Cyclosporin A and C

Summary Cyclosporin A and C are new antifungal antibiotics fromTrichoderma polysporum (Link ex Pers.) Rifai. The metabolites are produced in submerged culture and are extracted therefrom by organic solvents. Cyclosporin A is a nonpolar cyclic peptide with a molecular weight of 1202.6. The cyclosporins exhibit a narrow spectrum of antifungal activity and in addition have immunosuppressive properties.     

Cyclosporin A in cadaveric organ transplantation.

The use of cyclosporin A (CyA) with a protocol designed to avoid the effects of nephrotoxicity resulted in a one-year survival of 86% in recipients of renal allografts from unmatched cadaveric donors. The drug also controlled rejection of liver and pancreatic allografts. It was possible to change patients initially treated with CyA to azathioprine and corticosteroids and vice versa, thus enlarging the potential value of CyA in organ allografting. Of 34 recipients of renal allografts, 29 were currently receiving only CyA as immunosuppressive treatment. Twelve patients never required any adjuvant steroid treatment. These results suggest that CyA is an effective immunosuppressant, and if used with care side effects need not be severe.     

Cyclosporin A: a powerful immunosuppressant.

Cyclosporin A (CyA) is a powerful immunosuppressive agent whose lack of myelotoxicity makes it unique among nonsteroidal drugs currently given for immunosuppression. It has been used with initial success in recipients of kidney, liver, bone marrow and pancreas transplants, and it may also have clinical application in the treatment of autoimmune disorders. In regard to its use in transplant recipients, there are many remaining questions about its mechanism of action, the optimum dose, whether it should be used alone or with other immunosuppressants, whether it can suppress chronic rejection and what its long-term side effects may be. These questions can only be answered by further careful laboratory investigation and controlled clinical trials. Until then, CyA should only be administered in centres experienced in its use.     

Cyclosporin A blocks calcium-dependent pathways of gene activation.

We have used an interleukin-2 (IL-2) promoter-CAT fusion gene to study activation of IL-2 gene expression by IL-1, phytohemagglutinin (PHA), phorbol myristate acetate (PMA), and calcium ionophore in the murine thymoma line EL4 and the human lymphoma line Jurkat. The two cell lines respond differently to combinations of these stimuli. IL-1 in combination with suboptimal concentration of PMA induced chloramphenicol acetyltransferase (CAT) activity in EL4. In Jurkat cells, IL-1 failed to synergize with PMA or PHA. Cotransfection with the IL-2/CAT gene and a construct capable of expressing murine T-cell type IL-1 receptors converted Jurkat cells to IL-1 responsiveness. IL-1 in combination with PHA but not with PMA resulted in induction of CAT activity in these cells. Induction of IL-2/CAT activity by all stimuli in both cell lines was blocked by the presence of EGTA in the culture medium. EGTA did not inhibit IL-1/PMA activation of an SV40 early promoter-CAT fusion gene in either EL4 or Jurkat cells; therefore, calcium was not required for IL-1 or PMA signal transduction. Jurkat cells were shown to differ from EL4 in their requirement for calcium mobilization. Two different calcium-dependent pathways of gene activation were distinguished, both of which were blocked by the immunosuppressive drug cyclosporin A.     

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.     

Cyclosporin A modulates the course of woodchuck hepatitis virus infection and induces chronicity.

Immunosuppression is known to influence the state of chronic hepatitis B virus infection, and is thought to increase the risk of developing chronic infection in newly exposed individuals. Cyclosporin A (CsA), an immunosuppressive agent that inhibits Th cell function, was administered to woodchucks chronically infected with woodchuck hepatitis virus (WHV), and resulted in a decreased severity of chronic hepatitis and an increased viremia during the treatment. Adult woodchucks inoculated with WHV and given CsA for 14 wk had increased viremias, decreased acute phase liver injury, and developed chronic infections at a higher rate compared with immunocompetent woodchucks given virus alone (chronicity in seven of seven WHV + CsA + vs zero of nine WHV + CsA-; p less than 0.001). These results in a relevant animal model of hepatitis B virus infection indicate: 1) that liver injury in acute hepadnavirus infections is immune-mediated and not a direct cytopathic effect of virus replication; 2) that Th cells function in the inflammatory response and in the immunologic control of hepadnavirus infection; and 3) that suppression of Th cell function in acute hepadnavirus infection decreases liver injury but alters the outcome of infection in favor of chronicity. These results also suggest continued challenges in the application of CsA in liver transplantation for hepatitis B virus-induced diseases.     

Cyclosporin A and rabbit mammary prolactin receptors

The binding of cyclosporin A and ovine prolactin to rabbit mammary gland membranes was determined. CsA bound with a Kd of 2.2 X 10(-6)M whereas prolactin bound with a Kd of 2 X 10(-10)M. The binding of each ligand was an independent event and neither ligand influenced the binding of the other ligand showing that CsA does not inhibit the binding of prolactin to its specific receptor in this system.     

Immobilization of the fungus, Tolypocladium sp. for the production of Cyclosporin A

The fungus Tolypocladium sp. obtained from the culture collection of VCRC, Pondicherry, was immobilized in calcium alginate and used for the production of Cyclosporin A in a packed bed reactor under batch and continuous flow modes. The precursor amino acids, L-valine and L-leucine increased the yield of Cyc A when added individually; however when added together, the increase in the yield was no better than as added individually. The half-life of the biocatalyst was found to be around six months.     

