Design and synthesis of a folate-receptor targeted diazepine-ring-opened pyrrolobenzodiazepine prodrug conjugate.

Pyrrolobenzodiazepines (PBDs) and their dimers (bis-PBDs) have emerged as some of the most potent chemotherapeutic compounds and are currently under development as novel payloads in antibody-drug conjugates (ADCs). However, when used as stand-alone therapeutics or as warheads for small molecule drug conjugates (SMDCs), dose-limiting toxicities are often observed. As an elegant solution to this inherent problem, we designed and synthesized a diazepine-ring-opened bis-PBD prodrug (pro-PBD-PBD) folate conjugate lacking the one of the two imine moieties found in the corresponding free bis-PBD. Upon entering a targeted cell, cleavage of the linker system, including the hydrolysis of an oxazolidine moiety, results in the formation of a reactive intermediate which possesses a newly formed aldehyde as well as an aromatic amine. A fast and spontaneous intramolecular ring-closing reaction subsequently takes place as the aromatic amine adds to the aldehyde with the loss of water to give the imine, and as a result, the diazepine ring, thereby delivering the bis-PBD to the targeted cell. The in vitro and in vivo activity of this conjugate has been evaluated on folate receptor positive KB cells. Sub-nanomolar activity with good specificity and high cure rates with minimal toxicity have been observed.

Antiinflammatory Activity of a Novel Folic Acid Targeted Conjugate of the mTOR Inhibitor Everolimus.

Folate receptor (FR)-ß has been identified as a promising target for antimacrophage and antiinflammatory therapies. In the present study, we investigated EC0565, a folic acid-derivative of everolimus, as a FR-specific inhibitor of the mammalian target of rapamycin (mTOR). Because of its amphiphilic nature, EC0565 was first evaluated for water solubility, critical micelle formation, stability in culture and FR-binding specificity. Using FR-expressing macrophages, the effect of EC0565 on mTOR signaling and cellular proliferation was studied. The pharmacokinetics, metabolism and bioavailability of EC0565 were studied in normal rats. The in vivo activity of EC0565 was assessed in rats with adjuvant arthritis, a "macrophage-rich" model with close resemblance to rheumatoid arthritis. EC0565 forms micellar aggregates in physiological buffers and demonstrates good water solubility as well as strong multivalent FR-binding capacity. EC0565 inhibited mTOR signaling in rat macrophages at nanomolar concentrations and induced G0/G1 cell cycle arrest in serum-starved RAW264.7 cells. Subcutaneously administered EC0565 in rats displayed good bioavailability and a relatively long half-life (~12 h). When given at 250 nmol/kg, EC0565 selectively inhibited proliferating cell nuclear antigen expression in thioglycollate-stimulated rat peritoneal cells. With limited dosing regimens, the antiarthritic activity of EC0565 was found superior to that of etanercept, everolimus and a nontargeted everolimus analog. The in vivo activity of EC0565 was also comparable to that of a folate-targeted aminopterin. Folate-targeted mTOR inhibition may be an effective way of suppressing activated macrophages in sites of inflammation, especially in nutrient-deprived conditions, such as in the arthritic joints. Further investigation and improvement upon the physical and biochemical properties of EC0565 are warranted.

Folate-vinca alkaloid conjugates for cancer therapy: a structure-activity relationship.

Vintafolide is a potent folate-targeted vinca alkaloid small molecule drug conjugate (SMDC) that has shown promising results in multiple clinical oncology studies. Structurally, vintafolide consists of 4 essential modules: (1) folic acid, (2) a hydrophilic peptide spacer, (3) a disulfide-containing, self-immolative linker, and (4) the cytotoxic drug, desacetylvinblastine hydrazide (DAVLBH). Here, we report a structure-activity study evaluating the biological impact of (i) substituting DAVLBH within the vintafolide molecule with other vinca alkaloid analogues such as vincristine, vindesine, vinflunine, or vinorelbine; (ii) substituting the naturally (S)-configured Asp-Arg-Asp-Asp-Cys peptide with alternative hydrophilic spacers of varied composition; and (iii) varying the composition of the linker module. A series of vinca alkaloid-containing SMDCs were synthesized and purified by HPLC and LCMS. The SMDCs were screened in vitro against folate receptor (FR)-positive cells, and anti-tumor activity was tested against well-established subcutaneous FR-positive tumor xenografts. The cytotoxic and anti-tumor activity was directly compared to that produced by vintafolide. Among all the folate vinca alkaloid SMDCs tested, DAVLBH-containing SMDCs were active, while those constructed with vincristine, vindesine, or vinorelbine analogues failed to produce meaningful biological activity. Within the DAVLBH series, having a bioreleasable, self-immolative linker system was found to be critical for activity since multiple analogues constructed with thioether-based linkers all failed to produce meaningful activity both in vitro and in vivo. Substitutions of some or all of the natural amino acids within vintafolide's hydrophilic spacer module did not significantly change the in vitro or in vivo potency of the SMDCs. Vintafolide remains one of the most potent folate-vinca alkaloid SMDCs produced to date, and continued clinical development is warranted.

