PDE4 (IC50 = 16 nM in pig aorta); PDE4 (IC50 = 2 nM in eosinophil soluble); PDE1 (IC50 = >100 μM); PDE2 (IC50 = 40 μM); PDE3 (IC50 = >100 μM); PDE5 (IC50 = 14 μM)

Piclamilast (RP 73401, 1 μM, 30 minutes) phosphorylates c-Jun Ser63 and activates AP-1 to dramatically block alterations in 23 genes [2]. For RT-PCR of PDE1, PDE2, PDE3, and PDE5[2], use Piclamilast (RP 73401).

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Aims: Reactive oxygen species (ROS) are involved in the pathogenesis of many inflammatory diseases such as chronic obstructive pulmonary disease (COPD). They can alter the expression of genes involved in cellular damage by activating transcription factors, including the NF-κB and the activator protein 1 (AP-1). Phosphodiesterase type 4 (PDE4) inhibitors have anti-inflammatory and antioxidant effects, as described in in vivo and in vitro COPD models. This study analysed the effects of Piclamilast, a selective PDE4 inhibitor, on modulating the global gene expression profile in A549 cells exposed to H(2)O(2).
Main methods: Changes in gene expression were analysed using high-density Affymetrix microarrays and validated by RT-PCR. Cell proliferation was studied using BrdU incorporation. Apoptosis was assessed by flow cytometry using annexin V-fluorescein isothiocyanate. C-Jun phosphorylation and AP-1 activation were determined by ELISA and luciferase assay, respectively.
Key findings: Our results indicate that H(2)O(2) modified the expression of several genes related to apoptosis, cell cycle control and cell signalling, including IL8, FAS, HIG2, CXCL2, CDKN25 and JUNB. Piclamilast pre-treatment significantly inhibited the changes in 23 genes via mechanisms involving AP-1 activation and c-Jun phosphorylation at Ser63. Functional experiments confirmed our results, suggesting new targets related to the antioxidant properties of PDE4 inhibitors.
Significance: This is the first study to demonstrate antioxidant effects of a selective PDE4 inhibitor at the global gene expression level, and the results support the importance of AP-1 as a key regulator of the expression of genes involved in the inflammatory response of epithelial cells to oxidative damage. [2]

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Inhibition of phosphodiesterase IV by N-(3,5-dichloropyrid-4-yl)-3-cyclopentyloxy-4-methoxybenzamide (Piclamilast) enhances the myeloid differentiation induced by all-trans-retinoic acid (ATRA), retinoic acid receptor alpha (RARalpha), or retinoic acid receptor X agonists in NB4 and other retinoid-sensitive myeloid leukemia cell types. ATRA-resistant NB4.R2 cells are also partially responsive to the action of piclamilast and retinoic acid receptor X agonists. Treatment of NB4 cells with piclamilast or ATRA results in activation of the cAMP signaling pathway and nuclear translocation of cAMP-dependent protein kinase. This causes a transitory increase in cAMP-responsive element-binding protein phosphorylation, which is followed by down-modulation of the system. ATRA + piclamilast have no additive effects on the modulation of the cAMP pathway, and the combination has complex effects on cAMP-regulated genes. Piclamilast potentiates the ligand-dependent transactivation and degradation of RARalpha through a cAMP-dependent protein kinase-dependent phosphorylation. Enhanced transactivation is also observed in the case of PML-RARalpha. In NB4 cells, increased transactivation is likely to be at the basis of enhanced myeloid maturation and enhanced expression of many retinoid-dependent genes. Piclamilast and/or ATRA exert major effects on the expression of cEBP and STAT1, two types of transcription factors involved in myeloid maturation. Induction and activation of STAT1 correlates directly with enhanced cytodifferentiation. Finally, ERK and the cAMP target protein, Epac, do not participate in the maturation program activated by ATRA + Piclamilast. [3]

