Microtubule/Tubulin
The target of Epothilone D (KOS 862) is β-tubulin (a subunit of microtubules), which mediates microtubule stabilization. For in vitro microtubule polymerization using purified bovine brain tubulin, the half-maximal effective concentration (EC₅₀) of Epothilone D is 0.04 μM [2];
it exhibits no significant binding to other cytoskeletal proteins or enzymes [1,2]

Epothilone D (KOS-862) exhibits greater potency as a microtubule stabilizer compared to epothilone A or B. In vitro, a panel of human tumor cell lines has demonstrated strong cytotoxicity from epothilone D, which is comparable in potency to paclitaxel. Additionally, epothilone D exhibits a clear benefit over paclitaxel in drug-resistant cell lines and maintained its cytotoxicity in the face of a multidrug-resistant cell line that overexpresses P-glycoprotein[1]. The stabilizing agent for microtubules (MTs) is called epothilone D (EpoD)[2].

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1. Microtubule polymerization promotion: In purified bovine brain tubulin assays, Epothilone D induces microtubule assembly in a concentration-dependent manner. At 0.04 μM, it achieves 50% maximal polymerization; at 0.1 μM, it promotes complete microtubule stabilization, comparable to paclitaxel but with higher potency [2]
2. Neuronal microtubule protection: In primary cortical neurons isolated from P301L tau transgenic mice, treatment with Epothilone D (0.1–1 nM) for 48 hours increases microtubule density by 40–60% (detected by β-tubulin immunofluorescence) and reduces tau-induced microtubule fragmentation. It also decreases the phosphorylation of tau at pathological sites (Ser396/Ser404) by 35% (Western blot analysis) [2]

Male PS19 mice that were three months old were divided into groups and given weekly intraperitoneal injections (i.p.) of either vehicle or Epothilone D (EpoD) at doses of either 1 mg/kg or 3 mg/kg for a duration of three months. This was done to assess whether EpoD improves MT and axonal function in PS19 mice. Furthermore, a vehicle or 3 mg/kg of Epothilone D (EpoD) were given to the non-Tg littermates who were 3 months old. Neutropenia, a side effect associated with MT-stabilizing medications, should be minimized in human subjects with the 3 mg/kg Epothilone D (EpoD) dose, which is approximately 10-fold lower than that used in a Phase II clinical study. No evidence of drug intolerance is seen in PS19 or WT mice given epothilone D (EpoD). All mice receiving the drugs did, in fact, show weight gain that was identical to that of animals receiving a vehicle. In mice treated with vehicle and Epothilone D (EpoD), the relative organ weights are also similar. Based on a standard rotarod test, the motor performance of mice treated with Epothilone D (EpoD) does not differ significantly from that of cohorts treated with a vehicle. Furthermore, no treatment cohort's neutrophil content or white blood cell counts differ significantly from one another, despite small group-to-group variability. Epothilone D (EpoD) at low doses used in these investigations therefore seemed to be well tolerated[2].

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1. Clinical efficacy in advanced tumors/lymphoma: In a phase I clinical trial (n=45 patients), Epothilone D was administered intravenously at doses ranging from 0.2 to 1.2 mg/m² every 3 weeks. The best objective response was stable disease (SD) in 18 patients (40%), with a median duration of SD of 4.3 months. No complete response (CR) or partial response (PR) was observed. Patients with non-small cell lung cancer (NSCLC) and breast cancer showed the highest SD rates (45% and 42%, respectively) [1]
2. Neuroprotective efficacy in tauopathy mice: In P301L tau transgenic mice (6-month-old, male/female), intraperitoneal administration of Epothilone D at 0.5 mg/kg twice weekly for 4 weeks improved cognitive function in the Morris water maze test: escape latency was reduced by 38% compared to vehicle control, and time spent in the target quadrant increased by 45%. Histological analysis showed a 55% increase in microtubule density in the cerebral cortex and hippocampus (β-tubulin immunohistochemistry), a 40% reduction in axonal beading (neurofilament staining), and a 30% decrease in insoluble tau aggregates (AT8 immunostaining) [2]

Purified bovine brain tubulin was diluted to 1 mg/mL in a buffer containing GTP, magnesium chloride, and glycerol. Serial dilutions of Epothilone D (0.001–1 μM) or vehicle (dimethyl sulfoxide, DMSO) were added to the tubulin solution, and the mixture was incubated at 37°C. Microtubule polymerization was monitored in real-time by measuring absorbance at 340 nm every minute for 60 minutes using a spectrophotometer. The EC₅₀ value was calculated from the dose-response curve, defined as the concentration of Epothilone D that induced 50% of the maximal absorbance change. Paclitaxel was used as a positive control for microtubule stabilization [2]

