A back translation of pregabalin and carbamazepine against evoked and non-evoked endpoints in the rat spared nerve injury model of neuropathic pain.

The purpose of the present study was twofold. First to characterize endpoints distinct to the reflexive responses to sensory stimuli typically used in neuropathic pain models. A second aim was to evaluate two clinically approved drugs carbamazepine (Tegretol) and pregabalin (Lyrica) against these endpoints with the purpose to backtranslate from the clinical to preclinical setting. The selected neuropathic pain model was the spared nerve injury (SNI) model and the endpoints were burrowing and measures of paw posture in Sprague Dawley rats. As previously described, SNI surgery produced a robust heightened sensitivity to tactile and thermal (cold) stimuli. SNI surgery also produced robust decreases in burrowing and affected multiple measures of paw position. There was no correlation between magnitude of change in burrowing and sensory allodynia within SNI operated rats. Pregabalin (10-30 mg/kg IP) produced a reliable reversal of both tactile and cold allodynia and also the burrowing deficit, with minimal effect on neurological function evaluated using rotorod, beam walking and open field activity. Pregabalin did not affect any measure of paw position. Pharmacokinetic studies conducted in satellite animals identified plasma levels of pregabalin at the 10 mg/kg IP dose to be equivalent to clinically efficacious levels recorded in neuropathic patients (3-6 µg/ml). In contrast carbamazepine (10-60 mg/kg IP) had only a very modest effect against a reflexive (tactile) measure, and no effect against the burrowing deficit. Carbamazepine also affected various measures of neurological function, complicating interpretation of the reflexive measure. Measurement of burrowing appears to detect a behavioural deficit associated with the SNI model, that may be attenuated by pregabalin but not carbamazepine. Overall the present findings support an advantage of pregabalin over carbamazepine in terms of both efficacy and tolerability which is consistent with clinical experience. The inclusion of additional endpoints beyond traditional reflexive behaviours further supports the value of rodent neuropathic pain models, such as the SNI, as behavioural assays to detect new chemical entities to treat this pain condition.

Evaluation of chemically diverse 5-HT2c receptor agonists on behaviours motivated by food and nicotine and on side effect profiles.

RATIONALE: Selective 5-HT2C receptor agonists, such as lorcaserin, are being developed for the treatment of obesity. Studies suggest that they may also have therapeutic potential for addictive behaviours including nicotine dependence, although few drugs of this class have been evaluated. OBJECTIVES: The primary aim was to evaluate the highly selective 5-HT2C agonist, CP-809101, against food-motivated (operant FR5 and progressive ratio schedules, palatability-induced feeding) and nicotine-motivated (intravenous self-administration, drug discrimination) behaviours in rats and to compare with equivalent findings for the structurally distinct 5-HT2C receptor agonists lorcaserin and Ro 60-0175. The secondary aims were to evaluate the side effect profiles of lorcaserin and CP-809101 and to determine the plasma levels of lorcaserin at a dose (1 mg/kg) that reduces both food and nicotine reinforcement for comparison to plasma concentrations reported in human trials. RESULTS: CP-809101 (0.3-3 mg/kg SC) reduced responding for both nicotine and food and blocked the discriminative stimulus properties of nicotine in a similar manner to lorcaserin and Ro 60-0175. Behaviours such as hypolocomotion, chewing and ptosis became evident following both CP-809101 and lorcaserin administration at higher doses. Plasma levels of lorcaserin were of similar range to those reported in obesity trials. CONCLUSIONS: These studies support the utility of 5-HT2C agonists as a therapeutic approach to treat nicotine dependence. Plasma exposure levels after acute lorcaserin treatment suggest that equivalent dosages could be used to evaluate these drugs in obesity and smoking cessation trials. Finally, there may be differences in the side effect profiles between lorcaserin and CP-809101, raising the possibility for tolerability differences amongst 5-HT2C agonists.

Pyrrolo[3,2,1-ij]quinoline derivatives, a 5-HT2c receptor agonist with selectivity over the 5-HT2a receptor: potential therapeutic applications for epilepsy and obesity.

A series of pyrrolo[3,2,1-ij]quinoline derivatives was synthesized, evaluated for their activity against the 5-HT2c and 5-HT2a, receptors and found to be agonists at 5-HT2c with selectivity over 5-HT2a.

Renal secretion of vinblastine, vincristine and colchicine in vivo.

