Indian Patents. 279439:"NANOPARTICULATE FORMULATION FOR ORAL DELIVERY OF COENZYME Q10"

Title of Invention	"NANOPARTICULATE FORMULATION FOR ORAL DELIVERY OF COENZYME Q10"
Abstract	The present invention relates to the designing of nanoparticles that can effectively deliver the CoQIO leading to dose reduction and improved efficacy and further, to evaluate these formulations in hypertensive animal model. Nanoparticles were prepared by adopting an emulsion technique, which was further optimized for drug loading.

Full Text	Field of invention The present invention is in the field of nanoparticle drug delivery system and more particularly the invention discloses a nanoparticulate coenzyme Q10 (CoQIO) composition and preparation thereof. Background of the invention CoQIO is naturally occurring compound that plays a key role in energy metabolism as an integral part of electron transport system. Several studies have provided the evidence of the potentials of CoQIO in prophylaxis and therapy of various disorder related oxidative stress. CoQIO deficiency has been confirmed in patient with cardiovascular disorders like congestive heart failure coronary artery disease, angina pectoris, cardiomyopathy and hypertension. CoQIO deficiency also reported in cancer and neurodegenerative disorders like Parkinson's and Alzheimer's disease. CoQIO functions as antioxidant, scavenging free radicals and inhibiting free radicals. Despite of its potential pharmacological activity I many diseases, not much progress has been made in terms of formulation aspect owing to its poor aqueous and heigh molecular weight, leaving behind this molecule in the class of difficult to deliver molecules (class IV of Biopharmaceutical Classification System). The efficiency of CoQIO absorption and its bioavailability when given by oral route is very low from the current marketed products. Recently nanoparticles made of degradable and non-degradable materials have been actively explored as delivery system for both small and large drug molecules. Poorly water soluble and high molecular weight molecules are often difficult to process into dosage form that afford adequate oral bioavailability for clinical studies and commercialization of CoQIO is no exception with poor aqueous solubility and a high molecular weight of 863. Hence, there is a need for CoQIO formulation which has got higher bioavailability and the present invention has made an effort to provide alternative formulation which obviates the existing problem of using CoQIO. Objectives of the invention The primary object of the present invention is to develop a nanoparticle based drug delivery system. Another object of the present invention is to formulate nanoparticulate composition of CoQIO to increase its oral bioavailability. Brief description of the accompanying drawings Figure 1 is a bar graph showing the difference in uptake of 20 mg of CoQIO in the form of suspension and CoQIO loaded nanoparticles. The graph clearly shows that in case of CoQIO loaded nanoparticles percentage uptake is about 30% greater than suspension from thus suggesting enhanced uptake. Figure 2 shows the stability of CoQIO in presence of ethyl acetate and ethyl acetate in combination with PLGA (values are expressed as mean ± standard deviation [n=3]). Figure 3 shows the effect of initial drug loading on entrapment efficiency. Figure 4 (a) shows the effect of different CoQIO formulation on systolic blood pressure on 12th day after surgery in glodblatt hypertensive rats in sham operated group Vs. vehicle operated group. (Statistical analysis was performed by one way of ANOVA followed by Tukey's test for multipule comparisons. p Figure 4 (b) shows the effect of different CoQIO formulations on diastolic blood pressure on 12th day after surgery in glodblatt hypertensive rats in sham operated group Vs. vehicle operated group. (Statistical analysis was performed by one way of ANOVA followed by Tukey's test for multipule comparisons. p Figure 5 (a) shows the effect of different CoQIO formulations on systolic blood pressure on 15th day after surgery in glodblatt hypertensive rats in sham operated group Vs. vehicle operated group. (Statistical analysis was performed by one way of ANOVA followed by Tukey's test for multipule comparisons. p Figure 5 (b) shows the effect of different CoQIO formulations on diasystolic blood pressure on 15th day after surgery in glodblatt hypertensive rats in sham operated group Vs. vehicle operated group. (Statistical analysis was performed by one way of ANOVA followed by Tukey's test for multipule comparisons. p Figure 6 shows the effect of different Coenzyme Q10 formulations on lipid peroxidation of hypertensive rats in sham operated group Vs. vehicle operated group. (Statistical analysis was performed by one way of ANOVA followed by Tukey's test for multipule comparisons. *p Detailed description of the invention Accordingly, the present invention deals with designing of nanoparticles that can effectively deliver the CoQIO orally, and more particularly relates to dose reduction, improved bioavailability and efficacy. The main embodiment of the invention is Polymeric biodegradable CoQIO loaded nanoparticles for oral delivery was prepared by the emulsion-diffusion-evaporation method which consists of following steps: a) dissolving of CoQIO and polymer in organic phase b) addition of the organic phase of step (a) to aqueous system containing stabilizer /surfactant resulting into emulsion c) stirring the emulsion followed by homogenization of emulsion, d) addition of water to homogenized emulsion at constant stirring, and e) removal of organic solvent at controlled conditions thereby transforming the emulsion into a liquid-solid suspension, whereby the solid comprises of polymeric nanoparticles of PLGA loaded with CoQW for oral administration. The nanoparticles were optimized for drug loading. The effect of initial drug loading on particle size, particle size distribution, PDI, zeta potential and encapsulation efficiency was investigated. It was observed that there was no significant change in particle size with increasing CoQIO loading from 5 to 30 %w/w of polymer; however a slight increase in the particle size along with PDI was observed for 50% and 75% initial drug loading. Increase in encapsulation efficiency was found with increase in initial drug loading from 5 to 20% w/w of polymer (from 61.7±3.9 for 5% to 82.0±3.3 for 20%) and beyond 20% the entrapment efficiency was around 80%. There was no