Dr Patrick M Downey
email: patrick.downey@spcorp.com

Roberta Petrò

Jason S Simon

David Devlin

Gianluca Lozza

Alessio Veltri

Massimiliano Beltramo

Rosalia Bertorelli

Identification of single nucleotide polymorphisms of the human metabotropic glutamate receptor 1 gene and pharmacological characterization of a P993S variant

Keywords: metabotropic glutamate receptors, polymorphism, Pharmacogenetics, chronic pain

mGluR1 receptors are believed to play major roles in the pathophysiology of diseases such as anxiety and chronic pain and are being actively investigated as targets for drug development.

Sequence polymorphisms can potentially influence the efficacy of drugs in patient populations and are therefore an important consideration in the drug development process. To identify DNA sequence variants of the mGluR1 receptor, comparative DNA sequencing was performed on DNA samples (n=186) from apparently healthy subjects representing 2 ethnic groups. In total, 8 non synonymous single nucleotide polymorphisms (SNPs) were identified and one SNP (c2977>T) was found to be particularly common, this SNP results in a Proline to Serine substitution at residue 993 (P993S). The WT (P993) and S993 variants were expressed in an inducible system which allowed us to titrate gene expression to equivalent levels and were pharmacologically characterised. We determined the potency and affinity of standard antagonist compounds as well as the potency and efficacy of the endogenous ligand Glutamate and other agonist compounds at both receptor variants. Agonist evoked increases in intracellular Ca 2+ were measured by FLIPR. The potency of mGluR1 antagonists was evaluated by their ability to inhibit quisqualate induced increases in intracellular Ca 2+ , while their affinities were determined by radio-ligand binding studies. This study demonstrates that the Pro993Ser amino acid exchange is highly frequent in the human mGluR1 gene. This polymorphism however, does not appear to affect the potency of agonist compounds or the potencies or affinities of small molecule antagonist compounds.

A c c e p t e d M a n u s c r i p t

INTRODUCTION

Glutamate is the major excitatory neurotransmitter in the central nervous system, it produces its excitatory effects by acting on two classes of cell surface receptors, ligand gated cation channels known as ionotropic glutamate receptors and G protein coupled receptors termed metabotropic glutamate receptors [START_REF] Kew | Ionotropic and metabotropic glutamate receptor structure and pharmacology[END_REF]. Metabotropic glutamate receptors form a family of eight subtypes (mGluR1-8) which on the basis of structural homology, pharmacology and signal transduction mechanisms are subdivided into three groups [START_REF] Blasi | Molecular determinants of metabotropic glutamate receptor signaling[END_REF]. Group I receptors (mGluR1 and mGluR5) usually act postsynaptically to increase neuronal excitability [START_REF] Gasparini | Allosteric modulators of group I metabotropic glutamate receptors: novel subtype-selective ligands and therapeutic perspectives[END_REF]. Excessive glutamatergic neurotransmission has been shown to underlie many CNS diseases and to play an important role in the pathophysiology of diseases such as depression, anxiety, and chronic pain, which represent key areas of interest for drug development. Accordingly, mGluR group I antagonists could represent useful agents for the treatment of these conditions. Indeed, in preclinical models, mGluR I antagonists have been shown to be very effective agents in the treatment of prolonged and chronic pain [START_REF] Scheryantz | Prospects for metabotropic glutamate I receptor antagonists in the treatment of neuropathic pain[END_REF][START_REF] Bhave | Peripheral group I metabotropic glutamate receptors modulate nociception in mice[END_REF][START_REF] Neugebauer | Metabotropic glutamate receptors -important modulators of nociception and pain behaviour[END_REF] and to possess anxiolytic activity [START_REF] Steckler | Effects of mGlu1 receptor blockade on anxiety-related behaviour in the rat lick suppression test[END_REF][START_REF] Pietraszek | Anxiolytic-like effects of mGlu1 and mGlu5 receptor antagonists in rats[END_REF]. Recent data suggests that mGluR1 antagonists may also be useful therapeutic agents for treating drug dependence [START_REF] Dravolina | mGlu1 receptor blockade attenuates cue-and nicotine-induced reinstatement of extinguished nicotine self-administration behavior in rats[END_REF] and depression (10). However, the involvement of mGluR1 in human CNS disorders has yet to be demonstrated, it is therefore important to carry out clinical proof of concept studies to understand the functions of mGluR1 in man. As a prelude to this type of activity it is important to understand the genetic variation within this receptor gene among human populations and within different ethnic groups.

Single nucleotide polymorphisms (SNPs), substitutions of a novel nucleotide for the wild type nucleotide within genomic DNA are the most common type of genetic variation and occur at a frequency of greater than 1% in the general population [START_REF] Cargill | Characterization of single-nucleotide polymorphisms in coding regions of human genes[END_REF]. Within the coding region of a gene, non synonymous SNPs, those that change the encoded amino acid sequence, can profoundly effect protein folding and disrupt post translational modifications [START_REF] Wang | SNP's Protein structure and disease[END_REF]. As far as GPCRs are concerned, there are a number of reports which show that non synonymous SNPS may also alter the normal functioning of the receptor by disrupting ligand-receptor binding [START_REF] Green | A polymorphism of the human β2 Adrenergic receptor within the fourth transmembrane domain alters ligand binding and functional properties of the receptor[END_REF] or altering the normal coupling to G proteins [START_REF] Mason | A gain-of function polymorphism in a G-protein coupling domain of the human β1 Adrenergic receptor[END_REF][START_REF] Small | An Asn to Lys polymorphism in the third intracellular loop of the human A2A-Adrenergic receptor imparts enhanced agonist promoted G i coupling[END_REF]. In some cases, SNPs may lead to increased disease susceptibility or yield variable responses to therapeutic agents. Therefore, evaluating the pharmacogenetics of disease-related targets is an important step in the drug development process.

The current study reveals the frequency of polymorphisms in the human mGluR1 gene and pharmacologically characterizes the very frequent P993S receptor variant. Despite the fact that eight non synonymous SNPs were identified, most were very rare and only the P993S variant was present at a frequency of greater than 5% in both populations. We have previously described the use of an inducible system, the Ecdysone system, to functionally express representative mGluR subtypes [START_REF] Downey | Ecdysonebased system for controlled inducible expression of metabotropic glutamate receptor subtypes 2, 5, and 8[END_REF]. Expressing the WT and S993 mGluR1 variants in this system allowed us to tightly regulate gene expression, so that we were able to express the two forms of the receptor at almost

2.2.Research Subjects

To identify SNPs, the mGluR1 gene was sequenced from anonymous Caucasian (n=47) and African American (n=46) individuals. Genomic DNA samples were obtained from the Caucasian and African American human variation panels collected from the Human Genetic Cell Repository of the National Institute for General Medical Sciences (Coriell Cell Repository; Camden, NJ, USA). All samples came from individuals who provided informed consent to be part of the DNA polymorphism Discovery Resource. Information on geographic origin and gender was collected for each individual in order to assemble the DNA polymorphism Discovery Resource, but all identifying and phenotypic information has been removed from the individual samples.

Polymerase chain reaction

The general strategy for SNP discovery is as previously described [START_REF] Nickerson | Sequence based detection of single nucleotide polymorphisms[END_REF] Analysis parameters were all maintained at the software's default settings. TTACAGGGTGGAAGAGCTTTGCTTG 3' (3561-3588), the 5' and 3' fragments were cloned into pCRScript and fully sequenced on both DNA strands. After sequence confirmation the amplified products were joined together using the restriction enzyme Sac1 which has a single site within the mGluR1a coding sequence at nucleotide position 1890. The entire coding sequence was subcloned into pCRscript and the junction region was sequenced. To create the expression construct, the mGluR1 coding sequence was amplified using the following primers, Fwd 5' gAATTCgTCCTCACCACCATggTCgggCTCCTTTTgTTTTTTTTC 3', Reverse primer 5'

gCggCCgCggATCCTTCCCCCTTACAgggTggAAgAgCTTTgCTTgTAgTCCC which contain an EcoR1 site and a full kozak sequence (forward primer) and a Not1 site (Reverse primer). The mGluR1a coding sequence was sub-cloned into the expression vector pIND (Invitrogen, Paisley UK) as an EcoR1-Not1 fragment, the construct was fully sequenced on both DNA strands.

Creation of polymorphic variant

The following primer pair was used to amplify a 1260 bp fragment at the 3' end of the mGluR1a coding sequence from human hippocampal c-DNA, the amplified product extended from base pair 2340 to the end of the coding sequence 3588bp , FWD 5' CCgCCACgTgCCCgCCAACTTCA 3' was made by replacing the Nde1-Not1 restriction fragment from pINDP993 with the equivalent fragment from plasmid pCRScript-S993. The expression construct was completely sequenced in the region that had undergone restriction fragment swapping to confirm that only the intended mutation had been introduced.

Isolation of clonal cell lines

The UTRs and an average of 100 bp upstream and downstream flanking the intron/exon splice junctions from an anonymous panel of Caucasian (n=47) and African American (n=46) subjects identified 46 SNPs. 17 SNPs were identified in the protein coding region of the gene, eight of which resulted in an amino acid substitution (Table 1), of these, only one SNP c2977>T (rs6923492:

chr6:146797017 according to the March 2006 of the UCSC genome build) which results in an amino acid substitution P993S had an allele frequency of greater than 5% in both populations. This genetic variant occurred in exon 8 of the gene at a position corresponding to the C terminal intracellular tail of the receptor. The minor allele frequency was 28.3% in the African American samples, and 35.6% in the Caucasian samples.

Expression of WT and P993S variant mGluR1a receptors in HEK-293 cells

The coding sequence of the WT mGluR1a was isolated from hippocampal c-DNA and sub-cloned into the expression vector pIND. The C to T substitution at position 2977 of the mGluR1a cDNA clone (corresponding to 405804 ) was created by exchanging the Nde1-Not1 3' fragment of the WT pINDp993 expression construct with the equivalent fragment containing a thymine residue at position 2977. mGluR1 is a Gα q -coupled receptor; its activation thus leads to an increase in intracellular calcium which can be measured using a fluorometric imaging plate reader (FLIPR).

Clonal cell lines expressing the WT (P993) or the S993 variant of mGluR1 were initially prescreened for the stimulation of calcium mobilization in response to quisqualate following 24 h induction with 5µM Ponasterone A. A single clonal cell line for each receptor variant referred to as P993 or S993 respectively was chosen for further studies. To determine how receptor density can be modulated in these clones, cells were induced for 24 h with increasing concentrations (1-3-5 µM) of Ponasterone A and saturation binding studies were performed to determine relative expression levels. The WT clone (P993) gave mean B Max values of 0.1, 0,7, 1.6, 1.9 pmol/ml, while the variant gave mean B Max values of 0,1, 0.2, 0.7 and 1.55 pmol/ml respectively when induced with 0, 1, 3 and 5µM ponasterone A (See Figure 1 and Table 2). By varying levels of the inducer we were thus able to produce almost identical levels of expression for the WT and S993 cell line (mean B Max = 1603 fmol/mg protein WT at 3µM ponasterone A and 1550 fmol/mg protein S993 at 5uM ponasterone A). A functional characterization of the WT and variant clones was carried out under these conditions. SCH1041222 for the S993 variant (Table 3). Here again we did not observe significant differences in the potencies of these compounds at the two receptor variants. The focus of the present study was to determine if the S993 polymorphism had an effect on the affinity or potency of antagonist compounds. However, given the fact that the polymorphic residue resides in the C terminal tail of the protein, it could effect G protein/receptor coupling we therefore performed functional calcium assays with a number of agonist compounds. Again there were no significant differences, in either the potency or the efficacy of any of the compounds tested, ( Fig 4 for curves and table 3 ).

