in-vivo characterization of trans-resveratrol-loaded nanoparticulate drug delivery system for oral administration.

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Research Paper

Journal of Pharmacy And Pharmacology

In-vitro/in-vivo characterization of trans-resveratrol-loaded nanoparticulate drug delivery system for oral administration Gurinder Singh and Roopa S. Pai Department of Pharmaceutics, Faculty of Pharmacy, Al-Ameen College of Pharmacy, Bangalore, Karnataka, India

Keywords bioavailability; central composite design; enterohepatic recirculation; in-situ single-pass intestinal perfusion; in-vivo biodistribution Correspondence Roopa S. Pai, Faculty of Pharmacy, Department of Pharmaceutics, Al-Ameen College of Pharmacy, Near Lal Bagh Main gate, Hosur Road, Bangalore, 560027 Karnataka, India. E-mail: [email protected] Received November 26, 2013 Accepted January 18, 2014 doi: 10.1111/jphp.12232

Abstract Objectives The current studies entail successful formulation of systematically optimized (OPT) nanoparticulate drug delivery system to increase the oral bioavailability using Eudragit RL 100 of trans-resveratrol (t-RVT), and evaluate their in-vitro/in-vivo performance. Methods t-RVT-loaded Eudragit RL 100 nanoparticles (t-RVT NPs) were prepared by nanoprecipitation method. The nanoparticles (NPs) were systematically optimized using 32 central composite design and the OPT formulation located using overlay plot. The pharmacokinetic and in-vivo biodistribution of t-RVT NPs were investigated in rats, and various levels of in-vitro/in-vivo correlation (IVIVC) were established. Key findings The OPT formulation (mean particle size: 180 nm) indicated marked improvement in drug release profile vis-à-vis pure drug and marketed formulation (MKT). Augmentation in the values of Ka (5.64-fold) and AUC0–24 (7.25fold) indicated significant enhancement in the rate and extent of bioavailability by the optimized trans-resveratrol-loaded Eudragit RL 100 nanoparticles (OPT-tRVT NPs) compared with pure drug. Level A of IVIVC was successfully established. OPT-t-RVT NPs showed 4.11-fold rose in the values of t-RVT concentrations in liver. In-situ single pass intestinal perfusion studies construed remarkable enhancement in the absorptivity and permeability parameters of OPT-t-RVT NPs. Conclusions The results, therefore, insight into the role of solubility enhancement and trounce enterohepatic recirculation for improving the oral bioavailability of t-RVT.

Introduction Nano-based drug delivery systems, particularly the polymeric ones, have recently increased wide acceptance for augmenting the bioavailability of poorly soluble and permeable drugs. Of late, nanoparticles (NPs) have emerged as an effective delivery systems owing to their inherent meritorious visages. NPs are newer and are novel technological innovations with immense potential in oral bioavailability enhancement of lipophilic drugs.[1] It has been reported that by encapsulating drugs into NPs, the bioavailability, tissue distribution and half-life can be improved and the toxicity of the drugs can be minimized.[2] Several potential advantages of NPs include capability of bypassing hepatic portal route and promoting the lym-

phatic transport of lipophilic drugs, reducing metabolism by cytochrome-P450 family of enzymes present in the gut enterocytes and liver hepatocytes and/or inhibiting P-glycoprotein (P-gp) efflux.[3,4] NPs are highly sought-after owing to their myriad benefits like better portability, improved stability, higher drug-loading and encapsulation efficiency, coupled with ease and economy of their production.[5–7] Systematic optimization of NP formulations for various product variables viz. polymers, and surfactants using design of experiments (DoE), tends to reveal (any) synergism among the variables.[8,9] Plus, it yields the most promising NP formulations with advantages of economics in

© 2014 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, ••, pp. ••–••

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Bioavailability of trans-resveratrol

Gurinder Singh and Roopa S. Pai

terms of money, time and developmental exertion. As the formulation of any nanoparticulate system depends upon the composition of rational blends of such polymeric and emulsifying agents to yield the optimal nanoparticulate drug delivery system, the use of DoE studies is considered almost imperative for the purpose.[10,11] Trans-resveratrol (t-RVT) is a poorly water-soluble drug with a log P of 3.1.[12,13] Despite this poor water solubility, t-RVT exhibits high membrane permeability and can be considered a class-II compound in the Biopharmaceutical Classification System. t-RVT has a very short plasma halflife (∼8–14 min).[14] t-RVT suffers enterohepatic recirculation and extensive first-pass metabolism by CYP3A4 in the liver, resulting in very low bioavailability (almost zero) in humans as well as in animals.[15] To circumvent the afore-mentioned limitations, various formulation approaches of t-RVT have been reported, such as liposome,[16] solid dispersions,[17] β-cyclodextrins inclusion complex,[18] microspheres[19] and suspensions, but all with limited fruition. In recent years, several studies have focused on novel formulation approaches to stabilize and protect t-RVT from degradation, to increase its solubility in water to improve its bioavailability, to achieve a sustained release and ultimately to target t-RVT to specific locations via multiparticulate forms and colloidal carriers. Various polymers are being utilized for preparation of NPs.[20,21] Eudragit RL 100 polymer is a copolymer of poly(ethylacrylate, methyl-methacrylate) containing an amount of quaternary ammonium groups between 8.8% and 12%. It is insoluble at physiologic pH values and capable of limited swelling, thus representing a good material for the dispersion of drugs. Eudragit RL 100 polymer is commonly used for enteric coating and also for preparation of controlled-release dosage forms.[22] Thus, the aim of this work was to develop polymeric NPs of t-RVT using nanoprecipitation method solely involving the rational selection and optimization of polymer concentration and emulsifier concentration, thus circumnavigating the need of inert carriers. Further, the work also investigates the oral bioavailability enhancement potential of polymeric NPs using pharmacokinetic studies, and embarks upon in-vitro/in-vivo performance. In this communication, we present data on the effect of nanoparticulate drug delivery of t-RVT on in-vivo biodistribution in rats.

