Advances in nanotechnology-based carrier systems for targeted delivery of bioactive drug molecules with special emphasis on immunotherapy in drug resistant tuberculosis – a critical review

Figures & data

Figure 1. Pathogenesis of tuberculosis.

Table 1. Classification of anti-TB drugs and their mechanism of action.

Drug	Mechanism of action	Adverse or side effects	Role	BCS class
Isoniazid (INH) (1951)	It inhibits the synthesis of mycolic acid (an essential cell wall component) thus interferes with cell wall synthesis in mycobacterium.	Hepatitis, burning sensation in extremities, drug interaction, fatigue, fever etc. (Sensi, 1983; Kochi et al., 1993)	As a pro-drug conversion and as a drug target product (Zhang & Yew, 2009)	BCS-III class (Hari et al., 2010)
Rifampicin (RIF) (1966)	Inhibits the synthesis of bacterial RNA polymerase thus inhibits the nucleic acid synthesis.	Stomach upset, flu-like symptoms, bleeding (Sensi, 1983). Rashes and other hypersensitivity reactions, hepatitis-1 may also appear (Wade & Zhang, 2004).	As a drug target (Zhang & Yew, 2009)	BCS-II class (Hari et al., 2010)
Ethambutol (ETB) (1961)	Inhibits bacterium arabinogalactan synthesis and finally cell wall synthesis get inhibited.	Chances of visual disturbances. (Mikusová et al., 1995)	As a drug target (Zhang & Yew, 2009)	BCS-III class. (Hari et al., 2010)
Pyrazinamide (PYZ) (1952)	Depletes and inhibit the membrane energy.	Hepatitis, joint aches rashes and other hypersensitivity reactions. (Zhang et al., 2003)	As a drug target and as a pro-drug conversion (Zhang & Yew, 2009)	BCS-III class. (Hari et al., 2010)
Aminoglycosides Streptomycin (1944), kanamycin (1957), capreomycin (1960), amikamycin (1957)	Inhibition of protein synthesis irreversibly	Nephrotoxicity, ototoxicity, electrolyte abnormalities like hypokalemia etc. (Allen et al., 1983)	As a drug target (Zhang & Yew, 2009)	Amikacin-III BCS class. Kanamycin-Na (Hari et al., 2010)
Ethionamide (1956)	Inhibition of mycolic acid synthesis	Gastrointestinal upset, hepatotoxicity, neurotoxicity and anorexia etc. (Morlock et al., 2003)	As a pro drug conversion and drug target (Zhang & Yew, 2009)	BCS-II class. (Hari et al., 2010)
Amino salicylic acid (1946)	Inhibits the incorporation of PABA into folic acid and iron metabolism.	Gastrointestinal distress, hypothyroidism and other hypersensitivity reactions (Wade & Zhang, 2004).	Drug activation (Zhang & Yew, 2009)	Na (Hari et al., 2010)
Cycloserine	Inhibits the incorporation of d-alanine into peptidoglycan	CNS toxicity, Stevens–Johnson syndrome etc. (Caceres et al., 1997)	Drug target (Zhang & Yew, 2009)	BCS-IV/II class (Hari et al., 2010)

Figure 2. Problems with conventional drug delivery systems.

Figure 3. Advantages of nanotechnology-based drug delivery systems.

Figure 4. Classification of nanotechnology-based drug delivery systems.

Table 2. Examples of solid-lipid micro and nanoparticles in tuberculosis treatment.

Polymer used	Anti-TB drug used	Results obtained	References
Stearic acid and sodium taurocholate	Rifampicin	Good release kinetics was found and good flow properties of dry powder of rifampicin SLNs.	Maretti et al. (2014)
Compritol ATO 888, Tween 80	Rifampicin	The percentage degradation in combination of rifampicin solid-lipid nanoparticles and isoniazid solid-lipid nanoparticles was found decreased up to 12.35%	Singh et al. (2013)
Soya lecithin, compritol ATO 888, stearic acid, Tween 80	Isoniazid	In this study, the prepared SLNs give high entrapment efficiency (68%) and small size (48 nm) for isoniazid loaded SLNs. The marked improvement in bioavailability in both in plasma and brain was observed after giving isoniazid loaded SLNs and free/plain drug solution at the same dose.	Bhandari & Kaur (2013)
Cetyl palmitate, Tween 80, poloxamers 188	Rifampin	Rifampicin loaded SLNs were found spherical in shape with diameter up to 100 nm, low and negative zeta potential and encapsulation efficiency up to 82% and the anti-mycobacterial activity was found improved against Mycobacterium fortuitum. MIC of drug loaded SLNs were found to be eight times fewer than free rifampin solution administered.	Aboutaleb et al. (2012)
Phospholipon R 80 H, tristearin	Isoniazid	The prepared SLNs were of spherical in shape with diameter of 164 nm. Further the use of polysorbate80 and sonication time was found to be effective for entrapment efficiency and for reduction in size of particles.	Nair et al. (2011)
Mannose	Rifabutin	In this particular study, the cellular uptake studies was done which shows due to mannose coating, SLNs formulations for alveolar macrophages are better suited when compared to un-coated formulation (without mannose).	Nimje et al. (2009)

