Equimolar Doses Research Articles

Studies on Para-Methoxymethamphetamine (PMMA) Metabolite Pattern and Influence of CYP2D6 Genetics in Human Liver Microsomes and Authentic Samples from Fatal PMMA Intoxications.

Para-methoxymethamphetamine (PMMA) has caused numerous fatal poisonings worldwide and appears to be more toxic than other ring-substituted amphetamines. Systemic metabolism is suggested to be important for PMMA neurotoxicity, possibly through activation of minor catechol metabolites to neurotoxic conjugates. The aim of this study was to examine the metabolism of PMMA in humans; for this purpose, we used human liver microsomes (HLMs) and blood samples from three cases of fatal PMMA intoxication. We also examined the impact of CYP2D6 genetics on PMMA metabolism by using genotyped HLMs isolated from CYP2D6 poor, population-average, and ultrarapid metabolizers. In HLMs, PMMA was metabolized mainly to 4-hydroxymethamphetamine (OH-MA), whereas low concentrations of para-methoxyamphetamine (PMA), 4-hydroxyamphetamine (OH-A), dihydroxymethamphetamine (di-OH-MA), and oxilofrine were formed. The metabolite profile in the fatal PMMA intoxications were in accordance with the HLM study, with OH-MA and PMA being the major metabolites, whereas OH-A, oxilofrine, HM-MA and HM-A were detected in low concentrations. A significant influence of CYP2D6 genetics on PMMA metabolism in HLMs was found. The catechol metabolite di-OH-MA has previously been suggested to be involved in PMMA toxicity. Our studies show that the formation of di-OH-MA from PMMA was two to seven times lower than from an equimolar dose of the less toxic drug MDMA, and do not support the hypothesis of catechol metabolites as major determinants of fatal PMMA toxicity. The present study revealed the metabolite pattern of PMMA in humans and demonstrated a great impact of CYP2D6 genetics on human PMMA metabolism.

Oral 4-(N)-stearoyl gemcitabine nanoparticles inhibit tumor growth in mouse models.

In spite of recent advances in targeted tumor therapy, systemic chemotherapy with cytotoxic agents remains a vital cancer treatment modality. Gemcitabine is a nucleoside analog commonly used in the treatment of various solid tumors, but an oral gemcitabine dosage form remain unavailable. Previously, we developed the 4-(N)-stearoyl gemcitabine solid lipid nanoparticles (GemC18-SLNs) by incorporating 4-(N)-stearoyl gemcitabine (GemC18), an amide prodrug of gemcitabine, into solid lipid nanoparticles. GemC18-SLNs, when administered intravenously, showed strong antitumor activity against various human and mouse tumors in mouse models. In the present study, we defined the plasma pharmacokinetics of gemcitabine when GemC18-SLNs were given orally to healthy mice and evaluated the antitumor activity of GemC18-SLNs when given orally in mouse models of lung cancer. In mice orally gavaged with GemC18-SLNs, plasma gemcitabine concentration followed an absorption phase and then clearance phase, with a Tmax of ~2 h. The absolute oral bioavailability of gemcitabine in the GemC18-SLNs was ~70% (based on AUC0-24 h values). In mice with pre-established tumors (i.e. mouse TC-1 or LLC lung cancer cells), oral GemC18-SLNs significantly inhibited the tumor growth and increased mouse survival time, as compared to the molar equivalent dose of gemcitabine hydrochloride or GemC18 in vegetable oil or in Tween 20. Immunohistostaining revealed that oral GemC18-SLNs also have significant antiproliferative, antiangiogenic, and proapoptotic activity in LLC tumors. Formulating a lipophilic amide prodrug of gemcitabine into solid lipid nanoparticles may represent a viable approach toward developing a safe and efficacious gemcitabine oral dosage form.

Pharmacokinetics and Pharmacodynamics of Lisdexamfetamine Compared with D-Amphetamine in Healthy Subjects.

