A COMBINED MODEL FOR PREDICTING CYP3A4 CLINICAL NET DRUG-DRUG INTERACTION, BASED ON CYP3A4 INHIBITION - METHODOLOGY

 

Methodology

 

Plan of the Study:

            The whole study was divided into 5 phases.

 

First Phase:In this phase rats of one group (n=6) will be treated with Abraxane (10 mg/kg B.W.P.O.)¹⁰² for seven consecutive days and its influence on the blood glucose levels will be studied.

 

Second phase: In this phase, two groups are used (n=6). The rats of one group will be administered with Nateglinide (50 mg/kg B.W.P.O)¹⁰³, and the other group will be administered with Repaglinide(0.3 mg/kg B.W.P.O)¹⁰⁴. Thereafter the blood samples will be withdrawn at 0, 0.5, 1, 2, 4, 6, 8, 12, 18 and 24 hrs intervals and will be analyzed for blood glucose concentration determination by GOD/POD method.

 

Third phase: Here In this phase, the same animals of phase-2 study will be utilized. Thereafter, both the groups of animals will be administered with Abraxane (10 mg/kg B.W. P.O) for a period of one week. On 8^th day one hr after Abraxane administration to experimental animals’ one group will receive Nateglinide (50 mg/kg B.W.P.O.) and the other Repaglinide (0.3mg/kgB.W. P.O). Thereafter the blood samples are collected and analyzed for glucose concentration by GOD/POD method.This phase is expected to produce data regarding occurrence of interaction (if any) among administered combination.

 

Fourth phase: In this phase, the entire protocol of phase-2 and phase-3 will be repeated using Streptozotocin induced diabetic rats.

 

Fifth phase: In this phase, albino rabbits of either sex, weighing between 1.5-1.8kg will be divided into several groups. The entire protocol ranging from phase-1 to phase-3 will be repeated to understand the influence of onAbraxane anti diabetic effect of Nateglinide and Repaglinide.

4. 

 

4.1     General Principles in Each Study:

            Below mentioned are the common requirements needed for each of the experiment. However the additional specific requirements (if any) of each study will be mentioned under appropriate sections.

 

4.1.1 Common requirements:

1.            Vehicle: 2% w/v gum acacia suspension (this was prepared by triturating 2grams of high quality micronized gum acacia powder in 100 ml of double distilled water. This has been used as vehicle for suspending drugs to ensure correct dose administration throughout the study).

2.            Anticoagulant: sodium fluoride: potassium oxalate (1:3)

3.            Glucose estimation kit (Pathozyme diagnostic kit)

4.            Motor and pestle, alcohol, low voltage electric lamp, micropipette (5-50µl), 1 ml graduated pipettes, epindroff tubes, thin Aluminium foil, incubator and double distilled water etc.

5.            Semi auto analyzer (RMS BCA-201).

6.            Weighing balance(5mg-350gm) by  shimatzu

 

4.1.2. Various drugs used in the study:

Various drugs used in the study are the generous compliments from the esteemed pharmaceuticals mentioned against each drug below. The same may be considered as their source of procurement throughout the study.

1.            Abraxane: Cipla Pharmaceuticals Ltd. (Goa)

2.            Nateglinide: Dr. Reddy’s Laboratories Ltd. (Hyderabad)

3.            Repaglinide: MeproMax Lifesciences Pvt. Ltd. (Dehradun)

 

 

 

 

4.1.3 Mode of administration:

All the three drugs used in the study are administered orally in clinical practice. Hence the same route is followed. However, majority of the drugs used in the study are poorly water soluble. Hence in order to have proper administration, each drug was suspended individually in 2% w/v acacia suspension of each drug were freshly prepared in such a way to yield the required dose of the drug in maximum allowed volume of the vehicle for that particular animal species.

