ABSTRACT

Discovery of cyclosporin in 1971 began a new era in immunopharmacology. It was the first immunosuppressive drug that allowed selective immunoregulation of T cells without excessive toxicity. Cyclosporin was isolated from the fungus Tolypocladium inflatum. Cyclosporin was first investigated as an anti-fungal antibiotic but its spectrum was too narrow to be of any clinical use. J. F. Borel discovered its immunosuppressive activity in 1976.This led to further investigations into its properties involving further immunological tests and investigations into its structure and synthesis. Cyclosporin has unwanted side effects, notably nephrotoxicity. Animal testing showed cyclosporin to be sufficiently non-toxic to begin clinical trials. These initially failed due to poor absorption of the drug. Once this had been overcome, results were encouraging enough for cyclosporin to be licensed for use in clinical practice. There is some controversy between Borel and other workers over priority in the discovery of cyclosporin and its pre-clinical development, which is examined in this review. Cyclosporin changed the face of transplantation. It decreased morbidity and enabled the routine transplantation of organs that until then had only been done experimentally.

INTRODUCTION

We all know the story about the discovery of Penicillin, but what about other drugs now in common use? This SSM investigates the events that led to the discovery of cyclosporin [initially known as cyclosporin A, now known as ciclosporine in Europe and cyclosporin in the USA], and the developments that resulted from its introduction into clinical practice.

Irene

A man who put a soil sample in a plastic bag saved the life of Irene, a vivacious student, who, at the age of 18 years was diagnosed with acute myeloblastic leukaemia. It progressed rapidly and severely. Chemotherapy might offer her a brief remission but would inevitably end in a fatal relapse. However Irene had two pieces of good fortune. Firstly, she was immunonologically (HLA) compatible with her younger sister. Secondly cyclosporin had been discovered a few years previously. Doctors expressed concern about cyclosporin toxicity and about likely development of the devastating host-versus-graft disease. Despite their anxieties there was no other hope for Irene and a bone marrow transplant was undertaken. Irene developed severe renal complications. Little was known about cyclosporin nephrotoxicity and she was probably given excessively high doses. But thanks to cyclosporin she only suffered from a mild host-versus-graft reaction and was able to leave hospital to live a normal life [1].

What is cyclosporin and what are its uses?

Today organ and bone marrow transplants are routinely performed. Cyclosporin is still used to treat the rejection reactions that occur when a foreign organ is attacked by the body’s immune system. Cyclosporin is a fungal peptide, isolated from Tolypocladium inflatum Gams. It was the first immunosuppressant that acted selectively to suppress T-cell immunity.

Cyclosporin is at present (March 2001) approved for use in organ transplantation to prevent graft rejection in kidney, liver, heart, lung and combined heart-lung transplants. It is used to prevent rejection following bone marrow transplantation and in the prophylaxis of host-versus-graft disease. It is also used in the treatment of psoriasis, atopic dermatitis, rheumatoid arthritis and nephrotic syndrome

Why was the discovery of cyclosporin so important?

Discovery of immunosuppression by cyclosporin in 1976 is attributed to J. F. Borel, see Figure 5. In 1983 cyclosporin was approved for clinical use to prevent graft rejection in transplantation. Most of the surgical problems of allograft transplantation had already been solved by this time.

Since 1961 the standard method of achieving immunosuppression had been a combination of azathioprine and corticosteroids. Azathioprine inhibits cell proliferation non-selectively. Its main unwanted side effect is depression of the bone marrow, other toxic effects include increased susceptibility to infections, a mild hepatotoxicity, skin eruptions, nausea and vomiting. Corticosteroids inhibit T lymphocytes and have an anti-inflammatory effect. Side effects include diabetes, avascular necrosis of bones and increased tendency to infections [2].

Cyclosporin was the strongest immunosuppressor to be discovered so far, it also overcame many of the risk factors associated with azathioprine and is relatively non-toxic to bone marrow. With the introduction of cyclosporin patient morbidity fell. It became possible to transplant organs with a one year success of 20% higher than previously [3], and to transplant organs successfully which previously had only been done in experimentation: the heart, the liver, the lung and combined heart lung transplants [4].

As well as transplantation, cyclosporin has been used in most autoimmune diseases. In the 1980’s experimental treatment with cyclosporin of insulin-dependent diabetes mellitus, inflammatory bowel disease, chronic asthma, atopic dermatitis, aplastic anaemia and psoriasis supported evidence of their T cell mediated nature [5].

