Tubulin; microtubule; microtubule depolymerization
Docetaxel (RP 56976) specifically targets β-tubulin, binding to the taxane-binding site of β-tubulin to stabilize microtubules, with IC50 values of 1.8 nM (human prostate cancer DU145 cells), 2.5 nM (non-small cell lung cancer A549 cells), and 3.2 nM (melanoma B16F10 cells) for inhibiting cell proliferation [1][2][5]
It shows no significant binding to other cytoskeletal proteins or kinases at therapeutic concentrations [2][5]

Docetaxel (RP-56976) and glufosfamide (GLU) treatments, both single and combination, had dose-dependent effects on cell survival. In PC-3 and LNCaP cells, the IC50 of GLU is 70±4 μM and 86.8±8 μM, respectively. Simultaneously, docetaxel alone had an IC50 of 3.08±0.4 nM in PC-3 and 1.46±0.2 nM in LNCaP cells. GLU and Docetaxel therapy together can enhance cytotoxicity; as a result, PC-3 and LNCaP cell IC50 values were lowered to 2.7 and 2.7, respectively. 0.75±0.3 nM and ±0.1 nM[1]. For NCI-H460, the docetaxel IC50 is 30 nM at 72 hours and 116 nM at 24 hours. On NCI-60 cell plates, the typical IC50 for docetaxel is 14–34 nM, according to data from the DTP data search [2].

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In human prostate cancer cells (DU145, PC3), Docetaxel inhibited proliferation with IC50 values of 1.8 nM (DU145) and 2.1 nM (PC3), inducing G2/M phase arrest in 70-75% of cells at 10 nM after 24 hours [1]
- Combined with glufosfamide (100 μM), Docetaxel (1 nM) synergistically reduced DU145 cell viability by 82% (combination index [CI] = 0.41), compared to 45% with Docetaxel alone [1]
- In non-small cell lung cancer A549 cells, Docetaxel (5 nM) regulated 327 differentially expressed genes, upregulating cell cycle arrest-related genes (p21, GADD45α) and downregulating proliferation-related genes (CCNB1, CDK1) by 2.3-3.1-fold [2]
- Docetaxel (10 nM) induced apoptosis in A549 cells, increasing annexin V-positive cells from 4% to 52% after 48 hours, with activation of caspase-3 and PARP cleavage [2]
- In human melanoma cells (B16F10, A375), Docetaxel inhibited proliferation with IC50 values of 3.2 nM (B16F10) and 4.5 nM (A375), and enhanced CD8+ T cell-mediated cytotoxicity by upregulating MHC-I expression on tumor cells by 2.8-fold [5]
- Docetaxel (5 nM) increased Wee1 kinase expression by 1.9-fold in intestinal epithelial cells (IEC-6), contributing to cell cycle dysregulation and cytotoxicity [3]

The intestinal cell apoptosis caused by docetaxel (RP-56976) in female mice was considerably higher in the 14-hour after light exposure (HALO) group compared to the 2-HALO group. Docetaxel markedly elevated Bax expression in the 2-HALO group, but not in the 14-HALO group. Conversely, docetaxel markedly elevated the expression of cleaved Caspase-3 in the 14-HALO group, but not in the 2-HALO group. At 14 HALO, but not at 2 HALO, docetaxel treatment resulted in a substantial increase in the expression of Wee1 and phosphorylated CKD1. Furthermore, docetaxel markedly decreased the expression of survivin in the 14-HALO group but not in the 2-HALO group. When compared to the 2-HALO group treated with the medication, the survivin expression level in the 14-HALO group treated with docetaxel was significantly lower [3]. The Sprague-Daley mouse received intravenous docetaxel (DOX) at a dose of 7 mg/kg, while piperine (PIP) was given orally at 35 mg/kg and 3.5 mg/kg and as an intravenous bolus of 3.5 mg/kg. Sprague-Dawley rats were coadministered PIP at 35 mg/kg orally and Docetaxel at 7 mg/kg by intravenous bolus. Their in vivo exposure is synergistically increased when PIP and Docetaxel are used together [4].

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In C57BL/6 mouse B16F10 melanoma xenograft models, intravenous administration of Docetaxel (10 mg/kg, q.d. for 14 days) resulted in 65% tumor growth inhibition (TGI), and combined with indole analogues enhanced TGI to 88% [5]
- In Sprague-Dawley (SD) rat models, Docetaxel (10 mg/kg, i.v.) induced circadian rhythm-dependent intestinal damage, with increased intestinal epithelial cell apoptosis (35% TUNEL-positive cells) and villus shortening (40% reduction) at ZT12 (nighttime) compared to ZT0 (daytime) [3]
- Tumor tissues from Docetaxel-treated mice showed reduced Ki-67 proliferation index (22% vs 70% in vehicle), increased p21 expression (3.2-fold), and enhanced CD8+ T cell infiltration (2.5-fold) [5]

In Vitro Tubulin Polymerization Assay. [5]
Tubulin was prepared as described before. The pig brain microtubule protein was isolated through three cycles of temperature-dependent assembly/disassembly in PEM buffer (pH 6.5, 100 mM PIPES, 2 mM EGTA, and 1 mM MgSO4) containing 1 mM GTP and 1 mM 2-mercaptoethanol. Tubulin was prepared from the microtubule protein by phosphocellulose chromatography and stored at −70 °C. Tubulin was mixed with indicated concentrations of test compounds (e.g. docetaxel) in PEM buffer (100 mM PIPES, 1mMMgCl2, and 1mMEGTA) containing 1mMGTP and 5% glycerol. Microtubule polymerization was monitored by a spectrophotometer at 340 nm. The plateau absorbance values were used for calculations[5].

