Monoamine oxidase B (MAO-B) (IC50 = 98 nM)

The amplitude of peak sodium currents is lowered in a concentration-dependent manner by safinamide mesylate (1-300 µM). The IC50 value was 262 µM when currents were driven to a Vtest of +10 mV from a Vh of -110 mV. Rat cortical neurons have a depolarized holding potential of -53 mV due to the inhibitory effect of Safinamide mesylate, which has a lower IC50 value of 8 µM.

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Safinamide, (S)-N2-{4-[(3-fluorobenzyl)oxy]benzyl}alaninamide methanesulfonate, which is in phase III clinical trials as an anti-Parkinson drug, and a library of alkanamidic analogues were prepared through an expeditious solid-phase synthesis and evaluated for their monoamine oxidase B (MAO-B) and monoamine oxidase A (MAO-A) inhibitory activity and selectivity. (S)-3-Chlorobenzyloxyalaninamide (8) and (S)-3-chlorobenzyloxyserinamide (13) derivatives proved to be more potent MAO-B inhibitors than Safinamide (IC50 = 33 and 43 nM, respectively, vs 98 nM) but with a lower MAO-B selectivity (SI = 3455 and 1967, respectively, vs 5918). The highest MAO-B inhibitory potency (IC50 = 17 nM) and a good selectivity (SI = 2941) were displayed by (R)-21, a tetrahydroisoquinoline analogue of Safinamide. Structure-affinity relationships and docking simulations pointed out strong negative steric effects of alpha-aminoamide side chains and para substituents of the benzyloxy groups and favorable hydrophobic interactions of meta substituents. The significantly diverse MAO-B affinities of a number of R and S alpha-aminoamide enantiomers, including the two rigid analogues (21) of safinamide, indicated likely enantioselective interactions at the enzymatic binding sites. [1]

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Sodium Channel Inhibition in Rat Cortical Neurons. [3]
Voltage pulses to +10 mV evoked fast inward sodium currents from cortical neurons, whose amplitude was dependent on the voltage of the conditioning pulse (see Materials and Methods). The conditioning voltage at which maximal (resting state, Vrest) and 50% maximal sodium current (half maximal inactivation state, Vhalf) could be evoked were −110 and −53 mV, respectively (Fig. 6A). According to the observed steady-state inactivation curve, the effects of Safinamide on sodium currents and voltage/state dependence of the block were tested at preconditioning potentials of −110 mV (Vrest) and −53 mV (Vhalf). As shown in Fig. 6B, Safinamide (1–300 µM) reduced the amplitude of the peak sodium currents (tonic block) in a concentration-dependent manner. When currents were stimulated to a Vtest of +10 mV from a Vh of −110 mV, the IC50 value was 262 µM. The inhibitory effect of safinamide was voltage-dependent since a significantly lower IC50 value (8 µM) was obtained when the holding potential was depolarized to −53 mV. Washout resulted in complete reversal of the inhibition. The affinity constant for the inactivated state of the sodium channel (Ki) was 4.1 µM.

When administered intraperitoneally (90 mg/kg, once daily for 14 days), safinamide mesylate significantly reduces the volume of cerebral infarction caused by MCAO in mice, as well as the neurological deficit, disruption of the brain-blood barrier (BBB), and expression of ZO-1 and the tight junction protein occludin[3]. In vivo release of GABA and Glu is dose-dependently inhibited by safinamide mesylate (intraperitoneal injection; 5 mg/kg, 15 mg/kg, and 30 mg/kg). This effect is observed when veratridine is administered. Safinamide mesylate, at a dose of 30 mg/kg, blocks the effects of veratridine on the release of GABA (treatment F1,8=4.04; time F8,64=3.76, time×treatment interaction F8,64=2.83) and Glu (treatment F1,8=1.31; time×treatment interaction F8,64=2.4). In rats, safinamide mesylate completely inhibits veratridine-stimulated Glu release at doses of 5 and 15 mg/kg, while there is a minor but not statistically significant reduction at 0.5 mg/kg[3].

