The unit impulse response procedure for the pharmacokinetic evaluation of drug entry into the central nervous system

The unit impulse response theory has been adapted to characterize the transport profile of drugs into the central nervous system (CNS). From the obtained input function, the cumulative plasma volume (V) cleared by transport into the CNS in time can be calculated. Simulation studies demonstrated that transport governed by passive diffusion resulted in a linear relationship between V and time, while the slope of the line, the blood-brain barrier (BBB) clearance, proved to be an adequate and model independent parameter to characterize drug transport into the CNS. The error in the result of the numerical procedure could be limited to less than 10% of the theoretically predicted value. Superposition of 5 or 10% random noise on simulated data did not result in significant differences between the calculated and theoretically predicted clearance values. Simulations of carrier-mediated transport resulted in nonlinear transport curves; the degree of nonlinearity, and thus the detectability, was dependent on the initial degree of saturation of the system, the rate of desaturation, as caused by drug elimination processes and the noise level on the data. In vivo experiments in the rat were performed, using atenolol, acetaminophen, and antipyrine as model drugs. Linear transport relationships were obtained for all drugs, indicating that transport was dependent on passive diffusion or a low affinity carrier system. BBB-clearance values were 7 +/- 1 microliters/min for atenolol, 63 +/- 7 microliters/min for acetaminophen and 316 +/- 25 microliters/min for antipyrine. These experiments validate the applicability of the presented technique in in vivo studies.     

Stereoselective analysis of the disposition of tosufloxacin enantiomers in man

Summary The pharmacokinetics of tosufloxacin enantiomers after oral administration of racemic tosufloxacin were examined in healthy volunteers. Only small differences were observed in time to peak concentration (2.6±0.3 [mean ± SEM] h for (+)-tosufloxacin vs 2.4±0.2 h for (–)-tosufloxacin), elimination half-life (3.61±0.24 h vs 3.49±0.23 h), and area under the curve (2.78±0.19 h·g/ml vs 2.87±0.19 h·g/ml); however, peak concentration (0.40±0.03 g/ml vs 0.44±0.03 g/ml), renal clearance (226±10 ml/min vs 202±10 ml/min), and urinary recovery (35.4±2.2% vs 32.4±1.9%) differed significantly between enantiomers.     

Analysis of Nonlinear Hepatic Clearance of a Cyclopentapeptide,BQ-123, with the Multiple Indicator Dilution Method Using the Dispersion Model

Purpose. To bridge in vitro, in situ and in vivo kinetic analyses of the hepatic clearance of a cyclopentapeptide, BQ-123, by using dispersion models that assume nonlinear pharmacokinetics. Methods. Rat livers were perfused by the multiple indicator dilution method with doses of BQ-123 ranging from 1-1000 g. The outflow dilution curves were fitted to a two-compartment dispersion model that was solved numerically by the finite difference method. Further, in vivo plasma concentrations of BQ-123 after bolus injection were analyzed with a hybrid physiological model that incorporates the hepatic dispersion model. Results. The calculated Michaelis-Menten constants (K_m = 12.0 M, V_max = 321 pmol/min/10⁶ cells, P_dif = 1.2 l/min/10⁶ cells) were comparable to those obtained previously from the in vitro isolated hepatocyte experiment (K_m = 9.5 M, V_max = 517 pmol/min/10⁶ cells, P_dif =1.1 l/min/10⁶ cells). The plasma concentrations of BQ-123 at doses of 1-25 mg/kg were explained well by the hybrid physiological model. Conclusions. These results suggest that carrier-mediated transport on the sinusoidal membrane was responsible for the in vivo hepatic elimination of BQ-123.     