Design and regioselective synthesis of a new generation of targeted therapeutics. Part 3: Folate conjugates of aminopterin hydrazide for the treatment of inflammation.

Efficient syntheses of folate receptor (FR) targeting conjugates of the anti-inflammatory, aminopterin hydrazide, are described. 2-{4-Benzoylamino}-5-oxo-5-{N'-[2-(pyridin-2-yldisulfanyl)-ethoxycarbonyl]-hydrazino}-pentanoic acid is synthesized from commercially available 4-[(2-amino-4-imino-3,4-dihydro-pteridin-6-yl-methyl)-amino]-benzoic acid. Conjugation of this novel, activated aminopterin hydrazide to folic acid through cysteine-terminating (C-terminus), peptide/carbohydrate spacers results in highly water soluble conjugates which allow for the release of free aminopterin hydrazide within the endosomes of targeted cells.

Carbohydrate-based synthetic approach to control toxicity profiles of folate-drug conjugates.

To better regulate the biodistribution of the vinblastine-folate conjugate, EC145, a new folate-spacer that incorporates 1-amino-1-deoxy-D-glucitol-gamma-glutamate subunits into a peptidic backbone, was synthesized. Synthesis of Fmoc-3,4;5,6-di-O-isopropylidene-1-amino-1-deoxy-D-glucitol-gamma-glutamate 20, suitable for Fmoc-strategy solid-phase peptide synthesis (SPPS), was achieved in four steps from delta-gluconolactone. Addition of alternating glutamic acid and 20 moieties onto a cysteine-loaded resin, followed by the addition of folate, deprotection, and cleavage, resulted in the isolation of the new folate-spacer: Pte-gammaGlu-(Glu(1-amino-1-deoxy-D-glucitol)-Glu)(2)-Glu(1-amino-1-deoxy-D-glucitol)-Cys-OH (21). The addition of 21 to an appropriately modified desacetylvinblastine hydrazide (DAVLBH) resulted in a conjugate (25) with an improved therapeutic index. Treatment of 25 with DTT in neutral buffer at room temperature demonstrated that free DAVLBH would be released under the reductive environment of the internalized endosome.

Regioselective synthesis of folate receptor-targeted agents derived from epothilone analogs and folic acid.

Efficient regioselective syntheses of conjugates of folic acid and cytotoxic agents derived from natural epothilones are described. These folate receptor (FR) targeting compounds are water soluble and incorporate a hydrophilic peptide-based spacer unit and a reducible self-immolative disulfide-based linker system between the FR-targeting ligand and the parent drug.

Pre-clinical studies of EC2629, a highly potent folate- receptor-targeted DNA crosslinking agent.

Folate receptor (FR)-targeted small molecule drug conjugates (SMDCs) have shown promising results in early stage clinical trials with microtubule destabilizing agents, such as vintafolide and EC1456. In our effort to develop FR-targeted SMDCs with varying mechanisms of action, we synthesized EC2629, a folate conjugate of a DNA crosslinking agent based on a novel DNA-alkylating moiety. This agent was found to be extremely potent with an in vitro IC50 ~ 100× lower than folate SMDCs constructed with various microtubule inhibitors. EC2629 treatment of nude mice bearing FR-positive KB human xenografts led to cures in 100% of the test animals with very low dose levels (300 nmol/kg) following a convenient once a week schedule. The observed activity was not accompanied by any noticeable weight loss (up to 20 weeks post end of dosing). Complete responses were also observed against FR-positive paclitaxel (KB-PR) and cisplatin (KB-CR) resistant models. When evaluated against FR-positive patient derived xenograft (PDX) models of ovarian (ST070), endometrial (ST040) and triple negative breast cancers (ST502, ST738), EC2629 showed significantly greater anti-tumor activity compared to their corresponding standard of care treatments. Taken together, these studies thus demonstrated that EC2629, with its distinct DNA reacting mechanism, may be useful in treating FR-positive tumors, including those that are classified as drug resistant.

Properties influencing the relative binding affinity of pteroate derivatives and drug conjugates thereof to the folate receptor.

