Comparative HPLC and ELISA studies for CDT isoform characterization in subjects with alcohol related problems. Prospective application in workplace risk-prevention policy.

Serum carbohydrate-deficient transferrin (CDT) is the most specific marker of chronic alcohol abuse so far. The performance of commercial HPLC over the ELISA method for measurement of CDT was evaluated on a series of 105 serum samples obtained from subjects referred to the Toxicology Laboratory of Salvatore Maugeri Hospital for alcohol-related problems. Compared to ELISA, HPLC analysis was more valuable for determining alcohol-related patterns of CDT isoforms and quantifying serum levels of disialotransferrin that better reflect chronic heavy drinking. Other significant advantages of the HPLC method included reproducible separation and easier detection of glycoform types and genetic transferrin variants that are known to cause falsely high or low results in sera examined by immunoassay. Current scientific evidence indicates that disialotransferrin is the target analyte for CDT determination and HPLC the current CDT analysis reference method. Systematic studies for early assessment of excessive alcohol intake or abuse of alcoholic substances in workers are recommended by the Italian legislation in accordance with the European Alcohol Action Plan (EAAP) launched by the WHO Regional Committee for Europe. These studies are advisable given their potential role in preventing negative effects of alcohol abuse in workplace. A research strategy combining CDT and other laboratory markers with questionnaire and physician interview is recommended for examining subjects with alcohol related problems and the diagnosis of alcoholism. This approach can be applied for alcohol abuse in workplace surveillance.

Pharmacokinetic and pharmacologic properties of delapril, a lipophilic nonsulfhydryl angiotensin-converting enzyme inhibitor.

Delapril is a carboxy-alkyl-dipeptide mainly converted in animals and humans to an active diacid derivative (M-I), which in turn is converted to an active 5-hydroxy-indane diacid (M-III). In humans these metabolites are excreted in the urine. The presence of the indanyl-glycine moiety gives delapril a high lipophilicity, greater than several other angiotensin-converting enzyme (ACE) inhibitors, such as captopril and enalapril. Due to its greater lipophilicity, delapril has been shown to exert a more effective inhibition of vascular ACE than captopril and enalapril, both in vitro and in vivo. The activity of delapril on tissue ACE also lasts longer than on the circulating enzyme. At doses ranging from 1-10 mg/kg orally, delapril exerts a marked and long-lasting antihypertensive action in various experimental models of hypertension. The blood pressure reduction has been shown to be accompanied by suppression of angiotensin II release from the vascular wall. In stroke-prone spontaneously hypertensive rats (SHR-SP) and in SHR with chronic renal failure, besides reducing hypertension, delapril significantly improves survival rate and prevents the development of stroke, cardiac hypertrophy, and renal sclerosis. The ability of delapril to reduce hypertrophy in vascular and cardiac tissue has been demonstrated in both in vitro and in vivo experiments.

Phase I metabolism of ganstigmine. Rat, dog, monkey and human liver microsomal extracts investigated by liquid chromatography electrospray tandem mass spectrometry.

Ganstigmine, a new acetylcholinesterase inhibitor, was incubated with rat liver microsomes and the resulting metabolites were identified by high-performance liquid chromatographic/mass spectrometric (HPLC/MS) and HPLC/MS/MS analyses. The results showed the formation of eight main metabolites, among which geneseroline and molecules corresponding to mono-hydroxylated, demethylated and reduced ganstigmine. The metabolic profile drawn for humans, dog and monkey showed a pattern very similar to that of rat: only in the case of liver dog microsomes higher amounts of geneseroline and of a metabolite identified as demethylated and reduced drug were detected.

Comparison of the bioavailability and systemic effects of beclometasone dipropionate suspension for nebulization and beclometasone dipropionate via a metered-dose inhaler after single-dose administration in healthy male volunteers.

