Shelle Malkmus

Studies on the Safety of Chronically Administered Intrathecal Neostigmine Methylsulfate in Rats and Dogs

Background The spinal delivery of the cholinesterase inhibitor neostigmine yields analgesia in rats and augments the analgesic effects of alpha2 agonists in sheep. To assess its activity in humans, preclinical toxicology studies to define its safety were required in two species. Methods Rats with chronic intrathecal catheters received daily injections of saline (vehicle) or 5 micro gram/10 micro liter or 10 micro gram/10 micro liter neostigmine HCl (n = 6/group) for 4 days and were observed for general behavior and nociception (52.5 degrees Celsius hot plate). On day 6, rats were anesthetized and submitted to whole body perfusion/fixation. For dog studies, male beagles were prepared following rigid aseptic precautions with catheters passed from the cisterna magna to the lumbar intrathecal space. Catheters were connected to an external vest‐mounted pump. Based on preliminary studies, ten implanted dogs were randomly assigned to receive infusions of neostigmine for 28 days (4 mg/4 ml/day; n = 6) or saline (4 ml/day; n = 4). At 28 days, dogs were anesthetized, cisternal cerebrospinal fluid was obtained, and dogs were submitted to perfusion‐fixation. Rat and dog spinal cords were embedded, sectioned, stained, and assessed by the pathologist without knowledge of treatment. Results In rats, neostigmine produced a dose‐dependent increase in hot plate latency, and no tolerance was observed. Mild tremor was observed but was not debilitating. Histopathology revealed a mild fibrotic reaction to the catheter with mixed signs of moderate, acute, and chronic inflammation with no differences between saline or drug groups. In dogs, neostigmine had no effect on blood pressure or on the skin twitch response but produced bradycardia and an increase in muscle tone. At sacrifice, cerebrospinal fluid protein, specific gravity, and glucose were elevated in both saline and neostigmine groups. Histopathology displayed a local reaction to the spinal catheter and a mixed acute and chronic inflammatory reaction. No group differences were observed. These results suggest that, at the neostigmine concentration of 1 mg/ml in the rat and dog and in doses up to 4 mg/day in the dog, there is no evidence of spinal tissue toxicity that can be attributed to the drug. This result, observed in two species, suggests that intrathecal neostigmine given in this manner is without distinguishable toxicity in these two models.

Mutant dynein (Loa) triggers proprioceptive axon loss that extends survival only in the SOD1 ALS model with highest motor neuron death

Dominant mutations in cytoplasmic dynein (Loa or Cra) have been reported to provoke selective, age-dependent killing of motor neurons, while paradoxically slowing degeneration and death of motor neurons in one mouse model of an inherited form of ALS. Examination of Loa animals reveals no degeneration of large caliber α-motor neurons beyond an age-dependent loss (initiating only after 18 months) that was comparable in Loa and wild-type littermates. Absence of Loa-mediated α-motor neuron loss contrasted with dramatic, sustained, mutant dynein-mediated postnatal loss of lumbar proprioceptive sensory axons, accompanied by decreased excitatory glutamatergic inputs to motor neurons. In mouse models of inherited ALS caused by mutations in superoxide dismutase (SOD1), mutant dynein modestly prolonged survival in the one mouse model with the most extensive motor neuron loss (SODG93A) while showing marginal (SODG85R) or no (SODG37R) benefit in models with higher numbers of surviving motor neurons at end stage. These findings support a noncell autonomous, excitotoxic contribution from proprioceptive sensory neurons that modestly accelerates disease onset in inherited ALS.

Cancer-related bone pain is attenuated by a systemically available δ-opioid receptor agonist