Cyclosporin A as prophylaxis against graft-versus-host disease in 36 patients.

Oral cyclosporin A was used as prophylaxis against graft-versus-host disease in (a) 31 patients with acute leukaemia or aplastic anaemia given transplants of HLA-matched bone marrow and (b) five patients with inborn errors of metabolism given transplants of haplotype-identical (parental) bone marrow. Twenty-six patients survived longer than two months after the operation. Despite the cyclosporin A, 31 patients (86%) suffered an acute form of graft-versus-host disease and 22 (61%) a chronic form. Nevertheless, the disease was usually treatable with immunosuppressive agents and caused the death of only one patient. Cyclosporin A caused renal toxicity in all cases; occasionally this was associated with a "capillary leak" syndrome, fatal in two patients. In children hypertension, fits, and fluid retention were common side effects. Blood concentrations of cyclosporin A correlated with blood urea values and blood pressure but did not predict the occurrence of graft-versus-host disease. Four different dose schedules were used to find the optimum way to administer this drug. Oral cyclosporin A is extremely effective at reducing the severity of graft-versus-host disease, but prevention of the disease is limited by toxicity of the drug and variable absorption. Better results might be achieved with parenteral administration or by using the drug in combination with other methods.     

Cyclosporin A Disrupts Notch Signaling and Vascular Lumen Maintenance

Cyclosporin A (CSA) suppresses immune function by blocking the cyclophilin A and calcineurin/NFAT signaling pathways. In addition to immunosuppression, CSA has also been shown to have a wide range of effects in the cardiovascular system including disruption of heart valve development, smooth muscle cell proliferation, and angiogenesis inhibition. Circumstantial evidence has suggested that CSA might control Notch signaling which is also a potent regulator of cardiovascular function. Therefore, the goal of this project was to determine if CSA controls Notch and to dissect the molecular mechanism(s) by which CSA impacts cardiovascular homeostasis. We found that CSA blocked JAG1, but not Dll4 mediated Notch1 NICD cleavage in transfected 293T cells and decreased Notch signaling in zebrafish embryos. CSA suppression of Notch was linked to cyclophilin A but not calcineurin/NFAT inhibition since N-MeVal-4-CsA but not FK506 decreased Notch1 NICD cleavage. To examine the effect of CSA on vascular development and function, double transgenic Fli1-GFP/Gata1-RFP zebrafish embryos were treated with CSA and monitored for vasculogenesis, angiogenesis, and overall cardiovascular function. Vascular patterning was not obviously impacted by CSA treatment and contrary to the anti-angiogenic activity ascribed to CSA, angiogenic sprouting of ISV vessels was normal in CSA treated embryos. Most strikingly, CSA treated embryos exhibited a progressive decline in blood flow that was associated with eventual collapse of vascular luminal structures. Vascular collapse in zebrafish embryos was partially rescued by global Notch inhibition with DAPT suggesting that disruption of normal Notch signaling by CSA may be linked to vascular collapse. However, multiple signaling pathways likely cause the vascular collapse phenotype since both cyclophilin A and calcineurin/NFAT were required for normal vascular function. Collectively, these results show that CSA is a novel inhibitor of Notch signaling and vascular function in zebrafish embryos.     

The peripheral nerve allograft in the primate immunosuppressed with Cyclosporin A: I. Histologic and electrophysiologic assessment.

Nerve regeneration across peripheral nerve allografts and control autografts in primates immunosuppressed with Cyclosporin A was quantitatively evaluated by electrophysiologic and histologic methods. Twelve cynomolgus monkeys received 3-cm autografts and allografts in contralateral ulnar nerves. They were immunosuppressed with Cyclosporin A at 25 mg/kg per day or placebo vehicle. Morphometric analysis of nerve graft and distal nerve segments was assessed at 1 year after engraftment. Quantitative electrophysiologic studies were performed percutaneously at 6 and 12 months, and compound action potentials were measured directly across the nerve grafts at 1 year. Excellent regeneration was seen across autografts and allografts in Cyclosporin A-treated and placebo-treated recipients.     

Cyclosporin A regulates the expression of HLA-DR on human monocytes by two different mechanisms.

Cyclosporin A (CsA), but not its nonimmunosuppressive analog cyclosporin H (CsH), inhibited the expression of HLA-DR in human monocytes. Induction of HLA-DR by interferon (IFN)-gamma in fresh monocytes was also inhibited by CsA and not by CsH. However, when monocytes were pretreated with either CsA or CsH for 16 hr prior to the addition of IFN-gamma, HLA-DR expression was increased, probably because of a cyclosporin-induced increase in the number of IFN-gamma receptors. Down-regulation of the HLA-DR mRNA by CsA was found to be dependent on continuous protein synthesis. IFN-alpha also inhibited the IFN-gamma-induced HLA-DR mRNA expression and showed synergy with CsA at low concentrations but not at high concentrations of the drugs. A common mechanistic element in the pathways of CsA and IFN-alpha is proposed.