Effect of adrenergic stimulation on drug absorption via passive diffusion in Caco-2 cells

It is well known that the enteric nervous system (ENS) regulates the movement and function of the small intestine, but the effects of ENS on drug absorption from the small intestine still remain to be clarified. Focusing on adrenergic effect, we tried to evaluate how adrenergic stimulation influences the drug absorption via passive diffusion using Caco-2 cells as model epithelial cells, a terminal effector of ENS. Adrenaline, an adrenergic agonist, did not affect the transport of small molecules such as antipyrine, phenacetin and mannitol, but decreased the transport of large molecules such as FITC-dextran (FD)-20 and FD-40 without transepithelial electrical resistance (TEER) change. These results suggested that the transport of large molecules via paracellular route would be attenuated by adrenergic stimulation. Only clonidine, an alpha(2)-agonist, among selective adrenoceptor agonists decreased FD-40 transport across Caco-2 cell monolayers and the agonist also decreased intracellular cAMP. Furthermore, H-89, a protein kinase A inhibitor, significantly decreased FD-40 transport and dibutyryl cAMP, a cAMP derivative, increased it. These results suggest that the decrease in FD-40 transport would be mainly attributed to the decrease in intracellular cAMP and subsequent decrease in PKA activity via alpha(2)-receptor stimulation.     

Possible Regulation of P-glycoprotein Function by Adrenergic Agonists in a Vascular-luminal Perfused Preparation of Small Intestine

Although the functions of small intestine are largely regulated by enteric nervous system (ENS), an independent intrinsic innervation, as well as central nervous system (CNS), the neural regulation of drug absorption from the small intestine still remains to be clarified. To obtain some information on it, the effect of adrenergic agonists on P-glycoprotein (P-gp) function was investigated by utilizing a vascular-luminal perfused rat small intestine. Adrenaline significantly decreased the secretion of rhodamine-123 (R-123) into the intestinal lumen, but dibutyryl cAMP (DBcAMP) significantly enhanced R-123 secretion. The inhibition study with quinidine clearly indicated that the decrease in secretory clearance of R-123 by adrenaline or the increase by DBcAMP would be attributed to the decrease or increase in P-gp activity, respectively. Expression levels of P-gp in whole mucosal homogenates were not changed at all by any chemicals examined, but those on brush border membrane (BBM) of intestinal epithelial cells were significantly decreased or increased by adrenaline or DBcAMP, respectively. Furthermore, changes in P-gp activity caused by adrenergic agonists and DBcAMP were significantly correlated with changes in expression level of P-gp in BBM, suggesting that the trafficking of P-gp from cytosolic pool to BBM would be regulated by adrenergic agonists and DBcAMP.     

盐酸氯米帕明大鼠在体肠吸收动力学研究

目的研究盐酸氯米帕明在大鼠肠道的吸收特性。方法采用大鼠在体肠灌流实验方法,利用高效液相色谱法同时测定肠回流液中药物及酚红的浓度,通过酚红的浓度校正相应时刻供试液的体积。结果盐酸氯米帕明浓度为5、10、25μg/ml时小肠的吸收速率常数(ka)为(0.74±0.04)、(0.78±0.03)、(0.77±0.05)h-1;在十二指肠、空肠、回肠和结肠的ka分别为(0.590±0.026)、(0.670±0.032)、(0.680±0.030)和(0.560±0.031)h-1。结论盐酸氯米帕明在大鼠肠道均有良好的吸收。盐酸氯米帕明在小肠的吸收呈表观一级动力学过程,吸收机制为被动扩散。     

Montmorillonite adsorbs uric acid and increases the excretion of uric acid from the intestinal tract in mice

