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Critical Reviews in Therapeutic Drug Carrier Systems, 29(4), 299-353 (2012)
Colloidal Carriers: A Rising Tool for Therapy of Tuberculosis

Swati Gupta,1,2* Pankaj Kumar,1 Manish K. Gupta,3 & Suresh P. Vyas4
Nanomedicine 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
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*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; swatig25@gmail.com.
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 drawbacks 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 structure) 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 conventional 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 mycobacteria, 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.
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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 immunodeficiency 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 immunocompetent 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 subsequently 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 hosts cellular 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
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TABLE 1. Difference between latent TB infection and TB disease A person with latent TB infection Does not feel sick Cannot spread TB bacteria to others Usually has a skin test or blood test result indicating TB infection Has a normal chest X-ray and a negative sputum smear Needs treatment for latent TB infection to prevent active TB disease Has no symptoms A person with TB disease Usually feels sick May spread TB bacteria to others Usually has a skin test or blood test result indicating TB infection May have an abnormal chest X-ray, or positive sputum smear or culture Needs treatment to treat active TB disease 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
these droplets, but do not kill the pathogen. Specific T cells are stimulated in the draining 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 (approximately 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 immunologically 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 central 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 patient becomes infectious, and a person with active disease infects up to 15 people annually.7 The vicious circle then continues. There are three potential outcomes of infection of the human host in M. tuberculosis. (a) In the immunocompromised host, disease can develop directly after infection.
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Figure 2: Main features of tuberculosis: from infection to host defense
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(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 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- (IFN-) and tumor-necrosis factor- (TNF-), produced by T cells, are important macrophage activators. Macrophage activation permits phagosomal maturation and the production of antimicrobial molecules such as reactive nitrogen intermediates (RNI) and reactive oxygen intermediates (ROI), lymphotoxin-3 (LT- 3). General symptoms of active TB infection include weight loss, sweats, chills, fever, 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 disease 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 persons 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 expectorated.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.
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Figure 3: Mode of transmission of tuberculosis
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: 1025 times greater than the general population. Persons with diabetes mellitus have a risk for developing active TB that is 24 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).1416 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.
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TABLE 2. Persons at increased risk of TB Persons at increased risk Risk Increased risk of exposure to infectious cases Examples of persons with risk 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. Foreign-born persons from countries with a high prevalence of TB. Homeless persons. Persons living or working in facilities providing long-term care. 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.
Increased risk of TB infection
Increased risk of active TB, once infection has occurred
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 bodys defenses. Those that survive are engulfed by white blood cells called macrophages. 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 (including drugs) that weaken the immune system. In people with a fully functioning immune
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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 summarized in Fig. 4.
Figure 4: Stages of infection of TB
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 extracellular domain containing eight carbohydrate-recognition domains characteristic of Ctype (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 incompletely 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
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differentiation 14 (CD14), a phosphatidylinositol glycanlinked 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 interleukin-8.25 Macrophage scavenger receptors bind polyanionic macromolecules and particles, 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 endowed 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 initial interaction with surface receptors influences the subsequent fate of M. tuberculosis within the macrophage. Interactions with the constant regions of immunoglobulin receptors (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. tuberculosis 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 access 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 maturation 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-, 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--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 bacteria 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 respiration 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
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Figure 5: The intracellular lifestyle of M. tuberculosis. CR, complement receptor; FcR, receptor for the constant fragment of immunoglobulin; MR, mannose receptor; SPR, surfactant.
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specialized cell typesthe professional phagocyteswhereas virtually all eukaryotic cells are capable of engulfing small particles and fluids that end up in the endosomal pathway. 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 maturation of which is arrested at an early stage. The early phagosome-harboring mycobacterium 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 VH+ 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- 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 inconclusivediagnosis 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, microbiological 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 (ESAT6). 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 optimum conditions may detect 110 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
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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 active 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 containing fewer and slow-growing bacilli. Although TB can be cured with chemotherapy, the treatment is exceedingly lengthy and takes 69 months. Apart from significant toxicity, 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) bacteria.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. Antitubercular 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 antibiotics, 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) sulphate 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. tuberculosis; 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 TB5153 are summarized in Table 3 and Table 4, respectively, and mechanisms of action of various first-line drugs are summarized in Fig. 6.
TABLE 3. Various first-line drugs used against TB, their structure, mechanism of action, activity, adverse drug reactions, and typical adult dosage Mechanism of action Activity Rash, nephritis, thrombocytopenia, cholestasis, flu-like syndrome with intermittent dosing Inhibits DNAdependent RNA polymerase, thereby blocking production of RNA
O OH NH N N N O OH
Drug (ROA) & structure
Adverse drug reactions
RIF (oral)
Typical adult dosage 600 mg/d
Reference 51, 52
HO
H O
OH OH
Bactericidal activity against susceptible bacteria and mycobacteria resistance rapidly emerges when used as a single drug in the treatment of active infection
INH (oral) Bactericidal activity against susceptible strains of M. tuberculosis
Hepatotoxic, peripheral neuropathy
300 mg/d
52, 53
Inhibits synthesis of mycolic acids, an essential component of mycobacterial cell walls
PZA (oral)
CONHNH2
Hepatotoxicity, hyperuricemia
25 mg/ kg/d
51, 53
O
C
NH2
Not fully understood PZA is converted to the active pyrazinoic acid under acidic conditions of macrophage lysosomes
Bacteriostatic activity against susceptible strains of M. tuberculosis may be bactericidal against actively dividing organisms
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Table 3 Continued Bacteriostatic activity against susceptible mycobacteria Retrobulbar neuritis 1525 mg/ kg/d 51, 52
EMB (oral)
C2H5 NH C H CH2OH
CH2OH
NH (CH2)2
C2H5
Inhibits mycobacterial arabinosyl transferases, which are involved in the polymerization reaction of arabinoglycan, an essential component of the mycobacterial cell wall Bactericidal activity against susceptible mycobacterium Nephrotoxicity, ototoxicity 15 mg/ kg/d
SM (IM injection)
OH OH
Prevents bacterial protein synthesis by binding to the S12 ribosomal subunit

