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Research paper Acyclovir serum concentrations following peroral administration of magnetic depot tablets and the infl uence of extracorporal magnets to control gastrointestinal transit Ru diger Gro ning a, *, Michael Berntgena, Manolis Georgarakisb aInstitute for Pharmaceutical Technology, University of Mu nster, Mu nster, Germany bSection of Pharmaceutical Technology, Aritotelian University, Thessaloniki, Greece Accepted 25 May 1998 Abstract In the present investigations peroral acyclovir depot tablets with internal magnets were developed. An extracorporal magnet was used to prolong the gastric residence times of the dosage forms and to infl uence the duration of absorption of acyclovir. The magnetic depot tablets contained 200 mg acyclovir. In a three-way cross-over in vivo study with fi ve healthy male subjects, the plasma concentration-time profi les of acyclovir were determined. The acyclovir plasma concentrations following peroral administration of magnetic depot tablets in the presence and absence of an extracorporal magnet were determined. A commercially available immediate release preparation was used as a reference preparation. In the presence of an extracorporal magnet which was placed in the stomach region, the plasma concentrations of acyclovir were signifi cantly higher after 7, 8, 10 and 12 h (P , 0.05, U-test, Wilcoxon, MannWhitney). The mean area under the plasma concentration-time-curve (AUC024h), in the presence of the extracorporal magnet was 2802.7 ngh/ml. Without the extracorporal magnet a mean AUC024h of 1598.8 ngh/ml was achieved. Computer simulations were carried out to show the infl uence of the gastric residence time of acyclovir depot preparations on the plasma concentration-time profi les of acyclovir. 1998 Elsevier Science B.V. All rights reserved Keywords: Sustained release acyclovir; Gastric residence time; Magnetic tablets; Extracorporal magnet; Gastroretentive dosage forms; Bioavailability 1. Introduction The peroral route is most important for the delivery of drugs to the systemic circulation. After peroral administra- tion, the rate and extent of the absorption of drugs from depot preparations depend on the transit time of the drug carriers through the absorbing area of the gastrointestinal tract. Only a few drug substances like theophylline and metoprolol 1 are completely absorbed from lower parts of the gastrointestinal tract. Drugs like nitrofurantoin, allo- purinol, acyclovir or levodopa are only absorbed from the duodenum and small intestine 2,3. When slow release preparations of such drugs reach lower regions of the gas- trointestinal tract before the drug is completely released, its absorption is reduced. Different types of dosage forms with prolonged gastric residence time have been developed. The drug is released from these preparations to the absorbing areas of the duo- denum or small intestine. It is the aim of these investigations to develop preparations with a more complete, and longer lasting, absorption. Floating dosage forms remain in the sto- mach due to their low density, which enables them to fl oat on the gastric contents 4. Drug delivery devices have been developed which unfold in the stomach 5 or expand to form complex geometric shapes 6. Other attempts have been made using bioadhesive dosage forms 7 and swella- ble systems 8. Drug carriers have been investigated which release passage delaying excipients, like salts of myristic acid, in addition to the active constituent 9,10. Magnetic dosage forms with extracorporal magnetic guidance repre- European Journal of Pharmaceutics and Biopharmaceutics 46 (1998) 285291 0939-6411/98/$19.00 1998 Elsevier Science B.V. All rights reserved PII S0939-6411(98)00052-6 * Corresponding author. Corrensstrasse 1, D-48149 Mu nster, Germany. Tel.: +49 251 8339861; fax:+49 2518339308. sent a novel approach to optimize the rate and extent of absorption of drugs 11,12. The magnetic dosage forms contain a small internal magnet. An extracorporal magnet controls the gastrointestinal transit of the dosage form. The feasibility to control the transit of drug preparations by using magnets was investigated in animals 13,14 and in man 15,16. Using ribofl avin as a model drug, which is only absorbed from the upper part of the gastrointestinal tract, it could be shown that the renal excretion of the drug is enhanced and prolonged. The combination of a pH-telemetering capsule with a small magnet is a technique to determine the prolonged gastric residence time in the presence of an extracorporal magnet 17. 