Cyclosporins: Lack of Correlation between Antischistosomal Properties and Inhibition of Cyclophilin Isomerase Activity

EXPERIMENTAL PARASITOLOGY ARTICLE NO.

90, 103–109 (1998)

PR984307

Cyclosporins: Lack of Correlation between Antischistosomal Properties and Inhibition of Cyclophilin Isomerase Activity

A. Khattab, L. Pica-Mattoccia, M. Q. Klinkert,1 R. Wenger,*,2 and D. Cioli Istituto di Biologia Cellulare, Consiglio Nazionale delle Ricerche, 43 Viale Marx, 00137 Rome, Italy; and *Novartis Pharma AG, Basel, Switzerland

Khattab, A., Pica-Mattoccia, L., Klinkert, M. Q., Wenger, R., and Cioli, D. 1998. Cyclosporins: Lack of correlation between antischistosomal properties and inhibition of cyclophilin isomerase activity. Experimental Parasitology 90, 103–109. The immunosuppressive fungal products cyclosporin A (CsA) and FK506 bind with high affinity to intracellular receptor proteins: cyclophilin (CYP) is one of the receptors for CsA and FK506-binding protein (FKBP) is one of the receptors for FK506. These proteins catalyze the in vitro isomerization from a cis to a trans conformation of peptidyl-prolyl bonds in oligopeptides. The relative importance of the peptidyl-prolyl cis-trans isomerase (PPIase) activity of CYP compared to FKBP in schistosomes is not known. Here, we examine the effects of CsA and FK506 and show that the former inhibits PPIase activity in schistosome extracts, whereas the latter does not. Since CsA is specific for the CYP protein, this result is indicative of the fact that the PPIase activity in the parasite is mostly attributable to CYP. The observation that CsA was significantly more effective than FK506 as an antischistosomal agent, both in vivo and in vitro raises the possibility that killing of schistosomes is caused by the inhibition of schistosome CYP PPIase. We compared a number of Cs analogs for their antischistosomal effects and for the inhibition of CYP PPIase, but were unable to find a correlation between the two properties. We therefore conclude that the lethal effect of CsA is not directly linked to the inhibition of the enzymatic activity of schistosome CYPs. q 1998 Academic Press Index Descriptors and Abbreviations: Schistosoma mansoni; cyclosporins; FK506; PPIase.

1

To whom correspondence should be addressed at Mo-Quen Klinkert Consiglio Nazionale delle Ricerche, Istituto di Biologia Cellulare, Viale Marx 43, 00137 Rome, Italy. Fax: 139-6-8273287. 2 Present address: Wenger Chemtech, CH-4125 Riehen, Switzerland.

0014-4894/98 $25.00 Copyright q 1998 by Academic Press All rights of reproduction in any form reserved.

INTRODUCTION

Cyclosporin A (CsA) is a neutral lipophilic cyclic peptide of fungal origin consisting of 11 amino acids and exhibiting different biological properties. Its best known activity is the immunosuppressive effect exerted through inhibition of immunocompetent T lymphocytes (reviewed in Borel et al. 1996). Unexpectedly, CsA also possesses antiparasitic activities against schistosomes, plasmodia, nematodes, and cestodes (Bueding et al. 1981; Chappell and Thompson 1988; reviewed in Bell et al. 1996). It is well documented that the antiparasitic activity is not related to the immunosuppressive properties, based on the existence of CsA analogs that are poor immunosuppressants but exert antiparasitic effects (Chappell et al. 1987), and on the fact that parasites can be killed in vitro, that is, in the absence of host factors (Brannan et al. 1989). In contrast to the vast amount of knowledge on the biological and clinical aspects of CsA immunosuppressive activity, the mechanism through which the antiparasitic effect is mediated still remains elusive. We have approached the problem by examining the molecular interactions of CsA with parasite constituents. Proteins that bind CsA with high affinity (cyclophilins, CYPs) are present in schistosomes as in all eukaryotic cells and can be thought of as intracellular receptors for CsA. The molecular and biochemical properties of schistosome CYPs have been previously reported (Argaet and Mitchell 1992; Klinkert et al. 1995, 1996, Kiang et al. 1996).

