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Pharmacokinetic and Pharmacodynamic studies of PAMAM dendrimer based Simvastatin oral formulations for treatment of hypercholesterolemia Hitesh Kulhari, Deep Pooja Kulhari, Sunil Kumar Prajapati, and Abhay Singh Chauhan Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/mp300650y • Publication Date (Web): 21 May 2013 Downloaded from http://pubs.acs.org on May 24, 2013
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Molecular Pharmaceutics
Pharmacokinetic and Pharmacodynamic studies of PAMAM dendrimer based Simvastatin oral formulations for treatment of hypercholesterolemia ¥
Hitesh Kulhari , Deep Pooja Kulhari
¥ ,*
¥
, Sunil Kumar Prajapati , Abhay Singh Chauhan
¥
Institute of Pharmacy, Bundelkhand University, Jhansi, UP, India. School of Pharmacy, Concordia University, Mequon, Wisconsin, USA.
‡
*
Corresponding Author Deep Pooja Kulhari Institute of Pharmacy, Bundelkhand University, Jhansi (U.P.)-284128 Tel.: +91 510 2321035 E-mail address:
[email protected]
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ABSTRACT The aim of this investigation was to evaluate the in vivo potential of poly (amido) amine dendrimers (PAMAM) based simvastatin (SMV) formulations as nanoscale drug delivery units for controlled release action of simvastatin. Drug-dendrimer complexes were prepared and characterized by the Fourier transform infrared (FTIR) spectroscopy. In pharmacodynamic study, the percent increase in cholesterol was less with PAMAM dendrimer formulations as compared to pure drug. The cholesterol level was increased to 20.92% with pure SMV whereas 11.66% with amine dendrimer, 11.49% with PEGylated dendrimer and 10.86% with hydroxyl dendrimer formulations. Reduction in the increase in triglyceride and low density lipoprotein level was also more prominent with the drug-dendrimer formulations. The order of increase in high density lipoprotein level was PEGylated PAMAM-SMV (4.04%) > PAMAM-AmineSMV (2.57%) > PAMAM-Hydroxyl-SMV (1.48%) > pure SMV (1.09%). Dendrimer-SMV formulations showed better pharmacokinetic performances than pure SMV suspension. The peak plasma SMV concentration increased from 2.3 µg/ml with pure SMV to 3.8 µg/ml with dendrimer formulations. The dendrimer mediated formulation had 3-5 times more mean SMV residence time than pure SMV. Furthermore, SMV absorption and elimination rates were decreased significantly, showing controlled release of SMV from the dendrimer formulations.
Key Words: Poly (amidoamine) dendrimers, Simvastatin, Pharmacokinetic, Pharmacodynamic, Cholesterol
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GRAPHICAL ABSTRACT Dendrimers enhance the solubility and dissolution of SMV, enabling more uniform molecular dispersion in the gastrointestinal tract (GIT) fluid, which helps to carry drug to the apical surface of GIT membrane. Simultaneously, the effective surface area of SMV is also increased, which is further responsible for the enhancement in the bioavailability.
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INTRODUCTION
According to the World Health Report 2011, cardiovascular diseases (CVDs) are the most prevalent cause of death and disability in the world, claiming 17.1 million lives a year. Globally, south Asians have the highest rates of coronary artery disease (CAD).1 Hypercholesterolemia means the presence of high levels of cholesterol in blood. It is not a disease but a metabolic derangement which contributes to many forms of diseases, especially cardiovascular diseases. Statins (also known as 3-hydroxy-3-methylglutaryl coenzyme A [HMG-CoA] reductase inhibitors) are generally recognized as the treatment of choice in patients with hypercholesterolemia.2 They are easy to use, effective, well tolerated and have lesser interaction with other drugs; thus cause lesser adverse effects and drug interactions.3-7 Statins are the specific inhibitors of HMG CoA reductase, which catalyzes the reduction of HMG CoA to mevalonate. Thus, statins arrest a key step for cholesterol biosynthesis in liver, which leads to up-regulation of low density lipoprotein (LDL) receptors and an increase in catabolism of LDL cholesterol. Statins help to reduce coronary events through their antioxidant, antiinflammatory and antiproliferative effects on smooth muscle cells.8 For the patients who do not achieve their LDL goals and require more than 35% reduction of cholesterol, atorvastatin and simvastatin may be the best choices in initial therapy.9 But atorvastatin has a negative dose response for HDL.10-11 Moreover, atorvastatin is likely to have more adverse effects than people who take the highest available dose of simvastatin. In all statins, simvastatin is costeffective and the best choice for patients with stable CAD, diabetes and acute coronary syndrome.12-16 Thus, if it is not necessary to reduce cholesterol massively then SMV is the best option.
