Influence of quinidine on the binding of [3H]-ouabain and [3H]-digoxin by human lymphocytes

Summary To explore the molecular basis of the glycoside-quinidine interaction, the in vitro effect of quinidine on the binding of [³H]-ouabain and [³H]-digoxin to Na⁺ K⁺ ATPase receptors on human mononuclear cells was investigated. The maximum [³H]-ouabain binding capacity was 45.7±9.4×10³ molecules/cell in pure lymphocyte preparations (n=8) and 75.5±7.3×10³ molecules/cell in mixtures of mononuclear cells (n=8). These parameters were not influenced by 10⁻⁵ M quinidine. In eight equilibrium experiments with pure lymphocytes, the dissociation constant of [³H]-ouabain increased from 0.79±0.26×10⁻⁸ M in the absence of 10⁻⁵ M quinidine to 1.56±0.74×10⁻⁸ M in its presence (p<0.01), indicating that the affinity of the drug was decreased. Similar findings were observed using mixed mononuclear cells. In five uptake and release experiments, quinidine decreased the association rate constant of [³H]-ouabain from 3.15±0.36×10⁴ M⁻¹×s⁻¹ to 2.01±0.37×10⁴ M⁻¹ s⁻¹ (p<0.01), whereas the dissociation rate constant was not affected. A therapeutic concentration of quinidine does not affect the number of glycoside receptors on lymphocytes, but it does appear to reduce fractional receptor occupancy by both [³H]-ouabain and [³H]-digoxin at lower tracer concentrations. This finding is compatible with the clinical observation that quinidine reduces the distribution volume of digoxin.     

Involvement of an influx transporter in the blood–brain barrier transport of naloxone

Naloxone, a potent and specific opioid antagonist, has been shown in previous studies to have an influx clearance across the rat blood–brain barrier (BBB) two times greater than the efflux clearance. The purpose of the present study was to characterize the influx transport of naloxone across the rat BBB using the brain uptake index (BUI) method. The initial uptake rate of [³H]naloxone exhibited saturability in a concentration‐dependent manner (concentration range 0.5 µM to 15 mM ) in the presence of unlabeled naloxone. These results indicate that both passive diffusion and a carrier‐mediated transport mechanism are operating. The in vivo kinetic parameters were estimated as follows: the Michaelis constant, K_t, was 2.99±0.71 mM ; the maximum uptake rate, J_max, was 0.477±0.083 µmol/min/g brain; and the nonsaturable first‐order rate constant, K_d, was 0.160±0.044 ml/min/g brain. The uptake of [³H]naloxone by the rat brain increased as the pH of the injected solution was increased from 5.5 to 8.5 and was strongly inhibited by cationic H₁‐antagonists such as pyrilamine and diphenhydramine and cationic drugs such as lidocaine and propranolol. In contrast, the BBB transport of [³H]naloxone was not affected by any typical substrates for organic cation transport systems such as tetraethylammonium, ergothioneine or L ‐carnitine or substrates for organic anion transport systems such as p‐aminohippuric acid, benzylpenicillin or pravastatin. The present results suggest that a pH‐dependent and saturable influx transport system that is a selective transporter for cationic H₁‐antagonists is involved in the BBB transport of naloxone in the rat. Copyright © 2010 John Wiley & Sons, Ltd.     

Fatty Acid–Binding Protein 5 Mediates the Uptake of Fatty Acids,but not Drugs,Into Human Brain Endothelial Cells

The purpose of this study was to examine the involvement of fatty acid–binding protein 5 (FABP5), a lipid-binding protein expressed at the blood-brain barrier (BBB), in fatty acid and drug uptake into human brain endothelial cells. Following transfection with siRNA against hFABP5, human brain endothelial cell (hCMEC/D3) uptake of lipophilic ligands with varying affinity to FABP5 was assessed with intracellular concentrations quantified by liquid scintillation counting, HPLC, or LCMS/MS. The in situ BBB transport of [³H]-diazepam was also assessed in wild type and FABP5-deficient mice. hFABP5 siRNA reduced FABP5 expression in hCMEC/D3 cells by 39.9 ± 3.8% (mRNA) and 38.8 ± 6.6% (protein; mean ± SEM), leading to a reduction in uptake of [¹⁴C]-lauric acid, [³H]-oleic acid, and [¹⁴C]-stearic acid by 37.5 ± 8.8%, 41.7 ± 11.6%, and 50.7 ± 13.6%, respectively, over 1 min. No significant changes in [¹⁴C]-diazepam, pioglitazone, and troglitazone uptake were detected following FABP5 knockdown in hCMEC/D3 cells. Similarly, no difference in BBB transport of [³H]-diazepam was observed between wild type and FABP5-deficient mice. Therefore, although FABP5 facilitates brain endothelial cell uptake of fatty acids, it has limited effects on brain endothelial cell uptake and BBB transport of drugs with lower affinity for FABP5.     

