Cyclodextrins and drug permeability through semi-permeable cellophane membranes.

Determinations of drug fluxes through semi-permeable cellophane membranes are used to evaluate cyclodextrin complexes and cyclodextrin containing drug formulations. In the present study we investigated how the cyclodextrin concentration, the membrane thickness and the molecular weight cut off (MWCO) of the membrane influence drug fluxes. The cyclodextrin used was 2-hydroxypropyl-beta-cyclodextrin (HPbetaCD) and the sample drug was hydrocortisone. The MWCO of the membranes ranged from 500 to 14,000 and the HPbetaCD concentration ranged from 0 to 25% (w/v). The hydrocortisone flux from saturated solutions through the MWCO 500 membrane was unaffected by the cyclodextrin concentration. When MWCO of the membrane was greater than the molecular weight of the complex the flux from solutions saturated with hydrocortisone increased with increasing HPbetaCD concentration. This increase showed negative deviation from linearity. When the flux was corrected for the viscosity increase with increasing HPbetaCD concentration then the flux pattern could be described on the basis of Fick's first law and Stokes-Einstein equation. However, the flux did not correlate with the viscosity when it was increased by adding polymer to the saturated drug solutions. It was shown that the observed flux pattern was consistent with self-association of cyclodextrin complexes in the aqueous donor phase.     

Evidence that oxmetidine inhibits transmembrane-calcium flux in cardiac and vascular tissue.

Oxmetidine, at concentrations in excess of 1 X 10(-6)M, caused concentration-dependent negative inotropic and chronotropic responses in guinea-pig isolated heart preparations. Oxmetidine, at concentrations in excess of 1 X 10(-5)M, caused negative inotropic responses in guinea-pig papillary muscle preparations. The negative inotropic responses to oxmetidine were associated with shortening of the plateau phase of the action potential. Verapamil and nifedipine caused similar shortening of the plateau phase of the action potential at equivalent negative inotropic concentrations indicating that oxmetidine may also act as a calcium antagonist. In preparations partially depolarized by raising extracellular K+ concentration, oxmetidine also exhibited negative inotropic activity and reduced the calcium-dependent action potential. However, unlike verapamil and nifedipine, oxmetidine did not show voltage-dependent activity. Oxmetidine, at concentrations in excess of 1 X 10(-5)M, inhibited Ca2+-dependent contractions of dog saphenous vein preparations and inhibited 45Ca2+-uptake into veins depolarized by high extracellular K+. In vivo, these calcium antagonist actions of oxmetidine were demonstrated by vasodilatation, reduction in blood pressure, bradycardia and reduced cardiac output in anaesthetized cats. Oxmetidine, at concentrations of 1 X 10(-5)M and above, shows properties consistent with inhibition of transmembrane Ca2+ flux. This action can be distinguished from other calcium antagonists as the effects of oxmetidine are not voltage-dependent.     

Effect of bovine serum albumin on drug permeability estimation across Caco-2 monolayers.

The purpose of this study was to explore approaches to more accurately assess Caco-2 permeability of poorly water-soluble new chemical entities (NCEs) in an effort to determine their biopharmaceutics classification system (BCS) permeability class with a higher level of confidence. The transport of reference compounds and NCEs (Sch-Y, Sch 56592) was studied across Caco-2 monolayers in the absence or presence of varying percentage of bovine serum albumin (BSA) in the receiver chamber. The inclusion of 0.5-4% BSA in the receiver chamber caused a 4-5-fold increase in Sch-Y P(app), while Sch 56592 P(app) was not significantly influenced. Amongst reference solutes, the P(app) ratio (+BSA/ctrl) was significant (1.3-fold) only for diltiazem (log PC=2.7, plasma protein binding=78%), but the prediction of human oral absorption for such drugs was not affected by the presence of BSA in receiver. In summary, the use of 4% BSA in the receiver chamber during transport studies can dramatically affect the estimated Caco-2 P(app) and BCS permeability ranking of highly lipophilic NCEs, as in the case of Sch-Y with a log PC of 4.0. For Sch-Y, this is presumably due to improved sink conditions and/or a reduction in non-specific drug adsorption to plastic wells. In contrast, the permeability classification of Sch 56592 (log PC=2.4) based on estimated Caco-2 P(app) values is not affected by the presence of receiver BSA.     

