international journal of pharmaceutical innovations volume 2 issue 4 2014.pdf · 3 volume 1, 2014...

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INTERNATIONAL JOURNAL OF

PHARMACEUTICAL INNOVATIONS

Official Research Publication of

Nova college of Pharmaceutical Education and Research

Affiliated to Jawaharlal Nehru Technological University Kakinada,

Approved by Pharmacy Council of India and AICTE New Delhi.

Jupudi, Ibrahimpatnam- 521456, Krishna district, Andhra Pradesh.

Email id: [email protected]

Volume 1, 2014

mailto:[email protected]

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Volume 1, 2014 INTERNATIONAL JOURNAL OF PHARMACEUTICAL INNOVATIONS

Editorial Board

Editor in-Chief

Dr. P. Selvam M.Pharm, Ph.D., FNABS, (NCPER)

Associate editor

M. Tejaswi M. Pharm.(NCPER)

Editorial Board Members

Dr. G. Narasimhan M.Pharm, Ph.D (Harvard University, USA)

Dr. Andrea Brancle ,Ph.D (Cadiff University, UK)

Dr. C. N. Ramchand., Ph.D., ( Laila Pharmaceutical., Pvt Ltd. India)

Dr. D. Selvakumar , M.Pharm., Ph.D (Wayne StateUniversity, USA).

Dr. M. Chandramohan ,M.D ,Ph.D (Madurai)

Dr. N. Murugesh B Sc, MBBS., M.Sc., Ph.D (Madurai)

Dr. P. Vasanthraj, M.Pharm, Ph.D (AIMST, Malaysia)

Dr. R. Govindharajan, M.Pharm., Ph.D (Head Dabur R&D centre, Dubai)

Dr. K. Prabhu M.Pharm, Ph.D (Nova College of Pharmacy, Jangareddygudem)

Dr. Kasthur Reddy, M.Sc, PhD (Mako Research Labs, Hyderabad)

Dr. P. Venkatesh, M.Pharm ,Ph.D ( Senior Principal Scientist, Orchid R&D centre, Chennai)

Dr. Alagasamy, M.Pharm, Ph.D (Hyderabad)

Dr. D.Sriram, M.Pharm, Ph.D, ( BITS, Hyderabad)

Dr. Subashini M.Pharm, Ph.D, (MSU University, Kuala lumbur, Malaysia)

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INSTRUCTIONS TO AUTHORS

The International Journal of Pharmaceutical Innovations is a e-Journal, which publishes innovative

research papers, reviews, mini-reviews, short communications and notes dealing with Pharmaceutical

Sciences (Pharmaceutical Technology, Pharmaceutics, Biopharmaceutics, Pharmacokinetics,

Pharmaceutical/Medicinal Chemistry, Computational Chemistry and Molecular Drug Design,

Pharmacognosy and Phytochemistry, Pharmacology, Pharmaceutical Analysis, Pharmacy Practice,

Clinical and Hospital Pharmacy, Cell Biology, Genomics and Proteomics, Pharmacogenomics,

Bioinformatics and Biotechnology of Pharmaceutical Interest). All manuscripts are subject to rapid peer

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Introduction

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Journal Articles

Shashi A, Jain SK and Pandey M: In-vitro evaluation of antilthiatic activity of seeds of Dolichos

biflorus and roots of Asparagus racemosus . International Journal of Plant Sciences 2008; 1:67-71.

A Book

Kalia AN: A Text Book of Industrial Pharmacognosy. CBS Publishers & Distributors, First Edition

2005.

A Chapter in a Book

Nadkarni KM: Indian Materia Medica. Popular Prakashan, Mumbai, Edition 3, Vol. I, 2000:

242-246.

Illustrations

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should not be very large that they run more than one A4 sized page. Table format should be as follow:

Phytochemical Analysis of successive extract of............

Chemical Constituent Aqueous Extract Ethanolic Extract

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Abbrevations, Units Etc The journal strictly follows the rules defined in the IUPAC Manual of symbols and terminology for

physicochemical quantities and units. Short Communication The journal publishes exciting findings, preliminary data or studies that did not yield enough

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requirements are same as those applicable to full papers. Submission of Manuscript All manuscripts (must be in English and in MS Word format) and should be submitted via our online

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Institutional Review Board. Authors are requested to send their research articles strictly according to the given format

mentioned in the guidelines to the authors.

Email id: [email protected]

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Contents

Invited article:

DESIGN, MOLECULAR MODELLING STUDIES ON CHLOROQUINE AND ITS

ANALOGUES AS INHIBITORS OF HCV NS5B RNA POLYMERASE

M. ChandraMohan, P. Selvam , J. Pranitha, N. Saravanan Prabhu, M. Suresh Kumar Pg no: 5-20

Research article:

DISSOLUTION ENHANCEMENT OF PIOGLITAZONE HYDROCHLORIDE BY SOLID

DISPERSION TECHNIQUE

T. Divya , S. Pranav ----- Pg no : 21-41

AN ATTEMPT TO ISOLATE INDUSTRIALLY IMPORTANT MICROORGANISM FROM

DIVERSE SAMPLES COLLECTED FROM SEMELING, KEDAH, MALAYSIA

P. Vasanth Raj, S.A. Dhanaraj, Saw Pei Jie, Sim Poh Pei, Vanushya a/p Alagasan, Tien Ja She,

Loh Mon Ying, Tan Te Wei Pg no : 42-55

Short communications :

STUDIES OF HIV INTEGRASE INHIBITORY ACTIVITY OF WRIGHTIA TINCTORIA

Periyasamy Selvam, Kasthuraiah Maddali , Christophe marchand

, Yves Pommier Pg no : 55-63

DRUG DISCOVERY AND DEVELOPMENT-CURRENT STATUS AND FUTURE FOCUS

N. Somashekara., P. Jagadeesh., C.N. Ramchand Pg no : 64-67

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Invited Article



M. ChandraMohan 1, P. Selvam

2, J. Pranitha

3, N. Saravanan Prabhu

3, M. Suresh Kumar

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1 Bharat Ratna Kamarajar liver Hospital and Research Centre, Madurai-625020,

2Nova College of Pharmaceutical Education and Research, Jupudi, Krishna DT, AP,

3 Centre for Bioinformatics, School of Life Sciences, Pondicherry University, Puducherry-605014.

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ABSTRACT

Hepatitis C virus (HCV) a member of the Flavivirus genus is a negative strand RNA virus that is

estimated to have infected and rendering 170 million people as carrier of HCV globally. Ribavirin and

interferon combination therapy is now available to treat HCV viral infection. Due to high rate of viral

drug resistance, we need new targeted therapy to combat HCV. Structure-based drug design methods

utilize knowledge of three dimensional structure of an enzyme to develop some novel inhibitors of

HCV. NS5B protein encoded RNA dependent RNA polymerase activity has a critically important role in

HCV RNA replication and so considered as an attractive therapeutic target for designing newer classes of

antiviral compounds. Present work is to investigate the molecular modeling studies of quinoline

derivatives such as Chloroquine, Hydroxychloroquine and Primaquine with HCV RNA polymerase.

Modelling studies were done after the three quinoline derivatives 3D structures were converted from 2D;

using the Schrodinger Maestro the compounds were docked to the active site residues of HCV RNA

Polymerase which involves interaction of Lipophilic compounds. They formed hydrophobic interaction

with quinoline rings of Chloroquine and Primaquine and with the alkyl groups of hydroxy chloroquine.

Results of the molecular modelling studies indicate that the quinoline derivatives strongly interact with

the residues in the primer grip site of the polymerase.

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INTRODUCTION

The World Health Organization has estimated that there are 170 million HCV carriers in the world with a

substantial risk of developing liver cirrhosis and/or liver cancer. There is currently neither an effective

vaccine nor anti- HCV-specific therapeutics for HCV infection. Development of anti-HCV agents is thus

a matter of much importance. HCV is a positive single stranded RNA virus whose genome encodes one

polyprotein, which is processed to structural and non-structural proteins by host and viral proteases. A

non-structural protein at the polyprotein C terminus, NS5B, includes the RNA-dependent RNA

polymerase responsible for HCV replication. This RNA-dependent RNA polymerase is validated target

for the development of newer anti-HCV therapeutic agent.

Chloroquine is a versatile bioactive agent and reported to possess antiviral activity against human

immunodeficiency virus (Romanelli et al., 2004), Hepatitis A virus (Superti et al., 1987; Bishop, 1998),

and severe acute respiratory syndrome virus (Keyaerts et al, 2004) infections. The documented antiviral

activities of chloroquine are by the following mechanisms (1) Viral particles entry into target cells by

endocytosis is blocked by chloroquine (Carrillo et al.,1985 ; Zeichhardt, et al., 1985),

(2) As

lysosomotrophic agent chloroquine accumulates in lysosome of host cell and increases the lysosomal pH

and renders lysosomal enzymes inactive and prevent the viral decoating (Carrillo et al., 1984), (3)

Intercalating action; chloroquine intercalates the HBV-DNA polymerase and inhibits the enzyme activity

of the virus and blocks the multiplication of HBV (Hirschman, S. Z.1974; Garfinkel, E. 1978), (4)

Chloroquine inhibits Duck hepatitis B virus super coiled DNA (DHBV-sc DNA) (Civitico et al., 1990)

and Duck hepatitis B surface antigen (DHBsAg) (Offensperger, et al., 1991). Also Chloroquine inhibits

the replication of HIV-1 and 2 at clinically achievable concentration and the anti HIV activity is by (5)

inhibition of synthesis of surface antigen GP120 of HIV (Savarino et al., 2001) (6) by inhibition of HIV

Integrase (Fesen et al., 1993) (7) Chloroquine also inhibits the replication of Ribonucleic Acid (RNA) of

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Sindbis virus (Cassell et al., 1984). Inhibition of normal proteolytic processing of Flavivirus Pr.M Protein

(Randolph et al., 1990) is taken as the eighth mode of antiviral action of Chloroquine. The eight modes of

antiviral activity of Chloroquine made us to look for antiviral action for Primaquine an analogue of

Chloroquine and found that Primaquine has antiviral actions against HIV by blocking HIV Integrase, and

New Castle Virus by blocking the synthesis of its RNA and release of its progeny( Burdick et al, 1974).