Objectives The aim was to evaluate the adsorbing effect of montmorillonite on uric acid, promoting diffusion of uric acid from blood to intestine, preventing absorption of uric acid in intestine and reducing uric acid level in serum. Methods The adsorbing effect of montmorillonite on uric acid was observed in vitro. The intestine and blood vessel of rats were circularly perfused with intestinal perfusate and vascular perfusate, respectively. A model of hyperuricaemia in mice was prepared by intraperitoneal injection of hypoxanthine and potassium oteracil. The concentration of uric acid was determined by the method of urate oxidase and peroxide enzyme. Key findings The results showed that different concentrations of montmorillonite could adsorb uric acid in a concentration‐dependent manner. The adsorbing effect was fast. The adsorptive rate was high in acid solution and was low in alkaline solution. When blood vessels were circularly perfused by vascular perfusate containing uric acid, the concentration of uric acid in vascular perfusate was decreased and the concentration of uric acid in intestinal perfusate was increased, suggesting that uric acid in blood vessels diffused into the intestine. When the intestine was perfused with intestinal perfusate containing uric acid, the uric acid concentration in vascular perfusate was increased, but the uric acid concentration of intestinal perfusate was decreased, suggesting that uric acid was absorbed in the intestine. The uric acid concentrations of intestinal perfusate and vascular perfusate in montmorillonite 0.5 and 1.0 g/kg groups were lower than the control group. Concentrations of uric acid in serum and urine in the montmorillonite 1 and 2 g/kg groups were lower compared with mice in the hyperuricaemic group. Conclusions The results suggested that montmorillonite adsorbed uric acid and promoted diffusion of uric acid from blood vessels to intestine, prevented absorption of uric acid in intestine and decreased uric acid level in serum.     

隐丹参酮在小肠吸收机制的实验研究

目的研究隐丹参酮在大鼠小肠的吸收机制。方法用大鼠在体一过式肠灌流方法，通过肠系膜静脉插管采集血样，同时收集灌流流出液，以高效液相色谱法测定样品中隐丹参酮含量，计算其通透率。结果隐丹参酮在大鼠小肠的肠管通透率(Plumen)和血管通透率(Pbtood)随浓度的增加而下降；加入P-糖蛋白抑制剂维拉帕米后其通透率增高。结论隐丹参酮在大鼠小肠的转运机制可能为主动转运，隐丹参酮可能为P-糖蛋白底物。     

The use of a perfluorochemical emulsion as a vascular perfusate in drug absorption

In-vitro simultaneous luminal and vascular perfusion using the perfluorochemical emulsion, FC-43 emulsion, as a vascular perfusate, was examined for drug absorption in rat jejunum. The intestinal membrane in this system was found to retain its normal barrier functions for drug transport, as evident from the following: (i) stable absorption clearance of tritiated water and salicylic acid at steady-state, (ii) agreement of this clearance with that by in-situ single-pass luminal perfusion, (iii) active transport of D-glucose and its inhibition by phloridzin and (iv) normal glutamate pyruvate transaminase activity in the intestinal mucosa. FC-43 emulsion gave a more normal absorption site blood flow than the usual vascular perfusate containing erythrocytes and albumin in Krebs-Henseleit bicarbonate buffer solution. Consequently, this emulsion was useful for examining the contribution of blood flow resistance toward drug absorption. The rate-limiting steps of absorption of tritiated water, antipyrine and salicylic acid were examined by perfusion using FC-43 emulsion. The absorption of tritiated water was almost completely blood flow-limited and its absorption clearance may possibly be an approximated absorption site blood flow. The contribution of blood flow resistance to total resistance for antipyrine absorption exceeded that for salicylic acid absorption.     

A comparison of vasopressin and noradrenaline on oxygen uptake by perfused rat hindlimb, kidney, intestine and mesenteric arcade suggests that it is in part due to contractile work by blood vessels

1. The rat hindlimb, kidney and intestine were each perfused in a nonrecirculating mode at 25 degrees C using an artificial perfusate (initial pressure 85 +/- 5 mmHg) and the effects of vasopressin and noradrenaline on oxygen uptake and perfusion pressure determined. 2. Both vasopressin (K0.5 = 0.1 nM) and noradrenaline (K0.5 = 2 nM) increased oxygen uptake as well as perfusion pressure by the perfused hindlimb; changes in oxygen uptake were closely matched by changes in pressure. The maximum increase in oxygen uptake was approx. 9 mumol/hr per g wet wt of hindlimb. 3. The perfused kidney also responded to vasopressin and noradrenaline with parallel increases in oxygen uptake and perfusion pressure for each agent. The largest increase in oxygen uptake was approx. 30 mumol/hr per g wet wt but this was not maximal. 4. Vasopressin increased oxygen uptake and pressure by the perfused intestine over the range 0.01-2 nM, but the changes in pressure only became significant at doses greater than 0.1 nM. 5. Noradrenaline inhibited oxygen uptake and increased perfusion pressure in a dose-dependent manner at pharmacological concentrations (greater than 30 nM) when shunting of perfusate may have contributed to unperfused regions. 6. A network of mesenteric blood vessels estimated to contain approx. 6% vascular tissue by weight, with the remainder white fat cells, lymphatics and connective tissue, was also perfused. 7. Vasopressin (K0.5 = 0.3 nM) and noradrenaline (K0.5 = 30 nM) each increased oxygen uptake and perfusion pressure in a dose-dependent manner.(ABSTRACT TRUNCATED AT 250 WORDS)     

