cr8.pdf

Critical Reviews™ in Therapeutic Drug Carrier Systems, 29(4), 299-353 (2012)

0743-4863/12/$35.00 ©2012 Begell House, Inc. www.begellhouse.com 299

Colloidal Carriers: A Rising Tool for Therapy of Tuberculosis Swati Gupta,1,2* Pankaj Kumar,1 Manish K. Gupta,3 & Suresh P. Vyas4

1Nanomedicine Research Center, Department of Pharmaceutics, I.S.F. College of Pharmacy,Moga 142 001 (PB), India; 2Department of Pharmaceutical Sciences, University of South Florida Health, Tampa, FL, 33612, USA; 3Laboratory for Drug Design and Synthesis, Department of Pharmaceutical Chemistry, I.S.F. College of Pharmacy, Moga 142 001 (PB), India; 4Drug delivery Research Laboratory, Department of Pharmaceutical Sciences, Dr. H. S. Gour University, Sagar 470003, India

*Corresponding author: Swati Gupta, Nanomedicine Research Center, Department of Pharmaceutics I.S.F. College of Pharmacy, Moga 142 001 (PB), India, Telefax: +91-1636-236564; [email protected].

ABSTRACT: Tuberculosis (TB) is the second most deadly infectious disease, caused mainly by M. tuberculosis in humans, usually affecting the lungs; it also attacks other parts of the body. The design of novel antibiotics attempts to overcome drug resistance, to shorten the treatment course, and to reduce drug interactions with antiretroviral therapies. Overcoming technological draw-backs of these therapeutic agents as well as improving the effectiveness of the drugs by targeting the infection reservoirs remain the central aims of pharmaceutical technology. In this framework, colloidal carriers appear as one of the most promising approaches for the development of more effective and compliant medicines by releasing the drugs slowly over prolonged time periods and reducing the current costs of treatment. Due to unique physicochemical properties (ultrasmall and controllable size, large surface area to mass ratio, high reactivity, and functionalizable struc-ture) of colloidal carriers, they can facilitate the administration of antitubercular drugs, thereby overcoming some of the limitations in traditional antitubercular therapeutics. In recent years, encapsulation of antitubercular drugs in colloidal carrier systems is emerging as an innovative and promising alternative with enhanced therapeutic effectiveness and reduced undesirable side effects of the encapsulated drugs. The present review aims to describe the current conventional as well as combination drug therapy with special consideration towards the emerging role of novel colloidal carriers designed and targeted against TB. Colloidal carriers employing drugs alone or in combination targeted towards the site of action could lead to reduction in duration of conven-tional treatment, higher patient fulfillment, and prevention of antitubercular drug resistance or toxicity.

KEY WORDS: nanoparticles, liposomes, microparticles, tuberculosis

I. INTRODUCTION

TB is an infectious disease caused by mainly M. tuberculosis in humans. Other myco-bacteria, such as M. bovis, M. africanum, M. canetti, and M. microti, also cause TB, but these species are less common. TB is a leading health problem worldwide and remains one of the leading causes of death from infectious diseases. According to the World Health Organization, an estimated 2 billion people are infected with M. tuberculosis.

Critical Reviews™ in Therapeutic Drug Carrier Systems

300 S. Gupta et al.

The 5%–10% of people who are infected with TB bacilli become sick or infectious at some time during their life. In the year 2010, approximately 8.8 million new TB cases were identified worldwide. People with weakened immune systems, as in human im-munodeficiency virus (HIV) infection, are more prone to develop TB, and hence it is a major cause of death among people living with HIV. TB claimed 1.4 million deaths in 2010, including 0.35 million people with HIV.1,2

There are several outcomes associated with exposure to M. tuberculosis. Following close contact, 30% of individuals become infected, with about 40% of these individuals developing primary active TB and 60% developing latent infection; 2%–23% of immu-nocompetent patients with latent TB reactivate at a later date, while patients with HIV develop reactivation TB at a rate of ~5%–10% per year (Fig. 1).3

Figure 1: Outcomes associated with exposure to M. tuberculosis

Patients with latent TB infection have therefore been exposed to the organism, but the initial infection was controlled by the host defense mechanisms and can be sub-sequently traced only by the positive delayed hypersensitivity skin test response. The small numbers of remaining organisms are in a dormant or latent state, but they do pose a risk for reactivation at a later time, especially with any impairment in the host’s cel-lular immunity.

TB disease or active TB is defined by the presence of clinically active disease in one or more organ systems, ideally with confirmation of the diagnosis by isolation of the organism M. tuberculosis.4,5 Differences between latent TB infection and TB disease are summarized in Table 1.

I.A. Main Features of Tuberculosis: From Infection to Host Defense

TB can manifest itself at any tissue site; the lung represents both the main port of entry and an important site of disease manifestation. Extrapulmonary TB develops in less than 10% of all cases. Droplets containing minute numbers of bacilli are expelled by individuals suffering from active pulmonary TB. Alveolar macrophages (AMs) engulf

Volume 29, Number 4, 2012

Colloidal Carriers: A Rising Tool for Therapy of Tuberculosis 301

these droplets, but do not kill the pathogen. Specific T cells are stimulated in the drain-ing lymph nodes and induce bacterial containment in small granulomatous lesions of the lung, but fail to achieve complete microbial eradication (Fig. 2). So, a dynamic balance between bacterial persistence and host defense develops. This balance might be life long, so that the individual is infected but does not suffer from clinical disease. Less than 10% of infected individuals develop clinical disease during their lifetimes, but once disease does develop, if it remains untreated, it is fatal in 50% of patients (approxi-mately 2 million deaths annually). Disease outbreak is delayed because the progress of infection is very slow. In the adult, TB occurs typically as a result of a reactivation of existing foci, rather than as a direct outcome of primary infection (Fig. 2).6 Only in im-munologically incompetent individuals, such as newborns, the aged, and HIV-infected patients, primary infection ordinarily transforms into disease. In individuals in whom infection converts into disease, cavitary lesions develop and bacteria increase in number in the caeseous detritus. (As a result of cellular disintegration and destruction, the cen-tral material of a granuloma becomes caeseous. In TB, this lipid-rich material provides a nutrient-rich source for the pathogen. Further destruction might lead to liquefaction, thereby allowing microbial dissemination.) Once cavitation reaches the alveoli, the pa-tient becomes infectious, and a person with active disease infects up to 15 people annu-ally.7 The vicious circle then continues.

There are three potential outcomes of infection of the human host in M. tuberculo-sis. (a) In the immunocompromised host, disease can develop directly after infection.

TABLE 1. Difference between latent TB infection and TB disease

A person with latent TB infection A person with TB diseaseDoes not feel sick Usually feels sick

Cannot spread TB bacteria to others May spread TB bacteria to others

Usually has a skin test or blood test re-sult indicating TB infection

Usually has a skin test or blood test result indicating TB infection

Has a normal chest X-ray and a negative sputum smear

May have an abnormal chest X-ray, or positive sputum smear or culture

Needs treatment for latent TB infection to prevent active TB disease

Needs treatment to treat active TB disease

Has no symptoms Has symptoms that may include: •a bad cough that lasts 3 weeks or longer •pain in the chest •coughing up blood or sputum•weakness or fatigue •weight loss •no appetite •chills •fever •sweating at night


302 S. Gupta et al.

Figu

re 2

: Mai

n fe

atur

es o

f tub

ercu

losis

: fro

m in

fect

ion

to h

ost d

efen

se



(b) The frequency of abortive infection resulting in spontaneous healing is unknown, but is assumed to be minute. (c) In most cases, mycobacteria are initially contained and disease develops later as a result of reactivation. The granuloma is the site of infection, persistence, pathology, and protection. Effector T cells (including conventional CD4+ and CD8+ T cells, and unconventional T cells, such as γδ T cells, and double-negative or CD4/CD8 single-positive T cells, which recognize antigen in the context of CD1) and macrophages participate in the control of TB. Interferon-γ (IFN-γ) and tumor-necrosis factor-α (TNF-α), produced by T cells, are important macrophage activators. Macro-phage activation permits phagosomal maturation and the production of antimicrobial molecules such as reactive nitrogen intermediates (RNI) and reactive oxygen intermedi-ates (ROI), lymphotoxin-α3 (LT- α3).

General symptoms of active TB infection include weight loss, sweats, chills, fe-ver, coughing, fatigue, and loss of appetite. Other symptoms of active TB depend on the site of the infection. The lungs are the most common sites of TB disease, and approximately 85% of the TB cases are pulmonary. Patients with pulmonary TB dis-ease usually have a productive, prolonged cough and sometimes have chest pain or may cough up blood. Patients with chest radiographic evidence of TB are considered contagious. TB may also occur in the central nervous system and the bacterium can invade the bones and joints.8

I.B. Transmission and Pathogenesis of M. tuberculosis

TB is spread from person to person through the air by droplet nuclei, particles 1 to 5 mm in diameter that contain M. tuberculosis complex. Droplet nuclei are produced when per-sons with pulmonary or laryngeal TB cough, sneeze, speak, or sing. They also may be produced by aerosol treatments, sputum induction, aerosolization during bronchoscopy, and through manipulation of lesions or processing of tissue or secretions in the hospital or laboratory. Droplet nuclei, containing two to three M. tuberculosis organisms,9 are so small that air currents normally present in any indoor space can keep them airborne for long periods of time. Droplet nuclei are small enough to reach the alveoli within the lungs, where the organisms replicate. Although patients with TB also generate larger particles containing numerous bacilli, these particles do not serve as effective vehicles for transmission of infection because they do not remain airborne, and if inhaled, do not reach alveoli. Organisms deposited on intact mucosa or skin do not invade tissue. When large particles are inhaled, they impact on the wall of the upper airways, where they are trapped in the mucous blanket, carried to the oropharynx, and swallowed or expecto-rated.10,11 Four factors determine the likelihood of transmission of M. tuberculosis: (1) the number of organisms being expelled into the air, (2) the concentration of organisms in the air, determined by the volume of the space and its ventilation, (3) the length of time an exposed person breathes the contaminated air, and (4) presumably the immune status of the exposed individual.12 Modes of transmission of TB with their percentages13 are summarized in Fig. 3.


304 S. Gupta et al.

I.C. RIsk FACTORs

• Persons with silicosis have an approximately 30-fold greater risk for developing TB.• Persons with chronic renal failure who are on hemodialysis also have an increased

risk: 10–25 times greater than the general population.• Persons with diabetes mellitus have a risk for developing active TB that is 2–4 times

greater than persons without diabetes mellitus.• Gastrectomy with attendant weight loss and malabsorption, jejunoileal bypass, renal

and cardiac transplantation, carcinoma of the head or neck, and other neoplasms are also associated with active TB (e.g., lung cancer, lymphoma, and leukemia).14–16

• When people with a positive skin or blood test become infected with HIV, the risk of developing active infection is very high. Similarly, the risk is also high if people who have a latent infection take corticosteroids or other drugs that suppress the immune system (including some of the newer anti-inflammatory drugs).

• Children with immature immune systems, patients taking steroids or other forms of immunosuppressive therapy, diabetic patients, and patients under severe stress from major illness or major surgery and anesthesia are at significant risk of TB.17,18

• A vegetarian diet is an independent risk factor for TB in immigrant Asians.• Specific gene polymorphisms in IL12B have been linked to TB susceptibility.19,20 • Persons at increased risk of TB21 are summarized in Table 2.

Figure 3: Mode of transmission of tuberculosis



I.D. sTAges OF INFeCTION OF TB

There are several stages:• Primary infection• Latent infection• Active disease

Except for very young children and people with a weakened immune system, few people become sick immediately after TB bacteria enter their body (this stage is called primary infection). In most cases, TB bacteria that enter the lungs are immediately killed by the body’s defenses. Those that survive are engulfed by white blood cells called mac-rophages. The engulfed bacteria can remain alive inside these cells in a dormant state for many years, is walled off inside tiny scars (this stage is called latent infection). In 90% to 95% of cases, the bacteria never cause any further problems, but in about 5% to 10% of infected people, they eventually start to multiply and cause active disease. At this stage, infected people actually become sick and can spread the disease. More than half the time, dormant bacteria reactivate within the first 2 years after the primary infection, but they may not reactivate for a very long time, even decades. The progression of TB from latent infection to active disease varies greatly. Progression to active disease is far more likely and much faster in people with HIV infection and other conditions (includ-ing drugs) that weaken the immune system. In people with a fully functioning immune

TABLE 2. Persons at increased risk of TBPersons at increased risk

Risk Examples of persons with riskIncreased risk of exposure to infectious cases

Persons with recent close contact with persons known to have active TB.Health care workers who work at facilities where patients with TB are treated.

Increased risk of TB infection Foreign-born persons from countries with a high prevalence of TB.Homeless persons. Persons living or working in facilities providing long-term care.

Increased risk of active TB, once infection has occurred

HIV-infected persons.Persons with recent TB infection.Injection-drug users.Patients with end-stage renal disease.Patients with silicosis.Patients with diabetes mellitus.Patients receiving immunosuppressive therapy.Patients with hematologic cancers.Malnourished persons or those with a recent weight loss of more than 10% of their ideal body weight.Persons who have undergone gastrectomy or jejunoileal bypass.


306 S. Gupta et al.

system, active TB is usually limited to the lungs (pulmonary TB). TB that affects other parts of the body (extrapulmonary TB) comes from pulmonary TB that is spread from the lungs through the blood. As in the lungs, the infection may not cause disease, but the bacteria may remain dormant in a very small scar. Dormant bacteria in these scars can reactivate later in life, leading to symptoms related to the organs involved. In pregnant women, TB bacteria may spread to the fetus and cause disease (called congenital TB). However, such cases are extremely uncommon.17 Stages of infection of TB are sum-marized in Fig. 4.

II. ReCePTORs FOR MYCOBACTERIUM TUBERCULOSIS

Phagocyte complement receptors occur in two distinct structural forms. Complement receptor type 1 (CR1) is a monomeric transmembrane protein that binds C3b and C4b but not C3bi. CR3 and CR4 are heterodimeric proteins of the integrin superfamily. The macrophage mannose receptor is a monomeric transmembrane protein, with an extra-cellular domain containing eight carbohydrate-recognition domains characteristic of C-type (calcium-dependent) lectins. These are expressed on mature macrophages but not on fresh monocytes.22,23 Specific receptors for surfactant protein A (Sp-A) are present on macrophages; the number and specific molecular identity of Sp-A receptors are incom-pletely defined. Sp-A is a member of the collectin family of proteins, which includes serum mannose binding protein (MBP) and complement component C1q.24 Cluster of

Figure 4: Stages of infection of TB



differentiation 14 (CD14), a phosphatidylinositol glycan–linked membrane protein, is best known and characterized as the high-affinity receptor for lipopolysaccharides of Gram-negative bacteria. However, CD14 can also bind LAM (lipoarabinomannan) of M. tuberculosis (H37Ra), and this binding induces macrophages to secrete interleu-kin-8.25 Macrophage scavenger receptors bind polyanionic macromolecules and parti-cles, including lipopolysaccharides of Gram-negative bacteria and lipoteichoic acid of Gram-positive bacteria.26

III. THe INTRACeLLULAR LIFesTYLe OF M. TUBERCULOSIS

M. tuberculosis uses macrophages as its preferred habitat, which has two important implications for its survival. First, macrophages, as professional phagocytes, are en-dowed with several surface receptors that facilitate antigen uptake, thereby rendering specific host-invasion strategies dispensable.27 Cholesterol (CHOL) has been shown to act as a docking site for the pathogen, promoting receptor-ligand interactions. The ini-tial interaction with surface receptors influences the subsequent fate of M. tuberculosis within the macrophage. Interactions with the constant regions of immunoglobulin recep-tors (FcRs) and Toll-like receptors stimulate host-defense mechanisms, whereas those with complement receptors promote mycobacterial survival.28 The second consequence, which warrants more sophisticated coping mechanisms, is the survival of M. tuberculo-sis within the phagosome, as this constitutes a harsh environment that is detrimental to many microbes. M. tuberculosis solves this obstacle by arresting the phagosome at an early stage of maturation, and by preventing fusion of the phagosome with lysosomes (Fig. 5). Residing in the early recycling endosome, M. tuberculosis attains ready ac-cess to iron, which is essential for intracellular survival. Iron is also required in various host-defense mechanisms. To successfully compete for iron with its host, the pathogen possesses specialized iron-scavenging molecules. The arrest of phagosomal matura-tion is not an all-or-nothing event, and some mycobacterial phagosomes can proceed to develop to the more mature stages of the phagolysosome. Phagosomal maturation is promoted by activation with IFN-γ, which stimulates anti-mycobacterial mechanisms in macrophages, notably ROI and RNI (Fig. 5).29,30 The role of RNI in the control of human TB remains controversial, despite a body of evidence from animal experiments that supports its role in the control of M. tuberculosis. However, even IFN-γ-activated macrophages fail to fully eradicate their resident M. tuberculosis organisms. Persistent microbes might proceed to a stage of dormancy with a reduced metabolic activity that facilitates their survival under conditions of nutrient and oxygen deprivation. These bac-teria can persist without without any development of disease and therefore create a state of latency. Nevertheless, the risk of disease outbreak at a later time remains. In vitro experiments indicate that mycobacteria switch to lipid catabolism and nitrate respira-tion to ensure their survival.31,32 The abundant lipids present in the caeseous detritus of granulomas provide a rich source of nutrients during persistence.

Figure 5 illustrates phagosome and endosome maturation and the influence of M. tuberculosis on this process. Phagocytosis of larger particles is a unique property of


308 S. Gupta et al.

Figu

re 5

: The

intra

cellu

lar l

ifest

yle o

f M. t

uber

culo

sis. C

R, c

ompl

emen

t rec

epto

r; Fc

R, r

ecep

tor f

or th

e co

nsta

nt fr

agm

ent o

f im

mun

oglo

bulin

; MR

, m

anno

se re

cept

or; S

PR, s

urfa

ctan

t.



specialized cell types—the “professional” phagocytes—whereas virtually all eukaryotic cells are capable of engulfing small particles and fluids that end up in the endosomal path-way. Both the endosomal and phagosomal pathways undergo interconnected maturation processes that merge at a later stage, prior to fusion with lysosomes. Various surface receptors participate in the early encounter between M. tuberculosis and macrophages. CHOL serves as a docking site that facilitates interactions between mycobacteria and surface receptors.28 Once engulfed, M. tuberculosis ends up in a phagosome, the matu-ration of which is arrested at an early stage. The early phagosome-harboring mycobac-terium characteristically retains tryptophane aspartate containing coat protein (TACO), which apparently prevents its further maturation. M. tuberculosis inhibits phagosomal acidification (which occurs by means of a V–H+ ATPase) and prevents fusion with the endosomal pathway. The arrest of phagosomal maturation is, however, incomplete and some phagosomes mature to form phagolysosomes. Phagosome maturation is promoted in activated macrophages, particularly after IFN-γ stimulation.33 Although phagosome and endosome maturation form a continuum, distinct steps can be distinguished by means of different markers and tracers.