103

104 CYPs are enzymes with peptidyl-prolyl cis-trans isomerase (PPIase) activity that are capable of catalyzing the interconversion of peptide bonds on the amino side of proline residues that are in the cis conformation to the trans conformation (Fischer et al. 1989). The cis-trans isomerization of peptidyl-prolyl bonds is a relatively slow step in protein folding and PPIases are involved in the catalysis of this process. CsA binds into the proline-binding site of the PPIase to inhibit its activity. It could be imagined that the isomerase activity of cyclophilins is essential for schistosome survival and that its inhibition by CsA is the very cause of parasite death. In evaluating this hypothesis, however, one has to take into account the fact that another class of intracellular proteins is endowed with PPIase activity and, since these are not inhibited by CsA, they might in principle replace the function of CYPs in the presence of CsA. This second class of PPIases are the FK506-binding proteins (FKBPs), which, as the name suggests, bind an alternative immunosuppressive drug FK506 (Harding et al. 1989, Standaert et al. 1990). Not only are the molecular structures of the two drugs distinct from each other (Schreiber and Crabtree 1992), but the two protein families are not related at the amino acid level nor at the three dimensional level, even though they share the same catalytic activity and are uniquely and specifically inhibited by the respective immunosuppressive agent (Tradinh et al. 1992). The PPIase activity of the major FKBP member in humans, hFKBP12, is reported to be considerably lower than that of the abundant human CYP A present in the same cellular compartment (reviewed in Kay 1996). Since no equivalent information regarding the relative importance of CYPs versus FKBPs was available for schistosomes, we set out to clarify this point by determining the effects of the two inhibitors on schistosome PPIases and on schistosome survival. We report evidence indicating that FKBPs account for only a minor fraction of schistosome PPIase activity and that FK506 is not as effective as CsA as an antischistosomal agent. Since this leaves the cyclophilin PPIase inhibition as a viable hypothesis for explaining the antiparasitic action of CsA, we set out to comparatively evaluate the activity of a series of CsA analogs as antischistosomal agents and as PPIase inhibitors. In view of the finding that there is no correlation between the two activities for a number of compounds, we conclude that the antiparasitic effects of CsA are not primarily dependent on its inhibition of schistosome PPIases. MATERIALS AND METHODS Materials. CsA and CsA analogs were generous gifts of Novartis Pharma AG. Switzerland. FK506 was kindly supplied by Fujisawa

KHATTAB ET AL.

Pharmaceutical Co. Ltd., Osaka, Japan. Substrates N-succinyl-Ala-AlaPro-Phe-p-nitroanilide (succ-AAPF-pNA) and N-succinyl-Ala-LeuPro-Phe-p-nitroanilide (succ-ALPF-pNA) for the detection of PPIase activity were purchased from Bachem, Switzerland. Chymotrypsin and proteinase inhibitors were from Sigma, Germany. Schistosome homogenates. Adult S. mansoni extracts were prepared as described (Pica-Mattoccia et al. 1992) with the following modifications. Parasites were homogenized in 0.1 M Hepes (pH 7.4) containing 10% ethylene glycol and proteinase inhibitors (0.1 mM E64, 0.015 mM pepstatin A, and 0.1 U/ml aprotinin). The homogenate was centrifuged at 500g for 3 min and the supernatant was subjected to a further centrifugation step at 40,000g for 30 min. The resulting soluble fraction was used for enzymatic assays. Preparation of recombinant SmCYP B protein. Nonfused SmCYP B was synthesized in Escherichia coli and purified from other bacterial proteins by ammonium sulfate fractionation and exchange chromatography on a DEAE Fast Flow Sepharose column (Pharmacia), as described previously (Bugli et al. 1998). Purified protein was used in PPIase assays. Enzymatic activity measurements. PPIase activity was determined using synthetic peptides succ-AAPF-pNA and succ-ALPF-pNA as substrates (Kofron et al. 1991). A typical assay was carried out by incubating 100 nM of SmCYP B in PPIase buffer (50 mM Hepes, pH 8.0, 86 mM NaCl) in a 1-ml cuvette for 1 h on ice. Prior to starting the reaction, chymotrypsin (dissolved in 10 mM HCl), which cleaves the Ala-Pro or the Leu-Pro peptide bonds only when in the trans configuration, was added to a final concentration of 1.2 mM, followed by substrate (470 mM LiCl dissolved in trifluoroethanol) to a final concentration of 200 mM. Readings were taken at 88C using an HP 8452A spectrophotometer (390 nm). In the experiments where drugs are present, incubation of the enzyme in PPIase buffer was for 15 min on ice, after which drugs were added and the mixture was incubated for an additional hour on ice. IC50 values were determined using 100 nM SmCYP B and varying amounts of FK506, CsA, and CsA analogs (10 nM to 3 mM final concentrations from 10 mM stock solutions prepared in a 4:1 ratio of ethanol:Tween 80). In vivo antischistosomal effects of CsA and FK506. Equimolar amounts of CsA (18 mg/kg) and FK506 (12 mg/kg) were injected subcutaneously to eight mice per group on Days 3, 2, and 1 before infection with 110 cercariae of S. mansoni. Mice were perfused on Day 42 postinfection and the number of worms recovered was recorded. Eggs that were lodged in the liver were counted after an overnight digestion of liver tissues with 4% KOH. In vitro assays of schistosome killing. Cultures of 3-h schistosomula (obtained by mechanical transformation of cercariae) were set up in duplicate dishes containing approximately 200 organisms in 3 ml of Basch medium supplemented with 1% human serum (Basch 1981). A stock solution of CsA or FK506 in 4:1 ethanol:Tween 80 was added to the medium at the indicated final concentrations, with a maximum vehicle level of 0.25%. Schistosomula were left in contact with different concentrations of the drug for the whole duration of the experiment. Cultures of 7–10-week-old S. mansoni parasites (obtained by perfusion of infected mice) were maintained at 378C in an atmosphere of 5% CO2 in duplicate dishes containing 3 ml of Dulbecco modified Eagle’s medium and 10% newborn calf serum (10 parasites per dish). In initial experiments, we incubated male and female parasites in different dishes after their separation by gentle mechanical manipulation following exposure to 10 mg/ml of Neubutal. Female parasites

