Equimolar Doses Research Articles

Cell-targeted c(AmpRGD)-sunitinib molecular conjugates impair tumor growth of melanoma

Drug resistance and off-organ toxicity remain unsolved issues in chemotherapy of advanced-stage melanoma patients. Thus, the creation of new molecular conjugates able to combine a selective accumulation, high ability of internalization and signaling pathway inhibition, are highly requested. Recently, we reported a new class of molecular conjugates, compounds 1–3, where the anti-αVβ3 integrin peptidomimetic c(AmpRGD), which is a selective ligand for αVβ3 integrin, was covalently bound to the tyrosine kinase inhibitor sunitinib. Here, we report that these c(AmpRGD)-sunitinib conjugates and, in particular, compound 3, are selectively internalized by human melanoma cells through αVβ3 receptor-mediated endocytosis. Compound 3 is more effective than sunitinib in reducing in vitro melanoma cells proliferation, cloning efficiency, migration, and invasion. More interestingly, compound 3 is able to significantly reduce the growth of xenografted melanoma tumor developed in immune-compromised mice, more efficiently than an equimolar dose of sunitinib. Indeed, its targeting ability was demonstrated by the selective localization at the tumor level with respect to healthy tissues. Thus, c(AmpRGD)-sunitinib conjugates such as compound 3 could serve as intriguing multiple-target agents to selectively reach melanoma cells and interfere with the progression of the disease.

Hematologic safety of chronic brain-penetrating erythropoietin dosing in APP/PS1 mice

IntroductionLow blood-brain barrier (BBB) penetration and hematopoietic side effects limit the therapeutic development of erythropoietin (EPO) for Alzheimer's disease (AD). A fusion protein of EPO and a chimeric monoclonal antibody targeting the mouse transferrin receptor (cTfRMAb) has been engineered. The latter drives EPO into the brain via receptor-mediated transcytosis across the BBB and increases its peripheral clearance to reduce hematopoietic side effects of EPO. Our previous work shows the protective effects of this BBB-penetrating EPO in AD mice but hematologic effects have not been studied. Herein, we investigate the hematologic safety and therapeutic effects of chronic cTfRMAb-EPO dosing, in comparison to recombinant human EPO (rhu-EPO), in AD mice. MethodsMale APPswe PSEN1dE9 (APP/PS1) mice (9.5 months) were treated with saline (n = 11), and equimolar doses of cTfRMAb-EPO (3 mg/kg, n = 7), or rhu-EPO (0.6 mg/kg, n = 9) 2 days/week subcutaneously for 6 weeks, compared to saline-treated wild-type mice (n = 10). At 6 weeks, exploration and memory were assessed, and mice were sacrificed at 8 weeks. Spleens were weighed, and brains were evaluated for amyloid beta (Aβ) load and synaptophysin. Blood was collected at 4, 6 and 8 weeks for a complete blood count and white blood cells differential. ResultscTfRMAb-EPO transiently increased reticulocyte counts after 4 weeks, followed by normalization of reticulocytes at 6 and 8 weeks. rhu-EPO transiently increased red blood cell count, hemoglobin and hematocrit, and significantly decreased mean corpuscular volume and reticulocytes at 4 weeks, which remained low at 6 weeks. At 8 weeks, a significant decline in red blood cell indices was observed with rhu-EPO treatment. Exploration and cognitive deficits were significantly worse in APP/PS1-rhu-EPO mice. Both cTfRMAb-EPO and rhu-EPO decreased 6E10-positive brain Aβ load; however, cTfRMAb-EPO and not rhu-EPO selectively reduced brain Aβ1-42 and elevated synaptophysin expression. DiscussionChronic treatment with cTfRMAb-EPO results in better hematologic safety, behavioral, and therapeutic indices compared with rhu-EPO, supporting the development of this BBB-penetrable EPO analog for AD.

Oxidative stress and neurobehavioural changes in rats following copper exposure and their response to MiADMSA andd-penicillamine

An increase in copper concentration in body may lead to hepatolenticular degeneration which is considered as one clinical feature of Wilson’s disease. Chelation therapy using d-penicillamine is the preferred medical treatment for reducing the toxic effects of copper. However, a few shortcomings associated with d-penicillamine led us to search of an alternative antidote for copper toxicity. Monoisoamyl-2, 3-dimercaptosuccinic acid (MiADMSA), a potent arsenic chelator under clinical trial, has been reported to reduce system copper level. Thus, the present study was envisaged to explore the ameliorative effect of MiADMSA against copper toxicity. Copper pre-exposed animals (CuSO4.5H2O; 100 mg/kg; p.o., for 6 weeks) were segregated in different groups and were administered equimolar dose (0.3 mEq/kg/day; p.o.) of d-penicillamine and MiADMSA for 5 days. The effect of different treatments on spontaneous locomotor activity, muscle coordination, depression like behaviour and contextual fear memory was analysed using neurobehavioural battery test. Biochemical variables related to oxidative stress, zinc and copper concentration were determined in liver, kidney and brain. The results suggested that copper exposure led to oxidative stress in liver, kidney and blood, along with moderate effects in brain. Treatment with d-penicillamine and MiADMSA reduced liver copper load. MiADMSA produced more pronounced beneficial effect compared to d-penicillamine by increasing brain GPx activity. Our study suggests that MiADMSA might be equally effective as d-penicillamine in depleting body copper load. More detailed studies using different doses are required to suggest whether MiADMSA could be an alternative for d-penicillamine in reducing oxidative injury, neurobehavioural changes and depleting body copper burden.

