Transdermal fentanyl in opioid-naive cancer pain patients: an open trial using transdermal fentanyl for the treatment of chronic cancer pain in opioid-naive patients and a group using codeine.

To treat cancer pain, physicians often decide to jump directly from step 1 of the World Health Organization (WHO) analgesic ladder to step 3. The use of transdermal fentanyl in patients with cancer pain who had either used no opioid before, or only codeine, is evaluated in the present trial. Both opioid-naive (N = 14) and codeine-using (N = 14) patients started with transdermal fentanyl in the lowest available delivery rate (25 microg/hr). Immediate-release oral morphine was present as "rescue" medication. Transdermal fentanyl provided good to excellent pain relief in the majority (68%) of these patients. During the study, 5 patients continued with 25 microg/hr, and the others used a higher dose. Clinically relevant respiratory depression was not observed. The common side effects of opioids were found; constipation was mentioned by 3 patients (11%). Transdermal fentanyl appeared a safe analgesic in these opioid-naive cancer pain patients. In this study, WHO step 2 could be skipped without untoward complications.

Pharmacokinetic profile of alniditan nasal spray during and outside migraine attacks.

AIMS: To compare the pharmacokinetic profile of intranasal alniditan during and outside migraine attacks, and to investigate the relationship between initial rise of alniditan plasma concentration, and headache improvement. METHODS: Twenty-seven migraine patients (age: 18-65 years) were randomized to receive alniditan 2 mg or 4 mg, and investigated both during and outside a migraine attack. Maximal plasma concentrations (Cmax), time to Cmax (tmax), and the area under the curve over 2 h (AUC(0,2 h)), were calculated from the individual plasma concentration-time profile, obtained from 10 blood samples in each patient, during each of the two administrations. RESULTS: Alniditan was rapidly absorbed into the systemic circulation (tmax=11 min). All investigated pharmacokinetic parameters (Cmax, tmax, AUC(0,2 h)) were similar during and outside migraine attacks, both in the 2 mg (n = 13) and the 4 mg group (n = 14). In the 4 mg group, during attacks, mean plasma alniditan concentration at 5 min after administration (Ct=5) in responders (21+/-16 ng ml(-1); n=10) was significantly higher than the Ct=5 in nonresponders (3+/-3 ng ml(-1); P=0.01; n=4). However, the Cmax and AUC(0,2 h) in responders (33+/-18 ng ml(-1) and 12+/-6 ng ml(-1) h) were also significantly higher than the Cmax and AUC(0,2 h) in nonresponders (13+/-9 ng ml(-1); P=0.048 and 5+/-3 ng ml(-1) h; P=0.03). CONCLUSIONS: Absorption of alniditan nasal spray was not affected by migraine attacks, although 95% confidence intervals were wide. Early rise of plasma concentrations and the amount of drug in the circulation were related to headache improvement in the higher dose group.

DNA synthesis in isolated arteries of normotensive and hypertensive rats: effects of the endothelium.

OBJECTIVES: To compare DNA synthesis in isolated arteries of normotensive and hypertensive rats and to evaluate whether removal of the endothelium affects this process. DESIGN: Carotid and renal artery segments were isolated from normotensive Wistar, Wistar-Kyoto (WKY) and Sprague-Dawley rats, and from spontaneously hypertensive rats (SHR), transgenic Sprague-Dawley rats harbouring the mouse Ren-2 gene and from WKY rats rendered hypertensive by aortic coarctation. METHODS: Artery segments were exposed in vitro to serum with or without previous gentle removal of the endothelium. Nuclear incorporation of the thymidine analogue 5-bromo-2'-deoxyuridine was visualized by immunocytochemistry and the percentage of labelled medial nuclei was determined. RESULTS: In both types of artery, obtained from 6-week-old WKY rats and from 6-week-old SHR, removal of endothelium increased the percentage of 5-bromo-2'-deoxyuridine-labelled medial nuclei (L%). Also, in the arteries of 20-week-old Wistar rats, WKY rats and WKY rats rendered hypertensive by aortic coarctation and in vessels of 11-week-old Sprague-Dawley rats and Sprague-Dawley rats harbouring the mouse Ren-2 gene, removal of endothelium increased L%. Conversely, in the arteries of 20-week-old SHR removal of the endothelium did not alter L%. Furthermore, maximally stimulated DNA synthesis was considerably smaller in de-endothelialized arteries of adult SHR than in denuded vessels from the other strains and models. CONCLUSION: These findings confirm that the endothelium can reduce DNA synthesis in the intact rat arterial smooth muscle. This effect is not modified by hypertension, but is selectively reduced in the arteries of adult SHR.

