INTRODUCTION:

Nowadays the drugs are administrated orally about 74% and are not much valuable as most wanted. To improve such characters Transdermal Drug Delivery System (TDDS) was investigated. TDDS recognized itself as a major part of novel drug delivery systems with the creation of current time of pharmaceutical dosage forms. Transdermal dosage forms are becoming popular because of their beneficial advantages, still a costly alternative to conventional formulations.

Easy termination of the treatment is one of the potential advantages of transdermal drug delivery, TDDS is gaining more importance because it advantageous over conventional dosage forms and can improve bioavailability, control absorption, extra uniform plasma levels, painless and reduced adverse effects easy application and flexibility of terminating drug administration. ^[1]A transdermal patch or skin patch is a medicated adhesive patch that is placed on the skin to deliver a specific dose of medication through the Skin and into the bloodstream.^[2]Today the most common transdermal system present in the market mainly depends on semi-permeable membranes which were called as patches. Transdermal Drug Delivery Systems (TDDS) are self-contained, detachable dosage forms that, when applied to intact skin, are designed to deliver the drug(s) through the skin to the blood stream. Transdermal patch typically composed of an outer covering (barrier), a drug moiety that may have a membrane which can control drug release, a contact adhesive applied to some or all parts of the system and the system/skin interface for preventing the dislocation of system resulting in a termination of treatment, and a protective liner that is removed before the patient applies the system. This system is defined in terms of the release rate of the drug(s) from the system and surface area of the patch and is expressed as mass per unit time for a given surface area. With these drug products, the skin typically acts as the rate-controlling membrane for the drug input into the body. The system surface area and total duration of drug release from the system may also be stated. ^[13]TDDS work on the mechanism of diffusion: The diffusion drug occurs from the drug reservoir, through the rate-controlling membrane and/or contact adhesive if present, and then through the skin into the blood stream. Typically, the constant drug delivery and a steady state blood concentration can be achieved by developing modified release system. Following removal of the system, blood concentration decreases at a rate consistent with the pharmacokinetics of the drug. ^[3]

Figure 1: Transdermal patch showing its different components:

Hypertension (HTN or HT), also termed as high blood pressure, is a long term medical condition in which the blood pressure in the arteries is persistently increased. Blood pressure is expressed by two measurements, the systolic and diastolic pressures, which are the maximum and minimum pressures respectively in the arterial system. The systolic pressure occurs when the left ventricle is most contracted; the diastolic pressure occurs when the left ventricle is most relaxed prior to the next contraction. Normal blood pressure at rest is ranges between of 100– 140 millimeters mercury (mmHg) systolic and 60–90 mmHg diastolic. Hypertension is present if the resting blood pressure is persistently at or above 140/90 mmHg for adults; different numbers apply to children. Hypertension usually does show symptoms initially, but sustained hypertension over time is a major risk factor for hypertensive heart disease, coronary artery disease, stroke, aortic aneurysm, peripheral artery disease, and chronic kidney dysfunction. Cardiovascular diseases account for a large proportion of all deaths and disability worldwide. Global Burden of Disease (GBD) study reported that there were 5.2 million deaths due to cardiovascular diseases in economically developed countries and 9.1 million deaths due to the same causes in developing countries. Worldwide prevalence estimates or hypertension may be as much as 1 billion patients, and approximately 7.1 million deaths per year may be attributable to hypertension. 57% of all stroke deaths and 24% of all coronary heart disease deaths in India are caused by hypertension. Pooling of Indian epidemiological studies shows that hypertension is present in 25% urban and 10% rural individuals. Therefore cost effective approaches to optimally control blood pressure among Indians are very much needed. Transdermal systems are suitable for disease which demands chronic treatment like hypertension etc. Drugs for long term therapy of hypertension angiotensin converting enzyme (ACE) inhibitors are becoming first choice. ACE inhibitors are known for their reliable activity and their good tolerability. The high variation in the ACE inhibitors blood levels or their active forms leads however to uncertain courses of action. To make the action of ACE inhibitors independent of the metabolic condition of the patients, a pharmaceutical form which makes possible a reliable reproducible systemic supply of the active compounds would be desirable. A more reliable steady action can be achieved if ACE inhibitors are given in the form of their prod rugs or their active forms systemically through transdermally. In all grades of hypertension the angiotensin converting enzyme (ACE) inhibitors are one of the first choices of drugs. Most patients require relatively low doses which are well tolerated. When used alone, they control hypertension in 50-60% of individuals. Ramipril is a prodrug belongs to the class of ACE inhibitors. It is used in the treatment of hypertension and related cardiovascular diseases. It acts on the Renin–Angiotensin Aldosterone system. It inhibits the conversion of the inactive angiotensin-I to the highly potent vasoconstrictor, angiotensin-II, and also reduces the degradation of bradykinin. Ramipril, a potent antihypertensive agent has been used in the treatment of hypertensive disorders. The extent of absorption is at least 50-60% and is not significantly influenced by the presence of food in the GI tract, Protein binding of Ramipril is about 73%. It is a highly lipophilic and poorly water soluble drug, with absolute bioavailability of 28-35%.^{[4, 5]}

