ASP0028 in combination with suboptimal-dose of tacrolimus in Cynomolgus monkey renal transplantation model

Transplant Immunology 40 (2017) 57–65

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ASP0028 in combination with suboptimal-dose of tacrolimus in Cynomolgus monkey renal transplantation model Hao Dun a, Lijun Song a, Anlun Ma a, Yanxin Hu a, Lin Zeng b, Jieying Bai b, Guangzhou Zhang b, Liangyan Zhang b, Kumi Koide c, Yohei Okada c, Kaori Hanaoka c, Rie Yamamoto c, Jun Hirose c, Tatsuaki Morokata c, Pierre Daloze a, Huifang Chen a,⁎ a b c

Department of Surgery, Research Center, CHUM, Notre-Dame Hospital, University of Montreal, Montreal, Canada Laboratory Animals Center, the Academy of Military Medical Sciences, Beijing, China Drug Discovery Research, Astellas Pharma Inc., Japan

a r t i c l e

i n f o

Article history: Received 25 July 2016 Accepted 6 January 2017 Available online 7 January 2017 Keywords: Kidney transplantation Nonhuman primates Immunosuppressive drugs FTY720 Lymphocyte subsets

a b s t r a c t FTY720, a S1P-receptor modulator, has shown to be effective in several transplant and autoimmune disease models, via modulating lymphocyte homing into secondary lymphoid organs (SLOs), and thereby reducing these cells in peripheral blood. ASP0028, a newly developed S1P1/S1P5-selective agonist, presented comparable efficacy to FTY720 and wider safety margins than FTY720. In this study, we assessed the efficacy and safety of ASP0028 co-administered with suboptimal-dose of tacrolimus in the Cynomolgus monkey renal transplantation model. Seven animals in group-1 or group-2 received mono-tacrolimus 1.0 mg/kg once a day (QD), or ASP0028 0.6 mg/kg plus tacrolimus 1.0 mg/kg QD, respectively. Eight animals in group-3 received ASP0028 1.2 mg/kg plus tacrolimus 1.0 mg/kg QD. The allograft median survival time (MST) in group-2 and group-3 were significantly extended to 41 and 61.5 days, versus that of 28 days in group-1 (p = 0.036 and 0.001, respectively). ASP0028 administration remarkably reduced absolute numbers of peripheral lymphocytes, particularly subsets of CD4+/ or CD8+/naive and central memory cells, CD4+/Treg cells, and to a lesser extent on B cells, but not CD4+/ or CD8+/ effector memory cells and NK cells. These data show ASP0028 combined with suboptimal-dose of tacrolimus effectively prolongs renal allograft survival in nonhuman primates (NHPs) with well tolerated safety, supporting its further investigation to optimize CNI-sparing regimens. © 2017 Elsevier B.V. All rights reserved.

1. Introduction S1P is a sphingolipid mediator, which regulates multiple critical cellular processes through binding to five G protein-coupled S1P receptors (S1P1–5) [1,2]. S1P1 is dominantly expressed on lymphocytes and thymocytes, and regulates lymphocyte egress from SLOs and thymus [3]. FTY720 is an agonist at four of the five S1P receptors, including S1P1, S1P3, S1P4, and S1P5, but not S1P2 (4). After phosphorylation, FTY720 binding S1P1, causes internalization of receptors and consequently inhibits S1P/S1P1 recipient-dependent lymphocyte egress from SLOs. As a result, lymphocytes are sequestered in SLOs and thereby fail to recirculate into the peripheral blood and subsequently reach target tissues [5]. Because of these features, S1P1 appears to be a critical regulator of Abbreviations: AUC(0–24), the area under the plasma concentration-time curves from 0 to 24 h; Cmax, the maximum plasma concentration; CNI, calcineurin inhibitor; MS, multiple sclerosis; MST, median survival time; NHPs, nonhuman primates; PD, pharmacodynamic; PK, pharmacokinetic; QD, once daily; S1P, sphingosine-1-phosphate; SCr, serum creatinine; SLOs, secondary lymphoid organs; Tmax, the time to reach Cmax. ⁎ Corresponding author. E-mail address: [email protected] (H. Chen).

http://dx.doi.org/10.1016/j.trim.2017.01.002 0966-3274/© 2017 Elsevier B.V. All rights reserved.

lymphocyte trafficking, and therefore becomes a key target for development of immunosuppressive agents. FTY720 has been proven to significantly decrease the number of circulating T cells on selected lymphocyte subsets and B cells [6–9]. Based on this mechanism, FTY720 provided a variety of effects on autoimmune [10–12] and transplant models [13–17]. Moreover, due to distinct mechanisms of immunosuppressive action, FTY720 showed a synergistic effect on allograft survival when combined with cyclosporine or sirolimus in rodent and NHP models [15–17]. In September 2010, FTY720 was approved by the FDA for treatment relapsing multiple sclerosis (MS) [18]. In addition, two phase-3 clinical trials for renal transplant had been performed to evaluate the efficacy and safety of FTY720 in combination with suboptimal-dose of cyclosporine [19,20]. However, the trials failed to meet the endpoints, and therefore further study of the use of FTY720 was discontinued in this indication [2]. Of note, in all clinical trials of FTY720, bradycardia is one of the most commonly reported side effects [21–23]. This bradycardia is presumably because of agonistic activity of FTY720 toward S1P3, since FTY720 does not cause bradycardia in S1P3 knockout mice [24]. Based on these observations, S1P1-selective compound is presumed to be devoid of side-effects,