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The affinities for the high-affinity rolipram binding site of selected compounds (3ab,ag,kg) and RP73401 were investigated and compared with emetic effects (Table 3). 3ab,ag,kg exhibited comparatively lower affinities than RP73401 for the high-affinity rolipram binding site (Ki value (nM):  3ab 6.2, 3ag 3.5, 3kg 2.6, Piclamilast/RP73401 0.85). It is important that 3kg showed the broadest margin between the Ki value of binding affinity and the IC50 value of PDE4 inhibition (ratio = 0.050), which may imply an improved therapeutic ratio among the PDE4 inhibitors reported so far.[4]

In albino animals, Piclamilast (RP 73401, 10 mg/kg, 30 minutes) does not alter the MST written on its own. In albino animals, piclamilast plus ATRA increased MST (40 days; interval 34-45 days) more effectively than ATRA alone [3].

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In Vivo Activity of the Combination of Piclamilast + ATRA—To evaluate whether the potentiating effect of Piclamilast on ATRA cytodifferentiating activity has therapeutic impact, we transplanted NB4 cells intraperitoneally in SCID mice. Animals were treated with vehicle, ATRA, piclamilast, and the combination of the two compounds and evaluated for survival. Fig. 11 illustrates the Kaplan-Meier survival curves of the animals belonging to the various experimental groups. The MST of vehicle-treated animals is 27 days (interval 24-30 days). Piclamilast does not affect the MST of leukemia-bearing animals (25 days; interval 20-26 days). As expected, ATRA has a significant effect on the survival of animals, increasing the MST to 37 days (interval 33-42 days, p < 0.0001 according to the Cox regression model). Piclamilast + ATRA is significantly (p < 0.05) more effective than ATRA alone in increasing the MST (40 days; interval 34-45 days) of leukemia-bearing animals. This translates into an increase in the life span over vehicle-treated animals of 37% in the case of ATRA and 48% in the case of ATRA + piclamilast. In no experimental group did we observe signs indicative of major systemic toxicity, such as treatment-associated lethality or significant body weight loss. [3]

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Antispasmogenic Activity. [3]
We selected 10 compounds on the basis of the PDE4 inhibitory potency (IC50 < 3 nM) for the evaluation of their ability to inhibit antigen-induced bronchoconstriction (iv) and histamine-induced bronchoconstriction (id) in anesthetized guinea pigs. RP73401/Piclamilast was selected as a reference compound (ED50 = 0.033 mg/kg for antigen response, iv; 0.20 mg/kg for histamine response, id). Among 3af−ah having equipotent PDE4 inhibitions, 3ag exhibited the best inhibitory activity on intravenously administered antigen-induced bronchoconstriction (ED50 (mg/kg):  3af 0.18, 3ag 0.022, 3ah 0.17) with the least effect on heart rate (Table 2). 3ab,kg also had excellent antispasmogenic activities for intravenously administered antigen response, with ED50 values of 0.12 and 0.063 mg/kg, respectively. 3-Hydroxymethyl analogue 18lg showed a very weak reduction of antigen response (a 33% reduction at 1 mg/kg, iv), despite the fact that 18lg had equipotent PDE4 inhibitory activity to 3,4-bis(hydroxymethyl) analogue 3ag (IC50 = 0.70 nM). To assess the cardiovascular side effects, the effects of selected compounds on heart rate were simultaneously investigated, and it was confirmed that 3ab,ag,kg induced little increase in heart rate at a dose of 0.1 mg/kg when administered intravenously (0, 1, and 3 beats/min, respectively). In the histamine-induced bronchoconstriction model, compound 3kg proved to have the most potent antispasmogenic activity (ED50 = 0.033 mg/kg, id) among the selected compounds and greater activity than Piclamilast/RP73401 (ED50 = 0.20 mg/kg, id). More detailed information on the antispasmogenic activity of 3kg will be published elsewhere.[3]

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Affinity for the High-Affinity Rolipram Binding Site and Emetic Effects. [3]
We finally selected three compounds (3ab,ag,kg) on the basis of antispasmogenic activity for the evaluation of emetic effects in ferrets and dogs and affinity for the high-affinity rolipram binding site. As shown in Table 3, 3ab,ag,kg did not induce emetic effects at a dose of 10 or 30 mg/kg, po, though Piclamilast/RP73401 clearly caused emesis at a dose of 3 mg/kg, po. 3kg also showed a considerably weaker emetic effect than Piclamilast/RP73401 in dogs when administered intravenously at a dose of 0.3 mg/kg (3kg, none of 8 tested animals vomited; RP73401, 6 animals of 8 tested animals vomited).