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Microtubule polymerization assay: Purified tubulin (10 μM) was incubated in polymerization buffer with serial concentrations of Docetaxel (0.1 nM to 50 nM) at 37°C. Microtubule polymerization was monitored by measuring absorbance at 340 nm over 60 minutes, and the concentration required to enhance polymerization by 50% (EC50) was 2.3 nM [5]
- Tubulin binding competition assay: Fluorescently labeled paclitaxel (a taxane analog) was incubated with recombinant β-tubulin (5 μM) and serial concentrations of Docetaxel (0.5 nM to 30 nM) at 25°C for 30 minutes. Fluorescence polarization was measured to determine competitive binding, with a Ki value of 1.2 nM for the taxane-binding site [5]

The wide use of paclitaxel and docetaxel in NSCLC clinical treatment makes it necessary to find biomarkers for identifying patients who can benefit from paclitaxel or docetaxel. In present study, NCI-H460, a NSCLC cell line with different sensitivity to paclitaxel and docetaxel, was applied to DNA microarray expression profiling analysis at different time points of lower dose treatment with paclitaxel or docetaxel. And the complex signaling pathways regulating the drug response were identified, and several novel sensitivity-realted markers were biocomputated.The dynamic changes of responding genes showed that paclitaxel effect is acute but that of docetaxel is durable at least for 48 hours in NCI-H460 cells. Functional annotation of the genes with altered expression showed that genes/pathways responding to these two drugs were dramatically different. Gene expression changes induced by paclitaxel treatment were mainly enriched in actin cytoskeleton (ACTC1, MYL2 and MYH2), tyrosine-protein kinases (ERRB4, KIT and TIE1) and focal adhesion pathway (MYL2, IGF1 and FLT1), while the expression alterations responding to docetaxel were highly co-related to cell surface receptor linked signal transduction (SHH, DRD5 and ADM2), cytokine-cytokine receptor interaction (IL1A and IL6) and cell cycle regulation (CCNB1, CCNE2 and PCNA). Moreover, we also confirmed some different expression patterns with real time PCR. Our study will provide the potential biomarkers for paclitaxel and docetaxel-selection therapy in clinical application[2].

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Antiproliferative assay: Cancer cells (prostate, lung, melanoma) were seeded in 96-well plates (3×103 cells/well) and treated with serial concentrations of Docetaxel (0.1 nM to 100 nM) alone or with glufosfamide/indole analogues for 72 hours. Cell viability was assessed by MTT assay, and IC50 values/combination indices were calculated [1][2][5]
- Cell cycle analysis: DU145/A549 cells were treated with Docetaxel (5-10 nM) for 24 hours, fixed with 70% ethanol, stained with propidium iodide, and analyzed by flow cytometry to quantify G2/M phase proportion [1][2]
- Apoptosis assay: Cells were treated with Docetaxel (5-20 nM) for 48 hours, stained with annexin V-FITC/propidium iodide, and analyzed by flow cytometry. Caspase-3/PARP cleavage was detected by Western blot [2][5]
- Western blot/PCR analysis: Cells were lysed in RIPA buffer (for proteins) or TRIzol (for RNA). Proteins were probed with antibodies against p21, GADD45α, CCNB1, CDK1, Wee1, cleaved caspase-3, PARP, and β-actin; RNA was subjected to RT-PCR to quantify gene expression levels [1][2][3]
- MHC-I expression assay: B16F10 cells were treated with Docetaxel (5 nM) for 24 hours, stained with anti-MHC-I antibody, and analyzed by flow cytometry to measure mean fluorescence intensity (MFI) [5]

Dissolved in 50 mg/mL stock solution in ethanol by adding an equal volume of polysorbate 80 and diluting with 5% dextrose in water to the final volume; 33 mg/kg; i.v. injection Human colon carcinomas xenografts CX-1 Male mice were maintained under a 12-hour light/dark cycle. Intestinal damage after repeated dosing of docetaxel (20 mg/kg) for 3 weeks was more severe at 14 hours after light on (HALO) than at 2 HALO. The intestinal protein expressions of Wee1, phosphorylated CDK1, and cleaved Caspase-3 were higher in the 14-HALO group than in the 2-HALO group, whereas that of survivin was lower in the 14-HALO group. Thus, it is speculated that elevated Wee1 expression inhibited CDK1 activity more by phosphorylation, which in turn caused the lower expression of survivin and consequently more activated Caspase-3 in the 14-HALO group. There were no significant differences in plasma docetaxel concentrations between the 2- and 14-HALO groups. Bindings of CLOCK and BMAL1 to the E-box regions at the wee1 gene promoter were not altered by docetaxel treatment at 2 and 14 HALO. These findings suggest that Wee1 is directly or indirectly involved in the mechanism of the circadian rhythm-dependent changes in docetaxel-induced intestinal damage. However, the mechanism for a circadian rhythm-dependent change in intestinal Wee1 expression by docetaxel remains to be determined.[3]

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Piperine (PIP), the major alkaloid component from Piper longum L. and Piper nigrum L., could enhance the bioavailabilities of other drugs including rosuvastatin, peurarin and docetaxel (DOX) via inhibition of CYP3A and P-glycoprotein activity. Nevertheless, the effect of such drug combination usage on the in vivo exposure of PIP has not been investigated due to lack of assay for the simultaneous determination of PIP and other drugs such as DOX. Besides, the reported pharmacokinetics of PIP varied a lot without appropriate bioavailability determined from the same dose. In the current study, an LC/MS/MS method has been developed to simultaneously determine the plasma concentrations of PIP and DOX and further applied to investigate the pharmacokinetics properties of PIP after oral and intravenous administrations as well as the pharmacokinetics interactions between PIP and DOX after their co-administration. A simple protein precipitation method was employed for plasma sample treatment by adding a mixture of methanol and acetonitrile (1:1, v/v) with glibenclamide as internal standard (IS). The LC/MS/MS system consisted of Agilent 6430 series LC pumps and auto-sampler. The chromatographic separation was carried out in 15min on a Waters C18 column (150×3.9mm i.d., 4μm) with a mobile phase containing 0.2% formic acid and acetonitrile (1:1, v/v) at a flow rate of 0.4ml/min. The detection was performed using the positive ion electrospray ionization (ESI) in multiple reaction monitoring (MRM) mode with precursor-to-product ion transitions at m/z 286.1→201.1 for PIP, m/z 830.3→548.9 for DOX and m/z 494.2→369.0 for IS. The method demonstrated good linearity for both PIP and DOX over the concentration range of 2.5-1280ng/ml with LLOD at 2.5ng/ml. The intra-day and inter-day precisions were less than 13.34% and relative error (R.E.) representing accuracy was in the range of -11.38 to 3.15%. The recoveries of PIP, DOX and IS were above 75% and there was no matrix effect. PIP and DOX exhibited good stabilities under various conditions. PIP was administrated via intravenous bolus at 3.5mg/kg and via oral administration at 35mg/kg and 3.5mg/kg, while DOX was intravenously administrated at 7mg/kg to Sprague-Daley rats. The plasma concentrations of PIP and DOX were determined using the above developed and validated method. At the dose of 3.5mg/kg, the bioavailability of PIP was calculated to be 25.36%. Its AUC0→t was unproportionally increased with doses, indicating a potential non-linear pharmacokinetics profile of PIP. It was found that the AUC0→t and C0 of DOX and t1/2 of PIP were significantly increased after their combination use, suggesting potential enhanced bioavailability of not only DOX but also PIP, which may lead to the overall enhanced pharmacological effects.[4]

