SBIR-STTR Award

Following systemic administration, most anticancer drugs exhibit very limited uptake into the brain. The blood- brain barrier (BBB) is the single most important factor for restricting drug delivery to the brain. The BBB is formed by a continuous monolayer of cerebrovascular endothelial cells joined together by tight junctions and lacks fenestrations. To cross the BBB, solutes must diffuse across the lipid endothelial cell membrane or be carried across either by receptor-mediated transport or by membrane transport. The capillary endothelial cells of the BBB express an array of uptake and efflux transporters. Regulated expression and function of these transporters govern access of essential nutrients and therapeutic drugs to the central nervous system (CNS). Successful drug delivery to brain tumors includes not only penetration of the BBB, but also delivery to and uptake into tumor cells. The Large Amino Acid Transporter 1 (LAT1) fulfills all necessary requirements - expression at both sides of the BBB, high expression in almost all tumor cells regardless of the tissue of origin, and very low endogenous expression in non-proliferative cells. Furthermore, it is characterized by an appreciable capacity, which can be exploited to deliver therapeutically meaningful amounts of chemotherapeutic agents. LAT1 activity correlates with cell proliferation, tumor growth, and is inversely related to disease prognosis and patient survival. Quadriga BioSciences will use NIH/NCI funding to engineer novel anti-cancer agents by combining the properties of cytotoxic agents with LAT1-mediated delivery to cancerous cells. The aims of Phase 1 of the proposed SBIR research are geared to determine whether LAT1-transported anticancer agents are a viable option for the development of chemotherapeutics to treat brain tumors. Success of our strategy is defined as activity and high specificity for LAT1. In Aim 1 a set of compounds will be synthesized that are "hybrid" compounds in which a known cytotoxic moiety is chemically fused to a LAT1- specific recognition element. These compounds will be tested for LAT1 activity and specificity in Aim 2. LAT1 transport will be determined in vitro using [3H]-gabapentin uptake competition in HEK cells and direct uptake coupled with LC/MS/MS detection. The specificity for LAT1 vs. other amino acid transporters will be tested by [3H]-phenylalanine uptake competition in cells expressing those transporters. Possible BBB transport of the compounds will be assessed by and in vitro transwell transport assay. In Aim 3, cytotoxicity (LD50 and LD100) of compounds toward cancer cell lines and bone marrow progenitor cells is evaluated. To exclude that test compounds enter the cell via means other than LAT1, the effect of LAT1 inhibitors on the cytotoxicity will be determined. A successful compound will be cytotoxic to LAT1-expressing cancer cell lines through selective accumulation and exhibit no cytotoxicity to bone marrow progenitor cells. In projected Phase 2 studies, successful compounds will be tested in vivo in validated xenograft models for various brain tumors.

Public Health Relevance Statement:
This SBIR Phase 1 grant application is relevant to the mission of the National Institute of Health. The main objective is to develop novel anti-cancer agents for patients with primary or metastatic brain tumors. Small polar nutrients can cross the blood brain barrier (BBB) and reach brain cancer cells through specific influx transporters with expression at both the BBB and the tumor. Our strategy is to exploit these up-take mechanisms for an increased and targeted drug delivery. Engineered chemotherapeutics, mimicking endogenous nutrients are taken up from the circulation into the brain and the tumor. Key to our approach is the promise of high efficacy and low systemic toxicity. Transporter targeted chemotherapy provides a novel effective armamentarium against brain tumors with potential utility for treatment of other cancers.

