Faculté des Sciences
Institut de Recherches Microbiologiques
Heterologous expression systems for the production of metabolites
during early drug research
Project verricht met steun van het Vlaams Gewest – IWT
Thèse présentée en vue de l’obtention du grade légal de Docteur en Sciences
Promoteur de thèse: Pr. Dr. L. Droogmans
Co‐promoteurs de thèse: Pr. Dr. J.‐P. Simon & Dr. H. Bohets
Inneke Wynant
2010
Heterologous expression systems for the production of metabolites during early drug research
Thèse privée soutenue le 23 juin 2010, à Gosselies
Thèse publique soutenue le 30 septembre 2010, à Gosselies
Composition du jury:
Président du jury: Pr. Dr. E. Pays, ULB Vice‐président du jury: Pr. Dr. O. Leo, ULB
Secrétaire du jury: Pr. Dr. A. M. Marini, ULB Promoteur de thèse: Pr. Dr. L. Droogmans, ULB Co‐promoteur de thèse: Dr. H. Bohets, J&J
Co‐promoteur de thèse: Pr. Dr. J.‐P. Simon, UBT
Rapporteur: Pr. Dr. E. Dubois, ULB Rapporteur: Pr. Dr. V. Kruys, ULB Expert extérieur: Dr. M. Vinken, VUB Expert: Pr. Dr. P. Fickers, ULB
Voor Joeri, Dante en Storm
Amor omnia vincit. Liefde overwint alles.
TABLE OF CONTENTS
THANKS TO…
LIST OF ABBREVIATIONS SUMMARY
INTRODUCTION………..1
A. Facts on drug metabolism 1
A.1. Biotransformation of xenobiotics 1
A.2. Phase I enzymes and localization 1
A.3. Biochemistry of CYP oxidative metabolism 2
A.4. Genetics and CYP nomenclature 4
A.5. CYP variability 5
B. Pharmaceutical Research and Development 7
B.1. Drug metabolism studies 7
B.2. Reactive drug metabolites 8
B.3. Drug metabolite synthesis 10
B.3.1. Classical organic chemistry 10
B.3.2. Enzymatic synthesis 11
B.3.3 CYP mimicry 26
B.4. Purification of metabolites 26
B.5. Conclusion 27
SCOPE OF THE THESIS ………28
RESULTS ………... 30
Experimental outcome………..30
A. Recombinant hCYP/HR for metabolite production 30
A.1. Metabolite production on analytical scale 30
A.1.1. Recombinant E. coli co‐expressing hCYP3A4/HR 30
A.1.2. hCYP3A4/HR plasmid construct 31
A.1.3. Expression for small culture volumes 31
A.1.4. Two biocatalytic systems: cells and cell membranes 32
A.1.5 Stability of activity during conservation 32
A.1.6. Kinetic parameters of recombinant hCYP3A4 32
A.2. Metabolite production on preparative scale 33
A.2.1. Application of minimal medium 33
A.2.2. Stability of plasmid construct and expression potential 35
A.2.3. Metabolite production in a controlled fermenter 36
A.2.1. Stability of activity during substrate turnover 36
B. Flexible multiphasic bioreactor system 37
B.1. Application of organic solvents 37
B.1.1. Biocompatibility 37
B.1.2. Drug partitioning 38
B.1.3. n‐Hexadecane‐diethyl ether mixture in a bioreactor 39
B.2. Application of immobilization 39
B.2.1. Immobilization in Ca‐alginate microbeads 39
B.2.2. Initial proliferation and viability in alginate beads 40
B.2.3. hCYP/HR expression in alginate beads 40
B.2.4. Biomass evolution during culture/conservation in alginate beads 41 B.2.5. Metabolic potential of cells in alginate beads 41
B.2.6. Metabolic potential of cell membranes in alginate beads 43
B.2.7. Alternative cell immobilization techniques 44
C. Case study: Validation of the E. coli bioreactor system 49
C.1. Setting the scene: human versus animal metabolism 49
C.2. CYP identification 51
C.3. Selective metabolite production 51
C.4. Preparative purification 52
Discussion of results……….…………..……….……. 53
A.1 Lab‐scale drug metabolite synthesis with recombinant hCYP/HR 53
A.2. Preparative metabolite synthesis with recombinant hCYP/HR 56
B.1. Biphasic systems for improved drug solubility 58
B.2. Immobilized hCYP/HR for metabolite production 58
C.1. Validation of selective metabolite biosynthesis using bioreactor technology 64
GENERAL DISCUSSION, CONCLUSION & PERSPECTIVES 66
BIBLIOGRAPHY………..
APPENDIX 1: Materials & methods……….
A. Cooperation B. Molecular biology C. Microbiology D. Enzymology
E. Analytical techniques and statistics F. Fermentation technology
G. Purification H. Drug solubility
I. Immobilization in Ca‐alginate microbeads J. Alternative immobilization techniques
APPENDIX 2: Scientific manifestations……….
Promoveren is zeker geen solo-aangelegenheid. Dit proefschrift zou dan ook nooit tot stand gekomen zijn zonder hulp. Bijgevolg had ik iedereen willen bedanken die mij de afgelopen jaren gesteund &
geholpen heeft.
Ik wil graag de volgende personen bij naam noemen:
Joeri,
Jij deelde mijn kleinste & grotere successen, maar jij was tevens mijn steun en toeverlaat tijdens de grootste beproevingen. Ik zal onze geanimeerde, nachtelijke discussies niet snel vergeten. Je was steeds liefdevol & je begrip lijkt soms grenzeloos. Dank je, omdat je steeds in mij bent blijven geloven!
