The synergistic immunosuppressive potential of cyclosporin metabolite combinations

Int. J. hnmunopharmac., Vol. 14, No. 4, pp. 595-604, 1992. Printed in Great Britain.

0192-0561/92 $5.00 + .00 Pergamon Press Ltd. L~1992 International Society for Immunopharmacology.

THE SYNERGISTIC I M M U N O S U P P R E S S I V E P O T E N T I A L OF CYCLOSPORIN METABOLITE COMBINATIONS HEINFRIED H. RADEKE, *t UWE CHRISTIANS,* KARL-F. SEWING* a n d KLAUS RESCH* *Institut fiJr Molekularpharmakologie and *Institut fi.ir Allgemeine Pharmakologie, Medizinische Hochschule, Konstanty-Gutschow-Str. 8, D-3000 Hannover 61, F.R.G. (Received 20 July 1990 and in final form 12 November 1991)

-Out of the 29 cyclosporin (CS) metabolites defined so far seven representatives were isolated from the bile of liver grafted patients, purified by HPLC and characterized by FAB-MS and/or tH-NMR. These were used to determine the growth inhibitory effects on concanavalin A stimulated rat lymphocytes (LN). Metabolites diluted in culture medium at concentrations re-checked by HPLC at the respective assay time were added and proliferation determined by [~H]-thymidine incorporation after 48 h. A 50°70 growth inhibition of LN by single metabolites (AM) was achieved at the following concentrations (rag/l): CS: 0.023; primary metabolites AMI: 0.11; AMIc: 0.65; AM9: 1.05; secondary metaholites AM19: 1.02; AM4N9: 1.02; H355: 1.85; AM1A: 4.5. Although all metabolites were immunosuppressive at higher concentrations in vitro on a single metabolite level, only AMI with 20°7o of the activity of native CS seemed to play a role in vivo. However, when we tested the antiproliferative effects of double or triple metabolite combinations, we found a strong synergism not only of primary metabolites, but even with combinations including secondary metabolites. The concentration of the participating metabolites necessary to decrease LN growth by 50o70was far below the trough levels observed in vivo. Finally, to mimic to some extent the in vivo situation we determined the interaction of native CS with single metabolites or double combinations. In contrast to the clear synergism in the absence of CS the combinations of metabolites with native CS resulted in an additive growth inhibition. These results indicate an immunosuppressive potential of all metaholites tested and a clear synergism of metabolites in the absence of CS. Although up to double metabolite combinations did only additively enhance CS induced immunosuppression, the combination of 29 metabolites occurring in vivo might have significant immunosuppressive effects in situations where CS levels drop below active concentrations. Abstract

In a recent investigation K u n z e n d o r f , Brockm611er, J o c h i m s e n , R o o t s & O f f e r m a n n 0 9 8 9 ) described a direct c o r r e l a t i o n between a low incidence o f rejection episodes a n d high levels o f the cyclosporin metabolites, A M 9 a n d A M 1, in a g r o u p o f 78 kidney graft recipients. Since m e a s u r e m e n t of cyclosporin t r o u g h levels showed no differences between the n o n rejecting a n d the frequently rejecting patients, the observed discrepancy could be a t t r i b u t e d to the i m m u n o s u p p r e s s i v e activity o f cyclosporin m e t a b o lites. A l t h o u g h there is a n o n g o i n g discussion a b o u t the c o n t r i b u t i o n o f cyclosporin metabolites to i m m u n o s u p p r e s s i o n in vivo ( K a h a n , 1989), these results s u p p o r t recent in vitro studies o f m e t a b o l i t e effects o n h u m a n m o n o n u c l e a r cells (Wallemacq, Lh6est, L a t i n n e & De Bruy~re, 1989) a n d alloreactive T-cells g e n e r a t e d f r o m t r a n s p l a n t biopsies (Zeevi et al., 1988). Since CS metabolites are n o t easy to o b t a i n m o s t investigations have been suffering f r o m the limited

