http://informahealthcare.com/drd ISSN: 1071-7544 (print), 1521-0464 (electronic) Drug Deliv, Early Online: 1–8 ! 2013 Informa Healthcare USA, Inc. DOI: 10.3109/10717544.2013.853213
ORIGINAL ARTICLE
Enhanced oral bioavailability of acyclovir by inclusion complex using hydroxypropyl-b-cyclodextrin Anroop B. Nair1, Mahesh Attimarad1, Bandar E. Al-Dhubiab1, Jyoti Wadhwa2, Sree Harsha1, and Mueen Ahmed1 1
Department of Pharmaceutical Sciences, College of Clinical Pharmacy, King Faisal University, Al-Ahsa, Kingdom of Saudi Arabia and M.M. College of Pharmacy, MM University, Mullana, Ambala, India
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Abstract
Keywords
The therapeutic potential of acyclovir is limited by the low oral bioavailability owing to its limited aqueous solubility and low permeability. The present study was a systematic investigation on the development and evaluation of inclusion complex using hydroxypropylb-cyclodextrin for the enhancement of oral bioavailability of acyclovir. The inclusion complex of acyclovir was prepared by kneading method using drug: hydroxypropyl-b-cyclodextrin (1:1 mole). The prepared inclusion complex was characterized by Fourier transform infrared spectroscopy, differential scanning calorimetry, NMR spectroscopy and evaluated in vitro by dissolution studies. In vivo bioavailability of acyclovir was compared for inclusion complex and physical mixture in rat model. Phase solubility studies indicate the formation of acyclovir– hydroxypropyl-b-cyclodextrin complex with higher stability constant and linear enhancement in drug solubility with increase in hydroxypropyl-b-cyclodextrin concentration. Characterization of the prepared formulation confirms the formation of acyclovir–hydroxypropyl-b-cyclodextrin inclusion complex. Dissolution profile of inclusion complex demonstrated rapid and complete release of acyclovir in 30 min with greater dissolution efficiency (90.05 2.94%). In vivo pharmacokinetic data signify increased rate and extent of acyclovir absorption (relative bioavailability 160%; p50.0001) from inclusion complex, compared to physical mixture. Given the promising results in the in vivo studies, it can be concluded that the inclusion complex of acyclovir could be an effective and promising approach for successful oral therapy of acyclovir in the treatment of herpes viruses.
Acyclovir, bioavailability, dissolution, hydroxypropyl-b-cyclodextrin, pharmacokinetics
Introduction Acyclovir is a well-known antiviral agent used for the treatment of herpes viruses such as herpes simplex virus type I/II and varicella zoster (Spruance & Kriesel, 2002; Lin et al., 2003). Existing treatment modalities using acyclovir for herpes infections include oral, parenteral and topical therapy (Cortesi & Esposito, 2008). However, topical therapy is considered to be less effective due to the low skin permeability of acyclovir into the target site (Spruance et al., 2002). On the other hand, oral drug delivery is the most appropriate and patient compliant method as it offers several advantages and was preferred over other routes. Following oral therapy, the absorption of acyclovir was found to be slow, variable and incomplete with low oral bioavailability (15–30%) (Laskin, 1983; Fletcher & Bean, 1985; Arnal et al., 2008). Therefore the oral therapy of this drug required frequent administration of high dose of acyclovir, which leads to potential systemic adverse effects such as acute renal failure and neurotoxicity (Johnson et al., 1994). There has Address for correspondence: Anroop B. Nair, Assistant Professor, Department of Pharmaceutical Sciences, College of Clinical Pharmacy, King Faisal University, P.O. 400, Al-Ahsa-31982, Kingdom of Saudi Arabia. Tel: +966 536 219 868. Email: [email protected]
History Received 30 August 2013 Revised 5 October 2013 Accepted 5 October 2013
been intensive effort to improve the bioavailability of acyclovir following oral delivery. Several approaches have been attempted to develop effective drug delivery system for the successful delivery of acyclovir (Cortesi & Esposito, 2008). In one approach, vesicular carrier systems such as liposomes, niosomes, microparticles and nanoparticles were developed (Jain et al., 2005; Attia et al., 2007; Mukherjee et al., 2007; Caldero´n et al., 2013). On the other hand, drug discovery groups have synthesized prodrugs of acyclovir to improve the therapeutic efficacy (De Clercq & Field, 2006). Alternatively, microemulsions and self-emulsifying drug delivery systems were also attempted to augment the oral bioavailability of acyclovir (Cortesi & Esposito, 2008; Paul et al., 2013). Despite all these efforts, the successful oral therapy of acyclovir remains elusive. The poor efficiency of acyclovir in oral therapy is mainly due to its limited aqueous solubility and low permeability (Fletcher & Bean, 1985; Wagstaff et al., 1994; Friedrichsen et al., 2002; Bergstrom et al., 2003).The biopharmaceutics classification system (BCS) has categorized acyclovir under class 3 considering the highest possible strength up to 400 mg. However, this drug is also available at a high dose of 800 mg and falls under BCS class 4, suggesting that a distinct classification for this drug is not possible with the BCS
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system (Arnal et al, 2008). Aqueous solubility is a key property governs the dissolution and absorption. It is well known that the therapeutic efficiency of water-insoluble drugs could be increased by enhancing dissolution, which in turn improves the bioavailability. Several methods are reported in the literature to enhance the dissolution rate of low soluble drugs (Fasinu et al., 2013). Formation of inclusion complex is one of the promising approaches used to improve the apparent solubility, dissolution rates and consequently the bioavailability of poorly soluble drugs (Challa et al., 2005). Cyclodextrins are promising complexing agents consisting of cyclic oligosaccharides containing hydrophilic outer surface and hydrophobic inner cavity (Loftsson & Ducheˆne, 2007). This supramolecular host form stable inclusion complex with guest molecules without altering the molecular structure (Carrier et al., 2007; Mummidi & Jayanthi, 2013). Indeed, the inclusion of drug molecules into the cyclodextrin cavity could lead to modification of physical, chemical and biochemical properties of guest drug molecule (Taneri et al., 2010). The application of cyclodextrin in enhancing aqueous solubility of acyclovir by complexation has already been reported in the literature (Rossel et al., 2000; Tomar et al., 2010). Attempt has also been made to enhance the bioavailability of acyclovir by preparing inclusion complexes using b-cyclodextrin (Luengo et al., 2002). Selection of an appropriate complexing agent and the method of formulation are critical. Among the cyclodextrin derivatives, hydroxypropyl-b-cyclodextrin (HP-b-cyclodextrin) is most widely investigated for drug complexation and has captivated the drug delivery scientists probably owing to its high aqueous solubility, better complexation ability, greater stability, non-toxicity and its potential to enhance oral bioavailability of low aqueous soluble drugs (Irie & Uekama, 1997; Nasongkla et al., 2003). Recently, one attempt has been reported to enhance the aqueous solubility of acyclovir by preparing inclusion complex using HP-b-cyclodextrin (Koz´biał & Gierycz, 2013). The objective of this study was to carry out a systematic investigation on the effect of inclusion complex of acyclovir using HPb-cyclodextrin in enhancing the oral bioavailability. The inclusion complex of acyclovir-HP-b-cyclodextrin (1:1 molar ratio) was formulated by kneading method. Further the inclusion complex was characterized and evaluated in vivo for pharmacokinetics in Wistar rats.
