WO2023166528A1

WO2023166528A1 - "formulation for nasal delivery of insulin and other proteins and peptides"

Info

Publication number: WO2023166528A1
Application number: PCT/IN2023/050190
Authority: WO
Inventors: Dr. K. Koteswara RAO; Dr. Anil Kumar VEMUNOORI; Mohamed Arshad ALI
Original assignee: Transgene Biotek Limited
Priority date: 2022-03-02
Filing date: 2023-03-01
Publication date: 2023-09-07

Abstract

The present invention relates to a pharmaceutical preparation for nasal insulin administration in diabetic population comprising a polypeptide hormone insulin, or an analogue thereof, and a quaternised and thiolated Chitosan nano-particulate liquid formulation; wherein Insulin or its analogues is encapsulated in Trimethylated and thiolated chitosan.

Description

“FORMULATION FOR NASAL DELIVERY OF INSULIN AND OTHER PROTEINS AND PEPTIDES”

Field of Invention

The present invention in general relates to a pharmaceutical preparation and method thereof for an effective nasal administration of drug in both men and women. The invention particularly refers to liquid or semi liquid pharmaceutical formulation comprising insulin or its analogues, process thereof and a method of treating diabetes by administering a pharmaceutical preparation of the present invention.

Background of the Invention

With the advancement of recombinant DNA technology and largescale production of recombinant proteins, there is an ever increasing list of bio-pharmaceutical products that are available for administration to patients. Unfortunately, the administration of these molecules must generally be via injections either once, twice or thrice weekly, depending upon the molecule and dose, or, in the case of insulin, up to four times daily. The administration of these molecules via the sub-cutaneous route has many disadvantages, including pain and swelling at the site of injection, highly variable intra and inter-subject variability in dosing, and large fluctuations in the serum profile of the sub-cutaneous administered drugs.

Administration of these proteins via alternative routes is precluded by the very low bioavailability of these molecules when given orally, transdermally, nasal or via pulmonary administration. Despite this limitation, there are obvious benefits to nasal delivery of the aforementioned molecules over all the other routes, including the ability to maintain a flatter serum profile of the administered protein, an increase in patient comfort and an increase in patient compliance. As such, the oral or nasal administration of proteins would be regarded as much more “patient friendly” and clearly more desirable than subcutaneous administration and other routes of administration. Insulin is a polypeptide hormone of 51 amino acids. It is synthesized in the pancreas and it functions as a physiological regulator of the carbohydrate metabolism in the body. For many years insulin has been therapeutically used in patients with diabetes mellitus, to lower increased blood sugar levels in these patients. Oral administration of insulin, though possible, not effectively feasible because of its peptide structure getting broken down in the gastro-intestinal tract hence administered through parenteral route such as by subcutaneous (SC) or intramuscular (IM) or sometimes by intra-venous routes and further, even if administered through oral roue it requires high amount of AMI which in this case being Insulin in the formulation. However, these invasive methods or oral route of administration are likely to be associated with delayed and irregular absorption from the site of injection, probably due to hexamer formation apart from considerable inconvenience like pain and irritation at the site of injection.

There are a number of advantages in delivering Insulin through nasal route - the nasal cavity is easily accessible for drug administration; the nasal epithelial tissue has a rich vasculature; the nasal route avoids degradation of the drug as in the gastro-intestinal tract etc. Furthermore, intranasal insulin administration would not only circumvent the procedure of injection, but will probably result in more predictable blood levels.

In principle, intranasal administration of insulin may lead to poor bioavailability with Insulin possessing high molecular weight and being hydrophilic and therefore, is hardly capable of passing through nasal epithelial barrier. This relatively poor nasal bioavailability can be overcome by using different methods such as absorption enhancing adjuvants and some other methods.

In the literature, many absorption enhancers have been described for nasal administration of insulin, including ionic and non-ionic surfactants such as bile salts (Gordon et al., Proc. Natl. Acad. Sci. U.S.A. 82 (1985) 7419-7421; EP-A-0 ill 841) and polyoxyethylene alcohol ethers (Hirai et al.. Int. J. Pharm. 9 (1981) 165-172; GB-A-1 527 605), fatty acids and phospholipids (Mishima et al., J. Pharmacobio-Dyn. 10 (1987) 624-631; Ilium et al., Int. J. Pharm. 57 (1989) 49-54; PCT/DK-87.00158), chelating agents such as EDTA (USA-4 476 116) , and fusidate derivatives such as STDHF (Longenecker et al., J. Pharm. Sci. 76 (1987) 351-355; Deurloo et al., Pharm. Res. 6 (1989) 853-856; Kissel et al., Pharm. Res. 9 (1992) 52-57; US-A-4 548 922).

