Daunorubicins/Doxorubicins; Topoisomerase II
Aldoxorubicin (INNO-206) (0.27 to 2.16 μM) inhibits the formation of blood vessels and decreases the growth of multiple myeloma cells in a pH-dependent fashion[1].

Aldoxorubicin (INNO-206) (0.27 to 2.16 μM) inhibits the formation of blood vessels and decreases the growth of multiple myeloma cells in a pH-dependent fashion[1].

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The cytotoxicity of INNO-206 when conjugated to elastin-like polypeptide (ELP) carriers was evaluated. The conjugate SynB1-ELP-INNO-206 inhibited MCF-7 breast cancer cell proliferation, with an IC50 of 32.961 ± 4.092 µM at 42°C, while the carrier alone (SynB1-ELP) showed minimal toxicity. The acid-sensitive hydrazone linker in INNO-206 is crucial for activity, as its non-cleavable counterpart (EMC-DOXO) conjugated to SynB1-ELP showed no toxicity. [4]
Confocal fluorescence microscopy in MCF-7 cells showed that treatment with CPP-ELP-INNO-206 conjugates (Bac, Tat, SynB1) for 1 hour followed by a 4-hour incubation resulted in doxorubicin localization within the cell nucleus, similar to free doxorubicin, indicating successful intracellular release of the active drug from the acid-labile conjugate. [4]

Aldoxorubicin (INNO-206) (10.8 mg/kg, i.v.) is well tolerated; 90% of mice with the LAGκ-1A tumor survive until the study's conclusion[1]. It also exhibits significantly smaller tumor volumes and IgG levels on day 28. Phase I studies have shown that aldoxorubicin (INNO-206) can cause tumor regressions in sarcoma, small cell lung cancer, and breast cancer while maintaining a good safety profile at doses up to 260 mg/mL doxorubicin equivalents[2]. In models of breast carcinoma xenografts and murine renal cell carcinoma, aldoxorubicin (INNO-206) exhibits better efficacy than doxorubicin[3].

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In a syngeneic E0771 murine breast cancer model in C57BL/6 mice, the conjugate SynB1-ELP-INNO-206 (at 7 mg/kg doxorubicin equivalent dose, administered intravenously on days 0, 2, 4, and 6) combined with localized tumor hyperthermia (41°C) significantly inhibited tumor growth compared to free doxorubicin at the same dose under hyperthermic conditions. Tumor volume and weight on day 14 were significantly lower in the SynB1-ELP-INNO-206 + hyperthermia group. [4]
Animals treated with SynB1-ELP-INNO-206 and hyperthermia experienced less body weight loss during the treatment course compared to animals treated with free doxorubicin and hyperthermia, suggesting an improved therapeutic index. [4]

Cell viability assay[1]
Cells were seeded at 1 × 105 cells/100 μL/well in 96-well plates in RPMI-1640 media with FBS for 24 hours before treatment. Cells were cultured in the presence of medium, INNO-206 or doxorubicin for 48 hours. Next, cell viability was quantified using the CellTiter 96 AQueous Non-Radioactive Cell Proliferation Assay (Promega). Each well was treated with MTS for 1 to 4 hours, after which absorbance at 490 nm was recorded using a 96-well plate reader. The quantity of formazan product as measured is directly proportional to the number of living cells. Data graphed are means ± SEM using 3 replicates per data point.

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The anti–multiple myeloma effect of INNO-206 at different pH levels on multiple myeloma cell proliferation using multiple myeloma cell lines with the MTS assay and antiangiogenic activity using the chorioallantoic membrane/feather bud assay were determined. The anti–multiple myeloma effects and toxicity of INNO-206 were also compared with conventional doxorubicin and PEGylated liposomal doxorubicin (PLD) alone, and in combination with bortezomib, using our multiple myeloma xenograft models[1].

