Part I: Poly (ethylene sebacate) Nanoparticles of Anti...

Part I: Poly (ethylene sebacate) Nanoparticles of Anti-

Tubercular Drugs

1. Introduction

Poly (ethylene sebacate) Nanoparticles of Anti-Tubercular Drugs

1 Particulate Carriers as Drug Delivery Systems for Anti-Tubercular and Anti-Cancer Agents

1. INTRODUCTION:

1.1. TUBERCULOSIS

Tuberculosis (TB) is a chronic communicable disease caused by the bacterium

(Mycobacterium tuberculosis) and usually occurs in the lungs (pulmonary

tuberculosis), but it can also occur in other organs (extrapulmonary tuberculosis).

Pulmonary tuberculosis is the most common form of tuberculosis; involving alveolar

macrophages infected with Mycobacterium tuberculosis (Deol et al., 1997). TB

causes substantial mortality and morbidity. Most infections in humans result in an

asymptomatic, latent infection, and about one in ten latent infections eventually

progresses to active disease, which, if left untreated, kills more than 50% of its

victims. A third of the world's population are thought to be infected with M.

tuberculosis (Jasmer et al., 2002), and new infections occur at a rate of about one per

second. The proportion of people who become sick with tuberculosis each year is

stable or falling worldwide but, because of population growth, the absolute number of

new cases is still increasing. Each year, it leads to approximately 14.6 million chronic

active cases, 8.9 million new cases and 1.6 million deaths; new infections are

occurring at a rate of one per second according to the latest WHO report. Drugs or

vaccines have not been developed to rapidly prevent transmission to uninfected

individuals or to treat apparently healthy, recently infected individuals, in part

because of the requirement for early diagnosis of infection. These alarming figures

have put TB in a category, with AIDS and Malaria, of diseases that urgently require

attention to improve global public health.

a) Pathogenosis: Tuberculosis is spread by airborne droplet nuclei, which are

particles of 1–5 μm in diameter that contain Mycobacterium tuberculosis. Because of

their small size, the particles can remain airborne for minutes to hours after

expectoration by people with pulmonary or laryngeal tuberculosis during coughing,

sneezing, singing, or talking (Wells et al., 1934, Louden et al., 1966). The infectious

droplet nuclei are inhaled and lodge in the alveoli in the distal airways. M tuberculosis

is then taken up by alveolar macrophages, initiating a cascade of events that result in

either successful containment of the infection or progression to active disease

(primary progressive tuberculosis). Bacteria after pecked up by the alveolar

macrophages evolve to evade most of the host-defense mechanisms enabling

intracellular survival and also replicate within phagosomes. These phagosomes are



subjected to degradation by lysosomal enzymes (intra-lysosomal acidic hydrolases)

upon phagolysosomal fusion. However, in tuberculosis, phagosomes are not

digested by the lysosomes because their unification with lysosomes for destructive

action is inhibited by the mycobacterium. This is because of the generation of

ammonia by mycobacterium, which results in an alkaline pH, and, further, the

presence of mycobacterial sulphatides and derivatives of multiacylated trehalose 2-

sulphates have the ability to inhibit phagolysosomal fusion (Sturgill-Koszycki et al.,

1994). When the bacilli are not digested, there is a development of cellular-mediated

immunity by the activation of T cells and monocytes. This results in chemotaxis of

lymphocytes and activated macrophages, which result in lesions that progress into

granuloma formation. This granuloma is a circumscribed collection of inflammatory

mass of macrophages, which are further surrounded by lymphocytes and fibrous

tissue (fibroblasts). At initial stages of infection, granuloma mass is hard in nature, but

after 10 to 12 days, bacilli inside the granuloma is further attacked by activated T cells

(CD 4+ helper T cells via interferon [IFN-γ] and CD 8+ suppressor T cells directly)

and the toxicity of Mycobacterium tuberculosis (MTB) towards macrophages leads to

degradation of granular material at the core of granuloma. This forms a cheesy-like

liquefied material called caseum, which is soft in nature because of presence of

lipids, large concentration of nuclear debris, and dead macrophages and eosinophils

(Thontesh et al., 2007). Exponential growth of MTB is inhibited but bacilli may

remain dormant for many years within the granuloma. It replicates exponentially

during favorable conditions. Thus, MTB is protected from lysosomal digestion and

survives and proliferates in macrophages for a long period of time (Thontesh et al.,

2007).

b) Current chemotherapy of tuberculosis: Since the control measures for TB such as

Bacillus Calmette-Guérin (BCG) vaccination and chemoprophylaxis appear to be

unsatisfactory, treatment with anti-tubercular (anti-TB) drugs becomes the only option

available. The goals of the treatment are to ensure cure without relapse, to prevent

death, to impede transmission, and to prevent the emergence of drug resistance. Long

term treatment with multiple drug therapy is employed to delay or prevent emergence

of the organism resistant to particular agent during treatment.

As suggested by WHO DOTs (Directly Observed Therapy short-course) programme

the first-line therapy consists with Rifampicin (RIF), Isoniazid (INH), Pyrazinamide



(PZA), Ethambutol (ETH) and Streptomycin because of their efficacy and acceptable

degree of toxicity.

An initially intensive phase consist of three-four drugs (RIF, INH, PZA and

ETH) daily administered concurrently for 2 monts to reduce the rapidly

dividing bacilli load and

The continuation phase consists of two drugs (RIF and INH) either daily or

three times a week for 4-6 months to sterilize lesion containing fewer and

slow-growing bacilli

Intensive Phase-2 Months Under 50 kg Over 50 kg

AKT-3: (ETH-800mg, INH-300mg and RIF-

450mg)

AKT-4: (ETH-800mg, INH-300mg, PZA-

750mg and RIF-450mg)

3-4 tablets 4-5 tablets

Continuation Phase-4-6 Months Under 50 kg Over 50 kg

AKT-2: (INH-300mg and RIF-450mg) 3 tablets 2 tablets

Rifampicin is bactericidal for both intracellular and extracellular mycobacteria so it is

used in combination with Isoniazid as primary agents in a combination therapy and

acts against the metabolically dynamic mycobacteria that multiply perpetually and

rapidly, and also against to the quasi-dormant bacilli.

The second-line class of drug includes aminoglycoside antibiotics, cycloserin,

ethionamide and fluoroquinolones. Because they are no more effective and toxicity is

serious they considered as second-line drugs and are employed only if patient is not

responding to the first-line therapy and/or infected with drug resistant strains of

mycobacteria.

Current tuberculosis therapy administered by conventional oral route and consists of

minimum of six to nine months of treatment regimen and this prolongation of therapy

may lead to life threatening side effects, decreased bioavailability at the target sites

and most importantly non-compliance by the patient thus leading to multi-drug

resistance (MDR) lately extremely-drug resistant (XDR) strains of TB is of

particular concern. The MDR or XDR strains M. avium–M. intracellulare (MAC)

complex is the main cause of complications in immunodepressed patients(Mehta et

al., 1993, Gómez-Flores et al., 1996). MAC is resistant to most classic antitubercular



drugs owing to the low permeability of cells or rapid degradation, such that new drugs

have been developed, although in practice they do not provide very good results

(Bermudez et al., 1994, Leitzke et al., 1998). The use of delivery systems facilitates

the selective shuttling of antitubercular drugs to the site of infection and such systems

provide slow and sustained drug release, which allows administration over longer

intervals of time. However, once in the blood, most of the antibiotic administered is

captured by the liver and spleen cells but not so much by alveolar macrophages,

limiting efficiency in lung infections (Bermudez et al., 1994). Novel drugs or delivery

systems that are below a toxic threshold at the effective doses and act on the bacteria

by a different absorption mechanism are urgently needed to replace or supplement

drugs that have been lost as therapies to drug resistance. Treatment regimens that are

short and allow less frequent intake of drugs by patients would greatly benefit

compliance.

1.2. COLLOIDAL DRUG DELIVERY SYSTEMS FOR TUBERCULOSIS

THERAPY

Colloidal drug delivery systems with the aim of targeted drug delivery and controlled

release to better manage drug pharmacokinetics, pharmacodynamics, non-specific

toxicity, immunogenicity and biorecognition systems have been proven an important

cum effective strategy for complete eradication of mycobacterium tuberculosis from

intra cellular sites, in animal models (Labana et al., 2002, Pandey et al., 2003, 2004

and 2005, Vyas et al., 2004, Mullaicharam et al., 2004, Chono et al., 2007 and 2008,

Ohashi et al., 2009, Hirota et al., 2010, Saraogi et al., 2010 and 2011). The following

are among the important technological advantages of colloidal drug carriers: high

stability (i.e., long shelf life); high carrier capacity (i.e., many drug molecules can be

incorporated in the particle matrix); feasibility of incorporation of both hydrophilic

and hydrophobic substances; and feasibility of variable routes of administration,

including oral administration, intravenous and inhalation. These carriers can also be

designed to enable controlled (sustained) drug release from the matrix.

Colloidal carriers such as liposomes, niosomes, microparticles, polymeric

nanoparticles, solid lipid nanoparticles, nanosuspension, nanoemulsion, micellar

nanocarriers and porous particles explored for the delivery of antitubercular drugs

after oral, intravenous or inhalation administration as summarized in Table 1.1.



Table 1.1: Colloidal drug delivery systems containing anti-TB drugs Carrier Polymer/lipid Drugs Route of

administration/animal model

Study Ref.

Liposomes Lecithin Streptomycin Intravenous (mouse)

Significant decrease of the Mycobacterium count in the spleen, but not in the lungs. Prolonged mouse survival and reduced acute drug toxicity

Vladimirsky et al., 1982

ePC Gentamycin Intravenous (mouse)

Significantly reduced the bacterial count in spleen and liver. A dose-related reduction of the bacterial load, without sterilization was found.

Klemens et al., 1990

PC and phosphatidylglycerol

Sparfloxacin In-vitro cell culture

Reduction of the growth index to 30%. Duzgunes et al., 1996

PC, dicetylphosphate (O-SAP, Monosialogangliosides/DSPE-PEG 2000 ligand)

INH, RIF Intravenous (mice)

Significant decrease in the hepatoxic activity of the anti-TB agents. Within 30 min the accumulation of nanocarriers in the lungs was 31% with PEGylated systems.

Deol et al., 1997

PC, CH, dicetylphosphate (O-SAP, Monosialogangliosides/DSPE-PEG 2000 ligand)

INH, RIF Intravenous (mice)

A significant increase in the anti-TB activity was found. Deol et al., 1997 a

Lecithin Amikacin Intravenous (mice)

Significantly reduced bacterial replication in infected tissues and extended the survival time of infected mice.

Leitzke et al., 1998

PC Clofazimine Intravenous (mice)

Significant reduction of the in vitro and in vivo toxicity of the drug. The anti-TB activity in both acute and chronic models was enhanced.

Mehta et al., 1999, Adams et al., 1999

ePC, DSPE, PEG INH, RIF Intravenous (mice)

Reduced the mycobacterial load significantly in lungs, liver and spleen of infected mice.

Labana et al., 2002

DSPC, DPPC, HPC Capreomycin In vitro Demonstrated their suitability for use in inhaled formulations Giovagnoli et al., 2003

ePC (O-SAP and MBSA ligand)

RIF Inhalation (aerosol)

Percent viability of M. smegmatis inside macrophages in vitro and in vivo was significantly decrease with ligand-anchored liposomal aerosols.

Vyas et al., 2004



DSPC, DPPC, HPC Capreomycin Both freeze–thawing technique and a response surface methodology were used to improve the drug content.

Ricci et al., 2006

HSPC, DOPE (4-Aminophenyl-a-D-mannopyranoside ligand)

Inhalation (rat)

A pronounced increase in the uptake was observed with the mannosylated-nanocarriers in vitro and in vivo.

Chono et al., 2007

DPPC PZA Intravenous (mice)

High therapeutic efficacy of PZA liposomes was observed in the treatment of M. tuberculosis in mice.

El-Ridy et al., 2007

HSPC, DOPE (4-Aminophenyl-a-D-mannopyranoside ligand)

Ciprofloxacin Intravenous (rat)

High targeting efficiency towards rat AMs Manno-sylated liposomes exhibited potent antibacterial effects against many bacteria.

Chono et al., 2008

DPPC, DPPG RFB Intravenous (mice)

Lower bacterial loads in the spleen, liver and lung. Gaspar et al., 2008

DSPC (Man-C4-Chol ligand)

intratracheal (rat)

Higher uptake of mannosylated-nanocarriers, preferably (15–17-fold) by alveolar macro-phages over alveolar epithelial type II cells.

Wijagkanalan et al., 2009

Niosomes Span 85 RIF Intravenous (rat)

Up to 65% of the drug was localized in the lungs by adjusting the size of the carrier.

Jain et al., 1995

RIF Intravenous and intathoracic (rat)

After i.v. administration, niosomes preferentially accumulated in the lung, liver and kidney. After intathoracic (i.t.) administration, the lung and/or plasma ratios for niosomes and free drug represented a 145-fold increase in the accumulation capacity of RIF-loaded niosomes in the lungs as compared to the free drug.

Mullaicharam et al., 2004

Span 20, 40, 60, 80, 85 RIF Intravenous and intraperitoneal (rat)

Niosomal formulations attained substantially higher RIF concentrations in thoracic lymph nodes.

Jain et al., 2006

Nanoparticles and microparticles

PIBCA Ciprofloxacin Intravenous infusion (rabbit)

NPs led to increased AUC, t1/2 and Vd. NPs were more effective against M. avium complex in human macrophages.

Fawaz et al., 1998

PBCA, PIBCA RIF, INH, Streptomycin

In vitro Encapsulated INH, Streptomycin and RIF showed 4–8-, 7-and 22–25-fold increases in the intracellular concentration.

Anisimova et al., 2000

PLG RIF, INH, PZA

Inhalation-Nebulization (guinea pig)

Therapeutic drug concentrations in plasma were detected until day 6 for RIF and day 8 for both INH and PZA. No tubercle bacilli could be detected in the lungs after five doses of treatment (dosing at 10days interval)

O’Hara et al., 2000



PLG INH Intravenous (mice)

Porous and non-porous microparticles released INH in plasma for up to 2 days whereas hardened PLG microparticles up to 7 weeks. Concentrations of INH obtained were higher than the MIC of INH.

Dutt et al., 2001

PLG INH, RIF Intravenous (mice)

Single dose shows sustained release of INH and RIF for up to 7 and 6 weeks, respectively and cleared bacteria more effectively from lungs and liver in experimental murine TB model

Dutt et al., 2001a

PLG RIF, INH, PYZ, ETB

Oral (rat)

Drugs detected in circulation up to 72 h. Increased Cmax; AUC; t1/2(a) and t1/2 (e)

Ain et al., 2002

PLG RIF In vitro Monocyte cell lines showed release of RIF up to 7 days. Barrow et al., 1998 PLGA RIF, INH,

PZA Oral (mice)

Therapeutic concentrations in tissues were maintained for 9–11 days and cleared bacteria more effectively from different organs in experimental murine TB model lungs after only five doses of treatment (dosing at 10days interval)

Pandey et al., 2003

PLG RIF, INH, PZA

Subcutaneous (mice)

A single dose maintained drug plasma, lungs and spleen concentrations for more than 1 month and led to undetectable bacterial counts in the different organs.

Pandey et al., 2004

PLG (Wheat germ agglutinin ligand)

RIF, INH, PZA

Oral,Inhalation-aerosol (guinea pig)

Three doses administered fortnightly for 45 d were sufficient to produce a sterilizing effect in lungs and spleen.

Sharma et al., 2004

Stearic acid RIF, INH, PZA

Oral (mice)

Single oral showed drugs were maintained in the plasma for 8 days and in the organs (lungs, liver and spleen) for 10 days. No tubercle bacilli could be detected in the lungs/spleen after 5 oral doses of drug loaded SLNs administered at every 10th day.

Pandey et al., 2005

PLG RIF, INH, PZA

Oral (guinea pig)

Significantly reduced the bacterial count in lung following daily and every 10 days administration of NPs.

Johnson et al., 2005

Alginate RIF, INH, PZA

Inhalation-Aerosol (mice)

Drug levels above MIC were detected in the lungs, liver and spleen up to 15 days.

Zahoor et al., 2005

PLA INH intra-tracheal instillation (rat)

The particles provided sustained and targeted delivery to AMs with substantial reduction in the blood levels of a potential toxic metabolite acetylisoniazid (AcINH)

Zhou et al., 2005

PLGA RIF Inhalation-Aerosol (guinea pig)

A single administration of particles decreased the bacteria population in the spleen with increased drug residence time in the lungs.

Garcia-Contreras et al., 2006



PLGA RIF, INH, PZA, ETB

Oral (mice)

Therapeutic levels were maintained for 5–8 days in blood and 9 days in plasma; one administration every 10th day (5 doses) eliminated the bacteria in the meninges.

Pandey et al., 2006

PLG RIF, INH, PZA, ETB

Oral (mice)

Therapeutic levels were maintained for 5 days in plasma and 7-9 days in the organs (lungs, liver, spleen) after single oral dose while 3 oral doses of the 4-drug formulation administered at every 10th day resulted in undetectable bacilli in the organs.

Pandey et al., 2006a

Alginate RIF, INH, PZA, ETB

Oral (mice)

Encapsulated drugs were observed in plasma and in tissues until day 15.

Ahmad et al., 2006

PLGA RIF Insufflation or nebulization (guinea pig)

Single and double doses of microparticle reduced numbers of viable bacteria, inflammation and lung damage compared. Two doses of RIF-PLGA-reduced splenic enlargement.

Suarez et al., 2001

PLA RFB, INH Inhalation (mice)

Intracellular concentrations of respirable MPs were found to be four-fold.

Muttil et al., 2007

PBCA Moxifloxacin Intravenous (mice)

NPs were more toxic to the macrophages. Cellular uptake showed pronounced increase (2–3 fold) in the intracellular drug concentration. Anti-TB activity in infected mice showed a significant decrease in the total mycobacterial count in the lungs.

Kisich et al., 2007, Shipulo et al., 2008

PLG Econazole, Moxifloxacin, RIF

Intravenous (mice)

Only eight doses of NPs were sufficient to suppress bacterial clearance in infected mice. Third drug RIF to this combination showed complete bacterial clearance within 8 weeks.

Ahmed et al., 2008

Hyaluronan Ofloxacin Intratracheal and Intravenous (rat)

Ofloxacin-loaded hyaluronan particles resulted in 50% lower serum bioavailability with respect to intravenous or oral ofloxacin. This observation supported the view that inhaled MPs may reduce systemic side effects.

Hwang et al., 2009

PLGA, mannitol RIF Inhalation (rat)

NPs in mannitol improved the in vivo uptake of the drug by alveolar macrophages in rat lungs.

Ohashi et al., 2009

PLGA RIF Inhalation (rat)

Phagocytosis of PLGA MS does not generate the toxic humoral factors to AMs, such as TNF-a and NO, and the phagocytosis does not affect the viability of AMs.

Hirota et al., 2010

Gelatin RIF Intravenous (mice)

NPs sustained the plasma level with enhanced the AUC and MRT of the drug. Significant reduction in bacterial counts in the lungs and spleen of TB-infected mice was also found.

Saraogi et al., 2010



Gelatin (Mannose ligand)

INH Intravenous (mice)

NPs are potential carrier for safer and efficient management of TB.

Saraogi et al., 2011

PLG INH, RIF Inhalation (rat)

The intracellular drug concentrations resulting from particle inhalation were found to be higher than vascular delivery of soluble drugs.

Sharma et al., 2011

Polymeric micelles

INH-PEG-PAA INH In vitro The micelle-forming prodrug showed a 5.6-fold increase in antituberculous activity against M. tuberculosis in vitro when compared to the free drug

Silva et al., 2001

PYZ-PEG-PAA PYZ Intravenous (rat)

The size of the micelles prevented renal filtration, increased the residence time in the blood stream with improved antimicrobial activity.

Silva et al., 2006

MPEG-PLLA and MPEG-PDLA

RIF In vitro RIF loading capacity and encapsulation efficiency of the stereo-complexes were higher. There was a fast initial release (50% after 4–8 hours) and a more moderated one (100% after 48 hours) afterwards.

Chen et al., 2007

P(CL-GA)–PEG-P(CL-GA) RIF In vitro RIF sustained release was obtained over 32 days from 25% gel matrix.

Jiang et al., 2007

RIF-PEG-PAA RIF Intravenous (rat)

The size of the micelles prevented renal filtration, increased the residence time in the blood stream with improved antimicrobial activity.

Silva et al., 2007

INH lipid derivatives INH In vitro Micelles showed increased penetration of the drug into the pathogen leading to promising antibacterial activity

Jin et al., 2008

PLA-modified chitosan oligomers

RIF In vitro RIF-chitosan oligomer micelles showed initial burst drug release of 35% within 10 h followed by more sustained drug release till 5th day

Wu et al., 2009

Dendrimers PPI (Mannose ligand)

RIF In vitro Mannose on surface significantly reduced the hemolytic toxicity of the nanocarriers and drug and also sustained the drug release. Surface modification improved the selective uptake of the drug-loaded nanocarriers by cells of the immune system.

Kumar et al., 2006

PEGylated PPI RIF In vitro PEG grafted dendrimers showed significant increase in drug entrapment, sustained release of RIF and low hemolytic activity.

Kumar et al., 2007



1.3. ROUTES OF ADMINISTRATION:

Different routes of administration including oral, parenteral and pulmonary for

delivery of anti tubercular drugs have been actively investigated since last few

decades. Parenteral administration of colloidal DDS enable reduced toxicity of anti-

tubercular drugs through targeting to the alveolar macrophages. While the large

epithelial surface area, the high organ vascularization, the thin nature of the alveolar

epithelium and the immense capacity for solute exchange enable the lung to serve as

an ideal administration route for the application of drugs for treatment of tuberculosis.

1.3.1. Parenteral route

In contrast to microparticles with a diameter of more than 1 µm that cannot be

administered via intravascular routes, nanoparticles are small enough to allow

intracapillary passage followed by an efficient cellular uptake. When administered

intravenously, the nanoparticles follow the route of other foreign particulates,

including intracellular pathogens. They are endocytosed by resident macrophages of

the mononuclear phagocyte system and by circulating monocytes. On the other hand,

in the case of infections caused by intracellularly persisting microbes (e.g., Brucella,

Salmonella, Listeria, Mycobacterium), macrophages become reservoirs for pathogens,

thus representing one of the targets for delivery of antimicrobial agents.

Preferential uptake of nanoparticles by macrophages (mainly by Kupffer cells in the

liver) is achieved by the physicochemical properties of the carrier and by physiologic

opportunity, thus representing an example of passive delivery. This technology

improves drug delivery to macrophages, increasing the amount of the drug reaching

this target site, which allows reduction of the overall therapeutic dose and decrease of

the adverse effects. Accordingly, the enhanced efficacy of the nanoparticle-bound

antibiotics was demonstrated in a number of experimental infections (Pinto-

Alphandary et al., 2000, Kayser et al., 2003).