Materials and Methods Materials Trans-resveratrol was provided ex-gratis by M/s Sami Labs (Bangalore, Karnataka, India). Eudragit RL 100 was received as gift sample from M/s Evonik Industries, Mumbai, India. Tetradecyl trimethyl ammonium bromide (TTAB) and acetone were supplied ex-gratis by M/s SD Fine-Chem 2

Table 1 Factors and levels of an orthogonal design (A–C are the respective codes for each factor) Factors Levels

A

B

C

Low High

8 000 16 000

30 40

1 5

A: Homogenizer Speed (gyration (g)); B: Time for homogenization (min); C: Rate of addition from organic phase to aqueous phase (ml/ min).

Limited, Mumbai, India. The solvents, that is, methanol, acetic acid and other chemicals employed for liquid chromatographic studies, were all of HPLC grade. All other materials employed during the studies were of analytical grade and were used as obtained. Double-filtered (0.45 μm pore size, Millipore, Bangalore, Karnataka, India) deionized water was used in all sample preparations to avoid any possible contaminations.

Optimization of process parameters by Taguchi orthogonal array design (OA4 (23)) Taguchi’s optimization technique is a unique and powerful optimization discipline that allows optimization with minimum number of experiments.[23] In this work, the effect of three important factors including homogenizer speed, time for homogenization and rate of addition from organic to aqueous phase, and each factor at two levels on the particle size (Dnm), drug encapsulation efficiency (Dee) and polydispersity index were studied using Taguchi’s method. The used level setting values of the main factors (A–C) are shown in Table 1. OA4 orthogonal array scheme was adapted, which needs four experiments to complete the optimization process; results are portrayed in Figure 1. Further, the effect of composition of organic phase was also optimized (OPT). Organic phase has a marked influence on drug-loading capacity of NPs. The Dee was highest in case when acetone was used alone to form organic phase. Acetone and water give largest, and acetone and dichloromethane give intermediate, which in turn was lower than acetone alone.

Preparation of trans-resveratrol nanoparticles as per experimental design t-RVT-loaded Eudragit RL 100 NPs were prepared based on the method of nanoprecipitation as previously described, using Ultra Turrax IKA T25 digital high shear homogenizer.[24,25] In brief, 100 mg Eudragit RL 100 and weighed amount of t-RVT were dissolved in 30 ml acetone to form the organic phase. The resulting organic phase was poured drop wise into 50 ml aqueous phase containing 100 mg of TTAB while homogenizing at 16 000g for 30 min.




Homogenizer speed

Time of homogenization

Rate of addition from organic to aqueous phase 60

Response magnitude

40

20

0 Dee

Dnm

PDI

−20 −40 −60 Process parameters

Figure 1

Influence of selected parameters on the response magnitude.

The resulting emulsion was placed on the magnetic stirrer plate and continuously stirred at room temperature to evaporate acetone for 5 h. The NPs were collected by centrifugation at 12 000g for 30 min and washed four times with distilled water. The NPs were then lyophilized and stored at 4°C until further analysis. Blank (without drug) NPs were also prepared using the same method. The yield of the polymeric NPs was 94.7% with this protocol. For further formulation optimization work, Eudragit RL 100 (X1) and TTAB (X2), were chosen and finally selected as the two critical influential factors. A central composite design (CCD) was employed, where the amounts of Eudragit RL 100 (X1) and TTAB (X2) were studied at three levels each. Overall, a set of 13 experimental runs each were studied as per the experimental design matrix as depicted in Table 2. The formulation at the intermediate coded factor levels (i.e. 0,0) was studied in quintuplicate. The response variables considered for the current DoE optimization studies encompassed, Dnm, Dee and mean percentage drug release in 24 h (Rel24 h).

HPLC method development The chromatographic separation was performed on a Phenomenex C18 column (Phenomenex Inc., Hyderabad, India) (250 × 4.6 mm I.D., 5 μm particle size) at 30°C. The HPLC (Shimadzu, Kyoto, Japan) instrument was equipped with binary pump and SPD- 20AVP UV detector (Shimadzu, Kyoto, Japan). The mobile phase was composed of methanol: 10 mM potassium dihydrogen phosphate buffer (pH 6.8): 3% acetic acid solution (70 : 28 : 2, v/v/v).

Table 2 Preparation of trans-resveratrol-loaded Eudragit RL 100 nanoparticles as per experimental design Coded factor levels

Trial no.

Factor 1, Eudragit RL 100 (mg)

Factor 2, emulsifier (mg)

1 2 3 4 5 6 7 8 9 10 11 12 13

0.00 (200) 0.00 (200) −1.00 (100) −1.00 (100) 0.00 (200) 1.00 (300) −1.00 (100) 0.00 (200) 1.00 (300) 0.00 (200) 1.00 (300) 0.00 (200) 0.00 (200)

0.00 (300) 1.00 (500) 1.00 (500) −1.00 (100) 0.00 (300) −1.00 (100) 0.00 (300) 0.00 (300) 0.00 (300) −1.00 (100) 1.00 (500) 0.00 (300) 0.00 (300)

The flow rate was set at 1 ml/min in isocratic elution, and the injected sample volume was 20 μl. The assay was linear over the t-RVT concentration range of 0.010–3.2 μg/ml. The limits of detection and of quantification of t-RVT were 0.005 and 0.007 μg/ml, respectively.[26]

Formulation characterization Particle size (Dnm) and zeta (ζ) potential measurements The Dnm of the t-RVT NPs was determined using Malvern Zetasizer Nano S90 (Malvern Instruments Ltd.,


3



Worcestershire, UK) and the zeta potential was measured using Malvern Zetasizer Nano ZS (Malvern Instruments Ltd.). Samples were diluted in MilliQ (Bangalore, Karnataka, India) water before measurement. Drug encapsulation efficiency (Dee) The Dee of t-RVT NPs was calculated by determining the amount of pure drug using a filtration technique. The Dee was determined by the separation of drug-loaded NPs from the aqueous medium containing non-associated t-RVT by ultracentrifugation (REMI high speed, cooling centrifuge, REMI Corporation, Bangalore, Karnataka, India) at 12 000g for 30 min, at 4°C. The unencapsulated t-RVT was determined using HPLC. The total drug content in the t-RVT NPs was determined by dissolving the t-RVT NPs in methanol to release entrapped t-RVT. The resulting solution was analyzed using HPLC. The drug-loading content was the concentration of incorporated drug to polymer (w/w).

