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Sparingly water-soluble drugs such as candesartan cilexetil offer challenges in developing a drug product with adequate bioavailability. The objective of the present study was to develop a solid oral dosage form of candesartan cilexetil incorporating drug nanoparticles to increase its saturation solubility and dissolution velocity for enhancing bioavailability while reducing variability. Candesartan cilexetil nanoparticles were prepared using a wet bead milling technique. The milled nanosuspension was converted into solid intermediate using a spray drying process. The nanosuspensions were characterized for particle size before and after spray drying. The saturation solubility and dissolution characteristics of the nanoparticle formulation were investigated and compared with commercial candesartan cilexetil formulation to ascertain impact of particle size on drug dissolution. The drug nanoparticles were evaluated for solid-state transitions before and after milling using differential scanning calorimetry (DSC) and powder X-ray diffraction (PXRD). There was no solid-state transition upon milling indicating that crystallinity of drug was maintained. The rate and extent of drug dissolution in physiologically relevant dissolution medium for tablet formulation incorporating drug nanoparticles was significantly higher as compared to commercial tablet formulation. Systemic exposure studies in rats indicated a significant increase in the rate and extent of drug absorption. The pharmacokinetic results of nanoparticle formulation showed a 2.51 fold increase in area under the curve (AUC0-t) and 1.77 fold increase in maximum concentration (Cmax) and significant reduction in the time required (1.81 hours to 1.06 hours) to reach maximum plasma concentration (Tmax) when compared to the micronized formulation. The manufacturing process used is relatively simple and scalable indicating general applicability of the approach used herein to enhance dissolution and bioavailability of sparingly soluble compounds where bioavailability is either solubility or dissolution limited.
KEY WORDS: Nanoparticles; Poorly soluble drugs; Particle size; candesartan cilexetil
Candesartan cilexetil is an esterified prodrug of candesartan, a non-peptide angiotensin II type 1 (AT1) receptor antagonist used in the treatment of hypertension. Candesartan cilexetil is rapidly and completely bioactivated by ester hydrolysis during absorption form gastro intestinal tract to candesartan (figure 1). The major drawback in the therapeutic application and efficacy of candesartan cilexetil as an oral dosage form is its very low aqueous solubility (0.0003 mg/mL). Low solubility of candesartan cilexetil across the physiological pH range is reported to result in incomplete absorption from the gastrointestinal (GI) tract. Based on its solubility across physiologically relevant pH conditions and absorption characteristics, candesartan cilexetil is classified in the Biopharmaceutics Classification System (BCS) as a class II drug.
An approach that is commonly used to increase dissolution rate and impact saturation solubility of poorly soluble compounds like candesartan cilexetil is to formulate it as nanometer-sized particles, particles usually less than 1 µm in diameter. For example, when the particle size of the drug is reduced from 8 µm to 200 nm there is 40-fold increase in the surface area to volume ratio. This increase in surface area can provide substantial increase in the dissolution rate if the formulation disperses into discrete particles (Liversidge and Cundy, 1995).
Nanocrystalline dispersion or nanosuspension comprise of drug, water and stabilizers. Stabilizers used to aid the dispersion of particles are either polymers and /or surfactants. To be effective the stabilizers must be capable of wetting the drug crystals and providing steric and/or ionic barrier. In the absence of appropriate stabilizers, the high surface energy of the nanometer-sized particles would tend to agglomerate or aggregate drug crystals. Too little of stabilizers can cause aggregation and too high can cause Ostwald ripening on storage. Concentration of polymeric stabilizers may range from 1 - 10% w/v and concentration of surfactants is generally < 1 % w/v (Liversidge et al., 2003).
Nanocrystalline dispersions or nanosuspensions at high concentrations (> 20 % w/v) are suitable for granulating or spray drying can be prepared using the Bead mill. The manufacturing can be described as a simple procedure comprising of; attrition media, suspension and agitation. The extent of size reduction by wet bead milling is governed by amount of grinding energy which is determined by the intrinsic hardness of the drug, grinding media, and milling power. Using this process nanocrystalline dispersion at concentrations up to 40% w/v can be prepared easily (Liversidge et al., 2003).