We have previously reported antiviral activities of chloroquine and hydroxy chloroquine against

Hepatitis C virus in Huh-5-2 cells (Bartenschlager 2002; Chandramohan et al., 2006) and Primaquine

against Severe Acute Respiratory Syndrome (SARS-CoV) in Vero cells (Chandramohan et al., 2009) In

view of many modes of antiviral activities of chloroquine and primaquine against many human

pathogenic viruses; the present work is to evaluate the binding studies of quinoline derivatives against

HCV RNA polymerase activity by molecular modelling studies.

METHODOLOGY

Molecular docking studies were performed using Schrödinger Maestro v8.0, and Glide v4.5.19.

Chloroquine, Primaquine and Hydroxy chloroquine structures were obtained as 2D and then converted

into 3D structures and optimized using the Maestro. The three compounds were preprocessed with

LigPrep at pH 7. Generated 3D structures of ligand were used to dock to the active site residues of HCV

RNA polymerase.The 3D structure of protein chosen for molecular docking study is 1Z4U. The protein

was prepared and optimized using the Protein Preparation Wizard tool supplied with Schrödinger Glide

(Friesner et al., 2004; Halgren et al., 2004). Using this tool, hydrogen atoms were added to the RNA

polymerase, the protonation states for histidine residues were optimized, crystallographic waters not

deemed to be important for ligand binding were deleted, and the entire protein was minimized. The

OPLS2005 force field was used for all calculations. Previous to this process the missing residues of the

protein were modeled using the PrimeX module. Next, a grid was prepared for docking the three ligands

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into HCV RNA Polymerase using the Receptor Grid Generation tool in Glide. The binding site was

defined as the residues located within 10 Å from Tyr448 of the macromolecule. To compensate for the

rigid representation of the receptor, the Van der Waals radii of the atoms with partial charges lower than

0.25 were scaled by the factor 0.8 to generate the receptor grid. No constraints were defined for the

docking runs. For this single-ligand automated docking, the Simple Precision (SP) mode in Glide was

selected. Default settings were used for docking. The final ligand poses were selected based on the Glide

empirical docking scores (GlideScore). The highest-scoring docking pose returned for each of the ligand

was considered for interaction studies. Docked result was analyzed using Glide Pose Viewer and with

Pymol21

(DeLano 2002). All conformations of the docked compounds were compared with the preceding

reports ( Pfefferkorn et al., 2005a,b).

RESULT AND DISCUSSION

Interaction study of Lipophilic compounds with the Active residues of HCV RNA polymerase

Primaquine: Preceding reports suggest that amino acids in the binding pocket are hydrophobic in nature

which forms hydrophobic interaction with the lipophilic compounds. Due to the aromatic side chain and

non polar side chain groups of amino acids of binding pocket such as Tyr 448, Tyr 415, and Met 414 and

Leu 384 form hydrophobic interaction with the quinoline ring of Primaquine. The highest-scoring pose

for the Primaquine protein complex obtained from this SP Glide docking study had a docking score of -

6.66 kcal/mol. Other residues such as Cys 366 and Arg 200 also involve in hydrophobic interaction (

Pfefferkorn et al., 2005a,b) . The ligand has established 2 hydrogen bond interactions with the protein

residues. The H25 of the ligand has established a hydrogen bond with the hydroxyl oxygen in the residue

Tyr415.9 (Fig.1 and 4).

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Chloroquine: The highest scoring pose for the Chloroquine-protein complex obtained from the SP Glide

docking result had a score of -5.35kcal/mol. Amino acids such as Arg200, Cys366, Tyr448, Ile447,

Pro197, Leu384 and Tyr415 form hydrophobic interaction with quinoline ring of Chloroquine (Fig.2).

Hydroxy chloroquine: Hydroxyl oxygen of the Tyrosine 448 residue establishes a hydrogen bond with

the hydrogen moiety of Hydroxy chloroquine (Tyr448-OH…H26–Hydroxychloroquine) spanning of a

distance of 2.04Ao

(Fig.3). Other amino acids in the binding pocket show hydrophobic interaction with

the alkyl group of the hydroxy choloroquine ring. The interaction between the lipophilic compounds with

the active site residues of HCV RNA polymerase was preserved among the Lipophilic compounds (

Pfefferkorn et al., 2005a,b)

CONCLUSION

With the results on hand we may suggest or infer that the biochemical configuration of both the ligand

and the virus, along with their lipophilicity and hydrophobicity may determine the docking site and the

level of bonding. This may dictate the efficiency of the drug action and the susceptibility of the virus.

Possibly this docking and binding of pockets in the HCV RNA polymerase active site residues was the

mode of action in our work published in 2006. Now the new mode of action on HCV – NS5B polymerase

targeted action is being established.

Having noted “Eight” modes of antiviral activity for chloroquine against many human pathogenic

viruses, this molecular modelling and showing the binding of quinoline compound may add up the

modes of action to “Nine” and name chloroquine as a versatile, unique and promising antiviral agent. The

antiviral activity of lipophilic amine and acidotropic amine (both happen to be Chloroquine) had been

documented in 1987 and 1990 respectively. Chloroquine documented for anti-HCV activity with

inhibition of viral entry (Usman et al., 2011). Ferroquine (FQ), an antimalarial ferrocenic analog of

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chloroquine, is a novel inhibitor of HCV (Vausselin et al., 2013). FQ potently inhibited HCV infection of

hepatoma cell lines by affecting an early step of the viral life cycle. Recently our group also

demonstrated chloroquine inhibits the replication of Human Noro virus (HNV) at 24 + 2.2 ug/ml and

cytotoxicity was found to be 98 + 5.5 ug/ml (unpublished data).

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http://www.ncbi.nlm.nih.gov/pubmed?term=Lavillette%20D%5BAuthor%5D&cauthor=true&cauthor_uid=23348596

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Figure 1: Hydrogen bond interaction between (1) Tyr415–OH and H–Primaquine

(2) Gly449–H–HOH–H–Primaquine.

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Figure 2: Docked complex of HCV RNA polymerase with Chloroquine.

19

Figure 3: Hydrogen bond interaction between Tyr448-OH and H26-Hydroxy chloroquine.

20

Figure 4: Docked complex of HCV RNA polymerase with primaquine. Residues indicate the hydrophobic

interactions between the ligand and RNA polymer

21


DISSOLUTION ENHANCEMENT OF PIOGLITAZONE HYDROCHLORIDE BY

SOLID DISPERSION TECHNIQUE

T. DIVYA 1, S. PRANAV 1*

1Maliba Pharmacy College, Bardoli-Mahuva road,Dist. Surat (Gujarat), India - 394 350

Address for correspondence:

Maliba Pharmacy College, Bardoli-Mahuva road,

Dist. Surat (Gujarat), India - 394 350

[email protected]

22

ABSTRACT

The present study focuses on the formulation of immediate release tablets containing solid dispersion of

Pioglitazone hydrochloride (PGH), a poorly soluble drug used in the treatment of type II diabetes. The solid

dispersions (SDs) were prepared by melting method using hydrophilic carriers namely polyethylene glycol

(PEG) 6000 and Poloxamer 188 individually in different ratios. The prepared SDs were evaluated for

solubility, drug content, Fourier transform infra red spectroscopy (FTIR), differential scanning calorimetry

(DSC), X-ray diffraction (XRD), and scanning electron microscopy (SEM). The solid dispersions were

formulated into immediate release tablets by wet granulation method. The tablets were evaluated for weight

variation, thickness, hardness, friability, assay, in vitro disintegration time and in vitro drug release. The

hypoglycaemic response of the optimized formulation was also studied in Albino wistar rats. The solubility of

PGZ was enhanced Results of SDs showed enhanced solubility of in 0.1 N HCl. FTIR, DSC, XRD and SEM

studies confirmed the formation of solid dispersion and conversion of drug from crystalline to amorphous. He

optimized batch of SDs showed better in vitro dissolution profile compared to the marketed tablets. In vivo

studies exhibited better hypoglycaemic activity than marketed tablets. The formulations were proved to be

stable after three months accelerated stability studies.

Keyword(s): Pioglitazone hydrochloride, solid dispersion, solubility, dissolution, PEG 6000 and Poloxamer

188

23

INTRODUCTION

Oral administration is the most popular route for systemic effects due to its ease of ingestion, avoidance of

pain, versatility and most importantly patient compliance. Oral bioavailability of a drug depends on its solubility

and/or dissolution rate. Majority of the new chemical entities (NCE) and several existing drug molecules possess poor

solubility, thereby limiting their potential uses and increasing the difficulty of formulating bioavailable drug

delivery systems.1 There are various techniques employed to improve the solubility of poorly soluble

drugs. e.g. particle size reduction: micronization, nanosuspension; modification of the crystal habit:

polymorphs, pseudopolymorphs; drug dispersion in carriers: eutectic mixtures, solid dispersions, solid

solutions; complexation: use of complexing agents; solubilization by surfactants: microemulsions, self

microemulsifying drug delivery systems.2

Solid dispersion (SD) is one of the widely used approaches to improve the solubility of poorly water soluble

drugs. A solid dispersion can be defined as a dispersion of one or more drugs in an inert hydrophilic carrier or

matrix in the solid state. Once the solid dispersion is exposed to aqueous media, the hydrophilic carrier

dissolves spontaneously, releasing the drug as very fine, colloidal particles. This leads to greatly enhanced

surface area, as a result of which, the dissolution rate and bioavailability of poorly water-soluble drugs are

expected to be high.

SDs can be prepared by number of methods like melting method, solvent evaporation, spray drying, fusion

method, etc.3 Among all the methods, melting method seems quite advantageous compared to other methods.

