Pharmacodynamic comparison of single doses of IFN-beta1a and IFN-beta1b in healthy volunteers.

In a study in 75 volunteers, preparations of interferon-beta1b (IFN-beta1b) and IFN-beta1a were compared in terms of the resulting serum concentrations of three biologic markers, neopterin, human Mx protein, and 2',5' oligoadenylate synthetase. Each preparation was tested at five dose levels, the middle dose being that recommended for use in patients with multiple sclerosis on the basis of large clinical trials. Five randomly chosen volunteers each received a single subcutaneous dose of one of the IFN or of IFN-beta1a given intramuscularly. The amounts of each marker induced were dose related. There were no major differences between the results with the two IFN or in the duration of the changes in the markers after the two routes of injection. The data indicated that 8 million international units (MIU) of IFN-beta1b and 6 MIU of IFN-beta1a had very similar effects. Even after the highest single dose tested, the increase in the biologic markers were not sustained for a full week.

In vivo conversion of norethisterone and norethisterone acetate to ethinyl etradiol in postmenopausal women.

Previous studies with postmenopausal women receiving oral doses of norethisterone-containing preparations have shown that a small fraction of the dose is converted metabolically to ethinyl estradiol and may be detected in the peripheral blood. To investigate the extent and the dose dependence of this conversion in more detail, we performed a study with 24 postmenopausal women who received single oral doses of 5 mg norethisterone as well as 5 and 10 mg norethisterone acetate with a washout phase of 2 weeks between each treatment. After each treatment, blood was collected at regular intervals and the concentrations of norethisterone and ethinyl estradiol were analyzed in the serum samples by a specific radioimmunoassay and by gas chromatography/mass spectrometry, respectively. Ethinyl estradiol was present in the serum samples of all women following treatment with norethisterone acetate and, except for four cases, also after treatment with norethisterone. The conversion ratio of norethisterone acetate to ethinyl estradiol was 0.7 +/- 0.2% and 1.0 +/- 0.4% at doses of 5 and 10 mg, respectively. This corresponded to an oral dose equivalent of about 6 micrograms ethinyl estradiol per milligram of norethisterone acetate. For norethisterone, a conversion ratio of 0.4 +/- 0.4% was found at a dose of 5 mg, which corresponded to an oral dose equivalent of about 4 micrograms ethinyl estradiol per milligram of norethisterone. Although it cannot be excluded that in individual cases, even higher doses of ethinyl estradiol may be produced by conversion, it is concluded that at therapeutic doses of the progestogens, the exposure to metabolically derived ethinyl estradiol is probably of little clinical significance not only in fertile women using oral contraceptive combination preparations containing norethisterone and ethinyl estradiol, but also in postmenopausal women who receive oral doses of estradiol for estrogen replacement. The estrogenic effects of metabolically derived ethinyl estradiol on the liver (eg. synthesis of transport proteins) are very likely more than compensated due to the androgenic activity of norethisterone.

A single-dose and 3-month clinical-pharmacokinetic study with a new combination oral contraceptive.

The study was performed in 14 young women. The combination oral contraceptive contained 75 microgram gestodene (GSD) and 20 microgram ethinyl estradiol (EE2) per dosage unit. The volunteers received a single dose on day 21 of a treatment-free precycle (PCd21) and, after a washout period of 7 days, used the preparation in a 21 d/7 d schedule for three months. Daily drug serum level profiles were taken on PCd21 and on days 1 and 21 of treatment cycles 1 and 3. In addition, trough drug serum levels were followed every other day during treatment cycles 1 and 3. Serum levels of GSD, EE2, CBG, SHBG and testosterone (T) were determined by means of specifically developed or commercially available RIAs. Pharmacokinetic evaluation was carried out with TOPFIT and parameters were evaluated for differences with the t-test. Main target variables were Cmax, tmax and AUC for EE2, GSD and unbound GSD on day 21, cycle 3 vs. PCd21. EE2 pharmacokinetics were in agreement with a dose of 20 microgram/unit. Single-dose Cmax of 65 pg/ml and AUC of 612 pg h ml(-1) increased by 40-60% during treatment cycles as a result of accumulation EE2 induced basal SHBG (102nmol/L) and CBG (42 microgram/ml) serum levels to about 220 nmol/L and 87 microgram/ml, respectively, at the end of treatment cycles 1 and 3. Serum T levels dropped to 50% of baseline levels during treatment cycles and free T concentrations were reduced by 60-70%. GSD pharmacokinetics at the end of treatment cycles 1 and 3 were different from single-dose pharmacokinetics. Single-dose Cmax of 3.5 ng/ml and AUC 0-24 h of 22 ng h ml(-1) increased to steady-state levels of 8-8.7 ng/ml and 90-106 ng h ml(-1), respectively. The increase in GSD levels under treatment is the result of two parallel processes, i.e. accumulation and enlargement of the specific binding compartment. This was shown by protein-binding experiments, demonstrating an increase in specific (SHBG) binding from 69% to 80% and a reduction in the free fraction of GSD by 40% during treatment. The results of GSD and EE2 pharmacokinetics obtained in the present study confirm previous results with Femodene, when the reduction in the EE2 dose by 10 microgram/d is taken into account.

