Peter Galettis

Role of lipophilicity in determining cellular uptake and antitumour activity of gold phosphine complexes

Purpose: The lipophilic cation [Au(I)(dppe)2]+ [where dppe is 1,2-bis(diphenylphosphino)ethane] has previously demonstrated potent in vitro antitumour activity. We wished to determine the physicochemical basis for the cellular uptake of this drug, as well as of analogues including the 1:2 adducts of Au(I) with 1,2-bis(di-n-pyridylphosphino)ethane (dnpype; n=2, 3 and 4), and to compare in vitro and in vivo antitumour activity. Methods and results: Logarithmic IC50 values for the CH-1 cell line bore a parabolic dependence on drug lipophilicity, as measured either by high-performance liquid chromatography or by n-octanol-water partition. Cellular uptake of drug, as measured by inductively coupled plasma mass spectrometry, varied by over three orders of magnitude over the series. Logarithmic uptake had a parabolic dependence on drug lipophilicity but a linear relationship to logarithmic IC50 values. Free drug concentrations were determined under the culture conditions and logarithmic free drug IC50 values and uptake rates were linearly related to lipophilicity. Uptake of drug in vivo in tissue from murine colon 38 tumours was approximately proportional to the dose administered. Host toxicity varied according to lipophilicity with the most selective compound having an intermediate value. This compound was also the most active of those tested in vivo, giving a growth delay of 9 days following daily intraperitoneal dosing (10 days) at 4 μmol kg−1 day−1. It was also significantly more active than another lipophilic cation, MKT-077. Conclusions: Alteration of lipophilicity of aromatic cationic antitumour drugs greatly affects cellular uptake and binding to plasma proteins. Changes in lipophilicity also affect host toxicity, and optimal lipophilicity may be a critical factor in the design of analogues with high antitumour activity.

Relationships between hydrophobicity, reactivity, accumulation and peripheral nerve toxicity of a series of platinum drugs

Previous work has shown platinum drugs to differ in their effects on the peripheral nervous system. To test whether their differential toxicity was due to differences in their partitioning into the peripheral nervous system, we correlated the hydrophobicity, reactivity, tissue accumulation and neurotoxicity of a series of eight platinum analogues. Neurotoxicity was detected by measuring sensory nerve conduction velocity (SNCV) in Wistar rats treated twice per week at the maximum tolerated dose. Tissue platinum concentrations were measured by inductively coupled plasma mass spectrometry. Hydrophobicity (log P) was measured using an octanol-aqueous shake-flask method. The half-life of platinum drug binding to plasma proteins in vitro was determined. The cumulative dose causing altered SNCV ranged from 15 to > 2050 μmol kg–1. Ranking of the compounds by their neurotoxic potency in rats (oxaliplatin >R,R -(DACH)PtC4> ormaplatin >S,S -(DACH)PtCl4>S,S -(DACH)Pt oxalato > cisplatin > carboplatin > JM216) correlated with the frequency of neurotoxicity in patients (r> 0.99;P< 0.05). Ranking the compounds by their peripheral nerve accumulation was cisplatin > carboplatin > oxaliplatin >R,R -(DACH)PtCl4≈S,S -(DACH)PtCl4and did not correlate with neurotoxicity. Log P ranged from – 2.53 to –0.16 but did not correlate with neurotoxicity. Log P correlated inversely with platinum accumulation in dorsal root ganglia (r2= 0.99;P = 0.04), sural nerve (r2= 0.85;P = 0.025), sciatic nerve (r2= 0.98;P = 0.0012), spinal cord (r2= 0.97, P = 0.018) and brain (r2= 0.98, P = 0.001). Reactivity correlated with neurotoxicity potency in rats (r2= 0.89, P = 0.0005) and with the frequency of neurotoxicity in patients (r2= 0.99, P = 0.0002). The hydrophilicity of platinum drugs correlates with platinum sequestration in the peripheral nervous system but not with neurotoxicity. Differences in the reactivity of platinum complexes accounts for some of the variation in their neurotoxicity.

