J. vet. Pharmacol. Therap. 36, 399--407. doi: 10.1111/jvp.12019.
Ronidazole pharmacokinetics in cats following delivery of a delayedrelease guar gum formulation M. G. PAPICH* D. N. L E VINE
Papich, M. G., LeVine D. N., Gookin, J. L., Davidson, G. S., Stagner W. C., Hayes, R. B. Ronidazole pharmacokinetics in cats following delivery of a delayed-release guar gum formulation. J. vet. Pharmacol. Therap. 36, 399–407.
†
J. L. GOOKIN † G. S. DAVIDSON ‡ §
W. C. STAGNER & R. B. HAYES ‡ , ¶ *Department of Molecular Biomedical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, NC, USA; †Department of Clinical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, NC, USA; ‡ Clinical Pharmacy Services, College of Veterinary Medicine, North Carolina State University, Raleigh, NC, USA; §Department of Pharmaceutical Sciences, School of Pharmacy, Campbell University, Buies Creek, NC, USA; ¶Rex Inpatient Pharmacy, Rex University of North Carolina Health Care, Raleigh, NC, USA
Ronidazole (RDZ) is the only known effective treatment for feline diarrhea caused by Tritrichomonas foetus. This study aimed to develop guar gum-coated colon-targeted tablets of RDZ and to determine the pharmacokinetics of this delayed-release formulation in cats. Guar gum-coated tablets were administered orally once to five healthy cats (mean dose 32.3 mg/kg). The tablets were then administered once daily for 5 days to four cats (mean dose 34.5 mg/kg), and absorption studies repeated on day 5. Plasma was collected and analyzed for RDZ concentration, and pharmacokinetic noncompartmental and deconvolution analysis were performed on the data. There was negligible RDZ release until after 6 h, and a delayed peak plasma concentration (mean Cmax 28.9 lg/mL) at approximately 14.5 h, which coincides with colonic arrival in cats. Maximum input rate (mg/kg per hour) occurred between 6 and 16 h. This delayed release of ronidazole from guar gumcoated tablets indicates that release of RDZ may be delayed to deliver the medication to a targeted area of the intestine. Repeated dosing with guar gum tablets to steady-state did not inhibit drug bioavailability or alter the pharmacokinetics. Such targeted RDZ drug delivery may provide improved efficacy and reduce adverse effects in cats. (Paper received 18 June 2012; accepted for publication 14 September 2012) Mark G. Papich, College of Veterinary Medicine, North Carolina State University, 1060 William Moore Drive, Raleigh, North Carolina, USA, 27607. E-mail:
[email protected]
INTRODUCTION Ronidazole (RDZ) is the only effective published drug for the treatment of Tritrichomonas foetus intestinal infection in cats. Tritrichomonas foetus is a flagellated protozoan parasite that causes colitis and chronic, foul-smelling diarrhea (Gookin et al., 2006; Tolbert & Gookin, 2009). Tritrichomonas foetus resides in the distal ileum and colon (Gookin et al., 2006). But, work from our laboratory showed that immediate-release RDZ is completely absorbed from the upper small intestine (LeVine et al., 2011). It is unknown how much drug actually reaches the site of infection. Administration of immediate-release RDZ formulations at the reported effective dose (30–50 mg/kg q12 h) causes rapid release of the medication, which produces an immediate spike in plasma concentrations (LeVine et al., 2011). In some cases, this sudden burst of the drug into the systemic circulation has produced adverse neurologic events (Gookin et al., 2006; Rosado et al., 2007; Pham, 2009). By providing RDZ delivery to the site of infection, colon-targeted delivery of RDZ may allow for effective treatment of T. foetus © 2012 John Wiley & Sons Ltd
while avoiding systemic adverse effects. Our objective in this study was to develop a delayed-release colon-targeted drug delivery RDZ formulation consisting of guar gum as the formulation excipient. Guar gum is a nontoxic natural nonionic polysaccharide derived from seeds of Cyamopsis tetragonolobus. It has been used for colon-targeted drug delivery in people because it is not digested in the stomach or small intestine, but is degraded in the colon by resident bacteria (Krishnaiah et al., 1998). Drug release from guar gum-coated tablets occurs due to enzymatic degradation rather than time-dependent disintegration (Salyers et al., 1977; Krishnaiah et al., 1998; Prasad et al., 1998). Previous studies with tinidazole, another member of the nitroimidazole class of drugs to which RDZ belongs (Moreno et al., 1983), have shown that guar gum coating of tinidazole tablets successfully delivered tinidazole for local action in the colon in people for treatment of amebiasis (Krishnaiah et al., 2002, 2003). We decided to use guar gum as the matrix for targeted colon release in cats because a pH-dependent targeted RDZ drug delivery probably would not be successful because 399