Table 3. Examples of anti-tubercular drug loaded emulsion systems.

Polymer used	Anti-TB drug used	Results obtained	References
Brij 96, nile red, ruthenium dichloride	Rifampicin, Isoniazid, Pyrazinamide	The micro emulsion has been tested using different dye solutions. It was found that rifampicin has strong interaction with Nile red dye and pyrazinamide/isoniazid has interaction with ruthenium dichloride.	Kaur & Mehta (2014).
Oleic acid, ethanol, Tween 80	Rifampicin	An optically clear/stable microemulsion was obtained without phase separation. The phase changing between w/o microemulsion to o/w microemulsion and controlled/sustained release of the drug was achieved.	Mehta et al. (2007)
Oleic acid, ethanol, Tween 80	Rifampicin	In this method, the microemulsion-based system composed of oleic acid, Tween 80, ethanol and phosphate buffer was developed. A stable microemulsion was found after incorporating rifampicin in terms phase separation. The microemulsion get changes into o/w emulsion at infinite dilution conditions as depicted by particle size.	Mehta et al. (2007)
Sefsol, Tween 80	Rifampicin	In this study, different oil-in-water (o/w) nanoemulsions were prepared. The optimized formulation was also subjected further to stability studies as per the ICH guidelines. The formulation was found stable more than period of 19 months.	Ahmed et al. (2008)
Oleic acid, ethanol, Tween 80	Isoniazid	In this study, the microemulsion was found stable after the incorporation of isoniazid in terms of optical texture, phase separation and pH, as far as dissolution studies are concerns the sustained/controlled release of drug from o/w emulsion droplet was found.	Mehta et al. (2008)
Oleic acid, ethanol, Tween 80	Rifampicin, Isoniazid, Pyrazinamide	In this study, the degradation of rifampicin in the presence of isoniazid was seen. The results depicted that anti-tubercular drugs in single and mixed drug formulations was followed non-Fickian release behavior except for rifampicin in pH 7.4 release medium.	Mehta et al. (2010)

Table 4. Examples of liposomal-based preparations for tuberculosis.