A c c e p t e d M a n u s c r i p t

DISCUSSION

It is widely recognised that different patients respond in different ways to the same medication and there are now numerous examples of cases in which inter-individual differences in drug response are due to sequence variants in genes encoding drug metabolizing enzymes, drug transporters or drug targets. Studies of genetic variation originally focused on drug metabolism but have now been extended to encompass the full spectrum of drug disposition, including a growing list of transporters that influence drug absorption, distribution and excretion [START_REF] Evans | Pharmacogenomics -Drug disposition, drug targets, and side effects[END_REF]. Most drugs exert their pharmacological effects by interacting with specific target proteins, molecular studies have revealed that many of the genes which encode drug targets exhibit genetic polymorphism, which in many cases alters their sensitivity to specific medications. Well characterised examples include polymorphisms in β-adrenergic receptors and their sensitivity to β-agonists in asthmatics [START_REF] Martinez | Association between genetic polymorphisms of the ß2-Association between genetic polymorphisms of the ß2adrenoceptor and response to albuterol in children with and without a history of wheezing[END_REF], the angiotensin converting enzyme (ACE) and its sensitivity to ACE inhibitors (25) and the 5hydroxtryptamine receptor and response to neuroleptics such as clozapine [START_REF] Arranz | Association between clozapine response and allelic variation in 5-HT2A receptor gene[END_REF]. There is now an extensive and growing list of targets where polymorphisms have been linked to altered drug sensitivity, ongoing and future studies will undoubtedly expand the number of such pharmacogenetic relationships.

The metabotropic glutamate receptor family belongs to the class C GPCRs, and is the subject of intense investigation by pharmaceutical companies, as it is widely believed that the glutamatergic system plays a major role in the pathophysiology of clinically important diseases.

mGluR1 antagonists represent potential therapy in a wide range of disorders including chronic pain, depression and anxiety and the patent and literature databases suggest that the development of such compounds is a very active field. However, studies investigating the occurrence of genetic variation within this receptor gene and the comparison of agonist and antagonist potency at different mGluR1 variants have not been published. This is the first report of polymorphisms in the human metabotropic receptor 1 gene. Resequencing of mGluR1 in 93 DNA samples from apparently healthy subjects of African American or Caucasian ancestry identified 46 SNPs and 3 insertion/deletion polymorphisms. Of the 46 SNPS identified, 17 were in the coding region of the gene and 8 of those resulted in an amino acid change. This study demonstrates that while there are a number of non synonymous SNPs, most of these are very rare. Of the eight SNPs identified, four were only seen in the African American population and 2 were only observed in the Caucasian population, and all of these SNPs were present in less than 5% of their respective populations. Only 2 of the 8 identified SNPs were present in both populations and only the c2977>T (P993S) SNP was frequent in more than 5% of each population. This variant was observed in 28.3% of the African American and 35.6% of the Caucasian alleles respectively. The sample size used in this study was sufficiently powered to ensure identification of 99% of all SNPs present in either population at a minor allele freq of 5% or greater [START_REF] Cargill | Characterization of single-nucleotide polymorphisms in coding regions of human genes[END_REF]. The polymorphism described here, S993, occurs in the Intracellular C terminal part of A c c e p t e d M a n u s c r i p t 12 the receptor. mGluRs bind glutamate and other agonists at an extracellular amino terminal domain [START_REF] Costantino | Molecular dynamics simulation of the ligand binding domain of mGluR1 in response to agonist and antagonist binding[END_REF], while non competitive antagonists bind to a trans-membrane portion of the receptor [START_REF] Lavreysen | 3H]R214127: a novel high-affinity radioligand for the mGlu1 receptor reveals a common binding site shared by multiple allosteric antagonists[END_REF]. G protein/receptor coupling has been largely attributed to interactions between the second and third intracellular loops of the receptor and the C terminus of the G Protein [START_REF] Francesconi | Role of the second and third intracellular loops of metabotropic glutamate receptors in mediating dual signal transduction activation[END_REF][START_REF] Havlickova | The second intracellular loop of metabotropic glutamate receptors recognizes C termini of G-protein alpha-subunits[END_REF], Amino acids crucial for binding non competitive antagonists of the human mGluR1 receptor reside in the transmembrane segments V-VII [START_REF] Lavreysen | 3H]R214127: a novel high-affinity radioligand for the mGlu1 receptor reveals a common binding site shared by multiple allosteric antagonists[END_REF] and are thus located relatively far away from the polymorphism evaluated in this study. Although it cannot be excluded apriori that substitution of distant residues could evoke allosteric conformational changes in the antagonist binding pocket, it is not unduly surprising that the potency and affinity of these compounds does not differ between the S993 variant and the wild type receptor. Our results indicate that only the S993 polymorphism occurs frequently within the mGlu1 receptor and that this polymorphism does not significantly affect either antagonist affinities or potencies nor does it alter agonist potencies. However, it remains to be determined whether this SNP would play a role under pathological conditions or during neuronal plasticity. On balance, we believe that the identified genetic variations are unlikely to reduce the effectiveness of mGluR1 antagonists if and when such compounds eventually reach proof of concept studies in man

Position and allele frequencies of non synonymous SNPs identified in the human mGluR1 receptor gene.

A c c e p t e d M a n u s c r i p t 4 identical

 4 expression levels facilitating the functional analysis of the receptor variants. It has previously been demonstrated that non competitive antagonist compounds bind to the same binding pocket made from transmembrane helices IV-VII[START_REF] Lavreysen | 3H]R214127: a novel high-affinity radioligand for the mGlu1 receptor reveals a common binding site shared by multiple allosteric antagonists[END_REF]. All of the mGluR1 selective antagonists tested displayed similar potencies and affinities at the two receptor variants, it is therefore unlikely that the identified genetic variations would significantly modify the effectiveness of mGluR1 antagonists in man. (3,5-dihydrophenylglycine); L-Glutamate; Quisqualic acid ((L)-(+)-a-Amino-3,5dioxo-1,2,4-oxadiazolidine-2-propanoic acid). Antagonists, JNJ16259685, (3,4-dihydro-2H-pyrano[2,3]b-quinolinyl-7-yl)(cis-4-methoxycyclohexyl) methanone, LY456236, (4-methoxy-phenyl)-(6-methoxy-quinazolin-4-yl)-amine hydrochloride; All agonists and antagonists were purchased from Tocris Cookson (Bristol, UK), except for SCH1041222 which was synthesised by Schering Plough. Compounds were dissolved in HBSS or DMSO depending on their solubility. The final concentration of DMSO in all samples was compensated.

2. 5 .

 5 CloningCDNA was synthesised from human hippocampal mRNA (Clontech, Mountain View, CA ) using the superscript kit according to the manufacturers (Invitrogen, Paisley UK) instructions. The coding sequence of mGluR1a was amplified from hippocampal c-DNA by PCR using a proofreading enzyme Pfu Turbo (Stratagene, La Jolla, CA). The coding sequence for mGluR1a was isolated as two

A c c e p t e d M a n u s c r i p t 7 Rev 5 '

 75 gCggCCgcTTACAgggTggAAgAgCTTTgCTT 3', amplified products were cloned onto pCRScript (Stratagene, La Jolla, CA) and sequenced to determine clones with Thymine residues at position 2977 in the coding sequence, the P993 version contains a cytosine residue, the C to T change converts Proline into Serine. The coding sequence of mGluR1 contains a unique site for the restriction enzyme Nde1 at position 2724 and Plasmid pINDS993 containing mGluR1a-S993

A c c e p t e d M a n u s c r i p t 8 2. 9 . 1 .

 891 Measurement of intracellular calcium transients Cells from clonal cell lines were seeded into black clear-bottom 96-well plates at a density of 60,000 cells/well, in DMEM high glucose supplemented with 5% dialysed FBS, 3 U/mL Glutamic-Pyruvic Transaminase (GPT, Sigma, St. Louis, MO), 5 mM sodium pyruvate, 5 µM ponasterone A (Invitrogen, Paisley UK). Following 24 h incubation, the cells were loaded with a fluorescent calcium indicator (Molecular Devices, Sunnyvale, CA, USA). The dye was dissolved in the assay buffer which consisted of Hanks' balanced salt solution (Gibco Life Technologies) buffered with 20 mM Hepes solution (Sigma, St. Louis, MO). To avoid dye bleaching, 2.5 mM Probenecid (Sigma, St. Louis, MO) was added to the calcium indicator solution. Cells were loaded with the dye for 2 h at 37°C in a CO 2 incubator. A fluorometric imaging plate reader (FLIPR 384; Molecular Devices, Sunnyvale, CA, USA) was used to measure intracellular calcium by increases in fluorescence upon agonist stimulation following 30 sec-baseline measurement or 10 min antagonist administration. Genetic variation of the Human mGluR1 Gene. The Human mGluR1 gene consists of a 3582 nucleotide open reading frame (ORF) divided into 8 exons spanning 409 Kilo base pairs on 6q24.3. Re-sequencing of the mGluR1 ORF, 5' and 3'

A c c e p t e d M a n u s c r i p t 10 3 . 3 .

 33 Functional Characterization of the WT and S993 variant.Saturation binding experiments performed using the non competitive antagonist SCH1041222 ( 20), did not highlight significant differences in antagonist binding, mean K d values determined by saturation binding of this ligand were 0.873nM for the WT and 1.712nM for the S993 cell line at the chosen B Max values (1.60pmol/mg WT and 1.55pmol/mg S993), K d values for both the WT and S993 cell lines at different B Max values are shown in table2.Furthermore when heterologous displacement experiments were performed using standard non competitive antagonists LY456236[START_REF] Li | A novel series of potent and selective non competitive Antagonists of metabotropic glutamate receptor 1[END_REF] or JNJ16259685[START_REF] Lavreysen | JNJ16259685, a highly potent, selective and systemically active mGlu1 receptor antagonist[END_REF], no significant differences in the affinity of these ligands were observed, mean K i values of 1.6nM WT and 3,7nM S993 for the JNJ16259685 compound and 375nM WT and 264nM S993 for the LY456236 compound were determined (Figure 2 a and b). We also determined the potency of all of these non competitive antagonist compounds in functional calcium assays, quisqualate-induced Ca i 2+ release was tested in the presence or absence of LY456236, JNJ16259685 and SCH1041222 (Fig 3). When an agonist concentration corresponding to an EC 80 value was utilised to stimulate the receptor, the addition of these reference antagonists resulted in a concentration-dependent inhibition of quisqualate elicited calcium signal with calculated IC 50 values of 267nM LY456236, 2.3nM JNJ16259685 and 4.7nM SCH1041222 for the WT cell line and 445nM LY456236, 3.1nM JNJ16259685 and 5.2nM

Figure 1

 1 Figure 1 Saturation binding experiments for [ 3 H]SCH1041222, in the absence or presence of ponasterone A, a and b are saturation binding curves from representative experiments from the WT and S993 cell lines respectively, un-induced, 1µM, 3 µM and 5 µM of Ponasterone A.

Figure 2

 2 Figure 2 Competitive binding studies with selective mGluR1 antagonists, a JNJ 16259685 b LY456236, WT values are represented by an open square, and S993 values by an open triangle, results are the means of three independent experiments. Data are normalized to the specific binding of the radiolabeled ligand [ 3 H]SCH1041222 observed in the absence of competitor (% Control).

  Figure 3 Antagonism by LY456236, JNJ 16259685 and SCH1041222 of Quisqualate stimulated calcium mobilisation in the WT and S993 expressing cell lines. Inhibition curves were constructed from the percentage responses pooled from three independent experiments performed in quadruplicate, values are means ± SEM. Data are normalized to the response generated by the agonist in the absence of the antagonists.

Figure 4

 4 Figure 4 Concentration response curves for the effect of Quisqualate, Glutamate and DHPG on Ca 2+ mobilisation in WT and S993 cell lines, values are means ± SEM of quadruplicate determinations from a typical experiment.

