Intestinal Absorption of Stable Cyclic Glycylphenylalanine: Comparison with the Linear Form*

The absorption, especially the stability and transportability, of the cyclic peptide cyclic glycylphenylalanine (cyclo(Gly-Phe)) and the linear peptides glycylphenylalanine, glycyl-d -phenylalanine and phenylalanylglycine have been studied in rat small intestine. Linear peptides were degraded on the mucosal side and only glycyl-d -phenylalanine appeared on the serosal side. However, cyclo(Gly-Phe) was stable on the mucosal side and appeared on the serosal side. Furthermore, the absorption clearance of cyclo(Gly-Phe) was higher than that of glycyl-d -phenylalanine. In the presence of the peptidase inhibitor bestatin, the degradation of linear peptides was reduced and linear peptides appeared on the serosal side, but only phenylalanylglycine, which is transported by the oligopeptide transporter, was absorbed faster than cyclo(Gly-Phe). The absorption clearance of cyclo(Gly-Phe) was reduced as its concentration was increased from 125 μm to 500 μm . Furthermore, the absorption clearance of cyclo(Gly-Phe) at 125 μm was reduced at 4°C or in the presence of glycylsarcosine and cephalexin, which are transported by the oligopeptide transporter. These results indicated that cyclo(Gly-Phe) was stable enough to be absorbed and was transported in part by the oligopeptide transporter rather than completely by passive diffusion.     

Uptake of cyclic dipeptide by PEPT1 in Caco-2 cells: phenolic hydroxyl group of substrate enhances affinity for PEPT1

Uptake of cyclic dipeptides by H+/oligopeptide cotransporter (PEPT1) was studied in monolayers of the human intestinal cell line, Caco-2. The cyclic dipeptides studied were cyclic glycylphenylalanine (cyclo(Gly-Phe)), cyclic phenylalanylserine (cyclo(Phe-Ser)), cyclic seryltyrosine (cyclo(Ser-Tyr)) and cyclic glycyltyrosine (cyclo(Gly-Tyr)). These molecules have both peptide bonds and aromatic rings, and are similar in structure to cephalexin and cephadroxil, which are transported by PEPT1. Cellular uptake of these cyclic dipeptides was pH dependent, and was inhibited by the addition of PEPT1 substrates such as glycylsarcosine, indicating PEPT1-mediated transport. Michaelis constants (Km) for these cyclic dipeptides were cyclo(Ser-Tyr) < cyclo(Phe-Ser), and cyclo(Gly-Tyr) < cyclo(Gly-Phe), indicating that tyrosine possessing phenol moiety has higher affinity for PEPT1 than phenylalanine possessing benzen moiety. The Km for cephadroxil possessing phenol moiety was reportedly lower than that for cephalexin possessing benzen moiety. Therefore, it was concluded that the phenolic hydroxyl group of the substrate may enhance affinity for PEPT1.     

Concentration-dependent atypical intestinal absorption of cyclic phenylalanylserine: small intestine acts as an interface between the body and ingested compounds

Intestinal absorption of peptides in linear form has been studied extensively, but there is little knowledge of peptides in a cyclic form. In this report, intestinal absorption of cyclic phenylalanylserine (cyclo(Phe-Ser)), a precursor of gliotoxin, was studied in isolated rat small intestine as a model cyclic dipeptide. Absorption clearance (CLabs) decreased in the presence of glycylsarcosine, cephalexin or cephradine, substrates for H+/oligopeptide cotransporter (PEPT1). CLabs of cyclo(Phe-Ser) also decreased at 4 degrees C, thus indicating that cyclo(Phe-Ser) is in part transported by PEPT1. However, the Eadie-Hofstee plot of absorption revealed an atypical profile at lower concentrations of cyclo(Phe-Ser) (around 0.1 mM). Moreover, comparative experiments of absorptive and excretive transport showed that excretive transport from the serosal to mucosal side of isolated intestinal tissue at a 0.1 mM cyclo(Phe-Ser) was superior to absorptive transport from the mucosal side to the serosal side, and vice versa at a 1 mM cyclo(Phe-Ser). A kinetic model was constructed, in which cyclo(Phe-Ser) concentration for excretive transport was assumed to be at the binding site of excretive transporter, but not the unbound cytoplasmic concentration. These results as well as the results of kinetic analysis indicate that intestinal absorption consists of passive transport, carrier-mediated absorptive transport by PEPT1 and carrier-mediated excretive transport, resulting in atypical absorption. Although cyclic dipeptides have potentials as drugs, their intestinal absorption may be complex. The results of this study lead us to conclude that absorptive and excretive transport by the small intestine acts as an interface between the body and ingested compounds.     

