Research review paperCyclosporin A — A review on fermentative production, downstream processing and pharmacological applications
Introduction
Microorganisms are being used for thousands of years to supply fermented products such as bread, beer, wine, distilled spirits, vinegar, cheese, pickles and many other traditional regional products. The importance of microbes increased significantly during World War I during which development of fermentation, bioconversion, and enzymatic processes yielded many useful products such as amino acids, nucleotides, vitamins, organic acids, solvents, vaccines and polysaccharides. A major segment of these products are represented by secondary metabolites such as antibiotics. Many antibiotics have been used for purposes other than killing or inhibiting the growth of bacteria and/or fungi. These include hypocholesterolemic agents, immunosuppressants, anticancer agents, bioherbicides, bioinsecticides, coccidiostats, animal growth promoters, and ergot alkaloids (Demain, 2000).
Clinically, immunosuppression is defined as the inhibition of an immune response while avoiding the complications of immunodeficiency (Halloran, 1996). Patients who undergo solid organ transplantation require life-long immunosuppressive therapy to prevent allograft rejection. The success of post-transplantation patient care largely lies on the appropriate utilization of immunosuppressants. Immunosuppressants are a class of drugs which are capable of inhibiting the body's immune system. Many of the agents included in this category are also cytotoxic (cell poisons) and are used in the treatment of cancer. These drugs are used in organ transplant patients to prevent rejection of the organ by the body by decreasing the body's own natural defense to foreign bodies (such as the transplanted organs), and are also useful in the treatment of autoimmune diseases. The classification of immunosuppressants based on their primary sites of action is shown in Table 1.
Cyclosporins are a group of closely related cyclic undecapeptides produced as secondary metabolites by strains of fungi imperfecti, Cylindrocarpum lucidum Booth and Tolypocladium inflatum Gams isolated from soil samples (Dreyfuss et al., 1976, Borel et al., 1976). Both strains were isolated from soil samples collected in Wisconsin (USA) and Hardanger Vidda (Norway). CyA is the main component of this family of cyclic peptides containing 11 amino acids. CyA was isolated in the early 1970s on the basis of its ability to inhibit a mixed lymphocyte reaction (MLR), a measure of alloreactivity. CyA can be considered as the first of this kind of drug of a new generation of immunosuppressants. It is probably the first one to demonstrate the feasibility of an immunopharmacologic approach to the modulation of the immune response by drugs.
The introduction of CyA made an important advance in the immunotherapy of bone marrow and organ transplantations. CyA is one of the most commonly prescribed immunosuppressive drugs for the treatment of patients with organ transplantation and autoimmune diseases including AIDS owing to its superior T-cell specificity and low levels of myelotoxicity (Kahan, 1984, Schindler, 1985).
The organisms reported to produce CyA include T. inflatum (Agathos et al., 1986), Fusarium solani (Sawai et al., 1981), Neocosmospora varinfecta (Nakajima et al., 1988) and Aspergillus terreus (Sallam et al., 2003). CyA is reported to be produced by submerged culture fermentation (Agathos et al., 1986, Survase et al., 2009d), static fermentation (Balaraman and Mathew, 2006), solid state fermentation (Survase et al., 2009a), and also synthesized enzymically (Billich and Zocher, 1987).
Presently, CyA is available in the US market under brand names as Neoral®, Sandimmune®, Sandimmune® I.V by Novartis Pharmaceutical Corporation, USA; Gengraf® from Abbott Laboratories, USA; Restasis® from Allergan Inc USA; Apo-cyclosporin from Apotex Advancing Generics, Canada and Rhoxal-cyclosporin from Rhoxalpharma, USA. In India, Panium Bioral® by Panacea Biotech Ltd., Arpimune® from RPG Life Sciences and CyclophilME® from Biocon India Ltd. are available in the market.
Immunosuppressants which have gained considerable importance in the world market include cyclosporin A (CyA), tacrolimus, rapamycin and mycophenolate mofetil. In the present review, we discuss the chemical structure, pharmacological activities, biosynthetic pathway, fermentative production, downstream processing, pharmacokinetics and toxicity of CyA.
Section snippets
History
In March 1970, in the Microbiology Department at Sandoz Ltd., Basel, a Swiss pharmaceutical company, a fungus T. inflatum Gams was isolated by B. Thiele from two soil samples, the first from Wisconsin, USA and second from the Hardanger Vidda in Norway. In 1973, CyA was purified from the fungal extract of T. inflatum and in 1975 complete structural analysis was established (Wenger, 1982). CyA was first investigated as an anti-fungal antibiotic (Dreyfuss et al., 1976), but Borel et al. (1976)
Chemical structure
CyA is a neutral lipophilic cyclic polypeptide consisting of 11 amino acids and representative of this class which differs in their amino acid composition. It has molecular weight of 1202 and a molecular formula C62H111N11O12 (Fig. 1.). The acid hydrolysis of CyA showed that it is made up of eleven amino acids, ten of which are known aliphatic amino acids but the amino acid at position one was unknown (Wenger, 1982). All the amino acid residues have the 2S configuration, except for the alanine
Structure activity relationship
CyA to CyZ have been tested for antifungal activity as well as in many in vitro and in vivo assays for immunosuppressive activity (Borel et al., 1976, Borel et al., 1977, Wiesinger and Borel, 1979). The structure activity relationship was deduced from immunopharmacological data and is reviewed in detail elsewhere (Balakrishnan and Pandey, 1996a, Rehacek and De-xiu, 1991).