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Nonetheless, poor aqueous solubility and hence poor oral bioavailability (< 5%) of SMV is a limitation in formulation development. Thus, there is a need of a delivery system which not only delivers the drug efficiently but also improves the pharmacokinetic properties of the drug. Dendrimers are getting much attention in engineering, material science, biological and chemical sciences. The unique molecular architecture and presence of free surface groups allow the researchers to construct excellent nanoscale devices for efficient delivery of drugs and biologicals. Active ingredients from different categories like NSAIDs,17-24 anticancer,25-29 antimicrobials30 etc, with different properties have been successfully delivered using dendrimers. Poly (amido) amine (PAMAM) dendrimers have a well-defined, mono-dispersive and stable molecular architecture that is advantageous for targeted drug delivery. In our previous study,31 we evaluated the in vitro performance of PAMAM dendrimers with three different functionalities (NH2, OH and PEG) for the delivery of SMV. It was observed that PEGylation of the dendrimer leads to solubility enhancement, better dissolution, slow release of drug, biocompatibility and stability of the drug compared to the non-PEGylated dendrimers. The purpose of this study is to evaluate the in vivo potential of dendrimer-SMV formulations as nanoscale drug delivery units for controlled release action of SMV.
EXPERIMENTAL SECTION
Materials. G4-PAMAM-NH2, G4-PAMAM-OH and G4-PAMAM-PEG were purchased from Dendritic Nanotechnologies, USA. Simvastatin was obtained as gift sample from M/s Ranbaxy laboratory, (Gurgaon, India). Cellulose dialysis tubing (Mw~1000) and membrane filter of pore size 0.2 µm were purchased from Himedia Lab, (Mumbai, India). Diagnostic kit was purchased from Span Diagnostics Ltd., (Surat, India). Rest all chemicals were of analytical grade and were purchased from Central Drug House, (Gwaliar, India).
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Analytical method. The amount of SMV in distilled water and plasma were determined by HPLC method reported by Tan et al.32 HPLC (Shimadzu 10 Ai Japan) analysis was carried out in a binary mode with a photodiode array detector, SIL-10AD VP auto injector and a communication bus module. The analysis was performed at 25 °C on YMC, RP-C18, 250 × 4.6 mm, 5 µm HPLC column using a mobile phase of acetonitrile (60%) and water (40%) pumped at a flow rate of 1.0 ml/min, monitored at a wavelength of 239 nm. The calibration graph was rectilinear in the concentration range of 1µg/ml to 10 µg/ml with a correlation coefficient of 0.999. The inter-intraday accuracy and precision was within a relative standard deviation (RSD) of ≤ 5%. The extraction efficacy in case of spiked plasma samples was 98.1 ± 1.4%.
Preparation of drug-dendrimer complex and characterization. Simvastatin-dendrimer complexes were prepared by the method as described in our previous research paper.31 Briefly, an excess (10 mg) of SMV was added to the aqueous dendrimer solution (0.1 %w/v in deionized water). The suspension was sonicated for 2 minutes and incubated overnight at room temperature in shaking water bath. The vials were allowed to stand for 24 h at room temperature for equilibration of drug-dendrimer complex. The clear supernatant was filtered through 0.2 µm membrane filter to remove unentrapped drug. The filtrate was cooled using liquid nitrogen to form a solid thin layer and lyophilized (Modulyod freeze dryer-230) at -45 °C and 1.5 mbar. The obtained solid was characterized by HPLC and FTIR spectroscopy (FTIR- 8400 s Shimadzu, Japan). Drug-dendrimer formulations, containing 0.1% w/v of amine (DN2), hydroxyl (DO2) and PEGylated (DP2) PAMAM dendrimers, were prepared for in-vivo studies.