RIF loaded multilamellar vesicles consisting of lecithin dicetylphosphate and

cholesterol was administered parenterally, six times weekly to previously infected

mice, 7 days up to 8 weeks (Saito et al., 1989). Significant reduction in the colony

forming units (CFU) was observed by the intraperitoneal (i.p.) route as compared to

the subcutaneous (s.c.) and intramuscular (i.m.) routes (Saito et al., 1989). In another

study Deol and Khuller developed lung-specific Stealth® liposomes for the targeted

delivery of anti-TB drugs to the lung (Deol et al., 1997 and 1997a). Studies showed

that a prominent increase accumulation of the nanocarriers in lungs after



administration (5.1% with conventional liposomes to 31% with PEGylated systems

containing O-steroyl amylopectin) in healthy and tuberculosis infected mice. The

extent of accumulation was related to the composition of the vesicles. A statistically

significant decrease in the hepatotoxic activity of the antitubercular drugs was

observed upon encapsulation. Administration of sub-therapeutic doses (4 and 3 mg/kg

for INH and RIF respectively) led to a higher decrease in CFU, as compared to the

free drugs administered at a therapeutic concentration (Deol et al., 1997 and 1997a).

Overall, a significant increase in the anti-TB activity was found.

Clofazimine, a riminophenazine compound, is an agent considered for treating

patients with M. avium infection. However, use of this drug was restricted because of

its poor solubility. Drug-loaded liposomes of clofazimine were developed to improve

the antitubercular activity, to reduce toxicity and to enable the parenteral

administration of highly lipophilic drug (Mehta et al., 1999) and preclinically

evaluated in acute and chronic murine infections (Adams et al., 1999). Intravenous

injection of a nanocrystalline nanosuspension formulation of clofazimine (particle

size, 385 nm) resulted in a considerable reduction of bacterial loads in the liver,

spleen, and lungs of mice infected with M. avium (Peters et al., 2000). This result

correlated with the pharmacokinetic data. Drug concentrations in these organs reached

levels, well in excess of the minimal inhibitory concentration for most M. avium

strains. Interestingly, the effects of the nanocrystalline formulation of clofazimine

were similar to those of the liposomal formulation used as a control in this study

(Rabinow et al., 2004).

A single subcutaneous dose of PLG nanoparticles loaded with RMP, INH, and PZA

maintained therapeutic drug levels in plasma for 32 d and in lungs/spleen for 36 d

(Pandey et al., 2004). Moreover, this single subcutaneous injection produced a

sterilizing effect in lungs and spleen of the infected mice (36 d post treatment),

thereby demonstrating better chemotherapeutic efficacy, as compared with daily

treatment using free drugs (35 oral doses). As suggested by the authors, the

nanoparticles form a depot at the injection site to slowly release drugs into the

circulation.

1.3.2. Inhalation route (pulmonary delivery)

The potential advantages of direct delivery of the TB drug to the lungs include the

possibility of reduced systemic toxicity, as well as achieving higher drug

concentration at the main site of infection. A possible obstacle to using nanocarriers



for pulmonary delivery is that their mass median aerodynamic diameter, an essential

parameter for the particle deposition in the lungs, is often too small. Nevertheless, the

effectiveness of pulmonary drug delivery using nanoparticles was demonstrated in a

number of studies.

A single administration INH and RIF loaded multilamellar liposomes to guinea pigs

following nebulization showed plasma drug levels from 45 minutes onwards up to 48

hours, whereas no drug could be detected beyond 24 hours post-nebulization when

free drugs were used. The aerosol formulation also showed significantly enhanced

plasma half-life due to slower rate of elimination (Pandey et al., 2004). In vivo studies

in albino rats demonstrated a higher pulmonary delivery and better localization of

ligand-appended liposomes to alveolar macrophages compared with conventional

liposomes or free rifampicin, from 30 min to 24 h post-nebulization (Vyas et al.,

2004). A single nebulization of poly (DL-lactide-co-glycolide) (PLG) nanoparticles of

INH, RIF and PZA resulted in sustained therapeutic drug levels in plasma for 6-8

days and in lungs for 11 days.

Based on the experimental data, it is clear that respiratory drug delivery systems

certainly have the potential for antitubercular inhaled therapy. The requirements for

fewer drug doses as well as a low dosing frequency are definite advantages. However,

there are some key issues that still need to be addressed. The possibility of variable

deposition of an inhaled formulation in the lungs needs to be considered and is a

matter of concern because it could result in suboptimal drug concentrations in certain

lung regions. If this does occur to a significant extent then treatment response could

be impaired.

1.3.1. Oral administration

The oral route widely used today is the most patient friendly route. Development of a

drug delivery system for oral administration which could show enhanced pulmonary

uptake of anti-tubercular drugs could provide a promising effective alternative to

current therapy. Further targeting the drug to the macrophages the site of the

tuberculosis infection could provide the dual advantage of improved treatment with

the possibility of decreased side effects.

After a single oral administration of PLGA nanoparticles co-encapsulated with

rifampin (RMP), isoniazid (INH), and pyrazinamide (PZA) provided sustained release

with drugs detected in circulation for 4-9 days with tissue concentrations maintained

for 9-11 days (Pandey et al., 2003, 2004). No tubercle bacilli could be detected in the



tissues after 5 oral doses of treatment. Similar efficacy of the nanoparticle-bound

drugs was also observed in guinea pigs (Sharma et al., 2004). Anti-TB drug-loaded

alginate nanoparticles (235 nm diameter) were orally administered to mice and the

plasma concentrations monitored over time (Zahoor et al., 2006). Free drugs were

cleared from blood 12 to 24 h after administration and were detectable in tissues (e.g.

spleen, liver and lung) only until day 1. In contrast, encapsulated drugs were observed

in plasma up to 7, 9, 11 and 11 days after administration for ETB, RIF, INH and PYZ,

respectively, and in tissues until day 15 (Zahoor et al., 2006).

A single dose of RIF, INH and PYZ incorporated SLN administered orally in mice

resulted in drug concentrations detectable after 3 h and for up to 8 days (Pandey et al.,

2005). Moreover, plasma concentrations were equal to or above the MIC at all the

time points measured. The free drugs were cleared from circulation within 12h of

administration. Also, while drug-loaded SLN maintained detectable drug levels over

10 days in the lung, spleen and liver, free drugs were cleared from these organs 24–

48h after the oral administration. Finally, the initial CFU count 15 days after the

infection with M. tuberculosis H37Rv was 4.20 and 4.34 log in lungs and spleen,

respectively. Five doses of drug-loaded SLNs led to undetectable CFU. To attain a

similar effect with the free drugs, 46 daily doses were required.

Wheat germ agglutinin coated particles were administered to guinea pigs through

oral/aerosol route for pharmacokinetic and chemotherapeutic evaluation. The

presence of drugs in the plasma was detected for 6-7 days for RIF and 13-14 days for

INH and PZA. The lectin-modified formulations produced bacterial clearances in

lungs, liver and spleen after three oral doses administered every 14 days, which was

equivalent to 45 doses of oral free drugs (Sharma et al., 2004). Therapeutic efficacy of

the nanoparticle-based formulations of anti-tubercular drugs following oral and

inhalation route are compared in Table 1.2.



Table 1.2: Therapeutic efficacy of the nanoparticle-based formulations of RIF-INH-PZA combinations (Gelperina et al., 2005)

Delivery System

Animal Model

Administration Route

Duration of Drug Release

(d )

Regimen Producing Sterilizing Effect in

Lungs and Spleen

Plasma Organs

PLG nanoparticles

Mice Oral 6–9 9–11 5 doses every 10 d

Mice Subcutaneous 32 36 Single injectionGuinea

pigs Aerosol 4–9 up to 10

(each drug)

5 doses every 10 d

Guinea pigs

Oral 4–9 up to 10 d (each drug)

5 doses every 10 d

Lectin-functionalized PLG nanoparticles

Guinea pigs

Oral 7–13 up to 15 (each drug)

3 doses fortnightly

Guinea pigs

Aerosol 6–14 up to 15 (each drug)

3 doses fortnightly

Solid lipid nanoparticles

Guinea pigs

Aerosol 5 7 7 doses weekly

1.4. FATE OF ORAL NANOPARTICULATE DRUG DELIVERY:

Nanocarriers by virtue of their unique uptake mechanisms hold the advantage of

bypassing the first pass metabolism of the encapsulated drug prone to such

metabolism. Most evidences suggest that nanocarrier uptake occurs preferentially via

“M-Cells” in Peyer’s patches by process of endocytosis (lymphatic uptake) thereby

delivering the drug loaded particles directly into the systemic circulation through the

lymphatics and circumventing first pass metabolism. However uptake by transcellular

and paracellular pathways (non-lymphatic uptake) involving intestinal adsorptive

endocytosis cannot be ruled out (Florence et al., 1997; Jung et al., 2000). Additionally

nanocarriers can improve the bioavailability of orally administered drug by inhibiting

multidrug efflux pump transporter P-glycoprotein (P-gp) present in intestine, liver and

kidney (Kalaria et al., 2008).

It is generally accepted that smaller particles are more effectively absorbed than larger

particles. While larger particles are retained for longer period in the Peyer’s patches,



smaller particles are progressively transported to the major organs (Jani et al., 1990;

Jung et al., 2000). Further non-ionized carriers are reported to absorb at the greater

extent than charged carriers (Jani et al., 1989). Uptake of nanocarriers prepared from

hydrophobic polymers seems to be higher than from nanocarriers with hydrophilic

surfaces. Preferential uptake of polystyrene nanoparticles over PLA and PLGA

particles and negligible uptake of particles prepared from hydrophilic cellulose

matrices upon oral administration to mice has been observed by Eldridge et al., 1990.

Moreover coating of nanoparticles with hydrophilic polymers reduced their uptake

from the large intestine and inhibited their uptake in the small intestine (Hillery and

Florence et al., 1996). The other factors such as the vehicle used for oral

administration of nanocarriers and physiological factors including animal species, age

and food ingestion also have an influence on the uptake mechanisms of nanocarriers

(Galindo-Rodriguez et al., 2005).

Another strategy used for improving the bioavailability of poorly absorbed drugs is

based on developing bioadhesive nanocarriers to prolong gastrointestinal transit of

drugs. The intricacies and the factors influencing these interactions and uptake of

nanocarriers across GIT are extensively reviewed in the literature [Ponchel et al.,

1997; Chen and Langer et al., 1998; Hussain et al., 2001; Galindo-Rodriguez et al.,

2005; Rieux et al., 2006]. Tables 1.3-1.5 lists the nanocarriers evaluated for

improving oral bioavailability of drugs.



Table 1.3: Examples of in vivo studies with nanoparticles for improving oral bioavailability of drugs

Material Drug Comment Ref Nanoparticles Sulfobutylether-β-cyclodextrin/chitosan

Docetaxel Significantly increased the AUC(0→t) and decreased the clearance

Wu et al., 2012

PLGA Estradiol Significant increase in AUC values when compared to that of oral pure drug

Mittal et al., 2007

Doxorubicin Superior bioavilability and lower toxicity with nanoparticulate formulation

Kalaria et al., 2008

Estradiol Nanoparticles improved oral bioavailability/ efficacy of estradiol

Mittal et al., 2009

Curcumin 5.6 fold enhanced bioavalability with longer half life

Xie et al., 2011

Chitosan Insulin Nanoparticles maintained the serum glucose level of the diabetic rats at prediabetic levels (60 % of baseline) for at least 11h

Ma et al., 2005

Green tea catechins (+)-catechin and (-)-epigallocatechin gallate

nanoparticles enhances their intestinal absorption and bioavalibility

Dube et al., 2010

PEG-chitosan Calcitonin Chitosan–PEG nanocapsules enhanced and prolonged the intestinal absorption of salmon calcitonin

Prego et al., 2006

PVA/MA (Bioadhesive albumin coated nanoparticles )

5-fluorouridine Higher bioavailability with nanoparticles (79%) when compared to oral solution (11%)

Arbos et al., 2004



Table 1.4: Examples of in vivo studies with liposomes and proliposome for improving oral bioavailability of drugs

Material Drug Comment Ref

Liposomes polyelectrolyte-stabilized liposomes (layersomes)

Doxorubicin 5.94 fold increase in oral bioavailability with reduced toxicity

Jain et al., 2012

Chitosan–aprotinin coated

Calcitonin 11 fold and around 15 fold increase in AUC with chitosan coated liposomes and chitosan–aprotinin coated liposomes respectively compared to solution

Werle & Takeuchi 2009

Lectin coated Insulin Enhanced bioavilability with lectin coated liposomes in comparison with insulin solution administered subcutaneously

Zhang et al., 2005

Phosphotidylcholine (SPC) and sodium deoxycholate (SDC) and SPC/cholesterol (CL)

Fenofibrate Higher rates of fenofibrate absorption from both SPC/SDC and SPC/CL liposomes than micronized fenofibrate. 1.57-fold increase in bioavailability of SPC/SDC liposomes relative to SPC/CL liposomes,

Chen et al., 2009

Dipalmitoylphosphatidyl choline (DPPC) and cholesterol (CL)

Cyclosporine Significantly faster rate of absorption following liposome administration.

Al-Meshal et al. 1998

Proliposomes Phospholipids Silymarin Superior bioavailability of

SLN compared to drug suspension

Yan-yu et al., 2006



Table 1.5: Examples of in vivo studies with solid lipid nanoparticles (SLN) for improving oral bioavailability of drugs

Material Drug Comment Ref

Glyceryl monostearate and soya lecithin

Quercetin Superior bioavailability of SLN compared to drug suspension

Li et al., 2009

Lecithin Simvastatin Superior bioavailability of SLN compared to drug suspension

Zhang et al., 2010

Glyceryl monostearate

Vinpocetine Superior bioavailability of SLN compared with that of drug solution

Luo et al., 2006

Stearic acid Rifampicin, Isoniazid and Pyrazinamide

Enhanced therapeutic drug concentrations in plasma and organs with SLN compared to free drug Superior activity of SLN in M. tubercuIosis H37Rv infected mice compared to free drug

Pandey et al., 2005

Compritol 888 ATO Silymarin 2.79 fold higher bioavailability with SLN compared to drug suspension

He et al., 2007

1.5. OBJECTIVE

Targetted drug delivery to better manage drug pharmacokinetics, pharmacodynamics,

non-specific toxicity, immunogenicity and biorecognition systems have been proven

as an important strategy for complete eradication of mycobacterium tuberculosis from

intra cellular sites. Nanocarriers show great promise as carriers to mediate

intracellular drug delivery. Nanocarriers present the advantages of stability, high drug

loading, possibility of incorporating both hydrophilic and hydrophobic drugs, and

feasibility of variable routes of administration, including oral administration,

intravenous and inhalation. These carriers can also be designed to enable controlled

drug release to enable decreased dosing frequency.

Pulmonary tuberculosis is the most prevalent tuberculosis and specifically important

as it can be contagious. One of the major challenges in tuberculosis therapy is

achieving high drug concentration in the lungs and more specifically in the alveolar

macrophages the site of infection. A direct strategy to tackle pulmonary tuberculosis



is pulmonary delivery of anti-tubercular drugs. Direct pulmonary deposition of

micro/nanoparticles through inhalation/nebulization has been evaluated by various

research groups to deliver high concentration of anti tubercular drugs to the lungs

(Pandey et al., 2001, 2005, Sen et al., 2003, Zahoor et al., 2005, Sharma et al., 2001,

2007). Although promising, the dose that can be delivered by this route poses serious

constraints. Variable deposition of inhaled nanoparticles in the lungs, resulting in

suboptimal drug concentrations in certain lung regions is another limiting issue.

Oral drug administration with high bioavailability and sustained release, coupled with

enhanced pulmonary uptake presents an ideal drug delivery strategy for the treatment

of tuberculosis. Targetting to the lungs following oral administration is an attractive,

although difficulty strategy. Colloidal systems, because of their small size, are capable

of being absorbed intact from the gastrointestinal tract by passive diffusion via

transcellular or paracellular pathways, or via active processes mediated by membrane-

bound carriers or membrane-derived vesicles. Although these would results in rapid

sequester by liver and spleen. Lymphatic uptake by the M cells of the Peyer’s patches

is reported to be one of the major pathways for rapid absorption of nanoparticles

(O’Hagan et al., 1987). Moreover lymphatic uptake could also enable bypass of portal

circulation and thereby enhance lung deposition.

A promising approach for active targeting to infected macrophage is by receptor

mediated endocytosis. Macrophages express a number of receptors including mannose

(Taylor et al., 1992 and 1997, Lennartz 1987, Kaur et al., 2008), tuftsin(Gupta et al.,

2000), scavenger (Van der Laan et al., 1999, Fluiter et al., 1998),,

fibronectin(Yukihiko et al., 1995),, and folate(Low et al., 2004, Low et al., 2009).

Folate receptors which are overexpressed in infected macrophages are widely

investigated receptor for targeting.

The most common ligand investigated is folic acid a low cost, non immunogenic and

safe moiety which exhibits very high affinity for the folate receptor(Kd~ 10-10) (Low

et.al, 2004a, Fatemeh et.al, 2011, Sudimack et.al, 2000, Stella et.al, 2000, Patil et.al,

2008).

Design of Nanoparticulate Drug Delivery Systems for targeting the anti-tubercular

drugs with enhanced bioavailability can prove to be an effective and practical

approach. The present project deals with the design and evaluation of folate anchored



targeted nanoparticulate drug delivery system (NPDDS) of antitubercular drugs with

simultaneous entrapment of two drugs of different solubility, high entrapment

efficiency and desirable particle size for macrophage uptake and targeting. Poly

(ethylene sebacate) (PES) is a novel hydrolytically stable, nonionic, biocompatible

and biodegradable, non-mutagenic and non-genotoxic polymer was selected for the

present study (Deverajan et al., 2006, More et al., 2009).

The following specific NPDDS were evaluated.

A. PES-rifampicin nanoparticles with and without folic acid

B. PES-rifampicin-ethambutol combination nanoparticles with and without folic

acid

C. PES-rifampicin-MSDNC-22 (a new chemical entity) combination

nanoparticles with and without folic acid

2. Drug Profile



2 DRUG PROFILE 2.1. RIFAMPICIN

Rifampicin or rifampin is a bactericidal antibiotic of the rifamycin group used to

treat Mycobacterium infections, including tuberculosis and leprosy; and also has a

role in the treatment of methicillin-resistant Staphylococcus aureus (MRSA) in

combination with fusidic acid.

Rifampicin is a semisynthetic derivative of rifamycin antibiotics which are produced

by the fermentation of a strain of Streptomyces mediterranei, a species which was first

isolated in Italy in 1957 from a soil sample collected in France. The fermentation

produces rifamycin B. Rifamycin B is transformed by a series of reactions into 3-

formylrifamycin SV, which in turn is condensed with 1-amino-4-methylpiperazine in

peroxide-free tetrahydrofuran to give rifampicin.

It is used in the treatment of tuberculosis mainly in combination with other drugs like,

isoniazid, ethambutol,and pyrazinamide, as a fixed dose combination (FDC). This is

mainly due to the reason that the tubercule bacillus is reported to develop resistance

with single drugs. But the main disadvantage reported with fixed dose combinations is

poor/variable bioavailability of rifampicin.

2.1.1. Chemical structure:

Formula - C43H58N4O12 Molecular weight - 822.94 g/mol

2.1.2. Chemical name- 5,6,9,17,19,21-Hexahydroxy-23-methoxy-2,4,12,16,18,20, 22

-heptamethyl-8-[N-(4-methyl-1-piperazinyl)formimidoyl]-2,7(epoxypentadeca

[1,11,13]trienimino)-naphtho[2,1-b]furan-1,11(2H)-dione 21-acetate

2.1.3. CAS number - 13292-46-1

2.1.4. Physicochemical properties

The drug is red to orange, odourless and is available in crystalline form. It is very

slightly soluble in water (1 g in approximately about 762 mL water [pH < 6]),



acetone, carbon tetrachloride, alcohol, ether. Freely soluble in chloroform, DMSO;

soluble in ethyl acetate and methyl alcohol and tetrahydrofuran. Solubility in aqueous

solutions is increased at acidic pH. Melting point ranges from 138 to 188 °C.

Rifampicin has 2 pKa since it is a Zwitterion, pKa 1.7 related to 4-hydroxy and pKa

7.9 related to 3-piperazine nitrogen (Merck Index, 1989). A 1% suspension in water

has pH 4.5 to 6.5.

2.1.5. Analysis

Methods reported for analysis of rifampicin are HPLC (Panchgnula et al, 2004, Blain

et al, 1998, Singh et al, 2003), HPTLC (Shishoo et al, 1999, Shah et al, 2001, Argekar

et al, 1996), Dual wavelength UV vis spectrophotometry: (Shishoo et al, 1999),

Voltametric Assay: (Hammam et al, 2004), and Colorimetric analysis (Mariappan et

al, 2004).

2.1.6. Stability (Gallo and Radaelli et al., 1976)

Rifampicin is very stable in the solid state in sealedcontainers at room temperature as

well as temperature upto 70ºC. The stability of rifampicin in aqueous solution has

been widely investigated and the conditions and the transformation products are

reported below.

Conditions Transformed products

pH 2.3 at 20-22ºC and 0.1N HCl at 37ºC 3-Formyl-rifampicin SV

pH 8.2 at 20-22ºC Rifampicin-quinone

NaOH 5% in ethanol:Water (1:1) 20-22ºC 25-desacetyl rifampicin

pH 8.2 at 60-70ºC

25-desacetyl rifampicin

25-desacetyl-21-acetyl rifampicin

25-desacetyl-23-acetyl rifampicin

2.1.7. Indications

Rifampicin is typically used to treat Mycobacterium infections, including tuberculosis

and leprosy; and also has a role in the treatment of methicillin-resistant

Staphylococcus aureus (MRSA) in combination with fusidic acid. It is used in

prophylactic therapy against Neisseria meningitidis (meningococcal) infection.

2.1.8. Mechanism of action

Rifampicin inhibits DNA-dependent RNA polymerase in bacterial cells by binding its

beta subunit, thus preventing transcription of messenger RNA (mRNA) and

subsequent translation to proteins. Rifampicin may be bacteriostatic or bactericidal



depending on the concentration of drug attained at the site of infection. The

bactericidal actions are secondary to interfering with the synthesis of nucleic acids by

inhibiting bacterial DNA-dependent RNA polymerase at the B-subunit thus

preventing initiation of RNA transcription, but not chain elongation.

2.1.9. Pharmacokinetic properties:

It has the bioavailability of 90-95 % but it is erratic, and half life of 6-7 hrs.

Rifampicin is readily absorbed mostly from the upper gastrointestinal tract (90%).

