the 10μl final volume, and the treatment buffer occupied between 9.3μl and 5.6μl.

The treatment buffer was first reverse pipetted into the edge of the wells at the 3 o'clock position, such that the location of the buffer was known (see well A1 in Figure 7). Microsomes were subsequently reverse pipetted into the buffer. Reverse pipetting (over- depressing the pipette plunger to overfill the pipette tip) was used since it is believed to reduce bubble formation.

The lids of the microtitre plate(s) were replaced and the plates wrapped in a standard thick polypropylene bag before storage at -8O⁰C for 1 hour (this prevents water condensation forming on the microtitre plate after removal from the freezer).

After being unwrapped, the plates were immediately transferred (to prevent condensation forming) to a Heto Dry Winner machine (model no. CT/DW60E) fitted with a BOC- Edwards vacuum pump (model no. RV5), for drying. The condensing chamber of the drying machine had been pre-cooled for 1 hour. The system has a plastic chamber (approx 10 litre capacity) and has two metal tray racks. The microtitre plate lid(s) were placed on top, at an angle, with one side on the plate surface so as not to hinder the drying process. Depending on the number of microtitre plates being processed the plates remained in the machine for 2-4 hours.

The dried plates were vacuum-sealed into bags using a vacuum-sealing machine (Orved Eco Vacuum Pro which reaches pressure of 0.15 bar) to prevent the ingress of water vapour and potential oxidation of the dried microsomes. The 20x30cm bags have an airtight polyamide exterior with a food grade polyethylene interior that should not leach plasticisers. The plate(s) were stored in the dark to prevent potential photo-degradation (actual effects of light are unknown) at around 21⁰C (i.e. a normal laboratory environment will always have slight temperature variations). This makes the tests more realistic rather then using an incubator.

Rehydration

To use the microtitre plates, microsomes were resuspended in the same volume of

Ultrapure water (reverse pipetted at the 3 O'clock position) as the treatment buffer used.

Since microsomes are viscous, in a dried state it takes a long time to rehydrate them properly, otherwise the microsome surface area will be vastly reduced. Therefore, the experiment was conducted in the following order: rehydration, set-up of plate reader to warm to 37⁰C, dilution of control -8O⁰C microsomes (not lyophilised) and production of the reaction mix. This allows at least 15 minutes for rehydration which has been deemed sufficient.

Assays

CYP activity was assayed as described in Example 2. Since the plate reader is set to shake before each time point for assays lasting a minimum of 30 minutes, less than classical kinetic data may be produced as microsomes become more homogenous with the reaction over time.

Table 1

ω

Results

The effect of different buffers on microsome stability following lyophilisation was tested. Specifically, the stability of DMB Insect cell CYP2D6 microsomes in 10OmM potassium phosphate buffer, pH 7.5 with either 20% v/v glycerol or 20% w/v sucrose, was determined after lyophilisation.

Samples were plated in duplicate and the plate stored at -80⁰C for 1 hour and lyophilised for approximately 2 hours as described. Following storage at around 21⁰C for 40 hours, the activity of CYP2D6 was assayed. As a control, CYP2D6 enzyme was thawed from - 80⁰C without lyophilisation, and diluted in the glycerol or sucrose containing buffers before analysis.

The activities of insect cell-derived CYP2D6 with and without lyophilisation treatments in buffers containing glycerol or sucrose are shown in Figure 8. Microsomes treated with the glycerol buffer lost all CYP2D6 activity after lyophilisation, whereas CYP2D6 activity was retained in microsomes treated with the sucrose buffer.

The normal microtitre plate stability parameters developed for sucrose buffer were then used to test the stability of yeast-derived (DMB)₁ insect cell-derived (Invitrogen), insect cell-derived (BD Gentest), and bacterial cell-derived (Cypex) CYP enzymes after lyophilisation.

Figures 9-16 show that the lyophilisation treatment process itself has no significant detrimental effects upon Invitrogen CYP2D6 microsomes, DMB CYP2D6 microsomes, Invitrogen CYP3A4 microsomes, BD Gentest CYP3A4 microsomes, DMB CYP3A4 microsomes, BD Gentest CYP2C8 microsomes, Cypex CYP2C8 microsomes, and DMB CYP2C8 microsomes, respectively, after 20 hours storage at room temperature (-+21⁰C).