2. Materials and methods 2.1. Materials Acyclovir BP 93 (Ch. No. FP95320A; Fa hrhaus Pharma, Hamburg, Germany); NdFeB-disc magnet, 5 mm diameter, 2 mm thickness (Fehrenkemper, Lauenau, Germany); NdFeB-block magnet, 6.3 3.6 1.0 cm (Fehrenkemper, Lauenau, Germany); hydroxypropylmethylcellulose (Meth- ocel K4M CR Premium; Colorcon, Orpington, UK); lactose (Meggle,Wasserburg,Germany);magnesiumstearate (Caelo, Hilden, Germany); carnauba wax (Sigma, Deisen- hofen,Germany); hydrochloric acid 1 N (Merck, Darmstadt, Germany); perchloric acid 70% (Merck, Darmstadt, Ger- many); acetonitrile, supragradient HPLC grade (Scharlau, Barcelona, Spain). 2.2. Preparation of the press coated tablets with internal magnet Fig. 1 shows a schematic diagram of the depot tablets with an internal magnet. The coated magnet is embedded in the core of the tablet which contains 160 mg of acyclovir. The core is compressed and coated with an outer layer con- taining 40 mg of acyclovir. Each tablet contains 200 mg of acyclovir. Different preparations with various amounts of hydroxy- propylmethylcellulose were examined. Table 1 lists the dif- ferent depot tablet compositions. The press coated tablets with internal magnets were pro- duced in the following way: a commercial NdFeB disc- shaped magnet was dipped into melted carnauba wax at 90C and covered with a carnauba wax coating. The fi lm thickness is about 0.5 mm. The inner coat consists of the wax-coated magnet after compression with 177.8 mg of the tablet mixture. Non-magnetic compaction devices which consist of a die with a diameter of 7.5 mm and a faceted stamping-tool were used. The compacting was done with an IR-press (Perkin-Elmer, U berlingen, Germany). The inner coat is coated again with 400 mg of the tablet mixture for the outer coat. The diameter of the die of the non-magnetic compaction device is 10 mm. The coated tablets with inter- nal magnets have a diameter of 10 mm, a height of 6.8 mm and contain 200 mg acyclovir. 2.3. In vitro release experiments The in vitro release experiments were carried out accord- ing to the USP XXIII Paddle method at 37C and 100 rev./ min, using 0.01 N hydrochloric acid as the dissolution med- ium. Acyclovir was determined spectrophotometrically at 226 nm (Hitachi Spectrophotometer Model 100-40 UV- VIS, Hitachi, Tokyo, Japan) in a 0.2 cm fl ow cell. The measured values were continuously recorded using an IBM-compatible AT-computer (Friedrich, Mu nster, Ger- many). During the release experiments the magnetic tablets were located at the wall of the release vessel 5 cm under the surface of the liquid using an external magnet. The external magnet was the same as the extracorporal magnet in the in vivo study. The distance between the external magnet and the magnetic depot tablet was 8 cm. Additional in vitro investigations have shown that the external magnet does not have an infl uence on the release process of acyclovir from hydroxypropylmethylcellulose depot tablets with internal magnet. 2.4. Clinical investigations Table 2 shows the demographic data of the subjects and the sequence in which the dosage forms were administered. The subjects were fully informed of the nature of the study which was carried out under medical supervision with ethi- cal approval. On each subject, the clinical studies were con- ducted on three separate days according to a cross-over design, with a wash out interval of at least ten elimination half-lives. In experiments A and B, the subjects swallowed a press coated tablet with internal magnet. In experiment A an extracorporal magnet was positioned at the stomach level of Fig. 1. Schematic diagram of a press coated tablet with internal magnet; 1, magnet; 2, carnauba wax layer; 3, inner coat; 4, outer coat. 286R. Gro ning et al. / European Journal of Pharmaceutics and Biopharmaceutics 46 (1998) 285291 the subjects for a period of 12 h. The extracorporal magnet consists of three commercial block magnets with a total size of 6.3 3.6 3.0 cm. The magnet was fi xed to the body of the subject as described previously 15,16. During experi- ment B,no extracorporal magnet was used. In experiment C, the subjects swallowed an immediate release dosage form (Zovirax200, Ch. No. T2973A; Wellcome, Burgwedel, Germany). After an overnight fast for 10 h, on each study day at 0900 h, the subjects swallowed a dosage form containing 