Claims:1. A cationic particles comprising of:
(a) one or more microparticles consisting of a bioerodible polymer;
(b) optionally fluorescent dye incorporated in the one or more microparticles; and
(c) cationic polymer conjugated with surface of the one or more microparticles.

2. The cationic particles as claimed in the claim 1, wherein the bioerodible polymer is poly(lactic-co-glycolic) acid, polylactic acid (PLA), polyglycolid acid (PGA), or polycaprolactone.

3. The cationic particles as claimed in the claim 1, wherein the cationic polymer is poly-L-lysine (PLL) or poly-L-arginine.

4. The cationic particles as claimed in the claim 1, wherein the fluorescent dye is Cy5 dye, Cy3, Cy7, DiO, DiI, DiR, ATTO dyes, or rhodamine.

5. The cationic particles as claimed in any one of the claims 1 to 4, wherein particle size of the cationic particles is in the range of 100 nm to 5 µm.

6. A pharmaceutical composition comprising of:
(a) a cationic particles as claimed in any one of the claims 1 to 4;
(b) an anti-tuberculosis drug encapsulated in the cationic particles; and
(c) optionally pharmaceutically acceptable excipients.

7. The pharmaceutical composition as claimed in the claim 6, wherein the anti-tuberculosis drug is rifampicin, isoniazid, ethambutol, or pyrazinamide.

8. The pharmaceutical composition as claimed in the claim 6, wherein the composition is in the form of a dry powder formulation.

9. The pharmaceutical composition as claimed in the claim 6, wherein the dry powder formulation is dry powder inhalation.

10. The pharmaceutical composition as claimed in the claim 6, wherein the composition is in the form of sustained release formulation.

, Description:FIELD OF THE INVENTION
[0001] The present invention generally relates to materials for drug delivery in the pharmaceutical field. Specifically, the present invention relates to a pharmaceutical composition comprising of cationic particles consisting of one or more microparticles comprising a bioerodible polymer; fluorescent dye incorporated in the one or more microparticles; and cationic polymer conjugated with surface of the microparticles; an anti-tuberculosis drug encapsulated in the cationic particles; and optionally pharmaceutically acceptable excipients.

