PDE-4/phosphodiesterase

Apremilast (CC-10004) has an IC50 of 104 nM (pIC50=6.98±0.2) that inhibits TNF-α release by lipopolysaccharide (LPS). This is similar to the potency of Apremilast for PDE4 enzymatic inhibition (IC50=74 nM) and nearly exactly replicates the TNF-α inhibition that Apremilast has previously been shown to inhibit on peripheral blood mononuclear cells (PBMCs) (IC50=110 nM). With increased intracellular cAMP levels, apremilast suppresses TNF-α, and these results convincingly support this theory. Apremilast-induced IL-10 activation and TNF-α suppression were not observed in the presence of PKA, Epac1, or Epac2 knockdowns].

When taken orally at a dose of 5 mg/kg, apremilast (CC-10004) dramatically reduces the amount of TNF-α produced in the air pouch by 39% (61±6% of the vehicle, P <0.001) and decreases the number of leukocytes by 28% (72±12% of the vehicle, P <0.05). Immunohistologic investigation confirms that Apremilast significantly reduces neutrophil accumulation in the air pouch membrane. Both methotrexate (MTX) and apremilast considerably decrease leukocyte infiltration in the murine air pouch model, however only apremilast significantly inhibits TNF-α release. There is no greater suppression of leukocyte infiltration or TNF-α release when MTX (1 mg/kg) is added to Apremilast (5 mg/kg) than when Apremilast is used alone[1]. It has been demonstrated that the new oral PDE4 inhibitor apremilast controls inflammatory mediators. The mean maximum plasma concentration (Cmax) following oral administration of Apremilast is determined to be 67.00±14.87 ng/mL. Apremilast's plasma concentration drops quickly, and it eventually disappears from plasma with a terminal half-life of 0.92±0.46 h[2].

Luminex assay [1]
Quantification of cytokines and chemokines was performed using Luminex x-MAP technology. Tissue culture supernatants and mouse exudates were analyzed for expression of IL-1α, IL-6 and IL-10 using a Milliplex multi-analyte magnetic bead panel. Assays were performed according to the kit protocol using the appropriate matrix solution (culture media or PBS for supernatants and exudates, respectively). Data were collected on a Luminex 200 instrument and analyzed using Analyst 5.1 software with four-parameter logistic curve fitting. Samples were assayed in duplicate. All standard curves generated from the known reference cytokine concentrations supplied by the manufacturer had R2 values calculated at or close to 1 and percent recovery between 80 and 120 %. Quality controls included with each kit performed as expected.

cAMP measurements [1]
Intracellular cAMP was measured with the direct cAMP ELISA kit. Fifty-percent-confluent Raw 264.7 cells were starved for 24 h and stimulated at the indicated concentrations of Apremilast (CC-10004) for 30 minutes, and then with lipopolysaccharide (LPS) for 20 minutes, and cAMP was analyzed according to the manufacturer’s protocol.

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TNF-α measurement [1]
Raw 264.7 cells (100,000) were grown in 96-well plates. After 24 h, cells were stimulated with vehicle (final concentration of 0.025 % dimethyl sulfoxide (DMSO)) or with Apremilast (CC-10004) at the indicated concentrations. After 30 minutes cells were stimulated with LPS 1 μg/ml for 4 h. When studying CGS21680, SCH58261, ZM241385, BAY60-6583, or GS6201, the adenosine receptor ligands were added 15 minutes before apremilast. Methotrexate was added 24 h and 1 h before apremilast. Supernates were then collected and TNF-α levels were quantified with the Mouse TNF-α Quantikine ELISA Kit following the manufacturer’s instructions.

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Western blotting [1]
Seventy-percent-confluent Raw 264.7 cells were starved for 24 h and stimulated with Apremilast (CC-10004) for 30 minutes and then with LPS for different time points (n = 4), Cells were lysed with radioimmunoprecipitation assay (RIPA) buffer and protein concentration was determined by bicinchoninic acid (BCA). Protein (4 μg) was subjected to 7.5 or 10.0 % SDS-PAGE and transferred to a nitrocellulose membrane. Nonspecific binding was blocked with TBS/Tween-20 0.05−3 % BSA. Membranes where incubated overnight (4 °C) with primary rabbit polyclonal anti-pCREB, mouse monoclonal anti-CREB, rabbit polyclonal anti-PDE4 and mouse monoclonal anti-Actin (1:1000 each). Membranes were incubated with goat anti-rabbit IRDye 800CW 1:10000 and goat anti-mouse IRDye 680 RD 1:10000 in the dark. Proteins were visualized by Li-cor Odyssey equipment, which detects near-infrared fluorescence. As each secondary antibody emits a signal in a different spectrum, reprobing with actin (to check that all lanes were loaded with the same amount of protein) was performed simultaneously with primary antibody incubation. Intensities of the respective band were quantitated by densitometric analysis using Image Studio 2.0.38 software. Variations in band intensity were expressed as the percent of unstimulated controls, to minimize disparities among different experiments.

