[0001] The present invention relates to a novel formulation for treating neurodegenerative disorders, more particularly the present invention relates to nanoformulation of Saxagliptin (DPP-4 inhibitor) loaded polymeric nanomicelles, optionally in combination with one or more other inactive agents, for treatment of neurodegenerative disorders, by ocular to brain route of drug delivery.
BACKGROUND OF THE INVENTION

[0001] Neurodegenerative disorders (progressive loss of structure and/or function of neurons) are usually characterized by accumulation of abnormal protein aggregation that leads to inflammation as well as oxidative stress in the central nervous system (CNS). Nearly, 1.5 billion of population suffer from brain disorders. Alzheimer’s disease (AD) is the commonest cause of dementia in ageing adults. Alzheimer's, Parkinson's, Huntington's, and other neurodegenerative disorders share many common features at both cellular and subcellular levels. Intracellular and extracellular changes could be observed in neurodegenerative diseases. Alzheimer’s disease (AD), the commonest cause of dementia in ageing adults, is characterized by gradual cognitive impairment and severe functional disability.

[0002] Alzheimer's disease (AD) should be regarded as a degenerative metabolic disease caused by brain insulin resistance and deficiency, and overlapping with the molecular, biochemical, pathophysiological, and metabolic dysfunctions in diabetes mellitus, non-alcoholic fatty liver disease, and metabolic syndrome.

[0003] Insulin has functions in the brain and dysregulation of these functions may contribute to the expression of late-life neurodegenerative disease. Insulin dysregulation contributes to neurodegenerative disorders through disease-specific or general mechanisms.

[0004] Current pharmacotherapies of Alzheimer’s disease (AD) including achetylocholinesterase inhibitors and memantine fail to halt disease progression. Interestingly, type 2 diabetes mellitus (T2DM) and AD share several common characteristics, including Aß deposition, insulin resistance, degeneration, mitochondrial dysfunction, oxidative stress and excessive inflammation.

[0005] Gliptins have been demonstrated recently to display important neuroprotective effects, reverse pathophysiologic processes observed in AD, and improve cognitive performance of AD animal models and patients. Gliptins are a new class of blood glucose-lowering drug and used for treatment of type 2 diabetes.

[0006] Dipeptidyl peptidase-4 (DPP-4) is a serine exopeptidase widely expressed throughout the body. Inactivation of GLP-1 by DPP-IV is rapid and extensive, for this reason, DPP-IV inhibition has been estimated to significantly increase GLP-1 bioavailability and concentration. Studies in rodents provide evidence of both neuroprotective and neurotrophic effects of GLP-1, including improvement of learning and memory.

[0007] DDP-4 is present in human retina eye and highly expressed in retina, namely in retinal pigment epithelium (RPE) of patients suffering from diabetes. Retina is a thin, fragile layer of tissue lining at the back of the eye is an extension of the brain. This fact is highly significant because it means that a drug or biological therapy that helps will save retinal cells in cases of retinal degenerative diseases like retinitis pigmentosa (RP) or macular degeneration and preserve cells in the brain with a neurodegenerative condition.

[0008] Inhibitors of DPP-4 enzyme (acting through competitive enzymatic inhibition, such as sitagliptin, or being substrate-enzyme blockers, such as saxagliptin) have been tested and surprisingly they reached the retina when applied topically in the eye (i.e. in the cornea or conjunctival fornix or sclera, that is ophthalmic application to surface of the eye), despite their molecular weights and complexity of structures.

[0009] Saxagliptin is a second oral DPP-4 inhibitor developed for use in the treatment of type 2 diabetes (non-insulin dependent diabetes). Saxagliptin is a substrate-enzyme blocker that inhibits degradation of dipeptidyl dipeptidase-4 enzyme, thereby inhibiting the effects of incretin hormones, glucagon-like peptide-1 (GLP-1), and of glucose-dependent insulinotropic peptide (GIP).

[0010] The chemical designation of Saxagliptin is 3S, 5S)-2-[(2S)-2-amino-2-(3-hydroxy-1-adamantyl)acetyl]-2 azabicyclo [3.1.0]hexane-3-carbonitrile.

[0011] Drug targeting especially targeting of drugs by nanoformulation have been getting much attention by the researchers for treating various neurodegenerative disorders including Alzheimer’s disease. Drug delivery into brain using nanoformulation has several advantages which includes: (1) reducing the dose of a therapeutic drug which, when given peripherally, maintains the biological potency in the CNS, (2) allowing drugs that normally do not cross the blood-brain barrier to penetrate into the nervous system, (3) reducing the peripheral side effects by increasing the relative amount of the drug reaching the brain, and (4) the use of nanoformulations able to encapsulate molecules with therapeutic?value, while targeting specific transport processes in the brain vasculature, may enhance drug transport through the BBB in neurodegenerative disorders and target relevant regions in the brain for regenerative processes. ?
[0012] New delivery platforms based on polymer drug carriers along with a variety of methods to improve drug absorption through the nasal and ocular routes. Different types of nanoformulations such as polymeric nanomicelles, nanosuspension, nanoparticles, liposomes, emulsions, solid-lipid NPs, and dendrimers have been administered into living organisms by exploiting various routes for improved targeted therapies. Various conventional approaches such as chronic use of oral administration of DPP-4 inhibitors will result serious side effects as urinary tract infection, upper respiratory tract infection, nasopharyngitis, and abdominal pain and therefore to bypass such serious side effects there is a need to deliver the drug through a non-invasive route. The use of nanocarriers encapsulating molecules such as peptides and genomics may enhance drug transport through BBB in neurodegenerative diseases and target relevant brain regions for regenerative processes. Because of their mucoadhesive properties, polymeric nanosystems are able to adhere to the mucosa of the eye, leading to increased residence time at the cornea. This, along with the small nanosystem particle size, allows for increased drug permeation across the cornea.

[0013] Ocular route is preferred as an alternative route to deliver the drug to its target sites in the brain that is both efficient and effective. Ocular drug delivery to brain is non-invasive novel approach with increased patient compliance. Brain targeting through the ocular delivery route, as an non- invasive approach is a formidable task because eye is a highly sensitive and protected organ. The eye offers a natural window to the brain as the retina, the light-sensitive layer lining the interior of the eye is considered part of the CNS and the only optically accessible nervous tissue. Ocular drug delivery to brain is non-invasive novel approach to treat several neurodegenerative conditions such as Alzheimer's disease, Parkinson's disease and AD associated with diabetes. Topical drug application is useful in the treatment of many anterior segment disorders, but it is considered inefficient in delivering therapeutic concentrations of the drug to the posterior segment of the eye, owing to the unique anatomical, physiological and biochemical barriers of the eye. Ocular delivery plays a promising approach towards brain targeting. This route limits systemic exposure and distribution to peripheral sites of action, thus lessening unwanted side effects and the potential for toxicity. Thus toxicity and adverse effects can be minimized via the ocular to brain-targeted delivery system.