BACKGROUND OF THE INVENTION
[0002] Background description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.
[0003] Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis that primarily infects the lungs. It is one of the leading causes of death worldwide, claiming 1.2 million lives globally in 2018. In addition to that, TB inflicted death upon 251,000 HIV positive patients (WHO, Global Tuberculosis Report 2019, 2019). India accounts for 27% of the cases and has the highest burden of multi drug resistant TB (MDR-TB).
[0004] The conventional oral therapy for TB is 6 – 24 months long with high pill burden. The anti-TB drugs cause several adverse side effects (hepatoxicity, nephrotoxicity etc. (E.J. Forget, D. Menzies, Adverse reactions to first-line antituberculosis drugs, Expert Opin. Drug Saf. 5 (2006) 231–249; D. Yee et. al., Incidence of Serious Side Effects from First-Line Antituberculosis Drugs among Patients Treated for Active Tuberculosis, Am. J. Respir. Crit. Care Med. 167 (2003) 1472–1477)) and perturb the commensal microbiota because of the high administered dose. The concentration of anti-TB drugs needs to be high to overcome multiple biological barriers. This includes absorption from the gut, metabolism in the liver and finally, pervading the granuloma that contains cells harboring Mtb (B. Prideaux et. al., The association between sterilizing activity and drug distribution into tuberculosis lesions, Nat. Med. 21 (2015) 1223–1227). These drawbacks in current therapy can be circumvented by delivering the drugs through a different route - inhalation. For optimum deposition in the deep lung, particles with aerodynamic diameter 1-5 µm are preferred (N.R. Labiris, M.B. Dolovich, Pulmonary drug delivery. Part I : Physiological factors affecting therapeutic effectiveness of aerosolized medications, Br. J. Clin. Pharmacol. 56 (2003) 588–599). These microparticles can serve as carriers for anti-TB drugs. Once delivered, these particles can form depots in the lung, which is usually the primary site of infection, and allow high sustained local concentration (A. Misra et. al., Inhaled drug therapy for treatment of tuberculosis, Tuberculosis. 91 (2011) 71–81.). This approach is highly patient compliant and can potentially reduce pill burden and dosing frequency.
[0005] Inhalation based drug delivery also offers improved drug pharmacokinetics. When delivered through inhalation, Rifampicin (RIF) and Isoniazid (INH) loaded poly(lactic-co-glycolic) acid (PLGA) microparticles exhibited higher drug concentration of drugs compared to intravenously delivered drugs in cells recovered from bronchoalveolar lavage (BAL) and lung tissue in healthy mice. High and sustained drug concentrations were observed in both lung tissue and serum after inhalation delivery (R.K. Verma et. al., Intracellular time course, pharmacokinetics, and biodistribution of isoniazid and rifabutin following pulmonary delivery of inhalable microparticles to mice, Antimicrob. Agents Chemother. 52 (2008) 3195–3201). Similar biodistribution studies on rhesus macaques after delivering drug loaded polymeric microparticles through inhalation demonstrated improved bioavailability and longer elimination half-life of anti-TB drugs compared to intravenous delivery of an equivalent dose.
[0006] Thus, inhalation delivery has potential to be an effective route for drug delivery in tuberculosis. Besides, since TB is caused by an intracellular pathogen that infects alveolar macrophages, microparticles can facilitate intracellular delivery as macrophages are phagocytic. In THP-1 cells in vitro, it was observed that anti-TB drugs encapsulated in microparticles had higher intracellular concentration compared to equivalent concentration of free drug. This can potentially translate to increased bactericidal efficiency of the drug as shown by multiple groups with different carriers for TB drugs (S. Pandit et. al., Formulation and Intracellular Trafficking of Lipid − Drug Conjugate Nanoparticles Containing a Hydrophilic Antitubercular Drug for Improved Intracellular Delivery to Human Macrophages, ACS Omega. 5 (2020) 4433–4448).
[0007] Recently, it was also reported that M. bovis BCG infected macrophages have increased endocytic capacity. Microparticle based delivery of RIF was shown to attain higher intracellular accumulation compared to free drug (K. Hirota et. al., Delivery of rifampicin – PLGA microspheres into alveolar macrophages is promising for treatment of tuberculosis, J. Control. Release. 142 (2010) 339–346). In terms of surface charge, it has been previously shown that particle uptake by RAW 264.7 macrophages increases with corresponding increase in positive surface charge of polylactic acid microspheres. This also facilitated higher delivery of model antigen into the cells compared to non-modified microparticles and free antigen. In another study, polyethyleneimine (PEI) coated PLGA particles were shown to accumulate higher compared to non-modified particles. These PEI coated particles also displayed significantly higher intracellular concentration of RIF compared to non-modified particles and free drug (Z. Liu et. al., A novel and simple preparative method for uniform-sized PLGA microspheres : Preliminary application in antitubercular drug delivery, Colloids Surfaces B Biointerfaces. 145 (2016) 679–687). Additionally, PEI coated mesoporous silica nanoparticles demonstrated significant reduction of intracellular Mtb, hence improving bactericidal performance. Most of these studies have been conducted on uninfected cells which are not completely representative of the diseased state.
[0008] US20200138728A1 discloses cationic polymers for delivering anionic active agents, preferably in the form or nanoparticles and other nanostructures. The polymer can be a polycation homopolymer or a copolymer containing a polycation block. The polycations and polycation containing polymers can contain dicarboxylic acid ester units and units of (α-amino acid)-α,ω-alkylene diester units.
[0009] US20200179287A1 discloses novel drug delivery particles comprising an anionic polymer matrix and a cationic polymer, wherein the anionic polymer matrix and cationic polymer together form drug delivery particles bound by electrostatic interactions and wherein the drug delivery particles comprise at least one biologically active agent.
[0010] There is, therefore, an unmet need to an engineered polymeric microparticle (MP) contributing to increased uptake by macrophages infected with virulent Mycobacterium tuberculosis and a formulation comprising of anti-tuberculosis drugs encapsulated in the microparticles.

OBJECTS OF THE INVENTION
[0011] An object of the present invention is to provide an engineered polymeric microparticle (MP) contributing to increased uptake by macrophages infected with virulent Mycobacterium tuberculosis.
[0012] Another object of the present invention is to provide anti-tuberculosis drugs encapsulated in the microparticles that enhance intracellular concentration of the anti-tuberculosis drug and result in higher bactericidal efficiency compared to free drug and non-modified particles.
[0013] Another object of the present invention is to provide a pharmaceutical composition comprising of anti-tuberculosis drugs encapsulated in the microparticles.
[0014] Another object of the present invention is to provide a dry powder inhaler pharmaceutical composition comprising of anti-tuberculosis drugs encapsulated in the microparticles.