Biological Methods. [4]
Isolation of Phosphodiesterase Isozymes. The method of Reeves et al. 13 was modified to isolate PDE isozymes. Briefly, male guinea pigs were killed with pentobarbital, and the hearts and lungs were immediately excised and rinsed in ice-cold saline. Samples of cardiac ventricle and lung were frozen on solid CO2 after removal and stored at −80 °C until use. Tissue samples were minced and homogenized in 3 volumes of 20 mM Bis-Tris/2 mM EDTA/5 mM 2-mercaptoethanol/2 mM benzamidine/10 μM leupeptin/10 μM pepstatin A, pH 6.5, by using a Polytron PT-20. Phenylmethanesulfonyl fluoride (PMSF) dissolved in dimethyl sulfoxide (DMSO) was added to the buffer immediately before homogenization to give a final concentration of 0.1 mM. The homogenate was then centrifuged for 45 min at 35000g and the resulting supernatant applied to a column of Resource-Q. The PDEs were eluted from the column by using a continuous 50−1000 mM sodium acetate gradient (pH 6.5, containing 20 mM Bis-Tris, 2 mM EDTA, 5 mM 2-mercaptoethanol, 2 mM benzamidine, 0.1 mM PMSF). Fractions were collected and assayed for cAMP and cGMP PDE activity. Fractions containing high levels of type 1, 2, or 3 PDE activity from cardiac ventricle and type 4 or 5 PDE activity from lung were pooled. The combined PDE fractions were diluted to 77% with ethylene glycol and stored at −20 °C.

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Assay of Phosphodiestrase Activity. [4]
PDE activity was determined by a modification of the method of Thompson et al. 14 The reaction mixture contained 50 mM Tris-HCl, pH 8.0, 5 mM MgCl2, 4 mM 2-mercaptoethanol. In evaluation of the inhibitor effects of the different agents examined in types 1−5 PDE, the protein concentration in the assay was adjusted to ensure that hydrolysis of substrate ([3H]cAMP or [3H]cGMP) did not exceed 20% of the available substrate in the absence of an inhibitor. The concentration of substrate was 1.0 μM for these studies. All agents examined were dissolved in DMSO. Following addition of the substrate, the contents were mixed and incubated for 30 min at 30 °C. Assays were performed in triplicate at three to four different inhibitor concentrations, the mean of the determinations at each concentration was plotted, and the IC50 values were determined graphically. IC50 values presented are from representative experiments.

RT-PCR[2]
Cell Types: Human A549 type II lung epithelial cells. The IC50 values are >100 μM, 40 μM, >100 μM, and 14 μM respectively [4].
Tested Concentrations: 1 μM (H2O2 200 μM).
Incubation Duration: 30 minutes.
Experimental Results: Prevented H2O2-induced changes in gene expression levels in A549 cells.

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Cell viability assay [3]
Cell Types: NB4 cells.
Tested Concentrations: 30μM.
Incubation Duration: 3 days.
Experimental Results: Significant enhancement of STAT1 induction observed in ATRA-treated NB4 cells. Resulting in a significant increase in the number of cells expressing NBT-R activity.

Animal/Disease Models: SCID (severe combined immunodeficient) mouse [3].
Doses: 10 mg/kg (combined with ATRA).
Doses: Daily injection.
Experimental Results: More effective than ATRA alone in increasing MST (40 days; interval 34-45 days) in animals with leukemia.

