A Preclinical Safety Study of Thyroid Hormone Instilled into the Lungs of Healthy Rats-an Investigational Therapy for ARDS.

Acute respiratory distress syndrome (ARDS) is a severe, life-threatening form of respiratory failure characterized by pulmonary edema, inflammation, and hypoxemia due to reduced alveolar fluid clearance (AFC). Alveolar fluid clearance is required for recovery and effective gas exchange, and higher rates of AFC are associated with reduced mortality. Thyroid hormones play multiple roles in lung function, and L-3,5,3'-triiodothyronine (T3) has multiple effects on lung alveolar type II cells. T3 enhances AFC in normal adult rat lungs when administered intramuscularly and in normal or hypoxia-injured lungs when given intratracheally. The safety of a commercially available formulation of liothyronine sodium (synthetic T3) administered intratracheally was assessed in an Investigational New Drug Application-enabling toxicology study in healthy rats. Instillation of the commercial formulation of T3 without modification rapidly caused tracheal injury and often mortality. Intratracheal instillation of T3 that was reformulated and brought to a neutral pH at the maximum feasible dose of 2.73 µg T3 in 300 µl for 5 consecutive days had no clinically relevant T3-related adverse clinical, histopathologic, or clinical pathology findings. There were no unscheduled deaths that could be attributed to the reformulated T3 or control articles, no differences in the lung weights, and no macroscopic or microscopic findings considered to be related to treatment with T3. This preclinical safety study has paved the way for a phase I/II study to determine the safety and tolerability of a T3 formulation delivered into the lungs of patients with ARDS, including coronavirus disease 2019-associated ARDS, and to measure the effect on extravascular lung water in these patients. SIGNIFICANCE STATEMENT: There is growing interest in treating lung disease with thyroid hormone [triiodothyronine (T3)] in pulmonary edema and acute respiratory distress syndrome (ARDS). However, there is not any published experience on the impact of direct administration of T3 into the lung. An essential step is to determine the safety of multiple doses of T3 administered in a relevant animal species. This study enabled Food and Drug Administration approval of a phase I/II clinical trial of T3 instillation in patients with ARDS, including coronavirus disease 2019-associated ARDS (T3-ARDS ClinicalTrials.gov Identifier NCT04115514).

JZL184 is anti-hyperalgesic in a murine model of cisplatin-induced peripheral neuropathy.

Cisplatin has been used effectively to treat a variety of cancers but its use is limited by the development of painful peripheral neuropathy. Because the endocannabinoid 2-arachidonoyl-sn-glycerol (2-AG) is anti-hyperalgesic in several preclinical models of chronic pain, the anti-hyperalgesic effect of JZL184, an inhibitor of 2-AG hydrolysis, was tested in a murine model of cisplatin-induced hyperalgesia. Systemic injection of cisplatin (1mg/kg) produced mechanical hyperalgesia when administered daily for 7 days. Daily peripheral administration of a low dose of JZL184 in conjunction with cisplatin blocked the expression of mechanical hyperalgesia. Acute injection of a cannabinoid (CB)-1 but not a CB2 receptor antagonist reversed the anti-hyperalgesic effect of JZL184 indicating that downstream activation of CB1 receptors suppressed the expression of mechanical hyperalgesia. Components of endocannabinoid signaling in plantar hind paw skin and lumbar dorsal root ganglia (DRGs) were altered by treatments with cisplatin and JZL184. Treatment with cisplatin alone reduced levels of 2-AG and AEA in skin and DRGs as well as CB2 receptor protein in skin. Combining treatment of JZL184 with cisplatin increased 2-AG in DRGs compared to cisplatin alone but had no effect on the amount of 2-AG in skin. Evidence that JZL184 decreased the uptake of [(3)H]AEA into primary cultures of DRGs at a concentration that also inhibited the enzyme fatty acid amide hydrolase, in conjunction with data that 2-AG mimicked the effect of JZL184 on [(3)H]AEA uptake support the conclusion that AEA most likely mediates the anti-hyperalgesic effect of JZL184 in this model.

Increased anandamide uptake by sensory neurons contributes to hyperalgesia in a model of cancer pain.