The Predominant Contribution of Oligopeptide Transporter PepT1 to Intestinal Absorption of β-Lactam Antibiotics in the Rat Small Intestine

Although recent evidence suggests that certain β-lactam antibiotics are absorbed via a specific transport mechanism, its nature is unclear. To confirm whether peptide transport in the rat can be largely ascribed to the intestinal oligopeptide transporter PepT1, the transporter has been functionally characterized and its significance in the intestinal absorption of β-lactam antibiotics was evaluated. For evaluation of transport activity complementary RNA (cRNA) of rat PepT1 was synthesized in-vitro and expressed in Xenopus laevis oocytes. cRNA induced uptake of several β-lactam antibiotics and the dipeptide [¹⁴C]glycylsarcosine; this was specifically inhibited by various dipeptides and tripeptides but not by their constituent amino acids or by terra- or pentapeptides. The transport activity of PepT1 for β-lactam antibiotics correlated well with their in-vivo intestinal transport and absorption. Furthermore, mutual inhibitory effects on uptake were observed between glycylsarcosine and β-lactam antibiotics. Hybrid depletion of the functional expression of rat PepT1 in oocytes injected with rat intestinal epithelial total mRNA was studied using an antisense oligonucleotide corresponding to the 5′-coding region of PepT1. In oocytes injected with rat mRNA pre-hybridized with the antisense oligonucleotide against rat PepT1, the uptake of [¹⁴C]glycylsarcosine was almost completely abolished, whereas its uptake was not influenced by a sense oligonucleotide for the same region of PepT1. Similarly, the uptake of β-lactam antibiotics was also reduced by the antisense oligonucleotide against rat PepT1. These results demonstrate that the intestinal proton-coupled oligopeptide transporter PepT1 plays a predominant role in the carrier-mediated intestinal absorption of β-lactam antibiotics and native oligopeptides in the rat.     

Influence of oligopeptide transporter binding affinity upon uptake and transport of d-Asp(OBzl)-Ala and Asp(OBzl)-Sar in filter-grown Caco-2 monolayers

The oligopeptide transporter, which is responsible for the absorption of various di/tripeptides and several peptidomimetic drugs across the intestinal epithelia, is expressed in mature Caco-2 monolayers. Using certain enzymatically stable dipeptides containing either l- or d-aspartic acid at the amino terminus, we investigated the relationship between a side-chain modified dipeptide's degree of binding affinity for the apically expressed Caco-2 oligopeptide transporter and its ability to undergo uptake and/or apical-to-basal transport. Two β-esterified dipeptides, d-Asp(OBzl)-Ala and Asp(OBzl)-Sar, possess markedly different affinities for the Caco-2 oligopeptide transporter (IC₅₀=2.62±0.35 and 0.014±0.007 mM, respectively) as determined using a [¹⁴C]Gly-Sar cellular uptake displacement assay. d-Asp(OBzl)-Ala undergoes rapid internalization into Caco-2 monolayers (14.33±1.00 nmol/mg protein) during a 15-min uptake study; additionally, d-Asp(OBzl)-Ala is efficiently transported in the apical-to-basal direction across Caco-2 monolayers (14.41±0.91 nmol/h/cm²). Both uptake and transport of d-Asp(OBzl)-Ala are >90% inhibitable by the presence of a 20-fold molar excess of Gly-Pro in the apical chamber. Although Asp(OBzl)-Sar demonstrates a 187-fold lower IC₅₀ value than d-Asp(OBzl)-Ala, Asp(OBzl)-Sar does not achieve uptake or transport in parallel experiments. These data indicate that a side-chain modified, enzymatically stable dipeptide, d-Asp(OBzl)-Ala, is actively taken up into and transported across Caco-2 monolayers via the oligopeptide transporter. Additionally, the degree of affinity of a side-chain modified dipeptide for the Caco-2 oligopeptide transporter is not necessarily indicative of its ability to access the oligopeptide transporter-mediated uptake and transport pathway.     

