Cyclosporin A prodrugs: design, synthesis and biophysical properties.

The cyclic undecapeptide cyclosporin A (CsA) has a remarkable spectrum of diverse biological activities, including anti-inflammatory, antifungal, antiparasitic as well as immunosuppressive activities. However, the low water solubility of this drug is a serious problem causing undesirable pharmacological properties such as erratic oral absorption. In order to overcome this problem, the design and synthesis of water-soluble prodrugs of CsA are described. Using the OH-MeBmt-1-group as attachment site, we investigate dipeptide systems exhibiting differential tendencies for intramolecular cyclization [diketopiperazine (DKP) formation] for tailoring the chemoreversible release of the parent CsA. In modulating the chemical and structural features of the dipeptide esters (N-alkylation, side chains, C-terminal Pro), we find conversion rates at physiological conditions ranging from minutes to several days. Together with their thermodynamic stability in the solid state and strongly enhanced solubility in water, these chemoreversible CsA prodrugs represent versatile candidates for therapeutical use.

Association of cyclophilin A with renal brush border membranes: redistribution by cyclosporine A.

BACKGROUND: Administration of the immunosuppressive agent cyclosporine A (CsA) is associated with nephrotoxicity. The main target for CsA, cyclophilin A (CypA), was found in high levels in epithelial cells of renal proximal tubules. In the present study, CypA was immunodetected and characterized following CsA treatment in subcellular fractions of renal cortex. METHOD: The renal content and distribution of CypA was evaluated in untreated rats and in rats treated with a subcutaneous injection of CsA (10 mg. kg-1. day-1) for 10 days. RESULTS: In untreated rats, membrane-bound CypA represents 0.25% of total brush border membrane (BBM) proteins, similar to the proportion found in the soluble fraction. High ionic strength treatment was unable to extract CypA from BBMs, whereas alkaline treatment (Na2CO2, pH 11) and detergent 3 - [(3 - cholamidopropyl) - dimethyl - ammonio] - 1 - propanesulfate (CHAPS) released it from BBMs. These results indicate that CypA is associated with renal BBMs, and that hydrophobic interactions are involved in this association. The CypA distribution was strongly modified in both BBMs and the soluble fraction after CsA treatment, but its affinity for CsA estimated by photoaffinity labeling was unaffected. The CypA expression level decreased by 45% in BBMs, while it increased by 33% in the soluble fraction, compared with control rats. CypA remained associated with the membranes following in vitro incubation of renal BBMs with CsA. However, incubation of CypA with one of its substrates released CypA from renal BBMs. CONCLUSIONS: These experiments suggest that renal BBMs contain a significant amount of CypA and chronic exposure to CsA, and acute exposure to one of CypA substrates may modify its subcellular distribution.

Inhibition of P-glycoprotein by cyclosporin A analogues and metabolites.

The interaction between P-glycoprotein (P-gp) from membranes isolated from multidrug-resistant Chinese hamster ovary cells and cyclosporin A (CsA) analogues and its metabolites was characterized. Screening of these latter as chemosensitizers was performed using three different assays: (i) vinblastine uptake, (ii) photoaffinity labeling by [125I]iodoaryl azidoprazosin, and (iii) P-gp ATPase activity. Oxidation of the hydroxyl group at position I of CsA (200-096), CsG (215-834), or CsD (PSC-833) increased their inhibition of P-gp. CsA analogues (208-032, 208-183) modified at position 11 retained their ability to inhibit P-gp while analogues modified at position 2 (CsC and CsD) lost their efficiency. The inhibitions induced by metabolites of CsA were also compared to those obtained with CsG metabolites. From all the molecules tested, PSC-833 and 280-446 peptolide were the strongest inhibitors. Our results indicate that modifications of CsA analogues at position 1 and 2 are critical for their interaction with P-gp and that CsA metabolites retain a portion of the inhibitory activity of the parent drug.

Identification of the cyclosporin-binding site in P-glycoprotein.

