Characterization of pioglitazone cyclodextrin complexes: Molecular modeling to in vivo evaluation.

AIMS: The objective of present study was to study the influence of different β-cyclodextrin derivatives and different methods of complexation on aqueous solubility and consequent translation in in vivo performance of Pioglitazone (PE).
MATERIAL AND METHODS: Three cyclodextrins: β-cyclodextrin (BCD), hydroxypropyl-β-cyclodextrin (HPBCD) and Sulfobutylether-7-β-cyclodextrin (SBEBCD) were employed in preparation of 1:1 Pioglitazone complexes by three methods viz. co-grinding, kneading and co-evaporation. Complexation was confirmed by phase solubility, proton NMR, Fourier Transform Infrared spectroscopy, Differential Scanning Calorimetry (DSC) and X-Ray diffraction (XRD). Mode of complexation was investigated by molecular dynamic studies. Pharmacodynamic study of blood glucose lowering activity of PE complexes was performed in Alloxan induced diabetic rat model.
RESULTS: Aqueous solubility of PE was significantly improved in presence of cyclodextrin. Apparent solubility constants were observed to be 254.33 M(-1) for BCD-PE, 737.48 M(-1) for HPBCD-PE and 5959.06 M(-1) for SBEBCD-PE. The in silico predictions of mode of inclusion were in close agreement with the experimental proton NMR observation. DSC and XRD demonstrated complete amorphization of crystalline PE upon inclusion. All complexes exhibited >95% dissolution within 10 min compared to drug powder that showed <40% at the same time. Marked lowering of blood glucose was recorded for all complexes.
CONCLUSION: Complexation of PE with different BCD significantly influenced its aqueous solubility, improved in vitro dissolution and consequently translated into enhanced pharmacodynamic activity in rats.

Diabetes today has emerged as one of the major life-threatening diseases. A disease earlier prevalent in the developed countries, today is increasingly spreading among the third world countries, and a situation of global epidemic is arising with approximately 350 million people worldwide being diabetic.[1] Noninsulin-dependent diabetes mellitus is a chronic disorder characterized with insulin resistance caused by a defect in insulin secretion or insulin action on peripheral tissues. 90–95% cases of diabetes belong to this category.[234] One of the mainstay treatment involves administration of drugs that increase insulin sensitivity of peripheral tissues.[567] Pioglitazone (PE) is a potent activator of peroxisome proliferator activated receptor-γ agonist.[89] Pharmacological studies indicate that it improves the sensitivity of peripheral tissues viz., muscle and adipose tissue to insulin and inhibits hepatic gluconeogenesis thereby eliciting improved glycolic control at low circulating insulin levels.[567891011]

PE, a Biopharmaceutical Classification System Class II drug is characterized with low aqueous solubility (0.015 mg/ml).[12] This water insolubility results in highly erratic and poor dissolution profile in gastrointestinal fluids.[13] Absorption is dissolution rate limited which may translate in poor absorption, distribution, metabolism, and excretion profile with a reduction in pharmacological activity. Consequently, this may reflect in increased dose and dosage and associated adverse side effects. Hence, the goal of present study is to boost PE therapeutic efficacy by increasing its aqueous solubility through cyclodextrin (CD) complexation.

Solid dispersion of PE in polymeric carriers,[14] nanostructured lipid carriers,[15] microparticles of PE,[1617] self-emulsifying systems,[18] CD complexes[19202122] are some of the different formulation approaches reported in the literature. The interaction of PE with β-CD (BCD), hydroxypropyl-β-cyclodextrin (HPBCD), and dimethyl-β-cyclodextrin in the presence of water-soluble polymers was studied by Elbary et al.[20] They demonstrated that presence of CD increased solubility of PE, however, the addition of water soluble polymers like polyvinylpyrrolidone significantly improved PE dissolution. Alteration in physicochemical and biological properties of PE was more prominent in ternary systems.[20] Pandit et al. reported CD complexes of PE with BCD, methyl-BCD and γ-CD prepared by kneading and spray drying methods. They demonstrated that method of preparation greatly influenced in vitro performance of PE.[13] Similar reports have been cited in the literature that testify that both the type of CD and method of complexation synergistically impact dissolution.[2324] PE-sulfobutylether-7-BCD (SBEBCD) complexation has been less studied, and no detailed characterization studies of this complex are reported in the literature. The present study extensively investigates the inclusion complexation of PE with three different CDs viz., BCD, HPBCD, and SBEBCD. Similarly, methods of co-evaporation and co-grinding have not been evaluated for PE-CDs complexation. In methods involving aqueous medium (kneading and co-evaporation), the effect of pH on interaction was also deliberated. The ability of individual CD to interact with PE was assessed by physical mixing. Thus, this paper reports a comparative study of the influence of three CDs and three techniques: Co-grinding, kneading, and co-evaporation on in vitro and in vivo performance of PE. The complexes were characterized by Fourier-transform infrared (FTIR), differential scanning calorimetry (DSC), powder X-ray diffraction (PXRD), proton nuclear magnetic resonance (NMR). Molecular modeling studies for mode of the inclusion of PE in CDs cavity has been reported for the first time, and these in silico evaluations were in close correlation with proton NMR experimental results. Chemically, induced diabetic rat models were used to assess the pharmacodynamic efficacy of PE-CD complexes in comparison to PE suspension.

