Epothilone D accelerates disease progression in the SOD1G93A mouse model of amyotrophic lateral sclerosis.

AIMS: Degeneration of the distal neuromuscular circuitry is a hallmark pathology of Amyotrophic Lateral Sclerosis (ALS). The potential for microtubule dysfunction to be a critical pathophysiological mechanism in the destruction of this circuitry is increasingly being appreciated. Stabilization of microtubules to improve neuronal integrity and pathology has been shown to be a particularly favourable approach in other neurodegenerative diseases. We present evidence here that treatment with the microtubule-targeting compound Epothilone D (EpoD) both positively and negatively affects the spinal neuromuscular circuitry in the SOD1G93A mouse model of ALS. METHODS: SOD1G93A mice were treated every 5 days with 2 mg/kg EpoD. Evaluation of motor behaviour, neurological phenotype and survival was completed, with age-dependent histological characterization also conducted, using the thy1-YFP mouse. Motor neuron degeneration, axonal integrity, neuromuscular junction (NMJ) health and gliosis were also assessed. RESULTS: EpoD treatment prevented loss of the spinal motor neuron soma, and distal axon degeneration, early in the disease course. This, however, was not associated with protection of the NMJ synapse and did not improve motor phenotype or clinical progression. EpoD administration was also found to be neurotoxic at later disease stages. This was evidenced by accelerated motor neuron cell body loss, increasing gliosis, and was associated with detrimental outcomes to motor behaviour, clinical assessment and survival. CONCLUSIONS: The results suggest that EpoD accelerates disease progression in the SOD1G93A mouse model of ALS, and highlights that the pathophysiological involvement of microtubules in ALS is an evolving and underappreciated phenomenon.

The microtubule-stabilizing drug Epothilone D increases axonal sprouting following transection injury in vitro.

Neuronal cytoskeletal alterations, in particular the loss and misalignment of microtubules, are considered a hallmark feature of the degeneration that occurs after traumatic brain injury (TBI). Therefore, microtubule-stabilizing drugs are attractive potential therapeutics for use following TBI. The best-known drug in this category is Paclitaxel, a widely used anti-cancer drug that has produced promising outcomes when employed in the treatment of various animal models of nervous system trauma. However, Paclitaxel is not ideal for the treatment of patients with TBI due to its limited blood-brain barrier (BBB) permeability. Herein we have characterized the effect of the brain penetrant microtubule-stabilizing agent Epothilone D (Epo D) on post-injury axonal sprouting in an in vitro model of CNS trauma. Epo D was found to modulate axonal sprout number in a dose dependent manner, increasing the number of axonal sprouts generated post-injury. Elevated sprouting was observed when analyzing the total population of injured neurons, as well as in selective analysis of Thy1-YFP-labeled excitatory neurons. However, we found no effect of Epo D on axonal sprout length or outgrowth speed. These findings indicate that Epo D specifically affects injury-induced axonal sprout generation, but not net growth. Our investigation demonstrates that primary cultures of cortical neurons are tolerant of Epo D exposure, and that Epo D significantly increases their regenerative response following structural injury. Therefore Epo D may be a potent therapeutic for enhancing regeneration following CNS injury. This article is part of a Special Issue entitled 'Traumatic Brain Injury'.

Synthesis of stable isotope-labeled epothilone D using a degradation-reconstruction approach.