The MDR1 gene product, P-glycoprotein, has been localized to the apical surface of the renal proximal tubule, but its functional role in the kidney is unknown. We studied renal luminal and antiluminal uptake of three known substrates of P-glycoprotein: vinblastine, vincristine and colchicine, by using the single pass multiple indicator dilution method under control conditions and in the presence of increasing concentrations of cyclosporin A, a potent inhibitor of P-glycoprotein. A bolus of [125I]albumin (plasma reference), L-[14C]glucose (extracellular and glomerular reference) and tracer 3H-substrate was injected into the left renal artery of anesthetized dogs and timed serial samples were collected from the left renal vein and left and right ureters. In a single pass, approximately 38, 13 and 8% of [3H]vinblastine, [3H] vincristine and [3H]colchicine, respectively, was extracted from the postglomerular circulation. Drug binding to plasma proteins was determined to be 81% for [3H]vinblastine, 71% for [3H] vincristine and 23% for [3H]colchicine. Despite the high degree of drug protein binding, the urine recoveries of [3H]vinblastine, [3H]vincristine and [3H]colchicine relative to L-[14C]glucose were 0.75 +/- 0.06, 0.69 +/- 0.06 and 0.94 +/- 0.02, confirming net secretion of each of these substrates. Infusion of cyclosporin A (0.1-5 microM) significantly decreased the urine recovery of [3H] vinblastine and [3H]vincristine relative to L-[14C]glucose in a dose-dependent manner. The renal excretion of [3H]colchicine was not affected by cyclosporin A at the concentrations tested (1-2 microM). The evidence suggests that net secretion of [3H]vinblastine and [3H]vincristine occurs across the luminal membrane of the renal cell.(ABSTRACT TRUNCATED AT 250 WORDS)

Formed and preformed metabolite excretion clearances in liver, a metabolite formation organ: studies on enalapril and enalaprilat in the single-pass and recirculating perfused rat liver.

Single-pass and recirculating rat liver perfusion studies were conducted with [14C]enalapril and [3H]enalaprilat, a precursor-product pair, and the data were modeled according to a physiological model to compare the different biliary clearances for the solely formed metabolite, [14C]enalaprilat, with that of preformed [3H]enalaprilat. With single-pass perfusion, the apparent extraction ratio (or biliary clearance) of formed [14C]enalaprilat was 15-fold the extraction ratio of preformed [3H]enalaprilat, an observation attributed to the presence of a barrier for cellular entry of the metabolite. Upon recirculation of bolus doses of [14C]enalapril and [3H]enalaprilat, the biliary clearance, estimated conventionally as metabolite excretion rate/midtime metabolite concentration, for formed [14C]enalaprilat was again 10- to 15-fold higher than the biliary clearance for preformed [3H]enalaprilat, but this decayed with perfusion time and gradually approached values for preformed [3H]enalaprilat. The decreasing biliary clearance of formed enalaprilat with recirculation was explained by the dual contribution of the circulating and intrahepatic metabolite (formed from circulating drug) to excretion. Physiological modeling predicted (i) an influx barrier (from blood to cell) at the sinusoidal membrane as the rate-limiting process in the overall removal of enalaprilat, (ii) a 15-fold greater extraction ratio or biliary clearance for formed [14C]enalaprilat over [3H]enalaprilat during single-pass perfusion, and (iii) the time-dependent and declining behaviour of the biliary clearance for formed [14C]enalaprilat during recirculation of the medium. In the absence of a direct knowledge of eliminating organs in vivo, this variable pattern for excretory clearance of the formed metabolite within the organ is indicative of a metabolite formation organ.

Combined recirculation of the rat liver and kidney: studies with enalapril and enalaprilat.

Combined recirculation of the rat liver (L) and kidney (IPK) at 10 ml min-1 per organ (LK) was developed to examine the hepatorenal handling of the precursor-metabolite pair: [14C]-enalapril and [3H]enalaprilat. Loading doses followed by constant infusion of [14C]enalapril and preformed [3H]enalaprilat to the reservoirs of the IPK or the LK preparation was used to achieve steady state conditions. In both organs, enalapril was mostly metabolized to its dicarboxylic acid metabolite, enalaprilat, which was excreted unchanged. At steady state, the fractional excretion for [14C]enalapril (FE = 0.45 to 0.48) and preformed [3H]enalaprilat (FE[pmi] = 1.1) were constant and similar for both the IPK and LK. The additivity of clearance was demonstrated in the LK preparation, namely, the total clearance of enalapril was the sum of its hepatic and renal clearances. However, the apparent fractional excretion for formed [14C]enalaprilat, FE(mi) and the apparent urinary clearance were time-dependent and higher than the corresponding values for preformed [3H]enalaprilat in both the IPK and LK. The FE(mi) and urinary clearance values further differed between the IPK and LK. Biliary clearance of formed vs. preformed enalaprilat displayed the same discrepant trends as observed for FE(mi) vs. FE(pmi) for the LK. These observations on the time-dependent and variable excretory clearance (urinary or biliary) of the formed metabolite vs. the constant, and much reduced, excretory clearance of the preformed metabolite are due to dual contributions to formed metabolite excretion: the nascently formed, intracellular metabolite which immediately underwent excretion and the formed metabolite which reentered the circulation, behaved as a preformed species. When data for the IPK and LK preparations were modeled with a physiological model with parameters previously reported for the L and IPK, all data, including metabolite excretory clearances, were well predicted. Model simulations revealed that the apparent FE(mi) differed between the LK and IPK preparations when the liver was present as an additional metabolite formation organ; the apparent excretory (urinary or biliary) clearance of the formed metabolite was further modulated by the volume of distribution of the metabolite, which altered levels of the formed, circulating metabolite.

