MNK INHIBITORS AND EIF4E PHOSPHORYLATION MODULATION TO TREAT INFLAMMATORY PAIN IN THE AGED

Abstract

Disclosed is an important therapeutic strategy of targeting MNK to block eIF4E phosphorylation for the treatment of persistent inflammatory pain in later life. In one embodiment, a therapeutic drug that targets MNK and inhibits MNK is selected in order to block eIF4E phosphorylation and the therapeutic drug is administered to the subject with sufficient dosage to block eIF4E phosphorylation. The treatment is suitable for aged subjects experiencing inflammatory pain such as age-associated inflammation, age-associated low-grade inflammation, CFA-induced acute inflammation, spontaneous pain, mechanical hypersensitivity or thermal hypersensitivity.

Claims

1. A method of reducing inflammatory pain in a subject having age-associated inflammation, comprising administering to said subject an amount of a therapeutic sufficient to reduce said inflammatory pain, wherein the therapeutic effectively blocks eIF4E phosphorylation.

2. The method of claim 1, wherein the therapeutic targets MNK to block eIF4E phosphorylation.

3. The method of claim 1, wherein the inflammatory pain includes low-grade inflammation.

4. The method of claim 1, wherein the inflammatory pain includes CFA-induced acute inflammation, spontaneous pain, mechanical hypersensitivity, thermal hypersensitivity, or a combination thereof.

Description

DETAILED DESCRIPTION

[0021] The subject matter, which is regarded as the invention, is particularly pointed out and distinctly claimed at the conclusion of the specification. The foregoing and other objects, features, and advantages of the invention are apparent from the following detailed description.

[0022] To date, no one has taken advantage of the connection between eIF4E phosphorylation, aging, pain, and age-associated inflammation. Presented herein is an important therapeutic strategy of targeting MNK to block eIF4E phosphorylation for the treatment of persistent inflammatory pain in later life.

[0023] According to FIG. 1, a method of treatment for inflammatory pain in aged subjects. In step 101, eIF4E phosphorylation is blocked. In one embodiment, a therapeutic drug that targets MNK and inhibits MNK is selected in order to block eIF4E phosphorylation. Step 101 is not limited to selecting drugs that inhibit MNK, but allows for the use of other drugs that are known to block eIF4E phosphorylation. An example of a drug that inhibits MNK, suitable for the present invention, is eFT508 (Tomivosertib). In step 102, the treatment is targeted to a selected aged subject who is experiencing inflammatory pain. As used herein, “aged” means about 60% of average lifespan expectancy for the subject Inflammatory pain includes age-associated inflammation, age-associated low-grade inflammation, CFA-induced acute inflammation, spontaneous pain, mechanical hypersensitivity, thermal hypersensitivity, or a combination thereof. In step 103, the therapeutic drug is administered to the subject with dosage sufficient to block eIF4E phosphorylation and relieve inflammatory pain. In some embodiments, it is sufficient that the dosage be provided that inhibits eIF4E phosphorylation by at least 50% percentage over normal eIF4E phosphorylation in the subject, such as at least 60%, at least 70%, at least 80%, or at least 90%.

Materials and Methods

[0024] The experimental studies in support of the present disclosure used young and aged wild-type and eIF4E.sup.S209A mice, to examine eIF4E phosphorylation affected CFA-induced pain behaviors and levels of pro-inflammatory cytokines in the dorsal root ganglia (DRG) and spinal cord.

[0025] Animals. Young (6-9 months old) and aged (>22 months old) female and male eIF4E.sup.S209A phospho-mutants on a C57BL/6 background were generated in the Sonenberg laboratory as previously described (Furic et al., 2010) and gifted to us. These were further bred at the University of Texas at Dallas to generate experimental animals Wild-type (WT) C57BL/6 mice were used as controls; breeders were purchased from Jackson Laboratory and bred in-house as WT counterparts for all experiments. In-house bred eIF4E.sup.S209A mutant and WT mice were weaned between 3 and 4 weeks of age and tail-clipped to verify genotypes. All young mice weighed between 25 and 30 g at the time of experimental use, whereas all aged mice weighed between 30 and 40 g. Animals were group-housed in polypropylene cages maintained at 21° C. under a 12-h light/dark cycle with ad libitum access to water and rodent chow. The animal housing room lights were turned on at 6 AM and off at 6 PM daily. At the end of the study, mice were examined post mortem for gross signs of disease and any mouse found unhealthy (e.g., tumors or splenomegaly) was excluded from the dataset. Ethics approval: All procedures were in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals and were approved by the University of Texas at Dallas Institutional Animal Care and Use Committee protocols 15-15 and 16-07.