  Figure 1

Figure 2 - 13

 213 Figure 2

Figure 3 WT

 3 Figure 3

  Figure 4

  Unix workstation (DEC alpha, Compaq Corp), base calling was performed with the Phred software (version 0.990722.g), sequences were assembled with the Phrap software (version 3.01) and scanned with the Polyphred software (version 3.5),and the results were viewed with the Consed software (version 9.0) (Phred, Phrap and Consed available at http://www.genome.washington.edu, PolyPhred is available at http://droog.mbt.washington.edu).

	2.4. DNA sequencing and analysis
	Following DNA amplification, PCR amplicons were sequenced in the forward and reverse
	directions using ABI PRISM BigDye terminator v3.1 Cycle Sequencing DNA Sequencing Kit
	(Applied Biosystems, Foster City, CA) on an Applied Biosystems 3730XL DNA Analyzer.
	Chromatograms were transferred to a	M a n u s c r i p t
	with modifications as Primer3 software (http://www.genome.wi.mit.edu/cgi-bin/primer/primer3.cgi) to amplify 400-650 bp segments of the detailed. PCR primers were designed using the GRM1 coding region, as well as approximately 100 nucleotides flanking the intron/exon splice junctions as determined by gaped alignment of the GRM1 coding sequence (Genbank accession no. NM_000838) with a finished genomic contig from chromosome 6 using sequencher ( v 4.05 Gene codes Corp, Ann Arbor, MI, USA). Forward and reverse primers were 5' tailed with universal primers -21 M13 5' TGTAAAACGACGGCCAGT and M13REV; CAGGAAACAGCTATGACC, A c c e p t e d
	respectively. PCR reactions contained genomic DNA (24ng in the presence of Platinum PCR
	supermix (100mM dNTPs 1.5mM MgCl 2 , 0,1U Platinum Taq polymerase (Invitrogen Corp,
	Carlsbad, CA, USA) and 0.2pmol/ml forward and reverse primers in a 12µl total volume.
	Thermocycling was performed in 96 well microplates on a PTC.200 thermocycler (MJ Research;
	Waltham, MA, USA) with an initial denaturation of 94 o C for 5 min followed by 35 cycles of
	denaturation at 94 o c for 30S, primer annealing for 30s and primer extension at 72 o C for 1 min, after
		6

Table 2

 2 Receptor densities and K d values for the Wild type and S993 clones at varying ponasterone A concentrations, results were generated using the radiolabeled ligand [ 3 H]SCH1041222 and are expressed as means (± SEM) from three independent experiments

Table 3

 3 Functional activities of agonists and antagonists examined at the WT or S993 variant respectively, data are the mean EC 50 values of agonists and IC 50 values of antagonists determined from three independent experiments performed in quadruplicate, and are expressed as means ± SEM.

Table 1

 1

	African	African	Caucasian Caucasian
	American	American

Table 2 B

 2 Max (fmol/mg

			Kd (nM)	B Max (fmol/mg	Kd (nM)
		protein)		protein)
	Ponasterone A
	(µM)
	0	136.7 (± 67)	3.4 (± 0.76)	145.8 (± 50)	3.5 (± 0.47)
	1	799.4 (± 147)	3.8 (± 0.44)	217.2 (± 87)	4.5 (± 0.61)
	3	1603 (± 223)	4.0 (± 0.51)	707.7 (± 157)	4.8 (± 0.67)
	5	1951 (± 354)	4.9 (± 0.49)	1551 (± 270)	3.8 (± 0.51)


Karl Walter Bock
email: bock@uni-tuebingen.de

Christoph Köhle

Topological aspects of oligomeric UDP-glucuronosyltransferases in endoplasmic reticulum membranes: advances and open questions

Keywords: BaP, benzo[a]pyrene, ER, endoplasmic reticulum, 3-MC, 3methylcholanthrene, ROS, reactive oxygen species, T4, thyroxine, UDPGA, UDPglucuronic acid, UGT, UDP-glucuronosyltransferase UDP-glucuronosyltransferases, UGT topology in endoplasmic reticulum membranes, UGT oligomers, diglucuronide formation, β-glucuronidase

UDP-glucuronosyltransferases (UGTs) represent major Phase II enzymes involved in detoxification of endo-and xenobiotics, including many drugs. The intraluminal orientation of the active site of UGTs in endoplasmic reticulum membranes necessitates a number of transporters in these membranes, for example, for UDPglucuronic acid and glucuronides, the latter being insufficiently characterized. In addition, accumulating evidence suggests that UGTs are functional as homo-and hetero-dimers in monoglucuronide formation. They may form tetramers in diglucuronide formation. UGT oligomers probably serve to stabilize UGT monomers and fine-tune UGT activity. Glucuronide disposition may also be influenced by endoplasmic reticulum-localized β-glucuronidase, possibly involved in hydrolysis of hormone and drug glucuronides in target cells. The present commentary reviews recent advances and addresses open questions. Resolution of these questions may help to understand many problems of glucuronide synthesis and disposition in vivo, for example, under-prediction of the in vivo clearance of drugs mostly eliminated by glucuronidation by in vitro enzyme kinetic parameters of UGTs.

A c c e p t e d M a n u s c r i p t

Introduction

Glucuronidation represents a major Phase II biotransformation reaction. It is catalyzed by two evolutionary conserved UGT enzyme families, each including at least 8 enzymes which are adaptively regulated in a tissue-specific manner [START_REF] Dutton | Glucuronidation of drugs and other compounds[END_REF][START_REF] Mackenzie | The UDP-glucuronosyltransferase gene superfamily: recommended nomenclature based on evolutionary divergence[END_REF][START_REF] Tukey | Human UDP-glucuronosyltransferases: metabolism, expression, and disease[END_REF][START_REF] Miners | Prediction of human drug glucuronidation parameters: application of in vitro and in silico modeling approaches[END_REF][START_REF] Mackenzie | Nomenclature update for the mammalian UDP glycosyltransferase (UGT) gene superfamily[END_REF]. The review by Geoffrey Dutton [START_REF] Dutton | Glucuronidation of drugs and other compounds[END_REF] represents an invaluable source of the earlier literature.

UGTs are involved in detoxification of endobiotics including bilirubin, thyroxine, steroids, and xenobiotics including phytochemicals, carcinogenic polycyclic hydrocarbons and a variety of prescribed drugs from all therapeutic classes. The intraluminal orientation of the active site of UGTs and of β-glucuronidase in ER membranes necessitates a number of transporters in these membranes. Following transport of glucuronides between ER lumen and cytosol, conjugate transporters in the plasma membrane are required to prevent the accumulation of polar conjugates in cells [START_REF] König | Conjugate export pumps of the multidrug resistance protein (MRP) family: localization, substrate specificity, and MRP2-mediated drug resistance[END_REF][START_REF] König | Pharmacogenomics of human OATP transporters[END_REF][START_REF] Köhle | Coordinate regulation of drug-metabolizing enzymes and conjugate transporters by the Ah receptor, pregnane X receptor, and constitutive androstane receptor[END_REF]. Recently, accumulating evidence suggests that UGTs are functional in ER membranes as dimeric complexes, and may form tetramers in diglucuronide formation. The present commentary reviews recent advances and addresses open questions about the UGT topology in ER membranes and their oligomeric nature.

Glucuronide disposition may be influenced by ER-localized β-glucuronidase, possibly involved in futile cycling of glucuronides and hydrolysis of hormone and drug glucuronides in target cells. Resolution of these questions may help to understand many problems of glucuronide synthesis and disposition in vivo, for example underprediction of the in vivo clearance of drugs mostly eliminated by glucuronidation by in vitro enzyme kinetic parameters of UGTs.

Topology of UGTs in ER membranes

A number of advances have recently been made on the structure and topology of UGTs and β-glucuronidase in ER membranes (Fig. 1). The schematic representation should be viewed as an extension of the previously published hypothetical model of UGT dimers [START_REF] Miners | Prediction of human drug glucuronidation parameters: application of in vitro and in silico modeling approaches[END_REF]. UGTs are integral ER membrane proteins with a transmembrane segment and a short cytoplasmic tail. They are known to be active at the luminal side of the ER [START_REF] Tukey | Human UDP-glucuronosyltransferases: metabolism, expression, and disease[END_REF][START_REF] Miners | Prediction of human drug glucuronidation parameters: application of in vitro and in silico modeling approaches[END_REF]. This topology necessitates recently identified nucleotide sugar transporters (NSTs) to carry the cofactor UDP-glucuronic acid (UDPGA) to the ER lumen [START_REF] Kobayashi | Molecular and functional characterization of microsomal UDP-glucuronic acid uptake by members of the nucleotide sugar transporter (NST) family[END_REF]. They act as antiporters requiring countertransport of UDP-Nacetylglucosamine [START_REF] Bossuyt | Mechanism of stimulation of microsomal UDPglucurosyltransferase by UDP-N-acetylglucosamine[END_REF]. The reason for luminal orientation of UGTs is unknown but A c c e p t e d M a n u s c r i p t may be due to evolutionary constraints since many glycosyltransferase reactions including those involved in glycoprotein synthesis occur in the ER lumen. UGT monomers consist of the N-terminal aglycone substrate-binding half of the protein and the UDP-glucuronic acid-binding C-terminus, the latter containing one transmembrane sequence [START_REF] Mackenzie | The UDP-glucuronosyltransferase gene superfamily: recommended nomenclature based on evolutionary divergence[END_REF][START_REF] Tukey | Human UDP-glucuronosyltransferases: metabolism, expression, and disease[END_REF][START_REF] Miners | Prediction of human drug glucuronidation parameters: application of in vitro and in silico modeling approaches[END_REF]. The crystal structure of the UDPGA-binding Cterminal half of UGT2B7 has recently been determined at 1.8-Å resolution. Mutants at residues predicted to interact with UDPGA exhibited impaired catalytic activity, and mutants at predicted aglycone binding sites abrogated UGT activity [START_REF] Miley | Crystal structure of the cofactor-binding domain of the human Phase II drug-metabolizing enzyme UDP-glucuronosyltransferase 2B7[END_REF]. In addition, an internal signal sequence has been identified embedding part of the N-terminal half of UGTs in the ER membrane, a feature which may facilitate access of lipophilic aglycones to the active site [START_REF] Ouzzine | An internal signal sequence mediates the targeting and retention of the UDP-glucuronosyltransferase 1A6 to the endoplasmic reticulum[END_REF].

UGTs conjugate glucuronic acid to functional groups of a variety of aglycones mostly hydroxyl, amino or carboxyl groups. With hydroxyl groups (XOH) they catalyze the following reversible reaction: XOH + UDPGA  XOGA + UDP. Recent extensive kinetic analysis in microsomal preparations in vitro suggested an ordered bi bi mechanism in which the aglycone substrate may act as an inhibitor by binding to the enzyme-UDP complex and thus depleting the active enzyme pool [START_REF] Patana | Mutation analysis in UGT1A9 suggests a relationship between substrate and catalytic residues in UDPglucuronosyltransferases[END_REF]. However, the reversible reaction may only rarely occur in the intact cell since the two products of the UGT reaction are rapidly removed: (i) UDP is rapidly hydrolyzed to UMP by nucleoside diphosphatase in the hepatic ER lumen [START_REF] Finch | Nucleotide metabolism by microsomal UDPglucuronosyltransferase and nucleoside diphosphatase as determined by 31 P nuclear-magnetic-resonance spectroscopy[END_REF]. Rapid hydrolysis of UDP in microsomes may explain the lack of UDP-dependent inhibition of UGT reactions in liver microsomes in contrast to preparations from expressed UGTs [START_REF] Fujiwara | Product inhibition of UDP-glucuronosyltransferase (UGT) enzymes by UDP obfuscates the inhibitory effects of UGT substrates[END_REF]. (ii) Glucuronides appear to be rapidly translocated to the cytosol. Evidence for multiple ER-localized organic anion transporters (AT ER ) has been obtained which -in contrast to plasma membrane-localized ATP-dependent glucuronide transporters -do not need ATP but transport organic anions through ER membranes by facilitated diffusion [START_REF] Battaglia | A unique multifunctional transporter translocates estradiol-17β-glucuronide in rat liver microsomal vesicles[END_REF][START_REF] Csala | Evidence for multiple glucuronide transporters in rat liver microsomes[END_REF]. Some of these transporters may also be involved in transport of hormone glucuronides such as thyroxine glucuronide from the cytoplasm to ERlocalized β-glucuronidase (see chapter 5.3).

