Novel liposomal formulation encapsulating ripe drugs

Abstract

The present invention relates to a liposomal pharmaceutical composition encapsulating a combination of rifampicin, isoniazid, pyrazinamide, and ethambutol (RIPE). The composition comprises liposomes formed from dipalmitoylphosphatidylcholine and distearoylphosphatidylcholine, wherein rifampicin is associated with the lipid bilayer and isoniazid, pyrazinamide, and ethambutol are encapsulated within an aqueous core. The liposomal formulation exhibits a nanoscale particle size, controlled surface charge, and high encapsulation efficiency, enabling enhanced intracellular uptake by macrophages. The formulation demonstrates improved antimycobacterial activity against Mycobacterium tuberculosis, reduced bacterial regrowth at sub-inhibitory concentrations, preferential accumulation in lung tissue following oral administration, and reduced systemic toxicity in comparison with non-liposomal RIPE formulations. The disclosed liposomal RIPE formulation provides an improved drug delivery system for anti-tubercular agents.

Description

[0001] TECHNICAL FIELD

[0002] The present invention relates particularly, but not exclusively, to a novel liposomal formulation encapsulating the rifampicin, isoniazid, pyrazinamide, and ethambutol (RIPE) drugs. More specifically, the present invention relates to an innovative liposome formulation for enhanced delivery of RIPE in tuberculosis therapy.

[0003] BACKGROUND

[0004] Tuberculosis (TB), caused by Mycobacterium tuberculosis, remains a major global public health concern despite the availability of established therapeutic regimens. The disease predominantly affects the lungs but may also involve extrapulmonary sites, requiring prolonged and multi-drug treatment strategies. The current standard of care relies on a combination of first-line anti -tubercular drugs, namely rifampicin, isoniazid, pyrazinamide, and ethambutol (RIPE), administered over extended durations. While this combination therapy has proven effective, it is associated with several limitations that continue to hinder optimal treatment outcomes.

[0005] One of the primary challenges in TB therapy arises from the intracellular survival of Mycobacterium tuberculosis within host macrophages. Conventional formulations of anti-tubercular drugs often exhibit limited intracellular penetration, resulting in suboptimal drug concentrations at the site of infection. In addition, the physicochemical diversity of the RIPE drugs — particularly the hydrophobic nature of rifampicin in contrast to the hydrophilic properties of the remaining agents — poses formulation challenges that can adversely affect bioavailability, stability, and pharmacokinetic performance when administered as free drug combinations.

[0006] Further, systemic administration of free RIPE drugs is frequently associated with off -target distribution and dose-limiting toxicity, particularly affecting hepatic and renal function. These adverse effects, coupled with the prolonged duration of therapy, can contribute to poor patientadherence and inconsistent drug exposure, thereby increasing the risk of treatment failure and the emergence of drug-resistant strains. Although various drug delivery approaches have been explored to address these issues, there remains a need for a formulation strategy capable of simultaneously accommodating multiple anti -tubercular agents with differing solubility profiles while enhancing intracellular delivery and reducing systemic toxicity.

[0007] Liposomal drug delivery systems have been investigated in various therapeutic contexts due to their ability to encapsulate both hydrophobic and hydrophilic compounds, protect drugs from premature degradation, and modulate biodistribution. However, the development of a stable liposomal formulation capable of co-encapsulating the complete RIPE drug combination with controlled physicochemical properties and reproducible biological performance presents significant formulation and processing challenges.

[0008] Accordingly, there exists a need for an improved pharmaceutical composition that overcomes the limitations of conventional RIPE therapy by enabling efficient co-encapsulation of anti-tubercular drugs within a single carrier system, enhancing intracellular delivery to infected macrophages, and reducing off-target toxicity, while maintaining formulation stability and manufacturability. The present invention is directed toward addressing these unmet needs.

[0009] To address the above challenges, the present invention provides a liposomal pharmaceutical composition encapsulating a combination of rifampicin, isoniazid, pyrazinamide, and ethambutol (RIPE). The formulation employs liposomes as lipid-based nanocarriers, which enable stable incorporation of both hydrophobic (R) and hydrophilic (IPE) anti -tubercular agents within a single delivery system. Encapsulation of the RIPE drugs within the liposomal carrier facilitates intracellular uptake by macrophage cells, improves antimycobacterial activity, and limits off-target distribution, thereby addressing limitations associated with conventional RIPE formulations.

[0010] SUMMARY OF INVENTION

[0011] In order to address the limitations associated with conventional anti -tubercular drug delivery, the present invention provides a novel liposomal pharmaceutical composition encapsulating a combination of rifampicin, isoniazid, pyrazinamide, and ethambutol (RIPE). The liposomal formulation comprises a lipid bilayer formed from dipalmitoylphosphatidylcholine and

[0012] 3distearoylphosphatidylcholine, wherein rifampicin is associated with the lipid bilayer and the isoniazid, pyrazinamide, and ethambutol drugs are encapsulated within an aqueous core. The formulation is characterized by a nanoscale particle size, controlled surface charge, and high encapsulation efficiency, thereby enabling stable incorporation of both hydrophobic and hydrophilic anti -tubercular agents within a single carrier system.

[0013] The disclosed liposomal composition functions as an advanced drug delivery system capable of improving intracellular uptake by macrophage cells and enhancing antimycobacterial activity relative to non-liposomal RIPE formulations. The formulation demonstrates reduced bacterial growth and intracellular bacterial burden at lower drug concentrations, along with preferential accumulation in lung tissue and an improved systemic safety profile in preclinical evaluations. By integrating multiple first-line anti -tubercular drugs into a single liposomal carrier, the present invention overcomes challenges associated with poor bioavailability, drug hydrophobicity, and off-target toxicity, and provides an improved pharmaceutical composition for the delivery of RIPE drugs.

[0014] In one aspect of the present invention, there is provided a liposomal pharmaceutical composition comprising a lipid bilayer formed from dipalmitoylphosphatidylcholine (DPPC) and distearoylphosphatidylcholine (DSPC), wherein rifampicin is encapsulated within the lipid bilayer and isoniazid, pyrazinamide, and ethambutol are encapsulated within an aqueous core of the liposome. The liposomal composition has a mean hydrodynamic diameter in the range of 100-200 nm and a zeta potential in the range of -18 mV to -22 mV.

