cyclosporine A treatment promotes graft survival and lowers the peripheral lymphocyte count in DA to Lewis heart and skin transplantation models

Transplant Immunology 8 Ž2001. 267᎐277

Combined FTY720rcyclosporine A treatment promotes graft survival and lowers the peripheral lymphocyte count in DA to Lewis heart and skin transplantation models Zariana Nikolova, Akiko Hof, Yves Baumlin, Robert P. Hof U No¨ artis Pharma Research, No¨ artis Pharma AG, Basel CH-4002, Switzerland Received 6 December 2000; accepted 5 January 2001

Abstract Objecti¨ e: The immunomodulator, FTY720, lowers the peripheral lymphocyte count ŽPLC. by inducing migration of circulating lymphocytes to secondary lymphoid organs. We investigated the efficacy of mono- vs. combined-FTY720rCsA therapy on graft survival ŽGS. and on lowering the PLC in a solid organ and a skin graft model, using strains with strong MHC disparity. Methods: Heterotopic cardiac or tail skin grafting was performed using the DA ŽRT1a . to Lewis ŽRT11 . rat strain combination. FTY720 was administered as a single daily dose by gavage alone or in combination with subcutaneously delivered CsA. PLC, body weight and drug concentrations were determined on day 7, 28, or the day of rejection. Main findings: In placebo-treated animals the heart and skin allografts rejected after 6 and 8 days. FTY720 delayed rejection of both the solid organ and skin grafts. The maximal effect was achieved at 1 mg kgy1 dayy1 FTY720, resulting in a median survival time ŽMST. of 14 days for both allotransplants comparable to the effect achieved by 1 mg kgy1 dayy1 CsA in both models. In the cardiac graft experiment with CsA co-administration, doses of 0.3 and 1 mgrkg were used. Under these conditions very small doses of FTY720 were effective in maintaining grafts throughout the treatment period. Adding higher FTY720 doses to the 1 mg kgy1 dayy1 CsA was needed to effectively extend the skin GS, e.g. 0.3 mg kgy1 dayy1 FTY720 prolonged GS from 13 to 47.5 days MST, i.e. well beyond the 28 day-treatment period. CsA did not influence the PLC at clinically relevant doses. FTY720 lowered the PLC significantly and dose-dependently, at doses lower than those needed for the prolongation of both cardiac and skin GS with FTY720 monotherapy. In rats with skin grafts the PLC was markedly lowered up to 1 mg kgy1 dayy1 FTY720, whereas, in the heart model, it was lowered up to 0.1 mg kgy1 dayy1. Independently of the graft type, within the combination regimens 0.3 mg kgy1 dayy1 FTY720 achieved a maximal PLC depletion. Conclusions: Combining FTY720 and CsA was very well tolerated with respect to weight gain and lack of any clinically detectable infections. In the strain combination used FTY720 monotherapy was less effective than previously reported in maintaining grafts. The two-drug regimens extended strikingly the GS for both models. However, the prolongation of the heart GS was smoothly dose-related with FTY720 doses ranging from 0.01 to 1 mg kgy1 dayy1, whereas, the skin graft prolongation was modest at doses up to 0.1 mg kgy1 dayy1 and remarkably enhanced at 0.3 and 1 mg kgy1 dayy1 FTY720. 䊚 2001 Elsevier Science B.V. All rights reserved. Keywords: Immunomodulator; Heterotopic cardiac and tail skin grafting; Peripheral lymphocyte count

Abbre¨ iations: CsA: cyclosporine A; D: day; GS: graft survival; MNC: mononuclear cells; MST: median survival time; N: number of animals; PLC: peripheral lymphocyte count; WBC: white blood cell count. U Corresponding author. Tel.: q41-61-324-28-91; fax: q41-61-324-95-84. 0966-3274r01r$ - see front matter 䊚 2001 Elsevier Science B.V. All rights reserved. PII: S 0 9 6 6 - 3 2 7 4 Ž 0 1 . 0 0 0 3 1 - 4

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Z. Nikolo¨ a et al. r Transplant Immunology 8 (2001) 267᎐277

1. Introduction The current strategy for achieving sufficient immunosuppression in transplantation relies on combining substances with distinct mechanisms of action to minimize the side effects of each therapeutic agent. Cardiac allografting is frequently used to evaluate the therapeutic efficacy of novel immunosuppressive compounds, while skin grafting due to the strong immunogenicity of skin, selects for the most efficacious drug combinations. We investigated the contribution of FTY720 added to low CsA doses on graft survival ŽGS. and on lowering the peripheral lymphocyte count ŽPLC. in a solid organ vs. a skin graft model, using rat strains with strong MHC disparity. The novel immunomodulator FTY720, Ž2-amino-2Ž2- 4-octylphenyl4 ethyl.-1,3-propanediol hydrochloride., lowers the PLC by inducing migration of circulating lymphocytes to lymph nodes and Peyer’s patches w1,2x. FTY720 modulates lymphocyte recirculation and suppresses the recruitment of inflammatory lymphocytes into grafts w2x. An effect of FTY720 on the G protein coupled receptor-dependent, chemokine-driven cell trafficking, has been proposed w3x. Therapeutic doses of FTY720 do not impair T- and B- cell activation, expansion or memory w3,4x. FTY720 extends the survival of skin, cardiac, renal and liver allografts in rodent, canine and non-human primate models w5᎐8x. Combining FTY720 and subtherapeutic CsA doses results in remarkable synergism on GS prolongation in a number of species w9᎐17x. FTY720 decreases dose-dependently the PLC within hours after administration, inhibiting T-cell recruitment into allo-inflammatory sites, while CsA suppresses the intragraft cytokine production w2,4x. FTY720 application until day 3᎐5 post-transplantation w7x, when T-cell infiltration and activation in grafts occurs, could inhibit the development of graft vasculopathy w18x. With its specific mode of action, FTY720 promotes both long-term GS and mediates graft vessel disease prevention w15,16x. Solid organ transplant patients undergoing long-term current immunosuppression are at high risk of developing post-transplantation lymphoproliferative disorders, emphasizing the need for alternative therapeutic combinations w19,20x. Unlike inhibitors of cytokine transcription Že.g. FK506., FTY720 at therapeutic doses does not impair lymphocyte activation and function, which significantly reduces the risk of acquiring infections and virus-related malignancies due to immunosuppression w3,4x. The synergy of FTY720 and CsA also permits a CsA dose reduction, especially relevant for patients with renal dysfunction. FTY720 displays a completely novel mechanism of action that has not been observed with other immunosuppressive agents and offers a new alternative for

combined therapy with CsA in order to broaden the therapeutic window of clinical immunosuppression.

