Poly-guanidine shows high cytotoxicity in glioma cell cultures and glioma stem cells.

Glioblastoma multiforme (GBM) is a malignant CNS tumor with a poor prognosis. GBM shows aberrant glycosylation with hypersialylation. This property is a potential target for therapy. This study investigates the growth inhibitory efficacy of poly-guanidine (GuaDex), with an affinity for sialic acid (Sia). Glioma cell cultures and patient-derived glioma cell lines (PDGCLs) expressing Prominin-1 (CD133) were used. Human fibroblasts and astrocyte-derived cells were used as controls. Temozolomide (standard GBM drug, TMZ) and DMSO were used as a comparison. GuaDex at 1-10 µM concentrations, were incubated for 3.5-72 h and with PDGCLs cells for 6-24 h. The cytotoxicity was estimated with a fluorometric cytotoxicity assay (FMCA). Fluorescence-labelled GuaDex was used to study the cell interactions. Sia expression was confirmed with a fluorescence labelled Sia binding lectin. Expression of glial fibrillary acidic protein was determined. GuaDex induction of growth inhibition was fast, showing after less than 5 min incubation while the control cells were not affected even after 50 min incubation. The growth inhibitory effect on PDGCLs spheroids was persistent still showing after 4 weeks post-treatment. The growth inhibition of GuaDex was induced at low µM concentrations while TMZ induced only a slight inhibition at mM concentrations. GuaDex efficacy appears significant and warrants further studies.

Synthesis and Binding of a Novel PSMA-specific Conjugate.

BACKGROUND/AIM: Prostate-specific membrane antigen (PSMA) is emerging as a target for treatment of castration-resistant prostate cancer (CRPC) while its up-regulated in the majority of CRPC tumors. The most common approach is targeted radionuclide therapy. MATERIALS AND METHODS: The PSMA binding pharmacophore Glu-Urea-Lysine (GUL) and lysine were conjugated to oxidized dextran with reductive amination and subsequently labelled with fluorosceinisothiocyanate (FITC). Three prostate cancer cell lines were used for binding studies, 22Rv1 (PSMA positive), DU145 (PSMA negative) and PC3 (PSMA negative). Binding images were obtained by fluorescence microscopy. RESULTS: PDC binding was recorded on the 22Rv1 cell line while the negative cell lines showed no or slight background binding. PDC binding could be inhibited by pre-incubation with a molar excess of unlabelled PDC. CONCLUSION: This is a novel template for PSMA targeted CRPC therapy, either using cytostatics or radionuclides.

Development of a novel poly bisphosphonate conjugate for treatment of skeletal metastasis and osteoporosis.

Advanced stage prostate and breast cancer frequently metastasize to the skeleton (approximately 75%). An additional complication in these patients, that further affects the bones, is that their hormonal treatment, induces osteoporosis. Bisphosphonates (bpns) are standard drugs against osteoporosis and have been shown to have clinically significant anti-tumor effects. This study describes the development of a new polybisphosphonate conjugate (ODX) with enhanced dual efficacy i.e. with anti-bone resorption and anti-tumor properties. Zoledronic acid (Zometa) was used as a positive control (at equimolar concentrations). Alendronic acid and aminoguanidine were conjugated to oxidized dextran with subsequent reductive amination (on average approximately 8 alendronate and approximately 50 guanidine moieties per conjugate). ODX was tested in a bone resorption assay for its capacity to inhibit bone resorbing osteoclasts (bone organ culture from neonatal mice, 45Ca labelled bone mineral). Tumor cell toxicity was studied on prostate (PC3) and breast cancer (MDA231, MDA453) cell cultures. Two methods were employed, a fluorescent cytotoxicity assay (FMCA) and an apoptosis assay (Annexin V assay). In the bone resorption assay, Zometa and ODX showed very similar potency with 50% osteoclast inhibition at approximately 20 nM and 100% at 0.2 microM. In the FMCA, IC50 for ODX was at approximately 2 microM and 25 microM for Zometa (PC3). In the apoptosis assay, ODX induced approximately 85-97% apoptosis at 10 microM in both cell lines, while Zometa failed to induce any significant apoptosis in any of the cell lines at the tested concentration range (10 nM-10 microM). ODX appears to be a promising drug candidate with high dual efficacy for the treatment of bone metastasis and osteoporosis. It has both potent osteoclast inhibiting properties and enhanced anti-tumor efficacy.