Pharmacokinetic properties of a drug, and selection and correct usage of an appropriate delivery device, are factors that can affect the outcome of inhaled therapyThe use of nebulization can overcome problems that are associated with other delivery systems used for inhalation therapyThe objective of this open, randomized, single-dose study was to compare the systemic exposure and safety of beclometasone dipropionate (BDP) suspension for nebulization with BDP via metered-dose inhaler (MDI) in healthy subjects. Following a run-in period to assess basal 24-h serum cortisol levels and cortisol urinary excretion, 12 healthy males were administered BDP 1,600 microg given via MDI and were then randomized to receive a single dose of either 1,600 microg (n = 6) or 3,200 microg BDP (n = 6) suspension for nebulization given via a nebulizer Results with respect to systemic exposure to beclometasone-17-monopropionate (B17MP) (the active metabolite of BDP) and systemic effects on the hypothalamic-pituitary-adrenal (HPA) axis were determined by evaluation of a number of pharmacokinetic parameters for plasma B17MP and serum and urinary cortisol, respectively. A statistically significantly greater peak plasma concentration (Cmax) of B17MP was reported with BDP via MDI (1,587 pg ml(-1)) compared with BDP 1,600 microg (455 pg ml(-1)) and BDP 3,200 microg suspensions for nebulization (758 pg ml(-1)), and was achieved more rapidly (Tmax) (1.3 h, 3 h, and 2.5 h, respectively). In addition, elimination half-life (t 1/2(el)) was statistically significantly shorter with BDP via MDI (4.6 h) than with both dosages of BDP suspensions for nebulization (7.4 h and 6.3 h with 1600 microg and 3,200 microg, respectively), as was mean residence time (MRT) (5.4 h, 11.1 h, and 10.0 h, respectively). Total systemic exposure to B17MP (as determined by the area under the concentration-time curve: AUCinfinity) was comparable for BDP via MDI (6,883 pg ml(-1) h(-1)) and BDP 3,200 microg suspension for nebulization (8,201 pg ml(-1) h(-1)), but significantly greater than with BDP 1,600 microg suspension for nebulization (4,870 pg ml(-1); P < 0.05 vs BDP via MDI). All treatments were well tolerated, and no significant differences were found between them with respect to the serum or urinary cortisol pharmacokinetic parameters assessed. In conclusion, the results of this study demonstrate that BDP suspension for nebulization 3,200 microg given via a nebulizer and BDP 1,600 microg given via an MDI are equivalent in terms of systemic exposure to B17MP and systemic effects on the HPA axis, with BDP suspension for nebulization having a potentially more prolonged activity. It confirms that use of a double dose of BDP suspension for nebulization administered by nebulizer compared with BDP given via metered-dose inhalation is justified and poses no risk with regard to safety.

Modulite technology: pharmacodynamic and pharmacokinetic implications.

In the drive to replace chlorofluorinated hydrocarbons (CFCs) by alternative more environmentally friendly propellants in pressurized metered dose inhalers (pMDIs), Chiesi has developed new inhalers using Modulite technology. The aim was to obtain CFC-free pMDIs which are equivalent, in terms of safety and efficacy, to the previous CFC devices at the same dose. When beclometasone dipropionate (BDP) and budesonide Modulite formulations were compared to the equivalent CFC products there was no significant difference in morning serum cortisol or urinary cortisol excretion, at the maximum recommended daily dose (2000 micrograms or 1600 micrograms respectively). Single dose pharmacokinetic studies in both healthy volunteers and asthmatic patients compared systemic exposure (B17MP levels) for BDP-CFC with BDP Modulite and extrafine BDP-HFA (QVAR). B17MP levels for BDP-CFC and BDP Modulite were comparable, but substantially less than that seen with extrafine BDP-HFA. After 6 weeks of treatment in asthmatic patients, B17MP AUC after inhalation of BDP (1000 micrograms twice-daily) from BDP Modulite was comparable with that obtained after BDP-CFC (Becloforte). Plasma profile of BDP and B17MP were similar after inhalation from BDP Modulite with standard actuator or delivered via a spacer, suggesting that pulmonary delivery of BDP to the lung is similar with both actuators.

Influence of layer position on in vitro and in vivo release of levodopa methyl ester and carbidopa from three-layer matrix tablets.

