Optimization of Pharmacokinetic By Loading Bioenhancer in Optimized Nasal Mucoadhesive Microspheres of Insulin

 

Shinde AD1* and Bhise SB2

1S.V.P.M.’s College of Pharmacy, Malegaon (Bk II), Baramati, Pune, Maharashtra, India

2Govt. College of Pharmacy, Karad, Dist-Satara, Maharashtra. India

*Corresponding Author E-mail: ajayshinde2001us@yahoo.com

 

ABSTRACT:

Mucoadhesive Microspheres of Bovine Insulin containing a muchoadhesive polymers Sodium Alginate and Carbapol-934 were prepared by Ionic Gelation technique and Bovine Insulin encapsulated by lyophilization by utilizing 32 Factorial Drug Design and 9 formulation prepared. The formulation C-3 was selected as a final optimized formulation for its significant characteristics. The Optimized Mucoadhesive microspheres C-3 was loaded with bioenhancers such as Sodium deoxycholate, Sodium Taurocholate and Lecithin from soya in 1% w/w. Drug release study was carried out. The mucoadhesive microspheres loaded with 1% w/w Sodium deoxycholate indicate significant release profile. Hence the final optimized formulation loaded with1% w/w Sodium deoxycholate was investigate for animal study by selecting rabbit model. The diabetes was induced by using Alloxan 150 mg/kg to rabbits. The final optimized formulation loaded with and without 1% w/w Sodium deoxycholate. The formulation without bioenhancer was remarked as reference formulation; both formulations were given to animal by nasal route, with a dose 1.5 U/Kg by selecting Crossover drug design study. Prior to investigate a optimized and reference formulation to animal a standard dose of bovine insulin was given to each animal 0.5 U/Kg by subcutaneously. Serum blood concentration of bovine insulin was estimated by using previously calibrated and validated RP-HPLC method at predetermined time schedule and pharmacokinetic study was done. The % blood glucose levels were recorded during animal study.

 

KEYWORDS: Bovine Insulin, Mucoadhesive microsphere, Diabetes, Pharmacokinetic.

 


INTRODUCTION:

Diabetes mellitus is a heterogeneous disorder characterised by varying degrees of insulin resistance and insulin deficiency, which leads to a disturbance in glucose homeostasis. Symptoms of marked hyperglycemia include thirst ‘ polydipsia’, large volume of urine ‘polyuria’, frequent feeling of hunger ‘polyphagia’, feeling of tiredness, blurred vision, and weight gain or weight loss with ketoacidosis. Chronic diabetic patients are predisposed to cardiovascular disease and other organ damage such as retinopathy recognizes nephropathy.1, 2, 3.

 

Insulin dependent diabetes mellitus requires insulin, which is polypeptide in nature and lowers blood glucose level, facilitates glucose transport in cells and glucose utilization, increases lipid and glycogen synthesis, inhabits glycogenolysis and gluconeogenesis, increases amino acid transport into cells and prevents release of amino acids from muscle and increases movement of K + and Mg 2+    into cell 5.

 

Insulin, irrespective of its source, is classified according to its onset and duration of action into rapid, intermediate and long acting.4

 

Insulin is commonly administered by parenteral route, which is often complex, difficult, painful and occasionally dangerous. Insulin, when administered through oral controlled released dosage form shows poor bioavailability due to poor absorption and due to enzymatic degradation. The need for novel systems for an effective nasal delivery of Bovine Insulin has increased greatly. So mucoadhesive microspheres were prepared by selecting mucoadhesive polymers Sodium Alginate and Carbapol-934 in 1:1 % ratio, and drug Bovine insulin was encapsulate by using lyphilizer. For the preparation of microsphere Ionic Gelation Technique was utilized and characterization was done. The optimized formulation was loaded with bioenhancers and release profile was studied. The optimized formulation loaded with 1% Sodium Deoxycholate represents significant release profile. Hence it was selected for animal study. The final optimized formulation was given to rabbit animal model by nasal route, and pharmacokinetic study was done. The basic aim of this research work to study the pharmacokinetic of insulin in optimized formulation.