2. Objectives For the clinical use of FTY720 in combination with other drugs many questions are still open. Using the DA to Lewis strain combination the present report extends the FTY720 interaction studies to the classical immunosuppressant CsA. We investigated the following: Ž1. does combining low doses of FTY720 with clinically relevant CsA doses, result in an adequate survival prolongation of a solid organ andror a skin graft between strains with strong MHC disparity; Ž2. are doses of FTY720 causing only modest PLC lowering therapeutically useful in combination with CsA; and Ž3. whether the PLC decrease could be a marker for the biological activity of FTY720, at least over the steep part of the dose-effect relationship.

3. Materials and methods 3.1. Animals Male rats from DA ŽRT1a . and Lewis ŽRT11 . strains, approximately 250 g body weight, were purchased from Harlan Olac, AD Zeist, The Netherlands. The animals had unrestricted access to food and water and were allowed to adapt to the local environment for at least 1 week before surgery. Handling and care corresponded to the Swiss federal law for animal protection. 3.2. Heterotopic heart and tail skin allotransplantations All surgical interventions were performed under isoflurane anaesthesia ŽAbbott, Cham, Switzerland. using a Leica Wild M690 ŽLeica AG, Glattbrugg. or Zeiss OPMIMDU ŽCarl Zeiss AG, Zurich ¨ . operating microscope. Heterotopic cardiac transplantation was performed as described by Ono and Lindsey w21x and was also reported previously, in detail w22x. DA ŽRT1a . tail skin was transplanted into Lewis ŽRT11 . rats except for one group of animals, that received isografts ŽLewis skin.. The skin of the donor was disinfected with betadine solution, diluted with isotonic NaCl to 5 mgrml iodine. An incision was made around the tail 2᎐3 cm from the base and the tip, followed by a ventral incision between the two circular ones. The skin was grasped with strong forceps, peeled off, immersed immediately into cold isotonic NaCl and cut into squares of approximately 1 = 1 cm. A full-thickness square of 1 = 1-cm skin of the recipient was excised with a sharp scalpel taking care to

Z. Nikolo¨ a et al. r Transplant Immunology 8 (2001) 267᎐277

spare the capillary bed and to avoid bleeding. The transplant was fixed in its position with 8᎐12 sutures of size 6r0, either polyamide ŽDafilon, Braun-SSC AG, Emmenbrucke, Switzerland., or Polyglactin ŽVicryl, ¨ Ethicon GMbH, Norderstedt.. The skin was then again disinfected with betadine solution and sprayed with acutol sprayplast ŽGaba AG, Therwil, Switzerland. to apply a protective layer. An Alzet osmotic minipump ŽAlza Corp., Palo Alto, CA. filled with CsA or Placebo was implanted subcutaneously on the back of the animal w16x. After the completion of surgery, the animals were kept under an infrared lamp until fully awake. Heart graft function was assessed daily by palpation for the ventricular contraction. Hearts possessing a barely or not palpable impulse were considered rejected. The condition of the skin allografts was monitored from day 5 to rejection. Grafts were considered rejected when 90% or more of the graft was altered in color and consistency. 3.3. Study design Rats with heart grafts were subjected to one of the following treatments: vehicle Ž hP., monotherapy with CsA 0.3, 1.0 and 3.0, mg kgy1 dayy1 Ž hC0.3, hC1, hC3.; and monotherapy with FTY720 0.01, 0.03, 0.1, 0.3, 1.0 and 3.0 mg kgy1 dayy1 Ž hF0.01, hF0.03 hF0.1, hF0.3, hF1, hF3.. Rats also underwent combination therapy: with CsA 0.3 mg kgy1 dayy1 plus FTY720 0.03, 0.1, 0.3, 1.0 mg kgy1 dayy1 Ž hC0.3F0.03, hC0.3F0.1, hC0.3F0.3, hC0.3F1.; or with CsA 1 mg kgy1 dayy1 plus FTY720 0.01, 0.03, 0.1, 0.3 and 1.0 mg kgy1 dayy1 Ž hC1F0.01, hC1F0.03, hC1F0.1, hC1F0.3, hC1F1.. Rats with skin grafts were subjected to the following treatments: vehicle Žplacebo, sP., monotherapy with CsA 0.3, 1.0, 3.0, 10 mg kgy1 dayy1 Ž sC0.3, sC1, sC3, sC10.; and monotherapy with FTY720 0.1, 0.3, 1.0 mg kgy1 dayy1 Ž sF0.1, sF0.3, sF1.. Further group of rats were subjected to the combination of 1 mg kgy1 dayy1 CsA plus FTY720 0.03, 0.1, 0.3 and 1.0 mg kgy1 dayy1 Ž sC1F0.03, sC1F0.1, sC1F0.3, sC1F1.. The body weight was recorded on the day of surgery, weekly and on the day of rejection. Blood samples for hematological analysis and for determination of CsA and FTY720 concentrations were gained on day 7, on the last day of treatment Ž28., or on the day of rejection. The animals were killed on the day of rejection. 3.4. Drug administration CsA was dissolved in the commercial Sandimmun IV solution and administered by osmotic minipumps Ž2ML2., which were implanted subcutaneously on the back of the animal and replaced every 2nd week as reported w16x.