Materials

PE was obtained as gift sample from Macleods Pharmaceutical Ltd., (Mumbai, India. BCD (MW = 1135) and HPBCD (MW ≈1500) (average degree of substitution; 5.5) were kindly provided by (Cerestar, USA) and SBEBCD (MW = 2265) was purchased from Cydex, Inc., (Overland Park, KS). All chemicals and solvents used in this study were of analytical reagent grade. Freshly distilled water was used throughout the work.

Phase solubility study

Phase solubility studies were performed according to the method reported by Higuchi and Connors.[25] Briefly, PE (20 mg) was transferred to vials containing 10 ml of aqueous solution of BCD, HPBCD, and SBEBCD in different molar concentrations (0–15 mM for BCD, 0–20 mM for HPBCD and 0–69.3 mM for SBEBCD) and mixed on an orbital shaker for 24 h at room temperature. After 24 h, the solutions were centrifuged; the clear supernatant was collected and filtered through 0.45-micron nylon disk filter. The filtrate was suitably diluted, and the absorbance of resultant solutions was recorded on Jasco V-530 double beam ultraviolet/visible spectrophotometer.

Preparation of complexes

Complexes of BCD, HPBCD, and SBEBCD with PE were prepared in the molar ratio of 1:1 by three different methods: Co-grinding, kneading, and co-evaporation. For ease in discussion, the samples are designated with different abbreviations shown in Table 1.

Table 1

Abbreviations used to designate different complexes

Physical mixing

Physical mixtures of each of three CDs and PE were prepared by simply mixing the two powders with a spatula with no energy input.

Co-grinding

Appropriately, weighed quantities of PE and CDs were mixed and triturated. The resultant powder dispersion was collected, passed through 85 #BS sieve.

Co-evaporation

Complexes by co-evaporation method were prepared using two solvent systems: (a) Aqueous ethanol (ethanol:water: 1:1) and (b) acidified ethanol (ethanol: 0.1N HCl: 1:1). The required quantities of PE and CDs were dissolved in 20 ml of solvent, vortex mixed and evaporated under controlled heating at 45–50°C. The resultant solid was pulverized and then sieved through 85 #BS sieve.

Kneading method

Complexes by kneading method were prepared using two solvent systems: (a) Aqueous ethanol (ethanol:water: 1:1) and (b) acidified ethanol (ethanol: 0.1 N HCl: 1:1) used as kneading solvents. The mixture of PE and CDs was kneaded with solvent to form a paste. The kneaded mass was triturated in a mortar for about 15–20 min and the product was dried at 50°C for 3 h. The dry powder was sieved through 85 #BS.

Characterizations of complexes

Fourier-transform infrared

FTIR spectra of the PE, BCD, HPBCD, and SBEBCD and various solid dispersions were recorded on an FTIR–5300 Spectrophotometer (Jasco, Japan) over scanning range was 4000–400 cm−1 by KBr disk method using hydrostatic press to from a compact disc of samples.

Power X-ray diffraction

Powder X-ray diffraction patterns were recorded using Phillips P Analytical X' Pert PRO powder X-ray diffractometer using Ni-filtered, CuKα radiation, the voltage of 40 kV and a current of 30 mA. The scanning rate employed was 1/min and samples were analyzed at 2θ angles of over 10–40°.