The binding site of cyclosporin A to P-glycoprotein was characterized by using a multidrug-resistant Chinese hamster ovary cell line. P-glycoprotein photolabeled with diazirine-cyclosporin A analogue was purified by a two-step process involving continuous elution electrophoresis followed by wheat germ agglutinin-agarose precipitation. The cyclosporin A covalently bound to P-glycoprotein and to subsequent proteolytic fragments was detected by Western blot analysis using a monoclonal antibody against cyclosporin A. Proteolytic digestion of purified P-glycoprotein by V8 generated a major fragment of 15 kDa photolabeled by cyclosporin A, while proteolysis of P-glycoprotein photolabeled by [125I]-iodoaryl azidoprazosin generated a major fragment of 7 kDa. Limited proteolysis of cyclosporin A-photolabeled P-glycoprotein with trypsin indicated that the major binding site for cyclosporin A was in the C-terminal half of the protein. This cyclosporin A binding site was further characterized with chemical agents (N-chlorosuccinimide, cyanogen bromide, and 2-nitro-5-thiocyanobenzoate). These three chemical agents established a proteolytic profile of P-glycoprotein for fragments photolabeled with cyclosporin A and for fragments that contained the C494 and C219 epitopes. The smallest fragments generated by these chemical agents include the transmembrane domains (TMs) 10, 11, and 12 of P-glycoprotein. When the fragments generated by these chemical agents are aligned, the region that binds cyclosporin A is reduced to the 953-1007 residues. These combined results suggest that the major binding site of cyclosporin A occurs between the end of TM 11 and the end of TM 12.

Molecular interactions of cyclosporin A with P-glycoprotein. Photolabeling with cyclosporin derivatives.

The interaction between P-glycoprotein (140-180 kDa) from the multidrug-resistant Chinese hamster ovary cell line CHRC5 and cyclosporin A was characterized using three different photoactivable cyclosporin A analogs. Two monoclonal antibodies, which are able to discriminate between two major domains of cyclosporin A (the cyclophilin and calcineurin binding domains), were used to detect the photolabeled proteins. A protein of 155 kDa corresponding to P-glycoprotein was much more strongly photolabeled in membranes of CHRC5 cells than in membranes of their drug-sensitive parent cell line AuxB1. The antitumor drug vinblastine and the reversal agents verapamil and cyclosporin A inhibited the photolabeling, and the nonimmunosuppressive derivative PSC-833 caused a stronger inhibition than cyclosporin A. P-glycoprotein photolabeled with cyclosporin A analogs was only detected with the monoclonal antibody that recognizes cyclosporin A and its metabolites, indicating that the calcineurin binding domain recognized specifically by the other antibody is not exposed. These results suggest that the portion of cyclosporin A that binds to calcineurin plays a role in the interaction of cyclosporin A with P-glycoprotein.

Interactions of cyclophilin with the mitochondrial inner membrane and regulation of the permeability transition pore, and cyclosporin A-sensitive channel.

Mammalian mitochondria possess an inner membrane channel, the permeability transition pore (MTP), which can be inhibited by nanomolar concentrations of cyclosporin (CS) A. The molecular basis for MTP inhibition by CSA remains unclear. Mitochondria also possess a matrix cyclophilin (CyP) with a unique N-terminal sequence (CyP-M). To test the hypothesis that it interacts with the MTP, we have studied the interactions of CyP-M with rat liver mitochondria by Western blotting with a specific antibody against its unique N terminus. Although sonication in isotonic sucrose at pH 7.4 refraction sediments with submitochondrial particles at 150,000 x g. We show that the interactions of this CyP-M pool with submitochondrial particles are disrupted (i) by the addition of CSA, which inhibits the pore, but not of CSH, which does not, and (ii) by acidic pH condition, which also leads to selective inhibition of the MTP; furthermore, we show that the effect of acidic pH on CyP-M fully prevents the inhibitory effect of H+ on the MTP (Nicolli, A., Petronilli, V., and Bernardi, P. (1993) Biochemistry 32, 4461-4465). These data suggest that CyP-M inhibition by CSA and protons may be due to unbinding of CyP-M from its putative binding site on the MTP. A role for CyP-M in MTP regulation is also supported by a study with a series of CSA derivatives with graded affinity for CyP. We show that with each derivative the isomerase activity of CyP-M purified to homogeneity is similar to that displayed at inhibition of MTP opening, CyP-M (but not CyP-A) and decreased efficiency at MTP inhibition is obtained by substitution in position 8 while a 4-substituted, nonimmunosuppressive derivative is a as effective as the native CSA molecule, indicating that calcineurin is not involved in MTP inhibition by CSA.