Intestinal transport of bis(12)-hupyridone in Caco-2 cells and its improved permeability by the surfactant Brij-35

The objective of the present study was to elucidate the mechanisms of intestinal transport of bis(12)‐hupyridone (B12H) to predict its oral bioavailability. The effect of the B12H concentration and the contribution of the drug efflux transporters, P‐glycoprotein (P‐gp or ABCB1) and multidrug resistance‐associated proteins (MRPs or ABCC) on B12H absorption were measured and evaluated using the human intestinal epithelial Caco‐2 cell monolayer in the presence of transporter inhibitors. The results indicated that B12H was absorbed in a dose‐dependent manner at concentrations ranging from 132 to 264 µM . However, only apical efflux was observed in the directional transport studies for B12H below 88 µM (P_app(AP‐to‐BL): virtually zero; P_app(BL‐to‐AP): 1.591 ± 0.071 × 10^?5 cm s^?1). P‐gp and mixed P‐gp/MRP inhibitors significantly increased the absorptive transport (P_app(AP‐to‐BL)) to 0.619 ± 0.018 × 10^?5 and 0.608 ± 0.025 × 10^?5 cm s^?1, respectively, while decreasing secretory transport (P_app(BL‐to‐AP)) by >75%. A multiple‐MRP inhibitor, probenecid, increased the P_app(AP‐to‐BL) to 0.329 ± 0.015 × 10^?5 cm s^?1 while decreasing the P_app(BL‐to‐AP) by 50%. Another multiple‐MRP inhibitor, indomethacin, only modestly decreased the P_app(BL‐to‐AP) by ～30% and had no effect on the absorptive transport (P_app(AP‐to‐BL): virtually zero). In addition, the effect of various pharmaceutical excipients (e.g. Pluronic F‐68, Tween‐80 and Brij‐35) on B12H transport was determined and compared. Among them, Brij‐35 effectively enhanced B12H absorption at a concentration lower than its critical micelle concentration (CMC, 60 µM ). Therefore, Brij‐35 can be used as a potential enhancer to improve intestinal absorption of B12H for oral administration. Copyright © 2011 John Wiley & Sons, Ltd.     

β-Carboxylic acid esterified d-Asp-Ala retains a high affinity for the oligopeptide transporter in Caco-2 monolayers

d-Asp-Ala, a metabolically stable dipeptide, possesses a relatively high affinity for the Caco-2 oligopeptide transporter (IC₅₀ = 5.75 ± 0.09 mM) as demonstrated by its ability to compete with [¹⁴c]Gly-Sar in cellular uptake experiments. When the β-carboxylic acid of d-Asp-Ala is modified by esterification with a cyclohexyl group (d-Asp(OcHx)-Ala) or a benzyl group (d-Asp(OBzl)-Ala), the resulting compounds are still able to inhibit [¹⁴c]GlySar binding to the oligopeptide transporter, i.e., IC_so values for d-Asp(OcHx)-Ala and d-Asp(OBzl)-Ala were 2.80 ± 0.11 and 2.62 ±0.35 mM, respectively. HPLC analysis shows that both d-Asp-(OcHx)-Ala and d-Asp(OBzl)-Ala are fully resistant to degradation for up to 5 h when incubated in the apical media of confluent Caco-2 monolayers. These results demonstrate that it is possible to covalently modify the side chain of one amino acid in an enzymatically stabilized dipeptide with small, aromatic molecules while enabling them to retain their affinity for the oligopeptide transporter.     

