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In vitro and in vivo Evaluationof Floating Controlled Release DosageForms of Verapamil HydrochlorideSeham A. Elkheshen*, Alaa Eldeen B. Yassin, SaIeh Alsuwayeh, and Fayza A. AlkhaIedDepartment of Pharmaceutics, Collage of Pharmacy, King Saud University, Riyadh (Saudi Arabia) Present address: see address for correspondence

SummaryThe present work investigates the preparation of sustained release floatingsystems for verapamll hydrochlorideusing different hydrocolloid polymers including hydroxypropylmethylcellulose(HPMC),hydroxypropylcellulose (HPC),ethylcellulose (EC)and Carbopol (CP).Floating was achieved by adding an effervescent mixture of sodium bicarbonateand anhydrous citric acid. Some factorswere investigated concerning their effecton flotation and rate of drug release including the drug to polymer ratio, thegranulating agents, the incorporation ofrelease retardant, coating granules withethyl cellulose, and finally granule-compression into tablets.A formula composed of 1:1 of verapamil to HPMC, 5 % of gas generating mixture and prepared by wet granulationwith 96% alcohol followed by compression was chosen for in-vivo evaluationin comparison with a commercial controlled release product of verapamll hydrochloride. Investigations have been undertaken in beagle dogs to evaluate boththe intra-gastric retention performancesusing X-ray and the bioavallabllity of thedrug from both test and standard tablets.

ZusammenfassungUntersuchung von flotierenden Formullerungen zur kontrolllerten Freisetzungvon Verapamll-Hydrochlorid in vitro undin vivoUntersuchungen ilber die Zubereitungflotierender Systeme ffir die verzOgerteFreisetzung von Verapamil-Hydrochloridwerden beschrieben; verschledene Hydrokolloid-Polymere einschlieBllch Hydroxypropylmehtylcellulose (HPMC),Hyroxypropylcellulose, Ethylcellulose und Carhopol wurden eingesetzt. Flotation wurdedurch den Zusatz einer gasbildenden MIschung von Natrlumbicarhonat und wasserfreier Zltronensaure erreicht. Einige

Results showed that for powder-blendfilled into capsules, only HPMC-4000 formulations combined a good floating andreasonable delay in drug release. However granulation of the same formulations led to complete loss of both thefloating and the sustained release characteristics. Tableting of granules containingvarious verapamil:HPMC-4000 ratiosshowed excellent buoyancy and slow release prome. Results also revealed thatfloatation was able to delay the gastricemptying ofverapamil tablet in beagledogs for more than four hours comparedto almost one hour for a control tabletdevoid of the gel forming polymer andthe gas generating mixture. The floatingtablets showed bioequivalence with acommercial sustained release tablet withhigher mean AUCo~and Cm~ and longer1m~, however, non-significantly different.

Faktoren mit EinfluB auf Flotation undWirkstofffreisetzung wurden untersucht,u. a. Wirkstoff-Polymer-VerhaIU1is, Granuliermittel, Zusatz von frelsetzungsverwgernden Mlttein, Oberzlehen der Granula mit Ethylcellulose, Verpressen desGranulates zu Tabletten.

Filr die In-vivo-Priifung Im Vergleichmit einem kommerziell erhaItlichen Verapamll-Hydrochlorid-Produkt mit kontrollierter Freisetzung wurde eine Formulierung ausgewahlt, die Verapamil undHPMC Im VerhaItnls 1:1 sowie 5% gasbildende Agentlen enthaIt, mit 96%Alkohol na~ granullert und anschlie~nd verpreBt wurde. Von beiden Priiparaten

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wurde bei Beagle-Hunden die Bloverftigbarkeit sowie die Relentlon irn Magenronlgenologisch unlersuchl.Die Ergebnisse zeigen, da~ bei den inKapseln verftilllen Pulverrnischungennur soIche rnit HPMC-4000 sowohl Flolatlon als auch annehmbare Verwgerungder FreiselZung ergeben. Die Granulierung derselben PulverformulierungenfUhrle zu einem vollslandigen Verlusl so-

wohl der Rolalion als auch der Ver:rogerung der FreiselZung. Die Tablellierungvon Granulalen rnit verschiedenen Veraparnil-HPl\IC-4000-Verhiiltnissen ergabausgezeichneles Aufschwimrnen undlangsarne FreiselZungsprofile. Durch dieRotalion wurde die Entleerung der Veraparnil-Tablellen aus dern Magen von Beagle-Hunden urn mehr als vier Slundenverwgerl - irn Vergleich zu nahezu einer

Stunde bei einem KonlroUpriiparal ohnegelbildendem Polymer und ohne gasbildende Zusatze. Die Ootlerende Tablellenzubereitung zeigte Bioaquivalenz mit einer handelsiiblichen Tablelle mit ver:rogerler FreiselZung, wobei rniltlere AUCO~, Cmax und ~ nichl signiflkanl hahereWerle hallen.

