Development and validation of a bioanalytical method for the quantification of CHF6550 and its metabolite (CHF6671) in rat plasma and lung homogenate using LC-MS/MS.

A selective and sensitive liquid chromatography-tandem mass spectrometry method was developed and validated for accurate determination of CHF6550 and its main metabolite in rat plasma and lung homogenate samples. All biological samples were prepared by simple protein precipitation method using deuterated internal standards. The analytes were separated on a HSS T3 analytical column with 3.2 min run time at flow rate of 0.5 mL/min. The detection was performed on a triple-quadrupole tandem mass spectrometer equipped with positive-ion electrospray ionization by selected-reaction monitoring of the transitions at m/z 735.3 â†’ 98.0 for CHF6550 and m/z 638.3 â†’ 319.2 and 638.3 â†’ 376.2 for CHF6671. The calibration curves for plasma samples were linear between 50 and 50000 pg/mL for both analytes. The calibration curves for lung homogenate samples were linear within 0.1-100 ng/mL for CHF6550 and 0.3-300 ng/mL for CHF6671. The method was successfully applied to a 4-week toxicity study.

Validation of an LC-MS/MS method for the quantification IOA-289 in human plasma and its application in a first-in-human clinical trial.

IOA-289 is a novel small molecule inhibitor of autotaxin developed as a first-in-class therapy of fibrotic pathologies including cancer. A method for quantitation of IOA-289 in human plasma was developed using a stable isotope labeled compound ([13C4]IOA-289) as internal standard. The analytes were extracted from human plasma by protein precipitation and the analysis was performed by liquid chromatography coupled with tandem mass spectrometric detection (LCMS/MS). The chromatographic separation was performed with a gradient elution from a BEH C18 column and under these conditions the retention time and the run time were 1 and 2 min, respectively. The assay was fully validated over the range 3-3000 ng/mL, proved to be accurate, precise and selective and was successfully applied to quantitate IOA-289 in plasma samples from subjects in a first-in-humanclinical trial.

Bispecific antibody target pair discovery by high-throughput phenotypic screening using in vitro combinatorial Fab libraries.

Bispecific antibodies can uniquely influence cellular responses, but selecting target combinations for optimal functional activity remains challenging. Here we describe a high-throughput, combinatorial, phenotypic screening approach using a new bispecific antibody target discovery format, allowing screening of hundreds of target combinations. Simple in vitro mixing of Fab-fusion proteins from a diverse library enables the generation of thousands of screen-ready bispecific antibodies for high-throughput, biologically relevant assays. We identified an obligate bispecific co-targeting CD79a/b and CD22 as a potent inhibitor of human B cell activation from a short-term flow cytometry signaling assay. A long-term, high-content imaging assay identified anti-integrin bispecific inhibitors of human cell matrix accumulation targeting integrins ß1 and ß6 or αV and ß1. In all cases, functional activity was conserved from the bispecific screening format to a therapeutically relevant format. We also introduce a broader type of mechanistic screen whereby functional modulation of different cell subsets in peripheral blood mononuclear cells was evaluated simultaneously. We identified bispecific antibodies capable of activating different T cell subsets of potential interest for applications in oncology or infectious disease, as well as bispecifics abrogating T cell activity of potential interest to autoimmune or inflammatory disease. The bispecific target pair discovery technology described herein offers access to new target biology and unique bispecific therapeutic opportunities in diverse disease indications.

Evolutionary history of the Cameroon radiation of puddle frogs (Phrynobatrachidae: Phrynobatrachus), with descriptions of two critically endangered new species from the northern Cameroon Volcanic Line.