[0014] Most common adverse events (incidence =5%) with oral dose of saxagliptin 5 mg (monotherapy or combination therapy) are upper respiratory tract infection, urinary tract infection, headache, nasopharyngitis, pancreatitis and heart failure.

[0015] Saxagliptin due to its poor permeability, less protein binding (<10%) and its therapeutic use in chronic conditions necessitates its nanoformulations thus to limit its dose related side effects. There is urgent need to formulate better drug delivery systems with aim to improve bioavailability, show controlled & sustain effect in chronic diseases treatment, and reduce side effects of drugs.

SUMMARY OF THE INVENTION

[0016] In an aspect, the present invention relates to a nanoformulation of drug loaded polymeric nanomicelles for ocular to brain delivery for targeting and treating neurodegenerative disorders like dementia. The nanoformulation of drug loaded polymeric nanomicelles of the present invention has enhanced permeability and alleviates the side effect of free-form drug by increasing the encapsulation and loading efficiency. Further, it offers benefits of reduced dosing frequency, drug related side effects, improved patient compliance and brain targeted delivery systems.

[0017] In another aspect, the present invention provides a nanoformulation of drug loaded polymeric nanomicelles as a rapid, direct, efficient and non-invasive drug delivery approach to brain for treatment of neurodegenerative disorders.

[0018] In yet another aspect, the present invention provides nanoformulation of drug loaded polymeric nanomicelles for ocular to brain delivery/ocular administration comprising, a polymeric drug carrier nanomicelle, a drug encapsulated within the polymer core of said polymeric carrier nanomicelles and a surfactant.

[0019] In a further aspect, the drug is a gliptin, selected from linagliptin, sitagliptin, saxagliptin, vildagliptin, alogliptin.

[0020] In an alternate aspect, the polymer is a mucoadhesive polymer selected from chitosan, pullulan, hydroxyl propyl methyl cellulose, sodium alginate and starch.

[0021] In another aspect, the polymer is pullulan and surfactant is tween 80.

[0022] In a further aspect, the nanoformulation comprises of drug loaded nanomicelles of mean particle size of 152.8 nm.

[0023] In yet another aspect, the present invention provides a drug loaded polymeric nanomicelle wherein the drug is selected from saxagliptin or sitagliptin and polymer is pullulan.

[0024] In a further aspect, the % entrapment efficiency of the drug is 60% to 96% and % drug loading of polymeric nanomicelles is 11 % to 34% in the drug loaded polymeric nanomicelle.

[0025] In another aspect, the present invention also refers to a method of providing the nanoformulation of drug loaded polymeric nanomicelle for ocular to brain delivery/ocular administration for treatment of neurodegenerative diseases like dementia, Alzheimer’s.

[0026] The foregoing and other objects, features, and advantages of the invention will become more apparent from the following detailed description, which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

[0027] The foregoing summary, as well as the following detailed description of the invention, will be better understood when read in conjunction with the appended drawings. To assist in the explanation of the invention, there are shown in the drawings embodiments which are presently preferred and considered illustrative. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities shown therein. In the drawings:

[0028] FIG. 1 illustrates FESEM images for optimized saxagliptin loaded polymeric nanomicelles

[0029] FIG. 2 illustrates TEM images for optimized saxagliptin loaded polymeric nanomicelles

[0030] FIG. 3 (a) illustrates mean particle size of optimized saxagliptin loaded polymeric nanomicelles

[0031] FIG. 3 (b) illustrates zeta potential of optimized saxagliptin loaded polymeric nanomicelles

[0032] FIG. 4 illustrates the critical micelle concentration (CMC) of tween 80

[0033] FIG. 5 illustrates FTIR spectrum of saxagliptin loaded polymeric nanomicelles

[0034] FIG. 6 illustrates DSC results of saxagliptin loaded polymeric nanomicelles

[0035] FIG. 7(a) illustrates In-vitro drug release data of saxagliptin loaded polymeric nanomicelles

[0036] FIG. 7(b) illustrates In-vitro drug release kinetics of saxagliptin loaded polymeric nanomicelles.

[0037] FIG. 8(a) illustrates Ex-vivo drug release data of saxagliptin loaded polymeric nanomicelles

[0038] FIG. 8(b) illustrates Ex-vivo release kinetics of saxagliptin loaded polymeric nanomicelles

[0039] FIG. 9 (a)-(d) illustrates images of brain tissues of histopathological studies.

[0040] FIG. 10 (a)-(b) illustrates Gamma scintigraphy images in brain tissues.

DETAILED DESCRIPTION OF THE INVENTION

[0041] Embodiments of the invention are discussed in detail below. In describing embodiments, specific terminology is employed for the sake of clarity. However, the invention is not intended to be limited to the specific terminology so selected. A person skilled in the relevant art will recognize that other equivalent parts can be employed and other methods developed without parting from the spirit and scope of the invention.

[0042] In describing the invention, the following terminology will be used by the definitions set forth below. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described herein. As used herein, each of the following terms has the meaning associated with it in this section. Specific and preferred values listed below for individual process parameters, substituents, and ranges are for illustration only; they do not exclude other defined values or other values falling within the preferred defined ranges.

[0043] As used herein, the singular forms "a," "an," and "the" include plural reference unless the context dictates otherwise.

[0044] The terms "preferred" and "preferably" refer to embodiments of the invention that may afford certain benefits, under certain circumstances. However, other embodiments may also be preferred, under the same or different circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, and is not intended to exclude other embodiments from the scope of the invention.

[0045] When the term “about” is used in describing a value or an endpoint of a range, the disclosure should be understood to include both the specific value and end-point referred to.

[0046] As used herein, the terms "comprising" "including," "having," "containing," "involving," and the like are to be understood to be open-ended, i.e., to mean including but not limited to.

[0047] As used herein “polymeric nanomicelles” are vesicular nanometer sized, core-shelled carriers formed in water from amphiphilic monomer units with sizes between 5 to 200 nm in water. Surfactant and polymeric micellar nanocarriers provide an appropriate method for improving the solubilization of drugs. Due to its amphiphilic nature, the nanomicelles encapsulate both hydrophobic and hydrophilic drugs and helps in drug delivery. Monomers tend to initiate self-aggregation (i.e., formation of micelles in solvent system at certain concentration). The concentration at which monomer self- assembly is initiated is defined as the critical concentration of a micellar. The structure of polymeric micelles is often composed of an amphiphilic block copolymer and a CMC is directly related to the stability of the self-assembled structures, and a high CMC will imply disassembly in biological fluids upon dilution.