1. IntroductionVerapamil hydrochloride (VP), the first calcium channelblocker to be approved by FDAin 1981, is useful for thetreatment of angina pectoris, hypertension, and supraventricular tachyarrhythmia [I]. In atrial fibrillation, VPis more effective than digoxin for controlling ventricularrate [2]. As being a drug for chronic conditions the extended-release oral dosage form would increase the patient compliance and improve the therapeutic responses by reducing the peak-to-trough variation ofdrug plasma concentration. The already available extended-release VP per-oral dosage forms, given oncedaily, are as effective in lowering blood pressure over24 h as doses of conventional VP formulations giventhree times daily [3].VP is considered a good candidatefor incorporation in a gastro-retentive dosage form dueto its high solubility in the stomach medium comparedwith its solubility in the small intestine medium [4].This will allow the gradual release ofVP in solution formto the upper part of the small intestine, where it ismainly absorbed, which will maximize the absorptioncompared with other controlled release approaches.

Floating dosage forms are dosage forms with a bulkdensity lower than that of the gastric content. This allows them to remain buoyant on the surface of the gastric content for a certain period of time without affecting the intrinsic rate of emptying. They are also referredto as hydrodynamically balanced systems (HBS) as theyare able to maintain their low density while the polymerhydrates and builds a gelled barrier at the outer surface[5]. The drug is progressively released from the swollenmatrix as in the case of conventional hydrophilic matrices. In order to design successful floating dosage forms,three major conditions should be met: (i) They musthave sufficient structure to form a cohesive gel barrier;(ii) they must have an overall specific gravity lower thanthat of gastric contents (reported as 1.004-1.010 g/m\);(iii) and finally they should dissolve slowly enough toserve as a reservoir for the delivery system [61.

The selection of potential excipients that allow theformulation of matrices having sustained delivery characteristics and a bulk density of less than 1 glml is thekey point. After extensive work on the floating andswelling characteristics of commonly used excipients,Gerogiannis et al. [7J concluded that polymers withPharm. Ind. 66, Nr. 11, 1364-1372 (2004):e ECV . Editio Cantor Verlag. Aulendorf (Germany)

high molecular weight and less hydrophilic grades usually exhibit enhanced floating characteristics.

Floatation not only prolongs the gastrointestinal (GI)residence time, or obtains a sustained local action intothe stomach, but also does so in an area of the gastrointestinal tract (GIT) that would maximize the amount ofthe drug reaching its absorption site in solution, andhence ready for absorption. The retentive characteristics of the dosage form are most significant for drugsinsoluble in intestinal fluids, for those acting locally inthe upper GIT and those exhibiting site-specific absorption [6].

The techniques applied to achieve floatation are numerous. The design of an inflatable chamber containinga liquid that gasifies at body temperature is an example[8]. The preparation of an empty globular shell coveredby a solid formulation is another example [9]. Harrigan[10] designed a floating dosage form by the incorporation of a floatation chamber filled with air or a harmlessgas. The incorporation of fatty materials, having bulkdensity less than 1 g/ml, with hydrocolloid to decreasewater uptake and increase the duration of buoyancywas investigated [U]. The chemical generation of CO2by incorporation of an acid and a carbonate salt thatwill react in the presence of the aqueous stomach medium is a frequently used method for the induction offloatation [12, 13].

Different hydrocolloids of natural and semisyntheticorigin have been used for the formulations of HBSforms [14, IS]. Among the different hydrocolloids recommended for floating formulation, cellulose etherpolymers are the first choice, especially hydroxypropylmethylcellulose (HPMC), which is extensively usedby many authors [16-18].