MATERIAL AND METHODS:

a. Preparation of Standard Calibration Curve for the Bovine Insulin by RP-HPLC(In 0.01 M HC1/):5

i) Preparation of stock solution and various dilutions of Bovine Insulin:

The pure drug Bovine Insulin procured from Sigma Aldrich, Germany, was taken, accurately weighed 10 mg of pure powder on the digital balance and dissolved in 100 ml of 0.01M HCl in the 100 ml volumetric flask. The above solution was sonicated for 10 min. After that volume was adjusted up to 100ml with 0.01 M HCL solution. The above solution was then used for the further dilutions.

 

Form the above solution; the series of the dilutions were prepared. A series of 2 ml, 4ml.....10ml of stock solution was taken in Corning glass test tubes. Final volume was made up to 10 ml with 0.01 M HCl to prepare 20,40,,60,,80 and 100 µg/ml concentration of pure drug Bovine Insulin.  The tubes were sonicated for 10 min.

 

ii) Preparation of Mobile phase:

The 0.1 M sodium hydrogen orthophosphate solution was prepared which is mixed with acetonitrile in 72.5: 27.5 ratios respectively. The pH is adjusted 5.2 with ortho-phospheric acid. This solution was sonicate for 20 minutes and filtered thorough 0.22 µ membrane filter by using membrane filter assembly.

 

iii) RP-HPLC method:

The RP-HPLC method was carried for this study by doing some modification in the method which was described in Indian pharmacopoeia for more resolution and less retention time of response peak. The HPLC instrument is validated for its accuracy before run the mobile phase. The mobile phase sodium hydrogen orthophosphate and acetonitrile in combination 72.5: 27.5, the pH 5.2 was obtain with ortho-phospheric acid, was run as mobile phase in isocratic mode. The flow rate was adjusted 1.4 ml/min. A stationary phase was ODS- with average particle size 5 µ, Column-C-18.  and Column Size:- 250×4.60 mm. Response was obtained by using UV-detector (Table No.1) at wave length 214 nm by injecting 20 µl sample of various dilutions of pure drug Bovine Insulin. And a calibration curve was obtained (Fig. No.1). From observation the standard calibration curve of bovine insulin represent the equation y =6.5877x – 45.768 with R2 coefficient 0.9935 with linear absorbance. The regression line passes through eight points with slope -45.768.

 

Fig.No.1: Chromatogram of Bovine Insulin in concentration 100 µgm/ml.

 

Table No.1: Response of  RP-HPLC for Bovine Insulin at different Conc. Range.

Sr. No

Concentration in µ gm/ml

AUC in mV.s

ht in mV

Wo 5

1

20

100.51

1.275

1.29

2

40

200.549

2.301

1.33

3

60

355.676

4.375

1.2

4

80

462.372

5.762

1.25

5

100

628.372

8.755

1.1

 

 

 

 

 

 

 

b. Loading of Bioenhancers in Optimized Formulation of Insulin-Microsphere:

Optimized formulation C-3, were loaded with 1% bioenhancers such as Sodium Deoxycholate, Sodium Taurocholete and Lecithin from Soya. Which have possible mechanism of action that it disrupts membrane, open tight junction, enzyme inhibition and mucolytic activity.

 

c. Encapsulation of Bovine Insulin by using lyophilizer:6

The drug solution was prepared by dissolving 100 mg of bovine insulin in 0.01 M HCL and volume was adjusted 100 ml.  Preservative m-cresol with concentration 0.03 v/v was mixed in previously prepared solution. The drug solution was sonicated for 20 minutes. The drug solution 2 ml (2 mg Bovine Insulin) was pippet-out and placed sample tubes contain with, 50 mg of microspheres. The sample tubes were kept in lyophilizer for 18 hour for incubation period by maintaining temperature -420 C for complete encapsulation process. After the complete incubation period sample tubes are removed and kept for freeze temperature.

 

d. Drug Release study from Optimized Formulation loaded with Bioenhancers:7

Release of Bovine Insulin through isolated gout nasal mucosa, from the microspheres was studied. The nasal cells was fabricated in which donar chamber consist 60 ml, phosphate buffer saline pH 7.4 with 60 rpm was maintained with magnetic stirrer. Physiological condition was maintain to keep constant temperate  37° ± 1 °C through circulating warm water in each Nasal cell . A sample of microspheres equivalent to 20 mg of Bovine Insulin microspheres was used in each test. Samples of dissolution fluid were withdrawn through a sample outlet tube at different time intervals and were assayed at 276nm for Bovine Insulin content using a Shimadzu UV-150 double-beam spectrophotometer (Shimadzu Corporation, Japan).