[0015] In an embodiment of the invention, the molar ratio of DPPC to DSPC in the lipid bilayer is about 1:1.

[0016] In another embodiment of the invention, the liposomes have a polydispersity index in the range of 0.1 to 0.35.

[0017] In yet another embodiment of the invention, rifampicin is predominantly associated with the lipid bilayer, while isoniazid, pyrazinamide, and ethambutol are predominantly associated with the aqueous core of the liposome.

[0018] 4In a further embodiment of the invention, the encapsulation efficiency of rifampicin is in the range of 75-90%, the encapsulation efficiency of isoniazid is in the range of 65-80%, the encapsulation efficiency of pyrazinamide is in the range of 70-85%, and the encapsulation efficiency of ethambutol is in the range of 75-90%.

[0019] In another embodiment of the invention, the liposomes are spherical in nature.

[0020] In yet another embodiment of the invention, the liposomal pharmaceutical composition is provided in a lyophilized form capable of reconstitution in an aqueous medium.

[0021] In a further embodiment of the invention, the liposomal pharmaceutical composition is capable of enhanced intracellular uptake by macrophage cells.

[0022] In another embodiment of the invention, the liposomal pharmaceutical composition exhibits a lower minimum inhibitory concentration against Mycobacterium tuberculosis H37Rv relative to a non-liposomal RIPE formulation.

[0023] In yet another embodiment of the invention, the liposomal pharmaceutical composition is effective in reducing bacterial growth at sub-minimum inhibitory concentration levels against Mycobacterium tuberculosis H37Rv (in vitro growth kinetics).

[0024] In a further embodiment of the invention, the liposomal pharmaceutical composition is effective to reduce intracellular bacterial burden within macrophage cells infected with Mycobacterium tuberculosis H37Rv.

[0025] In another embodiment of the invention, the liposomal pharmaceutical composition preferentially accumulates in lung tissue following oral administration.

[0026] In yet another embodiment of the invention, the liposomal pharmaceutical composition exhibits reduced systemic toxicity relative to a non-liposomal RIPE formulation, while maintaining hepatic and renal biochemical parameters within normal physiological ranges at doses up to 2000 mg / kg in animal models.

[0027] 5In a further aspect of the present invention, there is provided a process for preparing the liposomal pharmaceutical composition, the process comprising dissolving DPPC, DSPC, and rifampicin in a chloroform-methanol solvent system, removing the solvent to form a thin lipid film, hydrating the lipid film at a temperature of 40-50°C with an aqueous medium comprising isoniazid, pyrazinamide, and ethambutol to form liposomal vesicles, subjecting the vesicles to probe sonication and filtration for size reduction, separating unencapsulated drug by centrifugation, and lyophilizing the resulting liposomal formulation.

[0028] In an embodiment of the process, the aqueous medium used for hydration comprises phosphate-buffered saline prepared using Milli-Q water.

[0029] It is to be understood that the aspects and embodiments of the invention described above may be employed individually or in combination with one another. The foregoing description is illustrative in nature and is not intended to limit the scope of the invention, which will be further apparent from the accompanying drawings and the detailed description that follows.

[0030] BRIEF DESCRIPTION OF DRAWINGS

[0031] In order that the present invention may be more readily understood and put into practical effect, reference will now be made to exemplary embodiments illustrated in the accompanying drawings. The drawings, together with the detailed description provided herein, form an integral part of this specification and serve to illustrate the structural, physicochemical, and functional features of the disclosed liposomal pharmaceutical composition. Like reference numerals, where used, refer to identical or functionally similar elements throughout the drawings. The embodiments described with reference to the drawings are provided solely for the purpose of explanation and are not intended to limit the scope of the present invention.

[0032] Figure 1 illustrates the physicochemical characterization of liposomal formulations, including

[0033] (a) particle size distribution,

[0034] (b) zeta potential measurements, and

[0035] (c) transmission electron microscopy (TEM) images depicting the morphology of liposomes and RIPE-loaded liposomes.

[0036] 6Figure 2 demonstrates Fourier-transform infrared (FTIR) spectra of the liposomal RIPE formulation, elucidating characteristic functional group interactions between lipid components and encapsulated drugs.

[0037] Figure 3 illustrates the biocompatibility and cellular uptake of liposomal formulations in macrophage cells, including

[0038] (a) and (b) cell viability assessment, and

[0039] (c) fluorescence microscopy and (d) flow-cytometry-based uptake of fluorescently labelled liposomes.

[0040] Figure 4 demonstrates comparative in vitro growth inhibition of Mycobacterium smegmatis and Mycobacterium tuberculosis H37Rv following exposure to free RIPE and liposomal RIPE formulations.

[0041] Figure 5 illustrates the growth kinetics of Mycobacterium tuberculosis H37Rv under treatment with liposomal RIPE, free RIPE, and control formulations at sub -minimum inhibitory concentration levels.

[0042] Figure 6 demonstrates intracellular bacterial burden in macrophage cells infected with Mycobacterium tuberculosis H37Rv following treatment with liposomal RIPE and comparator formulations.

[0043] Figure 7 illustrates the in vivo and ex vivo biodistribution of fluorescently labelled liposomal formulations in animal models following oral administration.

[0044] Figure 8 demonstrates the acute toxicity profile of liposomal RIPE, free RIPE, and liposome control formulations, as reflected by serum biochemical parameters indicative of hepatic and renal function.

[0045] It may be noted that, to the extent applicable, like reference numerals have been used to represent corresponding elements in the accompanying drawings. The drawings are provided for illustrative purposes and are not necessarily drawn to scale, and certain features may be shown in an enlarged or simplified manner to facilitate understanding of the present

[0046] 7invention. In particular, the drawings are intended to depict those structural and compositional features of the liposomal formulation that are pertinent to explaining the disclosed embodiments, while omitting details that would be readily apparent to a person skilled in the art in view of the description provided herein.

