Monitoring rFVIIa 90 µg kg⁻¹ dosing in haemophiliacs: comparing laboratory response using various whole blood assays over 6 h.

Recombinant FVIIa is a haemostatic agent administered to patients with severe FVIII or FIX deficiency with inhibitors. Although rFVIIa is effective at stopping bleeding, a reliable assay to monitor its effect is lacking. To characterize the pharmacokinetics and global coagulation effects of rFVIIa for 6 h following a IV dose of 90 µg kg⁻¹. Ten non-bleeding subjects with severe FVIII or FIX deficiency were infused with a single-dose of rFVIIa 90 µg k⁻¹ body weight and blood was collected before and at 0.5, 1, 2, 4 and 6 h postdose. Global haemostasis was characterized throughout the study utilizing whole blood analyses (Hemodyne HAS, TEG, ROTEM). The clearance and half-life of factor FVII:C was estimated as 39.0 ± 8.8 mL h⁻¹ kg⁻¹ and 2.1 ± 0.2 h respectively. There was good inter-assay agreement with respect to clot initiation parameters (R, CT and FOT) and these parameters all fell to a mean of approximately 9 min following rFVIIa dosing. The platelet contractile force (PCF) and clot elastic modulus (CEM) were positively correlated to FVII:C (P < 0.0001), and these parameters were dynamic throughout the 6-h period. The MA and MCF did not correlate to FVII:C nor did they significantly change during the study. Prothrombin F1 + 2 significantly increased following rFVIIa dosing (P < 0.001), but remained steady throughout the study. There was no change in D-dimer concentrations over time. The FOT, R and CT characterized clot initiation following rFVIIa dosing. The PCF and CEM were correlated to FVII:C and characterized the dynamics of platelet function and clot strength over the rFVIIa dosing interval. The clinical significance of these findings needs additional study.

Active transport of nitrofurantoin into human milk.

STUDY OBJECTIVE: To determine the extent to which nitrofurantoin is transferred into human milk. DESIGN: Prospective, single-dose pharmacokinetic study. SETTING: University-affiliated clinical research center. PATIENTS: Four healthy lactating women 8-26 weeks postpartum. INTERVENTION: All subjects received a single, oral, 100-mg dose of nitrofurantoin macrocrystals with food. Serial serum and milk samples were obtained and analyzed by high-performance liquid chromatography. MEASUREMENTS AND MAIN RESULTS: Milk pH, milk fat partitioning, and protein binding in serum and milk were determined. Predicted milk:serum ratio (M:S) was compared with the observed M:S. Nitrofurantoin M:S predicted was 0.28+/-0.05, whereas M:S observed was 6.21+/-2.71. Average milk concentration was 1.3 mg/L, and estimated suckling infant dosage was 0.2 mg/kg/day or 6% of maternal dose (mg/kg). CONCLUSIONS: Nitrofurantoin is actively transported into human milk, achieving concentrations in milk greatly exceeding those in serum. Concern is warranted for suckling infants younger than 1 month old, or for infants with a high frequency of glucose-6-phosphate dehydrogenase deficiency or sensitivity to nitrofurantoin.

Influence of lysine on cimetidine uptake and on excretion of cimetidine by the rat mammary gland.

Cimetidine is actively transported into human and rat milk. However, the transporters involved have not been characterized. It is possible that xenobiotics may be actively transported into milk by an amino acid transport system. The objective of these studies was to determine the influence of lysine on the uptake of cimetidine into rat mammary explants (study 1), and on the excretion of cimetidine into rat milk (study 2). In study 1, excised lactating rat mammary epithelial tissue fragments were exposed to 3H-cimetidine and 14C-lysine in the presence of 10 microM, 1 mM, or 1 M cold lysine, and the uptake of 3H-cimetidine and 14C-lysine were measured by liquid scintillation counting after 5 or 20 minutes of incubation. After 5 minutes of incubation, 1 M lysine inhibited 3H-cimetidine uptake by 47.7% (SD +/- 6.5%), compared with 10 microM lysine (P < 0.05), and 14C-lysine uptake was also inhibited by 54.1% (SD +/- 6.4%) (P < 0.05). Similar results were seen after 20 minutes of incubation. In a randomized crossover study (study 2), 6 lactating female rats were infused to steady state with cimetidine (0.5mg/h) in the presence or absence of lysine (360mg/h). Cimetidine concentrations in serum and milk were determined by high-performance liquid chromatography. Cimetidine systemic clearance (28.6+/-15.0mL/kg/min vs. 38.9+/-3.9 mL/kg/min, mean +/- SD) and milk to serum cimetidine ratio (M/S) (28.0+/-16.1 vs. 28.9+/-6.7), respectively, were not significantly altered by the presence or absence of lysine. Although 1 M lysine inhibited uptake of cimetidine in rat mammary explants, the concentrations of lysine used in this study, which approached toxicity in vivo, produced no significant effects on cimetidine transport into milk or the systemic clearance of cimetidine.

