Next-generation XPO1 inhibitor shows improved efficacy and in vivo tolerability in hematological malignancies.

The nuclear export receptor, Exportin 1 (XPO1), mediates transport of growth-regulatory proteins, including tumor suppressors, and is overactive in many cancers, including chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML) and aggressive lymphomas. Oral selective inhibitor of nuclear export (SINE) compounds that block XPO1 function were recently identified and hold promise as a new therapeutic paradigm in many neoplasms. One of these compounds, KPT-330 (selinexor), has made progress in Phase I/II clinical trials, but systemic toxicities limit its administration to twice-per-week and requiring supportive care. We designed a new generation SINE compound, KPT-8602, with a similar mechanism of XPO1 inhibition and potency but considerably improved tolerability. Efficacy of KPT-8602 was evaluated in preclinical animal models of hematological malignancies, including CLL and AML. KPT-8602 shows similar in vitro potency compared with KPT-330 but lower central nervous system penetration, which resulted in enhanced tolerability, even when dosed daily, and improved survival in CLL and AML murine models compared with KPT-330. KPT-8602 is a promising compound for further development in hematological malignancies and other cancers in which upregulation of XPO1 is seen. The wider therapeutic window of KPT-8602 may also allow increased on-target efficacy leading to even more efficacious combinations with other targeted anticancer therapies.

Antioxidants attenuate multiple phases of formalin-induced nociceptive response in mice.

An emerging theme in the study of the pathophysiology of chronic and persistent pain is the role of pro-oxidant substances. Reactive oxygen species (ROS) have been implicated in contributing to and/or maintaining conditions of chronic pain. Recent pre-clinical reports suggest that antioxidants are effective analgesics in neuropathic and inflammatory pain models. The present study extends this work by examining the effect of three antioxidants on tissue injury-induced nociception. C57BL6 mice (20-25 g) were pretreated with either phenyl-N-tert-butylnitrone (PBN; 50 mg/kg, i.p.), 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxy (TEMPOL; 200 or 50 mg/kg, i.p.), N-acetyl-L-cysteine (NAC; 200 or 100mg/kg, i.p.), or vehicle (0.5 ml/100 g), 5 min before intraplantar formalin (10%, 20 microl) injection. Nociceptive responding, indicated by licking or biting the affected hindlimb, was quantified for 30 min after formalin injection. Each drug was effective in attenuating two or more phases (acute, quiescent, and tonic) of the formalin response. To assess putative site of action, intrathecal TEMPOL (380 nmol/5 microl, i.t.) was given 5 min before intraplantar formalin. Intrathecal TEMPOL produced a 83% reduction in nociceptive responding in the tonic phase, but no significant attenuation of the acute phase response. To confirm that the antioxidant property of intrathecal TEMPOL was responsible for its analgesic effect on the formalin-induced pain response, intrathecal TEMPOL was coadministered with the free radical donor tert-butylhydroperoxide (tert-BuOOH). Tert-BuOOH coadminstration reversed the TEMPOL-induced analgesia in the tonic intraplantar formalin response reduction. The data suggest that pro-oxidant species may be important mediators of tissue injury-induced algesia in rodents, and that a spinal site of action is implicated in the tonic response.

GM1 enhances dopaminergic markers in the brain of aged rats.

A number of presynaptic markers are compromised in the dopaminergic neurons of aged Sprague-Dawley rats (22 months old) compared with young rats (3 months old). Indeed, in the striatum of the aged rats there is a diminished capacity to transport dopamine (DA), to bind the dopamine transporter (DAT) marker mazindol, to bind the vesicular monoamine transporter 2 (VMAT2) marker dihydrotetrabenazine, and to release DA under basal conditions or after induction by K(+) or amphetamine. Furthermore, the expression of DAT and VMAT2 mRNA in the midbrain is suppressed. GM1 ganglioside, 30 mg/kg ip daily, administered for 30 days, restores the afore-mentioned markers to values approaching those for young rats. Taken together with our published observations that GM1 partially restores tyrosine hydroxylase activity and DA metabolism in aged nigrostriatal and mesoaccumbal neurons and improves their morphology, our work suggests that GM1 might act as a dopaminergic neurotrophic factor in the aged brain and be a useful adjuvant for treating age-associated dopaminergic deficits.