Structural and Solution Chemistry of gold(I) and Silver(I) complexes of bidentate pyridyl phosphines: selective antitumour agents

Abstract The 1:2 adducts of Ag(I) and Au(I) with 1,2-bis(di- n -pyridylphosphino)ethane (d n pype) for n =2, 3 and 4 have been synthesised and solution properties characterised by multinuclear NMR spectroscopy. The complexes are hydrophilic analogs of the lipophilic Au(I) antitumour complex [Au(dppe) 2 ] + and the degree of hydrophilicity depends critically on the position of the N atom in the pyridyl ring. The complexes of d3pype and d4pype are simple monomeric [M(d3pype) 2 ] + and [M(d4pype) 2 ] + species which have a much higher water solubility than the 2-pyridyl complexes which crystallise in the solid state as dimeric [{M(d2pype) 2 } 2 ] 2+ . In solution these 1:2 M:d2pype species exist as equilibrium mixtures of monomeric, dimeric and trimeric (Ag) or tetrameric (Au) clusters. The Au(I) and Ag(I)pyridyl phosphine complexes have been evaluated for antitumour activity against a panel of cultured human ovarian carcinoma cell lines. The results show both potent and selective activity for the compounds with IC 50 values ranging from 0.18 to 1500 μM. There is a correlation between the degree of antitumour selectivity and the octanol/water partition coefficients with the greatest selectivity (500-fold range) found for the most hydrophilic complex [Au(d4pype) 2 ]Cl. Clinical development of the parent compound [Au(dppe) 2 ] + was halted by liver toxicity and the hydrophilic pyridylphosphine analogs are significantly less toxic than [Au(dppe) 2 ] + when exposed to isolated rat hepatocytes. Convenient synthetic routes to the bidentate pyridyl phosphines d2pype, d3pype and d4pype are also described.

Emerging roles for phospholipase A2 enzymes in cancer

Phospholipase A(2) (PLA(2)) enzymes (EC3.1.4.4) regulate the release of biologically active fatty acids and lysophospholipids from membrane phospholipid pools. These lipids are also substrates for intracellular biochemical pathways that generate potent autocrine and paracrine lipid mediators such as the eicosanoids and platelet activating factor. These factors, in turn, regulate cell proliferation, survival, differentiation, motility, tissue vascularisation, and immune surveillance in virtually all tissues, functions that are subverted by cancer cells for tumour growth and metastasis. Thus the relevance of PLA(2)-dependent pathways to the genesis and progression of cancer has been of interest since their discovery and with recent technological advances, their role in tumourigenesis has become more tractable experimentally. Limited human genetic studies have not yet identified PLA(2) enzymes as classical mutated oncogenes or tumour suppressor genes. However, there is strong evidence that of the 22 identified human PLA(2) enzymes, ten of which have been studied in cancer to date, most are aberrantly expressed in a proportion of tumours derived from diverse organs. Correlative and functional studies implicate the expression of some secreted enzymes (sPLA(2)s), particularly the best studied enzyme Group IIA sPLA(2) in either tumour promotion or inhibition, depending on the organ involved and the biochemical microenvironment of tumours. As in immune-mediated inflammatory pathologies, genetic deletion studies in mice, supported by limited studies with human cells and tissues, have identified an important role for Group IVA PLA(2) in regulating certain cancers. Pharmacological intervention studies in prostate cancer suggest that hGIIA-dependent tumour growth is dependent on indirect regulation of Group IVA PLA(2). Group VI calcium-independent PLA(2) enzymes have also been recently implicated in tumourigenesis with in vitro studies suggesting multiple possible roles for these enzymes. Though apparently complex, further characterization of the regulatory relationships amongst PLA(2) enzymes, lipid mediator biosynthetic enzymes and the lipid mediators they produce during tumour progression is required to define the biochemical context in which the enzymes modulate cancer growth and development.