Je zorgde tevens voor de nodig ontspanning in mijn spannende & drukke leven. Bovendien schonk jij mij in deze periode twee prachtige zonen, ónze allergrootste prestatie...
Ik heb de afgelopen jaren het geluk gehad om te mogen samenwerken met een aantal veelzijdige &
uiterst bekwame mensen:
Hilde,
Mijn voorbeeld. En een persoon met een warm hart voor haar medewerkers. Jij maakte me enthousiast voor de wondere wereld van drug metabolisme & gaf me de kans hiervoor een nieuwe technologie te ontwikkelen die nu reeds enige tijd zijn vruchten kon afwerpen binnen de vakgroep Drug metabolisme &
Preklinische farmacokinetiek. Het waren een aantal zware jaren, dank je om vol te houden. Jij schonk me de moed en het vertrouwen die ervoor zorgden dat dit project kon afgerond worden met een eervolle verdediging.
Jean-Paul,
Allereerst bedankt om je faciliteiten ter beschikking te stellen van Janssen. En verder, beroemd &
berucht om je scherpte. Je gaf me de nodige inzichten & ideeën om iets te kunnen betekenen. Je leerde me de kneepjes van de fermentatietechnologie & immmobilisatie kennen. Je gaf me de mogelijkheid mijn eigen kennis te toetsen & te delen met talrijke experten in het onderzoeksgebied.
Bovendien heb ik ook genoten van je talrijke jeugdavonturen uit het koloniale Afrika van weleer.
Karel,
Ik zal steeds blijven opkijken naar je genialiteit. Het ga je goed...
Alain,
Dank je voor onze talrijke waardevolle & leerrijke brain-storm sessies. Jij stond bovendien steeds klaar bij het samenstellen van de evaluatierapporten & resulterende publicaties. Ik kan nog veel van je leren.
Geert, Laurent, Mario,
Jullie gaven me de mogelijkheid te werken in een motiverende, professionele omgeving met dagelijks
talrijke uitdagingen & interessante figuren.Elke dag nog mag ik boeiende domeinen ontdekken.
wil bedanken voor de open & kritische feedback.
Bovendien had ik het genoegen in verschillende laboratoria te mogen werken:
Mijn UBT collega’s,
In het bijzonder, Angela, het was een voorrecht met jou te mogen samenwerken. Dank je voor de fijne
& gezellige (vroege en late) uren. Alhoewel soms de zenuwen gespannen stonden, hebben we heel wat gelachen. En ondanks je eerder technische functie, toonde je tijdens het praktische werk een duidelijke betrokkenheid, grote inzet & onbedwingbaar enthousiasme. We were in it together! Je leerde me bovendien een nieuwe cultuur & oude waarden kennen. Ik zal mijn Spaans blijven oefenen, want hopelijk leiden onze wegen ooit terug naar melkaar. Joseppe en Jean-Pierre, jullie hielden steeds een oogje in het zeil tijdens de fermentatieprocessen, terwijl, Anne-Marie, jij steeds klaarstond voor trouble- shooting tijdens de HPLC analyses. Zineb, Genevieve, Benedicte, Aimee, Sebastien, Xavier, Benjamin, Jeanine, Olivier, Laurent, Marc, Noelle, Elizabeth, Tibaut, jullie zorgden voor de aangename werksfeer
& droegen ertoe bij dat ik elke dag met veel plezier naar het lab vertrok.
Mijn Janssen-collega’s,
Jullie droegen belangeloos bij tot deze realisatie. Dank je, Ellen, mijn oprechte waardering. Het is fijn de passie voor metaboliet synthese te kunnen delen met iemand als jij. Filip & Maarten bedankt voor jullie extra inspanning bij het tot leven wekken van mijn introductie. Een extra uiting van dank gaat ook uit naar Jos, Vincent, Mark, Alex, Dirk, Veronica, Lieve, Helga, Gonda, Hilde & Peter voor jullie ondersteuning. Kelly, An & Veronique, dank je voor inspirerende tijden vol humor. Het is leuk vertoeven met jullie. Aan andere collega’s uit Preklinische farmackinetiek, dank je voor de prettige samenwerking van alledag & de steun tot de laatste loodjes (en die wogen- zoals altijd-het zwaarst). Ik ben blij dat jullie in mijn team zitten!
De mama’s & papa’s wil ik ook nog danken voor hun niet-aflatende interesse in het verloop van het onderzoek. Bovendien gaven jullie mij de kans om te studeren & legden jullie lang geleden reeds de fundamenten die dit proefschrift vandaag mogelijk hebben gemaakt. Dit is mijn kleine wederdienst...
Aan allen, van harte dank je wel!
Inneke
Dit project kon tot stand komen met financiële steun van het Vlaams Gewest-IWT.
