5. 5.1. 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
5.2. Materials and Methods 5.2.1. 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 5.2.2. Methods 5.2.2.1.Animals and grouping The pharmacokinetic and pharmacodynamic experiments were performed in both normal and insulin resistant rats. Healthy normal rats weighing around 150-200 grams and the insulin resistance (IR) rats (HCD+ 30% sucrose supplementation) weighing around 350-450 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 4.2.2.1. 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
Treatment groups Sl. No. Group Treatment No of animals Normal Rats 01 NG Glibenclamide alone 06 02 NGA Ashwagandha+ Glibenclamide 06 03 NGS Sallaki +Glibenclamide 06 04 NGPA 10 days pretreatment with Ashwagandha +Glibenclamide 06 05 NGPS 10 days pretreatment with Sallaki + Glibenclamide 06 Insulin Resistant (IR) Rats 06 DG Glibenclamide alone 06 07 DGA Ashwagandha+ Glibenclamide 06 08 DGS Sallaki +Glibenclamide 06 09 DGPA 10 days pretreatment with Ashwagandha +Glibenclamide 06 10 DGPS 10 days pretreatment with Sallaki + Glibenclamide 06 5.2.2.2.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 1.042 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 11.25 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
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. 5.2.2.3.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). 5.2.2.4.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. 5.2.2.5.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
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
5.2.2.6.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
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
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. 5.3.1. 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 150-225g. 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. 5.3.1.1.Effect of Ashwagandha C max of glibenclamide in NGA was 139.913± 11.554 ng/ml. This was lesser than that of control (NG) group (150.959±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 1203.623±71.404 and 1612.915±144.827 hr*ng/ml respectively, whereas those of control group were 1690.677±99.225 and 2174.487±156.583 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 3.1. 86
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 244.489±43.141 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 3.1 5.3.1.2.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 128.433±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 345.337±30.745 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 10.196±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 3.7 87
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 3.167 150.959 1690.677 2174.487 3.537 0.202 10.196 0.069 SEM 0.279 4.882 99.225 156.583 0.370 0.016 0.537 0.004 NGA Mean 3.000 139.913 1203.623 1612.915 5.069* 0.283 11.604 0.061 SE 0.000 11.554 71.404 144.827 0.199 0.023 0.743 0.004 NGPA Mean 2.833 244.489* 1964.361 2347.904 2.596 0.195 9.391 0.075 SE 0.105 43.141 143.573 178.859 0.431 0.016 0.506 0.004 NGS Mean 2.833 128.433 1315.641 1904.362 5.125* 0.270 12.285 0.061 SE 0.105 5.956 167.524 360.836 0.301 0.056 1.508 0.007 NGPS Mean 2.667 345.337* 2294.077 2485.462 2.167* 0.198 6.235* 0.114 SE 0.105 30.745 296.626 344.646 0.344 0.031 0.437 0.008 *P<0.05 vs NG; Oneway-ANOVA followed by Dunnett s test; n=6 C max AUC 0-t 400 300 200 * * NG NGA NGPA NGS NGPS 3000 2000 NG NGA NGPA NGS NGPS 100 1000 0 Treatment 0 Treatment 0.4 0.3 0.2 Clearance NG NGA NGPA NGS NGPS 0.1 0.0 Treatment Figure 3.1: Comparison of pharmacokinetic parameters of glibenclamide in normal rats. The data represents Mean±SEM 88
