Toll-like receptor 2 and 4 antagonism for the treatment of experimental autoimmune encephalomyelitis (EAE)-related pain.

Neuropathic pain is a major symptom of multiple sclerosis (MS) with up to 92% of patients reporting bodily pain, and 85% reporting pain severe enough to cause functional disability. None of the available therapeutics target MS pain. Toll-like receptors 2 and 4 (TLR2/TLR4) have emerged as targets for treating a wide array of autoimmune disorders, including MS, as well as having demonstrated success at suppressing pain in diverse animal models. The current series of studies tested systemic TLR2/TLR4 antagonists in males and females in a low-dose Myelin oligodendrocyte glycoprotein (MOG) experimental autoimmune encephalomyelitis (EAE) model, with reduced motor dysfunction to allow unconfounded testing of allodynia through 50+ days post-MOG. The data demonstrated that blocking TLR2/TLR4 suppressed EAE-related pain, equally in males and females; upregulation of dorsal spinal cord proinflammatory gene expression for TLR2, TLR4, NLRP3, interleukin-1ß, IkBα, TNF-α and interleukin-17; and upregulation of dorsal spinal cord expression of glial immunoreactivity markers. In support of these results, intrathecal interleukin-1 receptor antagonist reversed EAE-induced allodynia, both early and late after EAE induction. In contrast, blocking TLR2/TLR4 did not suppress EAE-induced motor disturbances induced by a higher MOG dose. These data suggest that blocking TLR2/TLR4 prevents the production of proinflammatory factors involved in low dose EAE pathology. Moreover, in this EAE model, TLR2/TLR4 antagonists were highly effective in reducing pain, whereas motor impairment, as seen in high dose MOG EAE, is not affected.

Targeted interleukin-10 plasmid DNA therapy in the treatment of osteoarthritis: Toxicology and pain efficacy assessments.

Osteoarthritis results in chronic pain and loss of function. Proinflammatory cytokines create both osteoarthritis pathology and pain. Current treatments are poorly effective, have significant side effects, and have not targeted the cytokines central to osteoarthritis development and maintenance. Interleukin-10 is an anti-inflammatory cytokine that potently and broadly suppresses proinflammatory cytokine activity. However, interleukin-10 protein has a short half-life in vivo and poor joint permeability. For sustained IL-10 activity, we developed a plasmid DNA-based therapy that expresses a long-acting human interleukin-10 variant (hIL-10var). Here, we describe the 6-month GLP toxicology study of this therapy. Intra-articular injections of hIL-10var pDNA into canine stifle joints up to 1.5 mg bilaterally were well-tolerated and without pathologic findings. This represents the first long-term toxicologic assessment of intra-articular pDNA therapy. We also report results of a small double-blind, placebo-controlled study of the effect of intra-articular hIL-10var pDNA on pain measures in companion (pet) dogs with naturally occurring osteoarthritis. This human IL-10-based targeted therapy reduced pain measures in the dogs, based on veterinary and owner ratings, without any adverse findings. These results with hIL-10var pDNA therapy, well-tolerated and without toxicologic effects, establish the basis for clinical trials of a new class of safe and effective therapies for OA.

Behavioral assessment of neuropathic pain, fatigue, and anxiety in experimental autoimmune encephalomyelitis (EAE) and attenuation by interleukin-10 gene therapy.

Relapsing-remitting multiple sclerosis is commonly associated with motor impairments, neuropathic pain, fatigue, mood disorders, and decreased life expectancy. However, preclinical pharmacological studies predominantly rely on clinical scoring of motor deficit as the sole behavioral endpoint. Thus, the translational potential of these studies is limited. Here, we have assessed the therapeutic potential of a novel anti-inflammatory interleukin-10 (IL-10) non-viral gene therapy formulation (XT-101-R) in a rat relapsing remitting experimental autoimmune encephalomyelitis (EAE) model. EAE induced motor deficits and neuropathic pain as reflected by induction of low-threshold mechanical allodynia, suppressed voluntary wheel running, decreased social exploration, and was associated with markedly enhanced mortality. We also noted that voluntary wheel running was depressed prior to the onset of motor deficit, and may therefore serve as a predictor of clinical symptoms onset. XT-101-R was intrathecally dosed only once at the onset of motor deficits, and attenuated each of the EAE-induced symptoms and improved survival, relative to vehicle control. This is the first pharmacological assessment of such a broad range of EAE symptoms, and provides support for IL-10 gene therapy as a clinical strategy for the treatment of multiple sclerosis.