The active heroin metabolite 6-acetylmorphine has robust reinforcing effects as assessed by self-administration in the rat

Previous studies have suggested that at least some of the behavioral effects of heroin might be mediated by its active metabolite 6-acetylmorphine (6-AM). The aim of the present study was to investigate the reinforcing effects of 6-AM and its role in mediating those of heroin. We used an intravenous self-administration procedure in male Sprague-Dawley rats including four phases: acquisition, extinction, reinstatement of drug-seeking, and re-acquisition. Independent groups of rats readily learned to self-administer equimolar doses (0.135 μmol/kg) of either 6-AM (44.3 μg/kg) or heroin (50 μg/kg). Under a fixed ratio 1 (FR1) schedule of reinforcement, the rate of responding was the same for 6-AM and heroin, but it was significantly higher for 6-AM than for heroin under a FR2 schedule. A non-contingent infusion (‘priming’) of 0.068 μmol/kg of either 6-AM or heroin reinstated non-reinforced drug-seeking (relapse). The rats readily re-acquired self-administration behaviour when given access to one of two doses (0.068 and 0.135 μmol/kg) of 6-AM or heroin. Pretreatment with a specific monoclonal antibody (mAb) against 6-AM blocked the priming effect of 6-AM, and modified the rate of lever-pressing on re-acquisition of 6-AM self-administration in a manner compatible with a shift to the right of the dose-effect curve. The mAb did not affect heroin responding. The present results show that 6-AM possesses reinforcing effects similar to those of heroin. The lack of effect of 6-AM mAb on heroin priming and heroin self-administration calls for further studies to clarify the role of heroin and its metabolites in heroin reward.This article is part of the Special Issue entitled ‘New Vistas in Opioid Pharmacology’.

Targeted radiotherapy of pigmented melanoma with 131I-5-IPN

PurposeThere has been no satisfactory treatment for advanced melanoma until now. Targeted radionuclide therapy (TRNT) may be a promising option for this heretofore lethal disease. Our goal in this study was to synthesize 131I-N-(2-(diethylamino)ethyl)-5-(iodo-131I)picolinamide (131I-5-IPN) and evaluate its therapeutic ability and toxicity as a radioiodinated melanin-targeting therapeutic agent.MethodsThe trimethylstannyl precursor was synthesized and labeled with 131I to obtain 131I-5-IPN. The pharmacokinetics of 131I-5-IPN was evaluated through SPECT imaging, and its biodistribution was assessed in B16F10 tumor models and in A375 human-to-mouse xenografts. For TRNT, B16F10 melanoma-bearing mice were randomly allocated to receive one of five treatments (n = 10 per group): group A (the control group) received 0.1 mL saline; group B was treated with an equimolar dose of unlabeled precursor; group C received 18.5 MBq of [131I]NaI; group D and E received one or two dose of 18.5 MBq 131I-5-IPN, respectively. TRNT efficacy was evaluated through tumor volume measurement and biology study. The toxic effects of 131I-5-IPN on vital organs were assessed with laboratory tests and histopathological examination. The radiation absorbed dose to vital organs was estimated based on biodistribution data.Results131I-5-IPN was successfully prepared with a good radiochemistry yield (55% ± 5%, n = 5), and it exhibited a high uptake ratio in melanin-positive B16F10 cells which indicating high specificity. SPECT imaging and biodistribution of 131I-5-IPN showed lasting high tumor uptake in pigmented B16F10 models for 72 h. TRNT with 131I-5-IPN led to a significant anti-tumor effect and Groups D and E displayed an extended median survival compared to groups A, B, and C. The highest absorbed dose to a vital organ was 0.25 mSv/MBq to the liver; no obvious injury to the liver or kidneys was observed during treatment. 131I-5-IPN treatment was associated with reduction of expression of proliferating cell nuclear antigen (PCNA) and Ki67 and cell cycle blockage in G2/M phase in tumor tissues. Decreased vascular endothelial growth factor and CD31 expression, implying reduced tumor growth, was noted after TRNT.ConclusionWe successfully synthesized 131I-5-IPN, which presents long-time retention in melanotic melanoma. TRNT with 131I-5-IPN has the potential to be a safe and effective strategy for management of pigmented melanoma.