change in zeta potential for any of the different drug loading batches (table 2). All the initial drug loading batches showed good reproducibility as observed from low standard deviation values. The nanoparticulate system was capable of entrapping small as well as large amount of drug without dramatic change in particle size and PDI. Such large range of initial drug loading provides us an option to produce different dose formulations without any change in the process. The high drug loading capacity along with high entrapment efficiency makes it a commercially feasible process. The prepared nanoparticles were evaluated for absorption characteristics by in situ studies and for its efficacy in hypertensive and breast cancer animal models (table 2). Table 1 shows the effect of initial drug loading on particle characteristics and encapsulation efficiency Initial Drug Size PDI Zeta Potential Entrapment (Table Removed) The zeta potential values reported are in the pH range 4.0- 4.85 (The values reported are mean±S.D. (n=3)) Table 2 shows the systolic and Diastolic blood pressure of hypertensive rats of various groups treated with different Coenzyme Q10 formulations (Table Removed) Table 3 shows the equilibrated solubility of CoQIO with different solvent system. (Table Removed) (Values are expressed as meant standard deviation [n=3]) Table 4 shows the effect of initial drug loading on particle size and PDI (Table Removed) (Values are expressed as meant standard deviation [n=3]) Yet another embodiment of the invention is nanoparticulate formulation of Coenzyme Q10, comprising of Coenzyme Q10, polymeric material and stabilizer/ surfactant. Still another embodiment of the invention is the polymer used in nanoparticulate formulation of Coenzyme Q10 belongs to polyester family, for example Polycaprolactone (PCL), Poly Lactide (PLA), Poly Glycolide (PGA) and copolymers of these like Poly Lactide-co-Glycolide (PLGA), PLA-PCL. Yet another embodiment of the invention is organic phase used to solubilize Coenzyme Q10 and polymer in nanoparticulate formulation of Coenzyme Q10, is selected from a group of organic solvents with partial to total water miscibility like ethyl acetate, dichloromethane, and acetone and any other such organic solvent and mixtures thereof. Still another embodiment of the invention is organic phase used to soluMise Coenzyme Q10 and polymer in nanoparticulate formulation of Coenzyme Q10, is a co-solvent system with organic solvents, wherein at least one of the solvents of the co-solvent system is from those mentioned above. Yet another embodiment of the invention is stabilizer forms a barrier between the continuous phase and the dispersed phase in the emulsion formed in step (b) in the nanoparticulate formulation of Coenzyme Q10. Still another embodiment of the invention is stabilizer dissolved in aqueous phase can be cationic (for example chitosan or quaternary ammonium salt like Didodecyldimethylammonium bromide), anionic (Polyvinyl alcohol) and nonionic (Vitamin ETPGS), or their combination in the nanoparticulate formulation of Coenzyme Q10. Yet another embodiment of the invention is different dose combinations can be obtained in the nanoparticulate formulation of Coenzyme Q10. Still another embodiment of the invention is Coenzyme Q10 loaded polymeric nanoparticles produced are in the range of 1 to 999 nm in size in the nanoparticulate formulation of Coenzyme Q10. Yet another embodiment of the invention is nanoparticles produced show in situ uptake of particles greater than 75% in the nanoparticulate formulation of Coenzyme Q10. Still another embodiment of the invention is homogenization is achieved by supplying energy to the biphasic system by means of a device selected from the group consisting of high shear mechanical stirrers, ultrasonic probe or a device capable of forcing the emulsion through a narrow space under normal or high pressures in the nanoparticulate formulation of Coenzyme Q10. Yet another embodiment of the invention is controlled heating in presence or absence of vacuum is used to remove the organic solvent during nanoparticulate preparation in the nanoparticulate formulation of Coenzyme Q10. Still another embodiment of the invention is formulation is used for treatment of hypertension. Yet another embodiment of the invention is formulation is able to reduce the systolic and diastolic blood pressure by 38 and 17 mmHg in renal hypertension model. Still another embodiment of the invention is the formulation is able to reduce MDA levels by scavenging free radicals. Yet another embodiment of the invention is nanoparticulate formulation of Coenzyme Q10 is used for treatment of cancer. Still another embodiment of the invention is nanoparticulate formulation of Coenzyme Q10 can be used as nutritional supplement. Yet another embodiment of the invention is nanoparticulate formulation of Coenzyme Q10 can be used for cosmetic applications. Still another embodiment of the invention is nanoparticulate formulation of Coenzyme Q10 is used for prophylaxis and therapy of various cardiovascular disorders like hypertension, cardiomyopathy, angina pectoris, congestive heart failure and coronary artery disease, and other disorders like neurodegenerative disorders (Parkinson's, Alzheimer's etc) and periodontal diseases. Yet another embodiment of the invention is matrix material of the nanoparticulate formulation of Coenzyme Q10 are degrades into harmless products in vivo. Still another embodiment of the invention is nanoparticulate formulation of Coenzyme Q10 contains very low concentration of stabilizer/surfactant which doesn't cause any inflammatory response and considered safe under in vivo conditions. Yet another embodiment of the invention is the final nanoparticulate formulation of Coenzyme Q10 is free of organic solvent. Still another embodiment of the invention is Coenzyme Q10 is dispersed uniformly throughout the matrix in the nanop articulate formulation of Coenzyme Q10. Yet another embodiment of the invention is nanoparticulate formulation of Coenzyme Q10 is route independent formulation. Now, the present invention will be described in detail in reference to examples, but the scope of the present invention should not be limited to these examples. In the examples, "part" and "parts" mean "part by weight" and "parts by weight", respectively, unless otherwise specified. The invention is illustrated by the following examples which are only meant to illustrate the invention and not act as