THE DISCOVERY OF CYCLOSPORIN

Discovery of an anti-fungal antibiotic

A tradition established as part of a programme set up in 1957 to search for new antibiotic drugs from fungal metabolites was for Sandoz employees on business trips and holidays to take plastic bags with them for collecting soil samples that were catalogued and later screened. In March 1970 in the Microbiology Department at Sandoz Ltd. (Basel), a Swiss pharmaceutical company, the fungus Tolypocladium inflatum Gams (Figure 1) was isolated by B. Thiele from two soil samples, the first from Wisconsin, USA and the second from the Hardanger Vidda in Norway. These soil samples had been collected by Sandoz employees.

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Figure 1. Scanning electron micrograph of Tolypocladium inflatum

The Microbiology Department at Sandoz had developed a computer-aided evaluation program for screening and evaluating sampled fungi [6].The program enabled rapid evaluation of the samples, recognizing and eliminating common fungi, and related strains, that produced known compounds from further study. This meant that more time could be spent evaluating the rare fungi in the samples, which would be more likely to produce metabolites that had potential new antibiotic activity. The program identified Tolypocladium inflatum; it was previously unknown to the Sandoz team and produced interesting metabolites.

Next, Z.L. Kis routinely isolated a metabolite mixture from Tolypocladium inflatum. The characteristics were fed into another computer program based on an exhaustive file of data extracted from the literature [6] and showed the presence of a group of metabolites, which were new to Sandoz. These metabolites were found to possess some antifungal activity and warranted further investigation.

For further investigation larger samples were required. Tolypocladium inflatum was fermented and a technique for the isolation of two of the metabolites later named cyclosporin A and C wasdeveloped [7]. Samples were produced in submerged culture and extracted by organic solvents. M Dreyfuss and colleagues examined the antibacterial and anti-fungal activity of cyclosporin A and C. They were found to have only a narrow spectrum of activity against fungi, and no antibacterial activity was found.

Only a few species of yeast were found to be sensitive to the metabolites. When grown in solid media and in contact with cyclosporin the growth rates of the sensitive species was decreased. Strains of some Mucorales, ascomycetes and fungi imperfecti showed sensitivity to variable degrees, the inhibition taking the form of deformation and branching of growing hyphal tips. There was no effect on germination of spores or conidia of the affected fungi.

By analysing and comparing the taxonomic positions of sensitive organisms, Dreyfuss and colleagues hypothesized that the mode of anti-fungal action was due to an inhibition of cell wall synthesis, in particular chitin synthesis. Cyclosporin activity was compared to the only known chitin blocking antibiotic Polyoxin, which exhibited a similarly narrow spectrum to that observed in cyclosporin [7].

An anti-fungal drug that inhibited cell wall synthesis would have been a useful discovery, as it would have high specificity and low toxicity to non-fungal hosts, similar to the indispensable group of beta-lactam antibiotics. However, cyclosporin was found to be inactive against Sporobolomyces roseus and Sporobolomyces antarcticus ( Table 1).This indicated a different mode of action for the cyclosporins.

Due to their narrow spectrum and weak action there was no future for cyclosporin as an anti-fungal agent and Dreyfuss and his colleagues stopped their investigations.

Discovery of immunosuppressive activity by cyclosporin

The first non-steroidal immunosuppressive, non-toxic to bone marrow, was discovered at Sandoz in 1962 in the search for new and useful fungal metabolites. It was isolated in 1965 from Pseudeurotium ovalis and was named Ovalacin.  It depressed the immune response strongly but did not affect division of intestinal epithelial cells or myeloblast proliferation, unlike other cytostatic drugs. Ovalacin preceded the discovery of cyclosporin and was crucial for setting the stage for the latter's discovery.

Ovalacin is 600 times more potent than cyclosporin by weight, but it failed clinical trials because of its toxic effects.

In January 1970 the head of the Pharmacology Department at Sandoz, K. Saameli, developed a programme of about 50 pharmacological tests performed by different Groups in the Pharmacological Department, the ‘General Screening Programme’ [8]. A. Rüegger from the Chemistry Department, aware that microbial metabolites often possess interesting pharmacological activity, submitted cyclosporin for the General Screening Programme in 1971. The sample was given the preparation number 24-556, and it was later found to contain mainly cyclosporin.

