Microbes An eternal source of innovative drugs

Refined fermentation process development strategies have geared up microbes as a sustained source of future drugs. Dr H Sivaramkrishna, President and Dr Girish Mahajan, Group Head, Anti-infective Screening & Prokaryote Isolation, Department of Natural Products, Piramal Life Sciences Limited (PLSL) illustrate the pre-eminence fo microbes in the pharma industry focusing on three prominent areas: anti-microbial (anti-bacterial and anti-fungus), anti-cancer and anti-diabetic drugs.

Today, microbiology is a major participant in the global pharmaceutical and neutraceutical industry. The microbes keep on producing novel metabolites as they move into the diverse ecological units. From the biologically active compounds that have been obtained so far from microbes, 45 percent are produced by actinomycetes, 38 percent by fungi and 17 percent by unicellular bacteria. However, the development of resistance in microbes and tumour cells has become a major problem and requires much research effort to combat it. Microbes have always been a better resource for getting lead molecule with novel scaffold to overcome any such limitation of existing drugs.

Mini medicine making factories

The versatility of microbial drugs is gigantic. Secondary metabolites of microbes are exceedingly important to our health and nutrition. These metabolites have tremendous economic importance. Per se the antiinfective market amounts to almost $62.5 billion and includes about 166 anti biotics and derivatives such as the -lactam peptide antibiotics, the macrolide polyketide erythromycin, tetracyclines, aminoglycosides, daptomycin, and others. Additional rudimentary pharma products produced by microrganisms are anti diabetic drugs, hypocholesterolemic agents, enzyme inhibitors, immunosuppressants, herbicides, antihelmintics, biopesticides and anti tumour compounds, some having markets of over $1 billion per year (Table 1). Microbial products comprising of actinomycetes, fungi and myxobacteria continued to play a highly significant role in the drug discovery and development process. The power of the microbial culture in the competitive world of commercial synthesis can be appreciated by the fact that even simple molecules (ie L-glutamic acid and L-lysine), are made by fermentation rather than by chemical synthesis. Most natural products are made by fermentation technology. Owing to technical improvements in screening programmes, and separation and isolation techniques, the number of natural compounds discovered exceeds one million by end of 2005. Of these five percent have a microbial origin. Of all the reported natural products, approximately 20-25 percent show biological activity, and of these, approximately 10 percent have been obtained from microbes. Furthermore, from these biologically active compounds, which have been obtained so far from microbes, 45 percent are produced by actinomycetes, 38 percent by fungi and 17 percent by unicellular bacteria.

Although, microbes are remarkably superior in presenting us with an amazing array of precious drugs, wild strains usually produce them in low amount, usually few micrograms to milligrams per litre of culture broth. The fermentation microbiologist, however, desires an 'extravagant' strain, which will overproduce and excrete the desired compound that can be isolated and marketed. During the screening stage, the microbiologist searches for microbes with weak regulatory mechanisms. Once a desired strain is found, a strain improvement programme is taken up to improve yields of desired products by modification of culture conditions, mutation and other molecular biology techniques. Thousand-fold increases have been recorded for small metabolites. Such refined fermentation process development strategies geared up microbes as sustained source of future drugs. To illustrate the preeminence of microbes in pharma industry we have focused on three prominent areas anti-microbial (anti-bacterial and antifungals), anti-cancer and anti-diabetic drugs.