As described in these publications all the said absorption enhancers resulted in an increased bioavailability of nasally administered insulin, but the reproducibility of the resulting insulin absorption profiles in the blood leaves to be desired. Moreover, many of these absorption enhancers are harmful to the nasal epithelial membranes (Wheatley et al., J. Controlled Rel. 8 (1988) 167-177; Ennis et al., Pharm. Res. 7 (1990) 468-475; Chandler et al., Int. J. Pharm. 76 (1991) 61-70), and various of these substances may seriously inhibit the natural movement of the cilia in the nose (Hermens et al., Pharm. Res. 7 (1990) 144-146; Merkus et al., J. Controlled Rel. 24 (1993), 201- 208). So far, the insulin absorption enhancers used, like bile acids and phospholipids have yielded results and side effects which were prohibitive for further development.

In order to provide a solution to the prior art problems, the present invention discloses a nasal formulation, methods, for administration of a pharmacologically active amount of drugs.

Summary of the Invention

The following presents a simplified summary of the different aspects of the invention. The summary is not to be construed as an extensive overview of the disclosure and it does not identify key/critical elements or delineate the scope of the invention. The sole purpose of the below summary is to present few concepts of the invention in a simplified form as a prelude to the more detailed description that is presented in detailed description of the invention.

The present invention provides a pharmaceutical preparation and methods for nasal delivery of drug.

One preferred aspect of the present invention is to provide a pharmaceutical preparation for nasal delivery of drug. In yet another aspect, the pharmaceutical preparation comprises insulin or an analogue thereof and a quaternised and thioloated chitosan nano-particulate liquid formulation.

In another preferred aspect, formulation of the present invention comprises of Insulin or its analogues encapsulated in Trimethylated and thiolated chitosan and its derivatives thereof having a specific particle size distribution profile. The formulation is provided as a clear aqueous solution containing nano particulates for intranasal administration to a subject in need thereof.

In yet another aspect, the pharmaceutical preparation of the present invention has polypeptide hormone insulin, or an analogue thereof at a concentration range of 50 to 100 IU and the quaternised and thiolated Chitosan nano-particulate has the concentration range of 50 to 100 pg.

In yet another aspect, the pharmaceutical preparation has polypeptide hormone insulin, or an analogue thereof and the quaternised thiolated Chitosan nano-particulate in the w/w ratio of 3.93:1.

Another preferred aspect of the invention is to provide a pharmaceutical preparation which comprises insulin such that insulin is substantially present as insulin monomer.

One more aspect of the present invention is to provide a pharmaceutical preparation comprising insulin analogue having a reduced capacity of self-association.

In a preferred aspect, the pharmaceutical preparation of the present invention is a liquid or semi liquid pharmaceutical preparation.

One more aspect of the present invention is the usage of a significantly less amount of Insulin during the preparation of formulation such that similar efficacy and bio-availability levels are derived as compared to the formulations using oral route of administration thereby cost of formulation is considerably reduced. One aspect of the present invention is to provide a process for the preparation of pharmaceutical preparation.

Another preferred aspect of the present invention is to provide a process for the preparation of the pharmaceutical preparation comprising the steps of (1) reductive methylation, (2) altering the degree of quaternization through a unique one-step method or by increasing the reaction time, (3) preparation of nano-particles thereof of a desired size.

One more preferred aspect of the process is that Tri-methylated Chitosan is having a degree of thiolation between 10 -500 mgs.

In a further aspect, the present invention provides for a kit.

In yet another aspect, the kit is utilized for delivering nasal formulation of insulin comprising insulin or an analogue thereof and a quatemised and thioloated chitosan nanoparticulate liquid formulation.

In one more aspect, a method of treating diabetes by administering a pharmaceutical preparation of the present invention is provided.

Another preferred aspect of the invention is a device for targeted delivery of the pharmaceutical preparation comprising of a dispenser for nasal administration containing a certain predetermined quantity of the pharmaceutical preparation.

DRAWINGS:

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. For the purpose of assisting 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. Figure 1. illustrates the comparative study between SC and intra-Nasal insulin delivery conducted on normal healthy adult SD rats.

Figure 2. illustrates the comparative study between SC and intra-Nasal Insulin delivery conducted on normal healthy adult SD rats.

Figure 3. illustrates comparative study between SC and intra-Nasal Insulin levels conducted on STZ induced diabetic adult SD rats.

Figure 4. illustrates a comparative study between SC and intra-Nasal glucose levels conducted on STZ induced diabetic adult SD rats.

Figure 5. illustrates comparative Octreotide concentration of SC and intra nasal formulation in animals.

Figure 6. illustrates glucose levels (mg%) following single Intranasal insulin (0.312 IU) and subcutaneous Insulin injection (0.125 IU).

Figure 7. illustrates comparative insulin level in comparison with subcutaneous (0.125 IU) with intra nasal insulin (0.312 IU).