[0026] Inflammatory Insult. Complete Freund's adjuvant (CFA) was purchased from Sigma Aldrich (St. Louis, Mo.) at a 1 mg/ml concentration. Equal parts of sterile phosphate-buffered saline (PBS) and CFA were thoroughly mixed via vortexing to make an emulsion. The emulsion was also actively vortexed prior to each intraplantar injection (20 μl) to ensure equal volume injections for all animals. The working solution administered contained 10 μg of CFA. The injection was done on the left hind paw, which was designated as ipsilateral and the right hind paw was considered as a control and designated as contralateral. After injection, the following time points were measured for each animal: 4 h and days 1, 2, 3, 5, and 7.

[0027] Behavioral tests. All behavioral tasks were done between 10 AM and 2 PM. Mice were acclimated to the experimental room and acrylic behavior racks for 4 h for a minimum of 2 days before any data acquisition. Spontaneous pain (Grimace Scale), paw temperature (FLIR), and mechanical allodynia (Von Frey) assessments were all conducted in the behavior racks that were 11 cm long by 10 cm wide and 4.5 cm in height. A total of 16 units were available at any given time for 16 mice to be housed individually and tested. The racks were cleaned with 70% ethanol to eliminate odor cues between each reading, baseline or experimental. Two consecutive baseline measures a day apart were taken in the behavior racks. One baseline day was taken for thermal hypersensitivity (Hargreaves) and paw edema (Plethysmometer) on the corresponding apparatus after animals baselined on the other three. Following completed baseline measures for all tests, animals were injected with emulsified complete Freund's adjuvant (CFA) at a dose of 10 μg into the left hind paw. After injection, the animals were tested at 4 h, and on days 1, 2, 3, 5, and 7 for the pain and inflammation measures listed below (FIGS. 2a-d). In order to diminish behavioral interference, tasks were done in the following order for the entirety of the experiment: spontaneous pain (Grimace), paw temperature (FLIR), mechanical allodynia (Von Frey), thermal hyperalgesia (Har-greaves), and paw edema (Plethysmometer). For mechanical allodynia, thermal hypersensitivity, paw temperature, and paw edema, readings were noted for stimuli applied to the contralateral paw and were averaged across all animals within a group. The mean value was used to provide a basis of comparison, indicated by a dashed line on the graphs and respective values. Experimenters were blind to the genotype of the animals during testing; individuals blind to the treatment and genotype analyzed data.

[0028] Assessment of spontaneous pain by grimace scale. Grimacing was assessed while the animals were in the acrylic racks before Von Frey testing. Assessment began approximately 1 h after being in the rack to ensure that the mice were calm, but not sleeping. Each mouse grimace score was measured as an average of the five components of rodent grimacing, i.e., orbital tightening or closing, ear position, whisker position, nose bulging, and cheek bulging. All five parameters measure the severity of perceived spontaneous pain. Each parameter was scored on a scale from 0 to 2, with 0 representing a normal non-painful state, 1 indicating moderate pain perceived, and 2 being representative of severe pain perceived (Langford et al., 2010).

[0029] Mechanical allodynia by Von Frey filament test. Mice were habituated to acrylic racks for approximately 1 h before testing at each experimental timepoint. Hind paw mechanical allodynia was measured by the up-down method using calibrated Von Frey filaments (Stoelting Co., Wood Dale, Ill.) as described by (Chaplan et al., 1994). Withdrawals in response to filaments up to 0.8 g were considered baseline.