Air pouch model[1]
As previously described, male mice were given weekly intraperitoneal injections of either MTX (1mg/Kg) or vehicle (phosphate-buffered saline; PBS) for 4 weeks. Air pouches were generated by subcutaneous injection of 3 ml of sterile air and reinflated with 1.5 ml of sterile air 2 days later. Vehicle (0.5 % carboxymethylcellulose and 0.25 % Tween 80) or Apremilast (CC-10004) (5 mg/Kg) were orally dosed, with a syringe through a blunt-ended curved feeding tube, 24 h and 1 h before inflammation was induced on day 6 by injection of 1 ml of 2 % carrageenan suspension. Four hours later, mice were killed by CO2 narcosis, and exudates harvested with 2 ml PBS. Leukocytes were counted in a hemocytometer chamber and concentrations of cytokines were measured by ELISA or by the Luminex platform as described below.

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Application to a pharmacokinetic study[2]
Male Sprague Dawley rats (180–220 g) were used to study the pharmacokinetics of Apremilast (CC-10004). Diet was prohibited for 12 h before the experiment, but water was freely available. Blood samples (0.3 mL) were collected from the tail vein into heparinized 1.5 mL polythene tubes at 0.25, 0.5, 0.75, 1, 1.5, 2, 3, 4, 6, 8 and 12 h after oral administration of Apremilast (CC-10004) (6.0 mg/kg). The samples were immediately centrifuged at 4,000 g for 8 min. The plasma obtained (100 µL) was stored at −20°C until analysis. Plasma apremilast concentration versus time data for each rat was analyzed by DAS (Drug and statistics) software.

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Dissolved in 0.5% carboxymethylcellulose and 0.25% Tween 80; 5 mg/kg; P.O.

Mouse xenograft model of psoriasis (Beige-SCID mice)

Absorption, Distribution and Excretion
Oral apromisit is well absorbed, with an absolute bioavailability of approximately 73%. The time to peak concentration (Tmax) is approximately 2.5 hours, and a pharmacokinetic study reported a peak plasma concentration (Cmax) of approximately 584 ng/mL. Food intake does not appear to affect apromisit absorption. Only 3% and 7% of the apromisit dose were detected unchanged in urine and feces, indicating extensive metabolism and high absorption. The mean apparent volume of distribution (Vd) is approximately 87 L, suggesting that apromisit is distributed in the extravascular space. In healthy patients, the plasma clearance of apromisit is approximately 10 L/hour. The human plasma protein binding of apromisit is approximately 68%. The mean apparent volume of distribution (Vd) is 87 L. This study evaluated milk excretion in lactating CD1 mice following oral administration of apromisit. In this study, female mice approximately 13 days postpartum were given a single oral dose of 10 mg/kg apromiscarriage via gavage (10 mL/kg). Milk and blood samples were collected from five mice at 1, 6, and 24 hours post-administration, and plasma and milk apromiscarriage concentrations were determined using liquid chromatography-tandem mass spectrometry (LC-MS/MS). At 1 and 6 hours post-administration, the average plasma apromiscarriage concentrations were 984 ng/mL and 138 ng/mL, respectively, while the average milk apromiscarriage concentrations were 1441 ng/mL and 186 ng/mL, respectively. The average milk-to-plasma concentration ratio ranged from 1.46 to 1.62, indicating that apromiscarriage can be transferred to mouse milk. At 24 hours, both plasma and milk concentrations were below the detection limit of 3 ng/mL. In monkeys, pregnant animals were administered apremilast orally daily from day 20 to day 50 of gestation, with a single oral dose on day 100 of gestation, at doses of 20, 50, 200, and 1000 mg/kg/day (n = 16 per group at the start of the study). Maternal and fetal blood were collected 5 hours after administration on day 100 of gestation. Fetal to maternal plasma concentration ratios were between 0.3 and 0.4 in all dose groups, indicating that apremilast can cross the monkey placenta. As part of a fertility and developmental toxicity study in female CD1 mice and an embryo-fetal development study in cynomolgus monkeys, the transplacental transport of apremilast was evaluated. In mice, apremilast was administered orally daily from day 15 before cohabitation until day 15 of the presumptive gestation at doses of 10, 20, 40, and 80 mg/kg/day. Blood samples were collected from pregnant mice (n=3 at each time point) at 0.5, 2, 4, 8, and 24 hours after administration on day 15 of gestation. Fetal blood samples were also collected from mice sacrificed 24 hours after administration. The increase in maternal plasma apromisin concentration was less than dose-proportional. Fetal plasma apromisin concentrations varied considerably at 24 hours; in 6 of the 10 litters evaluated, concentrations were below the limit of quantitation (1 ng/mL). Apromisin was detected in fetal plasma from 4 of the 10 litters evaluated, ranging from 14.5 to 2813 ng/mL. The average fetal-to-maternal plasma concentration ratio ranged from 0.3 to 1.07, indicating that apromisin can cross the mouse placenta.
For more complete data on the absorption, distribution, and excretion of apromisin (13 parameters), please visit the HSDB record page.

