Neighboring group-controlled hydrolysis: towards "designer" drug release biomaterials

To give the first demonstration of neighboring group-controlled drug delivery rates, a series of novel, polymerizable ester drug conjugates was synthesized and fully characterized. The monomers are suitable for copolymerization in biomaterials where control of drug release rate is critical to prophylaxis or obviation of infection. The incorporation of neighboring group moieties differing in nucleophilicity, geometry, and steric bulk in the conjugates allowed the rate of ester hydrolysis, and hence drug liberation, to be rationally and widely controlled. Solutions (2.5 x 10-5 mol dm-3) of ester conjugates of nalidixic acid incorporating pyridyl, amino, and phenyl neighboring groups hydrolyzed according to first-order kinetics, with rate constants between 3.00 +/- 0.12 x 10-5 s -1 (fastest) and 4.50 +/- 0.31 x 10- 6 s-1 (slowest). The hydrolysis was characterized using UV-visible spectroscopy. When copolymerized with poly(methyl methacrylate), free drug was shown to elute from the resulting materials, with the rate of release being controlled by the nature of the conjugate, as in solution. The controlled molecular architecture demonstrated by this system offers an attractive class of drug conjugate for the delivery of drugs from polymeric biomaterials such as bone cements in terms of both sustained, prolonged drug release and minimization of mechanical compromise as a result of release. We consider these results to be the rationale for the development of "designer" drug release biomaterials, where the rate of required release can be controlled by predetermined molecular architecture.     

The vinblastine binding site adopts high- and low-affinity conformations during a transport cycle of P-glycoprotein.

Conceptually one may envisage that substrate binding sites on the ABC transporter P-gp cycle between high- and low-affinity conformations in response to signals arising from nucleotide hydrolysis to effect active transport. A radioligand binding assay was used to characterize the interaction of [3H]vinblastine with P-gp and determine how drug binding site parameters are altered during a catalytic cycle of P-gp. In the absence of nucleotide, we show that [3H]vinblastine interacts with a single class of binding site with high affinity (K(d) = 80 +/- 18 nM). In the presence of the nonhydrolyzable ATP analogue AMP-PNP, the drug binding site was in a low-affinity conformation, manifest by a 9-fold increase in K(d) (K(d) = 731 +/- 20 nM). There was no alteration in the binding capacity, reflecting a complete shift in the high-affinity site to a low-affinity form. The posthydrolytic (Mg-ADP-V(i) bound) form of P-gp also exhibited low-affinity substrate binding (K(d) = 446 +/- 57 nM). Restoration of the high-affinity drug binding site conformation (K(d) = 131 +/- 32 nM) did not occur until release of phosphate from the posthydrolysis P-gp-Mg-ADP-P(i). complex. Our results suggest that alteration of the affinity of the vinblastine binding site involves only one nucleotide binding domain per transport cycle. The binding of ATP provides the signal to instigate this change, while release of phosphate post-ATP hydrolysis returns the transporter to its original state to complete the cycle.     

Drug Effects on the Release of Endogenous Acetylcholine In Vivo: Measurement by Intracerebral Dialysis and Gas Chromatography–Mass Spectrometry

Intracerebral microdialysis was combined with a sensitive and specific gas chromatographic-mass spectrometric assay to measure the release of endogenous acetylcholine in the rat striatum in vivo. In rats anesthetized with urethane (1.2 g/kg i.p.), the levels of striatal acetylcholine dialyzed into a Ringer's perfusate were: (a) reliably measurable only in the presence of physostigmine; (b) stable at between 3 and 8 h of perfusion (30-75 pmol/20 min in the presence of 75 microM physostigmine); (c) reduced by calcium-free Ringer's solution, tetrodotoxin (0.1 microM), and vesamicol (1.0 microM); and (d) increased by elevated potassium (100 mM), atropine (3-300 microM), and haloperidol (0.75 mg/kg i.p.). In conscious unrestrained rats, the spontaneous release of striatal acetylcholine was not altered significantly following the administration of urethane. The changes in acetylcholine release observed in this study are consistent with the known actions of some drugs or ionic conditions on striatal cholinergic neurotransmission and are evident under the condition of urethane anesthesia. The present results demonstrate the sensitivity and suitability of this method for monitoring endogenous striatal acetylcholine release in vivo.     