A versatile oral controlled release system for the simultaneous delivery of levodopa methyl ester and carbidopa, consisting of a three-layer matrix tablet, has been studied and developed. Each individual layer of the matrix exhibited a different release mechanism, i.e. the first layer was swellable (S), the second one was erodible (E) and the third one was disintegrating (D). The three layers have been assembled in the monolithic matrix in different relative positions. It was found that in the monolith the three layers could interact, producing in vitro release profiles depending on their relative position. The monoliths having the configurations DSE and SDE were administered to human volunteers in order to determine the plasma profiles. The pharmacokinetic data showed a significant difference between the early time plasma curves: the monolith DSE, having the fast release profile, gave rise to a rapid appearance of a high levodopa plasma level, whereas the slower releasing monolith SDE produced a smoothed plasma concentration profile.

Single dose pharmacokinetics of manidipine in hepatic impaired patients and healthy controls.

The pharmacokinetics and safety of a single oral dose of 20 mg manidipine dihydrochloride have been studied in 8 patients with mild to moderate hepatic impairment (grade A or B in Child's classification, or score < or = 7 in Pugh's modification of Child's classification), and in 12 healthy subjects. They received one 20 mg manidipine dihydrochloride tablet with 100 ml of tap water after a standard breakfast. Manidipine was determined using HPLC with electrochemical detection from plasma samples taken up to 24 or 36 h after dosing. The medication was well tolerated. A trend toward higher Cmax, AUC, and MRT was observed in patients with a more severe hepatic impairment, as a consequence of reduction in the liver metabolic function. Patients with grade A hepatic impairment did not exhibit significantly altered pharmacokinetics with respect to healthy subjects, while grade B impairment patients had significantly higher AUC and MRT. Tmax values pointed to reduced absorption rate in patients compared to healthy subjects; the changes were more evident in grade B than grade A patients, although statistical significance was not reached. The reduction in absorption rate in grade B patients is probably related to their higher mean age, since this effect has been reported for manidipine. The pharmacokinetics of manidipine seem only modified in patients with a certain degree of hepatic impairment (at least Pugh grade 6 and Child grade B); therefore, adaptation of the dosing regimen does not seem to be generally recommendable, but should be modulated according to the liver status of the patient.

Steady-state pharmacokinetics of ipriflavone and its metabolites in patients with renal failure.

Ipriflavone is a recently introduced anti-osteoporotic agent extensively metabolized to four major metabolites (M1, M2, M3, M5). Its pharmacokinetics, after repeated doses of the drug, was investigated in patients with renal failure and compared with those of healthy volunteers. Plasma levels at steady-state of the unchanged drug, and its metabolites M1, M2 and M5 were higher in the patient group, with the presence of secondary peaks, which could be explained by the biliary excretion of the substances. Evaluation of renal elimination in patients in respect to healthy volunteers was performed by a "relative renal elimination index". The index decreased with the increase of renal disease mainly when the renal failure was moderate to severe.

Pharmacokinetic study on piroxicam at the steady-state in elderly subjects and younger adults after administration of piroxicam beta-cyclodextrin.

The age effect on the kinetics of piroxicam at the steady-state after oral administration of piroxicam beta-cyclodextrin was evaluated. The mean plasma concentration of piroxicam at the steady-state was significantly higher in elderly subjects (9.30 +/- 0.69 micrograms/ml mean +/- s.e.) than in younger adults (6.24 +/- 0.58 micrograms/ml mean +/- s.e.). Even though the distribution volume tended to be lower in elderly subjects (106.37 ml/kg), it did not show substantial differences in the two groups whereas the correlation between clearance and age was significant (p less than 0.05). It is therefore confirmed that piroxicam beta-cyclodextrin has the same pharmacokinetic behaviour at the steady-state in elderly subjects, as the active principle in a non-complexed form.

Comparative bioavailability of two oral formulations of ipriflavone in healthy volunteers at steady-state. Evaluation of two different dosage schemes.