[0048] Polymeric nanomicelles can be either prepared from block copolymers or surfactants (ionic, non-ionic, zwitter-ionic). The ionic surfactants, e.g. sodium dodecyl sulphate and dodecyltrimethylammonium bromide as anionic and cationic surfactants, are referred to as amphiphilic monomers carrying a charge (anion or cation) on their head groups. On the contrary, non- ionic surfactants such as n-dodecyl tetra (ethylene oxide) (C12E4) are monomers which carry no charge on the head group. Amphiphilic monomers are known as zwitter ionic surfactants (e.g., dioctanoyl phosphatidyl choline) which carry both positive and negative charges on the head group.

[0049] Polymeric nanomicelles offers versatility in use due to its simplicity, ease of manufacturing, ease of sterilization, controlled size & shape, offers good encapsulation efficiency and loading capacity, with reproducibility at small and bulk scale. Polymeric nanocarriers are used to address key drug delivery issues: loading sufficient dosage of the active cargo, protecting it from the in-vivo environment surrounding it, and releasing it steadily at the target site without causing systemic toxicity. Monomers allow surface conjugation of moiety targeting or specific ligand allowing a targeted delivery of drugs. Monomers allow surface conjugation of moiety targeting or specific ligand allowing a targeted delivery of drugs. Because of their ability to translocate drugs across ocular tissues and significantly improve bioavailability, nanomicelles as carrier systems have drawn attention from scientists providing eye drugs.

[0050] As used herein “polymer” means high molecular weight substances comprising of a large number of repeating units called monomers. Biodegradable polymers are designed to degrade within the body into non- toxic components. Biodegradation of polymers involves hydrolytically or enzymatically cleavage of sensitive bonds in the polymer leading to polymer erosion. On the basis of occurrence, polymers can be categorized as synthetic and naturally occurring. The ideal polymer should be inert, biocompatible, mechanically strong, mucoadhesive, comfortable for the patient, safe from accidental release, ease with delivery and elimination, and easy to construct and sterilize. Mucoadhesive polymers in the ocular site prolong the time the formulation is in contact with the cornea and conjunctival epithelium, helping to lessen the challenges of fast clearance from the eye that is often experienced by topical ocular formulations. Further, polymer is selected from chitosan, pullulan, hydroxyl propyl methyl cellulose, sodium alginate and starch.

[0051] As used herein “surfactant” means compounds that lower the surface tension (or interfacial tension) between two liquids, between a gas and a liquid, or between a liquid and a solid. Surfactants may act as detergents, wetting agents, emulsifiers, foaming agents, and dispersants.

[0052] As used herein “drug” refers to Gliptins or dipeptidyl-peptidase-IV (DPP-4/ DPP-IV) inhibitors. These are a class of oral diabetes drugs introduced in 2006. Gliptins also known as adenosine deaminase complexing protein 2 or CD26 (cluster of differentiation 26) is a protein that, in humans, is encoded by the DPP4 gene. Examples of currently registered DPP-4 inhibitors are saxagliptin, sitagliptin, linagliptin, vildagliptin and alogliptin. DPP-4 inhibitors are administered orally.

[0053] Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described. All publications and other references mentioned herein are incorporated by reference in their entirety. Numeric ranges are inclusive of the numbers defining the range.

[0054] The use of examples anywhere in this specification including examples of any terms discussed herein is illustrative only, and in no way limits the scope and meaning of the invention or any exemplified term. Likewise, the invention is not limited to various embodiments given in this specification.

[0055] The present invention relates to a nanoformulation of drug loaded polymeric nanomicelles for ocular to brain delivery for targeting and treating neurodegenerative disorders like dementia. The nanoformulation of drug loaded polymeric nanomicelles of the present invention has enhanced permeability and alleviates the side effect of free-form drug by increasing the encapsulation and loading efficiency. Further, it offers benefits of reduced dosing frequency, drug related side effects, improved patient compliance and brain targeted delivery systems.

[0056] The present invention provides a nanoformulation of drug loaded polymeric nanomicelles as a rapid, direct, efficient and non-invasive drug delivery approach to brain for treatment of neurodegenerative disorders.

[0057] In a particular embodiment, the present invention provides nanoformulation of drug loaded polymeric nanomicelles for ocular to brain delivery/ocular administration comprising, a polymeric drug carrier nanomicelle, a drug encapsulated within the polymer core of said polymeric carrier nanomicelles and a surfactant.

[0058] In a further embodiment, the drug is a gliptin, selected from linagliptin, sitagliptin, saxagliptin, vildagliptin, alogliptin.

[0059] In an alternate embodiment, the polymer is a mucoadhesive polymer selected from chitosan, pullulan, hydroxyl propyl methyl cellulose, sodium alginate and starch.

[0060] In a further embodiment, the polymer is pullulan and surfactant is tween 80.

[0061] In yet another embodiment, the nanoformulation comprises of drug loaded nanomicelles of mean particle size of 152.8 nm.

[0062] In another embodiment, the present invention provides a drug loaded polymeric nanomicelle wherein the drug is selected from saxagliptin or sitagliptin and polymer is pullulan.

[0063] In alternate embodiment, the % entrapment efficiency of the drug is 60% to 96% and % drug loading of polymeric nanomicelles is 11 % to 34% in the drug loaded polymeric nanomicelle.

[0064] In yet another embodiment, the present invention also refers to a method of providing the nanoformulation of drug loaded polymeric nanomicelle for ocular to brain delivery/ocular administration for treatment of neurodegenerative diseases like dementia, Alzheimer’s.
[0065] The following examples are provided to illustrate the invention better and are not to be interpreted in any way as limiting the scope of the invention. All specific materials and methods described below, in whole or in part, fall within the scope of the invention. These specific compositions, materials, and methods are not intended to limit the invention, but merely to illustrate specific embodiments falling within the scope of the invention. One skilled in the art may develop equivalent materials and methods without the exercise of inventive capacity and without departing from the scope of the invention. It will be understood that many variations can be made in the procedures herein described while remaining within the bounds of the invention. The inventors intend that such variations are included within the scope of the invention.

EXAMPLES

[0066] Preparation of drug loaded polymeric nanomicelles for ocular delivery?
Polymeric nanomicelles of saxagliptin was prepared using pullulan, a mucoadhesive polymer, and tween 80 as surfactant for brain specific delivery.

In the present invention, polymeric nanomicelles were systematically and successfully formulated for gliptins, which finally composed of tween 80 and pullulan, as a polymer. The polymeric nanomicelles were prepared by direct dissolution method. 10 mg of pullulan was soaked for overnight in 15 ml of deionized water. 5 mg of drug, 0.5ml of tween 80 was added to the pre-soaked polymeric solution. Then, 0.5 ml of 5 % PVA solution prepared in 0.9 % NaCl solution and 0.005% of preservative (benzalkoniumchloride) was added dropwise to the solution under constant magnetic stirring (800 rpm). The solution was filtered using Whattman filter paper and sonicated for 40 min. Then solution was filtered by HPLC syringe filter (0.2 µm) and centrifuged at 10,000 rpm at 25°C for 45 min using ultracentrifuge (REMI International, Mumbai, India). The supernatant was analysed by UV spectrophotomer (UV 1800, Shimazdu, Japan) at 204 nm to calculate the % drug entrapment and drug loading. The nanomicelles formulations were analysed for particle size and particle size distribution with the help Malvern zetasizer analyser.