Evaluation of Renal Tubular Secretion and Reabsorption of Levofloxacin in Rats

Purpose. Levofloxacin, a quinolone antibacterial drug, is a zwitterion at physiological pH. We examined the effect of cationic and anionic drugs on renal excretion of levofloxacin by means of in vivo clearance to characterize the mechanisms of renal excretion of this drug. Methods. In vivo clearance was studied in male Wistar albino rats. A bolus dose of 2.85 mg/kg of levofloxacin was administered, followed by a constant infusion of 7.08 g/min. Cimetidine, tetraethylammonium, or p-aminohippurate was administered as a bolus and incorporated into the infusion solution. After reaching steady state, urine and blood concentrations were measured, and pharmacokinetic parameters were calculated. Results. Renal clearance was 2.56 ± 0.42 ml/min in control, which accounted for 34% of the total body clearance. Renal clearance was significantly decreased to 0.83 ± 0.25 ml/min by cimetidine (p<.05), corresponding to 32% of the control value. The cationic drug, tetraethylammonium also reduced the renal clearance of levofloxacin, but the effect of the anionic drug, p-aminohippurate, was slight. The clearance ratio of levofloxacin, which was calculated by renal clearance divided by the plasma unbound fraction and the glomerular filtration rate, was 1.60 ± 0.38 in the control and it was decreased to 0.68 ± 0.17 and 1.11 ± 0.22 by cimetidine and tetraethylammonium, respectively. Conclusions. The present results suggest that the renal excretion of levofloxacin in rats involves tubular secretion and reabsorption, in addition to glomerular filtration, and that tubular secretion is inhibited by cimetidine.     

Renal Secretion of the Antiviral Nucleoside Analog AM188 Is Inhibited by Probenecid,p-Aminohippuric Acid,and Cimetidine in the Isolated Perfused Rat Kidney

Purpose. To investigate the effects of potential inhibitors of membrane transport on the tubular secretion of AM188, an antiviral guanosine analog, in the isolated perfused rat kidney (IPK). Methods. AM188 was administered to the IPK perfusate as a bolus/infusion regimen. In inhibitor groups, probenecid, p-aminohippuric acid (PAH), cimetidine, or nitrobenzylthioinosine was added to the perfusing medium. Results. In control IPKs, the ratio of renal clearance of AM188 (CL_R) to GFR was 7.7 ± 0.51 (mean ± SD). The CL_R/GFR ratio for AM188 was 6.20 ± 0.41*, 2.85 ± 0.20*, 1.45 ± 0.07*, and 0.80 ± 0.01* when the concentration of probenecid in perfusate was 10, 50, 100, and 1000 M, respectively (*p < 0.05 compared to control group); the ratio was 7.71 ± 0.38, 6.02 ± 0.42*, 1.71 ± 0.15*, and 0.91 ± 0.07* for the PAH group and 6.42 ± 1.70*, 5.33 ± 1.53*, 3.16 ± 0.81*, and 1.21 ± 0.20* for the cimetidine group when the concentrations were 10, 100, 1000 and 10,000 M, respectively; and the ratio was 5.33 ± 0.21* when the concentration of nitrobenzylthioinosine was 5 M. Conclusions. These results suggest that renal tubular secretion of AM188 involves organic anion and cation transport systems.     

Effect of rifampicin treatment on the kinetics of mexiletine

Summary To study the effects of enzyme induction on its pharmacokinetics, a single oral dose of the new antiarrhythmic agent mexiletine hydrochloride 400 mg was administered to 8 healthy volunteers before and after treatment with rifampicin 300 mg b.i.d. for ten days. The absorption and distribution of mexiletine were not changed after rifampicin, but its elimination half-life fell from 8.5±0.8 h (mean±SE) to 5.0±0.4 h (p<0.01), and its nonrenal clearance increased from 435±68 ml/min to 711±101 ml/min (p<0.01). The mean renal clearance of mexiletine did not change, but it showed an exponential correlation with urinary pH. The amount of unchanged mexiletine excreted in urine over two days decreased from 32±7 to 18±3 mg (p<0.01). The half-life of antipyrine fell from 11.8±0.4 to 5.5±0.3 h and its clearance increased from 40±3 ml to 74±3 ml/min (p<0.01). There was a significant (p<0.05) positive linear correlation between both the half-lives and the clearances of antipyrine and mexiletine. The clearances were positively correlated with serum -glutamyl transpeptidase. The results suggest that the dosage of mexiletine should be adjusted when enzyme inducing drugs are started or stopped during therapy with it.     