Stereoselective peripheral sensory neurotoxicity of diaminocyclohexane platinum enantiomers related to ormaplatin and oxaliplatin

The diaminocyclohexane platinum (Pt(DACH)) derivatives ormaplatin and oxaliplatin have caused severe and dose-limiting peripheral sensory neurotoxicity in a clinical trial. We hypothesized that this toxicity could vary in relation to the biotransformation and stereochemistry of these Pt(DACH) derivatives. We prepared pure R,R and S,S enantiomers of ormaplatin (Pt(DACH)Cl4), oxaliplatin (Pt(DACH)oxalato) and their metabolites (Pt(DACH)Cl2 and Pt(DACH)methionine) and assessed their peripheral sensory neurotoxicity and tissue distribution in the rat and in vitro anti-tumour activity in human ovarian carcinoma cell lines. The R,R enantiomers of Pt(DACH)Cl4, Pt(DACH)oxalato and Pt(DACH)Cl2, induced peripheral sensory neurotoxicity at significantly lower cumulative doses (18 +/- 5.7 vs 32 +/- 2.3 micromol kg(-1); P < 0.01) and at earlier times (4 +/- 1 vs 6.7 +/- 0.6 weeks; P = 0.016) during repeat-dose treatment than the S,S enantiomers. Pt(DACH)methionine enantiomers showed no biological activity. There was no difference between Pt(DACH) enantiomers in the platinum concentration in sciatic nerve, dorsal root ganglia, spinal cord, brain or blood at the end of each experiment. Three human ovarian carcinoma cell lines (41 M, 41 McisR and SKOV-3) showed no (or inconsistent) chiral discrimination in their sensitivity to Pt(DACH) enantiomers, whereas two cell lines (CH-1 and CH-1cisR) showed modest enantiomeric selectivity favouring the R,R isomer (more active). In conclusion, Pt(DACH) derivatives exhibit enantiomeric-selective peripheral sensory neurotoxicity during repeated dosing in rats favouring S,S isomers (less neurotoxic). They exhibited less chiral discrimination in their accumulation within peripheral nerves and in vitro anti-tumour activity.

Biochemical and pharmacological studies of the mechanism of action of tenebrosin-C, a cardiac stimulatory and haemolytic protein from the sea anemone, Actinia tenebrosa

Tenebrosin-C is a protein of mol. wt 19,500 that displays potent cardiac stimulatory and haemolytic activities. Its haemolytic activity is inhibited by sphingomyelin but not phosphatidylcholine, and is not affected by Ca2+. The positive inotropic effect of tenebrosin-C on isolated guinea pig right atria is inhibited by the cyclooxygenase blockers indomethacin and aspirin, the lipoxygenase blocker and leukotriene antagonist RG5901, and the phospholipase A2 inhibitor mepacrine. This activity of tenebrosin-C therefore appears to be due to stimulation of the release of arachidonic acid and subsequent formation of prostaglandins and leukotrienes. Phospholipase A2-like activity was found with some tenebrosin-C preparations, but did not correlate with their positive inotropic or haemolytic activities and was too weak to account for either of these effects. Treatment of tenebrosin-C with various proteases in order to obtain active fragments showed that the protein is remarkably resistant to proteolysis.

Quantitative determination of platinum complexes in human plasma generated from the oral antitumour drug JM216 using directly coupled high-performance liquid chromatography-inductively coupled plasma mass spectrometry without desolvation

A new method was developed and validated for measuring platinum species generated from the clinical antitumour agent JM216 in methanol extracts of human plasma using high-performance liquid chromatography-inductively coupled plasma mass spectrometry (HPLC-ICP-MS). Good separation of JM216 and three of its biotransformation products (JM118, JM518 and JM383) was achieved with a run time of 20 min using a C8 column (4.6×150 mm) and a gradient methanol-water mobile phase (pH 2.5) at a flow rate of 1 ml min –1 . The presence of methanol in the mobile phase and in the sample matrix suppressed the platinum counts and the gradient step was associated with some base-line drift. However, the quantitation of JM216 and its biotransformation products (JM118, JM518 and JM383) was achieved with good intra-assay precision (range 1-12% RSD), inter-assay precision (range 2.3-11% RSD), accuracy (range 89-103%) and limits of quantitation (range 1-2 ng ml –1 ) without having to use a desolvation device. This new HPLC-ICP-MS technique has the advantages of greater sensitivity and efficiency compared to existing methods that use HPLC, fraction collection and the off-line detection of platinum by AAS.

