In-Vitro Release Kinetics and Stability of Anticardiovascular Drugs

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In-Vitro Release Kinetics and Stability of Anticardiovascular Drugs-Intercalated Layered Double Hydroxide Nanohybrids H. S. Panda,† R. Srivastava,‡ and D. Bahadur*,† Department of Metallurgical Engineering and Materials Science, Indian Institute of Technology, Bombay, Mumbai-400076, India, and School of Biosciences and Bioengineering, Indian Institute of Technology, Bombay, Mumbai-400076, India ReceiVed: June 10, 2009; ReVised Manuscript ReceiVed: September 13, 2009

We report the intercalation and characterization of pravastatin and fluvastatin drugs in MgII/AlIII layered double hydroxides (LDHs) to form novel nanohybrid hydroxides through the coprecipitation technique. powder X-ray diffraction, Fourier transform infrared spectroscopy, and thermal analysis techniques reveal that the drugs are accommodated within the brucite layers. Structural characterization, computed results, and atomic force microscopy image analysis demonstrate that the fluvastatin anions are attached with the brucite as a monolayer, whereas the pravastatin anions form a multilayer. The shift in the stretching frequency of carboxylate anion of statin drugs provides evidence that the drugs are electrostatically bonded to LDHs. X-ray diffraction and thermal analysis studies performed after keeping the nanohybrid particles at 75 ( 10% relative humidity atmosphere, indicate their physical stability due to proper confinement of drugs within the layers. In-vitro release study of developed nanohybrid particles suggests that the significant reduction in release rate of fluvastatin anions from fluvastatin intercalated LDHs is due to its hydrophobic nature and it can be further controlled by varying the concentration in physiological medium. After release, the data were fitted to the dissolution-diffusion kinetic model. The mechanism of drugs diffusion in hydrophobic nanohybrid is probably due to heterogeneous diffusion via anion exchange, while in a hydrophilic nanohybrid, it is due to intraparticle diffusion via anion exchange with the anions in the physiological medium. Introduction Insertion of organic/inorganic anions onto layered inorganic matrices offers an attractive route to nanohybrids which have incredible practical importance in catalysis, magnetic precursor and pharmaceuticals.1–4 Layered double hydroxides (LDHs) are a class of nanoporous material consisting of positively charged hydroxylated metal layers, which are neutralized by interlamellar anions. The general chemical formula is [MII1-xMIIIx(OH)2]x+[An]x/n · mH2O, where MII and MIII are divalent and trivalent cations and An- is an exchangeable anion. The positive charge in metal layers are constructed from edge-sharing of MII(OH)6 and MIII(OH)6 octahedra and compensated by interlayer anions. However, the charge density of the LDH layers and the content of anionic species and water molecules promote strong electrostatic interaction between the sheets. Because of intercalation properties of LDHs, it has received considerable attention for storing biologically active materials. Earlier Khan et al.4 have reported the intercalation of drugs such as diclofenac, gemfibrojil, ibuprofen, naproxen, and tolfenamic acid in LDHs and their potential as a sustained drug release carrier. Also, intercalation of several beneficial organic anions such as DNA, amino acids, cyclodextrin, pesticide, salicylate, citrate, aspartate, and glutamic acid have also been reported.5–8 However, there is a lack of information on the effect of molecular structure of bioactive anions on the physicochemical properties of LDHs based nanohybrid particles. These observations prompted us to investigate new drug intercalated layered double hydroxide * To whom correspondence should be addressed. Tel.: +91-22-25767632. Fax: 91-22-25723480. E-mail: [email protected]. † Department of Metallurgical Engineering and Materials Science. ‡ School of Biosciences and Bioengineering.

nanohybrid particles for the release of drugs more efficiently. Further, to our knowledge, there is no report on the use of statin family drug (antihypercholesterolemia) intercalated layered double hydroxide systems. Statin family drugs such as pravastatin and fluvastatin and their derivatives otherwise called 3-hydroxy-3 methyl glutarylcoenzyme A (HMG-CoA) reductase inhibitors are the most therapeutically effective drugs for reducing cholesterol level in the bloodstream.9 Elevated cholesterol level is a primary risk factor for coronary artery disease and it is a major problem in developed countries. These drugs exhibit a number of other effects, such as simulation of bone formation and nitric oxide mediated promotion of new blood vessel growth.10 However, pharmaceutically acceptable statin salts are generally unstable due to hygroscopic nature and they tend to become stable by changing conformation. But the arrangement and conformation of molecules in the crystal lattice strongly depend upon the intermolecular interaction. Therefore, conformational changes significantly affect their solubility in aqueous solution and this can have a potentially major effect on the bioavailability of the drugs. Additionally, statin, a m-hydroxy alkyl acid will easily form a lactone upon storage, which is a pharmaceutically inactive form. Again due to the highly unstable nature of statin drugs in acidic environment, delivery is mainly by solid dispersion within biodegradable polymers.11 However, because of their low stability it enhances the cost of the pharmaceutical product. Also, hydrophilic statin drugs show a cyto-toxic effect because of intracellular depletion of essential metabolites and destabilization of cell membrains.12 These observations encouraged us to formulate statin drugs with a class of inorganic material (LDHs) for stabilization and release of drugs more efficiently.

10.1021/jp905440e CCC: $40.75  2009 American Chemical Society Published on Web 10/15/2009

Anticardiovascular Drugs Intercalated LDHs

Figure 1. Schematic representation of statin drugs intercalated LDH nanohybrids.