limitations. All the embodiments apparent to a process their in the art are deemed to fall within the scope of the invention. Examples Example 1 Process for the preparation of CoQIO loaded polymeric nanoparticles Nanoparticles were prepared by adopting an emulsion-diffusion-evaporation technique, which was further optimized for drug loading. The increase in initial drug loading from 5 to 20% w/w of polymer led to increase in encapsulation efficiency and beyond 20% initial loading, the entrapment efficiency reached a plateau of 80%. There was no significant change in particle size and distribution with increasing drug loading from 5 to 30 %w/w of polymer; however a slight increase in the particle size and distribution was observed for 50% and 75% initial drug loading. The nanoparticles when evaluated for in situ uptake studies in Sprague-Dawley (SD) rats, showed better uptake in comparison to suspension form. The values of uptake were 45% for CoQIO suspension and 79% for CoQIO loaded nanoparticles. The nanoparticulate formulation was evaluated in hypertensive rats. The nanoparticles were compared with suspension form and . the commercial formulation. The ability of 3 doses of CoQIO (100 mg/kg) to treat hypertension was far more superior to 7 doses of CoQIO in suspension form and 3 doses of commercial formulation. CoQIO at 60% lower dose in nanoparticulate form was able to bring blood pressure to normal range on 12th day in comparison to the suspension form. The CoQIO nanoparticles were further investigated in breast cancer model and compared with the suspension form. The CoQIO nanoparticles showed better efficacy than the suspension form in reducing the tumor size. The in situ uptake studies along with evaluation in hypertension and cancer animal models clearly indicate the efficacy of CoQIO in the form of nanoparticles to that of suspension form and currently available commercial formulation. Example 2 Preparation of CoQIO loaded nanoparticles: 50 mg of polymer (of polyester family: Polycaprolactone (PCL), Poly Lactide (PLA), Poly Glycolide (PGA) and copolymers of these like Poly Lactide-co-Glycolide (PLGA), PLA-PCL etc) was dissolved in 2 ml organic phase (ethyl acetate, dichloromethane, acetone and co-solvent systems of different organic solvent) at room temperature for 2 hours. CoQIO was dissolved in 0.5ml of organic phase and added to the polymeric solution and stirred for 1 hour. The organic phase was then emulsified with 5 ml of an aqueous phase containing stabilizer/surfactant (PVA, DMAB or Vitamin ETPGS). The resulting o/w emulsion was stirred at room temperature for 1 hour before homogenizing at 15000 rpm for 5 min using a high-speed homogenizer (Polytron PT4000; Polytron Kinematica, Lucerne, Switzerland). To this emulsion, water was added with constant stirring which resulted into nanoprecipitation. The prepared nanoparticles have been optimized for drug loading. Example 3 Evaluation of CoQIO nanoparticles for its intestinal absorption and efficacy in animal models. Evaluation for intestinal absorption by in situ up takes study: Closed loop method was used to assess the uptake of CoQIO in suspension form and CoQIO loaded PLGA nanoparticles prepared using DMAB as stabilizer with 20 and 50% w/w of polymer as initial drug loading. Overnight fasted male Sprague Dawley (SD) rats were anaesthetized by intraperitoneal administration of thiopentone (50 mg/kg). The intestine was exposed through a midline incision and a closed loop of 10 cm length was prepared on the upper jejunum by ligation at both the ends. 1 ml of aqueous drug suspension or drug loaded nanoparticles containing 20 mg of CoQIO was injected into the loop with a syringe. Then entire intestine was restored to the abdominal cavity and body temperature was maintained during anesthesia. After 2 hours of drug administrationy the loops were rapidly isolated from the body. Contents of the loop were emptied into a container. The lumen of the intestine was rinsed with 10 ml water followed by 10 ml of methanol. The water and methanol solutions were collected and processed accordingly and diluted appropriately and the unabsorbed drug remaining in the solution was analyzed by HPLC. The uptake of CoQIO in the form of suspension was found to be 45%. It has been reported that around 60% of the CoQIO is excreted in feces in unchanged form and our uptake results closely match the reported values. The uptake of CoQIO nanoparticles was found to be 79% significantly greater than the suspension form clearly demonstrating the superiority of the nanoparticulate system for its absorption characteristics. Evaluation of efficacy of nanoparticulate system in breast cancer model: The prepared CoQIO nanoparticles were evaluated in breast cancer model. Female SD rats were administered DMBA (7,12-dimethylbenz[a]anthracene). Twenty two weeks after administration of DMBA the animals developed tumors. The animals were divided into 3 groups. One group received no treatment (untreated group); second group received 25 mg of CoQIO in suspension form and the last group was administered nanoparticles containing 25 mg of CoQIO. Frequency of administration was once a week for both the groups for 8 weeks. After administration of 8th dose (30th week from administration of DMBA) the animals were sacrificed and the tumors were removed and their volume measured. The nanoparticulate formulation was able to reduce the tumor burden by about 68% to that of untreated groups, whereas in the CoQIO suspension group the reduction was about 61 %. It was quite interesting to note the fact that in CoQIO suspension group there was a huge variability between the animals which is in agreement to the reported literature about variable bioavailability of CoQIO. The study demonstrated the efficacy of CoQIO in cancer treatment and incorporation of CoQIO into nanoparticles leads to further increase in efficacy, minimizing the variable bioavailability hence demonstrating the potential role of nanoparticulate system for cancer therapy. Example 4 Evaluation of CoQIO nanoparticles in hypertensive rats: Goldblatt method (2K1C) was used for induction of hypertension. The animals were divided into 5 groups. Group 1 was sham operated group; Group 2 was vehicle treated group. After surgery, Group 3 received 7 doses of CoQIO in suspension form (100 mg/Kg) while Group 4 and Group 5 received 3 doses of CoQIO (lOOmg/kg), as commercial formulation and prepared polymeric nanoparticles respectively. Blood pressure (systolic