Survival, neuron-like differentiation and functionality of mesenchymal stem cells in neurotoxic environment: the critical role of erythropoietin.

Mesenchymal stem cells (MSCs) can ameliorate symptoms in several neurodegenerative diseases. However, the toxic environment of a degenerating central nervous system (CNS) characterized by hypoxia, glutamate (Glu) excess and amyloid beta (Abeta) pathology may hamper the survival and regenerative/replacing capacities of engrafted stem cells. Indeed, human MSC (hMSC) exposed to hypoxia were disabled in (i) the capacity of their muscarinic receptors (mAChRs) to respond to acetylcholine (ACh) with a transient increase in intracellular [Ca(2+)], (ii) their capacity to metabolize Glu, reflected by a strong decrease in glutamine synthetase activity, and (iii) their survival on exposure to Glu. Cocultivation of MSC with PC12 cells expressing the amyloid precursor protein gene (APPsw-PC12) increased the release of IL-6 from MSC. HMSC exposed to erythropoietin (EPO) showed a cholinergic neuron-like phenotype reflected by increased cellular levels of choline acetyltransferase, ACh and mAChR. All their functional deficits observed under hypoxia, Glu exposure and APPsw-PC12 cocultivation were reversed by the application of EPO, which increased the expression of Wnt3a. EPO also enhanced the metabolism of Abeta in MSC by increasing their neprilysin content. Our data show that cholinergic neuron-like differentiation of MSC, their functionality and resistance to a neurotoxic environment is regulated and can be improved by EPO, highlighting its potential for optimizing cellular therapies of the CNS.

Nitric oxide restores impaired healing in normoglycaemic diabetic rats.

OBJECTIVE: Hyperglycaemia impairs wound healing. However, little is known about the underlying cellular mechanisms that lead to diminished wound repair in insulin-controlled and non-insulin-controlled diabetes. This study investigated the role of endogenous and exogenous nitric oxide on incisional wound healing in diabetic rats. METHOD: Groups of 10 wild-typeWistar control rats - 10 genetically diabetic BioBreeding rats and 10 genetically diabetic BioBreeding rats treated with subcutaneous insulin implants to render them normoglycaemic - underwent dorsal skin incision followed by subcutaneous insertion of polyvinyl alcohol sponges. The rats were sacrificed 10 days later to determine the wound-breaking strength and reparative collagen deposition. Nitric oxide, an important mediator in diabetic wound healing and collagen synthesis, was measured in wound fluid. Wound-derived fibroblasts were tested for ex vivo synthesis of nitric oxide and collagen. Exogenous nitric oxide was used for the therapeutic interventions. RESULTS: Wound-breaking strength and wound collagen deposition were significantly impaired in the hyperglycaemic diabetic animals (p<0.01). Wound nitric-oxide synthesis and ex vivo wound fibroblast nitric-oxide production were reduced in the hyperglycaemic rats (p<0.01). Insulin treatment partially reversed some of the effects of hyperglycaemia on wound repair (p<0.05). Exogenous nitric oxide further restored wound mechanical strength, collagen deposition and fibroblast collagen synthesis (p<0.01) in insulin-treated (normoglycaemic) diabetic animals. CONCLUSION: Wound healing is impaired in hyperglycaemic and normoglycaemic diabetic rats. This is reflected in impaired wound fibroblast nitric-oxide synthesis. Used in combination with insulin, exogenous nitric oxide further improves healing outcomes, making it a potential target for therapeutic intervention in insulin-treated normoglycaemic diabetes.

Effect of endothelin-1 on erythropoietin production in a rat model under normoxia and functional carbon monoxide-induced hypoxia.