We chose to synthesize statin drug-LDH nanohybrids by the coprecipitation route involving the in situ formation of double metal hydroxide layers. We believe that, because of the highly hygroscopic nature of statin drugs (less stable), this process is significantly superior than the intercalation into presynthesized LDHs by an ion exchange process. The abovementioned route to incorporate statin drugs such as pravastatin and fluvastatin through a coprecipitation technique in magnesium containing layered double hydroxide has several advantages: (a) use of sodium hydroxide during precipitation helps to convert lactones, an inactive impurity phase to active form,13 (b) intercalation of statin molecules in metal hydroxide sheets forms stable conformation due to both inter- and intramolecular interaction, and (c) the presence of magnesium cations with statin drugs may enhance the statin passive diffusion into hepatocytes.14 Furthermore, this technique has generated more interest because of low cost and possibly easy commercialization. In this work, we report the synthesis and characterization of novel statin-LDH hybrid materials for extended delivery. The hybrid particles thus obtained from pravastatin shows hydrophilic behavior, as reported earlier for other drug intercalated LDHs.4 However, fluvastatin upon intercalating in LDHs produces hydrophobic monolayers and we discuss the details of its effectiveness to drug loading and release kinetics. A distinct advantage of the drug-LDH hydrophobic monolayer is that it exhibits hydrophilic behavior in buffer (pH 7.4) and the kinetics of drug release involves the heterogeneous diffusion process. It also strongly depends upon the concentration of nanohybrid particles in the physiological medium. We further extend the LDHs technique to make statin drugs physically stable and for the stabilization of statin ions after decomposition in the physiological conditions. The schematic model of developed nanohybrids is shown in Figure 1. Experimental Section Synthesis of Statin Anion (Pravastatin and Fluvastatin) Intercalated LDHs Nanohybrid. Magnesium nitrate hexahydrate was purchased from Fluka with purity 99%, aluminum nitrate nonahydrate (98%) was purchased from Sigma-Aldrich. Sodium hydroxide was obtained from Thomas Beakar Ltd., India. All the chemicals were kept in vacuum before use. Pravastatin sodium and fluvastatin sodium was received from Lupin Pharmaceutical, Ltd., India, as a gift. In a typical experiment, 1 mmol individual statin drug and 60 mL M.Q water were kept in five-necked flat bottom flask by passing nitrogen gas for 15 min. Salt solution of magnesium nitrate hexahydrate (2.2 mmol, 569 mg) and aluminum nitrate nonahydrate (1.1 mmol, 418 mg) in 18 mL M.Q water was prepared and after complete dissolution, it was added dropwise to the above solution under stirring. Solution pH was adjusted to 8.5-9 by simultaneous drop by drop addition of freshly prepared NaOH

J. Phys. Chem. B, Vol. 113, No. 45, 2009 15091 solution (0.5 M). The whole solution was aged at 65 °C for 24 h under inert atmosphere. Following this, the precipitates were separated using centrifugation/filtration, and this was repeated four times to remove unreacted drugs and impurities. The separated solid particles were dried in vacuum at room temperature. Stability Study. Generally statin drugs such as pravastatin, fluvastatin, lovastatin, etc. are highly unstable in humid atmosphere, UV light, and also in solution. Therefore, a stability study for the developed nanohybrid materials was performed in two different environments. For the physical stability study, 75 ( 10% relative humidity was created artificially, wherein 3 cm of saturated salt solution was placed in the bottom of the glass desiccator, which is approximately 10 cm high. This was then placed in an oven and the temperature was maintained at 37 °C. After keeping samples in the desiccator, XRD patterns were recorded at different time intervals. Simultaneously, after removing the samples from humid atmosphere, TGA analysis was performed. Stability was also evaluated in pH 7.4 buffer solution, and the spectra were recorded for different time frames through UV-visible spectroscopy. The solutions were stored at ambient temperature without protection from light and tested at precalculated times. In Vitro Release Study. It has been reported that the pH values in the stomach before and after taking food are 1-2 and 4.5-5, respectively. In the lower part of the small intestine (jejunum and ileum), the pH is maintained at 7.5 ( 0.4 and then drops to 6.4 ( 0.6 in the ascending colon, and finally rises to 7.0 ( 0.7 in the distal colon.15 The ability of Mg/Al-prava or Mg/Al-fluva LDHs to act as an effective controlled release vehicle for intestinal drug delivery was investigated in a series of in vitro release experiments by monitoring the time dependence of the concentration of drug ions in phosphate buffer (pH 7.5), HCl buffer (pH 4.5), and simulated intestinal body fluids (pH 7). Phosphate buffer solution was prepared by following the standard protocol (0.68 g potassium dihydrogen phosphate and 0.1 g sodium hydroxide in 100 mL M.Q water). Simulated intestinal body fluid was manipulated by preparing phosphate buffer solution containing 10% fetal bovine serum (FBS). All the release experiments were performed by UV-visible spectroscopy. Further, to understand the release kinetics, dissolutiondiffusion kinetic models have been used and fit with the in vitro drug-LDHs release profiles.16–18 Characterization. Structural information of prepared samples was obtained by powder X-ray diffraction (PXRD) pattern using a Philips PW 3040/60 diffractometer. The measurement was performed by using 40 kV, 30 mA graphite filtered Cu KR radiation (λ ) 0.1506). The samples were scanned at a rate of 0.17°/minute and recorded every 0.02°. Fourier transform infrared (FTIR) spectra were recorded on a Magna-IR spectrometer-50 (Nicolet) instrument using the conventional KBr pellet technique (sample to KBr ratio 1:100). Elemental atomic weight percent of metal ions were analyzed using energy dispersive X-ray (EDX) analysis with a Hitachi-3400N apparatus having ultradry detector and operating at 30 kV. Small amounts of sample were placed in ethanol and sonicated in an ultrasonic bath sonicator. The dispersant was dropped onto a brass support and dried. The sample surface was coated with a thin carbon layer to reduce the charge. Atomic weight percent of carbon, nitrogen, and hydrogen was obtained with FLASH EA 1112 series CHN analyzer (Thermo Finnigan, Italy). Thermogravimetric analysis (TGA) was carried out on a SDT Q600 with the sample amount 5-10 mg. Samples were scanned at a heating rate of 10 °C per minute from 25-900 °C. Transmission electron