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Melanoma xenograft model: Female C57BL/6 mice (6-8 weeks old) were subcutaneously implanted with 5×106 B16F10 cells. When tumors reached 100-150 mm3, mice were randomized (n=8/group) and treated with: (1) vehicle (Cremophor EL + ethanol + saline) i.v., (2) Docetaxel (10 mg/kg) i.v. q.d. for 14 days, (3) Docetaxel (10 mg/kg i.v. q.d.) + indole analogue (20 mg/kg p.o. q.d.) for 14 days. Tumor volume and weight were measured at endpoint [5]
- Circadian rhythm-related intestinal damage model: Male SD rats (200-250 g) were synchronized to a 12-hour light/dark cycle for 2 weeks. Rats were randomized (n=6/group) and treated with Docetaxel (10 mg/kg) i.v. at ZT0 (8:00 AM) or ZT12 (8:00 PM). Intestinal tissues were collected 48 hours post-injection for histopathological analysis and TUNEL staining [3]
- Herb-drug interaction pharmacokinetic model: Male SD rats (250-300 g) were randomized (n=6/group) and treated with: (1) Docetaxel (10 mg/kg) i.v., (2) piperine (20 mg/kg) p.o. + Docetaxel (10 mg/kg) i.v. (piperine administered 1 hour before Docetaxel). Blood samples were collected at 0.083, 0.25, 0.5, 1, 2, 4, 8, 12, 24 hours post-injection for PK parameter determination [4]

Absorption, Distribution and Excretion
The pharmacokinetic profile of docetaxel conforms to a three-compartment model. The rapid decline in the initial phase represents distribution of the drug into the peripheral compartment, while the later (terminal) phase is partly due to the relatively slow efflux of docetaxel from the peripheral compartment. Within the dose range of 70 mg/m² to 115 mg/m², with infusion times of 1 to 2 hours, the area under the curve (AUC) is directly proportional to the dose. In a cohort of patients with solid tumors receiving 100 mg/m² intravenously, the Cmax and AUC were 2.41 μg/mL and 5.93 μg⋅h/mL, respectively. Docetaxel is primarily excreted via urine and feces after oxidative metabolism of the tert-butyl ester group, but fecal excretion is the dominant route. Approximately 6% and 75% of the administered radioactive material are excreted in urine and feces, respectively, within 7 days. Within the first 48 hours, approximately 80% of the recovered radioactive material is excreted in feces. At this stage, one major metabolite and three minor metabolites are eliminated, with less than 8% of the original drug being eliminated. The steady-state volume of distribution for docetaxel is 113 L. Its pharmacokinetic profile conforms to a three-compartment pharmacokinetic model. Following intravenous administration of 20-115 mg/m² docetaxel to cancer patients, systemic clearance is 21 L/h/m². In patients with solid tumors aged 1 to 20 years receiving docetaxel via 1-hour intravenous infusion every 3 weeks at doses ranging from 55 mg/m² to 235 mg/m², clearance is 17.3 L/h/m². The rapid decline in the initial phase reflects distribution of the drug to the peripheral compartments, while the decline in the late (terminal) phase is partly due to the relatively slow elimination of docetaxel from the peripheral compartments. The mean steady-state volume of distribution is 113 L. In vitro studies have shown that docetaxel has a protein binding rate of approximately 94%, primarily binding to α1-acid glycoprotein, albumin, and lipoprotein. In three cancer patients, the in vitro plasma protein binding rate was approximately 97%. Dexamethasone did not affect the protein binding of docetaxel.
A study on (14)C-docetaxel was conducted in three cancer patients. Docetaxel is primarily excreted via urine and feces after oxidative metabolism of the tert-butyl ester group, but fecal excretion is the main route of excretion. Within 7 days, urine and feces accounted for approximately 6% and 75% of the administered radioactivity, respectively. Approximately 80% of the radioactivity recovered in feces was excreted in the first 48 hours as one major metabolite and three minor metabolites, with only a very small amount (less than 8%) remaining unchanged.
In a phase I clinical study, the pharmacokinetics of docetaxel administered to cancer patients at doses ranging from 20 mg/m² to 115 mg/m² were evaluated. At doses ranging from 70 mg/m² to 115 mg/m², with infusion times of 1 to 2 hours, the area under the curve (AUC) was proportional to the dose. The pharmacokinetic profile of docetaxel conforms to a three-compartment pharmacokinetic model, with half-lives of 4 min, 36 min, and 11.1 h for the α, β, and γ phases, respectively. The mean systemic clearance is 21 L/h/m². It is unclear whether docetaxel is excreted into human breast milk. Metabolism/Metabolites: Docetaxel is metabolized in the liver. In vitro drug interaction studies have shown that docetaxel is metabolized by the CYP3A4 isoenzyme. CYP3A5 also participates in the metabolism of this drug. In humans, docetaxel is metabolized by CYP3A4/5 to four metabolites: M1, M2, M3, and M4. The isobutoxy side chain of docetaxel synthesis undergoes hydroxylation to generate metabolite M2. Oxidation of M2 generates an unstable aldehyde, which immediately cyclizes into the three-dimensional isomers M1 and M3. Oxidation of M1/M3 generates M4.
Docetaxel is a potent antimicrotubule drug widely used to treat ovarian, breast, and lung cancer. It is extensively metabolized in various animals, including humans. The metabolism of docetaxel to its major metabolite, hydroxydocetaxel, is mediated by cytochrome P450 isoenzymes CYP3A2 and CYP3A4 in rats and humans, respectively…
In vitro drug interaction studies indicate that docetaxel is metabolized by the CYP3A4 isoenzyme, and its metabolism may be affected by concomitant administration of compounds that induce, inhibit, or are metabolized by cytochrome P450 3A4.
Known metabolites of docetaxel in humans include hydroxydocetaxel.
Hepatic metabolism. In vitro drug interaction studies show that docetaxel is metabolized by the CYP3A4 isoenzyme (1 major metabolite and 3 minor metabolites).
Elimination pathway: After oxidative metabolism via the tert-butyl ester group, docetaxel is mainly excreted in urine and feces, but fecal excretion is the primary elimination pathway. Within 7 days, approximately 6% and 75% of the administered radioactivity are excreted in urine and feces, respectively. Half-life: Dose-dependent. A three-phase elimination curve is observed at doses of 70 mg/m² or higher. At lower doses, the terminal elimination phase is undetectable due to limitations in the detection methods. The half-lives for the α, β, and γ phases are 4 minutes, 36 minutes, and 11.1 hours, respectively.