supply o f pure a n d fully characterized metabolites. Thus, until recently reports have considered only individual effects o f a few m a i n metabolites (AM9, A M 1 9 , M9, A M 4 N 9 , A M 1 , A M l c , A M 4 N ) (Ryffel et al., 1986; Schlitt, Christians, Wonigeit, Sewing & P i c h l m a y r , 1987). A l t h o u g h these c o m p o u n d s are a m o n g the p r i m a r y p r o d u c t s o f CS m e t a b o l i z a t i o n , the in vivo situation is by far m o r e complex. In o u r d e p a r t m e n t 29 distinct metabolites have been detected in the bile o f liver grafted patients, a n d in a d d i t i o n to the 12 metabolites described previously the r e m a i n i n g 17 new metabolites have been structurally analyzed by F A B - M S (Sewing et al., 1990). Therefore, patients have to deal not only with the activities o f a few m a i n metabolites, but with a c o m b i n a t i o n o f a n i m m e n s e n u m b e r o f CS m e t a b o l i c p r o d u c t s as classified by us (Sewing et al., 1990) a n d W a l l e m a c q et al. (1989), as second or t h i r d generation metabolites. Only two recent investigations h a d the a d v a n t a g e o f using fairly well

tAuthor to whom correspondence should be addressed. 595

596

H. H. RADEKEet al.

characterized metabolites. Although only a limited number of metabolite combinations of unidentified HPLC peaks (Wallemacq et al., 1989) or CS + AM1 (Zeevi et al., 1988) were used in these studies, the authors claimed that the metabolites contribute to the efficiacy of CS or that the effectiveness of CS might be highly dependent on the immunosuppressive activity of metabolite combinations. By continuous collection of bile from liver grafted patients we were able to prepare purified, well characterized metabolites in sufficient quantities. This allowed us to perform systematic investigations to determine the activity of single primary and secondary metabolites, combinations thereof a n d - getting close to in vivo c o n d i t i o n s - - t o test the immunosuppressive effect of metabolite combinations together with the parent drug CS.

EXPERIMENTAL PROCEDURES

Cyclosporin and metabolites

Cyclosporin (CS) was a kind gift of Sandoz, Basle, Switzerland. The stock solution of 2 m g / m l was kept at - 2 0 ° C in dimethylsulfoxide (DMSO). Reference CS metabolites (AM9, AM19, AM4N9, A M I , A M l c

and A M 1 A ) were a kind gift from Dr G. Maurer (Sandoz, Basle, Switzerland). CS metabolites were purified from human bile, which was obtained from liver grafted patients usually receiving 2 m g / m l CS twice daily. For surgical reasons their bile was deviated and collected through a T-drain placed in the bile duct. Out of the bile metabolites were prepared according to our extraction procedure described recently (Christians et al., 1988) with the following modifications. In brief, 500 ml human bile were extracted using 500 ml dichloromethane. The dichloromethane phase containing cyclosporin and its metabolites was evaporated and the residue was dissolved in 150 ml acetonitrile/water p H 3.0 = 50/50 v / v . The sample was then cleaned by adding 300 ml hexane and was extracted from the aqueous phase by 1 5 0 m l dichloromethane. Dichloromethane was evaporated, the residue dissolved in 2 ml acetonitrile/water pI-I 3.0 = 50/50 v / v , cleaned by 4 ml hexane and 250 tal were injected into the H P L C system. The metabolites were eluted from preparative columns (three serially linked 250 x 10 m m columns filled with 10 taM, 100 .A, RP8 material) by a concave gradient as described earlier (Christians et al., 1988). Fractions are manually collected and stored in acetonitrile/water pH 3.0 = 50/50 v / v at 4°C. After separation by r p - H P L C the resulting peaks were pooled and the structures of the megabolites were

Table 1. Summary of metabolite concentrations (mg/1) resulting in a 50% inhibition of proliferation (lcs0) of Sprague Dawley rat lymphocytes (assay conditions see Experimental Procedures) compared with the chemical structure of the metabolites and the trough-level in blood of patients with liver transplantation Structure Trough level human blood

IC50 rat LN

AA9 R

AAI RI

AA3

CS

0.175 + 0.026

0.023 + 0.002

H

CH 3

CH 3

Primary metabolites AM1 (H370) AMlc (H400) AM9 (H390)

0.157 + 0.027 0.014 __.0.005 0.053 __.0.014

0.11 0.65 1.05

H H OH

CH2OH CH2OH CH3

CH~

0.152 _+ 0.029 0.021 + 0.006

1.02 1.02 1.85 4.50

OH CH:OH CH3 hydroxylation/N-demet hylation hydroxylated (both at AA1) H COOH CH3

Secondary metabolites AM19 (H250) AM4N9 (H320) -(H355) AMIA (H350)

0.022 _ 0.006

R2

CH3*

CH3

*AAI cyclization. n = 18; data from Christians et al. (1988). The amino acid (AA) residues (R) are numbered according to Sewing et al. (1990). The nomenclature of the metabolites proposed by the Consensus Conference in Hawks's Cay, U.S.A., June 1990 [Transplant. Proc., 22, 1357 - 1361 (1990)] and in parentheses according to Christians et al. (1988), was used.