IV. DIAgNOsIs

TB is diagnosed definitely by identifying the causative organism (M. tuberculosis) in a clinical sample (for example, sputum or pus). When this is not possible, a probable—although sometimes inconclusive—diagnosis may be made using imaging (X-rays or scans) and/or a tuberculin skin test (Mantoux test). A complete medical evaluation for TB must include a medical history, a physical examination, a chest X-ray, microbiologi-cal smears, and cultures. It may also include a tuberculin skin test and a serological test. Currently, latent infection is diagnosed in a non-immunized person by a tuberculin skin test.34,35 A Heaf gun is used to inject multiple samples of testing serum under the skin at once for a Heaf test. In a Mantoux test, a standard dose of 5 tuberculin units (0.1 mL) is injected intradermally (between the layers of dermis) and read 48 to 72 hours later. A person who has been exposed to the bacteria is expected to mount an immune response in the skin containing the bacterial proteins.36,37 The newer interferon gamma release assays (IGRAs) detect the release of interferon gamma in response to mycobacterial proteins such as the 6-kDa early secretory antigenic target of M. tuberculosis (ESAT-6). For rapid identification of M. tuberculosis and other mycobacteria, several DNA probes have been developed and are available. They are used for rapid confirmation of the identity of mycobacterial isolates.38,39 Ribosomal rRNA-based probes are 10,000 times more sensitive than DNA targeting and are used to directly confirm the diagnosis in clinical specimens. Gene amplification techniques are highly sensitive and under op-timum conditions may detect 1–10 organisms.40,41 Microscopy is the simplest and most rapid procedure currently available to detect acid fast bacilli in clinical specimens by the Ziehl-Neelsen staining method or its modifications. Its limitation is that it requires at least 10,000 to 100,000 bacilli per mL of sputum. Detection of lam in sputum is based on capture antibody derived from a murine source. The radiometric BACTEC 460 TB


310 S. Gupta et al.

method detects the presence of mycobacteria based on their metabolism rather than on visible growth.42,43 Insta test TB is a rapid in vitro assay for detection of antibody in ac-tive TB using whole blood or serum. Fast plaque TB uses mycobacteriophage to detect the presence of M. tuberculosis directly from sputum specimens.44,45

V. CONVeNTIONAL CHeMOTHeRAPY

Oral antibiotic treatment is the standard means of controlling and treating most cases of TB. For drug-susceptible TB disease, the initial intensive phase consists of a minimum of three drugs administered concurrently to reduce the rapidly dividing bacillary load; a minimum of two drugs is used in the continuation phase, aimed at sterilizing lesions con-taining fewer and slow-growing bacilli. Although TB can be cured with chemotherapy, the treatment is exceedingly lengthy and takes 6–9 months. Apart from significant toxic-ity, the lengthy therapy also creates poor patient compliance, which is a frequent cause for selection of drug-resistant and often deadly multidrug-resistant TB (MDR-TB) bacte-ria.46,47 The first-line therapy consists of isoniazid (INH), rifampicin (RIF), pyrazinamide (PZA), and ethambutol (EMB) in the United States. Streptomycin (SM) is sometimes substituted for EMB outside the US to reduce expense. INH and RIF are the primary agents in a combination therapy and act against the metabolically dynamic mycobacteria that multiply perpetually and rapidly, and also against the quasi-dormant bacilli. RIF has the added advantage of acting at a very early stage of bacillary propagation. Antituber-cular chemotherapy containing INH, RIF, and PZA has proved to be highly effective but hepatotoxic. Antitubercular drug-induced hepatotoxicity (DIH) is the most common side effect, leading to interruption of therapy. Risk of antitubercular DIH is increased when these drugs are combined.48 The second-line class of drugs includes: aminoglycoside an-tibiotics, cycloserine, ethionamide (ETH), and fluoroquinolones (FQs). Amikacin (AMI), kanamycin (KAN), and SM are the prominent aminoglycoside antibiotics; levofloxacin (LFX) and moxifloxacin (MFX) are the FQ antibiotics, while capreomycin (CAP) sul-phate is a cyclic polypeptide antibiotic effective against M. tuberculosis.49 Second-line antituberculosis drugs (ATDs) are employed only if the patient is not responding to the first-line therapy and/or is believed to be infected with drug-resistant strains of M. tu-berculosis; second-line agents are less effective and more toxic than the first-line drugs. The above antibiotics are sometimes administered by the parenteral route, but this is not preferred due to the associated or perceived discomfort. Blood-borne infections are also thought to originate from the re-use of needles in resource-poor nations.27

The current doses administered are high compared to the required minimum inhibitory concentration (MIC) of the drugs. This is because the drugs have poor bioavailability and low permeability, which is a function of degradation of drugs before reaching their target site. Thus the drawbacks of conventional chemotherapy necessitate the development of a carrier system that can release drugs slowly over prolonged time periods and reduce the current costs of treatment.50 The details of various first-line and second-line drugs against TB51–53 are summarized in Table 3 and Table 4, respectively, and mechanisms of action of various first-line drugs are summarized in Fig. 6.



TAB

LE 3

. Var

ious

firs

t-lin

e dr

ugs

used

aga

inst

TB

, the

ir st

ruct

ure,

mec

hani

sm o

f act

ion,

act

ivity

, adv

erse

dru

g re

actio

ns, a

nd ty

pi-

cal a

dult

dosa

ge

Dru

g (R

OA

) & s

truc

ture

Mec

hani

sm o

f ac

tion

Act

ivity

Adv

erse

dru

g

reac

tions

Typi

cal

adul

t do

sage

Ref

eren

ce

RIF

(ora

l)

Inhi

bits

DN

A-

depe

nden

t RN

A po

lym

eras

e, th

ereb

y bl

ocki

ng p

rodu

ctio

n of

RN

A

Bac

teric

idal

act

ivity

ag

ains

t sus

cept

ible

ba

cter

ia a

nd m

yco-

bact

eria

resi

stan

ce

rapi

dly

emer

ges

whe

n us

ed a

s a

sing

le d

rug

in th

e tre

atm

ent o

f act

ive

infe

ctio

n

Ras

h, n

ephr

itis,

th

rom

bocy

tope

nia,

cholestasis,flu-like

synd

rom

e w

ith in

ter-

mitt

ent d

osin

g

600

mg/

d51

, 52

INH

(ora

l)

Inhi

bits

syn

thes

is

of m

ycol

ic a

cids

, an

esse

ntia

l com

pone

nt

of m

ycob

acte

rial c

ell

wal

ls

Bac

teric

idal

act

ivity

ag

ains

t sus

cept

ible

st

rain

s of

M.

tube

rcul

osis

Hep

atot

oxic

, per

iph-

eral

neu

ropa

thy

300

mg/

d52

, 53

PZA

(ora

l)

Not

fully

und

erst

ood

PZA

is c

onve

rted

to th

e ac

tive

pyra

zi-

noic

aci

d un

der a

cidi

c co

nditi

ons

of m

acro

-ph

age

lyso

som

es

Bac

terio

stat

ic a

ctiv

-ity

aga

inst

sus

cep-

tible

stra

ins

of M

. tu

berc

ulos

is m

ay b

e ba

cter

icid

al a

gain

st

activ

ely

divi

ding

or

gani

sms

Hep

atot

oxic

ity,

hype

ruric

emia

25 m

g/kg

/d51

, 53

N CO

NH

NH

2

NNC

NH

2

O

OO

O

OH

NH

O

H

HO

O

OO

NN

N

OH

OH

OH


312 S. Gupta et al.

EM

B (o

ral)

In

hibi

ts m

ycob

acte

rial

arab

inos

yl tr

ansf

er-

ases

, whi

ch a

re

invo

lved

in th

e po

-ly

mer

izat

ion

reac

tion

of a

rabi

nogl

ycan

, an

esse

ntia

l com

pone

nt

of th

e m

ycob

acte

rial

cell

wal

l

Bac

terio

stat

ic a

ctiv

-ity

aga

inst

sus

cep-

tible

myc

obac

teria

Ret

robu

lbar

neu

ritis

15–2

5 m

g/kg

/d51

, 52

SM

(IM

inje

ctio

n)

Pre

vent

s ba

cter

ial

prot

ein

synt

hesi

s by

bi

ndin

g to

the

S12

rib

osom

al s

ubun

it

Bac

teric

idal

act

ivity

ag

ains

t sus

cept

ible

m

ycob

acte

rium

Nep

hrot

oxic

ity,

otot

oxic

ity15

mg/

kg/d

52, 5

3

Tabl

e 3

Con

tinue

d

C C2H

5

CH

2OH

HN

H(C

H2)

2N

HC

H

C2H

5

CH

2OH

O

HO

OH

O

NH

OH

H3C

O

CH

3

OH

O

H

O

OH

HO

N

N

OH

H2N

NH

2

NH

2

H2N



TAB

LE 4

. Var

ious

sec

ond

line

drug

s us

ed a

gain

st T

B, t

heir

stru

ctur

e, m

echa

nism

of a

ctio

n, a

ctiv

ity, a

dver

se d

rug

reac

tions

, and

ty

pica

l adu

lt do

sage

Dru

g (R

OA

) & s

truc

ture

Mec

hani

sm o

f ac

tion

Act

ivity

Adv

erse

dru

g

reac

tions

Typi

cal a

dult

dosa

geR

efer

ence

ETH

(ora

l)

inhi

bitio

n of

m

ycol

ic a

cid

bios

ynth

esis

and

co

nseq

uent

im-

pairm

ent o

f cel

l-w

all s

ynth

esis

Bact

erio

stat

icG

I dis

turb

ance

s; n

euro

toxi

c-ity

—vi

sual

dis

turb

ance

s,

olfa

ctor

y di

stur

banc

es, p

erip

h-er

al n

euro

path

y, c

onvu

lsio

ns;

hepa

toto

xici

ty; h

yper

sens

itivi

ty

reac

tions

; alo

peci

a; g

ynec

o-m

astia

15 m

g/kg

/d52

, 53

CA

P (IM

inje

ctio

n)In

hibi

ts p

eptid

e pr

otei

n sy

nthe

sis

Bac

teric

idal

Pai

n—in

dura

tion

at in

ject

ion

site

; oto

toxi

city

; nep

hrot

oxic

ity;

feve

r; ra

shes

; eos

inop

hilia

15–3

0 m

g/kg

/d

51, 5

2

Cyc

lose

rine

(ora

l)

Inhi

bits

cel

lw

all s

ynth

esis

(e

nol f

orm

is D

-al

anin

e an

alog

)

Bac

terio

stat

icM

ostly

neu

rolo

gica

l—he

adac

he,

dizz

ines

s, v

ertig

o, d

row

sine

ss,

trem

or, c

onvu

lsio

ns, d

epre

s-si

on, p

sych

osis

; abn

orm

al li

ver

func

tion;

meg

alob

last

ic a

nem

ia;

rash

es

0.5–

1 g/

d51

, 52

NH

5C2

CS

NH

2

HN

N

H NH N

H 2N

O

HNN HNH

2H N

N H

H 2N

HO

NH

OHO

ONH

2

H 2N

OO

HN O

O

NH

2


314 S. Gupta et al.

Tabl

e 4

Con

tinue

d

Am

inos

alic

ylic

Aci

d (P

AS

) (or

al)

Inhi

bits

fola

te

bios

ynth

esis

; in

terfe

res

with

in

corp

orat

ion

of

p-am

ino-

benz

oic

acid

Bac

terio

stat

icG

I dis

turb

ance

s; h

yper

sens

itiv-

ity re

actio

ns li

ke ra

sh, f

ever

, m

alai

se, a

rthra

lgia

, leu

cope

nia,

ag

ranu

locy

tosi

s, e

osin

ophi

lia,

lym

phoc

ytos

is, a

typi

cal m

ono-

nucl

eosi

s, th

rom

bocy

tope

nia,

he

mol

ytic

ane

mia

8–12

g/d

52

, 53

KA

N (I

M in

ject

ion)

Inhi

bit p

rote

in

synt

hesi

sB

acte

ricid

alO

toto

xici

ty; n

ephr

otox

icity

; ps

eudo

mem

bran

ous

colit

is;

neur

omus

cula

r blo

ckad

e at

hig

h do

ses

15 m

g/kg

/d

51, 5

3

AM

I (IM

inje

ctio

n)in

hibi

t pro

tein

sy

nthe

sis

Bac

teric

idal

Oto

toxi

city

; nep

hrot

oxic

ity;

pseu

dom

embr

anou

s co

litis

; ne

urom

uscu

lar b

lock

ade

at h

igh

dose

s

15 m

g/kg

/d51

, 52

Thia

ceta

zone

(ora

l)In

hibi

ts c

yclo

-pr

opan

atio

n of

ce

ll w

all m

ycol

ic

acid

s in

myc

o-ba

cter

ia

Bac

terio

stat

icG

I dis

turb

ance

s; s

kin

reac

tions

in

clud

ing

exfo

liativ

e de

rmat

itis;

hepa

toto

xici

ty;

mye

losu

ppre

ssio

n

2.5

mg/

kg/d

95, 9

6

CO

OH O

H

NH

2

OHO H 2N

OH

OH

O

HO

NH2

O

OHO

OH

OH NH2

H 2N

O

H2N

OH

OH

O

OH

OH

NH

2

NH

OO O

H

NH

2OH O

H

NH

2

O

HO

H

HN

N

H 3C

O

H NNH

2

S



Figu

re 6

:Mechanism

ofactionoffirst-linedrugs


316 S. Gupta et al.

V.A. Alternative second-Line Drugs for TB

The alternative drugs listed below are usually considered only (1) in case of resistance to first-line agents; (2) in case of failure of clinical response to conventional therapy; (3) in case of serious treatment-limiting adverse drug reactions; (4) when expert guidance is available to deal with the toxic effects.

The goals of TB control are to cure active disease, prevent relapse, reduce transmis-sion, and avert the emergence of drug resistance. However, since the 1960s, there have been few developments in available therapies. Currently available agents are compli-cated by numerous side effects, drug interactions, and the need for a long duration of therapy. RIF-containing regimes lead to hepatic enzyme induction, which can compli-cate or preclude the use of protease inhibitors and non-nucleoside reverse transcrip-tase inhibitors in patients infected with HIV. Furthermore, emerging drug resistance has complicated management for many patients and clinicians. Therefore, new chemo-therapeutic agents are urgently needed. Existing antimicrobials are emerging as potent antitubercular agents. Recent studies have demonstrated the antitubercular activity of newer fluoroquinolones (FQs), including LFX, MFX, and gatifloxacin (GFX). Their use as first line antitubercular agents is currently under investigation. Furthermore, the oxazolidinones, linezolid and PNU-100480, have been shown to have antitubercular activity in addition to their antibacterial effects.

Several other agents are currently being developed for the treatment of TB. These agents include diarylquinolones (R207910), nitroimidazopyrans (PA-824, OPC-67683), EMB analogs (SQ109), cerulenin, trans-cinnamic acid, macrolides, pyrroles (LL3858), long-acting RIFs, and inhaled interferon-gamma. Furthermore, vaccines are being ex-plored for pre-exposure and post-exposure use.54 Promising new antitubercular drug candidates with their class, mechanism of action, target, mechanism of resistance, de-velopment of stage, and activity55,56 are summarized in Table 5.

V.B. Challenges for Conventional Therapy

The most common treatment regimen in patients infected with drug-susceptible mycobac-terial strains consists of oral ingestion for a minimum of 6–9 months. Therapeutic drug concentrations are achieved in regions of the body having adequate blood circulation. In mice, approximately 99% of bacteria are killed within 2 weeks of drug treatment, but at least 3 more months of treatment are required to clear the remaining 1% of bacteria.57,58 The poorly vascularized lesions, granulomas or tubercles, in the lungs harbor bacilli in microenvironments where hypoxic conditions curb the treatment and extend therapy for many more months to be effective. Mycobacteria are protected in granulomas, and con-ventional therapy may not penetrate into them at therapeutic levels. The standard therapy can be extended for up to two or more years if the patient fails to respond to the initial treatment based on sputum conversion, or if the patient is believed to be infected with drug-resistant strains. Mycobacterial strains exhibiting resistance to one or more drugs



are arising at an alarming rate, requiring incorporation of more drugs in the increasingly complex combination therapy and consideration of extension of the duration of therapy. Prolongation of therapy may lead to reduced patient compliance and further increase the

TABLE 5. Promising new antitubercular drug candidates with their class, mechanism of action, target, mechanism of resistance, development stage, and activity

Drug ClassMechanism

of action

TargetMechanism

of resistance

Develop-ment stage

Activity Reference

PA-824 Nitroimid-azole-oxazine

Inhibition of protein and cell wall lipid synthesis, while anaerobic activity by generating reactive nitrogen species, including nitric oxide

F420-depen-dent nitrore-ductase

Rv0407, Rv3547, Rv3261, Rv3262 gene mutations

Phase II Bacteri-cidal

157, 158

OPC-67683

Nitroimid-azole-oxazine

Inhibition of cell wall lipid synthesis, inhibition of mycolic acid biosynthesis

a nitrore-ductase

Rv3547 gene mutations


159, 160

TMC207(R207910)

Diaryl-quinoline

Inhibition of ATP syn-thesis and membrane potential

F1F0 protonATP synthase

atpE gene mutation


161, 162

MFX

Fluoroqui-nolone

Inhibition of DNA synthesis

DNA gyrase

gyrA gene mutation

Phase III Bacteri-cidal

163, 164

GFX Fluoroqui-nolone

Inhibition of DNA synthesis

DNA gyrase

gyrA gene mutation

Phase III Bacteri-cidal

165, 166

SQ109 Diethyl-amine

Inhibition of cell wall synthesis

Un-known

Unknown Phase I/II Bacteri-cidal

167, 168

LL3858 Pyrrole Unknown Un-known

Unknown Phase I Bacteri-cidal

169, 170

Linezolid Oxazolidi-none

Inhibition of protein synthesis

Ribo-somal inhibi-tion

23SrRNA mutation

Phase I lead optimiza-tion

Bacteri-cidal

171, 172


318 S. Gupta et al.

chances of emergence of drug-resistant strains. The protracted duration of therapy is also limited by constraints on drug dosage, adverse drug reaction (which is exacerbated in HIV patients), and inadequate drug distribution at the sites of pathology.59 Conte et al. showed that PZA achieved greater concentrations in the lungs than in the plasma in AIDS patients when administered orally, and might be responsible for the effectiveness of this drug for treating pulmonary TB. Intrapulmonary concentration of oral INH was below the MIC for mycobacterial strains in AIDS patients and normal subjects, and might explain the rapid emergence of INH-resistant organisms when it was used alone to treat TB.60,61 Novel drugs or delivery systems that are below a toxic threshold at the effective doses and act on the bacteria by a different mechanism are urgently needed to replace or supplement drugs that have been lost as therapies due to drug resistance. Treatment regimens that are short and allow less frequent intake of drugs by patients would greatly benefit compliance.

