Indian Journal of Pharmaceutical Education & Research

Indian Journal of Pharmaceutical Education & Research

ijper Vol. 42(2), Apr –Jun, 2008

PAST EDITORS

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Dr. Nagavi B.G. Mysore 1997 - 2006 Dr. Rao M.N.A. Manipal 1995-1996 Dr. Gundu Rao P. Manipal 1985-1995

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Dr. Kasture A.V. Nagpur 1981 - 1984 Dr. Saoji A.N. Nagpur 1980 - 1980 Dr. Lakhotiya C. L. Nagpur 1979 - 1980

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Dr. Chopde C.T. Nagpur 1978 - 1978 Dr. Gundu Rao P. Manipal 1975 - 1978 Dr. Mithal B. M. Pilani 1967 – 1974

EDITOR–IN–CHIEF

Dr. Sanjay Pai P.N. [email protected]

ASSOCIATE EDITORS Dr. Srinivasa Murthy [email protected]

Dr. Mallikarjuna Rao C. [email protected]

Dr. Kulkarni P.K. [email protected]

Dr. Mueen Ahmed K. K. [email protected]

EDITORIAL OFFICE INDIAN JOURNAL OF PHARMACEUTICAL EDUCATION AND RESEARCH The Official Publication of Association of Pharmaceutical Teachers of India H.Q.: Al-Ameen College of Pharmacy, Opp. Lalbagh Main Gate, Hosur Road, Bangalore 560 027, INDIA Mobile : 91-9448207428 | 91-9242898028 | 91-9845655732 | 91-9880423041 | 91-9448445612 Fax: 080-22225834; 080-22297368 email: [email protected] | Website: www.ijper.org



EDITORIAL ADVISORY BOARD Dr. Betgeri G.V., USA. Dr. Mrs.Claire Anderson, UK. Mr. Frank May, USA. Dr. Gaud R.S., Mumbai. Dr. Goyal R.K., Ahmedabad. Dr. Harkishan Singh, Chandigarh. Dr. Hukkeri V.I., Bangalore. Dr. Jagdeesh G., USA. Dr. Katare O.P., Chandigarh. Dr. Khar R.K., New Delhi. Dr. Madan A. K., Rohtak. Dr. Madhusudhan Rao Y., Warangal. Dr. Manavalan R., Annamalai Nagar. Publication Committee • Pharmaceutics

Dr. Miglani B.D., New Delhi. Dr. Murthy R.S.R., Vadodara. Dr. Nagavi B.G., Dubai. Dr. Pulok K Mukherjee, Kolkata. Dr. Rao M.N.A., Hyderabad. Dr. Ravi T.K., Coimbatore. Prof. Shivananda B.G., Bangalore. Dr. Shivakumar H.G., Mysore Dr. Subrahmanyam C.V.S, Hyderabad. Dr. Suresh B., Ooty. Dr. Tipnis H.P., Mumbai. Dr. Udupa N., Manipal. Dr. Vyas S.P., Sagar.

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• Pharmaceutical Chemistry and Analysis • Pharmacology

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• Pharmacognosy

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• Pharmacy Practice

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• Pharmaceutical Education

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• Pharmaceutical Marketing

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Dr. Paradkar A.R., Dr. Sarasija Suresh, Dr. Vavia P.R. Dr. Gopal Krishna Rao, Dr. Raghurama Rao A. Dr. Valliappan K. Dr. Krishna D.R., Dr. Kshama Devi, Dr. Sreenivasan B.P. Dr. Ganapaty S., Dr. Salma Khanam, Dr. Swati S.Patil Dr. Nagappa A.N., Dr. Rajendran S.D, Dr. Shobha Rani R.H. Dr. Raman Dang, Dr. Unnikrishnan M.K., Dr. Bhise S.B. Dr. Burande M.D., Dr. Gayathri Devi S., Dr. Kusum Devi V.

Note: The Editor does not claim any responsibility, liability for statements made and opinions expressed by authors. INDIAN JOURNAL OF PHARMACEUTICAL EDUCATION AND RESEARCH The Official Publication of Association of Pharmaceutical Teachers of India H.Q.: Al-Ameen College of Pharmacy Opp. Lalbagh Main Gate, Hosur Main Road, Bangalore - 560027 INDIA Mobile : 91-9448207428 | 91-9242898028 | 91-9845655732 | 91-9880423041 | 91-9448445612 Fax: 080-22225834; 080-22297368; email: [email protected] | Website : www.ijper.org



CONTENTS •

Editorial…………………………………………………………………………………………………………………94

Review Articles • Hyaluronidase: Application of Enzyme as Pharmaceuticals Sahoo S., Ellaiah P., Mishra S.R., Nayak A. and Panda P.K. ………………………………………………96-103 • Therapeutic Protein Production and Delivery: An Overview Hajare A.A., Dange A.S. and Shetty. Y. T…………………………………………………………………….104-112 • Plant Made Pharmaceuticals (PMPs) - Development of Natural Health Products from Bio-Diversity Pulok K. Mukherjee, Satheesh Kumar N and Micheal Heinrich………………………………………….113-121 Research Articles • Simultaneous Determination of Simvastatin and Ezetimibe in Tablet Formulation by Derivative Spectrophotometry Anandakumar K., Kannan K.and Vetrichelvan T. ………………………………………………………….122-126 • Development of Fingerprints for an Ayurvedic formulation Bhaskar Lavan churna, via Piperine estimation by High Performance Liquid Chromatography Shukla Karunakar, Saraf Swarnlata and Saraf S. ………………………………………………………….127-132 • QSAR Analysis of 6-Aryl-2,4-Dioxo-5-Hexenoic Acids as HIV–1 Integrase Inhibitors Ravichandran V., Mourya V.K. and Agrawal R. K. ………………………………………………………...133-140 • Hepatotoxicity Studies of some Mycotoxins with Special Reference to Hepatoprotection against Mycotoxin induced Liver Damage. Papiya Mitra Mazumder and Sasmal D. ……………………………………………………………………141-146 • Study of Film Forming Properties of Hydroxy Propyl Guar And Its Use in Preparation of Medicated Transdermal Patches Swamy N.G.N, Dharmarajan T.S. and Paranjothi K.L.K …………………………………………………147-153 • Development and In-Vitro Drug Release Studies of Methotrexate from Modified Pulsatile Release Guar Gum based Enteric Coated Capsules for Colon Specific Delivery Lanjhiyana Sanjay Kumar and Dangi Jawahar Singh ……………………………………………………..154-160 • Solubility Behaviour of Rofecoxib in Individual Solvents using the Concept of Partial Solubility Parameters Sathesh Babu P.R., Subrahmanyam C.V.S., Manavalan R. and Valliappan K. ………………………...161-169 • Novel Preparation and Evaluation of Lotion containing Aloe Gel Beads Shika Srivastava, Shweta Kapoor, Swarnlata Saraf………………………………………………………...170-173 • Formulation and In Vitro Evaluation of Gastric Oral Floating Tablets of Glipizide Prabhakara Prabhu, Harish Nayari M, Gulzar Ahmed M, Brijesh Yadav, Narayana Charyulu R., Satyanarayana D. and Subrahmanayam E.V.S. …………………………………………………………….174-183 • A Study of Potential Drug-Drug Interactions in Prescriptions Received at Selected Community Pharmacies Adepu Ramesh, Rohit Singhal and Nagavi B.G. …….……………………………………………………...184-189 • Good Educational Practices in Pharmacy Chauhan Nitesh, Raveendra R., Jha Sajal, Sharma Uday, Karki Roopa and Goli Divakar …………190-194

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Introducing Molecular Bio-Pharmaceutics in the Post Graduate Level Curriculum Pattani Aditya and Patravale Vandana B ……………………………………………………………………195-198 Design of New Experiments in Pharmaceutical Instrumental Methods of Analysis- A Study on Undergraduate Students of Pharmacy Boyapati Shireesha, William Carey and Akkinepally Raghuram Rao …………………………………...199-204 ********

Editorial To accomplish great things, we must dream, as well as act. - Anatole France There is a greater need than ever for scientific research to guide changes in policy, programs and practice. This has not left the fraternity of education behind. Progressive improvement in education has a bearing through its quality, nature, and even the values. With about 800 odd institutions imparting the basic degree course in pharmacy in our country, several thousands of our youngsters would have taken admissions every year. The number too is on the rise every year. They are being taught the same curricula in different forms and instructional styles. The delivery of curricula is not harmonized and can range from the much needed research based instructional approach in certain areas to the most irrational notes reading style in certain other places. The fortunate few students benefit here, while the rest are denied even their basic rights of proper instructional learning. Many of our counterparts are on the search for the most appropriate mode of instructional delivery in teaching and learning. Unfortunately, teachers in many pharmacy institutions are neither trained nor encouraged to use research based learning, even though it is now proved in the education technology sector that students benefit the most out of it. In the absence of such training systems in our fraternity, teachers need professional development through support from their senior experienced colleagues in learning the process of teaching methodologies and thereby deliver the instructional materials to the satisfaction of students. For teaching undergraduate courses administrators are required to recruit post graduates and even doctoral candidates. As they are moulded researchers, it becomes a tedious exercise for them to understand the complexities of teaching and learning process. The empirically proven teaching methods are being forgotten today and are now replaced by power point presentations, which in many cases are copy and paste phrases out of a book. Does it make a sense of appointing a highly qualified person to present the same paragraphs published in the books via a computer? In fact the modern technology can be better utilized to simulate the concepts on computers for better understanding among the learners. It is here that ‘evidence based education practices’ can become a reality. We are now looking at a welcome change in the era of global competitiveness. There is an increased demand for a strong, education knowledge infrastructure that includes research, development, dissemination, technical assistance, professional development and evaluation. Our long range vision of quality education should not merely remain a dream. Time is ripe enough to couple vision with action. All of us should play a vital role in working towards our goals, thereby transforming our vision to a reality. Dr. Sanjay Pai P.N. Editor-in-Chief