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Cell Proliferation Assay (MTS): MCF-7 human breast carcinoma cells were plated in 96-well plates and allowed to grow for 24 hours. Cells were treated with varying concentrations of CPP-ELP-INNO-206 conjugates (expressed as doxorubicin equivalent concentration) or free doxorubicin for 1 hour at either 37°C or 42°C. After treatment, cells were washed and returned to the incubator with fresh medium. 72 hours later, cell proliferation was measured using an MTS assay. The absorbance was measured, and data were normalized to untreated control cells. Dose-response curves were fitted to determine IC50 values. [4]
Flow Cytometry for Cellular Uptake: MCF-7 cells were treated with 10 µM doxorubicin equivalent of CPP-ELP-INNO-206 conjugates for 1 hour at 37°C or 42°C. Cells were then washed, harvested using a non-enzymatic cell dissociation buffer, and resuspended in PBS. The total fluorescence associated with cells (from the doxorubicin moiety) was measured using a flow cytometer to quantify cellular uptake. [4]
Fluorescence Microscopy for Intracellular Localization: MCF-7 cells were plated on coverslips and grown to 50% confluence. Cells were treated with 20 µM doxorubicin equivalent of CPP-ELP-INNO-206 conjugates or free doxorubicin for 1 hour at 37°C or 42°C. After treatment, cells were washed, and fresh medium was added. 4 hours post-treatment, cells were rinsed, fixed with paraformaldehyde, and stained with Hoechst dye to visualize nuclei. The intracellular localization of doxorubicin fluorescence was visualized using an epifluorescence microscope. [4]

INNO-206 stock solutions (5.4 mg/mL) were prepared using 50% ethanol and 50% water and further diluted in sterile water. [1]
For the LAGκ-1A experiment, INNO-206 was administered to SCID mice at 10.8 mg/kg (doxorubicin equivalent dose of 8.0 mg/kg) once weekly. Mice were treated with conventional doxorubicin at 4.0 and 8.0 mg/kg once weekly. For the LAGκ-2 experiment, INNO-206 was administered once weekly (W) at doses of 2.7 and 5.4 mg/kg, or on 3 consecutive days (W-F) weekly at doses of 0.9 and 1.8 mg/kg. Bortezomib was administered twice weekly (W, F) at a dose of 0.5 mg/kg. Doxorubicin was administered to SCID mice at 2, 4, and 8 mg/kg, and PLD was administered to SCID mice at 2 mg/kg once weekly. Each drug was administered i.v. in a volume of 100 μL.[1]

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The (6-maleimidocaproyl)hydrazone derivative of doxorubicin (INNO-206) is an albumin-binding prodrug of doxorubicin with acid-sensitive properties that is being assessed clinically. The prodrug binds rapidly to circulating serum albumin and releases doxorubicin selectively at the tumor site. This novel mechanism may provide enhanced antitumor activity of doxorubicin while improving the overall toxicity profile. Preclinically, INNO-206 has shown superior activity over doxorubicin in a murine renal cell carcinoma model and in breast carcinoma xenograft models. In this work, we compared the antitumor activity of INNO-206 and doxorubicin at their respective maximum tolerated doses in three additional xenograft models (breast carcinoma 3366, ovarian carcinoma A2780, and small cell lung cancer H209) as well as in an orthotopic pancreas carcinoma model (AsPC-1). INNO-206 showed more potent antitumor efficacy than free doxorubicin in all tumor models and is thus a promising clinical candidate for treating a broad range of solid tumors[3].