The Cameroon Volcanic Line, a mountain chain located between West and Central Africa, is a region of numerous endemic diversifications, including of puddle frogs (Phrynobatrachus). This study reviews the phylogeny and taxonomy of puddle frogs of the "Cameroon radiation," which is a clade containing mainly montane but also at least three lowland species. Molecular data revealed a novel evolutionary lineage from high altitudes in the northern part of the mountains. Puddle frogs from the new, minute-sized (SVL < 20 mm) lineage are identified using molecular, morphological and acoustic data and described as two new species, Phrynobatrachus arcanus sp. nov. (Gotel Mountains, Cameroon-Nigeria) and P. mbabo sp. nov. (Tchabal Mbabo, Cameroon). The tadpole of the first species is also described. Phylogenetic analyses placed the new lineage to the proximity of the recently described lowland small-sized taxa (P. horsti, P. ruthbeateae). Based on the inferred phylogeny, we propose five species groups within the Cameroon radiation: P. arcanus, P. chukuchuku, P. ruthbeateae, P. steindachneri, and P. werneri. The taxonomically enigmatic P. hylaios is proposed to be a member of the P. ruthbeateae species group. The basal radiation evolved during the late Miocene with subsequent diversifications occurring during the Pliocene, while closely related terminal taxa originated during the Pleistocene. We recommend that the newly described species are categorized as Critically Endangered due to their limited ranges and because recent surveys did not identify any individuals at the type localities. This further supports the need for conservation interventions in the mountains of Cameroon and Nigeria.

Development and validation of a bioanalytical method for quantification of LNA-i-miR-221, a 13-mer oligonucleotide, in rat plasma using LC-MS/MS.

LNA-i-miR-221, a 13-mer oligonucleotide, is a new miR-221 inhibitor that could be used as a novel drug for multiple myeloma. Herein, an ion-pair reversed phase liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has been developed and validated for the quantification of LNA-i-miR-221 in rat plasma. Plasma samples were prepared with an initial phenol/chloroform/isoamyl alcohol liquid-liquid extraction followed by a solid phase extraction. Chromatographic separation was performed with a gradient system on a HALO C18 column using hexafluoro-2-propanol/triethylamine buffer and methanol as mobile phase at a flow rate of 0.4 mL/min. Under these conditions LNA-i-miR-221 and the analogue internal standard are co-eluted at 1.2 min. The detection was carried out in multiple reaction monitoring (MRM) mode using a negative electrospray ionization (ESI) interface. The assay showed a good linearity within the calibration range 10-10000 ng/mL. The precision, accuracy, and recovery values were found to be <15% (<20% at LLOQ), 100 ±â€¯15%, and 97.6-103.7%, respectively. This method was successfully applied to measure the concentrations of LNA-i-miR-221 in plasma samples following the intravenous administration during a 4-week toxicity study in rats.

Host preference, population growth and injuries assessment of Polyphagotarsonemus latus (banks) (ACARI: Tarsonemidae) on Capsicum annuum L. Genotypes.

Despite the continued efforts on the search for different genotypes, Capsicum annuum (L.) is quite susceptible to attack by pest arthropods, especially the broad mite Polyphagotarsonemus latus Banks. Thus, the host preference, population growth and the injuries assessment of P. latus was studied on six C. annuum genotypes used in Brazil (Atlantis, California Wonder, Impact, Palloma, Rubia and Tendence). Host preference was accessed in choice tests, pairing the several genotypes, and the population growth was observed through non-choice tests in laboratory. The injuries assessments were evaluated in the greenhouse, comparing the injury level among the six genotypes. The results indicate that California Wonder and Palloma genotypes were more preferred by P. latus, and Impact and Tendence were less preferred. P. latus presented positive population growth rates (ri) on all the genotypes, however, Palloma and California Wonder showed the highest values of population growth rate (ri = 0.344 and ri = 0.340, respectively), while Impact had the lowest value (ri = 0.281). All the evaluated C. annuum genotypes showed low tolerance to P. latus and exhibited several injuries, but there was no statistical difference between them. California Wonder had the highest average number of mites/leaf (57.15), while Impact and Tendence obtained the lowest values (36.67 and 35.12, respectively) at the end of the evaluation period. The total average of injuries notes at the end of the bioassay did not differ between the genotypes. The number of mites/leaf was growing for the injury scale to the note 3.0, but when the injury scale approached the note 4.0, there was observed a decrease in the number of mites/leaf for all the genotypes.

Biomarker responses to estrogen and androgen exposure in the brook stickleback (Culaea inconstans): A new bioindicator species for endocrine disrupting compounds.