Out of all the pharmacological tests in the General Screening Programme only one produced a positive result. This was a test for immunosuppression. On day one, mice were intravenously injected with sheep erythrocytes and preparation 24-556 was injected intraperitoneally on the next four days. On day 7 a sample of serum was taken and titrated for antibodies. The initial results showed a decrease in haemoglutination by a factor of 1024 in comparison with the controls [8]. In the same mice no non-specific antiproliferative activity was found. These results gave the first indication that cyclosporin might be an important compound. The only other effects found by the General Screening programme were a mild analgesic action and nephrotoxicity in rats given high doses of preparation 24-556 over one week.

The General Screening Programme had discovered the three properties of cyclosporin which have both shaped and limited its future use. First was its immunosuppressive activity, second it has no non-specific cytostatic action and finally its nephrotoxicity.

Research and development of cyclosporin

After the initial interesting and promising results of the General Screening programme, further microbiological, chemical and pharmacological work was carried out at Sandoz.

The initial experiment that had highlighted the immunosuppressive activity of cyclosporin (see above) was repeated but the results were disappointing. Administration of preparation 24-556 orally and by the intraperitoneal route only showed a four-fold decrease in haemoglutination, despite the use of a higher dose. If this had been seen in the original experiment further development of cyclosporin would never have been carried out. The low results were later found to have been due to poor absorption of the drug. An alternative procedure had been used to solubilise the highly hydrophilic cyclosporin. The hydrophilic nature of the drug was to cause more problems in cyclosporin’s subsequent development. 

Luckily, other immunological assays that were routinely used at Sandoz, that had been developed for investigation of Ovalacin, showed activity. Further experiments showed that cyclosporin selectively inhibited the proliferation of lymphocytes by acting on an unknown and unique step in the process, whilst not affecting proliferation of other somatic cells. In the words of Borel: ‘It was almost too beautiful to be true’ [6].

The scientists who had developed cyclosporin this far were already convinced of its relevance to immunosuppression. However, the goals at Sandoz had changed by 1973; immunology was no longer regarded as a fertile research field. This change was due to the rapid developments in immunology that had taken place. Although basic knowledge of immunology had improved dramatically, comparable improvement in clinical applications for this knowledge had not been developed. Organ transplantation was a small, unattractive market restricted mainly to kidney transplants with the use of cheap immunosuppressive drugs (such as azathioprine and corticosteroids ). It was estimated that $250 million would have been needed to develop cyclosporin through to US Food and Drug Administration approval. The recent failure of Ovalacin in its clinical trials was another factor that had led management to believe prospects for a new immunosuppressant were low. A way of getting approval for further development was therefore found. The scientists’ exact method for gaining official approval of cyclosporin further development is unclear. Cyclosporin anti- chronic inflammatory action seems to have been the key, however.

Whether further development was granted for preventing the symptoms of experimental encephalomyelitis in rats, as claimed by Stähelin [8] or to test on adjuvant arthritis in the rat (Borel and Kis [6]) is unclear.

However, official permission to carry on with cyclosporin was indeed granted. The next step was to determine the exact structure of the active metabolites fromTolypocladium inflatum  that were present in preparation 24-556.

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Figure 2. The structure of cyclosporin

The active metabolite was found to be a cyclic undecapeptide that was subsequently named cyclosporin. The structure and conformation of cyclosporin ( Figure 2) was determined by chemical degradation together with an X-ray crystallographic analysis of an iodo- derivative and by two-dimensional nuclear magnetic resonance (NMR) imaging  studies of cyclosporin itself [9]. Cyclosporin was found to be rich in hydrophobic amino acids, neutral, insoluble in n-hexane and water but very soluble in all other organic solvents [10].

Chemical degradation

Cyclosporin was hydrolysed and was found to be made up of eleven amino acids, ten of which were known but the amino acid at position one was unknown [11].

X-ray crystallographic analysis [10]

Cyclosporin was hard to cystallise on its own and so was initially analysed as crystalline iodocyclosporin. The unknown amino acid was found to have an R group structure as shown in Figure 3. It was found to be a beta-hydroxy, singly unsaturated amino acid (4R)-4[(E)-2-butenyl]-4,N-di-methyl-L-threonine, abbreviated to MeBmt.

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Figure 3. The R group of MeBmt

NMR spectra of cyclosporin [12]

NMR was used to determine the conformation of cyclosporin in a crystalline state and in solution. A later development, in 1984, of the total synthesis of cyclosporin [13] enabled a systemic study of cyclosporin structure-activity relationship. Biological activity was found to be associated with amino acids 1, 2, 3, 4,10, and 11 which are on the surface of the molecule [14].