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Indian J.Pharm. Educ. Res. 42(2), Apr-Jun, 2008

95


APTI

ijper

Hyaluronidase: Application of Enzyme as Pharmaceuticals Sahoo S.1*, Ellaiah P.2, Mishra S.R.1, Nayak A.1 and Panda P.K.1 1

University Department of Pharmaceutical Sciences, Utkal University, Bhubaneswar-751004. 2 Jeypore College of pharmacy, Jeypore, Orissa-764002 Author for Correspondence : [email protected]

Abstract Hyaluronidases (HD), the neglected group of enzymes possess panoramic utility in biotechnology processes and therapy. Hyaluronidase is a general term used to describe enzymes that are able to break down primarily hyaluronan and other glucoseaminoglycans which facilitates the invasion of the host tissues, by bacterial pathogen, mostly Gram-positive. Hyaluronic acid (HA) is the most abundant glucoseaminoglycan of the extracellular matrix (ECM) of connective tissues responsible for cellular attachment and locomotion. The present work encompasses therapeutic, pathophysiological, physiological and biological importance of the enzyme which aids in production of pure, potent, stable microbial hyaluronidases with its specific and selective inhibitors, as an alternative approach in designing and development of targeted drugs, potent vaccines and pharmaceuticals. Keywords : Hyaluronidases, Hyaluronic acid, Extracellular matrix, Physiological, Therapeutic importance. INTRODUCTION Hyaluronic acid (Hyaluronan, HA) Glucoseaminoglycans (GAGs), oligosaccharide chains of the extracellular matrix (ECM) of tissues, are a specific type of glycan. They differ in the type of disaccharides they utilize as building blocks, and in the linkage between the building blocks1. Such diversification led to their division into three structural groups: (i) cellbiose (e.g. hyaluronan), (ii) polylactosamine (e.g. dermatan, chondroitin and keratan sulphate) and (iii) polymaltose (heparin, heparan sulphate). Chondroitin, keratin sulphate and hyaluronan have a similar polymer backbone structure (Glcβ1→3Glcβ1→4). Hyaluronic acid (HA) is a ubiquitous component of the ECM of most vertebrates, was first found in the vitreous humor of bovine eyes by Meyer and Palmer in the year 19342. It is widely distributed in various other tissues such as aorta, blood, liver, synovial fluid, loose connective tissues and skin given in Table 1. The largest amount of HA (7-8 g per average adult human, about 50% of the total in the body) resides in the skin tissue.

Indian Journal of Pharmaceutical Education & Research Received on 16/04/2007 Modified on 04/12/2007 Accepted on 26/02/2008 © APTI All rights reserved

HA are linear polysaccharides of high molecular weight, composed of aminosugars (Nacetylglucosamine or N-acetylgalactosamine) and uronic acids (glucuronic acid or iduronic acid), along with chondroitin-, keratan- and dermatane sulfate, heparin and heparan sulfate. The chemical structure of HA is the simplest of all GAGs consisting of straightlined repeating disaccharide units of [→4)-β-Dglucuronic acid(1→3)-β-D-N-acetylglucosamine(1→]n, where n can be up to 25000 dependent on the tissue source (figure 1) It has enormous size up to 25,000 disaccharide units of molecular weight 107 Da. HA is highly hydrophilic and because of its ability to bind large quantities of water, it can form highly viscous solutions and thereby influence properties of the matrix. One of the main mechanical/ structural functions of HA is to lubricate joints and to absorb shock. Structure of Hyaluronan Primary structure of Hyaluronan When considering the energetically state the HA structure is very stable since the bulky groups (the hydroxyl groups, the carboxylate moiety, the N-acetyl residue and the adjacent sugars) are situated in sterically favorable equatorial positions while all of the 96


small hydrogen atoms occupy the axial positions. In contrast to the other glycosaminoglycanes, hyaluronan is not covalently bound to a protein core and is not sulfated. Lately, the term hyaluronan, suggested by Balasz et al.,3 has substituted the terms hyaluronic acid and hyaluronate reflecting the fact that it exists in vivo as a polyanion due to the mostly charged carboxyl groups of the glucuronic acid residues (pKa = 3-4, depending on ionic conditions). Secondary structure Nuclear magnetic resonance studies performed by Scott et al.,4 suggested an ordered structure of HA in solution This conformation is characterized by a gently undulating, tape-like, twofold helix which is formed as a result of 180°C rotations between alternating disaccharide units and is stabilized via internal hydrogen bonds and interactions with the solvent. The striking feature of this secondary structure of HA is an extensive hydrophobic patch (represented by the axial hydrogen atoms) of about 8 CH-groups of 3 carbohydrate units. Thus, HA is amphiphilic because it includes properties of highly hydrophilic material simultaneously with hydrophobic patches, which is characteristic of lipids. Tertiary structures In addition, the application of rotary shadowingelectron microscopy revealed that HA self-aggregates into strands of a honeycomb meshwork in an aqueous solution whereby the thickness of the strands increased with HA concentration. According to 13C-NMR studies, this aggregation is stabilized both by hydrophobic interactions between the hydrophobic patches and by hydrogen bonds between acetamido and carboxylate groups of neighboring HA chains arranged antiparallel to each other. Since such hydrophobic and hydrophilic “bonds” can be formed on both sides of the HA polymer higher order aggregates can assemble causing strands of increased thickness in a HA meshwork. Hyaluronidase Occurrence and Sources Hyaluronidase (HD) is a general term initially introduced by Karl Meyer in 1940 to describe enzymes that are able to break down primarily HA. HD activity was first identified in an extract of mammalian testes and other tissues as a ‘spreading factor’ that facilitated diffusion of antiviral vaccines, dyes and toxins injected subcutaneously5. These enzymes can be grouped into

two, hydrolases and lyases. Hydrolases degrade glycan via hydrolysis of glycosydic bond between sugars, and lyases degrade glycan using a b-elimination process. HD-like enzymes were identified from different sources. a) Tissues and organs, e.g. skin, liver, kidney, spleen, testes, uterus, placenta, b) The venoms of snakes, lizards, fish, bees, wasps, scorpions, spiders, c) Body liquids e.g. tear liquid, blood, sperm) d) Microorganisms including (bacteria, fungi) Bacterial strains of Micrococcus, Streptococcus, Peptococcus, Propionibacterium, Bacteroides, Streptomyces, Staphylococcus, Clostridium perfringens etc.. e) Invertebrate animals e.g. leech, crustacean. The HD from different sources vary in their molecular weight, substrate specificity, optimum activity at specific pH and catalytic mechanism6. Classification of Hyaluronidases K. Meyer established the first classification scheme for HDs in 1971. Based on detailed biochemical analysis of the enzymes and their reaction products, the HDs are subdivided into three main groups7 as shown in figure 2. The first group of HDs are hyaluronate 4glycanohydrolases degrading HA by cleavage of the β1,4-glycosidic bond to the tetrasaccharide as the main product. Apart from the preferred substrate HA, these enzymes depolymerize also chondroitin, chondroitin-4and chondroitin-6- sulfate and, to a small extent, dermatan sulfate. As a special characteristic, this class of enzymes reveals both hydrolytic and transglycosidase activity. The best-known enzymes are the testicular, the lysosomal and the bee venom HD. The second type is represented by HDs occurring in the salivary glands of leeches and hookworms. These hyaluronate 3-glycanohydrolases hydrolyze the β-1,3glycosidic bond of HA yielding sugar fragments bearing glucuronic acid at the reducing end. The main product of this reaction is a tetrasaccharide, too. The last group, the microbial HDs or hyaluronate lyases, act via a β-elimination reaction resulting in the unsaturated disaccharide 2-acetamido-2-deoxy-3-O-(βD-gluco-4-ene-pyranosyluronic acid)- D-glucose as main product8. The hyaluronate lyases, isolated from various microorganisms as e.g. strains of S. 97


pneumoniae, S. agalactiae, Staphylococcus aureus and Propionibacterium acnes, Clostridium, Micrococcus, Streptococcus or Streptomyces, are known at present9 and differ in substrate specificity. Based on molecular genetic analysis the HDs can be divided alternatively in two main families – the HDs from eukaryotes and from prokaryotes – according to amino acid sequence homology. Hyaluronidases from eukaryotes Mammalian hyaluronidases With the knowledge gained by the “human genome project” in the last years, six HD-like sequences in the human genome were identified with about 40% amino acid sequence identity. Three genes (HYAL1, HYAL2 and HYAL3) coding for Hyal-1, Hyal-2 and Hyal-3 are located tightly clustered on human chromosome 3p21.3.10 HYAL4, HYALP1 (a pseudogene) and PH20 (SPAM 1), coding for Hyal-4 and PH-20, are found on chromosome 7q31.3.11 Recently, the human plasma hyaluronidase (Hya1-1) was purified, cloned and expressed and over 40% sequence identity with PH-20, i.e. sperm-specific HD. Urine contains two HDs of 57 and 45 kDa; their isozymes recently purified and microsequenced Beevenom HD (66 kDa) has 40% homology with the mammalian PH-20 and greater than 50% sequence identity with other hymenoptera and with human lysosomal HD. The HDs of animal origin hydrolyse HA by adding a water molecule between the disaccharide unit or by a process of transglycosylation. The activity of the PH-20 protein (also known as SPAM 1 (sperm adhesion molecule 1)) was first found by Gmachl et al 12 due to the significant similarity to bee venom HD which was the first cloned eukaryotic HD13. Hyal-1 and Hyal-2 constitute the major HDs of somatic tissue. Thus, they are present in most tissues and body fluids. However, Hyal-2 is not existent in the adult brain. Both proteins are localized in lysosomes. Hyal-1 is also found in human urine14 and mammalian plasma15. Bovine testicular hyaluronidase (BTH) The bovine testicular hyaluronidase (BTH) was adopted as a spreading factor in several medical fields for a long time, e.g. orthopaedia, surgery, opthalmology, dermatology or internal medicine16. BTH acts as endoglycanohydrolase (EC 3.2.1.35) by cleaving the β-1,4-