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Animal model. Male albino rats of Sprague-Dawley strain with weight between 150-170 g were selected for in-vivo blood level and pharmacodynamic studies. The animals were kept in well-placed ventilated cages and maintained on a standard diet with free access to water. All the studies performed on the animals were in accordance with the guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) and study protocol was approved by the Institutional Animal Ethics Committee (IAEC) of the Institute of Pharmacy, Bundelkhand University, Jhansi, India (715/02/a/CPCSEA).
Pharmacokinetic studies. The animals were divided into four groups with each group comprising of six rats and were marked appropriately. Pure drug suspension was prepared by dispersing SMV in 0.5 %w/v Tween 80 solution. The dendrimer formulations were dissolved in one ml of normal physiological saline. All four formulations were administered by per oral route as gavage at a dose of 5 mg/kg body weight. The first group served as control and was given suspension of pure drug (SMV) in water. The remaining three groups were given the test formulations of drug–dendrimer complexes (DN2, DO2 and DP2, respectively). Following per oral administration of the formulations, blood samples were collected from the tail vein at predetermined time interval up to 24 h. The blood samples, after collection in anti-coagulated (EDTA-treated) glass vials, were centrifuged at 3000 rpm for 15 min to separate RBCs and plasma. The supernatant (plasma) was collected and SMV in plasma was extracted with cyclohexane-dichloromethane (3.5:1, v/v) and measured by HPLC.
Pharmacodynamic studies. The hyperlipidemia was induced by the method reported by Patil
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et al., giving 1.5 ml of coconut oil orally.33 The animals were housed into groups of six and maintained on a standard diet with free access to water. Animals were fasted overnight before starting the experiment, anesthetized, and bled by tail vein puncture to obtain baseline values of total cholesterol (CH), LDL, HDL and triglycerides (TG) in blood so that each animal served as its own control. The animals were divided into five groups with each group comprising of six rats and were marked appropriately. Formulations were administered by per oral route at a dose of 5 mg/kg body weight. The first group served as negative control. Group second was given suspension of pure drug (SMV) in water as reference formulation. The remaining three groups were administered the test formulations of drug–dendrimer complexes (DN2, DO2 and DP2, respectively). Blood samples were collected from the tail-vein puncture at predetermined time intervals, viz., before treatment (0 and 5 days), and after treatment (6, 7 and 8 days) in anti-coagulated (EDTA-treated) glass vials. Plasma was separated by centrifugation at 3000 rpm for 25 min and stored frozen until further use. Plasma samples were analyzed for total CH, HDL-CH and TG using in-vitro diagnostic kits.
Statistical Analysis. Data were analyzed by ANOVA-Dunnet’s test. The difference was considered significant when P < 0.05. Values were shown as mean of six observations ± the standard deviation (SD).
RESULTS AND DISCUSSION
Preparation of drug-dendrimer complexes and characterization. SMV is a biopharmaceutical classification system (BCS) class II drug with poor aqueous solubility and high permeability. Hence its formulation design is very much dependent upon its physicochemical and
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biopharmaceutical properties. The oral bioavailability of SMV is dissolution rate-limited which is affected by its solubility in aqueous gastric fluid. The pre-requisite for absorption of any drug is that it should be present in solution form at absorption site. Therefore, increase in water solubility of SMV may lead to sufficient and considerable increase in its oral bioavailability. Dendrimers, especially PAMAM dendrimers, have been reported to enhance the solubility of poorly water soluble drugs.18, 19, 22-25 The observed SMV concentration in different dendrimer-SMV formulations, containing 0.1%w/v dendrimer concentration, was 353.11, 306.04, 430.51 µM/L for DN2, DO2 and DP2 formulations, respectively. The enhanced SMV concentration with dendrimer formulations can be explained by the host-guest complex formation between SMV and dendrimer. We have proved the propensity of dendrimers to entrap hydrophobic drugs for enhanced aqueous solubilization.18 The solubilization property of dendrimer architecture makes them attractive drug delivery carrier for hydrophobic drugs. Guest molecules can be entrapped inside the dendrimer architecture along with the peripheral interactions with the polyvalent surface groups. The hydrophobic interior provides a lipophilic environment where a guest molecule can be housed by simple encapsulation or host-guest complex formation or weak hydrogen bonding. Non-covalent electrostatic interactions come in play for the surface group attachments with the guest molecules. Various drugs and vitamins have been solubilized and delivered by host-guest complex formation with dendrimers.34 Molar stoichiometry is an important tool to describe the quantitative relationship between these kinds of interactions. The observed molar stoichiometric ratio (SMV: dendrimers) was 11:1, 2:1 and 1.4:1 for DP2, DN2, and DO2, respectively. Thus, PEGylated dendrimers showed higher SMV encapsulation than amine and hydroxyl dendrimers. It may be due to the availability of large void spaces and
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entanglement potential for molecular encapsulation; and better drug-dendrimer interaction due to more spherical and symmetric shape with PEG chain on dendrimer surface.