The stability of microsomes in a 25OmM Tris buffer after lyophilisation was also tested but resulted in a slightly lower activity than rehydrated CYPs in a 10OmM potassium phosphate, pH 7.5 buffer (data not shown).

Conclusion After lyophilisation, sucrose-containing buffer afforded room-temperature stable microsomes. All CYP2D6 activity was lost after lyophilisation in glycerol containing buffer. Lyophilisation in sucrose-containing buffer maintains activity of CYP enzymes manufactured in diverse cell systems after 20 hours at +21⁰C (i.e. room temperature/lab temperature).

Example 3: Lyophilisation to prevent loss of recombinant CYP activity on storage for up to 24 days

Aim

We aimed to prevent the reduction of microsome activity on storage at room temperature

(about 21⁰C) for up to 24 days.

Materials and Methods

DMB yeast-derived CYP enzymes were tested for long term stability after lyophilisation in sucrose buffer as described in Example 3 and the lyophilised sample was stored for 7 or

24 days at room temperature (~+21 °C, RT). CYP activity was assayed as described in Example 2 and mean RFU slopes per minute recorded plus and minus one standard deviation.

Results

Figures 17-24 show activities of DMB (yeast-derived) CYP1A1 , CYP1B1, CYP2D6, CYP2C8, CYP1A2, CYP2A6, CYP2E1, and CYP3A4, respectively, before and after lyophilisation in sucrose-containing buffer. Lyophilised samples were kept at room temperature (~+21⁰C, RT) and stored for 7 days (part (A) of the figures) or 24 days (part (B) of the figures) before analysis of activity. In each case, activities of the lyophilised samples were the same as or higher than the non-lyophilised samples.

Conclusion

Lyophilisation in sucrose-containing buffer maintains activity (within the limits of experimental error as indicated by the standard deviations obtained from experiments performed in triplicate) of DMD (yeast-derived) CYP enzymes after storage for 7 and 24 days at +21⁰C (i.e. room temperature/lab temperature).

Example 4: Effect of different sugars in the treatment procedure with CYP1 A2.

Aim We aimed to determine whether the presence of different sugars in the treatment buffer has an effect on the stability afforded by lyophilisation. Materials and Methods

CYP1A2 obtained from DMB, BD Geπtest and Cypex was tested for stability after lyophilisation in 10OmM potassium phosphate buffer, pH 7.5, comprising different sugars at 20% w/v concentrations (sorbitol, trehalose, maltose, raffinose, sucrose, mannose, glucose and galactose) and storage of the lyophilised sample for 12 days at room temperature (-+21⁰C, RT). CYP activity was assayed as described in Example 2 and mean RFU slopes per minute recorded plus and minus one standard deviation.

Results Figures 25-27 show activities of lyophilised DMB (yeast-derived) CYP1A2, lyophilised BD Gentest (insect cell-derived) CYP1A2 and lyophilised Cypex (bacterial-derived) CYP1A2, respectively, in the presence of different sugars, after 12 days storage at room temperature (~+21 °C).

The work was replicated using a 25OmM Tris buffer; however, CYP activities were generally maintained less successfully than in the 10OmM Potassium Phosphate buffer (data not shown). This could be due to loss of activity on dehydration or rehydration.

Conclusion These experiments indicate that CYP enzymes may be stabilised using different sugars. Sucrose generally works well with all commercially available enzymes but is the best for the insect cell-produced enzymes. Trehalose appears to be the best for the yeast cell- produced enzymes and sorbitol for bacterial cell-produced enzymes.

Example 5: Confirmation of stability of lyophilised microsomes using liquid chromatography/mass spectrometry (LC/MS)

Aim

To confirm the stability of lyophilised microsomes using a non-fluorescence based LC- MS assay.

Materials and Methods

LC-MS was used to monitor the conversion of the substrate dextromethorphan hydrobromide (Sigma D9684-5g, MW 370.3) into the analyte dextrorphan, produced by a dextromethorphan O-demethylase reaction. A 1OmM substrate stock in ethanol was diluted to 100μM in water and 1.25μl used in a 100μl reaction to give a final substrate concentration of 125nM.

