Age-dependent Expression of P-Glycoprotein gp17O in Caco-2 Cell Monolayers

Purpose. To determine whether the expression and activity of the P-glycoprotein (P-GP) drug efflux pump vary with the culture age of Caco-2 cell monolayers. Methods. Caco-2 cell monolayers were grown for 3–27 days on tissue culture-treated Transwells. P-GP efflux function was determined by measuring transmonolayer fluxes of cyclosporin A (CsA) and verapamil, while P-GP expression level was evaluated by Western blot analysis using monoclonal antibody C219. Results. The apparent permeability coefficient (Papp) of CsA (0.5 µM) in the basolateral-to-apical (B A) direction increased with culture age and was higher than the apical-to-basolateral (A B) direction at all times. Net secretory Papp significantly increased from day 17 onward compared to that observed during day 3 through 13. Verapamil (100 µM) significantly inhibited CsA transport in the B A direction from day 17 to 27, while elevating CsA transport in the A B direction from day 6 to 27. Interestingly, the Papp of verapamil (0.5 µM) in the B A direction was significantly higher than in the A B direction from day 6 to 27, rendering increases in net secretory Papp of verapamil with culture age. Western analysis revealed that P-GP expression level was in the order of 4 weeks 1 week > 3 weeks > 2 weeks at equal loading of cell proteins. Conclusions. P-GP is continuously expressed throughout the culture period, but it may not be fully functional at an early age. Caco-2 cell monolayers of day 17 to 27 appear to be a good model to evaluate the functional role of P-GP in drug efflux.     

Effect of Experimental Diabetes Mellitus and Arthritis on the Pharmacokinetics of Hydroxychloroquine Enantiomers in Rats

Purpose. To study the effect of experimental diabetes and arthritis on the pharmacokinetics of hydroxychloroquine (HCQ) enantiomers in rats. Methods. The pharmacokinetic studies were carried out following administration of 40 mg/kg of racemic HCQ to diabetic, insulin-treated diabetic, adjuvant arthritic and control rats. Results. Renal (70% and 62% for R- and S-HCQ, respectively) and non-renal clearance (100% and 145% for R- and S-HCQ, respectively) of HCQ enantiomers were significantly increased in diabetic rats. Diabetes-induced alterations in the disposition of HCQ were reversed by insulin treatment. In arthritic rats, systemic clearance (CL) of HCQ enantiomers was significantly reduced (1.05 ± 0.15 and 1.3 ± 0.19 1/h/kg for R- and S-HCQ, respectively) compared to controls (1.69 ± 0.32 and 1.93 ± 0.34 1/h/kg for R- and S-HCQ, respectively). The fraction unbound of the R- and S-HCQ were 49.4% and 50.5% lower in platelet rich plasma of arthritic rats compared to healthy rats. Increased blood concentrations of HCQ enantiomers in arthritic rats were significantly related to the degree of inflammation. Conclusions. Diabetes significantly increased the CL of both R- and S-HCQ by increasing renal and non-renal clearance. Arthritis caused a significant decrease in CL of HCQ enantiomers through increased binding and a decreased intrinsic clearance. The effect of the diseases on the pharmacokinetics of HCQ, however, was not stereoselective.This work was presented, in part, at the Ninth Annual Meeting of the American Association of Pharmaceutical Scientists,     

Reversible metabolism of vitamin K-vitamin K epoxide: Modeling considerations and limitations