Immunosuppression by Cyclosporin Metabolites

100,

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0= 80.

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597

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10-

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0.1O0

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. . . . . . . .

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~ ,

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[rag/L]

[rag/L]

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A

120 t tlo

C

° o

•

0

1 0 0

' •

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,

0.010

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,

0.010

. . . . . . . .

,

0.100

. . . . . . . . . . . . . . . . .

1.000

,

10.000

[mg/L]

Fig. 1. Antiproliferative effects of CS and single metabolites. The ordinate gives the percent of proliferation of LN at the respective metabolite concentration compared with control. (A) Lymph node cells (LN) or Sprague Dawley spleen cells stimulated with 1 ~g/ml Con A were incubated with native CS for 48 h. Mean _+ S.D. of quadruplicates of a representative experiments out of a series of five and three, respectively, are shown. (B) The activities of the metabolite AM1 was determined in three independent experiments: bile from different patients, independent HPLC-purification and HPLCreassessment and different LN preparations. (C) Antiproliferative effects of the primary metabolites AM9, AMI, AM lc. Data points represent the mean of quadruplicates from two (AM9, AM 1c) or three (AM 1) independent assays, respectively. (D) The immunosuppressive effects of the secondary metabolites AM19, AM4N9, AMIA and H355 were determined as described in (1C).

determined and c o n f i r m e d by H - N M R and F A B - M S (Sewing et al., 1990). Since the Consensus C o n f e r e n c e in H a w k s ' s Cay, U.S.A., June 1990 [Transplant. Proc., 22, 1 3 5 7 - 1 3 6 1 (1990)], a new cyclosporin metabolite n o m e n c l a t u r e has been introduced. To allow

c o m p a r i s o n o f the " o l d " n o m e n c l a t u r e (Maurer, Loosli, Schreier & Keller, 1984; Christians et al., 1988) with the new " C o n s e n s u s N o m e n c l a t u r e " we like to give a brief translation (old = new): M1 = A M 9 ; M8 = A M I 9 ; M13 = AM4N9; M17 = A M I ; M18 = A M l c ; M21 = A M 4 N ,

598

H. H. RADEKE et al.

M25 = A M 1 4 N , M26 = AMlc9; M203-218 = AMIA. The new n o m e n c l a t u r e describes metabolic alterations o f the 11 a m i n o acids o f CS by special suffices following the a m i n o acid number: no suffix = oxidation; N = Nd e s m e t h y l a t i o n ; c = cyclization; A = oxidation to a n acid. A n a d d i t i o n a l secondary metabolite defined recently by us is indicated by a n H-labeled n u m b e r (H-355; for structure see Table I a n d Christians et al., 1988).

H P L C purified a n d lyophilized cyclosporin a n d cyclosporin metabolites were dissolved in 5/al D M S O a n d diluted in culture m e d i u m . D M S O never exceeded a c o n c e n t r a t i o n o f 0 . 1 6 % . To control the purity a n d the precise c o n c e n t r a t i o n of the c o m p o u n d s the highest a n d lowest c o n c e n t r a t i o n s o f the serial dilutions were re-determined by H P L C at the time o f the proliferation assay. This b e c a m e necessary as the recovery o f lyophilized metabolites in the final D M S O / m e d i u m solution varied extremely. One h u n d r e d microliters per well o f the C o n A cell suspension were pipetted into a 96-well plate a n d the test c o m p o u n d s were a d d e d to give a final volume o f 200/al. Cells were g r o w n for 44 h a n d for an a d d i t i o n a l 4 h with 0.5/aCi [3H]t h y m i d i n e / w e l l . The i n c u b a t i o n s were stopped by deep freezing, thawed, sonified a n d subsequently the cells were harvested with an a u t o m a t i c cell harvester (Titertek). Radioactivity was d e t e r m i n e d by liquid scintillation counting.

L y m p h o c y t e preparation

Rat l y m p h n o d e cells (LN) were p r e p a r e d f r o m 4 to 8 week old Sprague Dawley rats using inguinal, axillar, m a n d i b u l a r a n d mesenteric l y m p h n o d e s . Isolated l y m p h nodes were gently dissociated with a P o t t e r h o m o g e n i z e r , passed t h r o u g h a nylon filter a n d w a s h e d twice with culture m e d i u m . Sprague Dawley rat spleen cells were p r e p a r e d in a similar way. T h e culture m e d i u m used for the experiments consisted o f R P M I , s u p p l e m e n t e d with 2 m M glutamine, 100 U / m l penicillin, 100 tag/ml streptomycin, 10/0 non-essential a m i n o acids a n d 5 % fetal calf s e r u m (Gibco).