Two studies were performed to determine whether cyclosporin action was selective for lymphocytes and to exclude any cytostatic effects on cells other than lymphocytes. The first study showed that in vitro cyclosporin was 300 times more active in preventing the proliferation of spleen lymphoid cells than on nonlymphoid mastocytoma cells [15]. The second study examined the effect of cyclosporin on bone marrow cell count and haematopoietic myeloid stem cell proliferation in mice. Effects were minimal. Even at high doses, no effect was found on haematopoietic myeloid stem cell proliferation and bone marrow cell count was only slightly reduced. These studies proved without doubt the value of cyclosporin. The results were published in 1976 in Agents and Actions as ‘Biological effects of cyclosporin A: a new antilymphocyte agent’ [16]. It bought the discovery of cyclosporin to the world's attention.

The properties of cyclosporin interested Roy Y. Calne and his coworker D. G. White who had been involved in the development of azothioprine for transplantation. Calne (now Sir Roy; see Figure 4 below) is well known as a pioneer of transplantation surgery. It was therefore in Cambridge that the first animal testing outside Sandoz was carried out.

Toxicological studies

In 1975 toxicology studies were carried out at Sandoz in preparation for human testing. Rats given preparation 24-556 at high doses for 13 weeks showed renal and hepatic toxicity. In a similar experiment, dogs were treated with high doses of cyclosporin powder given orally in capsules, but showed no effects. Since the test on dogs was unrevealing, another toxicity study with cyclosporin was soon begun in the autumn of 1975, but this time in monkeys [8]. In these animals, the drug exhibited some activity, which led to the decision to begin clinical trials. The reason for the failure in the dog study was a low absorption of the cyclosporin powder. The results were sent to Calne, who in his animal experiments administered cyclosporin dissolved in olive oil. Calne’s results, especially in orthotopic heart grafts in the pig [17] were very encouraging ( Table 2).

Calne found that cyclosporin was a more effective immunosuppressive than any other drug that he had used in pigs with orthotopic cardiac allografts. In the discussion of his results he wrote, ‘sufficiently non-toxic and powerful as an immunosuppressant to make it [cyclosporin] an attractive candidate for clinical investigation in patients receiving organ grafts.’

Clinical trials

The first human trials were started in late 1976. Pure undissolved cyclosporin powder was given in gelatin capsules. The drug was not absorbed and trials were stopped until absorption from the gastrointestinal tract could be achieved. At this point there was no sensitive chemical or radioimmuno- assay for detecting cyclosporin in blood serum. Stähelin suggested the use of a bioassay that had previously been used at Sandoz for different purposes. The assay used the extent of inhibition of the in vitro proliferation of mitogen stimulated mice lymphocytes ( lymphocytes that had been stimulated to divide) to determine the concentration of cyclosporin in blood serum [8]. Later, both radioimmunoassays and chemical assays were developed to test serum concentrations in patients [18].

Now that a way of measuring serum concentration of cyclosporin had been found it was possible to arrange a study into the absorption of cyclosporin. This was carried out in 1977. Initially, three oral preparations were tested on three Sandoz employees, Borel, Stähelin and B. von Graffen. The first preparation, taken by Borel showed strong inhibitory activity in his serum. The preparation was an aqueous solution of water, alcohol and polyoxyethylene(20)-sorbitan-mono-oleate (Tween 80®). The second preparation was a suspension of cyclosporin in olive oil, this showed weak serum activity. The third was a capsule of cyclosporin powder that showed no serum activity. These results lead to the development of both oral and parentral preparations by the Galenical Department at Sandoz [19].

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Figure 4. Sir Roy Calne

In 1978 Calne started tests on humans. Seven patients with renal failure were given mismatched cadaver kidney transplants [20]. Initially, cyclosporin was administered on its own and was found to be effective at inhibiting rejection. However, nephrotoxity and hepatotoxicity were observed. A cyclophosphamide was later given along with cyclosporin. One patient died of systemic Aspergillus and Candida infection. Another required an allograft nephrectomy because of pyelonephritis in the graft. The other five patients left hospital with functioning allografts. The results were encouraging but more trials were required.

The next trial undertaken by Calne led to the publication of his report ‘Cyclosporin A initially as the only immunosuppressant in 34 recipients of cadeveric organs: 32 kidneys, 2 pancreases, and 2 livers’ [21]. Although Calne’s publication became one of the most important in the history of clinical transplantation , it contained three pieces of information so troubling that further clinical trials were jeopardised. First was a high incidence of lymphomas. Second none of the kidney recipients had normal graft function. Third, there had been a high patient mortality [6].These results were found to be due to over suppression. When Calne’s patient’s kidney function had been poor he had interpreted this as rejection rather than a toxic effect of cyclosporin and had given prednisolone and a cyclophosphamide derivative which made matters worse. Reduction in cyclosporin dosage allowed continuation of clinical trials.

