Determination of deferasirox plasma concentrations: do gender, physical and genetic differences affect chelation efficacy?

OBJECTIVES: Bioavailability of deferasirox (DFX) is significantly affected by the timing of administration relative to times and to composition of meals. Its elimination half-life is also highly variable - in some patients as a result of gene polymorphisms. Understanding whether deferasirox plasma levels are related to specific characteristics of patients could help physicians to devise a drug regimen tailored the individual patient. METHODS: We analyzed deferasirox plasma concentrations (CDFX ) in 80 patients with transfusion-dependent anemias, such as thalassemia, by a high performance liquid chromatography (HPLC) assay. We used a multivariate linear regression model to find significant associations between CDFX and clinical/demographical characteristics of patients. All patients were genotyped for UGT1A1. RESULTS: Fifty-six patients were female, 24 were male, the great majority (88%) affected by ß-thalassemia, and 15 were children and adolescents. No statistical correlation was detectable between CDFX and DFX dose (P = 0.6). Age, time from last drug intake to blood sampling, and ferritin levels in the 6 months before study initiation were significantly and inversely associated with CDFX in univariate analysis. In the multivariate analysis, the only two factors independently and inversely associated with CDFX levels were time from last drug intake to blood sampling and ferritin levels (P = 0.006). A significant inverse correlation (P = 0.03) was observed between CDFX and UGT1A1*28 gene polymorphism, but only in patients with levels of lean body mass (LBM) below the median (P for interaction = 0.05). CONCLUSIONS: The results could indicate that a higher plasma DFX concentration could be associated with greater chelation efficacy. As a correlation between dose and CDFX was not demonstrated, it seems useful to monitor the concentrations to optimize and determine the most appropriate dose for each patient. Interesting results emerged from the analysis of genetic and physical characteristics of patients: LBM was a borderline significant effect modifier of the relationship between UGT1A1 polymorphisms and CDFX . Individual patient-tailored dosing of DFX should help to improve iron chelation efficacy and to reduce dose-dependent drug toxicity.

HPLC determination of malondialdehyde as biomarker for oxidative stress: application in patients with alcohol dependence.

BACKGROUND: Chronic and excess ethanol exposure causes an increase in generation of free radicals which attack the polyunsaturated fatty acids in membranes to create lipid peroxides such as malondialdehyde (MDA) which is widely used as an indirect biomarker of oxidative stress. METHODS: In this study a sensitive and reproducible high performance liquid chromatography (HPLC) method for measurement of MDA was applied in a group of alcohol dependent patients who underwent detoxification treatment. RESULTS: Compared to the control group, mean MDA concentrations at baseline were significantly higher in alcohol dependent patients (1.28 +/- 0.58 microM vs. 0.9 +/- 0.21 microM; p < 0.02). However, MDA levels remained almost unchanged after three weeks of detoxification treatment (1.28 +/- 0.58 microM vs. 1.38 +/- 0.61 microM; p > 0.05). Among alcoholic patients, the MDA plasma concentration in smokers was higher than in non smokers both at baseline and after three weeks. CONCLUSIONS: The failure to reduce the levels of MDA after 3 weeks of detoxification treatment suggests that patients with chronic alcohol dependence have difficulty in compensating for alcohol-induced excessive production of free radicals. Furthermore, the possibility of cigarette smoke affecting the MDA plasma concentration cannot be ruled out.

Studies on genotoxicity and carcinogenicity of antibacterial, antiviral, antimalarial and antifungal drugs.