269

FTY720 was dissolved in distilled pyrogen-free water adjusted to 2 mlrkg body weight and administered as a single oral dose by gavage. Treatment started from the day of transplantation and continued until rejection or for 28 days. Pyrogen-free water served as placebo for FTY720, minipumps filled with vehicle only as controls for CsA. 3.5. Determination of white blood cell count (WBC) On day 7, 28 or the day of rejection, blood was obtained for hematological monitoring on an Technicon HU 1E counter ŽBayer Diagnostics, Zurich ¨ . or on a .. Tail MICROS 60, ŽAXON LAB AG, Baden-Dattwil ¨ blood was taken 24 h after the previous dose at trough time. CsA and FTY720 concentrations were also determined from the same blood. The 1-week WBC- and drug-concentration determination were omitted for the skin model as we did not wish to disturb the healing process of the freshly grafted upon tail with obtaining tail blood. 3.6. Determination of CsA and FTY720 blood le¨ els CsA blood levels were determined using the commercial Incstar ‘Cyclo-Trac SP-Whole Blood Radioimmunoassay for Cyclosporine’ ŽIncstar Corporation, Stillwater, Minnesota. and FTY720 concentrations by chromatographyrtandem mass spectrometry, as previously described w16x. 3.7. Histology (cardiac allografts only) All rats were allowed to reach rejection before being killed. At autopsy, grafted and recipient hearts were removed, fixed in 4% buffered formalin for 24 h at 4⬚C and processed for paraffin embedding. Four-␮m-thick sections were cut from three different levels through the paraffin block. Slides were routinely stained with hematoxylin and eosin and elastica van Gieson. The histologic assessment was conducted by light microscopy ŽZeiss. in a blinded fashion, based on the classification of the International Society of Heart Transplantation w23x. Sections from heart grafts were examined for myocyte destruction, interstitial and perivascular mononuclear cell ŽMNC. infiltrates, also semi-qualitatively scored for the severity of vessel injury or intimal thickening Žmodified after w24x. as described w22x. 3.8. Statistical analysis Statistical evaluation was carried out with Systat Version 8, Systat, Inc., Evanston, IL, using ANOVA, followed by multiple comparisons according to the Bonferoni test. The paired t-test was used, where appropri-

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Z. Nikolo¨ a et al. r Transplant Immunology 8 (2001) 267᎐277

ate. Correlation between PLC and FTY720 plasma concentrations was calculated using the Spearman rank correlation. In all test procedures probabilities P- 0.05 were considered statistically significant.

4. Results 4.1. Body weight and fate of the animals FTY720 alone as well as in combination with CsA was very well tolerated. The rats of all groups gained weight and showed no grossly abnormal behavior. Since change in body weight of these young, growing animals is a sensitive indicator, we have used a group of rats transplanted with isograft ŽLewis to Lewis. skin as control for the weight gain following transplantation surgery yet in the absence of immunosuppression. The weekly body weight was not significantly different between any of the groups during the 4 weeks treatment period. The highest dose of CsA, 10 mg kgy1 dayy1 , caused irritation at the pump implantation site. No clinically detectable infections or spontaneous mortality occurred in any of the rats. All animals were killed according to the protocol. 4.2. Effects of placebo treatment and FTY720 monotherapy on graft sur¨ i¨ al In placebo-treated animals, rejection of heart and skin allografts occurred after 6 and 8 days ŽFig. 1.. The maximal efficacy of FTY720 monotherapy was reached at a dose of 1 mg kgy1 dayy1 , resulting in a median survival time ŽMST. of 14 days for both heart and skin grafts, An increase to 3 mg kgy1 daysy1 FTY720 did not result in additional heart GS prolongation. 4.3. Effects of CsA monotherapy on graft sur¨ i¨ al CsA monotherapy prolonged GS in a dose-dependent fashion ŽFig. 2.. The lower CsA dose, 0.3 mg kgy1 dayy1 , prolonged the MST of heart and skin grafts only by 1 day, while 1 mg kgy1 dayy1 CsA extended GS similarly to the 1 mgrkg dose of FTY720. In contrast to FTY720, increasing CsA to 3 mg kgy1 dayy1 delayed cardiac graft rejection to 48 d MST. The 3.0 mg kgy1 dayy1 CsA dose prolonged skin GS to only 17 days and 10 mg kgy1 dayy1 CsA to 39 days MST, all grafts survived beyond the treatment phase ŽFig. 2b.. 4.4. Effects of combined FTY720 and CsA treatment on graft sur¨ i¨ al CsA at doses of 0.3 and 1 mg kgy1 dayy1 had small effect on GS ŽFig. 2a.. Even at the lower CsA dose,

Fig. 1. Survival of rat heart and skin grafts, placebo treatment and FTY720 monotherapy, administered daily orally by gavage. Ža. Survival of heterotopic heart grafts under placebo treatment and FTY720 monotherapy at the doses 0.01, 0.03, 0.1, 0.3, 1.0 and 3.0 mg kgy1 dayy1 , n s 6 for all groups. Žb. Survival of tail skin grafts under Placebo treatment and FTY720 monotherapy at the doses of 0.1, 0.3 and 1.0 mg kgy1 dayy1 , n s 8 for all groups.

adding 0.03 mgrkg FTY720 prolonged the MST from 7 to 22 days. The next higher doses, 0.1 and 0.3 mg kgy1 dayy1 FTY720 prolonged MST to 32.5 and 41 days, respectively, 2r6 animals rejected under treatment. A dose increase to 1 mg kgy1 dayy1 resulted in no further benefits in terms of GS Ž38.5 days MST. ŽFig. 3a.. Adding FTY720 to 1 mg kgy1 dayy1 CsA ŽFig. 3b. yielded excellent treatment effects, even 0.01 mg kgy1 dayy1 FTY720 reached 30 days MST, a single animal rejected during treatment. The next higher FTY720 doses, 0.03 and 0.1 mg kgy1 dayy1 , resulted in 36.5 and 39 days MST, none of the animals rejected under treatment. The two further dose levels indicate, that in this experiment an optimal effect was obtained using the combination containing 0.3 mg kgy1 dayy1 FTY720. It achieved 53.5 days MST, while a dose increase to 1 mg kgy1 dayy1 brought no further benefits in terms of GS Ž47 days MST.. Since animals with skin allografts needed more im-