[0030] Thermal hyperalgesia by Hargreaves test. Thermal hypersensitivity was measured using the Har-greaves heat plantar device (IITC Life Science Inc., Calif.) (Hargreaves et al., 1988). Mice were placed inside an acrylic enclosure on a glass surface heated evenly to 29° C., immediately after von Frey. Withdrawal latencies were measured after animals received a laser beam directed at the ipsilateral and contralateral hind paw. In order to obtain baseline withdrawal measures around 10-12 s, the inventor used the laser at an active intensity of 30%. The laser was preset to cutoff automatically at 25 s to avoid possible injury to the hind paw. A minimum of three trials of withdrawal latencies were recorded for each time point. The readings for the contralateral hind paws were averaged across all groups.

[0031] Inflammatory response tests. Paw edema measured by a plethysmometer: Paw edema was measured using the Plethysmometer apparatus (IITC Life Science Inc., Woodland Hills, Calif.). Both ipsilateral and contralateral hind paws were submerged in the water contained in the cell consistently, up to the heel of the animal throughout the experiment. The change in volume of the water level was calibrated to output a reading in milliliters (ml) shown on the electronic monitor and volume displacement was recorded.

[0032] Paw temperature recorded by forward-looking infrared camera: A forward-looking infrared (FLIR) camera model T650SC (Wilsonville, Oreg.) was used to measure the temperature of both ipsilateral and contralateral paws throughout the duration of the experiment immediately after grimace and before von Frey measurements. This was to ensure filament or laser application did not have a direct effect on paw temperature. The temperature across the inflamed plantar paw region was recorded at each time point and analyzed by a blind observer. (Moy et al., 2018a).

[0033] Western blots. A month after the inflammation and pain parameters were assessed, animals were deeply anesthetized with isoflurane and quickly decapitated. Lumbar spinal cord and dorsal root ganglia (L3-L5) ipsilateral to the site of injection were extracted from the animals and snap-frozen in liquid nitrogen before being stored at −80° C. These tissues were homogenized in protein extraction buffer (50 mM Tris, 150 mM NaCl, 1 mM EDTA, 0.5% Triton X-100, and 1% SDS). Protease inhibitor cocktail (Sigma P8340), phosphatase inhibitor cocktail 1 (Sigma P2850), and phosphatase inhibitor cocktail 2 (Sigma P5726) were added at 1% final concentration to the protein extraction buffer immediately before addition of buffer to tissue samples. Tissue was homogenized by sonication at 30 Hz for 20-40 s until no clumps remained followed by centrifugation at 13000×g for 15 min at 4° C. The clarified supernatant was used for western blotting. Total protein was estimated using the BCA protein Assay Reagent Kit (Pierce 23,223). Fifteen micrograms of protein was loaded per lane on 12% resolving, 4% stacking polyacrylamide gels. Proteins were transferred onto 0.22 μM nitrocellulose membranes and blocked with 5% nonfat dry milk. Membranes were probed with primary antibodies to TNFα (1:500, Abcam ab1793), IL-1β (1:1000, Abcam ab9722), phosphorylated eIF4E (1:1000, Abcam ab76256), total eIF4E (1:1000, Cell Signaling Technologies 9742), and β-actin (Cell Signaling Technologies 8H10D10, 1:3000) at 4° C. overnight followed by 3 washes with tris-buffered saline (50 mM Tris, 150 mM NaCl, 0.05% Tween-20—TBST), and 1 h incubation with horse radish peroxidase-conjugated secondary antibodies. Membranes were washed three more times using TBST, and target proteins detected via chemiluminescence. Levels were normalized according to β-actin signal.

[0034] Statistical analyses. GraphPad Prism software version 8.4 was used for all statistical analysis. For behavior tests, to determine differences between genotypes (WT and eIF4E.sup.S209A), two-way repeated measures ANOVA was used for young and aged cohorts, followed by Sidak's post hoc analysis.

[0035] For comparing all four cohorts, effect size was calculated by taking the difference of measures after CFA injection at every time point and baseline. Two-way ANOVA was used to analyze the generated values, followed by Tukey's post hoc test for multiple comparisons. Western blot analyses were performed in Image Lab software from BioRad. Target band intensities were normalized according to intensity value changes for β-actin. Two-way ANOVA was used for comparing groups based on age and genotypes followed by Tukey's post hoc. All data are graphically represented by the mean and standard error of the mean. A p value <0.05 was considered significant.