Based on the understanding that reduction in particle size can enhance dissolution rate, we wished to evaluate the affect of particle size on solubility, dissolution and bioavailability enhancement of candesartan. In this study, media milling was evaluated for production of candesartan cilexetil nanosuspension. The drug nanoparticles were evaluated for dissolution and solubility behavior to confirm theoretical enhancement predictions. Solid-state transitions were also evaluated before and after particle size reduction. Conversion of nanosuspension into solid intermediate was achieved by spray drying using mannitol as bulking agent. The recovery of particles from the powder was accessed following spray drying. Use of water-soluble career prevents particle agglomeration in powder state and further increases dissolution characteristics (Hecq et al., 2006) that can enhance bioavailability.
2. MATERIALS AND METHODS
Candesartan cilexetil was procured from Dr. Reddy's laboratories (Hyderabad, India). Mannitol was purchased from Roquette (Roquette Freres, Lestrem, France). Hydroxypropyl methylcellulose (HPMC, 6cps) was purchased from Colorcon (Mumbai, India). Sodium lauryl sulphate (SLS) was purchased from Qualigen chemicals (Delhi, India). Polyvinyl pyrolidone [PVP (K-30)] and Poloxamer 188 were procured from BASF (Germany) while Crospovidone (Polyplasdone) and Sodium starch glycolate (SSG) were obtained from ISP (USP) and Grain processing corporation (USA), respectively. All other chemicals were of analytical reagent grade.
2.2 Preparation of Nanosuspension on a Laboratory Scale
A glass apparatus mimicking the media-milling machine was fabricated in-house (figure 2). The apparatus comprised of a double walled jacketed cylinder having a volume of 250 mL. In this process the drug substance was dispersed uniformly in an aqueous medium containing dissolved stabilizers in the milling chamber using a Heidolph mixer (Model: RZR2051 Control, Rose Scientific Ltd., Alberta, Canada) operating at 500 rpm. The solid content of the suspension was 5 or 10% w/w. The milling media comprising of 0.2 mm yttrium stabilized zirconium beads and additional water were added into the milling chamber. The total volume of slurry (drug substance + stabilizer + water) was 100 mL. The batch size for these development trials was 100 mL and the temperature of the suspension was maintained at 20 - 25°C during milling by circulating cold water. The milling media was agitated using a Heidolph mixer operating at a speed of 1600 rpm. The milling time was fixed at 6 hours. Following milling, the beads were filtered, and the nanosuspension was collected and stored at refrigerated conditions (2 - 8°C) until further use. Generally, the choice and concentrations of stabilizer not only depends on its ability to facilitate particle size reduction but also its ability to produce suspensions with acceptable physical stability. Typically, a combination of steric and electrostatic stabilization is most effective for Nanosuspension stability (Marti-Mestres and Nielloud, 2000). The composition of surfactant and polymeric stabilizers used for production of candesartan cilexetil nanosuspension is summarized in Table 1. The stability of the nanosuspension was evaluated upon storage at refrigerated and 40°C temperature conditions for a period up to one week
2.3 Particle Size Analysis
Particle size and size distribution of the suspension before, during (at different milling times) and following milling were determined using a laser diffraction (LD) method, with a wet sampling system (Mastersizer S, Malvern Instruments, UK). The particle diameters reported were calculated using volume distribution. A refractive index of 1.5 was used for measurements. The particle size obtained with the different stabilizer compositions and their physical stability upon storage is summarized in Table 2. Based on particle size distribution obtained following milling and suspension stability a formula composition comprising of PVP (K-30) as the primary stabilizer and SLS as the secondary stabilizer (Formulation NS-9) were chosen for scale-up trials using the media-milling machine.
2.4 Scale-Up of Manufacturing Process for Production of Nanosuspension
The selected composition (Formula NS-9) was scaled-up using a bead mill (Model: Lab Star 1, Netzsch Mill, Netzsch, Germany). The milling chamber was charged with the milling or grinding media. The milling media comprised of 0.2 mm yttrium stabilized zirconium beads. The milling operation was performed in a re-circulation mode with the suspension fed at a rate of 150 mL per minute. The mill and pump speeds were operated at 2500 and 120 rpm, respectively. The suspension flowed axially through the milling chamber where the shear forces generated during impaction of the milling media with the drug provided the energy input to fracture the drug crystals into nanometer-sized particles. The temperature inside the milling chamber was controlled by circulating cooling water through the outer jacket. After milling, the suspension was collected and stored at a temperature below 25°C until further processing.
