Iontcho Radoslavov Vlahov

Evaluation of disulfide reduction during receptor-mediated endocytosis by using FRET imaging

Despite functional evidence for disulfide bond-reducing activity in endosomal compartments, the mechanistic details pertaining to such process (e.g., kinetics and sites of disulfide reduction) remain largely controversial. To address these questions directly, we have synthesized a previously uncharacterized fluorescent folate conjugate, folate-(BODIPY FL)-SS-rhodamine (folate-FRET), that changes fluorescence from red to green upon disulfide bond reduction. Using this construct, we have observed that disulfide reduction: (i) occurs with a half-time of 6 h after folate-FRET endocytosis, (ii) begins in endosomes and does not depend significantly on redox machinery located on the cell surface or within the lysosome or the Golgi apparatus, (iii) occurs independently of endocytic vesicle trafficking along microtubules, and (iv) yields products that are subsequently sorted into distinct endosomes and trafficked in different directions. Finally, colocalization of folate and transferrin receptors suggest that conclusions derived from this study may apply to other endocytic pathways.

Preclinical Evaluation of EC145, a Folate-Vinca Alkaloid Conjugate

We recently developed a new group of folate-conjugated Vinca alkaloids, one of which, EC145, emerged as a candidate for clinical development. Brief treatment of nude mice bearing approximately 100 mm(3) folate receptor-positive human xenografts led to complete response (CR) in 5/5 mice and cures (i.e., remission without a relapse for >90 days post-tumor implantation) in 4/5 mice. Multiple CRs and cures were also noted when EC145 was used to treat mice initially bearing tumors as large as 750 mm(3). Likewise, complete cures (5/5) resulted following the treatment of an aggressive folate receptor-positive J6456 lymphoma model. The activity of EC145 was not accompanied by noticeable weight loss or major organ tissue degeneration. Furthermore, no significant antitumor activity (0/5 CR) was observed in EC145-treated animals that were co-dosed with an excess of a benign folate ligand, thus demonstrating the target-specific activity of EC145. The enhanced therapeutic index due to folate conjugation was also evidenced by the fact that the unconjugated drug (desacetylvinblastine monohydrazide) was found to be completely inactive when administered at nontoxic dose levels and only marginally active when given at highly toxic dose levels. Subsequent dose regimen studies confirmed that EC145 given on a more frequent, qdx5 schedule resulted in the most effective antitumor response as compared with an equivalent total dose given on thrice- or single-injection-per-week schedule. Taken together, these studies show that EC145 has significant antiproliferative activity and tolerability, thus lending support to an ongoing phase 1 trial for the treatment of advanced malignancies.

Engineering Folate–Drug Conjugates to Target Cancer: From Chemistry to Clinic

The folate receptor (FR) is a potentially useful biological target for the management of many human cancers. This membrane protein binds extracellular folates with very high affinity and, through an endocytic process, physically delivers them inside the cell for biological consumption. There are now many examples of how this physiological system can be exploited for the targeted delivery of biologically active molecules to cancer. In fact, strong preclinical as well as emerging clinical evidence exists showing how FR-positive cancers can be (i) anatomically identified using folate conjugates of radiodiagnostic imaging agents and (ii) effectively treated with companion folate-targeted chemotherapies. While the biological results are compelling, it is of equal importance to understand the conjugation chemistries that were developed to produce these active molecules. Therefore, this review will focus on the methods utilized to construct folate-based small-molecule drug conjugates (SMDCs), with particular attention focused on modular design, hydrophilic spacers, and self-immolative linkers.

Characterization of the pH of Folate Receptor-Containing Endosomes and the Rate of Hydrolysis of Internalized Acid-Labile Folate-Drug Conjugates

Despite the widely accepted assumption that most endosomal compartments are acidic, evaluation of the efficiency of pH-dependent drug release from a ligand-targeted drug conjugate during receptor-mediated endocytosis is lacking. Therefore, we have characterized the kinetics of pH-dependent drug release from a model folate-drug conjugate during folate receptor (FR)-mediated endosomal trafficking. For this purpose, we synthesized an acid-labile folate-fluorescence resonance energy transfer reporter (ALFR) that emits green fluorescence (BODIPY FL, 6-((4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diazas-indacene-3-propionyl)amino)hexanoic acid) only after acid-catalyzed hydrolysis of the acyl hydrazone linker. In a cell-free system, cleavage of ALFR was found to be efficient only at acidic pH values (t1/2 = 1.95, 4.63, and 75 h at pH 4, 5, and 6, respectively) and essentially resistant to hydrolysis at pH 7. Curiously, when applied to folate receptor-expressing cancer cells, the acid-labile folate-linked probe exhibited little or no recovery of BODIPY FL fluorescence (green), even after 55 h of incubation, arguing very inefficient cleavage within the FR endocytic pathway. To understand this unanticipated observation, we measured the pH of FR-containing endosomes using ratiometric fluorescence microscopy and observed that most FR+ endosomes are only mildly acidic (average ∼pH 6.5). Taken together, these data argue that the FR-trafficking pathway does not involve acidic compartments and that acyl hydrazone linkers may constitute a poor option for FR-mediated drug delivery.

Comparative preclinical activity of the folate-targeted Vinca alkaloid conjugates EC140 and EC145

EC140 is a water soluble folate conjugate of desacetylvinblastine monohydrazide (DAVLBH), which is constructed with an endosome‐cleavable acyl hydrazone bond. This agent has proven to be active and specific against well established, subcutaneous folate receptor (FR)‐positive tumors in multiple animal models. Recent structure‐activity and optimization studies have yielded a disulfide bond‐containing counterpart to EC140, herein referred to as EC145. This new conjugate was found to retain high affinity for FR‐positive cells, and it produced specific, dose‐responsive activity in vitro. Comparative in vivo efficacy tests confirmed that, like EC140, EC145 displays activity against both syngeneic and xenograft tumor models. However, EC145 was found to be more active and better tolerated than EC140; hence, more durable complete responses were consistently observed in EC145‐treated tumor‐bearing animals. Furthermore, EC145 was not found to be active against a FR‐negative tumor model. Additional preclinical studies are therefore warranted to better understand EC145s breadth of activity against FR‐positive tumors.

Folate Targeting Enables Durable and Specific Antitumor Responses from a Therapeutically Null Tubulysin B Analogue

The membrane-bound high-affinity folate receptor (FR) is highly expressed on a wide range of primary and metastatic human cancers, such as those originating in ovary, lung, breast, endometrium, kidney, and brain. Because folate-linked conjugates bind to and become internalized within FR-expressing cells (similar to that of free folic acid), we explored the possibility of using the folate ligand to target a potent, semisynthetic analogue of the microtubule inhibitor tubulysin B to FR-enriched tumors. When tested in vitro, a novel folate conjugate, herein referred to as EC0305, was found to specifically inhibit the growth of a panel of FR-positive cell lines (IC50 range, 1-10 nmol/L) in a dose-dependent manner, whereas cells lacking FR expression were unaffected. The potency of EC0305 was also confirmed against a human KB xenograft-nu/nu mouse cancer model. Here, a brief three times per week, 2-week regimen yielded remarkable antitumor activity (100% tumor-free animals) without causing significant weight loss or major organ tissue degeneration. In contrast, antitumor activity was completely abolished in EC0305-treated animals that were co-dosed with an excess of a nontoxic folate-containing analogue, thereby confirming that the antitumor effect of this agent was mediated by FRs. The advantage provided by folate conjugation was further proved by the untargeted free drug, which was found to be completely inactive at both tolerable and highly toxic dose levels. Collectively, these results show that this potent antiproliferative tubulysin compound can be specifically delivered to FR-positive tumors to provide substantial therapeutic benefit using well-tolerable dosing regimens.

Folate receptor specific anti-tumor activity of folate–mitomycin conjugates

Purpose: Folate receptor (FR) targeted drug conjugates were prepared by covalently attaching the vitamin folate, to the potent anticancer drug, mitomycin C (MMC). One such conjugate, called EC72, was synthesized with an intramolecular disulfide bond, and it was found to exhibit efficacious anti-tumor activity against FR-expressing M109 tumors in a manner that yielded no gross or microscopic toxicity, even to FR-positive kidneys. Methods: EC72’s specificity was demonstrated by two methods: (1) blocking EC72’s activity with an excess of co-administered folic acid (FA) in M109 tumor bearing mice and (2) the absence of therapeutic activity in mice bearing FR-negative tumors. The importance of having a cleavable bond in the conjugate was also exemplified, since EC110 (a folate–MMC conjugate constructed with a more resilient amide bond) failed to produce anti-M109 tumor activity. EC72’s therapeutic potential was found to decrease with respect to the increasing size of subcutaneous tumor. However, a combination therapy with paclitaxel reproducibly improved the anti-tumor efficacy relative to either agent alone at well tolerated dose levels and with no apparent increase in toxicity. A more advanced folate–MMC conjugate was also synthesized in an effort to improve activity. Thus, EC118, a molecule constructed with both a reducible disulfide bond and an acid-labile hydrazone bond in the linker region, was tested and found to produce a significantly greater number of tumor regressions of more established M109 tumors than that achieved with EC72. Conclusion: Overall, these data indicate that folate-targeted drug therapy alone, or in combination with paclitaxel, may be a novel and effective clinical approach towards treating FR-positive cancers.