The stabilization of microtubules using epothilones represents a novel mechanism of action to treat Alzheimer's disease. Epothilone D is one such microtubule-stabilizing drug that has been investigated by Bristol-Myers Squibb. An important step in the development process was the synthesis of a stable isotope-labeled analog for use in bioanalytical assays to accurately quantify the concentration of the drug in biological samples. A novel synthetic route to stable isotope-labeled epothilone D is described. The synthetic route was based on a strategy to degrade epothilone B and then use that key intermediate to reconstruct stable isotope-labeled epothilone D. Epothilone B was treated with potassium osmate and sodium periodate. The thiazole moiety in epothilone B was efficiently cleaved to give (1S,3S,7S,10R,11S,12S,16R)-3-acetyl-7,11-dihydroxy-8,8,10,12,16-pentamethyl-4,17-dioxabicyclo[14.1.0]heptadecane-5,9-dione. The epoxide in the macrocyclic ring of that intermediate was cleanly removed by treatment with tungsten hexachloride and n-butyllithium to give the corresponding olefin (4S,7R,8S,9S,16S,Z)-16-acetyl-4,8-dihydroxy-5,5,7,9,13-pentamethyloxacyclohexadec-13-ene-2,6-dione. Bis(triethylsilyl) protection produced (4S,7R,8S,9S,16S,Z)-16-acetyl-5,5,7,9,13-pentamethyl-4,8-bis(triethylsilyloxy)-oxacyclohexadec-13-ene-2,6-dione. This intermediate was coupled to a stable isotope-labeled thiazole using a Wittig reaction as the key step to provide (13)C5, (15)N-labeled epothilone D. In summary, the synthesis was completed in nine total steps, only six of which involved isotopically labeled reagents. A total of 168 mg of (13)C5, (15)N-labeled epothilone D was prepared in an 8% overall yield from (13)C2, (15)N-labeled thioacetamide and (13)C3-labeled ethyl bromopyruvate.

Neuronal transport defects of the MAP6 KO mouse - a model of schizophrenia - and alleviation by Epothilone D treatment, as observed using MEMRI.

The MAP6 (microtubule-associated protein 6) KO mouse is a microtubule-deficient model of schizophrenia that exhibits severe behavioral disorders that are associated with synaptic plasticity anomalies. These defects are alleviated not only by neuroleptics, which are the gold standard molecules for the treatment of schizophrenia, but also by Epothilone D (Epo D), which is a microtubule-stabilizing molecule. To compare the neuronal transport between MAP6 KO and wild-type mice and to measure the effect of Epo D treatment on neuronal transport in KO mice, MnCl2 was injected in the primary somatosensory cortex. Then, using manganese-enhanced magnetic resonance imaging (MEMRI), we followed the propagation of Mn(2+) through axonal tracts and brain regions that are connected to the somatosensory cortex. In MAP6 KO mice, the measure of the MRI relative signal intensity over 24h revealed that the Mn(2+) transport rate was affected with a stronger effect on long-range and polysynaptic connections than in short-range and monosynaptic tracts. The chronic treatment of MAP6 KO mice with Epo D strongly increased Mn(2+) propagation within both mono- and polysynaptic connections. Our results clearly indicate an in vivo deficit in neuronal Mn(2+) transport in KO MAP6 mice, which might be due to both axonal transport defects and synaptic transmission impairments. Epo D treatment alleviated the axonal transport defects, and this improvement most likely contributes to the positive effect of Epo D on behavioral defects in KO MAP6 mice.

Acute microtubule changes linked to DMD pathology are insufficient to impair contractile function or enhance contraction-induced injury in healthy muscle.

Duchenne muscular dystrophy (DMD) is marked by the genetic deficiency of the dystrophin protein in striated muscle whose consequence is a cascade of cellular changes that predispose the susceptibility to contraction injury central to DMD pathology. Recent evidence identified the proliferation of microtubules enriched in post-translationally modified tubulin as a consequence of dystrophins absence that increases the passive mechanics of the muscle fiber and the excess mechanotransduction elicited reactive oxygen species and calcium signals that promote contraction injury. Motivated by evidence that acutely normalizing the disease microtubule alterations reduced contraction injury in murine DMD muscle (mdx), here we sought the direct impact of these microtubule alterations independent of dystrophins absence and the multitude of other changes consequent to dystrophic disease. To this end we used acute pharmacologic (epithiolone-D, EpoD; 4 hours) or genetic (vashohibin-2 and small vasohibin binding protein overexpression via AAV9; 2 weeks) strategies to effectively model the proliferation of detyrosination enriched microtubules in the mdx muscle. Quantifying in vivo nerve evoked plantarflexor function we find no alteration in peak torque nor contraction kinetics in WT mice modeling these DMD relevant MT alterations. Quantifying the susceptibility to eccentric contraction injury we show EpoD treatment proffered a small but significant protection from contraction injury while VASH/SVBP had no discernable impact. We conclude that the disease dependent MT alterations act in concert with additional cellular changes to predispose contraction injury in DMD.