[0047] DETAILED DESCRIPTION

[0048] 1. Introduction

[0049] Human tuberculosis (TB), caused by the bacterium Mycobacterium tuberculosis Mtb), continues to be a significant global health concern, affecting millions worldwide and ranking among the top causes of death from infectious diseases. Despite the availability of effective treatment regimens, TB management remains challenging due to several factors, including the prolonged duration of therapy, systemic toxicity, poor patient adherence, and the emergence of drug-resistant strains oiMtb. The cornerstone of TB therapy, the RIPE regimen — comprising Rifampicin (R), Isoniazid (I), Pyrazinamide (P), and Ethambutol (E) — has been the gold standard for decades. However, suboptimal bioavailability, off-target effects, and the inability to effectively penetrate intracellular Mtb reservoirs necessitate innovative therapeutic strategies to enhance efficacy and compliance while minimizing adverse effects.

[0050] TB primarily targets the lungs but can disseminate to extrapulmonary sites, requiring a systemic and targeted treatment approach. The intracellular survival of Mtb within macrophages poses a critical challenge, as conventional drugs often fail to achieve adequate concentrations within these host cells. Furthermore, the hydrophobic nature of many anti-TB drugs compromises their bioavailability and therapeutic index. Consequently, there is a pressing need for advanced drug delivery systems that can overcome these snags, ensuring efficient drug delivery, enhanced therapeutic outcomes, and reduced systemic toxicity.

[0051] The present invention accordingly relates to a novel liposomal pharmaceutical composition encapsulating a combination of rifampicin, isoniazid, pyrazinamide, and ethambutol (RIPE). The disclosed formulation employs liposomes as lipid-based nanocarriers to enable stable incorporation of both hydrophobic and hydrophilic anti -tubercular agents within a singledelivery system. The liposomal composition is configured to facilitate intracellular uptake by macrophage cells, improve antimycobacterial activity at reduced drug concentrations, and limit off-target distribution, thereby addressing key limitations associated with conventional RIPE formulations.

[0052] The materials employed, methods of preparation, in-vitro and in-vivo evaluations, and the results obtained are described in detail hereinafter.

[0053] 2. Materials

[0054] DPPC (Dipalmitoyl phosphatidyl choline) and DSPC (Distearoyl phosphatidyl choline) lipids (>99% purity) were procured from Lipoid (Germany) and used as received without further purification. The antitubercular drugs RIPE, fluorescein isothiocyanate (FITC), 4', 6-diamidino-2-phenylindole (DAPI), and resazurin were procured from Sigma Aldrich. High-performance liquid chromatography (HPLC) grade chloroform for stock solution preparation was obtained from E. Merck. RPMI media and Fetal Bovine Serum (FBS) were obtained from Gibco, Life Technologies Ltd. All aqueous solutions were prepared using Milli-Q water, which had a resistivity of 18.2 M’Q cm'1.

[0055] 3. Methods

[0056] 3.1 Preparation of LIPO-RIPE formulations

[0057] LIPO-RIPE were prepared using a modified thin-film hydration method. Lipids (ratio of DPPC / DSPC: 10 mg / 10 mg) and the hydrophobic drug ‘R’ (5 mg) were dissolved in a 3:1 chloroform-methanol mixture. A thin lipid film was formed after evaporating the solvents at 40 °C using a rotary evaporator. This film was hydrated at 45 °C with phosphate buffer saline (PBS), with continuous shaking to form vesicular structures. Hydrophilic drugs of each 5 mg of ‘I’, ‘P’, and ‘E’ were added to the aqueous phase during hydration for encapsulation into the LIPO core.

[0058] The dispersion underwent probe sonication and filtration for size reduction and uniformity. Purification by centrifugation removed unencapsulated drugs, and the resulting pellet was collected. Finally, the pellet was lyophilized to produce a stable, dry formulation suitable for storage and further application.

[0059] 93.1.1 Physicochemical characterization of Liposomal formulations

[0060] The synthesized void LIPO and LIPO-RIPE were characterized for their size, charge, morphology, and functional groups before proceeding with biological evaluation. Dynamic light scattering (Malvern Zetasizer) was used to determine the hydrodynamic size and zeta potential of both the void LIPO and LIPO-RIPE. Transmission electron microscopy (TALOS, 200 kV) was used to examine the size and surface morphology. The chemical composition of the LIPO-RIPE was analyzed using Fourier-transform infrared spectroscopy (FTIR).

[0061] 3.1.2 Encapsulation Efficiency (EE)

[0062] The LIPO-RIPE formulation was centrifuged at 25,000 x g for 15 min at 4 °C. The supernatant was analyzed for free drugs (RIPE) content using HPLC. The amount of drugs in the supernatant (w) was subtracted from the total drugs initially added during LIPO formulation (W). The difference (W-w) corresponded to the amount of drugs encapsulated within the LIPO-RIPE, which was collected in the pellet. Care was taken throughout the procedure to prevent drug precipitation or sedimentation, and the aqueous solubilities of the drugs were accounted for to minimize errors. The EE was calculated as a percentage of the total drugs entrapped using the formula:

[0063] 100

[0064]

[0065] 3.2 In-vitro Study

[0066] 3.2.1 Cell Culture Maintenance

[0067] RAW 264.7 cells were cultured in RPMI medium supplemented with 10% heat-inactivated FBS, 100 pM penicillin-streptomycin, 50 pM neomycin, and 200 mM glutamine. Cells were maintained at 37 °C with 5% CO2 and passaged every 2-3 days, maintaining density below 1 million cells / ml. Cultures were mycoplasma-free, and experiments were conducted at a density of 104cells / ml.

[0068] 3.2.2 In-vitro Cytotoxicity AssayLog-phase cells (104cells / well) were seeded in 96-well plates and treated with LIPO-RIPE, RIPE, and LIPO for 72 hr. After incubation, alamar Blue (10% of the media volume) was added, and the plates were incubated for 4-8 hr. Viability was assessed via absorbance at 570 nm using an ELISA plate reader. Assays were performed in biological duplicates.

[0069] 3.2.3 Cellular Uptake Assay

[0070] FITC-labelled LIPO (FITC-LIPO) was used to study cellular uptake. Cells seeded on coverslips for 24 hr were treated with FITC-LIPO, washed, fixed, stained with DAPI, and imaged via confocal microscopy. For flow cytometry, treated cells were washed, centrifuged, resuspended in PBS, and analysed using a BD FACS Aria II flow cytometer with Flowlo software.

