Pharmacokinetic modeling of hepatocyte growth factor in experimental animals and humans.

Hepatocyte growth factor (HGF) is under development for treatment of renal failure. This study was designed to clarify changes in HGF pharmacokinetics in renal failure and to establish a pharmacokinetic model applicable to single and repeated doses. The plasma concentration profile in mice with glycerol-induced acute renal failure was similar to that in normal mice, indicating a minimal contribution of kidney to systemic clearance of HGF. Nevertheless, accumulation of fluorescein-4-isocyanate-labeled HGF in renal tubules in both cases suggests the occurrence of efficient endocytosis of HGF in kidney. A pharmacokinetic model including plasma and liver compartments was constructed, incorporating both high- and low-affinity receptors for association and subsequent endocytosis of HGF because HGF is eliminated via specific receptor c-Met and heparin-like substance. The model well explained the plasma concentration profiles at all doses examined after bolus injection in animals and humans, and those during infusion in rodents. It includes externalization of receptors, which is negatively regulated by HGF, and can explain the gradual increase in trough concentration during repeated dosing in monkeys. Overall pharmacokinetic profiles of HGF are governed by at least two receptors and are well described by this pharmacokinetic model, which should assist in safe management of clinical trials.

Population pharmacokinetics of oseltamivir when coadministered with probenecid.

Oseltamivir is a potent, selective, oral neuraminidase inhibitor for the treatment and prophylaxis of influenza. Plasma concentrations of the active metabolite, oseltamivir carboxylate, are increased in the presence of probenecid, suggesting that the combination could allow for the use of reduced doses of oseltamivir. To investigate this proposal, we developed a population pharmacokinetic model and simulated the pharmacokinetics of candidate combination regimens of oral oseltamivir (45 mg and 30 mg twice a day) plus oral probenecid (500 mg/6 hourly). Probenecid plus oseltamivir 45 mg achieved all the pharmacokinetic parameters expected of oseltamivir alone, but combination with oseltamivir 30 mg and dose interval extension approaches did not. An oseltamivir-probenecid combination may compromise tolerability and enhance the potential for drug interactions. In addition, increased dosing requirements may affect compliance and attainment of optimal oseltamivir exposure, potentially facilitating the emergence of viral strains with reduced susceptibility to oseltamivir. These factors, set alongside increased capacity for oseltamivir production, should be carefully considered before an oseltamivir-probenecid combination is used.

A replicate study design for testing bioequivalence: a case study on two desmopressin nasal spray preparations.

OBJECTIVE: The present study was carried out to test bioequivalence between two different desmopressin nasal spray preparations. Due to the high variability of plasma pharmacokinetics of intranasally administered peptides like desmopressin, appropriate study designs are required to assess bioequivalence. Therefore, a single-dose, replicate study design was used to evaluate bioequivalence of two desmopressin nasal sprays. SUBJECTS AND METHODS: Thirty-two healthy male volunteers were enrolled in the study and were randomly assigned to receive the test- and reference drug on two occasions in a 4-period 2-sequence crossover study design. Subjects received a single dose of 20 microg (10 microg per nostril) of desmopressin-acetate per study day separated by wash-out periods of at least 1 week. Desmopressin blood concentrations were measured serially over a 14-h period using a validated radioimmunoassay method. Statistical analysis was initially performed using a complicated mixed-analysis model testing for individual bioequivalence according to recommendations by the Food and Drug Administration. This approach, however, failed to converge with all defined main PK parameters and, thus, a traditional mixed analysis of variance analysis based on population averages was definitely used for testing bioequivalence between study drugs. The procedure of selecting an appropriate statistical analysis for a replicate study design was predefined in the study protocol. RESULTS: The 90% confidence intervals (CI) were calculated for the area under the time-concentration curve (AUC), maximum concentration (C(max)) and the time to reach C(max) (t(max)) of test/reference drug ratios for a bioequivalence range from 0.80-1.25. The mean test/reference drug ratios were completely within the 90% CIs with values of 1.041 (CI: 0.892-1.216), 1.021 (CI: 0.913-1.140) and 1.068 (CI: 0.914-1.249) for AUC(0-14 h), C(max) and t(max), respectively. CONCLUSION: The rate and the extent of intranasal desmopressin absorption are identical for both study preparations. Thus, the desmopressin test preparation met all equivalence criteria and thereby was proven bioequivalent with a marketed reference nasal desmopressin spray.

