R. C. Elder

Liquid chromatography with an inductively coupled plasma mass spectrometric detector for simultaneous determination of gold drug metabolites and related metals in human blood

The use of inductively coupled plasma mass spectrometry (ICP-MS) for the analysis of blood samples from rheumatoid arthritis patients on gold drug therapy is demonstrated. Flow injection allows the determination of multiple metal ions (Au, Zn and Cu) in a single injection with detection limits that are more than adequate for the analysis. Matrix effects are significant with these samples. Chromatography is also possible with ICP-MS as the detector. Examples are given of high-performance liquid chromatography of patient blood samples using size-exclusion and weak anion-exchange columns.

Seleno-auranofin (Et3PAuSe-tagl): synthesis, spectroscopic (EXAFS, 197Au Mössbauer, 31P, 1H, 13C, and 77Se NMR, ESI-MS) characterization, biological activity, and rapid serum albumin-induced triethylphosphine oxide generation.

Seleno-auranofin (SeAF), an analogue of auranofin (AF), the orally active antiarthritic gold drug in clinical use, was synthesized and has been characterized by an array of physical techniques and biological assays. The Mössbauer and extended X-ray absorption fine structure (EXAFS) parameters of the solid compound demonstrate a linear P-Au-Se coordination environment at a gold(I) center, analogous to the structure of auranofin. The (31)P, (13)C, and (1)H NMR spectra of SeAF in chloroform solution closely resemble those of auranofin. The (77)Se spectrum consists of a singlet at 481 ppm, consistent with a metal-bound selenolate ligand. The absence of (2)J(PSe) coupling in the (31)P and (77)Se spectra may arise from dynamic processes occurring in solution or because the (2)J(PSe) coupling constants are smaller than the observed bandwidths. Electrospray ionization mass spectrometry (ESI-MS) spectra of SeAF in 50:50 methanol-water exhibited strong signals for [(Et(3)P)(2)Au](+), [(Et(3)PAu)(2)-mu-Se-tagl](+), and [Au(Se-tagl)(2)](-), which arise from ligand scrambling reactions. Three assays of the anti-inflammatory activity of SeAF allowed comparison to AF. SeAF exhibited comparable activity in the topically administered murine arachadonic acid-induced and phorbol ester-induced anti-inflammatory assays but was inactive in the orally administered carrageenan-induced assay in rats. However, in vivo serum gold levels were comparable in the rat, suggesting that differences between the in vivo metabolism of the two compounds, leading to differences in transport to the inflamed site, may account for the differential activity in the carrageenan-induced assay. Reactions of serum albumin, the principal transport protein of gold in the serum, demonstrated formation of AlbSAuPEt(3) at cysteine 34 and provided evidence for facile reduction of disulfide bonds at cysteine 34 and very rapid formation of Et(3)P=O, a known metabolite of auranofin.

Transport of the Dicyanogold(I) Anion

We have shown that dicyanogold(I), [Au(CN)2]- is a common metabolite found in blood and urine samples of patients treated with different gold based drugs. Some patients have high levels of gold within their red blood cells (RBCs). Size exclusion and C18 reversed phase chromatography show that the majority of the gold in RBC lysates is bound to protein, but small molecules such as dicyanogold(I) and gold-glutathione complexes are also present. Dicyanogold incubation with red blood cells in vitro leads to a rapid and complete uptake of gold. This uptake is unaffected by DIDS, an inhibitor of the anion channel, but is blocked by the addition of external cyanide. Dicyanogold is also readily taken up by H9 cells, a continuous CD4+ cell line. This uptake is significantly inhibited by N-ethylmaleimide, suggesting a requirement for sulfhydryl groups. Dicyanogold inhibits the replication of the AIDS virus, HIV, in a cell culture model.

Determination of Biotransformation Products of Platinum Drugs in Rat and Human Urine

Cisplatin is an extremely effective cancer chemotherapeutic agent, but its use is often accompanied by toxicity. Second generation drugs such as carboplatin are becoming more widely used because of reduced toxicity. Since biotransformation products have been implicated in the toxic responses, we have begun to investigate the reactions of cisplatin and carboplatin with potential biological ligands. Reaction products were characterized using HPLC with inductively coupled plasma - mass spectrometry (HPLC-ICP-MS), 1H and 13C NMR and fast atom bombardment - mass spectrometry (FAB-MS). Three Pt-creatinine complexes, cis-[Pt(NH3)2Cl(Creat)]+, cis-[Pt(NH3)2(H2O)(Creat)]2+ and cis-[Pt(NH3)2(Creat)2]2+, were synthesized and the platinum was shown to coordinate to the ring nitrogen, N(3). Human urine samples from patients on cisplatin chemotherapy were shown to contain cisplatin, its hydrolysis product and biotransformation products containing Pt-creatinine, Pt-urea and Pt-uric acid complexes. Urine from carboplatin patients shows fewer biotransformation products. Studies with control and diabetic (protected against cisplatin toxicity) rats showed systematic differences in the biotransformation products formed on administration of cisplatin.

Myochrysine solution structure and reactivity.

