Dispelling urban myths about default uncertainty factors in chemical risk assessment - Sufficient protection against mixture effects?

© 2013 Martin et al.; licensee BioMed Central LtdThis article has been made available through the Brunel Open Access Publishing Fund.Assessing the detrimental health effects of chemicals requires the extrapolation of experimental data in animals to human populations. This is achieved by applying a default uncertainty factor of 100 to doses not found to be associated with observable effects in laboratory animals. It is commonly assumed that the toxicokinetic and toxicodynamic sub-components of this default uncertainty factor represent worst-case scenarios and that the multiplication of those components yields conservative estimates of safe levels for humans. It is sometimes claimed that this conservatism also offers adequate protection from mixture effects. By analysing the evolution of uncertainty factors from a historical perspective, we expose that the default factor and its sub-components are intended to represent adequate rather than worst-case scenarios. The intention of using assessment factors for mixture effects was abandoned thirty years ago. It is also often ignored that the conservatism (or otherwise) of uncertainty factors can only be considered in relation to a defined level of protection. A protection equivalent to an effect magnitude of 0.001-0.0001% over background incidence is generally considered acceptable. However, it is impossible to say whether this level of protection is in fact realised with the tolerable doses that are derived by employing uncertainty factors. Accordingly, it is difficult to assess whether uncertainty factors overestimate or underestimate the sensitivity differences in human populations. It is also often not appreciated that the outcome of probabilistic approaches to the multiplication of sub-factors is dependent on the choice of probability distributions. Therefore, the idea that default uncertainty factors are overly conservative worst-case scenarios which can account both for the lack of statistical power in animal experiments and protect against potential mixture effects is ill-founded. We contend that precautionary regulation should provide an incentive to generate better data and recommend adopting a pragmatic, but scientifically better founded approach to mixture risk assessment. © 2013 Martin et al.; licensee BioMed Central Ltd.Oak Foundatio

Validation of the SCID-hu Thy/Liv mouse model with four classes of licensed antiretrovirals.

BackgroundThe SCID-hu Thy/Liv mouse model of HIV-1 infection is a useful platform for the preclinical evaluation of antiviral efficacy in vivo. We performed this study to validate the model with representatives of all four classes of licensed antiretrovirals.Methodology/principal findingsEndpoint analyses for quantification of Thy/Liv implant viral load included ELISA for cell-associated p24, branched DNA assay for HIV-1 RNA, and detection of infected thymocytes by intracellular staining for Gag-p24. Antiviral protection from HIV-1-mediated thymocyte depletion was assessed by multicolor flow cytometric analysis of thymocyte subpopulations based on surface expression of CD3, CD4, and CD8. These mice can be productively infected with molecular clones of HIV-1 (e.g., the X4 clone NL4-3) as well as with primary R5 and R5X4 isolates. To determine whether results in this model are concordant with those found in humans, we performed direct comparisons of two drugs in the same class, each of which has known potency and dosing levels in humans. Here we show that second-generation antiretrovirals were, as expected, more potent than their first-generation predecessors: emtricitabine was more potent than lamivudine, efavirenz was more potent than nevirapine, and atazanavir was more potent than indinavir. After interspecies pharmacodynamic scaling, the dose ranges found to inhibit viral replication in the SCID-hu Thy/Liv mouse were similar to those used in humans. Moreover, HIV-1 replication in these mice was genetically stable; treatment of the mice with lamivudine did not result in the M184V substitution in reverse transcriptase, and the multidrug-resistant NY index case HIV-1 retained its drug-resistance substitutions.ConclusionGiven the fidelity of such comparisons, we conclude that this highly reproducible mouse model is likely to predict clinical antiviral efficacy in humans

Antimalarial Iron Chelator, FBS0701, Shows Asexual and Gametocyte Plasmodium falciparum Activity and Single Oral Dose Cure in a Murine Malaria Model