Monoglucuronide formation by UGT dimers

Accumulating evidence suggests that UGTs mostly operate as dimeric complexes.

Using mutants and chimeric constructs Meech and Mackenzie demonstrated that

A c c e p t e d M a n u s c r i p t oligomerization of two inactive mutants could yield an active unit [START_REF] Meech | UDP-glucuronosyltransferase, the role of the amino terminus in dimerization[END_REF]. Nearest neighbor crosslinking studies followed by gel filtration provided evidence that UGTs form dimers in microsomes [START_REF] Ikushiro | Protein-protein interactions between UDPglucuronosyltransferase isozymes in rat hepatic microsomes[END_REF][START_REF] Ghosh | Homodimerization of human bilirubin-uridine-diphosphate glucuronosyltransferase-1 (UGT1A1) and its functional implications[END_REF]. Using a variety of techniques evidence was obtained that oligomers may function as homo- [START_REF] Ghosh | Homodimerization of human bilirubin-uridine-diphosphate glucuronosyltransferase-1 (UGT1A1) and its functional implications[END_REF][START_REF] Kurkela | Expression and characterization of recombinant human UDPglucuronosyltransferases (UGTs)[END_REF] or hetero-oligomers [START_REF] Kurkela | Expression and characterization of recombinant human UDPglucuronosyltransferases (UGTs)[END_REF][START_REF] Operana | Oligomerization of the UDP-glucuronosyltransferase 1A proteins[END_REF][START_REF] Finel | The UDP-glucuronosyltransferases as oligomeric enzymes[END_REF][START_REF] Kurkela | Interaction with other UDP-glucuronosyltransferases attenuate the consequences of the Y485D mutation on the activity and substrate affinity of UGT1A6[END_REF][START_REF] Fujiwara | Interactions between human UGT1A1, UGT1A4, and UGT1A6 affect their enzymatic activities[END_REF].

For example, in live cells intermolecular interactions among UGT1A proteins was demonstrated by fluorescently tagged UGT1A proteins and homo-and heterodimerization by co-immunoprecipitation analysis [START_REF] Kurkela | Expression and characterization of recombinant human UDPglucuronosyltransferases (UGTs)[END_REF]. Cotranslational insertion of UGTs into the membrane appears to be a requirement for oligomerization [START_REF] Kurkela | Expression and characterization of recombinant human UDPglucuronosyltransferases (UGTs)[END_REF].

UGTs as oligomeric enzymes were critically discussed [START_REF] Finel | The UDP-glucuronosyltransferases as oligomeric enzymes[END_REF]. Functional implications of dimer formation were studied in several ways: for example, co-expression of an inactive mutant of UGT1A6 (the only enzyme catalyzing serotonin glucuronidation [START_REF] Krishnaswamy | Validation of serotonin (5-hydroxytryptamine) as an in vitro substrate probe for human UDP-glucuronosyltransferase (UGT) 1A6[END_REF]) and of UGT1A4 restored UGT1A6-mediated serotonin glucuronidation, suggesting tight interaction between the two recombinant enzymes. Interestingly, these dimers did not share substrate binding sites [START_REF] Finel | The UDP-glucuronosyltransferases as oligomeric enzymes[END_REF][START_REF] Kurkela | Interaction with other UDP-glucuronosyltransferases attenuate the consequences of the Y485D mutation on the activity and substrate affinity of UGT1A6[END_REF]. Heterodimerization has been shown to up-or down-regulate UGT activity [START_REF] Fujiwara | Interactions between human UGT1A1, UGT1A4, and UGT1A6 affect their enzymatic activities[END_REF]. In addition, heterodimers may include recently identified inhibitory UGTs [START_REF] Girard | Genetic diversity at the UGT1 locus is amplified by a novel 3' alternative splicing mechanism leading to nine additional UGT1A proteins that act as regulators of glucuronidation activity[END_REF]. However, more work is needed to characterize these homo-and hetero-oligomers and their functional implications.

Diglucuronide formation by UGT tetramers

Some UGT dimers may form tetramers as suggested by radiation target analysis, a method which has been conceptually and experimentally established by Ellis Kempner, and frequently applied to determine the functional molecular mass of membrane proteins [START_REF] Kempner | Radiation inactivation of membrane components and molecular mass determination by target analysis[END_REF][START_REF] Kempner | Novel predictions from radiation target analysis[END_REF][START_REF] Vessey | In situ structural analysis of microsomal UDPglucuronosyltransferases by radiation inactivation[END_REF]. Studies of bilirubin glucuronidation suggested that diglucuronide formation may be carried out by UGT tetramers [START_REF] Peters | The molecular weights of UDPglucuronosyltransferase determined with radiation-inactivation analysis[END_REF]. Expressed human UGT1A1 efficiently converts the two bilirubin monoglucuronides (at either the C8 or C12 propionic acid group) to the diglucuronide [START_REF] Udomuksorn | Influence of mutations associated with Gilbert and Crigler-Najjar type II syndromes on the glucuronidation kinetics of bilirubin and other UDPglucuronosyltransferase 1A substrates[END_REF], and bilirubin is known to be mainly secreted in human bile as the diglucuronide.

Radiation inactivation analysis was also carried out in studies of 3,6-quinol monoglucuronide (MG) and diglucuronide (DG) formation of BaP and the chemically more stable chrysene (Fig. 2). We got interested in these reactions because of strong induction of these UGT activities in hepatic microsomes from 3-MC-treated rats [START_REF] Lilienblum | Protection against toxic redox cycles between benzo(a)pyrene-3,6-quinone and its quinol by 3-methylcholanthreneinducible formation of the quinol mono-and diglucuronide[END_REF][START_REF] Bock | Mono-and diglucuronide formation from chrysene and benzo(a)pyrene phenols by 3methylcholanthrene-inducible phenol UDP-glucuronosyltransferase (UGT1A1)[END_REF]. Induction factors were 10-and 40-fold for BaP-3,6-quinol MG and DG A c c e p t e d M a n u s c r i p t 6 formation, and 7-and 310-fold for chrysene MG and DG formation, respectively. 3-MC-inducible UGT1A7 efficiently converted the studied quinol monoglucuronides to their diglucuronides [START_REF] Bock | Mono-and diglucuronide formation from benzo[a]pyrene and chrysene diphenols by AHH-1 cell-expressed UDP-glucuronosyltransferase UGT1A7[END_REF], as suggested by much lower MG/DG ratios compared to microsomes from untreated controls (Table 1). However, enzyme induction alone may not explain the efficiency of DG formation. For example, 3-MC treatment of rats enhanced chrysene-3,6-DG formation about 310-fold in liver microsomes, suggesting -in addition to induction of UGT isoforms-an influence of topological features of UGT quaternary structure, discussed subsequently. Glucuronidation of quinols is toxicologically relevant since it probably prevents quinone-quinol redox cycles and associated oxidative stress [START_REF] Lilienblum | Protection against toxic redox cycles between benzo(a)pyrene-3,6-quinone and its quinol by 3-methylcholanthreneinducible formation of the quinol mono-and diglucuronide[END_REF][START_REF] Bock | Mono-and diglucuronide formation from chrysene and benzo(a)pyrene phenols by 3methylcholanthrene-inducible phenol UDP-glucuronosyltransferase (UGT1A1)[END_REF][START_REF] Bock | Mono-and diglucuronide formation from benzo[a]pyrene and chrysene diphenols by AHH-1 cell-expressed UDP-glucuronosyltransferase UGT1A7[END_REF][START_REF] Lorentzen | o POP. Benzo(a)pyrenedione/benzo(a)pyrenediol oxidationreduction couples and the generation of reactive reduced molecular oxygen[END_REF][START_REF] Lorentzen | Toxicity of metabolic benzo(a)pyrenediones to cultured cells and the dependence upon molecular oxygen[END_REF]. BaP quinol diglucuronides represent major conjugates in bile and urine after administration of BaP [START_REF] Bevan | Quinol diglucuronides are predominant metabolites found in bile of rats following intratracheal instillation of benzo(a)pyrene[END_REF][START_REF] Yang | Characterization of conjugated metabolites of benzo[a]pyrene in germ-free rat urine by liquid chromatography/electrospray tandem mass spectrometry[END_REF].

Radiation inactivation analysis was carried out using the described quinol MG and DG reactions [START_REF] Gschaidmeier | Radiation inactivation analysis of microsomal UDP-glucuronosyltransferases catalyzing mono-and diglucuronide formation of 3,6dihydroxybenzo[a]pyrene and 3,6-dihydroxychrysene[END_REF]. For monoglucuronide formation of BaP-and chrysene-3,6-quinol target sizes were 118 ± 33 and 109 ± 21 kDa, respectively (in agreement with the existence of dimers) whereas for diglucuronide formation target sizes were 218 ± 24 and 192 ± 34 kDa, respectively, suggesting the formation of tetramers. It should be noted that in early enzyme purification studies, gel filtration in presence of detergents revealed peaks at the position of tetrameric UGT complexes [START_REF] Tukey | Purification and properties of rabbit liver estrone and pnitrophenol UDP-glucuronyltransferases[END_REF][START_REF] Bock | Differential induction of UDPglucuronosyltransferases and their 'permanent induction' in pre-neoplastic rat liver[END_REF]. In diglucuronide formation some dimers may loosely interact in ER membranes to form tetramers (Fig. 1), findings which, however, need further validation. Loose interaction may explain why tetramers have not been detected in cross-linking studies [START_REF] Ghosh | Homodimerization of human bilirubin-uridine-diphosphate glucuronosyltransferase-1 (UGT1A1) and its functional implications[END_REF]. Nevertheless, as discussed previously [START_REF] Finel | The UDP-glucuronosyltransferases as oligomeric enzymes[END_REF], high molecular weight bands possibly corresponding to tetramers can be seen by close inspection of the cross-linking studies of Gosh et al.

[20 (Fig. 3, lane 3 and 4)]. Tetramers may generate a compartment between two dimers in which monoglucuronides reach high levels to facilitate diglucuronide formation, as evidenced in studies on BaP-3,6-quinol diglucuronide formation: while a high K M value (>70 µM) was detected when the diglucuronide was formed from synthesized monoglucuronides, a much lower K M of 10-20 µM was determined when starting the reaction from the quinol [START_REF] Hartung | Mono-und Diglucuronidbildung von Benzpyren-3,6-chinol durch Lebermikrosomen von Ratte and Mensch[END_REF][START_REF] Bock | UDP-glucuronosyltransferase 1A6: structural, functional, and regulatory aspects[END_REF]. However, it is acknowledged that the results could also reflect the ease of access of quinols versus quinol glucuronide across the ER membrane. Radiation target analysis of bilirubin glucuronidation using human microsomes and expressed UGT1A1 may be useful to substantiate formation of UGT tetramers.

In vitro-in vivo comparison of glucuronide formation

To guide future studies on synthesis and hydrolysis of glucuronides in ER membranes, the relevance of UGT activity is emphasized (i) by correlation studies using UGT polymorphism UGT1A1*28, and (ii) by comparative studies between in vitro kinetic parameters of UGTs and in vivo clearance of drugs mostly eliminated by glucuronidation.