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Metabolism/Metabolites
Apramistrast is primarily metabolized through multiple pathways, including oxidation, hydrolysis, and conjugation. It has approximately 23 metabolites. CYP3A4 mainly mediates the oxidative metabolism of this drug, while CYP1A2 and CYP2A6 enzymes contribute less. The major metabolite of apramistrast, M12, is an inactive glucuronide conjugate of the O-demethylated drug. Other major metabolites, such as M14 and M16, exhibit significantly lower activity in inhibiting PDE4 and inflammatory mediators than their parent drug, apramistrast. After oral administration, the primary metabolites detected in plasma are parent apramistrast (45%) and the inactive metabolite O-demethylapramistrast glucuronide (39%). Minor metabolites M7 and M17 are active, but their concentrations account for only 2% or less of the total apramistrast concentration, and may contribute little to its effect. In healthy subjects, the clearance of apromiscalcium in plasma is approximately 10 L/hr, with a terminal elimination half-life of approximately 6–9 hours. Following oral administration of radiolabeled apromiscalcium, approximately 58% and 39% of the radioactive material are recovered in urine and feces, respectively, with approximately 3% and 7% of the radioactive dose recovered in the form of apromiscalcium in urine and feces, respectively. Following oral administration in humans, apromiscalcium is the major circulating component (45%), followed by the inactive metabolite M12 (39%), which is an O-demethylated glucuronide conjugate of apromiscalcium. Apromiscalcium is extensively metabolized in humans, with up to 23 metabolites identified in plasma, urine, and feces. The metabolic pathways of apromiscalcium include cytochrome P450 (CYP) oxidative metabolism (followed by glucuronidation) and non-CYP-mediated hydrolysis. In vitro studies have shown that CYP metabolism of apromiscalcium is primarily mediated by CYP3A4, with smaller contributions from CYP1A2 and CYP2A6. In an oral study, the concentrations of total radioactivity (e.g., the parent compound and its metabolites) and the parent compound in plasma were higher in female rats than in male rats. The total radioactivity AUC was 25 to 96 times higher in male rats than in female rats, while the difference was only 2 to 3 times, indicating that male rats metabolized the drug more extensively than female rats. In the same study, after six consecutive days of administration, female mice showed drug accumulation in Cmax and AUC, while no accumulation was observed in male mice. In a bile duct cannulation study of male mice, after a single oral administration of 10 mg/kg (14)C-apromiscalcium, 54% and 16% of the radioactive dose were excreted via bile and urine, respectively, indicating that at least 70% of the radioactive dose was absorbed by the mice, suggesting moderate first-pass metabolism of apromiscalcium. Molecular toxicokinetics showed that exposure increased with increasing dose, but the trend toward increased exposure was less proportional to the dose at doses exceeding 100 mg/kg/day. No sex differences or conversion to its R enantiomer were observed in mice. For more complete data on the metabolism/metabolites of apramistrast (6 metabolites), please visit the HSDB record page. Biological Half-Life: The mean elimination half-life of this drug is 6–9 hours. The terminal elimination half-life is approximately 6–9 hours.