and diastolic) was recorded on 12th and 15th day after surgery using tail cuff blood pressure recorder (IITC INC., Life Science Instruments, Model No. 29,229; California, USA). Malondialdehyde (MDA) levels, an index of lipid peroxidation, were measured. MDA reacts with thiobarbituric acid (TBA) as a thiobarbituric acid reactive substance (TBARS) to produce a red complex that has a peak absorbance at 532 nm. On the 15th day after surgery, blood was collected from tail vein of rat under mild ether anesthesia into centrifuge tubes containing heparin and was used for estimation of MDA levels. On the 12th day after surgery, it was observed that renal clipping was able to induce hypertension in animals. The vehicle treated group had a higher average blood pressure (systolic 163.86±2.8 and diastolic 100.62±1.3 nun Hg) as compared to sham operated group (systolic 125.22±2.6 and diastolic 82.56±0.06 mm Hg). Administration of CoQIO was able to partially prevent the hypertension. The efficacy of 3 doses of nanoparticles in preventing hypertension (systolic 125.93±5.27and diastolic 83.36±2.39) was far more superior to 7 doses of CoQIO in suspension form (systolic 140.17±3.67 and diastolic 89.67±1.85). 3 doses of nanoparticles were able to reduce the blood pressure by 38 and 17 mm Hg for systolic and diastolic respectively in comparison to vehicle treated group while 7 doses were able to reduce it by 23 and 11 mm Hg (diastolic and systolic respectively). When the prepared nanoparticles were compared to commercial formulation, the nanoparticles were found to be more efficacious. The commercial formulation was able to reduce hypertension by 13 and 7 mm Hg for systolic and diastolic blood pressure (BP) respectively. The BP of nanoparticulate treated group was close to that of sham operated groups clearly demonstrating the efficacy of nanoparticulate systems. Similar results were obtained on 15th day after surgery. A marked difference in BP between vehicle treated group (systolic 164.53±1.50 and diastolic 100.31±0.99 mm Hg) was observed when compared to sham operated (systolic 119.50±2.18 and diastolic 78+1.92). The efficacy of 3 doses of nanoparticles in preventing hypertension (systolic 134.28±4.10 and diastolic 85.06±1.44) was again superior to 7 doses of CoQIO in suspension form (systolic 141.22±1.57 and diastolic 90.78±0.68) and 3 doses of commercial formulation. Three doses of nanoparticles were able to reduce the blood pressure by 30 and 17 mm Hg for systolic and diastolic in comparison to vehicle treated group while 7 doses were able to reduce 23 and 10 mmHg for diastolic and systolic BP and commercial formulation showed reduction by 15 and 8 mm Hg for systolic and diastolic respectively when compared to vehicle treated group. The efficacy of nanoparticulate system to reduce the MDA (malondialdehyde) levels by scavenging free radicals was also higher than compared to other formulations. From the results it was clear that incorporation of CoQIO into nanoparticles leads to an increase in efficacy of the drug for treatment of hypertension which may be due to increased bioavailability and sustained release from the nanoparticulate formulations. The nanoparticulate systems were able to reduce the dose by approximately 60%. So the nanoparticulate system is promising for therapeutic applications of CoQIO in hypertension. These nanoparticles formulations can also be used for various other therapeutic applications of CoQIO like in neurodegenerative disorders and other cardiovascular disorders. We Claim: 1. A nanoparticulate coenzyme Q10 drug delivery system comprising a co Q10 along with pharmaceutically acceptable adjuvant, diluent or carrier such as herein described. 2. A drug delivery system as claimed in claim 1, wherein the Coenzyme Q10 is encapsulated with polymeric materials. 3. A drug delivery system as claimed in claim 1, wherein the Coenzyme Q10 polymeric nanoparticles having particle size in the range of 1 to 999 nm. 3. A drug delivery system as claimed in claim 1, wherein the pharmaceutically acceptable diluent or carriers are polymeric material. 4. A composition as claimed in claim 3, wherein the polymer material is selected from polyester family such as Polycaprolactone (PCL), Poly Lactide (PLA), Poly Glycolide (PGA) and copolymers of these like Poly Lactide-co-Glycolide (PLGA), Poly Lactide- Polycaprolactone (PLA-PCL). 5. A composition as claimed in claim 1, wherein the stabilizer is quaternary ammonium salt like Didodecyldimethylammonium bromide, or anionic group such as Polyvinyl alcohol or nonionic group such as Vitamin ETPGS, or their combination. 7. A process for preparing a nanoparticulate co Q10 composition of claim 1, wherein said process comprising the steps: a) dissolving Coenzyme Q10 and polymer in an organic solvent optionally along with organic co-solvent to obtained organic phase such as herein described; b) adding the organic phase of step (a) to an aqueous system containing stabilizer and/ or surfactant to produce emulsion; c) reducing the size of emulsion to produce emulsion droplet. d) Removing residual organic solvent at controlled heating conditions such as herein described thereby transforming the emulsion into a liquid-solid suspension, wherein the solid phase comprises nanoparticulate Co Q10 composition. 8. A organic solvent as claimed in claim 7, wherein step (a), the organic solvent is selected from a group of organic solvents with partial to total water miscibility such as ethyl acetate, dichloromethane, acetone and mixture thereof.. 9. A process as claimed in claim 7, wherein step (c) reduction of the droplet size of emulsion is carried out by homogenization and wherein homogenization is achieved by supplying energy to the biphasic system by means of a device selected from the group consisting of high shear mechanical stirrers, ultrasonic probe or a device capable of forcing the emulsion through a narrow space under normal or high pressures. 10. A process as claimed in claim 7, wherein step (d) controlled heating is effected in presence or absence of vacuum. 11. A process as claimed in claim 7, wherein the final formulation is substantially free of organic solvent. 12. A process as claimed in claim 7, wherein the Coenzyme Q10 is dispersed uniformly throughout the matrix. 13. A nanoparticulate drug delivery system comprising a coenzyme Q10 and process for preparation of composition such as herein described with reference to the foregoing examples and accompanying drawings.