Bilirubin UGT polymorphism UGT1A1*28

Allelic variants often clearly demonstrate the relevance of proteins. Functional UGT polymorphisms are increasingly identified, and are updated in the UGT web site at http://som.flinders.edu.au/FUSA/ClinPharm/UGT. It is assumed that further analysis of bilirubin mono-and diglucuronide formation may help to answer open questions on UGT oligomers. Therefore, in vivo findings using the frequent bilirubin UGT polymorphism UGT1A1*28 are highlighted.

Early clinical studies of inherited hyperbilirubinemias identified the rare and fatal Crigler-Najjar syndromes I and II and the frequent mild form of Gilbert's syndrome.

Frequent occurrence of allelic variants is often due to 'balanced polymorphism' [START_REF] Beutler | Racial variability in the UDPglucuronosyltransferase 1 (UGT1A1) promoter: a balanced polymorphism for regulation of bilirubin metabolism?[END_REF],

i.e. balancing blood bilirubin between high neurotoxic levels and low levels acting as powerful antioxidant [46, for references]. Bilirubin is the final product of heme catabolism, as heme oxygenase cleaves the heme ring to form the water-soluble biliverdin, which is reduced by biliverdin reductase to bilirubin (Fig. 3). Why should mammals have evolved the potentially toxic and insoluble bilirubin? Recently, it was established that bilirubin is oxidized by reactive oxygen species (ROS) to biliverdin which is efficiently reduced back to bilirubin. This amplification cycle establishes a physiologic function of bilirubin as powerful antioxidant [START_REF] Baranano | Biliverdin reductase: a major physiologic cytoprotectant[END_REF]. Epidemiologic evidence indicates that homozygous UGT1A1*28 allele carriers with high serum bilirubin exhibit a strong association with lower risk of cardiovascular disease [START_REF] Lin | Association between UGT1A1*28 allele, bilirubin levels, and coronary heart disease in the Framingham Heart Study[END_REF]. Bilirubin and bilirubin monoglucuronides produced in extrahepatic tissues are efficiently taken up into hepatocytes by OATP1B1 and converted to bilirubin diglucuronide by UGT1A1.

Gilbert's syndrome in Caucasians is mostly due to an additional TA repeat in the TATA box of UGT1A1 (a promoter polymorphism termed UGT1A1*28), leading to reduced UGT1A1 expression and significantly decreased UGT activity. Liver [START_REF] Raijmakers | Association of human liver bilirubin UDP-glucuronosyltransferase activity with a polymorphism in the promoter region of the UGT1A1 gene[END_REF]. The frequency of homozygous carriers of UGT1A1*28 in Caucasians is approximately 10% and the allele frequency 30 to 40% [START_REF] Köhle | Frequent co-occurrence of the TATA box mutation associated with Gilbert's syndrome (UGT1A1*28) with other polymorphisms of the UDPglucuronosyltransferase-1 locus (UGT1A6*2 and UGT1A7*3) in Caucasians and Egyptians[END_REF]. Correlation studies between populations expressing UGT1A1*1 and UGT1A1*28 proteins are often used to associate UGT1A1 activity with in vivo responses. Examples include (i) adverse side effects of irinotecan. The topoisomerase inhibitor irinotecan has been approved for standard therapy of colorectal cancer. Its active metabolite SN-38 is mostly metabolized by UGT1A1 and 1A7 [START_REF] Tukey | Pharmacogenomics of human UDPglucuronosyltransferases and irinotecan toxicity[END_REF]. Poor glucuronidators, i.e., carriers of the promoter variant UGT1A1*28 have been suggested to be at higher risk to develop unwanted side effects such as diarrhea [START_REF] Ando | Polymorphisms of UDP-glucuronosyltransferase gene and irinotecan toxicity: a pharmacogenetic analysis[END_REF]. (ii) A strong correlation was observed between UGT1A1-mediated glucuronidation of SN-38 and of T4 [START_REF] Yoder Graber | UGT1A1*28 genotype affects in-vitro glucuronidation of thyroxine in human livers[END_REF]; there was a significant trend of decreasing T4 glucuronide levels and expression of UGT1A1*28.

Despite low levels of T4 glucuronide in serum, the role of T4 glucuronidation in vivo is supported by alteration of thyroid hormone homeostasis in antiepileptic drug-treated patients [START_REF] Benedetti | Alteration of thyroid hormone homeostasis by antiepileptic drugs in humans: involvement of glucuronosyltransferase induction[END_REF]. (iii) UGTs such as UGT1A1 and UGT2B7 appear to be involved in homeostasis and further metabobolism of estradiol in endometrium [START_REF] Lepine | Specificity and regioselectivity of the conjugation of estradiol, estrone, and their catecholestrogen and methoxyestrogen metabolites by human uridine-diphosphoglucuronosyltransferases expressed in endometrium[END_REF]. Estradiol is metabolized to genotoxic 4-hydroxy-or antiproliferative 2-hydroxy-catechol estrogen.

Interestingly, reducing the excretion of 2-hydroxyestradiol in carriers of the UGT1A1*28 allele has been suggested to be responsible for decreased endometrial cancer risk [START_REF] Duguay | The functional UGT1A1 polymorphism decreases endometrial cancer risk[END_REF]. These three examples may serve to highlight the relevance of in vitro microsomal UGT activity for a variety of in vivo functions.

Comparative studies between in vitro enzyme kinetic parameters of UGTs and in vivo drug clearance

Comparison of in vitro intrinsic clearance of drugs (represented by the enzyme kinetic parameter V max /K M ) with their hepatic clearance in vivo represents a challenging exercise. In the case of drugs mainly excreted by glucuronidation, it was found that the in vitro intrinsic clearance under-predicts in vivo hepatic clearance [START_REF] Miners | Prediction of human drug glucuronidation parameters: application of in vitro and in silico modeling approaches[END_REF][START_REF] Soars | In vitro analysis of human drug glucuronidation and prediction of in vivo metabolic clearance[END_REF][START_REF] Miners | In vitro-in vivo correlation for drugs and other compounds eliminated by glucuronidation in humans: pitfalls and promises[END_REF].

Many factors have been suggested to be responsible for this discrepancy. In addition to general factors such as the nutritional state, the following liver microsomal UGT assay conditions may be relevant: (i) In the case of the antiretroviral drug zidovudine (mainly eliminated via glucuronidation by UGT2B7 [START_REF] Court | Evaluation of 3'-azido-3'-deoxythymidine, morphine, and codeine as probe substrates for UDP-glucuronosyltransferase 2B7 (UGT2B7) in human liver microsomes: specificity and influence of the UGT2B7*2 polymorphism[END_REF]) inclusion of the physiologic carbonate buffer or Williams E medium increased V max and reduced K M in liver A c c e p t e d M a n u s c r i p t microsomes , thereby increasing the intrinsic clearance in vitro [START_REF] Engtrakul | Altered AZT (3'-azido-3'deoxythymidine) glucuronidation kinetics in liver microsomes as an explanation for underprediction of in vivo clearance: comparison to hepatocytes and effect of incubation environment[END_REF]. Determination of UGT activity in physiologic buffer may better preserve the topology of UGTs and associated proteins in the ER. (ii) Fatty acid-free human serum albumin and bovine serum albumin reduce the K M values for UGT2B7 substrates (but not UGT1A1 and UGT1A6 activities) by sequestering inhibitory long-chain fatty acids released by incubations of human liver microsomes and cell-expressed UGTs. This observation has been termed 'albumin effect' [START_REF] Rowland | The "albumin effect" and drug glucuronidation: bovine serum albumin and fatty acid-free human serum albumin enhance the glucuronidation of UDP-glucuronosyltransferase (UGT) 1A9 substrates but not UGT1A1 and UGT1A6 activities[END_REF]. (iii) As mentioned in chapter 2, rapid hydrolysis of UDP in the ER may explain the lack of product inhibition by UDP [START_REF] Fujiwara | Product inhibition of UDP-glucuronosyltransferase (UGT) enzymes by UDP obfuscates the inhibitory effects of UGT substrates[END_REF]. In addition, rapid translocation of glucuronides from ER into the cytoplasm may further decrease product inhibition in vivo. (iiii) Atypical enzyme kinetics, i.e. non-Michaelis-Menten kinetics, is frequently observed in studies of drug glucuronidation and often interpreted by evoking a two site model; in the light of UGT oligomers these sites could be present on separate molecules within the same oligomeric complex [START_REF] Finel | The UDP-glucuronosyltransferases as oligomeric enzymes[END_REF].

Atypical enzyme kinetics leading to autoactivation could in part be responsible for paradoxical effects observed with the over the counter analgesic drug paracetamol (acetaminophen) [START_REF] Court | Interindividual variability in acetaminophen glucuronidation by human liver microsomes: identification of relevant acetaminophen UDP-glucuronosyltransferase isoforms[END_REF]. Its metabolism has been intensely investigated, in particular due to its hepatotoxicity in suicidal overdose. Interestingly, paracetamol is a highaffinity substrate for UGT1A6 [START_REF] Court | Interindividual variability in acetaminophen glucuronidation by human liver microsomes: identification of relevant acetaminophen UDP-glucuronosyltransferase isoforms[END_REF][START_REF] Bock | Paracetamol glucuronidation by recombinant rat and human phenol UDPglucuronosyltransferases[END_REF]. However, it appears to be mainly conjugated in liver by UGT1A9 (and also by UGT1A1) [START_REF] Court | Interindividual variability in acetaminophen glucuronidation by human liver microsomes: identification of relevant acetaminophen UDP-glucuronosyltransferase isoforms[END_REF]. Of course, the presently unknown relative abundance of UGT isoforms may also play a role. In conclusion, understanding oligomeric UGTs and the influence of associated proteins may be necessary for the development of generalizable models for the in vitro-in vivo comparison of drug glucuronidation.

Role of ER-localized β-glucuronidase in glucuronide disposition

Glucuronide disposition may be influenced by ER-localized β-glucuronidase, possibly involved in futile cycling of glucuronides and hydrolysis of conjugated hormones in target cells. Murine β-glucuronidase is derived from a single gene but is located in two subcellular sites, the ER and lysosomes. On the way from synthesis in the rough ER to lysosomes part of the enzyme is retained in the lumen of the ER by associating with the carboxylesterase egasyn which contains an ER retention sequence [START_REF] Swank | Regulation of the subcellular localization and expression of glucuronidase[END_REF][START_REF] Zhen | The β-glucuronidase propeptide contains a serpin-related octamer necessary for complex formation with egasyn esterase and for retention within the endoplasmic reticulum[END_REF][START_REF] Islam | Human egasyn binds beta-glucuronidase but neither the esterase active site of egasyn nor the C terminus of beta-glucuronidase in involved in their interaction[END_REF].

ER-localized β-glucuronidase has been shown to form tetramers (Fig. 1). Its in vivo activity has been demonstrated in the hydrolysis of bilirubin monoglucuronides [START_REF] Whiting | Deconjugation of bilirubin-IXα glucuronides: a physiologic role of hepatic microsomal β-glucuronidase[END_REF].

The activity of β-glucuronidase in the lumen of the ER may lead to futile cycling of A c c e p t e d M a n u s c r i p t 10 glucuronides [START_REF] Kauffman | Regulation of drug conjugate production by futile cycling in intact cells[END_REF], which, however, appears to be limited since ß-glucuronidase activity is low. ER-resident β-glucuronidase may also be responsible for hydrolysis of hormone and drug glucuronides in cells, examplified by the hydrolysis of T4 glucuronide. T4 is widely used for treatment of hypothyroidism. It is mainly deiodinized to T3 (>70%) but is also metabolized by conjugation. T4 sulfate is very unstable because sulfation accelerates inner ring deiodination by approximately 200fold [START_REF] Yoder Graber | UGT1A1*28 genotype affects in-vitro glucuronidation of thyroxine in human livers[END_REF]. T4 glucuronidation (carried out by UGT1A1 and 1A3) leads to a stable T4 glucuronide which may serve as a mechanism of delivery of T4 into intracellular compartments, as discussed in chapter 5.1 [START_REF] Yoder Graber | UGT1A1*28 genotype affects in-vitro glucuronidation of thyroxine in human livers[END_REF][START_REF] Benedetti | Alteration of thyroid hormone homeostasis by antiepileptic drugs in humans: involvement of glucuronosyltransferase induction[END_REF]. However, the postulated uptake of circulating T4 glucuronide into target cells and its hydrolysis by cellular βglucuronidases needs to be further studied. In contrast, lysosomal glucuronidase is involved in the degradation of glycosaminoglycans, synthesized in the Golgi apparatus. Transit of lysosomal glucuronidase from the ER to lysosomes has been reviewed [START_REF] Swank | Regulation of the subcellular localization and expression of glucuronidase[END_REF].