[0071] 3.3 Bacterial Strains and Culture Conditions

[0072] All experimental procedures were conducted using two strains: the non-pathogenic Mycobacterium smegmatis mc2155 (Msmeg) and the virulent Mtb H37Rv. Bacterial cultures were maintained in Middlebrook 7H9 broth (Difco) supplemented with 10 % (v / v) Oleic acid, Albumin, Dextrose, and Catalase (OADC), 0.5% (v / v) glycerol, and 0.05 % (v / v) Tween-20. Cultures were incubated at 37 °C under shaking (100-120 rpm) to ensure optimal aeration and homogeneity.

[0073] 3.3.1 Minimum Inhibitory Concentration (MIC) Assay

[0074] The antimycobacterial efficacy of the liposomal RIPE formulation (LIPO-RIPE) and the free RIPE combination was assessed using the resazurin microtiter assay. Briefly, Msmeg and Mtb H37Rv log-phase cultures (ODeoo ~ 0.5) were resuspended by passing it for 10-15 times through a 26.5 gauze needle to obtain single-cell suspensions and diluted to an ODeoo of 0.001 (~2 x io4CFU / well). The bacterial suspensions were seeded into sterile 96-well flatbottom microtiter plates.

[0075] LIPO-RIPE formulations were reconstituted in sterile deionized water, and free RIPE drugs were prepared in sterile water at equivalent concentrations to match the RIPE content in

[0076] 11LIPO-RIPE. Both formulations were subjected to two-fold serial dilutions ranging from 50 pg / ml down to 0.39 pg / ml across the wells. Plates were incubated at 37 °C for 72 hr.

[0077] Following incubation, 0.02% resazurin solution was added to each well, and plates were further incubated for 12 - 24 hr. The MIC was determined as the lowest concentration at which no color change from blue (oxidized resazurin) to pink (reduced resorufin) was observed, indicating complete inhibition of bacterial growth.

[0078] 3.3.2 Growth Kinetics of Mtb H37Rv

[0079] To evaluate the growth kinetics, Mtb H37Rv was revived from glycerol stocks and inoculated into 10 ml of 7H9 broth supplemented with OADC. Cultures were grown to an ODeoo of 0.6, after which a large-scale primary culture was set up from the revival culture.

[0080] The primary culture was then further diluted to ODeoo = 0.05 and subjected to different treatments: (i) 0.25X void LIPO, (ii) free 0.25X RIPE, and (iii) 0.25X LIPO-RIPE. Bacterial growth was monitored over time by measuring ODeoo at regular intervals. Growth curves were plotted to determine the impact of each treatment.

[0081] 3.3.3 Determination of Intracellular Bacterial Burden (CFU Assay)

[0082] Differentiated THP-1 macrophages and primary macrophages (RAW 264.7 cells) were infected with Mtb H37RV at the predetermined multiplicity of infection (MOI: 15) under biosafety level-3 (BSL-3) conditions. After 4 hr of infection and removal of extracellular bacteria, cells were treated with different concentrations (12.5, 25, 50, 100, and 200 pg / ml) of LIPO, RIPE, and LIPO-RIPE formulations for 72 hr. Untreated (UT) infected cells served as controls. Following treatment, macrophages were lysed using 0.05% SDS to release intracellular bacteria, and appropriate serial dilutions of the lysates were plated on Middlebrook 7H10 agar supplemented with OADC. Plates were incubated until visible colonies appeared. The bacterial burden was calculated as CFU / ml using the formula:

[0083] (Number of colonies X Dilution factor')

[0084] CFU / ml = - - - — — - - - - Volume plated (ml)

[0085] 12Each sample was plated in duplicate, and the average CFU / ml was calculated from three independent experiments. Data were expressed as mean ± standard deviation (SD). All infection experiments were carried out in compliance with institutional biosafety and ethical guidelines within a certified BSL-3 facility.

[0086] 3.4 In Vivo Evaluation of LIPO-RIPE

[0087] 3.4.1 Biodistribution

[0088] To evaluate the biodistribution of the liposomal formulation, LIPO was labeled with the nearinfrared lipophilic dye DiR (l,l-dioctadecyl-3,3,3',3'-tetramethylindotricarbocyanine iodide). The labeled LIPO was administered to Balb / c mice via 25 mg / kg oral administration. In vivo fluorescence imaging was conducted using the in vivo imaging spectrum (IVIS) System at 4 hr intervals post-injection to monitor the real-time distribution of the formulation. Fluorescence intensity was observed in major organs, including the lungs, liver, spleen, heart, and kidney, using region-of-interest (ROI) analysis. Following imaging, mice were sacrificed, and ex vivo fluorescence imaging of excised organs was performed to validate in vivo findings.

[0089] 3.4.3 Acute Toxicity Studies

[0090] The acute toxicity profile of LIPO, RIPE, and LIPO-RIPE formulations was assessed in Balb / c mice following the Organization for Economic Cooperation and Development guideline 423 for acute oral toxicity testing. Mice were randomly divided into groups (n = 3 per group) and administered a single oral dose of each formulation at graded concentrations of 50, 100, 300, and 2000 mg / kg body weight. Animals were monitored continuously for the first 24 h post-administration and thereafter daily for 14 days for clinical signs of toxicity, including changes in body weight, grooming, feeding behaviour, posture, locomotor activity, and mortality. Blood samples were collected from the retro-orbital plexus on day 14 to perform biochemical assays to evaluate hepatic and renal function.

[0091] 4. Results and Discussion:

[0092] Synthesis and Characterization of LIPO-RIPE

[0093] 13The LIPO formulations used in this study, encapsulating four anti -tubercular drugs (RIPE), were synthesized using the thin-film hydration method. The successful loading of RIPE into the LIPO was evidenced by significant changes in both zeta potential and particle size.

[0094]

[0095]

[0096] Figure 1. Characterization of LIPO and LIPO-RIPE formulations, (a) Particle size and (b) zeta potential were measured using a Malvern Zetasizer Nano ZS. (c) Morphological features were visualized by transmission electron microscopy (TEM).

[0097] 14Dynamic light scattering measurements showed an increase in size from 95.34 ± 7.2 nm (PDI: 0.185 ± 0.02) to 127.2 nm ± 9.23 nm (PDI: 0.314 ± 0.13) post-loading (Figure la). Concurrently, the zeta potential shifted from -14.0 ± 1.9 to -20.4 ± 0.11 mV, as shown in Figure lb. The enhanced stability of LIPO under storage conditions likely facilitates macrophage uptake through interaction with scavenger receptors and negatively charged particles. Morphological analysis via transmission electron microscopy (TEM) confirmed the spherical structure and uniform distribution of the LIPO and LIPO-RIPE (Figure 1c).