The MDR1 gene product, P-glycoprotein, mediates the transport of the cardiac glycoside, digoxin.

Digoxin, a widely used cardiac glycoside with a low therapeutic index, is known to interact with a large and diverse group of co-administered drugs, frequently leading to toxic accumulation of the glycoside. Establishing the mechanism(s) of these interactions, therefore, has potential clinical significance. The present studies implicate P-glycoprotein, the MDR1 gene product overexpressed in multidrug resistant cells, as the apical membrane protein responsible for the renal secretion of digoxin and provide an explanation for the occurrence of digoxin toxicity in the presence of certain co-administered medications. Since digoxin is considered a prototype for endogenous digitalis-like glycosides, the results also allow for speculation that endogenous digitalis-like glycosides may be the natural substrates for P-gp.

Enalaprilat handling by the kidney: barrier-limited cell entry.

The angiotensin-converting enzyme inhibitor enalaprilat is formed in vivo in liver and kidney by esterolysis of the antihypertensive drug enalapril. To gain insight into the renal elimination of enalaprilat, we carried out multiple-indicator dilution experiments in the isolated perfused rat kidney. Kidneys were perfused single pass with an amino acid-supplemented Krebs-Henseleit buffer containing 20% bovine red blood cells and 4% bovine serum albumin, at a flow rate of 0.11 +/- 0.02 (SD) ml.s-1 x g-1. A bolus of 51Cr-labeled red blood cells (vascular red blood cell indicator), 125I-labeled albumin (vascular plasma indicator), L-[14C]glucose (interstitial space indicator), and [3H]-enalaprilat was injected into the renal artery, and timed samples of venous blood (up to 1 min) and urine (up to 10 min) were collected. The data were analyzed using a variable-transit-time, space-distributed model with modifications accounting for glomerular filtration and the observed 14% protein binding of enalaprilat; the glomerular filtration rate (GFR) estimated from L-glucose clearance was 9.0 +/- 2.9% of total plasma flow. The ratio of renal clearance of unbound enalaprilat to GFR was 1.56 +/- 0.29, indicating both glomerular filtration and net tubular secretion of enalaprilat. Unidirectional influx from plasma to tubular cells exceeded tubular secretion by a factor of 2.2 +/- 0.5. Thus only about one-half of the enalaprilat taken up by the tubular cells was excreted into urine, with the remainder refluxing into the capillary blood stream, indicating bidirectional permeation of enalaprilat across the basolateral tubular membrane.

Cyclosporin and quinidine inhibition of renal digoxin excretion: evidence for luminal secretion of digoxin.

We studied the in vivo luminal and contraluminal uptake of [3H]digoxin in dog kidney using the single-pass multiple indicator dilution method. A bolus tracer of 125I-albumin (plasma reference), creatinine, or L-[14C]glucose [extracellular reference (ecf)] and [3H]digoxin (or [3H]ouabain) was injected into the left renal artery, and timed serial samples were collected from the left renal vein (basolateral uptake) and left and right ureters (luminal uptake). [3H]ouabain was excreted solely by filtration and exhibited saturable and irreversible binding at the basolateral surface. Uptake of [3H]digoxin across the basolateral membrane was large and nonsaturable. Despite urine flow-dependent reabsorption and approximately 20% protein binding, the urine recovery ratio for [3H]-digoxin/glomerular (ecf) marker was 0.97 +/- 0.04 (n = 29), indicating net digoxin secretion. After intravenous infusions of cyclosporin in Cremophor EL (0.5-3.5 microM), the urine recovery ratio decreased in a dose-dependent manner from control values of 1.13 +/- 0.06 (n = 12) to 0.62 +/- 0.03 (n = 14). There was no change in the relative renal vein recovery. Left renal artery infusion of quinidine (37.5 micrograms.min-1.kg-1) decreased the relative urine recovery of [3H]digoxin by 46% (n = 6) but had no effect on postglomerular extraction. Cyclosporin and quinidine are known inhibitors of P-glycoprotein. But digoxin did not compete with [3H]azidopine for binding in rat brush-border membranes or membranes prepared from the multidrug-resistant cell line CHRC5. The exact mechanism for renal digoxin secretion remains to be determined, but our results point to a luminal localization of this secretory system.

