Transdermal Penetration of Vasoconstrictors—Present Understanding and Assessment of the Human Epidermal Flux and Retention of Free Bases and Ion-Pairs

Purpose. As reductions in dermal clearance increase the residence time of solutes in the skin and underlying tissues we compared the topical penetration of potentially useful vasoconstrictors (VCs) through human epidermis as both free bases and ion-pairs with salicylic acid (SA). Methods. We determined the in vitro epidermal flux of ephedrine, naphazoline, oxymetazoline, phenylephrine, and xylometazoline applied as saturated solutions in propylene glycol:water (1:1) and of ephedrine, naphazoline and tetrahydrozoline as 10% solutions of 1:1 molar ratio ion-pairs with SA in liquid paraffin. Results. As free bases, ephedrine had the highest maximal flux, Jmax = 77.4 ± 11.7 g/cm²/h, being 4-fold higher than tetrahydrozoline and xylometazoline, 6-fold higher than phenylephrine, 10-fold higher than naphazoline and 100-fold higher than oxymetazoline. Stepwise regression of solute physicochemical properties identified melting point as the most significant predictor of flux. As ion-pairs with SA, ephedrine and naphazoline had similar fluxes (11.5 ± 2.3 and 12.0 ± 1.6 g/cm²/h respectively), whereas tetrahydrozoline was approximately 3-fold slower. Corresponding fluxes of SA from the ion-pairs were 18.6 ± 0.6, 7.8 ± 0.8 and 1.1 ± 0.1 respectively. Transdermal transport of VC's is discussed. Conclusions. Epidermal retention of VCs and SA did not correspond to their molar ratio on application and confirmed that following partitioning into the stratum corneum, ion-pairs separate and further penetration is governed by individual solute characteristics.     

Pharmacokinetics of the Transdermal Reservoir Membrane System Delivering β-Estradiol: In Vitro/In Vivo-Correlation

Purpose. The aim of our study was to investigate the high fluctuations of Estradiol (E₂) plasma levels transdermally delivered in postmenopausal women by a commercially available membrane controlled reservoir system (MCRS). Methods. The transdermal E₂ flux either out of a complete MCRS or across its membrane out of defined ethanol water mixtures was determined, as well as E₂ plasma profiles in 6 postmenopausal women produced by a MCRS. Results. The transdermal in vitro E₂ flux rate out of a complete MCRS, claimed to deliver 25 g/day, increased steadily, reaching a maximum value of 2.06 ± 0.58 (g/h at 30 to 40 hours and decreased to a rate of about 0.5 (g/h from 60 to 90 hours. No statistically significant differences between plasma profiles calculated from the in vitro investigation and derived from a clinical study could be identified. The E₂ flux in defined ethanol/water mixtures across MCRS-membrane, adhesive and skin layer increased with increasing ethanol concentrations up to a maximum of 227 ± 34 ng/cm²/h at an ethanol concentration of 62.5% (V/V) and decreased with further increase in the volume fraction of ethanol. Conclusions. In vitro as well as in vivo investigations showed high fluctuation of E₂ plasma profiles in postmenopausal women produced by the MCRS. These fluctuations are caused by a non-constant input rate of E₂ which may be due to changing ethanol concentrations in the reservoir of the MCRS.     

Rate Control in Transdermal β-Estradiol Reservoir Membrane Systems: The Role of Membrane and Adhesive Layer