 

e Animal Study:8

i) Development of a Diabetic Rabbit Model:

The cross-over study with washout period of 3 days was undertaken by selecting long ears white Rabbit as animal model. Diabetes Induction was done by using Alloxan (Loba made) of dose-150 mg/kg by marginal ear vein to each animal. And standard diet was provided, which then kept in different cages. Seventy two hours later, stable hyperglycemic rabbits were formed and the diabetic model deemed successful when blood glucose in a fasted rabbit was above 15 mmol 1-1 .


Table-2: % Cumulative Drug Release of optimized formulation loaded with bioenhancer.

TIME  (hr)

C3

without bioenhancer

C3

1% Sodium Deoxycholate

C3

1% Sodium Taurocholate

C3

1% Lecitin from Soya

0.5

27.068

38.039

34.019

35.039

1

40.933

51.16

48.14

49.16

2

60.475

75.181

69.161

70.181

3

74.273

88.69

80.41

83.61

4

81.476

93.089

86.045

88.035

5

86.271

96.225

93.335

94.315

6

90.805

98.098

96.052

97.032

7

93.286

99.989

99.97

98.95

8

99.163

100.98

100.69

100.76

C=CARBAPOL-934.

 

Table.3: Average response of animal model for drug Bovine Insulin.

TIME (Minutes)

Insulin 0.5 U/Kg by Subcutaneous Route (microgm/L)

Ref. formulation -C3 Formulation without bioenhancer (microgm/L)

Optimized formulation C3 with 1% Bioenhancer (Sodium Deoxycholate) (microgm/L)

0

0.00

0.00

0.00

30

99.694

45.654

99.701

60

127.532

67.352

105.53

90

98.514

76.306

108.40

120

61.66

58.556

72.908

180

38.602

42.764

42.743

240

1.114

6.546

7.580

300

5.472

5.732

7.940

 

 

 

 

 

 

 

 

 

 

 

*p ‹ 0.05                                                                                                                                  

 

Table No.4: Estimated Blood Glucose in Percentages.

TIME (Minutes)

Insulin 0.5 U/Kg by Subcutaneous Route

Ref. formulation -C3 Formulation without bioenhancer (1.5 IU/Kg Insulin)

Optimized formulation C3 with 1% Bioenhancer (Sodium Deoxycholate) (1.5 IU/Kg Insulin)

 

Estimated Blood Glucose in Percentages

0

100.07

100.06

100.02

30

87.794

98.118

91.088

60

72.132

90.332

88.332

90

59.316

89.804

84.552

120

52.120

85.648

61.228

180

40.206

80.110

58.188

240

38.182

80.022

44.076

300

37.114

80.076

40.232

 

 

 

 

 

 

 

 

 

 

 

 

*p ‹ 0.03                                                                                                        

 

Table No.5: Pharmacokinetic study

Formulation

Dose

(IU)

Pharmacokinetic Parmeters of Bovine Insulin

Cmax-µgm/lit

t-max (minutes)

F (%)

AUC (µU.min./lit.)

t ˝ hr

K hr -1

Insulin by Subcutaneous

–Std. Dose

0.5

21.73µgm

127.53

60

100.00

251.546

0.93

0.74

Ref Formulation –C3 without bioenhancers

1.5

65.21µgm

76.306

90

25.02

190.749

1.17

0.59

Optimized formulation C3 ( 1% sodium deoxycholate)

1.5

65.21µgm

108.40

90

34.8

265.76

0.96

0.72

 


ii) Preclinical Study:

The standard Dose 0.5 IU/ml of Bovine Insulin was given by subcutaneous route to the animal model. The Reference formulation was  mucoadhesive microsphere of Bovine Insulin (C3 without Bioenhancer) and test was optimized formulation i.e. mucoadhesive microsphere of Bovine Insulin with 1% Bioenhancer (C3 with 1% Sodium Deoxycholate) formulations were given to animals model by nasal route with dose 1.5 IU/Kg , prior to nasal dose the tested diabetic rabbits were anaesthesthetised by using i.v. sodium pentobarbital injection solution into ear with dose 30mgkg-1  and animal were made to form appropriate angle with horizontal line one by one during delivery of dose. Before to dose of formulation to animals are subjected to fasting for 12 hours. After dosing standard food are given. Blood sample of 1.5 ml was taken from marginal ear vein in predecided schedule. Blood was centrifuged immediately and serum was separated and stored at –200c until analysis. Preclinical Assessment was done for Blood glucose.