LIST OF ABBREVIATIONS
δ‐ALA: δ‐aminolevulinic acid AscA: ascorbic acid
BM: Bacillus megaterium BAL: bioartificial liver BBB: blood brain barrier BSA: bovine serum albumin Caco: cancer colon
CMC: carboxymethyl cellulose CNS: central nervous system CHO: Chinese hamster ovary CFU: colony forming units
CDS: Chromatography Data System CYP: cytochrome P450
3‐D: 3‐dimensional DO: dissolved oxygen DTT: dithiotreitol DMSO: dimethyl sulfoxide EC: electrochemical ER: endoplasmic reticulum E. coli: Escherichia coli
EDTA: ethylene diamine tetra‐acetic acid EU: European union
FIH: First into Human
FDA: Food and Drug Administration GOI: gain from investment
GI: gastro‐intestinal G‐6‐P: glucose‐6‐phosphate
G‐6‐PD glucose‐6‐phosphate dehydrogenase GSH: glutathione
HPLC: high performance liquid chromatography hCYP: human cytochrome P450
HEK: human embryonic kidney HLM: human liver microsomes HR: human NADPH CYP reductase 6β‐OHT: 6β‐hydroxytestosterone ID: identification
ICH: International Conference on Harmonization IPTG: isopropyl‐β‐D‐thiogalactopyranoside
KP: Kansai Paint
LC: liquid chromatography LSC: liquid scintillation counting LLE: liquid‐liquid extraction LB: Luria‐Bertani
LBA: Luria‐Bertani with ampicillin MDCK: Madin‐Darby canine kidney MS: mass spectrometry
MCB: Master cell bank MTB: modified terrific broth MWCO: molecular weight cut‐off MIST: Metabolites in Safety Testing NCE: new chemical entity
NMR: nuclear magnetic resonance
OD660: optical density at wavelength 660 nm PD: pharmacodynamic
PK: pharmacokinetic
PMSF: phenylmethylsulfonyl fluoride PhD: philosophical degree
PDA: photo diode array PGA: poly galacturonic acid PVA: polyvinyl alcohol PVDF: polyvinylidene fluoride POP: Proof of Principle ROI: return on investment rpm: rotations per minute SPE: solid phase extraction SD: standard deviation
SAR: structure‐activity relationship THF: tetrahydrofurane
TSE: Tris‐sucrose‐EDTA TB: tuberculosis UV: ultraviolet
UDP: uridine diphosphate
UGT: UDP glucuronosyltransferase US: United States
WT: wild type WCB: Working cell bank
SUMMARY
Biotransformation of drug can produce toxic metabolites; information on metabolites is essential. An "in‐house" biological capacity to produce complex and in vivo relevant drug metabolites using one simple recombinant human cytochrome P450 isoenzyme (hCYP) co‐expressed with human reductase (HR) in E.
coli cells was realized. Using fermentation and bioreactor technology for propagation of cells and substrate bioconversion, the scale‐up of metabolite production was successful. Large amounts of functional hCYP3A4/HR complex were produced by intact cells, while whole cells as well as membranes were suitable as biocatalyst in a bioreactor system on a preparative scale for fast biosynthesis of milligram amounts of metabolite. However, the hydrophobic character of many molecules frequently resulted in low solubility in the aqueous bioconversion medium. A biphasic bioreactor was created: application of several organic solvents resulted in a suitable and flexible organic solvent mixture. But, since even the best tolerated solvent system affected biocatalysis, the biocatalytic system was immobilized in a protective polymer network. Ca‐alginate appeared to be an ideal immobilization matrix for the membranes, displaying hCYP/HR functionality together with the ability to preserve enzyme activity during long time storage. An appropriate cell immobilization method, that protected the cells but that did not compromise their hCYP/HR functionality, was not found. The combination of localization of the functional hCYP/HR complex in the inner membrane with reduced permeability of (immobilized) cells could be a logical explanation.
Meanwhile, this innovative bioreactor technology is routinely used to produce
“human” metabolites for actual drug candidates. The metabolites are successfully purified, demonstrating that the technology is competitive with conventional procedures and can aid in conscientious decision‐making. Still, recent advances in enzyme‐, cell‐ and reaction‐engineering are extremely exciting. Re‐engineered activity and stability open perspectives. For membranes, elimination of expensive co‐factors would be favorable. Seen the low permeability of cells, free as well as immobilized, surface expression of hCYP and HR might be considered for future bioreactor research. Additionally, solvent resistant CYP activity might circumvent the need for immobilization.
INTRODUCTION
The main aim of this thesis was the development and optimization of a bioreactor for the biological synthesis of milligram amounts of oxidative drug metabolites using a combination of
1. enzymatic bioconversion with recombinant human cytochrome P450 (hCYP)/human reductase (HR) enzymes expressed in Escherichia coli (E. coli), 2. fermentation (and immobilization) technology and
3. purification techniques.
The bioreactor was designed for innovative use in a drug development setting in the pharmaceutical industry.
Therefore, this introduction will particularly elaborate on the following:
‐ Relevant aspects of cytochrome P450 (CYP) related to the optimization of the bioreactor technology will be described. In addition, the importance of CYP‐
mediated drug metabolism will be demonstrated. Where appropriate, the link between characteristic features of CYP enzymes and the bioreactor technology will be highlighted (A).
‐ The importance of preparative synthesis and purification of oxidative drug metabolites will be illustrated, resulting in an extensive collection of available techniques to synthesize metabolites, enzymatically as well as non‐enzymatically (B).
A. Facts on drug metabolism
A.1. Biotransformation of xenobiotics
Xenobiotics are chemical compounds that are not part of the normal composition of the human body. The principal route of elimination of xenobiotics from the body is through biotransformation. Biotransformation of xenobiotics is controlled by metabolic reactions that can be divided in Phase I and Phase II (conjugations) reactions. Based on their evolutionary history, many of the enzymes involved in the metabolism of xenobiotics are principally involved in the metabolism of endogenous compounds such as steroids, bile and eicosanoids (Parkinson, 2001; Danielson, 2002).
A.2. Phase I enzymes and localization
Phase I reactions are functionalization reactions which can be broadly classified into 4 categories: oxidation (epoxidation, peroxidation, heteroatom oxidation and oxidation of aromatic rings), reduction, hydroxylation and oxidative N‐, O‐ or S‐dealkylation 1
CYP
Figure 1: In eukaryotes, CYPs are membrane bound enzymes (Bernhardt, 2006) mainly localized in the smooth ER (Werck‐Reichhart and Feyereisen, 2000; Danielson, 2002; Neve and Ingelman‐Sundberg, 2010; Preissner et al., 2010).