Purpose. The aim of our study was to clarify the kinetic performance of a membrane controlled reservoir system (MCRS) for -estradiol (E₂) under in vitroconditions by determination of the role of membrane and adhesive layer on E₂flux control. Methods. E₂and ethanol fluxes across EVA membrane or membrane coated with adhesive from saturated solutions in defined ethanol/PBS mixtures were measured in the symmetric and asymmetric configuration. Physicochemical parameters of the EVA membrane were determined. Results. The E₂flux across the 9% EVA membrane steadily increased with increasing ethanol concentrations in both configurations, due to enhanced uptake of E₂by the polymer and increasing membrane diffusivity. Permeation across the EVA membrane coated with an adhesive layer in the symmetric and asymmetric configuration increased up to maximum values of 0.80 ± 0.14 (g × cm^–2× h^–1and 0.37 ± 0.02 g × cm^–2× h^–1, respectively, at 62.5% (v/v) ethanol. The fluxes then decreased with further increase in the volume fraction of ethanol due to a dramatically reduced permeability of the adhesive layer. For the asymmetric case, a linear dependence of E₂on ethanol fluxes was observed. Conclusions. The E₂flux from MCRS is strictly dependent on reservoir ethanol concentrations, whereas the adhesive layer represents the rate controlling barrier at high ethanol levels (>70% v/v).     

Biotransformation of Estradiol in the Human Keratinocyte Cell Line HaCaT: Metabolism Kinetics and the Inhibitory Effect of Ethanol

Purpose. The aim of our study was to investigate the kinetics of -estradiol (E₂) metabolism in the human keratinocyte cell line HaCaT and to estimate the effect of the potential inhibitor ethanol on the biotransformation reaction. Methods. The formation rates of estrone (E₁) in dependence on substrate concentrations were determined in HaCaT cells using tritium labelled E₂. Experiments were conducted with and without addition of dehydroepiandrosterone (DHEA) and ethanol. Possible toxic effects on the cells due to ethanol were investigated by cytotoxicity tests. Results. The metabolism of E₂ in HaCaT cells exhibited Michaelis-Menten kinetics with K_m and V_max values of 3.5 M and 216 pmol × mg^–1 protein × h^–1, respectively. The reaction was inhibited by DHEA and ethanol. The alcohol showed a reversible competitive inhibition mechanism for concentrations of 4 to 8% (v/v). Lower ethanol concentrations had no effect, whereas levels 10% significantly decreased cell viability leading to a different inhibition mechanism. Conclusions. The HaCaT cell line seems to be a suitable model for studying enzyme kinetics equivalent to the human skin. The concentration dependent inhibitory effect of ethanol observed in this cell line may be relevant for the transdermal E₂ application in patients.     

Nuclear Transport of Oligonucleotides in HepG2-cells Mediated by Protamine Sulfate and Negatively Charged Liposomes

Purpose. The aim of this study was to characterize the intracellular fate and nuclear uptake kinetics of oligonucleotides (ON) that were complexed with protamine sulfate (PS) and negatively charged liposomes at different ratios of ON to PS. Methods. Double-fluorescence labelling of ON and liposomal lipid was applied to simultaneously monitor the interaction as well as the individual fate of active agent and carrier upon intracellular delivery using confocal laser scanning microscopy (CLSM). A DNA-analogue of a 68-mer intramolecular double-stranded RNA:DNA-hybridoligo- nucleotide (chimeraplasts) with unmodified phosphate backbone was employed. This construct was condensed with PS and coated with a liposomal formulation (AVE-3 = artificial viral envelope). Results. PS-ON complexes and AVE-3-coated complexes with a defined composition were very effective in nuclear transport of ON for a ON:PS charge ratio of 1:3. Nucleus:cytosol fluorescence ratios peaked at about 10 hrs and started to decrease again at 21 hrs. Conclusions. AVE associates with PS-condensed ON, and this complex is able to be taken up by cells and to deliver ON to the nucleus. PS-ON complexes are released from the liposomal formulation, mainly as an extranuclear enzymatic degradation of the liposomal phospholipids. The results of the kinetic analysis can be used to optimize transfection protocols with ON in HepG2 cells.     