DETAIUED DESCRIPTION OF THE INVENTION

In describing the invention, the following terminology will be used in accordance with 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. As used herein, the singular forms "a," "an," and "the" include plural reference unless the context clearly dictates otherwise.

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 other 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.

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.

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.

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. Numeric ranges are inclusive of the numbers defining the range.

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 of any exemplified term. Likewise, the invention is not limited to various embodiments given in this specification.

The detailed description of the present invention is not limited in its application merely to the exemplifications of construction and the arrangement of components set forth in the following description. The present disclosure is capable of encompassing other embodiments and of being practiced or of being carried out in various ways. Also, the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting.

Further, the use of terms “first”, “second”, and “third”, and the like, herein do not denote any order, quantity, or importance, but rather are used to distinguish one element from another.

The present invention relates to the delivery of drugs through nasal route to a targeted region of the nasal cavity preferably to the turbinate region. These drugs may include vaccines, Biologies and Hormones such as Insulin amongst others.

In one of the embodiments, the present invention relates to a nasal preparation.

In yet another embodiment, the nasal preparation comprises a pharmacologically active amount of insulin, or an analogue thereof, as the active agent.

In other embodiment, a pharmaceutical preparation for nasal insulin administration in diabetic population comprises a polypeptide hormone insulin, or an analogue thereof, and a quatemised and thiolated Chitosan nano-particulate liquid formulation; wherein Insulin or its analogues is encapsulated in Trimethylated and thiolated chitosan.

In other embodiment, the pharmaceutical preparation as claimed in present invention has polypeptide hormone insulin, or an analogue thereof at a concentration range of 50 to 100 IU and the quatemised and thiolated Chitosan nano-particulate has the concentration range of 50 to 100 pg.

In yet another embodiment, the pharmaceutical preparation as claimed in present invention has polypeptide hormone insulin, or an analogue thereof and the quatemised thiolated Chitosan nano-particulate in the w/w ratio of 3.93:1. In another embodiment, the pharmaceutical formulation comprises thiolated Chitosan is Tri-methylated Chitosan.

In another embodiment, the pharmaceutical formulation comprises Tri-methylated Chitosan having a degree of thiolation between 10 -500 mgs.

In another embodiment, the pharmaceutical preparation comprises Tri-methylated Chitosan which is 40 to 60 % quatemized.

In another embodiment, the pharmaceutical preparation has polypeptide hormone insulin, or an analogue thereof and the quaternised and thiolated Chitosan nano-particulate.

In another embodiment, the pharmaceutical preparation comprises insulin, which is present as insulin monomers having reduced capacity of self-association.

In another embodiment, the pharmaceutical preparation comprises insulin wherein the insulin is having a specific particle size (smaller and larger) ranging between 30 to 800 nm in size.

In another embodiment, the pharmaceutical preparation comprises insulin wherein insulin has two different particle size distribution profiles, one ranging between 30-80% of the drug substance particles having a particle size distribution between 30 to 110 nm, and another about 70-20% of the drug substance loaded particles have a particle size of 200- 800 nm.

In another embodiment, the pharmaceutical preparation further comprises excipient, wherein the excipient is selected from m-Cresol, Glycerol, Zinc oxide, Water for injections, Trehalose, Chitosan, STPP, Glutathione and Glacial acetic acid

In another embodiment, a process for preparing a pharmaceutical preparation for nasal administration is disclosed. In yet another embodiment, the process for preparation of pharmaceutical composition comprises the steps of:

(a) preparing a reduced chitosan solution (Chitosan high molecular weight,; MW = 310,000-375,000 Da; Viscosity 800-2000 cP, Deacetylation >75 %, Soluble in 1% w/w in 3% acetic acid) by dissolving a high molecular weight chitosan in glacial acetic acid, and reacting with sodium nitrite (NaNO₂) followed by adding Trehalose;

(b) sieving the product of step (a) to obtain a fraction of <0.4 micro meter of reduced chitosan;

(c) reacting the obtained reduced chitosan of step (b) by dissolved in 1 -methyl 2- pyrrolidinone (NMP) and synthesizing Trimethyl chitosan in the presence of sodium Iodide and Iodo methane in strong alkali conditions with 40% to 60% of quatemization;

(d) reacting the obtained Tri methyl Chitosan solution with thiolating substances to obtain tri-methyl and thiolated chitosan;

(e) process of reacting tri-methyl and thiolated chitosan solution of step (d) with sodium tripolyphosphate in 1:1 to 1:5 ratio to make nanoparticles of different sizes along with biologically active ingredient Insulin, and along with sodium caprate.

(f) the obtained nanoparticles were further curated by incubating and followed by addition of Trehalose.

In another embodiment, the process involves thiolating substances that are selected from glutathione, L-cysteine, co enzyme- A, selenocysteine, homo cysteine, lipoic acid, trypanothione, ergothioneine co enzyme- B and coenzyme-M. In another embodiment, a device for targeted delivery of the pharmaceutical preparation is disclosed.