Anti-microbial drugs

The eon of the drugs from microbes began in 1928, when Alexander Fleming discovered in a petri-plate seeded with a bacteria named Staphylococcus aureus, that a compound produced by a mould killed the bacteria. "When I woke up just after dawn on September 28, 1928, I certainly didn't plan to revolutionise all medicine by discovering the world's first antibiotic, or bacteria killer," Fleming would later say, "But I guess that was exactly what I did." Ernst Chain and Howard Florey became interested in his work, and isolated, characterised active compound from it. The mould, classified as Penicillium notatum, produced an active agent that was named penicillin. By history gramicidin, the first clinically tested antibiotic, was the first natural antibiotic discovered through a deliberate, systematic search for antibacterial compounds by René Dubos. This discovery helped revive the stalled interest in penicillin and launched the era of antibiotics. Later, penicillin was used as a potent antibacterial compound during World War II. Streptomycin, the first antibiotic remedy for tuberculosis, was first isolated from an actinobacterium Streptomyces griseus in 1943 by Albert Schatz, a graduate student, in the laboratory of Selman Abraham Waksman at Rutgers University. Chloramphenicol, a broad spectrum antibiotic was originally isolated from the soil microbe Streptomyces venezuelae in 1947. The tetracyclines are a large family of antibiotics produced my many species of streptomyces, which were discovered as natural products by Benjamin Minge Duggar and first described in 1948. Tetracycline was then discovered by Lloyd Conover in the research departments of Pfizer13,14.

Many small molecule drugs were approved in the antibacterial area from the beginning of 2003 to 2008. These included daptomycin which was launched as Cubicin by Cubist Pharmaceuticals in 2003, which was produced by Streptomyces roseosporus. It was the first of a new class of cyclic lipopeptides. It has been approved by FDA for the treatment of infections caused by MRSA and other Gram positive pathogens15. Wyeth had their modified tetracycline derivative, tigecycline, approved as a drug designed to overcome the tetracycline resistance pump in pathogenic bacteria. Recently launched Doripenem (Doribax, Johnson & Johnson) is a distant analogue of a carbapenem called theinamycin. The latter was produced by Streptomyces cattleya. Ceftobiprole (BAL5788) is the first, broad-spectrum, anti-MRSA fourth generation cephalosporin antibiotic with activity against a range of difficult-to-treat gram-positive and gram-negative hospital and community-acquired pathogens including methicillin-resistant S aureus (MRSA) and Pseudomonas aeruginosa16. In 2008 Ceftobiprole was licensed from and co-developed with Basilea Pharmaceutica. Janssen-Cilag will market ceftobiprole in Switzerland under the trade name Zevtera.

Fungal infections range from superficial conditions of the skin (e.g ringworm and athlete's foot) and nails (onychomycoses) to disseminated life threatening diseases. Serious invasive fungal infections caused by Candida spp, Cryptococcus neoformans, Aspergillus spp, Pneumocystis carinii and Histoplasma capsulatum, represent an increasing threat to human health. The prevalence of these systemic fungal infections has increased significantly during the past two decades. For nearly 30 years since 1955, amphotericin B, a polyene antibiotic (produced by Streptomyces nodosus), was the sole drug available to control serious fungal infections. To overcome its nephrotoxicity and solubility issues it has been marketed in different formulation brands such as Amphotec, Abelcet, Ambisome, fungisome, fungisome etc. Even today in the crowd of new antifungals this microbial product has unique market position. In the past decade in the antifungal area, of the five drugs approved, four were azoles and the semisynthetic echinocandin derivative, anidulofungin (microbial derived), which was approved for use in the US in early 2006. Micafungin (trade name Mycamine) is another echinocandin antifungal drug developed by Astellas Pharma and gained approval in the European Union in April, 2008. Discovery of echinocandins stemmed from studies on papulacandins isolated from a fungal strain of Papularia sphaerosperma.

Thus microbes have remained as consistent sources for new antimicrobial agents overcoming new snags and challenges.

Anti-cancer drugs

Dr H Sivaramkrishnan is President and Dr Girsh Mahajan is Group Head, Anti-infective Screening & Prokaryote Isolation, Department of Natural Products, Piramal Life Sciences Limited (PLSL). The address for correspondence is girish.mahajan@piramal.com

