DESC:CROSS-REFERENCE TO RELATED APPLICATIONS AND PRIORITY

The present application claims priority from Provisional Application number 201911034265 dated 26 August 2019.

TECHNICAL FIELD OF THE INVENTION

[0001] The present invention relates to a novel formulation for treating/preventing neurodegenerative disorders, more particularly the present invention relates to formulation of Linagliptin (DPP-4 inhibitor) nanosuspension for treating/preventing neurodegenerative disorders, by the nose to brain drug delivery.

BACKGROUND OF THE INVENTION

[0002] 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.

[0003] 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.

[0004] 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.

[0005] 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.

[0006] 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. Linagliptin is a dipeptidyl peptidase-4 (DPP-4) inhibitor that was approved for the treatment of type 2 diabetes in 2011.

[0007] 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.

[0008] Linagliptin is a xanthine-derived competitive and reversible dipeptidyl peptidase (DPP)-4 enzyme inhibitor that slows the breakdown of insulinotropic hormone glucagon-like peptide (GLP)-1.

[0009] The chemical designation of Linagliptin is 8-[(3R)-3-aminopiperidin-1-yl]-7-(but-2-yn-1-yl)-3-methyl-1-[(4 methylquinazolin-2-yl) methyl]-3, 7-dihydro-1H-purine-2, 6-dione.

[0010] 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, and (3) reducing the peripheral side effects by increasing the relative amount of the drug reaching the brain.
[0011] The oral bioavailability of poorly water-soluble drug can be improved using nanosuspension by increasing the dissolution rate. Nanosuspensions are distinguished from nanoparticles which are polymeric colloidal carriers of drug, and from solid lipid nanoparticles, which are lipidic carriers of drug. The major advantages of nanoparticles are its general applicability to most drugs and its simplicity. Nanosuspensions enhance dissolution and therefore the bioavailability of drugs; can achieve a higher drug loading; permit a possible dose reduction; and enhance physical and chemical stability of drugs.

[0012] Nose to brain drug delivery route provides a direct and non-invasive route which by-passes BBB, thereby increasing drug concentration in the brain and reducing systemic side effects. It is painless, patient-friendly and improves drug performance. Upper region of the nasal cavity (known as an olfactory region) remains directly connected to the brain (frontal cortex; especially olfactory bulb) via olfactory nerves. Along with this, the middle and the largest region of the nasal cavity (the respiratory region) remain supplied with the trigeminal sensory neurons and blood vessels. For treatment perspective, nose to brain drug delivery route is a direct, potential and convenient approach to treat neurodegenerative disorders with proper clinical investigations.

[0013] Adverse effects associated with use of Linagliptin are general pancreatitis, nausea and vomiting, loss of appetite, fast heart rate, fever, sore throat, and headache with a severe blistering, peeling, and red skin rash.

[0014] Linagliptin due to its poor permeability, low bioavailability (30%) and its therapeutic use in chronic conditions necessitates its nanoformulation development thus to limit its dose related side effects.

SUMMARY OF THE INVENTION

[0015] In an aspect, the present invention relates to a pharmaceutical formulation comprising of a nanosuspension of Linagliptin for nose to brain delivery for targeting and treating neurodegenerative disorders like dementia. The pharmaceutical formulation of nanosuspension of Linagliptin 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.

[0016] In another aspect, the present invention provides a pharmaceutical formulation comprising of a nanosuspension of Linagliptin as a rapid, direct, efficient and non-invasive drug delivery approach to brain for treatment of neurodegenerative disorders.

[0017] In yet another aspect, the present invention provides nanosuspension formulation for nose to brain delivery/nasal administration to a subject, the nanosuspension formulation comprising: a drug in nanoparticulate form; a polymer; and a surfactant.

[0018] In a further aspect, the drug is a gliptin, selected from linagliptin, sitagliptin, saxagliptin, vildagliptin, alogliptin.

[0019] In an alternate aspect, the polymer is a mucoadhesive polymer selected from chitosan, pullulan, hydroxyl propyl methyl cellulose, sodium alginate and starch.

[0020] In another aspect, the polymer is chitosan and surfactant is TWEEN 80.

[0021] In a further aspect, the drug comprises 0. 01 % wt to 0.10 % wt of the formulation.

[0022] In another aspect, the polymer comprises 0.01 % wt to 0.10 % wt of the formulation.

[0023] In yet another aspect, the surfactant comprises 0.005 % wt of the formulation.

[0024] In a further aspect, the formulation comprises of nanoparticulate drug of average size of 250.7 nm.

[0025] In another aspect, the present invention also refers to a method of providing the nanosuspension formulation for nose to brain delivery/nasal administration to a subject for treatment of a neurodegenerative disorder like dementia.

