Topoisomerase II; Daunorubicins/Doxorubicins
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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Aldoxorubicin HCl (INNO-206) inhibited the viability of multiple myeloma cell lines (RPMI8226, U266, MM1S) in a concentration- and pH-dependent manner. At pH 5, cell viability was markedly reduced compared to pH 7. For example, in RPMI8226 cells at pH 5, viability was essentially eliminated at INNO-206 concentrations ≥0.54 µmol/L (equivalent to ≥0.4 µmol/L doxorubicin). In contrast, under the same acidic conditions, the anti-myeloma effects of free doxorubicin were diminished.[1]
Aldoxorubicin HCl (INNO-206) exhibited antiangiogenic activity in a chorioallantoic membrane/feather bud (CAM/FB) assay. It inhibited feather bud development and reduced the expression of the endothelial cell marker Flk-1 gene in a concentration- and pH-dependent fashion, with more pronounced inhibition at pH 5.[1]

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 SCID mice bearing the LAGκ-1A human multiple myeloma xenograft, intravenous administration of Aldoxorubicin HCl (INNO-206) at 10.8 mg/kg (doxorubicin-equivalent dose of 8.0 mg/kg) once weekly significantly reduced tumor volume and serum human IgG levels compared to vehicle-treated controls on days 28, 35, and 42. 90% of mice survived until day 42.[1]
In SCID mice bearing the LAGκ-2 human multiple myeloma xenograft, Aldoxorubicin HCl (INNO-206) administered at 5.4 mg/kg once weekly or 1.8 mg/kg three times weekly significantly inhibited tumor growth compared to controls.[1]
The combination of Aldoxorubicin HCl (INNO-206) (2.7 mg/kg once weekly) with bortezomib (0.5 mg/kg twice weekly) in the LAGκ-2 model resulted in a more pronounced and sustained reduction in tumor volume compared to either agent alone, with tumors becoming non-palpable in some mice and showing delayed regrowth.[1]
Intravenous injection of Evans blue dye (which binds to albumin) into tumor-bearing mice demonstrated that albumin (and by extension, albumin-bound INNO-206) was taken up by multiple myeloma xenograft tumors within 4 hours.[1]

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.
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 viability was assessed using the MTS assay. Human multiple myeloma cell lines (RPMI8226, U266, MM1S) were seeded in 96-well plates. After 24 hours, cells were cultured with medium containing Aldoxorubicin HCl (INNO-206) or doxorubicin at various concentrations (INNO-206: 0.27 to 2.16 µmol/L; doxorubicin-equivalent: 0.2 to 1.6 µmol/L) for 48 hours. Drugs were pre-incubated at pH 5 or pH 7 for 45 minutes before addition. MTS reagent was added, and after 1-4 hours incubation, absorbance at 490 nm was measured. The quantity of formazan product is proportional to the number of living cells.[1]
The antiangiogenic effect was evaluated using a chorioallantoic membrane/feather bud (CAM/FB) coculture model. Fertilized chick eggs were incubated for 8 days. Dorsal skin feather buds (FBs) were excised, placed on culture inserts, and exposed to Aldoxorubicin HCl (INNO-206) or doxorubicin at different pH levels (5, 6, 7) for 48 hours. The FBs were then transferred onto the chorioallantoic membrane (CAM) of another 8-day-old embryo and incubated for an additional 4 days. Feather development was assessed microscopically.[1]
Gene expression analysis was performed on the FB tissue. Total RNA was isolated, reverse-transcribed to cDNA, and PCR was conducted using primers for the endothelial marker Flk-1 and GAPDH (as a loading control). PCR products were analyzed by gel electrophoresis and densitometry.[1]

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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For efficacy studies, human multiple myeloma xenografts (LAGκ-1A or LAGκ-2) were established in the gluteal muscle of 6-8 week old male CB17 SCID mice. Treatment began 7 days after tumor implantation.[1]
In the LAGκ-1A model, Aldoxorubicin HCl (INNO-206) was administered intravenously once weekly at a dose of 10.8 mg/kg (in 100 µL volume). Doxorubicin was administered intravenously once weekly at 4.0 and 8.0 mg/kg for comparison.[1]
In the LAGκ-2 model, Aldoxorubicin HCl (INNO-206) was administered intravenously either once weekly (5.4 mg/kg or 2.7 mg/kg) or three times per week on consecutive days (1.8 mg/kg or 0.9 mg/kg). Bortezomib was administered intravenously twice weekly at 0.5 mg/kg. Doxorubicin and PEGylated liposomal doxorubicin (PLD) were administered intravenously once weekly at 2 mg/kg in combination studies.[1]
Stock solutions: INNO-206 (5.4 mg/mL in 50% ethanol/50% water, diluted in sterile water); bortezomib (1 mg/mL stock diluted in 0.9% sodium chloride); doxorubicin (2 mg/mL in PBS); PLD (2 mg/mL diluted in sterile water).[1]
Tumor volume was measured weekly using calipers. Serum human IgG levels were determined by weekly retro-orbital bleeding and ELISA.[1]
To assess albumin uptake, a 2% Evans blue dye solution in sterile water was injected intravenously into tumor-bearing mice. Mice were sacrificed 4 hours post-injection, tumors were extracted, and dye accumulation was visually assessed and quantified using image analysis.[1]