The Improvement of Epothilone D Yield by the Disruption of epoK Gene in Sorangium cellulosum Using TALEN System.

Epothilones are a kind of 16-member macrolides with strong anticancer activity, which was produced by Sorangium cellulosum. Epothlione D shows better drug resistance and safety than taxol in clinical trials. However, the low yield of epothilone D in Sorangium cellulosum and thereof toxicity limited the application of epothilone D. In this study, the epoK gene in gene cluster for epothilone was firstly inactivated by the employment of TALEN gene knockout system. The qRT-PCR analysis and sequencing were performed to confirm the gene deletion of epoK, resulting in the epothilone D yield improvement by 34.9±1.6% and the decrease of epothilone B yield by 34.2±2.5%, which was demonstrated by LC-MS analysis. This study would lay a foundation for the yield improvement of epothilones D, B and thereof derivatives in S. cellulosum by genetic engineering, thus promoting the applications of epothilones in the field of anticancer.

Deleterious and protective effects of epothilone-D alone and in the context of amyloid ß- and tau-induced alterations.

Amyloid-ß (Aß) and hyperphosphorylated tau (P-tau) are Alzheimer's disease (AD) biomarkers that interact in a complex manner to induce most of the cognitive and brain alterations observed in this disease. Since the neuronal cytoskeleton is a common downstream pathological target of tau and Aß, which mostly lead to augmented microtubule instability, the administration of microtubule stabilizing agents (MSAs) can protect against their pathological actions. However, the effectiveness of MSAs is still uncertain due to their state-dependent negative effects; thus, evaluating their specific actions in different pathological or physiological conditions is required. We evaluated whether epothilone-D (Epo-D), a clinically used MSA, rescues from the functional and behavioral alterations produced by intracerebroventricular injection of Aß, the presence of P-tau, or their combination in rTg4510 mice. We also explored the side effects of Epo-D. To do so, we evaluated hippocampal-dependent spatial memory with the Hebb-Williams maze, hippocampal CA1 integrity and the intrinsic and synaptic properties of CA1 pyramidal neurons with the patch-clamp technique. Aß and P-tau mildly impaired memory retrieval, but produced contrasting effects on intrinsic excitability. When Aß and P-tau were combined, the alterations in excitability and spatial reversal learning (i.e., cognitive flexibility) were exacerbated. Interestingly, Epo-D prevented most of the impairments induced Aß and P-tau alone and combined. However, Epo-D also exhibited some side effects depending on the prevailing pathological or physiological condition, which should be considered in future preclinical and translational studies. Although we did not perform extensive histopathological evaluations or measured microtubule stability, our findings show that MSAs can rescue the consequences of AD-like conditions but otherwise be harmful if administered at a prodromal stage of the disease.

Epothilone D Modulates Autism-like Behaviors in the BTBR Mouse Model of Autism Spectrum Disorder.

Autism spectrum disorder (ASD) is a neurodevelopmental disorder, characterized by impaired social communication, abnormal repetitive behaviors and restricted interests and/or sensory behaviors. It has been widely accepted that ASD involves a complex interplay of both genetic and environmental risk factors. Existing medications are only symptomatic treatments, there are no effective treatments that can improve these core social behavior deficits. Recent studies indicated that synaptic development and abnormal myelination are linked to the pathogenesis of ASD. The stable tubule only polypeptide (STOP) protein, also known as microtubule-associated protein 6, plays an important role in neuronal development and synaptic plasticity. Our previous studies showed that STOP protein was significantly reduced in the plasma of autistic subjects and in the cortex of BTBR T+ Itpr3tf (BTBR) mouse model of ASD. Furthermore, studies have shown that Epothilone D, a taxol-like microtubule-stabilizing agent, could alleviate behavioral and synaptic deficits in STOP-null mice. Here, we further evaluate whether Epothilone D treatment is sufficient to modulate the autism-like behaviors in the BTBR mice, and explore the underlying mechanism. BTBR mice were treated either with Epothilone D dissolved in 99% dimethyl sulfoxide (DMSO) or with 99% DMSO vehicle. Our studies demonstrated that the restricted and repetitive behaviors of BTBR mice were improved after Epothilone D treatment, which could be achieved by improving microtubule stability and further regulating the expression of excitatory synapse-related and myelin-related proteins. These results indicate that microtubule stability may be a new and promising therapeutic target for treating patients with ASD.