Renal responses to chronic cold exposure.

The aim of this study was to assess our hypothesis that the release of antidiuretic hormone (ADH), the renal concentrating response to ADH, or both is decreased by prolonged cold exposure. Six groups (n = 6/group) of rats were used. Three groups were exposed to cold (5 degrees C), whilethe remaining three groups were kept at room temperature (25 degrees C). It was found that urine osmolality decreased significantly and serum osmolality increased significantly during cold exposure. The ratio of water/food intake was not affected by prolonged cold exposure. However, prolonged cold exposure increased the ratio of urine output/food intake in the cold-exposed rats, indicating that more urine flow is required by the cold-exposed rats to excrete the osmotic substance at a given food intake. The difference between water intake and urine output decreased significantly in the cold-exposed rats. Thus, prolonged cold exposure increases water loss from excretion. Renal concentrating responses to 24-h dehydration and Pitressin were decreased significantly in the cold-exposed rats. Plasma ADH levels remained unchanged, but renal ADH receptor (V2 receptor) mRNA was decreased significantly in the cold-exposed rats. The results strongly support the conclusion that cold exposure increases excretive water loss, and this may be due to suppression of renal V2 receptors rather than inhibition of ADH release.

Renal response to arginine vasopressin during the oestrous cycle in the rat: comparison of glucose and saline infusion using physiological doses of vasopressin.

The renal response to arginine vasopressin in the rat has been shown to depend on reproductive status. However there is no consensus as to when the kidney is most responsive. The varying results could depend on the protocol and the dose of hormone used. A study has been performed, with physiological doses of vasopressin, comparing the responses during infusion of hypotonic saline and glucose. After an equilibration period of 150 min, conscious rats were infused on each of the four days of the oestrous cycle with either isotonic saline (0.077 M) or 0.14 M glucose for a control period of 45 min. Vasopressin was then infused at 10-40 fmol min(-1) for 1 h, followed by a recovery period of 90 min. Timed urine samples were collected for determination of volume, sodium concentration and osmolality. During the control period urine flow was greatest at oestrus and dioestrus day 2 and sodium excretion on dioestrus day 2 irrespective of the infusate. Vasopressin concentrations achieved lay within the physiological range and no difference was observed between the different days for a given dose. Infusion of vasopressin in both saline and glucose produced a dose-dependent antidiuresis, the greatest responses being seen of pro-oestrus and dioestrus day 2. It was only with the highest rate of infusion that a significant increase in sodium excretion was seen on each day of the cycle and the greatest responses were seen on pro-oestrus and dioestrus day 1 for both infusates. Thus the kidney shows the greatest response to physiological doses of vasopressin at pro-oestrus and dioestrus day 1 irrespective of the infusate employed.

Preparation, purification, and characterization of a reversibly lipidized desmopressin with potentiated anti-diuretic activity.

PURPOSE: . To prepare and characterize a reversibly lipidized dipalmitoyl desmopressin (DPP), and to compare its anti-diuretic efficacy and biodistribution with that of unmodified desmopressin (DDAVP). METHODS: Dithiothreitol (DTT) was used to reduce the intramolecular disulfide bond in DDAVP, and the reduced DDAVP was treated with a thiopyridine-containing disulfide lipidization reagent, Pal-CPD. The product, DPP, was purified by acid precipitation and, subsequently, by size-exclusion chromatography. Reversed-phase HPLC was used to analyze the purity and to evaluate the hydrophobicity of the product. Mass spectrometry was employed to characterize its molecular structure. The biological activity of DPP was demonstrated by the antidiuretic effects in vasopressin-deficient Brattleboro rats. Preliminary pharmacokinetic and biodistribution studies of intravenously injected DDAVP and DPP were carried out in CF-1 mice. RESULTS: DDAVP was readily reduced by a 2-fold molar excess of DTT at 37 degrees C for 0.5 hr. DPP was formed by the reaction of reduced DDAVP with Pal-CPD. Each DPP molecule contains two palmitic acid moieties, which link to the peptide via two disulfide bonds. After acid precipitation and size-exclusion chromatography, the purity was found to be approximately 95%, and the overall yield was 57%. When DPP was administered subcutaneously to Brattleboro rats, the potency of the anti-diuretic activity of DDAVP was enhanced to more than 250-fold. The plasma concentration of intravenously injected DDAVP in mice decreased rapidly during the first 20 min and followed by a slow elimination rate. However, in DPP administered mice, the plasma concentration actually increased in the first 20 min, followed by a slow elimination with a rate similar to that in DDAVP-injected mice. The regeneration of DDAVP was detected in the plasma of mice treated with DPP. Studies of the organ distribution in mice indicated that the liver retention of DPP was longer than that of DDAVP. On the other hand, the intestinal excretion of DPP was significantly less than that of DDAVP. CONCLUSIONS: The 250-fold increase of the anti-diuretic potency in DPP is most likely due to a slow elimination and prolonged tissue retention, together with the regeneration of active DDAVP, in the animals. Our results indicate that reversible lipidization is a simple and effective approach for improving the efficacy of many peptide drugs.

