1. A drug loaded nanostructured lipid carrier comprising, a) clarithromycin (drug), b) compritol 888 ATO, c) surfactant, d) lipid material, e) stearic acid.

2. The composition as claimed in claim 1, wherein surfactant is selected from Pluronic F-127, Pluronic F-68 and Tween-80, preferably Pluronic F 68 (PF-68).

3. The composition as claimed in claim 1, wherein the lipid material is soya lecithin.

4. The composition as claimed in claim 1, wherein lipid material may comprise solid lipids preferably waxes, fatty acids (stearic acid, palmitic acid, myristic acid or oleic acid), triglycerides (tristearin, tripalmitin, trimyristine) or a mixture of mono, di and tri-glycerides i.e. compritol.

5. The composition as claimed in claim 1, wherein compritol 888 ATO and stearic acid is in a ratio varying from 1:2 to 2:1, drug to lipid is in a ratio varying from 1:25 to 1:8.33 and pluronic F-68 (0.25-1.0 % w/v).

6. A method of preparing the composition as claimed in claim 1, comprising the steps of: a) Melting compritol 888 ATO, stearic acid and soya lecithin at temperature exceeding 5 – 10 °C of their melting point and mixing them. b) Subsequently, dissolving clarithromycin in molten lipids to achieve oil phase. c) Simultaneously, preparing the aqueous phase by dissolving Pluronic F-68 and modulating to temperature 70 ± 2 °C. d) Adding the oil phase subsequently and emulsifying under high shear homogenization at 22000-24000 rpm for 20 min. e) Cooling down the resulting emulsion gradually to room temperature with continuous stirring. f) Centrifuging the obtained dispersion at 23,000-25000 rpm at 4 °C for 20-25 min to collect nanoparticles. g) Separating the supernatant to evaluate encapsulation efficiency. h) Dispersing the resultant pellet in distilled water and lyophilizing using a cryoprotectant. i) Storing the lyophilized CM-NLCs in desiccator at room temperature.

7. The method as claimed in claim 6, wherein lyophilisation is done at -52 to -55°C, 0.05-0.1 bar pressure for 48-50h.

8. The method as claimed in claim 6, wherein lyophilisation is done using 4-6 % w/v mannitol (cryoprotectant).

9. The method as claimed in claim 6, wherein nanostructured lipid carriers are prepared using modified hot homogenization method.

10. The composition as claimed in claim 1, wherein said composition is used to treat severe infections, septic shock, urinary tract infections or chronic respiratory infections.

TECHNICAL FIELD
[0001] The present invention belongs to the technical field of nanostructured lipid carriers, and in particular relates to development of a sustained release Clarithromycin loaded nanostructured lipid carriers (CM-NLCs) with improved bioavailability and antimicrobial activity against P. aeruginosa induced peritonitis.