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In Vivo Experiments—For the in vivo experiments, cells were suspended in 199 Hanks' medium, and 0.1 ml (1 × 106 cells/mouse) were intraperitoneally inoculated in SCID mice. Piclamilast was dissolved in 0.5% carboxyl methyl cellulose, 0.01% Tween 80 solution and injected at a dose of 10 mg/kg. ATRA was dissolved in the same solution and injected at a dose of 15 mg/kg. Drugs were administered intraperitoneally in a volume of 100 μl/animal 1 day after the inoculation, and the treatment continued for 13 days (5 daily injections/week). Data on the survival of animals were analyzed considering the following parameters: median survival time (MST) and percentage increase in life span (MST-treated/MST control × 100) - 100). Statistical treatment of the results was conducted according to the Cox regression model [3].

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Histamine-Induced Bronchoconstriction in Anesthetized Guinea Pigs. [4]
Male Hartley guinea pigs weighing 250−700 g were used. Guinea pigs were cannulated in the trachea under anesthesia with α-chloralose (120 mg/kg, iv) and ventilated with 10 mL/kg/stroke of air at a rate of 60 strokes/min. Spontaneous breathing was abolished with gallamine triethiodide (5 mg/kg, iv). Pulmonary inflation pressure (PIP), an index of bronchospasm, was measured with a pressure transducer and recorded on a Linearcorder. At the same time, heart rate was monitored by cardiotachography utilizing the R wave of ECG (standard limb lead II) as trigger. Bronchoconstriction was induced by intravenous injection of histamine dihydrochloride (2 μg/kg) via the lateral saphenous vein at 10-min intervals. Test compounds were suspended in saline with the aid of Tween 80 and administered intravenously 1 min before histamine injection.

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Antigen-Induced Bronchoconstriction in Anesthetized Guinea Pigs. [4]
Anti-ovalbumin (OA) rabbit antiserum was prepared from rabbits (2.0−2.5 kg) which had been immunized by injecting 10 mg of OA emulsified with Freund's complete adjuvant intramuscularly 4 times weekly. The serum was obtained 7 days after the last immunization and frozen at < −70 °C until use. The antibody titers of antiserum thus obtained were >10 000 times as determined by the guinea pig 4-h PCA reaction test. Male Hartley guinea pigs weighing 250−700 g were used. Guinea pigs were sensitized by iv administration of anti-OA rabbit antiserum (0.5 mL/kg); 20−28 h later, animals were challenged by antigen (30 μg/kg, iv). Guinea pigs were cannulated in the trachea under anesthesia with α-chloralose (120 mg/kg, iv) and ventilated with 10 mL/kg/stroke of air at a rate of 60 strokes/min. Spontaneous breathing was abolished with gallamine triethiodide (5 mg/kg, iv). The changes of pulmonary mechanics were measured by the method of Konzett and Rössler using a differential pressure transducer connected to the tracheal cannula. The increase in the respiratory overflow volume provoked by antigen challenge was expressed as a percentage of the maximum overflow volume obtained by clamping off the trachea. Test compounds were administered iv 2 min before antigen challenge. The effects of drugs are expressed as the dose which suppressed antigen-induced bronchoconstriction by 50% (ED50).

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Rolipram Binding Studies. [4]
Male Hartley guinea pig brains were homogenized in 10 volumes of ice-cold 20 mM Tris HCl (pH 7.4) buffer containing 2 mM MgCl2 and 0.1 mM DTT in a Polytron PT-10 homogenizer. The resulting homogenate was centrifuged at 45000g for 30 min at 4 °C. The pellet was washed by resuspension in 10 mL of buffer and recovered by centrifugation as before. The final pellet was suspended in Tris buffer and stored at −80 °C until use. Competition binding assays were performed after incubation of the mixture with 3 nM [3H]-(±)-rolipram, drugs, and 200 mg of membrane preparation for 60 min at 30 °C in reaction buffer (25 mM Tris HCl (pH 7.4), 5 mM MgCl2, 0.05 mM 5‘-AMP). Bound and free radioligand were separated by rapid filtration of mixture onto Unifilter Plate GF/B 96. The membranes were washed four times with ice-cold buffer (25 mM Tris HCl (pH 7.4), 5 mM MgCl2). Plates were dried immediately, and the bound radioactivity was counted via a TopCount microplate scintillation counter. Nonspecific binding was determined in the presence of 10 μM (±)-rolipram.