The second area where microbial drugs are very successful in making their unremitting influence is as cancer chemotherapeutic agents. The discovery of actinomycin has led to venture into microbial world in the quest for anticancer compounds. Among the approved products deserving special attention are actinomycin D, anthracyclines (daunorubicin, doxorubicin, epirubicin, pirirubicin and valrubicin), bleomycin, mitosanes (mitomycin C), anthracenones (mithramycin, streptozotocin and pentostatin), enediynes (calcheamycin), taxol and epothilones. Actinomycin A, was the first antibiotic isolated from actinomycetes, Actinomyces antibioticus (now Streptomyces antibioticus) by Waksman and Woodruff. Despite the toxicity, however, it has served well against Wilms tumour in children. The anthracyclines are some of the most effective antitumour compounds developed, and are effective against more types of cancer than any other class of chemotherapy agents. They are used to treat a wide range of cancers, including leukaemia, lymphomas, and breast, uterine, ovarian and lung cancers. Their main adverse effects are heart damage (cardiotoxicity), which considerably limits their usefulness, and vomiting.

The first anthracycline discovered was daunorubicin (daunomycin) in 1966, which is produced naturally by Streptomyces peucetius. Doxorubicin (adriamycin) was developed in 1967. Another anthracycline, epirubicin approved by the FDA in 1999, is favoured over doxorubicin in some chemotherapy regimens as it appears to cause fewer side effects. Epirubicin is primarily used against breast and ovarian cancer, gastric cancer, lung cancer and lymphomas. Valrubicin is a semisynthetic analog of doxorubicin approved as a chemotherapeutic drug in 1999 and is used to treat bladder cancer. Bleomycin is a non-ribosomal glycopeptide microbial metabolite produced as a family of structurally related compounds by the bacterium Streptomyces verticillus. It was first reported by Umezawa et al19 in 1966, and FDA approved it in 1973. Mitosanes are composed of several mitomycins that are formed during the fermentation of Streptomyces caespitosus. Although the mitosanes are excellent antitumour agents, they have limited utility owing to their toxicity. Mitomycin C was approved by the FDA in 1974, showing activity against several types of cancer (lung, breast, bladder, anal, colorectal, head and neck), including melanomas and gastric or pancreatic neoplasms20. Mithramycin (plicamycin) is an antitumour cum antibacterial aromatic polyketide produced by Streptomyces argillaceous. It is one of the classical chemotherapy drugs used in the treatment of testicular cancer, disseminated neoplasms and hypocalcaemia. Streptozotocin, a glucosamine-nitroso-urea, is a microbial metabolite with antitumour properties. It is produced by Streptomyces achromogenes. It is toxic to cells by causing damage to DNA, although other mechanisms may also contribute. The compound is selectively toxic to the ß-cells of the pancreatic islets. In 1982, FDA granted approval for streptozotocin as a treatment for pancreatic islet cell cancer.

Pentostatin (deoxycoformycin), a purine analogue is an anticancer drug produced by S antibioticus. It interferes with the cell's ability to process DNA.

The FDA granted approval for pentostatin in 1993. Calicheamicins are highly potent antitumour microbial metabolites of the enediyne family produced by Micromonospora echinospora. Their antitumour activity is apparently due to the cleavage of double-stranded DNA. They are highly toxic, but it was possible to introduce one such compound into the clinic by attaching it to an antibody that delivered it to certain cancer types selectively. This ingenious idea of the Wyeth Laboratories avoided the side effects of calicheamicin. It was approved by the FDA for use in patients over the age of 60 years with relapsed AML who are not considered candidates for standard chemotherapy.

Taxol (paclitaxel) is reported to be produced by the endophytic fungi Taxomyces andreanae and Nodulisporium sylviforme. This compound inhibits rapidly dividing mammalian cancer cells by promoting tubulin polymerisation and interfering with normal microtubule breakdown during cell division. In 1992, taxol was approved for refractory ovarian cancer, and today it is used against breast and advanced forms of Kaposi's sarcoma. Taxol sales amounted to $1.6 billion in 2006 for Bristol Myers-Squibb, representing 10 percent of the company's pharma sales and its third largest selling product. The epothilones are macrolides originally isolated from a myxobacterium, Sorangium cellulosum. This microbial product fuelled the hunt for novel class of microbes for drugs discovery. They have mode of action similar to taxol. Moreover, they are generally five to 25 times more potent than taxol in inhibiting cell growth in cultures. Five analogs are now undergoing investigation as candidate anticancer drugs, and their preclinical studies have indicated a broad spectrum of antitumour activity, including taxol-resistant tumour cells. One epothilone, ixabepilone, was approved in October 2007 by the FDA for use in the treatment of aggressive metastatic or locally advanced breast cancer no longer responding to currently available chemotherapies. The discovery of Salinosporamide A (NPI-0052) showed that the best has yet to come in cancer area. It is a novel anticancer compound from marine actinomycetes, Salinispora tropica. Salinosporamide A is a potent proteasome inhibitor used as an anticancer agent and it had recently entered phase I human clinical trials for the treatment of multiple myeloma only three years after its discovery.