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peak and trough levels for the osmotic pump administration. The CsA concentrations were not influenced by the administration of the second drug in both graft types. There were no significant differences between 1 and 4 week values for FTY720 concentrations in rats with grafted hearts ŽPaired t-test.. FTY720 blood levels

Fig. 2. Survival of rat heart and skin allografts, CsA monotherapy, administered by subcutaneously implanted osmotic minipumps. Ža. Survival of heterotopic heart grafts with CsA monotherapy at the doses of 0.3, 1.0, 3.0 mg kgy1 dayy1 , n s 6 for all groups. Žb. Survival of tail skin grafts under Placebo treatment and CsA monotherapy at the doses of 0.3, 1.0, 3.0 and 10.0 mg kgy1 dayy1 , n s 8 for all groups.

munosuppression than animals with heterotopic heart grafts only 1 mg kgy1 dayy1 CsA was used in the combination groups. A group treated with CsA and the FTY720-vehicle served as control for the two-drug regimens. Combining the low FTY720 doses of 0.03 and 0.1 mg kgy1 dayy1 with CsA achieved modest MST of 12 and 13.5 days. Adding 0.3 and 1.0 mg kgy1 dayy1 FTY720 to the fixed CsA dose prolonged GS remarkably to 47.5 and 54.5 days MST. The skin grafts of all animals from the two higher combination dose groups survived well beyond the 28-day-treatment period ŽFig. 3c.. 4.5. FTY720 and Cyclosporine A blood le¨ els Since CsA was administered by osmotic minipumps the 1st week measurement represents the concentrations in the middle of the pumping cycle, the 4th week measurement the levels at the end of the cycle when the pumps are nearly empty ŽTable 1, cardiac graft experiment.. The measurements thus represent the

Fig. 3. Survival of rat heart and skin allografts, combined FTY720rCsA therapy.FTY720 was administered daily orally by gavage and CsA-by subcutaneously implanted osmotic minipumps. Ža. Survival of heterotopic heart grafts under combined treatment of FTY720 at the doses of 0.03, 0.1, 0.3 and 1.0 mgrkgrd and CsA 0.3 mg kgy1 dayy1 , n s 6 for all groups. Žb. Survival of heterotopic heart grafts under combined treatment of FTY720 at the doses of 0.01, 0.03, 0.1, 0.3 and 1.0 mg kgy1 dayy1 and CsA 1.0 mg kgy1 dayy1 , n s 6 for all groups. Žc. Survival of tail skin grafts under Placebo treatment, CsA 1.0 mgrkgrd mono- and combined-therapy with FTY720 at the doses of 0.03, 0.1, 0.3 and 1.0 mg kgy1 dayy1 , n s 8 for all groups.

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272 Table 1 Blood levels of FTY720 and CsAa,b,c Drugs Žmgrkgy1 dayy1 . CsA

Blood Concentrations Žngrml. FTY

MST days

Heart 0 0.3 1 3 0 0 0 0 0 0 03 03 0.3 0.3 1 1 1 1 1

0 0 0 0 0.01 0.03 0.1 0.3 1 3 0.03 0.1 0.3 1 0.01 0.03 0.1 0.3 1

6 7 14 48 6 6.5 7.5 8 14 13 22 32.5 41 38.5 30 36.5 39 53.5 47

Skin 0 0.3 1 3 10 0 0 0 1 1 1 1 0

0 0 0 0 0 0.1 0.3 1 0.03 0.1 0.3 1 0

8 9 13 17 39 9 10 14 12 13.5 47.5 54.5 ) 100

CsA at 1 week

CsA at 4 weeks

Mean " S.E.M.

n

0 102 " 8.4 349 " 30 1121 " 98 0 0 0 0 0 0 82 " 9.8 88 " 14 78 " 6.8 131 " 13 321 " 19 349 " 28 316 " 40 390 " 28 311 " 75

6 6 6 6 6 6 6 6 6 6 6 5 6 5 6 6 6 6 3

ŽIsograft.

Mean " S.E.M.

97 229 " 20 655 " 77

FTY720 at 1 week n

1 6 6

72 " 6.8 86 " 11 101 " 28 64 " 5.2 254 " 9.8 229 " 13 191 " 12 237 " 31 189 " 18

6 6 6 6 6 6 6 6 6

0 73.3" 7.9 355 " 36 907 " 85 3505 " 234 0 0 0 317 " 37 328 " 48 343 " 30 332 " 25 0

8 8 8 8 8 8 8 8 8 8 8 8 6

FTY720 at 4 weeks

Mean " S.E.M.

n

0 0 0 0 0.3" 0.05 0.9" 0.1 2.2" 0.4 5.4" 0.6 21 " 3.2 76 " 11 1.9" 0.1U 5.7" 0.3U 20 " 2.0U 28 " 5.4 0.4" 0.02 1.3" 0.2 3.5" 0.3 8.8" 1.0 33 " 3.4

6 6 6 6 6 6 6 5 6 6 6 6 6 5 6 6 6 6 6

Mean " S.E.M.

n

0 0 0

6 6 6

1.2 1.5" 0.5 6.0" 1.5 23 " 3.1 77 " 10 1.5" 0.04 5.6" 0.5U 14 " 2.6U 35 " 6.4 0.4" 0.1 0.7" 0.1 3.5" 0.3U 9.1" 0.7 35 " 6.3

1 3 5 5 6 6 6 6 6 6 6 6 6 6

0 0 0 0 0 1.4" 0.2 5.2" 0.4 73 " 8.2 1.3" 0.3 4.0" 0.8 22 " 3.2 63 " 5.1 0

8 8 8 8 8 8 8 8 8 8 8 8 6

a

DA heart or skin was transplanted into Lewis rats. CsA was administered subcutaneously by minipumps, FTY720-orally by gavage. Graft survival is shown as median survival time ŽMST., blood concentrations in ngrml. Drug concentrations indicated as ‘0’ represent values below the limit of detection. The 4-week values represent values gained at 4 weeks or at the last day of treatment. b Abbre¨ iations: S.E.M. standard error of the mean; n: number of animals. c Asterisks ŽU .: Differences from the corresponding FTY720 monotherapy group. The last group consists of rats with isograft skin transplantation ŽLewis ª Lewis..