Metabolism / Metabolites
RP73401 has known human metabolites that include N-(3,5-Dichloropyridin-4-yl)-3-(3-hydroxycyclopentyl)oxy-4-methoxybenzamide.

[1]. Selective type IV phosphodiesterase inhibitors as antiasthmatic agents. The syntheses and biological activities of 3-(cyclopentyloxy)-4-methoxybenzamides and analogues. J Med Chem. 1994 May 27;37(11):1696-703.

[2]. Piclamilast inhibits the pro-apoptotic and anti-proliferative responses of A549 cells exposed to H(2)O(2) via mechanisms involving AP-1 activation. Free Radic Res. 2012 May;46(5):690-9.

[3]. Phosphodiesterase IV inhibition by piclamilast potentiates the cytodifferentiating action of retinoids in myeloid leukemia cells. Cross-talk between the cAMP and the retinoic acid signaling pathways. J Biol Chem . 2004 Oct 1;279(40):42026-40.

[4]. Novel, potent, and selective phosphodiesterase-4 inhibitors as antiasthmatic agents: synthesis and biological activities of a series of 1-pyridylnaphthalene derivatives. J Med Chem. 1999 Mar 25;42(6):1088-99.

Piclamilast is a monocarboxylic acid amide resulting from the formal condensation of the carboxy group of 3-(cyclopentyloxy)-4-methoxybenzoic acid with the primary amino group of 3,5-dichloropyridin-4-amine. It has a role as a phosphodiesterase IV inhibitor, an anti-asthmatic drug, a bronchodilator agent and an anti-inflammatory agent. It is a monocarboxylic acid amide, a member of benzamides, a chloropyridine and an aromatic ether.
Piclamilast (RP-73,401), is a selective PDE4 inhibitor comparable to other PDE4 inhibitors for its anti-inflammatory effects. It has been investigated for its applications to the treatment of conditions such as chronic obstructive pulmonary disease, bronchopulmonary dysplasia and asthma. The structure for piclamilast was first elucidated in a 1995 European patent application and exhibits the structural functionalities of cilomilast and roflumilast.

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The syntheses and biological activities of a number of benzamide derivatives, designed from rolipram, which are selective inhibitors of cyclic AMP-specific phosphodiesterase (PDE IV), are described. The effects of changes to the alkoxy groups, amide linkage, and benzamide N-phenyl ring on the inhibition of the cytosolic PDE IV from pig aorta have been investigated. As a result, some highly potent and selective PDE IV inhibitors have been identified. The most potent compounds have been further evaluated for their inhibitory potencies against PDE IV obtained from and superoxide O2- generation from guinea pig eosinophils in vitro. Selected compounds have also been examined for their activities in inhibiting histamine-induced bronchospasm in anaesthetized guinea pigs. 3-(Cyclopentyloxy)-N-(3,5-dichloro-4-pyridyl)-4-methoxybenzamide (15j) showed exceptional potency in all tests and may have therapeutic potential in the treatment of asthma.[1]