常咯啉,磷酸咯啶等药物对家兔心肌脂质易化钙离子转运的影响

已知心肌细胞膜微粒体脂质(CMML)能易化钙离子从水到脂相的转运。奎尼丁能抑制这一转运过程。为进一步证明其他带含氮碱性基团的心血管药物是否也能抑制CMML易化钙离子转运过程。本文选用了常咯啉(C),磷酸咯啶(P),奎尼下(Q),乌头碱(A),调微Ⅱ号(莨菪类,T),和非含氮碱性药物成脉安(V),研究了它们对这种易化钙转运过程的影响。结果表明C,P,Q和A在30μg/ml时对这一过程的抑制均超过70％。V和T在相同浓度时抑制作用分别为37.8％和微弱作用·C,P和Q的抑制作用可能是它们治疗作用的生化基础。V和T的药理机制可能与以上药物不同。     

Charge translocation by the Na+/K+ pump under Na+/Na+ exchange conditions: intracellular Na+ dependence

The effect of intracellular (i) and extracellular (o) Na+ on pre-steady-state transient current associated with Na+/Na+ exchange by the Na+/K+ pump was investigated in the vegetal pole of Xenopus oocytes. Current records in response to 40-ms voltage pulses from -180 to +100 mV in the absence of external Na+ were subtracted from current records obtained under Na+/Na+ exchange conditions. Na+-sensitive transient current and dihydroouabain-sensitive current were equivalent. The quantity of charge moved (Q) and the relaxation rate coefficient (ktot) of the slow component of the Nao+-sensitive transient current were measured for steps to various voltages (V). The data were analyzed using a four-state kinetic model describing the Na+ binding, occlusion, conformational change, and release steps of the transport cycle. The apparent valence of the Q vs. V relationship was near 1.0 for all experimental conditions. When extracellular Na+ was halved, the midpoint voltage of the charge distribution (Vq) shifted -25.3+/-0.4 mV, which can be accounted for by the presence of an extracellular ion-well having a dielectric distance delta=0.69+/-0.01. The effect of changes of Nai+ on Nao+-sensitive transient current was investigated. The midpoint voltage (Vq) of the charge distribution curve was not affected over the Nao+ concentration range 3.13-50 mM. As Nai+ was decreased, the amount of charge measured and its relaxation rate coefficient decreased with an apparent Km of 3.2+/-0.2 mM. The effects of lowering Nai+ on pre-steady-state transient current can be accounted for by decreasing the charge available to participate in the fast extracellular Na+ release steps, by a slowly equilibrating (phosphorylation/occlusion) step intervening between intracellular Na+ binding and extracellular Na+ release.     

Release of Purines from Postsynaptic Structures of Amphibian Ganglia

Isolated sympathetic paravertebral ganglia of the frog were incubated for 1 h with [3H]adenosine. Then, after washout of excess label, the contribution of pre- and post-synaptic activation on the release of 3H-labeled purines was studied. The ganglion was superfused with Ringer's solution at room temperature, and extracellular electrodes were used for stimulation and recording. Preganglionic stimulation enhanced overall release of 3H-labeled purines. At rest, the release of 3H-labeled purines per minute represented 0.62 +/- 0.02% of the total 3H-label in the ganglion, and this fraction increased depending on the frequency of orthodromic stimulation. Analyses of the effluent from resting and stimulated ganglia showed that in both cases the nonnucleotide fractions constituted greater than 97% of the total counts in the medium: adenosine (58.4 +/- 10.1%); inosine (31.7 +/- 12.9%); hypoxanthine (7.1 +/- 2.4%); and AMP, ADP, and ATP together (1.6 +/- 0.9%) (n = 11). Nucleotides were released, but their levels were not increased significantly during stimulation. Inclusion of ectophosphatase inhibitors slightly enhanced nucleotide release (from 1.1 +/- 0.5 to 1.8 +/- 0.7%; n = 5) but did not alter the amount of nucleosides. Hence, nucleosides are the main products released by the ganglion and do not arise from hydrolysis of extracellular ATP. Preganglionic stimulation enhanced release of labeled purines, which was frequency dependent from 1 to 20 Hz. Atropine (2 microM) and tubocurarine (150 microM) totally blocked the release of 3H-labeled purines associated with preganglionic stimulation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Independence of apical membrane Na+ and Cl- entry in Necturus gallbladder epithelium