Small-bodied freshwater fish are commonly used in regulatory testing for endocrine disrupting compounds (EDCs) but most lack a sensitive and quantifiable androgen-specific biomarker. Brook stickleback (Culaea inconstans) are a North American freshwater fish whose males produce an androgen-regulated glycoprotein in the kidney called spiggin. Although spiggin induction in females has been used as an androgen-specific biomarker of exposure in other stickleback species it has not been characterized in brook stickleback. Therefore, our objective was to develop a bioassay using brook stickleback to measure estrogenic and androgenic responses and establish the sensitivity of traditional and novel biomarkers of exposure. We first developed and optimized a qPCR assay to measure spiggin and vitellogenin transcript levels in kidney and liver tissue, respectively. Basal levels were differentially expressed in mature wild-caught male and female brook stickleback. To determine their sensitivity to EDCs, fish were exposed to nominal concentrations of 1, 10 and 100ng/L of 17α-methyltestosterone (MT) or 17α-ethinylestradiol (EE2) for 21days (sampled at 7 and 21days) under semi-static renewal conditions. MT and EE2 exposure induced spiggin and vitellogenin transcripts in female kidneys and male livers, respectively. Exposure to EE2 also increased hepatosomatic index in both sexes and decreased gonadosomatic index in females. Histopathological alterations were observed in the kidney of EE2-exposed fish and an increase in kidney epithelium cell height occurred in MT-exposed females. Given the sensitivity of these endpoints, the brook stickleback is a promising new freshwater fish model for EDC evaluation and a potential bioindicator for EDCs in North American freshwater environments.

Remote real-time monitoring of subsurface landfill gas migration.

The cost of monitoring greenhouse gas emissions from landfill sites is of major concern for regulatory authorities. The current monitoring procedure is recognised as labour intensive, requiring agency inspectors to physically travel to perimeter borehole wells in rough terrain and manually measure gas concentration levels with expensive hand-held instrumentation. In this article we present a cost-effective and efficient system for remotely monitoring landfill subsurface migration of methane and carbon dioxide concentration levels. Based purely on an autonomous sensing architecture, the proposed sensing platform was capable of performing complex analytical measurements in situ and successfully communicating the data remotely to a cloud database. A web tool was developed to present the sensed data to relevant stakeholders. We report our experiences in deploying such an approach in the field over a period of approximately 16 months.

Secretory state regulates Zn2+ transport in gastric parietal cell of the rabbit.

Secretory compartments of neurons, endocrine cells, and exocrine glands are acidic and contain high levels of labile Zn2+. Previously, we reported evidence that acidity is regulated, in part, by the content of Zn2+ in the secretory [i.e., tubulovesicle (TV)] compartment of the acid-secreting gastric parietal cell. Here we report studies focusing on the mechanisms of Zn2+ transport by the TV compartment in the mammalian (rabbit) gastric parietal cell. Uptake of Zn2+ by isolated TV structures was monitored with a novel application of the fluorescent Zn2+ reporter N-(6-methoxy-8-quinolyl)-para-toluenesulfonamide (TSQ). Uptake was suppressed by removal of external ATP or blockade of H+-K+-ATPase that mediates luminal acid secretion. Uptake was diminished with dissipation of the proton gradient across the TV membrane, suggesting Zn2+/H+ antiport as the connection between Zn2+ uptake and acidity in the TV lumen. In isolated gastric glands loaded with the reporter fluozin-3, inhibition of H+-K+-ATPase arrested the flow of Zn(2+) from the cytoplasm to the TV compartment and secretory stimulation with forskolin enhanced vectorial movement of cytoplasmic Zn2+ into the tubulovesicle/lumen (TV/L) compartment. Our findings suggest that Zn2+ accumulation in the TV/L compartment is physiologically coupled to secretion of acid. These findings offer novel insight into mechanisms regulating Zn2+ homeostasis in the gastric parietal cell and potentially other cells in which acidic subcellular compartments serve signature functional roles.

Differential involvement of Galpha16 in CC chemokine-induced stimulation of phospholipase Cbeta, ERK, and chemotaxis.