(Danielson, 2002; Baader and Meyer, 2004; Gillam, 2005; Wisniewska and Mazerska, 2009). This divers set of reactions is mainly catalyzed by CYP enzymes, the major xenobiotic metabolizing enzymes (Parkinson, 1996a). As a result, although also non‐CYP‐
mediated oxidations can play an important role in the metabolism of xenobiotics (Strolin et al., 2006), Phase I biotransformation of drugs is mainly catalysed by CYP enzymes (Guengerich, 1993b; Maurel, 1996; Danielson, 2002; Kumar, 2010), with aliphatic and aromatic hydroxylation (at C‐centres), N‐ and O‐dealkylation, heteroatom oxidation (at N‐ and S‐centres) and epoxide formation from alkenes and aromatic rings and oxidation of aldehydes to acids being the most commonly found in human drug metabolism (Guengerich, 2001a; Gillam, 2005; Isin and Guengerich, 2007; Gillam, 2008). This makes hCYP enzymes extremely valuable for production of in vivo relevant oxidative metabolites by the use of a bioreactor technology.
Since the overwhelming majority of CYPs is localized in mainly liver cells (about 2.5% of total hepatic microsomal protein), the primary organ of drug metabolism is the liver (Karlgren et al., 2004; Wilkinson, 2005). The liver thus acts as the gatekeeper of the body, the more since after absorption in the gut, all orally administered drugs are transported through the portal vein directly to the liver. However, some CYPs also occur extra‐hepatically (Parkinson, 1996a; Danielson, 2002; Ding and Kaminsky, 2003; Karlgren et al., 2004; Wilkinson, 2005; Daly, 2006; McLean et al., 2007).
Subcellularly, in eukaryotes, CYP are membrane bound enzymes (Bernhardt, 2006) mainly embedded in the smooth endoplasmic reticulum (ER) (microsomal type) (Werck‐
Reichhart and Feyereisen, 2000; Danielson, 2002; Neve and Ingelman‐Sundberg, 2010;
Preissner et al., 2010) (Figure 1). Axelrod (1955) and Brodie et al. (1955) were first to report on CYP enzymes. Both found that certain xenobiotics were oxidized by an enzyme system present in the ER of rat liver microsomes.
A.3. Biochemistry of CYP oxidative metabolism
In general, the CYP monoxygenase system constitutes a large superfamily of heme‐
thiolate enzymes, which catalyze mainly oxidative conversion reactions of a wide range of compounds (Porter and Coon, 1991), endogenous and exogenous (Guengerich, 1987;
Guengerich and Shimada, 1991; Gonzalez, 1992; Danielson, 2002). In a few instances, however, CYPs also carry out reductions (under anaerobic conditions) or peroxidations (Blake and Coon, 1980; Estabrook et al., 1984; Marnett and Kennedy, 1995).
Eukaryotic CYPs generally range in length from 480 to 560 amino acids. Even in those cases where global sequence identity is low, the overall fold topography appears to be 2
highly conserved (Sirim et al., 2009). Although crystallography data on full length membrane bound eukaryotic CYPs are rare, the big picture has emerged that all members of the CYP superfamily share a common globular to triangular framework that consist of a relatively alpha helix‐rich carboxy‐terminal half, a relatively beta sheet‐rich animo‐terminal half and a 'meander' region. Most of the beta sheets and alfa helices are thought to lie in roughly the same plane as the protestic heme group. Sequence alignments feature a number of conserved sequence motifs which play important roles in for example formation of the enzymes critical oxygen‐binding pocket or formation of the heme binding decapeptide loop. Further, microsomal CYPs are characterized by a transmembrane animo‐terminal signal‐anchor sequence, which along with other less well‐defined hydrophobic contacts, forms a hydrophobic core of 20‐25 residues that anchor the mature protein to the ER. The signal‐anchor domain is also characterized by the presence of a group of highly basic residues at the carboxy‐terminus and a negatively charged amino acid near the amino‐terminus. These charged residues are thought to serve a dual function of facilitating protein folding of microsomal CYP and ensuring that the anchor domain is properly oriented in the ER. The hydrophobic region is followed by a proline‐rich 'hinge' region, which imparts flexibility between the transmembrane region and the globular catalytic part of the protein that resides in the cytosolic region of the cell. This flexibility may be necessary to orient the cytosolic portion of the molecule with respect to the membrane for substrate access and for interaction with the appropriate electron transfer partner. For comprehensive and systematic comparison of protein sequences and structures, several databases are established. Most of the databases are interlinked resulting in readily accessible online biochemical information and provide a valuable tool to navigate in sequence space and analyze sequence‐structure‐function relationships (Sirim et al., 2009; Preissner et al., 2010).
Although CYP was initially perceived as a single enzyme, the functional complex consists of
(1) an electron‐transporting protein that contains a heme‐prosthetic group, a hemoprotein, where heme is an iron‐containing protoporphyrin IX liganded to a cysteine thiolate, and the protein is an apoprotein with a molecular mass of 45 000 – 55 000 Da (i.e. CYP, the substrate‐ and O‐binding site).