Growth responses in isolated elastic, muscular and resistance-sized arterial segments of the rat.

To evaluate whether intravascular phenomena contribute to local differences in growth responses of the arterial wall, we evaluated responses to organoid culture in a broad variety of arterial preparations. Arterial segments were isolated from adult, normotensive rats, sympathectomized, denuded from endothelium, and suspended in medium supplemented with serum. As judged from the nuclear incorporation of the thymidine analogue 5-bromo-2'-deoxyuridine (BrdUrd), this induced a transient stimulation of DNA synthesis in only a fraction of the arterial smooth muscle cells in all types of arteries. This intramedial DNA synthesis was more marked in renal arteries than in carotid arteries or aortae and was least pronounced in main pulmonary, femoral, and superior mesenteric artery and in mesenteric resistance-sized arteries. Organoid culture of isolated arteries did not increase the cross-sectional area of the media or the number of medial cells. It rather resulted in proliferation of smooth-muscle-like cells outside the media. In addition, smooth-muscle-like cells migrated out of the isolated arterial segments during culture. The rate of proliferation of these isolated cells did not differ between large arteries of different anatomical origin. However, isolated cells derived from mesenteric resistance arteries proliferated at a rate that was 4 times slower than that of large artery cells. The presence of endothelium significantly reduced medial DNA synthesis in carotid and renal artery segments, but not in mesenteric resistance-sized preparations. These data indicate that growth responses of the arterial wall differ quantitatively with the anatomical location and branching order of the vascular segment. In addition to the regional heterogeneity of endothelial effects on mitogenic responses of arterial smooth muscle, this seems to be due to regional differences in the susceptibility of arterial smooth muscle to exogenous growth factors. In this respect, we speculate that subsets of growth-resistant and growth-prone arterial smooth muscle cells could be heterogeneously distributed over the arterial tree.

Acute and long-term effects of tissue culture on contractile reactivity in renal arteries of the rat.

To evaluate long-term effects of contractile and mitogenic stimuli on the contractile reactivity of arterial smooth muscle, we measured the incorporation of the thymidine analogue 5-bromo-2'-deoxyuridine (BrdUrd) and mechanical responses in arterial segments that had been maintained in tissue culture. The experiments were performed on renal arteries that had been isolated from adult rats, chemically sympathectomized, mechanically denuded from endothelium and mounted under distension. Exposure of arterial segments for up to 3 weeks to culture medium supplemented with fetal calf serum resulted in the following consecutive changes: a strong acute contraction, selective pharmacological changes that included decreased contractile responses to phenylephrine and vasopressin and increased relaxing responses to isoproterenol, increased incorporation of BrdUrd, a progressive fall in contractile responses to all vasoconstrictor stimuli, and an increase in excitability. Serum-free medium resulted in a much smaller acute arterial contraction, induced less incorporation of BrdUrd, accelerated the occurrence of hyperexcitability, but did not affect early pharmacological changes or the subsequent fall in overall arterial contractility with tissue culture. Dialysis of the serum or addition of ketanserin abolished the contractile effect of serum but did not affect the incorporation of BrdUrd or the loss of contractility with tissue culture. Addition of serotonin to serum-free culture medium mimicked the contractile response to serum but not the stimulation of BrdUrd incorporation. These data indicate that tissue culture alters the properties of the arterial wall, that contraction does not underlie the proliferative response of arterial smooth muscle to serum-derived mitogens in vitro, and that stimulation of DNA synthesis does in itself not lead to selective changes in arterial contractility.

Direct measurement of vitamin K-dependent enzymes in various isolated and cultured tumor and non-tumor cells.

A modification of the assay for vitamin K-dependent carboxylase is described with which the enzyme could be detected in relatively low amounts of cells (n = 10(6)). Using this assay, we could demonstrate vitamin K-dependent carboxylase activity in hepatocytes, renal tubular cells, osteoblasts, endothelial cells and macrophages, but not in lymphocytes or platelets. The cultured tumor cells UMR-106, B16 and 5583 also contained vitamin K-dependent carboxylase activity. Vitamin K epoxide reductase activity was demonstrated only in cells where vitamin K-dependent carboxylase activity was present. The tumor cells possessed remarkably less K epoxide reductase activity than the normal cells. When cells were cultured in medium containing warfarin, the K epoxide reductase activity was found to be decreased and the amount of non-carboxylated precursor proteins had increased, suggesting an analogous vitamin K mechanism as in liver.