This study did not provide specific pharmacokinetic parameters (e.g., clearance, half-life, oral bioavailability) for adorocillin hydrochloride (INNO-206). The manuscript cited other studies showing that INNO-206 rapidly binds to cysteine-34 on serum albumin after intravenous injection and is released under acidic conditions. [1]
The study noted that the dose used in xenotransplantation studies (0.9–10.8 mg/kg/week) is clinically achievable and is significantly lower than the well-tolerated dose of 200 mg/m² (equivalent to 148.6 mg/m² of adorocillin) established in Phase I clinical trials. [1]

In the SCID mouse xenograft model, aldehyde-rubicin hydrochloride (INNO-206) was well tolerated at doses comparable to the toxic doses of free doxorubicin. In the LAGκ-1A model, once-weekly treatment with INNO-206 (10.8 mg/kg) resulted in a 90% survival rate on day 42, while all mice treated with once-weekly doxorubicin (4.0 or 8.0 mg/kg) died on day 42. [1] In the LAGκ-2 combination therapy study, once-weekly treatment with INNO-206 (2.7 mg/kg) in combination with bortezomib did not result in significant weight loss and resulted in a 70% survival rate. In stark contrast, all mice treated with equal or lower doses of free doxorubicin (2 mg/kg) or PLD (2 mg/kg) in combination with bortezomib died on day 28. [1]
The manuscript cites other studies indicating that the maximum tolerated dose (MTD) of INNO-206 in animals is 2 to 5 times that of free doxorubicin, and that its cardiotoxicity and accumulation in non-target organs such as the heart, liver, and kidneys are reduced. [1]

[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.

[5]. Hydrazone, Amide, Carbamate, Macromolecular and Other Prodrugs of Doxorubicin. The Open Drug Delivery Journal, 2008, 2, 77-85.

Aldoxorubicin is an antitumor drug and an albumin-bound prodrug of doxorubicin. Aldoxorubicin is a 6-maleimide hexanoyl hydrazone 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 rate, rapid angiogenesis, rich vascularity, and impaired lymphatic drainage of solid tumors, albumin tends to accumulate in solid tumors. This passive accumulation within the tumor may enhance the therapeutic effect of doxorubicin while minimizing systemic toxicity. Mechanism of action: INNO-206 is a (6-maleimide hexanoyl) hydrazone derivative of doxorubicin. INNO-206 is an doxorubicin prodrug that binds to endogenous albumin after administration. The bound doxorubicin is released through the cleavage of acid-sensitive linkers in the acidic environment of tumor cells. In preclinical models, INNO-206 demonstrated superior antitumor efficacy and toxicity compared to doxorubicin. Doxorubicin has shown efficacy, particularly in combination therapy for multiple myeloma; however, its side effects limit its application. INNO-206 is an albumin-bound doxorubicin prodrug that is released from albumin under acidic conditions. Since INNO-206 has not been previously evaluated in any hematologic malignancies, we investigated its anti-multiple myeloma activity. Experimental Design: We used the MTS assay to detect the effect of INNO-206 on the proliferation of multiple myeloma cells at different pH values and the chorioallantoic membrane/feather bud assay to detect its anti-angiogenic activity. Furthermore, using a multiple myeloma xenograft model, we compared the anti-multiple myeloma effects and toxicity of INNO-206 with those of conventional doxorubicin, pegylated liposomal doxorubicin (PLD), and in combination with bortezomib. Results: INNO-206 inhibited angiogenesis and reduced the growth of multiple myeloma cells in a pH-dependent manner. INNO-206 alone produced significant anti-multiple myeloma effects in vivo at doses comparable to the toxic doses of doxorubicin; the anti-multiple myeloma effect of INNO-206 in combination with bortezomib was enhanced compared to either drug alone. Conversely, all mice treated with bortezomib in combination with doxorubicin or PLD died. Conclusion: These results indicate that INNO-206 has anti-multiple myeloma effects both in vitro and in vivo, and can enhance the antitumor activity of bortezomib. These results suggest that INNO-206 may provide an anthracycline for patients with multiple myeloma, and that INNO-206 can be safely administered at higher doses compared to free doxorubicin, thus achieving superior efficacy compared to anthracyclines currently used to treat this B-cell malignancy. [1] Clin Cancer Res; 18(14); 3856–67. ©2012 AACR.