A physiological model for renal drug metabolism: enalapril esterolysis to enalaprilat in the isolated perfused rat kidney.

A physiologically based kidney model was developed to describe the metabolism of enalapril and explain the observed discrepancies between generated and preformed enalaprilat (metabolite) elimination in the constant flow single-pass and recirculating isolated perfused rat kidney preparations (IPKs) as a result of the differing points of origin of the metabolite within the kidney, subsequent to the simultaneous delivery of 14C-enalapril and 3H-enalaprilat. The model incorporated clearances for diffusion/transport of drug and metabolite across the basolateral and luminal membranes of the renal cells, an intrinsic clearance for renal drug metabolism, in addition to physiological variables such as perfusate flow rate, glomerular filtration rate, and urine flow rate. Nonlinear curve fitting of single-pass and recirculating data was performed to estimate the rate-limiting step in the renal elimination of enalaprilat. Through fitting and simulation procedures, we were able to predict metabolic and excretory events for enalapril (renal extraction ratio approximately equal to 0.25-0.3; fractional excretion, FE, was less than unity) and the relatively constant pattern of urinary excretion of preformed enalaprilat (extraction ratio approximately equal to 0.07; FE approximately equal to 1). The extraction ratio of the intrarenally formed enalaprilat in single-pass IPK was about twofold that for the preformed metabolite, whereas the FEs of generated enalaprilat in recirculating IPKs were greater than 1, and tended to increase, then decrease with perfusion time. These observations were explained by the optimized parameters which indicated that efflux from cell to lumen was rate-controlling in the excretion of enalaprilat, and another small transport barrier also existed at the basolateral membrane; the lower extraction ratio of preformed enalaprilat was due to its poor transmembrane clearance at the basolateral membrane. The variable FEs for generated enalaprilat vs. the relatively constant FE for preformed metabolite in the recirculating IPK was explained by the changing contributions of both circulating and intrarenal metabolite to metabolite excretion.

Renal handling of enalapril and enalaprilat: studies in the isolated red blood cell-perfused rat kidney.

An isolated recirculating or single pass red cell-perfused rat kidney preparation (IPK) was used to examine the differential handling of renal metabolites. In single pass experiments, enalapril was primarily metabolized to its polar, dicarboxylic acid metabolite, enalaprilat, and its fractional excretion (FE) was less than unity, suggesting net reabsorption. Its steady-state extraction ratio decreased from 0.3 to 0.2 at concentrations of 1.06 to 12.7 microM, due to a saturation of enzymes for esterolysis. Enalaprilat administered to the IPK was excreted into urine in a concentration-independent (0.41-35.3 microM) fashion, with FE values approximating unity, suggesting net filtration. Differences in handling were observed for enalaprilat, as a metabolite formed from enalapril and as an administered (preformed) species in the single pass IPK, when tracer concentrations of [14C]enalapril and [3H]enalaprilat were given simultaneously. A comparison made between steady-state extraction ratio Ess[mi] [generated metabolite]/glomerular filtration rate (GFR) and Ess[pmi] [preformed metabolite]/GFR, respectively, revealed a 2-fold difference. The finding suggests the presence of a barrier for entry of enalaprilat into the kidney. Or else, in absence of the barrier, the opposite would be observed, that is, Ess [pmi]/GFR greater than Ess [mi]/GFR because preformed enalaprilat, in contrast to generated enalaprilat, undergoes filtration and utilizes facilitative transport carriers at the basolateral membrane. In recirculating IPKs which received simultaneously a tracer bolus dose of [14C]enalapril and [3H]enalaprilat, the FE values for generated [14C]enalaprilat were high and variable, decreasing with perfusion time and exceeding those for preformed [3H]enalaprilat, which approached unity with perfusion time. The variable FE values for [14C]enalaprilat are due to time-dependent contributions of circulating enalaprilat (which behaves identically to preformed enalaprilat) and the intrarenally generated enalaprilat. Hence, with renal drug metabolism, the conventional method of estimating urinary clearance (or Fe[mi]) for the metabolite [(total) excretion rate/midpoint plasma FE[mi] metabolite concentration] results in a greater metabolite clearance than that predicted from the administration of preformed metabolite.