Materials and methods Acyclovir was gifted by Ind-Swift Laboratories (Chandigarh, India). HP-b-cyclodextrin was purchased from Hi Media, Mumbai, India. All other chemicals and reagents used were of analytical grade. Analytical method The amount of acyclovir in samples was quantified by high performance liquid chromatography (HPLC) system (Cyberlab, LC P100) consisting of a Symmetry C18 analytical column (4.6 mm 150 mm, 5.0 mm) with a detector LC UV-100. Mobile phase consisted of 0.1% acetic acid and acetonitrile (98:2). Elution was performed isocratically at 25 C at a flow rate of 1.2 ml/min. Injection volume was 25 ml
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and the column effluent was monitored at 254 nm (Volpato et al., 1997). The method was validated by the determination of linearity, precision and accuracy. Phase solubility studies The phase solubility of acyclovir was conducted according to Higuchi & Connors (1965). An excess amount of acyclovir (50 mg) was added to 5 ml of water or aqueous solutions of HP-b-cyclodextrin (up to 0.01 mol) in a glass vial. The system was stirred for 24 h at 37 C and kept at rest for 24 h to attain equilibrium. The solution was filtered using 0.45 mm Millex syringe driven filter unit (Millipore Corporation, Bedford, MA) and the solubility was determined by HPLC. The amount of drug dissolved against moles of carrier was plotted and the stability constant was calculated by the equation. Kc ¼
Slope Soð1 SlopeÞ
where So is the solubility of drug in the absence of carrier. Preparation of inclusion complexes The inclusion complex of acyclovir and HP-b-cyclodextrin was prepared by kneading method. Briefly, acyclovir and HPb-cyclodextrin were sieved through sieve No. 100 (149 mm). Weighed accurately the specified quantity of drug and carrier in a molar ratio of 1:1 in a glass mortar and water was added drop by drop with triturating until it become slurry. The mixture was further triturated and grinded for 3 h (optimized from a period of 30 min to 4 h by evaluating crystallinity and inclusion efficiency) and freeze dried for 12 h. Different from the normal method of drying, the inclusion complex was freeze dried to remove the water. Indeed the freeze drying of inclusion complex will avoid the long exposure of formulation to the atmosphere (happens with regular drying), provide storage at ambient temperatures, reduce contamination and is pretty simple and rapid. Moreover, there are several reports where in the inclusion complexes prepared using HP-b-cyclodextrin showed greater stability (Phillip Lee et al., 2009; Wang et al., 2009). The mass obtained after freeze drying was pulverized to obtain powder-like complex and stored in a desiccator over fused calcium chloride. The physical mixture of acyclovir and HPb-cyclodextrin was prepared using same molar ratio (1:1) by triturating in a glass mortar for 10 min and pulverized to obtain uniform mixing. Fourier transform infrared spectroscopy Fourier transform infrared (FTIR) spectroscopy was used to characterize the inclusion complex and physical mixture using Shimadzu model 8400S Spectroscope. The samples were prepared by grinding small amount of the dried sample and mixed with potassium bromide at 1:5 (sample: KBr) ratio and the discs were prepared using a hydraulic press. The discs were scanned in the range of 500– 4000 cm1 in order to assess structural changes that could have occurred in the drug or HP-b-cyclodextrin as a result of inclusion complex.
Enhanced oral bioavailability of acyclovir
DOI: 10.3109/10717544.2013.853213
NMR spectroscopy
In vivo studies
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In vivo experiments were carried out in male, 6–8 weeks old Wistar rats (100–150 g) which were maintained on a 12/12 h light/dark cycle in an animal facility with unlimited access to food and water (Institutional Animal Ethical Committee, M. M. University, Ambala, India). Two groups of rats (six animals in each group) fasted for 12 h with free access to water were used in the study. Inclusion complex or physical mixture was administered by peroral route (1 ml, dose of 20 mg/kg of acyclovir) by gavage to the first and second group of rats, respectively. The oral bioavailability of inclusion complex was determined by estimating the plasma acyclovir concentrations. The blood samples were collected (200 ml) from retro orbital plexus using dry heparinized tubes at predetermined time intervals (15, 30, 45, 60, 120, 180, 240, 360 min), under anesthesia (thiopentone sodium 30 mg/kg). Plasma (200 ml) was subjected to protein precipitation with equal volume of acetonitrile and was centrifuged at 10 000 rpm for 10 min. Supernatant was filtered and injected into high-performance liquid chromatography (HPLC). The sample withdrawn at time zero served as the baseline value. The pharmacokinetic parameters were calculated using non-compartmental pharmacokinetic model. Pharmacokinetic variables of interest included area under the concentration– time curve from time 0 to 1 (AUC0– ), peak concentration (Cmax), terminal elimination rate constant (Kel), half-life of the terminal phase (t1/2) and time to peak concentration (Tmax). A linear-up/log-down method of estimation was used for the calculation of AUC1 and was obtained by adding Clast/Kel to AUC0–t. The terminal elimination rate constant (Kel) was determined from the slope of terminal exponential phase of the logarithmic plasma concentration–time curve. The elimination half-life (t1/2) was calculated using 0.693/Kel.
H NMR experiments were carried out at 400 MHz on a Bruker spectrometer at 298 K in deutered water. The NMR spectra were recorded for pure acyclovir, HP-b-cyclodextrin, physical mixture and inclusion complex and were processed with the aid of Topspin 3. Differential scanning calorimetry
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Differential scanning calorimetry (DSC) was performed on inclusion complex, physical mixture, HP-b-cyclodextrin and pure drug. DSC measurements were done with a DSC-60 instrument (Shimadzu, Japan). Samples equivalent to 2 mg of acyclovir were sealed in an aluminium pan and heated at a temperature range of 0–350 C at the rate of 10 C/min using indium as reference. The change in endothermic peaks of acyclovir and HP-b-cyclodextrin was observed, which confirms the interaction between drug and excipients. Evaluation of drug content Weighed amount of inclusion complex or physical mixture (equivalent to 2 mg of acyclovir) were dispersed in water (50 ml) for a predetermined time (24 h) in screw-capped vials and stirred on a magnetic stirrer. The resulting dispersion was then centrifuged at 10 000 rpm (5 min) and the supernatant was collected and filtered using 0.2 mm Millex syringe driven filter unit (Millipore Corporation, Bedford, MA) and the sample was analyzed using HPLC. Dissolution studies In vitro drug release from pure acyclovir, physical mixture and inclusion complex were determined in simulated gastric fluid (pH 1.2, no enzyme) for 1 h. Weighed amount of inclusion complex, physical mixture or pure drug (equivalent to 100 mg of acyclovir) were filled into empty hard gelatin capsule shell and subjected to dissolution study. The samples were placed in a dissolution test apparatus USPXXIV Type II (Electro Lab, Mumbai, India) containing dissolution medium (900 ml) maintained at 37 0.5 C with paddle rotation maintained at 50 rpm (n ¼ 6). Aliquots of dissolution medium (1 ml) were withdrawn at appropriate time intervals and replaced with fresh media to compensate the loss of sample withdrawn. Further, samples were filtered using 0.2 mm Millex syringe driven filter unit (Millipore Corporation, Bedford, MA) and analyzed by HPLC. Dissolution efficiency studies The dissolution efficiency of the pure acyclovir, physical mixture and inclusion complex were calculated by the method mentioned by Khan (1975). It is defined as the area under the dissolution curve between time points t1 and t2 expressed as a percentage of the curve at maximum dissolution, y100, over the same time period or the area under the dissolution curve up to a certain time, t, expressed as a percentage of the area of the rectangle described by 100% dissolution in the same time. Rt 0 ydt Dissolution efficiency ¼ 100% y100ðt2 t1 Þ
Data analysis The data were expressed as mean of six trials SD. Statistical analysis was performed by unpaired t-test using Graphpad prism 5, graphpad software, Inc., CA, to test the drug concentration in various treatments. p50.05 was considered as the level of significance.