200 mg of acyclovir with 150 ml of water on an empty stomach. During the following 12 h, the subjects drank 150200 ml of water every hour and received a small standard meal consisting of one bread roll (75 g), 10 g butter and a 40 g slice of cheese after 2, 5 and 8 h. No other food or drink was allowed during the 12 h after administration. After collec- tion of the 12 h blood sample, no further restrictions of food and drink were made. Blood samples (7 ml) were collected from the subjects immediately prior to, and 0.33, 0.66, 1, 1.5, 2, 2.5, 3, 4, 5, 6, 7, 8, 10, 12 and 24 h after, the administration of the dosage form. The blood samples were heparinized using lithium- heparinized monovettes (Sarstedt, Nu mbrecht, Germany) and centrifuged for 10 min at 2500 rpm (Sigma centrifuge, model 203; Sigma, Osterode, Germany). The resulting plasma was directly transferred into 2.5 ml plastic tubes (safe-lock; Eppendorf, Hamburg, Germany). Immediately after pipetting, the samples were quickly frozen and stored in a freezer at 35C. The plasma samples were kept frozen until analysis. 2.5. Determination of acyclovir in the plasma The content of acyclovir in the plasma was determined by HPLC (pump model 655 A-11 liquid chromatograph, gra- dient control model L-5000 LC, fl uorescence spectrophot- ometer model F-1000, chromate-integrator model D-2000, Merck, Darmstadt, Germany). The HPLC method was based on a technique described in the literature 18. The thawed plasma samples were prepared in the follow- ing manner: 500 ml plasma were mixed with 150 ml 3 M perchloric acid. After intense shaking for 15 s (Vortex- Mixer, Bender and Hobein, Zu rich, Switzerland), the sam- ple was centrifuged for 2 min at 12000 rev./min (Zentrifuge 3200, Eppendorf, Hamburg, Germany). Twenty microliters of the clear supernatant was injected directly into the HPLC system. The analysis was conducted with a LiChroCart col- umn, packed with Superspher 100 RP-18 4 mm, 75 4 mm (Merck, Darmstadt, Germany) and a LiChroCart guard col- umn, packed with LiChrospher 100 RP-18 5 mm, 4 4 mm (Merck, Darmstadt, Germany). The mobile phase consisted of 0.02 M perchloric acid for the fi rst 3.5 min, 0.02 M perchloric acid:acetonitril 55:45 until 6.5 min to clean the column and 0.02 M perchloric acid for an additional 2.5 min to equalize the system. Every 9 min a new plasma sample could be injected. The fl ow rate was 1.5 ml/min. The col- umn temperature was room temperature. Acyclovir was detected fl uorimetrically with an excitation-wavelength of 260 nm and an emission-wavelength of 375 nm. With an USP-reference substance of acyclovir (acyclovir CRS, Cat. No. 01206, Lot H; USPC Inc. Rockville MD, USA), an acyclovir work standard (Ch. No. FP95320A; Fa hrhaus Pharma, Hamburg, Germany) was established. The standardization resulted from the UV-spectroscopic classifi cation at 254 nm in 0.1 N hydrochloric acid (n = 5). The acyclovir concentration of the work standard could be determined at 99.8%. The evaluation of the HPLC-chromatogram was con- ducted using the peak area. The retention time for acyclovir was 2.8 min. The validation of the method was carried out with pooled human plasma (frozen fresh plasma CPD; Deutsches Rotes Kreuz, Mu nster, Germany), which was mixed with aqueous acyclovir solution (reference standard) in different concentrations. In a range between 21.2 and 998.0 ng/ml, linearity (r = 0.999) was proven. The stability Table 1 Composition of compressed coated tablets with internal magnet 10%15% Outer coatAcyclovir4040 Methocel K4M4060 Lactose316296 Magnesium stearate44 Inner coatAcyclovir160 Methocel K4M16 Magnesium stearate1.8 Table 2 Demographic data of the subjects and the application scheme Subject no.SexAge (years) Height (m) Body weight (kg) Treatment on 1st day of the study Treatment on 2nd day of the study Treatment on 3rd day of the study 1M231.7675CAB 2M231.7058CAB 3M241.7575BCA 4M241.7865BCA 5M231.8794ABC A, magnetic depot dosage form in presence of an extracorporal magnet; B, magnetic depot dosage form in absence of an extracorporal magnet; C, immediate release dosage form. 287R. Gro ning et al. / European Journal of Pharmaceutics and Biopharmaceutics 46 (1998) 285291 of acyclovir during the freezing of the plasma samples for more than 3 weeks was tested. 