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Production Process Optimization of the Immunosupressant Drug Sirolimus

Production Process Optimization of the Immunosupressant Drug Sirolimus

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Published by: Akashdeep Sah on Dec 09, 2012
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INTRODUCTION Immunosuppressive drugs or immunosuppressive agents are drugs that inhibit or prevent activity of the immune system. They are used in immunosuppressive therapy to:
 

Prevent the rejection of transplanted organs and tissues (e.g., bone marrow, heart, kidney, liver) Treat autoimmune diseases or diseases that are most likely of autoimmune origin (e.g., rheumatoid arthritis, multiple sclerosis, myasthenia gravis, systemic lupus erythematosus, Crohn's disease, pemphigus, and ulcerative colitis). Treat some other non-autoimmune inflammatory diseases (e.g., long term allergic asthma control).

These drugs are not without side-effects and risks. Because the majority of them act nonselectively, the immune system is less able to resist infections and the spread of malignant cells. There are also other side-effects, such as hypertension, dyslipidemia, hyperglycemia, peptic ulcers, liver, and kidney injury. The immunosuppressive drugs also interact with other medicines and affect their metabolism and action. Actual or suspected immunosuppressive agents can be evaluated in terms of their effects on lymphocyte subpopulations in tissues using immunohistochemistry. Immunosuppressive drugs can be classified into five groups:
    

glucocorticoids cytostatics antibodies drugs acting on immunophilins other drugs

Sirolimus is one such Immunosuppressive drug acting on immunophilins. Sirolimus, also known as sirolimus, is a natural product found in a species of streptomycetes bacteria. It was originally used as an antifungal drug but was found to be more potent as an immunosuppressant for preventing organ transplant rejection. It also shows antitumor activity. Sirolimus is a macrolide derived from a product of the shikimate pathway. Researchers have found many ways to synthesize this natural product, allowing for its abundant use.

M S Lee et al found that different sources of nitrogen sources can largely impact the production of sirolimus. a 31-membered macro cyclic polyketide. When ingested. allowing for S. In a test. FKBP-12 further complexes with and deregulates the kinase enzyme. The hyphae eventually converts into “semi-dormant spores” that are heat resistant. FK506 binding protein-12 (FKBP-12).Fig. it enters the plasma membrane where it binds to the intracellular protein. S. hygroscopicus to survive and adapt to various soil environments.hygroscopicus can be found in most rich soil worldwide due to its complex fungi-like life cycle. OCCURRENCE Sirolimus was first isolated from the bacteria. As the mycelium grows. ammonium sulphate administered at 40mM resulted in the highest yield of the natural product in comparison to five other non-amino acid nitrogen compounds. it branches into the air into what is known as aerial hyphae.hygroscopicus depends on essential nutrients available. Another study found that L-lysine must be present for sirolimus production and that increases in lysine resulted in high yields of sirolimus. However. Brock suggests that Streptomyces tend to favour dryer alkaline and neutral soils. It grows under soil in branching filaments to form vegetative mycelium. BIOLOGICAL ACTIVITY Sirolimus is administered as an oral pill. The amount of sirolimus produced from S. Streptomyces hygroscopicus strain AY B994. commonly known as Easter Island. mammalian target of sirolimus (mTOR) that is responsible for regulating cell growth and proliferation through signal- . 1: Sirolimus. in a soil sample from Rapa Nui.