Due to the cyclical natural of the vitamin K-vitamin K epoxide system, a two-compartment reversible metabolism model was used to describe this interconversion. In attempting to apply this model to the vitamin K-vitamin K epoxide cycle using literature data from dogs, interconversion and elimination clearances were obtained which are not physiologic. Consequently, the assumptions of the model were reexamined with respect to their validity. One critical assumption of the twocompartment model for interconversion is that it can only be applied in the absence of flow limitations. To determine what effect flow limitations may exert on the vitamin K and vitamin K epoxide apparent blood clearances, a model separating the liver from the blood compartment was proposed assuming the interconversion and metabolism of vitamin K and its epoxide occurred only within the liver. Simulated data suggested that if the reversible metabolic clearance values exceeded the distributional clearance terms, all the apparent clearances calculated using blood concentration-time data were in error. It is suggested that a two-compartment interconversion model might be too simplistic for the vitamin K-vitamin K epoxide cycle where the reversible metabolism is efficient and the distributional clearance may be rate limiting.Glossary of symbols Dose ^E Dose of vitamin K epoxide - Dose ^K Dose of vitamin K AUCs calculated from blood concentration-time profile AUC _E ^E Area under curve for vitamin K epoxide after epoxide dose - AUC _E ^K Area under curve for vitamin K after epoxide dose - AUC _K ^K Area under curve for vitamin K after vitamin K dose - AUC _E ^K Area under curve for vitamin K epoxide after vitamin K dose AUCs calculated from liver concentration-time profile AUC _L,E ^E Area under curve for vitamin K epoxide after epoxide dose - AUC _L,K ^E Area under curve for vitamin K after epoxide dose - AUC _L,K ^K Area under curve for vitamin K after vitamin K dose - AUC _L,E ^K Area under curve for vitamin K epoxide after vitamin K dose Clearances calculated from blood AUCs Cl _E ^B Elimination clearance of vitamin K epoxide - Cl _K ^B Elimination clearance of vitamin K - Cl _EK ^B Conversion clearance from vitamin K epoxide to vitamin K - Cl _KE ^B Conversion clearance from vitamin K to vitamin K epoxide Clearances calculated from liver AUCs Cl _E ^L Elimination clearance of vitamin K epoxide - Cl _K ^L Elimination clearance of vitamin K - Cl _EK ^L Conversion clearance from vitamin K epoxide to vitamin K - Cl _KE ^L Conversion clearance from vitamin K to vitamin K epoxide Distributional clearances Cl _K ^BL Clearance of vitamin K from the blood to the liver - Cl _K ^LB Clearance of vitamin K from the liver to the blood - Cl _E ^BL Clearance of vitamin K epoxide from the blood to the liver - Cl _E ^LB Clearance of vitamin K epoxide from the liver to the blood Supported by a grant from The American Heart Association, Kentucky Affiliate.     

Mechanisms of Transport and Structure-Permeability Relationship of Sulfasalazine and Its Analogs in Caco-2 Cell Monolayers

Purpose. To investigate the mechanisms involved in transport of sulfasalazine in Caco-2 cells. Methods. Permeability coefficients of sulfasalazine and its analogs across Caco-2 cell monolayers were measured as a function of direction of transport, energy and concentration dependence, and in the presence of inhibitors of various cellular efflux pumps and transporters. Results. Permeability coefficients of sulfasalazine across Caco-2 cell monolayers were approximately 342-, 261-, and 176-fold higher from basolateral to apical direction (BLAP) than from apical to basolateral direction (APBL) at 100, 200, and 500 M, respectively. Carrier permeability coefficient, non-saturable membrane permeability coefficient, and Michaelis constant were estimated to be 1.4×10^–5 cm/s, 1.9×10^–8 cm/s, and 369 M, respectively. The efflux of sulfasalazine was completely blocked at 4°C and in the presence of an uncoupler of oxidative phosphorylation. Using cellular efflux inhibitors, the permeability of sulfasalazine was shown to depend on multidrug resistance-associated protein and anion sensitive transport mechanisms. Structure-permeability studies showed that the affinity of sulfasalazine for the cellular efflux pumps and transporters in Caco-2 cells depended strongly on the carboxylic acid functional group. Conclusions. The permeability of sulfasalazine across Caco-2 cell monolayer is very low due to its strong interaction with multiple cellular efflux pumps and transporters. This may partially explain its low absorption in vivo.     

Can the Enhanced Renal Clearance of Antibiotics in Cystic Fibrosis Patients Be Explained by P-Glycoprotein Transport?