Results and discussion The phase-solubility curve of acyclovir–HP-b-cyclodextrin complex in water was shown in Figure 1. It was apparent from Figure 1 that that the aqueous solubility of drug linearly increased in a concentration dependent manner with increases in HP-b-cyclodextrin concentration. The intrinsic solubility of acyclovir in water was found to be 1.61 0.28 mg/ml. However, solubility of acyclovir was significantly enhanced (2-fold; 3.09 0.46 mg/ml) when the HP-b-cyclodextrin concentration was 0.01 M. Increase in acyclovir solubility in aqueous HP-b-cyclodextrin solutions are consistent with the formation of inclusion complex between the drug and complexing agent. The possible mechanism is the hydrophobic interaction between acyclovir with HP-b-cyclodextrin. Moreover, the phase solubility curve was found to be a typical AL-type, described by Higuchi and Connors in solubility diagrams. Further, higher regression coefficient (r2 ¼ 0.99)
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and slope (0.66) value were observed in the specified concentration range assessed. Indeed, the linear curve observed in current experimental conditions indicates the formation of 1:1 (mol/mol) acyclovir–HP-b-cyclodextrin complex. Thus the possible association of guest–host molecule could be such that one molecule of acyclovir is included in the cavity of one molecule of HP-b-cyclodextrin. The calculated stability constant (Kc) value (276.67 12.48 M1) was found to be in the ideal range (100–1000 M1) (Shah et al., 2009), suggesting that acyclovir–HP-b-cyclodextrin complex is sufficiently stable in the aqueous media. The IR spectrum provides useful information for assessing the formation of complex or interaction between drug and polymers. Figure 2 shows the FTIR spectra of pure acyclovir, HP-b-cyclodextrin, physical mixture and inclusion complex. IR spectrum of HP-b-cyclodextrin showed broad absorption
Figure 1. Phase solubility diagram of acyclovir as function of HPb-cyclodextrin concentrations in water at 25 C.
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band at 3100–3345 cm1 for O–H stretching. Pure acyclovir was characterized by N–H stretching of NH2 and C ¼ O stretching at 3348 and 1727 cm1, respectively. FTIR spectrum of physical mixture shows all the absorption bands with same intensity indicating no interaction between drug and HP-b-cyclodextrin, whereas absorption band intensity in the range 3100–3500 is reduced for inclusion complex indicating the formation of complex. 1 H NMR spectra were recorded for pure acyclovir, HPb-cyclodextrin, physical mixture and inclusion complex and were depicted in Figure 3. It was evident from Figure 3 that acyclovir showed three signal for H8 ( 7.8), H10 ( 5.4) and H11/H12 ( 3.5). However, the protons of NH and NH2 interchanged with deuterium of deutered water, and were not appeared in the spectra. Indeed, the NMR spectra of physical mixture showed the signals at same delta as that of pure acyclovir whereas complex showed slight displacement of H8 ( 7.9) and H10 ( 5.5) proton signal, demonstrating the formation of complex. Further, variation in the shape of H10 signal was also observed, which confirm the involvement of H10 in complex formation. These observations were in agreement with the earlier reports in the literature (Rossel et al., 2000; S´wierzewski et al., 2002; Zielenkiewicz et al., 2010). The thermal behavior of pure drug, HP-b-cyclodextrin, physical mixture and inclusion complex was assessed using DSC and the corresponding thermograms were depicted in Figure 4. The observed data revealed that drug and polymers are compatible and the glass transition temperature was not influenced by the preparation procedure. Thermogram of pure acyclovir shows a characteristic endothermic peak at 253 C corresponding to its melting point, as well as suggesting its crystalline nature. On the other hand, the DSC curve for HP-b-cyclodextrin was characterized by broad endothermic event attributed to its dehydration process, which attained a maximum 95–120 C. This endothermic curve corresponds to the loss of water molecules in the HP-b-cyclodextrin existing as residual humidity (t5100 C) as well as those included in the cavity (t4100 C) (Castro-Hermida
Figure 2. FTIR spectra of acyclovir, HP-b-cyclodextrin, physical mixture and inclusion complex (drug: HP-b-cyclodextrin, 1:1).
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DOI: 10.3109/10717544.2013.853213
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Figure 3. 1H NMR spectra of acyclovir (A), HP-b-cyclodextrin (B), physical mixture (C) and inclusion complex (D; drug: HP-b-cyclodextrin, 1:1).
Figure 4. Differential scanning calorimetric curves of acyclovir (A), HP-b-cyclodextrin (B), physical mixture (C) and inclusion complex (D; drug: HP-b-cyclodextrin, 1:1).
et al., 2004). The thermogram of acyclovir and HPb-cyclodextrin (1:1 mole) physical mixture showed two peaks of reduced intensity, which were found in pure drug (253 C) and HP-b-cyclodextrin (95–120 C) thermograms, indicating that a true complex has not formed. The presence of specific drug peak also reveals the presence of crystalline form. On the other hand, the reduced intensity of peak
suggests the partial complexation and/or interaction between acyclovir and HP-b-cyclodextrin. The melting endotherm of acyclovir was completely absent in thermogram of inclusion complex, indicating the absence of crystallinity in the complex. On the other way, state of drug has been changed from crystalline to amorphous. In general, inclusion of drug molecules in cyclodextrin cavity or in the crystal lattice can cause shifting of their melting, boiling and sublimation points or disappear of drug peak. Thus, disappearance of thermal features of drug ascertained the formation of true complex of drug-HP-b-cyclodextrin, wherein the drug molecules penetrated into the cyclodextrin cavity by replacing the water molecules. The possible mechanism of the complex formation between acyclovir and HP-b-cyclodextrin was reported in the literature (S´wierzewski et al., 2002; Zielenkiewicz et al., 2010; Koz´biał & Gierycz, 2013). The authors suggest that the complexation of acyclovir by HP-b-cyclodextrin is likely due to the interaction/association between the hydroxyl ethoxy methyl group of acyclovir with the hydrophobic cavity of cyclodextrin. The percentage drug content was determined for the physical mixture and inclusion complex and was found to be comparable (96.26 3.82% and 98.60 1.37% for physical mixtures and solid dispersion, respectively). For oral formulations, in vitro dissolution data could be a key pharmaceutical parameter for poorly soluble drug because the rate limiting step for absorption of these molecules
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primarily depends on dissolution rate. In vitro dissolution studies were performed for pure drug (acyclovir), physical mixture (acyclovir: HP-b-cyclodextrin) and its inclusion complex in simulated gastric fluid (pH 1.2) at 37 0.5 C. A low dose of 100 mg of acyclovir was used to maintain sink condition. The observed dissolution profiles of pure drug, physical mixture and inclusion complex were depicted in Figure 5. It was apparent from Figure 5 that both inclusion complex and physical mixture exhibit faster dissolution rate than pure drug. The increase in dissolution rate of physical mixture was likely due to the local solubilizing effect of cyclodextrin in the microenvironment and/or improved wettability of drug due to hydrodynamic layer surrounding drug particles. However, release of drug from the inclusion complex was found to be rapid and complete when compared to the physical mixture and pure drug. Analysis of the dissolution data at two initial time intervals like Q5 and Q10 (i.e. percentage of drug dissolved in 5 and 10 min, respectively) reveals that dissolution rate decreased in the following pattern, inclusion complex4physical mixture4pure drug. For instance, the percentage of acyclovir released in 10 min was found to be 84.19 14.13% (p50.001), 53.40 8.28% and 39.16 8.13% from inclusion complex, physical mixture and pure drug, respectively. Indeed, complete drug release from the inclusion complex was observed in 30 min. Further, the highest dissolution efficiency was also observed with inclusion complex (90.05 2.94%; p50.0001) compared to physical mixture (80.44 1.62%) and pure drug (73.05 1.54%). The possible reasons for enhanced dissolution rate of acyclovir in inclusion complex are likely due to the formation of readily soluble hydrophilic inclusion complex, high energetic amorphous state (reduced degree of crystallinity) of the drug in the complex, increased drug particle wettability and/or reduced particle size (Loftsson & Brewster, 1996).