Motoric behavior in aged rats treated with GM1.

Aging is associated with impaired motor function. Nigrostriatal dopaminergic neurons, in part, regulate motoric behavior, and undergo degenerative changes during aging. GM1 ganglioside partially restores pre-synaptic dopaminergic markers and the number and morphology of dopaminergic neurons in the midbrain and striatum of Sprague--Dawley aged rats. These studies investigated whether GM1 treatment, 30 mg/kg, i.p. daily for 36 days, affects locomotor and stereotypic activity, as well as coordination, balance, and strength in aged rats. Under the treatment conditions used, GM1 did not improve the reduced locomotor and stereotypic behavior of the aged rats. While it partially improved performance on a square bridge test, GM1 had no effect on inclined screen and rod suspension tests. Although GM1 restored the decreased content of dopamine and homovanillic acid in the nigrostriatal neurons of the aged rats, it had no effect on the reduced D1 and D2 dopamine receptor binding and mRNA in the striatum. It appears, that despite the morphological and metabolic restoration of aged nigrostriatal neurons, GM1 has limited ability in improving age-associated motor deficits.

Boron neutron capture therapy of brain tumors: functional and neuropathologic effects of blood-brain barrier disruption and intracarotid injection of sodium borocaptate and boronophenylalanine.

Sodium borocaptate (BSH) and boronophenylalanine (BPA) are two drugs that have been used clinically for boron neutron capture therapy (BNCT) of brain tumors. We previously have reported that hyperosmotic mannitol-induced disruption of the blood-brain barrier (BBB-D), followed by intracarotid (i.c.) administration of BPA or BSH, either individually or in combination, significantly enhanced tumor boron delivery and the efficacy of BNCT in F98 glioma bearing rats. The purpose of the present study was to determine the short-term neuropathologic consequences of this treatment and the long-term effects on motor and cognitive function, as well as the neuropathologic sequelae 1 year following neutron capture irradiation. BBB-D was carried out in non-tumor bearing Fischer rats by infusing a 25% solution of mannitol i.c. followed by i.c. injection of BPA or BSH, either individually or in combination, immediately thereafter. Animals were euthanized 2 days after compound administration, and their brains were processed for neuropathologic examination, which revealed sporadic, mild, focal neuronal degeneration, hemorrhage, and necrosis. To assess the long-term effects of such treatment followed by neutron capture irradiation, non-tumor bearing rats were subjected to BBB-D after which they were injected i.c. with BPA (25 mg B/kg body weight (b.w)) or BSH (30 mg B/kg b.w.) either individually or in combination (BPA 12.5 mg and BSH 14 mg B/kg b.w.). Two and a half hours later they were irradiated at the Medical Research Reactor, Brookhaven National Laboratory, Upton, NY, with the same physical radiation doses (5.79, 8.10 or 10.06 Gy), delivered to the brain, as those that previously had been used for our therapy experiments. The animals tolerated this procedure well, after which they were returned to Columbus, Ohio where their clinical status was monitored weekly. After 1 year, motor function was assessed using a sensitive and reliable locomotor rating scale for open field testing in rats and cognitive function was evaluated by their performance in the Morris water maze, the results of which were similar to those obtained with age matched controls. After functional evaluation, the rats were euthanized, their brains were removed, and then processed for neuropathologic examination. Subtle histopathologic changes were seen in the choroid plexuses of irradiated animals that had received BPA, BSH or saline. Radiation related ocular changes consisting of keratitis, blepharitis, conjunctivitis and cataract formation were seen with similar frequency in most rats in each treatment group. Based on these observations, and the previously reported significant therapeutic gain associated with BBB-D and i.c. injection of BSH and BPA, the present observations establish its safety in rats and suggest that further studies in large animals and humans are warranted.

Retinal cholinergic and dopaminergic deficits of aged rats are improved following treatment with GM1 ganglioside.