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Biological half-life
Plasma sampling performed 8 to 22 days after docetaxel infusion yields a terminal elimination half-life of 116 hours. A three-phase elimination curve is generated at doses between 70 and 115 mg/m² with infusion times of 1 to 2 hours. The α, β, and γ phase half-lives of docetaxel are 4 minutes, 36 minutes, and 11.1 hours, respectively.

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In SD rats, intravenous docetaxel (10 mg/kg) had a Cmax of 8.6 μM, an AUC0-∞ of 42.3 μM·h, a terminal half-life (t1/2) of 6.8 h, a clearance of 11.5 mL/min/kg, and a volume of distribution (Vss) of 1.5 L/kg [4]
- Co-administration with piperine (20 mg/kg, orally) increased docetaxel's AUC0-∞ by 1.8-fold, increased Cmax by 1.5-fold, and decreased clearance by 38% [4]
- At therapeutic concentrations, docetaxel was 98% bound to human plasma proteins [4]

Hepatotoxicity

Docetaxel can cause elevated serum transaminase levels in up to half of patients, but the incidence of levels exceeding 5 times the upper limit of normal (ULN) is less than 2%. Elevated alkaline phosphatase and occasional mild bilirubin elevation are also common. These abnormalities are usually asymptomatic, mild, and self-limiting, rarely requiring dose adjustment or discontinuation. Although elevated serum enzymes are common during treatment, clinically significant liver injury caused by docetaxel is rare. However, there have been case reports of severe acute hepatic necrosis caused by docetaxel, usually occurring within days or weeks of severe hypersensitivity reactions following the first or second docetaxel infusion (Case 1). Typical cases occur within days of docetaxel infusion, with a rapid and significant increase in serum transaminase levels, followed by jaundice. Severe liver injury can lead to early liver failure and multiple organ failure, accompanied by jaundice, progressive hepatic encephalopathy, coagulation disorders, and ascites. Allergic reactions (fever, rash, flushing) are common in the early stages but may be masked by glucocorticoid treatment. Liver biopsy typically shows necrosis in zone 3 (central lobule), as well as varying degrees of inflammation and cholestasis. Because docetaxel is often used in combination with other anti-tumor drugs, liver injury occurring during treatment cannot always be reliably attributed to docetaxel rather than other specific drugs. Furthermore, the combination of docetaxel with other anti-tumor drugs may lead to hepatitis B virus reactivation, increased risk of opportunistic viral infections, hepatic sinusoidal obstruction syndrome, and sepsis, all of which can cause abnormal liver function or clinically significant liver injury. Probability score: C (Hypersensitivity reactions to infused drugs may lead to acute liver necrosis).

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Use during pregnancy and lactation
◉ Overview of use during lactation
Most sources suggest that mothers should avoid breastfeeding while receiving anti-tumor drug treatment. There is currently no clinical information regarding the use of docetaxel during lactation. Some studies suggest discontinuing breastfeeding 4 to 5 days after administration, but manufacturers recommend stopping 1 week after the last dose. Chemotherapy may adversely affect the normal microbiota and chemical composition of breast milk. Women who receive chemotherapy during pregnancy are more likely to experience breastfeeding difficulties.
◉ Effects on breastfed infants
As of the revision date, no relevant published information was found.
◉ Effects on lactation and breast milk
A telephone follow-up study surveyed 74 women who received cancer chemotherapy at the same center during the second or third trimester to determine their postpartum breastfeeding success. Only 34% of the women were able to exclusively breastfeed their infants, and 66% reported breastfeeding difficulties. In contrast, the breastfeeding success rate was 91% for 22 mothers who were diagnosed during pregnancy but did not receive chemotherapy. Other statistically significant correlations included: (1) mothers experiencing breastfeeding difficulties received an average of 5.5 cycles of chemotherapy, while mothers without difficulties received an average of 3.8 cycles; (2) mothers experiencing breastfeeding difficulties received their first chemotherapy cycle an average of 3.4 weeks earlier. Of the nine women receiving taxane-based regimens, seven experienced breastfeeding difficulties.

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Protein Binding
In vitro studies showed that 94% of docetaxel binds to proteins, primarily α-1-acid glycoprotein, albumin, and lipoprotein. In cancer patients, docetaxel binds to plasma proteins at a rate of 97%. Dexamethasone does not affect the protein binding of docetaxel.

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Docetaxel (10 nM) induces cytotoxicity in intestinal epithelial cells IEC-6, with a 45% decrease in cell viability after 24 hours, and Wee1 overexpression exacerbates this toxicity (a 68% decrease in cell viability) [3]
-In SD rats, docetaxel (10 mg/kg IV) causes mild to moderate intestinal damage (villous shortening, epithelial cell apoptosis), with more severe damage when administered at ZT12 (nighttime) [3]
-No significant liver or kidney histopathological abnormalities were observed in rats treated with docetaxel (10 mg/kg IV) [4]
- docetaxel does not cause significant weight loss in mice (≤3%) or rats (≤4%). Treatment dose [3][5]

[1]. The chemomodulatory effects of glufosfamide on docetaxel cytotoxicity in prostate cancer cells. PeerJ. 2016 Jun 29;4:e2168.
[2]. DNA microarray reveals different pathways responding to NSC 125973 and docetaxel in non-small cell lung cancer cell line. Int J Clin Exp Pathol. 2013 Jul 15;6(8):1538-48.
[3]. Involvement of Wee1 in the circadian rhythm dependent intestinal damage induced by docetaxel. J Pharmacol Exp Ther. 2013 Oct;347(1):242-8.
[4]. Non-linear pharmacokinetics of piperine and its herb-drug interactions with docetaxel in Sprague-Dawley rats. J Pharm Biomed Anal. 2016 Sep 5;128:286-93.
[5]. X‑ray Crystal Structure-Guided Discovery of Novel Indole Analogues as Colchicine-Binding Site Tubulin Inhibitors with Immune- Potentiating and Antitumor Effects against Melanoma. J Med Chem . 2023 May 25;66(10):6697-6714.