BACKGROUND ART
[0002] Complicated intra-abdominal infections (cIAIs) are the complex fatal clinical challenge. The most alarming causative biofilm forming pathogens like Staphylococcus aureus, Haemophilus influenzae, Streptococcus pneumoniae and Enterobacteriaceae are of particular concern as accountable for flare-up of infections. Thus, polymicrobial etiology and difficulty in diagnoses present an additional obstacle in cIAIs management.
[0003] Furthermore development of multidrug resistance drives cIAIs management formidable and posed a significant burden on quality of life and health care system.
[0004] The associated complications like uncontrolled immune responses leading to systemic inflammatory condition due to severe infection lead to organ dysfunction, sepsis or septic shock. Thus, peritonitis and coexisting alarming inflammatory responses are the vital cause of mortality in immunocompromised and critically ill patients. Therefore, an urgent need exists to develop alternative antimicrobial therapy for management of cIAIs.
[0005] Several studies recommend macrolides alone or in combined antibiotic therapy as the treatment of choice due to their broad spectrum of antimicrobial activity. Among macrolides, clarithromycin (CM) is a drug of choice due to higher stability in acidic milieu of stomach. It accumulates effectively in polymorphonuclear leukocytes and reaches to site of infection. However, biopharmaceutical impedance like poor aqueous solubility and systemic bioavailability, extensive hepatic first pass metabolism and higher dose frequency due to shorter half-life (3-5 h) limits its therapeutic efficacy in intracellular infections.
[0006] CM also hampers hepatic microsomal cytochrome P3A4 required for its metabolism contributing to elevated serum level of drug and hepatotoxicity. So far conventional formulations like tablets and suspension available in market have bestowed confined success to improve palatability, bioavailability and therapeutic efficacy along with reduction of hepatotoxicity of CM.
[0007] Recent expansions in nanotechnology have presented ample opportunities for efficient delivery of numerous therapeutic molecules. Nanostructured solid lipid carriers (NLCs) offers substantial benefits such as increased bioavailability, ability to retain structural integrity and drug payload with high perspective for commercial scalability compared to liposomes and nanoemulsions.
[0008] NLCs are composed of lipid (e.g., waxes, fatty acids, and glycerides) matrix having inherent propensity to solidify at room temperature. The characteristic features like nano size and lipid composition of NLCs enhances their permeability across gut wall by specific absorption mechanisms like endocytosis, pinocytosis or M-cell mediated transport and bypasses portal circulation.
[0009] However, NLCs composed of fatty acids or waxes usually exhibit higher crystalline structure, restricted drug loading capacity and drug expulsion during storage compared to triglycerides. The selection of a combination of fatty acids/ waxes and triglycerides can enhance physical stability and drug load due to blending of distinct constituents.
[0010] The lipophilicity of carrier systems can facilitate direct drug interaction to bacterial cell wall, complex biofilms formed by pathogens and inhibit different stages of biofilm formation. Lyophilization of NLCs in powder form also enables loading into capsules, pellets or tablets.
[0011] In order to overcome the aforementioned drawbacks, there is a need in the art to provide a novel clarithromycin loaded nanostructured carrier for preventing the first-pass metabolism and to enhance oral bioavailability of clarithromycin for the treatment of complicated intra-abdominal infections.
[0012] The information disclosed in this background of the disclosure section is only for enhancement of understanding of the general background of the invention and should not be taken as an acknowledgement or any form of suggestion that this information forms the prior art already known to a person skilled in the art

OBJECTS OF THE INVENTION
[0013] The principal object of the present invention is to overcome the disadvantages of the prior art by providing CM loaded nanostructured lipid carriers (CM-NLCs) and method of preparation thereof.
[0014] Another object of the present invention is to provide a nanostructured composition which has improved bioavailability.
[0015] Another object of the present invention is to provide a nanostructured composition which provides prolonged drug release.
[0016] Yet another object of the present invention is to provide a nanostructured composition which has antimicrobial activity against P. aeruginosa induced peritonitis.
[0017] The foregoing and other objects of the present invention will become readily apparent upon further review of the following detailed description of the embodiments as illustrated in the accompanying drawings.

SUMMARY OF THE INVENTION
[0018] The present invention relates to development of CM loaded nanostructured lipid carriers (CM-NLCs) and method of preparation thereof.
[0019] According to an embodiment of the present invention, the drug loaded nanostructured lipid carrier, comprises of a) clarithromycin (drug), b) mixture of mono, di and triglyceride, c) surfactant, d) lipid material and e) long chain saturated fatty acid.
[0020] According to an embodiment of the present invention, the method for synthesis of the drug loaded nano lipid carrier comprises the following steps of: a) melting mixture of mono, di and triglyceride, stearic acid and soya lecithin at temperature exceeding 5 – 10 °C of their melting point and mixing them, b) dissolving clarithromycin in molten lipids to achieve oil phase, c) simultaneously preparing the aqueous phase by dissolving Pluronic F-68 and modulating to temperature 70 ± 2 °C, d) adding the oil phase subsequently and emulsifying under high shear homogenization at 22000-24000 rpm for 20 min, e) cooling down the resulting emulsion gradually to room temperature with continuous stirring, f) centrifuging the obtained dispersion at 23,000-25000 rpm at 4 °C for 20-25 min to collect nanoparticles, g) separating the supernatant to evaluate encapsulation efficiency, h) dispersing the resultant pellet in distilled water and lyophilizing using a cryoprotectant and i) storing the lyophilized CM-NLCs in desiccator at room temperature.