Polymer-conjugated guanidine is a potentially useful anti-tumor agent.

Guanidine compounds have important biochemical properties. Aminoguanidine, as an example, is an anti-oxidant, a nitric oxide synthase inhibitor (NOS) which prevents nitric oxide formation, and an inhibitor of advanced glycosylation end products (AGEs). As an anti-oxidant, aminoguanidine may affect the formation of atherosclerotic lesions through protection from LDL oxidation. Inhibition of AGEs could have a preventive effect on the tissue damage caused by diabetes where AGEs are considered to be an important factor. The role of NO in cancer is complex and not fully understood, but it may have influence on growth and progression. In this study, the tumor growth inhibitory effect of conjugated guanidine (i.e. a polyguanidine) was investigated. The effect on tumor cell growth was studied in cultures of prostate, breast, bladder and renal cell cancer, and a fluorometric cytotoxicity assay was performed. Guanidine conjugates were prepared by reacting aminoguanidine or agmatine with periodate oxidized dextran followed by reductive amination. The cytotoxic effect was compared with an anthracycline (adriamycin). The dextran-guanidine conjugates were cytotoxic at low micromolar concentrations, and the dextran-aminoguanidine conjugate (GDC) had the highest efficacy, being more efficient than adriamycin, in all of the tested tumor cell lines. Breast and prostate cancer cells were the most sensitive. At 0.5 microM, GDC killed >95% of the breast cancer cells compared to 25% for Adriamycine. In prostate cancer cells, GDC killed approximately 55% of the cells at 0.1 microM and 100% of the cells at 0.5 microM compared to approximately 22 and approximately 62%, respectively, for adriamycin. Unconjugated aminoguanidine and agmatine did not seem to affect tumor cell growth even at high concentrations (mM). Polymer- conjugated guanidine is a potentially useful template for the construction of therapeutic tumor targeting cytotoxic agents.

The effect of intravenously administered mexiletine on tinnitus - a pilot study.

The effect of intravenously administered mexiletine on subjective tinnitus and hearing was studied in six patients, who initially responded positively to lidocaine. Distinct mexiletine-induced decreases in tinnitus loudness were demonstrated in three subjects, as reflected by maximum VAS (visual analogue scale) level reduction of 34%, 95%, and 100%, respectively. One subject reported change in tinnitus pitch, another one showed a slight (18% on VAS) tinnitus reduction, and one subject disclosed no effect. Side effects were seen only during one of seven infusions. Mexiletine induced shifts in pure-tone threshold, transient evoked otoacoustic emission, and acoustic reflex threshold, probably reflecting a reversible interference in the function of organ of Corti. The concentration effect relationship remained unclear and no general 'therapeutic' level could be identified. This study confirms the effect of mexiletine on the auditory function and its potential as a possible therapeutic agent or a model for further development in tinnitus pharmacotherapy.

Stability, pKa and plasma protein binding of roscovitine.

