Mitochondrial; neuroprotective

After being treated without neurotrophic factors derived from the brain, ciliary body, or glia, primary embryonic rat spinal MN were significantly protected against cell damage and death by exposure to Olesoxime (TRO 19622) at concentrations ranging from 0.1 to 10 µM one hour after inoculation. This protection persisted for three days in culture. Olesoxime (TRO 19622), at a concentration of 10 µM, sustains 74±10% neuronal survival by the action of a mixture of neurotrophic factors, including those produced from the brain, ciliary bodies, and glial cells. In this test, the average EC50 was 3.2±0.2 µM. Olesoxime (TRO 19622) not only shields MN cell bodies but also encourages neurite development. At a 1 µM concentration, olesoxime (TRO 19622) only slightly improved cell viability but significantly boosted neurite development per cell by 54% [1]. A novel class of cholesterol oximes known as olesoxime (TRO 19622) was discovered due to its ability to increase the survival of pure motor neurons in the absence of neurotrophic factors. Olesoxime (TRO 19622) selectively targets proteins in the outer membrane of the mitochondria, focusing on the mitochondria and inhibiting oxidative stress-mediated permeability transition pore opening, among other processes[2].

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Exposure to Olesoxime (ranging from 0.1 to 10 µM) at 1 h after plating significantly protected primary embryonic rat spinal MNs (that had been cultured for 3 days without brain-derived, ciliary and glia-derived neurotrophic factors) from cell death. At a concentration of 10 µM, olesoxime maintained survival of 74 ± 10% of the neurons supported by a combination of neurotrophic factors (brain-derived, ciliary and glia-derived neurotrophic factors). The mean EC50 value in this assay was 3.2 ± 0.2 µM. In addition to preserving MN cell bodies, olesoxime also promoted the outgrowth of neurites. At a concentration of 1 µM, which increased cell survival by only 38%, olesoxime increased overall neurite outgrowth per cell by 54%.
The chemotherapeutic camptothecin causes DNA strand breaks and increases production of ROS. Co-treatment of cultural cortical neurons with camptothecin and Olesoxime resulted in a dose-dependent increase in cell survival at 16 h and decreased levels of activated caspase-3 and -7. These effects were similar to those observed with brain-derived neurotrophic factor, although the neuroprotection with olesoxime was not associated with activation of ERK1/2 or PI3K.
In vivo dosing of microtubule-targeting agents is often restricted by development of peripheral neuropathy. In vitro, microtubule-targeting agents decreased neurite outgrowth in rat and human differentiated neuronal cells and triggered end binding protein (EB)1 and EB2 dissociation from the microtubules to the cytosol. EB distribution and neurite outgrowth was preserved with concomitant exposure to Olesoxime.
The myelination promoting activities of Olesoxime were tested in vitro in rodent central nervous system cell cultures. Olesoxime dose-dependently accelerated the differentiation of oligodendrocyte progenitor cells from neural progenitors. It also enhanced myelination in co-cultures of dorsal root ganglion neurons and oligodendrocyte progenitor cells [1].

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Olesoxime is a cholesterol-like neuroprotective compound that targets to mitochondrial voltage dependent anion channels (VDACs). VDACs were also found in the plasma membrane and highly expressed in the presynaptic compartment. Here, we studied the effects of olesoxime and VDAC inhibitors on neurotransmission in the mouse neuromuscular junction. Electrophysiological analysis revealed that olesoxime suppressed selectively evoked neurotransmitter release in response to a single stimulus and 20 Hz activity. Also olesoxime decreased the rate of FM1-43 dye loss (an indicator of synaptic vesicle exocytosis) at low frequency stimulation and 20 Hz. Furthermore, an increase in extracellular Cl- enhanced the action of olesoxime on the exocytosis and olesoxime increased intracellular Cl- levels. The effects of olesoxime on the evoked synaptic vesicle exocytosis and [Cl-]i were blocked by membrane-permeable and impermeable VDAC inhibitors. Immunofluorescent labeling pointed on the presence of VDACs on the synaptic membranes. Rotenone-induced mitochondrial dysfunction perturbed the exocytotic release of FM1-43 and cell-permeable VDAC inhibitor (but not olesoxime or impermeable VDAC inhibitor) partially mitigated the rotenone-driven alterations in the FM1-43 unloading and mitochondrial superoxide production. Thus, olesoxime restrains neurotransmission by acting on plasmalemmal VDACs whose activation can limit synaptic vesicle exocytosis probably via increasing anion flux into the nerve terminals [5].

