Novel controlled release of antihypertensive drug: preparation, in-vitro and in-vivo evaluation

1 Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmacy, King Abdulaziz University, Jeddah, KSA. 2 Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmacy, Al-Azhar University, Cairo Egypt. 3 Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmacy, Minia University, Minia, Egypt. 4 Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmacy, Beni Suef University, Beni Suef, Egypt.


INTRODUCTION
Coronary heart disease (CHD), one of the major causes of death and disability, attracts much attention from biomedical research community.Calcium antagonists are considered one of the first-line medical therapies for the treatment of coronary artery disease, along with betablockers and long-acting nitrates (Van Kesteren and Withagen, 1998).One of the calcium channel blockers is diltiazem (DLZ) HCl which is widely used in the treatment of angina pectoris and hypertension.Furthermore, its bioavailability has been reported to be about 40% due to the fact that it undergoes extensive first-pass hepatic metabolism.Peak plasma concentrations occur about 3 to 4 h after a dose by mouth.The half-life of DLZ is reported to be about 3 to 5 h (Sweetman, 2007).It is freely soluble in water, optimum oil-buffer partition coefficient (158 at pH7.4), and low molecular weight (450.98)make it an appropriate candidate for being incorporated into controlled-release formulations (Singh and Ahuja, 2002;Prabu et al., 2009;Dalvadi et al., 2011).During the past few decades, various types of oral controlled-release formulations have been developed to improve the clinical efficacy of drugs that have short half lives as well as to increase patient compliance.These formulations are designed to deliver drugs at a predetermined rate over a wide range of conditions and durations of therapeutic treatments (Löbenberg et al., *Corresponding author. E-mail dr_tarek_nour@yahoo.com;Tel.: +966 599828003. 2005;Li et al., 2007;Aamir and Ahmad, 2009;Sabitha et al., 2010;Lekshmi et al., 2012).
Microencapsulation is considered one of the most effective tools in formulating prolonged action dosage forms.It is used to modify drug release by delaying the time during which the drug is available and retard its attack by the gastrointestinal fluids.The preparation of microspheres results by the coating of the individual drug particles with polymeric inert materials, through which the drug would diffuse at a controlled and predictable rate to the surrounding medium.There are several techniques used to produce polymeric microspheres drug delivery systems, which include physicochemical processes such as solvent evaporation method (Li et al., 2007;Lekshmi et al., 2010;Jose et al., 2011) or phase separation method (Das et al., 2005;Lekshmi et al., 2012), mechanical processes such as spray drying (Davis and Illum, 1999), and a non-solvent addition process (Elbagory et al., 2007;Aamir and Ahmad 2009), or by crosslinking with glutaraldehyde using poly(vinyl alcohol)-guar gum interpenetrating network microspheres (Soppimath et al., 2000).The emulsion solvent evaporation method is commonly used for the preparation of controlled release drugs, for example, allopurinol microspheres (Arabi et al., 1996), Zidovudine sustained release (Abu-Izza et al., 1996) and nicardipine hydrochloride microspheres were prepared using acrylic polymers Eudragit RS and L, different concentration of sucrose stearate as a droplet stabilizer, and acetone-methanol mixture as a solvent (Yüksel and Baykara, 1997); and the amphiphilic block copolymers were used to prepare nifedipine microspheres by the solvent evaporation technique (Shelke and Aminabhavi, 2007).Also, the emulsion solvent evaporation method was optimized using ethyl acetate as a dispersing solvent to encapsulate nifedipine and verapamil hydrochloride with cellulose-based polymers (Soppimath et al., 2001).The processing parameters that influence the properties of DLZ microspheres were studied (Bhalerao et al., 2001).Also, the effect of plasticizers on various characteristics of Eudragit microspheres containing glipizide formulated by solvent evaporation method was also evaluated by Sahoo et al. (2011).So, the main purpose of the present work was to develop a controlled drug delivery system of DLZ for peroral administration using biocompatible ethyl cellulose and Eudragit ® RS polymers in order to increase its biological half and to determine the influence of formulation and preparation variables on microsphere characteristics, such as drug incorporation and in-vitro release and subsequently pharmacokinetic parameters.