Purpose. To investigate in vitro if P-glycoprotein (P-gp) transport can differentiate between antibiotic drugs exhibiting increased active renal clearance (CL_r) in cystic fibrosis (CF) patients (i.e., dicloxacillin, trimethoprim) and drugs that do not exhibit this phenomenon (i.e., cefsulodin, sulfamethoxazole). Methods. Transport studies were carried out in MDCK (wild type) and MDR1-MDCK (P-gp overexpressing) cells that were grown to confluence on Transwell inserts. [¹⁴C]-mannitol transport and transepithelial electrical resistance (TEER) were measured to validate the integrity of the cells. Drug concentrations were analyzed using HPLC. Results. Dicloxacillin and trimethoprim are substrates of P-gp (BA/AB ratios in MDR1-MDCK cells are 32 and 50, respectively). P-gp inhibitors (i.e., GG918, cyclosporine, ketoconazole, vinblastine) decreased the BA transport of dicloxacillin and trimethoprim and increased the AB transport of trimethoprim while non-P-gp inhibitors (e.g., PAH) had no effect. In contrast, cefsulodin and sulfamethoxazole are not substrates of P-gp (BA/AB values in MDCK and MDR1-MDCK cells are 1). Conclusions. Our in vitro studies suggest that P-glycoprotein may play a role in increasing renal clearance of drug substrates in CF patients. Dicloxacillin and trimethoprim, which are both substrates of P-gp, show increased active renal clearance in CF patients while cefsulodin and sulfamethoxazole, which are not P-gp substrates, do not show increased active renal clearance in CF patients.     

Relationship between the ocular and systemic disposition of flurbiprofen: The effect of altered protein dynamics at steady state

The differences in flurbiprofen disposition in the aqueous humor and the plasma were examined after systemic doses. Steady state plasma concentrations of flurbiprofen (20–60 g/mL) were achieved via intravenous infusion to albino rabbits. Flurbiprofen demonstrated linear systemic kinetics throughout the dosing range, with constant body clearance and unbound fraction in plasma. At steady state, aqueous humor drug concentrations depended on the corresponding plasma drug concentration. Two clearance terms—CL_so, the systemic clearance to ocular tissues, and CL_os, the ocular clearance to systemic circulation—were used. After systemic doses, the drug concentration in the aqueous humor was related to that in the plasma as well as to the ratio of these two clearances. Flurbiprofen was extensively bound to plasma proteins and showed limited ocular distribution; its CL_so to CL_so tratio was very small. Thus, the concentration of flurbiprofen in the aqueous humor after systemic doses was lower than that obtained after ophthalmic doses. A plasmapheresis technique was utilized to lower the plasma protein concentrations to 60% of normal levels. As a consequence, flurbiprofen demonstrated reduced aqueous humor protein concentrations, increased unbound fractions in the plasma and the aqueous humor, elevated aqueous humor drug concentrations, and elevated total body clearance. The unbound body clearance stayed unchanged. Our study indicated that a drug should present a significant CL_so/CL_os ratio in order to achieve therapeutic concentrations in the eye via systemic doses. The drug-protein binding kinetics can be different between the plasma and the aqueous humor circulations. Because the ocular compariment is very small compared to the overall systemic distribution of flurbiprofen, it has little effect on the steady state systemic concentrations.     

Stereoselective effects of mexiletine enantiomers on sodium currents and excitability characteristics of adult skeletal muscle fibers

The effects of the enantiomers of mexiletine were tested on sodium currents of frog skeletal muscle fibers recorded by means of the three vaseline gap voltage clamp method and compared with the effects produced by tocainide enantiomers. The R-( – ) mexiletine produced a tonic block of the sodium current, elicited by single depolarizing test pulses from the holding potential of – 100 mV to – 20 mV, with an IC₅₀ of 43.9 ± 1 M, whereas the corresponding S-( + ) enantiomer produced the same effects at about twofold higher concentrations. A similar stereoselectivity was observed with tocainide enantiomers, but at about 5 fold higher concentrations. Both the R-( – ) and S-( + ) enantiomers of mexiletine and tocainide produced a further use-dependent block of sodium currents when the test pulse was applied repetitively at a frequency of 2 Hz. The use dependent behaviour led to a significant lowering of the IC₅₀ values with respect to the tonic block but the eudismic ratios ([IC₅₀S-( + )]/[IC₅₀R( – )]) and the relative potency between mexiletine and tocainide were maintained. All the tested compounds produced a left shift of the steady state inactivation curves (h) , suggesting a high-affinity interaction with the inactivated sodium channels. Again a stronger potency of R-( – ) vs. S-( + ) enantiomers and of mexiletine vs. tocainide was observed. The excitability characteristics recorded from the semitendinosus muscle by the two microelectrode technique were modified by the tested drugs in agreement with their ability to block sodium current. Thus a concentration-related increase in the threshold current required to elicit an action potential was observed along with a decrease in the amplitude and a shortening of the latency of action potential and a decrease in the firing capability of the membrane. Again the R-( – ) isomers were more potent than the S-( + ) ones and mexiletine was more effective than tocainide. These data corroborate the presence of a stereospecific site for these drugs on adult skeletal muscle sodium channels. The constant eudismic ratios between the enantiomers during both tonic and use-dependent block suggest that the increase in the apparent affinity of the receptor during statedependent conformational changes of the channel does not enhance its stereospecificity. The decrease in effective concentration upon high frequency stimulation supports the potential usefulness of low doses of R-( – ) mexiletine in the treatment of the abnormal hyperexcitability of the myotonic muscles, with a likely reduction of unwanted side effects.     

