Estimating product bioequivalence for highly variable veterinary drugs.

The occurrence of drugs and drug formulations associated with large intrasubject pharmacokinetic (PK) variability has been well described in humans and is likewise encountered in veterinary medicine. The scaled average bioequivalence (SABE) approach adopted by CDER of the FDA for the determination of bioequivalence (BE) of highly variable drugs (HVD) needs to be considered when applied to veterinary dosage forms. However, because of some of the unique challenges that are encountered within the framework of veterinary medicine, variations of CDER's approach are presented. The present manuscript discusses HVD and highly variable veterinary drugs (HVVD) from the perspective of possible alternative approaches to support the assessment of product BE in veterinary medicine. Limitations in the use of 3- and 4-way crossover study designs are enumerated. In addition to a need for a statistical analysis of HVVD when using a parallel study design, the use of the secondary criteria (test-to-reference ratio), definition of σ(0) , and average BE with expanding limits are raised. A number of the details need to be finalized, from the selection of a regulatory constant to the determination of 'highly variable' in a veterinary drug product. Academicians, industrial scientists, and regulators should continue this discussion and resolve these details.

Osmotic drug delivery using swellable-core technology.

Swellable-core technology (SCT) formulations that used osmotic pressure and polymer swelling to deliver drugs to the GI tract in a reliable and reproducible manner were studied. The SCT formulations consisted of a core tablet containing the drug and a water-swellable component, and one or more delivery ports. The in vitro and in vivo performance of two model drugs, tenidap and sildenafil, formulated in four different SCT core configurations: homogeneous-core (single layer), tablet-in-tablet (TNT), bilayer, and trilayer core, were evaluated. In vitro dissolution studies showed that the drug-release rate was relatively independent of the core configuration but the extent of release was somewhat lower for the homogeneous-core formulation, particularly under non-sink conditions. The drug-release rate was slower with increasing coating thickness and decreasing coating permeability, and was relatively independent of the drug loading and the number and size of the delivery ports. The drug-release rates were similar for the two model drugs despite significant differences in their physicochemical properties. Tablet-recovery and pharmacokinetic studies conducted in beagle dogs showed that the in vivo release of drug from SCT formulations was comparable to the in vitro drug release.

Comparison of the gravimetric, phenol red, and 14C-PEG-3350 methods to determine water absorption in the rat single-pass intestinal perfusion model.

This study was undertaken to determine whether the gravimetric method provided an accurate measure of water flux correction and to compare the gravimetric method with methods that employ nonabsorbed markers (eg, phenol red and 14C-PEG-3350). Phenol red,14C-PEG-3350, and 4-[2-[[2-(6-amino-3-pyridinyl)-2-hydroxyethyl]amino]ethoxy]-, methyl ester, (R)-benzene acetic acid (Compound I) were co-perfused in situ through the jejunum of 9 anesthetized rats (single-pass intestinal perfusion [SPIP]). Water absorption was determined from the phenol red,14C-PEG-3350, and gravimetric methods. The absorption rate constant (ka) for Compound I was calculated. Both phenol red and 14C-PEG-3350 were appreciably absorbed, underestimating the extent of water flux in the SPIP model. The average +/- SD water flux microg/h/cm) for the 3 methods were 68.9 +/- 28.2 (gravimetric), 26.8 +/- 49.2 (phenol red), and 34.9 +/- 21.9 (14C-PEG-3350). The (average +/- SD) ka for Compound I (uncorrected for water flux) was 0.024 +/- 0.005 min(-1). For the corrected, gravimetric method, the average +/- SD was 0.031 +/- 0.001 min(-1). The gravimetric method for correcting water flux was as accurate as the 2 "nonabsorbed" marker methods.

Predicting injection site muscle damage. I: Evaluation of immediate release parenteral formulations in animal models.