[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 the visual appearance of optimized linagliptin loaded nanosuspension

[0029] FIG. 2 illustrates FESEM images for optimized linagliptin loaded nanosuspension

[0030] FIG. 3 illustrates TEM images for optimized linagliptin loaded nanosuspension

[0031] FIG. 4(a) illustrates mean particle size of optimized linagliptin loaded nanosuspension

[0032] FIG. 4(b) illustrates zeta potential of optimized linagliptin loaded nanosuspension

[0033] FIG. 5 illustrates FTIR spectrum of linagliptin loaded nanosuspension

[0034] FIG. 6 illustrates DSC results of optimized linagliptin nanosuspension

[0035] FIG. 7(a) illustrates In-vitro drug release data of optimized linagliptin nanosuspension

[0036] FIG. 7(b) illustrates In-vitro drug release kinetics of optimized linagliptin nanosuspension

[0037] FIG. 8(a) illustrates Ex-vivo drug release data of optimized linagliptin nanosuspension

[0038] FIG. 8(b) illustrates Ex-vivo release kinetics of optimized linagliptin nanosuspension

[0039] FIG. 9 illustrates images of brain tissues of histopathological studies.

[0040] FIG. 10 illustrates Gamma scintigraphic 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 “nanosuspension” means colloidal dispersions and biphasic systems consisting of drug particles that are suspended in an aqueous medium using a stabilizer to increase drug dissolution rate. The dispersed particles have a diameter of less than 1 µm. The nano-size particles (nanoparticulate form) in nanosuspensions provide a large drug surface area enhancing the rate of dissolution, improving overall bioavailability and causing rapid onset of action for poorly soluble medicines. Nanosuspension can be generated either from the bottom up, or from the top down. Bottom up consists of dissolving drug in a stabilizer containing organic solvent. For ocular delivery, it provides several advantages such as sterilization, ease of eye drop formulation, less irritation, increase precorneal residence time and enhancement in ocular bioavailability of drugs which are insoluble in tear fluid.

[0048] Drug preparation in the form of nanosuspensions has proven to be a more cost-effective and technically simpler alternative, yielding a more physically stable product than liposome dispersions. With this technique the drug, dispersed in water, is grounded in the range of nanometers (100–1000 nm) by shear forces to particles with a mean diameter. Due to their higher dissolution pressure, the fineness of the dispersed particles causes them to dissolve more quickly and leads to an increased solubility in saturation. This could improve drug bioavailability compared to other microparticular systems. If in-vivo dissolution velocity of the drug particles is low enough, the drug nanosuspensions will have the passive targeting advantages of colloidal drug carriers. Bhavna et al. 2014 developed a nanosuspension formulation of donepezil, a cholinesterase inhibitor, for enhancing brain exposure to treat AD. Mishra B et al. 2010 aimed to develop nanosuspension of lamotrigine by emulsification-solvent diffusion method and investigated its formulation characteristics using L9 orthogonal array.

[0049] 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.

[0050] 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.

[0051] 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 sitagliptin, linagliptin, saxagliptin, vildagliptin and alogliptin. DPP-4 inhibitors are administered orally.

[0052] 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.

[0053] 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.

[0054] The present invention relates to a pharmaceutical formulation comprising of a nanosuspension of Linagliptin for nose to brain delivery for targeting and treating neurodegenerative disorders like dementia. The pharmaceutical formulation of nanosuspension of Linagliptin 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.

[0055] The present invention also provides a pharmaceutical formulation comprising of a nanosuspension of Linagliptin as a rapid, direct, efficient and non-invasive drug delivery approach to brain for treatment of neurodegenerative disorders.

[0056] In a particular embodiment, the present invention provides nanosuspension formulation for nose to brain delivery/nasal administration to a subject, the nanosuspension formulation comprising: a drug in nanoparticulate form; a polymer; and a surfactant.

[0057] In a further embodiment, the drug is a gliptin, selected from linagliptin, sitagliptin, saxagliptin, vildagliptin, alogliptin.

[0058] In an alternate embodiment, the polymer is a mucoadhesive polymer selected from chitosan, pullulan, hydroxyl propyl methyl cellulose, sodium alginate and starch.

[0059] In a further embodiment, the polymer is chitosan and surfactant is TWEEN 80.

[0060] In yet another embodiment, the drug comprises 0.01 % wt to 0.10 % wt of the formulation.

[0061] In another embodiment, the polymer comprises 0.01 % wt to 0.10 % wt of the formulation.

[0062] In alternate embodiment, the surfactant comprises 0.005 % wt of the formulation.

[0063] In a further embodiment, the formulation comprises particles of the drug of average size of 250.7 nm.

[0064] In yet another embodiment, the present invention refers to a method of providing the nanosuspension formulation for nose to brain delivery/nasal administration to a subject for treatment of a neurodegenerative disorder like dementia.

[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, optimization and evaluation of Linagliptin loaded nanosuspension?Linagliptin (Mylan Laboratories) loaded nanosuspension was prepared using chitosan, as an mucoadhesive polymer, and tween 80 as surfactant.