Time Treatment Animal 0.00 0.33 0.67 1.00 1.50 2.00 2.50 3.00 4.00 8.00 12.00 24.00 NG NG1 0.00 33.08 103.74 47.30 62.90 97.00 134.62 129.30 128.91 63.89 50.78 34.76 NG2 0.00 55.05 87.14 51.10 66.76 79.97 84.24 143.49 107.42 66.96 45.05 23.82 NG3 0.00 37.04 116.16 52.97 78.44 88.62 114.75 132.78 168.35 95.54 79.26 46.92 NG4 0.00 28.13 92.80 51.79 67.34 118.42 153.43 142.57 111.34 87.56 75.00 32.10 NG5 0.00 61.64 97.57 43.78 64.84 87.63 131.94 159.09 128.62 85.54 74.44 26.68 NG6 0.00 31.50 79.12 50.00 73.97 87.82 108.14 96.46 146.76 100.44 69.90 28.74 N 6 6 6 6 6 6 6 6 6 6 6 6 Mean 0.000 41.073 96.088 49.490 69.043 93.243 121.187 133.948 131.903 83.323 65.739 32.171 SD 0.000 13.840 12.979 3.395 5.934 13.466 24.135 21.099 22.764 14.905 14.239 8.198 SE 0.000 5.650 5.299 1.386 2.422 5.498 9.853 8.614 9.293 6.085 5.813 3.347 Min 0.00 28.13 79.12 43.78 62.90 79.97 84.24 96.46 107.42 63.89 45.05 23.82 Max 0.00 61.64 116.16 52.97 78.44 118.42 153.43 159.09 168.35 100.44 79.26 46.92 NGA NGA1 0.00 48.30 37.12 93.82 91.54 117.26 137.41 153.24 81.83 50.40 41.36 20.37 NGA2 0.00 43.13 33.15 83.78 81.74 104.72 122.71 136.85 73.08 45.01 36.94 18.19 NGA3 0.00 25.97 39.72 36.92 48.01 56.48 85.60 176.54 93.93 71.84 61.15 33.98 NGA4 0.00 13.02 25.21 35.42 54.04 76.14 103.24 113.68 81.34 54.85 43.18 20.39 NGA5 0.00 23.19 35.47 32.97 42.87 50.44 76.44 157.65 83.88 64.15 54.61 30.34 NGA6 0.00 11.62 22.51 31.63 48.26 67.99 92.19 101.52 72.64 48.98 38.56 18.21 N 6 6 6 6 6 6 6 6 6 6 6 6 Mean 0.000 27.538 32.198 52.423 61.078 78.839 102.931 139.913 81.117 55.872 45.967 23.581 SD 0.000 15.228 6.858 28.417 20.351 26.750 23.229 28.302 7.858 10.197 9.703 6.816 SE 0.000 6.217 2.800 11.601 8.308 10.920 9.483 11.554 3.208 4.163 3.961 2.783 Min 0.00 11.62 22.51 31.63 42.87 50.44 76.44 101.52 72.64 45.01 36.94 18.19 Max 0.00 48.30 39.72 93.82 91.54 117.26 137.41 176.54 93.93 71.84 61.15 33.98 89 Table 3.2: Glibenclamide Concentration Table (ng/ml): Normal Rats
Time Treatment Animal 0.00 0.33 0.67 1.00 1.50 2.00 2.50 3.00 4.00 8.00 12.00 24.00 NGPA NGPA1 0.00 90.32 134.99 136.66 148.55 161.59 229.88 233.33 195.48 102.82 83.36 43.53 NGPA2 0.00 112.38 152.46 180.48 252.21 255.76 316.00 306.92 186.80 55.00 24.03 11.59 NGPA3 0.00 149.42 202.70 239.96 335.33 340.06 420.14 408.07 248.36 73.12 31.95 15.41 NGPA4 0.00 67.94 101.53 102.78 111.73 121.53 172.90 175.49 147.02 77.33 62.69 32.74 NGPA5 0.00 130.90 89.67 123.13 141.25 160.13 163.84 183.76 138.94 88.56 73.35 34.16 NGPA6 0.00 98.45 67.44 92.61 106.24 120.44 123.22 138.21 104.50 66.60 55.17 25.70 N 6 6 6 6 6 6 6 6 6 6 6 6 Mean 0.000 108.235 124.798 145.937 182.551 193.251 237.663 240.964 170.182 77.239 55.091 27.189 SD 0.000 29.215 48.995 55.400 91.535 87.184 111.566 100.447 50.704 16.775 23.195 12.088 SE 0.000 11.927 20.002 22.617 37.369 35.593 45.547 41.007 20.700 6.849 9.469 4.935 Min 0.00 67.94 67.44 92.61 106.24 120.44 123.22 138.21 104.50 55.00 24.03 11.59 Max 0.00 149.42 202.70 239.96 335.33 340.06 420.14 408.07 248.36 102.82 83.36 43.53 NGPS NGPS1 0.00 75.20 95.73 111.52 144.67 238.80 164.12 313.22 277.55 109.95 69.11 31.15 NGPS2 0.00 100.79 82.50 140.46 166.81 261.88 318.95 290.68 233.96 47.16 24.07 10.22 NGPS3 0.00 144.51 165.11 185.87 280.24 396.51 447.18 324.79 265.37 110.92 75.81 18.68 NGPS4 0.00 99.98 127.28 148.28 192.35 317.51 218.20 416.45 369.02 146.19 91.89 41.41 NGPS5 0.00 75.80 62.05 105.64 125.46 196.97 239.89 218.63 175.96 35.47 18.10 7.69 NGPS6 0.00 108.69 124.18 139.80 210.77 298.22 336.34 244.28 199.59 83.42 57.02 14.05 N 6 6 6 6 6 6 6 6 6 6 6 6 Mean 0.000 100.827 109.476 138.596 186.716 284.982 287.446 301.341 253.574 88.850 56.001 20.532 SD 0.000 25.509 36.863 28.817 55.269 69.402 101.158 69.500 68.358 42.040 29.357 13.156 SE 0.000 10.414 15.049 11.764 22.563 28.333 41.298 28.373 27.907 17.163 11.985 5.371 Min 0.00 75.20 62.05 105.64 125.46 196.97 164.12 218.63 175.96 35.47 18.10 7.69 Max 0.00 144.51 165.11 185.87 280.24 396.51 447.18 416.45 369.02 146.19 91.89 41.41 90 Table 3.2: Glibenclamide Concentration Table (ng/ml) : Normal Rats (contd.,)