Opioids do not effectively manage pain in many patients with advanced cancer. Because anandamide (AEA) activation of cannabinoid type-1 receptors (CB1R) on nociceptors reduces nociception, manipulation of AEA metabolism in the periphery may be an effective alternative or adjuvant therapy in the management of cancer pain. AEA is hydrolyzed by the intracellular enzyme fatty acid amide hydrolase (FAAH), and this enzyme activity contributes to uptake of AEA into neurons and to reduction of AEA available to activate CB1R. We used an in vitro preparation of adult murine dorsal root ganglion (DRG) neurons co-cultured with fibrosarcoma cells to investigate how tumors alter the uptake of AEA into neurons. Evidence that the uptake of [(3)H]AEA into dissociated DRG cells in the co-culture model mimicked the increase in uptake that occurred in DRG cells from tumor-bearing mice supported the utility of the in vitro model to study AEA uptake. Results with the fluorescent AEA analog CAY10455 confirmed that an increase in uptake in the co-culture model occurred in neurons. One factor that contributed to the increase in [(3)H]AEA uptake was an increase in total cellular cholesterol in the cancer condition. Treatment with the FAAH inhibitor URB597 reduced CAY10455 uptake in the co-culture model to the level observed in DRG neurons maintained in the control condition (i.e., in the absence of fibrosarcoma cells), and this effect was paralleled by OMDM-1, an inhibitor of AEA uptake, at a concentration that had no effect on FAAH activity. Maximally effective concentrations of the two drugs together produced a greater reduction than was observed with each drug alone. Treatment with BMS309403, which competes for AEA binding to fatty acid binding protein-5, mimicked the effect of OMDM-1 in vitro. Local injection of OMDM-1 reduced hyperalgesia in vivo in mice with unilateral tumors in and around the calcaneous bone. Intraplantar injection of OMDM-1 (5µg) into the tumor-bearing paw reduced mechanical hyperalgesia through a CB1R-dependent mechanism and also reduced a spontaneous nocifensive behavior. The same dose reduced withdrawal responses evoked by suprathreshold mechanical stimuli in naive mice. These data support the conclusion that OMDM-1 inhibits AEA uptake by a mechanism that is independent of inhibition of FAAH and provide a rationale for the development of peripherally restricted drugs that decrease AEA uptake for the management of cancer pain.

Peroxisome proliferator-activated receptor α mediates acute effects of palmitoylethanolamide on sensory neurons.

The amplitude of the depolarization-evoked Ca2+ transient is larger in dorsal root ganglion (DRG) neurons from tumor-bearing mice compared with that of neurons from naive mice, and the change is mimicked by coculturing DRG neurons with the fibrosarcoma cells used to generate the tumors (Khasabova et al., 2007). The effect of palmitoylethanolamide (PEA), a ligand for the peroxisome proliferator-activated receptor α (PPARα), was determined on the evoked-Ca2+ transient in the coculture condition. The level of PEA was reduced in DRG cells from tumor-bearing mice as well as those cocultured with fibrosarcoma cells. Pretreatment with PEA, a synthetic PPARα agonist (GW7647), or ARN077, an inhibitor of the enzyme that hydrolyzes PEA, acutely decreased the amplitude of the evoked Ca2+ transient in small DRG neurons cocultured with fibrosarcoma cells. The PPARα antagonist GW6471 blocked the effect of each. In contrast, the PPARα agonist was without effect in the control condition, but the antagonist increased the amplitude of the Ca2+ transient, suggesting that PPARα receptors are saturated by endogenous ligand under basal conditions. Effects of drugs on mechanical sensitivity in vivo paralleled their effects on DRG neurons in vitro. Local injection of ARN077 decreased mechanical hyperalgesia in tumor-bearing mice, and the effect was blocked by GW6471. These data support the conclusion that the activity of DRG neurons is rapidly modulated by PEA through a PPARα-dependent mechanism. Moreover, agents that increase the activity of PPARα may provide a therapeutic strategy to reduce tumor-evoked pain.

A decrease in anandamide signaling contributes to the maintenance of cutaneous mechanical hyperalgesia in a model of bone cancer pain.