Randomized Crossover Study Evaluating the Effect of Gemcitabine Infusion Dose Rate: Evidence of Auto-Induction of Gemcitabine Accumulation

PURPOSE Controversy exists over the optimal dose rate for administration of gemcitabine. There is a strong pharmacologic rationale for increased intracellular accumulation with prolonged infusions, but this failed to translate into a significant benefit in a large randomized study. The purpose of this study was to compare the intracellular pharmacokinetics of gemcitabine given for 30 minutes or for 100 minutes in a crossover design. PATIENTS AND METHODS We randomly assigned 33 patients to a standard dose of 1,000 mg/m2 over either 30 minutes or 100 minutes. At the second week, they were transferred to the alternate schedule. Blood samples were collected at various times after the gemcitabine infusion. Gemcitabine and difluorodeoxyuridine were measured in plasma by high-performance liquid chromatography (HPLC), and gemcitabine-triphosphate was measured by HPLC in leukocytes. RESULTS Intracellular accumulation was greater during the 100-minute infusion, which was consistent with previous data. This effect was confounded by an increase in gemcitabine-triphosphate accumulation between weeks 1 and 2, which was consistent with self-induction of gemcitabine accumulation. There was significant heterogeneity: 27% of patients had greater WBC accumulation during the 30-minute infusion (regardless of treatment order). Patients with relatively greater levels of gemcitabine-triphosphate in WBCs tended to have less under-dosing and a greater reduction in midcycle neutrophils. However, this observation did not correlate with plasma gemcitabine levels. CONCLUSION This work identifies significant variations in intracellular gemcitabine-triphosphate accumulation between and within individuals, and it provides evidence that this variation has potential clinical significance. The observed self-induction of gemcitabine metabolism has broad implications for the dosing of nucleoside analogs.

Combination of Albendazole and 2-Methoxyestradiol significantly improves the survival of HCT-116 tumor-bearing nude mice

BackgroundAlbendazole (ABZ) is a microtubule-targeting anthelmintic with a remarkable activity against a variety of human cancer cells. In this study, we examined if the antitumor activity of ABZ could be enhanced by its combination with other microtubule-binding agents.MethodsThe interactions between ABZ and microtubule-binding agents, paclitaxel, vinblastine, colchicine, and 2-methoxyestradiol were characterized using median effect analysis method in HCT-116 colorectal cancer cells and DU145 prostate cancer cell line. The mechanism underlying the synergistic interaction related to tubulin polymerization and apoptosis was then investigated. Finally, the effect of the combination therapy on the survival of HCT-116 tumor-bearing nude mice was evaluated.ResultsAmong the tested drugs, a synergistic anti-proliferative effect was observed with the combination of low concentrations of ABZ plus colchicine and ABZ plus 2-methoxyestradiol (2ME). Exploring the mechanism of the interaction between ABZ and 2ME revealed that the combination therapy synergistically activated the extrinsic pathway of apoptosis. Consistent with in vitro results, the combination of low concentration of ABZ with 2ME prolonged the survival of mice-bearing HCT-116 tumors. High concentration of ABZ in combination with 2ME, however, proved to be less effective than ABZ alone.ConclusionsThe combination of low doses of ABZ and 2ME has shown promising results in our pre-clinical model. Additionally, the finding that the combination of two microtubule-binding agents that share the same binding site can act synergistically may lead to the development of new therapeutic strategies in cancer treatment.

Enhancement of the activity of phenoxodiol by cisplatin in prostate cancer cells

Phenoxodiol is a novel isoflav-3-ene, currently undergoing clinical trials, that has a broad in vitro activity against a number of human cancer cell lines. Phenoxodiol alone inhibited DU145 and PC3 in a dose- and time-dependent manner with IC50 values of 8±1 and 38±9 μM, respectively. The combination of phenoxodiol and cisplatin was synergistic in DU145, and additive in PC3, as assessed by the Chou–Talalay method. Carboplatin was also synergistic in combination with phenoxodiol in DU145 cells. The activity of the phenoxodiol and cisplatin combination was confirmed in vivo using a DU145 xenograft model in nude mice. Pharmacokinetic data from these mice suggest that the mechanism of synergy may occur through a pharmacodynamic mechanism. An intracellular cisplatin accumulation assay showed a 35% (P<0.05) increase in the uptake of cisplatin when it was combined in a ratio of 1 μM: 5 μM phenoxodiol, resulting in a 300% (P<0.05) increase in DNA adducts. Taken together, our results suggest that phenoxodiol has interesting properties that make combination therapy with cisplatin or carboplatin appealing.