A colon-specific prodrug of metoclopramide ameliorates colitis in an experimental rat model.

BackgroundWe examined whether metoclopramide (MCP), a modulator of dopamine and serotonin receptors, alleviated colitis and had synergistic effects when coadministered with 5-aminosalicylic acid (5-ASA) in an experimental model of colitis.MethodsMCP azo-linked to 5-ASA (5-[4-chloro-2-{2-(diethylamino)ethylcarbamoyl}– 1-methoxyphenyl]azosalicylic acid, MCP-azo-ASA) was synthesized, where 5-ASA was used as a colon-targeting carrier and an anti-colitic agent, and the ability of MCP-azo-ASA to target the colon in vitro and in vivo was evaluated.ResultsOur results indicate that MCP-azo-ASA was cleaved to MCP and 5-ASA in the cecal contents, but not in the contents of the small intestine. Oral gavage with equimolar concentrations of MCP-azo-ASA and sulfasalazine (SSZ, a colon-specific prodrug of 5-ASA widely used clinically) demonstrated that the two prodrugs delivered comparable amounts of 5-ASA to the cecum. MCP was barely detected in the blood after oral gavage with MCP-azo-ASA. In a rat model of 2,4-dinitrobenzene sulfonic acid hydrate (DNBS)-induced colitis, MCP-azo-ASA alleviated colonic damage in a dose-dependent manner. Moreover, MCP-azo-ASA reduced the concentrations of inflammatory mediators in the inflamed colon. At low equimolar doses, MCP-azo-ASA, but not SSZ, resulted in significant anti-colitic effects, which indicates that MCP has anti-colitic activity. MCP-azo-ASA had anti-colitic effects equal to those of SSZ at high equimolar doses.ConclusionThus, our results indicate that MCP-azo-ASA is a colon-specific prodrug of MCP. Targeted delivery of MCP to the colon ameliorated DNBS-induced colitis in rats, and we did not observe any synergistic effects of MCP after co-delivery with 5-ASA.

BST-236, a Novel Cytarabine Prodrug, Is Safer and As Effective As Cytarabine in In Vivo Leukemia Models

Introduction: Acute myeloid leukemia (AML) is associated with poor outcomes in older and medically unfit patients, largely due to the severe toxicity associated with cytarabine treatment, which precludes the administration of effective cytarabine doses. BST-236 is a prodrug of cytarabine, inactive in its prodrug form and designed to deliver cytarabine to leukemia cells with reduced systemic toxicity, thus to enable delivery of high cytarabine doses to leukemia cells with relative sparing of normal tissues.Results: BST-236 is a conjugate of cytarabine and asparagine (Figure 1). It was demonstrated that BST-236 is inactive as an intact prodrug and that its activity is exerted by release of cytarabine via non-enzymatic hydrolysis. Unlike free cytarabine, the bound cytarabine in BST-236 is not phosphorylated into its active metabolite Ara-CTP and it is protected by the asparagine residue from deamination into its inactive form Ara-U. In vitro studies demonstrate that BST-236 enters into leukemia cells, accompanied by cellular accumulation of free cytarabine, which is released from BST-236 (Figure 2). Like cytarabine, treatment with BST-236 result in induction of cell death of various leukemia cell lines via apoptosis, an activity which is dependent on the human equilibrative nucleoside transporter 1 (hENT1). The in vitro kinetics of BST-236-induced toxicity were found to be delayed compared to administration of free cytarabine, correlating with an observed delayed cellular availability of cytarabine.In vivo studies in mice and dogs demonstrate that BST-236 concentrations in the plasma are dose-proportional, with a prodrug-typical profile and only ~5% of free cytarabine present in the plasma. The maximal tolerated dose of BST-236 was found to be several-fold higher than reported for cytarabine, with mainly hematological effects and no unexpected toxicities.In vivo head-to-head studies in human leukemia mouse models with equimolar doses of cytarabine and BST-236 demonstrate similar efficacy of complete elimination of the leukemia cells in the bone marrow, spleen, and peripheral blood by both molecules (Figure 3A). However, while cytarabine treatment was associated with significant toxicity including weight loss, dramatic reduction in spleen size and number of mouse spleen cells, and delayed normal murine white blood cell recovery, equimolar BST-236 doses enabled spleen and BM recovery with minimal weight loss and no observed clinical signs (Figure 3B, 3C).Summary: in vitro and in vivo studies demonstrate that BST-236 is a prodrug of cytarabine, which enables the delivery of cytarabine to target cells, resulting in elimination of the leukemia with reduced systemic toxicity compared to free cytarabine. The data also suggest that while the mechanism of cell death induced by BST-236 and cytarabine is similar, the observed differential kinetics of the delivery of cytarabine by BST-236 and its metabolism may explain its reduced systemic toxicity. Our nonclinical findings are in line with the clinical results of the BST-236 Phase 1/2 study (ASH 2017 abstract no 893, manuscript in preparation) and suggest that BST-236 may enable delivery of high cytarabine doses to older and medically-unfit patients who currently cannot benefit from an effective cytarabine therapy. This suggestion is to be confirmed by an ongoing Phase 2b study. [Display omitted] DisclosuresTessler:Biosight: Employment. Gengrinovitch:Biosight: Employment. Ben Yakar:Biosight: Employment. Peled:Cellect Biotherapeutics Ltd: Consultancy. Flaishon:Biosight: Employment.