PURPOSE: The current animal model generally accepted by the pharmaceutical industry and the FDA for assessment of muscle damage following intramuscular injection (IM) is the rabbit lesion volume model (RbLV). However, this model is resource intensive. The goal of this study was to find a resource sparing alternative to the rabbit lesion model for assessing injection site toleration in IM formulation screening. METHODS: Short term animal model alternatives to RbLV for evaluating IM formulations were examined. In addition to RbLV, myeloperoxidase (MPO), p-nitrophenyl N-acetyl-beta-glucosaminide (NA beta G) and/or plasma creatine phosphokinase (CK) activities were determined in rabbits (Rb) and rats (Rt) after injection of formulations (digoxin, azithromycin and danofloxacin). The edema from these formulations 24 hr after subcutaneous injection into the rat footpad (RFE) was also determined. RESULTS: MPO and NA beta G were not considered very useful as biochemical predictors of muscle damage for these formulations. Histology generally correlated with RbLV values. Compared to saline, RbLV was marked for all formulations within 1-3 days of injection. After day 3, lesions quickly resolved, and no significant differences were found. For these formulations, all CK animal models and RFE were generally predictive of RbLV. A formulation with RtCK > 1000 U/L or RbCK > 3000 U/L, was predicted to be poorly, tolerated. CONCLUSIONS: Due to ease, number of animals, time and intrinsic mechanism, we concluded that for most formulations, 2 and 4 hr RtCK data alone should be reasonably predictive of muscle damage.

Predicting injection site muscle damage. II: Evaluation of extended release parenteral formulations in animal models.

PURPOSE: The goal of this study was to find a resource sparing alternative to the rabbit lesion model (RbLV) for assessing injection site toleration in extended release (ER) intramuscular (IM) formulation screening. METHODS: ER formulations (danofloxacin oily and aqueous suspensions) were evaluated in RbLV, rat and rabbit plasma creatine phosphokinase (CK), and rat foot edema (RFE) models as described in the companion article. RESULTS: None of the short term models could consistently predict acute and chronic effects of the. For example, RFE predicted little muscle damage from aqueous vehicle (0.03 +/- 0.03 g) and 60 mg/ml (0.08 +/- 0.03 g) formulation; while RbLVdays1-3 was marked and greater (p < 0.05) for 60 mg/ml (6.0 +/- 3.1) than vehicle (2.2 +/- 2.9) formulations. Furthermore, RbLVdays 1-3) for vehicle (6.5 +/- 7.5) and 60 mg/ml (4.9 +/- 4.6) danofloxacin oily formulations were worse (p < 0.05) than oil alone (1.4 +/- 2.2); an observation not predicted by CK models, since they apparently reflected only the acute muscle damage of formulation components immediately available to surrounding tissue at the time of injection. CONCLUSIONS: The CK models may be useful to screen those ER formulations with unacceptable acute damage due to immediately available components. However, to evaluate potential delayed effects from ER formulations, the long-term model RbLV was still recommended.

Urinary and biliary disposition of the lactone and carboxylate forms of 20(S)-camptothecin in rats.

Recently, analytical methods have become available for determination of both the lactone (active form) and the carboxylate (inactive form) forms of 20(S)-camptothecin in biological fluids. Studies in our laboratory have shown that there are significant differences in the in vivo behavior of the two forms of camptothecin and that much higher plasma levels of the lactone form are present in rats after dosing with camptothecin (lactone) than after dosing with the sodium salt of the ring-opened camptothecin (carboxylate form). The present studies show that there are significant differences in the urinary and biliary elimination of the two forms and that the urinary excretion of the carboxylate form appears to be pH dependent. This apparent pH dependence of the urinary elimination of the carboxylate form may provide a method of reducing the bladder toxicity associated with the use of camptothecin. After administration of a 1 mg/kg iv dose of camptothecin (lactone) to rats, 10.1 +/- 4.2% of the dose was excreted into the urine and 7.5 +/- 4.2% of the dose was excreted into the bile. Following an equivalent intravenous dose of the carboxylate form, 39.5 +/- 10.4% of the dose was excreted into the urine and 26.4 +/- 8.9% of the dose was excreted into the bile.

Evaluation of sustained/controlled-release dosage forms of 3-hydroxy-3-methylglutaryl-coenzyme a (HMG-CoA) reductase inhibitors in dogs and humans.

Seven sustained/controlled-release dosage forms were designed for gastrointestinal delivery of lovastatin or simvastatin, two potent HMG-CoA reductase inhibitors for the treatment of hypercholesterolemia. The in vivo performance of these formulations was evaluated in dogs and healthy volunteers in terms of the cholesterol lowering efficacy and/or systemic concentrations of HMG-CoA reductase inhibitors. Results from the present and previous studies suggest that, through the controlled release of HMG-CoA reductase inhibitors, sustained lower plasma concentrations of HMG-CoA reductase inhibitors may result in an equal or better therapeutic efficacy.