Transepithelial fluid transport (Jv) and intracellular Na+ and Cl- activities (aNai, aCli) were measured in isolated Necturus gallbladders to establish the contribution of different proposed apical membrane entry mechanisms to transepithelial salt transport. In 10 mM HCO3- Ringer's, Jv was 13.5 +/- 1.1 microliter X cm-2 X h-1, and was significantly reduced by a low bicarbonate medium and by addition of amiloride (10(-3)M) or SITS (0.5 X 10(-3)M) to the mucosal bathing solution. Bumetanide (10(-5)M) was ineffective. Bilateral Na+ removal abolished Jv. The hypothesis of NaCl cotransport was rejected on the basis of the following results, all obtained during mucosal bathing solution changes: during Na+ removal, aNai fell 4.3 times faster than aCli; during Cl- removal, aCli fell 7.5 times faster than aNai; amiloride (10(-3) M) reduced aNai at a rate of 2.4 +/- 0.3 mM/min, whereas aCli was not changed; bumetanide (10(-5) M) had no significant effects on Jv or aCli. The hypothesis of Na-K-Cl cotransport was rejected for the same reasons; in addition, K+ removal from the mucosal bathing solution (with concomitant Ba2+ addition) did not alter aNai or aCli. The average rate of NaCl entry under normal transporting conditions, estimated from Jv, assuming that the transported fluid is an isosmotic NaCl solution, was 22.5 nmol X cm-2 X min-1. Upon sudden cessation of NaCl entry, assuming no cell volume changes, aNai and aCli should fall at an average rate of 4.8 mM/min. To compare this rate with the rates of Na+ and Cl- entry by ion exchange, the Na+ or Cl- concentration in the mucosal bathing solution was reduced rapidly to levels such that electroneutral cation or anion exchange, respectively, should cease. The rate of Na+ or Cl- entry before this maneuver was estimated from the initial rate of fall of the respective intracellular ionic activity upon the mucosal solution substitution. aNai and aCli decreased at initial rates of 3.7 +/- 0.4 and 5.9 +/- 0.8 mM/min, respectively. The rate of fall of aNai upon reduction of external [Na] was not affected by amiloride (10(-3) M), and the rate of fall of aCli upon reduction of external [Cl] was unchanged by SITS (0.5 X 10(-3) M), which indicates that net cation or anion exchange was, in fact, abolished by the changes in Na+ and Cl- gradients, respectively. I conclude that double exchange (Na+/H+ and Cl-/HCO-3) is the predominant or sole mechanism of apical membrane NaCl entry in this epithelium.     

Electrogenic uptake of nucleosides and nucleoside-derived drugs by the human nucleoside transporter 1 (hCNT1) expressed in Xenopus laevis oocytes

The concentrative pyrimidine-preferring nucleoside transporter 1 (hCNT1), cloned from human fetal liver, was expressed in Xenopus laevis oocytes. Using the two-electrode voltage-clamp technique, it is shown that translocation of nucleosides by this transporter generates sodium inward currents. Membrane hyperpolarization (from -50 to -150 mV) did not affect the K(0.5) for uridine, although it increased the transport current approximately 3-fold. Gemcitabine (a pyrimidine nucleoside-derived drug) but not fludarabine (a purine nucleoside-derived drug) induced currents in oocytes expressing the hCNT1 transporter. The K(0.5) value for gemcitabine at -50 mV membrane potential was lower than that for natural substrates, although this drug induced a lower current than uridine and cytidine, thus suggesting that the affinity binding of the drug transporter is high but that translocation occurs more slowly. The analysis of the currents generated by the hCNT1-mediated transport of nucleoside-derived drugs used in anticancer and antiviral therapies will be useful in the characterization of the pharmacological profile of this family of drug transporters and will allow rapid screening for uptake of newly developed nucleoside-derived drugs.     