Changes in the excretion of endogenous glycine conjugate as a possible artifact in toxicological experiments

Changes in the urinary excretion of hippuric acid (HIA) and phenaceturic acid (PUA) as well as their metabolic precursors, i.e. benzoic (BA) and phenylacetic acid (PAA), in rats housed in glass metabolic cages for 4 days were monitored using gas-liquid chromatography. The amount of HIA excreted was 128±63 mol/kg for female and 79±43 mol/kg for male rats in the first 24 h and decreased to 11±7 mol/kg (p< 0.01) for female and 3.2±2.4 mol/kg (p< 0.001) for male rats on the 2nd day. These values remained nearly at the same level until the end of the experiment. The amount of PUA decreased from 48±12 mol/kg on the 1st day to 22±9 mol/kg (p< 0.05) on the 2nd day by male rats, whereas by the females the decrease from 30±9 mol/kg to 21±8 mol/kg was not significant. The decrease in the excretion of glycine conjugates was compensated by a parallel increase in the level of unconjugated BA and PAA.     

The kinetics of methotrexate distribution in spontaneous canine lymphosarcoma

A mathematical model is presented to simulate the time-dependent uptake of methotrexate in spontaneous canine lymphosarcomas in vivo.Blood flow rates in these tumors are high so that transport to the tumor is limited by cell membrane resistance. A significant amount of rapidly exchangeable methotrexate appears to exist in extracellular space loosely bound to proteins or cell membranes. Transmembrane drug transport follows Michaelis-Menten kinetics, with the maximum facilitated transport ranging from 0.002 to 0.007 g/min/ml for the separate tumors studied and a Michaelis constant for transport equal to 0.2 g/ml. This is in the range of Michaelis constants reported for normal tissues in rats in vivoand in several cell lines in vitro.     

Responses of human bone marrow progenitor cells to fluoro-ara-AMP,homoharringtonine, and elliptinium

Summary The cytotoxicity of the investigational anticancer drugs fluoro-ara-AMP, homoharringtonine, and elliptinium on normal human granulocyte-macrophage colony-forming units in culture (GM-CFU) was investigated using a bilayer soft agar system. For each drug, the dose-dependent survival curve on a semilogarithmic plot formed a straight line. The Do were: 0.51 g/ml (fluoro-ara-AMP), 0.004 g/ml (homoharringtonine) and 0.026 g/ml (elliptinium). The in vitro toxicity of drugs on bone marrow progenitor cells did not correlate with the relative myelosuppressive potency observed in vivo.     

Characterization of P-glycoprotein Mediated Transport of K02, a Novel Vinylsulfone Peptidomimetic Cysteine Protease Inhibitor,Across MDR1-MDCK and Caco-2 Cell Monolayers

