Survase Et Al., 2011, Biotech Advances

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discussions, stats, and author profiles for this publication at: http://www.researchgate.net/publication/50906973
Cyclosporin A - A review on fermentative

production, downstream processing and
pharmacological applications
ARTICLE in BIOTECHNOLOGY ADVANCES MARCH 2011
Impact Factor: 9.02 DOI: 10.1016/j.biotechadv.2011.03.004 Source: PubMed
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4 AUTHORS, INCLUDING:
Shrikant Survase
Lalit D. Kagliwal
American Process Inc.GA, USA
Institute of Chemical Technology, Mumbai
48 PUBLICATIONS 590 CITATIONS
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Uday S. Annapure
Institute of Chemical Technology, Mumbai
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Available from: Shrikant Survase

Retrieved on: 27 September 2015
Biotechnology Advances 29 (2011) 418435
Contents lists available at ScienceDirect
Biotechnology Advances
j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / b i o t e c h a d v
Research review paper
Cyclosporin A A review on fermentative production, downstream processing and

pharmacological applications
Shrikant A. Survase, Lalit D. Kagliwal, Uday S. Annapure, Rekha S. Singhal
Food Engineering and Technology Department, Institute of Chemical Technology, Matunga, Mumbai 400 019, India
a r t i c l e
i n f o
Article history:
Received 10 October 2010
Received in revised form 5 March 2011
Accepted 15 March 2011
Available online 5 April 2011
Keywords:
Cyclosporin A
Fermentation
Immunosuppressants
Tolypocladium inatum
a b s t r a c t
In present times, the immunosuppressants have gained considerable importance in the world market.
Cyclosporin A (CyA) is a cyclic undecapeptide with a variety of biological activities including immunosuppressive, anti-inammatory, antifungal and antiparasitic properties. CyA is produced by various types of
fermentation techniques using Tolypocladium inatum. In the present review, we discuss the biosynthetic
pathway, fermentative production, downstream processing and pharmacological activities of CyA.
2011 Elsevier Inc. All rights reserved.
Contents
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
Introduction . . . . . . . . . . . . . . . . .
History. . . . . . . . . . . . . . . . . . . .
Chemical structure . . . . . . . . . . . . . .
Structure activity relationship . . . . . . . . .
Physical properties . . . . . . . . . . . . . .
Biosynthesis . . . . . . . . . . . . . . . . .
Mode of action . . . . . . . . . . . . . . . .
Fermentative production . . . . . . . . . . .
8.1.
Microorganisms . . . . . . . . . . . .
8.2.
Fermentation parameters . . . . . . . .
8.2.1.
Effect of carbon source(s) . . .
8.2.2.
Effect of nitrogen source(s) . .
8.2.3.
Effect of minerals . . . . . . .
8.2.4.
Effect of environmental factors .
8.2.5.
Effect of aeration and agitation .
8.3.
Strain improvement . . . . . . . . . .
8.4.
Effect of precursors . . . . . . . . . .
8.5.
Immobilization . . . . . . . . . . . .
8.6.
Production of CyA by SSF . . . . . . . .
Isolation and purication . . . . . . . . . . .
Methods of analysis. . . . . . . . . . . . . .
Pharmacokinetics . . . . . . . . . . . . . . .
Toxicity . . . . . . . . . . . . . . . . . . .
Drug interactions . . . . . . . . . . . . . . .
Therapeutic uses . . . . . . . . . . . . . . .
14.1. Use of CyA in organ transplantation . . .
14.2. CyA in parasitic infections . . . . . . .
14.3. CyA in autoimmune diseases . . . . . .
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Corresponding author. Tel.: + 91 22 3361 2512; fax: + 91 22 3361 1020.

E-mail address: rs.singhal@ictmumbai.edu.in (R.S. Singhal).
0734-9750/$ see front matter 2011 Elsevier Inc. All rights reserved.
doi:10.1016/j.biotechadv.2011.03.004
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419
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S.A. Survase et al. / Biotechnology Advances 29 (2011) 418435
14.4. CyA against hepatitis C . . . . . . .

14.5. CyA against human immunodeciency
14.6. CyA on eye infections . . . . . . . .
14.7. Use of CyA in cancer. . . . . . . . .
15.
Conclusions . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . .
. . . . . .
virus (HIV)
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1. 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 signicantly 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 dened as the inhibition of an
immune response while avoiding the complications of immunodeciency (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 classication 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 inatum 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 rst of this kind of drug of a new generation of
immunosuppressants. It is probably the rst 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 specicity and low
levels of myelotoxicity (Kahan, 1984; Schindler, 1985).
The organisms reported to produce CyA include T. inatum (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 Pharmaceu-
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429
430
430
430
431
431
tical 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.
2. History
In March 1970, in the Microbiology Department at Sandoz Ltd.,
Basel, a Swiss pharmaceutical company, a fungus T. inatum Gams was
isolated by B. Thiele from two soil samples, the rst from Wisconsin,
USA and second from the Hardanger Vidda in Norway. In 1973, CyA
was puried from the fungal extract of T. inatum and in 1975 complete structural analysis was established (Wenger, 1982). CyA was rst
investigated as an anti-fungal antibiotic (Dreyfuss et al., 1976), but
Borel et al. (1976) discovered its immunosuppressive activity. This led
to further investigations into its properties involving further immunological tests and investigations into its structure and synthesis.
CyA was approved by the USFDA for clinical use in 1983 to prevent
graft rejection in transplantation. It took 12 years for CyA to be
developed into a drug Sandimmune and was rst registered in
Switzerland. In 1984, synthetic CyA was produced. It was then possible
for the CyA to be chemically modied in every possible way. However,
none of the derivatives were found to have greater potency or lower
side effects than CyA itself.
Before the introduction of CyA, the immunosuppressants used
were methotrexate, azothioprine and corticosteroids. Beveridge
(1986) reported that they block cellular division non-specically and
thereby inhibit the proliferation of the immunocompetent cells too
which were attributed to their side effects. In contrast, CyA does not
cause myelotoxicity and/or impaired the proliferation of hemopoietic
stem cells (von Wartburg and Traber, 1986; Borel, 1981). Mcintosh
and Thomson (1980) reported that CyA suppresses lymphocytic function without damaging the phagocytic activity and migratory capacity
of the reticuloendothelial system which made its use in clinical
transplantation attractive. The discovery of CyA led the way to an era of
selective lymphocyte inhibition. It enabled expertise in clinical, technical and immunobiological aspects of transplantation to be put into
practice and changed the face of transplantation.
3. 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 conguration,
except for the alanine residue at position 8 which has the 2R
conguration and achiral sarcosine at position 3. Amino acid residues
420
Table 1
Classication of immunosuppressant antibiotics on the basis of site of action.
Site of action
Examples
Mechanism of action
Regulators of gene expression
Glucocorticoids
Cyclophosphamide
Inhibitors of de novo
purine synthesis
Methotrexate, Azathioprine, Mycophenolic acid
Inhibitors of de novo
pyrimidine synthesis
Leunomide
Inhibits the expression of genes for IL-2 and other mediators
Alkylating agents
Inhibitors of kinases
and phosphatases
Cyclosporin A,
FK-506 (tacrolimus), rapamycin
Inhibits the phosphatase activity of calcineurin, thereby suppressing the

production of IL-2 and other cytokines,
inhibits kinases required for cell cycling and responses to IL-2
at position 16 of the backbone adopt an antiparallel -pleated sheet

conrmation which contains 3 transannular H-bonds and is markedly twisted (Wenger, 1982). The remaining residues 711 form an
open loop which carries the only cis amide linkage between two
adjacent N-methyl leucine residues (position 9 and 10). The remaining H-bond of a 3-L type, serves to hold the backbone in a folded Lshape. Amino acids at positions 1, 3, 4, 6, 9, 10, and 11 are N-methylated
which is responsible for the lipophilic nature of the molecule. H-bond
formation by four available amide groups contributes to the rigidity of
the skeleton.
Petcher et al. (1976) studied the crystal and molecular structure
of an iodo-derivative of CyA by using X-ray crystallographic analysis.
As CyA was hard to crystallize on its own it was analyzed as
crystalline iodo-CyA. The unknown amino acid at position 1 was
found to be (2S,3R,4R,6E)-3-hydroxy-4-methyl-2-(methylamino)-6octenoic acid. According to amino acid nomenclature, it is now
designated as (4R)-4[(E)-2-butenyl]-4-[N-di-methyl-L-threonine]
and abbreviated as MeBmt. Nakajima et al. (1988) isolated a derivative of MeBmt, 2-acetylamino-3-hydroxy-4-methyloct-6-enoic
acid, from the fermentation medium of the fungus N. varinfecta
producing CyA. This was the rst report on isolation of the derivative
of the MeBmt. MeBmt and its derivative have been synthesized as
intermediates in the synthesis of CyA (Wenger, 1983; Evans and
Weber, 1986).
Several natural and synthetic structural congeners of CyA with
substitutions on the ring structure have been identied or synthesized.
All the natural cyclosporins isolated (B-I, K-Z, Cy26-Cy32) (Traber
et al., 1982; Traber et al., 1977a; 1977b; Kleinkauf and von Dhren,
1997; Kallen et al., 1997; Kleinkauf and von Dhren, 1999) so far are
given in Table 2. Twenty seven of the 32 analogs have a single alteration with respect to amino acid exchange or lack of N-methylation;
Alkylate DNA bases,

suppresses B-lymphocyte mediated response
Suppress inammatory responses through release of adenosine,
induces the apoptosis of activated T-lymphocytes,
inhibiting the synthesis of both purines and pyrimidines
Inhibits dihydroorotate dehydrogenase, thereby suppressing pyrimidine
nucleotide synthesis
only 5 compounds are doubly altered. The structures of the congeners