Ipriflavone (IP) is an isoflavone derivative with antiosteoporotic activity. This drug is extensively metabolized in humans and only negligible concentrations of unchanged IP can be detected in plasma. Metabolites M1 and M5 are predominant, while met abolites M2 and M3 are detected in minor amounts. The aim of this study was to compare the bioavailability of IP and its metabolites M1, M2, M3, and M5 at steady-state after administration of 200 mg tablets three times daily and 300 mg Scherer capsules twice daily during meals. IP plasma levels were below the limit of quantitation in 6 subjects out of 12 after administration of IP 200 mg tablets. On the other hand, after administration of the Scherer capsules IP plasma levels were quantifiable in all the volunteers. As regards IP metabolites, a mean increase in bioavailability, equal to 35%, was observed after administration of the Scherer capsules. Plasma level fluctuations, reflecting changes in absorption rate at steady-state, remained unvaried. The good bioavailability and fluctuation indexes of the Scherer capsules permit a simplification of the dosage scheme, reducing the daily administrations from three times to twice daily, thus improving the patients' compliance. In clinical practice this characteristic is not negligible, con sidering the mean age of the patients and the long-term treatment. Due to the high therapeutic index of IP, the increase in bioavailability does not cause any risk of accumulation or overdosage.

Bioavailability and pharmacokinetics of a fixed combination of delapril/indapamide following single and multiple dosing in healthy volunteers.

The study objective was to obtain detailed information on the bioavailability and pharmacokinetics of the new fixed combination of delapril and indapamide following single and multiple dosing. For this reason, the study was performed in two parts, separated by a medication-free period of at least 7 days. In the single dose part, one tablet, containing 30 mg delapril and 2.5 mg indapamide, was administered to 12 male volunteers; in the multiple dose part, the volunteers received one tablet of the test preparation, once daily over 7 days. Following single and on the last day of the multiple dosing regimen, blood samples were withdrawn and serum concentrations of delapril and its metabolites M1, M2 and M3 and whole blood concentrations of indapamide were quantified by means of HPLC methods. In addition, urine samples were collected following single and multiple dosing for evaluation of the cumulative amount of delapril and its metabolites M1-M3 excreted in urine. For the area under the curve, calculated from time 0 to infinity (AUC(0-infinity)) the study revealed, following single dosing, mean values of delapril and its metabolites M1, M2 and M3 of 281, 2178, 739 and 716 h.ng/ml, respectively; for indapamide the mean value was 1597 h.ng/ml. The corresponding mean values found after multiple dose administration were 272, 2071, 857 and 598 h.ng/ml for delapril and its metabolites, respectively and 1536 h.ng/ml for indapamide. Evaluation of the cumulative amount of delapril and its metabolites M1-M3 excreted in urine (Ae) demonstrated mean values following single dosing (observation period 36 h) of 705, 4521, 454 and 4203 micrograms, respectively; the corresponding values after multiple dose administration (observation period 24 h) of the test preparation were 655, 4679, 469 and 4801 micrograms, respectively. The most important pharmacokinetic parameters AUC(0-infinity) and Ae were statistically compared by analysis of variance (ANOVA) and 90% confidence intervals were calculated. It may be concluded from the results of this study, that the bioavailability and pharmacokinetic parameters of the test preparation after single dosing and after multiple doses correspond well. The undesired side effects observed are known to occur after administration of the test preparation. The occurrence was a little more frequent after multiple dose application in comparison with the single dose administration.

Food effect on the oral bioavailability of Manidipine: single dose, randomized, crossover study in healthy male subjects.

The effect of food on the oral bioavailability of a manidipine 20 mg tablet was studied after a single administration in 12 male healthy subjects. The clinical trial was conducted as an open, randomised, crossover study. In two different administration sessions, the subjects received a 20 mg manidipine tablet either in the fasting state or after a standardized breakfast. Plasma samples were collected before and at different times after each administration for up to 24 h. The concentrations of manidipine and its pyridine metabolite (M-XIII metabolite) were determined by HPLC with coulometric detection. The tolerability of manidipine was good. Only two cases of mild headache, one with each treatment, were reported. Food significantly improved the absorption, with an increase in AUC from 19.1 to 27.2 ng.h/ml (geometric mean, p<0.01) but did not modify the rate of absorption (tmax unchanged, median = 1.5 h). Peak plasma concentration was also increased (from 6.2 to 7.8 ng/ml), but the difference was not statistically significant (p=0.18). Other pharmacokinetic parameters (apparent elimination half-life and mean residence time) remained unchanged. The increase in bioavailability of manidipine administered with food is related to its high lipophilicity and may be explained through a solubilization effect produced by food and bile secretions.