Bioavailability and disposition kinetics of HI-6 in Beagle dogs

The absorption and disposition kinetics of HI-6 were determined in Beagle dogs given single doses (25 mg kg^?1) of the drug by the intravenous, intramuscular, and oral routes. Concentrations of the oxime in plasma and urine were measured by HPLC. A twocompartment open model was used to describe the disposition curve following intravenous drug administration while a one-compartment open model with first-order absorption adequately described the data following intramuscular or oral administration of the dose. Extravascular distribution of HI-6 was limited (V_ss 203 ml kg^?1) and the drug was eliminated rapidly after intravenous administration (t_1/2 46.5 min, MAT 55.4 min). Systemic clearance was 3.68 ml min^?1 × kg. A major fraction of the dose (63.7 per cent) was excreted in urine over a 24-h collection period. Following intramuscular drug administration, the absorption half-life (t_1/2(a), 5.3 min), MAT (17.1 min), C_max (70.37 μg ml^?1) and t_max (15.9 min) indicate that the drug was rapidly absorbed. Systemic availability was 83.43 per cent after oral drug administration, absorption was preceded by a lag time (23.2 min). The t_1/2(a) (41.5 min), MAT (81.6 min), C_max (4.30 μg ml^?1) and T_max (90.6 min) indicate somewhat delayed absorption. Systemic availability (11.38 per cent) and the fraction of dose excreted unchanged in the urine (9.3 per cent) show that the drug was poorly absorbed. The apparent half-life (58.0 min) and MRT (137.6 min) following oral administration were significantly longer (p <0.05) than following intravenous or intramuscular administration suggesting that the rate of absorption from the gastrointestinal tract decreases the elimination rate of the drug. In conclusion, HI-6 has limited distribution within the body, is rapidly eliminated mainly by renal excretion unchanged in the urine, and the bioavailability (i.e. rate and extent of absorption) of the drug varies with the route of administration.     

Mechanistic Insights into PEPT1-Mediated Transport of a Novel Antiepileptic, NP-647

The present study, in general, is aimed to uncover the properties of the transport mechanism or mechanisms responsible for the uptake of NP-647 into Caco-2 cells and, in particular, to understand whether it is a substrate for the intestinal oligopeptide transporter, PEPT1 (SLC15A1). NP-647 showed a carrier-mediated, saturable transport with Michaelis-Menten parameters K(m) = 1.2 mM and V(max) = 2.2 μM/min. The effect of pH, sodium ion (Na(+)), glycylsarcosine and amoxicillin (substrates of PEPT1), and sodium azide (Na(+)/K(+)-ATPase inhibitor) on the flux rate of NP-647 was determined. Molecular docking and molecular dynamics simulation studies were carried out to investigate molecular interactions of NP-647 with transporter using homology model of human PEPT1. The permeability coefficient (P(appCaco-2)) of NP-647 (32.5 × 10(-6) cm/s) was found to be four times higher than that of TRH. Results indicate that NP-647 is transported into Caco-2 cells by means of a carrier-mediated, proton-dependent mechanism that is inhibited by Gly-Sar and amoxicillin. In turn, NP-647 also inhibits the uptake of Gly-Sar into Caco-2 cells and, together, this evidence suggests that PEPT1 is involved in the process. Docking and molecular dynamics simulation studies indicate high affinity of NP-647 toward PEPT1 binding site as compared to TRH. High permeability of NP-647 over TRH is attributed to its increased hydrophobicity which increases its affinity toward PEPT1 by interacting with the hydrophobic pocket of the transporter through hydrophobic forces.     

The pharmacokinetic assessment of sodium cromoglycate

The plasma concentration of sodium cromoglycate (SCG) was measured in four healthy subjects by radioimmunoassay after a 4 mg intravenous dose and after inhaling from 20 mg capsules, and from 10 and 30 mg ml^?1 nebulizer solutions. The mean absorption constant (K₁) after inhalation was 0·43 h^?1. The mean elimination constant from the plasma (K_elim) after intravenous administration was 11·5 h^?1, and that after inhalation was similar. The apparent volume of distribution of SCG (V_dβ) was 0·2 litre kg^?1 and the mean plasma clearance was 0·35 litre h^?1kg^?1. The amount of SCG absorbed after inhalation varied according to the method of inhalation and dose. After the inhalation of powder from 20 mg capsules, 1·30–3·96 mg reached the plasma, after inhalation of SCG produced by nebulizing a 10 mg ml^?1 solution for 5 min at 10 psi using a Minineb nebulizer 0·19—0·31 mg reached the plasma and when the solution was increased to 30 mg ml^?1 the figure was 0·33—0·45 mg.     