have been determined by spectroscopic evidence, hydrolytic cleavage,
identication of amino acid prole, chemical correlation reactions, and
X-ray analysis.
The only common amino acid in all cyclosporins is D-alanine (D-Ala)
at position 8 of the ring whereas sarcosine at position 3 is common in 31
cyclosporins. At positions 6, 9 and 10, the only modication compared
to CyA consists of the lack of the N-methyl group in these positions.
The variability was greatest at position 2, which can be occupied
with L-aminobutyric acid (Abu), L-alanine (Ala), L-threonine (Thr),
L-valine (Val) or L-norvaline (Nva). In addition to these naturally
occurring cyclosporins, some cyclosporins differing in positions 1, 2,
and 8 from CyA could be produced by feeding amino acid precursors
to the fungus (Rehacek and De-xiu, 1991). The course of cyclosporin
biosynthesis is strongly inuenced by exogenous addition of amino
acid precursors (Traber et al., 1989; Lee and Agathos, 1989). Jegorov
et al. (1995) isolated new natural cyclosporins from Tolypocladium
terricola. The chemical structures were found to be (Leu4)CyA and
(MeLeul)CyA and were given the name CyJ.
The specic incorporation achieved by the addition of DL-allyl
glycine to the medium resulted in the production of (allyl gly 2) CyA.
Exogenous supply of L--cyclohexyl alanine led to the production of
(Me cyclohexyl ala) CyA. Substitution of D-alanine in position 8 by
D-serine gave new (D-ser 8) analogs of CyA, CyC, CyD and CyG as
well as (Allyl gly 2) CyA with high immunosuppressive property (Traber
et al., 1989). Lawen et al. (1994) reported the biosynthesis of ring
extend cyclosporins. The introduction of -alanine into position 7 or 8 of
the ring instead of the -alanine makes the 33 membered ring of the
cyclo undecapeptide to a 34 membered ring of CyA. Both -Ala at 7 CyA
and -Ala at 8 CyA showed impressive immunosuppressive activity.
Galpin et al. (1988) reported chemical synthesis of CyA analogs
containing (Me)Thr, (Me)Ser, hydroxy proline and di-amino butyric
acid at position l and amino butyric acid, Nva, Nle and Thr at position 2
by stepwise assembly of the undecapeptide fragments followed by
cyclization with a variety of reagents.
4. Structure activity relationship
Fig. 1. Chemical structure of CyA.
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 physicochemical properties
of the existing cyclosporins, whether natural or synthetic. Mutter et al.
(2004) reported on a method for the production of cyclosporin derivatives, where the peptide chain comprises at least one pseudoproline
421
Table 2
Amino acid composition of various cyclosporins (von Dhren, 2004).
Metabolite
CyA
CyB
CyC
CyD
CyE
CyF
CyG
CyH
CyI
CyK
CyL
CyM
CyN
CyO
CyP
CyQ
CyR
CyS
CyT
CyU
CyV
CyW
CyX
CyY
CyZ
Cy26
Cy27
Cy28
Cy29
Cy30
Cy31
Cy32
Amino acid composition

1
10
11
C9
C9
C9
C9
C9
desoxy-C9
C9
C9
C9
desoxy-C9
N-desmethyl-C9
C9
C9
MeLeu
N-desmethyl-C9
C9
C9
C9
C9
C9
C9
C9
C9
C9
Me-Amino octanoic
C9
N-desmethyl-C9
MeLeu
C9
MeLeu
C9
C9
Abu
Ala
Thr
Val
Abu
Abu
Nva
Abu
Val
Val
Abu
Nva
Nva
Nva
Thr
Abu
Abu
Thr
Abu
Abu
Abu
Thr
Nva
Nva
Abu
Nva
Val
Abu
Abu
Val
Abu
Abu
Sar
Sar
Sar
Sar
Sar
Sar
Sar
Sar
Sar
Sar
Sar
Sar
Sar
Sar
Sar
Sar
Sar
Sar
Sar
Sar
Sar
Sar
Sar
Sar
Sar
Sar
Sar
Sar
Sar
Sar
Sar
Gly
Meleu
MeLeu
MeLeu
MeLeu
MeLeu
MeLeu
MeLeu
MeLeu
MeLeu
MeLeu
MeLeu
MeLeu
MeLeu
MeLeu
MeLeu
Val
MeLeu
Val
MeLeu
MeLeu
MeLeu
MeLeu
MeLeu
MeLeu
MeLeu
MeLeu
MeLeu
MeLeu
MeILu
MeLeu
ILu
MeLeu
Val
Val
Val
Val
Val
Val
Val
Val
Val
Val
Val
Nva
Val
Val
Val
Val
Val
Val
Val
Val
Val
Val
Val
Val
Val
Leu
Val
Val
Val
Val
Val
Val
MeLeu
MeLeu
MeLeu
MeLeu
MeLeu
MeLeu
MeLeu
MeLeu
MeLeu
MeLeu
MeLeu
MeLeu
MeLeu
MeLeu
MeLeu
MeLeu
Leu(?)
MeLeu
MeLeu
Leu
MeLeu
MeLeu
MeLeu
Leu
MeLeu
MeLeu
MeLeu
MeLeu
MeLeu
MeLeu
MeLeu
MeLeu
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Abu
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
D-Ala
MeLeu
MeLeu
MeLeu
MeLeu
MeLeu
MeLeu
MeLeu
MeLeu
MeLeu
MeLeu
MeLeu
MeLeu
MeLeu
MeLeu
MeLeu
MeLeu
MeLeu
MeLeu
MeLeu
MeLeu
MeLeu
MeLeu
Leu
MeLeu
MeLeu
MeLeu
MeLeu
MeLeu
MeLeu
MeLeu
MeLeu
MeLeu
MeLeu
MeLeu
MeLeu
MeLeu
MeLeu
MeLeu
MeLeu
MeLeu
Leu
MeLeu
MeLeu
MeLeu
Leu
MeLeu
MeLeu
MeLeu
Leu(?)
MeLeu
Leu
MeLeu
MeLeu
MeLeu
MeLeu
MeLeu
MeLeu
MeLeu
MeLeu
MeLeu
MeLeu
MeLeu
MeLeu
MeLeu
MeVal
MeVal
MeVal
MeVal
Val
MeVal
MeVal
D-MeVal
MeVal
MeVal
MeVal
MeVal
MeVal
MeVal
MeVal
MeVal
MeVal
MeVal
MeVal
MeVal
MeVal
Val
MeVal
MeVal
MeVal
MeVal
MeVal
MeVal
MeVal
MeVal
MeVal
MeVal
D-Ala
D-Ala
D-Ala
D-Ala
D-Ala
D-Ala
D-Ala
D-Ala
D-Ala
D-Ala
D-Ala
D-Ala
D-Ala
D-Ala
D-Ala
D-Ala
D-Ala
D-Ala
D-Ala
D-Ala
D-Ala
D-Ala
D-Ala
D-Ala
D-Ala
D-Ala
D-Ala
D-Ala
D-Ala
D-Ala
D-Ala
Where, MeLeu methyl leucine, MeILu methyl isoleucine, ILu isoleucine, Abu L-aminobutyric acid, Ala L-alanine, Thr L-threonine, Val L-valine, Nva L-norvaline, Sar
sarcocine, Gly glycine.
type non-natural amino acid molecule. They synthesized derivatives

with improved biological activities and improved physicochemical
properties. They found that introduction of a pseudo-proline within
the cyclosporin chain allows preparation of a prodrug of the same
cyclosporin and introduction of highly water soluble polymer such as
polyethylene glycol suppresses the hydrophobic character of previous
cyclosporins.
It is stable in solution at temperatures below 30 C, but is sensitive

to light, cold, and oxidization (IARC, 1990). CyA is incompatible with
alkali metals, aluminum, and heat. Hazardous combustion or decomposition products include carbon monoxide, carbon dioxide, nitrogen
oxides, hydrogen chloride gas and phosgene (Sigma, 2000).
6. Biosynthesis
5. 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, exothermic solubility behavior characterized by an
inverse proportionality with respect to temperature. The solubility of
CyA in water at 5 C is at least 10 times higher than that at 37 C,
possibly as a result of stronger intramolecular hydrogen bonding at
higher temperature (Ismailos et al., 1991).
Malaekeh-Nikouei et al. (2007) found the aqueous solubility of CyA
to increase by 10 and 80 fold in the presence of -cyclodextrin (-CD)
and hydroxylpropyl--CD (HP--CD), respectively. They also reported
a mixture of 15% w/v -CD and 20% w/v HP--CD to be optimal for
increasing the aqueous solubility of CyA. Ismailos et al. (1994) found
solubility of CyA to increase in the presence of d-alphatocopherylpolyethylene-glycol-1000 succinate at temperatures 5 C, 20 C and
37 C.
The biosynthesis of cyclosporins is likely to proceed by a nonribosomal 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 directed by multienzyme thiotemplates is also
observed for other secondary metabolites such as actinomycin and ergot
alkaloids (Katz, 1974; Beacco et al., 1978). Biosynthesis of these compounds is directed from complex enzyme systems utilizing unusual
amino acids in addition to the known amino acids to generate peptides
differing from the linear mRNA-directed sequence of ribosomally
derived polypeptides. CyA and its homologues are synthesized by a
single multifunctional enzyme cyclosporin synthetase (CySyn) from
their precursor amino acids. Biosynthetic aspects have been reviewed by
Kleinkauf and von Dhren (1997, 1999), and Kallen et al. (1997).
Studies so far have been done by feeding experiments with 13C
(Kobel et al., 1983) and 14C-labeled precursors (Zocher et al., 1984).
Kobel et al. (1983) observed that by feeding 13C-labeled acetate and
methionine, the constituent amino acid MeBmt is built up by headto-tail coupling of four acetate units, whereas the C-methyl in the
422
carbon chain and the seven N-methyl groups in CyA originate from
the S-methyl of methionine. Kobel and Traber (1982) reported
exogenous supply of amino acid precursors in the fermentation
medium to strongly inuence the cyclosporin biosynthesis. This
can be seen from the fact that feeding of L--Abu, L-Ala, L-Thr, L-Val
and L-Nva gave enhanced yields of the CyA, CyB, CyC, CyD, and CyG,
respectively. Zocher et al. (1984) reported that 14C-labeled amino acid
feeding selectively incorporated L-Leu, L-Val, Gly and D- and L-Ala in
to CyA and CyC in the cultures of T. inatum. They also reported that all
N-methyl groups originate from methionine after performing experiments with L-(14methyl)-methionine. They proposed a possible mechanism of CyA synthesis as follows:
Synthesis of all 11 constituent amino acids