Project Terms:
Affect; Alkylators; Alkylating Agents; aminoacid; Amino Acids; essential aminoacid; Essential Amino Acids; inhibitor; inhibitor/antagonist; anticancer drug; anticancer agent; Tumor-Specific Treatment Agents; Neoplastic Disease Chemotherapeutic Agents; Cancer Drug; Antineoplastics; Antineoplastic Drugs; Anti-Cancer Agents; Antineoplastic Agents; Biologic Assays; Bioassay; Assay; Biological Assay; Circulation; Bloodstream; Blood Circulation; Hemato-Encephalic Barrier; Blood-Brain Barrier; Blood - brain barrier anatomy; Bone Marrow Reticuloendothelial System; Bone Marrow; Encephalon; Brain Nervous System; Brain; tumors in the brain; Brain Tumors; Brain Neoplasia; Brain Neoplasms; neoplasm/cancer; malignancy; Malignant Tumor; Cancers; Malignant Neoplasms; cultured cell line; Strains Cell Lines; CellLine; Cell Line; plasmalemma; Plasma Membrane; Cytoplasmic Membrane; Cell membrane; Cells; Cytotoxin; Diagnosis; Diffusion; disease/disorder; Disorder; Disease; drug/agent; Pharmaceutic Preparations; Medication; Drugs; Pharmaceutical Preparations; Elements; Engineering; Erythropoietic Stem Cells; Erythropoietic Progenitor Cells; Erythroid Stem Cells; Erythroid Precursor Cells; Erythroid Progenitor Cells; Exhibits; GABA; Aminalone; Aminalon; 4-amino-butanoic acid; 4-Aminobutyric Acid; 4-Aminobutanoic Acid; gamma-Aminobutyric Acid; Goals; Hybrids; In Vitro; Incidence; LD50; LD-50; Lethal Dose 50; L-Leucine; L-Isomer Leucine; Leucine; Lipids; Urea, N-(2-chloroethyl)-N'-cyclohexyl-N-nitroso-; N-(2-chloroethyl)-N'-cyclohexyl-N-nitrosourea; Lomustinum; Lomustine (CCNU); Chloroethylcyclohexylnitrosourea; Chloroethylcyclo- hexylnitrosourea; 1-Nitrosourea, 1-(2-chloroethyl)-3-cyclohexyl-; Lomustine; sarcolysin; phenylalanine nitrogen mustard; p-di(chloroethyl)amino-L-phenylalanine; melfalan; alanine nitrogen mustard; Phenylalanine Mustard; L-Sarcolysine; L-Sarcolysin Phenylalanine mustard; L-Phenylalanine mustard; L-PAM; DL-O-Sarcolysin; 4-[bis(2-chloroethyl)amino]-L-phenylalanine; 4-(Bis(2-chloroethyl)amino)phenylalanine; Melphalan; Methods; Mission; National Institutes of Health; NIH; United States National Institutes of Health; nitrosourea; Nitrosourea Compounds; Patients; Permeability; L-Phenylalanine; L-Isomer Phenylalanine; Phenylalanine; Prognosis; outcome forecast; Reagent; Research; Specificity; Progenitor Cells; Stem cells; Testing; Body Tissues; Tissues; Neurontin; 1-(aminomethyl)cyclohexaneacetic Acid; gabapentin; Drug Delivery; Drug Delivery Systems; Mean Survival Times; Mediating; Malignant Tumor of the Brain; Brain Cancer; Malignant neoplasm of brain; base; Organ; improved; Diffuse; Penetration; Phase; Evaluation; Endothelial Cells; Zonula Occludens; Occluding Junctions; Tight Junctions; Funding; uptake; Bone Marrow Suppression; Therapeutic; Cytotoxic drug; Cytotoxic agent; Normal Tissue; Normal tissue morphology; Malignant Cell; cancer cell; Myeloid; Myelogenous; Side; Cellular Proliferation; Cell Multiplication; Cell Growth in Number; Cell Proliferation; Lytotoxicity; cytotoxicity; membrane structure; Membrane; Tumor Cell; neoplastic cell; Receptor Protein; receptor; solute; success; tumor growth; Toxicities; Toxic effect; 3-isobutyl GABA; pregabalin; stereochemistry; Nutrient; novel; experimental study; experimental research; experiment; research study; chemotherapeutic agent; System L Transporter Proteins; System L Transporter; Large Neutral Amino Acid Transporter; Large Amino Acid Transporter; LNAA; Amino Acid Transport System L Proteins; Amino Acid Transport System L; Modeling; LOINC Axis 2 Property; Property; LC/MS; liquid chromatography mass spectrometry; monolayer; Central Nervous System; CNS Nervous System; Neuraxis; Amino Acid Transport Systems; Amino Acid Channel; Amino Acid Transporter; Molecular Interaction; Binding; Binding (Molecular Function); Capillary Endothelial Cell; Address; Affinity; cytotoxic; Data; Detection; Dose-Limiting; Grant Proposals; Applications Grants; N Domain; in vivo; Small Business Innovation Research; SBIRS (R43/44); SBIR; Small Business Innovation Research Grant; Membrane Transport; Transmembrane Transport; Xenograft Model; developmental; Development; cerebrovascular; Cancer cell line; Coupled; Cancerous; chemotherapy; tumor; public health relevance; in vitro testing; phase II study; phase 2 study

