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Tucatinib, a tyrosine kinase inhibitor of HER2, is approved in multiple regions for metastatic breast cancer and is being evaluated in metastatic colorectal and gastric cancers. During clinical development, quantification of tucatinib plasma concentrations for pharmacokinetic analysis was performed using MS/MS analysis by three laboratories using five different methods. Cross-validation was required to confirm data across laboratories were comparable. A five-way cross-validation procedure was developed where bioanalysis performed by one laboratory and method was used as a 'base' against which the other methods were validated. This cross-validation method provides an alternative to multiple head-to-head comparisons between two methods, and enabled combination of data from multiple tucatinib clinical trials for a single population pharmacokinetic analysis.

The purpose of the current investigation was to optimize an intranasal (IN) galantamine (an acetylcholinesterase inhibitor used for treatment of Alzheimer's disease) formulation using an in vitro tissue model, to correlate those results to in vivo bioavailability, and to compare emetic response to oral dosing. A design-of-experiments (DOE) based formulation screening employing an in vitro tissue model of human nasal epithelium was used to assess drug permeability, tight junction modulation, and cellular toxicity. In vivo studies in rats compared pharmacokinetic (PK) profiles of different formulations dosed intranasally. Finally, studies in ferrets evaluated PK and gastrointestinal (GI) related side effects of oral compared to nasal dosage forms. Galantamine permeation was enhanced without increasing cytotoxicity. Pharmacokinetic testing in rats confirmed the improved drug bioavailability and demonstrated an in vitro-in vivo correlation. Compared to oral dosing, IN galantamine resulted in a dramatically lowered incidence of GI-related side effects, e.g., retching and emesis. These findings illustrate that IN delivery represents an attractive alternative to oral dosing for this important Alzheimer's disease therapeutic. To our knowledge, the data herein represent the first direct confirmation of reducing GI-related side effects for IN galantamine compared to oral dosing.

The goal of the current study was to develop an intranasal (IN) formulation of the acetylcholinesterase inhibitor galantamine, an important therapeutic for treating Alzheimer's disease. To allow for delivering a therapeutically relevant dose, it was necessary to greatly enhance drug solubility. Various approaches were examined to this end, including adding co-solvents, cyclodextrins, and counterion exchange. Of these, the latter, for example, replacement of bromide ion with lactate or gluconate, resulted in a dramatic drug solubility increase, more than 12-fold. NMR confirmed the molecular structure of new drug salt forms. An in vitro epithelial tissue model was used to assess drug permeability and cellular toxicity. In vitro, galantamine lactate formulations performed as well as or better than their hydrobromide (HBr) counterparts with respect to drug permeation across the epithelial membrane with minimal toxicity. In vivo studies in rats compared pharmacokinetic (PK) profiles of different formulations. The in vivo studies confirmed that IN galantamine achieves systemic blood levels comparable to those of conventional oral administration. Both the in vitro and in vivo data support the feasibility of IN administration of this important drug., ((c) 2005 Wiley-Liss, Inc. and the American Pharmacists Association)

The purpose of this study was to assess whether proximal renal tubules generate excess hydroxyl radical (.OH) during hypoxia/reoxygenation or ischemia/reperfusion injury, thereby supporting the hypothesis that reactive oxygen species contribute to the pathogenesis of postischemic acute renal failure. In the first phase of the study, rat isolated proximal tubular segments (PTS) were subjected to hypoxia (95% N2- 5% CO2) for 15, 30, or 45 min, followed by 15 to 30 min of reoxygenation in the presence of sodium salicylate, a stable .OH trap. Cellular injury after hypoxia and reoxygenation was assessed by lactate dehydrogenase release; .OH production was gauged by hydroxylated salicylate by-product generation (2,3-, 2,5-dihydroxybenzoic acids (DHBA); quantified by HPLC/electrochemical detection). Continuously oxygenated PTS served as controls. Despite substantial lactate dehydrogenase release during hypoxia (8 to 46%) and reoxygenation (8 to 11%), DHBA production did not exceed that of the coincubated, continuously oxygenated control PTS. In the second phase of the study, salicylate-treated rats were subjected to 25 or 40 min of renal arterial occlusion +/- 15 min of reperfusion. No increase in renal DHBA concentrations occurred during ischemia or reperfusion, compared with that in sham-operated controls. To validate the salicylate trap method, PTS were incubated with a known .OH-generating system (Fe2+/Fe3+); in addition, rats were treated with antioxidant interventions (oxypurinol plus dimethylthiourea). Fe caused marked DHBA production, and the antioxidants halved in vivo DHBA generation. In conclusion, these results suggest that exaggerated .OH production is not a consequence of O2 deprivation/reoxygenation tubular injury.