Enhanced bioavailability of cefoxitin using palmitoylcarnitine. II. Use of directly compressed tablet formulations in the rat and dog.

The performance of tablets containing the absorption enhancer palmitoylcarnitine chloride (PCC) and the antibiotic cefoxitin (CEF) was determined by direct placement of tablets in the rat stomach, small intestine, and colon. While the bioavailability (F) of tablets containing 12 mg CEF without PCC ranged from 0.6 to 3.9%, the addition of 24 mg PCC resulted in an enhanced CEF bioavailability in the rat colon (mean +/- SD: F = 57 +/- 19%) and rat jejunum (F = 71 +/- 16%) but not in the rat stomach. Following oral administration to dogs, tablets of 200 mg CEF without or with 600 mg PCC resulted in the same low bioavailabilities (7.0 +/- 10.3 and 7.0 +/- 3.6%, respectively). However, when these tablets were enteric coated, PCC improved CEF bioavailability from 2.44 +/- 1.84 to 29.0 +/- 13.4%. Therefore, the use of enteric-coated direct compressed tablets containing PCC and direct compression excipients improved the peroral bioavailability of a poorly absorbed compound.

In vitro effects of long-chain acylcarnitines on the permeability, transepithelial electrical resistance and morphology of rat colonic mucosa.

Absorption-enhancing properties of the long-chain acylcarnitines (C12-C18) were examined in vitro utilizing a modified Sweetana-Grass diffusion cell system. Transepithelial electrical resistance (TEER) and drug permeability (P) of rat colonic mucosa were used as parameters to determine the potency, selectivity and reversibility of acylcarnitine-enhancing effects. All long-chain acylcarnitines induced a rapid, concentration-dependent decrease in mucosal TEER. The minimum or threshold concentration (Ct) which produced a rapid decrease in mucosal TEER was determined for each acylcarnitine. Tissues treated with either palmitoyl or lauroyl carnitine at their Ct showed significantly better recovery of TEER after removal of the enhancers from the diffusion cells as compared to the other long-chain acylcarnitines. In addition, palmitoyl and lauroyl carnitine treatment significantly enhanced the mucosal permeability to small, hydrophilic markers without adversely altering tissue morphology. Following enhancer replacement with buffer alone and the subsequent recovery of TEER, mucosal permeability was observed to return to near control levels. At higher concentrations of palmitoyl and lauroyl (5 x Ct), P increased nearly 2-fold over that observed at the Ct; however, epithelial barrier morphology was compromised. This study shows that colonic mucosal tissue mounted in modified Sweetana-Grass diffusion cells may be a useful model for examining the mechanism(s) of absorption enhancer activity. Moreover, the results present evidence that the long-chain acylcarnitines may enhance drug absorption via two different concentration-dependent pathways.

Simultaneous in vitro measurement of intestinal tissue permeability and transepithelial electrical resistance (TEER) using Sweetana-Grass diffusion cells.

A simple modification of the commercially available Sweetana-Grass (S-G) side-by-side diffusion cells, allowing the simultaneous measurement of tissue permeability and transepithelial electrical resistance (TEER), has been described and validated for rat excised, muscle-free intestinal tissue. The TEER-lowering effects of a series of acylcarnitines were shown to be correlated with previously reported in vitro (i.e., membrane perturbation) and in vivo (i.e., absorption enhancement) activity. The TEER-lowering effect of palmitoyl carnitine chloride (PCC) was also shown to be reversible. The effects of PCC on TEER and the permeability of poorly absorbed compounds (cefoxitin and lucifer yellow) were simultaneously determined. Compared to controls (mannitol-treated), PCC immediately produced a rapid drop in colon TEER. By 5 min post-PCC addition, colon TEER was 50% of control; by 10 min post-PCC addition, colon TEER was 17% of control. After a lag of about 5-10 min post-PCC addition, the cefoxitin or lucifer yellow permeability coefficient increased more than 20-fold. The modified S-G cells provide a simple and reproducible method whereby flux and TEER can be simultaneously determined, providing a valuable link between the effect of absorption enhancers on TEER measurements and the increased permeability of poorly absorbed compounds.

Enhanced bioavailability of cefoxitin using palmitoyl L-carnitine. I. Enhancer activity in different intestinal regions.

