Daunomustine is the lead compound of our hybrid anthracycline series, combining the anthracycline ring system, as in doxorubicin, with a nitrosoureido functionality found in alkylating agents and a demonstrated superiority over standard-of-care anthracycline drug, doxorubicin.

Relative to doxorubicin, daunomustine is:

– stronger in DNA binding and intercalation.
– more potent in producing direct DNA strand breaks and inhibiting both DNA topoisomerase II activity and RNA synthesis.
– comparable in inhibition of DNA synthesis.
– therapeutically superior to a simple admixture of anthracyclines and alkylating agents.

Daunomustine achieves these functions without the cardiotoxicity produced by doxorubicin and daunorubicin. The increased potency of daunomustine over doxorubicin and daunorubicin is significantly greater in multidrug-resistant cells.

Paradox Pharmaceuticals has completed toxicity and toxicology evaluation of daunomustine in a set of quality-assured GLP canine studies, as appropriate for FDA-delineated phase 1 clinical trials for ultimate drug approval and will be seeking conditional approval from the FDA for the treatment of canine lymphoma.

Pandey S, Bourn J, Cekanova M. Mutations of p53 decrease sensitivity to the anthracycline treatments in bladder cancer cells. Oncotarget. 2018; 9:28514-31. PMID: 29983877. (PubMed) (PDF)

Pawlik CA, Israel M, Sweatman TW, Lothstein L.  Cellular resistance against the novel hybrid anthracycline N-(2-chloroethyl)-N-nitrosoureidodaunorubicin (AD 312) is mediated by combined altered topoisomerase II and O6-methylguanine-DNA methyltransferase activities.  Oncol Res. 1998; 10:209-17.  PMID: 9778692 (PubMed)

Glaves D, Murray MK, Raghavan D. Novel bifunctional anthracycline and nitrosourea chemotherapy for human bladder cancer: analysis in a preclinical survival model. Clin Cancer Res. 1996; 2:1315-9. PMID: 9816302 (PubMed)

Benzarubicin (N-benzyladriamycin-14-valerate; AD 198), designed and developed by the researchers at Paradox Pharmaceuticals, is a unique, multifunctional anti-tumor compound with potential therapeutic superiority over conventional anthracycline drug formulations.

Novel Mechanism of Action

The combined N-benzyl and 14-O-acyl substitutions increase cell penetration and target the compound to the cytoplasm of cancer cells where it targets conventional and novel members of the protein kinase C (PKC) family of cell signaling enzymes.  Benzarubicin binds to the C1b regulatory domain of PKC, as well as other C1b-containing proteins, such as RasGRP and ß2-chimaerin to modify activity.  In tumor cells, benzarubicin activates the PKC-delta isoform to trigger rapid apoptotic cell death through a novel mitochondrial pathway. Benzarubicin also suppresses expression of the tumor promoting protein, c-Myc to inhibit tumor cell growth and induce cell death.

Overcomes Drug Resistance

Both its structure and mechanism of action, benzarubicin circumvents multiple mechanisms of clinically important cellular drug resistance produced by increased expression of multidrug transport proteins, anti-apoptotic proteins, and proliferative signaling proteins such as NF-kB and Bcr-Abl kinase.

Unique Bifunctional Drug

Benzarubicin exhibits additional multifunctional antitumor activity through gradual biotransformation to N-benzyladriamycin (AD 288). AD 288 possesses antitumor potency comparable to benzarubicin, but through catalytic inhibition of topoisomerase II rather than PKC activation.

Non-Cardiotoxic and Cardioprotective

In contrast to anthracyclines in current clinical use, benzarubicin is non-cardiotoxic after chronic dosing. Indeed, due to its ability to activate PKC-epsilon in cardiac myocyte cells, benzarubicin activates a cardioprotective response, protecting the heart against doxorubicin-induced damage and against damage inflicted by oxygen reperfusion into the heart after an ischemic event. In vivo rodent studies with benzarubicin have demonstrated greater systemic safety than doxorubicin, with no detectable pulmonary or hepatic toxicity at its maximum tolerated dose.

