Characterization of gastrointestinal absorption of digoxin involving influx and efflux transporter in rats: application of mdr1a knockout (−/−) rats into absorption study of multiple transporter substrate

Abstract

1.?This study was aimed to characterize gastrointestinal absorption of digoxin using wild-type (WT) and multidrug resistance protein 1a [mdr1a; P-glycoprotein (P-gp)] knockout (?/?) rats.

2.?In WT rats, the area under the plasma concentration–time curve (AUC) of oral digoxin increased after oral pretreatment with quinidine at 30?mg/kg compared with non-treatment, but the increasing ratio tended to decrease at a high dose of 100?mg/kg. In mdr1a (?/?) rats, however, quinidine pretreatment caused a dose-dependent decrease in the AUC.

3.?Quinidine pretreatment did not alter the hepatic availability of digoxin, indicating that the changes in the digoxin AUC were attributable to inhibition of the absorption process by quinidine; i.e. inhibition of influx by quinidine in mdr1a (?/?) rats and inhibition of efflux and influx by quinidine in WT rats.

4.?An in situ rat intestinal closed loop study using naringin implied that organic anion transporting peptide (Oatp) 1a5 may be a responsible transporter in the absorption of digoxin.

5.?These findings imply that the rat absorption behavior of digoxin is possibly governed by Oatp1a5-mediated influx and P-gp-mediated efflux. The mdr1a (?/?) rat is therefore a useful in vivo tool to investigate drug absorption associated with multiple transporters including P-gp.     

Down-regulation of mouse organic anion-transporting polypeptide 4 (Oatp4; Oatp1b2; Slc21a10) mRNA by lipopolysaccharide through the toll-like receptor 4 (TLR4).

Lipopolysaccharide (LPS) causes a systemic reaction known as sepsis, which is frequently associated with cholestasis. Many biological effects produced by LPS are thought to be mediated by Toll-like receptor 4 (TLR4). Organic anion-transporting polypeptide 4 (Oatp4; Slc21a10) mediates hepatic uptake of bile acids and other organic anions. The purpose of this study was to determine 1) whether LPS decreases Oatp4 mRNA levels; 2) the role of TLR4 in the LPS-induced down-regulation of Oatp4; and 3) the time course of serum concentrations of tumor necrosis factor alpha, interleukin (IL) 1beta, and IL-6 after LPS administration. For the dose-response study, LPS (1 mg/kg i.p.) produced a significant decrease in Oatp4 mRNA levels in TLR4-normal C3H/OuJ mice, and higher doses produced slightly greater decreases. However, none of the doses of LPS examined significantly decreased Oatp4 mRNA levels in TLR4-mutant C3H/HeJ mice. For the time-response study, LPS (5 mg/kg i.p.) produced a rapid decrease in Oatp4 mRNA levels in TLR4-normal C3H/OuJ mice. The maximal decrease in Oatp4 mRNA levels (80%) was observed 12 h after LPS administration and returned to control levels thereafter. In contrast, LPS did not produce a significant decrease in Oatp4 mRNA levels at any time in TLR4-mutant C3H/HeJ mice. These findings demonstrate that LPS decreases Oatp4 mRNA levels in mice, and the decrease is mediated through TLR4.     

Organic anion transporting polypeptide (Oatp) 1a1-mediated perfluorooctanoate transport and evidence for a renal reabsorption mechanism of Oatp1a1 in renal elimination of perfluorocarboxylates in rats

