Chapter 5 Herb-Drug Interactions: Glibenclamide

Transcription

1 Background Glibenclamide (GLI) is an oral hypoglycemic agent, belonging to the class of second generation sulfonylureas. Like all other sulfonylureas, it lowers the blood glucose by stimulating insulin release from pancreatic beta cells and may increase the insulin levels by reducing hepatic clearance of insulin. Glibenclamide is used in patients, who cannot control the blood glucose levels by diet and exercise alone. It is also established that about 5 to 10% of the patients, who respond to glibenclamide therapy initially would develop tolerance, which is mainly attributed to progressive loss of beta cells as the disease progresses and noncompliance of the patient with respect to dietary control. Some type-2 diabetic patients are given a combination of insulin and glibenclamide to ensure better glycemic control for which, reasonable functioning of beta cells is essential (Laurence, 2006). Some experiments performed on cultured human islets demonstrated apoptosis of beta cells by high doses of glibenclamide (Maedler et al., 2005). At the same time, comparative efficacy, safety and affordability of sulfonylureas, specifically glibenclamide cannot be underestimated. With the consideration of the available information, to keep insulin dependence at bay, optimization of sulfonylurea dosage can be a sensible approach. Such a strategy should aim at reducing the dose of sulfonylureas yet achieving required glycemic target. There are multiple ways of achieving it, primarily by modification of diet and exercise and other options include add-on drug therapy like metformin, glitazones, DPP-IV inhibitors or insulin(johnson et al., 1996, Marre et al., 2002, Goldstein et al., 2007, Riddle, 2002, Massi- Benedetti and Orsini-Federici, 2008). However, problem arises when patients are also on some herbal therapy, which may or may not be related to type-2 diabetes. Ashwagandha and sallaki are amongst such herbs, which are very popular in the community with high potential for their usage considering their spectrum of activity (Kulkarni and Dhir, 2008, Kimmatkar et al., 2003). Taking cue from such an approach, we designed a study to evaluate the influence of ashwagandha and sallaki on pharmacokinetic and pharmacodynamic actions of glibenclamide in normal and insulin resistant rats. 79

2 5.2. Materials and Methods Materials Drugs Material Details Manufacturer Glibenclamide 99.45% Sallaki Ashwagandha Consumables/Kits 81103RD AHWS9053 Glucose -- Tips (1mL, 200µL, 10 µl) Cadila Healthcare/Micro Labs, India The Himalaya Drug company, India The Himalaya Drug company, India Aspen Laboratories, New Delhi, India Tarsons, India Micro centrifuge tubes -- Tarsons, India Instruments Micropipette Eppendorf Eppendorf, Germany Refrigerated centrifuge Mikro Hettich, Germany Deep Freezer Carrier Carrier, India Analytical and statistical Tools Winnonlin Version 5.3 Pharsight Corporation, USA Graphpad Prism Version 5.0 GraphPad Software, USA Methods Animals and grouping The pharmacokinetic and pharmacodynamic experiments were performed in both normal and insulin resistant rats. Healthy normal rats weighing around grams and the insulin resistance (IR) rats (HCD+ 30% sucrose supplementation) weighing around grams were considered for the study. Six animals were allocated for each group. The procedures for housing, care and maintenance were same as elaborated in section All the animals were fasted overnight (12 hours) before the day of drug administration until 3 hours post dose. During the fasting period, for insulin resistant rats, 30% sucrose solution was replaced with normal drinking water. Water was allowed ad libitum for both normal and IR rats. 80