Statistics and validation o f synergism

Each point o f CS a n d single metabolite activity represents the m e a n o f four d e t e r m i n a t i o n s , respectively. In Fig. IB the d a t a points were raised in three completely i n d e p e n d e n t experiments, m e a n i n g t h a t the bile was f r o m different patients, the extraction procedures were f r o m different dates, as well as the re-assessment of the final c o n c e n t r a t i o n s by H P L C . C o n c e n t r a t i o n response curves calculated with the best curve fit are depicted. In all the r e m a i n i n g figures the d a t a points represent the m e a n

A s s a y procedures

Freshly p r e p a r e d L N (or in initial experiments spleen cells) were a d j u s t e d to a c o n c e n t r a t i o n of 4 X 1 0 6 cells/ml in culture medium and c o n c a n a v a l i n A ( C o n A ) was a d d e d (except the m e d i u m control) to give a final c o n c e n t r a t i o n o f 1 tag/ml, which resulted in o p t i m a l proliferation.

Table 2. Validation of synergistic CS metabolite activities Metabolite

Synergy index*

combinations

A

B

C

D

AM1 + A M 9

0.28 0.02

0.42 0.03

0.46 0.07

0.68 0.12

0.75 0.14

0.82 0.20

0.95 0.14

1.29 0.23

AMI + AM9 + AMlc AM4N9 + AMIA + H355

0.16 0.06

0.18 0.12

~ 0.23 0.22

CS CS CS CS

0.99 1.26 0.98 0.53

1.13 1.13 0.69 0.61

0.95 0.94 0.55 0.54

AMlc + AM4N9 AM4N9 + H355 AM1A + H355

+ + + +

AM1 AM1A AM1 + AM9 AM4N9 + H355

Referring to figure

E

F

G

~ 0.5 ~ 0.11

~ 0.5 ~ 0.27

< 0.75 ~ 0.47

1.34 0.37

0.72 0.60

< 0.85 1.06

2B 2C 2D

~ 0.26 0.44

~ 0.46 0.64

< 0.61 0.81

< 0.98 < 1.29

3A 3B

1.23 0.71 0.66 0.51

1.56 0.61 0.57 0.45

1.77 0.54 0.51 0.48

3.32 0.64 0.46 0.53

4A 4B 4C 4D

2A

*The indices of synergy were calculated as the "sum of fractions" (Berenbaum, 1977; see Experimental Procedures) for each combination of metabolite concentrations as depicted by "a, b , . . . g" throughout Figs 2A to 4D. Values < 1, = 1 or > 1 mean synergism, additivism or antagonism, respectively. Due to the experimental variation a clear synergism was considered, if the index was < 0.5 (indicated by bold, italic numbers).

Immunosuppression by Cyclosporin Metabolites

A

599

100SO

oC so

i:

}'1 7O

~

SO.

~• E

SO,

g

c --

:S i z

SO,

~

20

SO SO

I .=. IR

4O & - AM4Ng

3O

0

o

combination

20 10

[mgp.]

0 NIl9 : o.~ AM1 : 0.02

a

c

so

•u

50,

0.~ 0.04

b

O.U~ 0.00~

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o.3.1:1 o.1114 0.103 0.3,?,s

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1.328 0.18

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o.o~ A M 4 N g : 0.004

g

•

[,,s/t]

0

2.6Sa 1.3

o

o.ot O.Oll O.O~g 0.017

b

c

o,oH 0.034

o.o7o 0.0(i8

o.lu 0.135

d

•

f

g

':t

}'1

o.31 0.27

D

:t 50

E 4~ z

10 ¸

10,

[ms/q

0

0

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:

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o.~0

0.017

0.034

0.000

0,135

0.27

AM1A :

o.mm o.~37

o.27s

o.s4o

H355 :

o.o7|

o.1~

o.313

O.il2ib

1.251

2.503

5.005

H355 :

o.o711 o . l u

o.$13

o.12s

b

c

d

•

f

~14~

a

g

o

b

c

d

1.0it

•

l,q/t]

