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.

Prostate-Specific Membrane Antigen-Specific Antitumor Activity of a Self-Immolative Tubulysin Conjugate.

Prostate-specific membrane antigen (PSMA) is a biomarker that is overexpressed on prostate cancer, and it is also present on the neovasculature within many non-prostate solid tumors. Herein, we report on the construction and biological testing of novel tubulysin B-containing therapeutic agents for the treatment of PSMA-expressing cancer. One of these compounds, EC1169, emerged as a lead candidate for preclinical development and phase 1 clinical testing. This water-soluble conjugate was shown to have high affinity for PSMA-positive cells. When tested in vitro, EC1169 was found to inhibit the growth of PSMA-positive cells, but it displayed no activity against PSMA-negative cells. Brief treatment of nude mice bearing PSMA-positive LNCaP human xenografts with EC1169 led to complete remissions and cures. Furthermore, this activity occurred in the absence of weight loss. In contrast, the nontargeted tubulysin B drug proved to be inactive against the LNCaP tumor model when administered at doses near to or greater than the maximum tolerated level. PSMA-negative KB tumors did not appreciably respond to EC1169 therapy, thereby confirming this compound's targeted specificity for PSMA-positive cells. Finally, treatment of LNCaP-bearing mice with docetaxel (the most active chemotherapeutic agent approved for late stage prostate cancer therapy) was found to produce only modest anti-tumor activity, and this outcome was also associated with severe weight loss. Taken together, these results strongly indicate that PSMA-positive tumors may be effectively treated using highly potent, PSMA-targeted small-molecule drug conjugates using regimens that do not cause undesirable side effects.

Detecting Functional and Accessible Folate Receptor Expression in Cancer and Polycystic Kidneys.

Folate-based small molecule drug conjugates (SMDCs) are currently under development and have shown promising preclinical and clinical results against various cancers and polycystic kidney disease. Two requisites for response to a folate-based SMDC are (i) folate receptor alpha (FRα) protein is expressed in the diseased tissues, and (ii) FRα in those tissues is accessible and functionally competent to bind systemically administered SMDCs. Here we report on the development of a small molecule reporter conjugate (SMRC), called EC2220, which is composed of a folate ligand for FRα binding, a multilysine containing linker that can cross-link to FRα in the presence of formaldehyde fixation, and a small hapten (fluorescein) used for immunohistochemical detection. Data show that EC2220 produces a far greater IHC signal in FRα-positive tissues over that produced with EC17, a folate-fluorescein SMRC that is released from the formaldehyde-denatured FRα protein. Furthermore, the extent of the EC2220 IHC signal was proportional to the level of FRα expression. This EC2220-based assay was qualified both in vitro and in vivo using normal tissue, cancer tissue, and polycystic kidneys. Overall, EC2220 is a sensitive and effective reagent for evaluating functional and accessible receptor expression in vitro and in vivo.

Latent Warheads for Targeted Cancer Therapy: Design and Synthesis of pro-Pyrrolobenzodiazepines and Conjugates.

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 diazepine-ring-opened conjugated prodrugs lacking the imine moiety. Once the prodrug (pro-PBD) conjugate enters a targeted cell, cleavage of the linker system triggers the generation of a reactive intermediate possessing an aldehyde and aromatic amine. An 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. In our pro-PBDs, we mask the aldehyde as a hydrolytically sensitive oxazolidine moiety which in turn is a part of a reductively labile self-immolative linker system. To prove the range of applications for this new class of latent DNA-alkylators, we designed and synthesized several novel latent warheads: pro-PBD dimers and hybrids of pro-PBD with other sequence-selective DNA minor groove binders. Preliminary preclinical pharmacology studies showed excellent biological activity and specificity.

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 receptor-targeted aminopterin therapy is highly effective and specific in experimental models of autoimmune uveitis and autoimmune encephalomyelitis.

EC0746 is a rationally designed anti-inflammatory drug conjugate consisting of a modified folic acid-based ligand linked to a γ-hydrazide analog of aminopterin. In this report, EC0746's effectiveness was evaluated against experimental retinal S-antigen (PDSAg) induced autoimmune uveitis (EAU) and myelin-basic-protein induced autoimmune encephalomyelitis (EAE). In both models, functional FR-ß was detected on activated macrophages in local (retinal or central-nervous-system, respectively) and systemic (peritoneal cavity) sites of inflammation. In myelin-rich regions of EAE rats, an increased uptake of (99m)Tc-EC20 (etarfolatide; a FR-specific radioimaging agent) was also observed. EC0746 treatment at disease onset suppressed the clinical severity of both EAU and EAE, and it strongly attenuated progressive histopathological changes in the affected organs. In all parameters assessed, EC0746 activity was completely blocked by a benign folate competitor, suggesting that these therapeutic outcomes were specifically FR-ß mediated. EC0746 may emerge as a useful macrophage-modulating agent for treating inflammatory episodes of organ-specific autoimmunity.

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.

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.

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.