BIOSYNTHESIS Sirolimus is a triene macrocylic polyketide. Raps1 is responsible for the first four modules and contains a loading zone for the starter unit. In Streptomyces hygroscopicus. reported a 150% increase in sirolimus production by S.” This study reveals that sirolimus has very low toxicity with respect to the therapeutic dose. rapA. A reduction of the double bond occurs as the starter unit moves onto module 1. mainly the dephosphorylation and inactivation of the biochemical processes of P70 ribosomal S6 kinase (P70S6K).” The inhibition of mTOR halts downstream events. rats were given dosages up to 200 mg/kg intraperitoneally for 7 days. Cheng et al. three large polyketide synthase genes are responsible for the biosynthesis of sirolimus. rapB. At this point. the major role of sirolimus as an immunosuppressant.” By inactivating P70S6K. Damiao et al discovered that inhibiting mTOR “arrests the cell cycle in the G1 phase. This provides that sirolimus is a potent immunosuppressant. 4. P70S6K functions to induce “protein synthesis and cell-cycle progression. the pipecolate-incorporating enzyme (PIE) (produced by the RapP gene adjacent to Raps3). and Raps3. Raps2 adds three acetate units and three propionate units. These genes encode for 14 enzyme-containing modules. After module 14 is complete. This amount was “50-fold higher than the therapeutic dose of 5-10 mg/kg for mice” but only resulted in slight increases in “blood urea nitrogen levels” and “depressed body weight. binds to an activated L-pipecolate through a thioester linkage. three propionate units and one acetate unit is added with various oxidation states. derived from shikimate.” Raps3 adds three acetate units and a propionate unit. the sirolimus: FKBP-12: mTOR complex is able to inhibit the proliferation of T-cells. each having a specific role in elongating the sirolimus polyketide. In the first four modules. the shikimate derivative is latched onto CoA Ligase in the “loading domain” of Raps1. thereby deregulating cell growth. an enzyme complex consisting of six modules for further polyketide extension. The three polyketide synthase genes. . The resulting polyketide of Raps1 then enters Raps2. Note that L-pipecolate is a cyclization of L-Lysine. and rapC. 5-dihydroxycyclohex-1enecarboxylic acid. pre-sirolimus has a total of seven acetate and seven propionate units in addition to the shikimate derivative. At the start of the biosynthesis process.” the intermediate marocycle. The resulting fragment is transferred onto module 11 of Raps3 where four more successive rounds of elongation take place to complete “pre-sirolimus. encode the multienzyme polyketide synthases Raps1. respectively. The starter unit is a substituted cyclohexanecarboxylic acid. In another experiment conducted to test for potential side effects of sirolimus. Hygroscopicus upon addition of LLysine in a chemical defined medium. In its active form. Raps2. Leogrande et al found that sirolimus effectively reduces the levels of insulin-stimulated phosphorylated P70S6K in vivo and in vitro through measurements taken from human peripheral-blood mononuclear cells (PBMCs) of renal transplant recipients.transduction pathways.

Molecules 3. Three other syntheses were found in the same year.gif) TOTAL SYNTHESIS The total synthesis of sirolimus was first completed in 1993 by the Kyriacos Costa Nicolaou group. 5. Source:(http://openwetware. and 6 were the subtarget of this study. .Fig. 4. 5. The major disconnect of (-)-Sirolimus was at the triene carbon fragment that resulted in molecules 2 and 3. Molecule 2 was further broken down into fragments 4. sought a new convergent route to formulate sirolimus. 2: Life cycle of Streptomyces hygroscopicus. and 6. Maddess et al. In more recent times. A retrosynthesize was conducted as summarized in Figure 3.org/images/e/Streptomyces_Life_Cycle.

researchers are continuing to look at its structure in order to find additional analogs for treatment. .Fig. researchers aim to find other forms of sirolimus for treatment. Some highlights of this synthesis includes the “intramolecular trapping of oxonium ions” in 6. Although sirolimus has been studied extensively since its discovery in 1975. 3: Retrosynthesis of Sirolimus The actual process of total synthesis. is efficient in using a convergent route to chemically produce sirolimus. although long. and macroetherification/catechol tethering to form the macrocycle of sirolimus. use of BDA chemistry to protect and stereodirect certain reactions. Because sirolimus has antitumor activity.