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Figure 2. XRD patterns of different anion intercalated LDHs: (a) CO32-, (b) pravastatin, and (c) fluvastatin.

microscopy (TEM) images were taken on a Phillips-CM200 electron microscope operated at 200 kV. One drop of suspended samples (solvent-ethanol) was deposited on a carbon-coated copper grid and dried. Atomic force microscopic (AFM) images were recorded in a Nano Scope-IV multimode digital instrument operated in tapping mode (frequency ≈ 319.48 kHz, amplitude set point ≈ 0.50). The samples were prepared by coating from a dilute solution onto a freshly cleaved microscopic glass (12 mm) slide. Surface contact angles of 5 µL water drops were measured with a DIGIDROP contact angle meter GBX (Model DS) at ambient temperature. To study the electronic band structure and release kinetics, the samples were dissolved with phosphate buffer (pH 7.4) solution. The UV-vis spectra and absorbance at a particular wavelength were recorded with a GBC, Cintra 202 UV-vis spectrophotometer. Results and Discussion The XRD patterns of pristine CO3-LDHs and statin drugs intercalated LDHs are shown in Figure 2. The patterns exhibit the characteristic reflections of well-crystallized layered materials and these are sharp and symmetric at lower theta angles, but weaker nonbasal reflections at higher angle. The CO32- anion intercalated LDHs exhibit XRD patterns similar to those reported previously19 and demonstrate good crystallinity with d003 spacing of 7.6 Å. But intercalation of pravastatin and fluvastatin anions in LDHs demonstrates the shifting of d003 diffraction lines to lower theta angle with an increase of the basal spacing from 7.6 Å (pristine LDH) to 14.9 and 15.5 Å for pravastatin and fluvastatin intercalated LDHs, respectively. The d value of (003) plane represents the summation of thickness of the brucite-like layers and the interlayer anions. On comparing with the d-spacing of brucite layers, that is, 4.8 Å,19 the observed increase in interlayer space (10.1 Å for Mg/Al-prava and 10.7 Å for Mg/Al-fluva) confirms the presence of drugs within the brucite layer. Assuming 3R packing of the layers, lattice parameters for Mg/Al-prava LDHs and Mg/Al-fluva LDHs were calculated and given in Table 1. Further the highest dimension of pravastatin and fluvastatin molecules was calculated from the energy minimized 3D structure by using ChemDraw Ultra 8.0 software and is found to be 12.1 and 10.7 Å, respectively (Figure 3). On comparing the dimension of pravastatin with the XRD results, it has been observed that the simulated dimension (12.1 Å) is higher than the interlayer spacing (10.1 Å), which

Figure 3. Energy-minimized 3D molecular structure of (a) pravastatin and (b) fluvastatin.

TABLE 1: XRD Data and Structural Parameters of Different Anion-Intercalated LDHs parameters d003 (Å) d110 (Å) lattice parameter a (Å) lattice parameter c (Å)

Mg/Al-CO3 Mg/Al-prava Mg/Al-fluva 7.69 1.52 3.04 23.07

14.88 1.51 3.02 44.64

15.5 1.51 3.02 46.5

supported the tilting of molecular chains during intercalation as reported earlier20. However, the simulated dimension of free fluvastatin molecules is equal to the calculated XRD interlayer spacing (10.7 Å), suggesting that the conformation of the attached fluvastatin anions with brucite layers is similar to free fluvastatin. Also it has been observed from Figure 3 that the steric hindrance between fluvastatin molecules is higher than the pravastatin molecules. FTIR spectra of pure drug, physical mixture, and hybrid of drug-LDHs are compared in the range of 400-4000 cm-1 and shown in Figure 4 (400-2000 cm-1) and in the Supporting Information (2000-4000 cm-1). For CO3-LDHs, the broad adsorption bands at 3400-3500 cm-1 (Supporting Information, S1) are due to the stretching frequency mode of O-H groups in the brucite layer and the intercrystalline water. The band at 1381 cm-1 is due to stretching vibrations of intercalated CO32anions and the band at 554 cm-1 is attributed to M-O and M-O-H stretching vibrations. The stretching frequency peak at 450 cm-1 is due to O-M-O vibrations of metal hydroxides, which is characteristic of Mg2Al-LDHs materials.21 For drug-LDH hybrids, the stretching frequency bands of carboxylate anions of pure drugs appear at 1583 and 1576 cm-1 for pravastatin and fluvastatin sodium, respectively.22 It remains unchanged for the physical mixture, but it appears at 1576 and 1561 cm-1 for pravastatin- and fluvastatin-intercalated hybrid particles, respectively. The shifting is due to the combined electronegativity effect of Mg and Al; as a result, the covalency of C-O decreases. But the other characteristic bands in the


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Figure 5. TGA profile of (A) Mg/Al-prava LDHs, physical mixture, and pravastatin sodium, and (B) Mg/Al-fluva LDHs, physical mixture, and fluvastatin sodium.

