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Background: Exercise is often prescribed as a therapy for chronic pain. Short-term exercise briefly increases the production of endogenous analgesics, leading to transient antinociception. In limited studies, exercise produced sustained increases in endogenous opioids, sustained analgesia, or diminished measures of chronic pain. This study tests the hypothesis that regular aerobic exercise leads to sustained reversal of neuropathic pain by activating endogenous opioid-mediated pain modulatory systems.Methods: After baseline measurements, the L5 and L6 spinal nerves of male Sprague-Dawley rats were tightly ligated. Animals were randomized to sedentary or 5-week treadmill exercise-trained groups. Thermal and tactile sensitivities were assessed 23 h after exercise, using paw withdrawal thresholds to von Frey filaments and withdrawal latencies to noxious heat. Opioid receptor antagonists were administered by subcutaneous, intrathecal, or intracerebroventricular injection. Opioid peptides were quantified using immunohistochemistry with densitometry.Results: Exercise training ameliorated thermal and tactile hypersensitivity in spinal nerve-ligated animals within 3 weeks. Sensory hypersensitivity returned 5 days after discontinuation of exercise training. The effects of exercise were reversed by using systemically or intracerebroventricularly administered opioid receptor antagonists and prevented by continuous infusion of naltrexone. Exercise increased β-endorphin and met-enkephalin content in the rostral ventromedial medulla and the mid-brain periaqueductal gray area.Conclusions: Regular moderate aerobic exercise reversed signs of neuropathic pain and increased endogenous opioid content in brainstem regions important in pain modulation. Exercise effects were reversed by opioid receptor antagonists. These results suggest that exercise-induced reversal of neuropathic pain results from an up-regulation of endogenous opioids. [ABSTRACT FROM AUTHOR]

Invasive Staphylococcus aureus (S. aureus) infections are a leading cause of death and not effectively treated with prolonged standard of care antibiotics. A novel THIOMAB™ antibody antibiotic conjugate (TAC) was developed that uses a bacterial-wall specific antibody to deliver the antibiotic (dmDNA31, a rifamycin analogue) to bacteria to minimize toxicities typically seen with prolonged use of traditional antibiotics. The TAC nonclinical toxicology package included repeat dose rat and cynomolgus monkey toxicology studies for 8 weekly intravenous (IV) doses, a 7-day daily repeat dose IV toxicology study of dmDNA31 and an assessment of genotoxicity, cardiovascular toxicity, neurotoxicity and sperm parameters. TAC and dmDNA31 were well tolerated in rats and monkeys, and there was no evidence of genotoxicity, cardiovascular toxicity or neurotoxicity. Non-adverse findings were observed and included blue discoloration in skin, blood, etc. due to the blue color of dmDNA31, increased globulin due to the high doses of antibodies, and abnormal sperm morphology of small heads in male rats with no histopathology correlate in testis. This is an example of antibody-mediated delivery of an antibiotic that has the potential to offer a more effective way of eradicating infection while providing a better safety profile compared to traditional antibiotics., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Monomethyl auristatin E (MMAE) is a potent anti-cancer microtubule-targeting agent (MTA) used as a payload in three approved MMAE-containing antibody drug conjugates (ADCs) and multiple ADCs in clinical development to treat different types of cancers. Unfortunately, MMAE-ADCs can induce peripheral neuropathy, a frequent adverse event leading to treatment dose reduction or discontinuation and subsequent clinical termination of many MMAE-ADCs. MMAE-ADC-induced peripheral neuropathy is attributed to non-specific uptake of the ADC in peripheral nerves and release of MMAE, disrupting microtubules (MTs) and causing neurodegeneration. However, molecular mechanisms underlying MMAE and MMAE-ADC effects on MTs remain unclear. Here, we characterized MMAE-tubulin/MT interactions in reconstituted in vitro soluble tubulin or MT systems and evaluated MMAE and vcMMAE-ADCs in cultured human MCF7 cells. MMAE bound to soluble tubulin heterodimers with a maximum stoichiometry of ~1:1, bound abundantly along the length of pre-assembled MTs and with high affinity at MT ends, introduced structural defects, suppressed MT dynamics, and reduced the kinetics and extent of MT assembly while promoting tubulin ring formation. In cells, MMAE and MMAE-ADC (via nonspecific uptake) suppressed proliferation, mitosis and MT dynamics, and disrupted the MT network. Comparing MMAE action to other MTAs supports the hypothesis that peripheral neuropathy severity is determined by the precise mechanism(s) of each individual drug-MT interaction (location of binding, affinity, effects on morphology and dynamics). This work demonstrates that MMAE binds extensively to tubulin and MTs and causes severe MT dysregulation, providing convincing evidence that MMAE-mediated inhibition of MT-dependent axonal transport leads to severe peripheral neuropathy., (Published by Elsevier Inc.)