 

4.2     Study Design:

All the studies were of randomized, vehicle (2% w/v acacia vehicle) – controlled design. A generalized overview of the studies is given in table no. 1

                                

 

Table No. 1: General Scheme of the study:

Study	Pretreatment medication and dose	Duration of pretreatment	Study drug and dose	Wash-out period (w)
I	2% w/v gum acacia dose volume matched with the average of volume of drug treatments in the subsequent studies x 1	On day 1	2% w/v gum acacia on the same day.	1
II	2% w/v gum acacia dose volume matched with the average of volume of drug treatments in the subsequent studies x 1	On day 1	Nateglinide  50 mg rats/kg or 370 mg rabbits/kg, p.o. on the same  day	1
III	2% w/v gum acacia dose volume matched with the average of volume of drug treatments in the subsequent studies x 1	On day 1	Abraxane 10 mg rats/kg or 192 mg rabbits/kg, p.o. on the same  day	1
IV	2% w/v gum acacia dose volume matched with the average of volume of drug treatments in the subsequent studies x1	On day 1	Repaglinide 0.3 mg rats/kg or 45 mg rabbits/kg, p.o. on same day.	1
V	Abraxane 10 mg rats/kg, p.o. and 192 mg rabbits/kg, p.o.	7 days at 10:00 am   7 days at 10:00 am	Nateglinide 50mg rats/kg or 370 mg rabbits/kg, p.o. on the day  8 respectively at 11:00 am	1
VI	Abraxane 10 mg/kg rats/kg, p.o. and 192 mg rabbits/kg, p.o.	7 days at 10:00 am   7 days at 10:00 am	Repaglinide  0.3 mg rats/kg or 45 mg rabbits/kg, p.o. on day 8  at 11:00 am	1

 

During the days of Nateglinide administration, additional glucose solution for oral administration was made available in order to take care of any severe hypoglycemia so as to prevent the death.

 

4.2.1. Estimation of blood glucose

Enzyme, GOD-POD endpoint colorimetry ¹⁰⁵

            The older methods were based on the reducing property of glucose. But these methods do not measure the true glucose because of interferences of other reducing sugars. Subsequently other chemical and enzymatic methods were developed to overcome this problem. The GOD/POD method is one such evolved method by Trinder in 1964. This method is simple, single stepped, rapid, reliable and acceptable precision. Hence in the present study this method has been adopted⁹⁷. Trinder’s method (1964) utilizes two enzymes glucose oxidase (GOD) and peroxidase (POD) along with the chromogen L-amino antipyrine and phenol. This method is intended for in vitro quantitative determination of glucose in serum/plasma or cerebrospinal fluid. There was no interference due to the substances like creatinine, fructose, galactose, reduced glutathione, ascorbic acid and xylose. Hemoglobin or bilirubin upto 10 mg % does not affect the test.

 

Principle:

Glucose is determined after enzymatic oxidation in the presence of glucose oxidase. The hydrogen peroxide so formed reacts under catalysis of peroxidase with phenol and 4-amino antipyrine (4AAP) to form a red colored quinoneimine compound. The intensity of color is directly proportional to the glucose concentration.

                      Glucose + O₂ + H₂O    ^GODgluconic acid + H₂O₂

                      2H₂O₂ + 4AAP + Phenol ^PODQuinoneimine + 4H₂O₂

 

Reagents:      1. Glucose monoreagent                    --     5 x 100 ml bottles.

                        2. Glucose standard (100mg/dl)         --    1 x 5ml vial

 

Composition of reagents¹⁰⁶:

1.      Glucose monoreagent

a.       Phosphate buffer (pH 7.5)                         200mmol/L

b.      Glucose oxidase                                         ≥ 15 KU/L

c.       4- Amino antipyrene                                  0.3mmol/L

d.      Phenol                                                        5mmol/L

e.       Peroxidase                                                  ≥ 3K U/L

2.      Glucose standard (100mg/dl)

a.       Dextrose                                                    100mg/dl

b.      Benzoic acid                                              0.2%

 

 

Preparation of working reagents:

The mono reagent is ready for use.

 

Reagent storage and stability:

1.            Store the reagents at 2-8˚C and protect from light.

2.            Close the reagent bottle immediately after use.

3.            Avoid contamination of the reagents.

4.            The reagents are stable at 2-8˚C till the expiry date printed on the container label if contamination is avoided.

 

Specimen collection and handling:

1.               Serum or plasma collected in sodium fluoride: potassium oxalate (1:3).

2.               Serum or plasma should be separated within 30 mins of collection of sample.

3.               In separated and non heamolysed serum, the glucose concentration is generally stable for as long as 3 hours at room temperature and for up to 72hours at 2-8˚C.