Results

[0036] Validation of eIF4E.sup.S209A genotypes and eIF4E protein in all groups. After breeding animals in-house, the inventor checked the genotypes for successful mutants using clipped tails. A representative genotyping gel for WT, eIF4E.sup.S209A+/− or heterozygous, and eIF4E.sup.S209A−/− or homozygous mutant genotypes is shown in FIG. 2b. The inventor only used homozygous mutants compared with WT animals for all of the experiments. The inventor checked phosphorylated eIF4E in ipsilateral DRGs and spinal cord for all four groups (FIG. 2c representative image). The young and aged eIF4E.sup.S209A animals showed no presence of phosphorylated eIF4E, which was clearly seen in both young and aged WT groups. The inventor also confirmed that the expression of total eIF4E protein in the spinal cord and ipsilateral DRGs did not change with age or genotype (FIG. 2d).

[0037] Aged eIF4E.sup.S209A mice show faster resolution of inflammation at the injection site. The inventor assessed inflammation at the injection site over 7 days after CFA injection by recording paw temperatures with an infrared camera and measuring paw edema by a plethysmometer. Young mice, both WT and eIF4E.sup.S209A, showed consistently elevated paw temperatures and edema across 7 days, indicating ongoing inflammation in their ipsilateral paws (FIGS. 3a and 3b). Aged mice showed a decrease in ipsilateral paw temperature and edema, comparable with contralateral paw readings on day 7 after CFA injection; however, both were still higher than baseline (FIGS. 3a and 3b). Conversely, the aged eIF4E.sup.S209A mice had decreased ipsilateral paw temperature and edema starting day 2 post-injection, indicating that inflammation had reduced (FIGS. 3a and 3b). When the magnitudes of differences were compared for all four groups, the aged eIF4E.sup.S209A mice had significantly lower temperatures and reduced edema compared with young eIF4E.sup.S209A mice and aged WT (FIGS. 3a and 3b) and significantly less edema than aged WT mice. These results demonstrate that eIF4E phosphorylation differentially contributes to inflammation resolution in young versus old mice.

[0038] Aged eIF4E.sup.S209A mice have a higher threshold for spontaneous pain. The inventor then checked for spontaneous pain over 7 days post CFA injection in the four groups using the grimace scale to score grimacing behavior. The young WT and eIF4E.sup.S209A groups did not differ significantly from one another in that they both showed grimacing (FIG. 4a). The aged WT mice showed less grimacing on days 5 and 7, indicating reduced spontaneous pain (FIG. 4b). The aged eIF4E.sup.S209A group showed a trending decrease in levels of grimacing across the experiment (FIG. 4b). When the magnitudes of differences for all four groups were compared, an age-dependent effect of eIF4E phosphorylation was seen (Fig. 4c). From this data, the inventor suggests that eIF4E phosphorylation may contribute to spontaneous pain more strongly in aged mice.

[0039] Aged eIF4E.sup.S209A mice show reduced mechanical allodynia and less thermal hyperalgesia in response to CFA. Next, the inventor examined the response to evoked pain in all four groups using von Frey filaments for mechanical allodynia and the Hargreaves device for thermal hyperalgesia. Both young WT and eIF4E.sup.S209A mutant mice showed mechanical allodynia and thermal hyperalgesia in response to CFA injection (FIGS. 5a and 5d). The aged WT mice showed the same pattern as their young counterparts (FIG. 5 a-e); however, the aged eIF4E.sup.S209A mice showed reduced mechanical allodynia and thermal hyperalgesia compared with WT animals (FIGS. 5b and 5e). Interestingly, when the magnitudes of differences for all four groups were compared, the aged eIF4E.sup.S209A group was significantly different from the young mutant group and the aged WT group for mechanical allodynia and thermal hyperalgesia (FIGS. 5c and 5f). The inventor therefore concludes that, as shown for spontaneous pain, eIF4E phosphorylation contribution to evoked pain behavior is stronger in aged mice.