SUMMARY OF THE INVENTION
[0015] The present invention relates to a pharmaceutical composition comprising of cationic particles consisting of one or more microparticles comprising a bioerodible polymer; fluorescent dye incorporated in the one or more microparticles; and cationic polymer conjugated with surface of the microparticles; an anti-tuberculosis drug encapsulated in the cationic particles; and optionally pharmaceutically acceptable excipients.
[0016] In one aspect, the present invention relates to a cationic particles comprising of:
(a) one or more microparticles consisting of a bioerodible polymer;
(b) optionally fluorescent dye incorporated in the one or more microparticles; and
(c) cationic polymer conjugated with surface of the one or more microparticles.
[0017] In another aspect of the present invention, the bioerodible polymer is poly(lactic-co-glycolic) acid, polylactic acid (PLA), polyglycolid acid (PGA), or polycaprolactone.
[0018] In another aspect of the present invention, the the cationic polymer is poly-L-lysine (PLL) or poly-L-arginine.
[0019] In another aspect of the present invention, the fluorescent dye is Cy5 dye, Cy3, Cy7, DiO, DiI, DiR, ATTO dyes, or rhodamine .
[0020] In another aspect of the present invention, particle size of the cationic microparticles is in the range of 100 nm to 5 µm.
[0021] In another aspect, the present invention relates to a pharmaceutical composition comprising of:
(a) a cationic particle comprises of one or more microparticles consisting of a bioerodible polymer; fluorescent dye incorporated in the one or more microparticles; and cationic polymer conjugated with surface of the one or more microparticles;
(b) an anti-tuberculosis drug encapsulated in the cationic particles; and
(c) optionally pharmaceutically acceptable excipients.
[0022] In another aspect of the present invention, the anti-tuberculosis drug is rifampicin, isoniazid, ethambutol, or pyrazinamide.
[0023] In another aspect of the present invention, the composition is in the form of a dry powder formulation.
[0024] In another aspect of the present invention, the dry powder formulation is dry powder inhalation.
[0025] In another aspect of the present invention, the composition is in the form of sustained release formulation.
[0026] Various objects, features, aspects and advantages of the inventive subject matter will become more apparent from the following detailed description of preferred embodiments.

BRIEF DESCRIPTION OF THE FIGURES
[0027] Figure 1: (A) Representative fluorescence images of microparticles of sizes 500 nm,1 µm, and 2 µm. Images were acquired in the Cy5 channel under 100x magnification. Scale 5 µm. (B) Representative Scanning Electron Microscopy (SEM) images of PLGA particles of 500 nm, 1 μm, and 2 μm diameter (C) Size distribution of microparticles was determined using Dynamic Light Scattering. (D) Percentage of differentiated THP-1 macrophages with particles over 4 and 24 h. One-way ANOVA followed by Tukey’s multiple comparison test was used to detect statistical differences for each time point (*p = 0.0423, #p = 0.0487, ***p = 0.0053, n = 3). (E) Percentage of metabolic activity of THP-1 macrophages reported after 24 h incubation of particles at 50 μg/mL and 500 μg/mL concentration. Values were normalized to the values corresponding to the wells in which no particles were added (Untreated). All groups were found statistically insignificant using ordinary one-way ANOVA followed by Tukey’s multiple comparisons test. Data were represented as mean ± SD.