It has been hypothesized that autacoids, such as endothelin-1 (ET), may modulate erythropoietin (Epo) secretion. Therefore, we studied the effect of ET-1 infusion and of a nonselective ET(A/B) receptor antagonist on Epo secretion under carbon monoxide (CO) exposure. Anesthetized rats were supplied with room temperature air containing increasing concentrations of CO by an aerating cap. A CO-Epo dose-response curve over the range of 0.02-0.14 vol% CO was conducted. Subpressor doses of ET-1 (3 pmol/min/kg BW) and the ET(A/B) receptor antagonist LU302872 (LU; 30 mg/kg) were applied to anaesthetized rats under normoxia (controls CON, ET, LU) and following hypoxia (CO exposure; H-CON, H-ET, H-LU). Mean arterial blood pressure (MAP), glomerular filtration rate (GFR, inulin clearance), Epo and ET-1 serum concentrations (ELISA) and renal Epo mRNA (Light Cycler) were determined. The EC50 value for CO was 0.1 vol% with a 70-fold increase in Epo serum concentrations. CO exposure increased Epo serum and Epo mRNA concentrations in the expected range in all groups. None of the treatments with ET or LU influenced the effect of hypoxia on Epo serum concentrations and renal Epo mRNA content. Under hypoxia, administration of ET-1 as well as LU prevented the hypoxia-induced decrease in MAP (p<0.05). Under hypoxia, GFR was reduced by 50% except for H-LU with values comparable to normoxia. Taken together, the influence of hypoxia exceeds by far the effect of ET-1 on Epo production, irrespective of the presence or absence of exogenous ET-1. Thus, ET-1 does not appear to be a major modulator of Epo production.

Effect of acute hyperhomocysteinemia on methylation potential of erythrocytes and on DNA methylation of lymphocytes in healthy male volunteers.

Homocysteine is a precursor of S-adenosylmethionine (AdoMet) and a metabolite of S-adenosylhomocysteine (AdoHcy). The ratio of AdoMet to AdoHcy, defined as the methylation potential (MP), indicates the flow of methyl groups within the cells. Chronic elevations of total homocysteine (tHcy) in plasma correlate with increased AdoHcy concentrations, decreased MP, and impaired DNA methylation. However, the influence of acute hyperhomocysteinemia on MP is unknown. We induced acute hyperhomocysteinemia in 14 healthy volunteers by oral administration of l-homocysteine (65.1 micromol/kg body wt) in an open, randomized, placebo-controlled two-period crossover study. The kinetics of tHcy in blood and urine, MP in blood, and global DNA methylation in lymphocytes were studied systematically during 48 h. Plasma tHcy concentrations reached a peak at 34 +/- 11 min after an oral load with l-homocysteine and decreased with a half-life of 257 +/- 41 min (means +/- SD). Only 2.3% of the homocysteine dose were recovered in urine. AdoHcy concentrations and MP in whole blood and erythrocytes were not affected by the oral homocysteine load. Furthermore, global DNA methylation in lymphocytes did not change under these conditions. We found no difference between the genotypes of 5,10-methylenetetrahydrofolate reductase in response to the homocysteine load. However, AdoMet content in erythrocytes was significantly higher in the C677T carriers (CT; n = 7) compared with the CC genotype (n = 7). Although chronic elevation of tHcy has been shown to affect MP and DNA methylation, acute elevation of plasma tHcy above 20 micromol/l for 8 h is not sufficient to change MP and to induce DNA hypomethylation in lymphocytes.

Tacrolimus impairs wound healing: a possible role of decreased nitric oxide synthesis.

BACKGROUND: The effect of the immunosuppressant tacrolimus on wound healing is not known. Tacrolimus has been shown to decrease nitric oxide synthesis. The systemic inhibition of wound nitric oxide synthesis leads to impaired healing. METHODS: We studied the effect of systemic tacrolimus treatment on wound-breaking strength and collagen deposition 10 days after wounding in rats and to correlate the outcome of healing with wound nitric oxide synthesis. Beginning at the day of wounding, rats were treated once daily by intraperitoneal injections with 0.5, 1.0, or 2.0 mg tacrolimus/kg body weight. Nitrite and nitrate were measured in wound fluid as an index of wound nitric oxide synthesis. Expression of inducible nitric oxide synthase in the wound was investigated by immunohistochemistry. Splenic lymphocytes were tested for proliferative activity. Tacrolimus levels in blood and wound fluid were measured by enzyme-linked immunosorbent assay. RESULTS: Systemic tacrolimus treatment was well tolerated by all rats. Tacrolimus accumulated in wound fluid. Tacrolimus levels in wound fluid were found to be approximately 10-fold higher than blood levels (P < 0.001). Tacrolimus (2.0 mg/kg/day) reduced wound-breaking strength (P < 0.01) and collagen deposition (P < 0.05). This was paralleled by decreased wound nitrite + nitrate levels (P < 0.001) and wound-inducible nitric oxide synthase expression. Splenic lymphocyte proliferative activity was significantly decreased by 1.0 and 2.0 mg tacrolimus/kg body weight/day (P < 0.05), indicating that the tacrolimus doses used were immunosuppressive. CONCLUSION: Our data show for the first time that tacrolimus impairs wound healing, and this is reflected by diminished wound nitric oxide synthesis.