Interspecies differences in stereoselective protein binding and clearance of MK-571

(+)-3-(((3-(2-(7-chloro-2-quinolinyl)ethenyl)phenyl)((3-(dimethylamino)- 3-oxopropyl)thio)methyl)thio)propanoic acid (MK-571), is a potent and specific antagonist of leukotriene D4 action in vitro and in vivo. The compound, which is being developed for the treatment of asthma, contains a chiral center at the methine carbon of the dithio side chain and exists in two forms. The binding of MK-571 enantiomers to plasma protein was extensive (greater than 99.5%), stereoselective, and species dependent. The R-(-)-enantiomer was bound to rat plasma to a greater extent than the S-(+)-enantiomer, while in dog and monkey plasma the reverse was the case. The elimination clearance of the enantiomers was inversely related to the stereoselective plasma protein binding, that with the greater unbound fraction being cleared more rapidly. Thus, the pharmacologically more active S-(+)-enantiomer was cleared 3.7 times more rapidly than its antipode in rats following iv administration of the racemate (10 mg/kg), whereas in dogs and monkeys the R-(-)-enantiomer was cleared more rapidly. Kinetic analysis of the data revealed that the intrinsic clearances of the unbound enantiomers were similar within species, suggesting that stereoselectivity in elimination is not attributable to differences in metabolism and biliary excretion. Bioavailabilities of the S-(+)- and R-(-)-enantiomers in the rat were similar (75% and 71%, respectively) suggesting that MK-571 was not stereoselectively absorbed in that species.     

The Site of Absorption in the Small Intestine Determines Diltiazem Bioavailability in the Rabbit

Purpose. Since the ability of the small intestine to biotransform a drug may decrease in distal segments of the intestine, this study aimed to assess whether the site of administration in the small intestine could affect the systemic bioavailability of diltiazem and its two active metabolites, N-desmethyldiltiazem (MA) and desacetyldiltiazem (Ml). Methods. Five mg/kg of diltiazem were administered into the lumen of the proximal (0–30 cm, n = 9) or the distal (150–180 cm) small intestine (n = 7) of anesthetized New Zealand rabbits. Blood samples were drawn from the femoral artery for 6 hours, and diltiazem, MA and M1 were assayed by HPLC. Results. The area under the curve (AUC₀ )of diltiazem administered into the distal small intestine was larger than that estimated when diltiazem was given in the proximal segment (14.20 ± 2.82 vs 8.14 ± 0.88 µg.min/ml, p < 0.05), due to a lower diltiazem oral clearance (440 ± 78 vs 660 ± 55 ml/min/kg, p < 0.05). The AUC_{0 360} of MA was not affected by the site of diltiazem administration, but the AUC_{0 360} of M1 was increased when diltiazem was administered in the distal segment of the small intestine. When administered into the distal segment of the intestine, the molar sum of diltiazem and its active metabolites was 48% greater than when delivered into the 0–30 cm segment of the small intestine; as a consequence, absorption of diltiazem in distal segments of the small intestine may enhance its pharmacological response. Conclusions. The site of absorption into the intestine modulates the bioavailability of diltiazem and its two active metabolites.     