Toxicity Summary
Identification and Use: Apramisartil is a white to pale yellow powder. Apramisartil is used to treat adults with active psoriatic arthritis. It is also used to treat patients with moderate to severe plaque psoriasis who are suitable for phototherapy or systemic therapy. Human Exposure and Toxicity: The most common adverse reactions are diarrhea, nausea, upper respiratory tract infection, and headache, including tension headache. Apramisartil did not induce chromosomal aberrations in cultured human peripheral blood lymphocytes with or without metabolic activation. Animal Studies: Apramisartil has low acute toxicity. Repeated-dose oral toxicity studies lasting up to 6 months in mice (dose levels of 10, 100, and 1000 mg/kg/day), up to 12 months in monkeys (dose levels of 60, 180, and 600 mg/kg/day), and up to 90 days in rats have evaluated apramisartil. Apramisartil-related deaths have been observed in mice and rats, primarily attributable to vascular and/or perivascular inflammation. Dose-related inflammatory responses were observed primarily in mice and rats, including neutropenia, lymphopenia, and changes in serum proteins (decreased albumin, increased globulins, haptoglobin, C-reactive protein (CRP), and/or fibrinogen). These inflammatory responses were associated with arteritis and perivascular inflammation in various tissues and organs (e.g., mesentery, heart, lung, thymus, liver, skeletal muscle, mammary gland, skin, and pancreas) in mice and rats, but were not observed in monkeys, even though the systemic exposure in monkeys was higher than that in mice and rats. Complete or partial reversibility of the inflammatory responses was observed in mice and rats. Other target organs for apramiscut toxicity included non-adverse centrilobular hepatocyte hypertrophy in the liver (mice) and varying degrees of lymphopenia in lymphoid tissues (mice and rats). Long-term studies of apramiscut in mice and rats have been conducted to assess its carcinogenic potential. In mice, no evidence of apromiscalcium-induced tumors was observed at oral doses up to 8.8 times the maximum recommended human dose (MRHD) (based on AUC, i.e., 1000 mg/kg/day). In rats, no evidence of apromiscalcium-induced tumors was observed at oral doses up to approximately 0.08 and 1.1 times the MRHD (20 mg/kg/day for males and 3 mg/kg/day for females, respectively). In a male mouse fertility study, oral doses of apromiscalcium at 1, 10, 25, and 50 mg/kg/day did not affect male fertility. In a combined study of female mouse fertility and embryo-fetal developmental toxicity, oral doses of apromiscalcium at 10, 20, 40, and 80 mg/kg/day showed altered estrous cycles and prolonged mating time starting from 20 mg/kg/day. However, all mice mated successfully, and pregnancy rates were unaffected. Apremilast did not induce mutations in the Ames assay. At doses up to 2000 mg/kg/day, apremilast did not show chromosome breakage induction in the in vivo mouse micronucleus assay.

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Interactions
Otezila has not been evaluated and is not recommended for use in combination with biologics used to treat psoriasis (such as TNF antagonists and anti-IL-12/23 p40 antibodies). Otezila is not recommended for use in combination with these biologics.
Otezila has not been evaluated and is not recommended for use in combination with potent immunosuppressants (such as cyclosporine and tacrolimus). Otezila is not recommended for use in combination with potent immunosuppressants.
When used in combination with the CYP3A4 inducer rifampin, the exposure (AUC) and maximum concentration (Cmax) of apremilast were reduced by 72% and 43%, respectively, which may lead to a decrease in the clinical efficacy of apremilast. Therefore, it is not recommended to use olerazine in combination with rifampin or other CYP3A4 inducers (such as phenobarbital, carbamazepine, phenytoin sodium). St. John's wort is a CYP3A4 inducer, and its combination with olerazine may lead to reduced efficacy or weakened clinical response; therefore, such combination is not recommended.

[1]. Apremilast, a novel phosphodiesterase 4 (PDE4) inhibitor, regulates inflammation through multiple cAMP downstream effectors. Arthritis Res Ther. 2015 Sep 15;17:249.

[2]. Determination of Apremilast in Rat Plasma by UPLC-MS-MS and Its Application to a Pharmacokinetic Study. J Chromatogr Sci. 2016 Sep;54(8):1336-40.