Figure 4. (A) FTIR spectra of (a) Mg/Al-CO3 LDHs, (b) physical mixture, (c) pravastatin sodium, and (d) Mg/Al-prava LDHs; (B) FTIR spectra of (a) Mg/Al-CO3 LDHs, (b) physical mixture, (c) Mg/Al-fluva LDHs, and (d) fluvastatin sodium.

range of 2800-2990 cm-1 are attributed to C-H stretching vibration of intercalated statin drugs (2885 and 2985 cm-1 for Mg/Al-prava LDHs, 2932 and 2982 cm-1 for Mg/Al-fluva LDHs), and these are quite similar to pure drugs and physical mixture (Supporting Information S1), suggesting the lack of interaction of the unpolar C-H groups with the layers. For Mg/ Al-prava LDHs, the band at 1720-1750 cm-1 is due to the CdO stretching vibration of ester and is the same as in the physical mixture and pure drug. This confirms that the drug structure is retained after synthesis and only carboxylate anions are electrostatically bonded to the layers. The bands below 600 cm-1 are due to M-O vibrational modes of metal hydroxides. Further, the absence of NO3- or CO32- stretching frequency peak, that is, 1381 cm-1, in fluvastatin intercalated hybrid particles and the presence in pravastatin intercalated LDHs, suggest the existence of more intercalated fluvastatin anions. In addition, UV-vis studies suggest the retention of electronic bands of pravastatin (238 nm) and fluvastatin (236 and 305 nm) with metal double hydroxide, confirming the stability of drug ions in the interlayers of LDHs (Supporting Information S2). The thermal analysis of all samples are investigated by using TGA, and their derivative plots are shown in Figure 5. In the temperature range between ambient and 150 °C, both Mg/

Al-prava and Mg/Al-fluva LDHs exhibit weight loss, due to adsorbed and crystalline water. Therefore, the weight loss between 160-300 °C for drug-LDHs is due to the degradation of interlayer drugs. In contrast, pure drugs show a satellite degradation point centered at 200 °C which may be due to the presence of other impurity phase with the parent compound. However, broadening of the peak occurs in the case of the physical mixture probably due to the combined effect of carbonate and drugs. Further, the degradation peaks which are centered at 338 and 340 °C for Mg/Al-prava and Mg/Al-fluva LDHs, respectively, are due to the dehydroxylation of metal hydroxide layers. Therefore, it suggests the presence of a single polymorphic form of drugs in metal hydroxide sheets. Elemental analysis of fluvastatin intercalated hybrid particles was obtained using EDX and CHN analyzer and the details of their elemental atomic weight % are given in Table 2. The EDX results confirmed the absence of sodium in drug intercalated LDHs, but it is present in the physical mixture, indicating that the carboxylic group of drugs have been attached to the LDHs layers. Again, the atomic weight percent of C, H, and N obtained from the CHN analyzer suggests that ∼50% fluvastatin drugs have been loaded with LDHs. This is more than pravastatin intercalated LDHs (∼30%) and those reported earlier.4 Therefore, the above characterization results confirmed that both pravastatin and fluvastatin are present within the LDHs structure. However, the obtained fluvastatin-intercalated LDH particles, when dispersed in water, exhibit a hydrophobic behavior, in contrast to Mg/Al-prava LDHs which are hydrophilic in nature.

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Figure 6. Tapping mode AFM image of (a) 2D image of Mg/Al-fluva LDHs, (b) 3D image of panel a, (c) section analysis spectra of image a, and (d) 2D image of Mg/Al-prava LDHs.

TABLE 2: Elemental Analysis Data of Different Statin Drug-Intercalated LDHs EDX compounds Mg/Al-prava Mg/Al-fluva

CHN

Mg (atomic weight %)

Al (atomic weight %)

O (atomic weight %)

C (atomic weight %)

H (atomic weight %)

N (atomic weight %)

11 ( 0.7 8.1 ( 0.4

6.3 ( 0.3 4.2 ( 0.6

33( 0.2 25.1( 0.1

38 ( 2 46 ( 5

7.06 ( 0.01 5.4 ( 0.3

0.013 2.3 ( 0.1

This observation prompted us to further characterize and understand the mechanism of intercalation and stacking of layers. The particle size and morphology of the hybrid hydroxides were observed by AFM and TEM. From AFM micrograph analysis (Figure 6), the observed diameters of the platelets were around 65 and 16 nm for pravastatin and fluvastatin intercalated LDHs, respectively. The significant reduction in diameter is noteworthy and needs to be investigated in the following ways. A recent report23 on dodecyl sulphate (DDS)-intercalated LDHs suggests the formation of hydrophobic monolayers through a microemulsion technique, which was further confirmed through AFM image height. Therefore, upon comparing the height of individual particles, it is noted that for Mg/Al-fluva LDHs, the particles height (1.4 nm) is approximately similar to the observed d-spacing. In contrast, Mg/Al-prava LDHs hybrid shows particle heights around 3.2 nm, which is significantly higher than the corresponding d-spacing. This observation suggests that the hydrophobicity of Mg/Al-fluva LDHs hybrid particles is due to the formation of monolayers, where the hydrophobic end of fluvastatin provides the hydrophobic sphere, and this supports the formation of smaller diameter particles. Further, the larger particle diameter observed in Mg/Al-prava LDH nanohybrids may be due to the hydrophilic nature of the system, where the probability of coalescing during the growth is higher because of the presence of a water layer associated with the nucleus of interest. In contrast, the hydrophobic nature of Mg/Al-fluva LDH nanohybrids inhibits the coalescing of nuclei and further expels the aqueous phase, resulting in a reduction in diameter. Again, an AFM image indicates that the exfoliated monolayers self-assemble themselves as a triangular