14C-NaVP and 14C-PEV repeated dose study in rat. Pharmacokinetic study in rats after repeated oral administrations of 14C-valproic acid sodium salt and 14C-valproic acid pivaloyl oxymethyl ester.

The absorption, excretion and tissue distribution of radioactivity after repeated oral equimolar doses of 14C-valproic acid sodium salt (NaVP) or 14C-valproic acid pivaloyl oxymethyl ester (PEV) was investigated in male rats treated once a day for 14 consecutive days. The 14th day plasma time-course of radioactivity after PEV administrations was characterised by a slow absorption rate with a delayed peak (tmax 2 h, Cmax 7.52 +/- 1.35 microg eq./ml), followed by a plateau lasting up to 8 h. After NaVP treatment, the main peak of radioactivity was observed 0.5 h after administration (Cmax 8.30 +/- 1.26 microg eq./ml) followed by a secondary peak due to biliary enterohepatic recycling. Starting from 4 h onwards, radioactivity levels after PEV treatment were higher than those after NaVP (AUCtau = 113.3 h.microg eq./ml after PEV vs 71.9 h.microg eq./ml after NaVP), but concentrations declined with similar terminal half-lives (52.8 h for PEV and 49.7 h for NaVP). Radioactivity recovered (0-432 h interval) in urine accounted for 79.3% (PEV) and 56.1% (NaVP) while, in faeces accounted for 9.1% (PEV) and 26.1% (NaVP) of total administered dose (14 days). The difference is attributable to a higher excretion of radioactivity in the bile for NaVP. The missing fraction in the total radioactivity balance is probably excreted in expired air, as observed in single dose studies. Radioactivity excreted in bile (0-8 h interval of the last 14th day) accounted for 5.1% (NaVP) and 0.23% (PEV) of the total administered dose (14 days). A possible explanation of this difference may be a different metabolism pattern for the two compounds. The negligible biliary excretion observed after PEV administration is probably due to an inhibition of the glucuronation of valproic acid (or other metabolites) caused by the pivalic acid. Due to the presence of the enterohepatic recycle, the radioactivity levels in intestine, 0.5 and 2 h after administration, were higher after NaVP administration. According to higher plasma levels, the radioactivity concentrations in liver, kidneys and some fat tissues were found to be slightly higher after PEV administration. At 120 h after the last treatment of both compounds, relevant tissue concentrations were observed in mesenteric lymphnodes, perirenal and brown fat. The tissue-plasma radio activity ratio appeared quite similar for the two compounds.

Systemic exposure to inhaled beclometasone/formoterol DPI is age and body size dependent.

AIM: Prescription of inhaled corticosteroids to children with asthma is recommended at half the nominal dose of adults in order to reduce the risk of systemic side effects. However, there is a lack of pharmacokinetic trials supporting such dose reduction regimens. Therefore, we aimed to compare the systemic exposure to the active ingredients of a fixed dose combination of beclometasone-dipropionate (BDP) and formoterol after dry powder inhaler (DPI) administration in children, adolescents and adults. METHODS: The pharmacokinetic profiles of formoterol and beclometasone-17-monopropionate (B17MP; active metabolite of BDP) were evaluated over 8 h from two independent studies comprising children (6-11yrs, n = 27), adolescents (12-17 yrs, n = 28) and adults (≥18 yrs, n = 30) receiving a single, fixed dose of BDP/formoterol (children: 200 µg/24 µg, adolescents and adults: 400 µg/24 µg) via DPI. RESULTS: The systemic exposure (AUC) for children versus adults was almost doubled for formoterol and similar for B17MP despite the halved BDP dose administered in children. In adolescents the AUC for formoterol and B17MP were approximately one third higher than in adults for both compounds. Upon normalization for the BDP/formoterol dose in the three populations the AUC and peak concentration (C(max)) correlated inversely with age and body surface area of the patients (r ≤ -0.53; p < 0.0001). CONCLUSION: The systemic exposure to the active ingredients of BDP/formoterol administered as DPI correlates inversely with age and body size suggesting that dry powder dosage regimens should be adjusted for age and body size to avoid high systemic drug levels in children.

