| Size | Price | Stock | Qty |
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| 5mg |
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| 10mg |
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| Other Sizes |
Purity: ≥98%
PF-06380101 (Auristatin 0101; PF06380101; AUR-0101) is a novel and ultra-potent cytotoxic Dolastatin 10 analogue and a tubulin inhibitor with excellent anticancer activity. It exhibits differential ADME properties and high potencies in tumor cell proliferation assays when compared to other synthetic auristatin analogues used in ADC preparation. Clinically employed as payloads in antibody-drug conjugates (ADCs), auristatins are synthetic analogues of the naturally occurring antitumor compound Dolastatin 10. They are extremely potent cytotoxic microtubule inhibitors. Our lead auristatin, PF-06380101, is discovered and the design and synthesis of several new auristatin analogues with N-terminal modifications, including amino acids with α,α-disubstituted carbon atoms, are described. Comparing these novel peptide structure modifications to other synthetic auristatin analogues used in the creation of ADCs, the results were analogues with superior potencies in tumor cell proliferation assays and distinct ADME characteristics. Furthermore, tubulin and auristatin cocrystal structures that enable a thorough analysis of their binding mechanisms are presented. Surprisingly, in their functionally relevant tubulin bound state, all the analyzed analogues have a cis-configuration at the Val-Dil amide bond, whereas in solution this bond is exclusively in the trans-configuration. This astounding finding provides insight into the preferred binding mode of auristatins and is a useful resource for structure-based drug design.
| Targets |
Tubulin (microtubule) [1].
The compound binds at the interface of α,β-tubulin heterodimer, adjacent to the vinca binding site, and inhibits tubulin polymerization [1]. |
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| ln Vitro |
PF-06380101 demonstrated a mean systemic clearance (Cl) of 70 mL/min/kg and a volume of distribution (Vss) of 14.70 L/kg following an IV dose of 20a at 20 μg/kg to Wistar Han rats. This led to an approximate 6-hour terminal elimination half-life (t1/2). PF-06380101 is a P-glycoprotein (P-gp) substrate that preferentially distributes into human plasma as opposed to whole blood. It is expected that PF-06380101 will not cause pharmacokinetic drug interactions with substances whose main mechanism of clearance is mediated by CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and/or CYP3A4/5. Reports regarding the effectiveness of the newly developed auristatin analogues as ADC payloads, along with the creation of the lead analogue 20a (PF-06380101), will be provided eventually.
In tumor cell viability assays, PF-06380101 demonstrated potent antiproliferative activity across three cancer cell lines: BT474 (breast carcinoma) with GI₅₀ of 0.26 nM (mean; standard deviation ±0.37, n=16); MDA-MB-361-DYT2 (breast carcinoma) with GI₅₀ of 0.19 nM (±0.073, n=19); N87 (gastric carcinoma) with GI₅₀ of 0.27 nM (±0.16, n=18) [1]. In a cell-free tubulin polymerization assay, PF-06380101 showed good inhibition (data not shown) [1]. The compound exhibited low passive cell-membrane permeability (RRCK cell line: Papp AB < 0.5 × 10⁻⁶ cm/s) [1]. PF-06380101 is a P-glycoprotein (P-gp) substrate [1]. |
| ln Vivo |
The average systemic clearance (Cl) of PF-06380101 was 70 mL/min/kg, the volume of distribution (Vss) was 14.70 L/kg, and the terminal elimination half-life (t1/2) was approximately 6 hours following an intravenous injection of 20 μg/kg into Wistar Han rats for a period of 20 years. As a P-glycoprotein (P-gp) substrate, PF-06380101 distributes preferentially into human plasma as opposed to whole blood. PF-06380101 is anticipated to exhibit minimal pharmacokinetic drug interaction risk with substances whose principal clearance mechanism is metabolism mediated by CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and/or CYP3A4/5. We will report on the creation of lead analog 20a (PF-06380101) and the use of novel aristatin analogs as ADC payloads in due time.
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| Enzyme Assay |
Tubulin Polymerization Assay: A cell-free tubulin polymerization assay was used to assess compound activity, but detailed protocols were not provided in the manuscript (data not shown) [1].