In-vitro drug release studies In-vitro drug release studies were carried out for all the formulations in hexaplicate. The release kinetics of t-RVTloaded Eudragit RL 100 NPs, pure drug and marketed formulation (MKT) in phosphate buffered saline (PBS) were evaluated by a dialysis method for up to 24 h. The samples were individually dispersed in 1 ml of the releasing medium (PBS) and were placed into a cellulose membrane dialysis tube (molecular weight cut off 10 000–12 000). The dialysis tube was then placed into 200 ml of PBS and the release test was performed at 37°C with a stirring rate of 50 rpm. At predetermined time points, 1 ml release medium was taken, refilled with the same amount of the fresh medium and concentrations of the released drug were determined by HPLC as described in the ‘HPLC method development’ section. The absorbance was measured at 306 nm.[27]

Drug release studies of optimized trans-resveratrol-loaded Eudragit RL 100 nanoparticles vis-à-vis other products A drug release profile of the OPT-t-RVT NPs was compared with that of the pure drug and conventional MKT each containing 100 mg of t-RVT.

Optimization data analysis and validation of optimization model The response variables, which were considered for systematic DoE optimization studies, included Dnm, Dee and Rel24 h. For the studied design, the multiple linear regression analysis was applied to fit full second-order polynomial equation with added interaction terms to correlate the studied 4

responses with the examined variables using Design Expert ver. 8.0 software (M/s Stat-Ease, Minneapolis, Minnesota, USA). The polynomial regression results were demonstrated for the studied responses. Finally, the prognosis of OPT formulation was conducted using overlay plots, drawn using the Design Expert software.[8] Overlay plot was obtained by superimposing the values of various response variables to locate the OPT formulation. The observed and predicted responses were critically compared and the percent bias (i.e. prediction error) was also calculated with respect to the observed responses.

Differential scanning calorimetry The physical state of t-RVT entrapped in the NPs was characterized by differential scanning calorimetry (DSC-60, Shimadzu, Japan). Each sample was sealed in standard aluminium pans with lids and purged with air at a flow rate of 40 ml/min. A temperature ramp speed was set at 20°C/min, and the heat flow was recorded in the range of 30–300°C under inert nitrogen atmosphere. Thermograms were taken for pure drug, Eudragit RL 100 and OPT-t-RVT NPs.

Transmission electron microscopy The morphology of the OPT-t-RVT NPs was observed using transmission electron microscopy (TEM) attached with a mega view II digital camera (H 7500, Hitachi, Tokyo, Japan). A drop of sample diluted with water was placed on a copper grid and the excess was drawn off with a filter paper. Samples were subsequently stained with 1% of uranyl acetate solution for 1 min. The image was magnified and focused on a layer of photographic film.

In-vivo pharmacokinetic studies in rat Cognizance was taken that the research work, involving in-vivo pharmacokinetic studies, adheres to the guidelines for care and use of the laboratory animals. Thus, all the animal investigations were performed as per the requisite protocol approved by the Institutional Animal Ethics Committee (Letter no. AACP/IAEC/Jun-2012-02). The Committee is duly approved for the purpose of control and supervision of experiments on the animals by the Government of India. The pharmacokinetic study involved three groups. Six rats (male Wistar, weighing 250–300 g) were randomly distributed among each group. Group I received OPT-t-RVT NPs, group II received pure drug and group III received the MKT redispersed in 1 ml of water. All the animal groups received a dose equivalent to 20 mg of t-RVT per kg of body weight. Following oral drug administration, the rats kept in cages were allowed access to food and water ad libitum.




Serial aliquots of the blood samples (100 μl each) were withdrawn from the retro-orbital plexus under mild ether anaesthesia at 0, 0.083, 0.166, 0.25, 0.5, 1, 1.5, 2, 4, 6, 8 and 12 h in the heparinized microcentrifuge tubes (50 units heparin/ml of blood). Plasma was harvested by centrifugation at 15 000g for 15 min and stored at −20°C until analyzed. Acetonitrile was added to precipitate the plasma proteins. Thereafter, samples were vortexed and centrifuged at 15 000g for 15 min. Plasma samples were analyzed for t-RVT by HPLC method. Each of the plasma samples was thawed to room temperature. One hundred microlitre of acetonitrile was added to it and mixed using a vortex mixer (M/s Remi Equipment Pvt. Ltd, Bangalore, Karnataka, India) for 1 min. The upper organic phase was transferred and evaporated to dryness on a water bath at 50°C. To each dry residue, 100 μl of acetonitrile was added and mixed using the vortex mixer for 1 min to reconstitute the same. The reconstituted sample was injected into the HPLC system. The competence of nanoparticulate formulation was assessed by administering pure drug and MKT orally and measuring the blood levels at 0, 0.083, 0.166, 0.25, 0.5, 1, 1.5, 2, 4, 6, 8 and 12 h. Non-compartmental pharmacokinetic parameters for extravascular input, that is, Cmax, Tmax, AUC0–24, t1/2, Ke and MRT were computed by choosing Kinetica 5.0.11 version software (Thermo Fisher Scientific Inc., Waltham, Massachusetts, USA).

In-situ SPIP studies were performed in male Wistar rats. The animals were before made to abstain from solid food at least 24 h before the study. The SPIP studies were carried out on the OPT-t-RVT NPs, pure drug and MKT, each in hexaplicate, employing an in-house fabricated assembly. The animals were anaesthetized using thiopental sodium (50 mg/kg) intraperitoneally. Following midline incision of the abdomen, the proximal part of the jejunum, 2–5 cm below the Ligament of Trietz, was cannulated with a glass cannula and connected to a reservoir.[30] The second incision was made 10–15 cm below the first incision and connected with an outflow glass cannula. The intestinal segments were perfused with Krebs–Ringer’s solution until the perfusate was clear. The intestinal segments were consequently perfused with OPT-t-RVT NPs, pure drug and MKT, maintained at 37 ± 1°C at a perfusion rate of 0.25 ml/ min. Steady state was achieved within 30 min, after which aliquots of samples (1 ml each) were periodically withdrawn at a regular intervals of 10 min each. The volume of sample for each time interval was 2 ml. Samples were stored at −20°C until analysis. Samples were filtered and directly injected onto HPLC column and required no sample preparation before analysis.