Recent in vivo studies suggest that heme Fe causes proximal tubular lipid peroxidation and cytotoxicity, thereby contributing to the pathogenesis of myoglobinuric (Mgb) acute renal failure. Because hydroxyl radical (.OH) scavengers [dimethylthiourea (DMTU), benzoate, mannitol] can mitigate this injury, it is postulated that .OH is a mediator of Mgb-induced renal damage. The present study has tested these hypotheses using an isolated rat proximal tubular segment (PTS) system. An equal mixture of Fe2+/Fe3+ (4 mM total), when added to PTS, caused marked cytotoxicity [as defined by lactate dehydrogenase (LDH) release] and lipid peroxidation [assessed by malondialdehyde (MDA) increments]. Fe2+ or Fe3+ alone each induced massive MDA elevations, but only Fe2+ caused cytotoxicity. Although both DMTU and benzoate decreased LDH release during the Fe2+/Fe3+ challenge, mannitol and GSH did not, despite equivalent reductions in .OH (gauged by the salicylate trap method). GSH and catalase (but not DMTU, benzoate, or mannitol) decreased MDA concentrations, suggesting the Fe-driven lipid peroxidation was more H2O2 than .OH dependent. Deferoxamine totally blocked Fe-induced LDH release, even under conditions in which it caused an apparent increase in .OH generation. Mgb paradoxically protected against Fe-mediated PTS injury, an effect largely reproduced by albumin. In conclusion, these data suggest that: (a) Fe can cause PTS lipid peroxidation and cytotoxicity by a non-.OH-dependent mechanism; (b) Fe-mediated cytotoxicity and lipid peroxidation are not necessarily linked; and (c) Mgb paradoxically protects PTS against Fe-mediated injury, suggesting that: (i) Mgb Fe may require liberation from its porphyrin ring before exerting toxicity; and (ii) the protein residue may blunt the resulting injury.

We performed a prospective clinical evaluation of our newly developed fluorescence polarization procedure to predict fetal lung maturity (Clin Chem 1986;32:248-54). Net fluorescence polarization was measured at 34 degrees C after a 6.5-min incubation of amniotic fluid with fluorophore. For the 26 cases of neonatal respiratory distress syndrome encountered in 196 deliveries, the net polarization exceeded 0.287 for 22 (85%) of these, and exceeded 0.260 for all 26. The specificity of the polarization assay equaled or exceeded the specificity of the lecithin/sphingomyelin ratio for all sensitivities greater than 70%. Neither assay was a good predictor of the clinical severity of respiratory distress. For a separate group of 21 amniotic fluid specimens clinically contaminated with blood or meconium, the discriminatory power of the polarization assay was decreased, but six of seven respiratory-distress cases still had polarization values greater than 0.260. We conclude that this fluorescence polarization assay is a better overall predictor of fetal lung maturity than is the lecithin/sphingomyelin ratio, and that polarization values less than 0.260 are associated with little risk of respiratory distress.

One feature of fertilization is the alteration of the vitelline layer, by components released from the egg, to produce an elevated, covalently crosslinked, hard, insoluble, fertilization membrane. The following evidence indicates that crosslinking and hardening are caused by the production of diand trityrosyl residues, by oxidation of protein-bound tyrosyl residues in the presence of a peroxidase. Hardening of the fertilization membrane, as evidenced by its loss of solubility in 50 mM dithiothreitol, is inhibited by compounds known to inhibit many peroxidases. A peroxidase, here called the ovoperoxidase, is released from eggs at fertilization. This enzyme is inhibited by the same compounds that inhibit hardening and at similar concentrations. Inhibitors of the ovoperoxidase and the hardening reaction include KCN, 3-amino-1,2,4-triazole, NaN(3), phenylhydrazine, K(4)Fe(CN)(6), sodium sulfite, and glycine ethyl ester. In addition, tyramine and N-acetyltyrosine both inhibit hardening, but O-methyltyrosine does not. Dityrosyl and trityrosyl residues are found in acid hydrolysates of isolated, hardened fertilization membranes. These residues have been identified by cellulose phosphate column chromatography, thin-layer chromatography, and amino acid analysis. The amino acid data have been used to estimate that there is one dityrosine crosslink per 55,000 daltons of protein. We suggest that, by catalyzing the crosslinking of tyrosyl residues, the ovoperoxidase leads to the production of a hard fertilization membrane that blocks the entry of additional sperm. Because peroxidases are spermicidal, a secondary function of the enzyme could be to kill sperm in the vicinity of the fertilized egg.

Lipovitellin, very low density lipoproteins (VLDL), low density lipoproteins (LDL), high density lipoproteins (HDL), and proteins of d greater than 1.20 were isolated from blood plasma and egg yolks obtained from hens fed a normal diet or one containing 2.5% of crude cottonseed oil. The amounts and compositions of each fraction were determined. Hen blood plasma and egg yolk VLDL and LDL obtained from hens fed a normal diet contained similar levels of lipid, and the fatty acid compositions of those lipids were, for the most part, similar. The percentages of VLDL and LDL in total lipoproteins were similar for plasma and egg yolk obtained from hens fed the normal diet. Separation of VLDL from LDL was not clear-cut in eggs from hens fed the diet which contained crude cottonseed oil. Lipovitellin, as it is isolated from egg yolk, did not appear to be present in any appreciable amount in hen blood plasma. Hen plasma appeared to contain about 10% of a liproprotein d greater than 1.20, the lipid of which was similar in fatty acid composition to that of lipovitellin except for palmitic and oleic acids. Lipids of all the different lipoproteins isolated from plasma and egg yolks of hens fed diets which contained 2.5% of crude cottonseed oil contained more stearic acid and less palmitoleic and oleic acids than did those from normal hens. The increased content of stearic acid increased the density of the lipoproteins so that a larger proportion of the lipoproteins were in the LDL and a smaller proportion were in the VLDL than in lipoproteins from normal plasma and eggs.