hygroscopicus. the FDA approved safety labelling revisions for sirolimus to warn of the risk for decreased renal function associated with its use. of elaborating one or more products which possess substantial and varied antimicrobial activity. . under specific conditions. (1954). It is particularly advantageous in patients with kidney transplants for haemolytic-uremic syndrome. have made clear the necessity of examining a large assemblage of related forms as a prerequisite to establishing species boundaries. studying the same organism as well as other species of Streptomyces. aureofaciens. it is beyond the province of this paper to discuss such products or activities. it is felt that neither the capacity of a culture to produce such products nor the nature of such products themselves is of controlling significance in species differentiation in the case of S. However. (1954) emphasized the need for uniformity in methods of study and of reporting data relative to taxonomic studies. on October 7. this can be avoided by using sirolimus instead. while Jones (1954) pointed out the need for a better understanding of the organisms themselves before progress can be made in comprehending variability in Streptomyces.ADVANTAGE The chief advantage sirolimus has over calcineurin inhibitors is that it has low toxicity towards kidneys. 2008. a needlessly complex taxonomic system has arisen. Backus et al. as this disease is likely to recur in the transplanted kidney if a calcineurin-inhibitor is used. Furthermore. Burkholder and Sun (1954) have discussed criteria for speciation in Streptomyces and have stressed the need for a system of convenience in which a relatively few named species groups would be established. as a result of the indiscriminate granting of species status to numerous variants of already defined species. in their study of variability in S. (1954). and Duggar et al. While it is recognized that a majority of the cultures involved in this study are capable. STREPTOMYCES HYGROSCOPICUS It is becoming increasingly apparent that marked variability abounds in a great many of the species of Streptomyces and. Hesseltine et al. Transplant patients maintained on calcineurin inhibitors long-term tend to develop impaired kidney function or even chronic renal failure.

0). and tap water to 1 litre.4 litres of the same medium and autoclaved at 121 °C for 30 minutes." 20. The inoculated flasks were incubated for 18 hours at 25°C on a reciprocating shaker at 65 strokes per minute and 4"-throw. 500-m1 Erlenmeyer flasks were filled with 100 ml of an inoculum medium consisting of (g/litre): soybean meal ("Special X". The supernate was discarded and the mycelial pellet suspended in 250 ml of methanol and shaken vigorously.GENERAL METHOD HYGROSCOPICUS OF PRODUCTION OF SIROLIMUS FROM STREPTOMYCES The producing strain. as previously described1) Good growth and sporulation were obtained in 7-15 days of incubation at 25°C. New Brunswick Scientific Co.1 by addition of 10 N NH4OH solution. cooled to 25°C and pH of medium adjusted to 6. Streptomyces hygroscopicus. "Cerelose" (a pharmaceutical grade of glucose). 0.25 v/v/min. 20. was grown and maintained on tomato paste-oatmeal agar. 40. were dipped in the extract and placed on filter paper to dry.5 ml. The fermenters were inoculated with 3. The maximum titers were usually obtained in 96 hours. cooled to 25°C and inoculated with 78 ml (2 %) of the first-stage inoculum. These flasks were used to inoculate the production stage. cooled to 25°C and inoculated with 4 ml of the spore inoculum. Conventional paper disc-agar diffusion assays were used to determine the antibiotic titre. The extract was filtered. equipped with automatic antifoam addition system and pH recorder-controller. Unbaffled.). 1. Spores from one Roux bottle were suspended in 50 ml of sterile distilled water to constitute the spore inoculum.2 litres (2 %) of the second-stage inoculum. 13 mm in diameter. Archer Daniels Midland Co.. KH2PO4. 30.500 rev/min for 15 minutes. Sterile Mazer DF-143PXantifoam was added on demand. The flasks were agitated to resuspend the solids and autoclaved for an additional period of 1 hour at 121°C. The flasks were sterilized at 121'C for 30 minutes.). Minn. A 10-ml sample of fermentation broth was centrifuged at 2.35 hours of incubation the pH started to drop but was controlled at 6. a 40 % sterile solution of "Cerelose" was added continuously at the rate of 3. (NH 4)2SO4. Similar discs were dipped in . 250-liter capacity. The antibiotic titres were determined every 24 hours starting at 48 hours. 5. After 30. After 48 hours of incubation. "Cerelose. 5. 3. Minneapolis. CaCO3. Fermenters (model F-250. 2"-throw. Mazer DF-143PX (antifoam). and tap water to I litre (pH 7.0 by addition of 1ON NH4OH solution on demand. The fermentation was run at 25°C under an agitation of 200 rev/min and an aeration of 0. The fermenters were sterilized at 121'C for 30 minutes under an agitation of 150 rev/min. Unbaffled.5. were filled with 160 litres of the production medium consisting of (g/litre): soybean meal ("Special X"). (NH4)2SO4.85 % per day. to constitute the first-stage inoculum. 24-liter round bottom flasks were filled with 3. The inoculated flasks were incubated for 24 hours at 25°C on a gyrotory shaker at 240 rev/min. Filter paper discs.

