A NANOFORMULATION FOR COMBINATORIAL DRUG DELIVERY AND A METHOD OF PREPARATION THEREOF

Abstract

The present disclosure relates to the field of drug delivery systems. Particularly, the disclosure provides nanostructured lipid carriers (NLCs) formulation comprising a) a blend of a solid lipid and oil-based matrix with a globule size less than 200 nm; b) a non-ionic surfactant covering the lipid matrix; and c) a pharmaceutical active agent encapsulated in the lipidic matrix. The present disclosure also provides NLCs comprising: 2 to 4% w/w of the lipid matrix of the total weight of the formulation; 3 to 5% w/w of a non-ionic surfactant of the total weight of the formulation; and a therapeutic agent, wherein the therapeutic agent to total lipid ratio is in the range of 0.1:99.9 to 40:60. The present disclosure provides the delivery of an acetylcholinesterase (AChE) inhibitor, a butyrylcholinesterase enzyme inhibitor, a kinase inhibitor, and a combination thereof to the brain to achieve their combined synergistic effects against neurodegenerative disorder.

Specification

Description:FIELD OF INVENTION
[0001] The invention relates to a dual drug-loaded nanostructured lipid carrier (NLC) that is useful for the treatment of Alzheimer's disease (AD). More particularly, the present disclosure relates to the NLC for the intranasal delivery of drugs to the brain wherein the unique structure of NLC allows for increased bioavailability and therapeutic efficacy of the drugs. In the present invention, a drug combination of Rivastigmine Tartrate (RIV) and Nilotinib hydrochloride monohydrate (NIL) has been encapsulated in the pharmaceutical composition for the treatment of AD.
BACKGROUND OF INVENTION
[0002] Background description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.
[0003] AD is a neurological condition marked by memory loss, cognitive decline, behavioral abnormalities, and loss of functional abilities. The medial temporal lobe and neocortical structures of the brain, which are the most affected parts of the brain, accumulate amyloid-beta peptides (Aß), which causes the most prevalent form of dementia, which is described as a slowly progressing neurodegenerative illness with neuritic plaques and neurofibrillary tangles. Every ten years, the prevalence of the disease doubles. More than 50 million individuals worldwide are believed to have dementia today, with AD being the most common cause. This quantity is anticipated to exceed 150 million by 2050.
[0004] Four medications have been approved by the FDA (Food and Drug Administration) for AD. Donepezil (DPL), Rivastigmine (RIV), and galantamine are three of these acetylcholinesterase enzyme (AChE) inhibitors, while memantine (MM) is an antagonist of the N-methyl-D-aspartate (NMDA) receptor. However, the benefits of these medications in managing symptoms are merely marginal. Additionally, they are ineffective at halting the gradual decline in cognition caused by neuronal death, brain atrophy, or a combination thereof.
[0005] From the available treatment, a combination of RIV and NIL showed a promising therapeutic outcome, wherein RIV is a compound derived from physostigmine and acts as an inhibitor of both acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) enzymes, commonly used in AD treatment. It is administered orally but causes gastrointestinal side effects. Due to its short half-life of 1.5 hours, the typical dosage is 3 mg/day, divided into two doses. However, its oral bioavailability is relatively low at 40%. On the other hand, NIL is a kinase inhibitor primarily used to treat the chronic phase of Chronic Myeloid Leukemia (CML). NIL is a potent drug, with moderate brain penetration. The standard dosage for NIL is 150 mg. NIL has relatively low oral bioavailability, approximately 30%. This is due to extensive first-pass metabolism and poor gastrointestinal absorption. Notably, a study suggests that NIL may improve memory function by preventing the degeneration of dopaminergic neurons in AD.
[0006] The currently available formulations either have less site specificity or have sub-optimal therapeutic efficacy. Further, no combinational therapy of RIV and NIL loaded in nanostructured lipid carriers is used via the intranasal route.
[0007] The prior art of CN113599384A discloses a nano medicinal preparation comprised rosmarinic acid and nilotinib. The composition can be used for synergistically activating cell autophagy and has the effects of nerve protection and oxidative stress resistance. The medicine loaded with the rosmarinic acid and the nilotinib, can be used for effectively protecting dopaminergic neurons in nigra, so that the motion symptom of MPTP-induced Parkinson's Disease (PD) mice is improved, and a prospective scheme is provided for PD-targeted therapy.
[0008] Another prior art WO2024/081910A1 discloses liposome particles containing one or more active pharmaceutical ingredients (APIs), nilotinib, within the interior aqueous compartment of the particles, pharmaceutical compositions thereof, and the use of the drug-loaded liposomes and pharmaceutical compositions thereof for treatment of patients, including various types of cancer patients and neurodegenerative disease, to achieve synergistic therapeutic effects. However, none of the cited documents disclose a combination of RIV and NIL-loaded nanoformulation.
[0009] So far studies on RIV and NIL have not adequately addressed key challenges such as extensive metabolism, low solubility, limited permeability, low bioavailability, toxicity, and the dose-related burden on subjects. Moreover, there is a lack of developed formulations that enhance the bioavailability of RIV and NIL by bypassing hepatic first-pass metabolism and minimizing dose-related side effects through the intranasal route. Therefore, there is a requirement for a formulation that is capable of overcoming hepatic first-pass metabolism, reducing the frequency of the drugs, crossing the blood-brain barrier, providing controlled release, having minimal side effects, and enhanced bioavailability.