BRIEF DESCRIPTION OF DRAWINGS
[0021] The accompanying drawings are included to provide a further understanding of the present disclosure and are incorporated in and constitute apart of this specification. The drawings illustrate exemplary embodiments of the present disclosure and, together with the description, serve to explain the principles of the present disclosure.
[0022] Fig. 1 (A) illustrates SEM images of optimized CM-NLCs. (B) Drug release profile with respect to time from optimized CM-NLCs in pH progressive dissolution media in accordance with the present invention.
[0023] Fig. 2 illustrates Effect of storage conditions on the quality parameters like entrapment efficiency, particle size, zeta potential and polydispersity index (PDI).
[0024] Fig. 3 illustrates Inhibitory effect of CM and CM-NLCs on (A) growth of P. aeruginosa (MIC determination) and (B) ability of biofilm to rejuvenate P. aeruginosa (MBIC assay) at different concentrations respectively in accordance with the present invention
[0025] Fig. 4 illustrates Drug concentration versus time profile of CM and CM-NLCs in (A) plasma, (B) lung, (C) liver and (D) spleen respectively in accordance with the present invention.
[0026] Fig. 5 illustrates Effect of CM and CM-NLCs against P. aeruginosa induced oxidative stress biomarkers (A) LPO, (B) MPO and (C) carbonylated protein content respectively in accordance with the present invention.
[0027] Fig. 6 illustrates Effect of CM and CM-NLCs on level of immunological biomarkers (A) IL-6 and TNF-a, (B) NO (%), (C) Total leucocytes count and neutrophils count (%) respectively in P. aeruginosa induced model of peritonitis in accordance with the present invention.
[0028] Fig. 7 illustrates Effect of clarithromycin (A) and CM-NLCs (B) at a dose equivalent to 10 mg kg-1 body weight on liver tissues during repetitive oral dose toxicity study respectively in accordance with the present invention.
[0029] It should be noted that the figures are not drawn to scale, and the elements of similar structure and functions are generally represented by like reference numerals for illustrative purposes throughout the figures. It should be noted that the figures do not illustrate every aspect of the described embodiments and do not limit the scope of the present disclosure.
[0030] Other objects, advantages and novel features of the invention will become apparent from the following detailed description of the present embodiment when taken in conjunction with the accompanying drawings.