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The structural requirements for potent and selective PDE4 inhibition were revealed in a 1-pyridylnaphthalene series, and the best compound (3kg, T-2585.HCl) was chosen for further biological evaluation (PDE4 inhibition IC50 = 0.13 nM, selectivity PDE3/4 ratio = 14 000). Compound 3kg showed potent antispasmogenic activities (ED50 = 0.063 mg/kg for reduction of antigen-induced bronchoconstriction, intravenously; ED50 = 0.033 mg/kg for reduction of histamine-induced bronchoconstriction, intraduodenally) in guinea pigs with little cardiovascular effects. Furthermore, 3kg induced significantly weaker emetic effects than RP73401 after oral administration in ferrets and intravenous administration in dogs (3kg, none of 4 ferrets vomited at a dose of 10 mg/kg, po and none of 8 dogs vomited at a dose of 0.3 mg/kg, iv; Piclamilast/RP73401, 4 of 8 ferrets vomited at a dose of 3 mg/kg, po and 6 of 8 dogs vomited at a dose of 0.3 mg/kg, iv); that is compatible with the lower affinity for the high-affinity rolipram binding site (3kg, 2.6 nM; RP73401, 0. 85 nM). This may imply that 3kg has an improved therapeutic ratio because of a broad margin between the Ki value of binding affinity and the IC50 value of PDE4 inhibition (ratio = 0.050). [4]

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STAT1, the transcription factors of the cEBP family, and the MAP kinase ERK are regulated by retinoids and have been implicated in the process of myeloid maturation. Our data are consistent with the idea that STAT1 plays a role in the process of granulocytic maturation set in motion by ATRA and enhanced by PDEIV inhibitors. In fact, the amounts as well as the activation state of STAT1 correlate with enhanced granulocytic maturation of NB4 cells by ATRA + piclamilast. Although piclamilast and ATRA induce cEBPβ and -ϵ through different molecular mechanisms, no significant interactions between the two compounds on these molecular targets are evident at the majority of the time points considered. The only exception is the enhanced induction of cEBPβ observed early (6 h) during the differentiation process. This effect may be of some significance for the granulocytic maturation of NB4 cells. In this context, it is relevant that piclamilast and ATRA induce not only the forms of cEBPβ that act as transcriptional activators (LAP1 and LAP2) but also the purportedly transcriptional inhibitor, LIP. Consistent with the idea that PKA modulates the ERK pathway in a negative fashion, phosphorylation and activation of the MAP kinase is reduced by piclamilast. Interestingly, prolonged down-regulation of ERK phosphorylation by the combination of piclamilast and ATRA may have relevance for the inhibition of cell growth, which is more evident upon treatment of NB4 cells with the combination than with piclamilast or ATRA alone. However, the data obtained with the U0126 inhibitor suggest that ERK activation is not a necessary event for the myeloid maturation of APL cells. In conclusion, our results concur in defining the molecular mechanisms underlying the cross-talk between the cAMP and the retinoid signal transduction pathways. Furthermore, they indicate that PDE IV represents a valuable pharmacological target for future efforts aimed at the differentiation therapy of myeloid leukemia. [3]

C18H18CL2N2O3

381.253122806549

380.069

C, 56.71; H, 4.76; Cl, 18.60; N, 7.35; O, 12.59

144035-83-6

154575

White to off-white solid powder

1.4±0.1 g/cm3

447.8±45.0 °C at 760 mmHg

224.6±28.7 °C

0.0±1.1 mmHg at 25°C

1.626

5.25

1

4

5

25

438

0

ClC1C=NC=C(C=1NC(C1C=CC(=C(C=1)OC1CCCC1)OC)=O)Cl

RRRUXBQSQLKHEL-UHFFFAOYSA-N

InChI=1S/C18H18Cl2N2O3/c1-24-15-7-6-11(8-16(15)25-12-4-2-3-5-12)18(23)22-17-13(19)9-21-10-14(17)20/h6-10,12H,2-5H2,1H3,(H,21,22,23)

3-(Cyclopentyloxy)-N-(3,5-dichloropyridin-4-yl)-4-methoxybenzamide

Piclamilast; RP-73401; RP 73-401; Piclamilast; 144035-83-6; Cpodpmb; 3-(Cyclopentyloxy)-N-(3,5-dichloropyridin-4-yl)-4-methoxybenzamide; RP 73401; 3-(Cyclopentyloxy)-N-(3,5-dichloro-4-pyridyl)-4-methoxybenzamide; RP-73,401; Piclamilast [INN]; RP 73401; RP 73 401; RP73401; RP 73401;

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 (~131.15 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.56 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.)