Time Treatment Animal 0.00 0.33 0.67 1.00 1.50 2.00 2.50 3.00 4.00 8.00 12.00 24.00 NGS NGS1 0.00 25.96 34.24 37.17 64.54 88.05 107.54 75.32 56.69 36.12 32.06 11.37 NGS2 0.00 29.07 38.34 41.62 72.28 98.60 120.43 84.34 63.48 40.45 35.90 12.73 NGS3 0.00 33.00 39.90 41.18 69.10 92.68 102.26 133.15 102.42 50.23 41.36 25.16 NGS4 0.00 45.08 47.46 50.23 87.91 105.43 122.58 122.83 110.90 78.46 63.77 45.91 NGS5 0.00 36.95 44.68 46.11 77.37 103.78 114.51 149.10 114.68 56.25 46.32 28.17 NGS6 0.00 50.48 53.15 56.25 98.44 118.06 137.26 137.54 124.18 87.86 71.41 51.41 N 6 6 6 6 6 6 6 6 6 6 6 6 Mean 0.000 36.757 42.961 45.425 78.274 101.099 117.431 117.049 95.391 58.229 48.469 29.125 SD 0.000 9.460 6.838 6.949 12.722 10.594 12.369 30.169 28.309 20.787 15.766 16.609 SE 0.000 3.862 2.792 2.837 5.194 4.325 5.050 12.317 11.557 8.486 6.436 6.781 Min 0.00 25.96 34.24 37.17 64.54 88.05 102.26 75.32 56.69 36.12 32.06 11.37 Max 0.00 50.48 53.15 56.25 98.44 118.06 137.26 149.10 124.18 87.86 71.41 51.41 91 Table 3.2: Glibenclamide Concentration Table 9ng/mL) : Normal Rats (contd.,)
Figure 3.2: Glibenclamide Pharmacokinetic profiles in normal rats (Treatment NG) Figure 3.3: Glibenclamide Pharmacokinetic profiles in normal rats (Treatment NGA) 92
Figure 3.4: Glibenclamide Pharmacokinetic profiles in normal rats (Treatment NGPA) Figure 3.5: Glibenclamide Pharmacokinetic profiles in normal rats (Treatment NGPS) 93
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
5.3.2. 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 350-480g. 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. 5.3.2.1.Effect of Ashwagandha on Glibenclamide Pharmacokinetics C max of glibenclamide administered simultaneously with ashwagandha (DGA) was 274.301±21.096 ng/ml. This was lesser than that of control (DG) group (344.827±31.750 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 753.825±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 11.625±1.06 hrs as compared to that of control, 8.003±1.174 hrs. The details are presented in Table 3.3 5.3.2.2.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 990.091±61.361 ng/ml, AUC 0-t and AUC 0- were 8083.687±749.562 and 9147.349±1038.241 hr*ng/ml respectively. Whereas for DG (control), they were at 344.827±31.750 ng/ml (Cmax), 3788.392±504.483 hr*ng/ml (AUC 0-95
t) and 4362.247±504.483 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 718.59±35.902 ng/ml, AUC0-t at 6423.128±158.916 hr.ng/ml and AUC0- at 7369.614±270.672 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 2.917 344.827 3788.392 4362.247 3.001 0.259 8.003 0.097 SE 0.375 31.750 460.841 504.483 0.654 0.034 1.174 0.015 DGA Mean 2.333 274.301 2794.148 3617.339 5.269* 0.302 11.625* 0.062 SE 0.105 21.096 333.654 332.922 1.032 0.029 1.06 0.005 DGAP Mean 1.667 753.825* 5136.906 6079.678 2.512 0.199 9.052 0.077 SE 0.105 78.134 708.382 888.256 0.381 0.038 0.352 0.003 DGS Mean 2.167 990.091* 8083.687* 9147.349* 1.125 0.125* 6.992 0.121 SE 0.167 61.361 749.562 1038.241 0.113 0.019 1.168 0.026 DGSP Mean 2.333 718.59* 6423.128* 7369.614* 1.591 0.142* 7.85 0.092 SE 0.211 35.902 158.916 270.672 0.109 0.005 0.744 0.008 *P<0.05 vs DG; Oneway-ANOVA followed by Dunnett s test; n=6 96