Purpose. Here we characterized the transport properties of morpholine-urea-phenylalanine- homophenylalanine-vinylsulfone-phenyl (K02), a newly developed peptidomimetic cysteine protease inhibitor, across monolayers of P-gp-expressed MDR1 transfected MDCK cells (MDR1-MDCK) and Caco-2 cells. Methods. MDR1-MDCK, MDCK and Caco-2 cells, grown to confluence on Transwell insert membranes, were used to investigate transcellular transport of [¹⁴C]-K02. Results. The basolateral to apical (B-A) flux of 10 M [^I4C]-K02 across MDR1-MDCK cells was markedly greater than its apical to basolateral (A-B) flux (ratio = 39). This specific B-A transport was temperature dependent and saturable, with an apparent Michaelis-Menten constant and maximum velocity of 69.1 ± 19.5 M and 148.9 ± 16.3 pmol/min/cm², respectively. This B-A flux was significantly inhibited by cyclosporine (IC₅₀ = 17.1 ± 0.7 M), vinblastine (IC₅₀ = 75.9 ± 13.0 M) and verapamil (IC₅₀ = 236 ± 63 M). In Caco-2 cell monolayers, the B-A flux was reduced about 50% compared to that in MDR1-MDCK and the A-B flux was increased about 8-fold. The apparent Michaelis-Menten constant and maximum velocity values for the B-A transport were 71.8 ± 45.9 M and 35.3 ± 9.0 pmol/min/ cm². This B-A flux was also significantly inhibited by P-gp substrates/ inhibitors. Western blots showed that the P-gp expression in MDR1-MDCK cells was about 10-fold that in Caco-2 cells. Conclusions. K02 is transported by P-gp in both MDR1-MDCK and Caco-2 cells, and the in vitro interactions between K02 and various P-gp substrates may provide strategies to overcome the bioavailability barrier by intestinal P-gp. __     

Testosterone reinforcement: intravenous and intracerebroventricular self-administration in male rats and hamsters

Rationale Anabolic steroids are drugs of abuse. However, the potential for addiction remains unclear. Testosterone induces conditioned place preference in rats and oral self-administration in hamsters.Objectives To determine if male rats and hamsters consume testosterone by intravenous (IV) or intracerebroventricular (ICV) self-administration.Methods With each nose-poke in the active hole during daily 4-h tests in an operant conditioning chamber, gonad-intact adult rats and hamsters received 50 g testosterone in an aqueous solution of -cyclodextrin via jugular cannula. The inactive nose-poke hole served as a control. Additional hamsters received vehicle infusions.Results Rats (n=7) expressed a significant preference for the active nose-poke hole (10.0±2.8 responses/4 h) over the inactive hole (4.7±1.2 responses/4 h). Similarly, during 16 days of testosterone self-administration IV, hamsters (n=9) averaged 11.7±2.9 responses/4 h and 6.3±1.1 responses/4 h in the active and inactive nose-poke holes, respectively. By contrast, vehicle controls (n=8) failed to develop a preference for the active nose-poke hole (6.5±0.5 and 6.4±0.3 responses/4 h). Hamsters (n=8) also self-administered 1 g testosterone ICV (active hole:39.8±6.0 nose-pokes/4 h; inactive hole: 22.6±7.1 nose-pokes/4 h). When testosterone was replaced with vehicle, nose-poking in the active hole declined from 31.1±7.6 to 11.9±3.2 responses/4 h within 6 days. Likewise, reversing active and inactive holes increased nose-poking in the previously inactive hole from 9.1±1.9 to 25.6±5.4 responses/4 h. However, reducing the testosterone dose from 1 g to 0.2 g per 1 l injection did not change nose-poking.Conclusions Compared with other drugs of abuse, testosterone reinforcement is modest. Nonetheless, these data support the hypothesis that testosterone is reinforcing.     

Effect of Hydralazine on the Elimination of Antipyrine in the Rat

The concomitant administration of hydralazine with metoprolol or propranolol substantially increases the oral bioavailability of these beta-blockers, presumably via reduction of the first-pass effect. It has been suggested that this effect may be secondary to a decrease in the intrinsic clearance of propranolol, possibly by inhibition of oxidative metabolism. To examine the possibility that hydralazine alters oxidative metabolism in vivo, the effect of hydralazine on the pharmacokinetics of antipyrine was examined in the rat. The oral administration of hydralazine hydrochloride, 7.5 mg/kg, 15 min prior to antipyrine administration reduced antipyrine clearance from 9.66 ± 1.18 to 8.19 ± 0.76 ml/min/kg (P < 0.05). Hydralazine was observed to cause substantial hypothermia. The study was repeated in temperature-regulated animals and no alteration in antipyrine clearance was found. Two doses of hydralazine in temperature-regulated rats also failed to alter antipyrine clearance. Thus, it appears that the effect of hydralazine on antipyrine clearance is secondary to the hypothermic effect of hydralazine and not due to a direct inhibition of cytochrome P-450-mediated enzyme activity.     