Adult mice receiving daily subcutaneous injections of Olesoxime (TRO 19622) (3 or 30 mg/kg) for more than two months was well tolerated without toxicity or adverse effects [1]. Olesoxime (TRO 19622) increased motor neuron cell body survival in a dose-dependent manner when animals were treated orally for five days post-lesion; at this dose, motor neuron survival was 29 ±2% (n=18), a 42% increase in survival compared to vehicle-treated animals [3]. Paclitaxel-treated rats receiving prophylactic treatment with 3 mg/kg/d or 30 mg/kg/d Olesoxime (TRO 19622) had 239±17.6 and 247±14.4 IENF/cm, respectively. For both doses, the decrease was significantly smaller than the 46% seen in vehicle-administered paclitaxel-treated rats.

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Olesoxime is a small cholesterol-like molecule that was discovered in a screening program aimed at finding treatment for amyotrophic lateral sclerosis and other diseases where motor neurons degenerate. In addition to its neuroprotective and pro-regenerative effects on motor neurons in vitro and in vivo, it has been shown to have analgesic effects in rat models of painful peripheral neuropathy due to vincristine and diabetes. We used a rat model of painful peripheral neuropathy produced by the chemotherapeutic agent, paclitaxel, to determine whether olesoxime could reverse established neuropathic pain. In addition, we determined whether giving olesoxime during the exposure to paclitaxel could prevent the development of the neuropathic pain syndrome and the accompanying degeneration of the terminal arbors of sensory fibers in the epidermis. Olesoxime significantly reduced established mechano-allodynia and mechano-hyperalgesia. There was no indication of tolerance to the effect during five days of dosing and the analgesia persisted for 5-10 days after the last injection. Giving olesoxime during the exposure to paclitaxel significantly and permanently reduced the severity of mechano-allodynia and mechano-hyperalgesia and significantly reduced the amount of sensory terminal arbor degeneration. Olesoxime targets mitochondrial proteins and its effects are consistent with the mitotoxicity hypothesis for paclitaxel-evoked painful peripheral neuropathy. We conclude that olesoxime may be useful clinically for both the prevention and treatment of paclitaxel-evoked painful peripheral neuropathy. [4]

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Olesoxime was tested in a neonatal rat model of MN degeneration induced by axotomy of the facial nerve. At 7 days after nerve axotomy, rats administered olesoxime (100 mg/kg po) for 5 days had significantly more surviving MNs compared with animals administered vehicle.

To examine whether Olesoxime could enhance regeneration of peripheral nerves, adult mice underwent sciatic nerve crush and were then administered olesoxime (0.3, 3 or 30 mg/kg sc). Treatment resulted in a dose-dependent acceleration in regeneration beginning 2 weeks after injury and was significantly different for all doses compared with vehicle-treated mice by week 4 after injury. By week 6, mice administered olesoxime had recovered up to 80% of the neuromuscular function of sham-operated mice. Lesioned nerves from vehicle-treated mice demonstrated an overall reduction in axonal size compared with control mice. Olesoxime increased axonal cross-sectional area, with statistically significant differences compared with the vehicle group at the 30-mg/kg dose (mean axonal size = 7.6 ± 0.1 versus 6.0 ± 0.1 µm2; p < 0.05). At 4 weeks, all doses of olesoxime significantly reduced the number of 'poorly' militated fibers.

The efficacy of Olesoxime was also tested in a transgenic G93Ahigh-mSOD1 mouse model of ALS. Olesoxime (3 or 30 mg/kg sc, starting at post-natal day 60) improved motor performance, delayed disease onset and extended survival by 10%. There was a 15-day delay in the onset of decrease in body weight at the 3 mg/kg dose (p < 0.01) and a significant delay of approximately 11 days in decline in grid performance was observed at both doses (p < 0.01).

The neuroprotective and antinociceptive properties of Olesoxime were investigated in a rat model of diabetic neuropathy induced by injection of streptozotocin (55 mg/kg). Neuropathy was monitored using electrophysiological measures and the tail-flick test; nociception was measured using thermal allodynia and thermal and mechanical hyperalgesia tests. At oral doses of 30 and 300 mg/kg/day starting 10 days after diabetes induction, olesoxime significantly relieved pain in diabetic rats (p ≤ 0.05) and the effects were comparable with those after administration of 3 mg/kg of morphine. Olesoxime also significantly reduced compound muscle action potential latency, a measure of motor nerve conduction. A single oral administration of olesoxime (10, 30 or 100 mg/kg) dose-dependently reversed diabetic allodynia, with statistically significant differences compared with vehicle-treated rats at the highest dose (p ≤ 0.05). After dosing for 5 days, all doses of olesoxime significantly reversed tactile allodynia and the effect was comparable with that of gabapentin (50 mg/kg bid).