Nonclinical toxicology studies are conducted to characterize the potential toxicities and establish a safe starting dose for new drugs in clinical studies, but the question remains as to how predictable/translatable the nonclinical safety findings are to humans. In many cases, there is good concordance between nonclinical species and patients. However, there are cases for which there is a lack of predictivity or translatability that led to early termination of clinical studies due to unanticipated toxicities or early termination of programs before making it to the clinic due to unacceptable nonclinical toxicities assumed to be translatable. A few case examples of safety findings that are translatable versus safety findings that are not translatable and why they are not translateable were presented as a symposium at the 38th Annual Meeting of the American College of Toxicology in Palm Springs, California, and are discussed in this article.

The microtubule inhibitor (MTI) class of chemotherapeutics provide an effective treatment for several different types of cancers, however, severe chemotherapy-induced peripheral neuropathy (CIPN) is a major dose limiting toxicity in patients that limits their use. While CIPN was predicted with MTIs based on histopathology and functional effects in non-clinical toxicology studies, these investigations often require large numbers of animals and long term studies. As in vitro MT assays have been used for decades to study mechanisms of efficacy, we hypothesized that those same assays could be used to study mechanisms of peripheral neuropathy and predict severe CIPN. We analyzed published data on in vitro microtubule (MT) properties for different MTIs that cause varying levels of peripheral neuropathy in patients. Eribulin, vinorelbine and vinfluinine, which all have less severe CIPN than the vinca alkaloids or taxanes, have unique MT properties consisting of reduced affinity and limited binding to MTs (i.e. bind only to the ends and not along the length). Binding more potently to tubulin in the absence of neuronal BIII tubulin was also observed with eribulin and may suggest specificity for tumor tubulin over neuronal tubulin. These are possible mechanisms for causing less severe deleterious effects on MTs in peripheral nerves leading to reduced severity of CIPN. Our analyses demonstrated that in vitro tools used to study the mechanisms of action in inducing severe CIPN (i.e MTI interactions with MTs) warrant further investigation and may be useful for developing next generation MTIs with reduced CIPN., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Christine Genualdi and Nicola Stagg are employees of Genentech, USA and are working on cancer chemotherapy drugs. Stuart Feinstein, MaryAnn Jordan and Leslie Wilson are employees of the University of California, Santa Barbara, USA and consult for many drug companies that develop cancer chemotherapy drugs including Genentech, USA., (Copyright © 2019 Elsevier B.V. All rights reserved.)

This work discloses the first examples of antibody-drug conjugates (ADCs) that are constructed from linker-drugs bearing dimeric seco-CBI payloads (duocarmycin analogs). Several homogeneous, CD22-targeting THIOMAB antibody-drug conjugates (TDCs) containing the dimeric seco-CBI entities are shown to be highly efficacious in the WSU-DLCL2 and BJAB mouse xenograft models. Surprisingly, the seco-CBI-containing conjugates are also observed to undergo significant biotransformation in vivo in mice, rats, and monkeys and thereby form 1:1 adducts with the Alpha-1-Microglobulin (A1M) plasma protein from these species. Variation of both the payload mAb attachment site and length of the linker-drug is shown to alter the rates of adduct formation. Subsequent experiments demonstrated that adduct formation attenuates the in vitro antiproliferation activity of the affected seco-CBI-dimer TDCs, but does not significantly impact the in vivo efficacy of the conjugates. In vitro assays employing phosphatase-treated whole blood suggest that A1M adduct formation is likely to occur if the seco-CBI-dimer TDCs are administered to humans. Importantly, protein adduct formation leads to the underestimation of total antibody (Tab) concentrations using an ELISA assay but does not affect Tab values determined via an orthogonal LC-MS/MS method. Several recommendations regarding bioanalysis of future in vivo studies involving related seco-CBI-containing ADCs are provided based on these collective findings.