2.5 Conversion of Nanosuspensions into Solid Intermediate by Spray Drying Processes
The spray drying process (Model: LU 222, Labultima) was used to convert nanosupension into dried-powder for further processing. The nanoparticle or microparticle suspensions were spray dried at a spray rate of 15 mL / minute using 0.7mm fluid spray nozzle. The drying temperature was set at 120° C. Spray airflow was set at 1500 L/h and drying airflow was set at 50Nm3/h. The composition of nanosuspension used for spray drying incorporating mannitol as bulking agent is detailed in Table 3. The amount of mannitol used was 76.36 % (w/w) with respect to candesartan cilexetil content prior to the spray drying operation. The particle recovery from granules following spray drying is summarized in Table 4.
2.6 Solid-state Characterization
2.6.1 Differential Scanning Calorimetry (DSC)
Thermal properties of powder were investigated using a Perkin-Elmer DSC-7 differential scanning calorimeter / TAC-7 thermal analysis controller with an intracooler-2 cooling system (Perkin- Elmer Instruments, USA). About 3 to 5 mg of product was placed in perforated aluminum sealed 50 µL pans and the heat runs for each sample was set from 40 to 200°C at 5°C/minute, under an inert environment using nitrogen. The apparatus was calibrated using indium/cyclohexane.
2.6.2 Powder X-ray diffraction (PXRD)
PXRD diffractograms of un-milled and milled candesartan cilexetil formulations were recorded using a Panalytical Xpert Pro Diffractometer (PANalytical, The Netherlands) with a Cu line as the source of radiation. Standard runs using a 40 kV voltage, a 40mA current and a scanning rate of 0.02° minâˆ’1 over a 2Î¸ range of 3 - 40° were used.
2.7 Saturation Solubility
Saturation solubility evaluations were carried out in buffer media at different pH conditions using a shake flask method. In this method excess amount (100 mg/mL) of drug substance ("as is" and dried suspension containing microparticles or nanoparticles) was added to 25 mL of each buffer maintained at 37°C and shaken for a period up to 24 hours. The samples were filtered using 0.10µm Millex-VV PDVF filters (Millipore Corporation, USA) prior to analysis. Samples were diluted and concentrations were determined using an HPLC method described earlier.
2.8 Tablet Preparation
The drug was dissolved in a mixture of the isopropyl alcohol and dichloromethane in a suitable ratio, and layered on to the microcrystalline cellulose (Avicel 101). Then the drug layered micro crystalline cellulose is mixed with other intragranular components and granulate with media containing dissolved meglumine and SLS. Add extragranular materials, microcrystalline cellulose (Avicel 102), Sodium bicarbonate, crosscarmellose sodium (Ac-Di-Sol) and Sodium Stearyl fumerate blend in a double cone blender, for 10 minutes. The composition of tablet blend is summarized in Table 6. The blend was compressed into tablets using a mini compression machine (Model: Mini-II B, Rimek, India) fitted with 9.0 mm round, standard concave type D tooling. The physical properties of tablets, hardness, friability and disintegration time, were measured and are summarized in Table 7. The tablet hardness was measured using a hardness tester (Model: 8M, Dr Schleuniger Pharmatron, USA). Each hardness value reported is an average of ten measurements. The disintegration time was measured in purified water at 37 ± 0.5°C, using a disintegration tester (Model: ED2L, Electrolab, India), using sintered disks. The disintegration time reported is an average of six measurements. Tablet friability was calculated as the percentage weight loss of 20 tablets after 100 rotations using a friabilator (Model: EF2, Electrolab, India). The tablets were then with 10%w/w solution of Eudragit L100 in 70:30 Isopropyl Alcohol and Purified Water to 6 % weight build up.
2.9 Dissolution Studies
Dissolution rates of candesartan cilexetil were determined according to a method described by Nogami et al. (1969) with minor modifications. A USP dissolution apparatus (Model: DISSO 2000, Labindia, India) type II (paddle method) with a paddle operating at 50 rpm was used for dissolution studies. All dissolution tests were carried out on an equivalent of 16 mg of candesartan cilexetil (in suspension and powder state) and tablets. Dissolution studies on dispersions containing unmicronized, micronized and nanosized candesartan cilexetil were conducted in 0.1 N HCl (pH 1.2), acetate buffer (pH 4.5), phosphate buffer (pH 6.8) and purified water. Phosphate buffer (pH 6.8) was chosen as the dissolution medium based on its ability to discriminate the impact of changes in composition and particle size on dissolution rate. The volume and temperature of the dissolution medium were 500 mL and 37°C, respectively. Samples were withdrawn at predetermined time intervals, filtered in-line and assayed using an HPLC method (Waters Alliance HPLC system, USA). Chromatographic separation was accomplished using an Inertsil ODS-3, C18, 250 x 4.6mm 5Î¼m stainless steel column (Agilent Technologies, USA). The mobile phase consisted of a mixture of buffer (0.02M monobasic potassium phosphate), acetonitrile and triethylamine in the ratio of 40:60:0.2, pH adjusted to 6.0 with phosphoric acid. The mobile phase was pumped isocratically at a flow rate of 2.0 mL/minute during analysis and was maintained at a column temperature of 25°C. The amount of drug dissolved at each sampling time point was monitored at a UV wavelength of 254 nm.
