The discovery of cyclosporine started a new age of immunopharmacology of 1971. It was the first immunosuppressive drug to enable selective immunoregulation of T cells without excessive toxicity. Cyclosporin was isolated from Tolypocladium inflatum fungus. Cyclosporin was first studied as an anti-fungal antibiotic, but its scope was too limited for any clinical application. J. F. Borel discovered its immunosuppressive activity in 1976. This led to further study of its properties, including further immunological experiments and studies of its structure and synthesis. Cyclosporin has undesirable side effects , especially nephrotoxicity. Animal studies showed that cyclosporine was sufficiently non-toxic to start clinical trials. These were originally not attributed to poor absorption of the drug. Once this was reversed, the findings were convincing enough for cyclosporin to be approved for use in clinical practice. There is some dispute between Borel and other staff about the importance of the discovery of cyclosporine and its pre-clinical growth, which is discussed in this study. Cyclosporin has modified the face of the transplantation. It decreased morbidity and allowed the routine transplantation of organs that had only been done experimentally until then.
We all know the story of the discovery of Penicillin, but what about other medicines that are still widely used? This SSM explores the events that contributed to the discovery of cyclosporine [initially known as cyclosporine A, now known as ciclosporine in Europe and cyclosporine in the USA], and the changes that resulted from its introduction into clinical practice.
A man who placed a soil sample in a plastic bag saved the life of Irene, a lively student who was diagnosed with acute myeloblastic leukemia at the age of 18. It has evolved quickly and seriously. Chemotherapy may give her a brief recovery, but will eventually end in a fatal relapse. Yet Irene had two bits of good fortune. First, she was immunologically (HLA) compatible with her younger sister. The second cyclosporine was found a few years ago. Doctors expressed concern about the toxicity of cyclosporin and the possible development of catastrophic host-to-graft disease. Amid their anxieties, Irene had no other hope and a bone marrow transplant had been begun. Irene has had serious kidney problems. Nothing was known about cyclosporin nephrotoxicity, and unnecessarily large doses were possibly provided. Thanks to cyclosporin, however, she had a mild host-versus-graft reaction and was able to leave the hospital and live a normal life.
Table of Contents
- 1 What is cyclosporine and what is it used for?
- 2 Why was the discovery of cyclosporine so important?
- 3 THE CYCLOSPORIN DISCOVERY
- 4 SYNTHESIS
- 5 CONCLUSIONS
- 6 References
What is cyclosporine and what is it used for?
Organ and bone marrow transplants are regularly performed today. Cyclosporin is also used to treat rejection reactions that occur when the body’s immune system attacks a foreign organ. Cyclosporin is a fungal peptide that is isolated from Tolypocladium inflatum Gams. It was the first immunosuppressant to act selectively to suppress T-cell immunity.
Cyclosporin is currently (March 2001) licensed for use in organ transplantation to prevent grafting in kidney , liver, heart, lung and combined heart and lung transplants. It is used to avoid rejection after bone marrow transplantation and in the prophylaxis of host-versus-graft disease. It is also used for the treatment of psoriasis, atopic dermatitis, rheumatoid arthritis and nephrotic syndrome.
Why was the discovery of cyclosporine so important?
The discovery of cyclosporin immunosuppression in 1976 is credited to J. F. Borel, see Figure 5 above. Cyclosporin was approved for clinical use in 1983 in the prevention of graft rejection in transplantation. At this time, most of the medical complications of allograft transplantation have already been overcome.
Since 1961, the main procedure for achieving immunosuppression has been the combination of azathioprine and corticosteroids. Azathioprine prevents non-selective cell proliferation. Its major adverse side effects are bone marrow depression, other toxic effects include increased susceptibility to infections, moderate hepatotoxicity, skin rashes, nausea and vomiting. Corticosteroids suppress T lymphocytes and have anti-inflammatory effects. Side effects include diabetes, avascular bone necrosis and increased susceptibility to infection.