OBJECTIVES OF THE INVENTION
[0010] The main objective of the present invention is to provide a combination of RIV and NIL-loaded NLC for the treatment of AD.
[0011] Another object of the present invention is to develop a formulation with a high % entrapment efficiency.
[0012] Yet another object of the present invention is to provide an NLC loaded with drug combination for intranasal delivery to improve the bioavailability and therapeutic efficacy of the drugs in the brain.
[0013] Yet another object of the present invention is to provide an NLC loaded with drug combinations that exhibit improved bioavailability.
[0014] Still, another object of the present invention is to provide a method of preparation of the dual drug-loaded NLC for treating AD.
SUMMARY OF THE INVENTION
[0015] The present disclosure relates to a combination of cholinesterase inhibitor and kinase inhibitor-loaded NLC formulations for the treatment of neurodegenerative disease. More particularly, the present invention relates to an NLC formulation encapsulating matrixed RIV and NIL for targeted delivery of the drug to the brain via the intranasal route of administration.
[0016] In an embodiment, the present disclosure provides NLC comprising: a binary mixture; pharmaceutical active ingredients; a surfactant; and pharmaceutically acceptable excipients; wherein the binary mixture forms from a blend of solid and liquid lipids which is a core of the formulation; pharmaceutical active ingredients preferably RIV, NIL, or in combination; and the pharmaceutically active ingredient is dispersed in the matrix of binary mixture.
[0017] In another embodiment, the formulation is designed to enhance the delivery of therapeutic molecules via the intranasal route of administration leading to enhanced bioavailability and therapeutic efficacy; thus, offering a promising treatment for AD.
[0018] In yet another embodiment, the nanostructured lipid carriers were designed to improve bioavailability by making an imperfect crystal structure that accommodates more therapeutic molecules in the formulation. The prepared formulation can emerge as a promising delivery system for targeting therapeutic molecules in the brain via the intranasal route; thus, the formulation can create a direct connection between the nasal cavity and the brain, bypassing the blood-brain barrier.
[0019] In yet another embodiment, the present disclosure provides nanostructured lipid carriers comprising: 2 to 4% w/v of the binary mixture of the total weight of the formulation; 3 to 5% w/v of surfactant of the total weight of the formulation; pharmaceutical active ingredients; and sonication period for 80 to 140 seconds to sonicate the blend of a binary mixture, emulsifier, and pharmaceutical active ingredients.
[0020] In still yet another embodiment, the present disclosure provides a method of preparation for nanostructured lipid carrier formulations comprising: a) screening of the solid and liquid lipids based on the highest solubility of active ingredients; b) optimization of the binary lipid mixture (solid and liquid lipid) ratio based on the stability; c) the binary lipid mixture was melted and dissolved in half quantity of the surfactant followed by constant stirring on a magnetic stirrer resulting in the preparation of the lipid phase; d) simultaneously the aqueous phase was also prepared by adding the remaining half quantity of the selected surfactant in the required quantity of water stirred and maintained at the same conditions as that of the lipid phase; e) mix the aqueous phase into the lipid phase with continuous stirring at maintained temperature; and f) the resultant mixture was sonicated using an Ultra-Probe sonicator by keeping the mixture in the ice bath followed by cooling it down at room temperature to obtain a nanostructured lipid carriers.
[0021] The above objects and advantages of the present disclosure will become apparent hereinafter, and a brief description of the drawings, a detailed description of the invention, and claims are appended herewith.
BRIEF DESCRIPTION OF THE DRAWINGS
[0022] The accompanying drawings are included to provide further understanding of the present disclosure and are incorporated in and constitute a part of this specification. The drawings illustrate exemplary embodiments of the present disclosure and the description, explains the principles of the present disclosure.
[0023] Figure 1 illustrates molecular docking between therapeutic molecules and the binding site of the receptor, wherein (A) dock pose of Rivastigmine at the binding site of Acetylcholine esterase (ACE) showing hydrogen bond interaction (red dotted line) with TYR 130 and ASP 172 and pi-pi interaction (turquoise dotted line) with TRP 84 and PHE 330 amino acid residues (PDB ID: 1ACJ, Docking scores: -8.781 Kcal/mol), (B) dock pose of Nilotinib at the binding site of Acetylcholine esterase (ACE) showing pi-pi interaction (turquoise dotted line) with TRP 84, PHE 330, TYR 334 and TRP 279 amino acid residues (PDB ID: 1ACJ, Docking scores: -8.890 Kcal/mol), (C) dock pose of Tacrine (Cocrystal ligand) at the binding site of Acetylcholine esterase (ACE) showing hydrogen bond interaction (red dotted line) with HIS 440 and pi-pi interaction (turquoise dotted line) with TRP 84 and PHE 330 amino acid residues (PDB ID: 1ACJ, Docking scores: -10.024 Kcal/mol), and (D) the final structure alignment with favourable interaction or similar imitating interaction with tacrine co-crystal ligand shows the structure alignment with inhibitory effect towards the acetylcholine esterase enzyme. Superimposed dock poses of Nilotinib with Rivastigmine and Tacrine at the binding site of Acetylcholine esterase (ACE) showing hydrogen bond interaction (red dotted line) and pi-pi interaction (turquoise dotted line).
[0024] Figure 2 illustrates a 2D graph of % cell viability of N2a cell lines for (A) RIV, and (B) NIL performed for 24 hours, wherein the curve between the concentration and mean (%) cell viability revealed the IC50 concentration of RIV and NIL at 125 µg/ml and 62.5µg/ml. On increasing the concentration of RIV and NIL individually from 0.97 to 2000 µg/ml, the mean % cell viability was observed to be 68.42 ± 0.41 to 36.63 ± 0.34 whereas, in NIL it was observed to be 70.85 ± 0.44 to 36.51 ± 0.37, (C) 2D graph Dose effect Curve, and (D) 2D graph Combination index plot. CompuSyn version 1.0 was used to calculate the combination index (CI), which advised a 1:1 ratio of RIV and NIL for drug combination studies to have the greatest possible synergistic effect (CI was found to be 0.106) respectively, display the dose-effect curve, the combination index plot, and the logarithmic combination index plot. Therefore, it was suggested that NLC formulations be developed with a 1:1 combination ratio of RIV and NIL for the N2a cell line.
[0025] Figure 3 illustrates (A) the solubility of RIV and NIL in liquid lipids (medium-chain triglycerides and long-chain triglyceride) which showed that the maximum solubility of RIV and NIL was observed in Caproyl® PGMC of 52.62 ± 1.19 mg/ml and 54.47 ± 1.36 mg/ml, The drugs are usually more soluble in MCTs than LCTs because of the better emulsifying properties of MCTs over LCTs, (B) the solubility of RIV and NIL in solid lipids which showed that maximum solubility of RIV and NIL in Gelot®64 of 35.05 ± 2.55 mg/g and 36.40 ± 1.64 mg/g respectively, and (C) the selection of surfactant based on the emulsification capacity of the emulsifier for the selected BM, where the highest transmittance was observed with Sween 20 which was found to be 94.61 ± 2.08%.
[0026] Figure 4 illustrates the effect of the combination of independent variables i.e., the concentration of lipidic mixture and surfactant, sonication time and concentration of the lipidic mixture, and lastly sonication time and concentration of the surfactant on the globule size, polydispersity index (PDI), % entrapment efficiency (%EE) of RIV and NIL in RIV-NIL-NLC.
[0027] Figure 5 illustrates the (A) globule size and PDI of the optimized formulation of RIV-NIL-NLC showing that the size of RIV-NIL-NLC is within the desired range i.e., >200 nm for the targeted delivery of the drug to the brain via intranasal route; however, the PDI indicates that the developed formulation was homogenous and stable, (B) Zeta Potential of the optimized formulation of RIV-NIL-NLC was found to be 0.814 mV which observed to be near 1mV which must be due to the drug combination loaded in the NLC, and (C) transmission electron microscope of optimized RIV-NIL-NLC formulation showing the spherical-shaped globules appearing as dark, circular spots against a lighter background, possibly due to the differential scattering of electrons through the globule material.
[0028] Figure 6 illustrates the % cell viability of nanoformulations against Neuro2a cells, wherein % cell viability of RIV-NIL-NLC against Neuro2a cells shows that the combination loaded NLC showed the highest % cell viability i.e., 66.53% at a concentration of 500.00 µg/mL in comparison to the RIV-NLC and NIL-NLC, which showed % cell viability of 65.76% and 63.53% respectively signifying the synergistic effect of the drug combination loaded NLC over the single drug loaded NLC and highlighting the least cytotoxicity of this combination
[0029] Figure 7 illustrates the plasma drug concentration profile of RIV and NIL after intranasal administration of RIV-NIL-NLC and RIV-NIL-SUS and the data are expressed as mean ±SD (n = 3) showing that the maximum amount of RIV and NIL was available from the NLC at 4hr and 8hr, respectively. In contrast, in RIV-NIL-SUS, the maximum concentrations of RIV and NIL were observed at 2hr and 4hr. *** p< 0.001, ** p< 0.01, * p< 0.05= Statistically significant compared to plain drug.