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described in FTY720 clinical studies, e.g. bradycardia and macular edema. ASP0028 is a newly developed S1P1/S1P5-selective agonist in Astellas Pharma Inc. Unlike FTY720, in vitro much higher concentration of ASP0028 is required to stimulate S1P3 (unpublished data). The preliminary study showed that ASP0028 at 1.0 mg/kg level, similar to FTY720 at 0.1 mg/kg, significantly decreased the number of peripheral lymphocytes in rats. In addition, heart transplant studies in rats indicated that ASP0028 combined with suboptimal-dose of tacrolimus significantly prolonged allograft survival, comparable to that of FTY720 in combination with suboptimal-dose of tacrolimus (unpublished data). More importantly, our preliminary study showed that ASP0028 has a wider margin of safety than FTY720, in terms of side-effects of macular edema and bradycardia. Combination therapies of different immunosuppressive agents with distinct mechanisms of action are a valid strategy for transplant patients to minimize dose-related toxicities. In this Cynomolgus monkey renal transplantation model, tacrolimus at suboptimal-doses of 1.0 mg/kg was suggested in a tacrolimus-based combination therapy for testing a new compound [25]. Accordingly, in the present study, we evaluated the efficacy and safety of ASP0028 0.6 or 1.2 mg/kg in combination with tacrolimus 1.0 mg/kg in NHP renal transplantation model. Moreover, we investigated whether there is a differential inhibitory effect of ASP0028 on certain subsets of peripheral lymphocytes. In addition, we analyzed the pharmacokinetic (PK) profile of ASP0028 in combination with suboptimal-dose of tacrolimus. We assume that the selectivity of ASP0028 in NHPs is comparable in humans. Therefore, findings obtained from this study will contribute to a better understanding of the mechanism of action of ASP0028, which is a key for its further development as a candidate drug for optimizing CNI-sparing regimens in clinical transplant.

2. Materials and methods 2.1. Animals Twenty-two male Cynomolgus monkeys (Macaca fascicularis), aged 3–6 years, with body weight of 3.2–5.2 (4.1 ± 0.42) kg, and hepatitis B virus, hepatitis C virus, simian immunodeficiency virus, simian varicella virus, and herpes B virus free, were obtained from Laboratory Animals Center of the Academy of Military Medical Sciences (AMMS), Beijing, China. The experimental protocol was approved by the Ethical Committee for Animal Experimentation at Laboratory Animals Center of the AMMS, and all procedures were performed according to the Guide for the Care and Use of Laboratory Animals, National Institutes of Health Office of Animal Care and Use. Before study entry, all animals were quarantined for two weeks with general health screening. Each animal with a unique identification number was randomly assigned to an experimental group. All animals were housed in individual cages and allowed free access to water, monkey chows and fruits.

2.3. Experimental design and treatment protocols Animals were randomly divided into three experimental groups for a maximum of 90-day observation as shown in Table 1. Based on an earlier experiment [25], the suboptimal-dose of tacrolimus 1.0 mg/kg was chosen as a control. Accordingly, Animals in group-1 (n = 7) received mono-tacrolimus 1.0 mg/kg QD, in group-2 (n = 7) received ASP0028 0.6 mg/kg plus tacrolimus 1.0 mg/kg QD, and in group-3 (n = 8) received ASP0028 1.2 mg/kg plus tacrolimus 1.0 mg/kg QD, respectively. Tacrolimus (lot No. 701732K), manufactured by Astellas Pharma Inc., was orally given on day 0 (the day of kidney transplant) immediately after kidney transplantation until day 90, whereas ASP0028 was orally administered from 2 days before surgery (day -2) till day 90. 2.4. Biochemical and hematologic determinations Serum creatinine (SCr) levels were intensively monitored on day -7 and post-operative day 1, 3, 5 and 7 during the first week, and then at least twice per week for the first month, and thereafter weekly. The others were determined as described in our previous study [26]. 2.5. Lymphocyte subsets Lymphocyte phenotyping was performed on a subset of 4 animals of each group (Table 1). Blood samples were collected on day -7 and postoperative day 0, 7, 14, 28, 56 and 84 before dosing to determine the effects of ASP0028 on certain lymphocyte subsets. Lymphocyte subsets, including CD3+ (absolute number T cells), CD3+/CD4+ (T helper cells), CD4+ CD28+ CD95− (CD4+ naïve cells), CD4+ CD28+ CD95+ (CD4+ central memory cells), CD4+ CD28− CD95+(CD4+ effector memory cells), CD3+/CD8+ (T suppressor/killer cells), CD8+ CD28+ CD95− (CD8+ naïve cells), CD8+ CD28+ CD95+ (CD8+ central memory cells), CD8+ CD28− CD95+ (CD8+ effector memory cells), CD3+ CD4+ CD25+ CD127− (CD4+ Treg cells), CD3− CD20+ (B cells) and CD3− CD16+ (NK cells) were analyzed on the FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA) using the Becton Dickinson software CellQuest Pro. 2.6. Pharmacokinetic evaluations PK analyses for blood tacrolimus in all animals, and for plasma ASP0028 in animals of group-2 and -3 were conducted by a validated LC-Tandem Mass Spectrometry (LC/MS/MS) system, using Analyst Data Acquisition (Version 1.4.2) and Watson 7.3 LIMS software. The blood concentrations of tacrolimus were measured before and after 1 h administration on day 14, 28, 56 and 84. The plasma concentrations of ASP0028 were monitored before and after 4 h administration on day 0 and 7. Moreover, additional PK parameters of ASP0028 were evaluated on day 14, 28, 56 and 84. The Cmax of ASP0028 was achieved from observation of concentrations of trough (0), 2, 4, 8, 12 and 24 h after administration, and the Tmax was determined from above time points. In addition, AUC(0–24) for ASP0028 was analyzed. 2.7. Histopathologic determinations

2.2. Life supporting kidney transplantation In the present study, each animal served as both donor and recipient. Donor and recipient monkey pairs were selected by ABO blood-type compatibility, and the stimulation index of mixed lymphocyte reaction (MLR) ≥ 2.5. Life supporting kidney transplantation was performed as described previously [26,27]. Briefly, left kidneys were harvested en bloc and exchanged between paired animals for transplant. The allograft then was implanted into recipient abdomen by end-to-side anastomoses of renal artery to aorta and renal vein to vena cava, and by end-to-end anastomosis of donor and recipient ureters. After grafting, the right native nephrectomy was immediately performed.

All recipients underwent complete gross necropsies and histopathologic examinations as described in our previous study [26]. The presence and degree of renal allograft rejection was scored according to the Banff ‘97 criteria of renal allograft pathology [28]. 2.8. Statistical analysis All data were described as mean ± standard deviation (SD). Analyses of statistical differences among groups were performed using the twotailed t-test. Allograft survival times presented as MST were compared among groups by log-rank test. Statistical analyses were conducted using SPSS 13. A p value of b0.05 was considered statistically significant.