Cyclosporin has been the best immunosuppressant to be identified so far, has also resolved many of the risk factors associated with azathioprine and is relatively non-toxic to the bone marrow. Patient morbidity decreased with the advent of cyclosporin. It was possible to transplant organs with a one-year success of 20 per cent higher than before, and to successfully transplant organs that had only been performed in studies before: heart, liver , lung and mixed cardiac lung transplants.
In addition to transplantation, cyclosporin has been used in most autoimmune disorders. Experimental treatment with cyclosporine of insulin-dependent diabetes mellitus, inflammatory bowel disease, chronic asthma, atopic dermatitis, aplastic anaemia and psoriasis in the 1980s backed proof of their mediated T cell nature.
THE CYCLOSPORIN DISCOVERY
Discovery of anti-fungal antibiotics
The practice developed as part of a program set up in 1957 to look for new fungal metabolite antibiotics was for Sandoz workers on business trips and holidays to carry plastic bags with them to collect soil samples that were cataloged and then screened. In March 1970, the fungus Tolypocladium inflatum Gams (Figure 1) was isolated by B in the Department of Microbiology at Sandoz Ltd. (Basel), a Swiss pharmaceutical company. Thiele from two soil samples, the first from Wisconsin, USA, and the second from Hardanger Vidda, Norway. These soil samples were obtained by Sandoz employees.
Section 1. Figure 1. Tolypocladium inflatum electron scanning micrograph
The Department of Microbiology at Sandoz has established a computer-aided evaluation software for the screening and evaluation of sampled fungi. The software has allowed the rapid assessment of samples, the identification and elimination of common fungi and related strains, which have produced known compounds from further studies. This meant that more time could be spent analyzing rare fungi in the samples, which would be more likely to generate metabolites with potential for new antibiotic action. The software detected Tolypocladium inflatum, which was previously unknown to the Sandoz team and developed interesting metabolites.
First, Z.L. Kis systematically extracted a mixture of metabolites from Tolypocladium inflatum. The characteristics were integrated into another computer program on the basis of an exhaustive set of data collected from the literature and demonstrated the existence of a group of metabolites that were new to Sandoz. These metabolites were found to possess some antifungal activity and further study was required.
Large samples were needed for further investigation. Tolypocladium inflatum was fermented and a technique for the isolation of two of the metabolites later called cyclosporin A and C was developed. Samples were formed in submerged crops and extracted by organic solvents. M Dreyfuss and colleagues investigated the antibacterial and anti-fungal role of cyclosporine A and C. Only a small range of activity against fungi was detected, and no antibacterial activity was detected.
Only a few species of yeast have been found to be susceptible to metabolites. When grown in solid media and in contact with cyclosporine, the growth rate of the sensitive species has decreased. Strains of certain mucorals, ascomycetes and fungi imperfecti displayed susceptibility to varying degrees, inhibition taking the form of deformation and branching of rising hyphae tips. No effect was observed on the germination of the spores or conidia of the infected fungi.
By studying and comparing the taxonomic positions of sensitive species, Dreyfuss and colleagues believed that the anti-fungal mode of action was due to inhibition of cell wall synthesis, in particular chitin synthesis. Cyclosporin activity was contrasted with the only known chitin-blocking antibiotic Polyoxin, which had a similar narrow range to that seen in cyclosporin.
An anti-fungal drug that prevented cell wall synthesis would have been a valuable development, because it would have a high specificity and low toxicity to non-fungal hosts, comparable to an indispensable category of beta-lactam antibiotics. However, cyclosporine was found to be ineffective against Sporobolomyces roseus and Sporobolomyces antarcticus (Table 1). This suggested a different mode of action for cyclosporines.
Section 1. Table 1. Extension of inhibition zones in fungal growth (as measured in mm) caused by certain antifungal agents.
Owing to their limited spectrum and slow action, there was no hope for cyclosporine as an anti-fungal agent, and Dreyfuss and his colleagues stopped their investigations.