DETAILED DESCRIPTION OF THE INVENTION
[0031] While the present invention is described herein by way of example using embodiments and illustrative drawings, those skilled in the art will recognize that the invention is not limited to the embodiments of drawing or drawings described and are not intended to represent the scale of the various components. Further, some components that may form a part of the invention may not be illustrated in certain figures, for ease of illustration, and such omissions do not limit the embodiments outlined in any way. It should be understood that the drawings and the detailed description thereto are not intended to limit the invention to the particular form disclosed, but on the contrary, the invention is to cover all modifications, equivalents, and alternatives falling within the scope of the present invention as defined by the appended claim.
[0032] As used throughout this description, the word "may" is used in a permissive sense (i.e. meaning having the potential to), rather than the mandatory sense, (i.e. meaning must). Further, the words "a" or "an" mean "at least one” and the word “plurality” means “one or more” unless otherwise mentioned. Furthermore, the terminology and phraseology used herein are solely used for descriptive purposes and should not be construed as limiting in scope. Language such as "including," "comprising," "having," "containing," or "involving," and variations thereof, is intended to be broad and encompass the subject matter listed thereafter, equivalents, and additional subject matter not recited, and is not intended to exclude other additives, components, integers, or steps. Likewise, the term "comprising" is considered synonymous with the terms "including" or "containing" for applicable legal purposes. Any discussion of documents, acts, materials, devices, articles, and the like are included in the specification solely for the purpose of providing a context for the present invention. It is not suggested or represented that any or all these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention.
[0033] In this disclosure, whenever a composition or an element or a group of elements is preceded with the transitional phrase “comprising”, it is understood that we also contemplate the same composition, element, or group of elements with transitional phrases “consisting of”, “consisting”, “selected from the group of consisting of, “including”, or “is” preceding the recitation of the composition, element or group of elements and vice versa.
[0034] The present invention is described herein after by various embodiments with reference to the accompanying drawing, wherein reference numerals used in the accompanying drawing correspond to the like elements throughout the description. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiment set forth herein. Rather, the embodiment is provided so that this disclosure will be thorough and complete and will fully convey the scope of the invention to those skilled in the art. In the following detailed description, numeric values and ranges are provided for various aspects of the implementations described. These values and ranges are to be treated as examples only and are not intended to limit the scope of the claims. In addition, several materials are identified as suitable for various facets of the implementations. These materials are to be treated as exemplary and are not intended to limit the scope of the invention.
[0035] The present invention relates to development of CM loaded nanostructured lipid carriers (CM-NLCs) and method of preparation thereof.
[0036] According to an embodiment of the present invention, the drug loaded nanostructured lipid carrier comprises of a) clarithromycin (drug), b) mixture of mono, di and triglyceride, c) surfactant, d) lipid material, e) stearic acid
[0037] According to an embodiment of the present invention, the mixture of mono, di and triglyceride is Compritol 888 ATO.
[0038] According to an embodiment of the present invention, the surfactant is selected from Pluronic F-127, Pluronic F-68 and Tween-80, preferably Pluronic F-68.
[0039] According to an embodiment of the present invention, the lipid material is soya lecithin.
[0040] In an embodiment, composition may further comprise solid lipids preferably waxes, fatty acids (stearic acid, palmitic acid, myristic acid or oleic acid), triglycerides (tristearin, tripalmitin, trimyristine) or a mixture of mono, di and tri-glycerides i.e. compritol.
[0041] According to an embodiment of the present invention, CM-NLCs is produced utilizing compritol 888 ATO and stearic acid in a ratio varying from 1:2 to 2:1, drug to lipid ratio of 1: 25 to 1:8.33, pluronic F-68 (0.25-1 % w/v).
[0042] According to an embodiment of the present invention, the method of preparing the said nanostructured lipid carriers comprise of the following steps:
i. Compritol 888 ATO, stearic acid and soya lecithin were melted at temperature exceeding 5 – 10 °C of their melting point and mixed.
ii. Subsequently, CM was dissolved in molten lipids to achieve oil phase.
iii. Simultaneously, aqueous phase was prepared by dissolving Pluronic F-68 (0.5 % w/v) and modulated to temperature 70 ± 2 °C.
iv. Oil phase was subsequently added and emulsified under high shear homogenization at 22,000 rpm for 20 min.
v. The resulting emulsion was gradually cooled down to room temperature with continuous stirring. The obtained dispersion was centrifuged at 25,000 rpm at 4 °C for 20 min to collect nanoparticles.
vi. Supernatant separated was used to evaluate encapsulation efficiency.
vii. The resultant pellet was dispersed in distilled water and lyophilized at -55 °C and 0.07 bar pressure for 48 h using mannitol (5 % w/v) as cryoprotectant.
viii. Lyophilized CM-NLCs were stored in desiccator at room temperature.

[0043] According to another embodiment of the present invention, nanostructured lipid carriers were prepared using modified hot homogenization method.
[0044] Supernatants collected after separation of CM-NLCs pellet was estimated at 207 nm for CM content using UV/Visible spectrophotometer. Entrapment efficiency was estimated by using following equation:

[0045] According to another embodiment of the present invention, remarkable variation in particle size and encapsulation efficiency was noticed on varying amount of lipids due to difference in their HLB value. The average particle size and entrapment efficiency on varying compritol 888 ATO: stearic acid ratio from 1:2 to 2:1 ranged from 309.02 ± 37.89 nm to 426.66 ± 23.86 nm and 75.50 ± 3.68 % to 86.02 ± 3.26 % respectively.
[0046] According to another embodiment of the present invention, pluronic F-68 provided smaller sized (335.11 ± 25.40 nm) uniformly dispersed particles (PDI 0.19 ± 0.06) with higher entrapment efficiency (86.02 ± 3.26%). Pluronic F-68 might have facilitated the genesis of a closely packed mechanical and thermodynamic barrier at interface of micelles to enable the emergence of homogeneous nanosized particles with higher encapsulation efficiency in contrast to pluronic F-127 and tween 80. On increasing pluronic F-68 concentration from 0.1 % to 0.5 % w/v increase in encapsulation efficiency and decrease in particle size was observed. However, further increase in pluronic F-68 concentration (1% w/v) reduced size and entrapment efficiency.
[0047] According to another embodiment of the present invention, on varying ratio of drug to lipid content from 1:25 to 1:12.5, increase in encapsulation efficiency was observed. However, further increase in drug to lipid ratio to 1:8.33 led to decrease in encapsulation efficiency due higher diffusion of drug into continuous phase as relatively smaller proportion of lipid was present in comparison to drug amount.
[0048] According to another embodiment of the present invention, Zeta potential, a quantitative measurement of surface charge governs the physical stability of system. Zeta potential around ± 30 mV generates columbic forces of higher magnitude which overcome the Vander Walls forces of attraction and represents higher physical stability of colloidal system.
F.
code Soya Leci-thin (mg)
Com-pritol :
Stearic acid ratio
PF-68 (% w/v) PF-
127 (%w/v) Tween 80 (%w/v)
Drug (mg)
Homogenization Time (min) Exter-nal phase volume
(ml) Particle size
(nm±SD) PDI (PDI±SD) Zeta potential
(mV± SD) Entrap-ment efficiency (%±SD)
F1 50 1:2 0.5 - - 20 20 30 309.02± 37.89 0.25± 0.03 -27.31± 1.31 75.50± 3.68
F2 50 1:1 0.5 - - 20 20 30 335.11± 25.40 0.19± 0.06 -30.62± 2.08 86.02± 3.26
F3 50 2:1 0.5 - - 20 20 30 426.66± 23.86 0.36± 0.02 -26.62± 2.08 82.33± 4.83
F4 50 1:1 - - 0.5 20 20 30 226.60± 17.78 0.76± 0.12 -14.83±
2.70 74.04± 3.74
F5 50 1:1 - 0.5 - 20 20 30 420.06± 41.20 0.87± 0.11 -26.01± 2.32 81.13± 4.06
F6 50 1:1 0.5 - - 20 20 40 408.33± 34.07 0.38± 0.07 -19.64± 3.08 77.86± 4.38
F7 50 1:1 0.5 - - 20 20 50 496.33± 37.98 0.36± 0.03 -18.30± 3.08 68.8± 5.41
F8 50 1:1 0.5 - - 10 20 30 266.46± 25.01 0.36± 0.02 -14.71± 2.38 76.80± 6.94
F9 50 1:1 0.5 - - 30 20 30 322.13± 38.83 0.42± 0.01 -21.63± 3.78 78.33± 6.17
F10 50 1:1 0.25 - - 20 20 30 496.33±
29.64 0.49±
0.01 -22.62± 2.61 78.99±
4.02
F11 50 1:1 1.0 - - 20 20 30 301.02±
27.04 0.37± 0.12 -21.80± 3.03 72.1± 5.21
F12 50 1:1 0.5 - - 20 10 30 681.11±
34.80 0.68±
0.06 -17.81±
2.19 62.98±
9.78
F13 50 1:1 0.5 - - 20 30 30 486.20±
26.11 0.42±
0.12 -20.83±
2.53 71.21±
7.21
Table 1: Effect of diverse variables on quality parameters i.e. particle size, polydispersibility index, zeta potential and entrapment efficiency respectively.
Drug release studies
[0049] The assessment of CM release from CM-NLCs was determined in pH progressive media (HCl buffer pH 1.2 for 2 h followed by phosphate buffer pH 6.8) utilizing dialysis bag diffusion method. CM-NLCs equivalent to 10 mg of CM dispersed distilled water was filled in dialysis bag (12 – 14 kDa) and immersed in dissolution medium (200 ml) kept at 37 ± 2 °C and 100 rpm. Sink condition was maintained by adding 0.1 % w/v sodium lauryl sulfate. Sampling (3 ml) was executed at 0.5, 1, 2, 4, 6, 8, 12, 16 and 24 h with consecutive replacement with equal volume of fresh buffer maintained at 37 °C. Samples were centrifuged and supernatants collected were analyzed for CM content. Drug release kinetics was determined by plotting obtained data according to different release kinetics models. The best fit model was determined on the behalf of numerical value of regression coefficient calculated.
[0050] Fig. 1B represented biphasic drug release pattern i.e., an initial burst release (~24 %, lasted in 2 h) pursued by gradual drug release over 24 h under bio-relevant conditions of gastro-intestinal tract. Initial rapid release might be contributed to prompt diffusion and desorption of surface adsorbed or weakly bound drug or partial film defects emerged during lyophilization. The steady decline in drug release rate might be ascribed to intrinsic drug solubility, path length of drug depleted layers across which drug travelled, rigidity of lipoidal barrier and diffusion from dialysis membrane.
Stability studies
[0051] Optimized lyophilized CM-NLCs stored in glass vials screwed with polystyrene caps were examined for stability according to guidelines issued by ICH for zone III and IV. Samples were kept at different storage conditions like room temperature (25 ± 2 °C/ 60 ± 5 % RH) and cool conditions (4 ± 2 °C/ 65 ± 5 % RH) for 12 months. Samples were evaluated for colloidal stability (particle size, PDI and zeta potential) and entrapment efficiency at an interval of 0, 1.5, 3, 6, 9 and 12 months.
[0052] The results of stability study recommended storage of CM-NLCs at room conditions in order to retain their pharmaceutical properties for effective long-term use (Fig. 2). The physical appearance of CM-NLCs had not shown any noticeable change at different environmental conditions.
In vitro antimicrobial activity
A. Minimum inhibitory concentration (MIC) determination
[0053] The minimum amount of CM and CM-NLCs needed to inhibit growth of P. aeruginosa was determined by broth dilution method. Concisely, sterile nutrient broth was inoculated with bacterial suspension (100 µl, equivalent to 0.5 McFarland standard) with subsequent addition of series of serial dilutions of test samples equivalent to 50, 54, 58, 62, 66, 70 and 74 µg/ml of CM, respectively. The turbidity, an indication of bacterial growth in culture tubes was analyzed at 600 nm after an incubation period of 24 h at 37 °C.
B. Minimum biofilm inhibitory concentration (MBIC) evaluation
[0054] Briefly, broth inoculated with P. aeruginosa (adjusted to 0.5 Mc Farland standards) for 24 h at 37 °C was decanted to remove non-adherent bacteria. Biofilms adhered to walls and bottom of culture tubes were washed twice with sterile distilled water to draw out free bacteria. Subsequently, biofilm from the surface of tubes was detached, collected and treated with various concentrations of CM or CM-NLCs equivalent to 60, 70, 80, 100, 120, 140, 145 and 150 µg/ml of CM. Treated biofilms were withdrawn and incubated in fresh broth for 24 h at 37 °C respectively. The presence of turbidity measured at 600 nm indicated presence of viable organisms.
[0055] Referring to Fig. 3, Lower MIC value of CM-NLCs (58 µg/ ml) revealed higher effectiveness of formulation in inhibiting P. aeruginosa growth compared to free drug (74 µg/ ml). Similarly, 1.87-fold lower MBIC of CM-NLCs has assured its higher efficacy in inhibiting P. aeruginosa biofilm formation compared to pure drug. The enhanced bactericidal activity of CM-NLCs might be contributed by increased interaction i.e., diffusion or fusion of CM-NLCs with bacterial cellular membrane facilitating CM delivery directly to bacterial cytoplasm along with extended release of drug from CM-NLCs, working as drug depot.
In vivo studies
[0056] Wistar rats (200 ± 20 g) acquired from institutional animal house were kept on standard animal diet at 22 ± 2 °C and 55 ± 5% RH on a 12 h dark/light cycle. In vivo studies were previously sanctioned by Banasthali Vidyapith IAEC (574/GO/ReBi/S/02/CPCSEA) and performed according to CPCSEA guideline established for care and use of laboratory animals and performed.
Pharmacokinetic evaluation
[0057] Overnight fasted wistar rats were randomized into two groups (n=6) for single dose oral bioavailability study. Animals of group I and II received CM suspension (5 mg/kg) and CM-NLCs (equivalent to 5 mg/kg of CM) respectively. Blood samples (250 µl) were withdrawn from rat tail vein into heparinized tubes at 0.5, 1, 2, 4, 6, 8, 12 and 24 h post dosing. Blood loss was compensated by injecting equivalent volume of normal sterile saline solution. Later blood was centrifuged at 5000 rpm for 10 min at 4 °C to segregate plasma and analyzed for drug content by HPLC method.
[0058] Referring to Fig. 4, CM-NLCs attained 2.46-fold higher Cmax compared to CM suspension. Nano-size as well as hydrophobic surface of CM-NLCs might have aided their enhanced uptake and improved plasma drug concentration. Delayed Tmax i.e., 2 h too confirmed in vivo extended drug release profile of CM-NLCs alike in vitro release profile. CM plasma concentration declined after 1 h indicating rapid systemic clearance which was further ascertained by lower MRT. The elevated t1/2 (2.76-fold) and MRT (1.36-fold) of CM-NLCs further confirmed the extended absorption and release of drug from CM-NLCs in systemic circulation. The remarkable improvement in AUC0-24h (p<0.05) with CM-NLCs (~6.45-fold) compared to CM dispersion might be accredited to improved systemic absorption of CM-NLCs through paracellular and transcellular pathways via enterocytes, M-cells and gut associated lymphoid tissues or transport via triglyceride rich lipoproteins.