(2) a flavoprotein (NADPH cytochrome P450 reductase or NADH‐cytochrome b5 reductase, the e‐ shuttle) (Smith et al., 1994; Bernhardt, 2006). Since hCYPs are not self‐
sufficient and E. coli do not possess endogenous reductase activity, coexpression of CYPs and their redox partners in heterologous expression systems was introduced in order to
3
Figure 2: Proposed CYP‐mediated oxidative catalytic reaction cycle involving as well HR, cofactors and oxygen.
allow shuttling of electrons (Gillam et al., 1993; Blake et al., 1996; Pritchard, 1997;
Bernhardt, 2006). The latter E. coli cells were also used to optimize the bioreactor.
While the details of the catalytic cycle are still subject of ongoing research, the hydroxylation mechanism is well understood (Rabe et al., 2008) and the overall stochiometry of the mixed function oxidase function of CYP is as follows:
substrate (RH) +O2 +NADPH +H+ → product (ROH) + H2O + NADP+
(Ortiz de Montellano, 1989; Sono et al., 1996, Danielson, 2002;
Gillam, 2005; Bernhardt, 2006)
Essentially, one oxygen atom of dioxygen is incorporated into the substrate (Parkinson, 1996a), while the other oxygen atom is reduced by two electrons to result in water.
Therefore, molecular oxygen is an essential requirement for CYP dependent oxidation of drugs, as is the cofactor NADPH, who usually provides the two electrons needed to catalyze activation of molecular oxygen (Johansson et al., 2007). Based on the importance of oxygen for CYP‐mediated oxidative drug bioconversion, oxygen is often the rate limiting factor when applying the bioreactor technology. The oxidative catalytic cycle is demonstrated in Figure 2.
Additionally, if the catalytic cycle is interrupted and, therefore, not completed, uncoupling reactions can occur resulting in the formation of reactive oxygen species such as superoxide anion radicals and/or hydrogen peroxide (Parkinson, 1996a;
Schenkman and Jansson, 2003; Bernhardt, 2006; Myasoedova, 2008).
Furthermore, phospholipids, important components of biological membranes, are also necessary for the optimal interaction of hCYP and its electron transport partner reductase (Kumar, 2010). Thus, since E. coli do not possess an ER, but functionality depends on a lipid environment, hCYP/HR systems are generally directed to the hosts cell membrane (Figure 3). Additionally, it is imperative that hCYP and HR are close to each other in the membranar system allowing efficient channeling of the electron transport in the membrane from HR to hCYP.
For more reading on recent advances on structure, mechanism and biochemistry of CYP we refer to Ortiz de Montellano, 2005.
A.4. Genetics and CYP nomenclature
It is believed that the modern CYP gene superfamily originated from a single ancestral gene that existed approximately three and a half billion years ago (Danielson, 2002;
4
cytochrome P450 HR e‐, ...
substrate metabolite O2
out
in
cell membrane
Figure 3: Schematic drawing of the membrane topology of E. coli‐expressed CYP and HR, demonstrating that CYP and HR are both situated in the inner cell membrane, probably with their catalytic site facing the cytosol (based on unpublished LINK data; Friedberg et al., 1999; Hanlon et al., 2007; personal communication D. Leak, Editor of Biocatal Biotrans, 2009).
Omura, 2010). The nomenclature system for this superfamily of CYP isoforms, named after the typical absorption maximum of the reduced CO‐bound complex at 450 nm (Werch‐Reichhart and Feyereisen, 2000; Danielson, 2002; Bernhardt, 2006), was developed by Nelson et al. in 1996. Based on primary AA sequence homology, the CYP superfamily is devided in gene families (indicated with an arabic number e.g. CYP3) and gene subfamilies (indicated by a capital letter e.g. CYP3A). While numerous exceptions exist, within the same family the CYP proteins share more than 40% protein sequence homology (Danielson, 2002; Rabe et al., 2008). CYP proteins with a sequence homology greater than 55% belong to the same subfamily and those that are more than 97%
identical are considered to represent alleles unless there is evidence to the contrary (Danielson, 2002). Further, molecular techniques have allowed cloning, expression and improved characterization (generation of better antibodies, development of molecular probes) of individual members of CYP subfamilies. Each is indicated with an arabic number (e.g. CYP3A4, CYP3A5, CYP3A7, CYP3A43, Li et al., 1995; Daly, 2006) and is numbered sequentially as it is characterized. Each member represents a single gene and a single protein.
A.5. CYP variability
Gene duplication remains the most widely recognized mechanism responsible for driving the expansion and structural and functional diversification of the multigene family along with the evolution of living organisms (Danielson 2002; Omura, 2010). CYP are ubiquitous enzymes found from archaea to humans (Werck‐Reichhart and Feyereisen, 2000; Danielson, 2002; Urlacher et al., 2004; Gillam, 2005). Over 450 different CYP isoforms have been found in plants, bacteria, insects, yeast and mammals (Johansson et al., 2007). Although in bacteria some very interesting CYP activities are found lately (Gillam, 2008; Julsing et al., 2008; Rabe et al., 2008), E. coli lack CYP enzymes (Wolf, 1999; Guengerich, 2001b). This is one of the traits that make E. coli extremely attractive for this bioreactor technology, resulting in low background activity as well as clear metabolic profiles.
The CYPs display a tissue‐ as well as species‐specific expression (Ding and Kaminsky, 2003). Because of these interspecies differences it is discouraged to predict human oxidative metabolism of a drug based on animal models alone (Wrighton et al., 1993;
Nedelcheva and Gut, 1994; Ding and Kaminsky, 2003). Additionally, large intra‐species differences exist in the expression and catalytic capabilities of the CYP enzymes.