Dose dependent pharmacokinetics of nitroglycerin after multiple intravenous infusions in healthy volunteers

Evaluation of the pharmacokinetics of nitroglycerin has been hindered in the past by the lack of specific and sensitive analytical procedures, and the unavailability of parenteral nitroglycerin and infusion sets which did not adsorb nitroglycerin. The purpose for this present study was to determine the pharmacokinetic parameters of nitroglycerin and the dinitrate metabolites after multiple intravenous infusions of nitroglycerin in healthy volunteers. Six volunteers received variable infusion rates of nitroglycerin. Generally, at 0, 40, 80, and 120 min, the infusion rates were adjusted to 10, 20, 40, and 10 g/min, respectively. Plasma samples were drawn and analyzed for nitroglycerin and its 1,2-and 1,3-dinitraie metabolites using capillary GC. Steady-state nitroglycerin plasma concentrations attained at 10, 20, 40, and 10g/min were 0.44±0.31, 1.32±0.71, 4.23±1.50 and 1.04±0.43 ng/ml, respectively. As the infusion rate was increased, the steady-state concentrations increased disproportionately. When the dose was decreased from 40 to 10g/min, the steady-state nitroglycerin concentrations were always higher than those at the initial low infusion rate. Thus, in the majority of subjects, a hysteretic type of response was present. The hysteresis observed in the dose versus steady-state concentration curve may be explained by either end-product inhibition or saturable binding of nitroglycerin to blood vessels. The clearance values (5.5 to 71 l/min) were very high and far exceed the maximum possible hepatic clearance suggesting that nitroglycerin is metabolized by organs other than liver. Clearance was not directly related to plasma concentrations but was found to decrease to a constant value (approximately 11±6 l/min< as nitroglycerin concentrations initially increased.Supported in part by NIH Grant HL 32243 and by a grant from Key Pharmaceuticals, Inc., Miami, Florida. During the course of the work, PKN received support as an AFPE James F. Hoge Memorial Fellow.     

Pharmacokinetics and pharmacodynamics of nitroglycerin and its dinitrate metabolites in conscious dogs: Intravenous infusion studies

Intravenous infusions of nitroglycerin (GTN), 1,2-glyceryl dinitrate (1,2-GDN), and 1,3-glyceryl dinitrate (1,3-GDN) were given to four conscious dogs at 10 g/min, 30 g/min, 50 g/min, and 70 g/min of GTN and 20 g/min and 100 g/min of GDNs. The steady state plasma concentrations (Css)of GTN were reached after about 60 min whereas for 1,2-GDN and 1,3-GDN the Csswere reached at about 150 min after the infusion began. Except for one dog, the Cssof GTN were not proportional to infusion rate, however, all dogs together showed a good linear relationship between Cssof GTN and infusion rates with an average correlation coefficient of 0.917±0.102. Large variability in GTN clearance after various infusion rates was observed in all dogs. The Cssratios of 1,2-GDN/GTN and 1,3-GDN/GTN yield overall averages of 31.5 ±17.2 and 5.47 ±3.19,respectively. Average Cssratios of metabolites 1,2-GDN/1,3-GDN were 5.78±1.23. This ratio is different from those obtained after iv bolus and oral dosing indicating that the biotransformation of GTN to 1,2-GDN and 1,3-GDN differs for each dosing route. The clearances for 1,2-GDN and 1,3-GDN were not changed over the dose range of 20 g/min to 100 g/min. Terminal half-lives of 1,2-GDN and 1,3-GDN postinfusion were similar to those values obtained after a single bolus dose (45 min). It appears that all the GTN dose at steady state can be accounted for by the formation of measurable 1,2-GDN and 1,3-GDN. Large intra- and interdog variations in systolic blood pressure decrease (SPD) following infusions of GTN were observed, however, all dogs showed a clear systolic blood pressure decrease when the highest infusion rate (70 g/min) was given. No significant systolic blood pressure drop was detected following 20 g/min infusions of 1,2-GDN or 1,3-GDN. It was clear that systolic blood pressure in all dogs decreased following 100 g/min infusions of 1,2-GDN or 1,3-GDN. When SPD values were plotted vs. log GTN concentrations following the infusion of 70 g/min of GTN in all four dogs, a counterclockwise hysteresis was observed indicating the significant contribution of the active dinitrate metabolites to GTN pharmacodynamics.This work was supported in part by NIH grant HL32243.     