Organ distribution study
[0059] The quantitative tissue distribution pattern of CM and optimized formulation after oral dosing was evaluated in healthy Wistar rats. Animals (n= 36) were fasted over 24 h with free access to water ad libitum and randomly segregated into two groups (n=18). Rats from each group were administered with CM suspension (5 mg/kg) or CM-NLCs (equivalent to 5 mg/kg of CM) orally. Tissues like lungs, liver and spleen and plasma were collected after sacrificing rats (n=3 per group) at 1, 2, 4, 6, 8 and 12 h post dosing. The respective tissues were weighed and frozen at -70 °C until further analysis. Before proceeding to analysis, frozen tissues were thawed and homogenized to fine paste in a tissue homogenizer (Remi, Mumbai, India), centrifuged at 10,000 rpm for 15 min to obtain clear supernatant and analyzed by HPLC method.
[0060] Referring to Fig. 4, Peak CM concentration was achieved across 2 to 4 h. CM-NLCs attained comparatively higher concentration of drug in spleen and lung at all the time points (2-12 h). On the contrary, lymphatic uptake of CM-NLCs via endocytosis/ transcytosis through M-cells and gut associated lymphoid tissues or by partitioning into triglyceride rich lipoproteins secreted in intestinal lymph might had improved the drug absorption. This too had reduced the extent of drug concentration in liver from CM-NLCs compared to free drug.
Pharmacodynamic studies
[0061] Intra-abdominal infection was induced via intraperitoneal injection of inoculum of P. aeruginosa suspension in normal saline (1 X 1010 CFU/ml). Subsequently, Wistar rats were randomized to receive CM suspension (5mg/Kg), CM-NLCs (equivalent to 5mg/Kg CM) and normal saline orally (n=6) respectively. Animals were euthanized after 24 h of respective treatment to collect lungs, liver, spleen and blood samples via cardiac puncture aseptically. Blood samples were centrifuged at 5000 rpm and 4 °C to isolate plasma which was evaluated for lipid peroxidation (LPO), carbonylated protein, myeloperoxidase (MPO) activity, nitric oxide (NO), TNF-a and IL-6 level.
[0062] Microbial count in blood and tissue homogenates was determined by plating samples on agar plates followed by incubation at 37 °C for 48 h and counting colonies formed.
[0063] Referring to Fig. 5, The endotoxin administration manifested elevation of oxidative stress markers in lung, liver and spleen respectively. Pure drug and CM-NLCs were found remarkably efficacious in bringing down the LPO, MPO and carbonylated protein level in all tissues compared to control (p<0.05). The results revealed CM-NLCs were more effective in lowering oxidative stress markers and bacterial count compared to pure drug (p < 0.05) due to facilitated distribution of formulation in reticuloendothelial system rich organs like lungs, liver and spleen.
[0064] Referring to Fig. 6, significant elevation of serum total leukocyte count, neutrophils, lymphocytes, TNF-a, IL-6 and NO level in control group on intraperitoneal administration of LPS was observed. CM-NLCs treatment facilitated significant reduction in mean values of total leukocyte count, neutrophil, lymphocytes, TNF-a, IL-6 and NO level (p < 0.05) compared to drug and control.
[0065] The unique characteristics of CM-NLCs i.e., nanosize, ease of adherence or diffusion to bacterial cell and sustained drug release significantly inhibited bacterial growth and impeded the bacterial endotoxin induced systemic inflammatory conditions. In addition, CM-NLCs delivered higher CM plasma concentrations upto 24 h and better distribution of CM to tissues compared to pure drug. Thus, CM-NLCs significantly hampered the cascade of events induced by bacterial infection during peritonitis management.

