Paul J. Kleindl

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.

Ligand‐Targeted Delivery of Small Interfering RNAs to Malignant Cells and Tissues

Potential clinical applications of small interfering RNA (siRNA) are hampered primarily by delivery issues. We have successfully addressed the delivery problems associated with off‐site targeting of highly toxic chemotherapeutic agents by attaching the drugs to tumor‐specific ligands that will carry the attached cargo into the desired cancer cell. Indeed, several such tumor‐targeted drugs are currently undergoing human clinical trials. We now show that efficient targeting of siRNA to malignant cells and tissues can be achieved by covalent conjugation of small‐molecular‐weight, high‐affinity ligands, such as folic acid and DUPA (2‐[3‐(1, 3‐dicarboxy propyl)‐ureido] pentanedioic acid), to siRNA. The former ligand binds a folate receptor that is overexpressed on a variety of cancers, whereas the latter ligand binds to prostate‐specific membrane antigen that is overexpressed specifically on prostate cancers and the neovasculature of all solid tumors. Using these ligands, we show remarkable receptor‐mediated targeting of siRNA to cancer tissues in vitro and in vivo.

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.

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.

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.

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.

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 4735: Designing novel warheads for targeted therapies: SAR and efficient strategies for the synthesis of analogues of tubulysin

Background: Tubulysins are natural products isolated from myxobacterial species. They are potent mitotic poisons as they inhibit the polymerization of tubulin into microtubules. Structurally, tubulysins are linear tetrapeptides comprised of N-Me pipecolic acid (Mep), isoleucine (Ile), tubuvaline (Tuv) and tubutyrosine (Tut). All natural tubulysins possess an acid-, base-, and enzyme-sensitive N-acyloxymethyl substituent, as well as a labile acetate group. For the natural tubulysins, both of these functional groups are essential for their potent cytotoxicity. The focus of this presentation is on design and synthesis of chemically stable analogs of tubulysin with improved biological activity. Methods: To explore the influence of tubulysin9s functional groups on its cytotoxicity, we designed novel analogs, incorporating ether, thioether, amide and carbamate-based side chains. We also suggested basic SAR-rules for the activity of these synthetic analogs. Results: Structural features, critical for the tubulysin9s cytotoxicity, were investigated. e.g., changing the (R)-OAc group of Tuv to (S)-NHAc completely abolished cytotoxicity, while analogs possessing (R)-NHAc on the Tuv moiety still exhibited activity. On the other hand, exchange of Tuv9s (R)-OAc group with (R)-OMe had no influence activity. Replacement of the O-acyl part of N-acyloxymethyl substituent (dotted circle) with an alkyl or alkylthiol group resulted in N,O-acetals or N,S-thioacetals possessing similar or higher activity. Tup and Tut are interchangeable in terms of the activities of their respective tubulysin analogs. Conclusion: We designed and synthesized tubulysin analogs which are less prone to degradation under acidic and basic conditions, as well as enzymic influences. Structural features, essential for the cytotoxicity of the tubulysin analogs, were established. Some of the new compounds, especially those with N,S-thioacetal side chains, have increased cytotoxicity in comparison to natural tubulysins. These novel tubulysin analogs were used as warheads for folate receptor(FR)-targeted therapy. Citation Format: Iontcho Vlahov, Fei You, Paul Kleindl, Marilynn Vetzel, Joseph Reddy, Christopher Leamon. Designing novel warheads for targeted therapies: SAR and efficient strategies for the synthesis of analogues of tubulysin. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4735.