This method avoids the exposure to hazardous organic solvents. The carrier having low melting point can be

selected so that heat exposure to the drug can also avoided. The method is simple compared to others and has

industrial applicability.4

PGH is a thiazolidinedione class of antidiabetic agent that decreases insulin resistance in the periphery and in

the liver resulting in increased insulin-dependent glucose disposal and decreased hepatic glucose output. PGH

is a potent and highly selective agonist for peroxisome proliferator-activated receptor (PPAR)-gamma.

Activation of PPAR-gamma receptors regulates the transcription of insulin-responsive genes involved in the

control of glucose production, transport, and utilization. In this way, PGH enhances tissue sensitivity to

insulin. In general, rapid gastrointestinal (GI) absorption is required for oral hypoglycaemic drugs, in order to

prevent a sudden increase in blood glucose level after food intake in patient with diabetes mellitus. However,

PGH exhibits poor aqueous solubility and its absorption is dissolution rate limited. This eventually limits it

oral bioavaibility and therapeutic efficacy.5

24

The aim of the present investigation was to enhance the solubility of a poorly soluble drug, PGH, using solid

dispersion technique and thereby improve its dissolution characteristics. The solid dispersions were prepared

using hydrophilic carriers: PEG 6000, and Poloxamer 188. The solid dispersions were characterized by FTIR,

DSC and XRD techniques. The solid dispersions were compressed into immediate release tablets for oral

delivery. These tablets were evaluated for the quality control parameters and the dissolution rate. The

therapeutic efficacy of the prepared formulations was checked by in vivo in Albino wistar rats by measuring

blood glucose levels.

Materials and Methods

PGH was received as a gift sample from Torrent Research Centre, Ahmedabad, India. PEG 6000 and

Poloxamer 188 were purchased from Balaji drugs, Surat, India. Lactose monohydrate, starch, magnesium

stearate, talc and PVP K 30 were obtained from S.D Fine Chemicals Ltd., Mumbai, India. All other chemicals

and reagents used were of analytical grade.

Preformulation studies

The overall objective of preformulation testing is to generate information useful in developing the formulation

which is stable and acceptable. Physicochemical properties of the bulk drug like physical appearance,

solubility, bulk density, tapped density, compressibility index and drug-excipient compatibility were

studied.

Estimation of Pioglitazone hydrochloride

10 mg of PGH was dissolved in 10 ml of methanol and the volume was adjusted up to 100 ml with 0.1N

hydrochloric acid (100µg/ml)(Stock solution). The above solution was diluted with 0.1 N HCl to obtain the

series of dilutions containing 10, 15, 20, 25, 30, 35 and 40µg/ml of PGH. The absorbances of the above

solutions were measured on a Shimadzu UV-Visible Spectrophotometer at 269.5 nm. The calibration curve

was plotted and it was used for the estimation of PGH.

Preparation of solid dispersions

SDs of PGZ was prepared by melting method using PEG 6000 and Poloxamer 188 as hydrophilic carriers.

Three different weight ratios of drug: carrier of 1:1, 1:2, 1:3 and 1:4 were used (Table 1). A weighed amount

of carrier was taken in a porcelain dish and heated up to its melting point (60˚C for PEG 6000 and 55˚C for

Poloxamer 188) on a water bath. The drug was dispersed into molten carrier with continuous stirring. The

25

molten mass was removed from water bath and cooled on the ice bath till solidification. Then the solid mass

was pulverized and sifted through 40 # sieve.

Table I: Composition of Pioglitazone HCl solid dispersions

Batch Carrier Drug-carrier

ratio

B1

PEG 6000

1:1

B2 1:2

B3 1:3

B4 1:4

B5

Poloxamer 188

1:1

B6 1:2

B7 1:3

B8 1:4

Evaluation of SDs of PGH

Solubility studies

Solubility studies were performed as described by Higuchi and Connors. Excess amount of pure Pioglitazone

hydrochloride and the solid dispersions were added to 10 ml of 0.1 N HCl, taken in a stoppered conical flask

and the mixture was shaken (50 rpm) for 24 hrs on a rotary flask shaker. After, the equilibrium was attained;

the 2 ml aliquots were withdrawn at 1 hr intervals and filtered through the Millipore membrane filter (0.45 μ).

The filtrate was analysed spectrophotometerically at 269.5 nm. Shaking was continued until three consecutive

readings were the same. Results are indicated in table 2.

Fourier Transform infrared (FTIR) spectroscopy

FTIR spectroscopy was carried out for PGH, physical mixture (PM) of drug with PEG 6000 and Poloxamer

188 and SDs. Samples were scanned in the region of 4000-400 cm-1

using a Shimadzu FTIR spectrometer.

Differential scanning calorimetry (DSC) study

DSC analysis of PGH, PM, and SDs were carried out using a Shimadzu DSC 60 TSW 60 (Japan). Accurately

weighed samples were crimped in Aluminium pans and heated from 30 to 300 °C at a heating rate of 10

°C/min in N2 atmosphere. An empty sealed aluminium pan was used as reference.

26

X-ray diffraction (XRD) study

Crystallinity of PGH and SDs was determined using Analytical XRD (Bruker: D2 phaser). XRD patterns of

PGH and SDs were recorded at room temperature on Bruker’s D8 advance diffractometer (Bruker, Germany)

at 40 kV, 40 mA. Analysis was performed in a continuous mode with a step size of 0.02° and step time of 1

sec over an angular range of 10-80° 2θ. Obtained diffractograms were analyzed with DIFFRACplus EVA

(version 9.0) diffraction software.

Scanning electron microscopy

The surface morphology of PGZ and SDs were examined using a scanning electron microscope (JEOL Model

JSM - 6390LV, JEOL Ltd, Tokyo, Japan). The powders were fixed on a brass stub using double-sided

adhesive tape. The scanning electron microscope was operated at an acceleration voltage of 30 kV.

Percentage yield

Determination of % practical yield is useful to determine the efficiency of a preparation technique. The

practical yield is calculated by using following equation:

% Practical yield = (Weight of prepared solid dispersions × 100)/Theoretical weight

Estimation of drug content in solid dispersion

From each batch of SD, quantity equivalent to 15 mg of the Pioglitazone was taken into a 100 ml conical

flask containing 0.1 N HCl. The solution was diluted with 0.1 N HCl and filtered. The absorbance of this

solution was measured at 269.5 nm using UV spectrophotometer.

FORMULATION DEVELOPMENT

Preparation of immediate release tablets containing PGZ SDs

Wet granulation method was selected to formulate immediate release tablet containing SDs of PGZ. The

composition of different batches is shown in table II.

SDs, Lactose monohydrate and Starch were passed through 40 # mesh sieve prior to mixing. The ingredients

were mixed in a polythene bag for 5 minutes and granulated with binder solution of PVP K 30 in Isopropyl

alcohol. Dough mass was passed through 20 # sieve and granules were dried at 40ᵒC in Hot air oven. The

dried granules were passed through 30# mesh sieve. Talc and magnesium stearate were passed through 60#

sieve and then mixed with dried granules for 2 minutes. The lubricated blend was compressed into tablets

27

using a rotary tablet press (Rimek, Minipress I) equipped with 8 mm standard concave punches plain on both

sides.

.Table II: Formulation of immediate release tablets

Ingredients* Batch

F1 F2 F3 F4 F5 F6 F7 F8

Solid

dispersion** 33.068 45 66.136 82.67 33.068 45 66.136 82.67

Lactose

monohydrate 69.932 64 57.394 56.33 69.932 64 57.394 56.33

Starch 100 94 79.47 64 100 94 79.47 64

PVP K 30 2 2 2 2 2 2 2 2

Isopropyl

alcohol Q.S Q.S Q.S Q.S Q.S Q.S Q.S Q.S

Magnesium

stearate 3 3 3 3 3 3 3 3

Talc 2 2 2 2 2 2 2 2

Total weight 210 210 210 210 210 210 210 210

*All quantities are in mg

**Quantities of solid dispersion were taken equivalent to 15 mg of Pioglitazone

Evaluation of tablets

Weight variation test8

This is an important in-process quality control test. Corrections were made during the compression of tablets.

Any variation in the weight of tablet leads to either under medication or overdose. So, every tablet in each

batch should have uniformity of weight. Twenty tablets were weighed collectively and individually. Average

weight was calculated from the total weight of all the tablets. The individual weights were compared with the

average weight. The percentage weight variation is required to be within the permissible limits.

Thickness and diameter of tablet

Ten tablets were selected randomly and their thickness and diameter were measured using Vernier Callipers.

28

Hardness

Hardness (diametric crushing strength) is the force required to break a tablet across the diameter. The

hardness of a tablet is an indication of its strength. The tablet should be stable to mechanical stress during

handling and transportation. The hardness was tested using Monsanto hardness tester. The average of the five

determinations was determined and reported.

Friability8

Roche friabilator was used to measure the friability of the tablets. It was rotated at a rate of 25 rpm. Tablets

equivalent to weight 6.5 gms were weighed collectively and placed in the friabilator. In the friabilator, the

tablets were exposed to rolling, resulting from free fall of tablets within the chamber of the friabilator. After,

100 rotations (i.e. in 4 minutes), the tablets were taken out from the friabilator, dedusted and the intact tablets

were again weighed collectively.

In-vitro disintegration8

Disintegration time of the tablet was observed with the help of disintegration test apparatus (ED 2L

Electrolab) consisting of a basket rack assembly with 1000 ml beaker, a thermostatic arrangement for heating

the beaker between 35 ᵒC to 39 ᵒC, and a device for raising and lowering the basket in the immersion fluid at

a constant rate between 29 and 32 cycles per min. Six tablets were randomly selected and in vitro

disintegration was determined. using disintegration apparatus.

Assay

The prepared tablets were tested for their drug content. Ten tablets were finely powdered; quantities of the

powder equivalent to 30 mg of PGH were accurately weighed and transferred to a 100 ml of volumetric flask.