Lack of enantiomeric influence on the brain cytoprotective effect of ibuprofen and flurbiprofen

R(−) enantiomers of the 2-arylpropionic acid derivatives ibuprofen and flurbiprofen weakly inhibit cyclooxygenase (COX) activity. However, a possible cytoprotective effect has been proposed. The aim of the study is to investigate the possible mechanism of this effect. An in vitro hypoxia–reoxygenation model in rat brain slices was used (n = 6 rats per group). After reoxygenation, we measured LDH efflux (neuronal death), brain prostaglandin E₂ (PGE₂) concentration, interleukins (IL)-1β and 10, oxidative and nitrosative stress (lipid peroxides, glutathione, 3-nitrotyrosine, and nitrites/nitrates). Anti-COX activity was measured in human whole blood. Racemic, R(−), and S(+) enantiomers of ibuprofen and flurbiprofen were tested. All compounds had a cytoprotective effect with IC₅₀ values in the range of 10⁻⁵ M. R(−) enantiomers did not significantly inhibit brain PGE₂. The concentration of IL-1β was reduced by 53.1% by the racemic form, 30.6% by the S(+) and 43.2% by the R(−) enantiomer of ibuprofen. The IL-10 concentration increased significantly only with S(+)-flurbiprofen (33.1%) and R(−)-flurbiprofen (26.1%). Lipid peroxidation was significantly reduced by all three forms of flurbiprofen. Nitrite + nitrate concentrations were reduced by racemic, S(+), and R(−)-flurbiprofen. Peroxynitrite formation (3-nitrotyrosine) was significantly reduced by racemic and S(+)-ibuprofen. COX inhibition is not the main mechanism of cytoprotection for these compounds. Their influence on inflammatory mediators and oxidative and nitrosative stress could account for the potential cytoprotective effect of R(−) enantiomers.     

Assessment of Blood–Brain Barrier Permeability Using the <Emphasis Type="BoldItalic">In Situ</Emphasis> Mouse Brain Perfusion Technique

Purpose  To assess the blood–brain barrier (BBB) permeability of 12 clinically-used drugs in mdr1a(+/+) and mdr1a(−/−) mice, and investigate the influence of lipophilicity, nonspecific brain tissue binding, and P-gp-mediated efflux on the rate of brain uptake. Methods  The BBB partition coefficient (PS) was determined using the in situ mouse brain perfusion technique. The net brain uptake for 12 compounds, and the time course of brain uptake for selected compounds ranging in BBB equilibration kinetics from rapidly-equilibrating (e.g., alfentanil, sufentanil) to slowly-equilibrating (fexofenadine), was determined and compared. Results  There was a sigmoidal relationship in mdr1a(−/−) mice between the log-PS and clogD_7.4 in the range of 0–5. The brain uptake clearance was a function of both permeability and blood flow rate. The brain unbound fraction was inversely proportional to lipophilicity. Alfentanil achieved brain equilibrium approximately 4,000-fold faster than fexofenadine, based on the magnitude of PS×fu,brain. Conclusions   In situ brain perfusion is a useful technique to determine BBB permeability. Lipophilicity, ionization state, molecular weight and polar surface area are all important determinants for brain penetration. The time to blood-to-brain equilibrium varies widely for different compounds, and is determined by a multiplicity of pharmacokinetic factors.     