pattern to minimize their surface energy. Further, the morphology of nanohybrid particles is observed using TEM and is shown in Figure 7. Intercalation of different guest anions in the host LDHs changes the stacking force between the layers. As a consequence of CO32- intercalation, the LDH nanohybrid particles’ platelets fold to form nanoscrolls as reported earlier.24 But for pravastatin intercalated LDHs, the presence of more hydrogen-bonding interaction significantly reduces such folding effect and stacks one above the other. However, the distortion of layers in some region of micrograph in Figure 7a provides direct evidence of the presence of NO3- and/or CO32- with pravastatin. In contrast, fluvastatin-intercalated LDH layers are strongly attached to each other (Figure 7b,c). In support, contact angles of 5 µL water drops were measured at ambient temperature (Figure 7d). A typical carbonate-intercalated Mg/Al LDH surface demonstrates its hydrophilic property via a contact angle of 24° (image not given), whereas fluvastatin-intercalated LDH monolayers show a much higher surface contact angle of 86°, suggesting that the hydrophilic to hydrophobic phase transformation occurs for fluvastatin-intercalated LDHs. We note that the formation of the hydrophobic hybrid particle is due to a larger diameter of hydrophobic tail of fluvastatin (in the same reaction condition and carboxylate anions). Earlier Takagi et al.20 had argued that the actual arrangement of anions consists of two interpenetrating antiparallel half monolayers. As a result, it greatly reduces the repulsion between the anions and effectively retains the hydrophobic interaction between the hydrocarbon chains. However, in the case of fluvastatin, it causes steric repulsion (Figure 3) between the hydrocarbon chains because of the bulky hydrophobic tail, and hence the resulting



Figure 7. TEM images of (a) Mg/Al-prava LDHs, (b) Mg/Al-fluva LDHs, (c) HR images of panel b, and (d) contact angle microscopic image of water droplets in Mg/Al-fluva LDHs surface.

hydrophobic monolayers are arranged through two interpenetrating perpendicular half monolayers (head to tail). Evaluation of Physical and Solution Stability. Crystallization of statin drugs through lyophilization and dispersion of amorphous/crystalline drugs in different polymer matrix have been a subject of intensive research for decades since by this way a substantial stability during storage can be achieved.25 Several studies have demonstrated that “molecular mobility” is the most important factor, which affects the physical and chemical stability of pharmaceutical compounds during storage.26 It is well-known that the presence of moisture associated with hydrophilic drugs in the solid state can have significant effects on their physicochemical properties, such as chemical degradation and dissolution rate.27,28 However, other factors such as the dispersion of drugs in a polymer matrix29 and extent of drug-polymer interaction30 also affect the drug stability. Further,the moisture-absorbing ability of the drug’s carrier also has varied effects on drug stabilization. To minimize the above effects, Papageorgiou et al.22 recently suggested a solid dispersion technique in which fluvastatin molecules are dispersed homogeneously in various polymer matrices. For the amorphous fluvastatin molecules, owing to more number of hydrogen bondings, interaction remains stable in the amorphous form throughout the polymer matrix. We have investigated the physical stability of the nanohybrid particles (after keeping them for different time periods at 37 °C and 75 ( 10% relative humidity) through powder X-ray diffraction (PXRD), and these are depicted in Figure 8. PXRD patterns of Mg/Al-fluva LDH nanohybrid particles were taken after 4, 8, and 12 days, and this indicates that almost all the strong peaks remain unchanged with respect to as prepared nanohybrid particles. Hence, the similarity in diffraction peaks position clearly suggests the presence of hydrophobic tails, which enhances the water expulsion. Again, the similarity in d-spacing and lattice parameters of as-prepared nanohybrid particles and the particles put in a humid atmosphere support the presence of statin drugs within the brucite layers as an intercalated form, and these did not form corresponding salts. In contrast, pure fluvastatin sodium

salt did not withstand even a few days in the humid atmosphere and several structural modifications occurred as shown in Figure 8b. To further confirm, the TGA of nanohybrid particles and pure drug was carefully performed after putting the particles in a humid atmosphere and is shown in Figure 9. It was observed that nanohybrid particles after 12 days in humid atmosphere do not undergo any change in thermal degradation profile, and the spectra is superimposed with the pristine nanohybrid particles. In contrast pure drugs showed a new degradation peak, which is centered at ∼ 80 °C, and a shift of other degradation peaks toward higher temperatures. This is due to the presence of crystallized water and the crystallization of amorphous fluvastatin sodium, as reported earlier.31 On the other hand, stable behavior of the nanohybrid particles is due to the extent of electrostatic interactions (as found by FTIR) and inter- and/ or intramolecular interactions between the statin drugs and the used LDHs. These results indicate that the brucite is appropriate for maintaining physically stable statin drugs during storage. Similar stability results were also observed for the Mg/Al-prava LDHs nanohybrid particles and pravastatin sodium salt (figure not given). To investigate the chemical stability of fluvastatin in dissolution medium, the UV-vis electronic spectra of fluvastatin sodium and Mg/Al-fluva LDHs at pH 7.4 phosphate buffer solution are recorded at 37 °C at a precalculated time and are shown in Figure 10. For fluvastatin, the major byproduct is lactone, where as the minor byproducts are 3-hydroxy-5-keto fluvastatin, fluvastatin tert-butyl ester, and fluvastatin hydroxydiene. The formation of these inactive byproducts poses a serious problem for fluvastatin since it reduces the effectiveness of the drug considerably. However, the stability of fluvastatin improved significantly upon intercalating with an inorganic matrix. Antiisomer/lactone byproducts are formed after a few hours when fluvastatin sodium is dissolved in pH 7.4 buffer solution. But, this is absent in Mg/Al-fluva LDHs system. Generally, statin drugs are unstable in acidic medium and stable in alkaline medium (pH 8-13). Therefore, to stabilize statin drugs in solution, suitable stabilizing alkaline or alkaline earth metal salts

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Figure 8. Different time interval XRD patterns of (a) Mg/Al-fluva LDHs and (b) fluvastatin sodium after keeping in 75 ( 10% relative humidity atmosphere.