The 3D structure of a cyclosporin analogue in water is nearly identical to the cyclophilin-bound cyclosporin conformation.

The conformation of [D-MeSer3-D-Ser-(O-Gly)8]CS, a water soluble cyclosporin derivative, has been determined in (D6)DMSO and in water using NMR. In these polar solvents the conformation is identical and very similar to the structure found in the cyclophilin-cyclosporin complex. However, it differs significantly from its conformation in deuterated chloroform. This demonstrates unambiguously that the large structure change is induced primarily by the polar solvent rather than by complex formation with cyclophilin.

Mechanism of cyclosporin A biosynthesis. Evidence for synthesis via a single linear undecapeptide precursor.

Cyclosporin A is synthesized by cyclosporin synthetase, a multienzyme polypeptide. This enzyme catalyzes at least 40 reaction steps in an assembly belt-like mechanism. It activates all constituent amino acids of cyclosporin A to thioesters via amino acyladenylates and carries out specific N-methylation reactions. During elongation, the activated amino acids are linked by peptide bonds leading to enzyme-bound nascent peptide chains. Some of the linear peptides of the growing cyclosporin A chain were isolated and their N-terminal amino acid was determined. D-Alanine at position 8 of the cyclosporin A molecule was found to be a starting amino acid in the biosynthetic process of cyclosporin A formation. Four intermediate peptides of the growing peptide chain of cyclosporin A could be isolated and identified. All of them represent partial sequences of cyclosporin A starting with D-alanine. That these intermediate peptides were bound by thioester linkage to cyclosporin synthetase could be demonstrated by liberation of the peptides with performic acid. The peptides strongly suggest the stepwise synthesis of a single linear peptide precursor of cyclosporin A.

Identification of several cyclosporine binding proteins in lymphoid and non-lymphoid cells in vivo.

The immunosuppressant cyclosporine A (CSA) has been shown to bind to the ubiquitous cellular protein, cyclophilin, and to inhibit its rotamase activity. In the present study, 3H-cyclosporine diazirine analogue was used to photolabel viable human cells of lymphoid and fibroblast origin in order to identify the intracellular targets for the drug. While cyclophilin was strongly labeled in situ, additional minor cyclosporine-protein complexes of 25, 40, 46 and 60 kDa were identified in the T cell leukemia cell line Jurkat. These proteins bound specifically, since only active CSA but not inactive CSH or FK506 competed for binding. Photolabeling of MRC5 cells, a CSA resistant human fibroblast cell line, revealed a 25 kDa complex as the major product, while the 46 and 60 kDa bands were not detectable and cyclophilin labeling was only faint, even though both MRC5 and Jurkat cells contain similar cyclophilin concentrations. Thus, our data suggest that the intracellular targets of CSA and/or the accessibility to cyclophilin varies considerably in drug sensitive and resistant cell types, which may contribute to explaining the lymphocyte selectivity of the drug.

On the dependence of molecular conformation on the type of solvent environment: a molecular dynamics study of cyclosporin A.

The dependence of the conformation of cyclosporin A (CPA), a cyclic undecapeptide with potent immunosuppressive activity, on the type of solvent environment is examined using the computer simulation method of molecular dynamics (MD). Conformational and dynamic properties of CPA in aqueous solution are obtained from MD simulations of a CPA molecule dissolved in a box with water molecules. Corresponding properties of CPA in apolar solution are obtained from MD simulations of CPA in a box with carbontetrachloride. The results of these simulations in H2O and in CCl4 are compared to each other and to those of previous simulations of crystalline CPA and of an isolated CPA molecule. The conformation of the backbone of the cyclic polypeptide is basically independent of the type of solvent. In aqueous solution the beta-pleated sheet is slightly weaker and the gamma-turn is a bit less pronounced than in apolar solution. Side chains may adopt different conformations in different solvents. In apolar solution the hydrophobic side chain of the MeBmt residue is in an extended conformation with its hydroxyl group hydrogen bonded to the backbone carbonyl group. In aqueous solution this hydrophobic side chain folds over the core of the molecule and the mentioned hydrogen bond is broken in favor of hydrogen bonding to water molecules. The conformation obtained from the MD simulation in CCl4 nicely agrees with experimental atom-atom distance data as obtained from nmr experiments in chloroform. In aqueous solution the relaxation of atomic motion tends to be slower than in apolar solution.