2.11N~ 4 . ~ 5

f

g

Fig. 2. Antiproliferative effect of double CS metabolite combinations on LN. The upper regression curves of the following figures refer to the growth inhibition obtained with single metabolites. The X-axis shows the concentrations of the respective metabolites contributing to the combined effect and/or when given alone. In addition a - g refer to the calculation of synergism (see Table 2). Data points for the combinations are connected by the best fitting regression curves (n = 8). A combination of the primary metabolites AM9 + AM1 was added to Con A-stimulated LN for 48 h and compared with the activity of the metabolites alone (A). Under similar conditions (B), (C) and (D) show the antiproliferative effects of a combination of the metabolites AM4N9 + AMlc, the secondary metabolites AM4N9 + H355 and AM1A + H355, respectively. of two independent experiments (four d e t e r m i n a t i o n s , respectively) a n d for these points the best fitting regression curve is shown. T h e m e a n standard d e v i a t i o n (S.D.) o f the four-fold d e t e r m i n a t i o n s t h r o u g h o u t the e x p e r i m e n t a l series (n > 300) was 10.89 _+ 6.22 (not shown). The synergistic effects o f CS m e t a b o l i t e c o m b i n a t i o n s were calculated o n the basis o f a n algebraic m e t h o d p u b l i s h e d by B e r e n b a u m (1977). This m e t h o d is the m a t h e m a t i c a l surrogate o f the

well-known geometric description of drug interactions by isoboles. Essentially, the synergy indices s h o w n in T a b l e 2 were calculated with the following f o r m u l a , which determines the relative p a r t i c i p a t i o n o f drug A , B . . . . X in c o m b i n a t i o n s : Dose o f A Dose o f B Dose o f X + + ... Ao Be Xe < l for synergy = l for additivism > 1 for a n t a g o n i s m .

H. H. RADEKEet al.

600

Doses of A, B , . . . X were related to "equieffective" doses (A0, Be. . . . Xe, respectively) leading to the same effect as the combination, when A, B . . . . X were given alone. This equation becomes " 1 " , if A and B act additively, smaller than " 1 " in case of synergism and greater " 1 " , if A and B act antagonistically.

RESULTS

The concentrations depicted throughout the following figures refer to the actual CS metabolite concentrations measured by H P L C in the dilution medium. H P L C re-assessment in parallel to the respective experiment reconfirmed the purity of the respective metabolites excluding alterations caused by DMSO or culture medium (data not shown). DMSO controls (0.16o70, v/v) corresponding to the highest concentration reached with metabolites alone or with combinations caused a growth reduction by 9.13 _+ 6.23°7o (mean +_ S.D., n > 30) compared with medium controls.

Effect o f single cyclosporin metabolites Initially several series of experiments with native CS were performed to determine the sensitivity of rat spleen lymphocytes and rat lymph node cells stimulated with Con A concentrations ranging from

0.1 to 10/~g/ml for 24, 48 or 72 h (data not shown). These studies revealed that lymph node cells with a known higher proportion of T-lymphocytes were clearly more sensitive to CS, but that cell number, time of incubation or different Con A concentrations had no influence on the CS concentration causing a 50% growth reduction (I¢50) of the respective lymphocyte population. Representative for these experiments in Fig. 1A the concentration-dependent effect of the parent compound CS on spleen cells was compared with that on lymph node cells stimulated for 48 h with 1 /~g/ml Con A. The 1¢50 with lymphnode lymphocytes was 0.023 _+ 0.002 mg/l (n = 5) compared with 0.078 + 0.012 mg/l (n = 3) for spleen cells. As the sensitivity of Sprague Dawley rat lymph node cells (LN) is almost identical with that of peripheral human lymphocytes, the following experimental series with CS metabolites were performed using LN. Figure I B shows the concentration-dependent effects of metabolite A MI summarizing three independent experiments. Each data point is the mean of quadruplicates. Representative for this study this figure demonstrates that the antiproliferative effects of the respective metabolites were not significantly influenced by the bile of patients, metabolite preparation procedure, LN preparation or by H P L C re-assessments, because all of these parameters were different in these three experiments. The ICso of AM1 is 0.110 mg/1 (for

A

100'

II0

90'

!-1

oo.

7O

m

70.

(DO-

80. •o

SO.

E

E I -r

30.