The role of protein synthesis and degradation in the post-transcriptional regulation of rat multidrug resistance-associated protein 2 (Mrp2, Abcc2).

Multidrug resistance-associated protein 2 (Mrp2, Abcc2), an organic anion transporter present in the apical membrane of hepatocytes, renal epithelial cells, and enterocytes, is postulated to undergo post-transcriptional regulation. We hypothesized that Mrp2 protein undergoes altered rates of protein synthesis or degradation consistent with different Mrp2 protein expression. We analyzed Mrp2 synthesis, expression, and degradation in control female, 19- and 20-day pregnant, and pregnenolone-16alpha-carbonitrile (PCN)-treated rats using in vivo metabolic-labeling studies with [35S]cysteine/methionine or [14C]NaHCO3, polysomal distribution analyses and ribonuclease protection assays (RPA). Mrp2 protein was significantly increased in rats treated with PCN for 2 days but significantly decreased in 19-day pregnant rats relative to controls; no significant differences were observed in Mrp2 mRNA expression among these groups. The measured half-lives of 14C-labeled Mrp2 in control, pregnant, and PCN-treated rats were 27, 36, and 22 h, respectively, and were not significantly different. The rate of incorporation of 35S into Mrp2 was highest in PCN-treated rats. Polysomal distribution analysis of Mrp2 mRNA was consistent with increased Mrp2 protein synthesis after PCN treatment. The major transcription-initiation site for rat liver determined by RPA was -98 nucleotides (nt), with other start sites observed at -213, -163, -132, and -71 nt; use of transcription sites did not differ among the groups. Differences in the degradation of Mrp2 protein cannot explain the post-transcriptional regulation of Mrp2 in control, pregnant, and PCN-treated rats. Rather, the observed difference in protein synthesis suggests an intrinsic role for the translational regulation of rat Mrp2 protein.

Interactions between cimetidine, nitrofurantoin, and probenecid active transport into rat milk.

The purpose of these studies was to further elucidate the active mammary epithelial transport processes for the organic cation cimetidine and the organic anion nitrofurantoin and to determine which of the identified rat organic anion (rOATs) and organic cation (rOCTs) transporters may be responsible for transport of these drugs into milk. Milk-to-serum ratios (M/S) were predicted in vitro for nitrofurantoin, p-aminohippurate (PAH), and probenecid, and were compared with the observed M/S values. Groups of six lactating female rats received intravenous infusions of cimetidine, nitrofurantoin, PAH, or probenecid alone and with another agent. Steady-state milk and serum concentrations were measured by high performance liquid chromatography. Reverse transcriptase-polymerase chain reaction was performed to detect rOATs and rOCTs in livers, kidneys, and mammary glands of lactating rats. Nitrofurantoin and probenecid were actively transported into rat milk with an M/S 100- and 4.7-fold greater than predicted, respectively, but predicted and observed M/S values for PAH were similar. The cimetidine infusion did not alter nitrofurantoin M/S. Nitrofurantoin significantly decreased M/S of cimetidine (26.6 +/- 4.9 versus 17.7 +/- 5.6). Probenecid did not alter the M/S of nitrofurantoin, or PAH, but increased the M/S of cimetidine from 15.5 +/- 3.6 to 21.5 +/- 7.7. Of the six transporter genes, evidence of expression in lactating rat mammary tissue was found for only rOCT1 and rOCT3. The results suggest different secretory transport systems for cimetidine, nitrofurantoin, and probenecid, but that passive diffusion governs PAH passage into milk. The products of rOCT1 and rOCT3 might transport these drugs into milk.

Effects of naltrexone pellet implantation on morphine tolerance and physical dependence in the rat.

1. The effect of naltrexone pellets containing either 10 or 30 mg of naltrexone base on the development of tolerance and physical dependence on morphine was assessed in male Sprague-Dawley rats. Tolerance-dependence on morphine was induced by s.c. implantation of six morphine pellets, each containing 75 mg morphine base for 7 days. 2. Naltrexone pellet implantation blocked the development of tolerance to the analgesic and hyperthermic effects of morphine. Similarly, naltrexone pellet implantation reversed morphine withdrawal-induced body weight loss. The effect of pellets containing 10 and 30 mg naltrexone did not differ. 3. The effect of naltrexone (10 mg) pellet implantation on various signs of naltrexone-precipitated withdrawal such as body weight loss, hypothermia and increases in urinary and fecal output was investigated. Naltrexone pellet implantation did not alter the naltrexone-precipitated withdrawal-induced body weight loss. Concurrent naltrexone pellet implantation blocked the naltrexone-precipitated withdrawal-induced hypothermia, increased fecal and urinary output in morphine-dependent rats. 4. These results indicate that a single pellet of 10 mg of naltrexone can effectively block morphine tolerance and physical dependence in the rat. Such a procedure may be useful in studying biochemical, endocrinological and immunological mechanisms involved in opioid addiction processes.