The effects of Olesoxime on paclitaxel-induced neuropathic pain were studied in rats administered paclitaxel (2 mg/kg ip) on days 0, 2, 4 and 6. Olesoxime was administered from either day −1 to 15 for prevention studies or for 5 consecutive days beginning on day 25 to determine effects on paclitaxel-induced pain behavior. Olesoxime (3 or 30 mg/kg/day po) significantly reduced paclitaxel-induced allodynia and hyperalgesia until day 40 (25 days after the last dose of olesoxime). It also reduced the loss of intraepidermal nerve fibers in rats exposed to paclitaxel: these were decreased by 46% in paclitaxel-treated rats and by 22 to 25% in rats that also received olesoxime. At doses of 10 or 100 mg/kg/day, olesoxime significantly reduced hyperalgesia and allodynia from the second day of administration. Although reversible, this analgesic effect was maintained for 10 days following the last administration of olesoxime.

In a similar study, Olesoxime was assessed in a rat model of vincristine-induced (200 µg/kg iv, on days 1, 4, and 6) neuropathic pain. Olesoxime significantly decreased vincristine-induced allodynia 4 h after the first administration of the highest dose tested (100 mg/kg po; p < 0.001). Repeated treatment with 10, 30 and 100 mg/kg/day olesoxime significantly reduced vincristine-induced allodynia from day 11 to day 14 [1].

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by progressive death of cortical and spinal motor neurons, for which there is no effective treatment. Using a cell-based assay for compounds capable of preventing motor neuron cell death in vitro, a collection of approximately 40,000 low-molecular-weight compounds was screened to identify potential small-molecule therapeutics. We report the identification of cholest-4-en-3-one, oxime (Olesoxime/TRO19622) as a potential drug candidate for the treatment of ALS. In vitro, TRO19622 promoted motor neuron survival in the absence of trophic support in a dose-dependent manner[3].

Solutions and chemicals [5]
The nerve hemidiaphragm preparations were pinned to the bottom of Sylgard-coated chambers. The muscles were perfused at 5 ml·min−1 with physiological solution (129 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgSO4, 1 mM NaH2PO4, 20 mM NaHCO3, 11 mM glucose and 3 mM HEPES; pH – 7.4) saturated with a 5% CO2/95% O2 gas mixture. In some experiments a solution with high Cl− concentration (146 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 13.5 mM CholineCl and 3 mM HEPES; pH – 7.4) was used.
Pretreatment with 0.4 μM Olesoxime lasted 20 min prior to the nerve stimulation at 20 Hz or 5 Hz. Olesoxime was dissolved in DMSO and final concentration of the vehicle was 0.001%. At a concentration of 0.001–0.1% DMSO did not affect neuromuscular transmission in the mice diaphragm, thence the data from DMSO experiments were used as controls. DIDS (50 μM, 4,4′-Diisothiocyanatostilbene-2,2′-disulfonate; Tocris) and S-18 (1 μM, S-18 phosphorothioate randomer oligonucleotide) were used as inhibitors of VDACs and added to the bathing solution 5 min before the application of Olesoxime and remained in the perfusion throughout the experiment. Rotenone (10 μM, a 30 min-application) was used to induce mitochondrial dysfunction. (−)Vesamicol (2 μM) was used as inhibitor of vesicular acetylcholine transporter which is responsible for refilling of SVs with acetylcholine.

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Postsynaptic potential recordings [5]
End-plate potentials (EPPs) and miniature EPPs (MEPPs) were recorded using standard intracellular glass microelectrodes filled with 2.5 M KCl (tip resistance 5–10 MΩ). For the signal detection a Model 1600 amplifier and LA II digital I/O board were used. The recorded signals were filtered between 0.03 Hz and 10 kHz, digitized at 50 kHz and stored on PC for off-line analysis. Data were processed using a custom-developed program and analyzed to estimate mean amplitudes, rise (from 20% to 80% of the peak amplitude) and decay (from peak to 50% of the peak amplitude) times. The frequency of MEPPs was estimated in experiments after recording 150–200 signals. For MEPPs signal-to-noise ratio was >7:1 and threshold for the MEPP detection was set at level of 0.2 mV. The nerve was stimulated by rectangular supramaximal electrical 0.1-ms pulses at a frequency of 0.5 Hz or 20 Hz with a suction electrode connected to an isolated stimulator Model 2100. To prevent muscle contractions, the muscle-specific Na+ channel inhibitor μ-conotoxin-GIIIB (0.5 μM) was added to the perfusion 20 min prior to recording. EPPs were recorded at low frequency (0.5 Hz) stimulation during 20-min Olesoxime treatment, and then 20 Hz stimulation was applied for 3 min to the phrenic nerve of the pretreated with Olesoxime muscles.

