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].

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

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Olesoxime 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]

Treatment paradigm [4]
To 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.

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Preventive paradigm [4]
To 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.

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Effects of prophylactic treatment on paclitaxel-evoked intraepidermal nerve fiber degeneration [4]
The 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.

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Effects on paclitaxel-evoked spontaneous discharge [4]
Paclitaxel-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. Surgical 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.

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Olesoxime plasma levels [4]
Blood 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 has been administered to rats and mice by oral gavage as a suspension in hydroxypropylmethylcellulose or vegetable oil, and by subcutaneous injection as a mixture of Cremophor EL/dimethyl sulfoxide/ethanol/ phosphate-buffered saline (5:5:10:80, respectively). To determine bioavailability, adult mice received daily subcutaneous injections of olesoxime for 1 or 6 weeks at doses of 0.3, 3 and 30 mg/kg. Levels of olesoxime in plasma and brain were measured by high performance liquid chromatography with tandem mass spectroscopy detection. Plasma and brain olesoxime levels were dose-dependent, reached steady-state by 1 week and remained stable over the 6-week treatment period. Levels of olesoxime were approximately 1.25 and 0.5 µM in plasma and brain, respectively, at the 3-mg/kg/day dose.

Pharmacokinetic studies in rats demonstrated that olesoxime had an elimination t1/2 value of approximately 24 h, leading to accumulation with steady-state levels in plasma achieved after three daily oral administrations. In the diabetic and vincristine-treated rats, repeated oral administration of a 10-mg/kg/day dose of olesoxime resulted in steady-state plasma concentrations of between 2 and 4.5 µM. Single-dose oral olesoxime at 100 mg/kg resulted in plasma concentrations between 14.2 and 37.5 µM in both models.

In paclitaxel-treated rats, plasma levels after the first 10-mg/kg dose of olesoxime were 0.82 µM increasing to 1.39 µM after the fifth daily dose. For a 100-mg/kg dose, day 1 and day 5 plasma levels were 6.75 and 8.91 µM, respectively.
A phase I, randomized, double-blind, placebo-controlled, dose-escalation clinical trial assessed the pharmacokinetics of four doses of olesoxime (50, 150, 250 and 500 mg po, qd) administered for 11 consecutive days to healthy Caucasian volunteers (n = 48). The absorption and elimination of olesoxime were slow at all doses: the Tmax value was approximately 10 h and concentrations of olesoxime were measurable for up to 19 days after dosing. The mean t1/2 was comparable between doses at approximately 120 h. Dose increased in a ratio of 3, 5 and 10 (from 50 to 150, 250 and 500 mg, respectively), but day 1 Cmax increased in a ratio of 2.2, 4.4 and 10.2, and AUC0-τ increased in a ratio of 2.1, 4.6 and 10.8. At steady-state, which was reached on day 11 in all groups, Cmax increased with dose in a ratio of 2.1, 7.2 and 12.2, and AUC0-τ increased with a ratio of 2.0, 6.5 and 11.6. Mean accumulation ratios of Cmax and Ctrough observed between days 1 and 11 were approximately 4. Plasma pharmacokinetic profiles were similar between volunteers and across all doses: the coefficients of variation of Cmax and AUC0-τ were between 21 and 47% on day 11.

The pharmacokinetics of olesoxime (dosed just before a meal), co-administered with riluzole for 1 month, were assessed in a phase Ib clinical trial in patients (n = 36) with ALS. Median male and female Ctrough values were 512 and 742 ng/ml at a 125-mg dose of olesoxime, 979 and 1685 ng/ml at a 250-mg dose, and 2965 and 3310 ng/ml at a 500-mg dose; these values did not indicate a gender effect at any dose. The maximum Ctrough value was 5780 ng/ml and was observed on day 15 in the 500-mg dose group. Day 15 and day 30 olesoxime Ctrough values were similar, suggesting that steady-state was reached by day 15. Greater Ctrough plasma concentrations were observed in patients with ALS than in healthy volunteers, which may have been caused by co-administration with food or riluzole.

The pharmacokinetics of olesoxime were also assessed in a phase Ib trial in pediatric (n = 5) and adult (n = 3) patients with SMA. After a single dose of olesoxime (125 mg po), the Cmax and AUC values were comparable in children and adults after adjusting the dose to mg/kg; Tmax, t1/2 and total clearance were identical. Results after once-daily dosing were similar.

