Evofosfamide, formerly known as TH-302 is an

investigational new drug The United States Food and Drug Administration's Investigational New Drug (IND) program is the means by which a pharmaceutical industry, pharmaceutical company obtains permission to start human clinical trials and to ship an experimental drug inte ...

that is being evaluated for the treatment of multiple tumor types, including

pancreatic cancer Pancreatic cancer arises when cell (biology), cells in the pancreas, a glandular organ behind the stomach, begin to multiply out of control and form a Neoplasm, mass. These cancerous cells have the malignant, ability to invade other parts of ...

soft tissue sarcoma A soft-tissue sarcoma (STS) is a malignant tumor, a type of cancer, that develops in soft tissue. A soft-tissue sarcoma is often a painless mass that grows slowly over months or years. They may be superficial or deep-seated. Any such unexplained ...

, and

multiple myeloma Multiple myeloma (MM), also known as plasma cell myeloma and simply myeloma, is a cancer of plasma cells, a type of white blood cell that normally produces antibody, antibodies. Often, no symptoms are noticed initially. As it progresses, bone ...

, often in combination with other therapies. It is a hypoxia-activated

prodrug A prodrug is a pharmacologically inactive medication or compound that, after intake, is metabolized (i.e., converted within the body) into a pharmacologically active drug. Instead of administering a drug directly, a corresponding prodrug can be ...

designed to target and kill hypoxic cells within tumors. It functions by releasing the DNA crosslinking agent bromo-iso

phosphoramide Phosphoramide is a chemical compound with the molecular formula . It is a derivative of phosphoric acid in which each of the hydroxyl groups have been replaced with an amino group. In bulk, the compound is a white solid which is soluble in polar s ...

mustard under low oxygen conditions, making it potentially effective against tumor regions where standard

chemotherapy Chemotherapy (often abbreviated chemo, sometimes CTX and CTx) is the type of cancer treatment that uses one or more anti-cancer drugs (list of chemotherapeutic agents, chemotherapeutic agents or alkylating agents) in a standard chemotherapy re ...

and radiation therapies are less effective due to hypoxia. Commercialization has not been pursued due to the failure of several clinical trials.

Pharmacology

Evofosfamide is a 2-nitroimidazole prodrug of the

cytotoxin Cytotoxicity is the quality of being toxic Toxicity is the degree to which a chemical substance or a particular mixture of substances can damage an organism. Toxicity can refer to the effect on a whole organism, such as an animal, bacterium ...

bromo-isophosphoramide mustard (Br-IPM). Evofosfamide is activated by a process that involves a 1-electron (1 e⁻) reduction mediated by ubiquitous cellular reductases, such as the NADPH cytochrome P450, to generate a radical anion prodrug: *A) In the presence of oxygen (normoxia) the radical anion prodrug reacts rapidly with oxygen to generate the original prodrug and

superoxide In chemistry, a superoxide is a compound that contains the superoxide ion, which has the chemical formula . The systematic name of the anion is dioxide(1−). The reactive oxygen ion superoxide is particularly important as the product of t ...

. Therefore, evofosfamide is relatively inert under normal oxygen conditions, remaining intact as a prodrug. *B) When exposed to severe hypoxic conditions (< 0.5% O₂; hypoxic zones in many tumors), however, the radical anion undergoes irreversible fragmentation, releasing the active drug Br-IPM and an

azole Azoles are a class of five-membered heterocyclic compounds containing a nitrogen atom and at least one other non-carbon atom (i.e. nitrogen, sulfur, or oxygen) as part of the ring. Their names originate from the Hantzsch–Widman nomenclature. Th ...

derivative. The released cytotoxin Br-IPM alkylates DNA, inducing intrastrand and interstrand crosslinks. Evofosfamide is largely inactive under normal oxygen levels. In areas of hypoxia, evofosfamide becomes activated and converts to an alkylating cytotoxic agent resulting in DNA cross-linking. This renders cells unable to replicable their DNA and divide, leading to

apoptosis Apoptosis (from ) is a form of programmed cell death that occurs in multicellular organisms and in some eukaryotic, single-celled microorganisms such as yeast. Biochemistry, Biochemical events lead to characteristic cell changes (Morphology (biol ...

. This investigational therapeutic approach of targeting the cytotoxin to hypoxic zones in tumors may cause less broad systemic toxicity that is seen with untargeted cytotoxic

chemotherapies Chemotherapy (often abbreviated chemo, sometimes CTX and CTx) is the type of cancer treatment that uses one or more anti-cancer drugs ( chemotherapeutic agents or alkylating agents) in a standard regimen. Chemotherapy may be given with a cura ...

. The activation of evofosfamide to the active drug Br-IPM and the mechanism of action (MOA) via cross-linking of DNA is shown schematically below:

Drug development history

Phosphorodiamidate In organophosphorus chemistry, phosphoramidates (sometimes also called amidophosphates) are a class of phosphorus compounds structurally related to phosphates (or organophosphates) via the substitution of an group for an amine group (). They ...

-based, DNA-crosslinking, bis-alkylator mustards have long been used successfully in cancer chemotherapy and include e.g. the prodrugs

ifosfamide Ifosfamide, sold under the brand name Ifex among others, is a chemotherapy medication used to treat a number of types of cancer. This includes testicular cancer, soft tissue sarcoma, osteosarcoma, bladder cancer, small cell lung cancer, c ...

and

cyclophosphamide Cyclophosphamide (CP), also known as cytophosphane among other names, is a medication used as chemotherapy and to suppress the immune system. As chemotherapy it is used to treat lymphoma, multiple myeloma, leukemia, ovarian cancer, breast cancer ...