Volume was made up to 100 ml mark with 0.1 N HCl. Solution was then filtered to remove insoluble

excipients. The absorbance of the resulting solution was measured at 269.5 nm using a Shimadzu UV 1800

against 0.1 N HCl as blank. The linearity equation obtained from calibration curve was used for estimation of

PGH in the tablet formulation.

In vitro drug release8

In vitro dissolution studies of in-house tablets and marketed tablets (Piolem -15, Alembic Pharmaceuticals,

Vadodara) were carried out using IP dissolution apparatus 1, paddle (Electrolab Dissolution Tester TDT-08P)

at 75 rpm in 900 ml of 0.1 N HCl. Aliquots of 5 ml were withdrawn at specified time intervals of 5, 10, 15,

30, 45 and 60 min and replaced with fresh media. The samples were filtered, diluted with dissolution media

and analyzed spectrophotometerically at 269.5 nm. The dissolution studies were carried out in triplicate.

29

Similarity factor (f2 analysis):

Similarity factor between the reference and the test product was determined.

Stability study9

Accelerated stability studies of batches were carried out at 400C/75% RH for 3 months using stability

chamber Oswald JRIC 11B. Physical stability was analyzed by change in appearance and chemical stability

was analyzed by the change in the assay and % cumulative drug release after 3 month.

In vivo study

Animals

Male wistar rats of (250 - 300 g) were under standard conditions, maintained on a 12 h light/dark cycle and

had free access to food and water up to the time of experimentation. The animals were acclimatized to the

laboratory environment 1 h before the experiments. Animals were randomly distributed into groups of 6

animals each. All experiments were conducted during the light period (08.00-16.00 h). All the protocols were

approved by the Institutional Animal Ethical Committee (IAEC; Protocol No.: MPC/IAEC/10/2013.

Method 10

Fasted rats were divided into two groups with six rats each and were treated orally in the following

manner.

Group I (Reference): Treated with suspension of marketed PGZ tablets

Group II (Test): Treated with suspension of prepared PGZ tablets containing SDs

The rats were orally loaded with 1.25 g/kg of 25% (w/v) glucose solution. After glucose administration, the

rats were treated with PGZ equivalent to 6 mg/kg body weight from suspension of developed and marketed

immediate release tablets of PGZ. The suspension was administered orally to each group of rat using

stomach intubation. Blood samples were collected through tail vein immediately prior to commencement of

treatment and at 15, 30, 45, 60 and 75 minutes after glucose challenge. The blood glucose level was

determined using the glucose-measuring instrument Accu-Chek Sensor Comfort (Roche Diagnostics GmbH,

Germany).

30

RESULTS AND DISCUSSION

Preformulation studies

The drug appeared as white to off white crystalline powder, it is poorly water soluble (0.042 mg/ml), bulk

density and the tapped density of the pure drug (PGH) was found to be 0.491 g/ml and 0.748 g/ml

respectively. The compressibility index and the Hausner's ratio of the pure drug were found to be 34.36% and

1.52 respectively. These values suggested that poor flow characteristics ruling out the possibility of

employing direct compression for manufacturing of tablets. Wet granulation was employed as a method for

tablet manufacturing. No interaction between drug and excipients suggested the suitability of the selected

excipients.

Preparation of Solid Dispersions: The solid dispersions were prepared using PEG 6000 and Poloxamer 188

as the hydrophilic carriers by the melting method. All the prepared batches of solid dispersions were found to

be non-hygroscopic, free flowing powders. All the prepared solid dispersions exhibited uniformity of drug

content. The drug content of the solid dispersions was found in the range: 95% to102%. the % yield of all

formulations was found in the range of 93-99% (Table III).

Evaluations of SDs

Solubility studies

The aqueous solubility of Pioglitazone hydrochloride was found to be 0.042 mg/ml suggesting a strong need

to enhance the solubility of PGH. Our results are in agreement with the findings of other authors. Therefore,

the solid dispersion technique was employed for improving the solubility of PGH. Improvement of several

hundreds of folds in solubility of PGH was observed with the SDs (Table III). This could be attributed to the

formation of soluble complexes between the water soluble polymeric carriers and poorly water soluble

drugs. The values of solubilities suggested that as the amount of the carrier increased, the solubility was

found of PGH was found to increase.

31

Table III: Evaluation parameters of solid dispersions

Batch Polymer

Drug:

Polymer

ratio

Solubility

mg/ml

(n=3)

% Drug

content

(n=3)

%Yield

(n=3)

B1

PEG 6000

1:1 9.28±0.26 95.50±0.98% 93±1.2%

B2 1:2 15.23±0.09 96.28±1.04% 94±0.98%

B3 1:3 23.54±0.07 97.45±0.91% 96.66±0.12%

B4 1:4 29.12±0.15 98.95±1.1% 98.13±0.18%

B5

POLOXAMER

188

1:1 21.1±0.21 96.12±0.78% 94.87±0.15%

B6 1:2 30.2±0.06 95.87±1.02% 97.65±0.19%

B7 1:3 38.6±0.08 101.93±0.92% 98.63±0.13%

B8 1:4 39.2±0.31 98.45±0.71% 98.93±0.10%

Characterization of solid dispersions

Fourier transform infrared spectroscopy (FTIR)

The FTIR spectra of the PGH, PEG 6000, Poloxamer 188, PEG 6000, Poloxamer 188 and SDs (B4 and B7)

are shown in Figure I (a-e). FTIR peaks at 3084 cm-1

(aromatic C-H stretching), 2966 cm-1

(aliphatic C-H

stretching asymmetric), 1743 cm-1

(amide C = O stretching), 1610 cm-1

(C=C), 1460 cm-1

(ring C-N

stretching), 1243 cm-1

(C-S stretching), 1038 cm-1

(aliphatic C-O-C) and 850 cm-1

(para disubstituted

aromatic ring). The solid dispersion of PGH-PEG 6000 (Fig. I c) and PGH-Poloxamer 188 (Fig. I e) did not

show any new peaks and it is evident from spectra that the drug and polymer peaks are also present in the

solid dispersion spectra.

32

(a)

(b)

(c)

(d)

(e)

Figure I: FTIR spectra of (a) Pioglitazone HCl; (b) PEG 6000; (c) Solid dispersion (B4) (d) Poloxamer

188; (e) Solid dispersion (B7)

Differential Scanning Calorimetry

DSC thermograms obtained for PGH, PMs of drug with carrier and prepared solid dispersion formulation are

displayed in figure II (a-e). The DSC thermogram of PGZ (Figure II a) shows a single endothermic peak at

196.06°C due to the melting of drug. Figure II b &d shows DSC thermogram of physical mixture of PGH-PEG

33

6000 and PGH-Poloxamer 188 respectively. The endothermic peaks corresponding to the melting points of

PEG 6000 and PGH were observed. It also indicated absence of any interaction between drug and carriers. In

the thermogram of batch B4 & B7, disappearance of endothermic peak of PGH at 196.06° C, indicated the

formation of a solid dispersion system where the drug was dispersed in the amorphous state.

Figure II: DSC spectra of (a) Pioglitazone HCl; (b) PM of PGZ: PEG 6000; (C) SD of PGZ: PEG 6000

(B4); (d) PM of PGZ: Poloxamer 188; (e) SD of PGZ: Poloxamer 188 (B7)

(a)

(b)

(c)

(d)

(e)

34

X-Ray diffraction

The XRD spectra of PGZ, Poloxamer and Solid dispersions B4 and B7 are shown in Figure III (a-c). The X-

ray diffraction pattern of PGH (Fig III a) exhibited sharp, highly intense and less diffused peaks

indicating the crystalline nature of the drug. Numerous distinctive peaks were seen at 12.1°, 16.4°, 19.6°,

21.5°, 27.5° and 28.5°. The solid dispersions (Fig. III b & c) displayed less intense and highly diffused peaks

as compared to PGH. The XRD studies revealed the reduction in the crystalline nature of the drug in the solid

dispersion. The enhancement in the dissolution rate of the drug from the solid dispersion is ascribed to the

marked reduction in the crystallinity of the drug.

(a)

(b)

(c)

Figure III: XRD spectra of (a) Pioglitazone HCl; (b) Solid dispersion with PEG 6000 (B4); (c) Solid

dispersion with Poloxamer 188 (B7)

0

2000

4000

6000

0 10 20 30 40 50 60 70

0

2000

4000

6000

0 10 20 30 40 50 60 70

0

2000

4000

6000

8000

10000

0 10 20 30 40 50 60 70

35

Evaluations of immediate release tablets

Formulation studies

The composition of the prepared tablets is indicated in table. The tablet formulations (F1-F8) exhibited

uniform drug content. The drug content of all the tablet formulations was found in the range: 98% to 103%.

All the formulated tablets fulfilled the compendial limits of weight variation, hardness, friability and the

disintegration time (table 5).