Biliary Excretion of 17β-Estradiol 17β-d-Glucuronide Is Predominantly Mediated by cMOAT/MRP2

Purpose. The mechanism for the biliary excretion of 17-estradiol170-d-glucuronide (E₂17G), a cholestatic metabolite of estradiol, isstill controversial. The purpose of the present study is to examine thetransport of E₂17G across the bile canalicular membrane. Methods. We examined the uptake of [³H]E₂17G by isolatedcanalicular membrane vesicles (CMVs) prepared from Sprague-Dawley (SD)rats and Eisai Hyperbilirubinemic rats (EHBR) whose canalicularmultispecific organic anion transporter/multidrug resistance associatedprotein 2 (cMOAT/MRP2) function is hereditarily defective. Also,in vivo biliary excretion of intravenously administered [³H]E₂17Gwas examined. Results. In CMVs prepared from SD rats, but not from EHBR, amarked ATP-dependent uptake of [³H]E₂17G was observed.Moreover, E₂17G competitively inhibited the ATP-dependent uptake of[³H]2,4-dinitrophenyl-S-glutathione (DNP-SG). In addition, nosignificant inhibitory effect of verapamil (100 M) and PSC-833 (5 M) onthe uptake of [³H]E₂17G was observed. In vivo, the biliary excretionof intravenously administered [³H]E₂17G was severely impaired inEHBR while the biliary excretion of [³H]E₂17G in SD rats wasreduced by administering a cholestatic dose (10 mol/kg) unlabeledE₂17G, but not by PSC-833 (3 mg/kg). Conclusions. The transport of E₂17G across the bile canalicularmembrane is predominantly mediated by cMOAT/MRP2.     

Ion-Pair Partition of Quaternary Ammonium Drugs: The Influence of Counter Ions of Different Lipophilicity,Size, and Flexibility

Purpose. The ion-pair partition of quaternary ammonium (QA) pharmacons with organic counter ions of different lipophilicity, size, shape and flexibility was studied to elucidate relationships between ion-pair formation and chemical structure. Methods. The apparent partition coefficient (P) of 4 QAs was measured in octanol/pH 7.4 phosphate buffer system by the shake-flask method as a function of molar excess of ten counter ions (Y), namely: mesylate (MES), acetate (AC), pyruvate (PYRU), nicotinate (NIC), hydrogenfumarate (HFUM), hydrogenmaleate (HMAL), p-toluenesulfonate (PTS), caproate (CPR), deoxycholate (DOC) and prostaglandin E₁ anion (PGE₁). Results. Based on 118 of highly precise logP values (SD< 0.05), the intrinsic lipophilicity (without external counter ions) and the ion-pair partition of QAs (with different counter ions) were characterized. Linear correlation was found between the logP of ion-pairs and the size of the counter ions described by the solvent accessible surface area (SASA). The lipophilicity increasing effect of the counter ions were quantified and the following order was established: DOC ~ PGE₁ CPR ~ PTS NIC ~ HMAL PYRU ~ AC ~ MES ~ HFUM. Analyzing the lipophilicity/molar ratio (QA:Y) profile, the differences in the ion-pair formation were shown and attributed to the differences in the flexibility/rigidity and size both of QA and Y. Conclusions. Since the largest (in average, 300 ×) lipophilicity enhancement was found by the influence of DOC and PGE₁ and considerable (on average 40×) increase was observed by CPR and PTS, it was concluded that bile acids and prostaglandin anions may play a significant role in the ion-pair transport of quaternary ammonium drugs and caproic acid and p-toluenesulfonic acid may be useful salt forming agents to improve the pharmacokinetics of hydrophilic drugs.     

Covalent Binding of a Bucillamine Derivative with Albumin in Sera from Healthy Subjects and Patients with Various Diseases

Purpose. To investigate the difference of pharmacokinetics of thiol-containing drugs in various disease states, we studied the covalent binding of SA3786, a bucillamine derivative, with proteins in patient serum compared with that in healthy serum. Methods. Sera from healthy volunteers and patients of various diseases were supplied by the Japanese Red Cross Kumamoto Hospital. For the formation of conjugate experiments, SA3786 was added to a final concentration of 7 × 10^–4M. After 6h incubation at 37°C, HPLC analysis of 5 1 aliquots of each sample was performed using a column of N-methylpyridinium polymer (4VP-Me). Results. The extent of HSA-SA3786 conjugate formation was found to be lower in the sera from healthy volunteers (control) than those from patients of various diseases. Especially high reactivity with SA3786 was observed in sera from rheumatic patients and hepatic patients. With the exception of the fraction of mercaptoalbumin (f_HMA), none of the parameters showed a good correlation with conjugate formation. Conclusions. The parameter f_HMA must be considered to be one of the most important factors in formation of conjugates between plasma protein and thiol compounds. However, other factors may be involved in addition to f_HMA although the nature of these factors is not clear.     