MATERIALS AND METHODS:

Preparation of Transdermal patch:

In the present study, drug loaded transdermal patches of Ramipril were prepared by mercury substrate method. Weigh accurate quantity of Eudragit RL 100, Ethyl cellulose and Eudragit RS 100 (polymers) and Ramipril (drug) in the beaker and mix well. Dissolve the above mixture in the solution of Methanol & Chloroform and to this solution, a measured volume of Di-methyl Formamide (DMF permeation enhancer) and Di-butylpthalate (DBP plasticizer) should be added and stirred well. This solution was allowed to for 10min without disturbing until clear solution is formed. This solution is poured slowly in the center of the Petri dish which is previously filled with mercury to prevent sticking. To control the evaporation of solvent the funnel should be inverted on the Petri-dish and allow to evaporate in the room temperature for 24hrs. The films were cut into small pieces containing equivalent of 5mg of the drug per patch. ^[6]

Evaluation of Transdermal Patch:

Physical Appearance: ^[6]

The formulated patches were physically examined for color, clarity and surface texture.

Thickness Uniformity of the Patch: ^[7]

The thickness of the drug prepared patch is measured by using a digital micrometer at different point of patch and determines the average thickness and standard deviation for the same to ensure the thickness of the prepared patch.

Weight Uniformity: ^[8]

The patches were study for weight variation by individually weighing 10 selected patched randomly. Such variations studies were carried out for each formulated patches.

Folding Endurance: ^[8]

The folding endurance of patches was determined by repeatedly folding one film at the same place till it break. The number of times the film would be folded at the same place without breaking was taken as the value of folding endurance.

Drug Content Uniformity: ^[7]

A specified area of patch is to be dissolved in a suitable solvent in specific volume. Then the solution is to be filtered through a filter medium and analyses the drug contain with the suitable method (UV or HPLC technique).Then take the average of three different samples.

Scanning Electron Microscopy: ^[9]

The films were mounted onto stubs using double sided adhesive tape and sputter coated with platinum using a sputter coater (Edward S 150, UK). The coated films were observed under SEM (JEOL, JSM-6360, Kyoto, Japan) at the required magnification at room temperature. The acceleration voltage used was 10 kV with the secondary electron image as a detector.

X-RD Analysis: ^[9]

The spectra were recorded using a Philips, PW-171, X-ray diffractometer with Cu-NF filtered CuK radiation. Quartz was used as an internal standard for calibration. The powder x-ray diffractometer was attached to a digital graphical assembly and computer with Cu-NF 25kV/20mA tube as a CuK radiation source in the 2θ range 0-50 ºC.

Fourier Transform Infrared Spectroscopy (FTIR): ^[10]

The FTIR analysis of Ramipril was carried out for qualitative of the compound. The potassium bromide pellet of approximately 1 mm in diameter incorporating Ramipril was prepared by grinding 3-5 mg of Ramipril with 100-150 mg of potassium bromide in pressure compression machine. The resulting pellet was then mounted in the FTIR compartment and was scanned at a wavelength ranging from 4000 cm^-1. The observed peaks were reported for functional groups.

Differential Scanning Colorimetry (DSC) Studies: ^[11]

The prepared formulations were subjected to differential scanning calorimeter at a heating rate of 10ºC /min over a temperature range of 0-300 ºC. The sample of pure drug and prepared patches were hermetically sealed in an aluminum pen. Nitrogen gas was purged at the rate of 10 ml/min for maintained inert atmosphere.