Salvage Therapy for Metastatic Veterinary Tumors

Benzarubicin is currently being developed by Paradox Pharmaceuticals as a veterinary antitumor agent for the treatment of metastatic canine and feline tumors for which anthracycline-based therapy is currently indicated or has failed to achieve durable remission.

Pandey S, Bourn J, Cekanova M. Mutations of p53 decrease sensitivity to the anthracycline treatments in bladder cancer cells. Oncotarget. 2018; 9:28514-31. PMID: 29983877. (PubMed) (PDF)

Mittal NK, Mandal B, Balabathula P, Setua S, Janagam DR, Lothstein L, Thoma LA, Wood GC. Formulation, development, and in vitro evaluation of a CD22-targeted liposomal system containing a non-cardiotoxic anthracycline for B-cell malignancies. Pharmaceutics. 2018; 10. pii: E50. PMID: 29662041. (PubMed) (PDF)

Edwards SK, Han Y, Liu Y, Kreider BZ, Liu Y, Grewal S, Desai A, Baron J, Moore CR, Luo C, Xie P. Signaling mechanisms of bortezomib in TRAF3-deficient mouse B lymphoma and human multiple myeloma cells. Leuk Res. 2016; 41:85-95. PMID: 26740054. (PubMed) (PDF)

Smolensky D, Rathore K, Cekanova M. Phosphatidylinositol- 3-kinase inhibitor induces chemosensitivity to a novel derivative of doxorubicin, AD 198 chemotherapy in human bladder cancer cells in vitro. BMC Cancer. 2015; 15:927. PMID: 26597249. (PubMed) (PDF)

Rathore K, Cekanova M. A novel derivative of doxorubicin, AD 198, inhibits canine transitional cell carcinoma and osteosarcoma cells in vitro. Drug Des Devel Ther. 2015; 9:5323-35. PMID: 26451087. (PubMed) (PDF)

Moore CR, Edwards SK, Xie P. Targeting TRAF3 Downstream Signaling Pathways in B cell Neoplasms. J Cancer Sci Ther. 2015 7:67-74. PMID: 25960828. (PubMed) (PDF)

Mittal NK, Bhattacharjee H, Mandal B, Balabathula P, Thoma LA, Wood GC. Targeted liposomal drug delivery systems for the treatment of B cell malignancies. J Drug Target. 2014; 22:372-86. PMID: 24433007. (PubMed) (PDF)

Edwards SK, Moore CR, Liu Y, Grewal S, Covey LR, Xie P. N-benzyladriamycin-14-valerate (AD 198) exhibits potent anti-tumor activity on TRAF3-deficient mouse B lymphoma and human multiple myeloma. BMC Cancer. 2013; 13:481. PMID: 24131623. (PubMed)

Díaz Bessone MI, Berardi DE, Campodónico PB, Todaro LB, Lothstein L, Bal de Kier Joffé ED, Urtreger AJ. Involvement of PKC delta (PKCδ) in the resistance against different doxorubicin analogs. Breast Cancer Res Treat. 2011; 126:577-87. PMID: 20512658 (PubMed)

Cai C, Lothstein L, Morrison RR, Hofmann PA. Protection from doxorubicin-induced cardiomyopathy using the modified anthracycline N-benzyladriamycin-14-valerate (AD 198). J Pharmacol Exp Ther. 2010; 335:223-30. PMID: 20668052. (PubMed) (PDF)

Du Z, Fan M, Kim JG, Eckerle D, Lothstein L, Wei L, Pfeffer LM. An interferon-resistant Daudi cell line with a STAT2 defect is resistant to apoptosis induced by chemotherapeutic agents. J Biol Chem. 2009; 284:27808-15. PMID: 19687011 (PubMed) (PDF)

Lothstein L, Savranskaya L, Barrett CM, Israel M, Sweatman TW. N-Benzyladriamycin-14-valerate (AD 198) activates protein kinase C-delta holoenzyme to trigger mitochondrial depolarization and cytochrome c release independently of permeability transition pore opening and Ca2+ influx. Anticancer Drugs. 2006; 17:495-502. PMID: 16702805. (PubMed)