Activation of each amino acid
N-methylation and peptide bond formation, and nally
The cyclization reaction
Dittomann et al. (1994) showed that cyclosporin synthesis occurs as

a single linear undecapeptide precursor. They found D-alanine at
position 8 to be the starting amino acid in the biosynthetic process. All
the four intermediate peptides of the growing peptide chain isolated
represent partial sequences of CyA starting with D-alanine. This strongly
suggests the stepwise synthesis of a single linear peptide precursor of
CyA. CyA analogs could be prepared by precursor directed biosynthesis.
But incorporation of constituent and foreign amino acids demonstrates
low specicity of the biosynthesis (Zocher et al., 1982).
Attempts to characterize the enzyme system responsible for synthesis of cyclosporins rst led to the enrichment of an enzyme fraction
catalyzing the synthesis of the diketopiperazine cyclo-(D-Ala-MeLeu),
representing a partial sequence (positions 8 and 9) of CyA (Zocher et al.,
1986). This preparation was able to activate all constitutive amino acids
of CyA as thioesters via aminoadenylation; however, total synthesis of
CyA was not observed. Further efforts by Billich and Zocher (1987)
reported total in vitro synthesis of several cyclosporins by partially
puried CySyn fractions. The in vitro biosynthesis of several cyclosporins
which were not obtainable by fermentation has been reported by Lawen
et al. (1989). Billich and Zocher (1987) characterized the CySyn from
high producer mutants Tolypocladium niveum 7939/F and 7939/4547,
48. In these strains it was suggested that the presence of higher enzyme
levels was exerted by gene dosage, relaxed regulation at the
transcriptional level, or a reduced level of protein degradation.
Lawen and Zocher (1990) and Weber et al. (1994) reported that
the CySyn enzyme is composed of eleven modules, each being responsible for recognition, activation and modication of one substrate
and a small twelfth module putatively responsible for cyclization.
Marahiel et al. (1997) stated that each module of CySyn essentially
consists of a central adenylation domain for recognition and activation, thiolation domain for covalent binding of adenylated amino
acid on phosphopantetheine and condensation domain for elongation
step. Seven modules harbor an additional methyltransferase domain
for N-methylation (Husi et al., 1997). The unusual amino acid as Abu is
provided by main metabolic pathways of the cell, whereas D-Ala and
Bmt are synthesized by the Bmt polyketide synthase (Offenzeller
et al., 1993) and D-alanine racemase (Hoffmann et al., 1994). D-Ala is
synthesized by racemization of L-Ala, and is catalyzed by alanine
racemase with pyridoxal phosphate as cofactor.
Lawen and Zocher (1990) reported CySyn to be the most complex
enzymatically active multienzyme polypeptide chain of molecular
weight approximately 800 kDa. They found that 4-phosphopantetheine act as a prosthetic group of CySyn similar to other peptide
synthetases. This enzyme activates as thioesters via amino adenylation and carries specic N-methylation. Here S-adenosyl-L-methionine serves as methyl group donor. Methyl transferase activity is an
integral part of this enzyme which could be shown by a photoafnity
labeling method. It showed cross reactions with the monoclonal
antibodies directed against enniatin synthetase. Schmidt et al. (1992)

determined the molecular mass of CySyn by SDS-PAGE and CsCl
density gradient centrifugation and found it to be 1.4 MDa by both the
methods. The sedimentation coefcient of 26.3 S for CySyn indicates
an oblate overall shape of the enzyme. Weber et al. (1994) reported it
as a single chain polypeptide consisting of 15,281 amino acids with a
deduced molecular mass of 1.69 MDa.
Hoppert et al. (2001) reported on the structure and cellular localization of CySyn and alanine racemase in T. inatum. They observed
large globular complexes (25 nm in diameter) of native CySyn
assembled by smaller interconnected units by using electron microscopy. A signicant proportion of CySyn and D-alanine racemase was
detected at the vacuolar membrane and the cyclosporin was localized
in the fungal vacuole. They predicted a model for compartmentation
for cyclosporin synthesis. They reported that CySyn and alanine
racemase were attached to the outside of the vacuolar membrane and
synthesize cyclosporin from single amino acids where D-alanine was
the leading amino acid. Cyclosporin was subsequently deposited in the
vacuolar lumen.
Lawen and Zocher (1990) reported that with the exception of CyH
([D-MeVall1]CyA, i.e. CyA with D-methylvaline at position 11) all of the
cyclosporins are produced by CySyn. It catalyzes all the 40 reaction steps
necessary for the biosynthesis of CyA starting from the unmethylated
constituent amino acids.
A novel peptolide with several substitutions compared with CyA,
called SDZ 214103 was found. The main structural difference is a 2hydroxy acid instead of an amino acid at position 8. This novel drug
exhibited immunosuppressive, anti-inammatory, anti-fungal and
anti-parasitic activities similar to those of CyA. SDZ 214103 is produced by a multifunctional enzyme, peptolide synthetase (Lawen
et al., 1991a) with molecular mass similar to that of CySyn (Lawen
et al., 1991b) but its substrate specicity was found to be narrower
than that of CySyn (Lawen and Traber, 1993). This was conrmed by
Lawen et al. (1994) where they showed that peptolide synthetase was
not able to incorporate -alanine into position 7 or -hydroxy acid at
position 8. Lawen et al. (1994) showed that CySyn was capable of
introducing -alanine at position 8 instead of -alanine present in the
CyA ring. This leads to 34-membered in contrast to the 33-membered
ring of the undecapeptide CyA. Both [Ala7]CyA and [Ala8]CyA show
immunosuppressive activity.
The cloning of multienzyme structures eventually led to an understanding of the genetic organization of non-ribosomal templates.
Studies on peptide modifying enzymes may serve a signicant purpose
for the improvement of structureactivity relationships. The role of
peptides within the producing organisms and investigations on molecular genetics of regulatory controls of production should aid in
dening their role in nature.
Weber and Leitner (1994) reported on the manipulation of a giant
gene by DNA mediated transformation. They cloned cyclophilin gene
to establish a convenient transformation system. This gene encodes a
19,569 Da-protein with high similarity to the Neurospora crassa cyclophilin. The promoter region was combined with the Escherichia coli
hygromycin B phosphotransferase gene and the transcriptional
terminator of the Aspergillus nidulans trpC gene. This construct was
used to transform T. niveum which led to multiple and often tandem
integrations into the genome. Fragments of the CySyn gene inserted
into this vector were constructed and successfully used for gene
disruption with a high frequency, indicating a single copy of the synthetase. This was a rst step towards engineering the CySyn gene to
enable the production of new cyclosporins or cyclosporin derivatives.
Zocher et al. (1992) reported that cyclophilin is required to protect
the producer cell against the peptide by acting as acceptor of CyA.
Weber et al. (1994) cloned the synthetase gene by reverse genetics. They obtained sequence data by tryptic and proteinase Lys-C or
Glu-C digestion followed by N-terminal sequencing of isolated peptides. One of the 20 internal sequences obtained was used to screen a
genomic library of T. niveum. Regions of interest were selected by

Northern hybridization. The enormous size of mRNA involved only
permitted the detection of a heterogeneous population above 9.5 kb.
The respective clones were assembled to a 47 kb stretch containing an
intron-free reading frame of 45,823 bp. The ATG start codon was
deduced from other fungal genes. The positions of labeled fragments
matched the predicted domain pattern. Leitner et al. (1998) invented
the nucleotide sequence coding for an enzyme which possesses CySyn
like activity and in which at least one amino acid recognition unit is
different from that of CySyn. The invention also reported on a recombinant vector containing a nucleotide sequence and a suitable
promoter which was inserted in T. niveum. They used this culture to
produce cyclosporin derivatives. Schorgendorfer et al. (1999)
invented the method of altering the domains of CySyn to give
modied enzyme with altered amino acid recognition specicity and
to produce cyclosporin-like peptides or derivatives.
Velkov et al. (2006) developed a practical and reliable method suited
to large-scale processing to isolate CySyn for in vitro biosynthesis of
cyclosporins. They reported that a sequence of chromatographic steps
including ammonium sulfate precipitation, gel ltration, hydrophobic
interaction chromatography and anion exchange chromatography
yielded an electrophoretically homogeneous CySyn preparation. Isolated enzyme exhibited an optimal temperature range of 2429 C and
a pH optimum of 7.6. The native enzyme displayed a pI of 5.7, as
determined by isoelectric focusing.
7. 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 denes 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 molecular level. Behal (2006)
reviewed mode of action of various microbial bioactive metabolites.
Immunosuppressants affect various components of the immune
system such as T-helper, T-effector cell function, antigen presentation and B-lymphocyte cell function. The detailed mechanism of
action of CyA has been given elsewhere (Hamawy and Knechtle,
2003).
After entering the cell, CyA binds to an immunophilin called
cyclophilin (Marks, 1996; Handschumacher et al., 1984; Harding and
Handschumacher, 1988). Cyclophilin is reported to possess peptidylpropyl cis-trans isomerase (PPI) activity which catalyzes the folding of
ribonuclease (Harding et al., 1989; Takahashi et al., 1989; Fischer and
Bang, 1985). The binding of CyA to cyclophilin blocks its PPI activity
(Harding and Handschumacher, 1988; Takahashi et al., 1989; Fischer
et al., 1989; Schreiber, 1992). Hence, it was initially believed that CyA
mediates its immunosuppressive effects by blocking the PPI activity of
the cyclophilin. Some of the immunosuppressive effects of CyA have
also been attributed to the ability to induce the production of the
potent immunosuppressive cytokine TGF- (Khanna et al., 1997;
Prashar et al., 1995; Shin et al., 1998). TGF- is a powerful immunosuppressive molecule considered to be at least 10,000 times more
potent than CyA.
8. Fermentative production
8.1. Microorganisms
CyA is produced by many microorganisms (Table 3) which include
T. inatum (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. inatum has emerged as the most
423
widely used microorganism. The fungal genus, Tolypocladium, rst