2.10 In vivo Pharmacokinetic Study:
Male Wistar rats weighing 230 ± 20 g were fasted for 12 - 14 hours prior to dosing but allowed free access to water. Eight rats were assigned to two groups and they received suspensions comprising either spray dried drug micorparticles or drug nanoparticles, respectively at a dose level of 10 mg/kg. Rodent feed was returned 3 hours post dosing. Blood samples were collected from the orbital sinus at pre-determined intervals (0, 0.5, 1, 3, 5, 8, 10 and 24 hours) post-dosing. Plasma was separated immediately by centrifugation (13,000RPM for 2 minutes at 4°C) and stored in polypropylene vials below -10°C until analysis. The animal experiments were carried out in accordance with the guidelines provided by the Institutional Animal Care and Ethics Committee.
2.10.1 Plasma Sample Analysis:
Plasma (100 µL) was mixed with 2 mL of extraction solvent (1:1 ethyl acetate and dichloromethane) containing 100 ng/mL of internal standard and mixed for 3 minutes. It was then centrifuged at 3,000 rpm for 3 minutes. The organic supernatant (1.8 mL) was removed and evaporated to dryness under nitrogen in a clean test tube. The residue was reconstituted with 150 µL of mobile phase and an aliquot of 20 µL was injected into the LC-mass spectrometer with Agilent 1200 series model HPLC pump plus auto-sampler (Agilent, Waldbronn, Germany). Separation was achieved on a C18 column (Inertsil ODS-3, 100 Ã- 4.6 mm) using a mobile phase composed of acetonitrile and 10 mM ammonium acetate in the ratio of 90:10 at a flow rate of 1 mL/minute. MS (AB Sciex Ontario, Canada). Detection was performed using an electro spray ionization source in the negative mode and the following conditions: ion spray voltage 4500 V, dry gas temperature 600°C, nebulizer gas 40 psi, auxiliary gas 50 psi. MRM monitoring was done at m/z 609.3/ 521.10 and 439.2/ 309.1 for detection of candesartan cilexitil and candesartan, respectively. Analytical data were acquired by Analyst 1.4.1 software (AB Sciex Ontario, Canada). Standard curves were obtained from linear square regression analysis of drug/internal standard peak area ratio as a function of plasma concentration versus time data was analyzed, and the oral pharmacokinetic data were developed.
3. RESULTS AND DISCUSSION
3.1 Stabilizer screening for Production of Physically Stable Nanosuspensions
The compositions evaluated with different polymeric stabilizers (NS-7 to NS-9) showed differences in particle size distribution (Table 2). The median particle size (d50), for the milled suspensions for the three formula compositions (NS-7, NS-8 and NS-9) were, i.e. 0.128, 0.154, and 0.127 µm, respectively. The d90, which is indicative of large particles or aggregates, were 0.271, 0.693 and 0.269µm. Based on the particle size distribution obtained for the three formulae upon milling for a predetermined time interval, PVP (K-30) was selected as the primary stabilizer for steric stabilization of candesartan cilexetil nanoparticles. The better stabilization with this polymer may be attributed to its adsorption characteristics based on affinity as compared to Poloxamer 188. In addition, this composition provided finer particles in comparison to the other stabilizers investigated (Table 2). Although poloxamer 188 has been shown to be successful in stabilization of nanosuspensions, its use has been limited due to its low melting point that may pose processing problems during conversion to solid intermediates either using a fluid bed process or spray drying. Similar observation was made by Hecq et al. (2006), when comparing HPMC with polyvinyl alcohol, poloxamer 188 and acacia for stabilizing UCB-35440-3, a sparingly water soluble weak base that has a large dose.
