J Robert Lane
email: jrlane@chem.leidenuniv.nl

Elisabeth Klaasse

Judy Lin

John Van Bruchem

Margot W Beukers

Adriaan P Ijzerman

Characterization of [ 3 H]LUF5834: a novel non-ribose high-affinity agonist radioligand for the adenosine A 1 receptor

Keywords: adenosine receptor, G protein-coupled receptor, radioligand, partial agonist, inverse agonist cyclopentyladenosine, 8BCPA; C8-ethylamino-N 6 -cyclopentyladenosine, 8ECPA;

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

Abstract:

The adenosine A 1 receptor is a promising therapeutic target for neurological disorders such as cognition deficits and is involved in cardiovascular preconditioning. Classically adenosine receptor agonists were all derivatives of adenosine, and thought to require a Dribose moiety. More recently, however, the discovery of non-adenosine agonists for the human adenosine A 1 receptor (hA 1 R) has challenged this dogma [START_REF] Beukers | New, non-adenosine, high-potency agonists for the human adenosine A2B receptor with an improved selectivity profile compared to the reference agonist N-ethylcarboxamidoadenosine[END_REF]). In this study we characterize the tritiated form of one of these compounds, [ 3 H]LUF5834, as the first non-ribose partial agonist radioligand with nanomolar affinity for the hA 1 R. Due to its partial agonist efficacy, [ 3 H]LUF5834 labeled both G protein-coupled and uncoupled receptors with a similar high affinity. Using [ 3 H]LUF5834 we performed competition binding experiments to characterize a range of A 1 R ligands varying in efficacy from the full agonist CPA to the inverse agonist DPCPX. Surprisingly, in the control condition both agonists and inverse agonists displayed biphasic isotherms. With the addition of 1 mM GTP the high affinity isotherm of agonists or the low affinity isotherm of inverse agonists was lost revealing the mechanism of action of such inverse agonists at the A 1 R. Consequently, [ 3 H]LUF5834 represents a novel high affinity radioligand for the A 1 R and may prove a useful tool to provide estimates of inverse agonist efficacy at this receptor.

A c c e p t e d M a n u s c r i p t 1 Introduction

Adenosine is a ubiquitous local hormone that has been reported to play an important role in numerous tissues by acting mainly through four subtypes of adenosine receptors [START_REF] Fredholm | International Union of Pharmacology. XXV. Nomenclature and classification of adenosine receptors[END_REF]. These receptors (A 1 R, A 2A R, A 2B R and A 3 R) belong to the G protein-coupled receptor (GPCR) superfamily. This study focuses on the human adenosine A 1 receptor (hA 1 R) which primarily couples to G i/o G proteins which mediate the inhibition of adenylate cyclase. The A 1 R receptor is widely expressed, being present in the CNS and in peripheral systems such as cardiovascular tissue [START_REF] Fredholm | International Union of Pharmacology. XXV. Nomenclature and classification of adenosine receptors[END_REF][START_REF] Dixon | Tissue distribution of adenosine receptor mRNAs in the rat[END_REF]. The wide and abundant distribution of these receptors underlines their important role in the body and therefore they represent targets for useful therapeutic agents [START_REF] Jacobson | Adenosine receptors as therapeutic targets[END_REF][START_REF] Gao | Emerging adenosine receptor agonists[END_REF]. For example the abundance of the A 1 R in the atrioventricular node of the heart is the basis for clinical development of selective A 1 R agonists as antiarrythmic agents [START_REF] Peterman | Tecadenoson: a novel, selective A1 adenosine receptor agonist[END_REF]. Similarly, activation of A 1 Rs in the spinal cord may represent a novel treatment for neuropathic pain [START_REF] Elzein | A1 adenosine receptor agonists and their potential therapeutic applications[END_REF].

Historically, agonists for the adenosine receptors have all been derivatives of the endogenous ligand adenosine. For example chemical modification of the adenosine structure particularly at the N 6 , C2 and C5' positions have yielded selective high affinity agonists [START_REF] Muller | Adenosine receptor ligands-recent developments part I. Agonists[END_REF]. One such example is N 6 -cyclopentyladenosine (CPA), a high-affinity and selective agonist for the A 1 R. Furthermore the ribose moiety was thought to be essential for the agonistic efficacy of adenosine receptor ligands [START_REF] Soudijn | Medicinal chemistry of adenosine A1 receptor ligands[END_REF]. However, recent research has challenged this dogma. Rosentreter et al. patented a new class of adenosine receptor ligands, the 2-amino-4-(3,4 substituted phenyl)-6-(2-hydroxyethylsulfanyl)-pyridine-3,5dicarbonitriles [START_REF] Rosentreter | Substituted 2-thio-3,5-dicyano-4-phenyl-6-aminopyridines and their use as adenosine receptor-selective ligands[END_REF][START_REF] Rosentreter | Substituted 2-thio-3,5-dicyano-4-phenyl-6-aminopyridines and their use as adenosine receptor-selective ligands[END_REF]. These compounds were demonstrated to have both a significant A c c e p t e d M a n u s c r i p t affinity and efficacy towards different adenosine receptor subtypes [START_REF] Rosentreter | Substituted 2-thio-3,5-dicyano-4-phenyl-6-aminopyridines and their use as adenosine receptor-selective ligands[END_REF][START_REF] Rosentreter | Substituted 2-thio-3,5-dicyano-4-phenyl-6-aminopyridines and their use as adenosine receptor-selective ligands[END_REF][START_REF] Beukers | New, non-adenosine, high-potency agonists for the human adenosine A2B receptor with an improved selectivity profile compared to the reference agonist N-ethylcarboxamidoadenosine[END_REF][START_REF] Chang | A series of ligands displaying a remarkable agonisticantagonistic profile at the adenosine A1 receptor[END_REF]. One of these compounds, LUF 5831, was shown to be a partial agonist with a high affinity of 18 nM for the hA 1 R [START_REF] Heitman | Allosteric modulation, thermodynamics and binding to wildtype and mutant (T277A) adenosine A1 receptors of LUF5831, a novel nonadenosinelike agonist[END_REF]. Accordingly, this compound displayed binding characteristics typical of a partial agonist such as an insensitivity to GTP in radioligand binding studies and the ability to bind to a G protein-uncoupled mutant of the hA 1 receptor (hA 1 -T 277 A). Another high-affinity non-adenosine compound, LUF 5834, was characterized as an agonist at the hA 1 R in a cAMP assay [START_REF] Beukers | New, non-adenosine, high-potency agonists for the human adenosine A2B receptor with an improved selectivity profile compared to the reference agonist N-ethylcarboxamidoadenosine[END_REF] . Furthermore LUF5834 showed a moderate (10 fold) selectivity for the hA 1 R over the hA 2A R and hA 2B R and a higher selectivity (100 fold) over the hA 3 R [START_REF] Beukers | New, non-adenosine, high-potency agonists for the human adenosine A2B receptor with an improved selectivity profile compared to the reference agonist N-ethylcarboxamidoadenosine[END_REF]. Consequently it was of interest to further characterize this ligand at the hA 1 R. In this paper we confirm that LUF5834 indeed has a high (nanomolar) affinity for the hA 1 R. Using [ 35 S] GTPγS binding experiments to measure hA 1 R mediated G protein activation we demonstrate that that LUF5834 is a partial agonist as compared to the reference full agonist CPA. Given the high affinity of LUF5834 it was interesting to radioactively label this agonist with tritium. We demonstrate that [ 3 H]LUF5834 is a high affinity radioligand for the hA 1 R specifically labeling the hA 1 R expressed in CHO cells with negligible non-specific binding. Furthermore, due to its high affinity and partial agonist nature [ 3 H]LUF5834 was capable of labeling hA 1 R both coupled and uncoupled to G proteins. This allowed the characterization of high and low affinity binding for both agonists and antagonists at the hA 1 R. In summary then we describe a useful and novel high affinity radioligand for the hA 1 R. Radiolabeling was performed using a halogenation -dehalogenation approach yielding the desired labeled product. N 6 -cyclopentyladenosine (8ECPA) were synthesised as previously described [START_REF] Roelen | N6,C8-distributed adenosine derivatives as partial agonists for adenosine A1 receptors[END_REF].

Chinese hamster ovary (CHO) cells stably expressing the hA 1 R or the hA 2B R were obtained from Prof. Steve Hill (University of Nottingham, UK) or S.Rees (GSK, Stevenage, UK). HEK293 cells stably expressing the hA 2A R were kindly provided by Dr.

J Wang (Biogen/IDEC, Cambridge, MA) All other chemicals were of analytical grade and obtained from standard commercial sources.

Cell culture

Chinese hamster ovary (CHO) cells stably expressing the human adenosine A 1 receptor (CHO-hA 1 ) or the human A 2B receptor (CHO-hA 2B ) were cultured in a 1:1 mixture of Dulbecco's modified Eagle's medium (DMEM) and Ham's F12 medium containing 10% newborn calf serum, 50 µg/mL streptomycin, 50 IU/mL penicillin and 0.2 mg/mL neomycin (G418). Human embryonic kidney cells (HEK293) stably expressing the hA 2A R were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% newborn calf serum, 50 µg/mL streptomycin, 50 IU/mL penicillin and 0.2 mg/mL neomycin (G418). The cells were maintained in a humidified atmosphere at 37 ºC and 5% CO 2 .

Membrane preparation

Membranes of CHO or HEK293 cells stably expressing the hA 1 R, hA 2A or hA 2B were prepared as previously described [START_REF] Heitman | Allosteric modulation, thermodynamics and binding to wildtype and mutant (T277A) adenosine A1 receptors of LUF5831, a novel nonadenosinelike agonist[END_REF]. Membrane protein concentrations were measured using the BCA (bicinchoninic acid) method with BSA as a standard.

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

Radioligand-binding assays

For experiments using [ A c c e p t e d M a n u s c r i p t were performed as above using 30 µg of receptor per assay point and non-specific binding was determined using 10 µM DPCPX.

We performed similar studies on membranes expressing the hA 2A R or hA 2B R to assess the radioligand's potential to label these adenosine receptor subtypes as well.

hA

) (    (1)
where Y denotes the percent specific binding, top and bottom denote the maximal and minimal asymptotes respectively, x denotes the inhibitor potency (midpoint location)

parameter and n H denotes the Hill slope factor. Assuming simple competition IC 50 values were converted to K i values using the Cheng and Prusoff (1973) equation.

The CPA, DPCPX and N0840 displacement curves were best fitted to a two state/site binding model. For 8BCPA and 8ECPA one site binding isotherms were preferred. For experiments using [ 3 H]LUF5834 IC 50 -values were taken instead of calculated K i -values, since we did not attempt to attribute K d -values to each affinity state.