Rapid, fluorescence-based assay for microtiter plates to test drug influences on neutrophil transmigration through endothelial cell monolayers

Investigation of drug interactions between blood cells and endothelium is of high interest. The current study describes the development of a rapid fluorescence-based leukocyte transmigration system through endothelial cell monolayers for investigation of drug influences. To test the new assay, endothelial cells were cultured on microporous filters, pore size 3.0 microm, in 96-well-plates. Freshly isolated neutrophils were seeded on endothelial cell monolayers and transmigrated cells were measured after incubation for three hours. Migration of non-stimulated neutrophils through non-stimulated endothelial cell monolayer was used as control and set as 100%. The influence of the non-steroidal anti-inflammatory drug diclofenac was investigated. Assay precision tests were done using intraassay (within-day variability) and interassay (day-to-day variability) controls. Transmigration rate was decreased to 53 +/- 6.8% SD (diclofenac 0.7 microg/mL). Different concentrations showed a dose dependent effect (0.07 microg/mL: 97 +/- 9.5%, 7 microg/mL: 37 +/- 4.7%). Analysis of assay accuracy of the new 96-well-sized transmigration assay showed reliable results (coefficient of variation: intraassay 8.2 %; interassay 11.8%). In conclusion, this new, rapid, and sample saving 96-well-microtiter transmigration assay allows examination of drug influence on neutrophil migration through endothelial cell monolayers. Moreover, this assay can also be used for other cell-cell interactions.     

Controlled release from triple layer,donut-shaped tablets with enteric polymers

The purpose of this research was to evaluate triple layer, donut-shaped tablets (TLDSTs) for extended release dosage forms. TLDSTs were prepared by layering 3 powders sequentially after pressing them with a punch. The core tablet consisted of enteric polymers, mainly hydroxypropyl methylcellulose acetate succinate, and the bottom and top layers were made of a water-insoluble polymer, ethyl cellulose. Drug release kinetics were dependent on the pH of the dissolution medium and the drug properties, such as solubility, salt forms of weak acid and weak base drugs, and drug loading. At a 10% drug loading level, all drugs, regardless of their type or solubility, yielded the same release profiles within an acceptable level of experimental error. As drug loading increased from 10% to 30%, the drug release rate of neutral drugs increased for all except sulfathiazole, which retained the same kinetics as at 10% loading. HCl salts of weak base drugs had much slower release rates than did those of neutral drugs (eg, theophylline) as drug loading increased. The release of labetalol HCl retarded as drug loading increased from 10% to 30%. On the other hand, Na salts of weak acid drugs had much higher release rates than did those of neutral drugs (eg, theophylline). Drug release kinetics were governed by the ionization/erosion process with slight drug diffusion, observing no perfect straight line. A mathematical expression for drug release kinetics (erosion-controlled system) of TLDSTs is presented. In summary, a TLDST is a good design to obtain zero-order or nearly zero-order release kinetics for a wide range of drug solubilities.     

Raman spectroscopy and WAXS method as a tool for analysing ion-exchange properties of alginate hydrogels

Hydrogels are cross-linked three-dimensional macromolecular networks that contain a large fraction of water within their structure. One of the most important properties of alginate hydrogels, leading to their broad versatility, is their ability for controlled uptake, release and retention of molecules. This ability, in turn, is due to specific interactions of the macromolecular network with the diffusing or retained molecule. Raman spectroscopy has been employed to characterize the diffusion properties of solutes in hydrogels. Besides their application in the food sector, they are used in many biomedical, pharmaceutical and technical areas; for example, as a natural tissue or drug carriers. In the latter case, controlled release of drugs from a wound dressing is of particular interest-or ion exchange between the drug and the structure of the dressing. Raman active vibrations were used to show the areas responsible for the penetration of the model azo-dyes (based on non-genotoxic benzidine analogs) within Ca-alginate/carboxymethylcellulose Medisorb A wound dressing. In this case, the intensity of the stretching bands was used to obtain the concentration profiles of the model dye in alginate/carboxymethylcellulose gel (Medisorb A). The characteristic band at 1511 cm(-1) indicates that new band positions were observed following dye adsorption on wound dressing. The Raman spectra of alginate immersed for different times in Ringer's solution reveal peak shifts. Differences in peak shapes and the appearance of new bands are observed as the sodium content increased. Raman spectra give direct information on the exchange process. There are also new peaks appearing at 1034-1016 and 850 cm(-1) regions in the spectra after the release studies. This could, therefore, correspond to a partial bonding between sodium and oxygen atoms (the guluronic units originate a band at approximately 1025 cm(-1)). The aim of the examination in this paper also was to investigate the crystallinity index of Medisorb A wound dressing dyed (or undyed) and Medisorb A wound dressing after the release process in Ringer's solution (the crystallinity index is about 65%). In WAXS curves we can observed additional peaks (2theta at 32 degrees and 45 degrees ).     