 

iii) Validation of Analytical methods:

The suction filter and pump parts were thoroughly cleaned as per manufacturer’s directives. The efficiency of the pump was determined by evaluating the amount of mobile phase pumped in one min. the tubings were checked for any leaks. Flow rate setting range was 1.4 ml/min with accuracy - ± 2% or setting value ± 2 ml/min, Precision – RSD of 0.1 % or less, the instrument was Simatzu HPLC with UV detector in Isocratic Mode. The Mobile phase-0.1 M Sodium Hydrogen Orthophosphate solution was prepared which is mixed with Acetonitrile in 72.5: 27.5 ratios respectively. Stationary phase- ODS- with average particle size 5 µ, Column- C-18. pH adjusted 5.2 by using Ortho-posphoric acid.

 

f. Analysis of Blood Serum Bovine Insulin Concentration by using RP-HPLC:

The plasma concentration of Bovine insulin was estimated for each animal present in every group by using RP-HPLC method, and chromatograph are recorded by considering all chromatographic parameters. A statistical study was carried for the inter-subject variability by using one way ANOVA, and p-value was noted for significance of study. The Blood Serum samples were estimated for insulin concentration before and 30, 60, 90,120,180,240 and 300 min after the administration of the formulation.

 

g. Blood Glucose Study: 9

Subjects are checked for Blood glucose by using Robonik prietest, auto-analyzer for each blood sample after 30, 60, 90,120,180,240,300 and 360 min of dosing.

 

h. Pharmacokinetic study:10

The Pharmacokinetic parameters such as Cmax, t-max, Area Under Curve AUC, Elimination Half-life  t1/2 ,Bioavailability and Elimination Rate constant k are estimated by using the equation mentioned as bellow

i.. AUC= C2-C1/2  × T2-T1……∞- Trapezoidal Rule.

ii. t ˝  = 0.693/k

iii. F= (AUC) opz.formu./ (AUC) s.c × Dose s.c / Dose. opz.formu × 100%

 

RESULT AND DISCUSSION:

a. Drug Release study from Optimized Formulation loaded with Bioenhancers:

The optimized formulation of mucoadhesive microsphere C-3 was loaded with 1% bioenhancers such as Sodium Deoxycholate, Sodium Taurocholete and Lecithin from Soya and cumulative percentage release study was carried (Table.2.). This study indicate that optimized formulation C-3 loaded with 1% Sodium Deoxycholate represent significant cumulative release profile as compared with Optimized formulation loaded with  1% Sodium Taurocholete and Lecithin from Soya (Fig.No.2) . So it was decided that the optimized formulation C-3 loaded with 1% w/w Sodium Deoxycholate and investigated for animal study.

 

b. Preclinical study of Optimized formulation of Bovine Insulin:

i) Estimation of Blood serum Insulin concentration:

From observation Table No3., Average Blood serum Insulin concentration of Bovine Insulin for standard dose, Reference Formulation without bioenhancer and Optimized Formulation with 1% Sodium Deoxycholate. There was no inter-subject variability with significant p value p ‹ 0.0001 for each group of animal, during 5 hour animal study. There invariability appeared the process of significant rise of blood serum insulin concentration, followed by a gradual fall to the basal level at the end of the experiment (Fig.No.3). In contrast to the reference group the AUC’s of both the insulin subcutaneous and optimized formulation groups were significantly increased.

 

ii)  Blood Glucose Study:

Animal study represent there was no statically difference in the blood glucose level in all groups before treatment p>0.05. After treatment, there was no significant difference in the blood glucose level p>0.001 in all groups. After 60 minutes after administration of the formulation, the blood glucose level of the standard dose, and Optimized Formulation with 1% Sodium Deoxycholate were significantly lower than that of  reference formulation group with p value p ‹ 0.03. Ninety minutes after administration of the formulations, the blood glucose level of the optimized formulation groups were significantly lower than that of reference formulation group p ‹ 0.03 as compared with standard dose of bovine insulin (Fig.No.4). The data represent that optimized formulation plays a significant role in reducing the blood glucose level in diabetic rabbits. Although, there was no significant difference between the standard dose and reference formulation groups in their influence on the blood glucose level. The hypoglycemic effect lasted over 5 hr.