A reaction mixture consisting of: • 50μl 20OmM Potassium Phosphate pH 8.0,

• 10μl Solution A (20mg/ml NADP⁺, 20mg/ml Glucose-6-Phosphate and 13.3mg/ml MgCI₂),

• 2μl Solution B (5mM Sodium Citrate with 40 Units per ml of Glucose-6-Phosphate dehydrogenase), • 1.25μl substrate,

• 26.75μl Ultrapure water, was added to a microtitre plate containing 10μl of rehydrated microsomes (see Example 2) to give a total reaction volume of 100μl. The plate was incubated at 3O⁰C in a static incubator (Sanyo) with shaking (500rpm). Samples were removed after 10, 20 and 30 minutes and placed into neutralised glass tubes. The reaction was stopped by adding 100μl of 0.05% formic acid in acetonitrile and placing the sample on ice for 10 minutes. The tubes were then centrifuged at 300Og for 15 minutes at 4⁰C.

The supernatant was transferred to HPLC vials and 20μl injected for analysis. The HPLC analysis was performed on an Agilent LC/MS single quadrapole system (LC/MSD SL - G 1956B) under the following conditions:

• Column, Agilent Eclipse XDB Zorbax C18,

• Column Temperature 3O⁰C,

• Solvent A 0.05% formic acid in water, • Solvent B 0.05% formic acid in acetonitrile,

• Solvent flow rate 1 ml per minute,

• Max pressure 400psi.

Binary Solvent Gradient

Post Run 3 minutes at 10% B

Machine Settings

Retention times

Substrate retention time: 8.5 minutes. Analyte retention time: 7 minutes

Results The ability of lyophilised DMB CYP2D6 to convert dextromethorphan to dextrorphan, after overnight storage at room temperature, was analysed using the LC-MS assay described above. The activity of DMB CYP2D6 lyophilised in treatment buffer comprising trehalose or mannose (20% w/v) was measured. As controls, the activities of non-lyophilised DMB CYP2D6 in phosphate buffer directly thawed from -80⁰C, non- lyophilised DMB CYP2D6 in trehalose buffer and non-lyophilised DMB CYP2D6 in mannose buffer were determined.

Figure 28 shows DMB CYP2D6 microsomes demonstrate HPLC/MS activity after lyophilisation treatment. The amount of dextromethorphan converted per pMol of CYP2D6 is graphically represented.

Conclusion

These experiments indicate that lyophilisation-mediated stabilisation of microsomal CYP enzyme activity can be monitored either by fluorescence-based assays or by routine LC/MS assays (which are non-fluorescent). Example 6: Lyophilisation does not alter the active site of the CYP2D6 enzyme

Aim

To determine if lyophilisation treatment alters the active site of the CYP2D6 enzyme.

Materials and Methods

Inhibition of CYP2D6 by quinidine (a CYP2D6 inhibitor) was studied using AMMC (3-[2- (N,Ndiethyl-N-methylammonium)ethyl]-7-methoxy-4-methylcoumarin) as a substrate. The assay is based on demethylation of AMMC to AHMC (3-[2-(N,N-diethylamino)ethyl]- 7-hydroxy-4-trifluoromethylcoumarin). Three brands of CYP2D6 were tested (Cypex, BD Gentest and DMB). Microsomes were lyophilised from the 10OmM potassium phosphate buffer described in Example 3 in the presence of 3 different sugars.

IC₅₀ determinations were performed in a 96-well microtitre plate in duplicate rows of 12 wells (rows in the plate are designated A to H and columns are designated 1-12). The test compound (i.e. CYP2D6) was added to the wells in column 1 and serially diluted to the wells in column 8. Wells 9 and 10 were control wells which either contained no test compound or contained positive controls (therefore either no inhibition or full signal is detected). The wells in columns 11 and 12 were blanks. STOP solution was added prior to the addition of the enzyme/substrate mix to the NADPH regenerating system in columns 11 and 12. The only signal present in these wells was background noise. The assay was conducted in a final volume of 0.2 ml per well.

