Peter de Vries

Poly-(L)-Glutamic Acid-Paclitaxel (CT-2103) [XYOTAX™], a Biodegradable Polymeric Drug Conjugate

These data describing the preclinical and early clinical development of CT-2103 demonstrate the feasibility of using polyglutamic acid homopolymers to create macromolecular cytotoxic drug conjugates. PG has the characteristics of an ideal polymeric drug carrier including biodegradability, the ability to solubilise hydrophobic agents even at high loading, stability in circulation, and apparent lack of immunogenicity. Preliminary clinical data indicate that CT-2103 is well tolerated by short infusion and has what appears to be reduced toxicity to neural tissue and hair follicles compared with paclitaxel delivered in the standard formulation. In preclinical studies, the MTD was approximately twice that of standard paclitaxel and the antitumour efficacy in was improved. Preliminary clinical data from Phase II studies indicate that the MTD will be higher than that of paclitaxel and that CT-2103 has activity, even in patients who have failed prior taxane therapy. The potentially enhanced efficacy and apparently reduced toxicity of CT-2103 can be predominantly ascribed to its improved distribution to tumour tissue through the EPR effect and the reduced exposure of normal tissues. Taken together, these data suggest that PG is an excellent polymeric backbone for the delivery of oncologic therapeutics and is likely to improve the therapeutic indices of a number of other agents. A second PG conjugate designated CT-2106, a PG camptothecin with an interposed glycine linker, will enter clinical trials shortly.

Water-soluble poly-(L-glutamic acid)-Gly-camptothecin conjugates enhance camptothecin stability and efficacy in vivo

The therapeutic efficacy of 20(s)-camptothecin (CPT) is limited in humans by the instability of the active lactone form due to preferential binding of the carboxylate to serum albumin and by difficulty in formulation. Formation of an ester bond with an amino acid via the hydroxyl group at carbon 20 of CPT stabilizes the lactone. Linking CPT to a high molecular weight (MW) anionic polymer enhances solubility and improves distribution to the tumor through enhanced permeability and retention (EPR effect). Poly-(L-glutamic acid) (PG) is an anionic homo-polymer that can theoretically bind one molecule of a drug via the gamma carboxylic acid of each monomeric subunit. It has been used to make a water-soluble PG-paclitaxel conjugate currently in Phase II clinical trials that contains 37% paclitaxel by weight and is administered in a 10 min infusion without pre-medication. We evaluated the anti-tumor activity of PG conjugates of CPT after a single intraperitoneal injection using subcutaneous murine B-16 melanoma tumor growth as an indicator. Interposition of a glycine (gly) linker allowed CPT loading up to 50% w/w on the polymer. Increasing the PG MW from 33 to 49 kDa enhanced the efficacy without altering the maximum tolerated dose (MTD). In athymic mice bearing ectopic human colon or lung tumors, efficacy was enhanced compared to free camptothecin. Thus, as with paclitaxel, conjugation of CPT to PG enhanced pharmaceutical properties and preclinical efficacy.

In vitro and in vivo metabolism of paclitaxel poliglumex: identification of metabolites and active proteases

Background The efficacy and tolerability of paclitaxel is limited by its low solubility, high systemic exposure, and a lack of selective tumor uptake. Paclitaxel poliglumex (PPX; XYOTAX™) is a macromolecular drug conjugate that was developed to overcome these limitations; the 2′ hydroxyl group of paclitaxel is linked to a biodegradable polymer, poly-l-glutamic acid, to form an inactive polymeric conjugate. PPX was previously shown to accumulate in tumor tissue, presumably by taking advantage of the hyperpermeable tumor vasculature and suppressed lymphatic clearance in tumor tissue. Methods Because anti-tumor activity requires the release of paclitaxel from the polymer-drug conjugate, the current report characterizes PPX biodegradation and release of paclitaxel as determined by quantitative HPLC/mass spectral analysis. Results The identification of monoglutamyl-paclitaxel metabolites in tumor tissue confirmed the in vivo metabolism of PPX in a panel of mouse tumor models. In vitro characterization of the metabolic pathway suggests that PPX can enter tumor cells, and is metabolized to form both mono- and diglutamyl-paclitaxel cleavage products. The intracellular formation of these intermediate metabolites is at least partially dependent on the proteolytic activity of the lysosomal enzyme cathepsin B; PPX metabolism is inhibited by a highly selective inhibitor of cathepsin B, CA-074. Reduced metabolism of PPX in livers and spleens from cathepsin B deficient mice confirms that cathepsin B is an important mediator of PPX metabolism in vivo; however, other proteolytic enzymes may contribute as well. Conclusions The cathepsin B-mediated release of paclitaxel may have therapeutic implications as cathepsin B is upregulated in malignant cells, particularly during tumor progression.