石杉碱甲大鼠在体肠吸收动力学研究

目的研究石杉碱甲在大鼠小肠及各肠段的吸收动力学特征.方法采用大鼠在体方式对石杉碱甲进行了大鼠小肠及各肠段的吸收动力学研究;采用HPLC法测定石杉碱甲在大鼠体内肠吸收循环液中的药物浓度;采用UV法测定循环液中酚红浓度.结果石杉碱甲在小肠中吸收较好且没有特定吸收部位,各肠段吸收速率按十二指肠、空肠、回肠、结肠依次为0.001 0、0.000 9、0.001 1、0.000 9 h-1.结论结肠的吸收速率常数与小肠段相近,药物在肠道的吸收呈现一级吸收动力学,吸收机制为被动吸收,提示适于制备日服一次(24 h)缓释给药制剂.     

蒙脱石影响尿酸在血管和肠道间的扩散作用

目的研究蒙脱石促进血管中的尿酸向肠道的扩散作用和阻止肠道尿酸的吸收作用。方法大鼠肠道、血管分别用含尿酸140mg.L-1的血管灌流液和肠灌流液循环灌流,尿酸酶及过氧化物酶法测定灌流液中的尿酸含量。结果血管用含尿酸的血管灌流液循环灌流,肠道用不含尿酸的肠道灌流液循环灌流,同时肠道中加入蒙脱石时,蒙脱石0.25、0.5和1.0g.kg-1组的血管灌流液和肠道灌流液中的尿酸浓度均明显低于对照组。肠道用含尿酸的肠道灌流液循环灌流,血管用不含尿酸的血管灌流液循环灌流,同时肠道中加入蒙脱石时,蒙脱石0.5和1.0g.kg-1组肠道灌流液和血管灌流液中尿酸浓度明显低于对照组。结论蒙脱石能促进尿酸从血管向肠道的扩散,并减少尿酸在肠道的吸收。     

Intestinal absorption of rutin in free and conjugated forms.

Quercetin is one of the most common flavonoids in nature, occurring mainly in glycosidic forms such as rutin. Rutin has been reported to exert numerous biochemical and pharmacological activities, though information about its absorption and metabolism is scarce. The aim of this study was to investigate intestinal handling of luminally administered rutin in an isolated preparation of luminally and vascularly perfused rat small intestine. A synthetic perfusate free from blood components was used as vascular medium, with a perfluorocarbon as oxygen carrier. Luminal media consisted of a bicarbonate-buffered sodium chloride solution spiked with rutin (40.5 +/- 1.8 micromol/L). Viability was maintained during the entire perfusion; no differences between rutin and control perfusions for perfusion pressure, lactate-pyruvate ratio, oxygen uptake, and acid-base homeostasis were observed. About 10% of the administered rutin appeared at the vascular side, chiefly as free rutin (5.6%), but some rutin sulfate (2.5%) and glucuronide (2.0%) were also detected. The conjugates were preferentially absorbed to the vascular side, while only traces of the glucuronide (0.2%) were found in the luminal perfusate. Minute amounts of the rutin administered were located in the intestinal tissue (1.1%) in the form of unchanged rutin and its glucuronide and sulfate conjugates. The model used serves as a valuable tool for understanding intestinal handling of the bioactive flavonol glycoside rutin, and the obtained results confirm uptake of rutin in the rat small intestine.     