This grant application aims to radically transform the therapy of brain tumors by creating new efficacious agent with strong delivery to brain, robust activity against brain tumors, and decreased peripheral dose-limiting toxicity in other organs. While primary brain tumors represent a rare malignancy, high-grade gliomas are aggressive and most patients die of disease progression with a median survival is 9-15 months after diagnosis. The standard of care for newly diagnosed high-grade gliomas involves surgical resection, followed by fractionated radiation therapy (RT) and concurrent treatment with temozolomide (TMZ). However, 60% of patients are resistant to TMZ and suffer the consequences of chemotherapy without therapeutic benefit. Thus, there is a pressing need for a novel chemotherapeutic agent that is effective for brain cancers, exhibits low systemic toxicity, and is effective for TMZ-insensitive patients. Most anticancer drugs are ineffective for treating CNS tumors, in large part due to their inability to cross the blood-brain barrier (BBB). Although the BBB is frequently impaired in brain tumors, it is still a formidable obstacle for adequate drug delivery. The large amino acid 1 (LAT1), also known as Tumor Antigen 1 (TA1), is localized in the BBB and allows for the transport of essential amino acids and CNS therapeutics into the brain. LAT1 is highly over-expressed in almost all gliomas, but displays very low expression level in non-proliferative cells. Therefore, chemotherapeutic agents that can be transported by LAT1 are uniquely suited for crossing the BBB and specifically treating brain tumors. In Phase 1 of the SBIR research, we engineered novel anti-cancer compounds that combine the properties of a cytotoxic agent with a proprietary LAT1 recognition element. We identified QBS10072S as our lead candidate. QBS10072S demonstrated robust activity against brain tumors in an intracranial xenograft model of GBM and decreased peripheral dose-limiting toxicity in other organs. Median survival of QBS10072S treated mice was improved compared to vehicle- or standard of care (SOC)-treated animals. The aims of the proposed Phase II research are geared to determine whether QBS10072S can achieve investigational new drug (IND) status. In Aim 1, we will characterize the ADME properties of QBS10072S and analyze the biodistribution in healthy and intracranial tumor-bearing mice. In Aim 2, we will optimize the dose and dosing regimen for QBS10072S to develop a safe and effective treatment for GBM. In Aim 3, QBS10072S treatment will be evaluated against the SOC in TMZ-resistant and TMZ-sensitive animal models. We will conduct an exhaustive evaluation of QBS10072S efficacy in patient derived xenograft models to discover biomarkers, and identify patient responder and non-responder profiles. [Aim 4 explores synergistic/additive therapeutic effects of combining QBS10072S with other brain permeable drugs to reduce dose and toxicity and to minimize or delay the induction of drug resistance.] Single and repeat dose Good Laboratory Practice (GLP) toxicokinetic (TK) and toxicity studies will be performed in Aim 5 in support of an IND submission.

Public Health Relevance Statement:
Project Narrative This SBIR Phase 2 grant application is relevant to the mission of the National Institute of Health. The main objective is to develop novel anti-cancer agents for patients with brain tumors. Most chemotherapeutics are unsuccessful against brain tumors since they are unable to cross the blood brain barrier (BBB) effectively. Small polar nutrients can cross the BBB and reach brain cancer cells through specific influx transporters with expression at both the BBB and the tumor. We have engineered a novel chemotherapeutic agent that mimics endogenous nutrients, crosses the BBB, and enters tumors and suppresses tumor growth in animal models. Key to our targeted approach is high efficacy against brain tumors and low systemic toxicity. Transporter- targeted chemotherapy provides a novel effective armamentarium against brain tumors with potential utility for treatment of other cancers.

Project Terms:
Alkylators; Alkylating Agents; aminoacid; Amino Acids; essential aminoacid; Essential Amino Acids; Animals; inhibitor; inhibitor/antagonist; anticancer drug; anticancer agent; Tumor-Specific Treatment Agents; Neoplastic Disease Chemotherapeutic Agents; Cancer Drug; Antineoplastics; Antineoplastic Drugs; Anti-Cancer Agents; Antineoplastic Agents; astrocytic glia; Astroglia; Astrocytus; Astrocytes; postmortem; necropsy; Autopsy; Radioautography; Autoradiography; Behavior; Hemato-Encephalic Barrier; Blood-Brain Barrier; Blood - brain barrier anatomy; Bone Marrow Reticuloendothelial System; Bone Marrow; Encephalon; Brain Nervous System; Brain; tumors in the brain; Brain Tumors; Brain Neoplasia; Brain Neoplasms; neoplasm/cancer; malignancy; Malignant Tumor; Cancers; Malignant Neoplasms; multimodality therapy; combined treatment; combined modality treatment; combination therapy; Multimodality Treatment; Multimodal Treatment; Multimodal Therapy; Combined Modality Therapy; Diagnosis; Diffusion; domestic dog; canine; Dogs Mammals; Dogs; Canine Species; Canis familiaris; resistant to Drug; resistance to Drug; drug resistant; Drug resistance; drug/agent; Pharmaceutic