Anti-diabetic drugs

Actinomycetes are reported to produce potent anti-diabetic molecules, which target various glucosidase enzymes. Acarbose (produced by Actinoplanes strain SE 50) is an oral alpha-glucosidase and alpha-amylase inhibitor that was first launched by Bayer in Switzerland in 1989 for the oral treatment of type II diabetes mellitus. It is currently marketed in various countries worldwide, including the US, the UK, Canada, France, Germany, Italy and Japan. After intensive clinical development, acarbose (brand name Glucobay) was introduced into the market in Germany in 1990 for the treatment of diabetes and has since been successfully marketed in Europe and Latin America. In 1996, acarbose was introduced in the US under the brand name Precose.

Voglibose is an alpha-glucosidase inhibitor used for lowering post-prandial blood glucose levels in people with diabetes mellitus. It is produced and marketed in India by trade name Volix (Ranbaxy Labs) and Vocarb (Glenmark). It is produced by the actinomycete species, Stretomyces hygroscopicus subspecies limoneus.

Valielamine, a precursor of Voglibose and a new aminocyclitol has been isolated from the fermentation broth of Streptomyces hygroscopicus subspecies limoneus. It has more potent a-glucosidase inhibitory activity against porcine intestinal sucrase, maltase and isomaltase than valienamine, validamine and hydroxyvalidamine which were reported as building blocks of validamycins and microbial oligosaccharide a-glucosidase inhibitors. It is under preclinical development at Sikaqaku, Tokyo, Japan.

Tendamistat (produced by Streptomyces tendae 4158 and Streptomyces lividans) is an extracellular polypeptide containing 74 amino acids, shows significant biological activity as an a-amylase inhibitor.and has shown prominent application in treatment of diabetes mellitus. Due to its resistance against most hydrolytic enzymes, Tendamistat would be orally available for diabetes mellitus treatment. However, its immunogeneity could prevent its further developement. It is still under development at sanofi-aventis, Paris. Nojirimycin (produced by several strains of Bacillus, Streptomyces), a potent inhibitor of both a- and ß-glucosidases of different origins, Adiposin-1 (isolated from Streptomyces calvus), inhibitor of human a-amylase are few more examples of potential anti-diabetic compounds from microbes, with many more still at discovery stage.

In addition to being the source for such number of new molecules which are effective in different therapeutic areas, recent studies have shown that soil bacteria are so friendly to human being that , they alter the behaviour in a way similar to the effect of anti-depressants. A study published in Neuroscience in 2007, says levels of serotonin in brain were boosted by bacteria just as anti-depressants.

The representative data in three major therapeutic areas unanimously suggests that microbes form an importunate resource for discovering and developing new drugs that address the unmet medical needs of the masses. The concept has been exemplified by the recent discovery of a novel potent anti gram positive lead PM181104 by Piramal Life Sciences (PLSL) which has been isolated from an actinobacterium obtained from marine sponge34. PM181104 is under late toxicological studies at PLSL. Though one of the youngest research organisation, PLSL maintains the largest collection of diverse industrially important microbes (about 44,000 strains) and library of their extracts (about 43,000 extracts), and a state-of-the-art facility to develop drugs from microbes. Many promising leads from microbes and their semi-synthetic derivatives are in pre-clinical pipeline in anti-infective, anti-cancer, anti-diabetes and anti-inflammation area.

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