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| 1mg |
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| Other Sizes |
| Targets |
Pitavastatin-d5 sodium, like its non-deuterated parent, targets the enzyme HMG-CoA reductase, which is the rate-limiting enzyme in the cholesterol biosynthesis pathway. By inhibiting HMG-CoA reductase, Pitavastatin reduces the conversion of HMG-CoA to mevalonate, thereby decreasing de novo cholesterol synthesis in the liver. This leads to upregulation of the low-density lipoprotein (LDL) receptor on hepatocytes, resulting in increased clearance of LDL-cholesterol (LDL-C) from the blood. The deuterium labeling does not alter the mechanism of action but is used for analytical purposes.
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| ln Vitro |
Drug compounds have included stable heavy isotopes of carbon, hydrogen, and other elements, mostly as tracers for quantification throughout the drug development process. Due to its potential to alter the pharmacokinetic and metabolic characteristics of medications, deuteration has drawn attention[1].
In vitro, the deuterated compound exhibits the same activity as the parent compound, Pitavastatin sodium, due to the identical mechanism of action. Pitavastatin inhibits cholesterol synthesis from acetate in HepG2 cells with an IC50 of 5.8 nM. It is also an efficient inducer of the hepatocyte low-density lipoprotein-cholesterol (LDL-C) receptor and exhibits anti-cancer activity in research models. The presence of deuterium at specific positions does not significantly alter the in vitro potency but may affect metabolic stability if the labeled sites are involved in metabolism. |
| ln Vivo |
In vivo, Pitavastatin-d5 sodium is not typically administered for therapeutic efficacy; it is used as an internal standard for bioanalysis. However, the parent compound, Pitavastatin sodium, is an approved drug for the treatment of hyperlipidemia and dyslipidemia. It is known for its long half-life, minimal CYP metabolism, and low potential for drug-drug interactions. The in vivo efficacy of Pitavastatin includes significant reductions in LDL-C, total cholesterol, and triglycerides. The deuterated version behaves identically in vivo from a pharmacological standpoint but is used to differentiate endogenous or administered Pitavastatin from the labeled version in LC-MS/MS assays.
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| Enzyme Assay |
A cell-free enzyme inhibition assay is performed to assess potency. Recombinant human HMG-CoA reductase (the catalytic domain, 1-4 mU) is incubated in assay buffer (100 mM KH2PO4, 0.5 mM EDTA, 0.25 mM DTT, 0.1% BSA, pH 7.4) with varying concentrations (0.01-1000 nM) of Pitavastatin-d5 sodium (as an internal standard in a calibration curve) or unlabeled Pitavastatin sodium. The reaction is initiated by adding 50 microM NADPH and 100 microM HMG-CoA. After 30 minutes at 37degC, the reaction is stopped with 6% trichloroacetic acid. The amount of CoA-SH generated is measured using a fluorometric reagent (e.g., ThioGlo-1, ex/em = 380/505 nm). The IC50 is calculated. Note that the deuterated version is almost exclusively used for quantification by mass spectrometry, not for direct enzyme inhibition assays. To use Pitavastatin-d5 sodium as an internal standard for LC-MS/MS, a stock solution is prepared in methanol or acetonitrile. A known concentration of the d5 compound is added to the biological sample (e.g., plasma, urine) at a fixed concentration (e.g., 50-100 ng/mL). The sample is processed by protein precipitation (e.g., 3x volume of acetonitrile), vortexed, and centrifuged. The supernatant is injected into the LC-MS/MS system. The ratio of the peak area of the analyte (Pitavastatin) to the internal standard (Pitavastatin-d5) is used to calculate the concentration of the analyte in the sample. Standard curves are prepared using blank plasma spiked with known concentrations of unlabeled Pitavastatin and a fixed concentration of the d5 internal standard. LC-MS/MS is performed with a C18 column and a mobile phase of water/acetonitrile with 0.1% formic acid. Detection is performed in positive ion mode (ESI+), monitoring m/z 422.0 → 290.0 for Pitavastatin and m/z 427.0 → 290.0 for Pitavastatin-d5 (or appropriate transitions).
