Malarial PI4K inhibitor induced diaphragmatic hernias in rat: Potential link with mammalian kinase inhibition.

BACKGROUND: MMV390048 is an aminopyridine plasmodial PI4K inhibitor, selected as a Plasmodium blood-stage schizonticide for a next generation of malaria treatments to overcome resistance to current therapies. MMV390048 showed an acceptable preclinical safety profile and progressed up to Phase 2a clinical trials. However, embryofetal studies revealed adverse developmental toxicity signals, including diaphragmatic hernias and cardiovascular malformations in rats but not rabbits. METHODS: In vivo exposures of free plasma concentrations of compound in rats were assessed in relation to in vitro human kinase inhibition by MMV390048, using the ADP-Glo™ Kinase Assay. RESULTS: We demonstrate a potential link between the malformations seen in the embryofetal developmental (EFD) studies and inhibition of the mammalian PI4Kß paralogue, as well as inhibition of the off-target kinases MAP4K4 and MINK1. PI3Kγ may also play a role in the embryofetal toxicity as its in vitro inhibition is covered by in vivo exposure. The exposures in the rabbit embryofetal development studies did not reach concentrations likely to cause PI4K inhibition. Overall, we hypothesize that the in vivo malformations observed could be due to inhibition of the PI4K target in combination with the off-targets, MAP4K4 and MINK1. However, these relationships are by association and not mechanistically proven. CONCLUSIONS: Deciphering if the EFD effects are dependent on PI4K inhibition, and/or via inhibition of other off-target kinases will require the generation of novel, more potent, and more specific PI4K inhibitors.

A target safety assessment of the potential toxicological risks of targeting plasmepsin IX/X for the treatment of malaria.

The aspartic proteases plasmepsin IX/X are important antimalarial drug targets due to their specificity to the malaria parasite and their vital role as mediators of disease progression. Focusing on parasite-specific targets where no human homologue exists reduces the possibility of on-target drug toxicity. However, there is a risk of toxicity driven by inadequate selectivity for plasmepsins IX/X in Plasmodium over related mammalian aspartic proteases. Of these, CatD/E may be of most toxicological relevance as CatD is a ubiquitous lysosomal enzyme present in most cell types and CatE is found in the gut and in erythrocytes, the clinically significant site of malarial infection. Based on mammalian aspartic protease physiology and adverse drug reactions (ADRs) to FDA-approved human immunodeficiency virus (HIV) aspartic protease inhibitors, we predicted several potential toxicities including ß-cell and congenital abnormalities, hypotension, hypopigmentation, hyperlipidaemia, increased infection risk and respiratory, renal, gastrointestinal, dermatological, and other epithelial tissue toxicities. These ADRs to the HIV treatments are likely to be a result of host aspartic protease inhibition due a lack of specificity for the HIV protease; plasmepsins are much more closely related to human CatD than to HIV proteinase. Plasmepsin IX/X inhibition presents an opportunity to specifically target Plasmodium as an effective antimalarial treatment, providing adequate selectivity can be obtained. Potential plasmepsin IX/X inhibitors should be assayed for inhibitory activity against the main human aspartic proteases and particularly CatD/E. An investigative rodent study conducted early in drug discovery would serve as an initial risk assessment of the potential hazards identified.

Been There, Done That: A Practical Primer for Veterinarians Considering Inclusion of Small Animal Theriogenology Services in Their Practice.

Broadening your scope of practice to include theriogenology services offers a myriad of advantages. Theriogenology services are profitable, offer new revenue streams, and optimize the use of support staff and hospital. Offering reproductive services sets your practice apart from competitor practices. Breeder clients are demanding but loyal and return for repeat services; they also request and follow recommendations for "high-end" services. Your theriogenology clients often refer locally placed puppies and kittens to you for primary care and you gain new general practice clients. And it is fun!

Quantification of Drug-Induced Inhibition of Canalicular Cholyl-l-Lysyl-Fluorescein Excretion From Hepatocytes by High Content Cell Imaging.

We describe the use of a commercially available high content cell imaging algorithm (Cellomics Arrayscan Spot Detector) to quantify biliary excretion of the fluorescent probe substrate cholyl-l-lysyl-fluorescein (CLF) from rat hepatocytes cultured in collagen/matrigel sandwich configuration and to explore inhibition of this process by a variety of test compounds. The method provided robust, reproducible data. Twenty-nine pharmaceuticals inhibited biliary CLF efflux from hepatocytes and a broad range of potencies of inhibition were observed (IC50 values ranged between <1 and 794 µM). Thirteen drugs that inhibited CLF efflux also inhibited hepatocellular uptake of the probe substrate [(3)H]-taurocholate. Although no clear correlation between the potencies of inhibition of the 2 processes was evident, these data highlight the need to consider possible uptake transporter inhibition when interpreting hepatocyte CLF inhibition data. It has been reported that CLF is transported by MRP2. The CLF efflux inhibition data correlated closely with published data on inhibition by the drugs of the bile salt export pump (Bsep), which suggests that the tested drugs inhibit both Bsep and Mrp2. Calculation of the ratios between the maximum human plasma concentrations of the drugs and their CLF efflux inhibition IC50 values raised the possibility that for many, but not all, of them the in vitro effects may be functionally significant in vivo and that Mrp2 inhibition might be a drug-induced liver injury (DILI) risk factor. These data indicate that imaging hepatocyte CLF inhibition is a promising new method for quantification of biliary efflux inhibition by drugs, which could aid assessment of compound-related DILI risk.