Possible involvement of cationic‐drug sensitive transport systems in the blood‐to‐brain influx and brain‐to‐blood efflux of amantadine across the blood–brain barrier

The purpose of this study was to characterize the brain‐to‐blood efflux transport of amantadine across the blood–brain barrier (BBB). The apparent in vivo efflux rate constant for [³H]amantadine from the rat brain (k_eff) was found to be 1.53 × 10^‐2 min^‐1 after intracerebral microinjection using the brain efflux index method. The efflux of [³H]amantadine was inhibited by 1‐methyl‐4‐phenylpyridinium (MPP⁺), a cationic neurotoxin, suggesting that amantadine transport from the brain to the blood across the BBB potentially involves the rat plasma membrane monoamine transporter (rPMAT). On the other hand, other selected substrates for organic cation transporters (OCTs) and organic anion transporters (OATs), as well as inhibitors of P‐glycoprotein (P‐gp), did not affect the efflux transport of [³H]amantadine. In addition, in vitro studies using an immortalized rat brain endothelial cell line (GPNT) showed that the uptake and retention of [³H]amantadine by the cells was not changed by the addition of cyclosporin, which is an inhibitor of P‐gp. However, cyclosporin affected the uptake and retention of rhodamine123. Finally, the initial brain uptake of [³H]amantadine was determined using an in situ mouse brain perfusion technique. Notably, the brain uptake clearance for [³H]amantadine was significantly decreased with the co‐perfusion of quinidine or verapamil, which are cationic P‐gp inhibitors, while MPP⁺ did not have a significant effect. It is thus concluded that while P‐gp is not involved, it is possible that rPMAT and the cationic drug‐sensitive transport system participate in the brain‐to‐blood efflux and the blood‐to‐brain influx of amantadine across the BBB, respectively. Copyright © 2014 John Wiley & Sons, Ltd.     

Effects of ketoprofen and ibuprofen on platelet aggregation and prostanoid formation in man

Objective: In the present randomized, four-way crossover study we determined the effects of two oral doses each of ketoprofen and ibuprofen on platelet aggregation and prostanoid formation in man. Methods: Twelve healthy female volunteers received for 2 consecutive days, followed by a 5-day drug-free interval, one of the following: ketoprofen 3 × 25 mg per day, or ketoprofen 3 × 50 mg per day, or ibuprofen 3 × 200 mg per day, or ibuprofen 3 × 400 mg per day. The response criteria, determined before and on the 2nd day of each treatment period, were: maximal platelet aggregation in response to 1.0 mmol ⋅ l^–1 arachidonic acid measured by the method of Born and Cross, thromboxane B₂ (TXB₂) concentration in platelet-rich plasma after aggregation measured by radioimmunoassay, and PGE-M, the index metabolite of total body prostaglandin E₂ (PGE₂) production, assessed by gas chromatography/tandem mass spectrometry using ¹⁸O₂-PGE-M as internal standard. Results: Platelet aggregation was significantly reduced by ketoprofen 3 × 25 mg per day (−57%) and ketoprofen 3 × 50 mg per day (−85%) as compared to control, whereas ibuprofen 3 × 200 mg per day (−3%) and ibuprofen 3 × 400 mg per day (−22%) had no significant effects. TXB₂ synthesis was significantly decreased by ketoprofen 3 × 25 mg per day (−72%), ketoprofen 3 × 50 mg per day (−97%) and ibuprofen 3 × 400 mg per day (−48%) as compared to control; ibuprofen 3 × 200 mg per day did not reduce TXB₂ formation significantly (−23%). All four treatments reduced 24-h urinary excretion of PGE-M significantly in the range of−39% (ketoprofen 3 × 25 mg per day) to −53% (ibuprofen 3 × 400 mg per day) without significant differences between treatments. Conclusion: Our data show that both ketoprofen dosages were more effective in inhibition of platelet aggregation and platelet thromboxane synthesis than ibuprofen in low or high dosage. Total body synthesis of the E-prostaglandins was inhibited by all drug schedules without significant differences between treatments. Received: 26 January 1996;/Accepted in revised form: 11 June 1996     

Alterations in Neuronal Transport but not Blood-Brain Barrier Transport are Observed during Gamma-Hydroxybutyrate (GHB) Sedative/Hypnotic Tolerance