Characterization of the Regional Intestinal Kinetics of Drug Efflux in Rat and Human Intestine and in Caco-2 Cells

Purpose. The aim of the present study was to investigate the transport kinetics of intestinal secretory processes in the jejunum, ileum and colon of rats and humans and in Caco-2 cells, in vitro. Methods. Etoposide, vinblastine sulphate and verapamil hydrochloride were chosen as model substrates since they have been reported to undergo efflux in various other tissues. The concentration dependence, inhibition, directionality, temperature dependence, proton/sodium dependence, and ATP dependence of efflux were studied using side-by-side diffusion chambers and brush border membrane vesicles (BBMVs). Intestinal tissue from rats and humans and Caco-2 cells (passage no. 26) were used. Directional steady state effective permeabilities were calculated from drug appearance in the apical (AP) or basolateral (BL) chambers. Kinetic studies were carried out by investigating substrate efflux at concentrations ranging from 0.2 M to 1000 M. Since substrate efflux may be a result of more than one transporter, the hybrid efflux Km (Michaelis-constant), Pc (carrier-mediated permeability), and Pm (passive permeability) were determined as a function of intestinal region. Inhibitor studies were performed using quinidine (0.2 mM), a mixed inhibitor of P-glycoprotein (Pgp) and Multidrug Resistance-Associated Protein (MRP), and Leukotriene C₄(100 nM), an inhibitor of MRP and the canalicular multispecific organic anion transporter (cMOAT). Temperature dependent efflux was determined by investigating the BL to AP transport at temperatures ranging from 3°C to 37°C. Energies of activation (Ea) were determined from an Arrhenius analysis. Sodium, proton, and ATP dependence were determined using BBMVs. Immunoquantitation of Pgp, MRP and Lung Resistance Protein (LRP) in Caco-2 cells were carried out using Western blot analysis. Results. Active efflux of all substrates was observed in all regions of rat and human intestine and in Caco-2 cells. Directionality was observed with BL to AP transport exceeding AP to BL transport. The BL to AP/AP to BL permeability ratio, the efflux ratio, ranged from 1.4 to 19.8. Heal efflux was significantly higher (p < 0.001) than in other regions. Kinetic studies revealed that hybrid efflux Km values ranged from 4 to 350 M. In some cases, efflux was not saturable due to the solubility limits of the compounds utilized in this study. In presence of inhibitors, efflux ratios approached 1. BL to AP transport was temperature dependent in rat ileum for all substrates. Ea of intestinal efflux was found to be 11.6, 8.3, and 15.8 kcal/mole for etoposide, vinblastine and verapamil, respectively, suggesting an active, energy-dependent efflux mechanism. Substrate efflux was not sodium or proton dependent but was dependent on ATP. Using Western blot analysis the presence of Pgp, MRP, and LRP was demonstrated in Caco-2 cells and the amount of each transport protein varied as a function of passage number. Conclusions. Using multiple putative efflux substrates, the current results demonstrate that intestinal efflux was regionally dependent, mediated by multiple efflux transporters, the Kms were in the micro-molar range, and involved an energy dependent mechanism(s).     

Effect of the Flavonoids Biochanin A and Silymarin on the P-Glycoprotein-Mediated Transport of Digoxin and Vinblastine in Human Intestinal Caco-2 Cells

Purpose. The purpose of this study was to investigate the effects of flavonoids biochanin A and silymarin on intestinal absorption of P-gp substrates by determining their effects on P-gp-mediated efflux in Caco-2 cells. Methods. The cellular accumulation and bidirectional transport of digoxin and vinblastine in Caco-2 cells were determined in the presence and absence of flavonoids. Results. The 1.5-h accumulation of digoxin and vinblastine in Caco-2 cells was significantly increased by 50 M biochanin A or silymarin, and this effect was flavonoid-concentration dependent. The AP-to-BL transport of digoxin was significantly increased, whereas the BL-to-AP transport was significantly decreased by 50 M biochanin A or 75 M silymarin. At 150 M concentrations of biochanin A or silymarin, mean transport ratios (P _app,B-A/P _app,A-B) of 1.62 and 4.48, respectively, compared with the control ratio of 43.4, were obtained. Conclusion. These results indicate that biochanin A and silymarin can inhibit P-gp-mediated efflux in Caco-2 cells, suggesting they could potentially increase the absorption/bioavailability of coadministered drugs that are P-gp substrates.     