[0028] Figure 2: (A) Representative SEM image of rif-loaded 1 μm PLGA particle with PLL coating. (B) Drug release from rif loaded 1 µm PLGA microparticles was characterized by allowing the microparticles to incubate at 37 °C in 1X PBS (pH 7.4) and 0.1 M MES (pH 5.5). At specific time points, the particles were pelleted and dissolved in DMSO. The concentration of rif was determined by measuring absorbance at 335 nm. All values were normalized to the concentration measured at the first time point (day 0) and percentage of drug retained in the microparticles was plotted.
[0029] Figure 3: (A) Effect of surface charge on microparticle uptake in (A) H37Rv-infected THP-1 macrophages after 4 h incubation (***p = 0.0005, ****p < 0.0001, n = 6, N=2), (B) H37Rv-infected murine BMDMs after 2 h incubation (***p = 0.0035, **p = 0.0087, *p = 0.0316, #p = 0.0273, n = 3), (C) Median fluorescence intensity (MFI) plots (negatively charged particles in blue circles and positively charged particles in red squares) for H37Rv infected THP-1 macrophages, and (D) H37Rv infected BMDMs incubated with 500 nm particles, 1 µm particles and 2 µm particles. Analysis was done using two-tailed t-test and all groups had n = 3. Statistical test conducted was two tailed t-test between the negative and positive charge groups for each size and one-way ANOVA followed by Tukey’s multiple comparison test for analysis among sizes. Data were represented as mean ± SD (E) Representative confocal fluorescence images of 500 nm negatively and positively charged particle uptake in H37Rv-infected BMDMs. Nucleus = Blue, Bacteria = H37Rv-GFP green, Particles = Cy5 red. White arrows indicate instances of colocalization between particles and bacteria. Scale bar 20 µm.
[0030] Figure 4: Percentage of cells with particles is plotted for H37Rv infected (GFP positive, red squares) and non-infected cells (GFP negative, blue circles) in (A) THP-1 macrophages (n = 6, *p = 0.0314, **p = 0.001, ***p = 0.0002, ****p < 0.0001) and (B) BMDMs (n = 3, *p = 0.0368). Data were analysed using two-way ANOVA followed by Sidak’s multiple comparisons test. Data were represented as mean ± SD
[0031] Figure 5: (A) Intracellular rif quantification using HPLC after 4 h treatment with free rif and rif-MPs (n = 3, ****p <0.0001, ND = Not Detectable) (B) Intracellular bacteria from H37Rv infected THP-1 macrophages after 2 h treatment with free rif and encapsulated rif-MPs (n = 16, N = 2, ****p <0.001, ***p = 0.0007, *p = 0.0314). Data were combined from two separate biological replicates. (C) Intracellular bacterial counts (cfu/mL) from H37Rv-infected THP-1 macrophages reported after 2 h treatment with 500 nm microparticles – non-modified (Neg) and poly-l-lysine conjugated (Pos). All groups were found non-significant. Data were represented as mean ± SD. Ordinary one-way ANOVA followed by Tukey’s multiple comparisons test was used to determine significance. Data were represented as mean ± SD.
[0032] Figure 6: (A) Cy5-loaded MPs were delivered intratracheally and fluorescence intensity was measured immediately post-delivery from excised lung. (B) Microparticles deposition and distribution throughout the lung was assessed by fluorescence imaging of lung cryosections. Hoechst 33342 was used to stain nucleus and slides were visualized in DAPI and Cy5 channel. Red = Particles, Blue = Nucleus, Scale bar = 10 μm. Percentages of cells with particles reported for (C) total immune cells (*p = 0.0356) and (D) alveolar macrophages (**p = 0.0062) characterized using flow cytometry. Two-tailed t-test was used to determine significance between the two groups. Data were represented as mean ± SD. (E) Representative brightfield images of Hematoxylin and Eosin-stained lung sections after PBS and particle (negative and positive charge) exposure via intra-tracheal delivery. Mice were sacrificed after 1 and 7 days post-delivery. Scale bar = 100 μm.