In the functional [ 35 S]GTPγS assay, agonist concentration response curves were fitted to the following four-parameter Hill equation using Prism 5;

H n EC x bottom top response ) / 10 ( 1 ) ( 50 log    (2)
where top represents the maximal asymptote of the concentration response curves, bottom represents the lowest asymptote of the concentration-response curves, logEC 50

Results

Characterization of LUF5834, a novel non-ribose agonist at the hA 1 R

In a preliminary screen LUF5834 was shown to have a high (nanomolar) affinity for the hA 1 R ( These high and low affinity sites were only moderately affected by the addition of GTP, exhibiting 2 and 5-fold decreases in affinity respectively (K iH of 0.7 nM and a K iL of 18 nM), or when measured on pertussis toxin-treated membranes (K iH of 0.7 nM and a K iL of 34 nM). This relative insensitivity to GTP and pertussis toxin seems remarkable in terms of the observation that LUF5834 is a full agonist in a cyclic AMP assay. To assure ourselves that this effect was associated with the nature of the ligand rather than the receptor or indeed our experimental method, parallel experiments were performed on the same membranes using the typical agonist CPA as the competing ligand (Figure 1B, Table 1). As might be expected for a full agonist the displacement was best fit using a two site binding isotherm with a high affinity site (K iH = 7 nM ) and a low affinity site with a K iL of 389 nM. However, in the case of CPA the high affinity site was lost when an equivalent experiment was performed in the presence of 1 mM GTP. Furthermore, the high affinity site was not observed when the assay was performed using CHO-hA 1 membranes derived from pertussis toxin-treated cells.

Characterization of [ 3 H]LUF5834, a high affinity radioligand at the human A 1 adenosine receptor

The above data describing the characterization of LUF5834 underline its behavior as a novel high affinity agonist ligand for the hA 1 R with characteristics different from the membranes a small window (200 dpm) of specific binding was detected. However, the relatively high amounts of radioligand and membranes required and the high non-specific binding (1000 dpm) observed in these experiments kept us from further characterization of this probe as a radioligand for hA 2B Rs (data not shown).

[ 3 H]LUF5834 demonstrates biphasic association and dissociation kinetics

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

As a starting point for the characterization of the novel radioligand [ 3 H]LUF5834, kinetic association and dissociation experiments were performed (Figure 2). As summarized in Table 2, both the association and dissociation of [ 3 H]LUF5834 were best fit by biphasic curves, yielding two values for both k obs and k off . It is interesting to note that the dissociation rate observed when using CPA as the displacing ligand was not significantly different from that observed when using LUF5834 (Figure 2B). This suggests that the binding sites of the typical agonist CPA and LUF5834 are at the very least overlapping, and the binding of CPA and LUF5834 is mutually exclusive. To extrapolate these results to determine from k obs values both k on values and a kinetic K d is not straightforward. It is not possible to definitively state that the fast phase of association corresponds to the same site which is described by a slow phase of dissociation. Therefore, we decided to determine the affinity for both of these putative sites using radioligand saturation binding experiments.

Radioligand saturation binding experiments at hA 1 -CHO membranes using [ 3 H]LUF5834

Saturation experiments were performed with [ 3 H]LUF5834 on CHO-hA 1 membranes.

The binding of [ 3 H]LUF5834 was best characterized using a one-site model but with a variable Hill slope (Figure 3A, Table 3). With this fit a K d of 2.03 ± 0.52 nM and a B max of 10.70 ± 0.76 pmol/mg was determined. The Hill slope was shown to be 0.78 ± 0.02

A c c e p t e d M a n u s c r i p t which is significantly lower than unity. This is graphically demonstrated in the Scatchard plot (Figure 3Aii) where a distinct deviation from linearity occurred. A Hill slope of less than unity can be explained by a negatively co-operative binding mechanism or suggests the presence of multiple binding sites. Interestingly the competition of LUF5834 versus DPCPX was also best fit by a curve with a Hill slope of 0.77. Indeed, in three out of the seven experimental repeats of saturation binding, the curve was well fit by a two site binding isotherm. The two sites were shown to have K d values of 0.16 nM and 1.69 nM and gave B max values of 1.7 pmol/mg and 7.9 pmol/mg respectively (Table 3). These values correspond well to the values of K iH and K iL determined for LUF5834 in competition binding experiments with [ 3 H]DPCPX (Table 1). This gave a combined B max of 9.58 ± 0.32 pmol/mg protein, which is not significantly different from that determined using the one-phase curve with variable Hill slope. Parallel experiments using 4). As

shown in figure 4A, the addition of 1 mM GTP had negligible effect on the observed IC 50 . However, in the presence of 1 mM GTP a reduction in total counts (Figure 5Aii)

was observed. In contrast, the typical hA 1 R agonist CPA displayed a biphasic displacement of [ 3 H]LUF5834 with an IC 50H of 5 nM and an IC 50L of 340 nM (Figure 4B, Table 4). These values are consistent with the values of K ih and K il determined by competition of [ 3 H]DPCPX. With the addition of 1 mM GTP, the affinity of CPA for the hA 1 receptor was not affected but the percentage of high affinity binding sites decreased from 56 % in the absence to 22 % in the presence of GTP (Table 4). However, when radioligand and an inverse agonist radioligand respectively, was also tested (Figure 4C).

As might be expected, increasing concentrations of GTP had no significant effect on the binding of [ 3 H]DPCPX, although a slight increase in ligand binding was observed (Figure 4C). Conversely the binding of [ 3 H]CCPA was almost completely abolished with the addition of 1 mM GTP. Interestingly, increasing concentrations of GTP caused a maximum of 40 % displacement of LUF5834 binding (Figure 4C). 4). Unlike the full agonist CPA, the displacement of A c c e p t e d M a n u s c r i p t

[ 3 H]LUF5834 by 8BCPA and 8ECPA was best fit using a monophasic isotherm. The addition of 1 mM GTP produced a negligible reduction in affinity and a modest reduction in affinity for 8BCPA and 8ECPA respectively (pIC 50GTP -pIC 50control = 0.06 and 0.16).

In comparison the addition of 1 mM GTP caused a reduction in the percentage of receptors with a high affinity for the full agonist CPA. If we determine a value of affinity change for CPA in the presence and absence of GTP by subtracting the pK i value determined with GTP from the pK h value determined without it we observed a much more substantial change of 1.96.

Displacement of [ 3 H]LUF5834 by the inverse agonists /antagonists DPCPX and N0840

Next, it was of interest to investigate the ability of the previously well characterized inverse agonist, DPCPX, to displace [ 3 H]LUF5834 at the hA 1 R. Interestingly this displacement was best characterized by a biphasic curve with an IC 50H of 8.5 nM and an IC 50L of 182 nM (Figure 6A, Table 4). With the addition of 1 mM GTP, the low affinity binding site was lost. Furthermore, when identical experiments were performed on CHO-hA 1 membranes from cells which had been pre-treated with pertussis toxin DPCPX displaced [ 3 H]LUF5834 with a monophasic curve yielding an IC 50 value equivalent to the IC 50L observed in the control experiment (Figure 6A, Table 4). We next characterized the ability of the putative neutral antagonist N0840 to displace [ 3 H]LUF5834. If N0840 acted as a neutral antagonist we might expect to observe a monophasic displacement A c c e p t e d M a n u s c r i p t curve that is not sensitive to the inclusion of 1 mM GTP. In reality, though, we observed a biphasic curve with an IC 50L of 8 µM and an IC 50H of 455 nM (Figure 6B, Table 4).

With the addition of 1 mM GTP the low affinity site was lost demonstrating that this site is dependent on the coupling of the receptor to G protein. It is interesting to note the relative change in affinity with and without the presence of GTP as determined by pIC 50LGTP -pIC 50H was smaller for N0840 than that of DPCPX (-0.92 and -1.33 respectively).

[ 35 S]GTPγS binding experiments reveal LUF 5834 to be a partial agonist

To obtain an accurate measurement of ligand efficacy we investigated the ability of LUF5834 to stimulate hA 1 R-mediated guanine nucleotide exchange using a [ 35 S]GTPγS binding assay. [ 35 S]GTPγS binding experiments were performed on membranes derived from the same CHO-hA 1 R as used for all radioligand binding studies (figure 7, Table 5).

As compared to the reference full agonist CPA which had a potency of 5 nM, LUF5834 was a partial agonist with a higher potency (3 nM) and an E max of 70% of the maximal stimulation by CPA. Compared to LUF5834 the previously characterized partial agonists 8BPCA and 8ECPA demonstrated low potency at the hA 1 R (2 µM and 1 µM, respectively). It is interesting to note that 8BCPA which showed a similar insensitivity to the addition of GTP in radioligand displacement assays to LUF5834 also demonstrated a similar efficacy in the [ 35 S]GTPγS assay. DPCPX was shown to be an efficacious inverse agonist with a potency of 11 nM. In comparison N0840 had a much more modest for the A 1 R. This is consistent with the action of DPCPX as an inverse agonist having a high affinity for and therefore stabilizing the uncoupled or inactive state of the receptor but having a lower affinity for the G protein-coupled state of the receptor in agreement with the ternary complex model [START_REF] Samama | A mutation-induced activated state of the beta 2-adrenergic receptor. Extending the ternary complex model[END_REF][START_REF] Christopoulos | G protein-coupled receptor allosterism and complexing[END_REF]. N0840 has been previously characterized as a neutral antagonist at the hA 1 adenosine receptor [START_REF] Shryock | Inverse agonists and neutral antagonists of recombinant human A1 adenosine receptors stably expressed in Chinese hamster ovary cells[END_REF][START_REF] De Ligt | Intrinsic activity at adenosine A1 receptors: partial and inverse agonism[END_REF]. However, in this study, It should be noted however that the change in affinity with the addition of GTP was significantly smaller for N0840 than that observed for DPCPX (pIC 50LGTP -pIC 50H = 0.92 and -1.33 respectively). This more modest affinity change for N0840 compared to DPCPX is consistent with the partial inverse agonist efficacy of N0840 compared to that of DPCPX.

Consequently, competition studies using [ 3 H]LUF5834 in the presence and absence of guanine nucleotides provide useful information as regards to the efficacy of antagonists and inverse agonists at the hA 1 R receptor. Many studies have clearly shown the relationship between the observed change in binding affinity upon addition of guanine nucleotides and agonist efficacy [START_REF] Christopoulos | G protein-coupled receptor allosterism and complexing[END_REF][START_REF] Christopoulos | Qualitative and quantitative assessment of relative agonist efficacy[END_REF]. Indeed, such a relationship has even been demonstrated at the A 1 R [START_REF] De Ligt | Intrinsic activity at adenosine A1 receptors: partial and inverse agonism[END_REF]. In this study we also saw a relationship between the efficacy of the ligand in the Several studies have used partial agonist or neutral antagonist radioligands to characterise both agonist and inverse agonists at the 5-HT 1A receptor [START_REF] Newman-Tancredi | Labelling of recombinant human and native rat serotonin 5-HT1A receptors by a novel, selective radioligand, [3H]-S 15535: definition of its binding profile using agonists, antagonists and inverse agonists[END_REF][START_REF] Newman-Tancredi | Efficacy of antipsychotic agents at human 5-HT(1A) receptors determined by [3H]WAY100,635 binding affinity ratios: relationship to efficacy for G-protein activation[END_REF][START_REF] Newman-Tancredi | Differential modulation by GTPgammaS of agonist and inverse agonist binding to h5-HT(1A) receptors revealed by [3H]-WAY100,635[END_REF]. It should be noted that such changes in affinity in the presence of GTP are not observed for inverse agonists at the hA 1 R when the inverse agonist radioligand [ 3 H]DPCPX is used, despite this ligand labelling populations of both G protein-coupled and -uncoupled receptor [START_REF] De Ligt | Intrinsic activity at adenosine A1 receptors: partial and inverse agonism[END_REF]. If the radioligand and 'cold' ligand have a similar efficacy they will be modulated by the addition of nucleotide to a similar extent, and therefore a change in affinity will not be observed. In agreement with this finding, in competition experiments of [ 3 H]LUF5834

versus the partial agonist BCPA, no change in affinity was observed in the absence or presence of guanine nucleotide. This can be reconciled with both LUF5834 and BCPA having an identical efficacy in the [ 35 S]GTPγS assay. [ 3 H]LUF5834 therefore allows the discrimination of high and low affinity sites for both inverse agonists and agonists as demonstrated by displacement studies using CPA and DPCPX.