Mechanisms of action of plant sterols on inhibition of cholesterol absorption

Summary The effects of two different plant sterols on intestinal cholesterol absorption were compared in normal volunteers by an intestinal perfusion study during a control period followed by high dose infusion of sitosterol or sitostanol (3.6 mol/min), to which subjects were allocated in a randomized manner. Cholesterol absorption during the control period was similar in the two groups, averaging 0.88 ± 0.48 mol/min (32 ± 11%) for group I (sitosterol) and 0.68 ± 0.33 mol/min (29 ± 9%) for group II (sitostanol). The infusion of a high dose of sitosterol resulted in a significant reduction of cholesterol absorption to 0.47 mol/min (16%). Following the same dose of sitostanol, cholesterol absorption diminished significantly to 0.15 ± 0.11 mol/min (5.1 ± 2.9%). Overall cholesterol absorption declined during sitosterol infusion by almost 50%, whereas sitostanol infusion caused a reduction of cholesterol absorption by almost 85%. These findings of a more effective inhibition of cholesterol absorption by sitostanol might confirm the observation recorded by others that an increase in hydrophobicity of a plant sterol results in a higher affinity but lower capacity to mixed micells. This may cause an effective displacement of cholesterol from micellar binding and therefore diminished cholesterol absorption.     

Absorption of D-Glucose in the Rat Studied Using In Situ Intestinal Perfusion: A Permeability-Index Approach

Purpose. A permeability-index approach was developed and used to study the transport of D-glucose in the jejunum and ileum of rats. Methods. The effective permeability coefficient (P_e) of [³H]D-glucose and [¹⁴C]antipyrine (an internal standard) in jejunum and ileum of four rats was determined using an in situ rat intestinal perfusion technique. The permeability ratio of the test compound (D-glucose) to the internal standard was defined as the permeability-index (P_i), which was mathematically independent of the length and surface area of the intestinal segment perfused. Using this approach, the transport of [³H]D-glucose in jejunum and ileum of eight animals was investigated at concentrations ranging from 1 to 300 mM. The tissue/perfusate distribution of [³H]D-glucose and [¹⁴C]antipyrine at steady state was also determined. Results. The variability (%CV) in P_i of D-glucose was only 5%, compared with 23–36% in P_e values of D-glucose or antipyrine alone. The permeability and tissue distribution of [¹⁴C]antipyrine were unaffected by the presence of D-glucose. In contrast, the permeability and tissue distribution of [³H] D-glucose were concentration-dependent in both jejunum and ileum. The transport of D-glucose was studied assuming that the transport was mediated by a carrier (with maximum flux, V_max and dissociation constant, K_m) as well as by non-saturable transport (P_d). The maximum transport capacity for D-glucose in jejunum (0.522 mole/min/cm²) was twice that in ileum (0.199 mole/min/ cm²), but the affinity (l/K_m) was less than half of that in ileum (l/ (48.2 mole/mL) vs. 1/(21.4 mole/mL)), rendering a similar active transport efficiency (V_max/K_m) in these two regions. The non-saturable permeability (P_d) in jejunum (44.6 × 10^–4 cm/min) was approximately twice that in ileum (20.4 × 10^–4 cm/min). Conclusions. The permeability-index approach yielded parameters with reduced variability by eliminating potential imprecisions in length and surface area measurements of the intestinal segment perfused. D-glucose was transported via carrier-mediated systems in both jejunum and ileum, with different transport capacity and affinity in these two regions.     