A number of cytotoxic pyrrolobenzodiazepine (PBD) monomers containing various disulfide-based prodrugs were evaluated for their ability to undergo activation (disulfide cleavage) in vitro in the presence of either glutathione (GSH) or cysteine (Cys). A good correlation was observed between in vitro GSH stability and in vitro cytotoxicity toward tumor cell lines. The prodrug-containing compounds were typically more potent against cells with relatively high intracellular GSH levels (e.g., KPL-4 cells). Several antibody-drug conjugates (ADCs) were subsequently constructed from PBD dimers that incorporated selected disulfide-based prodrugs. Such HER2 conjugates exhibited potent antiproliferation activity against KPL-4 cells in vitro in an antigen-dependent manner. However, the disulfide prodrugs contained in the majority of such entities were surprisingly unstable toward whole blood from various species. One HER2-targeting conjugate that contained a thiophenol-derived disulfide prodrug was an exception to this stability trend. It exhibited potent activity in a KPL-4 in vivo efficacy model that was approximately three-fold weaker than that displayed by the corresponding parent ADC. The same prodrug-containing conjugate demonstrated a three-fold improvement in mouse tolerability properties in vivo relative to the parent ADC, which did not contain the prodrug.

Antibody drug conjugates (ADC) consist of potent cytotoxic drugs conjugated to antibodies via chemical linkers, which enables specific targeting of tumor cells while reducing systemic exposure to the cytotoxic drug and improving the therapeutic window. The valine citrulline monomethyl auristatin E (vcMMAE, conventional linker-drug) ADC platform has shown promising clinical activity in several cancers, but peripheral neuropathy (PN) is a frequent adverse event leading to treatment discontinuation and dose reduction. This was not predicted based on nonclinical toxicology studies in monkeys or rats treated with vcMMAE ADCs. We evaluated four hypotheses for the lack of translatability of PN with vcMMAE ADCs: 1) species differences in exposure; 2) insensitivity of animal models; 3) species differences in target biology and other vcMMAE ADC properties in peripheral nerves and 4) increased susceptibility of patient population. The result of this hypothesis-based approach identified opportunities to improve the predictivity of PN in our animal models by increasing duration of exposure and adding an expanded neurohistopathology assessment of peripheral nerves. The utility of a predictive animal model would be to provide possible mitigation strategies in the clinic with vcMMAE ADCs and help to screen the next generation microtubule inhibitor (MTI) ADCs for reduced PN., (Copyright © 2016 Elsevier Inc. All rights reserved.)

B7-H4 has been implicated in cancers of the female reproductive system and investigated for its possible use as a biomarker for cancer, but there are no preclinical studies to demonstrate that B7-H4 is a molecular target for therapeutic intervention of cancer. We provide evidence that the prevalence and expression levels of B7-H4 are high in different subtypes of breast cancer and that only a few normal tissues express B7-H4 on the cell membrane. These profiles of low normal expression and upregulation in cancer provide an opportunity for the use of antibody-drug conjugates (ADCs), cytotoxic drugs chemically linked to antibodies, for the treatment of B7-H4 positive cancers. We have developed an ADC specific to B7-H4 that uses a linker drug consisting of a potent antimitotic, monomethyl auristatin E (MMAE), linked to engineered cysteines (THIOMAB) via a protease labile linker. We will refer to ADCs that use the THIOMAB format as TDCs to help distinguish the format from standard MC-vc-MMAE ADCs that are conjugated to the interchain disulfide bonds. Anti-B7-H4 (h1D11)-MC-vc-PAB-MMAE (h1D11 TDC) produced durable tumor regression in cell line and patient-derived xenograft models of triple-negative breast cancer. It also binds rat B7-H4 with similar affinity to human and allowed us to test for target dependent toxicity in rats. We found that our anti-B7-H4 TDC has toxicity findings similar to untargeted TDC. Our results validate B7-H4 as an ADC target for breast cancer and support the possible use of this TDC in the treatment of B7-H4(+) breast cancer.