Full Text

Field of invention
The present invention is in the field of nanoparticle drug delivery system
and more particularly the invention discloses a nanoparticulate coenzyme Q10 (CoQIO) composition and preparation thereof.
Background of the invention
CoQIO is naturally occurring compound that plays a key role in energy metabolism as an integral part of electron transport system. Several studies have provided the evidence of the potentials of CoQIO in prophylaxis and therapy of various disorder related oxidative stress. CoQIO deficiency has been confirmed in patient with cardiovascular disorders like congestive heart failure coronary artery disease, angina pectoris, cardiomyopathy and hypertension. CoQIO deficiency also reported in cancer and neurodegenerative disorders like Parkinson's and Alzheimer's disease. CoQIO functions as antioxidant, scavenging free radicals and inhibiting free radicals. Despite of its potential pharmacological activity I many diseases, not much progress has been made in terms of formulation aspect owing to its poor aqueous and heigh molecular weight, leaving behind this molecule in the class of difficult to deliver molecules (class IV of Biopharmaceutical Classification System). The efficiency of CoQIO absorption and its bioavailability when given by oral route is very low from the current marketed products.
Recently nanoparticles made of degradable and non-degradable materials have been actively explored as delivery system for both small and large drug molecules. Poorly water soluble and high molecular weight molecules are often difficult to process into dosage form that afford adequate oral bioavailability for clinical studies and commercialization of CoQIO is no exception with poor aqueous solubility and a high molecular weight of 863.
Hence, there is a need for CoQIO formulation which has got higher bioavailability and the present invention has made an effort to provide
alternative formulation which obviates the existing problem of using CoQIO.
Objectives of the invention
The primary object of the present invention is to develop a nanoparticle based drug delivery system.
Another object of the present invention is to formulate nanoparticulate composition of CoQIO to increase its oral bioavailability.
Brief description of the accompanying drawings
Figure 1 is a bar graph showing the difference in uptake of 20 mg of CoQIO in the form of suspension and CoQIO loaded nanoparticles. The graph clearly shows that in case of CoQIO loaded nanoparticles percentage uptake is about 30% greater than suspension from thus suggesting enhanced uptake.
Figure 2 shows the stability of CoQIO in presence of ethyl acetate and ethyl acetate in combination with PLGA (values are expressed as mean ± standard deviation [n=3]).
Figure 3 shows the effect of initial drug loading on entrapment efficiency.
Figure 4 (a) shows the effect of different CoQIO formulation on systolic blood pressure on 12th day after surgery in glodblatt hypertensive rats in sham operated group Vs. vehicle operated group. (Statistical analysis was performed by one way of ANOVA followed by Tukey's test for multipule comparisons. *p Figure 4 (b) shows the effect of different CoQIO formulations on diastolic blood pressure on 12th day after surgery in glodblatt hypertensive rats in sham operated group Vs. vehicle operated group. (Statistical analysis was performed by one way of ANOVA followed by Tukey's test for multipule comparisons. *p Figure 5 (a) shows the effect of different CoQIO formulations on systolic blood pressure on 15th day after surgery in glodblatt hypertensive rats in sham operated group Vs. vehicle operated group. (Statistical analysis was performed by one way of ANOVA followed by Tukey's test for multipule comparisons. *p Figure 5 (b) shows the effect of different CoQIO formulations on diasystolic blood pressure on 15th day after surgery in glodblatt hypertensive rats in sham operated group Vs. vehicle operated group. (Statistical analysis was performed by one way of ANOVA followed by Tukey's test for multipule comparisons. *p Figure 6 shows the effect of different Coenzyme Q10 formulations on lipid peroxidation of hypertensive rats in sham operated group Vs. vehicle operated group. (Statistical analysis was performed by one way of ANOVA followed by Tukey's test for multipule comparisons. *p Detailed description of the invention
Accordingly, the present invention deals with designing of nanoparticles that can effectively deliver the CoQIO orally, and more particularly relates to dose reduction, improved bioavailability and efficacy.
The main embodiment of the invention is Polymeric biodegradable CoQIO loaded nanoparticles for oral delivery was prepared by the emulsion-diffusion-evaporation method which consists of following steps:
a) dissolving of CoQIO and polymer in organic phase
b) addition of the organic phase of step (a) to aqueous system containing
stabilizer /surfactant resulting into emulsion
c) stirring the emulsion followed by homogenization of emulsion,
d) addition of water to homogenized emulsion at constant stirring, and
e) removal of organic solvent at controlled conditions thereby transforming the emulsion into a liquid-solid suspension, whereby the solid comprises of polymeric nanoparticles of PLGA loaded with CoQW for oral administration.
The nanoparticles were optimized for drug loading. The effect of initial drug loading on particle size, particle size distribution, PDI, zeta potential and encapsulation efficiency was investigated. It was observed that there was no significant change in particle size with increasing CoQIO loading from 5 to 30 %w/w of polymer; however a slight increase in the particle size along with PDI was observed for 50% and 75% initial drug loading.
Increase in encapsulation efficiency was found with increase in initial drug loading from 5 to 20% w/w of polymer (from 61.7±3.9 for 5% to 82.0±3.3 for 20%) and beyond 20% the entrapment efficiency was around 80%. There was no change in zeta potential for any of the different drug loading batches (table 2). All the initial drug loading batches showed good reproducibility as observed from low standard deviation values. The nanoparticulate system was capable of entrapping small as well as large amount of drug without dramatic change in particle size and PDI. Such large range of initial drug loading provides us an option to produce different dose formulations without any change in the process. The high drug loading capacity along with high entrapment efficiency makes it a commercially feasible process. The prepared nanoparticles were evaluated for absorption characteristics by in situ studies and for its efficacy in hypertensive and breast cancer animal models (table 2).
Table 1 shows the effect of initial drug loading on particle characteristics and encapsulation efficiency Initial Drug Size PDI Zeta Potential Entrapment
(Table Removed)
The zeta potential values reported are in the pH range 4.0- 4.85 (The values reported are mean±S.D. (n=3))
Table 2 shows the systolic and Diastolic blood pressure of hypertensive rats of various groups treated with different Coenzyme Q10 formulations (Table Removed)
Table 3 shows the equilibrated solubility of CoQIO with different solvent system.
(Table Removed) (Values are expressed as meant standard deviation [n=3]) Table 4 shows the effect of initial drug loading on particle size and PDI (Table Removed)
(Values are expressed as meant standard deviation [n=3])
Yet another embodiment of the invention is nanoparticulate formulation of Coenzyme Q10, comprising of Coenzyme Q10, polymeric material and stabilizer/ surfactant.
Still another embodiment of the invention is the polymer used in nanoparticulate formulation of Coenzyme Q10 belongs to polyester family, for example Polycaprolactone (PCL), Poly Lactide (PLA), Poly Glycolide (PGA) and copolymers of these like Poly Lactide-co-Glycolide (PLGA), PLA-PCL.
Yet another embodiment of the invention is organic phase used to solubilize Coenzyme Q10 and polymer in nanoparticulate formulation of Coenzyme Q10, is selected from a group of organic solvents with partial to total water miscibility like ethyl acetate, dichloromethane, and acetone and any other such organic solvent and mixtures thereof.
Still another embodiment of the invention is organic phase used to soluMise Coenzyme Q10 and polymer in nanoparticulate formulation of Coenzyme Q10, is a co-solvent system with organic solvents, wherein at least one of the solvents of the co-solvent system is from those mentioned above.
Yet another embodiment of the invention is stabilizer forms a barrier between the continuous phase and the dispersed phase in the emulsion formed in step (b) in the nanoparticulate formulation of Coenzyme Q10.
Still another embodiment of the invention is stabilizer dissolved in aqueous phase can be cationic (for example chitosan or quaternary ammonium salt like Didodecyldimethylammonium bromide), anionic (Polyvinyl alcohol) and nonionic (Vitamin ETPGS), or their combination in the nanoparticulate formulation of Coenzyme Q10.
Yet another embodiment of the invention is different dose combinations can be obtained in the nanoparticulate formulation of Coenzyme Q10.
Still another embodiment of the invention is Coenzyme Q10 loaded polymeric nanoparticles produced are in the range of 1 to 999 nm in size in the nanoparticulate formulation of Coenzyme Q10.
Yet another embodiment of the invention is nanoparticles produced show in situ uptake of particles greater than 75% in the nanoparticulate formulation of Coenzyme Q10.
Still another embodiment of the invention is homogenization is achieved by supplying energy to the biphasic system by means of a device selected from the group consisting of high shear mechanical stirrers, ultrasonic probe or a device capable of forcing the emulsion through a narrow space under normal or high pressures in the nanoparticulate formulation of Coenzyme Q10.
Yet another embodiment of the invention is controlled heating in presence or absence of vacuum is used to remove the organic solvent during nanoparticulate preparation in the nanoparticulate formulation of Coenzyme Q10.