Pharmacokinetic studies were carried out by peroral administration of similar dose (1 ml, 20 mg/kg of acyclovir) of solutions of inclusion complex or suspensions of physical mixture in Wistar rats. Figure 6 compares the plasma level profiles of acyclovir following oral administration of inclusion complex and physical mixture (control). It was evident from Figure 6 that a significant amount of acyclovir plasma concentration was measured in 15 min (in both the cases) indicating immediate absorption of acyclovir following oral administration. Greater amount of acyclovir (measured from plasma acyclovir level) was observed in the absorptive phase following administration of inclusion complex when compared to physical mixture (Figure 6). This moderate enhancement in acyclovir plasma concentration with inclusion complex (observed in the current experimental condition) during the absorptive phase is probably because of acyclovirHP-b-cyclodextrin complex which ensured rapid and greater dissolution of drug in the aqueous gastrointestinal fluids which further diffused towards the mucosal surface and was available for immediate absorption (Loftsson et al., 2004). Thus it is likely that the complexation of acyclovir with HP-b-cyclodextrin has overcome one of the major issues (limited aqueous solubility) which have limited the efficiency of acyclovir in oral therapy. However, the partition of dissolved drug from aqueous exterior into the gastrointestinal mucosa would have been influenced by the low permeability of acyclovir which in turn narrowed the enhancement in absorption. On the other hand, the low absorption by physical mixture indicates less solubility of acyclovir and
Figure 5. Comparison of in vitro release profile of acyclovir, physical mixture and inclusion complex (drug: HP-b-cyclodextrin, 1:1) in 0.1 N HCl (pH 1.2) for a period of 1 h. The data represent the mean of six determinations.
Figure 6. Plasma drug profiles obtained following oral administration (dose of 20 mg/kg acyclovir) of physical mixture or inclusion complex (drug: HP-b-cyclodextrin, 1:1) for a period of 6 h in Wistar rats. The data represent mean SD of six determinations.
Enhanced oral bioavailability of acyclovir
DOI: 10.3109/10717544.2013.853213
Table 1. Mean pharmacokinetic parameters of acyclovir in plasma following oral administration of inclusion complex and physical mixture (1 ml, dose of 20 mg/kg of acyclovir) for a period of 5 h in Wistar rats (n ¼ 6). Parameter Tmax (min) Cmax (mg/ml) AUC0–a (mg h/ml) t1/2 (min) Kel
Inclusion complex
Physical mixture
45 3.37 0.75 483.68 52.82 132.35 24.18 0.005 0.001
45 1.97 0.51 298.17 36.13 131.19 28.62 0.005 0.001
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Cmax indicates maximum concentration; Tmax, time of maximum concentration; Kel, elimination rate constant; AUC0–a, area under the plasma concentration–time curve; t1/2, elimination half-life.
non-availability of dissolved drug for absorption. In the post absorptive phase, drug concentration in plasma declined rapidly in a similar way with both the cases suggesting rapid metabolism/quick distribution of drug into tissue compartments. The area under the curve (AUC0–a) is a measure of the bioavailability of drug. The maximum plasma concentration (Cmax) and time to reach maximum concentration (Tmax) indicate the rate of delivery of drug into the systemic circulation. AUC0–a was calculated using the trapezoid rule and Cmax, Tmax were determined from the concentration–time curves. The observed pharmacokinetic parameters were summarized in Table 1. It was apparent that the oral administration of inclusion complex significantly increased the values of Cmax and AUC0–a, compared to control. The maximum amount of acyclovir in the plasma (Cmax) after oral administration of inclusion complex was found to be 3.37 0.75 mg/ml, 2-fold higher (p ¼ 0.0036) than control (1.97 0.51 mg/ml). Further, significant enhancement in the AUC0–a was observed (1.5-fold, p50.0001) in the case of inclusion complex, compared to control. This noticeable enhancement in AUC values (in the case of inclusion complex) signifies increased rate and extent of acyclovir absorption (relative bioavailability 160%) from the inclusion complex when compared to control. The observed increase in systemic bioavailability of acyclovir by inclusion complex is likely due to the improved solubility of drug. However, t1/2, Tmax and Kel values (Table 1) were found to be comparable in both the cases suggesting the disposition of acyclovir in the body is not influenced by the inclusion complex, in the current experimental condition.
Conclusion The data observed in current study demonstrated the feasibility of enhancing oral bioavailability of acyclovir, an antiviral agent, by formulating inclusion complex using HP-b-cyclodextrin. The phase solubility study indicates linear increase in aqueous solubility of acyclovir in a concentration dependent manner with increases in HPb-cyclodextrin concentration. FTIR, NMR and DSC data indicated the true complex formation of acyclovir with HPb-cyclodextrin. The in vitro dissolution study reveals greater dissolution rate and higher dissolution efficiency by the highly soluble inclusion complex. The noticeable enhancement in AUC values following the administration of inclusion
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complex signifies increased rate and extent of acyclovir absorption.
Declaration of interest The authors report no conflict of interest.
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Mukherjee B, Patra B, Layek B, Mukherjee A. (2007). Sustained release of acyclovir from nano-liposomes and nano-niosomes: an in vitro study. Int J Nanomed 2:213–25. Mummidi V, Jayanthi V. (2013). Effect of hydrophilic polymers on isradipine complexation with hydroxypropyl b-cyclodextrin. Drug Dev Ind Pharm 39:970–7. Nasongkla N, Wiedmann AF, Bruening A, et al. (2003). Enhancement of solubility and bioavailability of beta-lapachone using cyclodextrin inclusion complexes. Pharm Res 20:1626–33. Paul S, Kumar A, Yedurkar P, Sawant K. (2013). Design and development of multiple emulsion for enhancement of oral bioavailability of acyclovir. Drug Dev Ind Pharm 39:1809–17. Phillip Lee YH, Sathigari S, Jean Lin YJ, et al. (2009). Gefitinib-cyclodextrin inclusion complexes: physico-chemical characterization and dissolution studies. Drug Dev Ind Pharm 35: 1113–20. Rossel CP, Carren˜o JS, Rodrı´guez-Baeza M, Alderete JB. (2000). Inclusion complex of the antiviral drug acyclovir with cyclodextrin in aqueous solution and in solid phase. Quı´mica Nova 23:749–52. Shah J, Vasanti S, Anroop B, Vyas H. (2009). Enhancement of dissolution rate of valdecoxib by solid dispersions technique with PVP K 30 & PEG 4000, preparation and in vitro evaluation. J Inclusion Phenom Macrocyclic Chem 63:219–24. Spruance SL, Kriesel JD. (2002). Treatment of herpes simplex labialis. Herpes 9:64–9.