In the present investigation, the binding of roscovitine (100, 500 and 1500 ng/mL) to plasma proteins was studied at 25 and 37 degrees C by ultrafiltration and equilibrium dialysis methods. Drug stability in plasma was assessed during a 48 h at 4, 25 and 37 degrees C. The effect of thawing and freezing on drug stability was studied. The pKa of roscovitine was measured using capillary electrophoresis coupled with mass spectrometry. Roscovitine was quantified utilizing liquid chromatography and tandem mass spectrometry. Roscovitine is highly bound to plasma proteins (90%). Binding of roscovitine to human serum albumin was constant (about 90%) within concentration range studied while the binding to alpha1-acid glycoprotein decreased with increasing drug concentration indicating that albumin is more important in clinical settings. However, alpha1-acid glycoprotein might be important when plasma proteins change with disease. Protein binding was higher at 25 degrees C compared to 37 degrees C. The results obtained by equilibrium dialysis were in good agreement with those obtained by ultrafiltration. Roscovitine was stable at all temperatures studied during 48 h. Roscovitine has a pKa of 4.4 showing that the drug mainly acts like a weak mono-base. The results obtained in our studies are important prior to clinical trials and to perform pharmacokinetic studies.

Tissue distribution, pharmacokinetics and identification of roscovitine metabolites in rat.

The pharmacokinetics, biodistribution and the metabolic pathway of roscovitine were investigated in Sprague-Dawley rats after a single intravenous dose of 25 mg/kg. Blood, lungs, kidney, liver, testis, adipose tissue, spleen and brain were removed at different time-points. Plasma and tissue samples were analyzed using high performance liquid chromatography. The metabolites were identified using liquid chromatography/tandem mass spectrometry and nuclear magnetic resonance spectroscopy. Roscovitine (MW=354) was cleared rapidly from circulation and highly distributed to the tissues. The elimination half-life of roscovitine in plasma and tissues was short (<30 min). A major metabolite (M1) was observed mainly in plasma and in lower levels in all other tissues. M1 was identified as conversion of the hydroxyl-group at C2 to carboxylic acid (MW=368). A second metabolite (M2) was observed mainly in liver and kidney and identified as a hydroxylation product of the C8 of the purine-ring (MW=370). A third metabolite (M3) was found in several organs and corresponded to N-dealkylation of the N9-isopropyl side-chain (MW=312). Roscovitine concentrations in the brain were 30% of that observed in plasma, however no metabolites were detected in brain. In this investigation, three major metabolites of roscovitine were isolated and identified. Also, it was shown that roscovitine eliminates rapidly from both blood and tissues.

Analysis of roscovitine using novel high performance liquid chromatography and UV-detection method: pharmacokinetics of roscovitine in rat.

Roscovitine (2-(R)-(1-ethyl-2-hydroxyethylamino)-6-benzylamino-9-isopropylpurine, is a potent and selective inhibitor of cyclin-dependent kinases (CDKs). It inhibits cdc2, cdk2, cdk5 and erk 1 and 2 by competing for the ATP binding domain of the kinases. It inhibits cell proliferation; induces DNA fragmentation and causes cell cycle arrest in S phase. Its stability and toxicity are not fully known. A liquid chromatography method was developed to measure roscovitine in human and rat plasma. The lower limit of quantitation (LLOQ) was 100ng/ml; the intra- and inter-day precision was below 10% at all control levels. Likewise, the accuracy between and within days was lower than 6% at all levels. The drug was stable at room temperature. Twenty-four hours at room temperature has result in a decrease of only 9% of the drug. The recovery of roscovitine from plasma was 84% at 750ng/ml. The present method was used to study the pharmacokinetics of the drug in a rat model. The present investigation, to the authors' knowledge, is the first analytical method reported and the first pharmacokinetics investigation of roscovitine in rat. Roscovitine was administered as a bolus injection (25mg/kg body weight). The pharmacokinetic analysis showed that roscovitine is fitted to a two-compartment open-mode with a biphasic elimination half-life (6 and 26min, respectively). The distribution volume was determined to 3.5l/kg and the clearance (Cl) was 29.5ml/min.

A simple pharmacokinetic method to evaluate the pulmonary dose in clinical practice--analyses of inhaled sodium cromoglycate.