Discovery of immunosuppressive activity by cyclosporine
The first non-steroidal immunosuppressive, non-toxic to bone marrow, was discovered in Sandoz in 1962 in the search for new and useful fungal metabolites. It was isolated from Pseudourotium ovalis in 1965 and was called Ovalacin. Immune response was highly depressed but did not impair intestinal epithelial cell division or myeloblast proliferation, unlike other cytostatic medicines. Ovalacin preceded the discovery of cyclosporine and was important for the discovery of cyclosporine.
Ovalacin is 600 times more potent than cyclosporin by weight, but due to its adverse effects, clinical trials have failed.
In January 1970, the head of the Department of Pharmacology at Sandoz, K. Saameli, established a program of about 50 pharmacological tests carried out by various groups in the Department of Pharmacology, the ‘General Screening Programme'. A. Rüegger of the Department of Chemistry, conscious that microbial metabolites also have important pharmacological activity, submitted cyclosporin to the General Screening Program in 1971. Preparation number 24-556 was given to the sample and was later found to contain mainly cyclosporin.
Of all the pharmacological studies carried out in the General Screening Programme, only one produced a positive result. This was an immunosuppression test. On day one, mice were injected intravenously with sheep erythrocytes and 24-556 was injected intraperitoneally for the next four days. On day 7 a serum sample was taken and titrated for antibodies. Initial findings showed a decrease of 1024 in haemoglutination relative to controls. No non-specific antiproliferative activity has been observed in the same mice. These findings provided the first hint that cyclosporine could be an effective compound. The only other effects detected by the General Screening program were mild analgesic activity and nephrotoxicity in rats given large doses of 24-556 over one week of training.
The General Screening Program has identified three characteristics of cyclosporine that have both influenced and restricted its potential use. First, it had its immunosuppressive behavior, second, it had no non-specific cytostatic action, and finally its nephrotoxicity.
Analysis and production of cyclosporine
Following the initial interesting and positive results of the General Screening program, further microbiological , chemical and pharmacological work has been carried out in Sandoz.
The initial experiment which highlighted the immunosuppressive activity of cyclosporin (see above) was repeated, but the results were disappointing. Administration of 24-556 oral and intraperitoneal preparation recorded just a four-fold decrease in haemoglutination despite the use of a higher dose. If this had been shown in the initial experiment, further production of cyclosporine may never have taken place. Low findings were later found to be due to poor absorption of the drug. An alternative method was used to solubilize the strongly hydrophilic cyclosporine. The hydrophilic aspect of the drug created further complications in the subsequent production of cyclosporine.
Luckily, other immunological tests that were regularly used in Sandoz, which had been developed for the investigation of Ovalacin, showed development. Further studies have shown that cyclosporin selectively inhibited the spread of lymphocytes by acting on an unknown and special stage in the process, while not affecting the spread of other somatic cells. In the words of Borel, ‘It was almost too good to be true'.
Scientists who have already produced cyclosporine have also been convinced of its importance to immunosuppression. However, the priorities of Sandoz had shifted after 1973; immunology was no longer seen as a promising area of study. This move was due to the rapid advances in immunology that have taken place. While basic knowledge of immunology has improved significantly, comparable improvements in clinical applications for this knowledge have not been made. Organ transplantation was a small, unattractive market confined primarily to kidney transplants using cheap immunosuppressive drugs (such as azathioprine and corticosteroids). It was estimated that $250 million would have been required to produce cyclosporin with the approval of the US Food and Drug Administration. The recent failure of Ovalacin in its clinical trials was another factor that led management to conclude that the potential for a new immunosuppressant was limited. Therefore, a way to gain permission for further progress has been identified. The exact procedure used by scientists to obtain official approval for further production of cyclosporin is unknown. Cyclosporin, however, seems to have been the precursor to persistent inflammatory action.
It is unclear if further progress has been made to prevent the symptoms of experimental encephalomyelitis in rats, as stated by Stähelin, or to test for adjuvant arthritis in rats (Borel and Kis).
