Research Article

SIMULTANEOUS IMPROVEMENT OF DISSOLUTION RATE AND STABILITY OF RAMIPRIL BY FORMATION OF UREA INCLUSION COMPLEXES

VIKAAS BUDHWAAR*, ARUN NANDA

Department of Pharmaceutical Sciences, Mahrshi Dayanand University, Rohtak, Haryana, India.

Received: 24 Nov 2011, Revised and Accepted: 05 Jan 2013

ABSTRACT

Ramipril is an angiotensin-converting enzyme (ACE) inhibitor, used to treat high blood pressure and congestive heart failure. In the present study, urea, a well-known adductor for linear compounds was successfully employed for inclusion of Ramipril—a substituted cyclic organic compound through a modified technique using oleic acid as rapidly adductible endocyte (RAE).The proportion of oleic acid and urea in the inclusion compounds was optimized for maximum dissolution of ramipril using 22 factorial design. Formation of urea inclusion compounds was confirmed by FTIR, DSC and XRD. Urea–Ramipril–RAE inclusion compounds containing varying proportions of guests were prepared and their thermal behavior studied by DSC. The inclusion compounds were also found to exhibit high content uniformity and markedly improved dissolution profile as demonstrated by increased dissolution efficiency in acidic , neutral and alkaline medium. Stability studies revealed that lesser amount of Ramipril in urea inclusion compounds degraded at 4oC, 25oC and 40oC than pure drug kept under same conditions. Studies reveal the possibility of exploiting co-inclusion of the drug in urea host lattice for simultaneous improvement of dissolution and stability.

Keywords: Adduction; Ramipril; Complexation; Dissolution; Dissolution rate; Inclusion compounds; Urea inclusion compounds, Normally Non-adductible endocyte, Rapidly adductible endocyte.

INTRODUCTION

Ramipril, inhibits the actions of angiotensin converting enzyme(ACE), thereby lowering the production of angiotensin II and also decreasing the breakdown of bradykinin which ultimately results in relaxation of arteriole smooth muscle leading to a decrease in total peripheral resistance, reducing blood pressure as the blood is pumped through larger diameter vessels. It is used in the management of mild to severe hypertension and myocardial infraction. It is a prodrug which after absorption, undergo rapid metabolism by ester hydrolysis to the active diacidic form ramiprilat[1,2] by liver esterase enzymes. Ramiprilat is mostly excreted by the kidneys. The half-life of ramiprilat is variable (3–16 hours), and is prolonged by heart and liver failure, as well as kidney failure.

Even though ramipril is without question one of the most important ACE inhibitors available today, current ramipril formulations show considerable problems like poor water solubility (3.5 mg/L) and wettability , poor bioavailability and susceptibility to undergo degradation. The degradation of ramipril is believed to occur mainly via two pathways: (a) hydrolysis to ramipril–diacid and (b) cyclization or condensation to ramipril–diketopiperazine[3]. The poor solubility and wettability of Ramipril leads to poor dissolution and hence, variations in bioavailability. It suffers from low bioavailability of 28%, two main reasons contributing towards the same being its poor aqueous solubility and high first pass metabolism[5]. Thus, increasing the aqueous solubility and shelf life of Ramipril is of therapeutic importance[4]. The complexation of drugs under hydrophilic carriers is one of the selective approaches to achieve this ideal therapy particularly for drugs with poor aqueous solubility and stability.

There are few works related with stability studies of ramipril: accelerated stability studies for the simultaneous determination of ramipril/moexipril[6], ramipril/ telmisartan[7], and ramipril/hydrochlorothiazide[8], a forced degradation study of ramipril in Altace capsules[9], a stability study of ramipril in solvents of different pH[10], a study of stability of ramipril over time in water apple juice and applesauc[11]and a comparative study of stability over time of marketed generics of ramipril tablets versus reference ramipril tablets[12] . Among some of the recent efforts to increase the solubility of ramipril tablets are formulation of ramipril containing solid self microemulsifying drug delivery system by adsorbent technique[13],formulation of inclusion complexes of ramipril using β-cylcodextrin (β-CD) and hydorxypropyl β-

cylcodextrin (HPβ-CD) [14], formulation and evaluation of Chitosan Loaded Mucoadhesive Microspheres of Ramipril to enhance its solubility[15], designed and development of oral oil in water ramipril nanoemulsion containing Ramipril[16], production of Multiple Unit Particle System (MUPS) of stabilized Ramipril pellets[17] etc.