Transport of Pregabalin in Rat Intestine and Caco-2 Monolayers

Purpose. The purpose of this study was to determine if the intestinal transport of pregabalin (isobutyl --aminobutyric acid, isobutyl GAB A), a new anticonvulsant drug, was mediated by amino acid carriers with affinity for large neutral amino acids (LNAA). Methods. Pregabalin transport was studied in rat intestine and Caco-2 monolayers. An in vitro Ussing/diffusion chamber model and an in situ single-pass perfusion model were used to study rat intestinal transport. An in vitro diffusion chamber model was used to evaluate Caco-2 transport. Results. In rat ileum, pregabalin transport was saturable and inhibited by substrates of intestinal LNAA carriers including neurontin (gabapentin), phenylalanine, and proline. Weak substrates of intestinal LNAA carriers (-alanine, --aminobutyric acid, and methyl aminoisobutyric acid) did not significantly change pregabalin transport. In Caco-2 mono-layers that showed a high capacity for phenylalanine transport, pregabalin transport was concentration- and direction-independent and equivalent in magnitude to the paracellular marker, mannitol. The in vitro and in situ rat ileal permeabilities of the LNAA carrier-mediated compounds neurontin, pregabalin, and phenylalanine correlated well with the corresponding in vivo human oral absorption. Conclusions. The transport of pregabalin was mediated by LNAA carriers in rat ileum but not in Caco-2 monolayers. Caco-2 was not an appropriate model for evaluating the in vivo human oral absorption of pregabalin and neurontin.     

Effects of Pluronic Block Copolymers on Drug Absorption in Caco-2 Cell Monolayers

Purpose. The present work characterizes the effects of Pluronic copolymers on the transport of a P-gp-dependent probe, rhodamine 123 (R123) in Caco-2 cell monolayers. Methods. The accumulation and efflux studies were performed on the confluent Caco-2 monolayers using fluorescent probes with and without Pluronic copolymers. Results. At concentrations below the critical micelle concentration single chains ("unimers) of Pluronic P85 enhanced the accumulation and inhibited the efflux of R123 in Caco-2 monolayers. The transport of the P-gp-independent probe, rhodamine 110 was not altered under these conditions. In contrast the micelles increased R123 accumulation to a much lower extent when compared to the unimers and enhanced R123 efflux in Caco-2 monolayers. Conclusions. Pluronic P85 unimers increase accumulation of a P-gp-dependent drug in Caco-2 monolayers through inhibition of the P-gp efflux system. The mechanism of the micelle effect is not known, however, it is very similar to the micelle effects in BBMEC. This has been previously shown to involve vesicular transport of the micelle-incorporated drug. The study suggests that Pluronic copolymers can be useful in increasing oral absorption of select drugs.     

Pluronic P85 Increases Permeability of a Broad Spectrum of Drugs in Polarized BBMEC and Caco-2 Cell Monolayers

Purpose. Previous studies demonstrated that inhibition of P glycoprotein (P-gp) by Pluronic P85 (P85) block copolymer increases apical (AP) to basolateral (BL) transport of rhodamine 123 (R123) in the polarized monolayers of bovine brain microvessel endothelial cells (BBMEC) and Caco-2 cells. The present work examines the effects of P85 on the transport of fluorescein (Flu), doxorubicin (Dox), etoposide (Et), taxol (Tax), 3-azido-3-deoxythymidine (AZT), valproic acid (VPA) and loperamide (Lo) using BBMEC and Caco-2 monolayers as in vitro models of the blood brain barrier and intestinal epithelium respectively. Methods. Drug permeability studies were performed on the confluent BBMEC and Caco-2 cell monolayers mounted in Side-Bi-Side diffusion cells. Results. Exposure of the cells to P85 significantly enhanced AP to BL permeability coefficients of Flu, Tax, Dox and AZT in both cell models. Further, P85 enhanced AP to BL transport of Et, VPA and Lo in Caco-2 monolayers. No changes in the permeability coefficients of the paracellular marker mannitol were observed in the presence of the copolymer. Conclusions. P85 increases AP to BL permeability in BBMEC and Caco-2 monolayers with respect to a broad panel of structurally diverse compounds, that were previously shown to be affected by P-gp and/ or multidrug resistance associated protein (MRP) efflux systems. Broad specificity of the block copolymer effects with respect to drugs and efflux systems appears to be a valuable property in view of developing pharmaceutical formulations to increase drug accumulation in selected organs and overcome both acquired and intrinsic drug resistance that limits the effectiveness of many chemotherapeutic agents.     