[0067] Preparation of linagliptin loaded nanosuspension for nose to brain delivery Chitosan nanosuspension of linagliptin (BCS III) was prepared by nanoprecipitation method. Different batches of linagliptin nanosuspension were prepared considering the solvent, drug, polymer and surfactant concentration to obtain nanosuspension. Various concentration of chitosan (0.1 %, 0.5%, 1 %, 1.5 %) were dissolved in deionized distilled water containing 1 mL acetic acid (1 % v/v glacial acetic acid) and kept for overnight soaking. 5mg drug was added to the polymeric solution and was filtered through Whattman filter paper and stirred for 15 min on magnetic stirrer (800 rpm). Stabilizer solution ( PVA+ TWEEN 80) and 0.005% of prservative was added dropwise while continuous stirring on magnetic stirrer. (800 rpm). The solution was filtered firstly through Whattman filter paper and then by HPLC syringe filter (0.2 µm). Then the solution was sonicated using bath sonicator for 15 cycles. The nanosuspension was centrifuged at 12,000 rpm and 25oC for 45 minutes. The supernatant was analyzed by UV spectrophotomery at 295 nm to calculate the % drug entrapment and drug loading. The nanosuspension formulations were analyzed for particle size and particle size distribution with the help Malvern zetasizer analyzer. The pre-optimization studies of prepared nanosuspension were carried out for determining the levels of the independent variables, drug concentration, polymer concentration. Entrapment Efficiency (%EE)
5ml of drug loaded nanosuspension 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 295 nm. The entrapment efficiency of drug loaded chitosan nanosuspension 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 nanosuspension, 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

[0069] In-vitro drug release study?
In-vitro dissolution studies was performed using drug concentration equivalent to 5 mg in comparison to pure drug. 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 linagliptin loaded nanosuspension 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.
The cumulative amount of drug (Linagliptin) was determined using sodium orthophosphate buffer (pH 6.3) used as a release medium was kept on a magnetic stirred water bath at 37°C at 100 rpm. Samples were withdrawn, filtered and analyzed by UV/VIS spectrophotometer at wavelength of 295 nm up to 48 hours. The best formulation resulting in highest entrapment efficiency, drug loading and drug release profile were selected for further characterization studies.
[0070] Optimization of linagliptin loaded nanosuspension?
[0071] Experimental design using CCD?
The present study adopted RSM technique with a two-factor, three-level, full central composite design. The effect of various independent variables was studied using Design-Expert (DOE) version 11.0.5.0 (Stat-Ease Inc, USA) on different formulation properties such as percentage drug loading (% DL) (R1), entrapment efficiency (% EE) (R2) and % drug release (R3) using Design- Expert (DOE) version 11.0.5.0 software (Stat-Ease Inc, USA). To optimize the drug-loaded nanosuspension, Design expert trial version 11.05.0 fitted the observed responses of 13 formulation to various models.?
The % entrapment efficiency of different formulation of linagliptin loaded nanosuspension was determined. The % entrapment efficiency of linagliptin loaded nanosuspension ranged from 60.67 ± 0.31% to 96.34 ± 0.67 %. The optimized linagliptin loaded nanosuspension (LS1) showed % entrapment efficiency of 95.8 ± 1.45 % .

The % drug loading of different formulation of linagliptin loaded nanosuspension was determined. The % drug loading of linagliptin loaded nanosuspension ranged from 11.89 ± 1.39 % to 33.89 ± 1.12 %. The optimized linagliptin loaded nanosuspension (LS1) showed % drug loading of 35.78 ± 0.19 %.