(e.g., potassium bicarbonate, sodium carbonate, sodium hydroxide, calcium carbonate, etc.) are used.32 Concerning the effect of LDHs on drug stability in solution, it was compared with the theoretical results of statin-lactone conversion and vice versa as reported earlier.33 Under basic conditions, the activation energy barriers for the hydrolysis of lactone is significantly lower (9 kcal/mol) than the reverse reaction (28 kcal/mol), making lactone unstable. Therefore, LDHs on decomposition in solution enhance the solution pH and hence the barrier energy and make statin drugs stable. In contrast, fluvastatin sodium at pH 7.4 buffer solution is unstable as shown in Figure 10b. From the above results it is inferred that the prepared statin-LDHs nanohybrid systems are highly stable with respect to their physical state and in the physiological medium. Release Study. Preliminary in vitro experiments were performed to observe the kinetics of drug release through UV-vis spectroscopy. For this, pure drugs, physical mixture, and hybrid particles were individually dispersed at pH 7.4 phosphate buffer, pH 4.5 HCl buffer medium, and simulated intestinal body fluids, and absorbance was recorded at different time intervals. The release patterns of pravastatin and fluvastatin from Mg/Al-prava LDHs and Mg/Al-fluva LDHs are shown in Figure 11. Although both the drugs contain single charged

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Figure 9. TGA curves of (a) Mg/Al-fluva LDHs and (b) fluvastatin sodium after keeping for 12 days in humid atmosphere.

anions and show a monophasic release pattern at pH 7.4 (phosphate buffer solution) as reported earlier for other drugs, the Mg/Al-fluva LDHs exhibit a gradual and slow release pattern. In both cases, there is an early fast release (for pravastatin 40% and fluvastatin 20%) of drug ions followed by a relatively slower release for fluvastatin. Also, on varying the concentration of materials in solution, there is a significant change of release patterns observed for both hydrophilic Mg/ Al-prava LDHs and hydrophobic Mg/Al-fluva LDHs nanohybrids. As an illustration, upon varying the concentration of nanohybrid particles in solution, a significant change in % of drug release was observed (for Mg/Al-fluva LDHs, 0.2 mg/ mLs100% in 16 h, 0.4 mg/mLs85% in 32 h and 0.55 mg/ mLs79% in 52 h; for Mg/Al-prava LDHs, 0.2 mg/mLs100% and 0.3 mg/mLs91%). Generally, the fluvastatin ions release from Mg/Al-fluva LDHs hydrophobic nanohybrid takes a much longer time than the release of other single charged drugs containing Mg/Al-LDHs at pH 7.4 as reported in the literature.34–36 There are various reports on the intercalation of drugs within brucite layers. The nanohybrid formed through intercalation of different drugs in same inorganic matrix showed anomalous release % with time. As an example, Zhang et al.34 reported that around 94% of captopril was released after 2.3 h, while Ambrogi et al.35,36 reported that


Figure 10. Time interval UV-vis spectra of (a) Mg/Al-fluva LDHs and (b) fluvastatin sodium at pH 7.4 phosphate buffer solution.

around 90% of diclofenac and 100% of ibuprofen were released after 9 and 1.6 h, respectively. Gu et al.37 reported that around 44% of low molecular weight Heparin (LMWH) from LMWH-LDHs was released after 120 h. More recently, Arco et al.38 claimed the suppression of release rate (compared to previous report) in anti-inflammatory drugs intercalated Mg/Al-LDHs on substituting Fe3+ in an Al3+ site, and it is due to the stronger interaction between anionic drugs and Fe containing brucite-type layers. However, there is a lack of systematic release studies on varying the concentration of nanohybrid particles in release medium and their rate kinetics. Gu et al.37 suggested that multianionic species such as LMWH show slower release due to strong electrostatic interaction of LDHs layers with ∼20 OSO3- and ∼10 COOgroups in each molecule. Therefore, the fast release response for pravastatin to release from Mg/Al-prava LDH nanohybrids is mainly due to hydrophilic nature and the single anion electrostatic interactions between the positively charged brucite layers and the negatively charged pravastatin anions in the LDHs interlayer. As a result, dissolutions Via leaking of metal ions and ion exchange take place simultaneously. However, fluvastatin shows extended release response from Mg/Al-fluva LDHs nanohybrids which is due to the hydrophobic nature and stronger electrostatic interactions with the positively charged LDH layers. It is interesting to note that the Mg/Al-fluva nanohybrid is hydrophobic in water and it slowly becomes hydrophilic in phosphate buffer solution.


Figure 11. Release profile of statin drugs for (a) pravastatin and (b) fluvastatin base systems at various physiological conditions.