Peak plasma concentration occurs at 1.5 to 4hours after an oral dose. Eighty nine per

cent of rifampicin in circulation is bound to plasma proteins. It is lipid soluble. It is

widely distributed in body tissues and fluids. Approximately 85% of rifampicin is

metabolised by the liver microsomal enzymes to its main and active metabolite-

deacetylrifampicin. Rifampicin undergoes enterohepatic recirculation but not the

deacetylated form. Rifampicin metabolite deacetylrifampicin is excreted in the bile

and also in the urine. Approximately 50% of the rifampicin dose is eliminated within

24 hours and 6 to 30% of the drug is excreted unchanged in the urine, while 15% is

excreted as active metabolite. Approximately 43 to 60% of oral dose is excreted in

the faeces.

2.1.10. Adverse effects

Adverse effects are chiefly related to the drug's hepatotoxicity, and patients receiving

rifampicin often undergo liver function tests including aspartate aminotransferase

(AST).The most common unwanted effects are fever, gastrointestinal disturbances,

rashes and immunological reactions. Liver damage, associated with jaundice, has also

been reported and in some rare cases has led to death.

2.1.11. Contraindications

Rifampicin is contraindicated in known cases of hypersensitivity to the drug. It may

be contraindicated in pregnancy (because of teratogenicity noted in animal studies and

since the effects of drugs on fetus has not been established) except in the presence of

a disease such as severe tuberculosis. It is contraindicated in alcoholics with severely

impaired liver function and with jaundice

2.1.12. Brand names/Trade names

Rifampicin capsule/tablet of 150mg, 300mg, 450mg and 600mg available with

different brand names are as follow:

FAMCIN (IDPL), KEMORIFA (Chemo), MACOX (Macleods), R-CIN (Lupin),

RIFACEPT (Concept), RIFACILLIN (PCI), RIFAMPILA (Alb. David),



RFAMYCIN (Biochem), RIFAPLUS CAPS (Redicura), RIMACTANE (Sandoz-

Novartis), RIMPACIN (Zydus Cadila), RIMPIN (Lyka Hetero), TICIN (Themis),

ZUCOX (GSK).

Also rifampicin Syrup – 100mg/5ml are available.

2.2. ETHAMBUTOL

Ethambutol (commonly abbreviated EMB, ETH or simply E) is a bacteriostatic

antimycobacterial drug prescribed to treat tuberculosis. It is usually given in

combination with other tuberculosis drugs, such as isoniazid, rifampicin and

pyrazinamide. It is sold under the trade names Myambutol and Servambutol.

2.2.1. Formula: C10H24N2O2

2.2.2. Mol. Wt: 204.31 g/mol

2.2.3. Chemical name: (2S,2′S)-2,2′-(ethane-1,2-diyldiimino)dibutan-1-ol

2.2.4. CAS number:

Ethambutol 74-55-5

Ethambutol hydrochloride 1070-11-7

Dose 15-20mg/kg body wt

2.2.5. Physicochemical Properties:

Colour - White

State/Form - Crystalline hygroscopic powder

Description - Odourless or almost odourless, Bitter taste

Melting point - 199 °C to 204 °C

Soluble 1 in 1 of water, 1 in 4 of alcohol, 1 in 850 of chloroform, and 1 in 9 of methyl

alcohol; very slightly soluble in ether. A solution in water is dextrorotatory. Solutions

are stable when heated at 121 °C for 10 minutes.

Storage conditions - Store in airtight containers between 15 to 30°C



2.2.6. Analysis

Methods reported for analysis of ethambutol are HPLC with fluorescence detection

(Breda et al., 1996,) HPLC with UV detection (Chenevier et al., 1998), HPLC with

mass detection (Conte et al., 2002), Gas chromatography (Wang et al., 1980), and Gas

chromatography with mass detection (Holdiness et al., 1981).

2.2.7. Mechanism of action

Ethambutol is bacteriostatic against actively growing TB bacilli. It works by

obstructing the formation of cell wall. Mycolic acids attach to the 5'-hydroxyl groups

of D-arabinose residues of arabinogalactan and form mycolyl-arabinogalactan-

peptidoglycan complex in the cell wall. It disrupts arabinogalactan synthesis by

inhibiting the enzyme arabinosyl transferase. Disruption of the arabinogalactan

synthesis inhibits the formation of this complex and leads to increased permeability of

the cell wall.

2.2.8. Pharmacokinetics

It is well absorbed from the gastrointestinal tract and well distributed in body tissues

and fluids, 50% is excreted unchanged in urine.

2.2.9. Side effects

Headache, loss of appetite, upset stomach, or nausea/vomiting may occur. If any of

these effects persist or worsen, tell your doctor or pharmacist promptly.Remember

that your doctor has prescribed this medication because he or she has judged that the

benefit to you is greater than the risk of side effects. Many people using this

medication do not have serious side effects.Tell your doctor immediately if any of

these unlikely but serious side effects occur: vision changes (such as

blurred/decreased vision, color blindness), symptoms of liver disease (such as

persistent nausea/vomiting, unusual tiredness/weakness, severe stomach/abdominal

pain, yellowing eyes/skin, dark urine), numbness/tingling of arms/legs, toe/joint pain.

Tell your doctor immediately if any of these rare but serious side effects occur:

mental/mood disorders (such as confusion, hallucinations), signs of a new infection

(such as fever, persistent sore throat), easy bleeding/bruising.A very serious allergic

reaction to this drug is rare. However, seek immediate medical attention if you notice

any symptoms of a serious allergic reaction, including: rash, itching/swelling

(especially of the face/tongue/throat), severe dizziness, trouble breathing.



2.2.10. Drug interactions

The effects of some drugs can change if you take other drugs or herbal products at the

same time. This can increase your risk for serious side effects or may cause your

medications not to work correctly. These drug interactions are possible, but do not

always occur. Your doctor or pharmacist can often prevent or manage interactions by

changing how you use your medications or by close monitoring. To help your doctor

and pharmacist give you the best care, be sure to tell your doctor and pharmacist

about all the products you use (including prescription drugs, nonprescription drugs,

and herbal products) before starting treatment with this product. While using this

product, do not start, stop, or change the dosage of any other medicines you are using

without your doctor's approval. Keep a list of all the products you use. Share the list

with your doctor and pharmacist to reduce your risk for serious medication problems.

2.2.11. Precautions

Before taking ethambutol, tell your doctor or pharmacist if you are allergic to it; or if

you have any other allergies. This product may contain inactive ingredients, which

can cause allergic reactions or other problems. Before using this medication, tell your

doctor or pharmacist your medical history, especially of: eye problems (such as optic

neuritis, cataracts, diabetic retinopathy), liver disease, kidney disease, alcohol use.

Before having surgery, tell your doctor or dentist about all the products you use

(including prescription drugs, nonprescription drugs, and herbal products).

Alcohol may increase the risk of liver disease. Avoid alcoholic beverages while using

this medication. During pregnancy, this medication should be used only when clearly

needed. Discuss the risks and benefits with your doctor. This medication passes into

breast milk but is unlikely to harm a nursing infant. Consult your doctor before breast-

feeding. If you have HIV disease, do not breast-feed because breast milk can transmit

HIV.

2.2.12. Contraindications:

Ethambutol hydrochloride is contraindicated in patients who are known to be

hypersensitive to his drug. Renal impairment, old age and optic neuritis are relative

contraindications.

2.2.13. Brand names/Trade names

Ethambutol (Argentina); Myambutol (Australia, Belgium, Canada, Denmark, France,

Germany, Netherlands, South Africa, Spain, Sweden, Switzerland, UK, USA), Mynah

(UK), Etibi (Canada), Dexambutol (France), EMS-Fasol (Germany), Etambutyl,



Etapiam, Miambutol, Mycobutol and Tibutolo (Italy), Afimocil, Anvital, Cidanbutol,

Etambin, Farmabutol, Fimbutol, Inagen and Tisiobutol (Spain)

2.3. MSDNC22 (Tawari et al., 2010)

2.3.1. Chemical structure:

OO2N

O

N

2.3.2. Molecular formula: C18 H18 N2 O4

2.3.3. Molecular weight: 326.35

2.3.4. Chemical name: (E)-3-(5-nitrofuran-2-yl)-1-(4-(piperidin-1-yl) phenyl) prop-

2-en-1-one

2.3.5. Minimum inhibitory concentration (MIC): 0.19 mcg/ml

2.3.6. Solubility: Soluble in tetrahydrofuran, dichloromethane, methyl ethyl ketone

and acetone, practically insoluble in methanol, ethanol and water

3. Analytical Method Development



3. ANALYTICAL METHOD DEVELOPMENT 3.1. RIFAMPICIN AND FOLIC ACID

The following two methods for the analysis of drug/s were developed:

UV Spectrophotometry

HPLC (High performance liquid chromatography) method

3.1.1. UV SPECTROPHOTOMETRY

UV spectrophotometric method were developed for routine analysis of

i) Rifampicin

ii) Folic acid

iii) Rifampicin in combination with Folic acid

Introduction

A UV spectrophotometric method was developed for the routine analysis of

rifampicin during dissolution studies and drug content analysis. Although the method

cannot be used easily for multicomponent formulations or formulations containing

interfering excipients, but ease of operation, low cost and low analysis time make it

the most popular analytical technique.

Materials

Rifampicin was obtained as a gift sample from Maneesh Pharma (India). Distilled

water was used during UV analysis. All other chemicals and solvents were analytical

– reagent grade and were purchased from s. d. fine chemicals, India. Instrumentation

UV1650PC, Schimadzu Corporation US spectrophotometer was used for analysis.

Preparation of standard curve

Approximately 5mg of rifampicin was accurately weighed and transferred to a 10mL

volumetric flask. It was dissolved in minimum amount of methanol (~1mL) and

volume was made up to 10mL with 0.1N HCl to obtain a primary stock solution of

concentration 500μg/mL. An aliquot 0.2mL was further diluted with 0.1N HCl to

10mL. This standard solutions of rifampicin (10μg/mL) were scanned in the range

from 200-600 nm on UV1650PC, Schimadzu Corporation US spectrophotometer.

Aliquots of the standard solution (500μg/mL) corresponding to 0.1, 0.2, 0.3, 0.4, 0.5,

0.6mL were diluted to 10mL in volumetric flasks with 0.1N HCl to obtain standard

solutions of concentrations 5, 10, 15, 20, 25, 30μg/mL respectively. The absorbance

of these solutions was recorded at 475 nm and plotted vs. the concentration to give the



Beer Lamberts plot for the standard curve. Similarly standard curve was developed in

methanol, pH 7.4, and PBS pH 7.4 by recording the absorbance at 475 nm.

Results and Discussion

The standard curves of RIF in 0.1N HCl, methanol, pH 7.4, and phosphate buffer

saline pH 7.4 are depicted in Figure 3.1. Standard curve parameters of UV-

Spectroscopic method for RIF are listed in Table 3.1.

std curve in 0.1N HCl

y = 0.0116x + 0.0008R2 = 0.9998

00.050.1

0.150.2

0.250.3

0.350.4

0 5 10 15 20 25 30

concentration (mcg/ml)

abso

rban

ce

std curve in methanol

y = 0.0181x + 0.0055R2 = 0.9995

0

0.1

0.2

0.3

0.4

0.5

0.6

0 5 10 15 20 25 30

concentration (mcg/ml)

abso

rban

ce

a) 0.1 N HCl b) Methanol

c) PBS pH 7.4 d) pH 7.4

Figure 3.1: Standard curves of rifampicin

Table 3.1: Standard curve parameters for rifampicin

Active agent Medium λmax Beer’s law range Slope Intercept r2

Rifampicin

0.1N HCl 475 nm 2.5-30 mcg/ml 0.0115 0.003 0.9999Methanol 475 nm 2.5-30 mcg/ml 0.0181 0.0055 0.9995

pH 7.4 475 nm 2.5-30 mcg/ml 0.018 0.001 0.999 PBS 7.4 475 nm 2.5-20 mcg/ml 0.017 0.007 0.999

The UV-spectroscopic method was precise and can be used for the routine analysis of

RIF.



ii) Folic acid

Preparation of standard curve: Approximately 10mg of folic acid was accurately

weighed and transferred to a 10mL volumetric flask. It was dissolved in 0.1N NaOH

and volume was made up to 10mL to obtain a primary stock solution of concentration

1000μg/mL. An aliquot of 1mL was further diluted with 0.1N NaOH to 10mL to give

solution of concentration 100μg/mL. Further an aliquot of 1mL was diluted with 0.1N

NaOH to 10mL to give solution of concentration 10μg/mL. This standard solutions of

folic acid (10μg/mL) was scanned in the range from 200-600 nm on UV1650PC,

Schimadzu Corporation US spectrophotometer.

Aliquots of the standard solution (100μg/mL) corresponding to 0.2, 0.5, 0.75, 1.0,

1.25, 1.5 and 1.75mL were diluted to 10mL in volumetric flasks with 0.1N NaOH to

obtain standard solutions of concentrations 2, 5, 7.5, 10, 12.5, 15, and 17.5μg/mL

respectively. The absorbance of these solutions was recorded at λmax 256.5 nm and

plotted vs. the concentration to give the Beer Lamberts plot for the standard curve.

Similarly standard curve was developed in PBS pH 7.4 by recording the absorbance at

λmax 256.5 nm.


The standard curves of folic acid in 0.1N NaOH, and phosphate buffer saline pH 7.4

are depicted in Figure 3.2. Standard curve parameters of UV-Spectroscopic method

for folic acid are listed in Table 3.2.

a) 0.1 N NaOH b) PBS pH 7.4

Figure 3.2: Standard curves of folic acid



Table 3.2: Standard curve parameters for folic acid

Active agent Medium λmax

Beer’s law range Slope Intercept r2

Folic Acid 0.1N NaOH 256.5nm 2-17.5 µg/mL 0.0547 0.0119 0.9999PBS pH 7.2 280 nm 0.25-10 µg/mL 0.065 0.005 0.999


folic acid.

iii) Rifampicin in combination with Folic acid

The concentrations of RIF and folic acid in combination were determined as follows.

Briefly, 10mg folic acid was dissolved in 0.5mL of 0.1 N NaOH, and volume made

~15 ml with water. To this solution 10mg of Rifampicin was added and dissolved by

sonication and volume made upto 20mL to obtain a concentration of 500µg/mL of

folic acid and rifampicin (Solution A).

Rifampicin analysis: Solution A (0.2mL) was diluted to 10mL with water to get

concentration 10µg/mL rifampicin and was analyzed by UV-Visible

spectrophotometry at λmax of 475nm without any interference.

Folic acid analysis: Dichloromethane (5mL) was added to 5mL of Solution A, mixed

on vortex mixer for 15mins, and allowed to separate for 1h. Dichloromethane (DCM)

and water are immiscible, RIF partitions into DCM and upper aqueous phase contains

folic Acid. The supernatant (0.2mL) was diluted to 10mL with 0.1 N NaOH and was

analyzed by UV-Visible spectrophotometry at λmax of 256.5nm without any

interference, from RIF.

The method for quantifying RIF and folic acid in combination was standardized as

follows:

i) Effect of Partition rate

After the addition of DCM (5mL) to the solution A containing mixture of RIF and

folic acid, the concentration of folic acid was monitored in the aqueous phase at

different time intervals viz. 1, 2 and 4 hrs to determine any interference from

unextracted RIF.

ii) Effect of surfactant and stabilizer concentration

The concentration of surfactant and stabilizer that is used in nanoparticles preparation

is added to the solution containing combination of RIF and folic acid, so as to check



for its/their interference in analysis of RIF and folic acid. The concentration of RIF

and folic acid was determined in presence of surfactants AOT and stabilizer PVA.


Drug content values ranged between 98-102% for RIF and 100-103% for folic acid

when analyzed in combinations with the developed extraction method.

i) Effect of Partition rate:

Drug content values at different time intervals viz. 1, 2 and 4 h revealed less than 2

percent relative standard deviation values (Table 3.3) and indicating no interference in

analysis of folic acid and hence it can be concluded that complete extraction of RIF is

achieved within 1h.

Table 3.3: Partition rate of RIF at different time intervals

Time (H) Absorbance at 256.5 nm Drug content of folic acid 1 0.554, 0.550 97.8 2 0.560, 0.565 99.7 4 0.555, 0.549 97.81

%RSD 1.11

ii) Effect of surfactant concentration:

Table 3.4: Effect of surfactant concentration on RIF analysis at different time intervals

Time (H) Absorbance at 256.5 nm Drug content of folic acid 1 0.548, 0.545 96.81 2 0.560, 0.540 97.45 4 0.558, 0.561 99.18

%RSD 1.25

Surfactant concentration did not affect in the analysis of folic acid as indicated by the

drug content values at the end of 4h revealing less than 2% relative standard deviation

values (Table 3.4). RSD value <2% suggest good precision of the method.

Conclusion


RIF and folic acid from combination.



3.1.2. STABILITY INDICATING HPLC METHOD

A stability indicating RP-HPLC method for the analysis of RIF as a bulk drug and in

pharmaceutical formulations was developed and validated.

Introduction

The objective of the present work was to develop and validate a stability indicating

RP-HPLC method for the assay of rifampicin as a bulk drug and in pharmaceutical

formulations.

Materials

Rifampicin was obtained as a gift sample from Maneesh Pharma (India). Glass

distilled water was used during HPLC analysis. All other chemicals and solvents were

analytical – reagent grade and were purchased from s.d. Fine Chemicals, India Instrumentation:

The HPLC system used was JASCO LC900 Intelligent pump (jasco japan) coupled

with UV detector and Rheodyne injector model (7725) fitted with 20μl sample loop.

Data integration was done by Borwin chromatography software version 1.21.

Chromatography

Chromatography was performed on a Waters Spherisorb® S5 ODS2 (250 × 4 mm i.d.,

5μm particle size) column. The mobile phase comprised of methanol: (0.01M) sodium

phosphate buffer pH 5.2 (pH adjusted by o-phosphoric acid) in the ratio 65:35 v/v.

The mobile phase was filtered through a nylon membrane (0.22 μm, Pall Gelman) and

degassed by sonication prior to use. Chromatography was performed at room

temperature under isocratic conditions at a flow rate of 1mL/min. UV detection was

done at a λmax of 254nm [Panchgnula et al., 1999].

Preparation of standard solutions

Rifampicin (10mg) was accurately weighed and transferred to a 10mL volumetric

flask. The volume was made up to 10 mL with methanol to obtain a stock solution

(1000μg/mL). From the above solution, 2mL was diluted upto 10mL with mobile

phase to get stock solution having concentration of 200μg/mL. Aliquots of this

solution corresponding to 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, and 0.6mL were diluted to

10mL with the mobile phase to obtain solutions in the concentration range of 1–

12μg/mL. Each solution was injected twice to obtain the output in duplicate. Average

of the peak areas were considered for calculation purposes.



Validation

a) Stability of analyte in solution:

The stability of rifampicin in mobile phase was assessed by injecting the standard

solution (4μg/mL) at interval of 0 and 24 hrs post preparation kept in amber colored

volumetric flasks at room temperature. The chromatograms were checked for

presence of peaks corresponding to degraded product.

b) Linearity

Standard solutions (1, 2, 4, 6, 8, 10 and 12μg/mL), each in three replicates, were

injected into the system. The method of linear regression was used for data

evaluation. Peak areas were plotted against theoretical concentrations of standards.

Linearity was expressed as a correlation coefficient.

c) Precision:

System precision (repeatability) was determined by performing four consecutive

injections of the 6μg/mL standard solution. Method precision was determined by

injecting three different samples (10μg/mL) prepared individually.

Forced Degradation Studies

It was necessary to perform forced degradation studies to verify and prove the

stability-indicating feature of the proposed method. Intentional degradation was

attempted by heating the drug in the presence of base, acid and hydrogen peroxide

and exposing to sunlight.

i) Acid degradation

To 0.6mL of standard stock solution (200μg/mL) of rifampicin, 1mL of 1N HCl was

added and the solution was placed in a boiling water bath for 30min. The sample was

allowed to cool to room temperature and neutralized using 1N NaOH. The volume

was adjusted to 10mL with distilled water, and this solution was injected in the HPLC

column.

ii) Base degradation

To 0.6mL of standard stock solution (200μg/mL) of rifampicin, 1mL of 1N NaOH

was added and the solution was placed in a boiling water bath for 30min. The sample

was allowed to cool to room temperature and neutralized using 1N HCl. The volume

was adjusted to 10mL with distilled water, and this solution was injected in the HPLC

column.



iii) Oxidation

To 0.6mL of standard stock solution (200μg/mL) of rifampicin, 1mL of 30% H2O2

was added and the solution was placed in a boiling water bath for 30min. The sample

was allowed to cool to room temperature. The volume was adjusted to 10mL with

distilled water, and this solution was injected in the HPLC column.

iv) Photodegradation

Drug solution (10μg/mL) in methanol was exposed to sunlight for 2h and this solution

was injected in the HPLC column.

The degraded samples were analyzed against an untreated control sample.


a) Stability of analyte in solution

Rifampicin was found to be stable in the mobile phase, when the standard solution of

strength 4μg/mL was analyzed at 0-24 h post preparation. No peaks corresponding to

the degradation products were observed. A low RSD value of 2.17 per cent indicated

that there was no significant change in the drug peak area (Table 3.5).

Table 3.5: Stability of rifampicin in solution

Time ( hr) Area (1) Area (2) Area (3) Average Mean concentration %RSD0 135480 133585 136874.1 134773.4 99.53784 2.17 24 131023.3 139025.8 132652.5 134233.8

b) Linearity

Figure 3.3: Standard curve of rifampicin by HPLC

Graph of the peak area vs concentration was plotted in order to check the linearity.

The developed method was found to be linear between concentration range of 1-

12μg/mL (Figure 3.3.) The regression coefficient was found to be 0.998.



c) Precision

Low RSD values of 1.84 per cent and 1.28 per cent were obtained for system and

method precision respectively (Table 3.6).

Table 3.6: Precision study of the RIF assay

Sample No. Area

System precision (6 mcg/ml)

Method precision (10 mcg/ml)

1 223765 390371 2 225596 386600 3 219346.5 396587 4 229312.75

%RSD 1.84 1.28


a) Standard rifampicin

b) Acid degraded rifampicin c) Base degraded rifampicin

d) H2O2 degraded rifampicin e) Photo degraded rifampicin

Figure 3.4: Forced degradation studies of RIF

The HPLC procedure was optimized with view to develop a stability indicating

method so as to resolve the degraded products from the drug. Various mobile phase

compositions were tried so as to obtain a sharp peak and also resolve the peaks of

degraded product from the peak of drug. The mobile phase consisting of sodium

phosphate buffer (0.01M): Methanol in the ratio of 35:65 per cent v/v resulted in a

retention time of 5.2 min for rifampicin. The chromatograms of rifampicin

RIF RT-5.2

RIF RT-5.2

RIF RT-5.2

RIF RT-5.2 RIF RT-5.2



(undegraded) and rifampicin degraded in the presence of acid, base, hydrogen

peroxide (H2O2) (oxidative degradation) and light (photo degradation) are shown in

Figure 3.4 and indicated a good separation of the undegraded drug from the

degradation products. Rifampicin is known to be degraded in presence of acid, base,

and light and also oxidized by peroxide. In our force degradation study same results

was observed. Acid degradation product was appeared around 3min, base degradation

products at 3min and 4min, peroxide degradation product at 3.5min and

photodegradation product was appeared at 4min. The peaks of all the degraded

products were resolved from the rifampicin peak.