ISOLATION OF SIROLIMUS The fermentation broth was adjusted to pH 4.25 µg rapamycin/ml. 1. 999). All the discs were deposited on agar plates seeded with the test strain of Candida albicans AY F-598. The inhibition zone diameters obtained for the standard solutions after overnight incubation were plotted against log concentration on semi-logarithmic paper and titre of fermentation broths read from the standard curve and corrected for dilution. dimethyl sulphoxide. The mixture was filtered and silica gel with adsorbed rapamycin washed onto a column with several volumes of 15 % acetone in hexane. dehydrated with anhydrous sodium sulfate and further concentrated to an oily residue. The residue was extracted twice with one volume of methanol. A typical 160-liter fermentation run yielded about 500 g of oily residue. The tri-chloroethane extracts were pooled and evaporated to a small volume under reduced pressure.9. containing the antibiotic. 8.2.5 and 1. dimethyl formamide. was extracted twice by stirring for 1 hour with 11 volume of trichloroethane. 1. 2 weights of silica gel G (Merck) per weight of oil were added and the mixture stirred gently for 50 minutes. The ultraviolet spectrum shows ymax at 288. N. The antibiotic was eluted with 25 % acetone in hexane and the eluant evaporated to dryness. N. The residue was dissolved in 10 v/w of a solvent mixture consisting of 15 % acetone in hexane. and practically insoluble in water. acetone.W. . 67. Found: C.93. 514 and 417 respectively. It is freely soluble in methanol. Calcd: C. H.standard solutions containing 10. The recoveries were about 40 % based on broth assay. 8. To this solution.39. methylene dichloride. ethanol. trichloroethane. 2. sparingly soluble in ether.4. 67. Rapamycin analysed for C56H89NO14 (E.24. chloroform. H. The residue was dissolved in ether from which pure rapamycin crystallized out.0 with a 30 % sulfuric acid solution and filtered on a vacuum rotary filter coated with Celite. 277 and 267 nm with E1%1cm 416. PHYSICAL AND CHEMICAL PROPERTIES OF SIROLIMUS Rapamycin is a white crystalline solid melting at 183-185'C. The mycelium. 5. The methanolic extracts were pooled and evaporated to dryness to yield about 50 g of oily residue containing rapamycin.

121oC. For the optimum production we basically focus on nitrogen sources used as nutrients Six non-amino acid nitrogen compounds were examined as nitrogen source for growth of Streptomyces hygroscopicus and biosynthesis of rapamycin. In the new chemically defined medium. Yeast extract 4. Sterilized distilled water was obtained from autoclaving the double distilled water at 15 psi. 5-6. CaCO3 2. Rapamycin forms a yellow chromophore when dissolved in 0.0g.1 . The growth media contains Glucose 4. Of the nitrogen sources studied. NMR spectrum (200 MHZ) of rapamycin shows vinylic protons between between 3. aspartate.5. Liquid broth culture was kept in the BOD shaker incubator. and Agar 25.1 N methanolic NaOH and heated at 60°C.0g. EFFECT OF NITROGEN SOURCE ON BIOSYNTHESIS OF RAPAMYCIN BY STREPTOMYCES HYGROSCOPICUS As we have discussed above the general method of production of Sirolimus.0g. The microbial strain received was in the lyophilized form.2 in methanol. First it was made active by the introducing the strain into sterilized distilled water. this property is the basis of a colorimetric assay. Two plates and two slant media was prepared to preserve the strain for the further use and for the current experimental purpose liquid broth culture was made. Materials and method The microbial strain Streptomyces hygroscopicus MTCC 4003 used for the optimization process of the Sirolimus production was obtained from ‘Institute of Microbial Technology. Chandigarh’ bearing MTCC code 4003. arginine plus histidine. an ammonium sulfate concentration of 40 mM was optimal for biosynthesis of rapamycin.The infrared spectrum shows OH at 3500. i. ammonium sulfate was the best with respect to formation of rapamycin. methoxyl Optical rotation is [α]25D-58.0g.6 and vinylic at 1. which is buffered with 200 mM 2-(N-morpholino) ethanesulfonic acid to prevent decline of pH during fermentation.. Malt extract 10. After this the growth media no 93 was made for the growth of the Streptomyces hygroscopicus. The growth condition is aerobic with incubation time of 2 days at 30oC.0g. Rapamycin production increased by more than 30% on both volumetric and specific bases as compared to the previous medium containing the three amino acids as nitrogen source.3. and supported cell growth comparable to the organic nitrogen sources used in the control chemically defined medium. .e. All the transfers were made in the laminar flow and the glass wares were autoclaved at 121°C and 15 psi to prevent the contamination of the strain. a band at 1730 (possibly lactone carbonyl) and at 1700 (carbonyl).8. and a band between 1610 and 1630 cm -1 (C=C).