H
52, 53
HO
O OH O O CH3 O O OH
H3C
NH
OH N H2N NH2
H2N
NH2HO
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TABLE 4. Various second line drugs used against TB, their structure, mechanism of action, activity, adverse drug reactions, and typical adult dosage Mechanism of action Activity Bacteriostatic 15 mg/kg/d Adverse drug reactions Typical adult dosage Reference 52, 53
Drug (ROA) & structure
ETH (oral)
H5C2
C NH2
inhibition of mycolic acid biosynthesis and consequent impairment of cellwall synthesis
GI disturbances; neurotoxicityvisual disturbances, olfactory disturbances, peripheral neuropathy, convulsions; hepatotoxicity; hypersensitivity reactions; alopecia; gynecomastia
NH2 O CAP (IM injection) H N N H H N H HN H2N N O HN NH2
HO O
H2N
N H O H N O NH
Inhibits peptide protein synthesis
Bactericidal
Paininduration at injection site; ototoxicity; nephrotoxicity; fever; rashes; eosinophilia
1530 mg/ kg/d
51, 52
H2N
O Cycloserine (oral) NH2

Inhibits cell wall synthesis (enol form is Dalanine analog)
Bacteriostatic
0.51 g/d Mostly neurologicalheadache, dizziness, vertigo, drowsiness, tremor, convulsions, depression, psychosis; abnormal liver function; megaloblastic anemia; rashes
51, 52
HN O
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Table 4 Continued Bacteriostatic 812 g/d 52, 53 Inhibits folate biosynthesis; interferes with incorporation of p-amino-benzoic acid GI disturbances; hypersensitivity reactions like rash, fever, malaise, arthralgia, leucopenia, agranulocytosis, eosinophilia, lymphocytosis, atypical mononucleosis, thrombocytopenia, hemolytic anemia
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Aminosalicylic Acid (PAS) (oral)
COOH OH
NH2
Inhibit protein synthesis
OH HO O NH2 NH2 HO O OH
KAN (IM injection)
Bactericidal
15 mg/kg/d
51, 53
OH
HO
Ototoxicity; nephrotoxicity; pseudomembranous colitis; neuromuscular blockade at high doses
H2N
OH
O H2N
AMI (IM injection)