Rationale: Lisdexamfetamine is a prodrug of D-amphetamine used for the treatment of attention-deficit/hyperactivity disorder (ADHD). Lisdexamfetamine is thought to have a prolonged pharmacokinetic profile compared with oral D-amphetamine, possibly associated with lower drug liking and a lower risk of oral misuse. However, differences in the pharmacokinetics and pharmacodynamics of lisdexamfetamine and D-amphetamine have not been directly compared.Methods: Equimolar doses of D-amphetamine (40 mg) and lisdexamfetamine (100 mg), and placebo were administered in 24 healthy subjects in a randomized, double-blind, placebo-controlled, cross-over study. Plasma concentrations of amphetamine, subjective effects, and vital signs were repeatedly assessed. The pharmacokinetic parameters were determined using compartmental modeling.Results: The increase in plasma concentrations of amphetamine had a 0.6 ± 0.6 h (mean ± SD) longer lag time and reached peak levels 1.1 ± 1.5 h later after lisdexamfetamine administration compared with D-amphetamine administration, but no differences in maximal concentrations or total exposure (AUC) were found between the two treatments. Consistent with the pharmacokinetics, the subjective and cardiovascular stimulant effects of lisdexamfetamine also occurred later compared with D-amphetamine. However, no differences in peak ratings of potentially abuse-related subjective drug effects (e.g., drug liking, drug high, stimulation, happy, well-being, and self-confidence) were observed after lisdexamfetamine administration compared with D-amphetamine administration. Lisdexamfetamine and D-amphetamine also produced similar peak increases in mean arterial blood pressure, heart rate, body temperature, pupil size, and adverse effects.Conclusion: The pharmacokinetics and pharmacodynamics of lisdexamfetamine are similar to D-amphetamine administered 1h later. Lisdexamfetamine is likely associated with a similar risk of oral abuse as D-amphetamine. The study was registered at ClinicalTrials.gov (NCT02668926).

The thermogenic effect of nesfatin-1 requires recruitment of the melanocortin system.

Nesfatin-1 is a bioactive polypeptide expressed both in the brain and peripheral tissues and involved in the control of energy balance by reducing food intake. Central administration of nesfatin-1 significantly increases energy expenditure, as demonstrated by a higher dry heat loss; yet, the mechanisms underlying the thermogenic effect of central nesfatin-1 remain unknown. Therefore, in this study, we sought to investigate whether the increase in energy expenditure induced by nesfatin-1 is mediated by the central melanocortin pathway, which was previously reported to mediate central nesfatin-1´s effects on feeding and numerous other physiological functions. With the application of direct calorimetry, we found that intracerebroventricular nesfatin-1 (25 pmol) treatment increased dry heat loss and that this effect was fully blocked by simultaneous administration of an equimolar dose of the melanocortin 3/4 receptor antagonist, SHU9119. Interestingly, the nesfatin-1-induced increase in dry heat loss was positively correlated with body weight loss. In addition, as assessed with thermal imaging, intracerebroventricular nesfatin-1 (100 pmol) increased interscapular brown adipose tissue (iBAT) as well as tail temperature, suggesting increased heat production in the iBAT and heat dissipation over the tail surface. Finally, nesfatin-1 upregulated pro-opiomelanocortin and melanocortin 3 receptor mRNA expression in the hypothalamus, accompanied by a significant increase in iodothyronine deiodinase 2 and by a nonsignificant increase in uncoupling protein 1 and peroxisome proliferator-activated receptor gamma coactivator-1 alpha mRNA in the iBAT. Overall, we clearly demonstrate that nesfatin-1 requires the activation of the central melanocortin system to increase iBAT thermogenesis and, in turn, overall energy expenditure.

Pharmacokinetics, excretion balance, and tissue distribution of [14C]-labeled glycolipids and long chain fatty acids from Dacryopinax spathularia in rats

The pharmacokinetics, excretion balance, and tissue distribution of [14C]-labeled glycolipids from Dacryopinax spathularia (herein referred to as “AM-1”) and [14C]-LCFA equivalents following single or repeated administration to Sprague Dawley rats were evaluated to support the safety assessment of these naturally derived jelly mushroom glycolipids for use as a food ingredient. Rats received equimolar doses of either [14C]-AM-1 or [14C]-LCFA via oral or intravenous administration followed by collection of biological samples at specified intervals. Approximately 88%–101% of the administered dose was recovered in expired air, urine, feces, and carcass following single or repeated oral administration of [14C]-AM-1 at 100 mg/kg or equimolar doses of [14C]-LCFA at 46 mg/kg. Cmax and AUClast for [14C]-AM-1- and [14C]-LCFA-equivalents-derived radioactivity detected by quantitative whole body autoradiography was highest in the tissues of the GI tract, as expected following oral administration. The remaining tissues had low concentrations of test article equivalents relative to the administered dose and no target tissues for residence or accumulation were identified. AM-1 and LCFA are poorly absorbed by the oral route and are primarily eliminated in the feces without absorption. Oral bioavailability of both AM-1 and LCFA including their metabolites is low at approximately 11%.

Targeting Fatty-Acid Amide Hydrolase with Prodrugs for CNS-Selective Therapy.