VI. DRUg ResIsTANCe IN M. TUBERCULOSIS

Drug-resistant TB was identified shortly after the first ATDs were introduced in the 1940s; the term refers to TB strains resistant to at least one antitubercular drug, usually determined by in vitro phenotypic methods (e.g., mycobacterial culture). Globally, resis-tance to a single antitubercular drug is the most common pattern of drug resistance. Rec-ognition of the relatively rapid onset of resistance to antitubercular monotherapy, usu-ally within months, led to the development of multidrug therapy as the standard of care in the 1960s.62,63 Drug resistance in TB arises from inadequate or inappropriate use of antimicrobial agents; however, the definitions used to classify drug resistance, as well as the public health control measures, vary. These differences sometimes lead to confusion and misinterpretation by those unfamiliar with either or both diseases. This confusion is compounded by the fact that each pathogen is increasingly resistant to more drugs, and new descriptive terms, such as multidrug-resistant (MDR) and, for TB, extensively drug-resistant (XDR), have been introduced to describe these changes. For TB, defini-tions of these new terms are now widely accepted.64 MDR-TB is defined as resistance of M. tuberculosis isolates to the two most effective first-line drugs, i.e., RIF and INH. Resistance to other ATDs, without resistance to both INH and RIF, is defined as poly-drug resistance. Extensively drug-resistant TB (XDR-TB) has additional resistance to any fluoroquinolone and at least one of the three injectable second-line ATDs (i.e., AMI, KAN, CAP).65,66 Mechanisms of antibiotic drug resistance are summarized in Fig. 7.

The design of colloidal carrier system of antibiotics attempts to overcome drug re-sistance, to shorten the treatment course, and to reduce drug interactions with antiretro-viral therapies; moxifloxacin (Phase III) and PA-824 (Phase II) are in advanced stages of clinical evaluation, and they could be available for use by 2012. On the other hand, first generation ATDs are still effective. Overcoming the main technological drawbacks of these therapeutic agents (e.g., limited aqueous solubility and stability, and bioavail-ability) to enhance compliance and adherence as well as improve the effectiveness of the drug by targeting the infection reservoirs (e.g., alveolar macrophages) remain the main goals of pharmaceutical technology. In this framework, colloidal carrier systems appear as one of the most promising approaches.



Figure 7: Mechanisms of antibiotic drug resistance

VII. COLLOIDAL CARRIeR sYsTeMs FOR TReATMeNT OF TUBeRCULOsIs

Colloidal carriers (nanocarrier systems) have led to increasing attention for pulmonary drug delivery. The lung is an attractive target for drug delivery due to non-invasive means to provide not only local lung effects but possibly high systemic bioavailabil-ity, avoidance of first-pass metabolism, more rapid onset of therapeutic action, and the availability of a huge surface area. Colloidal carrier systems in pulmonary drug delivery offer many advantages. These advantages include the following:

The potential to achieve relatively uniform distribution of drug dose among the alveoli; an achievement of enhanced solubility of the drug beyond its own aqueous solubility; the sustained release of drug, which consequently reduces the dosing fre-quency; suitability for delivery of macromolecules; decreased incidence of side effects; improved patient compliance; the potential of drug internalization by cells.67

Carriers are broadly classified as being synthetic or natural. Common examples of synthetic carriers used as drug delivery vehicles include poly (DL-lactide-co-glycolide) (PLGA), polylactic acid (PLA), polyglycolic acid (PGA), polyanhydrides, polymethyl acrylates, carbomer, etc. On the other hand, natural carriers include lipids (liposomes and solid lipid nanoparticles), alginic acid, chitosan, gelatin, dextrins, etc. Carriers not only allow designing of different delivery systems but also provide the flexibility of selecting the route of delivery. Thus, depending on the type of formulation, one may attempt to deliver ATDs as implants/injections or via the oral and respiratory routes.68 The role of natural drug carriers for the encapsulation and controlled release of antitubercular agents


320 S. Gupta et al.

need not be overemphasized. A wide range of ATDs have been successfully formulated into natural carrier systems. So far as ATDs are concerned, lipid-based formulations as well as polymer-based formulations such as alginate have been developed, with proven therapeutic efficacy in experimental TB. The drawback with lipid-based carriers is that most often one has to resort to the parenteral/inhalable route for successful delivery to the target organ.69 Schematic representations of carrier-based passive targeting and receptor-mediated active targeting to macrophages for the treatment of M. tuberculosis are illustrated in Figs. 8 and 9, respectively.

VII.A. Nanoparticles

Nanoparticles are defined as particles of less than 1000 nm in diameter and are colloidal structures composed of synthetic or semi-synthetic polymers. The bioactives are entrapped in the polymer matrix as enmeshed particulates or solid solution, or may be bound to the particle surface by physical adsorption or chemical reactions. Inhalation is the most signifi-cant route for the delivery of airborne nanoparticles. The human lungs contain about 2300 km of airways and 300 million alveoli (gas exchange areas). The surface area of lungs is estimated to be approximately 75–140 m² in adults. The large surface area of the al-veoli and the intimate air-blood contact in this region makes the alveoli less well protected against inhaled substances, such as nanoparticles, as compared to the airways.70,71

VII.A.1. Polymeric Nanoparticles

Biocompatible and biodegradable polymers have been used extensively in the clinic for controlled drug release. The annual worldwide market of polymer-based controlled release systems is about $60 billion and they are given to over 100 million patients each year. The first polymer-based drug-delivery system was developed by Langer and Folkman in 1976 for macromolecule delivery.72,73 However, the initial polymeric nanoparticles possessed poor therapeutic efficacy because of their rapid clearance by the reticuloendothelial system (RES) after intravenous administration. This limitation was overcome after the discov-ery of long-circulating stealth polymeric nanoparticles in 1994. Polymeric nanoparticles possess several unique characteristics for antimicrobial drug delivery. Firstly, polymeric nanoparticles are structurally stable and can be synthesized with a sharper size distribution. Secondly, particle properties such as size, zeta potentials, and drug-release profiles can be precisely tuned by selecting different polymer lengths, surfactants, and organic solvents during the synthesis. Thirdly, the surface of polymeric nanoparticles typically contains functional groups that can be chemically modified with either drug moieties or targeting ligands.74,75 There are currently two major types of polymeric nanoparticles for antimicro-bial drug delivery. One is formed via spontaneous self-assembly of diblock copolymers consisting of hydrophilic and hydrophobic segments. The hydrophobic segment forms a polymeric core containing the drugs while the hydrophilic segment shields the core from osponization and degradation. The rate of drug release can be tuned by varying the length of the hydrophobic chain. A variety of biodegradable polymers have been used to form the



Figu

re 8

: Sch

emat

ic re

pres

enta

tion

of c

arrie

r-bas

ed p

assiv

e ta

rget

ing

to m

acro

phag

es fo

r the

tre

atm

ent o

f M. t

uber

culo

sis


322 S. Gupta et al.

Figu

re 9

: Sch

emat

ic re

pres

enta

tion

of re

cept

or-m

edia

ted

activ

e ta

rget

ing

to m

acro

phag

es fo

r the

tre

atm

ent o

f M. t

uber

culo

sis



hydrophobic polymeric core, including PLA, PGA, PLGA, polycarprolactone (PCL), and poly(cyanoacrylate) (PCA), whereas polyethylene glycol (PEG) has been commonly used as a hydrophilic segment. Diblock copolymer nanoparticles are typically prepared through solvent displacement. In this process, polymers and drugs are first dissolved in a water-miscible organic solvent such as acetonitrile. The polymer-drug mixture is then added to an aqueous solution. As the organic solvent evaporates, the block copolymers and drugs undergo nanoprecipitation to form nanoparticles consisting of a hydrophobic core and a hydrophilic shell. Polymeric nanoparticles are primarily used to carry and deliver poorly water soluble drugs because of the hydrophobic nature of the nanoparticle core.76,77

In one study, Pandey et al. prepared the PLGA nanoparticle-based inhalable sus-tained drug-delivery system for experimental TB. To improve the bioavailability of ATDs as well as to assess the feasibility of administering ATDs via the respiratory route, the study reported the formulation of three frontline ATDs, i.e., RIF, INH, and PZA, encapsulated in PLGA nanoparticles suitable for nebulization. A single nebulization to guinea pigs resulted in sustained therapeutic drug levels in the plasma for 6–8 days and in the lungs for up to 11 days. The elimination half-life and mean residence time (MRT) of the drugs were significantly prolonged compared to the parent drugs, which were administered orally, resulting in an enhanced relative bioavailability (compared to oral administration) for encapsulated drugs (12.7-, 32.8-, and 14.7-fold for RIF, INH, and PZA, respectively). The absolute bioavailability compared to IV administration was also increased by 6.5-, 19.1-, and 13.4-fold for RIF, INH, and PZA, respectively. On nebulization of nanoparticles containing drugs to M. tuberculosis infected guinea pigs at every 10th day, no tubercle bacilli could be detected in the lung after 5 doses, whereas 46 daily doses of orally administered drug were required to obtain an equivalent thera-peutic benefit. The study suggested that nebulization of nanoparticles-based ATDs forms a sound basis for improving drug bioavailability and reducing the dosing frequency for better management of pulmonary TB.78

Similarly, Zahoor et al. developed the inhalable alginate nanoparticles as antitu-bercular drug carriers against experimental TB. Pharmacokinetic and chemotherapeutic studies were carried out with aerosolized alginate nanoparticles encapsulating INH, RIF, and PZA. The relative bioavailabilities of all drugs encapsulated in alginate nanopar-ticles were significantly higher compared with oral free drugs. All drugs were detected in organs (lungs, liver, and spleen) were above the MIC until 15 days after nebuliza-tion, while free drugs stayed up to day 1. The chemotherapeutic efficacy of 3 doses of drug-loaded alginate nanoparticles nebulized 15 days apart was comparable with 45 daily doses of oral free drugs. Thus, inhalable alginate nanoparticles can serve as an ideal carrier for the controlled release of ATDs.79 Moreover, Johnson et al. studied the oral therapy using nanoparticle-encapsulated ATDs (RIF, INH, and PZA) in guinea pigs infected with M. tuberculosis and evaluated the efficacy of nanoparticle-encapsulated ATDs administered every 10 days versus that of daily nonencapsulated drugs against M. tuberculosis aerosol infection in guinea pigs. Both treatments significantly reduced the bacterial count and lung histopathology, suggesting that the nanoparticle drug-delivery system had potential in intermitted treatment of TB.80


324 S. Gupta et al.

In another study, Pandey et al. developed and evaluated the potential of orally ad-ministered PLGA nanoparticle-encapsulated ATDs (RIF + INH + PZA + EMB) for cere-bral drug delivery in a murine model. A single oral dose of the formulation to mice could maintain sustained drug levels for 5–8 days in the plasma and for 9 days in the brain. There was significant improvement in the pharmacokinetic parameters such as MRT and relative bioavailability as compared with free drugs. The pharmacodynamic parameters such as the ratio of AUC/MIC and the time up to which MIC levels were maintained in plasma (TMIC) were also improved. In M. tuberculosis H37Rv–infected mice, five oral doses of the nanoparticle formulation administered every 10th day resulted in unde-tectable bacilli in the meninges, as assessed on the basis of colony forming units (cfu) and histopathology.81 Tripathi et al. developed RIF-loaded PLGA nanoparticles for IV administration to improve the therapeutic index of drug. The release behavior of RIF ex-hibited a biphasic pattern characterized by an initial burst (11.26% in 1 day) release fol-lowed by a slower and continuous release (more than 30 days). Therefore, RIF-loaded PLGA nanoparticles might be considered as an effective antitubercular drug-delivery system for therapy.82 Moreover, Pandey et al. evaluated the chemotherapeutic potential of oral PLGA-nanoparticle-encapsulated EMB in combination with PLGA-nanoparti-cle-encapsulated RIF + INH + PZA in a murine TB model. A single oral administration of the formulation to mice could maintain sustained drug levels in the plasma for 5 days and in the organs (lungs, liver, spleen) for 7–9 days. The relative/absolute bioavailability of the four ATDs was enhanced several fold. Repeated administration of the formulation did not produce any hepatotoxicity as assessed on a biochemical basis. In M. tuberculo-sis H37Rv–infected mice, just 3 oral doses of the four-drug formulation administered at every 10th day resulted in undetectable bacilli in the organs, replacing 28 conventional doses of free drugs. The study suggested that the polymeric nanoparticle-based oral four-drug combination bears significant potential to shorten the duration of TB chemo-therapy, besides reducing the dosing frequency.83

Saraogi et al. developed and characterized a RIF-loaded gelatin nanoparticulate de-livery system for the effective management of TB. The drug release showed the biphasic pattern of release, i.e., initial burst followed by a sustained release pattern. The cyto-toxicity studies revealed that nanoparticles are safe and nontoxic as compared to free drug. An in vivo biodistribution study showed higher localization of RIF-loaded GPs in various organs, as compared to plain RIF solution in PBS (pH 7.4). In contrast to free drug, the nanoparticles not only sustained the plasma level but also enhanced the AUC and MRT of the drug, suggesting improved pharmacokinetics of drug. RIF GPs addi-tionally resulted in significant reduction in bacterial counts in the lungs and spleen of TB-infected mice. Hence, GPs hold promising potential for increasing drug targetability vis-à-vis reducing dosing frequency, with the interception of minimal side effects, for efficient management of tuberculosis.84 Sharma et al. developed PLGA nanoparticles (encapsulating three frontline ATDs: RIF, INH, and PZA at 2/3rd therapeutic dose) for chemotherapeutic efficacy. PLGA nanoparticles were administered orally at 2/3rd thera-peutic dose to guinea pigs. A single oral administration of the formulation resulted in sustained drug levels in the plasma for 7–12 days and in the organs for 11–14 days, with



a significant improvement in MRT as well as in drug bioavailability. The administration of PLGA nanoparticles every 10 days (5 doses) to M. tuberculosis H37Rv–infected guin-ea pigs led to undetectable bacilli in the organs, as did 46 conventional doses. Therefore, nanoparticle-based antitubercular chemotherapy forms a sound basis for a reduction in dosing frequency and also offers the possibility of reducing the drug dosage.85

Similarly, Pandey et al. developed the formulation of three frontline ATDs, i.e., RIF, INH, and PZA, encapsulated in PLGA nanoparticles. Following a single oral adminis-tration of these preparations to mice, the drugs could be detected in the circulation for 6 days (RIF) and 9 days (INH/PZA), whereas therapeutic concentrations in the tissues were maintained for 9–11 days. Further, on oral administration of drug-loaded nanopar-ticles to M. tuberculosis–infected mice at every 10th day, no tubercle bacilli could be de-tected in the tissues after five oral doses of treatment. Therefore, nanoparticle-based ATD therapy forms a sound basis for reduction in dosing frequency for better management of TB.86 Ahmad et al. developed econazole (ECZ)- and MFX-loaded PLGA nanoparticles and evaluated them against murine TB (drug susceptible) in order to develop a more potent regimen for TB. PLGA nanoparticles were administered orally to mice. A single oral dose of PLGA nanoparticles resulted in therapeutic drug concentrations in plasma for up to 5 days (ECZ) or 4 days (MOX), while in the organs (lungs, liver, and spleen) it was up to 6 days. In comparison, free drugs were cleared from the same organs within 12–24 h. In M. tuberculosis–infected mice, 8 oral doses of the formulation administered weekly were found to be equipotent to 56 doses (MOX administered daily) or 112 doses (ECZ administered twice daily) of free drugs. Furthermore, the combination of MOX + ECZ was proved to be significantly efficacious compared with individual drugs. Ad-dition of RIF to this combination resulted in total bacterial clearance from the organs of mice in 8 weeks. Therefore, PLGA nanoparticles were found to have the potential for intermittent therapy of TB, and combination of MOX, ECZ, and RIF is the most potent.87 In another paper Ahmad et al. studied pharmacokinetic and pharmacodynamic behavior of ATDs (INH, RIF, PZA, and EMB) encapsulated in alginate nanoparticles at two doses. The formulation was orally administered to mice at two dose levels [D1 (INH = 91 mg/kg, RIF = 109.2 mg/kg, PZA = 227.5 mg/kg, and EMB = 145.6 mg/kg body weight) and D2 (INH = 10 mg/kg, RIF = 12 mg/kg, PZA = 25 mg/kg, and EMB = 16 mg/kg body weight)]. In the free drug groups, plasma levels of RIF and INH were higher and PZA and EMB levels were lower in the D1 group (per body surface area of mice) compared with the D2 group (recommended human dose). The plasma drug levels of all drugs were higher in the D1 encapsulated group compared with D2, resulting in higher values of area under the plasma drug concentration–time curve (AUC0–∞). The relative bioavailabilities of all drugs encapsulated in alginate nanoparticles were signifi-cantly higher compared with free drugs. Drug levels were maintained at or above the MIC90 until day 15 in organs after administration of encapsulated drugs, while free drugs stayed at or above the MIC90 up to day 1 only, irrespective of dose. The levels of drugs in various organs remained above the MIC at both doses for equal periods, demonstrating their equiefficiency. The study suggested that alginate nanoparticles hold great potential in reducing dosing frequency of ATDs.79


326 S. Gupta et al.

Also Pandey et al. developed PLGA nanoparticles encapsulating three frontline ATDs, i.e., INH, RIF, and PZA, and administered subcutaneously to mice for pharma-cokinetic/chemotherapeutic study. A sustained drug release was observed in the plasma for up to 32 days for each drug, following a single subcutaneous dose of PLGA-NP. Therapeutic drug concentrations were maintained for up to 36 days in the lungs (RIF 0.84 ± 0.1 mg/L, INH 0.99 ± 0.1 mg/L, and PZA 8.1 ± 1.2 mg/L homogenate) and spleen (RIF 0.8 ± 0.1 mg/L, INH 1.33 ± 0.3 mg/L, and PZA 8.33 ± 2.0 mg/L homogenate). The results indicated that a single subcutaneous injection of drug-loaded PLGA-NP demonstrated a better chemotherapeutic efficacy than 35 doses of oral free drugs.88 In another study, Pandey et al. developed PLGA nanoparticles encapsulating SM and administered orally to mice for biodistribution and chemotherapeutic studies. SM lev-els were maintained for 4 days in the plasma and for 7 days in the organs following a single oral administration of PLGA nanoparticles. There was a 21-fold increase in the relative bioavailability of PLGA-NP-encapsulated SM compared with intramuscular free drug. In M. tuberculosis H37Rv–infected mice, eight doses of the oral SM formula-tion (PLGA-NP-encapsulated SM) administered weekly were comparable to 24 intra-muscular injections of free SM. Further, the nanoparticle formulation did not result in nephrotoxicity as assessed on a biochemical basis.89