Effects of lisdexamfetamine on plasma steroid concentrations compared with d-amphetamine in healthy subjects: A randomized, double-blind, placebo-controlled study

The novel d-amphetamine prodrug lisdexamfetamine is applied to treat attention-deficit/hyperactivity disorder (ADHD). d-Amphetamine releases dopamine and norepinephrine and stimulates the hypothalamic-pituitary-adrenal (HPA) axis, which may contribute to its reinforcing effects and risk of abuse. However, no data is currently available on the effects of lisdexamfetamine on circulating steroids. This randomized, double-blind, placebo-controlled, cross-over study evaluated the effects of equimolar doses of d-amphetamine (40 mg) and lisdexamfetamine (100 mg) and placebo on circulating steroids in 24 healthy subjects. Plasma steroid and d-amphetamine levels were determined up to 24 h. Delayed increase and peak levels of plasma d-amphetamine concentrations were observed following lisdexamfetamine treatment compared with d-amphetamine administration, however the maximal concentrations and total exposure (area under the curve [AUC]) were similar. Lisdexamfetamine and d-amphetamine significantly enhanced plasma levels of adrenocorticotropic hormone, glucocorticoids (cortisol, cortisone, corticosterone, 11-dehydrocorticosterone, and 11-deoxycortisol), androgens (dehydroepiandrosterone, dehydroepiandrosterone sulfate, and Δ4-androstene-3,17-dione [androstenedione]), and progesterone (only in men) compared with placebo. Steroid concentration-time curves were shifted to later time points due to a non-significantly later onset following lisdexamfetamine administration than after d-amphetamine, however maximal plasma steroid concentrations and AUCs did not differ between the active treatments. None of the active treatments altered plasma levels of the mineralocorticoids aldosterone and 11-deoxycorticosterone or the androgen testosterone compared with placebo. The effects of the amphetamines on glucocorticoid production were similar to those that were previously reported for methylphenidate (60 mg) but weaker than those for the serotonin releaser 3,4-methylenedioxymethamphetamine (MDMA; 125 mg) or direct serotonin receptor agonist lysergic acid diethylamide (LSD; 0.2 mg). Lisdexamfetamine produced comparable HPA axis activation and had similar pharmacokinetics than d-amphetamine, except for a delayed time of onset. Thus, serotonin (MDMA, LSD) may more effectively stimulate the HPA axis than dopamine and norepinephrine (D-amphetamine).

Teratogenicity of valproic acid and its constitutional isomer, amide derivative valnoctamide in mice.

The anticonvulsant valproic acid (VPA) has a known teratogenic effect capable of inducing major congenital malformations and developmental disorders. A comparative teratogenicity study of VPA and its analog valnoctamide (VCD), which is a new generation candidate antiepileptic drug, was carried out using Swiss Vancouver (SWV) mice. Pregnant SWV dams were treated with either a single intraperitoneal injection of VPA (1.8 and 2.7 mmol/kg), VCD (1.8 and 2.7 mmol/kg), or vehicle on E8:12 (gestational day:hour). The numbers of implantation and resorption, viable and dead fetuses, and the presence of gross fetal visceral and skeletal abnormalities were determined (E18). Real-time Polymerase chain reaction (RT-PCR) arrays were used to analyze the expression of 84 genes related to the processes of neurogenesis and neural stem cell differentiation. Significant decreases in pregnancy weight gain and the number of live fetuses were observed when VPA was administered at the high dose, whereas the percentage of exencephalic fetuses was significantly increased in VPA treated compared with an equivalent VCD dosage group. There was a dose-related increase in visceral defects in the VPA-exposed fetuses. Missing skull bones and fused vertebrae in fetuses occurred at the high dose of VPA. Three genes (Mtap2, Bmp8b, and Stat3) were significantly upregulated and one (Heyl) was downregulated in samples from VPA-treated dams. The study demonstrates that the teratogenicity of VPA was significantly greater than that of an equimolar dose of VCD. Four genes (Mtap2, Bmp8b, Stat3, and Heyl) represent candidate target genes for the underlying teratogenic mechanism responsible for VPA-induced malformations.