Gene therapy prolongs survival and restores function in murine and canine models of myotubular myopathy.

Loss-of-function mutations in the myotubularin gene (MTM1) cause X-linked myotubular myopathy (XLMTM), a fatal, congenital pediatric disease that affects the entire skeletal musculature. Systemic administration of a single dose of a recombinant serotype 8 adeno-associated virus (AAV8) vector expressing murine myotubularin to Mtm1-deficient knockout mice at the onset or at late stages of the disease resulted in robust improvement in motor activity and contractile force, corrected muscle pathology, and prolonged survival throughout a 6-month study. Similarly, single-dose intravascular delivery of a canine AAV8-MTM1 vector in XLMTM dogs markedly improved severe muscle weakness and respiratory impairment, and prolonged life span to more than 1 year in the absence of toxicity or a humoral or cell-mediated immune response. These results demonstrate the therapeutic efficacy of AAV-mediated gene therapy for myotubular myopathy in small- and large-animal models, and provide proof of concept for future clinical trials in XLMTM patients.

In vitro inhibition of the bile salt export pump correlates with risk of cholestatic drug-induced liver injury in humans.

Inhibition of the activity of the human bile salt export pump (BSEP: ABCB11) has been proposed to play a role in drug-induced liver injury (DILI). To enhance understanding of the relationship between BSEP inhibition and DILI, inhibition of human BSEP (hBSEP) and its rat ortholog (rBsep) by 85 pharmaceuticals was investigated in vitro. This was explored using assays that quantified inhibition of ATP-dependent [(3)H]taurocholate uptake into inverted plasma membrane vesicles from Sf21 insect cells, which expressed the proteins. Of the pharmaceuticals, 40 exhibited evidence of in vitro transporter inhibition and overall a close correlation was observed between potency values for inhibition of hBSEP and rBsep activity (r(2) = 0.94), although 12 drugs exhibited >2-fold more potent inhibition of hBSEP than rBsep. The median potency of hBSEP inhibition was higher among drugs that caused cholestatic/mixed DILI than among drugs that caused hepatocellular or no DILI, as was the incidence of hBSEP inhibition with IC(50) <300 µM. All drugs with hBSEP IC(50) <300 µM had molecular weight >250, ClogP >1.5, and nonpolar surface area >180Å. A clear distinction was not evident between hBSEP IC(50) or unbound plasma concentration (C(max, u)) of the drugs in humans and whether the drugs caused DILI. However, all 17 of the drugs with hBSEP IC(50) <100 µM and C(max, u) >0.002 µM caused DILI. Overall, these data indicate that inhibition of hBSEP/rBsep correlates with the propensity of numerous pharmaceuticals to cause cholestatic DILI in humans and is associated with several of their physicochemical properties.

Cell based approaches for evaluation of drug-induced liver injury.

An improved understanding of mechanisms that underlie drug-induced liver injury (DILI) is required to enable design of drugs that have minimal potential to cause this adverse reaction in man. Available evidence suggests DILI arises in susceptible patients because of an imbalance between chemical insults (which are an inherent property of certain drugs and/or their metabolites) and the ability of the liver to mount compensatory/adaptive responses. In vivo safety testing in pre-clinical species ensures that drugs which enter clinical trials do not cause reproducible and dose-dependent liver injury in man, but is of limited value for exploration of underlying mechanisms and does not assess potential to cause rare idiosyncratic DILI. This review highlights the value that can be gained from in vitro studies using cultured hepatocytes and also hepatocyte-derived cell lines transfected with individual human cytochrome P450 (CYP450) isoforms. We have evaluated a range of mechanisms and endpoints (cell necrosis, mitochondrial injury, inhibition of biliary transporters and metabolite-mediated toxicity) using these model systems. Our data indicate that multiple mechanisms are likely to be involved in development of idiosyncratic DILI in man caused by numerous drugs, e.g. the anticonvulsant chlorpromazine.

Canopy effects droplet size distribution and meteorological change.