Improved transport apparatus for examining diffusion of drugs across isolated tissues and synthetic membranes.

A three-compartment diffusion apparatus that can accommodate synthetic as well as small isolated biological membranes such as rat intestine was developed and tested. The diffusion of drug from two outer donor cells into a central recipient cell allows for more membrane surface exposure and greater sensitivity in assaying as compared with previously published techniques. Reproducibility values for the synthetic membrane were equivalent to those obtained in more elaborate designs, while the reproducibility for biological membranes compared favorably with everted gut preparations and a similar diffusion apparatus. The clearance, per square centimeter of membrane, of salicylamide at pH 7.4 and 37 degrees was 0.288 ml/hr/cm2, which agreed with previous literature reports of different techniques. Although the diffusion through a synthetic membrane was more rapid, introduction of a nonionic surfactant enhanced the biological perfusion rate while having no significant effect on the synthetic barrier. The enhancement of the diffusion rate effected by 0.1 and 0.01% (w/v) surfactant was about twofold, rapid in onset, and not progressive, giving clearance values per square centimeter of 0.606 and 0.537 ml/hr/cm2, respectively.     

Paracellular drug transport across intestinal epithelia: influence of charge and induced water flux.

The influence of drug charge and transepithelial water flux on passive paracellular drug transport was investigated in Caco-2 cell monolayers and rat ileal mucosa in vitro. Three small hydrophilic compounds with different net charges (creatinine, erythritol and foscarnet) were used as model drugs. A hypotonic glucose-rich solution was applied apically to induce epithelial absorption of water. In the Caco-2 monolayers, permeability to creatinine (positively charged) was 25-fold greater than to foscarnet (negatively charged), indicating a pronounced cation selective paracellular permeability. During apical exposure to the hypotonic glucose-rich solution, transport of all model drugs increased in both the absorptive and secretory directions. This enhanced transport coincided with a decrease in transepithelial resistance. Further, fluorescence and transmission electron microscopy indicated dilatations of the paracellular spaces but no damage to the cell membranes. These findings suggested that the enhancement in drug transport was attributable to increased paracellular tight junction permeability rather than to "solvent drag". In the ileal segments, mucosal exposure to the hypotonic glucose-rich solution had no effect on transepithelial resistance and only a marginal increase in drug transport was observed. Taken together, the modest absorption enhancement demonstrated in the in vitro models agrees with results obtained in vivo, supporting the conclusion that a more pronounced disruption of the tight junction barrier than that obtained through stimulation of epithelial absorption of water is required for efficient enhancement of paracellular intestinal drug absorption.     

Iontophoretic and chemical enhancement of drug delivery. Part I: across artificial membranes

This paper reports on measurements of the release characteristics of the model drug salbutamol base from a liquid crystalline vehicle across a non-rate limiting synthetic membrane. The measured passive release rates were compared with analogous behaviour: (i) when a penetration enhancer such as oleic acid was incorporated into the vehicle; (ii) when the release was iontophoretically assisted; and (iii) when the penetration enhancer and iontophoretic assistance were used simultaneously. The effects of using isotonic phosphate buffer solution as the aqueous domain of the vehicle and in the receptor were also separately assessed. The passive release from the standard system was consistent with matrix diffusion control. The addition of oleic acid indicated association of the drug with the fatty acid so that its release into an aqueous medium was significantly retarded. With buffer ions present in the vehicle the release rate increased consistent with reduced association, and when phosphate buffer was used as a receptor medium the release rate exceeded that of the standard vehicle due to an ion exchange process. The delivery of salbutamol from the fatty acid containing systems was substantially enhanced by iontophoresis and the rates were shown to be approximately proportional to the assisting currents. The data clearly indicate the iontophoretic process to be significantly less efficient in the presence of buffer ions but with the iontophoretic delivery rates being enhanced by the presence of a fatty acid.     

Evidence that nitric oxide is an endogenous antiangiogenic mediator.