Chemokines are known to regulate the chemotaxis of leukocytes and play an important role in immunological processes. Chemokine receptors are widely distributed in hematopoietic cells and are often co-localized with the hematopoietic-specific G(16) and its close relative, G(14). Yet, many chemokine receptors utilize pertussis toxin-sensitive G(i) proteins for signaling. Given that both G(16) and G(14) are capable of linking G(i)-coupled receptors to the stimulation of phospholipase Cbeta, we examined the capacity of six CC chemokine receptors (CCR1, CCR2a, CCR2b, CCR3, CCR5 and CCR7) to interact with G(14) and G(16) in a heterologous expression system. Among the CC chemokine receptors tested, CCR1, CCR2b, and CCR3 were capable of mediating chemokine-induced stimulation of phospholipase Cbeta via either G(14) or G(16). The G(14)/G(16)-mediated responses exhibited CC chemokine dose-dependency and were resistant to pertussis toxin (PTX) treatment. In contrast, CCR2a, CCR5 and CCR7 were unable to interact with G(14) and G(16). Under identical experimental conditions, all six CC chemokine receptors were fully capable of inhibiting adenylyl cyclase via G(i) as well as stimulating phospholipase Cbeta via 16z44, a G(16/z) chimera that possesses increased promiscuity toward G(i)-coupled receptors. Moreover, CCR1-mediated ERK1/2 phosphorylation was largely PTX-insensitive in THP-1 monocytic cells that endogenously express Galpha(16). In addition, CCR1 agonist was less efficacious in mediating chemotaxis of THP-1 cells following the knockdown of Galpha(16) by overexpressing siRNA, indicating the participation of Galpha(16) in CCR1-induced cell migration. These results show that different CC chemokine receptors can discriminate against G(14) and G(16) for signal transduction.

Thiol-oxidant monochloramine mobilizes intracellular Ca2+ in parietal cells of rabbit gastric glands.

In Helicobacter pylori-induced gastritis, oxidants are generated through the interactions of bacteria in the lumen, activated granulocytes, and cells of the gastric mucosa. In this study we explored the ability of one such class of oxidants, represented by monochloramine (NH(2)Cl), to serve as agonists of Ca(2+) accumulation within the parietal cell of the gastric gland. Individual gastric glands isolated from rabbit mucosa were loaded with fluorescent reporters for Ca(2+) in the cytoplasm (fura-2 AM) or intracellular stores (mag-fura-2 AM). Conditions were adjusted to screen out contributions from metal cations such as Zn(2+), for which these reporters have affinity. Exposure to NH(2)Cl (up to 200 microM) led to dose-dependent increases in intracellular Ca(2+) concentration ([Ca(2+)](i)), in the range of 200-400 nM above baseline levels. These alterations were prevented by pretreatment with the oxidant scavenger vitamin C or a thiol-reducing agent, dithiothreitol (DTT), which shields intracellular thiol groups from oxidation by chlorinated oxidants. Introduction of vitamin C during ongoing exposure to NH(2)Cl arrested but did not reverse accumulation of Ca(2+) in the cytoplasm. In contrast, introduction of DTT or N-acetylcysteine permitted arrest and partial reversal of the effects of NH(2)Cl. Accumulation of Ca(2+) in the cytoplasm induced by NH(2)Cl is due to release from intracellular stores, entry from the extracellular fluid, and impaired extrusion. Ca(2+)-handling proteins are susceptible to oxidation by chloramines, leading to sustained increases in [Ca(2+)](i). Under certain conditions, NH(2)Cl may act not as an irritant but as an agent that activates intracellular signaling pathways. Anti-NH(2)Cl strategies should take into account different effects of oxidant scavengers and thiol-reducing agents.

An optimized experimental protocol based on neuro-evolutionary algorithms application to the classification of dyspeptic patients and to the prediction of the effectiveness of their treatment.

