Molecular Immunology, Vol. 29, No.9, pp. 1043-1047, 1992
`Printed in Great Britain.
`
`0161-5890/92 $5.00 + 0.00
`Pergamon Press Ltd
`
`NEW CYCLOSPORIN A ANALOGUE: SYNTHESIS AND
`IMMUNOSUPPRESSIVE ACTIVITY*
`
`JAN R6ZYCKI,t KRZYSZTOF ROLKA,t ZBIGNIEW WnkZOREK,t
`MICH~ ZIMECKIt and GOTFRYD KUPRYSZEWSKIt
`tlnstitute of Chemistry, University of Gdansk, PL-80-952 Gdansk, Poland and
`:j:Institute of Immunology and Experimental Therapy Polish Academy of Sciences,
`PL-53-114 Wroclaw, Poland
`
`(First received 4 October 1991; accepted in revised form 2 March 1992)
`
`Abstract-A synthetic analogue of cyclosporine A, in which an unusual amino acid (4R)-N -methyl-
`4-butenyl-4-methyl-L-threonine (MeBmt) is replaced with L-threonine (Thr), was synthesized by the
`solid phase method. Its activity in the humoral response to sheep red blood cells in vitro and in vivo
`in mice was practically the same as that of cycIosporine A used as a standard, whereas the analogue
`studied exerted a significantly stronger effect in the delayed type hypersensitivity to sheep red blood
`cells in mice.
`
`INTRODUCTION
`
`Cyclosporin A (Cs-A), a unique and remarkably effec(cid:173)
`tive immunosuppressive agent isolated from Tolypoc/a(cid:173)
`dium inflantum Gams fungal species by Dreyfuss et al.
`(1976) is a neutral, homodetic, hydrophobic, cyclic 11-
`peptide. Its amino acid sequence is presented below:
`
`centers, and can be synthesized by a multistep chemical
`procedure. The synthesis of Cs-A analogue, with im(cid:173)
`munosuppressive activity, with readily available amino
`acid, in this position could promote a production of a
`drug at a rather low price. Because of some similarities,
`we decided to substitute MeBmt with Thr.
`
`3
`4
`2
`MeBmt--Abu--Sar--MeLeu
`I
`I
`11
`5
`MeVal
`Val
`I
`I
`10
`7
`9
`8
`6
`Me-Leu-MeLeu-D-Ala-Ala-MeLeu
`
`MeBmt =
`
`H
`'"
`/
`C = C
`/
`'"
`H
`CH3
`
`CH2-CH
`I '"
`CH3 CH-CH-COOH
`I
`I
`OH NH-CH3
`
`MATERIALS AND METHODS
`
`Specific rotation was determined on a Perkin Elmer
`Model 141 spectrometer with an accuracy of 0.01°.
`HPLC analysis was performed with a Beckman Peptide
`System by reversed phase analytical column (ODS RP'8,
`4.6 mm x 30 cm) in a linear gradient from 35 to 50% of
`B in 40 min, (A) 0.1 % TFA, (B) acetonitrile, flow rate
`I mljmin. The homogeneity of pep tides was also checked
`by thin layer chromatography (TCL) using glass plates
`coated with 0.25 mm layer of silica gel (Kisselgel C,
`Merck). Chromatograms were developed in the systems:
`(A) n-butanol-acetic acid-water (4: I :5) upper layer; (B)
`n-butanol-pyridine-acetic acid-water (51: 12: 12:25); (C)
`ethyl acetate-methanol-water (10:2: I). Spots were
`visualised with iodine vapours. Fractions from chro(cid:173)
`matographic columns were analysed by means of a
`monitor equipped with a UV lamp (wavelength 254 nm).
`Molecular weights (molecular ions) were determined in
`a Varian type MAT 711 spectrometer using the field
`desorption technique. t-Butyloxycarbonylamino acids
`were obtained using di-t-butyldicarbonate according to
`Moroder et al. (1976). N-methylated amino acids
`(MeLeu, MeVal) were synthesized according to the
`procedure of Cheung and Benoiton (1977).