Toxicity study
[0066] Eighteen Wistar rats were randomly segregated into control group (PBS treated), CM treated and CM-NLC treated group (n=6). Animals were administered with test samples at a dose equivalent to 10mg/kg of CM once a day for 28 days respectively to determine the potential toxicity. All animals were hold onto standard normal diet and water ad libitum throughout the study. Animals were observed for behavioral change and mortality during this period. Rats were euthanized on 28th day to collect liver tissues for histopathological evaluation. Samples were stored at -80 ° C before analysis.
[0067] No behavioral change as well as mortality was observed during toxicity study. However, histological pictography of CM treated group indicated severe degeneration of hepatocytes, intense fatty changes, necrosis and ballooning injury. On the contrary, no remarkable toxicologically important changes in liver cells of CM-NLCs treated group was observed (Fig. 7). The results demonstrated safety of CM-NLCs upto an oral dose equivalent to 10 mg/kg.
[0068] According to another embodiment of the present invention, CM-NLCs may be used to treat severe infections, septic shock, urinary tract infections or chronic respiratory infections.
[0069] According to another embodiment of the present invention, CM-NLCs particularly for oral use are effective in treatment of infections caused by biofilm as well as non-biofilm forming microorganisms.
[0070] Various modifications to these embodiments are apparent to those skilled in the art from the description and the accompanying drawings. The principles associated with the various embodiments described herein may be applied to other embodiments. Therefore, the description is not intended to be limited to the 5 embodiments shown along with the accompanying drawings but is to be providing the broadest scope consistent with the principles and the novel and inventive features disclosed or suggested herein. Accordingly, the invention is anticipated to hold on to all other such alternatives, modifications, and variations that fall within the scope of the present invention and appended claims.