Busulfan pharmacokinetics in bone marrow transplant patients: is drug monitoring warranted?

Pharmacokinetics were studied in relation to hepatic side-effects in 20 patients (19 adults aged 18-53 years and one child of 11 years) undergoing BMT after conditioning with 1 mg/kg busulfan (every 6 hours for 16 doses). Busulfan was quantitated in plasma samples at 10 time points within the 6 h dosing interval using HPLC before and after dose numbers 1, 2, 5, 13 and 14. For 13 patients data on all five doses are available; for the remaining seven patients three to four doses were studied. Mean maximum concentrations were 1512 ng/ml; mean trough levels for second and subsequent doses were 615 ng/ml. Maxima (Cmax) tended to be lower and times of maxima (Tmax) were later when busulfan was taken with a meal. Correlation of the area under the concentration versus time curve (AUC0-6h) between different doses was low within patients. In several patients problems with compartmental fitting of concentration data were observed mainly caused by the short dosing interval, which made estimates of T1/2 and model derived AUCs unstable. Three patients experienced hepatic veno-occlusive disease; kinetic parameters were not helpful in describing a particulate risk constellation for this subgroup. In our experience, the role of drug monitoring in this setting needs to be defined more clearly.

Detection and separation of the anthrapyrazole CI-941 and its metabolites in serum and urine by high-performance liquid chromatography.

A high-performance liquid chromatographic method using ion-pairing chromatography on reversed-phase C18 material with a mobile phase of acetonitrile-water (19:81, v/v) containing 5 mM 1-pentanesulfonic acid was developed for the detection and separation of the anthrapyrazole CI-941 (I) and its metabolites. After sample clean-up with solid-phase extraction, I and its metabolites were measurable at a wavelength of 491 nm. A detection limit of 5 ng/ml was achievable for I. The dicarboxylic acid derivative and the isomers of the monocarboxylic acid derivative could be separated. Application of the method to a human pharmacokinetic study showed two and four metabolites of I in serum and urine respectively.

Intracellular cytosine arabinoside accumulation and cytosine arabinoside triphosphate formation in leukemic blast cells is inhibited by etoposide and teniposide.

Cytosine arabinoside (ara-C) is one of the most active compounds in the treatment of acute leukemias. In the majority of current protocols ara-C is combined with other cytotoxic agents in an attempt to increase antileukemic activity. The present study investigated the impact of etoposide, teniposide, amsacrine, mitoxantrone, anthracyclines, and asparaginase on the cellular accumulation of ara-C and its intracellular metabolism in order to provide a better rationale for combination therapy. Intracellular accumulation and phosphorylation of ara-C were determined in peripheral blast cells from twenty patients with acute leukemias after exposure to 1 and 10 mumol/l ara-C alone and after preincubation with 1 and 10 micrograms/ml etoposide, 10 and 100 micrograms/ml teniposide, 10 mumol/l amsacrine, 500 ng/ml mitoxantrone (or daunorubicin or doxorubicin) or 10 mumol/l asparaginase. Ara-C accumulation at 10 mumol/l was decreased by 1 microgram/ml etoposide (67 +/- 18% of control), 10 micrograms/ml etoposide (30 +/- 22%), 10 micrograms/ml teniposide (12 +/- 23%), 100 micrograms/ml teniposide (10 +/- 18%), and amsacrine (51 +/- 21%). Intracellular ara-CTP formation was determined at an extracellular concentration of 10 mumol/l and preincubation with these drugs. The intracellular formation of ara-CTP was decreased by 1 microgram/ml etoposide (77 +/- 15% of control), 10 micrograms/ml etoposide (32 +/- 22%), 10 micrograms/ml teniposide (10 +/- 9%), 100 micrograms/ml teniposide (0 +/- 0%), but not by amsacrine. These data indicate that prior exposure to etoposide and teniposide influence ara-C metabolism and possibly cytotoxicity, and thus should not immediately precede ara-C administration in clinical trials.