In vivo, TRO19622 rescued motor neurons from axotomy-induced cell death in neonatal rats and promoted nerve regeneration following sciatic nerve crush in mice. In SOD1G93A transgenic mice, a model of familial ALS, TRO19622 treatment improved motor performance, delayed the onset of the clinical disease, and extended survival. TRO19622 bound directly to two components of the mitochondrial permeability transition pore: the voltage-dependent anion channel and the translocator protein 18 kDa (or peripheral benzodiazepine receptor), suggesting a potential mechanism for its neuroprotective activity. TRO19622 may have therapeutic potential for ALS and other motor neuron and neurodegenerative diseases[3].\n

\nFor in vivo studies, Olesoxime/TRO19622 was administrated either by oral gavage as a suspension in hydroxypropylmethylcellulose or vegetable oil or by subcutaneous [3].\n

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\n\nOlesoxime was prepared fresh daily in corn oil. Olesoxime or the vehicle was administered via oral gavage in a volume of 5.0 ml/kg. The Olesoxime doses used here (3-100 mg/kg) were chosen based on prior reports of neuroprotective and analgesic activity.[4]

\n\nTreatment paradigm [4]
\nTo determine whether Olesoxime has an analgesic effect on established paclitaxel-evoked pain, we examined withdrawal responses in animals after daily oral dosing with olesoxime during the period of approximate peak pain severity. Baseline responses in the behavioral tests were done on D23 and D24 after the first administration of paclitaxel (the approximate beginning of the plateau phase of maximal pain severity), and three experimental groups were formed such that each had approximately equal mechano-allodynia and mechano-hyperalgesia. The groups (each n = 12) were then randomly assigned to receive Olesoxime (10 mg/kg or 100 mg/kg) or vehicle on 5 consecutive days, beginning on D27. Behavior was tested 4 h after each of the daily administrations. Behavior was also assessed during a washout period beginning 1 day after the last administration of olesoxime/vehicle (washout day 1; WD1), and on WD3, WD5, WD10, WD14, and WD18. Behavioral assays were done by an observer who was blind as to group assignment.\n\n

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\n\nPreventive paradigm [4]
\nTo determine whether Olesoxime could prevent the development of paclitaxel-evoked painful peripheral neuropathy, three experimental groups were compared (each n = 12). The groups were administered vehicle or Olesoxime at 3 mg/kg or 30 mg/kg daily for 17 consecutive days, starting the day prior to the first injection of paclitaxel (D-1) until 9 days after the last injection of paclitaxel (D15). Dosing was continued after the last paclitaxel injection because there is a delay of several days before the onset of statistically significant pain hypersensitivity [8] and the time of onset of the pain-producing pathology is thus uncertain. On those days when both drugs were to be administered, Olesoxime was given at 0900 h and paclitaxel at 1300 h. Behavioral assays were repeated every 3-5 days beginning on D16 until D40 by an observer who was blind as to group assignment.\n

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\n\n Effects of prophylactic treatment on paclitaxel-evoked intraepidermal nerve fiber degeneration [4]
\nThe paclitaxel model used here has been shown to be associated with a significant loss of intraepidermal nerve fibers (IENFs), i.e., the sensory terminal receptor arbors of the afferents that innervate the epidermis [11, 22]. To determine whether olesoxime prevents this degeneration, the prophylactic dosing protocol described above was repeated in three groups of rats (Olesoxime at 3 mg/kg or 30 mg/kg, or vehicle; each n = 12). Behavioral tests were done on D29 and D30 to confirm the presence of the expected paclitaxel-evoked pain hypersensitivity in the vehicle-treated group and the expected analgesic effects in the 3 mg/kg and 30 mg/kg groups. On D31, eight rats were randomly selected from each group and sacrificed for the immunocytochemical assessment of IENFs. An additional four naïve rats (i.e., neither paclitaxel nor olesoxime treatment) of the same age and weight were sacrificed as normal controls.\n

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\n\nEffects on paclitaxel-evoked spontaneous discharge [4]
\nPaclitaxel-evoked painful peripheral neuropathy is associated with an abnormal incidence of spontaneously discharging A-fibers and C-fibers. To determine whether the acute analgesic effects of Olesoxime were associated with suppression of this discharge, we surveyed the incidence of spontaneously discharging fibers in rats that had been treated with vehicle or 100 mg/kg olesoxime (each n = 6) on two consecutive days (the treatment paradigm study described above found significant anti-allodynic and anti-hyperalgesic effects after this treatment). All rats had confirmed paclitaxel-evoked pain (assessed on D23-D24) and subsequently received Olesoxime or vehicle treatment during the plateau phase of peak pain severity (D27-D44). Electrophysiological experiments began on the second day of treatment, 4 h after drug/vehicle administration. The paclitaxel-treated rats were compared to a group of four naïve rats (neither paclitaxel nor olesoxime exposure). The experimenter was blind as to the rat's group assignment.\n\nSurgical preparation for fiber recordings required about 1 h and data were collected over the next 2-3 h when plasma concentrations of Olesoxime are maximal after oral administration. Recording methods were identical to those described previously. Briefly, the number of individually-identifiable fibers in each microfilament was determined and the incidence of individually-identifiable fibers with spontaneous discharge was noted, as was their discharge frequency. The conduction velocity was determined for all individually-identifiable fibers. We did not differentiate between A-fibers with conduction velocities in the A□ and A□ ranges because it is impossible to differentiate functional classes of A-fibers on this basis. We purposely avoided characterizing the fibers’ responses to receptive field stimulation. To do so would require repeated application of noxious stimuli that might sensitize nociceptors. Sensitized nociceptors have an ongoing discharge that would be impossible to distinguish from paclitaxel-evoked spontaneous discharge.\n\n