CL (L/hr/Kg) Glibenclamide (ng/ml) AUC (hr*ng/ml) Chapter 5 C max AUC 0-t 1500 1000 500 * * * DG DGA DGAP DGS DGSP 10000 8000 6000 4000 2000 * * DG DGA DGAP DGS DGSP 0 Treatment 0 Treatment Clearance 0.4 0.3 0.2 0.1 * * DG DGA DGAP DGS DGSP 0.0 Treatment Figure 3.8: Comparison of pharmacokinetic parameters in IR rat. The data represents Mean±SEM 97
Time Treatment Animal 0.00 0.33 0.67 1.00 1.50 2.00 2.50 3.00 4.00 8.00 12.00 24.00 DG DG1 0.00 90.62 48.83 135.66 196.77 208.58 264.40 272.82 457.67 282.81 224.08 64.08 DG2 0.00 109.69 127.78 149.72 216.83 233.82 171.14 159.75 135.48 106.07 87.50 43.50 DG3 0.00 91.88 119.00 158.18 229.08 247.04 340.80 296.78 263.13 196.06 159.69 57.96 DG4 0.00 18.19 143.72 125.45 236.81 250.47 304.88 339.85 314.44 226.22 186.35 72.36 DG5 0.00 16.39 129.52 113.05 213.41 297.82 274.76 219.75 186.04 131.78 104.09 14.74 DG6 0.00 79.00 50.57 78.27 187.54 256.41 274.50 316.92 399.00 281.42 205.26 21.00 N 6 6 6 6 6 6 6 6 6 6 6 6 Mean 0.000 67.628 103.236 126.721 213.409 249.023 271.747 267.643 292.626 204.061 161.161 45.607 SD 0.000 40.211 42.225 28.757 18.713 29.352 56.674 67.009 123.254 74.254 55.180 23.547 SE 0.000 16.416 17.238 11.740 7.640 11.983 23.137 27.356 50.318 30.314 22.527 9.613 Min 0.00 16.39 48.83 78.27 187.54 208.58 171.14 159.75 135.48 106.07 87.50 14.74 Max 0.00 109.69 143.72 158.18 236.81 297.82 340.80 339.85 457.67 282.81 224.08 72.36 DGA DGA1 0.00 88.30 158.50 164.11 195.74 220.98 289.16 255.98 206.77 139.06 116.16 51.85 DGA2 0.00 40.62 55.90 65.24 84.17 188.69 121.25 107.21 91.10 73.20 65.68 38.44 DGA3 0.00 61.08 74.03 112.40 133.40 209.38 254.55 198.97 141.08 81.23 76.18 42.66 DGA4 0.00 57.07 150.02 132.62 158.77 190.23 266.12 204.43 195.71 131.62 114.60 49.08 DGA5 0.00 42.61 72.91 154.47 213.58 233.54 339.06 281.65 246.33 171.35 141.85 64.40 DGA6 0.00 37.15 63.56 169.54 255.94 308.22 295.59 280.41 249.62 184.26 159.41 56.14 N 6 6 6 6 6 6 6 6 6 6 6 6 Mean 0.000 54.472 95.821 133.063 173.599 225.173 260.956 221.441 188.435 130.120 112.314 50.426 SD 0.000 19.116 45.824 39.444 61.161 44.240 74.405 66.540 61.970 45.480 36.306 9.335 SE 0.000 7.804 18.708 16.103 24.969 18.061 30.376 27.165 25.299 18.567 14.822 3.811 Min 0.00 37.15 55.90 65.24 84.17 188.69 121.25 107.21 91.10 73.20 65.68 38.44 Max 0.00 88.30 158.50 169.54 255.94 308.22 339.06 281.65 249.62 184.26 159.41 64.40 98 Table 3.4: Glibenclamide Concentration Table (ng/ml): IR Rats
Time Treatment Animal 0.00 0.33 0.67 1.00 1.50 2.00 2.50 3.00 4.00 8.00 12.00 24.00 DGAP DGAP1 0.00 365.85 338.00 620.12 791.21 774.46 551.84 455.97 428.45 304.15 256.94 104.03 DGAP2 0.00 128.08 119.28 479.64 485.90 633.21 432.95 295.55 232.68 132.25 80.35 45.15 DGAP3 0.00 196.95 150.76 378.27 437.20 485.77 307.57 204.02 144.52 106.25 84.09 23.81 DGAP4 0.00 347.54 421.08 412.74 1011.46 594.32 571.15 457.43 389.67 281.04 235.16 82.33 DGAP5 0.00 277.15 343.43 371.96 911.52 535.60 514.72 412.23 351.16 253.27 205.57 74.20 DGAP6 0.00 318.94 294.66 540.62 689.78 675.17 481.10 397.52 373.52 265.16 231.98 90.70 N 6 6 6 6 6 6 6 6 6 6 6 6 Mean 0.000 272.418 277.868 467.227 721.179 616.420 476.555 370.455 320.002 223.685 182.348 70.036 SD 0.000 92.830 118.334 98.868 229.079 102.761 96.489 100.663 108.507 83.078 79.265 30.024 SE 0.000 37.898 48.310 40.363 93.521 41.952 39.392 41.096 44.298 33.917 32.360 12.257 Min 0.00 128.08 119.28 371.96 437.20 485.77 307.57 204.02 144.52 106.25 80.35 23.81 Max 0.00 365.85 421.08 620.12 1011.46 774.46 571.15 457.43 428.45 304.15 256.94 104.03 DGS DGS1 0.00 173.06 378.68 760.79 810.08 953.04 850.34 761.86 557.41 424.09 344.92 125.00 DGS2 0.00 163.80 358.43 720.11 728.90 674.91 804.86 861.21 830.49 