 

c. Pharmacokinetic study:

The pharmacokinetic study (Table.5) revels that, the Bioavailability of the Optimized Formulation loaded with 1% Sodium Deoxycholate is 34.8% and for Reference Formulation without bioenhancer, which is 25.02%. Which indicate that there in increase in bioavailability 9.78% for Optimized Formulation due to the presence of bioenhancer in optimized % strength i.e. 1% of sodium dexoycholate as compared with reference formulation without bioenhancer and also in contrast with standard dose of insulin, given by subcutaneous route of administration.

 

The maximum concentration achieved for Optimized Formulation loaded with 1% Sodium Deoxycholate is 108.40 µgm/lit and for Reference Formulation without bioenhancer, which is 76.306 µgm/lit within a time 1.5 hour. Which indicate that there in increase in Cmax 32.09 µgm/lit for Optimized Formulation as compared with reference formulation without bioenhancer and also in contrast with standard dose of insulin, given by subcutaneous route of administration.

 

The Area Under Curve AUC was estimated by using Trapezoidal Rule, which revels that, AUC of the Optimized Formulation loaded with 1% Sodium Deoxycholate is 265.76 µU.min./lit and for Reference Formulation without bioenhancer, which is 190.749 µU.min./lit Which indicate that there in increase in AUC 75.02 µU.min./lit for Optimized Formulation due to the presence of bioenhancer, as compared with reference formulation without bioenhancer and also in contrast with standard dose of insulin, given by subcutaneous route of administration. Which indicate there is increase in the blood serum concentration of insulin in a systemic circulation of animal due to the presence of  bioenhancer.

 

 


Fig. No.2. % Cumulative Drug Release Study of Optimized formulation loaded with   Bioenhancer

Fig. No.3: Blood serum Bovine Insulin Concentration Vs Time.

 

Fig. No.4: % Blood Glucose Vs Time.

 

 

 


The Half life and Elimination rate constant for the Optimized Formulation loaded with 1% Sodium Deoxycholate is 0.96 hour 0.72 hr -1 and for Reference Formulation without bioenhancer, which is 1.17 hour 0.59 hr -1 respectively. Which indicate that the half life of Optimized Formulation loaded with 1% Sodium Deoxycholate is less 0.21 hour as compared with reference formulation without bioenhancer. The elimination rate constant for Optimized Formulation loaded with 1% Sodium Deoxycholate is more 0.13 hr -1 as compared with reference formulation without bioenhancer. This represents that drug was released from Formulation in controlled release manner but due to the presence of bioenhancer 1% Sodium Deoxycholate in optimized formulation the drug insulin is rapidly absorbed and eliminate slowly as compared with reference formulation.

 

CONCLUSION:

The Blood serum Insulin concentration concentration of Bovine Insulin for standard dose, Reference Formulation without bioenhancer and Optimized Formulation with 1% Sodium Deoxycholate, represent no inter subject variability with significant p value p ‹ 0.0001 for each group of animal, during 5 hour animal study. There invariability appeared the process of significant rise of blood serum insulin concentration, followed by a gradual fall to the basal level at the end of the experiment. In contrast to the reference group the AUC’s of both the insulin subcutaneous and optimized formulation groups were significantly increased.

 

Animal study represent that optimized formulation plays a significant role in reducing the blood glucose level in diabetic rabbits. Although, there was no significant difference between the standard dose group in their influence on the blood glucose level. The hypoglycemic effect lasted over 5 hr.

 

The pharmacokinetic study represent that, the Bioavailability of the Optimized Formulation loaded with 1% Sodium Deoxycholate is 34.8% and for Reference Formulation without bioenhancer, which is 25.02%. Which indicate that there in increase in bioavailability 9.78% for Optimized Formulation due to the presence of bioenhancer in optimized % strength i.e. 1% of sodium dexoycholate .