IR

20,

~R

10,

0

¢omblnotlon

10-

,

0 Aids : Aid1 : AMIo:

20-

0.04 0.02 0.006

0.08 0.04 0.01

0.100 0.081 0.011

0,332 0,113 0.030

o

b

c

d

0.0114 0.325 0.078

e

1 . 3 2 0 2.835 0.i5 1.3 0.155 0.31

f

g

(,,,,oA)

0 Nd4NtP : AIIIA : H355:

(m~q 0.004 0.008 O.O~l 0.157 0.071 O.+M

a

b

0.017 0.270 0.313

0.034 0.540 0.820

0.018 1.009 1.2.51

0.130 2.~U 2.503

c

d

e

f

0.27 4.395 5.005

g

Fig. 3. Combinations of three metabolites resulting in a synergistic growth inhibition of Con A-stimulated LN. The effects of the combined primary metabolites AM9 + AM1 + AMlc (A) and secondary metabolites AM4N9 + AMIA + H355 (B) were tested on LN during a 48 h incubation period. For assay conditions see Experimental Procedures. Again, a - g refer to the calculation of synergism (see Table 2).

lmmunosuppression by Cyclosporin Metabolites

100-

A

601

IOO-

90-

9o.

• - AJdIA

~

B

80i

0-

=

IO-

CS

E

~

I '~'

so

I~

20.

'~-

3O

• - AM1 o - CS + AM1

IR

10-

lO , AMt:

0.011 0.021 0.04.1 ~

a

b

c

0.100

d

[mg/L]

0

O~Lq2 0.064

e

f

1

o - CS + AMIA

2O

,6,~II A :

g

a

C

10090-

90 -

b

c

d

•

f

g

~

100e" _ ~ S w ~ ~

, [mg/,]

0.034 0.0011 0.137 0.275 0.55 1.01HI 2.100

D

• - AId4N9 CS &

I0,

-3

so.

A - AMI

70.80. CS

E I z

30,

IR

20.

A - H355

--re~

~

~ 4o& O - ¢omblnoflon + CS

'~'

10.

O - combination + C$

20. 10-

, [mg/L] ~9:0.o,6 o.,1 0 ~ , 0.o4, o.**2 0 . , ~ , . - AM1: o.oos o.oos o.o~ o . o a o.o.~ o.o-~ o.~07 a

b

c

d

•

f

g

0

, [m0/L]

AM4N9 : H,,~55.*

o.oo3 o.oml o.o15 o.o25 o.o51 O.lO2 0.2o3 o,4~ o,ooo O,Oll 0.402,3 o,o441 o.ool o.11~ o

b

c

d

e

f

g

Fig. 4. The influence of a fixed CS concentration (0.02 mg/l = ics0) on the antiproliferative effects of single primary and secondary metabolites or on effects of double metabolite combinations. (A) The effect of CS on the action of the active AM1. (B) AM1A activity either alone (upper curve) or with the addition of CS. (C) Comparison of the effects of AM9 and AM1 alone with combination of these (see Fig. 2A) plus a fixed CS concentration. (D) Activity of the secondary metabolites AM4N9 and H355 alone compared with the combined activity in the presence of CS. a - g refer to the calculation of synergism (see Table 2). c o m p a r i s o n the ICs0's o f CS a n d seven tested metabolites are s u m m a r i z e d in T a b l e 1). T h e c o n c e n t r a t i o n - d e p e n d e n t effects o f three p r i m a r y metabolites A M 9 , A M I a n d A M I c are s h o w n in Fig. 1C. C o m p a r i n g these single metabolite activities with those o f s e c o n d a r y metabolites in Fig. 1D reveals t h a t i n d e p e n d e n t f r o m the degree o f m e t a b o l i z a t i o n the m a i n g r o u p o f metabolites ( A M 9 , A M 1 9 , A M 4 N 9 , A M I c a n d H355) exhibited at least a 50-fold lower activity t h a n native CS. H o w e v e r , A M I , as s h o w n previously, h a d a significantly higher

a n d A M I A a significantly lower activity t h a n the m a i n m e t a b o l i t e g r o u p (also see T a b l e 1).

Effect of metabolite combinations To get i n f o r m a t i o n c o n c e r n i n g the i n t e r a c t i o n o f CS metabolites o n rat L N we tested the g r o w t h i n h i b i t o r y action o f several c o m b i n a t i o n s o f two or three metabolites. Figure 2A d e m o n s t r a t e s the c o n c e n t r a t i o n - d e p e n d e n t effects o f a c o m b i n a t i o n o f two p r i m a r y metabolites, A M 9 + A M 1 , showing