 

Procedure:

            Pipette into clean, dry test tubes labeled as blank (B), standard (S) and test (t).

               

            General procedure of GOD/POD for the estimation of glucose in plasma (for 3ml cuvettes). Mix well; incubate at 37˚C for 10 min. measured the blood glucose concentration directly using Semi auto analyzer (RMS BCA-201)

                      

Pipette into tubes marked	Blank	Standard	Test
Serum	-	-	10ml
Standard	-	10ml	-
Glucose Reagent	1ml	1ml	1ml

 

4.3 Methodology:

            The studies were carried out in the Department of Pharmacology in our institution which is duly licensed by the CPCSEA (Committee for the Purpose of Control and Supervision of Experiments in Animals). The study protocols were approved according to current regulations of CPCSEA by the Institution Animal Ethics Committee for studies in experimental animals.

 

4.3.1. Animals:

All the animals (rats and rabbits) used in the study were procured from Mahavir Enterprises, Hyderabad. Registration number 346/CPCSEA and were housed under standard husbandry conditions in the institutional animal house. Hence the same may be considered as source of animal procurement in the subsequent sections.

            A total of 50 rats (either sex) and 18 rabbits (either sex) were selected for the current study.

                              

Inclusion criteria:

i.                 The animals which have normal behavioral parameters.

ii.               The animals which have healthy food consumption and excretory activities.

iii.             The animals with healthy body weight, temperature and heart rate.

iv.             The adult animals aging between 3 months to 9 months.

 

Exclusion Criteria:

i.                    The animals which do not exhibit above mentioned inclusion criteria are omitted.

ii.                  Female animals felt to be in gestational periods were excluded.

iii.                The animals exposed to insecticides (such as mosquito repellants used for maintenance of animal house) were excluded.

 

Preparation of the animals for the study:

i.                    The rabbits were not provided with vegetables and green leafy food for at least one week prior to each study.

ii.                  All the animals were given only standard pelleted animal diet (VRKBrand) from one week prior to the study till the completion of the study.

iii.                All the animals were fasted for 18 hrs prior to the study with water ad libitum.

 

4.3.2 Common Experimental Techniques

4.3.3 ALBINO RATS

Method for oral administration:

            Oral feeding administration was done by oral feeding needle (purchased from Space Labs, Nashik) and 1 ml glass syringe.

 

Method for blood sampling: ¹⁰⁷

The rat was anesthetized by anesthetic ether in anesthetic chamber. After small anesthesia rat was taken up from anesthetic chamber. Now put animal on operation table and tail is squeezed with Xylene to dilate the vein and cut the tip of tail and blood is collected in the epindroff tubes containing pinch of anticoagulant mixture (sodium fluoride and potassium oxalate in 1:3 ratio).

 

 

4.3.4 ALBINO RABBITS:

Method for oral administration:

            Oral feeding administration was done by oral feeding needle (purchased from Space Labs, Nashik) and 5 ml glass syringe.

 

Method for blood sampling:¹⁰⁸

            Blood samples were collected from the marginal ear vein. For this the rabbits were held in the wooden stall with their heads protruding out. The edges of the ears were depilated and the blood vessels were dilated by warming the ears by using low voltage electric bulb. The dilated marginal ear vein was punctured by means of sharp 26 gauge needle in the direction of venous blood flow. The blood samples (0.5 ml, each sample) were collected into epindroff tubes containing a pinch of anticoagulant powder.

 

Method for collection of plasma:¹⁰⁹

            The plasma was obtained by centrifuging the blood samples at 3000 rpm for 15min; decanting the supernatant fluid into the clean and dry test tubes.

 

Dose Selection:

Nateglinide:

Rat Dose         : 50mg/kg       

Rabbit Dose    : 370mg/kg

 

Repaglinide:

Rat Dose         : 0.3 mg/kg     

Rabbit Dose    : 45 mg/kg  

 

Abraxane:

Rat Dose         : 10 mg/kg      

Rabbit Dose    : 192 mg/kg

 

4.4.1 Effect of vehicle per se (2% w/v gum acacia) administration and long term fasting on blood glucose levels in albino rats.