Purpose To investigate if γ-Hydroxybutyrate (GHB) tolerance is mediated by alterations in GHB systemic pharmacokinetics, transport (blood brain barrier (BBB) and neuronal) or membrane fluidity.Materials and Methods GHB tolerance in rats was attained by repeated GHB administration (5.31 mmol/kg, s.c., QD for 5 days). GHB sedative/hypnotic effects were measured daily. GHB pharmacokinetics were determined on day 5. In separate groups, on day 6, in situ brain perfusion was performed to assess BBB transport alterations; or in vitro studies were performed (fluorescence polarization measurements of neuronal membrane fluidity or [³H]GABA neuronal accumulation).Results GHB sedative/hypnotic tolerance was observed by day 5. No significant GHB pharmacokinetic or BBB transport differences were observed between treated and control rats. Neuronal membrane preparations from GHB tolerant rats showed a significant decrease in fluorescence polarization (treated—0.320 ± 0.009, n = 5; control—0.299 ± 0.009, n = 5; p < 0.05). [³H]GABA neuronal transport V _max was significantly increased in tolerant rats (2,110.66 ± 91.06 pmol/mg protein/min vs control (1,612.68 ± 176.03 pmol/mg protein/min; n = 7 p < 0.05).Conclusions Short term GHB administration at moderate doses results in the development of tolerance which is not due to altered systemic pharmacokinetics or altered BBB transport, but might be due to enhanced membrane rigidity and increased GABA reuptake.Indranil Bhattacharya and Joseph J. Raybon have contributed equally to this work.     

Altered Brain Uptake of Therapeutics in a Triple Transgenic Mouse Model of Alzheimer’s Disease

Purpose

The purpose of this study was to systematically assess the impact of Alzheimer’s disease (AD)-associated blood–brain barrier (BBB) alterations on the uptake of therapeutics into the brain.

Methods

The brain uptake of probe compounds was measured in 18–20 month old wild type (WT) and triple transgenic (3×TG) AD mice using an in situ transcardiac perfusion technique. These results were mechanistically correlated with immunohistochemical and molecular studies.

Results

The brain uptake of the paracellular marker, [¹⁴C] sucrose, did not differ between WT and 3×TG mice. The brain uptake of passively diffusing markers, [³H] diazepam and [³H] propranolol, decreased 54–60% in 3×TG mice, consistent with a 33.5% increase in the thickness of the cerebrovascular basement membrane in 3×TG mice. Despite a 42.4% reduction in P-gp expression in isolated brain microvessels from a sub-population of 3×TG mice (relative to WT mice), the brain uptake of P-gp substrates ([³H] digoxin, [³H] loperamide and [³H] verapamil) was not different between genotypes, likely due to a compensatory thickening in the cerebrovascular basement membrane counteracting any reduced efflux of these lipophilic substrates.

Conclusion

These studies systematically assessed the impact of AD on BBB drug transport in a relevant animal model, and have demonstrated a reduction in the brain uptake of passively-absorbed molecules in this mouse model of AD.     

Monocarboxylate Transporter (MCT) Mediates the Transport of γ-Hydroxybutyrate in Human Kidney HK-2 cells

Purpose Previous studies in our laboratory have suggested that GHB may undergo renal reabsorption mediated by monocarboxylic acid transporters (MCT). The objectives of this study were to characterize the renal transport of GHB using HK-2 cells and the role of MCT in the renal transport of GHB. Materials and Methods Western blot was used to detect the protein expression of MCT1, 2, and 4. Cellular uptake and directional flux studies were conducted to investigate the transport of GHB and L-lactate. RNA interference assay was used to investigate the involvement of MCT isoforms in the transport of GHB. Results MCT1, 2 and 4 were present in HK-2 cells. The cellular uptake of L-lactate and GHB exhibited pH- and concentration-dependence (L-lactate: K _m of 6.5 ± 1.1 mM and V _max of 340 ± 60 nmol mg⁻¹min⁻¹; GHB: K _m of 2.07 ± 0.79 mM, V _max of 27.6 ± 9.3 nmol mg⁻¹min⁻¹, and a diffusional clearance of 0.54 ± 0.15 μl mg⁻¹min⁻¹), but not sodium-dependence. α-Cyano-4-hydroxycinnamate (CHC) competitively inhibited the uptake of GHB and L-lactate with inhibition constants (K _i) of 0.28 ± 0.1 mM, and 0.19 ± 0.03 mM, respectively. Using small-interference RNA (siRNA) for MCT1, the protein expression of MCT1 and the uptake of L-lactate and GHB were significantly decreased. The siRNA treatment of MCT2 in HK-2 cells inhibited the uptake of GHB by 17%, and the siRNA treatment of MCT4 demonstrated no inhibition of GHB uptake. GHB exhibited a directional flux across HK-2 monolayer from apical to basal chambers in the presence of a pH gradient of pH 6.0 to pH 7.4. Conclusion These data suggest that MCT1 represents an important transporter for GHB transport in renal tubule cells, responsible for the reabsorption of GHB in the kidney.     