 

The maximum concentration achieved for Optimized Formulation loaded with 1% Sodium Deoxycholate is 108.40 µgm/lit within a time 1.5 hour.  The Area Under Curve AUC of the Optimized Formulation loaded with 1% Sodium Deoxycholate is 265.76 µU.min./lit. There in increase in AUC 75.02 µU.min./lit for Optimized Formulation due to the presence of bioenhancer, as compared with reference formulation without bioenhancer and also in contrast with standard dose of insulin. Which indicate there is increase in the blood serum concentration of insulin in a systemic circulation of animal due to the presence of bioenhancer.

 

The Half life and Elimination rate constant for the Optimized Formulation loaded with 1% Sodium Deoxycholate is 0.96 hour 0.72 hr -1 and for Reference Formulation without bioenhancer, which is 1.17 hour 0.59 hr -1 respectively. Which indicate that the half life of Optimized Formulation loaded with 1% Sodium Deoxycholate is less 0.21 hour as compared with reference formulation without bioenhancer. The elimination rate constant for Optimized Formulation loaded with 1% Sodium Deoxycholate is more 0.13 hr -1 as compared with reference formulation without bioenhancer. This represents that drug was released from Formulation in controlled release manner but due to the presence of bioenhancer 1% Sodium Deoxycholate in optimized formulation the drug insulin is rapidly absorbed and eliminate slowly as compared with reference formulation.

 

From over all study conclude that the Optimized formulation of Bovine Insulin C-3 consist the composition of mucoadhesive polymer 1:1 % w/v sodium alginate and Carbapol-p-931 respectively containing 1% bioenhancer Sodium Deoxycholate ,  release the drug by controlled release manner during 5 hour study. For animal study after dosing the Optimized Formulation represent significant hypoglycemic effect.  which helps to optimize pharmacokinetic parameters such as Cmax, t-max, Area Under Curve- AUC, Elimination Half-life  t1/2 ,Bioavailability and Elimination Rate constant k significantly as compared with Reference Formulation without bioenhancer.

 

FUTURE PLAN:

From over all study the Optimized Formulation of Bovine Insulin C-3 with 1% bioenhancer Sodium Deoxycholate represents a significant release profile by nasal route with remarkable hypoglycemic effect. So it is necessary to investigate this Optimized Formulation of Bovine Insulin for clinical study on IDDM patient.

 

ACKNOWLEDGEMENT:

The authors would like to thanks the Principal Pof. R.N.Patil and Management SVPM’S College of Pharmacy, for providing research facility at department. Authors also would like to thanks Dr.S.R. Chapalkar  for her advice for fulfill the research work.

 

REFERENCES:

1)       Hillson R., Practical Diabetes Care., Oxford: Oxford University Press, 1996.

2)       Nattrass M (ed.), Malin’s Clinical Diabetes, 2nd ed. London: Chapman and Hall, 1996.

3)       MeReC Bulletin, Non-insulin dependent mellitus (part 1), MeReC Bull, 1996; 7: 21-  24.

4)       Editorial Advisory Board- Asia., ‘Current Index Of Medical Specialties’, CMP Medica India Private Limited: July-2005, 276.

5)       Indian Pharmacopoeia, 1996 edition, Volume I, 399-400.

6)       Michael Hinchcliffe and Lisbeth Illum, ‘Intranasal insulin delivery and therapy’, Advanced Drug Delivery Reviews, 35, 1999, 199–234.

7)       Catarina M. Silva, Antonino J. Ribeiro et.al., Alginate microspheres prepared by internal gelation, Development and effect on insulin stability, Int. Journ. Pharmaceutics, 311, 2006, 1-10.

8)       Gonjari I.D. and Kasture P.V. , Temperature Induced in situ Mucoadhesive Gel of Tramadol Hydrochloride for Nasal Drug Delivery, Journal of Pharmaceutical Research, Vol.6, No.2, 2007, 89-93.

9)       10.Hui-Bi Xu, Kai-Xun Huang et.al., Hypoglycaemic effect of a novel insulin buccal formulation on rabbits, Pharmaclogical Research, Vol.46.(5), 2002, 450-467.

10)    Malcolm Rowland and Thomas N. Tozer, ‘Clinical Pharmacokinetics Concept and Application, 3rd ed. William and wilkins PA, USA, 21.

 

 

 

Received on 20.01.2010                             Modified on 27.02.2010

Accepted on 28.03.2010                            © RJPT All right reserved

Research J. Pharm. and Tech. 3(2): April- June 2010; Page 613-618