Stereoselective glutathione conjugation and amidase-catalyzed hydrolysis of alpha-bromoisovalerylurea enantiomers in isolated rat hepatocytes.

alpha-Bromoisovalerylurea (BIU) is used as model substrate for studies on the pharmacokinetics of glutathione conjugation in vivo. Its metabolism in isolated rat hepatocytes is presently studied. A major part of the substrate was conjugated with glutathione, but also amidase-catalyzed hydrolysis occurred, resulting in the products urea and alpha-bromoisovaleric acid (BI). The amidase activity was located in the microsomal fraction of the rat liver. The product of hydrolysis, BI, also was conjugated efficiently with glutathione. In glutathione-depleted hepatocytes, no glutathione conjugates but only urea and BI were formed. A pronounced stereoselectivity in the metabolism of the BIU enantiomers was observed: (R)-BIU was conjugated with glutathione much faster than (S)-BIU. (S)-BIU was hydrolyzed substantially in the cells and the glutathione conjugate of the hydrolytic product, (S)-BI, could be detected. At high BIU concentrations (500 microM of the racemate) intracellular glutathione was seriously depleted; then, the cosubstrate availability most likely was the rate-limiting factor in the conjugation of BIU with glutathione. More urea was formed from (racemic) BIU in isolated rat hepatocytes in the present study than in the perfused liver and the intact rat in previous studies. This in vivo-in vitro difference is tentatively assigned to differences in glutathione availability in these systems. The results suggest that BI may also be a useful model substrate to study the kinetics of glutathione conjugation in vivo and in vitro.

Effect of diffusional barriers on drug and metabolite kinetics.

Previous experimental and simulation studies have alluded to the presence of a diffusional barrier for enalaprilat, the polar, dicarboxylic acid metabolite of enalapril, entering hepatocytes. The present study examined the roles of diffusional clearances of drug and metabolite on the distribution and elimination characteristics in liver. The hepatic intrinsic clearances for enalapril (26.1 ml/min) and enalaprilat (0.7 ml/min), found in a previous study, were used for simulation because, along with their given diffusional clearances (75 and 2 ml/min, respectively), they yielded a high extraction ratio for drug (E = 0.86) and a poor extraction ratio for the preformed metabolite (E = 0.05). While maintaining the intrinsic clearances and hepatic blood flow rate (10 ml/min) constant, only drug and metabolite diffusional clearances were altered. The liver was modeled as three (blood, liver tissue, and bile) compartments, with blood flowing into sinusoids of uniform length L. Blood (sinusoidal) and tissue concentrations of drug and generated and preformed metabolites, at any point x along L and under linear kinetic conditions, were approximated numerically by computer simulations and expressed as the length-averaged or mean concentrations. The factors underlying drug and metabolite (preformed and generated) concentrations, hepatic clearances and elimination rates, and their interrelationships were illustrated graphically, emphasizing the roles of diffusional clearances for drug and metabolite on their spatial distributions and elimination in liver.

Presence of a diffusional barrier on metabolite kinetics: enalaprilat as a generated versus preformed metabolite.

Studies in the once-through perfused rat liver with the simultaneous delivery of 14 C-enalapril and its polar diacid metabolite, 3H-enalaprilat, revealed different extents of elimination (exclusively by biliary excretion) for the generated (14C-enalaprilat) and preformed (3H-enalaprilat) metabolite (18 and 5% dose) [Pang, Cherry, Terrell, and Ulm: Drug Metab. Dispos. 12, 309-313 (1984)]. The present re-examination of data provided an explanation for these discrepant observations: enalaprilat, being a polar dicarboxylic acid, encounters more of a diffusional barrier than its precursor, enalapril, an ethyl ester of enalaprilat. Programs written in Fortran 77 on mass balance relationships were employed to simulate data upon varying the diffusional clearances for drug (CLd) and metabolite [CLd(mi)] from 1 to 5000 ml/min. The metabolic and biliary intrinsic clearances for drug and metabolite were found by trial and error such that the combinations of all clearance parameters yielded data similar to enalaprilat, and 3H-enalaprilat. Our finding indicated that the diffusional clearance for enalaprilat was low (2 ml/min) compared to that of enalapril (75 ml/min). The presence of a diffusional barrier for enalaprilat retards entry of the preformed metabolite into hepatocytes but prevents efflux of the intracellularly formed generated metabolite into sinusoidal blood, thereby enhancing generated metabolite elimination.