Uptake of ANG1005, A Novel Paclitaxel Derivative, Through the Blood-Brain Barrier into Brain and Experimental Brain Metastases of Breast Cancer

Purpose

We evaluated the uptake of angiopep-2 paclitaxel conjugate, ANG1005, into brain and brain metastases of breast cancer in rodents. Most anticancer drugs show poor delivery to brain tumors due to limited transport across the blood-brain barrier (BBB). To overcome this, a 19-amino acid peptide (angiopep-2) was developed that binds to low density lipoprotein receptor-related protein (LRP) receptors at the BBB and has the potential to deliver drugs to brain by receptor-mediated transport.

Methods

The transfer coefficient (K_in) for brain influx was measured by in situ rat brain perfusion. Drug distribution was determined at 30 min after i.v. injection in mice bearing intracerebral MDA-MB-231BR metastases of breast cancer.

Results

The BBB K_in for ¹²⁵I-ANG1005 uptake (7.3?±?0.2?×?10^-3 mL/s/g) exceeded that for ³H-paclitaxel (8.5?±?0.5?×?10^-5) by 86-fold. Over 70% of ¹²⁵I-ANG1005 tracer stayed in brain after capillary depletion or vascular washout. Brain ¹²⁵I-ANG1005 uptake was reduced by unlabeled angiopep-2 vector and by LRP ligands, consistent with receptor transport. In vivo uptake of ¹²⁵I-ANG1005 into vascularly corrected brain and brain metastases exceeded that of ¹⁴C-paclitaxel by 4–54-fold.

Conclusions

The results demonstrate that ANG1005 shows significantly improved delivery to brain and brain metastases of breast cancer compared to free paclitaxel.     

Interaction of Ibuprofen and Other Structurally Related NSAIDs with the Sodium-Coupled Monocarboxylate Transporter SMCT1 (SLC5A8)

Purpose Sodium-coupled monocarboxylate transporter 1 (SMCT1) is a Na⁺-coupled transporter for monocarboxylates. Many nonsteroidal anti-inflammatory drugs (NSAIDs) are monocarboxylates. Therefore, we investigated the interaction of these drugs with human SMCT1 (hSMCT1). Methods We expressed hSMCT1 in a mammalian cell line and in Xenopus laevis oocytes and used the uptake of nicotinate and propionate-induced currents to monitor its transport function, respectively. We also used [¹⁴C]-nicotinate and [³H]-ibuprofen for direct measurements of uptake in oocytes. Results In mammalian cells, hSMCT1-mediated nicotinate uptake was inhibited by ibuprofen and other structurally related NSAIDs. The inhibition was Na⁺ dependent. With ibuprofen, the concentration necessary for 50% inhibition was 64 ± 16 μM. In oocytes, the transport function of hSMCT1 was associated with inward currents in the presence of propionate. Under identical conditions, ibuprofen and other structurally related NSAIDs failed to induce inward currents. However, these compounds blocked propionate-induced currents. With ibuprofen, the blockade was dose dependent, Na⁺ dependent, and competitive. However, there was no uptake of [³H]-ibuprofen into oocytes expressing hSMCT1, although the uptake of [¹⁴C]-nicotinate was demonstrable under identical conditions. Conclusions Ibuprofen and other structurally related NSAIDs interact with hSMCT1 as blockers of its transport function rather than as its transportable substrates.     

P-glycoprotein mediates brain-to-blood efflux transport of buprenorphine across the blood–brain barrier

The involvement of P-glycoprotein (P-gp) in buprenorphine (BNP) transport at the blood–brain barrier (BBB) in rats was investigated in vivo by means of both the brain uptake index technique and the brain efflux index technique. P-gp inhibitors, such as cyclosporin A, quinidine and verapamil, enhanced the apparent brain uptake of [³H]BNP by 1.5-fold. The increment of the BNP uptake by the brain suggests the involvement of a P-gp efflux mechanism of BNP transport at the BBB. [³H]BNP was eliminated with an apparent elimination half-life of 27.5 min after microinjection into the parietal cortex area 2 regions of the rat brain. The apparent efflux clearance of [³H]BNP across the BBB was 0.154 ml/min/g brain, which was calculated from the elimination rate constant (2.52 × 10^{? 2} min^{? 1}) and the distribution volume in the brain (6.11 ml/g brain). The efflux transport of [³H]BNP was inhibited by range from 32 to 64% in the presence of P-gp inhibitors. The present results suggest that BNP is transported from the brain across the BBB via a P-gp-mediated efflux transport system, at least in part.     