Diverse effects of Ca2+ on the prostacyclin and corticotropin modulation of adenosine 3': 5'-monophosphate and steroid production in normal cat and mouse tumor cells of the adrenal cortex

Isolated cat adrenocortical cells exposed to corticotropin (ACTH) (2–50 μU) and prostacyclin (PGI₂) (10^?8 to 10^?4M) demonstrated dose- and time-dependent increases in cyclic AMP formation and steroid production. In the absence of extracellular Ca²⁺ ACTH (5 μU) did not augment cyclic AMP levels and steroid release. In contrast, Ca²⁺ deprivation did not affect the ability of PGI₂ (10^?6M) to augment cyclic AMP levels and only partially depressed PGI₂-induced steroid release, indicating that ACTH and PGI₂ affect the adenylate cyclase-cyclic AMP system differently. The distinctive effects of PGI₂ and ACTH were further demonstrated in mouse adrenal tumor (Y-1) cells, which responded to ACTH and cholera extertoxin (choleragen) (10^?9 M) with increases in cyclic AMP formation and steroidogenesis, whereas PGI₂ elicited a profound decrease in cyclic AMP levels and failed to enhance steroid production. The basis of these characteristic effects of ACTH and PGI₂on normal and adrenal tumor cells appears to be their particular actions on membrane receptors, which modulate cyclic AMP metabolism through different Ca²⁺ -mediated mechanisms.     

Relevance of PepT1 in the Intestinal Permeability and Oral Absorption of Cefadroxil

Purpose

To determine the contribution of intestinal PepT1 on the permeability and oral absorption of the β-lactam antibiotic drug cefadroxil.

Methods

The effective permeability (P _eff ) of cefadroxil was evaluated in wild-type and PepT1 knockout mice following in situ single-pass intestinal perfusions. The plasma concentration-time profiles of cefadroxil were also examined after oral gavage.

Results

The P _eff (cm/s) of cefadroxil in wild-type mice was 0.49?×?10^?4 in duodenum, 0.80?×?10^?4 in jejunum, 0.88?×?10^?4 in ileum and 0.064?×?10^?4 in colon. The P _eff (cm/s) in PepT1 knockout mice was significantly reduced in small intestine, but not in colon, as shown by values of 0.003?×?10^?4, 0.090?×?10^?4, 0.042?×?10^?4 and 0.032?×?10^?4, respectively. Jejunal uptake of cefadroxil was saturable (Km?=?2–4 mM) and significantly attenuated by the sodium-proton exchange inhibitor 5-(N,N-dimethyl)amiloride. Jejunal permeability of cefadroxil was not affected by L-histidine, glycine, cephalothin, p-aminohippurate or N-methylnicotinamide. In contrast, cefadroxil permeability was significantly reduced by glycylproline, glycylsarcosine, or cephalexin. Finally, PepT1 ablation resulted in 23-fold reductions in peak plasma concentrations and 14-fold reductions in systemic exposure of cefadroxil after oral dosing.

Conclusions

The findings are definitive in demonstrating that PepT1 is the major transporter responsible for the small intestinal permeability of cefadroxil as well as its enhanced oral drug performance.     

Interaction of green tea catechins with renal organic cation transporter 2

1.?Green tea extract (GTE) and EGCG have previously shown to increase the uptake of MPP⁺ into Caco-2 cells. However, whether GTE and its derivatives interact with renal basolateral organic cation transporter 2 (Oct2) which plays a crucial role for cationic clearance remains unknown. Thus, this study assessed the potential of drug-green tea (GT) catechins and its derivatives interactions with rat Oct2 using renal cortical slices and S2 stably expressing rat Oct2 (S2rOct2).

2.?Both GTE and ECG inhibited MPP⁺ uptake in renal slices in a concentration-dependent manner (IC₅₀?=?2.71?±?0.360?mg/ml and 0.87?±?0.151?mM), and this inhibitory effect was reversible. Inhibition of [³H]MPP⁺ transport in S2rOct2 by either GTE or ECG (IC₅₀?=?1.90?±?0.087?mg/ml and 1.67?±?0.088?mM) was also observed.

3.?The weak and reversible interactions of GTE and ECG with rOct2 indicate that consumption of GT beverages could not interfere with cationic drugs secreted via renal OCT2 in humans. However, the rise of therapeutic use of GTE and ECG might have to take into account the significant possibility of adverse drug–green tea catechins interactions which could alter renal organic cation drug clearance.     