Differential scanning calorimetry (DSC)
DSC studies were carried out using DSC instrument (DSC-60, having TA60 software, Shimadzu, Koyto, Japan).The instrument is very versatile as far interaction, and compatibility studies at preformulation stage were concerned and used to evaluate melting point, enthalpy changes and glass transition temperatures of drug with excipients and polymers.Approximately, 2 mg of samples were weighed into DSC aluminum pans and were crimped followed by heating under nitrogen flow (20 ml/min) at a heating rate 10°C/min from 40 -400°C.Aluminum pan containing the same quantity of indium was used as a reference.DSC was performed on DLZ, excipients and mixtures of the final formulation.

Fourier transform infrared spectroscopy (FTIR)
FTIR studies are very helpful in the evaluation of drug-polymer interaction studies.The incompatibility between the drug and excipients can be predicted by changes in the functional peaks (characteristic wave numbers).Drug and various polymers were thoroughly mixed with 300 mg of potassium bromide, compressed to a 2 mm semitransparent disk and placed in the light path for 2 min.The FTIR spectra were recorded over the wave length range from 400 to 4000 cm -1 using FTIR spectrometer (Perkin Elmer Spectrum One, Model 16 PC, Germany).

Preparation of DLZ microspheres
DLZ loaded microspheres were prepared by the emulsion solvent evaporation technique previously used by Obeidat and Price (2006) and Sahoo et al. (2011).The drug was dissolved in the polymeric solutions of ethyl cellulose (EC) or Eudragit RS 100 (ED) in dichloromethane at drug to polymer ratio of 1:3 forming the internal phase.The resultant dispersion was added drop wise to light liquid paraffin forming the external phase containing 3% Span 60 as an emulsifying agent during stirring at 800 rpm using a mechanical stirrer (Steady stir Digital, 855 Fisher Scientific, USA).The stirring was continued at room temperature until complete evaporation of the solvent.Liquid paraffin was decanted, and the formed microspheres were filtered off, washed with n-hexane several times to remove the remaining oily phase and then dried at room temperature for 24 h.

Determination of the entrapment efficiency
Fifty milligrams of microspheres was dissolved in dichloromethane and the drug content was determined using high performance liquid chromatography (HPLC) (Agilent 1200 series, equipped with a UV diode array detector, and an automatic sampling system, Germany) at 236 nm.The concentration was calculated using the standard calibration curve of DLZ in dichloromethane.The actual drug content of microspheres was determined and used to calculate the entrapment efficiency of the microspheres as follows: Entrapment efficiency = (Actual drug content / Theoretical drug content) × 100

Determination of micromeritic properties
The micromeritic properties of the prepared DLZ microspheres were studied using the following techniques:

Particle size analysis
The particle size distribution was evaluated by sieve analysis using a standard set of sieves (British Standard) with size range of 710 to 90 µm, and shaken for 10 minutes using an electric shaker.The weight of microspheres retained on each individual sieve was recorded, stored and used for further studies.The size distribution of the microspheres was determined and mean particle size of microspheres was calculated by using equation ( 1). (1) The logarithm of the particle size was plotted against the cumulative percent frequency on a probability scale and a linear relationship was observed.From this linear plot, both the logarithm of the particle size equivalent to 50% on the probability scale, that is, the 50% size that is known as the geometric mean diameter, dg, which is the nth root of the product of n terms and can be expressed in equation ( 2). (2) Where, dp is the mean particle diameter in µm, n is the number of fractions, and dg is the geometric mean diameter in µm.The slope of the obtained line is known as the geometric standard deviation, σg, which can be calculated by equation (3).Also, lumps ( 500 µm), fines ( 100 µm), and the mean particles (500 -100 µm) were determined (Martin et al., 1993). (3)