401.41 319.06 118.32 DGS3 0.00 141.70 338.94 658.81 929.99 1227.49 1187.66 992.73 674.04 281.53 196.95 9.26 DGS4 0.00 337.30 640.55 647.06 900.33 957.69 923.22 914.44 716.74 460.44 364.18 135.72 DGS5 0.00 416.08 597.43 593.71 838.10 1106.20 1070.30 822.54 499.29 109.52 50.70 8.34 DGS6 0.00 363.80 558.43 564.11 784.90 834.91 804.86 797.21 490.49 365.41 303.12 118.32 N 6 6 6 6 6 6 6 6 6 6 6 6 Mean 0.000 265.960 478.742 657.432 832.052 959.042 940.208 858.333 628.077 340.401 263.153 85.827 SD 0.000 119.755 134.649 74.149 74.344 194.884 156.944 84.312 135.243 128.478 119.277 60.005 SE 0.000 48.890 54.970 30.271 30.351 79.561 64.072 34.420 55.213 52.451 48.695 24.497 Min 0.00 141.70 338.94 564.11 728.90 674.91 804.86 761.86 490.49 109.52 50.70 8.34 Max 0.00 416.08 640.55 760.79 929.99 1227.49 1187.66 992.73 830.49 460.44 364.18 135.72 99 Table 3.4: Glibenclamide Concentration Table (ng/ml): IR Rats (Contd.,)
Time Treatment Animal 0.00 0.33 0.67 1.00 1.50 2.00 2.50 3.00 4.00 8.00 12.00 24.00 DGSP DGSP1 0.00 37.46 69.62 286.10 319.92 293.58 431.84 691.77 446.40 293.41 238.62 121.92 DGSP2 0.00 42.97 87.86 328.17 366.96 392.75 535.35 793.50 552.05 336.56 279.42 99.84 DGSP3 0.00 104.16 180.77 403.13 470.22 591.72 489.23 466.56 416.12 310.19 240.17 66.76 DGSP4 0.00 183.20 504.23 453.06 602.95 753.68 645.56 615.66 542.90 257.94 191.60 60.35 DGSP5 0.00 113.56 452.72 518.66 637.01 824.26 682.03 570.44 481.57 312.52 239.48 47.76 DGSP6 0.00 115.58 200.59 447.33 521.77 656.60 542.87 517.71 461.75 344.20 274.49 74.08 N 6 6 6 6 6 6 6 6 6 6 6 6 Mean 0.000 99.487 249.297 406.075 486.471 585.433 554.479 609.274 483.466 309.138 243.962 78.451 SD 0.000 53.905 185.358 86.088 126.304 206.280 94.193 119.202 54.073 31.184 31.604 27.468 SE 0.000 22.007 75.672 35.145 51.563 84.213 38.454 48.664 22.075 12.731 12.902 11.214 Min 0.00 37.46 69.62 286.10 319.92 293.58 431.84 466.56 416.12 257.94 191.60 47.76 Max 0.00 183.20 504.23 518.66 637.01 824.26 682.03 793.50 552.05 344.20 279.42 121.92 100 Table 3.4: Glibenclamide Concentration Table (ng/ml): IR Rats (Contd.,)
Figure 3.9: Glibenclamide Pharmacokinetic profiles in IR rats (Treatment DG) Figure 3.10: Glibenclamide Pharmacokinetic profiles in IR rats (Treatment DGA) 101
Figure 3.11: Glibenclamide Pharmacokinetic profiles in IR rats (Treatment DGAP) Figure 3.12: Glibenclamide Pharmacokinetic profiles in IR rats (Treatment DGS) 102
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
5.3.3. 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 75.227 ±0.903 mg/dl to 51.232±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, 23.558±1.86, 34.609±3.54, and 29.047±1.25 and 21.051±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 124.08±10.423, 586.997±51.122, 296.744±26.103, 159.887±69.597 hr*mg/dl were observed for NGA, NGPA, NGPS and NGS respectively as compared to NG (Control) 108.911±30.973 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 4.55. 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 123.543±9.09 mg/dl at 8th hour post dose and reduced to 106.185±1.96 mg/dl by 12 th hour. The details are presented in tables 4.1 to 4.3 and figures 4.1 and 4.2. 104
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 20 3 0-20 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 800 600 400 NG NGA NGPA NGPS NGS 10 5 NG NGA NGPA NGPS NGS 200 0 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