showed an increase Žheart grafts. in several groups treated concomitantly with CsA. No clear-cut dependence on the CsA dose was observed. The higher levels did not lead to a stronger PLC depletion than the observed in the corresponding FTY720 monotherapy groups Žsee ‘Effects on lymphocyte count’.. 4.6. Effects on the peripheral lymphocyte count in rats with grafted hearts CsA at 3 mg kgy1 dayy1 dose had a minimal effect

on the PLC. Even the smallest dose of FTY720 monotherapy had an almost half-maximal effect on the PLC, 0.1 mg kgy1 dayy1 elicited a near maximal effect and the dose of 1 mg kgy1 dayy1 FTY720 lowered the PLC only slightly further ŽFig. 4a.. Under combination therapy with 0.3 mg kgy1 dayy1 CsA, 0.1 mg kgy1 dayy1 FTY720 caused a near maximal and 0.3 mg kgy1 dayy1 a maximal PLC depletion ŽFig. 4b.. When FTY720 was co-administered with 1 mg kgy1 dayy1 CsA, the dose of 0.01 mgrkg caused PLC depletion by approximately one third. As in the other experiments 0.1 mg kgy1 dayy1 FTY720 was associated

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273

centrations in some groups of the heart model significantly, did not enhance PLC depletion. This is all the more noteworthy, since CsA monotherapy also tended to lower the PLC. 4.7. Effects on the peripheral lymphocyte count in rats with skin allografts The 0.3, 1.0 and 3.0 mg kgy1 dayy1 CsA doses did not influence the PLC, while 10 mg kgy1 dayy1 CsA lowered the PLC compared to placebo-treatment ŽFig. 4c, Table 2.. The PLC was lowered significantly and dose-dependently by FTY720 monotherapy. The maximal effect was achieved in the highest dose group, sF1.0 ŽFig. 4c.. At 4 weeks after surgery, the PLC under the two highest FTY720 doses Ž sF0.3, sF1. and all combination regimens was significantly lowered compared to PLC under Placebo- and the lower CsA-treated groups Ž sC0.3, sC1, sC3.. The PLC at 4 weeks under combination treatment, sC1F0.03, sC1F0.1, sC1F0.3 and sC1F1, was significantly lowered in comparison to PLC of sC1 group. The PLC under the combination treatment of sC1F0.1 and sC1F0.3 group was significantly lower than the PLC under the corresponding FTY720 monotherapy groups ŽFig. 4c.. Thus, independently of the graft type, the 0.3 mg kgy1 dayy1 FTY720 combined with 0.3 or 1 mg kgy1 dayy1 CsA achieved a maximal PLC depletion. 4.8. Effects on WBC and granulocyte count

Fig. 4. Effects on the peripheral lymphocytes. Lymphocyte count shown in units of 1000 per ␮l blood. FTY720 administered daily orally by gavage, CsA delivered by subcutaneously implanted osmotic minipumps. Bars show standard error of the mean. Ža. PLC in rats with heart grafts after 1 week of treatment with vehicle, doses of FTY720 and CsA monotherapy, n s 6 for all groups. Žb. PLC in rats with heart grafts after 1 week of treatment with vehicle, FTY720 combined with 0.3 or 1 mg kgy1 dayy1 of CsA, Ž噛. or Žq., significant differences from 0.3 or 1 mg kgy1 dayy1 CsA monotherapy group. N s 6 for all groups. Žc. PLC in rats with skin grafts after treatment with vehicle, FTY720 monotherapy at doses of 0.1, 0.3 and 1.0 mgrkgrd, CsA monotherapy and combined treatment with FTY720 at doses of 0.03, 0.1, 0.3 and 1.0 mg kgy1 dayy1 with CsA. Pluses Žq.: significant differences from 1 mg kgy dayy CsA monotherapy group; Asterisks ŽU .: differences from the corresponding FTY720 monotherapy group; n s 8 for all treatment groups.

with a near maximal and 0.3 mg kgy1 dayy1 with a maximal PLC depletion ŽFig. 4b.. Interestingly, CsA co-administration, which increased the FTY720 con-

All treatments of the cardiac graft experiment tended to decrease granulocyte counts, significantly so in four groups Žsee Table 2.. Four weeks post-surgery all combination regimens in the skin allografting set of experiments Žexcept sC1F0.03. and the sF1 monotherapy group showed lower WBC than the Placebo-treated group. The combination regimens Žexcept sC1F0.03. had also significantly lower WBC than the sC1 group. Four weeks post-transplantation, the granulocyte count showed no significant difference between the treatment groups. The WBC differences were thus attributable to PLC differences Žsee ‘Effects on lymphocyte count’ above.. 4.9. Histology Even though all rats with cardiac allografts were allowed to reach rejection before being killed, the two-drug regimens limited myocyte damage and reduced interstitial and perivascular MNC infiltration

274

Table 2 Effects of FTY720 and CsA on white blood cell, granulocyte and lymphocyte counts a,b,c Drugs Žmgrkgy1 dayy1 . CsA

Peripheral lymphocyte, granulocyte and white blood cell counts FTY

MST days

0 0 0 0 0.01 0.03 0.1 0.3 1 3 0.03 0.1 0.3 1 0.01 0.03 0.1 0.3 1

6 7 14 48 6 6.5 7.5 8 14 13 22 32.5 41 38.5 30 36.5 39 53.5 47

Skin 0 0.3 1 3 10 0 0 0 1 1 1 1

0 0 0 0 0 0.1 0.3 1 0.03 0.1 0.3 1

8 9 13 17 39 9 10 14 12 13.5 47.5 54.5

PLC 4 weeks n

8.3" 0.3 9.6" 0.4 8.7" 0.2 7.9" 0.5 4.6" 0.7U 1.9" 0.1U 1.7" 0.1U 1.7" 0.2U 1.3" 0.1U 1.3" 0.1U 2.3" 0.1U 䢇 1.8" 0.1U 䢇 1.3" 0.3U 䢇 1.4" 0.2U 䢇 6.6" 0.8噛⽧ 2.5" 0.1U ⽧ 1.6" 0.1U ⽧ 1.3" 0.2U ⽧ 1.3" 0.1U ⽧