The blood-brain barrier (BBB) can be a substantial impediment to achieving therapeutic levels of drugs in the CNS. Certain chemical functionality such as the carboxylic acid is a general liability for BBB permeability preventing significant CNS distribution of a drug from a systemic dose. Here, we report a strategy for CNS-selective distribution of the carboxylic acid containing thyromimetic sobetirome using prodrugs targeted to fatty-acid amide hydrolase (FAAH), which is expressed in the brain. Two amide prodrugs of sobetirome were shown to be efficient substrates of FAAH with Vmax/KM values comparable to the natural endocannabinoid FAAH substrate anandamide. In mice, a systemic dose of sobetirome prodrug leads to a substantial ∼60-fold increase in brain distribution (Kp) of sobetirome compared to an equimolar systemic dose of the parent drug. The increased delivery of sobetirome to the brain from the prodrug was diminished by both pharmacological inhibition and genetic deletion of FAAH in vivo. The increased brain exposure of sobetirome arising from the prodrug corresponds to ∼30-fold increased potency in brain target engagement compared to the parent drug. These results suggest that FAAH-targeted prodrugs can considerably increase drug exposure to the CNS with a concomitant decrease in systemic drug levels generating a desirable distribution profile for CNS acting drugs.

C53: A Novel CNP-Based Therapeutic Strategy Targeting Cardiac Fibrosis and Heart Failure

Background: Cardiac fibrosis is a characteristic feature of the failing heart for which there is an unmet need of fibro-inhibiting therapies targeting heart failure (HF). C-type natriuretic peptide (CNP), a human endothelial derived 22 amino acid (AA) peptide, is a guanylyl cyclase B (GC-B) activator that mediates potent anti-fibrotic actions via its second messenger, cGMP. However, the endogenous anti-fibrotic actions of CNP are limited, in part, due to its rapid metabolic clearance by the clearance receptor (NPRC), which has been reported to be up regulated in HF patients. Considering the fibro-suppressing properties of GC-B activation, we engineered a novel 53 AA peptide (C53) based upon the CNP and proCNP sequence with the goal of advancing C53‘s therapeutic potential including reduced NPRC binding. Hypothesis: We hypothesized C53 would have enhanced cGMP generating actions in vivo due to reduced NPRC binding compared to CNP and C53 would activate cGMP in human cardiac fibroblasts (hCFs). Methods: Therapeutic dose of CNP, equimolar dose of C53 and vehicle were IV infused into normal anesthetized rats (n = 12) and in vivo CNP and cGMP levels were assessed. NPRC binding with C53 and CNP were assessed using a modified AlphaScreen luminescent proximity assay (ALPA). In vitro cGMP activating property of C53 (10−8M and 10−6M) compared to no treatment in hCFs was assessed. Data are mean ± SE and significance was achieved with P < .05. Results: In vivo, C53 significantly increased plasma and urinary cGMP compared to equimolar CNP and vehicle (P < .05), which correlated with higher plasma CNP levels with C53 IV infusion (P < .05). Binding studies using the ALPA demonstrated that C53 had reduced binding to NPRC compared to CNP. In hCFs, C53 generated cGMP at 10−8M and markedly activated cGMP at 10−6M compared to no treatment (no treatment: 0.00 ± 0.00; 10−8M: 0.17 ± 0.03; 10−6M: 1.79 ± 0.02, pmol/well, P < .05). Conclusion: We report a newly engineered CNP-based peptide, C53, has cGMP activating actions in hCFs and has superior in vivo cGMP generation than CNP which may, in part, be related to reduced binding to the metabolic clearance receptor NPRC. These studies suggest that C53 may be a novel therapeutic strategy for the prevention and treatment of cardiac fibrosis in HF and further investigations are warranted.

Central effects of insulin detemir on feeding, body weight, and metabolism in rats.

Insulin detemir (DET) is a basal insulin analog that, in contrast to other long-acting forms of insulin, has significant weight-gain-sparing effects in diabetic patients. We hypothesized that this effect of DET may be due to its enhanced catabolic action in the central nervous system. We investigated the long-term effects of single third ventricular (3V) microinjections of equimolar doses of DET and regular insulin in normal male rats on feeding, body weight, energy expenditure (EE), and respiratory quotient (RQ). Also, in acute testing, we assessed the ability of lower doses of DET to alter feeding, EE, and RQ when microinjected directly into the paraventricular nucleus (PVN). The anabolic peptide ghrelin served as a positive control in acute testing. 3V administration of both DET (0.5-2.0 mU) and regular insulin (2.0-8.0 mU) significantly reduced feeding and body weight over 48 and 120 h, respectively, with DET yielding greater inhibitory effects. DET also stimulated greater elevations of EE and reductions of RQ over 72 and 48 h postinjection, respectively. In acute (4 h) testing, microinjections of DET (0.5 mU) into the PVN reduced feeding, increased EE, and reduced RQ, while ghrelin (100 pmol) had the opposite effects. When administered sequentially into the PVN, DET (0.25 and 0.5 mU) reversed ghrelin-induced feeding, EE, and RQ effects. These data support the notion that the weight-sparing effect of DET is at least in part based on its central catabolic action and that enhanced EE and reduced RQ may participate in this effect.