In summary then we describe the first non-ribose partial agonist radioligand for the A 1 R, LUF5834. This radioligand labels the hA 1 receptor with high, sub-nanomolar affinity, higher than any other radioligand available for that receptor. The high affinity of There is increasing interest in the potential of adenosine A 1 receptor partial agonists, e.g.

in the treatment of paroxysmal supraventricular tachycardia (PSVT) [START_REF] Gao | Emerging adenosine receptor agonists[END_REF]. Similarly, blockade of A 1 Rs may be useful in the treatment of neurological disorders such as cognitive decline [START_REF] Ribeiro | Adenosine receptors in the nervous system: pathophysiological implications[END_REF]. [ 3 H] LUF 5834 represents a new class of radioligand for the adenosine A 1 receptor and can be a versatile tool to allow estimations of efficacy for both agonists and antagonists at this increasingly attractive therapeutic target.

Acknowledgements

This study was performed within the framework of Top Institute Pharma, project number D1-105. A c c e p t e d M a n u s c r i p t The curves for LUF5834 were best fit using biphasic isotherms independent of the presence or absence of GTP. Contrastingly data from CPA experiments were best fit by biphasic isotherms in the control condition but monophasic isotherms in the presence of 1 mM GTP (F-test using Prism 5, statistical significance was regarded as P < 0.05). The effect of increasing concentrations of GTP on the binding of A c c e p t e d M a n u s c r i p t

  cyclopentyladenosine (CPA) was obtained from Research Biochemicals Inc. (Natick, MA, U.S.A.). 1,3-dipropyl-8-cyclopentylxanthine (DPCPX), bovine serum albumin (BSA) and 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) were from Sigma (St. Louis MO, U.S.A.). Adenosine deaminase (ADA) was purchased from Roche Biochemicals (Mannheim, Germany) and bicinchoninic acid (BCA) protein assay reagent was obtained from Pierce Chemical Company (Rockford, IL, U.S.A.). [ 3 H] 1,3-dipropyl-8-cyclopentylxanthine ([ 3 H]DPCPX -specific activity 124 Ci/mmol) and [ 3 H]CCPA (2-chloro-N6-cyclopentyladenosine, 55 Ci/mmol) was purchased from NEN (Du Pont Nemours, 's Hertogenbosch, The Netherlands). [ 3 H] 2-amino-4-(4hydroxyphenyl)-6-(1H-imidazol-2ylmethylsulfanyl)-pyridine-3,5-dicarbonitrile ([ 3 H]LUF5834specific activity 25 Ci/mmol) was labeled by Sibtech, Inc. (Newington, CT, U.S.A.). Unlabelled LUF5834 was provided as the precursor (synthesized in-house at University of Leiden, Netherlands).

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

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

  represents the logarithm of the agonist EC 50 , x represents the concentration of the agonist and n H represents the Hill slope. Data shown are the mean ± SEM of at least 3 separate experiments performed in duplicate. Comparisons between models were performed by applying the F-test in Prism 5. Unless otherwise stated values of p < 0.05 were taken as statistically significant.

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

  by the addition of 1 mM GTP. It should be noted that in three out of six experiments the data could be fit to a biphasic curve with a subnanomolar K iH (0.3 nM) and a K iL of 6 nM.

A c c e

  p t e d M a n u s c r i p t reference 'ribose' agonist CPA. Therefore LUF5834 could be a potentially useful and novel type of radioligand. Consequently, LUF5834 was radiolabelled giving [ 3 H]LUF5834 with a specific activity of 25 Ci/mmol. The conditions employed for the binding of [ 3 H]DPCPX to CHO-hA 1 membranes were used as a basis for an assay for [ 3 H]LUF5834. Membrane protein (10 μg) was incubated in a total volume of 400 μL Tris-HCL buffer, 50 mM, pH 7.4 as described in Methods. On control, untransfected, CHO cell membranes, [ 3 H]LUF5834 did not display any specific binding (data not shown). The use of GF/B versus GF/C filters with or without pre-soaking the filters with 0.25 % poly(ethyleneimine) solution (PEI) for 1 hour had no effect on the 'window' of specific binding observed. However, the addition of 0.1 % CHAPS increased the window of specific binding (data not shown). All subsequent assays were therefore performed in the above conditions with the addition of 0.1 % CHAPS and reactions were terminated by fast flow filtration through GF/C filters. In identical assay conditions the ability of [ 3 H ]LUF5834 to bind to the human A 2A receptor expressed in HEK293 cell membranes or to the human A 2B receptor expressed in CHO cell membranes was assessed using a final concentration of 20 nM [ 3 H]LUF5834. No specific binding was detected for the HEK-hA 2A membranes. For experiments with the CHO-hA 2B

[ 3 HA c c e p t e d M a n u s c r i p t 3 . 5

 335 ]DPCPX saturation binding on hA 1 -CHO membranes experiments were also performed. In this case the data was best fit using a single site model with a K d of 2.87 ± 0.39 nM and a B max of 12.8 ± 1.7 pmol/mg protein (Figure3B i). The total density of ligand binding sites observed for both [ 3 H]DPCPX and [ 3 H]LUF5834 was not significantly different. The monophasic nature of the curve is well demonstrated by the tight linear fit of the Scatchard plot in contrast to that of LUF5834 (Figure3Bii). This demonstrates that the low Hill slopes and biphasic nature of [ 3 H]LUF5834 saturation isotherms are most likely associated with the nature of the ligand, as this was the only variable in the experiments. The biphasic association and dissociation kinetics also suggests the presence of two distinct binding sites labelled by [3 H]LUF5834 (Table 3). Displacement of [ 3 H]LUF5834 by the agonists LUF 5834 and CPAFor the experiments describing the displacement of [ 3 H]LUF5834 by various hA 1 R ligands a concentration of 2.6 nM was used. This corresponds to the K d value determined by saturation analysis using a variable Hill slope plot. Using this concentration yields a window of specific binding of around 2000 dpm, a practical window for this type of experiment. However, we have also given considerable evidence that [ 3 H]LUF5834 labels two distinct binding sites with sub-nanomolar and nanomolar affinities. In addition, in displacement experiments of [ 3 H]DPCPX versus LUF5834, 1 mM GTP had negligible effect on either of these two sites suggesting that neither corresponds to a G protein-coupled state. Therefore, values of affinity for each of the displacing ligands are left as values of pIC 50 , i.e. not converted to apparent pK i values. Displacement of [ 3 H]LUF5834 by unlabelled LUF5834 was best characterized by a monophasic curve with a high affinity binding site with an IC 50 value of 5 nM (Figure 4A, Table

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

[ 3 H

 3 ]LUF5834 was used at a concentration of 2.6 nM the addition of 1 mM GTP gave a decrease of approximately 30% of [ 3 H]LUF5834 bound (Figure4Aii, 4Bii) . This would suggest then a fraction of bound [ 3 H]LUF5834 is sensitive to GTP. Therefore it was of interest to investigate the ability of increasing concentrations of GTP to displace 2.6 nM [ 3 H]LUF5834 binding at the hA 1 R. As a comparison the effect of increasing concentrations of GTP on the binding of [ 3 H]CCPA and [ 3 H]DPCPX, a full agonist

3. 6

 6 Displacement of specific [ 3 H]LUF5834 binding by the partial agonists C8ethylamino-N 6 -cyclopentyladenosine (8ECPA) or C8-butylamino-N 6cyclopentyladenosine (8BCPA) It was interesting to observe that LUF5834, both in competition with [ 3 H]DPCPX or indeed with [ 3 H]LUF5834, displayed no shift in affinity or loss of high affinity binding with the addition of 1 mM GTP. We assessed the ability of two reference partial agonists C8-butylamino-N 6 -cyclopentyladenosine (8BCPA) or C8-ethylamino-N 6cyclopentyladenosine (8ECPA) to displace [ 3 H]LUF5834 in the presence or absence of 1 mM GTP (Figure 5, Table

A c c e p t e d M a n u s c r i p t potency ( 20 4 Discussion

 204 µM) and was an inverse agonist, although with a lower efficacy than DPCPX. The 2-amino-4-(substituted phenyl)-6-(substituted sulfanyl)-pyridin-3,5-dicarbonitriles show no structural similarity to adenosine. However, it has been observed that several compounds of this class display a significant affinity and efficacy towards the different adenosine receptor subtypes. LUF5834 (2-amino-4-(4-hydroxyphenyl)-6-(1H-imidazol-2-ylmethylsulfanyl)-pyridine-3,5-dicarbonitrile) has previously been characterized as a novel high affinity non-ribose agonist for the hA 1 R (Beukers et al, 2004

A c c e

  p t e d M a n u s c r i p t incomplete. Guanine nucleotide exchange is a very early step in the signal transduction cascade, and hence is less subject to amplification or regulation by other cellular processes[START_REF] Milligan | Principles: extending the utility of [35S]GTP gamma S binding assays[END_REF]. Making use of a [ 35 S]GTPγS binding assay we demonstrated that, at the level of nucleotide exchange at the G protein, LUF5834 behaves as a partial agonist with greater potency for the hA 1 R than that of the typical agonist CPA.The competition binding profile of [ 3 H]LUF5834 at the hA 1 R was examined using a range of A 1 R ligands. The competition between [ 3 H]LUF5834 and unlabeled LUF5834 showed no shift in affinity in response to the addition of 1 mM GTP. The full agonist CPA displayed a biphasic curve with an IC 50H of 4.7 nM and an IC 50L of 338 nM. With the addition of 1 mM GTP or pre-treatment of CHO-hA 1 cells with pertussis toxin the high affinity (G protein-coupled) binding population decreased. This suggests that the high affinity site of CPA is dependent on G protein coupling and that in the case of the hA 1 R and CPA this coupling is to member(s) of the G i/o family of G proteins. This sensitivity to GTP of the reference agonist CPA was quite distinct from the relative nucleotide insensitivity of LUF5834. However, biphasic isotherms were not limited to full agonist ligands. DPCPX, shown to be a high-affinity inverse agonist in the [ 35 S] GTPγS experiments, also displayed a biphasic curve with an IC 50H of 8.5 nM and an IC 50L of 182 nM. Interestingly, in the case of DPCPX the low affinity site was shown to represent the G protein-coupled receptor population and that this G protein coupling is exclusively mediated by G i/o G proteins since this low affinity site was lost upon the addition of 1 mM GTP or by pretreatment of cells with pertussis toxin. Such behaviour by an inverse agonist has not been demonstrated before with other radioligands available A c c e p t e d M a n u s c r i p t

[

  35 S]GTPγS experiments revealed N0840 to be a partial inverse agonist with modest potency. Consistent with this finding, competition binding experiments of [ 3 H]LUF5834 versus N0840 reveal a biphasic curve with an IC 50H of 455 nM and an IC 50L of 8 µM. As for DPCPX, the low affinity binding site of N0840 disappeared with the addition of GTP.