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synergistic antiproliferative activities especially at low concentrations of the participating metabolites. This was validated by determining the synergy index mathematically (see Table 2). Similarly the combination of a primary and secondary metabolite, AMIc + AM4N9 (Fig. 2B) and two secondary metabolites, AM1A + H355 (Fig. 2D) resulted in more pronounced synergistic effects at lower compared with higher metabolite concentrations. The combination of AM4N9 + H355 (Fig. 2C) exhibited no higher activity than H355 alone, which on the basis of the calculation of the synergy indices might be explained by relatively low concentrations of one of the participating drugs (Berenbaum, 1977). Triple combinations of primary metabolites AM9 + AM1 + AMIc (Fig. 3A) and secondary metabolites AM4N9 + AM1A + H355 (Fig. 3B) induced an even more impressive synergistic growth inhibition of lectin stimulated LN. Again the synergism was more pronounced at low metabolite concentrations. When we compared the synergy indices (Table 2) of the double combinations AM1 + AM9 (Fig. 2A) and AM1A + H355 (Fig. 2D) with that of triple combinations (Figs 3A and B, respectively) there was a clear increase in the level of synergy at low metabolite concentrations ( a - e for Fig. 3A; a and b for Fig. 3B). Already the combined addition of 0.04 mg/1 AM9 + 0.02 mg/l AM1 + 0.005 mg/l AMlc caused a more than 50°70 reduction of LN proliferation, whereas single metabolites at these concentrations had exhibited no growth inhibitory effects at all (Fig. 1).

The influence o f native cyclosporin on metabolite combinations

To get close to the in vivo situation we performed a third series of experiments determining the effects of native CS at a fixed concentration of 0.02 mg/l 0c50) on concentration-response curves of single metabolites and double metabolite combinations. This CS concentration is 10 times below the trough level achieved in patients (Table 1). Figure 4A demonstrates that the highly active AM 1 (concentration- response curve obtained in parallel) did not increase the antiproliferative action of CS alone. Moreover, CS to some extent seemed to abolish the metabolite effect at higher concentrations, which also could be determined by calculating the synergy indices (Table 2). Similarly the secondary metabolite AM1A with the lowest activity when tested alone did not enhance the activity of native CS (Fig. 4B). Given the assay standard deviation of 10% the slight further decrease

of the CS induced 50°/0 growth inhibition caused by concentration of AM1A above 1 mg/l was not significant. Similar to the influence of CS on the effects of single metabolites CS also failed to enhance the antiproliferative effects of double combinations of primary metabolites AM9 + AM1 (Fig. 4C) and secondary metabolites AM4N9 + H355 (Fig. 4D). Only at high metabolite concentrations not occurring in vivo (Table 1) there was a slight tendency to decrease LN growth below the CS induced level, which resulted in a borderline synergism (Table 2).

DISCUSSION In this study we investigated the immunosuppressive effects of a defined group of primary and secondary CS metabolites in a rat model system. Based on the activities of single metabolites we demonstrated that metabolite combinations acted synergistically. Looking at our results in more detail reveals that the activities (IC~0) of CS and AMI obtained with lectin-stimulated rat lymphnode cells were almost identical with the ICs0S found in human anti-CD3 stimulated T-lymphocytes (Ryffel et al., 1986; Wallemacq et al., 1989). Polar metabolites like AMIA were able to suppress lectin-induced lymphocyte proliferation, but only metabolite AMI reached 20% of the immunosuppressive CS activity and therefore was regarded to be relevant in vivo by some (Schlitt et al., 1987; Wallemacq et al., 1989) but not by other authors (Ryffel et al., 1986). These statements, however, solely refer to single metabolite activities. Being aware of the far more complex situation in vivo we, like Wallemacq et al. (1989) and Zeevi et al. (1988), were interested in the way of interaction of several metabolites. Indeed, confirming and extending initial observations by Wallemacq et al. (1989), we could demonstrate that already double or triple metabolite combinations acted synergistically. The concentrations of the respective metabolites resulting in a more than 50°7o growth inhibition of lymphocytes are clearly below the trough blood levels of CS-treated patients (Fig. 3B vs Table 1). Not shown so far, secondary metabolites especially exhibited very high synergistic activities at low concentrations (confirmed by the algebraic determination of synergy indices, Table 2). In a parallel investigation examining the toxic effects of CS metabolites on renal glomerular mesangial cells we have made comparable observations (Radeke, Christians, Bleck, Sewing & Resch, 1991).