The detailed composition is described in Table 1.0.0. The washed cells were suspended in the same buffer to make a 10-ml cell suspension.Spores were produced in Petri dishes at 28°C for 14 days on oatmeal agar medium. MI.05% MgSO4·7H2O.USA) 4. The evaporative losses during autoclaving and fermentation amounted to 27% of the initial 25-ml volume. 5. we used a chemically defined medium (Medium 3) which had been developed previously. The resulting culture broth was centrifuged at 4°C for 15 min (5000 × g). and K2HPO4 1. pH 7.0.e. pH 6. Inoculum A seed culture was initiated by adding 0. Detroit.0.5 ml of the suspension was inoculated into 250-ml baffled Erlen-meyer flasks containing 25 ml of chemically defined fermentation medium for rapamycin production.5.0 g L−1 (= Medium 3A).3. Incubation was conducted at 30°C for 46 h on a rotary shaker (220 rpm).4 ml of the thawed spore suspension to a 250-ml baffled Erlenmeyer flask containing 30 ml of medium consisting of (g L−1): glucose 10. Fermentation was carried out in duplicate 250-ml baffled Erlenmeyer flasks containing 25 ml of medium at 28°C on a rotary shaker (220 rpm).. casamino acids (Difco)1.5% NaCl and 0. i. Fermentation For screening of nitrogen sources. but we modified the lysine-HCl concentration. Five millilitres of 20% glycerol were added to each dish to prepare a spore suspension which was stored at −80°C. yeast extract (Difco) 4. All assay values of growth and production have been corrected for this volume decrease. and the cells were washed once with 100 mM 2-(N-morpholino) ethanesulfonic acid buffer (MES.0 g L−1 instead of 10. Bacto-peptone (Difco Laboratories.5. Table 1: Composition of Chemically defined Media . and 0. Samples were taken at 5 and 7 days for assay. MgSO4·7H2O 0.0–7.0) containing 0.

yeast extract (Difco) 10. The reason for this discrepancy is unknown but is under active investigation. The maximum growth and rapamycin production values of the 5. It should be noted that all the values shown in the Tables and Figure were determined by the C. . Twenty microliters of the C.5 ml of methanol for 2 h at 30°C. The pooled extracts were added to the culture supernatant and assayed by the paper disc-agar diffusion method using Candida albicans ATCC 11651 as the assay microorganism. The culture supernatant fluid was transferred into a test tube and the pellet was extracted by shaking with 0.Assays for cell growth and rapamycin production Fermented whole broth (0. and the cells were suspended in the same medium to prepare a cell suspension which was stored at −80°C. Pradeep Srivastava and one of the faculties from our institution. albicans bioassay which are 2–3 times higher than those determined by HPLC. Bacto-peptone 2. The resulting culture broth was centrifuged at 4°C for 10 min. albicans cell suspension were seeded into 100 ml of assay medium before pouring of plates. The plates were incubated after addition of the paper discs saturated with extracts or the rapamycin standard for 16–18 h at 37°C. For the preservation of the strain. And as permit by our supervisor these experimental work will be continued further in our institution under the guidance of Dr. The assay medium consisted of (g L−1): glucose 5.0. albicans was cultured at 30°C for 2 days in YEPD medium consisting of (g L−1): glucose 20. petri plates and slant technique were used. Results and discussion The growth of the microbial strain streptomyces hygroscopicus MTCC 4003 was done successfully without any contamination and spores were formed after 14th day inoculation.and 7-day assays are taken into account. C. Cell growth was measured as dry cell weight (DCW) according to Kojima et al. The work could not be preceded further because of the lack of time. so that the strain must be used further for the experimental work.0 and agar 8.0.5 ml) was centrifuged at 9000 × g for 10 min. and Bacto-peptone (Difco) 20.

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