Sharma et al. developed lectin-functionalized PLGA nanoparticles as oral/aerosol-ized ATD carriers for treatment of TB. Upon administration of lectin-coated PLGA-NPs through the oral/aerosol route, the presence of drugs in plasma was observed for 6–7 days for RIF and 13–14 days for INH and PZA. However, upon administration of uncoated PLGA-NPs (oral/aerosolized) RIF was detectable in plasma for 4–6 days, whereas INH and PZA were detectable for 8–9 days. All three drugs were present in lungs, liver, and spleen for 15 days. Administration of wheat germ agglutinin (WGA)-coated PLGA-NPs caused a significant (P < 0.001) increase in the relative bioavail-ability of ATDs. Chemotherapeutic studies revealed that 3 doses of oral/nebulized lectin-coated nanoparticles fortnightly could yield undetectable mycobacterial cfu, which in contrast was achievable with 45 doses of oral free drugs.90 Moreover, Fawaz et al. studied the disposition of CPFX in free form and in polyisobutylcyanoacry-late (PIBCA) nanoparticles form after IV infusion to the rabbit. CPFX-loaded PIBCA nanoparticles led to increased AUC, t1/2, and Vd, and to a decreased Cl as compared with drug in solution. This could be due not only to the colloidal drug carrier but also to the pharmacokinetics of CPFX itself. Studies of efficacy against M. avium complex in human macrophages proved that CPFX-loaded PIBCA nanoparticles were more effective than free drug. The cytotoxicity of the polymeric material was observed at concentrations higher than 80 mg of PIBCA per mL with drastic reduction of viable macrophages.91

VII.A.2. Solid Lipid Nanoparticles (SLNs)

SLNs are another antimicrobial drug-delivery platform that has attracted much at-tention since the 1990s. SLNs are typically particulate systems with mean diameters



ranging from 50 nm up to 1000 nm for various drug-delivery applications.92 SLNs are mainly comprised of lipids that are in solid phase at room temperature and surfactants for emulsification. Solid lipids utilized in SLN formulations include fatty acids (e.g., palmitic acid, decanoic acid, and behenic acid), triglycerides (e.g., trilaurin, trimyristin, and tripalmitin), steroids (e.g., CHOL), partial glycerides (e.g., glyceryl monostearate and gylceryl behenate), and waxes (e.g., cetyl palmitate). Several types of surfactants are commonly used as emulsifiers to stabilize lipid dispersion, including soybean leci-thin, phosphatidylcholine, poloxamer 188, sodium cholate, and sodium glycocholate. The typical methods of preparing SLNs include spray drying,93 high-shear mixing, ul-trasonication,94 and high-pressure homogenization (HPH).95 In some severe cases, TB infection spreads from the lungs and affects the lymphatic systems. SLNs can facilitate the delivery of ATDs such as RIF, INH, and PZA to the lungs as well as to the lym-phatic systems.96 Once these SLNs enter the lungs, they are phagocytosed by AMs and subsequently transported to the lymphoid tissues.97 The SLN translocation mechanism and biodistribution through pulmonary delivery have been investigated using radio-labeled aerosolized SLNs in rats. Effective uptake of radio-labeled SLNs by the lungs after inhalation and considerable particle accumulation in the periaortic, axillary, and inguinal lymph nodes have been observed. The SLNs can provide a sustained release of the carried antimicrobial payloads, which then can effectively eliminate the infectious microbes harbored at these lymphatic sites.98

Pandey et al. developed the oral SLNs and evaluated the chemotherapeutic poten-tial of oral SLNs incorporating RIF, INH, and PZA against experimental TB. Following a single oral administration to mice, therapeutic drug concentrations were maintained in the plasma for 8 days and in the organs (lungs, liver, and spleen) for 10 days, whereas free drugs were cleared by 1–2 days. In M. tubercuIosis H37Rv–infected mice, no tu-bercle bacilli could be detected in the lungs/spleen after 5 oral doses of drug-loaded SLNs administered at every 10th day, whereas 46 daily doses of oral free drugs were required to obtain an equivalent therapeutic benefit. Thus, SLN-based antitubercular drug therapy forms a sound basis for reducing dosing frequency and improving patient compliance for better management of tuberculosis.99 Moreover, Nimje et al. developed mannosylated nanoparticulate carriers of RBU for alveolar targeting and evaluated the perspective of engineered nanoparticles for selective delivery of an ATD, RBU, to alve-olar tissues. In vitro release studies of RBU-loaded SLN showed a drug-release profile of 89.21 ± 3.8% after 120 h in PBS (pH 7.4), whereas RBU-loaded mannosylated SLN showed a drug-release profile of 65.23 ± 1.9% after 120 h in PBS (pH 7.4). Finally, it was concluded that mannose-conjugated SLNs can be exploited for effective and tar-geted delivery of RBU compared to its uncoated formulation, ultimately increasing the therapeutic margin of safety while reducing the side effects.100

In another study, Pandey et al. developed and evaluated the chemotherapeutic po-tential of nebulized SLNs incorporating RIF, INH, and PZA against experimental TB. Following a single nebulization to guinea pigs, therapeutic drug concentrations were maintained in the plasma for 5 days and in the organs (lungs, liver, and spleen) for 7 days, whereas free drugs were cleared by 1–2 days. In the case of nebulized drug-


328 S. Gupta et al.

loaded SLNs, the MRT was increased by 14- to 21-fold, 7- to 8-fold, and 13- to 21-fold compared to IV, oral, or nebulized free drugs, respectively. Thus, the relative bioavail-ability was increased by 10- to 16-fold, whereas the absolute bioavailability was in-creased by 8- to 10-fold. On nebulization of drug-loaded SLNs to infected guinea pigs at every 7th day, no tubercle bacilli could be detected in the lungs/spleen after 7 doses of treatment, whereas 46 daily doses of orally administered drugs were required to ob-tain an equivalent therapeutic benefit. Thus, nebulization of SLN-based ATDs forms a sound basis for improving drug bioavailability and reducing the dosing frequency for better management of pulmonary tuberculosis.101 Various SLN formulations are sum-marized in Table 6.

TABLE 6. Various nanoparticle formulations developed against TB

Carrier system

Drugs encapsu-

latedProperties Diseased

model usedRefer-ence

Polymeric nanopar-ticle

RIF, INH, PZA

PLGA nanoparticle having size 186–290 nm and entrapmentefficiency56.9%±2.7%forRIF,66.3%±5.8%forINH,and68%±5.6%forPZA

M. tuberculosis infected guinea pigs

78

RIF, INH, PZA

Alginatenanoparticleshavingsize235.5±0nmandentrapmentefficiency80%–90%forRIFand70%–90% for INH and PZA


79

RIF, INH, PZA

PLGA nanoparticles having size 186–290 nm and entrapmentefficiency60%—70%forallthreedrugs


80

RIF, INH, PZA EMB

PLGA nanoparticles having size 186–290 nm and entrapmentefficiency55.91%±2.70%forRIF,67.34%±3.8%forINH,68.32%±5.50%forPZA,and43.11%±4.21%forEMB

M. tuberculosis infected mice

81

RIF PLGA nanoparticles having size 380 nm and entrap-mentefficiency75.8%±2.16%

82

RIF, INH, PZA EMB

PLGA nanoparticles having size 186–290 nm andentrapmentefficiency55.91±2.70%forRIF,67.34±3.8%forINH,68.32±5.50%forPZAand43.11±4.21%forEMB

M. tuberculosis H37Rv infected mice

83

RIF Gelatinnanoparticleshavingsize264±11.2nmandentrapmentefficiency59.5±0.82%


84

RIF, INH, PZA

PLGA nanoparticle having size 186–290 nm and entrapmentefficiency58.99%±2.72%forRIF,68.02%±5.58%forPZA,66.31%±5.83%forINH


85

RIF, INH, PZA

PLGA nanoparticles having size 186–290 nm and entrapmentefficiency56.99%±2.7%forRIF,66.31%±5.83%forINH,and68.02%±5.58%forPZA


86

ECZ, MFX

PLGA nanoparticles having 217 nm and entrapment efficiency52.27%±3.80%forECZand33.69%±3.88% for MOX


87

INH, RIF, PZA, EMB

Alginatenanoparticleshavingsize235.5±0.0nmandentrapmentefficiency70%–90%forINHandPZA, 80%–90% for RIF, and 88%–95% for EMB


79

RIF, INH, PZA

PLGAnanoparticleshavingentrapmentefficiency56.9%±2.7%forRIF,66.3%±5.8%forINH,and68.0%±5.6%forPZA


88



TABLE 6 ContinuedSM PLGA nanoparticles having size 153.12 nm and

entrapmentefficiency32.12%±4.08%M. tuberculosis H37Rv infected mice

89

RIF, INH, PZA

PLGA nanoparticles having size 350–400 nm and entrapmentefficiency54%–66%

M. tuberculosis H37Rv infected guinea pigs.

90

CPFX PIBCA nanoparticles led to increased AUC, t1/2, and Vd, and to a decreased Cl as compared with drug in solution

Male albinos rabbits

91

Solid lipid nanopar-ticle

RIF, INH, PZA

SLNshavingentrapmentefficiency51%±5%forRIF,45%±4%forINH,and41%±4%forPZA

M. ubercuIosis H37Rv infected mice

99

RBU MannosylatedSLNshavingsize389±2.3nmforM-SLN-Rand251±5.1nmandentrapmentef-ficiencyofM-SLN-RandU-SLN-R82.6%±1.2%and87.8%±1.2%,respectively

macrophage cell lines (J774) and albino rats

100

RIF, INH, PZA

SLNshavingentrapmentefficiency51%±5%forRIF,45%±4%forINH,and41%±4%forPZA

M. tuberculosis H37Rv infected guinea pigs

101

VII.B. MICROPARTICLes

Microparticles are spherical particles with size range from 50 nm to 2 mm, containing a core substance. They are generally injected either intraperitoneally or directly to the tar-get organs, and because of their size they provide a sustained release depot of the drug. They entail the need for diversification of natural course of colloidal carrier biodisposi-tion, i.e., passive accumulation. They impart hydrophilicity to the surface and contribute a distinctive steric barrier.102

Dutt et al. investigated PLGA microparticles as carriers for INH. In vitro experi-ments showed a sustained release of INH up to 6 days from non-porous microparticles, while porous microparticles released INH over 3 days. Both porous and non-porous microparticles released INH in plasma for up to 2 days. Hardened PLGA microparticles sustained the release of INH for up to 7 weeks both in vitro and in vivo. The concentra-tions of INH obtained at all times were much higher than the MIC of INH. Controls in-jected with free INH showed release of INH in plasma for up to 12 h and in organs for up to 24 h. There was no hepatotoxicity induced as compared with control animals. Taken together these results suggested that PLGA-based ATDs may serve as ideal therapeutic agents for the treatment of TB infections.103 Moreover, Ain et al. developed an oral formulation based on PLGA microparticles for delivery of ATDs: INH, RIF, and PZA. PLGA-entrapped ATDs, when administered orally, were found to release the drugs in a sustained manner. This formulation was found to be stable in the acidic environment of gastric fluid, whereas in the intestinal fluid the drug release was obtained up to 20 days, as indicated by in vitro studies.104

Also, Dutt et al. developed PLGA microparticles containing a combination of INH and RIF as sustained-release carrier systems. A single dose of PLGA microparticles adminis-tered subcutaneously exhibited a sustained release of INH and RIF in vivo up to 7 and 6


330 S. Gupta et al.

weeks, respectively. Free drugs (in combination) injected in the same doses were detect-able in vivo up to 24 h only. One dose of PLGA microparticles cleared bacteria more effec-tively from lungs and liver in an experimental murine model of TB after low-dose PLGA combination drug therapy, and in liver after high-dose PLGA combination drug therapy, as compared with a daily administration of the free drugs. These results suggested that PLGA microparticles offer an improvement for TB chemotherapy over the conventional treatment.105 Suarez et al. developed RIF microparticles for the treatment of TB. A M. tuberculosis H37Rv–infected guinea pig model was used to screen for targeted delivery to the lungs by insufflation (with lactose excipient) or nebulization, of either RIF alone, RIF within PLGA microspheres (R-PLGA), or polymer microparticles alone (PLGA). Animals treated with single and double doses of R-PLGA microspheres exhibited significantly re-duced numbers of viable bacteria, and less inflammation and lung damage compared with lactose-, PLGA-, or RIF-treated animals 28 days after infection (P < 0.05). Two doses of R-PLGA resulted in reduced splenic enlargement. These studies supported the potential of R-PLGA delivered to the lung to treat pulmonary TB.106

Zhou et al. developed microparticles and evaluated their potential in targeting an ionizable prodrug of INH, INH methane sulfonate (INHMS), for sustained delivery of INH to AMs. The charged prodrug was ion-paired with two different hydrophobic cat-ions—tetrapentylammonium (TPA) and tetraheptylammonium (THA)-bromide)—and loaded separately into the PLA microparticles. Using a sensitive liquid chromatographic tandem mass spectrometric (LC-MS/MS) assay developed for INH, a high level of INH was detected in NR8383, a rat AM cell line, following exposure of these cells to drug-loaded microparticles. To confirm that the microparticles can target AMs in vivo, the INH levels in lavaged broncho AMs were compared by LC-MS/MS after administration of either INHMS-loaded PLA microparticles by intra-tracheal instillation or INH solu-tion by gavage or intra-tracheal instillation to Sprague-Dawley rats. As expected, only microparticles provided sustained and targeted delivery of INH to AMs. Most impor-tantly, this method of delivery led to substantial reduction in the blood levels of acetyl-isoniazid (AcINH), a major and potential toxic metabolite of INH.107

In another study, Ain et al. developed PLGA microparticles containing INH, RIF, and PZA, and used them as a sustained oral drug-delivery system to treat murine TB. Drug levels above the MIC were observed up to 72 h in plasma and for 9 days in vari-ous organs. Relative bioavailability of encapsulated drugs was greater than that of free drugs. Chemotherapy results showed better or equivalent clearance of bacilli in the PLGA-drug-administered group (weekly) than with free drugs (daily).108 Moreover, Sharma et al. developed inhalable microparticles containing drug combinations (INH; RIF) to target AMs for treatment of pulmonary TB. Large numbers of particles could be delivered to the bronchiopulmonary system through a 2-min exposure to fluidized particles. The intracellular drug concentrations resulting from vascular delivery of soluble drugs were found to be lower than those resulting from particle inhalation. Inhalable microparticles containing multiple ATDs offer promises of dose and dosing-frequency reduction, toxicity alleviation, and targeting macrophage-resident persis-tent mycobacteria.109 Muttil et al. developed microparticles containing large payloads



of two ATDs (INH and RBU) and evaluated them for suitability as a dry powder inha-lation for targeting AMs. About 70% of the payload was released in vitro in 10 days. Microparticles targeted only macrophages and not epithelial cells on inhalation. Drug concentrations in macrophages were ∼20 times higher when microparticles were in-haled than when drug solutions were administered. Microparticles were thus deemed suitable for enhanced targeted drug delivery to AMs.110 Various microparticulate for-mulations are summarized in Table 7.

VII.C. Liposomes

Liposomes are concentric bilayer vesicles in which an aqueous volume is entirely en-closed by a membranous lipid bilayer. These are the most extensively investigated systems for controlled delivery of drugs to the lungs, since they can be prepared with phospholipids endogenous to the lungs as surfactants. They can entrap a wide range of hydrophilic as well as hydrophobic drugs. Pulmonary delivery has been improved and even tested in animals and human subjects.111 Incorporation of drugs in liposome enhances bactericidal activity as compared to free drug, especially for the treatment of monocytes and macrophages.112 Drug distribution depends upon the drug release from the liposome, which could serve to retain drugs in the lungs and minimize their distribu-tion to other organs. Advantages of liposomes include: relatively low toxicity; prepared in wide size range (20 nm–1 mm); ability to solublize poorly water-soluble drugs, facili-tating their nebulization; serve as a biodegradable pulmonary reservoir with prolonged residence time; decrease mucociliary clearance of drugs due to their surface viscosity; can be exploited as a targeting device to individual population within the lung, specifi-cally to the infected or impaired AMs and the lung epithelium. Novel delivery systems can be administered to the lungs by various modes of delivery, i.e., nebulization, instil-lation, insufflations, etc.102

In a study, Vyas et al. prepared, characterized, and evaluated RIF-loaded aerosol-ized liposomes for their selective presentation to AMs, that being the most dense site of TB infection. In vitro airway penetration efficiency of the liposomal aerosols was determined by percent dose reaching the base of the lung, which was recorded to be 1.5–1.8 times higher as compared to plain drug solution-based aerosol. Percent viability of M. smegmatis inside macrophages (in vitro) after administration of drug (in vivo) was in the range of 7%–11% in the case of ligand-anchored liposomal aerosols, while it was recorded to be 45.7% and 31.6% in the case of plain drug and plain neutral liposomal aerosol (based on PC:Chol)-treated macrophages. Results suggested the preferential ac-cumulation of maleylated bovine serum albumin (MBSA)- and O-steroyl amylopectin (O-SAP)-coated formulations in the lung macrophages, which was further reflected in the periodically monitored in vivo tissue distribution studies. Higher lung drug concen-tration was recorded in the case of ligand-anchored liposomal aerosols and negatively charged liposomal aerosols (based on PC:Chol:DCP) as compared to plain drug and plain liposome-based aerosols. The drug was estimated in the lung in high concentration even after 24 h.113


332 S. Gupta et al.

TAB

LE 7

. Var

ious

mic

ropa

rtic

le fo

rmul

atio

ns d

evel

oped

aga

inst

TB

Car

rier

syst

em

Dru

gs

enca

psul

ated

Prop

ertie

sA

nim

al m

odel

/Infe

cted

m

odel

use

dR

efer

ence

Mic

ropa

rticl

esIN

HP

LGA

mic

ropa

rticl

es h

avin

g si

ze 6

2.11

, 71.

95, a

nd

11.7

5 µm

for p

orou

s, n

on-p

orou

s, a

nd h

arde

ned

PLG

A microparticles,respectively,andentrapm

entefficiency

of IN

H in

the

vario

us fo

rmul

atio

ns o

f PLG

A m

icro

parti

-cl

es w

as 1

6%, 1

1%, a

nd 1

0%–1

1% in

por

ous,

non

-po-

rous

, and

har

dene

d P

LGA

mic

ropa

rticl

es, r

espe

ctiv

ely.

D

ose

give

n—75

mg/

kg b

ody

wt.

Mic

e (L

aca

stra

in)

103

INH

, RIF

, PZA

P

LGA

mic

ropa

rticl

es h

avin

g si

ze o

f 1.1

1, 1

.4, a

nd 2

.20

µm fo

r IN

H, R

IF, &

PZA

mic

ropa

rticl

es, r

espe

ctiv

ely.

Thedrugencapsulationefficiencyofthemicroparticles

was

foun

d to

be

8–10

% fo

r PZA

; 10–

11%

for I

NH

, and

12

–18%

for R

IF.

Mic

e (L

aca

stra

in)

104

INH

, RIF

Har

dene

d PL

GA

mic

ropa

rticl

es h

avin

g si

ze 1

1.75

and

11

.64

µm fo

r IN

H- a

nd R

IF-c

onta

inin

g m

icro

parti

cles

, respectively,andentrapm

entefficiency10%

–11%for

INH

and

12%

–14

% fo

r RIF

.