However, official permission to continue cyclosporin has also been given. The next move was to determine the exact structure of the active metabolites of Tolypocladium inflatum present in preparation 24-556.
Chart 2. Figure 2. The composition of the cyclosporine
A cyclic undecapeptide, subsequently called cyclosporin, was found to be the active metabolite. The structure and conformation of cyclosporine (Figure 2) was determined by chemical degradation, along with an X-ray crystallographic examination of the iodine derivative and a two-dimensional nuclear magnetic resonance ( NMR) imaging study of cyclosporine itself. Cyclosporin has been found to be abundant in hydrophobic amino acids, neutral, insoluble in n-hexane and water, but very soluble in all other organic solvents.
Degradation of chemicals
Cyclosporin was hydrolyzed and was found to consist of eleven amino acids, 10 of which were known but the amino acid at position one was unknown.
Cristalographic examination of X-rays
Cyclosporin was difficult to cystallize on its own and was initially studied as crystalline iodoclosporin. An unknown amino acid has been found to have an R-group structure as shown in Figure 3. It was identified to be beta-hydroxy, a single unsaturated amino acid (4R)-4[(E)-2-butenyl]-4,N-di-methyl-L-threonine, abbreviated to MeBmt.
Section 3. Figure 3. The MeBmt R Party
NMR spectra of cyclosporine
NMR was used to determine the conformation of cyclosporine in a crystalline state and in a solution. Later, in 1984, the complete synthesis of cyclosporine allowed a systemic analysis of the structure-activity relationship of cyclosporine. Biological activity was found to be correlated with amino acids 1 , 2, 3, 4, 10 and 11 on the surface of the molecule.
Two experiments were conducted to determine whether cyclosporin activity was selective for lymphocytes and to eliminate any cytostatic effects on cells other than lymphocytes. The first study showed that in vitro cyclosporin was 300 times more involved in preventing the spread of spleen lymphoid cells than in non-lymphoid mastocytoma cells. The second study looked at the effect of cyclosporin on bone marrow cell count and haematopoietic myeloid stem cell proliferation in mice. Effects are small. Even at high doses, no effect on haematopoietic myeloid stem cell proliferation was observed and the bone marrow cell count was only marginally reduced. Without hesitation, these studies have shown the importance of cyclosporine. The findings were published in 1976 in Agents and Acts as’ Biological Effects of Cyclosporin A: A New Anti-Lymphocyte Agent'. It purchased the discovery of cyclosporine for the benefit of the world.
Roy Y. Calne and his coworker D were interested in the properties of cyclosporin. G. White who was interested in the production of azothioprine for transplantation. Calne (now Sir Roy; see Figure 4 below) is well regarded as a pioneer in transplantation surgery. It was also in Cambridge that the first animal study outside of Sandoz was carried out.
In 1975, toxicology tests were performed at Sandoz in preparation for clinical research. Rats administered 24-556 at elevated doses for 13 weeks showed renal and hepatic toxicity. In a related experiment, dogs were treated with high doses of cyclosporin powder given orally in capsules, but had no effect. Since the dog research was unrevealed, a further toxicity analysis with cyclosporin started soon in the autumn of 1975, but this time in monkeys. The drug showed some activity in these animals, which contributed to the decision to launch clinical trials. The explanation for failure in the dog study was a poor absorption of cyclosporine material. The findings were sent to Calne, who administered cyclosporine dissolved in olive oil in his animal experiments. Calne ‘s results, particularly in the orthotopic heart grafting of the pig, were very promising (Table 2).
Section 2. Table 2. Survival times for orthotopic heart grafting in pigs with different immunosuppressive therapies
Calne found that cyclosporin was a more effective immunosuppressive agent than any other drug used in pigs with orthotopic cardiac allografts. In the review of his observations, he wrote, ‘Sufficiently non-toxic and effective as an immunosuppressant to make [cyclosporin] an attractive candidate for clinical investigation in patients receiving organ grafts.’