described by Gams (1971), belongs to the class fungi imperfecti,
occurring in soil or litter habitats. The species are characterized by
white slow growing cottony colonies that belong to the family of
conidiospore generating Ascomycota. The conidiophores are usually
short and bear lateral or terminal whorls of phialides which have a
swollen, sometimes cylindrical base and thin, often bent necks. The
conidia are one celled, hyaline, and formed in slimy heads (Samson
and Soares, 1984).
T. inatum is also known as Trichoderma polysporum (Rifai, 1969) and
Beauveria nivea (von Arx, 1986). But there is ongoing discussion on the
taxonomy of the genera Tolypocladium and Beauveria, classied by some
authors as one genus (von Arx, 1986). The two genera have been
distinguished through enzyme analyses (Mugnai et al., 1989), comparison of rRNA sequences (Rakotonirainy et al., 1991) as well as
hybridization with a mitochondrial DNA probe (Hegedus and Khachatourians, 1993). Furthermore, Tolypocladium strains are also differentiated by the production of highly specic cyclosporins (Jegorov et al.,
1990) and siderophore peptides (Jegorov et al., 1993). Stimberg et al.
(1992) established electrophoretic karyotypes to easily distinguish
T. inatum from related strains of the genera Tolypocladium or Beauveria.
They reported all strains to display similar morphologies, but showed
chromosomal length polymorphism. Kadlec et al. (1994) reported on
chemotaxonomic discrimination among the fungal genera Tolypocladium, Beauveria and Paecilomyces. They showed that fungi of the genera
Beauveria and Paecilomyces but not of genus Tolypocladium produced
cyclotetradepsipeptides. Todorova et al. (1998) studied the utilization
prole of 49 carbohydrates, based on API 50 CH biochemical tests and
used it for the identication and the discrimination of 75 isolates of
Beauveria and Tolypocladium. The API 50 CH system is a standardized
method used to study the capability of microorganisms to ferment,
assimilate and oxidize various carbon sources.
Tolypocladium species produce a wide range of metabolites
including cyclosporins, efrapeptins, elvapeptins and the antibiotic
LP237-F8 (Dreyfuss et al., 1976; Bullough et al., 1982; Krasnoff et al.,
1991; Rehacek, 1995; Tsantrizos et al., 1996). A new Tolypocladium sp.
fungus, Cs-HK1 isolated from wild Cordyceps sinensis has antitumor
effects. It can be a promising medicinal fungus and an effective,
economical substitute for the wild C. sinensis in health care (Leung
et al., 2006).
Aarnio and Agathos (1989) studied the production of extracellular
enzymes and CyA by T. inatum and morphologically related fungi
such as Beauveria, Fusarium and Neocosmospora. They found that all of
them produce CyA and extracellular lipase and chitinase in variable
amounts, but entomopathogenically important protease activity was
not detected. Aarnio and Agathos (1990) isolated four distinct colony
types of T. inatum which were morphologically normal white, red,
and orange colonies and morphologically diverse tiny brown colonies.
They found that in liquid cultures, normal white and brown colonies
developed into yellow broths. The broth of the brown colony had a
low nal pH and low CyA production, whereas orange and red
colonies had dark brown and even black broths with higher nal pH
and high CyA production. The specic production of CyA by the red
colony was three times more than that of the normal white colonies.
8.2. Fermentation parameters
8.2.1. Effect of carbon source(s)
Dreyfuss et al. (1976) for the rst time reported CyA and CyC as
metabolites of T. polysporum and also the taxonomy, fermentation
conditions, isolation, characterization, and antimicrobial activity of
these compounds. They used glucose (40 g/l) as carbon source and
found it to produce 180 mg/l of CyA using industrial strain of T. inatum.
Glucose has also been reported to be a better carbon source for CyA
production by Sallam et al. (2003) and Survase et al. (2010b).
Balakrishnan and Pandey (1996b) isolated T. inatum strain from soil
424
Table 3
Microorganisms reported to produce CyA.
Microorganism
Type of fermentation
Reference
T. inatum ATCC 34921
SmF
T. inatum NRRL 8044

T. terricola
Indigenous T. inatum
Aspergillus terreus NRC FA 200
Fusarium solani
Fusarium roseum
T. cyclindrosporum
Beauveria bassiana
Neocosmospora varinfecta
SmF
SmF
SmF
SmF
SmF
SmF
SmF
SmF
SmF
Fusarium oxysporum NRC

T. inatum DSM 63544
T. niveum VHb9
Beauveria nivea CA411
T inatum DSMZ 915
T inatum VCRC F21
T. inatum MTCC 557
Trichoderma polysporum (Link ex Pers.) Rifai
Nectria sp. F-4908
T. cyclindrosporum F21
T. inatum ATCC 34921
T. inatum MTCC 557
T. inatum DRCC 106
SmF
SmF
SmF
SmF
SmF
SmF (static)
Smf
SmF
SmF
SSF
SSF
SSF
SSF
Lee and Agathos (1989)

Chun and Agathos (2001)
Foster et al. (1983)
Jegorov et al. (1995)
Balakrishnan and Pandey (1996b)
Sallam et al.(2003)
Sawai et al. (1981)
Ismaiel et al. (2010)
Aarnio and Agathos (1989)
Aarnio and Agathos (1989)
Nakajima et al.(1988),
Nakajima et al. (1989)
El-Refai et al. (2004)
Zhao et al.(1991)
Lee et al. (2009)
Xiao-xian et al.(2009)
Abdel-Fattah et al.(2007)
Balaraman and Mathew (2006)
Survase et al. (2009a)
Dreyfuss et al. (1976)
Goto et al.(1995)
Sekar et al. (1997)
Nisha et al. (2008)
Survase et al. (2009b)
Ramana Murthy et al.(1999)
Where, SmF submerged fermentation; SSF solid state fermentation.
and found maltose, glucose and starch to be suitable carbon sources for
culture growth but obtained maximum production with combination of
glucose and maltose.
Zhao et al. (1991) found carbon source to not only affect the
magnitude of CyA production, but also the proportion of individual
components of the cyclosporin mixture. The best specic production of
cyclosporins was achieved using glucose, whereas the highest yield of
CyA was obtained by maltose. There was no remarkable relationship
between the biomass formation and the intensity of cyclosporin
synthesis. Glucose, sucrose and maltose favored biomass production
but provided a different physiological state necessary for the biosynthesis of cyclosporins.
Margaritis and Chahal (1989) developed fructose based medium
for the production of CyA using B. nivea. They used fructose to
minimize the catabolite repression and oxygen limitation in the pellets
formed during the production stage to get maximum CyA yields.
Agathos et al. (1986) found sorbose (30 g/l) to produce maximum CyA
(105 mg/l) using T. inatum ATCC 34921. Increasing the concentration
of sorbose did not increase the CyA yields, but feeding two carbon
sources sequentially gave higher yields. Addition of maltose (2%) after
8 days of fermentation improved the CyA production (Survase et al.,
2010b). Abdel-fattah et al. (2007) used glucose (10 g/l), sucrose (20 g/
l) and starch (20 g/l) in combination to give maximum CyA (110 mg/l)
production using T. inatum DSMZ 915.
8.2.2. Effect of nitrogen source(s)

Agathos et al. (1986) screened different organic nitrogen sources
as bactopeptone, soytone and corn steep liquor at various concentrations and found bactopeptone at 10 g/l to give maximum production
of CyA. Agathos et al. (1987) found casamino acids as best nitrogen
source among the complex nitrogen sources and the extract produced
was also the cleanest. Abdel-fattah et al. (2007) reported ammonium
sulfate to support maximum production. They also reported yeast
extract to have positive effect on CyA production. Balaraman and
Mathew (2006) used casein acid hydrolysate (30 g/l), malt extract
(20 g/l) and peptone (10 g/l) in static fermentation to produce maximum CyA after 21 days fermentation using Tolypocladium sp. (VCRC
F21 NRRL No.18950). Balakrishnan and Pandey (1996b) and Survase
et al. (2009d) reported maximum production with casein acid

hydrolysate as nitrogen source.
8.2.3. Effect of minerals
Increased production of CyA by supplementation of salts could be
due to the supporting effect of divalent ions in enhancing the production of CyA by mushrooms (Ramana Murthy et al., 1993). Zinc is known
to provide a more complete pattern of glucose utilization, a more
stable pH and higher CyA production (Agathos et al., 1986). Ramana
Murthy et al. (1999) and Survase et al. (2009a) supplemented various
minerals such as FeCl3, ZnSO4, and CoCl2 to solid substrates for
supporting the CyA production.
8.2.4. Effect of environmental factors
pH plays an important role in the nal CyA titers in T. inatum
fermentation (Aarnio and Agathos, 1990). Low nal pH results in low
CyA production, whereas higher nal pH gives higher product titers.
Balakrishnan and Pandey (1996b) reported that the soil isolate of
T. inatum tolerated pH in the range 56 and gave maximum mycelial
growth at pH 5. They showed that a 1 day old culture transferred at 2%
(v/v) supported maximum mycelial growth. The synthesis of CyA was
found to increase only after maximum mycelial growth was attained
and was higher when the pH of the culture broth was above 7.
In SSF, Sekar et al. (1997) reported lower initial pH to give higher
production, and found the pH of the substrate to increase with the
progress of fermentation. Similar results were reported by Ramana
Murthy et al. (1999) and Survase et al. (2009a) who reported an initial
pH 2 to give better production as compared to higher pH.
Isaac et al. (1990) reported a higher spore density to give higher
production of CyA in SmF using T. inatum UAMH 2472. Ramana Murthy
et al. (1999) and Sallam et al. (2003) used 72 h old seed culture for
maximum production of CyA. The spore inoculum plays a critical role in
the maximization of CyA production (Lee et al., 2008). They reported
that 3% of the spore inoculum gives the highest CyA productivity in a
15 day T. niveum production culture. A spore inoculation below 3% in the
production culture prolonged the lag phase and hence delayed the
mycelial growth; this eventually lowered CyA productivity. However,
spore inoculation above 3% stimulated germination too profoundly in a
xed culture volume, thereby resulting in the limitation of both oxygen

and nutrients.
Xiao-xian et al. (2009) developed a fermentation process for
production of CyA using B. nivea CA411, where water feeding was
used to increase the culture growth and fermentation period was
decreased by 2 days.
8.2.5. Effect of aeration and agitation
Agitation and aeration are involved to different extents in overall
mass and oxygen transfer in the process uid. Agitation controls
nutrient transfer and the distribution of air and oxygen, while aeration
determines the oxygenation of the culture and also contributes to bulk
mixing of the fermentation uid, especially where mechanical
agitation rates are low (McNeil and Harvey, 1993).
El-Refai et al. (2004) studied the kinetics of growth and CyA
production by Fusarium oxysporum NRC in both shake ask and
bioreactor (5 l capacity). They found 38% increase in the fungal dry
weight in fermentor at an agitation of 300 rpm and aeration of 1 vvm.
The volumetric and specic production of CyA was 73.5% and 57.1%
higher in the fermentor. The increased biomass and CyA production
could be due to the agitation and aeration which permit higher
oxygen transfer rate as compared to the ask culture technique. Similar results were observed by El Enshasy et al. (2008) where 90%
higher volumetric production of CyA was found in the bioreactor than
in shake ask cultures.
Chun and Agathos (2001) studied oxygen uptake rate as a parameter for estimation of growth rate and cell concentration in cell-free
system as well as immobilized cells. They reported high oxygen
uptake rate during initial growth phase followed by a decrease during
the stagnant phase, reecting both slowing down of the cell proliferation and a decline in the cell viability. They also reported specic
oxygen uptake rates of the immobilized cells to be slightly lower than
that of the free cells shortly after the start of fermentation, but these
rates increased rapidly with increasing cell concentrations during the
exponential phase.
Benchapattarapong et al. (2005) evaluated and compared the
rheological properties, mixing and mass transfer performance in a
stirred tank bioreactor of the T. inatum fermentation broth with the
simulated pseudoplastic fermentation (SPF) broth and carboxymethyl
cellulose solution. They found a higher solid content to have a strong
negative effect on KLa, gas hold-up, and mixing time in the SPF broth,
which closely simulated the behavior of the mycelial fermentation
broth.
8.3. Strain improvement
Improvement of microbial strain to maximize the productivity
of metabolite(s) is important in microbial fermentations. Genetic
engineering has been applied for many suitable systems in addition to
the conventional mutagenesis techniques such as chemical and
physical mutations. Traditional techniques are especially used for
strains with little available genetic information or to those that are
recalcitrant to genetic manipulation.
Agathos et al. (1986) for the rst time isolated and regenerated
protoplasts as a step towards genetic studies to improve the production
of CyA. Agathos and Parekh (1990) treated the conidia of T. inatum with
0.15 M epichlorohydrin and isolated a mutant strain named M6 which
exhibited a growth rate that was similar to the parent organism but
exhibited more extensive conidiation and several-fold higher overall
CyA production.
Swidinsky (1998) used classical methods of mutation and selection for strain improvement. He reported that increased CyA producing mutants showed decreased glucose consumption and slower
biomass build-up than the parent strain suggesting slower rate of
growth to support higher production. He observed decreased acti-
425
vities of hexokinase, phosphofructokinase and pyruvate kinase in