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\n\nOlesoxime plasma levels [4]
\nBlood was drawn from the tail vein in animals enrolled in the behavioral studies or via cardiac puncture in the animals sacrificed for the anatomical and electrophysiological studies, collected in lithium-heparin tubes, centrifuged at 3000 rpm for 10 min and the plasma frozen on Dry Ice. Quantification was performed via high-performance liquid chromatography with MS/MS detection. The detection limit of the assay was 0.01 μM.

Metabolism and Pharmacokinetics [1]
Olesoxime was administered orally to rats and mice in the form of a suspension of hydroxypropyl methylcellulose or vegetable oil, and subcutaneously in a mixture of Cremophor EL/dimethyl sulfoxide/ethanol/phosphate buffer (in a ratio of 5:5:10:80). To determine bioavailability, adult mice were subcutaneously injected daily for 1 or 6 weeks at doses of 0.3, 3, and 30 mg/kg, respectively. The concentrations of olexoxime in plasma and brain tissue were determined by high performance liquid chromatography-tandem mass spectrometry. The concentrations of olexoxime in plasma and brain tissue were dose-dependent, reaching steady state after 1 week and remaining stable during the 6-week treatment period. At a dose of 3 mg/kg/day, the concentrations of olexoxime in plasma and brain tissue were approximately 1.25 µM and 0.5 µM, respectively.
Pharmacokinetic studies in rats showed that oleexocoxime has an elimination half-life of approximately 24 hours, leading to drug accumulation, with steady-state plasma concentrations reached after three consecutive oral administrations. In diabetic and vincristine-treated rats, repeated oral administration of 10 mg/kg/day of oleexocoxime resulted in steady-state plasma concentrations ranging from 2 to 4.5 µM. A single oral administration of 100 mg/kg oleexocoxime resulted in plasma concentrations ranging from 14.2 to 37.5 µM in both models.
In paclitaxel-treated rats, the plasma concentration was 0.82 µM after the first administration of 10 mg/kg oleexocoxime, increasing to 1.39 µM after the fifth administration. For a 100 mg/kg dose, the plasma concentrations on days 1 and 5 were 6.75 µM and 8.91 µM, respectively. A phase I randomized, double-blind, placebo-controlled, dose-escalation clinical trial evaluated the pharmacokinetics of olexoxidine in 48 healthy white volunteers who received four doses (50, 150, 250, and 500 mg once daily) orally for 11 consecutive days. Olexoxidine was slowly absorbed and eliminated at all doses: the time to peak concentration (Tmax) was approximately 10 hours, and olexoxidine concentrations were detectable up to 19 days post-dose. The mean half-life (t1/2) was similar across the dose groups, approximately 120 hours. While doses were increased by 3-, 5-, and 10-fold (from 50 mg to 150 mg, 250 mg, and 500 mg), peak concentrations (Cmax) on day 1 increased by 2.2-, 4.4-, and 10.2-fold, respectively, and AUC0-τ increased by 2.1-, 4.6-, and 10.8-fold, respectively. At steady state on day 11 in all groups, Cmax increased by 2.1-fold, 7.2-fold, and 12.2-fold with increasing dose, respectively, while AUC0-τ increased by 2.0-fold, 6.5-fold, and 11.6-fold, respectively. The mean cumulative ratio of Cmax and Ctrough observed from day 1 to day 11 was approximately 4. Plasma pharmacokinetic characteristics were similar across volunteers and all dose groups: the coefficients of variation for Cmax and AUC0-τ on day 11 ranged from 21% to 47%.
In a phase Ib clinical trial in ALS patients (n = 36), the pharmacokinetics of oleexocorticoid (administered before meals) in combination with riluzole for 1 month were evaluated. At a dose of olaxoxim at 125 mg, the median trough was 512 and 742 ng/ml in men and women, respectively; at a dose of 250 mg, it was 979 and 1685 ng/ml; and at a dose of 500 mg, it was 2965 and 3310 ng/ml, respectively; these values did not show any sex difference at any dose. In the 500 mg dose group, a peak trough concentration of 5780 ng/ml was observed on day 15. The trough concentrations of olaxoxim on days 15 and 30 were similar, indicating that steady state was reached on day 15. The plasma trough concentrations in ALS patients were higher than in healthy volunteers, which may be due to co-administration with food or riluzole. The pharmacokinetics of olaxoxim were also evaluated in a phase Ib clinical trial in pediatric (n = 5) and adult (n = 3) patients with SMA. Following a single oral dose of olexocoxime (125 mg), dose adjustments to mg/kg yielded comparable Cmax and AUC values in children and adults; Tmax, t1/2, and total clearance were also similar. Results were similar with once-daily dosing. The brain permeability of olexocoxime in mice and rats was investigated using multiple methods. The brain permeability of olexocoxime relative to known brain-permeable compounds was assessed using the orthotopic rat brain perfusion technique originally developed by Q. Smith. Six 5- to 6-week-old rats were perfused with C4-labeled 3H-olexocoxime. The blood-brain barrier permeability coefficient (Kin) was 5.9 ± 3.0 µl/g/s on a scale ranging from 0.01 to 60. Compared to the reference compound, the permeability of olexocoxime was intermediate between that of colchicine (a permeability-dependent compound) and flumazenil (a flux-dependent compound). In addition, an extraction and analysis method was developed for detecting and quantifying oleexocoxime in plasma and brain tissue. In multiple pharmacokinetic and efficacy studies, the concentration of oleexocoxime in mouse brain tissue was measured, and the AUC of plasma samples collected in the same study was compared. Chronic administration studies in mouse models of neurological injury showed that oleexocoxime accumulated in brain tissue over time, while plasma concentrations remained constant (Table 4). Combining these different methods, it can be concluded that oleexocoxime can enter the brain, and efficacy-related tissue concentrations can be correlated with plasma concentrations. [2]