Organic anion transporting polypeptide (Oatp) 1a1 has been hypothesized to play a key role in rat renal reabsorption of perfluorooctanoate (PFO). We have investigated PFO uptake kinetics in Chinese Hamster Ovary (CHO) cells that have been stably transfected with the cDNA encoding Oatp1a1. The Oatp1a1-expressing CHO cells have been validated by their Oatp1a1 gene expression, estrone-3-sulfate (E3S) uptake kinetics, and the correlation between Oatp1a1 gene expression and E3S uptake activity that were both induced by the treatment of sodium butyrate. Oatp1a1-mediated PFO uptake underwent a saturable process with a K_m value of 162.2 ± 20.2 μM, which was effectively inhibited by known Oatp1a1 substrates sulfobromophthalein and taurocholate, and a major flavonoid in grapefruit juice, naringin. The inhibition of Oatp1a1-mediated E3S uptake has been compared for linear perfluorocarboxylates with carbon chain lengths ranged from 4 to 12. There was no apparent inhibition by perfluorobutanoate and perfluoropentanoate at 1 mM. Inhibition was observed for perfluorohexanoate at 1 mM and the level of inhibition increased as the increase of the chain length up to perfluorodecanoate. The values of apparent inhibition constant (K_i,app) were determined for perfluorocarboxylates with chain lengths between 6 and 10. The log values of K_i,app exhibited a negative linear relationship to the chain lengths and a positive linear relationship to the log values of the total clearance of perfluorocarboxylates in male rats. This in vitro-to-in vivo correlation strongly supports a tubular reabsorptive role of Oatp1a1 in rat renal elimination of perfluorocarboxylates. Due to the sex-dependent expression of Oatp1a1 in rat kidney, Oatp1a1-mediated tubular reabsorption is suggested to be the mechanism for the sex-dependent renal elimination of PFO in rats.     

Expression and Functional Involvement of Organic Anion Transporting Polypeptide Subtype 3 (Slc21a7) in Rat Choroid Plexus

Purpose. It has been shown that transport(s) are involved in the uptake of estradiol 17 glucuronide (E217G) by the choroid plexus (CP). The purpose of this study is to compare the substrate specificity of the transporter in the CP with those of rat organic anion transporting polypeptide 1 (rOatp1) and rOatp3. Methods. The expression of rOatp1 and rOatp3 in rat CP was confirmed by RT-PCR and Western blot analyses. The substrate specificity of rOatp1 and rOatp3 was compared using cDNA-transfected LLC-PK1 cells. The uptake of E217G by rat isolated CP was determined by centrifugal filtration technique. Results. PCR analyses demonstrated that the mRNA expression of rOatp3 was abundant in the CP, whereas that of rOatp1 was low. Immunohistochemical staining revealed that rOatp3 is expressed on the apical membrane of the CP. Kinetic parameters (K_m and K_i values) of rOatp3 were similar to those for rOatp1. The results of mutual inhibition study suggest that E217G and taurocholate share the same mechanism in the CP. Corticosterone, estrone-3-sulfate and indomethacin are moderate inhibitors, but no effects by digoxin, p-aminohippurate, benzylpenicillin and cimetidine were observed. Conclusions. rOatp3 is most possible candidate transporter involved in the uptake of organic anions on the brush border membrane of the choroid epithelial cells.     

Involvement of Organic Anion Transporting Polypeptide 1a5 (Oatp1a5) in the Intestinal Absorption of Endothelin Receptor Antagonist in Rats

Purpose To assess the contribution of organic anion transporting polypeptide 1a5 (Oatp1a5/Oatp3) in the intestinal absorption of an orally active endothelin receptor antagonist, (+)-(5S,6R,7R)-2-butyl-7-[2-((2S)-2-carboxypropyl)-4-methoxyphenyl]-5-(3,4-methylene-dioxyphenyl)cyclopenteno[1,2-b]pyridine-6-carboxylic acid (compound-A) in rats. Methods Uptakes of [¹⁴C]compound-A by Oatp1a5-expressing Xenopus laevis oocytes and isolated rat enterocytes were evaluated. Results The uptake of compound-A by Oatp1a5-expressing oocytes was significantly higher than that by water-injected oocytes and Oatp1a5-mediated uptake was saturable with K _m value of 116 μM. Compound-A was taken up into isolated enterocytes in time- and concentration-dependent manners and the estimated K _m value was 83 μM, which was close to that for the Oatpt1a5-mediated uptake in oocytes. Both uptakes of compound-A by Oatp1a5-expressing oocytes and enterocytes were pH-sensitive with significantly higher uptake at acidic pH than those at neutral pH. Uptakes of compound-A into Oatp1a5-expressing oocytes and enterocytes were significantly decreased in the presence of Oatp1a5 substrates such as bromosulfophthalein and taurocholic acid. Conclusions These results consistently suggested that Oatp1a5 is contributing to the intestinal absorption of compound-A at least in part, and the transporter-mediated absorption seems to be maximized at the acidic microenvironment of epithelial cells in the small intestine in rats.     