Determination of flow properties
The flow properties of DLZ microspheres were described using the following methods:

Bulk density method
The bulk density (dB) of the microspheres was determined by pouring a sample of 50 g into a 100-ml graduated cylinder.The cylinder was then dropped from a height of 1 inch onto a hard surface three times at 2-second intervals.The volume of the powder was then recorded and used to calculate the bulk density by dividing the weight in (g) by volume in (cm 3 ) (Martin et al., 1993).The tapped density (dT) of the microcapsules was determined by tapping onto a hard surface until a constant volume was achieved.This volume was taken and used to calculate the tapped density of the microspheres.Hausner ratio is the ratio between tapped density and bulk density and Carr's index (C.I.) can be estimated from equation ( 4).The obtained value gives an idea about the flow behavior of the microspheres (4)

Angle of repose method
The flow properties were investigated by determining the angle of repose of the microspheres using the fixed-base cone method.Microspheres were allowed to fall freely through a funnel fixed 1 cm above the horizontal flat surface until the apex of the conical pile just touches the tip of the funnel.The height and diameter of the cone was measured and angle of repose was calculated by using the following formula (Tan θ = h / r), where h = cone height, r = radius of a circular base formed by the microspheres on the ground (Martin et al., 1993).Each experiment was carried out in triplicate (n = 3).

Surface morphology of microspheres
The surface morphology of the microspheres was examined by scanning electron microscope (Philips XL30, Netherlands) operated at 4-25 kV on samples gold-sputtered for 120 s at 10 mA, under argon at low pressure.

Formulations of DLZ controlled release capsule
Microspheres equivalent to 10 mg DLZ according to its DLZ content were mixed with Carbopol (CRB), hydroxypropyl methyl cellulose (HPMC) or chitosan (CS) at different ratios of 1:1, 1:2, and 1:3.The obtained mixtures were further mixed with the calculated amount of Avicel and magnesium stearate for 10 min and then filled into hard gelatin capsules (size 0).Twenty formulae were formulated along with the formulation codes as shown in Tables 1 and 2.

In-vitro dissolution studies
Dissolution studies were performed in a USP Dissolution Tester Apparatus II (ERWEKA DT 700, Germany) at 37 ± 0.5°C.The paddles were rotated at a speed of 100 rpm.Each of the formulated capsules was placed in the cylinder with 900 ml distilled water as a dissolution medium.An aliquot of samples were withdrawn and replaced with fresh dissolution media at time intervals of 0.25, 0.5, 1, 2, 3, 6, 9, and 12 h.The drug content in the filtered samples was analyzed using HPLC (Agilent 1200 series equipped with UV, diode array, detector, and an automatic sampling system, Germany).Reversed-phase column 25 cm × 4.6 mm (i.d.) C18, 5 m -Intersil® ODS-3 (GL Sciences Inc, Japan).The mobile phase consists of a mixture of (28:72) acetonitrile/0.05Mpotassium dihydrogen orthophosphate (pH 3.5), and the flow rate was 1 ml/min and DLZ content was determined at 236 nm.An injection volume of 10 μl and DLZ could be detected at a retention time of 3.5 min.Assay performance was evaluated through determination of specificity, recovery, linearity, the limit of quantification (LOQ), the limit of detection (LOD), precision, accuracy as reported in the International Conference on Harmonization guidelines.The release experiments were done in triplicate.This drug release profile was fitted into several mathematical models to explain the release mechanism.

Kinetic analysis of release data
The amount of DLZ released was calculated from the regression line of the standard curve.The release data obtained were fitted to different kinetic models; zero-order, first-order, Higuchi, and Korsmeyer-Peppas models.The best fitness of the model was chosen to determine the corresponding drug release mechanism for the formulated capsules.

In-vivo evaluation
Formulated DLZ capsules showed the desired release characteristics were chosen for the in-vivo studies in comparison to the commercial sustained release capsules.