Time Treatment Animal 0 0.33 0.67 1 1.5 2 2.5 3 4 8 12 NG Mean 70.043 47.07 55.397 74.46 73.59 59.082 68.108 79.135 54.417 92.037 104.358 SD 2.421 6.795 5.459 5.394 1.673 12.699 8.317 3.472 19.39 6.179 9.808 SE 0.988 2.774 2.229 2.202 0.683 5.185 3.396 1.417 7.916 2.522 4.004 Min 66.97 40.55 50.61 69.84 71.82 51.19 53.03 73.94 44.98 84.93 94.1 Max 73.94 57.27 65.45 84.85 76.36 84.55 75.11 82.13 93.94 98.46 116.79 NGA Mean 75.277 70.667 57.592 80.645 73.565 72.463 65.568 73.515 75.808 89.297 115.452 SD 2.212 1.915 4.757 7 3.099 4.426 6.038 4.135 20.915 12.706 9.307 SE 0.903 0.782 1.942 2.858 1.265 1.807 2.465 1.688 8.539 5.187 3.799 Min 72.58 67.9 53.94 69.39 70.3 66.67 57.88 68.48 39.39 77.58 105.76 Max 79.09 73.3 66.67 90.3 77.88 77.5 76.06 78.43 96.67 111.21 133.03 NGPA Mean 52.03 39.023 33.728 41.692 72.853 76.943 75.978 79.678 112.763 123.543 106.185 SD 4.644 1.507 2.704 4.544 10.514 8.949 8.141 9.233 7.983 22.267 4.807 SE 1.896 0.615 1.104 1.855 4.292 3.653 3.323 3.769 3.259 9.09 1.962 Min 47.06 37.57 29.41 33.24 53.24 62.65 62.35 62.65 99.71 86.76 100.29 Max 58.82 41.47 36.88 45.52 81.4 87.06 84.12 87.58 123.46 151.47 114.43 NGPS Mean 51.232 39.367 36.538 45.45 80.823 74.693 76.205 72.538 88.992 67.693 97.632 SD 2.121 3.076 2.177 3.647 3.423 3.205 4.291 4.048 6.307 12.285 6.934 SE 0.866 1.256 0.889 1.489 1.397 1.308 1.752 1.653 2.575 5.015 2.831 Min 48.53 36.76 33.82 40.59 76.13 70.88 71.07 68.09 79.12 52.06 85.88 Max 53.81 43.37 39.25 49.41 85.94 78.18 81.51 78.03 98.13 84.41 104.01 NGS Mean 71.36 62.735 90.087 61.548 67.647 63.7 67.992 61.175 96.402 96.848 76.873 SD 11.878 8.286 13.582 5.571 1.919 8.053 2.178 3.75 4.964 8.634 5.453 SE 4.849 3.383 5.545 2.274 0.783 3.288 0.889 1.531 2.027 3.525 2.226 Min 52.73 52.12 72.73 57.27 64.77 50.3 65.22 57.71 91.3 82.42 72.73 Max 87.27 77.27 111.21 72.12 70.04 75.15 71.21 66.97 103.64 107.88 87.27 Chapter 5 106 Table 4.1: Plasma glucose levels (mg/dl) of normal rats : 0-12 hours post dose
Figure 4.2: Plasma glucose profile obtained simultaneously with PK profile in normal rats. The data represents Mean±SEM 107
NGA Mean 75.277 81.862 57.592 1.667 0.67 1.756 14.554-12.798 0.615 2.385 SD 2.212 5.046 4.757 1.033 0 1.114 1.535 0.506 0.104 0.104 SE 0.903 2.06 1.942 0.422 0 0.455 0.627 0.207 0.043 0.043 Median 74.75 81.07 55.78 1 0.67 1.86 14.64-12.9 0.63 2.37 NGPA Mean 52.03 80.022 33.728 2.667 0.67 40.219 13.244 26.976 1.821 1.179 SD 4.644 9.505 2.704 0.516 0 10.367 4.335 11.583 0.096 0.096 SE 1.896 3.88 1.104 0.211 0 4.232 1.77 4.729 0.039 0.039 Median 51.34 82.91 33.78 3 0.67 41.27 12.09 29.52 1.84 1.16 NGPS Mean 51.232 80.823 36.342 1.5 0.613 43.169 10.125 33.044 1.922 1.078 SD 2.121 3.423 2.008 0 0.139 4.165 2.148 5.165 0.036 0.036 SE 0.866 1.397 0.82 0 0.057 1.7 0.877 2.109 0.015 0.015 Median 51.62 81.59 36.61 1.5 0.67 42.26 9.38 30.47 1.93 1.07 NGS Mean 71.36 92.51 55.043 0.558 1.555 12.347 22.952-10.605 0.785 2.215 SD 11.878 10.898 3.753 0.274 1.312 21.115 23.031 39.095 1.103 1.103 SE 4.849 4.449 1.532 0.112 0.535 8.62 9.402 15.96 0.45 0.45 Median 69.56 90.77 55.22 0.67 1.5 3.19 15.43-8.41 0.45 2.55 108 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 70.043 79.64 46.363 2.667 0.692 5.685 18.857-13.172 1.235 1.765 SD 2.421 4.103 5.645 0.816 0.886 1.644 5.007 5.893 0.126 0.126 SE 0.988 1.675 2.305 0.333 0.362 0.671 2.044 2.406 0.051 0.051 Median 69.79 80.57 45.92 3 0.33 5.03 18.95-13.57 1.19 1.81