In-vivo biodistribution of optimized trans-resveratrol-loaded Eudragit RL 100 nanoparticles

The OPT-t-RVT NPs were subjected to stability studies carried out at 25 ± 2°C/ 60 ± 5% RH, as per the ICH guidelines[31] for the climatic zone IV. The formulation was kept in airtight glass vials and assayed periodically, at the time points of 0, 1, 3 and 6 months, for Dnm, Dee, Rel24 h and ζ potential.[32]

The animals and dosing protocol were the same as in pharmacokinetic study. To study in-vivo biodistribution, 45 rats were randomly divided into three groups of 15 rats per group. Group I was treated with OPT-t-RVT NPs, Group II was treated with pure drug and Group III was treated with MKT. At time 1, 3, 6, 12 and 24 h after dosing, three rats of each group were sacrificed by cervical dislocation under general pentobarbital anaesthesia. Brain, heart, liver, lungs, kidneys and spleen were collected and stored at −16°C until analysis. The extent of in-vivo biodistribution and retention following oral administration of NPs was analyzed for 24 h.

In-vitro/in-vivo correlations Attempts were made to correlate the in-vivo plasma level data obtained for OPT-t-RVT NPs, pure drug and MKT with the corresponding in-vitro drug release data and establish in-vitro/in-vivo correlations (IVIVC). For establishing Level A IVIVC, percent drug absorbed data at various time points were obtained using modified Wagner–Nelson method and correlated with percent drug release data.[28,29]

In-situ SPIP studies

Stability studies

Statistical analysis For pharmacokinetic analysis, the plasma concentrations of each rat were analyzed with the Kinetica 5.0.11 version software (Thermo Fisher Scientific Inc.), using a noncompartmental model. All results were expressed as means ± standard deviation (SD). For in-vivo biodistribution analysis, the concentrations of drug in each organ were performed by one-way ANOVA using GraphPad Prism software ver 5.0 (M/s GraphPad Software Inc., San Diego, CA, USA). The results were confirmed by Bonferroni’s as a post hoc test.

Results Dnm and ζ potential Dnm of all the 13 NP formulations, prepared as per the experimental design, ranged between 130 and 210 nm. Remarkably small Dnm was observed at the higher levels of


5



the TTAB and intermediate levels of the Eudragit RL 100. The ζ-potential was found to be positive of all the 13 NP formulations ranged between +18.52 and +22.17.

Drug encapsulation efficiency Dee is drug-loading capacity of any formulations. In the current study, the centrifugation method was used to determine Dee. The Eudragit RL 100 concentration has a marked influence on drug-loading capacity of NPs. The Dee of all the 13 NP formulations ranged between 35.72 and 60.31% as per experimental design. The drug loading was ranged between 79.24 and 92.16% of all the 13 NP formulations.

Drug release studies of OPT t-RVT NPs vis-à-vis other products

In-vitro drug release studies All the formulations tended to release ∼75.41% of drug within 24 h, with relatively miniscule amount of drug release after 24 h.

Response surface analyses The coefficients of the polynomial equation (Eq. 1), for Dnm, Dee and Rel24 h formed excellent fits to the data, with the values of r2 ranging between 0.9956 and 0.9985 (P < 0.001 in all the cases).

Y = β0 + β1X1 + β2 X 2 + β3 X1X 2 + β4 X12 + β5 X 22 + β6 X1X 22 + β7 X 2 X12

(1)

As observed in Figure 2a, a somewhat linear increase in Dnm was observed with rise in the levels of polymer concentration (Eudragit RL 100). With increase in emulsifier (TTAB) concentration, however, a curvilinear descending trend was observed. Figure 2b shows a ‘canopy type’ of response surface, characterizing initial non-linear increase, followed by a decline in the values of Dee with further rise in the levels of polymer. Analogous observation, although of less magnitude, was noticeable in case of emulsifier except at the higher levels of polymer. Figure 2c depicts a non-linear reduction in percentage Rel24 h values with augmenting levels of TTAB, the effect being much more pronounced at the higher levels of polymer. As the polymer levels rise, Rel24 h values tend to increase non-linearly.

Optimization data analysis and validation The optimum formulation was selected by ‘trading off’ various response variables while adopting the following criteria: Dnm ∼200 nm; Dee >80.00%; Rel24 h >90% 6

The OPT-t-RVT-loaded freeze-dried Eudragit RL 100 NPs (containing emulsifier 243 mg and polymer 220 mg) was found to fulfil maximal criteria for optimal performance. The said formulation exhibited Dnm of 180 nm, ζ potential of +22.61, Dee of 83.69%, drug loading of 98.52% and Rel24 h of 97.21%. Linear correlation plots drawn between the predicted and observed responses after forcing the line through the origin, also demonstrated high values of r, ranging between 0.9973 and 0.9998, indicating excellent goodness of fit (P < 0.001 in each case). The corresponding residual plots also showed nearly uniform and random scatter around the zero-axis.

Marked improvement was observed in the release profile of OPT-t-RVT NPs vis-à-vis the pure drug and MKT (Figure 3). Drug dissolution was nearly completed within 24 h (drug release 97.21%) in case of OPT-t-RVT NPs, as compared with that of the pure drug and MKT, where drug release was only 87.14% and 94.02%, respectively within 4 h.

Differential scanning calorimetry DSC thermograms of pure drug, Eudragit RL 100 and OPTt-RVT NPs showed characteristic endothermic peaks at 270.2, 89.9 and 62.7°C are shown in Figure 4.

Transmission electron microscopy The TEM image (Figure 5a) unambiguously reveals that most of the emulsion particles of OPT-t-RVT NPs were below 250 nm in size and were spherical in shape. Figure 5b depicting the Dnm distribution distinctly reveals the particles size of the optimum formulation as 180 nm.

