Folate targeting

Folate targeting is a method utilized in biotechnology for drug delivery purposes. This Trojan Horse process, which was created by Drs. Christopher P. Leamon and Philip S. Low, involves the attachment of the vitamin, folate (folic acid), to a molecule/drug to form a "folate conjugate". Based on the natural high affinity of folate for the folate receptor protein (FR), which is commonly expressed on the surface of many human cancers, folate-drug conjugates also bind tightly to the FR and trigger cellular uptake via endocytosis. Molecules as diverse as small radiodiagnostic imaging agents to large DNA plasmid formulations have successfully been delivered inside FR-positive cells and tissues.

Background
Folic acid (FA, folate or vitamin B9), is a vital nutrient required by all living cells for nucleotide biosynthesis and for the proper metabolic maintenance of 1-carbon pathways. Aside from its cofactor role for intracellular enzymes, FA also displays high affinity for the folate receptor (FR), a glycosylphosphatidyinositol-linked protein that captures its ligands from the extracellular milieu and transports them inside the cell via a non-destructive, recycling endosomal pathway. The FR is also a recognized tumor antigen/biomarker. Because of this, diagnostic and therapeutic methods which exploit the FR’s function are being developed for cancer.

From a mechanistic perspective, the FR functions to concentrate exogenous ligands (e.g. folates and folate-drug conjugates) into the cell cytosol by endocytosis. The term endocytosis refers to the process whereby the plasma membrane invaginates and eventually forms a distinct intracellular compartment. The endocytic vesicles (endosomes) rapidly become acidified to allow the FR to release its ligand. Afterwards, the empty FR returns to the cell surface where is can participate in another round of ligand-mediated endocytosis.

FR-positive cancer
Elevated expression of the FR occurs in many human malignancies, especially when associated with aggressively growing cancers. Recently, it was proposed that this relationship may possibly be used for prognostic purposes. Non-mucinous ovarian cancer (the majority of ovarian cancers) was the first tumor type to be associated with FR “over-expression”, and it was later shown that this antigen was identical to that found on KB tumor cells and in placental tissue. Several studies confirmed that ~80-90% of ovarian tumors over-express the FR. Other gynecological cancers also over-express the receptor  as well as pediatric ependymal brain tumors, mesothelioma, and  breast, colon, renal and lung tumors. The FR may also be found associated with cancer, particularly when related to myeloid leukemia and perhaps head and neck carcinomas. Taken together, the total number of tumors that express the FR is very large; therefore, FR-targeted strategies could have significant impact on cancer treatment for patients diagnosed with FR-positive disease.

Diagnostics
The FR is expressed on many different types of malignant tissues and in large quantities. But, not all human cancers within a particular indication will express the FR. Because novel FR-targeted therapies are now being tested clinically  , having the ability to screen patients for FR-positive disease could certainly increase the efficiency of and decrease the time for clinical investigations of these novel agents.

Currently, there are two principal methods that have been utilized for assessing a patient’s “FR status”. These include an invasive tissue-based immunohistochemical assay, and a non-invasive radiodiagnostic approach. The latter method is now being tested clinically using 99mTc-EC20.

Folate-targeted chemotherapy
To date, four distinct FA-drug conjugates have entered clinical trials for the treatment of cancer:

Vintafolide (EC145) represents a novel water soluble FA conjugate of the powerful microtubule destabilizing agent, desacetylvinblastine monohydrazide (DAVLBH; a derivative of the natural product, vinblastine). EC145 was found to produce marked anti-tumor effect against well-established, subcutaneous FR-positive tumor xenografts using well tolerated regimens. EC145 also represents the first FA-drug conjugate to be evaluated in clinical trials, and it is currently (2009) being tested in a multi-national randomized Phase 2b trial in combination with pegylated liposomal doxorubicin (Doxil).

EC0225 represents the “first in class” multi-drug, FA-targeted agent to be reported. It is a molecule constructed with a single FA moiety and extended by a hydrophilic peptide-based spacer that is, in turn, linked to Vinca alkaloid and mitomycin units via 2 distinct disulfide-containing linkers. Animals bearing well-established human tumor xenografts were found to completely respond to EC0225 therapy with dosing regimens that were approximately 3-fold less intensive to that required for EC145. A Phase 1 trial for EC0225 is in progress.

BMS-753493 is a molecule born from a collaboration between scientists at Endocyte Inc. and Bristol Myers Squibb (BMS). It represents a FA conjugate that was constructed with a semi-synthetic analog of Epothilone A. BMS-753493 is currently being evaluated for safety and efficacy in Phase 2 clinical trials sponsored by BMS.

EC0489 is the latest folate-targeted chemotherapeutic to enter clinical trials sponsored by Endocyte. This molecule is actually a derivative of EC145 (see above) that was designed to have limited non-specific clearance properties through the liver. By reducing hepatic clearance, less drug will transit through the biliary excretion route; as a consequence, less off-target toxicities (predicted from preclinical tests) are expected.

Activated macrophage targeting
Activated, TNF-alpha producing macrophages express the beta isoform of the FR, and they are targetable with folate conjugates in vivo. For example, 99mTc-EC20 was reported to concentrate in the livers, spleens and arthritic extremities of adjuvant-induced arthritic rats via a folate-dependent mechanism. Development of folate-drug conjugates for inflammation therapy is underway. . It is expected that ailments which harbor activated macrophages (such as arthritis, psoriasis and inflammatory bowel disease) may someday be treatable with folate-targeted medicines.