Thermophilic Fungus Rhizomucor Pusillus Biology Essay

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An anti-hypertension drug losartan was biotransformed employing thermophilic fungi Rhizomucor pusillus in search of novel metabolites of medicinal importance. Methods and Results: R. pusillus transformed losartan to five metabolites at pH 6.0, temp of 45°C in four days incubation. The metabolites were detected by high performance liquid chromatography (HPLC) and the structures were elucidated by liquid chromatography- tandem mass spectrometry (LCMS/MS) analysis as glucuronic acid compound (M1), 3-hydroxy-N-acetyl losartan (M2), N-acetylated carboxylic acid metabolite of losartan (M4), and two novel metabolites M3 and M5. Conclusion: The metabolites produced were similar to mammalian metabolites indicating that this fungus can be used as tool for producing mammalian metabolites of losartan. Further, the production of novel metabolites of losartan reveals the possibility of producing compounds with novel structures and biological activities.

Significance and impact of the study: Current study implies that employing thermophilic fungus R.pusillus both mammalian and novel metabolites of losartan not possible or difficult by synthetic methods can be produced effectively for pharmacological studies.

Key words: Biotransformation, HPLC, Losartan, LC-MS/MS, drug metabolism studies

Introduction

Biotransformation reactions are an important route for introducing chemical functions into inaccessible sites of molecules and are very useful in the production of intermediates of medicinal, agricultural and chemicals from both active and inactive materials. The transformation reactions performed by microorganisms involve high regio - and stereo-specificity and require mild reaction conditions (Prasad et al. 2008). These reactions are very attractive for the preparation of metabolites of drug candidate compounds, which are required for testing biological activity or toxicity and also as analytical standards. Biotransformation studies carried out using fungi proved to be an efficient and ecofriendly means of achieving large-scale metabolite production of a range of drugs.

Losartan (I, 2-n-butyl-4-chloro-1-[p-(o-1H-tetrazol-5-ylphenyl) benzyl]-imidazole-5 methanol monopotassium salt) is a potent, orally active, angiotensin II receptor antagonist used as antihypertensive agent. This is metabolized hepatically to 2-n-butyl-4-chloro-1-[(29-(1H-tetrazol-5-yl) biphenyl-4-yl) methyl] imidazole-5-carboxylic acid (EXP3174) which contributes to the overall in vivo activity of losartan in rats and humans (Yoshitani et al. 2002) and has longer half-life. An aldehyde metabolite was also recorded in human as an intermediate in the oxidation of losartan to carboxylic acid metabolite (Stearns et al. 1995) which was also excreted in urine and feces as conjugate (Boris and Bernahard 2003;Yun et al.1995). C-1', C-3' hydroxylations and N-2 tetrazole glucuronidation are other routes of metabolism of losartan ( ).

Microbial species are found in many environments that experience extremes of temperature, pH, chemical content and/or pressure. This occurrence is due to certain genetic and/or physiological adaptations. Thermophilic fungi are a small group in eukaryota having a unique mechanism of growing at higher temperature extending up to 60 to 62°C. These fungi are commonly found in soil and in habitats where organic matter gets heated up for different reasons. Among eukaryotes, thermophilic fungi have the exceptional ability to grow at an elevated temperature of 50°- 60°C (Cooney and Emerson 1964). Enzymes of these microorganisms are highly stable and catalyse biochemical reactions at a higher temperature which is advantageous because of a higher reactivity (higher reaction rate, lower diffusional restrictions), higher stability, higher process yield (increased solubility of substrates and products and favorable equilibrium displacement in endothermic reactions), lower viscosity and fewer contamination problems (Mozhaev 1993). Thermostable biocatalysts are therefore, highly attractive. Thermophily in fungi is not as extreme as in eubacteria or archaea which are able to grow near or above 100°C in thermal springs, solfatara fields, or hydrothermal vents (Blohl et al. 1997; Brock 1995) hence; they have not received much attention.

Owing to immense potential of thermophilic fungi in biocatalysis, biotransformation of losartan an anti-hypertension drug using Rhizomucor pusillus was performed in search of novel metabolites of medicinal importance.

Materials and methods

Microorganisms

Rhizomucor pusillus NRRL 28626 were collected from culture deposit of Microbiology Research Laborataory, Kakatiya University, Warangal.

Chemicals

Losartan was gifted by Dr.Reddy's Laboratories, Hyderabad, India. Methanol and acetonitrile were of HPLC grade obtained from Ranbaxy, New Delhi, India. Peptone, yeast extract, glucose, starch and all other chemicals were obtained from Himedia, Mumbai, India.