Inclusion compounds were first observed by Mylius in 1886[18]as unusual complexations occuring between hydroquinone and several volatile compounds. Urea inclusion compounds were first reported by Bengen from experiments on the effect on the addition of urea to pasteurized milk[19]. Bengen was able to separate quantitavely the butter fat by formation of its urea inclusion compounds. In the conventional urea inclusion compounds, the host structure comprises a hydrogen-bonded arrangement of urea molecules that contain hexagonal, non-intersecting, linear, parallel tunnels [20] of diameter varying between about 5.5 and 5.8Å according to the position of guest along the tunnel and this structure is stable only when the tunnels are filled with a dense packing of guest molecules. Linear molecules are included along the tunnel in an extended planar zigzag conformation[18,21]

As the cross-section of the channels in urea inclusion compounds (defined by the van der Waals surface of the tunnel wall) is 5.5–5.8 A ° , only guest molecules that have a molecular size smaller than this area can fit into these cavities. More specifically, the molecules having a sufficiently long n-alkane chain with a limited degree of substitution can fit within these channels[22,23] .There are two main criteria which determine whether or not a particular urea inclusion compound will be formed, a) the length of the carbon backbone of the guest, and b) the degree of branching or substitution in the guest carbon skeleton. More specifically the adductible guests must have a carbon skeleton consisting of six or more atoms with little or no branching or substitution is usually a requirement for urea inclusion compound formation[24]. In general, molecules containing benzene or cyclohexane rings do not form inclusion compounds with urea, presumably because these structural components are too wide to fit comfortably inside the tunnel[25]. If however benzene carries a long chain substituent, then an inclusion compound may be formed because the long chain of this compound is readily adducted and apparently the unit cell can easily withstand the distortion caused by an occasional benzene group[26]. Similarly normally a non-adductible endocyte (NNAE) like 3-methyl heptane forms an adduct with urea only when a more slender hydrocarbon (e.g. n-C6H14)—a rapidly adductible endocyte (RAE) serves as a ‘‘pathfinder’’[26,27]. The endocytes possessing a

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sufficiently long n-alkane chain and hence easily adductible within urea channels are named herein rapidly adductible endocytes (RAE) while sufficiently substituted and/or cyclic endocytes which are known to be non-adductible in urea due to their large molecular size and/or shorter alkyl chains are named normally non-adductible endocytes (NNAE).Generally the drugs of BCS class II contain aeromatic or cyclic rings within their molecules which contribute towards their lipophilic character and low water solubility. Such rings also make these drugs non-adductable under small cavities of urea channels. In the present study, the possibility of coinclusion of low doses of NNAE drug, that is, Ramipril in the presence of a suitable RAE has been investigated[28]. A number of long straight chain compounds can be employed as RAE. However, fatty acid-urea adducts exhibit improved stability as compared to other compounds mainly due to dimerization of fatty acids in hexagonal urea lattice[29]. Hence, oleic acid was selected as the RAE for the purpose of co-inclusion of Ramipril in urea.

MATERIALS AND METHODS

Materials

Ramipril was supplied as gift sample by Ranbaxy, Gurgaon, India; the following materials were of analytical grade, urea crystals, extra pure (E. Merck, Mumbai, India), oleic acid (Rankem, New Delhi, India), methanol A.R. grade (Rankem). All other reagents were of analytical grade.

Methods

Excipients Compatibility Studies

This study was carried out by comparing the FTIR graphs of ramipril and its mixture in urea in the ratio of 1:1 using FTIR spectrophotometer (IR 200 ThermoNicolet, Madison, USA) employing KBr disc technique and all samples were scanned over a range of 400–4000 cm_1.The graphs were compared for the presence of any new peak in the mixture. The powdered mixture of urea and ramipril was dissolved in methanol, dried in air and kept in dessicstor for 24 hours before conducting the FTIR study[5,30].

Preparation of urea inclusion compounds of ramipril with RAE: 1 g of ramipril was dissolved in 30 ml methanol containing 3 g urea by gentle heating. Subsequently 0.5 g oleic acid was added as RAE to the above solution; this led to immediate precipitation of crystals. The solution was allowed to stand at room temperature for 2–3 h. Crystals were separated from the mother liquor by vacuum filtration, dried and packed in suitable containers[31,32].

Determination of entrapment efficiency of ramipril-urea inclusion compounds

10 µg/ml ramipril solution was scanned in the wavelength region 190 to 800 nm to determine the wavelength of maximum absorption using 5 % aqueous methanol as blank .linearity calibration plot between the absorption and the concentration was prepared within the range of 1 – 38 µg/ml using 210 nm as λmax.