`
`According to Borel (1986), Cs-A is widely used follow(cid:173)
`ing organ (kidney, bone marrow, liver, heart) transplant
`surgery in general and in diabetic patients receiving
`transplanted segments of pancreas or isolated pancreatic
`islets in particular. All cyclosporines isolated to date
`contain a unique N -methylated amino-acid in position
`I, (2S, 3R, 4R, 6E)-3-hydroxy-4-methyl-2(N -methyl(cid:173)
`amino )-6-octeonic acid, initially celled C-9-ene and more
`recently - MeBmt 1(4R)-N -methyl-4-butenyl-4-methyl-L(cid:173)
`threoninel. As suggested by Ritch et al. (1986, 1990) and
`Traber et al. (1988), this amino acid residue is essential
`for high biological activity. It contains three asymetric
`
`>I< Abbreviations: The symbols of the amino acids and peptides
`are in accordance with the 1983 Recommendations of the
`IUPAC-IUB Joint Commission on Biochemical Nomencla(cid:173)
`ture (1984), Eur. J. Biochem. 138, 9-37.
`
`Peptide synthesis and purification
`I Thr'l Cs-A was synthesized by
`the solid-phase
`method, using chi oro methylated resin (substitution
`0.7meqjg). C-terminal amino acid (Ala) was coupled to
`the resin using the Horiki et al. (1978) KF procedure. By
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`1044
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`JAN R6ZYCKI et al.
`
`this method, a substitution of 0.67 meq/g was achieved.
`Reversible protection of the amino groups was accomp(cid:173)
`lished with t-butyloxycarbonyl (Boc-) group and, in
`addition, the hydroxyl group of Thr was protected as
`benzyl ether. Deblockings were performed with 33%
`trifluoroacetic acid (TF A) in dichloromethane (CH2CI2)
`in the presence of 1 % anisole or dimethyl sulfide (DMS).
`Double couplings in CH 2CI2 were achieved using 0.25 M
`dicyclohexylcarbodiimide (DCCI) in CH 2C!2' Coupling
`efficiency was monitored according to tests of Kaiser
`et al. (1970) or Christensen (1979). Peptide was cleaved
`from the resin by the standard HF procedure in the
`presence of 10% anisole according to Sandberg and
`Ragnarsson (1974).
`Crude linear peptide was purified on a Sephadex
`LH-20 column (1.8 x 37 cm) using MeOH as an eluent.
`Finally, the peptide was cyclized by the azide method
`according to Varga et al. (1979) using diphenylphospho(cid:173)
`razidate (DPPA) in dimethylformamide (DMF) as a
`coupling reagent. Crude IThr1ICs-A was purified under
`the same conditions as its linear precursor. The purity of
`the final product was checked by HPLC method.
`
`Materials
`(Cs-A)-commercially available
`Cyclosporine A
`50 mg/ml ampoules (Sandoimmun, Sandoz, Switzer(cid:173)
`land).
`
`Animals: CBA/Iiw mice 8-10 weeks old.
`Antigen: Sheep red blood cells (SRBC).
`RPMI: culture medium.
`Solvents: mixture of Cremophore-EL (Sigma, St
`Louis, MO) and 94% ethanol (6.5:3.5 ratio). In(cid:173)
`tralipid (100 mg/ml) (Kabi, Sweden) and apyrogenic
`solution of 0.9% NaCI.
`
`The influence of IThr1ICs-A on the humoral immune
`response was performed by plaque-forming cells (PFC)
`test according to Mishell and Dutton (1967).
`
`Determination of primary humoral immune response
`in vivo
`solution: 2 mg of
`IThr1ICs-A
`Preparation of
`IThr1ICs-A was dissolved in 0.1 ml of the mixture of
`Cremophor-ethanol. Treatment of mice with reagents:
`IThr1ICs-A, dissolved in the solvent was diluted at a
`desired concentration with 0.9% NaCI and 0.2 ml of the
`solution was twice introduced intraperitoneally (i.p.)