Such changes from hydrophilic to hydrophobic states may be used for triggered drug release. Similar observation has been made in a layered microgel assembly where, by changing the temperature, it can transform from hydrophilic swollen to hydrophobic squeezed state and is suitable for drug release.39 This signifies that the phosphate ions in solution compete to exchange with the fluvastatin anion which is present in the interlayer space of LDHs. Further, this behavior was more obvious by varying the nanohybrid particles concentration in solution and changing the phosphate concentration to make 10% FBS buffer solution. In both the cases, there is a significant enhancement of release time, and hence the rate of drug release can only be due to the replacement of binding COO- by phosphate anions. Similarly, on varying the concentration of hydrophilic Mg/Al-prava LDHs nanohybrid concentration from 0.2 to 0.3 mg/mL, there is an enhancement of release time, and around 91% pravastatin has been released at 10 h. Further, Mg/Al-fluva LDH shows very slow release response at pH 4.5 HCl buffer solution, suggesting the stability of hydrophobic nanohybrid particles in acidic environment (stomach). Therefore, the above results suggest that the strength of ionic interaction between anions and cations concentration and the hydrophobic nature of nanohybrid particles control the release time and hence the rate kinetics. Again, the hydrophobic nature in an acidic (pH 4.5) environment of Mg/Al-fluva LDH

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Figure 12. Dissolution-diffusion kinetic models plot of (a) first-order model of Mg/Al-prava LDHs, (b) first-order model of Mg/Al-fluva LDHs, (c) parabolic diffusion model of Mg/Al-prava LDHs, (d) parabolic diffusion model of Mg/Al-fluva LDHs, (e) modified Freundlich model of Mg/Al-prava LDHs and (f) modified Freundlich model of Mg/Al-fluva LDHs.

nanohybrids could benefit therapeutic treatment. In addition, the initial fast release at pH 7.4 quickly allows establishment of a therapeutic dose, and the subsequent sustained release allows maintenance of this dose over a long period of time. In contrast with statin-LDHs, the pure drug and physically mixed powder exhibit a burst release within the first few min, followed by a steady decline of statin drugs in solution. To understand the Mg/Al-fluva LDHs release behavior (0.2 mg/ mL), the resultant powder was recovered after the release test (14 h) and washed with M.Q water, and further characterized through XRD and FTIR (Supporting Information S3). The amorphous XRD pattern was observed and there are no other Mg/Al-fluva LDH peaks, suggesting the detonation of layers after drug release. The FTIR spectra show broadband centered at 1070 cm-1 and is assigned to δ (P-OH) and 03 (P-O).40 Further, the diminishing of bands, a characteristic of drug suggests that the release of fluvastatin from Mg/Al-fluva nanohybrids is mainly via exchange with phosphate anions and simultaneous dissolution of the LDH layers.

Release Kinetics. To understand the drug release mechanism from statin-LDHs nanohybrid systems, three types of dissolution-diffusion kinetic models16–18 were applied and are stated as follows: 1. First-order model demonstrates the release system where dissolution rate depends on the amount of drug present in the nanohybrids and can be expressed as

log(Xt/X0) ) -K1t

(1)

2. Parabolic diffusion model express the diffusion controlled phenomena of drug from clay nanohybrids and mathematically written as

(1 - Xt /X0)/t ) Kdt-0.5 + a

(2)

3. Modified Freundlich model suggested the fitting of experimental data on ion exchange and diffusion-controlled process with the following equation:

(X0-Xt)/X0)Kmtb

(3)

In equations 1-3, X0 and Xt are the amount of drugs in the LDHs matrix at release time 0 and t, respectively, K is the rate



TABLE 3: Linear Correlation Coefficients (R2) Value of the Dissolution-Diffusion Kinetic Models Applied to Different Statin Drug Release from Mg/Al LDHs at pH 7.4 Phosphate Buffer Solution Mg/Al-prava kinetic models first-order model parabolic diffusion model modified Freundlich model

Mg/Al-fluva

0.2 mg/mL

0.3 mg/mL

0.2 mg/mL

0.4 mg/mL

0.55 mg/mL

0.96 0.99 0.79

0.85 0.97 0.95

0.92 0.42 0.98

0.97 0.95 0.99

0.97 0.98 0.99

constant, and a and b are the constants whose chemical significance is not clearly understood.41 The release profiles are fitted to the above equations and the corresponding linear correlation coefficient (R2) is evaluated. Generally in LDH systems the drug release rate is controlled either by dissolution of LDH particles or by diffusion through the LDH particles. Therefore, only the first-order model is not suitable to explain the whole release of statin drugs from statin-LDH nanohybrid systems, which is reflected in Figure 12a and b, that do not form a straight line for all concentrations in a particular system (R2 ) 0.85-0.96 for Mg/Al-prava LDHs and 0.92-0.97 for Mg/Al-fluva LDHs). However, the kinetic models are best fitted with the parabolic diffusion and modified Freundlich model for Mg/Al-prava and Mg/Al-fluva LDHs, respectively (Figure 12c-f). The parabolic diffusion model describes the intraparticle diffusion, while the Freundlich model describes heterogeneous diffusion from the flat surfaces via ion exchange.16 For hydrophilic Mg/Al-prava LDH nanohybrid particles, linearity was obtained for all concentrations in the parabolic diffusion model (R2 ) 0.97-0.99), compared with that of the first-order model (R2 ) 0.85-0.96) and Freundlich model (R2 ) 0.79-0.95). But for hydrophobic Mg/Al-fluva LDH nanohybrid particles, the Freundlich model was more satisfactory (R2 ) 0.98-0.999) than the first-order model (R2 ) 0.92-0.97) and parabolic diffusion model (0.42-0.98). These simulation results suggest that the release for both the systems is diffusion-controlled because, (i) for hydrophilic Mg/Al-prava LDHs, most pravastatin anions diffuse into the medium solution through intraparticle diffusion via anion exchange and, (ii) in hydrophobic Mg/Al-fluva LDHs, diffusion of fluvastatin occurs through heterogeneous diffusion via anion exchange. In both the cases diffusion takes place, together with the leaking of metal ions and the dissolution of brucite layers. But the kinetic model simulation data in Table 3 suggest the difference in the release of Mg/Al-prava and Mg/ Al-fluva LDHs. This may be due to the formation of monolayers (smaller dimension) in Mg/Al-fluva LDHs than Mg/ Al-prava LDHs, which is formed as multilayers as discussed earlier. Due to smaller dimension and a hydrophobic nature, it provides a shorter diffusion path and hence controls the release rate. In addition, our finding differentiates the diffusion phenomena of LDHs when they transform from hydrophilic to hydrophobic. Conclusion New nanohybrid materials were developed by intercalating anticardiovascular drugs in layered double hydroxide by using the coprecipitation method. XRD patterns suggested the expansion of interlayer spacing from 7.6 Å for Mg/Al-CO3 LDHs to 14.9 Å for Mg/Al-prava and 15.5 Å for Mg/Al-fluva LDHs. The electronic interaction between drugs and brucite layers was confirmed through FTIR. The hydrophobic monolayer is seen through AFM image analysis for Mg/Al-fluva LDHs. On the basis of the elemental analysis results, the novel hydrophobic drug-LDH monolayers have shown to provide maximum drug loading. The physical and solution stability results suggested