Still another embodiment of the invention is formulation is used for treatment of hypertension.
Yet another embodiment of the invention is formulation is able to reduce the systolic and diastolic blood pressure by 38 and 17 mmHg in renal hypertension model.
Still another embodiment of the invention is the formulation is able to reduce MDA levels by scavenging free radicals.
Yet another embodiment of the invention is nanoparticulate formulation of Coenzyme Q10 is used for treatment of cancer.
Still another embodiment of the invention is nanoparticulate formulation of Coenzyme Q10 can be used as nutritional supplement.
Yet another embodiment of the invention is nanoparticulate formulation of Coenzyme Q10 can be used for cosmetic applications.
Still another embodiment of the invention is nanoparticulate formulation of Coenzyme Q10 is used for prophylaxis and therapy of various cardiovascular disorders like hypertension, cardiomyopathy, angina pectoris, congestive heart failure and coronary artery disease, and other disorders like neurodegenerative disorders (Parkinson's, Alzheimer's etc) and periodontal diseases. Yet another embodiment of the invention is matrix material of the nanoparticulate formulation of Coenzyme Q10 are degrades into harmless products in vivo.
Still another embodiment of the invention is nanoparticulate formulation of Coenzyme Q10 contains very low concentration of stabilizer/surfactant which doesn't cause any inflammatory response and considered safe under in vivo conditions.
Yet another embodiment of the invention is the final nanoparticulate formulation of Coenzyme Q10 is free of organic solvent.
Still another embodiment of the invention is Coenzyme Q10 is dispersed uniformly throughout the matrix in the nanop articulate formulation of Coenzyme Q10.
Yet another embodiment of the invention is nanoparticulate formulation of Coenzyme Q10 is route independent formulation.
Now, the present invention will be described in detail in reference to examples, but the scope of the present invention should not be limited to these examples. In the examples, "part" and "parts" mean "part by weight" and "parts by weight", respectively, unless otherwise specified.
The invention is illustrated by the following examples which are only meant to illustrate the invention and not act as limitations. All the embodiments apparent to a process their in the art are deemed to fall within the scope of the invention.
Examples
Example 1
Process for the preparation of CoQIO loaded polymeric nanoparticles
Nanoparticles were prepared by adopting an emulsion-diffusion-evaporation technique, which was further optimized for drug loading. The increase in initial drug loading from 5 to 20% w/w of polymer led to increase in encapsulation efficiency and beyond 20% initial loading, the entrapment efficiency reached a plateau of 80%. There was no significant change in particle size and distribution with increasing drug loading from 5 to 30 %w/w of polymer; however a slight increase in the particle size and distribution was observed for 50% and 75% initial drug loading. The nanoparticles when evaluated for in situ uptake studies in Sprague-Dawley (SD) rats, showed better uptake in comparison to suspension form. The values of uptake were 45% for CoQIO suspension and 79% for CoQIO loaded nanoparticles. The nanoparticulate formulation was evaluated in hypertensive rats. The nanoparticles were compared with suspension form and
. the commercial formulation. The ability of 3 doses of CoQIO (100 mg/kg) to treat hypertension was far more superior to 7 doses of CoQIO in suspension form and 3 doses of commercial formulation. CoQIO at 60% lower dose in nanoparticulate form was able to bring blood pressure to normal range on 12th day in comparison to the suspension form. The CoQIO nanoparticles were further investigated in breast cancer model and compared with the suspension form. The CoQIO nanoparticles showed better efficacy than the suspension form in reducing the tumor size. The in situ uptake studies along with evaluation in hypertension and cancer animal models clearly indicate the efficacy of CoQIO in the form of nanoparticles to that of suspension form and currently available commercial formulation.
Example 2
Preparation of CoQIO loaded nanoparticles: 50 mg of polymer (of polyester family: Polycaprolactone (PCL), Poly Lactide (PLA), Poly Glycolide (PGA) and copolymers of these like Poly Lactide-co-Glycolide (PLGA), PLA-PCL etc) was dissolved in 2 ml organic phase (ethyl acetate, dichloromethane, acetone and co-solvent systems of different organic solvent) at room temperature for 2 hours. CoQIO was dissolved in 0.5ml of organic phase and added to the polymeric solution and stirred for 1 hour. The organic phase was then emulsified with 5 ml of an aqueous phase containing stabilizer/surfactant (PVA, DMAB or Vitamin ETPGS). The resulting o/w emulsion was stirred at room temperature for 1 hour before homogenizing at 15000 rpm for 5 min using a high-speed homogenizer (Polytron PT4000; Polytron Kinematica, Lucerne, Switzerland). To this emulsion, water was added with constant stirring which resulted into nanoprecipitation. The prepared nanoparticles have been optimized for drug loading.
Example 3
Evaluation of CoQIO nanoparticles for its intestinal absorption and efficacy in
animal models.
Evaluation for intestinal absorption by in situ up takes study: Closed loop method was used to assess the uptake of CoQIO in suspension form and CoQIO loaded PLGA nanoparticles prepared using DMAB as stabilizer with 20 and 50% w/w of polymer as initial drug loading. Overnight fasted male Sprague Dawley (SD) rats were anaesthetized by intraperitoneal administration of thiopentone (50 mg/kg). The intestine was exposed through a midline incision and a closed loop of 10 cm length was prepared on the upper jejunum by ligation at both the ends. 1 ml of aqueous drug suspension or drug loaded nanoparticles containing 20 mg of CoQIO was injected into the loop with a syringe. Then entire intestine was restored to the abdominal cavity and body temperature was maintained during anesthesia. After 2 hours of drug administrationy the loops were rapidly isolated from the body. Contents of the loop were emptied into a container. The lumen of the intestine was rinsed with 10 ml water followed by 10 ml of methanol. The water and methanol solutions were collected and processed accordingly and diluted appropriately and the unabsorbed drug remaining in the solution was analyzed by HPLC. The uptake of CoQIO in the form of suspension was found to be 45%. It has been reported that around 60% of the CoQIO is excreted in feces in unchanged form and our uptake results closely match the reported values. The uptake of CoQIO nanoparticles was found to be 79% significantly greater than the suspension form clearly demonstrating the superiority of the nanoparticulate system for its absorption characteristics.
Evaluation of efficacy of nanoparticulate system in breast cancer model:
The prepared CoQIO nanoparticles were evaluated in breast cancer model. Female SD rats were administered DMBA (7,12-dimethylbenz[a]anthracene). Twenty two weeks after administration of DMBA the animals developed tumors. The animals were divided into 3 groups.
One group received no treatment (untreated group); second group received 25 mg of CoQIO in suspension form and the last group was administered nanoparticles containing 25 mg of CoQIO.
Frequency of administration was once a week for both the groups for 8 weeks. After administration of 8th dose (30th week from administration of DMBA) the animals were sacrificed and the tumors were removed and their volume measured. The nanoparticulate formulation was able to reduce the tumor burden by about 68% to that of untreated groups, whereas in the CoQIO suspension group the reduction was about 61 %. It was quite interesting to note the fact that in CoQIO suspension group there was a huge variability between the animals which is in agreement to the reported literature about variable bioavailability of CoQIO. The study demonstrated the efficacy of CoQIO in cancer treatment and incorporation of CoQIO into nanoparticles leads to further increase in efficacy, minimizing the variable bioavailability hence demonstrating the potential role of nanoparticulate system for cancer therapy.
Example 4
Evaluation of CoQIO nanoparticles in hypertensive rats:
Goldblatt method (2K1C) was used for induction of hypertension. The animals were divided into 5 groups. Group 1 was sham operated group; Group 2 was vehicle treated group. After surgery, Group 3 received 7 doses of CoQIO in suspension form (100 mg/Kg) while Group 4 and Group 5 received 3 doses of CoQIO (lOOmg/kg), as commercial formulation and prepared polymeric nanoparticles respectively. Blood pressure (systolic and diastolic) was recorded on 12th and 15th day after surgery using tail cuff blood pressure recorder (IITC INC., Life Science Instruments, Model No. 29,229; California, USA). Malondialdehyde (MDA) levels, an index of lipid peroxidation, were measured. MDA reacts with thiobarbituric acid (TBA) as a thiobarbituric acid reactive substance (TBARS) to produce a red complex that has a peak absorbance at 532 nm. On the 15th day after surgery, blood was collected from tail vein of rat under mild ether anesthesia into centrifuge tubes containing heparin and was used for estimation of MDA levels. On the 12th day after surgery, it was observed that renal clipping was able to induce hypertension in animals. The vehicle treated group had a higher average blood pressure (systolic 163.86±2.8 and diastolic
100.62±1.3 nun Hg) as compared to sham operated group (systolic 125.22±2.6 and diastolic 82.56±0.06 mm Hg). Administration of CoQIO was able to partially prevent the hypertension. The efficacy of 3 doses of nanoparticles in preventing hypertension (systolic 125.93±5.27and diastolic 83.36±2.39) was far more superior to 7 doses of CoQIO in suspension form (systolic 140.17±3.67 and diastolic 89.67±1.85). 3 doses of nanoparticles were able to reduce the blood pressure by 38 and 17 mm Hg for systolic and diastolic respectively in comparison to vehicle treated group while 7 doses were able to reduce it by 23 and 11 mm Hg (diastolic and systolic respectively). When the prepared nanoparticles were compared to commercial formulation, the nanoparticles were found to be more efficacious. The commercial formulation was able to reduce hypertension by 13 and 7 mm Hg for systolic and diastolic blood pressure (BP) respectively. The BP of nanoparticulate treated group was close to that of sham operated groups clearly demonstrating the efficacy of nanoparticulate systems. Similar results were obtained on 15th day after surgery. A marked difference in BP between vehicle treated group (systolic 164.53±1.50 and diastolic 100.31±0.99 mm Hg) was observed when compared to sham operated (systolic 119.50±2.18 and diastolic 78+1.92). The efficacy of 3 doses of nanoparticles in preventing hypertension (systolic 134.28±4.10 and diastolic 85.06±1.44) was again superior to 7 doses of CoQIO in suspension form (systolic 141.22±1.57 and diastolic 90.78±0.68) and 3 doses of commercial formulation. Three doses of nanoparticles were able to reduce the blood pressure by 30 and 17 mm Hg for systolic and diastolic in comparison to vehicle treated group while 7 doses were able to reduce 23 and 10 mmHg for diastolic and systolic BP and commercial formulation showed reduction by 15 and 8 mm Hg for systolic and diastolic respectively when compared to vehicle treated group. The efficacy of nanoparticulate system to reduce the MDA (malondialdehyde) levels by scavenging free radicals was also higher than compared to other formulations. From the results it was clear that incorporation of CoQIO into nanoparticles leads to an increase in efficacy of the drug for treatment of hypertension which may be due to increased bioavailability and sustained release from the nanoparticulate formulations. The nanoparticulate
systems were able to reduce the dose by approximately 60%. So the nanoparticulate system is promising for therapeutic applications of CoQIO in hypertension. These nanoparticles formulations can also be used for various other therapeutic applications of CoQIO like in neurodegenerative disorders and other cardiovascular disorders.