In human, 57 putative functional CYP isoenzymes (Meyer, 1996; Danielson, 2002; Miller et al., 2009; Wisniewska and Mazersk, 2009; Kumar, 2010), divided over 18 families and 5
CYP2B6 (0,2%) CYP2D6 (2%) Figure 4: CYP3A4 is the most important CYP involved in drug metabolism. Left: In human liver the CYP3A4 isoform is most predominantly present. Right: Most drugs are mainly metabolized by CYP3A4.
CYP2A6 (4%) CYP2E1 (7%) CYP1A2 (13%) CYP2C (18%) unknown (27%)
CYP3A (29%)
CYP2E1 (1%) CYP1A2 (5%) CYP2C (20%) CYP2D6 (25%) CYP3A (55%)
43 subfamilies (Danielson, 2002; Preissner et al., 2010), occur. The most important hCYP isoenzymes are CYP1A2, 2A6, 2B6, 2C, 2D6, 2E1 and 3A (Shimada et al., 1994). The percentage of different CYP isoforms present in the human liver is displayed in Figure 4.
Three hCYP gene families CYP1, CYP2 an CYP3 (Ding and Kaminsky, 2003; Karlgren et al., 2004; Wilkinson, 2005), including especially hCYPs CYP1A2, 2C9, 2C19, 2D6 and 3A4, are involved in ~95% of the CYP‐mediated metabolism of drugs in clinical use (Wisniewska and Mazersk, 2009) (about ~75% of drug metabolism (Lamb et al., 2007)). The percentage of CYP isoforms that metabolize existing drugs is given in Figure 4. This clearly illustrates the importance of CYP3A in drug metabolism. Especially CYP3A4 is qualitatively and quantitatively the most important CYP involved in drug metabolism (Wrighton et al., 1996; Anzenbacher and Anzenbacherova, 2001; Danielson, 2002; Daly, 2006; Sawayama et al., 2009), followed by CYP2D6 and CYP2C9 (Wisniewska and Mazersk, 2009). CYP3A4 and CYP2D6 are responsible for 80% of all CYP‐related drug metabolism (Guengerich, 2003; Gillam, 2008). Therefore, development of the current bioreactor technology focused mainly on CYP3A4 activity (and to a lesser extent on CYP2C9 and CYP2D6).
CYPs are characterized by unique, but broad and frequently overlapping substrate specificity and regio‐ and stereoselectivity (Crespi et al., 1993; Danielson, 2002;
Wilkinson, 2005), reflecting the wide variety of compounds they have to cope with, thus underscoring their catalytic versatility (Danielson, 2002; Gillam, 2005). Therefore, recombinant (human) CYP isoenzymes are particularly valuable for selective generation of (potentially human‐)specific oxidative metabolites of drugs in development. The details of the mechanism underlying the unusual substrate diversity of these CYPs are currently not fully understood (Urlacher et al., 2004).
Finally, extensive inter‐ (Danielson, 2002) and intra‐individual differences in the expression and activity of CYP exist. For example, there are no individuals lacking CYP3A, although high inter‐individual variation (>10‐fold in vivo) is not exceptional (Danielson, 2002; Wilkinson, 2005). These differences are largely caused by environmental factors such as sex, age (e.g. CYP3A5 and CYP3A7 in human fetal liver; Wilkinson, 1996), diet (caffeine), hormonal influences, disease state (cirrhosis, hepatitis), drug consumption (induction and inhibition), chemicals (smoke, alcohol), but also genetic and epigenetic factors (genetic polymorphism: CYP2C9, CYP2C19 and 2D6; London et al., 1996;
Ingelman‐Sundberg, 2005; Wilkinson, 2005) play an important role (Wrighton et al., 1996; Danielson, 2002; Wilkinson, 2005). Therefore, genetic variations such as polymorphisms and regulatory mechanisms at mainly the transcriptional (Guengerich, 2001b), but also translational and posttranslational (e.g. mRNA stabilization, 6
Figure 5: The whole process of drug development takes more than a decade and costs hundreds of millions. During this process the efficacy of a NCE as well as its safety are rigorously investigated.
phosphorylation) levels of these enzymes have been extensively explored (Lim and Huang, 2008). Several nuclear receptors (such as the constitutive androstane receptor, pregnane X receptor, peroxisome proliferator activated receptor alpha, retinoid X receptor alpha, liver X receptor alpha, farnesol X receptor, aryl hydrocarbon receptor and glucocorticoid receptor) function as key regulators of CYP expression upon binding of foreign chemical inducers and endogenous hormones, growth factors and cytokines (Guengerich, 2001b; Zordoky and El‐Kadi, 2009; Yang et al., 2010). Pelkonen et al. (2008) and Hengstler and Bolt (2008) provided excellent articles on CYP regulation with emphasis on extensive lists of inducers and inhibitors (down‐regulation) and a systematic survey of Yang et al. (2010) provides a comprehensive view of the functionality, genetic control and interactions of hCYPs. Another previously unappreciated source of genetic variation in drug metabolism are variants in CYP oxidoreductase (Hart et al., 2008; Miller et al., 2009).
B. Pharmaceutical Research and Development
The following overview will be presented in:
‐ Publication: “Metabolite synthesis in drug development: a partnership between biological and chemical methodologies”, Wynant I., Cuyckens F., Vliegen M., Scheers E., Bohets H. (for Current Drug Metabolism) (Accepted abstract in Appendix 2, manuscript in preparation).