O NH2 NH H OH
inhibit protein synthesis
Bactericidal
15 mg/kg/d
51, 52
NH2
Ototoxicity; nephrotoxicity; pseudomembranous colitis; neuromuscular blockade at high doses
OH
OH
OH O O OH OH NH2 OH
OH
H2N
Thiacetazone (oral)
Bacteriostatic
2.5 mg/kg/d GI disturbances; skin reactions including exfoliative dermatitis; hepatotoxicity; myelosuppression
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O S
H N NH2
Inhibits cyclopropanation of cell wall mycolic acids in mycobacteria
H3C
HN
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Figure 6: Mechanism of action of first-line drugs
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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 transmission, and avert the emergence of drug resistance. However, since the 1960s, there have been few developments in available therapies. Currently available agents are complicated 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 complicate or preclude the use of protease inhibitors and non-nucleoside reverse transcriptase inhibitors in patients infected with HIV. Furthermore, emerging drug resistance has complicated management for many patients and clinicians. Therefore, new chemotherapeutic 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 explored for pre-exposure and post-exposure use.54 Promising new antitubercular drug candidates with their class, mechanism of action, target, mechanism of resistance, development 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 mycobacterial strains consists of oral ingestion for a minimum of 69 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 conventional 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
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TABLE 5. Promising new antitubercular drug candidates with their class, mechanism of action, target, mechanism of resistance, development stage, and activity
Drug PA-824 Class Nitroimidazoleoxazine Mechanism of action Inhibition of protein and cell wall lipid synthesis, while anaerobic activity by generating reactive nitrogen species, including nitric oxide Inhibition of cell wall lipid synthesis, inhibition of mycolic acid biosynthesis Inhibition of ATP synthesis and membrane potential Inhibition of DNA synthesis Inhibition of DNA synthesis Inhibition of cell wall synthesis Unknown Target F420dependent nitroreductase Mechanism of resistance Rv0407, Rv3547, Rv3261, Rv3262 gene mutations Development stage Phase II Activity Bactericidal Reference 157, 158
OPC67683
Nitroimidazoleoxazine
a nitroreductase
Rv3547 gene mutations
Phase II
Bactericidal
159, 160
TMC207 (R207910)
Diarylquinoline
F1F0 proton ATP synthase DNA gyrase DNA gyrase Unknown Unknown Ribosomal inhibition
atpE gene mutation
Phase II
Bactericidal
161, 162
MFX
Fluoroquinolone Fluoroquinolone Diethylamine Pyrrole
gyrA gene mutation gyrA gene mutation Unknown
Phase III
Bactericidal Bactericidal Bactericidal Bactericidal Bactericidal
163, 164
GFX
Phase III
165, 166
SQ109
Phase I/II
167, 168
LL3858
Unknown
Phase I
169, 170
Linezolid
Oxazolidinone
Inhibition of protein synthesis
23SrRNA mutation
Phase I lead optimization
171, 172
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
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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, resistance to a single antitubercular drug is the most common pattern of drug resistance. Recognition of the relatively rapid onset of resistance to antitubercular monotherapy, usually 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, definitions 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 polydrug 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 resistance, to shorten the treatment course, and to reduce drug interactions with antiretroviral 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 bioavailability) 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.
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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 bioavailability, 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 frequency; 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
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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 significant 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 75140 m in adults. The large surface area of the alveoli 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 discovery 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 antimicrobial 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
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Figure 8: Schematic representation of carrier-based passive targeting to macrophages for the treatment of M. tuberculosis
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Figure 9: Schematic representation of receptor-mediated active targeting to macrophages for the treatment of M. tuberculosis
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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 watermiscible 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 sustained 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 68 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 therapeutic 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 antitubercular 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 nanoparticles 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 nebulization, 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
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In another study, Pandey et al. developed and evaluated the potential of orally administered PLGA nanoparticle-encapsulated ATDs (RIF + INH + PZA + EMB) for cerebral drug delivery in a murine model. A single oral dose of the formulation to mice could maintain sustained drug levels for 58 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 H37Rvinfected mice, five oral doses of the nanoparticle formulation administered every 10th day resulted in undetectable 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 exhibited a biphasic pattern characterized by an initial burst (11.26% in 1 day) release followed 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-nanoparticle-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 79 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. tuberculosis H37Rvinfected 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 chemotherapy, besides reducing the dosing frequency.83 Saraogi et al. developed and characterized a RIF-loaded gelatin nanoparticulate delivery 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 cytotoxicity 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 additionally 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--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 therapeutic dose to guinea pigs. A single oral administration of the formulation resulted in sustained drug levels in the plasma for 712 days and in the organs for 1114 days, with