NGA Mean 75.277 115.452 55.065 12 1.225 149.841 25.761 124.08 8.604 3.396 SD 2.212 9.307 8.91 0 1.359 33.178 18.837 25.532 1.21 1.21 SE 0.903 3.799 3.637 0 0.555 13.545 7.69 10.423 0.494 0.494 Median 74.75 113.04 55.78 12 0.67 140.91 17.3 122.37 9.23 2.77 NGPA Mean 52.03 127.613 33.728 7.333 0.67 600.24 13.244 586.997 10.821 1.179 SD 4.644 15.359 2.704 1.633 0 121.923 4.335 125.222 0.096 0.096 SE 1.896 6.27 1.104 0.667 0 49.775 1.77 51.122 0.039 0.039 Median 51.34 129.07 33.78 8 0.67 637.36 12.09 623.97 10.84 1.16 NGPS Mean 51.232 98.662 36.342 10.667 0.613 306.869 10.125 296.744 10.922 1.078 SD 2.121 5.04 2.008 3.266 0.139 62.071 2.148 63.938 0.036 0.036 SE 0.866 2.058 0.82 1.333 0.057 25.34 0.877 26.103 0.015 0.015 Median 51.62 100.02 36.61 12 0.67 297.84 9.38 288.77 10.93 1.07 NGS Mean 71.36 102.863 55.043 6.112 1.555 189.076 29.189 159.887 8.893 3.107 SD 11.878 5.621 3.753 3.109 1.312 144.786 32.927 170.477 1.993 1.993 SE 4.849 2.295 1.532 1.269 0.535 59.108 13.442 69.597 0.814 0.814 Median 69.56 101.1 55.22 8 1.5 160.9 16.42 144.48 9.14 2.86 109 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 70.043 104.358 45.34 12 1.915 154.652 45.742 108.911 7.898 4.102 SD 2.421 9.808 4.889 0 1.821 57.444 18.908 75.869 1.321 1.321 SE 0.988 4.004 1.996 0 0.743 23.451 7.719 30.973 0.539 0.539 Median 69.79 102.48 44.4 12 1.42 148.63 49.28 98.89 7.45 4.55
5.3.4. Pharmacodynamic Interaction in IR rats The baseline plasma glucose values were 160.917±4.57, 143.811±3.88, 62.558±1.887, 139.279 ±4.391 and 72.891±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 72.597±1.649 mg/dl for DGAP and 74.059±4.013mg/dL for DGSP as compared to control (DG) at 249.91±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 52.582±2.772 and 53.986±2.69 mg/dl, which was significantly low when compared to DG with 140.786±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 91.002±4.18 mg/dl for DGAP and 105.296±3.822mg/dL for DGSP as compared to control (DG) at 249.91±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 52.582±2.772 and 53.986±2.69 mg/dl, which was significantly less when compared to DG with 123.635±12.267 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 150.091±47.452 and 116.414±47.526 hr/mg/dl for DGAP and DGSP respectively as compared to 246.005±34.081 hr*mg/dl for control (DG). Net_AUC_B changes in simultaneous herb administration groups were variable and stood at 315.164±105.518, 461.759±25.021 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 3.5 110
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 0.33-2.0 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 200 150 100 50 DG DGA DGAP DGS DGSP 3.5 3.0 2.5 2.0 1.5 1.0 DG DGA DGAP DGS DGSP 0 0.5-50 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 600 400 200 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
Time Treatment 0 0.33 0.67 1 1.5 2 2.5 3 4 8 12 DG Mean 160.917 146.23 175.634 202.622 213.606 169.944 238.188 228.984 144.609 178.05 201.109 SD 11.193 6.497 11.457 11.112 36.497 36.579 18.695 19.981 50.126 12.849 18.051 SE 4.57 2.652 4.677 4.536 14.9 14.933 7.632 8.157 20.464 5.246 7.369 Min 146.69 139.67 152.48 190.08 159.09 127.27 214.46 204.96 84.3 162.81 166.94 Max 176.89 154.98 182.35 217.95 255.71 212.81 262.17 252.02 196.96 196.06 219.42 DGA Mean 143.811 136.089 126.465 177.285 180.009 173.688 182.009 215.773 214.072 149.975 145.437 SD 9.504 7.474 12.266 10.883 15.742 18.117 35.633 18.503 13.859 38.406 22.511 SE 3.88 3.051 5.008 4.443 6.427 7.396 14.547 7.554 5.658 15.679 9.19 Min 132.07 125.06 112.4 160.64 165.7 149.85 136.78 196.77 192.23 92.15 107.02 Max 154.96 144.4 143.72 189.75 205.79 194.63 222.69 242.09 233.52 193.39 176.45 DGAP Mean 62.558 54.747 54.587 67.408 69.57 70.143 62.358 64.133 88.983 73.952 79.233 SD 4.621 9.096 4.378 3.938 3.376 3.852 5.331 7.75 8.902 24.344 16.712 SE 1.887 3.713 1.787 1.608 1.378 1.573 2.176 3.164 3.634 9.938 6.823 Min 55.96 42.91 48.66 61.18 64.21 65.25 56.49 55.81 75.99 43.14 59.57 Max 70.02 68.4 61.27 73.23 73.01 76.8 71 77.45 100.75 97.07 102.43 DGS Mean 139.279 161.861 185.515 156.416 216.925 226.928 194.606 205.881 218.486 151.233 152.029 SD 