Lipid used	Anti-TB drug used	Results obtained	References
Di-palmitoyl phosphatidyl choline (DPPC)	Isoniazid, Rifampicin, Pyrazinamide, Ethambutol	In this study, the liposomal formulation shows high entrapment efficiency, effective sustained/prolonged drug release, improved recovery/uptake of drugs from affected/target site.	Bhardwaj et al. (2013).
Soybean phosphatidylcholine, porous mannitol, cholesterol	Pyrazinamide	Pyrazinamide loaded pro-liposomes were developed and 4.26–4.39 µm aerodynamic diameter and up to 45% encapsulation efficiency was observed.	Rojanarat et al. (2012)
Di-palmitoyl phosphatidyl choline (DPPC)	Isoniazid	Controlled/sustained release of isoniazid from liposomes was observed over period of 1 day.	Chimote & Banerjee (2010)
DNA-hsp65 vaccine	DNA-hsp65 vaccine	The study shows that vaccine can be entrapped or complexed within liposomal system and can be used to immunize the animal (mice) by intra-nasal or intra-muscular routes.	Rosada et al. (2008).
Dipalmitoyl phosphatidyl choline, Dipalmitoyl phosphatidyl glycerol	Rifabutin	Significant decline in CFUs count was measured/observed in organs like lungs, liver etc. when compare to free/plain rifabutin in infected mice.	Gaspar et al. (2008)
Di-palmitoyl phosphatidyl choline, cholesterol	Pyrazinamide	High therapeutic efficacy was observed in infected mice when pyrazinamide loaded liposomes were injected twice weekly.	El-Ridy et al. (2007)
Phosphatidyl choline, cholesterol, dicetyl phosphate	Isoniazid, Rifampicin	The loading capacity of rifampicin was found better as compared to the loading of isoniazid in liposomal system.	Pandey et al. (2004)
Di-palmitoyl phosphatidylcholine (DPPC), hydrogenated phosphatidylcholine (HPC) and distearoyl phosphatidyl choline (DSPC)	Capreomycin	Capreomycin loaded liposomes were found suitable for inhalable formulations as with diameter of 200 nm or less. The liposome was found to provide the high capreomycin sulfate content.	Ricci et al. (2006)
Di-stearoyl phosphatidyl choline and cholesterol	Kanamycin	In this study, the encapsulation efficiency up to 63% was obtained for kanamycin. The lipidic vesicles were found with mean diameter of at least 132 nm.	Justo & Moraes (2005)
Di-stearoyl phosphatidyl choline and cholesterol	Isoniazid, Rifampicin, Ethionamide, Pyrazinamide, Streptomycin	In this study, mean vesicle diameters were found varied from 286 to 329 nm. No drug leakage or mean vesicle diameter changes were observed for 3 weeks.	Justo & Moraes (2003)
Phosphatidyl choline, cholesterol, dicetyl phosphate	Isoniazid, Rifampicin	In this study, the liposomal formulation encapsulating rifampicin and isoniazid for at least 6 weeks was found to reduce the mycobacterial load in lungs and other organs of infected mice when compared with untreated animals after administered once in a week.	Labana et al. (2002)
Phosphatidyl choline and phosphatidyl serine	Rifabutin	In this study, the rifabutin was incorporated in liposomal system which results in significant enhancement of its anti-tubercular activity against M. avium infection when compared to rifabutin in free form or plain solution.	Gaspar et al. (2000)
Bristol	Amikacin	In this particular study, the liposomal entrapment of amikacin was found high. The minimum inhibitory concentration (MICs) for M. avium was found for at least 28 days.	Leitzke et al. (1998)
Egg phosphatidyl choline, cholesterol, dicetyl phosphate, O-SAP and monosialogangliosides, di-stearyl phosphatidyl ethanolamine-poly(ethylene glycol)	Isoniazid, Rifampin	In this study, biodistribution studies shows that liposomes containing O-SAP were 15% accumulated in lungs when compared to conventional liposome which was 5.1% accumulated in lungs preparation after 30 min of injection.	Deol & Khuller (1997)
Phosphatidyl glycerol, phosphatidyl choline, cholesterol	Sparfloxacin	In this method, treatment with free or sparfloxacin loaded liposome for 24 h was studied in animals, which shows reduction in the growth index up to 25% for untreated group and 30% for control groups. Free sparfloxacin reduced the growth index up to 6% of the untreated control group, while liposome-encapsulated sparfloxacin was found to reduce it up to 8% to that of the control group when cultures were treated for 4 days.	Düzgüneş et al. (1996)
Poly ethylene glycol, distearoyl phosphatidyl ethanolamine, distearoyl phosphatidyl choline, cholesterol, phosphatidylinositol, phosphatidyl glycerol, phosphatidyl choline	Streptomycin	In this study, liposomal system was prepared which encapsulates streptomycin. They were found to reduce the level of MAC (Mycobacterium avium complex) infection in high level when compared to MAC infection in un-treated (Control groups) and they were also found bacteriostatic in lungs.	Gangadharam et al. (1995)

Table 5. Examples of niosomes preparations in tuberculosis.