[0072] Evaluation of Linagliptin loaded nanosuspension

[0073] Visual Appearance
Prepared nanoformulation was observed visually and three different systems were identified namely: Clear solution, opalescent suspension and aggregates.
The optimized linagliptin loaded nanosuspension (LS1) was found to be clear, transparent and devoid of any particulate matter as shown in FIG. 1.
[0074] Field emission scanning electron microscope (FESEM)
The particle shape and morphology of optimized nanoformulation was studied using FE-SEM to determine their average particle size and morphology. FESEM from the external surface of the nanoformulation provided the possibility to observe the structural situation depending upon the designed parameters during the preparation.
The optimized nanosuspension formulation (LS1) was found to be visually clear and devoid of any visible particles through naked eye. The FESEM images for optimized nanosuspension showed spherical shape with smooth surface morphology and absence of aggregation as shown in FIG. 2.
[0075] Transmission electron microscopy
The particle size of optimized linagliptin loaded nanosuspension was determined using TEM (Hitachi, H-7500, 120 kV). 5–10 microlitre of optimized linagliptin loaded nanosuspension 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.
TEM images for optimized linagliptin loaded nanosuspension (LS1) (FIG. 3) were observed in nanometric range of 20 nm with smooth and regular spherical shaped particles.
[0076] Mean particle size, particle size distribution and zeta potential?
The particle size distribution, polydispersity index (PDI) and zeta potential were determined by Malvern Zetasizer (Nano ZS90 model). PDI gives the physical stability of nanosuspensions and should be as low as possible for the long-time stability of nanosuspensions. Zeta potential was to study the surface charge properties of the nanosuspensions. The value of particle surface charge indicates the stability of nanosuspensions at the macroscopic level. The zeta potential values are calculated by determining electrophoretic mobility of particle.
The mean particle size of optimized linagliptin loaded nanosuspension (LS1) was found to be 250.7 nm with a polydispersity index 0.188 as shown in Figure 4(a). The zeta potential of optimized linagliptin loaded nanosuspension (LS1) was found to be -16.3 mV as shown in Figure 4(b).
[0077] Viscosity measurement
The viscosity was measured for optimized linagliptin loaded nanosuspension 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 optimized linagliptin loaded nanosuspension for measurement are to be immersed in it and viscosity of the sample was determined.
The observed viscosity of optimized linagliptin loaded nanosuspension (LS1) was found to be i.e. 0.882 ± 0.23 cps. Viscosity less than 2.0 centipoise (cP) is necessary for retention of formulation in the cul-de-sac and this does not affect the rate of tear drainage.
[0078] pH value determination
The rate and extent of absorption of ionizable drugs can be influenced by the pH inside the nasal cavity. The average baseline pH of human nasal cavity is 6.3. The pH was measured for optimized linagliptin loaded nanosuspension at 25±1 °C using pH meter (Systronic, Delhi). Difference in pH can cause nasal stinging and higher pH can lead to fungal infections.
The pH value for optimized linagliptin loaded nanosuspension (LS1) was found to be 6.33 ± 0.53. All the formulations are within acceptable range 5.85 and 6.33 and hence would not cause any irritation upon administration of the formulation.
[0079] Saturation solubility studies
The saturation solubility of the particles is essential as it affects the bioavailability of the drug, the rate of drug release into dissolution medium and consequently, the therapeutic efficiency of the pharmaceutical product. The saturation solubility studies were measured for both optimized linagliptin loaded nanosuspension and pure drug. The sample was stirred in phosphate buffer pH 7.2 for 48 hrs on magnetic stirrer with 100 rpm at 25 ± 1°C. Then the sample was centrifuged for 10 min at 10,000 rpm. Clear supernatant was collected using 0.22 µm syringe filter and analysed using UV spectrophotometer at 295 nm.
The observed saturation solubility was found to be 0.987 mg/ml and 0.002 ± 0.07 mg/ml for LS 1 and pure drug respectively. The improvement in saturation solubility is due to a reduction in particle size and subsequent increase in surface area.
[0080] % 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 10 µg/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 linagliptin loaded nanosuspension (LS1) was found to be 98.39 %.
[0081] Fourier Transform Infrared Spectroscopy (FTIR)
The FTIR spectra for optimized linagliptin loaded nanosuspension (LS 1) was compared with FTIR spectra of linagliptin, chitosan, and physical mixture (drug:polymer) using FTIR spectrophotometer (Agilent Technologies, USA) by KBr method. Samples were previously prepared with KBr at 1 : 5 (sample: KBr, w/w). KBr disks were prepared by compressing the powders at a pressure of 5 tons for 3 min in a hydraulic press and scanned against a blank KBr disk at wave numbers ranging from 500 to 4000 cm-1 with a resolution of 1.0 cm-1 .
The characteristic peaks linagliptin were observed at 3358 cm-1 for N-H stretching, 2237 cm-1(–C=C–), 1659 (C=O) as shown in fig.5.39. These characteristic peaks of the drug were also observed in the FTIR spectrum of linagliptin loaded nanosuspension (LS1) as shown in FIG. 5. at wavelength of 3405 cm-1, 1342.86 cm-1, without any distinct shift of vibration bands. Also LS1 showed characteristic absorption peak at 3405.24 cm-1 (O-H), 2942.35 cm-1(C–H), 1641.99 cm-1(C=0),1561.8 cm-1 (NH) which corresponds the absorption peak of chitosan. It indicates that no chemical interaction between the drug and the polymer occurred and linagliptin retained its chemical nature after formulation into the nanosuspension.
[0082] Differential Scanning Calorimetry (DSC)?
DSC technique was used for thermal analysis of optimized linagliptin loaded nanosuspension (LS 1) when compared with thermograms of polymer and drug:polymer mixture. Samples were sealed separately in aluminium cells and DSC apparatus was set between 25°C and 300°C. Thermal analysis was performed at a heating rate maintained at 10°C/min in a nitrogen atmosphere. An empty alumina pan was used as the reference DSC study measures the temperatures and heat flows associated with the transition in drugs from crystalline to amorphous state as a function of time and temperature in a controlled atmosphere.