Apparent Lack of Mrp1-Mediated Efflux at the Luminal Side of Mouse Blood-Brain Barrier Endothelial Cells

Purpose. The purpose of this work was to determine mrp1-mediated efflux across the luminal membrane of endothelial cells at the blood-brain barrier (BBB) in mice. Methods. The transport of radiolabeled etoposide, 17-estradiol-D-17-glucuronide (E₂17G), vincristine, and doxorubicin across the BBB of mrp1(–/–) and wild-type mice was evaluated by in situ brain perfusion. Etoposide transport was also determined in P-glycoprotein-deficient mdr1a(–/–) mice perfused with both etoposide and mrp1 inhibitors like probenecid or MK571. Cerebral vascular volume was determined by co-perfusion with labeled sucrose. Results. Sucrose perfusion indicated that the vascular space was close to normal in all the studies, indicating that the BBB remained intact. The transport of etoposide, E₂17G, vincristine, and doxorubicin into the brain was not affected by the lack of mrp1. Trans-efflux studies in mrp1-deficient mice with etoposide and E₂17G confirmed that mrp1 was not involved in the efflux of these substrates across the BBB. There was also a significant P-gp-mediated efflux of etoposide in studies with P-glycoprotein-deficient mdr1a(–/–) mice. Perfusion of mdr1a(–/–) mice etoposide plus probenecid or MK571 did not affect the brain transport of etoposide. Conclusion. Efflux mediated by mrp1 does not seem to occur across the luminal membrane of the endothelial cells forming the mouse BBB.     

Involvement of Multidrug Resistance Associated Protein 1 (Mrp1) in the Efflux Transport of 17β Estradiol-D-17β-Glucuronide (E217βG) Across the Blood-Brain Barrier

Purpose.The purpose of present study is to investigate the involvement of multidrug resistance-associated protein 1 (Mrp1), Mrp2, and P-glycoprotein (Mdr1a) in the efflux transport of 17—estradiol-D-17-glucuronide (E₂17G) across the blood—brain barrier (BBB). Methods. The expression of Mrp1 and Mrp2 at the BBB was investigated by RT-PCR and Western blot analyses. The time profiles of the remaining radioactivity of [³H]E₂17G in the brain were compared in wild-type, Mdr1a/Mdr1b and Mrp1 knockout mice and normal and Mrp2-deficient mutant rats [Sprague-Dawley and Eisai hyperbilirubinemic rats (EHBR), respectively] after intracerebral microinjection. Results. RT-PCR and Western blot analyses revealed the expression of Mrp1 in isolated rat brain capillary; however, RT-PCR was unable to detect any expression of Mrp2. Significant elimination of E₂17G was observed in wild-type mice at a rate constant of 0.007 min^–1, which was significantly decreased (0.004 min^–1) in Mrp1 knockout mice. In contrast, there was no difference in the efflux of E₂17G from the brain in wild-type and Mdr1a/Mdr1b knockout mice and in normal and EHBR. No significant difference was observed in the accumulation of E₂17G by brain slices prepared from wild-type and Mrp1 knockout mice. Conclusion. Mrp1, but not Mrp2, is involved in the excretion of E₂17G at the BBB and provides a barrier function by extruding conjugated metabolites into the blood.     