Influence of bile salts and lipids on intestinal absorption of griseofulvin in man

Summary The influence of bile salts and lipids on the intestinal absorption of griseofulvin has been studied in 11 healthy male volunteers by the intestinal perfusion technique. The drug in a nutrient solution (Realmentyl) was perfused into the second part of duodenum at 5 ml/min. Intestinal samples were taken continuously at 1 ml/min, 20 cm (at the angle of Treitz) and 45 cm distal to the perfusion point. To study the effect of lipids on griseofulvin absorption, the drug was perfused with solutions A and B, of which B contained a total lipid and caloric load three times that of A. The influence of bile salts on griseofulvin absorption was examined by perfusing the drug on Day 1 with bile salts and again on the following day after bile salt depletion. Bile salts and a varying quantity of lipid perfusate had no significant influence on the duodeno-jejunal griseofulvin absorption rate per cm of intestine. Lipids, however, may still play a role in griseofulvin absorption along the entire intestine.     

Effect of angiotensin on the response to norepinephrine and peri-arterial stimulation of the isolated perfused cat terminal ileum

The interaction of angiotensin and cocaine with norepinephrine and peri-arterial stimulation was studied in the isolated terminal ileum of the cat perfused through the mesenterial artery at constant flow with tyrode solution. Norepinephrine and peri-arterial stimulation decreased the spontaneous motility of intestine and increased vascular perfusion pressure. Angiotensin further augmented the rise of perfusion pressure and the inhibition of ileal motility induced by peri-arterial stimulation without changing the effect of norepinephrine. Cocaine, however, caused a significant increase in response to both norepinephrine and peri-arterial stimulation. These results suggest that angiotensin potentiates the effect of peri-arterial stimulation probably by facilitating the release of norepinephrine from adrenergic nerve endings and not by inhibiting the re-uptake of neurotransmitter.     

Inhibition by carbapenem antibiotic imipenem of intestinal absorption of valproic acid in rats.

The concomitant use of carbapenem antibiotics with valproic acid has been prohibited because panipenem induced a decrease in plasma concentration of valproic acid in epileptic patients during valproic acid therapy. To clarify the possible mechanism of the carbapenem-valproic acid interaction, we investigated the effect of imipenem on the pharmacokinetic behaviour of valproic acid in rats. Co-administration of imipenem (30 mg kg(-1), i.v.) induced a decrease in the peak plasma concentration of valproic acid after oral administration. However, the imipenem-induced decrease in plasma concentrations of valproic acid was not observed within 60 min after intravenous injection of valproic acid. By utilizing in-situ vascular and luminal perfused small intestine, it was confirmed that absorption of valproic acid from the luminal to the vascular perfusate was decreased in the presence of imipenem (0.5 mM) in the vascular perfusate. The everted gut sac method was used to determine the effect of imipenem on active transport of valproic acid. The accumulation of valproic acid on the serosal side of the intestinal sac against the concentration gradient was reduced by lactic acid that inhibits the carrier-mediated transport of valproic acid across the intestinal brush-border membrane. However, imipenem did not affect the active transport of valproic acid. Therefore, the inhibition by imipenem of valproic acid absorption may be caused by a mechanism different from that of lactic acid. In conclusion, imipenem inhibits the intestinal absorption of valproic acid, which contributes to the decrease in plasma concentration of valproic acid after oral administration.     

Application of the axial dispersion model of hepatic drug elimination to the kinetics of diazepam in the isolated perfused rat liver.