The effect of SLS concentration (0.75 and 1.00 %w/v) on particle size distribution of nanosuspension indicated that the d90 values decreased with increasing SLS concentration and were 0.655 and 0.580µm at 0.75 and 1.00% (w/v) SLS concentrations, respectively. A larger fraction of finer particles (d90) was obtained when SLS concentration was 1.00 % w/v.
The effect of PVP (K-30) concentrations (1 & 2 % w/v) on particle size distribution of nanosuspension indicated that there was no effect on the median particle size. The median particle size (d50), for compositions with 1% and 2% w/v PVP (K-30) were 0.130 and 0.127 µm, respectively. Similar trend was also obtained for large particles (d90). Surprisingly low concentration of polymeric stabilizer was required to form stable nanosuspensions as reported by other investigators (Shah et al., 2006). This could be attributed to the fact that higher concentration of polymers would result in increased viscosity that could hinder particle attrition at same milling energy (Hecq et al., 2005). A formulation containing 2% w/v PVP (K-30) was selected as it provided the right balance for stabilization without affecting viscosity required for effective milling.
The physical stability of nanosuspension was evaluated following storage in refrigerated (2 - 8°C) and at 40°C for a period up to 1 week. The particle size distribution of nanosuspension before and after storage for compositions is detailed in Table 2. There was no significant change in particle size distribution of nanosuspensions incorporating PVP (K-30) and HPMC as the primary stabilizer. However, in Poloxamer stabilized nanosuspensions, an increase in particle size was observed, which is more pronounced at higher temperature (40°C). This could be attributed to the fact that heterogeneity in particle size distribution could result due to difference in saturation solubility based on PSD. These differences could have resulted in Ostwald ripening leading to crystal growth with concomitant increase in particle size (Schott, 1955; Van den Mooter et al., 2001). Ostwald ripening did not occur in suspension containing HPMC and PVP (K-30) as the primary stabilizers for two reasons. In the first instance, the drug candidate is poorly soluble thus leading to insignificant changes in the dissolved concentration during preparation and storage. Secondly, the particles are relatively homogenous in size thus avoiding larger differences in the saturation solubility between differently sized crystals (Nystrom, 1998). The effect of suspension concentration or solids content on particle size distribution of nanosuspension at identical processing conditions indicated that the more concentrated suspension showed smaller particle size distribution (Table 2). The smaller particle size obtained with concentrated nanosuspensions may be attributed to increased collision of particles at higher concentrations during processing (Krause and Muller, 2001). Alternatively, smaller particles can also be obtained by increasing the applied total disintegration energy (Krause and Muller, 2001). The nanosuspension obtained with different solids content showed good physical stability upon storage for a week at 40oC and refrigerated conditions. The particle size distribution before and after storage is shown in Table 2. The primary stabilizer (PVP K-30) and secondary stabilizer (SLS) used at concentrations of 2% and 1.00%, respectively, were able to maintain the physical stability of the nanosuspension even at higher drug concentration. This stabilizer composition was selected for scale-up using the agitator bead mill. Physical stability of nanosuspensions upon storage for a period up to 3 years has been reported (Peters, 1996), this could be explained based on absence of Ostwald ripening (Jacobs et al., 2000). Long-term stability of nanosuspensions were not studied in the present case since the prepared nanosuspensions are only intermediate products and was subsequently converted to solid intermediates for solid dosage forms.
3.2 Preparation of Nanosuspension Using Media Milling Process
Particle size reduction kinetics using wet bead milling process is shown in Figure 3. As reported by other investigators (Lee, 2003), the particle size was found to decrease with increase in milling time. Particle size reduction generally depends on the fragmentation of drug crystals due to impaction with the milling media (Ploehn and Russel, 1990). Following 3 hours of bead milling, the particle size reached a plateau value (281 nm), and a Gaussian particle size distribution was observed. The final particle size obtained was dependent on the stabilizers/drug ratio. A composition comprising of PVP (K-30) and SLS at concentrations of 2.0% and 1.0% (w/w), respectively, relative to candesartan cilexetil content (16 mg) were found optimal in stabilizing the drug nanoparticles (130 nm @ d50). The results from these studies and others indicate that wet bead milling is a versatile technology capable of producing drug nanosuspension at high concentrations (> 10% solids content) for poorly soluble drugs (Liversidge et al., 1996; Liversidge et al., 2003).
