No abstract

No abstract

Antibody-drug conjugates (ADCs) are being actively pursued as a treatment option for cancer following the regulatory approval of brentuximab vedotin (Adcetris) and ado-trastuzumab emtansine (Kadcyla). ADCs consist of a cytotoxic agent conjugated to a targeting antibody through a linker. The two approved ADCs (and most ADCs now in the clinic that use a microtubule disrupting agent as the payload) are heterogeneous conjugates with an average drug-to-antibody ratio (DAR) of 3-4 (potentially ranging from 0 to 8 for individual species). Ado-trastuzumab emtansine employs DM1, a semisynthetic cytotoxic payload of the maytansinoid class, which is conjugated via lysine residues of the antibody to an average DAR of 3.5. To understand the effect of DAR on the preclinical properties of ADCs using maytansinoid cytotoxic agents, we prepared a series of conjugates with a cleavable linker (M9346A-sulfo-SPDB-DM4 targeting folate receptor α (FRα)) or an uncleavable linker (J2898A-SMCC-DM1 targeting the epidermal growth factor receptor (EGFR)) with varying DAR and evaluated their biochemical characteristics, in vivo stability, efficacy, and tolerability. For both formats, a series of ADCs with DARs ranging from low (average of ∼2 and range of 0-4) to very high (average of 10 and range of 7-14) were prepared in good yield with high monomer content and low levels of free cytotoxic agent. The in vitro potency consistently increased with increasing DAR at a constant antibody concentration. We then characterized the in vivo disposition of these ADCs. Pharmacokinetic analysis showed that conjugates with an average DAR below ∼6 had comparable clearance rates, but for those with an average DAR of ∼9-10, rapid clearance was observed. Biodistribution studies in mice showed that these 9-10 DAR ADCs rapidly accumulate in the liver, with maximum localization for this organ at 24-28% percentage injected dose per gram (%ID/g) compared with 7-10% for lower-DAR conjugates (all at 2-6 h post-injection). Our preclinical findings on tolerability and efficacy suggest that maytansinoid conjugates with DAR ranging from 2 to 6 have a better therapeutic index than conjugates with very high DAR (∼9-10). These very high DAR ADCs suffer from decreased efficacy, likely due to faster clearance. These results support the use of DAR 3-4 for maytansinoid ADCs but suggest that the exploration of lower or higher DAR may be warranted depending on the biology of the target antigen.

Excessive cervical facet capsular ligament stretch has been implicated as a cause of whiplashassociated disorders following rear-end impacts, but the pathophysiological mechanisms that produce chronic pain in these cases remain unclear. Using a rat model of C6/C7 cervical facet joint capsule stretch that produces sustained mechanical hyperalgesia, the presence of neuronal hyperexcitability was characterized 7 days after joint loading. Extracellular recordings of spinal dorsal horn neuronal activity between C6 and C8 (117 neurons) were obtained from anesthetized rats, with both painful and non-painful behavioral outcomes established by the magnitude of capsule stretch. The frequency of neuronal firing during noxious pinch (p<0.0182) and von Frey filaments applications (4-26 g) to the forepaw was increased (p<0.0156) in the painful group compared to the non-painful and sham groups. In addition, the incidence and frequency of spontaneous and afterdischarge firing were greater in the painful group (p<0.0307) relative to sham. The proportion of cells in the deep laminae that responded as wide dynamic range neurons also was increased in the painful group relative to non-painful or sham groups (p<0.0348). These findings suggest that excessive facet capsule stretch, while not producing visible tearing, can produce functional plasticity of dorsal horn neuronal activity. The increase in neuronal firing across a range of stimulus magnitudes observed at day 7 post-injury provides the first direct evidence of neuronal modulation in the spinal cord following facet joint loading, and suggests that facet-mediated chronic pain following whiplash injury is driven, at least in part, by central sensitization.

The cervical facet joint and its capsule are a common source of neck pain from whiplash. Mechanical hyperalgesia elicited by painful facet joint distraction is associated with spinal neuronal hyperexcitability that can be induced by transmitter/receptor systems that potentiate the synaptic activation of neurons. This study investigated the temporal response of a glutamate receptor and transporters in the dorsal root ganglia (DRG) and spinal cord. Bilateral C6/C7 facet joint distractions were imposed in the rat either to produce behavioral sensitivity or without inducing any sensitivity. Neuronal metabotropic glutamate receptor-5 (mGluR5) and protein kinase C-epsilon (PKCε) expression in the DRG and spinal cord were evaluated on days 1 and 7. Spinal expression of a glutamate transporter, excitatory amino acid carrier 1 (EAAC1), was also quantified at both time points. Painful distraction produced immediate behavioral hypersensitivity that was sustained for 7 days. Increased expression of mGluR5 and PKCε in the DRG was not evident until day 7 and only following painful distraction; this increase was observed in small-diameter neurons. Only painful facet joint distraction produced a significant increase (p<0.001) in neuronal mGluR5 over time, and this increase also was significantly elevated (p ≤ 0.05) over responses in the other groups at day 7. However, there were no differences in spinal PKCε expression on either day or between groups. Spinal EAAC1 expression was significantly increased (p<0.03) only in the nonpainful groups on day 7. Results from this study suggest spinal glutamatergic plasticity is selectively modulated in association with facet-mediated pain.