Polymers used	Anti-TB Drug used	Results obtained	References
Cholesterol, dicetyl phosphate, stearyl amine	Ethambutol	The drug-loading efficiency of ethambutol was found increased from 12.20% to 25.81% and the zeta potential was found up to neutral values. Drug targeting towards lungs was also got increased by niosomal system.	El-Ridy et al. (2015)
Sorbitan monostearate, cholesterol, dicetyl phosphate diethyl ether	Isoniazid	In this study, drug entrapment efficiency gets increased up to an optimum level by enhancing the cholesterol content and there after decrease in encapsulation efficiency was found. Study also proves that isoniazid loaded niosomes are capable of providing reduced drug dose as well as dose frequency. The reduced toxicity was observed when the formulation was given through parenteral route or via any other suitable routes	Singh et al. (2011)
Cholesterol, dicetyl phosphate, stearyl amine, span 60 and span 85	Pyrazinamide	In this study, the highest percentage of pyrazinamide-loading efficiency was found using surfactant span-60.	El-Ridy et al. (2011)
Triton X 100, polyethylene glycol 2000, span 80	Rifampicin, Isoniazid	In this method, the prepared niosomal particles was found in range of 200–300 nm size and high drug entrapment efficacy was found for rifampicin and isoniazid.	Mehta et al. (2011)
Cholesterol, triton-X-100, diethyl ether, span-80 and span-85	Rifampicin	This study depicts that effective targeting of the rifampicin mainly happens in the lymphatic regions. Rifampicin was entrapped in niosomes successfully and was found useful for treatment/management of disease conditions.	Jain et al. (2006)

Table 6. Dendrimer-based anti-tuberculosis preparations.

Polymer used	Anti-TB drug used	Results obtained	References
Poly ethylene glycol, poly propyleneimine	Rifampicin	In this study, it was found that dendrimer-based system helps to increase the drug encapsulation capacity and reduce the drug release rate etc. The systems were found more suitable for sustained/controlled delivery of rifampicin in tuberculosis treatment.	Vijayaraj Kumar et al. (2007)
Poly ethylene glycol, poly propyleneimine	Rifampicin	In this study, rifampicin loaded dendrimers were found to reduce release rate of drug in 7.4 pH. The cytotoxicity/hemolytic toxicity were also found reduced. The dendrimer-based system was found effective in delivery of rifampicin in tuberculosis treatment.	Kumar et al. (2006)

Table 7. Examples of microparticles/nanoparticles/polymeric nanoparticle-based anti-TB preparations.