[0030] Figure 8 illustrates the drug concentration profile of RIV and NIL in the brain after intranasal administration of RIV-NIL-NLC and RIV-NIL-SUS where the data are expressed as mean ±SD (n = 3) showing that the maximum amount of RIV and NIL released from the NLC at 4hr and 8hr, respectively. In contrast, in RIV-NIL-SUS, the maximum concentrations of RIV and NIL were observed at 2hr and 4hr. *** p< 0.001, ** p< 0.01, * p< 0.05= Statistically significant compared to plain drug.
[0031] Figure 9 illustrates the drug concentration profile of RIV and NIL in different organs after intranasal administration of RIV-NIL-NLC and RIV-NIL-SUS in the rats. *** p< 0.001, ** p< 0.01, * p< 0.05= Statistically significant compared to plain drug.
DETAILED DESCRIPTION OF THE INVENTION
[0032] The embodiments herein and the various features and advantageous details thereof are explained more comprehensively concerning the non-limiting embodiments that are detailed in the following description. Descriptions of well-known components and processing techniques are omitted so as not to unnecessarily obscure the embodiments herein. The examples used herein are intended merely to facilitate an understanding of how the embodiments herein may be practiced and to further enable those of skill in the art to practice the embodiments herein. Accordingly, the examples should not be construed as limiting the scope of the embodiments herein.
[0033] Unless otherwise specified, all terms used in disclosing the invention, including technical and scientific terms, have the meaning as commonly understood by one of the ordinary skills in the art to which this invention belongs. Using further guidance, term definitions may be included to appreciate the teaching of the present invention better.
[0034] As used in the description herein, the meaning of "a," "an," and "the" includes plural reference unless the context dictates otherwise. Also, as used in the description herein, the meaning of "in" includes "in" and "on" unless the context dictates otherwise.
[0035] As used herein, the terms "comprise", "comprises", "comprising", "include", "includes", and "including" are meant to be non-limiting, i.e., other steps and other ingredients which do not affect the end of result can be added. The above terms encompass the terms "consisting of" and "consisting essentially of".
[0036] As used herein, the terms "blend", and "mixture" are all intended to be used interchangeably.
[0037] The term "Alzheimer's disease" is used synonymously and interchangeably throughout the specification. Alzheimer's disease is a neurodegenerative disorder that encompasses a progressive brain disorder that causes memory and thinking skills to decline over time. Alzheimer's disease is characterized by dementia - a gradual decline in memory, thinking, behavior, and social skills.
[0038] The terms "weight percent", "vol-%", "percent by weight", "% by weight", and variations thereof, as used herein, refer to the concentration of a substance as the weight of that substance divided by the total weight of the composition and multiplied by 100. It is understood that, as used here, "percent", "%", and the like are intended to be synonymous with "weight percent", "vol-%", etc.
[0039] In some embodiments, the numbers expressing quantities of ingredients, properties such as concentration, reaction conditions, and so forth, used to describe and claim certain embodiments of the invention are to be understood as being modified in some instances by the term "about". Accordingly, in some embodiments, the numerical parameters outlined in the written description are approximations that can vary depending on the desired properties sought to be obtained by a particular embodiment. In some embodiments, the numerical parameters should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the invention are approximations, the numerical values outlined in the specific examples are reported as precisely as practicable.
[0040] The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each value is incorporated into the specification as if it were individually recited herein.
[0041] The headings and abstract of the invention provided herein are for convenience only and do not interpret the scope or meaning of the embodiments.
[0042] The following discussion provides many embodiments of the inventive subject matter. Although each embodiment represents a single combination of inventive elements, the inventive subject matter is considered to include all possible combinations of the disclosed elements. Thus, if one embodiment comprises elements A, B, and C, and a second embodiment comprises elements B and D, then the inventive subject matter is also considered to include other remaining combinations of A, B, C, or D, even if not explicitly disclosed.
[00043] With the aid of the nanotechnology approach, the present disclosure develops a nanostructured lipid carrier (NLC) that has emerged as a promising delivery system for targeting drugs to the brain via the intranasal route; thus, creating a direct connection between the nasal cavity and the brain, bypassing the blood-brain barrier.
[0044] To increase the bioavailability of cholinesterase inhibitors and kinase inhibitors, in particular, RIV and NIL, nanostructured lipid carriers were prepared wherein RIV and NIL were entrapped in the lipid matrix. Till now, hardly any studies have been carried out on the intranasal delivery of RIV and NIL. The present invention is the first study on the development of nanostructured lipid carriers for intranasal delivery of RIV and NIL. Thus, this work has an appreciable novelty concerning the increase in bioavailability of RIV and NIL after its intranasal administration of nanostructured lipid carriers.
[0045] In this embodiment, the present disclosure generally relates to the field of novel drug delivery systems. Particularly, the present disclosure provides a nanostructured lipid carrier formulation. The present disclosure also provides a method of preparation of a nanostructured lipid carrier formulation.
[0046] In another embodiment, the present disclosure provides a nanostructured lipid carriers formulation comprising a) a matrix of solid lipid and oil-based with an average size ranging from 100 to 200 nm; b) a non-ionic surfactant covering NLCs; and c) therapeutic agents encapsulated in the lipidic matrix.
[0047] In yet another embodiment, the solid lipid is solid at both room temperature and body temperature having a carbon chain length of 14 to 20 carbons. In this embodiment, the solid lipid is selected from a group consisting of Gelot® 64, Gelucire® 50/13, GeleolTM mono and diglyceride NF, Emulcire TM 61 WL 2659, Gelucire® 43/01, Gelucire® 33/01, Gelucire® 39/01, Tefose® 1500, stearic acid, Glyceryl monostearate, Labrafil® M 2130 CS, Compritol® 888 ATO, and a combination thereof.Preferably, the solid lipid is Gelot® 64, Glyceryl monostearate, and macrogol-75 stearate or combination thereof.
[0048] The oil is vegetable or synthetic oil. In yet another embodiment, the oil is selected from a group consisting of Caproyl® PGMC, Capmul® MCM, Capryol® 90, Labrafac® WL 1349, Capmul® PG8 NF, Lauroglycol® 90, Labrasol, Captex® 355, Neobee® M20, Captex® 300, Hemp oil, Sesame oil, Labrafil® M1944, wheat germ oil, olive oil, Jojoa oil, castor oil, grape seed oil, Plurol® oleique CC 497, Rice bran oil, Canola oil, Captex® 8000 and a combination thereof. Preferably, the oil is Caproyl® PGMC, medium-chain triglycerides, or a combination thereof.
[0049] In yet another embodiment, the non-ionic surfactant is selected from a group consisting of Sween® 20, Tween® 80, Cremophor® EL, Cremophor® RH 40, Solutol, Poloxamer® 188, Transcutol®, Labrasol®, Span® 80, Span® 20 and a combination thereof. Preferably, the non-ionic surfactant is Sween® 20, a polysorbate or a combination thereof.
[0050] In yet another embodiment, the therapeutic agent is selected from the cholinesterase inhibitor, kinase inhibitor, and combination thereof. Preferably, the therapeutic molecule is RIV, NIL, and a combination thereof.
[0051] In yet another embodiment, the nanostructured lipid carriers are prepared by ultrasound dispersion, hot and cold high-pressure homogenization, film hydration, solvent emulsification-evaporation/diffusion method, modified emulsiosonication, solvent injection, microemulsification or double emulsification or any other suitable method (s) involving centrifugation, dialysis, column separation or any other suitable method for unentrapped drug separation and also involving lyophilization, drying or any other method for obtaining powder form. Preferably, the modified emulsiosonication method is used for the preparation of nanostructured lipid carriers.
[0052] In yet another embodiment, the present disclosure is to provide a nanostructured lipid carriers formulation comprising: 2 to 4% w/w of a binary mixture of the total weight of the formulation; 3 to 5 % w/w of a non-ionic surfactant of the total weight of the formulation; and a therapeutic agent, wherein the ratio of solid lipid to liquid lipid in the binary mixture is in the range of 4:6 to 7:3.
[0053] In still another embodiment of the present disclosure is to provide a method of preparation of a nanostructured lipid carriers formulation comprising following steps: a) mixing of a solid lipid and an oil to obtain a lipid oil mixture; b) heating the binary mixture under condition to obtain a molten binary mixture; c) adding a therapeutic agents to the molten binary mixture to obtain a dispersion; d) adding a non-ionic surfactant to the dispersion under condition to obtain a coarse emulsion; and e) sonicating the coarse emulsion followed by cooling to obtain a nanostructured lipid carriers.