Conclusion

The RP-HPLC method developed for rifampicin was found to be precise, rapid,

accurate, and stability indicating. Thus the method could be used for determining the

stability of rifampicin.

3.1.3. HPLC METHOD FOR DETERMINATION OF RIFAMPICIN FROM

MACROPHAGE CELLS

Instrumentation




Chromatography


5 μm particle size) column. The mobile phase comprised of methanol: (0.01M)

sodium phosphate buffer pH 5.2 (pH adjusted by o-phosphoric acid) in the ratio 65:35

v/v. The mobile phase was filtered through a nylon membrane (0.22 μm, Pall

Gelman) and degassed by sonication prior to use. Chromatography was performed at

room temperature under isocratic conditions at a flow rate of 1 mL/min. UV detection

was done at a λmax of 254 nm.[Panchgnula R, et al, 1999]


Rifampicin was accurately weighed (10mg) and transferred to a 10 mL volumetric

flask. The volume was made up to 10mL with methanol to obtain a stock solution

(1000μg/mL). From the above solution, 0.2mL was diluted upto 10mL with mobile

phase to get concentration of (200μg/mL)(Stock solution). Aliquots of this solution



corresponding to 0.1, 0.2, 0.3, 0.4, 0.5 and 0.6mL were diluted to 10mL to get

concentration range from 2 to 12μg/mL. Each solution was injected twice to obtain

the output in duplicate. Average of the peak areas were considered for calculation

purposes.

Recovery of RIF from macrophage cells

A suspension of U-937 of histiocytic lymphoma origin macrophage like cells at a cell

density 2*106 cells/mL in RPMI media containing 10% fetal calf serum was prepared.

A solution of 0.5% sodium dodecyl sulphate (in PBS) was added to the cell

suspension in a ratio of (1:1) and the dispersion sonicated to enable cell lysis. The

dispersion was than centrifuged, the supernatant served as blank. To determine RIF

recovery from macrophage cells, RIF solution (100µL) (20–1200µg/mL) was spiked

to drug-free blank macrophage cells (400µL) and vortexed vigorously for 2min.

Methanol (500µL) was added to precipitate protein and the dispersion vortexed for

2min followed by centrifuged at 20,000 rpm for 20min at 25ºC. The supernatant was

injected into the HPLC system. Recovery of RIF from macrophage cells was

calculated by comparing the peak heights of standard RIF solution.

Validation

The chromatographic method was validated for linearity, specificity, sensitivity,

precision, accuracy and stability.

i) Linearity: All validation runs were performed in triplicate to assess variation.

Calibration curves were constructed in macrophage cells over the concentration range

2 – 12 μg/ml for RIF.

ii) Sensitivity: Limit of quantification (LOQ) of standard drug and spiked

macrophage cells was determined at a signal to noise ratio of 1:10.

iii) Precision: System precision (repeatability) was determined by performing four

consecutive injections of the 2μg/ml for RIF. Method precision was determined by

injecting three different samples of 4μg/ml for RIF prepared individually.


Rifampicin from macrophage cells

The developed method showed good resolution of the drug with retention time (RT)

of 7.2 mins with no interference. The chromatograms indicate that RIF peaks are well

separated from other peaks of cells protein (Figure 3.5). The HPLC analytical method

was found to be linear between concentration range of 2-12 μg/ml in the presence of



macrophage cells and other media supplements with high correlation coefficient of

0.9995 (Figure 3.6). The standard curve of RIF after extraction from macrophage cells

is shown in Fig 3.6.

Figure 3.5: Chromatogram of RIF after recovery from macrophage cells

Figure 3.6: Standard curve of rifampicin after recovery from macrophage cells

Table 3.7: Recovery data of RIF from macrophage cells

Conc. In ng/ml Avg std area Avg spiked area %recovery 1000 35210 31828

91.68 34257 30512 33589 32781

8000 307766 281687 94.7 315488 295674

294567 291874 Average % recovery 93.19

Recovery was greater than 90% (Table 3.7) and low RSD values (<5%) were obtained

for system and method precision (Table 3.8).

Blank cells

RIF from macrophage cells



Table 3.8: Precision data of RIF from macrophage cells

Sample No. Area

System precision(2000 ng/ml)

Method precision (4000 ng/ml)

1 88013 183159 2 84793.5 173120 3 89796.75 171012.5 4 89312.75 %RSD 2.56 3.69

HPLC method was validated for linearity, specificity, sensitivity, precision, &

stability thus establishing that the method can be efficiently used for uptake study of

RIF from macrophage cells.

Conclusion

The RP-HPLC method developed for rifampicin was found to be specific, precise,

rapid and accurate. Thus the method could be efficiently used for uptake study of

rifampicin in macrophage cells.

3.1.4. HPLC METHOD FOR DETERMINATION OF RIFAMPICIN FROM

PLASMA AND LUNG HOMOGENATE

Instrumentation:




Chromatography


5 μm particle size) column. The mobile phase comprised of methanol: (0.01M)

sodium phosphate buffer pH 5.2 (pH adjusted by o-phosphoric acid) in the ratio 65:35

v/v. The mobile phase was filtered through a nylon membrane (0.22 μm, Pall

Gelman) and degassed by sonication prior to use. Chromatography was performed at

room temperature under isocratic conditions at a flow rate of 1 mL/min. UV detection

was done at a λmax of 254 nm.[Panchgnula R, et al, 1999]


Rifampicin (10mg) was accurately weighed and transferred to a 10 mL volumetric

flask. Rifampicin was dissolved by sonication, the volume was made up to 10 mL

with methanol (1000μg/mL) and 0.1 ml was diluted to 10 ml with mobile phase to



obtain concentration of 10μg/mL (Stock solution). Aliquots of this solution

corresponding to 0.05, 0.1, 0.25, 0.5, 1 and 5mL were diluted to 10 mL with mobile

phase to get concentrations of 50, 100, 250, 500, 1000, and 5000 ng/mL. Each

solution was injected twice to obtain the output in duplicate. Average of the peak

areas were considered for calculation purposes.

i) Recovery from plasma: Rat blood was collected in tubes containing 4.1% EDTA,

centrifuged and the plasma separated. To determine RIF recovery from plasma, RIF

solution (100µL) (20–1200µg/mL) was spiked to drug-free plasma (400µL) and

vortexed vigorously for 2min. Methanol (500µL) was added to precipitate protein and

the dispersion vortexed for 2min followed by centrifuged at 20,000 rpm for 20min at

25ºC. The supernatant was injected into the HPLC system. Recovery of RIF from

plasma was calculated by comparing the peak heights of standard RIF solution.

ii) Recovery from lung homogenate: Rats were sacrificed by cervical dislocation

and the lungs placed in phosphate buffered saline PBS, pH 7.4 and homogenized

using a tissue homogenizer. To determine RIF recovery from lung homogenate, RIF

solution (100µL) (20–1200µg/mL) was spiked to drug-free blank lung homogenate

(400µL) and vortexed vigorously for 2min. Methanol (500µL) was added to

precipitate protein and the dispersion vortexed for 2min followed by centrifuged at

20,000 rpm for 20min at 25ºC. The supernatant was injected into the HPLC system.

Recovery of RIF from lung homogenate was calculated by comparing the peak

heights of standard RIF solution.

Validation




Calibration curves were constructed in plasma over the concentration range 50 –

5000ng/ml and in lung homogenate over the concentration range 50-5000ng/ml.

ii) Sensitivity: Limit of quantification (LOQ) of standard drug and spiked plasma and

spiked lung homogenate was determined at a signal to noise ratio of 1:10.


consecutive injections of the 250ng/ml plasma and lung homogenate extracted drug

samples. Method precision was determined by injecting three different samples

(500ng/ml) prepared individually.




a) Plasma

The retention time of RIF was found to be 10.4 mins (Figure 3.7). The method was

found to be specific as blank plasma showed no interference with RIF. LOQ of RIF

was found to be 50ng/ml for plasma. HPLC chromatograms of blank plasma and RIF

recovered from plasma are as shown in Figure 3.7. The chromatograms indicate that

RIF peaks are well separated from other peaks in rat plasma. The method was found

to be linear over the concentration range 50-10000 ng/ml in rat plasma. The slope of

the equation was found to be 32.14 with r2 of 0.999. Figure 3.8 shows the linearity

curve of RIF recovered from rat plasma.

Figure 3.7: Chromatogram of RIF after recovery from rat plasma

Figure 3.8: Standard curve of RIF after recovery from rat plasma

Recovery of RIF from rat plasma was optimized greater than 90% as given in Table

3.9. Recovery data of RIF from rat plasma and precision data of RIF in rat plasma is

shown in Table 3.10.

Blank Plasma

RIF from Plasma RT-10.4 min



Table 3.9: Recovery data of RIF from rat plasma

Conc. (ng/ml) Avg. std area Avg. spiked area % Recovery 100 4151.6 3848

91.65 4271.5 3963 4396.9 3939.25

1000 35210 31828 92.3 34257 30512


Table 3.10: Precision data of RIF from rat plasma

Sample No. Area



1 9134 15666.25 2 10053 15123.5 3 9346.5 16012.5 4 9312.75

%RSD 4.28 2.87

The HPLC method was well validated for linearity, specificity, sensitivity, precision,

and stability thus establishing that the method can be efficiently used for

pharmacokinetic study of RIF from rat plasma.

b) Lung homogenate

Figure 3.9: Chromatogram of RIF after recovery from lung homogenate

Figure 3.10: Standard curve of RIF after recovery from lung homogenate

Blank lung homogenate

RIF from lung homogenate RT-10.4 min



The retention time of RIF was found to be 10.5 mins (Figure 3.9). The method was

found to be specific as blank lung homogenate showed no interference with RIF. LOQ

of RIF was found to be 50ng/ml for lung homogenate. HPLC chromatograms of blank

lung homogenate and RIF recovered from lung homogenate are as shown in Figure

3.9. The chromatograms indicate that RIF peaks are well separated from other peaks

in rat lung homogenate. The method was found to be linear over the concentration

range 50-1000ng/ml in rat lung homogenate. The slope of the equation was found to

be 30.40 with R2 of 0.999. Figure 3.10 show the standard curve of RIF after extraction

from rat lung homogenate.

Recovery was optimized greater than 85%. Recovery data of RIF from rat lung

homogenate and precision data of RIF in rat lung homogenate is shown in Table 3.11

and Table 3.12 respectively.

Table 3.11: Recovery data of RIF from lung homogenate


87.27 4271.5 3853 4396.9 3687.3

1000 35210 29828 87.48 34257 28512


Table 3.12: Precision data of RIF from lung homogenate

Sample No. Area



1 8634 14857.25 2 9173 14356.25 3 8146.5 15123.5 4 8696.25

RSD 4.84 2.63

Conclusion:


and stability thus establishing that the method can be efficiently used for lung uptake

study of RIF from rat.



3.2. RIFAMPICIN AND ETHAMBUTOL

3.2.1. UV SPECTROPHOTOMETRIC METHOD FOR DETERMINATION OF

RIFAMPICIN IN COMBINATION WITH ETHAMBUTOL

Introduction

UV spectrophotometry method was developed for the routine analysis of rifampicin

and ethambutol in combination during dissolution studies and drug content analysis.

Although the method cannot be used easily for multicomponent formulations or

formulations containing interfering excipients, but ease of operation, low cost and low

analysis time make it the most popular analytical technique.

Materials

Ethambutol was obtained as a gift sample from Themis Medicare (India). Distilled

water was used during UV analysis. All other chemicals and solvents were analytical

– reagent grade and were purchased from s. d. fine chemicals, India. Instrumentation

UV1650PC, Schimadzu Corporation US spectrophotometer was used for analysis

Method

RIF was analyzed from the combination by UV-Visible spectrophotometry at λmax

475 nm as described in chapter II, without any interference. For analysis of ETH, RIF

was extracted from the combination solution by using dichloromethane (DCM). ETH

remaining in the aqueous phase was then determined by UV- spectrophotometry at

λmax of 412nm after derivatization with acetyl acetone reagent by a method

developed in our laboratory.

Active agent Medium λmax Beer’s law range Slope Intercept r2

Rifampicin Water 475 nm 5-30 mcg/ml 0.0116 0.0008 0.999

Ethambutol Water 412 nm 1-14 mcg/ml 0.054 0.002 0.999


Drug content values ranged between 97.5-101.5% for RIF and 97-102% for ETH

when analyzed in combinations with the developed extraction method

Conclusion


RIF and ETH from combination of both.



3.2.2. HPLC METHOD FOR DETERMINATION OF ETHAMBUTOL FROM

MACROPHAGE CELLS

Chromatography


5 μm particle size) column. The mobile phase comprised of 1 mM CuSO4 .5H2O :

Tetrahydrofuran (THF) [75:25]. The mobile phase was filtered through a nylon

membrane (0.22 μm, Pall Gelman) and degassed by sonication prior to use.

Chromatography was performed at room temperature under isocratic conditions at a

flow rate of 1 mL/min. UV detection was done at a λmax of 260 nm.


ETH (10mg) was accurately weighed and transferred to a 10 mL volumetric flask.

The volume was made up to 10 mL with glass distilled water to obtain a stock

solution (1000μg/mL). From the above solution, 1 ml was diluted upto 10 ml with

mobile phase to get concentration of (100μg/mL)(Stock solution). Aliquots of this

solution corresponding to 0.1, 0.25, 0.5, 0.75, and 1mL were diluted to 10 mL with

mobile phase to get concentration range from 1 to 10μg/mL. Each solution was

injected twice to obtain the output in duplicate. Average of the peak areas were

considered for calculation purposes.

Recovery of ETH from macrophage cells





dispersion was than centrifuged, the supernatant served as blank. To determine ETH

recovery from macrophage cells, ETH solution (100µL) (20–1200µg/mL) was spiked

to drug-free lysed macrophage cells (400µL) and vortexed vigorously for 2min

followed by addition of equal volume (500µL) of methanol. The resulting mixture

was vortexed vigorously for 2 min and centrifuged at 20,000 rpm for 20 min at 25 ºC.

The supernatant containing ETH was kept with mobile phase containing CuSO4.5H2O

for 45mins to form a derivative quantifiable/detected by HPLC. The supernatant was

injected into the HPLC system. Recovery of ETH from macrophage cells was

calculated by comparing the peak heights of standard ETH solution.



Validation





2 – 12 μg/ml for RFM and 1-10 μg/ml for ETH.




consecutive injections of the 2μg/ml for RFM and 1μg/ml for ETH. Method precision

was determined by injecting three different samples of 4μg/ml for RFM and 2.5μg/ml

for ETH prepared individually.

RESULTS AND DISCUSSION:

Figure 3.11: Chromatogram of ETH after recovery from macrophage cells

Figure 3.12: Standard curve of ETH after recovery from macrophage cells


of 8.5 mins with no interference. The chromatograms indicate that ETH peaks are

well separated from other peaks after recovery from macrophage cells (Figure 3.11).

Blank macrophage cells

ETH from macrophage cells RT- 8.5min



The HPLC analytical method was found to be linear between concentration range of

1-10 μg/ml in the presence of macrophage cells and other media supplements with

high correlation coefficient of 0.997 (Figure 3.12).

Recovery was greater than 90% (Table 3.13) and low RSD values (<5%) were

obtained for system and method precision (Table 3.14).

Table 3.13: Recovery data of ETH from macrophage cells

Conc. In ng/ml Avg std area Avg spiked area %recovery 1000 25356.79 22710.8

92.6 27487 26570 27987 25570

7500 136546 120821 91.66 141987 131950


Table 3.14: Precision data of ETH from macrophage cells

Sample No. Area



1 25356.79 43842.4 2 27487 41143 3 27987 44123.5 4 26312.75

%RSD 4.41 3.82

Further the HPLC method was well validated for linearity, specificity, sensitivity, precision, and stability thus establishing that the method can be efficiently used for uptake study of ETH from macrophage cells.

3.2.3. HPLC METHOD FOR DETERMINATION OF ETHAMBUTOL FROM PLASMA AND LUNG HOMOGENATE

Chromatography


5 μm particle size) column. The mobile phase comprised of 1 mM CuSO4 .5H2O :

Tetrahydrofuran (THF) [75:25]. The mobile phase was filtered through a nylon

membrane (0.22 μm, Pall Gelman) and degassed by sonication prior to use.

Chromatography was performed at room temperature under isocratic conditions at a

flow rate of 1 mL/min. UV detection was done at a λmax of 260 nm.




ETH (10mg) was accurately weighed and transferred to a 10 mL volumetric flask.

The volume was made up to 10 mL with glass distilled water (1000μg/mL). From the

above solution, 0.1mL and 1mL was diluted to 10mL with glass distilled water to get

concentration of 10μg/mL and 100μg/mL respectively (Stock solutions). Aliquots of

this solution corresponding to 0.05, 0.2, 0.5, 1 and 5mL were diluted to 10mL with

mobile phase to get concentrations of 50, 200, 500, 1000, and 5000ng/mL. The

samples were kept for 45mins for derivatization with mobile phase prior to injection.

Each solution was injected twice to obtain the output in duplicate. Average of the

peak areas were considered for calculation purposes.

i) Recovery from plasma: Rat blood was collected in tubes containing 4.1% EDTA,

centrifuged and the plasma separated. In order to determine recovery, ETH solution

(100µL) (0.5–100µg/mL) was spiked to drug-free plasma (400µL) and vortexed

vigorously for 2 min followed by addition of equal volume (500µL) of methanol to

induce protein precipitation. The resulting mixture was vortexed vigorously for 2min

and centrifuged at 20,000 rpm for 20min at 25 ºC. Supernatant was evaporated to

dryness under nitrogen gas. The residue obtained was reconstituted with 100μL

mobile phase, vortex mixed for 1 min, kept for 45mins and injected into HPLC

system. Recovery of ETH from plasma was calculated by comparing the peak heights

of standard ETH solution.

ii) Recovery from lung homogenate: Rats were sacrificed by cervical dislocation

and the lungs placed in phosphate buffered saline PBS, pH 7.4 and homogenized

using a tissue homogenizer in order to determine recovery, ETH solution (100µL)

(0.5–100µg/mL) was spiked to drug-free lung homogenate (400µL) and vortexed

vigorously for 2min followed by addition of equal volume (500µL) of methanol to

induce protein precipitation. The resulting mixture was vortexed vigorously for 2min

and centrifuged at 20,000 rpm for 20 min at 25 ºC. Supernatant was evaporated to

dryness under nitrogen gas. The residue obtained was reconstituted with 100μL

mobile phase, vortex mixed for 1 min, kept for 45mins and injected into HPLC

system. Recovery of RIF from lung homogenate was calculated by comparing the

peak heights of standard RIF solution.



Validation:




Calibration curves were constructed in plasma over the concentration range 50 –

5000ng/ml and in lung homogenate over the concentration range 50-5000ng/ml.

ii) Sensitivity: Limit of quantitation (LOQ) of standard drug and spiked plasma and

spiked lung homogenate was determined at a signal to noise ratio of 1:10.


consecutive injections of the 250ng/ml plasma and lung homogenate extracted drug

samples. Method precision was determined by injecting three different samples

(500ng/ml) prepared individually.

Results and Discussion:

a) Plasma method

Figure 3.13: Chromatogram of ETH after recovery from plasma

Figure 3.14: Standard curve of ETH after recovery from plasma

Blank Plasma

ETH from plasma RT- 8.8min



The retention time of ETH was found to be 8.8mins. The method was found to be

specific as blank plasma showed no interference with ETH. LOQ of ETH was found

to be 50ng/ml for plasma. HPLC chromatograms of ETH in mobile phase, and rat

plasma spiked with ETH are as shown in Figure 3.13. The chromatograms indicate

that ETH peaks are well separated from other peaks in rat plasma. The method was

found to be linear over the concentration range 50-10000 ng/ml in rat plasma. The

slope of the equation was found to be 62.17 with r2 of 0.998 (Figure 3.14).

Extraction efficiency was optimized greater than 80% as given in Table 3.15.

Recovery data of ETH from rat plasma and precision data of ETH in rat plasma is

shown in Table 3.15 and Table 3.16 respectively.

Table 3.15: Recovery data of ETH from plasma


84.33 8496.3 6822 8910 8630

1000 52687 39934 89.65 53815 52877


Table 3.16: Precision data of ETH from rat plasma

Sample No. Area



1 18500 48924 2 19000 46683.5 3 16881 51265.75 4 18156.5

%RSD 4.98 4.68


and stability thus establishing that the method can be efficiently used for

pharmacokinetic study of ETH from rat plasma.

b) Lung homogenate method

The retention time of ETH was found to be 7.7mins. The method was found to be

specific as blank lung homogenate showed no interference with ETH as shown in

Figure 3.15. LOQ of ETH was found to be 50ng/mL for lung homogenate. HPLC



chromatograms of standard ETH in mobile phase, and rat lung homogenate spiked

with ETH are as shown in Figure 3.15. The chromatograms indicate that ETH peaks

are well separated from other peaks in rat lung homogenate. The method was found to

be linear over the concentration range 50-1000ng/mL in rat lung homogenate. The

slope of the equation was found to be 58.68 with r2 of 0.998 (Figure 3.16).

Figure 3.15: Chromatogram of ETH after recovery from lung homogenate

Figure 3.16: Standard curve of ETH after recovery from lung homogenate

Recovery was optimized greater than 80%. Recovery data of ETH from rat lung

homogenate and precision data of ETH in rat lung homogenate is shown in Table 3.17

and Table 3.18 respectively.

Table 3.17: Recovery data of ETH from lung homogenate

Conc. In ng/ml Avg std area Avg spiked area %recovery 200 7903.5 6578

80.62 8696.3 6136 8830 7789

1000 51516 44123 85.96 56715 48578


Blank lung homogenate

ETH from lung homogenate RT- 7.7min



Table 3.18: Precision data of ETH from lung homogenate

Sample No. Area



1 17932 48578 2 18124 45568.5 3 16981 48896.25 4 16556.5

%RSD 4.31 3.85


and stability thus establishing that the method can be efficiently used for lung uptake

study of ETH from rat.