Delivery system	Anti-TB drug used	Results obtained	References
Gelatin nanoparticles (mannosylated)	Isoniazid	In this particular study, the nanoparticles (both plain and mannosylated gelatin nanoparticles) was prepared and the particles was found to be in size ranging from 261 to 380 nm. The drug loading was found up to 55%. The nanoparticles were found effective in targeting isoniazid to alveolar regions as the considerable drop in bacterial counts within the infected mice after IV administration was observed. The decline in the hepatic toxicity was also observed.	Saraogi et al. (2011)
Chitosan nanoparticles	Isoniazid	In this method, isoniazid loaded chitosan/tripolyphosphate nanoparticles was found to decrease the MIC against Mycobacterium avium infection. The nanoparticles were finally spray dried. Reduction in size of particles was observed as the concentration of leucine was increased.	Pourshahab et al. (2011)
PLGA nanoparticles	Ethionamide	In this study, ethionamide loaded nanoparticles was prepared. The size was found 287 nm. The drug entrapment efficiency was found 35% and a nanoparticle also found to generate controlled drug release for at least 6 days within plasma.	Kumar et al. (2011)
PLGA microspheres	Rifampicin	Rifampicin loaded PLGA microspheres were prepared and spherical shaped particles was observed with a diameter of 1.9 μm. It was found that they were effectively taken up by the NR-8383 cells were found localize in phago-lysosomes.	Onoshita et al. (2010), Feng et al. (2014)
PLGA oral nanoparticles	Ethionamide	The oral nanoparticles were found 286 ± 26 nm in size; with zeta-potential was −13 ± 2.5 mV. The drug encapsulation efficiency and loading capacity were found 35.2 ± 3.1% and 38.6 ± 2.3%.	Kumar et al. (2011)
PLGA microspheres	Rifampicin	In this case, rifampicin loaded microspheres was found 1.72 ± 0.16 μm in diameter.41% release of rifampicin over 4–5 days was also observed.	Doan & Olivier (2009)
PLGA microparticles	Rifampicin	In this study, it was found that the uptake of rifampicin from the PLGA microspheres was larger (about 4%) at 1 h and 9.3% at 4 h. An in vivo imaging study was done which depicts that the micron sized particles were rapidly excreted from the lungs but nanosized particles were found retained within lungs.	Ohashi et al. (2009)
Porous nanoparticle-aggregates particles (PNAPs) of PLGA	Rifampicin	The porous nanoparticle aggregates was found effectively deposited within the lungs. Initial rapid release of rifampicin was observed, when in vitro release study was employed. Rifampicin concentrations were found to be measurable in tissue and cells (Lungs) for 8 h.	Sung et al. (2009)
Inhalable microparticles	Isoniazid, Rifabutin	The inhalable microparticles incorporating first line anti-tubercular drugs were found to maintain the intra-cellular concentration of isoniazid for 24 h and rifabutin for 96 h period of time.	Verma et al. (2008)
Inhalable microparticles of poly(lactic acid) (l-PLA)	Isoniazid, Rifabutin	In this study, the prepared inhalable microparticles of isoniazid/rifabutin were found to have drug content up to 50%. Particle size was found up to 5 μm. Aerodynamic diameter was found up to 3.58 μm. About 70% of the drug load was found released in 10 days. Macrophage targeting was found effectively.	Muttil et al. (2007)
Oral nanoparticles of poly-lactide-co-glycolide (PLG)	Isoniazid, Rifampicin, Pyrazinamide, Ethambutol	It was found that after administration of the formulation to animal (mice) via oral route it maintains controlled drug levels for at least 5–8 days within plasma. Significant improvement in the pharmacokinetic parameters was found as compared with drug solution (Plain or free).	Pandey & Khuller (2006a)
PLG nanoparticles	Streptomycin	The prepared nanoparticles were found to be 153 nm in size. Drug encapsulation was found 32.12% and drug loading was found to be 14.28%. Streptomycin levels were found to be maintained for 4 days in the plasma and for 7 days in the different organs after single oral administration of streptomycin loaded nanoparticles.	Pandey & Khuller (2006b)
Alginate nanoparticles	Rifampicin, Ethambutol, Isoniazid, Pyrazinamide	In this study, high drug entrapment efficiency was achieved from 70% to 90%. It was found that after a single oral dose administration, therapeutic drug concentrations were found to be retained in the plasma for 7–11 days and in the organs like lungs etc. for at least 15 days when compared to free drug solutions.	Ahmad et al. (2006)
Alginate nanoparticles	Isoniazid, Rifampicin, Pyrazinamide	In this study, the alginate nanoparticles were prepared. The particles were found 235 nm in size. The drug entrapment efficiencies were found upto70–90% for pyrazinamide and isoniazid, respectively, and 80–90% for Rifampicin. The majority of particles were found in the respirable range. The aerodynamic diameter was found around 1.1 μm.	Zahoor et al. (2005)
Inhalable microparticles of poly(d,l-lactic acid)	Isoniazid, Rifampicin	This study depicted that large amount of particles can be delivered to the lungs by 2 min exposure to fluidized particles.	Sharma et al. (2001)
PLGA microspheres	Rifampicin	In this study, animals were treated with PLGA microspheres encapsulating rifampicin which shows significantly reduction in numbers of bacteria, inflammation and lung damage.	Suarez et al. (2001)
PLG microparticles	Isoniazid, Rifampicin	In this study, the PLG microparticles was found to be effective to clear bacteria more from lungs when administered to an experimental murine model of tuberculosis.	Dutt & Khuller (2001)
PLGA microspheres	Rifampicin	In this study the rifampicin loaded PLGA-microparticles was prepared. Finally, spray drying was done and the particles were found 3.45 μm in size.	O'Hara & Hickey (2000)
PLGA microspheres	Rifabutin	Rifabutin loaded microspheres were prepared and in vivo studies were conducted in J774 murine and Mono Mac 6 (MM6) human monocytic cell lines. The rifabutin caused significant reduction of intra cellular replicating Mycobacterium avium complex (MAC).	Barrow et al. (2007)

Figure 5. Factors responsible for drug resistance in tuberculosis treatment.

Table 8. First line drugs and their resistance.

Drugs	Gene involved in resistance	Gene function	Mechanism of drug resistance
Isoniazid	katG inhA	Catalase-peroxidase Enoyl ACP reductase	In this type of drug resistance, the Kat GS315T mutation is the most common mutation in most of the strains. This resistance was seen 94% of all isoniazid resistant clinical isolates that were reported. Further mutations in the mabA or inhA (promotor region) can also induce resistance (Zhang & Yew, 2009).
Rifampicin	rpoB	β subunit of RNA polymerase	Generally in this case of drug resistance, the mutation occurs in the 81 base pair region of the rpoB. It was found in about 96% of rifampicin resistant M. tuberculosis isolates reported. Mutations in rpoB generally results in high level resistance and cross-resistance (Palomino & Martin, 2014)
Ethambutol	embB	Arabinosyl transferase	This particular kind of resistance occurs due to the mutations in the embCAB operon particular in emb band and occasionally in embC but the embB codon 306 mutation is most frequent in clinical isolates resistant to ethambutol as it was found in 68% of the resistance strains (Zhang & Yew, 2009).
Pyrazinamide	pncA	Nicotinamidase, pyrazinamidase	Most of pyrazinamide resistant M. tuberculosis strains have mutations in pncA region as it accounts for 72–97% of the resistance cases reported. However, in some resistant, strains do not have pncA type mutation (Zhang & Yew, 2009).