The application of the axial dispersion model to diazepam hepatic elimination was evaluated using data obtained for several conditions using the single-pass isolated perfused rat liver preparation. The influence of alterations in the fraction unbound in perfusate (fu) and perfusate flow (Q) on the availability (F) of diazepam was studied under steady conditions (n = 4 in each case). Changes in fu were produced by altering the concentration of human serum albumin (HSA) in the perfusion medium while maintaining diazepam concentration at 1 mg L-1. In the absence of protein (fu = 1), diazepam availability was 0.011 +/- 0.005 (mean +/- SD). As fu decreased, availability progressively increased and at a HSA concentration of 2% (g/100 ml), when fu was 0.023, diazepam availability was 0.851 +/- 0.011. Application of the axial dispersion model to the relationship between fu and F provided estimates for the dispersion number (DN) of 0.337 +/- 0.197, and intrinsic clearance (CL(int)) of 132 +/- 34 ml min-1. The availability of diazepam during perfusion with protein-free media was also studied at three different flow rates (15, 22.5, and 30 ml min-1). Diazepam availability always progressively increased as perfusate flow increased, with the axial dispersion model yielding estimates for DN of 0.393 +/- 0.128 and CL(int) of 144 +/- 38 ml min-1. The transient form of the two-compartment dispersion model was also applied to the output concentration versus time profile of diazepam after bolus input of a radiolabeled tracer into the hepatic portal vein (n = 4), providing DN and CL(int) estimates of 0.251 +/- 0.093 and 135 +/- 59 ml min-1, respectively. Hence, all methods provided similar estimates for DN and CL(int). Furthermore, the magnitude of DN is similar to that determined for noneliminated substances such as erythrocytes, albumin, sucrose, and water. These findings suggest that the dispersion of diazepam in the perfused rat liver is determined primarily by the architecture of the hepatic microvasculature.     

Histamine H(3) receptor-mediated modulation of perivascular nerve transmission in rat mesenteric arteries

The rat mesenteric artery has been shown to be innervated by adrenergic vasoconstrictor nerves and calcitonin gene-related peptide (CGRP)-containing (CGRPergic) vasodilator nerves. The present study was designed to investigate the involvement of histamine H(3) receptors in the neurotransmission of perivascular adrenergic and CGRPergic nerves. The mesenteric vascular beds without an endothelium isolated from male Wistar rats were perfused with Krebs solution and perfusion pressure was measured. In preparations with resting tension, the selective H(3) receptor agonist (R)-α-methylhistamine (α-methylhistamine; 10-100nM) significantly reduced periarterial nerve stimulation (2-8Hz)-induced vasoconstriction and noradrenaline release in the perfusate without an effect on the vasoconstriction induced by exogenously injected noradrenaline (0.5, 1.0nmol). In preparations with active tone produced by methoxamine (2μM) and in the presence of guanethidine (5μM), the periarterial nerve stimulation (1, 2Hz)-induced vasodilator response was inhibited by α-methylhistamine (0.1-1μM) perfusion without affecting vasodilation induced by exogenously injected CGRP (5pmol). Clobenpropit (histamine H(3) receptor antagonist, 1μM) canceled the α-methylhistamine-induced decrease in the periarterial nerve stimulation-induced vasoconstriction and noradrenaline release and periarterial nerve stimulation-induced vasodilation. These results suggest that the stimulation of H(3) receptors located in rat perivascular nerves inhibits presynaptically the neurotransmission of not only adrenergic nerves, but also CGRP nerves, by decreasing neurotransmitters.     

适于研究药物相互作用的肝脏灌流模型

目的:改进和建立大鼠离体肝脏灌流模型.方法:采用Krebs-Henseleit 缓冲液(pH7.4)为港灌流液基液,含2%透析48h的牛血清白蛋白组分V、20%(V:V)洗过的人红细胞和0.3%葡萄糖.灌流速度1.5ml·min~(-1)·g~(-1),温度(37±0.5)℃,灌流压力1.7～1.33pKa.测定大鼠灌流过程中胆汁分泌量、耗氧量及灌流液pH、Na~ 、K~ 水平,并观察肝脏外观变化和组织切片的细胞形态学,评定肝脏功能.结果:本系统灌流中大鼠的各项考察指标正常,离体肝脏的存活力可达3h.结论:该模型适用于研究药物在肝脏代谢中的相互作用及其发生机制,也可用于研究某些药物特殊的代谢动力学特征.     

Griseofulvin absorption from different sites in the human small intestine

The site-dependent small-intestinal absorption pattern of griseofulvin was investigated in man. Griseofulvin was chosen as a model substance having extremely low water solubility and moderate lipid solubility. A conventional steady-state perfusion technique (triple-lumen tubing system with a 20 cm test segment) was applied. Dissolved griseofulvin (10.0 mg L^?1) was perfused (10 mL min^?1) during 160 min into different parts of the small intestine with the middle of the test segment between 85 cm and 270 cm beyond the teeth. Each of the ten healthy volunteers was examined twice with the test segment localized in different regions to allow for intraindividual comparisons. Mean drug absorption rates calculated from intestinal aspirate concentrations were similar in the two intestinal parts (proximal, 15.0 ± 5.9 μg (20cm min)^?1; distal, 16.2 ± 4.3 μg (20 cm min)^?1; mean ± SD). Absorption rate was strongly correlated to the amount of griseofulvin offered to the test segment per unit time. Extrapolating these findings it follows that an amount of griseofulvin, once dissolved, would be absorbed completely (>99%) along 100 cm of the small intestine. A significant, positive correlation between the rate of transmucosal fluid transport and the absorption rate of griseofulvin was observed in the distal parts investigated.     