Differential scanning calorimetry

DSC studies were performed using the scanning rate of 10°C/min over a temperature range of 30–300° Con Shimadzu DT-40 Thermal Analyzer.

1H Nuclear magnetic resonance

The NMR spectra of PE, the carriers and complexes prepared by kneading method (BCDK2, HPBCDK2, and SBEBCDK2) were recorded in deuterated water using a Bruker AVANCE 500 DRX (500 MHz) Fourier-transform nuclear magnetic resonance instrument at 298 K.

Molecular modeling studies

Computer simulations were performed on a high-performance cluster built on Rocks Cluster 6.1 (The Rocks Group, University of California) with Intel Xeon Processor.

Structure preparation

The three-dimensional structure for BCD was retrieved from Protein Data Bank (PDB ID: 3CGT)[26] and HPBCD was built by adding 2-hydroxypropyl groups at appropriate positions of BCD. The structure of SBEBCD was built as reported in our previous paper.[27] All the structures were energy minimized in Schrodinger Suite 2012 (Schrödinger LLC, New York, NY, 2012), and atom types were defined as per OLPS2005 force field.

Preparation of host-guest complexes

Drug complexes with different BCD analogs were prepared by manually placing the ligand in the hydrophobic cavity of the CDs in different orientations. Since, the exact binding mode was not known various combinations of ligand in the cavity were tried. All the resultant complexes were subjected to molecular dynamics (MD) simulations using Desmond v3.1 (D. E. Shaw Research120 W. 45th St., 39th Fl. New York, NY 10036).

Molecular dynamics simulations

Each of the aforementioned PE-CD complexes, free PE and unbound CD were solvated with TIP3P water model in an orthorhombic box such that a 10 A water shell between the system and the boundary was maintained.[28] These solvated complexes were relaxed with restrains on solute and subsequently equilibrated at 300 K and 1 atm. Pressure over NVT and NPT ensembles. The productive MD simulations were performed for 10 ns using NPT ensemble. Langevin Barostat and Thermostat were used to maintain the pressure and temperature, respectively. The structural states and the corresponding energies were sampled from the trajectories after every 10 picoseconds.[293031]

In vitro dissolution study

Dissolution studies were performed using USP type II apparatus in 900 ml of 0.01N HCl-0.3M KCl buffer pH 2.0.[32] Dissolution was studied at 37°C ± 0.5°C at 75 ± 2 rpm for 1 h. Samples were withdrawn at fixed time intervals, filtered, and spectrophotometrically assayed for drug content. Dissolution efficiency (DE) was calculated from the area under the dissolution curve at time t (measured using the trapezoidal rule) and expressed as percentage of the area of the rectangle described by 100% dissolution in the same time.[3334]

In vivo pharmacodynamic study

The antidiabetic activity was evaluated in Alloxan induced diabetic male Wistar rats (250–300 g).[353637] Powder PE and PE-CDs prepared by kneading method (BCDK2, HPBCDK2, and SBEBCDK2) were studied or glucose-lowering efficacy. The institutional animal ethics committee clearance was obtained prior to conducting these studies. The animals were housed into six groups of six animals each and maintained on a standard diet with free access to water in a clean room in a temperature controlled environment. Diabetes was induced by injecting Alloxan at a dose of 130 mg/kg intraperitoneally 7 days prior to the study. Blood was withdrawn by retro-orbital puncture and blood glucose levels were monitored. Estimation of blood glucose was done using in vitro diagnostic glucose kit wherein hydrogen peroxide generated by enzymatic action on glucose reduces dye giving a colored solution. The intensity of color produced is proportional to the glucose concentration. This method is known as Trinder's method and is also known as GOD-POD method.[38] Animals having blood glucose level in the range 200–300 mg/dL were considered diabetic and were used for the study. They were fasted overnight and the food was restored after 4 h of start of the experiment. PE-CD complexes (equivalent to 1.55 mg/Kg of PE) and plain drug (1.55 mg/Kg) as aqueous suspensions in 0.5%w/v carboxymethyl cellulose (CMC) were administered orally. 0.25% CMC aqueous dispersion was administered to control groups. A normal control consisting of nondiabetic animals showing normal blood glucose levels served as negative control while untreated diabetic animals with elevated blood glucose levels served as positive control.