The mother liquor obtained after the separation of crystals of Ramipril-urea inclusion compounds was evaporated under vacuum and the powder obtained was dissolved in 50 ml of absolute methanol, shaken for 15 min in a vortex mixer and diluted to the 100 ml mark with the same solvent. It was then filtered using Whatman number. 1 filter paper to obtain stock solution. Five milliliters of this solution was further diluted to 100 ml with distilled water and then analysed in UV-visible spectrophotometer at 210 nm. A solution containing 12.5 µg/ml of pure ramipril was used as standard for comparison. All analyses were carried out in triplicate[33].

Characterization of urea inclusion compounds

Ftir Analysis

The FTIR spectra of the samples was recorded using FTIR spectrophotometer (IR 200 ThermoNicolet, Madison, USA) employing KBr disc technique and all samples were scanned over a range of 400–4000 cm_1.

Differential scanning calorimetry

Thermal analysis of the crystals was performed using a DSC Q10 V 9.0 (275), Waters Ltd., Vienna, Austria. TA system with a differential scanning calorimeter equipped with a computerized data station. 2 mg sample were heated in crimped closed aluminum pan at scanning rate of 108oC/min from 40 to 160oC in an atmosphere of nitrogen gas by passing at a flow rate of 60 ml/min. An empty aluminum pan was used as the reference pan. DSC was calibrated using Indium metal with a melting endotherm at 156.898oC.

Powder X-ray diffraction study

X-ray diffractograms of the crystals were recorded using X-ray Diffractometer (Philips, X’Pert Pro, PW 3050/PW 3071, Lelyweg, The Netherlands). Experimental settings were: Nickel filtered Cu Ka radiations (λ=1.540598 A ° ), voltage 40 kV, current 30 mA and scanning rate 2o/min over a 2θ range of 10–80o.

Preparation of urea inclusion compounds containing varying proportions of ramipril and rae

A number of urea Ramipril–RAE–co-inclusion compounds containing varying proportions of Ramipril and RAE(oleic acid) were prepared by the above method. The contents included 10 gm urea, 30 ml methanol and a total of 2 gm of endocytes, that is, RAE and Ramipril in the varying proportions of RAE:AH as per details outlined in Table 1. DSC scans of these inclusion compounds were conducted at temperature range of 40–200oC as mentioned above.

Optimization of inclusion compounds

Optimization refers to the art and science of allocating available resources to the best possible effect[34]. In order to design the best formulation of inclusion complexes that would give maximum dissolution rate in the entire pH range a 22 factorial design was used to optimize the amount of oleic acid and urea against a fixed amount of ramipril in the inclusion compounds. Amount of urea and oleic acid was taken each at two levels i.e., maximum (3 mg for urea and 0.5 mg for oleic acid) and minimum (2 mg for urea and 0.25 mg for oleic acid) for 1 mg of drug, to obtain four formulations[35].

Dissolution Rate Studies

Dissolution study was conducted using IP dissolution apparatus I, in 500 ml of 0.1 N Hcl, phosphate buffer 5 and phosphate buffer 6.8 maintained at 37±0.5oC at a basket speed of 75 rpm [34]. The quantity of ramipril (30 mg) and corresponding batches of inclusion complexes containing amount of drug equivalent to 40 mg, was added to the dissolution medium. At predetermined time intervals (10, 20, 30, 40, 50 and 60 min) 10 ml of the samples were withdrawn with volume replacement. The samples were filtered through 0.45 mm membrane filter, the solutions were suitably diluted with corresponding mediums and assayed spectrophotometrically (225 nm in 0.1 N Hcl,215 nm in phosphate buffer pH 6.8 and 208 nm in pH 5 phosphate buffer). A cumulative correction was made for the removed samples while determining total amount of drug dissolved. All experiments were run in triplicate.

Stability Studies

Stability of a dosage form refers to the chemical and physical integrity of the dosage unit throughout its shelf life and when appropriate, the ability of the dosage unit to maintain protection against microbiological contamination. Ramipril is a very sensitive and unstable molecule. It was necessary to check the relative stability of the Ramipril inclusion compounds and pure drug under stress conditions. Stability was checked when the optimized formulation and pure drug was kept at different temperatures (40 ±2°C, 25 ± 2°C, and 5 ± 2°C)3

Nine batches of inclusion complexes were prepared, out of which, three batches were kept at three different temperatures 40 ± 2°C (stability chamber), 25 ± 2°C (room temperature), and 5 ± 2°C (refrigerator) at ambient humidity.