Nocturnal enuresis: a suggestion for a European treatment strategy.

The objective of this study was to review the published literature on aetiology and treatment of nocturnal enuresis, with the aim of providing a treatment strategy which is easy for the patient and their family to follow. Results from European studies conducted over the last 15 y were included in this review. It can be concluded from the results of these studies that enuresis is the cause and not the result of a psychiatric disorder. However, there is still considerable variation in success rates, from 28 to 90%. It is of vital importance that a caring approach from the doctor and a positive family and patient attitude are present for successful treatment. The first choice of treatment should be the one most acceptable to the family, e.g. alarm, desmopressin and combination treatment.

Improved extraction procedure and RIA for determination of arginine8-vasopressin in plasma: role of premeasurement sample treatment and reference values in children.

We optimized an RIA for measurement of arginine8-vasopressin (AVP) in plasma by use of 100-mg Isolute C18 columns for extraction, addition of a preincubation step, and use of maximal dilution of a commercially available antiserum. The detection limit was 0.06 ng/L when 0.5 mL of acidified plasma was extracted. The within- and between-run CVs (n = 16) at physiological concentrations were 5.8-10. 2% and 6.5-11.7%, respectively. Prolonged storage of blood at 25 degreesC, but not at 4 degreesC, led to a significant increase in measured plasma AVP concentrations. When plasma samples were prepared at several centrifugation speeds, plasma AVP was significantly correlated with the platelet count (r = 0.899; P <0. 001). This emphasizes the need for careful sample preparation to avoid contamination of plasma with platelet-bound AVP. Basal plasma AVP in 203 children and adolescents (105 males and 98 females; ages, 1 day to 18 years) averaged 1.1 +/- 0.6 ng/L. There was no significant difference between males and females and no correlation with age. In 16 healthy adult controls, plasma AVP averaged 1.0 +/- 0.5 ng/L.

Pharmacokinetics and antidiuretic effect of intravenous administration of desmopressin in orally overhydrated male volunteers.

In order to study the antidiuretic activity of desmopressin in healthy human subjects, a study design has previously been used with subjects orally hydrated. The objective of the present study was to investigate the pharmacokinetics and the antidiuretic activity of desmopressin administered as an intravenous infusion to eight orally hydrated male volunteers. After initial ingestion of water corresponding to 15 ml.kg-1 body weight, the overhydration was maintained by replacing the urinary fluid loss by drinking-water. Desmopressin (4 micrograms) was administered intravenously over 10 min. 1.5 hr after the start of the hydration procedure. Blood was sampled and urine was collected at intervals throughout the study day (9.5 hr). The terminal half-life of elimination (t1/2,) of desmopressin was calculated to be 2.97 +/- 0.24 (S.E.M.) hr while the clearance was 1.77 +/- 0.10 ml.min.-1.kg-1 and the volume of distribution at steady state was 373 +/- 30 ml.kg-1. The infusion caused a marked and long-lasting reduction of the urine flow rate. Four hr after the start of the infusion the mean urine osmolality was 909 +/- 46 mOsm.kg-1.

Effect of food intake on the pharmacokinetics and antidiuretic activity of oral desmopressin (DDAVP) in hydrated normal subjects.