Iron chelators for the treatment of malaria have proven therapeutic activity in vitro and in vivo in both humans and mice, but their clinical use is limited by the unsuitable absorption and pharmacokinetic properties of the few available iron chelators. FBS0701, (S)3”-(HO)-desazadesferrithiocin-polyether [DADFT-PE], is an oral iron chelator currently in Phase 2 human studies for the treatment of transfusional iron overload. The drug has very favorable absorption and pharmacokinetic properties allowing for once-daily use to deplete circulating free iron with human plasma concentrations in the high µM range. Here we show that FBS0701 has inhibition concentration 50% (IC50) of 6 µM for Plasmodium falciparum in contrast to the IC50 for deferiprone and deferoxamine at 15 and 30 µM respectively. In combination, FBS0701 interfered with artemisinin parasite inhibition and was additive with chloroquine or quinine parasite inhibition. FBS0701 killed early stage P. falciparum gametocytes. In the P. berghei Thompson suppression test, a single dose of 100 mg/kg reduced day three parasitemia and prolonged survival, but did not cure mice. Treatment with a single oral dose of 100 mg/kg one day after infection with 10 million lethal P. yoelii 17XL cured all the mice. Pretreatment of mice with a single oral dose of FBS0701 seven days or one day before resulted in the cure of some mice. Plasma exposures and other pharmacokinetics parameters in mice of the 100 mg/kg dose are similar to a 3 mg/kg dose in humans. In conclusion, FBS0701 demonstrates a single oral dose cure of the lethal P. yoelii model. Significantly, this effect persists after the chelator has cleared from plasma. FBS0701 was demonstrated to remove labile iron from erythrocytes as well as enter erythrocytes to chelate iron. FBS0701 may find clinically utility as monotherapy, a malarial prophylactic or, more likely, in combination with other antimalarials

Phase 1 trial of the antiangiogenic peptide ATN-161 (Ac-PHSCN-NH2), a beta integrin antagonist, in patients with solid tumours

To evaluate the toxicity, pharmacological and biological properties of ATN-161, a five –amino-acid peptide derived from the synergy region of fibronectin, adult patients with advanced solid tumours were enrolled in eight sequential dose cohorts (0.1–16 mg kg−1), receiving ATN-161 administered as a 10-min infusion thrice weekly. Pharmacokinetic sampling of blood and urine over 7 h was performed on Day 1. Twenty-six patients received from 1 to 14 4-week cycles of treatment. The total number of cycles administered to all patients was 86, without dose-limiting toxicities. At dose levels above 0.5 mg kg−1, mean total clearance and volume of distribution showed dose-independent pharmacokinetics (PKs). At 8.0 and 16.0 mg kg−1, clearance of ATN-161 was reduced, suggesting saturable PKs. Dose escalation was halted at 16 mg kg−1 when drug exposure (area under the curve) exceeded that associated with efficacy in animal models. There were no objective responses. Six patients received more than four cycles of treatment (>112 days). Three patients received 10 or more cycles (⩾280 days). ATN-161 was well tolerated at all dose levels. Approximately, 1/3 of the patients in the study manifested prolonged stable disease. These findings suggest that ATN-161 should be investigated further as an antiangiogenic and antimetastatic cancer agent alone or with chemotherapy

Potent Activity of the HIV-1 Maturation Inhibitor Bevirimat in SCID-hu Thy/Liv Mice