Stability of suspension prepared using the bead milling process was evaluated to reconfirm the stabilizers selected. The nanosuspension with the selected formula showed good physical stability following storage for a period up to 1 week at accelerated temperature (40°C) and refrigerated conditions (2 - 8°C). A physically stable nanosuspension is obtained when the weight ratio of drug to stabilizer was in the range of 20:1 to 2:1 (Liversidge et al., 2003). Liversidge et al (2003) reported that for a poorly soluble drug such as naproxen, when the particle size was reduced from 20 - 30 µm to the nanometer range (d50 - 270 nm) following wet milling using, the ratio of drug to the stabilizer, PVP (K-15), was 5:3. The naproxen nanoparticles did not aggregate and its physical and chemical stability was maintained for a period up to 4 weeks at 4°C (Liversidge, 1995; Liversidge and Cundy, 1995). In another study, nanosuspension of paclitaxel containing 2% w/v paclitaxel and 1% w/v pluronic F127 as stabilizer and prepared using wet bead milling showed that relatively larger amount of higher molecular weight polymeric stabilizer was needed for effective particle size reduction and to maintain physical stability (Liversidge et al., 1996; Liversidge et al., 2003; Liversidge and Cundy, 1995).
3.3 Spray Drying Processes for Conversion of Nanosuspensions into Solid Intermediate.
Drying nanosuspensions using conventional processes, such as spray drying and freeze-drying has been utilized extensively for further processing into various dosage forms (Lee, 2003; Hecq et al., 2005; Hecq et al., 2006; Lee and Cheng, 2006). The solid intermediate (powder) should be able to quickly disperse into nanometer-sized drug crystals upon dissolution. Otherwise, the surface area enhancement will not be fully utilized to achieve the desired bioavailability. Therefore, a key property of dried nanoparticles is their recovery, i.e., whether they can reconstitute to nanometer-sized particles when dispersed in an aqueous medium or physiologically relevant media. Table 4 shows particle size distribution from the solid intermediates following re-dispersion in an aqueous medium. The solid intermediates obtained showed good recovery, indicating mannitol chosen as water-soluble carrier for spray drying prevented agglomeration. (Hecq et al., 2005; Hecq et al., 2006). Mannitol, due to high aqueous solubility offers the advantage of creating a highly hydrophilic environment around candesartan cilexetil nanoparticles thus mitigating agglomeration upon dispersion.
The solid state transitions of candesartan cilexetil following milling studies using DSC and X-ray diffraction techniques indicated that drug crystallinity was maintained and no significant solid-state transitions were observed. The DSC thermogram of candesartan cilexetil; Placebo physical mix (SLS+PVP K-30+Mannitol); physical mix (Drug+SLS+PVP K-30+Mannitol); spray dried nanoparticles are shown in Figure 4. The x-ray diffractograms for spray dried nanoparticles, physical mix (drug + SLS + PVP K-30 + Mannitol), Placebo physical mix (SLS + PVP K-30 + Mannitol) and unmilled drug is shown in Figure 5. There are instances where solid-state transitions has been observed when processing is not done in a controlled environment I (Byrn et al., 1995). The absence of any solid-state transitions in these studies may be attributed to the fact that milling was performed under controlled temperature conditions and the aqueous phase effectively dissipated the heat generated during processing (Liversidge et al., 2003).
After spray drying, the DSC and X-ray thermograms show no change in crystallinity when compared with the unmilled physical mix of the formulation. The extra peaks observed in the spray-dried powder are attributed to the presence of mannitol that has been used as a diluent to aid processing.
The scanning electron micrographs from commercial candesartan cilexetil and spray dried nanosuspension, as shown in figure 6, illustrate the recrystallization of water soluble carrier (mannitol) around the nanoparticles during the water-removal operation, thus creating a highly hydrophilic environment around candesartan cilexetil nanoparticles and preventing particle interaction. (Hecq et al., 2005)
3.4 Solubility Studies
Saturation solubility of solid intermediates containing drug nanoparticles was evaluated in 0.1N HCl, acetate buffer pH 4.5, Phosphate buffer pH 6.8 and water at physiological temperature (37°C) and compared with "as is" and jet-milled drug. The results from these studies summarized in Table 5 indicated a decrease in drug solubility with increasing pH. This observation was found in agreement with observations made by Carlson et al. (1983). The higher solubility in acidic pH (0.1N HCl) condition as compared to phosphate buffer pH 6.8 or water could be attributed to the weakly basic nature of candesartan cilexetil. The saturation solubility of spray dried drug nanoparticles was significantly higher than jet-milled microparticles at all pH conditions. These results clearly demonstrate that reduction in particle size to sub-micron or nanometer range affects saturation solubility that may result in enhancement in dissolution velocity and concomitantly higher bioavailability (Muller et al., 2001; Liversiedge et al., 2003).