[0067] Determination of Entrapment Efficiency (%EE)
5ml of drug loaded polymeric nanomicelles was taken and centrifuged for 5 min at 10,000 rpm using ultracentrifuge (Remi International, Mumbai, India). Aqueous layer was separated and concentration of free drug was calculated in the supernatant liquid using the UV/visible spectrophotometer at ?max 204 nm . The %EE of drug loaded nanomicelles was calculated using following:
%EE = (Total amount of the drug- Amount of free drug) × 100
Total drug

[0068] Determination of Drug Loading (%DL)
The drug loading refers to the percentage amount of drug that is entrapped in nanoparticulate formulations. To determine the loading efficiency of drug loaded nanomicelles, 5ml of sample was taken and centrifuged for 5 min at 10,000 rpm. The aqueous layer was separated from the residual sample. The concentration of free drug was found in the supernatant fluid and was determined using the UV/VIS spectrophotometer at ?max 204 nm. The % drug loading was calculated using formula given below:
%DL = (Amount of the drug- Unentrapped drug) × 100
Weight of nanomicelles recovered

The % entrapment efficiency and % drug loading of saxagliptin loaded polymeric nanomicelles ranged from 60.67 ± 0.31% to 96.34 ± 0.67 % and 11.89± 1.39 % to 33.89 ± 1.12 %.

[0069] Optimization of Saxagliptin Loaded Polymeric Nanomicelles?

[0070] Experimental design using CCD
The prepared polymeric nanomicelles of saxagliptin were optimized using a statistical model. The present study adopted RSM technique with a two-factor, three-level, full central composite design. The effect of different independent variables was studied using Design-Expert (DOE) version 11.0.5.0 (Stat-Ease Inc, USA) on various formulation properties such as percentage drug loading (Percent DL) (R1), trapping efficiency (Percent EE) (R2) and percent drug release (R3). On the basis of optimization results by Design expert software, optimized saxagliptin polymeric nanomicelles were chosen for further studies.

[0071] Evaluation Parameters of Saxagliptin Loaded Polymeric Nanomicelles?

[0072] Visual Appearance?
Prepared nanoformulations were observed visually and were identified for clear solution, opalescent and aggregates. Optical clarity of the nanoformulation was compared with distilled water as blank.

Visual appearance for all the nanomicellar formulations was found to be clear, transparent and devoid of any particulate matter. Optical clarity of the formulation was compared with distilled deionized water as blank. This study indicates the formulation is similar to water with no particulate matter present in the optimized polymeric nanomicelles (PNM 12).

[0073] Field emission scanning electron microscope (FESEM)?
External morphology of optimized drug loaded polymeric nanomicelles was determined using a field emission scanning electron microscope (FE-SEM, Hitachi-PU model, Japan). The samples were coated with platinum before observation at an acceleration voltage of 5 kV.

The optimized polymeric nanomicellar formulation (PNM 12) showed smooth surface morphology, with spherical shape and no aggregation as shown in FIG 1.

[0074] Transmission electron microscopy
The particle size of optimized drug loaded polymeric nanomicelles was determined using TEM (Hitachi, H-7500, 120 kV). 5–10 microliter of optimized polymeric nanomicelles were placed on a paraffin sheet with a carbon coated grid placed over it and the sample left was for one min for adherences of the nanoparticles on the carbon substrate. For 10 sec the grid was placed on the phosphotungstate drop. The sample was air-dried and examined using TEM at 40-120 kV at different magnifications.

The FIG 2 showed the particles in the narrow size range of 20nm.

[0075] Average particle size, polydispersity index and Zeta Potential analysis
The mean particle size and zeta potential of polymeric nanomicelles were measured by Malvern Zetasizer's (Nano ZS90 model) and a fixed angle of 90°. Particle size distribution showed mean diameter (nm) of particle and polydispersity index (PDI) indicates the size distribution in the sample. The zeta potential, which reflects charges present on the surface of nanoparticles, is responsible for the stability of formulation and interaction with cellular membranes. The measurements were performed in triplicate at 25°C under appropriate dilution conditions. Saxagliptin-loaded polymeric nanomicelles displayed a mean particle size of 152.8 nm and PDI of 0.250 as shown in FIG 3(a). A PDI value of 0.1 to 0.25 shows a fairly narrow size distribution. The zeta potential values of optimized formulation was found to be -15.1 mV as shown in FIG 3(b). The negative zeta potential may be due to presence of various unreacted OH groups on the final conjugates. This residual surface charge indicates stable polymeric nanomicelles with a zeta potential of - 15.1 mV.

[0076] Critical Micelle Concentration (CMC) determination of tween 80
The non-ionic surfactant CMC (tween-80) was determined using iodine probe (IPM) method. Specific quantity of iodine equivalent to 2.6 gm and 3 gm of potassium-iodide was solubilized in 100 ml distilled water. This solution was scanned from 200 to 400 nm to determine the wavelength of maximum absorption. The prepared stock solution of iodine was then diluted to obtain an 80 per cent transmittance (T) of iodine solution. A series of both surfactants solution was prepared by diluting with 80 percent transmittance (T) iodine solution. Solubilized I2 prefers to participate in the surfactant system's hydrophobic microenvironment which causes the conversion of I3 to I2 from the excess potassium iodide in the solution to maintain the saturated aqueous I2 concentration. The absorption intensity of I2 was plotted as a function at surfactant. The plot break point was taken CMC of tween 80. The triplet experiment was conducted to inspect reproductively.

The intensity of absorption of I2 was plotted as a function of the concentration of surfactants as shown in FIG 4. The plot break point was taken as a twenty-eight CMC. The CMC of the optimum formula of tween 80 micelles was 3 mcg/ml, indicating the high stability of the mixed micelles and their ability to maintain integrity even after extreme dilution by body fluids. Aside from its surfactant-like property, tween 80 helps to increase the association of nanomicellar with the target cells.

[0077] Nanomicellar viscosity
Viscosity of polymeric nanomicellar formulations was determined using Brookfield viscometer (Brookfield engineering laboratories, USA). The Brookfield Dial Viscometer measures fluid viscosity at different shear rates at different temperatures. The sample room of instrument must be maintained at 37°C by thermobath and the polymeric nanomicelles for measurement are to be immersed in it and viscosity of the sample was determined.

The observed viscosity of the optimized saxagliptin loaded polymeric nanomicelles (PNM 12) was found to be 0.794 ± 0.16 cps. Optimum viscosity helps to enhance the mean residence time of formulation at its administration site.