glycosidic bond of hyaluronan. The structurally related GAGs chondroitin, chondroitin-4- and -6-sulfate are accepted as substrate, too. Hyaluronidases from prokaryotes The bacterial enzymes capable of breaking down HA were reviewed by Suzuki17 and Hynes and Walton18. The amino acid sequences of a variety of HDs from prokaryotes are known. Among the bacterial HDs, the hyaluronate lyases from Streptococcus pneumoniae and from S. agalactiae (Group B Streptococcus) are the best characterized ones. Physiological Importance of Hyaluronan and Hyaluronidases a) Fibrotic healing of adult and late gestational wounds correlates with increased HD activity and removal of HA. b) HD is thought to play a role in HA homeostasis and metabolism, e.g. in the turnover of anterior chamber HA, and in the degradation of highly concentrated HA used as viscoelastic. c) HA as an essential structural element in the matrix plays an important role for tissue architecture by immobilizing specific proteins (aggrecan, versican, neurocan, brevican, CD44 etc.) in desired locations within the body. d) HA is implicated in many biological processes including fertilization, embryonic development, cell migration and differentiation, wound healing, inflammation, growth and metastasis of tumor cells and whenever rapid tissue turnover and repair are occurring19. e) The function of HA may be partly regulated and dependent on its chain length, e.g. angiogenesis is presumably induced by small HA oligosaccharides, whereas high molecular weight HA exerts inhibitory effects20. f) At physiological concentrations HA molecules form a random network of chains. Such a network may act as a sieve and regulate the distribution and transportation of plasma proteins. g) HA interacts with a variety of receptors and hyaluronan binding proteins called hyaladherins. The great number of hyaladherins known so far can be hyaladherins grouped into i) Structural HA -binding proteins of the ECM, such as link protein and the aggregating proteoglycanes, ii) Cell surface HA receptors, 98


iii) Intracellular HA binding proteins. The most studied HA receptor to date is CD44 (lymphocyte homing receptor), which is responsible for a wide variety of cellular functions, e.g. receptor mediated internalization/degradation of HA, cell migration and cell proliferation. Several other cell membrane-localized receptors have been identified and their function is indicated in Table 2. Medical Applications of Hyaluronan and Hyaluronidases a) HA has found applications in various medical and pharmaceutical areas owing to its high waterbinding capacity and the viscoelasticity of its solutions. In the late 1950s, HA was probably applied for the first time to humans, in fact as vitreous humor supplement/replacement during eye surgery, an application which has proved therapeutically useful up to now (e.g. in cataract surgery)21. During surgical intervention in the eye, HA is often injected intraoperationally to keep the anterior eye chamber intact or to protect the corneal endothelium during lens transplantation. b) Since HA retains moisture it is used in some cosmetics to keep skin young and fresh-looking22. c) Sodium hyaluronate and a covalently cross-linked form of HA are successfully applied for the treatment of osteoarthritis23. d) Anabolic effects of HA on degraded bovine articular cartilages suppress their degeneration24. e) HA normalizes the properties of synovial fluids and produces an analgesic effect25. f) A rapid increase of HA levels can occur in many clinical situations, e.g. shock incidents, septicaemia and in burn patients26. g) The therapeutical benefit of HDs is based on the cleavage of HA in tissues resulting in increased membrane permeability, a reduced viscosity and a facilitated diffusion of injected fluids. These phenomena are referred to as spreading effect of HDs. The ability to promote penetration and spread are used to accelerate and increase absorption of injected drugs, e.g. antibiotics, to promote resorption of excess fluids, to improve the effectiveness of local anaesthesia and to diminish pain due to subcutaneous or intramuscular injection of fluids19. HD facilitates of diffusion antiviral drugs, dyes and toxins.

h) For many years, HDs, especially BTH preparations, are widely used in many fields like orthopaedia, surgery, ophthalmology, internal medicine, oncology, dermatology and gynaecology 27 . i) Testicular HDs have significant homology with the protein PH-20 (64 kDa) present on the posterior head and the acrosomal membrane of mammalian sperm that plays an essential role in fertilization28. j) HD has been investigated as an additive to chemotherapeutic drugs for augmentation of the anticancer effect 29. There is evidence that HD may have intrinsic anticancer effects and can suppress tumor progression. Furthermore, Zahalka et al.30 showed in an animal model of T cell lymphoma that hyaluronidase blocks lymph node invasion by tumor cells. k) Moreover, Hyal-1 and Hyal-2 seem to be involved in tumor formation. Hyal-1 is a candidate tumor suppressor gene that is inactivated in many tobacco-related lung tumors and was found to promote tumor cell cycling. Overexpression of Hyal-2 is reported to accelerate tumor formation of murine astrocytoma cells and on the other hand, Hyal-2 seems to speed up apoptosis. With the exception of PH-20, which is displaying activity at neutral pH, all known mammalian HDs are active at acidic pH. l) The concomitant increase in intraocular pressure can be efficiently counteracted by injection of HD. HD can be used as an alternative or adjunct to conventional mechanical vitrectomy. m) Bacterial hyaluronate lyases are considered as virulence factors that facilitate the spreading of bacteria in host tissues by degradation of HA. Commercial Importance Generally, HD is extracted from bovine and sheep testis or from bacteria. For therapeutic purposes, highly purified HDs from animal origin have already been marketed i.e, bovine and ovine HDs [Hynidase: Ovine testicular hyaluronidase, Hylenex: Recombinant human hyaluronidase, Wydase, Vitragan (TM): Ovine hyaluronidase, Vitrase (R)]. Highly purified preparations include testicular hyaluronidase from bovine testes in (lyophilized powder), essentially saltfree form and HD from sheep testes (lyophilized powder) have already been marketed. However, HD 99


Fig. 1: Chemical structure of hyaluronic acid. H: axial hydrogens that contribute to the hydrophobic face. CO O H O

CO O H O

C H 2O H O

O

OH

C H 2O H O

O

O

OH

O

H

HO OH

OH OH

n = 2 0 -1 2 5 0 0 NHCO CH3

OH

NHCO CH3

h ya l u r o n i c a c i d

b o vin t e st i c u l a r h y a l u ro n i d a se

CO O H O

C H 2O H O

O

OH

OH

OH

OH OH

C H 2O H O OH

OH

O OH

NHCO CH3

E C 4 .2 .2 .1

CO O H O OH

O

OH

O

NHCO CH3

C H 2O H O

CO O H O

OH

b a c t e ri a l h y a l u ro n i d a se

OH

NHCO CH3

E C 3 .2 .1 .3 6

C H 2O H O OH

O

OH

O

E C 3 .2 .1 .3 5

le e ch h y a l u ro n i d a se

CO O H O

CO O H O

NHCO CH3

OH

C H 2O H O OH

O

CO O H O

OH

C H 2O H O OH

O

OH

OH

OH

OH

OH OH

NHCO CH3

OH

NHCO CH3

Fig. 2 : Classification scheme of hyaluronidases according to Meyer7.

Table 1: Concentration of hyaluronan in human tissues/ fluids and other sources. Tissues/ fluids Concentration (mg/l) 14,100 Umbilical cord Synovial fluid 1,420–3,600 Vitreous body 140–338 8.5–18 Thoracic lymph Urine 0.1–0.5 Serum 0.01–0.1 Dermis (0.5 mg/g wet tissue) (0.1 mg/g wet tissue) Epidermis Other sources Rooster comb 7500 500 Matrix of cumulus cells around oocyte

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Hyaluronan receptors CD44 (lymphocyte homing receptor)

Table 2: The function of hyaluronan receptors HA Receptor- protein Function interaction Noncovalent cellular functions, e.g. lymphocyte homing, endocytosis of extracellular HA, mediate cell attachment to the ECA and metastatically spread tumor cell, receptor mediated internalization/degradation of hyaluronan, cell migration and cell proliferation

RHAMM (receptor for hyaluronic acid mediates motility) ICAM-1 (intercellular adhesion molecule-1)

Noncovalent

motility

Noncovalent

motility

LEC (Liver Endothelial Cell clearance receptor LYVE-1 (Lymphatic endothelial hyaluronan receptor) SHAP(Serum-derived Hyaluronan-Associated Protein)

Noncovalent

motility

Noncovalent

motility

covalent

construction/maintenance of ECM

from microbial sources viz. Streptomyces hyalurolyticus (lyophilized powder) with specific activity (U/mg) is limitedly available in the market. Inhibitors of Hyaluronidases Certain chemicals inhibit31 HD activity (vitamin C, salicylate, heparin, dicumarene, antihistamines and flavonoids). The inhibitory effect shows that vitamin C is probably directly involved in bacterial invasion, in addition to its antioxidant and free radical scavenger properties. During 1952, inhibitory effects on bovine testicular HD were reported for iron, copper and zinc salts, heparin, polyphenols and flavonoids. Based on the structural similarity to HA, heparin and heparan sulfate have been investigated as inhibitors of HD. Structurally related compounds Inhibit HDs was also described for instance, alginic acids comprising L-glucuronic acid and D-mannuronic acid, O-sulfated glycosaminoglycans in which fully sulfated substances showed the highest inhibitory activity, fully O-sulfated HA oligosaccharides or dextran sulfate. Some flavones and flavone analogs like apigenin and kaempferol inhibit HD. Other natural products like saponins and sapogenins, norlignans and extracts of plants or feces32 reveal likewise weak inhibitory activity.