Guanidine Transport in a Human Choriocarcinoma Cell Line (JAR)

Purpose. Many endogenous substances and xenobiotics are organic cations. Transplacental transport of organic cations is an important determinant of the delivery of these compounds to the fetus. The aim of this study was to determine the mechanisms of organic cation transport using the human choriocarcinoma cell line (JAR) as a model system with [¹⁴C]guanidine as a ligand. Methods. Uptake studies of [^l4C]guanidine were carried out in JAR cell monolayers on day 2 after plating. Results. [¹⁴C]guanidine uptake was temperature dependent, saturable (K_m = 167 M) and inhibited by many organic cations including amiloride, cimetidine, quinine, quinidine and nicotine. [^l4C]guanidine uptake exhibited a counterflux phenomenon indicative of a carrier-mediated process. The uptake of [¹⁴C]guanidine was sodium and pH-independent and could be driven by an inside-negative membrane potential difference. Conclusions. This is the first demonstration of an electrogenic guanidine transporter in a human cell culture model. This transporter may play a role in the transplacental transport of many clinically used drugs and xenobiotics.     

Effect of mdr1a P-glycoprotein gene disruption, gender, and substrate concentration on brain uptake of selected compounds

Purpose. This study assessed the influence of mdr1a P-glycoprotein (P-gp) gene disruption, gender and concentration on initial brain uptake clearance (Cl _up) of morphine, quinidine and verapamil. Methods. Cl _up of radiolabeled substrates was determined in P-gp-competent and deficient [mdr1a(–/–)] mice by in situ brain perfusion. Brain:plasma distribution of substrates after i.v. administration was determined in both strains. Results. Genetic disruption of mdr1a P-gp resulted in 1.3-, 6.6- and 14-fold increases in Cl _up for morphine, verapamil and quinidine, respectively. With the exception of small differences for verapamil, gender did not affect Cl _up. Saturable transport of verapamil and quinidine was observed only in P-gp-competent mice, with apparent IC ₅₀ values for efflux of 8.6 ± 2.3 M and 36 ± 2 M, respectively. VerapamilCl _up was 50% higher in mdr1a(+/–) vs. mdr1a(+/+) mice; no such difference was observed for quinidine. In P-gp-competent mice, uptake of verapamil and quinidine was unaffected by organic vehicles. Plasma decreased VER Cl _up to a greater extent in the presence of P-gp. The influence of P-gp in situ was lower than, but correlated with, the effect in vivo. Conclusions. P-gp decreases Cl _up of morphine, verapamil and quinidine in situ with little or no influence of gender, but this effect cannot fully account for the effects of P-gp in vivo. P-gp is the only saturable transport mechanism for verapamil and quinidine at the murine blood-brain barrier. The influence of protein binding on Cl _up may be enhanced by P-gp-mediated efflux.     

Differential Contributions of rOat1 (Slc22a6) and rOat3 (Slc22a8) to the <Emphasis Type="Italic">in Vivo</Emphasis> Renal Uptake of Uremic Toxins in Rats