[0098]

[0099] Figure 2. FTIR spectra of LIPO-RIPE formulation. The FTIR spectra were recorded to assess the molecular interactions and confirm the encapsulation of RIPE components within the liposomal matrix. Characteristic absorption bands corresponding to functional groups of both the lipids and RIPE constituents were observed. Shifts or changes in peak intensities, compared to individual components, indicate potential interactions between the drug and lipid bilayer, confirming successful incorporation of RIPE into the liposomal formulation.The average particle size of the LIPO-RIPE formulations (100-200 nm) is ideal for deep lung deposition and effective uptake by macrophages. The FTIR spectrum of LIPO-RIPE (Figure 2) confirmed the presence of the lipid bilayer, encapsulated drugs, and their interactions. Broadening or shifting of O-H and N-H bands in the 3200-3500 cm'1region, indicate hydrogen bonding between the lipid bilayer and drug molecules (RIPE). Shifts in the C=O (1730-1740 cm'1) and PO2' (1240-1250 cm'1) stretching bands, suggesting interactions between the drugs and lipid head groups. The O-H Stretch (Hydration Layer), a broad band around 3200-3500 cm'1, reflects water molecules associated with the LIPO surface, crucial forLIPO stability. Fingerprint Region (600-1500 cm'1), revealed complex vibrations from C-C, C-O, and N-N bonds within both the lipid bilayer and RIPE components, providing structural validation.

[0100] Drug loading and encapsulation performance

[0101] EE represents the percentage of the drugs encapsulated relative to the initial total drugs in solution. Among the four anti-tubercular drugs tested, ‘E’ and ‘R’ demonstrated the highest EE, followed by ‘I’, with ‘P’ showing the lowest. Specifically, ‘R’ encapsulation was around 82.12 % ± 3.27 %, "I" entrapment reached 71.12 % ± 4.15 %, ‘E’ and ‘P’ achieved 81.32 % ± 7.16 % and 74.28 % ± 3.54 % respectively, with values presented as mean ± standard deviation (n = 3). The thin-film hydration method yielded multilamellar LIPO capable of encapsulating significant amounts of hydrophobic drugs within the lipid bilayers. ‘R’, a highly hydrophobic drug with a high partition coefficient, preferentially integrates into the lipid bilayer, avoiding the aqueous phase. In contrast, ‘I’, the most hydrophilic drug with the lowest partition coefficient, remains primarily in the aqueous phase. ‘E’, with lower solubility than ‘I’ and a relatively higher partition coefficient, achieves greater EE than both ‘I’ and ‘P’. These variations highlight the influence of drug properties on EE.

[0102] Biocompatibility Study

[0103] The cytotoxicity of the LIPO-RIPE on RAW 264.7 cells was assessed using the Alamar Blue assay. After 72 hr of treatment, the percent cell viability ranged between 80% and 90% for all concentrations of LIPO-RIPE, void LIPO, and free RIPE, as shown in Figures 3a and 3b. These results indicated that the LIPO exhibited minimal cytotoxicity, suggesting their potential as safe and effective carriers for RIPE in a targeted drug delivery approach.

[0104] 16Efficient cellular uptake of LIPO

[0105] Fluorescently labelled LIPO was used to study cellular uptake in RAW 264.7 cells (macrophage cell line). Confocal microscopy and flow cytometry analysis were also performed to qualitatively and quantitatively assess the efficient uptake of FITC-labelled LIPO by RAW 264.7 cells. According to confocal images, DAPI staining of the nucleus emits blue light, and FITC-loaded LIPO emits green fluorescence. After the different time points of incubation (2, 4, 6 hr), the FITC-loaded LIPO were effectively internalized and localized inside the cytoplasm and near the nucleus in comparison to the successive time points, as shown in Figure 3c. Flow cytometry analysis further confirmed the complete uptake of FITC-loaded LIPO at successive time points (Figure 3d). These findings confirmed that LIPO-RIPE has targeted delivery capabilities.

[0106]

[0107] Figure 3. Biocompatibility and cellular uptake of FITC-labeled LIPO by macrophages.

[0108] Results are expressed as mean ± SD from three independent experiments, each with three

[0109] 17technical replicates (n = 3). (a) Cell viability of RAW 264.7 cells were evaluated in the presence of LIPO, LIPO-RIPE, and RIPE using the Alamar blue assay, (b) confirming minimal cytotoxicity of the LIPO and LIPO-RIPE formulation. The cells were incubated with FITC-labeled LIPO to assess uptake efficiency using (c) fluorescence microscopy and (d) flow cytometry, which revealed efficient internalization of FITC-LIPO, as evidenced by the green fluorescence localized within the cytoplasm of macrophage cells, indicating successful cellular uptake.

[0110] In vitro Release Profile and Characterization of LIPO-RIPE

[0111] To ensure an accurate comparison, the concentration of the free RIPE drugs was standardized to match the equivalent amount of free RIPE drugs loaded within the LIPO. Serial dilutions of both formulations were prepared over a concentration range of 50 pg / ml to 0.39 pg / ml, ensuring uniform exposure of bacterial cultures to equivalent drug doses in both free RIPE and RIPE in LIPO forms (LIPO-RIPE). This design allowed for the precise evaluation of therapeutic efficacy attributable solely to liposomal encapsulation rather than dosage variations.

[0112] To evaluate cytotoxicity and antimicrobial specificity, an in vitro assay was performed against two bacterial models: Msmeg, a non-virulent, fast-growing homologue, and H37Rv, a virulent clinical strain. The Alamar Blue assay, which measures bacterial metabolic activity as an indicator of growth and viability, was employed to determine drug-mediated inhibition across the tested concentration range.