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Spruance SL, Nett R, Marbury T, et al. (2002). Acyclovir cream for treatment of herpes simplex labialis: results of two randomized, double-blind, vehicle-controlled, multicenter clinical trials. Antimicrob Agents Chemother 46:2238–43. S´wierzewski R, Poznan´ski J, Golankiewicz B, Zielenkiewicz W. (2002). NMR investigations of the stacking-like self-association of acyclovir, bromoacyclovir and ganciclovir. J Mol Liq 95:339–45. Taneri F, Ozcan I, Guneri T. (2010). In vitro and in vivo evaluation of oral tablet formulations prepared with ketoconazole and hydroxypropyl-beta-cyclodextrin. Drug Deliv 17:152–7. Tomar V, Garud N, Kannojia P, et al. (2010). Enhancement of solubility of acyclovir by solid dispersion and inclusion complexation methods. Der Pharmacia Lett 2:341–52. Volpato NM, Santi P, Laureri C, Colombo P. (1997). Assay of acyclovir in human skin layers by high-performance liquid chromatography. J Pharm Biomed Anal 16:515–20. Wagstaff AJ, Faulds D, Goa KL. (1994). Acyclovir: a reappraisal of its antiviral activity, pharmacokinetic properties and therapeutic efficacy. Drugs 47:153–205. Wang S, Ding Y, Yao Y. (2009). Inclusion complexes of fluorofenidone with beta-cyclodextrin and hydroxypropyl-beta-cyclodextrin. Drug Dev Ind Pharm 35:808–13. Zielenkiewicz W, Koz´biał M, Golankiewicz B, Poznan´ski J. (2010). Enhancement of aqueous solubility of tricyclic acyclovir derivatives by their complexation with hydroxypropyl-b-cyclodextrin. J Therm Anal Calorim 101:555–60.
ORIGINAL ARTICLE
Enhanced oral bioavailability of acyclovir by inclusion complex using hydroxypropyl-b-cyclodextrin Anroop B. Nair1, Mahesh Attimarad1, Bandar E. Al-Dhubiab1, Jyoti Wadhwa2, Sree Harsha1, and Mueen Ahmed1 1
Department of Pharmaceutical Sciences, College of Clinical Pharmacy, King Faisal University, Al-Ahsa, Kingdom of Saudi Arabia and M.M. College of Pharmacy, MM University, Mullana, Ambala, India
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Abstract
Keywords
The therapeutic potential of acyclovir is limited by the low oral bioavailability owing to its limited aqueous solubility and low permeability. The present study was a systematic investigation on the development and evaluation of inclusion complex using hydroxypropylb-cyclodextrin for the enhancement of oral bioavailability of acyclovir. The inclusion complex of acyclovir was prepared by kneading method using drug: hydroxypropyl-b-cyclodextrin (1:1 mole). The prepared inclusion complex was characterized by Fourier transform infrared spectroscopy, differential scanning calorimetry, NMR spectroscopy and evaluated in vitro by dissolution studies. In vivo bioavailability of acyclovir was compared for inclusion complex and physical mixture in rat model. Phase solubility studies indicate the formation of acyclovir– hydroxypropyl-b-cyclodextrin complex with higher stability constant and linear enhancement in drug solubility with increase in hydroxypropyl-b-cyclodextrin concentration. Characterization of the prepared formulation confirms the formation of acyclovir–hydroxypropyl-b-cyclodextrin inclusion complex. Dissolution profile of inclusion complex demonstrated rapid and complete release of acyclovir in 30 min with greater dissolution efficiency (90.05 2.94%). In vivo pharmacokinetic data signify increased rate and extent of acyclovir absorption (relative bioavailability 160%; p50.0001) from inclusion complex, compared to physical mixture. Given the promising results in the in vivo studies, it can be concluded that the inclusion complex of acyclovir could be an effective and promising approach for successful oral therapy of acyclovir in the treatment of herpes viruses.
Acyclovir, bioavailability, dissolution, hydroxypropyl-b-cyclodextrin, pharmacokinetics
Introduction Acyclovir is a well-known antiviral agent used for the treatment of herpes viruses such as herpes simplex virus type I/II and varicella zoster (Spruance & Kriesel, 2002; Lin et al., 2003). Existing treatment modalities using acyclovir for herpes infections include oral, parenteral and topical therapy (Cortesi & Esposito, 2008). However, topical therapy is considered to be less effective due to the low skin permeability of acyclovir into the target site (Spruance et al., 2002). On the other hand, oral drug delivery is the most appropriate and patient compliant method as it offers several advantages and was preferred over other routes. Following oral therapy, the absorption of acyclovir was found to be slow, variable and incomplete with low oral bioavailability (15–30%) (Laskin, 1983; Fletcher & Bean, 1985; Arnal et al., 2008). Therefore the oral therapy of this drug required frequent administration of high dose of acyclovir, which leads to potential systemic adverse effects such as acute renal failure and neurotoxicity (Johnson et al., 1994). There has Address for correspondence: Anroop B. Nair, Assistant Professor, Department of Pharmaceutical Sciences, College of Clinical Pharmacy, King Faisal University, P.O. 400, Al-Ahsa-31982, Kingdom of Saudi Arabia. Tel: +966 536 219 868. Email: [email protected]
History Received 30 August 2013 Revised 5 October 2013 Accepted 5 October 2013
been intensive effort to improve the bioavailability of acyclovir following oral delivery. Several approaches have been attempted to develop effective drug delivery system for the successful delivery of acyclovir (Cortesi & Esposito, 2008). In one approach, vesicular carrier systems such as liposomes, niosomes, microparticles and nanoparticles were developed (Jain et al., 2005; Attia et al., 2007; Mukherjee et al., 2007; Caldero´n et al., 2013). On the other hand, drug discovery groups have synthesized prodrugs of acyclovir to improve the therapeutic efficacy (De Clercq & Field, 2006). Alternatively, microemulsions and self-emulsifying drug delivery systems were also attempted to augment the oral bioavailability of acyclovir (Cortesi & Esposito, 2008; Paul et al., 2013). Despite all these efforts, the successful oral therapy of acyclovir remains elusive. The poor efficiency of acyclovir in oral therapy is mainly due to its limited aqueous solubility and low permeability (Fletcher & Bean, 1985; Wagstaff et al., 1994; Friedrichsen et al., 2002; Bergstrom et al., 2003).The biopharmaceutics classification system (BCS) has categorized acyclovir under class 3 considering the highest possible strength up to 400 mg. However, this drug is also available at a high dose of 800 mg and falls under BCS class 4, suggesting that a distinct classification for this drug is not possible with the BCS
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system (Arnal et al, 2008). Aqueous solubility is a key property governs the dissolution and absorption. It is well known that the therapeutic efficiency of water-insoluble drugs could be increased by enhancing dissolution, which in turn improves the bioavailability. Several methods are reported in the literature to enhance the dissolution rate of low soluble drugs (Fasinu et al., 2013). Formation of inclusion complex is one of the promising approaches used to improve the apparent solubility, dissolution rates and consequently the bioavailability of poorly soluble drugs (Challa et al., 2005). Cyclodextrins are promising complexing agents consisting of cyclic oligosaccharides containing hydrophilic outer surface and hydrophobic inner cavity (Loftsson & Ducheˆne, 2007). This supramolecular host form stable inclusion complex with guest molecules without altering the molecular structure (Carrier et al., 2007; Mummidi & Jayanthi, 2013). Indeed, the inclusion of drug molecules into the cyclodextrin cavity could lead to modification of physical, chemical and biochemical properties of guest drug molecule (Taneri et al., 2010). The application of cyclodextrin in enhancing aqueous solubility of acyclovir by complexation has already been reported in the literature (Rossel et al., 2000; Tomar et al., 2010). Attempt has also been made to enhance the bioavailability of acyclovir by preparing inclusion complexes using b-cyclodextrin (Luengo et al., 2002). Selection of an appropriate complexing agent and the method of formulation are critical. Among the cyclodextrin derivatives, hydroxypropyl-b-cyclodextrin (HP-b-cyclodextrin) is most widely investigated for drug complexation and has captivated the drug delivery scientists probably owing to its high aqueous solubility, better complexation ability, greater stability, non-toxicity and its potential to enhance oral bioavailability of low aqueous soluble drugs (Irie & Uekama, 1997; Nasongkla et al., 2003). Recently, one attempt has been reported to enhance the aqueous solubility of acyclovir by preparing inclusion complex using HP-b-cyclodextrin (Koz´biał & Gierycz, 2013). The objective of this study was to carry out a systematic investigation on the effect of inclusion complex of acyclovir using HPb-cyclodextrin in enhancing the oral bioavailability. The inclusion complex of acyclovir-HP-b-cyclodextrin (1:1 molar ratio) was formulated by kneading method. Further the inclusion complex was characterized and evaluated in vivo for pharmacokinetics in Wistar rats.