No HeadingPurpose. Evidence suggests that uremic toxins such as hippurate (HA), indoleacetate (IA), indoxyl sulfate (IS), and 3-carboxy-4-methyl-5-propyl-2-furanpropionate (CMPF) promote the progression of renal failure by damaging tubular cells via rat organic anion transporter 1 (rOat1) and rOat3 on the basolateral membrane of the proximal tubules. The purpose of the current study is to evaluate the in vivo transport mechanism responsible for their renal uptake.Methods. We investigated the uremic toxins transport mechanism using the abdominal aorta injection technique [i.e., kidney uptake index (KUI) method], assuming minimal mixing of the bolus with serum protein from circulating serum.Results. Maximum mixing was estimated to be 5.8% of rat serum by measuring estrone sulfate extraction after addition of 0–90% rat serum to the arterial injection solution. Saturable renal uptake of p-aminohippurate (PAH, K_m = 408 M) and benzylpenicillin (PCG, K_m = 346 M) was observed, respectively. The uptake of PAH and PCG was inhibited in a dose-dependent manner by unlabeled PCG (IC₅₀ = 47.3 mM) and PAH (IC₅₀ = 512 M), respectively, suggesting that different transporters are responsible for their uptake. A number of uremic toxins inhibited the renal uptake of PAH and PCG. Excess PAH, which could inhibit rOat1 and rOat3, completely inhibited the saturable uptake of IA, IS, and CMPF by the kidney, and by 85% for HA uptake. PCG inhibited the total saturable uptake of HA, IA, IS, and CMPF by 10%, 10%, 45%, and 65%, respectively, at the concentration selective for rOat3.Conclusions. rOat1 could be the primary mediator of the renal uptake of HA and IA, accounting for approximately 75% and 90% of their transport, respectively. rOat1 and rOat3 contributed equally to the renal uptake of IS. rOat3 could account for about 65% of the uptake of CMPF under in vivo physiologic conditions. These results suggest that rOat1 and rOat3 play an important role in the renal uptake of uremic toxins and the induction of their nephrotoxicity.     

Immobilized Artificial Membrane (lAM)-HPLC for Partition Studies of Neutral and Ionized Acids and Bases in Comparison with the Liposomal Partition System

Purpose. To study the partitioning of model acids ((RS)-warfarin and salicylic acid), and bases (lidocaine, (RS)-propranolol and diazepam), with immobilized artificial membrane (lAM)-HPLC, as compared to partitioning in the standardized phosphatidylcholine liposome/buffer system. Methods. The pH-dependent apparent partition coefficients D were calculated from capacity factors (k_IAM) obtained by IAM-HPLC, using a 11-carboxylundecylphosphocholine column. For lipophilic compounds k_IAM, values were determined with organic modifiers and extrapolation to 100% water phase (k_IAMw) was optimized. Temperature dependence was explored (23 to 45° C), and Gibbs free energy (G), partial molar enthalpy (H) and change in entropy (S) were calculated. Equilibrium dialysis was used for the partitioning studies with the liposome/buffer system. Results. For extrapolation of k_IAMw, linear plots were obtained both with the respective dielectric constants and the mole fractions of the organic modifier. All tested compounds showed a similar pH-D diagram in both systems; however, significant differences were reproducibly found in the pH range of 5 to 8. In all cases, G and H were negative, whereas S values were negative for acids and positive for bases. Conclusions. In both partitioning systems, D values decreased significantly with the change from the neutral to the charged ionization state of the solute. The differences found under physiological conditions, i.e. around pH 7.4, were attributed to nonspecific interactions of the drug with the silica surface of the IAM column.     

Efflux Ratio Cannot Assess P-Glycoprotein-Mediated Attenuation of Absorptive Transport: Asymmetric Effect of P-Glycoprotein on Absorptive and Secretory Transport Across Caco-2 Cell Monolayers