Superior efficacy of HI-6 dimethanesulfonate over pralidoxime methylsulfate against Russian VX poisoning in cynomolgus monkeys (Macaca fascicularis)

Organophosphorus nerve agents still represent a serious risk to human health. In the French armed forces, the current emergency treatment against OP intoxications is a fully licensed wet-dry dual-chambered autoinjector (Ineurope ®), that contains pralidoxime methylsulfate (2-PAM) to reactivate inhibited acetylcholinesterase (AChE), atropine sulfate (AS) and avizafone chlorhydrate (AVZ). While this treatment is effective against several of the known nerve agents, it shows little efficacy against the Russian VX (VR), one of the most toxic compounds. HI-6 dimethanesulfonate (HI-6 DMS) is an oxime able to reactivate in vitro and in vivo VR-inhibited AChE. To confirm the superiority of HI-6 DMS towards 2-PAM prior to licensing, we compared the two 3-drug-combinations (HI-6 vs 2-PAM, 33 and 18 mg/kg respectively, equimolar doses; AS/AVZ 0.25/0.175 mg/kg respectively) in VR-poisoned cynomolgus macaques, the model required by the French drug regulatory agency. In parallel we performed HI-6 pharmacokinetics analysis using a one compartment model. A better efficacy of the HI-6 DMS combination was clearly observed: up to 5 LD50 of VR (i.m.), a single administration of the HI-6 DMS combination, shortly after the onset of clinical signs, prevented death of the four intoxicated animals. Conversely 2-PAM only prevented death in one out of three subjects exposed to the same amount of VR. As expected with V agents, reinhibition of blood AChE was observed but without any apparent impact on the clinical recovery of the animals. A single administration of the HI-6 DMS combination was still but partially effective at 15 LD50 of VR, allowing a 50% survival rate.

Anti-tumour activity of a first-in-class agent NUC-1031 in patients with advanced cancer: results of a phase I study

BackgroundGemcitabine is used to treat a wide range of tumours, but its efficacy is limited by cancer cell resistance mechanisms. NUC-1031, a phosphoramidate modification of gemcitabine, is the first anti-cancer ProTide to enter the clinic and is designed to overcome these key resistance mechanisms.MethodsSixty-eight patients with advanced solid tumours who had relapsed after treatment with standard therapy were recruited to a dose escalation study to determine the recommended Phase II dose (RP2D) and assess the safety of NUC-1031. Pharmacokinetics and anti-tumour activity was also assessed.ResultsSixty-eight patients received treatment, 50% of whom had prior exposure to gemcitabine. NUC-1031 was well tolerated with the most common Grade 3/4 adverse events of neutropaenia, lymphopaenia and fatigue occurring in 13 patients each (19%). In 49 response-evaluable patients, 5 (10%) achieved a partial response and 33 (67%) had stable disease, resulting in a 78% disease control rate. Cmax levels of the active intracellular metabolite, dFdCTP, were 217-times greater than those reported for equimolar doses of gemcitabine, with minimal toxic metabolite accumulation. The RP2D was determined as 825 mg/m2 on days 1, 8 and 15 of a 28-day cycle.ConclusionsNUC-1031 was well tolerated and demonstrated clinically significant anti-tumour activity, even in patients with prior gemcitabine exposure and in cancers not traditionally perceived as gemcitabine-responsive.

The active compounds derived from Psoralea corylifolia for photochemotherapy against psoriasis-like lesions: The relationship between structure and percutaneous absorption

8‑Methoxypsoralen (8-MOP) in combination with ultraviolet A (PUVA) is a photochemotherapy for management of psoriasis. 8-MOP is a natural compound from Psoralea corylifolia. The present work was undertaken to evaluate the percutaneous absorption of five compounds derived from P. corylifolia, and to further explore the inhibitory effect on psoriasis-like lesions generated by imiquimod stimulation in a mouse model. 8-MOP, psoralen, isopsoralen, psoralidin, and bakuchiol were comparatively tested for in vitro skin permeation, keratinocyte apoptosis, and in vivo antipsoriatic potency. The pig ear skin deposition of 8-MOP, isopsoralen, and bakuchiol at an equimolar dose was 0.47, 0.58, and 0.50 nmol/mg, respectively, which was comparable and higher than that of psoralen (0.25 nmol/mg) and psoralidin (0.14 nmol/mg). Psoralidin and bakuchiol were absorbed into the skin without further penetration across the skin. Besides experimental data of physicochemical properties, the hydrogen bond number, total polarity surface, and stratum corneum lipid docking calculated could explain the correlation of the penetrant structure with the skin permeability. The antiproliferative activity against keratinocytes was stronger for 8-MOP and isopsoralen than the others. Topical application of PUVA by using 8-MOP and isopsoralen on imiquimod-induced plaque significantly reduced transepidermal water loss from 55 to 33 and 38 g/m2/h, respectively. The epidermal thickening elicited by imiquimod (117 μm) was decreased to 62 and 26 μm by 8-MOP and isopsoralen application. IL-6 expression in psoriasiform skin was downregulated by isopsoralen but not 8-MOP. Isopsoralen may be a potential candidate for PUVA therapy.