`into the animals. The first dose was administered 3 hr
`before the immunization, the second dose 24 hr later.
`The immunosuppressive activity of I Thr11 Cs-A was com(cid:173)
`pared with that of Cs-A diluted with 0.9% NaC!. An
`appropriate concn of the solvent was used as a control.
`Immunization of mice: mice were injected i.p. with
`0.2 ml of 10% SRBC suspension in 0.9% NaCI 3 hour
`after the introduction of the first dose of the reagents.
`After 4 days, the number of PFC producing IgM
`antibodies in the spleen was determined according to
`Mishell and Dutton (1967). The magnitude of the hu(cid:173)
`moral response was expressed as PFC number per 106 of
`
`viable splenocytes. The group of mice tested consisted of
`six animals.
`
`Determination of the humoral immune response in vitro
`Preparation oflThr1lCs-A solution: 2 mgoflThr1lCs-A
`was dissolved in 0.15 ml of95% ethanol, then 0.35 ml of
`Intralipid was added and adjusted to a desired concen(cid:173)
`tration with culture medium (RPM I) supplemented with
`10% fetal calf serum (FCS). The immunosuppressive
`activity of IThr1ICs-A was compared with that of Cs-A:
`to 0.04 ml of Cs-A, 0.35 ml of Intralipid was added, then
`diluted with culture medium to a desired concn. An
`appropriate amount of Intralipid, diluted with culture
`medium, was used as a control. Mice were primed i.v.
`with 0.2 ml of 1% SRBC suspension in PBS. Four days
`later the animals were sacrificed, their spleens were
`minced and pressed through a plastic screen into 0.83%
`NH4Cl, buffered with 0.017 M Tris, to remove erythro(cid:173)
`cytes. Then, the cells were washed three times with
`phosphate buffer solution (PBS) and finally resuspended
`in RPMI medium, supplemented with 10% FCS.
`To I ml of spleen cell suspension (5 x 106 cells/ml),
`0.1 ml of the reagent was added, followed by the ad(cid:173)
`dition of 0.1 ml of SRBC suspension in the culture
`medium, and incubated for 4 days at 37°C, 5% CO2 and
`100% humidity (cell culture incubator). PFC number
`was determined according to Mishell and Dutton (1976).
`The experiments were performed using Falcon 24-well
`tissue culture plates.
`
`Delayed type hypersensitivity (DTH) in mice
`For dissolving oflThr1lCs-A see "determination of the
`primary immune response in vivo". Mice were treated
`i.p. with two doses of the reagents, the first dose 3 hr
`before sensitization with SRBC, the second dose 48 hr
`later. DTH was induced in mice by the procedure
`described by Lagrange et al. (1975). Mice were sensitized
`i.v. with 106 ofSRBC in 0.1 ml of NaCI. After 4 days the
`effector reaction was elicited by an intradermal introduc(cid:173)
`tion of 108 SRBC into the left hind foot pad. The
`magnitude of the reaction was measured as an increase
`of the foot pad thickness at 24 hr following the adminis(cid:173)
`tration of the antigen challenging dose.
`
`RESULTS
`
`Physiochemical properties of IThr1ICs-A and its linear
`precursor are presented below.
`
`IThr1ICs-A linear precursor: lex 120 = -127.0° (c = 1,
`methanol).
`TLC: Rra = 0.47, Rib = 0.65, Rrc = 0.20. HPLC analy(cid:173)
`sis: retention time (RT) = 35.3 min. Molecular ion
`(molecular weight): calc. 1138: found 1140.
`IThr1ICs-A: lexl 20 = -173.4° (c = I, methanol). TLC:
`Rra = 0.72, Rrb = 0.77, Rrc = 0.75. HPLC analysis:
`RT = 39.3 min. Molecular ion (molecular weight):
`calc. 1120: found 1120.