3.2.4. RIFAMPICIN AND ETHAMBUTOL COMBINATION

Rifampicin and Ethambutol combination was analysed by HPLC in 2 separate

injections. The collected macrophage cells/plasma/lung homogenate was divided into

2 parts and processed separately for RIF and ETH and analysed by HPLC as

described above.

3.3. RIFAMPICIN AND MSDNC-22 COMBINATION

3.3.1. UV SPECTROPHOTOMETRIC METHOD FOR RIFAMPICIN AND

MSDNC-22 COMBINATION

Introduction

UV spectrophotometric method was developed for the routine analysis of rifampicin

and MSDNC-22 in combination during dissolution studies and drug content analysis.

Although the method cannot be used easily for multicomponent formulations or

formulations containing interfering excipients, but ease of operation, low cost and low

analysis time make it the most popular analytical technique.

Materials

MSDNC-22 is a new cell wall modulator synthesized. Distilled water was used during

UV analysis. All other chemicals and solvents were analytical – reagent grade and

were purchased from s. d. fine chemicals, India.

Instrumentation

UV1650PC, Schimadzu Corporation US spectrophotometer was used for analysis.




Rifampicin: Rifampicin (10mg) was accurately weighed and transferred to a 10mL

volumetric flask. The volume was made up to 10mL with tetrahydrofuran (THF) to

obtain a stock solution (1000μg/mL). Aliquots of this solution corresponding to 0.01,

0.05, 0.1, 0.15, 0.2, 0.25 and 0.3mL were diluted to 10mL with mixture of THF:water

(1:1) to obtain solutions in the concentration range of 1–30 μg/mL. The absorbance of

these solutions was recorded at 425nm and 475 nm and plotted vs. the concentration

to give the Beer Lamberts plot for the standard curve.

MSDNC-22: MSDNC-22 (10mg) was accurately weighed and transferred to a 10mL

volumetric flask. The volume was made up to 10mL with THF to obtain a stock

solution (1000μg/mL). Aliquots of this solution corresponding to 0.01, 0.05, 0.1, 0.15,

0.2, 0.25 and 0.3mL were diluted to 10mL with mixture of THF:water (1:1) to obtain

solutions in the concentration range of 1–30 μg/mL. The absorbance of these

solutions was recorded at 425nm and 475 nm and plotted vs. the concentration to give

the Beer Lamberts plot for the standard curve.

Simultaneous equation

The two drugs in combination were estimated using the simultaneous equation

method and the equations are as follow.

Cx = (A2*ay1- A1*ay2)/(ax2*ay1-ax1*ay2)-----------------------------------------1

Cy = (A2*ay1- A1*ay2)/(ax2*ay1-ax1*ay2)-----------------------------------------2

For MSDNC-22 in combination with Rifampicin

Where: Cx=Concentration of MSDNC-22

Cy=Concentration of Rifampicin

A1= Absorbance of unknown sample at λmax 425 nm and

A2= Abs. of unknown sample at λmax 475 nm

ax1 ax2 ay1 ay2

0.0399 0.0263 0.0079 0.0196



Results and Discussion UV spectroscopic/colorimetric methods was developed for routine analysis of RIF

and MSDNC-22 alone and in combination and are summarized in table 3.23.

Table 3.23: UV spectroscopic/ colorimetric analysis data RIF-MSDNC-22

Active

agent Medium λmax Beer’s law

range Slope Intercept r2

Rifampicin THF:Water

(1:1)

425 5-30 mcg/ml 0.0079 0.0005 0.9998

475 5-30 mcg/ml 0.0196 0.0003 0.9996

MSDNC-22 THF:Water

(1:1)

425 5-25 µg/ml 0.0399 0.0043 0.9997

475 5-25 µg/ml 0.0263 0.0005 0.9999

Figure 3.17: Standard curves of MSDNC-22 in THF:water (1:1) at a) 425nm b) 475nm

Figure 3.18: Standard curves of rifampicin in THF:water (1:1) at

a) 425nm b) 475nm

Conclusion


RIF and MSDNC-22 from combination of both.



3.3.2. STABILITY INDICATING HPLC METHOD

A stability indicating RP-HPLC method for the analysis of RIF and MSDNC-22

combination as a bulk drug and in pharmaceutical formulations was developed and

validated.

Introduction

The objective of the present work was to develop and validate a stability indicating

RP-HPLC method for the assay of rifampicin and MSDNC-22 as a bulk drug and in

pharmaceutical formulations.

Materials

Rifampicin was obtained as a gift sample from Maneesh Pharma (India). MSDNC-22

is a new chemical entity synthesized in Prof. M.S. Degani laboratory at Institute of

Chemical Technology. Glass distilled water was used during HPLC analysis. All

other chemicals and solvents were analytical – reagent grade and were purchased

from s.d. Fine Chemicals, India Instrumentation:




Chromatography


5μm particle size) column. The mobile phase comprised of THF:Methanol: water in

the ratio 30:30:40 v/v. The mobile phase was filtered through a nylon membrane

(0.22μm, Pall Gelman) and degassed by sonication prior to use. Chromatography was

performed at room temperature under isocratic conditions at a flow rate of 1 mL/min.

UV detection was done at a λmax of 254 nm.


Rifampicin (10mg) and MSDNC-22 (10mg) was accurately weighed and transferred

to a 10 mL volumetric flask. The volume was made up to 10 mL with methanol to

obtain a stock solution (1000μg/mL). Aliquots of this solution corresponding to 0.01,

0.05, 0.1, 0.15, 0.2, 0.25 and 0.3 were diluted to 10 mL with the mobile phase to

obtain solutions in the concentration range of 1–30 μg/mL. Each solution was injected

in triplicate. Average of the peak areas were considered for calculation purposes.



Validation

a) Stability of analyte in solution: The stability of rifampicin and MSDNC-22

combination in mobile phase was assessed by injecting the standard solution

(30μg/ml) at interval of 0, 3 and 6 hrs post preparation kept in amber colored

volumetric flasks at room temperature. The chromatograms were checked for

presence of peaks corresponding to degraded product.

b) Linearity: Standard solutions (1, 5, 10, 15, 20, 25 and 30 μg/mL), each in three

replicates, were injected into the system. The method of linear regression was used for

data evaluation. Peak areas were plotted against theoretical concentrations of

standards. Linearity was expressed as a correlation coefficient.

c) Precision: System precision (repeatability) was determined by performing five

consecutive injections of the 30 μg/ml standard solution. Method precision was

determined by injecting three different samples (30 μg/ml) prepared individually.


It was necessary to perform forced degradation studies to verify and prove the

stability-indicating feature of the proposed method. Intentional degradation was

attempted by heating the drug in the presence of base, acid and hydrogen peroxide

and exposing to sunlight.

i) Acid degradation: To 1mL of standard stock solution (300μg/mL) of rifampicin

and MSDNC-22 combination, 1 mL of 0.1N HCl was added and the solution was

placed in a boiling water bath for 2h. The sample was allowed to cool to room

temperature and neutralized using 0.1N NaOH. The volume was adjusted to 10 ml

with distilled water, and this solution was injected in the HPLC column.

ii) Base degradation: To 1mL of standard stock solution (300μg/mL) of rifampicin

and MSDNC-22, 1 mL of 0.1N NaOH was added and the solution was placed in a

boiling water bath for 2h. The sample was allowed to cool to room temperature and

neutralized using 0.1N HCl. The volume was adjusted to 10 ml with glass-distilled

water, and this solution was injected onto the HPLC column.

iii) Oxidation: To 1mL of standard stock solution (300μg/mL) of rifampicin and

MSDNC-22, 1 mL of 3% H2O2 was added and the solution was placed in a boiling

water bath for 2h. The sample was allowed to cool to room temperature. The volume



was adjusted to 10 ml with glass-distilled water, and this solution was injected onto the

HPLC column.

iv) Photodegradation: Drug solution (30μg/mL) in methanol was exposed to

sunlight for 2h and this solution was injected onto the HPLC column.

The degraded samples were analyzed against an untreated control sample.


a) Stability of analyte in solution

Rifampicin and MSDNC-22 was found to be stable in the mobile phase, when the

standard solution of strength 10μg/mL was analyzed at 0-6h post preparation. No

peaks corresponding to the degradation products were observed. A low RSD (<2)

value indicated that there was no significant change in the drug peak area (Table

3.20).

Table 3.20: Stability of rifampicin and MSDNC-22 in mobile phase

RIF Area 1 Area 2 Average SD SE %RSD

0 hr 739019.3 747143.5 743081.4 5744.712 4062.125 0.7730933 hr 749146 743750.3 746448.1 3815.364 2697.87 0.5111366 hr 746895.5 746751.8 746823.6 101.6466 71.875 0.013611

Average %RSD 0.4326 MSDNC-22 Area 1 Area 2 Average SD SE %RSD

0 hr 1133610 1140362 1136986.0 4774.385 3376.0 0.4199163 hr 1120521 1083644 1102082.1 26076.15 18438.6 2.3660816 hr 1118988 1075519 1097253.3 30737.58 21734.8 2.80132

Average %RSD 1.8624

b) Linearity

Figure 3.19: Standard curve of rifampicin by HPLC



Figure 3.20: Standard curve of MSDNC-22 by HPLC

Graph of the peak area vs concentration was plotted in order to check the linearity.

The developed method was found to be linear between concentration range of 1-

30μg/mL (Figure 3.19 and 3.20). The regression coefficient was found to be 0.9987

for RIF and 0.9994 for MSDNC-22.

c) Precision

Low RSD values of 1.84 per cent and 1.28 per cent were obtained for system and

method precision respectively (Table 3.21)

Table 3.21: Precision study of the RIF and MSDNC-22 assay

Sample No.

Area for Rifampicin Area for MSDNC-22System

precision (30 µg/ml)

Method precision

(30 µg/ml)

System precision

(30 µg/ml)

Method precision

(30 µg/ml)1 750446 750446 1114051 1114051 2 743081 739328 1136986 1139463 3 734956 740387 1124987 1126433 4 743872 - 1142386 - 5 739845 - 1122674 -

% RSD 0.7658 0.8254 1.0100 1.1278


The HPLC procedure was optimized with view to develop a stability indicating

method so as to resolve the degraded products from the drugs. Various mobile phase

compositions were tried so as to obtain a sharp peak and also resolve the peaks of

degraded product from the peak of drug. The mobile phase consisting of

THF:Methanol: water in the ratio of 30:30:40 per cent v/v resulted in a retention time

of 7.7 min for rifampicin and 15.3 min for MSDNC-22. The chromatograms of

rifampicin and MSDNC-22 (undegraded) and rifampicin and MSDNC-22 degraded in



the presence of acid, base, hydrogen peroxide (H2O2) (oxidative degradation) and

light (photo degradation) are shown in Figure 3.21 and indicated a good separation of

the undegraded drug from the degradation products. The peaks of all the degraded

products were resolved from the rifampicin and MSDNC-22 peak. The

chromatograms after forced degradation are shown in Figure 3.21.

a) Standard RIF and MSDNC-22

b) Acid degraded RIF and MSDNC-22 c) Base degraded RIF and MSDNC-22

d) H2O2 degraded RIF and MSDNC-22 e) Photo degraded RIF and MSDNC-22

Figure 3.21: Forced degradation studies of RIF and MSDNC-22

Conclusion

The RP-HPLC method developed for rifampicin was found to be precise, rapid,

accurate, and stability indicating. Thus the method could be used for determining the

stability of rifampicin.

3.3.3. HPLC METHOD FOR DETERMINATION OF RIFAMPICIN AND

MSDNC-22 FROM MACROPHAGE CELLS

Instrumentation




Chromatography


5μm particle size) column. The mobile phase comprised of THF:Methanol: water in



the ratio 30:30:40 v/v. The mobile phase was filtered through a nylon membrane

(0.22μm, Pall Gelman) and degassed by sonication prior to use. Chromatography was

performed at room temperature under isocratic conditions at a flow rate of 1 mL/min.

UV detection was done at a λmax of 254 nm.


Rifampicin and MSDNC-22 was accurately weighed (10mg) and transferred to a

10mL volumetric flask. The volume was made up to 10mL with methanol to obtain a

stock solution (1000μg/mL). Aliquots of this solution corresponding to 0.01, 0.05,

0.1, 0.2 and 0.3mL were diluted to 10mL to get concentration range from 1-30μg/mL.

Each solution was injected twice to obtain the output in duplicate. Average of the

peak areas were considered for calculation purposes.

Recovery of RIF and MSDNC-22 from macrophage cells





dispersion was than centrifuged, the supernatant served as blank. To determine RIF

and MSDNC-22 recovery from macrophage cells, RIF-MSDNC-22 solution (100µL)

(20–1200µg/mL) was spiked to drug-free lysed macrophage cells (400µL) and

vortexed vigorously for 2 min followed by addition of equal volume (500µL) of

methanol. The resulting mixture was vortexed vigorously for 2 min and centrifuged at

20,000 rpm for 20 min at 25 ºC. The supernatant was injected into the HPLC system.

Recovery of RIF and MSDNC-22 from macrophage cells was calculated by

comparing the peak heights of standard RIF and MSDNC-22 solutions.

Validation





1 – 30μg/mL for RIF and MSDNC-22.






consecutive injections of the 10μg/ml for RIF and MSDNC22. Method precision was

determined by injecting three different samples of 10μg/ml for RIF and MSDNC22

prepared individually.


Rifampicin and MSDNC-22 from macrophage cells


of 7.7 mins for RIF and 15.3min for MSDNC-22 with no interference. The

chromatograms indicate that RIF and MSDNC-22 peaks are well separated from other

peaks in macrophage cells. The HPLC analytical method was found to be linear over

concentration range of 1-30 μg/ml in the presence of macrophage cells and other

media supplements with high correlation coefficient of 0.9995 (Figure 3.23). The

standard curve of RIF after extraction from macrophage cells is shown in Figure 3.22.

a) b)

Figure 3.22: Chromatogram a) Blank macrophage cells b) RIF and MSDNC22 after

recovery from macrophage cells

Rifampicin MSDNC22

Figure 3.23: Standard curve after recovery from macrophage cells

Recovery was greater than 90% for both RIF and MSDNC22 (Table 3.22 and 3.23)

and low RSD values (<5%) were obtained for system and method precision (Table

3.24).

Blank macrophage cells RIF and MSDNC-22 from macrophage cells

RIF RT-7.7min

MSDNC-22 RT-15.3min



Table 3.22: Recovery data of RIF from macrophage cells

Concentration (µg/mL)

Average Area of

Standard

Average Area of extracted RIF

Extraction Efficiency/ recovery

1 7620.8 6796.9 89.18 5 53173.4 49171.1 92.47 10 150327.3 130365.5 86.72 20 296432.8 281359.6 94.91 30 469845.3 452355.8 96.27

Average Extraction efficiency 91.91

Table 3.23. Recovery data of MSDNC22 from macrophage cells

Concentration (µg/mL)

Average Area of

Standard

Average Area of extracted

MSDNC22

Extraction Efficiency/ recovery

1 32863.1 30708.0 93.44 5 173521.6 167801.7 96.70 10 378521.5 367331.5 97.04 20 774322.9 762150.2 98.42 30 1280367.2 1195225.5 93.35

Average Extraction efficiency 95.79

Table 3.24: Precision data of RIF and MSDNC22 from macrophage cells

Sample No.

System precision Area (10 µg/mL)

Method precision Area (10 µg/mL)

RIF MSDNC22 RIF MSDNC22 1 130832 367060 130832 367060 2 129899 367603 132157 368921 3 131443 370234 128930 371243 4 128842 369952 - -

Average 130254 368712 130639 369074 RSD 0.87 0.44 1.24 0.57

HPLC method was validated for linearity, specificity, sensitivity, precision, &

stability thus establishing that the method can be efficiently used for macrophage

uptake study of RIF and MSDNC22.

Conclusion

The RP-HPLC method developed for rifampicin and MSDNC22 was found to be

specific, precise, rapid and accurate. Thus the method could be efficiently used for

macrophage uptake study of RIF and MSDNC22.

4. Experimental methods



4. EXPERIMENTAL METHODS 4.1. MATERIALS

Poly (ethylene sebacate) [PES] synthesized in our laboratory (Mw= 11300) earlier,

was used, rifampicin (RIF), ethambutol (ETH), poly vinyl alcohol (PVA), and

trehalose 100 (Hayashibara Co. Ltd., Japan) were kindly gifted by Maneesh Pharma

(Mumbai, India), Themis medicare, Colorcon Asia Pvt ltd and Gangwal Chemicals

Pvt. Ltd. (Mumbai, India) respectively. Lutrol-F-68 (polyoxyethylene

polyoxypropylene block co-polymer) was a gift sample from BASF. Folic acid(FA),

dichloromethane AR, methanol AR, methyl ethyl ketone AR, dioctyl sodium

sulphosuccinate AR (Aerosol OT , AOT), tetrahydrofuran AR (THF), stanneous

chloride, disodium hydrogen phosphate AR and sodium chloride AR were purchased

from s. d. fine-chem limited (Mumbai, India). Ethyl alcohol AR (99.9% pure) was

purchased from Changshu Yangyuan Chemical (China). Filtered (0.45 µ membrane

filter) doubled distilled water was used for preparation of nanoparticles. All other

chemicals and solvents were either spectroscopic or analytical grade.

4.2. PREPARATION AND OPTIMIZATION OF NANOPARTICLES

A. PES-RIF nanoparticles with and without folic acid

Rifampicin loaded PES nanoparticle were prepared by nanoprecipitation (Fessi et al

1992). Briefly, rifampicin (20mg) and PES (100mg) were dissolved in

tetrahydrofuran: methanol (9:1ml). The non solvent phases comprised an aqueous

solution of lutrol-F-68 (10mg) in 25ml water. The organic phase was added to the non

solvent phase under magnetic stirring. The dispersion was kept under continuous

stirring on a magnetic stirrer at room temperature till complete evaporation of organic

solvent (approx. 2-3 hrs). The nanoparticle suspension was centrifuged at 15000 rpm

for 30 min and the supernatant analyzed for drug to determine entrapment efficiency.

a) Effect of Lutrol-F-68 concentration

The surfactant concentration evaluated was 0.04, 0.1 and 0.2% in order to find the

optimum concentration for maximum entrapment efficiency (Table 4.1).

b) Effect of drug: polymer ratio

Effect of RIF:PES ratio (drug:polymer) on entrapment efficiency and particle size was

evaluated. Ratio of RIF:PES evaluated include 1:2, 1:3, 1:4 and 1:5 (Table 4.2).



Table 4.1: Effect of Lutrol-F-68 concentration on %EE and particle size

RPES/1 RPES/2 RPES/3

Rifampicin (mg) 20 20 20

PES (mg) 100 100 100

Tetrahydrofuran (ml) 9 9 9

Methanol 1 1 1

Water (ml) 25 25 25

Lutrol-F-68 (mg) 10 (0.04% w/v)

25 (0.10% w/v)

50 (0.20% w/v)

Table 4.2: Effect of drug: polymer ratio on %EE and particle size

RPES/1 RPES/4 RPES/5 RPES/6

Rifampicin (mg) 20 20 20 20

PES (mg) 100 80 60 40

Tetrahydrofuran (ml) 9 9 9 9

Methanol 1 1 1 1

Water (ml) 25 25 25 25

Lutrol-F-68 (mg) 10 (0.04% w/v)

10 (0.04% w/v)

10 (0.04% w/v)

10 (0.04% w/v)

c) Folate anchored nanoparticles (RPESFA/1)

Folate anchoring was achieved by addition of a solution of 0.5% folic acid (0.5ml) in

0.1N NaOH to the preformed nanoparticle dispersion of RPES/1, prior to solvent

removal.

B. PES-RIF-ETH combination nanoparticles with and without folic acid

PES nanoparticles of rifampicin-ethambutol were prepared by the multiple emulsion

technique. Briefly, 0.5ml of an aqueous Ethambutol (20mg) solution was first

emulsified in 10ml dichloromethane (DCM) containing rifampicin (2.5 mg), PES

(100mg) and Aerosol® OT (20mg) by probe sonication for 2minutes (20sec on/10sec

off cycle) to form a primary emulsion. The primary emulsion was poured into 24.5ml

of 0.2% aqueous PVA solution and probe sonicated for 5minutes (20sec on/10sec off

cycle) to form the second water-in-oil-in-water emulsion. The dispersion was kept



under continuous stirring on a magnetic stirrer till complete evaporation of organic

solvent (approx. 2-3 hrs) at room temperature. The nanoparticle suspension was

obtained and evaluated for entrapment efficiency and particle size. RIF-ETH PES

nanoparticles with different ratios of RIF:ETH were prepared by varying the

concentration of RIF and ETH during the method of preparation.

a) Effect of PVA concentration

The PVA concentration evaluated was 0.2, 0.5 and 1% w/v in order to find the

optimum concentration for maximum entrapment efficiency (Table 4.3).

Table 4.3: Effect of PVA concentration on %EE and particle size

REPES/1 REPES/2 REPES/3

Rifampicin (mg) 10 10 10

Ethambutol 20 20 20

PES (mg) 100 100 100

Dichloromethane 10 10 10

Water (ml) 25 25 25

Aerosol OT (mg) 20 20 20

PVA (mg) 50 (0.2% w/v)

125 (0.5% w/v)

250 (1% w/v)

b) Optimization of RIF:ETH ratio

Table 4.4: Optimization of RIF:ETH ratio

REPES/4 REPES/5 REPES/6 REPES/7 REPES/8

Rifampicin (mg) 20 15 2.5 20 20

Ethambutol 20 20 20 5 2.5

PES (mg) 100 100 100 100 100

Dichloromethane 10 10 10 10 10

Water (ml) 25 25 25 25 25

Aerosol OT (mg) 20 20 20 20 20

PVA (mg) 50 (0.2% w/v)

50 (0.2% w/v)

50 (0.2% w/v)

50 (0.2% w/v)

50 (0.2% w/v)

Nanoparticles with RIF:ETH in ratios from 10:1, 1:1, and 1:10 were optimized by

varying the ratios of ETH and RIF in REPES/1 batch during their preparation based



on their entrapment efficiency observed. Various batches evaluated are reported in

(Table 4.4).

c) Folate anchored nanoparticles (REPES6FA/1)


0.1N NaOH to the preformed nanoparticle dispersion of REPESFA/6 prior to solvent

removal.

C. PES-RIF-MSDNC-22 combination nanoparticles with and without folic acid

RIF-MSDNC-22 loaded PES nanoparticle were prepared by nanoprecipitation

method. Briefly, rifampicin (2-20mg), MSDNC-22 (10mg) and PES (100mg) were

dissolved in tetrahydrofuran: methanol (9:1ml). The non solvent phases comprised an

aqueous solution of lutrol-F-68 (10mg) in 25ml water. The organic phase was added

to the non solvent phase under magnetic stirring. The dispersion was kept under

continuous stirring on a magnetic stirrer at a room temperature till complete

evaporation of organic solvent (approx. 2-3 hrs). The nanoparticle suspension was

obtained and evaluated for entrapment efficiency and particle size. RIF-MSDNC-22

PES nanoparticles with different ratios of RIF:MSDNC-22 were prepared by varying

the concentration of RIF and MSDNC-22 during the method of preparation (Table

4.5).

a) Folate anchored nanoparticle (RMPES3FA/1)


0.1N NaOH to the preformed nanoparticle dispersion of RMPES/3, prior to solvent

removal.