Proteomic analysis of 2-monochloropropanediol (2-MCPD) and 2-MCPD dipalmitate toxicity in rat kidney and liver in a 28-days study

2-monochloropropanediol (2-MCPD), 3-monochloropropanediol (3-MCPD) and their fatty acid esters have recently been identified as heat-induced contaminants in fat- and salt-containing foodstuff. Toxicity of 3-MCPD has been studied previously in some detail. Disturbance of glycolysis and cellular redox functions appear to be involved in 3-MCPD toxicity. By contrast, only very few toxicological data are available for 2-MCPD or 2-MCPD esters, especially at the molecular level. This study was therefore aimed to provide a comprehensive overview of proteomic alterations induced in rat kidney and liver by 2-MCPD and 2-MCPD dipalmitate, a representative 2-MCPD fatty acid ester. Sub-toxic doses of 10 mg/kg body weight 2-MCPD, or equimolar doses of 2-MCPD dipalmitate were applied in a 28-day in vivo gavage oral toxicity study in male rats. Two-dimensional gel electrophoresis and mass-spectrometric protein identification using material from 5 animals per treatment group were employed together with bioinformatic data mining to obtain information about the molecular basis of the observed proteomic alterations. Obtained data indicate toxic consequences of 2-MCPD exposure in the kidney and provide evidence that 2-MCPD exerts its cellular effects in rat kidney by mechanisms different from 3-MCPD.

Potent therapeutic efficacy of an alphavirus replicon DNA vaccine expressing human papilloma virus E6 and E7 antigens

ABSTRACTCervical cancer develops as a result of infection with high-risk human papillomavirus (HPV) through persistent expression of early proteins E6 and E7. Our group pioneered a recombinant viral vector system based on Semliki Forest virus (SFV) for vaccination against cervical cancer. The most striking benefit of this alphavirus vector-based vaccine platform is its high potency. DNA vaccines on the other hand, have a major advantage with respect to ease of production. In this study, the benefits associated with both SFV-based vaccines and DNA vaccines were combined with the development of a DNA-launched RNA replicon (DREP) vaccine targeting cervical cancer. Using intradermal delivery followed by electroporation, we demonstrated that DREP encoding for E6,7 (DREP-E6,7) induced effective, therapeutic antitumor immunity. While immunizations with a conventional DNA vaccine did not prevent tumor outgrowth, immunization with a 200-fold lower equimolar dose of DREP (0.05 µg of DREP) resulted in approximately 85% of tumor-free mice. To overcome the safety concern of potential malignant transformation at the vaccination site, we evaluated the anti-tumor effect of a DREP vaccine encoding a shuffled version of E7 (DREP-E7sh). DREP-E7sh delayed tumor growth yet not to the same extent as DREP-E6,7. In addition, inclusion of a helper cassette and an ER targeting signal (sigHelp) did not significantly further enhance the suppression of tumor outgrowth in the long term, albeit exhibiting better tumor control early after immunization. Collectively, this study points towards the clinical evaluation of DREP encoding HPV antigens as a potent immunotherapy for patients with HPV16 (pre)-malignancies.

Comparative analysis of metabolism of trichloroethylene and tetrachloroethylene among mouse tissues and strains

Trichloroethylene (TCE) and tetrachloroethylene (PCE) are structurally similar chemicals that are metabolized through oxidation and glutathione conjugation pathways. Both chemicals have been shown to elicit liver and kidney toxicity in rodents and humans; however, TCE has been studied much more extensively in terms of both metabolism and toxicity. Despite their qualitative similarities, quantitative comparison of tissue- and strain-specific metabolism of TCE and PCE has not been performed. To fill this gap, we conducted a comparative toxicokinetic study where equimolar single oral doses of TCE (800 mg/kg) or PCE (1000 mg/kg) were administered to male mice of C57BL/6J, B6C3F1/J, and NZW/LacJ strains. Samples of liver, kidney, serum, brain, and lung were obtained for up to 36 h after dosing. For each tissue, concentrations of parent compounds, as well as their oxidative and glutathione conjugation metabolites were measured and concentration-time profiles constructed. A multi-compartment toxicokinetic model was developed to quantitatively compare TCE and PCE metabolism. As expected, the flux through oxidation metabolism pathway predominated over that through conjugation across all mouse strains examined, it is 1,200–3,800 fold higher for TCE and 26–34 fold higher for PCE. However, the flux through glutathione conjugation, albeit a minor metabolic pathway, was 21-fold higher for PCE as compared to TCE. The degree of inter-strain variability was greatest for oxidative metabolites in TCE-treated and for glutathione conjugation metabolites in PCE-treated mice. This study provides critical data for quantitative comparisons of TCE and PCE metabolism, and may explain the differences in organ-specific toxicity between these structurally similar chemicals.