In-vivo pharmacokinetic studies in rats The mean plasma concentration–time profile (Figure 6a) depicts significant higher plasma levels from OPT-t-RVT NPs with respect to pure drug and MKT (P < 0.001). Further, the lower magnitude of standard error means of OPT-t-RVT NPs indicated that the pharmacokinetic profile was more reproducible than that of pure drug and MKT. Plasma drug profile of OPT-t-RVT NPs exhibited more sustained release vis-à-vis of pure drug and MKT. The absorption rate was markedly enhanced, as revealed by distinct improvement in various pharmacokinetic parameters, that is, Tmax and AUC0–24 (Figure 6b). Maximal change in the pharmacokinetic parameter was observed in case of absorption rate constant (Ka), which is known to reflect the



(b)

400.0

80.0

266.7

66.7

133.3

53.3

Dee

Dnm

(a)


40.0

0.0

1.00 0.00 B: TTAB

1.00

1.00

(c)

0.00 B: TTAB

0.00 A: Eudragit RL 100

−1.00 −1.00

1.00

−1.00 −1.00

0.00 A: Eudragit RL 100

100.0

Rel24h

66.7

33.3

0.0

1.00

B: TTAB

1.00

0.00 0.00 −1.00 −1.00

A: Eudragit RL 100

Figure 2 Response surface plot showing the influence of tetradecyl trimethyl ammonium bromide (TTAB) (emulsifier) and Eudragit RL 100 (polymer) on (a) particle size (Dnm), (b) drug encapsulation efficiency (Dee) and (c) mean percent drug release in 24 h (Rel24 h) for nanoparticles (NPs) formulations of trans-resveratrol (t-RVT).

first-order rate of drug absorption. Nearly 5.64-fold augmentation in the magnitude of Ka was perceptible with the OPT-t-RVT NPs. On the whole, the values of Cmax, Tmax, AUC0–24 and MRT rose considerably vis-à-vis pure drug to 1.29, 12.0, 7.25 and 8.82-fold, respectively, in case of the OPT-t-RVT NPs formulation ratifying distinct improvement in extent of bioavailability too (P < 0.001). Subsequent one-way ANOVA carried out on various pharmacokinetic parameters viz. AUC0–24, Ka and Tmax connoted highly statistically significant variation (P < 0.001) in the drug absorption potential of t-RVT from OPT NPs visà-vis pure drug.

In-vivo biodistribution Results demonstrate that OPT-t-RVT NPs of various physiochemical properties mainly accumulated in the brain, heart, liver, lungs, kidneys and spleen over a period of 24 h is shown in Figure 7. It was observed that liver and spleen accumulated major portion of the administered OPT-tRVT NPs. Nevertheless, liver is one of the major organs of reticuloendothelial system, which is known to accumulate and metabolize NPs.[33] However, very low concentrations were observed in the plasma over the same period. The biodistribution data revealed initial rapid uptake and


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OPT-t-RVT NPs

Pure drug

MKT

80

Mean percent drug release

Mean percent drug release

100

60

40

20

100 75 50 25 0 0

1

2 3 Time (h)

4

0 0

4

8

12

16

20

24

Time (h) Figure 3 In-vitro release profile of trans-resveratrol (t-RVT)-loaded Eudragit RL 100 NPs with tetradecyl trimethyl ammonium bromide (TTAB) as emulsifier in pH 7.4 phosphate buffer saline (PBS). Data points shown are mean ± standard deviation (SD) (n = 3).The inset shows the mean percent drug release in 4 h.

7b). At the end of 3 h, faster elimination of pure drug as well as MKT was observed in all the organs vis-à-vis OPT-tRVT NPs. For instance, the fractions of the concentrations which reached these organs with drug and MKT were (27.33 ± 0.052 ng/gm) and (34.66 ± 0.078 ng/gm) in the liver and (25.04 ± 0.031 ng/gm) and (27.34 ± 0.044 ng/gm) in the spleen, respectively, 1 h after the administration. In comparison, these values were higher considering the OPT-t-RVT NPs (heart: 33.02 ± 0.017 ng/gm, lungs: 29.67 ± 0.011 ng/gm and kidneys: 100.04 ± 0.052 ng/gm) during the first 3 h vis-à-vis pure drug and MKT (Figure 7c, 7d and 7e). No t-RVT was found in the brain of rats during the first 1 h treated by OPT-t-RVT NPs, pure drug and MKT. At 6 h, miniscule concentrations were found in brain (18.67 ± 0.23 ng/gm) of OPT-t-RVT NPs (Figure 7f). After 24 h, concentrations of t-RVT were not detectable and reached below detectable limits.

216 J/g (a)

270.2Cel −44.94(J/g) 58.4 J/g (b) 89.9Cel −12.47(J/g) 108 J/g (c)

50.0

62.7Cel −24.43(J/g) 100.0

150.0 200.0 Temp Cel

250.0

300.0

In-vitro/in-vivo correlation

Figure 4 Differential scanning calorimetry (DSC) thermograms of (a) pure drug, (b) Eudragit RL 100 and (c) optimized trans-resveratrolloaded Eudragit RL 100 nanoparticles (OPT-t-RVT NPs).

showed significantly higher concentrations of t-RVT by liver and spleen, which were 268.13 ± 2.71 ng/gm and 180.56 ± 1.68 ng/gm, respectively, for OPT-t-RVT NPs during the first 3 h of the experiment than those observed in groups treated with pure drug and MKT (Figure 7a and 8

Both linear and non-linear Level A correlations were investigated between the two variables. Overall, the quadratic relationship was found to be better fitted than the linear and cubic non-linear relationship.

In-situ SPIP In the current study (Figure 8), OPT-t-RVT NPs significantly improved the magnitude of absorption number by




(b) 20 Intensity (%)

(a)

Size distribution by intensity

15 10 5 0 0.1

500 nm

1

10 100 1000 10000 Size (Dnm)

Figure 5 (a) Transmission electron microscopy (TEM) image shows particle size of optimized trans-resveratrol-loaded Eudragit RL 100 nanoparticles (OPT-t-RVT NPs); (b) size distribution of OPT-t-RVT NPs.

6.60-fold, with reference to the pure drug. Nearly identical values of absorption number for the pure drug and MKT suggest analogous absorption mechanisms. Another dimensionless absorption parameter, fraction absorbed, provides an estimate of the extent of drug absorption.[34] As depicted in Figure 8, the marked increase in the magnitude of fraction absorbed in case of OPT-t-RVT NPs (5.37-fold) vis-àvis pure drug indicates high bioavalability improvement potential of the OPT-t-RVT NPs. The effective permeability provides a direct measurement of absorption rate across the intestinal epithelium. As shown in Figure 8, Nearly 4.43fold enhancement in the values of effective permeability of t-RVT was noticeable upon formulating it as NPs.

Stability studies Table 3 shows miniscule variation in the formulation parameters during 6 months of storage at the stability conditions of 25 ± 2°C/60 ± 5% RH. The values of the similarity factor (f2) ranged between 92.36 and 97.58.