The present work involves tile use of several polymers such as HPMC-4000 (hydroxypropylmethylcellulose), HPC-MF (hydroxypropylcellulose), EC-25 (ethylcellulose), CP-934p (Carbo po\) and a number of formulation metllOds as gran ulation, film coating of the granu~es and compression of granules into tablets in orderto develop a floating solid dosage form for controllingthe release of verapamil hydrochloride (VP). A formulathat combined both excellent floating and sustained release characteristics was chosen for farther in vivoevaluation in comparison with a commercial controlledrelease product of verapamil hydrochloride.

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2. Experimental2.1. MaterialsVerapamil HCl from Adwic Chemical Co. (Cairo, Egypt),Hydroxypropylmethylcellulose 4000 from Shin-Etsu chemicals(Tokyo, Japan). hydroxypropylcellulose, Kiucel MF from AquaIon Co. (Delaware, USA), ethylcellulose USP/NF from SpectrumQuality Products (Gardena, New Brunswick, USA). poly acrylicacid, Carbopol 934p from BF Goodrich Co. (Cleveland, Ohio,USA), anhydrous citric acid and sodium hydrogen carbonatefrom Winlab (Edgware, UKJ, magnesium stearate, glycerylmono stearate, and pepsin powder from BDH Chemicals Ltd.(Poole, England) were used as received. Commercial, 120 mg,extended-release verapamil tablets, each containing 120 mgverapamil as hydrochloride, lot No. M-lO, manufactured May2000, expiry May 2003 (Knoll, Ludwigshafen, Germany). dihydroergotamine mesylate (Sandoz, East Hanover, NI. USA)and empty hard gelatin capsules (Feton, Coni-Snap~, Bruxelles,Belgium) were obtained from National Guard Hospital.

2.2. Methods2.2.1. Preparation of floating dosage formsAiming to prepare a sustained release floating systems for verapamil hydrochloride, the hydrocolloid polymers, HPMC-4000,HPC, EC and CP were employed. Floatation was achieved byadding an effervescent mixture of sodium bicarbonate and anhydrous citric acid. Some factors were investigated includingthe drug to polymer ratio, the granulating solution, coatinggranules with ethyl cellulose, and finally granule compressioninto tablets.

2.2.1.1. Preparation of verapamil capsulesfilled with powder or granulated-mixThe ingredients of each formula as described in Table 1 weremixed in a cubic mixer (Erweka KUl, Erweka Apparatebau,Heusenstamm, Germany) for 15 min and either filled directlyinto capsules or granulated manually using 80% or 96 % alcohol by sieve granulation method where the wet mass was passed through 1.25 mm sieve followed by complete drying, sieving and mling into capsules. In each case a portion from eachformula (passed through a sieve of pore size 1.25 mm and retained on a sieve ofpore size 0.8 mm) containing VPequivalentto 120 mg was weighed and filled manually into hard gelatincapsules of suitable size.

2.2.1.2. Preparation ofethylcellulose-coated granulesAiming to investigate the effect of coating granules on sustaining the release of verapamil from the granulated matrices aportion of a formula containing 1:1VP:HPMCwas subjected tomm coating by EC using the pan coater (AR-4000, Erweka).The coating solution (l0 % w/v of EC in absolute ethanol) wassprayed over the granules in the coating pan and the solventwas allowed to evaporate after each spray using a hot-airblower. Four different coat percent (l0.8, 20.4, 33.24 and 40 %)were prepared byvarying the application time and determinedbased on the overall weight gain afrer complete drying. Portions from each coated granule formula equivalent to 120 mgVPwere weighed and filled manually into hard gelatin capsulesof suitable size.

1qhh "I~hp

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2.2.3. In vivo evaluation of a selectedfloating formula2.2.3.1. Evaluation of gastric retentionusing X-ray imagingThe selected tablet formula for in vivo investigation wasreformulated with 12% BaS04 as opaqueing agent and prepared by wet granulation with 96 % alcohol followed by compression (ErwekaAR400 tableting machine, f itted with 9 mmflat-faced punches) . A control tablet, devoid of HPMC and thegas generating mixture, and containing Avicel PH 101 in addition to the dmg was prepared using the same proportion ofBaS04'The X-ray studies were carried out using 6 male beagle dogs

having weight range of 11.13 to 12.5 kg. In each experiment,the animals were allowed to fast overnight with free access towater and a radiograph was made just before the administ ration of the tablet to ensure the absence of radio-opaque material in the stomach. The two formulations (test and contro!)were administered by natural swallowing each to a group ofthree dogs followed by 50 ml of water. The radiographic imaging was taken to animals in a standing position and the distance between the source of X-rays and the animal was keptconstant for all imaging, thus the observation of the tabletmovements could be easily noticed. Radiographic imaging ofthe abdomen was taken using an X-ray machine (Model No.3064581-B-531OJ. connected to a video cassette recorder SVO9500MD and a video graphic printer UP960-EC (Siemens, Germany) every 30-min intervals for a period of 6 h.