Table V: Evaluation parameters of immediate release tablets

Batch

Weight

Variation

(mg)

(n=20)

Thickness

(mm)

(n=10)

Hardness

(kg/cm2)

(n=10)

Friability

(%)

In vitro

disintegration

time (min)

(n=10)

Assay (%)

(n=10)

F1 210.58 ±

3.26

3.75 ±

0.04 3- 3.5

0.12 ±

0.05 6-7

96.24 ±

1.34

F2 210.82 ±

4.18

3.79 ±

0.03 3.5- 4

0.08 ±

0.03 6-7

97.62 ±

2.12

F3 211.58 ±

3.12

3.73 ±

0.03 3.5- 4

0.06 ±

0.01 7-8

98.73 ±

1.93

F4 210.97 ±

2.96

3.78 ±

0.05 4- 4.5

0.05 ±

0.03 8-9

97.28 ±

1.05

F5 209.55 ±

4.07

3.72 ±

0.04 3- 3.5

0.09 ±

0.02 6-7

96.72 ±

2.65

F6 210.18 ±

3.83

3.73 ±

0.05 3.4- 4

0.07 ±

0.015 6-7

96.98 ±

1.45

F7 210.27 ±

3.26

3.72 ±

0.04 4- 4.5

0.04 ±

0.02 7-8

99.03 ±

1.21

F8 210.43 ±

4.14

3.70 ±

0.06 4- 4.5

0.03 ±

0.01 8-9

98.88 ±

1.34

In vitro drug release

The in-vitro drug release profile of pure drug, in-house and marketed tablets were performed in 0.1 N HCl for

60 minutes and the plot of the cumulative % drug released versus time (min) was plotted (Figure ). The

release of the pure drug was found to be only 23.9% (data not shown). This can be attributed to the poor

solubility of the drug. The results of the dissolution study of tablets indicated an improvement in the

dissolution rate of PGH. The rate of dissolution was found to increases as the concentration of the hydrophilic

polymeric carriers in the solid dispersions increased. The improvement in the dissolution rate is possibly

caused by several factors. Such factors are :the strong hydrophilic property of the polymeric carriers, which

36

improves the water penetration, wettability of the hydrophobic drug and forms a hydrophilic interfacial

layer between the drug particles and the dissolution medium; the optimal dispersion of the drug in the

hydrophilic polymeric carrier; the absence of crystals (amorphous dispersions) corresponds to lower energy

required to break the crystal lattice during the dissolution process; the molecular dispersion of drug on the

polymeric carrier improves the hydrophilic characteristics of the hydrophobic drug (the insoluble drug is

in an extremely fine state of subdivision). Also, the decrease in the particle size, increase in surface area and

the improvement in the dispersibility of the hydrophobic drug particles improves the dissolution rate of the

drug. The dissolution of marketed tablets of PGH was also compared with in-house tablets containing SDs.

The dissolution of PGH was improved with the solid dispersions as compared to marketed tablets.

Figure VI In vitro release profile of immediate release tablets (n=6)

0.0

20.0

40.0

60.0

80.0

100.0

120.0

0.0 20.0 40.0 60.0 80.0

% C

um

ula

tive

dru

g re

leas

e

Time (min)

F1

F2

F3

F4

F5

F6

F7

F8

0

20

40

60

80

100

120

0 20 40 60 80 100

% C

um

ula

tive

dru

g re

leas

e

Time (min)

Piolem 15

F4

F7

37

Figure VII Comparison of batch F4 and F7 with Piolem 15 (n=6)

In vivo studies

In-house tablets (batch F4 and F7) and marketed tablets (Piolem 15) were administered to the rats in a

suspension form. There was a significant difference observed between the in-house and marketed tablets in

terms of % reduction in blood glucose levels. The in-house batches provided greater % reduction in blood

glucose levels as compared to marketed tablets. This can be attributed to the faster dissolution rate of the in-

house tablets, which may in turn lead to improvement in bioavailability.

Above results (Figure 5.24) demonstrated that batches F4 and F7 exhibited similar hypoglycaemic activity.

The significant difference observed between prepared (F4 and F7) and marketed formulation.

Figure VII: Reduction in blood glucose level in rats (n=6)

Accelerated stability studies

The formulations F4 & F7 were stored at 40 ºC /75%RH for three months. After 3 months, the tablets were

evaluated for drug content, dissolution rate, weight variation, disintegration time, hardness, diameter and

thickness (Table and Figure ). All the parameters of the tablets were found within the limits. The results of

stability study indicated that prepared formulations were stable.

0

5

10

15

20

25

30

35

40

0 20 40 60 80

% R

ed

uct

ion

in b

loo

d g

luco

se le

vel

Time (min)

Piolem 15

Batch F4

Batch F7

38

Table VI Evaluation of tablets after exposure to accelerated stability conditions

Parameters

Batch F4 Batch F7

Initial After 3

months

Initial After 3

months

Physical change No change No change No change No change

Weight variation

(mg)

(n=20)

210.97 ± 2.96 210.56 ± 2.68 210.27 ± 3.26 209.87 ± 2.47

Thickness (mm)

(n=10)

3.78 ± 0.05 3.69 ± 0.07 3.72 ± 0.04 3.70 ± 0.06

Hardness

(kg/cm2) (n=10)

4-4.5 4-4.2 4-4.5 4-4.2

Disintegration

time (min) (n=10)

8-9 7-8 7-8 6-7

Assay (%)

(n=10)

97.28 ± 1.05 96.87 ± 1.92 99.03 ± 1.21 98.56 ± 1.32

Figure IX Comparative dissolution profile of batch F4 and F7 before and after stability studies

0

20

40

60

80

100

120

0 20 40 60

% C

DR

Time (min)

F4

Initial

After 3months

0

20

40

60

80

100

120

0 20 40 60

%C

DR

Time (min)

F7

Initial

After 3months

39

There were no significant changes observed in the physical parameters, assay and dissolution of the tablets

after 3 months storage at 400C/75% RH.

Conclusion:

The solid dispersions of PGH were successfully formulated by melting method using hydrophilic carriers like

PEG 6000 and Poloxamer 188 in various ratios. The characterization studies of SDs indicated the conversion

of PGH from crystalline to amorphous form. The prepared solid dispersions were further formulated into

immediate release tablets by wet granulation method. The in vitro drug release profiles of developed tablets

were compared to marketed tablets of PGH. The tablets containing SDs of PGH showed higher dissolution

rate than pure drug and marketed tablets (Piolem 15). The dissolution rate enhancement of the in-house

tablets based on solid dispersions was further substantiated by the in vivo studies in Albino wistar rats.

Pharmacodynamic evaluation suggested greater reduction in % blood glucose by the in-house tablets as

compared to marketed tablets. The in-house tablet formulations were found to be stable after accelerated

stability studies. Thus, the dissolution rate improvement of PGH by solid dispersions may be useful in

enhancing the oral bioavailability of the drug, which may in turn lead to reduction in dose; dose related side

effects; cost of the therapy and improved patient compliance.

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2. Gribbon P, Andreas S. High-throughput drug discovery: what can we expect from HTS? Drug

Discovery Today. 2005; 10(1):17-22.

3. Chiou WL, Riegelman S. Pharmaceutical applications of solid dispersion systems. J Pharm Sci. 1971;

60:1281-1302.

4. Patel T, Patel LD. Enhancement of dissolution of Fenofibrate by Solid dispersion Technique. Int. J.

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42

Volume 1, 2014 NTERNATIONAL JOURNAL OF PHARMACEUTICAL INNOVATIONS

An Attempt to isolate industrially important microorganism from diverse samples

collected from Semeling, Kedah, Malaysia

P. Vasanth Raj, S.A. Dhanaraj, Saw Pei Jie, Sim Poh Pei, Vanushya a/p Alagasan,

Tien Ja She, Loh Mon Ying, Tan Te Wei

Faculty of Pharmacy, AIMST University, Semeling, Bedong, Malaysia 08100

Corresponding Author:

Dr. P. Vasanth Raj

Faculty of Pharmacy

AIMST University

Semeling, Bedong, Malaysia 08100

Phone: +604- 429 8000, Fax: +604- 429 8009

Email: [email protected]

mailto:[email protected]

43

Abstract:

An attempt was made to isolate industrially important microorganisms from diverse samples collected

from Semeling, Kedah, Malaysia. Samples were aseptically collected from cafeteria soil, lake water,

rotten wood soil, mangrove swamp, plastic bag and sewage water. Primary screening experiments such as

antibiotic screening, cellulose utilization, oil utilization and plastic degrading properties were checked.

All the nine isolates were investigated for biochemical test like indole test, methyl red test, Macconkey

test, Celluose utilization test. Morphological testing such as gram staining, motility test, Mannitol semi

solid agar method were investigated. An attempt for optimization of cellulose medium was also made.

Total of nine isolates were obtained from primary screening. Eight samples were able to utilize cellulose,

one sample produced zone of inhibition (antibiotic screening). One sample was able to utilize plastic and

three samples showed positive result in oil medium. This Project has covered the major concerns about

the nine different isolates, their morphology, biochemical characteristics, capability of producing specific

enzymes, identification tests and uses. Further studies on cellulose optimization, batch level and scale up

have to be explored. On long term studies, this project will contribute in the field of environmental and

industrial biotechnology.

1. Introduction

Microorganism was known to have undiscovered benefits to the industrial sectors. However, the lack of

initiative to discover deeper on the earthly microbes has become a hindrance for the industries to

improve. Thus in this research project, we are motivated to learn more on their usage. There are various

applications of microbes in industry. We selected few important areas of research such as.

1.1 Plastic biodegradation:

Lack of degradability and the closing of landfill sites as well as growing water and land pollution

problems have led to concern about plastics. With excessive use of plastics and increasing pressure being

placed on capacities available for plastic waste disposal, the need for biodegradable plastics and

biodegradation of plastic wastes has assumed increasing importance in the last few years. (Aamer Ali

Shah et al 2008) In view of this, the biodegradation of plastics has been studied extensively for the past

three decades. Some types of plastic have shown to be biodegradable and their degradation mechanisms

have progressively become clearer. (Masayuki Shimao 2001) It is possible to say that if any

microorganism that able to grow in plastic medium has the ability to degrade the plastic.

1.2 Oil biodegradation:

Crude oil continues to be used as the principle source of energy and play an important role in the global

environment pollutant consideration. On the other hand, oil remains as a major source of energy in the

next several decades, because reliable alternative energy consumption has not yet been substituted.

(Trindade et al, 2005 Al-Saleh and Obuekwe.2005). The biodegradation of crude oil by microorganism is

one of the primary ways for eliminating crude oil from contaminated sites and appears to be the most

environmentally friendly method of removal oil pollutant. (Korda et al 1997, Kapley 1999,Del Arco and

44

Dabe Franca, 2001: Barathi and Vasudevan, 2001 ). It shows that any microorganism that able to grow

in oil-medium able to utilize oil for growth.