Discussion This is the first pharmacokinetic, IVIVC and in-vivo biodistribution studies of OPT-t-RVT NPs to assess the potential for a NPs stabilized Eudragit RL 100 based formulation to improve the absorption of a poorly water-soluble drug for ratifying its biopharmaceutical superiority. The OPT formulation provided enhanced bioavailability when compared with the pure drug and MKT. Optimization of a pharmaceutical formulation or a process using this traditional approach involves studying the influence of the corresponding composition and process variables by changing one single variable at a time (COST), while keeping others as constant. Screening involves the selection of factors that really have an effect on the system being studies, is distinguishable unequivocally from the background noise.

The influence of process parameters also plays a significant role in the preparation of the NPs. From the information obtained from the bar plot in Figure 1, we could infer that the homogenizer speed and rate of addition of organic to aqueous phase were positively significant process parameters and that, by contrast, the time for homogenization was negatively significant. Finally, the process parameters time for homogenization was considered as not significant at all and was omitted for the optimization step. Finally, homogenizer speed (16 000g) and rate of addition of organic to aqueous phase (2 ml/min) were selected and kept constant in further optimization studies. Further, Organic to aqueous phase ratio and composition of organic phase were altered to study the effect of these variables on physicochemical properties of NPs. As the aqueous phase volume was increased, Dnm decreased. This is due to higher diffusion of non-solvent into the aqueous phase. Because as the aqueous phase volume is increased, it becomes dilute. So diffusion increased and results into smaller Dnm. Smallest NPs size was observed with acetone as solvent system. NPs prepared with acetone and dichloromethane were intermediate and largest in the case of acetone and water. Finally, acetone was opted as solvent system. Solubility of drug has a marked influence in Dee. Dee was higher in solvent system in which the drug is highly soluble. t-RVT solubility is 6.3 mg/ml in acetone. The CCD is documented as the most efficient design. In the present studies, fitting a non-linear model was considered better, as the values of the response surfaces were not known from the previous findings. This design, that is, a two-factor at three levels with α = 1 has an added advantage of determining the non-linear (e.g. quadratic) in the factor– response relationship. The linearity of the correlation plots between predicted and observed responses (P < 0.001), and nearly uniform and random scatter of the corresponding residual plots indicate high prognostic ability of the postulated model.


9


Plasma drug conc. (μg/ml)


Plasma drug conc. (μg/ml)

(a) 0.2

0.15

0.1

Pure drug MKT OPT-t-RVT NPs

0.16 0.12 0.08 0.04 0 0

0.5 1 1.5 Time (h)

2

0.05

0 0

10

Percentage change in pharmacokinetic parameters

(b)

4

8

Pure drug

12 Time (h) MKT

16

20

24

OPT-t-RVT NPs

8 6 4 2 0 Cmax (μg/ml)

Tmax (h)

MRT (h) AUC (μg/ml)*h

Ka

Figure 6 (a) Plasma drug level profiles of optimized trans-resveratrolloaded Eudragit RL 100 nanoparticles (OPT-t-RVT NPs), pure drug and marketed formulation (MKT). Each point represents mean of six replicates and each cross bar indicates standard error of mean (SEM); (b) bar chart depicting comparative change in pharmacokinetic parameters of optimized trans-resveratrol-loaded Eudragit RL 100 nanoparticles (OPT-t-RVT NPs) relative to pure drug and MKT.

We next investigated the in-vitro release profiles of all the 13 NP formulations in pH 7.4 PBS at 37°C. A sustained release pattern is a key issue in the development of colloidal drug delivery systems used in the field of nanomedicine. As the drug content in the formulation was higher, the release rate also increased. This may be due to the saturation of quaternary ammonium group present in Eudragit RL 100 polymer by drug molecules, occurred at a high drug content, which results in increased drug release from the formulations. None of the formulations showed burst release pattern. This indicates that the drug is homogeneously dispersed in polymer matrix. Some other factors, including the polymer composition, percentage of drug loading, Dnm, as well as the characteristics of the 10

encapsulated drug also affect the drug release from Eudragit RL 100 NPs. All the formulations, prepared as per the experimental design, exhibited nano Dnm, ranging between 130 nm and 210 nm. As we all know, the Dnm plays an important role in the alternation of pharmacokinetics through adjusting the in-vivo distribution and clearance. The polydispersity of all NP formulations was less than 0.25, demonstrating a unimodal size distribution. ζ-Potential is also of interest for the characterizations of these NPs because it reflects the surface charge of NPs and has been usually used to predict and control stability. Positive surface charge noted for the NPs were due to the polymer. It is a cationic polymer having quaternary ammonium groups. Dee is also an important characteristic, which can directly influence the amount of the drug-loaded NPs at a given dose level. The Dee of all the 13 NP formulations ranged between 35.72 and 60.31% as per experimental design. A high Dee of 83.69 ± 1.2% for OPT-t-RVT NPs was obtained. Eudragit RL 100 NPs have been reported to give high Dee for water-insoluble drugs.[35] Present investigation also reveals that nanoprecipitation method can lead to NPs with high entrapment with water insoluble drug such as t-RVT. Marked improvement was observed in the drug release profiles (Figure 3) of the OPT-t-RVT NPs as compared with that of pure drug and MKT. This significant augmentation in the values of dissolution time for OPT-t-RVT NPs is quite indicative of higher bioavailability potential of t-RVT, a Biopharmaceutical Classification System (BCS) class-II drug-exhibiting dissolution-limited absorption. Analogous in-vitro dissolution profiles from OPT-t-RVT NPs construe that the Eudragit RL 100 seem to sustain the drug release from the NPs. DSC characteristic peak for t-RVT in NPs was found to be reduced in intensity and shifted to 62.7°C, probably because of encapsulation in Eudragit RL 100 and TTAB NPs. However, peak at 270.2°C exhibited by t-RVT was not visible in OPT-t-RVT NPs, indicating that t-RVT was encapsulated by the polymers in the NPs. It can thus be indicated that t-RVT in NPs was in amorphous or disordered crystalline phase, which inhibited crystal growth, thereby leading to enhanced stability of formulation. OPT-t-RVT NPs were designed to improve the oral bioavailability of the drug. One study reported in the literature demonstrating the in-vivo performance of t-RVTloaded Eudragit RL 100 NPs.[36] To our knowledge, there was no comprehensive study reported in the literature demonstrating the in-vivo biodistribution, IVIVC and in-situ SPIP studies of t-RVT-loaded Eudragit RL 100 NPs. We believe that no single parameter can alone influence the fate of the formulations in vivo; rather, it is a collective influence of many parameters. Blood levels after oral