[Protective effect of Epothilone D against traumatic optic nerve injury in rats].

OBJECTIVE: To investigate the therapeutic effect of Epothilone D on traumatic optic neuropathy (TON) in rats. METHODS: Forty-two SD rats were randomized to receive intraperitoneal injection of 1.0 mg/kg Epothilone D or DMSO (control) every 3 days until day 28, and rat models of TON were established on the second day after the first administration. On days 3, 7, and 28, examination of flash visual evoked potentials (FVEP), immunofluorescence staining and Western blotting were performed to examine the visual pathway features, number of retinal ganglion cells (RGCs), GAP43 expression level in damaged axons, and changes of Tau and pTau-396/404 in the retina and optic nerve. RESULTS: In Epothilone D treatment group, RGC loss rate was significantly decreased by 19.12% (P=0.032) on day 3 and by 22.67% (P=0.042) on day 28 as compared with the rats in the control group, but FVEP examination failed to show physiological improvement in the visual pathway on day 28 in terms of the relative latency of N2 wave (P=0.236) and relative amplitude attenuation of P2-N2 wave (P=0.441). The total Tau content in the retina of the treatment group was significantly increased compared with that in the control group on day 3 (P < 0.001), showing a consistent change with ptau-396/404 level. In the optic nerve axons, the total Tau level in the treatment group was significantly lower than that in the control group on day 7 (P=0.002), but the changes of the total Tau and pTau-396/404 level did not show an obvious correlation. Epothilone D induced persistent expression of GAP43 in the damaged axons, detectable even on day 28 of the experiment. CONCLUSION: Epothilone D treatment can protect against TON in rats by promoting the survival of injured RGCs, enhancing Tau content in the surviving RGCs, reducing Tau accumulation in injured axons, and stimulating sustained regeneration of axons.

Upregulation of Apol8 by Epothilone D facilitates the neuronal relay of transplanted NSCs in spinal cord injury.

BACKGROUND: Microtubule-stabilizing agents have been demonstrated to modulate axonal sprouting during neuronal disease. One such agent, Epothilone D, has been used to treat spinal cord injury (SCI) by promoting axonal sprouting at the lesion site after SCI. However, the role of Epothilone D in the differentiation of neural stem cells (NSCs) in SCI repair is unknown. In the present study, we mainly explored the effects and mechanisms of Epothilone D on the neuronal differentiation of NSCs and revealed a potential new SCI treatment. METHODS: In vitro differentiation assays, western blotting, and quantitative real-time polymerase chain reaction were used to detect the effects of Epothilone D on NSC differentiation. Retrograde tracing using a pseudotyped rabies virus was then used to detect neuronal circuit construction. RNA sequencing (RNA-Seq) was valuable for exploring the target gene involved in the neuronal differentiation stimulated by Epothilone D. In addition, lentivirus-induced overexpression and RNA interference technology were applied to demonstrate the function of the target gene. Last, an Apol8-NSC-linear ordered collagen scaffold (LOCS) graft was prepared to treat a mouse model of SCI, and functional and electrophysiological evaluations were performed. RESULTS: We first revealed that Epothilone D promoted the neuronal differentiation of cultured NSCs and facilitated neuronal relay formation in the injured site after SCI. Furthermore, the RNA-Seq results demonstrated that Apol8 was upregulated during Epothilone D-induced neuronal relay formation. Lentivirus-mediated Apol8 overexpression in NSCs (Apol8-NSCs) promoted NSC differentiation toward neurons, and an Apol8 interference assay showed that Apol8 had a role in promoting neuronal differentiation under the induction of Epothilone D. Last, Apol8-NSC transplantation with LOCS promoted the neuronal differentiation of transplanted NSCs in the lesion site as well as synapse formation, thus improving the motor function of mice with complete spinal cord transection. CONCLUSIONS: Epothilone D can promote the neuronal differentiation of NSCs by upregulating Apol8, which may provide a promising therapeutic target for SCI repair.