Samples were withdrawn periodically at predetermined time intervals (0, 15, 30, 45, 90, and 180 days) and evaluated for any

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physical change in the formulation and drug content. Zero time samples were used as controls. Analysis was carried out at each time interval using Shimadzu UV-1601 UV/Vis double beam spectrophotometer at 210 nm , taking 95% aqueous methanol as blank.

RESULTS AND DISCUSSION

Characterization of urea inclusion compounds

Preparation of inclusion compounds of Ramipril in urea in presence of oleic acid as a suitable RAE was attempted. Addition of small amount of oleic acid as the RAE to a methanolic solution of urea and AH led to immediate precipitation of fine needle-shaped, white crystals. The formation of inclusion compound was confirmed as follows.

DSC: Thermograms for pure Ramipril and crystals of inclusion of Ramipril are shown in Figure1. Pure Ramipril exhibits a melting endotherm at 112oC whereas the thermogram of Ramipril-Urea inclusion compounds shows that the urea crystals melt in two steps, a characteristic of hexagonal form of complexed urea[36]. The first step (126.9oC) involves the collapse of the hexagonal form of urea inclusion compound to yield the guest moiety and tetragonal solid urea while the second step (131.61oC) is attributed to melting of tetragonal urea[38]. The disappearance of sharp melting endotherm of Ramipril at 293.5oC clearly indicates that AH may be present in an amorphous state.

FTIR

Fig. 2 demonstrates the FTIR spectra of pure Ramipril and its urea inclusion complexes. In the Ramipril spectra absorption peaks were observed at 3400 cm-1 due to -NH and -OH stretching of acid, peaks at 3000 cm-1and 2900 cm-1are due to –CH aromatic stretching. Peak at 2800 cm-1is due to –CH aliphatic stretching, peak at 1730 cm-1is due to –C=O of acid Peak at 1680 cm-1is due to presence of ester group, peaks at 1500 cm-1-1460cm-1is due to –CH aromatic bending and peaks at 1360- 1320 cm-1is due to –CH aliphatic bending vibrations.

The IR spectra of urea inclusion complexes of Ramipril crystals indicate out-of-phase vibrations at 3413.9cm-1and in-phase vibrations at 3224.8cm-1which are characteristic of hexagonal channel structure of urea[37,38](the same vibrations reported at 3445 and 3347 cm-1for tetragonal uncomplexed form of urea[39]. Similar observations were made regarding occurrence of four bands between 1675 and 1596.5 cm-1 (due to CO stretching and NH2 bending vibrations), slight increase of the skeletal out-of-phase bending frequency at 7491.4 cm-1 and the symmetric C–N frequency increased from 1000 to 1013.2cm-1, which are characteristic of hexagonal channel structure of urea[37,38].These findings indicate transformation of uncomplexed tetragonal urea to hexagonal form containing guest moieties.

X-Ray diffraction studies

Prominent peaks at 4.09179 Å, 3.54247 Å, 3.83583 Å, 7.04696 Å, 3.835 Å and 3.25086 Å in the decreasing order of their intensities[39] reveal the presence of hexagonal urea forming adducts. In addition the presence of some uncomplexed urea also is revealed by comparatively feeble peaks at 3.975 Å, 3.604 Å, 3.0339 Å, 2.81 Å, 2.52 Å and 2.415 Å (fig 3).

Characterization of inclusion compounds containing varying proportions of rae and ramipril

DSC thermograms of different Ramipril adduct containing varying proportions of RAE and AH are shown in Fig. 4. In all these thermograms, a low-temperature endotherm corresponding to release of tetragonal urea was observed. The plot clearly indicates that an increase in amount of RAE in the adduct, makes the adduct more stable . Since Ramipril is an aromatic moiety with substitutions in the ring, it is presumably too wide. However, in presence of the oleic acid , Ramipril is assumed to get co-included with it. This would in turn lead to local distortion of the urea tunnel structure in the vicinity of aromatic ring, the extent of steric strain on host lattice being proportional to the amount of oleic acid incorporated.

Dissolution rate studies

When co-inclusion compounds of urea comes in contact with an aqueous dissolution medium, the urea lattice dissolves almost instantaneously and results in immediate release of the included drug at a molecular level. Also, inclusion of a drug along with RAE in urea leads to weakening/distortion of urea host lattice, manifested as subsequent increase in the dissolution rate.