`
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`Cyc1osporin A analogue, with immunosuppressive activity
`
`1045
`
`Table 1. PFC number in the spleen cell cultures treated with
`IThr1ICs-A or Cs-A
`
`Compound
`
`Control 0.9% NaCI
`
`Control intralipida
`
`IThr1ICs-A
`
`Cs-A
`
`Dose
`( Il g/culture) PFCb /106 cells ± SE
`
`0.1
`1.0
`10.0
`
`0.1
`1.0
`10.0
`
`0.1
`1.0
`10.0
`
`1.510 ± 100
`
`1.332 ± 92
`1.220 ± 55
`964 ± 112
`
`1.518 ± 101
`1.296 ± 71
`137 ± 36**
`912 ± 65'"
`308 ± 10 ... •
`99 ± 12"'*
`
`• p < 0.05, "P < 0.001.
`aConcentration of intralipid in control cultures corresponded
`to concn of intralipid in cultures containing 0.1, 1.0 or 10 Il g
`of the preparations per m!.
`bThe results are expressed as a mean of 6 determinations
`(wells) + SE (standard error).
`
`Results of IThr1ICs-A activity in the humoral immune
`response to SRBC in vitro, in vivo, and its effect on the
`development of DTH to SRBC in mice are summarized
`in Tables 1-5.
`
`Activity oflThr1lCs-A in the humoral immune response to
`SRBC in vitro and in vivo
`To investigate a potential suppressive activity of
`IThr1ICs-A, the preparation was added to splenocyte
`cultures at a concn of 0.1, 1.0 and 10.0 jig/ml culture.
`Cyc1osporin A (Cs-A) was used as a standard immuno-
`
`Table 2. PFC number in the spleen cell cultures treated with
`IThr1ICs-A or Cs-A
`
`Compound
`
`Control 0.9% NaCI
`Control of Cremophora
`
`Cs-A
`
`Dose
`( Il g/culture)
`
`PFCb/106 cells ± SE
`
`1990 ± 181
`
`2445 ± 395
`
`846 ± 175·
`ISO ± 68**
`66 ± 15*·
`
`810 ± 76*
`280 ± 50**
`86 ± 26**
`
`0.1
`1.0
`5.0
`
`0.1
`1.0
`5.0
`
`*P < 0.01.
`.* P < 0.001.
`IThr1ICs-A was dissolved in the mixture ofethanol-cremophor
`than diluted to desired concn in 0.9% NaC!. 0.1 ml of the
`reagent was added to I ml of spleen cell culture.
`aConcentration of cremophor in control cultures corresponded
`to concn of cremophor in cultures containing 5.0 Ilg/ml of
`the preparations.
`bThe results are expressed as a mean of 6 determinations
`(wells) ± SE.
`
`MIMM 29;9--8
`
`Table 3. PFC number in the spleen cells of mice treated
`intraperitoneally with two doses of IThr1ICs-A or Cs-A 3 hr
`before and 24 hr after the antigen administration
`
`Compound
`Controla
`
`IThr1ICs-A
`
`Cs-A
`
`Dose
`(llg/mouse) PFCj106 cells ± SE
`
`1.875 ± 184
`
`510 ± 58*
`371±41*
`
`386 ± 32*
`126 ± 21 *
`
`10
`100
`
`10
`100
`
`* P < 0.001.
`aConcentration of cremophor in the control inoculum was the
`same as in the inocula containing IOOllg of the prep(cid:173)
`arations .
`The results are expressed as a mean of 6 determinations
`(mice) ± SE.
`
`suppressive drug at the same doses. The control cultures
`contained Intralipid, serving as a solvent for the prep(cid:173)
`arations, at appropriate concn. The results (Table I)
`show that IThr1ICs-A inhibits the formation of PFC, but
`only at a concn of 10.0 jig/ml. The suppression is
`comparable to that of Cs-A, Cs-A however, is able to
`suppress the humoral immune response in vitro also at
`a lower concn (1.0 jig/mi). More interesting results con(cid:173)
`cerning the activity of IThrIICs-A in the in vitro exper(cid:173)
`iments were obtained with the substance dissolved in the
`mixture of cremophor-ethanol. It was shown (Table 2)
`that the activity of I Thr11 Cs-A dissolved in cremophor
`was practically the same as the activity of Cs-A. We
`suggest that the discrepancy between the in vitro activity
`of IThrIICs-A and commercially available cyc1osporine A
`(in the first experiment (Table I) was caused by the
`intralipid used as a solvent.