3 Treatment groups Sl. No. Group Treatment No of animals Normal Rats 01 NG Glibenclamide alone NGA Ashwagandha+ Glibenclamide NGS Sallaki +Glibenclamide NGPA 10 days pretreatment with Ashwagandha +Glibenclamide NGPS 10 days pretreatment with Sallaki + Glibenclamide 06 Insulin Resistant (IR) Rats 06 DG Glibenclamide alone DGA Ashwagandha+ Glibenclamide DGS Sallaki +Glibenclamide DGPA 10 days pretreatment with Ashwagandha +Glibenclamide DGPS 10 days pretreatment with Sallaki + Glibenclamide Dosing Selection of Dose: As per the approved product monograph (dia eta,usfda), Glibenclamide(GLI) is available in different unit dose formulations from 1.25 to 10 mg and can be administered up to 20 mg daily (Coppack et al., 1990). Based on the routinely used dose and sensitivity of the bioanalytical method, a dose of mg/kg equivalent to 10 mg human dose was selected for IR rats. A lower dose of 0.45 mg/kg glibenclamide was selected for normal rats because of possibility of profound hypoglycemia during first 03 hours of continued fasting post dose. Ashwagandha dry extract was administered at the dose of 22.5 mg/kg b.w. and Sallaki was administered at the dose of mg/kg b.w. The doses were selected based upon recommended human dosage of ashwagandha (250 mg/day) and Sallaki (125 mg/day) (as per the label recommendations; Himalaya pure herbs). The herbs were administered either as a single dose, simultaneously with the test drug or as pretreatment for 10 days with once daily dosage depending on the treatment group. Preparation of Dose: Glibenclamide: The weighed quantity was triturated with 0.05% tween 80 in a clean dry glass mortar and the volume was made up to achieve a concentration of 0.35 mg/ ml and vortexed. The doses were administered to the rats depending on their body weight. Ashwagandha: Weighed amount was dissolved in 0.05% tween 80, volume was made up to achieve a concentration of 7.5 mg/ml and vortexed. Though ashwagandha dry extract was miscible with water, tween-80 was used to keep the vehicle constant. 81

4 Sallaki: Weighed amount was dissolved in 0.05% tween 80, volume was made up to achieve a concentration of 3.75 mg/ml and vortexed. Drug administration: The drugs were administered by gavage Blood Sample Collection For pharmacokinetic evaluation of GLI, about 12 blood samples of approximately 200 µl were collected by retro-orbital sinus bleeding technique. Zero hour sample was collected before dosing and post dose samples were collected at 0.33, 0.67, 1.0, 1.5, 2.0, 2.5, 3.0, 4.0, 8.0, 12.0 and 24.0 hours in micro centrifuge tubes containing K 2 EDTA. The time points were designed based on reported C max, T max, t 1/2 of the drug. Extent of blood loss was considered upon carefully reviewing other PK experiments performed on albino rats. (Makino et al., 2004, Atack et al., 2006) Sample Preparation and analysis The blood samples were centrifuged at 3000 rpm for 15 minutes, 100 µl plasma was separated and frozen at -20 o C till further analysis. The remaining plasma was taken for glucose estimation by glucose oxidase-peroxidase method with commercially available kit Assessment of Pharmacokinetic Interaction Pharmacokinetic evaluation included estimation of C max, t max, AUC 0-t, AUC 0-, V d, Cl, t 1/2 and K el by non-compartmental analysis using Winnonlin version 5.3. Model No 200 was selected from Winnonlin model library, which is designed for noncompartmental analysis of plasma concentration Vs data obtained after extravascular input and uses dose and time of last dose as constants. Following parameters were assessed for evaluating the pharmacokinetic variation in presence and absence of concomitant herbal medicines. 82

5 Sl.No Notation Pharmacokinetic Parameters Description 1 C max Maximum observed concentration, occurring at T max 2 t max Time of maximum observed concentration 3 AUC 0-t Area under the curve from the time of dosing to the last measurable concentration calculated using linear trapezoidal rule 4 AUC 0- Area under the curve from the time of dosing extrapolated to infinity, based on the last observed concentration. 5 V d Volume of distribution based on the terminal phase. 6 Cl Total body clearance for extravascular administration. 7 t 1/2 Terminal half-life 8 K el First order rate constant associated with the terminal (log-linear) portion of the curve. Estimated by linear regression of time vs. log concentration. Extent of bioavailability as measured by C max and AUC and the derived parameters, volume of distribution and total body clearance were compared to assess the extent of pharmacokinetic interaction with one-way analysis of variance followed by suitable post-test to assess the extent of interaction. Graph pad Prism version 5.0 was used to perform all statistical calculations. 83