[0113] 18

[0114]

[0115] Figure 4. Comparative growth inhibition of Msmeg and Mtb by RIPE in LIPO and free RIPE, assessed using the Alamar Blue assay. The antimicrobial efficacy of the RIPE in LIPO was compared to that of free RIPE against Msmeg and Mtb H37Rv strains. Bacterial cultures were treated with equivalent concentrations of each formulation, and metabolic activity was evaluated using the Alamar Blue assay. A decrease in colour intensity indicated reduced bacterial viability. LIPO-RIPE demonstrated enhanced inhibitory effects compared to free RIPE, suggesting improved delivery and potency of the encapsulated drugs. Results are expressed as mean ± SD from three independent experiments, each with three technical replicates (n = 3).

[0116] (LIPO-RIPE) RIPE in LIPO exhibited approximately 75% greater efficacy in H37Rv cultures and 50% enhanced efficacy in Msmeg compared to the free RIPE combination (Figure 4). The observed antimicrobial activity reflected a pronounced reduction in the MIC when drugs were delivered in liposomal form, indicating a significant improvement in potency.

[0117] This superior performance of LIPO-RIPE can be attributed to the unique pharmacological advantages conferred by liposomal encapsulation. Liposomes enhance drug stability, prolong circulation time, and facilitate efficient penetration into host cells harboring intracellular mycobacteria. Moreover, the lipid bilayer aids in improved accumulation and retention within the mycobacterial cell wall, a major barrier to conventional free drugs. Collectively, these

[0118] 19factors synergistically optimize drug delivery, resulting in enhanced bactericidal efficacy compared to free RIPE.

[0119] Growth Kinetics of Mtb under RIPE and LIPO-RIPE Treatment

[0120] To evaluate the impact of liposomal encapsulation on the efficacy of anti-TB drugs, we assessed the growth kinetics of H37Rv under various treatment conditions: (i) LIPO alone; (black) (ii) free RIPE at 0.25X MIC (red), (iii) LIPO-RIPE at 0.25X MIC (blue), and (iv) untreated wild-type H37Rv control (green) as shown in Figure 5.

[0121] As expected, the untreated control exhibited a typical logarithmic growth curve, indicating the robust viability and proliferation of the strain under standard in vitro conditions. Similarly, the LIPO-only group showed no inhibitory effect on bacterial growth, confirming the biocompatibility and inertness of the LIPO carrier in the absence of drug loading. In contrast, the LIPO-RIPE formulation at just 0.25X MIC exerted a significant and sustained inhibitory effect on bacterial growth, with a marked decline observed from the first day of treatment. The growth remained staggered and consistently suppressed throughout the 18-day observation period.

[0122]

[0123] Figure 5. Growth kinetics of Mtb H37Rv under different treatment conditions. Results are expressed as mean ± SD from three independent experiments, each with three technical replicates (n = 3). Statistical significance: *p<0.05, **p<0.01, ***p<0.001 vs Control; ###p<0.001 between RIPE and LIPO-RIPE (One-way ANOVA with Tukey’s post hoc test).This outcome suggests enhanced intracellular delivery and retention of anti-TB drugs via liposomal encapsulation, resulting in early and sustained bactericidal activity, even at sub-therapeutic concentrations.

[0124] On the other hand, the free RIPE treatment at the same 0.25X MIC exhibited only marginal growth suppression. Although a slight reduction in bacterial proliferation was noted initially, the culture gradually resumed growth over time, indicative of a limited bacteriostatic response. The stark contrast in outcomes between free RIPE and LIPO-RIPE at identical sub-MIC doses highlights the potent therapeutic efficacy of the liposomal formulation, which may allow for dose sparing without compromising antibacterial potency.

[0125] Quantification of Intracellular Bacterial Survival after Treatment

[0126] To evaluate the intracellular bactericidal potential of the formulations, CFU assays were performed after 72 hr of treatment in Mtb H37Rv-infected THP-1 and RAW 264.7 macrophages, as shown in Figure 6.

[0127]

[0128] Figure 6. Intracellular bacterial survival in THP-1 and RAW 264.7 macrophages following treatment with different formulations. Mtb H37Rv-infected THP-1 and RAW 264.7 macrophages were treated with LIPO, RIPE, or LIPO-RIPE formulations (12.5-200 pg / ml) for 72 hr. Intracellular bacterial burden was quantified by CFU assay and expressed as CFU / ml. The untreated (UT) group showed the highest bacterial load, whereas LIPO-RIPE treatment significantly reduced CFU counts compared to LIPO and RIPE groups (p < 0.05). Results are expressed as mean ± SD from three independent experiments, each with three technical replicates (n = 3).

[0129] 21In the UT control group, bacterial burden remained high, averaging approximately (3.6 ± 2.1) x IO10CFU / ml in THP-1 macrophages and (6.8 ± 1.7) x 104CFU / ml in RAW 264.7 macrophages. Treatment with the only LIPO resulted in a marginal, concentration-dependent reduction in bacterial load, reaching (3.25 ± 0.2) x 1O10CFU / ml in THP-1 cells and (5.7 ± 0.9) x io4CFU / ml in RAW 264.7 cells at 200 pg / ml. In contrast, treatment with the conventional RIPE formulation exhibited greater bactericidal efficacy, decreasing CFU counts to approximately (1.3 ± 0.2) x 108CFU / ml in THP-1 cells and (1.0 ± 0.2) x 104CFU / ml in RAW 264.7 macrophages at the same concentration. Notably, the LIPO-RIPE formulation demonstrated the most pronounced intracellular killing effect, reducing bacterial counts to (1.25 ± 0.3) x 105CFU / ml in THP-1 macrophages and (1.5 x 103CFU / ml) in RAW 264.7 macrophages, corresponding to an approximate 6 to 7 fold reduction compared to the UT group. The decline in CFU was statistically significant for LIPO-RIPE treatment at concentrations >25 pg / ml compared with both LIPO and RIPE formulations (p < 0.05). Collectively, these findings indicate that the LIPO-RIPE formulation significantly enhances intracellular bacterial clearance in macrophages, underscoring its superior synergistic antimicrobial efficacy against Mtb compared to LIPO or RIPE alone.

[0130] Biodistribution Profile of DiR-Labeled LIPO

[0131] To assess the in vivo distribution and organ-specific accumulation of the LIPO formulation, DiR-labeled LIPO was administered orally, and fluorescence imaging was conducted both in vivo and ex vivo using the IVIS system (Figure 7). At 4 hr post-injection, ex vivo fluorescence imaging of excised organs revealed a predominant accumulation of the DiR signal in the lungs, indicating a strong tropism of the LIPO formulation toward pulmonary tissue. This preferential localization suggests that the LIPO formulation possesses inherent or formulation-induced lung-targeting capabilities, which are particularly advantageous for the treatment of TB.

