D Nardini

Clinical and experimental pharmacokinetic interaction between 6-mercaptopurine and methotrexate

Clinical and experimental pharmacokinetic interaction between 6-mercaptopurine (6-MP) and methotrexate (MTX) was investigated in patients as well as in rats and in HL-60 human leukemic cells. Ten children affected by acute lymphoblastic leukemia (ALL) in remission received daily doses of 6-MP given at 25 mg/m2 and i.v. infusion of high-dose MTX at 2 or 5 g/m2 once every other week. When 6-MP was given alone, the mean peak plasma concentration (Cmax) and area under the curve (AUC) of 6-MP were 72.5 ng/ml and 225.3 hngml-1. Concurrent treatment with MTX at 2 or 5 g/m2 resulted in a mean increase of 108% and 121% in the Cmax and of 69% and 93% in the AUC, respectively. In rats treated with an oral dose of 6-MP at 75 mg/m2, MTX given i.p. at 5 g/m2 produced mean increases of 110% and 230% in the Cmax and AUC of 6-MP, respectively. In HL-60 human leukemic cells incubated with 6-MP at 250 ng/ml, the cumulative intracellular concentration of 6-thioguanine and 6-MP nucleotides was not significantly modified by treatment with 20 ug/ml of MTX. The present findings indicate that high-dose MTX enhances the bioavailability of 6-MP as evidenced by the observed increases in the plasma Cmax and AUC of 6-MP in humans and animals.

Manumycin inhibits ras signal transduction pathway and induces apoptosis in COLO320-DM human colon tumour cells

The aim of the present study was to assess the cytotoxicity of manumycin, a specific inhibitor of farnesyl:protein transferase, as well as its effects on protein isoprenylation and kinase-dependent signal transduction in COLO320-DM human colon adenocarcinoma which harbours a wild-type K- ras gene. Immunoblot analysis of isolated cell membranes and total cellular lysates of COLO320-DM cells demonstrated that manumycin dose-dependently reduced p21 ras farnesylation with a 50% inhibitory concentration (IC50) of 2.51 ± 0.11 μM and 2.68 ± 0.20 μM, respectively, while the geranylgeranylation of p21 rhoA and p21 rap1 was not affected. Manumycin dose-dependently inhibited (IC50= 2.40 ± 0.67 μM) the phosphorylation of the mitogen-activated protein kinase/extracellular-regulated kinase 2 (p42MAPK/ERK2), the main cytoplasmic effector of p21 ras, as well as COLO320-DM cell growth (IC50= 3.58 ± 0.27 μM) without affecting the biosynthesis of cholesterol. Mevalonic acid (MVA, 100 μM), a substrate of the isoprenoid synthesis, was unable to protect COLO320-DM cells from manumycin cytotoxicity. Finally, manumycin 1–25 μM for 24–72 h induced oligonucleosomal fragmentation in a dose- and time-dependent manner and MVA did not protect COLO320-DM cells from undergoing DNA cleavage. The present findings indicate that the inhibition of p21 ras processing and signal transduction by manumycin is associated with marked inhibition of cell proliferation and apoptosis in colon cancer cells and the effect on cell growth does not require the presence of a mutated ras gene for maximal expression of chemotherapeutic activity.

Inhibitory effect of the somatostatin analogue SMS 201–995 and cytokines on the proliferation of human colon adenocarcinoma cell lines

The activity of the synthetic somatostatin analogue SMS 201-995 was investigated in vitro on the growth of SW480 and SW620 human colon adenocarcinoma cell lines. The inhibition of cell proliferation was significant in SW480 cells (-19.6 +/- 1.4% at SMS 201-995 10-9 M, P < 0.05), but not in SW620 cells (-5.5 +/- 0.8% at SMS 201-995 10-8 M) as compared to untreated cultures. Moreover, SMS 201-995 10-8 M decreased the mitogenic effect of epidermal growth factor (EGF) on the SW480 cell line (-26.6 +/- 3.4% vs. cells exposed to EGF 10 ng ml-1 alone, P < 0.05). The effect of combining SMS 201-995 plus the cytokines interleukin-2 (IL-2) or gamma-interferon (gamma-IFN) on SW480 and SW620 cancer cell growth was also evaluated. The treatment produced a synergistic antiproliferative effect against SW620 cells as compared to untreated cultures, with growth inhibition being -20.2 +/- 1.2 and -19.3 +/- 1.3%, at SMS 201-995 10-8 M plus IL-2 or gamma-IFN 100 IU ml-1, respectively, but did not increase the activity of SMS 201-995 against the SW480 cells. In conclusion, the effect of SMS 201-995 on colon cancer cell growth can be enhanced by its combination with cytokines in SW620 but not in SW480 colon adenocarcinoma cells.