Doxycycline and its quaternary ammonium derivative for adjuvant therapies of chondrosarcoma.

This study was conducted during the development of innovative treatment targeting the microenvironment of chondrosarcoma. In this context, MMP inhibitors were conjugated with a quaternary ammonium (QA) function as a targeting ligand to proteoglycans of chondrosarcoma extracellular matrix. Here we report the proof of concept of this strategy applied to the MMP13 inhibitor, doxycycline (Dox). A quaternary ammonium derivative of the MMP13 inhibitor doxycycline (QA-Dox) was synthesized, and its anticancer activity was evaluated in the Swarm rat chondrosarcoma (SRC) model compared with the parent drug doxycycline, in vitro and in vivo. In vivo, dox and QA-Dox efficiency was assessed at equimolar doses according to a q4dx4 schedule by monitoring tumour volume by MRI and PG-targeted scintigraphy. Molecular mechanism (MMP13 expression, proteoglycan level) and histology studies were performed on tumours. The link of QA targeting function to Dox maintained the MMP13 inhibitory activity in vitro. Interestingly, the bacteriostatic activity was lost. SRC cells incubated with both drugs were blocked in S and G2M phases. Tumour growth inhibition (confirmed by histology) was observed for both Dox and QA-Dox. Undesirable blood effects (leukocyte decrease) were reduced when Dox was targeted to tumour tissue using the QA function. In the SRC model, the MMP13 inhibitor Dox and its QA derivative are promising as adjuvant therapies for chondrosarcoma management.

Abstract 3857: Patient-derived tumor xenografts as pharmacological model of human pancreatic ductal adenocarcinoma

Abstract INTRODUCTION and AIM Pancreatic ductal adenocarcinoma is a lethal disease with an overall survival rate of 5% and a median survival time of less than 6 months. Current systemic treatments offer only a modest benefit in symptom control and survival, thus surgery remains the most effective treatment. The exploitation of new therapeutics is an urgent need, but the research is braked by the limited value of the current preclinical models as predictors of patients response. The aim of our study is to develop patient-derived primary pancreatic ductal adenocarcinoma xenografts (PDAC-PDX) growing into the pancreas of recipient mice that retain features of the patient’s tumors and mimic the biological heterogeneity of human pancreatic cancer. METHODS Cancer specimens from patients undergoing cephalic duodenopancreatectomy were transplanted in NSG mice and orthotopically propagated by their re-implantation into the pancreas of SCID mice. Tumor burden was measured by abdominal palpation and by non invasive magnetic resonance imaging (MRI). PDAC-PDXs were characterized histologically, for mutational status (KRAS, TP53 and SMAD4), and responsiveness to gemcitabine. Gemcitabine was administered alone or combined with nab-paclitaxel, to mice bearing tumor in their pancreas; drugs were given intravenously on days 1 and 8 of each 21-day cycle to copy as much as possible patient’s recommendations. To determine the antitumor effects of the treatments, changes in tumor growth over time were evaluated by MRI and plasma level of Ca19-9. RESULTS Surgically resectable pancreatic adenocarcinoma (histologically confirmed) engrafted successfully in 5 cases (out of 11) and retained the molecular, morphological and biological features of the original patient tumors. Mutational landscape of the PDAC-PDXs reflect the clinical scenario: 80% are mutated in KRAS (G12D, G12R), 80% carry a TP53 mutation (I162F, R213*, Y220C, R248Q) and 20% show the deletion of SMAD4. Histological features recapitulate the human disease, the PDAC-PDXs exhibit a robust amount of host derived stroma with collagen deposition, constant through generations (up to five). Orthotopic growing PDAC-PDXs were marginal responsive to gemcitabine treatment, while significant response (stable disease and regression) was observed with the gemcitabine and nab-paclitaxel combination regimen, which was more efficacious than paclitaxel (equimolar dose). CONCLUSION PDAC-PDXs growing orthotopically in the pancreas of mice provide a valuable tool for the preclinical exploitation of novel therapeutics for pancreatic cancer. . Supported by Associazione Italiana per la Ricerca sul Cancro (AIRC 5 per mille n. 12182) and Fondazione “Eugenio Morandi” ONLUS per lo studio e la cura dei tumori del pancreas. Citation Format: Alessia Anastasia, Andrea Resovi, Carmen Ghilardi, Roberta Avigni, Edoardo Micotti, Lucia Minoli, Eugenio Scanziani, Carlo Corbellini, Eugenio Morandi, Dorina Belotti, Raffaella Giavazzi, MariaRosa Bani. Patient-derived tumor xenografts as pharmacological model of human pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3857. doi:10.1158/1538-7445.AM2017-3857