Toxicity[1]
Adult mice were well tolerated with daily subcutaneous injections of oleexoxime (3 or 30 mg/kg) for more than 2 months, with no toxicity or adverse reactions observed. No toxicity was also observed in animals receiving doses 40 times higher than the expected therapeutic dose for 4 weeks. As of the time of this publication, no other toxicity data were available.

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The early central nervous system safety of oleexoxime was assessed by culturing the generation/conduction of action potentials in cortical neurons. Spontaneous action potentials of cortical neurons were recorded using a multi-electrode array approximately 10 days after inoculation. The firing frequency (Hz/s) was measured for 10 minutes under control conditions (standard saline) followed by 10 minutes of perfusion with oleexoxime. No change in the firing frequency of cortical neurons was observed after exposure to 10 µM oleexoxime. In contrast, complete inhibition of firing was observed immediately after perfusion with tetrodotoxin (100 nM TTX) (Fig. 8A). Similarly, acute exposure of rat E14 motor neurons to 10 µM olexocoxime (3 min) did not alter the action potential patterns evoked by 20 Hz stimulation (data not shown). [2]

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Side effects and contraindications [1]
No serious adverse events were reported in the Phase I clinical trial of olexocoxime (50, 150, 250 and 500 mg, orally, once daily for 11 days) in healthy volunteers. A total of 69 treatment-related adverse events (TEAEs) were reported, of which 18 were considered likely to be related to olexocoxime, 22 were considered unlikely to be related, and 27 were determined to be unrelated to olexocoxime. Of the likely related treatment-related adverse events (TEAEs), 2 occurred in the 50 mg dose group, 2 in the 250 mg dose group, 7 in the 500 mg dose group, and 7 in the placebo group. Most treatment-associated adverse events (TEAEs) were mild (48 cases) or moderate (21 cases). The most frequently reported TEAEs were diarrhea (9 cases), headache (7 cases), constipation (4 cases), pharyngitis (4 cases), and back pain (4 cases). TEAEs were dose-independent. No relevant changes were observed in vital signs, ECG parameters, laboratory tests, or physical examinations. In a phase Ib clinical trial of olexocoxime (125, 250, or 500 mg once daily, orally) in combination with riluzole (50 mg twice daily, orally) for the treatment of amyotrophic lateral sclerosis (ALS), all doses were well tolerated. A total of 69 treatment-associated adverse events (TEAEs) were reported, of which 2 were considered possibly related to olexocoxime, 13 were possibly related, 21 were unlikely to be related, and 33 were not related to olexocoxime; both of the TEAEs considered possibly related occurred in the placebo group. Of the potentially relevant treatment-associated adverse events (TEAEs), 1 occurred in the control group, 6 in the 125 mg group, 3 in the 250 mg group, and 3 in the 500 mg group. The severity of TEAEs was categorized as mild (n = 55), moderate (n = 13), and severe (n = 2). The most frequently reported TEAEs were fatigue (12 cases, 9 of which occurred after olarexime treatment), diarrhea (6 cases, 4 of which occurred after olarexime treatment), muscle cramps (4 cases, 3 of which occurred after olarexime treatment), and constipation (3 cases, 1 of which occurred after olarexime treatment). The frequency, severity, and duration of TEAEs were not dose-dependent. No changes in vital signs, ECG parameters, laboratory tests, or physical examinations were observed in any of the dose groups. No safety issues were reported during the Phase Ib clinical trial of olexocoxime (125 mg orally) in children and adults with spinal muscular atrophy (SMA) and during the 1-month follow-up period.