Concentration (μg/gm)

(b)

Liver

0.30


0.24 0.18 0.12 0.06


(a)

1

3

(c)

6 Time (h)

12

0.12 0.08 0.04

1

24


0.024 0.018 0.012 0.006 0.000

6 12 Time (h)

24

Lungs

0.035 Concentration (μg/gm)

0.030

3

(d)

Heart 0.036



0.16

0.00

0.00


0.028 0.021 0.014 0.007 0.000

3

(e)

6 Time (h)

12

24 (f)

Kidneys

0.12


0.09

1

0.06

0.03

3

6 12 Time (h)

24

Brain

0.024


1


Spleen

0.20


0.018

0.012

0.006

0.000

0.00 1

3

6 Time (h)

12

24

1

3

6 Time (h)

12

24

Figure 7 In-vivo biodistribution of pure drug, marketed formulation (MKT) and optimized trans-resveratrol (t-RVT)-loaded Eudragit RL 100 nanoparticles (OPT-t-RVT NPs) to rats. Concentration of t-RVT (y axis) was determined in different organs (a) Liver, (b) Spleen, (c) Heart, (d) lungs, (e) kidneys and (f) Brain at 1, 3, 6, 12 and 24 h after oral administration.

administration of pure drug and MKT were compared with those after oral administration of OPT-t-RVT NPs. The mean plasma levels of t-RVT after oral administration of OPT-t-RVT NPs, pure drug and MKT are shown in Figure 6a. From the graphs obtained by plotting plasma drug concentration versus time, it was observed that OPT-t-RVT

NPs showed the release for 16 h with Cmax of 0.192 ± 2.91 μg/ml and AUC0–24 of 1.813 ± 1.67 μg/ml at Tmax of 6.02 h. Furthermore, Dnm was also influence the performance in vivo. Oral administration of pure drug exhibited only 2 h plasma profile, where oral pure drug suspension showed a Cmax of 0.144 ± 1.82 μg/ml at Tmax of 30 min. Low values of AUC0–24 and Cmax after oral adminis-


11

Percent increase in permeability and absorption parameters


250


Effective permeability Absorption number

Wall permeability Fraction absorbed

200 150 100

50 0 MKT

Pure drug

OPT-t-RVT NPs

Figure 8 Percent increase in permeability parameters and absorption parameters in optimized trans-resveratrol-loaded Eudragit RL 100 nanoparticles (OPT-t-RVT NPs) vis-à-vis pure drug and marketed formulation (MKT) using in-situ single-pass intestinal perfusion technique.

Table 3 Various parameters of the optimized trans-resveratrol-loaded Eudragit RL 100 nanoparticles formulation analyzed at different time points during stability studies Time (month) 25 ± 2°C/60 ± 5% 0 1 3 6

Dnm RH 200 199.72 199.45 198.97

Dee

Rel24 h

ζ potential

83.69 83.22 82.64 82.13

93.52 93.08 92.86 92.05

+22.61 +22.78 +22.05 +21.46

tration of adequate dose of pure drug (20 mg/kg) also point towards incomplete absorption of drug from rats.[37] The higher Cmax levels coupled with higher values of AUC0–24 observed in that OPT-t-RVT NPs indisputably vouch distinct improvement in rate and extent of drug bioavailability. This augmentation in bioavailability would finally result in an increase in the intensity of therapeutic effect of t-RVT. Exclusively higher values of Tmax, AUC0–24, MRT and Ka for OPT-t-RVT NPs were observed vis-à-vis pure drug and MKT as portrays in Figure 6b. The oral bioavailability of t-RVT is less than 1% owing to various factors, which includes partial solubility in water, extensive metabolism in the intestine, intense hepatic firstpass metabolism, colonic bacterial metabolism and poor absorption. Further, following rapid and extensive metabolism various metabolites of t-RVT are formed viz. t-RVT glucuronides and t-RVT sulfates.[38] Extremely rapid sulfate conjugation by the intestine/liver appears to be the ratelimiting step in t-RVT bioavailability. Second peak at 2 h indicates enterohepatic recirculation of the drug that has been well documented and enterohepatic recirculation is 12

susceptible to circadian variation[14,39] (as is evident from the Figure 6a where two peaks of Cmax and Tmax were observed). A major portion of the bile acids secreted is reabsorbed from the intestines and returned via the portal circulation to the liver, thus completing the enterohepatic cycle.[40,41] The parent compound and its conjugated metabolite undergo enterohepatic recirculation, resulting in multiple peaks in the plasma concentration-time profile. This effect can be explained by reduced enterohepatic recirculation of t-RVT in case of NPs due to inhibition of the multidrug resistance-associated protein 2 enzyme, thus avoidance of enterohepatic recirculation, preventing their reabsorption.[42] On the other hand, due to the existence of amino groups, Eudragit RL 100 (non-adhesive) possesses positive charge, so it can bind negatively charged bile acids in the intestine.[40,43] The positively charged NPs will bind and thus remove negatively charged bile acids from the enterohepatic circulation and excrete them into the faeces.[44,45] The increase in bioavailability observed with the OPT-t-RVT NPs can be attributed to the absence of first-pass metabolism and enterohepatic recirculation, resulting in single peak in the plasma concentration-time profile without any sharp peaks. Thus, besides increasing the patient compliance owing to reduced dosing and frequency of administration (once a day). These results are in consonance with literature findings, where significant enhancement in oral bioavailability of antioxidant (baicalein) has been attained using nanoparticulate drug delivery system to overcome enterohepatic recirculation.[45] This low absorption rate potential can be attributed to slower and incomplete dissolution of this BCS class-II drug in GI tract. Thus, besides poor aqueous solubility of t-RVT, decreased extent of bioavailability in rat may be due to a multitude of other reasons. The potential causes for decreased absorption include thicker GI barriers, higher hepatic first-pass metabolism, lower residence time in GI tract and lower absorptive surface area. A significant increase (P < 0.001) in the AUC values of OPT-t-RVT NPs vis-à-vis pure drug distinctly indicates the improved bioavailability with the NPs. IVIVC allows prediction of the in-vivo performance of a drug based on the in-vitro drug release profiles. This relationship facilitates the rational development and evaluation of immediate/extended release dosage forms as a tool for formulation screening, in setting dissolution conditions and as a surrogate for bioequivalence testing. Excellent Level A correlation was observed with OPT-t-RVT NPs. Because t-RVT is a poorly water-soluble drug exhibiting nearly complete and dissolution limited absorption, it is anticipated to establish good point-to-point correlation between in-vitro and in-vivo performance (i.e. Level A). Apart from being an excellent product development tool, IVIVC can be exploited for obtaining biowaivers of such systems too. For class-II