Aluminum absorption by rat duodenum: further evidence of energy-dependent uptake

Duodenums of intact anaesthetised rats have been perfused with saline-bicarbonate buffer (pH 8.5), containing AlCl3, across a range of concentrations 0-300 microM. A linear relation between rate of uptake and perfusate Al concentration was found. The clearance of Al from the lumen was also studied with the mean perfusate concentration of 60 microM, in the presence of certain inhibitors. The clearance was significantly reduced by NaCN(1 mM) or dinitrophenol (0.1 mM) in the perfusate, and by prior perfusion with vanadate (10 microM) in saline. No effect was observed after colchicine (25 micrograms) was administered i.v. 3 h before perfusion, but vincristine (10 micrograms) given similarly reduced clearance significantly. Verapamil (25 micrograms/ml) in the perfusate caused a small, just significant, reduction in clearance. These data further characterise duodenal absorption of Al under physiological conditions and suggest that energy-dependent transport plays an important role in its uptake. Calcium channels may provide an additional entry site.     

Mechanism of nasal absorption of drugs I: Physicochemical parameters influencing the rate of in situ nasal absorption of drugs in rats

The effect of rate of perfusion, volume, pH of the perfusate, and partition coefficient of the drug on the rate of in situ nasal absorption in rats was examined. The studies showed that the rate constant for the nasal absorption of phenobarbital was independent of the rate of perfusion above a value of 2 mL/min. The nasal absorption of benzoic acid was found to depend on the pH of the perfusate with the benzoate anion being absorbed at a rate one-fourth of that of benzoic acid. The effect of lipid solubility on the extent of nasal absorption was studied using a series of barbiturates. The rate and extent of absorption was found to be dependent on the chloroform-water partition coefficient of the barbiturate. The effect of the volume of the perfusate on the absorption rate constant of phenobarbital, phenol red, tyrosine, and propranolol was studied. The data obtained showed that a linear relationship existed between the rate constants of absorption and the reciprocal of the volume of the perfusate. Using this in situ relationship it was possible to predict in vivo absorption rate constants for propranolol and L-tyrosine when volumes of 0.1 mL were administered. The calculated values for these compounds were found to be close to those determined in in vivo experiments. This indicates that the in situ technique can be used to predict in vivo absorption rate constants.     

环糊精包合作用对在体大鼠肠道P-糖蛋白药泵的影响

目的研究环糊精包合作用对P-糖蛋白底物药物体内吸收的影响。方法以P-糖蛋白底物盐酸小檗碱为模型药物,使用大鼠在体单向肠灌流装置,采用高效液相色谱法分别测定灌流液中盐酸小檗碱和酚红的浓度变化;研究2-羟丙基-β-环糊精(HP-β-CyD)对盐酸小檗碱肠道吸收的影响,以此评价环糊精包合作用对P-糖蛋白药泵的影响。结果盐酸小檗碱、盐酸小檗碱/HP-β-CyD物理混合物及盐酸小檗碱/HP-β-CyD包合物在大鼠空肠的吸收速率常数(Ka)分别为(0.45±0.029)、(0.70±0.087)、(2.39±0.119)×10-2·min-1,有效渗透系数(Peff)分别为(0.43±0.028)、(0.63±0.098)、(2.17±0.145)×10-3min·cm-1。HP-β-CyD对盐酸小檗碱的包合作用促进了大鼠肠道对盐酸小檗碱吸收。HP-β-CyD与盐酸小檗碱混合给药也能促进药物的吸收,但其作用远低于包合作用。结论环糊精的包合作用将有可能成为提高P-糖蛋白底物药物生物利用度的有效手段。