Electrophysiological responses of frog skin to the peptides bombesin, caerulein, and physalaemin

Isolated skins from the frog Rana pipiens were mounted on Ussing-type chambers and bathed with Amphibian Ringer's solution on both sides. Electrical potential difference, resistance, and short-circuit current (SCC) were measured by using calomel electrodes, Ag-AgCl electrodes, Ringer's-agar bridges, and Tektronix digital multimeters. Under the conditions employed, SCC is a measure of net Na+ transport. The frog skin peptides bombesin, caerulein, and physalaemin were administered to the serosal side at concentrations of 0.5, 5.0, and 50 ng/ml. Control electrophysiological parameters were: potential difference, 23 +/- 2 mV; resistance, 738 +/- 59 ohms cm2; and SCC 32 +/- 3 microA/cm2. Although bombesin and caerulein had no significant, reversible effect on potential difference, resistance, or SCC, physalaemin significantly, and reversibly, depressed SCC by 22%. Caerulein did significantly depress SCC, but the response was not reversible.     

Detecting the cytogenetic effects in workers occupationally exposed to vincristine with four genetic tests

To study the human genetic damage induced by vincristine (VCR), the cytogenetic effects in workers occupationally exposed to vincristine were studied with micronucleus (MN) test, comet assay, hypoxantinepho-guanine phosphoribosyl-transferase (hprt) gene mutation assay and T-cells receptor (TCR) gene mutation assay. Fresh peripheral blood samples were collected from the workers and controls. Fifteen workers from a plant producing antineoplastic drug (vincristine) and 15 controls were matched according to age, gender and smoking. The results of MN test showed that the mean micronuclei rate (MNR) and mean micronucleated cells rate (MCR) in 15 workers were 17.80+/-1.88 per thousand and 13.67+/-1.56 per thousand, respectively, which were significantly higher than those (3.73+/-0.80 per thousand and 3.13+/-0.59 per thousand) in controls (P<0.01). It was found in the comet assay that the mean tail length (MTL) of 15 workers and 15 controls were 1.72+/-0.15 microm and 0.71+/-0.01 microm, respectively, there was significant difference between workers and controls for MTL (P<0.05), but the difference between the mean tail moment (MTM, 0.29+/-0.03) of 15 workers and MTM (0.17+/-0.05) of 15 controls was not significant (P>0.05). The results of hprt gene mutation assay showed that the average mutation frequency of hprt (Mf-hprt) in workers was 1.03+/-0.02 per thousand, which was significantly higher than that (0.87+/-0.01 per thousand) in controls (P<0.05). Meanwhile, the results of TCR gene mutation assay indicated that Mfs-TCR of workers and controls were 2.52+/-0.34 x 10(-4) and 1.51+/-0.11 x 10(-4), respectively, there was a significant difference between workers and controls (P<0.01). It is found in the results of our study that the genetic damage is detectable in 15 workers occupationally exposed to vincristine.     

Deuterium oxide and temperature effects on the properties of endplate channels at the frog neuromuscular junction

The effects of deuterium oxide (D2O) and temperature on the properties of endplate channels were studied in voltage-clamped muscle fibers from the frog Rana pipiens. Studies were performed at temperatures of 8, 12, 16, and 20 degrees C. The single channel conductance (gamma) and mean channel lifetime (tau) were calculated from fluctuation analysis of the acetylcholine-induced end-plate currents. The reversal potential was determined by interpolation of the acetylcholine-induced current-voltage relation. The mean reversal potential was slightly more negative in D2O Ringer's (-7.9 +/- 0.1 mV [+/- SEM]) compared with H2O Ringer's (-5.2 +/- 0.6 mV, P less than 0.01). The single channel conductance was decreased in D2O. This decrease was greater than could be accounted for by the increased viscosity of D2O solutions, and the amount of the decrease was greater at higher temperatures. For example, gamma was 38.4 +/- 1.3 pS (+/- SEM) in H2O Ringer's and 25.7 +/- 1.0 pS in D2O Ringer's for a holding potential of -70 mV at 12 degrees C. The mean channel lifetime was significantly shorter in D2O, and the effect was greater at lower temperatures. There was not a strong effect of solvent on the temperature dependence of gamma. On the other hand, the temperature dependence of the reciprocal mean channel lifetime, alpha (where alpha = 1/tau), was strongly dependent upon the solvent. The single channel conductances showed no demonstrable voltage dependence over the range of -90 to -50 mV in both solvents. The reciprocal mean channel lifetime showed a voltage dependence, which could be described by the relation alpha = B exp(AV). The slope A was not strongly affected by either temperature or the solvent. On the other hand, the intercept B was a strong function of temperature and was weakly dependent upon the solvent, with most values greater in D2O. The D2O effects on alpha were what would be expected if they were due to the properties of D2O as a solvent (solvent isotope effects), while the D2O effects on gamma must also include the exchange of D for H in the vicinity of the selectivity filter (primary and/or secondary kinetic isotope effects).     