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Safinamide has been recently approved as an add-on to levodopa therapy for Parkinson disease. In addition to inhibiting monoamine oxidase type B, it blocks sodium channels and modulates glutamate (Glu) release in vitro. Since this property might contribute to the therapeutic action of the drug, we undertook the present study to investigate whether safinamide inhibits Glu release also in vivo and whether this effect is consistent across different brain areas and is selective for glutamatergic neurons. To this aim, in vivo microdialysis was used to monitor the spontaneous and veratridine-induced Glu and GABA release in the hippocampus and basal ganglia of naive, awake rats. Brain levels of safinamide were measured as well. To shed light on the mechanisms underlying the effect of safinamide, sodium currents were measured by patch-clamp recording in rat cortical neurons. Safinamide maximally inhibited the veratridine-induced Glu and GABA release in hippocampus at 15 mg/kg, which reached free brain concentrations of 1.89-1.37 µM. This dose attenuated veratridine-stimulated Glu (but not GABA) release in subthalamic nucleus, globus pallidus, and substantia nigra reticulata, but not in striatum. Safinamide was ineffective on spontaneous neurotransmitter release. In vitro, safinamide inhibited sodium channels, showing a greater affinity at depolarized (IC50 = 8 µM) than at resting (IC50 = 262 µM) potentials. We conclude that safinamide inhibits in vivo Glu release from stimulated nerve terminals, likely via blockade of sodium channels at subpopulations of neurons with specific firing patterns. These data are consistent with the anticonvulsant and antiparkinsonian actions of safinamide and provide support for the nondopaminergic mechanism of its action [3].

In Vitro Enzyme Activity Assay. The enzyme activities were assessed with a radioenzymatic assay using the selective substrates 14C-serotonin (5-HT) and 14C-phenylethylamine (PEA) for MAO-A and MAO-B, respectively. The mitochondrial pellet (500 μg protein) was resuspended in 200 μL of 0.1 M phosphate buffer, pH 7.40, and was added to 50 μL of the solution of the inhibitor (transformed to the methanesulfonate salt upon addition of a stoichiometric amount of 0.01 M methanesulfonic acid to the aqueous solution of the free base) or of buffer and incubated for 30 min at 37 °C (preincubation). Then the substrate in 50 μL of buffer (5 μM 14C-5-HT or 0.5 μM 14C-PEA) was added and the assay mixture was incubated at 37 °C for 30 min (5-HT) or for 10 min (PEA). [1]
The reaction was stopped by adding 0.2 mL of HCl or perchloric acid for 5-HT or PEA, respectively. After centrifugation, the acidic radioactive metabolites were extracted with 3 mL of diethyl ether (for 5-HT) or toluene (for PEA) and the radioactivity of the organic phase was measured by liquid scintillation spectrometry at 90% efficiency. [1]
The enzymatic activity was expressed as nanomoles of substrate transformed per milligram of protein per minute (nmol mg-1 min-1). [1]
The drug inhibition curves were obtained from five to eight different concentrations (10-10−10-5 M), each in duplicate, and the IC50 was determined using nonlinear regression analysis (GraphPad best-fitting computer program). For very low active inhibitors, the percent of enzyme inhibition was determined in duplicate at the concentrations indicated in Table.1.

Whole-Cell Patch-Clamp Recording. [3]
The experiments were carried out according to standard whole-cell patch-clamp recording techniques (Hamill et al., 1981) at room temperature (25°C). Neuronal cells were continuously superfused with an extracellular solution containing (in millimolars) NaCl (60), choline chloride (60), CaCl2 (1.3), MgCl2 (2), CdCl2 (0.4), NiCl2 (0.3), TEACl (20), glucose (10), and HEPES (10). Patch pipettes were pulled using a Sutter P-87 electrode puller and filled with an internal solution consisting of (in millimolars): CsF (65), CsCl (65), NaCl (10), CaCl2 (1.3), MgCl2 (2), EGTA (10), HEPES (10), and MgATP (1). Patch electrodes had a tip resistance of 2–3 MΩ. Membrane currents were recorded and filtered at 5 kHz using an Axopatch 200B amplifier, and data were digitized using an Axon Digidata 1322A. Voltage command protocols and data acquisitions were controlled using Axon pClamp8 software. Measuring and reference electrodes were AgCl-Ag electrodes. Access resistance ranged from 5 to 10 MΩ; linear leakage and capacitative currents were eliminated using a P/4 leak subtraction protocol. Safinamide (20 mM stock solution in distilled water) was diluted in external solution and applied for 2 minutes to reach an equilibrium response.

Animal/Disease Models: Focal cerebral ischemia C57/BL6 male mouse Model[3]
Doses: 90 mg/kg
Route of Administration: intraperitoneal (ip)injection; one time/day; 14 days
Experimental Results: Dramatically diminished infarction volume in brain areas.

