Dechun Wu

Novel Prodrugs of SN38 Using Multiarm Poly(ethylene glycol) Linkers

CPT-11, also known as irinotecan, is a prodrug that is approved for the treatment of advanced colorectal cancer. The active metabolite of CPT-11, SN38 (7-ethyl-10-hydroxy-camptothecin), has 100- to 1000-fold more potent cytotoxic activity in tissue cell culture compared with CPT-11. However, parental administration of SN38 is not possible because of its inherently poor water solubility. It is reported here that a multiarm poly(ethylene glycol) (PEG) backbone linked to four SN38 molecules (PEG-SN38) has been successfully prepared with high drug loading and significantly improved water solubility (400- to 1000-fold increase). Three different protecting strategies have been developed in order to selectively acylate the 20-OH of SN38 to preserve its E-ring in the lactone form (the active form of SN38 with cytotoxic activities) while PEG is still attached. One chemical process has been optimized to make a large quantity of the PEG-SN38 conjugate with a high yield that can be readily adapted for scale-up production. The PEG-SN38 conjugates have shown excellent in vitro anticancer activity, with potency similar to that of native SN38, in a panel of cancer cell lines. The PEG-SN38 conjugates also have demonstrated superior anticancer activity in the MX-1 xenograft mice model compared with CPT-11. Among the four conjugates, PEG-Gly-(20)-SN38 (23) has been selected as the lead candidate for further preclinical development.

Camptothecin-20-PEG ester transport forms: the effect of spacer groups on antitumor activity.

An improved synthesis of the hindered PEG-camptothecin diester transport form has been achieved using the Mukaiyama reagent. We have also assessed the effect of changing the electronic configuration of the (d-position of PEG-camptothecin transport forms on the rates of hydrolysis of the pro-moiety, and attempted to correlate these differences to efficacy in two animal models. In addition to the simple substitution of N for O, other synthetic modifications of these atoms were accomplished by employing heterobifunctional linker groups. The half lives by disappearance (rates of hydrolysis) of the transport forms in buffer and rat plasma were determined. It was established that anchimeric assistance to hydrolytic breakdown of the pro-moiety occurs in a predictable manner for some of these compounds. Results for the new derivatives in a P388 murine leukemic model and HT-29 human colorectal xenograft study are also presented. The use of a glycine linker group was found to provide similar efficacy in rodent models to that of simple camptothecin 20-PEG ester, and displayed enhanced pharmacokinetics.

Anticancer drug delivery systems: multi-loaded N4-acyl poly(ethylene glycol) prodrugs of ara-C. II. Efficacy in ascites and solid tumors.

The synthesis of branched PEG (40,000) acids has been achieved using aspartic acid (Asp) and AspAsp dendrons. Complete conjugation of these dendritic acids with cytosine arabinoside (ara-C) was achieved by the use of spacers that allowed a greater separation of the branches to accommodate several large ara-C molecules in proximity to each other. The tetrameric and octameric PEG-ara-C amide prodrugs were much more effective in the treatment of solid and ascites tumors compared to the native drug. The greater loading of the PEG backbone appears to have achieved a minimum threshold concentration for the therapeutic delivery of ara-C.

Anticancer drug delivery systems: N4-acyl poly(ethyleneglycol) prodrugs of ara-C. I. Efficacy in solid tumors.

A systematic study of N(4) amino PEG-prodrugs of ara-C (1) was conducted and provided a series of disubstituted amides, as well as a carbamate derivative. These conjugates showed hydrolysis half lives in rat plasma from about 1 h to 3 days, but were stable for >24 h in phosphate buffer, pH 7.4. In an LX-1 solid lung tumor model some of the PEG prodrugs exhibited superior activity to ara-C when compared on a molar basis. One problematic issue that was identified in this investigation was the need to increase the loading of ara-C onto PEG in order to avoid highly viscous solutions.

Drug delivery of anticancer agents: water soluble 4-poly (ethylene glycol) derivatives of the lignan, podophyllotoxin.