6 Assessment of Pharmacodynamic Interaction Pharmacodynamic evaluation included estimation of plasma glucose concentrations at the scheduled time along with PK sampling. The data assessment involved plotting of response (plasma glucose) Vs time and subsequent group wise comparison of the obtained data in both actual form and percent-normalized form. The analysis of response Vs time data was performed by non-compartmental pharmacodynamics analysis WinNonlin version 5.3. Model No 220 was selected from WinNonlin model library, which is designed for noncompartmental analysis of response Vs time data. The parameters for the evaluation included comparison of AUC above the baseline, AUC below the baseline, Net AUC, Maximum observed response value, minimum observed response value, duration of response above baseline, duration of response below baseline. The parameters related to threshold were not employed, as a specific threshold for plasma glucose level decrease was not applicable. 84

7 Sl.No Pharmacodynamic Parameters Notation Description 1 Baseline (B) Response values at the time of dosing (0 hour plasma glucose level) 2 AUC_Above_B Area Under the curve that is above baseline (Linear trapezoidal rule) 3 AUC_Below_B Area that is below the baseline and above the response curve 4 AUC_Net_B AUC_Above_B - AUC_Below_B. This is lower or negative value for inhibitory effects 5 R max Maximum observed response 6 R min Minimum observed response 7 T max Time of maximum observed response value (Rmax). 8 T min Time of minimum observed response value (Rmin). 9 Time_Above_B Total time that Response >= Baseline 10 Time_ Below_B Total time that Response < Baseline Plasma glucose values were obtained simultaneously with the PK profiling. Glucose level was response variable and time was an independent variable. As the study involved single dose, dosing time was considered as 0 hr. The plasma glucose level obtained at 0 hour (predose plasma glucose level) was taken as baseline value for each rat. Extent of influence of glibenclamide on the blood glucose levels was assessed in presence or absence of herbal drug either as simultaneous administration or as pretreatment in both normal and insulin resistance conditions. The obtained response data was analyzed in two ways. First, the data up to 03 hour post dose (0 to 3 hr) was evaluated and the second, entire profile (0-12 hr) was analyzed. This was done to understand and differentiate the responses to the drug during fasting conditions and overall response. The extent of pharmacodynamics interaction was assessed with one-way analysis of variance with suitable post-test. Graphpad Prism version 5.0 was used to perform all statistical calculations. 85

8 5.3. Results Pharmacokinetic and pharmacodynamic evaluations were performed in both normal and insulin resistant rats. Glibenclamide PK parameters, C max, t max, AUC 0-t, AUC 0-, V d, Cl, t 1/2 and K el and the PD parameters post glibenclamide treatment, viz., baseline glucose levels (B), Rmin, Rmax, Tmin, Tmax, Net AUC_B, Time_Above_B and Time Below_B for plasma glucose profile were calculated to estimate the herb-drug interaction with Ashwagandha and Sallaki. The average values of measures of bioavailability as estimated by Cmax, AUCs and extent of removal of the drug from the body as estimated by CL along with average Rmin, Net_AUC_B, and median Time_Above_B/Time_Below_B of plasma glucose profile post glibenclamide were primarily considered for statistical comparison Pharmacokinetic interaction in normal rats Healthy, inbred albino rats of Wistar strain were selected for this study. The animals were about 06 months old, weighing g. The dosing and subsequent pharmacokinetic profiling was initiated after an overnight fast. The animals were provided with food 3 hours after dosing. Drinking water was not restricted. In pretreatment groups, the allocated herbal pretreatment was given 02 hours before glibenclamide dosing in order to avoid absorption related issues Effect of Ashwagandha C max of glibenclamide in NGA was ± ng/ml. This was lesser than that of control (NG) group ( ±4.882 ng/ml). However, the change was not statistically significant. The AUCs upon comparison did not show statistically significant difference with respect to NG. However, NGA showed decreased AUC as compared to control with mean AUC 0-t and AUC 0- at ± and ± hr*ng/ml respectively, whereas those of control group were ± and ± hr*ng/ml. This shows that there is a marked decrease in bioavailability to an extent of about 7.32% with C max and about 26% reduction in AUC after simultaneous administration of glibenclamide with Ashwagandha. There was a noticeable increase in total systemic clearance. This may be due to decrease in AUC as non-compartmental model uses dose and AUC to calculate total systemic clearance. The graphical comparisons are presented in figure

