1. INTRODUCTION:

Clonazepam is a potent anticonvulsant (5-(2-chlorophenyl)-7-nitro-2, 3-dihydro-1,4-benzodi-azepin -2-one) that controls some types of myoclonus. Its primary mode of action is to facilitate gamma amino butyric acid transmission in the brain by a direct effect on benzodiazepine receptors. GABA receptors lie on the cell bodies of dorsal raphe neurons, and GABA acts to
inhibit raphe cell firing, an action potentiated by benzodiazepines. CZP is practically insoluble in water (B.P, 2007). Because water-insoluble drugs often show low absorption and weak bioavailability, improvement in dissolution rate and /or solubility are important for such drugs preparations (Hirasawa et al., 2003). Over the years a variety of solubilization techniques have been studied to improve the dissolution rate of water-insoluble drugs and to obtain more rapid and complete absorption such as using adsorbents (Bogdanova et al., 2007), surfactant (Krasowska, 1980), hydrotopes and cosolvents (Etman and Nada, 1999), solid dispersion (valizadeh et al., 2004) or complexation with cyclodextrins (Bandi et al., 2004).

A solid dispersion may be defined as a dispersion of one or more active ingredients in an inert carrier or matrix in the solid state prepared by the melting, solvent, or melting-solvent method (Chiou and Riegelman, 1971).

It has been widely used to improve the dissolution rate, solubility and oral absorption of poorly water-soluble drugs (Thybo et al., 2007). In solid dispersion, the particle size of the drugs was reduced, the wettability and the dispersibility were enhanced; therefore, drug dissolution was markedly improved (Craig, 2002).

PEG and PVP are among the several carriers which have been employed in preparing solid dispersions (Leuner and Dressman, 2000). PEG polymers are widely used for their low melting point, low toxicity, wide drug compatibility and hydrophobicity (Ahuja et al., 2007). Low molecular weight polyvinyl-pyrrolidone (PVP K30), is another commonly utilized polymer that provides fast or regular release in oral dosage forms (Karavas et al., 2007).

Pentylenetetrazole (PTZ) is a convulsant used to model epileptic seizures in animals (Cremer et al., 2009 and Bertram, 2007).

Bioavailability determinations are performed to ensure that the given formula will get the therapeutic agent to its site of action in an adequate concentration. Bioavailability studies are also carried out to compare the availability of a drug from different dosage forms (Makoid, 1999).

The aim of the present study was to characterize the solid state properties of the solid dispersion system of clonazepam in PEG 4000 and PVP K30 prepared at different ratios. The methods of characterization were achieved through using different tools as differential scanning calorimetry (DSC), powder X-ray diffractory (XRD) and Fourier transform infrared (FTIR). Also, solubility and dissolution rate study were performed to qualify the solid dispersion in comparison to the drug alone or as a physical mixture (PM). Moreover, the formulations of CZP which showed the highest dissolution rate and release were selected to study the bioavailability and anticonvulsant activity.

2. EXPERIMENTAL:

2.1. Materials:

Clonazepam (kindly supplied by EIPICO Co. Egypt), Diazepam (kindly supplied by El-Nile Co., Egypt), Methanol, Ethanol, PVP K30 (Kindly supplied by El. Gomhouria Co., Egypt), and Polyethylene glycol (PEG) 4000 (Hoechest Chemikalien, Werk Gendort, Germany), dichloromethane, diethyl ether, sodium hydroxide were of pharmaceutical grade and were purchased from El. Gomhouria Co., Egypt. Methanol-HPLC grade (Honil Limited, London, U.K), Acetonitrile-HPLC grade (BDH laboratory Supplies, England), Cal-Heparine 5000 I.U. (Amoun Pharmaceutical Co., El-Obour City, Cairo, Egypt), Thiopental 0.5 gm (SANDOZ GmbH, Kundl- Austria), Pentylenetetrazole (SIGMA ALDRISH, China) and Commercial product of Clonazepam (Amotril^®) (AMOUN Pharmaceutical Co., Cairo, Egypt). All other materials and reagents were of analytical grade.