B.1. Drug metabolism studies
The history of the pharmaceutical industry shows that many important drugs have been discovered by a combination of fortuity and luck. Today, however, new chemical entities (NCEs) are designed to achieve an improved efficacy (pharmacological activity) in combination with limited side effects (toxicology). A large number of potential NCEs are generated via combinatorial chemistry and high‐throughput screening techniques (Guengerich and MacDonald, 2007). The process of drug development takes more than a decade and costs hundreds of millions and besides the efficacy of a NCE, its safety is rigorously investigated (Figure 5). Since lots of drug failures result from lack of efficacy, toxicity (Guengerich and MacDonald, 2007; Guengerich, 2008) and/or inadequate pharmacokinetics (PK), the toxicological and PK properties are examined to have a better understanding of the human drug safety profile. Therefore, it is important to develop in vitro drug metabolism models which allow rapid and flexible screening of unfavorable properties from a series of drug candidates (Figure 6). Reactive or toxic metabolites can be one of the potential cases of failure. Gathering information on metabolites in an early stage of development helps in judgment of potential safety and efficacy outcome.
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Figure 6: Early elimination of drug candidates with unfavorable characteristics is important in order to facilitate lead optimization and ultimately attempt reduction in the overall development cost of new drug substances. Bringing new or better drugs to the market is the main objective of the pharmaceutical industry, although elimination of drug candidates at an early stage that would eventually not make it to the clinic is just as important.
One of the selection criteria in early phase of development (discovery) is the metabolic stability of a NCE. Therefore, the metabolism is investigated in vitro in the different toxicological and/or pharmacological animal species and man, supported by in vivo metabolism studies in animals. Resulting data primarily give information on the relevance of the toxicological and/or pharmacological species and on the (semi‐)quantitative occurrence of potentially (re)active and/or human‐specific metabolites. Next to the metabolism, data on the enzymes involved in the metabolism are gathered. Therefore, investigation of the metabolism in human liver microsomes (HLM) in the presence of specific CYP inhibitors (Halpert et al., 1994; Bourrie et al., 1996; Guengerich, 1996) and in heterologous expression systems expressing a single hCYP are conducted (Ono et al., 1996; Guengerich, 1996; Van ‘t Klooster and Lavrijsen, 1997; McGinnity et al., 2000) to identify CYPs involved. Metabolism data allow assessing the impact of toxicological and pharmacological characteristics of the NCE and can drive initiation of specific metabolite synthesis. The scope of the current research is the development of a bioreactor to produce metabolites. As mentioned before, these metabolites are mainly needed to assess the pharmacological and toxicological properties of metabolites formed. The basis of our bioreactor system is E. coli expressing recombinant hCYP.
B.2. Reactive drug metabolites
The ultimate goal of xenobiotic metabolism is the ''elimination'' of xenobiotics (Porter and Coon, 1991; Ioannides, 1996) by altering their physical properties from those favouring absorption (lipophylicity) to those favouring excretion in urine or faeces (hydrophilicity) (Taavitsainen, 2001; Gillam, 2005). Therefore, the drug metabolism reaction cascade generally tends to increase hydrophilicity, which can influence the pharmaco‐toxicological drug properties. Therefore, biotransformation does not always stand for bioinactivation or detoxification, especially among oxidative reactions (Blaauboer, 1996). Numerous examples are known of compounds which are biotransformed into pharmacologically or toxicologically active metabolites (bioactivation) with desirable (prodrugs) or adverse effects, including hepatotoxicity, cellular necrosis, hypersensitivity, carcinogenicity, teratogenicity and mutagenicity (Wrighton and Stevens, 1992; Guengerich, 1993a; Park et al., 1995; Blaauboer, 1996;
Parkinson, 1996a; Friedberg and Wolf, 1996; Parkinson, 2001; Purnapatre et al., 2008) due to the production of electrophylic species that can react with cellular nucleophiles, such as glutathione, lipids, DNA and proteins (Gillam, 1998; van Bladeren, 2000). Over the last few years, several high profile late stage failures and post‐approval drug recalls caused by toxicity or negative drug‐drug interactions have led to an increased focus on metabolite studies, including metabolite synthesis.
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Regulatory documents in this perspective include the Food and Drug Administration (FDA) guidance for industry on safety testing of drug metabolites, published in February 2008. The guidance mainly focuses on Phase I metabolites. The guidance provides recommendations to industry on how to deal with safety of drug metabolites (MIST or Metabolites in Safety Testing) and especially deals with human unique metabolites, i.e.
metabolites which are not present in the toxicology species used to support the clinical development program. Disproportionately higher levels in humans than in any of the animal species used during standard nonclinical toxicology testing are discussed as well.
Based on the FDA guidance, coverage of a human metabolite (and nonclinical characterization) is only warranted in the toxicity studies when that metabolite is observed at exposures greater than 10% of the total parent drug at steady state concentration. This guidance triggered a lot of discussions as referred to in the following review articles: Smith and Obach, 2005; Vishwanathan et al., 2009; Smith et al., 2009;
Leclercq et al., 2009; Frederick and Obach, 2010. One of the major concerns by the different scientists authoring the reviews and by industry was the “10% of parent drug AUC exposure rule and the lack of dose relationship”. Further discussions between industry and regulators resulted in an updated International Conference on Harmonization (ICH) M3 (R3) guidance “Non‐clinical safety studies for the conduct of human clinical trials and marketing authorization for pharmaceuticals.” The guidance is intended to be applicable in the EU, Japan and the US and is less conservative compared to the MIST guidance of FDA. In the M3 guidance coverage (and nonclinical characterization) of human metabolites only needs to be assessed for compounds with a recommended daily dose exceeding 10 mg with metabolites at exposures greater than 10% of total drug‐related material and at significantly greater levels in humans than the maximum exposure seen in the toxicity studies.