9 C max of glibenclamide in groups pretreated with ashwagandha were significantly higher (p<0.05) as compared to control group. C max of NGPA was ± ng/ml, which was a 61% increase as compared to control. The mean systemic exposure (AUC) was higher. The clearance values were almost similar to that of control. There were no variations with respect to time taken to achieve C max (average) and mean plasma half-life (t 1/2 ) irrespective of simultaneous or prior administration (pretreatment). Considering the median values, the T max was 3 hours and half-life was around 10 hours across the treatment groups NG, NGA and NGPA. The details are presented in table Effect of Sallaki In a similar approach, Sallaki (SAL) was administered simultaneously with glibenclamide and as pretreatment for 10 days. A marked difference in pharmacokinetics of glibenclamide was observed. In case of simultaneous administration of SAL, the observations were similar to that of ASH with C max much lesser than control with ±5.956 ng/ml, a mean decrease of about 15% as compared to control. The observed AUC was also low indicating reduced systemic exposure. However, the test of significance did not confirm the same statistically. The clearance values across the treatments were comparable. The pretreatment group showed marked increase in C max values as compared to control. The mean observed C max of NGPS was ± ng/ml with about 129% increase as compared to control. The increase in AUC was not statistically significant. The clearance values were similar to that of control group. The average T max values did not clearly indicate the variation but the median T max was 3 hours for NG. NGS and NGPS showed 2.5 hours. This indicates that peak plasma values of glibenclamide were advanced by about 30 min upon sallaki pretreatment. The t 1/2 was significantly lower in case of NGPS at 6.235±0.437 hours as compared to control at ±0.537 hours. This may be explained by lower apparent volume of distribution (Vd) 4.119±0.587 L/Kg as compared to control which had 7.151±0.428 L/Kg. However, clearance values were comparable to control group. The details of concentration vs time profile, pharmacokinetic parameters and associated plots are presented in Table 3.1 to 3.2 and Figure 3.1 to

10 CL (L/hr/Kg) Glibenclamide (ng/ml) AUC (hr*ng/ml) Chapter 5 Table 3.1: Pharmacokinetic parameters of glibenclamide (Normal Rats) Treatment Estimate T max C max AUC 0-t AUC 0- Vd CL T 1/2 K el Units hr ng/ml hr*ng/ml hr*ng/ml L/kg L/hr/kg hr 1/hr NG Mean SEM NGA Mean * SE NGPA Mean * SE NGS Mean * SE NGPS Mean * * * SE *P<0.05 vs NG; Oneway-ANOVA followed by Dunnett s test; n=6 C max AUC 0-t * * NG NGA NGPA NGS NGPS NG NGA NGPA NGS NGPS Treatment 0 Treatment Clearance NG NGA NGPA NGS NGPS Treatment Figure 3.1: Comparison of pharmacokinetic parameters of glibenclamide in normal rats. The data represents Mean±SEM 88

11 Time Treatment Animal NG NG NG NG NG NG NG N Mean SD SE Min Max NGA NGA NGA NGA NGA NGA NGA N Mean SD SE Min Max Table 3.2: Glibenclamide Concentration Table (ng/ml): Normal Rats

12 Time Treatment Animal NGPA NGPA NGPA NGPA NGPA NGPA NGPA N Mean SD SE Min Max NGPS NGPS NGPS NGPS NGPS NGPS NGPS N Mean SD SE Min Max Table 3.2: Glibenclamide Concentration Table (ng/ml) : Normal Rats (contd.,)