6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6

GC 1 week

Mean " S.E.M.

n

7.1 8.0" 0.2 5.4" 0.4

1 6 6

2.7 2.0" 0.3 2.0" 0.4 1.5" 0.1 2.1" 0.4 1.7" 0.2 䢇 1.5" 0.1䢇 1.0" 0.1䢇 1.1" 0.1䢇 4.1" 0.4 1.8" 0.2 1.3" 0.1 0.9" 0.1 1.2" 0.1

8.7" 0.7 10.0" 0.6 7.8" 0.7 9.2" 0.6 5.8" 0.3U 6.1" 0.4 5.0" 0.9U 3.6" 0.4U 5.0" 0.7U ⽧ 2.9" 0.5U 噛⽧ 2.1" 0.3U 噛⽧ 2.2" 0.4U ⽧

1 3 5 5 6 6 6 6 6 6 6 6 6 6

8 8 8 8 8 8 8 8 8 8 8 8

GC 4 weeks

Mean " S.E.M.

n

5.0" 0.7 4.7" 0.7 3.5" 0.3 3.6" 0.3 3.0" 0.3 3.0" 0.5 3.6" 0.4 3.0" 0.3 2.2" 0.2U 2.8" 0.4U 3.2" 0.3 3.7" 0.4 2.4" 0.3U 䢇 2.6" 0.2U 3.3" 0.6 2.8" 0.3U 3.2" 0.5 3.1" 0.3 2.9" 0.3

6 6 6 6 6 6 6 5 6 6 6 6 6 5 6 6 6 6 6

WBC 1week

Mean " S.E.M.

n

3.8 3.9" 0.5 4.4" 0.4

1 6 6

4.0 3.4" 0.5 2.6" 0.5 1.6" 0.1 2.8" 0.5 2.0" 0.3 3.1" 0.4 2.3" 0.4 2.5" 0.5 2.9" 0.6 2.2" 0.3 2.4" 0.3 1.9" 0.3 2.5" 0.3

1 3 5 5 6 6 6 6 6 6 6 6 6 6

1.4" 0.4 1.3" 0.3 1.5" 0.6 2.2" 0.3 1.0" 0.2 1.1" 0.2 1.2" 0.2 1.8" 0.4 2.1" 0.2 1.3" 0.2 1.0" 0.2 1.1" 0.3

8 8 8 8 8 8 8 8 8 8 8 8

WBC 4 weeks

Mean " S.E.M.

n

15 " 1.1 15 " 0.8 13 " 0.4 12 " 0.6 8.5" 1.1U 5.7" 0.7U 6.1" 0.6U 5.5" 0.5U 4.1 " 0.4U 4.9" 0.6U 6.2" 0.3U 6.5" 0.6U 4.3" 0.6U 4.6" 0.4U 11 " 0.8U 5.8" 0.2U 5.3 " 0.6U 4.8" 0.4U 4.6" 0.3U

6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6

Mean " S.E.M.

n

12 13 " 0.6 11 " 0.9

1 6 6

7.3 6.1" 0.7 5.2" 1.0 3.4" 0.2 5.5" 1.0 4.1" 0.6 5.1" 0.5 3.7" 0.5 3.9" 0.6 7.5" 0.7 4.4" 0.4 4.0" 0.3 3.1" 0.4 4.0" 0.3

1 3 5 5 6 6 6 6 6 6 6 6 6 6

12 " 1.6 13 " 1.0 11 " 1.0 13 " 0.9 8.0" 0.6 8.4" 0.8 7.6" 1.1 6.9" 0.9U 8.9" 0.7 5.5" 0.7U ⽧ 4.0" 0.5U ⽧ 4.4" 1.0U ⽧

8 8 8 8 8 8 8 8 8 8 8 8

a DA heart or skin was transplanted into Lewis rats. CsA was administered subcutaneously by minipumps, FTY720, orally by gavage. Graft survival is shown as median survival time ŽMST., White blood cell ŽWBC., peripheral lymphocyte ŽPLC. and granulocyte ŽGC. counts are shown in units of 1000 per ␮l blood. The 4-week values represent values gained at 4 weeks or at the last day of treatment. b Abbre¨ iations: N: number of animals; S.E.M. standard error of the mean. c Asterisks ŽU .: Differences from the control ŽPlacebo. group. Dots Ž 䢇 ., Diamonds Ž⽧. or Ž噛.: Differences from the group treated with 0.3 and 1 mgrkgrd CsA or FTY720 monotherapy, respectively.

Z. Nikolo¨ a et al. r Transplant Immunology 8 (2001) 267᎐277

Heart 0 0.3 1 3 20 0 0 0 0 0 0.3 0.3 0.3 0.3 1 1 1 1 1

PLC 1 week Mean " S.E.M.

Z. Nikolo¨ a et al. r Transplant Immunology 8 (2001) 267᎐277

compared with the inflammatory manifestations observed after CsA monotherapy. While CsA did not prevent coronary vasculitis or intravascular MNC accumulation, FTY720 within the wo-drug regimens had clear beneficial effects.