Pharmacological characterisation of human cerebral cortex somatostatin SRIF1 and SRIF2 receptors

Radioligand binding studies were performed in membranes of human cerebral cortex using [¹²⁵I]Tyr³-octreotide in the presence of 5 mM MgCl₂, [¹²⁵I]SRIF-14 ([¹²⁵I]Tyr¹¹-SRIF-14) and [¹²⁵I]CGP 23996 ([¹²⁵I]c[Asu-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Tyr-Thr-Ser]) both in the presence of 120 mM NaCl, to characterise the nature of the somatostatin (SRIF) receptors. The pharmacological profile of human brain SRIF recognition sites was compared with that of recombinant human SRIF₁ (sst₂-sst₃-sst₅) or SRIF₂ receptors (sst₁-sst₄) and with that of native rat sst₁, sst₂ and sst₄ receptors. [¹²⁵I]Tyr³-octreotide labelled binding sites in human cerebral cortex: B _max = 238 ± 36 fmol/mg protein and pK_d = 9.73 ± 0.08. The pharmacological profile of [¹²⁵I]Tyr³-octreotide labelled sites correlated very significantly with that of recombinant human sst₂ receptors (r = 0.98) and much less with those of recombinant human sst₃ (r = 0.65) or sst₅ receptors (r = 0.72). The correlation between [¹²⁵I]Tyr³-octreotide binding to native sst₂ receptors in human and rat cerebral cortex was also highly significant (r = 0.97). [¹²⁵I]SRIF-14 and [¹²⁵I]CGP 23996 binding (performed in the presence of 120 mM NaCl) in the human cerebral cortex identified very similar populations of sites B _max = 44 ± 7 and 36 ± 5 fmol/mg protein and pK_d = 9.44 ± 0.08 and 9.48 ± 0.10, respectively. The pharmacological profiles of the sites labelled with [¹²⁵I]SRIF-14 and [¹²⁵I]CGP 23996 correlated highly significantly with those of recombinant human sst₁ (r = 0.97–0.99) or sst₄ receptors (r = 0.91–0.94). Similarly, the correlations between [¹²⁵I]SRIF-14 or [¹²⁵I]CGP 23996 binding in human cortex and [¹²⁵I]SRIF-14 binding to native sst₁ sites in rat cerebral cortex were also highly significant (r = 0.97 and 0.94, respectively). Finally, the pharmacological profile of native rat lung sst₄ sites determined with [¹²⁵I]LTT-SRIF-28 ([Leu⁸,D-Trp²²,¹²⁵I-Tyr²⁵]SRIF-28) correlated with [¹²⁵I]SRIF-14 and [¹²⁵I]CGP 23996 binding in human cortex; r = 0.91 and 0.87, respectively. The present data show that in human cerebral cortex, [¹²⁵I]Tyr³-octreotide labels SRIF₁ receptor sites which are best characterised as of the sst₂ type, whereas [¹²⁵I]SRIF-14 and [¹²⁵I]CGP 23996 (both in the presence of 120 mM NaCl), label sites which fit almost equally well with sst₁ or sst₄ receptors and therefore are best described as of the SRIF₂ type. Under the conditions used, there was no evidence that either of these ligands would label sst₃ or sst₅ receptors in human cerebral cortex. Received: 8 August 1996 / Accepted: 8 November 1996     

Binding of some non-steroidal anti-inflammatory drugs to glucocorticoid receptors in vitro