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Table 1 Clinical Characteristics and PK Profile of ASP0028 in Cynomolgus Monkey Renal Allograft Recipients Administrated with ASP0028 and Tacrolimus. Animal ID

Group

Survival days

0606651a 0501803a 0505545 0407461 0502225a 0502831a 0502697 0510591a 0509123a 0503759 0503447 0502155 0404745a 0507281a 0503661 0510823 0505193a 0411415a 0501571 0408147 0509135a 0501833a

1 1 1 1 1 1 1 2 2 2 2 2 2 2 3 3 3 3 3 3 3 3

35 10 28 36 35 7 17 35 59 N90 41 N90 24 17 67 N90 64 52 42 59 31 N90

Mean ASP0028 trough (ng/ml)

27.4 ± 6.8 30.5 ± 11.0 21.3 ± 9.8 48.3 ± 22.8 36.5 ± 17.8 57.5 ± 21.5 31.2 ± 20.3 34.6 ± 8.7 80.5 ± 25.5 113.5 ± 25.7 66.1 ± 31.3 106.4 ± 25.2 80.4 ± 29.7 113.0 ± 49.5 79.2 ± 13.7

Mean ASP0028 Cmax (ng/ml)

88.2 ± 25.7 69.8 ± 2.1 63.7 ± 16.5 83.2 ± 23.7 80.7 ± 32.9 50.1 ± 0.0 30.7 ± 0.0 113.8 ± 20.4 141.1 ± 19.8 266.4 ± 104.6 158.7 ± 54.7 231.4 ± 54.7 106.1 ± 21.6 181.5 ± 7.5 138.4 ± 11.1

Median ASP0028 AUC(0–24) (ng·h/mg)

Graft pathology (Banff 1997)

1429.19 1034.88 1017.86 1597.60 1031.62 884.00 445.05 1280.04 2452.20 4022.90 2877.66 3602.34 1709.73 3442.96 2451.33

AR IIA AR III CAN IIB No rejection AR IIA AR IIA AR IIA AR IIA CAN IIA CAN IA CAN IA CAN IIA AR IIA AR IIB CAN IB CAN IB CAN IB CAN IB AR IIA CAN IIB with AR IIA CAN IB No rejection

AR: acute rejection; CAN: chronic allograft nephropathy. a Animals assigned for PD assessment.

3. Results 3.1. Renal allograft survival 3.1.1. ASP0028 combination-treatment significantly prolongs allograft survival Survival times and pathology at necropsy for all of 22 treatedanimals are summarized in Table 1. Renal allograft survival days were determined according to definitions of our previous study [29]. Tacrolimus 1.0 mg/kg in group-1 yielded the allograft MST of 28 days. Moreover, tacrolimus 1.0 mg/kg in addition to ASP0028 0.6 mg/kg in group-2 or 1.2 mg/kg in group-3 significantly prolonged the allograft MST to 41 or 61.5 days, respectively, compared to that of group-1 (p = 0.036 and 0.001, respectively) (Fig. 1). In combination-therapy groups, high-dose of ASP0028-treatment was associated with a longer allograft MST, although the difference did not reach significant levels

(p = 0.60). Two animals in each of combination-therapy groups completed the 90-day follow-up and were euthanized with a nearly normal renal function and limited histologic lesions on final pathology (Table 1). 3.2. Renal graft function 3.2.1. ASP0028 combination-treatment improves renal graft function Changes of SCr and BUN levels after transplantation are shown in Fig. 2A and B, respectively. SCr levels in group-1 began to increase at around 1 week after transplantation, whereas SCr levels in group-2 and -3 were maintained at nearly normal levels up to 2 or 3 weeks. When acute allograft rejection occurred, SCr and BUN levels were rapidly elevated. However, SCr and BUN values in combination-therapy groups, particularly group-3, were maintained at relatively lower levels during study course.

Fig. 1. Kaplan-Meier survival curves for transplanted animals. Combination-therapy in group-2 (ASP0028 0.6 mg/kg + FK506) and -3 (ASP0028 1.2 mg/kg + FK506) significantly improved allograft survival over mono-therapy FK506 in group-1 (Log Rank, p = 0.036 and 0.001, respectively). Comparing between two combination-therapy groups, high-dose of ASP0028 trended toward a longer allograft MST, although this difference did not reach significant levels (p = 0.6). FK506: Tacrolimus.

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Fig. 2. Mean (±SD) SCr levels (A) and BUN levels (B) versus time profile in each group.

3.3. Body weight, clinic symptoms and biochemistry Body weight, clinic symptoms and biochemistry parameters were unaffected by the administration of ASP0028 plus suboptimal-dose of tacrolimus (data not shown). 3.4. Hematologic determinations 3.4.1. ASP0028 combination-treatment remarkably decreases the absolute numbers of lymphocytes Changes of the absolute number of lymphocytes are shown in Fig. 3A. In group-1, a slight reduction in lymphocyte counts was observed on day 7 after transplantation, and gradually returned to baseline level thereafter during follow-up period. In comparison with baseline values, animals in gorup-2 and -3 demonstrated a marked reduction (a maximum of around 76%) in lymphocyte counts, which was observed at day 0 and sustained till 90 day. As illustrated, this reduction from baseline value was more severe in group-3 than that in group-2, and reached statistical significance on day 28 (p = 0.01). Of note, the time course pattern of white blood cell counts was similar to that of lymphocyte counts in three groups (data not shown). 3.5. Lymphocyte phenotyping 3.5.1. ASP0028 combination-treatment significantly decreases CD3+ T lymphocytes Changes of circulating CD3+ cells are shown in Fig. 3B. CD3+ cells were significantly reduced in both combination-therapy groups of group-2 and -3, throughout the post-operative course, compared to that in group-1 (p b 0.05). Mean CD3+ cell counts in both combination-therapy groups were significantly decreased on day 0 by approximately 80%, from baseline values (p b 0.001). Comparing two combination-therapy groups, there were slight variations evident

Fig. 3. Changes of mean (±SD) numbers of peripheral lymphocytes (A) and lymphocyte subsets of CD3+ cells (B) in each group. Both (A) and (B) values in combination-therapy groups decreased significantly observed early on day 0, when compared to group-1 (*p b 0.05, **p b 0.01), except for (A) on day 28 in group-2 not reaching statistical significance. To compare two combination-therapy groups, both values only differed significantly on day 28 (#p b 0.05).