Pharmacokinetic studies. Pharmacokinetic studies were carried to determine the release and performance of the formulations in-vivo (Fig. 1). Various pharmacokinetic parameters (Cmax, Tmax, Ka, and Ke) were calculated and reported (Table 1). The maximum plasma concentration (Cmax) observed was 2.3 µg/ml, 3.1 µg/ml, 3.3 µg/ml and 3.8 µg/ml for pure SMV suspension, DN2, DO2 and DP2 formulations, respectively. The increase in peak plasma concentration with dendrimer formulations may attribute to increase in oral bioavailability of the drug. The time for peak plasma concentration (tmax) was also increased for drug-dendrimer formulations, 2.95.2 h, compared to SMV (1.5 h). The faster tmax for SMV suspension is due to faster absorption of SMV from SMV suspension (Ka=1.38) compared to dendritic formulations (Ka=0.334 to 0.465) (Table 1). Area under the plasma concentration-time profile (AUC0→t) with DN2, DO2 and DP2 formulations was found to be 19.03 µg/ml/h, 11.14 and 25.43 µg/ml/h, respectively, which was significantly higher than the pure drug (3.4 µg/ml/h). Thus the amount of drug absorbed with PAMAM dendrimer formulation was 5.6x (DN2), 3.2x (DO2) and 7.4x (DP2) higher compared to pure SMV. This is due to increase in drug dissolution at the absorption site in the presence of dendrimers (Fig.2). The lower tmax and AUC for DO2 formulation compared to other two dendrimer formulations were due to stoichiometrically less drug encapsulation in DO2. Secondly, in DO2, the drug was loosely bound to the dendrimers so it was released faster compared to DN2 and DP2.31 All three dendrimer formulations showed increase in mean residence time (MRT) of the drug. MRT for SMV suspension was 1.5 h whereas for DN2, DO2 and DP2 were 4.3, 7.2 and 6.6 h, respectively. Higher AUC and MRT values of dendrimer
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formulations indicate that the solubilization of SMV by PAMAM dendrimers enhance its bioavailability and allows prolonged release time. The absorption rate constant Ka was less with different dendrimer formulations compared to pure SMV suspension (Table 1). The SMV absorption rate was decreased 2.96, 4.13 and 3.81 times with DN2, DO2 and DP2 formulations; respectively. This shows the slow absorption of the drug in dendrimer formulations. These results were in quite agreement with slow and controlled release of SMV from these formulations during in vitro studies.31 The elimination rate constant Ke for pure SMV suspension was 2.33 whereas; Ke for DN2, DO2 and DP2 formulations was 1.84, 0.114 and 0.576, respectively. Data suggested that there was significant decrease in elimination rate of the drug with dendrimer formulations. Hydroxy and PEG terminated dendrimer formulations (DO2 and DP2) showed slower drug elimination (p DP2 (13.60%) > DO2 (12.92%) (Fig. 3b). A significant decrease in CH and LDL levels was found with all three dendrimer formulations. This could be attributed to slow release of the drug from the dendrimer formulations (decrease in absorption rate) which maintain a steady-state SMV concentration in the blood for a longer duration (more MRT) as compared to its conventional suspension formulation. Most of the health professionals and guidelines emphasize on LDL cholesterol level reduction in the treatment of CVDs. But, CVDs are multifactorial and also contributed by high TG and low HDL levels especially in patients with type 2 diabetes mellitus. The order of increase in triglyceride level was control (18.74%) > pure SMV (17.12%) > DO2 (14.08%) > DP2 (13.34%) > DN2 (13.16%) (Fig. 3c). Thus, after three days treatment, no significant (p>0.05) reduction in TG level was found with all three dendrimer formulations as compared to the control. High HDL level is reported to inhibit atherogenesis through reverse cholesterol transport, antioxidant and anti-inflammatory activities. Generally, the effect of statins on raising HDL level is between 5 to 15%.35 Here, the maximum increase in HDL level was found with DP2 (4.04%) followed by DN2 (2.57%), DO2 (1.48%) and pure SMV (1.09%) (Fig. 3d). A little increase in HDL level can be attributed to two factors-shorter duration of treatment and simultaneous administration of coconut oil. However, % increase with DP2 was nearly 4 times compared to pure SMV suspension which indicated the increased efficacy of the formulation and justified the primary objective of the study. Finally, improved pharmacokinetic and pharmacodynamic profiles with dendrimer formulation may help in decrease in total drug requirement, dosing frequency and hence potential toxicities of SMV.