Purpose. The purpose of this work was to determine whether P-glycoprotein (P-gp) modulates absorptive and secretory transport equally across polarized epithelium (i.e., Caco-2 cell monolayers) for structurally diverse P-gp substrates, a requirement for the use of the efflux ratio to quantify P-gp-mediated attenuation of absorption across intestinal epithelium. Methods. Studies were performed in Caco-2 cell monolayers. Apparent permeability (P _app) in absorptive (P _app,AB) and secretory (P _app,BA) directions as well as efflux ratios (P _app,BA / P _app,AB) were determined for substrates as a function of concentration. Transport of these compounds (10 M) was measured under normal conditions and in the presence of the P-gp inhibitor, GW918 (1 M), to dissect the effect of P-gp on absorptive and secretory transport. Apparent biochemical constants of P-gp-mediated efflux activity were calculated for both transport directions. Results. Efflux ratios for rhodamine 123 and digoxin were comparable (approx. 10). However, transport studies in the presence of GW918 revealed that P-gp attenuated absorptive transport of digoxin by approx. 8-fold but had no effect on absorptive transport of rhodamine 123 (presumably because absorptive transport of rhodamine 123 occurs via paracellular route). The apparent K _m for P-gp-mediated efflux of digoxin was >6-fold larger in absorptive vs. secretory direction. For structurally diverse P-gp substrates (acebutolol, colchicine, digoxin, etoposide, methylprednisolone, prednisolone, quinidine, and talinolol) apparent K _m was approximately 3 to 8-fold greater in absorptive vs. secretory transport direction, whereas apparent J _max was somewhat similar in both transport directions. Conclusions. P-gp-mediated efflux activity observed during absorptive and secretory transport was asymmetric for all substrates tested. For substrates that crossed polarized epithelium via transcellular pathway in both directions, this difference appears to be caused by greater apparent K _m of P-gp-mediated efflux activity in absorptive vs. secretory direction. These results clearly suggest that use of efflux ratios could be misleading in predicting the extent to which P-gp attenuates the absorptive transport of substrates.     

Organic Anion Transporter oatp2-Mediated Interaction between Digoxin and Amiodarone in the Rat Liver

Purpose. The interaction between amiodarone and digoxin has been known to increase serum concentrations of digoxin in humans and rats. In this study, we assessed the molecular mechanism(s) of that drug interaction, focusing on digoxin transport mediated by P-glycoprotein (Pgp) and by rat liver organic anion transporter (oatp2). Methods. Digoxin transport by Pgp and oatp2 was assessed using Pgp-overexpressing transfectant LLC-GA5-COL150 monolayers and oatp2-expressing Xenopus oocytes, respectively. The digoxin uptake into the isolated rat hepatocytes was also examined. Results. Amiodarone (10 M) inhibited slightly the transcellular transport of digoxin in LLC-GA5-COL150 monolayers, whereas itraconazole (10 M), a potent Pgp inhibitor, markedly blocked the transport. The digoxin uptake by the isolated rat hepatocytes and by the oatp2-expressing Xenopus oocytes was decreased markedly in the presence of amiodarone but not in the presence of itraconazole. In addition, amiodarone inhibited the oatp2-mediated digoxin uptake in a competitive manner with an apparent inhibition constant value of 1.8 M. Conclusion. These findings suggest that rat oatp2 rather than Pgp may be one of the interaction sites for digoxin and amiodarone in the liver.     

Thermodynamic Study of Sulfanilamide Polymorphism: (I) Monotropy of the α-Variety

Purpose. Sulfanilamide was chosen as a model compound in order to gain insights on the stability hierarchy of drug polymorphs from structural and thermodynamic criteria. Despite numerous studies, disagreements remained on the reported enthalpies associated with the mutual interconvertions of the -, -, and -forms of sulfanilamide. Therefore, the unambiguous determination of these enthalpies was the purpose of this work. Methods. Samples, free of solvent inclusions and made of only one form, were prepared, and analyzed combining X-ray powder diffraction and Differential Scanning Calorimetry (DSC). Results. The enthalpy values associated with the - to - and - to -transitions were found to be + 10.2 and + 10.9 J g^–1, respectively. The calculated enthalpy of the - to -transition is consistent with the experimental one ( + 1 J g^–1). Conclusions. The monotropy of the -form was ascertained over the explored temperature range at ordinary pressure.     

Lipopolysaccharide-induced down-regulation of organic anion transporting polypeptide 4 (Oatp4; Slc21a10) is independent of tumor necrosis factor-alpha, Interleukin-1beta, interleukin-6, or inducible nitric oxide synthase.