We Claim:
1. A nanoparticulate coenzyme Q10 drug delivery system comprising a co Q10 along with pharmaceutically acceptable adjuvant, diluent or carrier such as herein described.
2. A drug delivery system as claimed in claim 1, wherein the Coenzyme Q10 is
encapsulated with polymeric materials.
3. A drug delivery system as claimed in claim 1, wherein the Coenzyme Q10
polymeric nanoparticles having particle size in the range of 1 to 999 nm.
3. A drug delivery system as claimed in claim 1, wherein the pharmaceutically
acceptable diluent or carriers are polymeric material.
4. A composition as claimed in claim 3, wherein the polymer material is selected
from polyester family such as Polycaprolactone (PCL), Poly Lactide (PLA), Poly Glycolide (PGA) and copolymers of these like Poly Lactide-co-Glycolide (PLGA), Poly Lactide- Polycaprolactone (PLA-PCL).
5. A composition as claimed in claim 1, wherein the stabilizer is quaternary
ammonium salt like Didodecyldimethylammonium bromide, or anionic
group such as Polyvinyl alcohol or nonionic group such as Vitamin ETPGS, or
their combination.
7. A process for preparing a nanoparticulate co Q10 composition of claim 1, wherein said process comprising the steps:
a) dissolving Coenzyme Q10 and polymer in an organic solvent optionally along with organic co-solvent to obtained organic phase such as herein described;
b) adding the organic phase of step (a) to an aqueous system containing stabilizer and/ or surfactant to produce emulsion;
c) reducing the size of emulsion to produce emulsion droplet.
d) Removing residual organic solvent at controlled heating conditions such as herein described thereby transforming the emulsion into a liquid-solid suspension, wherein the solid phase comprises nanoparticulate Co Q10 composition.
8. A organic solvent as claimed in claim 7, wherein step (a), the organic solvent is
selected from a group of organic solvents with partial to total water miscibility
such as ethyl acetate, dichloromethane, acetone and mixture thereof..
9. A process as claimed in claim 7, wherein step (c) reduction of the droplet size of
emulsion is carried out by homogenization and wherein homogenization is achieved by supplying energy to the biphasic system by means of a device selected from the group consisting of high shear mechanical stirrers, ultrasonic probe or a device capable of forcing the emulsion through a narrow space under normal or high pressures.
10. A process as claimed in claim 7, wherein step (d) controlled heating is effected
in presence or absence of vacuum.
11. A process as claimed in claim 7, wherein the final formulation is substantially
free of organic solvent.
12. A process as claimed in claim 7, wherein the Coenzyme Q10 is dispersed
uniformly throughout the matrix.
13. A nanoparticulate drug delivery system comprising a coenzyme Q10 and process for preparation of composition such as herein described with reference to the foregoing examples and accompanying drawings.