dependent on the ASP0028 dose-levels. The mean decrease by 81.40% and 69.89% from baseline values in group-3 verses group-2 reached statistical significance on days 28 (p b 0.05). 3.5.2. ASP0028 combination-treatment significantly decreases CD3+ CD4+, CD4+/naïve and central memory T lymphocytes, but not CD4+/effector memory T lymphocytes Changes of circulating CD3+ CD4+ cells are shown in Fig. 4A. After initiation of ASP0028, there was a remarkable reduction in CD3+ CD4+ cells for both combination-therapy groups, observed early on day 0 (Supplementary Fig. 2) and sustained throughout the post-operative course. By contrast, the number of CD3+ CD4+ cells in group-1 was significantly increased after initiation treatment. Consistently, the time courses of CD4+/naïve cells (Fig. 4B) and CD4+/central memory cells (Fig. 4C) in three groups were similar to that of total CD4+ cells. Thus, at a low-dose level of ASP0028 in group2, the profound lymphocyte depletion for total CD4+, CD4+/naïve and central memory cells was already achieved. Surprisingly, compared with baseline values, there were no appreciable changes observed for either the absolute number or percentage of circulating CD4+/effector memory cells in three groups (Fig. 4D, E and Supplementary Fig. 3). 3.5.3. ASP0028 combination-treatment decreases CD3+ CD8+, CD8+/naïve and central memory T lymphocytes, but has no clear effect on CD8+/effector memory T lymphocytes Changes of circulating CD3+ CD8+ cells are shown in Fig. 5A. In group-1, compared with baseline values, there was a mean decrease for CD3+ CD8+ cells by 42.81% on day 7 with a quick return to baseline

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Fig. 4. Changes of mean (±SD) numbers of CD4+ cells (A), CD4+/naive cells (B), CD4+/central memory cells (C), and numbers (D) and percentage (E) of CD4+/effector memory cells in each group. All values in combination-therapy groups decreased significantly observed early on day 0, when compared to group-1 (*p b 0.05, **p b 0.01), except for both (D) and (E), for which no significant changes were seen.

level on day 14, and a moderate increase of 180% on day 28. By contrast, in both combination-therapy groups, the numbers of CD3+ CD8+ cells were significantly decreased by 47.57% to 84.63% from baseline values, with a time course similar to that of CD3+ CD4+ cells. The mean decrease in CD8+ cell counts by 71.19% and 47.57% from baseline values in group-3 verses group-2 reached statistical significance on days 28 (p b 0.01). Similar to the behavior of CD4+/naïve and central memory cells, a marked drop from baseline values in both CD8+/naïve cells (Fig. 5B) and CD8+/central memory cells (Fig. 5C) was observed on day 0, without a recovery through all time-points in both combination-therapy groups. As illustrated, this drop was also comparable between two combination-therapy groups. Similar to the pattern of CD4+/effector memory cells, there were no clear changes on both the percentage and absolute numbers of circulating CD8+/effector memory subset observed in this study (data not shown).

3.5.4. ASP0028 combination-treatment remarkably decreases CD4+/Treg lymphocytes and shows a decreasing trend in B lymphocytes, but not NK cells Changes of circulating CD4+/Treg cells are shown in Fig. 6A. Similar to the behavior of total CD4+ cells, a remarkable decrease of CD4+/Treg cells from baseline values was observed in both combination-therapy groups throughout the time-course. By contrast, they were moderately increased in group-1. A trend toward reduced B cell numbers was seen in combination-therapy groups (Fig. 6B), however, which was not doserelated. In group-2, by day 0 there was an approximately 75% reduction in the absolute numbers of B cells compared with baseline values. This reduction persisted throughout day 56 in the follow-up period. In group-3, the numbers of B cells were only moderate decreased by 45% from baseline value on day 0, and reached a nadir on day 28, with a reduction of 82%. However, after day 56, the numbers of B cells in group-3 returned to near baseline level. Because the data were derived from only two available animals in this time-point, it thereby should be

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Fig. 6. Changes of mean (±SD) numbers of CD4+/Treg cells (A) and B cells (B) in each group. Values of (A) in both combination-therapy groups decreased significantly observed early on day 0, except for that of group-2 on 28 not reaching statistical significance, when compared to that in group-1 (*p b 0.05, **p b 0.01). A trend toward reduction of B cells (B) was seen in combination-therapy groups, reached significant levels on day 0 and day 7 in group-2 (#p b 0.01), and on day 14 and day 28 in group-3 (*p b 0.05, **p b 0.01) when compared to that in group-1. However, after day 56, the B cell counts in group-3 recovered to near baseline level.

Fig. 5. Changes of mean (± SD) numbers of CD8+ cells (A), CD8+/naive cells (B) and CD8+/central memory cells (C) in each group. All values of (A), (B) and (C) in both combination-therapy groups decreased early on day 0, and reached significant levels on most of time points, when compared to that in group-1 (*p b 0.05, **p b 0.01). To compare two combination-therapy groups, only values of (A) on day 28 differed significantly (#p b 0.05).

interpreted with caution. In addition, NK cells were unaffected after ASP0028-treatment (data not shown). 3.6. Pharmacokinetic studies PK parameters of ASP0028 are summarized in Table 1. Corresponding to the increase in ASP0028 from 0.6 to 1.2 mg/kg, mean ASP0028 trough concentration, Cmax and AUC(0–24) values were significantly enhanced from 36.09 to 84.21 ng/ml, from 66.62 to 167.17 ng/ml, and from 1062.89 to 2729.90 ng.h/ml, respectively (p b 0.001). The Cmax showed higher correlations with AUC(0–24) (r = 0.97) than the trough concentration (r = 0.90). There were no correlations found between either mean Cmax or median AUC(0–24) values and the allograft survival time (r b 0.1). The plasma concentrations of ASP0028 versus time curves on day 14 are shown in Fig. 7A, similar to that observed on day 28, 56 and 84 (data not shown). The plasma concentrations of ASP0028 increased modestly after dosing, reaching a first maximum at approximately 2–4 h,