400 M. G. Papich et al.
the feline duodenal pH (5.7) overlaps with the feline colonic pH (5.8) (Brosey et al., 2000). The present study set out to determine if compression coating immediate-release RDZ tablets with guar gum could modify the RDZ release to target the colon in healthy cats using pharmacokinetic methods after a single dose. Because metronidazole, another nitroimidazole drug, significantly decreases aerobic and anaerobic bacterial counts in the duodenum of healthy cats, we were concerned that repeated RDZ dosing could sufficiently alter colonic flora with repeated administration, which would alter guar gum digestion and prevent RDZ release (Johnston et al., 2000). Previous in vitro studies with rat caecal contents have shown that administration of either metronidazole or tinidazole interferes with degradation of guar gum matrix tablets by colonic bacteria (Krishnaiah et al., 2001). For this reason, a repeated dosage study with guar gum-coated RDZ tablets was also conducted, with RDZ pharmacokinetics determined on the fifth day of once daily administration. MATERIALS AND METHODS
Table 1. Composition of Ronidazole (RDZ) core tablets and guar gum granules for compression coating of RDZ core tablets Quantity (mg) present in
Ingredients Ronidazole (Spectrum, Gardena, CA, USA) Fast flo lactose (Foremost NF Fast Flo Lactose®, Foremost Farms, Baraboo, WI, USA) Sodium starch glycolate (Spectrum, New Brunswick, NJ, USA) Magnesium stearate (Spectrum, New Brunswick, NJ, USA) Guar gum (Spectrum, New Brunswick, NJ, USA) Hydroxypropyl methylcellulose (HPMC) (Methocel K100M®, Professional Compounding Centers of America, Houston, TX, USA) Starch (Spectrum, New Brunswick, NJ, USA)
Core RDZ tablets
Guar gum granules
150
–
38
–
10
–
2
4
–
260
–
96
–
40
Drug formulation Preparation of guar gum-based colon-targeted ronidazole tablets. Core RDZ tablets containing 150 mg of RDZ (Spectrum, Gardena, CA, USA) [(1-methyl-4-nitroimadazol-2-yl)methyl carbamate] were prepared by direct compression. Core tablet ingredients are listed in Table 1. The mixture was compressed into tablets at an average applied force of 681 kg (range 591– 909 kg) using 9-mm round, flat punches on a hydraulic tablet press (Carver Press, Carver, Wabash, IN, USA). Core tablets had an average hardness of 42.2 kg/ms2, as assessed by a tablet hardness tester (VK 200 Tablet Hardness Tester; VanKel, Cary, NC, USA). A coat formulation of guar gum granules containing 65% guar gum for compression coating of RDZ core tablets was prepared by wet granulation technique according to the methods published previously (Krishnaiah et al., 2003, Table 1). Briefly, the guar gum, hydroxypropyl methylcellulose (HPMC), and starch (added as a paste 10% w/ w in water) were combined. Additional water was added to the granulation mixture (6 mL). The mixture was passed through a #12 mesh sieve (1.7 mm; W.S. Tyler, Mentor, OH, USA) and vacuum dried to 36% moisture at 50 °C for 5 h in a vacuum oven (Napco Vacuum Oven Model 5831; Precision Scientific, Chicago, IL, USA). Magnesium stearate was added after the drying process, and the dry mixture passed through a #16 mesh sieve (1.18 mm; Torpac, Fairfield, NJ, USA). The core RDZ tablets were then guar gum coated by direct compression. About 30% (120 mg) of the guar gum granules was placed in an 11-mm die cavity. A core RDZ tablet was centered in the die cavity over the guar gum mixture. The tablet was then covered with the remaining guar gum granule mixture (280 mg). The tablet was then compressed at an applied force of 2273 kg (Carver). The guar gum-coated RDZ core tablets had an average hardness of 84.3 kg/ms2 (VK 200 Tablet Hardness Tester; VanKel).
