INTRODUCTION:

In recent years many drugs have been shown to achieve better systemic bioavailability through nasal route than by oral administration. In recent years, the nasal mucosa has been considered as an administration route to achieve faster and higher level of drug absorption. The rich vascular nature of the nasal mucosa coupled with its high drug permeation makes the nasal route of administration attractive for many drugs, including proteins and peptides. In addition, absorption of drug at the olfactory region of the nose provides a potential for a pharmaceutical compound to be available to the central nervous system (Chien et al., 1989).

The nasal delivery of vaccines is another very attractive application in terms of efficacy and patient acceptance. The replacement of injection therapy by "nonparenteral application routes" is an area of intensive research efforts.

The rapid onset of action and relatively high bioavailabilities favour this route of application for these drug substances (Kadam et al., 1993).

Insulin is a peptide hormone composed of 51 amino acid residues and has a molecular weight of 5808 Da. It is produced in the Islets of Langerhans in the pancreas. It is a hormone that has extensive effects on metabolism and other body functions. It causes cells in the liver, muscle and fat tissue to take up glucose from the blood, storing it as glycogen in the liver and muscle, and stopping the use of fat as an energy source. It is used medically to treat some forms of Diabetes Mellitus. Patients with type1 diabetes mellitus depend on external insulin for their survival because the hormone is no longer produced internally. Some patients with type2 diabetes may eventually require insulin when other medications fail to control blood glucose levels adequately.

Gels are advantageous as compared to other semisolid formulations due to reasons like ease of application, good spreadability, greaseless and can be easily removed, nonstaining, greater dissolution of a drug possible due to higher aqueous component, superior in terms of use and patient acceptability and superior optical clarity

Currently, insulin administration requires subcutaneous (sc) injection, which even in the simplest form (Nova-Pen system, Novo Nordisk, Bagsvaerd, Denmark) is cumbersome and unacceptable to many patients with diabetes. However it is every patient’s dream to have access to insulin without the pain of injection. This desire has motivated the search for novel therapeutic approaches to replace the present parenteral insulin delivery (William et al., 2004).

Ideally, an oral insulin dosage form would be preferred over the currently available parenteral route of administration, but this novel approach is confronted by common biological and physicochemical problems such as luminal degradation, particle aggregation, and polypeptide degradation in the absorptive area of the gastrointestinal tract. Several new and alternative routes including nasal, pulmonary, buccal, ocular, rectal, vaginal, transdermal, and others have also been explored for noninvasive delivery of insulin. Out of them nasal route of administration is one of the promising noninvasive routes of administration owing to its high vasculature and other associated advantages like absence of first pass and pH related degradation of Insulin (Heinemann et al., 2001).

MATERIALS AND METHODS:

Materials:

Human insulin was a gift from Torrent pharmaceuticals ltd, Ahmedabad. Chitosan (low molecular weight) was kindly given by Indian Fisheries department, Kerala. Glucose oxidase kit, Ethylene diamine tetra acetic acid, sodium hydroxide flakes, sodium chloride were purchased from Himedia chemicals ltd. Glycerol, ethanol (absolute), acetic acid and concentrated hydrochloric acid were purchased from sd-fine chemicals ltd.

Equipment:

HPLC, Shimadzu, LC 10 AT, Japan,

UV- Visible Spectrophotometer (Elico instruments),

Centrifuge (Remi instruments),

pH meter (Global instruments),

Micro pipettes (Finn pipettes, Mumbai),

Magnetic stirrer (Remi instruments),

Bath sonicator-Model D150H of mrc-Ultrasonic cleaner, Israel.

Preparation of the gel:

Weighed quantity of Insulin was dissolved in an aqueous 3% (v/v) acetic acid solution. To the clear solution the polymer i.e. chitosan was added gradually and stirred gently for 20 min and it was kept aside for 1hr to allow the polymer to swell. Then to this the permeation enhancer i.e. Hydroxy prophyl beta cyclodextrin was added and stirred gently for 10min. Then this preparation was sonicated on a bath sonicator to remove the air bubbles if any. In order to optimize the formulation three formulations were prepared differing in the concentration of the polymer chitosan (Jaleh et al., 2006).

Invitro drug release studies of the gels:

1 ml of the gel was taken into a small test tube. The open end of the test tube was closed with the dialysis membrane (DM 70) with molecular weight cut off 12000, by tying it with a thread. Then this was placed in a beaker containing the diffusion media i.e. phosphate buffer of pH-6 of volume 100ml such that the membrane was dipped into the media. Then at regular intervals of time i.e. 0.25hr, 0.5hr, 1hr, 1.5hr, 2hr, 3hr, 4hr, 6hr and 8hr samples of volume 2ml were withdrawn using tuberculin syringe and then replaced using the media. The samples then were analyzed using HPLC at the wavelength of 214nm (Reshma et al., 2005).