He Y, Liu J, Durrant D, Yang HS, Sweatman T, Lothstein L, Lee RM. N-benzyladriamycin-14-valerate (AD 198) induces apoptosis through protein kinase C-delta-induced phosphorylation of phospholipid scramblase 3. Cancer Res. 2005; 65:10016-23. PMID: 16267027. (PubMed) (PDF)

Bilyeu JD, Panta GR, Cavin LG, Barrett CM, Turner EJ, Sweatman TW, Israel M, Lothstein L, Arsura M. Circumvention of nuclear factor kappaB-induced chemoresistance by cytoplasmic-targeted anthracyclines. Mol Pharmacol. 2004; 65:1038-47. PMID: 15044634. (PubMed) (PDF)

Panta GR, Kaur S, Cavin LG, Cortés ML, Mercurio F, Lothstein L, Sweatman TW, Israel M, Arsura M. ATM and the catalytic subunit of DNA-dependent protein kinase activate NF-kappaB through a common MEK/extracellular signal-regulated/p90(rsk) signaling pathway in response to distinct forms of DNA damage. Mol Cell Biol. 2004; 24:1823-35. PMID: 14966265 (PubMed) (PDF)

Roaten JB, Kazanietz MG, Caloca MJ, Bertics PJ, Lothstein L, Parrill AL, Israel M, Sweatman TW. Interaction of the novel anthracycline antitumor agent N-benzyladriamycin-14-valerate with the C1-regulatory domain of protein kinase C: structural requirements, isoform specificity, and correlation with drug cytotoxicity. Mol Cancer Ther. 2002; 1:483-92. PMID: 12479266. (PubMed) (PDF)

Barrett CM, Lewis FL, Roaten JB, Sweatman TW, Israel M, Cleveland JL, Lothstein L. Novel extranuclear-targeted anthracyclines override the antiapoptotic functions of Bcl-2 and target protein kinase C pathways to induce apoptosis. Mol Cancer Ther. 2002; 1:469-81. PMID: 12479265. (PubMed) (PDF)

Roaten JB, Kazanietz MG, Sweatman TW, Lothstein L, Israel M, Parrill AL. Molecular models of N benzyladriamycin-14-valerate (AD 198) in complex with the phorbol ester-binding C1b domain of protein kinase C-delta. J Med Chem. 2001; 44:1028-34. PMID: 11297449. (PubMed)

Lothstein L, Israel M, Sweatman TW. Anthracycline drug targeting: cytoplasmic versus nuclear–a fork in the road. Drug Resist Updat. 2001; 4:169-77. Review. PMID: 11768330 (PubMed)

Sweatman TW, Seshadri R, Israel M. Pharmacology of N-benzyladriamycin-14-valerate in the rat. Cancer Chemother Pharmacol. 1999; 43:419-26. PMID: 10100599. (PubMed)

Lothstein L, Suttle DP, Roaten JB, Koseki Y, Israel M, Sweatman TW. Catalytic inhibition of DNA topoisomerase II by N-benzyladriamycin (AD 288). Biochem Pharmacol. 2000; 60:1621-8. PMID: 11077044 (PubMed)

Pivarubicin (N-Benzyladriamycin-14-pivalate; AD 445) was developed as a hydrolytically stable alternative to benzarubicin for specific indications. The tertiary trimethyl arrangement (pivalate) sterically hinders esterase-mediated cleavage of the 14-O-acyl linkage with any apparent effect on the PKC-mediated of pivarubicin. In all studies performed thus far, pivarubicin possesses the functional characteristics of benzarubicin, including potency against an array of human tumor cell types and circumvention of multiple mechanisms of drug resistance and non-cardiotoxicity at an experimental therapeutic dose.