Abstract Patients with bone cancer report severe pain and receive &mgr;‐opioids. We developed a family of peptidomimetic &dgr;‐agonists, one of which H2N‐Tyr‐dVal‐Gly‐Phe‐Ala‐OH Symbol binds with a 1700× affinity at the &dgr; versus &mgr; receptor. To examine the systemic analgesic efficacy of this &dgr;‐agonist versus morphine in osteosarcoma pain, osteosarcoma cells are injected into one femur of the anesthetized mouse. After 10–18 days, a decalcification of the injected femur occurs along with a pronounced tactile allodynia. IP morphine and Symbol produced a dose‐dependent reversal of allodynia with the respective ED50 values being 5.3 ± 1.9 mg/kg for morphine and 1.3 ± 0.3 mg/kg for [Symbol. Plotting peak effect versus area under the analgesic curve for doses of morphine and [Symbol revealed overlapping curves suggesting that for a given effect, [Symbol produced a similar duration of action as morphine. These effects were reversed by IP naloxone (3 mg/kg). IP naltrindole (1 mg/kg) preferentially reversed [Symbol. The upper dose effects of morphine but not [Symbol were limited by pronounced hyperactivity. No other effects were noted. These results show that IP [Symbol through a &dgr; receptor produces analgesia equal in efficacy to that of morphine but with a 4.5‐fold greater potency. Over the doses examined, morphine actions were side effect limited. The &dgr; side effects were not so limited, suggesting a favorable therapeutic ratio for &dgr;‐agonists in this pain model. These studies suggest that a systemically delivered &dgr;‐opioid agonist has pronounced analgesic properties on a preclinical cancer pain model. Figure. No Caption available. Figure. No Caption available. Figure. No Caption available. Figure. No Caption available. Figure. No Caption available. Figure. No Caption available. Figure. No Caption available. Figure. No Caption available.

Sensory neuronal phenotype in galanin receptor 2 knockout mice: focus on dorsal root ganglion neurone development and pain behaviour.

Galanin is a 29‐amino‐acid peptide expressed in dorsal root ganglion (DRG) neurones and spinal dorsal horn neurones. It affects pain threshold and has developmental and trophic effects. Galanin acts at three G‐protein‐coupled receptors, galanin receptors (GalR1–3), each expressed in the DRGs as suggested by in situ hybridization and/or reverse transcriptase‐polymerase chain reaction. The GalR2 knockout (–/–) mice permit studies on the contributions of this receptor subtype to the role of galanin at the spinal level. At 1 week after sciatic nerve transection (axotomy), there were 16–20% fewer neurones in intact and contralateral DRGs of –/– mice as compared with wild‐type (WT) mice. In addition, a significant neurone loss (26% reduction) was found in the ipsilateral DRGs of WT mice, whereas no further neurone loss was seen in –/– mice. Expression of several peptides has been examined after axotomy, including galanin, neuropeptide Y and two of its receptors as well as substance P, and no significant differences were found between –/– and WT mice in either ipsi‐ or contralateral DRGs, respectively. After thermal injury and spinal nerve ligation, onset and duration of hyperalgesia in the injured paw were similar in GalR2–/– and WT animals. Recovery from spinal nerve ligation‐caused allodynia had the same kinetics in –/– and WT animals. These data are in line with earlier observations from the peripheral and central nervous system, suggesting that galanin actions mediated by GalR2 subtype are of importance in neurodevelopment and neuroprotection.

Spinal Botulinum Neurotoxin B: Effects on Afferent Transmitter Release and Nociceptive Processing

Botulinum neurotoxin B (BoNT-B) mediates proteolytic cleavage of VAMP I/II (synaptobrevins I/II), which prevents vesicle-membrane fusion and blocks neurotransmitter release. In the present study, we investigated the effects of BoNT-B on neurotransmitter release in vivo from spinal primary afferent sensory fibers and the effects of this blockade on nociception. With intrathecally (IT) delivered BoNT-B in C57B/l6 mice, we characterized the effects of such block on the release of substance P (SP) from spinal afferent nociceptors (as measured by neurokinin-1 receptor, NK1-R, internalization), spinal neuronal activation (as indicated by spinal C-Fos expression) and nociceptive behavior after intraplantar (IPLT) formalin. In addition, we investigated the effect of IT BoNT-B on spinal nerve ligation-induced tactile allodynia. A single percutaneous IT injection of BoNT-B 0.5 U at 2 or 5 days prior to IPLT formalin reduced NK1-R internalization and C-Fos expression. These effects correlated with BoNT-B cleavage of VAMPI/II protein in tissue lysate. IT BoNT-B also produced a corresponding reduction in phase 2 of formalin-evoked flinching behavior for over 30 days after IT injection. In mice with spinal nerve ligation (SNL), tactile allodynia was observed, which was attenuated by IT BoNT-B 0.5 U over the next 15 days, as compared to vehicle animals. These effects were observed without effects upon motor function. The specificity of the IT BoNT-B effect is indicated by: i) IT co-injection of BoNT-B and anti-BoNTB antibody prevented effects on SP release, and ii) IT BoNT-B 50 U in the Sprague Dawley rats showed no effect on formalin-evoked flinching or SNL-induced tactile allodynia, which is consistent with rat resistance to BoNT-B. IT BoNT-B blocks transmitter release from spinal primary afferents, and attenuates inflammatory nociceptive response and spinal nerve injury-induced neuropathic pain, in the absence of motor impairment. These observations provide an initial assessment of the ability of IT BoNT-B to regulate spinal nociceptive processing.