Table 1: Formulation Table of Ramipril Patches:

Formulation	Code	RM 1	RM 2	RM 3	RM 4	RM 5	RM 6	RM 7	RM 8	RM 9
Drug (mg)	Ramipril	50	50	50	50	50	50	50	50	50
	Eudragit RL 100	100	200	100	200	-	200	150	150	100
Polymer (mg)	Ethyl cellulose	200	100	100	200	200	-	150	100	150
	Eudragit RS 100	100	100	200	-	200	200	100	150	150
Solvent (ml)	Chloroform	5	5	5	5	5	5	5	5	5
	Methanol	5	5	5	5	5	5	5	5	5
Penetration enhancer (% w/v)	DMF	2	2	2	2	2	2	2	2	2
Plasticizer (% w/v)	DBP	3	3	3	3	3	3	3	3	3

Water Vapour Transmission (WVT) Rate: ^[12]

Glass vials of equal diameter were used as transmission cells. These transmission cells were washed thoroughly and dried in an oven. About 1gm anhydrous calcium chloride was placed in the cells and the brim. The cells were accurately weighed and kept in a closed desiccators containing saturated solution of potassium chloride to maintain a humidity of 84%.The cells were taken out and weighed after 6, 12, 24, 36, 48 and 72 hrs of storage. The amount of water vapour transmitted was found using the formula.

Water vapour transmission rate is usually expressed as the number of grams of moisture gained/h/cm².

In-Vitro Drug Release study: ^[12,13]

Preparation of mice skin: The Swiss albino mice (weight range of 20‐25gm) were decapitated. The abdominal skin of excised hairless mice skin was separated along the epidermal junction and it was kept in water bath, which was maintained at 60 ºC for 50s. The heat‐treated skin was cleared of its subcutaneous fatty substances and immediately kept in normal saline solution for flattering and smoothing. The cell consists of the donor and the receptor chambers. The donor compartment is open at the top and is exposed to the atmosphere. The receptor compartment is surrounded by a water jacket for maintaining the temperature at 37 ºC ± 2 ºC and is provided with a sampling port. The excised abdomen rat skin containing patch was mounted between donor and receptor compartment. The diffusion medium was phosphate buffer of pH 7.4, which was stirred with the help of magnetic bead (operated by a magnetic stirrer). Diffusion media was stirred to prevent the formation of concentrated drug solution just beneath the membrane. Samples from the receptor compartment were taken at various intervals of time over a period of 24 hours and the concentration of the drug was determined by UV Spectrophotometric method using the standard curve. Amount of drug diffused that is cumulative % of drug permeated at various time intervals was calculated and plotted against time.

Skin Irritation Study: ^[14]

A primary skin irritation test was performed on six healthy rabbits, (weight between 2 to3.5 kg). The patch of area 3.14cm² was used as a test patch. The dorsal surface of rabbits was cleared well and the hair was removed by using a depilatory preparation. The skin was cleared using rectified spirit. The transdermal patch was placed on the dorsal surface of the abdominal skin by using an adhesive tape. The patches were removed after 24 hr and the skin was examined for erythema and edema.

Stability Studies: ^[15]

The stability studies of the formulated transdermal patches were studied on prepared film at different temperature and humidity 25-30°C (60%RH) and 45-50°C (75%RH) over a period of60 days. The patches were wrapped in aluminum foil and stored in desiccators for stability study. The patches were tested for drug content and other parameters at regular intervals (0, 15, 30, 45and 60 days).

RESULTS:

Table 2: Spectroscopic estimation of Ramipril in 7.4 P^H phosphate buffer at 210nm:

Concentration (mcg/ml)	0	2	4	6	8	10
Absorbance	0	0.059	0.104	0.158	0.189	0.237

Figure 2: Calibration curve for the estimation of Ramipril in phosphate buffer P^H 7.4:

Spectra 3: DSC Thermogram of Ramipril (RM):

Spectra 4: DSC Thermogram of Drug free patch of RM-7:

Spectra 5: DSC Thermogram of Drug loaded patch of RM-7:

Spectra 6: XRD diffraction of Ramipril (RM):

Spectra 7: XRD diffraction of drug free RM-7 patch:

Spectra 8: XRD diffraction of drug loaded RM-7 patch:

Table 5: Physicochemical properties of Ramipril patches:

Sr. No.	Formulation	Physical Appearance	Weight (gm)	Thickness (mm)	Drug Content	Folding Endurance
1	RM-1	++	0.4396 (0.013)	0.1534 (0.004)	82.46	>229
2	RM-2	++	0.4842 (0.014)	0.1146 (0.003)	93.36	>241
3	RM-3	++	0.4878 (0.014)	0.1188 (0.003)	92	>244
4	RM-4	++	0.5909 (0.017)	0.0816 (0.002)	86.32	>251
5	RM-5	++	0.538 (0.016)	0.115 (0.003)	90.32	>248
6	RM-6	++	0.5727 (0.017)	0.1404 (0.004)	88.8	>238
7	RM-7	++	0.528 (0.015)	0.1362 (0.003)	91.12	>262
8	RM-8	++	0.4696 (0.014)	0.1282 (0.003)	87.84	>222
9	RM-9	++	0.5278 (0.015)	0.0824 (0.002)	85.28	>245

Table 6: Water Vapor Transmission (WVT):

Formulation

RM-1

RM-2

RM-3

RM-4

RM-5

RM-6

RM-7

RM-8

RM-9

WVT Rate constant (gm.cm/cm² 24 hrs.)