[1]. Olesoxime, a cholesterol-like neuroprotectant for the potential treatment of amyotrophic lateral sclerosis. IDrugs. 2010 Aug;13(8):568-80.

[2]. Olesoxime (TRO19622): A Novel Mitochondrial-Targeted Neuroprotective Compound. Pharmaceuticals (Basel). 2010 Jan 28;3(2):345-368.

[3]. Identification and characterization of cholest-4-en-3-one, oxime (TRO19622), a novel drug candidate for amyotrophic lateral sclerosis. J Pharmacol Exp Ther. 2007 Aug;322(2):709-20.

[4]. Olesoxime (cholest-4-en-3-one, oxime): analgesic and neuroprotective effects in a rat model of painful peripheral neuropathy produced by the chemotherapeutic agent, paclitaxel. Pain. 2009 Dec 15;147(1-3):202-9.

[5]. Olesoxime, a cholesterol-like neuroprotectant restrains synaptic vesicle exocytosis in the mice motor nerve terminals: Possible role of VDACs. Biochim Biophys Acta Mol Cell Biol Lipids . 2020 Sep;1865(9):158739.

Olexoxime is a cholesterol-like small molecule that has demonstrated significant neuroprotective effects in a range of in vitro and in vivo preclinical models. For example, it can prevent neurodegeneration, enhance nerve function, and accelerate nerve regeneration after nerve injury. Drug Indications It is under investigation for the treatment of neurological diseases. Treatment of Spinal Muscular Atrophy Mechanism of Action Olexoxime interacts with a physiologically relevant target: the mitochondrial permeability transition pore (mPTP). Mitochondria are a core mediator of cell death and are associated with most (if not all) neurodegenerative diseases, regardless of their initiating factors: gene mutations, excitotoxicity, reactive oxygen species, ischemia, chemical toxicity, etc. Mitochondria play multiple roles in all cells. In neurons, especially near synapses, mitochondria are important calcium-buffering organelles where membrane excitability leads to a large influx of calcium ions through calcium channels. Mitochondria also produce microtubule-mediated axoplasmic transport and ATP, which is necessary to maintain the activity of ion and nutrient transport proteins. If neurons fail to establish or maintain their function, mitochondria eliminate them by releasing apoptotic factors. Orexoxime protects neurons by interacting with protein components of the mitochondrial permeability transition pore (mPTP), preventing the release of these apoptotic factors. This mechanism of action may have universal neuroprotective activity and could be used for other therapeutic indications. Amyotrophic lateral sclerosis (ALS) is a disabling and fatal motor neuron disease requiring effective treatments. Cellular and animal models of ALS are gradually revealing the biological mechanisms of selective vulnerability in motor neurons, in which mitochondria and the mitochondrial permeability pore (mPTP) play crucial roles. Proteins associated with mPTP are known to be enriched in motor neurons, and gene deletion of key regulators of mPTP has significant effects in ALS transgenic mice, delaying disease onset and prolonging survival. Therefore, mPTP is a plausible, mechanism-based drug development target for ALS. Trophos SA has discovered a small molecule compound with a cholesterol-like structure called orexoxime (TRO-19622), which has shown significant neuroprotective effects on motor neurons in both cell culture and rodent models. Orexoxime appears to act on mitochondria and may act on the mPTP. Phase I clinical trials of orexoxime have been successfully completed. Oral orexoxime is well tolerated and reaches the expected clinically effective concentrations. In the United States, orexoxime has been granted orphan drug designation for the treatment of amyotrophic lateral sclerosis (ALS); in the European Union, it has also been granted orphan drug designation for the treatment of spinal muscular atrophy. Phase II/III clinical trials are currently underway in Europe. [1] The development of orexoxime as a potential therapy for ALS is a major advance in the field of mitochondrial diseases. The rationale for using orexoxime to treat ALS is quite strong and is based on its mechanism of action. The rationale for targeting mitochondria, particularly the mitochondrial permeability transition pore (mPTP), is based on fundamental research in cell and animal models. The specific targets of olexoxime are believed to be TSPO and VDAC: TSPO is considered a regulator of mPTP, while VDAC is considered a non-essential component of mPTP. Another study using a different TSPO ligand (Ro5-4864) showed that this ligand could protect newly generated motor neurons from axonotomy-induced cell death, but no positive effect was observed in G93Ahigh-mSOD1 mice, suggesting that the binding of olexoxime to VDAC may be a more therapeutically significant neuroprotective mechanism against pathological mPTP opening in adult motor neurons. The properties of olexoxime make it an ideal neurotherapeutic agent: it can be taken orally, crosses the blood-brain barrier, and is well-tolerated. However, more data are needed regarding the effects and safety of this compound on damaged biological systems. Further basic biological research is required to elucidate the subcellular and molecular mechanisms of action of olexoxime. First, it needs to be clarified that olexoxime is a drug that acts on mitochondria. VDAC has three subtypes, some located in the plasma membrane, endoplasmic reticulum, and outer mitochondrial membrane (OMM). If oleexoxime does indeed target mitochondria and modulate the mitochondrial permeability transition pore (mPTP), then its mechanism of action on mitochondrial calcium retention needs further clarification. In addition, oleexoxime may also indirectly act on mPTP by regulating the production of mitochondrial reactive oxygen species (ROS). Furthermore, it is necessary to clarify the cell types protected by oleexoxime. For example, in vivo oleexoxime may directly protect motor neurons (MNs) or indirectly protect motor neurons by acting on microglia, astrocytes, Schwann cells, or skeletal muscle cells. Patients with amyotrophic lateral sclerosis (ALS) urgently need effective treatments. Exploring oleexoxime as a novel small molecule therapy offers hope. [1]