When the expected effect of an inhaled drug is not achieved, the cause could be poor inhalation technique and consequently a low pulmonary dose. A simple in vivo test to evaluate the pulmonary dose would be a benefit. This study evaluates the relative and systemic bioavailability following inhalation of nebulized sodium cromoglycate (SCG) in healthy subjects. Blood samples were collected during 240 min and urine was collected in two portions, up to 6 h post-inhalation. Two exposures were performed and comparisons based on the quantification of drug in plasma and urine by a high-performance liquid chromatography (HPLC) procedure were done. In one of the exposures, a pulmonary function test was performed to study if an expected effect of increased absorption could be detected. There was a good correlation between the two exposures shown in the plasma concentrations, but not in the urine analyses. The forced exhaled volume manoeuvres were associated with a higher Cmax and plasma concentrations up to 60 min post-inhalation (P<0.01). This effect was not detected in the urine analyses. We conclude that this pharmacokinetic method with inhaled SCG and plasma analyses could be used to evaluate individual inhalation technique. The HPLC method used was rapid and had adequate sensitivity.

Effect of ketoconazole on venlafaxine plasma concentrations in extensive and poor metabolisers of debrisoquine.

OBJECTIVE: To study the influence of CYP3A4 inhibition by ketoconazole on the disposition of venlafaxine in individuals with different CYP2D6 pheno- and genotypes. METHODS: In an open two-phase study, 21 healthy volunteers with known CYP2D6 pheno- and genotype [14 extensive metabolisers (EMs), 7 poor metabolisers (PMs)] were given a single oral dose of venlafaxine (50 mg to EMs and 25 mg to PMs). Plasma and urine levels of venlafaxine and its three metabolites were measured and the pharmacokinetics of venlafaxine were determined. After a 2-week washout period, subjects were treated for 2 days with ketoconazole (100 mg twice daily) starting 1 day before the administration of venlafaxine; and the same parameters as for the administration of venlafaxine only were measured. RESULTS: Data were evaluated from 20 subjects (14 EMs and 6 PMs) who completed the study. The dose-corrected AUC of venlafaxine was on average 2.3 times higher ( P<0.01) and that of its active metabolite O-desmethylvenlafaxine 3.4 times lower ( P<0.0001) in PMs than EMs. There was a good correlation between the debrisoquine metabolic ratio and the ratio between the AUC of venlafaxine and that of O-desmethylvenlafaxine ( Rs=0.93, P<0.002). The majority of subjects showed higher plasma levels of venlafaxine and O-desmethylvenlafaxine upon co-administration of ketoconazole. AUC of venlafaxine significantly increased by 36% and that of O-desmethylvenlafaxine by 26% ( P<0.01). C(max) values increased by 32% and 18%, respectively. The elimination half-life of venlafaxine was unaltered. Three of the PMs displayed marked increases in AUC (81, 126 and 206%) and C(max) (60, 72, 119%) of venlafaxine while the other three showed small or no changes. CONCLUSIONS: Ketoconazole consistently affected the disposition of venlafaxine in EMs of debrisoquine while the response in PMs was erratic. The precise mechanisms underlying this interaction remain to be elucidated.

Simple method based on fluorescent detection for the determination of 4-hydroxycyclophosphamide in plasma.

Cyclophosphamide is a prodrug used both as a single drug and in combination chemotherapy. Cyclophosphamide is converted to its active metabolite (4-hydroxycyclophosphamide) by the cytochrome P450 enzymes. A liquid chromatography method including liquid-liquid extraction and protein precipitation in one step was developed to measure 4-hydroxycyclophosphamide in plasma. The 4-hydroxycyclophosphamide was stabilized and converted to a fluorescent dansylhydrazone derivative, which was chromatographed on a reverse-phase column and detected using a spectrofluorometric detector at excitation of 350 nm and emission of 550 nm. The limit of quantitation was 60 ng/mL and the between-day accuracy and precision were less than 9%. The method was applied to the analysis of plasma from patients who had received an intravenous infusion of 1 g/m(2) cyclophosphamide.