Folate receptor mediated drug delivery system

During the study of tumor cells, researchers have found a series of over-expressed receptors in tumor cells and vascular surface, which are closely related to the growth and proliferation of tumor cells.^[2] These tumor-specific receptors provide a target for tumor therapy. The receptor specific

binding of targeted anticancer drugs with the tumor-specific receptor ligand can induce tumor specific cellular uptake, thus improving selectivity and reducing side effects of the drug.^[82]

The folate receptor (FR) is a very promising target for anti-cancer tumor therapy; FR is over expressed on the cellular membrane of tumor cells, such as ovarian, breast, cervical, colorectal and nasopharyngeal tumor cell; and its expression is restricted in most of the normal human tissues.^[83-85]

(Table 10)

Table 10. Dependence of Folate receptor (FR) expression on tumor type. ^[83-85]

Cancer Estimated new cases in the US Estimated % FR-positive

Ovarian 23300 93%

Brain 17000 75%

Mesothelinma 2500 73%

Kidney 31800 50%

Head and neck 28900 38%

Lung 169400 37%

Breast 20500 32%

Colorectal 152200 22%

Folic acid also known as folate or vitamin M, vitamin B9, is essential to numerous bodily functions.

The human body needs folate to synthesize DNA, repair DNA, and methylate DNA as well as to act as a cofactor in certain biological reactions.^[86-89] Folate is especially important in aiding rapid cell division and growth, such as in infancy and pregnancy. Children and adults both require folic acid to produce healthy red blood cells and prevent anemia. Compared to normal cells, rapidly proliferating tumor cells have a higher demand of folic acid.

Folic acid has several features: inexpensive, non-immunogenic and high affinity to folate receptor;

furthermore, it has small molecule volume, thus favoring rapid tumor cell extravasation and complete cell infiltration. Folic acid consists of L-Glutamic acid, p-aminobenzoic acid and pteridine, and can be coupled with functional molecules through classic coupling reactions, such as anticancer drugs, macromolecule proteins, haptens, imaging agents, etc.^[90-91] (Figure 24)

Figure 24. Structure of folic acid.

To construct a folate conjugates, there are four essential elements: the carrier element (Folate), the (linker), possibly with a cleavable bond (CB), and the therapeutic agent (Drug).^[92] (Figure 25)

Figure 25. Schematic sketch of a folate-drug conjugate.^[92]

In the last decades, the main focus was on folate-based diagnostic agents like radiopharmaceuticals (e.g., folate-based Ga, ⁹⁹mTc and In complexes).^[93] In recent years, research on folic acid as targeting structure has been mainly focused on its macromolecular conjugates bearing anti-cancer agents, like proteins, polymeric micelles, liposomes, synthetic polymers and nanoparticles.In contrast to this, only a few reports were published on folic acid-drug conjugates of low molecular weight for tumor targeting.

In 2009, Pang from Wuhan University reported the use of folate-conjugated fluorescent quantum dots (QDs) as the cell targeting fluorescent nanobioprobes.^[94] (Figure 26)

Figure 26. Folate-conjugated fluorescent quantum dots.^[94]

They coupled folate onto water dispersible PEG coated QDs (PEG-QDs) to produce FA-coupled PEG-QDs which demonstrated to specifically recognize folate receptors overexpressed in human nasopharyngeal cells (KB cells), but not in an FR-deficient lung carcinoma cell line (A549 cells).

(Figure 26)

Leamon's group reported another folate-targeted drug EC145 to enter clinical trials. In this molecule, a short peptide linker to desacetylvinblastine hydrazide via a disulfide bond connects the folic acid. This disulfide is positioned in the molecule so that intermolecular reduction will trigger traceless bond cleavage and the release of the desacetylvinblastine hydrazide. EC145 shows a good activity in both preclinical and clinical trials. It meets all phase I objectives and is now approaching completion of phase II trials.^[95-96] (Figure 27)

O OH

Figure 27. Reductive activation of EC145 to release the targeting tetra-peptide conjugated FA, the fragments of the linker group and the cytotoxic desacetyl vinblastine hydrazide.^[96]

In 2006, Low’s group synthesized a folate receptor targeted camptothecin prodrug, which is using a hydrophilic peptide spacer linked to folate via a releasable disulfide carbonate linker, i.e. in principle following a similar approach as before. The linking group can be cleaved off in a traceless manner.

The conjugate was found to possess high affinity for folate receptor-expressing cells and inhibited

cell proliferation in human KB cells with an IC50 of 10 nM. Activity of the prodrug was completely blocked by excess folic acid, demonstrating receptor-mediated uptake.^[97] (Figure 28)

O OH

Figure 28. Disulfide mediated release of camptothecin from conjugate. ^[97]

2002, Schibli and co-workers from ETH reported the folate-based PET radiotracer 3’-aza-2’-[¹⁸ F]-fluoro-folic acid which may serve as an appropriate diagnostic tool for imaging FR-positive tissue.

This radiotracer revealed favorable characteristics both in vitro and in vivo despite chemical modification of the folate-backbone. Visualization of FR-positive tumors in mice was achieved with high image contrast and with only minor accumulation of radioactivity in non-targeted tissue.(See Figure 29) In vivo PET imaging and biodistribution studies with mice demonstrated a high and specific uptake in FR-positive KB tumor xenografts (12.59 ± 1.77% ID/g, 90 min p.i.). A high and specific accumulation of radioactivity was observed in the kidneys (57.33 ± 8.40% ID/g, 90 min p.i.) and salivary glands (14.09 ± 0.93% ID/g, 90 min p.i.), which are known to express the FR and nonspecific uptake found in the liver (10.31 ± 2.37% ID/g, 90 min p.i.). Pre-injection of folic acid

resulted in a >85% reduced uptake of tracer in FR-positive tissues (xenografts, kidneys, and salivary glands). While increased uptake of the radiotracer was found in the intestinal tract and the liver. ^[98]

O OH

Figure 29. (Up) Folate-based F¹⁸ PET radiotracer. (Down) Maximum intensity projection of PET/CT in vivo scans of 30 min duration (120-150 min p.i.) performed with KB tumor bearing mice of the radio tracer. (A):The scan of a mouse which received only the radiotracer (13 MBq), which demonstrate that the visualization of the tracer in tumor xenografts was excellent, and the accumulation of radioactivity in nontargeted regions was generally low. (B): The scan of a mouse which received folic acid (100 μg) prior to the radiotracer (18 MBq), which shows that the uptake of the radiotracer in the tumors, kidneys, and salivary glands was blocked, while increased uptake of the radiotracer was found in the intestinal tract and the liver. (Tu = tumor, Li = liver, Ki = kidney, Bl = bladder, SGS = salivary glands, GB = gallbladder, and Int = intestine.)^[98]