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The (6-maleimide hexanoyl)hydrazone derivative of doxorubicin (INNO-206, formerly known as DOXO-EMCH) is a prodrug of the anticancer drug doxorubicin, which was planned to be administered intravenously in 2011 and selectively bind to cysteine-34 of endogenous albumin within minutes. Preclinical and clinical studies have shown that the albumin-bound form of INNO-206 has a larger AUC, a smaller volume of distribution, and a lower clearance rate compared to doxorubicin. Its uptake in solid tumors is mediated by the pathophysiological mechanisms of tumor tissue, characterized by angiogenesis, angiogenesis, vascular structural defects, and impaired lymphatic drainage. This prodrug contains an acid-sensitive hydrazone linker, enabling extracellular release of doxorubicin in the weakly acidic environment common in tumor tissue, or intracellular release in acidic endosomes or lysosomal compartments after tumor cells take up the albumin conjugate. In various preclinical tumor models, INNO-206 demonstrated significantly superior antitumor efficacy compared to free doxorubicin. In a Phase I study, INNO-206 showed good safety at doses up to 260 mg/m² doxorubicin equivalents. Although not the primary endpoint of this Phase I study, INNO-206 induced tumor regression in breast cancer, small cell lung cancer, and sarcoma. Phase II clinical trials for gastric cancer, pancreatic cancer, and sarcoma are planned to begin at the end of 2010. [2] Elastin-like peptides (ELPs) are thermoresponsive macromolecular carriers that passively accumulate in solid tumors and further aggregate in tumor tissue upon exposure to high temperatures. This study conjugated ELPs with the anticancer drug doxorubicin (DOXO) and three different cell-penetrating peptides (CPPs) to inhibit tumor growth in mice compared to free doxorubicin. Fluorescence microscopy studies of MCF-7 breast cancer cells showed that all three different CPP-ELP-DOXO conjugates could deliver doxorubicin to the cell nucleus. All CPP-ELP-DOXO conjugates exhibited cytotoxicity at 42 °C, with IC50 values ranging from 12 to 30 μM, but the ELP carrier using SynB1 as the cell-penetrating peptide showed the lowest inherent cytotoxicity. Therefore, the antitumor efficacy of SynB1-ELP-DOXO versus doxorubicin was compared under hyperthermic conditions. Female C57BL/6 mice carrying mammary tumors from the E0771 mouse were treated with either free doxorubicin or the SynB1-ELP-DOXO conjugate, with or without local hyperthermia targeting the tumor. Under hyperthermic conditions, SynB1-ELP-DOXO showed twice the tumor inhibition rate of an equivalent dose of free doxorubicin, making it an ideal candidate for optimizing thermoresponsive drug polymer conjugates. [4]

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Doxorubicin hydrochloride (INNO-206) is a prodrug designed to covalently bind to circulating albumin via a pH-sensitive linker. It can specifically release the active drug doxorubicin in acidic microenvironments, such as tumors (due to the Wahlberg effect) and bone marrow near osteoclasts in multiple myeloma. [1]
This study is the first to evaluate the efficacy of INNO-206 in hematologic malignancies (multiple myeloma). The acid-dependent release mechanism may provide a therapeutic advantage for the acidic bone marrow microenvironment in myeloma patients. [1]
In preclinical models, doxorubicin hydrochloride (INNO-206) combined with the proteasome inhibitor bortezomib showed enhanced anti-myeloma efficacy without increased toxicity, suggesting it may be a potential clinical combination therapy strategy. [1]
This study was funded by CytRx. [1]

C37H43CLN4O13

787.209329843521

786.251

C, 56.45; H, 5.51; Cl, 4.50; N, 7.12; O, 26.42

480998-12-7

Aldoxorubicin;1361644-26-9;MC-DOXHZN;151038-96-9

10056071

Red solid powder

8

15

12

55

1510

6

Cl.O(C1CC(C(C(C)O1)O)N)C1C2C(=C3C(C4C(=CC=CC=4C(C3=C(C=2C[C@@](C(CO)=NNC(CCCCCN2C(C=CC2=O)=O)=O)(C1)O)O)=O)OC)=O)O

NGKHWQPYPXRQTM-UKFSEGPMSA-N

InChI=1S/C37H42N4O13.ClH/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);1H/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;hydrochloride

INNO-206 HCl; MC-DOXHZN hydrochloride; INNO206; DOXO-EMCH; EMCH-Doxo; EMCH-doxorubicin; INNO-206; INNO 206; MC-DOXHZN HCl; INNO-206 hydrochloride; Aldoxorubicin?HCl; 1361563-03-2; Aldoxorubicin (hydrochloride); Aldoxorubicin Hydrochloride [USAN];Aldoxorubicin.

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light.

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

DMSO: ~25 mg/mL (~31.8 mM)
H2O: ~12.5 mg/mL (~15.9 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (2.64 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 2: ≥ 2.08 mg/mL (2.64 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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 (Please use freshly prepared in vivo formulations for optimal results.)