We Claim:

1. A drug loaded nanostructured lipid carrier comprising, a) clarithromycin (drug), b) compritol 888 ATO, c) surfactant, d) lipid material, e) stearic acid.
2. The composition as claimed in claim 1, wherein surfactant is selected from Pluronic F-127, Pluronic F-68 and Tween-80, preferably Pluronic F 68 (PF-68).
3. The composition as claimed in claim 1, wherein the lipid material is soya lecithin.
4. The composition as claimed in claim 1, wherein lipid material may comprise solid lipids preferably waxes, fatty acids (stearic acid, palmitic acid, myristic acid or oleic acid), triglycerides (tristearin, tripalmitin, trimyristine) or a mixture of mono, di and tri-glycerides i.e. compritol.
5. The composition as claimed in claim 1, wherein compritol 888 ATO and stearic acid is in a ratio varying from 1:2 to 2:1, drug to lipid is in a ratio varying from 1:25 to 1:8.33 and pluronic F-68 (0.25-1.0 % w/v).
6. A method of preparing the composition as claimed in claim 1, comprising the steps of:
a) Melting compritol 888 ATO, stearic acid and soya lecithin at temperature exceeding 5 – 10 °C of their melting point and mixing them.
b) Subsequently, dissolving clarithromycin in molten lipids to achieve oil phase.
c) Simultaneously, preparing the aqueous phase by dissolving Pluronic F-68 and modulating to temperature 70 ± 2 °C.
d) Adding the oil phase subsequently and emulsifying under high shear homogenization at 22000-24000 rpm for 20 min.
e) Cooling down the resulting emulsion gradually to room temperature with continuous stirring.
f) Centrifuging the obtained dispersion at 23,000-25000 rpm at 4 °C for 20-25 min to collect nanoparticles.
g) Separating the supernatant to evaluate encapsulation efficiency.
h) Dispersing the resultant pellet in distilled water and lyophilizing using a cryoprotectant.
i) Storing the lyophilized CM-NLCs in desiccator at room temperature.
7. The method as claimed in claim 6, wherein lyophilisation is done at -52 to -55°C, 0.05-0.1 bar pressure for 48-50h.
8. The method as claimed in claim 6, wherein lyophilisation is done using 4-6 % w/v mannitol (cryoprotectant).
9. The method as claimed in claim 6, wherein nanostructured lipid carriers are prepared using modified hot homogenization method.
10. The composition as claimed in claim 1, wherein said composition is used to treat severe infections, septic shock, urinary tract infections or chronic respiratory infections.