PURPOSE: Using in vitro competition assays, determine salient chemical features of pteroates and pteroate-drug conjugates which afford high affinity to the folate receptor. MATERIALS AND METHODS: Both folate binding protein-coated polystyrene plates and adherent human cell-based assays were used to evaluate the effects of assay temperature and buffer composition on pteroate/pteroate-drug conjugate binding affinity. Following assay selection and optimization, the relative binding affinities of ten vitamers and derivatives as well as seven pteroate-drug conjugates were evaluated. RESULTS: Compared to polystyrene plates containing immobilized folate binding protein, adherent KB cells were determined to be an equally effective, more desirable source of folate receptor for such analyses. Using the latter method, we discovered that a charged group positioned in close proximity to the pteroate's aryl moiety is critical for retaining high binding affinity. We also found that a diverse set of bioactive small molecule agents can be attached to folic acid in a manner that does not appreciably disturb this vitamin's intrinsic high affinity for the folate receptor. However, conjugation of lipophilic, high protein-binding agents to folate was sometimes found to dramatically reduce affinity, which is a finding that best exemplifies the need for having a reliable in vitro assay for determining a compound's RA. CONCLUSION: Molecules which bind best to the human folate receptor are those that contain hydrophilic regions distal to the ligand's aryl group, and for drug conjugates, an extended hydrophilic spacer placed in-between the pteroate and drug cargo moieties.

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.

Tumor targeting using 67Ga-DOTA-Bz-folate--investigations of methods to improve the tissue distribution of radiofolates.

INTRODUCTION: Use of folic acid radioconjugates for folate receptor (FR) targeting is a promising strategy for imaging purposes as well as for potential therapy of cancer and inflammatory diseases due to the frequent FR overexpression found on cancer cells and activated macrophages. Herein, we report on preclinical results using a novel DOTA-Bz-EDA-folate conjugate radiolabeled with [(67)Ga]-gallium. METHODS: DOTA-Bz-EDA-folate was prepared by conjugation of ethylenediamine-(γ)-folate with 2-(p-isothiocyanobenzyl)-DOTA. Radiolabeling was carried out with (67)GaCl(3) according to standard procedures. Biodistribution studies of the tracer were performed in mice bearing FR-positive KB tumor xenografts. The effects on radiofolate biodistribution with coadministered renal uptake-blocking amino acids, diuretic agents, antifolates as well as different routes of administration were likewise investigated. Supportive imaging studies were performed using a small-animal single photon emission computed tomography (SPECT)/CT scanner. RESULTS: (67)Ga-DOTA-Bz-EDA-folate showed a high and specific accumulation in tumors (6.30%±0.75% ID/g, 1 h pi and 6.08%±0.89% ID/g, 4 h pi). Nonspecific radioactivity uptake in nontargeted tissues was negligible, but significant accumulation was found in FR-positive kidneys, which resulted in unfavorably low tumor-to-kidney ratios (<0.1). Coadministered amino acids or diuretics did not effectively reduce renal accumulation; in contrast, predosed pemetrexed did significantly reduce kidney uptake (<29% of control values). The SPECT/CT studies confirmed the excellent tumor-to-background contrast of (67)Ga-radiofolate and the favorable reduction in kidney uptake (with improved imaging quality) resulting from pemetrexed administration. CONCLUSION: Conventional methods to reduce kidney uptake of radiofolates fail. However, the novel (67)Ga-radiolabeled DOTA-Bz-EDA-folate can effectively be used to image FR-positive cancer and potentially inflammatory diseases. Due to its rapid blood clearance properties, this tracer is also a promising candidate for positron emission tomography imaging if radiolabeled with the short-lived [(68)Ga]-gallium radionuclide.

Synthesis and biological analysis of prostate-specific membrane antigen-targeted anticancer prodrugs.

Ligand-targeted therapeutics have increased in prominence because of their potential for improved potency and reduced toxicity. However, with the advent of personalized medicine, a need for greater versatility in ligand-targeted drug design has emerged, where each tumor-targeting ligand should be capable of delivering a variety of therapeutic agents to the same tumor, each therapeutic agent being selected for its activity on a specific patient's cancer. In this report, we describe the use of a prostate-specific membrane antigen (PSMA)-targeting ligand to deliver multiple unrelated cytotoxic drugs to human prostate cancer (LNCaP) cells. We demonstrate that the PSMA-specific ligand, 2-[3-(1, 3-dicarboxy propyl)ureido] pentanedioic acid, is capable of mediating the targeted killing of LNCaP cells with many different therapeutic warheads. These results suggest that flexibility can be designed into ligand-targeted therapeutics, enabling adaptation of a single targeting ligand for the treatment of patients with different sensitivities to different chemotherapies.

Prostate-specific membrane antigen targeted imaging and therapy of prostate cancer using a PSMA inhibitor as a homing ligand.