Investigation of Low Dose Cabazitaxel Potential as Microtubule Stabilizer in Experimental Model of Alzheimer's Disease: Restoring Neuronal Cytoskeleton.

BACKGROUND: Neuronal Microtubule (MT) tau protein, providing cytoskeleton to neuronal cells, plays a vital role, including maintenance of cell shape, intracellular transport, and cell division. Tau hyperphosphorylation mediated MT destabilization results in axonopathy, additionally neurotransmitter deficit and ultimately causing Alzheimer's disease. Pre-clinically, streptozotocin (3mg/kg, 10µl/ unilateral, ICV) stereotaxically mimics the behavioral and neurochemical alterations similar to Alzheimer's tau pathology resulting in MT assembly defects further lead to neuropathological cascades. OBJECTIVE: Clinically approved medications such as Donepezil (DNP), rivastigmine, and Memantine (MEM) are responsible for symptomatic care only, but there is no specific pharmacological intervention that directly interacts with the neuronal microtubule destabilization. METHODS: The current study focused on the involvement of anti-cancer agent microtubule stabilizer cabazitaxel at a low dose (0.5 and 2 mg/kg) alone and in combination with standard drugs DNP (5 mg/kg), MEM (10 mg/kg) and microtubule stabilizer Epothilone D (EpoD) (3 mg/kg) in the prevention of intracerebroventricular streptozotocin (ICV-STZ) intoxicated microtubule-associated tau protein hyperphosphorylation. RESULTS: Chronic treatment of CBZ at a low dose alone and in combination with standard drugs showing no side effect and significantly improve the cognitive impairment, neurochemical alterations along with reducing the level of hyperphosphorylated tau by preventing the breakdown of the neuronal cytoskeleton, respectively. CONCLUSION: The above findings suggested that CBZ at low dose show neuroprotective effects against ICV-STZ induced microtubule-associated tau protein hyperphosphorylation in rats and may be an effective agent for the preventive treatment of AD.

Potential for Treatment of Neurodegenerative Diseases with Natural Products or Synthetic Compounds that Stabilize Microtubules.

No effective therapeutics to treat neurodegenerative diseases exist, despite significant attempts to find drugs that can reduce or rescue the debilitating symptoms of tauopathies such as Alzheimer's disease, Parkinson's disease, frontotemporal dementia, amyotrophic lateral sclerosis, or Pick's disease. A number of in vitro and in vivo models exist for studying neurodegenerative diseases, including cell models employing induced-pluripotent stem cells, cerebral organoids, and animal models of disease. Recent research has focused on microtubulestabilizing agents, either natural products or synthetic compounds that can prevent the axonal destruction caused by tau protein pathologies. Although promising results have come from animal model studies using brainpenetrant natural product microtubule-stabilizing agents, such as paclitaxel analogs that can access the brain, epothilones B and D, and other synthetic compounds such as davunetide or the triazolopyrimidines, early clinical trials in humans have been disappointing. This review aims to summarize the research that has been carried out in this area and discuss the potential for the future development of an effective microtubule stabilizing drug to treat neurodegenerative disease.

Oxidative Stress-Induced Axon Fragmentation Is a Consequence of Reduced Axonal Transport in Hereditary Spastic Paraplegia SPAST Patient Neurons.

Hereditary spastic paraplegia (HSP) is a group of inherited disorders characterized by progressive spasticity and paralysis of the lower limbs. Autosomal dominant mutations in SPAST gene account for ∼40% of adult-onset patients. We have previously shown that SPAST patient cells have reduced organelle transport and are therefore more sensitive to oxidative stress. To test whether these effects are present in neuronal cells, we first generated 11 induced pluripotent stem (iPS) cell lines from fibroblasts of three healthy controls and three HSP patients with different SPAST mutations. These cells were differentiated into FOXG1-positive forebrain neurons and then evaluated for multiple aspects of axonal transport and fragmentation. Patient neurons exhibited reduced levels of SPAST encoded spastin, as well as a range of axonal deficits, including reduced levels of stabilized microtubules, lower peroxisome transport speed as a consequence of reduced microtubule-dependent transport, reduced number of peroxisomes, and higher density of axon swellings. Patient axons fragmented significantly more than controls following hydrogen peroxide exposure, suggesting for the first time that the SPAST patient axons are more sensitive than controls to the deleterious effects of oxidative stress. Treatment of patient neurons with tubulin-binding drugs epothilone D and noscapine rescued axon peroxisome transport and protected them against axon fragmentation induced by oxidative stress, showing that SPAST patient axons are vulnerable to oxidative stress-induced degeneration as a consequence of reduced axonal transport.