Targeting human liver cancer cells with lactobionic acid-G(4)-PAMAM-FITC sorafenib loaded dendrimers

Reported here is the synthesis and biological evaluation of the asialoglycoprotein receptor (ASGP-R) targeted fourth generation poliamidoamine dendrimer (G(4)-PAMAM) loaded with sorafenib. The ASGP-R targeted dendrimer was obtained by conjugation of Lactobionic acid (La) to the G(4)-PAMAM dendrimer, followed by acetylation (Ac) of the free amino groups in order to reduce the non-specific interactions with the cell membrane. Moreover, by additionally grafting fluorescein (FITC), it was easy to characterize the internalization pathway and the intracellular fate of the targeted dendrimer Ac-La-G(4)-PAMAM-FITC. In vitro experiments performed on HepG-2 and HLE cell lines, allowed to study the ability of the dendrimers to affect the cell vitality. Confocal microscopy and cytofluorimetric analysis confirmed higher binding and uptake ability of the Ac-La-G(4)-PAMAM-FITC dendrimer in well differentiated and ASGP-R expressing human liver cancer cell line HepG-2 compared non-expressing HLE cells. Ac-La-G(4)-PAMAM-FITC dendrimer loaded with sorafenib was stable and showed sustained sorafenib release. As evidenced by the cytotoxicity studies, sorafenib included in the dendrimer maintained its effectiveness, and was able to produce a longer lasting effect over the time compared to molar equivalent doses of free sorafenib. This new targeted dendrimer appears to be a suitable carrier for the delivery of sorafenib to liver cancer cells expressing ASGP-R.

Nesfatin-1 employs the central melanocortin pathway to stimulate non-shivering thermogenesis in brown adipose tissue (BAT)

Nesfatin-1 is a bioactive polypeptide involved in the regulation of food-intake and insulin secretion. Recently, our group has demonstrated that nesfatin-1 also increases energy expenditure; however, the underlying mechanism is still to be identified. Therefore, in this study, we sought to investigate whether this increase is mediated by the central melanocortin (MC) pathway, which had previously been reported to underlie the anorexigenic effect of nesfatin-1. Male Wistar rats (˜300 g) were stereotaxically implanted with intracerebroventricular cannula to receive central injections. With the application of direct calorimetry, we show that nesfatin-1 (25 pmol/rat) elicits a substantial increase in dry heat loss (p < 0.01; peak effect at 5h) over the 8h time-course, and that this effect is completely precluded by the co-administration with equimolar dose of SHU9119, a potent MC3/4 receptor antagonist (p < 0.01). The increased dry heat loss induced by nesfatin-1 correlates positively with body weight loss (r = 0.879, p < 0.001), which does not occur after the co-administration with SHU9119. Moreover, with the use of thermography, we report that nesfatin-1 (100 pmol/rat) specifically increases interscapular BAT (iBAT) (p < 0.01; peak effect at 5h), as well as inner ear temperature (p < 0.05, peak effect at 5h). The RT-PCR analysis reveals that centrally administered nesfatin-1 promotes POMC and MC3 receptor mRNA expression (p < 0.05, respectively) in the hypothalamus, accompanied with a trend of increase in UCP1 and PGC-1α in the iBAT. Overall, we herein demonstrate that the increase in dry heat loss induced by nesfatin-1 is a neuroendocrine process mediated via the central melanocortin system that elicits activity in the iBAT.

B7-33 replicates the vasoprotective functions of human relaxin-2 (serelaxin)

Recombinant H2 relaxin (serelaxin) has gained considerable attention as a new vasoprotective drug, largely due to its potential therapeutic effects in heart failure and fibrosis. However, serelaxin is laborious and costly to produce. A single-chain peptidomimetic, B7-33, has been developed to overcome these problems but little is known about its biological actions in the vascular system. This study first compared the rapid vascular effects of an acute bolus injection of B7-33 compared with serelaxin. Male Wistar rats received a tail vein injection of placebo (20mM sodium acetate), B7-33 (13.3μg/kg) or serelaxin (26.6μg/kg). Three hours later vascular function in the mesenteric artery, small renal artery and abdominal aorta was assessed by wire myography. B7-33 and serelaxin selectively enhanced bradykinin-mediated endothelium-dependent relaxation in the rat mesenteric artery by increasing endothelium-derived hyperpolarization, but had no overall effects on relaxation in the small renal artery or aorta. We then compared the actions of B7-33 and serelaxin in an ex vivo model of vascular disease using virgin female mouse mesenteric arteries pre-incubated in placental trophoblast conditioned media to induce endothelial dysfunction characteristic of preeclampsia. Co-incubation of these arteries in trophoblast conditioned media with B7-33 or serelaxin (15, 30nM) prevented the development of endothelial dysfunction. In conclusion, equimolar doses of B7-33 replicated the acute beneficial vascular effects of serelaxin in rat mesenteric arteries and also prevented endothelial dysfunction induced by placental trophoblast conditioned media in mouse mesenteric arteries. Therefore, B7-33 should be considered as a cost-effective vasoactive therapeutic in cardiovascular diseases, including preeclampsia.

