Antibody-conjugatable maytansinoid; tubulin; microtubules

When applied to more than 60 different types of cancer cell lines, mertansine is a potent antiproliferative chemotherapy[3].
Mertansine (0–1 μg/mL) has antitumor activity in malignant B16F10 melanoma cells and, when combined with DTX, inhibits tumor cell growth by blocking mitosis[3].

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Maytansine is a potent microtubule-targeted compound that induces mitotic arrest and kills tumor cells at subnanomolar concentrations. However, its side effects and lack of tumor specificity have prevented successful clinical use. Recently, antibody-conjugated maytansine derivatives have been developed to overcome these drawbacks. Several conjugates show promising early clinical results. We evaluated the effects on microtubule polymerization and dynamic instability of maytansine and two cellular metabolites (S-methyl-DM1 and S-methyl-DM4) of antibody-maytansinoid conjugates that are potent in cells at picomolar levels and that are active in tumor-bearing mice. Although S-methyl-DM1 and S-methyl-DM4 inhibited polymerization more weakly than maytansine, at 100 nmol/L they suppressed dynamic instability more strongly than maytansine (by 84% and 73%, respectively, compared with 45% for maytansine). However, unlike maytansine, S-methyl-DM1 and S-methyl-DM4 induced tubulin aggregates detectable by electron microscopy at concentrations ≥2 μmol/L, with S-methyl-DM4 showing more extensive aggregate formation than S-methyl-DM1. Both maytansine and S-methyl-DM1 bound to tubulin with similar K(D) values (0.86 ± 0.2 and 0.93 ± 0.2 μmol/L, respectively). Tritiated S-methyl-DM1 bound to 37 high-affinity sites per microtubule (K(D), 0.1 ± 0.05 μmol/L). Thus, S-methyl-DM1 binds to high-affinity sites on microtubules 20-fold more strongly than vinblastine. The high-affinity binding is likely at microtubule ends and is responsible for suppression of microtubule dynamic instability. Also, at higher concentrations, S-methyl-DM1 showed low-affinity binding either to a larger number of sites on microtubules or to sedimentable tubulin aggregates. Overall, the maytansine derivatives that result from cellular metabolism of the antibody conjugates are themselves potent microtubule poisons, interacting with microtubules as effectively as or more effectively than the parent molecule.[1]

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Maytansine and the DM1 (S-methyl-DM1; ((N2’-deacetyl-N2’-(3-thiomethyl-1-oxopropyl)-maytansine; Fig. 1) suppress microtubule dynamics at very low drug concentrations. Specifically, using video-enhanced differential interference contrast microscopy, researchers showed that maytansine as well as the DM1 derivative strongly suppress the dynamic instability parameters of microtubules assembled from MAP-free tubulin in vitro (Fig. 2). Maytansine and S-methyl-DM1 suppressed almost all dynamic instability parameters, including the growth rate, the shortening rate, the catastrophe frequency, and the rescue frequency, with the DM1 derivative showing stronger suppression of dynamics than the parent macrolide. The molecular mechanism of action of S-methyl-DM1 was found to be microtubule end poisoning. That is, S-methyl DM1 binds to the tips of microtubules and thereby inhibits the growth and the shortening of microtubules, leading to suppression of microtubule dynamics. Specifically, the maytansinoid showed high-affinity binding (KD, 0.1 μmol/L ) to approximately 37 sites per microtubule. The researchers also suggested that S-methyl-DM1 binds to high-affinity sites on microtubules 20 times more strongly than vinblastine and that the high-affinity binding of the maytansinoid at the tips might have suppressed the dynamics. S-methyl DM1 is also reported to bind to soluble tubulin. Although its affinity for soluble tubulin is approximately ten times less than that for microtubules, the possibility of tubulin-S-methyl-DM1 complex binding at the tips and thereby contributing to the suppression of microtubule dynamics cannot be ruled out.[2]

The minimal maximum tolerated dose (MTD) of mertansine (DM1) is 1 mg/kg[3].