Carnosine-LVFFARK-NH2 Conjugate: A Moderate Chelator but Potent Inhibitor of Cu2+-Mediated Amyloid β-Protein Aggregation.

Aggregation of amyloid-β (Aβ) protein stimulated by Cu2+ has been recognized as a crucial step in the neurodegenerative process of Alzheimer's disease. Hence, it is of significance to develop bifunctional agents capable of inhibiting Aβ aggregation as well as Cu2+-mediated Aβ toxicity. Herein, a novel bifunctional nonapeptide, carnosine-LVFFARK-NH2 ( Car-LK7), was proposed by integrating native chelator carnosine ( Car) and an Aβ aggregation inhibitor, Ac-LVFFARK-NH2 (LK7). Results revealed the bifunctionality of Car-LK7, including remarkably enhanced inhibition capability on Aβ aggregation as compared to LK7 and a moderate Cu2+ chelating affinity ( KD = 28.2 ± 2.1 μM) in comparison to the binding affinity for Aβ40 ( KD = 1.02 ± 0.13 μM). The moderate Cu2+ affinity was insufficient for Car-LK7 to sequester Cu2+ from Aβ40-Cu2+ species, but it was sufficient to form ternary Aβ40-Cu2+- Car-LK7 complexes. Formation of the ternary complexes directed the aggregation into small, unstructured aggregates with little β-sheet structure. Car-LK7 also showed higher activity on arresting Aβ40-Cu2+-catalyzed reactive oxygen species production than Car. Cell viability assays confirmed the prominent protection activity of Car-LK7 against Cu2+-mediated Aβ40 cytotoxicity; Car-LK7 could almost eliminate Aβ40 cytotoxicity at an equimolar dose (cell viability increased from 59% to 99%). The research has thus provided new insight into the design of potent bifunctional agents against metal-mediated amyloid toxicity by conjugating moderate metal chelators and existing inhibitors.

Molecular docking and pharmacological/toxicological assessment of a new compound designed from celecoxib and paracetamol by molecular hybridization.

Nonsteroidal anti-inflammatory drugs are commonly used worldwide; however, they have several adverse effects, evidencing the need for the development of new, more effective and safe anti-inflammatory and analgesic drugs. This research aimed to design, synthesize and carry out a pharmacological/toxicological investigation of LQFM-102, which was designed from celecoxib and paracetamol by molecular hybridization. To evaluate the analgesic effect of this compound, we performed formalin-induced pain, hot plate and tail flick tests. The anti-inflammatory effect of LQFM-102 was evaluated in carrageenan-induced paw oedema and pleurisy tests. The biochemical markers indicative of toxicity-AST, ALT, GSH, urea and creatinine-as well as the index of gastric lesion after prolonged administration of LQFM-102 were also analyzed. In addition, the interaction of LQFM-102 with COX enzymes was evaluated by molecular docking. In all experimental protocols, celecoxib or paracetamol was used as a positive control at equimolar doses to LQFM-102. LQFM-102 reduced the pain induced by formalin in both phases of the test. However, this compound did not increase the latency to thermal stimuli in the hot plate and tail flick tests, suggesting an involvement of peripheral mechanisms in this effect. Furthermore, LQFM-102 reduced paw oedema, the number of polymorphonuclear cells, myeloperoxidase activity and TNF-α and IL-1β levels. Another interesting finding was the absence of alterations in the markers of hepatic and renal toxicity or lesions of gastric mucosa. In molecular docking simulations, LQFM-102 interacted with the key residues for activity and potency of cyclooxygenase enzymes, suggesting an inhibition of the activity of these enzymes.

Abstract 758: Head-to-head comparison of ADC and SMDC products, alone and in combination with antibody-IL2 fusion proteins

Abstract In an attempt to improve the therapeutic index of cancer chemotherapy, monoclonal antibodies and small organic ligands have been proposed as delivery vehicles, allowing the construction of antibody-drug conjugates (ADCs) and of small molecule-drug conjugates (SMDCs). Four ADC products have gained marketing authorization for cancer therapy, while SMDCs are still being investigated in clinical trials. SMDCs could in principle offer certain advantages compared to ADCs, such as a more rapid and uniform diffusion into the tumor mass, lower cost-of-goods and lack of immunogenicity, but a direct head-to-head comparison has not yet been reported. Here we describe a novel small molecule-drug conjugate, based on a high-affinity ligand specific to carbonic anhydrase IX. The product features a peptide linker, suitable for cleavage in the tumor extracellular environment, and monomethyl auristatin E as cytotoxic payload. We compared the therapeutic efficacy of the novel SMDC and of an anti-CAIX ADC, which were injected at equimolar doses in nude mice bearing SKRC-52 renal cell carcinoma xenografts. Both products showed a potent anti-cancer activity, although no complete tumor eradication was observed. Instead, when the SMDC was administered together with L19-IL2 (a clinical-stage fusion protein capable of delivering interleukin-2 to the tumor neo-vasculature), all treated mice in the combination group could be rendered tumor-free, in a process which favored the influx of natural killer cells into the tumor mass. The combination of L19-IL2 and the new small molecule-drug conjugate also eradicated cancer in 100% of immunocompetent mice, bearing subcutaneously-grafted CT26 colorectal cancer cells, which stably expressed carbonic anhydrase IX. These findings may be of clinical significance, since carbonic anhydrase IX is over-expressed in the majority of clear-cell renal cell carcinomas and in approximately 30% of colorectal cancers. The targeted delivery of interleukin-2 helps potentiate the action of targeted cytotoxics leading to cancer eradication in models that cannot be cured by conventional chemotherapy. Citation Format: Samuele Cazzamalli, Alberto Dal Corso, Fontaine Widmayer, Dario Neri. Head-to-head comparison of ADC and SMDC products, alone and in combination with antibody-IL2 fusion proteins [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 758.