5. Discussion FTY720 synergises efficiently with immunosuppressive agents that directly suppress T cell activation, thus blocking the T cell response at different levels, i.e. induction᎐proliferation and emigration-homing to the periphery, resulting in very potent immunosuppression w4x. Several investigators reported synergism between CsA and FTY720 to prolong the survival of heart or skin allografts in rodents w9,11,13᎐16x. However, the scanty data on the pharmacokinetic properties of FTY720 andror of CsA administered via minipumps, on the PLC level and the limited dose-range in many of the reports prohibited a thorough evaluation of the therapeutic efficacy of the drugs. The present study includes detailed analysis of the compounds’ effects on the PLC, their blood levels and impact on survival of a solid organ vs. a skin allograft. Independently of the transplant system, CsA showed the characteristic dose-effect relationship observed in the DA to Lewis strain combination when administered by osmotic minipump. One milligram kilogramy1 dayy1 CsA was slightly more potent than 0.3 mg kgy1 dayy1 FTY720 and equipotent to 1 mg kgy1 dayy1 FTY720. The 3 mg kgy1 dayy1 CsA was markedly more potent than the highest FTY720 dose in the heart experiment Ž48 vs. 13 days MST. but only slightly more efficient then the highest FTY720 dose in the skin model. In a WKAH to ACI heart graft model, 0.1 and 1 mg kgy1 dayy1 FTY720 alone achieved 21 and 23 days MST, respectively, combining FTY720 with CsA prolonged further the GS w9x. Similarly, in a BUF to WF heart graft study, 0.1 and 1 mg kgy1 dayy1 FTY720 resulted in 18 and 29 days MST. Combining FTY720 0.1q 1 mg kgy1 dayy1 CsA lead to ) 100 days MST w14x. Accordingly, in a WKAH to F344 skin allograft model, 0.1 and 1 mg kgy1 dayy1 FTY720 resulted in 10.5 and 21 days MST w2x. Combining FTY72 0 0.1q 10 mg kgy1 dayy1 CsA resulted in ) 70 days MST. In the same skin graft model, 27 days MST was achieved with 10 mg kgy1 dayy1 FTY720 alone and ) 70 days for 2r8 rats when combined with 3 mg kgy1 dayy1 CsA w11x. These findings differ Žas far as this can be judged in the absence of blood levels. from those obtained in the current study. FTY720 delayed the allograft rejection

275

of heart and skin in the DA to Lewis strain combination only modestly. The maximal effect was achieved at 1 mg kgy1 dayy1 FTY720, resulting in 14 days MST for both models. CsA monotherapy showed higher efficacy, but at blood levels that are not tolerated in man. Adding 0.3 mg kgy1 dayy1 FTY720 to the 1 mg kgy1 dayy1 CsA dose prolonged the heart and skin GS to 53.5 and 47.5 days MST, respectively. The two-drug regimens enhanced significantly and remarkably the GS compared to the corresponding monotherapy groups. The maximal effect was achieved in hC0.3F0.3, hC1F0.3 as well as in sC1F0.3 and sC1F1 treatment groups. Our findings on GS under combined therapy differ from those obtained in other reports w2,9,11,13,14x. This discrepancy could be attributed to strain differences, as DA to Lewis strain combination represents a very strong mismatch. It appears that the strain combination used in this study responds relatively poorly to FTY720. This could be due to the fact that in Lewis rats the maximal PLC depleting efficacy of FTY720 was limited to a minimum of 1000 PLC per ␮l of blood or approximately 12᎐15% of the normal value ŽTable 2., which may not be enough to suppress the rejection process permanently in this strain combination. An important consideration for the use of FTY720 concerns the relationship between the PLC and the therapeutic efficacy. The PLC was lowered significantly and dose-dependently by FTY720, at doses lower than those needed for prolongation of cardiac and skin GS under FTY720 monotherapy. In the skin graft model, the PLC was lowered dose-dependently up to 1 mg kgy1 dayy1 FTY720 monotherapy. This differs from the heart transplantation, where PLC was dose-dependently lowered up to 0.1 mg kgy1 dayy1 . Independently of the graft type, the 0.3 mg kgy1 dayy1 FTY720 combined with 0.3 or 1 mg kgy1 dayy1 CsA achieved a maximal PLC depletion. The steep, log-linear part of the dose-effect relationship for the PLC decrease was observed between 0.01 and 0.1 mg kgy1 dayy1 in the cardiac graft model ŽFig. 4 a,b.. The correlation between PLC and plasma concentration was tight over this dose-range Ž r s y0.82, Fig. 5. when animals receiving CsA were excluded and somewhat less tight Ž r s y0.72. when all animals receiving FTY720 in this dose-range were included. In the skin model, PLC correlated well with FTY720 concentration for the combination regimens, Ž n s 32, r s y0.64., or less tight when all rats treated with FTY720 were included, Ž n s 56, r s y0.54.. A comparison of the FTY720 effect on PLC and GS indicates that these two dose-effect relationships are not superimposable. The heart experiment suggests that the GS plateaus between 0.3 and 3 mg kgy1 dayy1 , depending on the experimental situation. GS was strikingly enhanced by combining FTY720 and CsA in the

276

Z. Nikolo¨ a et al. r Transplant Immunology 8 (2001) 267᎐277

w3x

w4x

w5x

Fig. 5. Lymphocyte count Ž1000 cellsr␮l. as a function of the blood concentration of FTY720 Žngrml. over the steep part of the dose᎐response curve Ž0.01᎐0.1 mg kgy1 dayy1 . observed after 1 week of monotherapy in the heart model.

w6x

w7x

absence of a relevant enhancement of the PLC depletion. The dissociation between effect on PLC and GS appears larger for the heart than for the skin where the PLC curve was less steep and extended to 1 mg kgy1 dayy1 . The variability of the FTY720 effects on PLC were very small even on the steep part of the dose᎐response curve, smaller than the SEM of the FTY720 blood concentrations themselves. However, the differences in the effects of FTY720 on PLC between heart and skin transplantation models were large. Fully effective combination regimens were well tolerated in terms of wellbeing, weight gain and lack of any clinically detectable infections. This is in agreement with the efficacy and good tolerability of therapies involving FTY720 and sub-therapeutic CsA doses in various transplant models in rodents, dogs, non-human primates w9᎐17x and in a Phase I clinical trial w25x. FTY720, which alters circulation and homing of lymphocytes to peripheral lesions, without affecting the induction and expansion of immune responses in secondary lymphoid organs, offers a new alternative for combined therapy with CsA to broaden the therapeutic window of clinical immunosuppressants.