1. The involvement of nitric oxide (NO) in the regulation of angiogenesis was examined in the in vivo system of the chorioallantoic membrane (CAM) of the chick embryo and in the matrigel tube formation assay. 2. Sodium nitroprusside (SNP) (0.37-28 nmol/disc), which releases NO spontaneously, caused a dose-dependent inhibition of angiogenesis in the CAM in vivo and reversed completely the angiogenic effects of alpha-thrombin (6.7 nmol/disc) and the protein kinase C (PKC) activator 4-beta-phorbol-12-myristate-13-acetate (PMA) (0.97 nmol/disc). In addition, SNP (28 x 10(-6) M) stimulated the release of guanosine 3'-5'-cyclic monophosphate (cyclic GMP) from the CAM in vitro. 3. In the matrigel tube formation assay, an in vitro assay of angiogenesis, both SNP (1-3 x 10(-6) M) and the cell permeable cyclic GMP analogue, Br-cGMP (0.3-1.0 x 10(-3) M) reduced tube formation. 4. The inhibitors of NO synthase, NG-monomethyl-L-arginine (L-NMMA) (3.8-102 nmol/disc) and NG-nitro-L-arginine methylester (L-NAME) (1.3-34.2 nmol/disc) stimulated angiogenesis in the CAM in vivo, in a dose-dependent fashion. D-NMMA and D-NAME on the other hand had no effect on angiogenesis in this system. 5. L-Arginine (10.9 nmol/disc), although it had a modest antiangiogenic effect by itself, was capable of abolishing the angiogenic effects of L-NMMA (34.2 nmol/disc) and of L-NAME (3.8 nmol/disc). 6. Dexamethasone, an inhibitor of the induction of NO synthase, at 0.2-116.1 nmol/disc, stimulated angiogenesis in the CAM, whereas at 348.4-1161 nmol/disc it inhibited this process. Combination of 38.7 nmol/disc dexamethasone with L-NAME (9.3 nmol/disc) resulted in a potentiation of the angiogenic effect of the former. It appears therefore that both the constitutive and the inducible NO synthase may contribute to the NO-mediated inhibition of angiogenesis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mechanisms regulating cell resistance to adriamycin. Evidence that drug accumulation in resistant cells is modulated by phosphorylation of a plasma membrane glycoprotein

Incubation of adriamycin resistant Chinese hamster lung cells with the calmodulin inhibitor trifluoperazine (TFP) resulted in a significant increase in the cellular accumulation of drug. When resistant cells were prelabeled with 32Pi and then treated with TFP, a major increase also occurred in the phosphorylation of a plasma membrane glycoprotein (P-180). The concentration of TFP required for inducing the superphosphorylation of this protein correlated well with the TFP concentration required for inducing an increase in drug accumulation in resistant cells. In addition to TFP, the Ca2+ channel blocker verapamil also induced drug uptake and enhanced the phosphorylation level of P-180. Additional studies showed that, when resistant cells reverted to drug sensitivity, there was a parallel loss in the TFP-induced P-180 phosphorylation. The results of this study indicate that the trifluoperazine-induced uptake of drug in resistant cells is mediated by a mechanism which involves an enhanced phosphorylation of P-180. It is suggested that, when this protein is superphosphorylated, it becomes biologically inactive, and that this results in the conversion of the resistant cell to one having a drug sensitive phenotype.     

Mitochondrial permeability transition induced by the anticancer drug etoposide.

Etoposide (VP-16) is widely used for the treatment of several forms of cancer. The cytotoxicity of VP-16 has been assigned to the induction of apoptotic cell death but the signaling pathway for VP-16-induced apoptosis is essentially unknown. There is some evidence that this process depends on events associated with the loss of mitochondrial membrane potential (Delta Psi) and/or release of apoptogenic factors, putatively as a consequence of mitochondrial permeability transition (MPT) induction. This work evaluates the interference of VP-16 with MPT in vitro, which is characterized by the Ca(2+)-dependent depolarization of Delta Psi, the release of matrix Ca(2+) and by extensive swelling of mitochondria. Delta Psi depolarization and Ca(2+) release were measured with ion-selective electrodes, and mitochondrial swelling was monitored spectrophotometrically. Incubation of rat liver mitochondria with VP-16 results in a concentration-dependent induction of MPT, evidenced by an increased sensitivity to Ca(2+)-induced swelling, depolarization of Delta Psi, Ca(2+) release by mitochondria and stimulation of state 4 oxygen consumption. All of these effects are prevented by preincubating the mitochondria with cyclosporine A, a potent and specific inhibitor of the MPT. Therefore, VP-16 increases the sensitivity of isolated mitochondria to the Ca(2+)-dependent induction of the MPT. Together, these data provide a possible mechanistic explanation for the previously reported effects of VP-16 on apoptosis induction.     