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Oleexoxime (TRO19622) is a novel mitochondrial-targeting neuroprotective compound that is currently undergoing pivotal clinical efficacy studies in amyotrophic lateral sclerosis (ALS) and is being developed for the treatment of spinal muscular atrophy (SMA). It belongs to a new class of cholesterol oxime compounds and was discovered for its activity in promoting cell survival in purified motor neurons lacking neurotrophic factors. Olexoxime targets mitochondrial outer membrane proteins, accumulates in mitochondria, and prevents the opening of mitochondrial permeability transition pores mediated by factors such as oxidative stress. Olexoxime has shown strong neuroprotective effects in a variety of in vitro and in vivo models. In particular, olexoxime provides significant protection in animal models of motor neuron diseases, especially ALS. Olexoxime is orally available, crosses the blood-brain barrier, and is well tolerated. Overall, its pharmacological properties suggest that olexoxime is a promising candidate drug for the treatment of motor neuron diseases. [2] Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the progressive death of cortical and spinal motor neurons, for which there is currently no effective treatment. We screened approximately 40,000 small molecule compounds using a cell-based in vitro assay to find potential small molecule therapeutics. We report the discovery of cholesterol-4-en-3-one oxime (TRO19622) as a potential ALS treatment candidate. In vitro experiments showed that TRO19622 promoted the survival of motor neurons in a dose-dependent manner in the absence of nutritional support. In vivo experiments showed that TRO19622 rescued motor neurons that died from axonal transection in newborn rats and promoted neurogenesis after sciatic nerve injury in mice. In SOD1(G93A) transgenic mice (a familial ALS model), TRO19622 treatment improved motor function, delayed the onset of clinical disease, and prolonged survival. TRO19622 binds directly to two components of the mitochondrial permeability transition pore: a voltage-dependent anion channel and an 18 kDa transporter (or peripheral benzodiazepine receptor), suggesting a potential mechanism of its neuroprotective effect. TRO19622 may have the potential to treat ALS and other motor neuron diseases and neurodegenerative diseases. [3]

C27H45NO

399.6523

399.35

C, 81.14; H, 11.35; N, 3.50; O, 4.00

22033-87-0

76971721

Typically exists as white to off-white solids at room temperature

1.1

510ºC at 760mmHg

145-148ºC

341ºC

1.56E-12mmHg at 25°C

1.583

7.858

1

2

5

29

663

7

C[C@@]12C(CC[C@]3([H])[C@]2([H])CC[C@@]4(C)[C@@]3([H])CC[C@@]4([C@]([H])(C)CCCC(C)C)[H])=CC(CC1)=NO

QNTASHOAVRSLMD-SIWSWZRQSA-N

InChI=1S/C27H45NO/c1-18(2)7-6-8-19(3)23-11-12-24-22-10-9-20-17-21(28-29)13-15-26(20,4)25(22)14-16-27(23,24)5/h17-19,22-25,29H,6-16H2,1-5H3/b28-21+/t19-,22+,23-,24+,25+,26+,27-/m1/s1

(8S,9S,10R,13R,14S,17R,E/Z)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-1,2,6,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-3H-cyclopenta[a]phenanthren-3-one oxime

E/Z-olesoxime; NSC 21311; NSC-21311; NSC21311; TRO-19622; TRO19622; TRO19622; RG6083; RG 6083; RG-6083;Olesoxime; Olesoxime, Z-; 22033-87-0; UNII-I2QN18P645; I2QN18P645; 66514-00-9; TRO 19622; (NE/Z)-N-[(8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-[(2R)-6-methylheptan-2-yl]-1,2,6,7,8,9,11,12,14,15,16,17-dodecahydrocyclopenta[a]phenanthren-3-ylidene]hydroxylamine;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~50 mg/mL (~125.11 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.26 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (6.26 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (6.26 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)