Molecular characteristics of cyclophilin-cyclosporine interaction.

The ability of cyclophilin to react with derivatives of cyclosporine (CsA) was studied. Cyclophilin was found to interact preferentially with CsA-residues 1, 2, 10 and 11, which, together with residue 3, are the residues known to contribute to the immunosuppressive activity of CsA. The recognition of different CsA-derivatives by cyclophilin was correlated with their immunosuppressive activity in vitro. All CsA-derivatives showing a significant activity did bind to cyclophilin, although some of the CsA-derivatives able to bind cyclophilin exhibited only low activities. The results suggest that binding to cyclophilin might be one requirement for immunosuppressive activity of CsA derivatives. When tested with CsA-derivatives showing various conformational changes, the binding of cyclophilin was strongly specific for the peptide-ring conformation of CsA. No binding of calmodulin to CsA could be detected in several formats of solid-phase enzyme- or radioimmunoassay, suggesting that, in contrast to cyclophilin, calmodulin does not possess sufficient affinity for CsA to bind to it when immobilized on the solid phase.

Study of the conformation of cyclosporine in aqueous medium by means of monoclonal antibodies.

The three-dimensional structure of the immunosuppressive cyclic peptide cyclosporine (Cs), determined in crystal by X-ray analysis and in solution in aprotic solvents by n.m.r., differs mainly by the orientation of the 7 carbon side chain of residue 1. Because of its poor solubility in water, the conformation of Cs in aqueous medium cannot be studied by n.m.r. methods, which require concentrations of the substance of the order of milligram/mL but can be analyzed by immunochemical methods in which concentrations in the nanogram/mL range are detected. In the present study, the ability of a series of monoclonal antibodies (McAbs) raised against Cs to recognize different parts of residue 1 of Cs was determined from the cross-reactivity of different Cs-analogues modified in residue 1. The results show that when Cs is dissolved in aqueous buffer, the terminal atoms of residue 1 side chain are not available for binding to antibodies recognizing the face of the molecule defined by residues 1, 2, 3, 10, 11, suggesting that the chain is probably folded back under the molecule, as observed in the crystal structure. Binding of McAbs to Cs was also affected by conformational modifications of the peptide ring that occur in some Cs-analogues. The results illustrate the potential of McAbs for probing the conformation of Cs-derivatives for which no structural data are available.

Fine specificity and cross-reactivity of monoclonal antibodies to cyclosporine.

More than 180 monoclonal antibodies (McAbs) to the cyclic undecapeptide cyclosporine (Cs) have been prepared. Several immunization protocols and antibody screening processes were compared. Two main groups of McAbs recognizing different "sides" of the Cs molecule could be differentiated. The antibodies belonged to the IgG and IgA classes and showed high affinity for Cs (up to 10(-10) -10(-11) mol/l). Based on their ability to discriminate Cs-derivatives modified singly at each of the 11 residues of the Cs molecule, the antigenic recognition pattern of different McAbs was studied at the level of individual residues. Closely related recognition patterns were found in each of the two main McAb groups. The apparent size of the Cs antigenic sites recognized by different McAbs varied from four to ten residues and did not correlate with antibody affinity.

Cyclophilin binds to the region of cyclosporine involved in its immunosuppressive activity.

Although several cytosolic proteins including calmodulin and cyclophilin have been shown to bind cyclosporine, the direct involvement of these proteins in the immunosuppressive activity of cyclosporine remains to be established. In the present study, a quantitative immunoassay for cyclophilin was developed which made it possible to compare its relative affinity for cyclosporine and any of its analogues. The binding of cyclophilin to cyclosporine coated on a solid phase was revealed by anti-cyclophilin rabbit antiserum followed by antiglobulin-enzyme conjugate. This reaction could be inhibited by addition of free cyclosporine or certain cyclosporine analogues. By studying the binding of cyclophilin to more than fifty cyclosporine derivatives modified singly on each of the eleven amino acid residues, it could be shown that cyclophilin binds to the residues of cyclosporine known to be critical for its immunosuppressive activity. These data identify cyclophilin as a highly discriminating stereospecific binding protein for cyclosporine.