2.6. Pharmacokinetic simulations A computer program was developed to simulate plasma concentration-time profi les after peroral administration of dosage forms and to calculate the infl uence of the gastro- intestinal transit on the plasma levels. Numerical mathema- tical procedures 19 were used for the mathematical calculations. First order kinetics describe the transfer of the drug substances between the compartments in the math- ematical model. The absorption-rate constant is decreased parallel with the assumed transport of the drug preparation through the gastrointestinal tract 20. The gastric residence time of the dosage form infl uences the calculated drug absorption. The decrease of the absorption-rate-constant was calculated with the following equation: ka(t)=ka ekn t where ka is the fi rst order absorption rate constant, ka(t), the fi rst order absorption rate constant at time t, and kn , the fi rst order decrease rate constant. The simulations of the plasma concentration-time pro- fi les, after peroral administration of acyclovir, were con- ducted with the following rate constants: ka= 0.0133/min, k12= 0.0065/min, k21= 0.0013/min, k10= 0.006/min and kn= 0.0085/min. The volume of distribution amounted to 154 l. The profi les were calculated for the following gastric residence times: 1.25 and 12 h. The simulated plasma con- centration-time profi les were compared to the average pro- fi les of the in vivo study using a non-linear correlation analysis. The results of the simulations show the infl uence of different gastric transit times which can be expected in the case of acyclovir. 3. Results 3.1. In vitro investigations In the present investigations peroral acyclovir depot tablets with internal magnets were developed. Each mag- netic tablet contained 200 mg of acyclovir. The internal magnets were coated with carnauba wax. A schematic dia- gram of the tablet is shown in Fig. 1. The layer surrounding the magnet contained 160 mg of acyclovir and the outer layer of the compression coated tablet contained 40 mg. For controlling the release of the active substance, the tablet layers contained hydroxypropyl- methylcellulose. Two preparations with different amounts of hydroxypropylmethylcellulose in the outer coat were developed (Table 1). The in vitro release profi les of the two formulations are shown in Fig. 2. A higher amount of hydroxypropylmethylcellulose in the outer coat causes a delay in the drug release. By using 10% hydroxypropyl- methylcellulose, 80% of the acyclovir dose is released within 5 h. A higher amount of hydroxypropylmethylcellu- lose (15%) in the outer coat leads to a drug release of 80% within 8 h. Additional experiments showed that an external magnet has no infl uence on the release of acyclovir from depot tablets containing hydroxypropylmethylcellulose. In the in vivo study, a magnetic depot tablet with a 10% hydroxypropylmethylcellulose in the outer coat was used. A reference preparation in the in vivo studies was a commer- cially available normal tablet. It could be shown that the release of the total dose of acyclovir takes place within 5 min. 3.2. In vivo study Five healthy male subjects took part in the in vivo study. Fig. 2. In vitro release of acyclovir from press coated tablets with internal magnet (200 mg acyclovir; n = 5, arithmetic mean SD). X, 10% hydro- xypropylmethylcellulose 4000 in the outer coat; W, 15% hydroxypropyl- methylcellulose 4000 in the outer coat. Fig. 3. Plasma concentration of acyclovir after peroral administration (200 mg, subjects (1) to (5); arithmetic mean SD). *Statistically signifi cant differences between the applications of depot tablets with and without extracorporal magnet (P , 0.05). X, Magnetic depot tablet in presence of an extracorporal magnet; W, magnetic depot tablet in absence of an extracorporal magnet; B, immediate release dosage form. 288R. Gro ning et al. / European Journal of Pharmaceutics and Biopharmaceutics 46 (1998) 285291 On different days according to a three-way cross-over plan, the subjects swallowed a magnetic depot tablet or the com- mercially available fast releasing tablet. The administration took place at 0900 h after a 10-h fast with 200 ml water. The intake of food and drink took place over a 12 h time period at specifi c times. The plasma concentrations of acyclovir were determined over 24 h. The average plasma concentration-time profi les, which were determined after peroral administration of the dosage forms are shown in Fig. 3. The results show that, through the use of magnetic tablets and external magnets in the stomach region, average plasma concentrations can be obtained which are higher and longer las