Therapeutic Uses

Nonsteroidal anti-inflammatory drug; Phosphodiesterase inhibitor
Otezla is indicated for the treatment of adult patients with active psoriatic arthritis. /US product label includes/
Otezla is indicated for the treatment of patients with moderate to severe plaque psoriasis who are eligible for phototherapy or systemic therapy. /US product label includes/
Exploratory Treatment/This study aims to/evaluate the efficacy and safety of oral phosphodiesterase 4 inhibitor apremilast for the treatment of ankylosing spondylitis (AS) through a preliminary study monitoring symptoms and signs. This study includes an exploratory investigation of the effects of PDE4 inhibition on bone biological blood biomarkers. In this double-blind, placebo-controlled, single-center phase II study, patients with symptoms of active ankylosing spondylitis (AS) as shown by MRI were randomized to receive apremilast 30 mg twice daily (BID) or placebo for 12 weeks. Bath index was continuously monitored during the study. Patients were followed up for 4 weeks after discontinuation of treatment. Bone biomarkers were assessed at baseline and day 85. A total of 38 participants were randomized, and 36 completed the study. Although the primary endpoint (change in BASDAI score at week 12) was not met, the apremilast group showed greater improvement from baseline on all clinical assessment endpoints compared to the placebo group, with mean changes in BASDAI (-1.59 ± 1.48 vs -0.77 ± 1.47), BASFI (-1.74 ± 1.91 vs -0.28 ± 1.61), and BASMI (-0.51 ± 1.02 vs -0.21 ± 0.67) being significantly greater than in the placebo group; however, these differences did not reach statistical significance. Four weeks after discontinuation of apremilast, all clinical endpoints returned to baseline. Six patients (35.3%) in the apremilast group achieved ASAS20 remission, compared to only three (15.8%) in the placebo group (p=0.25). Serum RANKL, RANKL/osteoplastin ratio, and plasma scleroprotein levels showed statistically significant decreases, but serum DKK-1, bone alkaline phosphatase, TRAP5b, MMP3, osteoprotegerin, or osteocalcin levels showed no significant changes. Although this is a small, preliminary study, these results suggest that apremilast may be effective and well-tolerated in ankylosing spondylitis and can modulate bone biomarkers. These data support further investigation into the role of apremilast in axial inflammation.
Exploring Treatment: Discoid lupus erythematosus (DLE) is a chronic inflammatory disease mediated by Th1 cells. Apremilast is a novel oral PDE4 enzyme inhibitor that blocks the production of IL-12, IL-23, TNF-α, and INF-12 by leukocytes, thereby inhibiting Th1 and Th17-mediated immune responses. It has been shown to have clinical efficacy in psoriasis, as well as rheumatoid arthritis and psoriatic arthritis. In 8 patients with active discoid lupus erythematosus, after 85 days of treatment with apromiscalcium 20 mg twice daily, the disease area and severity index (CLASI) of lupus cutaneum was significantly reduced (P<0.05). Drug-related adverse events were mild and transient. This is the first open-label study of apromiscalcium for the treatment of discoid lupus erythematosus. Our observations suggest that apromiscalcium may be a safe and effective treatment option for discoid lupus erythematosus.

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Drug Warnings
Use of Otezla is associated with an increased risk of adverse reactions related to depression. Prescribing physicians should carefully weigh the risks and benefits of Otezla treatment in patients with a history of depression and/or suicidal ideation or behavior before administering it. Patients, their caregivers, and families should be informed to be alert for the onset or exacerbation of depression, suicidal ideation, or other mood changes, and to contact their healthcare provider immediately if such changes occur. Prescribing physicians should carefully assess the risks and benefits of continuing Otezla treatment if such conditions occur.
The safety and efficacy of octazine in children under 18 years of age have not been established.
It is currently unknown whether octazine or its metabolites are present in human milk; however, apromiscarriage has been detected in the milk of lactating mice. Because many drugs are present in human milk, caution should be exercised when octazine is taken by breastfeeding women.
FDA Pregnancy Risk Category: C/Risk cannot be ruled out. There is a lack of adequate, well-controlled clinical studies, and animal studies have not shown any risk to the fetus or lack relevant data. Taking this drug during pregnancy may cause harm to the fetus; however, the potential benefits may outweigh the potential risks. /
For more complete data on drug warnings for apromiscarriage (11 in total), please visit the HSDB record page.

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Pharmacodynamics
Apramistrastem can reduce, but not completely inhibit, a variety of inflammatory cytokines, such as IL-1α, IL-6, IL-8, IL-10, MCP-1, MIP-1β, MMP-3, and TNF-α, thereby alleviating symptoms of psoriasis and Behcet's disease caused by increased levels of these inflammatory mediators. This drug has also been shown to effectively relieve pain caused by oral ulcers in Behcet's disease. Apramistrastem may cause weight loss and exacerbation of depressive symptoms, and may even trigger suicidal thoughts or behaviors. Monitoring for depressive symptoms is recommended, and patients should seek medical attention promptly if symptoms occur, especially those with a history of depression. The necessity of using apramistrastem and the risk of exacerbation of depression and suicide should be carefully evaluated. If weight loss occurs, the degree of weight loss should be assessed, and the need to discontinue apramistrastem should be considered.