A typical plate set up for a high throughput assay is depicted in Figure 31. • Columns 1 -8 are serial dilutions (3-fold) of the test compound or positive control.

• Columns 9 and 10 contain no test compound.

• Columns 11 and 12 are blank controls (STOP solution added prior to initiation of the reaction).

• Rows A/B, C/D, EΞ/F and G/H are replicates.

This set up allows the study of one enzyme and four test compounds and/or positive controls or four enzymes and one test compound and/or positive control.

The specific procedure used to determine the IC50 values is as follows: 1. A multi-channel pipette was used to dispense 0.144 ml Serial Dilution Buffer that contains the NADPH regenerating system (1.3 mM NADP+, 3.3 mM Glucose-6- Phosphate, 3.3 mM Magnesium Chloride and 0.4 Units/ml Glucose-6-Phosphate Dehydrogenase) and 100 mM Potassium Buffer, pH 7.4, into the wells in column 1.

2. A multi-channel pipette was used to dispense 0.1 ml of Serial Dilution Buffer into the wells in columns 2 - 12.

3. 0.006 ml of the test compound (i.e. the inhibitor quinidine for the CYP2D6 assay) or positive control was added to the desired well(s) in column 1.

4. A multi-channel pipette was used to serially dilute 0.05 ml from the wells in column 1 to the wells in columns 2 through 8. The contents in each well were mixed by pipetting 3 to 5 times. The tips were changed during the serial dilution. The (extra) 0.05 ml in the wells in column 8 were removed and discarded.

5. A lid was placed on the plate and the plate incubated in a 37°C incubator for at least 10 min (to pre-warm the buffer and plate).

6. A multi-channel pipette was used to dispense 0.1 ml enzyme/substrate mix (2.5 pmol of enzyme and 0.0025 mM of substrate in 100 mM Potassium Phosphate buffer, pH 7.4) to columns 1 through 10. The liquid was dispensed in a stream, not dropwise. Mixing of the components in the wells is dependent upon dispensing rapidly.

7. The lid was replaced and the plate was incubated at 37°C for 30 min.

8. A multi-channel pipette was used to dispense 0.075 ml of STOP Solution (80% acetonitrile/20% 0.5 M Tris base). 100 ml 0.5 M Tris base (prepared by dissolving 60.55 g of solid Tris base in 1000 m deionised water; pH was unadjusted) was added to 400 ml acetonitrile. The elevation in pH provided by the Tris base improves the signal to noise ratio for AHMC fluorescence and is critical for adequate signals in all wells. The solution was dispensed in a stream, not dropwise.

9. A multi-channel pipette was used to dispense 0.1 ml of the respective enzyme/substrate mix to the wells in columns 11 and 12.

10. The plate was scanned with a fluorescent plate scanner (Biotek) after an inactivation time of 30 min. The recommended excitation/emission filters for the specific assays are 390 nm (20 nm) and 460 nm (40 nm) respectively. For the quinidine solutions used in the CYP2D6 inhibition assay, a 5 mM stock solution was prepared by adding 3.78 mg quinidine to 2.0 ml acetonitrile and a working solution (0.025 mM) prepared by diluting 0.01 ml 5 mM quinidine stock solution in 2.0 ml acetonitrile.

Results

Figures 29 and 30 show quinidine inhibition of lyophilised and non-lyophilised CYP2D6 obtained from Cypex, BD Gentest and DMB₁ expressed as IC_5O values in μM depicted in a tabular form and graphical form respectively. The IC₅₀ values are an average of three different experiments.

Conclusion

The active sites of CYP2D6 enzymes produced in different cell systems remain unaltered after the lyophilisation process. The minor differences in the IC₅₀ values for inhibition of CYP2D6, as seen above, are within the limits of experimental error. Similar results have been obtained with other CYP enzymes (data not shown).

Example 7: Alterations in sugar concentrations

Aim

To determine an optimal sugar concentration range for lyophilisation using methods described in the previous studies with CYP3A4. This study used escalating concentrations of sucrose and mannose.

Materials and Methods

Identical methods to the previous CYP3A4 studies in Example 1 were used, except that the plate reader settings had Gain 60, and each well was read every 45 seconds. The buffer was always 100 mM Potassium Phosphate, pH 7.5, which contained varying amounts of sugar.