Study design
Single dose one period parallel design was chosen.The dose was 3.6 mg and the sampling points were determined as per USFDA guidance "the sampling times should extend to at least 3 multiples of the drug's apparent terminal elimination half-life, beyond the time when maximum blood concentrations are achieved".It was stated that the t1/2 for DLZ is 3-5 h and tmax is 3-4 h (Sweetman 2007).Wistar rats with average weight 300 g were used for the in-vivo studies.The rats were acclimatized for one week before the study in a temperature controlled room.The rats were kept with free access to food and water.During this week, the animals were maintained with a 12 h light/darkness cycles.The study was started during the light cycle.Animals were divided into three groups (n = 3); group I administered F7, group II administered F17 and group III administered the commercial DLZ capsule as control.The dose was administered to the rat in the form of oral suspension using gastric tube.

Sample collection
Blood samples were collected in glass tubes before administration of the dosage form, and at 0.25, 0.5, 1, 3, 6, 9, 12, 16, 20 and 24 h after drug administration.All samples were collected through the orbital plexus.Plasma was immediately separated from the blood cells by centrifugation at 6000 rpm for 10 min and stored frozen at -20°C till HPLC analysis.

HPLC determination of DLZ in rat plasma
The concentrations of DLZ in rat plasma samples were determined using a modified HPLC method (Yeung et al., 1996).A volume of 100 µl acetonitrile, 100 µl of (0.02 mg/ml) of cetrizine HCl dissolved in acetonitrile and 30 µl of 0.1N HCl were added to 200 µl of each rat plasma sample in a clean glass test tube.The mixture was vortexed for 10 s and centrifuged at 3000 rpm for 10 min.An aliquot (30 µl) of the clear supernatant was injected directly onto the HPLC system (Agilent 1200 series equipped with UV, diode array, detector, and an automatic sampling system, Germany).Reversed-phase column 25 cm × 4.6 mm (i.d.) C18, 5 m -Intersil® ODS-3(GL Sciences Inc, Japan).The column effluent was monitored by UV detector at 236 nm and the eluent flow was 1.3 ml/min.The mobile phase consisted of 35 % acetonitrile and 65 % of 0.05M ammonium acetate buffer.The retention time for cetrizine HCl and DLZ were 3.1 and 6.1 min, respectively.Blank rat plasma was spiked with known amounts of DLZ to produce standard samples with concentrations in the range of 1-50 µg/ml.Calibration curves were constructed from the concentration of DLZ in each standard sample and the resulting peak area.The concentrations of DLZ in the unknown samples were determined from the calibration curves.Linearity, selectivity, precision and accuracy were determined to validate the HPLC method of analysis.The calibration curves were linear over the entire range, with correlation coefficient always greater than 0.999.There was no interference between the endogenous peaks in the rat plasma and the peaks for DLZ.Withinday precision was determined from the analysis of three calibration curves on the same day and the coefficient of variation (CV) ranged from 0.058% to 1.18%.Between-day precision was determined from the analysis of three different calibration curves on three different days during the study period, and the CV was in the range 0.068% to 1.16%.The accuracy of the assay, determined from the predicted DLZ concentration in each standard, was in the range 98.86-103% within-day and 82.6-119.15%between-day.

Pharmacokinetic parameters
The following pharmacokinetic parameters were determined; maximum plasma concentration (Cmax), time point of maximum plasma concentration (tmax), area under the plasma concentrationtime curve from zero h to the last measurable concentration (AUC 0- t), and area under the plasma concentration-time curve from zero h to infinity (AUC0-).Similarly, the elimination half life (t1/2), the mean residence time (MRT), and the total renal clearance (TRC) were determined using pharmacokinetic software Kinetica TM (version 4, Thermo Scientific, MA, USA).

Statistical analysis
The pharmacokinetic parameters obtained from the three groups of rats were analyzed by oneway ANOVA using SPSS version 18 software.