The effects of epothilone D on microtubule degradation and delayed neuronal death in the hippocampus following transient global ischemia.

Disruption of microtubule cytoskeleton plays an important role during the evolution of brain damage after transient cerebral ischemia. However, it is still unclear whether microtubule-stabilizing drugs such as epothilone D (EpoD) have a neuroprotective action against the ischemia-induced brain injury. This study examined the effects of pre- and postischemic treatment with different doses of EpoD on the microtubule damage and the delayed neuronal death in the hippocampal CA1 subfield on day 2 following reperfusion after 13-min global cerebral ischemia. Our results showed that systemic treatment with 0.5 mg/kg EpoD only slightly alleviated the microtubule disruption and the CA1 neuronal death, while treatment with 3.0 mg/kg EpoD was not only ineffective against the CA1 neuronal death, but also produced additional damage in the dentate gyrus in some ischemic rats. Since the pyramidal cells in the CA1 subfield and the granule neurons in the dentate gyrus are known to be equipped with dynamically different microtubule systems, this finding indicates that the effects of microtubule-disrupting drugs may be unpredictably complicated in the central nervous system.

Synthesis and Activity of Epothilone D.

Epothilones are a class of macrolide compounds. Their activities of tubulin polymerization and microtubule depolymerization inhibition like paclitaxel make them a new generation of antimitotic drugs. The mechanism of action is similar to that of paclitaxel, which can bind to tubulin and cause cancer cells to fail to undergo mitosis, thereby causing apoptosis in cancer cells. Epothilone is superior to paclitaxel in anti-tumor spectrum, anti-tumor activity, safety, water solubility and synthetic methods. It is expected to develop into a more effective anti-tumor drug than paclitaxel. Herein, the synthesis methods and activity of epothilone D were summarized and analyzed.

Systemic administration of epothilone D improves functional recovery of walking after rat spinal cord contusion injury.

Central nervous system (CNS) injuries cause permanent impairments of sensorimotor functions as mature neurons fail to regenerate their severed axons. The poor intrinsic growth capacity of adult CNS neurons and the formation of an inhibitory lesion scar are key impediments to axon regeneration. Systemic administration of the microtubule stabilizing agent epothilone B promotes axon regeneration and recovery of motor function by activating the intrinsic axonal growth machinery and by reducing the inhibitory fibrotic lesion scar. Thus, epothilones hold clinical promise as potential therapeutics for spinal cord injury. Here we tested the efficacy of epothilone D, an epothilone B analog with a superior safety profile. By using liquid chromatography and mass spectrometry (LC/MS), we found adequate CNS penetration and distribution of epothilone D after systemic administration, confirming the suitability of the drug for non-invasive CNS treatment. Systemic administration of epothilone D reduced inhibitory fibrotic scarring, promoted regrowth of injured raphespinal fibers and improved walking function after mid-thoracic spinal cord contusion injury in adult rats. These results confirm that systemic administration of epothilones is a valuable therapeutic strategy for CNS regeneration and repair after injury and provides a further advance for potential clinical translation.

Systemic epothilone D improves hindlimb function after spinal cord contusion injury in rats.