  [ 35 S]GTPγS experiments and the affinity change with the addition of GTP in competition experiments. The changes of affinity for agonists and A c c e p t e d M a n u s c r i p t inverse agonists were inversely related. Furthermore, affinity changes observed for partial agonists such as BCPA and ECPA and partial inverse agonists such as N0840 were more modest than those observed for the full agonist CPA and the efficacious inverse agonist DPCPX.

[ 3 H

 3 ]LUF5834, plus its partial agonist nature allows it to bind to both G protein-coupled A c c e p t e d M a n u s c r i p t and -uncoupled receptors with similar high affinity. Competition experiments using [ 3 H]LUF5834 revealed a population of receptors with a low affinity for inverse agonists such as DPCPX. To our knowledge this study is the first to reveal the affinity of A 1 R inverse agonists such as DPCPX for the G protein-coupled population of receptors.

Figure 1 Displacement of [ 3 H

 13 Figure 1 Displacement of [ 3 H]DPCPX bound to the hA 1 R expressed on CHO cell membranes

Figure 2 Association

 2 Figure 2 Association (A) to and dissociation (B) of [ 3 H]LUF5834 from the hA 1 R stably expressed on CHO cell membranes. Dissociation of [ 3 H]LUF5834 was achieved by

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

Figure 3 Saturation radioligand binding at the hA 1 R

 31 Figure 3

Figure 4 Displacement of [ 3 H

 43 Figure 4 Displacement of [ 3 H]LUF5834 bound to the hA 1 R expressed on CHO cell

  [ 3 H]LUF5834, the full agonist [ 3 H]CCPA or the inverse agonist [ 3 H]DPCPX was also tested (C). [ 3 H]CCPA, A c c e p t e d M a n u s c r i p t

[ 3 H

 3 ]LUF5834 and [ 3 H]DPCPX were used at concentrations of 1.6 nM, 2.6 nM and 2 nM respectively, values approximately equal to the respective K d values at this receptor.

Figure 5 Displacement of [ 3 H

 53 Figure 5 Displacement of [ 3 H]LUF5834 bound to the hA 1 R expressed on CHO cell

Figure 6 Displacement of [ 3 H

 63 Figure 6 Displacement of [ 3 H]LUF5834 bound to the hA 1 R expressed on CHO cell

Figure 7 [

 7 Figure 7 [ 35 S]GTPγS binding experiments reveal LUF5834 to be a partial agonist at the

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

Figure 1 A

 1 Figure 1

Figure 2 A c c e p t e d M a n u s c r i p

 2 Figure 2

Figure 3 A

 3 Figure 3

Figure 4 A

 4 Figure 4

Figure 5 A

 5 Figure 5

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

 6 Figure 6

Figure 7 A c c e p t e d M a n u s c r i p

 7 Figure 7

  of two populations of receptor with distinct affinities for the receptor. Indeed, three out of the seven experiments could be well fit with a two binding site equation with values of K d similar to those values of K i determined using [ 3 H]DPCPX competition experiments. Kinetic experiments demonstrated that both association and dissociation of [ 3 H]LUF5834 were biphasic. The rate of dissociation observed remained unchanged when either the reference agonist CPA or the cold ligand LUF5834 was used as the displacing ligand.This indicates then that the binding site of LUF5834 overlaps greatly with that of the typical A 1 receptor agonist CPA, and provides evidence that LUF5834 is an orthosteric rather than an allosteric ligand. It is interesting to note that the number of receptors labeled by [ 3 H]LUF5834 was similar to that labeled by the inverse agonist [ 3 H]DPCPX.

	M a n u s c r i p t
	assay conditions described above, we have demonstrated that [ 3 H]LUF5834 selectively labels A 1 Rs over A 2A Rs and A 2B Rs, and that [ 3 H]LUF5834 does not represent a displaced by GTP whereas increasing concentrations of GTP could only partially displace bound [ 3 H]LUF5834. This suggests that [ 3 H]LUF5834 can label both G protein-coupled A c c e p t e d and -uncoupled receptors, an ability difficult to reconcile with LUF5834 being a full
	radioligand for the latter receptors. In all saturation experiments the data was well fit agonist as was shown in an initial study using a whole cell cAMP assay [11]. Similar
	using a single site binding model with a variable Hill slope and a K d of approx. 2 nM and experiments have been carried out for [ 3 H]lisuride, [ 3 H]S15535 and [ 3 H]DOI,
	a B max of approx, 10 pmol/mg. The Hill slope (0.78 ± 0.02) was significantly less than radiolabelled partial agonists at the 5HT 1A , and 5HT 2A receptors, respectively [18-20]. In
	unity which is suggestive of either negative co-operative binding or indeed the presence

). We now demonstrate that, in more elaborate studies, the compound displayed two binding sites with a high sub-nanomolar and nanomolar affinity for this receptor, respectively. The high affinity of LUF5834 made it a suitable candidate for labeling with tritium to provide a new radioligand for the A 1 R. Current adenosine A 1 receptor radioligands are confined to adenosine derivative full agonists such as [

3

H]CCPA or xanthine derivative inverse agonists such as [ 3 H]DPCPX [15, 16]. Initial experiments demonstrated that [ 3 H]LUF5834 could label A 1 Rs with low non-specific binding. Furthermore, using the This is in stark contrast to a previous study using the radiolabelled agonist [ 3 H]CCPA. In this study Kourounakis et al. found that [ 3 H]CCPA labeled only 20% of the binding sites labeled by [ 3 H]DPCPX [17]. This suggests then, that while [ 3 H]DPCPX and [ 3 H]LUF5834 can label both G protein-coupled and uncoupled receptors with high affinity, [ 3 H]CCPA as a full agonist will only label G protein-coupled receptors. In agreement with the above conclusion [ 3 H]CCPA binding in our hands was completely all cases displacement of these partial agonist radioligands by GTP analogues was

Table 1 Estimates of affinity (K i values) derived from the competition of [ 3 H]DPCPX with the agonists CPA and LUF5834 at the hA 1 R expressed in CHO cell membranes.

 1 Values represent the means ± S.E.M. obtained from three to seven experiments conducted in duplicate. Values of pK h and pK l are expressed as -log values with the corresponding values of K i shown in parentheses.

		LUF 5834	LUF 5834	LUF 5834	CPA	CPA	CPA
			+ GTP	+ PTX		+ GTP	+ PTX
	pK h				8.15 ± 0.39
	(K h , nM)				(7 nM)
	pK i or pK l	8.59 ± 0.11	8.33 ± 0.10	8.47 ±0.10	6.41 ± 0.07	6.44 ± 0.07	6.41 ± 0.08
	(K i or K l ,	(3 nM)	(5 nM)	(4 nM)	(389 nM)	(363 nM)	(389 nM)
	nM)
	Hill Slope	0.74 ± 0.06 0.77 ± 0.06 0.79 ± 0.15
	% K h				27 ± 6	0	0

Table 2 Association

 2

	and dissociation kinetic parameters (± S.E.M.) of [ 3 H]LUF5834 binding
	to hA 1 Rs stably expressed in CHO cell membranes. Dissociation was induced with
	the addition of a large excess of either CPA (10 μM) or LUF5834 (1 μM). Both
	association and dissociation curves were best fit using biphasic models. No significant
	difference was observed between the values of k off1 or k off2 when either LUF5834 or CPA
	was used as the displacing ligand (un-paired t test using Prism 5, where statistic
	significance was taken as p< 0.05)
	Association
	k obs1 ,

min -1 .M -1 Half-life 1 , min k obs2 , min -1 .M -1 Half-life 2 , min

	[ 3 H]LUF5834	0.078 ± 0.03	14.37 ± 0.08	1.63 ± 0.24	0.45 ± 0.08

Dissociation Isotopic ligand k off1 , min -1 Half-life 1 , min k off2 , min -1 Half-life 2 , min

	M a n u s c r i p t
	LUF 5834 CPA A c c e p t e d 1.07 ± 0.37 0.93 ± 0.43 1.96 ± 1.24 0.98 ± 0.61	0.05 ± 0.01 0.06 ± 0.02	15.83 ± 4.12 12.77 ± 4.38

Table 3 Saturation binding of [ 3 H]LUF5834 and [ 3 H]DPCPX to hA 1 -CHO membranes

 3 Saturation binding experiments were carried out using at least 12 concentrations of radioligand at hA 1 -CHO membranes in duplicate at least three times. Data for the saturation binding of [ 3 H]LUF5834 was best fit using a monophasic binding curve with a variable Hill slope. Three experiments from this series were also well fitted by a biphasic saturation curve. The saturation binding curve for [ 3 H]DPCPX was best fitted by a monophasic curve (F-test using Prism 5, p < 0.05 was taken as statistically significant).

	Radioligand	K D1 , nM	B max1 , pmol/mg	Hill	K D2 , nM	B max2 ,
				slope		pmol/mg
	[ 3 H]LUF5834	2.03 ± 0.52	10.70 ± 0.66	0.78 ±	-	-
	n = 7			0.02
	[ 3 H]LUF5834	0.16 ± 0.03	1.70 ± 0.02	-	1.69 ± 0.02 9.58 ± 0.32
	n = 3
	[ 3 H]DPCPX	2.87 ± 1.03	12.8 ± 1.7	1	-	-

Table 4 Estimates of affinity (IC 50 values) derived from the competition of [ 3 H]LUF5834 versus various A 1 R agonists and antagonists, in the absence and presence of 1 mM GTP, at the hA 1 R expressed in CHO cell membranes.

 4 Values represent the means ± S.E.M. obtained from three to seven experiments conducted in duplicate. Values of pK h and pK l are expressed as -log values with the corresponding values of K i shown in parentheses. The affinity change was calculated by subtracting the pIC 50low value determined in the presence of GTP by the pIC 50high value determined in its absence.When no pIC 50high could be determined then affinity change was calculated by subtracting the pIC 50low value determined in the presence of GTP by the pIC 50low value determined in its absence.

				Fraction	Affinity change
	Compound	pIC 50 , high	pIC50 or pIC 50 low
				High affinity, %
	LUF5834		8.26 ± 0.03 (5.4 nM)
	LUF5834 + GTP		8.18 ± 0.04 (12.3 nM)		-0.08
	CPA	8.32 ± 0.06 ( 4.7 nM)	6.47 ± 0.13 ( 338 nM)	56 ± 2
	CPA + GTP	8.14 ± 0.11 ( 7 nM)	6.36 ± 0.04 (435 nM)	22 ± 3	1.96
	BCPA		4.94 ± 0.07 (12 µM)
	BCPA + GTP		4.89 ± 0.11 (13µM)		0.06
	ECPA		5.17 ± 0.12 (6.7 µM)
	ECPA + GTP		5.01 ± 0.12 (9.8 µM)		0.16
	DPCPX	8.07 ± 0.12 (8.5 nM)	6.74 ± 0.14 (182 nM)	53 ± 8	-1.33
	DPCPX + GTP	8.34 ± 0.13 (4.5 nM)		100

Table 5 : The efficacy and potency of agonists, partial agonists and inverse agonists at the hA 1 R in a [ 35 S] GTPγS binding assay.