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Brain penetration of olesoxime was studied in mice and rats by a variety of methods. The relative level of olesoxime brain penetration compared to known brain-penetrating compounds was evaluated using the in situ rat brain perfusion technique initially developed by Q. Smith. A total of six rats, 5 to 6 weeks old, were perfused with 3H-Olesoxime labelled in the C4 position. The permeation coefficient of the blood brain barrier (Kin) was found to be 5.9 ± 3.0 µl/g/s in a scale ranging from 0.01 to 60. When compared to reference compounds, olesoxime penetration falls between colchicine, a permeability-dependent compound, and flumazenil, a flux-dependent compound. An extraction and analytical method was also developed to detect and quantify olesoxime in both plasma and brain tissue. The amount of olesoxime in brains of mice used in various pharmacokinetic and efficacy studies allowed to compare brain levels to the AUC measured from plasma samples collected in the same study. Chronic administration performed in the nerve crush study in mice demonstrates accumulation in brain tissue over time while plasma concentrations remain constant (Table 4). Using these different approaches it can be concluded that olesoxime enters the brain and that tissue levels corresponding to efficacy can be correlated with plasma concentrations. [2]

Toxicity [1]
Daily administration of olesoxime (3 or 30 mg/kg sc) to adult mice for more than 2 months was well tolerated without toxicity or adverse effects. Toxicity was not observed in animals exposed to doses 40-fold greater than the expected therapeutic dose for 4 weeks. At the time of publication, no further toxicity data were available.

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Early CNS safety of olesoxime was assessed on the generation/propagation of action potentials in cultured cortical neurons. About 10 days after plating, spontaneous action potentials of cortical neurons were recorded on Multi-Electrode Arrays. The firing rate (Hz/s) was measured in control conditions (standard saline solution) for 10 minutes, and then olesoxime was perfused for an additional 10 minute period. When exposed to 10 µM olesoxime, cortical neurons did not show any modification of their firing rate. In comparison, complete firing inhibition was observed immediately after tetrodotoxin (100 nM TTX) perfusion (Figure 8A). Similarly acute exposure of rat E14 motoneurons to 10 µM olesoxime (3 min) did not modify the action potential profiles evoked by a 20 Hz stimulation (data not shown). [2]

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Side effects and contraindications [1]
In the phase I clinical trial of olesoxime (50, 150, 250 and 500 mg po, qd for 11 days) in healthy volunteers, no serious adverse events were reported. There were 69 treatment-emergent adverse events (TEAEs) reported, of which 18 were considered possibly related, 22 were considered unlikely to be related and 27 were judged unrelated to olesoxime. Of the possibly related TEAEs, two occurred after the 50-mg dose, two occurred after the 250-mg dose, seven occurred after the 500-mg dose and seven occurred after placebo. Most TEAEs were mild (48 events) or moderate (21 events) in intensity. The most frequently reported TEAEs were diarrhea (9 episodes), headache (7 episodes), constipation (4 episodes), pharyngitis (4 episodes) and back pain (4 episodes). TEAEs were not dose-related. There were no relevant changes in vital signs, ECG parameters, laboratory tests or physical examinations.
In the phase Ib clinical trial of olesoxime (125, 250 or 500 mg po, qd) plus riluzole (50 mg po, bid) in patients with ALS, all doses were well tolerated. There were 69 TEAEs reported, of which 2 were considered probably related, 13 possibly related, 21 unlikely to be related and 33 unrelated to olesoxime; the 2 considered probably related both occurred in the placebo group. Of the possibly related TEAEs, one occurred in the control group, six in the 125-mg group, three in the 250-mg group and three in the 500-mg group. The TEAEs were mild (n = 55), moderate (n = 13) or severe (n = 2) in intensity. The most frequently reported TEAEs were asthenia (12 episodes, 9 after olesoxime), diarrhea (6 episodes, 4 after olesoxime), muscle spasms (4 episodes, 3 after olesoxime) and constipation (3 episodes, 1 after olesoxime). The frequency, severity and duration of TEAEs were not dose-related. No relevant changes in vital signs, ECG parameters, laboratory tests or physical examinations were observed in any dose group.
No safety issues were reported during the phase Ib clinical trial of olesoxime (125 mg po) in pediatric and adult patients with SMA, or 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.

Olesoxime is a cholesterol-like small molecule that has demonstrated a remarkable neuroprotective profile in a battery of both in vitro and in vivo preclinical models. For example, it has demonstrated the ability to prevent neurodegeneration, enhance nerve function and accelerate neuroregeneration following nerve trauma.