Influence of leucine on intestinal baclofen absorption as a model compound of neutral α-aminoacids

The inhibitory effect of the essential α-aminoacid L-leucine on the intestinal absorption of the antispastic drug baclofen was examined by means of an in situ rat gut perfusion technique. When 0.5 mM baclofen solutions were perfused in the presence of increasing concentrations of the aminoacid (5–100 mM), the apparent absorption rate constant of the drug decreased as the initial leucine concentration increased. Higher leucine concentrations, however, did not completely abolish the absorption of the drug (at 100 mM of leucine, only 76% inhibition was observed). The interaction can be mathematically described as a complete competitive inhibition with a second component, K = 0.35 (±0.08)h^?1, K_i = 0.25 (±0.09)mM, AIC= ?97.02. In the light of some of the absorption features of the drug, however, the residual absorption of baclofen in the presence of high leucine concentrations should be attributed to another transport system not used by leucine. Apparent parameters characterizing absorption of leucine in the presence of baclofen (0.5mM) were V_m=61.02 (±5.46)mM h^?1; K_m=8.04 (±0.89)mM, and AIC=62.25. The results indicate that baclofen and leucine share some carriers in the intestinal absorption processes. Since leucine is an essential dietary aminoacid, and therefore a normal food component, this finding could be relevant in preventing interactions that would lead to a reduced oral bioavailability during baclofen therapy.     

Chronic terbutaline treatment desensitizes β-adrenergic inhibition of lymphocyte activation in healthy volunteers

1 A substantial body of evidence has accumulated that β-adrenoceptor mediated increases in human lymphocyte cyclic AMP can inhibit activation of resting lymphocytes. The aim of this study was to determine whether this effect might desensitize during chronic β-adrenoceptor agonist treatment. We assessed the effects of 2 weeks treatment with the β₂-adrenoceptor agonist terbutaline (3 × 5 mg day^?1 p.o.) on isoprenaline-induced inhibition of concanavalin A-evoked lymphocyte activation in nine healthy male volunteers. Lymphocyte activation was determined by [³H]-thymidine incorporation (as a measure of proliferation), and inositol phosphate formation was assessed in [³H]-myo-inositol prelabelled lymphocytes in the presence of 10 mm LiCl. 2 Terbutaline treatment caused a significant reduction in isoprenaline (1 nm – 10 μm )-induced increases in lymphocyte cyclic AMP content; the maximal increase was 14 ± 3 pmol/10⁶ cells before and 7 ± 2 pmol/10⁶ cells (n= 9, P < 0.05) after terbutaline treatment. 3 The mitogen concanavalin A (Con A, 1–32 μg ml^?1)-induced increase in inositol phosphate formation was significantly enhanced after terbutaline treatment (max. increase before treatment: 255 ± 25% above basal; after treatment 453 ± 16% above basal; n= 9, P < 0.001), while isoprenaline (1 nm – 10 μm )-induced inhibition of Con A (16 μg ml^?1)-evoked increases in inositol phosphate formation was significantly reduced after the terbutaline treatment (max. inhibition before treatment: 22 ± 4%; after treatment 9 ± 1%, n= 9, P < 0.01). 4 Con A (1.25 – 10 μg ml^?1)-induced increases in [³H]-thymidine incorporation into the lymphocytes (as a measure of proliferation) was not affected by the terbutaline treatment. On the other hand, isoprenaline (1 nm – 1 μm )-induced inhibition of Con A (5 μg ml^?1)-evoked lymphocyte proliferation (max. inhibition: 33 ± 7%, n= 9) was almost completely abolished after the terbutaline treatment. 5 We conclude that chronic treatment with terbutaline desensitizes lymphocyte β₂-adrenoceptors and, therefore, the inhibitory effect of cyclic AMP on lymphocyte activation.     