Polyester Nanocapsules as New Topical Ocular Delivery Systems for Cyclosporin A

Purpose. Nanocapsules composed of an oily core (Migliol 840) (MG) surrounded by a poly--caprolactone (PECL) coat were evaluated as potential vehicles for the. topical ocular administration of cyclosporin A (CyA). Methods. A 2³ experimental factorial design was applied to optimize the coating of the oily nanodroplets by a solvent displacement tecnique and to encapsulate a high dose of CyA. The variables investigated were: volume of oil (MG), amount of polymer (PECL), and volume of the organic solvent (acetone) used to dissolve the polymer. Results. Nanocapsules had a mean size in the range of 210–270 nm, a negative zeta potential (between –55 and –60 mV) and a maximum loading capacity of 50% (CyA/PECL ratio). These highly loaded nano-capsules displayed a thick spongeous polymer coating around the oily nanodroplets. The corneal levels of CyA were up to 5 times higher for the encapsulated CyA than for the oily solution of CyA. In addition, these levels remained significantly higher than those of the control group (oily solution) for up to 3 days. Furthermore, the area-under-the-curve (AUC) values were significantly increased for the encapsulated CyA (319.98) with respect to the oily control (74.34). Conclusions. The CyA-loaded nanocapsules are shown to be interesting vehicles for the improvement of the ocular penetration of CyA.     

Pharmacokinetics and Biodistribution of Oligonucleotide Adsorbed onto Poly(isobutylcyanoacrylate) Nanoparticles After Intravenous Administration in Mice

Purpose. The goal of this study was to evaluate the ability of nanoparticles to be used as a targeted delivery system for oligonucleotides. Methods. Pharmacokinetic and tissue distribution were carried out in mice by measuring the radioactivity associated to the model oligothymidylate ³³P-pdT₁₆ loaded to poly(isobutylcyanoacryrate) (PIBCA) nanoparticles. In addition, we have used a TLC linear analyzer to measure quantitatively on a polyacrylamide gel electrophoresis, the amount of non degraded pdT₁₆ Results. Organ distribution study has shown that nanoparticles deliver ³³P-pdT₁₆ specifically to the liver reducing its distribution in the kidney and in the bone marrow. Nanoparticles could partially protect pdT₁₆ against degradation in the plasma and in the liver 5 min after administration, whereas free oligonucleotide was totally degraded at the same time. Conclusions. Nanoparticles protect oligonucleotides in vivo against degradation and deliver them to the liver.     

Transport Activity of Human MRP3 Expressed in Sf9 Cells: Comparative Studies with Rat MRP3

Purpose. Multidrug resistance-associated protein 3 (MRP3) was initially cloned as a hepatic transporter induced under cholestatic/hyperbilirubinemic conditions. In the present study, transport property of human MRP3 (hMRP3) was compared with that of rat MRP3 (rMRP3). Methods. Adenosine 5 triphosphate (ATP)-dependent uptake of several organic anions into the membrane vesicles isolated from the Sf9 cells expressing hMRP3 and rMRP3 was measured by rapid filtration technique. Results. ATP-dependent uptake of glucuronide conjugates, glutathione conjugates, and [³H]methotrexate (MTX) was stimulated by infection of cDNAs for hMRP3 and rMRP3. The mean (± SE) K_m values for the uptake of 17 estradiol 17-D-glucuronide ([³H]E₂17 G) by hMRP3 and rMRP3 were 42.9 ± 4.3 M and 33.4 ± 2.2 M, respectively. Although the K_i values of glucuronides on the uptake of E₂17G were similar in humans and rats, hMRP3 exhibited higher K_i values toward MTX. In addition, although glycocholate and taurolithocholate 3-sulfate (TLC-S) were transported by both hMRP3 and rMRP3, taurocholate was only transported to a significant degree by rMRP3. Moreover, the inhibitory effect of taurocholate and glycocholate on the transport of E₂17G was much more potent in rMRP3 compared to hMRP3. Conclusion. Collectively, the substrate specificity of hMRP3 resembles that of rMRP3 although differences were observed, particularly in bile acid transport.     