Evidence for oxidative activation of mitoxantrone in human, pig, and rat.

A new metabolite of mitoxantrone in human, rat, and pig urine has been discovered by means of HPLC. The metabolite has been isolated by preparative HPLC from patient urine and is characterized by tandem mass spectrometry and UV-visible spectroscopy as 8,11-dihydroxy-4-(2-hydroxyethyl)-6-[[2-[(2-hydroxyethyl)amino]ethyl] amino]-1,2,3,4,7,12-hexahydronaphtho-[2,3-f]-chinoxaline-7,1 2-dione. Final structural proof has been obtained by independent synthesis. The new metabolite is a product of the enzymatic oxidation of the phenylenediamine substructure of mitoxantrone. An important biological consequence of the oxidative biotransformation is the possibility of covalent binding to intracellular targets via a highly electrophilic intermediate. Thus, alkylation may be an important mode of action of mitoxantrone. Incubation of mitoxantrone with horseradish peroxidase/hydrogen peroxide in the presence of glutathione led to the formation of two glutathione conjugates of mitoxantrone. Their structures have been elucidated by combination of IonSpray (Sciex, Canada) ionization and tandem mass spectrometry. Radioactive mitoxantrone, synthesized from sodium [14C]cyanide, was used to determine interspecies variations between human and rat. The collected rat urine was analyzed by HPLC using a radioactivity monitoring detector and revealed significant differences in the biotransformation of mitoxantrone in rat compared to human. The main metabolites thus far described in human urine are not observed in rat urine.

Isolation and structure elucidation of urinary metabolites of mitoxantrone.

Three 13C-labeled 1,4-dihydroxy-5,8-bis(2-[(2-hydroxyethyl)amino]-ethyl)amino-9,10- anthracenedione dihydrochloride (mitoxantrone) isotopomers were synthesized to prove the proposed chemical structures of human urinary metabolites by means of nuclear magnetic resonance spectroscopy. After application of labeled mitoxantrone to an anesthetized pig, urine was collected by way of a vesicourethral catheter. The urinary metabolites were isolated by liquid chromatography using a new procedure developed for extraction of mitoxantrone metabolites. Structural elucidation by nuclear magnetic resonance spectroscopy and tandem mass spectrometry confirmed the proposed mono- and dicarboxylic acid structures of the metabolites. High-performance liquid chromatography of native pig urine showed an additional metabolite detected by its UV-visible absorption. The new metabolite was identified as a glucuronic acid conjugate of mitoxantrone by means of nuclear magnetic resonance spectroscopy and tandem mass spectrometry. Incubation with beta-glucuronidase under high-performance liquid chromatography control revealed mitoxantrone as the sole product. Quantitative high-performance liquid chromatography analyses showed that the new urinary metabolite represents the main biotransformational pathway of mitoxantrone in pigs, indicating significant interspecies variation in mitoxantrone biotransformation. Expressed in equivalents of mitoxantrone, the new metabolite amounts to 25% and 31%, respectively, of urinary excreted drug-related material. Extraction of patient urine using the same procedure led to the isolation of pure metabolite B. Tandem mass spectrometric data delivered definitive evidence for the structure of metabolite B as monocarboxylic acid of mitoxantrone.

Unaltered pharmacokinetics after the administration of high-dose etoposide without prior dilution.

The pharmacokinetics of etoposide following a new method of administration was determined. Undiluted etoposide was given at a dose of 30 mg/kg as part an intensified conditioning regimen prior to bone marrow transplantation. A terminal half-life of 3.4 +/- 0.7 h and a volume of distribution of 15.4 +/- 9.6 l were found (n = 8); the AUC was 764 +/- 302 micrograms h ml-1. As compared with those obtained in other pharmacokinetic studies using etoposide diluted in normal saline, our data reflect full systemic bioavailability and unaltered pharmacokinetics. The application of undiluted etoposide makes the therapy easier and less time-consuming and avoids a high fluid volume and a high saline load.