The first clinical trials began at the end of 1976. Pure undissolved cyclosporine powder was delivered in gelatin capsules. The drug was not absorbed and the experiments were suspended before absorption from the gastrointestinal tract could be achieved. At this point, there was no sensitive chemical or radioimmuno-assay to detect cyclosporine in the blood serum. Stähelin proposed the use of a bioassay that had previously been used in Sandoz for a number of purposes. The assay used the level of inhibition of the in vitro distribution of mitogen-stimulated mice lymphocytes (lymphocytes that had been stimulated to divide) to assess the concentration of cyclosporine in the blood serum. Subsequently, both radioimmunoassays and chemical assays were developed to assess serum concentrations in patients.
Now that a way was found to measure the serum concentration of cyclosporine, a report on the absorption of cyclosporine could be planned. This was done in 1977. Three oral preparations were initially checked on three Sandoz staff, Borel, Stähelin and B. It’s von Graffen. The first test, taken by Borel, showed significant inhibitory activity in the serum. The preparation was an aqueous solution of water , alcohol and polyoxyethylene(20)-sorbitan monooleate (Tween 80 ®). The second preparation was a suspension of cyclosporine in olive oil, which showed poor serum activity. The third was a cyclosporin powder capsule that displayed no serum activity. These findings have led to the production of oral and parenteral preparations by the Galenic Department of Sandoz.
Chart 4. Figure 4. Sir Roy Calne’s
Calne began experiments on humans in 1978. Seven patients with renal failure were treated with irregular renal transplants. Initially, cyclosporin was administered on its own and was found to be effective in inhibiting rejection. However, nephrotoxicity and hepatotoxicity have been reported. Later, cyclophosphamide was given along with cyclosporine. One patient died of systemic infection with Aspergillus and Candida. Another needed allograft nephrectomy due to pyelonephritis in the graft. The remaining five patients left the hospital with a working allograft. The results were promising, but more trials were required.
Calne’s next trial led to the publication of his report ‘Cyclosporin A’ initially as the only immunosuppressant in 34 cadeveric organ recipients: 32 kidneys, 2 pancreases and 2 livers. While Calne ‘s publication became one of the most significant in the history of clinical transplantation, it included three pieces of information so alarming that further clinical trials were at risk. Next, there was a high rate of lymphomas. Second, none of the recipients of the kidney had natural graft activity. Third, there was a high patient mortality rate. These findings were found to be due to over-repression. Because Calne’s kidney function was low, he viewed it as a rejection rather than a toxic effect of cyclosporin and gave prednisolone and a cyclophosphamide derivative that made things worse. Reduction in cyclosporin dosage allowed clinical trials to proceed.
Following further research, cyclosporin with a combination of steroids has been shown to have improved control of rejection, conserved renal function, and decreased morbidity. In November 1983, 13 years after its discovery, cyclosporin was approved by the US Food and Drug Administration for the prevention of transplantation rejection.
Borel vs. Stehelin
The widely accepted discoverer and founder of cyclosporine is Jean Francois Borel (Figure 5). Borel began working at Sandoz in 1970, replacing S. Lazaray as Director of the Department of Immunology. After earning his PhD in 1958, Borel worked primarily in the area of veterinary immunogenetics. In 1965, he moved to the Swiss Research Institute, where he studied immunology and inflammation at the Department of Medicine. Then he moved to Sandoz in 1970.
Section 5. Figure 5. Jean François Borel
It was in the Borel-led immunology department that the immunosuppressive role of cyclosporin was discovered. However, Stähelin refutes the widely held view that Borel had discovered its work. Stähelin was in charge of the Department of Pharmacology, which was part of the Immunology Division. ‘The research [immunizing sheep erythrocyte mice] was carried out in my [Stähelin’s] laboratory, except for the titration performed in the Borel laboratory …… Borel, not involved in the General Screening Method, did not see the results until later.’