M. t

uber

culo

sis

H37

Rv

infe

cted

mic

e10

5

RIF

SingledoseR-PLG

A(12mg/kg)insufflationsignificantly

reducedthelungburdenofbacteria(logcfu/mL=3.7±

0.3)com

paredwithRIF(logcfu/mL=4.17±0.1),PL

GA

(logcfu/mL=4.4±0.32)orthelactosegroup(logcfu/

mL=4.33±0.16).D

oubledoseR-PLG

A(12mg/kgby

insufflationfollowedby5mg/kgbynebulization)treat-

mentsignificantlyreducedthelungandspleenbacterial

burd

en c

ompa

red

with

an

equi

vale

nt d

ose

of R

IF o

r PL

GA,

or u

ntre

ated

gro

ups,

resp

ectiv

ely.

M. t

uber

culo

sis

(H37

Rv)

-in

fect

ed g

uine

a pi

g10

6

INH

MS

PLA

mic

ropa

rticl

es b

etw

een

1 an

d 3

µm in

dia

met

er. T

he

char

ged

prod

rug

was

ion-

paire

d w

ith tw

o di

ffere

nt h

y-dr

opho

bic

catio

ns (T

PA- a

nd T

HA-

brom

ide)

, and

load

ed

sepa

rate

ly in

to th

e PL

A m

icro

parti

cles

.

NR

8383

, a ra

t AM

cel

l lin

e10

7



Car

rier

syst

em

Dru

gs

enca

psul

ated

Prop

ertie

sA

nim

al m

odel

/Infe

cted

m

odel

use

dR

efer

ence

INH

, RIF

,an

d PZ

APL

GA

mic

ropa

rticl

es h

avin

g si

ze v

arie

d fro

m 1

.11

to 2

.20

µmforINH,R

IF,andPZA

,andentrapm

entefficiency8%

to 1

0% fo

r IN

H, 2

0% to

30%

for R

IF, a

nd 9

% to

13%

for

PZA.

Dos

e gi

ven:

INH

, 10

mg/

kg o

f bod

y w

eigh

t; R

IF, 1

2 m

g/kg

; and

PZA

, 25

mg/

kg, o

rally

.

M. t

uber

culo

sis

(H37

Rv)

-in

fect

ed m

ice

(laca

stra

in)

108

INH

, RIF

PLA

mic

ropa

rticl

es h

avin

g si

ze 0

.5–3

mic

ron.

Wis

tar r

ats,

mur

ine

mac

roph

age

line

J774

A

109

INH

and

RB

UP

LA m

icro

parti

cles

hav

ing

size

5µm

and

ent

rapm

ent

efficiency50%.

Fem

ale

Sw

iss

mic

e11

0

Tabl

e 7

Con

tinue

d


334 S. Gupta et al.

Moreover, Agarwal et al. developed the tuftsin-bearing liposomes as RIF vehicles in the treatment of TB in mice. The antitubercular activity of RIF was considerably increased when it was encapsulated in egg phosphatidylcholine (EPC) liposomes. A further increase in the activity was observed when the macrophage activator tetrapeptide tuftsin was grafted on the surface of the drug-loaded liposomes. Intermittent treatments (twice weekly) with these preparations were significantly more effective than the con-tinuous treatments. RIF delivered twice weekly for 2 weeks in tuftsin-bearing liposomes was at least 2,000 times more effective than the free drug in lowering the load of lung bacilli in infected animals. However, pretreatment with drug-free tuftsin-bearing lipo-somes did not render the pretreated animals resistant to the M. tuberculosis infections; neither did it appreciably increase the chemotherapeutic efficacy of the liposomized RIF. These results clearly demonstrated that liposome targeting to macrophages could considerably increase the antitubercular activity of liposomized drugs such as RIF. Also, it shows that immunoprophylactic treatment with macrophage activators such as tuftsin does not afford any advantage in treatment of TB infections, presumably because of inactivation of the primed macrophages by the mycobacterial sulfatides.114

Furthermore, Gaspar et al. developed and characterized several RBU liposomal formulations and compared their in vivo profile with free RBU following IV admin-istration. With the RBU liposomal formulations tested, higher concentrations of the antibiotic were achieved in liver, spleen, and lungs 24 h after administration compared with free RBU. The concentration of RBU in these organs was dependent on the ri-gidity of liposomal lipids. The liposomal RBU formulation prepared with dipalmitoyl phosphatidylcholine:dipalmitoyl phosphatidylglycerol (DPPC:DPPG) was the most effective and was selected for biological evaluation in a mouse model of disseminated TB. Compared with mice treated with free RBU, mice treated with the DPPC: DPPG RBU formulation exhibited lower bacterial loads in the spleen (5.53 log10 vs. 5.18 log10) and liver (5.79 log10 vs. 5.41 log10). In the lung, the level of pathology was lower in mice treated with encapsulated RBU. These results suggested that liposomal RBU is a promising approach for the treatment of extrapulmonary TB in HIV co-infected patients.115

Chimote et al. developed and evaluated INH-entrapped liposomes of DPPC (the most abundant lipid of lung surfactant and exogenous surfactant). Sustained release of INH from DPPC liposomes was observed over 24 h. In vitro alveolar deposition ef-ficiency using the twin impinger exhibited ∼25%–27% INH deposition in the alveolar chamber upon 1 minute nebulization using a jet nebulizer. At 37°C, the DPPC liposomes had better pulmonary surfactant function with quicker reduction of surface tension on adsorption (44.67 ± 0.57 mN/m at the first second and reached a lower value of 36.62 ± 0.37 mN/m at the end of 30 min), and 87% airway patency was exhibited by the formulation in a capillary surfactometer. The formulation was biocompatible and had antimycobacterial activity. The INH-entrapped DPPC liposomes could fulfill the dual purpose of pulmonary drug delivery and alveolar stabilization due to the antiatelectatic effect of the surfactant action, which can improve the reach of antitubercular drug INH to the alveoli.116 Similarly, Deol et al. studied the therapeutic efficacies of INH and



RIF encapsulated in lung-specific stealth liposomes against M. tuberculosis infection induced in mice. Liposome-encapsulated drugs at and below therapeutic concentrations were more effective than free drugs against TB, as evaluated on the basis of cfu detect-ed, organomegaly, and histopathology. Furthermore, liposomal drugs showed marginal hepatotoxicities, as determined from the levels of total bilirubin and hepatic enzymes in serum. The elimination of mycobacteria from the liver and spleen was also higher with liposomal drugs than with free drugs. The encapsulation of ATDs in lung-specific stealth liposomes seems to be a promising therapeutic approach for the chemotherapy of TB.117

Furneri et al. developed OFX-loaded liposomes and determined their antimicro-bial activities in comparison to those of the free drug by means of MIC determinations with both American Type Culture Collection standards and wild-type bacterial strains (six strains of Enterococcus faecalis, seven strains of Escherichia coli, six strains of Staphylococcus aureus, and six strains of Pseudomonas aeruginosa). Encapsulated fluo-roquinolone yielded MICs that were at least two-fold lower than those obtained with the free drug. In particular, liposomes made up of dimyristoylphosphatidylcholine-choles-terol-dipalmitoylphosphatidylserine and dimyristoylphosphatidylcholine-cholesterol-dihexadecylphosphate (4:3:4 molar ratio) provided the best improvement in antimicro-bial activity against the various bacterial strains investigated. The liposome formulation produced higher intracellular fluoroquinolone concentrations than those achieved si-multaneously with the free drug in both E. coli and P. aeruginosa.118 Likewise, Karki et al. formulated and evaluated coencapsulated RIF and INH liposomes using different lipids. Coencapsulated liposomal formulations elevated plasma elimination half-life and decreased elimination rate constants for RIF and INH. In vivo studies suggested that coencapsulation retarded the release of drug from circulation compared to free drug due to slow drug release into systemic circulation. This formulation also reduced the accumulation of drug in the liver, kidney, and lungs. It is evident from this study that liposomes could be promising delivery systems for RIF and INH, with prolonged drug-release profiles and reasonably good stability characteristics.119

Pandey et al. carried out the nebulization of liposome-encapsulated ATDs in guinea pigs. Dunkin-Hartley guinea pigs were administered free drugs once IV and free drugs/liposome-encapsulated drugs once via nebulizer. The drug doses were selected accord-ing to the surface area of the animals, i.e., RIF 46.5 and INH 23.25 mg/kg body weight. A single nebulization of liposomal drugs could maintain therapeutic drug levels in the plasma from 45 min onwards up to 48 h, whereas no drugs could be detected beyond 24 h after nebulization when free drugs were used. It was observed that aerosolized liposo-mal drugs were present in the lungs (RIF: 0.25 ± 0 µg/mL; INH: 0.29 ± 0.03µg/mL ho-mogenate) as well as in the AMs (RIF: 0.15 ± 0.02 µg/105 cells; INH: 0.1 ± 0.03µg/105 cells; approximately 2 × 105 viable cells were isolated from each animal) until day 5 after nebulization. Aerosolized/IV free drugs, however, were undetectable in the lungs/macrophages beyond 48 h. The data clearly show the ability of nebulized liposomes to target the AMs, which are the residence of tubercle bacilli.120

Ridy et al. developed and evaluated PZA liposomes for treatment of M. tuberculosis. Liposomal PZA resulted in highly significant reduction in bacterial counts (cfu/g lung),


336 S. Gupta et al.

10, 20, and 30 days after the last treatment dose. Histopathological examination of mice lungs showed highest severity of infection in drug-free liposomes (control) group > PZA liposomes > free PZA 6 days/week. The results indicated high therapeutic efficacy of PZA liposomes, injected twice weekly, in treatment of M. tuberculosis in mice.121 Wiens et al. developed a liposome formulation of EMB. In vitro efficacy of developed lipo-somes was found to be equivalent to that of the free drug, suggesting that encapsulation of EMB had the potential to shorten the current regimens for TB.122 Various liposomal formulations are summarized in Table 8.

VII.D. Microspheres

Microspheres are small spherical particles, with diameters in the micrometer range [typically 1 μm to 1000 μm (1 mm)]. These drug carriers can be prepared over a wide range of particle sizes, which is a decisive factor in the in vivo deposition of particu-late carriers. Drugs can be easily incorporated with relatively high efficiency, and by the manipulation of the synthetic process procedure, different drug-release rates can be achieved. Microspheres are more physicochemically stable both in vitro and in vivo. Drugs entrapped have a slower release rate and a longer duration of action. The higher stability allows easy formulation. A number of biodegradable microspheres have proved to be non-toxic, biodegradable, and non-immunogenic following sys-temic injection.123

In a study, Pandey et al. developed alginate-chitosan microspheres as drug carriers to reduce dose/dosing frequency in the management of TB, which otherwise demands prolonged chemotherapy. Alginate-chitosan microspheres encapsulating three frontline ATDs—RIF, INH, and PZA—were formulated. A therapeutic dose and a half-thera-peutic dose of the microsphere-encapsulated ATDs were orally administered to guinea pigs for pharmacokinetic/chemotherapeutic evaluations, respectively. Administration of a single oral dose of the microspheres to guinea pigs resulted in sustained drug levels in the plasma for 7 days and in the organs for 9 days. The half-life and MRT of the drugs were increased 13- to 15-fold by microsphere encapsulation, along with an enhanced relative/absolute bioavailability. The sustained release and increase in bioavailability were also observed with a sub-therapeutic dose of the microspheres. In M. tuberculosis H37Rv–infected guinea pigs, administration of a therapeutic dose of microspheres every 10 days (three oral doses) produced a clearance of bacilli equivalent to conventional treatment for 6 weeks. The results suggested that alginate-chitosan microspheres hold promise as a potential natural polymer-based oral ATD carrier for better management of TB.124

Barrow et al. used microsphere technology to developed formulations of RIF for targeted delivery to host macrophages. Release characteristics were examined in vitro and also in two monocytic cell lines, the murine J774 and the human Mono Mac 6 cell lines. Bioassay assessment of cell culture supernatants from monocyte cell lines showed release of bioactive RIF during a 7-day experimental period. Treatment of M. tuberculosis H37Rv–infected monocyte cell lines with RIF-loaded microspheres



TABLE 8. Various liposomes formulations developed against TBCarrier system

Drugs encapsulated Properties Disease

model used Reference

Liposomes RIF MBSA-coated liposomes having size 3.64±0.65µmandentrapmenteffi-ciency47.4%±2.7%,whileO-SAP-coated vesicles 3.85±0.59 µm in size and entrap-mentefficiencyof47.4%±2.7%

Albino rats 113

RIF Liposomes having size 25 and 65 nm and entrapmentefficiency28%to32%forRIF.

M. tuber-culosis (H37Rv)-infected

Swiss albino mice

114

RBU RBU liposomes having size 0.1 µm and entrapmentefficiency72%±5%.

BALB/c mice

115

INH DPPC liposomes having size 750 nm and entrapmentefficiency36.7%±1.8%.

HimediaVR LA 387 di-

alysis mem-brane-50,

India

116

INH, RIF Liposomeshavingentrapmentefficiency8% to 10% for INH and 44% to 49% for RIF.

M. tuber-culosis (H37Rv)-infected

mice

117

OFX OFX-loaded unilamellar vesicles having size200nmandentrapmentefficiencyofliposomes made up of dimyristoylphospha-tidylcholine-cholesterol-dipalmitoylphosphatidylserine and dimyristoylphosphatidylcholine-choles-terol-dihexadecylphosphate21.0±3.7%and15.7%±1.9%respectively.

American Type Culture

Collection standards and wild-

type bacte-rial strains

118

RIF, INH Size of the vesicles (varied with type of the lipid used; EPC: CHOL, 50:50; EPC: CHOL, 40:60; DPPC: CHOL, 50:50; DPPC:CHOL,40:60)was3.165±0.002µm;2.895±0.001µm;2.8075±0.001µm;2.7452±0.005µmandentrapmentefficiency(RIFandINH)was68.25%and57.85%; 69.23% and 59.36%; 70.92% and 61.11%; 72.98% and 63.22%, respectively.

albino rats 119

RIF and INH Liposomeshavingentrapmentefficiency40%–45% for RIF and 8%–12% for INH, dose given RIF 46.5 and INH 23.25 mg/kg body weight.

Dunkin-Hartley

guinea pigs

120

PZA Entrapmentefficiencyofliposomesmadeup of dipalmitoyl phosphatidyl choline (7):cholesterol (2) neutral anddipalmitoyl phosphatidyl choline (7):cho-lesterol (2):dicetyl phosphate (1) nega-tivelychargedwasfoundtobe8.833%±2.4%and7.883%±1.9%respectively.

Mice in-fected by M. tuberculosis

121

EMB EMB liposomes having size 125–145 nm andentrapmentefficiency76%–92%.

M. bovis BCG

122


338 S. Gupta et al.

resulted in a significant decrease in numbers of cfu at 7 days following initial infec-tion, even though only 8% of the microsphere-loaded RIF was released. The levels of RIF released from microsphere formulations within monocytes were more effective at reducing M. tuberculosis intracellular growth than equivalent doses of RIF given as a free drug. These results demonstrated that RIF-loaded microspheres can be for-mulated for effective sustained and targeted drug delivery to host macrophages.125 Similarly, Quenelle et al. reported the use of RIF-loaded microspheres to effectively treat M. tuberculosis–infected macrophages and mice. Formulations were evaluated individually and in combination with oral regimens of INH for the treatment of M. tuberculosis H37Rv–infected mice. Groups (10 mice per group) consisted of mice that received (i) oral dosages of INH (25 to 0.19 mg/kg of body weight/day), (ii) two in-traperitoneal injections of RIF-loaded microspheres on days 0 and 7, (iii) a combina-tion of small RIF-loaded microspheres on days 0 and 7 and INH orally for 25 days (12.5 to 0.39 mg/kg/day), (iv) placebo injections, and (v) no treatment. Treatment with RIF-loaded microspheres alone resulted in significant reductions in the numbers of cfu in the lungs and spleens by day 26. A bioassay revealed that plasma RIF levels from the microspheres exceeded the MICs by more than two-fold throughout the 26-day experimental period. Susceptibility testing demonstrated continued sensitivity to RIF during the treatment period. Whereas INH alone significantly reduced the num-bers of cfu for dosages ranging from 12.5 to 1.56 mg/kg, combination therapy with RIF-loaded microspheres increased the effective range to 0.39 mg/kg. In many cases, complete elimination of cfu was obtained with the combination therapy, something not achieved with most of the single therapies. These results demonstrated that the ability to use small microsphere formulations alone to achieve significant results in a murine TB model and also the ability to use them safely in combination with another antimycobacterial agent.126

Furthermore, Contreras et al. studied the efficacy of RIF-loaded polymeric mi-crospheres (RPLGA) delivered to guinea pigs infected with M. tuberculosis (H37Rv), which was compared with a daily dose of nebulized RIF suspension. Drug and poly-mer–treated multiple dose groups exhibited significantly lower wet lung weights than untreated animals. Spleen wet weights and viable bacterial counts (VBCs) were much lower for drug microsphere–treated animals than for all other groups. In multiple-dose studies with RIF-only suspensions, wet lung weights for 10-RIF and 20-RIF treated animals were much smaller than controls. Likewise, wet spleen weights of 10-RIF and 20-RIF treated animals were much smaller than controls, consistent with reduced inflammation. Spleen VBC of 20-RIF treated animals was much smaller than controls. No statistical differences were observed in the lung VBC among single dose groups. However, a trend similar to that of the wet weights was observed.127 Hasega-wa et al. studied phagocytic activity of AMs towards polystyrene latex microspheres (PSL MS) and PLGA microspheres loaded with RIF. Phagocytic activity of cell line NR8383, derived from rat AMs, towards PSL MS of various diameters was examined by incubating the cells for 4 h at 37°C with various numbers of PSL MS per macro-phage cell (MS/macrophage = 0.1–10). The results were then compared with those of



the phagocytosis towards RIF-PLGA MS. Hasegawa et al. determined the phagocytic activity by counting the population of macrophage cells that had phagocytosed mi-crospheres (N) and the number of particles phagocytosed (n) in microscopic fields. Both N and n for PSL and RIF-PLGA MS increased in general with an increase in microspheres/macrophage, but both of these values for PSL MS were smaller than those for RIF-PLGA microspheres. Phagocytosis of the particles was dependent on the particle size; i.e., of the PSL MS the 6-µm ones were taken up by macrophage the most, and the RIF-PLGA MS 3 µm in diameter seemed to be phagocytosed the most efficiently, although Hasegawa et al. were not able to determine exactly the phagocytosis of 6- and 10-µm RIF-PLGA MS. From the changes in N and n values with MS/macrophage, the phagocytosis of RIF-PLGA MS was likely to enhance the phagocytic activity of macrophage cells, but this effect did not seem to be significant for PSL MS.128

Samad et al. designed RIF-containing microspheres by using a biodegradable and biocompatible polymer, gelatin B, using a thermal gelation method. The microspheres were cross-linked with a natural cross-linker, sucrose, to avoid the toxicities due to the synthetic di- and poly-aldehydes. This formulation was found to be controlled release for drug in the gastro-intestinal tract. The formulation was subjected to in vitro release studies using the USP paddle method. After 24 h at 37C, the release of RIF from the microspheres was ∼80% of the total amount of the encapsulated drug. Microspheres could be observed in the intestinal lumen at 4 h and were detectable in the intestine 24 h after oral administration, although the percentage of radioactivity had significantly decreased (t1/2 of 99mTc = 4–5 h).129 Likewise, Suarez et al. developed respirable PLGA microspheres containing RIF for the treatment of TB. RIF alone (RIF, 1.03–1.72 mg/kg), within PLGA microspheres (R-PLGA, equivalent to 1.03–1.72 mg/kg) or polymer microparticles alone (PLGA) were administered to male Dunkin-Hartley guinea pigs by insufflation or nebulization, 24 h before bacterial aerosol exposure. RIF and R-PLGA exhibited action in limiting the growth of MTB in this culture system. RIF inhibited MTB growth at both concentrations employed (1 and 4 mg/mL). R-PLGA limited MTB growth by approximately 50% at the lowest concentration (2 mg/mL) and by 100% at both of the higher concentrations (20 and 100 mg/mL). R-PLGA was not as effective as RIF, as the total drug load was available only on dissolution. Indeed, the release rate of drug from R-PLGA was not sufficient to inhibit growth at the low dose of 2 mg/mL. PLGA treatments did not have any effect on the growth of MTB.130 Moreover, Zhang et al. developed RIF PLA microspheres for lung targeting. Pharmacokinetic and tissue dis-tribution of the RIF PLA microspheres of 5–15 µm, after IV administration, were carried out on rabbits. Compared with RIF injection, the microspheres were mainly localized in the lungs. Drug content in the lungs was drastically enhanced, while in plasma and other tissues (liver, spleen, kidney, heart) it was much lower. For the redistribution of RIF in vivo, drug content in the lungs was gradually reduced. Prolonged retention could also be seen 3 days after the administration of the microspheres, and the drug content in the lungs was still 3.18 µg/g.131 Various microsphere-based formulations are summarized in Table 9.