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| Cell Assay |
For a cellular activity assay, HepG2 cells are seeded in 24-well plates (2×10⁵ cells/well) in DMEM + 10% FBS. After 48 h, the medium is replaced with fresh medium containing Pitavastatin-d5 sodium (or non-labeled Pitavastatin as a control) at concentrations of 0.1-100 nM. Cells are incubated for 16-24 h. For cholesterol synthesis measurement, [14C]-acetate (1 microCi/mL) is added during the final 4 h of incubation. Cells are harvested, lipids are extracted with chloroform/methanol (2:1), and the organic phase is collected and dried. The radioactivity incorporated into cholesterol is measured by liquid scintillation counting. The IC50 for inhibition of cholesterol synthesis is calculated. For LDL receptor induction, cells are treated with Pitavastatin (0.1-100 nM) for 16 h. LDL receptor expression is measured by Western blot (anti-LDLR antibody) or by a fluorescent LDL uptake assay using Dil-LDL. The experiment can be repeated 3 times for statistical analysis.
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| ADME/Pharmacokinetics |
Pitavastatin-d5 sodium is used as an internal standard for PK studies. The following describes the PK study design: Male Sprague-Dawley rats (200-300 g, n=5 per time point) are administered a single oral (p.o., 0.5-1 mg/kg in 0.5% methylcellulose) or intravenous (i.v., 0.1-0.5 mg/kg in saline) dose of Pitavastatin sodium. Blood samples (200-300 uL) are collected via the jugular vein at pre-dose and at 0.083, 0.25, 0.5, 1, 2, 4, 6, 8, 12, and 24 h post-dose. Plasma is separated by centrifugation. A fixed concentration of Pitavastatin-d5 sodium (e.g., 50 ng/mL) is added to each plasma sample as an internal standard. Samples are processed and analyzed by LC-MS/MS. Plasma concentration-time data are analyzed using non-compartmental methods (e.g., Phoenix WinNonlin) to calculate Cmax, Tmax, AUC(0-t), AUC(0-∞), t½, CL, Vd, and bioavailability F (%). In clinical PK studies, a similar protocol is used for human subjects.
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| Toxicity/Toxicokinetics |
Pitavastatin is generally well-tolerated in both preclinical and clinical settings. Common adverse effects of statins include myalgia (muscle pain) and elevated liver transaminases, though Pitavastatin has a lower incidence of drug-drug interactions due to its minimal CYP metabolism (primarily via CYP2C9, with minimal involvement of CYP3A4). The deuterated version is considered to have the same safety profile as the parent drug. No specific toxicity data for the d5 labeled version are available, as it is used only as an analytical standard in trace amounts.
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| References |
[1]. Russak EM, et al. Impact of Deuterium Substitution on the Pharmacokinetics of Pharmaceuticals. Ann Pharmacother. 2019;53(2):211-216. ;Morikawa S, et al. Relative induction of mRNA for HMG CoA reductase and LDL receptor by five different HMG-CoA reductase
[2]. Morikawa S, et al. Relative induction of mRNA for HMG CoA reductase and LDL receptor by five different HMG-CoA reductase inhibitors in cultured human cells. J Atheroscler Thromb. 2000;7(3):138-44. [3]. Russak EM, et al. Impact of Deuterium Substitution on the Pharmacokinetics of Pharmaceuticals. Ann Pharmacother. 2019 Feb;53(2):211-216. |
| Additional Infomation |
Pitavastatin-d5 sodium is a stable isotope-labeled compound used as an internal standard for LC-MS/MS quantification of Pitavastatin in biological samples. The parent compound, Pitavastatin (NK-104), is a potent HMG-CoA reductase inhibitor. The deuterated version is not intended for therapeutic use; it is strictly an analytical tool for research and development. The product can be used for analytical method development, validation (AMV), and quality control applications. Store as a solid at -20degC, tightly closed and protected from light and moisture.
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| Molecular Formula |
C25H18D5FNNAO4
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| Related CAS # |
Pitavastatin;147511-69-1
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| Appearance |
Typically exists as solid at room temperature
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
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
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| Solubility (In Vivo) |
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.
Injection Formulations
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *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). View More
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)] Oral Formulations
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). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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