In vitro drug release. The guar gum-coated colon-targeted RDZ tablets were evaluated for their integrity in the physiologic environment of the stomach and small intestine under conditions mimicking the pH and temperature of these organs. Similarly, the core RDZ tablets without guar gum coating were evaluated to confirm that these tablets behave as immediaterelease tablets in the physiologic environment of the stomach and small intestine. Both guar gum-coated RDZ tablets and RDZ core tablets without coating were subjected to dissolution tests in simulated gastric and intestinal environments. The simulated gastric environment consisted of 400 mL of 0.1 N hydrochloric acid (HCl) (pH 2) (Fisher Scientific, Fair Lawn, NJ, USA) in a 500mL beaker kept between 37 and 40 °C and stirred at 50 rpm with a stir bar. This HCl volume corresponds to feline gastric volume (Kararli, 1995). The HCl solution for simulated gastric conditions was prepared as specified by the standards of the United States Pharmacopeia (United States Pharmacopeial Convention). The simulated intestinal environment was similar, except the HCl solution was replaced by a phosphate buffer (pH 6.8), again prepared according to the standards in General Chapter of the United States Pharmacopeia (USP, 2010a). Drug release was monitored at various time intervals by withdrawing 1 mL of the dissolution sample and assessing drug concentration using a spectrophotometer (Spectronic GenesysTM 2; Spectronic Instruments, Rochester, NY, USA) set at a wavelength of 313 nm. RDZ concentration in each sample of dissolution solution was determined by comparing absorbance of a 1-mL 1:4 dilution of the beaker solution to calibration curves of absorbance plotted against RDZ concentration. The calibration curves were generated from five RDZ dilutions (from 0 to 100 lg/mL) prepared in
© 2012 John Wiley & Sons Ltd
Delayed-release ronidazole in cats 401
both the HCl and phosphate solutions. The calibration curves were linear with a R2 value 0.99. Cats. Five healthy 1-year-old male intact cats (Liberty Research Inc., Waverly, NY, USA) were used in the study (4.53 kg ± 0.38 kg body weight). Cats were housed in the institutional laboratory animal facility, and all cats were adopted as pets at study completion or were enrolled in the NCSU CVM blood donor program and subsequently adopted. All protocols were approved by the Institutional Animal Care and Use Committee of North Carolina State University. Ronidazole administration and sample collection. In the single-dose study, 15 hours prior to drug administration, a 19-gauge catheter (BD Intracath, Becton Dickinson, Sandy, UT, USA) was placed in the medial saphenous vein of each cat to facilitate sample collection. To minimize the stress of catheter placement, cats were sedated with a combination of IM ketamine (Fort Dodge Laboratories Inc., Fort Dodge, IA, USA) and medetomidine (Dormitor®; Pfizer Animal Health, Exton, PA, USA). Food was withheld for 24 h prior to and 8 h after drug administration. Water was available at all times. In the repeated dose study, medial saphenous catheters were similarly placed on day 4 of drug administration, the day prior to blood sampling. Food was withheld 16 h prior to and 8 h after administration of the 5th dose of RDZ. All cats recovered uneventfully. In the single-dose study, each cat received one 150-mg guar gum-coated RDZ tablet (range 30.18–37.30 mg/kg; mean dose 32.3 ± 2.9 mg/kg) followed by 6 mL of tap water orally to ensure that the medication was swallowed. After a 3-week washout period, four of the cats entered the repeated dose administration study. In the repeated dose study, each cat received one 150-mg (mean dose 34.5 mg/kg, ±2.6, range 31.91–38.07 mg/kg) guar gum-coated RDZ tablet followed by a water flush once daily for 5 days, and blood samples were collected on day 5. Blood samples (3 mL each) were collected via the IV catheter prior to, and 1, 2, 3, 4, 5, 6, 8, 10, 12, 16, 24, 30, and 48 h after drug administration, with drug administration being the single dose (single dose study) or the 5th dose (repeated dose study). Cumulative blood collection volumes were within published guidelines to avoid adverse hemodynamic effects (Diehl et al., 2001). Blood was immediately transferred to tubes containing lithium heparin (BD Vacutainer, Becton Dickinson, Franklin Lakes, NJ, USA) and centrifuged to harvest plasma. Plasma was frozen and stored at 80 °C until analysis. Ronidazole assay. The quantitative determination of RDZ in feline plasma samples was performed by high-pressure liquid chromatography (HPLC) using a method developed in our laboratory (LeVine et al., 2011). The reference standard of RDZ was obtained from the same source as that used for drug formulations (Spectrum) and confirmed to be 101% pure by supplier certificate of analysis. RDZ was dissolved in methanol (Sigma–Aldrich, St. Louis, MO, USA) to make up a 1-mg/mL © 2012 John Wiley & Sons Ltd