Anti-allergic drugs such as disodium cromoglycate (DSCG), tranilast and traxanox possess inhibitory effects on HD. Anti-inflammatory drug indomethacin was found to inhibit HD in vivo. Other antiinflammatory agents like glycyrrhizin, phenylbutazone and oxyphenbutazone are also mentioned to weakly inhibit HDs. Recently, vitamin C, L-arginine derivatives and cis-unsaturated fatty acids were reported to inhibit a streptococcal HD. Stimulators of hyaluronidases The activity of HD is modulated by various activators including adrenaline, histamine and acid phosphatase formed in the prostrate, spleen, kidney, erythrocytes and platelets. Identification Of Hyaluronidase Producing Microorganisms The plate test by Winkle (1979) The plate test for the detection of HD positive isolates was performed according to Winkle (1979)33. For this the isolates were cultivated in close proximity of the mucoid growing S. equi subsp. zooepidemicus strain W60. A growth of the indicator strain in non mucoid colonies in close proximity of the isolate to be tested indicated a positive reaction.

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Mucin clot prevention assay Turbidity reaction assay-It is a simple method to determ -ine the enzyme activity of the bacterial cultures, the mucin clot prevention test34 of McClean was employed. Specific concentration of substrate HA was diluted with substrate diluent was mixed with definite concentration of the supernatant of an 18-24 h broth culture of microorganism to be tested and incubated at 37° for 30 minutes. At the end of incubation time the tubes were cooled in ice water and desired strength to acetic acid was added to precipitate the remaining HA. Tubes containing sterile broth or broth from inactive cultures became turbid while tube containing broth from HD producing organism remained clear on addition of the acid. Assay methods for hyaluronidase activity Various assay methods were devised for the determination of HDs activity. Hynes and Ferretti35 gave an overview on the applied methods, which should be applicable to HDs from either microbial or mammalian sources. They classified the different assays into spectrophotometric, radiochemical, fluorogenic, enzymoimmunological, plate (solid media) assays as well as chemical (Colorimetric assay-MorganElson assay), physicochemical (Turbidimetric assay) and zymographic analyses. Stern and Stern discussed an ELISA-like assay for HD and HD inhibitors. Recently, new assay methods for instance, a combination of fluorescently labeled HA and gel filtration on high-performance liquid chromatography (HPLC) are used to examine the degree of digestion for rapid determination of HD activity. Moreover, a flow cytometric method detecting the decrease in fluorescence of substrate-coated beads, a fluorimetric Morgan-Elson assay and quartz crystal impedance technique were reported for measuring HD activity. Turbidimetric assay and a colorimetric assay is used to determine the potency of inhibitors. CONCLUSION Certain proteins or enzymes displayed on the surface of Gram-positive organisms significantly contribute to pathogenesis and might be involved in the disease process caused by these pathogens. Often, these proteins are involved in direct interaction with host tissues or in concealing the bacterial surface from the host defence mechanism. If antibodies to these proteins could offer better protection to humans, they could

provide an alternative targeted drug or approach in vaccine preparations by antagonizing the pathogens caused by the respective microorganisms. The availability of three-dimensional structural information about microbial proteins with tetra and hexasaccharide substrate and inhibitors will most probably facilitate the elucidation of the function and detailed mechanism involved in such functions. Such knowledge will aid the development of treatment strategies by designing specific and selective inhibitors as pharmacological tools and potential drugs against specific microbial infections. ACKNOWLEDGEMENTS The authors are thankful to A.I.C.T.E. for sanction of RPS project to one of the author Prof. P. K. Panda, U.D.P.S., Utkal University to carry out research work. The authors are thankful to HOD, U.D.P.S., Utkal University and Director RRL, Bhubaneswar for providing research facility. REFERENCES 1. Scott JE, Heatley F. Hyaluronan forms specific stable tertiary structures in aqueous solution: A 13C NMR study. Proc Natl Acad Sci USA 1999;96:4850–5. 2. Meyer K, Palmer JW. The polysaccharide of the vitreous humor. J BiolChem 1934;107:629-34. 3. Balazs EA, Laurent TC, Jeanloz RW. Nomenclature of hyaluronic acid. Biochem J 1986;235: 903. 4. Scott JE. Secondary structures in hyaluronan solutions: chemical and biological implications. Ciba Found Symp 1989;143:6-15; discussion 1520,281-5. 5. Duran-Reynals F. Exaltationde l'activité du virus vaccinal par les extraits de certains organes. CR Séances Soc Biol Fil 1928;99:6-7. 6. Frost GI, Csoka T, Stern R. The hyaluronidases: a chemical, biological and clinical overview. Trends Glycosci Glycotechnol 1996;8:419-34. 7. Meyer K. Hyaluronidases. The Enzymes; 3 rd ed.; Academic Press: NewYork, 1971;pp 307-20. 8. Kreil G. Hyaluronidases-a group of neglected enzymes. Protein Sci 1995;4:1666-9. 9. Hynes WL, Hancock L. Analysis of a second bacteriophage hyaluronidase gene from Streptococcus pyogenes: Evidence for a third hyaluronidase involved in extracellular enzymatic activity. Infect Immunol 1995;63:3015–20.

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Stern R. Devising a pathway for hyaluronan catabolism: are we there yet? Glycobiology 2003;13:105R-15R. Csoka AB, Frost GI, Stern R. The six hyaluronidase-like genes in the human and mouse genomes. Matrix Biol 2001;20:499-508. Gmachl M, Sagan S, Ketter S, Kreil G. The human sperm protein PH-20 has hyaluronidase activity. EBS Lett 1993;336:545-8. Gmachl M, Kreil G. Bee venom hyaluronidase is homologous to a membrane protein of mammalian sperm. Proc Natl Acad Sci U S A 1993;90:356973. Csoka AB, Frost GI, Wong T, Stern R. Purification and microsequencing of hyaluronidase isozymes from human urine. FEBS Lett 1997;417: 307-10. Frost GI, Csoka AB, Wong T, Stern R. Purification, cloning, and expression of human plasma hyaluronidase. Biochem Biophys Res Commun 1997;236:10-15. Menzel EJ, Farr C. Hyaluronidase and its substrate hyaluronan: biochemistry, biological activities and therapeutic uses. Cancer Lett 1998;131:3- 11. Suzuki S. Microbial hyaluronan lyases http://www.glycoforum.gr.jp/science/hyaluronan/H A14/HA14E.html 2000. Hynes WL, Walton SL. Hyaluronidases of Grampositive bacteria. FEMS Microbiol Lett 2000;183:201-7. Csoka TB, Frost GI, Stern R. Hyaluronidases in tissue invasion. Invasion Metastasis 1997; 17:297311. West DC, Chen H. Is hyaluronan degradation an angiogenic/ metastatic switch? New Frontiers in Medicinal Sciences: Redefining Hyaluronan; Elsevier Science, 2000; pp 77-86. Liesegan g TJ. Viscoelastic substances in ophthalmology. Surv Ophthalmol 1990; 34:268-93. Andre P. Hyaluronic acid and its use as a "rejuvenation" agent in cosmetic dermatology. Semin Cutan Med Surg 2004; 23:218-22. Balazs EA, Denlin ger JL. Clinical uses of hyaluronan. Ciba Found Symp 1989;143:265-75; discussion 275-80, 281-265. Fukuda K, Dan H, Takayama M, Kumano F, Saitoh M et al. Hyaluronic acid increases proteoglycan synthesis in bovine articular cartilage in the presence of interleukin-1. J Pharmacol Exp Ther 1996;277:1672-75. Gotoh S, Onaya J, Abe M, Miyazaki K, Hamai A et al. Effects of the molecular weight of hyaluronic

acid and its action mechanisms on experimental joint pain in rats. Ann Rheum Dis 1993;52: 817-22. 26. Stern R, Csoka AB. Mammalian hyaluronidases http://www.glycoforum.gr.jp/science/hyaluronan/H A15/HA15E.html 2000. 27. Farr C, Menzel J, Seeberger J, Schweigle B. [Clinical pharmacology and possible applications of hyaluronidase with refere nce to Hylase "Dessau"]. Wien Med Wochenschr 1997;147:34755. 28. Primakoff P, Lathrop W, Woolman L, Cowan A, Myles DG. Fully effective contraception in male and female guinea pigs immunized with the sperm protein PH-20. Nature 1988; 335:543–6. 29. Muckenschnabel I, Bernhardt G, Spruss T, Buschauer A. Pharmacokinetics and tissue distribution of bovine testicular hyaluronidase and vinblastine in mice: an attempt to optimize the mode of adjuvant hyaluronidase administration in cancer chemotherapy. Cancer Lett 1998; 131:7184. 30. Zahalka MA, Okon E, Gosslar U, Holzmann B, Naor D. Lymph node (but not spleen) invasion by murine lymphoma is both CD44- and hyaluronatedependent. J Immunol 1995; 154:5345-55. 31. Houck JC. The competitive inhibition of hyaluronidase. Arch Biochem Biophys 1957;71:336-41. 32. Kawagishi H, Tonomura Y, Yoshida H, Sakaki S, Inoue S. Orirubenones A, B and C, novel hyaluronan-degradation inhibitors from the mushroom Tricholoma orirubens. Tetrahedron 2004; 60:7049-52. 33. Winkle S. 1979. Mikrobiologiche und Serologische Diagnostik. 3. Auflage, Gustav Fischer Verlag, Stuttgart/New York. 34. Gochnauer TA, Wilson JB. The production of hyaluronidase by Lancefield’s group B streptococci. J Bacteriology 1951; 62(4):405-14. 35. Hynes WL, Ferretti JJ. Assays for hyaluronidase activity. Methods Enzymol 1994; 235:606-16.