340 S. Gupta et al.

VII.e. Niosomes

Non-ionic surfactant-based vesicles (niosomes) are the structures formed from the self-assembly of non-ionic amphiphiles in an aqueous medium, resulting in a closed bilayer structure. The assembly is rarely spontaneous and usually involves some input of en-ergy, such as physical agitation or heat. The result is an assembly in which the hydropho-bic parts of the molecule are shielded from the aqueous solvent and the hydrophilic head

TABLE 9. Various microsphere formulations developed against TBCarrier system

Drugs encapsulated Properties Disease model

used Reference

Microspheres RIF, INH, PZA Alginate-chitosan micro-sphereshavingsizeof70±4µmandentrapmentefficiency83%±2%forRIF,65%±6%forINHand69%±6%forPZA. Dose given RIF 12 mg/kg + INH 10 mg/kg + PZA 25 mg/kg body weight—orally.

M. tuberculosis H37Rv–infected Dunkin-Hartley guinea pigs

124

RIF Microspheres having size 3–4 µm.

Murine J774 and the human Mono Mac 6 cell lines

125

RIF, INH Microspheres having size 3.98±2.74µmforRIFand4.2±3.2µmforINH.RIF-loaded microspheres were injected intraperitoneally, and INH was given orally.

M. tuberculosis H37Rv–infected mice

126

RIF Microspheres having size 2.83 µm.

Guinea pigs infected with M. tuberculosis (H37Rv)

127

RIF RIF-PLGA particles having size1.50±0.28,3.38±0.45,6.31±0.67,10.28±0.78µm.

Cell line NR8383

128

RIF Microspheres having size 53.02±31.44mmanden-trapmentefficiency70.42%±14.11%.

White rabbits (3–3.5 kg)

129

RIF Dose given, RIF (1.03–1.72 mg/kg), within poly(lactide-co-glycolide) microspheres (R-PLGA, equivalent to 1.03–1.72 mg/kg) or polymer microparticles alone (PLGA) byinsufflationornebulization,24 h before bacterial aerosol exposure.

Dunkin-Hartley guinea pig

130

RIF Microspheres having size 7.90±4.07µm.

rabbits 131



groups enjoy maximum contact with same. These structures are analogous to phospho-lipid vesicles (liposomes) and are able to encapsulate aqueous solutes and serve as drug carriers. The low cost, greater stability, and resultant ease of storage of non-ionic surfac-tants has led to the exploitation of these compounds as alternatives to phospholipids.132

In one study, Jain et al. developed niosomes containing RIF using span-85 and CHOL in various molar fractions. In vivo distribution studies of the prepared nio-somes found that 30% of the drug was recovered from the lungs after 24 h, where-as only 4% was obtained in the case of plain drug solution administration via the same route.133 In another study, Jain et al. prepared the niosomes containing RIF us-ing various nonionic surfactants of sorbitan ester class and CHOL in 50:50 percent mol fraction ratios. In vitro release rate studies revealed that the cumulative percent RIF released was maximum for span-20-based niosomes and minimum for span-85-based niosomes. The maximum lymph concentration achieved was 46.2% of the ad-ministered dose, when the drug was administered as a niosomal formulation via the intraperitoneal route, while only 7.3% of the administered dose was recovered in the thoracic lymph when the niosomal formulation was administered by the IV route, and only 13.1% and 11.5% of the administered dose could be recovered from the thoracic lymph after administration of plain drug solution via the intraperitoneal and IV route respectively.134 Similarly, Moazeni et al. formulated and evaluated in vitro CPFX-containing niosomes for pulmonary delivery. Formulations composed of span 60 and tween 60 in combination with 40 mol% of CHOL exhibited high encapsulation efficacy and stability, and also had fine particle fraction and nebulization efficiency of about 61.9 ± 1.0% and 77.9 ± 2.8%, respectively. MIC of the niosomal CPFX against some pulmonary pathogens were lower than free CPFX. Using the MTT assay in hu-man lung carcinoma cell line (A549), niosome- entrapped CPFX showed significantly lower cytotoxicity in comparison to the free drug. These results indicated that niosome can be used as a carrier for pulmonary delivery of CPFX via nebulization.135 Various niosomal formulations are summarized in Table 10.

VII.F. Dendrimer

Dendrimer represents a novel type of polymeric material. It is also known as starburst,136 cascade,137 molecular trees,138 arborols, or polymers. They attract increasing attention because of their unique structure, high degree of control over molecular weight, and their shape, which has led to the synthesis of unimolecular micelles. Among the plethora of possible dendrimers, poly (propylene imine) (PPI) dendrimers have been synthesized on a relatively large scale and characterized both experimentally and by molecular simu-lation only in recent years.139,140 The potential applications of the PPI dendrimers are generally based on one or more of the following characteristics: regular size and shape; larger number of readily accessible end groups; either nitrile or amine; possibility of end group modification in order to tailor properties such as solubility, reactivity, toxic-ity, stability, glass transition temperature, polyelectrolyte character; and possibility of encapsulating guest molecules. Considering the use of dendrimers for drug delivery, it is


342 S. Gupta et al.

necessary that they be non-toxic and biocompatible. However, it has been demonstrated that widely used dendrimers, such as PAMAM and PPI dendrimers bearing primary amino group termini, are quite cytotoxic, and also these dendrimers were cleared rapidly from the circulation when administered IV.141,142

Kumar et al. developed and explored the use of PEGylated PPI dendritic architec-ture for the delivery of an ATD, RIF. In vitro release studies among the four formula-tions PEGylated dendrimers showed relatively slower release of RIF when compared with non-PEGylated dendrimers. While the 4.0G EDA-PPI dendrimers-(NH2)32 released 97.3% of RIF in 36 h, PEGylated 4.0G EDA-PPI dendrimers released only 46.3% in 36 h and 93.1% in 96 h. The 5.0G EDA-PPI dendrimers-(NH2)64 released 94.6% of RIF in 48 h, while PEGylated 5.0G EDA-PPI dendrimers released only 43.3% and 93.4% in 48 and 120 h, respectively. The cumulative % release of RIF in the PEGylated EDA-PPI dendrimers was decreased with increase of dendrimer generation. This may be due to greater hydrophobic interaction between the drug and the core of higher generation den-drimer (5.0G). The PEGylation of the systems was found to increase their drug-loading capacity and to reduce their drug-release rate and hemolytic toxicity. The systems were found suitable for prolonged delivery of RIF.143 In another study, Kumar et al. developed and explored the use of mannosylated dendritic architecture for the selective delivery of RIF to AMs. RIF-loaded mannosylated dendrimer reduced the release rate of drug in pH 7.4, hemolytic toxicity, and cytotoxicity; whereas enhanced drug release in pH 5.0 and AM uptake were observed.144 Various dendrimer formulations are summarized in Table 11.

TABLE 10. Various niosomes formulations developed against TB

Carrier system

Drugs encapsulated Properties

Disease model used

Reference

Niosomes RIF Niosomes having size 8–15 µm. albino rats

133

RIF Niosomes having size containing RIF 1.8 µm (span 20), 1.8 µm (span 40), 1.7 µm (span 60), 1.8 µm (span 80), 1.9µm(span85)andentrapmenteffi-ciency 20.6% (span 20), 25.3% (span 40), 28.5% (span 60), 31.7% (span 80), 35.4% (span 85).

Wistar rats

134

CPFX Niosomeshavingsize8.44±0.87,7.26±0.33,4.21±0.14,8.79±0.73,7.08±0.33,4.54±0.24µmanden-trapmentefficieny54.39%±3.13%,65.18%±2.8%,73.42%±2.01%,58.52%±1.01%,71.24%±1.28%,75.65%±2.28%forformulationsF1,F2, F3, F4, F5, F6 respectively.

Human lung car-cinoma cell line (A549)

135



VII.g. Dry Powder

Pulmonary drug delivery by dry powder inhalers (DPIs), by virtue of its propellant-free nature, high patient compliance, high dose carrying capacity, drug stability, and pat-ent protection, has encouraged rapid development in the recent past to realize the full potential for local and systemic treatment of lung diseases. But DPIs are complex in nature and their performance relies on many aspects, including the design of inhaler, the powder formulation, and the airflow generated by the patient. In the last decade, perfor-mance of DPIs has improved significantly through the use of engineered drug particles and modified excipient systems.145,146

Sung et al. formulated PA-824, a nitroimidazopyran with promise for the treatment of TB, for efficient aerosol delivery to the lungs in a dry powder porous particle form. The physical, aerodynamic, and chemical properties of the dry powder were stable at room temperature for 6 months and under refrigerated conditions for at least 1 year. Oral and inhaled delivery of PA-824 achieved equivalent systemic delivery at the same body dose within the first 12 h of dosing. However, animals dosed by the pulmonary route showed drug loads that remained locally in the lungs for 32 h after exposure, whereas those given the drug orally cleared the drug more rapidly.147 Moreover, Sawatdee et al. developed INH as a dry powder aerosol for delivery to the lower airways and studied the susceptibility of M. bovis and M. tuberculosis to the formulations. The content of all INH dry powder formulations was almost 100% of the theoretical content and found to be stable over 3 months’ storage. Drug susceptibility testing of M. tuberculosis by broth microdilution showed that the MIC of INH dry powder formulations were 1.7 to 3.4 times lower than standard INH. Flow cytometry analysis of viable M. bovis revealed that MIC of INH dry powder formulations and standard INH had no significant difference.148

Garcia-Contreras et al. formulated and characterized nitroimidazo-oxazine PA-824 for efficient aerosol delivery as dry powder porous particles and the subsequent dis-position in guinea pigs after pulmonary administration. Four weeks after infection by the pulmonary route, animals received daily treatment for 4 weeks of either a high or a low dose of PA-824 dry powder aerosol. Animals received PA-824 cyclodextrin/leci-thin suspensions orally as positive controls, and those receiving placebo particles or no

TABLE 11. Various dendrimer formulations developed against TB

Carrier system

Drugs encapsulated Properties Reference

Dendrimer RIF Among the four formulations PEGylated dendrimers showed relatively slower release of RIF when compared with non-PEGylated dendrimers.

143

RIF Mannosylated dendrimer having size <5 mm andentrapmentefficiency37.34%±1.32%.

144


344 S. Gupta et al.

treatment were negative controls. The lungs and spleens of animals receiving the high dose of inhaled PA-824 particles exhibited a lower degree of inflammation (indicated by wet tissue weights), bacterial burden, and tissue damage (indicated by histopathology) than those of untreated or placebo animals. Treatment with oral PA-824 cyclodextrin/lecithin suspension resulted in a more significant reduction in the bacterial burden of lungs and spleen, consistent with a dose that was larger than inhaled doses (eight times the inhaled low dose and four times the inhaled high dose). However, histopathological analysis revealed that the extent of tissue damage was comparable in groups receiving either the oral or inhaled dose.149 Likewise, Fiegel et al. formulated dry powder aerosols containing CAP to enable efficient delivery of large drug masses to the lungs of guinea pigs. Aerosols loaded with 73% CAP sulfate were shown to possess good aerosoliza-tion properties and physical-chemical stability for up to 3 months at room temperature. Upon insufflation into guinea pigs, the amount of CAP sulfate reaching the bloodstream was significantly lower compared to IV or intramuscular administration, but resulted in a significantly longer drug half-life.150

VII.H. Nanoemulsions

Nanoemulsions are thermodynamically stable oil-in-water (o/w) dispersions displaying drop sizes between 10 and 100 nm.151 The main advantage of these systems is that they are generated spontaneously and can be produced on a large scale without the need of high homogenization energy. In addition, they can be sterilized by filtration. The en-hanced uptake of nanoemulsions by cells of the phagocytic system reported elsewhere gives these nanocarriers passive targeting features. Also, they are taken up by lipopro-tein receptors in the liver after oral administration.152,153

Ahmed and co-workers developed different o/w nanoemulsions of RIF for IV ad-ministration using pharmaceutically acceptable excipients: Sefsol® 218 as the oil phase, Tween® 80, Tween® 85, and saline water as the surfactant, the cosurfactant, and the aqueous phase. In vitro drug-release studies indicated an initial burst effect ranging from 40% to 70% after 2 h, followed by a more moderate release afterwards. Finally, stability assays over 3 months indicated slight increases in the droplet size and the viscosity of the systems at 4°C and 25°C.154

VII.I. Nanosuspensions

Nanosuspensions are sub-micron colloidal dispersions of pure drugs stabilized with surfactants.155 Nanonization (reduction of the average size of solid drug particles to the nano-scale, generally by top milling or grinding) is a useful methodology to im-prove the solubility of drugs displaying strong solute-solute interactions and high melting points, as well as, in general, both poor water and lipid solubility. The solid and dense states of the pure drug particles confer a maximal mass per volume ratio, es-pecially critical in systems demanding high drug loadings. Despite its potential, only a few studies aiming to optimize the pharmacotherapy of TB have been reported. Pe-



ters and collaborators developed a clofazimine nanocrystalline suspension in order to overcome the toxicity and the low solubility (0.3 μg/mL) of the drug. To evaluate the suitability of the formulation for IV administration they compared the effectiveness of the nanosuspension in the treatment of murine M. avium infection to that of drug-loaded liposomes. In vivo assays were conducted with nano-formulations containing drug concentrations between 0.16% and 0.18%. Findings showed drug concentrations above the MIC of the pathogen following the administration of the nanoparticles: 81.4, 72.5, and 35.0 mg/kg tissue in spleen, liver, and lung, respectively. Moreover, continued treatment led to a significant reduction of bacterial counts in all the organs evaluated. Effectiveness levels were comparable to those of liposomal clofazimine; however, the ease of preparation and the higher physical stability of the nanosuspen-sion were distinguishing.156

Among all the discussed colloidal carriers, polymeric nanoparticles are the most extensively studied colloidal carrier system. The above literature review suggests that polymeric nanoparticles have a considerable potential for treatment of TB. Their major advantages, such as improvement of drug bioavailability and reduction of the dosing frequency, may create a sound basis for better management of the disease, making di-rectly observed treatment more realistic and affordable. Another important advantage of the nanoparticles as compared to other colloidal carriers is the possibility of the ver-satile routes of drug administration, including oral, inhalation, and intravascular routes. Moreover, the potential advantages of direct delivery of the nanoparticulate antituber-cular drug to the lungs include the possibility of reduced systemic toxicity, as well as achieving higher drug concentration at the site of infection. Furthermore, in contrast to the oral route of administration, inhaled drugs are not subjected to first-pass metabolism. In addition, the high stability of the nanoparticles suggests long shelf life. Therefore, based on the above-mentioned advantages of nanoparticles and routes of administration, nanoparticles alone can be considered as an ideal colloidal formulation, and nanapar-ticles in the form of dry powder inhalers can be considered as an ideal drug delivery system. Finally, the success of this technology will probably depend on toxicological issues associated with understanding of the fate of nanocarriers and their polymeric con-stituents in the body, as well as elimination of the risk of the residual organic solvents. In this respect, the possibility of using drug carriers made from natural polymers (e.g., chitosan or alginate) represents an attractive perspective.

VIII. FUTURe PROsPeCTs

Drug resistance remains an important obstacle to better outcomes in the treatment of TB by conventional drug therapy. Resistance that arises in the case of conventional therapy can be overcome by the newer approach of these colloidal carriers. Although plain col-loidal carriers are largely unsuccessful in drug targeting against TB due to their diffi-culties in gaining access to targeted tissues, penetrating vascular barriers, and evading phagocytic capture by the reticuloendothelial system, they are able to modify the dis-tribution of an associated drug substance, which can overcome the drug resistance. A


346 S. Gupta et al.

common practice can be to use ligands such as glycoproteins or polysaccharides ending in mannose or fucose radicals, and polyanionic macromolecules such as acetylated LDL lipoproteins, having affinity for macrophage receptors. Another alternative is the fixation of specific antibodies of the agent responsible for the infection to these colloidal carriers, such that the selectivity for infected cells is increased. Generic production of antituber-cular drugs should be promoted to increase competition and remedy the vulnerable situa-tion in which TB drugs and colloidal carriers are produced by only one manufacturer. To guard against unregulated use, the cash value of antitubercular drugs and carriers needs to be minimized by the provision of free treatment to patients. Prevention of resistance also needs to include monitoring of the effectiveness of treatment, relapse rates, and cure rates of relapsed patients, and the establishment of a method for in vitro drug susceptibility testing. The introduction of the novel colloidal carriers holds the key for the prevention of TB. It can be expected that future research will concentrate on the development of the vectorized delivery systems combining advantages of the colloidal carriers, such as large payloads of a drug, with ligand-mediated active targeting to the infection sites.