            The study was aimed to understand the influence of vehicle (2% w/v gum acacia) as well as long term fasting (18 hrs) on blood glucose levels in healthy albino rats.

 

Materials and Methods:

1.      Vehicle: 2% w/v gum acacia

2.      Albino rats.

 

Experimental procedure:

            Six albino rats of either sex weighing between 150-180 gm were randomly selected for the study. They were marked suitably for ready identification. The animals were housed in colony cages under standard husbandry conditions.

 

On the previous day of experimentation, the food was withdrawn 18-hours advance. However water was allowed ad libitum. The fasting was continued till the completion of the experiment. On next day, the blood samples were withdrawn from tail vein (0.5 ml, each) for determination of basal glucose concentration. Then the animals were administered with plain 2% w/v acacia suspension (volume matched with the average volume of drugs administrated in the subsequent studies). Thereafter the blood samples (0.5 ml,each) were collected at 0, 0.5, 1, 2, 4, 6, 8, 12, 18 and 24  hours and analyzed for the determining the glucose concentration using GOD/POD method

            The percentage reduction in blood glucose levels at time ‘t’ was calculated by using the following equation.

 

Where, A = Initial blood glucose level before drug administration.

            B = Blood glucose levels at time ‘t’ after the drug administration.

 

4.4.2 Influence of Abraxane on blood glucose levels in healthy albino rats:

            The aim of this study was to understand the individual influence ofAbraxane(10mg/kg) therapeutic dose, on the blood glucose levels in healthy albino rats.

 

Materials and methods:

1.            Abraxane

2.            Gum acacia: 2%.

3.            Glucose estimation kit (Pathozyme diagnostic kit).

4.            Albino rats: Obtained from Mahavir Enterprizes, Hyderabad.

5.            sodium fluoride and potassium oxalate in 1:3 ratio

6.            Motor and pestle, alcohol, low voltage electric lamp, micropipette     (5-50µl), 1 ml graduated pipettes, epindroff tubes, thin aluminium foil, incubator and double distilled water etc.

7.            Semi autoanalyzer (RMS BCA-201).

8.            weighing balance (5mg-350gm) by shimatzu

 

Experimental procedure:

Six albino rats of either sex weighing between 150-180 gm were randomly selected for the study. They were marked suitably for ready identification. The animals were housed in colony cages under standard husbandry conditions.

 

On the previous day of experimentation, the food was withdrawn 18-hrs advance. However water was allowed ad libitum. The fasting was continued till the completion of the experiment. On next day, the blood samples were withdrawn from tail vein (0.5 ml, each) for determination of basal glucose concentration. Then the animals were administered with plain 2% w/v acacia suspension of Abraxane (volume matched with the average volume of drugs administrated in the subsequent studies). Thereafter the blood samples (0.5 ml,each) were collected at 0, 0.5, 1, 2, 4, 6, 8, 12, 18 and 24 hours and analyzed for the determining the glucose concentration using GOD/POD method

 

The percentage reduction in blood glucose levels at time ‘t’ was calculated by using the following equation.

Where, A = Initial blood glucose level before drug administration.

             B = Blood glucose levels at time ‘t’ after the drug administration.

 

4.4.3        Effect of Abraxane pre-treatment on the hypoglycemic activity of Nateglinide and Repaglinide in healthy albino rats:

            In the first part of this study, the hypoglycemic effect of Nateglinide and Repaglinidewas established in healthy albino rats.

           

            In the next part of this experiment, the effect of dose oAbraxanef 10mg/kg per day for one week on the hypoglycemic activity of Nateglinide and Repaglinidewas carried out in the same animals.

 

Materials and methods:

1.            Nateglinide: A solution was prepared by using 2% w/v gum acacia as a suspending agent to represent 370 mg/ml.

2.            Repaglinide: A suspension was prepared by using 2% gum acacia as a suspending agent to represent 45 mg/ml.

3.            Abraxane: The suspensions of Abraxane were prepared in 2% gum acacia to represent 192 mg/ml.

4.            Albino Rats: Obtained from Mahavir Enterprises, Hyderabad

5.            Glucose estimation Kit (Pathozyme diagnostic kit).

6.            Motor and pestle, alcohol, low voltage electric lamp, micropipette (5-50µl), 1 ml graduated pipettes, epindroff tubes, thin Aluminium foil, incubator and double distilled water etc.