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a significant improvement in MRT as well as in drug bioavailability. The administration of PLGA nanoparticles every 10 days (5 doses) to M. tuberculosis H37Rvinfected guinea 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 administration 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 911 days. Further, on oral administration of drug-loaded nanoparticles to M. tuberculosisinfected mice at every 10th day, no tubercle bacilli could be detected 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 1224 h. In M. tuberculosisinfected 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. Addition 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 concentrationtime curve (AUC0). The relative bioavailabilities of all drugs encapsulated in alginate nanoparticles were significantly 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
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Also Pandey et al. developed PLGA nanoparticles encapsulating three frontline ATDs, i.e., INH, RIF, and PZA, and administered subcutaneously to mice for pharmacokinetic/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 levels 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 H37Rvinfected mice, eight doses of the oral SM formulation (PLGA-NP-encapsulated SM) administered weekly were comparable to 24 intramuscular 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/aerosolized ATD carriers for treatment of TB. Upon administration of lectin-coated PLGANPs through the oral/aerosol route, the presence of drugs in plasma was observed for 67 days for RIF and 1314 days for INH and PZA. However, upon administration of uncoated PLGA-NPs (oral/aerosolized) RIF was detectable in plasma for 46 days, whereas INH and PZA were detectable for 89 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 bioavailability 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 polyisobutylcyanoacrylate (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 attention since the 1990s. SLNs are typically particulate systems with mean diameters
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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 lecithin, phosphatidylcholine, poloxamer 188, sodium cholate, and sodium glycocholate. The typical methods of preparing SLNs include spray drying,93 high-shear mixing, ultrasonication,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 lymphatic 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 radiolabeled 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 potential 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 12 days. In M. tubercuIosis H37Rvinfected mice, no tubercle 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 alveolar 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 targeted 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 potential 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 12 days. In the case of nebulized drug-
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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 bioavailability was increased by 10- to 16-fold, whereas the absolute bioavailability was increased 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 obtain 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 summarized in Table 6.
TABLE 6. Various nanoparticle formulations developed against TB
Carrier system Polymeric nanoparticle Drugs encapsulated RIF, INH, PZA RIF, INH, PZA RIF, INH, PZA RIF, INH, PZA EMB RIF RIF, INH, PZA EMB RIF RIF, INH, PZA RIF, INH, PZA ECZ, MFX INH, RIF, PZA, EMB RIF, INH, PZA Properties PLGA nanoparticle having size 186290 nm and entrapment efficiency 56.9% 2.7% for RIF, 66.3% 5.8% for INH, and 68% 5.6% for PZA Alginate nanoparticles having size 235.5 0 nm and entrapment efficiency 80%90% for RIF and 70%90% for INH and PZA PLGA nanoparticles having size 186290 nm and entrapment efficiency 60%70% for all three drugs PLGA nanoparticles having size 186290 nm and entrapment efficiency 55.91% 2.70% for RIF, 67.34% 3.8% for INH, 68.32% 5.50% for PZA, and 43.11% 4.21% for EMB PLGA nanoparticles having size 380 nm and entrapment efficiency 75.8% 2.16% PLGA nanoparticles having size 186290 nm and entrapment efficiency 55.912.70% for RIF, 67.343.8% for INH, 68.325.50% for PZA and 43.11 4.21% for EMB Gelatin nanoparticles having size 26411.2 nm and entrapment efficiency 59.50.82 % PLGA nanoparticle having size 186290 nm and entrapment efficiency 58.99% 2.72% for RIF, 68.02% 5.58% for PZA, 66.31% 5.83% for INH PLGA nanoparticles having size 186290 nm and entrapment efficiency 56.99% 2.7% for RIF, 66.31% 5.83% for INH, and 68.02% 5.58% for PZA PLGA nanoparticles having 217 nm and entrapment efficiency 52.27% 3.80% for ECZ and 33.69% 3.88% for MOX Alginate nanoparticles having size 235.5 0.0 nm and entrapment efficiency 70%90% for INH and PZA, 80%90% for RIF, and 88%95% for EMB PLGA nanoparticles having entrapment efficiency 56.9% 2.7% for RIF, 66.3% 5.8% for INH, and 68.0% 5.6% for PZA Diseased model used M. tuberculosis infected guinea pigs M. tuberculosis infected guinea pigs M. tuberculosis infected guinea pigs M. tuberculosis infected mice Reference 78 79 80 81
82 M. tuberculosis H37Rv infected mice M. tuberculosis H37Rv infected mice M. tuberculosis infected guinea pigs M. tuberculosis H37Rv infected mice M. tuberculosis H37Rv infected mice M. tuberculosis H37Rv infected mice M. tuberculosis H37Rv infected mice 83
84 85 86
87 79 88
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TABLE 6 Continued
SM RIF, INH, PZA CPFX Solid lipid nanoparticle RIF, INH, PZA RBU PLGA nanoparticles having size 153.12 nm and entrapment efficiency 32.12% 4.08% PLGA nanoparticles having size 350400 nm and entrapment efficiency 54%66% PIBCA nanoparticles led to increased AUC, t1/2, and Vd, and to a decreased Cl as compared with drug in solution SLNs having entrapment efficiency 51% 5% for RIF, 45% 4% for INH, and 41% 4% for PZA Mannosylated SLNs having size 389 2.3 nm for M-SLN-R and 251 5.1 nm and entrapment efficiency of M-SLN-R and U-SLN-R 82.6% 1.2% and 87.8% 1.2%, respectively SLNs having entrapment efficiency 51% 5% for RIF, 45% 4% for INH, and 41% 4% for PZA M. tuberculosis H37Rv infected mice M. tuberculosis H37Rv infected guinea pigs. Male albinos rabbits M. ubercuIosis H37Rv infected mice macrophage cell lines (J774) and albino rats M. tuberculosis H37Rv infected guinea pigs 89 90 91 99 100
RIF, INH, PZA
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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 target 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 biodisposition, 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 experiments 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 concentrations of INH obtained at all times were much higher than the MIC of INH. Controls injected 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 administered subcutaneously exhibited a sustained release of INH and RIF in vivo up to 7 and 6