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E0771 Murine Breast Cancer Model: Female C57BL/6 mice were implanted with 1×10^6 E0771 murine breast cancer cells into the mammary fat pad. When tumors reached approximately 150 mm³ in volume, treatment began (Day 0). Mice were injected intravenously via the femoral vein with either saline, free doxorubicin (7 mg/kg), or SynB1-ELP-INNO-206 (7 mg/kg doxorubicin equivalent) on Days 0, 2, 4, and 6 under isoflurane anesthesia. This dose was based on the maximum tolerated dose of the ELP carrier. [4]
Hyperthermia Application: For hyperthermia groups, immediately after drug injection, the tumor region was subjected to localized heating. An infrared laser probe was placed 2-3 mm above the skin over the tumor, while surrounding areas were shielded. A thermal cycling protocol was used: 20 minutes of heating (raising tumor temperature to ~41°C) followed by 10 minutes of cooling, repeated for a total of 2 hours. The animals were anesthetized during this procedure. [4]
Monitoring: Tumor dimensions were measured daily using calipers, and animal body weight was recorded. On Day 14, animals were euthanized, tumors were excised and weighed, and statistical analysis was performed. [4]

INNO-206 is an acid-sensitive doxorubicin prodrug designed to rapidly and selectively bind to cysteine-34 of circulating albumin after intravenous administration, thereby prolonging its plasma half-life and promoting tumor accumulation through enhanced permeability and retention (EPR) effects. [4] The carboxyhydrazone linker in INNO-206 is stable under physiological pH conditions, but it cleaves in the acidic environments of endosomes (pH 5.0–6.5) and lysosomes (pH 4–5), resulting in the release of active doxorubicin into the cells. [4]

The active drug doxorubicin released from INNO-206 has serious side effects, including neutropenia, mucositis, alopecia, nausea, myelosuppression, and cardiotoxicity. Prodrug strategies using albumin-bound and macromolecular carriers such as ELP aim to improve the therapeutic index by reducing systemic exposure and toxicity to normal tissues. [4] In animal studies, mice treated with SynB1-ELP-INNO-206 experienced less weight loss compared to mice treated with free doxorubicin, suggesting that the bound form may have reduced systemic toxicity. [4] The maximum tolerated dose (MTD) of the SynB1-ELP carrier in mice is 800 mg/kg when used alone. Higher doses combined with local hyperthermia near the femoral artery can lead to carrier accumulation, vascular occlusion, and limb necrosis. At the maximum tolerated dose of the SynB1-ELP-INNO-206 carrier, the equivalent dose of doxorubicin is approximately 7 mg/kg. [4]

[1]. Anti-Myeloma Effects of the Novel Anthracycline Derivative INNO-206. Clin Cancer Res.2012 18; 3856.

[2]. INNO-206 (DOXO-EMCH), an Albumin-Binding Prodrug of Doxorubicin Under Development for Phase II Studies. Current Bioactive Compounds, 2011, 7(1): 33-38(6).

[3]. INNO-206, the (6-maleimidocaproyl hydrazone derivative of doxorubicin), shows superior antitumor efficacy compared to doxorubicin in different tumor xenograft models and in an orthotopic pancreas carcinoma model. Invest New Drugs. 2010 Feb;28(1):14-9.

[4]. Cell penetrating peptides fused to a thermally targeted biopolymer drug carrier improve the delivery and antitumor efficacy of an acid-sensitive doxorubicin derivative. Int J Pharm. 2012 Oct 15;436(1-2):825-32.

Aldoxorubicin is an antitumor drug and an albumin-bound prodrug of doxorubicin. Aldoxorubicin is a 6-maleimide hexanoylhydrazone derivative prodrug of the anthracycline antibiotic doxorubicin (DOXO-EMCH) and possesses antitumor activity. After intravenous injection, Aldoxorubicin selectively binds to cysteine-34 of albumin via its maleimide moiety. In the acidic environment of the tumor, the acid-sensitive hydrazone linker breaks, releasing doxorubicin from the albumin carrier. Once inside the cell, it intercalates into DNA, inhibiting DNA synthesis and inducing apoptosis. Due to the high metabolic turnover, rapid angiogenesis, rich vascularity, and impaired lymphatic drainage of solid tumors, albumin tends to accumulate in them. This passive accumulation within the tumor may enhance the therapeutic effect of doxorubicin while minimizing systemic toxicity.
Drug Indications
Investigations are underway for the treatment of solid tumors.