7.            Semi autoanalyzer (RMS BCA-201).

8.            weighing balance (5mg-350gm) by shimatzu

 

 

Experimental procedure:

            Six albino rats of either sex weighing between 150-180 gm were selected for the study. The animals were randomly distributed into four groups (I, II, III, and IV); each group was consisting of 6 animals. They were marked suitably for ready identification. The animals were housed in colony cages under standard husbandry conditions.

 

On the previous day of experimentation, the food was withdrawn 18-hrs advance. However water was allowed ad libitum. The fasting was continued till the completion of the experiment. On next day, the blood samples were withdrawn from tail vein (0.5 ml,each) for determination of basal glucose concentration. Thereafter, the animals of first group were administrated with suspension of Nateglinide 50 mg/kg trough oral route. Blood sample were withdrawn from the tail vein at intervals of at 0, 0.5, 1, 2, 4, 6, 8, 12, 18 and 24 hours and analyzed for blood glucose concentration by GOD/POD method.

 

Animals of second group were administrated with suspension of Repaglinide0.3 mg/kg trough oral route. Blood sample were withdrawn from the tail vein at intervals of at 0, 0.5, 1, 2, 4, 6, 8, 12, 18 and 24 hours and analyzed for blood glucose concentration by GOD/POD method.

 

Animals of third group were administrated with suspension ofAbraxane 10mg/kg trough oral route for 7 days,on 7^th day, 6hours after the dose of in 2% Abraxaneacacia suspension of administration; the animals were fasted for 18 hours. This fasting was continued till the end of experiments. However water supplied ad libitum. On 8^th day 1 hour after the dose administration, after 60 min of respective treatment, Nateglinide 15 mg/kg, p.o. was administered to the same animals.  Thereafter the blood samples (0.5 ml, each) were collected at 0, 0.5, 1, 2, 4, 6, 8, 12, 18 and 24  hours and analyzed for the determining the glucose concentration using GOD/POD method.

 

Animals of fourth group were administrated with suspension of Abraxane10 mg/kg trough oral route for 7 days, on 7^th day, 6hours after the second dose of Abraxanein 2% acacia suspension of administration; the animals were fasted for 18 hours. This fasting was continued till the end of experiments. However water supplied ad libitum. On 8^th day 1 hour after the dose administration, after 60 min of respective treatment, R

paglinide0.3 mg/kg, p.o. was administered to the same animals.  Thereafter the blood samples (0.5 ml, each) were collected at 0, 0.5, 1, 2, 4, 6, 8, 12, 18 and 24  hours and analyzed for the determining the glucose concentration using GOD/POD method.

           

The percentage reduction in blood glucose levels at time‘t’ was calculated by using the following equation.

 

Where, A = Initial blood glucose level before drug administration.

             B = Blood glucose levels at time ‘t’ after the drug administration.

 

4.4.4Influence of Abraxane on blood glucose levels in healthy albino rabbits:

The experiment was carried out to find the effect of Abraxane single dose treatment (192 mg/kg) on blood glucose levels in healthy albino rabbits.

 

Materials and methods:

1.         Abraxane

2.         Gum acacia: 2%

3.         Glucose estimation kit (Pathozyme diagnostic Kit)

4.         Albino rabbits: obtained from Mahavir Enterprises, Hyderabad.

5.         Motor and pestle, alcohol, low voltage electric lamp, micropipette (5- 50µl), 1 ml graduated pipettes, epindroff tubes, thin Aluminium foil, incubator and double distilled water etc.

6.         Semi auto analyzer (RMS BCA-201).

7.         weighing balance (5mg-350gm) by shimatzu

 

Experimental procedure:

Abraxane suspension was prepared by using 2% gum acacia as suspending agent. The suspension was prepared to represent Abraxane 192 mg/kg for each rabbit.

 

            A group of 6 albino rabbits of either sex weighing between 1.5-1.8 kg were used in the study. They were suitably marked and kept in different cages. The animals were kept in colony cages at standard husbandry conditions. The animals were fasted for 18hours before commencing the experiment. During this period the rabbits were allowed to take adequate water and the fasting was continued till the end of the experiment.