Preparations; Medication; Drugs; Pharmaceutical Preparations; Investigational New Drugs; Investigational Drugs; Elements; Engineering; Exhibits; spongioblastoma multiforme; glioblastoma multiforme; Grade IV Astrocytoma; Grade IV Astrocytic Tumor; Grade IV Astrocytic Neoplasm; Glioblastoma; neuroglia tumor; neuroglia neoplasm; glial-derived tumor; Neuroglial Tumor; Neuroglial Neoplasm; Glial Tumor; Glial Neoplasm; Glial Cell Tumors; Glioma; Goals; Modern Man; Human; In Vitro; Incidence; Lead; heavy metal lead; heavy metal Pb; Pb element; Lomustine; Lomustinum; Mechlorethamine; chloromethine; Nitrogen Mustard; Mustine; Methylchlorethamine; HN2; HN-2; Chlormethine; Chlorethazine; Chloramin; Melphalan; sarcolysin; phenylalanine nitrogen mustard; melfalan; alanine nitrogen mustard; Phenylalanine Mustard; L-Sarcolysine; L-PAM; DL-O-Sarcolysin; Mission; mortality; Mus; Murine; Mice Mammals; Mice; Mustard; United States National Institutes of Health; National Institutes of Health; NIH; Nitrosourea Compounds; nitrosourea; Pain; Painful; Palliative Care; comfort care; Palliative Treatment; Palliative Therapy; Pathology; Patients; Permeability; Prodrugs; Pro-Drugs; Drug Precursors; Radiation therapy; treatment with radiation; radio-therapy; radiation treatment; Radiotherapy; Radiotherapeutics; Rattus; Rats Mammals; Rat; Common Rat Strains; Research; research and development; R&D; R & D; Development and Research; Risk Factors; Stem cells; Progenitor Cells; Testing; Time; Drug or chemical Tissue Distribution; Tissue Distribution; Tissues; Body Tissues; Toxicology; tumor-specific antigen; Tumor-Associated Antigen; Tumor Antigens; methazolastone; Temodar; Temodal; temozolomide; Drug Delivery; Drug Delivery Systems; tumors in the central nervous system; Central Nervous System Tumors; CNS neoplasm; CNS Tumor; Central Nervous System Neoplasms; Mean Survival Times; Schedule; Malignant Tumor of the Brain; Brain Cancer; Malignant neoplasm of brain; Investigational New Drug Application; base; Organ; improved; Peripheral; Clinical; Malignant; Malignant - descriptor; Phase; Evaluation; excretion; Excretory function; Distress; Disease Progression; Toxicokinetics; Therapeutic; Cytotoxic agent; Cytotoxic drug; Metabolic; Normal tissue morphology; Normal Tissue; cancer cell; Malignant Cell; exhaustion; Frequencies; brain tissue; Xenograft procedure; xeno-transplantation; xeno-transplant; Xenotransplantation; Xenograft; Heterologous Transplantation; Heterograft; Operative Surgical Procedures; surgery; Surgical Procedure; Surgical Interventions; Surgical; Operative Procedures; Membrane; membrane structure; lipophilicity; tumor growth; Animal Model; model organism; model of animal; Animal Models and Related Studies; Patient Agents; Toxic effect; Toxicities; Nutrient; novel; Modality; Excision; resection; Surgical Removal; Removal; Extirpation; Abscission; chemotherapeutic agent; Primary Brain Neoplasms; Primary Brain Tumors; Maximum Tolerated Dose; Maximally Tolerated Dose; Maximal Tolerated Dose; Amino Acid Transport System L; System L Transporter Proteins; System L Transporter; Large Neutral Amino Acid Transporter; Large Amino Acid Transporter; LNAA; Amino Acid Transport System L Proteins; Modeling; Property; drug development; Adverse effects; treatment adverse effect; therapy adverse effect; side effect; Treatment Side Effects; Dose; cytotoxic; Data; Dose-Limiting; Applications Grants; Grant Proposals; Newly Diagnosed; Scheme; Small Business Innovation Research; SBIR; Small Business Innovation Research Grant; Xenograft Model; Monitor; Preparation; Therapeutic Effect; developmental; Development; Intracranial Tumor; Intracranial Central Nervous System Tumors; Intracranial Central Nervous System Neoplasms; Intracranial Neoplasms; cancer research; anticancer research; Biodistribution; Cancer cell line; Impairment; Resistance; resistant; clinically relevant; clinical relevance; translational study; chemotherapy; tumor; overexpression; overexpress; over-expression; standard of care; effective therapy; effective treatment; Biological Markers; biomarker; biologic marker; bio-markers; efficacy testing; good laboratory practice; Regimen; predictive marker; predictive molecular biomarker; predictive biomarkers; tumor heterogeneity; intratumor heterogeneity; intra-tumoral heterogeneity; heterogeneity in tumors; Intratumoral heterogeneity; responders and non-responders; responders vs non-responders; responders versus non-responders; responders or non-responders; responders from non-responders; novel anticancer drug; novel cancer drug; novel antineoplastic; novel anticancer agent; novel anti-cancer drug; novel anti-cancer agent; new cancer drug; new antineoplastic; new anticancer drug; new anticancer agent; new anti-cancer agent; systemic toxicity; biomarker identification; marker identification