Invitro permeation studies of gels:

Invitro permeation of insulin from chitosan gel through pig nasal membrane was studied. Pig nasal portion was obtained and nasal membrane was isolated. 1 ml of the gel was taken into a small test tube. The open end of the test tube was closed with the nasal membrane of the pig by tying it with a thread. Then this was placed in a beaker containing the diffusion media i.e. phosphate buffer of pH-6 of volume 50ml such that the membrane was dipped into the media. Then at regular intervals of time i.e.0.25hr, 0.5hr, 1hr, 1.5hr, 2hr, 3hr, 4hr, 6hr and 8hr samples of volume 2ml were withdrawn using tuberculin syringe and then replaced using the media. The samples then were centrifuged using Microcentrifuser to separate tissue components and analyzed by HPLC at the wavelength of 214nm (Vamshi et al., 2010).

Chromatographic Conditions:

Mobile Phase: Sodium sulphate Buffer: Acetonitrile: Triethylamine- 70:30:0.2

Flow rate : 1ml/min.

Sensitivity : 0.0008 AUFC

Wave length : 214nm.

Retention time : 4.85min.

Column : C18 ODS RP

Shimadzu HPLC Unit:

Solvent delivery system: LC-10AT

Detector: SPD-10AVP UV-Visible

Data processor: LC-10 Software

Injector port: Reodyne20µl capacity loop

Column: Phenomenex C₁₈ ODS (SS) 10cm length, 4.6mm ID packed with porous silica Spheres of 5µ diameter 100A⁰ pore diameter.

Invivo bioavailability studies:

The invivo studies were conducted in rabbits for which the permission was obtained from the Animal Ethical Committee of Kakatiya University. The invivo studies were conducted in rabbits. Rabbits were selected for this study because of the ease of administration of the nasal preparation and to avoid the sacrifice of the animal. The six animals that were procured were divided into two groups each group containing three animals. To one of these groups the nasal gel (150μl containing 6IU of insulin) was administered and to the other group a subcutaneous marketed product (75μl containing 3IU of insulin) was injected. The marketed product was Human Actrapid solution. Then the blood samples before the treatment and after the treatment were collected from the marginal ear vein of the rabbit. The serum of these blood samples was obtained by centrifugation at 3000rpm for 0.5hr. The blood glucose levels of these samples were estimated by glucose oxidase method using glucose oxidase kits. The color that was produced was analyzed using spectrophotometer at 505nm wavelength (Abdol et al., 2004).

RESULTS AND DISCUSSION:

Standard graph of insulin in glacial acetic acid by HPLC:

Standard graph of insulin was constructed in glacial acetic acid by taking the concentrations in the range of 0.2µg/ml to 28 µg/ml and the solutions were injected in to HPLC unit with C₁₈analytical column. The peak height and area are measured. The standard graph was plotted with concentration on X-axis and peak area on Y-axis as shown in the graph-1.

Graph-1

Invitro release of Insulin from Chitosan gels:

In vitro release study was conducted by taking 100 ml of diffusion media in a receptor compartment and 1 ml of gel in donor compartment. Samples are collected at a different time points and subjected for analysis, the amount of insulin release at different time points was calculated using the straight-line equation of the standard graph.

Optimization of the gels was mainly based on viscosity of the formulation and their invitro drug release studies. With 1, 2 and 3% of chitosan the prepared gel was found to be very less viscous and there is no three dimensional network. So the optimization was carried out with 4, 5 and 6% chitosan. Insulin solution was taken as control which released 97% of insulin within 3hrs.

As shown in the graph-2, 4% gel formulation released about 97% of the drug in the gel, whereas 5% gel formulation released about 93% of the drug in the gel and 6% gel formulation released about 84% of the drug in the gel within 8hr of the time respectively.

All these release profile were fitted into Higuchi model of drug release with correlation coefficient values of 0.9831, 0.9916 and 0.9941 for 4%, 5% and 6% gels respectively.

5% gel was selected as the best formulation. 4% gel was ruled out because it has low viscosity and also do not have convenience while handling and administering. 6% gel was ruled out because of its high viscosity and high thickness, even though it sustained the drug release up to 8hr.

Graph-2

Invitro Permeation of Insulin through pig nasal membrane from Chitosan gel:

Invitro permeation study was conducted by taking the 50ml of diffusion media in a donor compartment and 1 ml gel in receptor compartment, Samples are collected at a different time points and subjected for analysis, the amount of insulin permeated at different time points was calculated using the straight-line equation of the standard graph.