The conditions under which the absorption enhancer palmitoyl L-carnitine chloride (PCC) improved the bioavailability of the poorly absorbed antibiotic cefoxitin throughout the rat intestine has been studied. Cefoxitin alone was appreciably absorbed only in the duodenum (31% vs less than 7% elsewhere). PCC solutions (3 mg/rat, pH 4.0) enhanced cefoxitin bioavailability (F) by 0-, 22-, 16-, and greater than 32-fold in the duodenum, jejunum, ileum, and colon regions, respectively. The inability of PCC to improve F in the duodenum could not likely be attributed to enzymatic degradation of the enhancer, since coadministration with protease and esterase inhibitors produced similar results (F = 30%). Coadministration of PCC solution with cefoxitin in the unligated or ligated colon, increased F to 33 and 76%, respectively. Qualitatively similar results were seen with PCC suspensions (3 mg/rat, pH 6.0). Maintaining a high concentration of cefoxitin and PCC in a restricted region (i.e., by ligating a 2- to 3-cm section of the colon) afforded a two- to threefold advantage over an unligated colon section. The difference in cefoxitin bioavailability between ligated and unligated colon was probably due to sample spreading and subsequent/simultaneous dilution.

Relationship between drug absorption enhancing activity and membrane perturbing effects of acylcarnitines.

Acylcarnitines with chain lengths of 2 to 18 carbon atoms were tested for their effects on rat intestinal brush border membrane order (S) by fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH). These results were compared to the previously reported effectiveness of the acylcarnitines as absorption enhancers of the poorly absorbed antibiotic cefoxitin. Acylcarnitines with fatty acids less than 12 carbon units in length were ineffective in increasing drug absorption and perturbing brush border membrane order. Long-chain acylcarnitines (12-18 carbons) significantly increased the bioavailability of cefoxitin and decreased the lipid order of brush border membranes. The results suggest that, in order to promote drug absorption, the acylcarnitines must surpass a critical chain length (10 carbon units) to partition effectively into the membrane and, in addition, must perturb the lipid order beyond a threshold value (15-20%). Membrane perturbing capacity may serve as an indicator of the absorption enhancing potential of other aliphatic-type compounds.

The solubility-modulated osmotic pump: in vitro/in vivo release of diltiazem hydrochloride.

A generalized method was investigated for conversion of controlled-porosity osmotic pump release profiles from first-order to zero-order kinetics using diltiazem.HCl as a model drug. Diltiazem.HCl has an aqueous solubility greater than 590 mg/ml (37 degrees C) and was released from controlled-porosity osmotic pump devices with first-order kinetics. This high solubility was markedly reduced (155 mg/ml; 37 degrees C) in the presence of NaCl (1 M). Based on theory for osmotically actuated drug release, this reduced solubility would be expected to result in a zero-order release profile of greater than 80% of an initial diltiazem.HCl load. Devices were prepared with cores that contained diltiazem.HCl and sufficient NaCl granules coated with a microporous cellulose acetate butyrate 381-20 film to maintain a 1 M NaCl concentration within the drug compartment over a 16-hr period. This resulted in release of approximately 75% of the initial diltiazem.HCl load with zero-order kinetics over a 14- to 16-hr period. The in vivo performance of these devices in beagle dogs was analyzed. The in vivo percentage diltiazem absorbed profiles were superimposable with the in vitro release profile. These results suggest that diltiazem release and absorption from the solubility modulated osmotic pump occur throughout the GI tract in a fashion predictable from in vitro dissolution data.

Performance of diltiazem tablet and multiparticulate osmotic formulations in the dog.

The in vivo performance of two extended-release (ER) osmotic formulations of diltiazem were evaluated in the beagle dog. Both ER formulations had similar bioavailabilities (F) as the diltiazem solution. Although F was somewhat variable following ER administration, this variability may be related to the drug entity since intra- and interanimal variability of orally administered diltiazem solutions was substantial. Deconvolution of the ER plasma diltiazem data with absorption data from the orally administered diltiazem solutions provided an estimate of the in vivo drug release from the ER formulations. The two ER formulations, designed with different in vitro release profiles, reflected these differences in vivo, with nearly identical respective in vivo and in vitro release profiles.

Absorption of magnesium from orally administered magnesium sulfate in man.