. To demonstrate that known drugs of proven efficacy could serve as the basis of efficacious hypoxia-activated prodrugs, the 2-nitroimidizole HAP of the active phosphoramidate bis-alkylator derived from ifosfamide was synthesized. The resulting compound, TH-281, had a high HCR (hypoxia cytotoxicity ratio), a quantitative assessment of its hypoxia selectivity. Subsequent structure-activity relationship (SAR) studies showed that replacement of the chlorines in the alkylator portion of the prodrug with bromines improved potency about 10-fold. The resulting, final compound is evofosfamide (TH-302) which was developed by Threshold Pharmaceuticals Inc. . Threshold Pharmaceuticals Inc. applied for a patent on evofosfamide in 2006 which was granted in 2011. In 2012, Threshold signed a global license and co-development agreement for evofosfamide with

Merck KGaA The Merck Group, branded and commonly known as Merck, is a German Multinational corporation, multinational science and technology company headquartered in Darmstadt, with about 60,000 employees and a presence in 66 countries. The group include ...

. Threshold was responsible for the development of evofosfamide in the soft tissue sarcoma indication in the United States. In all other cancer indications, Threshold and Merck KGaA developed evofosfamide together. After evofosfamide failed to improve longevity in patients in phase three clinical trials, Merck abandoned attempts to commercialize evofosfamide in 2015.

Chemistry

Synthesis

Evofosfamide synthesis involves several steps, starting with the preparation of 2-nitroimidazole derivatives: # Preparation of 2-nitroimidazole: This is the key bioreductive group used in the synthesis. # Formation of the prodrug: The 2-nitroimidazole is linked to a brominated derivative of isophosphoramide mustard. # Activation under hypoxic conditions: In low oxygen environments, typical of solid tumors, the prodrug is activated to release the cytotoxic agent. The activation under hypoxic conditions allows evofosfamide to target hypoxic tumor cells selectively, making it a candidate in for cancer treatment.

Clinical trials

Overview and results

Evofosfamide (TH-302) was evaluated in clinical studies as a monotherapy and in combination with chemotherapy agents and other targeted cancer drugs. The indications were a broad spectrum of solid tumor types and blood cancers. Evofosfamide clinical trials (as of 16 March 2025):

Soft tissue sarcoma

Evofosfamide was tested in combination with

doxorubicin Doxorubicin, sold under the brand name Adriamycin among others, is a chemotherapy medication used to treat cancer. This includes breast cancer, bladder cancer, Kaposi's sarcoma, lymphoma, and acute lymphocytic leukemia. It is often used toge ...

in patients with advanced

. The study TH-CR-403 was a single arm trial investigating evofosfamide in combination with doxorubicin. Evofosfamide was further tested in the Phase 3 clinical trial TH-CR-406/SARC021 with results published in 2017 indicating no improvement in patient mortality rates.

Metastatic pancreatic cancer

Evofosfamide was studied in combination with

gemcitabine Gemcitabine, sold under the brand name Gemzar, among others, is a chemotherapy medication used to treat cancers. It is used to treat testicular cancer, breast cancer, ovarian cancer, non-small cell lung cancer, pancreatic cancer, and bladder ca ...

in patients with metastatic pancreatic cancer. The study TH-CR-404 compared gemcitabine with gemcitabine plus evofosfamide. The study showed comparable efficacy profiles for evofosfamide and nab-paclitaxel when combined with gemcitabine; however, the hematologic toxicity was higher for patients given evofosfamide vs. nab-paclitaxel. In the Phase 3 MAESTRO study, patients with previously untreated, locally advanced unresectable or metastatic pancreatic adenocarcinoma treated with evofosfamide in combination with gemcitabine did ''not'' demonstrate a statistically significant improvement in overall survival.

Nasopharyngeal Carcinoma

Oxygen deficient conditions are linked to tumor progression throughout the body and poses an issue in cancer treatments such as chemotherapy and radiation. Hypoxia-activated prodrugs (HAPs) function in hypoxic conditions and inhibit the growth of tumor cells. Evofosfamide is a HAP that targets tumor progression in nasopharyngeal carcinoma (NPC) tissues by inhibiting the overexpression of hypoxia-inducible factor-1α (HIF-1α). In this study , the efficacy of Evofosfamide along with cisplastin (DDP) in blocking cell progression was measured. "The combination of evofosfamide with DDP had a synergistic effect on cytotoxicity in the NPC cell lines by combination index values assessment. Cell cycle G2 phase was arrested after treated with 0.05 μmol/L evofosfamide under hypoxia. Histone H2AX phosphorylation (γH2AX) (a marker of DNA damage) expression increased while HIF-1α expression suppressed after evofosfamide treatment under hypoxic conditions". These findings allow for evidence for Evofosfamide to be pushed towards clinical trials to further investigate the potential to be developed as an FDA approved anticancer drug.

References

{{Reflist, 3 Experimental cancer drugs Prodrugs Alkylating antineoplastic agents Drugs developed by Merck Nitrogen mustards Phosphorodiamidates Nitroimidazoles Organobromides Bromoethyl compounds