Intrathecal injection of naked plasmid DNA provides long-term expression of secreted proteins.

Therapeutic benefit has been reported to result from intrathecal (i.t.) injection of transgene vectors, including naked DNA. However, most studies using naked DNA have measured only the transgene expression of intracellular proteins. Here we demonstrate that i.t. injection of naked DNA can result in long-term expression of secreted proteins. Plasmids expressing either secreted alkaline phosphatase (SEAP) or human interleukin-10 (hIL-10) were injected into the i.t. space in rats, and transgene products were repeatedly measured in the cerebrospinal fluid (CSF). Both SEAP and hIL-10 were maximal at 1 and 2 days after the injection and still detectable at 4 months. The utilization of a plasmid having two features that are hypothesized to increase gene expression (matrix attachment regions (MARs) and lack of CpG dinucleotides) resulted in a significant increase in gene expression. Reinjection of SEAP or hIL-10 plasmids after 4 months significantly increased protein levels at 1 and 14 days after the reinjection. SEAP was uniformly distributed between the DNA delivery site (approximately vertebral level T13) and the lumbar puncture site (L5/L6 inter-vertebral space), was reduced at the cisterna magna, and was detectable, though at much lower levels, in serum. These data suggest that naked DNA has the potential to be used as a therapeutic tool for applications that require long-term release of transgenes into the CSF.

Immunogenicity of intrathecal plasmid gene delivery: cytokine release and effects on transgene expression.

BACKGROUND: One method for the delivery of therapeutic proteins to the spinal cord is to inject nonviral gene vectors including plasmid DNA into the cerebrospinal fluid (CSF) that surrounds the spinal cord (intrathecal space). This approach has produced therapeutic benefits in animal models of disease and several months of protein expression; however, there is little information available on the immune response to these treatments in the intrathecal space, the relevance of plasmid CpG sequences to any plasmid-induced immune response, or the effect of this immune response on transgene expression. METHODS: In the present study, coding or noncoding plasmids were delivered to the intrathecal space of the lumbar spinal region in rats. Lumbosacral CSF was then collected at various time points afterwards for monitoring of cytokines and transgene expression. RESULTS: This work demonstrates, for the first time, increased tumor necrosis factor-alpha and interleukin-1 in response to intrathecal plasmid vector injection and provides evidence indicating that this response is largely absent in a CpG-depleted vector. Transgene expression in the CSF is not significantly affected by this immune response. Expression after intrathecal plasmid injection is variable across rats but correlates with the amount of tissue associated plasmid and is increased by disrupting normal CSF flow. CONCLUSIONS: The data obtained in the present study indicate that plasmid immunogenicity may affect intrathecal plasmid gene therapy safety but not transgene expression in the CSF. Furthermore, the development of methods to prevent loss of plasmid via CSF flow out of the central nervous system through the injection hole and/or natural outflow routes may increase intrathecal plasmid gene delivery efficacy.

PEGylation of interleukin-10 for the mitigation of enhanced pain states.

The anti-inflammatory cytokine interleukin-10 (IL-10) shows promise for the treatment of neuropathic pain, but for IL-10 to be clinically useful as a short-term therapeutic its duration needs to be improved. In this study, IL-10 was covalently modified with polyethylene glycol (PEG) with the goal of stabilizing and increasing protein levels in the CSF to improve the efficacy of IL-10 for treating neuropathic pain. Two different PEGylation methods were explored in vitro to identify suitable PEGylated IL-10 products for subsequent in vivo testing. PEGylation of IL-10 by acylation yielded a highly PEGylated product with a 35-fold in vitro biological activity reduction. PEGylation of IL-10 by reductive amination yielded products with a minimal number of PEG molecules attached and in vitro biological activity reductions of approximately 3-fold. In vivo collections of cerebrospinal fluid after intrathecal administration demonstrated that 20 kDa PEG attachment to IL-10 increased the concentration of IL-10 in the cerebrospinal fluid over time. Relative to unmodified IL-10, the 20 kDa PEG-IL-10 product exhibited an increased therapeutic duration and magnitude in an animal model of neuropathic pain. This suggests that PEGylation is a viable strategy for the short-term treatment or, in conjunction with other approaches, the long-term treatment of enhanced pain states.