[0054] In an embodiment, the method includes: i) in step b), the heating is carried out at a temperature in the range of 40 to 90 °C, preferably at a temperature in the range of 60 to 80 °C, and more preferably at a temperature in the range of 70 °C; ii) in step d), the condition includes temperature in the range of 50 to 100 °C, preferably at a temperature in the range of 60 to 80 °C, and more preferably at a temperature in the range of 70 °C; and iii) in step e) cooling is carried out at a temperature in the range of 5 to 20 °C for a period in the range of 10 min to 30 min, preferably at a temperature in the range of 6 to 18 °C for a period in the range of 10 min to 20 min. More preferably, at a temperature in the range of 8 to 15 °C for a period in the range of 15 min.
[0055] In another embodiment, the nanostructured lipid carrier is administered by the intranasal route. In some embodiment, the suitable dosage form of nanostructured lipid carrier dosage in dispersion or solution or powder (or any other solid) or semisolid and the like. The delivery system is to be intranasally administered and will be taken up by getting absorbed via olfactory epithelium and trigeminal nerve pathways, which would provide direct access to the brain. This pathway allows for the delivery of the drug combination to the central nervous system (CNS) without needing to cross the BBB. In addition, dual drug-loaded NLC meaningfully augmented the solubility and bioavailability of poorly soluble drugs. The lipid-based composition allows to encapsulate hydrophobic as well as hydrophilic drugs, ensuring efficient transport to the brain.
[0056] In another embodiment, drug-loaded NLC is optimized using response surface methodology (RSM), which quantifies the relationship amidst controllable independent variables and the responses obtained. A three-factor three-level Central Composite Rotatable Design (CCRD) utilizing Design-Expert 13 software (Stat-Ease Inc., Minneapolis, MN, USA) was used. Various independent variables were involved in the development of the drug-loaded NLC by using CCRD which resulted in producing 20 experimental runs and a quadratic polynomial equation (equation 1) describing the relationships amongst the evaluated response (R) and the number of experimental independent variables A, B and C, where b0 was the constant and b were the regression coefficients. These regression coefficients described the quantitative measures of effects of the factors that were either linear or curvilinear and their interactions that were denoted by b12AB, b13AC, and b23BC. This equation was considered as a statistical model rather than a physical model.
R = b0 + b1A + b2B + b3C + b12AB + b13AC + b23BC + b11A2 + b22B2 + b33C2-----(Equation 1)
The relationships among the evaluated response (R) and three experimental independent variables A, B, and C are described by a statistical model.
[0057] The foregoing description of the specific embodiments will so fully reveal the general nature of the embodiments herein that others can, by applying current knowledge, readily modify and/or adapt for various applications such specific embodiments without departing from the generic concept, and, therefore, such adaptations and modifications should and are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments. It is to be understood that the phraseology or terminology employed herein is for description and not for limitation. Therefore, while the embodiments herein have been described in terms of preferred embodiments, those skilled in the art will recognize that the embodiments herein can be practiced with modification within the spirit and scope of the embodiments as described herein.
EXAMPLES
[0058] The present invention is further explained in the form of the following examples. However, it is to be understood that the following examples are merely illustrative and are not to be taken as limitations upon the scope of the invention.
Example 1
[0059] The glide dock method from the Schrodinger suite was used to execute docking with test molecules of NIL and RIV and compare the outcomes with the tacrine co-crystal ligand inside the active pocket site of the Acetylcholine Esterase Enzyme Protein 1ACJ.
The testing molecule NIL is inhibited by the target of the acetylcholine esterase enzyme while maintaining residues like PHE 330, TYR 334, TRP 84, and TRP 279 with a representation of the pi-pi interaction as a turquoise dotted line. Additionally, the testing molecule (NIL) has a similar association to that of the co-crystal ligand tacrine, which mimics the inhibitory activity of the acetylcholine esterase enzyme by conserving residues of PHE 330 and TRP 84. Additionally, the docking scores demonstrate the closeness of the binding energy between the tacrine co-crystal and NIL, which was found to be -8.890 Kcal/mol. Therefore, these interpretations may point toward the tacrine co-crystal ligand where NIL has the most chance of working. To simulate the same residue interaction and structural similarities for the final ligand structure alignment, tacrine and NIL was superimposed. Furthermore, RIV exhibits the same docking interaction as the tacrine co-crystal ligand after NIL, with a similar docking score. Examples include interactions with TYR 130 and ASP 72, as well as pi-pi interactions with TRP 84 and PHE 330. The docking score for these interactions was -8.781 Kcal/mol. Where the contact was discovered, residues TRP 84 and PHE 330 were involved with a pi-pi interaction that exhibits properties comparable to those of the co-crystal ligand tacrine, with the same pocket site suggesting potential for an analogous manner of inhibition of the acetylcholine esterase enzyme. To simulate the same residue interaction and structural similarities for the final ligand structure alignment, RIV and tacrine were superimposed.
[0060] N2a (neuroblastoma) cells (N2a) lines were used to evaluate the % cell viability of the RIV and NIL, which were harvested from T-25 cm2 tissue culture flasks, and a stock suspension was prepared. The cells were seeded into a 96-well plate with 0.1 mL of DMEM in each well. The plate was incubated at 37 °C, 5% CO2 for 24 h. Before the experiment, the stock solution (2mg/mL) of each sample was prepared in DMEM. After 24 h, the cells were treated with 100 µL of different concentrations ranging from 1-1000 µg/mL of each sample, and the plate was further incubated for 24 h. Thereafter, the old media was replaced with 100 µL of without phenol red DMEM followed by the addition of 10 µL of the MTT reagent (5 mg/mL) and the plate was incubated further for 3 h. Afterward, the supernatant was removed, and the formed formazan crystals were dissolved in 100 µL of solubilizing agent and the absorbance was measured at 570 nm using a microplate ELISA reader. After this, the IC50 was determined for each drug which was observed to be at a concentration of 125 µg/mL and 62.5µg/mL of RIV and NIL respectively. Feasibly, on increasing the concentration of RIV and NIL individually from 0.97 to 2000 µg/mL, the mean % cell viability was observed to be 68.42 ± 0.41 to 36.63 ± 0.34 whereas, in NIL it was observed to be 70.85 ± 0.44 to 36.51 ± 0.37 as shown in Figure 2. All the measurements were taken in triplicate and the percentage cell viability was determined using the equation:
% Cell viability=(Absorbance of the treated cells)/(Absorbance of the untreated cells) x 100---- Equation 2
[0061] Later, the combination index (CI) of RIV and NIL was calculated, where the comparison between different ratios 1:1, 1:2, 1:3, 1:4, 1:5, 1:6 & and 1:7 was done based on the synergistic action of this combination i.e., RIV and NIL. So, it was discovered that a ratio of 1:1 produced the highest N2a cell viability in 24 hours as shown in Figure 2 suggesting that RIV and NIL in a 1:1 ratio of drug combination had the greatest possible synergistic effect where CI was found to be 0.106.
Example 2
[0062] In this example, the method of preparation of NLC containing cholinesterase inhibitor and kinase inhibitor, or combination therefore is explained.
[0063] The process for the production of a combination of RIV and NIL loaded NLC was prepared by using a modified emulsiosonication method followed by ultra probe sonication, in which the optimized binary lipid mixture (3% w/v) was melted and dissolved in half of the surfactant at 75±5°C. The lipid phase was then prepared by constant stirring on a magnetic stirrer at 500 rpm. The remaining half of the chosen surfactant was added to the necessary amount of water, mixed, and kept under the same conditions as the lipidic phase to create the aqueous phase concurrently. While maintaining a temperature of 75±5°C for 45 minutes, thoroughly agitate the lipid phase and aqueous phase at 800 rpm. The aqueous phase was mixed into the lipid phase to form a emulsion system and the resulting mixture was sonicated using an Ultra-Probe sonicator (Hielscher, Germany).
[0064] Design-Expert® software statistically was used to optimize several independent factors involved in NLC preparation. The selected independent variables were the concentration of binary mixture (A), concentration of surfactant (B), and sonication time (C). The selected dependent variables were globule size (R1), polydispersity index (PDI) (R2), and entrapment efficiency (EE) of RIV (R3) and NIL (R4). The desirable range of independent variables chosen for the study is shown in Table 1.
[0065] The responses obtained after putting the values of independent variables in the CCRD, 20 runs were suggested as shown in Table 2. The details of experimental batches of NLC formulation in CCRD are given in Table 2 along with the summary of results of regression analysis for responses R1, R2, R3, and R4 for fitting to the quadratic model in Table 3.