Immunosuppression by Cyclosporin Metabolites Imaging the in v i v o situation we added the parent compound CS to single or combinations of metabolites at concentrations occurring in v i v o . Surprisingly, and making the situation more complex, the addition of metabolites resulted in an enhancement of CS-induced immunosuppression only at high metabolite concentrations, which are not relevant in v i v o . Especially the effect of AM1 at high concentrations seemed to be abolished by CS. It has to be mentioned that the normal trough levels of CS, which are achieved during optimal immunosuppressive treatment, are in the range of 0.1 - 0.3 mg/1 (Table 1) thus almost 10 times higher than the CS concentration used in our experiments. In contrast to our findings, Zeevi et al. (1988), who used almost identical concentrations, showed a positive interaction of CS and AMI. The reasons for this discrepancy are unclear. However, this group measured extraordinary high metabolite activities for all metabolites compared with our results and those of Wallemacq et al. (1989) and Schlitt et al. (1987). This might possibly be due to the use of a special subpopulation of human lymphocytes propagated from a heart biopsy and due to the special activation by an MHC class II dependent alloreactive stimulation. Supporting our results Schlitt et al. (1990) also failed to demonstrate a major influence of AM1 on the activity of native CS on human lymphocytes in vitro. It might be hazardous to compare our in vitro investigations in a rat system with the findings of Kunzendorf et al. (1989), who showed a correlation of high AM9 and AMI levels with a low incidence of rejection episodes. In this respect the correlation between rare rejection episodes and high levels of two metabolites might reflect only one part of the whole story. It is tempting to speculate that high levels of AM9 and AM1 are indicating high metabolite levels in general. Subsequently, this would be very compatible with our results as combinations of secondary metabolites, even in the presence of native CS (Fig. 4D), are able to compensate for low CS levels. Thus, although recent publications, mainly based on the examination of single metabolites, doubt the clinical significance of CS metabolites (reviewed in Yatscoff, Rosano & Bowers, 1991) our results might encourage studies of synergistic activities of metabolite combinations in v i v o . The precise mechanism of the immunosuppressive action of CS is still a matter of debate, although considerable progress has been made by elucidating the key events as modifications of transcription factors like N F - A T (Emmel, Verweij, Durand, Higgins, Lacy & Crabtree, 1989; Fischer, Wittmann-

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Liebold, Lang, Kiefhaber & Schmid, 1989; Flanagan, Corthesy, Bram & Crabtree, 1991). Our experiments with CS metabolites were not designed to investigate such a mechanism. However, as CS abolished the synergistic inhibition of T-cell activation by its metabolites, it appears that CS as well as the metabolites act through a common cellular target, to which CS has a much higher binding affinity. This cellular binding protein, cyclophilin, has been identified (Tropschug, Barthelmess & Neupert, 1989). Very recent results have suggested that binding of CS to cyclophilin is not sufficient for immunosuppression and, furthermore, metabolite affinity to cyclophilin is not linearly correlated with immunosuppressive activity (reviewed in Fahr, Hiestand & Ryffel, 1990). Nevertheless, in case of CS the binding represents a prerequisite for the formation of a complex with other proteins, which might finally mediate the inhibition of T-cell activation. One candidate for such an effector protein, calcineurin, a Ca 2~dependent phosphatase, has now been described (Liu, Farmer, Lane, Friedmann, Weissman & Schreiber, 1991). Considering the synergistic activity of metabolite combinations, which formally requires two interaction sites, our data might fit well into the current concept. However, the detailed mechanism, by which metabolite combinations mediate this kind of complex function, is unknown at present. In conclusion, we determined activities of single CS metabolites and confirmed the immunosuppressive potential of AM1 (21°70 of CS activity) and have described the moderate effects of AM9, AMIc, AM19, AM4Ng, H 3 5 5 ( 3 . 6 - 1 . 3 % of CS) and the low potential of AM1A (0.5°7o of CS). Combinations of metabolites, especially secondary, acted synergistically at concentrations occurring in v i v o . On the other hand, up to double metabolite combinations failed to enhance the immunosuppressive effect of the parent compound CS at relevant concentrations. However, there exists the possibility that the combination of 29 metabolites with the expected high combined immunosuppressive activity might contribute to immunosuppressive treatment in situations, where the actual concentrations of the parent drug CS drop below active concentrations. added in p r o o f - - During processing of this publication a study with a similar experimental design has been published (Schultz et al., 1991).

Note

A c k n o w l e d g e m e n t s - - We would like to thank Mrs Juliane

yon der Ohe, Mrs Renate Schottmann and Mrs Claudia Bovenkerk for their excellent technical assistance. This work was supported by Deutsche Forschungsgemeinschaft grant SFB 244/B1 and Pi 48/11-2, project DS.

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