Table 9. Newer drug candidates in tuberculosis treatment (Mohan et al., 2013).

Drugs under pre-clinical development stages	Drugs under clinical development
Nitromidazole (TBA-354)	Phase-I	Phase-II	Phase-III
Capuramucin (SQ-641)	Oxazolidinone AZD5847	Rifamycin: (Rifapentine)	Fluoroquinolones: (Gatifloxacin) (Moxifloxacin)
Benzothiazone (BTZ-043)		Oxazolidinone: (Sutezolid (PNU-100480) (Linezolid)	4-Thioureido-iminomethylpyridinium: (Perchlorate) (Perchlozone)
Imidazopyridine (Q201)		Diarylquinoline: (Bedaquiline TMC207)	Nitro-dihydro-imidazooxazole: (Delamanid -OPC67683)

Table 10. Second line anti-tubercular drugs used in the treatment of MDR-TB and XDR-TB (Prasad, 2007).

	Daily dosage (mg)
Anti-TB drugs	Minimum dosage (mg)	Maximum dosage (mg)	Type of anti-TB activity	Duration of treatment
Fluoroquinolone Ciprofloxacin Levefloxacin Ofloxacin Gatifloxacin	1500 mg 500 mg 600 mg 400 mg	1500 mg 750 mg 800 mg 400 mg	Weak bactericidal action	Minimum duration: 6 months. Maximum duration: 12–18 months.
Polypeptides: Capreomycin	500–750 mg	1000 mg	Bactericidal action	Minimum duration: 6 months. Maximum duration: 12–18 months.
Analog of d-alanine Cycloserine	500 mg	750 mg	Bacteriostatic action	Minimum duration: 6 months. Maximum duration: 12–18 months.
Thioamides Ethionamide	500 mg	750–1000 mg	Bacteriostatic action	Minimum duration: 6 months. Maximum duration: 12–18 months.
Amino glycosides Kanamycin Amikacin	500–750 mg 500 mg	1000 mg 750–1000 mg	Bactericidal Bactericidal against actively multiplying organisms.	Minimum duration: 6 months. Maximum duration: 12–18 months.

Table 11. Examples of immunostimulant (Patil et al., 2012).

Immunostimulant	Mechanism of action
Interferon’s: alpha, beta, gamma	They are able to induce certain enzymatic reactions, inhibition of cell proliferation and enhancement of immune activities like increased phagocytosis by macrophages and augmentation of specific cytotoxicity by T lymphocytes.
Interleukins: aldesleukin, des-alanyl-1, serine-125 human IL-2	They mainly help to activate cellular immunity profoundly with lymphocytosis, eosinophilia and thrombocytopenia and by release of multiple cytokines (e.g. TNF, IL-1 and interferon-g).
Levamisole, immuvac, reimun	Others

Table 12. Examples of immunosupressants (Patil et al., 2012).

Immunosupressant drugs	Mechanism of action
Cyclosporine, tacrolimus sirolimus, everolimus	Inhibitors of lymphocyte signaling (a) Calcineurin inhibitors (b) mTOR inhibitors
Glucocorticoids	Inhibitors of lymphocyte gene expression
Anakinra, daclizumab, basiliximab, etanercept, infliximab, adalimumab	Cytokine inhibitors
Efalizumab (LFA-1 inhibitor)	Inhibitors of immune cell adhesion
Antithymocyte globulin (ATG), alemutuzmab (antiCD-52 antibodies), muromunab (anti CD-3 antibodies, OKT-3)	Antibodies against specific immune cell molecules
Rho (D) immune globulin	Others

Table 13. Vaccines used in immunotherapy in tuberculosis.