Table 4.5: Optimization of RIF:MSDNC-22 ratio

RMPES/1 RMPES/2 RMPES/3

Rifampicin (mg) 15 20 2.5

MSDNC-22 10 2 10

PES (mg) 100 100 100

Tetrahydrofuran 9 9 9

Methanol 1 1 1

Water (ml) 25 25 25

Lutrol-F-68 (mg) 50 (0.2% w/v)

50 (0.2% w/v)

50 (0.2% w/v)



4.3. ISOLATION AND PRESERVATION OF NANOPARTICLES

4.3.1. Freeze-thaw (FT) study

The Rifampicin PES nanoparticles dispersion obtained was centrifuged at 15000 rpm

for 30 min and the supernatant separated. The pellet was re-dispersed in 10 ml water

to obtain nanoparticle concentration 11-12mg/ml, cryoprotectants added and

subjected to freezing at a temperature -70°C, for 12 hrs in deep freezer (Eclipse 400,

RS Biotech, UK) followed by thawing at 28°C. The particle size and polydispersity

index (P.I) before freezing and after thawing was determined by PCS. Selected

batches were subjected to freeze drying using the LABCONCO freeze dryer system

(FreeZone 4.5, USA).

a) Effect of cryoprotectant - type and concentration: Two cryo-protectants namely

mannitol and trehalose and Lutrol-F-68 as stabilizer were evaluated. The ratio of

nanoparticle to cryo-protectant was 1:5, 1:10 and 1:20, while the ratio of nanoparticle

to Lutrol-F-68 was 1:0.05, 1:0.1 and 1:0.2. Based on the freeze thaw batches with

trehalose and lutrol-F-68 were selected for freeze drying and the size obtained after

freeze drying are shown in table 1.3. The optimum composition was then selected for

freeze drying of the nanoparticles of all the other batches.

4.3.2. Freeze-drying of nanoparticles

Freeze-drying of various nanoparticle batches were carried out using trehalose (10:1

by weight of nanoparticles) and lutrol-F-68 (0.1:1 by weight of nanoparticles), which

showed minimal change in particle size during the freeze thaw study. Samples of 10

mL dispersion of nanoparticles were dispensed in 250mL freeze drying glass vessels,

frozen at -70 °C for 12 h and then subjected to freeze-drying using Labconco freeze-

drying system (FreeZone 4.5, USA). Sublimation lasted for 36-48 h at a vacuum

pressure of 10-50×10-3 bar, with the condenser surface temperature maintained at less

than -50 °C. Lyophilized samples were collected under anhydrous conditions and

stored in a dessicator until re-hydrated. Re-hydration of lyophilized nanoparticles was

carried with 0.2 µm filtered water by simple manual shaking. Particle size and PI of

the re-hydrated samples were determined by PCS to assess the cryoprotection

provided by the cryoprotectant.



4.4. EVALUATION AND PHYSICAL CHARACTERIZATION

4.4.1. Entrapment Efficiency

Nanoparticle dispersion was centrifuged at 15,000 rpm for 30 min at 20 ºC. The

resultant supernatant was analysed for free drug.

i) Entrapment Efficiency of RIF: The concentration of rifampicin in the supernatant

was determined by UV-Visible spectrophotometry at λmax of 475nm without any

interference of folic acid. Entrapment efficiency was calculated using equation 1:

Entrapment efficiency (%) = (RIFinitial – RIFsupernatant) / RIFinitial ×100 (1)

ii) Entrapment Efficiency of ETH: Entrapment efficiency of ETH was determined by

partitioning the supernatant with dichloromethane, in which RIF completely partitions

while the ethambutol remains in the aqueous phase, and this aqueous phase containing

ethambutol was derivatized and analyzed by UV-Visible spectrophotometry at λmax

of 412nm without any interference. The method used for analysis of ethambutol was

validated (chapter 2). Entrapment efficiency was calculated using equation 2.

Entrapment efficiency (%) = (ETHinitial – ETHsupernatant) / ETHinitial ×100 (2)

iii) Entrapment Efficiency of folic acid: Entrapment efficiency of folic acid was

determined by partitioning the supernatant with dichloromethane, in which RIF

completely partitions while the folic acid remains in aqueous phase, and this aqueous

phase can be analyzed for folic acid by UV-Visible spectrophotometry at λmax of

256.5nm without any interference. The method used for analysis of folic acid was

validated (chapter 2). Entrapment efficiency was calculated using equation 3.

Entrapment efficiency (%) = (FAinitial – FAsupernatant) / FAinitial ×100 (3)

iv) Entrapment Efficiency of MSDNC-22: The concentration of RIF and MSDNC-22

in the supernatant was determined using simultaneous equation method developed by

UV-Visible spectrophotometry at λmax of 425 and 475nm. Entrapment efficiency was

calculated using equation 4.

%EE = (MSDNC-22initial – MSDNC-22supernatant) / MSDNC-22initial ×100 (4)

4.4.2. Particle Size

Particle size was determined by Photon Correlation Spectroscopy using N4 plus

submicron particle size analyzer (Beckman Coulter, USA). The analysis was

performed at a scattering angle of 90º at a temperature of 25 ºC. All the

nanoparticulate dispersions were sonicated using ultrasonic probe system (DP120,

Dakshin, Mumbai, India) for 5 min with 10 sec pulse at 200 voltages over an ice bath.



Dispersions were then appropriately diluted with filtered water (0.2 µm filter,

Millipore India Pvt. Ltd.) to obtain 5 ×104 to 1×106 counts per second. Each sample

was analyzed in triplicate and average particle size and polydispersity index (PI)

measured.

4.4.3. Drug loading

Measured quantity of freeze dried nanoparticles were dissolved in THF:water (1:1) by

sonication for 5 mins and assayed for drug content by developed HPLC method.

Percent Drug loading (DL%) was calculated using the equation:

DL (%) = WDL/WNP×100 ………………………………………….. (5)

where WDL = weight of Drug in Np and WNP = weight of Np

4.4.4. Zeta Potential

Zeta potential of nanoparticle dispersion was measured using Malvern Zetasizer

Nanoseries using DTS Nano software. Nanoparticle dispersion was centrifuged at

15,000 rpm for 30 min at 20ºC. The resultant pellet was washed and redispersed with

distilled deionized water (nanoparticles 100µg/ml) by sonication. Samples were filled

in to the folded capillary cell and zeta potential was measured. Each sample was

analyzed in triplicate.

4.4.5. Scanning Electron Microscopy (SEM)

The morphology/shape of nanoparticles was determined by SEM (JSM-6380-LA,

JEOL, Tokyo, Japan). A drop of colloidal dispersion was deposited onto a carbon tape

and dried under vacuum. The samples were sputtered with platinum using an auto fine

coater prior to analysis (JFC-1600, JEOL, Tokyo, Japan).

4.4.6. Fourier Transform Infrared (FTIR) Spectroscopy

FT-IR for rifampicin, ethambutol, MSDNC-22, folic acid, PES, the nanoparticles and

excipients were recorded on a Perkin-Elmer FTIR spectrophotometer by the KBr disk

method from 4000 to 500 cm-1. Samples were crushed to a fine powder, mulled with

anhydrous potassium bromide, compressed to form a thin transparent pellet and

subjected to FTIR.

4.4.7. Differential Scanning Calorimetry (DSC)

DSC thermograms of rifampicin, ethambutol, MSDNC-22, folic acid, PES, the

nanoparticles and excipients were recorded on a Perkin Elmer Pyris 6 DSC

(PerkinElmer, Netherlands) system in the temperature range 40 -300°C at a heating

rate of 10°C /min in a dynamic nitrogen atmosphere (20 mL/min). A sample of 5-6



mg was sealed in an aluminum pan and an empty sealed aluminum pan was used as

the reference.

4.4.8. Powder X-Ray Diffraction (PXRD)

Powder XRD patterns were obtained for rifampicin, ethambutol, MSDNC-22, folic

acid, PES and the nanoparticles were recorded using a Rigaku Miniflex

diffractometer, with Cu Kα target tube, NaI detector, variable slits, a 0.050 step size,

operated at a voltage of 30 kV, 15mA current, at 2θ/min scanning speed, and scanning

angles ranged from 8-60º(2θ).

4.4.9. Hydrophobicity evaluation of nanoparticles (contact angle measurement)

Hydrophobicity of RIF, ETH, PES, RIF PES, RIF PES FA nanoparticles, RIF-ETH

PES nanoparticles and RIF-ETH PES FA nanoparticles was evaluated by measuring

the static contact angle of nanoparticles/drug pellet. Briefly, nanoparticle pellets were

prepared in KBr press using 25 mg freeze dried nanoparticles and press it at 10 tone

pressure for 1 minute. Drop water contact angles were measured on a Kruss contact

angle measuring instrument G10, Germany. Milli-Q water was used with a drop

volume of approximately 5µl. Results are presented as an average of 3 measurements.

4.4.10. In-Vitro Release

i) Rifampicin release from RIF-PES nanoparticles

Drug release studies were performed by a modified dialysis method (Li, Y., et al.,

2005). Nanoparticles (equivalent to 10 mg drug) were loaded into a pre-treated

dialysis bag (Sigma, molecular weight cut-off 12-14 kDa,) and introduced into the

basket of USP apparatus-I. Phosphate-buffered saline(900ml) containing 1%w/v

ascorbic acid and 0.05%w/v sodium azide, at a pH of 7.2 was used as the dissolution

medium (Muttil, P., et al., 2007). Aliquots (5 mL) were withdrawn at specific time

intervals, and analyzed for RIF by UV spectroscopy at λmax475 nm. Percent drug

release versus time profiles were plotted. Negligible leakage of the particles from the

dialysis tube was confirmed by testing the blank Np, which showed no absorbance in

the release medium.

ii) Folic acid release from RIF-PES FA nanoparticles

Release of folic acid from the nanoparticles was evaluated as above excluding sodium

azide and ascorbic acid from dissolution medium. Aliquots (5 mL) were withdrawn at

specific time intervals, and extracted with dichloromethane (DCM) to separate RIF.

Folic acid was quantified at λmax256.5 nm. Additionally release of folic acid was



studied in media having various pH such as pH-1.2, pH-4.5, and pH-6.8. Percent folic

acid release versus time profiles were plotted.

iii) RIF and ETH release from RIF-ETH PES nanoparticles

In-vitro release of RIF and ETH from RIF-ETH PES nanoparticles was performed by

a modified dialysis method (Li, Y., et al., 2008). Nanoparticles (equivalent to 10 mg

drug) were loaded into a pre-treated dialysis bag (Sigma, molecular weight cut-off 12-

14 kDa,) and introduced into the basket of USP apparatus-I. Phosphate-buffered

saline (900ml) at a pH of 7.4 was used as the dissolution medium. Aliquots (5 mL)

were withdrawn at specific time intervals. RIF was analysed by UV spectroscopy at

λmax412 nm. RIF was extracted out and ETH was analyzed after derivatization with

copper sulphate by UV spectroscopy at λmax412 nm. Percent drug release versus time

profiles were plotted.

vi) Kinetic model study

In vitro release data for RIF was fitted to kinetic models such as zero-order, first-

order, Higuchi equation, Korsemeyer–Peppas equation, and Hixson–Crowell

equation. The regression analysis was performed.

• Qt versus t (zero order)

• log Qt versus t (first order)

• Qt versus square root of t (Higuchi)

• log %Qt versus log %t (Korsmeymer-Peppas)

• Qt versus cube root of t (Hixson–Crowell)

Where Qt is the amount of drug released at time t. The criteria for selecting the most

appropriate model were sum of square of residuals (SSR) (Shivkumar et. al, 2007,

Shoaib et.al, 2006, Thakkar et,al. 2009).

4.5. STABILITY STUDY

The nanoparticles were freeze dried, packed and sealed in amber glass vials and

subjected to stability studies as per the ICH guidelines at 300C/65RH and 400C/75RH.

4.6. MACROPHAGE UPTAKE STUDY IN U937 CELL LINE

4.6.1. Maintenance of cell line

Human macrophage cell line U-937 of histiocytic lymphoma origin was procured

from the National Centre for Cell Science (NCCS), Pune, India. Cells were cultured at

37o C and 5% CO2 in RPMI 1640 medium with growth supplements, gentamycin and



10% bovine fetal calf serum. Cells were screened for their viability and change in

morphology by trypan blue dye exclusion technique. 100 µl of the cells from each

well were mixed with 100 µl of Trypan Blue solution (0.16%w/v) and cells were

counted in a WBC chamber of the Neubauer hemocytometer.

4.6.2. Cell uptake study

Macrophage uptake of the following formulations was evaluated:

i) RIF solution, RIF PES nanoparticles and RIF PES FA nanoparticles

ii) RIF-ETH solution, RIF-ETH PES nanoparticles and RIF-ETH PES FA

nanoparticles

iii) RIF-MSDNC-22 solution, RIF-MSDNC-22 PES nanoparticles and RIF-MSDNC-

22 PES FA nanoparticles

Free drug/drugs and nanoparticles (equivalent to 10µg drug/drugs) were incubated,

with the human macrophage cells containing 10% fetal calf serum, at 370C in

presence of 5% CO2 at a cell density of 2 x 106 cells/ml. After incubation for 1 h, the

cells were separated from the uninternalized nanoparticles by centrifugation at 1000

rpm for 3 mins. Methanol was added to the supernatant. The precipitated protein was

separated by centrifugation and drug/drugs in the supernatant quantified by HPLC

method. The cell pellet was lysed by sonication following addition of 0.5% sodium

dodecyl sulphate solution (in PBS). Methanol was added to precipitate the proteins,

the suspension centrifuged and the supernatant analyzed by HPLC method to

determine drug/drugs concentration in the cells. Drug was quantified both in

supernatant and cell pellet to ensure mass balance.

4.7. ORAL BIODISTRIBUTION STUDY BY GAMMA SCINTIGRAPHY

4.7.1. Instruments

In vivo studies were carried out by gamma scintigraphy. The radioactivity in each

organ was counted using CAPINTEC radioisotope calibrator (CRC®-127 R) and

expressed as percent injected dose per gram organ. Dynamic images were acquired in

64 X 64 matrix for 30 minutes (4 fr/sec for 1 minute followed by frame/minute for 29

minutes) using Millenium MP5 single head Gamma camera mounted with pin-hole

collimator (GE). The data was acquired in Genie Acquisition Station. Dynamic

images were processed on eNTEGRA workstation employing temporal filter. Static

images were acquired in 256 x 256 matrix at 140 kev and 20% window for 1 minute.



The acquisition of images (dynamic and static) was performed by using GENIE

acquisition station and transferred to eNTEGRA workstation for further processing.

4.7.2. Radiolabeling

Rifampicin and RIF nanoparticles were radiolabelled using 99mTc direct labeling

method. Briefly 0.1 ml of stannous chloride stock solution was added to 0.5 ml of

solution containing 8mCi of radioactivity and was shaken for 10 minutes. RIF/RIF

nanoparticles (0.6ml) were added to the above mixture followed by shaking for 20

minutes at room temperature.

4.7.3. Radiolabeling- efficiency

Radiolabeled RIF/RIF nanoparticles (50 µl) were loaded onto precoated TLC plates

and chromatography was carried out using acetone as the mobile phase. The spots

were eluted and counted in the gamma counter and radiolabeling efficiency was

calculated using the equation:

4.7.4. Stability of Radiolabeled RIF/RIF nanoparticles

All the radio-conjugates were tested for in-vivo and in-vitro stability. For

ascertaining in-vivo stability, the radiolabeled drug or the nanoparticulates were

administered to rats under gamma camera and whole body images were acquired.

The radioactivity counts appearing in thyroid over a time period up to 2 hours is an

indication of in vivo hydrolysis and reflects the in-vivo stability of the radio-

conjugate. Stability of the radioconjugates was also tested in-vitro where the

radiolabeled conjugates were tested for their stability in normal saline and rat serum.

Radiolabeled RIF/RIF nanoparticles (0.2ml) were taken and mixed with 5 ml rat

serum/normal saline. This mixture was shaken for one hour and the aliquot of the

mixture run on a TLC plate. The stability was determined over 6 hours.

4.7.5. Biodistribution study

Healthy male wistar rats with average body weight (BW) 200-250 g and were used for

the study. The animals were maintained under standard laboratory conditions (14h:

10h dark/light cycle, a temperature of 22±2°C and 50-70% humidity). Pelleted feed

and water were provided ad libitum. The animal ethics committee, Bombay veterinary

College, Mumbai, India had approved the experimental protocol for study. Animals



were anesthetized by injecting a ketamine hydrochloride (50 mg kg-1) and xylazine

hydrochloride (10 mg kg-1) cocktail intramuscularly 20 min prior to study.

Male wistar rats (n=4) were fasted for 12 hrs. RIF (10 mg/kg) as radiolabeled RIF

solution/ RIF nanoparticles (approx 500 µCi) was administered by oral gavage. The

animals were placed prone on a Millennium MPS Acquisition System, (Multipurpose

Single Head Square Detector) Gamma Camera fitted with Low Energy General

Purpose (LEGP) collimator. The distance of collimator to table was maintained at 95

cm for all acquisitions. For the purpose of determining total injected dose the

radioactivity of syringe before and after dosing was determined using gamma counter.

Total injected dose was estimated by subtracting radioactivity after dosing from

radioactivity before dosing. At intervals of 0.5h, 1 h, 2 h, 4 h, and 6 h post dosing,

static images were recorded for a period of 1 min each at 1.33x zoom. All the static

images were stored digitally in a 256 x 256 matrix. At the end of 6 hours animals

were sacrificed by cervical dislocation, and different organs of interest including

heart, spleen, lungs, stomach, liver, and small intestine were isolated. The organs were

washed three times with saline, blot dried and weighed and radioactivity was measure

in gamma counter. Administered dose was calculated by subtracting the activity of

empty syringe from the full syringe image. The radioactivity counts per minute (cpm)

in each organ were determined using gamma counter (Capnitech) and corrected for

physical decay. The % injected dose g-1 of organ was calculated.

4.8. PHARMACOKINETIC AND LUNG UPTAKE OF NANOPARTICLES

4.8.1. Pharmacokinetic study

Male Wistar rats (200-250gms) (n=6) were fasted 12-18h prior to dosing. Rats were

divided into six groups, one group received plain drug solution, the second group

received RIF PES Np’s, third group received RIF PES FA Np’s (equivalent to

10mg/kg body weight of RIF) administered by oral gavage. While fourth group

received RIF-ETH solution, the fifth group received RIF-ETH PES Np’s and sixth

group received RIF-ETH PES Np’s (equivalent to 20mg/kg body weight of ETH)

administered by oral gavage. Blood samples were withdrawn from the retro-orbital

plexus prior to dosing and at 1, 2, 4, 6, 12 and 24h post dosing and collected in tubes

containing 4.1% EDTA. Plasma was separated, drug extracted and quantified by

HPLC. The peak plasma concentration (Cmax) and peak plasma time (Tmax) were



obtained by visual data inspection. The area under plasma drug concentration over

time curve (AUC0–t) and t1/2 were calculated using BASICA software.

4.8.2. Lung uptake study

Rats mentioned above in pharmacokinetic were euthanized at 24h by excessive

carbon dioxide, lungs, liver, spleen and kidney were isolated and placed in phosphate

buffered saline (PBS), pH 7.4 and homogenized using a tissue homogenizer. Drug

was extracted and evaluated for drug content by HPLC.

5. Results and Discussion



5. RESULTS AND DISCUSSION 5.1. PREPARATION AND OPTIMIZATION OF NANOPARTICLES

5.1.1. PES Nanoparticles of Rifampicin

Nanoprecipitation method first developed by Fessi et al., 1992 is an easy and

reproducible technique to produce nanoparticles with size < 500nm and has been used

by several research groups to prepare drug loaded polymeric nanoparticles. Hence

nanoprecipitation method was selected for the preparation of RIF loaded PES

nanoparticles.

Studies were initiated with a prototype formula consisting of 100mg PES, 25ml

aqueous phase, 10ml organic phase (THF:MeOH-9:1), 20mg RIF, and 50mg lutrol-f-

68.

a) Effect of lutrol-F-68 concentration

Table 5.1 : Effect of lutrol-F-68 concentration on %EE and particle size

RPES/1 RPES/2 RPES/3

Particle size (nm) 405.9 ± 15.9 287.15 ± 18 252.75 ± 14.5

PI 0.328 ± 0.083 0.347 ± 0.062 0.297 ± 0.092

%EE RIF 74.18 ± 2.35 % 72.37 ± 2.13 72.37 ± 2.13

% RIF loading 12.56 ± 0.186 12.3 ± 0.211 10.9 ± 0.143

Figure 5.1: Effect of lutrol-F-68 concentration on %EE and particle size

As shown in figure 5.1 lutrol-f-68 concentration from 0.04% w/v (RPES/1) to 0.1%

w/v (RPES/2) revealed no significant change in entrapment efficiency of RIF



(P>0.05), with further increase in lutrol-F-68 concentration from 0.1% w/v (RPES/2)

to 0.2% w/v (RPES/3) significant decrease in entrapment efficiency (P<0.05) was

observed (table 5.1). The decrease in entrapment efficiency with lutrol-f-68

concentration 0.2% w/v was attributed to the low CMC value of lutrol-F-68 (0.1 %)

(Youan et al., 2003) resulting in enhanced solubilisation of RIF. A significant

decrease in particle size (P<0.05) was seen with increase in lutrol-F-68 concentration.

Surfactants bring about reduction in particle size by two mechanisms; (i) reduction in

interfacial tension between organic and aqueous phases due to surfactants promotes

rapid diffusion of two phases and allows formation of smaller droplet and thus small

mean size of nanoparticles, (ii) increase in viscosity of the aqueous phase promotes

hydrodynamic stabilization by preventing coalescence and aggregation of droplets

formed (Schubert et al., 2003; Galindo-Rodriguez et al., 2004). This effect is more

pronounced above the critical micelle concentration (CMC) of the surfactant. Above

CMC, excess surfactant present in the bulk solution is available for droplet coverage

resulting in better hydrodynamic stability and thereby lower particle size. This

explains the role of lutrol-F-68 on particle size (figure 5.1). Similar data is reported in

literature with other drugs (Lamprecht et al., 2001, Patil et al., 2008).

b) Effect of RIF: PES ratio

Figure 5.2: Effect of Drug:Polymer ratio on %EE and particle size

Effect of different RIF:PES ratio on entrapment efficiency and particle size is shown

in figure 5.2. Varying the RIF:PES ratio from 1:2 to 1:5 significantly increased the

entrapment efficiency of RIF with increase in polymer concentration (table 5.2). This



increase in entrapment efficiency can be related to higher amount of polymer

available for entrapment. Increase in particle size can be related to increased viscosity

of the organic phase at higher polymer concentration. At higher polymer

concentration solvent diffusing into the aqueous phase carries with it higher amount

of polymer which forms relatively larger nanoparticles. Moreover, increasing polymer

concentration increases polymer-polymer interaction which means that more polymer

remains associated during the diffusion process to contribute relatively larger particle

size [Quintanar-Guerrero et al., 2005, Galindo-Rodriguez et.al, 2004].