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Therapeutic Protein Production and Delivery: An Overview Hajare A.A.*, Dange A.S. and Shetty Y.T. Bharati Vidyapeeth College of Pharmacy, Kolhapur- 416 013, M. S., INDIA Author for Correspondence: [email protected] ; [email protected] Abstract Therapeutic proteins are used to treat patients suffering from cancers, heart attacks, strokes, cystic fibrosis, diabetes, anemia and heamophilia. These proteins are produced by using microbial fermentation on cell cultures, in transgenic animals and in transgenic plants. Being delicate compounds most of the commercially available therapeutic proteins are administered mainly by parentral route. Other routes include pulmonary, oral, transdermal, nasal and mucosal. Attempts have been made to improve the pharmacodynamic and pharmacokinetic properties of proteins by making changes in formulation types or by pegylation. Many new approaches are designed to increase duration of activity, decrease in adverse effects, increase in patient compliance and quality of life of patients. Keywords: Therapeutic protein, production, delivery systems, commercial challenges.

INTRODUCTION Until recently, pharmaceuticals used for treatment of diseases were small molecules such as antibiotics, analgesics, hormones and other pharmaceuticals which have been based on their production by microorganisms or by organic synthesis. Currently, pharmaceutical industry is undergoing remarkable changes with the discovery and introduction of biotech drugs. Proteins that are engineered in the laboratories and in plants for pharmaceutical use are known as biotechnological therapeutic proteins.1 The structure and functionality of given protein is determined by its sequence of amino acids which determines its three dimensional conformation. Internal bonds among the amino acids give the proteins its final shape and form. Complex proteins undergo modifications such as addition of phosphate group (phosphorylation) or carbohydrate molecule (glycosylation) modifying its functions.2 Since protein play critical roles in cell biology, they have many potential therapeutic uses in preventing and curing diseases and disorders. The protein biopharmaceuticals marketed to date are recombinant therapeutic protein drugs produced by living microorganisms, in animals and in plants to fight various diseases.3 Today therapeutic proteins are used to relieve patients suffering from many conditions Indian Journal of Pharmaceutical Education & Research Received on 19/07/2007; Modified on 05/11/2007 Accepted on 14/1/2008 © APTI All rights reserved

including various cancers (monoclonal antibodies, interferons), heart attacks, strokes, cystic fibrosis (enzymes and blood factors), diabetes (insulin), anemia (erythropoietins) and heamophilia (blood clotting factor). Currently, most of the commercially available protein products are for parentral administration. They suffer from short half-lives and they would benefit from development of controlled release parentrals. Alternative to this route is pulmonary delivery and at same time lot of research is going on other routes of administration such as oral, transdermal, nasal and other mucosal routes. 4 Production Unlike other medicines therapeutic proteins are not synthetically produced but are usually produced through microbial fermentation on by cell cultures, in transgenic animals and in transgenic plants (Table 1). Proteins are macromolecules composed of long chains of subunits called amino acids. Briefly, Proteins are the ultimate players of the cellular functions. Information stored in DNA direct the protein synthesizing machinery of the cell to produce the specific protein require for its structure and metabolism.5 The first therapeutic protein used to treat disease was insulin, a small peptide that revolutionized the treatment of diabetes.6 Antigens used in vaccinations to induce immune responses are often proteins. Short peptide 104


chains (less than 30 amino acids) synthesized chemically, larger proteins produced by living cells and in some cases proteins are secreted by cells have been used in therapeutics.7 Producing a biotechnological drug is a complicated and time-consuming process. Many years can be spent in just identifying the proteins, determining its gene sequence and working out a process to make the molecule. Once the method is devised and scaled up they can be produced in large batches. This is done by growing host cells that have been transformed to contain the gene of interest in carefully controlled conditions in large stainless steel tanks. The cells are kept alive and stimulated to produce the target proteins through precise culture conditions that include a balance temperature, oxygen, acidity and other variables. After careful culture the proteins are isolated from culture, stringently tested at every step of purification. Purification Proteins are purified to remove non-protein contaminants without affecting their biological activities. Isolated proteins are purified by analytical and preparative methods depending upon the amount of protein that can practically be purified. Analytical methods include which aim to detect and identify a protein in a mixture; where as preparative methods aim to produce large quantities of the protein for structural biology or industrial use. Several methods are used for purification includes; extraction, precipitation and differential solubilization, ultracentrifugation and chromatographic methods viz. size exclusion chromatography, ion exchange chromatography, affinity chromatography, metal binding, immunoaffinity chromatography, hydrophobic interaction chromatography, HPLC and buffer exchanges. After purification process the proteins are concentrated by lyophilisation that simply removes all volatile components leaving the proteins behind or by ultrafiltration which concentrates a protein solution using selective permeable membranes.8 Purified proteins upon concentration are formulated into products. All of these procedures are in strict compliance with FDA regulations.9 Most proteins are purified to where they compose 99.99% or more of the material in the sample.

Traditional cell culture methods require significant capital and labour investment. A cell culture facility on average takes 3-5 years to construct and costs $250 million to $450 million and also required to be approved and certified by FDA prior to their full scale operation.10 Production of Low Complex Proteins Microbial cell culture Hybridoma technology and subsequent technological expansion has provided molecular tool for protein production in microorganisms including bacteria, yeast and fungi. The best protein-expressing hosts include Escheria coli, Staphylococcus cerevisiae and Pseudomonas pastoris. Scalability and wellcharacterized genetics of microbial host systems made it as the best option for low complex and moderate volume protein production at relatively low cost.11 Major obstacle in this type of production is postproduction recovery of proteins. The strategies for improved bioprocess include use of secretory signal peptide fused to N-terminal of target protein and addition of charged acid residues or mutation of amino acids of protein for improving purification.12 Transgenic plants In the past 20 years plants have been used for the production of specific therapeutic protein. Plants have many advantages over established production technologies for large-scale expression of recombinant proteins. A small number of plant-derived biologics are approaching commercialization. Use of biotechnology now days also referred as pharming, biopharming or molecular farming. The first pharmaceutically relevant protein made in plants was human growth hormone, which was expressed, in transgenic tobacco in 1986.13 Since then; many other human therapeutic proteins have been produced in an increasingly diverse range of crops (Table 2). To produce a specific molecule in plants a novel gene is inserted into its chromosomes. A regulatory code inserted with that gene dictates to the plants to produce the desired protein in its parts. Transgenic plants are suitable for large volume production of proteins.14 This type of production is most effective than conventional microbial systems, which has drawbacks in terms of cost, scalability, product safety, authenticity and quality (Table 3).

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Production of Complex Proteins Complex proteins undergo processing such as phosphorylation or glycosylation that modify protein function. Some human proteins lack glycosylation or phosphorylation and can be produced by a simple bacterial host. Over 70% of all human proteins are glycosylated while some are phosphorylated and their successful production as therapeutic protein relies on host system that can mimic the structures synthesized by humans.15 Animal cell culture Animal cell lines have been utilized increasingly as host for the production of therapeutic proteins such as erythropoietin and Factor VIII. Many of them used for large production of therapeutic proteins undergo apoptotic death due to deprivation of key nutrients such as amino acids, glucose, serum, oxygen, protein expression systems and cytostatic agents in the bioreactor environment.16 Although this technique is well characterized, production volume constraints call for accurate manufacturing strategy and hence limited to low volume production. Significant research inputs are required in areas of cell line selection, bioreactor design, online bioreactor controls and various production modes to reduce duration and for increased yield. Productions using animal cell cultures are required to be monitored carefully to avoid unintentional transmission of viral diseases that could infect humans. Transgenic animals Transgenic animals can be used to make large quantities of complex human proteins. These animals are genetically engineered by the addition of gene designed to direct the synthesis of a foreign protein without affecting the health of the animal. Some common applications of these medicinal proteins include the treatment of congenital deficiencies or augmentation of the naturally occurring protein during life threatening diseases states such as sepsis (blood poisoning) or trauma from accidents and surgery. For example, transgenic animals may become a major source of therapeutics used to treat the common forms of hemophilia.17 The process involves microinjection of the DNA solution into the pronucleus of embryo using a very fine glass needle. The injected zygote is then transferred in to a hormonally prepared recipient and brought to term.