Plasma amino acids and metabolic profiling of dairy cows in response to a bolus duodenal infusion of leucine.

Leucine (Leu), one of the three branch chain amino acids, acts as a signaling molecule in the regulation of overall amino acid (AA) and protein metabolism. Leucine is also considered to be a potent stimulus for the secretion of insulin from pancreatice β-cells. Our objective was to study the effects of a duodenal bolus infusion of Leu on insulin and glucagon secretion, on plasma AA concentrations, and to do a metabolomic profiling of dairy cows as compared to infusions with either glucose or saline. Six duodenum-fistulated Holstein cows were studied in a replicated 3 × 3 Latin square design with 3 periods of 7 days, in which the treatments were applied at the end of each period. The treatments were duodenal bolus infusions of Leu (DIL; 0.15 g/kg body weight), glucose (DIG; at Leu equimolar dosage) or saline (SAL). On the day of infusion, the treatments were duodenally infused after 5 h of fasting. Blood samples were collected at -15, 0, 10, 20, 30, 40, 50, 60, 75, 90, 120, 180, 210, 240 and 300 min relative to the start of infusion. Blood plasma was assayed for concentrations of insulin, glucagon, glucose and AA. The metabolome was also characterized in selected plasma samples (i.e. from 0, 50, and 120 min relative to the infusion). Body weight, feed intake, milk yield and milk composition were recorded throughout the experiment. The Leu infusion resulted in significant increases of Leu in plasma reaching 20 and 15-fold greater values than that in DIG and SAL, respectively. The elevation of plasma Leu concentrations after the infusion led to a significant decrease (P<0.05) in the plasma concentrations of isoleucine, valine, glycine, and alanine. In addition, the mean concentrations of lysine, methionine, phenylalanine, proline, serine, taurine, threonine, and asparagine across all time-points in plasma of DIL cows were reduced (P<0.05) compared with the other groups. In contrast to the working hypothesis about an insulinotropic effect of Leu, the circulating concentrations of insulin were not affected by Leu. In DIG, insulin and glucose concentrations peaked at 30–40 and 40–50 min after the infusion, respectively. Insulin concentrations were greater (P<0.05) from 30–40 min in DIG than DIL and SAL, and glucose was elevated in DIG over DIL and SAL from 30–75 min and 40–50 min, respectively. Multivariate metabolomics data analysis (principal component analysis and partial least squares discriminant analysis) revealed a clear separation when the DIL cows were compared with the DIG and SAL cows at 50 and 120 min after the infusion. By using this analysis, several metabolites, mainly acylcarnitines, methionine sulfoxide and components from the kynurenine pathway were identified as the most relevant for separating the treatment groups. These results suggest that Leu regulates the plasma concentrations of branched-chain AA, and other AA, apparently by stimulating their influx into the cells from the circulation. A single-dose duodenal infusion of Leu did not elicit an apparent insulin response, but affected multiple intermediary metabolic pathways including AA and energy metabolism by mechanisms yet to be elucidated.

Characterization of a recombinant humanized anti-cocaine monoclonal antibody produced from multiple clones for the selection of a master cell bank candidate

We have generated a humanized anti-cocaine monoclonal antibody (mAb), which is at an advanced stage of pre-clinical development. We report here in vitro binding affinity studies, and in vivo pharmacokinetic and efficacy studies of the recombinant mAb. The overall aim was to characterize the recombinant antibody from each of the three highest producing transfected clones and to select one to establish a master cell bank. In mAb pharmacokinetic studies, after injection with h2E2 (120 mg/kg iv) blood was collected from the tail tip of mice over 28 days. Antibody concentrations were quantified using ELISA. The h2E2 concentration as a function of time was fit using a two-compartment pharmacokinetic model. To test in vivo efficacy, mice were injected with h2E2 (120 mg/kg iv), then one hour later injected with an equimolar dose of cocaine. Blood and brain were collected 5 min after cocaine administration. Cocaine concentrations were quantified using LC/MS. The affinity of the antibody for cocaine was determined using a [3H] cocaine binding assay. All three antibodies had long elimination half-lives, 2–5 nM Kd for cocaine, and prevented cocaine's entry into the brain by sequestering it in the plasma. Pharmacokinetic and radioligand binding assays supported designation of the highest producing clone 85 as the master cell bank candidate. Overall, the recombinant h2E2 showed favorable binding properties, pharmacokinetics, and in vivo efficacy.