Toxicokinetics ofN-nitrosodimethylamine in the Syrian golden hamster

The single-dose toxicokinetics ofN-nitrosodimethylamine (NDMA) has been characterized in 8-week-old male Syrian golden hamsters by analysis using high performance liquid chromatography of serial blood samples. An i.v. bolus dose of 4.2 mol/kg [¹⁴C]NDMA revealed biphasic first-order elimination with a terminal half-life of 8.7±1.0 min (mean±SE) for unchanged NDMA and 31.5±5.5 min for total radioactivity, and evidence for conversion to polar metabolites was seen in the chromatographic assays. The systemic blood clearance and apparent steady-state volume of distribution for unchanged NDMA were 51.2±3.0 ml/min/kg and 582±60 ml/kg, respectively. No unchanged NDMA was detected in the urine following an i.v. bolus dose of 15 mol/kg [¹⁴C]NDMA, but 31% of the total radioactivity was eliminated by that route. A dose of 38 mol/kg given by gavage indicated a systemic bioavailability of 11±4% for unchanged NDMA. Reversible binding of NDMA to hamster plasma proteins was found to be negligible. Estimation of the intrinsic hepatic clearance (Cl_I) in the hamster produced a value of 648 ml/min/kg, which is greater than that previously obtained for the rat, and indicates that the metabolic capacity of the hamster liver is greater than that of the rat. These results suggest that this difference in Cl_I may play a role in the previously reported (Lijinsky et al. 1987) switch in organotropism from almost exclusivity for liver tumors in hamsters dosed by gavage to additional high incidences of lung and kidney tumors in the rat.     

X-ray microanalysis of cryopreserved human skin to study the effect of iontophoresis on percutaneous ion transport

Purpose. To study at the ultrastructural level which part of the skin is associated with percutaneous iodide transport by passive diffusion and iontophoresis. Methods. Following passive diffusion or iontophoresis of iodide, the morphology and the ion distribution of the skin was preserved by rapid freezing. The skin was kept frozen until and during examination by transmission electron microscopy (TEM) and X-ray microanalysis (XRMA). The intrinsic electron absorbing characteristics of cryopreserved skin allow direct TEM examination without additional staining. XRMA can be used to obtain in a relatively nondestructive way in situ information on ion distributions across the skin. Results. After passive diffusion, iodide was mainly found in the stratum corneum (SC), whereas there was little iodide in the viable epidermis. Iontophoresis up to 300 A/cm² did not significantly affect this distribution. With iontophoresis at 1000 A/cm², the amount of iodide increased dramatically and was equally distributed over the SC and viable epidermis. The presence of iodide in the SC suggests that iodide is present inside corneocytes. Conclusions. Iontophoresis up to 300 A/cm² does not significantly perturb skin structures in contrast to iontophoresis at 1000 A/cm². The presence of iodide inside corneocytes suggests the possibility of transcellular percutaneous iodide transport.     

Characterization and presynaptic modulation of stimulation-evoked exocytotic co-release of noradrenaline and neuropeptide Y in guinea pig heart