In yet another embodiment, the device for targeted delivery of pharmaceutical formulation comprises a dispenser containing the pharmaceutical preparation.

In another embodiment, a method of treating diabetes is disclosed.

In another preferred embodiment, the method of treating diabetes involves administration of a pharmaceutical preparation wherein said method significantly reduces the Insulin usage.

In another embodiment, a kit for delivering a nasal pharmaceutical preparation is disclosed.

Furthermore, it was found that an excellent absorption can be obtained in case the insulin molecules are administered substantially as insulin monomers. Accordingly, it turned out that the quatemisation of Chitosan and preparation of nano-particles of a desired size played their part in the successful delivery of Insulin into systemic circulation after administering through nasal route without inducing aggregation.

The invention further provided evidence that liquid particulate formulation can be a successful delivery route, contrary to the belief that only dried or lyophilized powder particles can be used for successful delivery. Preparations in the form of a liquid particulate formulation suitable for nasal administration may for instance contain 2 to 200 IU of the active agent per nasal dose.

The nasal preparations according to the invention may also contain one or more adjuvants conventionally used in nasal drug formulations, such as preservatives, stabilizers, excipients (e.g.m-Cresol, Glycerol, Zinc oxide, Water for injections, Trehalose, Chitosan, STPP, Glutathione, Glacial acetic acid), pH-controlling compounds and complexing agents etc. Agents suitable for these and other purposes are known in the pharmaceutical literature and to men skilled in the art of nasal drug delivery. Number of nasal drug delivery devices are available to deliver the novel drug formulation to the nasal cavity in the form of drops or liquid particulate spray. If it is a requirement that drug is to be absorbed into systemic circulation, it is preferable to deposit the liquid particulate formulation around the turbinate region of the nasal passage more preferably on to turbinate region of the nasal cavity.

The present invention relates to a nasal formulation in the form of particulate liquid comprising of a drug substance having a specific particle size (smaller and larger) ranging between 30 to 800 nm in size. The final liquid particulate formulation is delivered through intra-nasal route using a specific delivery device.

In a preferred embodiment, formulation of the present invention comprises of Insulin or its analogues encapsulated in Trimethylated and thiolated chitosan and its derivatives thereof having a specific particle size distribution profile. The formulation is provided as a clear aqueous solution containing nano particulates for intranasal administration to a subject in need thereof.

Methods of making finely divided drugs have been studied and efforts have been made to control the size and size range of the encapsulated drug particulates in pharmaceutical compositions.

It is known that inhaled nasal insulin formulation are one of the most effective antidiabetic medications used in the treatment of uncontrolled diabetic mellitus to reduce and maintain blood glucose levels at optimal levels.

Like most hormones or biologically active peptides, in this case Insulin can undergo degradation due to proteolytic enzymes and pH changes if administered through gastrointestinal route. When these substances are administered intranasally via conventional nasal spray, less than optimal amount of drug substance is absorbed by the target tissue i.e., nasal mucosa, with the remainder being swallowed or expelled from the nasal cavity. To overcome this problem, active pharma ingredient is encapsulated in quatemised and thiolated chitosan derivative naturally possessing high mucosal adhesion and thiolated particles having specific particle size distribution resulting in increased bioavailability, increased bio-efficacy and prolonged therapeutic effect of the drug substance delivered intra nasally.

In the present invention, nanoparticles are generated by employing encapsulating agent as Trimethylated and thiolated modified chitosan derivatives. The formulation of the present invention comprises of a drug substance (Insulin-active ingredient) having following two different particle size distribution profiles, one ranging between 30-80% of the drug substance particles having a particle size distribution between 30 to 110 nm, and another about 70-20% of the drug substance loaded particles have a particle size of 200-800 nm.

Delivery efficiency seems strongly dependent on the nanoparticle sizes, small size rapid absorption through transcytosis and also lesser size, more surface area favours rapid absorption. Larger size nanoparticles absorb slowly, due its larger size and less surface area, which favours in increased residence time in the nasal cavity, increased efficiency of drug release, and improved bioavailability through the enhancement of transport of active ingredients across biological membranes.

As used herein particle size refers to an average particle size measured by conventional measuring techniques such as Zetasizer etc.

Mode of administration:

The pharmaceutical preparation of the present invention is administered as in any conventional manner, preferably by using a nasal device and preferably in such a way as to deliver a fixed, predetermined dose of the drug substance (e.g., active ingredient) directed at a specific intra-nasal area.

The following examples are provided to better illustrate the invention 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 still remaining within the bounds of the invention. It is the intention of the inventors that such variations are included within the scope of the invention.