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weeks, respectively. Free drugs (in combination) injected in the same doses were detectable in vivo up to 24 h only. One dose of PLGA microparticles cleared bacteria more effectively 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 H37Rvinfected 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 reduced 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 cationstetrapentylammonium (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 drugloaded 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 solution by gavage or intra-tracheal instillation to Sprague-Dawley rats. As expected, only microparticles provided sustained and targeted delivery of INH to AMs. Most importantly, this method of delivery led to substantial reduction in the blood levels of acetylisoniazid (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 various 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 dosingfrequency reduction, toxicity alleviation, and targeting macrophage-resident persistent mycobacteria.109 Muttil et al. developed microparticles containing large payloads
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of two ATDs (INH and RBU) and evaluated them for suitability as a dry powder inhalation 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 inhaled than when drug solutions were administered. Microparticles were thus deemed suitable for enhanced targeted drug delivery to AMs.110 Various microparticulate formulations are summarized in Table 7. VII.C. Liposomes Liposomes are concentric bilayer vesicles in which an aqueous volume is entirely enclosed 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 distribution to other organs. Advantages of liposomes include: relatively low toxicity; prepared in wide size range (20 nm1 mm); ability to solublize poorly water-soluble drugs, facilitating 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, specifically 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, instillation, insufflations, etc.102 In a study, Vyas et al. prepared, characterized, and evaluated RIF-loaded aerosolized 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 1.51.8 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 accumulation 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 concentration 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
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TABLE 7. Various microparticle formulations developed against TB Drugs encapsulated Properties PLGA microparticles having size 62.11, 71.95, and 11.75 m for porous, non-porous, and hardened PLGA microparticles, respectively, and entrapment efficiency of INH in the various formulations of PLGA microparticles was 16%, 11%, and 10%11% in porous, non-porous, and hardened PLGA microparticles, respectively. Dose given75 mg/kg body wt. PLGA microparticles having size of 1.11, 1.4, and 2.20 m for INH, RIF, & PZA microparticles, respectively. The drug encapsulation efficiency of the microparticles was found to be 810% for PZA; 1011% for INH, and 1218% for RIF. Hardened PLGA microparticles having size 11.75 and 11.64 m for INH- and RIF-containing microparticles, respectively, and entrapment efficiency 10%11 % for INH and 12%14 % for RIF. Single dose R-PLGA (12 mg/kg) insufflation significantly reduced the lung burden of bacteria (log cfu/mL = 3.7 0.3) compared with RIF (log cfu/mL = 4.17 0.1), PLGA (log cfu/mL = 4.4 0.32) or the lactose group (log cfu/ mL = 4.33 0.16). Double dose R-PLGA (12 mg/kg by insufflation followed by 5 mg/kg by nebulization) treatment significantly reduced the lung and spleen bacterial burden compared with an equivalent dose of RIF or PLGA, or untreated groups, respectively. PLA microparticles between 1 and 3 m in diameter. The charged prodrug was ion-paired with two different hydrophobic cations (TPA- and THA-bromide), and loaded separately into the PLA microparticles. Mice (Laca strain) Mice (Laca strain) INH Animal model/Infected model used Reference 103
Carrier system
Microparticles
INH, RIF, PZA
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INH, RIF
M. tuberculosis H37Rv infected mice
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RIF
M. tuberculosis (H37Rv)infected guinea pig
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INHMS
NR8383, a rat AM cell line
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Table 7 Continued Drugs encapsulated Properties PLGA microparticles having size varied from 1.11 to 2.20 m for INH, RIF, and PZA, and entrapment efficiency 8% to 10% for INH, 20% to 30% for RIF, and 9% to 13% for PZA. Dose given: INH, 10 mg/kg of body weight; RIF, 12 mg/kg; and PZA, 25 mg/kg, orally. PLA microparticles having size 0.53 micron. INH, RIF, and PZA Animal model/Infected model used M. tuberculosis (H37Rv)infected mice (laca strain) Reference 108
Carrier system
INH, RIF
Wistar rats, murine macrophage line J774 A Female Swiss mice
109
INH and RBU
PLA microparticles having size 5m and entrapment efficiency 50%.
110
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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 continuous 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 liposomes 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 administration. 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 rigidity 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 coinfected 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 efficiency using the twin impinger exhibited 25%27% INH deposition in the alveolar chamber upon 1 minute nebulization using a jet nebulizer. At 37C, 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
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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 detected, 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 antimicrobial 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 fluoroquinolone yielded MICs that were at least two-fold lower than those obtained with the free drug. In particular, liposomes made up of dimyristoylphosphatidylcholine-cholesterol-dipalmitoylphosphatidylserine and dimyristoylphosphatidylcholine-cholesteroldihexadecylphosphate (4:3:4 molar ratio) provided the best improvement in antimicrobial activity against the various bacterial strains investigated. The liposome formulation produced higher intracellular fluoroquinolone concentrations than those achieved simultaneously 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 drugrelease 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 according 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 liposomal drugs were present in the lungs (RIF: 0.25 0 g/mL; INH: 0.29 0.03g/mL homogenate) as well as in the AMs (RIF: 0.15 0.02 g/105 cells; INH: 0.1 0.03g/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),
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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 liposomes 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 m to 1000 m (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 particulate 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 systemic 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 ATDsRIF, INH, and PZAwere formulated. A therapeutic dose and a half-therapeutic 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 H37Rvinfected 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 H37Rvinfected monocyte cell lines with RIF-loaded microspheres
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TABLE 8. Various liposomes formulations developed against TB