DETAILED DESCRIPTION OF THE INVENTION
[0033] The following is a detailed description of embodiments of the disclosure. The embodiments are in such detail as to clearly communicate the disclosure. However, the amount of detail offered is not intended to limit the anticipated variations of embodiments; on the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the present disclosure as defined by the appended claims.
[0034] Unless the context requires otherwise, throughout the specification which follow, the word “comprise” and variations thereof, such as, “comprises” and “comprising” are to be construed in an open, inclusive sense that is as “including, but not limited to.”
[0035] Reference throughout this specification to “one embodiment” or “an embodiment” means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, the appearances of the phrases “in one embodiment” or “in an embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.
[0036] As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the content clearly dictates otherwise. It should also be noted that the term “or” is generally employed in its sense including “and/or” unless the content clearly dictates otherwise.
[0037] In some embodiments, the numbers expressing quantities of ingredients, properties such as concentration, reaction conditions, and so forth, used to describe and claim certain embodiments of the invention are to be understood as being modified in some instances by the term “about.” Accordingly, in some embodiments, the numerical parameters set forth in the written description are approximations that can vary depending upon the desired properties sought to be obtained by a particular embodiment. In some embodiments, the numerical parameters should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as practicable.
[0038] The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein.
[0039] All processes described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g. “such as”) provided with respect to certain embodiments herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.
[0040] The headings and abstract of the invention provided herein are for convenience only and do not interpret the scope or meaning of the embodiments.
[0041] The following discussion provides many example embodiments of the inventive subject matter. Although each embodiment represents a single combination of inventive elements, the inventive subject matter is considered to include all possible combinations of the disclosed elements. Thus if one embodiment comprises elements A, B, and C, and a second embodiment comprises elements B and D, then the inventive subject matter is also considered to include other remaining combinations of A, B, C, or D, even if not explicitly disclosed.
[0042] All publications herein are incorporated by reference to the same extent as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. Where a definition or use of a term in an incorporated reference is inconsistent or contrary to the definition of that term provided herein, the definition of that term provided herein applies and the definition of that term in the reference does not apply.
[0043] Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member can be referred to and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group can be included in, or deleted from, a group for reasons of convenience and/or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified thus fulfilling the written description that follows, and the embodiments described herein, is provided by way of illustration of an example, or examples, of particular embodiments of the principles and aspects of the present disclosure. These examples are provided for the purposes of explanation, and not of limitation, of those principles and of the disclosure.
[0044] It should also be appreciated that the present invention can be implemented in numerous ways, including as a system, a method or a device. In this specification, these implementations, or any other form that the invention may take, may be referred to as processes. In general, the order of the steps of the disclosed processes may be altered within the scope of the invention.
[0045] Various terms as used herein are shown below. To the extent a term used in a claim is not defined below, it should be given the broadest definition persons in the pertinent art have given that term as reflected in printed publications and issued patents at the time of filing.
[0046] The term, “pharmaceutically acceptable excipients” as used herein refers an inactive ingredient in the composition which has no role in the therapeutic effect of the composition however helps in controlling the bio-availability, and improving the stability of the composition.
[0047] In a general embodiment, the present invention relates to a pharmaceutical composition comprising of cationic particles consisting of one or more microparticles comprising a bioerodible polymer; fluorescent dye incorporated in the one or more microparticles; and cationic polymer conjugated with surface of the microparticles; an anti-tuberculosis drug encapsulated in the cationic particles; and optionally pharmaceutically acceptable excipients.
[0048] In one aspect, the present invention relates to a cationic particles comprising of:
(a) one or more microparticles consisting of a bioerodible polymer;
(b) optionally fluorescent dye incorporated in the one or more microparticles; and
(c) cationic polymer conjugated with surface of the one or more microparticles.
[0049] In an embodiment, the present invention relates to a cationic particles comprising of:
(a) one or more microparticles consisting of a poly(lactic-co-glycolic) acid;
(b) optionally fluorescent dye, Cy5 dye incorporated in the one or more microparticles; and
(c) poly-L-lysine (PLL) conjugated with surface of the one or more microparticles.
[0050] In an embodiment of the present invention, particle size of the cationic microparticles is in the range of 100 nm to 5 µm. Preferably in the range of 500 nm to 2 µm.