 

Then Abraxane 192 mg/kgwas administered orally to all the rabbits. After that blood samples were collected at 0, 0.5, 1, 2, 4, 6, 8, 12, 18 and 24 hours after the administration of Abraxane and analyzed for glucose levels by using GOD/POD method. The blood glucose levels were expressed as mg/100ml of blood.

           

The % reduction in blood glucose levels at time ‘t’ was calculated by using the equation.

 

Where, A = Initial blood glucose level before drug administration.

             B = Blood glucose levels at time ‘t’ after the drug administration.

 

4.4.5 Effect of Abraxane pre-treatment on the hypoglycemic activity of Nateglinide and Repaglinide in healthy albino rabbits:

 

Materials and methods:

1. Nateglinide: A solution was prepared by using 2% w/v gum acacia as a suspending agent to represent 370 mg/ml.
2. Repaglinide: A suspension was prepared by using 2% gum acacia as a suspending agent to represent 45 mg/ml.
3. Abraxane: The suspensions of Abraxane prepared in 2% gum acacia to represent 192 mg/ml.
4. Albino Rabbits: obtained from Mahavir Enterprises, Hyderabad.
5. Glucose kit (Pathozyme diagnostic kit).
6. Motor and pestle, alcohol, low voltage electric lamp, micropipette           (5-50µl), 1 ml graduated pipettes, epindroff tubes, thin aluminium foil, incubator and double distilled water etc.
7. Semi-auto analyzer (RMS BCA-201).
8. Weighing balance (5mg-350gm) by Shimatzu.

 

Experimental procedure:

Albino rabbits of either sex weighing between 1.5-1.8 kg were divided into two groups (group I and II) of 6 animals each and they were marked conveniently. The animals were randomly distributed into different groups. The animals were kept in colony cages standard husbandry conditions. The animals were fasted for 18 hrs before commencing the experiment. During this period, rabbits were allowed to take sufficient water. Fasting was continued throughout the experiment. After 18 hours of fasting, the blood samples were collected for fasting blood glucose estimation from marginal ear vein of rabbits.

           

The animals in group I received suspension of Nateglinide 370 mg/kg and the animals in the group II received Repaglinide45 mg/kg through oral route. Blood samples were collected at 0, 0.5, 1, 2, 4, 6, 8, 12, 18 and 24 hours and blood glucose levels were estimated by GOD/POD method. Blood glucose levels were expressed as mg/dl of blood.

 

In the next part of this experiment, animals in the group III received suspension of Abraxane 192 mg/kg per day orally for one week. Group IV also received Abraxane 192 mg/kg per day orally for one week. On the 7^th day, 6 hours after administration of Abraxane the rabbits were fasted for 18 hrs. On the 8^th day, Abraxane 192 mg/kg was administered orally to groups III and IV respectively. After 60 minutes Nateglinide 370 mg/kg was administered to group III and Repaglinide 45 mg/kg was administered to group IV. Blood samples were collected thereafter at different time intervals up to 24 hrs. Blood glucose levels were estimated by GOD/POD method and expressed as mg/dl of blood.

 

Then the hypoglycemic activity of Nateglinide and Repaglinide at time‘t’ was calculated and the % of blood glucose reduction at various time intervals were calculated before and after Abraxane treatment.

 

 

Where, A = Initial blood glucose level before drug administration.

             B = Blood glucose levels at time ‘t’ after the drug administration.

           

4.5     Effect of Abraxane pre-treatment on the antidiabetic activity of Nateglinide and Repaglinide in diabetic rats :

 

The earlier experiments in this project have revealed that the Abraxane has got potential interaction with antidiabetic drugs like Nateglinide and Repaglinide in healthy albino rats and rabbits.

 

However, it is not clear whether Abraxane has got any influence on antidiabetic drugs in the pathophysiological conditions like diabetes mellitus in various species of animals.

 

Hence, in the present study, we have planned to use diabetic rats as experimental animals to clarify this aspect. In the earlier part of this study it was observed that pre-treatment with Abraxaneat dose (10 mg/kg) has significant influence at the peak hypoglycemia induced by Nateglinide andRepaglinide. Therefore the dose of Abraxane was selected for the study to verify the interactions in diabetic albino rats.