[0078] pH determination
pH determination is crucial parameter needed to be maintained in physiological range to avoid adverse effects of instilled formulation in the eye. The tear of pH ranges from 7.31 to 7.62. Variations in the pH may have harmful effects on the eye. Therefore, the pH of the formulations were adjusted near to the tear pH of ~7.2 ± 0.1 using 0.1 N sodium hydroxide or 0.1 N hydrochloric acid solution. The pH was measured for optimized nanomicellar formulations at 25 ± 1°C using pH meter (Systronic, Delhi).

The pH of the optimized saxagliptin loaded polymeric nanomicelles (PNM 12) was found to be 7.23 ± 0.73 which is within acceptable range of 7.07 to 7.40 and hence would not cause any irritation upon administration of the formulation.

[0079] % Drug content
Assay was carried out by taking 5 mg of pure drug dissolved in 0.5 ml of methanol in 50 ml dry volumetric flask and then volume was made up using water. About 5 mg of drug was taken and dilution was made to 10µg/ml. The dilutions were filtered and analyzed using UV-Visible spectrophotometer at 295 nm wavelength for their content uniformity.

The % drug content of the optimized saxagliptin loaded polymeric nanomicelles (PNM 12) was found to be 99.70 %.

[0080] Fourier transform infrared spectroscopy (FTIR)
The FTIR spectra for optimized saxagliptin loaded polymeric nanomicelles (PNM 12) was compared with FTIR spectra of drug, polymer, and physical mixture (drug:polymer) using FTIR spectrophotometer (Agilent Technologies, USA) by KBr method.

FTIR spectrum of saxagliptin showed the characteristic peaks at 3444.98 cm-1 for N-H stretching, 2925.32 cm-1 for C-H stretching as shown in FIG 5. Also peaks appeared at 3304 cm-1 and 1618.67 cm-1 corresponding to the OH stretching and C-N stretching respectively. These characteristic peaks of the drug were also observed in the FTIR spectrum of saxagliptin loaded polymeric nanomicelles as shown in fig. 5.18. at wavelength of 3353.87 cm-1, 2941.45 cm-1, 1640.69 cm-1 and 1225.88 cm-1 without any distinct shift of vibration bands. Also PNM 12 showed characteristic absorption peak at 2913.64 which corresponds the absorption peak of C–H bond in pullulan at 2929 cm-1 thus indicating no chemical interactions between drug and polymer. Saxagliptin retained its chemical nature even after formulation into the polymeric nanomicelles.

[0081] Differential Scanning Calorimetry analysis
DSC technique was used for thermal analysis of optimized saxagliptin loaded pullulan nanomicelles formulation (PNM12) when compared with thermograms of polymer and drug:polymer mixture.
The pullulan thermogram displayed an endothermic peak at 100.11°C. A sharp endotherm (Tpeak = 106.69 °C) was observed for optimized polymeric nanomicelles (PNM 12) as given in FIG 6, showing no distinct peak in the optimized formulation which was due to the entrapment of the drug in the polymer matrix. A sharp endothermic peak of pure saxagliptin observed at 96.82°C was significantly reduced or almost absent, due to the reduced crystallinity in the nanoformulation or drug solvation in the amorphous carrier. During nanoformulation preparation, change in crystalline structure of formulation occurs which may be converted to either amorphous or other polymorphic forms.

[0082] In-vitro drug release study?
In-vitro dissolution studies was performed using drug concentration equivalent to 5 mg in comparison to pure drug (saxagliptin). In vitro release studies were performed using dialysis bag membrane method by UV spectrophotometric analysis at 204 nm.?Dialysis membrane (Visking®, MWCO 12,000–14,000; SERVA, Heidelberg, Germany) was soaked overnight in distilled water before the experiment. 5 ml of drug loaded polymeric micellar formulation was placed in the dialysis membrane bag which was sealed from both the ends. The bag was sunk fully in 15 ml of release medium (phosphate buffer solution, pH 7.2) under constant stirring at 37°C. At specified time intervals, the samples were withdrawn for 48 hrs and the amount of drug released in the medium was determined using UV/VIS spectrophotometer till the plateau phase was reached. The best formulation resulting in highest entrapment efficiency, drug loading and drug release profile were selected for further characterization studies.

The optimized saxagliptin loaded nanomicelles showed highest release of 92.4 ± 0.89 % up to 48 hrs when compared with control (plain saxagliptin) that showed release for 12 hrs as shown in FIG 7 (a) and (b). The release profile showed a biphasic release pattern as an initial fast release (burst effect) due to the fraction of drug which was weakly absorbed or bound to the surface of nanomicelles and then followed by a slow release phase (sustained release) which is the effect of the polymer-entrapped fraction of the drug. An initial fast release pattern helps to achieve the therapeutic concentration of the drug in minimum time, secondly it showed slow sustained and controlled release effect.

[0083] Ex-vivo transcorneal permeation study?
Ex-vivo transcorneal permeation studies were carried out using Franz diffusion cell (EMFDC-06, Orchid Scientifics, Maharashtra India).

i) Procedure for excising the corneal layer
The fresh, whole eyeballs of goats were obtained from a local butcher’s shop and transported to the laboratory at a chilled condition (4°C) in normal saline. The cornea was then carefully excised along with 2 to 4 mm of surrounding scleral tissue and was washed with normal saline until the washing was free from protein.

ii) Procedure for permeation studies
The excised cornea was fixed between the clamped donor and receptor compartments of Franz diffusion cell in such a way that the donor compartment faced its epithelial surface. The area of the cornea used for distribution was 0.50 cm2. The receptor compartment was filled with 10 ml simulated ocular buffer (pH 7.2) that was freshly prepared, and all air bubbles were expelled from the compartment. The aliquot (2 ml) of the prepared nanomicelle was placed on the cornea and the donor cell opening was sealed with a glass cover slip; the receptor fluid was kept at 37° C, using a Teflon-coated magnetic stirring bead. The permeation study was carried out for 24 hrs, and samples were withdrawn from the receptor compartment. The samples were analyzed for drug content by absorbance measurement at 204 nm in a UV/Visible spectrophotometer.

The permeation release across the freshly excised goat cornea was found to be 78.45 ± 0.34% in 5 hrs and 82.23 ± 0.57% of release in 24 hrs, as compared with the standard drug solution which showed release of 71.16 ± 0.89% in 12-16 hrs as shown in FIG 8(a). Enhanced permeation across the corneal layer was due to the agglomeration of nanomicelles as a depot near the cornea from which the drug is released slowly ocular area. Transcorneal transport of saxagliptin followed Fickian diffusion and Korsemeyer-Peppas (K-P) model was best fitted model, with values of n= 0.2437 and k= 4.0233 and R2= 0.9390 as shown in FIG 8 (b). Permeation through goat cornea using in polymeric nanomicelles demonstrated a harmless ocular delivery of saxagliptin.