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Drug Indication
Investigated for use/treatment in neurologic disorders.
Treatment of spinal muscular atrophy

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Mechanism of Action
Olesoxime interacts with a physiologically relevant target: the mitochondrial permeability transition pore (mPTP). Mitochondria are central mediators of cell death and are implicated in most if not all neurodegenerative diseases regardless of the initiating factor: genetic mutations, excitotoxicity, reactive oxygen species, ischemia, chemical toxicity, etc. Mitochondria play diverse roles in all cells. In neurons, especially near synaptic sites, mitochondria are essential calcium-buffering organelles in areas where membrane excitability leads to large influx of calcium through calcium channels. Mitochondria also produce the ATP necessary for microtubule-based axoplasmic transport and maintaining the activity of ion and nutrient transporters. If a neuron fails to establish or maintain its functional role, mitochondria are responsible for eliminating it by releasing apoptotic factors. Olesoxime, by interacting with protein components of the mPTP, prevents the release of these apoptotic factors and therefore protects the neuron. This mechanism of action may lead to a general neuroprotective activity with utility in other therapeutic indications.

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Effective therapies are needed for amyotrophic lateral sclerosis (ALS), a debilitating and fatal motor neuron disease. Cell and animal models of ALS are beginning to reveal possible principles governing the biology of motor neuron-selective vulnerability that implicate mitochondria and the mitochondrial permeability pore (mPTP). Proteins associated with the mPTP are known to be enriched in motor neurons and the genetic deletion of a major regulator of the mPTP has robust effects in ALS transgenic mice, delaying disease onset and extending survival. Thus, the mPTP is a rational, mechanism-based target for the development of drugs designed to treat ALS. Trophos SA has discovered Olesoxime (TRO-19622), a small-molecule with a cholesterol-like structure, which has remarkable neuroprotective properties for motor neurons in cell culture and in rodents. Olesoxime appears to act on mitochondria, possibly at the mPTP. Phase I clinical trials of olesoxime have been completed successfully. Olesoxime is well tolerated and achieves levels predicted to be clinically effective when administered orally. It has been granted orphan drug status for the treatment of ALS in the US and for the treatment of spinal muscular atrophy in the EU. Phase II/III clinical trials are in progress in Europe.[1]

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The development of Olesoxime as a potential treatment for ALS is a major step forward for the field of MN disease. The rationale for the use of olesoxime in ALS is reasonably strong and mechanism-based. The theory behind targeting mitochondria and, specifically, the mPTP is based firmly on cell and animal model basic research. The specific targets of olesoxime have been suggested to be the TSPO and VDAC: TSPO is believed to be a modulator of the mPTP and VDAC is believed to be a dispensable component of the mPTP. Another study using a different TSPO ligand (Ro5–4864) demonstrated protection against neonatal MN cell death induced by axotomy, but no positive effects were noted in G93Ahigh-mSOD1 mice, suggesting that olesoxime binding to VDAC is perhaps the more therapeutically relevant interaction for neuroprotection against pathologic mPTP opening in adult MNs. The properties of olesoxime are propitious for a neurotherapeutic compound: it can be administered as an oral capsule, it passes the blood-brain barrier and it is well tolerated. However, more data are needed on the actions and safety of this compound on injured or damaged biological systems. More basic biological studies need to be undertaken to define the mechanisms of action of Olesoxime at subcellular and molecular levels. First, the identification of olesoxime as a drug acting on mitochondria needs to be established firmly. There are three isoforms of VDAC and some are at the plasma membrane and endoplasmic reticulum as well as on the OMM. If olesoxime does target mitochondria and does modulate the mPTP, then its actions on mitochondrial calcium retention need to be clarified. Alternatively, olesoxime could indirectly act on the mPTP by modulating mithochondrial ROS products. Furthermore, the types of cells that olesoxime protects need to be identified. For example, in vivo olesoxime could be exerting protective actions directly on MNs or it could be acting on microglia, astrocytes, Schwann cells or skeletal muscle cells to indirectly protect MNs. Patients with ALS desperately need an effective treatment for their disease. The exploration of olesoxime as a new small-molecule therapy offers hope. [1]

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Olesoxime (TRO19622) is a novel mitochondrial-targeted neuroprotective compound undergoing a pivotal clinical efficacy study in Amyotrophic Lateral Sclerosis (ALS) and also in development for Spinal Muscular Atrophy (SMA). It belongs to a new family of cholesterol-oximes identified for its survival-promoting activity on purified motor neurons deprived of neurotrophic factors. Olesoxime targets proteins of the outer mitochondrial membrane, concentrates at the mitochondria and prevents permeability transition pore opening mediated by, among other things, oxidative stress. Olesoxime has been shown to exert a potent neuroprotective effect in various in vitro and in vivo models. In particular olesoxime provided significant protection in experimental animal models of motor neuron disorders and more particularly ALS. Olesoxime is orally active, crosses the blood brain barrier, and is well tolerated. Collectively, its pharmacological properties designate olesoxime as a promising drug candidate for motor neuron diseases. [2]

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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 (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. 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 SOD1(G93A) 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]

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.)