Madduri Srinivasarao

Principles in the design of ligand-targeted cancer therapeutics and imaging agents

Most cancer drugs are designed to interfere with one or more events in cell proliferation or survival. As healthy cells may also need to proliferate and avoid apoptosis, anticancer agents can be toxic to such cells. To minimize these toxicities, strategies have been developed wherein the therapeutic agent is targeted to tumour cells through conjugation to a tumour-cell-specific small-molecule ligand, thereby reducing delivery to normal cells and the associated collateral toxicity. This Review describes the major principles in the design of ligand-targeted drugs and provides an overview of ligand–drug conjugates and ligand–imaging-agent conjugates that are currently in development.

Ligand-Targeted Drug Delivery

Safety and efficacy constitute the major criteria governing regulatory approval of any new drug. The best method to maximize safety and efficacy is to deliver a proven therapeutic agent with a targeting ligand that exhibits little affinity for healthy cells but high affinity for pathologic cells. The probability of regulatory approval can conceivably be further enhanced by exploiting the same targeting ligand, conjugated to an imaging agent, to select patients whose diseased tissues display sufficient targeted receptors for therapeutic efficacy. The focus of this Review is to summarize criteria that must be met during design of ligand-targeted drugs (LTDs) to achieve the required therapeutic potency with minimal toxicity. Because most LTDs are composed of a targeting ligand (e.g., organic molecule, aptamer, protein scaffold, or antibody), spacer, cleavable linker, and therapeutic warhead, criteria for successful design of each component will be described. Moreover, because obstacles to successful drug design can differ among human pathologies, limitations to drug delivery imposed by the unique characteristics of different diseases will be considered. With the explosion of genomic and transcriptomic data providing an ever-expanding selection of disease-specific targets, and with tools for high-throughput chemistry offering an escalating diversity of warheads, opportunities for innovating safe and effective LTDs has never been greater.

Design, Synthesis, and Evaluation of a Neurokinin-1 Receptor-Targeted Near-IR Dye for Fluorescence-Guided Surgery of Neuroendocrine Cancers

The neurokinin-1 receptor (NK1R) is implicated in the growth and metastasis of many tumors, including cancers of the brain (e.g., gliomas, glioblastomas, and astrocytomas), skin (e.g., melanomas), and neuroendocrine tissues (cancers of the breast, stomach, pancreas, larynx, and colon). Because overexpression of NK1R has been reported in most of these malignancies, we have undertaken designing an NK1R-targeted near-infrared (NIR) fluorescent dye for fluorescence-guided surgeries of these cancers. We demonstrate here that an NK1R-binding ligand linked to the NIR dye LS288 selectively accumulates in NK1R-expressing tumor xenografts with high affinity (Kd = 13 nM), allowing intraoperative imaging of these cancers in live mice. Because tumor accumulation is nearly quantitatively blocked by excess unlabeled ligand, and because NK1R-negative tumors and normal tissues display virtually no uptake, we conclude that the observed tumor retention is NK1R-mediated. Results on the synthesis, in vitro characterization, and animal testing of NK1R-targeted NIR dye are presented.

Studies on the synthesis of apoptolidin: synthesis of a C1-C27 fragment of apoptolidin D.

Synthesis of a C(1)-C(27) fragment, a key intermediate in the synthesis of apoptolidin D, is reported. The synthesis involves a combination of Heck coupling and Horner-Wadsworth-Emmons reaction for the C(1)-C(7) trienoate portion and an efficient Suzuki cross-coupling protocol for the C(10)-C(13) diene portion.

Fluorescence-guided surgery of cancer: applications, tools and perspectives

Thousands of patients die each year from residual cancer that remains following cytoreductive surgery. Use of tumor-targeted fluorescent dyes (TTFDs) to illuminate undetected malignant tissue and thereby facilitate its surgical resection shows promise for reducing morbidity and mortality associated with unresected malignant disease. TTFDs can also improve i) detection of recurrent malignant lesions, ii) differentiation of normal from malignant lymph nodes, iii) accurate staging of cancer patients, iv) detection of tumors during robotic/endoscopic surgery (where tumor palpation is no longer possible), and v) preservation of healthy tissue during resection of cancer tissue. Although TTFDs that passively accumulate in a tumor mass provide some tumor contrast, the most encouraging TTFDs in human clinical trials are either enzyme-activated or ligand-targeted to tumor-specific receptors.