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Experimental Protocols and Design. [3]
Ninety-five rats were used for the microdialysis experiments, 84 for the study of veratridine-stimulated neurotransmitter release and 11 for the study of spontaneous release. The experimental protocols were approved by the Italian Ministry of Health (licenses 170/2013B and 714/2016-PR-B). As for the design of the experiments (Fig. 1, C and D and Fig. 2), each rat was randomized to saline/veratridine or Safinamide/veratridine (0.5, 5, or 15 mg/kg, Fig. 1, C and D; 5 or 15 mg/kg, Fig. 2) in the first and second microdialysis sessions, ensuring that no rat received the same treatment in the two sessions. Rats underwent two microdialysis sessions (i.e., at 24 and 48 hours after probe implantation), after which they were sacrificed with an isoflurane overdose, and placement of the probes was verified histologically. For study of veratridine-stimulated release (Fig. 1, A and B, 3, 4, and 5), each animal implanted with a single microdialysis probe was randomized to saline/veratridine or Safinamide/veratridine (30 mg/kg, Fig. 1, A and B; 15 mg/kg, Figs. 3–5) in the first microdialysis session, and treatments crossed in the second session. For the study on spontaneous Glu and GABA release, rats implanted with one probe in STN and another in the contralateral SNr were randomized to saline or veratridine 15 mg/kg in the first microdialysis session, and treatments crossed in the second session. Overall, seven animals were discarded for probe misplacement or probe clogs during microdialysis.

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In Vivo Microdialysis. [3]
Intracerebral microdialysis was performed as previously described (Morari et al., 1996; Paolone et al., 2015). One probe of concentric design was stereotaxically implanted under isoflurane anesthesia in five different brain regions according to the following coordinates (in millimeters) from the bregma and the dural surface (Paxinos and Watson, 1986): hippocampus (1-mm dialyzing membrane, antero-posterior (AP) −3.14, medio-lateral (ML) ± 1.8, dorso-ventral (DV) −4.2.), STN (1-mm dialyzing membrane, AP −3.7, ML ± 2.5, DV −8.6), SNr (1-mm dialyzing membrane, AP −5.5, ML ± 2.2, DV −8.3), DLS (3-mm dialyzing membrane, AP +1.0, ML ± 3.5, DV −6.0) and GP (2-mm dialyzing membrane, AP −1.3, ML ± 3.3, DV −6.5). When veratridine-stimulated neurotransmitter release was studied, each animal was implanted with one probe at the time. Conversely, when spontaneous neurotransmitter release was studied, each animal was implanted with two probes at the same time, one in the STN and another in the contralateral SNr. Probes were secured to the skull with dental cement. The wound was infiltrated with local anesthetic (lidocaine 2%) before surgery completion. Twenty-four hours after surgery, the probes were perfused with a modified Ringer’s solution (1.2 mM CaCl2, 2.7 mM KCl, 148 mM NaCl, and 0.85 mM MgCl2) at a flow rate of 3.0 μl/min, and sample collection (every 20 minutes) began after 6 hours of rinsing. At least four baseline samples were collected before systemic (i.p.) administration of saline or Safinamide. Thirty minutes later, veratridine (10 μM) was perfused for 30 minutes through the probe by reverse dialysis; at the end of veratridine perfusion, sample collection was continued for 80 minutes.

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Brain Pharmacokinetic Analysis. [3]
Twenty-seven rats were used for pharmacokinetic analysis.Safinamide was administered at three dose levels (5, 15, and 30 mg/kg, i.p.), and brains were removed 40, 60, and 80 minutes later to match the veratridine perfusion time in the microdialysis study. Brain samples were homogenized by sonication; after protein precipitation, the total Safinamide concentration was measured by HPLC-tandem mass spectrometry on a Sciex API4000 mass spectrometer (AB Sciex, Framingham, MA). Samples (5 µl) were injected using a CTC analytics HTS Pal autosampler (Zwingen, Switzerland) onto a Synergi MAX-RP 30 ×2.0 mm, 4-µm column at an eluent flow rate of 1.5 ml/min. Analytes were eluted using a high-pressure linear gradient program by an HP1100 binary HPLC system. To calculate the free brain concentration, the fraction of unbound Safinamide in the brain (fu,b) was determined by in vitro equilibrium dialysis (Summerfield et al., 2007). The fu,b percent was 3.27.