This paper reports on the synthesis and in vivo oncolytic activity of a series of water-soluble acyl derivatives of polyethylene glycol (PEG) conjugated podophyllotoxin. Some analogs of the polymer conjugate showed significantly better activity in a murine leukemia model than native podophyllotoxin suspended in an intralipid emulsion. Additionally, when tested intravenously against a solid lung tumor (A549) model, some conjugated analogs were equivalent to the podophyllotoxin/intralipid emulsion, while those compounds demonstrating slower rates of plasma hydrolysis (in vitro) appeared to cause greater toxicity. There appeared to be an overall correlation between the in vivo antitumor activity of the conjugate and its rate of hydrolysis in vitro, with those showing faster release possessing greater antitumor activity. In conclusion, the solubilization and predictable release of podophyllotoxin from a PEG carrier was achieved and resulted in some derivatives demonstrating, at a minimum, equivalency with podophyllotoxin when administered on an equal molar basis. Further studies may be warranted to assess the PEG-conjugates pharmacokinetics and therapeutic indices in leukemic models.

Selective phenolic acylation of 10-hydroxycamptothecin using poly (ethylene glycol) carboxylic acid.

Selective acylation of the phenolic hydroxyl group of 10-hydroxycamptothecin has been accomplished using phenyl dichlorophosphate. Additional modification of the 10-OH as an ether permits a 20-acyl derivative to be synthesized. This result along with data from a 6-hydroxyquinoline model strongly suggests that powerful intermolecular hydrogen bonding exists in the parent molecule.

Abstract 2645: Customized PEG linkers improve the pharmaceutical properties of cytotoxic small molecules

Introduction: PEGylation is an established delivery technology for proteins with benefits such as decreased immunogenicity and prolonged circulating half-life. The application of PEGylation to small molecules may improve the poor pharmaceutical properties including poor solubility, suboptimal pharmacokinetic (PK) profiles, and unwanted toxicities. Here we utilized Customized PEG Linkers to enhance the therapeutic index of several cytotoxic agents including doxorubicin, ara-C, gemcitabine, and SN38. Experimental procedures: drug molecules were either reacted with proper linker moieties first before PEGylation, or conjugated directly with PEG linkers. For those compounds thathave more than two reactive functional groups, the unwanted reactive sites were first protected before conjugation with PEG linkers. The PEG conjugates were incubated in PBS and plasma to study their stability, followed by in vivo PK studies in mice. Additionally, in vitro anti-proliferation assays with different human cancer cell lines were conducted to evaluate the PEG conjugates with different half-lives. Furthermore, the PEG conjugates were evaluated in vivo in multiple human cancer models in mice for their anticancer activities. Summary of data: a series of novel PEG conjugates were synthesized with different Customized PEG Linkers. In general, while these PEG conjugates were stable in PBS buffer, they demonstrated a broad range of half-lives in rat and human plasma, varying from minutes to days. PEG conjugates with longer half-lives have shown prolonged circulation time and increased AUC compared to native drugs in the PK studies in mice. In addition, PEGylation also greatly increased the water solubility for those insoluble molecules, for instance by about 1000-fold for SN38. In the cellular based studies, PEG conjugates showed different degrees of anticancer activities against a panel of human cancer cells. Remarkably, enhanced anticancer activities compared to the non-pegylated agent have been observed for many of the conjugates in a variety of cancer models, including both solid tumors and hematological malignancies. Conclusions: Customized PEG Linkers have greatly improved the solubility of certain small molecule drugs and enabled the systemic administration of these drugs. It allowed us to generate a variety of PEG-drug conjugates with broad half-life ranges in plasma which are clinically relevant. The combined effect of increased solubility, optimized PK profile, and passive accumulation of macromolecular PEG conjugates in tumors due to the enhanced permeation and retention effect may all contributed to the greatly enhanced anticancer efficacy of these PEG conjugates in animal models. In summary, customized PEG Linkers represent a promising technology to improve current cytotoxic agents. One agent, PEG-SN38 is undergoing Phase II evaluation in cancer patients with metastatic colorectal and breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2645.