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were found to be less sensitive to the drugs, an observation made also by Liu and Chappell (1992). In subsequent experiments, we examined only male parasites, since they were more susceptible to the drugs and also more easily available from single-sex infections. Parasite viability was evaluated according to movements and morphological appearance under the light microscope (40X enlargement). Parasites were considered dead when they ceased in their movements, detached from the Petri dish, and had undergone certain morphological changes, like shortening, curling up, and thickening. In order to determine the antischistosomal activities of CsA and analogs, two protocols which gave essentially the same results were used. Schistosomes were exposed to 20 mM of the drug for 2 days and subsequently washed and further maintained in a drug-free medium. Alternatively, parasites were exposed continuously to 5 mM of the drugs. In both cases, the parasites were observed over a period of at least 2 weeks.

RESULTS We first sought to assess the contribution of CYPs in comparison with FKBPs in providing the PPIase activity of schistosomes, by measuring the reduction in activity caused by the specific inhibitors CsA and FK506, respectively. We assayed schistosome extracts for PPIase activity in the presence and absence of the drugs and found that most of the activity was inhibitable by CsA, while FK506 had practically no effect (Fig. 1). This led us to argue that most of the PPIase activity in the extract can be ascribed to CYP. This was true whether the substrate used in the assay was succAAPF-pNA (the most commonly used substrate for CYP assays) or succ-ALPF-pNA (the standard substrate for FKBP

assays) (Kofron et al. 1991). It appears from these results that FKBP activity is hardly detectable in schistosome extracts, despite the use of the recommended FKBP substrate. When drug effects were assayed on purified recombinant SmCYP B instead of schistosome extracts, using the two different substrates, CsA was also found to specifically inhibit PPIase activity. As expected, FK506 exerted no inhibitory effect on SmCYP B. The concentration of CsA required to inhibit 50% of the PPIase activity (IC50) of SmCYP B is 20 nM, while FK506 did not inhibit the enzyme at the concentrations tested (up to 3 mM). We then proceeded to examine the in vivo antischistosomal effects of CsA and FK506. Drugs were administered to mice before infection with S. mansoni cercariae. Infected mice were perfused and the number of worms as well as the number of eggs/liver were determined. As shown in Table I, the administration of CsA to S. mansoni-infected mice resulted in a significant reduction in the number of worms and in the number of eggs. On the contrary, FK506 had hardly any antischistosomal properties in vivo, as evidenced by the finding that both worm and egg recoveries from the FK506-treated mice were similar to those of the vehicletreated control. Similar results were obtained with schistosomula and adult schistosomes maintained in culture. As shown for adult schistosomes in Table II, CsA kills in a concentration-dependent manner and a lethal effect is observed on Day 11 at the lowest concentration tested (5 3 1026 M). Most notable is the observation that killing by FK506 is totally absent at this concentration. At a 10-fold higher dose, FK506 appears to be schistosomicidal, but this level is likely to have nonspecific toxic effects. The inferior efficacy of FK506 in comparison to CsA was also observed using schistosomulum cultures (data not shown). In agreement with previous reports (Liu and Chappell 1992), schistosomula were more susceptible to CsA than adult worms. The results of these experiments