Following a spinal cord injury (SCI) a growth aversive environment forms, consisting of a fibroglial scar and inhibitory factors, further restricting the already low intrinsic growth potential of injured adult central nervous system (CNS) neurons. Previous studies have shown that local administration of the microtubule-stabilizing drug paclitaxel or epothilone B (Epo B) reduce fibrotic scar formation and axonal dieback as well as induce axonal growth/sprouting after SCI. Likewise, systemic administration of Epo B promoted functional recovery. In this study, we investigated the effects of epothilone D (Epo D), an analog of Epo B with a possible greater therapeutic index, on fibrotic scarring, axonal sprouting and functional recovery after SCI. Delayed systemic administration of Epo D after a moderate contusion injury (150 kDyn) in female Fischer 344 rats resulted in a reduced number of footfalls when crossing a horizontal ladder at 4 and 8 weeks post-injury. Hindlimb motor function assessed with the BBB open field locomotor rating scale and Catwalk gait analysis were not significantly altered. Moreover, formation of laminin positive fibrotic scar tissue and 5-HT positive serotonergic fiber length caudal to the lesion site were not altered after treatment with Epo D. These findings recapitulate a functional benefit after systemic administration of a microtubule-stabilizing drug in rat contusion SCI.

The Microtubule-Modulating Drug Epothilone D Alters Dendritic Spine Morphology in a Mouse Model of Mild Traumatic Brain Injury.

Microtubule dynamics underpin a plethora of roles involved in the intricate development, structure, function, and maintenance of the central nervous system. Within the injured brain, microtubules are vulnerable to misalignment and dissolution in neurons and have been implicated in injury-induced glial responses and adaptive neuroplasticity in the aftermath of injury. Unfortunately, there is a current lack of therapeutic options for treating traumatic brain injury (TBI). Thus, using a clinically relevant model of mild TBI, lateral fluid percussion injury (FPI) in adult male Thy1-YFPH mice, we investigated the potential therapeutic effects of the brain-penetrant microtubule-stabilizing agent, epothilone D. At 7 days following a single mild lateral FPI the ipsilateral hemisphere was characterized by mild astroglial activation and a stereotypical and widespread pattern of axonal damage in the internal and external capsule white matter tracts. These alterations occurred in the absence of other overt signs of trauma: there were no alterations in cortical thickness or in the number of cortical projection neurons, axons or dendrites expressing YFP. Interestingly, a single low dose of epothilone D administered immediately following FPI (and sham-operation) caused significant alterations in the dendritic spines of layer 5 cortical projection neurons, while the astroglial response and axonal pathology were unaffected. Specifically, spine length was significantly decreased, whereas the density of mushroom spines was significantly increased following epothilone D treatment. Together, these findings have implications for the use of microtubule stabilizing agents in manipulating injury-induced synaptic plasticity and indicate that further study into the viability of microtubule stabilization as a therapeutic strategy in combating TBI is warranted.

Use of a carboxylesterase inhibitor of phenylmethanesulfonyl fluoride to stabilize epothilone D in rat plasma for a validated UHPLC-MS/MS assay.

A sensitive, accurate and rugged UHPLC-MS/MS method was developed and validated for the quantitation of Epothilone D (EpoD), a microtubule stabilizer in development for treatment of Alzeimer's disease, in rat plasma. The ester group in EpoD can be hydrolyzed by esterases in blood or plasma, which creates a stability concern for the bioanalysis of EpoD. Species differences in the stability of EpoD in plasma were observed. Carboxylesterases were identified as the likely esterases responsible for the hydrolysis of EpoD in plasma ex vivo, and the cause of the species different stability. Phenylmethanesulfonyl fluoride, a carboxylesterase inhibitor, was used to stabilize EpoD in rat blood during sample collection, processing, and storage. A systematic method screening and optimization strategy was used to improve the assay sensitivity and minimize potential bioanalytical risks. The stabilized plasma samples were extracted by liquid-liquid extraction. Chromatographic separation was achieved on an Acquity UPLC BEH Phenyl column with a gradient elution. EpoD and its stable isotope labeled internal standards were detected by positive ion electrospray tandem mass spectrometry. The standard curve, which ranged from 0.100 to 100ng/mL was fitted to a 1/x(2) weighted linear regression model. The intra-assay precision was within ±3.6% CV and inter-assay precision was within ±4.2% CV. The assay accuracy was within ±8.3% of the nominal values. Assay recovery of EpoD was high (∼90%) and matrix effect was minimal (1.02-1.05). EpoD was stable in stabilized rat plasma for at least 30h at room temperature, 180 days at -20°C, and following three freeze-thaw cycles. The validated method was successfully applied to sample analysis in toxicology studies.