higher CyA producing strains.
Gharavi et al. (2004) carried out UV mutation of T. inatum and an
auxotroph dependent on -Abu was prepared, which gave increased
production of CyA. Bakhtiari et al. (2007) found protoplast fusion
technique to result in 21% regeneration and 38% recombination frequencies. One of the recombinants produced 2.8 times more CyA than
the parent strain. Lee et al. (2009) used a combination strategy to
increase CyA productivity by T. niveum ATCC 34921 using random
mutagenesis by UV treatment and protoplast transformation. They
rst performed random mutagenesis and got 9-fold increase in CyA
yield. Subsequently, a foreign bacterial gene, Vitreoscilla hemoglobin
gene (VHb), was transformed via protoplast regeneration and an
additional 33.5% increase of CyA production was observed. UV
radiation and EMS are known to increase the yields of CyA by about
33% and 37.5%, respectively (Ibrahim et al., 2009).
Weber and Leitner (1994) transformed the protoplasts with
plasmid vector constructed with promoter region of the T. niveum
cyclophilin gene and bacterial hygromycin phosphotransferase gene.
Using this transformation system, mutants of T. niveum with disrupted
versions of the cyclosporin synthetase gene (simA) were engineered
by DNA-mediated transformation but the disruption of the cyclosporin synthetase gene resulted in loss of the ability to produce
cyclosporins. Kempken et al. (1995) found repeated DNA sequences
named CPA element (cyclosporin production associated) in recombinant lambda clones isolated from the CyA T. inatum (ATCC 34921) by
differential hybridization with total fungal DNA and rDNA probes. This
sequence was strain specic, since it was absent in recombinant
lambda clones from other related strains or fungi.
8.4. Effect of precursors
The biosynthesis of CyA and analogs is known to involve sequential activation of all amino acids, their N-methylation and eventual
peptide formation by a multifunctional enzyme, CyA synthetase. Addition of DL-Abu and Nval exclusively produces CyA and CyG, respectively (Kobel and Traber, 1982). Addition of L-Thr led to a 5-fold
increase in total cyclosporin level with a specic yield of 59% CyA and
41% CyC. Addition of L-valine showed a 5.7 fold increase in the yield of
total cyclosporins with a specic yield of CyA (43%), CyC (20%) and
CyD (37%), whereas addition of L-valine to the synthetic medium did
not support the production of CyC and CyD (Lee and Agathos, 1989).
L-methionine or sarcosine lowers the cyclosporin production.
When L-methionine is added along with L-valine, the stimulatory
effect of L-valine is completely reversed. DL-valine does not increase
the product titer as that of L-valine, whereas D-valine does not show
any stimulatory effect. L-leucine and glycine enhances the CyA production in synthetic medium containing inorganic nitrogen source.
This is due to the modulation of the transport system of the fungal cell
(transinhibition) by some compounds in peptone (Lee and Agathos,
1989). Methionine does not take part in the biosynthesis, as methylated amino acids interfere with the biosynthesis of cyclosporin in
vivo (Zocher et al., 1984). The methylation step may not be rate
limiting in a low production environment but it can be a bottleneck in
physiological states involving large methionine pools.
A combination of L-valine and L-leucine improves the production and
seem to act independently of each other with different modes of action.
Experiments with different times of addition of L-valine indicate that the
amino acid may need to be present in the exponential growth phase for
optimal production (Lee and Agathos, 1989; Balakrishnan and Pandey,
1996c; Nisha et al., 2008). Survase et al. (2009b,2009d) found a
combined addition of L-valine and L-leucine in exponential growth
phase to be benecial.
When the medium was externally supplemented with L-valine, the
concentration of intracellular L-valine increased four times from the
end of the exponential phase to the beginning of the stationary phase
426
(Lee and Agathos, 1991). Agathos and Lee (1993) developed a

mathematical model to describe the kinetics of fungal growth, CyA
production and nutrient consumption with special emphasis on utilization of L-valine. The model assumed that L-valine acts as a precursor
as well as an inducer for the CyA synthetase.
Sekar and Balaraman (1996) found that addition of L-valine and
DL--Abu on day 5 signicantly increased the yield of CyA in SSF.
Survase et al. (2009b) reported that addition of L-valine and L-leucine
in combination after 20 h of fermentation resulted in maximum CyA
production. Nisha et al. (2008) reported that L-valine, L-leucine and
-amino butyric acid showed an increase of 26%, 17% and 16%,
respectively, in production of CyA when added in SSF.
H-ATPase stimulators are also reported as stimulators for CyA
biosynthesis. An analogy between the mechanisms of action of phytohormones on plant cells and cells of the fungus producing CyA has
been found. Fusicoccin and cytokinin stimulates the biosynthesis of
CyA (Bibikova et al., 1994). The activities of polyphosphatase and
pyrophosphatase during the culture growth and CyA biosynthesis are
higher in the highly productive strain (Sotnikova et al., 1990).
8.5. Immobilization
Many researchers have used different carrier materials for immobilization of spores as well as mycelia for the production of CyA. Foster
et al. (1983) reported CyA production in a low foaming semi-synthetic
media by carrageenan entrapped T. inatum in an airlift bioreactor
with an external circulation loop.
Chun and Agathos (1989) studied the immobilization of T. inatum
conidia into porous celite beads. They demonstrated a strong increase
in volumetric as well as specic production of CyA in immobilized cell
cultures as compared to free cell culture. They also found an altered
metabolic pattern in the form of pink pigmentation in immobilized
cell culture as well as better utilization of nutrients. They observed
smaller beads to give better CyA production as bead size may be acting
as a diffusion limiting parameter. Chun and Agathos (1991) compared
the physiological and environmental effects of CyA production by
suspended and immobilized cells of T. inatum, and found a signicant
difference in precursor ow between the immobilized and free cell
systems.
Chun and Agathos (1993) tested a feeding strategy for L-valine
for the production of CyA in celite-immobilized cells of the fungus
T. inatum. They observed signicant increase in CyA biosynthesis when
L-valine was added at 108 h and at 156 h i.e. during the exponential
growth phase. However, no stimulating effect of L-valine was observed
when supplemented at hour 60 (lag phase) or when the L-valine was
present from the beginning of the fermentation.
An efcient sporulation/immobilization procedure to shorten the
time and number of steps of sporulation was developed by Lee (1996).
They used this method for an immobilized cell perfusion bioprocess
for continuous production of CyA. They found that a large number of
spores in the fermentation broth in the reactor were entrapped in-situ
into the newly supplemented celite beads and then germinated, thus
forming new immobilized cells. Lee (1996) developed an efcient
immobilized cell separator for continuous operation of immobilized
fungal cell cultures, and applied it to actual fermentation process for
the production of CyA.
Sekar and Balaraman (1998a) immobilized Tolypocladium sp. using
sodium alginate as carrier material for the production of CyA in a
packed bed reactor under batch and continuous ow modes. They
found L-valine and L-leucine to increase the yield of CyA when added
individually, but not when added in combination. The half life of
immobilized catalyst was found to be six months.
Sallam et al. (2005) studied immobilization of a local isolate of A.
terreus spores and mycelia with the objective to increase the capacity
of the A. terreus to produce CyA by cell immobilization and found best
CyA yields with Ca-alginate (3% w/v), mycelial weight 15% (w/v) at
pH 4.5 and for four repeated cycles. They also found the CyA productivity to be markedly accelerated in the presence of L-valine and a
combination of L-valine and L-leucine.
Survase et al. (2010a) studied the immobilization of T. inatum MTCC
557 in different carriers and found gellan gum as an immobilization
carrier to give 274 mg/l of CyA. Additionally, they also found that the
addition of L-valine and L-leucine after 48 h of fermentation increases
the production to 1338 mg/l of CyA using gellan gum as an immobilization matrix. These immobilized beads could be repeatedly used
up to four cycles and thus enhanced their potential for semicontinuous
production of CyA.
8.6. Production of CyA by SSF

In recent years, there has been an increasing trend towards
efcient utilization and value addition of agro-industrial residues
(Pandey et al., 2000). There are several recent publications describing
bioprocesses that have been developed utilizing these raw materials
for the production of bulk chemicals and value-added ne products.
The application of agro-industrial residues in bioprocess has provided alternative substrates, and also alleviated pollution problems.
SSF could be a perfect technology for value-addition of agro products and their residues. SSF offers an alternative to solve many problems encountered in SmF for the production of CyA like uncontrollable
foaming. It also utilizes a cost-effective media with reduced energy
input.
Ramana Murthy et al. (1999) reported T. inatum DRCC 106 to
produce 4843 mg CyA/kg of wheat bran under optimum fermentation
conditions in 10 days when grown on wheat bran medium containing
millet our 20%, jowar our 10%, zinc sulfate 0.15%, ferric chloride
0.25% and cobaltous chloride 0.05%. The optimal fermentation
conditions were an inoculum of 60% v/w initial moisture content of
70%, initial bran pH 20, and incubation temperature of 25 C.
Sekar et al. (1997) also attempted CyA production by SSF using a
local isolate of Tolypocladium sp. and reported the yield to be 10-fold
higher than that obtained by SmF. Hydrolysis of wheat bran with dilute
HCl could further increase the yields. They further studied the effect of
several parameters such as tray fermentation with and without
perforation, thickness of solid substrate bed, type of inoculum, size
of inoculum and relative humidity for the optimum production of CyA
by SSF using Tolypocladium sp. (Sekar and Balaraman, 1998b). Nisha
and Ramasamy (2008) screened different indigenously available and
cost effective solid substrates and found wheat bran to support
maximum production of 179 mg/kg and biomass production of 22 g/
kg. L-valine, L-leucine and amino butyric acid increased the CyA yield
(Nisha et al., 2008).
Survase et al. (2009a) evaluated the effect of different fermentation parameters in SSF on production of CyA by T. inatum MTCC 557.
They found a combination of hydrolyzed wheat bran our and coconut oil cake (1:1) at 70% initial moisture content to support
maximum production of 3872 156 mg CyA/kg substrate. Supplementation with salts, glycerol (1% w/w) and ammonium sulfate (1%
w/w) further increased the production of CyA to 5454 75 mg/kg
substrate. Inoculation of 5 g solid substrate with 6 ml of 72 h old seed
culture resulted in maximum production of 6480 mg CyA/kg substrate.
Survase et al. (2009b) employed statistical designs such as Plackett
Burman and response surface methodology to optimize the SSF
parameters. They also found that the combined addition of L-valine
and L-leucine after 20 h of fermentation results in maximum production
of CyA to 8166 mg/kg.
Survase et al. (2009c) evaluated coconut coir as an inert support for
the production of CyA using T. inatum MTCC 557 by SSF and found
coconut coir impregnated with medium containing glycerol as carbon
source, pH 6, at 80% moisture content and inoculum size of 2.5 ml/2.5 g
support to produce 2641 mg/kg of CyA after 12 days. The yields were
further increased to 3597 mg/kg substrate on addition of L-valine and