Carrier system Liposomes Drugs encapsulated RIF Properties MBSA-coated liposomes having size 3.64 0.65 m and entrapment efficiency 47.4% 2.7%, while O-SAP-coated vesicles 3.85 0.59 m in size and entrapment efficiency of 47.4% 2.7% Liposomes having size 25 and 65 nm and entrapment efficiency 28% to 32% for RIF. Disease model used Albino rats Reference 113
RIF
M. tuberculosis (H37Rv)infected Swiss albino mice BALB/c mice HimediaVR LA 387 dialysis membrane-50, India M. tuberculosis (H37Rv)infected mice American Type Culture Collection standards and wildtype bacterial strains albino rats
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RBU INH
RBU liposomes having size 0.1 m and entrapment efficiency 72% 5%. DPPC liposomes having size 750 nm and entrapment efficiency 36.7% 1.8%.
115 116
INH, RIF
Liposomes having entrapment efficiency 8% to 10% for INH and 44% to 49% for RIF. OFX-loaded unilamellar vesicles having size 200 nm and entrapment efficiency of liposomes made up of dimyristoylphosphatidylcholinecholesterol-dipalmitoylphosphatidylserine and dimyristoylphosphatidylcholine-cholesterol-dihexadecylphosphate 21.0 3.7% and 15.7% 1.9% respectively. 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) was 3.165 0.002 m; 2.895 0.001 m; 2.8075 0.001 m; 2.7452 0.005 m and entrapment efficiency (RIF and INH) was 68.25% and 57.85%; 69.23% and 59.36%; 70.92% and 61.11%; 72.98% and 63.22%, respectively. Liposomes having entrapment efficiency 40%45% for RIF and 8%12% for INH, dose given RIF 46.5 and INH 23.25 mg/kg body weight. Entrapment efficiency of liposomes made up of dipalmitoyl phosphatidyl choline (7):cholesterol (2) neutral and dipalmitoyl phosphatidyl choline (7):cholesterol (2):dicetyl phosphate (1) negatively charged was found to be 8.833% 2.4% and 7.883% 1.9% respectively. EMB liposomes having size 125145 nm and entrapment efficiency 76%92%.
117
OFX
118
RIF, INH
119
RIF and INH
DunkinHartley guinea pigs Mice infected by M. tuberculosis
120
PZA
121
EMB
M. bovis BCG
122
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resulted in a significant decrease in numbers of cfu at 7 days following initial infection, 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 formulated 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. tuberculosisinfected macrophages and mice. Formulations were evaluated individually and in combination with oral regimens of INH for the treatment of M. tuberculosis H37Rvinfected 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 intraperitoneal injections of RIF-loaded microspheres on days 0 and 7, (iii) a combination 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 26day experimental period. Susceptibility testing demonstrated continued sensitivity to RIF during the treatment period. Whereas INH alone significantly reduced the numbers 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 microspheres (RPLGA) delivered to guinea pigs infected with M. tuberculosis (H37Rv), which was compared with a daily dose of nebulized RIF suspension. Drug and polymertreated 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 microspheretreated animals than for all other groups. In multipledose 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 Hasegawa 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 37C with various numbers of PSL MS per macrophage cell (MS/macrophage = 0.110). The results were then compared with those of
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the phagocytosis towards RIF-PLGA MS. Hasegawa et al. determined the phagocytic activity by counting the population of macrophage cells that had phagocytosed microspheres (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-m ones were taken up by macrophage the most, and the RIF-PLGA MS 3 m 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-m 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 = 45 h).129 Likewise, Suarez et al. developed respirable PLGA microspheres containing RIF for the treatment of TB. RIF alone (RIF, 1.031.72 mg/ kg), within PLGA microspheres (R-PLGA, equivalent to 1.031.72 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 distribution of the RIF PLA microspheres of 515 m, 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 g/g.131 Various microsphere-based formulations are summarized in Table 9.
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TABLE 9. Various microsphere formulations developed against TB