PEGylation of brain-derived neurotrophic factor for preserved biological activity and enhanced spinal cord distribution.

Brain-derived neurotrophic factor (BDNF) was covalently attached to polyethylene glycol (PEG) in order to enhance delivery to the spinal cord via the cerebrospinal fluid (intrathecal administration). By varying reaction conditions, mixtures of BDNF covalently attached to one (primary), two (secondary), three (tertiary), or more (higher order) PEG molecules were produced. The biological activity of each resulting conjugate mixture was assessed with the goal of identifying a relationship between the number of PEG molecules attached to BDNF and biological activity. A high degree of in vitro biological activity was maintained in mixtures enriched in primary and secondary conjugate products, while a substantial reduction in biological activity was observed in mixtures with tertiary and higher order conjugates. When a biologically active mixture of PEG-BDNF was administered intrathecally, it displayed a significantly improved half-life in the cerebrospinal fluid and an enhanced penetration into spinal cord tissue relative to native BDNF. Results from these studies suggest a PEGylation strategy that preserves the biological activity of the protein while also improving the half-life of the protein in vivo. Furthermore, PEGylation may be a promising approach for enhancing intrathecal delivery of therapeutic proteins with potential for treating disease and injury in the spinal cord.

Intrathecal interleukin-10 gene therapy attenuates paclitaxel-induced mechanical allodynia and proinflammatory cytokine expression in dorsal root ganglia in rats.

Paclitaxel is a commonly used cancer chemotherapy drug that frequently causes painful peripheral neuropathies. The mechanisms underlying this dose-limiting side effect are poorly understood. Growing evidence supports that proinflammatory cytokines, such as interleukin-1 (IL-1) and tumor necrosis factor (TNF), released by activated spinal glial cells and within the dorsal root ganglia (DRG) are critical in enhancing pain in various animal models of neuropathic pain. Whether these cytokines are involved in paclitaxel-induced neuropathy is unknown. Here, using a rat neuropathic pain model induced by repeated systemic paclitaxel injections, we examined whether paclitaxel upregulates proinflammatory cytokine gene expression, and whether these changes and paclitaxel-induced mechanical allodynia can be attenuated by intrathecal IL-1 receptor antagonist (IL-1ra) or intrathecal delivery of plasmid DNA encoding the anti-inflammatory cytokine, interleukin-10 (IL-10). The data show that paclitaxel treatment induces mRNA expression of IL-1, TNF, and immune cell markers in lumbar DRG. Intrathecal IL-1ra reversed paclitaxel-induced allodynia and intrathecal IL-10 gene therapy both prevented, and progressively reversed, this allodynic state. Moreover, IL-10 gene therapy resulted in increased IL-10 mRNA levels in lumbar DRG and meninges, measured 2 weeks after initiation of therapy, whereas paclitaxel-induced expression of IL-1, TNF, and CD11b mRNA in lumbar DRG was markedly decreased. Taken together, these data support that paclitaxel-induced neuropathic pain is mediated by proinflammatory cytokines, possibly released by activated immune cells in the DRG. We propose that targeting the production of proinflammatory cytokines by intrathecal IL-10 gene therapy may be a promising therapeutic strategy for the relief of paclitaxel-induced neuropathic pain.

Pharmacological, pharmacokinetic, and primate analgesic efficacy profile of the novel bradykinin B1 Receptor antagonist ELN441958.