followed by a shallow dip at 8 h and a second maximum at 12 h, thereafter decreased to trough level. On day 14, after the low- and high-dose of ASP0028 administration, the Cmax of ASP0028 ranged from 30.67 to 67.34 ng/ml, and from 91.36 to 176.68 ng/ml, respectively. The Tmax was highly variable, reached between 2–12 h after dosing, and appeared unrelated to dose. The mean trough concentrations of ASP0028 versus time-point curves are shown in Fig. 7B. ASP0028 trough levels significantly increased in a dose-dependent fashion. As illustrated, mean trough level-time point curves of ASP0028 were nearly parallel between two combination-therapy groups. Mean trough levels of the peak (on day 28) and the nadir (on 14 and 56) were essentially unaltered in two groups, suggesting a constant change in the rate and extent of ASP0028 absorption with time. There was a wide variation in trough levels of ASP0028 ranged from 8.88 to 82.87 ng/ml and from 26.02 to 198.48 ng/ml for low- and high-dose groups, respectively. The blood trough levels of tacrolimus was unaffected by co-administration with ASP0028 (data not shown). 4. Discussion Untreated naive control animals, which were not included in the present study for limiting the number of animals, constantly showed an MST of around 7 ± 1 days [29–31]. In this animal model, tacrolimus 1.0 mg/kg has been reported to achieve the renal allograft MST of 21 [25], 27 [32] and 28.5 days [29], respectively, in previous studies. These are similar to our present study (MST: 28 days) in animals received mono-tacrolimus 1.0 mg/kg treatment. When combined with ASP0028 at doses of 0.6 and 1.2 mg/kg, the allograft MST was significantly extended to 41 and 61.5 days, respectively. We documented for

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Fig. 7. (A) Mean (±SD) plasma ASP0028 concentrations versus time profile on day 14 in two combination-therapy groups. (B) Mean (±SD) plasma ASP0028 trough levels versus timepoints in two combination-therapy groups (*p b 0.05, **p b 0.01).

the first time that ASP0028 in combination with suboptimal-dose of tacrolimus shows a potential immunosuppressive effect in NHP renal transplantation model without obvious evidence of serious side-effects. Similar to that observed in NHP treated with FTY720 [17], a dramatic decrease in the number of peripheral lymphocytes was observed after ASP0028 administration (Fig. 3A). The observed decrease likely reflects the agonistic activity of ASP0028 on S1P1. The mechanism responsible for this effect is an accelerated homing and sequestration of peripheral blood lymphocytes into SLOs, resulting in a remarkable reduction of circulating lymphocytes [17]. The lymphocyte depletion was evident for both the CD4+ and CD8+ T cells. However, the reduction in CD4+ cells was more profound than that in CD8+ cells, which is in good agreement with previous studies with FTY720-treatment in NHPs and patients [17,33,34]. These results indicated that CD4+ cells are more susceptible to S1P1 agonistic activity of ASP0028, compared to CD8+ cells. This feature may be particularly advantageous in preventing transplant rejection, since CD4+ but not CD8+ subsets are considered to be major contributors to initiate allograft rejection [35].

Interestingly, both CD4+/ or CD8+/naive and central memory cells decreased to a greater extent after ASP0028-administration. However, ASP0028 did not have any measureable effect on the numbers of both CD4+/ or CD8+/effector memory cells. The present result supports the notion that lymphocyte trafficking between blood and SLOs is regulated and dependent on the expression of lymphocyte homing-receptors, CCR7 and CD62L, in response to their ligands of CCL21 and CCL19 within the lymph nodes [9,36–39]. Due to lack of the expression of such receptors, effector memory cells are unable to migrate to SLOs, consequently allowing for them to immediately accumulate in peripheral tissues and exert effector function [37]. Consistently, there is increasing evidence that FTY720 only modulates the sequestration of naive and central memory cells, but not effector memory cells into SLOs [6,7]. As a result, FTY720-treatment can not completely deplete circulating blood lymphocytes, and the remaining cells are considered to be effector memory T cells [40,41], which supports our present findings. Based on these observations, we presume that a S1P1 agonist alone may not sufficiently interfere with immune functions mediated by peripheral effector

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memory T cells. Since effector memory T cells are known to be a major cause of allograft rejection, we expect that ASP0028 is combinable with various immunosuppressants to enhance potency without toxicity. CD4+ CD25+ Treg cells comprise 5–10% of the total CD4+ population [42], also express the same pattern of S1P1 and are mediated by S1P in circulating blood [43]. On the other hand, according to expression profile of CD62L, CD4+/Treg cells can be divided into two subsets, CD62L+ and CD62− cells [42]. However, only CD62L+ subsets preferentially CCL19-driven migrate to SLOs [42]. As noted, in this study there was a dramatic decrease in the number of peripheral CD4+/Treg cells after ASP0028-treatment. Therefore, we presume that the majority of CD4+/Treg population are CD62L+ subsets, since lymphocyte homing and sequestration into SLOs is dependent on the expression of CD62L molecule. Regarding effect of FTY720 on CD4+/Treg cells, there were conflicting results from previous studies. For example, most researchers reported that CD4+/Treg cells were significantly enhanced not only in total number but also in functional activity under FTY720 treatment [44–47]. A possible explanation is that FTY720 could convert the conventional antigen-stimulated T cells to FoxP3+ Treg cells [47]. Conversely, Wolf et al. [48] found that FTY720 potentially impairs Treg cell activity via trapping them in inflammatory SLOs, and inhibits its expansion via the inhibition of IL-2-induced STAT-5 activation. Considering that Treg cells-mediated suppression of immune responses is a key element in inducing allograft tolerance, implication of such an inhibitory effect of ASP0028 on CD4+/Treg cells remains to be investigated with respect to the influence of emergence of allograft-rejection. Consistent with previous studies in FTY720 [49], NK cells are not affected by ASP0028-treatment. Although NK cells also express S1P1 [50], they appear not to react with S1P agonist. However, Johnson et al. [51] found that there was a marked decrease in the proportion of CCR7expresing subsets (CD56brighNK cells) of peripheral NK cells in MS patients under FTY720-treatment, indicating that these cells are likely redistributed into lymphoid organs. Because this CCR7+ subset is a minor circulating population (~ 10%) of NK cells, their redistribution may not obviously alter absolute numbers of peripheral NK cells. However, these CCR7-expressing NK cells present predominantly at sites of inflammation and are a major contributor to cytokine production [51]. These cells may be also important for clinical transplant. Therefore, its redistribution under ASP0028 treatment needs to be considered in further studies. Studies showed that effect of FTY720 was seen both on T and B lymphocytes, although the latter was less affected than CD4+ and CD8+ T lymphocytes [34]. Similarly, there was a trend suggestive of a moderate decrease in the absolute number of B cells in combination-therapy groups in our present study. It was previously reported that S1P1 is crucial for both T and B lymphocytes retention in SLOs [52,53]. However, some studies found that S1P3 but not S1P1 mediates B lymphocytes chemotaxis to S1P [8,54], suggesting that S1P3 may play an important role in S1P-mediated B cell egress. Unlike FTY720, which displays agonistic activity at all of S1P receptors, except S1P2 [4], ASP0028 affects S1P3activity at much higher concentration (unpublished data). Our finding showed that B cells in group-3 returned to the baseline level on day 56. Due to the extremely small sample size of only two animals in this time point, this result may not be reliable to represent true effect of ASP0028. However, whether less effect on S1P3 accounts for such a result in B cells remains to be investigated. The mean Cmax and the systemic exposure (AUC(0–24)) of ASP0028 showed a dose-dependent increase. Because there are only two dose groups designed in this study, the dose-linearity cannot be determined currently. Further investigation with more dose groups regarding doselinearity is warranted. Of note, the degree of lymphopenia showed only minor differences between low- and high-dose groups, especially for CD4+/or CD8+/naive and central memory, and CD4+/Treg cells. Thus, the pharmacodynamic (PD) response to the ASP0028 is unlikely predicted by PK profile. Similarly, in NHPs and humans with FTY720treatment, a poor correlation between extent of lymphodepletion and