Selected cholinergic and dopaminergic markers were compared in the retina of aged (20-22-months-old) and young (3-months-old) rats before and after treatment with GM1 ganglioside. The dopaminergic markers, tyrosine hydroxylase, aromatic L-amino acid decarboxylase, dopamine, 3,4-dihydroxyphenylacetic acid and homovanillic acid were comparable in the young and aged animals and GM1 treatment did not alter them. In contrast, mazindol binding, a marker for the dopamine transporter, was diminished in the aged retina and treatment with GM1 restored binding to values found in the young animals. The cholinergic markers choline acetyltransferase and hemicholinium-3 binding, a marker for the high-affinity choline transport, were depressed in aged rats and GM1 corrected the deficits.

GM1 ganglioside restores abnormal responses to acute thermal and mechanical stimuli in aged rats.

We investigated the effect of aging on the responses to thermal and mechanical stimuli in rats. Young (3-5 months old) and aged (22-24 months old) male Sprague-Dawley rats were tested in the hot plate, high- and low-intensity radiant heat tail flick, and von Frey hair assays. Compared to young rats, aged rats displayed longer latencies in the hot plate and the high-intensity tail flick assays (hypoalgesia), but there was no difference in the low-intensity tail flick assay. In addition, aged rats had decreased thresholds to mechanical stimuli produced by von Frey hairs compared with young rats (mechanical allodynia). Administration of GM1 ganglioside, 30 mg/kg, i.p., once daily for 30 days, to aged rats partially restored the responses in the hot plate and von Frey hair assays. GM1 had no effect on the altered responses in the tail flick test in aged rats, and in general, had no effect on any sensory modality tested in young rats.

GM1 ganglioside restores dopaminergic neurochemical and morphological markers in aged rats.

The monosialoganglioside GM1 exerts neurotrophic-like activity in vitro and in vivo. In particular, it improves cholinergic neuron morphology and chemistry and learning abilities of cognitively impaired aged rats and young animals with cholinergic lesions, and restores neurochemical, pharmacological, morphological and behavioral parameters in animal models of Parkinson's disease. Our studies present evidence that GM1 reverses dopaminergic deficits in the nigrostriatal neurons of aged rats. GM1 administered to aged Sprague-Dawley rats for 30 days reversed the decreased activity of tyrosine hydroxylase in the midbrain and striatum, elevated the reduced protein content and mRNA levels of the enzyme in the midbrain, and reversed the decrements of dopamine and 3,4-dihydroxyphenylacetic acid content in both the midbrain and striatum. Tyrosine hydroxylase activity of the midbrain, but not of the striatum, remained elevated for 15 days after discontinuing GM1. The count profiles of tyrosine hydroxylase-immunopositive neurons, the size of tyrosine hydroxylase-immunopositive neurons and the number of tyrosine hydroxylase-immunopositive fibers were decreased in the substantia nigra pars compacta and the ventral tegmental area of aged rats. GM1 corrected the morphology of dopaminergic neurons in the substantia nigra pars compacta and partially improved it in the ventral tegmental area. These findings support the notion that the aged striatal dopaminergic neurons respond to GM1, and strengthen the utility of using this compound for combating age-associated neuronal deficits.

Decreased neuropeptide content in the spinal cord of aged rats: the effect of GM1 ganglioside.

This study investigated the status of substance P (SP), methionine-enkephalin (Met-Enk) and dynorphin A(1-13) (Dyn A) in the spinal cord of aged Sprague-Dawley rats and the effect of GM1 ganglioside on these neuropeptides. SP and Met-Enk, but not Dyn A, were decreased in both dorsal and ventral horns of the aged spinal cord. Treatment with GM1 ganglioside (30 mg/kg i.p., daily for 30 days) restored, in part, the neuropeptide deficits in the ventral horns, but not in the dorsal horns. This information might be important for understanding the sensory and motor deficits associated with ageing, and how the spinal cord neuropeptides might be amplified in the aged spinal cord.