Mercapturic acid formation in cultured opossum kidney cells.

We investigated the last step of mercapturic acid formation, the N-acetylation of cysteine S-conjugates, in the established opossum kidney (OK) cell line which exhibits characteristics of the proximal tubule. S-Benzyl-L-cysteine was used as a model substance for such a cysteine S-conjugate. We succeeded in showing that OK cells absorb S-benzyl-L-cysteine via an active transport system which is inhibitable by phenylalanine. This transport follows Michaelis-Menten kinetics and the two characterizing parameters were determined: the Michaelis-Menten constant Km = 1.8 mmol/l, and the maximum of the difference between the intracellular and the extracellular concentration of S-benzyl-L-cysteine delta Cmax = 19.4 mmol/l. S-Benzyl-L-cysteine is converted to N-acetyl-S-benzyl-L-cysteine at a constant rate, which is independent of the extracellular S-benzyl-L-cysteine concentration. Under the tested experimental conditions this is probably due to saturation of the microsomal N-acetyltransferase catalyzing this reaction. In conclusion, we have shown that OK cells are a suitable model for studying mercapturate formation. They take up S-benzyl-L-cysteine mainly via the same carrier as phenylalanine, which is known to be transported in the rat by the high-capacity, low-affinity neutral amino acid carrier, and convert it to N-acetyl-L-benzyl-S-cysteine.

Localization and capacity of the last step of mercapturic acid biosynthesis and the reabsorption and acetylation of cysteine S-conjugates in the rat kidney.

We investigated the capacity and the localization of N-acetylation of the mercapturic acid precursor S-benzyl-L-cysteine (BC), as well as the tubular reabsorption of this compound in the rat kidney in vivo et situ by renal clearance and continuous microinfusion and microperfusion experiments. In renal clearance experiments. 450 mumol BC was infused intravenously for 180 min. During the time of BC infusion and the following 180 min, the two kidneys excreted 400 mumol or 90% of the infused BC dose as the mercapturate N-acetyl-S-benzyl-L-cysteine (AcBC). Comparison of the amounts of BC and AcBC entering the left kidney via the renal artery with those leaving it via the renal vein and the ureter showed that 0.13 +/- 0.04 mumol BC/min (mean +/- SEM) was extracted and 0.24 +/- 0.08 mumol AcBC/min was formed by one kidney. The intrarenal acetylation can account for the formation of 38% of the mercapturate excreted in the final urine. In additional experiments, 50 pmol/min [14C]BC was microinfused into single superficial tubules at three different sites. During microinfusion into early proximal tubules, the final urine contained 16.3 +/- 1.8% of the microinfused radioactivity as AcBC, but no BC. When [14C]BC was microinfused into late proximal tubules, 13.0 +/- 2.3% of the infused label was recovered as BC, 28.1 +/- 2.3% as AcBC. During microinfusion into early distal tubules, the final urine contained no AcBC, but 90.3 +/- 2.1% of the infused [14C]BC was recovered.(ABSTRACT TRUNCATED AT 250 WORDS)

pHi-dependent membrane conductance of proximal tubule cells in culture (OK): differential effects on K(+)- and Na(+)-conductive channels.

Confluent monolayers of the established opossum kidney cell line were exposed to NH4Cl pulses (20 mmol/liter) during continuous intracellular measurements of pH, membrane potential (PDm) and membrane resistance (R'm) in bicarbonate-free Ringer. The removal of extracellular NH4Cl leads to an intracellular acidification from a control value of 7.33 +/- 0.08 to 6.47 +/- 0.03 (n = 7). This inhibits the absolute K conductance (gK+), reflected by a decrease of K+ transference number from 71 +/- 3% (n = 28) to 26 +/- 6% (n = 5), a 2.6 +/- 0.2-fold rise of R'm, and a depolarization by 24.2 +/- 1.5 mV (n = 52). In contrast, intracellular acidification during a block of gK+ by 3 mmol/liter BaCl2 enhances the total membrane conductance, being shown by R'm decrease to 68 +/- 7% of control and cell membrane depolarization by 9.8 +/- 2.8 mV (n = 17). Conversely, intracellular alkalinization under barium elevates R'm and hyperpolarizes PDm. The replacement of extracellular sodium by choline in the presence of BaCl2 significantly hyperpolarizes PDm and increases R'm, indicating the presence of a sodium conductance. This conductance is not inhibited by 10(-4) mol/liter amiloride (n = 7). Patch-clamp studies at the apical membrane (excised inside-out configuration) revealed two Na(+)-conductive channels with 18.8 +/- 1.4 pS (n = 10) and 146 pS single-channel conductance. Both channels are inwardly rectifying and highly selective towards Cl-. The low-conductive channel is 4.8 times more permeable for Na+ than for K+. Its open probability rises at depolarizing potentials and is dependent on the pH of the membrane inside (higher at pH 6.5 than at pH 7.8).