2.2. Methods:

2.2.1. Preparation of physical mixture

Physical mixtures were prepared by triturating appropriate quantities of CZP and carriers using a mortar and pestle, at 1:1, 1:2, 1:3 and 1:6 weight ratio of CZP:carrier then transferred to a vaccum dessicator until ready for use. Powder of PMs were sieved to obtain particles passing through 355 μm and retained on 150 μm sieve.

2.2.2. Preparation of solid dispersions

Solid dispersions (SDs) at various weight ratios were prepared by solvent evaporation method. Accurately weighed quantities of CZP and the respective dispersion carrier (PEG 4000 or PVP K30) were transferred into a flask. A sufficient quantity of the solvent was added to dissolve the ingredients. The solution was stirred at room temperature, and the solvent was then removed under vacuum at a maximum temperature of 40^oC. Solid residue was dried in a vacuum oven for 24 h at room temperature, pulverized and sieved. Powdered samples below 420 um (40 mesh) were stored in closed containers away from light and humidity until use.

2.2.3. Fourier transform infrared spectroscopy (FTIR):

FTIR spectroscopy was employed to characterize the possible interactions between the drug and the carrier in the solid state on a FTIR spectrophotometer using KBr disk method. The scanning range was 200-4000cm^-1 and the resolution was 1 cm^-1.

2.2.4. Differential Scanning Calorimetry (DSC):

The possibility of any interaction between the drug and the carriers during preparation of physical mixture and solid dispersion was assessed by carrying out thermal analysis of drug and polymer alone as well as physical mixture and solid dispersion using DSC. The DSC thermograms were recorded on a Shimadzu-Dsc 50. Samples (1.5 mg) were heated in hermetically sealed aluminum pans over the temperature of 30-300 ºC at a constant rate of 10 ºC /min under a nitrogen purge (30 ml/min).

2.2.5. X-ray diffraction (XRD):

To determine powder characteristics, X-ray powder diffraction studies of drug and polymer alone as well as physical mixture and solid dispersion was performed. X-ray diffraction patterns were obtained using a Siemens Kristallofex D-5000 powder diffractometer with CuKα radiation. Diffractograms were run at a scanning speed of 8º/ min over the 2θ range of 0-80º.

2.2.6. Solubility measurements:

An excess amount of CZP was placed into a 25-ml glass vial containing various concentrations of each carrier in 10 ml water. The glass vials were closed with stopper. The content of the suspension was equilibrated by shaking in a thermostatically controlled water bath at 25 ºC for all the carriers for 24 hrs. After attainment of equilibrium, the content of each vial was then filtered through a double layered filter paper (Whatman 42). The filtrate was suitably diluted and assayed spectrophotometrically at λ_max309 nm to measure the amount of dissolved drug. All experiments were conducted in duplicate.

2.2.7. Release rate studies:

The dissolution of CZP from pure drug, the prepared (SDs) and (PMs) was carried out according to the USP-24, rotating paddle method. Dissolution medium consisting of 250 ml of distilled water was used. The stirring rate was 100 rpm and the temperature was maintained at 37+ 0.5 ºC. A sample of 2 mg of CZP or its equivalent of the (SDs) or the (PMs) was placed on the surface of the dissolution medium. At appropriate time intervals (5, 10, 20, 30, 45, 60, 90, 120 min), 5 ml samples were withdrawn and replaced with an equivalent amount of the fresh dissolution medium kept at 37 ºC. The samples were filtered rapidly through a double layered filter paper (Whatman 42), diluted with dissolution medium and assayed spectrophotometrically at λ_max309 nm without the interference from the carriers. All experiments were conducted in duplicate.

2.2.8. Pharmacological study:

All experiments were performed on adult male Swiss mice weighing 22-26 g. The animals were randomly assigned to experimental groups comprising 8 mice per group. All animal experiments were approved by the ECAHZU (Ethical Committee for Animal Handling at Zagazig University).