Carrier system Microspheres Drugs encapsulated RIF, INH, PZA Properties Alginate-chitosan microspheres having size of 70 4 m and entrapment efficiency 83% 2% for RIF, 65% 6% for INH and 69% 6% for PZA. Dose given RIF 12 mg/ kg + INH 10 mg/kg + PZA 25 mg/kg body weightorally. Microspheres having size 34 m. Microspheres having size 3.98 2.74 m for RIF and 4.2 3.2 m for INH. RIFloaded microspheres were injected intraperitoneally, and INH was given orally. Microspheres having size 2.83 m. RIF-PLGA particles having size 1.50 0.28, 3.38 0.45, 6.31 0.67, 10.28 0.78 m. Microspheres having size 53.02 31.44 mm and entrapment efficiency 70.42% 14.11%. Dose given, RIF (1.031.72 mg/kg), within poly(lactideco-glycolide) microspheres (R-PLGA, equivalent to 1.031.72 mg/kg) or polymer microparticles alone (PLGA) by insufflation or nebulization, 24 h before bacterial aerosol exposure. Microspheres having size 7.90 4.07 m. Disease model used M. tuberculosis H37Rvinfected Dunkin-Hartley guinea pigs Reference 124
RIF
RIF, INH
Murine J774 and the human Mono Mac 6 cell lines M. tuberculosis H37Rvinfected mice
125
126
RIF
RIF RIF
Guinea pigs infected with M. tuberculosis (H37Rv) Cell line NR8383 White rabbits (33.5 kg) Dunkin-Hartley guinea pig
127
128 129
RIF
130
RIF
rabbits
131
VII.e. Niosomes Non-ionic surfactant-based vesicles (niosomes) are the structures formed from the selfassembly 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 energy, such as physical agitation or heat. The result is an assembly in which the hydrophobic parts of the molecule are shielded from the aqueous solvent and the hydrophilic head
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groups enjoy maximum contact with same. These structures are analogous to phospholipid 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 surfactants 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 niosomes found that 30% of the drug was recovered from the lungs after 24 h, whereas 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 using 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-85based niosomes. The maximum lymph concentration achieved was 46.2% of the administered 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 human 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 simulation 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, toxicity, stability, glass transition temperature, polyelectrolyte character; and possibility of encapsulating guest molecules. Considering the use of dendrimers for drug delivery, it is
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TABLE 10. Various niosomes formulations developed against TB Carrier system Niosomes Drugs encapsulated RIF RIF Properties Niosomes having size 815 m. Niosomes having size containing RIF 1.8 m (span 20), 1.8 m (span 40), 1.7 m (span 60), 1.8 m (span 80), 1.9 m (span 85) and entrapment efficiency 20.6% (span 20), 25.3% (span 40), 28.5% (span 60), 31.7% (span 80), 35.4% (span 85). Niosomes having size 8.44 0.87, 7.26 0.33, 4.21 0.14, 8.79 0.73, 7.08 0.33, 4.54 0.24 m and entrapment efficieny 54.39% 3.13%, 65.18% 2.8%, 73.42% 2.01%, 58.52% 1.01%, 71.24% 1.28%, 75.65% 2.28% for formulations F1, F2, F3, F4, F5, F6 respectively. Disease model used albino rats Wistar rats Reference 133 134
CPFX
Human lung carcinoma cell line (A549)
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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 architecture for the delivery of an ATD, RIF. In vitro release studies among the four formulations 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 dendrimer (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.
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TABLE 11. Various dendrimer formulations developed against TB Carrier system Dendrimer Drugs encapsulated RIF Properties Among the four formulations PEGylated dendrimers showed relatively slower release of RIF when compared with non-PEGylated dendrimers. Mannosylated dendrimer having size <5 mm and entrapment efficiency 37.34% 1.32%. Reference 143
RIF
144
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 patent 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, performance 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 disposition 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/lecithin suspensions orally as positive controls, and those receiving placebo particles or no
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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 aerosolization 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 enhanced uptake of nanoemulsions by cells of the phagocytic system reported elsewhere gives these nanocarriers passive targeting features. Also, they are taken up by lipoprotein receptors in the liver after oral administration.152,153 Ahmed and co-workers developed different o/w nanoemulsions of RIF for IV administration 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 4C and 25C.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 improve 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, especially 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-
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ters and collaborators developed a clofazimine nanocrystalline suspension in order to overcome the toxicity and the low solubility (0.3 g/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 drugloaded 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 nanosuspension 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 directly observed treatment more realistic and affordable. Another important advantage of the nanoparticles as compared to other colloidal carriers is the possibility of the versatile routes of drug administration, including oral, inhalation, and intravascular routes. Moreover, the potential advantages of direct delivery of the nanoparticulate antitubercular 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 nanaparticles 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 constituents 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 colloidal carriers are largely unsuccessful in drug targeting against TB due to their difficulties in gaining access to targeted tissues, penetrating vascular barriers, and evading phagocytic capture by the reticuloendothelial system, they are able to modify the distribution of an associated drug substance, which can overcome the drug resistance. A
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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 antitubercular drugs should be promoted to increase competition and remedy the vulnerable situation 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 nanoparticles, solid lipid nanoparticles, microparticles, liposomes, microspheres, niosomes, dendrimers, microemulsions, dry powder, nanoemulsions, and nanosuspensions are able to overcome drug-resistance issues and facilitate antitubercular drug delivery to M. tuberculosis 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 carriers 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
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Critical Reviews in Therapeutic Drug Carrier Systems, 29(4), 299-353 (2012)