w8x

w9x

w10x

w11x

w12x

w13x

w14x

w15x

References

w1x Chiba K, Yanagawa Y, Masubuchi Y 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 1998;160:5037᎐5044. w2x Yanagawa Y, Sugahara K, Kataoka H, Kawaguchi T, Masubuchi Y, Chiba K. FTY720, a novel immunosuppressant, induces sequestration of circulating mature lymphocytes by acceleration of lymphocyte homing in rats. II. FTY720 prolongs skin

w16x

w17x

w18x

allograft survival by decreasing T cell infiltration into grafts but not cytokine production in vivo. J Immunol 1998;160:5493᎐5499. Brinkmann V, Pinschewer D, Chiba K, Feng L. FTY720: a novel transplantation drug that modulates lymphocyte traffic rather than activation. TIPS 2000;21:49᎐52. Pinschewer DD, Ochsenbein AF, Odermatt B, Brinkmann V, Hengartner H, Zinkernagel RM. FTY720 immunosuppression impairs effector T cell peripheral homing without affecting induction, expansion and memory. J Immunol 2000; 164:5761᎐5770. Suzuki S, Enosawa S, Kakefuda T et al. A novel immunosuppressant, FTY720, with a unique mechanism of action, induces long-term graft acceptance in rat and dog allotransplantation. Transplantation 1996;61:200᎐205. Yanagawa Y, Hoshino Y, Kataoka H et al. FTY720, a novel immunosuppressant, prolongs rat skin allograft survival by decreasing T-cell infiltration into grafts. Transplant Proc 1999;31:1227᎐1229. Yanagawa Y, Hoshino Y, Chiba K. The significance of timing of FTY720 administration on the immunosuppressive effect to prolong rat skin allograft survival. Int J Immunopharmacol 2000;22:597᎐602. Yamashita K, Nomura M, Omura T et al. Effects of a novel immunosuppressant, FTY720, on heart and liver transplantations in rats. Transplant Proc 1999;31:1178᎐1179. Hoshino Y, Suzuki C, Ohtsuki M, Masubuchi Y, Amano Y, Chiba K. FTY720, a novel immunosuppressant possessing unique mechanisms. II. Long-term graft survival induction in rat heterotropic cardiac allografts and synergistic effects in combination with cyclosporine A. Transplant Proc 1996;28:1060᎐1061. Troncoso P, Stepkowski SM, Wang M-E et al. Prophylaxis of acute renal allograft rejection using FTY720 in combination with subtherapeutic doses of cyclosporine. Transplantation 1999;67:145᎐151. Chiba K, Hoshino Y, Suzuki C et al. FTY720, a novel immunosuppressant possessing unique mechanisms. I. Prolongation of skin allograft survival and synergistic effects in combination with cyclosporine in rats. Transplantat Proc 1996;28:1056᎐1059. Kawaguchi T, Hoshino Y, Rahman F et al. FTY720, a novel immunosuppressant possessing unique mechanisms. III. Synergistic prolongation of canine renal allograft survival in combination with cyclosporine A. Transplant Proc. 1996; 28:1062᎐1063. Stepkowski SM, Wang M-E, Qu X et al. Synergistic interaction of FTY720 with cyclosporine or sirolimus to prolong heart allograft survival. Transplant Proc 1998;30:2214᎐2216. Wang M-E, Tejpal N, Qu X et al. Immunosuppressive effects of FTY720 alone or in combination with cyclosporine andror sirolimus. Transplantation 1998;65:899᎐905. Hwang M-W, Matsumori A, Furukawa Y et al. FTY720, a new immuno-suppressant, promotes long-term graft survival and inhibits the progression of graft coronary artery disease in a murine model of cardiac transplantation. Circulation 1999;100:1322᎐1329. Nikolova Z, Hof A, Rudin M, Baumlin Y, Kraus G, Hof RP. Prevention of graft vessel disease by com bined FTY720rCyclosporine A treatment in a rat carotid artery transplantation model. Transplantation 2000;69:2525᎐2530. Furukawa H, Suzuki T, Jin MB et al. Prolongation of canine liver allograft survival by a novel immunosuppressant, FTY720. Effect of monotherapy and combined treatment with conventional drugs. Transplantation 2000;69:235᎐241. Tori M, Kitagawa-Sakakida S, Li Z-Z et al. Initial T-cell activation required for transplant vasculopathy in retransplanted rat cardiac allografts. Transplantation 2000;70:737᎐746.

Z. Nikolo¨ a et al. r Transplant Immunology 8 (2001) 267᎐277 w19x Dotti G, Fiocchi R, Motta T et al. Epstein-barr virus-negative lymphoproliferative disorders in long-term survivors after heart, kidney, and liver transplant. Transplantation 2000;69:827᎐833. w20x Opelz G, Henderson R. Incidence of non-Hodgkin lymphoma in kidney and heart transplant recipients. Lancet 1993;342:1514᎐1516. w21x Ono K, Lindsey ES. Improved technique of heart transplantation in the rat. J Thorac Cardiovasc Surg 1969;57:225᎐229. w22x Nikolova Z, Hof A, Baumlin Y, Hof RP. The peripheral lymphocyte count predicts graft survival in DA to Lewis heterotopic heart transplantation treated with FTY720 and SDZ RAD. Transplant Immunol 2000;8:115᎐124.

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w23x Billingham ME, Cary NRB, Hammond ME et al. A working formulation for the standardization of nomenclature in the diagnosis of heart and lung rejection: heart rejection study group. J Heart Transplant 1990;9:587᎐593. w24x Adams DH, Tilney NL, Collins Jr. JJ, Karnovsky MJ. Experimental Graft Arteriosclerosis. I. The Lewis-to-F344 allograft model. Transplantation 1992;53:1115᎐1119. w25x Kahan BD, Chodoff L, Leichtman AB et al. Safety and pharmacodynamics of multiple doses of FTY720. Transplantation 2000;69:S132.