TABLE I Effects of Cyclosporin A and FK506 Treatment on Schistosoma mansoni in Vivo

FIG. 1. PPIase activity of a S. mansoni extract using the synthetic substrate succ-AAPF-pNA. Curve (a) represents the thermal isomerization of the peptide in the absence of the enzyme; (b) the thermal isomerization of the substrate in the presence of the schistosome extract; (c) the activity of the extract preincubated with FK506 (shown for 1mM); and (d) inhibition of the activity of the extract after incubation with CsA (shown for 300 nM).

Control Cyclosporin A FK506

Number of worms 1 SD

Eggs/liver (3 103) 1 SD

23.3 6 8.4 13.6 6 7.1* 25.8 6 11.8†

10.33 6 3.2 2.8 6 2.5** 10.53 6 5.7†

Note. Each experiment was performed with eight mice per group. * P 5 0.02; ** P 5 0.002; †P 5 not significant.

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TABLE II Effects of Cyclosporin A and FK506 on Schistosomes in Vitro Days in culture

3

5

7

9

11

Medium control Vehicle control (0.25%) CsA: 5 3 1025 M 2 3 1025 M 1 3 1025 M 5 3 1026 M FK506: 5 3 1025 M 2 3 1025 M 1 3 1025 M 5 3 1026 M

0 0 15 0 0 0 0 0 0 0

0 0 70 20 0 0 0 0 0 0

0 0 100 60 40 0 10 0 0 0

0 0 100 100 100 0 65 0 0 0

0 0 100 100 100 40 100 0 0 0

Note. Antischistosomal activities are given in percentage. 0 5 no killing of parasites and 100 5 100% killing.

allow us to conclude that CsA displays antischistosomal activity, whereas FK506 is far less active. Taken together, all the above results strongly suggest that CYPs represent the major source of PPIase activity in schistosomes and that their inhibition by CsA might conceivably represent a major factor in schistosome death. We next examined a series of CsA analogs to see whether their relative ability to inhibit the PPIase activity of schistosome CYPs was correlated with their schistosomicidal properties. PPIase assays were performed using a purified recombinant schistosome CYP, SmCYP B. We chose to study SmCYP B based on previously published biochemical and molecular data, which demonstrated that SmCYP B is the predominant CYP class in schistosomes and accounts for most of the PPIase activity in an extract. Thus, not only was SmCYP B a more active PPIase than SmCYP A, but also mRNA of the former was more abundant in schistosomes (Bugli et al. 1998). In addition, we also showed that SmCYP B is the main target protein in schistosomes for CsA, since it was more sensitive than SmCYP A to CsA inhibition. Our choice of analogs takes into account previous observations on differences in their immunosuppressive activities (Wenger 1986), and also in their reported antimalarial activities (Bell et al. 1994). The concentrations of compound that inhibited 50% of PPIase activity (IC50) are reported in Table III. To assess the schistosomicidal properties of each compound we adopted an in vitro assay in which adult parasites were maintained in culture with 20 mM of the drug for 2 days prior to transfer to a drug-free medium and observed for morphological changes. The survival curves of worms treated with different CsA analogs are shown in Fig. 2. Based on their action when compared to CsA, we have classified drugs into three groups, namely those that have

TABLE III Inhibition of PPIase Activity by CsA and CsA Analogs Is Not Correlated with Schistosome Killing

Compound Group A SDZ 27-400 (CsA) SDZ 33-804 SDZ 34-271 SDZ 37-325 SDZ 205-549 SDZ 210-039 SDZ 214-103 SDZ 204-718 SDZ 207-256 Group B SDZ 209-313 SDZ 211-810 SDZ 220-384 Group C SDZ 37-839 SDZ 90-004 SDZ 208-032 SDZ 215-918 Group D PSC-833