1μl (~1 pmol) of DMB's yeast CYP3A4 in 9μl of buffer containing different concentrations of sugars (i.e. sucrose or mannose), as detailed in Table below, aliquoted into wells of a 96-well microtitre plate, was lyophilised. This forms a i/θ"¹ dilution of the stock buffer. (Sucrose has MW = 342; Mannose has MW = 180).

The samples were stored for 18 days at ambient room temperature (-21⁰C) in the laboratory before activity was measured.

Each test plate was laid out in triplicate, and contained CYP3A4 lyophilised in the presence of various concentrations of sucrose; CYP3A4 lyophilised in the presence of various concentrations of mannose; and exact replicates of fresh (i.e. not stored) -8O⁰C stocks in the presence of various concentrations of either sucrose or mannose.

Results

Figure 33 is a graph demonstrating the log mean slope per min of CYP3A4 with increasing concentrations of mannose after lyophilisation as well as fresh samples stored at -80C. 1xSD is indicated. Lyo = Lyophilised samples; M80 = -80⁰C samples.

In both cases, low concentrations of sugar (2.2% w/v) increased the stability of the CYP3A4, and the activity on storage increased with an increasing concentration of sugar. From these results, 9%w/v or more of sugar is preferred.

Conclusion

With sucrose, stability increases with the concentration of added sucrose, and perhaps would continue to rise if the percentage was increased above 36%. With mannose, again stability is improved as the mannose concentration increases. However, mannose is noticeably less effective at retaining CYP3A4 activity than sucrose. Example 8: Lyophilisation maintains activity of CYP1A2 after 95 days at room temperature.

Aim

Using methods described in previously for CYP1 A2, activity was tested after 3 months (95 days) storage at ambient laboratory temperature (~+21⁰C).

Materials and Methods Thee methods were essentially the same as those used on CYP1A2 in Example 1. DMB yeast CYP1A2 samples were lyophilised using 1 pmol and 9μl of buffer per well of a 96- well microtitre plate. The buffer used was 100 mM Potassium Phosphate, pH 7.5. After lyophilisation, the samples were stored for 95 days at ambient room temperature (~+21°C) in the lab. The only alteration to previous studies was linked to plate reader settings: Gain, 82. Each well was read every 42 seconds.

Plate Layout in Triplicates

1 μl CYP1A2 microsomes lyophilised in 100 mM Potassium Phosphate. 1μl CYP1A2 microsomes lyophilised in 100 mM Potassium Phosphate + 20% (w/v) sucrose.

1 μl CYP1 A2 microsomes lyophilised without the addition of any buffer.

1 μl CYP1A2 microsomes thawed directly from -80°C in 100 mM Potassium Phosphate buffer was used as a control.

Results

Figure 34 is a graph demonstrating the mean RFU slope per min of DMB yeast-derived CYP1A2 lyophilised in the presence of no buffer, phosphate buffer, 20% w/v sucrose + phosphate buffer, and a fresh sample stored at -80⁰C. Almost full CYP1A2 activity was retained in CYP1A2 lyophilised in the presence sucrose, whereas CYP1A2 lyophilised in the presence of phosphate buffer or no buffer retained only about 1 % activity.

Conclusion

Membrane-associated CYP enzymes do not have the inherent ability to be lyophilised without gross loss of activity. While the presence potassium ions assists in this process, it is not the main factor but the sugar is.

References

Baillie (2008) Chem. Res. Toxicol. 21, 129-137 Bertz (1997) CHn. Pharmacokinet. 32, 210-258.

Bruno & Njar (2007) Bioorg. Med. Chem. 15, 5047-5060.

Frye (2004) Molecular Interventions 4(3), 157-162.

Guengerich (2006) The AAPS Journal 8(1) Article 12, E101-E111.

McGinnity & Riley (2001) β/oc/)em. Soc. Trans. 29, 135-139.

Sistonen et a/ (2007) Pharmacogenet. Genomics. 17(2), 93-101.

Spatzenegger & Jaeger (1995) Drug Metabolism Reviews 27, 397-417.