The HIV-1 maturation inhibitor, 3-O-(3',3'-dimethylsuccinyl) betulinic acid (bevirimat, PA-457) is a promising drug candidate with 10 nM in vitro antiviral activity against multiple wild-type (WT) and drug-resistant HIV-1 isolates. Bevirimat has a novel mechanism of action, specifically inhibiting cleavage of spacer peptide 1 (SP1) from the C-terminus of capsid which results in defective core condensation.Oral administration of bevirimat to HIV-1-infected SCID-hu Thy/Liv mice reduced viral RNA by >2 log(10) and protected immature and mature T cells from virus-mediated depletion. This activity was observed at plasma concentrations that are achievable in humans after oral dosing, and bevirimat was active up to 3 days after inoculation with both WT HIV-1 and an AZT-resistant HIV-1 clinical isolate. Consistent with its mechanism of action, bevirimat caused a dose-dependent inhibition of capsid-SP1 cleavage in HIV-1-infected human thymocytes obtained from these mice. HIV-1 NL4-3 with an alanine-to-valine substitution at the N-terminus of SP1 (SP1/A1V), which is resistant to bevirimat in vitro, was also resistant to bevirimat treatment in the mice, and SP1/AIV had replication and thymocyte kinetics similar to that of WT NL4-3 with no evidence of fitness impairment in in vivo competition assays. Interestingly, protease inhibitor-resistant HIV-1 with impaired capsid-SP1 cleavage was hypersensitive to bevirimat in vitro with a 50% inhibitory concentration 140 times lower than for WT HIV-1.These results support further clinical development of this first-in-class maturation inhibitor and confirm the usefulness of the SCID-hu Thy/Liv model for evaluation of in vivo antiretroviral efficacy, drug resistance, and viral fitness

Pre-Clinical Evaluation of a 213Bi-Labeled 2556 Antibody to HIV-1 gp41 Glycoprotein in HIV-1 Mouse Models as a Reagent for HIV Eradication

Any strategy for curing HIV infection must include a method to eliminate viral-infected cells. Based on our earlier proof-of-principle results targeting HIV-1 infected cells with radiolabeled antibody (mAb) to gp41 viral antigen, we embarked on identifying a suitable candidate mAb for preclinical development.Among the several human mAbs to gp41 tested, mAb 2556 was found to have high affinity, reactivity with multimeric forms of gp41 present on both the surface of virus particles and cells expressing HIV-1 Env, and recognition of a highly conserved epitope of gp41 shared by all HIV-1 subtypes. Also, mAb 2556 was the best in competition with HIV-1+ serum antibodies, which is an extremely important consideration for efficacy in the treatment of HIV patients. When radiolabeled with alpha-emitting radionuclide 213-Bismuth ((213)Bi) - (213)Bi-2556 efficiently and specifically killed ACH-2 human lymphocytes chronically infected with HIV-1, and HIV-1 infected human peripheral blood mononuclear cells (hPBMCs). The number of binding sites for (213)Bi-2556 on the surface of the infected cells was >10(6). The in vivo experiments were performed in two HIV-1 mouse models--splenic and intraperitoneal. In both models, the decrease in HIV-1 infected hPBMCs from the spleens and peritoneum, respectively, was dose-dependent with the most pronounced killing of hPBMCs observed in the 100 µCi (213)Bi-2556 group (P = 0.01). Measurement of the blood platelet counts and gross pathology of the treated mice demonstrated the lack of toxicity for (213)Bi-2556.We describe the preclinical development of a novel radiolabeled mAb reagent that could potentially be part of an HIV eradication strategy that is ready for translation into the clinic as the next step in its development. As viral antigens are very different from "self" human antigens - this approach promises high selectivity, increased efficacy and low toxicity, especially in comparison to immunotoxins

Oral Pre-Exposure Prophylaxis by Anti-Retrovirals Raltegravir and Maraviroc Protects against HIV-1 Vaginal Transmission in a Humanized Mouse Model