IX. CONCLUsIONs

Extensive studies have demonstrated that colloidal carriers such as polymeric nanopar-ticles, solid lipid nanoparticles, microparticles, liposomes, microspheres, niosomes, den-drimers, microemulsions, dry powder, nanoemulsions, and nanosuspensions are able to overcome drug-resistance issues and facilitate antitubercular drug delivery to M. tubercu-losis infection sites. Thus the drugs, which are well known for their effectiveness, however compromised due to their contraindicated manifestations, can safely be administered for effective cure of TB in the form of carriers. However, the new side effects generated from the use of these carriers need to be studied. While most of these antitubercular drug carri-ers are currently in preclinical development, several have been approved for clinical use. With the ongoing efforts in this field, there is no doubt that carrier-based drug delivery will continue to improve the treatment of M. tuberculosis infections.

ReFeReNCes

1. Somani RR, Gavarkar PS, Shirodkar PY, Bhanushali UV, Kadam VJ. Tropical diseases: an unsolved challenge. Int J PharmTech Res. 2010;2(3):1691–98.

2. http://www.who.int/gho/tb/en/ (Accessed on Dec 23, 2011 )3. Nikki M, James D, William R, Parrish NM, Dick JD, Bishai WR. Mechanisms of latency in Mycobac-

terium tuberculosis. Trends Microb. 1998;6(3):107–12.4. Amin Z. Clinical tuberculosis problems and management. Indones J Intern Med. 2006;38(2):109–16.5. Centers for Disease Control and Prevention (CDC), Division of Tuberculosis Elimination. Core

Curriculum on Tuberculosis: What the Clinician Should Know. 4th edition (2000). Updated August 2003. Available from http://www.cdc.gov/tb/topic/basics/default.htm.

6. Manabe YC, Bishai WR. Latent Mycobacterium tuberculosis—persistence, patience, and winning by waiting. Nature Med. 2000;6:1327–29.

7. Kaufmann SHE. Is the development of a new tuberculosis vaccine possible? Nature Med. 2000;6:955–60.



8. Reddy JR, Kwang J, Lechtenberg KF, Khan NC, Reddy BP, Chengappa MM. An immunochro-matographic serological assay for the diagnosis of Mycobacterium tuberculosis. Comp Immunol Microbiol Inf Dis. 2002;25:21–27.

9. Riley R. Transmission and environmental control of tuberculosis. In Reichman L, Hershfield E, editors. Tuberculosis. New York: Marcel Deckker; 1993.

10. Riley R. Airborne infection. Am. J. Med. 1974;57:466–75.11. Murray J. Defense mechanisms. In Murray JF, editor. The normal lung: the basis for diagnosis and

treatment of pulmonary disease. Philadelphia: Saunders WB; 1986. p. 313–38.12. Horsburgh CR. Tuberculosis without tubercles. Tuberc. Lung Dis. 1996;77:197–98.13. Bhatt CP, Bhatt AB, Shrestha B. Nepalese people’s knowledge about tuberculosis. Saarc J Tuber

Lung Dis HIV/AIDS. 2009;6(2):31–37.14. CDC. Targeted Tuberculin Testing and Treatment of Latent Tuberculosis Infection. 2000;49(RR-

6):38–39.15. Lee JH. The diagnosis of tuberculosis in the presence of silicosis. Can Med Assoc J. 1948;58(4):349–53.16. http://emedicine.medscape.com/article/302027-overview.17. http://www.merckmanuals.com/home/sec17/ch193/ch193a.html.18. Griffith De, Kerr CM. Tuberculosis: disease of the past, disease of the present. J Perianesth Nurs.

1996;11(4):240–45.19. Strachan DP, Powell KJ, Thaker A, Millard FJC, Maxwell JD. Vegetarian diet as a risk factor for

tuberculosis in immigrant south London Asians. Thorax.1995;50:175–80.20. Tso HW, Lau YL, Tam CM, Wong HS, Chiang KS. Associations between IL12B polymorphisms and

tuberculosis in the Hong Kong Chinese population. J Infect Dis. 2004;190(5):913–19.21. Jasmer RM, Nahid P, Hopewell PC. Clinical practice. Latent tuberculosis infection. N Engl J Med.

2002;347(23):1860–66.22. Ahearn JM, Fearon DT. Structure and function of the complement receptors, CR1 (CD35) and CR2

(CD21). Adv Immunol. 1989;46:183–219.23. Taylor ME, Drickamer K. Structural requirements for high affinity binding of complex ligands by

the macrophage mannose receptor. J Biol Chem. 1993;268:399–404.24. Nepomuceno RR, Henschen-Edman AH, Burgess WH, Tenner AJ. cDNA cloning and primary

structure analysis of C1qR(P), the human C1q/MBL/SPA receptor that mediates enhanced phagocy-tosis in vitro. Immunity. 1997;6:119–29.

25. Pugin J, Heumann ID, Tomasz A, Kravchenko VV, Akamatsu Y, Nishijima M, Glauser MP, Tobias PS, Ulevitch RJ. CD14 is a pattern recognition receptor. Immunity. 1994;1:509–16.

26. Dunne DW, Resnick D, Greenberg J, Krieger M, Joiner KA. The type I macrophage scavenger receptor binds to gram-positive bacteria and recognizes lipoteichoic acid. Proc Natl Acad Sci USA. 1994;91:1863–67.

27. Ernst JD. Macrophage receptors for Mycobacterium tuberculosis. Infect Immun. 1998;66:1277–81. 28. Gatfield J, Pieters J. Essential role for cholesterol in entry of mycobacteria into macrophages. Scien-

ce. 2000;288:1647–50.29. Lounis N, Truffot-Pernot C, Grosset J, Gordeuk VR, Boelaert JR. Iron and Mycobacterium tubercu-

losis infection. J Clin Virol. 2001;20:123–26. 30. Schaible UE, Sturgill-Koszycki S, Schlesinger PH, Russell DG. Cytokine activation leads to aci-

dification and increases maturation of Mycobacterium avium-containing phagosomes in murine macrophages. J Immunol. 1998;160:1290–96.

31. Nathan C, Shiloh MU. Reactive oxygen and nitrogen intermediates in the relationship between mammalian hosts and microbial pathogens. Proc Natl Acad Sci USA. 2000;97:8841–48.

32. Weber I, Fritz C, Ruttkowski S, Kreft A, Bange FC. Anaerobic nitrate reductase (narGHJI) activity of Mycobacterium bovis BCG in vitro and its contribution to virulence in immunodeficient mice. Mol Microbiol. 2000;35:1017–25.

33. Ferrari G, Langen H, Naito M, Pieters J. A coat protein on phagosomes involved in the intracellular survival of mycobacteria. Cell. 1999;97:435–47.

34. Konstantinos A. Testing for tuberculosis. Australian Prescriber. 2010;33:12–18.35. Kumar V, Abbas AK, Nelson F, Mitchell RN. Robbins basic pathology, 8th ed. Philadelphia: Saun-

ders Elsevier; 2007:516–22.36. Ormerod LP. Joint Tuberculosis Committee of the British Thoracic Society, control and prevention

of tuberculosis in the United Kingdom: Code of Practice 2000. BMJ. 2000;55:887–901.


348 S. Gupta et al.

37. http://www.immunisation.nhs.uk/files/mantouxtest.pdf.38. Nahid P, Pai M, Hopewell P. Advances in the diagnosis and treatment of tuberculosis. Proc Am Thorac

Soc. 2006;3(1):103–10. 39. McFadden JJ, Kunze Z, Seechum P. DNA probes for detection and identification. In: McFadden J,

editor. Molecular biology of the mycobacteria. Surrey, UK: Surrey University Press. 1990; p. 139–72.40. Kaminski DA, Hardy DS. Selective utilization of DNA probes for identification of Mycobacte-

rium species on the basis of cord formation in primary BACTEC cultures. J Clin Microbiol. 1995;33:1548–50.

41. Katoch VM, Sharma VD. Advances in the diagnosis of mycobacterial diseases. Ind J Med Microbiol. 1997;15:49–55.

42. Mathur JN. What is new in the diagonosis of tuberculosis. ICMR Bull. 2002;32(8).43. Siddiqi SH, Libonati JP, Middlebrook G. Evaluation of rapid radiometric method for drug suscepti-

bility testing of Mycobacterium tuberculosis. J Clin Microbiol. 1981;13:908.44. Chan ED, Heifets L, Iseman MD. Immunologic diagnosis of tuberculosis: a review. Tuber Lung Dis.

2000;80:131.45. Albert H, Heydenrych A, Brookes R, Mole RJ, Harley B, Subotsky E, Henry R, Azevedo V. Perfor-

mance of a rapid phage-based test, AST Plaque TB to diagnose pulmonary tuberculosis from sputum specimens in South Africa. Int J Tuber Lung Dis. 2002;6:529.

46. Muttil P, Wang C, Hickey AJ. Inhaled drug delivery for tuberculosis therapy. Pharm Res. 2009;26(11):2401–16.

47. Somani RR, Gavarkar PS, Shirodkar PY, Bhanushali UV, Kadam VJ. Tropical diseases: an unsolved challenge. Int J PharmTech Res. 2010;2(3):1691–98.

48. Singla R, Sharma SK, Mohan A, Makharia G, Sreenivas V, Jha B, Kumar S, Sarda P, Singh S. Evaluation of risk factors for antituberculosis treatment induced hepatotoxicity. Ind J Med Res. 2010;132:81–86.

49. Venable SJ. Drugs treating mycobacterial infections. In: Aschenbrenner DS, Venable SJ, editors. Drug therapy in nursing. Baltimore: Lippincott Williams & Wilkins; 2008. p. 828–41.

50. Smith PJ, van Dyk J, Fredericks A. Determination of rifampicin, isoniazid and pyrazinamide by high performance liquid chromatography after their simultaneous extraction from plasma. Int J Lung Dis. 1999;3(11):S325–S328.

51. Gumbo T. Chemotherapy of tuberculosis, Mycobacterium avium complex disease, and leprosy. In: Goodman & Gilman’s the pharmacological basis of therapeutics. Brunton L, Chabner B, Knollman B, editors. Columbus, OH: McGraw-Hill. 2006.

52. Katzung BG, Masters SB, Trevor AJ. Basic & clinical pharmacology. 11th ed. Columbus, OH: Mc-Graw-Hill; 2009:1041–50.

53. Hazre A, Laha B. Chemotherapy of osteoarticular tuberculosis. Ind J Pharmacol. 2005; 37(1):5–12. 54. Barry PJ, O’Connor TM. Novel agents in the management of Mycobacterium tuberculosis disease.

Curr Med Chem. 2007;14:2000–8.55. Lalloo UG, Naidoo R, Ambaram A. Recent advances in the medical and surgical treatment of multi-

drug resistant tuberculosis. Curr Opin Pulm Med. 2006;12:179–85.56. Shi R, Sugawara I. Development of new anti-tuberculosis drug candidates. Tohuku J Exp Med.

2010;221:97–106.57. Nuermberger EL, Yoshimatsu T, Tyagi S, O’Brien RJ, Vernon AN, Chaisson RE, Bishai WR, Gros-

set JH. Moxifloxacin-containing regimen greatly reduces time to culture conversion in murine tuber-culosis. Am J Respir Crit Care Med. 2004;169(3):421–26.

58. Tyagi S, Nuermberger E, Yoshimatsu T, Williams K, Rosenthal I, Lounis N, Bishai W, Grosset J. Bactericidal activity of the nitroimidazopyran PA-824 in a murine model of tuberculosis. Antimicrob Agents Chemother. 2005;49(6):2289–93.

59. Sacks LV, Pendle S, Orlovic D, Andre M, Popara M, Moore G, Thonell L, Hurwitz S. Adjunctive salvage therapy with inhaled aminoglycosides for patients with persistent smear-positive pulmonary tuberculosis. Clin Infect Dis. 2001;32(1):44–49.

60. Conte JE, Golden JA, Duncan S, McKenna E, Zurlinden E. Intrapulmonary concentrations of pyra-zinamide. Antimicrob Agents Chemother. 1999;43(6):1329–33.

61. Conte JE, Golden JA, McQuitty M, Kipps J, Duncan S, McKenna E, Zurlinden E. Effects of gender, AIDS, and acetylator status on intrapulmonary concentrations of isoniazid. Antimicrob Agents Che-mother. 2002;46(8):2358–64.

62. Crofton J, Mitchison D. Streptomycin resistance in pulmonary tuberculosis. BMJ. 1948;2:1009–15.



63. Mitchinson DA. The diagnosis and therapy of tuberculosis during the past 100 years. Am J Respir Crit Care Med. 2005;171:699–706.

64. Wongsrichanalai C, Varma JK, Juliano JJ, Kimerling ME, MacArthur JR. Extensive drug resistance in malaria and tuberculosis. Emerg Infect Dis. 2010;16(7):1063–67.

65. Shah NS, Wright A, Bai GH, Barrera L, Boulahbal F, Martin-Casabona N, Drobniewski F, Gilpin C, Havelkova M, Lepe R, Lumb R, Metchock B, Portaels F, Rodrigues MF, Rusch-Gerdes S, Deun AV, Vincent V, Laserson K, Wells C, Cegielski JP. Worldwide emergence of extensively drug-resistant tuberculosis. Emerg Infect Dis. 2007;13:380–87.

66. Centers for Disease Control and Prevention. Revised definition of extensively drug-resistant tuber-culosis. MMWR. 2006;55:1176.

67. Mansour HM, Rhee YS, Wu X. Nanomedicine in pulmonary delivery. Int J Nanomed. 2009;4:299–319.68. Pandey R, Khuller GK. Nanotechnology based drug delivery system for the management of tuber-

culosis. Ind J Exper Biol. 2006;44:357–66.69. Ahmad Z, Pandey R, Sharma S, Khuller GK. Alginate nanoparticles as antituberculosis drug car-

riers: formulation development, pharmacokinetics and therapeutic potential. Ind J Chest Dis Allied Sci. 2006;48:171–76.

70. Hoet PH, Bruske-Hohlfeld I, Salata OV. Nanoparticles—known and unknown health risks. J Nano-biotechnol. 2004;2:12.

71. Courrier HM, Butz N, Vandamme TF. Pulmonary drug delivery systems: recent developments and prospects. Crit Rev Ther Drug Carrier Sys. 2002;19:425–98.

72. Venugopal J, Prabhakaran MP, Low S, Choon AT, Deepika G, Dev VR, Ramakrishna, S. Continuous nanostructures for the controlled release of drugs. Curr Pharm Des. 2009,15:1799–1808.

73. Langer R, Folkman J. Polymers for the sustained release of proteins and other macromolecules. Nature. 1976;263:797–800.

74. Gref R, Minamitake Y, Peracchia MT, Trubetskoy V, Torchilin V, Langer R. Biodegradable long-circulating polymeric nanospheres. Science. 1994; 263:1600–3.

75. Umamaheshwari RB, Jain NK. Receptor mediated targeting of lectin conjugated gliadin nanoparti-cles in the treatment of Helicobacter pylori. J Drug Target. 2003;11:415–23.

76. Cheng J, Teply BA, Sherifi I, Sung J, Luther G, Gu FX, Levy-Nissenbaum E, Radovic-Moreno AF, Langer R, Farokhzad OC. Formulation of functionalized PLGA-PEG nanoparticles for in vivo tar-geted drug delivery. Biomaterials. 2007;28:869–76.

77. Gu FX, Zhang L, Teply BA, Mann N, Wang A, Radovic-Moreno AF, Langer R, Farokhzad OC. Pre-cise engineering of targeted nanoparticles by using self-assembled biointegrated block copolymers. Proc Natl Acad Sci USA. 2008;105:2586–91.

78. Pandey R, Sharma A, Zahoor A, Sharma S, Khuller GK. Poly (DL-lactide-co-glycolide) nanopar-ticle-based inhalable sustained drug delivery system for experimental tuberculosis. J Antimicrob Chemother. 2003;52:981–86.

79. Ahmad Z, Pandey R, Sharma S, Khuller GK. Pharmacokinetic and pharmacodynamic behaviour of antitubercular drugs encapsulated in alginate nanoparticles at two doses. Int J Antimicrob Agents. 2006;27:409–16.

80. Johnson CM, Pandey R, Sharma S, Khuller GK, Basaraba RJ, Orme IM, Lenaerts AJ. Oral therapy using nanoparticle-encapsulated antituberculosis drugs in guinea pigs infected with Mycobacterium tuberculosis. Antimicrob Agents Chemother. 2005;49(10):4335–38.

81. Pandey R, Khuller GK. Oral nanoparticle-based antituberculosis drug delivery to the brain in an expe-rimental model. J Antimicrob Chemother. 2006;57(6):1146–52.

82. Tripathi A, Gupta R, Saraf SA. PLGA nanoparticles of anti tubercular drug: drug loading and release studies of a water in-soluble drug. Int J PharmTech Res. 2010;2(3):2116–23.

83. Pandey R, Sharma S, Khuller GK. Chemotherapeutic efficacy of nanoparticle encapsulated antituber-cular drugs. Drug Deliv. 2006;13:287–94.

84. Saraogi GK, Gupta P, Gupta UD, Jain NK, Agrawal GP. Gelatin nanocarriers as potential vectors for effective management of tuberculosis. Int J Pharm. 2010;385:143–49.

85. Sharma A, Pandey R, Sharma S, Khuller GK. Chemotherapeutic efficacy of poly (DL-lactide-co-gly-colide) nanoparticle encapsulated antitubercular drugs at sub-therapeutic dose against experimental tuberculosis. Int J Antimicrob Agents. 2004;24:599–604.

86. Pandey R, Zahoor A, Sharma S, Khuller GK. Nanoparticle encapsulated antitubercular drugs as a po-tential oral drug delivery system against murine tuberculosis. Tuberculosis. 2003;83:373–78.


350 S. Gupta et al.

87. Ahmad Z, Pandey R, Sharma S, Khuller GK. Novel chemotherapy for tuberculosis: chemothera-peutic potential of econazole- and moxifloxacin-loaded PLG nanoparticles. Int J Antimicrob Agents. 2008;31:142–46.

88. Pandey R, Khuller GK. Subcutaneous nanoparticle-based antitubercular chemotherapy in an experi-mental model. J Antimicrob Chemother. 2004;54:266–68.

89. Pandey R, Khuller GK. Nanoparticle-based oral drug delivery system for an injectable antibiotic—streptomycin evaluation in a murine tuberculosis model. Chemotherapy. 2007;53:437–41.

90. Sharma A, Sharma S, Khuller GK. Lectin-functionalized poly (lactide-co-glycolide) nanoparticles as oral/aerosolized antitubercular drug carriers for treatment of tuberculosis. J Antimicrob Chemother. 2004;54:761–66.

91. Fawaz F, Bonini F, Maugein J, Lagueny AM. Ciprofloxacin-loaded polyisobutylcyanoacrylate nano-particles: pharmacokinetics and in vitro antimicrobial activity. Int J Pharm. 1998;168:255–59.

92. zur Mühlen A, Schwarz C, Mehnert W. Solid lipid nanoparticles (SLN) for controlled drug delivery—drug release and release mechanism. Eur J Pharm Biopharm. 1998;45:149–55.