Investigation of potential interaction of ciprofloxacin with cyclosporine in bone marrow transplant recipients.

The effect of the 4-quinolone antimicrobial agent ciprofloxacin on the concentration in plasma and the pharmacokinetics of the immunosuppressive agent cyclosporine was studied in 10 bone marrow transplant recipients. There were no statistically or clinically significant changes in cyclosporine trough concentrations or areas under the concentration-time curve following oral doses of 500 mg of ciprofloxacin every 12 h for 4 days. The data suggest a lack of relevant pharmacokinetic interaction of ciprofloxacin with cyclosporine. There was no indication of an enhanced nephrotoxicity for this drug combination.

Pharmacokinetics and metabolism of mitoxantrone. A review.

Mitoxantrone, a cytotoxic anthracenedione derivative, has given clinical evidence of beneficial activity in breast cancer, lymphoma and leukaemia. Several different mechanisms of action have been suggested to account for this. In addition to intercalation, biological effects such as electrostatic interactions with DNA, DNA-protein cross-links, immunosuppressive activities, inhibition of topoisomerase II, prostaglandin biosynthesis and calcium release have been described. Various methods of drug monitoring in biological fluids and tissues are available: the highest sensitivity has been achieved with high performance liquid chromatography with electrochemical detection, radioimmunoassay and enzyme linked immunosorbent assay. Early pharmacokinetic studies of mitoxantrone in experimental animals using radioactive material showed an extensive tissue distribution and a long terminal plasma half-life. The best fit for the plasma concentration-time curve in humans is achieved in a 3-compartment model. All studies reported a short absorption half-life of between 4.1 and 10.7 minutes, with the distribution phase being between 0.3 and 3.1 hours. In contrast, the values of the terminal half-life are quite variable, ranging from 8.9 hours to 9 days. Differences might be attributed to assay sensitivity, number and weighting of data points beyond 24 hours and coadministration drugs. Many studies showed a very large volume of distribution with sequestration of mitoxantrone in a deep tissue compartment. In autopsy studies, relatively high tissue concentrations have been measured in liver, bone marrow, heart, lung, spleen and kidney. Bile is the major route for the elimination of mitoxantrone, with lesser amounts excreted in the urine. Several metabolites have been separated, 2 of which were identified as the monocarboxylic and dicarboxylic acid derivatives. Mitoxantrone is usually administered by rapid intravenous infusion at 3-weekly intervals; other regimens include continuous infusion, daily repeated doses or weekly administration. In peritoneal carcinosis, the pharmacological advantage of intraperitoneal administration is clear. The optimal regimen for different disease categories with respect to efficacy and side-effects remains to be determined in future clinical trials.

Detection and separation of intracellular 1-beta-D-arabinofuranosylcytosine-5-triphosphate by ion-pair high-performance liquid chromatography.

An ion-pair high-performance liquid chromatographic method, using a reversed-phase C18 column, was developed to provide an isocratic, sensitive, fast and reproducible separation of intracellular 1-beta-D-arabinofuranosylcytosine-5-triphosphate and its measurement at a low limit of 5 pmol by ultraviolet absorbance at 280 nm with a coefficient of variation lower than 10%. A rapid separation is achieved by using a backflush procedure at 16 min and the retention time is 14 min.

Escalating dose regimen of intraperitoneal mitoxantrone: phase I study--clinical and pharmacokinetic evaluation.