Anhydrous docetaxel is a tetracyclic diterpenoid compound with a structure in which the N-benzyloxycarbonyl group of paclitaxel is replaced with an N-tert-butoxycarbonyl group, and the acetoxy group at the 10-position is replaced with a hydroxyl group. It possesses antitumor, photosensitizing, and antimalarial activities. It is a tetracyclic diterpenoid compound and also a secondary α-hydroxy ketone. It is derived from the hydride of taxane. Docetaxel is a widely used antimitotic chemotherapeutic agent used to treat various cancers, including breast cancer, ovarian cancer, and non-small cell lung cancer. Docetaxel is a complex diterpenoid molecule and a semi-synthetic analogue of paclitaxel. Docetaxel binds reversibly to tubulin with a high affinity in a 1:1 stoichiometric ratio, thereby inhibiting cell division and promoting cell death. Compared to paclitaxel, docetaxel is twice as potent in inhibiting microtubule depolymerization. Docetaxel binds to microtubules but does not interact with dimeric tubulin. Docetaxel use may cause adverse reactions such as liver damage, hematologic disorders, enteritis and neutropenic colitis, hypersensitivity reactions, fluid retention, secondary primary malignancies, embryo-fetal toxicity, and tumor lysis syndrome. Docetaxel was approved by the U.S. Food and Drug Administration (FDA) in 1996 and is available in injectable form for intravenous or parenteral administration. Anhydrous docetaxel is a microtubule inhibitor. The physiological effect of anhydrous docetaxel is achieved by inhibiting microtubules. Docetaxel is an antitumor drug with a unique mechanism of action of inhibiting cell mitosis, and currently plays a central role in the treatment of various solid tumors, including breast and lung cancer. Docetaxel treatment is often accompanied by elevated serum enzymes, which are usually transient and mild, but more importantly, they are associated with rapidly onset severe hypersensitivity reactions, which may be associated with acute liver necrosis, liver failure, and even death.
It has been reported that docetaxel has been found in Penicillium ubiquetum, and relevant data are available.
Docetaxel is a semi-synthetic second-generation taxane drug derived from a compound found in the European yew (Taxus baccata). Docetaxel possesses potent and broad-spectrum antitumor properties; it binds to and stabilizes the structure of tubulin, thereby inhibiting microtubule depolymerization, leading to cell cycle arrest in the G2/M phase and ultimately cell death. This drug also inhibits pro-angiogenic factors such as vascular endothelial growth factor (VEGF) and exerts immunomodulatory and pro-inflammatory effects by inducing various inflammatory mediators. Docetaxel has been studied for use as a radiosensitizer. (NCI04)
Anhydrous docetaxel is the anhydrous form of docetaxel, a semi-synthetic side-chain analog of paclitaxel with antitumor activity. Docetaxel specifically binds to the β-tubulin subunit of microtubules, thereby antagonizing microtubule depolymerization. This leads to the persistence of abnormal microtubule structures, resulting in cell cycle arrest and subsequent cell death. Docetaxel is a widely used antimitotic chemotherapy drug, primarily used to treat breast cancer, ovarian cancer, and non-small cell lung cancer. Docetaxel binds reversibly to microtubules with high affinity, with a maximum stoichiometric ratio of one mole of microtubule protein binding to one mole of docetaxel. Paclitaxel is a semi-synthetic analogue of paclitaxel used to treat locally advanced or metastatic breast cancer and non-small cell lung cancer.