[0084] Sterility test
The tests for sterility were done to detect the presence of viable types of fungi, bacteria and yeast in/on the formulations that were carried out under strict aseptic environment to avoid contamination of the nanoformulation. Optimized polymeric nanomicelles was sterilized by autoclaving at a pressure of 15l lb and a temperature of 121°C for 15 minutes. The sterilized optimized formulations was further evaluated for sterility test according to Indian pharmacopoeia 2007 by using fluid thioglycollate medium and soybean casein medium at 20-25°C for 14 days. The media were examined visually for microbial growth at specific time intervals during the incubation period.
The results showed no growth of microorganisms in optimized formulation (PNM12) during the 14 days of incubation period. Thus, optimized formulation passes the test for sterility for microorganism which indicates optimized formulation can be used for ophthalmic purpose.

[0085] In-vivo pharmacodynamics studies for optimized saxagliptin loaded polymeric nanomicelles?

The in-vivo pharmacodynamics studies were approved by Institutional animal ethical committee (IAEC) of Pinnacle biomedical research institute, Bhopal (Regd. No. 1842/PO/Rc/S/15/CPCSEA).

i) Animals
Twenty four (24) Sprague Dawley rats (equal sex ratio; weighing about 200-250 gm) were divided into four groups each containing six rats.?
a) ‘Group I’ or ‘control’ served ‘healthy rats’ that received normal saline intra-ocularly.?
b) ‘Group II’ or ‘negative control’ served ‘dementia induced rats’ received normal saline
c) ‘Group III’ animals served ‘dementia induced rats’ treated with plain saxagliptin solution (0.5 mg/ml).?
d) ‘Groups IV’ animals served ‘dementia induced rats’ treated with saxagliptin loaded polymeric nanomicelles.?In the groups II, III, and IV dementia was induced in rats using aluminium chloride (AlCl3). All rats were kept under controlled conditions of temperature (25 ± 2oC) and humidity (55 ± 5 % RH). They were given food and drinking water ad libitum for 12 h day and night cycle in the animal house. The experimental protocol met the national guidelines on the proper care and use of animals in the laboratory research.

ii) Determination of animal dose
Dose for the rats was calculated by human equivalent dose formulae. The dose to be given to a 250 g rat on the basis of surface area ratio was determined by multiplying the human dose by a correction factor of 0.018.?
a) Adult dose of saxagliptin is 5 mg
b) Therefore, for 250 g rat, saxagliptin dose calculated = 5 x 0.018 mg. = 0.09 mg
c) Saxagliptin dose for rats was calculated to be 0.45 mg/kg body weight.

iii) In-vivo pharmacodynamics tests?
The in-vivo pharmacodynamics studies for spatial learning, memory deficit and behavioural study using the Morris water maze test, Y-maze and Open field test in rat model (Sprague dawley rats, 200-225gm). The best-optimized nanoformulation was selected for performing in vivo studies.

[0086] Morris-maze water test (Behavioural Assay)
Saxagliptin (0.5 mg/ml) and saxagliptin loaded polymeric nanomicelles (0.5 mg/ml) was injected by intraocular route to group II and group III animals respectively before training. In the Morris water maze (MWM) test, the animal is required to find a hidden platform to escape from swimming in a pool of water. To accomplish this task, the animal forms a “spatial orientation map” in the brain using visual stimuli from extra-maze cues in the testing room. Circular water tank (100 cm in diameter) filled to a depth of 30 cm with water (25°C) was used as apparatus. Four points were equally distributed along the tank’s perimeter that served as starting locations. The tank was arbitrarily divided into four equal quadrants and at the centre of one of the quadrants was a small platform (5 cm width). During the training days, the platform remained in the same position. The rats were released into the water and were allowed to find the platform for 90 seconds. Each rat received four trials per day for 5 days with an interval of 5 minutes between trials. Escape latency was further recorded.?
The data depicted in Table 1 showed improved spatial learning behaviour in all animals throughout all days of the experiment; animals that showed any motor disabilities or difficulties in the learning of were excluded. The latency time was significantly higher in the control group (P < 0.01). Group III and IV animals which are treated with plain saxagliptin and saxagliptin nanomicelles had significantly shorter escape latency times (58.2 ± 5.037, 55.2 ± 4.492 on day 4 respectively) than animals in the negative control group (P < 0.01) as shown in Table 1. Collectively, these data are interpreted as spatial learning because the rat require less time to find the platform and spend more time and path length in the target quadrant, indicating they have learned the platform location relative to the extra-maze visual cues.
Table 1: Data showing escape latency time (sec.) in rat model
S.No.
Groups
Treatment
Day 1 Day 2 Day 3 Day 4
1 I or Control (Vehicle) Normal saline 56.3 ± 4.367 49.2 ± 6.616 43.5 ± 6.124 38.5 ± 5.394

2 II or Negative Control
Normal saline 79.8 ± 4.021 74.7 ± 4.033 68.2 ± 4.262 61.7 ± 3.933
3 III Plain saxagliptin 71.3 ± 3.983 68.3 ± 4.502 63.7 ± 5.279 58.2 ± 5.037
4 IV Saxagliptin nanomicelles (PNM 12) 69.0 ± 3.742
61.0 ± 4.899 55.2 ± 4.492 65.3 ± 5.610

[0087] Open-field test (Behavioural Assay)
Open field test is common tests to monitor general motor activity and exploratory behaviour. In open field test, when animals were placed in a different environment from home cage, they express their fear and anxiety by reduction in exploratory behaviour and ambulation. Rats were placed individually in an open arena equipped with infrared photo-beams to monitor mice behaviour for 30 minutes without the experimenter in the room being present. Trial starts immediately and ends when the defined duration is over. Animal is returned to the home cage and it records the number of faecal pellets. Arena is cleaned in between trials with Virkon. The following activities were recorded with photo-beams– (i) counts of ambulatory (walking), (ii) counts of stereotypical (repetitive activity, eg, grooming) and (iii) counts of vertical (rearing up) movements; and (iv) number of faecal drops.

The results given in Table 2 indicated that the saxagliptin-loaded nanomicelles treated rats showed a significant decrease in rearing, self-grooming and faecal drop on comparison with negative control group. Locomotion and rearing measurements are parameters used to determine habituation learning in the open field test. Open field exploration and habituation are closely related to the hippocampus and its cholinergic input. Behavioural activation noted by the novel environment is positively correlated with increased hippocampal ache levels; however, behavioural habituation to this environment is not paralleled by a decreased activation of cholinergic activity. Additionally, lesions in the hippocampus or pharmacological blockade of the cholinergic input strongly affect the exploratory behaviour and its habituation. In our experimental work, saxagliptin loaded nanomicelles treated rats evidenced more exploratory activity i.e. ambulations (64.29 ±1.776) as compared to control group (31.51±1.202) as shown in table 2. A significant (p < 0.01) reduction in rearing and faecal drop was noted in group II (negative control) as compared to control rats. However, on treatment of these dementia induced rats with PNM 12 a significant (p < 0.05) increase in ambulatory activity was noted.