Clonic convulsions were induced in mice by s.c. administration of PTZ (98mg/kg). Following PTZ administration, mice were placed separately into cages (24 cm x 15 cm x 10 cm) and observed for 30 min for the occurrence of clonic seizures. Clonic seizure activity was defined as clonus of whole body lasting over 3 s, with an accompanying loss of righting reflex.

The number of animals convulsing out of the total number of the mice tested was noted for control animals. A group of mice (28 mice) were injected with PTZ s.c. at a dose of 98 mg/kg, then the total number of the mice convulsing out from the total animals tested was noted and taken as the control group. The anticonvulsant activity of CZP formulations against the clonic phase of PTZ-induced seizures were determined after s.c. administration of PTZ. All formulations were administered orally in a volume 0.008 ml/g body weight. The selected formulations were orally administered 30 min before PTZ administration. PTZ administered subcutaneously (s.c.) into a loose fold of skin in the midline of the neck in a volume 0.005 ml/g body weight.

2.2.9. Bioavailability studies:

A parallel design comprising three groups with 30 mice (Swiss mice weighing 22-28 gm) in each group was adopted. All groups received an equivalent of 0.2 mg drug/kg body weight. Group A received “CZP” alone; Group B received formula 1; Group C received formula 2 orally.

Formulae no.	Formulae
1	CZP-PVP (1:4) SDs
2	CZP-PEG 4000 (1:4) SDs

2.2.10. Determination of CZP concentration by HPLC:

CZP and diazepam (Internal standard) were eluted with acetonitrile-water mixture (40:60, v/v). Temperature column oven 480, fixed with reverse phase C 18 column (Chromonith® Performance RP-18E, 100x4.6mm. Merck, Germany). The operating temperature was ambient, the flow rate was 1 ml /min which generated a pressure of approximately 1100 bar. The effluent was monitored at 215 nm. Under these conditions, both CZP and diazepam could be detected and a baseline separation was readily achieved. The retention time for CZP was 1.7 min and for diazepam was 2.5 min (Christopher and Stewart, 1998).

Blood samples were withdrawn from heart into heparinized tubes at different time points following drug administration (0.5, 1, 2, 4, 6, 8, 10, 12, and 24 hours), the blood samples were immediately centrifuged for 10 min at 3000 rpm and plasma was stored at -20ºC. Internal standared in a conc. of 10 μl was added then, 80 μl of methanol and 0.6 ml of distilled water were added to plasma samples and mixed. The sample was alkalinized with 8 μl of NaoH (5M) to adjust the pH to 12. The basified solution was extracted twice with 2.8 ml of dichloromethane/diethyl ether (1:3) by mixing for 15 min. After centrifugation for 10 min at 3000 rpm, the organic extracts were combined and evaporated to dryness under vaccum at 45 ºC. The residue was dissolved in 20 μl of methanol. A volume of 10 μl solution was automatically injected into the sample loop. The concentration of CZP in samples was determined from calibration curves of the peak area ratio versus plasma CZP concentration (Jung et al., 1997).

3. RESULTS AND DISCUSSION:

3.1. X-ray powder diffraction (XRD)

XRD patterns for different samples are displayed in fig.1 and 2 for CZP-PEG4000 and CZP-PVP K30 systems, respectively. CZP was a highly crystalline powder with characteristic diffraction peaks at 2θ of 11.79^o, 22.84^o, 23.05^o and 23.75^o, in addition there were some other peaks of lower intensity.

In case of untreated PEG 4000 (fig. 1), there were sharp peaks at 19.23^o, 23.14^o and 23.53^o,while in case of PEG 4000 solid dispersion, the diffraction peaks were traced at 19.18^o and 23.32^o. The peaks of CZP were completely missing thus indicating that CZP was in amorphous form. While PVP K30 in pure form revealed high degree of crystallinity. In case of PVP K30 solid dispersions and physical mixtures, the diffraction peaks of CZP were not observed whereas the diffraction peaks of PVP K30 were noted (fig.2). This indicated that CZP was in amorphous state (Okonogi et al., 1997). No new peaks could be observed suggesting the absence of the chemical interaction between the drug and the carrier (Ahuja et al., 2007).

Figure (1): XRD diffractograms for: (CZP) clonazepam; (PEG) polyethylene glycol 4000, and their different systems prepared at different ratios; (PM) physical mixture; (SD) solid dispersion.

Figure (2): XRD diffractograms for: (CZP) clonazepam; (PVP) polyvinyl pyrrolidone K30, and their different systems prepared at different ratios; (PM) physical mixture; (SD) solid dispersion.

3.2. Differential scanning calori-metry (DSC)

Fig. 3 depicted thermograms of CZP, PEG 4000, their PMs and SDs. DSC curves of pure CZP exhibited a sharp endothermic peak at 235.35^oC (table 1), which is corresponding to its melting point.

Mura et al., 1999, studied the DSC scans of SD of naproxen in binary systems with different PEG molecular weights, they observed the disappearance of the drug melting peak which indicated the dissolution of the naproxen in the melted carrier. A slight change occurs in the shape of PEGs endothermic peaks which appeared broadened in solid dispersions.

The DSC thermograms of CZP-PEG 4000 solid dispersions and corresponding physical mixtures showed no CZP endothermic peak but did exhibit the endothermic peaks due to the fusion of the carriers. This result indicated that CZP might be in amorphous state. Fig. 4 illustrates the DSC thermograms of CZP -PVP K30 systems and the absence of CZP peak and the predominance of PVP K30 peaks. This suggests that CZP is completely soluble in liquid phase of PVP K30 (Domain et al., 2000).

Table (1): Fusion temperature and Heat of Fusion (ΔHF) of some CZP solid dispersions and physical mixtures compared with individual components.

System	Fusion temperature (^oC)	Heat of fusion (ΔHF) (J/g)
CZP	235.35	106.72
PEG 4000	61.79	174.20
CZP -PEG 4000 (PM) (1:4)	62.30	152.56
CZP -PEG 4000 (SD) (1:4)	61.34	142.79
PVP	82.02	283.32
CZP – PVP (PM) (1:4)	71.32	118.07
CZP – PVP (SD) (1:4)	64.96	198.46

Figure (3): DSC thermograms for; (CZP) clonazepam; (PEG) polyethylene glycol 4000, and their different systems prepared at different ratios; (PM) physical mixture; (SD) solid dispersion.

Figure (4): DSC thermograms for; a) (CZP) clonazepam; b) (PVP) polyvinyl pyrrolidone K30, and their different systems prepared at different ratios; c) (PM) physical mixture; d) (SD) solid dispersion.

3.3. Fourier Transform Infrared spectroscopy (FTIR):

The FTIR of CZP, PVP K30, PEG 4000, (PMs and, SDs) as shown in fig 5, 6 respectively. In case of PVP K30, each pyrrolidone moiety of PVP has two groups (=N- and C=O) that might interact with the drug. If the drug and these carriers interact, then the functional groups in FTIR spectra will show band changes and broadening compared to the spectra of the plain carriers. Pure CZP spectra showed sharp characteristic peaks at N-H (stretching), C=O (group), C=C and NO₂at 3105.2 cm^-1, 1693.4 cm^-1, 1616.2 cm^-1 and, 1531.9,1338.5 cm^-1 respectively. The spectrum of PVP K30 showed, among others, important bands at 2954.7 (C-H stretch) and 1658.7 (C-O) cm^-1. A visible band was detected at 3454.3 cm^-1which was attributed to the presence of interaction (Ahire et al., 2010).

In case of PEG 4000 (fig. 6), the carbonyl stretching band of CZP that appeared at 1693.4 cm^-1decreased in intensity with the disappearance of C-H stretching band and N-H stretching band and predominance of O-H band corresponding to PEGs. It was concluded from the chemical structures that an interaction of a significant magnitude could be present between the aromatic hydrogen of the drug and the hydroxyl groups of PEG.

Figure (5): FTIR spectra for; a) (CZP) clonazepam; b) (PVP) polyvinyl pyrrolidone K30, and their different systems prepared at different ratios; c) (PM) physical mixture; d) (SD) solid dispersion.

Figure (6): FTIR spectra for; a) (CZP) clonazepam; b) (PEG) polyethylene glycol 4000, and their different systems prepared at different ratios; c) (PM) physical mixture; d) (SD) solid dispersion.

3.4. Solubility determination:

The aqueous solubility of a drug is a prime determinant of its dissolution rate and compounds with aqueous solubility less than 0.1 mg/ml often present dissolution limitation to absorption. The solubility of CZP in distilled water at 25^oC was found to be 0.0012 + 0.007 mg/ml (Hammad and Müller, 1998).

Figure (7) depicted the effect of different carriers on CZP solubility in distilled water at 25ºC. In case of PEG 4000, PVP K30, the solubility of CZP linearly increased as the carrier concentration increased, showing the feature of an A_L-type solubility phase diagram (Higuchi and Connor, 1965). This result illustrates that the complex formed was soluble and did not form precipitate over the range of the carrier concentration.

On the other hand, the solubility plot of PVP showed a B_s-type curve (Higuchi and Conners, 1965). The initial rising portion was followed by a decrease in total concentration of CZP. Consequently, these carriers can be ranked according to its effect on increasing the solubility of CZP as PVP > PEG 4000. The increased solubility of CZP in carrier’s solution may be attributed to both complex formation and reduction in interfacial tension of water and hence intermolecular forces and polarity caused by the presence of those carriers (Al-Angary et al., 1996).

Figure (7): Phase solubility diagram of CZP in water at 25ºC in presence of PEG 4000 and PVP K30

3.5. Release rate studies:

The dissolution profiles of pure CZP, its PMs and SDs with different carriers are shown in (Figs. 8 and 9). Data are average of three measurements.

The calculated dissolution parameters revealed that, pure CZP yielded the slowest dissolution rate with only about 9.09% of the drug dissolved in 120 min. The hydrophobic property of CZP prevented its contact with the dissolution medium (distilled water) which led to a slow dissolution rate (Tantishaiyakul et al., 1996). As shown in tables 2 and 3, all PMs released the CZP at faster rate than the drug alone as reflected by higher initial dissolution rate (IDR) and greater extent of dissolution after 120 min. These results can be explained on the basis that dry mixing brings the drug in close contact with the hydrophilic polymer (Van den Mooter et al., 1998). Also this may be due to a possible solubilization effect by the carrier operating the microenvironment (diffusion layer) that immediately surrounds the drug particles in the early stages of solubilization (Arias et al., 1996).

During dissolution experiments, it is apparent that, the rate and the extent of dissolution of CZP from (SDs) exceeded those of pure CZP or the corresponding (PMs). The dissolution efficiency (DE%) of (1:4) CZP- PEG 4000 co-precipitate (table 2) was 27.25 %, while the DE% of the corresponding physical mixture was only 19.06%. In case of PVP K30, the DE% of (1:4) CZP-PVP K30 co-precipitate (table 3) was 40.01%, while for its PM was 21.6%. The observed higher dissolution of the prepared (SDs) could possibly be due to the solubilizing effect of the carriers that may operate in the diffusion layer immediately surrounding the drug particles. Also, each single crystallite of the drug was very intimately encircled by the soluble carrier particles which can readily dissolve and cause the aqueous medium to contact and wet the drug particles easily (Etman, 2000). Moreover, it can be generally assumed that the increased dissolution via (SDs) could be explained on the basis of alterations in the solid-state structures of the carriers and the drug particles. These structural changes include the formation of solid solution, eutectic mixtures or soluble complex between the drug and the carriers and formation of amorphous drug particles or loss of crystallinity of the drug. For most (SDs), more than one of these factors may probably be responsible for the dissolution enhancement (Trapani et al., 1999 and Mura et al., 1999).

Figure (8): Dissolution profile for CZP-PEG 4000 systems in pure distilled water prepared at different ratios of CZP:PEG 4000.(CZP) clonazepam; (PM) physical mixture; (SD) solid dispersion.

Figure (9): Dissolution profile for CZP-PEG 4000 systems in pure distilled water prepared at different ratios of CZP:PEG 4000.(CZP) clonazepam; (PM) physical mixture; (SD) solid dispersion

Table (2): Dissolution parameters (±SD) of clonazepam in distilled water from different clonazepam-PEG 4000 systems.

Composition (w/w)

IDR

(% dissolved /min)

PD20

(%)

PD60

(%)

DE*100

(%)

Clonazepam Powder

0.21+0.08

4.19+1.68

7.27 +0.33

4.66+1.13

Clonazepam-to- PEG 4000

PM 1:1

SD 1:1

0.44+0.08

0.95+0.01

8.84+1.72

18.93+0.20

12.79+0.04

25.90+2.47

10.11+0.48

19.83 + 0.67

PM 1:2

SD 1:2

0.46+0.03

1.09+0.07

9.18+0.76

21.89+0.01

17.48+0.24

31.86+2.84

13.24+0.17

22.77+ 0.03

PM 1:3

SD 1:3

0.56+0.19

1.11+0.97

11.24+3.88

22.32+1.93

20.49+0.24

31.78+2.48

13.56+0.24

23.36+0.43

PM 1:4

SD 1:4

0.91+0.139

1.19+0.03

18.13+2.79

23.90+0.76

24.58+1.94

38.34+0.53

19.06+2.1

27.25+0.32

PM 1:5

SD 1:5

0.59+ 0.05

0.80+ 0.06

11.90+0.99

16.01+1.39

18.93+1.26

26.34+2.26

14.58+0.48

18.73+ 0.68

PM 1:6

SD 1:6

0.67+0.87

0.87+ 0.03

13.47+1.75

17.49+0.79

19.48+1.58

27.63+0.57

14.9+1.2

20.10+ 0.60

IDR = Initial dissolution rate. PD20 = Extent of dissolution after 20 min

PD60 = Extent of dissolution after 60 min DE% = Dissolution efficiency after 60 min

Table (3): Dissolution parameters (±SD) of clonazepam in distilled water from different clonazepam-PVP systems.

Composition (w/w)

IDR

(% dissolved /min)

PD20

(%)

PD60

(%)

DE*100

(%)

Clonazepam Powder

0.21+0.08

4.19+1.68

7.27+0.33

4.66+1.13

Clonazepam-to- PVP

PM 1:1

SD 1:1

0.89+0.05

1.09+0.06

17.76+1.02

21.94+1.28

23.84+0.57

32.16+1.67

19.52+0.75

25.03+0.12

PM 1:2

SD 1:2

1.24+0.15

1.38+0.01

24.86+3.045

27.61+0.23

27.02+2.46

32.94+4.98

23.39+0.74

27.32+1.30

PM 1:3

SD 1:3

1.27+0.07

1.45+0.01

25.39+1.50

29.00+0.22

30.62+0.81

37.11+0.78

25.96+0.72

29.54+0.66

PM 1:4

SD 1:4

1.28+0.04

2.016+0.08

25.59+0.98

40.32+1.61

32.40+0.56

49.51+0.36

21.60+0.88

40.01+1.00

PM 1:5

SD 1:5

1.15+ 0.06

1.82+ 0.01

23.04+1.25

36.32+0.20

20.83+0.72

43.43+1.28

23.45+1.03

36.52+0.56

PM 1:6

SD 1:6

1.42+ 0.07

1.61+ 0.07

28.46+1.57

32.22+1.50

26.68+0.77

39.12+0.40

21.60+0.56

33.15+0.63

IDR = Initial dissolution rate. PD20 = Extent of dissolution after 20 min

PD60 = extent of dissolution after 60 min DE% = Dissolution efficiency after 60 min

3.6. Pharmacological studies:

3.6.1. Threshold for PTZ-induced clonic seizures

PTZ at a dose of 98 mg/kg induced clonic seizure in 85.7% of mice (24 out of 28 mice injected).

3.6.2. Anticonvulsant effect of CZP formulations:

CZP formulations (CZP-PEG 4000 1:4 and CZP-PVP K30 1:4) were administered orally at dose of 0.02 mg/kg (Jastrzebska et al., 2009) 30 min before the convulsive dose of PTZ and the animals were monitored for 30 min and the number of the convulsive animals was recorded in each group (table 4).

Table (4): Anticonvulsant effect of CZP formulations against PTZ induced clonic seizures.

Group	Treatment (mg/kg)	Number of animals protected /group	(% of animals protected)
1	Distilled water	0/8	0
2	(1:4) CZP-PVP	7/8	87.5%
3	(1:4) CZP-PEG 4000	8/8	100 %
4	Commercial formulae (Amotryl®)	1/8	12.5%

In group 1 (vehicle treated group), all animals showed clonic seizures lasting for over 3 s with loss of righting reflex at a time ranging from 5-7 min which was taken as the end point.

In group 2 (receiving CZP-PVP), one animal showed the convulsion at 11.36 min.

In group 3 (receiving CZP-PEG 4000), all animals were protected against clonic convulsion.

In group 4 (receiving CZP- commercial product (Amotril^®), seven animals showed clonic convulsions at 3.01, 3.50, 4.52, 5.20, 5.58, 11.30 and 15.37 min.

These results demonstrated that Clonazepam formulations show better and more efficient pharmacological effect than commercial formulae against PTZ-induced seizures. This may be attributed to the enhanced dissolution of the drug from SDs (Arias et al., 1996).

3.7. Bioavailability studies:

3.7.1. HPLC analysis of CZP in plasma:

Figure (10) shows the chromatogram of mouse plasma containing CZP and diazepam as an internal standard. The retention times (R.T.) of CZP and diazepam were 1.75 and 2.30 min respectively. Diazepam was selected as the internal standard because it has a suitable molecular similarity, simple bioavailability and low cost.

Figure (10): HPLC chromatogram of mouse plasma containing CZP and diazepam

Data are presented as means ± SD, n= 3.

3.7.2. Bioavailability of CZP after oral administration of tested formulae in mice

The mean plasma concentration as a function of time for the different formulations of CZP after oral administration is summarized in (table 5) and (fig.11 -13). From these data, it is clear that, there was a difference between the mean plasma concentration as a function of time for CZP after oral administration of all tested formulae at all time intervals compared to CZP alone. Also, there is a notable difference in the C_max and T_maxbetween CZP alone and the tested formulae.

Table (5): Comparison between the mean plasma concentration after oral administration of CZP formulae to mice.

Time	Clonazepam	CZP –PVP (1:4)	CZP-PEG4000 (1:4)
0	0	0	0
0.5	1.99+1.23	13.25+5.23	17.64+2.21
1	2.07+2.43	16.04+6.47	20.87+3.44
2	1.92+1.82	20.41+5.85	26.09+2.83
4	1.89+6.90	22.91+2.87	29.68+5.89
6	1.51+4.21	24.01+0.17	30.67+3.20
8	1.50+0.94	24.07+1.97	33.34+4.99
10	1.314+2.43	24.44+9.46	34.34+3.44
12	0.68+1.21	22.36+0.18	30.76+3.20
24	0.49+2.84	17.18+1.81	21.49+4.83

Data are presented as means ± SD, n= 3.

Figure (11): Mean plasma concentration of Clonazepam after oral administration of Clonazepam solution.

Data are presented as means ± SD, n= 3.

Figure (12): Mean plasma concentration of Clonazepam after oral administration of CZP-PEG 4000 (SD 1:4)

Data are pesented as means ± SD, n= 3.

Figure (13): Mean plasma concentration of Clonazepam after oral administration of CZP-PVP K30 (SD 1:4)

Data are presented as means ± SD, n= 3.

The pharmacokinetic parameters of CZP represented by the value of C_max (ng/ml), T_max (hr), K_e (hr^-1), t₁_/₂(hr), AUC_0-24(ng.hr.ml^-1) and AUC_0-α (ng.hr.ml^-1) are illustrated in table 6.

From the obtained results, it is obvious that, CZP alone has a very poor and slow absorption, where the peak plasma concentration was 2.12±0.09 ng/ml that was reached within 0.96±0.27 hr, whereas, following oral administration of formulae containing CZP-PEG 4000 and CZP-PVP the C_maxwas; 32.43±1.68 and 24.17±0.87 hr respectively. While in the same time 0.96 hr of clonazepam alone , the C_max for PVP K30 and PEG 4000 were, 5.74± 0.55 and 7.24±0.73 respectively. In addition, the mean time T_max was 4.30±0.62 and 4.04±0.42, hr, respectively. These results were correlated with the in vitro release results and may be attributed to the enhanced dissolution of solid dispersions formulations due to the solubilizing effect of carriers (Arias et al., 1996) while hydrophobic property of Clonazepam prevented its contact with the dissolution medium which led to a slow dissolution rate and low bioavailability (Tantishaiyakul et al., 1996).

As a result, the bioavailability of CZP was increased for these formulations compared to CZP alone. Also these results were compared with standard CZP (Rivotril^®) whose C_maxwas 15.87 ng/ml, T_max was 1.72 hr and AUC_0-α was 405.96 ng.hr.ml^-1(Cavedal et al., 2007). So, it is clear that the bioavailability was significantly increased in case of CZP-PEG 4000 and CZP-PVP compared to the commercial CZP formulation and there was a significant difference (P<0.001) between all formulations and pure CZP in C_max value, T_max, K_ele, K_abs, AUC_0-24, AUC_0-α, t₁_/_{2 abs} and t₁_/_2
ele.

Table (6): Pharmacokinetic parameters after oral administration of Clonazepam in various formulations (0.2 mg/kg CZP) in mice.

Parameter	Formulae
Parameter	CZP	CZP-PVP (SD) (1:4)	CZP-PEG 4000 (SD) (1:4)
C_max(ng/ml)	2.12±0.09	24.17±0.87*	32.43±1.68*
T_max(hr)	0.96±0.27	4.04±0.42*	4.30±0.62*
K_ele (hr^-1)	0.067±0.44	0.014±0.038*	0.015±0.031*
t₁_/₂(hr)	10.25±1.57	48.77±17.78*	44.40±22.06*
K_abs (hr^-1)	4.41±11.55	1.08±6.88*	0.97± 4.33*
t₁_/_2abs(hr)	0.15±0.06	0.63±0.10*	0.70±0.15*
AUC_0-24(ng.hr.ml-1)	25.65±890	499.83±770*	664.05± 930*
AUC_0-α (ng.hr.ml-1)	33.59±3.80	1801.89±579.34*	2221.99±961.08*

Data are presented as means ± SD, n= 3, *:significantly different at P<0.001 using one way ANOVA and ? post hock test.

4. CONCLUSION:

The study has demonstrated that dispersion of CZP into water-soluble carriers like PEG 4000 or PVP K30 changed the crystallinity of CZP according to type and amount of the polymer. The formation of CZP-PEG 4000 or CZP-PVP K30 represents a suitable modification for improving its availability. Many factors contributed to faster release rate such as a decrease in particle size, a decrease in agglomeration of particles, an increase in wetability and a decrease in crystallinity of the drug.

Pharmacological results have proven that Clonazepam formulations show faster, better and more efficient pharmacological effects than commercial formulae against PTZ-induced seizures.

Bioavailability studies have demonstrated that, Clonazepam formulations containing PVP and PEG 4000 SDs show better release of Clonazepam with high concentration level in the plasma which is indicated by high C_max and T_maxwhen compared with drug solution alone and commercial formula. Also, these formulations showed higher bioavailability in comparison with Clonazepam solution alone and commercial formulae.

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Received on 06.02.2012 Modified on 26.02.2012

Asian J. Research Chem. 5(4): April 2012; Page 446-455