Fermentation

Microbial fermentation was carried out in a liquid broth containing (per liter) glucose (20 g), peptone (5.0 g), yeast extract (5.0 g), K2HPO4 (5.0 g), and NaCl (5.0 g). The pH of the broth was adjusted to 6.0 with 0.1 N HCl or 0.1 N NaOH. The prepared media were autoclaved and cooled to room temperature before inoculation. For each of the culture, the first stage fermentation was initiated by inoculating a 100 ml culture flask containing 20 ml of broth with a loopful of spores (about 20 spores) obtained from a freshly growing 7 days old culture. After incubating for 48h at 120 rpm and 45oC, 1.0 ml portion from the first-stage culture was used to inoculate second stage 20 ml medium in a 100 ml culture flask. The culture was incubated for 24 h before the substrate losartan (2mg in 200µl dimethylsulfoxide) was added. The flasks were incubated under similar conditions for 4 days. Two types of controls were run simultaneously with the fermentation. Culture controls consisted of a fermentation blank in which the microorganism was grown under identical conditions and no substrate was added. Substrate control comprised, losartan added to the sterile medium and incubated under similar conditions with no fungus.

Extraction of metabolites and analysis

After the incubation period, the contents of the flasks were transferred to separating funnel and extracted with three volumes of ethyl acetate. The combined organic extracts were evaporated using a rotary vacuum evaporator and dried over a bed of sodium sulfate. The resultant residues were analyzed by high performance liquid chromatography (HPLC) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the presence and identification of metabolites.

HPLC analysis was performed according to the method described by Vidyavathi et al. (2008) with modification to gradient elusion. Methanol was included as one of the component of mobile phase to prevent tailing of the peaks. The samples were analyzed using Water's PDA 2595 system by injecting 100 µl of sample into the syringe-loading sample injector. The column used was Water's, Symmetry shield, C18, 4.6x250mm, 5µm. A three component mobile phase pumped at 1ml/min with a gradient system containing the following, solvent A: 0.01M ammonium acetate (pH 5), solvent B: acetonitrile, and solvent C: methanol. The solvent flow was programmed as shown in table.1. Metabolites were detected by absorbance at 225 nm.

LC-MS/MS analysis was carried out using system MDS SCIEX API-4000, Q-TRAP, Canada with MS/MS API-4000, Q-Trap detector. Chromatographic separation was achieved by Waters column C18, 4.6Ã-250mm, 5 µm, with enhanced mass scan (EMS) and enhanced product ion scan (EPI) detector mode. The scan range was 50 amu to 600 amu for both EMS and EPI. The mobile phase and all other conditions were same as described for HPLC analysis. The data was acquired and processed by means of Analyst 1.4.2 software. The transformed compounds were identified from the masses of the fragmentation products obtained.

RESULTS

Biotransformation & identification of losartan metabolites

Rhizomucor pusillus transformed losartan to five metabolites in 4 days incubation. The ethyl acetate extracts of culture broth of R.pusillus with losartan and control samples when analyzed by HPLC the substrate control (containing medium and losartan) had shown single peak of losartan which clearly indicates the stability of the drug throughout the experimental conditions and the chromatogram of culture control (fungus without drug) showed no coincidental peaks eluting with identified metabolite peaks. The losartan metabolites detected in test sample was designated as M1, M2, M3, M4 and M5 and the structural elucidation was done with the help of mass values (m/z) of fragmentation ions obtained in LC-MS-MS spectra (Fig.1), HPLC retention times and fragmentation pattern of the metabolites (table.2).

The mass spectrum of losartan metabolite M1 showed an apparent molecular ion at m/z 600 [M+H] (addition of 177 units to parent compound losartan) with fragment ions at m/z 405.2 and 207.2. This indicates conjugation of glucuronic acid to losartan resulting in formation of glucuronic acid conjugate of losartan. The product ion at m/z=405 might be arising by loss of C6H10O7 (194) from M1. Further loss of C9H13N3Cl (198) from m/z 405 fragment might result in fragment 207.

Metabolite M2 eluting at 9.8 min gave m/z 481 [M+H] (addition of 58 units to losartan) which was supported by fragment ions m/z 337, m/z 310, m/z 278 and m/z 241. The fragment ion peak at m/z 241 suggests hydroxylation of alkyl group of losartan and fragments 337 and 278 might be arising by diazole ring cleavage of m/z 480 and benzyl cleavage of m/z 337 respectively supports N-acetylation of hydroxy losartan. Based on this assumption the metabolite was identified as 3-hydroxy-N-acetyl losartan.

The compound M3 had a retention time of 16.0 min and showed a molecular ion at m/z 430 [M+H] with fragment ions at m/z 310, m/z 278 and m/z 241. The fragment m/z 310 may be arising by loss of propyl group and triazole ring from parent compound losartan. Fragment m/z 278 may be derived from benzylic cleavage. This metabolite was assumed to be produced by sequential oxidation and acylation of demethylated and dechlorinated parent compound, losartan. This is the novel metabolite of losartan detected in the present study.