An antagonist of substance P N-terminal fragments, D-substance P(1-7), reveals that both nociceptive and antinociceptive effects are induced by substance P N-terminal activity during noxious chemical stimulation.

Substance P (SP) N-terminal fragments are known to alter nociception when injected intrathecally or when released in response to capsaicin. However, it is not known whether a sufficient concentration of SP N-terminal metabolites accumulate during noxious stimulation to modulate nociception. To test this, we examined the effect of the SP(1-7) antagonist, D-SP(1-7), injected intrathecally in mice, on two nociceptive assays that are differentially affected by exogenous SP(1-7): acetic acid-induced writhing that is inhibited and formalin-induced behaviors that are enhanced by SP(1-7). One nmol of D-SP(1-7) is sufficient to block the acute (30 min) antinociceptive effects of SP(1-7) on writhing. When injected alone at much higher doses (10-100 nmol), D-SP(1-7) inhibited writhing. In the formalin assay, SP(1-7) had no acute effect (30 min) on responses during Phase 1 at any dose tested, but D-SP(1-7) increased responses 5 min after injection of low (2-1000 pmol), but not high doses (10 and 100 nmol). Twenty-four hours after injection of SP(1-7), writhing was inhibited and formalin responses were increased. D-SP(1-7) prevented these effects of SP(1-7) but had no effect when injected alone, indicating that there is no tonic SP N-terminal activity in mice not exposed to noxious stimuli. Thus, acetic acid and formalin each induce endogenous SP N-terminal activity, respectively, producing a pro-nociceptive effect that is relatively insensitive to D-SP(1-7) and antinociception that is very sensitive to inhibition by D-SP(1-7).

Inhibition of substance P release from spinal cord tissue after pretreatment with capsaicin does not mediate the antinociceptive effect of capsaicin in adult mice.

Substance P (SP) is released from primary afferent fibers in response to nociceptive stimuli. Capsaicin, which produces an initial hyperalgesic response followed by persistent antinociception, also elicits release of SP from primary afferent fibers. Capsaicin pretreatment decreases the content and release of SP from primary afferent fibers. This effect on SP has been hypothesized to mediate the antinociceptive effect of capsaicin. To test this hypothesis, mice were injected intrathecally (i.t.) with antinociceptive doses of capsaicin or SP(1-7) before superfusion of spinal cord tissue with 3 microM capsaicin 24, 48, 96 or 168 h later. N-terminal metabolic fragments of SP that accumulate after capsaicin-induced SP release and are involved in the antinociceptive effect of capsaicin, were also tested. Like capsaicin SP(1-3), SP(1-4) and SP(1-7) were each antinociceptive when injected 24 h before nociceptive testing. However, at this time there was no decrease in capsaicin-evoked release of SP in tissue from capsaicin- and SP(1-7)-pretreated animals compared to those injected with vehicle. In contrast, capsaicin-evoked SP release decreased significantly in tissue from mice pretreated with capsaicin or SP(1-7) 48 h prior to testing. D-Substance P(1-7), which prevents antinociception, blocked capsaicin- and SP(1-7)-induced decreases in SP release, indicating that these effects are mediated by SP N-terminal activity. Total spinal cord content of SP did not differ amongst treatment groups. These data indicate that antinociception does not appear to depend on decreases in SP release or content as antinociception precedes decreases in SP release.

Nitric oxide mediates long-term hyperalgesic and antinociceptive effects of the N-terminus of substance P in the formalin assay in mice.

