PRODUCTION PROCESS OPTIMIZATION OF THE IMMUNOSUPRESSANT DRUG SIROLIMUS

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 non-selectively, 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.

Fig. 1: Sirolimus, a 31-membered macro cyclic polyketide.

OCCURRENCE

Sirolimus was first isolated from the bacteria, Streptomyces hygroscopicus strain AY B994, in a soil sample from Rapa Nui, commonly known as Easter Island. However, S.hygroscopicus can be found in most rich soil worldwide due to its complex fungi-like life cycle. It grows under soil in branching filaments to form vegetative mycelium. As the mycelium grows, it branches into the air into what is known as aerial hyphae. The hyphae eventually converts into “semi-dormant spores” that are heat resistant, allowing for S. hygroscopicus to survive and adapt to various soil environments. Brock suggests that Streptomyces tend to favour dryer alkaline and neutral soils.

The amount of sirolimus produced from S.hygroscopicus depends on essential nutrients available. M S Lee et al found that different sources of nitrogen sources can largely impact the production of sirolimus. In a test, ammonium sulphate administered at 40mM resulted in the highest yield of the natural product in comparison to five other non-amino acid nitrogen compounds. Another study found that L-lysine must be present for sirolimus production and that increases in lysine resulted in high yields of sirolimus.

BIOLOGICAL ACTIVITY

Sirolimus is administered as an oral pill. When ingested, it enters the plasma membrane where it binds to the intracellular protein, FK506 binding protein-12 (FKBP-12). FKBP-12 further complexes with and deregulates the kinase enzyme, mammalian target of sirolimus (mTOR) that is responsible for regulating cell growth and proliferation through signal-

transduction pathways. Damiao et al discovered that inhibiting mTOR “arrests the cell cycle in the G1 phase, thereby deregulating cell growth.” The inhibition of mTOR halts downstream events, mainly the dephosphorylation and inactivation of the biochemical processes of P70 ribosomal S6 kinase (P70S6K). In its active form, P70S6K functions to induce “protein synthesis and cell-cycle progression.” By inactivating P70S6K, the sirolimus: FKBP-12: mTOR complex is able to inhibit the proliferation of T-cells, the major role of sirolimus as an immunosuppressant. 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. This provides that sirolimus is a potent immunosuppressant. In another experiment conducted to test for potential side effects of sirolimus, rats were given dosages up to 200 mg/kg intraperitoneally for 7 days. 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.” This study reveals that sirolimus has very low toxicity with respect to the therapeutic dose.

BIOSYNTHESIS

Sirolimus is a triene macrocylic polyketide. In Streptomyces hygroscopicus, three large polyketide synthase genes are responsible for the biosynthesis of sirolimus. These genes encode for 14 enzyme-containing modules, each having a specific role in elongating the sirolimus polyketide. The three polyketide synthase genes, rapA, rapB, and rapC, encode the multienzyme polyketide synthases Raps1, Raps2, and Raps3, respectively. Raps1 is responsible for the first four modules and contains a loading zone for the starter unit. The starter unit is a substituted cyclohexanecarboxylic acid, 4, 5-dihydroxycyclohex-1-enecarboxylic acid, derived from shikimate. At the start of the biosynthesis process, the shikimate derivative is latched onto CoA Ligase in the “loading domain” of Raps1. A reduction of the double bond occurs as the starter unit moves onto module 1. In the first four modules, three propionate units and one acetate unit is added with various oxidation states. The resulting polyketide of Raps1 then enters Raps2, an enzyme complex consisting of six modules for further polyketide extension. Raps2 adds three acetate units and three propionate units. The resulting fragment is transferred onto module 11 of Raps3 where four more successive rounds of elongation take place to complete “pre-sirolimus,” the intermediate marocycle.” Raps3 adds three acetate units and a propionate unit. At this point, pre-sirolimus has a total of seven acetate and seven propionate units in addition to the shikimate derivative. After module 14 is complete, the pipecolate-incorporating enzyme (PIE) (produced by the RapP gene adjacent to Raps3), binds to an activated L-pipecolate through a thioester linkage. Note that L-pipecolate is a cyclization of L-Lysine. Cheng et al. reported a 150% increase in sirolimus production by S. Hygroscopicus upon addition of L-Lysine in a chemical defined medium.