Uncoupled basal sodium absorption and chloride secretion in prairie dog (Cynomys ludovicianus) gallbladder.

1. Prairie dog gallbladders mounted in a Ussing-type chamber and bathed with symmetrical Ringer's solutions exhibited a transepithelial resistance (Rt) of 51 +/- 5 omega cm2, a lumen negative potential difference (Vms) of 11.5 +/- 0.7 mV and a short-circuit current (Isc) of 6.9 +/- 0.3 microEq/hr/cm2. 2. Radioisotopic ion flux experiments revealed that the basal Isc of 6.9 +/- 0.3 microEq/hr/cm2 was mostly accounted for by net Na+ absorption of 3.2 +/- 0.5 microEq/hr/cm2 and net Cl- secretion of 2.9 +/- 0.3 microEq/hr/cm2. 3. In HCO3- free Ringer's, net Na+ flux was virtually abolished, net Cl- flux decreased by 50% and Isc was reduced by 77%. 4. 10(-3) M mucosal amiloride and DIDS reduced Isc by 28 and 24%, respectively. 5. Mucosal NaCl diffusion potentials indicated that the paracellular pathway was cation selective. 6. Thin section electron micrographs showed a single cell population in this epithelium suggesting that net Na+ absorption and Cl- secretion may emerge from the same cells. 7. We conclude that prairie dog gallbladder epithelium is an electrogenic tissue and, in contrast to gallbladders of most other species, simultaneously but independently absorbs Na+ and secretes Cl-.     

Antioxidant defense mechanisms of human mesothelioma and lung adenocarcinoma cells

The development of drug resistance of tumors is multifactorial and still poorly understood. Some cytotoxic drugs generate free radicals, and, therefore, antioxidant enzymes may contribute to drug resistance. We investigated the levels of manganese superoxide dismutase (Mn SOD), its inducibility, and its protective role against tumor necrosis factor-alpha and cytotoxic drugs (cisplatin, epirubicin, methotrexate, and vindesin) in human pleural mesothelioma (M14K) and pulmonary adenocarcinoma (A549) cells. We also studied other major antioxidant mechanisms in relation to oxidant and drug resistance of these cells. A549 cells were more resistant than M14K cells toward both oxidants (hydrogen peroxide and menadione) and all the cytotoxic drugs tested. M14K cells contained higher basal Mn SOD activity than A549 cells (28.3 +/- 3.4 vs. 1.8 +/- 0.3 U/mg protein), and Mn SOD activity was significantly induced by tumor necrosis factor-alpha only in A549 cells (+524%), but the induction did not offer any protection during subsequent oxidant or drug exposure. Mn SOD was not induced significantly in either of these cell lines by any of the cytotoxic drugs (0.007-2 microM, 48 h) tested when assessed by Northern blotting, Western blotting, or specific activity. A549 cells contained higher catalase activity than M14K cells (7.6 +/- 1.3 vs. 3.6 +/- 0.5 nmol O(2). min(-1). mg protein(-1)). They also contained twofold higher levels of glutathione and higher immunoreactivity of the heavy subunit of gamma-glutamylcysteine synthetase than M14K cells. Experiments with inhibitors of gamma-glutamylcysteine synthetase and catalase supported our conclusion that mechanisms associated with glutathione contribute to the drug resistance of these cells.     