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The in vivo therapeutic performance of cRGD-MMP was evaluated using MDA-MB-231 triple-negative breast tumor-bearing mice. cRGD-MMP, MMP or free Maytansine/mertansine/DM1 was administered every 3 days for a total of four injections at a dosage of 0.8 or 1.6 mg Maytansine/DM1 equiv./kg. As expected, mice treated with PBS showed rapid tumor growth (Figure 5A). Free DM1 showed modest tumor growth inhibition at 0.8 mg/kg. Considering that free DM1 has an maximum-tolerated dose of 1 mg/kg, no higher dosage was attempted. cRGD-MMP exhibited better tumor suppression at 0.8 mg/kg than free DM1 and MMP controls under otherwise the same conditions. Moreover, cRGD-MMP showed a dose-dependent tumor growth inhibition, in which tumor progression was greatly enhanced at 1.6 mg DM1 equiv./kg. The images of tumors isolated on day 16 showed clearly that mice treated with cRGD-MMP at 1.6 mg DM1 equiv./kg had almost complete growth inhibition (Figure 5B). The tumor weights revealed that cRGD-MMP at 0.8 mg DM1 equiv./kg yielded a TIR of 67.3%, which was significantly higher than that for MMP (TIR: 46.2%) and free DM1 (TIR: 17.5%; Figure 5C). A high TIR of 87.6% was achieved with 1.6 mg DM1 equiv./kg cRGD-MMP. The significantly enhanced tumor growth inhibition of cRGD-MMP as compared to free DM1 is most likely because cRGD-MMP has a longer circulation time, better tumor selectivity and higher drug accumulation in tumor than free DM1. No obvious body weight loss was observed for all treatments over the entire experimental period (Figure 5D). Notably, formyl peptide receptor-targeting and paclitaxol-encapsulated human serum albumin nanoparticles were reported to cause more effective tumor inhibition though similar toxicity as compared to Taxol in MDA-MB-231 tumor-bearing mice Johnstone et al reported that mitaplatin-loaded Poly (lactide-co-glycolide)-PEG nanoparticles with a long-term controlled drug release behavior exhibited a similar TIR to free mitaplatin in MDA-MB-468 tumor-bearing mice. DTX-loaded PEG-poly(epsilon-caprolactone) nanoparticles displayed a comparable in vivo antitumor efficacy and survival rate in MDA-MB-231 TNBC animal model to DTX commercial formulation (Taxotere®). H&E staining displayed that cRGD-MMP at 0.8 and 1.6 mg DM1 equiv./kg did not cause significant damage to the major organs, while free DM1 induced obvious increased Kupffer cells in liver tissues, fat vacuoles, liver cell cord derangement and dilated intercellular spaces (Figure 6). All the abovementioned results demonstrate that cRGD-MMP has better tumor selectivity and enhanced treatment of MDA-MB-231 TNBC. These cRGD-decorated, polycarbonate-based, reduction-sensitive mertansine prodrug micelles have proven to be an effective platform for TNBC chemotherapy.[5]

Effects of the Maytansinoids on Microtubule Polymerization[1]
The ability of maytansine, S-methyl DM1 and S-methyl DM4 to inhibit microtubule assembly was determined by incubating MTP (3 mg/mL) with a range of maytansinoid concentrations (0 – 20 μmol/L) in the presence of 1 mmol/L GTP in PEM buffer (30°C, 45 min). For sedimentation assays, the polymers formed were centrifuged (35000 × g, 1 h, 30°C). The microtubule pellets were depolymerized at 0°C overnight and the protein concentrations determined. The sedimentation assay for each compound was performed at least twice. For observing microtubule morphology, samples were fixed in 0.2% glutaraldehyde, and stained with 0.5% uranyl acetate. The images were acquired at 50000 × or 100000 × magnification using a JEOL 1230 transmission electron microscope at 80 KV.

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Effects of Maytansine and Its Derivatives on Microtubule Dynamic Instability[1]
Microtubule dynamic instability parameters were measured as previously described. Briefly, tubulin (1.5 mg/mL) was assembled on the ends of sea urchin (Strongylocentrotus purpuratus) axoneme fragments at 30°C in 87 mmol/L Pipes, 36 mmol/L Mes, 1.4 mmol/L MgCl2, 1 mmol/L EGTA, pH 6.8 (PMME buffer) containing 2 mmol/L GTP for 30 min to achieve steady state. We used a 100 nmol/L concentration of each compound, to analyze their individual effects on dynamic instability. Time-lapse images of microtubule plus ends were obtained at 32°C by video-enhanced differential interference contrast microscopy using an Olympus IX71 inverted microscope with a 100 × (NA = 1.4) oil immersion objective. We identified plus ends of the microtubules by their faster growth rate, greater length changes, and larger number of microtubules per axoneme end as compared with the minus ends. Microtubule dynamics were recorded for 40 min at 30°C, capturing 10 min long videos for each area under observation. The rates and durations of growing and shortening and the transition frequencies were determined after tracking the microtubules using RTM-II software (23) and analyzing using IgorPro software. Microtubules were considered as growing if they increased in length >0.3 μm at a rate >0.3 μm/min. Shortening events were identified by a >1 μm length reduction at a rate >2 μm/min. Fifteen to 25 microtubules were analyzed per condition. The catastrophe (a transition from a growing or attenuated state to shortening) frequency was calculated as the total number of catastrophes divided by time spent growing and attenuated (paused). The rescue (transition from shortening to growing) frequency was calculated as the total number of rescue events divided by total time spent shortening. The dynamicity of microtubules was derived as the sum of the total growth length and the total shortening length divided by the total time.