Kinetic and Safety Studies on Intrathecally Infused Recombinant-Methionyl Human Brain-Derived Neurotrophic Factor in Dogs☆

To define the kinetics and safety of spinally infused recombinant-methionyl human brain-derived neurotrophic factor (r-metHuBDNF), beagle dogs were prepared with lumbar intrathecal catheters passed through the cisternal membrane to the L1-L4 lumbar level. For kinetic studies, r-metHuBDNF was delivered by bolus or infusion through one catheter and lumbar CSF was sampled periodically through a second. As a lumbar bolus, r-metHuBDNF displayed a biphasic clearance with t(1/2)a = 0.7 hr and t(1/2)b = 7. 9 hr. Lumbar to cisternal concentrations after bolus delivery were approximately 60:1. For safety studies, dogs received continuous intrathecal infusion (2.4 ml/day) for 28 days of saline (n = 6), r-metHuBDNF at 200 (n = 6), 800 (n = 6), or 2000 (n = 7) microg/day. Control dogs showed no changes. Intrathecally infused r-metHuBDNF produced a dose-dependent increase in muscle tone and decreased coordination. Low-dose r-metHuBDNF was associated with moderate increases in muscle tone after 22-28 days of infusion. No clinically important changes were noted in rectal temperature, arterial pressure, respiration and heart rate, body weight, food consumption, stool or urine output, or change in blood chemistries measured throughout the study. Cisternal CSF protein and glucose sampled at 28 days were not different between dose groups and all cultures were negative. Histopathological examination of the spinal cord typically revealed some degree of chronic inflammation around the catheter, including fibrotic adhesions and focal accumulations of lymphoid and plasma cells, but these effects were not dose dependent. In other dogs receiving r-metHuBDNF (2000 or 4000 microg/day), termination of infusion resulted in significant recovery.

Toxicology Profile of N -Methyl-d-aspartate Antagonists Delivered by Intrathecal Infusion in the Canine Model

Background:Intrathecal N-methyl-d-aspartate antagonists have antihyperalgesic efficacy. The authors examined toxicity in a canine model of chronic lumbar intrathecal infusion. Methods:Dogs (10–16 kg) were prepared with lumbar intrathecal catheters connected to vest-mounted pumps (100 &mgr;l/h). In phase 1, stepwise incrementations in infusion concentration were performed at 48- to 72-h intervals to determine an infusion dose with minimal but detectable behavioral effects. In phase 2, the dose/concentration defined in phase 1 was infused for 28 days. Behavioral function during infusion and histopathology at sacrifice was assessed. Drugs examined were 2-amino-5-phosphono-valorate (AP5), MK801, memantine, amitriptyline, S-methadone, and saline. Results:In the phase 1 dose ranging, the minimum effect doses for the several agents were as follows: AP5, 1 mg/day; amitriptyline, 1 mg/day; ketamine, 10 mg/day; MK801, 1 mg/day; and memantine, 4 mg/day. In phase 2, infusion of these doses typically resulted in mild hind limb weakness by 3–5 days after initiation of infusion, which progressed over the 28-day infusion interval. In a limited number of animals, a similar effect was observed with S-methadone. Histopathologically, vehicle-infused animals displayed a minor local catheter reaction. With the drug treatments, a gradient of increasing pathology from cervical to lumbar segments was noted. Pathology ranged from local demyelination to necrotizing lesions of spinal parenchyma near the catheter tip. All drugs given at their respective doses produced pathology scores significantly worse than saline controls. Conclusions:These drugs given for 28 days at acutely tolerable doses lead to spinal pathology. These data suggest a reevaluation of the use of these agents in chronic spinal delivery.