Fig. 2: Life cycle of Streptomyces hygroscopicus. Source:(http://openwetware.org/images/e/Streptomyces_Life_Cycle.gif)

TOTAL SYNTHESIS

The total synthesis of sirolimus was first completed in 1993 by the Kyriacos Costa Nicolaou group. Three other syntheses were found in the same year. In more recent times, Maddess et al. sought a new convergent route to formulate sirolimus. A retrosynthesize was conducted as summarized in Figure 3. 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, 5, and 6. Molecules 3, 4, 5, and 6 were the subtarget of this study.

Fig. 3: Retrosynthesis of Sirolimus

The actual process of total synthesis, although long, is efficient in using a convergent route to chemically produce sirolimus. Some highlights of this synthesis includes the “intramolecular trapping of oxonium ions” in 6, use of BDA chemistry to protect and stereodirect certain reactions, and macroetherification/catechol tethering to form the macrocycle of sirolimus.

Although sirolimus has been studied extensively since its discovery in 1975, researchers are continuing to look at its structure in order to find additional analogs for treatment. Because sirolimus has antitumor activity, researchers aim to find other forms of sirolimus for treatment.

ADVANTAGE

The chief advantage sirolimus has over calcineurin inhibitors is that it has low toxicity towards kidneys. Transplant patients maintained on calcineurin inhibitors long-term tend to develop impaired kidney function or even chronic renal failure; this can be avoided by using sirolimus instead. It is particularly advantageous in patients with kidney transplants for haemolytic-uremic syndrome, as this disease is likely to recur in the transplanted kidney if a calcineurin-inhibitor is used. However, on October 7, 2008, the FDA approved safety labelling revisions for sirolimus to warn of the risk for decreased renal function associated with its use.

STREPTOMYCES HYGROSCOPICUS

It is becoming increasingly apparent that marked variability abounds in a great many of the species of Streptomyces and, as a result of the indiscriminate granting of species status to numerous variants of already defined species, a needlessly complex taxonomic system has arisen. 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. Hesseltine et al. (1954) emphasized the need for uniformity in methods of study and of reporting data relative to taxonomic studies, 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. Backus et al. (1954), in their study of variability in S. aureofaciens, and Duggar et al. (1954), studying the same organism as well as other species of Streptomyces, have made clear the necessity of examining a large assemblage of related forms as a prerequisite to establishing species boundaries.

While it is recognized that a majority of the cultures involved in this study are capable, under specific conditions, of elaborating one or more products which possess substantial and varied antimicrobial activity, it is beyond the province of this paper to discuss such products or activities. Furthermore, 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. hygroscopicus.

GENERAL METHOD OF PRODUCTION OF SIROLIMUS FROM STREPTOMYCES HYGROSCOPICUS

The producing strain, Streptomyces hygroscopicus, was grown and maintained on tomato paste-oatmeal agar, as previously described1) Good growth and sporulation were obtained in 7-15 days of incubation at 25°C. Spores from one Roux bottle were suspended in 50 ml of sterile distilled water to constitute the spore inoculum.

Unbaffled, 500-m1 Erlenmeyer flasks were filled with 100 ml of an inoculum medium consisting of (g/litre): soybean meal ("Special X", Archer Daniels Midland Co., Minneapolis, Minn.), 40; "Cerelose" (a pharmaceutical grade of glucose), 20; (NH4)2SO4, 3; CaCO3, 1.5; and tap water to I litre (pH 7.0). The flasks were sterilized at 121'C for 30 minutes, cooled to 25°C and inoculated with 4 ml of the spore inoculum. The inoculated flasks were incubated for 24 hours at 25°C on a gyrotory shaker at 240 rev/min, 2"-throw, to constitute the first-stage inoculum.

Unbaffled, 24-liter round bottom flasks were filled with 3.4 litres of the same medium and autoclaved at 121 °C for 30 minutes. The flasks were agitated to resuspend the solids and autoclaved for an additional period of 1 hour at 121°C, cooled to 25°C and inoculated with 78 ml (2 %) of the first-stage inoculum. The inoculated flasks were incubated for 18 hours at 25°C on a reciprocating shaker at 65 strokes per minute and 4"-throw. These flasks were used to inoculate the production stage.