The pure linagliptin showed melting endothermic peak at 206°C. DSC thermogram of chitosan showed an endothermic peak between 77.78°C - 80.44°C and an exothermic peak between 303.77 - 304.28oC as indicated in FIG. 6. By analyzing the DSC results of optimized linagliptin nanosuspension as indicated in FIG. 6, it was found that an endothermic peak of chitosan was observed at 113.25 °C characteristic of increase in chitosan Tg (glass transition temperature), followed by another endothermal event of fusion at 169.46°C. The peak of drug loaded nanosuspension was reduced due to significant decrease in drug crystallinity when formulated as nanosuspension.

[0083] In-vitro drug release study?
In-vitro drug release study was performed for the optimized nanosuspension formulae (LS1) using an amount of drug equivalent to 5 mg in comparison to pure drug (Linagliptin) using dialysis bag diffusion technique. Drug diffusion and drug dissolution are two process of transportation of drug molecules from formulation inside the dialysis bag to the recipient compartment through the dialysis membrane. Drug release from polymeric nanosuspension may involve diffusion, polymer erosion or a combination of both based on the physicochemical characteristics of the polymer.?The drug was released from the linagliptin-loaded nanosuspension (LS1), in a biphasic pattern with an initial burst release (42.81 %) followed by sustained release (89.65 %) after 48 hrs as shown in Figure 7(a). Immediate burst effect was due to the weakly absorbed fraction of drug on the surface of nanoparticles and extended release was due to the influence of the polymer- entrapped drug fraction. Thus, the reduced particles enhances the adsorption of drugs on the polymer which leads to increased surface area and hence increased dissolution profile as compared to pure drug that showed drug release upto 12 hrs.

The drug release data obtained from various in-vitro release experiments were subjected to various kinetics equations to evaluate the drug release mechanism and kinetics using Bit Software 1.12. as shown in Figure 7(b). The in-vitro release kinetics of optimized linagliptin-loaded nanosuspension (LS1) was studied for zero order, first order, Higuchi model, and Korsemeyer- Peppas model. The kinetics data showed that in-vitro release from LS 1 formulation is best explained by the Korsemeyer-Peppas (K-P) model (R=0.9740, K= 4.053, n=0.3601) as shown in and Figure 7(b). The optimized nanosuspension follows the fickian diffusion of drug release.

[0084] Ex- vivo permeation study?
Ex-vivo permeation study was conducted using a Franz diffusion cell containing 100 ml of phosphate buffer (pH 6.3, 0.1 M) using an excised goat nasal mucosa maintained at 37 ± 1°C with water bath. The goat nasal cavity was obtained from local slaughterhouse and kept in cool phosphate buffer (pH 6.3); the nasal mucosa was carefully removed using forceps and surgical scissors from the exposed septum of the goat’s nasal cavity. The mucosal tissues were immediately immersed in Ringer's solution. The freshly excised nasal mucosa was mounted on the diffusion cell, and 1ml of nanosuspension was placed on it. The samples withdrawn at predetermined time intervals were appropriately diluted, filtered and absorbance was measured spectrophotometrically at 295 nm using UV Spectrophotometer, taking phosphate buffer (pH 6.3) as the blank for 24 hrs.

Ex-vivo studies of optimized nanosuspension (LS1) showed 82.23± 1.25 % of drug permeation for 24 hrs as compared to 68.23 ± 1.03 % of drug permeated from plain drug solution in 16 hrs and after that it remained constant for 24 hrs as shown in Figure 8 (a). Thus the sustained effect of linagliptin loaded chitosan nanosuspension showed enhanced penetration effect due to presence of chitosan, which has an effect of opening the tight junctions of the nasal mucosa. Thus the LS1 formulation showed sustained release of the drug for 24 hrs periods.

The ex-vivo release kinetics data showed that linagliptin-loaded nanosuspension was best fitted to Korsemeyer-Peppas (K-P) model (r2=0.9961, K= 4.1947, n=0.3508) with fickian diffusion release pattern as shown in Figure 8(b).

[0085] Sterility test
The tests for sterility were done by detecting the presence of viable forms of bacteria, fungi and yeast in or on preparations. The tests were carried out under strict aseptic techniques to avoid accidental contamination of the preparation. Optimized linagliptin loaded nanosuspension 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 sterility studies concluded that after 14 days of incubation period; there was no appearance of turbidity, which indicated that the optimized formulation LS1 was sterile. The formulation passes the test for and hence it can be used for nasal administration.
[0086] In-vivo studies for optimized linagliptin loaded chitosan nanosuspension