The mechanism of action of non-steroidal anti-inflammatory drugs (NSAID) is felt to be via prostaglandin synthetase inhibition. Yet several clinical findings suggest these drugs may possess glucocorticoid agonist activity as well. To evaluate this possibility, a variety of non-steroidal antiinflammatory drugs was examined to determine whether they compete for rat kidney glucocorticoid receptor sites in vitro. Meclofenamic acid, MK 410 (an indomethacin analogue) and indomethacin were the most potent competitors requiring 7 × 10³-fold, 2 × 10⁴-fold and 3 × 10⁴-fold molar excess, respectively, to inhibit 50 per cent of the binding of 10 nM [³H]dexamethasone to kidney cytosol binding sites. The same drugs also inhibited the binding of [³H]dexamethasone in kidney slices requiring about 3-fold higher concentrations to achieve 50 per cent inhibition. At a 10⁵-fold molar ratio, sulfinpyrazone reduced cytosol binding to 60 per cent, phenylbutazone to 85 per cent and ibuprofen to 89 per cent of control levels; aspirin and naproxen did not compete even at this concentration. Similarly, prostaglandins failed to compete with [³H]dexamethasone for kidney receptors. Indomethacin inhibition of [³H]dexamethasone binding appeared competitive when analyzed by a double reciprocal plot. As expected, inhibition of [³H]dexamethasone cytosol binding by indomethacin prevented the appearance of the nuclear complex. Of the NSAID examined, none competed for plasma binding of [³H]corticosterone except indomethacin which reduced the binding to 50 per cent of control at a 10⁵-fold molar excess. In conclusion, these studies support the possibility that some NSAID may possess intrinsic glucocorticoid agonist activity. Furthermore, they indicate the glucocorticoid receptor site is not as specific as once thought and suggest that non-steroidal compounds may potentially serve as glucocorticoid agonists or antagonists.     

In Vivo Transport of a Dynorphin-like Analgesic Peptide,E-2078, Through the Blood–Brain Barrier: An Application of Brain Microdialysis

In vivo transport through the blood–brain barrier (BBB) has been demonstrated for a dynorphin-like analgesic peptide, CH₃-[¹²⁵I]Tyr-Gly-Gly-Phe-Leu-Arg-CH₃Arg-D-Leu-NHC₂H₅ ([¹²⁵I]E-2078). A remarkable time-dependent increase in the distribution volume of [¹²⁵I]E-2078 in the brain parenchyma separated from blood vessels and capillaries was observed during a brain perfusion. The distribution volume of [¹²⁵I]E-2078 in the brain parenchyma after 20 min of perfusion was 2.18 ± 0.09 µl/g brain (mean ± SE) and was significantly greater than the distribution volume of [³H]inulin (0.994 ± 0.138 (µl/g brain), providing in vivo evidence for the penetration of [¹²⁵I]E-2078 into the brain parenchyma. Brain microdialysis was carried out to collect directly the brain interstitial fluid (ISF) during the brain perfusion of [¹²⁵I]E-2078. No metabolite of [¹²⁵I]E-2078 in the brain ISF was found by high-performance liquid chromatographic analysis of the brain dialysate. The concentrations of [¹²⁵I]E-2078 and [¹⁴C]sucrose in the brain ISF were estimated based on an in vitro evaluation of dialysis clearance. The concentration ratio of [¹²⁵I]E-2078 between the brain ISF and the brain perfusate was determined to be 2.92 × 10^–l ± 0.50 × 10^–l and was approximately 100 times higher than that of [¹⁴C]sucrose (2.71 × 10^–3 ± 1.43 × 10^–3), demonstrating transport of [¹²⁵I]E-2078 through the BBB in vivo. On the other hand, no remarkable difference in the cerebrospinal fluid (CSF)-to-perfusate concentration ratios of [¹²⁵I]E-2078 and [¹⁴C]sucrose was observed, indicating little contribution of the blood–CSF barrier (BCSF barrier) transport to the penetration of [¹²⁵I]E-2078 into the brain.     

Pharmacokinetics and pharmacodynamics of a premixed formulation of soluble and protamine-retarded insulin aspart