[0040] Levels of inflammatory mediators vary based on age and genotype. The inventor examined the levels of pro-inflammatory cytokines IL-1β (FIGS. 6a-b) and TNFα (FIGS. 7a-b) in the ipsilateral DRGs and the spinal cord, 30 days post CFA injection. The inventor found that the aged mice (both WT and eIF4E.sup.S209A) had elevated IL-1β (FIG. 6a) and TNFα (FIG. 7a) in their ipsilateral DRGs and spinal cord, respectively. The inventor found no difference between cytokine levels in aged WT mice and those lacking eIF4E phosphorylation. Overall, the aged groups had higher levels of IL-1β and TNFα than their young counterparts which is consistent with an age-associated pro-inflammatory phenotype.

[0041] Levels of DRG ion channel proteins change with age. Given the dissociation of higher levels of pro-inflammatory mediators in aging and pain behaviors, the inventor examined the effects of eIF4E phosphorylation on nociceptor plasticity in aging. The inventor probed ipsilateral DRG samples from all four groups for transient receptor potential ankyrin 1 (TRPA1) and small conductance calcium-activated potassium channel 2 (SK2) two cap-dependent ion channels. While the inventor did not find a genotype effect for either protein (FIGS. 8a and 8b), the inventor found that levels of TRPA1 were decreased in samples taken from aged mice (FIG. 8a), indicating that aging may affect ion channel expression for certain TRP channels in the DRG.

Discussion

[0042] The inventor found that in the absence of phosphorylated eIF4E, CFA-induced acute inflammation, spontaneous pain, and mechanical and thermal hypersensitivity are reduced in aged eIF4E mutant mice. However, the levels of pro-inflammatory cytokines IL-1β and TNFα in the ipsilateral DRGs and spinal cord were comparable with aged WT animals. Similar to previous studies, the young mutant mice did not differ significantly in assessed pain behaviors, but the inventor reports inflammatory protein levels in specific peripheral nervous system tissue and observed a genotype effect in the levels of IL-1β in their ipsilateral DRGs compared with young WT mice. However, the aged mutant mice showed a clear increase of IL-1β in the spinal cord and TNFα in ipsilateral DRGs. This data suggests that loss of eIF4E phosphorylation modulates the levels of different pro-inflammatory cytokines, which is dependent upon tissue type. Interestingly, how these inflammatory molecules engender changes in pain behavior in the aged may be less relevant as nociceptor plasticity takes over.

[0043] The aged mutants demonstrated faster recovery from CFA-induced inflammatory insult across 7 days when compared with their WT counterparts and young mice of either genotype (FIGS. 3a-f). It has been documented that young adult mice lacking eIF4E phosphorylation show decreased pain responses in hyperalgesic priming, a model of the transition to chronic pain (Moy et al., 2017; Moy et al., 2018b). These mice also show blunted spontaneous pain immediately after inflammatory injury, as measured by paw guarding, but no change in acute peak inflammatory responses (Moy et al., 2018a), consistent with these results. The inventor's data adds novel perspectives to the eIF4E phosphorylation literature revealing that inflammatory responses are attenuated and resolve faster along with spontaneous and evoked pain responses in the aged. To reveal inflammatory mechanisms, the inventor assayed relevant tissue-specific levels of inflammatory mediators and showed they are increased (FIGS. 6a, 6b, 7a and 7b). This suggests that longer-lasting pro-inflammatory cytokine-mediated activation of cells in the peripheral as well as the central nervous system may not be as important for the behavioral effects seen in aging. This leads us to ascertain that eIF4E phosphorylation-dependent translation may play a crucial role in nociceptor plasticity during aging and that this nociceptor plasticity supersedes inflammatory mediators in terms of pain outcomes. To delve into this possibility, the inventor probed DRG tissue for TRPA1 and SK2, ion channels implicated in neuron plasticity, and aging. Interestingly, there is an age-dependent decrease in TRPA1 levels that could explain some of the inventor's observations, but further investigation is needed (FIGS. 8a-b).

[0044] This work builds on previous research showing that eIF4E phosphorylation is involved in pain behavior (Moy et al., 2017; Moy et al., 2018b). The present findings illustrate that this pathway plays an even more important role in inflammation and inflammatory pain in aged subjects.

[0045] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

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