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.

Toward Orally Absorbed Prodrugs of the Antibiotic Aztreonam. Design of Novel Prodrugs of Sulfate Containing Drugs. Part 2.

Aztreonam, first discovered in 1980, is an FDA approved, intravenous, monocyclic beta-lactam antibiotic. Aztreonam is active against Gram-negative bacteria and is still used today. The oral bioavailability of aztreonam in humans is less than 1%. Herein we describe the design and synthesis of potential oral prodrugs of aztreonam.

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.

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.

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.

Prevention of drug-induced memory impairment by immunopharmacotherapy.

One approach to treating drug abuse uses antidrug antibodies to immunize subjects against the illicit substance rather than administering therapeutics that target the specific CNS site of action. At present, passive vaccination has recognized efficacy in treating certain gross symptoms of drug misuse, namely, motor activation, self-administration, and overdose. However, the potential for antibodies to prevent drug-induced changes involving finer cognitive processes, such as benzodiazepine-associated amnesia, remains unexplored. To address this concept, a flunitrazepam hapten was synthesized and employed in the generation of a panel of high affinity monoclonal antibodies. Anti-flunitrazepam mAb RCA3A3 ( K d,app = 200 nM) was tested in a mouse model of passive immunization and subsequent mole-equivalent challenge with flunitrazepam. Not only was flunitrazepam-induced sedation prevented but immunization also conferred protection to memory consolidation as assessed through contextual and cued fear conditioning paradigms. These results provide evidence that immunopharmacotherapeutic blockade of drug intoxication also preserves complex cognitive function.

Delta9-tetrahydrocannabinol immunochemical studies: haptens, monoclonal antibodies, and a convenient synthesis of radiolabeled delta9-tetrahydrocannabinol.

Immunopharmacotherapy as an approach to combat drugs of abuse has become an active area of investigation. Marijuana is the most commonly used illicit drug in the U.S. The main active chemical in marijuana is delta9-tetrahydrocannabinol (delta9-THC); hence, monoclonal antibodies with high affinity and specificity for delta9-tetrahydrocannabinol could be valuable immunopharmacotherapeutic intervention and diagnostic tools. We have synthesized immunoconjugates that induce an effective immune response to delta9-THC and describe a convenient synthesis of radiolabeled delta9-THC. We demonstrate the value and use of this probe to select anti-delta9-THC antibodies that bind delta9-THC with good affinity. The synthetic route to radiolabeled delta9-THC has enabled the correct assessment of the affinity of these antibodies to their ligand and may facilitate future binding studies between delta9-THC and its analogues and the cannabinoid receptors.

Quorum sensing in Vibrio harveyi: probing the specificity of the LuxP binding site.

Quorum sensing activity was investigated in the bacterium Vibrio harveyi using a series of both natural and nonnatural analogs of DPD, the penultimate precursor to autoinducer AI-2. The progression of molecules that were both synthesized and investigated includes enantiomeric variants, carbon-chain extension, and hydroxyl-functional group addition/deletions of DPD. The compilation of these studies reveals a binding cleft that can accommodate a number of different structural variants of DPD, albeit with invariably lower activities.

Fluorescent cocaine probes: a tool for the selection and engineering of therapeutic antibodies.

Cocaine is a highly addictive drug, and despite intensive efforts, effective therapies for cocaine craving and addiction remain elusive. In recent years, we and others have reported advances in anti-cocaine immunopharmacotherapy based on specific antibodies capable of sequestering the drug before it reaches the brain. In an effort to obtain high affinity therapeutic anti-cocaine antibodies, either whole IgGs or other antibody constructs, fluorescence spectroscopic techniques could provide a means of assisting selection and engineering strategies. We report the synthesis of a series of cocaine-fluorophore conjugates (GNC-F1, GNC-F2, GNC-I) and the functional evaluation of these compounds against single-chain Fv antibodies obtained via crystallographic analysis/engineering and against commercially available anti-cocaine monoclonal antibodies with a wide range of cocaine-binding affinities. From these studies, we determined that the GNC-F2 fluorophore reproduced affinity constants obtained using [(3)H]-labeled cocaine. We anticipate that the readily synthesized and nonradioactive GNC-F2 will find use both as a tool for bioimaging and in the high-throughput selection and engineering of potential therapeutic antibodies against cocaine.

Solid-phase synthesis of anandamide analogues.

The endocannabinoids are amides and esters of arachidonic acid that can mimic the pharmacological properties of Delta(9)-tetrahydrocannabinol (Delta(9)-THC). Anandamide, the most prominent of the endocannabinoids, has been implicated in both metabolic/physiological roles of the central nervous system, making it an attractive medicinal target. As such, we report the first solid-phase methodology that expedites access to various anandamide analogues. Our synthesis features a repetitive Cu-mediated coupling reaction between terminal alkynes and propargyl halides or allylic halides.