Vaccines used	Results obtained	References
Ag 85 (A) DNA vaccine	In this study, the effect of Ag85 (A) DNA vaccine alone and in combination with rifampin and pyrazinamide was observed in mice. Ag85A DNA vaccine was found to be effective in production of IFN-gamma but it lowered the production of interleukins-4. Ag85 (A) DNA vaccine alone and in combination with rifampin and pyrazinamide was also found to lower the pulmonary and spleen bacterial loads by 1.03–1.38 logs.	Liang et al. (2011)
RUTI (which is a vaccine)	This study was based on principle of correlation between ESAT-6-specific interferon (IFN)-γ secretion and pulmonary lesions development. In this study animals were infected using Mycobacterium caprae. The animals were taken in three different groups. Group 1 is Untreated group (CT). Group 2 was treated with Isoniazid (INH). Group 3 was treated with Isoniazid (INH) and RUTI. RUTI vaccine was found effective to enhance the interferon-γ production.	Domingo et al. (2009)

Table 14. Antibodies in tuberculosis immunotherapy.

Antibody used	Results obtained	References
Polyclonal antibodies	In this study, SCID mouse model was taken for aerosol infection by M. tuberculosis, which was followed by 3–8 weeks of treatment of chemotherapy with isoniazid and rifampicin (INH-25 mg/kg + RIF-10 mg/kg). The animals (mice) were divided in two groups. Both the groups were treated with intra-peritoneal passive immunization using hyper-immune serum (HS) for 10 weeks. The infected mice were treated with chemotherapy (INH + RIF) for 8 weeks and inoculated with RUTI (HS group) or with normal serum (CT group). Significant differences were found between HS and CT groups in terms of number of bacilli in the lungs.	Guirado et al. (2006)
Heparin-binding hemagglutinin adhesin (HBHA)	In this study, the heparin binding hemagglutinin adhesin (HBHA) was identified as an antigen. Lymphocytes from 60% of healthy infected individuals (n = 25) produced interferon (IFN)-γ after stimulation with HBHA, compared with only 4% of patients with active tuberculosis (n = 24) was found. Most of the individual (82%) in study was found to produce anti-HBHA antibodies as compared with 36% of the infected healthy subjects. These observations indicate that HBHA is recognized differently by the immune systems of patients with tuberculosis and by healthy individuals.	Masungi et al. (2002)

Table 15. Studies showing role of immunomodulators as adjuvant to chemotherapy in the treatment of tuberculosis.

