Jeremy F. Vaughn

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 vintafolides 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.

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.

Antiinflammatory activity of a novel folic acid targeted conjugate of the mTOR inhibitor everolimus

Folate receptor (FR)-β 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, EC0746s 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-β 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-β mediated. EC0746 may emerge as a useful macrophage-modulating agent for treating inflammatory episodes of organ-specific autoimmunity.

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.

Abstract 4495: Small molecule approach for targeting tumor-associated macrophages via a functional FRβ

Tumor-associated macrophages (TAMs) are considered an attractive target for oncotherapy because of their important role in tumor microenvironment. While TAMs mostly arise from inflammatory blood monocytes, they become quickly programmed to the pro-tumor phenotype. In response to tumor-derived factors, these M2-biased TAMs are essentially tumor helper cells, as both cancer cells and TAMs promote each another through a paracrine signaling loop involving epidermal growth factor and colony stimulating factor-1. This important cellular interaction is critical at all stages of tumor progression including angiogenesis, invasion, growth and metastasis. In recent years, TAMs (CD68+, CD163+) have been found to express a functional folate receptor-β (FRβ) that displays high-affinity ligand binding and subsequent ligand-initiated endocytosis. FRβ-expressing TAMs are present in various cancer subtypes, the perivascular region, and the tumor invasive front. The abundance of FRβ+ TAMs has been linked to poor patient survival in pancreatic ductal adenocarcinoma. In the present study, we first characterized the phenotype of FRβ+ TAMs ex-vivo from both syngeneic and xenographic tumor models (breast, lung, melanoma, and pancreatic cancers). Using thioglycollate-elicited macrophages as positive controls, we estimated FRβ levels on TAMs with monoclonal antibodies and functional folate ligands. Using a ligand conjugation approach, we focused on targeting FRβ+ TAMs in tumors arising from cancer cells that do not express functional FR themselves. Preliminary proof-of-concept was established in the highly aggressive FR-negative 4T1 mammary tumor with a folate-targeted DNA alkylating agent. In addition, immunohistochemical analysis showed high FRβ expression in human anaplastic thyroid cancer, a TAM-rich disease with very poor prognosis. Collectively, our data suggest that targeting a TAM-rich cancer via FRβ may be a viable therapeutic approach with potential applications in FR-negative tumors. Citation Format: Leroy W. Wheeler, Yingjuan Lu, Vicky Cross, Alex Lloyd, Nikki Parker, Longwu Qi, Kevin Wang, Ian Wang, Spencer Hahn, Jeremy Vaughn, Iontcho P. Vlahov, Philip S. Low, Christopher P. Leamon. Small molecule approach for targeting tumor-associated macrophages via a functional FRβ. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4495. doi:10.1158/1538-7445.AM2015-4495

Abstract 4462: Structure-activity relationship studies for PSMA-targeted tubulysin conjugates

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Prostate-specific membrane antigen (PSMA) is a cell-surface receptor expressed on prostate cancer cells. Recent findings suggest that PSMA is also abundant on newly formed blood vessels which supply most non-prostatic solid tumors; including lung, colon, breast, renal, liver, and pancreatic carcinomas. These reports have provided solid support for the concept of designing drug conjugates for PSMA-targeted cancer therapy. Here we describe our structure-activity relationship analyses resulting in the discovery of highly potent PSMA-targeted tubulysin conjugates. In our study, more than thirty ligand-drug constructs were designed and synthesized based on strategically placing a variety of chemical groups responsible for evoking a targeted biological effect in vitro and in vivo. The optimized molecular architecture is comprised of a high affinity PSMA-binding urea-type ligand, a water soluble peptide- or carbohydrate-based spacer unit, a cleavable disulfide-containing self-immolative linker system, and the highly potent chemotherapy drug tubulysin which inhibits microtubule activity . One of the conjugates possessed superior targeted activity and was selected for further preclinical development. Citation Format: Kevin Wang, Spencer Hahn, Hari Santhapuram, Longwu Qi, Paul Kleindl, Jeremy Vaughn, Fei You, Joe Reddy, Ryan Dorton, Christopher Leamon, Iontcho Vlahov. Structure-activity relationship studies for PSMA-targeted tubulysin conjugates. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4462. doi:10.1158/1538-7445.AM2014-4462