[0051] In another embodiment, the present invention relates to a pharmaceutical composition comprising of:
(a) a cationic particles comprises of microparticles consisting of a bioerodible polymer, optionally fluorescent dye and cationic polymer conjugated with surface of the one or more microparticles;
(b) an anti-tuberculosis drug encapsulated in the cationic particles; and
(c) optionally pharmaceutically acceptable excipients.
[0052] In another embodiment of the present invention, the pharmaceutically acceptable excipients are diluent, lubricant or mixture thereof.
[0053] In another embodiment of the present invention, the diluent can be selected from lactose, xylitol, arabinose, dextran, and mannitol.
[0054] In another embodiment of the present invention, the lubricant is selected from sodium benzoate, magnesium stearate, colloidal silica, hydrogenated oils and fatty bases.
[0055] In another embodiment of the present invention, the anti-tuberculosis drug is rifampicin, isoniazid, ethambutol, or pyrazinamide. Preferably, the anti-tuberculosis drug is rifampicin.
[0056] In another embodiment of the present invention, the composition is in the form of a dry powder formulation.
[0057] In another embodiment of the present invention, the dry powder formulation can be dry powder inhalation.
[0058] In another embodiment of the present invention, the composition is in the form of sustained release formulation.
[0059] In another embodiment, the present invention relates to a pharmaceutical composition comprising of:
(a) a cationic particles comprises of microparticles consisting of poly(lactic-co-glycolic) acid, cy5 dye and poly-L-lysine conjugated with surface of the one or more microparticles;
(b) rifampicin encapsulated in the cationic particles; and
(c) optionally pharmaceutically acceptable excipients.
[0060] In another embodiment, the present invention relates to a pharmaceutical composition comprising of:
(a) a cationic particles comprises of microparticles consisting of poly(lactic-co-glycolic) acid, cy5 dye and poly-L-lysine conjugated with surface of the one or more microparticles; and
(b) rifampicin encapsulated in the cationic particles.
[0061] In another embodiment, the present invention relates to a dry powder composition comprising of:
(a) a cationic particles comprises of microparticles consisting of poly(lactic-co-glycolic) acid, optionally cy5 dye and poly-L-lysine conjugated with surface of the one or more microparticles; and
(b) rifampicin encapsulated in the cationic particles.
[0062] In another embodiment, the present invention relates to a dry powder inhaler pharmaceutical composition comprising of:
(a) a cationic particles comprises of microparticles consisting of poly(lactic-co-glycolic) acid, optionally cy5 dye and poly-L-lysine conjugated with surface of the one or more microparticles;
(b) an anti-tuberculosis drug encapsulated in the cationic particles; and
(c) optionally pharmaceutically acceptable excipients.
[0063] In another embodiment of the present invention, the anti-tuberculosis drug encapsulated in the cationic particles of dry powder inhaler pharmaceutical composition can be rifampicin, isoniazid, ethambutol, or pyrazinamide.
[0064] In another embodiment, the present invention relates to a dry powder inhaler pharmaceutical composition comprising of:
(a) a cationic particles comprises of microparticles consisting of poly(lactic-co-glycolic) acid, optionally cy5 dye and poly-L-lysine conjugated with surface of the one or more microparticles; and
(b) rifampicin encapsulated in the cationic particles.
[0065] According to the present invention, the surface charge of MPs is a major factor contributing to increased uptake by macrophages infected with virulent Mycobacterium tuberculosis. Positive surface charge was imparted by covalent conjugation of cationic polymers such as poly-L-lysine.
[0066] In another embodiment of the present invention, the cationic microparticles facilitated enhanced intracellular concentration of the anti-tuberculosis drug and resulted in higher bactericidal efficiency compared to free anti-tuberculosis and non-modified particles.
[0067] In another embodiment of the present invention, the engineered drug-loaded micro carriers deposit efficiently in the lung via inhalation and can be administered to TB-patients as dry powders through pulmonary delivery.
EXAMPLES
[0068] The present invention is further explained in the form of following examples. However, it is to be understood that the following examples are merely illustrative and are not to be taken as limitations upon the scope of the invention.
[0069] Synthesis of Poly (lactic-co-glycolic) acid microparticles:
Chemicals used:
Poly(lactic-co-glycolic) acid (PLGA) (50:50 LA:GA, Mn: 10,000-15,000 Da)
Akina Cat# AP-041
Dichloromethane (DCM)
Cyanine 5 amine (Cy5) (Lumiprobe Cat# 130C0)
Polyvinyl alcohol (PVA) (Mw 13000 - 23000 Da)
Double distilled MilliQ water
[0070] Steps:
1. 100 mg PLGA was weighed and dissolved in 2 mL DCM and mixed on a rotor for 10 minutes at room temperature.
2. 8 µL Cy5 was added to the mixture of PLGA and DCM to encapsulate the fluorescent dye within the microparticles. This is referred to as “organic phase”.
Note: 50 mg Rifampicin was added at this step instead of Cy5 to encapsulate the anti-TB drug within these particles.
3. The “organic phase” was added to 10 mL of 1% PVA solution (“aqueous phase”) and homogenized for 2 minutes at 1000 rpm to synthesize 2 µm particles and 1200 rpm to obtain 1 µm particles.
4. To synthesize 500 nm particles, the organic phase was added to aqueous phase and homogenized using a probe sonicator at the following conditions: 30% amplitude for 2 minutes with 10 second pulses.
5. The homogenized mixture was immediately added to 100 mL of 1% PVA in a beaker kept on a magnetic stirrer at 300 rpm.
6. The solvent was allowed to evaporate from the mixture by leaving the mixture continuously stirring for 4 hours.