RESULTS AND DISCUSSION
Pre-formulation studies are known to be an essential prerequisite to develop a successful formulation.DSC and FTIR spectroscopic analysis were used for the evaluation of physicochemical compatibility and interactions.They helped in the prediction of possible interaction of DLZ with the polymers used in microsphere formulations.It was recommended that the ratio of the drug to polymers used in this study was 1:1 to maximize the possibility of interaction and helps in easier detection of incompatibilities as investigated previously (Sultana et al., 2009).

Thermal analysis of DLZ and polymers using DSC
DSC thermograms of DLZ, EC, DLZ-loaded EC micro-spheres, ED and DLZ-loaded ED microspheres are presented in Figures 1 and 2. The DSC thermogram of DLZ showed an endothermic peak indicating the melting point 226°C which is very close to the literature value of drug (Kulkarni et al., 2011).The DSC thermograms of DLZ with EC or ED mixtures exhibited the same type of endothermic peaks of DLZ.From this observation, it is concluded that no possible interaction detected for the drug with the used polymers.

Fourier transform infrared spectroscopy (FTIR)
FTIR spectrum of DLZ showed two characteristic peaks around 1681 and 1743 cm -1 due to the presence of lactam carbonyl group and ester carbonyl group, respectively.In addition to the peaks at 2966 cm -1 (C-H stretching), 2387 cm -1 (C-S), and 1475 cm -1 (O-CH 3 ) are observed (Figures 3 and 4).Similar peaks were also seen in DLZ loaded EC and ED microspheres spectra indicating the stable nature of DLZ during microencapsulation process.

Entrapment efficiency of DLZ microspheres
The percentage of drug entrapment in EC microspheres was found to be 67.45 % while that of ED microspheres were 75.83 %.These results revealed acceptable entrapment efficiency for DLZ in the prepared microspheres, regardless of DLZ water solubility.

Particle size analysis
The particle size analysis curves of both types of the prepared microspheres are presented in Figures 5 and 6.The arithmetic mean diameter is 425 and 158 µm for DLZ-loaded EC and DLZ-loaded ED microspheres, respectively.Figure 5 shows that mean microsphere size represents 74.93 and 95.52% for EC and ED microspheres, respectively.The size distribution can be specified by two values; the geometric mean diameter (d g ) and the geometric standard deviation (σ g ).From the observed data, d g of EC microspheres was 330 μm, and its σ g was 0.67 which in agreement with the previous work (Agnihotri and Aminabhavi, 2005).On the contrary, the frequency distribution data of ED microspheres revealed that, d g is 72 μm and σ g is 0.51.The obtained particle size analysis data illustrated in Figure 5 indicates a wide particle size distribution of EC microspheres and a narrow size distribution of ED microspheres while Figure 6 presented the percentage of fines, means, and lumps

Flow properties
The flow properties of the DLZ microspheres are shown in Table 3.The ED microspheres were found to be denser than EC microspheres which improved its flowability.This finding was confirmed by the values of Hausner ratio, Carr's index, and angle of repose.Hausner ratio was 1.3, Carr's index was 23.12 %, and the angle of repose was 43.8° for EC microspheres.According to USP 29-NF24, passable to flow materials have Hausner ratio ranged from 1.26 -1.34; Carr's index from 21 -25 and angle of repose from 41 -45°.On the other hand, the flow parameters of ED microspheres were 1.15 for Hausner ratio, 13 % for Carr's index, and 34.6° for the angle of repose.These results indicate good flowability of ED microspheres.

Surface morphology by SEM
Figure 7 shows SEM pictures taken from dried microspheres prepared by emulsion solvent evaporation     technique.The surface of the microspheres prepared with ethyl cellulose was rather smooth and porous as can be seen in the photomicrograph; there are many pores and cavities in the microspheres (Figure 7A and B). Figure 7B shows the surface morphology of microsphere at a higher magnification which displays the porous structure of the microspheres.This could explain the low bulk density of EC microspheres.Similar observations were made for microspheres prepared with ED (Figure 7C and D) with the exception that these microspheres appeared rough with less porous surface but were mostly spherical in appearance.The pictures also show that ED microspheres are not aggregates.