Finally, positive transgenic animals are matured and the level of expression of transgene is determined. The cost of making one transgenic animal ranges from $20,000 to $30,000 with very low success rate. The transgenesis rate is 5% to 25% of line birth, but the unit cost per protein should be significantly less if animals are used as bioreactors.18 Cow, sheep, goat, pig, rabbits are in use as transgenic species due to several advantages such as higher expression level, larger outputs, low initial investment, low operational cost and reproducible production facility.19 However, transgenic animal has its own constraints such as regulatory approval, protein purification and extensive testing. Therapeutic Protein Delivery Systems Since therapeutic proteins are delicate compounds, are usually administered mainly by injections. A number of systems that alter the delivery of injectable protein have been developed in attempts to improve pharmacodynamic and pharmacokinetic properties of therapeutic agents. New drug delivery systems can be produced either through the change in formulation type such as continuous release liposomes (cyclosporine, insulin, catalase, interleukin2) or an addition to the protein molecule called as pegylation which may minimize many limitations associated with the delivery of proteins.20 Potential advantages of new delivery mechanisms include an increased or prolonged duration of pharmacologic activity, decrease in adverse effects, increased patient compliance and quality of life. Injectable continuous release systems deliver drug in controlled predetermined fashion and are particularly appropriate when it is important to avoid large fluctuations in the plasma drug concentrations. Encapsulating protein molecule within a liposome can produce a prolonged half-life and a shift of distribution toward tissues with increased capillary permeability (tumors, infected tissues). Among non-invasive methods pulmonary delivery is most promising one.21 Some of the examples of products in the pipeline are given in Table 4. Techniques used for controlled release / pulsed release of intact proteins includes; Use of a pump with a catheter and fixed needle to administer drugs for local or systemic delivery, liposomal dispersions, formulation in amorphous form or as crystals (for example, insulin) to ensure release over a short period 106


of up to two days and hydrogel-based technologies. Compared to conventional, small molecule drugs therapeutic proteins are more susceptible to chemical, enzymatic and mechanical degradation, have much lower oral bioavailability due to rapid digestion to peptide fragments with no residual therapeutic properties and have shorter in vivo half-lives. They are poor candidates for dermal delivery and require more substantial reformulation for compatibility with deep lung delivery mechanisms. Delivery systems should be sharply defined in terms of their chemical and physical properties and for its capability of being tracked in complex biological environments.22 Advances in delivery are unlikely to bear fruits unless they overcome the major losses via non-specific routes and lack sufficiently bio-specific targeting and triggering methods. New approaches that are designed to overcome the principle biological problems may include and combine: New methods to increase the payload, exposure or potency of the drug at the target, new approaches to amplify specific Drug Delivery of drug quantities at particular sites or times, closed loop delivery, release or activation where the trigger is specifically bio-responsive to target or disease condition and approaches to realize in-vivo high specificity of targeting by coupling trigger mechanisms. Factors Affecting Therapeutic Delivery Factors that affect protein or peptide delivery include the large and unstable (structure is held together by weak non-covalent forces) molecules, hydrophilicity, physical and chemical liability (easily destroyed by relatively mild storage conditions) and short half-lives (easily destroyed by proteolysis: by endo or exo peptidases and eliminated by the body: elimination by B and T cells) of the drug molecule.23 Small proteins ( 99.9% with F values F =14.10. Cross validated squared correlation coefficient of this model was 0.633, which shows the good internal prediction power of this model. The best equation received for HIV intergrase strand transfer (ST) inhibitory activity when we considered single parameter is pIC50 (ST) = 0.960 (± 1.599) + 0.014 (± 0.009) TE (5) n = 12, r2 = 0.213, r2adj = 0.135, SEE = 0.369, F = 2.71, P < 0.05, q2 = 0.096. The above equation is not statistically significant one. So we considered the best equation containing two parameters is Eq. 6. pIC50 (ST) = 1.113 (± 1.127) – 0.636 (± 0.315) logP + 0.013 (± 0.006) HF (6) 2 2 n = 12, r = 0.406, r adj = 0.274, SEE = 0.338, F = 3.07, P < 0.05, q2 = 0.268 The above two parameter equation is also not statistically significant one. Then, we considered three parameters: pIC50 (ST) = 2.800 (± 1.020) – 0.891 (± 0.250) logP + 0.748 (± 0.259) LUMO + 0.018 (± 0.005) HF (7) n = 12, r2 = 0.709, r2adj = 0.600, SEE = 0.251, F = 6.50, P < 0.05, q2 = 0.401, SDEP = 0.322, SPRESS = 0.339. pIC50 (ST) = 18.440 (± 5.840) – 0.790 (± 0.235) logP + 1.878 (± 0.626) HOMO + 0.018 (± 0.005) HF (8) n = 12, r2 = 0.720, r2adj = 0.615, SEE = 0.246, F = 6.86, P < 0.001, q2 = 0.597 135


Table No.1 - Structure, Observed Biological Activity and Physico-Chemical Parameters of Hexenoic Acid Derivatives O

R

O

N O

X

Comp. NO

X

IC50 (µM)

R

3’-proc

HOMO

S.T.

1 H H 7.9 7.0 -8.834 2 H Et 8.9 7.5 -8.86 3 Cl H 50 65 -8.814 4 Cl Et 85 90 -8.779 5 F H 61 72 -8.763 6 F Et 87 95 -8.923 7 Me H 56 67 -8.829 8 Me Et 73 88 -9.169 9 OMe H 22 41 -8.934 10 OMe Et 38 50 -8.9 11 NO2 H 76 92 -8.805 12 NO2 Et 57 45 -9.076 3’-Proc – 3’ processing, S. T. – strand transfer, HOMO – Highly occupied molecular orbital, logP heat of formation.

Comp. No

logP

HF

1.817 -90.153 2.709 -93.786 2.284 -99.678 1.938 -126.31 2.191 -89.734 1.956 -133.247 2.658 -96.369 1.77 -98.597 2.335 -98.174 2.33 -131.169 1.564 -128.467 2.144 -96.743 – partition coefficient, HF –

Table No.2 - Observed, Calculated and Predicted (LOO) Activities of Hexenoic Acid Derivatives Calculated activity Predicted activity (LOO) Model Model pIC50 (3’pIC50 P)

(ST)

3

4

1 -0.897 -0.845 -.949 -1.012 2 -0.949 -0.875 -1.206 -1.176 3 -1.699 -1.813 -1.631 -1.747 4 -1.929 -1.954 -1.842 -1.853 5 -1.785 -1.857 -1.801 -1.872 6 -1.939 -1.978 -2.082 -2.109 7 -1.748 -1.826 -1.521 -1.486 8 -1.863 -1.944 -1.814 -1.783 9 -1.342 -1.613 -1.283 -1.248 10 -1.580 -1.699 -1.557 -1.479 11 -1.881 -1.964 -1.716 -1.752 12 -1.756 -1.653 -1.968 -1.853 3’-P – 3’ processing, ST – strand transfer, pIC50 = -log IC50.

7

8

3

4

7

8

-.982 -1.226 -1.655 -1.840 -1.938 -2.198 -1.558 -1.824 -1.433 -1.685 -1.725 -1.958

-1.038 -1.194 -1.764 -1.851 -2.006 -2.225 -1.522 -1.794 -1.397 -1.610 -1.765 -1.857

-.994 -1.313 -1.622 -1.799 -1.807 -2.176 -1.488 -1.794 -1.240 -1.550 -1.494 -2.147

-1.095 -1.283 -1.755 -1.816 -1.912 -2.226 -1.442 -1.750 -1.174 -1.442 -1.536 -1.896

-1.104 -1.372 -1.633 -1.785 -1.973 -2.244 -1.518 -1.775 -1.302 -1.581 -1.601 -2.215

-1.177 -1.144 -1.756 -1.801 -2.074 -2.395 -1.672 -1.732 -1.326 -1.578 -1.732 -1.347

136


Table No.3 - Correlation Matrix and Variance Inflation Factor of Descriptors in Best QSAR Models pIC50 (3’P/ST)

HUMO

logP

VIF

HF

Model-2

Model-3

3’ processing pIC50 (3’-P) HUMO logP HF

1 0.472 -0.459 0.249

1 0.159 -0.119

1.026 1.026 --

1 0.372 1 Strand transfer

1.066 1.220 1.206

Residual Value

pIC50 (ST) 1 Model-5 Model-6 HUMO 0.386 1 1.161 1.066 logP -0.343 0.159 1 -1.220 HF 0.370 -0.119 0.372 1 1.161 1.206 VIF – Variance inflation factor, HOMO – Highest occupied molecular orbital, logP – partition coefficient, HF – heat of formation.

0.3 0.25 0.2 0.15 0.1 0.05 0 -0.05 0 -0.1 -0.15

1

2

3

4

5

6

7

8

9

10 11

12

13

-0.2 -0.25

-0.3 Compound Number Fig.1

O AcO AcO

O O

O

OAc OAc

137


0.4

Residual Value

0.3 0.2 0.1 0 -0.1 0

2

4

6

8

10

12

14

-0.2 -0.3 -0.4 Compound Number Fig.2 O HO

O NH

OH

O

HO

OH

O HO

O O

HO

SDEP = 0.259, SPRESS = 0.333. Model - 7 shows good correlation coefficient (r) of 0.842 between descriptors (HF, logP and LUMO) and HIV intergrase strand transfer inhibitory activity. Squared correlation coefficient (r2) of 0.709 explains 70.9% variance in biological activity with F values F = 6.50. Cross validated squared correlation coefficient of this model was 0.401, which shows the poor internal prediction power of this model. Model - 8 shows good correlation coefficient (r) of 0.849 between descriptors (HF, logP and HOMO) and HIV intergrase strand transfer inhibitory activity. Squared correlation coefficient (r2) of 0.720 explains 72% variance in biological activity with F values F = 6.86. Cross validated squared correlation coefficient of this model was 0.597, which shows the good internal prediction power of this model.