Absorption, Distribution and Excretion
Rapid with peak plasma concentrations ranging from 2 to 4 h, total bioavailability is 95%. Food prolonged the rate and did not affect the extent of absorption of safinamide.
76% renal, 1.5% faeces
1.8 litres/kg
total oral clearance of plasma , which accounts for parent safinamide as well as metabolites, was on average only 17.53 ± 2.71 ml/h × kg

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Metabolism / Metabolites
The principal step is mediated by amidases which have not been identified, and produces safinamide acid. It is also metabolized to O-debenzylated safinamide and N-delkylated amine. The N-dealkylated amine is then oxidized to a carboxylic acid and finally glucuronidated. Dealkylation reactions are mediated by cytochrome P450s (CYPs), especially CYP3A4. Safinamide acid binds to organic anion transporter 3 (OAT3), but no clinical relevance of this interaction has been determined. Safinamide also binds to ABCG2 transiently. No other transporter affinities have been found in preliminary studies.

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Biological Half-Life
22 h

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Safinamide Brain Levels. [3]
In a separate group of rats, the brain levels of safinamide were measured 40, 60, and 80 minutes after the administration of 5, 15, or 30 mg/kg safinamide. Free brain concentrations, derived by taking into account the brain-binding tissue of safinamide, correlated with doses, being highest for the 30 mg/kg dose and lowest for the 5 mg/kg dose at any time points examined (Table 1). In addition, for all doses, a gradual and linear decline was observed from the first through the last time point examined. During veratridine perfusion (100–120 minutes), safinamide-free brain levels for the 5, 15, and 30 mg/kg doses were in the 0.70–0.44, 1.89–1.70, and 4.77–3.04 µM concentration ranges, respectively.

Effects During Pregnancy and Lactation
◉ Summary of Use during Lactation
No information is available on the use of safinamide during breastfeeding. Because of liver toxicity in nursing rat pups, the manufacturer recommends that the drug not be used in nursing mothers. Alternate agents are preferred.
◉ Effects in Breastfed Infants
Relevant published information was not found as of the revision date.
◉ Effects on Lactation and Breastmilk
Relevant published information was not found as of the revision date. Hepatotoxicity
Safinamide has been reported to cause serum enzyme elevations in a small proportion of patients treated long term, although the abnormalities were usually mild and self-limiting and were usually no more frequent than with placebo or comparator agents. Safinamide has not been implicated in cases of acute liver injury, but such instances have been reported with nonspecific MAO inhibitors.
Likelihood score: E (unlikely cause of clinically apparent liver injury).

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Protein Binding
88–90%

[1]. Solid-phase synthesis and insights into structure-activity relationships of safinamide analogues as potent and selective inhibitors of type B monoamine oxidase. J Med Chem, 2007, 50(20), 4909-4916.

[2]. Safinamide: from molecular targets to a new anti-Parkinson drug. Neurology. 2006 Oct 10;67(7 Suppl 2):S18-23.

[3]. Safinamide Differentially Modulates In Vivo Glutamate and GABA Release in the Rat Hippocampus and Basal Ganglia.J Pharmacol Exp Ther. 2018 Feb;364(2):198-206.

See also: Safinamide (has active moiety).

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Drug Indication
Xadago is indicated for the treatment of adult patients with idiopathic Parkinson's disease (PD) as add-on therapy to a stable dose of Levodopa (L-dopa) alone or in combination with other PD medicinal products in mid-to late-stage fluctuating patients.

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Safinamide is an amino acid amide.
Safinamide is for the treatment of parkinson's disease. It was approved in Europe in February 2015, and in the United States on March 21, 2017.
Safinamide is a Monoamine Oxidase Type B Inhibitor. The mechanism of action of safinamide is as a Monoamine Oxidase-B Inhibitor, and Breast Cancer Resistance Protein Inhibitor.
Safinamide is an inhibitor of monoamine oxidase used as adjunctive therapy in combination with levodopa and carbidopa in the management of Parkinson’s disease. Safinamide has been associated with a low rate of serum enzyme elevations during treatment, but has not been linked to instances of clinically apparent acute liver injury.
See also: Safinamide Mesylate (active moiety of).

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Drug Indication
Safinamide is indicated as an add-on treatment to levodopa with or without other medicines for Parkinson’s disease
Xadago is indicated for the treatment of adult patients with idiopathic Parkinson's disease (PD) as add-on therapy to a stable dose of Levodopa (L-dopa) alone or in combination with other PD medicinal products in mid-to late-stage fluctuating patients.

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Mechanism of Action
Safinamide is a unique molecule with multiple mechanisms of action and a very high therapeutic index. It combines potent, selective, and reversible inhibition of MAO-B with blockade of voltage-dependent Na+ and Ca2+ channels and inhibition of glutamate release. Safinamide has neuroprotective and neurorescuing effects in MPTP-treated mice, in the rat kainic acid, and in the gerbil ischemia model.