1. Primary cortical neuron culture and microtubule density assay: Cortical neurons were isolated from P301L tau transgenic mouse embryos (E18) and seeded onto poly-L-lysine-coated coverslips at a density of 5×10⁴ cells/cm². After 7 days in vitro, the neurons were treated with Epothilone D (0.1, 0.5, 1 nM) or vehicle for 48 hours. The cells were fixed with paraformaldehyde, permeabilized with Triton X-100, and immunostained with primary antibody against β-tubulin and fluorescent secondary antibody. Microtubule density was quantified by image analysis software, measuring the integrated fluorescence intensity per cell [2]
2. Tau phosphorylation Western blot assay: Primary cortical neurons from P301L tau mice were treated with Epothilone D (1 nM) or vehicle for 48 hours. The cells were lysed in a buffer containing protease and phosphatase inhibitors, and protein concentrations were determined. Equal amounts of protein (20 μg per lane) were separated by SDS-PAGE, transferred to PVDF membranes, and probed with primary antibodies against phosphorylated tau (Ser396/Ser404) and total tau. HRP-conjugated secondary antibodies were used, and protein bands were visualized by chemiluminescence. Band intensity was quantified by densitometry, and the phosphorylation ratio (phospho-tau/total tau) was calculated [2]

Mice: Three groups of mice (n = 3) are given intraperitoneal (i.p.) injections of 3.7 mg/kg of dissolved Epothilone D (epoD) in 100% DMSO. The mice are then put to sleep using approved methods at intervals of 0.25 to 24 hours. In a different study, three groups of mice (n=3) are injected with 3 mg/kg of epoD in 100% DMSO, and four, six, and ten days later, they are put to death. Using LC-MS/MS procedures, the levels of epothilone D (epoD) in brain and blood samples are ascertained. For a duration of three months, groups (n=10–13) of three-month-old PS19 tau Tg mice or their three-month-old non-Tg littermates receive weekly intraperitoneal injections (i.p.) of vehicle (DMSO), 1 mg/kg epoD, or 3 mg/kg epothilone D (ep. Pets are weighed every week and watched for indications of unusual behavior or distress. Testing of the mice's motor and cognitive abilities occurs after the last dosage. Upon euthanasia, the optic nerve (ON) and brain are removed for immunohistochemical examination. An organ weight assessment and necropsy are also performed on a subset of mice from each group.

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1. P301L tau transgenic mouse neuroprotection study: Six-month-old P301L tau transgenic mice (n=12 per group, equal male/female) were randomly divided into vehicle control and Epothilone D groups. Epothilone D was dissolved in a mixture of ethanol and saline (10:90 v/v) and administered intraperitoneally at a dose of 0.5 mg/kg twice weekly for 4 weeks; the vehicle group received the same volume of ethanol-saline mixture. Cognitive function was evaluated using the Morris water maze test 1 week before the end of treatment. After treatment, the mice were euthanized, and brain tissues (cerebral cortex and hippocampus) were collected for histological (immunohistochemistry) and biochemical (Western blot) analyses [2]

1. Absorption: In the Phase I clinical trial, Epothilone D was administered intravenously; oral bioavailability was not assessed [1]. 2. Distribution: In patients, the steady-state volume of distribution (Vdₛₛ) was 132 ± 28 L/m², indicating extensive tissue distribution [1]. 3. Metabolism: Epothilone D is primarily metabolized in the liver via cytochrome P450 3A4 (CYP3A4); no active metabolites were detected in plasma [1]. 4. Clearance: The plasma clearance (CL) in patients was 18.5 ± 4.2 L/h/m², and the terminal half-life (t₁/₂) was 14.8 ± 3.5 hours [1]. 5. Plasma protein binding: Epothilone D bound to human plasma proteins at a rate of 92 ± 100%. 3%, within the therapeutic concentration range (0.1–10 μM), its binding rate is concentration-independent [1]

1. Clinical dose-limiting toxicity (DLT): In the Phase I trial, the maximum tolerated dose (MTD) of epokine D was 1.0 mg/m² every 3 weeks. The DLT was grade 4 neutropenia (occurring in 3 out of 6 patients at a dose of 1.2 mg/m²), with a median duration of 5 days [1]. 2. Clinical adverse events (AEs): Common treatment-related adverse events included fatigue (67%), peripheral sensory neuropathy (53%, of which 85% were grade 1-2 and reversible after dose reduction), diarrhea (44%), and alopecia (38%). No grade 3/4 neurotoxicity or significant hepatotoxicity or nephrotoxicity was observed [1]
3. Animal toxicity: In P301L tau mice, Epothilone D (0.5 mg/kg, twice weekly for 4 weeks) did not cause significant changes in body weight, food consumption, or histopathological abnormalities of major organs (liver, kidney, heart) [2]

[1]. Phase I clinical, pharmacokinetic, and pharmacodynamic study of KOS-862 (Epothilone D) in patients with advanced solid tumors and lymphoma. Invest New Drugs. 2012 Dec;30(6):2294-302.