EXAMPLES

Preparation of thiolated Trimethyl Chitosan solution (Stock)

High molecular weight chitosan was dissolved in Glacial acetic acid (50-100%) based on specific particle size profile requirement, with continuous stirring from 1-12 hours. 0.55 - 1 % Sodium nitrite (NaNO₂) is added to the solution till the desired particle size is achieved.

The Chitosan as in the above step reacting in sieved fractions of <0.4 micro meter reduced chitosan dissolved in 1 -methyl 2- pyrrolidinone (NMP) at 60 degrees Celsius and Trimethyl chitosan is synthesized as a single step process in the presence of sodium Iodide and Iodo methane in strong alkali conditions.

As another embodiment of the present invention, is one step method achieved in deriving Tri methyl chitosan possessing 10 to 40% of quaternization and its charge density, resulting in absorption enhancement hence requiring lesser amounts of peptide drugs in the present case being Insulin in slightly neutral environments (Nasal anionic epithelial cells).

Thiolation of Chitosan

The aim of the present investigation is to evaluate the effect of the degree of quaternization of TMC (high) on the in vivo absorption of insulin.

Tri methyl Chitosan (10 to 40 % quaternized) solution is treated with thiolating substances selected from glutathione, L-cysteine, co enzyme- A, selenocysteine, homo cysteine, lipoic acid, trypanothione, ergothioneine co enzyme- B and coenzyme-M. 2-10 ml of reduced Trimethyl chitosan solution from the stock (prepared in preceding paragraph) was solubilized by treating with l-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) (3 - 30 M) and N-Hydroxy succinimide (NHS) (10-40 M) for 30 to 60 minutes for activation and then glutathione (1-10 M) was added. The reaction is continued for 2-12 hours between 4° Celsius to 16° Celsius temperature achieving 40 to 60 % of thiolation on carboxylic ends of chitosan.

Quatemized derivative of chitosan, N-trimethyl chitosan chloride (TMC) is able to increase the paracellular transport and an effective absorption enhancer of peptide drugs such as Insulin. TMC has proven to be very soluble over a wide pH range (pH 1-9) up to 10% w/v concentrations, it was found that the charge density of TMC, as determined by its degree of quaternization, plays an important role in its ability to act as an absorption enhancer in neutral environments (Small intestinal area). The charge density of highly quatemized TMC is sufficient for interaction with the negative charged sites on the cell membranes and tight junctions. Thiolated chitosans have numerous advantageous features in comparison to unmodified chitosan, such as significantly improved mucoadhesive and permeation enhancing properties, make them highly suitable excipients for controlled drug release dosage forms. This contributes to increase in Bioavailability of Insulin in systemic circulation.

Preparation of Biologically Active Ingredient

Biologically Active Ingredient (Insulin) is to be weighted according to body weight/kg and dissolved in 0.1 N HCL prior to formulation process, store at 2 to 10 degrees Celsius.

Preparation of Sodium caprate

Sodium caprate (3 mM-18 mM /kg body weight) was dissolved in milli-Q water and added during the formulation preparation.

Preparation of STPP

Prepare freshly Img /ml of sodium tri-poly phosphate in Milli-Q water and filter through 0.2-micron filter to remove any sediments or particulates appearing in the solution. Keep it in refrigerator until further use.

Preparation of chitosan Nanoparticles encapsulating Biologically Active Ingredient such as Insulin

Thiolated/Tri-methyl chitosan solution prepared earlier was added to sodium tripolyphosphate (STPP) in 1:1 to 1:5 ratio (w/w) to make nanoparticles along with biologically active ingredient such as Insulin and along with sodium caprate solution prepared in the preceding paragraph added in a drop wise manner for 4 to 10 minutes with continuous stirring for 30 - 60 min at low temperatures on cooling magnetic stirrer. The nanoparticles were further curated by incubating at 4°-16° C for 4-18 hours, followed by addition of 0.02 to 0.05 mg /ml of Trehalose.

According to the outcome of numerous in-vitro experiments, pH 6.2 was investigated as most suitable for the polymer solution whereas pH 5 appears to be most favourable for the TPP solution. The ideal polymer: crosslinker ratio was 1:1 to 1:5 with a positive zeta potential of +24 + 2. Positive zeta potential of these particles can give rise to a strong electrostatic interaction with negatively charged mucus layer.

Methodology

(a) Prepare a reduced chitosan (90 to 250 nm) solution by dissolving a high molecular weight chitosan in glacial acetic acid, based on specific particle size profile requirement, continuous stirring for up to 12 hours and reacting with sodium nitrite (NaNO2) for 16 hours at 1200 rpm add Trehalose.