[0132] In comparison, the fluorescence intensity detected in the liver was significantly lower, likely due to partial hepatic uptake via the reticuloendothelial system (RES), a common clearance route for nanocarriers. Negligible signal was observed in other major organs, including the heart, spleen, and kidneys, indicating minimal off-target distribution.

[0133] 22Interestingly, in vivo whole-body imaging showed a prominent DiR signal in the abdominal region, which raised the possibility of gut-associated accumulation or nonspecific signal interference. However, this observation was not substantiated upon ex vivo analysis, as the gastrointestinal organs did not exhibit significant fluorescence intensity. This disparity suggests that the abdominal fluorescence observed in vivo may be due to transient systemic circulation, background tissue absorption, or artifacts associated with skin and fur autofluorescence in live imaging.

[0134] Taken together, the biodistribution profile clearly supports preferential accumulation of the LIPO formulation in lung tissue, reinforcing its potential utility as a targeted drug delivery system for TB. The limited distribution to other organs further underscores the safety of formulations and organ-specific targeting, which could reduce systemic toxicity and enhance therapeutic efficacy.

[0135]

[0136] Figure 7. In vivo and ex vivo biodistribution of DiR labelled LIPO in Balb / c mice. In vivo fluorescence imaging was performed on Balb / c mice to visualize real-time biodistribution of fluorescently labelled LIPO 4 hr post-administration. (A-D) Correspond to individual mice imaged in vivo, followed by ex vivo imaging of excised organs from the same animals. Organs are labeled as follows: 1 - Kidney, 2 - Lungs, 3 - Spleen, 4 - Liver, 5 - Heart. All experiments were performed once, and data represent mean ± SD obtained from four biological replicates (individual animals) within the same experiment.

[0137] Acute Toxicity Analysis: Serum Biochemical Markers (Urea, Creatinine, SGOT, and SGPT)To assess the acute toxicity profile, BALB / c mice were orally administered a single dose of RIPE, LIPO, and LIPO-RIPE formulations at graded concentrations (50, 100, 300, and 2000 mg / kg body weight). Animals were monitored for 14 days for survival, physiological behavior, and biochemical alterations. All mice treated with LIPO and LIPO-RIPE formulations survived the entire observation period without any behavioural abnormalities such as tremors, lethargy, or appetite loss, indicating excellent tolerability up to 2000 mg / kg. In contrast, mild signs of slowness were noted in the RIPE -treated group at the highest dose, though no mortality occurred, confirming that the LD50 value for all formulations was greater than 2000 mg / kg body weight. Serum biochemical analyses were performed to evaluate hepatic and renal function, using urea, creatinine, SGOT (serum glutamic-oxaloacetic transaminase), and SGPT (serum glutamic-pyruvic transaminase) as indicators, as shown in Figure 8. The normal physiological ranges reported for BALB / c mice are: urea (20-45 mg / dL), creatinine (0.2-0.8 mg / dL), SGOT (45-120 U / L), and SGPT (25-90 U / L).

[0138] 24

[0139]

[0140] Figure 8. Acute Toxicity Profile of LIPO, RIPE, and LIPO-RIPE in BALB / c Mice.

[0141] Serum biochemical markers were analyzed to assess acute toxicity, (a-c) Urea levels decreased in all treated groups compared to the Control, (d-f) Creatinine levels significantly increased in the RIPE group compared to LIPO and LIPO-RIPE. (g-i) SGPT and (j-1) SGOT levels were elevated in RIPE, while LIPO-RIPE showed minimal changes, indicating better hepatic and renal tolerance. Five animals were used per group (n = 4). All experiments were performed once, and data represent mean ± SD obtained from five biological replicates (individual animals) within the same experiment.

[0142] Compared to the untreated control group, the serum urea levels declined across all treated groups (RIPE, LIPO, and LIPO-RIPE), suggesting that the treatments did not induce renaloveractivity or hypercatabolic effects. However, a significant increase in serum creatinine was observed in the RIPE-treated mice, particularly at higher doses, indicating a mild renal stress response likely due to the systemic exposure of free drug components (Figure 8a, 8b, 8c). In contrast, creatinine levels in the LIPO and LIPO-RIPE groups remained within the normal range, comparable to untreated controls, demonstrating effective renal protection conferred by liposomal encapsulation (Figure 8d, 8e, 8f).

[0143] Similarly, hepatic enzymes (SGOT and SGPT) showed marked elevation in the RIPE-treated group, signifying mild hepatocellular stress. This elevation was notably attenuated in LIPO and LIPO-RIPE groups, where enzyme levels remained close to the baseline physiological range. The minimal alteration in transaminase levels for LIPO and LIPO-RIPE indicates reduced hepatic burden and improved systemic tolerance (Figure 8g, 8h, 8i, 8j, 8k, 81).

[0144] Overall, the biochemical findings corroborate the behavioral observations, revealing that LIPO and LIPO-RIPE formulations exhibit better biocompatibility and negligible hepatic or renal toxicity compared to free RIPE. The encapsulation of RIPE drugs in liposomes effectively stabilized enzymatic and metabolic profiles, maintaining near-physiological homeostasis. Collectively, these results confirm that the LD50 values for RIPE, LIPO, and LIPO-RIPE exceed 2000 mg / kg, underscoring the high safety margin and reduced systemic toxicity of the liposomal formulations.

[0145] 5. Conclusion and Implications:

[0146] The LIPO-RIPE formulation was designed to enhance the efficacy of conventional RIPE therapy by improving targeted biodistribution and intracellular delivery. In vitro studies demonstrated potent antimycobacterial activity of liposomal formulations against Mtb H37Rv and Msmeg, showing significant growth inhibition compared to standard treatments. Preliminary in vivo assessments indicated efficient biodistribution with preferential lung accumulation and, importantly, no signs of hepatic or renal toxicity at the tested dose, confirming the formulation’s safety profile. Future studies will focus on determining the therapeutic potential of LIPO-RIPE in infected models to validate its efficacy in vivo and assess its ability to reduce treatment duration and improve patient compliance.