Mitoxantrone, a recent anthracenedione derivative, is a potentially useful drug for direct intraperitoneal (i.p.) application because of its high tissue binding and therapeutic index. We have carried out studies to establish maximum tolerated doses as well as pharmacokinetic studies with i.p. mitoxantrone in 21 patients (5 male, 16 female) with gastrointestinal (9), ovarian (6), unknown (2) and other (4) primary cancers and peritoneal carcinomatosis. Increasing doses (10-40 mg/m2) were given i.p. every 4 weeks. Five partial remissions (2-8+ months) and 7 stable disease courses (2-6+ months) were achieved. A reduction or disappearance of ascites was seen in an additional 3 patients. Severe toxicity (leucopenia) was observed in 4 patients only after 35 and 40 mg/m2 i.p. Pharmacokinetic analysis using high performance liquid chromatography yielded the following data: The mean ratio of area under curve peritoneal fluid to plasma was 1109. The peritoneal clearance rate was 680 ml/min and the mean disappearance half life was 13.1 h. Mean urinary excretion within 24 h was 0.42% of the i.p. dose. These data indicate that mitoxantrone is sequestered in the intraperitoneal tissue compartment and only slowly released. Based on the outcome of this phase I study we recommend phase II studies at a dose of 30 mg/m2 i.p., repeated every 3-4 weeks.

A case of accidental cyclosporin overdose with pharmacokinetic analysis.

In a case of oral overdose of cyclosporin we measured plasma concentrations by radioimmunoassay and high pressure liquid chromatography. We observed unchanged pharmacokinetic parameters after the overdose indicating normal renal and hepatic function. Laboratory findings showed no renal or hepatic damage. As toxic symptoms, only emesis and a short, mild drowsiness were noted.

Drug monitoring of quinine by HPLC in cerebral malaria with acute renal failure treated by haemofiltration.

The monitoring of quinine by HPLC in 3 patients suffering from cerebral malaria with acute renal failure and treated by haemofiltration is reported. The recommended dose of quinine in this situation is reduced to 10 to 15 mg.kg-1.day-1. However, in the first patient, when given quinine 10 mg kg-1.day-1 the plasma concentration was mainly below the recommended therapeutic range of 5 to 15 mg/l. In consequence, the dose of quinine in the second patient was elevated to quinine dihydrochloride 15.1 mg.kg-1.day-1 which produced plasma concentrations in the low therapeutic range. In the third patient, an unreduced dose of quinine dihydrochloride 25.7 mg.kg-1.day-1 was employed, resulting in plasma concentrations above 15 mg/l, which is generally assumed to be toxic, although, no sign of acute quinine toxicity was seen. The antimalarial effect in all three patients was satisfactory. Quinine was estimated in the haemofiltrate in two patients and was found to be below the limit of sensitivity (0.25 mg/l). Plasma quinine did not change during or shortly after haemofiltration. It is concluded that in case of acute renal failure in cerebral malaria the dose of quinine should be reduced, but that the common recommendation of 10 to 15 mg.kg-1.day-1 may be too low, and that haemofiltration has no marked influence on the total body clearance of quinine.

Clinical pharmacology of mitoxantrone.

A pharmacokinetic study on mitoxantrone was performed within the framework of a phase II clinical trial. Serum concentrations and urinary excretion were measured using a high-performance liquid chromatography method. Four metabolites were separated in urine and three in serum. The two major metabolites cochromatographed with the synthesized monocarboxylic and dicarboxylic acids of mitoxantrone. Within 48 hours, 4.4% of the administered dose was excreted in urine as mitoxantrone, 0.5% as Metabolite 1, and 0.3% as Metabolite 2. The pharmacokinetic parameters are adequately described by a three-compartment model with a terminal half-life of 214.8 hours and a volume of distribution of 2248 L/m2. The total-body clearance was 217 ml/min/m2 and the renal clearance was 15 ml/min/m2. These results suggest that mitoxantrone is sequestered in a deep tissue compartment and only slowly released.

Detection and separation of mitoxantrone and its metabolites in plasma and urine by high-performance liquid chromatography.

A high-performance liquid chromatographic method using ion-pair chromatography on reversed-phase C18 material was developed. After sample clean-up on XAD columns, mitoxantrone at concentrations below 1 ng/ml in serum and 0.2 ng/ml in urine were measurable with a coefficient of variation of less than 9.3% at a wavelength of 658 nm. Four metabolites were separated in urine. The two major metabolites co-chromatographed with the synthesized mono- and dicarboxylic acid derivatives of mitoxantrone. The method allowed the measurement of mitoxantrone and its metabolites in serum up to more than one week and in urine up to four weeks after administration of the drug.