Targeted Tubulysin B Hydrazide Conjugate for the Treatment of Luteinizing Hormone-Releasing Hormone Receptor-Positive Cancers

The targeted delivery of chemotherapeutic agents to receptors that are overexpressed on cancer cells has become an attractive strategy to concentrate drugs in cancer cells while avoiding uptake by healthy cells. Luteinizing hormone-releasing hormone receptor (LHRH-R) has attracted considerable interest for this application, since LHRH-R is upregulated in ∼86% of prostate cancers, ∼80% of endometrial cancers, ∼80% of ovarian cancers, and ∼50% of breast cancers, but virtually absent from normal tissues. Although LHRH and related peptides have been used to deliver cytotoxic drugs to LHRH-R overexpressing cancer cells, they have suffered from off-target delivery of the therapeutic agents to the liver and kidneys. To reduce such unwanted uptake by peptide scavenger receptors in the liver and kidneys, we have explored the use of a nonpeptidic LHRH-R antagonist (NBI42902) to construct an LHRH-R targeted tubulysin conjugate (BOEPL-L3-TubBH). In vitro studies with BOEPL-L3-TubBH demonstrate that the conjugate can kill LHRH-R expressing triple-negative breast cancer cells (MDA-MB-231 cells) with low nanomolar IC50. Related studies on tumor-bearing mice further reveal that the same conjugate can eradicate MDA-MB-231 solid tumors without any measurable side-effects, yielding mice that gain weight during therapy and show no evidence of tumor recurrence for at least 5 weeks after termination of treatment. That these complete responses are LHRH-R targeted was then established by showing that identical treatment of receptor-negative (SKOV3) tumors yields no antitumor response. Overall, these data provide a proof-of-concept that LHRH-R specific targeting of an extremely toxic drug like tubulysin B can treat LHRH-R positive tumors without causing significant toxicity to healthy cells.

New Mechanism for Release of Endosomal Contents: Osmotic Lysis via Nigericin-Mediated K+/H+ Exchange

Although peptides, antibodies/antibody fragments, siRNAs, antisense DNAs, enzymes, and aptamers are all under development as possible therapeutic agents, the breadth of their applications has been severely compromised by their inability to reach intracellular targets. Thus, while macromolecules can often enter cells by receptor-mediated endocytosis, their missions frequently fail due to an inability to escape their entrapping endosomes. In this paper, we describe a general method for promoting release of any biologic material from any entrapping endosome. The strategy relies on the fact that all nascent endosomes contain extracellular (Na+-enriched) medium, but are surrounded by intracellular (K+-enriched) fluid in the cytoplasm. Osmotic swelling and rupture of endosomes will therefore be facilitated if the flow of K+ down its concentration gradient from the cytosol into the endosome can be facilitated without allowing downhill flow of Na+ from the endosome into the cytosol. While any K+ selective ionophore can promote the K+ specific influx, the ideal K+ ionophore will also exchange influxed K+ for an osmotically inactive proton (H+) in order to prevent buildup of an electrical potential that would rapidly halt K+ influx. The only ionophore that catalyzes this exchange of K+ for H+ efficiently is nigericin. We demonstrate here that ligand-targeted delivery of nigericin into endosomes that contain an otherwise impermeable fluorescent dye can augment release of the dye into the cell cytosol via swelling/bursting of the entrapping endosomes. We further show that nigericin-facilitated escape of a folate-targeted luciferase siRNA conjugate from its entrapping endosomes promotes rapid suppression of the intended luciferase reporter gene. Taken together, we propose that ionophore-catalyzed entry of K+ into endosomal compartments can promote the release of otherwise impermeable contents from their encapsulating endosomes.