PPIase (IC50 nM)

Schistosome killing

Immunosuppressive hCYP A activity binding

28 200 153 300 45 150 80 110 24

1 1 1 1 1 1 1 11 11

*** *** ** *** ** ** *** *** ***

yes yes yes yes yes yes yes yes yes

46 22 17

6 6 6

*** * *

yes yes yes

.1000 .1000 .1000 .1000

1 1 1 11

* * * *

no no no no

.1000

6

*

no

Note. Inhibition concentrations at 50% (IC50) of PPIase activity are divided into two large groups to simplify interpretation of our results, namely values below 300 nM are considered inhibitory, those above 1000 nM are noninhibitory. Data on schistosome survival are derived from Fig. 2 and tentatively classified into three classes; stronger than CsA activity (11); similar to CsA activity (1); no significant or weak killing (6). Immunosuppressive activity in pharmacological models previously used to test CsA (IL-2, MLR) is determined previously (Wenger 1986) as follows: no immunosuppressive activity or no significant activity (*); average activity (**); strong immunosuppressive activity (***).

stronger antischistosomal properties than CsA (designated 11; see also Table III), those that are similar in action to CsA (1), and a third group with no significant or weak antiparasitic activities (6). The majority of the derivatives tested is found in the second group. In parallel experiments, parasites were maintained in culture medium containing 5 mM of the drug. Results obtained under both conditions were similar. We made an overall comparison of their capacities to inhibit PPIase activity with their antischistosomal effects, which resulted in a classification of the compounds into four groups (Table III). Amongst the compounds tested, we have those that strongly inhibit SmCYP B PPIase activity and are clearly schistosomicidal (Group A). SmCYP B PPIase

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are consistent with previous findings that they do not bind human CYP A (Wenger 1986). From these results we conclude that the inhibition of PPIase activity is not sufficient for mediating the antiparasitic effects of cyclosporins.

DISCUSSION

FIG. 2. Survival of schistosomes in the presence of CsA and analogs. Parasites were exposed to 20 mM drug for 2 days and subsequently washed and further maintained in a drug-free medium. Results are presented as three graphs, compounds that are antiparasitically stronger than CsA (11), compounds that are similar in action to CsA (1), and compounds that exhibit no or lower killing than CsA (6). CsA is shown in all graphs for comparative purposes (dashed line).

at an enzyme concentration of 100 nM was inhibited with IC50 values ranging from 17 to 300 nM. Killing of schistosomes was stronger than or like CsA and assigned 11 or 1, respectively. A second group of derivatives are inhibitory for PPIase but lack antischistosomal activity of significance (6) (Group B) and vice versa, a third group are non-PPIase inhibitors (IC50 of . 1000 nM) but efficient schistosomicides (1 or 11) (Group C). Finally, we have one compound that does not inhibit PPIase activity (IC50 greater than 1000 nM) and is least active in killing parasites (6) (Group D). The high IC50 values of Cs derivatives in the two latter groups

It is known in the literature that bovine cyclophilin of the A subtype is the predominant PPIase present in the cytoplasm of mammalian cells (Bergsma et al. 1991; Price et al. 1991), and that the PPIase activity of FKBP is only 1–5% that of CYP A (Kay 1996). On the other hand, little is known about the PPIase activity of schistosomal FKBPs. Previous work published by Osman et al. (1995) has identified a protein Smp50, which exhibits 56% identity to p59, a heat-shock protein binding immunophilin and approximately 40% identity with various FKBPs. As an extension of these results, we would expect the PPIase activity attributed to FKBP to be distinguishable from that of a CYP PPIase in a homogenate of schistosome proteins, and as a consequence, CsA and FK506 were used to inhibit CYP and FKBP PPIase activities, respectively. The results of our enzymatic assays indicate that the bulk of PPIase activity in schistosomes is due to CYPs and that FKBPs have a minor role. An alternative explanation for the lack of FK506-inhibitable activity is that the assay conditions used are not optimal for FKBP PPIase; for example, FKBPs might be more active with physiological protein substrates rather than peptide substrates (Gething and Sambrook 1992). Also, one cannot rule out the possibility that FKBP is preferentially inactivated during extraction. Our conclusion, however, is indirectly reinforced by the observation that CsA is a schistosomicidal compound, while FK506 has significantly lower effects. The above observations would invoke the hypothesis that the lethal effects of CsA on schistosomes are due to its inhibition of parasite PPIase activities. To further explore this hypothesis, we took advantage of some reagents that were available in our laboratory. The cloning and expression of the major schistosome CYP cDNA in E. coli and its purification in sufficient quantities (Bugli et al. 1998) have enabled us to characterize its interaction with the cyclosporins and to investigate whether the inhibition of the PPIase activity of CYP is directly responsible for the action of cyclosporin-related drugs. Our data indicate that the mechanism of antischistosomal action is apparently not linked to the inhibition of PPIase activity, since a comparison of the antischistosomal and anti-PPIase properties of CsA analogs