13 Time Treatment Animal NGS NGS NGS NGS NGS NGS NGS N Mean SD SE Min Max Table 3.2: Glibenclamide Concentration Table 9ng/mL) : Normal Rats (contd.,)

14 Figure 3.2: Glibenclamide Pharmacokinetic profiles in normal rats (Treatment NG) Figure 3.3: Glibenclamide Pharmacokinetic profiles in normal rats (Treatment NGA) 92

15 Figure 3.4: Glibenclamide Pharmacokinetic profiles in normal rats (Treatment NGPA) Figure 3.5: Glibenclamide Pharmacokinetic profiles in normal rats (Treatment NGPS) 93

16 Figure 3.6: Glibenclamide Pharmacokinetic profiles in normal rats (Treatment NGS) Figure 3.7: Mean glibenclamide pharmacokinetic profiles in normal rats. The data represents Mean±SEM 94

17 Pharmacokinetic Interaction in Rats with Insulin Resistance The rats with impaired glucose tolerance and strikingly increased insulin levels post glucose challenge mimicking the insulin resistance were randomized for this study. The rats were on HCD since weaning at least for 6 months and were on 30% additional sucrose supplementation since at least one month. The rats weighed around g. The pharmacokinetic profiling was initiated after overnight fast followed by dosing. The animals were provided with food 3 hours after dosing. During fasting until 03 hours post dose, drinking water was allowed. In pretreatment groups, the allocated herbal pretreatment was given 02 hours before glibenclamide dosing in order to avoid drug absorption related issues Effect of Ashwagandha on Glibenclamide Pharmacokinetics C max of glibenclamide administered simultaneously with ashwagandha (DGA) was ± ng/ml. This was lesser than that of control (DG) group ( ± ng/ml). However, the change was not statistically significant. The AUCs upon comparison did not show statistically significant difference with respect to control. C max of glibenclamide in groups pretreated with ashwagandha were significantly higher (p<0.05) as compared to control group. C max of DGAP was ±78.134ng/mL, which was an 118% increase as compared to control. The mean systemic exposure (AUC) was high accordingly. The clearance values did not vary significantly. Some variations in T max were observed with decrease in T max of about 0.5 to 01 hour across different groups. The variations in elimination half-life were not significant except for DGA where elimination half-life appeared to be prolonged by 2 to 2.5 hours with ±1.06 hrs as compared to that of control, 8.003±1.174 hrs. The details are presented in Table Effect of Sallaki on Glibenclamide Pharmacokinetics Sallaki (SAL) was administered simultaneously with glibenclamide and as a pretreatment for 10 days before glibenclamide PK assessment. Upon simultaneous administration of SAL, overall bioavailability increased significantly. The average peak plasma concentration of GLI with simultaneous SAL (DGS) was ± ng/ml, AUC 0-t and AUC 0- were ± and ± hr*ng/ml respectively. Whereas for DG (control), they were at ± ng/ml (Cmax), ± hr*ng/ml (AUC 0-95

18 t) and ± hr*ng/ml(auc 0- ). The increase in Cmax was about 187% and percentage increase in systemic exposure was about 113% Total systemic clearance (CL_F) decreased significantly with 0.125±0.019 L/hr/Kg as compared to that of DG 0.259±0.034 L/hr/Kg. No significant changes were observed with Tmax and elimination half-life. Significant increase in bioavailability was observed in SAL pretreated group (DGSP) with mean Cmax at ± ng/ml, AUC0-t at ± hr.ng/ml and AUC0- at ± ng/ml as compared to DG. The clearance values were significantly lower (0.142±0.005 L/hr/Kg) when compared to DG. No significant changes were observed with Tmax and elimination half-life. The details of concentration Vs time profile, pharmacokinetic parameters and associated plots are presented in Table 3.3 to 3.4 and Figure 3.8 to 3.14 Table 3.3: Pharmacokinetic parameters of glibenclamide: IR Rats Treatment Estimate Tmax Cmax AUC0-t AUC0- Vd CL T1/2 Kel Units hr ng/ml hr*ng/ml hr*ng/ml L/kg L/hr/kg hr 1/hr DG Mean SE DGA Mean * * SE DGAP Mean * SE DGS Mean * * * * SE DGSP Mean * * * * SE *P<0.05 vs DG; Oneway-ANOVA followed by Dunnett s test; n=6 96