Wang et al (1997) Proc Natl Acad Sci U S A. 94(1.6), 8411-8416.

Claims

1. A dried composition comprising a membrane-associated recombinant cytochrome P450 (CYP) enzyme, wherein the CYP enzyme is stable at 21⁰C for a period of at least 10 days.

2. A dried composition according to Claim 1 wherein the recombinant CYP enzyme has been produced in bacterial cells, yeast cells, insect cells, or mammalian ceils.

3. A dried composition according to Claim 2 wherein the yeast cells are Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pichia pastoris or Yarrowia lipolytica cells.

4. A dried composition according to Claim 2 wherein the bacterial cells are E. coli cells.

5. A dried composition according to Claim 2 wherein the insect cells are Sf9, Sf21 , Tni, High Five or Tn368 cells.

6. A dried composition according to Claim 2 wherein the mammalian cells are lymphoblastoid cells.

7. A dried composition according to any of the preceding claims wherein the recombinant CYP enzyme is stable at 21⁰C for a period of at least 24 days.

8. A dried composition according to Claim 7 wherein the recombinant CYP enzyme is stable at 21⁰C for a period of at least 3 months.

9. A dried composition according to any of the preceding claims wherein the recombinant CYP enzyme retains at least 30% of its activity compared to its activity prior to drying.

10. A dried composition according to any of Claims, 1-8 wherein the recombinant CYP enzyme retains at least 30% of its activity compared to its activity after storage at -8O⁰C for the same time period.

11. A dried composition according to Claim 9 or 10 wherein the recombinant CYP enzyme retains at least 50% of its activity.

12. A dried composition according to Claim 11 wherein the recombinant CYP enzyme retains at least 90% of its activity.

13. A dried composition according to any of the preceding claims wherein the recombinant CYP enzyme is a recombinant mammalian, preferably human, CYP enzyme.

14. A dried composition according to any of the preceding claims wherein the recombinant CYP enzyme is selected from CYP1B1, CYP7A1, CYP7B1 , CYP8B1, CYP11A1 , CYP11B1 , CYP11 B2, CYP17A1 , CYP19A1 , CYP21A2, CYP27A1 , CYP39A1 , CYP46A1 , CYP51A1 , CYP1A1, CYP1A2, CYP2A6, CYP2A13, CYP2B6, CYP2C8, CYP2C9, CYP2C18, CYP2C19, CYP2D6, CYP2E1 , CYP2F1 , CYP3A4, CYP4A5, CYP3A7, CYP2J2, CYP4A11 , CYP4B1 , CYP4F12, CYP3F2, CYP4F3, CYP4F8, CYP5A1 , CYP8A1 , CYP2R1 , CYP24A1 , CYP26A1 , CYP26B1 , CYP26C1 , CYP27B1 , CYP2A7, CYP2S1 , CYP2U1, CYP2W1 , CYP3A43, CYP4A22, CYP4F11 , CYP4F22, CYP4V2, CYP4X1 , CYP4Z1 , CYP20A1 and CYP27C1.

15. A dried composition according to Claim 14 wherein the recombinant CYP enzyme is selected from CYP3A4, CYP2D6, CYP2C8, CYP2C9, CYP1A1, CYP1 B1 , CYP2E1 and CYP1A2.

16. A dried composition according to Claim 15 wherein the recombinant CYP enzyme is CYP3A4, CYP2D6, CYP2C9, CYP1A2 or CYP2E1.

17. A dried composition according to any of the preceding claims wherein the composition further comprises a recombinant cytochrome P450 reductase (CPR) enzyme.

18. A dried composition according to Claim 17 wherein the recombinant CPR enzyme is mammalian, preferably human or rabbit, CPR enzyme.

19. A dried composition according to any of the preceding claims wherein the composition further comprises cytochrome b₅.

20. A dried composition according to any of the preceding claims wherein the composition further comprises a sugar.

21. A dried composition according to Claim 20 wherein the sugar is selected from sorbitol, trehalose, maltose, raffinose, sucrose, mannose, glucose and galactose.