The cervical facet joint and its capsule have been reported to be injured during whiplash scenarios and are a common source of chronic neck pain from whiplash. Both the metabotropic glutamate receptor 5 (mGluR5) and the excitatory amino acid carrier 1 (EAAC1) have pivotal roles in chronic pain. In this study, spinal mGluR5 and EAAC1 were quantified following painful facet joint distraction in a rat model of facet-mediated painful loading and were evaluated for their correlation with the severity of capsule loading. Rats underwent either a dynamic C6/C7 joint distraction simulating loading experienced during whiplash (distraction; n = 12) or no distraction (sham; n = 6) to serve as control. The severity of capsular loading was quantified using strain metrics, and mechanical allodynia was assessed after surgery. Spinal cord tissue was harvested at day 7 and the expression of mGluR5 and EAAC1 were quantified using Western blot analysis. Mechanical allodynia following distraction was significantly (p < 0.001) higher than sham. Spinal expression of mGluR5 was also significantly (p < 0.05) greater following distraction relative to sham. However, spinal EAAC1 was significantly (p = 0.0003) reduced compared to sham. Further, spinal mGluR5 expression was significantly positively correlated to capsule strain (p < 0.02) and mechanical allodynia (p < 0.02). Spinal EAAC1 expression was significantly negatively related to one of the strain metrics (p < 0.003) and mechanical allodynia at day 7 (p = 0.03). These results suggest that the spinal glutamatergic system may potentiate the persistent behavioral hypersensitivity that is produced following dynamic whiplash-like joint loading; chronic whiplash pain may be alleviated by blocking mGluR5 expression and/or enhancing glutamate transport through the neuronal transporter EAAC1.

Facet joint injury induces persistent pain that may be maintained by structural plasticity in the spinal cord. Astrocyte-derived thrombospondins, especially thrombospondin-4 (TSP4), have been implicated in synaptogenesis and spinal sensitization in neuropathic pain, but the TSP4 response and its relationship to synaptic changes in the spinal cord have not been investigated for painful joint injury. This study investigates the role of TSP4 in the development and maintenance of persistent pain following injurious facet joint distraction in rats and tests the hypothesis that excitatory synaptogenesis contributes to such pain. Painful facet joint loading induces dorsal horn excitatory synaptogenesis along with decreased TSP4 in the DRG and increased astrocytic release of TSP4 in the spinal cord, all of which parallel the time course of sustained tactile allodynia. Blocking injury-induced spinal TSP4 expression with antisense oligonucleotides or reducing TSP4 activity at its neuronal receptor in the spinal cord with gabapentin treatment both attenuate the allodynia and dorsal horn synaptogenesis that develop after painful facet joint loading. Increased spinal TSP4 also facilitates the development of allodynia and spinal hyperexcitability, even after non-painful physiologic loading of the facet joint. These results suggest that spinal TSP4 plays an important role in the development and maintenance of persistent joint-mediated pain by inducing excitatory synaptogenesis and facilitating the transduction of mechanical loading of the facet joint that leads to spinal hyperexcitability.

The cervical facet joint is implicated as one of the most common sources of chronic neck pain, owing to its rich nociceptive innervation and susceptibility to injurious mechanical loading. Injuries to the facet joint and its ligament can induce inflammation in the joint and spinal cord. Inflammatory molecules which are known to have a role in pain can also stimulate the integrated stress response (ISR). Therefore, we hypothesize that ISR is activated by facet joint injury in a rodent model of pain. To address this hypothesis, we assessed the expression of binding protein (BiP) (also known as growth-related protein 78 (GRP78)), a marker of endoplasmic reticulum stress response, in the dorsal root ganglion (DRG) after painful facet joint injury. In a rodent model of facet joint injury, dynamic distraction of the C6/C7 joint (injury, n = 12) was imposed; sham procedures were performed separately (sham, n = 8). Forepaw mechanical allodynia was assessed postoperatively for 7 days as a quantitative measure of pain symptoms. The C6 DRG was harvested and assessed for BiP expression using triple label immunofluorescent confocal microscopy and immunoblot analyses. BiP was significantly higher (p < 0.001) in the DRG after injury than sham and was expressed predominantly in neurons. Similarly, quantification of BiP by immunoblot demonstrated a significant 2.1-fold increase (p = 0.03) in injury compared to sham at day 7. Findings suggest neuronal stress activation is associated with painful facet joint injury, and that joint loading may directly mediate the behavior of DRG neurons in this class of injury. Inflammation contributes to persistent pain [5,34]. Cytokines and neurotrophic factors are upregulated in dorsal root ganglion (DRG) neurons in models of facet joint inflammation [20,21]. Lumbar neuropathic pain models exhibit glial cell activation, macrophage reactivity, and cytokine upregulation in the DRG and spinal cord [8,[32][33][34]. Glial activation can alter neuronal signaling and can also induce secretion of several pain mediators, including glutamate, pro-inflammatory cytokines and chemokines [19]. The release of glutamate and cytokines in the inflammatory cascade can also cause neuronal damage [2], and may trigger the integrated stress response (ISR). The integrated stress response is also known as the endoplasmic reticulum (ER) stress response or the unfolded protein response and is a protective cellular response that establishes protein homeostasis in response to a variety of injurious stimuli by increasing transcription and translation of gene products that regulate protein folding, degradation, delivery, and oxidation [16,18]. The ISR binding protein (BiP) is an ERresident chaperone that acts as a major regulator of the stress response [9,26]. While the mechanisms of activation of the ISR and the specific pathways of the neuronal stress response are receiving attention, their potential role in painful injury remains unexplored. KeywordsFacet joint distraction in the rat produces sustained spinal astr...