93. Eldem T, Speiser P, Hincal A. Optimization of spray-dried and -congealed lipid micropellets and cha-racterization of their surface morphology by scanning electron microscopy. Pharm Res. 1991;8:47–54.

94. Domb AJ. Liposphere parenteral delivery system. Proc Intern Symp Control Rel Bioact Mater. 1993;20:346–47.

95. Mullera RH, Maaenb S, Weyhersa H, Spechtb F, Lucksb JS. Cytotoxicity of magnetite-loaded polylac-tide, polylactide/glycolide particles and solid lipid nanoparticles. Int J Pharm. 1996;138:85–94.

96. Pandey R, Khuller GK. Solid lipid particle-based inhalable sustained drug delivery system against experimental tuberculosis. Tuberculosis. 2005;85:227–34.

97. Gelperina S, Kisich K, Iseman MD, Heifets L. The potential advantages of nanoparticle drug delivery systems in chemotherapy of tuberculosis. Am J Respir Crit Care Med. 2005;172:1487–90.

98. Videira MA, Botelho MF, Santos AC, Gouveia LF, de Lima JJ, Almeida AJ. Lymphatic uptake of pul-monary delivered radiolabelled solid lipid nanoparticles. J Drug Target. 2002;10:607–13.

99. Pandey R, Sharma S, Khuller GK. Oral solid lipid nanoparticle-based antitubercular chemotherapy. Tuberculosis. 2005;85:415–20.

100. Nimje N, Agarwal A, Saraogi GK, Lariya N, Rai G, Agrawal H, Agrawal GP. Mannosylated nano-particulate carriers of rifabutin for alveolar targeting. J Drug Target. 2009;17(10):777–87.

101. Pandey R, Khuller GK. Solid lipid particle-based inhalable sustained drug delivery system against experimental tuberculosis. Tuberculosis. 2005;85:227–34.

102. Kaur K, Gupta A, Narang RK, Murthy RSR. Novel drug delivery systems: desired feat for tubercu-losis. J Adv Pharm Tech Res. 2010;1(2):145–63.

103. Dutt M, Khuller GK. Sustained release of isoniazid from a single injectable dose of poly (DL-lactide-co-glycolide) microparticles as a therapeutic approach towards tuberculosis. Int J Antimicrob Agents. 2001;17:115–22.

104. Ain Q, Sharma S, Garg SK, Khuller GK. Role of poly [DL-lactide-co-glycolide] in development of a sustained oral delivery system for antitubercular drug(s). Int J Pharm. 2002;239:37–46.

105. Dutt M, Khuller GK. Chemotherapy of Mycobacterium tuberculosis infections in mice with a com-bination of isoniazid and rifampicin entrapped in Poly (DL-lactide-co-glycolide) microparticles. J Antimicrob Chemother. 2001;47:829–35.

106. Suarez S, O’Hara P, Kazantseva M, Newcomer CE, Hopfer R, McMurray DN, Hickey AJ. Airways delivery of rifampicin microparticles for the treatment of tuberculosis. J Antimicrob Chemother. 2001;48:431–34.

107. Zhou H, Zhang Y, Biggs DL, Manning MC, Randolph TW, Christians U, Hybertson BM, Ng Ka-yun. Microparticle-based lung delivery of INH decreases INH metabolism and targets alveolar macrophages. J Contr Rel. 2005;107:288–99.

108. Ain Q, Sharma S, Khuller GK. Chemotherapeutic potential of orally administered poly(lactide-co-glycolide) microparticles containing isoniazid, rifampin, and pyrazinamide against experimental tu-berculosis. Antimicrob Agents Chemother. 2003;47(9):3005–7.

109. Sharma R, Saxena D, Dwivedi AK, Misra A. Inhalable microparticles containing drug combi-nations to target alveolar macrophages for treatment of pulmonary tuberculosis. Pharm Res. 2001;18(10):1405–10.

110. Muttil P, Kaur J, Kumar K, Yadav AB, Sharma R, Misra A. Inhalable microparticles containing large payload of anti-tuberculosis drugs. Eur J Pharm Sci. 2007;32:140–50.



111. Kellaway IW, Farr SJ. Liposomes as drug delivery systems to the lungs. Adv Drug Del Rev. 1990;5:149–61.

112. Couvreur P, Fattal E, Andremont A. Liposomes and nanoparticles in the treatment of intracellular bacterial infections. Pharm Res. 1991;8:1079–86.

113. Vyas SP, Kannan ME, Jain S, Mishra V, Singh P. Design of liposomal aerosols for improved delivery of rifampicin to alveolar macrophages. Int J Pharm. 2004;269:37–49.

114. Agarwal A, Kandpal H, Gupta HP, Singh NB, Gupta CM. Tuftsin-bearing liposomes as rifampin vehicles in treatment of tuberculosis in mice. Antimicrob Agents Chemother. 1994;38(3):588–93.

115. Gaspar MM, Cruz A, Penha AF, Reymao J, Sousa AC. Eleuterio CV, Domingues SA, Fraga AG, Filho AL, Cruz MEM, Pedrosa J. Rifabutin encapsulated in liposomes exhibits increased therapeutic activity in a model of disseminated tuberculosis. Int J Antimicrob Agents. 2008;31:37–45.

116. Chimote G, Banerjee R. In vitro evaluation of inhalable isoniazid-loaded surfactant liposomes as an adjunct therapy in pulmonary tuberculosis. J Biomed Mater Res B: Applied Biomaterials. 2010;94b(1):1–10.

117. Deol P, Khuller GK, Joshi K. Therapeutic efficacies of isoniazid and rifampin encapsulated in lung-specific stealth liposomes against Mycobacterium tuberculosis infection induced in mice. Antimi-crob Agents Chemother. 1997;41(6):1211–14.

118. Furneri PM, Fresta M, Puglisi G, Tempera G. Ofloxacin-loaded liposomes: in vitro activity and drug accumulation in bacteria. Antimicrob Agents Chemother. 2000;44(9):2458–64.

119. Karki R, Subramanya G, Udupa N. Formulation and evaluation of coencapsulated rifampicin and isoniazid liposomes using different lipids. Acta Pharmaceutica Sciencia. 2009;51:177–88.

120. Pandey R, Sharma S, Khuller GK. Nebulization of liposome encapsulated antitubercular drugs in guinea pigs. Int J Antimicrob Agents. 2004;24:93–94.

121. El-Ridy MS, Mostafa DM, Shehab A, Nasr EA, El-Alim SA. Biological evaluation of pyrazinamide liposomes for treatment of Mycobacterium tuberculosis. Int J Pharm. 2007;330:82–88.

122. Wiens T, Redelmeier T, Av-Gay Y. Development of a liposome formulation of ethambutol. Antimi-crob Agents Chemother. 2004;48(5):1887–88.

123. Martin GP, Zeng XM, Marriott C. The controlled delivery of drugs to the lungs. Int J Pharm.1995;124:149–64.

124. Pandey R, Khuller GK. Chemotherapeutic potential of alginate-chitosan microspheres as anti-tuber-cular drug carriers. J Antimicrob Chemother. 2004;53:635–40.

125. Barrow ELW, Winchester GA, Staas JK, Quenelle DC. Use of microsphere technology for targeted delivery of rifampin to Mycobacterium tuberculosis-infected macrophages. Antimicrob Agents Che-mother. 1998;42(10):2682–89.

126. Quenelle DC, Winchester GA, Staas JK, Barrow ELW, Barrow WW. Treatment of tuberculosis using a combination of sustained-release rifampin-loaded microspheres and oral dosing with isoniazid. Antimicrob Agents Chemother. 2001;45(6):1637–44.

127. Garcia-Contreras L, Sethuraman V, Kazantseva M, Godfrey V, Hickey AJ. Evaluation of dosing regimen of respirable rifampicin biodegradable microspheres in the treatment of tuberculosis in the guinea pig. J Antimicrob Chemother. 2006;58:980–86.

128. Hasegawa T, Hirota K, Tomoda K, Ito F, Inagawa H, Kochi C, Soma G-I, Makino K, Terada H. Phago-cytic activity of alveolar macrophages toward polystyrene latex microspheres and PLGA microsphe-res loaded with anti-tuberculosis agent. Colloids and Surfaces B: Biointerfaces. 2007;60:221–28.

129. Samad A, SultanaY, Khar RK, Chuttani K, Mishra AK. Gelatin microspheres of rifampicin cross-linked with sucrose using thermal gelation method for the treatment of tuberculosis. J Microen-capsulation. 2009;26(1):83–89.

130. Suarez S, O’Hara P, Kazantseva M, Newcomer CE, Hopfer R, McMurray DN, Hickey AJ. Respi-rable PLGA microspheres containing rifampicin for the treatment of tuberculosis: screening in an infectious disease model. Pharm Res. 2001;18(9):1315–19.

131. Zhang W, Jiang X, Hu J, Fu C. Rifampicin polylactic acid microspheres for lung targeting. J Micro-encapsulation. 2000;17(6):785–88.

132. Uchegbu IF, Vyas SP. Non-ionic surfactant based vesicles (niosomes) in drug delivery. Int J Pharm. 1998;172:33–70.

133. Jain CP, Vyas SP. Preparation and characterization of niosomes containing rifampicin for lung targe-ting. J Microencapsulation. 1995;12(4):401–7.

134. Jain CP, Vyas SP, Dixit VK. Niosomal system for delivery of rifampicin to lymphatics. Ind J Pharm


352 S. Gupta et al.

Sci. 2006;68(5):575–78.135. Moazeni E, Gilani K, Sotoudegan F, Pardakhty A, Najafabadi AR, Fazeli MR, Jamalifar H. For-

mulation and in vitro evaluation of ciprofloxacin containing niosomes for pulmonary delivery. J Microencapsulation. 2010;27(7):618–27.

136. Tomalia DA, Naylor AM, Goddar WA. Starburst dendrimers: control of size, shape, surface, chemis-try, and topology. Angew Chem. 1990;102:119–57.

137. Tomalia DA, Durst HD. Supramolecular chemistry—directed synthesis and molecular recognition. Top Curr Chem. 1993;165:193–313.

138. Frechet JMJ. Functional polymers and dendrimers: reactivity, molecular architecture, and interfacial energy. Science.1994;263:1710–15.

139. Voit BI. Dendritic polymers: from aesthetic macromolecules to commercially interesting materials. Acta Polym. 1995;46(2):87–99.

140. de Brabender-Van Den Berg EMM, Meijer EW. Poly(propylene imine) dendrimers: large scale synt-hesis by heterogeneously catalyzed hydrogenation. Angew Chem Int Ed Engl. 1993;32(a):1308–11.

141. Pricl S, Fermeglia M. Molecular simulations of host-guest inclusion compounds: an approach to the lactodendrimers case. Carbohydrate Polymers. 2001;45:23–33.

142. Roberts JC, Bhalbat MK, Zera RTJ. Preliminary biological evaluation of polyamidoamine (PAMAM) Starburst dendrimers. Biomed Mater Res. 1996;30:53–65.

143. Kumar PV, Agashe H, Dutta T, Jain NK. PEGylated dendritic architecture for development of a pro-longed drug delivery system for an antitubercular drug. Current Drug Deliv. 2007;4:11–19.

144. Kumar PV, Asthana A, Dutta T, Jain NK. Intracellular macrophage uptake of rifampicin loaded man-nosylated dendrimers. J Drug Target. 2006;14(8):546–56.

145. Frijlink HW, Boer AD. Dry powder inhalers for pulmonary drug delivery. Expert Opinion Drug Deliv. 2004;1(1):67–86.

146. Maa YF, Prestrelski SJ. Biopharmaceutical powders: particle formation and formulation considera-tions. Curr Pharm Biotech. 2000;1(283–302):1389–2010.

147. Sung JC, Garcia-Contreras L, VerBerkmoes JL, Peloquin CA, Elbert KJ, Hickey AJ, Edwards DA. Dry powder nitroimidazopyran antibiotic PA-824 aerosol for inhalation. Antimicrob Agents Che-mother. 2009;53(4):1338–43.

148. Sawatdee S, Worakul N, Srichana T. Preparation of isoniazid as dry powder formulations for inhala-tion by physical mixing and spray drying. Malays J Pharm Sci. 2006;4(1):43–63.

149. Garcia-Contreras L, Sung JC, Muttil P, Padilla D, Telko M, VerBerkmoes JL, Elbert KJ, Hickey AJ, Edwards DA. Dry powder PA-824 aerosols for treatment of tuberculosis in guinea pigs. Antimicrob Agents Chemother. 2010;54(4):1436–42.

150. Fiegel J, Garcia-Contreras L, Thomas M, VerBerkmoes J, Elbert K, Hickey A, Edwards D. Pre-paration and in vivo evaluation of a dry powder for inhalation of capreomycin. Pharm Res. 2008;25(4):805–11.

151. Constantinides PP, Chaubal MV, Shorr R. Advances in lipid nanodispersions for parenteral drug delivery and targeting. Adv Drug Deliv Rev. 2008;60:757–67.

152. Seki J, Sonoke S, Saheki A, Fukui H, Sasaki H, Mayumi T. A nanometer lipid emulsion, lipid nano-sphere (LNS®), as a parenteral drug carrier for passive drug targeting. Int J Pharm. 2004;273:75–83.

153. Singh KK, Vingkar SK. Formulation, antimalarial activity and biodistribution of oral lipid nano-emulsion of primaquine. Int J Pharm. 2008;347:136–43.

154. Ahmed M, Ramadan W, Rambhu D, Shakeel F. Potential of nanoemulsions for intravenous delivery of rifampicin. Pharmazie. 2008;63:806–11.

155. Rabinow BE. Nanosuspensions in drug delivery. Nat Rev Drug Discov. 2004;3:785–96.156. Peters K, Leitzke S, Diederichs JE, Borner K, Hahn H, Muller RH, Ehlers S. Preparation of a clo-

fazimine nanosuspension for intravenous use and evaluation of its therapeutic efficacy in murine Mycobacterium avium infection. J Antimicrob Chemother. 2000;45:77–83.

157. Stover CK, Warrener P, VanDevanter DR, Sherman DR, Arain TM, Langhorne MH, Anderson SW, Towell JA, Yuan Y, McMurray DN, Kreiswirth BN, Barry CE, Baker WR. A small molecule nitroi-midazopyran drug candidate for the treatment of tuberculosis. Nature. 2000;405:962–66.

158. Lenaerts AJ, Gruppo V, Marietta KS, Johnson CM, Driscoll DK, Tompkins NM, Rose JD, Rey-nolds RC, Orme IM. Preclinical testing of the nitroimidazopyran PA-824 for activity against My-cobacterium tuberculosis in a series of in vitro and in vivo models. Antimicrob Agents Chemother. 2005;49:2294–2301.



159. Matsumoto M, Hashizume H, Tomoshige T, Kawasaki M,Tsubouchi H, Sasaki H, Shimokawa Y, Komatsu M. OPC-67683, a nitro-dihydro-imidazooxazole derivative with promising action against tuberculosis in vitro and in mice. Plos Med. 2006;3:466.

160. Sasaki H, Haraguchi Y, Itotani M, Kuroda H, Hashizume H, Tomishige T, Kawasaki M, Matsumoto M, Komatsu M, Tsubouchi H. Synthesis and antituberculosis activity of a novel series of optically active 6-nitro-2,3-dihydroimidazo [2,1-b] oxazoles. J Med Chem. 2006;49:7854–60.

161. Andries K, Verhasselt P, Guillemont J, Gohlmann HW, Neefs JM, Winkler H, Van Gestel J, Timmer-man P, Zhu M, Lee E, Williams P, de Chaffoy D, Huitric E, Hoffner S, Cambau E, Truffot-Pernot C, Lounis N, Jarlier V. A diarylquinoline drug active on ATP synthase of Mycobacterium tuberculosis. Science. 2005;307:223–27.

162. Gelber R, Andries K, Paredes RM, Andaya CE, Burgos J. The diarylquinoline R207910 is bacterici-dal against Mycobacterium leprae in mice at low dose and administered intermittently. Antimicrob Agents Chemother. 2009;53:3989–91.

163. Shandil RK, Jayaram R, Kaur P, Gaonkar S, Suresh BL, Mahesh BN, Jayashree R, Nandi V, Bharath S, Balasubramanian V. Moxifloxacin, ofloxacin, sparfloxacin, and ciprofloxacin against Mycobac-terium tuberculosis: evaluation of in vitro and pharmacodynamic indices that best predict in vivo efficacy. Antimicrob Agents Chemother. 2007;51:576–82.

164. Yoshimatsu TE, Nuermberger E, Tyagi S, Chaisson R, Bishai W, Grosset J. Bactericidal activity of increasing daily and weekly doses of moxifloxation in murine tuberculosis. Antimicrob Agents Chemother. 2002;46:1875–79.

165. Paramasivan CN, Sulochana S, Kubendiran G, Venkatesan P, Mitchison DA. Bactericidal action of gatifloxacin, rifampin, and isoniazid on logarithmic- and stationary-phase cultures of Mycobacte-rium tuberculosis. Antimicrob Agents Chemother. 2005;49:627–31.

166. Shindikar AV, Viswanathan CL. Novel fluoroquinolones: design, synthesis, and in vivo activity in mice against Mycobacterium tuberculosis H37Rv. Bioorg Med Chem Lett. 2005;15:1803–6.

167. Jia L, Tomaszewski JE, Hanrahan C, Coward L, Noker P, Gorman G, Nikonenko B, Protopopova M. Pharmacodynamics and pharmacokinetics of SQ109, a new diamine-based antitubercular drug. Br J Pharmacol. 2005;144:80–87.

168. Protopopova M, Hanrahan C, Nikonenko B, Samala R, Chen P, Gearhart J, Einck L, Nacy CA. Identification of a new antitubercular drug candidate, SQ109, from a corabinatorial library of 1,2-et-hylenediamines. J Antimicrob Chemother. 2005;56:968–74.

169. Arora S. Eradication of Mycobacterium tuberculosis infection in 2 months with LL3858: a preclini-cal study. Int J Tuber Lung Dis. 2004;8(Suppl.1):S29.

170. Protopopova M, Bogatcheva E, Nikonenko B, Hundert S, Einck L, Nacy CA. In search of new cures for tuberculosis. Med Chem. 2007;3:301–16.

171. Alcala L, Ruiz-Serrano MJ, Peres-Fernandez Turegano C, Carcia De Viedma D, Diaz-Infantes M, Marin-Arriaza M, Bouza E. In vitro activities of linezolid against clinical isolates of Mycobacterium tuberculosis that are susceptible or resistant to first-line antituberculous drugs. Antimicrob Agents Che-mother. 2003;47:416–17.

172. Fortun J, Martin-Davila P, Navas E, Peres-Elias MJ, Cobo J, Tato M, de la Pedrosa EG, Gomez-Mam-paso E, Moreno S. Linezolid for the treatment of multidrug-resistant tuberculosis. J Antimicrob Che-mother. 2005;56:180–85.

Documents

cr8.pdf