 5 The ability of increasing concentrations of compound to stimulate or inhibit [ 35 S] GTPγS binding at CHO membranes expressing hA 1 R was determined. E max was determined as a percentage of the maximal stimulation exerted by the full agonist CPA ((maximal stimulationbasal stimulation)/(maximal CPA stimulationbasal stimulation)). Values of pEC 50 are expressed as -log values with the corresponding values of EC 50 shown in parentheses.

		pEC 50	E max
		(EC 50 )
	CPA	8.31 ± 0.02	100
		(4.84 nM)
	LUF 5834	8.48 ± 0.02	70 ± 3
		(3.29 nM)
	8BCPA	5.75 ± 0.04	65 ± 1
		(1.78 µM)
	8ECPA	6.12 ± 0.05	94 ± 4
		(0.79 µM)
	DPCPX	7.98 ± 0.14	-18 ± 3
		(11 nM)
	N0840	4.88 ± 0.3
		(19.7 µM)


Zuzana ; Zelinkova
email: z.zelinkova@erasmusmc.nl

Colin De Haar

Lissy De Ridder

Marie Pierik

Ernst J Kuipers

Maikel Peppelenbosch

C Janneke Van Der Woude

Colin De Ridder

Lissy Pierik

Marie ; Mumc

Gastroenterology Kuipers

De Haar

Pierik Mj

Kuipers Ej

Peppelenbosch Mp

Woude Van Der

Cj

High Intra-Uterine Exposure to Infliximab Following Maternal Anti-Tnf Treatment During Pregnancy

Keywords:

anti-TNF agents. [START_REF] Zelinkova | Reproductive wish represents an important factor influencing therapeutic strategy in inflammatory bowel diseases[END_REF] Therefore, the safety of anti-TNF use during conception and pregnancy has become a major concern for patients and their clinicians.

Of the various anti-TNF agents, infliximab (IFX), a chimeric monoclonal antibody against TNF, is the best characterised with respect to safety during human reproduction. Short-term data from two safety registries found no important teratogenicity issues [START_REF] Katz | Outcome of pregnancy in women receiving infliximab for the treatment of Crohn's disease and rheumatoid arthritis[END_REF][START_REF] Lichtenstein | Safety of infliximab in Crohn`s disease: data from 5000-patients TREAT registry[END_REF] . However, intrauterine exposure to IFX resulting from placental antibody transfer was previously reported in children born to mothers treated with IFX during pregnancy [START_REF] Vasiliauskas | Case report: evidence for transplacental transfer of maternally administered infliximab to the newborn[END_REF][START_REF] Mahadevan | Infliximab levels in infants born to women with inflammatory bowel disease[END_REF] . Like all IgG antibodies, IFX crosses the placenta beginning in the second trimester, probably reaching its maximal transport capacity during the third trimester [START_REF] Kane | Placental transport of immunoglobulins: a clinical review for gastroenterologists who prescribe therapeutic monoclonal antibodies to women during conception and pregnancy[END_REF] . To limit this placental transfer stopping IFX treatment prior to gestational week 30 was proposed [START_REF] Gisbert | Safety of immunomodulators and biologics for the treatment of inflammatory bowel disease during pregnancy and breast-feeding[END_REF][START_REF] Van Assche | The second European evidence-based Consensus on the diagnosis and management of Crohn`s disease: Special situations[END_REF] . Nevertheless, in view of the strong immunomodulatory properties of IFX and the lack of long-term outcome data, the safety of terminating IFX therapy at gestational week 30 is difficult to take for granted without data

showing that this is sufficient to prevent transfer of significant amounts of anti-TNF to the unborn child.

Concern about the unpredictable outcomes of early exposure of the developing immune system to powerful TNF-alpha neutralization and the immunomodulatory potential of anti-TNF molecules is, at least on a theoretical basis, substantial. This concern, together with an absence of data in which the level of IFX transfer to the unborn child is actually assessed The respective gestational weeks at delivery are shown in Table 1. The decision of discontinuation of IFX was at the discretion of treating gastroenterologist who discussed all pros and cons of the treatment continuation with the patients at an early stage of their pregnancy. The timing of discontinuation was motivated by the current approach advising not to extend the use of IFX beyond 30 weeks of pregnancy. The exact date of the last infusion was based on the 8-weeks schedule with which patients entered the pregnancy so that the last infusion would be given prior or at the week 30 at the latest. At delivery, cord blood was collected from the newborns, and peripheral blood serum was obtained within two days after delivery from the patients.

All children received routine childhood vaccinations according to recommendations in the Netherlands, i.e. a combined vaccine for diphteria, tetanus, pertussis, and polio;

Haemophilus influenzae b; and pneumococcus at the ages of 2, 3, and 4 months. The response to bacterial vaccines was assessed at month 6 of life in two of the three children born with significant IFX levels.

The IFX serum levels were measured by ELISA, the assay has been adapted and validated in our laboratory based on the previously reported and validated assay [START_REF] Ternant | An enzyme-linked immnosorbent assay for therapeutic drug monitoring of infliximab[END_REF] . High binding during one hour at room temperature and subsequently washed four times with 0.05% PBST.

The reaction was revealed using 100µl of 3,3',5,5'tetramethylbenzidine (eBioscience) solution at room temperature and stopped with 100µl of 1M H 2 SO 4 . Reading was performed at two wave lengths; 450 and620 nm using Biorad 680 plate reader. The samples were run in different concentrations as mentioned above and the final infliximab serum concentrations were interpolated from the standard curve. The results were rejected if difference between the calculated concentration of the serial dilution was higher the 20%, and the accepted measurements were considered as triplicates to calculate the mean.

The assay was tested and validated using serum samples from patients receiving IFX and those who were naïve to this drug. For the average inter-assay coefficient of variation calculation, 10 samples were run on different days and the standard deviation per patients sample was expressed as a percentage of the mean. The average inter-assay coefficient was 4%. The inter-assay coefficient was calculated using the serial dilutions of samples that were used instead of triplicates. For this calculation 3 dilutions of 10 samples were measured with the intra-assay variability coefficient of 4.4%.

Since the treatment protocol is considered a standard care, no approval of ethical committee has been solicited.

Results

Pregnancy outcomes

Patients gave birth at gestational weeks 37, 36, 41, and 38, respectively (Table 1). Three

IFX levels

Only one patient (patient 1) had undetectable levels of IFX in her peripheral blood at delivery and undetectable levels in her infant's cord blood. This patient received her last IFX infusion at gestational week 21. In three other patients, therapeutic levels of IFX were found in the cord blood at levels of 13, 5.5 and 13.7 µg/mL, respectively. The levels in cord blood were higher than the levels measured in peripheral blood of mothers (p=0.032 with one-tailed paired t-test), which were 4.9, 2.4, and 5 µg/mL respectively.

Patient follow-up

Discussion

In this case series, we show that the currently advocated approach of stopping IFX at gestational week 30 does not prevent the intra-uterine exposure of the fetus to significant levels of IFX. This finding is important in clinical practice because of the potential short-term as well as long-term complications of early exposure to anti-TNF.

Data from the safety registries [START_REF] Katz | Outcome of pregnancy in women receiving infliximab for the treatment of Crohn's disease and rheumatoid arthritis[END_REF][START_REF] Lichtenstein | Safety of infliximab in Crohn`s disease: data from 5000-patients TREAT registry[END_REF] , as well as a recently published first prospective cohort on the outcomes of pregnancies exposed to infliximab [START_REF] Schnitzler | Outcome of pregnancy in women with inflammatory bowel disease treated with antitumor necrosis factor therapy[END_REF] did not reveal significant teratogenicity issues. In one out of the four patients presented in our study, polydactyly of the hand was diagnosed in a child born to a mother using IFX at conception and during the first two trimesters of the pregnancy. However, this patient also conceived only two months after stopping methotrexate. Digital malformations have been reported with the use of methotrexate but almost exclusively in combination with other birth defects [START_REF] Finley | Birth defect surveillance: Jefferson County, Alabama, and Uppsala County, Sweden[END_REF] which were not present in this child. To our knowledge, no isolated polydactyly has been reported in the case of the pre-natal exposure to infliximab. In addition, polydactyly is a very common birth defect in general population [START_REF] Lloyd | The effects of methotrexate on pregnancy, fertility and lactation[END_REF] . Thus, no clear association with the medication can been concluded, however, the role of the pre-conceptional use of methotrexate remains a subject of discussion in this patient.

Intra-uterine exposure to IFX of children born to mothers receiving IFX treatment was first suggested by Vasiliauskas et al. in 2006 [START_REF] Vasiliauskas | Case report: evidence for transplacental transfer of maternally administered infliximab to the newborn[END_REF] . In this case report, the mother used IFX during the entire pregnancy, and clinically significant levels of IFX were detected in the child's serum sample. The hypothesis of placental transfer of IFX was further supported by a study by Mahadevan et al. [START_REF] Mahadevan | Infliximab levels in infants born to women with inflammatory bowel disease[END_REF] where authors reported clinically significant levels in the cord blood of the infant and serum samples taken directly postpartum from five children born to mothers treated with IFX. In this case series, the mean time between birth and last IFX infusion was 30 days. Kane et al. [START_REF] Kane | Absence of infliximab in infants and breast milk from nursing mothers receiving therapy for Crohn's disease before and after delivery[END_REF] measured the levels of IFX in two children born to mothers treated with IFX until gestational week 32 and 24, respectively. In both children, IFX was undetectable; The kinetics of IgG antibodies in general are governed by the neonatal Fc receptor (FcRn) [START_REF] Roopenian | The MHC class I-like IgG receptor controls perinatal IgG transport, IgG homeostasis, and fate of IgG-Fc-coupled drugs[END_REF] .

Antibodies are passively taken up by endothelial cells among other cell types and subsequently sequestered through binding to intracellularly expressed FcRn then shuttled back to the extracellular milieu. This recycling helps to increase the life time of the antibodies by protecting them from catabolism [START_REF] Baker | Immune and non-immune functions of the (not so) neonatal Fc receptor, FcRn[END_REF] . The same mechanism is used by maternal IgG antibodies that cross the placenta beginning in the second trimester [START_REF] Simister | Placental transport of immunoglobulin G[END_REF] . IFX, being an IgG1 class antibody with a functional Fc, very probably crosses the placenta in the same way [START_REF] Pentsuk | An interspecies comparison of placental antibody transfer: new insights into developmental toxicity testing of monoclonal antibodies[END_REF] and is subject to the efficient FcRn-mediated protection from break down in the newborn.

The biological half time of IFX in the newborn is also expected to be longer than in adults because of the high expression of the FcRn during the first months of life. Indeed, the series of 5 children born with detectable IFX levels showed the persistence of IFX in the peripheral blood of the children for as long as 6 months [START_REF] Mahadevan | Infliximab levels in infants born to women with inflammatory bowel disease[END_REF] . Thus, the placental transfer of IFX not only raises the issue of intra-uterine exposure but, most importantly, the concern about consequences for immune system functioning and development during the first weeks of life.