Enhanced permeability of phenylalanyl-glycine (Phe-Gly) across the intestinal membranes by chemical modification with various fatty acids

We synthesized four novel lipophilic derivatives of phenylalanyl-glycine (Phe-Gly), C4-Phe-Gly, Phe-Gly-C4, C6-Phe-Gly and C8-Phe-Gly by chemical modification with butyric acid (C4), caproic acid (C6) and octanoic acid (C8). The effect of the acylation on the stability, permeability and accumulation of Phe-Gly in the intestine was investigated by in vitro studies. The stability of Phe-Gly in homogenates of duodenal and colonic membranes was low, but was significantly improved by the acylation except for Phe-Gly-C4. In the transport studies, a modified Ussing chamber was used for the intestinal permeability experiments with Phe-Gly and its acyl derivatives. The permeability of native Phe-Gly and Phe-Gly-C4 across the intestinal membrane was not observed during the transport studies. However, the permeability of Phe-Gly was much improved by chemical modification with various fatty acids to its N-terminal portion. The permeability of acyl-Phe-Gly derivatives across the intestinal membrane decreased with increasing the chain length of fatty acids. In addition, the intestinal tissue accumulation of acyl-Phe-Gly derivatives at the end of the transport studies was much higher than that of native Phe-Gly. The intestinal tissue accumulation of acyl-Phe-Gly in the duodenum increased as the chain length of fatty acids increased. Furthermore, intestinal permeability of C4-Phe-Gly was slightly inhibited in the presence of 5 mM ceftibuten and was significantly reduced under low temperature condition. We observed a directional difference in the transport of C4-Phe-Gly (the mucosal to serosal transport of C4-Phe-Gly was higher than its serosal to mucosal transport) suggesting that C4-Phe-Gly might be transported by a carrier-mediated process as well as other dipeptides. These findings indicate that acylation might be useful approach to enhance the transport of Phe-Gly, a model dipeptide, transported by a carrier-mediated process.     

Evidence that physical dependence on morphine is mediated by the ventral midbrain

Rats were given daily injections of increasing doses of morphine sulfate (40-100 mg/kg, s.c.), for 4 days. Twenty hours after the last injection of morphine, the animals received bilateral injections of naloxone (1-10 micrograms) into the substantia nigra, ventral tegmental area or sites 2 mm rostral, caudal or dorsal to the site in the nigra. Withdrawal signs were monitored for 20 min after the intracerebral injection. Naloxone administered into the nigra in morphine-dependent rats produced dose-dependent significant increases in wet dog shakes, irritability to touch, teeth chattering, diarrhea and locomotion, compared to morphine-dependent animals that received injections of saline into the nigra. The injection of naloxone (3 micrograms) into the ventral tegmental area of morphine-dependent animals, produced irritability to touch and diarrhea, compared to morphine-dependent controls that received saline in this region of the brain. Significant differences in withdrawal signs were observed between morphine-dependent animals, that received injections of naloxone (3 micrograms) into the nigra and those that received naloxone (3 micrograms) into the ventral tegmental area or rostral or caudal sites. No differences between the substantia nigra and the dorsal sites were observed. However, withdrawal symptoms were produced by injections of naloxone into the substantia nigra and ventral tegmental area, even when the guide cannulae were angled to avoid penetration of sites dorsal to these regions of the brain. Naloxone, injected into the ventral midbrain of non-dependent animals, produced no signs of withdrawal. These studies suggest that the ventral midbrain mediates physical dependence on morphine.     