22. A dried composition according to Claim 21 wherein the sugar is sucrose or trehalose.

23. A dried composition according to Claim 20 wherein if the recombinant CYP enzyme has been produced in bacterial cells the sugar is sorbitol, wherein if the recombinant CYP enzyme has been produced in yeast cells the sugar is trehalose, or wherein if the recombinant CYP enzyme has been produced in insect cells the sugar is sucrose.

24. A dried composition according to any of the preceding claims wherein the composition does not comprise glycerol.

25. A dried composition according to any of the preceding claims which has been lyophilised.

26. A dried composition according to any of the preceding claims, wherein the membrane-associated recombinant CYP is attached to a microsome.

27. A dried composition according to any of the preceding claims wherein the composition comprises more than one recombinant CYP enzyme.

28. A dried composition according to Claim 27 wherein the composition comprises any two or more of CYP1A1 , CYP1A2, CYP1 B1, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A4.

29. A solid support comprising therein or thereon a dried composition according to any of the preceding claims.

30. A solid support according to Claim 29 wherein the solid support is a multiwell plate.

31. A kit of parts comprising a dried composition according to any of Claims 1-28, or a solid support according to Claim 29 or 30, and any one, two, three, four, five or all six of: a sterile liquid for rehydrating the dried composition, a substrate for the one or more CYP enzymes in the dried composition, an inhibitor of the one or more CYP enzymes in the dried composition, NADPH, a negative control dried membrane preparation without CYP activity, and a positive control CYP enzyme stored at -80⁰C.

32. A kit of parts according to Claim 31 wherein: if the recombinant CYP is CYP1B1 , CYP7A1 , CYP7B1 , CYP8B1 , CYP11A1 , CYP11 B1 , CYP11B2, CYP17A1 , CYP19A1 , CYP21A2, CYP27A1 , CYP39A1, CYP46A1 or CYP51 A1 , the substrate is a sterol; if the recombinant CYP is CYP1A1 , CYP1A2, CYP2A6, CYP2A13, CYP2B6,

CYP2C8, CYP2C9, CYP2C18, CYP2C19, CYP2D6, CYP2E1 , CYP2F1 , CYP3A4, CYP4A5 or CYP3A7, the substrate is a xenobiotic; if the recombinant CYP is CYP2J2, CYP4A11 , CYP4B1 or CYP4F12, the substrate is a fatty acid; if the recombinant CYP is CYP3F2, CYP4F3, CYP4F8, CYP5A1 or CYP8A1, the substrate is a eiconasoid; and if the recombinant CYP is CYP2R1 , CYP24A1 , CYP26A1 , CYP26B1 , CYP26C1 or CYP27B1 , the substrate is a vitamin.

33. A kit of parts according to Claim 31 wherein the sterile liquid is water.

34. A kit of parts according to Claim 31 wherein the sterile liquid does not contain a detergent.

35. A kit of parts according to any of Claims 31-34 wherein the kit further comprises a CPR enzyme.

36. A kit of parts according to any of Claims 31-35 wherein if the recombinant CYP is CYP2A6, CYP2B6, CYP2C10, CYP2C8, CYP2C9, CYP3A4, CYP3A5, CYP3A7, CYP4F12, CYP4F2, CYP4F3A, CYP4F3B or CYP2E1 , the kit further comprises cytochrome 65.

37. A method of stabilising a recombinant membrane-associated CYP enzyme, the method comprising freeze-drying the CYP enzyme.

38. A method according to Claim 37 wherein the membrane-associated CYP enzyme is freeze-dried in the presence of a sugar.

39. A method according to Claim 38 wherein the sugar is selected from sorbitol, trehalose, maltose, raffinose, sucrose, mannose, glucose and galactose.

40. A method according to any of Claims 37-39 wherein the recombinant CYP enzyme is selected from CYP1 B1 , CYP7A1 , CYP7B1 , CYP8B1 , CYP11A1 , CYP11B1 , CYP11 B2, CYP17A1 , CYP19A1 , CYP21A2, CYP27A1 , CYP39A1, CYP46A1 , CYP51A1 , CYP1A1, CYP1A2, CYP2A6, CYP2A13, CYP2B6, CYP2C8, CYP2C9, CYP2C18, CYP2C19, CYP2D6, CYP2E1 , CYP2F1 , CYP3A4, CYP4A5, CYP3A7, CYP2J2, CYP4A11 , CYP4B1, CYP4F12, CYP3F2, CYP4F3, CYP4F8, CYP5A1 , CYP8A1 , CYP2R1 , CYP24A1, CYP26A1 , CYP26B1, CYP26C1, CYP27B1 , CYP2A7, CYP2S1 , CYP2U1 , CYP2W1 , CYP3A43, CYP4A22, CYP4F11 , CYP4F22, CYP4V2, CYP4X1 , CYP4Z1 , CYP20A1 and CYP27C1.