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Patent Number

279439

Indian Patent Application Number

18/DEL/2006

PG Journal Number

04/2017

Publication Date

27-Jan-2017

Grant Date

23-Jan-2017

Date of Filing

02-Jan-2006

Name of Patentee

NATIONAL INSTITUTE OF PHARMACEUTICAL EDUCATION AND RESEARCH

Applicant Address

SECTOR 67, S.A.S. NAGAR, MOHALI-160062, PUNJAB, INDIA

Inventors:

#	Inventor's Name	Inventor's Address
1	MAJETI NAGA VENKATA RAVIKUMAR	DEPARTMENT OF PHARMACEUTICS, NIPER, SECTOR 67, S.A.S. NAGAR, MOHALI-160062, PUNJAB, INDIA
2	DHAWAL D. ANKOLA	DEPARTMENT OF PHARMACEUTICS, NIPER, SECTOR 67, S.A.S. NAGAR, MOHALI-160062, PUNJAB, INDIA
3	VISWANAD BHOOMI	DEPARTMENT OF PHARMACEUTICS, NIPER, SECTOR 67, S.A.S. NAGAR, MOHALI-160062, PUNJAB, INDIA
4	VIVEKANAND BHARDWAJ	DEPARTMENT OF PHARMACEUTICS, NIPER, SECTOR 67, S.A.S. NAGAR, MOHALI-160062, PUNJAB, INDIA
5	PODURI RAMARAO	DEPARTMENT OF PHARMACEUTICS, NIPER, SECTOR 67, S.A.S. NAGAR, MOHALI-160062, PUNJAB, INDIA

PCT International Classification Number

A61K 9/51

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA