INTRODUCTION:

Diltiazem hydrochloride is a calcium channel blocker used in the treatment of arrhythmia, angina pectoris and hypertension¹. The literature survey reveals that it undergoes variable and extensive first pass metabolism before entering into systemic circulation^2,3 and varies with species^4,5. It is well recognized that the transdermal administration of drugs, which undergoes first passm etabolism, can improve the bioavailability, reduces the dosing frequency compared to the oral route⁶. Hence in the present study, the in vivo performance of orally and transdermally administered Diltiazem hydrochloride was evaluated in rabbits in a randomized cross over design. Membrane-moderated transdermal systems of Diltiazem hydrochloride were selected for the transdermal administration based on the invitro studies⁷. The pharmacokinetic parameters of both oral and transdermal formulation analysis were compared statistically to reveal the relative bioavailability and to justify relative rate of extensive first pass metabolism of diltiazem hydrochloride.

MATERIALS AND METHODS:

Diltiazem hydrochloride (gift sample from NATCO Pharm. Pvt. Ltd. Hyderabad), cellulose acetate (S.D Fines chemicals Ltd. Mumbai), Ethyl acetate (Qualigens), Dibutyl phthalate (Ranbaxy Laboratories), Sodium carboxy methyl cellulose (S.D Fines chemicals Ltd. Mumbai), Poly ethylene glycol-6000(S.D Fines chemicals Ltd. Mumbai) were of pharmaceutical grade and obtained commercially.

Animals: New Zealand male rabbits (1.2 - 1.8 kg) maintained at 25 + 1^oC were used for the study. The animals were housed in stainless steel metabolic cages and provided standard diet and water ad libitum.

Preparation of membrane-moderated transdermal systems

Membrane-moderated transdermal systems of Diltiazem hydrochloride were prepared by incorporating the drug reservoir within a shallow compartment moulded from a drug-impermeable backing membrane and 2% w/v cellulose acetate rate-controlling membrane casted with ethyl acetate-methanol (8:2)employing dibutyl phthalate (40% w/w of dry polymer) as plasticizer .

Solvent evaporation technique was employed for the preparation of cellulose acetate films. The polymer solutions were prepared by dissolving the polymer (2% w/w cellulose acetate) in 20 ml of ethyl acetate-methanol (8:2). Dibutyl phthalate at a concentration of 40% w/w of the polymer was used as a plasticizer. 20 ml of the polymer solution was poured in a Petri plate (9.4 cm diameter) placed on a horizontal flat surface. The rate of evaporation was controlled by inverting a funnel over the petri plate. After 24 hours the dried films were taken out and stored in a desiccator. A circular silicon rubber ring with an internal diameter of 2.5cm and a thickness of 3mm was fixed on to a backing membrane (an unperforated adhesive strip; supplied by Johnson and Johnson Limited ,Mumbai). This serves as a compartment for drug reservoir.

Drug reservoir gels were formulated as per the composition given in Table 1. The required quantities of polymer was weighed and transferred separately into a mortar. It was triturated with 5 ml of water. Specified amount of Diltiazam hydrochloride, methyl paraben, and propyl paraben were weighed accurately and dissolved in glycerin. The resulting drug solution was incorporated into the polymer dispersion slowly with continuous trituration to obtain a gel. The gel was transferred in to a measuring cylinder and the volume was made up to 20 ml with distilled water. 1 g of medicated gel was taken into the compartment as a drug reservoir. Cellulose aceate membrane of known thickness was fixed on the ring with glue to form a membrane moderated therapeutic systems.

Table 1 : Reservoir of the Diltiazem hydrochloride membrane-controlled Transdermal systems

Ingredients	Quantity
Diltiazem hydrochloride (mg)	5000
Sodium carboxy methyl cellulose: Polyehylene glycol6000(1:1)(mg)	4000
Methylparaben(mg)	100
Propylparaben(mg)	50
Tween 20(ml)	1.805
Glycerin (ml)	10
Distilled water (ml) up to	100

In-Vivo Evaluation:

Prior approval by Institutional animals ethics committee was obtained for conduction of experiments (Ref:IAEC/I-3/BCOP/2007-2008).

Pharmacokinetic evaluation:

The pharmacokinetic performance of Diltiazem hydrochloride following oral and transdermal administration was studied in a randomized cross over design in rabbits. Animals were fasted 24 hrs prior to the administration of the drug formulation, but had free access to water. One day before the experiment, hair on the abdominal area was clipped by applying depilatory for 10 min and washed with distilled water. On the day of experiment the animals were anaesthetized with urethane (1 gm/kg, i.p.). Following anaesthesia, animals were secured in a supine position.

Subject selection:

Twelve healthy rabbits with a mean age of 10 ± 2 weeks and with a mean body weight of 3 ± 0.2 kg included in the above investigation for oral versus transdermal formulations of Diltiazem hydrochloride.

Study design:

The study was of a non-blinded, open-label design. Subjects were fasted for at least 24 hrs prior to timing of dose. One day before the experiment, hair on the abdominal area was clipped by applying depilatory for 10 min and washed with distill water. Overnight-fasted rabbits, whose hair had been removed prior to the experiment, were divided into two treatment groups (n = 6 per group) as follows:

(1) Group I (Diltiazem hydrochloride solution (5 ml) containing 7 mg of drug)

(2) Group II (Transdermal formulation containing 7 mg of drug )

Blood sampling:

About 1 ml of blood samples were drawn at 0 (before drug administration), 0.5, 1.0, 2.0, 3.0, 4.0 and 6.0 hrs after oral administration and 2, 4, 6, 8, 12, 16, 20, 22,24,26,28,30 and 32 hrs after administration of transdermal formulation involved in the study at a dose equivalent to 7 mg of Diltiazem hydrochloride. Blood sample volume was replaced by administration of isotonic saline. Blood samples were collected into heparinized tubes and centrifugation at 3000 rpm for 10 min and plasma samples were stored at –20°C until analysis by a known LC-MS/MS method⁸.

Estimation of Diltiazem hydrochloride in plasma

The estimation of Diltiazem hydrochloride in plasma was carried out by LC-MS/MS method. A summary of the chromatographic and mass spectrometric conditions are shown in Table 2 and Table 3.

Table 2: Summary of the Chromatographic and Mass spectrometric conditions

Chromatographic and Mass spectrometric conditions
HPLC	Agilent Series 1200
Mass spectrometer	API 4000 QTRAP
Ion source	Heated nebulizer
Polarity	Positive ion mode
Detection ions
Diltiazem Verapamil	455.5 amu (parent), 165.1 amu (Product) 415.3* amu (parent), 178.0* amu (product)
Column	Purospher Star RP-18, 4.6x150mm, 5µ
Mobile phase	Ammonium acetate buffer (pH 4.5 ±0.1): Acetonitrile (20:80)
Flow rate	1 ml/min
Retention time	Diltiazem 1.6 to 2.4 minutes ISTD 1.5 to 2.3 minutes
Run time	3.00 minutes
Curtain Gas (CUR)	20.0 PSI
Nebulizer Current (NC)	3.0
Ion Spray Voltage (IS)	5500V
Temperature (TEM)	475.0 °C

Table 3: Summary of the MRM parameters and

Parameter	Diltiazem	Verapamil
Declustering Potential (DP)	70.0	70.0
Entrance Potential	10.0	10.0
Collision Energy (CE)	36.0	36.0
Collision Cell Exit Potential (CXP)	12.0	12.0

Standard solutions

A standard stock solution of Diltiazem hydrochloride (100 mg/mL) were prepared by dissolving an accurately weighed samples in methanol. The standard solutions used to construct the calibration curve were prepared by adding known amounts of Diltiazem hydrochloride (0.101 to 201.846 ng/ml) to blank plasma.

Extraction procedure:

Step 1: Blank, calibration curve standards and the subject samples were withdrawn from the deep freezer and allowed them to thaw. The thawed samples were vortexed to ensure complete mixing of contents. To 0.25 ml of plasma sample in a ria vial, 25ul of Verapamil standard (719.880ng/ml) was added. To plasma blank, 25 ul of 60%methanol in water solution was added and vortexed the samples to ensure complete mixing of contents.

Step 2: Oasis HLB 30mg/lcc cartridge were taken, (new cartridge for each sample) on to a positive pressure processor and the following procedure was followed. 1 ml methanol and followed by 1 ml of water was added. (Taken care not to dry the cartridge). The sample was applied and allowed it to dry for about 5 minutes under positive pressure. The cartridge was rinsed twice with 1ml of water and was allowed to dry under positive pressure for about 5 minutes. The cartridge was rinsed with 1ml of 5% methanol in water solution and was allowed to dry under positive pressure for about 5 minutes. The drug was eluted into 1 ml of methanol and was allowed to dry under positive pressure for approximately 2 minutes. The organic layer was evaporated under a stream of nitrogen gas at 45°C. The residue was reconstituted with 0.5 ml of mobile phase and vortexed. The samples were transferred in to auto-injector vials and loaded the vials in to auto sampler. 20 ul of sample was injected in to LC-MS/MS system.

Figure 1 : Calibration curve for estimation of diltiazem hydrochloride in plasma

Calibration curve:

Analyte concentrations of stock dilutions of standard Diltiazem hydrochloride solution with plasma were shown in Table 4.A calibration curve (Figure:1) was obtained by plotting peak area ratios of Diltiazem to Verapamil (y-axis) against Diltiazem concentration (x-axis).

Determination of Pharmacokinetic Parameters:

Various pharmacokinetic parameters such as peak plasma concentration (C_max), time at which peak occurred (T_max), area under the curve (AUC), elimination rate constant (K_el), biological half-life (t_½) and mean residence time (MRT) were calculated using the noncompartmental pharmacokinetics data analysis software PK Solutions 2.0™(Summit Research Services, Montrose, CO, USA).

Statistical analysis of the pharmacokinetic parameters:

The pharmacokinetic parameters of the tested formulations were statistically analyzed using paired sample’s t-test for normal distributed results of C_max, K_a, AUC_0-24 and AUC_0-α values. All tests were performed at 0.001 level of significance.

Pharmacodynamic evaluation :

Pharmacodynamic response in animals after oral and transdermal administration of Diltiazem hydrochloride was assessed by the degree of reduction of adrenaline induced arrhythmia⁹. The animals were fasted for 24 hrs prior to the administration of drug formulations but had free access to water. Animals were anaesthetized with urethane (1gm/kg i.p.). Following anesthesia normal electrocardiogram for all the animals was recorded. Before administering the drug formulations, adrenaline (30 µg/kgi.v.) in normal saline was administered into the marginal ear vein, electrocardiogram was recorded immediately and these cardiograms served as control. Animals were left for one hour and the drug formulations were administered as described in pharmacokinetic studies. Adrenaline was re administered after 1.0, 3.0 and 6.0 hrs after oral and 4.0, 8.0, 12.0, 20.0 and 24.0 hrs after transdermal administration of Diltiazem hydrochloride. The reduction in number of ectopic beats before and after adrenaline injection at each time interval was calculated and taken as response to the adrenaline challenge at that time. Net reduction in number of ectopic beats after the administration of drug formulations following adrenaline challenge was compared with the per se adrenaline response.

RESULTS AND DISCUSSION:

Plasma Diltiazem hydrochloride concentrations following oral and transdermal administration at different times were calculated and are shown in Fig 1 and 2. Pharmacokinetic parameters such as absorption rate constant, elimination rate constant, half life, AUC, AUMC and MRT were calculated from the plot of time versus plasma concentration and reported in Table 5 and subjected to statistical analysis. The results indicated that the parameters significantly differed following transdermal administration, compared to oral administration.

Table 4 :Analyte concentrations of stock dilutions of standard diltiazem hydrochloride solution with plasma

Sample name	Analyte concentration	Analyte peak area	IS Peak Area	Area Ratio	Calculated Concentration (ng/ml)	Accuracy (%)
Aqueous mixture	N/A	2247	1940259	0.001160	0.094	N/A
Plasma blank	0	0	0	0	N/A	N/A
Blank+ISTD	0	0	1217732	0	N/A	N/A
CC1	0.101	1325	1102974	0.0012	0.098	97.03
CC2	0.202	2696	1132350	0.00238	0.197	97.52
CC3	0.506	7223	1258131	0.005740	0.481	95.06
CC4	2.022	30281	1277746	0.0237	1.995	98.66
CC5	10.11	150940	1281233	0.118	9.933	98.25
CC6	40.443	584605	1221929	0.478	40.351	99.77
CC7	101.107	1494406	1242395	1.2	101.455	100.34
CC8	202.213	2956131	1271509	2.32	196.099	96.98

Table 5: Statistical treatment of pharmacokinetic parameters (Mean ± S.D.) of Diltiazem hydrochloride obtained with oral and transdermal formulations

Pharmacokinetic parameter	Oral solution	Transdermal formulation	Calculated value of ‘t’
C_max (ng/ml)	71.4 ± 0.31	54.6 ± 0.41	36.70***
MRT (h)	2.4 ± 0.01	16.5 ± 0.14	75.50***
t_1/2 (h)	1.33 ± 0.011	2.09 ± 0.014	40. 75***
K_el (h^-1)	0.52 ± 0.012	0.33 ± 0.014	8.87***
K_a (h^-1)	1.44 ± 0.01	0.59 ± 0.02	19.67***
AUC_0-_ (ng h/ml)	192 ± 1.43	1405.1. ±2.07	256.60***

Values are presented in Mean  SD (n = 6); *p<0.05, ** p<0.01,*** p<0.001

Table 6: Percent reduction of ectopic beats after oral and transdermal administration of diltiazem hydrochloride in rabbits.

Formulation	Percent reduction of ectopic beats at
Formulation	1h	4h	6h	8h	12h	16h	20h	24h
Oral	99.8 ±6.2	58.2 ±4.6	28.4 ±2.3	---	---	---	---	---
Transdermal	---	56.2 ±5.6	77.4 ±4.2	97.6 ±3.8	96.8 ±3.2	96.6 ±3.4	96.4 ±3.6	96.2 ±3.5

The results from the oral administration of Diltiazem hydrochloride indicated the maximum plasma concentration (C_max) 71.40 ± 0.31 ng/ml at 1 hrs (t_max) while transdermal administration exhibited the steady state concentration of 54.60 ± 0.41 ng/ml after an initial lag time 12 hrs. The oral administration of Diltiazem hydrochloride resulted in a low and quite variable AUC of 192 ± 1.43 ng.hr/ml, where as the transdermal resulted in AUC of 1405.1± 2.07 ng.hr/ml. The MRT of transdermal administration (16.5 ± 0.14 hrs) was found to be more than oral administration (2.4 ± 0.01 hrs).

Figure 2: Plasma concentration-time curves of diltiazem hydrochloride following oral administration

The concentration of Diltiazem hydrochloride in plasma was found to be stabilized and maintained in a narrow range over the study period up to 24 hrs for transdermal formulation where as the concentration was decreased rapidly up on oral administration (Fig. 3). The maximum plasma concentration (C_max) was attained at 1 hrs after oral administration (7 mg) and it was observed after 12 h upon application of transdermal formulation of same dose.

The mean residence time (MRT) was found to be increased significantly (p<0.001) for transdermal application on comparison with oral administration. Though both the formulations containing an equivalent amount of Diltiazem Hydrochloride (7 mg), the AUC_0-∞ values observed with transdermal formulations (p<0.001) was found to be seven fold than that of oral solution. The low t_max and high C_maxvalues following oral administration was due to rapid absorption from the gastro intestinal tract, in contrast the low C_maxand prolonged t_maxafter transdermal administration was due to barrier properties of skin that leads to accumulation of drug into skin tissues in intial stages followed by continuous delivery into the systemic circulation.

Thus, the transdermal drug delivery system of Diltiazem hydrochloride indicated an enhancement in bioavailability significantly over oral formulations. The observed significant increase in bioavailability may be due to the reduced extensive first pass metabolism of Diltiazem hydrochloride up on transdermal administration.

The results of the present study concluded that the route of administration has significant influence on bioavailability of Diltiazem hydrochloride. Transdermal administration may influence the bioavailability by hindering the extensive first pass metabolism by organ such as intestine and liver.

Figure 3: Plasma concentration-time curve of diltiazem hydrochloride following transdermal administration

The percent reduction of ectopic beats after oral and transdermal administration are given in Table 6. In agreement with pharmacokinetic data maximum protection against adrenaline challenge was obtained at 1 hr after oral administration and is declined to one-third after 6 hrs. Whereas in case of transdermal administration a steady state antiarrhythmic activity was observed after 10.0 hrs and was prolonged over a period of 24 hrs. The low antiarrhythmic activity after oral administrations in good agreement with pharmacokinetic data which indicates that the plasma concentration of diltiazem declined rapidly and hence shorter duration of antiarrhythmic action. The in vivo pharmacokinetic studies revealed that the transdermal formulation of Diltiazem hydrochloride exhibited controlled release and absorption kinetics over longer periods of time which in turn the plasma concentrations was maintained in a narrow range over longer periods of time and leading to the conclusion that of Diltiazem hydrochloride transdermal formulations are suitable for once a day administration.

REFERENCES:

1. Buckley MMT, Grant, S, Goa, KL, McTavish D, Sorkin EM. Diltiazem. A reappraisal of its pharmacological properties and therapeutic use. Drugs. 1990; 39:757-806.

2. Chaffman M, Brogden RN. Diltiazem : A review of its pharmacological properties and therapeutic efficacy. Drugs.1985; 29:387-454.

3. Hermann P, Rodger SD, Remoner G, Thenot JP, London DR, Morselli PL. Pharmacokinetics of diltiazem after intravenous and oral administration. Eur J Clin Pharmacol. 1983; 24: 349-52.

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6. Chien YW. Transdermal controlled systemic medications. Marcel Dekker Inc, New York. 1987.

7. Sai Kishore V, Murthy T E. G K. Effect of Permeation Enhancers on the release and permeation kinetics of diltiazem Hydrochloride gels through mouse skin. Adv. Pharmacol. Toxicol.2008; 9 (1): 63-69.

8. Molden E, Helen Boe G, Christensen H, Reubsaet, High-performance liquid chromatography –mass spectrometry analysis of diltiazem and 11 of its phase I metabolites in human plasma. J.Pharm. Biomed. Anal 2003;33:275-85.

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Received on 23.06.2010 Modified on 03.07.2010

Research J. Pharm. and Tech. 4 (1): January 2011; Page 150-154

Ingredients

Quantity

Diltiazem hydrochloride (mg)

5000

Sodium carboxy methyl cellulose:

Polyehylene glycol6000(1:1)(mg)

100

50

Tween 20(ml)

1.805

Glycerin (ml)

10

Distilled water (ml) up to

100

Subject selection:

Figure 1 : Calibration curve for estimation of diltiazem hydrochloride in plasma

1h

4h

6h

8h

12h

16h

20h

24h

99.8 ±6.2

58.2 ±4.6

28.4 ±2.3

---

---

---

---

---

---

56.2 ±5.6

77.4 ±4.2

97.6 ±3.8

96.8 ±3.2

96.6 ±3.4

96.4 ±3.6

96.2 ±3.5