Effect of Probenecid on the Enantioselective Pharmacokinetics of Oxprenolol and Its Glucuronides in the Rabbit

Purpose. To study the effect of probenecid on the stereoselective pharmacokinetics of oxprenolol and its glucuronides in the rabbit. Methods. An oral dose of 50 mg/kg racemic oxprenolol was given to nine rabbits twice, in random sequence with and without the concurrent administration of probenecid. Oxprenolol enantiomers were determined in plasma and urine by an enantioselective HPLC method. Oxprenolol glucuronides were measured in plasma and urine after enzymatic hydrolysis. Results. The disposition of the oxprenolol enantiomers in rabbits is stereoselective, mainly due to a difference in metabolism. Renal excretion is only a minor elimination route for unchanged oxprenolol, and the renal clearances of the enantiomers are similar. Pre-treatment with probenecid did not affect the plasma concentrations of the oxprenolol enantiomers, but there was a slight decrease in their urinary excretion. The plasma concentrations of the oxprenolol glucuronides are much higher than those of the parent enantiomers, and those of (S)-glucuronide are about twice those of its antipode. About 10% of the oxprenolol dose is excreted in the urine as glucuronides. The renal clearances of both glucuronides are similar, and markedly higher than the creatinine clearance. After probenecid, the mean glucuronide plasma levels were markedly higher, with for both glucuronides a more than twofold increase in mean AUC. Probenecid decreased the renal clearance of both glucuronides to about 30%. Moreover, it decreased slightly the formation clearance of (S)-glucuronide, while the formation clearance of (R)-glucuronide was not significantly influenced. Conclusions. Our results show that in the rabbit, both oxprenolol glucuronide diastereomers are actively secreted by the kidney, and that this process is inhibited by probenecid.     

Glucuronidation Kinetics of R,S-Ketoprofen in Adjuvant-Induced Arthritic Rats

Purpose. A pharmacokinetic study was carried out in rats to investigate the effect of arthritis on the glucuronidation of the nonsteroidal anti-inflammatory drug ketoprofen. Methods. An iv bolus dose of R,S-ketoprofen (10 mg/kg) was administered to control (n = 6) and adjuvant-induced arthritic rats (n = 6). All experiments were carried out in bile-exteriorized animals. Concentrations of R- and S-ketoprofen in plasma, bile and urine, and of their glucuronides in bile and urine were determined by HPLC. In a separate series of experiments, the ex vivo plasma protein binding of R- and S-ketoprofen was measured in control and arthritic rats following iv administration of R,S-ketoprofen. Results. As a result of a significant decrease in plasma albumin concentrations in arthritic rats, the unbound fraction of R- and S-ketoprofen was significantly increased (approximately 2-fold) in rats with adjuvant-induced arthritis. Total (i.e., bound plus unbound) plasma clearances of R- and S-ketoprofen were not different in arthritic rats. Unbound plasma clearances of both ketoprofen enantiomers, however, were significantly reduced (by 53% and 61%, respectively). This was due to a significant impairment in the formation of the R- and S-ketoprofen glucuronides. There was no apparent effect of adjuvant-induced arthritis on the chiral inversion of R- to S-ketoprofen. Conclusions. Adjuvant-induced arthritis in the rat leads to a significant impairment in the in vivo glucuronidation of R- and S-ketoprofen.     

Enantioselective Synthesis and Pharmacological Evaluation of a New Type of Verapamil Analog with Hypotensive and Calcium Antagonist Activities

Purpose. The syntheses and evaluation for cardiovascular activity in the rat of both enantiomers of a verapamil analog in which the cyano group has been replaced by hydroxyl. Methods. ( + )- and (–)--[3-[[2-(3,4-Dimethoxyphenyl)ethyl]methylamino]propyl]-3, 4-dimethoxy--(l-methyl ethyl)benzyl alcohol were prepared from chiral sulfoxides produced by microbial biotransformations using Mortierella isabellina ATCC 42613 or Helminthsporium species NRRL 4671, and were examined for hypotensive and calcium antagonist activity using anaesthetized normotensive rats and isolated rat aorta and atria. Results. The analogs showed a pharmacological profile similar to that exhibited by verapamil, possessing a remarkable hypotensive activity, accompanied by a significant bradycardia, in anaesthetized normotensive rats. In vitro, these analogs displayed clear inhibitory effects: in isolated rat aorta they inhibited, in a concentration-dependent fashion, the contractions and ⁴⁵Ca²⁺ uptake induced by norepinephrine and high KC1, and in isolated rat atria the analogs considerably decreased the rate of contraction (negative chronotropic effects). No significant differences between the quantitative cardiovascular effects produced by the two enantiomers of the verapamil analogs were observed. Conclusions. The results suggest that, like that of verapamil, the cardiovascular activity exhibited by the new compounds seems to be due, at least in part, to a blockage of transmembrane calcium channels present in vascular smooth muscle cells.     