41. A method according to Claim 40 wherein the CYP enzyme is selected from CYP3A4, CYP2D6, CYP2C8, CYP1A1 , CYP1B1 , CYP2E1 and CYP1A2.

42. A method according to any of Claims 37-41 wherein the CYP enzyme is a human CYP enzyme.

43. A method according to any of Claims 37-41 wherein the CYP enzyme is a plant CYP enzyme, other than a plant CYP74 enzyme.

44. A method according to any of Claims 37-43 wherein the membrane-associated CYP enzyme is freeze-dried in the presence of a CPR enzyme.

45. A method of storing a recombinant membrane-associated CYP enzyme, the method comprising: providing a recombinant membrane-associated CYP enzyme; freeze-drying the CYP enzyme; and storing the dried CYP enzyme at greater than O⁰C.

46. A method according to Claim 45 wherein the membrane-associated recombinant CYP enzyme is freeze-dried in the presence of a sugar.

47. A method according to Claim 46 wherein the sugar is selected from sorbitol, trehalose, maltose, raffinose, sucrose, mannose, glucose and galactose.

48. A method according to Claim 47 wherein the sugar is trehalose or sucrose.

49. A method according to any of Claims 45-48 wherein the recombinant CYP enzyme is selected from CYP1B1, CYP7A1 , CYP7B1 , CYP8B1 , CYP11A1 , CYP11B1 ,

CYP11B2, CYP17A1 , CYP19A1, CYP21A2, CYP27A1 , CYP39A1 , CYP46A1 , CYP51A1, CYP1A1 , CYP1A2, CYP2A6, CYP2A13, CYP2B6, CYP2C8, CYP2C9, CYP2C18, CYP2C19, CYP2D6, CYP2E1, CYP2F1, CYP3A4, CYP4A5, CYP3A7, CYP2J2, CYP4A11 , CYP4B1, CYP4F12, CYP3F2, CYP4F3, CYP4F8, CYP5A1 , CYP8A1 , CYP2R1, CYP24A1 , CYP26A1, CYP26B1, CYP26C1, CYP27B1 , CYP2A7, CYP2S1, CYP2U1 , CYP2W1 , CYP3A43, CYP4A22, CYP4F11 , CYP4F22, CYP4V2, CYP4X1 , CYP4Z1 , CYP20A1 and CYP27C1.

50. A method according to Claim 49 wherein the CYP enzyme is selected from CYP3A4, CYP2D6, CYP2C8, CYP1A1, CYP1B1, CYP2E1 and CYP1A2.

51. A method according to any of Claims 45-50 wherein the CYP enzyme is a mammalian, preferably human, CYP enzyme.

52. A method according to any of Claims 45-50 wherein the CYP enzyme is a plant CYP enzyme, other than a plant CYP74 enzyme.

53. A method according to any of Claims 45-52 wherein the step of providing the CYP enzyme further comprises providing CPR enzyme.

54. A method according to any of Claims 45-53 wherein the dried CYP enzyme is stored at refrigeration temperature (about 4-8°C) or above.

55. A method according to Claim 54 wherein the dried CYP enzyme is stored at room temperature.

56. A method according to any of Claims 45-55 wherein the dried CYP enzyme is stored for at least 10 days.

57. A method according to Claim 56 wherein the dried CYP enzyme is stored for at least 3 months.

58. A method according to any of Claims 45-57 wherein the dried CYP enzyme retains at least 30% of its activity after storage.

59. A method according to Claim 58 wherein the dried CYP enzyme retains at least 50% of its activity after storage.

60. A method according to Claim 59 wherein the dried CYP enzyme retains at least 90% of its activity after storage.