This review provides a compendium of retrievable results of genotoxicity and animal carcinogenicity studies performed of antibacterial, antiviral, antimalarial and antifungal drugs of long-term or intermittent frequent use. Of the 48 drugs considered, 9 (18.75%) do not have retrievable data, whereas the other 39 (81.25%) have at least one genotoxicity or carcinogenicity tests result. Of these 39 drugs, 24 tested positive in at least one genotoxicity assay and 19 in at least one carcinogenicity assay; 14 of them gave a positive response in both at least one genotoxicity assay and at least one carcinogenicity assay. Concerning the predictivity of genetic toxicology findings for the results of long-term carcinogenesis assays, of 23 drugs with both genotoxicity and carcinogenicity data: 2 (8.7%) were neither genotoxic nor carcinogenic, 2 (8.7%) tested positive in at least one genotoxicity assay but were non-carcinogenic, 4 (17.4%) tested negative in genotoxicity assays but were carcinogenic, and 15 (65.2%) gave a positive response in at least one genotoxicity assay and in at least one carcinogenicity assay. Only 18 (37.5%) of the 48 drugs examined had all data required by present guidelines for testing of pharmaceuticals, but a fraction of them (49%) were developed and marketed prior to the present regulatory climate. In the absence of compelling indications, the prescription of the 19 drugs that are animal carcinogens should be avoided.

Update of carcinogenicity studies in animals and humans of 535 marketed pharmaceuticals.

This survey is a compendium of information retrieved on carcinogenicity in animals and humans of 535 marketed pharmaceuticals whose expected clinical use is continuous for at least 6 months or intermittent over an extended period of time. Of the 535 drugs, 530 have the result of at least one carcinogenicity assay in animals, and 279 (52.1%) of them gave a positive response in at least one assay. Only 186 drugs (34.8%) have retrievable information on carcinogenicity in humans, and 104 of them gave to a variable extent evidence of a potential carcinogenic activity. Concerning the correlation between results obtained in animals and epidemiological findings, 58 drugs gave at least one positive result in carcinogenicity assays performed in animals and to a variable extent displayed evidence of carcinogenicity in humans, but 97 drugs tested positive in animals and were noncarcinogenic in humans or vice versa. Our findings, which are in agreement with previous studies, indicate that the evaluation of the benefit/carcinogenic risk ratio should be always made in prescribing a drug.

Exposure to asbestos: correlation between blood levels of mesothelin and frequency of micronuclei in peripheral blood lymphocytes.

Inhalation of asbestos, a mineral extensively used in a variety of applications, is strongly associated with malignant mesothelioma (MM), a fatal cancer of the pleura. Soluble mesothelin-related peptides (SMRP) are a promising biomarker suggested for the screening of MM in healthy asbestos-exposed subjects. In the present study a comparison of micronucleus (Mn) frequencies in peripheral blood lymphocytes (PBL) between 44 asbestos-exposed and 22 control individuals has been performed, and the correlation with serum SMRP has been examined. SMRP levels were found to be significantly higher in subjects exposed to asbestos and in their various subgroups than in controls. Concerning micronucleated lymphocytes, a statistically significant difference from controls was seen in the percentages of both micronucleated mononucleated lymphocytes (MnMNL) and micronucleated binucleated lymphocytes (MnBNL), but the difference was markedly higher for the percentage of micronucleated polynucleated lymphocytes (MnPNL). With respect to the correlation between the frequency of the three types of micronucleated lymphocytes and serum-SMRP values of asbestos-exposed subjects, it was statistically significant for MnMNL, but not for MnBNL and MnPNL.

Genotoxicity and carcinogenicity studies of antihistamines.

This review provides a compendium of the results of genotoxicity and carcinogenicity assays performed on marketed antihistamines. Of the 70 drugs examined, 29 (41.4%) have at least one genotoxicity and/or carcinogenicity test result: 12 tested positive in at least one genotoxicity assay, six in at least one carcinogenicity assay, and four gave a positive response in both at least one genotoxicity assay and at least one carcinogenicity assay. Of 19 drugs with both genotoxicity and carcinogenicity data, eight were neither genotoxic nor carcinogenic, two were carcinogenic in at least one sex of mice or rats but tested negative in genotoxicity assays, five tested positive in at least one genotoxicity assay but were non-carcinogenic, and four gave a positive response in at least one genotoxicity assay and in at least one carcinogenicity assay. Only 12 (17.1%) of the 70 drugs examined have all data required by present guidelines for testing of pharmaceuticals, but it should be considered that a large fraction of them were developed and marketed prior the present regulatory climate.