Effects of verapamil on the pharmacokinetics of puerarin in rats

Abstract

1. The oral bioavailability of puerarin is poor which hindered its clinical performance.

2. This study investigates the effects of verapamil on the pharmacokinetics of puerarin in rats.

3. The pharmacokinetics of orally administered puerarin (50?mg/kg) with or without verapamil pretreatment (10?mg/kg/day for 7?days) were investigated. The plasma concentration of puerarin was determined using LC-MS/MS method, and the pharmacokinetics profiles were calculated and compared. Caco-2 cell transwell model was also used to investigate the effects of verapamil on the transport pf puerarin.

4. The results showed that when the rats were pretreated with verapamil, the maximum concentration (C_max) of puerarin increased from 683.7?±?51.2 to 933.5?±?75.8?ng/mL (p?<?0.05), and the area under the concentration-time curve from zero to infinity (AUC_0-inf) also increased from 3687.3?±?444.6 to 5006.1?±?658.6?μg·h/L (p?<?0.05). The Caco-2 cell transwell experiments indicated that verapamil could decrease the efflux ratio of puerarin from 1.90 to 1.19 through inhibiting the activity of P-gp.

5. In conclusion, these results indicated that verapamil could affect the pharmacokinetics of puerarin, possibly by increasing the systemic exposure of puerarin by inhibiting the activity of P-gp.

     

The effects of verapamil on the pharmacokinetics of curculigoside in rats

Context: Clarifying the potential mechanism of the poor oral bioavailability of curculigoside would be helpful for for investigating pharmacological effects and clinical applications.

Objective: To clarify the main mechanism for poor oral bioavailability.

Materials and methods: First, the pharmacokinetics of curculigoside (20?mg/kg) in rats with and without pretreatment with verapamil (10?mg/kg) was determined using a sensitive and reliable LC-MS method. Then the effects of verapamil on the transport and metabolic stability of curculigoside were investigated using Caco-2 cell transwell model and rat liver microsome incubation systems.

Results: The results showed that verapamil could significantly increase the peak plasma concentration (from 60.17?ng/mL to 93.66?ng/mL) and AUC_0?t (from 289.57 to 764.02?ng·h/mL) of curculigoside. The Caco-2 cell experiments indicated that the efflux ratio of curculigoside was 3.92 (P_appAB 6.43?±?0.57?×?10?^?7?cm/s; P_appBA 2.52?±?0.37?×?10?^?36?cm/s), P-gp might be involved in the transport of curculigoside, and verapamil could inhibit the efflux of curculigoside and increase the absorption of curculigoside significantly in the Caco-2 cell monolayer. Additionally, the rat liver microsome incubation experiments indicated that verapamil could significantly decrease the intrinsic clearance rate of curculigoside (from 38.8 to 23.6?μL/min/mg protein).

Discussion and conclusion: These results indicated that verapamil could significantly change the pharmacokinetic profiles of curculigoside in rats, the poor absorption due to P-gp mediated efflux in intestine and high intrinsic clearance rate in rat liver may be the main reason for the poor oral absolute bioavailability of curculigoside.     

Triterpenoid saponins from cultural plants of Stenocereus stellatus (Cactaceae)

A new triterpenoid saponin, named stellatoside A (1), and a known saponin (2), were isolated from cultural plants of the cactus Stenocereus stellatus (Ludwig G. K. Pfeiffer) V. Riccobono. The structure of 1 was determined to be a stellatogenin 3-O--l-rhamnopyranosyl(14)--l-rhamnopyranosyl(12)--d-glucuronopyranoside by spectroscopic methods. This is the first study to examine the substances of the cultural plants of cacti.