We have determined the framework structure of Myochrysine (disodium gold(I)thiomalate) in the solid state and extremely concentrated aqueous solution, previously. It consists of an open chain polymer with linear gold coordination to two thiolates from the thiomalic acid moieties which bridge between pairs of gold atoms providing an Au-S-Au angle of 95°. The question remained: was this structure relevant to the dilute solutions of drugs administered and the still lower concentrations of gold found in the bodies of patients (typically 1 ppm Au in blood and urine or 5 μM in Au). We have provided an answer to that question using extended X-ray absorption spectroscopy (EXAFS) and capillary zone electrophoresis (CZE). EXAFS studies confirm that the polymeric structure with two sulfur atoms per gold atom persists from molar concentrations down to millimolar concentrations. CZE is able to separate and detect Myochrysine at millimolar levels. More importantly, at micromolar levels Myochrysine solutions exhibit identical CZE behavior to that measured at millimolar levels. Thus, aqueous solutions of the drug remain oligomeric at concentrations commensurate with those found in patient blood and urine. The reactivity of Myochrysine with cyanide, a species especially prevalent in smoking patients, was explored using CZE. Cyanide freely replaces thiomalic acid to form [Au(CN)2]- and thiomalic acid via a mixed ligand intermediate. The overall apparent equilibrium constant (Kapp) for the reaction is 6×10-4M-1. Further reaction of [Au(CN)2]- with a large excess of L, where L is cysteine, N-acetylcysteine, or glutathione, shows that these amino acids readily replace cyanide to form [AuL2]-. These species are thus potential metabolites and could possibly be active forms of gold in vivo. That all of these species are readily separated and quantified using CZE demonstrates that capillary electrophoresis is an accessible and powerful tool to add to those used for the study of gold-based antiarthritis drugs.

Chromatography with Metal Specific Detection of Urine Samples from an Arthritis Patient on Auranofin Therapy

Abstract Six sequential urine samples obtained from a patient on auranofin therapy were analyzed for total gold, zinc, copper and creatinine levels. They were separated by high performance liquid chromatography (HPLC) on a weak anion exchange column. The detector utilized was an inductively coupled plasma mass spectrometer (ICP-MS). Five, gold-containing metabolites as veil as multiple zinc- and copper-containing materials were eluted. The distribution of all three metals changed with time on therapy.

Preparation, characterization and electrochemical properties of technetium(II) complexes with 1,2-bis(diethylphosphino)ethane (depe) and dithiocarbamate ligands. Single-crystal structural analysis of [Tc((CH3)2NCS2)(DEPE2)](PF6)

Abstract Three technetium(II) complexes of the type [Tc(dtc)(DEPE)2]+ have been newly prepared and isolated, where DEPE denotes 1,2-bis(diethylphosphino)ethane and dtc represents dimethyldithiocarbamate (dmdc), diethyldithiocarbamate (dedc) or pentamethylenedithiocarbamate (pmdc). The complexes have been characterized by elemental analysis, UV-vis spectroscopy. FAB mass spectroscopy, electrochemistry and high performance liquid chromatography. Bidentate coordination of the dithiocarbamate ligand is confirmed by a single-crystal structural analysis of the prototypical complex [Tc(dmdc) (DEPE)2](PF6). Bond lengths around the approximately octahedral technetium(II) centre are TcS = 2.449(6), TcP (trans to P) = 2.43(2) and TcP (trans to S) = 2.38(2) A. The TcP length trans to the sulphur atom is significantly shorter than that which is trans to a phosphorus atom. Electrochemical measurements for the [Tc(dtc)(DEPE)2]+ complexes show a reversible technetium(II/I) couple between −0.517 and −0.544 V (vs Ag/AgCl) and another reversible technetium(III/II) couple ranging from +0.298 to +0.312 V (vs Ag/AgCl) in 0.5 M TEAP/DMF. The relevance of this chemistry to the design and development of new technetium radiopharmaceuticals is briefly discussed.

Nearly regular tetrahedral geometry in a gold(I)-phosphine complex. X-Ray crystal structure of tetrakis(methyldiphenylphosphine)gold(I) hexafluorophosphate

An X-ray crystal structure determination shows the gold(I) complex with four methyldiphenylphosphine ligands to have symmetry with all Au–P bond lengths equal and nearly tetrahedral angles; further, 31P n.m.r. spectroscopy at –80 °C indicates that the nearly tetrahedral species persists in solution.

Dicyanogold Effects on Lymphokine Production

Having identified dicyanogold(I) as a common metabolite of gold-based antiarthritis drugs, we are investigating the effects of the compound on the production of lymphokines. Handel, et al. 1 suggested that the transcription factor AP-1, critical to the production of a number of cytokines, might be the target for gold compounds because of a critical cysteine within its DNA binding region. Using Jurkat cells, an established cell line as a model for CD4+ lymphocytes, we have shown that dicyanogold inhibits the binding of AP-1 to DNA and inhibits the synthesis of IL-2 mRNA and protein. In a macrophage line, THP-1, which synthesizes IL-1β in response to mitogen, we have shown that dicyanogold inhibits the binding of a second transcription factor, CREB to DNA. Incubation of THP-1 cells with dicyanogold inhibits the production of IL-1β mRNA. These results suggest that the mechanism of action of gold drugs may be through their interaction with transcription factors necessary for the immune activation seen in Rheumatoid Arthritis.

EXAFS Studies of Film Coated Electrodes

EXAFS has been used to study structural and redox changes in transition metal complexes as films coating electrodes or contained in films. We have used copper complexes of 2,9-dimethylphenanthroline embedded in Nafion films to study the change from four to five coordination which occurs on oxidation of Cu(I) to Cu(II). The increase in coordination number presumably occurs from the addition of a water ligand on rearrangement from tetrahedral to trigonal bipyramidal geometry. When similar experiments are performed with the analogous bathocuproine ligand, with phenyl sulfonate substituents, in a poly(dimethyldiallylammonium) chloride film, the copper can still be oxidized from I to II. However in this case, there is no increase in the coordination number. Other studies were made on Prussian Blue and Ruthenium Purple films. In these films multiple oxidation states are also present and the EXAFS were measured for each. In the latter case the iron centers are redox active whereas the ruthenium centers are not. EXAFS studies were performed at both the Fe and Ru K edges. These studies were carried out at SSRL, NSLS and CHESS.