Borel also deserves a considerable amount of credit for the further production and promotion of cyclosporine, claiming to have supported its production throughout. Stähelin believes that Borel tried to minimize the production of cyclosporin following failure to detect immunosuppressive activity in dogs dosed with cyclosporin.
It is widely agreed that ‘Borel will lose his life later … by experimenting on himself before cyclosporin toxicity was known … ‘. This is in the sense of the absorption experiment in which Borel and others participated. Stähelin maintains that ‘this was an experiment organized by B. Von Gaffenried, guy. …The treatment was, of course, a controlled clinical trial, not self-administration.’
Inevitably, envy may occur when credit for major discoveries is credited to a single individual. The contribution of others was equally essential to the production of cyclosporine. With so much tedious and repetitive work being performed at Sandoz (1,000 preparations were fed into the General Screening program each year), it is likely that there will be some debate about the exact nature of the discovery and production of cyclosporine.
Perhaps much of the credit should go to the K scientists. Saameli, who set up the General Screening Program and S. Lazary, who had developed immunology tests before Borel had even arrived in Sandoz. The software ensured that the immunosuppressive properties of any compound entered would be identified. Luck, however, has also played a part. If A. Rüegger had not entered the sample in the cyclosporin method, it could only have been identified as a poor antifungal agent of no clinical benefit. But the blend of hard work and good luck bore fruit.
Synthetic cyclosporine was produced in 1984 (Figure 6). It was then possible for cyclosporine to be chemically modified in every possible way. However, none of the derivatives have been found to have greater efficacy or reduced side effects than cyclosporin itself. Today , the two major drawbacks of cyclosporin treatment remain its nephrotoxicity and poor regulation of chronic rejection.
Chart 6. Figure 6. SEM of cyclosporine crystals in the purest form
In 1996, students of mycology at Cornell University on a field trip to Ithaca, New York, were told to pick up something that seemed interesting. Among the results, there was a strange fungal fruiting body in an eviscerated beetle grub (Figure 7). The fungus was later known as Cordyceps subsessilis, an extremely rare fungus that is the sexual condition of Tolypocladium inflatum. So far, all cyclosporine had been produced from Tolypocladium inflatum cultures without reaching sexual status. Cordyceps is a wide family of about 280 species and may be a good place to start searching for a new and improved transplant drug in an estimated 90 percent of the world’s fungi that have yet to be identified.
Figure 7. Figure 7. Beetle larva containing the subsessilis of Cordyceps
The discovery of cyclosporine contributed to a period of selective lymphocyte inhibition. It has made it possible to bring the experience in clinical, scientific and immunobiological aspects of transplantation into practice and to transform the face of transplantation. Its contribution to autoimmune therapy is less well documented, but is likely to be of comparable significance in the long term.
Cyclosporin did not solve all transplantation issues. Chronic rejection is the biggest problem today. It is not well known and there is no cure for it, although it is thought to have a significant immunological portion. Most transplant patients need long-term care with large doses of immunosuppressive agents that increase vulnerability to infection and malignancies.
Thanks to the discovery and development of cyclosporine, patients are still alive years after their surgery. They wouldn’t have lasted without cyclosporin.
- Borel, J. F. 1986. Ciclosporin and Its future. Progress in Allergy, 38: 9-18.
- Rang, H. P., Dale, M. M., Ritter, J. M.1999. Pharmacology. 4th ed. Churchill Livingstone.
- Kahan, B. D.1984. Cyclosporine: nursing and paraprofessional aspects. Grune and Stratton.
- Kahan, B. D. 1999. Cyclosporine: a revolution in transplantation. Transplantation Proceedings, 31: (Suppl 1/2A), 14S-15S.
- Bach, J. F. 1999. The contribution of cyclosporine A to the understanding and treatment of autoimmune diseases. Transplantation Proceedings, 31: (Suppl 1/2A), 16S-18S.
- Borel, J. F, Kis, Z. L.1991.The discovery and development of cyclosporine (Sandimmune). Transplantation Proceedings, 23:1867-1874.