Conditions such as hyperalgesia can occur days or months after the noxious insult. Substance P (SP) is released in response to noxious stimuli. Given the long-term effects of the N-terminus of SP on putative nociceptive transmitters, we investigated changes in formalin-induced nociception following an accumulation of SP N-terminal metabolites in mice. Pre-treatment with the N-terminal metabolite of SP, SP(1-7), was without effect when injected intrathecally (i.t.) 5 or 30 min before formalin. However, at 24 h, SP(1-7) increased behaviors during Phase 1, indicating hyperalgesia, and attenuated Phase 2 responses, consistent with antinociception. The nitric oxide (NO) synthase inhibitor, N omega-nitro-L-arginine methyl ester HCl (L-NAME), blocked both hyperalgesic and antinociceptive effects when co-injected with SP(1-7). Consistent with a NO-mediated pathway, L-arginine (L-arg), the N-terminal amino acid of SP and precursor to NO, mimicked the antinociceptive effect of SP(1-7) on Phase 2. The hyperalgesic effect of SP(1-7) in Phase 1, which was not mimicked by L-arg, was prevented by D-SP(1-7), a SP(1-7) antagonist. Thus, SP(1-7) modulates nociception via two distinct NO-mediated pathways. When injected for 7 days, tolerance developed to the antinociceptive effect of SP(1-7) on Phase 2, but not to the hyperalgesic effect on Phase 1. Intraperitoneally injected SP(1-7) also produced hyperalgesia during Phase 1, to which tolerance developed following seven daily injections. Together, these data support the hypothesis that an accumulation of SP N-terminal metabolites, either peripherally or within the spinal cord area, is sufficient for long-term modulation of multiple types of nociception with hyperalgesic responses being most persistent.

Substance P N-terminus inhibits C-fiber-dependent facilitation of the rat flexor reflex.

Substance P injected intrathecally or a conditioning stimulus (1 Hz, 20 s) at C-fiber strength, which releases substance P from intraspinal primary afferent terminals, each enhance the hamstring muscle flexor reflex elicited by electrical stimulation of the sural nerve in decerebrate, spinalized rats. This suggests a role for substance P in pain transmission. Since substance P N-terminal metabolites are biologically active, we examined the effect of the metabolite substance P-(1-7) on the flexor reflex and the enhancement of the flexor reflex following a conditioning stimulus of the sural nerve. In contrast to the excitatory effect of substance P, intrathecal injection of substance P-(1-7) had no effect on the flexor reflex. However, 10 and 30 min after injection, 0.6 or 6 micrograms of substance P-(1-7) attenuated the facilitation of the flexor reflex by the conditioning stimulus. These data indicate that substance P-(1-7) may modulate the expression of nociception involving repetitive firing of C-fibers while having no significant effect on acute or phasic responses.

Activity at phencyclidine and mu opioid sites mediates the hyperalgesic and antinociceptive properties of the N-terminus of substance P in a model of visceral pain.

Substance P, a putative neurotransmitter or neuromodulator of nociception or pain in the spinal cord, exhibits both antinociceptive and hyperalgesic properties. Investigators have shown that the N-terminal metabolite of substance P, substance P(1-7), produces naloxone-reversible antinociception when given supraspinally and systemically in mice and hyperalgesia when injected intrathecally in rats. The goal of our investigation was to identify the receptors mediating these actions of substance P(1-7) at the initial site of release of substance P, i.e. in the spinal cord. Thirty minutes after intrathecal injection, substance P(1-7) produced naloxone-reversible antinociception in a dose-dependent manner in the abdominal stretch assay. When administered with naloxone, substance P(1-7) produced hyperalgesia 5 and 10 min after injection, which was inhibited by dizocilpine (MK-801), a phencyclidine ligand and non-competitive antagonist of N-methyl-D-aspartate. Antinociception was inhibited by the mu-selective opioid antagonist beta-funaltrexamine, but not by the mu 1-selective opioid antagonist naloxonazine or the delta-selective antagonist naltrindole, indicating a mu 2-opioid receptor-mediated effect. These findings suggest that the N-terminal portion of substance P may modulate nociception or pain, as demonstrated in the acetic acid abdominal stretch (writhing) assay, via activation of two different receptor systems. Substance P(1-7)-induced hyperalgesia is mediated by a phencyclidine-sensitive mechanism and antinociception involves activity at mu-opioid, most likely mu 2, receptors.

Antinociception induced by 3-((+-)-2-carboxypiperazin-4-yl)-propyl-1- phosphonic acid (CPP), an N-methyl-D-aspartate (NMDA) competitive antagonist, plus 6,7-dinitroquinoxaline-2,3-dione (DNQX), a non-NMDA antagonist, differs from that induced by MK-801 plus DNQX.