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Binding of Maytansine or S-methyl DM1 to Soluble Tubulin[1]
Maytansine or S-methyl DM1 (0–20 μmol/L) was incubated with 3 μmol/L tubulin in PEM buffer for 45 min at 30°C. The relative intrinsic fluorescence intensity of tubulin was monitored at 335 nm in a Perkin Elmer LS 50B spectrofluorometer using a 0.3–cm path length cuvette at an excitation wavelength of 295 nm. The fluorescence emission intensity of S-methyl DM1 and maytansine at this excitation wavelength was negligible. The inner filter effects were corrected using the formula Fcorrected = Fobserved.antilog [(Aex + Aem)/2], where A is the absorbance at the excitation wavelength and Aem is the absorbance at the emission wavelength. The dissociation constant (KD) was determined by the formula: 1/a = KD/[free ligand] + 1, where a is the fractional occupancy of the drug and [free ligand] is the concentration of free maytansine or S-methyl DM1. The fractional occupancy (a) was determined by the formula a = ΔF/ΔFmax, where ΔF is the change in fluorescence intensity when tubulin and its ligand are in equilibrium and ΔFmax is the value of maximum fluorescence change when tubulin is completely bound with its ligand. Experiments were performed three times.

Cell Line: Malignant B16F10 melanoma cells[3]
Concentration: 0, 0.01, 0.1, and 1 μg/mL
Incubation Time: 4 h
Result: demonstrated antitumor activity, with an IC50 of 0.092 μg/mL, in malignant B16F10 melanoma cells. To achieve a combinatorial anticancer effect, co-delivering DTX and DM1, which both inhibit tumor cell growth by inhibiting mitosis, is a useful tactic.

Combinatorial chemotherapy, which has emerged as a promising treatment modality for intractable cancers, is challenged by a lack of tumor-targeting, robust and ratiometric dual drug release systems. Here, docetaxel-loaded cRGD peptide-decorated redox-activable micellar mertansine prodrug (DTX-cRGD-MMP) was developed for targeted and synergistic treatment of B16F10 melanoma-bearing C57BL/6 mice. DTX-cRGD-MMP exhibited a small size of ca. 49 nm, high DTX and DM1 loading, low drug leakage under physiological conditions, with rapid release of both DTX and DM1 under a cytoplasmic reductive environment. Notably, MTT and flow cytometry assays showed that DTX-cRGD-MMP brought about a synergistic antitumor effect to B16F10 cancer cells, with a combination index of 0.37 and an IC50 over 3- and 13-fold lower than cRGD-MMP (w/o DTX) and DTX-cRGD-Ms (w/o DM1) controls, respectively. In vivo studies revealed that DTX-cRGD-MMP had a long circulation time and a markedly improved accumulation in the B16F10 tumor compared with the non-targeting DTX-MMP control (9.15 versus 3.13% ID/g at 12 h post-injection). Interestingly, mice treated with DTX-cRGD-MMP showed almost complete growth inhibition of B16F10 melanoma, with tumor inhibition efficacy following an order of DTX-cRGD-MMP > DTX-MMP (w/o cRGD) > cRGD-MMP (w/o DTX) > DTX-cRGD-Ms (w/o DM1) > free DTX. Consequently, DTX-cRGD-MMP significantly improved the survival rates of B16F10 melanoma-bearing mice. Importantly, DTX-cRGD-MMP caused little adverse effects as revealed by mice body weights and histological analyses. The combination of two mitotic inhibitors, DTX and DM1, appears to be an interesting approach for effective cancer therapy [3].