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Mechanism of Action
INNO-206 is a (6-maleimide hexanoyl)hydrazone derivative of doxorubicin. INNO-206 is a prodrug of doxorubicin that binds to endogenous albumin after administration. The bound doxorubicin is released by the cleavage of acid-sensitive linkers in the acidic environment of tumor cells. In preclinical models, INNO-206 was superior to doxorubicin in both antitumor efficacy and toxicity.

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INNO-206 (formerly DOXO-EMCH) is a (6-maleimide hexanoyl)hydrazone derivative of doxorubicin. It represents a clinically validated technology that allows maleimide prodrugs to be in situ bound to endogenous albumin. [4]
This study explored its conjugation with a thermoresponsive elastin-like peptide (ELP) carrier fused with a cell-penetrating peptide (CPP). The combination of passive targeting (EPR effect), active thermotargeting (local hyperthermia-induced intratumoral ELP aggregation), and enhanced cellular uptake (via CPP) aims to optimize doxorubicin delivery. [4]
INNO-206 is currently undergoing a phase II clinical trial in soft tissue sarcoma at the time of publication. [4]

C37H42N4O13

750.748

750.274

C, 59.19; H, 5.64; N, 7.46; O, 27.70

1361644-26-9

MC-DOXHZN hydrochloride;480998-12-7;MC-DOXHZN;151038-96-9;Aldoxorubicin hydrochloride;1361563-03-2

9810709

Purple to purplish red solid powder

1.6±0.1 g/cm3

1.708

3.08

7

15

12

54

1510

6

[H][C@@]1(O[C@H]2C[C@@](/C(CO)=N/NC(CCCCCN3C(C=CC3=O)=O)=O)(O)CC(C2=C4O)=C(O)C5=C4C(C6=C(OC)C=CC=C6C5=O)=O)O[C@@H](C)[C@@H](O)[C@@H](N)C1

OBMJQRLIQQTJLR-USGQOSEYSA-N

InChI=1S/C37H42N4O13/c1-17-32(46)20(38)13-27(53-17)54-22-15-37(51,23(16-42)39-40-24(43)9-4-3-5-12-41-25(44)10-11-26(41)45)14-19-29(22)36(50)31-30(34(19)48)33(47)18-7-6-8-21(52-2)28(18)35(31)49/h6-8,10-11,17,20,22,27,32,42,46,48,50-51H,3-5,9,12-16,38H2,1-2H3,(H,40,43)/b39-23+/t17-,20-,22-,27-,32+,37-/m0/s1

N-[(E)-[1-[(2S,4S)-4-[(2R,4S,5S,6S)-4-amino-5-hydroxy-6-methyloxan-2-yl]oxy-2,5,12-trihydroxy-7-methoxy-6,11-dioxo-3,4-dihydro-1H-tetracen-2-yl]-2-hydroxyethylidene]amino]-6-(2,5-dioxopyrrol-1-yl)hexanamide

EMCH-doxorubicin; MC-DOXHZN hydrochloride; INNO 206; INNO-206 HCl; DOXO-EMCH; EMCH-Doxo; INNO206; INNO-206; Doxorubicin-EMCH; 151038-96-9; MC-Doxhzn; N-[(E)-[1-[(2S,4S)-4-[(2R,4S,5S,6S)-4-amino-5-hydroxy-6-methyloxan-2-yl]oxy-2,5,12-trihydroxy-7-methoxy-6,11-dioxo-3,4-dihydro-1H-tetracen-2-yl]-2-hydroxyethylidene]amino]-6-(2,5-dioxopyrrol-1-yl)hexanamide; Aldoxorubicin

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: (1). Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture.  (2). This product is not stable in solution, please use freshly prepared working solution for optimal results.

Ship with dry ice

DMSO: ≥ 50 mg/mL (~66.6 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (3.33 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 (3.33 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 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.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (3.33 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.)