19 CL (L/hr/Kg) Glibenclamide (ng/ml) AUC (hr*ng/ml) Chapter 5 C max AUC 0-t * * * DG DGA DGAP DGS DGSP * * DG DGA DGAP DGS DGSP 0 Treatment 0 Treatment Clearance * * DG DGA DGAP DGS DGSP 0.0 Treatment Figure 3.8: Comparison of pharmacokinetic parameters in IR rat. The data represents Mean±SEM 97

20 Time Treatment Animal DG DG DG DG DG DG DG N Mean SD SE Min Max DGA DGA DGA DGA DGA DGA DGA N Mean SD SE Min Max Table 3.4: Glibenclamide Concentration Table (ng/ml): IR Rats

21 Time Treatment Animal DGAP DGAP DGAP DGAP DGAP DGAP DGAP N Mean SD SE Min Max DGS DGS DGS DGS DGS DGS DGS N Mean SD SE Min Max Table 3.4: Glibenclamide Concentration Table (ng/ml): IR Rats (Contd.,)

22 Time Treatment Animal DGSP DGSP DGSP DGSP DGSP DGSP DGSP N Mean SD SE Min Max Table 3.4: Glibenclamide Concentration Table (ng/ml): IR Rats (Contd.,)

23 Figure 3.9: Glibenclamide Pharmacokinetic profiles in IR rats (Treatment DG) Figure 3.10: Glibenclamide Pharmacokinetic profiles in IR rats (Treatment DGA) 101

24 Figure 3.11: Glibenclamide Pharmacokinetic profiles in IR rats (Treatment DGAP) Figure 3.12: Glibenclamide Pharmacokinetic profiles in IR rats (Treatment DGS) 102

25 Figure 3.13: Glibenclamide Pharmacokinetic profiles in IR rats (Treatment DGSP) Figure 3.14: Mean glibenclamide pharmacokinetic profiles in IR rats. The data represents Mean±SEM. 103

26 Pharmacodynamic interaction in normal rats Analysis of 0-3 hour plasma glucose profile: The baseline values of fasting normal rats were in the range of ±0.903 mg/dl to ±0.866 mg/dl. Both the pretreated groups (NGPA and NGPS) had lower baseline values at 50 mg/dl whereas NG, NGA, NGS had approximately 70 mg/dl.. Upon glibenclamide administration, the plasma glucose levels further lowered with % decrease (with respect to base line) of 33.75±3.35, ±1.86, ±3.54, and ±1.25 and ±5.68 % for NG, NGA, NGPA, NGPS and NGS respectively. Net AUC_B demonstrated profound hypoglycemia in NG, NGA and NGS for the first 03 hours. However, the mean Net_AUC_B for NGPA and NGPS demonstrated reduced extent of hypoglycemia in the first 03 hours. Analysis of 0-12 hour plasma glucose profile: The profile showed continued hypoglycemic effect of the drug even after providing food ad libitum after 03 hours of dosing. Net_AUC_B of ±10.423, ±51.122, ±26.103, ± hr*mg/dl were observed for NGA, NGPA, NGPS and NGS respectively as compared to NG (Control) ± hr*mg/dl. The median duration of hypoglycemia (with respect to baseline) was 2.77, 1.16, 1.07 and 2.86 hours as compared to that of NG, which stood at Considering the entire profile, it can be pointed out that approximately up to 8 hours post dose, the plasma glucose levels continued to be below 100 mg/dl across all the treatments except NGPA, which went up to ±9.09 mg/dl at 8th hour post dose and reduced to ±1.96 mg/dl by 12 th hour. The details are presented in tables 4.1 to 4.3 and figures 4.1 and