Conformation and complexation with metal ions of cyclic hexapeptides: cyclo (l-Leu-l-Phe-l-Pro)2 and cyclo (l-Cys(Acm)-l-Phe-l-Pro]2

Cyclic hexapeptides, cyclo (l -Leu-l -Phe-l -Pro)₂ and cyclo[l -Cys(Acm)-l -Phe-l -Pro]₂, in which Acm represents an acetoamide-methyl group, were synthesized, and the conformation and complexation with metal ions were investigated. Cooperation of the carbonyl groups of the Cys(Acm) side chains with those of the cyclic skeleton in complexation was especially examined. Cyclo(l -Leu-l -Phe-l -Pro)₂, which possesses no functional groups on side chains, was taken as the reference compound. ¹³C- and two-dimensional n.m.r. measurements revealed that cyclo(l -Leu-l -Phe-l -Pro)₂ and cyclo[l -Cys(Acm)-l -Phe-l -Pro]₂ took a C₂-symmetric conformation contaIntng cisl -Phe-l -Pro bonds in chloroform and acetonitrile. Both cyclic hexapeptides were found to complex selectively with Ba²⁺ and Ca²⁺ in acetonitrile. On complexation the conformation of either cyclic hexapeptide changed into a similar one. However, the binding constant of cyclo[l -Cys(Acm)-l -Phe-l -Pro]₂ was higher than that of cyclo(l -Leu-l -Phe-l -Pro)₂. The n.m.r. measurements showed that the amide carbonyl groups of Cys(Acm) side chains as well as those of cyclic skeleton in cyclo[l -Cys(Acm)-l -Phe-l -Pro], cooperatively bound the cations.     

Age related changes in the clearance and oral absorption of sodium cromoglycate in the developing albino rat

A dual radioisotope method was used to investigate the clearance and oral abso***rption of sodium cromoglycate. Radiolabelled sodium cromoglycate was administered orally at a dose of 100 mg kg ¹ (¹⁴ C-labelled) and simultaneously subcutaneously at a dose of 2 mg kg^?1 (³ H-labelled) to rat pups 5, 9, 14, 20, 29 and 75 days old. Blood concentrations of ¹⁴ C and ³ H were measured at intervals for 24 h after dosing. Since the compound is not metabolized the blood concentrations of ¹⁴ C were taken as a measure of the sodium cromoglycate absorbed orally and the blood concentrations of ³ H as a measure of the subcutaneously administered material. Using the area under the oral ¹⁴ C blood curve (AUC) as an index of bioavailability, the calculated bioavailability of sodium cromoglycate (692·9?945·9 min μg ml^?1) in 5, 9 and 14 day old pups was 4–8 times greater than that observed (61·0-118·8 min μg ml^?1) in 20, 29 and 75 day old pups. The blood clearance of sodium cromoglycate was increased four-fold in 75 day old animals (43·9 ml min^?1 kg^?1) and three-fold in 20 and 29 day old pups when compared to the clearance in 5, 9 and 14 day old pups. The clearance in 5, 9 and 14 day old pups was relatively constant (10·8 ? 9·9 ml min^?1 kg^?1). In rats less than 14 days old the systemic absorption of sodium cromoglycate after oral administration was 2–3 times greater (6·8-9·2%) than in rats aged 20, 29 or 75 days old (2·7-3·3%). The reduction in oral bioavailability of sodium cromoglycate as the pups grew older was, therefore, due to both an increased blood clearance and a decreased absorption of the compound.     

The In Vitro Permeability Coefficient and Short-Term Absorption Rates for Vinyl Toluene Using Human Cadaver Skin Mounted in a Static Diffusion Cell Model

Vinyl toluene is one of several methylstyrene monomers that provide improved performance in resins for specialty paints, hydrocarbon resins for adhesives, specialty polymers, and unsaturated polyester resins. The purpose of this study was to determine a permeability coefficient (Kp) and short-term absorption rate for vinyl toluene using human cadaver skin mounted in an in vitro static diffusion cell model with an exposure area of 0.64 cm². For the Kp determination, vinyl toluene was applied at a rate of 100 μL/cm² to 6 skin replicates representing 4 human subjects. Serial receptor fluid samples were collected at 1, 2, 4, 8, 12, 24, 36, and 48 h postapplication and analyzed for vinyl toluene by HPLC-UV. Based on the slope at steady-state (203.3 μg cm^?2 h^?1 ± 120.0 μg cm^?2 h^?1) and the concentration of the applied dose of vinyl toluene, taken as its density (894,600 μg/cm³), the Kp was calculated to be 2.27 × 10^?4 cm/h (±1.34 × 10^?4 cm/h). For the short-term absorption experiments, 12 skin replicates representing 3 human subjects were employed. Following 10- and 60-min exposures to a finite dose of vinyl toluene (10 μL/cm²), the respective short-term absorption rates were calculated to be 66.0 (±29.9) and 104.2 (±63.0) μg cm^?2 h^?1. These data provide industrial hygienists and safety personnel values to estimate the amount of vinyl toluene that may be absorbed via the dermal exposure route, given a variety of exposure scenarios, and the time it takes (skin absorption time) to reach a body burden equal to the Occupational Safety and Health Administrative permissible exposure level (OSHA PEL) or ACGIH TLV.     