OBJECTIVE: This paper aims to present a specific optimized experimental protocol (EP) for classification and/or prediction problems. The neuro-evolutionary algorithms on which it is based and its application with two selected real cases are described in detail. The first application addresses the problem of classifying the functional (FD) or organic (OD) forms of dyspepsia; the second relates to the problem of predicting the 6-month follow-up outcome of dyspeptic patients treated by helicobacter pylori (HP) eradication therapy. METHODS AND MATERIAL: The database built by the multicentre observational study, performed in Italy by the NUD-look Study Group, provided the material studied: a collection of data from 861 patients with previously uninvestigated dyspepsia, being referred for upper gastrointestinal endoscopy to 42 Italian Endoscopic Services. The proposed EP makes use of techniques based on advanced neuro-evolutionary systems (NESs) and is structured in phases and steps. The use of specific input selection (IS) and training and testing (T and T) techniques together with genetic doping (GenD) algorithm is described in detail, as well as the steps taken in the two benchmark and optimization protocol phases. RESULTS: In terms of accuracy results, a value of 79.64% was achieved during optimization, with mean benchmark values of 64.90% for the linear discriminant analysis (LDA) and 68.15% for the multi layer perceptron (MLP), for the classification task. A value of 88.61% was achieved during optimization for the prediction task, with mean benchmark values of 49.32% for the LDA and 70.05% for the MLP. CONCLUSIONS: The proposed EP has led to the construction of inductors that are viable and usable on medical data which is representative but highly not linear. In particular, for the classification problem, these new inductors may be effectively used on the basal examination data to support doctors in deciding whether to avoid endoscopic examinations; whereas, in the prediction problem, they may support doctors' decisions about the advisability of eradication therapy. In both cases the variables selected indicate the possibility of reducing the data collection effort and also of providing information that can be used for general investigations on symptom relevance.

Simultaneous determination of estramustine phosphate and its four metabolites in human plasma by liquid chromatography-ionspray mass spectrometry.

A sensitive and selective method, using liquid chromatography-ionspray mass spectrometry, was developed and validated for the simultaneous determination of Estracyt (estramustine phosphate) and its four metabolites, estramustine, estromustine, estrone and estradiol, in human plasma. Deuterated internal standards were available for all analytes. The five compounds were extracted from plasma by protein precipitation with acetonitrile. The chromatographic separation was performed using a Zorbax SB C18, (150 x 4.6 mm i.d., 5 microm) reversed-phase column under gradient conditions with a mobile phase containing 2 mm ammonium acetate buffer (pH 6.8) and acetonitrile. MS detection was by electrospray ionization with multiple reaction monitoring in the positive ion mode for estramustine phosphate, estromustine and estramustine, and in the negative ion mode for estrone and estradiol. The limit of quantitation was 10 ng/mL for estramustine phosphate, 3 ng/mL for estromustine, estramustine and estrone and 30 ng/mL for estradiol. Linearity was verified from these LLOQs up to about 4000 ng/mL for the parent drug and 2000 ng/mL for the metabolites. Inter-day precision and accuracy values were all less than 15%. This assay was applied successfully to the routine analysis of human plasma samples collected in cancer patients administered estramustine phosphate intravenously.

A phase I and pharmacokinetic study of MAG-CPT, a water-soluble polymer conjugate of camptothecin.

Polymeric drug conjugates are a new and experimental class of drug delivery systems with pharmacokinetic promises. The antineoplastic drug camptothecin was linked to a water-soluble polymeric backbone (MAG-CPT) and administrated as a 30 min infusion over 3 consecutive days every 4 weeks to patients with malignant solid tumours. The objectives of our study were to determine the maximal tolerated dose, the dose-limiting toxicities, and the plasma and urine pharmacokinetics of MAG-CPT, and to document anti-tumour activity. The starting dose was 17 mg m(-2) day(-1). Sixteen patients received 39 courses at seven dose levels. Maximal tolerated dose was at 68 mg m(-2) day(-1) and dose-limiting toxicities consisted of cumulative bladder toxicity. MAG-CPT and free camptothecin were accumulated during days 1-3 and considerable amounts of MAG-CPT could still be retrieved in plasma and urine after 4-5 weeks. The half-lives of bound and free camptothecin were equal indicating that the kinetics of free camptothecin were release rate dependent. In summary, the pharmacokinetics of camptothecin were dramatically changed, showing controlled prolonged exposure of camptothecin. Haematological toxicity was relatively mild, but serious bladder toxicity was encountered which is typical for camptothecin and was found dose limiting.