Summary The relationship between noradrenaline and neuropeptide Y (NPY) release was investigated in the in situ perfused guinea pig heart with intact sympathetic innervation. For determination of NPY concentrations in the perfusate, a specific radioimmunoassay was employed and further characterized. Electrical stimulation of the left stellate ganglion (4, 8, 12, and 50 Hz; for 10 min) evoked a calcium-dependent and frequency-related overflow of noradrenaline and NPY, which was positively correlated (r = 0.83; p < 0.001; n = 25). When two subsequent stimulations (12 Hz; each for 1 min) were performed in the same heart, addition of noradrenaline (10 M) 5 min prior to the second stimulation reduced NPY overflow by 43 ± 10%. The stimulated release of noradrenaline and NPY was increased by the alpha₂-adrenoceptor antagonist yohimbine (1 M) to 170 ± 10% and 199 ± 26%, and attenuated by the alpha₂-adrenoceptor agonist B-HT 920 (1 M) to 70 ± 9% and 68 ± 9%, respectively. The adenosine analogue cyclohexyladenosine (1 M) significantly reduced the stimulated overflow of both noradrenaline (to 57 ± 5%) and NPY (to 73 ± 8%). Exogenous NPY (100 nM) attenuated the stimulated overflow of noradrenaline by 30 ± 6%. Uptake₁ blockade with desipramine (100 nM) or nisoxetine (100 nM) prior to the second stimulation significantly increased noradrenaline overflow and attenuated that of NPY; the attenuation of the stimulation-evoked overflow of NPY was abolished by yohimbine (1 M).Our results indicate that electrical stimulation induces a calcium-dependent, exocytotic co-release of noradrenaline and NPY. The co-release of both transmitters is regulated by presynaptic receptors in a parallel manner; furthermore, both transmitters, noradrenaline and possibly NPY, modulate their own release by a presynaptic negative feedback mechanism via presynaptic alpha₂-adrenoceptors and NPY-receptors.This work was supported by a grant of the Deutsche Forschungsgemeinschaft (SFB 320 - Herzfunktion und ihre Regulation). Presented in part at the 61st Scientific Sessions of the American Heart Association. Washington DC, November 1988 Send offprint requests to M. Haass at the above address     

pH-Metric Solubility. 2: Correlation Between the Acid-Base Titration and the Saturation Shake-Flask Solubility-pH Methods

Purpose. The objective of this study was to compare the results of anormal saturation shake-flask method to a new potentiometricacid-base titration method for determining the intrinsic solubility and thesolubility-pH profiles of ionizable molecules, and to report thesolubility constants determined by the latter technique. Methods. The solubility-pH profiles of twelve generic drugs (atenolol,diclofenac.Na, famotidine, flurbiprofen, furosemide,hydrochlorothiazide, ibuprofen, ketoprofen, labetolol.HCl, naproxen, phenytoin, andpropranolol.HCl), with solubilities spanning over six orders ofmagnitude, were determined both by the new pH-metric method and by atraditional approach (24 hr shaking of saturated solutions, followed byfiltration, then HPLC assaying with UV detection). Results. The 212 separate saturation shake-flask solubilitymeasurements and those derived from 65 potentiometric titrations agreed well.The analysis produced the correlation equation:log(1/S)_titration = ±0.063(± 0.032)+ 1.025(±0.011) log(1/S)_shake-flask,s = 0.20, r² = 0.978.The potentiometrically-derived intrinsic solubilities of the drugs were:atenolol 13.5 mg/mL, diclofenac.Na 0.82 g/mL, famotidine 1.1mg/mL, flurbiprofen 10.6 g/mL, furosemide 5.9 g/mL,hydrochlorothiazide 0.70 mg/mL, ibuprofen 49 g/mL, ketoprofen 118 g/mL,labetolol.HCl 128 g/mL, naproxen 14 g/mL, phenytoin 19 g/mL, andpropranolol.HCl 70 g/mL. Conclusions. The new potentiometric method was shown to be reliablefor determining the solubility-pH profiles of uncharged ionizable drugsubstances. Its speed compared to conventional equilibriummeasurements, its sound theoretical basis, its ability to generate the fullsolubility-pH profile from a single titration, and its dynamic range (currentlyestimated to be seven orders of magnitude) make the new pH-metricmethod an attractive addition to traditional approaches used bypreformulation and development scientists. It may be useful even todiscovery scientists in critical decision situations (such as calibratingcomputational prediction methods).