O

OH OH

The robustness of the selected model (4 and 8) was checked by Y – randomization test (Data not given). The low r2 and q2 values indicate that the good results in our original model are not due to a chance correlation or structural dependency of the training set. The predictive ability of these models (4 and 5) was also confirmed by leave 33 % out cross validation. All the three models showed good predictivity. The equations received from the leave 33 % out cross validation technique are Leave 33% out crossvalidation equation for model – 4 (number of cycles 3) pIC50 (3’-P) = 39.120 (± 12.057) – 1.943 (± 0.446) logP + 4.001 (± 1.303) HOMO + 0.029 (± 0.009) HF (9) r2CVextLSO = 0.561, r2LSO = 0.659 Leave 33% out crossvalidation equation for model –8 (number of cycles 3) 138


pIC50 (ST) = 33.681 (± 18.341) – 1.821 (± 0.686) logP + 3.362 (± 1.978) HOMO + 0.035 (± 0.013) HF (10) r2CVextLSO = 0.537, r2LSO = 0.612 Consequently Eq. 4 can be considered as most suitable model for 3’ processing inhibitory activity with both high statistical significant and excellent predictive ability. Eq. 8 was selected as best model for strand transfer inhibition activity. The variables used in the selected models have no mutual correlation (Table No.3). These models showed good correlation coefficient between descriptors and HIV integrase 3’ processing and integration inhibitory activity. In model – 4, the negative contribution of logP on the biological activity showed that the increase of hydrophobicity lead to reduce HIV integrase 3’ processing inhibitory hexenoic acid compounds. The positive coefficient of HOMO showed that the substitution with high electro positive groups is conducive for the inhibitory activity of hexenoic acid. The less electro positive groups are detrimental to biological activity. The third parameter HF is positively contributed to biological activity. In model -8, all the above three parameters showed the same type of contribution to HIV integrase strand transfer inhibition activity of hexenoic acid. In both the models logP contributed negatively. Based on the developed QSAR model, new HIV integrase inhibitors of hexenoic acid derivatives can be designed with caution. ACKNOWLEDGMENTS One of the authors V. Ravichandran is thankful to AICTE, New Delhi for providing (QIP) Senior Research Fellowship. REFERENCES 1. De Clercq E. Toward improved anti-HIV chemotherapy: therapeutic strategies for intervention with HIV infections. J Med Chem 1995;38:2491-517. 2. Craigie R, Fujiwara T, Bushman F. The IN protein of Moloney murine leukemia virus processes the DNA viral ends and accomplishes their integration in vitro. Cell 1990; 62:829-35. 3. Katz RA, Merkel G, Kulkosk T, Leis J, Salka AM. The avian retroviral IN protein is both necessary and sufficient for integrative recombination in vitro. Cell 1990;63:87-91.

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Chen IJ, Neamati N, Nicklaus MC et al. Identification of HIV-1 integrase inhibitors via three-dimensional database searching using ASV and HIV-1 integrases as targets. Bioorg Med Chem 2000;8: 2385-90. Neamati N. Patented small molecule inhibitors of HIV-1 integrase: a 10-year saga. Expert Opin Ther Patents 2002;12:709. Parril AL. HIV-1 integrase inhibition: binding sites, structure activity relationships and future perspectives. Curr Med Chem 2003;10:1811-15. Carteau S, Mouscadet JF, Goulaouic H, Subra F, Auclair C. Effect of topoisomerase inhibitors on the in vitro HIV DNA integration reaction. Biochem Biophys Res Commun 1993;192:140913. Fesen MR, Kohn KW, Leteurtre F, Pommier Y. Inhibitors of human immunodeficiency virus integrase. Proc Natl Acad Sci USA 1993;90:239903. Mouscadet JF, Carteau S, Goulaouic H, Subra F, Auclair C. Triplex-mediated inhibition of HIV DNA integration in vitro. J Biol Chem 1994;269:21635-8. Eich E, Pertz H, Kaloga M, Schulz J, Fesen MR, Mazumder A, Pommier Y. (–)-Arctigenin as a lead structure for inhibitors of human immunodeficiency virus type-1 integrase. J Med Chem 1996;39:86-92. Mazumder A, Raghawan K, Weinstein J, Kohn KW, Pommier Y. Inhibition of human immunodeficiency virus type-1 integrase by curcumin. Biochem Pharmacol 1995;49:1165-70. Cushman M, Sherman P. Inhibition of HIV-1 integration protein by aurintricarboxylic acid monomers, monomer analogs, and polymer fractions. Biochem Biophys Res Commun 1992;185:58-63. Robinson WE Jr, Cordeiro M, Abdel-Malek S et al. Dicaffeoylguinic acid inhibitors of human immunodeficiency virus integrase: inhibition of the core catalytic domain of human immunodeficiency virus integrase. Mol Pharmacol 1996;50:846-50. Robinson WE Jr, Reineck MG, Abdel-Malek S, Jia Q, Chow SA. Inhibitors of HIV-1 replication inhibit HIV integrase. Proc Natl Acad Sci USA 1996;93:6326-32. 139


15. Mazumder A, Neamati N, Sommadossi JP, Gosseli G, Schinazi RF, Pommier Y. Effects of nucleotide analogues on human immunodeficiency virus type 1 integrase. Mol Pharmacol 1996;49:621-6. 16. Hansch C. A quantitative approach to biochemical structure-activity relationships. Acc Chem Res 1969;2:232-8. 17. Pungpo P, Hannongbua S. Three-dimensional quantitative structure-activity relationships study on HIV-1 reverse transcriptase inhibitors in the class of dipyridodiazepinone derivatives, using comparative molecular field analysis. J Mol Graphics & Model 2000;18:581-90. 18. Ravichandran V, Agrawal RK. Predicting anti-HIV activity of PETT derivatives:CoMFA approach. Bioorg Med Chem Lett 2007;17:2197-02.

19. Barreca ML, Carotti A, Chimirri A, Monforte AM. Comparative molecular field analysis (CoMFA) and docking studies of nonnucleoside HIV-1 RT inhibitors (NNIS), Bioorg Med Chem 1999;7:228392. 20. Ravichandran V, Mourya VK, Agrawal RK. QSAR study of novel 1, 1, 3 – trioxo [1, 2, 4] - thiadiazine (TTDs) analogues as potent anti-HIV agents. Arkivoc 2007;XIV:204-12. 21. Costi R, Santo RD, Artico M et al. 6-Aryl-2,4dioxo-5-hexenoic acids, novel integrase inhibitors active against HIV-1 multiplication in cell-based assays. Bioorg Med Chem Lett 2004;14:1745-9.

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Hepatotoxicity Studies of some Mycotoxins with Special Reference to Hepatoprotection against Mycotoxin Induced Liver Damage. Papiya Mitra Mazumder* and Sasmal D. Department of Pharmaceutical Sciences, Birla Institute of Technology Mesra, Ranchi- 835215, Jharkhand. * Author for correspondence : Email: [email protected] Abstract The two potent mycotoxins Citrinin and Fumonisins obtained from the fungus Penicillium citrinum and Fusarium moniliforme respectively mostly contaminate food and agricultural products. The hepatotoxic effects were induced by Citrinin and the crude extract of Fusarium moniliforme containing Fumonisins in mice. The hepatoprotective activity of Liv 52 (a polyherbal formulation) against Citrinin and fumonisin induced experimental hepatotoxicity was investigated and evaluated. Citrinin extracted from Penicillium citrinum and the crude extract of Fusarium moniliforme containing a mixture of Fumonisins were injected intraperitoneally at doses of 5mg/Kg body weight and 50 and 100mg/Kg body weight respectively to mice. The polyherbal formulation was administered orally at a dose level of 5mg/kg body weight to the toxin treated groups. Body and liver weights were significantly decreased in Citrinin and crude extract of Fusarium moniliforme treated groups and these changes were reversed more or less to normal in Liv 52 treated animals. The liver transaminases, serum transaminases and reduced glutathione levels have been determined. It was observed that liver transaminases (LGOT and LGPT) and GSH levels decreased whereas SGOT, SGPT levels increased considerably in the toxin treated groups. All these effects were reversed nearly to normal in the groups treated with Liv 52. The results show that the changes in hepatological parameters are due to liver damage, because the enzymes leak out into the serum due to hepatocyte injury. These effects can be reverted with the use of the polyherbal formulation Liv 52. Keywords – Mycotoxin, Citrinin, Fumonisin, Liv 52, Hepatotoxicity. INTRODUCTION Citrinin, a potent mycotoxin has been isolated from the fungus Penicillium citrinum, which has been identified as one of the major causative agents of the various toxic effects of Penicillium citrinum. It contaminated agricultural products including corn, maize, rice, wheat etc. It is a dihydroisocoumarin metabolite produced by several species of Penicillium1. The distribution of this fungus in the natural environment is extensive. The presence of this toxin has been demonstrated in sorghum, corn, wheat, rice, and peanuts2-4. This mycotoxin has been reported to be very toxic for ducklings, rats, chicks, rainbow trouts, beagle dogs and swine5. Humans may be exposed to this toxin via two routes:- (a) consumption of foodstuffs directly Indian Journal of Pharmaceutical Education & Research Received on 15/03/2007 Modified on 30/06/2007 Accepted on 14/12/2007 © APTI All rights reserved

contaminated by the mycotoxin- producing fungi and (b) consumption of meat of slaughtered animals fed on mycotoxin contaminated feed. Fumonisins are a group of mycotoxins produced by species of Fusarium, mainly Fusarium moniliforme. The discovery of Fumonisins in 1988 (by researchers from the South African medical research council) received worldwide attention6-8. Chemically, fumonisins are diesters of propane 1,2,3 tricarboxylic acid and structurally similar to sphingosine produced by Fusarium moniliforme and related species in agricultural commodities, in the field as well as during storage. Although different Fumonisins namely Fumonisin A1, A2, B1, B2, B3, B4 & C1 have been reported, only fumonisins B1, B2 and B3 were found to occur naturally in foods9. Among different varieties, Fumonisin B1 is the major metabolite occurring naturally, mostly in maize. Fumonisins have been shown to be hepatotoxic, hepatocarcinogenic and 141