[0066] Contour plots in Figure 4 show the effect of total lipid, surfactant concentration, and sonication time on (a) globule size (nm), (b) polydispersity index, and (c) entrapment efficiency (%). As the total lipid amount was increased, globule size, PDI, and entrapment efficiency were significantly (p<0.05) increased. As the concentration of surfactant increased, the globule size and PDI decreased however, the EE of both the drugs was observed to increase. In addition, with an increase in the sonication time, the globule size and EE of both drugs decreased, but PDI increased.
Table 1: CCRD variables for the preparation of RIV-NIL-NLC.
FACTORS LEVELS
Independent Variables Axial
(-) Low
(-1) Medium
(0) High
(+1) Axial
(+)
A - Binary lipid concentration (% w/v) 1.31 2 3 4 4.68
B - Surfactant concentration (% v/v) 2.31 3 4 5 5.68
C - Sonication time (seconds) 59.54 80 110 140 160.45
Dependent Variable Constraints Used
R1 - Globule size (nm) Minimum
R2 - PDI Minimum
R3 - % EE of NIL Maximum
R4 - % EE of RIV Maximum

Table 2: Responses of formulation runs of RIV-NIL-NLCs.

Runs Factor 1: Binary lipid concentration (% w/v) Factor 2: Surfactant concentration (% v/v) Factor 3: Sonication time (seconds) R1: Globule size
(nm) R2:
PDI R3:
%EE of RIV R4:
%EE of NIL
1 3 4 59.5 134.1 0.232 84.2 89.6
2 3 5.6 110 44.9 0.227 89.2 88.8
3 3 4 110 126.1 0.197 85.2 89.2
4 3 4 110 128.3 0.188 86.4 90.3
5 3 2.3 110 159.9 0.257 83.3 85.6
6 3 4 110 124.5 0.192 83.1 87.6
7 4 5 80 136.5 0.256 88.8 93.2
8 3 4 110 148.9 0.187 83.4 87.6
9 2 3 80 139.1 0.175 78.2 82.5
10 3 4 160.4 92.3 0.265 81.3 88.1
11 3 4 110 130.6 0.214 83.7 87.9
12 2 5 80 34.1 0.261 77.7 81.5
13 4.6 4 110 180.1 0.350 91.3 95.2
14 4 5 140 97.6 0.263 89.5 93.2
15 4 3 140 174.2 0.374 89.1 93.3
16 3 4 110 122.1 0.191 84.8 89.1
17 2 5 140 30.2 0.253 76.1 80.2
18 2 3 140 122.2 0.202 79.2 83.7
19 4 3 80 159.9 0.334 88.3 92.4
20 1.3 4 110 41.9 0.184 69.8 73.9
Volume of dispersion = 10 mL
Table 3: Summary of regression analysis for responses, R1, R2 & R3, for fitting to the quadratic model.