Folate Receptor–Specific Antitumor Activity of EC131, a Folate-Maytansinoid Conjugate

EC131, a new folate receptor (FR)-targeted drug conjugate, was prepared by covalently attaching the vitamin folic acid (FA) to a potent microtubule-inhibiting agent, maytansinoid DM1, via an intramolecular disulfide bond. When tested on cells in culture, EC131 was found to retain high affinity for FR-positive cells and to provide FR-specific cytotoxicity with an IC(50) in the low nanomolar range. The activity of EC131 was completely blocked in the presence of an excess of free FA, and no activity was detected against FR-negative cells. When evaluated against s.c. FR-positive M109 tumors in BALB/c mice, EC131 showed marked antitumor efficacy. Furthermore, this therapeutic effect occurred in the apparent absence of weight loss or noticeable organ tissue degeneration. In contrast, no significant antitumor activity was observed in EC131-treated animals that were codosed with an excess of FA, thus demonstrating the targeted specificity of the in vivo activity. EC131 also showed marked antitumor activity against FR-positive human KB tumors, but not against FR-negative A549 tumors, in nude mice with no evidence of systemic toxicity during or after the therapy. In contrast, therapy with the free maytansinoid drug (in the form of DM1-S-Me) proved not to be effective against the KB model when administered at its maximum tolerated dose (MTD). Taken together, these results indicate that EC131 is a highly potent agent capable of producing therapeutic benefit in murine tumor models at sub-MTD levels.

Treatment of experimental adjuvant arthritis with a novel folate receptor-targeted folic acid- aminopterin conjugate

IntroductionFolate receptor (FR)-expressing macrophages have been shown to accumulate at sites of inflammation, where they promote development of inflammatory symptoms. To target such a macrophage population, we designed and evaluated the biologic activity of EC0746, a novel folic acid conjugate of the highly potent antifolate, aminopterin.MethodsUsing a FR-positive subclone of murine macrophage-derived RAW264.7 cells and rat thioglycollate-elicited macrophages, we studied the effect of EC0746 on dihydrofolate reductase activity, cell proliferation, and cellular response towards bacterial lipopolysaccharide as well as IFNγ activation. The EC0746 anti-inflammatory activity, pharmacokinetics, and toxicity were also evaluated in normal rats or in rats with adjuvant-induced arthritis; that is, a FR-positive macrophage model that closely resembles rheumatoid arthritis in humans.ResultsEC0746 suppresses the proliferation of RAW264.7 cells and prevents the ability of nonproliferating rat macrophages to respond to inflammatory stimuli. In the macrophage-rich rat arthritis model, brief treatment with subcutaneously administered EC0746 is shown to mediate an FR-specific anti-inflammatory response that is more potent than either orally administered methotrexate or subcutaneously delivered etanercept. More importantly, EC0746 therapy is also shown to be ~40-fold less toxic than unmodified aminopterin, with fewer bone marrow and gastrointestinal problems.ConclusionsEC0746 is the first high FR-binding dihydrofolate reductase inhibitor that demonstrates FR-specific anti-inflammatory activities both in vitro and in vivo. Our data reveal that a relatively toxic anti-inflammatory drug, such as aminopterin, can be targeted with folic acid to inflammatory macrophages and thereby relieve inflammatory symptoms with greatly reduced toxicity.

Folate-Conjugated Rapamycin Slows Progression of Polycystic Kidney Disease

Activation of the mammalian target of rapamycin (mTOR) signaling pathway is aberrant in autosomal-dominant polycystic kidney disease (ADPKD). The mTOR inhibitors, such as rapamycin, ameliorate PKD in rodent models, but clinical trials have not shown benefit, possibly as a result of low tissue concentrations of rapamycin at clinically tolerable doses. To overcome this limitation, we synthesized a folate-conjugated form of rapamycin (FC-rapa) that is taken up by folate receptor-mediated endocytosis and cleaved intracellularly to reconstitute the active drug. We found that renal cyst-lining cells highly express the folate receptor in ADPKD and mouse models. In vitro, FC-rapa inhibited mTOR activity in a dose- and folate receptor-dependent manner. Treatment of a PKD mouse model with FC-rapa inhibited mTOR in the target tissue, strongly attenuated proliferation and growth of renal cysts and preserved renal function. Furthermore, FC-rapa inhibited mTOR activity in the kidney but not in other organs. In summary, these results suggest that targeting the kidney using FC-rapa may overcome the significant side effects and lack of renal efficacy observed in clinical trials with mTOR inhibitors in ADPKD.