Ion-Pairing Interactions between 99MTc-Based Myocardial Imaging Agents and Oleic Acid

Purpose. The purpose was to test the hypothesis that ion-paired facilitated transport is of importance in successful myocardial uptake of cationic imaging complexes. In vitro ion-pairing interactions between oleic acid and seven cationic technetium-99m complexes based on the ligands l,2-bis[bis(2-ethoxyethyl) phosphino ethane] (tetrofosmin), l,2-bis(dimethyl phosphino ethane) (DMPE) and l,2-bis(diethyl phosphino ethane) (DEPE) has been studied. The complexes studied were: [^99mTc O₂ (tetrofosmin)₂]⁺ (commercially available as myocardial perfusion imaging kit, Myoview^®), [^99mTc O₂ (DMPE)₂]⁺, [^99mTc O₂ (DEPE)₂]⁺, [^99mTc C1₂ (DMPE)₂]⁺, [^99mTc C1₂ (DEPE)₂]⁺, [^99mTc (DMPE)₃]⁺ and [^99mTc (DEPE)₃]⁺. Methods. Ion-pairing interactions were monitored using a rotating diffusion cell containing a solid supported liquid membrane and by formation of lipid monolayers. Results. Depletion of complex from the donor phase into an isopropyl myristate model membrane was generally in proportion to distribution coefficient and transfer to the receptor compartment was in all cases very small. However, by the inclusion of 5%w/v oleic acid, which is used in myocardial metabolism, partitioning was enhanced by amounts which varied depending on the tendency to form complex/oleate ion-pairs. Transfer to the receptor compartment was increased for most complexes when given sufficient time to diffuse through the membrane. The complex [^99mTc O₂ (tetrofosmin)₂]⁺ appeared to form particularly stable ion-pairs with oleic acid. Monolayer formation also indicated ion-pairing interactions. Conclusions. The results suggested that whether or not a complex is taken up by the myocyte may depend on its ability to hitch a ride by ion-pairing with the myocytes energy source—a molecule of long chain fatty acid.     

Probe Calibration in Transient Microdialysis In Vivo

Purpose. We examine the theoretical basis for calibrating microdialysis probes in vivo for pharmacokinetic experiments in which the extracellular analyte concentrations vary in time. Methods. A software package, MICRODIAL, was used to simulate microdialysis for illustrative transient situations with linear concentration dependence. Results. For a constant distant extracellular analyte concentration, the calibration factor (extraction fraction, E _d) exhibits a mass transfer transient associated with the development of spatial concentration profiles within the tissue and the probe. Processes clearing the analyte from the extracellular fluid (ECF) strongly influence the rapidity of approach to steady-state and affect the magnitude of the steady-state calibration factor, E _d ^ss . For situations in which the distant ECF concentration varies in time as a result of exchange with the plasma compartment, different time profiles of the distant ECF and plasma concentrations yield different transient E _d . For the linear, transient cases examined, the area-under-the-curve (AUC _0-) time integral of the distant ECF concentration was found to be proportional to the outflow dialysate concentration-time integral with E _d ^ss being the proportionality constant. Conclusions. The options for calibrating microdialysis probes in solid tissues appear limited under non-steady state conditions; however, AUC integrals for linear systems may be determined by continuous microdialysis sampling and steady-state probe calibration approaches.     