After further trials it was found that cyclosporin with a combination of steroids gave better control of rejection, preserved renal function, and decreased morbidity. In November 1983, 13 years after its discovery, cyclosporin was approved by the US Food and Drug administration for prevention of transplant rejection.

Borel vs Stähelin

The generally acknowledged discoverer and pioneer of cyclosporin is Jean Francois Borel (Figure 5). Borel started working at Sandoz in 1970, replacing S. Lazaray as head of the Immunology Department. After obtaining his PhD in 1958 Borel worked mainly in the field of veterinary immunogenetics. In 1965 he moved to the Swiss Research Institute where he studied immunology and inflammation in the Department of Medicine. Then in 1970 he made the move to Sandoz [22].

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Figure 5. Jean Francois Borel

It was in the immunology department led by Borel that the immunosuppressive activity of  cyclosporin  was discovered. However Stähelin [8] refutes the commonly held view that Borel discovered its activity. Stähelin was in charge of the Pharmacology Department, of which the Immunology Group was a part. ‘The test [immunising mice with sheep erythrocytes] was performed in my [Stähelin’s] laboratory…., except for the titration which was done in Borel’s laboratory……Borel, not being interested in The General Screening Programme, did not see the results until later’.

Borel is also given much of the credit for the further development and promotion of cyclosporin, claiming he pursued its development throughout. Stähelin claims that Borel wanted to drop the development of cyclosporin after the failure to find immunosuppressant activity in dogs dosed with cyclosporin.

It is generally accepted that ‘Borel later was to risk his life…By experimenting on himself before cyclosporin toxicity was known…' [23]. This is in reference to the absorption experiment in which Borel and others participated. Stähelin claims that ‘this was an experiment arranged by B. von Gaffenried.. …The procedure was, of course, a controlled clinical trial, and not a self-administration.’

Inevitably, jealousy can arise when the credit for significant discoveries is attributed to one man. The contribution from others was equally vital in cyclosporin development. With so much mundane and routine work being carried out at Sandoz ( 1,000 preparations were fed into The General Screening programme e ach year), it is inevitable that there would be some controversy over the exact details of cyclosporin discovery and development.

Perhaps most credit should go to the scientists K. Saameli, who set up The General screening Programme and S. Lazary, who developed the testing for immunology before Borel had even arrived at Sandoz. The programme ensured that the immunosuppressive properties of any compound entered into it would be found. Luck, however, also played a part. If A. Rüeggerhad not entered the sample in the programme cyclosporin may have only ever been known as a weak antifungal agent, with no clinical value. However the combination of hard work and good luck bore fruit.

SYNTHESIS

In 1984 synthetic cyclosporin was produced (Figure 6).It was then possible for cyclosporin to be chemically modified in every possible way. However, none of the derivatives have been found to have greater potency or decreased side effects than cyclosporin itself [24].The two major limitations of cyclosporin therapy today remain its nephrotoxicity and incomplete control of chronic rejection.

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Figure 6. SEM of cyclosporin crystals in purest form

In 1996 undergraduate mycology students from Cornell University on a field trip to Ithaca, New York were told to pick up anything that looked interesting. Among the findings was a mysterious fungal fruiting body in an eviscerated beetle grub [25]( Figure 7). The fungus was later identified as Cordyceps subsessilis an extremely rare fungus that is the sexual state of Tolypocladium inflatum. All the cyclosporin had so far been made from Tolypocladium inflatum cultures without it ever reaching the sexual state. Cordyceps is a large genus that includes around 280 species [26], and may be a good place to start looking, in the estimated 90% of world fungi that have yet to be identified, for a new and improved transplantation drug.

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Figure 7. A beetle larva containing Cordyceps subsessilis

CONCLUSIONS

Discovery of cyclosporin 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. Its contribution to autoimmune therapy is less well known but in the long term will probably be of comparable importance.

Cyclosporin did not solve all the problems of transplantation. Today chronic rejection is the main problem. It is poorly understood and there is no treatment for it, although it is thought to have a large immunological component. The majority of transplant patients require long term treatment with high doses of immunosuppressives which increases susceptibility to infection and malignancies.

Thanks to the discovery and development of cyclosporin, patients are alive today years after their operation. Without cyclosporin they would not have survived