Abstract 4499: Total synthesis of tubulysins: A new chemical reaction leads to analogues with enhanced cytotoxicity.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Tubulysins are natural products isolated from myxobacterial species and are mitotic poisons which exceed the cell growth inhibition of any clinically relevant traditional chemotherapeutic. Structurally, tubulysins are linear tetrapeptides comprised of N-methyl pipecolic acid (Mep), isoleucine (Ile), the novel amino acid tubuvaline (Tuv), and the novel tyrosine analogue tubutyrosine (Tut). All isolated tubulysins possess an acid- and base-sensitive N-acyloxymethyl substituent not previously found in nature. This N,O-acetal of formaldehyde is attached as a side chain to the amide N-atom of the Tuv fragment. The isolation of natural tubulysins from culture extracts provides only limited quantities. Recently, we reported a large scale total synthesis of natural tubulysin B. Among the multiple synthetic and stereochemical challenges, the most striking were: a) the incorporation of the labile N,O-diacyl N,O-acetal and b) the regioselective hydrolysis of the C-terminal methyl ester (OMe) in the tripeptide Mep-Ile-Tuv-OMe. Herein, we present a convergent total synthesis of tubulysin analogues incorporating an alkoxymethyl side chain. Such molecular architecture allows for compounds that are more base and esterase inert, thus providing additional metabolic/catabolic stabilization. The key step in our synthetic strategy relies on use of a novel reagent for the efficient conversion of the N-acyloxymethyl substituent to novel side chain groups with concomitant facilitated hydrolysis of the C-terminal methyl ester. Following LiOH-based hydrolysis of the C-terminal ester, the Tut fragment is added resulting in novel tubulysin analogues. This novel process opens the door to tubulysin analogs with improved potency for treating cancer. Citation Format: Iontcho Vlahov, Longwu Qi, Fei You, Spencer Hahn, Hari K. Santhapuram, Kevin Wang, Paul Kleindl, Jeremy Vaughn, Marilynn Vetzel, Joseph Reddy, Christopher Leamon. Total synthesis of tubulysins: A new chemical reaction leads to analogues with enhanced cytotoxicity. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4499. doi:10.1158/1538-7445.AM2013-4499

Abstract 2518: Structure-activity relationships of folate-vinca alkaloid conjugates

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL EC145 is a potent folate-targeted vinca alkaloid conjugate that has been evaluated in 2 single agent phase 2 trials (ovarian and non small cell lung cancers) and 1 randomized open labeled phase 2 study in combination with DOXIL® in women with platinum-resistant ovarian cancer. Results from all of these trials have looked promising, and they support the continued evaluation of EC145 in a planned phase 3 trial thats expected to begin in early 2011. Structurally, EC145 consists of 4 essential modules: 1) folic acid, 2) a hydrophilic peptide spacer, 3) a disulfide-containing, self-immolative linker, and 4) the microtubule destabilizer, desacetylvinblastine hydrazide (DAVLBH). Due to EC145s potential for success, recent effort was given to determine the impact of i) substituting the DAVLBH vinca alkaloid unit in EC145 with analogs of vincristine, vindesine or vinorelbine, ii) substituting the naturally (S)-configured Asp-Arg-Asp-Asp-Cys peptide spacer in EC145 with (R)-configured amino acids placed in selective locations, and iii) varying the composition of the linker module to evaluate the impact of having a readily releasable, self-immolative linker system (e.g. disulfide-based as found in EC145) versus a more stable linker system (e.g. a thioether). New conjugates were screened in vitro against folate receptor-positive cells, and their cytotoxic activities were directly compared to that produced by EC145. Our data show that only DAVLBH-containing folate conjugates were active; thus, conjugates built with vincristine, vindesine or vinorelbine failed to produce activity when cells were exposed to concentrations as high as 1 µM. Within the DAVLBH series, having a bio-releasable, self-immolative linker system was found to be critical for activity since multiple analogs constructed with thioether-based linkers failed to produce meaningful activity both in vitro and in vivo. Substitutions of some or all of the natural amino acids within EC145s hydrophilic spacer module was not found to significantly change the in vitro potency of the conjugate; however, some enhancement in anti-tumor activity was measurable when tested in vivo against well-established subcutaneous folate receptor-positive tumor xenografts. Despite these modest improvements, EC145 remains as one of the more potent folate-vinca alkaloid conjugates produced to date, and continued clinical development is therefore warranted. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2518. doi:10.1158/1538-7445.AM2011-2518

Abstract 5515: Design and synthesis of releasable folate-drug conjugates possessing optimized carbohydrate-based spacer units

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC The spacer region of releasable folate-drug conjugates may play an important role in “tailoring” the properties of the molecules for targeted therapies by: 1) modulating the affinity toward the folate receptor, 2) providing desirable hydrophilicity, and 3) changing dramatically the biological properties. Herein we describe the design and synthesis of a variety of novel carbohydrate-based spacers mimicking a peptidic backbone. After carefully optimizing the chemical structure of the carbohydrate units, we incorporated such spacers in a folate-containing molecular systems using standard solid phase synthesis (SPS). The synthetic utility of this elegant and innovative approach was exemplified by creating novel releasable folate conjugates of single cytotoxic agents, e.g. Tubulysin-Folate conjugate (currently in preclinical development) and Vinblastine-Folate conjugate, (already in clinical development). Furthermore, using the same powerful method, we synthesized several constructs containing a rational combination of two drugs (e.g. Vinblastine and Doxorubicin) attached to a single folate-targeting ligand via a dual-headed releasable Spacer-Linker system. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5515.