L-leucine in combination after 48 h of fermentation.
9. Isolation and purication

Fungi produce a variety of cyclosporins with varying amino acid
composition of which CyA is the most potent. Various purication
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 ow chart for isolation and purication of CyA.
Sekar and Balaraman (1998b) and Survase et al. (2009a,b,c,d) used
butyl acetate for the extraction of CyA from fermentation broth or
fermented solid substrate. Ramana Murthy et al. (1999) extracted
fermented matter using ethyl acetate and puried it using silica gel and
Sephadex LH20 resin. Silica gel and Sephadex LH20 columns were
eluted with hexane:chloroform:methanol (10:9:1) and methanol,
respectively. They characterized the CyA using NMR and IR.
Bakhtiari et al. (2003) used ethyl acetate:isopropanol (95:5, v/v) to
elute silica gel-40 column and methanol to elute Sephadex LH20 resin
to obtain 98% pure CyA. HPLC and IR spectrometry conrmed the
purity and identity of the product. Therwil and Ruegger (1978) and
Rudat et al. (1993) used gel ltration by Sephadex LH20 and/or silica
gel or alumina columns.
Szanya et al. (1995) reported favorable separation achieved on
heat treatment of solid mixture/evaporative residue to 80115 C
prior to chromatography on silica gel. A mixture of chloroform
dichloromethaneethanol or chloroformethyl acetateethanol was
used as eluent. The product obtained was subjected to further chromatography and recrystallization. Lee and Agathos (1989) reported
treatment of fermentation broth with a concentrated solution of
NaOH in order to reach a concentration of 1 N and heated at 60 C for
30 min for recovery of CyA. This mixture was extracted with equal
volume of n-butyl acetate on rotary shaker (250 rpm) for 24 h.
Fermented substrate or broth + solvent for extraction

Solvent extract
Concentrated to dryness
Suspended in solvent & washed with other solvent

to remove lipids
Solvent layer
Lipid containing solvent layer

discarded
Silica gel/ alumina

chromatography
Re-chromatography on
silica gel/ Sephadex LH 20
Re-crystallization and
confirmation of purity by
HPLC, IR
Fig. 2. General protocol for isolation and purication of CyA.
427
Ly et al. (2007) studied solvent concentration and the kinetics of

solidliquid extraction and extraction yields of CyA from the mycelia of
T. inatum and found acetone at 50% v/v concentration to be the best
solvent among methanol, acetone, and isopropanol at different concentrations in aqueous mixtures at room temperature. A linear relationship was found between extraction yield of CyA and methanol
concentration with 100% CyA extraction at 90% v/v methanol. The
effective diffusivities of CyA were found to be between 4.41 10 15 and
6.18 10 14 m2/s for all the three solvents. Ly and Margaritis (2007)
studied the effect of temperature on the extraction kinetics of CyA from
the mycelia of T. inatum. A linear relationship was found between the
extraction yield of CyA and temperature. As the temperature increased,
the yield of CyA increased with a maximum CyA yield of 18.3% obtained
at 45 C, which was 21.3% higher than the yield at 25 C.
10. Methods of analysis
Various methods such as immunoassays (Tredger et al., 2000), HPLC
(Kreuzig, 1984), liquid chromatographytandem mass spectrometry
(Simpson et al., 1998) etc. have been used for CyA measurement in
clinical samples. Although immunoassays fulll 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 routine monitoring of CyA in
transplant recipients. Its wide dynamic range has utility for pharmacokinetic studies of CyA (Range et al., 2002; Salm et al., 2005). It must be
noted that HPLC has remained the method of choice for CyA analysis in
fermentation broths.
Kreuzig (1984) developed HPLC method for analysis of CyA for
separation and determination of the closely related cyclosporins viz.
CyA, CyB, CyC and CyD in fermentation broths. They used 3 nm Nucleosil
C8 column and acetonitrilewaterphosphoric acid (70:30:0.01) as
eluent at 70 C column temperature. George et al. (1992) optimized
mobile phase composition, temperature, stationary phase and UV
detection wavelength for analysis of different cyclosporins. They found
that CyA, CyB and CyC were well separated with a Supelco C8, column
(7.5 cm 4.6 mm I.D.) at 60 C using acetonitrilewater (50:50) containing 0.01% of orthophosphoric acid at a ow rate of 1 ml/min with UV
detection at 202 nm. Husek (1997) also evaluated different columns and
conditions for the HPLC analysis of CyA, its congeners and degradation
products.
A simple and reliable HPLC method was developed and validated
for the evaluation of four CyA degradation products (ID-005-95, CyH,
IsoCyH and IsoCyA) and two related compounds (CyB and CyG).
Elution was performed at a ow rate of 1 ml/min on C18 analytical
column maintained at 75 C with a tetrahydrofuran: phosphoric acid
(0.05 M) (44:56, v/v) as mobile phase. The UV detection was
performed at 220 nm (Bonifacio et al., 2009).
Sekar and Balaraman (1998b) used C18 column maintained at 60 C
for analysis of CyA. The column was eluted with acetonitrile:water
(80:20 containing 0.1% orthophosphoric acid) at a ow rate of 2 ml/min
and detected at 214 nm. Agathos et al. (1986) and Sallam et al. (2003)
analyzed cyclosporins using C8 column maintained at 72 C with
acetonitrile:methanol:water (42.5:20:37.5) as mobile phase and detection at 210 nm. Survase et al. (2009a) analyzed CyA using C18 column at
70 C using acetonitrile:water (70:30) at 210 nm. The pH of mobile phase
was adjusted to 3 using orthophosphoric acid. High column temperature
resulted in a low eluent viscosity, sharper peaks, and minimized or
eliminated temperature gradients which could arise by viscous or
frictional heating in microparticulate columns (Abbott et al., 1981).
11. Pharmacokinetics
Pharmacokinetics has been used for many years to relate immunosuppressant dose to drug exposure in vivo. It is the primary
428
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 interpretation of blood CyA concentrations must be done carefully (Freeman, 1991). Due to large
inter- and intra-patient pharmacokinetic variability, the use of CyA
has become complicated (Kahan, 1986). Variability in CyA pharmacokinetics has been observed after oral and/or intravenous administration of the drug. This variability is related to the patient's disease
state, the type of organ transplant, the age of the patient, and therapy
with other drugs that interact with CyA. Yee (1991) and Fahr (1993)
have reviewed in detail about the clinical pharmacokinetics of CyA,
whereas Christians and Sewing (1995) reviewed alternative CyA
metabolic pathways and toxicity.
12. 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 and tubular cells and on kidney and systemic hemodynamics.
CyA produces afferent arteriolar vasoconstriction when given to animals,
resulting in increased vascular resistance, decreased renal blood ow,
and decreased glomerular ltration. Castello et al. (2005) studied the
pathways of glomeruli damage. They reported that CyA releases
endothelin-1 (ET) and angiotensins independently and glomerular CyA
toxicity is mediated by recruitment of vasoconstricting peptides and
modulated by relative ETA and ETB receptor occupancy. Vascular injury is
a common factor in all types of CyA-induced organ damage (Gallego
et al., 1994; Meyer-Lehnert and Schrier, 1989; Zoja et al., 1986). CyA
therapy increases hypertension in transplant patients. This in turn
increases the thickness of the arteriolar walls and decreases the size of
the vessel lumen leading to ischemia and glomerulosclerosis. Hypertension can directly damage the glomeruli by increasing the intraglomerular
hydrostatic pressure. Benediktsson et al. (1996) suggested antihypertensive drug treatment to improve graft survival by decreasing the
urinary protein excretion rate. The nephrotoxicity caused by CyA
treatment varies with animal models (Sekar, 1991). In present times,
the nephrotoxicity of CyA is manageable and is achieved by dosage
adjustments based on the monitoring of CyA blood concentrations.
Neurotoxicity described in CyA administration is generally mild,
most commonly consisting of involuntary ne tremors, headache,
tinnitus and nervousness that respond to dose reduction. However,
more complex types of neurotoxicity including motor and cerebellar syndromes, seizures, cortical blindness and coma have rarely
been described in bone marrow, renal and liver transplant patients
(Nussbaum et al., 1995; Palmer and Toto, 1991; de Groen et al., 1987;
Kutlay et al., 1997). Magnesium is stored in bone marrow under normal
circumstances. CyA treatment increases Mg content in organs like
kidney and liver (Barton et al., 1989). CyA-induced hypomagnesaemia
may lead to hypertension and neurotoxicity (Thomson et al., 1984).
Another common side effect of CyA treatment is hyperlipidemia.
Pirsch et al. (1997) found higher incidence of hyperlipidemia and
hypercholesterolemia in CyA treated patients as compared to treatment with tacrolimus. Klintmalm et al. (1981) reported incidence of
hepatotoxicity in CyA treated patients. Changes in several liver enzymes like serum glutamate oxaloacetate transaminase, serum
glutamate pyruvate transaminase and alkaline phosphatase also in
the level of serum bilirubin have been observed.
13. 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 modication of dosage are essential. Wadhwa et al.
(1987) have systematically compiled the CyA drug interactions.
Zylber-Katz (1995) reported on multiple drug interactions with
CyA in a heart transplant patient. She reported that drugs such as
rifampin and erythromycin, which are known to be inducers or substrates of cytochrome P-450 IIIA, have the potential to alter CyA blood
concentrations. Coadministration of rifampin/isoniazid and CyA for a
week and erythromycin for the last 4 days is shown to lower the CyA
blood concentration, probably because of microsomal induction by
rifampin. Wright et al. (1999) observed a nearly 10-fold increase in
whole blood CyA concentrations in a cardiac transplant patient after
the addition of nefazodone, an antidepressant drug. They suggested
the drugdrug interaction between nefazodone and CyA to be due to
inhibition of cytochrome P-450 IIIA4 isoenzymes by nefazodone. Both
non-nucleoside reverse transcriptase inhibitors and protease inhibitors give rise to substantial drug-to-drug interactions with immunosuppressive drugs such as tacrolimus and CyA (Vogel et al., 2004).
Non-steroidal anti-inammatory drugs alone can have an adverse
effect on renal function. Addition of these drugs to CyA therapy or an
increase in their dosage may lead to complete renal failure (Kovarik
et al., 1997). So, there should be a close monitoring of renal function.
Diclofenac concentration was found to be doubled in the presence of
CyA. Similar ndings are reported by Altman et al. (1992). Cheyron
et al. (1999) reported that co-administration of sulphasalazine increased the bioavailability of CyA in kidney transplant patients.
Hermann et al. (2002) reported co-administration of grapefruit juice
with CyA to affect the formation and/or elimination of the metabolites.
In addition, administration of CyA with juice induced a moderate but
signicant increase in systemic exposure of CyA in renal transplant
recipients. Grapefruit juice should be avoided owing to its possible
interference with the P450 enzyme system which may increase the
bioavailability of CyA.
As CyA and many HMG-CoA reductase inhibitors are metabolized
by the same cytochrome P450 IIIA4 enzyme system in the liver,
possible drug interactions have to be expected during a combination
therapy with CyA and statins (Christians et al., 1998). In transplant
patients receiving the HMG-CoA reductase inhibitor (lovastatin) in
combination with CyA, there have been reports of severe rhabdomyolysis that precipitated acute renal failure (Meier et al., 1995).
Felipe et al. (2009) found that the magnitude of the effect of CyA
on sirolimus blood concentration is higher than that of sirolimus on
CyA blood concentrations. They emphasized the need for therapeutic
drug monitoring using this drug combination.
14. Therapeutic uses