 

Various methods for induction of experimental diabetes¹⁰¹:

In experimental animals permanent diabetes can be produced by:

• Repeated injection of pituitary extract.
• Total pancreatectomy.
• Injection of insulin antibodies.
• Selective destruction of the beta cells by single injection of alloxan or closely allied compounds such as alloxantin, dialuric acid which are possibly converted to alloxan can also induce permanent diabetes.
• Streptozotocin destroys cells of the islet of Langerhans (in rats when given by I.P. in the dose of 35 to 55 mg which is dissolved in ice cold saline solution.
• Streptozotocin induced beta cell in the pancrease is due to the alkylation of DNA thereby producing hyperglycemia. Streptozotocin (50mg/kg i,p) induced diabetes in rats was selected for the present study.¹¹⁰

 

Materials and methods:

1.         Nateglinide: A solution was prepared by using 2% w/v Gum accacia as a suspending agent to represent 125 mg/ml.

2.         Repaglinide: A suspension was prepared (as explained earlier) to represent 7.5 mg/ml.

3.         Streptozotocin: (Hi Media laboratories Bombay). A solution of streptozotocin was prepared in ice cold saline to represent 100 mg/ml. (dose-50 mg/kg).

        Route: Intraperitonially.

4.         Albino Rats: Obtained from Mahavir Enterprises, Hyderabad.

5.         Glucose estimation kit (Pathozyme diagnostic kit).

6.         Motor and pestle, alcohol, low voltage electric lamp, micropipette (5 to 50µl), 1 ml graduated pipettes, epindroff tubes, thin aluminium foil, incubator and double distilled water etc.

7.         Semi-autoanalyzer (RMS BCA-201).

8.         Weighing balance (5mg-350gm) by Shimatzu.

 

Induction of diabetes:

Rats of either sex weighing between 150–250 gm were selected and fasted for 18 hours and water ad-libitum. The animals were randomly distributed into different groups. The animals were kept in colony cages at standard husbandry condition. The rats were administered with 50 mg/kg of Streptozotocin intraperitonially. After 48 hours, the blood samples were collected and analyzed for blood glucose level. It was found that diabetes was induced in about 48 hours. In our experiment the diabetes was characterized by weight loss and hyperglycemia. The blood samples were collected and analyzed for seven more days for stabilization of blood glucose levels. These animals were further used for our antidiabetic study. 

 

Experimental procedure:

Diabetic rats (blood glucose level more than 250mg/dl after 48hrs of STZ injection) of either sex were divided in to two groups (groups I and II) of 6 animals and they were marked conveniently. The animals were randomly distributed into different groups. The animals were kept in colony cages at standard husbandry condition. The animals were fasted for 18 hrs before commencing the experiment. During this period the rats were supplied with water ad libitum. Fasting was continued throughout the experiment. The blood samples were collected for fasting blood glucose estimation.

 

In the first part of this anti-diabetic study, the animals in group I, II and III received suspension of Nateglinide 50 mg/kg, Repaglinide 0.3 mg/kg and Abraxane 10 mg/kg respectively through oral route. Blood samples were collected at pre-determined time intervals and blood glucose levels were estimated by GOD/POD method.

 

In the next part of this experiment, all the animals in group I and II were treated with Abraxane at dose 10 mg/kg. On the 7^th day, 6 hrs after administration of Abraxane, the rats were fasted for 18hrs. On the 8^th day, Abraxane 15 mg/kg was administered orally to all the animals in group I and II. 60 minutes later, Nateglinide 50 mg/kg andRepaglinide 0.3 mg/kg was administered to group I and II respectively.  Blood samples were collected thereafter at different time intervals at 0, 0.5, 1, 2, 4, 6, 8, 12, 18 and 24 hours and were analyzed by GOD/POD method. Blood glucose levels were expressed as mg/dl of blood.

 

Then the antidiabetic activity of Nateglinide and Repaglinide at time ‘t’ was calculated and the % blood glucose reduction at various time intervals were calculated before and after Abraxane treatment.

 

                                               

 

Where, A = Initial blood glucose level before drug administration.

             B = Blood glucose levels at time ‘t’ after the drug administration.