Materials and methods Acyclovir was gifted by Ind-Swift Laboratories (Chandigarh, India). HP-b-cyclodextrin was purchased from Hi Media, Mumbai, India. All other chemicals and reagents used were of analytical grade. Analytical method The amount of acyclovir in samples was quantified by high performance liquid chromatography (HPLC) system (Cyberlab, LC P100) consisting of a Symmetry C18 analytical column (4.6 mm 150 mm, 5.0 mm) with a detector LC UV-100. Mobile phase consisted of 0.1% acetic acid and acetonitrile (98:2). Elution was performed isocratically at 25 C at a flow rate of 1.2 ml/min. Injection volume was 25 ml
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and the column effluent was monitored at 254 nm (Volpato et al., 1997). The method was validated by the determination of linearity, precision and accuracy. Phase solubility studies The phase solubility of acyclovir was conducted according to Higuchi & Connors (1965). An excess amount of acyclovir (50 mg) was added to 5 ml of water or aqueous solutions of HP-b-cyclodextrin (up to 0.01 mol) in a glass vial. The system was stirred for 24 h at 37 C and kept at rest for 24 h to attain equilibrium. The solution was filtered using 0.45 mm Millex syringe driven filter unit (Millipore Corporation, Bedford, MA) and the solubility was determined by HPLC. The amount of drug dissolved against moles of carrier was plotted and the stability constant was calculated by the equation. Kc ¼
Slope Soð1 SlopeÞ
where So is the solubility of drug in the absence of carrier. Preparation of inclusion complexes The inclusion complex of acyclovir and HP-b-cyclodextrin was prepared by kneading method. Briefly, acyclovir and HPb-cyclodextrin were sieved through sieve No. 100 (149 mm). Weighed accurately the specified quantity of drug and carrier in a molar ratio of 1:1 in a glass mortar and water was added drop by drop with triturating until it become slurry. The mixture was further triturated and grinded for 3 h (optimized from a period of 30 min to 4 h by evaluating crystallinity and inclusion efficiency) and freeze dried for 12 h. Different from the normal method of drying, the inclusion complex was freeze dried to remove the water. Indeed the freeze drying of inclusion complex will avoid the long exposure of formulation to the atmosphere (happens with regular drying), provide storage at ambient temperatures, reduce contamination and is pretty simple and rapid. Moreover, there are several reports where in the inclusion complexes prepared using HP-b-cyclodextrin showed greater stability (Phillip Lee et al., 2009; Wang et al., 2009). The mass obtained after freeze drying was pulverized to obtain powder-like complex and stored in a desiccator over fused calcium chloride. The physical mixture of acyclovir and HPb-cyclodextrin was prepared using same molar ratio (1:1) by triturating in a glass mortar for 10 min and pulverized to obtain uniform mixing. Fourier transform infrared spectroscopy Fourier transform infrared (FTIR) spectroscopy was used to characterize the inclusion complex and physical mixture using Shimadzu model 8400S Spectroscope. The samples were prepared by grinding small amount of the dried sample and mixed with potassium bromide at 1:5 (sample: KBr) ratio and the discs were prepared using a hydraulic press. The discs were scanned in the range of 500– 4000 cm1 in order to assess structural changes that could have occurred in the drug or HP-b-cyclodextrin as a result of inclusion complex.
Enhanced oral bioavailability of acyclovir
DOI: 10.3109/10717544.2013.853213
NMR spectroscopy
In vivo studies
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In vivo experiments were carried out in male, 6–8 weeks old Wistar rats (100–150 g) which were maintained on a 12/12 h light/dark cycle in an animal facility with unlimited access to food and water (Institutional Animal Ethical Committee, M. M. University, Ambala, India). Two groups of rats (six animals in each group) fasted for 12 h with free access to water were used in the study. Inclusion complex or physical mixture was administered by peroral route (1 ml, dose of 20 mg/kg of acyclovir) by gavage to the first and second group of rats, respectively. The oral bioavailability of inclusion complex was determined by estimating the plasma acyclovir concentrations. The blood samples were collected (200 ml) from retro orbital plexus using dry heparinized tubes at predetermined time intervals (15, 30, 45, 60, 120, 180, 240, 360 min), under anesthesia (thiopentone sodium 30 mg/kg). Plasma (200 ml) was subjected to protein precipitation with equal volume of acetonitrile and was centrifuged at 10 000 rpm for 10 min. Supernatant was filtered and injected into high-performance liquid chromatography (HPLC). The sample withdrawn at time zero served as the baseline value. The pharmacokinetic parameters were calculated using non-compartmental pharmacokinetic model. Pharmacokinetic variables of interest included area under the concentration– time curve from time 0 to 1 (AUC0– ), peak concentration (Cmax), terminal elimination rate constant (Kel), half-life of the terminal phase (t1/2) and time to peak concentration (Tmax). A linear-up/log-down method of estimation was used for the calculation of AUC1 and was obtained by adding Clast/Kel to AUC0–t. The terminal elimination rate constant (Kel) was determined from the slope of terminal exponential phase of the logarithmic plasma concentration–time curve. The elimination half-life (t1/2) was calculated using 0.693/Kel.