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Safinamide, (S)-N2-{4-[(3-fluorobenzyl)oxy]benzyl}alaninamide methanesulfonate, which is in phase III clinical trials as an anti-Parkinson drug, and a library of alkanamidic analogues were prepared through an expeditious solid-phase synthesis and evaluated for their monoamine oxidase B (MAO-B) and monoamine oxidase A (MAO-A) inhibitory activity and selectivity. (S)-3-Chlorobenzyloxyalaninamide (8) and (S)-3-chlorobenzyloxyserinamide (13) derivatives proved to be more potent MAO-B inhibitors than safinamide (IC50 = 33 and 43 nM, respectively, vs 98 nM) but with a lower MAO-B selectivity (SI = 3455 and 1967, respectively, vs 5918). The highest MAO-B inhibitory potency (IC50 = 17 nM) and a good selectivity (SI = 2941) were displayed by (R)-21, a tetrahydroisoquinoline analogue of safinamide. Structure-affinity relationships and docking simulations pointed out strong negative steric effects of alpha-aminoamide side chains and para substituents of the benzyloxy groups and favorable hydrophobic interactions of meta substituents. The significantly diverse MAO-B affinities of a number of R and S alpha-aminoamide enantiomers, including the two rigid analogues (21) of safinamide, indicated likely enantioselective interactions at the enzymatic binding sites.[1]

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Ideal treatment in Parkinson's disease (PD) aims at relieving symptoms and slowing disease progression. Of all remedies, levodopa remains the most effective for symptomatic relief, but the medical need for neuroprotectant drugs is still unfulfilled. Safinamide, currently in phase III clinical trials for the treatment of PD, is a unique molecule with multiple mechanisms of action and a very high therapeutic index. It combines potent, selective, and reversible inhibition of MAO-B with blockade of voltage-dependent Na+ and Ca2+ channels and inhibition of glutamate release. Safinamide has neuroprotective and neurorescuing effects in MPTP-treated mice, in the rat kainic acid, and in the gerbil ischemia model. Safinamide potentiates levodopa-mediated increase of DA levels in DA-depleted mice and reverses the waning motor response after prolonged levodopa treatment in 6-OHDA-lesioned rats. Safinamide has excellent bioavailability, linear kinetics, and is suitable for once-a-day administration. Therefore, safinamide may be used in PD to reduce l-dopa dosage and also represents a valuable therapeutic drug to test disease-modifying potential. [2]

C17H19FN2O2.CH4O3S

398.45

398.131

C, 54.26; H, 5.82; F, 4.77; N, 7.03; O, 20.08; S, 8.05

202825-46-5

Safinamide;133865-89-1

3038502

White to off-white solid powder

476.7ºC at 760 mmHg

210° (dec)

242.1ºC

2.98E-09mmHg at 25°C

4.044

3

7

7

27

438

1

C[C@@H](C(=O)N)NCC1=CC=C(C=C1)OCC2=CC(=CC=C2)F.CS(=O)(=O)O

YKOCHIUQOBQIAC-YDALLXLXSA-N

InChI=1S/C17H19FN2O2.CH4O3S/c1-12(17(19)21)20-10-13-5-7-16(8-6-13)22-11-14-3-2-4-15(18)9-14;1-5(2,3)4/h2-9,12,20H,10-11H2,1H3,(H2,19,21);1H3,(H,2,3,4)/t12-;/m0./s1

(S)-2-((4-((3-fluorobenzyl)oxy)benzyl)amino)propanamide methanesulfonate

PNU-151774E, FCE28073; Safinamide mesylate; Safinamide mesylate; 202825-46-5; (S)-2-((4-((3-Fluorobenzyl)oxy)benzyl)amino)propanamide methanesulfonate; Safinamide mesilate; PNU-151774E; NW-1015; safinamide methanesulfonate; Xadago; NW 1015; PNU 151774E; EMD 1195686; FCE-28073; Safinamide mesilate; FCE 28073; NW1015; NW-1015; EMD-1195686; EMD1195686; PNU-151774E;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

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

Solubility in Formulation 1: ≥ 2.08 mg/mL (5.22 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 20.8 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: ≥ 2.08 mg/mL (5.22 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 20.8 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: ≥ 2.08 mg/mL (5.22 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 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: Saline: 30 mg/mL

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