Species-differences in disposition and reductive metabolism of methoxymorpholinodoxorubicin (PNU 152243), a new potential anticancer agent.

Plasma pharmacokinetics, excretion balance and urinary metabolites of methoxymorpholino doxorubicin (MMDX) were investigated in male and female rats and in female dogs after i.v. administration of the(14)C-labelled drug. The mean total recovery of radioactivity in 96 h (urine plus faeces) was approximately 74 and 60% dose in male and female rats, respectively, while in female dogs approximately 72% dose was recovered in 336 h. Most of the radioactivity was present in faeces, with the urinary elimination accounting for only 3-4% dose in rats and dogs. These data suggest that biliary excretion is an important route of elimination of MMDX and/or its metabolites in both species. No differences were observed in the urinary metabolic profile of male and female rats. Two main peaks were present in radiochromatograms of urine from rats and dogs, i.e. MMDX and its 13-dihydro metabolite (MMDX-ol), accounting for approximately 25 and 20% of total radioactivity in 0-24-h urine in rats and 30 and 36% in dogs. The MMDX-ol/MMDX ratio in dog urine was higher than that observed in rat urine. No aglycones were detected in the urine samples from either species. In the rat, the plasma concentration-time profile suggested that the disposition of MMDX, MMDX-ol and total radioactivity is not sex-dependent. MMDX was the major species present in the systemic circulation; its AUC (0-96 h) accounted for 70% of total plasma radioactivity with the sum of AUC (MMDX) plus AUC (MMDX-ol) accounting for 77% of total radioactivity. In the dog, the sum of AUC (MMDX) plus AUC (MMDX-ol) amounted to 8% of radioactivity AUC(0-t(z) indicating that an important proportion of other(s) unknown metabolite(s) is present in dog plasma. Plasma levels of MMDX-ol in the rat were approximately 10-fold lower than those of the parent compound, whereas they were three times higher than those of MMDX in the dog. These data show that the reduction of the 13-keto group of MMDX is species-dependent, and occurs preferentially in the dog compared to the rat.

Determination of 4-demethoxy-3'-deamino-3'-aziridinyl-4'-methylsulphonyldaunorubicin+ ++ and its 13-hydroxy metabolite by direct injection of human plasma into a column-switching liquid chromatography system with mass spectrometric detection.

A selective, sensitive and fully automated column-switching LC system using direct injection of human plasma followed by mass spectrometry (MS) detection was developed and validated to determine the concentrations of 4-demethoxy-3'-deamino-3'-aziridinyl-4'-methylsulphonyldaunorubicin++ + (PNU-159548) and its 13-hydroxy metabolite (PNU-169884). A 50-microl human plasma sample was directly introduced into a C4-alkyl-diol silica clean-up column separating analytes from proteins and polar endogenous compounds using water and methanol as the mobile phase. The fraction containing PNU-159548 and its metabolite was back-flushed and transferred to the analytical column. The compounds were separated using a Zorbax SB C8 column (150x4.6 mm, 5 microm) under gradient conditions with the mobile phase containing acetonitrile and 2 mM ammonium formate, pH 3.5. MS detection was by atmospheric pressure ionisation with multiple reaction monitoring in positive ion mode. Linearity was demonstrated over the calibration range of 0.051-10.291 ng/ml for PNU-159548 and 0.104-10.434 ng/ml for PNU-169884. The assay was validated with respect to accuracy, precision and analyte stability. On the basis of the validation data, the developed analytical method was found to be suitable for use in Phase I clinical studies.

Phase I clinical and pharmacokinetic study of 3'-deamino-3'-(2-methoxy-4-morpholinyl)doxorubicin (FCE 23762).