1.3 Cellulose utilisation:

The cellulose constitutes the major form of stocking glucose obtained through photosynthesis and in the

same time the major component of solar energy conversion to the biomass. Because of its highly ordered

structure, the cellulose is very hard to be degraded and that is why it is unusable and stocked in nature as

waste. The capacity to degrade the natural cellulose implies the synthesis of the entire cellulolytic system.

Cellulose has been used by man for centuries, however, its enormous potential as renewable source of

energy was recognized only after cellulose degrading enzymes “cellulases” has been identified (Bhat &

Bhat, 1997) These components act synergistically in the conversion of cellulose to glucose (Eveleigh

1987). It is possible to say that any microorganism that able to grow in cellulose medium has the ability

to degrade cellulose.

1.4 Antibiotic Screening:

The activity of antibiotic may be demonstrated under suitable condition by inhibitory effect of

microorganisms. A reduction of antimicrobial activity will also reveal the subtle changes not

demonstrable by chemical method. Accordingly, microbial and biology assays remain generally the

standard for resolving doubt with respect to possible loss of activity.

2. Methodology

2.1 Collection of Samples

The samples were aseptically collected from cafeteria soil, AIMST lake water, cafeteria water, rotten

wood soil, mangrove swamp, plastic bag, and sewage water.

2.2 Test for Plastic Degrading Microorganism

Plastics are man-made long chain polymeric molecules (Scott, 1999). More than half a century ago

synthetic polymers started to substitute natural materials in almost every area and nowadays plastic have

become an indispensable part of our life. The plastic we use today are made from inorganic and organic

raw materials, such as carbon, silicon, hydrogen, nitrogen, oxygen and chloride. The basic materials used

for making plastics are extracted from oil, coal and natural gas. (Syemour, 1989). In 1980’s, scientist

started to look if plastic could be designed to become susceptible to microbial attack, making them

degradable in a microbial active environment. Biodegradable plastic opened the way for new

considerations of waste management strategies since these materials are designed to degrade under

environmental conditions or in municipal and industrial biological waste treatment facilities (Augusta et

al, 1992; Witt et al., 1997).

45

2.3 Test for Oil Degrading Bacteria

Biosurfactant or surface-active compounds are a heterogeneous group of surface active molecules

produced by microorganisms, which either adhere to cell surface or are excreted extracellular in the

growth medium (Fietcher 1992; Zajic and Stiffens, 1994; Makker and Cameotra, 1998). These molecules

reduce surface tension and Critical Micelle Dilution (CMD) in both aqueous solutions and hydrocarbon

mixtures. These properties create microemulsion in which micelle formations occur where hydrocarbons

can solubilize in water or water in hydrocarbons (Banat, 1995). Several types of biosurfactant have been

isolated and characterized, including glycolipids, phospholipids, lipopeptides, natural lipids, fatty acids,

lipopolysacharides and other fully characterized. The majority of known biosurfactants are synthesized

by microorganisms grown on water immiscible hydrocarbons, but some have been produced on such

water-soluble substrates as glucose, glycerol and ethanol (ABU-Ruwaida et al., 1991). Chemically-

synthesized surfactants have been used in the oil industry to aid cleanup of oil spills, as well as to

enhance oil recovery from oil reservoirs. Biosurfactant have special advantage over their commercially

manufactured counterparts because of their lower toxicity, biodegradable nature and effectiveness at

extreme temperature, pH, salinity and ease of synthesis.

2.4 Test for Cellulose Degrading Bacteria

A key role in the decomposition and transformation of organic matter is attributed to bacteria, especially

of relatively complex and recalcitrant plant residues, such as polysaccharides as well as aromatic

compounds in different terrestrial or semi-terrestrial habitats. According to the complexities of habitats

and physiological capabilities of organisms, a range of approaches is available in order to assess the

composition, diversity and activities of soil microbial communities, including 16S rDNA analysis, assays

of substrate utilization profiles and assays of enzyme activities. As there is still a general need to cultivate

micro-organisms isolated from soil in order to study their role in soil biochemical processes, a

combination of both culture-dependent and culture-independent methods is suggested. Most studies of

aerobic cellulolytic soil bacteria involve techniques based on dilution plating, which is a quick,

inexpensive and reliable technique, though necessarily selective since an unknown part of the indigenous

microbial population is considered to be non-culturable. Moreover, a range of different cellulose

substrates is used to detect cellulolytic activity in vitro, such as soluble, substituted cellulose derivatives,

colloidal or amorphous cellulose, insoluble micro-crystalline cellulose and cellulose acetate films.

Several reports have shown incomplete cellulose systems of soil bacteria such as Bacillus species with

the main activity being that of endo-cellulase (carboxymethyl-cellulase).

2.5 Antimicrobial Test

There are two general methods employed, the cylinder plate assay and the turbidimetric assay. The first

depends upon the diffusion upon the antibiotic from a vertical cylinder through a solidified agar layer in a

petri dish or plate to extent such that growth of the added microorganism is prevented entirely in a

circular area or “zone” around the cylinder containing a solution of antibiotic. The turbidimetric method

depends upon the inhibition of growth of a microbial culture in a uniform solution of an antibiotic in a

46

fluid medium that is favourable to its rapid growth in the absence of the antibiotic. (Brian D. Gilbert, vol

30)

2.6 Optimization of Cellulose

Cellulose is the most abundant carbohydrate found in plants that provides structural integrity to their cell

walls. It is composed of a linear polymer of D-glucose linked by β-1,4 bonds which can be degraded by

the cellulase enzyme complex. Bacterial and fungal plant pathogens utilize cellulases to breach the plant

cell wall for nutrient acquisition and colonization. Spectrophotometric methods previously used to detect

cellulase activity were based on the reducing sugar method described by Miller (1959). The main

objective of these studies was to determine the presence of cellulase-producing enzyme in bacteria, and

the amount of cellulose utilized. This can be done by determining the glucose present in cellulose broth

with the help of UV Spectrometer.

3. Results and Discussion:

3.1 Collection of Samples

The samples were aseptically collected from cafeteria soil, AIMST lake water, cafeteria water, rotten

wood soil, mangrove swamp, plastic bag, and sewage water.

Table 1: Different types of isolates

ISOLATE

NUMBER

NAME Sample code

1 Cafeteria soil antimicrobial CSA 1

2 AIMST lake water near guardhouse ALWG2

3 Cafeteria water CW 3

4 Rotten wood soil RWS 4

5 Mangrove soil MS 5

6 Big plastic BP 6

7 Sewage water SW 7

8 Small plastic soil SPS(A) 8

9 Small plastic soil SPS(O) 9

3.2 Test for Plastic Degrading Microorganism

As the results show after a few days of incubation, growth was observed in the medium that even consists

of only basal salt nutrient medium, therefore it is possible to say that the microorganism able to utilize the

plastic bag for growth. Day 5 result shows that isolates number 2 able to grow in petri dish with only

plastic bag. While the petri dish number 5,6, and 7 shows grow of fungi. Therefore it is possible to say

that isolate number 2 had the ability to grow in plastic bag medium, it should be further studied to

confirm the properties of the microorganism. While for the medium that consists of toothbrush fiber as

the main source for bacteria growth shows no contamination. Day 5 results of the isolate number 3 also

showed growth.

47

It shows that growth of fungus in the coffee dusk for petri dish that consists of plastic bag that not treated

with UV light. Yet the actual result of bacterial degrading plastic unable to identify because the holes and

pores unable to identify under microscope, it should be studied under scanning electro-microscope

(SEM).

Figure 1: Plastic Degrading Activity

3.3 Test for Oil Degrading Bacteria

The samples from the various sources carried several oil degrading microorganisms. Utilization of crude

oil as a substrate by isolated microorganism is shown by increasing the number of cells. Observations of

both turbidity and viable cell count during the experimental period indicate that isolated microorganism

can utilize crude oil as carbon substrate. The growth dynamics of the organisms was determined by the

optical densities, and total viable count.This marked variation is obvious due to the difference in the

samples sites, environmental condition and nutrient availability and also difference in time of exposure

with the pollutants. The results were shown in Figures and reflect the ability of isolated microorganism to

degrade and utilize crude oil as a source of carbon and energy. This technique was used in several studies

to show the ability of bacteria for utilizing crude oil. (Bola, 2006; Emtiazi and Shakarami, 2004).

In the similar investigation by Rahman (2002) in England, the total viable count method used to confirm

the ability of different kind of bacteria for mineralizing crude oil. The comparison of the statistics

obtained in this study and other similar study shows that isolated microorganism can use crude oil and

degrading that better than many kind of bacterial that isolated previously.

The isolates 1, 2, 6 and 7 found to produce biosurfactants. As mentioned before, the work forms part of

an ongoing program. The figure obtained so far suggest that the inoculums would be potentially useful

for treating wastewaters containing oils of both plant and animal origins. Isolation of the responsible gene

and subsequent PCR amplification may prove useful to artificially produce environmentally friendly

surfactants. In future studies involving biosurfactants and bioremediation technologies, these four isolates

may be helpful. Further understanding of the metabolic process of this organism on the crude oil will

48

increase possibilities of developing models and strategies for removing crude oil pollutants from oil-

impacted environments.

Figure 2: Oil Degrading Activity

3.4 Test for Cellulose Degrading Bacteria

The samples from the various sources carried several cellulose degrading microorganisms. Utilization of

cellulose as a substrate by isolated microorganism is shown by increasing the number of cells.