Tumors in bone are associated with pain in humans. Data generated in a murine model of bone cancer pain suggest that a disturbance of local endocannabinoid signaling contributes to the pain. When tumors formed after injection of osteolytic fibrosarcoma cells into the calcaneus bone of mice, cutaneous mechanical hyperalgesia was associated with a decrease in the level of anandamide (AEA) in plantar paw skin ipsilateral to tumors. The decrease in AEA occurred in conjunction with increased degradation of AEA by fatty acid amide hydrolase (FAAH). Intraplantar injection of AEA reduced the hyperalgesia, and intraplantar injection of URB597, an inhibitor of FAAH, increased the local level of AEA and also reduced hyperalgesia. An increase in FAAH mRNA and enzyme activity in dorsal root ganglia (DRG) L3-L5 ipsilateral to the affected paw suggests DRG neurons contribute to the increased FAAH activity in skin in tumor-bearing mice. Importantly, the anti-hyperalgesic effects of AEA and URB597 were blocked by a CB1 receptor antagonist. Increased expression of CB1 receptors by DRG neurons ipsilateral to tumor-bearing limbs may contribute to the anti-hyperalgesic effect of elevated AEA levels. Furthermore, CB1 receptor protein-immunoreactivity as well as inhibitory effects of AEA and URB597 on the depolarization-evoked Ca(2+) transient were increased in small DRG neurons cocultured with fibrosarcoma cells indicating that fibrosarcoma cells are sufficient to evoke phenotypic changes in AEA signaling in DRG neurons. Together, the data provide evidence that manipulation of peripheral endocannabinoid signaling is a promising strategy for the management of bone cancer pain.

Chemical interactions between fibrosarcoma cancer cells and sensory neurons contribute to cancer pain.

In an experimental model of cancer pain, the hyperalgesia that occurs with osteolytic tumor growth is associated with the sensitization of nociceptors. We examined functional and molecular changes in small-diameter dorsal root ganglion (DRG) neurons to determine cellular mechanisms underlying this sensitization. The occurrence of a Ca2+ transient in response to either KCl (25 mM) or capsaicin (500 nM) increased in small neurons isolated from murine L3-L6 DRGs ipsilateral to fibrosarcoma cell tumors. The increased responses were associated with increased mRNA levels for the Ca2+ channel subunit alpha2delta1 and TRPV1 receptor. Pretreatment with gabapentin, an inhibitor of the alpha2delta1 subunit, blocked the increased response to KCl in vitro and the mechanical hyperalgesia in tumor-bearing mice in vivo. Similar increases in neuronal responsiveness occurred when DRG neurons from naive mice and fibrosarcoma cells were cocultured for 48 h. The CC chemokine ligand 2 (CCL2) may contribute to the tumor cell-induced sensitization because CCL2 immunoreactivity was present in tumors, high levels of CCL2 peptide were present in microperfusates from tumors, and treatment of DRG neurons in vitro with CCL2 increased the amount of mRNA for the alpha2delta1 subunit. Together, our data provide strong evidence that the chemical mediator CCL2 is released from tumor cells and evokes phenotypic changes in sensory neurons, including increases in voltage-gated Ca2+ channels that likely underlie the mechanical hyperalgesia in the fibrosarcoma cancer model. More broadly, this study provides a novel in vitro model to resolve the cellular and molecular mechanisms by which tumor cells drive functional changes in nociceptors.

Neurotrophin activation of NFAT-dependent transcription contributes to the regulation of pro-nociceptive genes.

Nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) play key roles in the development of inflammation-induced hyperalgesia by triggering the expression of pro-nociceptive genes within primary afferent and spinal neurons. However, the mechanisms by which neurotrophins elicit gene expression remain largely unknown. Recently, neurotrophins have been shown to activate members of the calcineurin (CaN)-regulated, nuclear factor of activated T-cells (NFATc) family of transcription factors within brain. Thus, we hypothesized that NFATc transcription factors couple neurotrophin signaling to gene expression within primary afferent and spinal neurons. In situ hybridization revealed NFATc4 mRNA within the dorsal root ganglion and spinal cord. In cultured dorsal root ganglion cells, NGF triggered NFAT-dependent transcription in a CaN-sensitive manner. Further, increased BDNF expression following NGF treatment relied on CaN, thereby suggesting that NGF regulates BDNF transcription via activation of NFATc4. Within cultured spinal cells, BDNF also activated CaN-dependent, NFAT-regulated gene expression. Interestingly, BDNF stimulation increased the expression of the pro-nociceptive genes cyclooxygenase-2, neurokinin-1 receptor, inositol trisphosphates receptor type 1, and BDNF itself, through both NFAT-dependent and NFAT-independent transcriptional mechanisms. Our results suggest that regulation of pro-nociceptive genes through activation of NFAT-dependent transcription is one mechanism by which NGF and BDNF signaling contributes to the development of persistent pain states.