Type of study	Description and results obtained	References
To determine effect of Dzherelo on HIV and TB co-infected patients.	In this study, the clinical trial (open label) was conducted in 40 patients. The main aim of the trial was to determine the effect of oral immune modulator i.e. Dzherelo on the immune and other different viral parameters. The patients under the study were taken in two equal groups. In the group A (control group) patients was given first line anti-tubercular drugs, i.e. isoniazid, rifampicin, pyrazinamide, streptomycin and ethambutol and in the group B patients received Dzherelo as an immunomodulator twice in a day along with first line HRZSE regimens. After two months of the treatment in 40 patients, it was found that the total lymphocytes (CD3+) were increased upto728–920 cells/μl in group B (Dzherelo receivers) but in the group A the lymphocytes were found to reduce from 650 to 585 cells/μl.	Nikolaeva et al. (2008b)
Therapy for interferon gamma for pulmonary tuberculosis treatment.	In this trial study, total nine trials were conducted with interferon-γ. In 1 trial: patients were aerosolized or subcutaneously administration, In 5 trial: patients were only aerosolized, In 3 trials: patients were intramuscularly administration In these trials, patients were aerosolized by IFN-γ which was found effective in sputum negative conversion in patients.	Gao et al. (2011)
Adjuvant immune-therapy for XDR-TB management and treatment	In this study, the different trials were conducted on seven patients having XDR-TB. In this treatment, the patients received Immunoxel (Dzherelo) during trials. An increase in hemoglobin up to 104.1–118 g L^−1 was found. The time for mycobacterium clearance in infected patients was found to 25–28 days which is effective result.	Prihoda et al. (2009)
Role of recombinant interferon-α2b in MDR-tuberculosis treatment	In this trial study, five patients were taken to study the effect of recombinant-interferon-α2b. The patients under the study were given of recombinant-interferon-α2b. The subcutaneous administration was done in every week to the patients under the study till 12 weeks period. The patients under the trial study were observed for clinical examination, radiological examination etc. before treatment and after treatment also. Two patients out of total five patients were found to be negative sputum smear after recombinant-IFN-α2b therapy. One patient was found to show clinical improvement and negative smear after therapy after examination. The other two patients showed no response in this trial study.	Palmero et al. (1999)
Immuno-modulation therapy with recombinant interferon-γ1b in management and treatment of pulmonary tuberculosis	In this study, it was found that in the immune-adjunctive therapy with nebulized r-interferon-γ1b along with DOTS treatment can significantly reduce various lung inflammatory cytokines in 16 weeks. The immune modulation-based treatment was done on5 MDR-patients. In all five patients, the sputum was found negative with improved symptoms.	Dawson et al. (2009)
Adjunct immune therapy for relapsed TB, first-diagnosed TB, treatment-failed TB and multidrug-resistant TB and TB and HIV co-infected patients.	This study trial was done to check the effectiveness of the (V-5) immunomodulator in different TB infected cases when Immunitor, an immunomodulator which is also known as V-5 was given as an adjunct immunotherapy along with DOTS regimens, i.e. HRSZE. The trial study was conducted after dividing patients into two groups. One group (group A) in which 62 patients was taken in trial and was given above immunomodulator once in a day. The control group (group B) which contains 61 patients who received adjunct treatment for at least 30 days along with DOTS therapy regimens. After therapy period about 1 month with the treatment with immunomodulator, i.e. V-5 the following results were obtained. Fifty-five patients from group A become negative sputum smear testing and this result accounts for (88.7%) of the trial study results. Whereas in the case of the (group B) 9 patients out of 61 patients was found converted as it accounts for (14.8%). Thirty-three TB infected patients in (group B) gained 0.8 kg weights on average basis which is less effective result. Fifty-seven patients in (group A) gained at least 2.8 kg weight during trial study which accounts for (91.9%). No adverse or side effects were found. The chance of TB reactivation was observed. Therapy period up to 1 month with improved patient compliance.	Dawson et al. (2009)
Immunotherapy using IL-2 and GM-CSF in potential management and treatment for MDR-tuberculosis	This study mainly investigated the therapeutic effects of interleukin (IL)-2 and granulocyte-macrophage colony-stimulating factor (GM-CSF) in co-administration with anti-tubercular agents, i.e. isoniazid and rifampin to treat animal model (mice) of tuberculosis. The TB strain, i.e. H37Rv was used to cause tubercular infection in the mice. To check the effectiveness of IL-2 and GM-CSF. The animals (mice) who was receiving immunotherapy in trial study, a very few lungs lesions was developed as compared with animals (mice) who were receiving anti-bacterial drug therapy or immune therapy and anti-bacterial drug therapy both as adjunct. Survival rate up to 30% was found in animals (mice) was found under the trial study who received immune-therapy and less CFUs in the lungs was found, i.e. was 1.02 log CFUs. In the case of spleen 1.34 log CFUs was found. Therefore, from the study, it is clear that both interleukin-2 and GM-CSF may be effective to treat tuberculosis cases and some other secondary complications too.	Zhang et al. (2012)
To study the effect of adjuvant Immunoxel and Anemin immunomodulators in HIV and Pulmonary tuberculosis co-infected patients.	In this study, when Immunoxel and Anemin as an immunomodulators in combination with anti-tubercular therapy (ATT) were given to patients under trials. Total 60 patients were taken in the study which was taken into three groups. Group 1: ATT. Group2: ATT + ImmunoxelGroup3: ATT + Immunoxel + Anemin both as immunomodulators. Blood samples from the patients were taken and were measured in order to check plasma levels of tumor necrotic factor-α, interferon-γ, interleukins-2, inter leukins-6 and interferon-α. After the therapy time, i.e. 6 months period it was found that group 2 (ATT + Immunoxel) produce 61% and group 3 (Immunoxel + Anemin combinations) developed 44.4% higher levels of interleukin-2. Whereas in group 1 (ATT) it was declined or 33.1%. The levels of interleukin-6 were found increased by 17% in group 1 (ATT group) The interleukins-6 levels were found to be reduce up to 26.2% in group 2 (ATT + Immunoxel). The levels of interleukins-6 were to reduced up to 21.3% in group 3 (ATT + Immunoxel + Anemin) Tumor necrotic factor-α was suppressed in two immunotherapy groups by 19.1 and 76.3% in groups 2 and 3, respectively, but had risen by 14% in group 1 patients.	Nikolaeva et al. (2008a)

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