The metabolite M4 produced by R.pusillus was eluted at 19.5 min showed molecular ion at m/z 479 [M+H] supported by fragments 429,391,260,167 and 149. This indicates formation of N-acetylated carboxylic acid metabolite, arising by oxidation and acylation of parent compound losartan. The fragment ion peaks at m/z 429,260 and 149 indicates oxidation of primary alcoholic group to carboxylic acid. The fragment m/z 391 indicates formation of decarboxylated product.

Metabolite M5 eluted at 28.5 min in present study showed a molecular ion peak at m/z 392 [M+] (decrease of 44 units from carboxylic acid metabolite) with 207.2 and 71 fragments. This metabolite might be arising by decarboxylation of carboxylic acid metabolite of losartan. This is another novel metabolite detected in our study.

The compound eluting at 24.0min was identified as losartan by comparision of the retention time with pure drug. Losartan has generated a molecular ion [M+] at m/z 423 with fragment ion peaks at m/z 405,279 and 207 in its mass spectrum.

The fragmentation pattern of the drug, losartan with that of the metabolites was found to be similar. The proposed metabolic pathway of losartan in culture broth of R.pusillus was presented in fig.2.

DISCUSSION

In the present investigation, a successful biotransformation of losartan using thermophilic fungus R.pusillus was reported which produced a total of five metabolites. This might be due to induction of enzymes by the fungus or presence of readymade enzymes required to transform losartan.

The transformation of losartan was identified by HPLC analysis compared to controls. The metabolite peaks showed similar UV spectral pattern compared to parent compound losartan using photodiode array detector which clearly indicates that losartan has undergone slight structural alternation in biotransformation process by fungi. The metabolites viz. M1 to M4 was found to be polar compounds eluting before losartan and M5 after losartan in HPLC analysis.

The metabolite M1, is a glucuronic acid conjugate of losartan. Conjugation has been well recognized as an important metabolic pathway of many compounds both in mammals and in microorganisms (Bin et al. 2007). Glucuronic acid compound of losartan using mesophilic microbial culture was recorded by Chen et al. 1993. This metabolite was reported to catalyze by UGT super family of enzymes in animals and is also detected in monkeys, rats, dogs and humans (Zhang et al. 2006; Huskey et al.1993; Krieter et al.1995). This compound was also reported to exhibits angiotensin II receptor antagonist activity and is also useful in treating hypertension, in management of congestive heart failure and has other wide applications (Chen et al.1993).

N-acetylation of hydroxy losartan resulted in metabolite M2 formation. The N-acetylation reactions catalyzed by N-acetyl transferase in mammals are well known. This metabolite of losartan using mesophilic bacteria was reported earlier (Vidyavathi et al.2008) via an intermediate compound 3-hydroxy losartan. In the present study, 3-hydroxy losartan was not detected in culture broth of R.pusillus. This might be due to instantaneous conversion of 3-hydroxy losartan to 3-hydroxy-N-acetyl losartan at high incubation temperature. Similar type of N-acetylation reactions using microbial cultures were reported previously (Foster et al. 1998; Milliken et al.2004; Huang et al.1998). The metabolite M3 is a novel metabolite of losartan which might be resulted by sequential oxidation and acylation of demethylated and dechlorinated parent compound, losartan. This type of demethylation and dechlorination of chlorinated hydroquinones using bacteria was reported earlier (Milliken et al.2004).

N-acetylation and carboxylation of losartan resulted in metabolite M4. This was also recorded in mammalian metabolic pathway of losartan catalyzed by CYP3A4 (Yun et al.1995). This metabolite of losartan was also recorded previously (Vidyavathi et al.2008) in culture broth of mesophilic bacteria Proteus vulgaris, which clearly indicates that similar enzyme system exists in mammals, bacteria and fungi.

Carboxylic acid compound of losartan was not recorded in culture broth of R.pusillus but, was reported in mesophilic bacteria Proteus vulgaris (Vidyavathi et al.2008) and in mammalian metabolism of losartan (Stearns et al.1995) indicating carboxylic acid metabolite was not stable in culture broth of R.pusillus but instantaneously converted to metabolite M5. Milliken et al. (2004) recorded similar type of decarboxylation reaction using microbial cultures.

Rhizomucor pusillus, a thermophilic and saprophytic zygomycete could efficiently transform anti-hypertension drug losartan to five metabolites both novel and previously reported. The study also provided the first evidence of effective biotransformation of losartan. Further, desired amount of metabolites can be produced in large quantities; isolated and biological activity studies can be performed. The ability to produce metabolites which have novel structures in the study reveals the possibility of producing compounds with novel structures and biological activities.

Acknowledgements

The authors are thankful to Council of Scientific and Industrial Research (CSIR), New Delhi, India for financial assistance and Mrs. Akshaya for interpretation of LCMS/MS spectra.

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