Nonlinear elimination and hepatic concentration of conjugation- metabolite of valproate in guinea-pigs

The plasma clearance and metabolic rate characteristics of valproic acid (VPA) were studied using guinea-pigs placed on various (0.08-9 μmol ml^?1 = 11–1303 μg ml^?1) steady-state plasma concentrations (C_ss) by constant intravenous (i.v.) infusion. The total clearance (CL) was significantly decreased at plasma concentration of 0.61 μmol ml^?1 (88 μg ml^?1). The metabolic clearance of VPA was apparently biphasic. The maximum metabolic rate (V_max) and the Michaelis-Menten constant (K_m) for the primary (V_maxl, K_ml) and the secondary (V_max2, K_m2) pathways were V_maxl = 1.52 μmol min ^?1kg^?1, K_ml = 0.15 μmol ml^?1, V_max2 = 24.98 μmol min ^?1 kg^?1 and K_m2 = 11.70 μmol ml^?1, respectively. The K_ml value was within clinical therapeutic concentration range. The formation of conjugated VPA (cjVPA) metabolite in liver was shown to be saturable. Plasma protein binding of VPA was also nonlinear. The dose-dependent decrease in metabolic clearance was counterbalanced by the increased unbound fraction (f_u), resulting in a relatively constant apparent clearance of VPA over a wide concentration range. The hepatic concentration of VPA was not significantly different from the plasma unbound concentration, again over a wide concentration range. The biliary and hepatic concentrations of VPA were not significantly different; but the concentration ratio of cjVPA in bile compared with that of VPA in liver decreased against hepatic concentration of VPA, which suggests a saturable conjugation rate. The K_m value estimated from hepatic cjVPA production as a function of plasma VPA concentration was comparable with the K_ml value. These results implied that the primary metabolic parameters may describe the conjugation pathway which is nonlinear within the clinical therapeutic concentration range.     

Effects of morphine on the rat striatal dopamine metabolism.

Concomitant with total suppression of the spontaneous unitary discharges of neurons in the rat corpus striatum, intracarotidly injected morphine (5 mg/kg) was also found to increase the levels of dopamine, homovanillic acid and cyclic AMP by 80, 65 and 46 per cent respectively, measured 5 min after injection. This provides further support to the hypothesis that the nigrostriatal dopaminergic pathway is stimulated by acutely administered morphine. Morphine (10^?5-10^?3 M) did not alter the activity of striatal tyrosine aminotransferase. The drug, added in vitro (10^?6-10^?4 M) or by intracarotid injection (5 mg/kg) did not affect the activity of striatal tyrosine hydroxylase. Moreover, morphine (10^?4 M) did not interfere with the inhibitory effects of dopamine (10^?6-10^?4 M) on striatal tyrosine hydroxylase. However, it significantly potentiated the stimulatory effects of cyclic AMP on this enzyme. Morphine (10^?5-10^?4) was also found to have no effect on the spontaneous or K⁺-stimulated release of dopamine from striatal homogenate and synaptosomes. However, in the presence of 5 × 10^?5 M and 10^?4 M morphine, the uptake of dopamine by striatal homogenate was inhibited by 14 and 33 per cent respectively. With synaptosomal preparations, dopamine uptake was inhibited by 17 per cent in the presence of 10^?4 M morphine—the inhibition being competitive with dopamine with an apparent K_i of 0.41 mM. The inhibition of dopamine uptake caused by 10^?4 M morphine in either preparation was not reversed by the addition of 10^?4 M naloxone. It was concluded that the increase in dopaminergic activity following acute treatment of morphine is probably due to (1) prolongation of the effect of dopamine on the post-synaptic neurons resulting in increased production of cyclic AMP which in turn potentiates dopamine synthesis and (2) decrease in presynaptic cystosol dopamine which is normally a feedback inhibitor of tyrosine hydroxylase thus leading to increased synthesis of dopamine.