GLUT1-mediated selective tumor targeting with fluorine containing platinum(II) glycoconjugates.

Increased glycolysis and overexpression of glucose transporters (GLUTs) are physiological characteristics of human malignancies. Based on the so-called Warburg effect, 18flurodeoxyglucose-positron emission tomography (FDG-PET) has successfully developed as clinical modality for the diagnosis and staging of many cancers. To leverage this glucose transporter mediated metabolic disparity between normal and malignant cells, in the current report, we focus on the fluorine substituted series of glucose, mannose and galactose-conjugated (trans-R,R-cyclohexane-1,2-diamine)-2-flouromalonato-platinum(II) complexes for a comprehensive evaluation on their selective tumor targeting. Besides highly improved water solubility, these sugar-conjugates presented improved cytotoxicity than oxaliplatin in glucose tranporters (GLUTs) overexpressing cancer cell lines and exhibited no cross-resistance to cisplatin. For the highly water soluble glucose-conjugated complex (5a), two novel in vivo assessments were conducted and the results revealed that 5a was more efficacious at a lower equitoxic dose (70% MTD) than oxaliplatin (100% MTD) in HT29 xenograft model, and it was significantly more potent than oxaliplatin in leukemia-bearing DBA/2 mice as well even at equimolar dose levels (18% vs 90% MTD). GLUT inhibitor mediated cell viability analysis, GLUT1 knockdown cell line-based cytotoxicity evaluation, and platinum accumulation study demonstrated that the cellular uptake of the sugar-conjugates was regulated by GLUT1. The higher intrinsic DNA reactivity of the sugar-conjugates was confirmed by kinetic study of platinum(II)-guanosine adduct formation. The mechanistic origin of the antitumor effect of the fluorine complexes was found to be forming the bifunctional Pt-guanine-guanine (Pt-GG) intrastrand cross-links with DNA. The results provide a rationale for Warburg effect targeted anticancer drug design.

Lanthanum chloride precipitation-based toxicoproteomic analysis of 3-monochloropropane-1,2-diol toxicity in rat kidney reveals involvement of extracellular signal-regulated kinase 2.

The heat-induced food contaminant 3-monochloropropane-1,2-diol (3-MCPD) and its fatty acid esters exert nephrotoxicity in rodents. Previous studies including a non-targeted toxicoproteomics approach using samples from a 28-day oral toxicity study in rats with 10mg/kg body weight (b.w.) of 3-MCPD, an equimolar dose of 53mg/kg b.w. 3-MCPD dipalmitate and a lower dose of 13.3mg/kg b.w. of 3-MCPD dipalmitate, revealed substance-induced alterations in metabolic pathways, especially for glycolysis and energy metabolism. In order to obtain deeper insight into mechanisms of 3-MCPD toxicity, samples from the above-mentioned study were reanalyzed using a lanthanum chloride precipitation-based toxicoproteomics approach in order to increase the yield of phosphorylated proteins, crucial players in cellular signaling. A comparison of standard 2D-gel-based proteomics and lanthanum chloride precipitation was performed, thus providing a comprehensive case study on these two methods using in vivo effects of an important food toxicant in a primary target organ. While resulting in similar 2D-gel electrophoresis pherograms and spot counts, data analysis demonstrated that lanthanum precipitation yielded more significantly deregulated proteins thus considerably improving our knowledge on 3-MCPD-dependent proteomic alterations in the kidney. 3-MCPD-induced deregulation of the phosphorylated, active version of extracellular signal-regulated kinase 2 (ERK2) in rat kidney was demonstrated using mass spectrometry and immunohistochemistry. In summary, this paper for the first time links 3-MCPD effects to deregulation of the ERK/mitogen-activated protein kinase signaling pathway in rat kidney and demonstrates that lanthanum chloride precipitation is suited to support the gain of mechanistic knowledge on organ toxicity using 2D-gel-based proteomics.

Feasibility of gadoteric acid for delayed gadolinium-enhanced MRI of cartilage (dGEMRIC) at the wrist and knee and comparison with Gd-DTPA.