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Introduction: This study aims to elucidate the intracellular signaling pathway of the PDE4 inhibitor apramistrastem and to explore the interaction between apramistrastem, methotrexate, and adenosine A2A receptor (A2AR). Methods: In the Raw264.7 mononuclear cell line, the levels of intracellular cAMP, TNF-α, IL-10, IL-6, and IL-1α were detected after incubation with apremilast and LPS. PKA, Epac1/2 (an intermediate in the cAMP signaling pathway), and A2AR were knocked down by shRNA transfection, and their interactions with A2AR, A2BR, and methotrexate were investigated in vitro and in a mouse air sac model. Statistical differences were determined using one-way or two-way ANOVA or Student's t-test. The nominal α level for all tests was set at 0.05. A p-value <0.05 was considered statistically significant. Results: In vitro experiments showed that apremilast increased intracellular cAMP levels and inhibited TNF-α release (IC50 = 104 nM), while the specific A2AR agonist CGS21680 (1 μM) enhanced the potency of apremilast (IC50 = 25 nM). In this cell line, apramisartine increased IL-10 production. Knockdown of PKA, Epac1, and Epac2 prevented apramisartine's inhibitory effect on TNF-α and its stimulatory effect on IL-10. In a mouse air sac model, both apramisartine and MTX significantly inhibited leukocyte infiltration, while apramisartine (but not MTX) significantly inhibited TNF-α release. Adding methotrexate (MTX, 1 mg/kg) to apramisartine (5 mg/kg) did not enhance its inhibitory effect on leukocyte infiltration or TNF-α release compared to apramisartine alone. Conclusion: The immunomodulatory effects of apramisartine appear to be mediated through cAMP and its downstream effector molecules PKA, Epac1, and Epac2. A2AR agonists enhanced the inhibitory effect of apramisartine on TNF-α, consistent with the cAMP-enhancing effect of this receptor. Since A2AR is also involved in the anti-inflammatory effect of MTX, the mechanisms of action of both drugs involve the cAMP-dependent pathway and are essentially partially overlapping. [1]

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We developed and validated a rapid, sensitive and selective ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS-MS) method for determining the concentration of apromiscal in rat plasma and for pharmacokinetic studies. Sample preparation was performed using a simple single-step deproteinization method, adding 0.2 mL of acetonitrile to 0.1 mL of plasma sample. Plasma samples were separated by ultra-high performance liquid chromatography (UPLC) using an Acquity UPLC BEH C18 column and a mobile phase of acetonitrile-0.1% formic acid aqueous solution, with gradient elution. The total run time was 3.0 min, and the elution time for apromiscal was 1.27 min. The detection was performed using a triple quadrupole tandem mass spectrometer in multiple reaction monitoring (MRM) mode. The ion pairs for apromisci were m/z 461.3 → 257.1, and for carbamazepine (internal standard) were m/z 237.2 → 194.2. The calibration curves showed a linear relationship in the range of 0.1–100 ng/mL, with a limit of quantitation of 0.1 ng/mL. The average recovery rate of apromisci in plasma was 83.2%–87.5%. The intra-day and inter-day precision were both less than 9.6%. This method has been successfully applied to the pharmacokinetic study of rats after oral administration of 6.0 mg/kg apromisci. [2]

C22H24N2O7S

460.500164985657

460.13

253168-86-4

Apremilast-d5;1258597-47-5; (R)-Apremilast; 608141-44-2;(Rac)-Apremilast-d5; 1258597-61-3; 253168-86-4

10151715

Typically exists as solid at room temperature

3.525

1

7

8

32

825

0

S(C)(CC(C1C=CC(=C(C=1)OCC)OC)N1C(C2C=CC=C(C=2C1=O)NC(C)=O)=O)(=O)=O

IMOZEMNVLZVGJZ-UHFFFAOYSA-N

1S/C22H24N2O7S/c1-5-31-19-11-14(9-10-18(19)30-3)17(12-32(4,28)29)24-21(26)15-7-6-8-16(23-13(2)25)20(15)22(24)27/h6-11,17H,5,12H2,1-4H3,(H,23,25)

Acetamide, N-(2-(1-(3-ethoxy-4-methoxyphenyl)-2-(methylsulfonyl)ethyl)-2,3-dihydro-1,3-dioxo-1H-isoindol-4-yl)-

CC-10004 CC 10004 CC10004 Apremilast (+/-)-,

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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