Organic anion transporting polypeptide 4 (Oatp4; Slc21a10) is expressed almost exclusively in liver, where it mediates uptake of a variety of compounds, including bile acids, as well as other endo- and xenobiotics, across hepatic sinusoidal membranes in a Na+-independent manner. Lipopolysaccharide (LPS) has been shown to decrease Oatp4 mRNA levels in a dose- and time-dependent manner in Toll-like receptor 4 (TLR4)-normal (C3H/OuJ) mice, but not in TLR4-mutant (C3H/HeJ) mice. Moreover, after LPS administration, serum concentrations of tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), and interleukin-6 (IL-6) are markedly lower in TLR4-mutant mice than in TLR4-normal mice. Thus, TLR4 is considered an upstream mediator of LPS-induced decrease in mouse Oatp4 mRNA. LPS is thought to alter liver gene expression through LPS-induced cytokines or nitric oxide (NO). TNF receptor p55 (TNFRp55) and type I IL-1 receptor (IL-1RI) mediate the biological functions of TNF-alpha and IL-1beta, respectively. Therefore, to determine whether endogenous cytokines or NO are mediators of LPS-induced down-regulation of Oatp4, Oatp4 mRNA levels were determined in mice deficient in the TNFRp55, IL-1RI, IL-6, or inducible nitric oxide synthase (iNOS) after LPS administration. Mice homozygous for a targeted deletion of genes for TNFRp55, IL-1RI, IL-6, or iNOS exhibited similar decreases in Oatp4 mRNA levels as wild-type mice after LPS administration. Moreover, in mouse hepatoma cells, treatment with TNF-alpha, IL-1beta, or IL-6 individually or in combination did not suppress activity of mouse Oatp4 promoter (-4.8 kb to +30). Therefore, LPS-induced down-regulation of Oatp4 appears to be independent of TNF-alpha, IL-1beta, IL-6, or iNOS.     

Transport of Biologically Active Interferon-gamma Across Human Skin in Vitro

Purpose. Several studies have suggested epidermal uptake of cytokines, such as interferons, can be facilitated using topical liposomal formulations. We have evaluated the in vitro transport of biologically active recombinant human interferon- (rhIFN-) into and through split-thickness human skin to assess this possibility. Methods. Skin samples were exposed to rhIFN- under various conditions involving hydrated and dry surface conditions in the presence and absence of liposomes. A new low-level ELISA and an anti-viral bioassay were used to quantitate transported rhIFN-. Immunohistochemical staining for ICAM-1 expression by keratinocytes was used to visualize the extent and distribution of rhIFN- transport. Results. Apparent steady-state transport of rhIFN- occurred within the first 5 hours of exposure with approximately 10% of transported rhIFN- demonstrating bioactivity. While the permeability of rhIFN- across human skin under drying conditions was enhanced by the presence of liposomes, no augmentation of permeability was observed when the skin was kept hydrated. Liposomal formulations of rhIFN-; had greater transport rates than aqueous formulations when the applied formulations were allowed to dry after dosing. Conclusions. Our results demonstrate the transport of biologically active rhIFN- across human skin in vitro and suggest a role for stratum corneum hydration as one possibility for the augmented cytokine transport.     

Pharmacokinetics of quinidine related to plasma protein binding in man

Summary The disposition and plasma protein binding of quinidine after intravenous administration were studied in 13 healthy subjects. Plasma protein binding, expressed as the fraction of quinidine unbound ranged from 0.134–0.303 (mean 0.221). Elimination rate constant () varied from 0.071 to 0.146 h^–1 (mean 0.113), and apparent volume of distribution (V) varied from 1.39–3.20 l · kg^–1 (mean 2.27). Total body clearance was 2.32–6.49 ml min^–1 · kg^–1. There was a positive linear correlation between the plasma fraction of unbound quinidine and both V (r=0.885, p<0.01) and total body clearance (r=0.668, p<0.05). No significant correlation existed between the fraction of unbound quinidine in plasma and the elimination rate constant. The results show that both the apparent volume of distribution and total body clearance of quinidine are proportional to the unbound fraction in plasma. This implies that the total plasma concentration of quinidine at steady state will change with alterations in plasma binding, whilst the concentration of unbound compund and its elimination rate will remain unaffected.     