Table 5.2 : Effect of Drug:Polymer ratio on %EE and particle size

RPES/1 RPES/4 RPES/5 RPES/6 Particle size

(nm) 405.9 ± 15.9 349.5 ± 30 330.2 ± 20 302.3 ± 25

PI 0.328 ± 0.083 0.239 ± 0.09 0.281 ± 0.102 0.197 ± 0.12 %EE RIF 74.18 ± 2.35 60.32 ± 2.3 43.57 ± 1.95 28.94 ± 3.04

% RIF loading 12.56 ± 0.186 10.9 ± 0.140 7.83 ± 0.12 5.66 ± 0.11

5.1.2. Folate anchored PES Nanoparticles of Rifampicin

Folic acid is a known ligand for the folate receptors over-expressed on infected

macrophages. Folic acid was included to increase uptake of nanoparticles by active

targeting to the macrophages and to increase the efficacy. Folate anchored PES

nanoparticles of RIF (RPESFA/1) was successfully prepared by simple physical

adsorption technique. Entrapment efficiency ~42% was obtained for folic acid which

did not influence the particle size.

Literature reports folic acid anchored to the nanocarriers by covalent conjugation to

the polymer prior to nanoparticle preparation (Zhao et al., 2008; Lee et al., 2003; Han

et al., 2009; Lee et al., 1994; Low et al., 1991), alternatively preformed polymeric

nanoparticles are covalently conjugated to folic acid (Stella et al., 2000). A common

approach is the use of carbodimide derivatives to activate folic acid and conjugate it

to the polymer (Lee et al., 2003; Chen et al., 2007). Couvreur et al. have coupled folic

acid to poly[aminopoly(ethylene glycol)cyanoacrylate-co-hexadecyl cyanoacrylate,

by preparing its N-hydroxysuccinimide ester. Conjugation methods include the need

for purification, and moreover toxicity evaluation, as the conjugate could be

considered as a new excipient. This could increase the cost (You et al., 2008; Chen et

al., 2007; Turk et al., 2004; Leamon et al., 1991). Physical adsorption techniques to

anchor folic acid could provide a significant advantage in the design of folate



anchored nanoparticles. Physical adsorption techniques as an approach for folate

anchoring therefore presents a simple, practical and viable non-covalent method for

the design of folate anchored nanoparticles that could facilitate folate receptor

mediated targeting.

Table 5.3 shows optimized batch for RIF loaded PES nanoparticles with and without

folic acid using nanoprecipitation method.

Table 5.3: Optimized batch for RIF PES Np’s and RIF PES FA Np’s

RIF-PES NPs

(RPES/1)RIF-PES FA NPs

(RPESFA/1)

Mean Particle size (nm) 405.9 ± 15.9 416.6 ± 10.1

Polydispersity index 0.328 ± 0.083 0.298 ± 0.059

%EE for RIF 74.18 ± 2.35 % 73.07 ± 1.69 %

%EE for Folic acid - 42.28 ± 2.5 %

%Drug Loading 12.56 ± 0.186% 12.48 ± 0.167%

5.1.3. PES Nanoparticles of Rifampicin-Ethambutol

The challenge here was preparation of nanoparticles of combination of two drugs with

contrasting solubility i.e RIF having poor water solubility and EMB being highly

water soluble. Therefore we evaluated multiple emulsion method (double emulsion

method). Method is advantageous for entrapment of both hydrophilic as well as

hydrophobic drugs especially for good entrapment efficiency of water soluble small

drugs, protein and peptide with retention of bioactivity (Soppimath et al., 2001, Bala

et al., 2004, Moinard-Checot et al., 2006). In this method highly water soluble drug(s)

is present in innermost aqueous phase (w/o/w) and hydrophobic molecule present in

middle oil phase and hence good entrapment for both hydrophilic and hydrophobic

drugs achieved.

Multiple emulsion method necessitates selection of a water immiscible solvent. Hence

DCM was selected. Studies were initiated with a prototype formula consisting of

100mg PES, 25ml aqueous phase, 10ml dichloromethane 10mg RIF, EMB 20mg,

20mg dioctyl sodium sulfosuccinate (AOT) and 50mg polyvinyl alcohol (PVA).

Although AOT has HLB >25 it was selected as surfactant as it is reported to form

reverse micelles. Amount of PVA and RIF to EMB ratio were varied in this prototype

formula.



a) Effect of PVA concentration

As shown in figure 5.3 significant decrease in RIF entrapment efficiency (P>0.05)

was observed from 0.2% w/v (REPES/1) to 1% w/v (REPES/3) of PVA whereas no

significant change in entrapment efficiency for EMB was evident (table 5.4).

Decrease in RIF entrapment efficiency was attributed to partitioning of RIF from the

oil phase to secondary aqueous phase at higher PVA concentration. No change in

entrapment efficiency of EMB was attributed to the poor partitioning of EMB from

primary aqueous phase to oil phase and subsequently to the secondary aqueous phase.

Decrease in particle size was seen with increase in PVA concentration attributed to

the lower interfacial tension during particle formation. PVA exerts its stabilizing

effect by adsorbing at the droplet interface thus reducing surface tension and

promoting mechanical and steric stabilization. Similar data is reported in literature

with other drugs (Lamprecht et al., 2001, Patil et al., 2008).

Figure 5.3 Effect of PVA concentration on %EE and particle size

Table 5.4 : Effect of PVA concentration on %EE and particle size

REPES/1 REPES/2 REPES/3 Particle size (nm) 395.25 ± 25 265.8 ± 30 213.4 ± 22

PI 0.401 ± 0.13 0.362 ± 0.102 0.338 ± 0.121 %EE RIF 62.34 ± 1.79 47.25 ± 3.25 29.18 ± 3.42

%EE EMB 49.84 ± 2.36 53.82 ± 4.19 43.92 ± 4.18 % Drug loading 13.79 ± 0.197 13.04 ± 0.203 11.11 ± 0.175

b) Optimization of RIF:EMB ratio

To optimize nanoparticles with RIF:EMB in ratios from 10:1, 1:1, and 1:10 we

initiated nanoparticles preparation with 20mg of RIF and 20mg of EMB. As shown in



table 5.5 RIF has entrapment efficiency in the range of 57-64% and EMB 44-51%.

Based on the entrapment efficiency of RIF and EMB initially we obtained

nanoparticles in a ratio of 1:0.7 (RIF:EMB) (REPES/4). For example based on the

ratio the amount of RIF and EMB was varied during nanoparticle preparation to

obtain the desire ratio of RIF and EMB. To obtain RIF:EMB ratio 1:1 and 10:1

amount of RIF was modified as 15mg (REPES/5) and 2.5mg (REPES/6) respectively.

Similarly to obtain RIF:EMB ratio 1:10 amount of RIF kept same in REPES/4 and

amount of EMB was modified as 2.5mg (REPES/8). All the nanoparticles having the

particle size ranging from 350 to 450 nm.

Table 5.5: Optimized batch for RIF-EMB PES Np’s at different ratio

REPES/4 REPES/5 REPES/6 REPES/7 REPES/8 Average particle size (nm) 397.3 ± 25 401.05 ± 30 391.2 ± 40 399.8 ± 10 385.0 ± 45

%EE RIF 63.98 ± 1.06 63.42 ± 1.5 57.33 ± 1.93 63.51 ± 1.24 62.6 ± 2.05 %EE EMB 44.55 ± 0.33 47.93 ± 2.59 50.93 ± 2.34 51.34 ± 0.82 48.75± 3.1 Drug Entrapped RIF (mg) 12.79 ± 0.12 9.51 ± 0.28 1.14 ± 0.24 12.60 ± 0.09 12.52 ± 0.29 EMB (mg) 8.90 ± 0.16 9.58 ± 0.35 10.18 ± 0.31 2.57 ± 0.19 1.22 ± 0.36 RIF:EMB ratio ~1:0.7 ~1:1 ~1:10 ~1:0.2 ~1:0.1 (10:1)

5.1.4. PES Nanoparticles of Rifampicin-Ethambutol with ligand folic acid

In-vitro studies on RIF-EMB combinations revealed maximum efficacy against

Mycobacterium tuberculosis at RIF:EMB (1:10) hence nanoparticles of the same ratio

of the two drugs were evaluated with ligand folic acid.

RIF-EMB PES FA nanoparticles (REPESFA/1) were readily prepared by adding

ligand folic acid to RIF-EMB PES nanoparticles (REPES/6) and the concentration of

folic acid optimized in section 5.1.2 (RPESFA/1) was used. Folate anchored PES

nanoparticles of RIF-EMB (RPESFA/1) was successfully prepared with entrapment

efficiency ~42% was obtained for folic acid which did not influence the particle size.

5.1.4. PES Nanoparticles of Rifampicin-MSDNC-22 with and without folic acid

This study was intended of a physibility study of an entrapment of MSDNC-22 with

RIF. RIF-MSDNC-22 PES and RIF-MSDNC-22 PES FA nanoparticles were prepared

by nanoprecipitation method optimized in section 5.1.1 and 5.1.2 was used (RPES/1).

MSDNC-22 was included in organic phase. Nanoparticles with RIF:MSDNC-22 in

ratios from 10:1, 1:1, and 1:10 and particle size ranging from 350 to 450nm were



readily prepared by varying the ratios of MSDNC-22 and RIF during their preparation

based on their entrapment efficiency observed. Table 5.6 shows optimized batch for

RIF- MSDNC-22 loaded PES nanoparticles at different ratio.

Table 5.6: Optimized batch for RIF- MSDNC-22 PES Np’s at different ratio

RMPES/2

RMPES/1

RMPES/3

Average particle size (nm) 421.05 ± 30 430.0 ± 20 425.2 ± 20 %EE RIF 59.32 ± 1.85 75.3 ± 2.25 35.33 ± 2.43 %EE MSDNC-22 87.53 ± 0.66 86.17± 0.37 86.74 ± 0.79 Drug Entrapped RIF (mg) 8.72 ± 0.13 15.24 ± 0.41 0.85 ± 0.25 MSDNC-22 (mg) 8.75 ± 0.21 1.71 ± 0.29 8.75 ± 0.12 RIF:MSDNC-22 (1:1) (10:1) (1:10)

RIF-MSDNC-22 PES FA nanoparticles (RMPESFA/1) were readily prepared by

adding ligand folic acid to RIF-MSDNC-22 PES nanoparticles (REPES/6) and the

concentration of folic acid optimized in section 5.1.2 (RPESFA/1) was used. Folate

anchored PES nanoparticles of RIF-MSDNC-22 (RMESFA/1) was successfully

prepared with entrapment efficiency ~42% was obtained for folic acid which did not

influence the particle size.

5.2. ISOLATION AND PRESERVATION OF NANOPARTICLES

5.2.1. Freeze-Thaw study

The RIF PES nanoparticle (RPES/1) was selected for freeze thaw study as it has

entrapment efficiency >70% with desire particle size 350-450nm. The

physicochemical properties of nanoparticles have an effect on their efficiency in drug

delivery (Bozdag et.al, 2005). If particles are stored as a suspension in an aqueous

medium, degradation and/or solubilization of the polymer, drug leakage, drug

desorption and/or drug degradation may occur. Lyophilization represents one of the

most useful methodology to ensure the long term conservation of unloaded or drug-

bound polymeric nanoparticles (Roy et.al, 1997, Abdelwahed et.al, 2006, Kuleshova

et.al, 1999). After freeze-drying, easy and rapid reconstitution and unchanged particle

size of the product are important features. Freeze thaw represent a quick and

economical method to assess effect of cryoprotectant on particle aggregation.

Carbohydrates are favored as freeze-drying excipients since they are chemically

innocuous and can be easily vitrified during freezing (Bozdag et.al, 2005).



Carbohydrates as cryoprotectant have proved to be superior to polyalcohols. Hence a

disaccharide trehalose and a sugar alcohol mannito were selected for the study.

(Schwarz, et.al, 1997, Chasteigner et.al, 1996, Franks et.al, 1998).

a) Effect of type and concentration of cryoprotectant

Table 5.7: Freeze Thaw study of RPES/1 nanoparticle using Mannitol

Lutrol-F-68 (1:0) Lutrol-F-68 (1:0.05) Lutrol-F-68 (1:0.1) Lutrol-F-68 (1:0.2)

Si Sf Sf/Si

ratio Si Sf

Sf/Si

ratio Si Sf

Sf/Si

ratio Si Sf

Sf/Si

ratio

Mannitol

(1:5) 385.5 570.5 1.468 434 499.6 1.151 415.5 482.6 1.161 390.2 513.2 1.468

Mannitol

(1:10) 401.4 >1000 2.491 428.6 602.4 1.405 442 592.2 1.339 424.8 959.2 2.258

Mannitol

(1:20) 427.2 >1000 2.339 388.9 >1000 2.902 391 819 2.052 423.3 843.8 1.988

Table 5.8: Freeze Thaw study of RPES/1 nanoparticle using Trehalose

Lutrol-F-68 (1:0) Lutrol-F-68 (1:0.05) Lutrol-F-68 (1:01) Lutrol-F-68 (1:0.2)

Si Sf Sf/Si

ratio Si Sf

Sf/Si

ratio Si Sf

Sf/Si

ratio Si Sf

Sf/Si

ratio

Trehalose

(1:5) 395.9 >1000 3.120 450.6 622.2 1.380 447.1 495.3 1.107 455.5 464.4 1.019

Trehalose

(1:10) 421.1 742.3 1.762 383.2 392.2 1.023 432.3 419.6 0.970 449.9 467.8 1.039

Trehalose

(1:20) 420.1 621.4 1.479 462 452.9 0.980 442.5 447.3 1.010 439.8 455.8 1.036

Samples with cryoprotectants showed concentration dependent cryoprotection and

revealed greater increase in particle size at lower cryoprotectant concentrations

(Sugrue et.al, 1992). Large aggregates were clearly observed in samples frozen

without cryoprotectant, and hence their particle size was not determined. Absence of

lutrol-f-68 revealed poor cryoprotection with both trehalose and Mannitol (Table 5.7

and 5.8). Similar kind of results was observed by Guhagarkar et al., 2009 and 2010

for PES as a polymer. At equivalent concentration in combination with lutrol-f-68,

trehalose proved to be a better cryoprotectant than mannitol (Table 5.8). Trehalose

(1:10, 1:20 by weight of nanoparticles) in combination with lutrol-F-68 (1:0.05, 1:0.1,

1:0.2 by weight of nanoparticles) revealed the best cryo-protection with a Sf/Si (Sf-



final particle size after freeze thaw, Si- initial particle size) ratio of <1.3 (Table 5.8) is

considered acceptable(Saez, A et.al, 2000), while mannitol at the same concentration

revealed a high Sf/Si ratio of >1.3 after the freeze thaw study.

When the nanoparticle dispersion containing cryoprotectant is frozen below glass

transition temperature, the cryoprotectants form a glassy/vitreous coating around the

nanoparticles protecting them against stresses like mechanical stress of ice crystals

thereby preventing aggregation. Insufficient concentration of cryoprotectant leads to

incomplete coating of glassy matrix around nanoparticles favoring aggregation.

Further according to the Particle Isolation Hypothesis the spatial separation of

particles within the unfrozen fraction results in insufficient cryoprotectant at higher

concentration of nanoparticles, leading to aggregation. Particle isolation hypothesis

suggest that the separation of individual particles within the unfrozen fraction

prevents aggregation during freezing. According to this hypothesis, sufficient

quantities of virtually any excipient should offer similar protection during freezing.

Furthermore the concentrated suspensions employed in clinical trials may be difficult

to preserve by lyophilization (Allison et.al, 2000).

Freezing temperature below Tg′ of cryoprotectant has no effect on the glassy

protective matrix of cryoprotectant formed around the nanoparticles. Freeze thaw

study was carried out at temperatures below the Tg′ of cryoprotectants used (Franks

et.al, 1998) and hence it is expected to have no effect on stability, aggregation and

redispersion properties of freeze dried nanoparticle preparation. It has been reported

that there was a negligible increase in Sf/Si ratio of poly(D,L-lactic acid)

nanoparticles during freeze thawing at 2 different temperatures i.e -55°C and at -

196°C with trehalose (Guerrero et.al, 1998).

Trehalose showed better cryoprotectant effect than mannitol because, trehalose is a

non-reducing sugar and exists only in closed ring form. Moreover trehalose has one of

the highest glass transition temperatures (Tg) of all saccharides commonly used

(TREHATM, trehalose by Hayashibara, Japan) and disaccharides like trehalose appear

to have a greater influence on the vitrification properties or they are more active than

monosaccharides like mannitol. The lower the Tg, the more the lyophilizates tend to

collapse. Trehalose is very often considered as the best cryoprotective agent among

the available carbohydrates (Abdelwahed, et.al, 2006, Chasteigner et.al, 1996, Franks

et.al, 1998). Our study supports this observation.



5.2.2. Freeze-drying of nanoparticles

Table 5.9: Freeze drying of RIF PES nanoparticle

Sr. No RIF PES Np’s (RPES/1) Size before

FD (Si)

Size after

FD (Sf)

Sf/Si ratio

1 Trehalose (1:10) + Lutrol-F-68 (1:0.05) 389.7 602.1 1.54 2 Trehalose (1:20) + Lutrol-F-68 (1:0.05) 421.5 575.8 1.36 3 Trehalose (1:5) + Lutrol-F-68 (1:0.1) 416.5 598.3 1.43 4 Trehalose (1:10) + Lutrol-F-68 (1:0.1) 413.4 501.2 1.21

Various concentrations of trehalose along with lutrol-F-68 showed marginal increase

in particle size with a Sf/Si ratio <1.3 hence selected for freeze drying of RIF PES

nanoparticle. As shown in table 5.9 trehalose (10:1 by weight of nanoparticles) in

combination with lutrol-F-68 (0.1:1 by weight of nanoparticles) revealed the best

cryo-protection with a Sf/Si (Sf- final particle size after freeze thaw, Si- initial particle

size) ratio of <1.3 during freeze drying. Hence was selected for freeze drying of all

other nanoparticles, the data is shown in table 5.10.

Table 5.10: Sf/Si ratio of various freeze dried nanoparticles

Freeze dried NPDDS Size beforeFD (Si)

Size after FD (Sf)

Sf/Si ratio

RIF PES Np (RPES/1) 413.4 501.2 1.21 RIF PES FA Np (RPESFA/1) 421.3 518.1 1.23 RIF-EMB PES Np (REPES/6) 403.9 484.6 1.20 RIF-EMB PES FA Np (REPES6FA/1) 410.6 480.4 1.17 RIF-MSDNC-22 PES Np (RMPES/3) 425.3 514.6 1.21 RIF-MSDNC-22 PES FA Np (RMPES3FA/1) 416.8 495.9 1.19

5.3. EVALUATION AND PHYSICAL CHARACTERIZATION

5.3.1. Scanning Electron Microscopy (SEM)

a) b)



c) d)

e) f)

Figure 5.4: SEM images of a) RIF PES Nps b) RIF PES FA Nps c) RIF-EMB PES Nps d) RIF-EMB PES FA Nps e) RIF-MSDNC-22 PES Nps f) RIF-MSDNC-22 PES FA Nps

Scanning Electron Microscopy (SEM) revealed polydispersed nanoparticles with

spherical morphology (Figure 5.4).

5.3.2. Zeta Potential

Table 5.11: Zeta potential of nanoparticles

NPDDS Zeta Potential mV RIF PES Np (RPES/1) -33.22 RIF PES FA Np (RPESFA/1) -29.85 RIF-EMB PES Np (REPES/6) -31.79 RIF-EMB PES FA Np (REPES6FA/1) -32.07 RIF-MSDNC-22 PES Np (RMPES/3) -29.11 RIF-MSDNC-22 PES FA Np (RMPES3FA/1) -30.95

Nanoparticles exhibited a negative zeta potential due to free terminal hydroxyl group

of PES (Table 5.11). Zeta potential values ranged from -25 to – 35 mV which is an

indicator of good colloidal stability (Sugrue et al., 1992). Anchoring folic acid did not

cause significant change in zeta potential.



5.3.3. Fourier Transform Infrared spectroscopy (FTIR)

FTIR studies are rapid method of accessing drug: excipient interaction in the

formulation. FTIR of RIF, EMB, MSDNC-22, folic acid, PES and their respective

nanoparticles along with excipients revealed all the characteristic peaks of RIF, EMB,

MSDNC-22. RIF has principal peaks at wavenumbers 1250, 1567, 976, 1098, 1064,

1650 cm−1which are also found in RIF PES nanoparticles with and without folic acid.

EMB has principal peaks at wavenumbers 1090, 1061, 1142, 987, 1050, 1009 cm−1.

RIF-EMB PES nanoparticles with and without folic acid revealed no change in the

wavenumbwe of RIF and EMB. No drug: excipient interaction was evident in the

spectra (Figure 5.5).

Rifampicin Ethambutol

MSDNC-22 Folic acid

Poly (ethylene sebacate) Lutrol-F-68



Poly vinyl alcohol Aerosol OT

RIF PES Nps RIF PES FA Nps

RIF-EMB PES Nps RIF-EMB PES FA Nps

RIF-MSDNC-22 PES Nps RIF-MSDNC-22 PES FA Nps

Figure 5.5: FTIR of drugs, excipients and nanoparticles



5.3.4. Differential Scanning Calorimetry (DSC)

a) b)

c) d)

e) f)

Figure 5.6: DSC thermogram of a) RIF PES Nps b) RIF PES FA Nps c) RIF-EMB PES Nps d) RIF-EMB PES FA Nps e) RIF-MSDNC-22 PES Nps f) RIF-MSDNC-22 PES FA Nps

DSC enables detection of all the processes in which energy is required or produced

(i.e. endothermic and exothermic phase transformations). The thermograms of RIF,

EMB, MSDNC-22, folic acid, PES and their respective nanoparticles along with

excipients are shown in figure 5.6. Pure RIF, EMB and MSDNC-22 revealed a sharp

melting endotherm corresponding to their melting point indicating crystalline nature.