Ramipril and its co-inclusion complexes were subjected for dissolution in 0.1 N Hcl, phosphate buffer pH 5 and phosphate buffer pH 6.8 as per the IP 2007 monograph[34]. Four preparations containing varying porporations of urea and oleic acid against a fixed proportion of ramipril according to 22 factorial design were dissolved along with the pure drug (Table_).On the basis of the %drug released plotted against time it was revealed that although the presence of both urea and oleic acid is significant for enhancing the dissolution rate of Ramipril, the presence of urea was more significant than that of oleic acid. On the basis of the results obtained , it was concluded that the formulation containing 2.5 mg of urea and 0.8 mg of oleic acid would show the maximum dissolution rate in all these three mediums. This was confirmed by comparing the dissolution of pure ramipril and this formulation in all these mediums according to IP 2007[34]. The dissolution rate of inclusion complexes increased upto 96.91%, 78.69% and 91.03% which was 21.2%, 21.24% and 93.41% for the pure drug in 0.1N Hcl, phosphate buffer pH 5 and phosphate buffer pH 6.8 respectively, in 1 hour.

Stability Studies

The degradation of ramipril occurs mainly via two pathways: the hydrolysis to rampiril diacid (impurity E described in the European Pharmacopoeia) and the cyclization to rampiril diketopiperazine (impurity D described in the European Pharmacopoeia).Both these processes are accelerated in the presence of stress conditions like moisture temperature and light [40]. It was predicted that ramipril being a photosensitive drug would be protected from the deteriorating effect of moisture, temperature and light if covered by some carrier molecules. When any drug is entrapped within the channels formed by loose association of urea molecules in its co-inclusion compounds, its functional groups remain apart to prevent them to react with each other on one hand and it is protected by the covering of urea lattice by deleterious effects of external factors like light rays on the other hand.

Formulations kept at 40°C, 25°C, and 5°C were analyzed individually. The percentage of undecomposed Ramipril remaining in urea co-inclusion complexes formulations was 42.80%, 85.53%, and 98.87% at 40°C, 25°C, and 5°C, respectively, after 180 days of storage whereas the corresponding samples pure drug showed 22.5%, 57% and 94.3% of undecomposed drug, after 180 days of storage3.

CONCLUSION

The formation of co-inclusion compounds of Ramipril in urea in the presence of oleic acid was studied. The higher dissolution rate displayed by inclusion compounds in the mediums of varying pH may imply enhanced oral bioavailablity of the drug. The urea co-inclusion compounds were also found to show good Entrappment efficiency. The accelerated stability studies showed that these prepar8ations could protect the included drug from the harmful environmental effects. The overall process of urea inclusion complex formation involves relatively simple preparation steps, reduced processing cost, minimal energy consumption and short processing time. Urea inclusion compounds of Ramipril in the presence of a suitable RAE may be a promising alternative for formulation of potent poorly soluble and instable drugs into immediate release products with enhanced stability.

Table 1: Entrappment efficiencies of ramipril urea co-inclusion compounds with varying proportions of oleic acid

Ratio of ramipril, urea and oleic acid in co-inclusion compounds

Entrapment Efficiency

1:3:0.2 55.6% 1:3:0.4 62% 1:3:0.6 68% 1:3:0.8 69.2% 1:3:1 66%

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Fig. 1: FTIR Spectra of ramipril-urea inclusion compounds, ramipril urea mixture, urea and ramipril

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Fig. 2: DSC Thermograph of urea co-inclusion compounds of ramipril

Fig. 3: X-Ray diffractographgraph of urea co-inclusion compounds of ramipril

Fig. 4: DSC Thermograms of ramipril–urea co-inclusion compounds containing varying proportions of oleic acid

Position [°2Theta] (Copper (Cu))

10 20 30 40

Counts

0

2000

4000

6000

8000 UIC

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Table 2: A comparison between % cumulative release of ramipril and that of ramipril urea co-inclusion compounds in different media.

Time (min)

% Cumulative Release of Ramipril % Cumulative Release of Ramipril Urea Co-inclusion compounds 0.1N Hcl Phosphate Buffer pH 5 Phosphate Buffer pH 6.8 0.1N Hcl Phosphate Buffer pH 5 Phosphate Buffer pH 6.8

0 10 20 30 40 50 60

0 1.667 14.67619 27.35 34.55714 37.93571 39.4119

0 4.60094 5.819718 12.78873 16.42066 19.1831 21.24131

0 2.564103 5.435897 14.00513 19.41026 20.12308 21.20256

0 12.05128 50.19744 62.08974 80.09744 87.60256 96.91282

0 15.11905 39.39524 60.88571 75.52857 80.80714 91.03571

0 14.74178 49.7784 59.21878 65.63568 71.95681 78.69296

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