2.2.3.2. Comparative bioavailabiIity study2.2.3.2.1. Treatment protocol and sample analysisThe selected floating formula containing 120 mg of VP wascompared with a commercial sustained release VP product,which contains 120 mg VP.Five healthy beagle dogs (3 malesand 2 females) , having a body weight range of 9.5-12 kg, wereused in this study. The single dose randomized design was applied where all dogs received the same treatment at the sametime and a washout period of at least two weeks was allowedbetween treatments. The sequence of the trials was the standard commercial tablets followed by the test formula. The dogswere allowed to fast over night prior to and 4 h after administration of each treatment; thereafter, they resumed a normalunrestricted diet. They were placed in normal cages during thecourse of the experiment without using a restrainer . The treatments were given by normal swallowing followed by 50 mlwater. 5 ml blood samples were collected, in heparinizedevacuated glass tubes through an indwelling polyethylene cannula into the cephalic vein, at 0, I, 2, 3, 4, 5, 6, 9, 12, and 24 hfrom the time of administration. The plasma was immediatelyseparated by aspiration after centrifugation at 3000 rpm for5 min and frozen at -20 QCuntil analyzed. Animal 4 was excluded from the second round (floating tablet) due to its lowheal th condition. The concentration of VP in the test sampleswas determined using the HPLC method introduced by Niazyand Jun [19].

2.2.3.2.2. Calculation and statistical treatmentof pharmacokinetic parametersThe pharmacokinetic parameters were calculated from theplasma level data obtained for the individual dogs and presented as mean SO.

Phann. tnd. 66, Nr. 11, 1364-1372 (2004)Cl ECV . Editio Cantor Veria", Aulendorf (Germany)

From the data of plasma concentration, the maximumplasma concentration (Cm"", ng/m!) and the correspondingtime (tma.vh) were directly extracted for the two treatments ineach individual animal. A plot of the mean plasma concentration versus time has been constructed for each treatment. Thearea under the plasma concentration-time curve from timezero to 24 h (AUCo-24h'ng . hIm!) was obtained by applyingthe trapezoidal mle. The AUC24_ was estimated by adding thearea under the tail to AUCO-24h' The area under the tail wascalculated bydividing the last measurable concentration bytheelimination rate constant obtained by l inear regression of theelimination phase of the plasma concentration versus timecurve. The mean residence time (MRT,hJ. which is a non-compartmental pharmacokinetic parameter was obtained using thesuitable equation [20] after the calculation of the area underthe first-moment curve (AUMCo.~, ng . h2/m!). The relativebioavailabiliry (Frol)of the tested formula compared with thereference product was calculated as:Frol = [AUCo.~(tested formula) / AUCo.~

(reference product)] . lOOThe significance of the difference between the two .treatmentswas evaluated by one-way analysis of variance (AI\lOVA)usingDuncan's multiple comparison test on the computer program,Stat lOO, version 1.24, Biosoft (1996). Differences were considered signif icant at p $ 0.05.3. Results and discussionAnhydrous citric acid (CA) and sodium hydrogen carbonate in the ratio of 1:1 (w/w) and a concentrationof 5 % per formula (based on preliminary experiments)were used as gas generating mixture (GG) during thecourse of this work. Ethanol 80 % (v/v) was used as agranulating agent for all the prepared granules as it produced more firm granules. However, granules meant tobe compressed into tablets were prepared with 96 %ethanol as they were more compressible.3.1. In vitro evaluation3.1.1. Effect of the polymer type andthe drug to polymer ratiosThe capsulation of a well-blended powder of differenttypes of polymers (HPMC-4000, HPC. and EC) and different drug to polymer ratios was investigated. OnlyHPMC-4000 formulations combined a good floatingand reasonable delay in drug release (Fig. 1). The threeformulations containing 1:1 (Ft), 2:1 (F4), and 1:2 (Fs) ofVP:HPMC showed exceIlent floating properties as theyremained buoyant for more than 12 h. The formulaecontaining HPC (Fz) and EC (F3) showed fast releaseprofile with 82.9 0.38 % and 86.80 2.43 % of the dosebeing released within 30 min, respectively. They bothexhibited immediate floatation, however only the formulae containing EC remained floating during thecourse of the experiment (12 h). while that containingHPC sunk after 15 min only.The incorporation of CP was thought to improve thesustaining profile of VPfrom the powder-fiIled capsules

containing HPC as a hydrophilic polymer. The results

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Fig. 1: Release profile of verapamil hydrochloride from powderfilled capsules formulated with different polymers and drug topolymer ratios.