Pharmacological activity and/or toxicity of metabolites can be put into different categories as described by Park et al. (1998). The different categories and how to put this in the context of MIST was further discussed in the review of Frederick and Obach (2010). Some examples of drugs with metabolites that can potentially result in toxicity are acetaminophen, clozapine, carbamazepine, troglitazone (Park et al., 2009; Smith and Obach, 2006). Still clinical relevance of toxic metabolites will depend on specific clinical situations. Acetaminophen is seen as a safe drug in clinical use but can result in severe hepatotoxicity at overdose. Reactive metabolites which are not trapped by glutathione is one of the causes of this type of toxicity. Troglitazone was withdrawn from the market due to safety issues. Examples of drugs with active metabolites, such as atorvastatin, amitryptyline (active metabolite nortriptiline) codeine (active metabolite morphine), propranolol, risperidone, tolbutamide, loratidine, zolmitriptan, tramadol are described in reviews of Gad (2003), Fura (2006) and Smith and Obach (2005, 2009). Both the 9
occurrence of reactive or active metabolites can significantly affect the drug development process, both in terms of developmental activities and cost. As mentioned earlier, it is therefore important to understand the specific features of a metabolite in an early phase of development. The CYP bioreactor technology can play a major role in this process.
B.3. Drug metabolite synthesis
These documents state that already in the early phase of development of a NCE, information about the identity and pharmacological or toxicological activity of drug metabolites can be useful in the further development process. Therefore, sufficient amounts (in the order of milligrams) of drug metabolites are desired (Kumar, 2010):
1) to identify metabolite structure via Nuclear Magnetic Resonance (NMR), 2) to screen the metabolites for their pharmacological activity,
3) as reference material for the optimization of bioanalytical methods, 4) for screening in receptor binding studies,
5) for PK tests (such as drug‐drug interaction studies at the level of CYP) and 6) for in vitro investigation of potential (cyto)toxicity
7) data on drug metabolites will be used for selection of the toxicological species and
8) to support regulatory filings.
Early identification (ID) of potential therapeutic problems related to drug metabolites is vital to drive a more profound evaluation of metabolite safety and reduce the development costs of NCEs.
B.3.1. Classical organic chemistry
Hitherto drug metabolites are still mainly produced by traditional chemical synthesis because this approach generally allows large quantities to be synthesized. However, depending on the nature of the metabolite, this process is often time consuming, frequently in the order of weeks or even months and expensive, depending on the raw materials. The technique is extra complicated when dealing with a chiral mixture and/or when several steps are involved, requiring development of specific purification methods (Griffin et al., 1998). Additionally, the exact structure of the metabolite to be synthesized is not always known from previous in vitro and in vivo experiments reducing chemical synthesis often to a play of trial and error. Sometimes it is simply impossible to produce metabolites by chemical synthesis (Zöllner et al., 2010). Complementary to chemical synthesis, stress‐induced degradation of product to generate drug metabolites is currently gaining more interest, since some metabolites are sometimes also formed
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Table 1: General advantages and limitation of in vitro liver preparations, adopted from Guillouzo, 1998.
during natural degradation, although generally in smaller amounts. Further reading is supplied in Appendix 2.
B.3.2. Enzymatic synthesis
Since animals provide the most physiologically relevant test system for evaluation of drug metabolism (Tingle and Helsby, 2006), isolation and purification of metabolites from animal biological fluids (excreta such as urine, faeces and bile and blood or plasma) and tissues (e.g. liver, brain), following in vivo studies, has been common practice for decades. Some of the possible limitations are difficult purification from complex matrices resulting in considerable losses, need for human‐specific metabolites and insufficient yield of one particular metabolite due to multiple metabolic pathways.
Therefore, although a combination of in vivo animal and in vitro human studies will remain a foreseeable future (Tingle and Helsby, 2006), a whole area of alternative in vitro methods are used to evaluate human drug metabolism. Since this domain is too extensive to describe in detail, we will give a brief overview of interesting in vitro models to study drug metabolism (not drug‐drug interaction) and mimic human metabolism and their respective evolutions, but especially elaborating on drug metabolite synthesis. Well‐established methods using biological matrices are liver‐
derived models and pure enzymes:
Liver‐derived models
Since the liver is the key organ involved in drug biotransformation (Lohmann and Karst, 2008), human liver tissues (from liver subcellular fractions, cultured hepatocytes, liver slices to perfused liver, Table 1) are valuable in vitro models for drug metabolism studies (Donato et al., 2008). Although all kinds of cell‐based assays are beginning to replace traditional liver subcellular fractions (Sinz and Kim, 2006), liver microsomes are still used extensively as one the simplest forms (Tingle and Helsby, 2006). A recent review on the essential role of HLM in in vitro metabolism of drugs was written by Asha and Vidyavathi (2009). For complex metabolite profiles involving sequential or competing pathways, freshly isolated hepatocytes, mainly primary hetapocyte cultures, are of important value (Allen et al., 2005; Tingle and Helsby, 2006) to qualitatively predict in vivo metabolic profiles (Marianthi et al., 2009) but are also the “gold standard” to study induction (Sinz and Kim, 2006). Since the optimization of precision‐cut technology, freshly isolated liver slices ‐where the physiological liver micro‐architecture is maintained (Vermeir et al., 2005)‐ can be added to the battery of in vitro assays of drug metabolism (Thohan and Roosen, 2002; Gebhardt et al., 2003). Together with perfused liver, these systems can be used for short time studies (Allen et al. 2005). Besides all being very interesting test systems, in vitro phenotypic instability of hepatocytes, high functional variability in the 11