Oxyfluorfen is a herbicide that is not genotoxic and produces liver toxicity in rodents, following repeated administration at high dose levels. Lifetime rodent feeding studies reported in 1977 with low-purity oxyfluorfen (85%) showed no increase in any tumor type in rats (800 ppm, high dose) and only a marginally increased incidence of hepatocellular tumors in male CD-1 mice at the highest dose (200 ppm). To evaluate the potential carcinogenicity of the currently registered oxyfluorfen (> 98% purity), we conducted a series of short-term liver mode of action (MOA) toxicology studies in male CD-1 mice administered dietary doses of 0, 40, 200, 800, and 1600 ppm for durations of 3, 7, 10, or 28 days. MOA endpoints examined included liver weight, histopathology, cell proliferation, nuclear receptor-mediated gene expression, and other peroxisome proliferator-specific endpoints and their reversibility. Minimal liver effects were observed in mice administered doses at or below 200 ppm for up to 28 days. Increased liver weight, single-cell necrosis, cell proliferation, and peroxisomal acyl-CoA oxidase (ACO) were observed at 800 ppm after 28 days, but there was no increase in peroxisomes. Expression of Cyp2b10 and Cyp4a10 transcripts, markers of constitutive androstane receptor and peroxisome proliferator activated receptor α nuclear receptor activation, respectively, were increased at 800 and 1600 ppm after 3 or 10 days. Collectively, these data along with the negative genotoxicity demonstrate that oxyfluorfen (> 98% purity) has the potential to induce mouse liver tumors through a nongenotoxic, mitogenic MOA with a clear threshold and is not predicted to be carcinogenic in humans at relevant exposure levels.

DAS-40278-9 maize (corn) plants have been genetically modified by the insertion of the aad-1 gene (aryloxyalkanoate dioxygenase), which confers tolerance to 2,4-dichlorophenoxyacetic acid (2,4-D) and aryloxyphenoxypropionate (AOPP) acetyl coenzyme A carboxylase (ACCase) inhibitors ("fop" herbicides) to enable the effective use of these herbicides on maize. The aad-1 gene, derived from Sphingobium herbicidovorans, encodes the aryloxyalkanoate dioxygenase (AAD-1) enzyme. As part of the safety assessment of the AAD-1 protein expressed in maize, acute and repeated dose mammalian toxicology studies were conducted. AAD-1 protein (heterologously produced) was orally administered to mice at a dose of 2000mg/kg, and no acute lethality or adverse effects were observed. Similarly, no adverse effects were observed in mice in a 28-day repeated-dose dietary toxicity study that incorporated the AAD-1 protein into diets at concentrations up to 1000-fold greater than the highest estimate of human exposure to maize. These results support the conclusion that the AAD-1 protein, as expressed in biotechnology derived DAS-40278-9 maize, represents a negligible risk to human health., (Copyright © 2011. Published by Elsevier Inc.)

Unlabelled: Several factors were studied as affecting protein degradation and texture of skipjack tuna muscle following ambient pressure thermal processing (precooking). These included degree of mushy tuna syndrome (MTS) evidenced in the raw meat, raw meat pH, abusive thawing/holding, and precooking temperature/time. Slurries and intact pieces from frozen skipjack tuna, either tempered for 2 h or thawed and held at 25 °C for 22 h (abusive treatment) were heated at temperatures ranging from 40 to 80 °C for up to 2 h, and also at 90 °C for 1 h, with or without prior adjustment of pH to 5 or 7 to favor cathepsin or calpain activity, respectively. Proteolysis of precooked samples was monitored by Lowry assay and SDS-PAGE; cooked texture of intact meat was measured using a Kramer shear press and by sensory profile analysis. Proteolysis maximally occurred in slurries of skipjack tuna muscle that had been abusively stored (22 h at 25 °C) and adjusted to pH 5 prior to heating at 55 °C. Intact pieces of tuna abusively thawed/held for 22 h with subsequent heating at 55 °C also evidenced the most proteolysis and were the least firm in texture. Raw fish that evidenced higher severity of MTS when raw displayed higher levels of proteolysis prior to cooking, which were further increased after cooking at 55 °C., Practical Application: The kinetic data presented here can be used to optimize processing conditions for skipjack tuna canning to minimize textural degradation and optimize quality., (© 2012 Institute of Food Technologists®)