Genotoxicity and carcinogenicity testing of pharmaceuticals: correlations between induction of DNA lesions and carcinogenic activity.

This survey is a compendium of the results of DNA lesions assays (DNA adducts, DNA strand breaks, DNA repair synthesis) and of the results of carcinogenicity assays of 146 pharmaceuticals. Of these drugs, 55 (37.7%) tested negative in both DNA lesions assay(s) and in carcinogenicity assay(s); 65 (44.5%) tested negative in DNA lesions assay(s), but gave a positive response in at least one carcinogenicity assay; 6 (4.1%) tested positive in at least one DNA lesions assay, but negative in carcinogenicity assay(s); 20 (13.7%) tested positive in at least one DNA lesions assay and in at least one carcinogenicity assay. Concerning the predictivity of DNA lesions assays findings for the results of long-term carcinogenesis assays performed in mice, rats or other species, concordance was found to exist for the 46.2% of pharmaceuticals in the case of DNA adducts, for 63.1% in the case of DNA strand breaks, and for 47.3% in the case of DNA repair synthesis (UDS).

Co-Occurring Psychiatric and Substance Use Disorders: Clinical Survey Among a Rural Cohort of Italian Patients.

PURPOSE: Dual diagnosis (DD) is the co-occurrence of both a mental illness and a substance use disorder (SUD). Lots of studies have analysed the integrated clinical approach, which involves both psychiatry and toxicology medical experts. The purpose of this study is to analyse the socio-demographic characteristics and treatment strategies of patients with DD in a rural area of Italy. PATIENTS AND METHODS: Clinical data of 750 patients were collected in 2016 through the analysis of health plan records. RESULTS: The rate of co-occurring disorders is highly variable among people with SUD. In the considered area, patients with DD are 24%, of these only 46.1% have been treated with an integrated clinical program. Moreover, this percentage is further reduced (35.8%) if only patients with heroin use disorder are considered. CONCLUSION: A comprehensive revision of DD treatment is needed, especially for people suffering from heroin use disorder and living in remote areas. Meticulous data analysis from other addiction health services of rural areas could be necessary to identify a science-based clinical intervention.

Combined evaluation of genotype and phenotype of thiopurine S-methyltransferase (TPMT) in the clinical management of patients in chronic therapy with azathioprine.

Background The thiopurine S-methyltransferase (TPMT)/azathioprine (AZA) gene-drug pair is one of the most well-known pharmacogenetic markers. Despite this, few studies investigated the implementation of TPMT testing and the combined evaluation of genotype and phenotype in multidisciplinary clinical settings where patients are undergoing chronic therapy with AZA. Methods A total of 356 AZA-treated patients for chronic autoimmune diseases were enrolled. DNA was isolated from whole blood and the samples were analyzed for the c.460G>A and c.719A>G variants by the restriction fragment length polymorphism (RFLP) technique and sequenced for the c.238G>C variant. The TPMT enzyme activity was determined in erythrocytes by a high-performance liquid chromatography (HPLC) assay. Results All the patients enrolled were genotyped while the TPMT enzyme activity was assessed in 41 patients. Clinical information was available on 181 patients. We found no significant difference in the odds of having adverse drug reactions (ADRs) in wild-type patients and variant allele carriers, but the latter had an extra risk of experiencing hematologically adverse events. The enzyme activity was significantly associated to genotype. Conclusions TPMT variant allele carriers have an extra risk of experiencing hematologically adverse events compared to wild-type patients. Interestingly, only two out of 30 (6.6%) patients had discordant results between genotype, phenotype and onset of ADRs.

Formation of DNA-damaging N-nitroso compounds from the interaction of calcium-channel blockers with nitrite.