In-vitro dissolution
The dissolution profiles of DLZ-loaded EC microspheres (F1), DLZ-loaded ED microspheres (F11), F7, F17, and the marketed DLZ sustained release capsules are presented in Figure 8.The release from DLZ-loaded EC microspheres was found to be faster in comparison to DLZ-loaded ED microspheres; this could be related to the porous nature of EC microspheres which allow the release of DLZ more easily than less porous ED microspheres.By mixing the microspheres with different polymers CRB, HPMC, and CS, the release rate was remarkably extended particularly at higher polymer concentrations.This could be attributed to the increase in the length of the diffusional pathway developed by the increase in polymer concentration (Prasanth et al., 2011).
On the other hand, by increasing the polymer concentration, large amount of drug got bind in the polymer matrix that leads to decrease in the rate of release (Chinna et al., 2010).The results shown in Figure 9 revealed that both CRB and CS, even at higher ratios, did not control the release of DLZ from formulated capsules to meet the desired criteria of sustained release formulation which is clear from the dissolution profiles of (F4, F14) and (F10, F20), for CRB and CS, respectively.On the basis of sustained release criteria, the drug should reveal less than 30% release in the first hour, that is, no dose dumping, about 60% release in 5-7 h, and more than 85% release of drug in more than 12 h, while maintaining smooth release for in between time points (Carrico, 1996).Similar criteria were specified for bioequivalence studies of modified oral dosage formulations during approval (Cartwright and Matthews, 1994).Formulae (F2-4, F8-10, F12-16 and F18-20) were rejected because 30% of the drug was released during   the first hour which is considered to be not satisfactory for the desired criteria for sustained release formulations.This could be attributed to the low polymer concentrations in those capsules in addition to the high solubility of DLZ.On the other hand, F7 and F17 which contain HPMC, at a ratio of 1:3 microspheres: HPMC fulfilled the desired criteria required in a controlled release product.HPMC hydrate upon contact with water forms a rate controlling gel layer around the solid microspheres.This layer acts as a physical and diffusion barrier to the rate of waterentrance and the diffusional/erosional release of drug (Colombo et al., 1996).Moreover, the presence of HPMC in the formulae at higher concentration increased the viscosity in the gel layer which led to further controlling of DLZ release.This finding was in a good agreement with the previous work confirmed that, the drug release is controlled by the rate of diffusion through such a gel layer formed by HPMC (Katzhendler et al., 1997;Mitchell et al., 1993;Gao et al., 1995).According to these results, F7 and F17 were selected for further in-vivo study.