Results and Discussion

Phase solubility analysis evaluates affinity between guest and host molecules in water. The phase solubility curve of PE in the presence of CDs is shown in Figure 1a and b. The curves indicated a linear increase in solubility of PE with a gradient increase in concentrations of CDs. Such solubility curves are classified as AL type. Linear nature of the plots with a slope <1 suggests 1:1 stoichiometric ratio of optimum complexation over the range of CD concentration investigated. The apparent stability constants (Ks) for the complexes assuming a 1:1 stoichiometry, calculated from the slope of the phase solubility diagram were 254.33 M−1 for BCD-PE, 737.48 M−1 for HPBCD-PE and 5959.06 M−1 for SBEBCD-PE. Thus, solubility enhancing the ability of CDs was observed in the order SBEBCD >> HPBCD > BCD. A similar trend in solubility enhancement potential of different CD for Quercetin was reported by Yousaf et al.[39] The high efficiency of SBEBCD in improving solubility of poorly soluble drugs is attributed to its unique chemical structure. The presence of the butyl chain coupled with the repulsion of the negatively charged end group allows for an “extension” of the hydrophobic region of CD cavity.[40] De Chasteigner et al. findings in the year 1996 suggest that longer the hydrophobic chain linked to β-CD, the higher the association of drug within the complex.[4041] Elbary et al. also observed solubility plots of AL type with solubility enhancing the potential of CDs decreasing in the order DMBCD > HPBCD > BCD and solubility constants of 567, 464.92 and 381.44 M−1 respectively.[20] Similar results were reported by Pandit et al. who observed AL type plots of PE with methyl BCD, BCD, and γ CD and solubility constants of 2747, 1452, and 359 M−1 respectively.[13] It is reported that drug-CD complexes having stability constants in the range of 200–5000 M−1 show improved dissolution properties and hence better bioavailability.[25] The influence of each CD (at fixed concentration of 15 mM) on the solubility of PE is summarized in Table 2.

Figure 1

(a) Phase solubility curves of β-cyclodextrin-pioglitazone and hydroxypropyl-β-cyclodextrin - pioglitazone (n = 3). (b) Phase solubility curve of sulfobutylether-7-ß-cyclodextrin-pioglitazone (n = 3)

Table 2

Increase in solubility of PE in presence of CD. Data expressed as mean of three determinations and RSD <5%)

Characterization of complexes

FTIR has been used to assess the interaction between CD and guest molecules in the solid state. In case of any interactions, the principal absorption bands of functional groups in the guest molecule may get affected.[42] The principal absorption bands of PE corresponding to the structural features are N-H stretching at 3418 cm−1, C-H stretching at 3086, 2928 cm−1, C=O at 1750 cm−1 and C=C stretching at 1693,1610,1552,1510 cm−1.[43] All the dispersions showed a combination of the bands of PE and carrier with no significant changes in the principal absorption bands. The FTIR spectra of different PE complexes are shown in Figure 2.

Figure 2

Overlay of fourier-transform infrared spectra of different pioglitazone

Powder X-ray diffraction analysis is a commonly employed technique used to assess the degree of crystallinity of the given sample. Crystalline samples exhibit sharp, intense peaks in diffractograms while broad diffuse peaks are obtained for amorphous materials. The inclusion of crystalline drug into amorphous carrier results in a decrease in crystallinity and increase in amorphous nature of the system. In the present study, the CDs showed increasing amorphization of PE in the order SBEBCD ≥ HPBCD > BCD. Overlay of the diffractograms of the different complexes is shown in Figure 3. In case HPBCD and SBEBCD, a similar degree of amorphization of PE was observed irrespective of the method employed. However, BCD showed a distinction in the order of amorphization kneading ~ co-evaporation > co-grinding >> physical mixing. In a study, comparing different methods of complexation of fenofibrate by Yousaf et al. spray drying and solvent evaporation methods were found to yield true complexes in comparison to kneading method while physical mixing showed presence of both drug and CD peaks in the diffractograms.[39] Similar observations were observed which suggested that solvent evaporation, spray drying, freeze drying methods resulted in complete amorphization of crystalline drugs owing to intimate contact between drug and CD molecules.[1316394445] Interestingly in the present study, kneading method was found to be equally efficient as co-evaporation method. A lower degree of amorphization was seen in samples prepared in acidic condition compared to those prepared in aqueous ethanol.