We clinically evaluated, retrospectively, our improved fluorescence polarization assay for fetal lung maturity. The procedure requires 0.5 mL of amniotic fluid and a standard clinical laboratory fluorescence polarimeter (TDx Analyzer, Abbott Laboratories). We measured the L/S ratios for 93 freshly collected amniotic fluids, uncontaminated with blood or meconium, collected within three days of delivery. The fluids were stored frozen for eight to 32 months, then thawed and assayed for net fluorescence polarization. Fourteen of the infants developed respiratory distress syndrome; five, transient tachypnea of the newborn; and 74, no respiratory distress. The polarization assay and lecithin/sphingomyelin ratio had equivalent receiver operating characteristic curves, indicating no difference in their clinical performance. Although a prospective study with fresh amniotic fluid specimens will be necessary to establish a definitive reference range, the present study shows that this assay can be used to rapidly predict fetal lung maturity.

At the time of fertilization, sea urchin eggs release a peroxidase which, together with H2O2 generated by a respiratory burst, is responsible for hardening of the fertilization membrane. We demonstrate here that the ovoperoxidase of unfertilized eggs is located in cortical granules and, after fertilization, is concentrated in the fertilization membrane. Fertilization of sea urchin eggs or their parthenogenetic activation with the ionophor A23187 also results in (a) the conversion of iodide to a trichloroacetic acid-precipitable form (iodination), (b) the deiodination of eggs exogenously labeled with myeloperoxidase and H2O2, (c) the degradation of thyroxine as measured by the recovery of the released radioiodine at the origin and in the inorganic iodide spot on paper chromatography, and (d) the conversion of estradiol to an alcohol-precipitable form (estrogen binding). The iodination reaction and the binding of estradio occurs predominantly in the fertilization membrane where the ovoperoxidase is concentrated. From the estimation of the kinetics of incorporation of iodine, we determine that the peroxidative system is active for 30 min after fertilization, long after hardening of the fertilization membrane is complete. Most of the bound iodine is lost during the hatching process. Iodination of albumin is catalyzed by the material released from the egg during fertilization, when combined with H2O2 and iodide. Iodination, thyroxine degradation, and estradiol binding are inhibited by azide, cyanide, aminotriazole, methimazole, ascorbic acid and ergothioneine, all of which can inhibit peroxidase-catalyzed reactions. These responses of the sea urchin egg to fertilization are strikingly similar to the changes induced in polymorphonuclear leukocytes by phagocytosis and, in both instances, a peroxidative mechanism may be involved.

We assessed the analytical performance of an improved fluorescence polarization assay for use in evaluating fetal lung maturity and compared results with the lecithin/sphingomyelin ratio. During a three-month period 150 patients' samples were assayed by clinical laboratory personnel with both techniques. Values for the lecithin/sphingomyelin ratio correlate closely with net fluorescence polarization values (r = -0.85), less closely with net fluorescence intensity (r = 0.65). Background fluorescence intensity and polarization varied widely, indicating a need to correct measurements for endogenous fluorescence. Net fluorescence polarization values have a CV of 0.32% within-run, 1.07% between-day. A comparison of two amniotic fluid centrifugation procedures showed no significant difference in such values. For both methods, however, such values are slightly but significantly higher than those obtained for amniotic fluids without prior centrifugation. Short-term storage (less than 30 days) of uncentrifuged amniotic fluid samples at -20 degrees C does not significantly affect results.

After fertilization of the sea urchin, Strongyl-ocentrotus purpuratus, a crosslinked fertilization membrane is formed; the crosslinks (dityrosine residues) are synthesized in a reaction catalyzed by an ovoperoxidase that is released from the cortical granules during fertilization. The substrate for ovoperoxidase activity, hydrogen peroxide, is generated by the egg coincident with the "respiratory burst" that follows parthenogenetic activation by the divalent ionophore A23187 or fertilization. This burst of oxygen consumption may be almost quantitatively accounted for by hydrogen peroxide evolution, as measured by the peroxidase-catalyzed quenching of scopoletin fluorescence. Neither the burst of oxygen consumption nor hydrogen peroxide production occurs when the inhibitor of cortical granule discharge, procaine, is present at fertilization. Fertilization or parthenogenetic activation with A23187 also is associated with a burst of light emission. This chemiluminescence is inhibited in vivo by inhibitors of the ovoperoxidase, such as 3-amino-1,2,4-triazole, phenylhydrazine, sulfite, or azide. A crude ovoperoxidase preparation catalyzes hydrogen peroxide-dependent chemiluminescence that is similarly inhibited. Thus, the bursts of oxygen uptake, peroxide production, and chemiluminescence appear to be several manifestations of the peroxidative system released at fertilization. This system may additionally be responsible for spermicidal activity and thus may act as a component of the block to polyspermy.