nephrotoxic in rats, atherogenic to monkeys and also lead to poor weight gain, diarrhoea and rickets in boiler poultry, equine leukoencephalomalacia, porcine pulmonary edema10. Fumonisins are shown to cause developmental toxicity in rats, mice and hamsters and are toxic to chick embryos. Studies have shown that Fumonisins are cytotoxic to different mammalian cell lines in vitro, although the sensititvity differs between different cell lines. Fumonisin B1 has been found to inhibit the sphingolipid synthesis both in vitro and in vivo11-13. International agency for research on Cancer (IARC) has classified Fumonisin B1 under the 2B group of carcinogens i. e, possibly carcinogenic14. The polyherbal preparation Liv 52 is formulated of several plant principles. It is manufactured by Himalaya Drug Co. Pvt. Ltd and is reputed for it’s hepatoprotective activity. Liv 52 contains plant extracts of the following herbs:- Capparis spinosa, Cichorium intybus, Solanum nigrum, Cassia occidentalis, Terminalia arjuna, Achillea millefolium, Tamarix gallica and Phyllanthus amarus15. Mycotoxins are found to contaminate agricultural products including animal feed and they produce considerable liver damage. Thus, the aim of this present study is to evaluate the hepatoprotective activity of Liv 52 against liver damage induced by Citrinin and the crude extract of Fusarium moniliforme in mice. MATERIALS AND METHODS Chemicals and Reagents Bovine Serum Albumin, Glutathione, DTNB (Sigma Chemical Co., USA), Transaminase detection Kits (Span Diagnostics, India). All other chemicals were of analytical grade. Herbal formulation Liv 52 (Himalaya Drug Co. Pvt. Ltd., India). Toxin Citrinin was isolated and purified from the culture medium of Penicillium citrinum by the method of Raistrick & Hetherington16-17. Sterile Potato Dextrose media (200gm potato and 20gm dextrose for 1 lit) at pH 6 was taken in 50ml flasks and inoculated with Penicillium citrinum. The liquid medium was fermented in a rotary shaker at 28 ± 0.5° C for 7 days. The culture filtrate was collected from each flask and was made acidic to pH 1.5 by the addition of concentrated hydrochloric acid. Citrinin was crystallized from the chloroform-extracted fraction of

acidic broth and was recrystallized from ethyl alcohol. The purity was verified by melting point, HPTLC (CAMAG, Switzerland) and Spectrophotometric analysis (Beckmann UV-VIS spectrophotometer (Model No. 108), India). It was chemically pure. The pure culture of Fusarium moniliforme MTCC 2088 was inoculated in a fresh sterile Potato Dextrose Agar slant (PDA slant), which was incubated at 25º - 28 ± 0.5°C under aerobic conditions and the incubation time required was 7 days. After 15 days of inoculation, the spores were harvested with 1ml of distilled water and the spore density was adjusted. 100 g of maize were distributed equally into 500ml Erlenmeyer flasks and autoclaved for 1 hour for two consecutive days. After adjusting the moisture to 50%, sterile substrates were inoculated with 2ml of spore suspension, incubated in dark for 29 days at 25 ± 0.5° C. The cultures were harvested by pouring acetone and drying overnight at 50° C after removing acetone. Dried culture material was firmly powdered and stored at –20° C till further analyzed. This dried and powdered crude culture material was taken as a whole, which contained Fumonisins which was confirmed by Reversed Phase T.L.C 18-19. Animals Eighty inbred adult Swiss Albino male mice weighing between 25 - 35gms were raised in the animal house of Birla Institute of Technology, Mesra, Ranchi, and were housed in polypropylene cages. They were divided into eight groups with 10 mice in each group. They were kept under controlled environmental conditions (30° C + 2 ° C and normal humidity) with natural light / dark cycle and allowed free access to food (standard pellet diet, Hindustan Lever Ltd., India) and water and acclimatized for at least a week before the commencement of the experiment. All experiments were performed as per the norms of the ethical committee and the studies were approved and clearance obtained by the ‘Institutional Animal Ethical Committee’. Acute Toxicity Studies Acute toxicity was determined according to the method of Litchfield and Wilcoxon 20. The LD50 value of Citrinin was found to be 41.69 mg/kg body weight and the LD50 value of crude culture material of Fusarium moniliforme MTCC 2088 was found to be 562.34 142


mg/kg body weight. Treatment Protocol The isolated and purified Citrinin from Penicillium citrinum MTCC 1751 was dissolved in propylene glycol for intraperitoneal administration at a dose of 5mg/Kg-body weight. The crude extract from Fusarium moniliforme MTCC 2088 containing fumonisins was dissolved in sterile normal saline (NaCl 0.9%w/v), at a dose of 50 mg / kg and 100 mg / kg body weight intraperitoneally. All animals were kept in fasting condition 18 hours prior to the start of the experiment. Treatments of the various groups of animals are given as below: Group – 1 served as normal saline control and received 0.9% NaCl w/v at a dose of 0.1ml/10gm intraperitoneally. Group – 2 served as vehicle control and received propylene glycol at a dose of 0.1ml/10gm intraperitoneally. Group – 3 received the toxin Citrinin dissolved in propylene glycol at a dose of 5mg/Kg-body weight intraperitoneally. Group – 4 received the toxin Citrinin dissolved in propylene glycol at a dose of 5mg / Kg-body weight intraperitoneally and Liv 52 orally at a dose of 5ml / Kg body weight. Group – 5 received the crude extract of Fusarium moniliforme containing Fumonisins dissolved in normal saline at a dose of 50 mg/ Kg body weight intraperitoneally. Group – 6 received the crude extract of Fusarium moniliforme containing Fumonisins dissolved in normal saline at a dose of 50 mg/ Kg body weight intraperitoneally and Liv 52 orally at a dose of 5ml / Kg body weight. Group – 7 received the crude extract of Fusarium moniliforme containing Fumonisins dissolved in normal saline at a dose of 100 mg / kg body weight intraperitoneally. Group – 8 received the crude extract of Fusarium moniliforme containing Fumonisins dissolved in normal saline at a dose of 100 mg / kg body weight intraperitoneally and Liv 52 orally at a dose of 5ml / Kg body weight. Treatment was done for six weeks once a week. Twenty-four hours after the last dose, the animals from each group were sacrificed under mild ether anesthesia.

Assay of Transaminases Blood was collected by cardiac puncture. The blood was allowed to clot and the serum was separated on the top. This serum was collected for the estimation of Serum glutamic oxaloacetic transaminase (SGOT) and Serum glutamic pyruvic transaminase (SGPT) 21, by using transaminase kits supplied by Span Diagnostics Pvt. Ltd. For the estimation of liver GOT and GPT, the liver from the sacrificed animal was immediately removed, weighed, and taken in a beaker containing precold distilled water. 1 gm from the liver was weighed and homogenized with 10 ml of precold KCl-phosphate buffer (1.17%, 0.1M, pH 7.4) in a Teflon homogeniser plunged in ice to obtain a 10% homogenate. The homogenate was centrifuged in cold (at 2000 rpm, 5 min followed by 10,000 rpm, 15 min, 4°C). The post mitochondrial supernatant (PMS) thus obtained was collected for the estimation of liver transaminase enzymes22, by using transaminase kits supplied by Span Diagnostics Pvt. Ltd. Determination of reduced glutathione (GSH): The post mitochondrial supernatant (PMS) was prepared as above. GSH was estimated using 5,5dithiobis – 2- nitrobenzoic acid (DTNB). The tissue content of reduced glutathione 23, and total proteins24 were estimated following the methods of Ellman23 and Lowry24 respectively. The absorbance for glutathione was read in the spectrophotometer at 412 nm. RESULTS Body and Liver Weights The results are summarized in Table 1. Body and liver weights were significantly decreased in Citrinin and crude extract of Fusarium moniliforme treated groups. The body weights of the mice increased in control groups and progressively declined in toxin treated hosts. These changes were reversed more or less to normal in Liv 52 treated animals. The liver weights that sharply decreased in the Citrinin and crude extract of Fusarium moniliforme treated groups, recovered on treatment with Liv 52. The mean liver somatic index significantly decreased in the Citrinin and crude extract of Fusarium moniliforme treated groups but it becomes more or less normal in toxin + Liv 52 treated groups. Biochemical Changes The Serum glutamic oxaloacetic transaminase (SGOT), Serum glutamic pyruvic transaminase (SGPT) levels, 143


Table 1. Effect of Citrinin and the crude extracts of Fusarium moniliforme with Liv 52 on the body weight and liver weights of mice. Treatment Mean Body Weight Mean Liver Weight Mean liver somatic index (mg) (mg) Group 1 28.6 ± 1.01 1.65 ± 0.91 5.77 ± 0.24 Group 2 27.9 ± 1.27* 1.58 ± 0.14 5.66 ± 0.14* Group 3 20.9 ± 1.11** 0.90 ± 0.11 4.31 ± 0.22*** Group 4 26.4 ± 1.03* 1.40 ± 0.09 5.30 ± 0.28* Group 5 20.6 ± 1.98** 0.8 ± 0.06 4.11 ± 0.50** Group 6 27.5 ± 1.50* 1.46 ±0.08 5.5 ± 1.54* Group 7 18.1 ± 1.05*** 0.55 ± 0.18 3.95 ± 0.5** Group 8 26.8 ± 1.23* 1.5± 0.15 5.21 ± 0.62* Values ( mean + SEM), (n=10) ; The statistical significance of difference between means was calculated by student’s unpaired ‘t’ test. * Insignificant as compared to normal control; ** P