The establishment and characterization of two new cell lines derived from a single human colonic adenocarcinoma.

Two cell lines with different in vitro growth characteristics were established from a single mucinous colonic adenocarcinoma. Epithelial cells of the line 5583-E demonstrated anchorage-dependent growth while those of line 5583-S were anchorage-independent and grew as multicellular floating spheroids. Both cell lines shared common characteristics with respect to the expression of differentiation markers (secretory component, carcinoembryonic antigen), mucins and karyotype (trisomy 12 and 14, marker chromosome) but also showed consistent differences. In nude mice 5583-S cells formed moderately differentiated mucinous adenocarcinomas with high carcinoembryonic antigen and mucin production, whereas 5583-E xenografts were poorly differentiated and almost entirely failed to produce carcinoembryonic antigen and mucins. The plating efficiency of 5583-E cells appeared to be greater and doubling time shorter than those of 5583-S cells. Furthermore, 5583-E cells showed an extra isochromosome, 1q. The cell lines were genotypically and phenotypically stable over a period of 2 years. Our results reemphasize that multiple cell lines with heterogeneous phenotypic and genotypic characteristics can be obtained from a single primary tumor.

B16 tumor cells contain a warfarin sensitive vitamin K1 2,3 epoxide reductase.

Using an adapted assay that requires an enzyme aliquot that forms only 5 pmoles vitamin K, we were able to demonstrate vitamin K1 2,3 epoxide reductase activity in cultured B16 mouse melanoma cells. The enzyme uses dithiothreitol, but not NADH as a reducing cofactor and is sensitive to inhibition by warfarin (2% residual activity at 10 micrograms/ml warfarin). Incubation of B16 cells in culture with 30 micrograms/ml warfarin leads to an 45% residual reductase as compared to normally cultured B16 cells. Combined with the reported presence of vitamin K dependent carboxylase in B16 cells and the cytotoxicity of warfarin towards B16 cells this suggests an active vitamin K cycle in these melanoma cells that may be essential for survival.

Influence of concomitant infusion of thymidine and inosine on methotrexate activity in normal and P388-bearing mice.

Combinations of thymidine and inosine (ranging from 0 to 7.5 mg/hr) were co-administered during a 72-hr continuous i.v. infusion of 3 micrograms/hr methotrexate in normal and P388 solid tumor-bearing DBA/2 mice. Methotrexate alone was lethal to all normal mice. Inosine at 1.0-7.5 mg/hr could reverse toxicity up to 100% while thymidine at 0.5-7.5 mg/hr was less effective (less than or equal to 86% survival). Combinations of the nucleosides averted toxicity more effectively than either compound alone and in a synergistic manner. From P388 tumor-bearing mice 27% survived methotrexate and eventually died of tumor. Co-infusion of thymidine or inosine decreased the percentage of toxic deaths and caused an increase in life span of more than 50% as compared to untreated tumor-bearing mice. However, the diminishing effect on survival with thymidine and inosine doses above 0.5 mg/hr indicated loss of the antitumor effect of methotrexate. This was also observed with combinations of the nucleosides. The influence of thymidine on the antitumor effect of methotrexate was compared in L1210- and P388-bearing mice (both in ascites) with 13 micrograms/hr methotrexate for 48 hr and 4 mg/hr thymidine for 96 hr. The increase in life span for L1210-bearing mice (8.6 days, 96%) was significantly longer than that with P388-bearing mice (6.1 days, 56%), probably due to biochemical differences between these tumors. It is concluded that co-administration of inosine and/or thymidine allows the use of methotrexate doses otherwise not tolerated, though with loss of anti-tumor effect. The choice of the tumor model may greatly influence the outcome of in-vivo studies on the modulation of methotrexate action by nucleosides.

Human tumour clonogenicity in agar is improved by cell-free ascites.