Prostate cancer (PCa) is a major cause of mortality and morbidity in Western society today. Current methods for detecting PCa are limited, leaving most early malignancies undiagnosed and sites of metastasis in advanced disease undetected. Major deficiencies also exist in the treatment of PCa, especially metastatic disease. In an effort to improve both detection and therapy of PCa, we have developed a PSMA-targeted ligand that delivers attached imaging and therapeutic agents selectively to PCa cells without targeting normal cells. The PSMA-targeted radioimaging agent (DUPA-(99m)Tc) was found to bind PSMA-positive human PCa cells (LNCaP cell line) with nanomolar affinity (K(D) = 14 nM). Imaging and biodistribution studies revealed that DUPA-(99m)Tc localizes primarily to LNCaP cell tumor xenografts in nu/nu mice (% injected dose/gram = 11.3 at 4 h postinjection; tumor-to-muscle ratio = 75:1). Two PSMA-targeted optical imaging agents (DUPA-FITC and DUPA-rhodamine B) were also shown to efficiently label PCa cells and to internalize and traffic to intracellular endosomes. A PSMA-targeted chemotherapeutic agent (DUPA-TubH) was demonstrated to kill PSMA-positive LNCaP cells in culture (IC(50) = 3 nM) and to eliminate established tumor xenografts in nu/nu mice with no detectable weight loss. Blockade of tumor targeting upon administration of excess PSMA inhibitor (PMPA) and the absence of targeting to PSMA-negative tumors confirmed the specificity of each of the above targeted reagents for PSMA. Tandem use of the imaging and therapeutic agents targeted to the same receptor could allow detection, staging, monitoring, and treatment of PCa with improved accuracy and efficacy.

Strategy to prevent drug-related hypersensitivity in folate-targeted hapten immunotherapy of cancer.

Cancer vaccine/immunotherapy rarely involves systemic administration of an immunogenic compound to an actively immunized host. We have developed such a strategy that utilizes folate to deliver antigenic haptens [e.g., fluorescein (FITC) and dinitrophenyl] to folate receptor-positive tumors in a hapten-pre-vaccinated host. Here, we investigated the safety of this novel approach and developed strategies to prevent drug-related hypersensitivity. Using FITC as the model hapten, we identified a potential source of allergic species in folate-FITC preparations by LC-MS/MS. In mice and guinea pigs, we tested the significance of this impurity by passive cutaneous anaphylaxis and active systemic anaphylaxis assays. We studied the effect of immunogen (e.g., KLH-FITC) dose and derived a desensitization regimen that was further evaluated in a murine tumor model. Administration of folate-FITC with low multi-haptenated contaminants (e.g. bis-FITC) resulted in hypersensitivity in underimmunized animals. However, this drug-related hypersensitivity may be independently prevented by (1) increasing the immunogen dose and/or (2) desensitizing animals with folate-FITC during vaccination. In addition, such manipulation in vivo did not appear to negatively alter the effectiveness of immunotherapy. This study provided confidence on the safety of folate-hapten-targeted cancer immunotherapy in an actively immunized host.

Preclinical antitumor activity of a novel folate-targeted dual drug conjugate.

We have designed a new type of tumor-targeted agent by tethering two different drug molecules, with distinct biological mechanisms of action, to the same ligand. This compound, named EC0225, represents the "first in class" multidrug, folate receptor (FR)-targeted agent to be disclosed. It was constructed with a single folate molecule, extended by a hydrophilic peptide-based spacer, which was in turn attached to mitomycin and Vinca alkaloid units via two separate disulfide-containing linkers. EC0225 produced potent, dose-responsive activity in vitro, and curative activity was observed against FR-positive syngeneic and xenograft tumors following the administration of well-tolerated dosing regimens. Multiple complete responses and cures were also noted when EC0225 was used to treat mice initially bearing tumors as large as 750 mm (3) in volume. Overall, EC0225's impressive preclinical activity allowed for its selection as a development candidate and for the start of Phase 1 clinical trials, which began in March of 2007, for the treatment of advanced malignancies.

An assembly concept for the consecutive introduction of unsymmetrical disulfide bonds: synthesis of a releasable multidrug conjugate of folic acid.

We describe the development of methodology which allows for the introduction of a second disulfide bond into a molecular framework with a pre-existing disulfide linker system. Compounds which contain an S-9-fluorenylmethyl-protected thiol and an additional disulfide linkage are deprotected in situ and trapped with an activated thiophile. This methodology allowed for the synthesis of the first molecule possessing two different biologically active agents covalently attached to a folate receptor targeting ligand unit via two disulfide-based release systems.

Design and regioselective synthesis of a new generation of targeted chemotherapeutics. Part 1: EC145, a folic acid conjugate of desacetylvinblastine monohydrazide.

An efficient synthesis of the folate receptor (FR) targeting conjugate EC145 is described. EC145 is a water soluble derivative of the vitamin folic acid and the potent cytotoxic agent, desacetylvinblastine monohydrazide. Both molecules are connected in regioselective manner via a hydrophilic peptide spacer and a reductively labile disulfide linker.