Gregg Caldwell

Antineoplastic activity of polyaspartamide–ferrocene conjugates †

The ferrocene/ferricenium redox system plays a significant role in biological oxidation, reduction and free-radical reactions. Of particular interest are the findings of earlier investigations which showed certain water-soluble ferricenium salts to possess appreciable antiproliferative activity against various murine tumor lines and a xenografted human colorectal adenocarcinoma. Solubility in water, a prerequisite for efficacious transport and dissipation in central circulation, was then proposed as a principal requirement for the ferrocene complex system to exert antineoplastic activity irrespective of the oxidation state in which it is administered. In order to shed more light on this question, we decided to investigate the antiproliferative properties of polymer–ferrocene conjugates containing the metal complex in the non-oxidized (ferrocene) form while fulfilling the critical requirement of water solubility. To this end, five selected, water-soluble conjugates, synthesized by reversible coupling of 4-ferrocenylbutanoic acid to variously structured polyaspartamides featuring pendant primary amino groups as coupling sites, were tested in vitro against cultured HeLa cells at concentrations up to 50 µg Fe ml−1. Optimal antiproliferative activities, with IC50 in the range of 2–7 µg Fe ml−1, were determined for three compounds possessing tertiary-amine functions susceptible to protonation at physiological pH. Lower activities (IC50 = 45–60 µg Fe ml−1) were demonstrated for two poly(ethylene oxide)-containing conjugates. However, no reasonable structure–performance relationships can be derived at this stage from the small number of compounds tested. Copyright

Water soluble polyamides as potential drug carriers. IX. Polyaspartamides grafted with amine‐terminated poly(ethylene oxide) chains

The synthesis of side-chain-functionalized polyaspartamides as potential carrier polymers for medicinal agents is described. The nucleophilic ring opening in poly-D,L-succinimide, mediated by O,O′-bis(2-aminopropyl)poly(ethylene oxide) (nominal molecular mass is 600) and ethanolamine under carefully controlled experimental conditions leads to the formation of aspartamide polymers bearing hydrosolubilizing hydroxyethyl side groups in addition to variable proportions of poly(ethylene oxide) (PEO) side chains terminated with primary amino groups. The side chain terminals represent functionalities for drug binding, whereas the PEO constituents contribute to overall hydrophilicity and biocompatibility of the carriers and to enhance their biomedical performance by imparting resistance to protein binding and increasing central circulation lifetime. The water-soluble polymeric products are isolated by dialysis (molecular mass cutoff: 25,000) and freeze-drying in typical yields of 40–60%, with inherent viscosities in the range of 10–18 mL g−1. Polymer compositions are determined by 1H NMR spectroscopy and microanalysis. A selected carrier is modified by N-acylation with 4-ferrocenylbutanoic acid as a model drug, giving a ferrocene-containing, water-soluble conjugate, thus demonstrating the accessibility of the terminal amino groups on the PEO side chains to acylating agents and other potential reactants.

Antiproliferative Activity of Polymer-Bound, Monoamine-Coordinated Platinum Complexes Against LNCaP Human Metastatic Prostate Adenocarcinoma Cells

The chemotherapeutic treatment of secondary, i.e., disseminated cancers, has until now remained an unsatisfactory modality. The LNCaP human metastatic prostate adenocarcinoma cell line lends itself as a useful tool to probe the efficaciousness of novel antineoplastic agents for the control of metastatic cell growth. In this paper we report on a series of cell culture tests assessing the antiproliferative activity of several water-soluble polymer–platinum conjugates in which the metal is tied (anchored) to various polymeric carriers through coordination by a single carrier-attached primary amine ligand. The conjugates are based on polyaspartamide carriers composed, within the backbone, of a majority fraction of subunits bearing water-solubilizing tertiary amine side-group functions and a minority fraction of subunits featuring side chain-terminating primary amino groups for metal binding. Two similarly structured conjugates in which the platinum center is coordinated by two amine ligands in cisoid orientation mimicking the structural skeleton of cisplatin are included in the study for comparison. In all structures cleavage of a side-chain segment is required for release of the monomeric bioactive platinum complex. The growth of LNCaP human metastatic prostate adenocarcinoma cells in RPMI 1640 medium in the presence and absence of the conjugates in a range of concentrations is assessed by a protein assay, and the IC50 values, representing the drug concentrations required for 50% cell growth inhibition relative to untreated control, are determined. The results show both classes of conjugates, those comprising monoamine-coordinated platinum and those featuring cis-diamine-coordinated metal, to be well comparable in antiproliferative activity. A major program of synthesis and evaluation of polymer-bound monoamineplatinum complexes, prompted by these findings, is forthcoming in this laboratory.