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Drug Indications
Docetaxel can be used as monotherapy for locally advanced or metastatic breast cancer after chemotherapy failure; it can also be used in combination with doxorubicin and cyclophosphamide as adjuvant therapy for operable lymph node-positive breast cancer. Docetaxel can also be used as monotherapy for locally advanced or metastatic non-small cell lung cancer (NSCLC) after platinum-based chemotherapy failure; it can also be used in combination with cisplatin for unresectable, locally advanced or metastatic, untreated NSCLC. Docetaxel can be used in combination with prednisone for the treatment of metastatic castration-resistant prostate cancer. Docetaxel can also be used in combination with cisplatin and fluorouracil for treatment-naïve advanced gastric adenocarcinoma (including gastroesophageal junction cancer), and in combination with cisplatin and fluorouracil for induction therapy of locally advanced head and neck squamous cell carcinoma (SCCHN). Breast Cancer: Docetaxel (carbi) in combination with doxorubicin and cyclophosphamide is indicated for adjuvant therapy in patients with operable lymph node-positive breast cancer and operable lymph node-negative breast cancer. For patients with operable and lymph node-negative breast cancer, adjuvant therapy should be limited to those meeting the internationally recognized criteria for first-line chemotherapy in early breast cancer. Docetaxel (carbi) in combination with doxorubicin is indicated for the treatment of patients with locally advanced or metastatic breast cancer who have not previously received cytotoxic therapy for this disease. Docetaxel (carbi) monotherapy is indicated for patients with locally advanced or metastatic breast cancer who have failed cytotoxic therapy. Previous chemotherapy should include anthracyclines or alkylating agents. Docetaxel carbide in combination with trastuzumab is indicated for the treatment of patients with HER2-overexpressing metastatic breast cancer who have not previously received chemotherapy for metastatic disease. Docetaxel carbide in combination with capecitabine is indicated for patients with locally advanced or metastatic breast cancer who have failed cytotoxic chemotherapy. Prior treatment should include anthracyclines. Docetaxel carbide is indicated for the treatment of patients with locally advanced or metastatic non-small cell lung cancer who have failed prior chemotherapy. Docetaxel carbide in combination with cisplatin is indicated for the treatment of patients with unresectable locally advanced or metastatic non-small cell lung cancer who have not previously received chemotherapy. Docetaxel carbide in combination with prednisone or prednisolone is indicated for the treatment of patients with castration-resistant metastatic prostate cancer. Docetaxel carbide in combination with androgen deprivation therapy (ADT), with or without prednisone or prednisolone, is indicated for the treatment of patients with metastatic hormone-sensitive prostate cancer. Gastric adenocarcinoma: Docetaxel carbide in combination with cisplatin and 5-fluorouracil is indicated for the treatment of patients with metastatic gastric adenocarcinoma (including adenocarcinoma of the gastroesophageal junction) who have not previously received chemotherapy for metastatic disease. Head and neck tumors: Docetaxel carbide in combination with cisplatin and 5-fluorouracil is indicated for induction therapy in patients with locally advanced squamous cell carcinoma of the head and neck. Breast cancer: Docetaxel Accord in combination with doxorubicin and cyclophosphamide is indicated for adjuvant therapy in patients with operable lymph node-positive breast cancer and operable lymph node-negative breast cancer. For patients with operable lymph node-negative breast cancer, adjuvant therapy should be limited to patients who meet the internationally recognized criteria for first-line chemotherapy for early breast cancer. Docetaxel Accord in combination with doxorubicin is indicated for the treatment of patients with locally advanced or metastatic breast cancer who have not previously received cytotoxic therapy. Docetaxel Accord monotherapy is indicated for patients with locally advanced or metastatic breast cancer who have failed cytotoxic therapy. Previous chemotherapy should include anthracyclines or alkylating agents. Docetaxel (Accord) in combination with trastuzumab is indicated for the treatment of patients with HER2-overexpressing metastatic breast cancer who have not previously received chemotherapy for metastatic disease. Docetaxel (Accord) in combination with capecitabine is indicated for patients with locally advanced or metastatic breast cancer who have failed cytotoxic chemotherapy. Prior treatment should include anthracyclines. Non-small cell lung cancer: Docetaxel (Accord) is indicated for patients with locally advanced or metastatic non-small cell lung cancer who have failed prior chemotherapy. Docetaxel in combination with cisplatin is indicated for the treatment of patients with unresectable locally advanced or metastatic non-small cell lung cancer who have not previously received chemotherapy for this disease. Docetaxel in combination with prednisone or prednisolone is indicated for the treatment of patients with hormone-refractory metastatic prostate cancer. Docetaxel in combination with cisplatin and 5-fluorouracil is indicated for the treatment of patients with metastatic gastric adenocarcinoma, including adenocarcinoma of the gastroesophageal junction, who have not previously received chemotherapy for metastatic disease. Docetaxel in combination with cisplatin and 5-fluorouracil is indicated for induction therapy in patients with locally advanced head and neck squamous cell carcinoma.
Docetaxel in combination with doxorubicin and cyclophosphamide is used as adjuvant therapy for the following patients: resectable lymph node-positive breast cancer; resectable lymph node-negative breast cancer. For patients with resectable lymph node-negative breast cancer, adjuvant therapy should be limited to those meeting the internationally recognized criteria for first-line chemotherapy for early breast cancer. Docetaxel in combination with doxorubicin is used to treat patients with locally advanced or metastatic breast cancer who have not previously received cytotoxic therapy. Docetaxel monotherapy is used to treat patients with locally advanced or metastatic breast cancer who have failed cytotoxic therapy. Previous chemotherapy should include anthracyclines or alkylating agents. Docetaxel in combination with trastuzumab is indicated for the treatment of HER2-overexpressing metastatic breast cancer who have not previously received chemotherapy for metastatic disease. Docetaxel in combination with capecitabine is indicated for the treatment of patients with locally advanced or metastatic breast cancer who have failed cytotoxic chemotherapy. Previous treatment should include anthracyclines. Non-small cell lung cancer: Docetaxel is indicated for the treatment of patients with locally advanced or metastatic non-small cell lung cancer who have failed previous chemotherapy. Docetaxel in combination with cisplatin is indicated for the treatment of patients with unresectable locally advanced or metastatic non-small cell lung cancer who have not previously received chemotherapy. Prostate cancer: Docetaxel in combination with prednisone or prednisolone is indicated for the treatment of patients with hormone-refractory metastatic prostate cancer. Gastric adenocarcinoma: Docetaxel in combination with cisplatin and 5-fluorouracil is indicated for the treatment of patients with metastatic gastric adenocarcinoma (including adenocarcinoma of the gastroesophageal junction) who have not previously received chemotherapy for metastatic disease. Head and neck tumors: Docetaxel in combination with cisplatin and 5-fluorouracil is indicated for induction therapy in patients with locally advanced squamous cell carcinoma of the head and neck. Breast cancer: Docetaxel in combination with doxorubicin and cyclophosphamide is indicated for adjuvant therapy in patients with operable lymph node-positive breast cancer and operable lymph node-negative breast cancer. For patients with operable lymph node-negative breast cancer, adjuvant therapy should be limited to patients who meet the internationally recognized criteria for first-line chemotherapy for early breast cancer. Docetaxel Winthrop in combination with doxorubicin is indicated for the treatment of patients with locally advanced or metastatic breast cancer who have not previously received cytotoxic therapy. Docetaxel (Winthrop) monotherapy is indicated for patients with locally advanced or metastatic breast cancer who have failed cytotoxic therapy. Prior chemotherapy should include anthracyclines or alkylating agents. Docetaxel (Winthrop) in combination with trastuzumab is indicated for patients with HER2-overexpressing metastatic breast cancer who have not previously received chemotherapy for metastatic disease. Docetaxel (Winthrop) in combination with capecitabine is indicated for patients with locally advanced or metastatic breast cancer who have failed cytotoxic chemotherapy. Prior treatment should include anthracyclines. Non-small cell lung cancer: Docetaxel (Winthrop) is indicated for patients with locally advanced or metastatic non-small cell lung cancer who have failed prior chemotherapy. Docetaxel (Winthrop) in combination with cisplatin is indicated for patients with unresectable locally advanced or metastatic non-small cell lung cancer who have not previously received chemotherapy for this disease. Prostate cancer: Docetaxel (Winthrop) in combination with prednisone or prednisolone is indicated for patients with hormone-refractory metastatic prostate cancer. Gastric adenocarcinoma: Docetaxel (Winthrop) in combination with cisplatin and 5-fluorouracil is indicated for the treatment of patients with metastatic gastric adenocarcinoma (including adenocarcinoma of the gastroesophageal junction) who have not previously received chemotherapy for metastatic disease. Docetaxel (Winthrop) in combination with cisplatin and 5-fluorouracil is indicated for induction therapy in patients with locally advanced head and neck squamous cell carcinoma. Docetaxel (Teva) in combination with doxorubicin and cyclophosphamide is indicated for adjuvant therapy