10.755 6.682 26.717 30.68 10.598 39.453 13.535 33.405 19.397 13.214 20.126 SE 4.391 2.728 10.907 12.525 4.326 16.106 5.526 13.638 7.919 5.395 8.217 Min 129.41 153.26 146.69 116.53 200.56 183.91 173.55 167.77 195.87 132.82 127.53 Max 157.48 167.86 217.36 199.44 227.88 286.54 214.88 255.86 249.09 171.93 182.76 DGSP Mean 72.891 61.697 53.986 69.103 68.756 64.974 60.976 68.908 103.574 88.093 99.812 SD 10.471 13.964 6.589 6.197 9.372 8.753 8.918 4.552 10.113 5.371 6.186 SE 4.275 5.701 2.69 2.53 3.826 3.574 3.641 1.859 4.129 2.193 2.525 Min 63.32 50.13 48.05 63.25 56.58 53.43 48.52 61.71 93.57 80.29 89.89 Max 89.61 82.75 64.71 79.54 82.2 77.35 71.04 75.24 120.37 95.22 107.82 112 Table 4.4: Plasma glucose levels (mg/dl) of IR rats: 0-12 hours post dose
Figure 4.4: Plasma glucose profile obtained simultaneously with PK profile in IR rats. The data represents Mean±SEM Chapter 5 113
DGA Mean 143.811 218.805 125.57 2.667 0.613 83.203 7.582 75.621 2.185 0.815 SD 9.504 16.749 11.576 0.606 0.139 26.379 6.636 31.841 0.271 0.271 SE 3.88 6.838 4.726 0.247 0.057 10.769 2.709 12.999 0.111 0.111 Median 145.94 217.77 126.2 3 0.67 85.36 6.42 80.61 2.22 0.78 DGAP Mean 62.558 72.597 52.582 1.917 0.557 10.809 6.491 4.317 1.944 1.056 SD 4.621 4.04 6.79 0.585 0.176 6.206 4.566 10.674 0.579 0.579 SE 1.887 1.649 2.772 0.239 0.072 2.534 1.864 4.358 0.236 0.236 Median 62.16 72.07 53.94 1.75 0.67 12.54 6.61 5.93 2.03 0.97 DGS Mean 139.279 235.656 137.006 1.667 0.167 157.267 0.14 157.127 2.979 0.021 SD 10.755 32.196 14.013 0.258 0.408 6.634 0.343 6.529 0.05 0.05 SE 4.391 13.144 5.721 0.105 0.167 2.708 0.14 2.665 0.021 0.021 Median 137.2 222.94 137.2 1.5 0 160.28 0 159.86 3 0 DGSP Mean 72.891 74.059 53.986 1 0.67 0.849 25.485-24.636 0.324 2.676 SD 10.471 9.829 6.589 1.549 0 2.039 9.897 11.393 0.745 0.745 SE 4.275 4.013 2.69 0.632 0 0.832 4.04 4.651 0.304 0.304 Median 67.9 70.54 51.22 0 0.67 0 26.18-26.18 0 3 114 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 160.917 249.91 140.786 2.167 0.887 108.923 5.095 103.828 2.4 0.6 SD 11.193 9.592 11.205 0.516 0.862 15.322 2.662 17.666 0.083 0.083 SE 4.57 3.916 4.574 0.211 0.352 6.255 1.087 7.212 0.034 0.034 Median 161.99 250.71 139.67 2.5 0.33 111.65 5.12 106.2 2.41 0.59 Time_ Below_B
DGA Mean 143.811 220.456 118.888 3.25 3.723 373.014 57.85 315.164 8.849 3.151 SD 9.504 15.784 18.374 0.612 5.025 188.97 90.277 258.466 2.829 2.829 SE 3.88 6.444 7.501 0.25 2.052 77.147 36.855 105.518 1.155 1.155 Median 145.94 219.42 119.72 3 0.67 319.38 24.79 287.08 9.54 2.46 DGAP Mean 62.558 91.002 52.582 6.667 0.557 167.224 17.133 150.091 9.319 2.681 SD 4.621 10.238 6.79 4.131 0.176 101.256 16.901 116.234 2.402 2.402 SE 1.887 4.18 2.772 1.687 0.072 41.338 6.9 47.452 0.98 0.98 Median 62.16 91.93 53.94 4 0.67 185.56 11.97 178.5 10.12 1.88 DGS Mean 139.279 237.635 136.348 2.5 4.167 462.368 0.609 461.759 11.504 0.496 SD 10.755 30.414 14.127 1.183 6.08 60.771 0.699 61.288 0.722 0.722 SE 4.391 12.417 5.768 0.483 2.482 24.81 0.285 25.021 0.295 0.295 Median 137.2 226.04 136.17 2 0.5 461.83 0.42 461.41 11.94 0.06 DGSP Mean 72.891 105.296 53.986 6.667 0.67 191.439 26.712 164.728 9.131 2.869 SD 10.471 9.362 6.589 4.131 0 105.103 11.495 116.414 0.88 0.88 SE 4.275 3.822 2.69 1.687 0 42.908 4.693 47.526 0.359 0.359 Median 67.9 105 51.22 4 0.67 204.87 26.22 178.65 8.95 3.05 Chapter 5 115 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 160.917 249.91 123.635 2.167 2.165 296.706 50.701 246.005 9.696 2.304 SD 11.193 9.592 30.047 0.516 2.01 49.89 54.895 83.481 1.826 1.826 SE 4.57 3.916 12.267 0.211 0.821 20.367 22.411 34.081 0.745 0.745 Median 161.99 250.71 139.4 2.5 2.17 286.32 25.46 223.86 9.97 2.03