Hydrolysis of glucuronides is also important for enterohepatic and entero-enteric recirculation of many important drugs including SN-38 (the metabolite of irinotecan (mentioned in chapter 5.1) and in the action of the cholesterol-lowering drug ezetimide which is mainly conjugated by UGT1A1 [START_REF] Oswald | Intestinal expression of P-glycoprotein (ABCB1), multidrug resistance associated protein 2 (ABCC2), and uridine diphosphate-glucuronosyltransferase 1A1 predicts the disposition and modulates the effects of cholesterol absorption inhibitor ezetimibe in humans[END_REF]. It has been suggested that ezetimibe glucuronide represents a storage form for long lasting recycling of the active ezetimibe to the intestinal absorption compartment via the systemic circulation.

However, it is unknown to what extent glucuronide hydrolysis contributes to the clearance of drugs mainly eliminated by glucuronidation.

Conclusions

UDP-glucuronosyltransferases (UGTs) represent major Phase II biotransformation enzymes involved in detoxification of endobiotics such as bilirubin, thyroxin and steroid hormones and xenobiotics including a variety of phytochemicals, environmental pollutants such as benzo[a]pyrene, and many drugs. UGTs represent integral membrane proteins. Accumulating evidence suggests that UGTs operate in ER membranes mostly as homo-and heterodimeric complexes which may fine-tune UGT activity and stabilize the enzyme. Evidence includes recombinant technology [START_REF] Meech | UDP-glucuronosyltransferase, the role of the amino terminus in dimerization[END_REF], cross-linking studies [START_REF] Ghosh | Homodimerization of human bilirubin-uridine-diphosphate glucuronosyltransferase-1 (UGT1A1) and its functional implications[END_REF], co-immunoprecipitation, and fluorescence resonance technology in live cells [START_REF] Operana | Oligomerization of the UDP-glucuronosyltransferase 1A proteins[END_REF]. The results of radiation target analysis suggest that tetramers are involved in diglucuronide formation, a finding which, however, needs

  A c c e p t e d M a n u s c r i p t7

A

  c c e p t e d M a n u s c r i p t homogenate bilirubin UGT activity in Caucasian carriers of the UGT1A1*28 genotype was only 48% (ranges 27-75; n=6) of the UGT1A1*1 reference genotype (ranges 47-170; n=17)

A

  c c e p t e d M a n u s c r i p t further validation. Orientation of the active site toward the intraluminal space of the ER necessitates a number of transporters (i) for the cofactor UDP-glucuronic acid synthesized in the cytosol, and (ii) for glucuronides between the ER lumen and the cytosol, the latter being insufficiently characterized. Glucuronide hydrolysis by ERlocalized β-glucuronidase may also require ER-localized glucuronide transporters involved in transport of glucuronides from cytoplasm to ER lumen. Many advances have been made in recent years on the topology and quaternary structure of oligomeric UGTs in ER membranes; but a lot of open questions are remaining. It is hoped that resolution of these questions may help to understand many problems of glucuronide formation and disposition in vivo, for example, under-estimation of in vivo clearance of drugs mainly eliminated by glucuronidation by in vitro enzyme kinetic parameters of UGTs.

Fig. 1 .

 1 Figure legends

Fig. 2 .

 2 Fig.2. (A) Structures of benzo[a]pyrene (BaP)-and chrysene-3,6-quinols. (B) Role of glucuronidation in detoxification of BaP quinones (Q). QOH, quinols; QOGA and QO(GA) 2 , quinol monoglucuronides and diglucuronide, respectively; NQO1, NAD(P)H quinone oxidoreductase-1, which bypasses the semiquinone step; GSTs, glutathione S-transferases.

Fig. 3 .

 3 Fig.3. Scheme of heme catabolism to bilirubin and its diglucuronide in hepatocytes and the physiologic role of bilirubin as antioxidant. Bilirubin can be oxidized by ROS to biliverdin which is efficiently reduced back to bilirubin by biliverdin reductase. This magnifying cycle represents the basis for the role of bilirubin as antioxidant[START_REF] Baranano | Biliverdin reductase: a major physiologic cytoprotectant[END_REF].

Figure 1 - 3 A

 13 Figure 1-3

Table 1. Formation of moglucuronides (MG) and diglucuronides (DG) of benzo[a]pyrene (BaP)-and chrysene-3,6-quinol. For calculation of the MG/DG ratio the sum of the two MGs are used. Data for BaP-3,6-quinol UGT activity in microsomes and expressed UGT1A6 were taken from [START_REF] Lilienblum | Protection against toxic redox cycles between benzo(a)pyrene-3,6-quinone and its quinol by 3-methylcholanthreneinducible formation of the quinol mono-and diglucuronide[END_REF] and [START_REF] Bock | Mono-and diglucuronide formation from chrysene and benzo(a)pyrene phenols by 3methylcholanthrene-inducible phenol UDP-glucuronosyltransferase (UGT1A1)[END_REF], respectively; data for chrysene-3,6-quinol UGT activity in liver microsomes of untreated controls and 3-MC-treated rats and expressed UGT1A6 were taken from [START_REF] Bock | Mono-and diglucuronide formation from chrysene and benzo(a)pyrene phenols by 3methylcholanthrene-inducible phenol UDP-glucuronosyltransferase (UGT1A1)[END_REF] and for expressed UGT1A7 from [START_REF] Bock | Mono-and diglucuronide formation from benzo[a]pyrene and chrysene diphenols by AHH-1 cell-expressed UDP-glucuronosyltransferase UGT1A7[END_REF]. MG/DG ratios are operationally used as inverse parameters for the efficiency of diglucuronide formation.

Substrate

UGT


Huub H Van Rossum

Fred P H T M Romijn

Nico P M Smit

Johan W De Fijter

Johannes Van Pelt

Everolimus and Sirolimus antagonize Tacrolimus based calcineurin inhibition via competition for FK-binding protein 12

Keywords: tacrolimus, mTOR, mammalian target of rapamycin, MTIs, mTOR inhibitors, FKBP, FK506-binding protein, PBMCs, peripheral blood mononuclear cells, CsA, cyclosporin A, SRL, sirolimus, EVL, everolimus, PHA, Calcineurin, tacrolimus, everolimus, sirolimus, transplantation

Abstract

The calcineurin inhibitors cyclosporin A and tacrolimus and the inhibitors of the mTOR, sirolimus and everolimus bind immunophilins that are required for their immunosuppressive action. In contrast to cyclosporin A, tacrolimus and the mTOR inhibitors (MTIs) share common immunophilins, the FK506-binding proteins (FKBPs). We investigated the immunosuppressive interactions of MTIs on tacrolimus based immune suppression, since insights in immunological drug-drug interactions can be very relevant for optimization of immunosuppressive regimens in allograft transplantation medicine.

Isolated peripheral blood mononuclear cells from healthy volunteers were incubated with combinations of MTIs and calcineurin inhibitors and when monitored for calcineurin activity and IL-2 excretion after mitogen stimulation, tacrolimus IC 50 concentrations shifted to higher concentrations in the presence of MTIs. This antagonism was absent for cyclosporin A, reproducible for 10 healthy volunteers (p<0.001) and stronger for sirolimus than for everolimus. When cell lysate was treated with and without MTI, tacrolimus and FKBP12, FKBP12 could increase calcineurin inhibition by tacrolimus and reverse the MTI antagonism for both MTIs. These results demonstrate that FKBP12 can be rate limiting for calcineurin inhibition at high tacrolimus concentrations and that the antagonism of sirolimus and everolimus on tacrolimus based immune suppression is mediated via saturation of FKBP12.

Introduction

After organ transplantation, immune suppression to prevent allograft rejection is crucial for successful graft survival. Several immunosuppressive agents, such as the calcineurin (CN) inhibitors tacrolimus (TRL) or cyclosporin A (CsA) and the mammalian target of rapamycin (mTOR) inhibitors sirolimus (SRL) or its derivative everolimus (EVL), are used and combinational therapies are applied to achieve optimal immunosuppressive effect and reduce individual drug toxicity [START_REF] Halloran | Immunosuppressive drugs for kidney transplantation[END_REF]. CsA and TRL inhibit CN, a calcium/calmodulin dependent serine/threonine protein phosphatase that plays an important role in mediating T cell receptor stimuli into cytokine (IL-2, IFNγ) transcription. SRL and EVL inhibit mTOR, a kinase with a key function in cell cycle progression [START_REF] Halloran | Immunosuppressive drugs for kidney transplantation[END_REF][START_REF] Feske | Gene regulation mediated by calcium signals in T lymphocytes[END_REF]. Both classes of drugs inhibit enzyme activity only after binding to specific intracellular binding proteins, so called immunophilins [START_REF] Liu | Calcineurin Is A Common Target of Cyclophilin-Cyclosporine-A and Fkbp-Fk506 Complexes[END_REF][START_REF] Dumont | Mechanism of action of the immunosuppressant rapamycin[END_REF]. CsA binds selectively to the cyclophilin class of immunophilins that are cytosolic peptidyl-prolyl isomerases, while TRL and the mTOR inhibitors (MTIs) bind to the FK506-binding proteins (FKBP) that also exert peptidyl-prolyl isomerase activity [START_REF] Liu | Calcineurin Is A Common Target of Cyclophilin-Cyclosporine-A and Fkbp-Fk506 Complexes[END_REF]. SRL, EVL and TRL bind similar FKBPs [START_REF] Dumont | The Immunosuppressive Macrolides Fk-506 and Rapamycin Act As Reciprocal Antagonists in Murine T-Cells[END_REF][START_REF] Schuler | SDZ RAD, a new rapamycin derivative -Pharmacological properties in vitro and in vivo[END_REF], while distinct and selective inhibition of respectively mTOR and CN is observed [START_REF] Dumont | Distinct Mechanisms of Suppression of Murine T-Cell Activation by the Related Macrolides Fk-506 and Rapamycin[END_REF]. Since similar FKBPs are required for successful CN inhibition by TRL and mTOR inhibition by SRL or EVL, pharmacological drug-drug interactions have been investigated by several groups. Dumont et al. has shown that SRL and TRL are reciprocal antagonists [START_REF] Dumont | The Immunosuppressive Macrolides Fk-506 and Rapamycin Act As Reciprocal Antagonists in Murine T-Cells[END_REF][START_REF] Dumont | Quantitative and Temporal Analysis of the Cellular Interaction of Fk-506 and Rapamycin in T-Lymphocytes[END_REF], while others have demonstrated immunosuppressive synergism of these drugs [START_REF] Vu | Tacrolimus (FK506) and sirolimus (rapamycin) in combination are not antagonistic but produce extended graft survival in cardiac transplantation in the rat[END_REF][START_REF] Barten | Synergistic effects of sirolimus with cyclosporine and tacrolimus: Analysis of immunosuppression on lymphocyte proliferation and activation in rat whole blood[END_REF].