FTY-exposure has been observed, although PK profile of FTY720 showed the linearity [21,34]. The mechanism is unknown yet. Some limitations of the study were the relatively small sample size, and only 4 animals were assigned in each group for PD assessments. After post-operative day 28, there were extremely small numbers of animals available for PD examination, which may not reliable to achieve significance. In addition, single-dose ASP0028 was not included in the study. Consequently, we could not answer the question whether tacrolimus has any effect on ASP0028 PK parameters. In addition, although S1P3 receptor is associated with bradycardia in mice [24], a recent study reported that BAF312, a S1P3-sparing S1P modulator, induces bradycardia in humans, indicating species-specific effects of S1P receptor on cardiac function [55]. In this study, heart rates were not monitored, and thus the effect of ASP0028 on transient asymptomatic bradycardia could not be ascertained. This should be taken into account in further studies. In conclusion, we show that ASP0028, a S1P1/S1P5-selective agonist, in combination with suboptimal-dose of tacrolimus significantly prolong renal allograft survival in NHPs with well tolerated safety. ASP0028 treatment-induced lymphocyte distribution appears to be specific for CD4+/ or CD8+/naïve and central memory cells, CD4+/Treg cells and to a lesser extent on B cells, but lacks selectivity for CD4+/ or CD8+/effector memory cells and NK cells. Implication of such a selective effect of ASP0028 on certain lymphocyte subsets remains to be established. Our data suggest that ASP0028 might represent an innovative adjuvant for transplant immunotherapy, and finally support its further investigation of use in CNI-sparing regimens to provide potent effects without increasing toxicities. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.trim.2017.01.002. Disclosure Authors of K. Koide, Y. Okada, K. Hanaoka, R. Yamamoto, J. Hirose and T. Morokata are employees of Astellas Pharma Inc. Other authors have no conflicts of interest regarding the publication of this paper. Acknowledgements This work was supported by Astellas Pharma Inc., Japan. References [1] J. Chun, E.J. Goetzl, T. Hla, Y. Igarashi, K.R. Lynch, W. Moolenaar, et al., International union of pharmacology. XXXIV. Lysophospholipid receptor nomenclature, Pharmacol. Rev. 54 (2002) 265–269. [2] K. Takabe, S.W. Paugh, S. Milstien, S. Spiegel, “Inside-out” signaling of sphingosine1-phosphate: therapeutic targets, Pharmacol. Rev. 60 (2008) 181–195. [3] M. Matloubian, C.G. Lo, G. Cinamon, M.J. Lesneski, Y. Xu, V. Brinkmann, et al., Lymphocyte egress from thymus and peripheral lymphoid organs is dependent on S1P receptor 1, Nature 427 (2004) 355–360. [4] V. Brinkmann, M.D. Davis, C.E. Heise, R. Albert, S. Cottens, R. Hof, et al., The immune modulator FTY720 targets sphingosine 1-phosphate receptors, J. Biol. Chem. 277 (2002) 21453–21457. [5] J.G. Cyster, Chemokines, sphingosine-1-phosphate, and cell migration in secondary lymphoid organs, Annu. Rev. Immunol. 23 (2005) 127–159. [6] M. Mehling, V. Brinkmann, J. Antel, A. Bar-Or, N. Goebels, C. Vedrine, et al., FTY720 therapy exerts differential effects on T cell subsets in multiple sclerosis, Neurology 71 (2008) 1261–1267. [7] M. Mehling, R. Lindberg, F. Raulf, J. Kuhle, C. Hess, L. Kappos, et al., Th17 central memory T cells are reduced by FTY720 in patients with multiple sclerosis, Neurology 75 (2010) 403–410. [8] R.K. Sinha, C. Park, I.Y. Hwang, M.D. Davis, J.H. Kehrl, B lymphocytes exit lymph nodes through cortical lymphatic sinusoids by a mechanism independent of sphingosine-1-phosphate-mediated chemotaxis, Immunity 30 (2009) 434–446. [9] K. Chiba, Y. Yanagawa, Y. Masubuchi, H. Kataoka, T. Kawaguchi, M. Ohtsuki, et al., FTY720, a novel immunosuppressant, induces sequestration of circulating mature lymphocytes by acceleration of lymphocyte homing in rats. I. FTY720 selectively decreases the number of circulating mature lymphocytes by acceleration of lymphocyte homing, J. Immunol. 160 (1998) 5037–5044. [10] J.A. Cohen, F. Barkhof, G. Comi, H.P. Hartung, B.O. Khatri, X. Montalban, et al., Oral fingolimod or intramuscular interferon for relapsing multiple sclerosis, N. Engl. J. Med. 362 (2010) 402–415.