[0147] 26One of the advantages of the present invention is that the disclosed liposomal pharmaceutical composition enables stable co-encapsulation of both hydrophobic and hydrophilic anti-tubercular drugs within a single carrier system, thereby improving formulation uniformity and stability. The lipid-based encapsulation further facilitates intracellular uptake by macrophage cells and limits premature drug degradation and off-target distribution.

[0148] Another advantage of the invention lies in the nanoscale size and controlled surface characteristics of the liposomal formulation, which contribute to improved interaction with infected cells and reduced bacterial regrowth at lower drug concentrations. The formulation thereby enables enhanced antimycobacterial activity while potentially reducing the overall drug burden required to achieve the desired biological effect.

[0149] Still another advantage of the invention is the improved systemic safety profile exhibited by the liposomal RIPE formulation. Encapsulation of the RIPE drugs within liposomes reduces direct exposure of free drugs to non-target tissues, thereby mitigating hepatic and renal toxicity relative to non-liposomal formulations, as reflected by maintained biochemical parameters within physiological ranges in preclinical studies.

[0150] While specific language has been used herein to describe the disclosed embodiments, no limitation is intended thereby. As will be apparent to a person skilled in the art, various modifications, substitutions, and variations may be made to the composition and process described herein without departing from the inventive concept disclosed.

[0151] Although particular emphasis has been placed on certain features of the disclosed embodiments, it will be appreciated that various changes and modifications may be made without departing from the principles of the invention. Such modifications and variations will be apparent to those skilled in the art upon reading the present disclosure, and the foregoing description is to be construed as illustrative rather than limiting.

[0152] The embodiments described herein and the associated advantageous features are explained with reference to non-limiting examples. Descriptions of well-known materials, components, and processing techniques have been omitted where appropriate so as not to unnecessarily obscure the disclosed embodiments. The examples provided herein are intended solely to

[0153] 1facilitate understanding and enable practice of the invention and should not be construed as limiting the scope of the invention.

[0154] The foregoing description of specific embodiments fully reveals the general nature of the invention such that others skilled in the art can, by applying current knowledge, readily modify and adapt the disclosed embodiments for various applications without departing from the inventive concept. Such adaptations and modifications are intended to fall within the scope and spirit of the invention and its equivalents. The terminology employed herein is for the purpose of description and not limitation.

[0155] Any discussion of documents, materials, methods, or techniques in this specification is included solely to provide context for the present disclosure and should not be construed as an admission that such material forms part of the prior art or was common general knowledge before the priority date of this application.

[0156] The numerical values and ranges specified for various parameters, properties, and quantities are intended to be approximate, and it is envisaged that values higher or lower than those expressly stated may fall within the scope of the invention, unless the specification expressly indicates otherwise.

Claims

WE CLAIM:

1. A liposomal pharmaceutical composition comprising:(a) a lipid bilayer comprising dipalmitoylphosphatidylcholine (DPPC) and distearoylphosphatidylcholine (DSPC);(b) rifampicin encapsulated within the lipid bilayer; and(c) isoniazid, pyrazinamide, and ethambutol encapsulated within an aqueous core of the liposome,wherein the liposomal composition has:(i) a mean hydrodynamic diameter in the range of 100-200 nm; and(ii) a zeta potential in the range of -18 mV to -22 mV.

2. The liposomal pharmaceutical composition as claimed in claim 1, wherein the DPPC to DSPC molar ratio is about 1:1.

3. The liposomal pharmaceutical composition as claimed in claim 1, wherein the liposomes have a poly dispersity index (PDI) of 0.1 to 0.35.

4. The liposomal pharmaceutical composition as claimed in claim 1, wherein rifampicin is predominantly associated with the lipid bilayer, and isoniazid, pyrazinamide and ethambutol are predominantly associated with the aqueous core.

5. The liposomal pharmaceutical composition as claimed in claim 1, wherein the encapsulation efficiency of:(a) rifampicin is 75-90%;(b) isoniazid is 65-80%;(c) pyrazinamide is 70-85%; and(d) ethambutol is 75-90%.The liposomal pharmaceutical composition as claimed in claim 1, wherein the liposomes are spherical.

7. The liposomal pharmaceutical composition as claimed in claim 1, wherein the composition is in a lyophilized form capable of reconstitution in an aqueous medium.

8. The liposomal pharmaceutical composition as claimed in claim 1, wherein the composition is capable of enhanced intracellular uptake by macrophage cells.

9. The liposomal pharmaceutical composition as claimed in claim 1, wherein the composition exhibits a lower minimum inhibitory concentration against Mycobacterium tuberculosis H37Rv relative to a non-liposomal RIPE formulation.

10. The liposomal pharmaceutical composition as claimed in claim 1, wherein the composition is effective to reduce bacterial growth at sub-minimum inhibitory concentration levels against Mycobacterium tuberculosis H37Rv.

11. The liposomal pharmaceutical composition as claimed in claim 1, wherein the composition is effective to reduce intracellular bacterial burden within macrophage cells infected with Mycobacterium tuberculosis H37Rv.

12. The liposomal pharmaceutical composition as claimed in claim 1, wherein the composition preferentially accumulates in lung tissue following oral administration.

13. The liposomal pharmaceutical composition as claimed in claim 1, wherein the composition exhibits reduced systemic toxicity relative to a non-liposomal RIPE formulation, while maintaining hepatic and renal biochemical parameters within normal physiological ranges at doses up to 2000 mg / kg in animal models.

14. A process for preparing the liposomal pharmaceutical composition as claimed in claim 1, comprising:(a) dissolving DPPC, DSPC and rifampicin in a chlorofornrmethanol solvent system; (b) removing the solvent to form a thin lipid film;(c) hydrating the lipid film at 40-50°C with an aqueous medium comprising isoniazid, pyrazinamide and ethambutol to form liposomal vesicles;(d) subjecting the vesicles to probe sonication and filtration for size reduction;(e) separating unencapsulated drug by centrifugation; and(f) lyophilizing the resulting liposomal formulation.3015. The process as claimed in claim 14, wherein the aqueous medium comprises phosphate-buffered saline prepared using Milli-Q water.Dated this 21st day of January, 2025.Registration No. IN-PA / 810 OF KUMAR & SARDANA ASSOCIATES ATTORNEY FOR THE APPLICANTS

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