In clinical practice, pharmacokinetic drug-drug interactions on metabolic and drugtransporter proteins are therapeutically avoided or adjusted for by therapeutic drug A c c e p t e d M a n u s c r i p t 5 monitoring. When drug-drug interactions occur on the pharmacologic level and thus inhibit each others effectiveness, concentration measurement becomes less relevant since it is unable to detect this interaction [START_REF] Barten | Synergistic effects of sirolimus with cyclosporine and tacrolimus: Analysis of immunosuppression on lymphocyte proliferation and activation in rat whole blood[END_REF]. In addition, combinations of immunosuppressive drugs might be associated with immunosuppressive synergism [START_REF] Oellerich | Biomarkers -The link between therapeutic drug monitoring and pharmacodynamics[END_REF]. Pharmacodynamic monitoring strategies that can detect these interactions may prove to be useful to further optimize immunosuppressive regimen [START_REF] Burkhart | Pharmacodynamics in the development of new immunosuppressive drugs[END_REF].

In this respect we studied the interaction of EVL and SRL on TRL based immunosuppression in peripheral blood mononuclear cells (PBMCs) isolated from healthy individuals. Pharmacological interaction was investigated by measurement of CN enzyme activity. For immunological monitoring, the IL-2 downstream product of CN was used.

A c c e p t e d M a n u s c r i p t 6

Materials and Methods

Materials

The following materials and reagents were used: Buffy coats were purchased from Sanquin (Amsterdam, NL). RPMI 1640, heat inactivated fetal calf serum (iFCS), penicillin/streptomycin, phytohemagglutinin (PHA) and IL-2 EASIA kit from Invitrogen (Carlsbad, CA), NP-40, soybean trypsin inhibitor, cyclosporin A, tacrolimus, sirolimus (rapamycin), FKBP12 and phorbol-12-myristate-13-acetate (PMA) from Sigma-Aldrich (Steinheim, Germany). Phenylmethylsulfonyl fluoride from MERCK (Darmstadt, Germany), leupeptin from Roche (Basel, Switzerland), aprotinin from Bayer (Leverkusen, Germany), phosphate standard concentrate and everolimus from Fluka (Buchs, Switzerland) and the malachite green calcineurin activity assay kit from Biomol (Plymouth Meeting, PA). All other chemicals and reagents were of the highest available grade.

Immunosuppressants

The immunosuppressants CsA (MW 1203), TRL (MW 804), EVL (MW 958) and SRL (MW 914) were dissolved in ethanol and serial diluted in ethanol. These immunosuppressant stock solutions were diluted in the experiments resulting in final CsA, EVL and SRL concentrations of 0.025, 0.25, 2.5, 25, 250 and 2500 μg/L and final TRL concentrations of 0.00625, 0.0625, 0.625, 6.25, 62.5 and 625 μg/L. Final ethanol concentrations were 0.25% for single immunosuppressant experiments and 0.5% for the interaction experiments. For the CsA inhibition curve in figure 2, CsA ranged from 0.1 to A c c e p t e d M a n u s c r i p t 7 3600 μg/L with final ethanol concentrations of 1 %. The ethanol concentrations resulted in a 5% increase in CN activity when compared to an ethanol blank (n=4, p=0.04).

PBMC isolation and cell culture

Peripheral blood mononuclear cell (PBMC) fraction of healthy volunteer blood was isolated from buffy coats by ficoll density gradient centrifugation. Remaining of erythrocytes and thrombocytes were lysed and washed out by NH 4 Cl lysis buffer (8.4 g/L NH 4 Cl, 1.0 g/L KHCO 3 , pH 7.3) and resuspended in phosphate buffered saline (PBS, 154 mmol/L NaCl, 1.4 mmol/L phosphate pH 7.5) for cell count on a Sysmex XE2100. Cells were centrifuged (10 min., 350g, 4 ºC) and resuspended at a concentration of 2•10 6 cells/mL in RPMI 1640 cell culture containing 10% iFCS, 100 U/mL penicillin and 100 μg/mL streptomycin.

For CN activity measurement, cells were incubated for 1 hr at 37 ºC with immunosuppressant(s). After incubation, samples were centrifuged (10 min., 350g, 4 ºC) and cell pellets were washed once with 400 μL HEPES buffered saline (9.0 g/L NaCl, 10 mmol/L Hepes pH 7.5), resuspended in 400 μL lysis buffer (50 mmol/L Tris HCl pH 7.7, 1.0 mmol/L dithiothreitol, 5.0 mmol/L ascorbic acid, 0.02% (v/v) NP-40, 50 mg/L soybean trypsin inhibitor, 50 mg/L phenylmethylsulphonyl fluoride, 5.0 mg/L leupeptin and 5.0 mg/L aprotinin) and lysed by 3 freeze thaw cycles (liquid N 2 / 30 ºC). Cell debris was centrifuged (10 min., 10000g, 4 ºC) and supernatants were snap frozen in liquid N 2 before storage at -80 ºC till CN activity measurement [START_REF] Sellar | Spectrophotometric assay for calcineurin activity in leukocytes isolated from human blood[END_REF].

For IL-2 excretion measurement, cells were stimulated by PHA/PMA 1 hour after incubation with immunosuppressant(s), and incubated overnight at 37 ºC in a humidified A c c e p t e d M a n u s c r i p t 8 environment (5%, CO 2 ). PMA stock solution was 4000 times diluted in cell cultures resulting in a final PMA and DMSO concentration of respectively 5.3 nM and 0.025 %, and PHA was 100 times diluted according to manufacturers' protocol. The next day, cells were centrifuged (10 min., 350g, 4 ºC) and supernatant was stored at -20 ºC until IL-2 concentration measurement.

Cell lysate experiments

In order to investigate the role of FKBPs, we isolated PBMCs from buffycoats obtained from healthy volunteers. Untreated PBMCs were lysed as described before and aliquoted lysate was incubated in duplicate (1 hr, 37 ºC) with and without TRL, MTI and FKBP12 resulting in final lysate concentrations of 25 μg/L TRL, 50 μg/L of SRL/EVL and 2000 μg/L and 10000 μg/L of extra FKBP12. At these concentrations the molar concentrations of the MTIs, TRL and FKBP12 were of the same order of magnitude. Directly after incubation CN activity was determined.

Calcineurin assay

Calcineurin phosphatase activity was measured as described before [START_REF] Sellar | Spectrophotometric assay for calcineurin activity in leukocytes isolated from human blood[END_REF]. In brief, CN phosphatase was quantified by measurement of the dephosphorylation of the phosphorylated RII substrate in the presence of okadaic acid. [START_REF] Bierer | 2 Distinct Signal Transmission Pathways in T-Lymphocytes Are Inhibited by Complexes Formed Between An Immunophilin and Either Fk506 Or Rapamycin[END_REF]

Cytokine concentration measurement

IL-2 concentrations were determined with the EASIA kit (Invitrogen) and performed according to manufacturers' protocol.

Data analysis and statistics

Values are presented as mean of duplicate and blank experiments were performed in quadruple. CN activity and IL-2 concentrations were expressed as % of blank measurement in order to remove inter-individual variation in absolute CN activity and IL-2 excretion.

CN inhibition curves and IL-2 cytokine inhibition curves were fitted by sigmoidal doseresponse curves using GraphPad software and IC 50 concentrations, E max values and 95% confidence intervals (CI) were obtained from these regression analysis. Student t-tests were used to test significance and statistical significance was defined as p<0.05. The MTIs inhibited IL-2 excretion in the PHA/PMA stimulated PBMCs themselves at the concentration range used and this inhibition increased with increasing concentrations of MTI, though no clear sigmoidal concentration response relationship was visible as well as no inhibition of total IL-2 excretion. When high TRL concentrations were used in combination with EVL or SRL, low concentration of the MTIs did not significantly effect IL-2 inhibition by TRL. However at higher concentrations (250 μg/L), significant shifts in IL-2 IC 50 values were observed. MTI concentrations of 25 μg/L and higher reversed IL-2 inhibition by TRL though the occurrence and magnitude of this effect is also dependent on the TRL concentration and seems to be less for EVL when compared to SRL (figure 1A and 1B). No increasing IC 50 values and significant differences in IC 50 values were observed when CsA was used in combination with EVL and SRL (figure 1C and1D). A concentration dependent effect of EVL and SRL on CN inhibition by TRL was observed and significant higher TRL IC 50 values were found when 25 μg/L SRL and 250 μg/L EVL or higher, were present in culture medium (figure 3A and3B). This pattern was not detected for the CsA CN inhibition curves; here no differences in IC 50 values were observed by increasing concentration of MTIs (figure 3C and3D).

CN inhibition by CNIs, MTIs and combinations thereof

Antagonism of MTIs on TRL based immune suppression in 10 healthy volunteers

To determine whether the TRL/MTI interactions are reproducible for other individuals we investigated the interaction on PBMCs isolated from 10 healthy volunteers. healthy volunteers and treated the PBMCs with 0.625 μg/L TRL and 25 μg/L of EVL or SRL, concentrations that previously (figure 1) showed to be characteristic for the interaction. The results are shown in figure 4.

All three drugs inhibited IL-2 excretion in our cell culture system, though TRL caused a larger inhibition than SRL (p=0.001) and EVL (p=0.002) that partially inhibited the IL-2 excretion in these concentrations. When combinations of TRL and MTIs were tested, SRL significantly reversed IL-2 excretion inhibition by TRL (p=0.004), while for EVL no difference in IL-2 levels was found (p=0.53). The IL-2 concentrations observed for the EVL/TRL combination was also significant lower when compared to the combination of SRL/TRL (p=0.01).

Next, we determined CN activity in the presence/absence of TRL (6.25 μg/L) and/or EVL/SRL (25 μg/L), concentrations that were previously illustrative for the interaction (figure 3).

Here no CN inhibition was found for SRL and EVL. TRL caused a mean CN inhibition of 44%±11% (SD) in the PBMC fractions isolated from these individuals. When MTIs were co-incubated with TRL in the culture medium, CN inhibition by TRL was partially lost and final mean inhibition in the presence of SRL and EVL was 18%±14% and 26%±10%, respectively. Both were significant lower (p<0.001) when compared to TRL alone.

Antagonism of SRL on TRL mediated CN inhibition was significantly larger than that of EVL for these concentrations (p=0.01).

Role of FKBPs

A c c e p t e d M a n u s c r i p t 13

To investigate whether the mechanism of antagonism between both mTOR inhibitors and TRL was mediated via competition for saturated FKBP binding sites, we prepared PBMC lysate from untreated cells of 4 healthy volunteers in duplicate and incubated (1 hr, 37 ºC) these with and without mTOR inhibitor, TRL and FKBP12. CN activity was determined directly after incubation. The results of these experiments are shown in figure 5.

In this test system no inhibition of CN was found by the MTIs, FKBP12 and combinations thereof (data not shown). Inhibition by TRL in cell lysate was observed though it was much smaller (± 10%) compared to the cell systems. Since the addition of 2000 μg/L FKBP12 increased TRL inhibition (± 35%, p<0.001) it seemed that the FKBP12 pool from the intracellular compartment was diluted in the cell lysate, limiting CN inhibition by TRL.

Addition of SRL to TRL reduced TRL inhibition of CN (p=0.005), while for EVL antagonism under these conditions could not be detected (p=0.7). In the presence of 2000 μg/L FKBP12 antagonism for both SRL and EVL was found (for both p<0.001) and when 10000 μg/L FKBP12 was used, antagonism of both MTIs was no longer observed.