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CONCLUSION Unique architecture of dendrimer nanoconstruct provides an effective platform for multitasking such as entrapment of bioactive molecule, preformulation and formulation efficacy, stability and smart maneuvering potential for in vivo applications. Results suggest that the drugdendrimer formulations have better in-vivo performance than the pure SMV. Significant difference was observed in peak plasma concentration, tmax, and extent of drug absorbed. This could be attributed to high drug loading and enhanced aqueous solubility of the drug translating to higher dissolution at absorption site in the presence of dendrimers. Increases in tmax with dendrimer formulations indicate the slow and controlled release ofthe drug from drugdendrimer complexes. Further, slow absorption (decrease in absorption rate constant) and decreased elimination rate maintain the better plasma drug level in the blood as compared to suspension formulation that leads to enhanced pharmacodynamic performance as well as therapeutic efficacy of the drug.
ACKNOWLEDGEMENTS We are highly grateful to M/s Ranbaxy laboratory, (Gurgaon, India) for providing SMV. Hitesh Kulhari is thankful to the Head, Institute of Pharmacy, Bundelkhand University, Jhansi, India for providing the facilities and moral support to complete this work. The authors also wish to thank Dr. Sistla Ramakrishna, Principal Scientist, Indian Institute of Chemical Technology, Hyderabad, for helpful discussion on assessment of pharmacokinetic parameters.
DECLARATION OF INTEREST Authors report no competing financial interest.
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of ketoprofen by in-vitro and in-vivo studies. Eur J Med Chem2006, 41, 670-4. 24) Svenson, S.; Chauhan, A. S. Dendrimers for enhanced drug solubilization. Nanomedicine 2008, 3(5), 679-702. 25) Bhadra, D.; Bhadra, S.; Jain, S.; Jain, N.K. A PEGylated dendritic nanoparticulate carrier of fluorouracil. Int J Pharm2003, 257, 111-24. 26) Kukowska-Latallo, J. F.; Candido, K. A.; Cao, Z. Y.; Nigavekar, S. S.; Majoros, I. J.; Thomas, T. P.; Balogh, L. P.; Khan, M. K.; Baker, J. R. Nanoparticle targeting of anticancer drug improves therapeutic response in animal model of human epithelial cancer. Cancer Res 2005, 65, 5317–24. 27) Malik, N.; Evagorou, E. G.; Duncan, R. Dendrimer-platinate: a novel approach to cancer chemotherapy. Anticancer Drugs 1999, 10, 767-76. 28) Padilla De Jesu’s, O. L.; Ihre, H. R.; Gagne, L.; Fre’chet, J. M. J.; Szoka Jr., F. C. Polyester dendritic systems for drug delivery applications: in vitro and in vivo evaluation. BioconjugChem2002, 13, 453–61. 29) Tripathi, P. K.; Khopade, A. J.; Nagaich, S.; Jain, S.; Jain, N. K. Dendrimer grafts for delivery of 5-fluorouracil. Pharmazie2002, 57, 261-4. 30) Bhadra, D.; Yadav, A. K.; Bhadra, B.; Jain, N. K. Glycodendrimericnanoparticulate carriers of primaquine phosphate for liver targeting. Int J Pharm2005, 295, 221-33. 31) Kulhari, H.; Deep Pooja; Prajapati, S. K.; Chauhan, A. S. Performance evaluation of PAMAM dendrimer based Simvastatin formulations. Int J Pharm 2011, 405(1-2), 203-9. 32) Tan, L.; Yang, L. L.; Zhang, X.; Yuan, Y. S.; Ling, S. S.Determination of simvastatin in human plasma by high performance liquid chromatography. Se Pu2000, 18(3), 232-4. 33) Patil, P.; Patil, V.; Paradkar, A. Formulation of a self-emulsifying system for oral delivery