3.5 Compression into Tablets
The physical properties of the tablets are summarized in Table 7. Three compositions with different disintegrants were compressed into tablets with different hardness, 9 - 11 Kp. The friability of all the tablets was < 0.1% indicating good mechanical strength. The disintegration results indicated that among the different disintegrants, crospovidone showed the fastest disintegration (10 minutes) and cornstarch the slowest disintegration (>20 minutes). The superior disintegrant property of crospovidone with candesartan cilexetil may be attributed to its nonionic nature. It has been demonstrated that crospovidone, a nonionic disintegrant shows better disintegration with cationic drugs. Other disintegrants such as croscarmellose sodium and sodium starch glycolate being anionic can retard disintegration and concomitantly dissolution of cationic drugs (Pharmaceutical Technical Bulletin, ISPCorp.com, 2007), which may impact bioavailability.
3.6 Dissolution Rate Evaluation
The comparative dissolution of suspension containing drug nanoparticles, micronized and un-micronized drug in pH 6.8 phosphate buffer is shown in Figure 7. There was a significant increase in the rate of drug dissolution for nanoparticulate suspension as compared to suspension incorporating micronized and un-micronized drug. The increase in dissolution velocity may be attributed to its smaller particle size and increased surface area (Diaz et al., 1999). The simultaneous increase in saturation solubility and decrease in diffusion distance also leads to increase in dissolution velocity in addition to the surface effect (Muller and Bohn, 1998; Muller et al., 2001).
Composition of tablet formulations incorporating nanoparticle and micronized candesartan cilexetil are shown in Table 6. The dissolution characteristic of these tablet formulations in phosphate buffer (pH 6.8) is shown in Figure 8. The rate and extent of drug dissolution from tablets incorporating candesartan cilexetil nanoparticles was significantly higher (84%) as compared to micronized (32%) and commercial formulation (2.3%) at the end of 60 minutes. However, the drug dissolution from all tablet formulations was incomplete as compared to the drug suspension. This may be attributed to the fact that compression of powder blend would resulted in formation of aggregates or larger particles with reduced surface area that could have impacted dissolution rate.
3.7 In vivo evaluation:
Following per oral intraduodenal administration, the average peak plasma concentration obtained with micronized and nanoparticulate dispersion of candesartan cilexetil were 0.09 ± 0.03 and 0.16 ± 0.10µg /mL (figure 9). The pharmacokinetic results show a 2.51 fold increase in area under the curve (AUC0-t) and 1.77 fold increase in maximum concentration (Cmax) and significant reduction in the time required (1.81 hours to 1.06 hours) to reach maximum plasma concentration for the nanoparticle formulation over the micronized formulation (Table 8). The increase in rate and extent of absorption for the nanoparticle dispersion could be attributed to the increase in the rate and extent of drug dissolution.
The approach of reducing particle size to nanometer range in the presence of stabilizers for enhancing oral bioavailability is attractive approach for BCS II compounds (drugs with dissolution rate limited absorption). The use of wet bead milling technology coupled with spray drying process is a viable approach capable of resolving many of the current issues associated with developing formulation of poorly water soluble drugs. Enhancing dissolution velocity of sparingly soluble compounds generally correlates with faster absorption rates. The faster absorption rates can correlate into better bioavailability, reduction in fed- and fast effects and inter-subject variability with concomitantly improved therapeutic outcome. The same approach can also be extended to other class of BCS (drugs with dissolution and permeation limited absorption) if combined with agents that can enhance permeation.
Table 1: Formula Composition of Candesartan cilexetil Nanosuspensions
Formulation Composition (% w/v)
HPMC (6 cps)
NS-Nanosuspensions manufactured using commercial drug substance;
MS- Microparticulate suspension manufactured using micronized drug substance
*Scaled-up batch manufactured by Netzsch bead mill; other suspensions (NS- 1 to NS- 5)
manufactured were Lab scale batch using in house fabricated apparatus
Table 2: Particle Size (nm) of Nanosuspension Prepared by Media milling.
