Synthesis and Antitumor Properties of BQC-Glucuronide, a Camptothecin Prodrug for Selective Tumor Activation.

Major limitations of camptothecin anticancer drugs (toxicity, nonselectivity, water insolubility, inactivation by human serum albumin) may be improved by creating glucuronide prodrugs that rely on beta-glucuronidase for their activation. We found that the camptothecin derivative 5,6-dihydro-4H-benzo[de]quinoline-camptothecin (BQC) displays greater cytotoxicity against cancer cells than the clinically used camptothecin derivatives SN-38 and topotecan even in the presence of human serum albumin. We synthesized the prodrug BQC-glucuronide (BQC-G), which was 4000 times more water soluble and 20-40 times less cytotoxic than BQC.

Mast cells mediate malignant pleural effusion formation.

Mast cells (MCs) have been identified in various tumors; however, the role of these cells in tumorigenesis remains controversial. Here, we quantified MCs in human and murine malignant pleural effusions (MPEs) and evaluated the fate and function of these cells in MPE development. Evaluation of murine MPE-competent lung and colon adenocarcinomas revealed that these tumors actively attract and subsequently degranulate MCs in the pleural space by elaborating CCL2 and osteopontin.

Versatile online SPE-HPLC method for the analysis of Irinotecan and its clinically relevant metabolites in biomaterials.

Monitoring levels of Irinotecan and its metabolites during cancer therapy could help link broad interpatient variations in antitumor activity and toxicity to the patient's metabolic status. We have developed and validated a versatile and highly sensitive method for the simultaneous determination of Irinotecan and its clinically relevant metabolites 7-ethyl-10-hydroxy-camptothecin (SN-38) and SN-38 glucuronide. Sample clean-up involves precipitation by acetone/methanol/0.

The B7-1 cytoplasmic tail enhances intracellular transport and mammalian cell surface display of chimeric proteins in the absence of a linear ER export motif.

Membrane-tethered proteins (mammalian surface display) are increasingly being used for novel therapeutic and biotechnology applications. Maximizing surface expression of chimeric proteins on mammalian cells is important for these applications. We show that the cytoplasmic domain from the B7-1 antigen, a commonly used element for mammalian surface display, can enhance the intracellular transport and surface display of chimeric proteins in a Sar1 and Rab1 dependent fashion.

ECSTASY, an adjustable membrane-tethered/soluble protein expression system for the directed evolution of mammalian proteins.

We describe an adjustable membrane-tethered/soluble protein screening methodology termed ECSTASY (enzyme cleavable surface tethered all-purpose screening system) which combines the power of high-throughput fluorescence-activated cell sorting of membrane-tethered proteins with the flexibility of soluble assays for isolation of improved mammalian recombinant proteins. In this approach, retroviral transduction is employed to stably tether a library of protein variants on the surface of mammalian cells via a glycosyl phosphatidylinositol anchor. High-throughput fluorescence-activated cell sorting is used to array cells expressing properly folded and/or active protein variants on their surface into microtiter culture plates.

A humanized immunoenzyme with enhanced activity for glucuronide prodrug activation in the tumor microenvironment.

Antibody-directed enzyme prodrug therapy (ADEPT) utilizing β-glucuronidase is a promising method to enhance the therapeutic index of cancer chemotherapy. In this approach, an immunoenzyme (antibody-β-glucuronidase fusion protein) is employed to selectively activate anticancer glucuronide prodrugs in the tumor microenvironment. A major roadblock to the clinical translation of this therapeutic strategy, however, is the low enzymatic activity and strong immunogenicity of the current generation of immunoenzymes.

Antiangiogenesis targeting tumor microenvironment synergizes glucuronide prodrug antitumor activity.

Purpose: This study is aimed at investigating the in vivo antitumor activity of a novel cell-impermeable glucuronide prodrug, 9-aminocamptothecin glucuronide (9ACG), and elucidating the synergistically antitumor effects of antiangiogenesis therapy by targeting the tumor microenvironment.

Experimental Design: We analyzed the antitumor effects of 9ACG alone or combined with antiangiogenic monoclonal antibody DC101 on human tumor xenografts by measuring tumor growth and mouse survival in BALB/c nu/nu nude and NOD/SCID mice. The drug delivery, immune response, and angiogenesis status in treated tumors were assessed by high performance liquid chromatography, immunohistochemistry, and immunofluorescence assays.

Local enzymatic hydrolysis of an endogenously generated metabolite can enhance CPT-11 anticancer efficacy.

Irinotecan (CPT-11) is a clinically important anticancer prodrug that requires enzymatic hydrolysis by carboxyesterase to generate the active metabolite SN-38. However, SN-38 is further metabolized to inactive SN-38 glucuronide (SN-38G), thus diminishing the levels of active SN-38. Although exogenously administered glucuronide drugs are being investigated for cancer therapy, it is unknown if endogenously generated camptothecin glucuronide metabolites can be used for tumor therapy.

Directed evolution of a lysosomal enzyme with enhanced activity at neutral pH by mammalian cell-surface display.

Human beta-glucuronidase, due to low intrinsic immunogenicity in humans, is an attractive enzyme for tumor-specific prodrug activation, but its utility is hindered by low activity at physiological pH. Here we describe the development of a high-throughput screening procedure for enzymatic activity based on the stable retention of fluorescent reaction product in mammalian cells expressing properly folded glycoproteins on their surface. We utilized this procedure on error-prone PCR and saturation mutagenesis libraries to isolate beta-glucuronidase tetramers that were up to 60-fold more active (k(cat)/K(m)) at pH 7.

Effect of pH and human serum albumin on the cytotoxicity of a glucuronide prodrug of 9-aminocamptothecin.

Purpose: 9-aminocamptothecin glucuronide (9ACG) is a prodrug of 9-aminocamptothecin (9AC) that displays potent antitumor activity against human tumor xenografts in nude mice. Camptothecins exist in a pH dependent equilibrium between active lactone and inactive carboxy forms that can be altered by binding to human serum albumin (HSA). Here we investigated the influence of pH and HSA on the lactone-carboxy equilibrium, HSA binding, and cytotoxicity of 9ACG.

A simple method for the production of recombinant proteins from mammalian cells.

Expression of recombinant proteins in mammalian cells is useful for obtaining products with normal post-translational modifications. We describe a simple and economical method for the production of milligram levels of proteins in murine fibroblasts. Retroviral or LIPOFECTAMINE (Gibco Laboratories) transduction was employed to generate stable murine-fibroblast producer cells.

Stability of the new prodrug 9-aminocamptothecin glucuronide (9ACG) in the presence of human serum albumin.

9-aminocamptothecin glucuronide (9ACG) is a new water-soluble prodrug of 9-aminocamptothecin (9AC) that is a substrate for beta-glucuronidase and displays potent antitumor activity against human tumor xenografts. The lactone ring of camptothecins (CPTs) is required for antitumor activity but spontaneously opens under physiological conditions to an inactive carboxy form. The carboxy form of many CPTs, including 9AC, preferentially binds to human serum albumin (HSA), which further reduces the equilibrium amount of active lactone and greatly decreases antitumor efficacy.