compounds, dissolution is the rate-limiting step in absorption, therefore the establishment of IVIVC is expected vis-àvis other BCS classes. The IVIVC established in the current studies has a definitive novel nuance, as hardly any study on fruitful establishment of level A IVIVC has been reported for a BCS class-II drug t-RVT encapsulated in Eudragit RL 100 nanoparticulate drug delivery system. Figure 7 reported t-RVT concentrations found in different organs of the rats after oral administration of the OPTt-RVT NPs, pure drug and MKT over a period of 24 h. As shown Figure 7, significant amount of OPT t-RVT NPs was found in the different organs including the brain, heart, liver, lungs, kidneys and spleen. The concentrations varied with time and were greatly influenced by the type of oral formulation viz. OPT-t-RVT NPs, pure drug and MKT. t-RVT concentrations found in liver of rats treated with OPT-t-RVT NPs were significantly higher than that found in animals of the group treated with pure drug and MKT. The biodistribution results show that the majority of OPT t-RVT NPs accumulated in the liver. This is due to the fact that the discontinuous gaps in the endothelium, which lines the sinusoidal walls of liver allow the passive entrapment of foreign particulates.[46,47] The high organ concentration for liver indicates that NPs were internalized post-physical sequestration because of the prevalent presence of macrophages in this organ. Thus, the liver, where most macrophages reside, showed the most extensive OPT-t-RVT NPs accumulation in the biodistribution study. After 24 h of treatment, neither OPT-t-RVT NPs nor pure drug and MKT were detected in brain, heart, liver, lungs, kidneys and spleen samples of rats. Several factors, such as Dnm, ζ-potential and opsonization may influence the organ distribution of orally administrated NPs. In addition, the lower concentration of NPs detected in brain and heart as opposed to the higher proportion in the liver and the spleen may be due to an enhanced lymphatic uptake (representing particles that are taken up by the M cells of the Peyer’s patches via opsonization) and the liver (representing particles that are taken up by the Kupffer cells of the liver). Thus, from the results obtained, it could be anticipated that the rapid uptake of the OPT-t-RVT NPs by macrophages of the MPS and possibility to target the associated drug in high concentrations in the liver. It was observed that uptake was faster for OPT-t-RVT NPs, which may be due to higher particle size. Although there was no statistically significant difference between pure drug and MKT after administration (P > 0.05), significantly higher levels were observed for OPT-t-RVT NPs formulation (P < 0.01). Significant improvement in the magnitudes of fraction absorbed and absorption number exhibited by the OPT-tRVT NPs (Figure 8) indicated distinct improvement in the amount of drug transported across the intestine. This cor-


roborated their high bioavailability improvement potential vis-à-vis pure drug and MKT. Much higher values of the absorptivity parameters from the Eudragit RL 100 NPs can be ascribed to the transport of drug via M cells and the Peyer’s patches, in accordance with an earlier literature report.[48,49] Size is also an important characteristic for efficient uptake. Numerous studies have been conducted for investigating the effect of size in various animal models and experimental systems, with the common consensus being that particles less than 1 μm in size can be transcytosed by M cells.[50,51] Also, the improvement in permeability of OPTt-RVT NPs may be attributed to the high concentration of emulsifier present in the formulations. Analogous results of increased intestinal permeability on increasing the emulsifier concentration have been reported in literature.[52] The in-situ SPIP studies, in this regard, are known to provide tangible inkling on the absorption and permeation potential of a drug when administered as nanoparticulate system and may be useful to determine the enteric and enterohepatic recycling of drugs.[49] The in-situ SPIP studies in rats, therefore, corroborate the biopharmaceutical superiority of the NP formulations over the MKT. The close analogy of the in-vivo results with that of the SPIP studies ratifies the usefulness of this in-situ technique in the estimation of bioavailability and/or permeation parameters.

Conclusions Despite the bioavailability enhancement potential of nanoparticulate drug delivery systems, it is highly desirable to have these as solid dosage form(s) owing to their stellar merits. The novelty of the current work is the development of nanoparticulate drug delivery system solely through the judicious selection of apt blends of Eudragit RL 100 and emulsifier, and optimizing their composition using systematic ‘DoE’ methodology. Considerable enhancement in the rate and extent of oral drug absorption ratified the superior performance of the nanoparticulate drug delivery systems in enhancing the bioavailability of t-RVT. Besides drug release enhancement and nano Dnm, significant improvement in oral bioavailability of t-RVT from nanoparticulate drug delivery system may be attributed ostensibly to reduced metabolism. This study showed that conformation of Eudragit RL 100 decorating the NP surface is influencing the pharmacokinetic and in-vivo biodistribution of NPs. The rather short circulation half-life was explained by the rapid accumulation of the NPs in organs over 24 h, which occurred in parallel with the reduction in the plasma concentration of the drug. Thus, the OPT-t-RVT NPs are in a blood with a short circulation time. In-vivo biodistribution study showed that t-RVT concentration in liver for OPT-tRVT NPs was higher than pure drug. Based on these findings it can be concluded that t-RVT-loaded Eudragit RL 100


13



NPs developed in this study may be considered as a potential targeting drug delivery to liver. The composition of the Eudragit RL 100 affected significantly the blood residence time and in-vivo biodistribution of the NPs in liver and spleen.

Declarations

Acknowledgements The authors gratefully acknowledge financial support and granting research fellowship (45/38/2011/Nan-BMS) from ICMR (Indian Council of Medical Research, Govt of India, New Delhi). Authors are also grateful to Sami Labs, Bangalore, India for providing the gift sample of trans-resveratrol.

Conflict of interest The Author(s) declare(s) that they have no conflicts of interest to disclose.

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15

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