Children born with detectable levels of IFX do not seem to have an increased risk of infections in their first year of life and have normal responses to vaccinations with bacterial non-live vaccines. This was documented in the eight-patient series reported by Mahadevan et al. [START_REF] Mahadevan | The effect of maternal peripartum infliximab use on neonatal immune response[END_REF] and is also supported by the findings in the current study. However, a fatal case of disseminated mycobacterial infection after BCG administered at month 3 to a child born to a mother treated with IFX during the entire pregnancy was recently reported [START_REF] Cheent | Case Report: Fatal case of disseminated BCG infection in an infant born to a mother taking infliximab for Crohn`s Disease[END_REF] . In this case, the likely mechanism of insufficient immune control of the live attenuated vaccine would be the neutralization of TNF-alpha by IFX, although no measurements of IFX levels in the child were performed. The specific infectious complications resulting from the use of anti-TNF agents are well known and, therefore, vaccinations with live antigens are also prohibited in patients on anti-TNF treatment. Because significant levels of IFX may persist for several months in Early exposure of the immune system to powerful TNF-alpha blockade also raises concerns about the long-term consequences for the maturation of the immune system. In the context of the increasing number of reports of hepatosplenic T-cell lymphoma in young adolescents treated with combined immunosuppressive therapy containing, in most cases, anti-TNF [START_REF] Rosh | Hepatosplenic T-cell lymphoma in adolescents and young adults with Crohn's disease: a cautionary tale?[END_REF] , and keeping in mind the lack of long-term outcome data, we believe the actual approach should be to try to limit the intra-uterine and postnatal exposure of children to anti-TNF in general.

The decision to stop treatment with a drug that keeps the mother's underlying disease in remission during pregnancy is a difficult decision for the treating physician. A disease flare may represent a substantial risk not only for the patient, but also for the unborn child. On the other hand, in the absence of convincing long-term safety data of the perinatal exposure to IFX, the continuation of the treatment remains a subject of discussion. Simultaneously, the decision to stop IFX is difficult in the current absence of predictors for the disease course during pregnancy in this specific setting. In addition, in case of treatment with anti-TNF, allergic reactions during retreatment which had been postponed for more than 12 weeks represent another concern. In this limited series of patients, we did not observe any of these problems; all patients remained in remission after stopping IFX and no allergic reactions occurred during retreatment after delivery. Our observation is also supported by the results of the first prospective cohort of 35 IBD patients treated with IFX during first two trimesters of the pregnancy that did not show negative impact of this approach nor on the disease course neither on the pregnancy outcomes [START_REF] Schnitzler | Outcome of pregnancy in women with inflammatory bowel disease treated with antitumor necrosis factor therapy[END_REF] . Interestingly, in three patients with detectable levels of IFX at delivery, the interval between the last IFX infusion was 10, 15, and 9 weeks, respectively. The elimination half-time of IFX for women being 18 days [START_REF] Ternant | Infliximab pharmacokinetics in inflammatory bowel disease patients[END_REF] , one would expect no detectable levels in these patients at delivery.

  to clinicians to treat several autoimmune conditions including inflammatory bowel disease (IBD). Potential candidate patients for anti-TNF treatment are often young and in their reproductive years, which is also reflected in the observation that 20% of IBD patients with active reproductive wishes require treatment with

  well ELISA plates (Nunc-Immuno TM Plates) were coated overnight at 4°C with 50µl of 2µg/mL recombinant human TNF-alpha (Invitrogen) diluted in phosphate buffered saline (PBS). The plates were washed twice with PBS 0.05% Tween 20 (PBST). The remaining protein-binding sites were blocked during two hours at room temperature with 250µl of 3% non-fat dry milk (Elk Campina, Eindhoven, The Netherlands) diluted in PBST.The plates were washed five times with PBST and 100µl of serum, standard and control samples were added. For the negative control, a pooled human serum, obtained from random 40 different healthy individuals was used. To generate standard curve, serial concentrations of infliximab (Remicade®, Merck Sharp&Dohme) were used. The serum samples were serially diluted at concentrations of 1:1000 to 1: 32000. All samples were diluted in PBST containing 1% bovine serum albumine (BSA). The samples were incubated

  children were healthy without congenital malformations. One baby girl, born to a 19 year old mother (patient Nr 3), experienced respiratory depression after birth owing to anesthetics used during delivery, and which resolved spontaneously. This child was also diagnosed with polydactyly of her left hand. Apart from infliximab, the mother had used methotrexate till two months prior to conception and had omitted to use the recommended folic acid supplementation during the first weeks of the pregnancy. Other risk factors for congenital malformations, such as alcohol consumption, smoking or consanguinity were not revealed.During the follow-up of 4 to 11 months, all children developed normally, there were no signs of infections, and no abnormal reactions to vaccinations were observed. The antibody response to vaccinations against pneumococcus and Haemophilus influenzae b were assessed in two of the three children born with detectable IFX levels. Both children had protective levels of vaccine antibodies when checked at month 6.

  patients remained in remission throughout their pregnancy and resumed IFX treatment within 4 weeks after delivery. The re-treatment with IFX did not result in allergic reactions and all patients remained in remission during the 4 to 11 months of follow-up after delivery.

  samples were taken postpartum on days 15 and 57, respectively, which makes the assumption of a lack of intra-uterine and early postnatal exposure to IFX uncertain.

  mothers treated with IFX, vaccinations with attenuated vaccines should be postponed until IFX levels are undetectable.

Figure 1 .Figure 1 .

 11 Figure 1.Infliximab levels in cord blood of newborns (black bars) and in the peripheral blood of the mothers (grey bars) at delivery. In the three patients with detectable IFX levels, the levels in newborn were higher than the levels at the peripheral blood of the mothers (one-sided paired t-test, p=0.032) * Levels undetectable

  of the newborn to IFX in this case series of four IBD patients intentionally treated with IFX until gestational week 30. We found that this approach is not sufficient to limit the placental transfer of IFX, resulting in IFX levels in the cord blood that exceed the therapeutic level for adults.

	F o r Patients and Methods Four patients (three with Crohn`s disease and one with ulcerative colitis, aged 19, 29, 29, and 31 years, respectively) were intentionally treated with IFX during pregnancy. All patients continued the remission-maintaining preconception dose regimen. In one patient, methotrexate was advised to be discontinued following her expressed wish to reproduce. Three patients were receiving IFX at a stable dose of 5 mg/kg every eight weeks, and one patient received 10 mg/kg every eight weeks. Two patients had periconceptional IFX monotherapy, one patient was using azathioprine. The patient using methotrexate conceived determine the exposure F within two months after stopping methotrexate and continuing IFX monotherapy. The other o r three patients had stable medication during the six months prior to conception. One patient
	P P stopped IFX at gestational week 21, two patients at week 26, and one patient at week 30.
	e e
	e r e r
	R R
	e e
	v i e v i e
	w w
	4

following termination of anti-TNF therapy during different gestational weeks, prompted us to

Table 1 .

 1 a longer biological half-life of IFX and, therefore, possibly may provide a safe way to stop treatment earlier in the pregnancy.In conclusion, the use of IFX at the end of the second trimester of pregnancy leads to intrauterine exposure of the fetus to IFX. This exposure does not seem to have a negative impact on the child in terms of increased infection rate and vaccination failure, but in specific situations, such as vaccinations with attenuated live antigens, the presence of IFX may have fatal consequences. Therefore, we propose that the levels of IFX be assessed in every child born to mothers treated with IFX during pregnancy, and to administer attenuated live vaccines only to children with undetectable IFX levels. Additionally, the long-term effects of this early exposure of the immune system to IFX are hard to predict. Therefore, for patients in whom the quiescent disease during pregnancy allows interruption of treatment, intrauterine and postnatal exposure of newborns to IFX may probably be avoided by stopping IFX at the beginning of the second trimester. In order to determine the exact timing of the discontinuation of IFX during pregnancy, further studies on the pharmacokinetics in this setting need to be conducted. . De Ridder had support from MSD for consultancy C. van der Woude had support from Abbott, Shering-Plough and Shire for consultancy and received payment for lectures including service on speakers bureaus from Falk and Abbott Pregnancy outcomes and medication during pregnancy

	would lead to
	F o r
	P
	e
	e r
	R
	e
	v i e
	w
	12

That these patients still had detectable levels of IFX may indicate changes in the pharmacokinetics of IFX during pregnancy that LF o r P e e r R e v i e w *Methotrexate two months prior to conception

Alimentary Pharmacology & Therapeutic


M.Sc Merlijn Hutteman

J Sven

M.D. D Mieog

Joost R Vorst

M.D Gerrit-Jan Jan Liefers

Ph.D Hein Putter

Ph.D Clemens W G M Löwik

M.D John V Frangioni

Cornelis J H Velde

M.D Alexander L Vahrmeijer
email: a.l.vahrmeijer@lumc.nl

M.D Joost R Van Der Vorst

M.D J H Van De Velde

Ph.D Albinusdreef

None Gerrit-

M.D Jan Liefers

Ph.D None Hein Putter

Ph.D : None

W G M Clemens

Ph.D None Löwik

V John

M.D Frangioni

Ph.D : Mini-Flare

M.D Cornelis J H Van De Velde

Ph.D None Alexander

M.D L Vahrmeijer

Randomized, double-blind

Keywords: Near-Infrared Fluorescence Imaging, Image-Guided Surgery, Breast Cancer, Sentinel Lymph Node Mapping, Indocyanine Green, Albumin

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

Sentinel lymph node (SLN) mapping is currently regarded as standard of care in staging of the axilla in breast cancer patients with clinically negative axillary lymph nodes [START_REF] Cox | Guidelines for sentinel node biopsy and lymphatic mapping of patients with breast cancer[END_REF]. In general, a combination of radioactive colloid and blue dye is used for SLN mapping. Using this combination, identification rates of 95% to 97% are achieved [START_REF] Goyal | Factors affecting failed localisation and falsenegative rates of sentinel node biopsy in breast cancer--results of the ALMANAC validation phase[END_REF][START_REF] Krag | Technical outcomes of sentinel-lymph-node resection and conventional axillary-lymph-node dissection in patients with clinically node-negative breast cancer: results from the NSABP B-32 randomised phase III trial[END_REF][START_REF] Zavagno | A Randomized clinical trial on sentinel lymph node biopsy versus axillary lymph node dissection in breast cancer: results of the Sentinella/GIVOM trial[END_REF][START_REF] Straver | Sentinel Node Identification Rate and Nodal Involvement in the EORTC 10981-22023 AMAROS Trial[END_REF]. The use of only one of these two detection methods results in significantly lower identification rates [START_REF] Goyal | Factors affecting failed localisation and falsenegative rates of sentinel node biopsy in breast cancer--results of the ALMANAC validation phase[END_REF][START_REF] Krag | Technical outcomes of sentinel-lymph-node resection and conventional axillary-lymph-node dissection in patients with clinically node-negative breast cancer: results from the NSABP B-32 randomised phase III trial[END_REF][START_REF] Zavagno | A Randomized clinical trial on sentinel lymph node biopsy versus axillary lymph node dissection in breast cancer: results of the Sentinella/GIVOM trial[END_REF][START_REF] Straver | Sentinel Node Identification Rate and Nodal Involvement in the EORTC 10981-22023 AMAROS Trial[END_REF]. Both detection methods possess certain disadvantages. Radioactive colloids require involvement of a nuclear medicine physician, can be difficult to localize with a handheld gamma probe, and the time-window for SLN identification is limited due to the short half-life (6 hours) of 99m Technetium. Blue dyes cannot be seen through skin and fatty tissue, and permit only limited visualization of afferent lymphatic vessels and the SLN.