Table 2: Y maze test data in rat model

Groups
Treatment
Activity on open field
Ambulations
Rearings
Self- Grooming
Faecal drop

I or Control Normal saline 80.18 ± 2.101 7.06 ± 0.350 12.58 ± 0.213 3.2 ± 0.141

II or Negative Control Normal saline
31.51 ± 1.202 13.25 ± 0.441 2.38 ± 0.231 8.15±0.372

III Plain saxagliptin
46.36 ± 0.909 14.68 ± 0.263 3.05 ± 0.234 7.25±0.344

IV Saxagliptin nanomicelles (PNM 12)
64.29 ± 1.776 11.283 ± 0.778
7.33 ± 0.250 4.91 ± 0.440

[0088] Y- maze Spontaneous Alternation Test (Behavioural Assay)
Y-maze test is used to measure spatial working memory and evaluate exploratory behaviour in hippocampus, basal forebrain, septum, and prefrontal cortex areas of rat brain.

For monitoring normal exploratory behaviour, healthy animals released in arm #1 will alternate entries into arm #2 and arm #3, for an 8 min period. Re-entry to a just visited arm is seen in rodents with frontal cortical lesions. The number of arm entries served as an indicator for locomotor and movement activity. Percentage of same arm returns (%SAR) and alternate arm returns (%AAR) were recorded as a response parameters.

The total number of arm entries (NAEs) were not significantly (p < 0.05) different among all groups. There was a decrease in the percentage of same arm returns (%SAR) in saxagliptin nanomicelles treated group (group IV). A notable differences were observed among the control (group I) and other groups. The results obtained with the Y-maze test indicated an increased spontaneous alternate arm returns (%AAR) compared to negative control (group II) and group III, suggesting effects on short term-memory comparable to control group animals. A significant difference was observed between the saxagliptin nanomicelles and negative control groups. In Y- maze test, a significant (p < 0.05) increase in of spatial memory in animal treated with saxagliptin-loaded nanomicelles were observed after three days as shown in table 3. Thus saxagliptin nanomicelles treated animal group confirmed an ability to enhance memory impaired by inducer in the spontaneous alternation performance in the Y-maze.?
Table 3: Open field test data in rat model
S.No. Groups Treatment
Arm return Same arm returns Alternate arm returns
1
I or Control (Vehicle) Normal saline
24.16 ± 0.752 6.5 ± 0.547 25.16 ± 1.169
2 II or Negative Control Normal saline 5.0 ± 0.894 25 ± 1.414 7.83 ± 2.316

3 III
Plain saxagliptin 10.16 ± 0.752
20 ± 2.190
9 ± 1.264

4 IV Saxagliptin nanomicelles (PNM 12) 15.5±1.048 11.33 ± 1.211 17.66 ± 0.816

[0089] Histopathological studies
The brain tissues and ocular tissue were collected, allowed to set in 10 % formalin and embedded in paraffin. Sections of 5 µm were cut from ocular tissue and middle brain lobes of each group from similar positions. The brain tissue sections were deparaffinised using ethanol and xylene. The deparaffinised sections were stained with haematoxylin and eosin. The histopathological changes were analysed under 20 X magnification. Microscopic changes in the ocular tissues and brain tissues of treated animals (group III & IV) were compared with the control (group I).

The images of brain tissues were observed for any changes in the animals of group II, III, IV and compared with group I animals as shown in FIG 9 (a)-(d). Histopathological images showed absence of microscopic changes in the brain tissues, of treated animals (Group III and IV) when compared with the control animals (Group I) and negative control (Group II). Thus, the saxagliptin loaded polymeric nanomicelles showed no toxicity or structural damage to brain region.

[0090] Haematological analysis
Blood samples were collected in a tube containing EDTA (anticoagulant) for analysis of haematological parameters directly from the cardiac puncture. Values of Red blood cells (RBC), White blood cells (WBC), Haemoglobin (Hb), Differential leucocyte counts (DLC), Total leucocyte counts (TLC) and platelet counts were determined and controlled using an auto- analyser (Roche Integra, 400 Plus, Diagnostic Systems, IN, United States). Hb, RBC, WBC, DLC, TLC and platelet values in treated animals were compared with animal control values.

For haematological studies, blood samples of groups ( II, III, IV) animals were compared with the control group (Group I) animals. The values of Hb, RBC, WBC, DLC, TLC and platelets were found to be within normal range in treated animals (Group III and IV) and no significant changes (p>0.05) was found on comparison to control animals (Group I) as shown in Table 4.
Table 4: Hematological data in rat model
Groups Treatments Hb (gm/dL) RBC (106/µL) WBC (103/µL) DLC (%) TLC (%) Plateletes (103/µL)
I or Control (Vehicle) Normal saline 13.88 ± 0.938 8.08 ± 0.371 7.95 ± 0.557 33.3 ± 1.198 67.03 ± 1.057 819.3 ± 5.125
II or Negative Control Normal saline 10.06 ± 0.763 5.183 ± 0.278 6.05 ± 0.361 19.43 ± 0.643
53.21 ± 1.190
553.4 ± 3.223
III Plain saxagliptin 11.08±0. 306
6.00±0.2 82
6.26±0.2 80 22.4±0.74 7 57.93±0. 47 682.8±2.4 83
IV Saxagliptin nanomicelles (PNM 12) 11.31±0. 263 6.65 ±0.242 6.816±0. 271
27.03±1.2 61 62.61±1. 881
710.16±2. 483

[0091] Statistical analysis
The data were presented as mean ± SEM values. One-way ANOVA followed by “t”-test was performed. A probability level of 0.05 or less was accepted as significant. Pearson’s correlation coefficient and regression analysis were used to evaluate the connection between the working memory errors and some parameters like locomotion, grooming and rearing in the in-vivo pharmacodynamics models.

[0092] Gamma scintigraphy studies
In order to visualize localization of drug in brain via ocular administrations, gamma scintigraphy studies were performed on Albino New Zealand rabbits, in the Institute of Nuclear Medicine and Allied Science (INMAS), Defence Research and Development Organisation Delhi (DRDO), Delhi, India (Regd. No. INM/IAEC/19/01).