The effect of vanadate on glucose transport and metabolism in rat small intestine

The effect of sodium orthovanadate on the absorption, transmural transport and metabolism of glucose was studied by perfusion of isolated loops of rat jejunum in vitro. The presence of 1 mM vanadate in the serosal medium diminished absorption from 539 +/- 19 (n = 12) to 246 +/- 19 (P less than 0.001) mumol/h per g dry weight and transmural transport from 333 +/- 17 to 14 +/- 19 (P less than 0.001) mumol/h per g dry weight, whereas glucose utilisation was unaffected. The rate of release of lactate into the serosal medium was also diminished from 168 +/- 14 to 75 +/- 5 mumol/h per g dry weight (P less than 0.001). The observed rates were linear with respect to time and vanadate was effective within 5 min. In contrast, the rate of release of lactate into the luminal perfusate was strongly enhanced. Moreover, the progress curve showed a positive transient with an apparent lag time of 18.0 +/- 0.3 min, during which the rate increased to a value 9.2-times that of the control. Under the final steady-state conditions, the ratio of mucosal to serosal lactate production was 5.2 +/- 0.2 compared with 0.25 +/- 0.06 for the control, so that the effect of vanadate was to reverse the vectorial disposition of lactate. The concentration dependence of the effect of vanadate on absorption and metabolism was similar to that observed for the inhibition by vanadate of Na+/K+-ATPase activity in mucosal homogenates. The results are discussed in terms of the dissipation of transmembrane Na+ gradients as a result of the inhibition of the Na+/K+-ATPase.     

Diffusion-limited binding explains binary dose response for local arterial and tumour drug delivery

Background:  Local drug delivery has transformed medicine, yet it remains unclear how drug efficacy depends on physicochemical properties and delivery kinetics. Most therapies seek to prolong release, yet recent studies demonstrate sustained clinical benefit following local bolus endovascular delivery.
Objectives:  The purpose of the current study was to examine interplay between drug dose, diffusion and binding in determining tissue penetration and effect.
Methods:  We introduce a quantitative framework that balances dose, saturable binding and diffusion, and measured the specific binding parameters of drugs to target tissues.
Results:  Model reduction techniques augmented by numerical simulations revealed that impact of saturable binding on drug transport and retention is determined by the magnitude of a binding potential, B_p , ratio of binding capacity to product of equilibrium dissociation constant and accessible tissue volume fraction. At low B_p (< 1), drugs are predominantly free and transport scales linearly with concentration. At high B_p (> 40), drug transport exhibits threshold dependence on applied surface concentration.
Conclusions:  In this paradigm, drugs and antibodies with large B_p penetrate faster and deeper into tissues when presented at high concentrations. Threshold dependence of tissue transport on applied surface concentration of paclitaxel and rapamycin may explain threshold dose dependence of in vivo biological efficacy of these drugs.     

Release of non-neuronal acetylcholine from the isolated human placenta is affected by antidepressants

Non-neuronal acetylcholine (ACh) is released from the human placenta into the extracellular space via organic cation transporters (OCTs). The present experiments investigated whether ACh release from epithelial cells is affected by drugs which are substrates of OCTs. The antidepressant drugs amitriptyline and doxepine were tested as both substances are not approved for pregnant women but frequently used. Release of ACh was measured in 10 min intervals over a period of 100 min. Test substances were added from t=50 min of incubation onwards. The effect was calculated by comparing the ACh release of the last three samples (t=70-100 min; B2) with that immediately before the application of the test substances (t=20-50 min; B1). The baseline ACh release amounted to 2.07+/-0.17 nmol/10 min (n=29; villus). Under control conditions a B2/B1 ratio of 0.78+/-0.02 was obtained. The following B2/B1 ratios were found, when therapeutic drugs were added: 0.54+/-0.04 (n=7; P<0.05) in the presence of 10 microM amitriptyline; 0.44+/-0.04 (10; P<0.01) in the presence of 10 microM doxepin; 0.73+/-0.04 (13) in the presence of 10 microM metformin; 0.76+/-0.06 (7) in the presence of 10 microM minoxidil; 0.63+/-0.03 (10) in the presence of 1 microM theophylline. The results demonstrate that antidepressants reduce the release of non-neuronal ACh at least in the human placenta, most likely by intracellular substrate competition at the polyspecific organic cation transporters (OCTs) but only at concentrations roughly 30-fold above the therapeutic range. Theophylline may also interfere with the release of non-neuronal ACh.