Optical imaging using the near-infrared (NIR) fluorescence lymphatic tracer indocyanine green (ICG) enables real-time transcutaneous and intraoperative visualization of lymphatic channels and SLNs [START_REF] Kitai | Fluorescence navigation with indocyanine green for detecting sentinel lymph nodes in breast cancer[END_REF][START_REF] Murawa | Sentinel lymph node biopsy in breast cancer guided by indocyanine green fluorescence[END_REF][START_REF] Tagaya | Intraoperative identification of sentinel lymph nodes by near-infrared fluorescence imaging in patients with breast cancer[END_REF][START_REF] Hirche | ICG fluorescence-guided sentinel node biopsy for axillary nodal staging in breast cancer[END_REF][START_REF] Hojo | Evaluation of sentinel node biopsy by combined fluorescent and dye method and lymph flow for breast cancer[END_REF][START_REF] Troyan | The FLARE intraoperative near-infrared fluorescence imaging system: a first-inhuman clinical trial in breast cancer sentinel lymph node mapping[END_REF][START_REF] Sevick-Muraca | Imaging of lymph flow in breast cancer patients after microdose administration of a near-infrared fluorophore: feasibility study[END_REF][START_REF] Tanaka | Image-guided oncologic surgery using invisible light: completed pre-clinical development for sentinel lymph node mapping[END_REF].

Therefore, NIR fluorescence imaging could provide an alternative for, or an addition to, conventional techniques used for SLN mapping. Recently, our group has demonstrated that NIR fluorescence performed equally well as the combination of radioactive colloid and blue dye in SLN mapping of breast cancer patients [14].

ICG is currently the only clinically available NIR lymphatic tracer. However, due to its relatively low fluorescence brightness and its small hydrodynamic diameter, which permits flow through the SLN to higher tier nodes, it is not an optimal lymphatic tracer. Preclinical work has demonstrated that adsorption of ICG to human serum albumin (HSA, complex is ICG:HSA), by simply mixing it, increases the fluorescence intensity and the hydrodynamic diameter, thereby providing improved detection and better retention in the SLN in certain anatomical sites, such as the intestine [START_REF] Ohnishi | Organic alternatives to quantum dots for intraoperative near-infrared fluorescent sentinel lymph node mapping[END_REF]. Another parameter that must be considered when using ICG or ICG:HSA is the effect of fluorescence quenching, which results in a decrease in fluorescence intensity as the concentration of ICG (or ICG:HSA) is increased above 50 M. The use of 50 M ICG for SLN imaging, however, is suboptimal, because ICG will be diluted once taken up by the lymphatic system. To assess the magnitude of this in vivo dilution effect, our group has conducted a dose-finding study to demonstrate that the optimal concentration of ICG:HSA for NIR-based SLN mapping in breast cancer patients lies between 400 µM and 800 µM [14].

A theoretical disadvantage of the use of ICG alone is poor retention of the lymphatic tracer in the SLN, which as a consequence results in fluorescent staining of higher tier nodes and background staining of the axilla. Although not compared directly, studies using ICG alone reported a higher average number of identified However, comparison of these data is difficult because the concentration of ICG used was significantly higher (typically 6.4 mM) than in the trials using ICG:HSA (10 µM to 1000 µM).

Although we have obtained good results with the use of ICG:HSA for SLN mapping in breast cancer patients, the use of albumin adds cost and complexity to the procedure. Moreover, the use of human blood products, such as HSA, poses regulatory hurdles in certain countries, such as the United States. Therefore, the use of ICG alone would be favorable. After intravenous administration, ICG binds rapidly and completely to plasma proteins [START_REF] Cherrick | Indocyanine green: observations on its physical properties, plasma decay, and hepatic extraction[END_REF]. Lymph fluid has a similar protein constitution as serum, albeit in a lower concentration (20.6 g/L for lymph fluid versus 73.7 g/L for plasma) [START_REF] Fogh-Andersen | Composition of interstitial fluid[END_REF]. After intradermal or subcutaneous injection, ICG could theoretically bind to these proteins, eliminating the need for premixing ICG and HSA. We therefore hypothesized that ICG alone could render the same fluorescence intensity in SLNs as ICG:HSA, and tested this hypothesis in a double-blind randomized trial.

METHODS

Preparation of Indocyanine Green adsorbed to Human Serum Albumin: ICG (25 mg vials) was purchased from Pulsion Medical Systems (Munich, Germany) and was resuspended in 10 cc of sterile water for injection to yield a 2.5 mg/ml (3.2 mM) stock solution. To obtain a 500 µM dilution, 7.8 mL of the 3.2 mM ICG solution was diluted in 50 cc vial of sterile water for injection or 50 cc vial of Cealb (20% human serum albumin, Sanquin, Amsterdam, The Netherlands) for the preparation of ICG alone or ICG:HSA, respectively. Prior to the addition of ICG, 7.8 mL was drawn from the 50 cc vials. In a previous study, we determined that the optimal dose of ICG:HSA lies between 400 µM and 800 µM [14]. A dose of 500 µM was chosen because it requires minimal manipulation of ICG and albumin volumes.

Intraoperative Near-Infrared Imaging System (Mini-FLARE): SLN mapping was performed using the Mini-Fluorescence-Assisted Resection and Exploration (Mini-FLARE) image-guided surgery system as described earlier [14]. Briefly, the system consists of two wavelength isolated light sources: a "white" light source, generating 26,600 lx of 400-650 nm light, and a "near-infrared" light source, generating 7.7 mW/cm 2 of 760 nm light. Color video and NIR fluorescence images are simultaneously acquired and displayed in real-time using custom optics and software that separate the color video and NIR fluorescence images. A pseudo-colored (lime green) merged image of the color video and NIR fluorescence images is also displayed. The imaging head is attached to a flexible gooseneck arm, which permits positioning of the imaging head at extreme angles virtually anywhere over the surgical field. For intraoperative use, the imaging head and imaging system pole stand are wrapped in a sterile shield and drape (Medical Technique Inc., Tucson, AZ).

Clinical Trial:

The double-blind, randomized, single-institution, non-inferiority trial comparing ICG:HSA with ICG alone was approved by the Medical Ethics Committee of the Leiden University Medical Center and was performed in accordance with the ethical standards of the Helsinki Declaration of 1975. All patients planning to undergo a sentinel lymph node procedure whether for invasive breast cancer or for high-risk carcinoma in situ were eligible for participation in the study. Patients had clinically negative axillary nodes as assessed by palpation and ultrasonography. Exclusion criteria were pregnancy, lactation or an allergy to iodine, shellfish, or indocyanine green.

RESULTS

Eighteen consecutive breast cancer patients undergoing standard-of-care SLN mapping were randomized to ICG:HSA or ICG alone for the NIR-based SLN imaging. Patient, tumor, and treatment characteristics were equally distributed over the treatment groups (Table 1). Use of the Mini-FLARE during surgery did not interfere with the standard of care. Average time between lymphatic tracer injection and skin incision was 15.6 ± 2.2 minutes (Table 2). In all patients (N = 18), NIR fluorescence imaging enabled visualization of one or more SLNs (Figure 1). In the ICG:HSA group (N = 8 subjects), a total of 11 SLNs were identified (average per patient = 1.4 ± 0.5); 9 (82%) were radioactive, 8 (73%) were blue and 11 (100%) were NIR fluorescent. In the ICG alone group (N = 10 subjects), a total of 14 SLNs were identified (average per patient = 1.4 ± 0.5); 14 (100%) were radioactive, 10 (71%) were blue and 14 (100%) were NIR fluorescent.

The average number of SLNs identified was not significantly different between both groups (P = 0.74).

The primary endpoint of this study was the average brightness of the SLN in both groups, expressed in signal-to-background ratio (SBR). The results are presented in Table 2. The average SBR of ICG:HSA (8.4 ± 3.6) and ICG alone (11.3 ± 4.8) was not significantly different (P = 0.18). However, in the ICG alone group, the afferent lymphatics were significantly better visualized percutaneously compared to the ICG:HSA group (P = 0.004; Table 2 and Figure 1). In all patients, the NIR fluorescence signal in the SLN was detected earlier in the procedure before patent blue was visualized. Average time between skin incision and resection of the first SLN was 11.0 ± 4.1 minutes and was not different between both groups (t-test, P = 0.74). No adverse reactions associated with the use of ICG, ICG:HSA, or Mini-FLARE occurred.

DISCUSSION

The use of NIR fluorescence for SLN mapping has several advantages over conventional methods, such as better tissue penetration when compared to blue dyes, and lack of ionizing radiation and real-time visualization when compared to radiotracers. A number of clinical studies have been published on the use of NIR fluorescence in the SLN procedure in breast cancer, all of which use ICG, which is currently clinically available [START_REF] Kitai | Fluorescence navigation with indocyanine green for detecting sentinel lymph nodes in breast cancer[END_REF][START_REF] Hirche | ICG fluorescence-guided sentinel node biopsy for axillary nodal staging in breast cancer[END_REF][START_REF] Troyan | The FLARE intraoperative near-infrared fluorescence imaging system: a first-inhuman clinical trial in breast cancer sentinel lymph node mapping[END_REF][START_REF] Sevick-Muraca | Imaging of lymph flow in breast cancer patients after microdose administration of a near-infrared fluorophore: feasibility study[END_REF]14]. Preclinical studies indicated that adsorption of ICG to human serum albumin (HSA, complex is ICG:HSA), by simply mixing it, increases the fluorescence intensity and the hydrodynamic diameter, thereby providing improved detection and better retention in the SLN [START_REF] Ohnishi | Organic alternatives to quantum dots for intraoperative near-infrared fluorescent sentinel lymph node mapping[END_REF]. Our group has subsequently conducted a dose-finding study and demonstrated that the optimal concentration of ICG:HSA for NIR-based SLN mapping in breast cancer patients lies between 400 µM and 800 µM [14]. Indeed, above 800 µM ICG:HSA, the fluorescent intensity dropped due to quenching. Based on these results a concentration of 500 µM ICG:HSA, which requires minimal manipulation of albumin volumes and uses only one vial of ICG, was chosen for further studies to identify whether premixing with albumin indeed increases the fluorescent intensity of the node in a clinical setting. In the current study, SLN mapping after ICG or ICG:HSA injection was successful in all patients. ICG showed a comparable or even slightly increased (though not significantly) brightness than ICG:HSA while identifying an equal average number of SLNs. Although no macrometastases in the SLNs were observed in the current study, our previous study showed that tumor-positive SLNs were also detected by NIR fluorescence, signifying ICG uptake [14]. However, lymph node macrometastases continue to be a contraindication for SLN mapping and preoperative staging of the axilla remains pivotal to minimize false negatives.