Replacement of enriched CMRL 1066 medium by cell-free ascites from tumour patients in the human tumour clonogenic assay described by Hamburger and Salmon (1977) increased plating efficiency for ovarian cancer cells by a median of 8-fold (range 0.4-1012 fold). In 40 experiments, two cases had a lower plating efficiency when cultured in cell-free ascites, 10 grew neither in standard medium nor in cell-free ascites and in two cases, growth was only observed in cell-free ascites. With standard medium, we observed 53% growth (greater than 5 colonies/dish) and 41% evaluable for chemosensitivity testing (greater than 30 colonies/dish). With cell-free ascites as culture medium, these figures were 71% and 63%, respectively. While under standard conditions the highest plating efficiency obtained was 0.25%, in 21% of the experiments done with cell-free ascites a plating efficiency higher than 1% was reached. We conclude that cell-free ascites is able to stimulate proliferation of ovarian cancer cells in agar and that the use of it extends the applicability of the clonogenic assay.

Reversal of methotrexate inhibition of colony growth of L1210 leukemia cells in semisolid medium.

L1210 mouse leukemia cells were grown in a methylcellulose-based medium, and the inhibitory effect of methotrexate (MTX) on colony formation and its reversal were examined. The effect on colony formation was studied in order to compare the results with those obtained with normal mouse bone marrow cells grown in a similar manner in previous studies and in additional experiments presented. Light microscopy could not be used for colony counting of L1210 cells because MTX did not inhibit colony formation and only affected further colony growth. Therefore, it was necessary to evaluate the toxic effect of MTX by analysis of colony size distribution using an electronic image analyzer. Results show that the reversal of MTX toxicity to L1210 cells with leucovorin is competitive and is similar to that with normal mouse bone marrow cells. Thymidine in combination with a purine prevents MTX toxicity as well. The optimal concentration of thymidine is 10(-5) M, whereas at least 10(-4) M purine is required. Reversal of MTX toxicity by thymidine and purines is independent of the MTX concentration and is possible at drug concentrations as high as 10(-4) M. Compared to mouse bone marrow cells, L1210 cells appear to require more purines to prevent the toxic effects of MTX. MTX toxicity towards bone marrow myeloid precursor cells can be reversed by 10(-4) M inosine alone. These cells are better protected against MTX toxicity when 10(-6) to 10(-5) M thymidine is added. The results suggest that the use of a high-purine-low-thymidine combination has advantages over the use of leucovorin in controlling toxicity over a wide range of MTX concentrations and in providing some degree of selective protection to normal proliferating cells.

An ultramicrochemical test for mycoplasmal contamination of cultured cells.

A sensitive ultramicrochemical enzyme test for mycoplasmal contamination of cultured cells, based on the determination of the activity of adenosine phosphorylase, is described. The test was performed by assaying the enzymatic conversion of [8-14C]adenine and ribose-1-phosphate to [8-14C]adenosine by incubating a plastic leaflet carrying a counted number of cells (1 to 10). These leaflets were isolated from the bottom of the same plastic film dish in which the cells were cultured for experimental or diagnostic purposes, e.g. prenatal diagnosis or inborn errors of metabolism. The present test should be several 1000-fold more sensitive than the originally reported enzymatic method because (a) the adenosine-phosphorylase reaction is measured in the nucleoside forming direction which is by far the most active; and (b) the assay is performed with the cells and not with the culture medium. The latter is of special importance for the detection of those low-grade contamination in which most of the mycoplasma particles are attached to cell membranes.

Molecular and tissue-specific heterogeneity in HPRT deficiency.

In several patients with different degrees of HPRT deficiencies, residual activities have been determined in both lysed and intact erythrocytes. No close correlation could be found between the degree of HPRT deficiency and the severity of the clinical expression. Unless HPRT activity in both intact and lysed erythrocytes was below detection level, the residual activity in intact red blood cells was higher than in lysates. Tissue-specific heterogeneity was illustrated with a patient suffering from X-linked gout. Lysates from erythrocytes, leukocytes, and cultured fibroblasts showed 1%, 8%, and 100% of normal HPRT activity, respectively. Characterization of the erythrocyte and fibroblast HPRT from this patient showed no kinetic abnormalities. However, there was a decreased heat stability. It is concluded that for a better understanding of the pathophysiology in HPRT deficiency studies on nucleated cells from the different tissues are needed.

Characterisation of purine nucleoside phosphorylase from fibroblasts using ultra-microchemical methods.

A new technique to quantitate nucleoside phosphorylase (NP) activity in single or small numbers of counted visually selected cells is presented. Fibroblasts were cultivated on the plastic film bottom of culture dishes. After lyophilisation in situ, plastic film leaflets carrying a counted number of cells were cut out and tested for NP activity. Some properties of NP, including temperature stability, pH optimum and substrate affinity, have been studied. The data obtained suggest that Np might play a regulatory role in the purine interconversion pathway.