The clinically important opioid fentanyl, administered acutely, enhances mechanical hypersensitivity in a model of surgical pain induced by plantar incision. Activity of neurokinin-1 (NK-1) receptor-expressing ascending spinal neurons, descending pathways originating in the rostral ventromedial medulla (RVM), and spinal dynorphin are necessary for the development and maintenance of hyperalgesia during sustained morphine exposure, suggesting that these mechanisms may also be important in opioid enhancement of surgical pain. Therefore, we examined the roles of these mechanisms in sensory hypersensitivity produced by acute fentanyl administration in rats not undergoing surgical incision and in rats undergoing plantar incision. In non-operated rats, fentanyl induced analgesia followed by immediate and long-lasting sensory hypersensitivity, as previously described. Fentanyl also enhanced pain sensitivity induced by plantar incision. Ablation of NK-1-expressing spinal neurons by pre-treatment with substance P-Saporin reduced sensory hypersensitivity in fentanyl-treated rats and, to a lesser extent, in fentanyl-treated rats with a surgical incision. Microinjection of lidocaine into the RVM completely reversed fentanyl-induced sensory hypersensitivity and fentanyl enhancement of incision-induced sensory hypersensitivity. RVM lidocaine injection resulted in a slight reduction of incision-induced sensory hypersensitivity in the absence of fentanyl pre-treatment. Spinal dynorphin content increased by 30 +/- 7% and 66 +/- 17% in fentanyl- and fentanyl/incision-treated rats. Spinal administration of antiserum to dynorphin attenuated sensory hypersensitivity in fentanyl-treated rats. These data support a partial role of NK-1 receptor-containing ascending pathways and a crucial role of descending facilitatory pathways in fentanyl-induced hyperalgesia and in the enhanced hyperalgesia produced by fentanyl treatment following surgical incision.

Management of acute pain remains a significant clinical problem. In preclinical studies, CB2 cannabinoid receptor-selective agonists inhibit nociception without producing central nervous system side effects. The CB2 receptor-selective agonist AM1241 produces antinociceptive effects that are antagonized by CB2, but not CB1, receptor-selective antagonists, suggesting that activation of CB2 receptors results in antinociception. However, it has not been possible to definitively demonstrate that these effects are mediated by CB2 receptors, because we have lacked the pharmacological tools to confirm the in vivo receptor selectivity of the antagonists used. Further, recent evidence for cannabinoid-like receptors beyond CB1 and CB2 raises the possibility that AM1241 exerts its antinociceptive effects at uncharacterized CB2-like receptors that are also inhibited by AM630. The experiments reported here further test the hypothesis that CB2 receptor activation inhibits nociception. They evaluated the antinociceptive actions of AM1241 and the less-selective CB2 receptor agonist WIN55,212-2 in wild-type (CB2+/+) mice and in mice with genetic disruption of the CB2 receptor (CB2-/- mice). AM1241 inhibited thermal nociception in CB2+/+ mice, but had no effect in CB2-/- littermates. WIN55,212-2 produced equivalent antinociception in CB1+/+ and CB1-/- mice, while its antinociceptive effects were reduced in CB2-/- compared to CB2+/+ mice. The effects of morphine were not altered in CB2-/- compared to CB2+/+ mice. These data strongly suggest that AM1241 produces antinociception in vivo by activating CB2 cannabinoid receptors. Further, they confirm the potential therapeutic relevance of CB2 cannabinoid receptors for the treatment of acute pain.