A c c e p t e d M a n u s c r i p t 14

Discussion

In transplant recipients, multiple immunosuppressive agents are normally used in order to optimally prevent acute rejection episodes and to reduce the side effects of the individual drugs [START_REF] Halloran | Immunosuppressive drugs for kidney transplantation[END_REF]. The CNI and MTI class of drugs require intracellular binding proteins (immunophilins) to exert their immunosuppressive action. TRL binds to its intracellular binding proteins (FKBPs) and especially the 12 kDa protein FKBP12 is considered critical for the immunosuppressive action of TRL [START_REF] Bram | Identification of the Immunophilins Capable of Mediating Inhibition of Signal-Transduction by Cyclosporine-A and Fk506 -Roles of Calcineurin Binding and Cellular Location[END_REF]. The MTIs SRL and its analog EVL require binding to the same protein though an other immunosuppressive mechanism is observed for these drugs [START_REF] Schuler | SDZ RAD, a new rapamycin derivative -Pharmacological properties in vitro and in vivo[END_REF][START_REF] Bierer | 2 Distinct Signal Transmission Pathways in T-Lymphocytes Are Inhibited by Complexes Formed Between An Immunophilin and Either Fk506 Or Rapamycin[END_REF]. Where TRL inhibits CN and block T-cell receptor induced activation/proliferation of T cells, the mTOR inhibitors inhibit IL-2 induced T-cell proliferation [START_REF] Dumont | Distinct Mechanisms of Suppression of Murine T-Cell Activation by the Related Macrolides Fk-506 and Rapamycin[END_REF][START_REF] Bierer | 2 Distinct Signal Transmission Pathways in T-Lymphocytes Are Inhibited by Complexes Formed Between An Immunophilin and Either Fk506 Or Rapamycin[END_REF]. In this study, using a CN assay, we confirmed that CN enzyme activity is not inhibited by EVL and SRL but selectively by TRL and CsA (figure 2), which is in agreement with previous observations. In studies by Dumont et al., reciprocal antagonism was observed for SRL and TRL which was explained by the use of similar intracellular binding sites of the drugs [START_REF] Dumont | The Immunosuppressive Macrolides Fk-506 and Rapamycin Act As Reciprocal Antagonists in Murine T-Cells[END_REF][START_REF] Dumont | Quantitative and Temporal Analysis of the Cellular Interaction of Fk-506 and Rapamycin in T-Lymphocytes[END_REF]. Here we show that both SRL and EVL antagonize TRL mediated CN inhibition and TRL inhibition of IL-2 excretion in PHA/PMA stimulated PBMCs, an effect which is not observed for the CNI CsA. In addition, we show that the antagonism between MTIs and TRL in cell lysate is reversed by the addition of excess of FKBP12 (10000 μg/L), indicating that the antagonism is mediated via this protein.

The in vitro antagonism observed in our cellular system is significant starting at SRL The MTIs themselves reduce IL-2 excretion in our cell culture system, without showing a clear sigmoidal concentration-response relationship. This effect is probably best explained to be indirect and mediated through inhibition of proliferation/activation of IL-2 producing cells. In our system the lowest MTIs concentrations used already significantly decreased IL-2 excretion. Under these conditions of low MTI concentration, immunosuppressive additivity or even synergism could be very well achieved in patients in combination protocols with CsA, but also TRL; although MTIs are mechanistically antagonists of TRL, antagonism only occurs at saturating conditions for FKBP. Immunosuppressive synergy has been demonstrated for SRL/TRL and SRL/CsA combinations on lymphocyte proliferation and lymphocyte activation markers [START_REF] Barten | Synergistic effects of sirolimus with cyclosporine and tacrolimus: Analysis of immunosuppression on lymphocyte proliferation and activation in rat whole blood[END_REF].

25 μg/L of both EVL and SRL partially reversed CN inhibition by 6.25 μg/L TRL though the interference by SRL was significant larger when compared to EVL in 10 healthy volunteers. In addition, only SRL could significantly reverse IL-2 inhibition by 0.625 μg/L TRL. These results indicate relevant differences between both MTIs that could be explained by the weaker binding of EVL to FKBP12 [START_REF] Schuler | SDZ RAD, a new rapamycin derivative -Pharmacological properties in vitro and in vivo[END_REF].

A c c e p t e d M a n u s c r i p t 16 Kung and Halloran showed that at high concentrations of CNIs, immunophilins limit CN inhibition for both CsA and TRL [START_REF] Kung | Immunophilins may limit calcineurin inhibition by cyclosporine and tacrolimus at high drug concentrations[END_REF]. It is however not known whether this is relevant for CNI treated patients. When using combination immunosuppressant therapy of both MTI and TRL, saturation of FKBPs and thus pharmacological drug-drug interaction is even more likely and this interaction could therefore be clinically relevant. The MTI concentrations for which TRL antagonism was observed (25 μg/L) are of the same magnitude of MTI blood concentrations in allograft recipients treated with these drugs [START_REF] Oellerich | The role of therapeutic drug monitoring in individualizing immunosuppressive drug therapy: Recent developments[END_REF].

In addition, also the TRL concentrations at which the interference was observed are found in allograft recipients treated with this drug [START_REF] Oellerich | The role of therapeutic drug monitoring in individualizing immunosuppressive drug therapy: Recent developments[END_REF]. The drug concentrations that are used in our in vitro model can however not directly be translated to full blood drug concentrations, since drug concentrations from cell culture experiments do not necessarily show similar cellular responses compared to drug concentrations in blood due to the presence of irrelevant binding sites as has been demonstrated for CsA [START_REF] Batiuk | Cyclosporine inhibition of leukocyte calcineurin is much less in whole blood than in culture medium[END_REF]. The true clinical relevance remains therefore unclear.

If CN inhibition is limited by the immunophilin content in patients receiving CNI, this could be of great importance; higher CNI concentrations are ineffective. Especially early after graft implantation, when the blood concentrations are highest, this might become relevant. Consequently, under condition of immunophilin saturation, inter-individual variation in the content of immunophilins could be of great importance for allowing different maximal CN inhibitions.

  μL of sample lysis was added to 40 μL of assay buffer (0.313 µmol/L calmodulin, 0.375 mmol/L RII phosphopeptide substrate, 75 mmol/L Tris pH 7.5, 150 mmol/L NaCl, 9 mmol/L MgCl 2 , 0.75 mmol/L dithiothreitol, 0.0375 % NP-40, 0.625 µmol/L okadaic acid, 0.75 mmol/L CaCl 2 and 5 mmol/L ascorbic acid) and substrate blanks were used to correct for A c c e p t e d M a n u s c r i p t 9 background absorbance. The assay was run for 30 min. at 30 ºC and stopped by addition of 100 μL of malachite green reagent. Colour was developed the next 50 min. before absorbance measurement at 620nm, and phosphate was quantified by means of a 0-5 nmol calibration curve.

  A c c e p t e d M a n u s c r i p t 10 3. Results 3.1 IL-2 inhibition by TRL and CsA in the presence of EVL and SRL To gain insights whether and under which conditions the immunological interaction between the MTIs and TRL occurred, we first measured the effect of the MTIs on IL-2 inhibition by TRL. PBMCs isolated from healthy volunteers were stimulated using PHA/PMA and IL-2 excretion was measured in the presence of TRL or CsA and various concentrations of EVL or SRL. The results of these experiments are shown in figure 1 and the obtained IC 50 values of CsA and TRL in the presence of various concentrations of EVL and SRL are listed in table 1.

A

  c c e p t e d M a n u s c r i p t 11To further investigate the origin of the observed IL-2 antagonism, we measured calcineurin activity that is the pharmacological target enzyme of TRL. First, the individual effect of the immunosuppressants on CN activity was investigated and therefore CN inhibition curves were determined for CsA, TRL, EVL and SRL. The data in figure2shows that SRL and EVL do not inhibit CN activity, while for TRL and CsA a clear dose-response curve was found with an IC 50 value of 1.0 μg/L (0.51 -2.1 μg/L, 95% CI) and an E max of 53% (48 -58%, 95% CI) for TRL and an IC 50 value of 14 μg/L (9.6 -21 μg/L, 95% CI) and an E max of 31% ( 26 -36%, 95% CI) for CsA.Next, we incubated five concentrations of TRL (0.0625 -625 μg/L) and a TRL blank, each in combination with five concentrations (0.25 -2500 μg/L) and a blank of either EVL or SRL. As control experiment TRL was replaced by CsA (0.25 -2500 μg/L). The obtained CN inhibition curves are shown in figure3and the calculated IC 50 concentrations for TRL and CsA in the presence of various concentrations of EVL and SRL are listed in table 2.

A c c e p t e d M a n u s c r i p t 12 For

 12 reproduction of the immunological antagonism we reproduced the experiment for 10

  concentrations of 25 μg/L for CN activity and 250 μg/L for IL-2 excretion and at EVL A c c e p t e d M a n u s c r i p t concentrations of 250 μg/L for both CN inhibition and IL-2 excretion inhibition by TRL. These relative high concentrations of MTIs in combination with TRL seem to saturate FKBP binding sites that are required for CN inhibition by TRL. Competition between the MTIs and TRL for these binding sites is clearly illustrated by the IL-2 interaction experiments in figure 1. Here it is shown that 0.625 μg/L TRL itself inhibits IL-2 excretion, but when increasing concentrations of SRL are introduced, above a certain threshold (25 μg/L SRL), IL-2 inhibition by TRL is lost and the IL-2 response is more comparable to the 25 μg/L SRL response itself.

Finally, blood drug

  concentrations and also intracellular drug concentrations are not able to detect the limitation of CN inhibition by immunophilins. Only pharmacodynamic strategies A c c e p t e d M a n u s c r i p t are theoretically capable of detecting these. This also holds for detecting the interaction between the MTI and TRL in individuals receiving both drugs.In conclusion, it is shown that mTOR inhibitors can antagonize TRL and not CsA mediated immunosuppression. FKBP12 that is bound by EVL and SRL, and required for TRL based CN inhibition is a key mediator; saturation of this protein by high concentrations of MTIs and TRL results in competition for FKBP12.

Figure 1 .

 1 Figure 1. IL-2 inhibition by TRL and CsA in the presence of MTIs. IL-2 inhibition by

Figure 2 .

 2 Figure 2. CN inhibition by CsA, TRL, SRL and EVL. Data are normalized and

Figure 3 .

 3 Figure 3. CN inhibition by CsA and TRL in the presence of MTIs. CN inhibition by

A c c e p t e d M a n u s c r i p t 22 Figure 4 .

 224 Figure 4. MTI antagonism in 10 healthy volunteers. Left: IL-2 excretion after

Figure 5 .

 5 Figure 5. CN activity after FKBP12, TRL and MTI addition to cell lysate. CN activity

Table 2 .

 2 CN IC 50 values of TRL and CsA in the presence of various concentrations of SRL and EVL. IC 50 values and 95% Confidence Intervals (CI) were obtained from the sigmoidal concentration-response regressions presented in figure 3 by Graphpad software.

Figure 2 A

 2 Figure 2

Table 1 .

 1

	23

Table 1 . IL-2 IC 50 values of TRL and CsA in the presence of various concentrations of SRL and EVL. IC

 1 50 values and 95% Confidence Intervals (CI) were obtained from the sigmoidal concentration-response regressions when the data in figure1is presented with the CNI inhibitor on the x-axis and the individual curves are variations in EVL or SRL concentrations.

	M a n u s c r i p t
	A c c e p t e d
	24

Table 2 .

 2

		[mTOR inhibitor]	Tacrolimus	Cyclosporin A
		in μg/L	IC 50 in μg/L (95% CI)	IC 50 in μg/L (95% CI)
	Sirolimus	0	1.0 (0.32 -3.4)	30 (12 -74)
		0.25	1.5 (0.69 -3.4)	13 (7.8 -23)
		2.5	3.8 (1.6 -8.8)	31 (15 -63)
		25	11 (6.7 -18)	29 (11 -79)
		250	125 (25 -620)	13 (5.3 -31)
		2500	290 (0.08 -1.1•10 6 )	16 (8.5 -31)
	Everolimus 0	0.54 (0.15 -1.9)	14 (5.1 -41)
		0.25	0.70 (0.34 -1.4)	7.6 (3.7 -16)
		2.5	1.2 (0.39 -3.9)	13 (4.0 -43)