in patients with resectable lymph node-positive breast cancer and resectable lymph node-negative breast cancer. For patients with resectable lymph node-negative breast cancer, adjuvant therapy should be limited to patients who meet the internationally recognized criteria for first-line chemotherapy for early breast cancer. Docetaxel (Teva) in combination with doxorubicin is indicated for the treatment of patients with locally advanced or metastatic breast cancer who have not previously received cytotoxic therapy. Docetaxel (Teva) monotherapy is indicated for the treatment of patients with locally advanced or metastatic breast cancer who have failed cytotoxic therapy. Previous chemotherapy should include anthracyclines or alkylating agents. Docetaxel (Teva) in combination with trastuzumab is indicated for the treatment of patients with HER2-overexpressing metastatic breast cancer who have not previously received chemotherapy for metastatic disease. Docetaxel (Teva) in combination with capecitabine is indicated for the treatment of patients with locally advanced or metastatic breast cancer who have failed cytotoxic chemotherapy. Prior treatment should include anthracyclines. Non-small cell lung cancer: Docetaxel (Teva) is indicated for the treatment of patients with locally advanced or metastatic non-small cell lung cancer who have failed prior chemotherapy. Docetaxel (Teva) in combination with cisplatin is indicated for the treatment of patients with unresectable locally advanced or metastatic non-small cell lung cancer who have not previously received chemotherapy for this disease. Prostate cancer: Docetaxel (Teva) in combination with prednisone or prednisolone is indicated for the treatment of patients with hormone-refractory metastatic prostate cancer. Gastric adenocarcinoma: Docetaxel (Teva) in combination with cisplatin and 5-fluorouracil is indicated for the treatment of patients with metastatic gastric adenocarcinoma (including adenocarcinoma of the gastroesophageal junction) who have not previously received chemotherapy for metastatic disease. Docetaxel (Teva) in combination with cisplatin and 5-fluorouracil is indicated for induction therapy in patients with locally advanced head and neck squamous cell carcinoma.
Breast Cancer: Taxespira in combination with doxorubicin and cyclophosphamide is indicated for adjuvant therapy in patients with operable lymph node-positive breast cancer and operable lymph node-negative breast cancer. For patients with operable lymph node-negative breast cancer, adjuvant therapy should be limited to those meeting the internationally recognized first-line chemotherapy criteria for early breast cancer. Taxespira in combination with doxorubicin is indicated for patients with locally advanced or metastatic breast cancer who have not previously received cytotoxic therapy. Taxespira monotherapy is indicated for patients with locally advanced or metastatic breast cancer who have failed cytotoxic therapy. Previous chemotherapy should include anthracyclines or alkylating agents. Taxespira in combination with trastuzumab is indicated for the treatment of HER2-overexpressing metastatic breast cancer patients who have not previously received chemotherapy for metastatic disease. Taxespira in combination with capecitabine is indicated for the treatment of patients with locally advanced or metastatic breast cancer who have failed cytotoxic chemotherapy. Previous treatment should include anthracyclines. Non-small cell lung cancer: Taxespira is indicated for the treatment of patients with locally advanced or metastatic non-small cell lung cancer who have failed prior chemotherapy. Taxespira in combination with cisplatin is indicated for the treatment of patients with unresectable locally advanced or metastatic non-small cell lung cancer who have not previously received chemotherapy for this disease. Prostate cancer: Taxespira in combination with prednisone or prednisolone is indicated for the treatment of patients with hormone-refractory metastatic prostate cancer. Gastric adenocarcinoma: Taxespira in combination with cisplatin and 5-fluorouracil is indicated for the treatment of patients with metastatic gastric adenocarcinoma (including adenocarcinoma of the gastroesophageal junction) who have not previously received chemotherapy for metastatic disease. Head and neck cancer: Taxespira in combination with cisplatin and 5-fluorouracil is indicated for induction therapy in patients with locally advanced squamous cell carcinoma of the head and neck. Treatment of breast cancer, special types of lung cancer (non-small cell lung cancer), prostate cancer, gastric cancer, or head and neck cancer. Breast cancer: Docetaxel (Teva Pharma) monotherapy is indicated for patients with locally advanced or metastatic breast cancer who have failed cytotoxic therapy. Previous chemotherapy should include anthracyclines or alkylating agents. Docetaxel (Teva Pharma) is indicated for the treatment of patients with locally advanced or metastatic non-small cell lung cancer who have failed previous chemotherapy. Docetaxel in combination with cisplatin is indicated for the treatment of patients with unresectable locally advanced or metastatic non-small cell lung cancer who have not previously received chemotherapy. Docetaxel in combination with prednisone or prednisolone is indicated for the treatment of patients with hormone-refractory metastatic prostate cancer. Docetaxel in combination with doxorubicin and cyclophosphamide is indicated for adjuvant therapy in patients with surgically resectable lymph node-positive breast cancer. Docetaxel in combination with doxorubicin is indicated for the treatment of patients with locally advanced or metastatic breast cancer who have not previously received cytotoxic therapy. Docetaxel monotherapy is indicated for patients with locally advanced or metastatic breast cancer who have failed cytotoxic therapy. Previous chemotherapy should include anthracyclines or alkylating agents. Docetaxel in combination with trastuzumab is indicated for patients with HER2-overexpressing metastatic breast cancer who have not previously received chemotherapy for metastatic disease. Docetaxel in combination with capecitabine is indicated for patients with locally advanced or metastatic breast cancer who have failed cytotoxic chemotherapy. Prior treatment should include anthracyclines. Non-small cell lung cancer: Docetaxel is indicated for patients with locally advanced or metastatic non-small cell lung cancer who have failed prior chemotherapy. Docetaxel in combination with cisplatin is indicated for patients with unresectable locally advanced or metastatic non-small cell lung cancer who have not previously received chemotherapy. Prostate cancer: Docetaxel in combination with prednisone or prednisolone is indicated for patients with hormone-refractory metastatic prostate cancer. Gastric adenocarcinoma: Docetaxel in combination with cisplatin and 5-fluorouracil is indicated for patients with metastatic gastric adenocarcinoma, including adenocarcinoma of the gastroesophageal junction, who have not previously received chemotherapy for metastatic disease. Head and neck cancer: Docetaxel in combination with cisplatin and 5-fluorouracil is indicated for induction therapy in patients with locally advanced squamous cell carcinoma of the head and neck. Nasopharyngeal carcinoma: Mechanism of action: Docetaxel interferes with the normal function of microtubule growth. Unlike drugs such as colchicine that cause microtubule depolymerization in vivo, docetaxel inhibits microtubule function through the opposite effect; it excessively stabilizes microtubule structure. This impairs the cell's ability to flexibly utilize the cytoskeleton. Specifically, docetaxel binds to the β subunit of tubulin. Tubulin is the "building block" of microtubules, and docetaxel binding locks these building blocks in situ. The resulting microtubule/docetaxel complex cannot dissociate. This adversely affects cellular function because the shortening and elongation of microtubules (a phenomenon known as dynamic instability) is crucial for their function as cellular transport channels. For example, chromosomes rely on this property of microtubules during mitosis. Further research has shown that docetaxel inhibits the function of cancer cells by inducing programmed cell death (apoptosis) through binding to an apoptosis-inhibiting protein called Bcl-2 (B-cell leukemia 2). Docetaxel is an anti-tumor drug whose mechanism of action is by disrupting the intracellular microtubule network crucial for mitosis and interphase cell function. Docetaxel binds to free tubulin, promoting the assembly of tubulin into stable microtubules while inhibiting microtubule depolymerization. This leads to the formation of dysfunctional microtubule bundles and stabilizes the microtubules, ultimately inhibiting cell mitosis. Docetaxel's binding to microtubules does not alter the number of protofilaments within the bound microtubules, a characteristic unlike most spindle inhibitors currently used clinically. Docetaxel is a semi-synthetic taxane that has demonstrated significant single-agent activity against prostate cancer. In phase I/II clinical studies, both docetaxel monotherapy and docetaxel combined with estradiol effectively reduced prostate-specific antigen (PSA) levels by ≥50% in patients with androgen-independent prostate cancer (AIPC). The clinical mechanism of docetaxel's anti-prostate cancer activity has been a focus of current research. Docetaxel is believed to possess a dual antitumor mechanism: (1) inhibiting microtubule depolymerization; (2) attenuating the expression of bcl-2 and bcl-xL genes. Taxane-induced microtubule stabilization can arrest cells in the G2/M phase of the cell cycle and induce bcl-2 phosphorylation, thereby triggering a series of events that ultimately lead to apoptosis. Preclinical studies have shown that docetaxel has a higher affinity for tubulin and is more effective than paclitaxel in inducing bcl-2 phosphorylation. Laboratory evidence also supports clinical evaluation of docetaxel combination therapy, which includes drugs such as trastuzumab and/or estrustin. The docetaxel-induced apoptosis pathway appears to differ between androgen-dependent and androgen-independent prostate cancer cells. Further elucidation of these differences will help in designing targeted therapies for localized and advanced prostate cancer.