The results of our study are discordant with preclinical work in intestine, which suggested an improvement in fluorescent brightness and retention in the SLN by premixing ICG with HSA [START_REF] Ohnishi | Organic alternatives to quantum dots for intraoperative near-infrared fluorescent sentinel lymph node mapping[END_REF]. Although the current study was powered to determine non-inferiority in SBR of ICG alone when compared to ICG:HSA, it was not formally powered to assess the secondary endpoint, average number of SLNs identified. However, power analysis using data from our previous study [14] demonstrated that a difference of at least one additional SLN identified per patient could be detected with 90% power with the current sample size. The discrepancy between the current clinical results and these preclinical experiments could be caused by the increased distance that the injected dye has to travel, as the preclinical studies were performed in the bowel of healthy pigs [START_REF] Ohnishi | Organic alternatives to quantum dots for intraoperative near-infrared fluorescent sentinel lymph node mapping[END_REF].

Lymph fluid contains a high concentration of albumin, among other proteins; therefore, a longer traveling distance could aid ICG in adsorbing to albumin or other proteins, as it would after intravenous injection [START_REF] Moody | Non-covalent labeling of human serum albumin with indocyanine green: a study by capillary electrophoresis with diode laser-induced fluorescence detection[END_REF], diminishing the need for premixing ICG with HSA. This observation implies that premixing might prove to be useful in other cancer types (such as colon cancer, for example), where ICG is less likely to completely adsorb to proteins before the SLN is reached. Therefore, the use of ICG:HSA or ICG alone should be tested for every anatomical site.

Although the fluorescent brightness did not differ significantly between both groups, ICG alone showed significantly improved percutaneous visibility of lymphatic channels when compared to ICG:HSA (Figure 1). It has been shown that in plasma, ICG preferably binds to 1 -lipoprotein and -globulin, despite the higher concentration of albumin [START_REF] Sauda | Determination of protein in human serum by high-performance liquid chromatography with semiconductor laser fluorometric detection[END_REF]. Previous experiments have shown a higher increase in quantum yield when ICG is mixed with serum, in comparison to HSA [START_REF] Ohnishi | Organic alternatives to quantum dots for intraoperative near-infrared fluorescent sentinel lymph node mapping[END_REF]. Therefore, the observed differences in visualization of lymphatics could likely be attributable to the protein constitution of lymph fluid. The high albumin content (20%) of ICG:HSA could also be a contributing factor, as the increased hydrodynamic diameter and higher viscosity could diminish ICG uptake in lymphatic channels. It should be noted that the anatomical variation (amount of tissue overlying the lymphatic channels) is also a major influencing factor and may be primarily responsible for the observed difference.

An optimal lymphatic tracer is non-toxic, has a high quantum yield (i.e., brightness), migrates quickly to the SLN, and does not migrate to higher tier nodes. If a tracer migrates to higher tier nodes, non-sentinel lymph nodes could incorrectly be identified as SLNs, causing more nodes than necessary to be resected. ICG is far from optimal; in aqueous solution the quantum yield is relatively low and due to its small hydrodynamic diameter, it can flow to higher tier nodes, as is the case with blue dyes. The synthesis and clinical introduction of an optimal probe will be the subject of future studies and will greatly help to confirm the clinical benefit of NIR fluorescence imaging in the SLN procedure [START_REF] Ohnishi | Organic alternatives to quantum dots for intraoperative near-infrared fluorescent sentinel lymph node mapping[END_REF][START_REF] Hutteman | Clinical Translation of Ex Vivo[END_REF].

In the current study, NIR fluorescence after ICG injection could consistently be visualized before the blue dye could be observed, which is consistent with earlier findings [14]. Therefore, NIR fluorescence imaging has the potential to replace blue dyes in SLN mapping of breast cancer patients. Furthermore, as NIR fluorescence light penetrates relatively deep into tissue, it can potentially replace radiocolloids in SLN mapping in a selected group of patients, for example those with a low body mass index. A clinical trial on omitting blue dyes and using NIR fluorescence without the need for radiocolloids is currently ongoing (NTR2674).

In conclusion, this double-blind, randomized trial showed no advantage of ICG:HSA in comparison to ICG alone for the SLN procedure. To reduce the cost and complexity of the procedure, a dose of 500 µM ICG alone (1.6 ml) is recommended for NIR fluorescence SLN mapping in breast cancer patients. Therefore, this study has determined the optimal parameters that can be used to validate this technique in a larger series in order to investigate patient benefit. a Partial percutaneous visualization was noted when lymphatic channels could be visualized percutaneously from the injection site, but did not reach the axilla.

TABLES

FIGURE CAPTIONS

Color NIR Fluorescence

Color-NIR Merge

Fig. 1 -

 1 Fig. 1 -NIR fluorescence imaging during sentinel lymph node mapping in breast cancer patients a. ICG:HSA. In the upper panel, the periareolar injection site (arrow) is shown, but percutaneously, no lymphatic channel can be visualized. In the lower panel, identification of the SLN (arrow) and an afferent lymphatic channel (open arrowhead) with NIR fluorescence imaging is demonstrated 27 min after injection of 1.6 mL of 500 µM ICG:HSA. Camera exposure times were 60 msec (top row) and 150 msec (bottom row). Scale bars represent 1 cm. b. ICG alone. In the upper panel, the periareolar injection site (arrow) and a lymphatic channel (arrowhead) are clearly visualized. In the lower panel, identification of the SLN (arrow) and an afferent lymphatic channel (open arrowhead) with NIR fluorescence imaging is demonstrated 28 min after injection of 1.6 mL of 500 µM ICG. Camera exposure times were 30 msec (top row) and 100 msec (bottom row). Scale bars represent 1 cm.

  Figure 1 Hutteman and Mieog et al.

  Figure 1 Hutteman and Mieog et al.

Table 1 -Patient and Tumor Characteristics

 1

		ICG:HSA (N = 8)	ICG alone (N = 10)
	Characteristic	N	%	N	%	P
	Age (Median, Range)	59.5 (38-72)	57.5 (40-73)	0.99
	Menopausal State					0.74
	-Premenopausal	3	37.5	3	30
	-Postmenopausal	5	62.5	7	70
	Body Mass Index (median, range)	26 (20-41)	23.5 (21-30)	0.26
	Skin Type					0.40
	-II	2	25	1	10
	-III	6	75	9	90
	Previous Procedure of Breast					0.12
	-Breast Implants	1		0
	-Breast Reduction	0		1
	-Lumpectomy	2		0
	-Radiotherapy	0		1
	-Neoadjuvant Chemotherapy	1		0
	Multifocality	0		0		1.00
	Tumor side					0.81
	-Left	2	25	3	30
	-Right	6	75	7	70
	Tumor localization					0.23
	-Upper Outer	6	75	4	40
	-Lower Outer	0	0	1	10
	-Lower Medial	1	12.5	2	20
	-Upper Medial	0	0	3	30
	-Central	1	12.5	0	0
	Type of Operation					0.15
	-Mastectomy	3	37.5	1	10
	-Wide Local Excision	4	50	9	90
	-SNB Only	1	12.5	0	0
	Pathological Tumor Size (Median, Range)	7 (5-11)		12 (8-15)		0.13
	Histological Type					0.62
	-Infiltrating Ductal Adenocarcinoma	7	87.5	7	70
	-Infiltrating Lobular Adenocarcinoma	0	0	1	10
	-Ductal Carcinoma In Situ	1	12.5	1	10
	-Other	0	0	1	10
	Histological Grade					0.15
	-I	1	16.7	3	33.3
	-II	2	33.3	5	55.6
	-III	3	50	1	11.1
	Abbreviations: ICG:HSA = indocyanine green adsorbed to human serum albumin
	Skin type = American Academy of Dermatology Skin Types I-VI:
	I. Pale, white skin: always burns easily; never tans (Celtic, Scandinavian, and infants)
	II. White: usually burns easily; tans minimally (Northern European)
	III. White (average): sometimes burns; tans gradually to light brown (Central European)
	IV. Beige or lightly tanned: burns minimally; always tans to moderately brown (Mediterranean, Asian)

V. Moderate brown or tanned: rarely burns; tans well (South American, Indian, Native American) VI. Dark brown or black: never burns; deeply pigmented (African, African-American, Aborigine)

Table 2 -SLN Identification Results

 2

		Total		ICG:HSA	ICG alone
	Characteristic	(18 Subjects)	(8 Subjects)	(10 Subjects)	P
		N	%	N	%	N	%
	Number of SLNs Identified	25		11		14
	Number of SLNs Identified per Patient							0.91
	-One SLN	11	61	5	63	6	60
	-Two SLNs	7	39	3	37	4	40
	Average Number of SLNs Identified ±
	S.D.	1.4 ± 0.5	1.4 ± 0.5	1.4 ± 0.5	0.92
	Method of Detection
	-Radioactive	23	92	9	82	14	100	0.18
	-Blue	18	72	8	73	10	71	1.00
	-NIR Fluorescent	25	100	11	100	14	100	1.00
	Signal-to-Background Ratio	10.0 ± 4.4	8.4 ± 3.6	11.3 ± 4.8	0.18
	Percutaneous NIR Fluorescent Lymph
	Drainage Visualization							0.004
	-Yes	10	56	1	13	9	90
	-Partially a	3	17	3	38	0	0
	-No	5	28	4	50	1	10
	Average Time between Injection and
	Skin Incision ± S.D. (minutes)	15.6 ± 2.2	15.3 ± 1.7	15.9 ± 2.6	0.55
	Average Time between Skin Incision and
	SLN Resection ± S.D. (minutes)	11.0 ± 4.1	10.6 ± 5.1	11.3 ± 3.3	0.74
	Histology							0.44
	-Negative	24	96	10	91	14	100
	-Isolated Tumor Cells	1	4	1	9	0	0
	-Macrometastases	0	0	0	0	0	0
	Axillary Lymph Node Dissection							1.00
	-No	18	100	8	100	10	100
	-Yes	0	0	0	0	0	0

Abbreviations: ICG:HSA = indocyanine green adsorbed to human serum albumin; S.D. = standard deviation; SLN = sentinel lymph node.

Sentinel Lymph Node Mapping for Colorectal Cancer Using Invisible Near-Infrared Fluorescence Light. Ann Surg Oncol. Epub ahead of print

ACKNOWLEDGMENTS

We thank the following individuals for the contribution to this study: Gemma Ranke, Elly Krol-Warmerdam, Annemarie Voet-van den Brink, Gerlinda van Bruijn (Breast Cancer Unit) and Linda van der Hulst (Central Pharmacy). We thank Lindsey Gendall for editing. This work was supported in part by NIH grants R01-CA-115296 and R21-CA-130297, the Dutch Cancer Society grant UL2010-4732, the Nuts Ohra Fund, the "Maurits and Anna de Kock" Foundation and the American Women's Club of The Hague. J.S.D.

Mieog is a MD-medical research trainee funded by The Netherlands Organisation for Health Research and Development (grant nr. 92003526).

This work was supported in part by NIH grants R01-CA-115296 and R21-CA-130297, the Dutch Cancer Society grant UL2010-4732, the Nuts Ohra Fund, the "Maurits and Anna de Kock" Foundation and the American Women's Club of The Hague. J.S.D. Mieog is a MD-medical research trainee funded by The Netherlands Organisation for Health Research and Development (grant nr. 92003526).