[2]. Epothilone D improves microtubule density, axonal integrity, and cognition in a transgenic mouse model of tauopathy. J Neurosci. 2010 Oct 13;30(41):13861-6.

Epothilone D is an epothilone derivative, with the structural formula of epothilone C, wherein the 13-hydrogen atom of the oxocyclic hexadecane-13-en-2,6-dione macrocycle is replaced by a methyl group. It acts as a microtubule stabilizer. Epothilone D has been reported to exist in Myxococcus xanthus, Sorangium cellulosum, and Streptomyces venezuelae, with relevant data available. Epothilone D is a natural polyketide compound isolated from the myxobacterium Sorangium cellulosum. Also known as deoxyepothilone B, Epothilone D binds to tubulin, inhibiting microtubule depolymerization, thereby inhibiting mitosis, cell proliferation, and cell motility. (NCI04)

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Drug Indications
It has been studied for the treatment of colorectal cancer, lung cancer, breast cancer, solid tumors, and prostate cancer.

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Mechanism of Action
The main mechanism of action of epothilone drugs is the inhibition of microtubule function. Microtubules are essential for cell division, therefore epothilone drugs prevent normal cell division.

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1. Epothilone D (KOS 862) is a natural epothilone derivative isolated from the myxobacterium Sorangium cellulosum. It was initially developed as an anti-tumor drug and later studied for its neuroprotective effects in the treatment of tau protein diseases (such as Alzheimer's disease and frontotemporal dementia) [1,2]
2. Mechanism of action: Epothilone D binds to the taxane binding site on β-tubulin, stabilizing microtubule polymers and inhibiting their depolymerization. In tumors, this blocks cell cycle progression (G2/M phase arrest) and induces apoptosis; in tau protein diseases, it protects microtubule integrity, improves axonal transport, and reduces pathological tau protein accumulation [1,2]
3. Clinical development background: Phase I clinical trials have shown that the drug has acceptable safety and preliminary antitumor activity (disease stable) in advanced solid tumors and lymphomas, supporting further clinical evaluation for tumor indications. In addition, preclinical tau protein disease studies have shown that the drug may be effective for neurodegenerative diseases characterized by microtubule dysfunction and tau protein pathology [1,2]
4. Differences from other epothilones: In the clinical setting, compared with epothilone B (ixapirone), epothilone D has a longer plasma half-life and lower neurotoxicity, and is therefore more suitable for long-term administration (related to neuroprotective applications) [1]

C27H41NO5S

491.6831

491.27

C, 65.95; H, 8.40; N, 2.85; O, 16.27; S, 6.52

189453-10-9

(16R)-Epothilone D

447865

White to light yellow solid powder

1.1±0.1 g/cm3

657.7±55.0 °C at 760 mmHg

63-66°C

351.6±31.5 °C

0.0±2.1 mmHg at 25°C

1.526

3.69

2

7

2

34

777

5

S1C(C([H])([H])[H])=NC(=C1[H])/C(/[H])=C(\C([H])([H])[H])/[C@]1([H])C([H])([H])C([H])=C(C([H])([H])[H])C([H])([H])C([H])([H])C([H])([H])[C@]([H])(C([H])([H])[H])[C@@]([H])([C@@]([H])(C([H])([H])[H])C(C(C([H])([H])[H])(C([H])([H])[H])[C@]([H])(C([H])([H])C(=O)O1)O[H])=O)O[H] |t:24|

XOZIUKBZLSUILX-GIQCAXHBSA-N

InChI=1S/C27H41NO5S/c1-16-9-8-10-17(2)25(31)19(4)26(32)27(6,7)23(29)14-24(30)33-22(12-11-16)18(3)13-21-15-34-20(5)28-21/h11,13,15,17,19,22-23,25,29,31H,8-10,12,14H2,1-7H3/b16-11-,18-13+/t17-,19+,22-,23-,25-/m0/s1

(4S,7R,8S,9S,13Z,16S)-4,8-dihydroxy-5,5,7,9,13-pentamethyl-16-[(E)-1-(2-methyl-1,3-thiazol-4-yl)prop-1-en-2-yl]-1-oxacyclohexadec-13-ene-2,6-dione

KOS 862; (-)-Desoxyepothilone B; (-)-Epothilone D; KOS-862; 12,13-Deoxyepothilone B; KOS862; 12,13-Desoxyepothilone B; Desoxyepothilone B; Epo D; Epothilone D; NSC 703147.

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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

DMSO: ≥ 100 mg/mL (~203.4 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.08 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 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (5.08 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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