Cellular Delivery of Oligonucleotides by Synthetic Import Peptide Carrier

Purpose. Inefficient cellular uptake and endosomal entrapment are among the obstacles impeding the therapeutic use of oligonucleotides (ONs). The objectives of this study are to investigate the feasibility of utilizing a synthetic import peptide as a drug carrier for cytoplasmic delivery of ONs and to study its transport mechanisms. Methods. A molecular conjugate consisting of a signal import peptide (IP) derived from Kaposi fibroblast growth factor (K-FGF) and a polycationic ON linker, polylysine (PL), was synthesized and complexed with 5 fluorescently-labeled ON. Complex formation was verified by spectral shift assay and cellular uptake of the ON complex was studied fluorometrically. Microscopic studies were performed to visualize the intracellular distribution of the ON. Results. Cells treated with the ON:IP-PL complex exhibited a dose-dependent increase in ON uptake over free ON-treated controls. The uptake of the complex was shown to occur via an energy-independent, non-endocytic, process since metabolic and endocytic inhibitors and low temperature did not prevent the uptake. Microscopic studies revealed a non-punctate fluorescence pattern, consistent with the non-endocytic transport process. Intense nuclear fluorescence was observed in cells treated with the complex but not with free ON, suggesting enhanced cytoplasmic delivery and nuclear accumulation of the ON by the conjugate. Efficient complex uptake was shown to require both the ON-binding moiety PL and the IP moiety. The delivery system was found to be non-toxic at the concentrations used. Conclusions. The peptide carrier was effective in promoting the cellular uptake of ON. The mechanism by which the peptide facilitates ON uptake appears to involve a direct translocation of ON via a non-endocytic process. The peptide carrier has the potential to overcome the problem of ON endosomal entrapment and degradation.     

Mechanism-Based Modeling of Adaptive Changes in the Pharmacodynamics of Midazolam in the Kindling Model of Epilepsy

Purpose. A mechanism-based model is proposed for the analysis of adaptive changes in the pharmacodynamics of benzodiazepines in vivo. Methods. The pharmacodynamics of midazolam was studied in the kindling model of experimental epilepsy. Concentration-EEC effect data from kindled rats and their controls were fitted to the operational model of agonism. A stepwise procedure was used, allowing changes in the parameters efficacy () and tissue maximum (E_m) either separately or in combination. The results were compared to data obtainedin vitro in a brain synaptoneurosomal preparation. Results. The relationship between midazolam concentration and EEC effect was non-linear. In kindled rats the maximum EEC effect was reduced by 27± 8.3µV from the original value of 94± 4.4µV. Analysis on the basis of the operational model of agonism showed that this decrease could be explained by a difference in the parameter system maximum (E_m) rather than efficacy (). In the in vitro receptor binding assay no changes in density, affinity or functionality of the benzodiazepine receptor were observed, consistent with the lack of a change in efficacy (). Conclusions. The operational model of agonism provides a mechanistic basis to characterise adaptive changes in the pharmacodynamics of midazolam.     

Sex Specificity in Methadone Analgesia in the Rat: A Population Pharmacokinetic and Pharmacodynamic Approach

Purpose. To quantify the extent to which a sex-specific dichotomy in the temporal evolution of the analgesic effect, after intravenous (i.v.) methadone injection in the rat, relates to the pharmacokinetics (PK) and pharmacodynamics (PD) that mediate the dose-to-effect pathway. Methods. Tail-flick analgesia was measured after i.v. methadone injection (0.35 mg/kg) in female (n = 16) and male (n = 16) Sprague-Dawley rats. The PK were evaluated in separate female (n = 56) and male (n = 56) rats after they had received the same dose of methadone i.v. (0.35 mg/kg). A bicompartmental model described the kinetics and a sigmoid E_max model-related drug effect vs. simulated concentrations (pharmacodynamics) at the times of effect measurement. All model parameters as well as interanimal and assay variabilities were estimated with a mixed-effects population method using the program NONMEM. Results. The area under the effect-time curve (AUCE_0-120) was (mean ± interanimal SD) 1859 ± 346 min in the females, which was significantly lower than the 4871 ± 393 min in the males (P < 0.0001). On the contrary, the profiles of concentration vs. time were higher in females and, therefore, corresponded inversely to the effect vs. time-relative magnitudes. The central volume of distribution, V1, was 1.94 ± 0.37 l/kg for female rats and 3.01 ± 0.33 l/kg for male rats. Also, the central clearance was 0.077 ± 0.006 l/min/kg and 0.102 ± 0.005 l/min/kg, respectively, for female and male rats. Both parameters differed significantly between sexes (P < 0.0001). The pharmacodynamic maximum observed effect parameter (E_max) was 37% ± 29% in female rats and 85% ± 16% in male rats, and these values were significantly different (P < 0.0001). The parameter for the concentration eliciting half of E_max (EC₅₀) was 24.1 ± 7.5 g/l in female rats and 20.3 ± 2.9 g/l in male rats, and the Hill-related exponent, , was 6.3 ± 3.9 in female rats and 5.5 ± 4.1 in male rats. These parameters did not differ significantly (at the P < 0.05 level). Conclusions. A sex-specific dichotomy in the methadone antinociceptive effect, in the rat, was not proportionally related to plasma concentrations. Each sex corresponded to a distinct subpopulation of the PK parameters and one of the pharmacodynamic parameters (E_max). When the course of a drug involves PK or PD subpopulations, PK/PD modeling can afford the safest prediction of the effect-time evolution for a particular dose.     