OBJECTIVE: The effect of food ingestion on the gastrointestinal absorption and antidiuretic action of oral desmopressin. An oral preparation of desmopressin, a synthetic analogue of vasopressin, has recently become available for clinical use. DESIGN: A randomized, single-blind, crossover study with four treatment arms. Day A, no meal + placebo; B, no meal + 400 micrograms oral desmopressin; C, standard meal + 400 micrograms oral desmopressin; D, standard meal + 400 micrograms oral desmopressin after 1.5 hours. Plasma desmopressin was measured every 15-30 minutes for 6 hours after drug intake. An intravenous hydration regimen was employed on each study day. SUBJECTS: Sixteen healthy, non-smoking, mean aged 20-35 years (mean 27.8 years). MEASUREMENTS: Plasma desmopressin concentrations were measured throughout each study day to calculate the area under the desmopressin plasma-concentration-time curve to infinity (AUCinf), the maximum plasma desmopressin concentration (Cmax), the time at which Cmax was reached (Tmax) and the time at which plasma desmopressin was first detected (Tlag). Urine volume, urine osmolality and plasma sodium concentrations were also measured at specified times on each study day. RESULTS: The total absorption of oral desmopressin, reflected by the AUCinf, was significantly higher when taken during the fasting state (day B) compared with its administration with or 1.5 hours after a standard meal (days C and D). In addition, Cmax was higher and both Tmax and Tlag were shorter on day B compared with days C and D. No effect of food ingestion was observed on the pharmacodynamics of oral desmopressin: urine volume was decreased and urine osmolality was increased to similar extents on all active treatment days (B, C and D). No significant reductions in plasma sodium concentrations (a safety parameter) was observed during the trial. CONCLUSIONS: The gastrointestinal absorption of desmopressin is reduced and delayed if administered with or 1.5 hours after a meal. This decreased absorption of desmopressin did not have an impact on the antidiuretic action of the drug since all treatment regimens elicted a maximal response. It is possible that administration of desmopressin in the fasting state may prolong its duration of action.

A comparative study of pharmacodynamics and bioavailability of 2 different desmopressin nasal sprays.

The antidiuretic effect and pharmacokinetics were investigated in 16 healthy, male overhydrated volunteers after intranasal administration of 20 microg desmopressin. The antidiuretic activity was measured by determination of urine osmolality and diuresis every 15 minutes over a period of 8 hours. Both study preparations were equally effective regarding a rapid onset of activity and a highly reproducible magnitude of effect. Urine osmolalities, analyzed as area under the time curve (AUCosm) and maximum urine osmolalities were similar for both nasal sprays. Urine volume, analyzed as area under the time curve, was raised after treatment with the test preparation. Bioequivalence was assessed for the primary criterion AUCosm by a calculated mean ratio (test/reference) of 102.8% and a 90% confidence interval ranging from 95.4% to 110.8%. Plasma levels of desmopressin, measured by a specific and sensitive radio-immunoassay method, were already detectable 20 minutes after administration. The mean time curves were parallel at different concentration levels. The maximum desmopressin plasma concentrations of both preparations were comparable, showing high interindividual variability. The times of reaching maximum plasma concentrations were similar. Desmopressin bioavailability was increased after treatment with the test preparation (mean ratio of 130.8% and a 90% confidence interval ranging from 109.9% to 155.7%). Both preparations showed a pronounced biological effect with similarly raised urine osmolalities. The detected differences in bioavailability seem to have no direct correlation to the biological response.

The efficacy of DDAVP is related to the circadian rhythm of urine output in patients with persisting nocturnal enuresis.