showed that CP did not affect the release characteristicsof the drug even upon the addition of 40 % CP per totalpolymer content (F7). None of the tested formulationsshowed any retardation to the release and the floatingcharacteristics were poor as well.The release kinetic data (Table 2) revealed that therelease mechanism from Fl (1:1 of VP:HPMC) and Fs(1:2 of VP:HPMC) followed zero-order kinetics that wasconfirmed by their release exponent (n) which de'scribes the mechanism of drug release as introducedby Korsmeyer et aI. [21]. However, F4 (2:1 ofVP:HPMC)followed Higuchi diffusion model as indicated by thehighest correlation coefficient for the diffusion modeland the closeness of the n value to 0.5 (r = 0.999,n = 0.504). Besides changing the mechanism of drug

__ F1(1 :1 . VP:HPMC-4(00 )___ F2 (1:1. VP:HPC)-.!o- F3 (1:1. VP:EC)-*-F4 (2:1. VP:HPMC-4(00 )- il l- FS (1:2. VP:HPMC-4(00)_ _ F6 (1: 0 .8: 0. 2. VP:HPC:CP)-+-F7 (1: 0.6: aA, VP:HPC:CP)

3.1.2. Effect of granulationThe granulation of two of the formulations that showedperfect performance in powder form concerning thesustained release and long floating period (F1 and Fs)lead to complete loss of the sustained release pattern(TSO%of 45 and 30 min, respectively) and the floatingduration (1.5 and 0.25 h, respectively). Trying to retardthe release after granulation, magnesium stearate, glyceryl mono stearate and CP were incorporated in 10 to15% per formula. These additives only improved tllefloating properties (3.5 to 12 h duration), while the release rate was even mildly accelerated (tSO%of 15 to30 min).The slow release of the drug from the powder-filled

capsules compared to the granule-filled capsules of thesame composition was due to the congealing of the externallayer of the powder during the dissolution of thecapsule shells in the former case. The congealed layeracted as a barrier for farther penetration of the dissolution medium or the diffusion of tlle drug. While, granules spread all over the dissolution medium once thegelatin shell dissolved exposing greater surface area incontact with the dissolution medium. Further more,being soluble in alcohol, which is used as granulatingagent, VP concentrates more at the surface of granulesupon evaporation of alcohol leading to further increasein the dissolution rate. Moreover, the granulating agent(ethanol) allowed the formation of porous structures inthe granule formulations through which the dissolutionmedium can diffuse rapidly to extract the drug.

release from simple diffusion to concentration independent (zero order) by increasing the ratio of the polymer to the drug, it is obvious that increasing this ratioretarded the release rate of the drug significantly (Fig.1). The tsO%for tlle formula containing 2:1 drug:HPMCwas 2.17 0.14 h compared to 5.53 0.31 hand 7.95 0.19 h for the formulations containing 1:1 and 1:2 ofVP:HPMC, respectively. A significant difference (p $0.05) was detected using one-way ANOVAbetween thethree formulations.

30505Time (hours)

10

100

90000

CD

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Fig.2: Release proftle of verapamil hydrochloride from capsulesfilled with granules coated with different ethylcellulose coat percent.

3.1.4. Effect of tableting the granulesThe objectives of the present study (good floating properties and sustaining drug release) were achieved bytableting the prepared granules of FJ, F4' and Fs containing different VP:HPMC ratios of 1:1, 2:1 and 1:2, respectively. Allof the compressed granules showed buoyancy during the entire time course of the experiment(12 h). Only F4 showed immediate flotation while F1and F5 showed a lag period of 4 and 18 min, respec-

30505

___ F1(1:1 VP:HPWC-4000)--e- F4(2:1VP:HPWC-4000)~ F5(1:2VP:HPWC-4000)

Time (hours)10

oo

40

20

.,60VIj.,a:g