环孢素的研发历史-

1、环孢素的研发历史ABSTRACTDiscovery 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 invest

2、igated 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

3、. 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 lice

4、nsed 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 routi

5、ne transplantation of organs that until then had only been done experimentally.INTRODUCTIONWe 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

6、, now known as ciclosporine in Europe and cyclosporin in the USA, and the developments that resulted from its introduction into clinical practice.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 myeloblast

7、ic 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 discov

8、ered 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 complicatio

9、ns. 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

10、 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 bodys immune system. Cyclosporin is a fungal peptide, isolated from Tolypocladium inflatum Gams. It was the first immunosuppressant that act

11、ed 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 pr

12、ophylaxis of host-versus-graftdisease. It is also used in the treatment of psoriasis, atopic dermatitis, rheumatoid arthritis and nephrotic syndromeWhy was the discovery of cyclosporin so important?Discovery of immunosuppression by cyclosporin in 1976 is attributed to J. F. Borel, see Figure 5. In 1

13、983 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 corti

14、costeroids. 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

15、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

16、-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

17、 lung and combined heart lung transplants 4.As well as transplantation, cyclosporin has been used in most autoimmune diseases. In the 1980s experimental treatment with cyclosporin of insulin-dependent diabetes mellitus, inflammatory bowel disease, chronic asthma, atopic dermatitis, aplastic anaemia

18、and psoriasis supported evidence of their T cell mediated nature 5.THE DISCOVERY OF CYCLOSPORINDiscovery of an anti-fungal antibioticA 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

19、 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 s

20、amples, the first from Wisconsin, USA and the second from the Hardanger Vidda in Norway. These soil samples had been collected by Sandoz employees.Figure 1. Scanning electron micrograph of Tolypocladium inflatumThe Microbiology Department at Sandoz had developed a computer-aided evaluation program f

21、or 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 w

22、ould 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.

23、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 werenew to Sandoz. These metabolites were found to possess some antifungal activity and warranted further investigat

24、ion.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 co

25、lleagues 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

26、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 germ

27、ination 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

28、 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-fung

29、al 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.Table 1. Extent of inhibition zones in fungal g

30、rowth (measured in mm caused by some antifungal agentsOrganism P olyoxin A Cyclosporin A Cyclosporin CNeurospora crassa 25 20 20Neurospora crassa slime mutant0 0 0Rhodotorula rubra 40 20 17Sporobolomyces roseus 23 0 0Sporobolomycesantarcticus 18 0 0Due to their narrow spectrum and weak action there

31、was no future for cyclosporin as ananti-fungal agent and Dreyfuss and his colleagues stopped their investigations.Discovery of immunosuppressive activity by cyclosporinThe first non-steroidal immunosuppressive, non-toxic to bone marrow, was discovered at Sandoz in 1962 in the search for new and usef

32、ul 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 cyclosp

33、orin 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 a

34、bout 50 pharmacological tests performed by different Groups in thePharmacological Department, the General Screening Programme 8. A. Rüegger from t he Chemistry Department, aware that microbial metabolites often possess interesting pharmacological activity, submitted cyclosporin for the General

35、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 w

36、ere 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 contr

37、ols 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 do

38、ses 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.Resea

39、rch and development of cyclosporinAfter 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 (se

40、e 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 cyclos

41、porin 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 cyclosporins subsequent

42、 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 t

43、h e 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

44、; 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 develop

45、ed. 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 Ad

46、ministration 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 thereforefound. The scientists exact method for gaining official ap

47、proval 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

48、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.Figure 2. The structure of cy

49、closporinThe 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-dimensiona

50、l 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 degradationCyclosporin was hydrolysed and was found to be made u

51、p of eleven amino acids, ten of which were known but the amino acid at position one was unknown 11.X-ray crystallographic analysis 10Cyclosporin 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 structu

52、re 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.Figure 3. The R group of MeBmtNMR spectra of cyclosporin 12NMR was used to determine the conformation of cyclosporin in a crystalline state and i

53、n solution.A later development, in 1984, of the total synthesis of cyclosporin 13 enabled a systemic studyof 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

54、 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 nonlymph

55、oid 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 cou

56、nt 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 propertie

57、s 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; seeFigure 4 below is well known as a pioneer of transplantation surgery. It was therefore in Cambridge that the first animal testing

58、 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 cyc

59、losporin 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 exp