OBJECTIVE: Desmopressin may be a useful treatment in some, but not all, patients with nocturnal enuresis. We have evaluated a relation between nocturnal urine output in patients with primary monosymptomatic nocturnal enuresis and the treatment response to synthetic vasopressin. DESIGN: Adolescent or adult enuretics and normal subjects were enrolled in the study and admitted to hospital for a 24 hour investigation of the diurnal variation in urine output, plasma vasopressin (AVP) and plasma atrial natriuretic peptide (ANP). The enuretics were characterized prior to investigation as either 1-desamino-8-D-arginine vasopressin (DDAVP) responders or non-responders. During admission the fluid intake was restricted to 25 ml/kg per day. PATIENTS: Twenty-four patients (15-37 years) with primary monosymptomatic nocturnal enuresis and 9 normal subjects (24-31 years). MEASUREMENTS: Circulating levels of AVP, ANP, plasma electrolytes and plasma osmolality were measured (1400, 2000, 2300, 0200, 0500 and 0800 hours) together with urine volume, urine osmolality and urine electrolytes during daytime and nighttime. Tubular reabsorptive capacity for water, osmoles and creatinine were assessed as well as urinary and fractional excretion rates of sodium and potassium. RESULTS: Controls and DDAVP non-responders had a significant decrease in urine output at night concomitant with a significant plasma AVP amplitude in peak/nadir values although both groups lacked a significant nocturnal increase in AVP. In contrast, in DDAVP responders there was no circadian variation in urine output and thus a nocturnal polyuria together with no oscillation in plasma AVP. The DDAVP responding group had a nocturnal urine production significantly larger than the two other groups. However, the mean 24 hour AVP levels were similar in all groups. The excessive urine production at night in DDAVP responders was accompanied by nocturnal natriuresis due to an increased fractional excretion of sodium. In contrast, nocturnal antidiuresis in controls and DDAVP non-responding enuretics coincided with diminished sodium excretion. Average ANP levels were elevated in both enuretic groups compared to normals, whereas a circadian variation was detected only in the latter. CONCLUSION: It is concluded that DDAVP responsiveness is linked to the nocturnal urine production and that no pathophysiological role can be ascribed to AVP or ANP in DDAVP refractory adolescent and adult enuretics. Moreover, it is suggested that an abnormal tubular handling of sodium may contribute to the nocturnal polyuria seen in DDAVP responders.

Pathophysiology of hyponatremia after transsphenoidal pituitary surgery.

Hyponatremia after pituitary surgery is presumed to be due to antidiuresis; however, detailed prospective investigations of water balance that would define its pathophysiology and true incidence have not been established. In this prospective study, the authors documented water balance in patients for 10 days after surgery, monitored any sodium dysregulation, further characterized the pathophysiology of hyponatremia, and correlated the degree of intraoperative stalk and posterior pituitary damage with water balance dysfunction. Ninety-two patients who underwent transsphenoidal pituitary surgery were studied. To evaluate posterior pituitary damage, a questionnaire was completed immediately after surgery in 61 patients. To examine the osmotic regulation of vasopressin secretion in normonatremic patients, water loads were administered 7 days after surgery. Patients were categorized on the basis of postoperative plasma sodium patterns. After pituitary surgery, 25% of the patients developed spontaneous isolated hyponatremia (Day 7 +/- 0.4). Twenty percent of the patients developed diabetes insipidus and 46% remained normonatremic. Plasma arginine vasopressin (AVP) was not suppressed in hyponatremic patients during hypoosmolality or in two-thirds of the normonatremic patients after water-load testing. Only one-third of the normonatremic patients excreted the water load and suppressed AVP normally. Hyponatremic patients were more natriuretic, had lower dietary sodium intake, and had similar fluid intake and cortisol and atrial natriuretic peptide (ANP) levels compared with normonatremic patients. Normnonatremia, hyponatremia, and diabetes insipidus were associated with increasing degrees of surgical manipulation of the posterior lobe and pituitary stalk during surgery. The pathophysiology of hyponatremia after transsphenoidal surgery is complex. It is initiated by pituitary damage that produces AVP secretion and dysfunctional osmoregulation in most surgically treated patients. Additional events that act together to promote the clinical expression of hyponatremia include nonatrial natriuretic peptide-related excess natriuresis, inappropriately normal fluid intake and thirst, as well as low dietary sodium intake. Patients should be monitored closely for plasma sodium, plentiful dietary sodium replacement, mild fluid restriction, and attention to symptoms of hyponatremia during the first 2 weeks after transsphenoidal surgery.

Intracranial volume receptors: possible role on ADH homeostatic control.