The use of magnesium sulfate (Epsom salt) as a cathartic in patients with impaired renal function can lead to severe toxicity due to hypermagnesemia. Although toxicity is uncommon in healthy subjects, little is known concerning the extent of absorption of magnesium after a cathartic dose of magnesium sulfate. The bioavailability of magnesium following a large oral dose of magnesium sulfate in normal volunteers was examined in the present investigation. Baseline 24-hour urinary excretion rates of magnesium and creatinine were determined over 3 consecutive days in 6 healthy men. The oral administration of 13.9 g (56.5 mmoles) magnesium sulfate U.S.P., in 4 equal hourly increments, resulted in the urinary excretion (corrected for baseline excretion rate) of 4.0 +/- 2.9% (mean +/- SD) of the dose of magnesium during the first 24 hours and 6.9 +/- 7.0% of the dose during a 72-hour interval. Magnesium sulfate administration had no effect on the 24-hour urinary excretion rate of creatinine. The baseline excretion rate of magnesium was significantly correlated with that of creatinine (r = 0.875) and inorganic sulfate (r = 0.921). All of the subjects experienced mild or moderate diarrhea. Therefore, magnesium is absorbed to a limited and variable extent in healthy adults following a cathartic dose of magnesium sulfate.

Blood vessel uptake and metabolism of organic nitrates in the rat.

Recent reports have suggested that the unusual pharmacokinetics observed for nitroglycerin (NTG) and isosorbide dinitrate (ISDN) may be partially explained by extensive uptake and/or metabolism of these drugs by vascular and other extrahepatic tissues. Using the rat as an animal model, this hypothesis was examined by in vivo intravessel NTG and [14C]ISDN infusion and injection into various vessel segments, viz. the femoral vein, inferior vena cava [IVC: lower, middle and upper) and the aorta. NTG and [14C]ISDN concentrations were determined in these blood vessels and in plasma. Blood vessel segments nearest the input site had the greatest amounts of nitrate, whereas segments further away from the input site had progressively less nitrate, with the exception of aorta, which appeared to take up NTG less extensively, on a per weight of vessel basis, than the IVC. Blood vessel NTG concentrations (nanogram per gram) were generally higher (10-fold) and declined about twice as slowly as NTG plasma concentrations (nanograms per milliliter). [14C]NTG and [14C]ISDN were also incubated with cofactors in IVC, aorta, abdominal muscle, lung and liver. The amounts of nitrate metabolites formed from parent drug were larger in each extrahepatic tissue incubation than in the controls (P less than .05). The results are consistent with the hypothesis that vascular and other extrahepatic tissues can take up and/or metabolize organic nitrates. The data appear to provide a partial explanation for the large systemic clearance seen with nitrates and appear consistent with existing mechanistic hypotheses for the vascular action of these compounds.

Effect of dosage regimen on the development of tolerance to nitroglycerin in rats.

The recent introduction of several sustained delivery systems of nitroglycerin (NTG) raises the question whether the mode of drug input (e.g., sustained versus intermittent) may be a critical determinant in the development of nitrate tolerance. This hypothesis was tested in an animal model. Sixty male Sprague-Dawley rats (weighing 240-260 g) were administered a total intravenous dose of 2.5 mg NTG either as a continuous 6-h infusion (6.8 micrograms/min) or as six hourly pulse injections of 425 micrograms each. Animals were sacrificed 5 min following the termination of the infusion and 65 min following the last injection. A blood sample was taken from a central vein for plasma NTG determination, and the aorta and portal vein were isolated. Dose-response curves to NTG were determined on some of these blood vessels and on controls using an isolated tissue bath apparatus. Other blood vessels were incubated with 94 ng [14C]NTG for 60 min, and the incorporation of [14C]NTG into these tissues was determined after thin-layer chromatographic separation of NTG from its metabolites. There was no difference between the plasma NTG concentration measured at the time of sacrifice following either regimen. For the artery preparation, there was also no difference in the dose response to NTG or in the incorporation of [14C]NTG into the blood vessel from rats treated by either regimen. For the portal vein preparation, however, rats treated by continuous infusion had a similar sensitivity to NTG as controls, but there was a marked downward shift in the dose response to NTG in the veins of rats treated by the intermittent regimen.(ABSTRACT TRUNCATED AT 250 WORDS)