27 AUC (min*g/dl) Hours (hr) Net AUC (hr*mg/dl) Hours (hr) Chapter 5 Net AUC with respect to baseline 03 hours post dose 40 4 Plasma glucose profile 03 hour post dose Median Time NG NGA NGPA NGPS NGS 2 1 NG NGA NGPA NGPS NGS -40 Treatment 0 Tmax Tmin Time_Above_B Time_Below_B Parameters Plasma glucose profile 12 hour post dose Median Time 15 Net AUC with respect to baseline 12 hours post dose NG NGA NGPA NGPS NGS 10 5 NG NGA NGPA NGPS NGS Treatment 0 Tmax Tmin Time_Above_B Time_Below_B Parameters Figure 4.1: Comparison of pharmacodynamic parameters in normal rats. AUC represents Mean±SEM and time as Median. 105

28 Time Treatment Animal NG Mean SD SE Min Max NGA Mean SD SE Min Max NGPA Mean SD SE Min Max NGPS Mean SD SE Min Max NGS Mean SD SE Min Max Chapter Table 4.1: Plasma glucose levels (mg/dl) of normal rats : 0-12 hours post dose

29 Figure 4.2: Plasma glucose profile obtained simultaneously with PK profile in normal rats. The data represents Mean±SEM 107

30 NGA Mean SD SE Median NGPA Mean SD SE Median NGPS Mean SD SE Median NGS Mean SD SE Median Table 4.2: Plasma glucose profile parameters of normal rats: 0-3 hours post dose Treatment Animal Baseline Rmax Rmin Tmax Tmin AUC_ Above_B AUC_ Below_B AUC_ Net_B Time_ Above_B Units mg/dl mg/dl mg/dl hr hr hr*mg/dl hr*mg/dl hr*mg/dl hr hr Time_ Below_B NG Mean SD SE Median

31 NGA Mean SD SE Median NGPA Mean SD SE Median NGPS Mean SD SE Median NGS Mean SD SE Median Table 4.3: Plasma glucose profile parameters of normal rats: 0-12 hours post dose Treatment Animal Baseline Rmax Rmin Tmax Tmin AUC_ AUC_ AUC_ Time_ Time_ Above_B Below_B Net_B Above_B Below_B Units mg/dl mg/dl mg/dl hr hr hr*mg/dl hr*mg/dl hr*mg/dl hr hr NG Mean SD SE Median

32 Pharmacodynamic Interaction in IR rats The baseline plasma glucose values were ±4.57, ±3.88, ±1.887, ±4.391 and ±4.275 mg/dl for DG, DGA, DGAP, DGS and DGSP respectively. Similar to the observations made in normal animals, the 0 hour plasma glucose level concentrations were significantly less in pretreated rats as compared to control. Analysis of 0-3 hour plasma glucose profile: R max was significantly less in both the pretreatment group with ±1.649 mg/dl for DGAP and ±4.013mg/dL for DGSP as compared to control (DG) at ±3.916 mg/dl. Simultaneous treatment showed marginal decrease in both simultaneous herbal treatments (DGA and DGS). In case of DGAP and DGSP, R min was ±2.772 and ±2.69 mg/dl, which was significantly low when compared to DG with ±4.574 mg/dl. The other groups showed marginal decrease in blood glucose level. Median T min for DGA, DGAP and DGSP was 0.67 hrs whereas for DGS, it was 0 hr. and for control, it was 0.33hrs. Analysis of 0-12 hour plasma glucose profile: R max was significantly less in both the pretreatment groups with ±4.18 mg/dl for DGAP and ±3.822mg/dL for DGSP as compared to control (DG) at ±3.916 mg/dl. Simultaneous treatment showed marginal reduction in both simultaneous herbal treatments (DGA and DGS). In case of DGAP and DGSP, R min was ±2.772 and ±2.69 mg/dl, which was significantly less when compared to DG with ± mg/dl. Median T min for DGA, DGS, DGAP and DGSP was 0.67 hrs and for control, it was 2.17hrs. Median T max was 2.5, 3.0, 2.0 for DG, DGA and DGS where as DGAP and DGSP had median T max of about 4 hours. AUC analysis revealed that Net_AUC_B was significantly less in both the pretreated groups with ± and ± hr/mg/dl for DGAP and DGSP respectively as compared to ± hr*mg/dl for control (DG). Net_AUC_B changes in simultaneous herb administration groups were variable and stood at ± , ± hr/mg/dl for DGA and DGS respectively. The treatment differences were evident with the 12 hour plasma glucose profile in IR rats. The profile reveled that in both pretreated groups, the blood glucose levels were regulated similar to normal rats with plasma glucose levels hardly crossing 100 mg/dl. Where as in DGA and DGS groups, the plasma glucose level reduction was significant after 4 th hour and continued to maintain the baseline plasma glucose levels till 12 th hour. This was not the same with glibenclamide alone (DG) where significant fall of glucose levels were observed at