Objective: With the aim to obtain a premixed rapid-acting insulin with a serum insulin profile more closely resembling the endogenous meal-stimulated serum insulin profiles, a 30/70 (rapid/intermediate-acting) premixed suspension of the rapid-acting insulin analogue insulin aspart (BIAsp30) was compared with a similar premixed suspension of biphasic human insulin 30/70 (BHI30) after a single subcutaneous injection. Methods: The study had a randomised, double-blind, two-period crossover design. Twenty-four healthy male subjects received a single subcutaneous dose of either 0.2 U · kg⁻¹ bodyweight of BIAsp30 or BHI30 on two study days. Results: BIAsp30 was absorbed faster than BHI30, as reflected in the area under the insulin concentration-time curve from 0 to 90 min after dosing [AUC_{(0–90 min)}]. This was significantly larger for BIAsp30 than for BHI30 (1403 ± 372 versus 752 ± 191 mU · l⁻¹ · min⁻¹ [mean ± SD]; P < 0.0001). Furthermore, the time to maximum serum insulin concentration (t_max) of BIAsp30 was approximately half the t_max of BHI30 (60 [45–70] versus 110 [90–180] min [median, interquartile range]; P=0.0001) and the maximum insulin concentration (C_max) was significantly higher for BIAsp30 than for BHI30 (23.4 ± 5.3 versus 15.5 ± 3.7 mU · l⁻¹ [mean ± SD]; P < 0.0001). The serum glucose profiles showed a significantly earlier onset of the glucose-lowering effect following BIAsp30 than following BHI30. Conclusions: The improved absorption properties of soluble insulin aspart in its premixed formulation provide a basis for a more efficient meal-related glucose control and immediate pre-meal delivery when compared with a similar human premixed insulin in the treatment of diabetes mellitus. Received: 22 November 1999 / Accepted in revised form: 7 April 2000     

Efavirenz Does Not Interact with the ABCB1 Transporter at the Blood—Brain Barrier

Purpose This work characterizes the interactions between efavirenz (EFV) and P-glycoprotein (P-gp/ABCB1) at the blood–brain barrier (BBB) and predicts the possible consequences on the brain uptake of coadministered P-gp substrates. Methods The uptake of EFV was measured in whole brains of rat and mdr1a^−/− and mdr1a^+/+ mice, and in GPNT cells (rat brain endothelial cell line) with and without P-gp inhibitors (PSC833, S9788, Quinidine). The effect of a single dose or multiple doses of EFV on the P-gp functionality was evaluated in vivo and in vitro by measuring the brain and cell uptake of digoxin, completed by the analysis of the P-gp expression at the rat BBB after repeated administrations of EFV. Results Inhibition of P-gp did not alter the uptake of EFV in rat brain and GPNT cells. The EFV brain/plasma ratio in mdr1a^−/− mice, lacking the expression of P-gp, was not different from that in mdr1a^+/+ mice. Moreover, a single dose of EFV did not modify the uptake of digoxin in rat brain and GPNT cells. Finally, the 3-day exposure of GPNT cells to EFV did not have any effect on the uptake of digoxin. Similarly, the 7-day treatment with EFV did not change the uptake of digoxin in rat brain nor the expression of P-gp at the BBB. Conclusion EFV is strongly distributed in the brain, but is neither a substrate nor an inhibitor of the P-gp at the blood–brain barrier. On the other hand, EFV did not induce P-gp, allowing to sustain the brain accumulation of associated P-gp substrates such as protease inhibitors. These findings make EFV suitable for combinations circumventing the brain HIV-1 residency.     

Glucose Partition Coefficient and Diffusivity in the Lower Skin Layers

Purpose This work aims to estimate the diffusivity and partitioning of glucose in the dermis and the viable epidermis of human skin. Methods The partition coefficient of glucose between phosphate-buffered saline and dermis, tape-stripped epidermis (TSE), stratum corneum (SC), and split-thickness skin, was measured in vitro using human cadaver skin. Glucose permeability across dermis and tape-stripped split-thickness skin (TSS) was measured using side-by-side diffusion cells. Glucose desorption from TSE and human epidermal membrane (HEM) was measured. All measurements were conducted at 32°C. Results The partition coefficient for glucose [mean ± SD (no. of samples)] was 0.65 ± 0.09 (n = 25) for dermis, 0.81 ± 0.06 (n = 10) for TSE, and 0.53 ± 0.12 (n = 9) for SC. Glucose diffusivity in dermis was calculated to be 2.64 ± 0.42 × 10⁻⁶ cm²/s (n = 14). Glucose diffusivities in the viable epidermis estimated from TSS permeation, TSE desorption, and HEM desorption were 0.075 ± 0.050 × 10⁻⁶ cm²/s (n = 5), 0.037 ± 0.018 × 10⁻⁶ cm²/s (n = 4), and 1.0 ± 0.6 × 10⁻⁶ cm²/s (n = 4), respectively. Conclusion The tissue/buffer partition coefficient of glucose in all skin layers was found to be less than unity, suggestive of excluded volumes in each layer. Glucose diffusivity in human dermis was found to be one third of its value in water, indicative of hindered diffusion related to the structural components of the tissue. A substantially lower value for glucose diffusivity in viable epidermis is suggested.