`The results obtained in the in vivo experiments con(cid:173)
`cerning the effect of IThrIICs-A on the humoral immune
`response are presented in Table 3. In those experiment
`suppressive action of IThrIICs-A is a little weaker but
`comparable with that of Cs-A.
`
`Table 4. The effect of IThrIICs-A on the delayed type hypersen(cid:173)
`sitivity (DTH) to SRBC. The peptides were administered twice
`i.p., first dose at 3 hr before sensitization of mice with SRBC,
`second 48 hr after the antigen injection
`
`Compound
`Controla
`
`IThrIICs-A
`
`Cs-A
`
`Dose
`(llg/mouse) DTH unitsb + SE
`16.84 ± 1.60
`
`10
`
`10
`
`8.12 ± 1.08*
`
`10.74 ± 0.94*
`
`*P < 0.01.
`aConcentration of cremophor in the control inoculum was the
`same as in the inocula containing the preparations.
`bOne unit = 0.1 mm.
`The results are expressed as a mean of 10 determinations
`(mice) ± SE.
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`JAN R6ZYCKI et al.
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`Table 5. The effect of IThrIICs-A on the delayed type hypersen(cid:173)
`sitivity (DTH) to SRBC. The peptides were administered twice
`i.p., first dose at 3 hr before sensitization of mice with SRBC,
`second 48 hr after the antigen injection
`
`compared with Cs-A which contains the unusual MeBmt
`amino acid residue.
`
`Compound
`
`Controla
`
`Cs-A
`
`Dose
`(Jig/mouse)
`
`I
`10
`100
`
`I
`10
`100
`
`DTH unitsb ± SE
`12.0 ± 0.92
`8.88 ± 0.40*
`6.33 ± 0.77***
`4.10 ± 0.82***
`10.60 ± 0.84
`8.30 ± 0.94**
`5.40 ± 0.38***
`
`*P < 0.05.
`**P < 0.01.
`*** P < 0.001.
`aConcentration of cremophor in the control inoculum was the
`same as in the inocula containing the preparations.
`bane unit = 0.1 mm.
`The results are expressed as a mean of 10 determinations
`(mice) ± SE.
`
`Effect oflThrllCs-A on the development of DTH to SRBC
`in mice
`
`Mice were treated with the preparations as indicated
`under Tables 4 and 5. The results reveal that the intensity
`of the DTH reaction was significantly suppressed by
`IThrIICs-A (more than 65% Table 5). The suppression
`caused by Cs-A was slightly weaker. The immuno(cid:173)
`suppressive activity is dose dependent.
`
`DISCUSSION
`
`The results presented clearly indicate that the ana(cid:173)
`logue of Cs-A with MeBmt substituted with Thr exerts
`immunosuppressive activity both on humoral and cellu(cid:173)
`lar immune response. In the case of development of
`DTH to SRBC the activity is even stronger than that for
`cyclosporine A. Our results are in contradiction with
`previous reports of Ritch et al. (1986, 1990) and Traber
`et al. (1968) who are of the opinion that MeBmt
`is essential for biological function of cyclosporine A.
`to Wenger (1983),
`According
`the substitution of
`MeBmt with N -methyl-L-threonine (MeThr) signifi(cid:173)
`cantly decreased or even caused the unexpected decay
`of immunosuppressive activity.
`
`CONCLUSION
`
`According to our experiments, I Thrll Cs-A is an active
`immunosuppressor and was synthesized with a good
`yield by methods commonly used in peptide chemistry.
`Synthesis of this analogue is quite inexpensive as
`
`Acknowledgement ~ This work was supported by the Polish
`Scientific Research Committee (KBN), research grant no.
`DS/S-300-7-016-1.
`
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