At present, there is still a need to modify the CyA structure in order to improve the biological activity and/or
Physical properties
CyA consists of 11 amino acids with a molecular weight of 1202.6 and occurs as a white solid with a melting point of 148 °C to 151 °C (natural) and 149 °C to 150 °C (synthetic) (IARC, 1990). It is slightly soluble in water and soluble in organic solvents (Budavari et al., 1996). The solubility of CyA at 25 °C (in mg/g) is 0.04 in water, 1.6 in n-hexane and greater than 500 in methanol, ethanol and acetonitrile (Rosenthaler and Keller, 1990). In aqueous solution, CyA exhibits pH independent,
Biosynthesis
The biosynthesis of cyclosporins is likely to proceed by a non-ribosomal process involving multifunctional enzyme as indicated by the cyclic structure, presence of N-methylated amino acids and several unusual amino acids in their structure (Lawen and Zocher, 1990). Similar processes are reported for fungal depsipeptides enniatin (Zocher et al., 1986), gramimicidin H (Kleinkauf and Koischwitz, 1978) and beauvericin (Peeters et al., 1988). This characteristic non-ribosomal biosynthetic pathway
Mode of action
Many of the microbial metabolites have toxic side effects along with medicinal value, and hence cannot be used in clinical practice. To eliminate the side effects of these metabolites, their mode of action should be studied in detail. This knowledge gives an insight into the mode of damage to the microorganisms and also defines the sequence of enzyme reactions in some metabolic pathways. Various molecular biology techniques can be used to estimate the mode of action of bioactive substances at
Microorganisms
CyA is produced by many microorganisms (Table 3) which include T. inflatum (Agathos et al., 1986), F. solani (Sawai et al., 1981), Fusarium roseum (Ismaiel et al., 2010), N. varinfecta (Nakajima et al., 1988) and A. terreus (Sallam et al., 2003), but T. inflatum has emerged as the most widely used microorganism. The fungal genus, Tolypocladium, first described by Gams (1971), belongs to the class fungi imperfecti, occurring in soil or litter habitats. The species are characterized by white slow
Isolation and purification
Fungi produce a variety of cyclosporins with varying amino acid composition of which CyA is the most potent. Various purification processes to isolate pharmacopoeial grade CyA are reported in the literature. Conventionally, researchers extract fermented biomass with an organic solvent, evaporate the solvent, reextract the residue, concentrate and then subject the residue to various chromatographic processes to separate CyA from other cyclosporins and impurities. Fig. 2 describes the flow chart
Methods of analysis
Various methods such as immunoassays (Tredger et al., 2000), HPLC (Kreuzig, 1984), liquid chromatography–tandem mass spectrometry (Simpson et al., 1998) etc. have been used for CyA measurement in clinical samples. Although immunoassays fulfill the criteria of fast analysis, the cross-reactivity of the antibodies with inactive CyA metabolites is its main concern. On the other hand, HPLC is more time consuming. HPLC-tandem mass spectrometry assay is a realistic alternative to immunoassay for the
Pharmacokinetics
Pharmacokinetics has been used for many years to relate immunosuppressant dose to drug exposure in vivo. It is the primary method to measure drug absorption, distribution, metabolism, routes of excretion and interactions with other drugs. Concentration of CyA in blood and serum is monitored as a means of reducing the risk of nephrotoxicity or rejection associated with inappropriate drug concentrations. However, the pharmacokinetics of CyA in humans can be quite unpredictable and the
Toxicity
The main disadvantage in the therapeutic use of CyA is its toxic effects. Apart from the general risks of immunosuppression (opportunistic infection, malignancy), nephrotoxicity and hypertension are most relevant among the undesirable effects. Other side effects found occasionally are neurotoxicity, hepatoxicity, hyperlipidemia, anorexia, nausea, vomiting, paresthesia, hypertrichosis, gingival hyperplasia and tremor.
Renal toxicity of CyA is encircled by multiple effects on different glomerular
Drug interactions
There are various drug interactions reported with CyA. Caution should be exercised in patients receiving drug treatment with nephrotoxic drugs, cytotoxic drugs, immunosuppressants or radiation and drug affecting metabolism/absorption of CyA. If combined administration is unavoidable, careful monitoring of blood CyA concentration and appropriate modification of dosage are essential. Wadhwa et al. (1987) have systematically compiled the CyA drug interactions.
Zylber-Katz (1995) reported on
Therapeutic uses
CyA has a range of pharmacological activities including suppression of antibody-and cell-mediated responses, inhibition of chronic inflammatory reactions, fungicidal and antiparasitic activities, anti-HIV and anti-hepatitis C virus.
CyA potentiates the effect of some cytostatic drugs in both tumor and normal cells but it should also be noted that any form of immunosuppression of sufficient duration and intensity can lead to the development of certain forms of cancer. CyA may result in
Conclusions
As evident from the foregoing review, CyA is among the most important immunosuppressants used. In more than 35 years of CyA related research great insight has been gained regarding the production, purification, mechanism of action as well as applications of CyA. The numerous applications so far identified, together with several novel ones will surely result in a growing worldwide commercial demand for CyA. In the last few years, this fact has led to a multiplication of efforts to improve their
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