Fermenters (model F-250, New Brunswick Scientific Co.), 250-liter capacity, equipped with automatic antifoam addition system and pH recorder-controller, were filled with 160 litres of the production medium consisting of (g/litre): soybean meal ("Special X"), 30; "Cerelose," 20; (NH4)2SO4, 5; KH2PO4, 5; Mazer DF-143PX (antifoam), 0.5 ml; and tap water to 1 litre. The fermenters were sterilized at 121'C for 30 minutes under an agitation of 150 rev/min, cooled to 25°C and pH of medium adjusted to 6.1 by addition of 10 N NH4OH solution. The fermenters were inoculated with 3.2 litres (2 %) of the second-stage inoculum. The fermentation was run at 25°C under an agitation of 200 rev/min and an aeration of 0.25 v/v/min. Sterile Mazer DF-143PXantifoam was added on demand. After 30.35 hours of incubation the pH started to drop but was controlled at 6.0 by addition of 1ON NH4OH solution on demand. After 48 hours of incubation, a 40 % sterile solution of "Cerelose" was added continuously at the rate of 3.85 % per day. The antibiotic titres were determined every 24 hours starting at 48 hours. The maximum titers were usually obtained in 96 hours.

Conventional paper disc-agar diffusion assays were used to determine the antibiotic titre. A 10-ml sample of fermentation broth was centrifuged at 2,500 rev/min for 15 minutes. The supernate was discarded and the mycelial pellet suspended in 250 ml of methanol and shaken vigorously. The extract was filtered. Filter paper discs, 13 mm in diameter, were dipped in the extract and placed on filter paper to dry. Similar discs were dipped in

standard solutions containing 10, 5, 2.5 and 1.25 µg rapamycin/ml. 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.

ISOLATION OF SIROLIMUS

The fermentation broth was adjusted to pH 4.0 with a 30 % sulfuric acid solution and filtered on a vacuum rotary filter coated with Celite. The mycelium, containing the antibiotic, was extracted twice by stirring for 1 hour with 11 volume of trichloroethane. The tri-chloroethane extracts were pooled and evaporated to a small volume under reduced pressure, dehydrated with anhydrous sodium sulfate and further concentrated to an oily residue. A typical 160-liter fermentation run yielded about 500 g of oily residue. The residue was extracted twice with one volume of methanol. The methanolic extracts were pooled and evaporated to dryness to yield about 50 g of oily residue containing rapamycin. The residue was dissolved in 10 v/w of a solvent mixture consisting of 15 % acetone in hexane. To this solution, 2 weights of silica gel G (Merck) per weight of oil were added and the mixture stirred gently for 50 minutes. The mixture was filtered and silica gel with adsorbed rapamycin washed onto a column with several volumes of 15 % acetone in hexane. The antibiotic was eluted with 25 % acetone in hexane and the eluant evaporated to dryness. The residue was dissolved in ether from which pure rapamycin crystallized out. The recoveries were about 40 % based on broth assay.

PHYSICAL AND CHEMICAL PROPERTIES OF SIROLIMUS

Rapamycin is a white crystalline solid melting at 183-185'C. It is freely soluble in methanol, ethanol, acetone, chloroform, methylene dichloride, trichloroethane, dimethyl formamide, dimethyl sulphoxide; sparingly soluble in ether, and practically insoluble in water.

Rapamycin analysed for C56H89NO14 (E.W. 999). Calcd: C, 67.2; H, 8.9; N, 1.4; Found: C, 67.24; H, 8.93; N, 1.39.

The ultraviolet spectrum shows ymax at 288, 277 and 267 nm with E1%1cm 416, 514 and 417

respectively.

The infrared spectrum shows OH at 3500, a band at 1730 (possibly lactone carbonyl) and at 1700 (carbonyl), and a band between 1610 and 1630 cm-1 (C=C).

NMR spectrum (200 MHZ) of rapamycin shows vinylic protons between 5-6.5, methoxyl

between 3.1 - 3.6 and vinylic at 1.8.

Optical rotation is [α]25D-58.2 in methanol. Rapamycin forms a yellow chromophore when

dissolved in 0.1 N methanolic NaOH and heated at 60°C; this property is the basis of a colorimetric assay.