(b) Progress to reacting the resultant sieved fractions of <0.4 micro meter reduced chitosan dissolved in 1 -methyl 2- pyrrolidinone (NMP) at 60° C and synthesized Trimethyl chitosan in the presence of sodium Iodide and Iodo methane in strong alkali conditions in a step method achieved Tri methyl chitosan, one with 10 to 40% of quaternization and its charged density. (c) Tri methyl Chitosan solution was treated with thiolating substances selected from glutathione, L-cysteine, co enzyme- A, selenocysteine, homo cysteine, lipoic acid, trypanothione, ergothioneine co enzyme- B and coenzyme -M. 2-10 ml of reduced Trimethyl chitosan solution from the stock (prepared in preceding paragraph) was solubilized by treating with l-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) (3 -30 M) and N-Hydroxy succinimide (NHA) (10-20 M) for 30 to 60 minutes for activation and then glutathione (1-10 M) was added. The reaction was continued for 2-12 hours at 4° Celsius or 16° Celsius temperature achieved 40 to 60 % of thiolation on carboxylic ends of chitosan.

(d) Progress to reacting tri-methyl and thiolated chitosan solution of step *(c) with sodium tripolyphosphate in 1:1 to 1:5 ratio) to make nanoparticles different sizes along with biologically active ingredient such as Insulin, prepared in the preceding paragraph added in a drop wise manner along with sodium caprate for 4 to 10 minutes with continuous stirring for 30 - 60 min at low temperatures on cooling magnetic stirrer. The nanoparticles were further curated by incubating at 4°-16° Celsius for 4-18 hours, followed by addition of 0.02 to 0.05 mg /ml of Trehalose.

Final formulation after completion has been subjected to a number of studies as outlined below.

STUDY -1

Intra Nasal delivery of Insulin

Nasal Formulation of Insulin was prepared using recombinant human insulin (Sigma Aldrich) as per the approved study protocol.

Normal healthy animals were selected and acclimatized for a minimum five days prior to initiation of study. The animals had ad libitum access to a certified rodent diet and water. Environmental controls for the animal room were set to maintain a temperature of 22 to 25°C, humidity of 40-70% RH, and a 12-hour light /12-hour dark cycle. Animals were dosed intra nasally 50 to 100 μL of the nasal formulation) was administered via a PE 10 tube attached to a microliter syringe inserted half cm into right nostril of rats. After collection of blood samples at each time points 5 min, 15 min, 30 min, 1, 2, 4, 6, 8 hours, collected blood samples were stored on ice, prior to centrifugation. Blood samples were centrifuged to separate plasma. Centrifugation conducted at 2500 x g for 15 minutes at 4°C. Insulin levels in the plasma were measured using respective Rat Insulin ELISA kits (Mercodia).

Conclusion:

In Intra Nasal Insulin administered rats, there is a significant reduction in blood glucose levels corresponding to increased serum insulin levels within the first 15 minutes of administration with a further fall at the end of 30 minutes and sustained for 4 hours, longer time than SC administered Insulin.

TABLES

Table: 1

[Figure 1]

Table 2:

[Figure 2]

STUDY-II:

Plasma pharmacokinetic study of Nozulin Intra nasal formulation: Single dose Subcutaneous and Intra-nasal administration of Intra nasal formulation in male STZ induced diabetic Sprague dawley rats and observe, changes in the blood glucose concentration verses insulin bioavailability

SD rat (200-250g) were housed at a room temperature of 24+2 Oc with 12 h light /dark cycle and 40-50% relative humidity. The animals had ad libitum access to a standard diet and water except, wherever indicated. After randomization into various groups, the rats are acclimatized for a period of 3-4 days in a new environment, before initiation of the experiment.

Induction of diabetes

Strep tozotocin is dissolved as 45-55 mg/ml in 0.1 M citrate buffer PH 4.0 and sonicated to make sure of even dissolution. To induce diabetes, freshly prepared streptozotocin solution is administered intraperitoneally to the rats (200 to 250 gm) at a dose range of 45-55 mg/kg.

A series of biochemical indicators were measured including serum glucose and insulin levels. Administration of STZ (45-55 mg/kg b.w. I.P.) has induced hyperglycemia (blood glucose level > 450 mg/dl) in almost all treated rats. The blood glucose level remained fairly stable between 400 and 450 mg/dl prior to dosing.

STZ induced diabetic SD rats were selected and acclimatized for a minimum five days prior to initiation of study. The animals had ad libitum access to a certified rodent diet and water. Environmental controls for the animal room were set to maintain a temperature of 22 to 25°C, humidity of 40-70% RH, and a 12-hour light /12-hour dark cycle.

Animals were dosed intra nasally 50 to 100 pL of the nasal formulation) was administered via a PE 10 tube attached to a microliter syringe inserted half cm into right nostril of rats. After collection of blood samples at each time points 5 min, 15 min, 30 min, 1, 2, 4, 6, 8 hours, collected blood samples were stored on ice, prior to centrifugation. Blood samples were centrifuged to separate plasma. Centrifugation conducted at 2500 x g for 15 minutes at 4°C. Insulin levels in the plasma were measured using respective Rat Insulin ELISA kits (Mercodia).