Formulation Response R2 Adjusted R2 Predicted R2 S.D. % C.V. Adequate precision
RIV-NIL-NLC R1 = Globule Size 0.964 0.932 0.745 11.87 10.23 18.85
R2 = PDI 0.984 0.971 0.934 0.009 4.07 29.87
R3 = %EE of RIV 0.965 0.934 0.807 1.34 1.53 20.80
R4 = %EE of NIL 0.947 0.900 0.689 1.70 2.03 16.52
Equation
RIV-NIL-NLC R1 = +128.79 +34.78 A -35.91 B -8.47 C +12.13 AB -0.4750 AC -5.03 BC -5.47 A² -8.52 B² -4.70 C²
R2 = +0.1946 +0.0450 A -0.0075 B +0.0089 C -0.0407 AB +0.0035 AC -0.0085 BC +0.0274 A² +0.0185 B² +0.0208 C²
R3 = +84.43 +5.91 A +0.5289 B -0.2912 C +0.5625 AB +0.2625 AC -0.3375 BC -1.32 A² +0.6961 B² -0.5413 C²
R4= +88.59 +5.86 A +0.1158 B -0.1261 C + 0.6500 AB +0.1250 AC -0.4250 BC -1.28 A² -0.3448 B² +0.2385 C²
C.V. = Coefficient of deviation, S.D = Standard Deviation, R2 = Coefficient of correlation