33 AUC (min*g/dl) Hours (hr) AUC (min*g/dl) Hours (hr) Chapter 5 hour till 04 hours, but 08 hour and 12 hour samples showed upward trend in plasma glucose levels. The details are presented in tables 4.4 to 4.6 and figures 4.3 and 4.4. Analysis of hour plasma insulin profile: The obtained insulin levels demonstrated the changed insulin secretion pattern in presence of ASH and SAL especially after pretreatment. DG and DGA demonstrated similar profiles. Pretreated groups either demonstrated a steady insulin level or increased magnitude of spike in DGAP and DGSP groups respectively. However, as the data was limited and gathered for qualitative purpose, no tests of significance were applied. Figure 4.5 gives the graphical presentation of results. 4.0 Plasma glucose profile 03 hour post dose Median Time Net AUC with respect to baseline 03 hours post dose DG DGA DGAP DGS DGSP DG DGA DGAP DGS DGSP Treatment 0.0 Tmax Tmin Time_Above_B Time_Below_B Parameters Plasma glucose profile 12 hour post dose Median Time 15 Net AUC with respect to baseline 12 hours post dose DG DGA DGAP DGS DGSP 10 5 DG DGA DGAP DGS DGSP 0 Treatment 0 Tmax Tmin Time_Above_B Time_Below_B Parameters Figure 4.3: Comparison of pharmacodynamic parameters in IR rats. The data represents Mean±SEM for AUC and Median for time. 111

34 Time Treatment DG Mean SD SE Min Max DGA Mean SD SE Min Max DGAP Mean SD SE Min Max DGS Mean SD SE Min Max DGSP Mean SD SE Min Max Table 4.4: Plasma glucose levels (mg/dl) of IR rats: 0-12 hours post dose

35 Figure 4.4: Plasma glucose profile obtained simultaneously with PK profile in IR rats. The data represents Mean±SEM Chapter 5 113

36 DGA Mean SD SE Median DGAP Mean SD SE Median DGS Mean SD SE Median DGSP Mean SD SE Median Table 4.5: Plasma glucose profile parameters of IR rats: 0-3 hours post dose Treatment Baseline Rmax Rmin Tmax Tmin AUC_ Above_B AUC_ Below_B AUC_ Net_B Time_ Above_B Units mg/dl mg/dl mg/dl hr hr hr*mg/dl hr*mg/dl hr*mg/dl hr hr DG Mean SD SE Median Time_ Below_B

37 DGA Mean SD SE Median DGAP Mean SD SE Median DGS Mean SD SE Median DGSP Mean SD SE Median Chapter Table 4.6: Plasma glucose profile parameters of IR rats: 0-12 hours post dose Treatment Baseline Rmax Rmin Tmax Tmin AUC_ AUC_ AUC_ Time_ Time_ Above_B Below_B Net_B Above_B Below_B Units mg/dl mg/dl mg/dl hr hr hr*mg/dl hr*mg/dl hr*mg/dl hr hr DG Mean SD SE Median