Wind speed fluctuations measured via a 3-dimensional sonic anemometer recording at 10 Hz returned detailed information both above and within the canopy. The information returned facilitated detailed descriptions of atmospheric energy. In short, large energetic motions equal spray transfer into the target zone, the plant canopy. Data are presented on the physical and biological characterization of spray flux. When nontarget mortality was high the conditions were stable, and large volumes of pesticide descended via aircraft vortices and sedimentation. On the neutral night where there was turbulence in the atmosphere a large proportion of the spray was transported from the target, by winds at altitude. Therefore nontarget mortality and the overall volume entering the canopy were low. That chemical, however, which did enter the canopy was well mixed and transported horizontally as opposed to the more vertical sedimentation on the stable night creating more consistent control.

Field tests of malathion and permethrin applied via a truck-mounted cold fogger to both open and vegetated habitats.

Field tests of formulated permethrin (Permanone 30:30) and malathion applied by ground ultra-low volume equipment were conducted using caged, laboratory-reared, adult Ochlerotatus taeniorhynchus. Cages were placed in an open field and also in semi-dense scrub pine. Two doses, a high and a medium dose of each compound, were applied as suggested by the label. Regardless of application rate, neither chemical provided satisfactory control under the canopy. The average mortality count at the open site and vegetated site was 86.6% and 49.1%, respectively (F.pr. < 0.001, SED 3.87). Malathion for both open and canopied areas achieved significantly greater mortality compared to permethrin.

A comparison of two ultra-low-volume spray nozzle systems by using a multiple swath scenario for the aerial application of fenthion against adult mosquitoes.

Two hydraulic spray nozzle systems, a flat fan and a high-pressure hollow cone, were used for ultra-low-volume application of the mosquito adulticide fenthion under a multiple swath scheme. Eight swaths at 322-m intervals were applied from a height of 91 m to simulate operational conditions. Deposition, effects on nontarget organisms (fiddler crabs), aerial flux, and mosquito (Ochlerotatus taeniorhynchus) mortality were monitored for 8,230 m downwind, including the area under all 8 swaths. The flat-fan nozzle system deposited 88 times the amount of fenthion deposited by the high-pressure system in a lightly vegetated zone directly beneath the application area (0-2,134 m). Further downwind (2,286-4,420 m) in the 2nd semiopen urban zone, 10.5 times more chemical was deposited with the flat-fan nozzles than with the high-pressure nozzles, and in the 3rd highly vegetated zone (4,572-8,230 m), 25 times more was deposited compared with high-pressure nozzles. The corresponding nontarget mortalities with the flat-fan nozzle were 80, 12, and 17% at 2,438, 3,658, and 4,572 m, respectively. No treatment-induced mortality was observed with high-pressure nozzles. Similar amounts of fenthion residue were recovered from yarn samples for both nozzle systems, with the exception of the zone directly under the flight paths, where the flat-fan system deposited 2.5 times the amount recovered with the high-pressure system. Mosquito mortality was similar between the 2 nozzle types except in the farthest zone, where the average mortalities for the high-pressure system and the flat-fan system were 73.4 and 34.8%, respectively. Regression analysis of the mosquito mortality and yarn samples showed that the high-pressure hollow-cone application could control mosquitoes with half the amount of chemical compared to flat-fan nozzles.

A comparison of two spray nozzle systems used to aerially apply the ultra-low-volume adulticide fenthion.

Field experiments with the mosquito adulticide fenthion (Baytex) compared the conventional flat-fan nozzle system (Tee Jet 8002SS) and a new high-pressure hollow-cone nozzle system (1/8 MISS). Ground deposition and aerial flux of the mosquito adulticide fenthion were measured up to 4.83 km downwind by using filter paper and yarn collectors, respectively. Biological efficacy was investigated by using caged salt-marsh mosquitoes (Ochlerotatus taeniorhynchus), and caged fiddler crabs (Uca pugilator) were exposed to quantify nontarget impact. Peak deposits to the ground were 1,729 microg/m2 and 240 microg/m2 for the flat-fan nozzles and high-pressure cones, respectively. Deposits from the flat-fan nozzles resulted in a cumulative fiddler crab mortality of 80%, whereas no deaths were recorded with the high-pressure system. The range of fenthion flux detected in the air when using the yarn collectors was similar for the 2 systems, with both showing drift through 4.83 km. For the flat-fan spray nozzle system, the aerosol flux ranged from 3.02 to 67.33 microg/yarn collector. The range of aerosol flux for the high-pressure nozzle spray system was 0.15-50.66 microg/yarn collector. Although the 2 systems produced comparable ranges of flux, the high-pressure system provided higher control efficacy against mosquitoes. Maximum mosquito control when using the flat-fan spray nozzle system against female salt-marsh mosquitoes was 26.6%, whereas maximum control with the high-pressure spray system was 92.9%.