0.001457857

0.001270952

0.001513929

0.002149405

0.002728809

0.002336309

0.0011775

0.001476548

0.001775595

Table 7: In-vitro drug release:

Formulation		RM-1	RM-2	RM-3	RM-4	RM-5	RM-6	RM-7	RM-8	RM-9
%Drug Release		92.84 %	81.43 %	89.51 %	78.86 %	72.20 %	73.98 %	67.58 %	87.33 %	95.38 %

Figure 4: WVT profile of Ramipril patch:

Figure 5: In-vitro permeation of Ramipril transdermal patch:

Table 8: SKIN IRRITATION TEST OF RAMIPRIL PATCHES ON RABBITS:

FORMULATIONS	ERYTHEMA	EDEMA
Control patch	++	+
RM-1	-	-
RM-2	-	-
RM-3	-	-
RM-4	-	-
RM-5	+	-
RM-6	-	-
RM-7	-	-
RM-8	-	-
RM-9	-	-

-Nil, +Mild, ++Severe, +++Very severe

RESULT AND DISCUSSION:

The appearances of prepared transdermal patches of Ramipril were thin, flexible, elastic, smooth and transparent/translucent. The thickness was measured by a Digital micrometer; there is no difference in the thickness within the formulations. The dried patches of 1cm² were rate weighed on electrical single plan balance (Shimadzu Corporation). The results indicate lesser SD value more uniform are the patches. The folding endurance result indicates the increase in Ethyl cellulose concentration decreases folding endurance. The drug contents indicate all the Ramipril patches uniform drug content and the values ranged from 82.46 % to 93.42 %. The increase in the Ethyl cellulose concentration increases the drug content. The SEM of formulation of RM-7 reveals that the surface of the film was porous, smooth and free from air bubbles. The DSC thermograph of pure drug has shown and sharp endothermic peak of 133.76 ^◦C due to its melting point, but this peak is not seen in the other thermographs of drug loaded formulations. This indicates that the drug has got uniformly dispersed in an amorphous from in the formulations. The result indicates that the increase in the concentration of Ethyl cellulose shows the better WVT. Formulation RM-5 shows highest WVT and RM-7 shows least WVT value indicating that increase in the concentration of Eudragit RS 100 decreases the WVT. In-vitro release studies were performed as the Ethyl cellulose concentration increase the drug release also increases. The results indicates that RM-5 patch produced mild erythema as compared to control patch, whereas all other patches shows no evidence of erythema and edema, this indicates skin acceptability of the test patches for topical application. The film were subjected for stability studies for one month and observed for change in appearance and flexibility at a temperature 40 ºC. There were no physical changes in appearance, flexibility. The percentage of degradation with respect to drug content of the patch was observed was low (2-3%). Hence, the formulations were stable.

CONCLUSION:

It can be concluded that Ramipril with Eudragit RS 100, Eudragit RL 100 and Ethyl cellulose with incorporation of DBP produced smooth and flexible film. Characterization and evaluation showed that RM-7 formulation is found to be optimum. The overall results reported in the present study, it is proposed that Ramipril can be used in the formulation of matrix type transdermal drug delivery system to prolong the drug release.

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Received on 01.08.2016 Modified on 21.08.2016

Research J. Pharm. and Tech 2016; 9(12):2127-2134.

DOI: 10.5958/0974-360X.2016.00432.7

Sr. No.	Water Solubility (mg/ml)	Partition coefficient (log P)	Melting point	P^H	Drug polymer interaction
1.	3.5	0.92	109⁰C	5.2	No

Drug/polymer	C=O Stretch	O-H Stretch	N-H Stretch	C-H Stretch	C-C Stretch	C-O Stretch
Ramipril	1783.14	3279.10	700.18	2931.90	1695.05	1033.88
Ramipril + Polymers	1738.42	3252.09	752.26	2936.16	1651.12	1064.74