Ultrastructural and biochemical evidence of apoptosis induced by a novel inhibitor of protein geranylgeranylation in human MIA PaCa-2 pancreatic cancer cells.

Analogs of geranylgeranyl diphosphate (GGdP) have been demonstrated to inhibit the geranylgeranylation of proteins, producing cytotoxic activity in human prostate cancer cells. A detailed study is reported on the programmed cell death in vitro of human exocrine pancreas cancer cells (MIA PaCa-2) induced by the most active compound of this series of geranylgeranylation inhibitors, the dipotassium salt of (E,E,E)[2-oxo-2-[[(3,7,11,15-tetramethyl-2, 6,10,14-hexadecatetraenyl)-oxy]amino]ethyl] phosphonic acid (BAL 9504), using transmission and scanning electron microscopy (SEM). The results show that, after 72 h of treatment with BAL 9504, 25 microM, most MIA PaCa-2 cells display the typical morphological features of apoptosis, including condensation of nuclear chromatin, dilation of endoplasmic reticulum, and fragmentation of both nucleus and cytoplasm, giving rise to small membrane-bound vesicles (apoptotic bodies); surface protrusions and blebs are well demonstrated by SEM. The electrophoresis showed the presence of various bands corresponding to fragmented DNA of 180 base pairs, or multiples of this length, thus indicating that BAL 9504 effectively induces apoptosis. The present study provides the first evidence that inhibition of protein geranylgeranylation produces apoptosis in human MIA PaCa-2 exocrine pancreas cancer cells.

Ex-vivo MR imaging of liver intracellular contrast agents

The objective is to evaluate whether an ex-vivo model can be used to test intracellular contrast agents for MR imaging of the liver. T1 weighted inversion recovery, proton density spin echo and T2* weighted gradient echo images of the liver were acquired at 0.5 T in 10 rats before and 30 min after intravenous injection of 0.075 mmol/kg Gadolinium benzyloxypropionictetraacetate (Gd-BOPTA, n = 5) or 0.015 mmol/kg dextran magnetite (DM, n = 5), Four additional animals served as controls. After exsanguination and perfusion with saline and formalin, specimens of the liver and brain were embedded in an agar gel and examined with MR imaging one to three weeks later using the same protocol. In-vivo, the mean liver signal enhancement caused by Gd-BOPTA in T1, proton density and T2* weighted images was +23%, +28% and -70%, respectively. The mean liver signal enhancement caused by DM was -71%, -76% and -94%. In-vitro, no signal change was seen in the brain of animals injected with Gd-BOPTA and DM as compared to controls. Liver signal was increased by Gd-BOPTA and decreased by DM. Mean liver enhancement rate induced by Gd-BOPTA was +22%, +5% and +27% for T1, proton density and T2* weighted images, respectively. Mean liver enhancement rate induced by DM was -27%, -19% and -31%. MR imaging signal changes induced by liver intracellular contrast agents are still appreciable in an ex-vivo model. The latter might be useful for for preliminary investigation of intracellular contrast agents for MR imaging of the liver.

Chronic administration of suramin induces neurotoxicity in rats

In the present study, the ability of suramin 18 mg/kg i.p. twice a week to induce chronic neurotoxicity in rats was investigated. After 20 weeks of suramin treatment, morphological analysis of nerve fibers demonstrated that 57.7+/-3.2% of them presented vesicular disruption of myelin sheaths; their thickness was 0.23+/-0.07 microm in suramin-treated rats with respect to 0.43+/-0.07 microm of controls (P<0.05). To investigate the interaction between suramin and nerve tissue, the binding of the drug to partially purified myelin P0 protein obtained from sciatic nerves was analysed. The percentage of suramin bound to rat myelin P0 protein was 94.0+/-9.5%; this value was decreased to 55.0+/-7.6% when heparan sulfate was added to the myelin protein suspension before suramin. The analysis of tissue drug concentrations at 5, 10 and 20 weeks of treatment showed that suramin accumulated into the sciatic nerve in a time-dependent fashion (130.8+/-18.1, 219.7+/-17.1 and 449.3+/-15.6 microg/g of tissue, respectively). In conclusion, suramin induces a chronic peripheral neurotoxicity in rats characterized by myelin damage and high tissue levels of the drug. The high affinity of suramin for partially purified myelin P0 protein suggests a possible mechanism for drug-induced toxicity.