Excitatory amino acid receptors have been implicated in mediating pain. 3-((+-)-2-Carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP), a competitive N-methyl-D-aspartate (NMDA) antagonist and MK-801, a phencyclidine (PCP) ligand and non-competitive NMDA antagonist, were injected intrathecally in mice alone or in combination with 6,7-dinitroquinoxaline-2,3-dione (DNQX), a non-NMDA antagonist. When tested in the formalin model of pain, antinociception following CPP plus DNQX was greater than that after MK-801 plus DNQX in both the acute and tonic phases. These dissimilarities are not consistent with activity of CPP and MK-801 at the same sites in the spinal cord.

Porcine malignant hyperthermia: halothane effects on force generation in skeletal muscles.

Halothane-induced malignant hyperthermia (MH) is thought to result from a defect in the regulation of cytosolic calcium concentration in MH-susceptible (MHS) skeletal muscle. Such a defect might be expected to alter the time course of contractile responses. To test this hypothesis, isolated intact cell bundles from external intercostal and common digital extensor muscles of normal and MHS pigs were stimulated electrically to elicit twitch and tetanic tension in the presence and absence of halothane (2.5%). Time intervals measured for both twitches and tetani were (1) the latent period between the stimulus and tension increase, (2) the time to peak tension, and (3) the half-relaxation time. In contrast to previous reports, halothane had no effect on any measured time course parameter of twitches of either type of normal or MHS muscle, nor did the twitches of MHS and normal muscles differ in any parameter in the absence of halothane. However, the tetanic tension relaxation in both types of MHS muscle was markedly slowed by halothane, whereas in normal muscles there was little change. The slower rate of relaxation induced by halothane in MHS muscles suggests that halothane, either directly or indirectly, enhances the release or slows the removal of calcium in intact MHS muscles following maximal activation. This slowed tetanus relaxation could be of use in identification of MHS individuals.

Porcine malignant hyperthermia: cell injury enhances halothane sensitivity of biopsies.

A possible relationship between muscle cell injury or deterioration and enhanced halothane sensitivity was studied by monitoring mechanical responses of skeletal muscles from normal pigs and pigs susceptible to malignant hyperthermia (MH). Increased time postbiopsy and decreased maximum control tetanic tension both correlated significantly with enhanced sensitivity to halothane. In both normal and MH-susceptible (MHS) muscles, greater halothane sensitivity was observed in cut cell than in intact cell bundles and in low tetanic tension as compared to high tension preparations. Subsequent to halothane exposure, twitches of high tension (greater than or equal 1.75 kg . cm-2) intact bundles of both normal and MHS muscles were potentiated. Tetani of normal intact bundles were not altered, whereas those of MHS bundles were depressed after halothane exposure. Control twitch-to-tetanus ratios (twitch ratios) were higher in MHS (0.23) than in normal (0.12) intact bundles. According to discriminant analysis, the best distinction between normal and MHS muscles, either cut or intact, was obtained by comparing halothane-induced changes in tetanic tension and control twitch ratios.

Effects of calcium antagonists on mechanical responses of mammalian skeletal muscles.

Rodent muscles were exposed to several organic calcium antagonists, and mechanical responses to direct electrical stimulation were recorded. Verapamil and D600, at 25 microM, depressed twitch and tetanus tension and caused fading of the tetanus plateau. These effects increased with frequency of stimulation, and were not reversed by doubled extracellular calcium. Depression of tension progressed to complete paralysis after 60-90 min exposure to verapamil. Bepridil and diltiazem both caused depression of tension and tetanus fade. Nifedipine caused marked, and nitrendipine caused slight, potentiation of twitch tension but did not alter tetanic tension. The magnitude of the observed effects on tension (either depression or potentiation) correlated with neither the relative calcium antagonist potencies of the drugs in other tissues nor with the ability of the drugs to cross the cell membrane. The continued decline in tension observed on prolonged exposure indicates that chronic exposure to low levels in vivo might lead to significant muscle weakness.