The bradykinin B(1) receptor plays a critical role in chronic pain and inflammation, although efforts to demonstrate efficacy of receptor antagonists have been hampered by species-dependent potency differences, metabolic instability, and low oral exposure of current agents. The pharmacology, pharmacokinetics, and analgesic efficacy of the novel benzamide B(1) receptor antagonist 7-chloro-2-[3-(9-pyridin-4-yl-3,9-diazaspiro[5.5]undecanecarbonyl)phenyl]-2,3-dihydro-isoindol-1-one (ELN441958) is described. ELN441958 competitively inhibited the binding of the B(1) agonist ligand [(3)H]desArg(10)-kallidin ([(3)H]DAKD) to IMR-90 human fibroblast membranes with high affinity (K(i) = 0.26 +/- 0.02 nM). ELN441958 potently antagonized DAKD (but not bradykinin)-induced calcium mobilization in IMR-90 cells, indicating that it is highly selective for B(1) over B(2) receptors. Antagonism of agonist-induced calcium responses at B(1) receptors from different species indicated that ELN441958 is selective for primate over rodent B(1) receptors with a rank order potency (K(B), nanomolar) of human (0.12 +/- 0.02) approximately rhesus monkey (0.24 +/- 0.01) > rat (1.5 +/- 0.4) > mouse (14 +/- 4). ELN441958 had good permeability and metabolic stability in vitro consistent with high oral exposure and moderate plasma half-lives in rats and rhesus monkeys. Because ELN441958 is up to 120-fold more potent at primate than at rodent B(1) receptors, it was evaluated in a primate pain model. ELN441958 dose-dependently reduced carrageenan-induced thermal hyperalgesia in a rhesus monkey tail-withdrawal model, with an ED(50) approximately 3 mg/kg s.c. Naltrexone had no effect on the antihyperalgesia produced by ELN441958, indicating a lack of involvement of opioid receptors. ELN441958 is a novel small molecule bradykinin B(1) receptor antagonist exhibiting high oral bioavailability and potent systemic efficacy in rhesus monkey inflammatory pain.

Repeated intrathecal injections of plasmid DNA encoding interleukin-10 produce prolonged reversal of neuropathic pain.

Neuropathic pain is a major clinical problem unresolved by available therapeutics. Spinal cord glia play a pivotal role in neuropathic pain, via the release of proinflammatory cytokines. Anti-inflammatory cytokines, like interleukin-10 (IL-10), suppress proinflammatory cytokines. Thus, IL-10 may provide a means for controlling glial amplification of pain. We recently documented that intrathecal IL-10 protein resolves neuropathic pain, albeit briefly (approximately 2-3 h), given its short half-life. Intrathecal gene therapy using viruses encoding IL-10 can also resolve neuropathic pain, but for only approximately 2 weeks. Here, we report a novel approach that dramatically increases the efficacy of intrathecal IL-10 gene therapy. Repeated intrathecal delivery of plasmid DNA vectors encoding IL-10 (pDNA-IL-10) abolished neuropathic pain for greater than 40 days. Naked pDNA-IL-10 reversed chronic constriction injury (CCI)-induced allodynia both shortly after nerve injury as well as 2 months later. This supports that spinal proinflammatory cytokines are important in both the initiation and maintenance of neuropathic pain. Importantly, pDNA-IL-10 gene therapy reversed mechanical allodynia induced by CCI, returning rats to normal pain responsiveness, without additional analgesia. Together, these data suggest that intrathecal IL-10 gene therapy may provide a novel approach for prolonged clinical pain control.

Controlling pathological pain by adenovirally driven spinal production of the anti-inflammatory cytokine, interleukin-10.

Gene therapy for the control of pain has, to date, targeted neurons. However, recent evidence supports that spinal cord glia are critical to the creation and maintenance of pain facilitation through the release of proinflammatory cytokines. Because of the ability of interleukin-10 (IL-10) to suppress proinflammatory cytokines, we tested whether an adenoviral vector encoding human IL-10 (AD-h-IL10) would block and reverse pain facilitation. Three pain models were examined, all of which are mediated by spinal pro-inflammatory cytokines. Acute intrathecal administration of rat IL-10 protein itself briefly reversed chronic constriction injury-induced mechanical allodynia and thermal hyperalgesia. The transient reversal caused by IL-10 protein paralleled the half-life of human IL-10 protein in the intrathecal space (t(1/2) approximately 2 h). IL-10 gene therapy both prevented and reversed thermal hyperalgesia and mechanical allodynia, without affecting basal responses to thermal or mechanical stimuli. Extra-territorial, as well as territorial, pain changes were reversed by this treatment. Intrathecal AD-h-IL10 injected over lumbosacral spinal cord led to elevated lumbosacral cerebrospinal fluid (CSF) levels of human IL-10, with far less human IL-10 observed in cervical CSF. In keeping with IL-10's known anti-inflammatory actions, AD-h-IL10 lowered CSF levels of IL-1, relative to control AD. These studies support that this gene therapy approach provides an alternative to neuronally focused drug and gene therapies for clinical pain control.