Preliminary toxicological studies of selected water‐soluble polymer–platinum conjugates

The objective of this preliminary investigation of a number of water-soluble carrier-bound platinum(II) complexes for potential use in cancer chemotherapy was to assess the toxicological behavior of representative platinum coordination compounds anchored to, or incorporated into, polymeric carriers via polymer-attached amine ligands. The conjugates included linear polyaspartamides (1-4, 6, 7), each composed of a major fraction of subunits featuring side-chain-attached tertiary amino groups as water-solubilizing entities, and a minor fraction of subunits comprising the anchored platinum complexes, again as side-chain components. Whereas in 1-4 the platinum atom was polymer-bound through a single amino group, both 6 and 7 contained polymer-attached cis-diamine-chelating ligands coordinating to the metal center. Also included in this study was a linear polyamidoamine (5), which contained a poly(ethylene oxide) segment in the backbone in addition to intrachain ethylenediamine segments acting as cis-diamine chelating ligands for coordination to the platinum center. The compounds were injected as aqueous (phosphate-buffered saline) solutions into the tail veins of CD-1 mice (four to eight mice per conjugate), and the maximally tolerated dose was determined for each compound. For polyaspartamides 1-4 the dose levels ranged from about 25 mg Pt (kg body weight -1 ) (in conjugate 4) to 500 mg Pt kg -1 (in compound 1), the latter conjugate proving some 100-fold less toxic than cisplatin (3-4 mg Pt kg -1 ), which was included in this study for comparison. Low toxicity (tolerated dose 160 mg Pt kg -1 ) was also observed for the intrachain cis-diamineplatinum complex polymer (5). The polyaspartamide conjugates 6 and 7, on the other hand, both characterized by a cis-diamineplatinum complex system in the side chain, were toxic even below the dose level of 20-25 mg Pt kg -1 . The preliminary findings of this study, while providing a basis for more extensive and broad-based toxicological studies, will serve to direct and optimize structural conjugate designs in forthcoming synthetic programs.

Cytotoxicity of Selected Water-Soluble Polymer–cis-Diaminedichloroplatinum(II) Conjugates Against the Human HeLa Cancer Cell Line

Several polymer-platinum conjugates comprising the square-planar cis-diaminedichloroplatinum(II) complex system of cisplatin-type anticancer drugs are screened for antiproliferative activity in cell culture tests. The water-soluble conjugates prepared in this study or taken from preceding investigations are obtained by platination of aliphatic polyamide carriers containing ethylenediamine segments as side-group or main-chain components. These segments, coordinating to the metal as cis-diamine chelating ligands, are bound to, or into, the carrier backbone through biofissionable amide links permitting drug release from the carrier. In vitro tests are performed against a HeLa human cervix carcinoma cell line. IC50 data, expressed as the concentration of Pt in the conjugates (μg/ml), causing 50% inhibition of cell growth, show the highest activity, with IC50=14 μg/ml, to be associated with a conjugate derived from a polyaspartamide carrier that contains the platinum complex as a side group in proximity to the main chain and, additionally, contains dimethylaminopropyl side groups as solubilizing functions. At the low end of the performance spectrum is a conjugate, with IC50>120 μg/ml, possessing a similar backbone and metal-binding structure, yet comprising long poly(ethylene oxide) grafts. The latter apparently shield the complex-binding tether from enzymatic attack and thus prevent efficient intracellular release of the monomeric complex. Selected conjugates will be submitted for toxicological evaluation.

Cytotoxic activity of two polyaspartamide‐ based monoamineplatinum(II) conjugates against the HeLa cancer cell line

In this screening study in vitro, two polymer-conjugated, square-planar platinum(II) complexes bound to the carrier via a single primary amine ligand were tested for antineoplastic activity against the HeLa human cervical epithelioid carcinoma cell line. In the first of these conjugates, 1-Pt, the spacer connecting the metal complex with the carrier backbone is a short oligo(ethylene oxide) segment, whereas a long poly(ethylene oxide) chain represents the spacer unit in the second conjugate, 2-Pt. IC 50 data, expressed as conjugate concentration at 50% cell growth inhibition, are 48 μg Pt ml -1 for 1-Pt and 120 μg Pt ml - (estimated) for 2-Pt, the long tether in the latter conjugate presumably causing retarded enzymic release and lysosomal membrane crossing of the monomeric complex. The IC 50 value of 1-Pt is close to that (44 μg Pt ml -1 ) of a similar conjugate of an earlier investigation, 3-Pt, in which the metal is chelated by two carrier-attached, cis-oriented amino groups in conformance with the ligand arrangement in cisplatin. It thus appears that, in the carrier-bound state, both monoamine- and cis-diamine-coordinated platinum(II) complexes of suitable structures may well show similar biological performance patterns.

cis-Diaminedichloroplatinum(II) complexes reversibly bound to water-soluble polyaspartamide carriers for chemotherapeutic applications. II: Platinum coordination to ethylenediamine ligands attached to poly(ethylene oxide)-grafted carrier polymers