stock solution. From this stock solution, further dilutions were prepared in distilled water to use as fortifying solutions for plasma in order to generate calibration curves in plasma. The stock solution was kept at 4 °C in a tightly sealed dark vial, which we determined to be stable throughout the duration of the study. RDZ spiking solutions were added to blank (control) plasma, to make up eight calibration standards (range 0.025– 75 lg/mL). The mobile phase for HPLC analysis consisted of acetonitrile (15%) (Fisher Scientific) and 0.1% trifluoroacetic acid (85%) (Fisher Scientific). The HPLC system consisted of a quaternary solvent delivery system (Agilent Technologies, Wilmington, DE, USA) set at a flow rate of 1 mL/min, an autosampler, (1100 Series Autosampler; Agilent Technologies) and UV detector set at a wavelength of 313 nm (1100 Series UV Detector; Agilent Technologies). The chromatograms were integrated with a computer program (1100 Series Chemstation software; Agilent Technologies). A C8 reverse-phase column (Zorbax RX-C8, 4.6 9 150 mm reverse phase column; Agilent Technologies) was used for separation and kept at a constant temperature of 40 °C. All experimental plasma samples, calibration samples, and blank (control) plasma samples were prepared identically. Solidphase extraction cartridges (Oasis® HLB, hydrophilic-lipophilicbalanced, 1 cc extraction cartridges, Waters Corporation, Milford, MA, USA) were attached to a vacuum manifold and initially conditioned with 1-mL methanol followed by 1-mL distilled water. Each plasma sample was then extracted (0.5 mL), and the cartridge was washed with 1 mL of a 95:5 (vol:vol) mixture of distilled water: methanol. Samples were then eluted by the use of 1 mL of 100% methanol into clean glass tubes. The resulting eluate was evaporated under a stream of compressed air at 40 °C for 20 min. Evaporated samples were reconstituted with 200 lL of mobile phase and vortexed. Twenty-five microliter of each reconstituted solution were then injected into the HPLC system. Retention time for peak of interest was 4.10–4.20 min. A fresh set of calibration and blank samples were prepared for each day’s run. All calibration curves were linear with a R2 value of 0.99 or greater. Limit of quantification (LOQ) for RDZ in feline plasma was 0.025 lg/mL, which was determined from the lowest point on a linear calibration curve that produced an acceptable signal-to-noise ratio of 10:1. Quality control samples were run each day that samples were run (0.1, 5, and 50 lg/mL RDZ) and compared against the calibration curve. All quality control samples analyzed were within 10% of their nominal concentrations. The laboratory used guidelines published by the United States Pharmacopeia, General Chapter for method validation (USP, 2010b). Pharmacokinetic calculations Plasma drug concentrations of RDZ following single and repeated dose administration were plotted on linear and semilogarithmic graphs for analysis. The average of each cat’s normalized RDZ concentration (cat’s RDZ concentration at a given time point divided by the ratio of the cat’s dose to the mean
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dose) was calculated and used for plotting on concentration versus time graphs. Using a commercial software package, (Phoenix Software, Version 6.0; Pharsight Corporation, Mountain View, CA, USA) pharmacokinetic parameters were derived from the single-dose individual cat data using noncompartmental analysis. A compartmental analysis was performed initially, but there was no consistent compartmental model that would be suitable for all cats. The area under the plasma concentration vs. time curve (AUC) from time 0 to the last measured concentration, (defined by the sensitivity of the assay) was calculated using the log-linear trapezoidal method. The AUC from time 0 to infinity was calculated by adding the terminal portion of the curve, estimated from the relationship Cn/kz, to the AUC0cn, where kz is the terminal slope of the curve, and Cn is the last measured concentration point. Half-lives were calculated from the terminal slope: T½ = ln 2.0/(terminal rate constant), where ln 2.0 is the natural logarithm of 2.0. Pharmacokinetic parameters were reported as the geometric mean and percent coefficient of variation (CV%) of the geometric mean. Deconvolution analysis (Phoenix software, version 6.0.; Pharsight Corp.) was used to evaluate the in vivo drug release and delivery from the input obtained from the pharmacokinetic study. A unit impulse response function (UIR) was obtained from the intravenous pharmacokinetic study published previously (LeVine et al., 2011). The UIR provides exact linkage between drug level response (plasma concentration) and the input rate function. The drug appearance from the single oral dose in each cat was evaluated to obtain input response rates. The extent of absorption (F) was estimated from a comparison of the AUC values from single-dose oral guar gum tablet administration with IV administration in these same cats previously determined in our laboratory (LeVine et al., 2011) using the following formula: %F ¼
AUCORAL DOSEIV AUCIV DOSEORAL
Fraction of dose absorbed (fraction input) was also obtained from the deconvolution analysis. RESULTS In vitro drug release In vitro drug release studies showed that no RDZ was released from guar gum-coated colon-targeted tablets after 3 h in a simulated gastric environment (average feline gastric half-emptying time in fed cats) (Steyn et al., 1995; Goggin et al., 1998). In contrast, core RDZ tablets without the guar gum coating released all of their RDZ content within 10 min. Similarly, at the end of 4 h in the simulated intestinal fluids (longer than feline small intestinal transit time) (Chandler et al., 1997), there was