Colloidal Carriers: A Rising Tool for Therapy of Tuberculosis

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

Figure 1: Outcomes associated with exposure to M. tuberculosis

Critical Reviews in Therapeutic Drug Carrier Systems

Colloidal Carriers: A Rising Tool for Therapy of Tuberculosis

Volume 29, Number 4, 2012

Critical Reviews in Therapeutic Drug Carrier Systems

Figure 2: Main features of tuberculosis: from infection to host defense

Colloidal Carriers: A Rising Tool for Therapy of Tuberculosis

Volume 29, Number 4, 2012

Figure 3: Mode of transmission of tuberculosis

Critical Reviews in Therapeutic Drug Carrier Systems

Colloidal Carriers: A Rising Tool for Therapy of Tuberculosis

Increased risk of TB infection

Increased risk of active TB, once infection has occurred

Volume 29, Number 4, 2012

Figure 4: Stages of infection of TB

Critical Reviews in Therapeutic Drug Carrier Systems

Colloidal Carriers: A Rising Tool for Therapy of Tuberculosis

Volume 29, Number 4, 2012

308 S. Gupta et al.

Critical Reviews in Therapeutic Drug Carrier Systems

Colloidal Carriers: A Rising Tool for Therapy of Tuberculosis

Volume 29, Number 4, 2012

Critical Reviews in Therapeutic Drug Carrier Systems

Drug (ROA) & structure

Adverse drug reactions

Volume 29, Number 4, 2012

Typical adult dosage 600 mg/d

Colloidal Carriers: A Rising Tool for Therapy of Tuberculosis

INH (oral) Bactericidal activity against susceptible strains of M. tuberculosis

Hepatotoxic, peripheral neuropathy

Inhibits synthesis of mycolic acids, an essential component of mycobacterial cell walls

Prevents bacterial protein synthesis by binding to the S12 ribosomal subunit

Critical Reviews in Therapeutic Drug Carrier Systems

Drug (ROA) & structure

Volume 29, Number 4, 2012

inhibition of mycolic acid biosynthesis and consequent impairment of cellwall synthesis

NH2 O CAP (IM injection) H N N H H N H HN H2N N O HN NH2

Colloidal Carriers: A Rising Tool for Therapy of Tuberculosis

Inhibits peptide protein synthesis

Paininduration at injection site; ototoxicity; nephrotoxicity; fever; rashes; eosinophilia

1530 mg/ kg/d

O Cycloserine (oral) NH2

Aminosalicylic Acid (PAS) (oral)

KAN (IM injection)

Ototoxicity; nephrotoxicity; pseudomembranous colitis; neuromuscular blockade at high doses

AMI (IM injection)

inhibit protein synthesis

Ototoxicity; nephrotoxicity; pseudomembranous colitis; neuromuscular blockade at high doses

Critical Reviews in Therapeutic Drug Carrier Systems

Inhibits cyclopropanation of cell wall mycolic acids in mycobacteria

Colloidal Carriers: A Rising Tool for Therapy of Tuberculosis

Volume 29, Number 4, 2012

Figure 6: Mechanism of action of first-line drugs

Critical Reviews in Therapeutic Drug Carrier Systems

Colloidal Carriers: A Rising Tool for Therapy of Tuberculosis

Rv3547 gene mutations

atpE gene mutation

Fluoroquinolone Fluoroquinolone Diethylamine Pyrrole

gyrA gene mutation gyrA gene mutation Unknown

Bactericidal Bactericidal Bactericidal Bactericidal Bactericidal