Electrical properties of cultured renal tubular cells (OK) grown in confluent monolayers.

UNLABELLED: OK cells grown to confluent monolayers were investigated by microelectrode techniques and microinjection. Cell membrane potential difference (PDm) in bicarbonate-free solution is -61.8 +/- 0.6 mV (n = 208), cell membrane resistance (Rm) amounts to 1.4 +/- 0.2 k omega. cm2 (n = 8). The apparent transference number for potassium (t'k+) is 71 +/- 3% (n = 28) and can be reduced by 3 mmol/l BaCl2 to 7.5 +/- 4.0%; (n = 8). In the presence of extracellular CO2 and HCO3- (pH 7.4) the cells acidify by 0.34 +/- 0.05 pH units (n = 12). This leads to a depolarization of PDm by 8.4 +/- 1.8 mV (n = 8), an increase in Rm by 49 +/- 10% (n = 10), and a reduction of K+-conductance to 63 +/- 5% (n = 13). Intracellular acidification by the NH4Cl-prepulse technique also inhibits K+-conductance and depolarizes the membrane. Recovery from an intracellular acid load is reflected by cell membrane repolarization. This recovery can be inhibited by amiloride (10(-3) mol/l). Na+- and Cl- -conductances could not be detected. The transepithelial resistance (Rte) of OK cell monolayers 1 day after plating is 41 +/- 6 omega.cm2 and decreases with time after plating. Intercellular communication (electrical or dye coupling) was not observed. CONCLUSIONS: 1. The membrane potential of OK cells is largely determined by a pH-sensitive, barium-blockable K+-conductance. 2. Amiloride-blockable Na+/H+-exchange is reflected by membrane potential changes via this K+-conductance. 3. Monolayers of OK cells are electrically leaky.

Electrogenic transport of neutral and dibasic amino acids in a cultured opossum kidney cell line (OK).

A study has been made of electrogenic cellular uptake of amino acids resulting in the depolarization of cell membrane potential (PDm) in confluent monolayers of an established opossum kidney (OK) cell line using conventional and pH-selective microelectrodes. Apical superfusion of neutral and dibasic amino acids rapidly depolarized the cell membrane, while application of acidic amino acids had no effect on PDm. The depolarization in response to L-phenylalanine and L-arginine was stereoselective, dose-dependent and saturable. 10 mmol/l of L-phenylalanine reduced PDm by 4.8 +/- 0.4 mV (n = 51) in a completely sodium-dependent way and the concentration necessary for half-maximal depolarization (C1/2) was about 1.5 mmol/l. On the other hand, the C1/2 for L-arginine was about 0.02 mmol/l. The maximal depolarization produced by L-arginine (measured at 10 mmol/l) amounted to 6.8 +/- 1.2 mV (n = 10) and this was not affected when extracellular sodium was replaced by choline (6.3 +/- 1.2 mV; n = 10). The depolarizations induced by L-phenylalanine and L-arginine were significantly additive (p less than 0.001). The intracellular pH of OK cells was 7.09 +/- 0.03 (n = 11) and did not change during L-arginine application. We conclude that (1) carrier-mediated uptake of neutral and dibasic amino acids into OK cells is at least partially electrogenic. (2) L-Phenylalanine is transported by a Na+-symport. (3) In contrast, L-arginine depolarizes PDm independently of extracellular sodium. (4) Electrogenic uptake of acidic amino acids is not detectable in OK cells.

Renal tubular transport of glutathione in rat kidney.

Filtered glutathione (gamma-glutamyl-cysteinyl-glycine or GSH) is rapidly hydrolyzed by brush-border enzymes facing the tubular lumen and is reabsorbed in the form of the constituent amino acids. The first step of hydrolysis is catalyzed by gamma-glutamyltransferase (gamma-GT). We investigated localization and capacity of the rat renal glutathione degradation/reabsorption during elevation of the filtered load (intravenous infusion of 12 resp. 18 mumol GSH/min). Fractional excretion went up from about 0.003 to 0.31 +/- 0.02 SEM during infusion of the lower and to 0.49 +/- 0.03 SEM during infusion of the higher glutathione dose. GSH degradation/reabsorption took place along the entire proximal tubule and was partially saturated by a 150-200-fold elevation of the normal filtered load. Net reabsorption of GSH up to the last accessible superficial loop was significantly lower during infusion of 18 mumol GSH/min (0.3 mumol/min) than during infusion of 12 mumol GSH/min (1.6 mumol/min). In further experiments, infusion of 18 mumol GSH/min was preceded by the i.v. administration of acivicin (0.5 mmol/kg body wt.), an inhibitor of gamma-GT. In these experiments, fractional glutathione deliveries to late proximal and early distal tubules did not significantly differ from 1, fractional excretion of GSH at the same time was 1.46 +/- 0.11 SEM, revealing net secretion of GSH with the final urine. Tubular secretion of GSH in the acivicin-treated animals occurred either in distal tubules and/or collecting ducts or in the proximal tubules of deep nephrons which are not accessible to micropuncture.(ABSTRACT TRUNCATED AT 250 WORDS)