The experimental protocol for in-vivo pharmacodynamic studies was 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 Sprague Dawley rats (equal sex ratio; weighing about 200-250 gm) were divided into four groups each containing six rats and were kept under controlled conditions of temperature (25 ± 2oC) and humidity (55 ± 5 % RH).?
a) ‘Group I’ or ‘control’ served ‘healthy rats’ that received normal saline intraocularly.?
b) ‘Group II’ or ‘negative control’ served ‘dementia induced rats’ received normal saline?c) ‘Group III’ animals served ‘dementia induced rats’ treated with plain linagliptin solution (0.5 mg/ml).?
d)‘Groups IV’ animals served ‘dementia induced rats’ treated with linagliptin loaded nanosuspension (0.5 mg/ml).
Dementia was induced in animal groups II, III, and IV using aluminium chloride (AlCl3). They were given food and drinking water ad libitum. A 12 h day and night cycle was maintained 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 factor of 0.018.
a) Adult dose of linagliptin is 5 mg?
b) Therefore, for 250 g rat, linagliptin dose calculated = 5 x 0.018 mg. = 0.09 mg
c) Linagliptin dose for rats was calculated to be 0.45 mg/kg body weight.
[0087] In-vivo pharmacodynamic studies?
The in-vivo pharmacodynamic studies for spatial learning, memory deficit and behavioral study was done using the Morris water maze test, Y-maze and open field test in rat model (Sprague dawley rats, 200-225gm).
[0088] Morris-maze water test (Behavioral Assay)
Linagliptin (0.5 mg/ml) and linagliptin loaded nanosuspension(0.5 mg/ml) was injected by intranasally to group II and group III animals respectively. 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 center 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.?
Morris water maze was performed to evaluate spatial memory in dementia-induced rats. Animals that showed any motor disabilities or difficulties in the learning of were excluded. The latency time was significantly higher in the negative control group (P < 0.01) than group III and IV animals which are treated with plain linagliptin and linagliptin nanosuspension. Group III and IV animals had significantly shorter escape latency times (44.2±7.278 and 34.7 ± 5.391 on day 4 respectively) than animals in the negative control group (P < 0.01) as shown in Table 1. Collectively, these data interpreted 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 (V ehicle) 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 Linagliptin
57.0 ± 5.586
53.2 ± 5.565 48.2 ± 5.269
44.2 ± 7.278

4
IV Linagliptin loaded nanosuspension (LS1) 49.3 ± 5.241 45.5 ± 4.806 40.5 ± 5.577 34.7 ± 5.391

[0089] Open-field test (Behavioral Assay)
Open field test is other test to monitor general motor activity, exploratory behavior in rat model to determine habituation learning in the open field by measuring locomotion and rearing measurements. Rats were placed individually in an open arena equipped with infrared photobeams to monitor mice behavior 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 fecal pellets. Arena is cleaned in between trials with virkon. The following activities were recorded with photobeams– (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 fecal drops.
Locomotion and rearing measurements are conventionally used to determine habituation learning in the open field exploration which are closely related to the hippocampus and its cholinergic input. Linagliptin nanosuspension treated animals evidenced more exploratory activity i.e. ambulation (72.46 ± 1.946) as compared to negative control group (31.51 ± 1.202) as tabulated in Table 2 correlating to increased activity in novel environment by rodents indicating anxiolytic effect which may act as additive factor in enhancing cognition. Behavioural activation noted by the novel environment is positively correlated with increased hippocampal ache levels.
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 LS1 a significant (p < 0.05) increase in ambulatory activity was noted.
Table 2. Open field test data in rat model

Group
Treatment
Activity on open field
Ambulations
Rearings

Self- Grooming

Faecal drop


Control or I Vehicle (normal) 80.18 ± 2.101 7.06 ± 0.350 12.58 ± 0.213 3.2 ± 0.141
Negative control or II Negative Control 31.51 ± 1.202 13.25 ± 0.441 2.383 ± 0.231 8.15 ± 0.372

III Plain Linagliptin 53.00 ± 2.131
12.28 ± 0.803
5.51 ± 0.331
5.15 ± 0.187
IV
Linagliptin nanosuspension (LS 1) 72.46 ± 1.946 8.00 ± 0.346
9.183 ± 0.343
4.1 ± 0.544

[0090] Y- maze Spontaneous Alternation Test (Behavioral Assay)
Y-maze test is a measure of spatial working memory and are used to evaluate exploratory behavior in rats. The Y-maze is three-arm horizontal maze with an angle of 120 degrees, with length of 28 cm, 6 cm width, and height of 18 cm. White polyvinyl plastic is used to construct maze walls and floor. 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 justvisited 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.
In Y-maze test, after three days administration a significant increase of spatial memory in animal treated with linagliptin loaded nanosuspension are shown Table 3. The total number of arm entries (NAEs) were not significantly different among all four groups. There was a decrease in the percentage of same arm returns (%SAR) linagliptin nanosuspension treated groups i.e group IV animals as compared to group II and III animals. linagliptin nanosuspension treated animals or group IV animals had the lowest %SAR which was more or less the same as that of vehicle group (positive control). The %AAR for linagliptin nanosuspension treated was high and comparable to that for the positive control group. 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 group IV animals (treated with linagliptin nanosuspension) and negative control groups which demonstrated an ability to enhance memory impaired by inducer in the spontaneous alternation performance in the Y-maze.
Table 3. Y maze test data in rat model
S.No. Groups Treatment
Arm entries Same arm entries Alternate arm returns
1 Control or I Normal saline
24.16 ± 0.752
6.5 ± 0.547 25.16 ± 1.169
2. Negative control or II Normal saline 5 ± 0.894
25.0 ± 1.414 7.83 ± 2.316
3. III Plain Linagliptin 11.5 ± 1.048
16 ± 1.414 13.66 ± 1.032
4. IV Linagliptin nanosuspension (LS1) 20.83 ± 1.471
7 ± 0.894
22.16 ± 1.940