Figure 3

The overlay of powder X-ray diffraction diffractograms of pioglitazone and various solid dispersions

The results of DSC were in accordance and supported PXRD results. The overlay of DSC thermograms of PE, carriers, and various dispersions are shown in Figure 4. The thermograms of PE and PE complexes obtained in the present study are analogous to the ones reported in the literature by Pandit et al., Elbary et al., Gajare et al.[132021] Sharp melting endotherm at 195.6°C corresponding to PE melting was observed.[132021] The endotherms at 80–120°C in all the samples containing CDs corresponded to dehydration of the CDs. The presence of melting endotherm of PE at the same temperature in the physical mixtures implied partial complexation while it is absent in case of other dispersions was indicative of complete inclusion and formation of the true complex. A similar observation has been well documented in the literature.[162733394445] BCD samples prepared in acidic conditions did show slight depression near the melting temperature of PE in their thermograms. This could be due to charge induction on PE under an acidic condition which impacted PE inclusion into BCD cavity. Loftsson et al. presented that complexation efficiency of basic drugs decreased when complexed under low pH.[46]

Figure 4

The overlay of differential scanning calorimetry thermograms of pioglitazone and various dispersions

Proton nuclear magnetic resonance

1H NMR is an effective tool that not only confirms complexation but also provides insight into the mode of inclusion of host into the guest cavity. The changes in the chemical shift patterns of the complexes are indicative of host-guest interaction. Upon inclusion, host may affect H3 and H5 protons of CD (H atoms located in the interior of the cavity) which may appear upfield/downfield depending on the changes in the microenvironment whereas the protons on the exterior surface of the torus (H1, H2, H4, and H6) will either be unaffected or experience a marginal shift when compared to that of empty CD cavity. Alternately, if association takes place at the exterior of the torus (surface interaction) H1, H2, H4, and H6 shall be strongly affected.

The different protons of PE have been labeled as shown in Figure 5. Proton signals of BCD and HPBCD NMR spectra in D2O has been summarized in Tables 3a and b.[47] The complexation of PE with BCD and HPBCD caused upfield shifts of different magnitude while causing a downfield shift of the aromatic protons of PE depicted as Ha (benzene) and Hb (pyridine) in Figure 5. These results are in close agreement with the observations by Ali and Upadhyay. They performed 1D and 2D NMR analysis in D2O and concluded that the phenoxy group of PE was embedded in the BCD cavity.[48]

Figure 5

Graphical representation of mode of inclusion as interpreted from proton nuclear magnetic resonance

Table 3a

Changes in chemical Shift values of BCD protons and PE aromatic protons

Table 3b

Changes in chemical Shift values of HPBCD protons and PE aromatic protons

In case of SBEBCD, owing to the structural complexity it was difficult to assign protons of SBEBCD; the changes in the spectrum of PE postinclusion were monitored.

The changes in the chemical shift for the CD protons and that of the aromatic region of PE are summarized in Tables 3a and c. It is evident from proton NMR that the aromatic region of PE is included into the CD cavity. The NMR spectra of kneaded complexes in shown in Figure 6.

Table 3c

Changes in the chemical shift of aromatic proton of PE complexed with SBEBCD

Figure 6

Overlay of nuclear magnetic resonance spectra of kneaded complexes

Computer simulations

Computer simulations provide excellent insight into binding of the host and the guest and can thus potentially channelize the experiment in fruitful directions. In the present study, we modeled PE with different CDs in an attempt to determine the best host molecule for PE. The free energy of binding for the drug CD complexes was computed from the energetic of the MD trajectory.

A common observation in the modeling studies of PE with all three CDs was that the drug could be embed into the CD cavity in two orientations, and there was no significant difference in the free energies of binding between the two cases as shown in Figure 7 and Table 4. Furthermore, it can be inferred from these simulations that the phenoxy part of the PE was involved in interaction with the hydrophobic region of each of the CDs. This explains the shift in the protons of the phenoxy part of the drug as observed in 1H NMR spectrum.

Figure 7

Three-dimensional structures of the complexes of Pioglitazone with different cyclodextrins obtained by molecular docking. Case 1 and Case 2 depict the two orientations in which pioglitazone can be embedded into the cyclodextrin cavity

Table 4

Summary of free energy of binding calculated from the energetic of the MD simulations for the three cyclodextrins

RMSD of the configuration of PE in the cavity of different CD was also constructed from the MD trajectory in water in order to study the dynamics of inclusion of PE into different CDs (data not shown). The plot of heavy atom RMSD and radius of gyration throughout the MD trajectory also indicated that the system was stable throughout the simulation. The hydrogen bonding plot over the MD trajectory suggested that one hydrogen bond was consistent throughout the simulations, and intermittently two and rarely three hydrogen bonds were seen.