Sexual HIV-1 transmission by vaginal route is the most predominant mode of viral transmission, resulting in millions of new infections every year. In the absence of an effective vaccine, there is an urgent need to develop other alternative methods of pre-exposure prophylaxis (PrEP). Many novel drugs that are currently approved for clinical use also show great potential to prevent viral sexual transmission when administered systemically. A small animal model that permits rapid preclinical evaluation of potential candidates for their systemic PrEP efficacy will greatly enhance progress in this area of investigation. We have previously shown that RAG-hu humanized mouse model permits HIV-1 mucosal transmission via both vaginal and rectal routes and displays CD4 T cell loss typical to that seen in the human. Thus far systemic PrEP studies have been primarily limited to RT inhibitors exemplified by tenofovir and emtricitabine. In these proof-of-concept studies we evaluated two new classes of clinically approved drugs with different modes of action namely, an integrase inhibitor raltegravir and a CCR5 inhibitor maraviroc as potential systemically administered chemo-prophylactics. Our results showed that oral administration of either of these drugs fully protects against vaginal HIV-1 challenge in the RAG-hu mouse model. Based on these results both these drugs show great promise for further development as orally administered PrEPs

Evaluation of the cancer chemopreventive efficacy of rice bran in genetic mouse models of breast, prostate and intestinal carcinogenesis

Brown rice is a staple dietary constituent in Asia, whereas rice consumed in the Western world is generally white, obtained from brown rice by removal of the bran. We tested the hypothesis that rice bran interferes with development of tumours in TAg, TRansgenic Adenocarcinoma of the Mouse Prostate (TRAMP) or ApcMin mice, genetic models of mammary, prostate and intestinal carcinogenesis, respectively. Mice received rice bran (30%) in AIN-93G diet throughout their post-weaning lifespan. In TAg and TRAMP mice, rice bran did not affect carcinoma development. In TRAMP or wild-type C57Bl6/J mice, dietary rice bran increased kidney weight by 18 and 20%, respectively. Consumption of rice bran reduced numbers of intestinal adenomas in ApcMin mice by 51% (P<0.01), compared to mice on control diet. In parallel, dietary rice bran decreased intestinal haemorrhage in these mice, as reflected by increased haematocrit. At 10% in the diet, rice bran did not significantly retard ApcMin adenoma development. Likewise, low-fibre rice bran (30% in the diet) did not affect intestinal carcinogenesis, suggesting that the fibrous constituents of the bran mediate chemopreventive efficacy. The results suggest that rice bran might be beneficially evaluated as a putative chemopreventive intervention in humans with intestinal polyps

Characterisation of metabolites of the putative cancer chemopreventive agent quercetin and their effect on cyclo-oxygenase activity

Quercetin (3,5,7,3′,4′-pentahydroxyflavone) is a flavone with putative ability to prevent cancer and cardiovascular diseases. Its metabolism was evaluated in rats and human. Rats received quercetin via the intravenous (i.v.) route and metabolites were isolated from the plasma, urine and bile. Analysis was by high-performance liquid chromatography and confirmation of species identity was achieved by mass spectrometry. Quercetin and isorhamnetin, the 3′-O-methyl analogue, were found in both the plasma and urine. In addition, several polar peaks were characterised as sulphated and glucuronidated conjugates of quercetin and isorhamnetin. Extension of the metabolism studies to a cancer patient who had received quercetin as an i.v. bolus showed that (Quercetin removed) isorhamnetin and quercetin 3′-O-sulphate were major plasma metabolites. As a catechol, quercetin can potentially be converted to a quinone and subsequently conjugated with glutathione (GSH). Oxidation of quercetin with mushroom tyrosinase in the presence of GSH furnished GSH conjugates of quercetin, two mono- and one bis-substituted conjugates. However, these species were not found in biomatrices in rats treated with quercetin. As cyclo-oxygenase-2 (COX-2) expression is mechanistically linked to carcinogenesis, we examined whether quercetin and its metabolites can inhibit COX-2 in a human colorectal cancer cell line (HCA-7). Isorhamnetin and its 4′-isomer tamarixetin were potent inhibitors, reflected in a 90% decrease in prostaglandin E-2 (PGE-2) levels, a marker of COX-2 activity. Quercetin was less effective, with a 50% decline. Quercetin 3- and 7-O-sulphate had no effect on PGE-2. The results indicate that quercetin may exert its pharmacological effects, at least in part, via its metabolites