Observations of both turbidity and viable cell count during the experimental period indicate that isolated

microorganism can utilize cellulose (isolates 1, 2, 3, 4, 5, 6, 8, 9). The growth dynamics of the organisms

was determined by the optical densities and total viable count.This marked variation is obvious due to the

difference in the samples sites, environmental condition and nutrient availability and also difference in

time of exposure with the pollutants. The results were shown in isolates 1, 2, 3, 4, 5, 6, 8, 9 and reflect

the ability of isolated microorganism to degrade and utilize cellulose. The cellulolytic activities of

bacteria are mostly screened using agar-plate techniques with a variety of substrates, such as

carboxymethyl-cellulose (CMC). This technique was used in several studies to show the ability of

bacteria for utilizing cellulose. (Bola, 2006; Emtiazi and Shakarami, 2004). The comparison of the

statistics obtained in this study and other similar study shows that isolated microorganism can use

cellulose and degrading that better than many kind of bacterial that isolated previously.

As mentioned before, the work forms part of an ongoing program. The figure obtained so far suggest that

the inoculums would be potentially useful. Isolation of the responsible gene and subsequent PCR

amplification may prove useful. More surveys of the degradation potentials of cellulolytic soil bacteria

are required to further validate this function. In future studies involving cellulose, these eight isolates

may be helpful.

http://www.bioline.org.br/showimage?se/photo/se08027f1.jpg




49

Figure 3: Cellulose Degrading Activity

3.5 Antimicrobial Test

We have used the agar cup method to evaluate the antibacterial activity. It is one of the non-automated in

vitro bacterial susceptibility tests. This classic method yields a zone of inhibition in mm result for the

amount of antimicrobial agents that is needed to inhibit growth of specific microorganisms. It is carried

out in petri plates. Agar cup plate method is simple and less sensitive. They are tremendously advantages

in cases of analyses a large number of sample. The basic principle involves diffusion of the vitamins

though the agar medium and can be adapted from the crude to more sophisticated and quantitative assay.

In cup plate method a number of cups a definite diameter are bored (with the use of glass cylinders or

specially design apparatus) in the agar plate made with the proper assay medium and seeded with the

vitamin requiring organisms. Aliquots of standards and isolates are pipette into the cups as quickly as

possible and the plates are incubated overnight at 37 °C. The vitamins in the cups diffuse out into the

surrounding agar producing a zone of growth proportional to the concentration. At the end of the

incubation period the growth zones are measured either with a ruler or needle-pointed vernier calipers.

Isolate 1 showed positive result in this test, means it produces zone of inhibition.

50

Figure 4: Antimicrobial Activity

3.6 Optimization of Cellulose

Based on graph (1), the highest absorbency of glucose is shown at the concentration of 250µg/ml, and the

lowest absorbency is at 62.5µg/ml. The aim of finding out standard glucose plot was for comparison

purposes. Due to some problem related to our instrumentation, the obtained graph was not shown in a

perfect straight line. Another concern was that the glucose concentration in cellulose broth was too high,

thus we were unable to compare with standard glucose plot. However, it was proven that cellulose was

utilized, and degraded into glucose.

The utilization of cellulose broth to detect the presence of glucose was successfully shown. The amount

of glucose produced was detected using UV Spectrometer, and was identified through the standard

glucose graph.

51

Graph 1: Standard plot of absorbance vs concentration of glucose

Graph 2: Absorbance of glucose in cellulose medium

0

0.02

0.04

0.06

0.08

0.1

0.12

1 2 3 4 5 6 7 8 9

Ab

sorb

ance

at

47

0n

m

Isolates Number

Absorbance at 470nm

Absocbance at 470nm

0

0.002

0.004

0.006

0.008

0.01

0.012

0 200 400 600 800 1000 1200

Ab

sorb

ance

at

47

0n

m

Concentration (µg/ml)

Standard glucose plot

absorbance

52

Table 2: Summary of results of various tests

Isolates Plastic Degradability Oil Degradability

Cellulase

Activity

PB PT

1 - - + +

2 + - + +

3 - + - +

4 - - - +

5 - - - +

6 - - + +

7 - - + -

8 - - - +

9 - - - +

Table 3: Summary of results of Plastic Degradation, Oil Degradation and Cellulase Activity

Isolates

Antimicrobial

Activity

Gram

Staining

Mac-

Conkey

Test

Motility

Test

Indole

Test

Methyl

Red Test

P.A. S.A.

1 + + + - + - +

2 - - + - + - -

3 + + - + + - -

4 - - - - + - +

5 - - + + + - -

6 - - + + + - -

7 - - + + + - -

8 - - - - + - +

9 - - + + + - +

53

4. Conclusion

This Project has covered the major concerns about the nine different isolates, their morphology,

biochemical characteristics, capability of producing specific enzymes, identification tests and uses. There

are a large number of tests which are used to determine their morphology, namely gram staining and

hanging drop method. These methods have brought great results as the morphology of all the isolates

were identified clearly. Moving on to the biochemical tests, we adopted methods like Mac-conkey test,

methyl red and Indole test which has given us a clearer picture on how the isolates react with different

chemicals. Followed by that was some identification test such as motility test, and antimicrobial test.

Next, utilizing different mediums like cellulose medium, plastic medium, and basal oil medium enabled

us to find out more details on their capability of degrading cellulose, plastics, and oil. This will also lead

us to find out their uses and benefits to our environment. We basically worked on Primary and Secondary

screening of samples. Due to time constrain, we leave the secondary screening experiments to be

explored further. With our output, increased understanding and contribution this project will contribute

in the field of environmental and industrial biotechnology for the society.

5. Acknowledgement:

We thank AIMST University, Malaysia for financial support and infrastructure provided to carry out this

study.

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Witt U, Muller RJ, Deckwer WD. Biodegradation behavior and material properties of aliphatic/aromatic

polyesters of commercial importance. J Environ Poly Degrade 1997; 15:81-9

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Short Communication

Studies on HIV Integrase inhibitory activity of Wrightia tinctoria (Roxb) R.Br

P. Selvam1, Kasthuraiah Maddali

2, Christophe marchand

2, Yves Pommier

2

1Nova College of Pharmaceutical Education and Research, Jupudi-515 721, Krishna DT.A.P

2Laboratory of Molecular Pharmacology, National Cancer Institute, Bethesda, Maryland 20892, USA

* For correspondance

Email: [email protected]

57

Wrightia tinctoria (WT) leaf extracts have been studied against inhibition of HIV-1 integrase activity. Chloroform,

ether, ethanol, methanol and aqueous extracts of Wrightia tinctoria were investigated for both 3’processing (3’P)

and strand transfer (ST) process of HIV-1 integrase enzymatic activity. All extracts exhibited significant inhibitory

activity against HIV-1 integrase enzyme (3’P 1.9-12 µg/ml and ST: 2.2-12 µg/ml). The aqueous extract (AWT)

displayed potent inhibitory activity against both step of HIV Integrase enzymatic activity (3’P IC50:1.9±0.451

µg/ml and ST IC50: 1.4 ± 0.3 µg/ml).

Keywords: Wrightia tinctoria, HIV Integrase activity, Aqueous extract

INTRODUCTION

Acquired immunodeficiency syndrome (AIDS) is a life threatening and debilitating disease state caused by

retrovirus HIV. Three different classes of chemotherapeutic agents are generally combined to block the replication

of human immunodeficiency virus type 1 (HIV-1) responsible for AIDS and to prevent the occurrence of

resistance: reverse transcriptase inhibitors (RTI), protease inhibitors (PRI), and fusion inhibitors. This widespread

triple combination therapy is referred to as HAART [highly active antiretroviral therapy] ( Richman DD. 2001).

HAART effectively inhibits HIV replication to such an extent that the virus becomes undetectable in the blood.

However, it fails to eradicate viruses that are integrated in the host genome or that persist in cellular and

anatomical “reservoirs”. In addition, prolonged drug exposure led to HIV drug resistance, thus reducing patients’

therapeutically available options (Cohen J. 2002). Based on above fact and the toxicity profile of available of

antiretroviral agents have fueled the discovery of drugs against additional molecular targets. Among them, HIV

integrase (IN), which has no cellular counterpart and crucial enzyme HIV replication, has been intensely studied

over the past 15 years (Fesen et al., 1993; Pommier, et al., 2005; Dayam et al., 2008).

Integration occurs via a sequence of reactions, which start with the IN-mediated cleavage of terminal dinucleotide

from the 3'- end of the viral cDNA (termed “3'-processing”, 3'-P) shortly after reverse transcriptions in the

cytoplasm. Following transfer of the resulting processed viral cDNA into the nucleus, IN catalyzes the insertion of

both ends into target cellular host DNA. That second reaction is referred as “strand transfer” (ST). In the past 15

years, a range of natural and synthetic compounds have been identified as inhibitors of recombinant IN enzyme in

58

biochemical assays. Interestingly, polyhydroxylated aromatics and diketo compounds were among the first

inhibitors identified. (Fesen et al., 1993 & 1994). HIV integrase has recently been fully validated as a therapeutic

target with the first FDA approved IN inhibitor Raltegravir (Evering T. H, Markowitz M.2007)

Wrightia tinctoria (Roxb) R.Br is an important medicinal plant used in the Indian system of medicine for the

treatment of variety of diseases (Bigoniya et al., 2008) and it reported to possess analgesic (Reddy et al., 2002)

antifertility ( Keshri et al., 2008), cytotoxicity (Kawamoto et al., 2003), hemostasis (Rajesh et al., 2003), anti-ulcer

activity (Bigoniya et al., 2006) and Hepatoprotective (Selvam et al., 2011). Indole derivatives such as isatin,

indirubin, indigotin and tryphanthrin are the principle active constituents of wrightia tinctoria (Muruganandhan

et al., 2000) which may responsible for board spectrum biological activity. Review of literature revealed that

antiviral activity of Wrightia tinctoria against the replication of HIV is relatively less explored. The present study

is designed to find the inhibitory activity of different extracts of Wrightia tinctoria (WT) against the HIV-1

integrase enzymatic activity.

Extraction: Leaf parts of Wrightia tinctoria (Apocynaceae) were collected in and around Tenkasi, Tamilnadu,

India and were dried in shade, subjected to hot continuous percolation using ether, chloroform, methanol, ethanol

and water. The ether (EWT), chloroform (CWT), methanol (MWT) and ethanol (ETWT) and aqueous extracts

(AWT) of Wrightia tinctoria were concentrated by distillation and dried under vaccum.