108 tested revealed no consistent correlation. We found compounds that kill schistosomes with high efficiency while not inhibiting the PPIase activity, as well as compounds that are PPIase-inhibitory but have no identifiable effect on parasite survival. For the former compounds, it could still be argued that they are killing schistosomes, for example, as a result of nonspecific toxic effects. Conversely, for those compounds that do not kill, it could be that some of them have low uptake rates, are inactivated upon entry, or are metabolically transformed. We addressed the above possibilities by measuring the inhibition of PPIase activity of schistosome homogenates derived from parasites exposed for 2 days to analogs devoid of schistosomicidal activity (Group B) and subsequently washed. Our results showed a strong reduction of PPIase activity with respect to that of untreated controls, clearly demonstrating that the CsA analogs were present and active within the schistosome (data not shown). In addition, we noticed that PPIase activity was restored to control levels 1 day after drug removal in schistosomes that had been exposed to the chemical during the 2 previous days. Despite this return to normal levels of PPIase activity, worms exposed to CsA or to other schistosomicidal analogs would die a few days later, a phenomenon we interpret as further evidence for a lack of correlation between PPIase inhibition and parasite killing. A previous attempt to determine whether antimalarial and anti-isomerase activities were linked also revealed no such correlation in these two properties (Bell et al. 1994). We have also noted differences between the antischistosomal and antimalarial activities of some CsA analogs. For example, PSC-833 does not readily kill schistosomes, a finding which is in strong contrast with the results reported by Bell et al. (1994), in which the drug exhibited superior activity against Plasmodium falciparum. In this study, we have been able to identify one CsA analog (SDZ 220-384) that is an even more active antiparasitic reagent than CsA, but is inactive as an immunosuppressant. Three others have schistosomicidal activities similar to CsA, but with no significant immunosuppressive activity. The potency of some of these derivatives might be taken as a starting basis for the development of new antischistosomal compounds. In general, there is no correlation of parasite killing with immunosuppressive properties of the analogs nor with their capacities to bind human CYP (refer to Table III). Not unexpectedly, only those analogs that can bind to human CYP A are capable of inhibiting PPIase activity. CsA is a very tight binding inhibitor of the enzymatic activity of CYPs, and although the drug-protein complex lacks PPIase activity, it is known to be capable of associating

KHATTAB ET AL.

specifically with other cell proteins that are involved in processes of cell regulation such as signal transduction and cell cycle control (Walsh et al. 1992; Liu 1993; Bram et al. 1993). One such target of the CYP-CsA complex has been shown to be calcineurin, which is a serine/threonine phosphatase (Liu et al. 1991; Fruman 1992). Inhibition of the phosphatase activity is the basis of the immunosuppressive activity of CsA (Clipstone and Crabtree 1992), and it is quite possible that the CsA-CYP complex is mediating its lethality in schistosomes through the interaction with a critical cell component such as calcineurin. A more precise biochemical and cell biological analysis of the immunosuppressant-receptor protein and their downstream targets will therefore be very useful in understanding those interactions that are relevant to killing and for obtaining fundamental insight into the mode of action of the drugs.

ACKNOWLEDGMENTS

We thank Novartis Pharma AG, Switzerland, and Fujisawa Pharmaceutical Co. Ltd., Japan for their kind gifts of CsA/CsA analogs and FK506, respectively. This work received the partial support of the CMT - Italian Ministry for Foreign Affairs - DGSC.

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