1 week @ 40°C/75%RH
1 week @ 2-8°C
NS-Nanosuspensions manufactured using commercial drug substance;
* Scaled-up batch manufactured by Netzsch bead mill; other suspensions (NS- 1 to NS- 5)
manufactured were Lab scale batch using in house fabricated apparatus
Table 3. Formula Composition of Solid Intermediate*
Spray Dried Suspension (Nanosuspension)
Water qs to
Water soluble carrier
*Nanosuspension was spray dried after dissolving mannitol to
obtain dry powder
Table 4: Particle Size of Nanoparticles before and after Drying
(Spray Drying Process)
Particle Size Distribution
Formulation NS- 9: Nanoparticle suspension
Formulation MS-1: Microparticle suspension
Table 5: Saturation Solubility of Un-milled, Micronized and Spray Dried
Commercial candesartan cilexetil*
Spray Dried candesartan cilexetil nanoparticles@
0.1 N HCl
Acetate buffer pH 4.5
Phosphate buffer pH 6.8
* Solubility was tested in respective solvents containing PVP (K-30) and SLS, 2 and 1.0% w/v, respectively.
@ nanoparticles were containing PVP (K-30) 2.0% and SLS (1%)
Table 6: Formulation Composition of Tablets
Formula Composition (mg/tablet)
Spray dried formulation*
Sodium Starch Glycolate
Colloidal Silicon Dioxide
* Spray dried powder containing 16 mg candesartan cilexetil, 1.6mg SLS, 3.2 mg PVP (K-30) and 67.20 mg
@ Formulation contains jet-milled candesartan cilexetil and other formulations (NS-1 to NS-5) were containing
Table 7: Physical Properties of Tablets
11.0 ± 0.24
18 ± 2.4
10.6 ± 0.46
9.8 ± 0.42
10 ± 1.2
10.2 ± 0.44
9 ± 1.0
@ Formulation contains jet-milled candesartan cilexetil and other formulations were
Table 8. Pharmacokinetic parameters (mean ± SD, n=4) of candesartan cilexetil
0.31 ± 0.07
0.78 ± 0.22
0.09 ± 0.03
0.16 ± 0.10
1.81 ± 1.13
1.06 ± 0.38
2.01 ± 0.41
3.87 ± 0.94
*Cmax, maximum plasma concentration; Tmax , time to reach Cmax; AUC, area under the
plasma concentration-time curve; T1/2 elimination half-life.
Figure 1: The chemical structure of candesartan cilexetil indicating the site of ester hydrolysis
Figure 2: Fabricated in-house media milling apparatus with double walled jacketed cylinder
Figure 3: Particle size distribution of Formulation NS-9 as a function of milling time
(Diameters d10, d50 and d90 in µm)
(Stabilizer combination: SLS @ 1.00 % and PVP (K-30) @ 2.0% and Drug content @ 10%)
Figure 4: DSC thermograms for; A-Candesartan cilexetil (CC); B-Spray dried nanoformulation (CC + SLS + PVP (K-30) + mannitol); Placebo mix (SLS + PVP (K-30) + mannitol); Physical mix (CC + SLS + PVP (K-30) + mannitol)
Figure 5: PXRD diffractograms for: A-Physical mix (CC + SLS + PVP (K-30) + mannitol); B-Placebo mix (SLS + PVP (K-30) + mannitol); C-Spray dried nanoformulation (CC + SLS + PVP (K-30) + mannitol); D-Candesartan cilexetil (CC)
Figure 6: SEM micrographs of commercial candesartan cilexetil (left) ; spray-dried nanosuspension (right)
(magnification 5000 x Scale=10.0 µm)
Figure 7: Dissolution profile comparison of candesartan cilexetil nanoparticulate dispersion with
micronized candesartan cilexetil dispersion .
Figure 8: Dissolution profile comparison of tablets of nanoparticles with tablet containing
Micronized and commercial candesartan cilexetil
Figure 9: Plasma concentration-time profiles after oral administration of micronized and
nanosized suspension to Wister rats. Each value represents the mean±SD. (n = 4).
A-Candesartan cilexetil (CC); B-Spray dried nanoformulation; C-Placebo mix (SLS + PVP (K-30) + mannitol); D-Physical mix (CC + SLS + PVP (K-30) + mannitol)
A-Physical mix (CC + SLS + PVP (K-30) + mannitol); B-Placebo mix (SLS + PVP (K-30) + mannitol); C-Spray dried nanoformulation; D-Candesartan cilexetil (CC)
SEM micrographs of commercial candesartan cilexetil (left); spray-dried nanosuspension (right) (magnification 5000 x Scale=10.0 µm)