Comparative pulmonary toxicity assessment of pristine and functionalized multi-walled carbon nanotubes intratracheally instilled in rats: morphohistochemical evaluations.

Increasing interest in safety evaluation of carbon nanotubes (CNTs) has risen in relation to their wide applications, together with the evidence of their cytotoxic effects. It has been shown that chemical functionalization extends the applications of CNTs, conferring them new functions that cannot otherwise be acquired by pristine CNTs, but also impacts on biological response to CNTs, modifying their toxicological profile. We assessed the onset of pulmonary toxic effects caused by pristine MW-CNTs and functionalized MW-NH2 or MW-COOH, 16 days after intratracheal instillation (1 mg/kg b.w.); major endpoints tested included (i) histopathology of lung (Haematoxylin/Eosin Staining), (ii) apoptotic/proliferating features examined by TUNEL and PCNA immunostaining, and (iii) presence/distribution of (1) Transforming Growth Factor-beta1 (TGFß1), (2) Interleukin-6 (IL-6) and (3) Collagen (Type I) investigated by immunochemical methods, as markers of lung toxicity, inflammation, and fibrosis, respectively. Lung histopathology from exposed animals showed dark, particulate-laden macrophages, reflecting carbon nanomaterial engulfing, both at alveolar and bronchiolar levels, after treatment with all the tested CNTs. Alteration of lung architecture was also observed in several areas showing collapsed thick-walled alveoli and the presence of micro-haemorrhagic foci. TUNEL and PCNA, indicative of apoptosis and cell proliferation respectively, showed a significant increase of immunopositive cells at bronchiolar, alveolar and macrophagic levels, as expression of an improved cellular turnover. Increased immunoreactivity for pulmonary TGFß1 and IL-6 was observed in treated rats, particularly in bronchiolar areas, collapsed alveoli and at stromal level, while evident changes for collagen were not detected. Taken together these findings demonstrated the general pulmonary toxicity coupled with inflammatory response after in vivo exposure to CNTs, without overt signs of fibrosis and granuloma formation, irrespectively of nanotube functionalization.

Comparative in vitro and ex-vivo myelotoxicity of aflatoxins B1 and M1 on haematopoietic progenitors (BFU-E, CFU-E, and CFU-GM): species-related susceptibility.

Haemato- and myelotoxicity are adverse effects caused by mycotoxins. Due to the relevance of aflatoxins to human health, the present study, employing CFU-GM-, BFU-E- and CFU-E-clonogenic assays, aimed at (i) comparing, in vitro, the sensitivity of human vs. murine haematopoietic progenitors to AFB1 and AFM1 (0.001-50microg/ml), (ii) assessing whether a single AFB1 in vivo treatment (0.3-3mg/kgb.w.) alters the ability of murine bone marrow cells to form myeloid and erythroid colonies, and (iii) comparing the in vitro with the in vitro ex-vivo data. We demonstrated (i) species-related sensitivity to AFB1, showing higher susceptibility of human myeloid and erythroid progenitors (IC(50) values: about 4 times lower in human than in murine cells), (ii) higher sensitivity of CFU-GM and BFU-E colonies, both more markedly affected, particularly by AFB1 (IC(50): 2.45+/-1.08 and 1.82+/-0.8microM for humans, and 11.08+/-2.92 and 1.81+/-0.20microM for mice, respectively), than the mature CFU-E (AFB1 IC(50): 12.58+/-5.4 and 40.27+/-6.05microM), irrespectively of animal species, (iii) regarding AFM1, a species- and lineage-related susceptibility similar to that observed for AFB1 and (iv) lack of effects after AFB1 in vivo treatment on the proliferation of haematopoietic colonies.