[0091] Histopathological studies
The brain tissues and nasal mucosa were collected and fixed in 10 % formalin and embedded in paraffin. Sections (5 µm) were cut from the nasal mucosa and middle lobes of brain of each group more or less from similar positions. The paraffin embedded brain tissue sections (5 µm) were deparaffinized using xylene and ethanol. The deparaffinized sections were stained with haematoxylin and eosin. The histopathological changes were analysed under 20 X magnification. Microscopic change in the nasal tissues and brain tissues of treated animals were compared with the control animals.
The images of brain tissues of all animals from groups (II, III, IV) were observed for and compared for any changes with positive control group or group I. The obtained images of histopathological studies in brain are shown in Figure 9. Histopathological images showed that there was no microscopic change in the brain tissues, of group animals (Group III and IV) as similar to the control animals (Group I). The group III and IV animals showed improved brain microscopic images as compared to negative control (group II). Thus, the linagliptin loaded nanosuspension showed no toxicity or structural damage to brain region.
[0092] Hematological analysis
Blood samples were collected directly from the cardiac puncture in a tube containing EDTA (anticoagulant) for analysis of hematological parameters. Values of red blood cells (RBC), white blood cells (WBC), hemoglobin (Hb), Differential leucocyte counts (DLC), Total leucocyte counts (TLC) and platelets counts were determined and compared with control using an auto analyzer (Roche Integra, 400 Plus, Diagnostic Systems, IN, USA). The values of RBC, WBC, Hb, DLC, TLC and platelets in treated animals were compared to control animal.
Blood samples of group II and III animals were collected and compared with the controlled group for performing haematological studies. The values of Hb, RBC, WBC, DLC, TLC and platelets were found to be within normal range in treated animals (Group II and III) and no significant changes (p>0.05) was found on comparison to control animals (Group I) as shown in Table 4.
Table 4. Haematological 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±1.19 8 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 Linagliptin 10.6 ± 0.089 5.63 ± 0.175
5.95±0.4 03
20.38±0.3 18
54.83 ± 1.432

609.5 ± 3.619

IV Linagliptin nanosuspension (LS1) 12.11 ± 0.470 7.06 ± 0.350 7.2±0.06 3
30.66± 1.861 64.75 ±1.179
789.5 ± 3.391

[0093] Statistical analysis
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 pharmacodynamic models.
[0094] Gamma scintigraphy studies
In order to visualize localization of drug in brain via intranasal route administrations, gamma scintigraphic studies were done on Albino New Zealand rabbits which were carried out in in the Institute of Nuclear Medicine and Allied Science (INMAS), Defence Research and Development Organisation Delhi (DRDO), India (Regd. No. INM/IAEC/19/01). Linagliptin and linagliptin loaded nanosuspension was labeled with 99mTc by the stannous reduction method. Rabbits for gamma scintigraphic studies were divided into two groups
a) Group I animals served as standard and received 99mTc labelled linagliptin (0.5 mg/ml)?
b) Groups II animals served as test and received 99mTc labelled linagliptin loaded nanosuspension
The nasal cavity has about a total volume of 15–20 ml with a total surface area of 150 cm2; the volume that can be delivered to the nasal cavity is therefore limited to 25–200 microlitre. Group I animals received 0.1 ml (100 microlitre) of solution in each nostrils and this was compared with Group II animals.
Gamma scintigraphic images in Figure 10 (a) and Figure 10 (b), gives a clear distinction between the intranasal administration of 99mTc labelled linagliptin solution and linagliptin loaded nanosuspension. Study showed less amount of drug reached to brain region in Tc-99m labeled linagliptin solution as shown in in Figure 10 (a). Whereas the amount of entrapped drug in Tc-99m labeled nanosuspension (LS1) was enhanced in target area i.e., to brain. Thus results demonstrated better localization of the radiolabeled drug complex compared to radiolabelled plain drug solution.
[0095] Accelerated stability studies according to ICH Q1A (R2) guidelines
As per the ICH guidelines, three batches of optimized chitosan nanosuspension 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 linagliptin loaded nanosuspension exhibited good physical stability over a storage period of 6 months (180 days). The samples showed clear- colored appearance with no significant changes in the evaluation parameters of the optimized nanoformulation under any of the storage conditions as shown in Table 8. Stability parameters were statistically analysed by one way analysis of variance (ANOVA) and multiple comparison test using GraphPad Instat software (GraphPad Software Inc., CA, USA). Results confirmed that changes in the observed parameters were not found to be statistically significant (i.e. P > 0.05) that indicates good stablity of optimized formulation (LS1) for period of 6 months.
Table 5. Stability studies parameters on optimized linagliptin loaded nanosuspension at different temperature and humidity
Time (Days) Storage conditions % Drug Content
% Drug entrapment % Drug loading
% Drug release
0 4oC 98.39 ± 1.24
95.8 ± 1.45 35.78 ± 0.19
89.65 ± 0.82
30 98.16 ± 1.32
95.33 ± 1.71
35.81±1.44
89.77 ± 2.03