[0067] The optimized drug-loaded NLC was evaluated and characterized by using Zetasizer Nano ZS, (Malvern Instrument Ltd, Worcestershire, UK) was used to measure the globule size and PDI of RIV-NIL-NLC at 25°C. The volume of the sample to be determined was kept constant i.e. 1mL. As it is well known that the globules exhibit brownian motion, this causes the intensity of light to scatter from the particles, which is then detected as intensity with time with suitable optics and photomultiplier. The instrument is equipped with apt software for analysis of globule size and PDI.
[0068] Zeta potential is the electrostatic potential that exists as the shear plane of a globule, which is related to both surface charge and the local environment of the globule. The zeta potential of a sample determines whether the globules within a liquid will tend to flocculate or not. Particles with zeta potential in the range of +30mV to -30mV are normally considered stable. Surface charge (zeta potential) was measured using zetasizer (Nano ZS, Malvern Instruments, Worcestershire, UK).
[0069] The prepared RIV-NIL-NLC were centrifuged for 30 minutes at 10,000 rpm at Centrifugation apparatus, Sigma, Germany. After separating the supernatant, the supernatant was dissolved into the solvent with methanol. Ten milliliters of methanol were used to further dilute after dilution, thoroughly mix the mixture before using UV spectroscopy to examine it. As a result, the amount of drug was calculated to determine the free drugs. Each of the parameters was determined using the following formulas:
EE%=((Total drug - Free drug))/((Total drug)) x 100-----Equation 3
[0070] The morphology of RIV-NIL-NLC was determined by Transmission electron microscopy (TEM) of Morgani 268D, Eindhoven, Netherlands. The sample was negatively stained by placing a drop of 1 mL of the vesicular suspension on a carbon-coated grid. The suspension was left for 2 min, to allow its absorption in the carbon film, and the excess liquid was drawn off with filter paper. Subsequently, a drop of 1% phosphotungstic acid was placed on the grid. The excess was removed with distilled water and the samples were visualized using a soft imaging viewer software.
[0071] Globule size, PDI, Zeta Potential, and % EE studies: On performing the characterization parameters of the RIV-NIL-NLC formulation, it was perceived that globule size and PDI were observed to be 148.66 ± 3.45 nm and 0.184 ± 0.007 as shown in Figure 5, demonstrating that the developed formulation was within the desired range, i.e., >200 nm, for the targeted delivery of the drug combination to the brain via intranasal route and homogenous and stable. However, the Zeta Potential was observed to be 0.819 ± 0.021 mV as shown in Figure 5. In addition, the lipids used for the development of the NLC formulations have no charge over them i.e., they are neutral. The EE% of RIV and NIL in the RIV-NIL-NLC was found to be 84.73 ± 4.68 % and 86.31± 3.85% respectively.
Example 3
[0072] Wistar albino rats weighing 100-150 gm were selected for pharmacokinetic studies. Studies were performed after getting approval from the Institutional Animal Ethics Committee of Jamia Hamdard, New Delhi (Protocol no: 2086), and committee guidelines for animal handling were followed. The animals were housed six per cage at 20-24°C with free access to food and water with a 12-h light-dark cycle. Rats were divided into different groups (Table 5) used in the pharmacokinetic and biodistribution study. The formulation was given intranasally. The rats were anesthetized with carbon dioxide, and blood samples (1 mL) along with various organs were collected at 0.5, 2, 4, 8, 16, and 24 hours, wherein the blood samples collected were then centrifuged at 10,000 rpm for 10 minutes. Additionally, the collected organs were stored at -80oC for further study.
[0073] In this example, the pharmacokinetic study was performed along with the biodistribution study after giving the RIV-NIL-SUS and RIV-NIL-NLC to the different groups of the rats as shown in Table 5 via intranasal route and different pharmacokinetic parameters were calculated by using PK solver software as shown in Table 6 showing that the Cmax(ng/ml) of RIV and NIL from NLC was 2.2 to 2.6-fold higher than that from SUS in the plasma signifying the significant (p<0.001) increase in the concentration of RIV and NIL in the plasma.
[0074] The drug concentration profile of RIV and NIL in the brain following IN administration of RIV-NIL-NLC and RIV-NIL-SUS is shown in Figure 14. The maximum amount of RIV and NIL released from the NLC occurred at 4 and 8 hours, while the release of both drugs was observed at 2 and 4 hours in the SUS, and Cmax (ng/mL) of RIV and NIL in the brain from NLC was 3.6 to 5.5-fold higher than from SUS signifying the significant (p<0.001) increase in the concentration of RIV and NIL in the brain.
Table 5: Animal groups used in pharmacokinetic & biodistribution study.