Volume receptors are situated in many organs and are capable of modulating ADH secretion. We have evaluated the variation of plasma ADH concentration after an experimentally induced increase of cerebrospinal fluid (CSF) pressure (PCSF). The experiment was performed in controlled environmental conditions to avoid pain or stress-related ADH release. In 15 rats (10 experimental, 5 control) a cannula was positioned in the left cerebral ventricle: in the experimental group artificial CSF was infused at a rate of 0.6 (microliter/min for 6h: this manoeuvre, in a separate set of animals obtained an increase from 13.03 +/- 0.8 to 25.4 +/- 2.5 cmH2O of PCSF. The same conditions were reproduced in the control group without infusion into lateral ventricle. At the end of the experiment, plasma ADH had fallen significantly in the experimental group from 18.9 +/- 4.8 to 11.9 +/- 2.3 pg/ml (p < 0.05), while it was not changed in the control group (from 25.5 +/- 13.7 to 23.7 +/- 16.2 pg/ml). Heart rate, arterial pressure, plasma Na+ and osmolality, did not change significantly. Plasma K+ fell significantly in both groups: from 5.5 +/- 0.6 to 4.3 +/- 0.3 (p < 0.05) and from 5.4 +/- 0.7 to 4.3 +/- 0.15 mEq/l (p < 0.05) in the experimental and control group respectively. Plasma creatinine was normal, checked only at the end of the experiment. Our results demonstrate that a relationship exists between PCSF variations and plasma ADH concentration. We believe this relationship is due to the pressure receptors in the cerebral ventricles or in structures connected to it, such as the inner ear, and we hypothesize the existence of a control system of body fluids, more diffused than though to be, up till now.

Urinary excretion of aquaporin-2 in humans: a potential marker of collecting duct responsiveness to vasopressin.

The vasopressin-sensitive water channel (aquaporin 2; AQP-2) mediates water transport across the apical plasma membrane of the renal collecting ducts and is excreted in human urine. This study presents the hypothesis that measurements of the AQP-2 excretion rate might be used as a marker of collecting-duct responsiveness to vasopressin, and therefore could be useful in the clinical evaluation of various water-balance disorders. This study presents information about the development of an antibody to human AQP-2, and measures the urinary excretion of AQP-2 by quantitative Western analysis. A standard curve of band densities was generated by using known quantities of the modified immunizing peptide to derive the amount of AQP-2 contained in aliquots of urine. AQP-2 urinary excretion changed with short-term alterations in hydration status produced either by water loading (76% decrease, P < 0.01) or by 3% sodium chloride (760% increase, P < 0.01). Steady-state 24-h urinary excretion of AQP-2 was 43 +/- 10 nmol/24 h (or 28.5 +/- 6.9 pmol/mg creatinine), and 20 +/- 6 nmol/24 h (or 18.3 +/- 7.9 pmol/mg creatinine) in men and women, respectively. Therefore, urinary AQP-2 excretion can be quantified by using Western analysis, and may serve as a marker of collecting-duct responsiveness to vasopressin in different physiologic settings.

Screening of potential transport systems for methyl mercury uptake in rat erythrocytes at 5 degrees by use of inhibitors and substrates.

The current study was designed to screen the potential transport systems for methyl mercury (MeHg) uptake by isolated erythrocytes from rats at 5 degrees. Several inhibitors and substrates were used to test which potential transport system might be involved in MeHg uptake. Probenecid was used to test the organic anion transport system, valinomycin was used to test the effect of the membrane potential, D-glucose and cytochalasin B were used to test the facilitated diffusive D-glucose transport system and colchicine and vinblastine were used to test the microtubule system. The effects of Ca++, Mg++ and Na+ on MeHg uptake have been examined. Ouabain, ATP and glucose were used to test the active transport system, cysteine for the cysteine-facilitated transport system, glycine for system Gly, DL-methionine for system L, and MeHgCl and 4',4-diisothiocyano-2',2-stilbenedisulfonic acid (DIDS) for the Cl- ion transport system. The results showed that MeHg uptake might be involved in the following transport systems at 5 degrees: 1) organic anion transport system; 2) facilitated diffusive D-glucose transport system; 3) cysteine-facilitated transport system; 4) Cl- ion transport system. Moreover, the transport systems for MeHg uptake were sensitive to the membrane potential. Although the mechanisms of interaction of transport systems have not been fully clarified, evidence has been presented which support the existence of several simultaneous transport systems for MeHg uptake.