7. The mixture was collected in 50 mL centrifuge tubes and centrifuged at 10,000 g for 5 minutes to pellet the microparticles.
8. The pellet was resuspended in distilled MQ water and centrifuged again to remove residual PVA. This washing step was repeated twice.
9. The final pellet was resuspended in 2-3 mL distilled MQ water, snap frozen and lyophilized.
[0071] Covalent conjugation of Poly-L-lysine to microparticle surface using EDC-NHS chemistry:
Chemicals used:
N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) Sigma Cat # E7750
N-Hydroxysuccinimide (NHS) Sigma Cat# 130672
2-(N-morpholino)ethanesulfonic acid hydrate (MES hydrate)
Poly-L-Lysine Sigma Cat# P8920
[0072] Steps:
1. Weighed 2 mg of particles in 2 mL Lobind tubes.
2. Weighed required amount of EDC and NHS for 1 mL reaction volume (Final concentrations: 40 mM EDC and 50 mM NHS) and dissolved in MES buffer maintained at pH 5.6
3. Washed the particles once by resuspending in MQ water, sonicating and spinning them down at 10,000 g.
4. Resuspended in MES buffer (pH 5.6) and added calculated respective volumes of EDAC (40 mM) and NHS (50 mM) for a 1 mL reaction.
5. Incubated on a rotor at room temperature for 1 hour.
6. Washed twice with MES buffer at pH 7.4 and resuspend in 0.1% Poly-L-Lysine solution prepared in MES buffer (pH 7.4).
7. Allowed incubation in rotating condition at room temperature for 3 hours.
8. Washed thrice with MQ water by centrifuging and resuspending. Sonicate in low volume to properly resuspend pellet.
9. Proceeded with snap freezing and lyophilization
[0073] Microparticles (MPs) were synthesized using a widely used bioerodible polymer poly(lactic-co-glycolic) acid (PLGA). Oil-in-water single emulsion method was used to obtain spherical particles of different diameters – 500 nm, 1 µm and 2 µm. This was achieved by modulating the homogenization speed. Fluorescent dye Cy5 was incorporated in the MPs to enable detection of the particles using microscopy (Figure 1A) and flow cytometry. SEM analysis confirmed that the particles were smooth and spherical (Figure 1B). The size distribution of the particles was also confirmed using dynamic light scattering (Figure 1C).
[0074] Cytocompatibility of the particle formulations
[0075] WST-8 assay was performed with THP-1 macrophages. Concentrations tested at 50 µg/mL and 500 µg/mL were found to be cytocompatible (Figure 1E). Particle concentration of 50 µg/mL was used for all subsequent experiments.
[0076] Effect of microparticle size on uptake by macrophages
[0077] THP-1 macrophages were incubated with particle formulations and at specific time points, cells were washed thrice to remove adsorbed particles, and analysed using flow cytometry. It was observed that 500 nm particle had slightly higher uptake as compared to 1 µm and 2 µm MPs at both 4 h (38.9 ± 3.5% of cells, p = 0.0423) and 24 h (80.5 ± 0.98% of cells) post incubation (Figure 1D). MPs of size 1 µm and 2 µm were also phagocytosed by ~30% cells in 4 h and by ~65% cells by 24 h. Overall, no major increase in percentage of cells taking up particles of different sizes suggesting that all particles in these size ranges have similar uptake by macrophages.
[0078] Rifampicin (rif), an anti-TB drug, was encapsulated within these polymeric particles which allowed sustained release of drug upon hydrolysis of the polymeric matrix. Particle morphology remained smooth and spherical after Rif encapsulation and PLL coating as confirmed using SEM imaging (Figure 2A). 1 µm particles was encapsulated with rif (60 µg/mg particle) and observed drug release over 21 days. 40% of the drug is released in 3 d, because the polymer used was low molecular weight acid-capped that degrades quickly (Figure 2B). In addition, assessed the release of Rif at a lower pH of 5.5 to mimic acidified vacuoles where the particles might reside. Release at pH 5.5 was quicker due to acid-calalyzed degradation of the polymer (Figure 2B).
[0079] The particle surface was modified by conjugating a cationic polymer, poly-L-lysine (PLL), using EDC-NHS chemistry. The zeta potential measured before the conjugation was -21.53 ± 5.14 mV which was modified to 21.73 ± 3.1 mV after attachment of PLL.
[0080] Thus, we were able to engineer microparticle size and surface charge and encapsulate drug within MPs so that they can be used as a sustained delivery formulation for treatment of tuberculosis.
[0081] Study of the micro carriers on optimal delivery into mycobacteria infected macrophages.
[0082] An in vitro infection model was established with PMA differentiated THP-1 cells and primary murine Bone Marrow Derived Macrophages (BMDMs) that are infected with virulent mycobacterial strain H37Rv. These infected macrophages were incubated with the different types of MPs and particle uptake was assessed using flow cytometry. In H37Rv infected THP-1 macrophages and H37Rv infected BMDMs, 500 nm and 1 µm particles were most efficiently phagocytosed (Figure 3A-B). Decrease in particle uptake was observed with increasing particle size. However, remarkable improvement in particle uptake was observed when the MP surface charge was modified by poly-L-lysine conjugation (PLL-MPs). Upon incubation with PLL-MPs, particle internalization doubled across all sizes in both H37Rv infected THP-1 and BMDMs, with ~90% H37Rv-infected cells with particles within 4 h of incubation for each size. This could be explained by the fact that mammalian cell surface has a slightly negative charge and the interaction between PLL-MPs and cells could have been enhanced due to electrostatic forces. It was also evident from the Median Fluorescence Intensity values that in addition to higher number of cells that internalized particles, the number of particles getting internalized were also significantly higher in both H37Rv infected THP-1 (Figure 3C) and BMDMs (Figure 3D). This was confirmed using fluorescence microscopy where higher accumulation of cationic 500 nm particles is clearly visible in contrast to sparingly internalized non-modified particles (Figure 3E). Additionally, higher incidence of colocalization events was also apparent with LL-MPs. Another interesting observation across all infected groups was that bacteria-harbouring macrophages (GFP+ cells) had higher Cy5+ population compared to cells that were exposed to bacteria but had not taken them up (uninfected macrophages), for both H37Rv infected THP-1 and BMDMs (Figure 4A-B). This indicates high phagocytic ability of macrophages infected with virulent Mtb. Thus, Mtb-infected macrophages were capable of phagocytosis and surface charge of MPs played a major role in enabling higher uptake.