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of simvastatin: in-vitro and in-vivo evaluation. Acta Pharm2007, 57, 111-22. 34) Wolowiec, S.; Laskowski, M.; Laskowska, B.; Magon, A.; Mysliwiec, B.; Pyda, M. Dermatological Application of PAMAM - Vitamin Bioconjugates and Host-Guest Complexes. Vitamin C Case Study. In Stoichiometry and Research - The Importance of Quantity in Biomedicin, A., Innocenti. INTECH, 2012; pp 195-220. 35) Baigent, C.; Keech, A.; Kearney, P. M. et al. Efficacy and safety of cholesterol-lowering treatment: prospective meta-analysis of data from 90,056 participants in 14 randomised trials of statins. Lancet 2005, 366, 1267–78.
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Legends for Tables
Table 1. Pharmacokinetic parameters of SMV and different SMV-PAMAM dendrimer formulations (Mean ± SD, n=6) Cmax, Maximum plasma concentration; Tmax, Time for peak plasma concentration; Ka, Absorption rate constant; Ke, Elimination rate constant
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Legends for Figures
Fig. 1. Plasma concentration of SMV released from suspension and dendrimerSMVformulations (Mean ± SD, n=6)
Fig. 2. Schematic diagram showing how dendrimers may facilitate the absorption of thewater insoluble drug SMV Dendrimers enhance the solubility and dissolution of SMV, enabling more uniform molecular dispersion in the gastrointestinal tract (GIT) fluid, which helps to carry drug to the apical surface of GIT membrane. Simultaneously, the effective surface area of SMV is also increased, which is further responsible for the enhancement in the bioavailability.
Fig. 3. Plasma-lipid levels: (a) Cholesterol, (b) LDL, (c) TG and (d) HDL;during the treatment with SMV suspension and SMV-PAMAM dendrimer formulations (Mean ± SD, n=6)
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TABLES
Table 1. Pharmacokinetic parameters of SMV and different SMV-PAMAM dendrimer formulations (Mean ± SD, n=6) Cmax
Tmax.
AUC
MRT
(µg/ml)
(h)
(µg/ml/h)
(h)
SMV
2.3±0.08
1.5±0.12
3.40±0.74
DN2
3.1±0.11
5.2±0.15
DO2
3.3±0.2
DP2
3.8±0.14
Formulation
Ka
Ke
1.6±0.24
1.380
2.331
19.03±1.28
5.9±0.37
0.465
1.840
2.9±0.06
11.14±1.51
7.2±0.11
0.334
0.114
4.3±0.22
25.43±2.16
6.6±0.18
0.362
0.576
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FIGURES
Fig. 1. Plasma concentration of SMV released from suspension and drug-dendrimer formulations (Mean ± SD, n=6)
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Fig. 2. Schematic diagram showing how dendrimers may facilitate the absorption of the water insoluble drug SMV: Dendrimers enhance the solubility and dissolution of SMV, enabling more uniform molecular dispersion in the gastrointestinal tract (GIT) fluid, which helps to carry drug to the apical surface of GIT membrane. Simultaneously, the effective surface area of SMV is also increased, which is further responsible for the enhancement in the bioavailability.
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a
b
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c
d
Fig. 3. Increase in plasma-lipid levels:(a) Cholesterol, (b) LDL, (c) Triglyceride and (d) HDL; during the treatment with SMV suspension and SMV-PAMAM dendrimer formulations (Mean
± SD, n=6)
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