Recombinant CYP Inhibition Assays: Initial reaction phenotyping experiments suggested that CYP3A4 is the predominant enzyme involved in the metabolism of PF-06380101, with hydroxylation occurring on the N-terminus. Based on in vitro drug-drug interaction studies (direct inhibition, metabolism-dependent inhibition, and induction), the compound was anticipated to be of low risk for perpetuating pharmacokinetic drug interactions with compounds cleared by CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and/or CYP3A4/5 [1]. |
| Cell Assay |
Tumor Cell Viability Assay (Cytotoxicity Assay): Cells (N87, BT474, MDA-MB-361-DYT2) were seeded in 96-well plates at low density and treated the following day with compounds in 3-fold serial dilutions at 10 concentrations in duplicate. Cells were incubated for 4 days in a humidified 37°C / 5% CO₂ incubator. Plates were harvested by incubating with a tetrazolium compound (MTS) solution for 1.5 hours, and absorbance was measured at 490 nm on a plate reader. IC₅₀ values were calculated using a four-parameter logistic model [1].
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| Animal Protocol |
Rat Pharmacokinetic Studies: Male Wistar Han rats were used for pharmacokinetic evaluation. For single-dose PK, PF-06380101 was administered intravenously (IV) as a bolus at 20 μg/kg (n=3). For repeat-dose PK, the compound was administered IV once daily for 29 days at doses of 20, 60, and 120 μg/kg/day (n=3 per dose). Blood samples were collected at specified time points, and plasma concentrations were analyzed by LC-MS/MS [1].
Rat Excretion Studies: After a single 36 μg/kg IV bolus dose in Wistar Han rats (n=3) and Sprague-Dawley rats (n=3), urine and bile were collected. Approximately 8% of the dose was recovered in urine and approximately 4% in bile [1]. |
| ADME/Pharmacokinetics |
Rat PK (Single IV Dose): After a single IV dose of 20 μg/kg in male Wistar Han rats (n=3), PF-06380101 showed: Cmax of 19.4 ± 2.16 ng/mL; Tmax of 0.14 ± 0.050 h; AUCt of 4.51 ± 1.14 ng·h/mL; systemic clearance (Cl) of 70.0 ± 12.8 mL/min/kg; volume of distribution at steady state (Vss) of 14.70 ± 3.05 L/kg; terminal elimination half-life (t₁/₂) of 5.85 ± 1.17 h [1].
Repeat IV Dose in Rats: Following repeat IV dosing of PF-06380101 to Wistar Han rats (20, 60, 120 μg/kg/day for 29 days), mean systemic exposure increased with increasing dose. Mean Cmax and AUC ratios on day 29 relative to day 1 were generally <2.0, suggesting no accumulation over the dosing interval [1]. Plasma Protein Binding: The unbound fraction (fᵤ) at 0.1 μM dose was 0.24 for monkey, 0.10 for rat, and 0.07 for human plasma [1]. Metabolism: In vitro studies with rat, monkey, and human liver S9 fractions and human recombinant CYP enzymes showed that all metabolites were oxidative and NADPH-dependent, with no amide hydrolysis products or glucuronide conjugates observed. There was no evidence of human-specific metabolites. CYP3A4 was identified as the predominant enzyme involved in metabolism [1]. |
| Toxicity/Toxicokinetics |
Plasma Protein Binding: PF-06380101 was moderately to highly bound to plasma proteins (fᵤ: monkey 0.24, rat 0.10, human 0.07 at 0.1 μM) [1].
Drug-Drug Interaction Potential: Based on in vitro DDI studies, PF-06380101 is anticipated to be of low risk for perpetuating pharmacokinetic drug interactions with compounds cleared by CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and/or CYP3A4/5. However, because it is primarily metabolized by CYP3A4, there is potential for DDIs if coadministered with moderate and/or potent clinical inhibitors and/or inducers of CYP3A4 [1]. |
| References | |
| Additional Infomation |
PF-06380101 (compound 20a) is a novel auristatin analogue with an N-terminal modification incorporating 2-aminoisobutyric acid (Aib), an α,α-disubstituted amino acid. This modification is unprecedented in auristatin chemistry [1].