Results:

[Figure 3]

[Figure 4]

Conclusion:

PK study suggests that a significant increase of serum Insulin concentration within 15 minutes and Cmax was observed at 30 minutes caused reduction of blood glucose levels appeared until 4 hours. In the control group, their blood glucose level declined remarkably until 2 hours. 70.3 % Insulin Bioavailability is observed at 30 minutes.

STUDY -III

Plasma pk study of Intra-nasal Octreotide formulation: single dose sub-cutaneous and Intra-nasal administration of Intra nasal Octreotide in male healthy SD rats.

Nasal Formulation of Octreotide was prepared using recombinant Octreotide (MSD, USA) as per the approved study protocol.

Normal healthy animals were selected and acclimatized for a minimum five days prior to initiation of study. The animals had ad libitum access to a certified rodent diet and water. Environmental controls for the animal room were set to maintain a temperature of 22 to 25°C, humidity of 40-70% RH, and a 12-hour light /12-hour dark cycle.

Animals were dosed intra nasally 50 to 100 pL of the nasal formulation) was administered via a PE 10 tube attached to a microliter syringe inserted half cm into right nostril of rats. After collection of blood samples at each time points 5 min, 15 min, 30 min, 1, 2, 4, 6, 8 hours, collected blood samples were stored on ice, prior to centrifugation. Blood samples were centrifuged to separate plasma. Centrifugation conducted at 2500 x g for 15 minutes at 4°C. Octreotide levels in the plasma were measured using respective Octreotide ELISA kits (BMA Biomedicals).

[Figure 5]

Conclusion:

Octreotide Intra-nasal formulation provided markedly enhanced intranasal absorption of Octreotide peptide, revealing 2-3-fold improvement through Nasal route using our novel formulation with relative pharmacological availability as compared to subcutaneous injection.

STUDY IV

In-Vivo Studies to estimate PK and Bio-availability profile following single dose of nasal Insulin formulation in male and female STZ induced diabetic Sprague Dawley rats and observe, changes in the blood glucose and serum Insulin concentrations with simultaneous sub-cutaneous route of administration to be used as controls.

Nasal Formulation of Insulin was prepared using recombinant human insulin (Sigma Aldrich) as per the approved study protocol. SD rats (200-250g) were housed at a room temperature of 24+2 Oc with 12 h light /dark cycle and 40-50% relative humidity. The animals had ad libitum access to a standard diet and water except, wherever indicated. After randomization into various groups, the rats are acclimatized for a period of 3-4 days in a new environment, before initiation of the experiment.

Induction of diabetes

STZ induction has been carried out as per the established protocols and a series of biochemical indicators were employed to measure serum glucose and insulin levels. The blood glucose levels have been stabilised to range between 400 and 450 mg/dl prior to initiation of dosing.

Finally, 10 STZ induced diabetic SD rats comprising of two sets of equal number of male and female rats were selected and acclimatized for a minimum five days prior to initiation of dosing. The animals had ad libitum access to a certified rodent diet and water. Environmental controls for the animal room were set to maintain a temperature of 22 to 25°C, humidity of 40-70% RH, and a 12-hour light /12-hour dark cycle.

Blood samples from all animals were collected prior to administration of dosing.

On the day of study 4 (four) animals were administered Insulin formulation comprising of 0.125 IU/ (4.3 mcg/dose) by sub-cutaneous route and 6 (six) animals, comprising of two sets of equal number of male and female rats were administered insulin formulation comprising of 0.312 IU (10.7 mcg/dose) , dosed intra nasally in the form of 50 to 100 pL of the nasal formulation) was administered via a PE 10 tube attached to a microliter syringe inserted half cm into right nostril of rats. After collection of blood samples at each time points 15 min, 30 min, 1, 2, 4, 6, 8 hours, collected blood samples were stored on ice, prior to centrifugation. Blood samples were centrifuged to separate plasma. Centrifugation conducted at 2500 x g for 15 minutes at 4°C. Insulin levels in the plasma were measured using respective Rat Insulin ELISA kits (Mercodia). On the day of study, second group of STZ induced 6 diabetic rats included in the study with 3 males and 3 females were administered Insulin formulation comprising of 0.312 IU/ (10.7 mcg/dose) by intra-nasal route in the form of 50 to 100 pL of the nasal formulation) administered via a PE 10 tube attached to a microliter syringe inserted half cm into right nostril of rats. After collection of blood samples at each time points 15 min, 30 min, 1, 2, 4, 6, 8 hours, collected blood samples were stored on ice, prior to centrifugation. Blood samples were centrifuged to separate plasma. Centrifugation conducted at 2500 x g for 15 minutes at 4°C. Insulin levels in the plasma were measured using respective Rat Insulin ELISA kits (Mercodia).