[1]. Maytansine and Cellular Metabolites of Antibody-Maytansinoid Conjugates Strongly Suppress Microtubule Dynamics by Binding to Microtubules.

[2]. Antibody-DM1 conjugates as cancer therapeutics. Cancer Lett. 2011 Aug 28;307(2):113-8.

[3]. cRGD-installed docetaxel-loaded mertansine prodrug micelles: redox-triggered ratiometric dual drug release and targeted synergistic treatment of B16F10 melanoma. Nanotechnology. 2017 Jul 21;28(29):295103.

[4]. MAPN: First-in-Class Reagent for Kinetically Resolved Thiol-to-Thiol Conjugation. Bioconjug Chem. 2015 Sep 16;26(9):1863-7.

[5]. αvβ3 integrin-targeted micellar mertansine prodrug effectively inhibits triple-negative breast cancer in vivo. Int J Nanomedicine. 2017; 12: 7913–7921.

Mertansine is an organic heterotetracyclic compound and 19-membered macrocyclic lactam that is maytansine in which one of the hydrogens of the terminal N-acetyl group is replaced by a sulfanylmethyl group. It has a role as an antineoplastic agent and a tubulin modulator. It is an alpha-amino acid ester, a carbamate ester, an epoxide, an organic heterotetracyclic compound, an organochlorine compound, a thiol and a maytansinoid. It is functionally related to a maytansine.
An ansa macrolide isolated from the MAYTENUS genus of East African shrubs.

C35H48CLN3O10S

738.29

737.274

C, 56.94; H, 6.55; Cl, 4.80; N, 5.69; O, 21.67; S, 4.34

139504-50-0

139504-50-0;

11343137

White to off-white solid powder

1.33±0.1 g/cm3

937.1±65.0 °C at 760 mmHg

190-192 ºC

520.5±34.3 °C

0.0±0.3 mmHg at 25°C

1.599

4.76

3

11

8

50

1340

8

ClC1C(=C([H])C2C([H])([H])C(C([H])([H])[H])=C([H])C([H])=C([H])[C@]([H])([C@]3(C([H])([H])[C@@]([H])([C@@]([H])(C([H])([H])[H])[C@@]4([H])[C@](C([H])([H])[H])([C@]([H])(C([H])([H])C(N(C([H])([H])[H])C=1C=2[H])=O)OC([C@]([H])(C([H])([H])[H])N(C(C([H])([H])C([H])([H])S[H])=O)C([H])([H])[H])=O)O4)OC(N3[H])=O)O[H])OC([H])([H])[H])OC([H])([H])[H] |c:13,17|

ANZJBCHSOXCCRQ-GCRZMMRQSA-N

InChI=1S/C35H48ClN3O10S/c1-19-10-9-11-26(46-8)35(44)18-25(47-33(43)37-35)20(2)31-34(4,49-31)27(48-32(42)21(3)38(5)28(40)12-13-50)17-29(41)39(6)23-15-22(14-19)16-24(45-7)30(23)36/h9-11,15-16,20-21,25-27,31,44,50H,12-14,17-18H2,1-8H3,(H,37,43)/b11-9-,19-10+/t20-,21+,25+,26-,27-,31?,34+,35+/m1/s1

(14S,16S,33S,2R,4R,10E,12Z,14R)-86-chloro-14-hydroxy-85,14-dimethoxy-33,2,7,10-tetramethyl-12,6-dioxo-7-aza-1(6,4)-oxazinana-3(2,3)-oxirana-8(1,3)-benzenacyclotetradecaphane-10,12-dien-4-yl N-(3-mercaptopropanoyl)-N-methyl-L-alaninate

DM1; DM-1; DM 1; DM1; Compound DM1; [Maytansinoid]; Maytansinoid DM 1; Maytansinoid DM1; Maytansinoid DM-1; UNII-DDZ29HGH0E; maytansine; Mertansine (DM1); N2'-deacetyl-N2'-(3-mercapto-1-oxopropyl)-maytansine; DDZ29HGH0E; DM 1; Mertansine; emtansine

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 is not stable in solution, please use freshly prepared working solution for optimal results.

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

DMSO : 50~62.5 mg/mL ( 67.72~84.66 mM )
Ethanol : 2 mg/mL

Solubility in Formulation 1: 2.17 mg/mL (2.94 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 sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 21.7 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 2: ≥ 2.08 mg/mL (2.82 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 3: ≥ 2.08 mg/mL (2.82 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 corn oil and mix evenly.

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