that the statin drugs are much more stable within brucite layers. In addition, transformation of hydrophobic to hydrophilic state in buffer solution often induces the release of drug ions in a significantly controlled manner. The rate kinetics by using the dissolution-diffusion kinetic models indicated that the diffusion behavior of hydrophobic Mg/Al-fluva nanohybrid particles is different than hydrophilic Mg/Al-prava LDHs, and it mainly involves the heterogeneous diffusion of loaded fluvastatin. Overall, the results suggested the potential applicability of LDHs for making statin drugs pure and stable, and as a sustained release carrier. Acknowledgment. We gratefully acknowledge Nano Mission of Department of Science and Technology, Government of India, for financial support and SAIF and CRNTS, IIT, Bombay, for providing instrumental facilities. Supporting Information Available: Additional FTIR spectra, UV-vis spectra, and XRD patterns. This material is available free of charge via the Internet at http://pubs.acs.org. References and Notes (1) Han, J. B.; Lu, J.; Wei, M.; Wang, Z. L.; Duan, X. Chem. Commun. 2008, 5188. (2) Panda, H. S.; Srivastava, R.; Bahadur, D. J. Phys. Chem. C 2009, 113, 9560. (3) Li, F.; Liu, L.; Evans, D. G.; Duan, X. Chem. Mater. 2004, 16, 1597. (4) Khan, A. I.; Lei, L.; Norquist, A. J.; O’Hare, D. Chem. Commun. 2001, 2342. (5) Desigaux, L.; Belkacem, M. B.; Richard, P.; Cellier, J.; Leone, P.; Leroux, F.; Taviot-Gueho, C.; Pitard, B. Nano Lett. 2006, 6, 199. (6) Zhao, H.; Vance, G. V. J. Chem. Soc., Dalton Trans. 1997, 1961. (7) Mohanambe, L.; Vasudevan, S. Inorg. Chem. 2005, 44, 2128. (8) Choy, J.; Junga, J.; Oh, J.; Park, M.; Jeong, J.; Kang, Y.; Han, O. Biomaterials 2004, 25, 3059. (9) Sankyo. U.S. Patent 4346227, 1981. (10) Istvan, E. S.; Deisenhofer, J. Science 2001, 292, 1160. (11) Taylor, L. S.; Zografi, G. Pharm. Res. 1997, 14, 1691. (12) Evans, M.; Rees, A. Drug Safety 2002, 25, 649. (13) Kathawala, F. G. U.S. Patent 4739073, 1988. (14) Sarr, F. S.; Guillaume, Y. C.; Andru, C. J. Pharm. Biomed. Anal. 2008, 47, 651. (15) Wei, M.; Pu, M.; Guo, J.; Han, J.; Li, F.; He, J.; Evans, D. G.; Duan, X. Chem. Mater. 2008, 20, 5169. (16) Yang, J. H.; Han, Y. S.; Park, M.; Park, T.; Hwang, S. J.; Choy, J. H. Chem. Mater. 2007, 19, 2679. (17) Li, Z. H. Langmuir 1999, 15, 6438. (18) Kodama, T.; Harada, Y.; Ueda, M.; Shimizu, K.; Shuto, K.; Komarneni, S. Langmuir 2001, 17, 4881. (19) Panda, H. S.; Srivastava, R.; Bahadur, D. J. Nanosci. Nanotechnol. 2008, 8, 4218. (20) Takagi, K.; Shichi, T.; Usami, H.; Sawaki, Y. J. Am. Chem. Soc. 1993, 115, 4339. (21) Xu, Z. P.; Kurniawan, N. D.; Bartlett, P. F.; Lu, G. Q. Chem.sEur. J. 2007, 13, 2824. (22) Papageorgiou, G. N.; Papadimitriou, S.; Karavas, E.; Georgarakis, E.; Docoslis, A.; Bikiaris, D. Curr. Drug DeliVery 2009, 6, 101. (23) Hu, G.; Wang, N.; O’Hare, D.; Davis, J. Chem. Commun. 2006, 287. (24) Panda, H. S.; Srivastava, R.; Bahadur, D. Mater. Res. Bull. 2008, 43, 1448. (25) Karavas, E.; Georgarakis, E.; Bikiaris, D.; Thomas, T.; Katsos, V.; Xenakis, A. Progr. Colloid Polym. Sci, 2001, 118, 149. (26) Yoshioka, S.; Aso, Y. J. Pharm. Sci. 2007, 96, 960.

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JP905440E

In-Vitro Release Kinetics and Stability of Anticardiovascular Drugs

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