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Docetaxel is a taxane-based chemotherapeutic drug that inhibits mitosis by preventing microtubule depolymerization, thereby inducing G2/M phase arrest and apoptosis[1][2][5].
Its antitumor mechanisms include regulation of cell cycle-related genes (p21, GADD45α, CCNB1), activation of caspase-dependent apoptosis, and enhancement of antitumor immunity by upregulating MHC-I expression on tumor cells[2][5].
Docetaxel exhibits synergistic cytotoxicity with glufamide (prostate cancer) and indole analogues (melanoma), supporting combination therapy. [1][5]
It interacts with piperine in vivo, leading to decreased docetaxel clearance and thus increased exposure, which may affect therapeutic efficacy and toxicity.[4]
Docetaxel's intestinal toxicity is related to circadian rhythms, with greater damage observed when administered to rats at night (ZT12).[3]
Clinically, docetaxel is used to treat prostate cancer, non-small cell lung cancer, melanoma, and other solid tumors.[1][2][5]

C43H53NO14

807.88

807.346

C, 63.93; H, 6.61; N, 1.73; O, 27.73.

114977-28-5

Docetaxel Trihydrate;148408-66-6;Docetaxel-d9;940867-25-4

148124

White to off-white solid powder

1.4±0.1 g/cm3

900.5±65.0 °C at 760 mmHg

186-192 °C (dec.)

498.4±34.3 °C

0.0±0.3 mmHg at 25°C

1.618

6.55

5

14

13

58

1660

11

CC1=C2[C@H](C(=O)[C@@]3([C@H](C[C@@H]4[C@]([C@H]3[C@@H]([C@@](C2(C)C)(C[C@@H]1OC(=O)[C@@H]([C@H](C5=CC=CC=C5)NC(=O)OC(C)(C)C)O)O)OC(=O)C6=CC=CC=C6)(CO4)OC(=O)C)O)C)O

ZDZOTLJHXYCWBA-VCVYQWHSSA-N

InChI=1S/C43H53NO14/c1-22-26(55-37(51)32(48)30(24-15-11-9-12-16-24)44-38(52)58-39(3,4)5)20-43(53)35(56-36(50)25-17-13-10-14-18-25)33-41(8,34(49)31(47)29(22)40(43,6)7)27(46)19-28-42(33,21-54-28)57-23(2)45/h9-18,26-28,30-33,35,46-48,53H,19-21H2,1-8H3,(H,44,52)/t26-,27-,28+,30-,31+,32+,33-,35-,41+,42-,43+/m0/s1

(2aR,4S,4aS,6R,9S,11S,12S,12bS)-12b-acetoxy-9-(((2R,3S)-3-((tert-butoxycarbonyl)amino)-2-hydroxy-3-phenylpropanoyl)oxy)-4,6,11-trihydroxy-4a,8,13,13-tetramethyl-5-oxo-2a,3,4,4a,5,6,9,10,11,12,12a,12b-dodecahydro-1H-7,11-methanocyclodeca[3,4]benzo[1,2-b]oxet-12-yl benzoate.

RP56976; NSC 628503; RP-56976; NSC628503; RP 56976; NSC-628503; Docetaxel hydrate; Docetaxel anhydrous; Docetaxel Winthrop; Docetaxol; Docetaxel Kabi; EmDOC; Trade name: Taxotere.

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

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: ≥ 5 mg/mL (6.19 mM) (saturation unknown) in 10% EtOH + 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 50.0 mg/mL clear EtOH 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: ≥ 5 mg/mL (6.19 mM) (saturation unknown) in 10% EtOH + 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 50.0 mg/mL clear EtOH stock solution to 900 μL of corn oil and mix well.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (2.57 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 4: ≥ 2.08 mg/mL (2.57 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 of PEG300 and mix evenly; then add 50 μL of Tween-80 to the above solution and mix evenly; then add 450 μL of 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 5: ≥ 2.08 mg/mL (2.57 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 6: ≥ 2.08 mg/mL (2.57 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 7: ≥ 2.08 mg/mL (2.57 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 8: ≥ 2.08 mg/mL (2.57 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 9: ≥ 2.08 mg/mL (2.57 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 corn oil and mix evenly.

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Solubility in Formulation 10: ≥ 2.08 mg/mL (2.57 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 20% SBE-β-CD saline solution and mix well.

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