From these studies, it can be said that SBEBCD, HPBCD, and BCD, all three CDs, serve as equally good carriers for PE, as exemplified by their equal free energies of binding.

In vitro dissolution study

The objective of complexation was to accelerate dissolution of PE by improving its aqueous solubility employing CD as hydrophilic carriers. Hence, dissolution profile of each PE-CD complex was compared to that of PE powder using two parameters viz., (a) percent dissolved at 10 min (DP10) (b) DE at 60 min (DE60). The comparative dissolution profiles of the PE-CD dispersions have been summarized in Table 5.

Table 5

DE60 and DP10 values for BCD, HPBCD and SBEBCD complexes. Measurements expressed as mean of three determinations with RSD <5%

Elbary et al. reported that dissolution rate of PE and its CD complexes were found to decrease with increase in pH in the order rate at 1.2 >4.6 >6.8 buffered medium. Hence, in the present study, buffered medium of pH 2 was employed for dissolution. It was observed that presence of CDs significantly improved the rate and extent of dissolution of PE (P < 0.001) as was reported by other researchers in the literature. Enhanced dissolution is attributed to combined effect of significant amorphization of PE and molecular dispersion of PE in CD via inclusion complexation.

Owing to its low solubility, PE powder showed slow dissolution profile with <40% release at the end of 60 min. The influence of BCD and the method of complexation on solubility of PE were distinctively observed. BCDPM showed slight increment reaching ~70% release by 60 min while all other complexes: Co-ground, co-evaporated, and kneaded showed a maximum cumulative release of about 95–100%, that is, a 2.5-folds increase in release in the first 10 min.

In case of binary mixtures of HPBCD and SBEBCD, it was interesting to observe all dispersions including physical mixture exhibited superior dissolution profiles of about >95% release in the first 10 min. The most soluble DMBCD PE spray dried complex reported by Pandit et al. exhibited slower and lower rate and extent of dissolution in comparison to the present study. This could be due to different parameters such as slower stirring speed, low volume, and the composition of the dissolution medium.[13]

In vivo pharmacodynamic study

Enhancement of pharmacodynamic activity of PE, when complexed with CDs, was evaluated in diabetic rat models. We observed that PE-CD exhibited better blood glucose control in comparison to PE suspension.

Diabetes was induced by alloxan, a classical diabetogen used to effect B-cell destruction and type 1 diabetes due to its selective cytotoxic effect on pancreatic β-cells.[4950] The changes in blood glucose level preprandial and postprandial conditions are shown in Figure 8a and b.

Figure 8

(a) Preprandial changes in blood glucose levels (n = 6). (b) Postprandial changes in blood glucose levels (n = 6)

In the fasted state HPBCD-PE and SBEBCD-PE exhibited a maximum reduction in blood glucose levels in comparison to PE at the end of 4 h. Extremely high solubility of these complexes resulted in faster and better activity. Though PE complexed with BCD showed lower activity than HPBCD, it was nevertheless better than PE.

The consumption of food resulted in a sudden increase in blood glucose level at 8 and 24 h. Interestingly, BCD and HPBCD were found to exhibit better blood glucose control than PE in the postprandial state too. The reduction in therapeutic efficacy of SBEBCD and HPBCD could be attributed to high first pass metabolism of PE. The slower dissolution rate of BCD translated in an extended period of action.

Conclusion

Present study describes successful application of CD complexation in improving dissolution of PE and consequently enhancing its pharmacological activity. All three CDs employed viz., BCD, HPBCD, and SBEBCD augmented rate and extent of dissolution of PE, and this effect was independent of the method of complexation. All PE-CDs complexes exhibited >95% dissolution within 10 min vis à vis PE powder exhibiting <40% at the same time. Molecular association and inclusion were confirmed by1H NMR, DSC, and PXRD and supported by computational studies. Pharmacodynamic evaluation for antidiabetic activity in rats provided evidence for enhanced in vivo activity of PE-CDs complexes with PE-BCD being most effective.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.