Reducing undesirable hepatic clearance of a tumor-targeted vinca alkaloid via novel saccharopeptidic modifications.

During a phase I trial of EC145 (a folate-targeted vinca alkaloid conjugate), constipation was identified as the dose-limiting toxicity, probably from a nonfolate receptor-related liver clearance process capable of releasing unconjugated vinca alkaloid from EC145 and shuttling it to the bile. Here, we report on the selective placement of novel carbohydrate segments (1-amino-1-deoxy-glucitolyl-γ-glutamate) spaced in-between the folate and vinca alkaloid moieties of EC145, which yielded a new agent (EC0489) that is equipotent but less toxic than EC145. Whereas both compounds could cure tumor-bearing mice reproducibly, EC0489 differed from EC145 with i) a shorter elimination half-life, ii) approximately 70% decrease in bile clearance, iii) a 4-fold increase in urinary excretion, and iv) improved tolerability in rodents. This combination of improvements justified the clinical evaluation of EC0489 where currently administered dose levels have exceeded the maximal tolerated dose of EC145 by approximately 70%, thereby reflecting the translational benefits to this new approach.

Acid mediated formation of an N-acyliminium ion from tubulysins: a new methodology for the synthesis of natural tubulysins and their analogs.

Tubuylsins are extremely potent cytotoxic agents which inhibit tubulin polymerization and lead to cell cycle arrest and apoptosis. Tubulysins have been isolated from fermentation mixtures and have been chemically synthesized; however, these efforts have been hampered by poor yields and arduous purifications. In contrast, treatment of a mixture of natural tubulysins A, B, C, G, and I, obtained from a fermentation batch with trifluoroacetic acid results in the formation of a single N-acyliminium ion. Subsequent addition of butyric, isopentyl, or acetic acid results in the formation of tubulysin B, A, or I, respectively, as a single species. New tubulysin analogs can be formed upon treatment of the acyliminium ion with other nucleophiles such as alcohols, thiols, and nitriles, resulting in corresponding N-acyl-N,O-acetals, N-acyl-N,S-thioacetals, and N,N'-diacyl-aminals. Carbon-carbon bond formation is also possible with a modification of this protocol. The cytotoxicies of the natural tubulysins and tubulysin analogs synthesized by this method were evaluated on KB cells.

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.

Targeting folate receptor beta on monocytes/macrophages renders rapid inflammation resolution independent of root causes.

Provoked by sterile/nonsterile insults, prolonged monocyte mobilization and uncontrolled monocyte/macrophage activation can pose imminent or impending harm to the affected organs. Curiously, folate receptor beta (FRß), with subnanomolar affinity for the vitamin folic acid (FA), is upregulated during immune activation in hematopoietic cells of the myeloid lineage. This phenomenon has inspired a strong interest in exploring FRß-directed diagnostics/therapeutics. Previously, we have reported that FA-targeted aminopterin (AMT) therapy can modulate macrophage function and effectively treat animal models of inflammation. Our current investigation of a lead compound (EC2319) leads to discovery of a highly FR-specific mechanism of action independent of the root causes against inflammatory monocytes. We further show that EC2319 suppresses interleukin-6/interleukin-1ß release by FRß+ monocytes in a triple co-culture leukemic model of cytokine release syndrome with anti-CD19 chimeric antigen receptor T cells. Because of its chemical stability and metabolically activated linker, EC2319 demonstrates favorable pharmacokinetic characteristics and cross-species translatability to support future pre-clinical and clinical development.

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.

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.

Design and regioselective synthesis of a new generation of targeted chemotherapeutics. Part II: Folic acid conjugates of tubulysins and their hydrazides.

Efficient syntheses of folate conjugates of tubulysins and their hydrazides 1a-d are described. These water soluble folate receptor (FR) targeted conjugates are derivatives of folic acid and the potent cytotoxic agents: tubulysin A, B, or their respective hydrazides, connected in regioselective manner via a hydrophilic peptide spacer and a reducible disulfide linker.

Pre-clinical evaluation of EC1456, a folate-tubulysin anti-cancer therapeutic.

EC1456 is a folate-tubulysin conjugate constructed with an all-D enantiomeric spacer/linker configuration. When tested against folate receptor (FR)-positive cells, EC1456 demonstrated dose-responsive activity with an approximate 1000-fold level of specificity. Treatment of nude mice bearing FR-positive human xenografts (as large as 800 mm3) with non-toxic doses of EC1456 led to cures in 100% of the mice. Combinations of low dose EC1456 with standard of care agents such as platins, taxanes, topotecan and bevacizumab, safely and significantly augmented the growth inhibitory effects of these commonly used agents. When tested against FR-positive human tumor xenograft models having confirmed resistance to a folate-vinca alkaloid (vintafolide), cisplatin or paclitaxel, EC1456 was found to generate partial to curative responses. Taken together, these studies demonstrate that EC1456 has significant anti-proliferative activity against FR-positive tumors, including models which were anticancer drug resistant, thereby justifying a Phase 1 trial of this agent for the treatment of advanced human cancers.