Lothstein L, Savranskaya L, Barrett CM, Israel M, Sweatman TW. N-Benzyladriamycin-14-valerate (AD 198) activates protein kinase C-delta holoenzyme to trigger mitochondrial depolarization and cytochrome c release independently of permeability transition pore opening and Ca2+ influx. Anticancer Drugs. 2006; 17:495-502. PMID: 16702805. (PubMed)

Bilyeu JD, Panta GR, Cavin LG, Barrett CM, Turner EJ, Sweatman TW, Israel M, Lothstein L, Arsura M. Circumvention of nuclear factor kappaB-induced chemoresistance by cytoplasmic-targeted anthracyclines. Mol Pharmacol. 2004; 65:1038-47. PMID: 15044634. (PubMed) (PDF)

FDA-Approved

Valrubicin (N-Trifluoroacetyladriamycin-14-valerate; AD 32), currently FDA-approved for intravesicular use against superficial bladder cancer (distributed by Endo Pharmaceuticals), exemplifies the successful clinical development, by members of the Paradox Pharmaceuticals scientific team, of anthracyclines that are functionally distinct from and superior to conventional anthracyclines, such as doxorubicin and daunorubicin.

Novel Biofunctional Agent

In vivo pre-clinical analyses have shown that valrubicin is superior to doxorubicin in the control of hematologic tumors as well as lung tumors, osteosarcoma, and melanoma. In pre-clinical studies, valrubicin enters cells more rapidly than doxorubicin, resulting in more extensive solid tumor penetration. The mechanism of action of valrubicin, while still not fully elucidated, is restricted to the cytoplasm, producing G2/M blockade and cell death following marked inhibition of DNA and RNA synthesis, but without direct nuclear DNA interaction and topoisomerase II inhibitory activity. The valerate side chain is susceptible to hydrolysis, resulting in the generation of N-trifluoroacetyladriamycin (AD 41), a mixed topoisomerase I/II inhibitor.

Systemic Superiority to Doxorubicin

– no cumulative cardiotoxicity
– no contact toxicity following administration into various body cavities or inadvertent venous extravasation
– reduced gastrointestinal toxicity and alopecia than comparable doses of doxorubicin

Novel Radiation Enhancing Agent

The pursuit of new therapeutic applications has led to our discovery that valrubicin synergizes with ionizing radiation (IR). Preliminary studies from our research team indicate that low-dose, sub-cytotoxic valrubicin synergizes with low-dose, sub-cytotoxic IR to achieve persistent inhibition in vitro squamous carcinoma cells and in vivo of oral tumors. The synergistic effects of valrubicin and IR is through a novel, non-nuclear mechanism.

Valrubicin and other related compounds being developed by Paradox will satisfy an unmet clinical need for effective radiation enhancing agents: 1) through efficient delivery of a non-contact toxic lipophilic drug directly to the tumor site coupled with rapid drug permeation into the tumor cells and 2) with drugs that are intrinsically less toxic to normal tissue but whose mechanism of action potentiates IR therapeutic effects. Benefits to patients would include more effective control of tumor and reduction in severe and often irreversible adverse effects produced by high-dose IR.

Sabnis N, Nair M, Israel M, McConathy WJ, Lacko AG. Enhanced solubility and functionality of valrubicin (AD-32) against cancer cells upon encapsulation into biocompatible nanoparticles.  Int J Nanomedicine. 2012; 7:975-83. Epub 2012 Feb 22. PMID: 22393294 (PubMed) (PDF)

Wani MK, Koseki Y, Yarber RH, Sweatman TW, Ahmed A, Samant S, Hengesteg A, Israel M, Robbins KT. Rationale for intralesional valrubicin in chemoradiation of squamous cell carcinoma of the head and neck. Laryngoscope. 2000; 110:2026-32. (PubMed)

Greenberg RE, Bahnson RR, Wood D, Childs SJ, Bellingham C, Edson M, Bamberger MH, Steinberg GD, Israel M, Sweatman T, Giantonio B, O’Dwyer PJ. Initial report on intravesical administration of N-trifluoroacetyladriamycin-14-valerate (AD 32) to patients with refractory superficial transitional cell carcinoma of the urinary bladder. Urology. 1997; 49:471-5. PMID: 912372. (PubMed)