H. Dun et al. / Transplant Immunology 40 (2017) 57–65 [11] G. Comi, P. O'Connor, X. Montalban, J. Antel, E.W. Radue, G. Karlsson, et al., Phase II study of oral fingolimod (FTY720) in multiple sclerosis: 3-year results, Mult. Scler. 16 (2010) 197–207. [12] L. Kappos, E.W. Radue, P. O'Connor, C. Polman, R. Hohlfeld, P. Calabresi, et al., A placebo-controlled trial of oral fingolimod in relapsing multiple sclerosis, N. Engl. J. Med. 362 (2010) 387–401. [13] V. Brinkmann, D.D. Pinschewer, L. Feng, S. Chen, FTY720: altered lymphocyte traffic results in allograft protection, Transplantation 72 (2001) 764–769. [14] S. Suzuki, S. Enosawa, T. Kakefuda, T. Shinomiya, M. Amari, S. Naoe, et al., A novel immunosuppressant, FTY720, with a unique mechanism of action, induces longterm graft acceptance in rat and dog allotransplantation, Transplantation 61 (1996) 200–205. [15] M.E. Wang, N. Tejpal, X. Qu, J. Yu, M. Okamoto, S.M. Stepkowski, et al., Immunosuppressive effects of FTY720 alone or in combination with cyclosporine and/or sirolimus, Transplantation 65 (1998) 899–905. [16] S.M. Stepkowski, M. Wang, X. Qu, J. Yu, M. Okamoto, N. Tejpal, et al., Synergistic interaction of FTY720 with cyclosporine or sirolimus to prolong heart allograft survival, Transplant. Proc. 30 (1998) 2214–2216. [17] H.J. Schuurman, K. Menninger, M. Audet, A. Kunkler, C. Maurer, C. Vedrine, et al., Oral efficacy of the new immunomodulator FTY720 in cynomolgus monkey kidney allotransplantation, given alone or in combination with cyclosporine or RAD, Transplantation 74 (2002) 951–960. [18] V. Brinkmann, A. Billich, T. Baumruker, P. Heining, R. Schmouder, G. Francis, et al., Fingolimod (FTY720): discovery and development of an oral drug to treat multiple sclerosis, Nat. Rev. Drug Discov. 9 (2010) 883–897. [19] M. Salvadori, K. Budde, B. Charpentier, J. Klempnauer, B. Nashan, L.M. Pallardo, et al., FTY720 versus MMF with cyclosporine in de novo renal transplantation: a 1-year, randomized controlled trial in Europe and Australasia, Am. J. Transplant. 6 (2006) 2912–2921. [20] H. Tedesco-Silva, M.D. Pescovitz, D. Cibrik, M.A. Rees, S. Mulgaonkar, B.D. Kahan, et al., Randomized controlled trial of FTY720 versus MMF in de novo renal transplantation, Transplantation 82 (2006) 1689–1697. [21] K. Budde, R.L. Schmouder, R. Brunkhorst, B. Nashan, P.W. Lücker, T. Mayer, et al., First human trial of FTY720, a novel immunomodulator, in stable renal transplant patients, J. Am. Soc. Nephrol. 13 (2002) 1073–1083. [22] H. Tedesco-Silva, G. Mourad, B.D. Kahan, J.G. Boira, W. Weimar, S. Mulgaonkar, et al., FTY720, a novel immunomodulator: efficacy and safety results from the first phase 2A study in de novo renal transplantation, Transplantation 79 (2005) 1553–1560. [23] L. Kappos, J. Antel, G. Comi, X. Montalban, P. O'Connor, C.H. Polman, et al., Oral fingolimod (FTY720) for relapsing multiple sclerosis, N. Engl. J. Med. 355 (2006) 1124–1140. [24] M.G. Sanna, J. Liao, E. Jo, C. Alfonso, M.Y. Ahn, M.S. Peterson, et al., Sphingosine 1phosphate (S1P) receptor subtypes S1P1 and S1P3, respectively, regulate lymphocyte recirculation and heart rate, J. Biol. Chem. 279 (2004) 13839–13848. [25] F. Kinugasa, I. Nagatomi, H. Ishikawa, T. Nakanishi, M. Maeda, J. Hirose, et al., Efficacy of oral treatment with tacrolimus in the renal transplant model in cynomolgus monkeys, J. Pharmacol. Sci. 108 (2008) 529–534. [26] H. Chen, J. Peng, H. Luo, M. Loubeau, X. Wan, D. Xu, et al., Compromised kidney graft rejection response in Vervet monkeys after withdrawal of immunosuppressants tacrolimus and sirolimus, Transplantation 69 (2000) 1555–1561. [27] S. Qi, D. Xu, J. Peng, M.D. Vu, J. Wu, I. Bekersky, et al., Effect of tacrolimus (FK506) and sirolimus (rapamycin) mono- and combination therapy in prolongation of renal allograft survival in the monkey, Transplantation 69 (2000) 1275–1283. [28] L.C. Racusen, K. Solez, R.B. Colvin, S.M. Bonsib, M.C. Castro, T. Cavallo, et al., The Banff 97 working classification of renal allograft pathology, Kidney Int. 55 (1999) 713–723. [29] L. Song, A. Ma, H. Dun, Y. Hu, L. Zeng, J. Bai, et al., Effects of ASKP1240 combined with tacrolimus or mycophenolate mofetil on renal allograft survival in cynomolgus monkeys, Transplantation 98 (2014) 267–276. [30] D.C. Borie, P.S. Changelian, M.J. Larson, M.S. Si, R. Paniagua, J.P. Higgins, et al., Immunosuppression by the JAK3 inhibitor CP-690,550 delays rejection and significantly prolongs kidney allograft survival in nonhuman primates, Transplantation 79 (2005) 791–801. [31] T. Aoyagi, K. Yamashita, T. Suzuki, M. Uno, R. Goto, M. Taniguchi, et al., A human anti-CD40 monoclonal antibody, 4D11, for kidney transplantation in cynomolgus monkeys: induction and maintenance therapy, Am. J. Transplant. 9 (2009) 1732–1741. [32] G. Chen, P.P. Luke, H. Yang, L. Visser, H. Sun, B. Garcia, et al., Anti-CD45RB monoclonal antibody prolongs renal allograft survival in cynomolgus monkeys, Am. J. Transplant. 7 (2007) 27–37.