Kinetic treatment of the in-vitro release data
The release behaviors of DLZ from the different formulae were fitted to various kinetic orders and models for prediction of their release behavior.Zero-order (Wagner, 1969) is expressed in equation (5) as: (5) While the first-order (Desai et al., 1966) is expressed in the equation ( 6): (6) According to Higuchi relationship (Higuchi, 1963), the amount of drug released were plotted as cumulative drug release versus square-root time as in the equation ( 7).The equation explains diffusional release rate as indicated below: Where, Q t is the amount of drug released or dissolved at time t, Q 0 is amount of drug released or dissolved at a time t = 0, and k 0 , k 1 , and k H are the release rate constant for zero, first, and Higuchi's equations, respectively.Two factors diminish the applicability of Higuchi's equation to matrix systems.This model fails to allow the influence of swelling of the matrix (upon hydration) and gradual erosion of the matrix.Therefore, the dissolution data were also fitted according to the well-known exponential equation ( 8) of Ritger and Peppas (Korsmeyer et al., 1983;Ritger and Peppas, 1987), which is often used to describe the drug release behavior from polymeric systems. (8) Where, M t is the amount of drug released at time t, M ∞ The model that best fits the release data was evaluated by the correlation coefficient (r) except in Peppas model which was evaluated by (n) value.The (r) value was used as criteria to select the best model that describe drug release from the controlled release capsules.The calculated (r) and the exponent (n) values for the in-vitro release of DLZ from capsules based on different kinetic orders or systems are shown in Table 4. F1 and F11 were the DLZ-loaded microspheres with EC and ED, respectively, without incorporation of polymers.F7 and F17 were the same microspheres incorporated with HPMC in ratio 1: 3 that met the desired criteria of controlled release among all formulations.
Kinetic treatment results of the prepared microspheres revealed that the (n) values were 0.123 and 0.079 for DLZ-loaded EC (F1) and DLZ-loaded ED microspheres (F11), respectively.These results indicated that the release followed Fickian diffusion mechanism.On the other hand, incorporation of HPMC to the prepared microspheres increased the (n) value to 0.698 and 0.498 for F7 and F17, respectively, which subsequently changed the mechanism to non-fickian one.The change in mechanism could be related to the nature of HPMC as a swellable polymer.So, in addition to diffusion mechanism, other factors are included in the release mechanism attributed to relaxation of HPMC chains, imbibitions of water causing HPMC to swell and changing it from initial glassy to rubbery state (Siepmann and Peppas, 2001).The increase in volume as a result of HPMC swelling leads to diffusion boundaries shift that necessitate further calculation in addition to Fick's second law of diffusion (Siepmann and Peppas, 2001).So, data were further treated by equation ( 8) that revealed the non-fickian (anomalous) mechanism.

In-vivo study
The in-vivo studies were carried out using two formulae F7, and F17 as it showed the optimum controlled release behavior among all formulations.The study was carried out in comparison with the marketed DLZ capsule.The mean plasma concentration-time data of DLZ following the oral administration of the aforementioned formulae is presented in Table 5 and shown in Figure 10.The mean peak plasma concentrations (C max ) were 0.236, 0.269, and 0.272 µg/ml for F7, F17, and the commercial DLZ, respectively, with the same t max .The mean AUC (0-24) was found to be 18.48, 13.07, and 14.93 µg-h/ml for F7, F17, and the marketed, respectively.Similarly, the t 1/2 was found to be 5.35, 14.80, and 5.83 h, while, the mean residence time (MRT) was found to be 7.72, 12.33, and 7.78 h for F7, F17, and the marketed capsules, respectively.From the obtained results, we can conclude that capsules containing ED microspheres mixed with HPMC in ratio 1: 3 (F17) offer the good criteria for controlled DLZ effect.The statistical results between the studied three groups indicated that there was no significant difference between the prepared microspheres and the commercial sustained release product (P-value = 0.767) which indicates the usefulness of the technique used for sustaining the release of diltiazem.

Conclusions
The results of this study revealed that the incorporation of retardant polymers, HPMC, with DLZ-loaded ED microspheres leads to controlling the release of DLZ.In addition to, the pharmacokinetic parameters of the developed formulation will provide a longer period of residence time required for achieving once or twice dosing daily of DLZ in the management of coronary heart diseases especially in treatment of angina and hypertension.

Figure 5 .
Figure 5. Frequency distribution curve of both types of DLZ microspheres.

Figure 6 .
Figure 6.Percentage of fines, means, and lumps of both types of DLZ microspheres.

Figure 10 .
Figure 10.Means of plasma concentrations-time profiles of DLZ from F7, F17 and the marketed DLZ capsule.

Table 1 .
Composition of the formulated capsules containing DLZ loaded-EC microspheres.

Table 2 .
Composition of the formulated capsules containing DLZ loaded-ED microspheres.

Table 4 .
Release kinetics of DLZ from different controlled release formulations.

Table 5 .
Mean bioavailability and pharmacokinetic parameters of DLZ following the administration of a single dose of F7, F17 and marketed sustained release DLZ capsule.