Methoxymorpholinyldoxorubicin (FCE 23762) is a novel, highly lipophilic doxorubicin analogue. It possesses potent in vitro and in vivo antitumor activity including efficacy in multidrug-resistant tumor cell lines. It is also metabolically activated in vivo resulting in an 80-fold increase in potency over the parent drug. In this phase I study the drug was administered by i.v. bolus injection at 3-week intervals. Fifty-three patients with refractory solid tumors were treated; 133 courses of FCE 23762 were administered at doses ranging from 30 to 2250 micrograms/m2. The dose limiting toxicity was reversible myelo-suppression (granulocytopenia and thrombocytopenia), demonstrating a delayed nadir and recovery in comparison to doxorubicin. Other toxicities included transient elevation of hepatic transaminases, delayed and prolonged nausea and vomiting, mucositis, anorexia, fatigue, and diarrhea. Heavily pretreated patients demonstrated more myelosuppression than previously untreated patients at 1250 micrograms/m2. No cardiotoxicity was observed. Four objective tumor responses were seen: one complete response in a patient with pelvic recurrence of cervical cancer; one partial response in a patient with cutaneous and lymph gland metastases from head and neck cancer; and two minor responses in patients with liver metastases from colorectal cancer. Plasma concentrations of FCE 23762 and its 13-dihydro metabolite, FCE 26176, were measured in 20 patients at doses > or = 675 micrograms/m2, using HPLC with fluorescence detection. The area under the plasma concentration-time curve ranged from 30 to 80 ng/h/ml; plasma data suggested linear kinetics in the range of tested doses (although there was considerable interpatient variability). The maximum tolerated dose defined in this study using this schedule is 1500 micrograms/m2. A safe phase II dose for previously untreated patients using this schedule is 1250 micrograms/m2; however, this may actually be below the optimal dose for this patient population.

Successful long-term preservation of the neonatal heart with a modified intracellular solution.

UNLABELLED: Current methods of myocardial preservation for transplantation are suboptimal. A newly developed intracellular cardioplegic and storage solution (modified University of Wisconsin solution, group 1) was compared in a randomized, blinded fashion with our present clinical protocol, Stanford cardioplegic solution and saline storage (group 2) in an isolated neonatal pig model. After arrest and storage for 12 hours at 4 degrees C, biopsy specimens were taken from six group 1 hearts and five group 2 hearts for examination under an electron microscope and assessment of high-energy phosphate levels and water content. The remainder (group 1, n = 7; group 2, n = 6) were reperfused with blood for 50 minutes, after which function curves were obtained at left ventricular end-diastolic pressures of 3 to 12 mm Hg and biopsy tissue was taken. Eight control hearts (group 3) were cannulated in situ and perfused on the circuit without arrest or intervening ischemia. Stroke and minute work index curves were approximately threefold and fivefold higher for group 1 (modified University of Wisconsin solution) than for group 2 (Stanford), respectively (p less than 0.01). The hearts preserved with University of Wisconsin solution did not differ in function from unpreserved control hearts (group 3). High-energy phosphate levels were better maintained in group 1 than group 2 (p less than 0.05), and water content was lower (p less than 0.01). Semiquantitative grading of electron micrographs paralleled the functional and biochemical results. CONCLUSION: Modified University of Wisconsin intracellular solution provides markedly better heart preservation than conventionally used cardioplegic and storage solutions.

Determination of 3'-deamino-3'-[2(S)-methoxy-4-morpholinyl]doxorubicin, a new morpholinyl anthracycline, in plasma by high-performance liquid chromatography with fluorescence detection.

A sensitive and selective high-performance liquid chromatographic method for the determination of 3'-deamino-3'-[2(S)-methoxy-4-morpholinyl]doxorubicin and its possible 13-dihydro metabolite in human plasma has been developed. The plasma samples were buffered and the drugs and internal standard (doxorubicin) were extracted with diethyl ether-n-butanol, back-extracted into 0.3 M phosphoric acid, then analysed by reversed-phase liquid chromatography. Quantitation was achieved by fluorescence detection of the eluate. The linearity, precision and accuracy of the method were evaluated. No interference from blank plasma sample was observed. The suitability of the method for in vivo samples was checked by analysis of plasma samples drawn from female rats that had received repeated intravenous doses of the test compound.