- Dreyfuss, M., Härri, H., Hofmann, H., Kobel, H., Pache, W., Tscherter, H. 1976. Cyclosporin A and C. European Journal of Applied Microbiology, 3:125-133.
- Stähelin, H. F. 1996. The history of cyclosporin A (Sandimmune®) revisited: another point of view. Experientia, 52:5-13.
- Wenger, R. M. 1986. Synthesis of Ciclosporin and analogues: structural and conformational requirements for immunosuppressive activity. Progress in Allergy, 38:46-64.
- 10. Petcher, T. J., Weber, H. P., Rüegger, A. 1976. Crystal and molecular structure of an iodo-derivative of the cyclic undecapeptide cyclosporin A. Helvetica Chimica Acta, 59:1480:157.
- Wenger, R. 1989. Pharmacology of cyclosporin. Pharmacological Reviews, 41:2 43-247.
- Kessler, H., Loosli, H. R., Oschkinat, H. 1985. Assignment of the 1H-, 13C, and 15N-NMR spectra of cyclosorin A in CDCl3 and C6D6 by a combination of homo- and heteronuclear two-dimensional techniques. Helvetica Chimica Acta, 68: 661-682.
- Wenger, R. M. 1984. Total synthesis of ‘cyclosporin A’ and ‘cyclosporin H’, two fungal metabolites isolated from species Tolypocladium Inflatum Gams. Helvetica Chimica Acta, 67: 503:515.
- Quesniaux, V., Tees, R., Schreier, M. H., Wenger, R. M., Donatsch, P., Van Regenmortel, M. H. V. Monoclonal antibodies to ciclosporin. Progress in Allergy, 38:108-122.
- Borel, J. F., Wiesinger, D. 1977. Regulatory mechanisms in lymphocyte activation. New York Press 56:716-721.
- Borel, J. F., Feurer, C., Gubler, H.U. & Stähelin, H. 1976. Biological effects of cyclosporin A: a new antilymphocytic agent. Agents and Actions, 6: 468-475.
- Calne, R. Y., White D. J. G., Rolles, K., Smith, D. P. 1978. Prolonged survival of pig orthotopic heart grafts treated with cyclosporin A. The Lancet, 1182: 1185.
- Schran, H. F., Robinson, W. T., Abisch, E., Niederberger, W. 1986. Bioanalytical considerations. Progress in Allergy, 38: 73-92.
- Cavanak, T., Sucker, H. 1986. Formulation of dosage forms. Progress in Allergy, 38: 65-72.
- Calne, R. Y., White, D. J., Thiru, S., Evans, D. B., McMaster, P., Dunn, D. C., Craddock, G. N., Pentlow, B. D., Rolles, K. Cyclosporin A in patients receiving renal allografts from cadaver donars. Lancet, 2(8104-5):1323-1327.
- Calne, R. Y. ., White, D. J., Thiru, S., Evans, D. B., McMaster, P., Dunn, D. C., Craddock, G. N., Pentlow, B. D., Rolles, K. 1979. Cyclosporin A initially as the only immunosuppressant in 34 recipients of cadeveric organs: 32 kidneys, 2 pancreases, and 2 livers. Lancet 2: 1327
- Stiller, C. R.1999. Tribute to Jean Francois Borel: A gentleman and a scholar. Transplantation Proceedings, 31: (Suppl 1/2A), 3S-8S.
- Bernard, J. 1986. Ciclosporin: foreward. Progress in Allergy, 38:1-8.
- Wenger, R. M.1982. Cyclosporin A. Biomedical Journal, 3:19-31
- Cornell University Page. Science News. Last updated 1996. Available from http://www.news.cornell.edu/releases/Sept96/cyclosporine.hrs.html
- Hodge, K. T., Krasnoff, S. B., Humber, R. A. 1996. Tolypocladium inflatam is the anamorph of Cordyceps subsessilis. Mycologia 88:715:719.