Group No. of rats Period (hours) Treatment Route
1 06 - For bioanalytical method development and Validation Not Applicable
2 18 0.5, 2, 4, 8, 16, 24 RIV-NIL-SUS Intranasal
3 18 0.5, 2, 4, 8, 16, 24
RIV-NIL-NLC Intranasal

Table 6: Pharmacokinetic Parameters of RIV and NIL in Wistar Rats in Plasma after intranasal administration of the RIV-NIL-SUS and RIV-NIL-NLC.
Parameters RIV-NIL SUS RIV-NIL NLC
RIV NIL RIV NIL
Tmax (h) 2 4 4 8
Cmax(ng/ml) 196.22 ± 23.74 231.16 ± 61.62 596.56 ± 85.23† 782.75 ± 53.12†
AUC0?t (ng.hr/ml) 2511.39 ± 214.82 3678.46 ± 888.12 7486.89 ±310.82† 13195.18 ± 451.55†
AUC0?8 (ng.hr/ml) 3022.24 ± 362.13 4953.79 ± 747.01 12970.21 ± 901.12† 26506.48 ± 357.77†
T1/2 11.09 ± 3.64 12.37 ± 8.32 19.57 ± 9.75 20.62 ± 7.28
† sign represents a significant difference at p < 0.05.
Table 7: Pharmacokinetic Parameters of RIV and NIL in Wistar Rats in Brain after intranasal administration of the RIV-NIL-SUS and RIV-NIL-NLC.
Parameters RIV-NIL SUS RIV-NIL NLC
RIV NIL RIV NIL
Tmax (h) 2 4 4 8
Cmax(ng/ml) 256.88 ± 46.72 394.42 ± 87.01 1292.94 ± 293.37† 1414.87 ± 512.81†
AUC0?t (ng.hr/ml) 3054.99 ± 213.93 5521.85 ± 357.63 19452.17 ± 618.13† 24153.02 ± 541.83†
AUC0?8 (ng.hr/ml) 4620.93 ± 246.84 7176.78 ± 801.12 47906.35 ± 996.33† 58835.58 ± 863.99†
T1/2 15.54 ± 3.94 11.04 ± 5.01 31.04 ± 11.12 26.02 ± 12.91
† sign represents a significant difference at p < 0.05.
The foregoing examples are merely illustrative and are not to be taken as limitations upon the scope of the invention. Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art. Such changes and modifications may be made without departing from the scope of the invention.
, Claims:We Claim:

1. A combination of cholinesterase inhibitor and kinase inhibitor encapsulated nanostructured lipid carrier (NLC) formulation comprising:
a) a solid and liquid lipids-based binary mixture;
b) a non-ionic surfactant covering said binary mixture; and
c) a therapeutic agent is a combination of said cholinesterase inhibitor and said kinase inhibitor

2. The formulation as claimed in claim 1, wherein said solid lipid is selected from a group of GelotTM 64, Gelucire® 50/13, GeleolTM mono and diglyceride NF, Emulcire TM 61 WL 2659, Gelucire® 43/01, Gelucire® 33/01, Gelucire® 39/01, Tefose® 1500, stearic acid, Glyceryl monostearate, Labrafil® M 2130 CS, Compritol® 888 ATO, and a combination thereof.

3. The formulation as claimed in claim 1, wherein said liquid lipid is selected from a group of Capryol® PGMC, Capmul® MCM, Capryol® 90, Labrafac® WL 1349, Capmul® PG8 NF, Lauroglycol® 90, Labrasol, Captex® 355, Neobee® M20, Captex® 300, Hemp oil, Sesame oil, Labrafil® M1944, wheat germ oil, olive oil, Jojoa oil, castor oil, grape seed oil, Plurol® oleique CC 497, Rice bran oil, Canola oil, Captex® 8000 and a combination thereof.


4. The formulation as claimed in claim 1, wherein said non-ionic surfactant is selected from a group of Sween® 20, Tween® 80, Cremophor® EL, Cremophor® RH 40, Solutol, Poloxamer® 188, Transcutol®, Labrasol®, Span® 80, Span® 20 and a combination thereof.

5. The formulation as claimed in claim 1, wherein said kinase inhibitor is Nilotinib hydrochloride monohydrate (NIL).


6. The formulation as claimed in claim 1, wherein said cholinesterase inhibitor is Rivastigmine Tartrate (RIV).
7. A nanostructured lipid carrier formulation comprising:
a) 2 to 4% w/w of a binary mixture of the total weight of the formulation;
b) 3 to 5% w/w of non-ionic surfactant of the total weight of the formulation; and
c) a therapeutic agent,
wherein the binary mixture comprises solid and liquid lipids in a 7:3 ratio.

8. A method of preparation of a nanostructured lipid carrier formulation comprising:
a) mixing of GelotTM 64 as a solid lipid and Caproyl® PGMC as a liquid lipid to obtain a lipid matrix;
b) heating the lipid matrix under the condition to obtain a molten lipid matrix;
c) adding a therapeutic agent to the molten lipid matrix;
d) the molten lipid matrix dissolved in half of the non-ionic surfactant acts as a lipid phase, the remaining half of the non-ionic surfactant added to the necessary amount of water as an aqueous phase;
e) adding said aqueous phase to the lipid phase to obtain a coarse emulsion; and
f) sonication of said coarse emulsion in the ice bath followed by cooling to obtain a nanostructured lipid carrier.

9. The method as claimed in claim 8, wherein the heating is carried out at a temperature in the range of 40 to 90 °C.

10. The formulation as claimed in claims 1 to 9, wherein said nanostructured lipid carrier is administered by an intranasal route.

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