Molecular Mobility of Protein in Lyophilized Formulations Linked to the Molecular Mobility of Polymer Excipients,as Determined by High Resolution 13C Solid-State NMR

Purpose. The mobility of protein molecules in lyophilized protein formulations was compared with that of excipient molecules based on the spin-lattice relaxation time (T₁) of each molecule determined by high resolution ¹³C solid-state NMR. The relationship between molecular mobility and protein stability is discussed. Methods. Protein aggregation of lyophilized bovine serum --globulin (BGG) formulation containing dextran was measured by size exclusion chromatography. The T₁ of the BGG carbonyl carbon and dextran methin carbon in the formulation was determined by high resolution ¹³C NMR, and subsequently used to calculate the correlation time (_C) of each carbon. The spin-spin relaxation time (T₂) of BGG and dextran protons was measured by pulsed NMR spectrometry, and the critical temperature of appearance of Lorentzian relaxation due to liquid BGG and dextran protons (T_mc) was determined. Results. The _C of dextran methin carbon in BGG-dextran formulations exhibited a linear temperature dependence according to the Adam-Gibbs-Vogel equation at lower temperatures, and a nonlinear temperature dependence described by the Vogel-Tamman-Fulcher equation at higher temperatures. The temperature at which molecular motion of dextran changed was consistent with the T_mc. The _C of BGG carbonyl carbon exhibited a similar temperature dependence to the _C of the dextran methin carbon and substantially decreased at temperatures above T_mc in the presence of dextran. The temperature dependence of BGG aggregation could be described by the Williams-Landel-Ferry equation even at temperatures 20°C lower than T_mc. Conclusions. High resolution ¹³C solid-state NMR indicated that the molecular motion of BGG was enhanced above T_mc in association with the increased global segmental motion of dextran molecules.     

Long-Circulating Emulsions (Oil-in-Water) as Carriers for Lipophilic Drugs

Purpose. Rapid clearance of parenterally administered oil-in-water emulsions from blood by the reticuloendothelial system (RES), mainly macrophages of the liver and spleen, has been one of the major obstacles for delivering lipophilic drugs to cells other than those in the RES. The purpose of this study therefore is to overcome this problem and develop emulsions that will have prolonged blood circulation time. Methods. A series of amphipathic polyethylene-glycol (PEG) derivatives have been included as co-emulsifier into emulsions composed of Castor oil and phosphatidylcholine. The effect of amphipathic PEG on reducing the RES uptake and prolonging the blood circulation of the emulsion particles has been tested in vivo using mice as an animal model. Results. Inclusion of PEG derivatives such as Tween-80 or dioleoyl N-(monomethoxy-polyethyleneglycol succinyl)phosphotidylethanolamine (PEG-PE) into emulsions composed of Castor oil and phosphatidylcholine decreases the RES uptake and increases blood residence time of the emulsions. The activity of PEG derivatives in prolonging the circulation time of emulsions depends on the PEG chain length (PEG2000PEG5000>PEG1000, Tween-80) and the PEG density on emulsion surface. Conclusions. Inclusion of amphipathic PEG as emulsifier into oil-in-water emulsions is a very effective method to prolong the blood half life of the emulsions. Emulsions with long circulating half life in blood should be very useful as a delivery vehicle for lipophilic drugs.