MEDI0382, a GLP-1/Glucagon Receptor Dual Agonist, Acutely Inhibits Food Intake in Leptin-Deficient ob/ob Mice and Leptin Receptor–Deficient fa/fa Rats

MEDI0382 is a potent glucagon-like-peptide-1 (GLP-1) and glucagon receptor dual agonist that inhibits feeding and induces weight loss in mice. To test whether MEDI0382-mediated improvements in metabolism require leptin signaling, we determined its acute effects on food intake in rodents lacking either leptin (ob/ob mice) or the leptin receptor (fa/fa rats). An acute dose response study in ob/ob mice was performed with mice dosed subcutaneously (SC) with GLP-1 receptor agonist liraglutide (30 nmol/kg), glucagon receptor agonist G1437 (30 nmol/kg), or MEDI0382 (1, 3, 10 or 30 nmol/kg). At 24 h, liraglutide and G1437 treatment reduced food intake by 50% and 16% vs. controls, respectively. MEDI0382 inhibited food intake in a dose-dependent manner, and exerted superior inhibition of food intake compared to equimolar doses of liraglutide and G1437. The fa/fa rats were also dosed once with liraglutide, G1437 (both at same doses as above), or MEDI0382 (3, 10 and 30 nmol/kg) and food intake measured. Liraglutide and G1437 treatment decreased 24 h food intake by 55% and 27% respectively and MEDI0382 dose-dependently decreased food intake (32%, 67% and 77%). Also, fa/fa rats treated with MEDI0382 had an additional reduction in food intake than equimolar doses of either liraglutide or G1437. Finally, lean C57BL/6 mice housed in the BioDaq food intake monitoring system were dosed SC once with MEDI0382 (0.3, 1, 3, 10 and 30 nmol/kg), and a dose-dependent effect on inhibition of food intake was observed. Further analysis showed MEDI0382 decreased total meal number and increased satiety ratio. In summary, MEDI0382 exerted potent anorexigenic effects in mice independent of the leptin pathway, characterized by decreased number of meals consumed and increased satiety. Disclosure V.G. Howard: Employee; Self; AstraZeneca. Stock/Shareholder; Self; Regeneron Pharmaceuticals, Inc., AstraZeneca. S. Oldham: Stock/Shareholder; Self; AstraZeneca. Employee; Self; MedImmune. A. Konkar: Employee; Self; Sanofi. Other Relationship; Self; MedImmune. J. Trevaskis: Employee; Self; MedImmune. Stock/Shareholder; Self; AstraZeneca. S. Will: Employee; Self; MedImmune. Stock/Shareholder; Self; AstraZeneca.

A comparative study of edetate calcium disodium and dimercaptosuccinic acid in the treatment of lead poisoning in adults

Background: We evaluated the efficacy of two antidotes, edetate calcium disodium (CaNa2EDTA) and dimercaptosuccinic acid (DMSA), for the treatment of lead poisoning in adults.Methods: Thirty-seven patients with blood lead concentrations >40 μg/dL and positive CaNa2EDTA lead mobilization were randomized to receive 1050 mg/m2/day of oral DMSA (n = 21) or 500 mg/m2/day of intravenous CaNa2EDTA (n = 16) over two five-day courses separated by a 10-day rest period. Efficacy of treatment was evaluated by blood lead assays on the first day of the two courses and 14 days after the end of treatment and baseline CaNa2EDTA lead mobilization test and 14 days after the end of treatment.Results and conclusion: Both treatments significantly reduced the prevalence of clinical symptoms, blood lead levels and CaNa2EDTA lead mobilization and were well tolerated. DMSA had a greater impact on reducing blood lead concentrations (p = .005) and CaNa2EDTA on lead mobilization (p = .04). Comparison of equimolar doses showed that CaNa2EDTA was more effective than DMSA (p < .001).