H NMR experiments were carried out at 400 MHz on a Bruker spectrometer at 298 K in deutered water. The NMR spectra were recorded for pure acyclovir, HP-b-cyclodextrin, physical mixture and inclusion complex and were processed with the aid of Topspin 3. Differential scanning calorimetry
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Differential scanning calorimetry (DSC) was performed on inclusion complex, physical mixture, HP-b-cyclodextrin and pure drug. DSC measurements were done with a DSC-60 instrument (Shimadzu, Japan). Samples equivalent to 2 mg of acyclovir were sealed in an aluminium pan and heated at a temperature range of 0–350 C at the rate of 10 C/min using indium as reference. The change in endothermic peaks of acyclovir and HP-b-cyclodextrin was observed, which confirms the interaction between drug and excipients. Evaluation of drug content Weighed amount of inclusion complex or physical mixture (equivalent to 2 mg of acyclovir) were dispersed in water (50 ml) for a predetermined time (24 h) in screw-capped vials and stirred on a magnetic stirrer. The resulting dispersion was then centrifuged at 10 000 rpm (5 min) and the supernatant was collected and filtered using 0.2 mm Millex syringe driven filter unit (Millipore Corporation, Bedford, MA) and the sample was analyzed using HPLC. Dissolution studies In vitro drug release from pure acyclovir, physical mixture and inclusion complex were determined in simulated gastric fluid (pH 1.2, no enzyme) for 1 h. Weighed amount of inclusion complex, physical mixture or pure drug (equivalent to 100 mg of acyclovir) were filled into empty hard gelatin capsule shell and subjected to dissolution study. The samples were placed in a dissolution test apparatus USPXXIV Type II (Electro Lab, Mumbai, India) containing dissolution medium (900 ml) maintained at 37 0.5 C with paddle rotation maintained at 50 rpm (n ¼ 6). Aliquots of dissolution medium (1 ml) were withdrawn at appropriate time intervals and replaced with fresh media to compensate the loss of sample withdrawn. Further, samples were filtered using 0.2 mm Millex syringe driven filter unit (Millipore Corporation, Bedford, MA) and analyzed by HPLC. Dissolution efficiency studies The dissolution efficiency of the pure acyclovir, physical mixture and inclusion complex were calculated by the method mentioned by Khan (1975). It is defined as the area under the dissolution curve between time points t1 and t2 expressed as a percentage of the curve at maximum dissolution, y100, over the same time period or the area under the dissolution curve up to a certain time, t, expressed as a percentage of the area of the rectangle described by 100% dissolution in the same time. Rt 0 ydt Dissolution efficiency ¼ 100% y100ðt2 t1 Þ
Data analysis The data were expressed as mean of six trials SD. Statistical analysis was performed by unpaired t-test using Graphpad prism 5, graphpad software, Inc., CA, to test the drug concentration in various treatments. p50.05 was considered as the level of significance.
Results and discussion The phase-solubility curve of acyclovir–HP-b-cyclodextrin complex in water was shown in Figure 1. It was apparent from Figure 1 that that the aqueous solubility of drug linearly increased in a concentration dependent manner with increases in HP-b-cyclodextrin concentration. The intrinsic solubility of acyclovir in water was found to be 1.61 0.28 mg/ml. However, solubility of acyclovir was significantly enhanced (2-fold; 3.09 0.46 mg/ml) when the HP-b-cyclodextrin concentration was 0.01 M. Increase in acyclovir solubility in aqueous HP-b-cyclodextrin solutions are consistent with the formation of inclusion complex between the drug and complexing agent. The possible mechanism is the hydrophobic interaction between acyclovir with HP-b-cyclodextrin. Moreover, the phase solubility curve was found to be a typical AL-type, described by Higuchi and Connors in solubility diagrams. Further, higher regression coefficient (r2 ¼ 0.99)
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and slope (0.66) value were observed in the specified concentration range assessed. Indeed, the linear curve observed in current experimental conditions indicates the formation of 1:1 (mol/mol) acyclovir–HP-b-cyclodextrin complex. Thus the possible association of guest–host molecule could be such that one molecule of acyclovir is included in the cavity of one molecule of HP-b-cyclodextrin. The calculated stability constant (Kc) value (276.67 12.48 M1) was found to be in the ideal range (100–1000 M1) (Shah et al., 2009), suggesting that acyclovir–HP-b-cyclodextrin complex is sufficiently stable in the aqueous media. The IR spectrum provides useful information for assessing the formation of complex or interaction between drug and polymers. Figure 2 shows the FTIR spectra of pure acyclovir, HP-b-cyclodextrin, physical mixture and inclusion complex. IR spectrum of HP-b-cyclodextrin showed broad absorption
Figure 1. Phase solubility diagram of acyclovir as function of HPb-cyclodextrin concentrations in water at 25 C.
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band at 3100–3345 cm1 for O–H stretching. Pure acyclovir was characterized by N–H stretching of NH2 and C ¼ O stretching at 3348 and 1727 cm1, respectively. FTIR spectrum of physical mixture shows all the absorption bands with same intensity indicating no interaction between drug and HP-b-cyclodextrin, whereas absorption band intensity in the range 3100–3500 is reduced for inclusion complex indicating the formation of complex. 1 H NMR spectra were recorded for pure acyclovir, HPb-cyclodextrin, physical mixture and inclusion complex and were depicted in Figure 3. It was evident from Figure 3 that acyclovir showed three signal for H8 ( 7.8), H10 ( 5.4) and H11/H12 ( 3.5). However, the protons of NH and NH2 interchanged with deuterium of deutered water, and were not appeared in the spectra. Indeed, the NMR spectra of physical mixture showed the signals at same delta as that of pure acyclovir whereas complex showed slight displacement of H8 ( 7.9) and H10 ( 5.5) proton signal, demonstrating the formation of complex. Further, variation in the shape of H10 signal was also observed, which confirm the involvement of H10 in complex formation. These observations were in agreement with the earlier reports in the literature (Rossel et al., 2000; S´wierzewski et al., 2002; Zielenkiewicz et al., 2010). The thermal behavior of pure drug, HP-b-cyclodextrin, physical mixture and inclusion complex was assessed using DSC and the corresponding thermograms were depicted in Figure 4. The observed data revealed that drug and polymers are compatible and the glass transition temperature was not influenced by the preparation procedure. Thermogram of pure acyclovir shows a characteristic endothermic peak at 253 C corresponding to its melting point, as well as suggesting its crystalline nature. On the other hand, the DSC curve for HP-b-cyclodextrin was characterized by broad endothermic event attributed to its dehydration process, which attained a maximum 95–120 C. This endothermic curve corresponds to the loss of water molecules in the HP-b-cyclodextrin existing as residual humidity (t5100 C) as well as those included in the cavity (t4100 C) (Castro-Hermida
Figure 2. FTIR spectra of acyclovir, HP-b-cyclodextrin, physical mixture and inclusion complex (drug: HP-b-cyclodextrin, 1:1).
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DOI: 10.3109/10717544.2013.853213
Enhanced oral bioavailability of acyclovir
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Figure 3. 1H NMR spectra of acyclovir (A), HP-b-cyclodextrin (B), physical mixture (C) and inclusion complex (D; drug: HP-b-cyclodextrin, 1:1).
Figure 4. Differential scanning calorimetric curves of acyclovir (A), HP-b-cyclodextrin (B), physical mixture (C) and inclusion complex (D; drug: HP-b-cyclodextrin, 1:1).