Conjugation of Camptothecins to Poly‐(l‐Glutamic Acid)

Abstract: Conjugation of water‐insoluble cancer chemotherapeutic drugs to macromolecular polymers can lead to improved pharmaceutical properties and improved therapeutic ratios due to accumulation of the polymer‐drug conjugate in tumor tissue through the enhanced permeability and retention (EPR) to macromolecules associated with tumor vasculature. Pharmaceutical shortcomings of certain active camptothecins including difficulty in formulation and instability of the active lactone form due to interactions with human albumin might be improved by conjugation to polymers. In this report, conjugations of camptothecin (CPT), 10‐hydroxy‐CPT, and 9‐amino‐CPT to poly‐(l‐glutamic acid) (PG) are described; coupling was accomplished either through the 20(S)‐hydroxyl or 9 and 10 substituents with and without the use of a glycine linker. Studies using a PG paclitaxel conjugate (PG‐TXL), which is currently in Phase I testing, demonstrated that PG enhanced aqueous solubility, prolonged plasma residence time, and greatly increased the distribution of paclitaxel to tumor tissue in a murine model. In this report, we describe the use of similar conjugation technology for CPT derivatives and demonstrate that these difficult to formulate compounds can be rendered water soluble, that their maximum tolerated doses are increased, and that they retain substantial anti‐tumor activity in syngeneic and xenogeneic tumor models. Preliminary data suggest that PG with molecular weights between 37 and 50 kDa with CPT loading between 14% and 37% with or without glycine linkers display enhanced efficacy compared with nonconjugated camptothecins administered at their maximum tolerated dose.

Lysophosphatidic acid acyltransferase-β: a novel target for induction of tumour cell apoptosis

Phosphatidic acid (PA) is a component of cellular membranes that is also a mediator of certain cell signalling functions associated with oncogenesis. These include ras/raf/Erk and Akt/mTor [1-3]. The authors have investigated whether it would be possible to interrupt these known oncogenic pathways through the inhibition of lysophosphatidic acid acyltransferase (LPAAT), an enzyme that catalyses the biosynthesis of PA. The expression and activity of the LPAAT-β isoform are elevated in human tumours, and the respective gene displays transforming capacity when overexpressed in vitro. Inhibition by either genetic means or by isoform-specific small molecules results in a block to cell signalling pathways and apoptosis. Furthermore, the small-molecule inhibitors of LPAAT-β are not cytotoxic to a number of normal cell types, including primary bone marrow progenitors, indicating a differential dependence of tumour cells on LPAAT-β function. These discoveries indicate that LPAAT-β represents a potential novel cancer therapy target.

Lisofylline suppresses ex vivo release by murine spleen cells of hematopoietic inhibitors induced by cancer chemotherapeutic agents

Many cytotoxic cancer therapeutic drugs activate stress response signaling pathways that transcriptionally activate a variety of genes. We decided to determine if cytotoxic therapies induce inflammatory cytokines with inhibitory effects on hematopoiesis and if lisofylline (LSF), a novel antiinflammatory compound, suppresses this induction. Mice were treated with cytosine beta-d-arabinofuranoside (AraC), cis-platinum(II)diammine-dichloride (CisP), etoposide (VP-16), or melphalan at clinically relevant doses, with or without LSF. Spleen cell conditioned media (CM) derived from mice treated with cytotoxic agents, but not from control or LSF treated mice, reduced colony formation by murine bone marrow progenitors belonging to the myeloid, erythroid, megakaryocytic, and B-lymphoid lineages. LSF (100 mg/kg), administered either simultaneously with or up to 48 hours before the cytotoxic agents, suppressed the release of this inhibitory activity. Treatment of inhibitory CM with neutralizing antibodies against known growth inhibitory cytokines, including tumor necrosis factor alpha, transforming growth factor beta, and macrophage inflammatory protein-1alpha, resulted in enhanced colony growth. We conclude that treatment of mice with chemotherapeutic drugs induces the ex vivo production of multilineage hematopoietic inhibitors and that induction of these inhibitors could be abrogated by treatment with LSF. These findings suggest a mechanism whereby LSF can accelerate recovery of hematopoiesis following cytotoxic therapies.

Antileukemic Activity of Lysophosphatidic Acid Acyltransferase-β Inhibitor CT32228 in Chronic Myelogenous Leukemia Sensitive and Resistant to Imatinib

Purpose: Lysophosphatidic acid acyltransferase (LPAAT)-β catalyzes the conversion of lysophosphatidic acid to phosphatidic acid, an essential component of several signaling pathways, including the Ras/mitogen-activated protein kinase pathway. Inhibition of LPAAT-β induces growth arrest and apoptosis in cancer cell lines, implicating LPAAT-β as a potential drug target in neoplasia. Experimental Design: In this study, we investigated the effects of CT32228, a specific LPAAT-β inhibitor, on BCR-ABL-transformed cell lines and primary cells from patients with chronic myelogenous leukemia. Results: CT32228 had antiproliferative activity against BCR-ABL-positive cell lines in the nanomolar dose range, evidenced by cell cycle arrest in G2-M and induction of apoptosis. Treatment of K562 cells with CT32228 led to inhibition of extracellular signal-regulated kinase 1/2 phosphorylation, consistent with inhibition of mitogen-activated protein kinase signaling. Importantly, CT32228 was highly active in cell lines resistant to the Bcr-Abl kinase inhibitor imatinib. Combination of CT32228 with imatinib produced additive inhibition of proliferation in cell lines with residual sensitivity toward imatinib. In short-term cultures in the absence of growth factors, CT32228 preferentially inhibited the growth of granulocyte-macrophage colony-forming units from chronic myelogenous leukemia patients compared with healthy controls. Conclusion: These data establish LPAAT-β as a potential drug target for the treatment of BCR-ABL-positive leukemias.