Anti-HIV Integrase assays.

Integrase Catalytic Assay (Marchand et al., 2001): Integrase reactions were carried out by mixing 20 nM DNA

with 400 nM IN in a buffer containing 50 mM MOPS, pH 7.2, 7.5 mM MgCl2, 14 mM 2-mercaptoethanol, and the

drug of interest or 10% DMSO. Reactions were incubated at 37°C for 1 h and quenched by addition of an equal

volume of gel loading dye (formamide containing 1% SDS, 0.25% bromophenol blue, and xylene cyanol).

Reaction products were separated in 20% polyacrylamide denaturing sequencing gels. Dried gels were visualized

using the Typhoon 8600 (GE Healthcare, Piscataway, NJ). Densitometry analyses were performed using

ImageQuant software from GE Healthcare

59

RESULTS AND DISCUSSION

The ether (EWT), chloroform (CWT), methanol (MWT) and ethanol (ETWT) and aqueous(AWT) leaf extracts

of Wrightia tinctoria have been evaluated for HIV Integrase enzyme inhibitory activity (Table 1; Fig 1.). All the

extracts were investigated for both 3’ processing (3’P) and strands transfer reaction (ST) of HIV integrase

enzymatic activity (Fig. 1). All extracts exhibited inhibitory activity against HIV-1 integrase enzyme (3’P 1.9-12

µg/ml and ST: 2.2-12 µg/ml). The aqueous extract (AWT) displayed significant activity against both step of HIV

In enzymatic activity (3’P IC50:1.9 ± 0.451 µg/ml and ST IC50:1.4 ± 0.3 µg/ml). Indirubin-3'-monoxime (Heredia et

al.,2005) a derivative of a Chinese antileukemia medicine isatis tinctoria inhibits HIV-1 replication and another

isatin containing medicinal plant Isatis indigotica also reported to posses anti HIV activity (Liu et al., 2007).

Anti-HIV and Cytotoxicity of Wrightia tomentosa also demonstrated by our preliminary studies (Selvam et al.,

2012). To our best knowledge, this is the first time to report the anti-HIV-1 Integrase activities of these Indian

plants locally known as wrightia tinctoria. The isolation of active compounds possessing anti-HIV-1 activities

from wrightia tinctoria are now in progress.

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of Wrightia tinctoria bark in experimentally induced acute gastric ulcers on rat; Nig. J Nat. Pro. Med., 10, 36-

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Hydro-Alcoholic Extract. Asian J. Exp. Sci.,22: 235-244

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Dayam R, Gundla R, Al-Mawsawi LQ, Neamati, N. 2008. HIV-1 integrase inhibitors: 2005-2006 update.

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Evering T. H, Markowitz M.2007. Raltegravir (MK-0518): an integrase inhibitor for the treatment of HIV-1.

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Fesen MR, Pommier Y, Leteurtre F, Hiroguchi S, Yung J, Kohn KW. 1994. Inhibition of HIV-1 integrase

by flavones, caffeic acid phenethyl ester (CAPE) and related compounds. Biochem. Pharmacol. 48:595–608.

Heredia A., Davis C., Bamba D., Le N., Gwarzo, MY, Sadowska M., Gallo RC, and Redfield RR. Indirubin-

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Wrightia tinctoria in Sprague-Dawley rats: a preliminary study. Contraception 78: 266-70

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Table1: Anti-HIV integrase activity of wrightia tinctoria

Extracts IC50 3’P, g/ml IC50 ST, g/ml

CWT 13 ± 3 12 ± 2

MWT 8.7 ± 1.2 4.6 ± 0.4

AWT 1.9 ± 0.45 1.4 ± 0.3

ETWT 2.3 ± 0.4 2.2 ± 0.5

EWT 5.3 ± 0.6 4.6 ± 1

The results are IC50 ±S.D, n = 4 for HIV-1 IN inhibitory activity

63

Fig 1. HIV Integrase Inhibitory activity of wrightia tinctoria

64


Drug Discovery and Development-Current status and Future Focus

N. Somashekara., P. Jagadeesh., C.N. Ramchand

Laila Pharmaceuticals Pvt.Ltd., Vijayawada, A.P

65

Short Note

Drug is “a substance intended for use in the diagnosis, cure, mitigation, treatment, or prevention of

disease or as a component of a medication” (The Merriam-Webster online dictionary). Drugs generally

affect the biological processes and must be present in the body at an adequate concentration for it to act at

the specific site, where it can exert its effect. Drug must be easily absorbed, metabolized, and eliminated

from the body without causing any injury. The process of developing a new drug consists of two main

stages called discovery and development. The objective of drug discovery is to find synthetic compounds

or natural products that will cure or ameliorate diseases (Mart et al., (2006). The discovery stage is key

step which includes target selection, lead identification, while the development stage includes Preclinical

studies, clinical trials, manufacturing and marketing of the developed drug and managing the life cycle of

the drug. Target may be a biomolecule such as gene or protein, which can potentially be affected by a

drug molecule. Once the target is identified, a promising lead molecule can be developed which in turn

can be used therapeutically. The lead is optimized by further analyzing and screening through in vitro , ex

vivo and in vivo techniques to get the drug molecule, which will further go for preclinical development to

access pharmaco kinetics, pharmaco dynamics and toxicity. Once the preclinical studies are over, the

drug will go through phase-I, phase-II and Phase III development program. After starting with thousands

of compounds, only a few drug candidates continue into the development stage for clinical trials and

manufacturing (Lifenget al., 2008)

The process of discovering drug by serendipitous approach was improved to a more rational approach.

Rational drug design is a method for developing new pharmaceuticals that typically involves the

elucidation of fundamental physiological mechanisms. Rational drug design is aimed at the targeted

discovery and optimization of new drugs based on detailed structural and chemical information of the

protein. Unlike the earlier approaches to drug discovery, by trial-and-error testing of chemical substances

http://www.bambooweb.com/articles/d/r/Drug_discovery.html

http://www.bambooweb.com/articles/t/r/Trial-and-error.html

66

on animals, and matching the apparent effects to treatments, rational drug design begins with a

knowledge of specific chemical responses in the body, and tailoring combinations of these to fit a

treatment profile. It thus combines the quest for a scientific understanding of natural phenomena with the

design of useful technology and hence integrates epistemic and practical aims of research and

development. The rational method usually involves a theoretical understanding of which protein is

targeted by the drug, how the drug acts on it, and which mechanisms lead to the desired therapeutic

effects (Matthias Adam, 2005). Another different approach to drug development was to use the body’s

own biological molecules as disease treatments. Antibodies, cytokines, and other immune system

substances can be produced in the laboratory for use in disease treatment.

There are different technologies which enabled successful drug discovery process. The technologies such

as High throughput screening, Virtual screening, combinatorial chemistry. Although there was little

success, there is a steep fall the in identifying and developing new chemical entities (NCE’s). The recent

trend on development of NCE’s is declining year by year ending with only few NCE’s and the future

graph of NCE’s may be worse.

The future focus in the drug discovery and development are mainly concentrated on Biologics,

Biosimilars, Analogs , Cell therapy, Antibodies. Also reverse pharmacology approach to drug discovery

is taking leading path. The resources required for new drug discovery are enormous. The time to bring a

drug to market averaging 14 -15 years and at the same time the average cost to bring a drug to market is

more than $1billion (Dimasi., 2001a). Pharmaceuticals companies are trying hard to reduce the time to

market the drugs. Result of once such effort is contract research, which will enable pharmaceutical

companies to outsource the part of drug discovery and development to contract research organizations.

Contract research has benefitted pharmaceutical companies to have accesses to skills sets of contract

research organization and also achieve quicker time to market.

http://www.bambooweb.com/articles/a/n/Animal.html

67

References:

1. Marta Artal-Sanz, Liesbeth de Jong and NektariosTavernarakis ,(2006), Caenorhabditiselegans: A

versatile platform for drug discovery, Biotechnol. J. 2006, 1, 1405–1418

2. Lifeng Kang, Bong GeunChung,Robert Langer and Ali Khademhosseini, (2008) Microfluidics for

drug discovery and development: From target selection to product lifecycle management, Drug

Discovery Today _ Volume 13, Numbers 1/2 January 2008

3. Matthias Adam, Studies in History and Philosophy of Biological and Biomedical Sciences 36,

(2005)

4. Dimasi, J.A. (2001a). Risks in new drug development is approval success rates for investigational

drugs. Clin.Pharmacol.Ther. 69, 297-307 .

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Contents

Invited article:



M. ChandraMohan, P. Selvam , J. Pranitha, N. Saravanan Prabhu, M. Suresh Kumar Pg no: 5-20

Research article:

DISSOLUTION ENHANCEMENT OF PIOGLITAZONE HYDROCHLORIDE BY SOLID

DISPERSION TECHNIQUE

T. Divya , S. Pranav ----- Pg no : 21-41

AN ATTEMPT TO ISOLATE INDUSTRIALLY IMPORTANT MICROORGANISM FROM

DIVERSE SAMPLES COLLECTED FROM SEMELING, KEDAH, MALAYSIA

P. Vasanth Raj, S.A. Dhanaraj, Saw Pei Jie, Sim Poh Pei, Vanushya a/p Alagasan, Tien Ja She,

Loh Mon Ying, Tan Te Wei Pg no : 42-55

Short communications :

STUDIES OF HIV INTEGRASE INHIBITORY ACTIVITY OF WRIGHTIA TINCTORIA

Periyasamy Selvam, Kasthuraiah Maddali , Christophe marchand

, Yves Pommier Pg no : 55-63

DRUG DISCOVERY AND DEVELOPMENT-CURRENT STATUS AND FUTURE FOCUS

N. Somashekara., P. Jagadeesh., C.N. Ramchand Pg no : 64-67

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