60 98.66 ± 1.54 95.81 ± 2.33 35.83 ± 2.71 89.43 ± 1.05
90 98.18 ± 1.28
95.33 ± 1.21 35.44 ± 1.33
89.88 ± 1.25
120 97.11 ± 1.36
94.61 ± 1.39
35.74 ± 1.74
88.41 ± 2.34

150 97.21 ± 2.06 94.45 ± 2.01 34.10 ±1.37 88.62 ± 1.81
180 97.64 ± 2.73
94.71 ± 2.74 34.26 ± 2.36
88.27 ± 2.83
0 40 ± 2 oC and?75 ± 5% RH 98.39 ± 1.24
95.8 ± 1.45
35.78 ± 0.19
89.65 ± 0.82

30 98.84 ± 2.10 95.29 ± 1.66 35.33 ± 1.32
89.44 ± 1.73
60 98.55 ± 1.94 95.19 ± 2.32
35.77 ± 2.43 89.37 ± 1.47

90 98.34 ± 2.35 95.92 ± 1.53 35.76 ± 1.14 89.65 ± 1.17
120 97.50 ± 2.17
94.39 ± 1.16
34.28 ± 1.75
88.63 ± 1.32

150 97.18 ± 1.88 94.83 ± 2.08 34.11 ± 1.16 88.73 ± 2.31
180 97.48 ± 1.75 94.19 ± 1.01
34.65 ± 2.74 88.56 ± 2.01
0 25 ± 2 oC and?60 ± 5% RH 98.39 ± 1.24
95.8 ± 1.45
35.78 ± 0.19
89.65 ± 0.82

30 98.66 ± 2.96 95.32 ± 1.27 35.17 ±1.73 89.63± 1.03
60 98.53 ± 2.67 95.17 ± 2.63
35.53±1.54 89.31 ± 2.34

90

98.46 ± 1.05
95.43 ± 1.14 35.81 ±2.03 89.73 ± 1.34
120 97.54 ± 2.53 94.67± 1.53 34.13 ±1.30 88.76 ± 2.54
150 97.76 ± 2.38
94.91± 2.28 34.79 ±2.64
88.35 ± 2.15
180 97.14 ± 1.05
94.63 ± 1.10
34.79 ±1.86
88.23 ± 1.32

,CLAIMS:I CLAIM:

1. A nanosuspension formulation for nose to brain delivery/nasal administration to a subject, the nanosuspension formulation comprising:
a drug in nanoparticulate form;
a polymer; and
a surfactant.
2. The formulation as claimed in claim 1, wherein the drug is a gliptin, selected from linagliptin, sitagliptin, saxagliptin, vildagliptin, alogliptin.
3. The formulation as claimed in claim 1, wherein the drug is linagliptin.
4. The formulation as claimed in claim 1, wherein the polymer is a mucoadhesive polymer selected from chitosan, pullulan, hydroxyl propyl methyl cellulose, sodium alginate and starch.
5. The formulation as claimed in claim 1, wherein the polymer is chitosan.
6. The formulation as claimed in claims, wherein the surfactant is TWEEN 80.
7. The formulation as claimed in claim 1, wherein the drug comprises 0.01 % wt to 0.10 % wt of the formulation.
8. The formulation as claimed in claim 1, wherein the polymer comprises 0. 01 % wt to 0.10 % wt of the formulation.
9. The formulation as claimed in claim 1, wherein the surfactant comprises 0.005 % wt of the formulation.
10. The formulation as claimed in claim 1, wherein the formulation comprises of nanoparticulate drug of average size of 250.7 nm.
11. A method of providing the nanosuspension formulation as claimed in claim1 for nose to brain delivery/nasal administration to a subject for treatment of a neurodegenerative disorder like dementia.

Dated this 24th day of August 2020


Aayushi Mishra
Agent for the applicant
IN/PA- 2368