To assess the feasibility of gadoteric acid for delayed gadolinium-enhanced magnetic resonance imaging of cartilage (dGEMRIC) and to compare the dGEMRIC values obtained using gadoteric acid with those obtained by an equimolar dose of Gd-DTPA. At 3T, dGEMRIC of the wrist was performed twice using a T1 -weighted 3D-volumetric interpolated breath-hold examination sequence in 16 healthy volunteers (10 women; mean age 26.0 years) using gadoteric acid first and Gd-DTPA 3 weeks later. In addition, 24 patients with knee pain were examined using gadoteric acid (n = 12; seven women; mean age 45.8 years) or Gd-DTPA (n = 12; four women; mean age 47.1 years). T1 values, the relative decrease in T1 , and the delta R1 were compared using t-tests. Interobserver agreement was assessed using the intraclass correlation (ICC) between two independent readers. At the wrist, there was no significant difference in delta R1 values (0.34 ± 0.10/s, 95% confidence interval [0.30;0.38]/s for gadoteric acid and 0.32 ± 0.09 [0.29;0.35]/s for Gd-DTPA, P = 0.24) or the relative decrease in T1 (0.25 ± 0.06 [0.29;0.35] msec for gadoteric acid and 0.24 ± 0.05 [0.22;0.27] msec for Gd-DTPA, P = 0.35). High observer agreement was found at precontrast (ICC = 0.87, P < 0.001) and postcontrast (ICC = 0.89, P < 0.001). Similarly, at the knee, there was no significant difference in delta R1 (0.39 ± 0.18 [0.32;0.47]/s for gadoteric acid and 0.41 ± 0.09 [0.38;0.45]/s for Gd-DTPA, P = 0.59) or the relative decrease in T1 (0.30 ± 0.10 [0.26;0.34] msec for gadoteric acid and 0.33 ± 0.05 [0.30;0.35] msec for Gd-DTPA, P = 0.28). High ICCs of 0.96 (P < 0.01) were noted both at precontrast and postcontrast. dGEMRIC using gadoteric acid is feasible and yields comparable values when compared with Gd-DTPA. 2 Technical Efficacy: Stage 2 J. Magn. Reson. Imaging 2017;46:1433-1440.

Anti-melasma codrug of retinoic acid assists cutaneous absorption with attenuated skin irritation

Melasma treatment with combined retinoic acid (RA) and hydroquinone (HQ) usually causes unsatisfactory outcomes and safety concerns. This study attempted to evaluate the cutaneous absorption and skin tolerance of the codrug conjugated with RA and HQ via ester linkage. The codrug’s permeation of the pig skin was estimated using Franz diffusion cell. The codrug and parent drugs were comparatively examined for anti-inflammatory activity and tyrosinase inhibition. In vivo cutaneous irritation was assessed on nude mouse skin. Chemical conjugation of RA with HQ increased the lipophilicity and thus the skin absorption. The codrug absorption produced a 5.5- and a 60.8-fold increment compared to RA skin deposition at an equimolar (1.2mM) and saturated solubility dose, respectively. The cumulative amount of HQ derived from the codrug in the receptor was comparable to or less than that of topically applied HQ. The RA-HQ codrug was partly hydrolyzed on penetrating the skin. The hydrolysis rate in intact skin was significantly lower than that in esterase medium and skin homogenates. The codrug showed an interleukin (IL)-6 inhibition activity comparable to RA. A therapeutic index 6-fold greater than RA was obtained with the topical codrug. The tyrosinase inhibition percentage of the codrug and HQ was 13% and 21%, respectively. The skin tolerance test determined by transepidermal water loss (TEWL), redness, and histopathology had exhibited minor skin irritation caused by the codrug compared to the physical mixture of RA and HQ at an equivalent dose. Topical codrug delivery not only promoted RA absorption, but also diminished the adverse effects of the parent agents.

Extra-nuclear effects of estrogen on cortical bone in males require ERαAF-1.

Estradiol (E2) signaling via estrogen receptor alpha (ERα) is important for the male skeleton as demonstrated by ERα inactivation in both mice and man. ERα mediates estrogenic effects not only by translocating to the nucleus and affecting gene transcription but also by extra-nuclear actions e.g., triggering cytoplasmic signaling cascades. ERα contains various domains, and the role of activation function 1 (ERαAF-1) is known to be tissue specific. The aim of this study was to determine the importance of extra-nuclear estrogen effects for the skeleton in males and to determine the role of ERαAF-1 for mediating these effects. Five-month-old male wild-type (WT) and ERαAF-1-inactivated (ERαAF-10) mice were orchidectomized and treated with equimolar doses of 17β-estradiol (E2) or an estrogen dendrimer conjugate (EDC), which is incapable of entering the nucleus and thereby only initiates extra-nuclear ER actions or their corresponding vehicles for 3.5 weeks. As expected, E2 treatment increased cortical thickness and trabecular bone volume per total volume (BV/TV) in WT males. EDC treatment increased cortical thickness in WT males, whereas no effect was detected in trabecular bone. In ERαAF-10 males, E2 treatment increased cortical thickness, but did not affect trabecular bone. Interestingly, the effect of EDC on cortical bone was abolished in ERαAF-10 mice. In conclusion, extra-nuclear estrogen signaling affects cortical bone mass in males, and this effect is dependent on a functional ERαAF-1. Increased knowledge regarding estrogen signaling mechanisms in the regulation of the male skeleton may aid the development of new treatment options for male osteoporosis.