Midazolam Exhibits Characteristics of a Highly Permeable P-Glycoprotein Substrate

Purpose. The purpose of this study was to investigate whether midazolam exhibits characteristics of a highly permeable P-glycoprotein (P-gp) substrate and to evaluate the potential influence of P-gp inhibition on 1-OH midazolam formation during midazolam transport. Methods. P-gp interaction was investigated by P-gp ATPase assay, efflux inhibition studies, and transport studies of midazolam across MDR1-MDCK and 1-,25-dihydroxy vitamin D₃-induced Caco-2 monolayers with and without the P-gp inhibitor GF120918. Results. Midazolam was highly permeable and transport appeared essentially unpolarized. In MDR1-MDCK, the basolateral-to-apical (B-to-A) permeability was slightly higher (16%) than apical-to-basolateral (A-to-B) permeability (p = 0.04); GF120918 increased A-to-B permeability by 27% (p = 0.01), and increased cellular midazolam accumulation during A-to-B transport by 45% (p = 0.01). Midazolam (200 M) decreased rhodamine123 and vinblastine B/A ratios 3-fold (p < 0.006), while increasing their cellular accumulation (p < 0.003). P-gp ATPase activation by midazolam was dose-dependent and saturable [K _m=11.5(±4.0) M; V _max = 41.1(±7.4) nmol/mg/min]. P-gp inhibition increased 1-OH midazolam formation in A-to-B studies 1.3-fold when midazolam donor M (p < 0.03). In B-to-A studies, P-gp inhibition did not significantly increase metabolite formation (p = 0.06). Midazolam's extraction ratio was not influenced by P-gp (p = 0.2). Conclusions. The results indicate that midazolam exhibited characteristics of a highly permeable P-gp substrate. 1-OH midazolam formation during A-to-B midazolam transport increased slightly when P-gp was inhibited.     

Wirkung von cyclischem Adenosin-3′,5′-Monophosphat (3′,5′-AMP) und seinem Dibutyrylderivat (DBA) auf Lipolyse,Glykogenolyse und Corticosteronsynthese

Zusammenfassung 1. Am isolierten Fettgewebe von Ratten hatte das Dibutyrylderivat des cyclischen Adenosin-3,5-Monophosphat (DBA) eine etwa 100 mal stärkere lipolytische Wirkung als das nicht substituierte cyclische Adenosin-3,5-Monophosphat (3,5-AMP). Hormone (ACTH, Noradrenalin) waren an diesem Testobjekt 10000 mal wirksamer als DBA. Durch Hemmung der Phosphodiesterase mit Theophyllin ließ sich auch die Wirkung des DBA verstärken.2. An isolierten Nebennieren von Ratten stimulierte DBA die Corticosteronsynthese etwa 100 mal stärker als 3,5-AMP; ACTH war aber 500 mal wirksamer als DBA. Durch Theophyllin ließ sich die Wirkung von ACTH, DBA bzw. 3,5-AMP nicht verstärken. Hohe Konzentrationen des Xanthinderivates hemmten die Corticosteronsynthese.3. An Ratten war die hyperglykämische Wirkung des DBA wesentlich stärker als diejenige des 3,5-AMP: Für eine Erhöhung des Blutzuckerspiegels um 40 mg/100 ml benötigten wir von DBA weniger als 1 mol/kg, von 3,5-AMP aber 30 mol/kg. Diese Wirkung der Nucleotide ließ sich durch Theophyllin nicht verstärken. Der Fettsäuren- und Glyceringehalt des Plasmas wurde durch Injektion von DBA bzw. 3,5-AMP nicht erhöht, sondern erniedrigt. — Die Ergebnisse wurden im Zusammenhang mit dem Second Messenger Concept von Sutherland u. Mitarb. diskutiert.Über einen Teil der Ergebnisse wurde auf der 8. Frühjahrstagung der Deutschen Pharmakologischen Gesellschaft (Stock u. Westermann, 1967; Bieck u. Westermann, 1967) sowie in einer kurzen Mitteilung (Bleck et al., 1968) berichtet.     