Results:

[Figure 6]

Serum Insulin Levels:

[Figure 7]

Conclusion

The study demonstrated that the plasma glucose has dropped within 15 minutes of the nasal administration similar to SC administered controls.

Plasma Glucose reduction has sustained without swings for 4 hr period following intranasal administration unlike raising of glucose levels from 2 hr period in SC controls.

Peak serum Insulin levels have been observed at 30 min time point in nasal administered rats compared to 15 min time point in those SC administered controls.

Results of the administrations.

As will be clear from all the above studies on different models using Insulin (Tri- Methylated Chitosan Thiolated nano-particulate formulations demonstrated blood glucose reduction following nasal administration is comparable to that of sub-cutaneously administered Insulin preparation. Further, nasal instillation of the liquid particulate formulation resulted in remarkable increased plasma insulin levels.

Administration of the liquid particulate formulation is highly effective resulting in peak Insulin concentration within 30 minutes of administration and gradual, sustained reduction of serum glucose commencing within 15 minutes of nasal administration, the level of such reduction comparable to sub-cutaneous route of administration without any fluctuations or swings.

The combination of all the factor mentioned above results in usage of extreme low amount of API within the limits more preferably between 1.5x to 3x compare to Subcutaneous 0.5 x/per kg body weight. We achieved rapid and long-acting insulin bioavailability in the systemic circulation, which helped in significant reduction of blood sugar levels for longer time, which is depicted in experimental results It is evident contrary to the common belief; liquid particulate delivery is highly effective with comparable bio-efficacy to that of sub-cutaneous administration of Insulin. No morbidities (side effects) or mortality in rats were reported.

Claims

CLAIMS:

1. A pharmaceutical preparation for nasal insulin administration in diabetic population comprising a polypeptide hormone insulin, or an analogue thereof, and a quaternised and thiolated Chitosan nano-particulate liquid formulation; wherein Insulin or its analogues is encapsulated in Trimethylated and thiolated chitosan.

2. The pharmaceutical preparation as claimed in claim 1, wherein the thiolated Chitosan is Tri-methylated Chitosan.

3. The pharmaceutical preparation as claimed in claim 2, wherein the Tri-methylated Chitosan is having a degree of thiolation between 10 -500 mgs.

4. The pharmaceutical preparation as claimed in claim 2, wherein the Tri-methylated Chitosan is 40 to 60 % quatemized.

5. The pharmaceutical preparation as claimed in claim 1, wherein said pharmaceutical preparation has polypeptide hormone insulin, or an analogue thereof at a concentration range of 50 to 100 IU and the quaternised and thiolated Chitosan nano-particulate has the concentration range of 50 to 100 pg.

6. The pharmaceutical preparation as claimed in claim 1, wherein said pharmaceutical preparation has polypeptide hormone insulin, or an analogue thereof and the quaternised thiolated Chitosan nano-particulate in the w/w ratio of 3.93:1.

7. The pharmaceutical preparation as claimed in 1, wherein the insulin is present as insulin monomers having reduced capacity of self-association.

8. The pharmaceutical preparation as claimed in 1, wherein the insulin is having a specific particle size (smaller and larger) ranging between 30 to 800 nm in size.

9. The pharmaceutical preparation as claimed in 1, wherein insulin may contain two different particle size distribution profiles, one ranging between 30-80% of the drug substance particles having a particle size distribution between 30 to 110 nm, and another about 70-20% of the drug substance loaded particles have a particle size of 200-800 nm.

10. The pharmaceutical preparation as claimed in claims 1-10, further comprising excipient, wherein the excipient is selected from m-Cresol, Glycerol, Zinc oxide, Water for injections, Trehalose, Chitosan, STPP, Glutathione and Glacial acetic acid or combinations thereof.

11. A process for preparing a pharmaceutical preparation for nasal administration as claimed in claim 1, comprising the steps of:

(a) preparing a reduced chitosan solution by dissolving a high molecular weight chitosan in glacial acetic acid, and reacting with sodium nitrite (NaNO2) followed by adding Trehalose;

(e) process of reacting tri-methyl and thiolated chitosan solution of step (d) with sodium tripolyphosphate in 1:1 to 1:5 ratio to make nanoparticles of different sizes along with biologically active ingredient Insulin, and along with sodium caprate;

12. The process as claimed in claim 11, wherein thiolating substances are selected from glutathione, L-cysteine, co enzyme- A, selenocysteine, homo cysteine, lipoic acid, trypanothione, ergothioneine co enzyme- B and coenzyme-M.

13. A device for targeted delivery of the pharmaceutical preparation comprising a dispenser containing the pharmaceutical preparation as claimed in claims 1-10.

14. A method of treating diabetes by administering a pharmaceutical preparation as claimed in claims 1-9, wherein said method significantly reduces the Insulin usage.

15. A kit for delivering a nasal pharmaceutical preparation as claimed in claims 1-10.