The disappearance of RIF, EMB and MSDNC-22 melting endotherm in their

respective nanoparticle suggests remarkable decrease in crystallinity (Date et al., 2012

Guhagarkar et al., 2010).

5.3.5. Powder X-ray Diffraction (P-XRD)

Poly (ethylene sebacate)

Rifampicin Ethambutol

MSDNC-22 Folic acid

RIF PES Nps RIF PES FA Nps

RIF-EMB PES Nps RIF-EMB PES FA Nps

RIF-MSDNC-22 PES Nps RIF-MSDNC-22 PES FA Nps

Figure 5.7: pXRD spectra of RIF, EMB, MSDNC-22, folic acid, PES and their respective nanoparticles



Crystallinity in the sample is reflected by a characteristic fingerprint region in the

diffraction pattern. The XRD spectra of RIF, EMB, MSDNC-22, folic acid, PES and

their nanoparticles are shown in figure 5.7. RIF, EMB, MSDNC-22, folic acid and

PES are highly crystalline powders showing characteristic sharp diffraction peaks.

These sharp diffraction peaks disappeared in the respective nanoparticles indicating

amorphization/remarkable decrease in crystallinity of the drug (Date et al., 2012

Guhagarkar et al., 2010).

5.3.6. In-vitro release

i) RIF PES FA nanoparticles

a) Rifampicin

RIF PES FA nanoparticles (RPESFA/1) showed sustained release of RIF upto 12h

with t50% 3.1h and t90% 10.13h which may be due to slow release of drug from the

nanoparticle matrix (figure 5.8). Ascorbic acid in the medium served as a stabilizer

(Muttil, et al., 2007).

Figure 5.8: In-vitro release of RIF from RIF PES FA nanoparticles (RPEFA/1)

Kinetic model study

The results of curve fitting of in-vitro release of RIF from RIF PES FA nanoparticles

into different mathematical models are given in table 5.12.

Table 5.12: Model fitting of RIF from RIF PES FA nanoparticles (RPESFA/1)

Model r2 Slope Intercept

Zero order 0.935 7.933 12.25

First order 0.993 -0.101 2.024

Higuchi 0.987 32.47 -12.41

Korsmeyer-Peppas 0.990 0.745(n) 1.233



The plots of Log percent cumulative drug release vs. time were found to be linear

with higher correlation coefficient value (r2 = 0.993), and following the first order

model, and the mechanism of drug release from the nanoparticles is suggested as

diffusion controlled (Thakkar et al., 2009; Shivakumar et al., 2007; Shoaib et al.,

2006).

b) Folic acid

RIF PES FA nanoparticles (RPESFA/1) revealed slow release of folic acid upto 12h

in phosphate buffered saline pH 7.4 (Figure 5.9) with no initial burst release. Slower

release of folic acid observed at pH 1.2 and pH 4.5 is attributed to the low solubility

of folic acid at acidic pH. Relatively faster release were seen at pH 6.8, pH 7.4 and its

related to enhance solubility of folic acid. Folic acid release however was sustained

with 100% release occuring over 12h even in the alkaline medium. Tabe 5.13 shows

t50% and t90% values for release of folic acid at different pH.

Figure 5.9: Effect of pH on In-vitro release of folic acid from RPESFA/1 Nps

Table 5.13: t50% and t90% release of folic acid at different pH

Buffer Medium t50% t90%

pH 1.2 15.86h 48.27h

pH 4.5 10.27h 31.35h

pH 6.8 4.25h 10.96h

pH 7.4 4.68h 10.32h



Table 5.14: Model fitting of folic acid from RPESFA/1 nanoparticles


pH 1.2 pH 4.5 pH 1.2 pH 4.5 pH 1.2 pH 4.5

Zero order 0.933 0.940 0.683 0.689 6.466 9.008

First order 0.958 0.975 13.51 16.74 1.973 1.965

Higuchi 0.991 0.995 -0.018 -0.026 -3.84 -3.73 Korsmeyer-

Peppas 0.989 0.961 3.292 4.087 0.928 1.038

As showen in Table 5.14 the plots of percent cumulative folic acid release vs. SQRT

of time were found to be linear with higher correlation coefficient value (r2 = 0.991 at

pH 1.2 and 0.995 at pH 4.5), and following the Higuchi model, and the mechanism of

folic acid release from the nanoparticles is suggested as diffusion and erosion

controlled (Thakkar et al., 2009; Shivakumar et al., 2007; Shoaib et al., 2006).

ii) RIF-EMB PES FA nanoparticles

Figure 5.10: In-vitro release of RIF and EMB from REPES6FA/1 nanoparticles

RIF-EMB PES FA nanoparticles (REPES6FA/1) showed sustained release of RIF and

EMB upto 12h for both RIF and ETH which may be due to slow release of drug from

the nanoparticle matrix (figure 5.10). EMB however exhibited faster release in the

initial time period up to 4h (P<0.05) beyond which a release of both drugs were

comparable (P>0.05). RIF and EMB revealed t50% release at 3.49h and 2.93h

respectively whereas t90% release at 9.69h and 8.59h respectively. Ascorbic acid in the

medium served as a stabilizer (Muttil, et al., 2007).



Kinetic model study

The results of curve fitting of in-vitro release of RIF and EMB from RIF-EMB PES

FA nanoparticles into different mathematical models are given in table 5.15.

Table 5.15: Model fitting of RIF and EMB from REPES6FA/1 nanoparticles


RIF EMB RIF EMB RIF EMB

Zero order 0.934 0.874 7.818 7.839 14.47 19.53 First order 0.964 0.905 -0.114 -0.175 2.046 2.129

Higuchi 0.992 0.966 32.09 32.82 -10.01 -6.276 Korsmeyer-

Peppas 0.983 0.889 0.732 0.864 1.263 1.213

The plots of percent cumulative drug release vs. SQRT of time were found to be

linear with higher correlation coefficient value (r2 = 0.992 for RIF and 0.966 for

EMB), and following the Higuchi model, and the mechanism of drug release from the

nanoparticles is suggested as diffusion and erosion controlled (Thakkar et al., 2009;

Shivakumar et al., 2007; Shoaib et al., 2006).

5.4. STABILITY OF NANOPARTICLES

Freeze dried NPDDS were stable for 12 months at 30°C/60%RH and for 6 months at

40°C/75%RH (Table 5.16-5.21). All the NPDDS revealed good redispersibility, no

significant change in particle size (as indicated by Sf/Si ratio<1.3) and drug content

>90% suggest stability. No significant difference in drug release was observed as the

F2 >70 (Figure 5.11 and Table 5.16-5.17)

a



Figure 5.11: In-vitro release stability results of RPESFA/1 nanoparticles

a) 300C/65% RH, b) 400C/75% RH

Table 5.16: Stability results of RPES/1 and RPESFA/1 at 300C/65%RH

Drug content Sf/Si ratio F2 value

RPES/1 RPESFA/1 RPES/1 RPESFA/1 RPESFA/1

Initial 97.17±2.31 97.48±1.55 1.276 1.257 -

1 month 96.93±0.33 97.20±1.82 1.228 1.260 97

3 month 97.58±1.05 97.92±1.61 1.236 1.267 99

6 month 95.83±1.23 96.03±2.31 1.254 1.271 82

12 month 96.21±1.87 95.82±2.01 1.249 1.253 78

Table 5.17: Stability results of RPES/1 and RPESFA/1 at 400C/75%RH

Drug content Sf/Si ratio F2 value

RPES/1 RPESFA/1 RPES/1 RPESFA/1 RPESFA/1

Initial 97.17±2.31 97.48±1.55 1.276 1.257 -

1 month 97.94±0.25 98.09±0.95 1.246 1.248 88

3 month 98.54±0.65 97.74±1.42 1.223 1.240 82

6 month 96.29±0.61 96.11±1.61 1.258 1.255 82

b



Table 5.18: Stability results of REPES/6 and REPES6FA/1 at 300C/65%RH

Drug content Sf/Si ratio

REPES/6 REPES6FA/1 REPES/6 REPES6FA/1

RIF EMB RIF EMB

Initial 98.32±1.51 99.83±2.05 99.27±2.01 99.19±0.97 1.193 1.210

1 month 97.78±1.97 99.12±0.97 98.89±1.32 98.87±1.49 1.216 1.218

3 month 96.64±1.35 98.54±1.61 98.07±1.46 98.34±1.67 1.177 1.215

6 month 97.13±2.07 96.65±1.29 96.22±1.24 97.53±2.01 1.220 1.218

12 month 97.58±1.05 96.12±1.71 95.96±2.13 96.12±1.71 1.239 1.227

Table 5.19: Stability results of REPES/6 and REPES6FA/1 at 400C/75%RH


REPES/6 REPES6FA/1 REPES/6 REPES6FA/1

RIF EMB RIF EMB

Initial 98.32±1.51 99.83±2.05 99.27±2.01 99.19±0.97 1.193 1.210

1 month 97.34±1.43 99.43±1.17 98.14±1.25 98.35±1.24 1.184 1.186

3 month 97.02±1.68 98.17±1.46 98.26±1.12 98.01±1.92 1.201 1.196

6 month 95.64±1.03 96.78±2.09 97.32±1.24 95.83±1.01 1.210 1.221

Table 5.20: Stability results of RMPES/3 and RMPES3FA/1 at 300C/65%RH


RMPES/3 RMPES3FA/1 RMPES/3 RMPES3FA/1

RIF MSDNC22 RIF MSDNC22

Initial 97.87±2.05 101.8±2.39 98.67±2.31 99.87±1.97 1.228 1.1769

1 month 97.73±1.17 99.67±1.97 98.19±1.72 99.45±1.84 1.204 1.1580

3 month 97.27±1.49 99.11±1.83 97.83±1.39 98.89±1.92 1.218 1.1767

6 month 98.56±1.22 98.18±1.78 95.82±2.24 97.94±2.09 1.2398 1.1924

12 month 97.18±1.59 98.22±1.75 95.96±2.73 96.92±1.83 1.231 1.217



Table 5.21: Stability results of RMPES/3 and RMPES3FA/1 at 400C/75%RH


RMPES/3 RMPES3FA/1 RMPES/3 RMPES3FA/1

RIF MSDNC22 RIF MSDNC22

Initial 97.87±2.05 101.8±2.39 98.67±2.31 99.87±1.97 1.228 1.176

1 month 97.14±1.56 99.44±1.67 97.23±1.59 99.41±1.49 1.214 1.172

3 month 96.45±1.79 99.02±1.91 96.63±1.66 98.34±1.67 1.243 1.187

6 month 95.43±2.22 96.23±1.89 95.19±1.47 97.49±2.01 1.234 1.200

5.5. MACROPHAGE UPTAKE STUDY IN U937 CELL LINE

Table 5.22: In vitro macrophage uptake of NPDDS

NPDDS

Supernatant Cell pellets

RIF (%)

EMB/ MSDNC-22

(%)

RIF (%)

EMB/ MSDNC-22

(%)

RIF Solution 90.68 ±1.94 - 7.3

±2.86 -

RIF-EMB solution (RIF to EMB ratio 1:10)

87.08 ±1.01

89.34 ±0.96

10.01 ±0.95

8.93 ±1.45

RIF-MSDNC-22 solution (RIF to MSDNC-22 ratio 1:10)

88.98±1.22

86.59 ±0.92

9.32 ±1.42

10.23 ±1.35

RIF PES Nps (RPES/1) 60.61±0.76 - 39.27

±0.36 -

RIF PES FA Nps (RPESFA/1) 39.24±0.53 - 59.57

±0.64 -

RIF-EMB PES Nps (REPES/6) (RIF to EMB ratio 1:10)

54.72±1.25

57.34 ±1.23

43.12 ±3.27

42.06 ±0.87

RIF-EMB PES FA Nps (REPES6FA/1) (RIF to EMB ratio 1:10)

35.87±1.48

36.34 ±0.93

62.94 ±1.53

62.71 ±1.10

RIF-MSDNC-22 PES Nps (RMPES/3) (RIF to MSDNC-22 ratio 1:10)

56.86±1.95

55.35 ±1.14

44.68 ±2.80

44.48 ±0.76

RIF-MSDNC-22 PES FA Nps (RMPES3FA/1) (RIF to MSDNC-22 ratio 1:10)

35.89±2.20

36.18 ±1.74

64.36 ±2.62

64.80 ±0.71



Quantification of drug was done both in the supernatant and the cell pellet.

Quantitative uptake study was carried out by incubating nanoparticles equivalent to

10μg/ml of RIF as this was the maximum non-cytotoxic concentration. In-vitro

studies on RIF-EMB combinations revealed maximum efficacy against MTb at

RIF:EMB (1:10). Hence nanoparticles of the same ratio of the two drugs were

evaluated for macrophage uptake.

Uptake of drugs by macrophages was in the order drug solution< nanoparticles < folic

acid anchored nanoparticles for RIF, RFM-EMB and RIF-MSDNC-22 combination

(Table 5.22). The mass balance was >90% in all cases when total drug concentration

was evaluated in supernatant and in cell pellets. The plain drug revealed ≤10% uptake

irrespective of their hydrophobicity. PES nanoparticles of RIF, EMB and MSDNC-22

revealed ~43% uptake which further increase to ~60% with folate anchoring (figure

5.12). Macrophage are known to express folate receptor and our study confirmed the

role of folate receptor on enhance macrophage uptake. This enhancement in uptake is

reflected in table 5.22 and figure 5.12.

a) b)

c)

Figure 5.12: In vitro macrophage uptake of a) RPES/1 and RPESFA/1, b) REPES/6 and REPES6FA/1, c) RMPES/3 and RMPES3FA/1



Despite contrasting solubilities of RIF (slightly water soluble) and EMB (highly water

soluble), the comparable uptake from RIF and EMB from the combination

nanoparticles suggests stability/integrity and rapid internalization of nanoparticles by

macrophages, as EMB being highly water soluble could have leached out of the

nanoparticles prior to uptake. As combination drug therapy is important for the

efficient treatment of tuberculosis due to rapid development of drug resistance with

single drugs, the comparable uptake of RIF and EMB from combination nanoparticles

suggests NPDDS of drug combinations as a promising approach for targeted delivery

to macrophage for the treatment of tuberculosis.

Premature release of folate from nanoparticles could result in decreased uptake of

folate anchored nanoparticles due to competitive inhibition (Reddy et al., 2002; Patil

et al., 2008; Gabizon et al., 1999; Aronov et al., 2003; Goren et al., 2000). Although

folic acid was anchored by simple physical adsorption folate mediated enhanced

macrophage uptake was observed suggesting physical adsorption as a simple strategy

for folate anchoring.

5.6. ORAL BIODISTRIBUTION STUDY BY GAMMA SCINTIGRAPHY

Figure 5.13: Organ distribution profile of RPES/1 and RPESFA/1 post 6h oral administration by gamma scintigraphy

The organ distribution of RIF nanoparticle is shown in figure 5.13. After 6h of RIF

PES nanoparticle with and without folic acid administration maximum radioactivity

was observed in stomach (50-60%) and intestine (20-30%) (Table 5.23). Significantly

higher concentrations of RIF were seen in the liver, the lungs and kidney following



oral administration of nanoparticles compared to drug solution. Surprisingly the

concentration of RIF in the lung with the nanoparticles was significantly higher than

from the solution. While an approximately 13 fold increase in lung concentration was

seen with RIF PES Np’s, the folate anchored RIF PES Np’s revealed a 35 fold

increase.

Table 5.23: Organ distribution of RIF nanoparticles expressed as % injected dose

% Distribution

RIF Solution RPES/1 RPESFA/1 Heart 0.12 0.14 0.17 Liver 0.53 1.47 1.42 Lung 0.09 1.08 3.52 Left Kidney 0.10 0.16 0.21 Right Kidney 0.11 0.15 0.21 Spleen 0.25 0.25 0.14 Blood 0.22 0.23 0.25 Bone 0.15 0.26 0.13 Muscle 0.14 0.25 0.25 Stomach 55.87 51.10 64.02 Intestine 32.40 34.20 21.17 Body 7.13 5.00 5.57 TOTAL 97.47 94.29 97.06

Polymeric nanoparticles, because of their small size, are capable of being absorbed

intact from the gastrointestinal tract by transcellular pathways, or via active processes

mediated by membrane-bound carriers or membrane-derived vesicles (Devissaguet et

al., 1992, Jiao et al., 2002). Most evidences suggest that nanoparticle uptake occurs

preferentially via “M-Cells” in Peyer’s patches by the process of endocytosis

(lymphatic uptake). Nanoparticles below 1 μm are taken up by M cells and delivered

in the basal medium, while particles larger than 5 μm are taken up by M cells but

remain entrapped in Peyer's patches. Our nanoparticles are in the size range of 350-

450nm which enable absorption of nanoparticle by M-cells of Peyer’s patches and

avoid portal circulation. Subsequently particles enter mesenteric lymph nodes and are

drained in to the blood via junction of the left jugular and sub clavian vain through

thoracic duct to pass to the heart (Swartz et al., 2001). Bypass of portal circulation and



direct transport to the lungs via heart by the lymph could have enabled enhanced lung

concentration with the nanoparticles.

Further the high blood concentration of RIF from the nanoparticle is suggestive of

longer circulation time and possible controlled release. Folic acid is a known ligand

for macrophage which express the folate receptor. This suggests the higher

concentration with folic acid anchored nanoparticles in the lungs due to alveolar

macrophage uptake.

5.7. PHARMACOKINETIC AND LUNG UPTAKE STUDY

The plasma concentration versus time profile of RIF solution, RIF PES Np’s and RIF

PES FA Np’s following oral administration is depicted in figure 5.14.

Figure 5.14: Pharmacokinetic profile of RIF PES nanoparticles

Rifampicin nanoparticles with and without ligand folic acid exhibited higher Cmax

and Tmax and sustained plasma drug concentration as compared to rifampicin

solution. The pharmacokinetic parameters are reported in table 5.24. A significant

increase in T1/2 was seen with the RIF PES Np’s compared to plain drug, however a

lower T1/2 was revealed in presence of folate suggesting more rapid clearance from

the plasma, due to probable enhanced uptake by the RES. High plasma concentrations

were observed even at 12 hours with the Np’s which were comparable to the Cmax of

the solution of RIF. Bioavailability increased upto 178% with RIF PES NPs and,

210% with RIF PES FA NPs compared with plain drug.



Table 5.24: Pharmacokinetic parameters of RIF PES nanoparticles

Parameters RIF Solution RPES/1 RPESFA/1

Cmax (μg/ml) 6.53 ± 1.2 7.90 ± 0.78 8.17 ± 0.88 Tmax (h) 2 4 4

Slope - 4.34 -2.41 -2.81 Kel(h-1) 0.1001 0.0055 0.0064 T ½ (h) 6.92 ± 1.32 12.48 ± 0.59 10.67 ± 0.73

AUC0-24h (μg/ml*h) 55.51 ± 2.57 98.55 ± 2.89 115.17 ± 3.12Bioavailability enhancement Ref. 178% 210%

PES nanoparticles revealed significantly higher lung concentration compared to

rifampicin solution (figure 5.15). Folic acid anchoring further enhanced lung uptake

significantly (P<0.05). The reported MIC of RIF is 0.25µg/ml. The concentration of

RIF in the lungs following oral administration of Np’s is significantly greater than

then MIC (P<0.05), ~70 times higher with RIF PES Np’s and ~100 times higher with

folate anchoring.

Figure 5.15: Lung concentration of RPES/1 and RPESFA/1 nanoparticles at 24h

Similar results were obtained with RIF-EMB PES combination nanoparticles. RIF

bioavailability increased upto 132% and 146% with RIF-EMB PES NPs and RIF-

EMB PES FA NPs respectively compared to plain RIF. EMB bioavailability

increased upto 237% and 247% with RIF-EMB PES NPs and RIF-EMB PES FA NPs

respectively compared with plain EMB (table 5.25). The nanoparticles revealed

sustained release with significant increase in half life for both RIF and EMB. Folate

anchoring did not influence the PK behavior of the nanoparticles (figure 5.16). The

Cmax for RIF and EMB was higher and the Tmax delayed compared to RIF and EMB

solutions respectively.



Figure 5.16: Pharmacokinetic profile of REPES/6 and REPES6FA/1 nanoparticles

Table 5.25: Pharmacokinetic parameters of RIF-EMB PES nanoparticles

Parameters RIF-EMB Solution REPES/6 REPES6FA/1

RIF EMB RIF EMB RIF EMB

Cmax

(μg/ml) 1.31 12.62 1.24 12.38 1.31 12.46

T max

(h) 2 1 4 4 4 4

Slope -4.36 -5.38 -3.53 -3.03 -3.65 -3.77

Kel(h-1) 0.10 0.124 0.0081 0.0085 0.0084 0.0086

T ½ (h) 6.89 5.58 8.5 8.13 8.24 7.96

AUC0-24h (μg/ml*h) 11.13 62.33 14.68 147.57 16.25 153.43

Bioavailability enhancement factor

Ref Ref 132% 237% 146% 246%

Figure 5.17: Lung concentration a) RIF and b) EMB from REPES/6 and

REPES6FA/1 nanoparticles at 24h



Lung uptake study revealed significantly high lung concentration of RIF and EMB

from the nanoparticles compared to the respective solutions. Moreover the lung

concentration at the end of 24 hrs for both drugs from the nanoparticles translated to

concentrations many fold higher than the MIC for RIF and EMB (MIC of RIF-

0.25μg/ml, EMB-1.5μg/ml). Further folate anchoring resulted in enhanced lung

deposition (figure 5.17). Increased lung uptakes following oral administration of

nanoparticles can be explained as discussed earlier in section 5.6.

5.8. HIGHLIGHTS A promising and effective oral drug delivery system for pulmonary targeting

using a new biodegradable polymer PES, which will be useful not only for

treatment of tuberculosis but also other potential lung infections, has been

developed.

Nanoparticles with RIF:EMB in varying ratios could be readily prepared

despite contrasting solubilities of RIF and EMB.

A simple physical adsorption approach for anchoring ligand folic acid on

nanoparticles prepared using polymer PES has been developed thereby

precluding the need for covalent bonding.

High and comparable uptake of both RIF and EMB from combination

nanoparticles indicated integrity/stability of RIF-EMB combination

nanoparticles and rapid macrophage uptake, as EMB being highly water

soluble could have leached out of the nanoparticles prior to uptake

A significantly enhancement in bioavailability was observed following oral

administration of RIF PES and RIF-EMB PES nanoparticles

RIF PES and RIF-EMB PES nanoparticles exhibited lung concentrations

much higher than MIC of RIF and EMB at 24h.

Bypass of portal circulation and direct transport to the lungs via heart by the

lymph could have enabled enhanced lung concentration with the nanoparticles.

Macrophages are known to express folate receptor, hence higher lung

concentration with folate anchored nanoparticle suggests folate receptor

mediated uptake.

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