65

[33] K. Budde, R.L. Schmouder, B. Nashan, R. Brunkhorst, P.W. Lücker, T. Mayer, et al., Pharmacodynamics of single doses of the novel immunosuppressant FTY720 in stable renal transplant patients, Am. J. Transplant. 3 (2003) 846–854. [34] V. Quesniaux, L. Fullard, H. Arendse, G. Davison, N. Markgraaff, R. Auer, et al., A novel immunosuppressant, FTY720, induces peripheral lymphodepletion of both T- and B cells and immunosuppression in baboons, Transpl. Immunol. 7 (1999) 149–157. [35] N.R. Krieger, D.P. Yin, C.G. Fathman, CD4+ but not CD8 + cells are essential for allorejection, J. Exp. Med. 184 (1996) 2013–2018. [36] E.C. Butcher, L.J. Picker, Lymphocyte homing and homeostasis, Science 272 (1996) 60–66. [37] F. Sallusto, D. Lenig, R. Förster, M. Lipp, A. Lanzavecchia, Two subsets of memory T lymphocytes with distinct homing potentials and effector functions, Nature 401 (1999) 708–712. [38] M.D. Gunn, S. Kyuwa, C. Tam, T. Kakiuchi, A. Matsuzawa, L.T. Williams, et al., Mice lacking expression of secondary lymphoid organ chemokine have defects in lymphocyte homing and dendritic cell localization, J. Exp. Med. 189 (1999) 451–460. [39] A.E. Denton, E.W. Roberts, M.A. Linterman, D.T. Fearon, Fibroblastic reticular cells of the lymph node are required for retention of resting but not activated CD8+ T cells, Proc. Natl. Acad. Sci. U. S. A. 111 (2014) 12139–12144. [40] V. Brinkmann, J.G. Cyster, T. Hla, FTY720: sphingosine 1-phosphate receptor-1 in the control of lymphocyte egress and endothelial barrier function, Am. J. Transplant. 4 (2004) 1019–1025. [41] M.A. Morris, D.R. Gibb, F. Picard, V. Brinkmann, M. Straume, K. Ley, Transient T cell accumulation in lymph nodes and sustained lymphopenia in mice treated with FTY720, Eur. J. Immunol. 35 (2005) 3570–3580. [42] S. Fu, A.C. Yopp, X. Mao, D. Chen, N. Zhang, D. Chen, et al., CD4+ CD25+ CD62+ Tregulatory cell subset has optimal suppressive and proliferative potential, Am. J. Transplant. 4 (2004) 65–78. [43] W. Wang, M.H. Graeler, E.J. Goetzl, Physiological sphingosine 1-phosphate requirement for optimal activity of mouse CD4+ regulatory T cells, FASEB J. 18 (2004) 1043–1045. [44] P.J. Zhou, H. Wang, G.H. Shi, X.H. Wang, Z.J. Shen, D. Xu, Immunomodulatory drug FTY720 induces regulatory CD4(+)CD25(+) T cells in vitro, Clin. Exp. Immunol. 157 (2009) 40–47. [45] E. Sawicka, G. Dubois, G. Jarai, M. Edwards, M. Thomas, A. Nicholls, et al., The sphingosine 1-phosphate receptor agonist FTY720 differentially affects the sequestration of CD4+/CD25 + T-regulatory cells and enhances their functional activity, J. Immunol. 175 (2005) 7973–7980. [46] C. Daniel, N. Sartory, N. Zahn, G. Geisslinger, H.H. Radeke, J.M. Stein, FTY720 ameliorates Th1-mediated colitis in mice by directly affecting the functional activity of CD4 + CD25+ regulatory T cells, J. Immunol. 178 (2007) 2458–2468. [47] S. Sehrawat, B.T. Rouse, Anti-inflammatory effects of FTY720 against viral-induced immunopathology: role of drug-induced conversion of T cells to become Foxp3+ regulators, J. Immunol. 180 (2008) 7636–7647. [48] A.M. Wolf, K. Eller, R. Zeiser, C. Dürr, U.V. Gerlach, M. Sixt, et al., The sphingosine 1phosphate receptor agonist FTY720 potently inhibits regulatory T cell proliferation in vitro and in vivo, J. Immunol. 183 (2009) 3751–3760. [49] L.M. Vaessen, N.M. van Besouw, W.M. Mol, J.N. Ijzermans, W. Weimar, FTY720 treatment of kidney transplant patients: a differential effect on B cells, naïve T cells, memory T cells and NK cells, Transpl. Immunol. 15 (2006) 281–288. [50] L. Kveberg, Y. Bryceson, M. Inngjerdingen, B. Rolstad, A.A. Maghazachi, Sphingosine 1 phosphate induces the chemotaxis of human natural killer cells. Role for heterotrimeric G proteins and phosphoinositide 3 kinases, Eur. J. Immunol. 32 (2002) 1856–1864. [51] T.A. Johnson, B.L. Evans, B.A. Durafourt, M. Blain, Y. Lapierre, A. Bar-Or, et al., Reduction of the peripheral blood CD56(bright) NK lymphocyte subset in FTY720-treated multiple sclerosis patients, J. Immunol. 187 (2011) 570–579. [52] G. Cinamon, M. Matloubian, M.J. Lesneski, Y. Xu, C. Low, T. Lu, et al., Sphingosine 1phosphate receptor 1 promotes B cell localization in the splenic marginal zone, Nat. Immunol. 5 (2004) 713–720. [53] L.R. Shiow, D.B. Rosen, N. Brdicková, Y. Xu, J. An, L.L. Lanier, et al., CD69 acts downstream of interferon-alpha/beta to inhibit S1P1 and lymphocyte egress from lymphoid organs, Nature 440 (2006) 540–544. [54] I. Girkontaite, V. Sakk, M. Wagner, T. Borggrefe, K. Tedford, J. Chun, et al., The sphingosine-1-phosphate (S1P) lysophospholipid receptor S1P3 regulates MAdCAM-1+ endothelial cells in splenic marginal sinus organization, J. Exp. Med. 200 (2004) 1491–1501. [55] P. Gergely, B. Nuesslein-Hildesheim, D. Guerini, V. Brinkmann, M. Traebert, C. Bruns, et al., The selective sphingosine 1-phosphate receptor modulator BAF312 redirects lymphocyte distribution and has species-specific effects on heart rate, Br. J. Pharmacol. 167 (2012) 1035–1047.