CyA has a range of pharmacological activities including suppression of antibody-and cell-mediated responses, inhibition of chronic
inammatory reactions, fungicidal and antiparasitic activities, antiHIV 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 sufcient duration and intensity can lead to the
development of certain forms of cancer. CyA may result in hypomagnesaemia which in turn may mediate some of the undesirable
effects such as hypertension and may contribute to neurotoxicity. CyA
is also reported to prevent onset of diabetes in rat. CyA and CyC only
have a narrow spectrum of antifungal and no antibacterial activity.
14.1. Use of CyA in organ transplantation

CyA was rst registered as Sandimmune for use in organ transplantation. Prior to the development of CyA, side effects of high-dose
steroids including bone marrow suppression with recurrent infections
made the immunosuppressive therapy for organ transplantation complex. Most of the early immunosuppressive drugs such as azathioprine
act by blocking all cells in mitosis. Because of selective immunosuppressive activities, CyA signicantly reduces rejection rates and
improves patient and graft survival in solid organ, bone marrow transplants and the main post-transplant complications (Van Buren et al.,
1984; Borel, 1983). It is generally used in combination with other
immunosuppressive agents which have the advantage of exploiting
additive and synergistic drug effects while minimizing the adverse
reactions. By 1996, some 200,000 transplant patients relied on use of
CyA.
CyA has made many important contributions to transplantation.
The organs successfully grafted under CyA treatment include skeletal
muscles (Gulati and Zalewski, 1982; Watt et al., 1981), lung (Norin
et al., 1982; Beveridge, 1983), small bowel (Craddock et al., 1983),
cornea (Hunter et al., 1981), skin (Balaraman et al., 1991), heart (Reitz
and Stinson, 1982) and liver (Starzl et al., 1982). Donor-specic
immunologic tolerance and clonal detection have been suggested as
the mechanism for prolonged allograft survival in patients treated
with CyA. Ferguson and Fidelus-Gort (1983) reported the presence of
CyA in plasma to be necessary for its blockage of lymphocyte responsiveness and hence the prevention of allograft rejection.
Borel et al. (1998) found signicant improvement in survival rate
of patients with CyA treatment. They reported that before the
introduction of CyA in transplantation therapy, the overall one-year
graft survival rate was about 60% which depending on the center
increased to 8090%. In case of liver transplantation, the 5-year
survival of patients, increased from 20% to 60%. The 5-year survival
rate of heart transplantations was approximately 70% with CyA.
Heartlung and lung transplantation was never successful without
CyA. The one-year survival of the heartlung transplantation was 60
65% with CyA. A one-year graft survival of about 80% can be achieved
in simultaneous transplantation of pancreas and kidney.
In bone marrow transplantation, CyA prevents rejection of the
transplanted bone marrow and is also used for prevention and treatment of graft-versus-host disease (GVHD) (Borel, 1976; Van Bekkum
et al., 1980; Gratwohl et al., 1982).
14.2. CyA in parasitic infections
Malaria, leishmaniasis, trypanosomiasis, schistosomiasis and lariasis along with a number of other human diseases caused by
protozoans and helminths continue to trouble mankind throughout
the world. In the context of drug resistance exhibited by parasites
against many known drugs, discovery of new drugs is also important.
CyA displays pronounced antiparasitic properties (High and
Handschumacher, 1995; Page et al., 1995). The antiparasitic activities
of CyA include schistosomiasis (Bueding et al., 1981; Munro and
Mclaren, 1990), toxoplasmosis (Mack and McLeod, 1984), cystic
hydatidosis (Colebrook et al., 2002), leshminiasis (Behforouz et al.,
1986; Adinol and Bonventre, 1990), malaria (Grau et al., 1987) and
strongyloidiasis (Armson et al., 1995).
CyA acts as an immunosuppressant, causing enhanced infection or
delayed elimination of parasites (McCabe et al., 1985; Wastling et al.,
1990). Protozoan infections were seen to be exacerbated by CyA
include Giardia muris (gut), Trypanosoma cruzi and Trypanosoma
musculi (blood), Leishmania donovani (blood macrophages), Eimeria
adenoeides, Eimeria meleagrimitis, and Eimeria gallopavonis (gut).
Helminth parasitic infections that are prolonged or exacerbated with
CyA include Hymenolepis diminuta (mouse gut) and Echinococcus
multilocularis (mouse and human liver).
429
Chappell and Wastling (1992) reviewed antiparasitic activity

against various infections in laboratory models reducing survival,
growth and multiplication of protozoans and helminthes. They reported a reduction and/or elimination with protozoan infections like
Trypanosoma brucei (blood), Leishmania tropica and Leishmania major
(macrophages), Eimeria vermiformis and Eimeria mitis (gut), and
Plasmodium berghei, Plasmodium chabaudi, Plasmodium yoelii and
Plasmodium falciparum (blood). Helminthes infections like schistosomes (blood), liver ukes (liver), the tapeworms Hymenolepis
microstoma (mouse bile duct), Echinococcus granulosus (mouse body
cavity) and Mesocestoides corti (mouse liver, body cavity), and the
nematodes Acanthocheilonema (Dipetalonema) viteae, Brugia pahangi,
Strongyloides stercoralis and Strongyloides ratti in laboratory models
and man are all variously inhibited by drug treatment.
In a small number of cases, including Toxoplasma gondii, Eimeria
tenella, Paragonimus myazakii and Paragonimus ohirai, Litomosoides
carinii, and Heligmosomoides polygyrus, CyA may act in different ways
on different stages of the parasite or respond to varying treatment
regimens. Nickell et al. (1982) and Somasundaram et al. (1989)
showed that malaria infected mice recovered from the infection when
treated with CyA. Since CyA is cytotoxic, it may act directly on the
parasite and kill it. Cyclophilins have been identied in P. falciparum,
the principal agent for malaria. CyA inhibits calcineurin activity in P.
falciparum only in the presence of cyclophilin (Bell et al., 1994;
Dobson et al., 1999).
The anti-leishmanial effect of CyA is independent of effector
mechanisms employed by macrophage-activating cytokines (Meissner et al., 2003). As far as antiparasitic effects are concerned, the role of
drug metabolites is not clearly established, but it is clear that residual
parent drug or possibly metabolites can have a long-lasting action on
some parasites such as Schistosoma mansoni in mice (Bout et al., 1986;
Chappell et al., 1987). Bell et al. (1996) reviewed the antiparasite
effects of CyA, the possible drug targets and clinical applications.
14.3. CyA in autoimmune diseases
A signicant number of diseases are caused by the body's natural
defense mechanisms. As with the immune system, immunosuppressive therapy may be used to treat patients with these types of
diseases. Since 1987, CyA has also been registered for the treatment of
several autoimmune disorders. CyA is reportedly efcacious for autoimmune diseases in humans such as uveitis (autoimmune, Behqet
disease) (Binder et al., 1987), psoriasis (Finzi et al., 1993), idiopathic
nephrotic syndrome (Niaudet and Habib, 1994), rheumatoid arthritis
(Cranney and Tugwell, 1998), severe aplastic anemia (Bern et al.,
1986; Porwit et al., 1987) and autoimmune hepatitis type 2 (Debray
et al., 1999). Severely affected patients resistant to conventional
therapy benet from CyA therapy.
It is also used in some diseases but the benets achieved are
unclear. These include Crohn disease (Nicholls et al., 1994; Lmann
et al., 1998), atopic dermatitis (Sowden et al., 1991; Van Joost et al.,
1994), asthma (Evans et al., 2000; Alexander et al., 1995), primary
biliary cirrhosis (Gong et al., 2007), myasthenia gravis (Bonifati and
Angelini, 1997) and insulin-dependent diabetes mellitus (Bach, 1987).
14.4. CyA against hepatitis C
Hepatitis C virus (HCV) infection characterized by chronic liver
inammation and brogenesis affects millions of people worldwide
(Alter, 1997). One of the reasons for failure in complete eradication of
this disease is unavailability of suitable treatment options. The
therapies which are available have serious side effects. Watashi
et al. (2003) and Nakagawa et al. (2004) reported CyA to substantially
and specically inhibit intracellular HCV replication in vitro. Inoue
et al. (2003) reported a combination of CyA with interferon to be more
effective than interferon monotherapy, especially in patients with a
430
high viral load. Despite the clinical effectiveness of CyA, little is

understood about its anti-viral mechanisms in patients with chronic
hepatitis C.
Nakagawa et al. (2005) reported the anti-HCV effect of CyA to be
different from its immunosuppressive activity. They showed that the
antiviral action of CyA is mediated by blocking the action of cellular
CyA-binding proteins, the cyclophilins. A cyclosporin analog, CyD,
which lacks immunosuppressive activity but exhibits cyclophilin
binding, induced a similar suppression of HCV replication. Watashi
et al. (2005) reported that cyclophilin B, a cellular target of CyA, also
facilitated viral replication via the regulation of the RNA binding
ability of NS5B. Thus cyclophilin (in addition to viral proteins including NS3 protease and NS5nn B polymerase) can also be useful as a
molecular target for antiviral strategies.
Goto et al. (2009) established and characterized the replicon resistant to cyclophilin inhibitors using the subgenomic replicon system
to deepen the understanding of the anti-HCV actions of cyclophilin
inhibitor so as to maximize the efcacy of the agent. Their results are
important for elucidating additional mechanisms of the regulation of
HCV replication by cyclophilin and also for designing novel and
specic anti-HCV strategies with cyclophilin inhibitors.
14.5. CyA against human immunodeciency virus (HIV)