et al., 2004). The thermogram of acyclovir and HPb-cyclodextrin (1:1 mole) physical mixture showed two peaks of reduced intensity, which were found in pure drug (253 C) and HP-b-cyclodextrin (95–120 C) thermograms, indicating that a true complex has not formed. The presence of specific drug peak also reveals the presence of crystalline form. On the other hand, the reduced intensity of peak
suggests the partial complexation and/or interaction between acyclovir and HP-b-cyclodextrin. The melting endotherm of acyclovir was completely absent in thermogram of inclusion complex, indicating the absence of crystallinity in the complex. On the other way, state of drug has been changed from crystalline to amorphous. In general, inclusion of drug molecules in cyclodextrin cavity or in the crystal lattice can cause shifting of their melting, boiling and sublimation points or disappear of drug peak. Thus, disappearance of thermal features of drug ascertained the formation of true complex of drug-HP-b-cyclodextrin, wherein the drug molecules penetrated into the cyclodextrin cavity by replacing the water molecules. The possible mechanism of the complex formation between acyclovir and HP-b-cyclodextrin was reported in the literature (S´wierzewski et al., 2002; Zielenkiewicz et al., 2010; Koz´biał & Gierycz, 2013). The authors suggest that the complexation of acyclovir by HP-b-cyclodextrin is likely due to the interaction/association between the hydroxyl ethoxy methyl group of acyclovir with the hydrophobic cavity of cyclodextrin. The percentage drug content was determined for the physical mixture and inclusion complex and was found to be comparable (96.26 3.82% and 98.60 1.37% for physical mixtures and solid dispersion, respectively). For oral formulations, in vitro dissolution data could be a key pharmaceutical parameter for poorly soluble drug because the rate limiting step for absorption of these molecules
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primarily depends on dissolution rate. In vitro dissolution studies were performed for pure drug (acyclovir), physical mixture (acyclovir: HP-b-cyclodextrin) and its inclusion complex in simulated gastric fluid (pH 1.2) at 37 0.5 C. A low dose of 100 mg of acyclovir was used to maintain sink condition. The observed dissolution profiles of pure drug, physical mixture and inclusion complex were depicted in Figure 5. It was apparent from Figure 5 that both inclusion complex and physical mixture exhibit faster dissolution rate than pure drug. The increase in dissolution rate of physical mixture was likely due to the local solubilizing effect of cyclodextrin in the microenvironment and/or improved wettability of drug due to hydrodynamic layer surrounding drug particles. However, release of drug from the inclusion complex was found to be rapid and complete when compared to the physical mixture and pure drug. Analysis of the dissolution data at two initial time intervals like Q5 and Q10 (i.e. percentage of drug dissolved in 5 and 10 min, respectively) reveals that dissolution rate decreased in the following pattern, inclusion complex4physical mixture4pure drug. For instance, the percentage of acyclovir released in 10 min was found to be 84.19 14.13% (p50.001), 53.40 8.28% and 39.16 8.13% from inclusion complex, physical mixture and pure drug, respectively. Indeed, complete drug release from the inclusion complex was observed in 30 min. Further, the highest dissolution efficiency was also observed with inclusion complex (90.05 2.94%; p50.0001) compared to physical mixture (80.44 1.62%) and pure drug (73.05 1.54%). The possible reasons for enhanced dissolution rate of acyclovir in inclusion complex are likely due to the formation of readily soluble hydrophilic inclusion complex, high energetic amorphous state (reduced degree of crystallinity) of the drug in the complex, increased drug particle wettability and/or reduced particle size (Loftsson & Brewster, 1996).
Pharmacokinetic studies were carried out by peroral administration of similar dose (1 ml, 20 mg/kg of acyclovir) of solutions of inclusion complex or suspensions of physical mixture in Wistar rats. Figure 6 compares the plasma level profiles of acyclovir following oral administration of inclusion complex and physical mixture (control). It was evident from Figure 6 that a significant amount of acyclovir plasma concentration was measured in 15 min (in both the cases) indicating immediate absorption of acyclovir following oral administration. Greater amount of acyclovir (measured from plasma acyclovir level) was observed in the absorptive phase following administration of inclusion complex when compared to physical mixture (Figure 6). This moderate enhancement in acyclovir plasma concentration with inclusion complex (observed in the current experimental condition) during the absorptive phase is probably because of acyclovirHP-b-cyclodextrin complex which ensured rapid and greater dissolution of drug in the aqueous gastrointestinal fluids which further diffused towards the mucosal surface and was available for immediate absorption (Loftsson et al., 2004). Thus it is likely that the complexation of acyclovir with HP-b-cyclodextrin has overcome one of the major issues (limited aqueous solubility) which have limited the efficiency of acyclovir in oral therapy. However, the partition of dissolved drug from aqueous exterior into the gastrointestinal mucosa would have been influenced by the low permeability of acyclovir which in turn narrowed the enhancement in absorption. On the other hand, the low absorption by physical mixture indicates less solubility of acyclovir and
Figure 5. Comparison of in vitro release profile of acyclovir, physical mixture and inclusion complex (drug: HP-b-cyclodextrin, 1:1) in 0.1 N HCl (pH 1.2) for a period of 1 h. The data represent the mean of six determinations.
Figure 6. Plasma drug profiles obtained following oral administration (dose of 20 mg/kg acyclovir) of physical mixture or inclusion complex (drug: HP-b-cyclodextrin, 1:1) for a period of 6 h in Wistar rats. The data represent mean SD of six determinations.
Enhanced oral bioavailability of acyclovir
DOI: 10.3109/10717544.2013.853213
Table 1. Mean pharmacokinetic parameters of acyclovir in plasma following oral administration of inclusion complex and physical mixture (1 ml, dose of 20 mg/kg of acyclovir) for a period of 5 h in Wistar rats (n ¼ 6). Parameter Tmax (min) Cmax (mg/ml) AUC0–a (mg h/ml) t1/2 (min) Kel
Inclusion complex
Physical mixture
45 3.37 0.75 483.68 52.82 132.35 24.18 0.005 0.001
45 1.97 0.51 298.17 36.13 131.19 28.62 0.005 0.001
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Cmax indicates maximum concentration; Tmax, time of maximum concentration; Kel, elimination rate constant; AUC0–a, area under the plasma concentration–time curve; t1/2, elimination half-life.
non-availability of dissolved drug for absorption. In the post absorptive phase, drug concentration in plasma declined rapidly in a similar way with both the cases suggesting rapid metabolism/quick distribution of drug into tissue compartments. The area under the curve (AUC0–a) is a measure of the bioavailability of drug. The maximum plasma concentration (Cmax) and time to reach maximum concentration (Tmax) indicate the rate of delivery of drug into the systemic circulation. AUC0–a was calculated using the trapezoid rule and Cmax, Tmax were determined from the concentration–time curves. The observed pharmacokinetic parameters were summarized in Table 1. It was apparent that the oral administration of inclusion complex significantly increased the values of Cmax and AUC0–a, compared to control. The maximum amount of acyclovir in the plasma (Cmax) after oral administration of inclusion complex was found to be 3.37 0.75 mg/ml, 2-fold higher (p ¼ 0.0036) than control (1.97 0.51 mg/ml). Further, significant enhancement in the AUC0–a was observed (1.5-fold, p50.0001) in the case of inclusion complex, compared to control. This noticeable enhancement in AUC values (in the case of inclusion complex) signifies increased rate and extent of acyclovir absorption (relative bioavailability 160%) from the inclusion complex when compared to control. The observed increase in systemic bioavailability of acyclovir by inclusion complex is likely due to the improved solubility of drug. However, t1/2, Tmax and Kel values (Table 1) were found to be comparable in both the cases suggesting the disposition of acyclovir in the body is not influenced by the inclusion complex, in the current experimental condition.
Conclusion The data observed in current study demonstrated the feasibility of enhancing oral bioavailability of acyclovir, an antiviral agent, by formulating inclusion complex using HP-b-cyclodextrin. The phase solubility study indicates linear increase in aqueous solubility of acyclovir in a concentration dependent manner with increases in HPb-cyclodextrin concentration. FTIR, NMR and DSC data indicated the true complex formation of acyclovir with HPb-cyclodextrin. The in vitro dissolution study reveals greater dissolution rate and higher dissolution efficiency by the highly soluble inclusion complex. The noticeable enhancement in AUC values following the administration of inclusion
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complex signifies increased rate and extent of acyclovir absorption.
Declaration of interest The authors report no conflict of interest.
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