EFFECT OF NITROGEN SOURCE ON BIOSYNTHESIS OF RAPAMYCIN BY STREPTOMYCES HYGROSCOPICUS

As we have discussed above the general method of production of Sirolimus. 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. Of the nitrogen sources studied, ammonium sulfate was the best with respect to formation of rapamycin, and supported cell growth comparable to the organic nitrogen sources used in the control chemically defined medium, i.e., aspartate, arginine plus histidine. In the new chemically defined medium, which is buffered with 200 mM 2-(N-morpholino) ethanesulfonic acid to prevent decline of pH during fermentation, an ammonium sulfate concentration of 40 mM was optimal for biosynthesis of rapamycin. 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.

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. The microbial strain received was in the lyophilized form. First it was made active by the introducing the strain into sterilized distilled water. Sterilized distilled water was obtained from autoclaving the double distilled water at 15 psi, 121oC. After this the growth media no 93 was made for the growth of the Streptomyces hygroscopicus. The growth media contains Glucose 4.0g, Yeast extract 4.0g, Malt extract 10.0g, CaCO3 2.0g, and Agar 25.0g. The growth condition is aerobic with incubation time of 2 days at 30oC. 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. Liquid broth culture was kept in the BOD shaker incubator. 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.

Spores were produced in Petri dishes at 28°C for 14 days on oatmeal agar medium. Five

millilitres of 20% glycerol were added to each dish to prepare a spore suspension which was stored at −80°C.

Inoculum

A seed culture was initiated by adding 0.4 ml of the thawed spore suspension to a 250-ml baffled Erlenmeyer flask containing 30 ml of medium consisting of (g L−1): glucose 10.0, Bacto-peptone (Difco Laboratories, Detroit, MI,USA) 4.0, yeast extract (Difco) 4.0, casamino acids (Difco)1.5, MgSO4·7H2O 0.5, and K2HPO4 1.0, pH 7.0–7.3. Incubation was conducted at 30°C for 46 h on a rotary shaker (220 rpm). The resulting culture broth was centrifuged at 4°C for 15 min (5000 × g), and the cells were washed once with 100 mM 2-(N-morpholino) ethanesulfonic acid buffer (MES, pH 6.0) containing 0.5% NaCl and 0.05% MgSO4·7H2O. The washed cells were suspended in the same buffer to make a 10-ml cell suspension, and 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.

Fermentation

For screening of nitrogen sources, we used a chemically defined medium (Medium 3) which had been developed previously, but we modified the lysine-HCl concentration, i.e., 5.0 g L−1 instead of 10.0 g L−1 (= Medium 3A). The detailed composition is described in Table 1. 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). Samples were taken at 5 and 7 days for assay. The evaporative losses during autoclaving and fermentation amounted to 27% of the initial 25-ml volume. All assay values of growth and production have been corrected for this volume decrease.

Table 1: Composition of Chemically defined Media

Assays for cell growth and rapamycin production

Fermented whole broth (0.5 ml) was centrifuged at 9000 × g for 10 min. The culture

supernatant fluid was transferred into a test tube and the pellet was extracted by shaking

with 0.5 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. C. albicans was cultured at 30°C for 2 days in YEPD medium consisting of (g L−1): glucose 20, yeast extract (Difco) 10, and Bacto-peptone (Difco) 20. The resulting culture broth was centrifuged at 4°C for 10 min, and the cells were suspended in the same medium to prepare a cell suspension which was stored at −80°C. The assay medium consisted of (g L−1): glucose 5.0, Bacto-peptone 2.0 and agar 8.0. Twenty microliters of the C. 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. It should be noted that all the values shown in the Tables and Figure were determined by the C. albicans bioassay which are 2–3 times higher than those determined by HPLC. The reason for this discrepancy is unknown but is under active investigation. Cell growth was measured as dry cell weight (DCW) according to Kojima et al. The maximum growth and rapamycin production values of the 5- and 7-day assays are taken into account.

Results and discussion

The growth of the microbial strain streptomyces hygroscopicus MTCC 4003 was done successfully without any contamination and spores were formed after 14 th day inoculation. For the preservation of the strain, petri plates and slant technique were used, so that the strain must be used further for the experimental work. The work could not be preceded further because of the lack of time. And as permit by our supervisor these experimental work will be continued further in our institution under the guidance of Dr. Pradeep Srivastava and one of the faculties from our institution.