Fate of the predominant phospholipid component of DepoFoamTM drug delivery matrix after intrathecal administration of sustained-release encapsulated cytarabine in rats

The distribution, metabolism, and excretion of dioleoylphosphatidylcholine (DOPC), the predominant phospholipid component of DepoFoam (DF) drug delivery matrix, was determined after lumbar intrathecal injection of double-radiolabeled ((14)CDOPC, (3)H-cytarabine) sustained-release encapsulated cytarabine (DF-cytarabine) in rats prepared with chronic spinal catheters. Radioactivity was quantitated in central nervous system (CNS) and peripheral tissues, cerebrospinal fluid (CSF), blood, urine, and feces at various time points up to 504 h. The distribution of (14)C radiolabel among lipid classes was also determined in selected body fluid samples. Both radiolabels distributed rapidly throughout the neuraxis after injection. Levels of both labels declined in a biphasic manner from CSF and plasma, with an initial rapid decline over the first 96 h, followed by a much slower rate of decline out to 504 h. Greater than 90% of the (3)H (drug) label was estimated to be excreted in urine. In contrast, the data suggest that most of the (14)C (phospholipid) label was expired as (14)CO(2); small percentages of the dose remained incorporated in CNS (7%) and peripheral tissues (8%) or were excreted in urine (6%). Characterization of lipidic (14)C in plasma confirmed metabolism of the parent lipid. The data confirm the sustained-release nature of the DF-cytarabine multivesicular liposomal preparation. Moreover, the results indicate that the DOPC lipid component enters standard catabolic path-ways after breakdown of the DF particles in the intrathecal space. Similar CSF and plasma kinetic profiles of drug and lipid radio-labels support the hypothesis that release of drug is related directly to breakdown of the lipid particles.

Role of p38 mitogen activated protein kinase in a model of osteosarcoma-induced pain.

The focus of this work was to examine the potential role of p38 mitogen activated protein kinase (p38) in a mouse model of bone cancer (osteosarcoma) pain. To generate osteosarcoma and sham animals, osteosarcoma cells or medium were injected into the medullary canal of the femur. Initially, ipsilateral tactile allodynia was observed in both groups, but by 12 days post-surgery, thresholds in the sham group returned towards baseline while hypersensitivity in the osteosarcoma group lasted throughout the study. An increase in phosphorylated p38 was detected by western blotting in dorsal root ganglia (DRG) and spinal cord day 14 after surgery. Immunohistochemistry showed that p38 was phosphorylated in DRG and spinal dorsal horn neurons at this time point. Two doses of a selective p38 inhibitor, SCIO-469, were administered in the chow starting 5 days post-surgery and continued throughout the study. Treatment with SCIO-469 led to a decrease in osteosarcoma-induced clinical score but had no effect on the allodynia. Bone erosion and tumor growth were also examined but no significant reduction of bone erosion or tumor growth was observed in the SCIO-469 treated mice. These data suggest that the p38 signaling pathway does not play a major role in bone cancer-mediated pain.

Increased hyperalgesia after tissue injury and faster recovery of allodynia after nerve injury in the GalR1 knockout mice

Evidence suggests that galanin and its receptors including GalR1 are involved in the modulation of nociception. To understand the contributions of this galanin receptor subtype to the analgesic effect of galanin, we systematically examined the nociception phenotype of the GalR1 knockout (KO) mice. (1) Baseline thresholds: Thermal escape latencies and tactile thresholds of the hind paws were not different between the GalR1 KO and wild type (WT) mice. (2) Thermal injury evoked hyperalgesia: Thermal injury (52 degrees C, 45 s) to one hind paw resulted in a reduction in the thermal escape latency as compared to the uninjured paw. The right/left difference score was significantly greater in the KO (5.9 +/- 0.8 s) than for the WT (2.8 +/- 0.7 s) indicating a greater hyperalgesia. (3) Formalin-induced flinching: Formalin paw injection (2.5%/20 microl) produced a two-phase flinching in both GalR1 KO and WT groups, that was detected by an automated flinching sensor device. Phase II flinching of KO (1510 +/- 90) was slightly greater than that observed for WT (1290 +/- 126), but the difference is not statistically significant. (4) Nerve injury evoked allodynia: Tactile thresholds were assessed prior to and at intervals up to 21 days after left L5 spinal nerve ligation and transection. In both GalR1 KO and WT mice, nerve injury caused thresholds to fall to 0.2-0.3g though 11 days. On days 14-21, GalR1 KO animals showed a significant recovery as compared to WT. In summary, GalR1 KO mice showed no difference from WT with respect to acute nociception, but showed a modest tendency towards increased hyperalgesia after tissue injury and inflammation. These results are consistent with a regulatory effect of galanin at GalR1 receptors on nociceptive processing.