Evidence that 5β-progestins produce uterine relaxation by diminishing cellular calcium permeability

This report presents the relaxant effect of progesterone and some of its 5β-derivatives on the tonic contractions induced in isolated rat uterus by high K⁺ (40 mM) and high K⁺? Na⁺-free depolarizing solutions. Experiments were recorded with a polygraphic system. Contractions were countered by the action of progesterone (101 μM), pregnanedione (41 μM), pregnanolone (25 μM), and epipregnanolone (47 μM). The relaxant effect of progestins was, however, significantly more effective against the contraction produced by high K⁺ alone than that produce by high K⁺? Na⁺-free solutions. These different results suggest the ability of 5β-reduced progesterone metabolites to block the sensitive-to-voltage Ca²⁺ channels (which are activated by K⁺) and also agree with the existence of a Na⁺/Ca²⁺ interchanger in the myometrial membrane. It can be concluded that 5β-progestins induce relaxation of the uterus probably by blocking the calcium entrance into the cell.     

Evidence that the inhibition of ATP release from sympathetic nerves by adenosine is a physiological mechanism.

1. Perfusion with the P1-purinoceptor agonist adenosine (1-500 microM) greatly reduced the stimulation-induced release of ATP and the initial contractile phase of the response of the guinea pig vas deferens to field stimulation. 2. The inhibitory effects of adenosine (100 microM) were readily antagonised by the P1-purinoceptor antagonist, 8-phenyltheophylline (10 microM). 3. Dipyridamole (10 microM), inhibited the stimulation-evoked release of ATP from the guinea pig vas deferens and reduced the initial component of contraction. 4. These results support the view that adenosine, resulting from ectoenzymatic breakdown of ATP released as a cotransmitter from sympathetic nerve terminals, acts as a physiological prejunctional regulator of transmitter release.     

Evidence that taste aversion learning induced by l-5-hydroxytryptophan is mediated peripherally

Rats learned to avoid a saccharin solution if their initial consumption of it was followed by intraperitoneal (IP) administration of 25 mg/kg l-5-hydroxytryptophan (l-5-HTP); this taste aversion learning did not occur in rats pretreated with 50 mg/kg (IP) of the aromatic l-amino acid decarboxylase inhibitor RO 4-4602 (benserazide). RO 4-4602 antagonized the l-5-HTP-induced elevation of 5-hydroxytryptamine (5-HT) in the mesentery but significantly increased the l-5-HTP-induced elevation of 5-HT in the brain. These results indicate that l-5-HTP-induced taste aversion is correlated with peripheral, but not central, elevation of 5-HT.     

Evidence that hydroxysafflor yellow A protects the heart against ischaemia-reperfusion injury by inhibiting mitochondrial permeability transition pore opening

1. The present study was conducted to investigate whether hydroxysafflor yellow A (HSYA) has a protective effect against heart injury after ischaemia-reperfusion and to determine the possible mechanism involved. 2. Hearts isolated from male Sprague-Dawley rats were perfused on a Langendorff apparatus and subjected to 30 min global ischaemia, followed by 120 min reperfusion. Infarct size and the level of lactate dehydrogenase (LDH) in the coronary effluent were determined. In mitochondria from isolated perfused hearts, Ca(2+)-induced swelling was observed. In isolated ventricular myocytes, depolarization of the mitochondrial membrane was determined by tetramethyl-rhodamine ethyl ester (TMRE) fluorescence. Furthermore, levels of phosphorylated endothelial nitric oxide synthase (eNOS) protein were measured by western blot. 3. Pretreatment with HSYA for 5 min before ischaemia reduced infarct size and the release of LDH. Administration of 20 micromol/L atractyloside, an opener of the mitochondrial permeability transition pore, and 10 micromol/L N(G)-nitro-L-arginine methyl ester (L-NAME), an inhibitor of NOS, attenuated the protective effects of HSYA. In mitochondria isolated from hearts pretreated with 0.1 mmol/L HSYA for 5 min, a significant inhibition of Ca(2+)-induced swelling was observed and this inhibition was attenuated by l-NAME. In isolated ventricular myocytes, pretreatment with HSYA prevented ischaemia-induced cell death and depolarization of the mitochondrial membrane, whereas atractyloside or l-NAME attenuated the effects of HSYA. Levels of phosphorylated eNOS protein were significantly enhanced in the HSYA-treated group. 4. The findings of the present study indicate that HSYA protects the myocardium against ischaemia-reperfusion injury by inhibiting mitochondrial permeability transition pore opening. The effect of HSYA on mitochondrial permeability transition pore opening may be mediated through enhanced nitric oxide production by eNOS activation.