The compound was designed based on analysis of the tubulin co-crystal structure of soblidotin (TZT-1027), which revealed a binding void around the N-terminus, suggesting that α,α-dialkyl substitutions could be tolerated [1]. In the co-crystal structure of PF-06380101 with tubulin (PDB 4X11, 3.1 Å resolution), the Val-Dil amide bond was observed in the cis-configuration in the functionally relevant bound form at the β1/α2 tubulin interface, whereas in solution this bond is exclusively in the trans-configuration. This finding revealed a preferred binding mode for auristatins [1]. The compound showed significantly higher human liver microsome (HLM) apparent intrinsic clearance values (Cl(int, app) = 84 μL/min/mg) compared to MMAD (2) and MMAE (4). Human liver hepatocyte (HHEP) clearance was 212 μL/min/million cells [1]. PF-06380101 was selected as a lead auristatin for development as an antibody-drug conjugate (ADC) payload. The utility of the compound as an ADC payload will be reported in a future publication [1]. |
| Molecular Formula |
C39H62N6O6S
|
|---|---|
| Molecular Weight |
743.0112
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| Exact Mass |
742.445
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| Elemental Analysis |
C, 63.04; H, 8.41; N, 11.31; O, 12.92; S, 4.31
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| CAS # |
1436391-86-4
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| Related CAS # |
PF-06380101-d8
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| PubChem CID |
71569947
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| Sequence |
XV
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| Appearance |
White to off-white solid powder
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| Density |
1.1±0.1 g/cm3
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| Boiling Point |
903.1±65.0 °C at 760 mmHg
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| Flash Point |
500.0±34.3 °C
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| Vapour Pressure |
0.0±0.3 mmHg at 25°C
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| Index of Refraction |
1.546
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| LogP |
5.05
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| Hydrogen Bond Donor Count |
3
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| Hydrogen Bond Acceptor Count |
9
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| Rotatable Bond Count |
19
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| Heavy Atom Count |
52
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| Complexity |
1170
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| Defined Atom Stereocenter Count |
8
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| SMILES |
S1C([H])=C([H])N=C1[C@]([H])(C([H])([H])C1C([H])=C([H])C([H])=C([H])C=1[H])N([H])C([C@]([H])(C([H])([H])[H])[C@]([H])([C@]1([H])C([H])([H])C([H])([H])C([H])([H])N1C(C([H])([H])[C@]([H])([C@]([H])([C@@]([H])(C([H])([H])[H])C([H])([H])C([H])([H])[H])N(C([H])([H])[H])C([C@]([H])(C([H])(C([H])([H])[H])C([H])([H])[H])N([H])C(C(C([H])([H])[H])(C([H])([H])[H])N([H])[H])=O)=O)OC([H])([H])[H])=O)OC([H])([H])[H])=O
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| InChi Key |
QAAFNSMAIAVCHE-BZLYQNAUSA-N
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| InChi Code |
InChI=1S/C39H62N6O6S/c1-11-25(4)33(44(8)37(48)32(24(2)3)43-38(49)39(6,7)40)30(50-9)23-31(46)45-20-15-18-29(45)34(51-10)26(5)35(47)42-28(36-41-19-21-52-36)22-27-16-13-12-14-17-27/h12-14,16-17,19,21,24-26,28-30,32-34H,11,15,18,20,22-23,40H2,1-10H3,(H,42,47)(H,43,49)/t25-,26+,28-,29-,30+,32-,33-,34+/m0/s1
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| Chemical Name |
(2S)-2-[(2-amino-2-methylpropanoyl)amino]-N-[(3R,4S,5S)-3-methoxy-1-[(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-[[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino]propyl]pyrrolidin-1-yl]-5-methyl-1-oxoheptan-4-yl]-N,3-dimethylbutanamide
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| Synonyms |
AUR-0101; PF06380101; PF-06380,101; 1436391-86-4; Auristatin 0101; Q8020AX34E; AUR-0101; Auristatin 0101; AUR 0101; PF-06380101; AUR0101; PF 06380101
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| HS Tariff Code |
2934.99.03.00
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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) |
DMSO: ≥ 65 mg/mL (~87.5 mM)
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|---|---|
| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (3.36 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. Solubility in Formulation 2: 2.5 mg/mL (3.36 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication. 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 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. View More
Solubility in Formulation 3: ≥ 2.5 mg/mL (3.36 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.3459 mL | 6.7294 mL | 13.4588 mL | |
| 5 mM | 0.2692 mL | 1.3459 mL | 2.6918 mL | |
| 10 mM | 0.1346 mL | 0.6729 mL | 1.3459 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
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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