There has been a long standing controversy as to whether CyA
treatment may be benecial to HIV-infected humans or AIDS patients.
Cyclophilin A is the cellular target of CyA as well as the binding protein
of the human immunodeciency virus type 1 (HIV-1) related Gag
polyprotein p55 (Luban et al., 1993).
Thali et al. (1994) examined the Pr55gag-cyclophilin interaction on
the life-cycle of HIV-1, and found HIV-1 to incorporate a substantial
amount of cyclophilin A. They detected approximately equimolar
amounts of the viral envelope glycoprotein and cyclophilin A in virions.
Cyclophilin inhibitors face challenges such as side effects and drug
resistance which are barriers to successful treatment in cases of HIV
(Cordes et al., 2006; Shulman and Winters, 2003). Schwarz et al. (1993)
showed the incidence of AIDS to be signicantly lower in the group of
patients who were treated with CyA than in the group that was treated
with other immunosuppressants.
Evers et al. (2003) described the regioselective and stereoselective
synthesis and the pharmacological properties of a novel series of CyA
analogs. The [2-(dimethyl or diethylamino)-ethylthio-Sar]3-[(4-OH)
MeLeu]4-CyA derivatives 3k and 3l displayed potent in vitro anti-HIV-1
and low immunosuppressive activities. Other cyclosporin analogs that
are active against HIV-1 replication possess either one modication on
the [MeBmt] or [MeLeu], two modications on the [MeBmt]1 or [Abu]
and [MeLeu], or three modications on the [Abu], [Sar] and [MeLeu]
residues.
Saini and Potash (2006) investigated cyclophilin A and CyA activities
in HIV-1-infected primary human macrophages, compared with
primary human lymphocytes. They demonstrated that the major
distinction among host cell types in these elements of HIV-1 infection
lies between transformed cells on the one hand and both primary
lymphocytes and macrophages on the other. They reported that
cyclophilin AGag interactions, CyA sensitivity, and the biology of
mutations that disrupted these effects were different in primary cells
than was reported previously in various transformed human cell lines.
Thali (1995) reviewed cyclosporins as immunosuppressive drugs
with anti-HIV-1 activity. They reported that although immunosuppressive and antiviral activities are different functions of cyclosporins,
both do require an interaction of the drug with cyclophilins. Cron
(2001) presented evidence supporting a role for NFAT proteins in
augmenting HIV-1 transcription. In addition, they reviewed other
mechanisms of HIV-1 inhibition by CyA and the rationale for the use of
CyA to treat AIDS.
14.6. CyA on eye infections

Ophthalmic emulsion of CyA (0.05%) is available as an FDAapproved treatment for dry eye disease since 2003. CyA formulation
has been used for topical treatment of a number of ocular inammatory diseases such as posterior blepharitis (Rubin and Rao, 2006),
ocular rosacea (Schechter et al., 2009), post-LASIK dry eye (Salomo
et al., 2009), contact lens intolerance (Hom, 2006), venral keratoconjunctivitis (BenEzra et al., 1988; Bleik and Tabbara, 1991), atopic
keratoconjunctivitis (Hingorani et al., 1999), meibomian gland
dysfunction (Perry et al., 2006) and herpetic stromal keratitis (Yoon
et al., 2008). Calcineurin and NFAT are present in retinoblastoma cells,
and CyA treatment of retinoblastoma cell lines reduce proliferation
and induce apoptosis (Eckstein et al., 2005). Strong et al. (2005)
reported CyA to signicantly reduce apoptosis of conjunctival epithelial cells, as assessed by DNA fragmentation and levels of activated
caspase-3, in an experimental murine model of dry eye.
The hydrophobic nature of CyA has presented a challenge to
developing an effective ophthalmic formulation. To overcome this,
Tang-Liu and Acheampong (2005) developed a novel ophthalmic CyA
formulation prepared in castor, corn, olive, and peanut oils. However,
burning, redness, itching, and epithelial keratitis hindered the use of
such oils. Lee et al. (2007) investigated the pharmacokinetics of an
episcleral CyA implant as an alternative treatment option to topical
CyA in preventing corneal allograft rejection. Donnenfeld and
Pugfelder (2009) reviewed pharmacology and clinical applications
of topical CyA formulation. They discussed the mechanism of action
for CyA at the molecular level, challenges in developing an effective ophthalmic formulation of CyA and reviewed in detail the
studies evaluating the effectiveness of topical CyA treatment for
ocular disorders.
14.7. Use of CyA in cancer

Several studies have reported CyA to be selectively cytotoxic and/
or growth inhibitory to the T-cell phenotypic cells (Twentyman, 1988;
Twentyman et al., 1990). CyA and its derivatives are reported to direct
reverse the multidrug resistance of cancer cell lines associated with
increased expression of the transport glycoprotein gp170. Since the
report by Zwitter (1988) stating the uses of CyA in chemotherapyresistant Hodgkin's disease, interest in its use in cancer has widened
in several areas. Various mechanisms are predicted for resistancemodier effect which include inhibition of polyamine synthesis,
correction of altered plasma membrane potentials (Vayuvegula et al.,
1988) or enhancement of the R23 nuclear protein translocation
(Sweet et al., 1989).
Ledermann et al. (1988) analyzed the potential of CyA for inhibiting immune response to therapeutic anticancer mAb. In patients
treated with radiolabelled mAb to carcino-embryonic antigen for
colonic cancer, administration of CyA resulted in higher mAb concentration because of the lower clearance and lower human antimouse antibody responses than in non-CyA-treated controls.
Van de Vrie et al. (1993) reported on the chemosensitizing effect
of CyA in colon tumors mediated through P glycoprotein. They
reported on the reversibility of intrinsic multidrug resistance in a
syngeneic, solid tumor model where the sensitivity to doxorubicin,
daunorubicin and colchicine was enhanced by the addition of the
chemosensitizers verapamil and CyA. CyA may be used as an integral
part of the chemotherapy for acute myeloid leukemia (AML) due to
its ability to signicantly diminish the multidrug resistance in K562/
ADM cells and enhance the complete remission rates in patients with
AML (Li et al., 2009). Use of CyA as a reverter of multidrug resistance
may produce short-term improvement of antitumor activity but may
also induce enhancement of tumor metastasis (Van de Vrie et al.,
1997).
15. 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, purication, mechanism of action as well as applications
of CyA. The numerous applications so far identied, 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 production from various
strains. Although, a number of microbial sources exist for the efcient
production of CyA, commercial production of CyA has been limited to
only a few selected strains of fungi. Thus, commercially viable processes with improved yields should be developed to reduce the cost of
production.
Discovery of cyclosporins led the way to an era of selective lymphocyte inhibition. It enabled the expertise in clinical, technical and
immunobiological aspects of transplantation to be put into practice
and changed the face of transplantation. CyA did not solve all the
problems of transplantation. Its limitation to chronic rejection is less
understood and there is no treatment for it. The majority of transplant
patients require long term treatment with high doses of immunosuppressants which increases susceptibility to infection and malignancies. The discovery and development of cyclosporins have enabled
many patients to survive after their operation.
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Cyclosporin A - A review on fermentative

American Process Inc.GA, USA

Institute of Chemical Technology, Mumbai

48 PUBLICATIONS 590 CITATIONS

Available from: Shrikant Survase

Biotechnology Advances 29 (2011) 418435

Contents lists available at ScienceDirect

Research review paper

Cyclosporin A A review on fermentative production, downstream processing and

Corresponding author. Tel.: + 91 22 3361 2512; fax: + 91 22 3361 1020.

S.A. Survase et al. / Biotechnology Advances 29 (2011) 418435

14.4. CyA against hepatitis C . . . . . . .

tical Corporation, USA; Gengraf from Abbott Laboratories, USA;

S.A. Survase et al. / Biotechnology Advances 29 (2011) 418435

Regulators of gene expression

Methotrexate, Azathioprine, Mycophenolic acid

Inhibits the expression of genes for IL-2 and other mediators

Inhibits the phosphatase activity of calcineurin, thereby suppressing the

inhibits kinases required for cell cycling and responses to IL-2

at position 16 of the backbone adopt an antiparallel -pleated sheet

Alkylate DNA bases,

only 5 compounds are doubly altered. The structures of the congeners

4. Structure activity relationship

Fig. 1. Chemical structure of CyA.

CyA to CyZ have been tested for antifungal activity as well as in

S.A. Survase et al. / Biotechnology Advances 29 (2011) 418435

Amino acid composition

type non-natural amino acid molecule. They synthesized derivatives

It is stable in solution at temperatures below 30 C, but is sensitive

S.A. Survase et al. / Biotechnology Advances 29 (2011) 418435

Synthesis of all 11 constituent amino acids

Dittomann et al. (1994) showed that cyclosporin synthesis occurs as

antibodies directed against enniatin synthetase. Schmidt et al. (1992)

S.A. Survase et al. / Biotechnology Advances 29 (2011) 418435

genomic library of T. niveum. Regions of interest were selected by

widely used microorganism. The fungal genus, Tolypocladium, rst

S.A. Survase et al. / Biotechnology Advances 29 (2011) 418435

T. inatum ATCC 34921

T. inatum NRRL 8044

Fusarium oxysporum NRC

Lee and Agathos (1989)

Where, SmF submerged fermentation; SSF solid state fermentation.

8.2.2. Effect of nitrogen source(s)

et al. (2009d) reported maximum production with casein acid

S.A. Survase et al. / Biotechnology Advances 29 (2011) 418435

xed culture volume, thereby resulting in the limitation of both oxygen

vities of hexokinase, phosphofructokinase and pyruvate kinase in

S.A. Survase et al. / Biotechnology Advances 29 (2011) 418435

(Lee and Agathos, 1991). Agathos and Lee (1993) developed a

8.6. Production of CyA by SSF

S.A. Survase et al. / Biotechnology Advances 29 (2011) 418435

further increased to 3597 mg/kg substrate on addition of L-valine and

9. Isolation and purication

Fermented substrate or broth + solvent for extraction

Suspended in solvent & washed with other solvent

Lipid containing solvent layer

Silica gel/ alumina

Ly et al. (2007) studied solvent concentration and the kinetics of

S.A. Survase et al. / Biotechnology Advances 29 (2011) 418435

method to measure drug absorption, distribution, metabolism, routes

13. Drug interactions

14. Therapeutic uses

S.A. Survase et al. / Biotechnology Advances 29 (2011) 418435