In continuation of previous investigations aiming at the development of macromolecular metal complexes for biomedical use, this communication describes poly(alkylene oxide)-grafted polymeric platinum complexes. The platinum-containing macromolecules are obtained from presynthesized polyaspartamide carriers bearing poly(ethylene/propylene oxide) side chains and hydroxyethyl side groups as hydrosolubilizing units in addition to ethylenediamine side group terminals for metal coordination. Platination is brought about by treatment of the carriers with tetrachloroplatinate(II) ion in aqueous solution at 25–60°C. pH 4–6. The polymeric products, purified by dialysis in aqueous solution, are isolated by freeze-drying in yields of 60–80%. Platinum contents are in the range of 4–15%. The metal is bound to the carrier through chelation with the ethylenediamine ligands, forming square-planarcis-dichloroethylenediamine-platinum(II) complex species as side-chain terminals. Initially, the product polymers dissolve smoothly in water. Although on room-temperature storage in the solid state they gradually turn insoluble as a consequence of intermolecular solid-state interaction, solubility is retained on low-temperature storage and in frozen aqueous solutions.

cis-Diaminedichloroplatinum(II) Complexes Reversibly Linked into the Main Chain of Water-Soluble Polyamides

As a follow-up study to previous work involving the platination of polyamide carriers through metal chelation by side group-incorporated ethylenediamine ligands, the present investigation is concerned with the synthesis of platinum-containing polymers in which the metal-coordinating ethylenediamine segments are components of the main chain. Two chloro groups in cis geometry are attached to each Pt atom as additional ligands, complementing a square-planar cis-diaminedichloroplatinum(II) complex system. The water-soluble polymeric carriers are synthesized by Michael-type addition polymerization, interfacial polymerization, and high-temperature solution polycondensation techniques and are crudely fractionated by stepwise aqueous dialysis, ultimately in tubing with a molecular mass cutoff of 25,000. Carrier platination is brought about by treatment with tetrachloroplatinate(II) anion in aqueous solution, care being taken to exercise strict control of reaction variables and workup conditions in an effort to restrict platination to the given ligands and avoid metal aquation or hydroxylation. The platinum conjugates, with Pt contents ranging from about 11 to 23% by mass, are completely soluble in aqueous media when freshly prepared, although long-term storage at room temperature in the solid state is conducive to gradual loss of solubility. The conjugates are of interest as carcinostatic agents.

A Preliminary Study of the Toxicological Properties of Selected Polymer–Ferrocene Conjugates

Prompted by early observations of the cytotoxic and antineoplastic properties of certain ferrocene and ferricenium derivatives, efforts in this laboratory were focused on the synthesis of carrier-bound ferrocene compounds. Subsequent cell culture tests carried out with selected conjugates obtained in that program showed these polymers to be highly active antiproliferative agents. In the present project toxicological work has been performed in vivo on several ferrocene conjugates in an effort to assess their toxic effects in experimental animals (CD-1 mice). The conjugates, all based on an α,β-DL-polyaspartamide backbone structure, comprise the ferrocene drug model as a terminal on short side chains containing biofissionable amide or ester links for intracellular drug release. The polymers, dissolved in phosphate-buffered saline, have been injected in predetermined concentrations into the vein of the mice, and the maximum tolerated dose (MTD) levels have been determined, the latter referring to the highest dose levels administered that would allow long-term survival of the test animals. For the five conjugates tested, MTD levels range from about 3 to 30 mg Fe/kg or 0.05–0.66 mmol Fe/kg. Compared on a molar metal-to-metal basis with similarly structured platinum conjugates tested previously (MTD, ∼0.14–2.66 mmol Pt/kg), these values are indicative of comparatively high toxicity of the ferrocene polymers. Some implications of these findings are discussed.

Poly(ethylene Oxide)-Modified Polyaspartamide–Ferrocene Conjugates

In continuation of earlier investigations of polymer–ferrocene conjugates for biomedical applications, this article deals with conjugates prepared by N-acylation of linear, amine-functionalized polyaspartamide carriers with 4-ferrocenylbutanoic acid. Acylation is brought about both by mediation of HBTU coupling agent and by the N-hydroxysuccinimide active ester method. The polymeric carriers contain oligo- or poly(ethylene oxide) side chains introduced here for enhancement of water solubility. The longer side chains, in addition, are to impart such biomedically important properties as increased resistance to uptake by the reticuloendothelial system and to protein binding, extended circulation life time, and lowered immunogenicity. The conjugates comprise from 10 to 25 mol% ferrocenylated subunits, corresponding to ca. 2–5% Fe by mass. Freshly prepared and isolated in the solid state, they dissolve smoothly in aqueous media, with upper concentration limits (>0.2g/ml) dictated solely by their viscosity behavior. The conjugates are of interest in biomedical applications.