[0083] Assessment of cationic particles in improving treatment of TB:
[0084] To determine if positively charged particles can deliver higher drug amounts in cells compared to negatively charged particles and free drug, THP-1 macrophages were incubated with non-modified and PLL-coated rif-MPs (100 μg/mL). After 4 h of incubation, the cells were washed, lysed and intracellular rif content was quantified using HPLC. It was observed that rif-MPs encapsulating equivalent rif concentration allowed a log-fold higher amount of rif to accumulate within cells (Figure 5A). PLL-coated rif-MP further resulted in ~2-fold higher intracellular rif amount compared to non-modified rif-MP (p <0.0001) (Figure 5A). Thus, rif loaded microparticles create an intracellular reservoir of the drug and high concentrations can be achieved within infected cells. To test bactericidal efficacy inside mammalian cells, THP-1 macrophages were infected with H37Rv followed by 2 h incubation with free rif (0.65 μg/mL) as well as both negative and positive surface charge rif-MPs with equivalent amount of rif. Only PLL-rif MP were able to reduce the intracellular bacterial counts after 72 h compared to untreated (p = 0.0007), free rif (p = 0.0314) and non-modified rif-MP (p <0.001), which indicates that prompt uptake of PLL-coated MPs leads to higher accumulation of intracellular drug and higher bactericidal efficiency (Figure 5B). Blank particles had no effect on the intracellular bacterial counts (Figure 5C).
[0085] Thus, microparticle parameters were optimized to maximize delivery to Mtb-infected macrophages. Cationic microparticles improved bactericidal action owing to the higher intracellular accumulation of drug due to rapid uptake of rif-MPs in high numbers.
[0086] Optimization to maximize delivery to alveolar macrophages in vivo
[0087] Particles (1.5 mg per mouse) were resuspended in PBS and delivered intratracheally into mice lungs using a custom made intratracheal tip attached to a 3 mL syringe. The particles deposited efficiently throughout the lungs using this method (Figure 6A). The particles were majorly dispersed in the alveolar region (Figure 6B). Next, effect of surface charge on particle uptake by immune cells in vivo was studied. After 1.5 h of particle delivery, mice were euthanized and lungs were excised and digested with collagenase. Various immune cell populations (CD45+) such as neutrophils (CD11b+ Ly6G+), dendritic cells (CD24+ CD11c+), alveolar macrophages (CD24- CD11b- SiglecF+) and interstitial macrophages (CD24- CD11b+ SiglecF-) were identified using flow cytometry and particle uptake by each cell type was analyzed. Particle delivery to immune cells and AMs was significantly enhanced with PLL-MPs. For all immune cells, non-modified particles were taken up by 4.89 ± 2.3% cells while PLL-MPs exhibited two-fold higher uptake with 10.18 ± 4.5% cells with particles (p = 0.0356) (Figure 6C). Similarly for alveolar macrophages, drastic improvement in delivery was observed with percentage of cells with particles increasing from 26.25 ± 16.27% to 61.18 ± 17.78% with PLL-MPs (p = 0.0062) (Figure 6D). Safety of the formulation was determined by histological analysis 1 and 7 d after particle administration, which revealed no undesired pathology in mice lungs administered with PBS, non-modified and PLL-MPs (Figure 6E). Thus, PLL-MPs represent an effective and safe platform to enhance delivery to alveolar macrophages.
[0088] Hence, in summary, a MP platform engineered to increase internalization by Mtb-macrophages improves treatment of intracellular Mtb infection. This is achieved by prompt uptake by more infected cells and increased accumulation of payload (anti-TB drugs such as rifampicin) inside infected cells. The charge-dependent enhanced uptake was also observed in vivo, with PLL-MPs demonstrating two-fold higher uptake by immune cells and alveolar macrophages after pulmonary delivery in mice. The PLL-MPs were also found cytocompatible and biocompatible based on in vitro WST-8 assay and in vivo histological analysis.
[0089] A skilled artisan will appreciate that the quantity and type of each ingredient ingredients can be used in different combinations or singly. All such variations and combinations would be falling within the scope of present disclosure
[0090] The foregoing examples are merely illustrative and are not to be taken as limitations upon the scope of the invention. Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art. Such changes and modifications may be made without departing from the scope of the invention.

ADVANTAGES OF THE PRESENT INVENTION
[0091] The present invention provides cationic particles that encapsulate the anti-TB drug which allowed sustained release of drug upon hydrolysis of the polymeric matrix.
[0092] The present invention provides microparticles contributing to increased uptake by macrophages infected with virulent Mycobacterium tuberculosis.
[0093] The present invention provides pharmaceutical composition comprising of anti-tuberculosis drugs encapsulated in the microparticles that enhance intracellular concentration of the anti-tuberculosis drug and result in higher bactericidal efficiency compared to free drug and non-modified particles.
[0094] The present invention provides a composition which shows two-fold increase in microparticle delivery to alveolar macrophages in vivo.