Plain saxagliptin solution and saxagliptin loaded polymeric nanomicelles was labelled with 99mTc by the stannous reduction method. Technetium-99m (Tc-99m) was chosen as radionuclide for radiolabelling due to a short half-life (6 hours), cost effective and ease of administration in millicurie amounts. This results in a very low dose of radiation which benefits the animals. Rabbits for gamma scintigraphy studies were divided into two groups

a) Group I animals served as standard and received 99mTc labelled saxagliptin (0.5 mg/ml)

b) Groups II animals served as test and received 99mTc labelled saxagliptin loaded polymeric nanomicelles (0.5 mg/ml)

The radiolabeled formulations (both nanoparticulate and drug solution) was administered via intraocular route, and the localization of drug was visualized using Single Photon Emission Computerized Tomography (SPECT). A syringe containing 100 µL solution aliquot to be tested was placed near the rabbit's eye before and after installation as a tracer of position. The eyelids were kept closed for 5 seconds after instillation, to prevent loss of the instilled solution. The rabbit was kept on a table without a restraining box, with the experimenter's hand with his left eye supporting his head at a distance of 6 cm in front of the collimator aperture. After instillation of 100 µL radiolabelled solutions (plain drug, polymeric nanomicelles formulation) to group I and II, gamma camera (GE) was adjusted to detect the radiation of 99mTc (MBp) and amount of drug reached to the brain via ocular route.

Gamma scintigraphy images shown in FIG 10 (a) and (b), gives a clear differentiation between the intraocular administration of 99mTc labelled drug solution and drug loaded polymeric nanomicelles formulation respectively. Study revealed that the entrapped drug reached to the target area i.e., to brain and also showed the localization of the radiolabeled drug complex comparatively more than radiolabelled plain drug solution. The drug comes into the systemic circulation after few minutes as shown in FIG 10 (b). Radiolabelled drug loaded polymeric nanomicelles as shown in FIG 10 (b) enhances the drug delivery directly to the brain via ocular administration on comparison with radiolabelled plain drug solution which is shown in FIG 10 (a).

[0093] Accelerated stability studies according to ICH Q1A (R2) guidelines
As per the ICH guidelines, three batches of optimized polymeric nanomicellar formulations was subjected to accelerated stability study by storing the formulations at high-density polyethylene bottles and stored at 4 ± 1 °C, 25 ± 2 °C/60 ± 5 % RH %, 40 ± 2°C/75 ± 5 % RH for 180 days. Samples were withdrawn at 0, 30, 60 and 90, 120, 150, 180 days and evaluated for various parameters (% drug content, % entrapment, % drug loading and drug release).

The formulated optimized saxagliptin loaded polymeric nanomicelles exhibited good physical stability over a storage period of 6 months (180 days). The samples showed clear-coloured appearance with no significant changes in the evaluation parameters of the optimized nanoformulation under any of the storage conditions. The stability data is given in table 5. and were analyzed for statistical significance by ANOVA and Tukey-Kramer multiple comparison test using GraphPad Instat software (GraphPad Software Inc., CA, USA). The changes of the observed parameters were not found to be statistically significant (P > 0.05) which indicated that the optimized formulation (PNM 12) was stable for six months.
Table 5: Stability studies for optimized saxagliptin loaded polymeric nanomicelles at different temperature and humidity
Time (Days) Storage conditions % Drug Remaining % Drug entrapment
% Drug loading % Drug release
0 4oC 99.70 ± 1.56
96.34 ± 0.67
33.89± 1.12 92.40 ± 0.89
30 99.41 ± 1.67 96.12 ± 1.47
33.28±1.37
92.17 ± 1.83
60 99.21 ± 1.05
96.33 ± 1.33

33.66±1.77
92.43 ± 1.55
90 99.24 ± 2.32 96.28 ± 1.95 33.38±1.41 92.65 ± 2.32
120 98.24 ± 1.26
95.99 ± 1.66
32.74±2.37 91.31 ± 1.84
150 98.74 ± 1.61 95.12 ± 1.41
32.51±1.51

91.22 ± 2.31

180 98.48 ± 2.27 95.77 ± 1.87 32.92±2.37 91.67 ± 2.81
0 40 ± 2 oC and?75 ± 5% RH 99.70 ± 1.56
96.34 ± 0.67
33.89± 1.12 92.4 ± 0.89
30 99.20 ± 1.31
96.42± 1.43

33.77 ±1.55
92.74 ± 0.67
60 99.70 ± 1.39 96.19 ± 1.35 33.53±2.74 92.68 ± 2.54
90 99.93 ± 1.73 96.92 ± 2.51
32.21±2.01
92.95 ± 1.21
120 99.33 ± 1.11 95.99 ± 2.65
32.52±2.37

91.63 ± 2.47

150 98.81 ± 2.71 95.28 ± 1.08 32.83±2.12 91.57 ± 1.31
180 98.29 ± 1.27
95.31 ± 2.61
32.72±1.64 91.11 ± 1.01

0 25 ± 2 oC and?60 ± 5% RH
99.70 ± 1.56 96.34 ± 0.67 33.89± 1.12 92.4 ± 0.89

30

99.70 ± 1.11
96.55± 2.32 33.23 ±0.57 92.76± 2.67
60 99.70 ± 2.86 96.21 ± 1.57 33.45±1.04 92.31 ± 1.04
90 99.31 ± 1.55
96.84 ± 2.51
33.38±2.17 92.88 ± 2.12

120

98.54 ± 2.85 95.56± 2.65 32.21±2.70 91.76 ± 2.71
150 98.37 ± 1.53 95.29± 1.08 32.72±1.63 91.73 ± 2.15

180 98.31 ± 2.55 95.16 ± 2.61 32.29±1.84 91.11 ± 1.17

CLAIMS:I CLAIM:

1. A nanoformulation of a drug loaded polymeric nanomicelle for ocular to brain delivery/ocular administration comprising:
a. a polymeric drug carrier nanomicelle;
b. a drug encapsulated within the polymer core of said polymeric carrier nanomicelles; and
c. a surfactant.
2. The nanoformulation as claimed in claim 1, wherein said polymer is selected from pullulan, chitosan, hydroxy propyl methyl cellulose, sodium alginate and starch.
3. The nanoformulation as claimed in claim 2, wherein said polymer is pullulan.
4. The nanoformulation as claimed in claim 1, wherein said drug is gliptin selected from saxagliptin, sitagliptin, lingliptin, vildagliptin, alogliptin.
5. The nanoformulation as claimed in claim 4, wherein said drug is saxagliptin or sitagliptin.
6. The nanoformulation as claimed in claim 1, wherein surfactant is tween 80.
7. The nanoformulation as claimed in claim 1, wherein mean particle size of the drug loaded nanomicelle is 152.8 nm.
8. A drug loaded polymeric nanomicelle wherein the drug is selected from saxagliptin or sitagliptin and polymer is pullulan.
9. The drug loaded polymeric nanomicelle as claimed in claim 8 wherein % entrapment efficiency of the drug is 60% to 96% and % drug loading of polymeric nanomicelles is 11 % to 34%.
10. A method of providing the nanoformulation of polymeric nanomicelle as claimed in claim 1 for ocular to brain delivery/ocular administration for treatment of neurodegenerative diseases like dementia, Alzheimer’s.