Overall pharmacokinetics during prolonged treatment of healthy volunteers with digoxin andβ-methyldigoxin

Summary Five healthy volunteers received digoxin 0.4 mg or -methyldigoxin 0.4 mg i. v., daily for 14 days, in a randomized cross-over arrangement. By monitoring minimal plasma concentrations during multiple dosing, it was found that the steady state pharmacokinetics of digoxin and -methyldigoxin could be estimated even better by a one-compartment than by a two-compartment model. The following mean parameters were calculated: the half life of digoxin of 1.54±0.31 days was significantly shorter than the half life of 2.29±0.34 days for -methyldigoxin. The distribution volume of 807±187 liters for digoxin was not significantly larger than the 735±227 liters for -methyldigoxin. Renal digoxin clearance of 191±25 ml/min was significantly higher than both the renal clearance of -methyldigoxin of 111±23 ml/min and also the creatinine clearance, which indicates tubular secretion of digoxin. There was a 2.8-fold accumulation of -methyldigoxin injected once a day, which was significantly higher than the 1.8-fold accumulation of digoxin.Abbreviations of parameters D dose - EST estimated disposition rate constant (day^–1) semilog curve fit - KTOT total disposition rate constant (day^–1) NONLIN fit - KREN renal disposition rate constant (day^–1) NONLIN fit - VOL volume of distribution (liters) NONLIN fit - CTOT total clearance (ml/min) - CREN renal clearance (ml/min) - BMIN body stores (µg) before next dose in steady state - CCR creatinine clearnace (ml/min) - CR/CT fraction renally excreted - CR/CCR renal drug clearance versus creatinine clearance - T/2 half life (days) - BMIN/D extent of accumulation This study was supported by Bundesministerium für Jugend, Familie und Gesundheit, Federal Republic of Germany.     

Antifungal Activity of a New Triterpenoid Glycoside from Pithecellobium racemosum (M.)

Purpose. In a continuation of our search for novel antifungal compounds from higher plants, the standard extract of the bark of Pithecellobium racemosum was found to have good activity against important AIDS-related opportunistic yeasts. Methods. The extract was subjected to bioguided fractionation using silica gel column chromatography which led to purification of triterpene glycosides. The structures of these compounds were determined by a combination of spectroscopic (IR, NMR, HRMS) and chemical methods. Results. Compound 1 is a new glycoside, 3-O[-L-arabinopyranosyl (1 -2)][-L arabinopyranosyl (1 -6)]2-acetoamido-2-deoxy--D-gluco-pyranosyl oleanolic acid and Compound 2 was identified as the known compound 3-O-[-L-arabinopyranosyl (l-2)]-L-arabinopyranosyl (1-6)] 2-acetamido-2-deoxy--D-glucopyranosyl echinocystic acid. Conclusions. Compound 1 is a new glycoside, 3-O-[-L-arabinopyranosyl (1-2)]-L-arabinopyranosyl (l-6)]-2-acetoamido-2-deoxy--D-glucopyranosyl oleanolic acid and exhibits moderate antifungal activity against T. mentogrophytes, C. albicans and S. cerevisiae with MIC values of 6.25, 12.5 and 12.5 g/ml respectively.     

Naltrexone-3-salicylate (a Prodrug of Naltrexone): Synthesis and Pharmacokinetics in Dogs

Naltrexone-3-salicylate (3), a prodrug of naltrexone (1), was prepared by a simple procedure from naltrexone-3-acetylsalicylate (2). The plasma (dog and human) hydrolysis half-life of 3 was found to be approximately 30 min. Compound 2 was previously shown to hydrolyze in dog and human plasma with a fast deacetylation step to 3, followed by slower hydrolysis of 3 to 1 (t _1/2, 30 min). Oral naltrexone bioavailability was greatly improved (30-fold) after oral administration of 3 to dogs, similar to the improvement observed after oral administration of 2. The half-life of naltrexone in dogs after oral administration of 3 was similar to that observed after oral administration of 2 (1 hr).