Department of Biotechnology, Faculty of Life Sciences and Informatics, Balochistan University of Information Technology Engineering and Management Sciences, (BUITEMS), Quetta, Pakistan
Received Date: July 12, 2017; Accepted Date: August 04, 2017; Published Date: August 10, 2017
Citation: Khan NT (2017) Cyclosporin A Production from Tolipocladium inflatum. Gen Med (Los Angeles) 5:294. doi:10.4172/2327-5146.1000294
Copyright: © 2017 Khan NT. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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The demand for this drug in the US market is estimated at $450 million. Cyclosporin A demand is increasing worldwide because of its enhanced anti-fungal and anti-viral potency that it can be used to treat diseases like canine skin disease and rheumatoid arthritis. Cyclosporin A is easily produced from the fungus Tolipocladium inflatum by the process of fermentation in a bioreactor under optimum reaction conditions to obtain maximum yield of the antibiotic. Currently attempts have been made to increase its solubility to enhance its absorption in the body.
Tolipocladium inflatum; Cyclosporin A; Submerged culture fermentation
Tolipocladium inflatum is an ascomycete fungus, a pathogen of beetle larvae and best known as the producer of the anti-fungal antibiotic cyclosporin A. This fungus is a prolific producer of bioactive secondary metabolites with potential applications in medicine and agriculture. It was originally isolated from Norwegian soil sample. Tolipocladium inflatum is the asexual state whereas the sexual state Elaphocordyceps subsessilis is the pathogen of beetles and is widely distributed in the soil  (Table 1).
Table 1: Classification of Tolipocladium inflatum.
During the asexual phase of its life cycle, Tolipocladium inflatum lives as saprotrophs in soil. Asexual mode of reproduction is by means of conidia, positioned on phialides with inflated bases. While reproduce sexually by producing ascospores through the fusion of opposite mating type spores .
This drug possesses low toxicity to mammals, anti-viral and antifungal properties. It is the most commonly prescribed immunosuppressive drug for the treatment of patients with organ transplantation, autoimmune diseases including AIDS, owing to its superior T-cell specificity and low levels of myelotoxicity. Organisms like Tolipocladium inflatum , Fusarium solani , Neocosmospora varinfecta, and Aspergillus terreus are well known producers of cyclosporin A. It possesses anti-fungal properties. Cyclosporin A consisted of 11 hydrophobic amino acids. It is neutral and very soluble in all organic solvents. Molecular weight of cyclosporine A 1202.6 g/mol with UV absorption of 215 nm  (Figure 1).
Mode of action
Cyclosporin A is a non-polar cyclic oligopeptide produced by the fungus Tolipocladium inflatum. Cyclosporin A is an important immunosuppressive agent that inhibits the functioning of several proteins that are involved in the activation of T-cells at the level of mRNA transcription thus affecting T-lymphocytes. Cyclosporin A binds with its intracellular receptor cyclophilin to form a unique compex. This complex then binds to calcineurin which is a calcium and calmodulin dependant protein phosphatases, inhibiting its enzymatic activity. It was found that cyclosporin A suppresses the replication of hepatitis C virus genome in cultured hepatocytes. It can also inhibit IL2 resulting from T cell activation through calcineurin inhibition and also block cytochrome c release from mitochondria  (Figure 2).
Methods of production
Cyclosporin A is commonly produced by the following given methods:
• Submerged culture fermentation
• Static fermentation
• Solid state fermentation
• Enzymatic synthesis 
Requirements of fermentation process for Cyclosporin A production
• Carbon source i.e., glucose, sucrose, maltose, glycerol etc. It influences morphological differentiation of the fungus Tolipocladium inflatum.
• Organic and inorganic nitrogen sources i.e., casein peptone, bactopeptone, yeast extract, xylose, galactose, beef extract, malt extract etc.
• Amino acids like L-valine, DL amino butyric acid, L-leucine, glycine, DL methionine etc.
• Solvents like concentrated hydrochloric acid, acetonitrile, sodium hydroxide, n-butyl acetate, sulphuric acid etc.
• Salts like sodium chloride, calcium chloride, zinc sulphate, magnesium sulphate, magnesium chloride, ferric chloride, Tween 20 etc.
• Organism subculture incubated at 25 degree centigrade for 7 or 12 days
• Fungal agar disc from 7 days old culture or 12 days old culture
• 100 ml of autoclaved broth medium
• Incubation conditions for cultured flask at 30 degree centigrade for 7 days
• Agitation rates at 150 and 200 rpm
• pH range of the medium 2 to 9
Fungal agar disc from 7 days old culture or 12 days old culture was inoculated into 100 ml of autoclaved broth medium in a rotatory shaker that contains all the essential nutrients required for the fungal growth under appropriate incubation conditions of 30 degree centigrade for 7 days.
Then it is transferred to a fermentation bioreactor with proper aeration i.e., dissolved oxygen supply since Tolipocladiun inflatum grows under aerobic conditions. Probe indicators and pH meter detect temperature and pH changes respectively and is automatically adjusted to optimum conditions .
During the theoretical phase of the fermentation reaction when maximum production of cyclosporin takes place as indicated by the curve analysis techniques that monitors the ongoing fermentation reaction (Figure 3).
After that it is followed by a decline phase in which cyclosporin production slows down because of depletion of one or more critical carbon sources used which act as limiting reagents.
The desired drug is then separated and purified by filtration and distillation i.e., downstream processing .
Current research and development of Cyclosporin A
Attempts have been made in order to increase the level of absorption of Cyclosporin A in the body. Therefore different approaches had been used to solubilize the highly hydrophobic cyclosporin A. So extensive study of cyclosporin A structure will help the researchers to understand its highly hydrophobic nature which in turn will aid in devising strategies or methods to solubilize this drug. Work has been carried out that highlighted the immunosuppressive nature of this drug by using different dosage levels in different model organisms . Further it was revealed that Cyclosporin A selectively inhibits lymphocytes proliferation at the same time does not affects somatic cells proliferation. Series of experiments were conducted by using applications of genetic engineering to genetically transform the fungus in order to increase its growth rate for large scale production of cyclosporin A [13,14]. For this purpose various genetically modified strains of Tolipocladium are under study. Besides, optimization of fermentation process by controlling and maintaining optimum reaction conditions is another effort to increase the yield of the obtained product. Currently new oral cyclosporin A has been developed for patients with rheumatoid arthritis [15-17].
Uses of Cyclosporin A
• Prevention of tissue rejection in case of organ transplantation e.g. Kidney, liver, heart and bone marrow transplants.
• Acts as T-lymphocytes suppresses and also inhibits interleukins.
• Used in combination with other immunosuppressant and steroid medications.
• Use to treat canine skin disease and rheumatoid arthritis .
Side effects of Cyclosporin A
• Causes metabolic alterations in liver, muscle and adipose tissue, which may contribute to the development of dyslipidemia and insulin resistance 
• Acute kidney injury
• Hyperlipidemia (especially hypertriglyceridemia)
• Gastrointestinal upset and headaches
• Hypertrichosis 
• Increase testosterone level 
• Gingival overgrowth 
Cyclosporin A is one of the mostly used drugs that are easily produced from a biological organism such as fungi. This drug is used to treat patients with organ transplantation, canine skin disease and rheumatoid arthritis.