From the cumulative % drug release profile of these gels, we can notice that the 4%, 5% and 6% gel formulations released about 97%, 93% and 84% of the drug content in the gel within 8hr of time respectively. According there release pattern and viscosity 5% gel was selected as the best formulation and permeation study was conducted for this formulation.

As shown in the graph-3, the cumulative amount permeated from 5% gel formulation was about 330.89µg/ml and from the control solution, it was about 466.76 µg/ml within 8hrs. That is about 70% of drug permeated from the gel formulation when compared to that of control solution.

Viscosity of the gel formulation:

The viscosity of the 5% chitosan nasal insulin gel was determined by Brookfield viscometer, which is spindle type of viscometer with a spindle size of 62 at a torque level of 15% and an rpm of 2.5. The viscosity determined was 750cps.

Graph-3

In-vivo bioavailability study:

A relative bioavailability study between the prepared nasal insulin gel and human actrapid solution containing crystalline zinc insulin was performed. As shown in the graph-4, there was a sudden and drastic decline in the serum glucose levels with subcutaneous injection whereas there was a gradual decrease in the serum glucose levels with nasal administration was observed. The time required for the nasal gel to attain the lowest serum glucose levels was around 1.5hr to 2hr whereas for the subcutaneous formulation it was 3hr.

Even though the in vitro release of the insulin from the gel was 6 hrs, the sustained release was not correlated in vivo because of the influence of various factors like the rapid passage of the drug into the systemic circulation because of the high blood capillary network of nasal mucosa, mucociliary clearance of nasal cavity which clears the drug and prevents it from entering the systemic circulation, thickness of nasal mucosa and other physiological conditions of the animal.

Comparative In-vivo study of nasal Insulin gel with subcutaneous marketed product in rabbits

Table-1

Time (hrs)	mg % Glucose
Time (hrs)	Subcutaneous	Nasal
0	95.56 ± 3.00	91.70 ± 5.15
0.5	58.88 ± 29.15	66.40 ± 14.49
1	48.27 ± 16.69	54.17 ± 4.73
1.5	37.15 ± 5.38	46.68 ± 2.72
2	35.91 ± 10.49	78.32 ± 12.96
2.5	45.02 ± 9.64	80.98 ± 11.49
3	53.66 ± 8.64	85.95 ± 9.07
4	73.65 ± 10.57	88.88 ± 7.70
5	81.11 ± 10.57	91.16 ± 5.84

From the % lowering of serum glucose levels by the nasal insulin gel and human actrapid solution, as shown in the graph-5, the relative bioavailability of nasal insulin gel was calculated and it was found to be 20.65% of the human actrapid solution.

Graph-4

Graph-5

CONCLUSION:

Based on this study it can be concluded that nasal route is one of the important non-invasive routes of administration for protein and peptide drugs like insulin because of enhanced patient compliance when compared with that of invasive routes of administration and relatively high bioavailability than oral administration.

REFERENCES:

1. Vamshi Krishna T, Madhusudan Rao Y, 2010. Formulation and evaluation of Nasal Insulin Gel. Int. J. Pharmacy Pharm. Sci. 2 (3), 161-164.

2. Jaleh Varshosaz, Hassan Sadrai, Alireza Heidari, 2006. Nasal Delivery of Insulin using Bioadhesive Chitosan Gels; Drug Delivery, 13, 31-38.

3. Reshma D’ Souza, Srinivas Mutalik, Sudha Vidyasagar, Madhavacharya Venkatesh, Nayanabhirama Udupa, 2005. Nasal insulin gel as an alternate to parenteral insulin, AAPS PharmSciTech, 6(2), Article 27.

4. Abdol Hossein Najefabadi, Payam Moslemi, Hosnieh Tajerzadeh, 2004. Intranasal bioavailability of insulin from carbopol based gel spray in rabbit; Drug Delivery, 11,295-300.

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8. Beht et al, 1998. Optimization of systemic nasal drug delivery with pharmaceutical excipients, Adv. Drug. Del. Rev, 29, 117-133

9. Kadam, S.S, Mahadik, K.R, Pawar, A.P, Paradkar, A.R, 1993. Transnasal delivery of peptides – a review, The East Pharm, 47 – 49.

10. Lee, W. A, Narog, B.A, Patapoff T. W, Wang, Y.J, 1991. Intranasal bioavailability of insulin powder formulation: Effect of permeation enhancer to protein ratio, J. Pharm. Sci. 80, 725-729.

11. Schipper et al, 1991. The nasal mucociliary clearance: relevance to nasal drug delivery” Pharma. Res., 7, 807-814.

Received on 31.05.2011 Modified on 25.06.2011

Research J. Pharm. and Tech. 4(9): Sept. 2011; Page 1418-1421