A large number of drugs have been shown to react with nitrite to give genotoxic-carcinogenic N-nitroso compounds (NOC). However, the majority of drugs remain to be examined in this respect, among which calcium-channel blockers, all theoretically nitrosatable and widely used in the therapy of hypertension and other cardiovascular diseases. In this preliminary investigation, seven calcium-channel blockers have been examined either for their in vitro nitrosation according to the procedure recommended by the WHO, or for occurrence of liver DNA fragmentation, as detected by the Comet assay, in rats given by gavage 1/2 LD50 of the drug and 80 mg/kg of sodium nitrite. After 6h incubation the yields of NOC formed in vitro from nicardipine, nifedipine, nimodipine and nitrendipine ranged from 37 to 45% of the theoretical one, whereas the yields of NOC formed from diltiazem, gallopamil and verapamil ranged from 2 to 5%. In vivo, as compared with the effect of the same dose of the drug alone, a significant increase of both tail length and tail moment, indicative of an increased frequency of DNA single-strand breaks and alkali-labile sites, was produced in rat liver DNA by the administration with nitrite of gallopamil, nifedipine, nimodipine and nitrendipine, the ratio [tail length of drug+NaNO(2)/tail length of drug alone] being 3.2 for nimodipine, 3.1 for gallopamil 2.2 for nifedipine, and 2.1 for nitrendipine. Even if present, the increase in the degree of DNA fragmentation did not reach the statistical significance in rats given with nitrite nicardipine, diltiazem and verapamil. Further studies should be performed to investigate the formation of NOC in conditions simulating those occurring in the stomach of humans treated with a therapeutic dose, and to quantitate their genotoxic potency.

Measurement of sorafenib plasma concentration by high-performance liquid chromatography in patients with advanced hepatocellular carcinoma: is it useful the application in clinical practice? A pilot study.

Pharmacokinetics and dose-finding studies on sorafenib were conducted on heterogeneous groups of patients with solid tumors. Portal hypertension, gut motility impairment and altered bile enterohepatic circulation may explain different sorafenib toxicological profile in cirrhotic patients. This study evaluated sorafenib plasma concentration in a homogeneous group of cirrhotic patients with hepatocellular carcinoma (HCC). Sorafenib concentrations were determined by liquid chromatography in 12 consecutive patients. Data have been evaluated by the generalized estimating equations method (p value statistical level was set at α = 0.05). (1) There were not significant differences between sorafenib concentrations in patients who tolerate the full dose versus patients with reduced dose due to toxicity; (2) the average sorafenib concentrations measured 3 h after the morning dosing were lower than those measured 12 h after the evening dosing (p = 0.005); (3) sorafenib concentrations decrease overtime (p < 10(-4)); (4) it has been found an association between the development of severe adverse reactions and sorafenib concentrations (p < 10(-5)). The relationship between dose and concentration of sorafenib in HCC patients is poor and not clinically predictable, confirming the variability both in the maximum tolerated dose and in plasma concentrations. Several factors may influence the pharmacokinetics in patients with liver disease. This may explain the inter-patient variability of concentrations and the lack of differences in concentration at different dosages. It could be interesting to extend the series of HCC patients to enhance information on the kinetics of the drug; furthermore, to establish a threshold of plasma sorafenib concentrations to predict severe adverse reactions would be clinically useful.

Correlation between induction of DNA fragmentation in urinary bladder cells from rats and humans and tissue-specific carcinogenic activity.

Seven chemicals, six of which are known to induce epithelial neoplasms of the urinary bladder in rats, were assayed for their ability to induce DNA damage in primary cultures of rat and human cells from urinary bladder mucosa, and in urinary bladder, liver and kidney of intact rats. Significant dose-dependent increases of DNA fragmentation, as measured by the Comet assay, were obtained in cells from both rats and humans with the following concentrations of five test compounds: 2-naphthylamine and N-nitrosodi-n-butylamine 0.5 and 1 mM, phenacetin 2 and 4 mM, cyclophosphamide from 2 to 8 mM, and o-toluidine 16 and 32 mM. Nitrilotriacetic acid (1-4 mM), a rat bladder carcinogen, and 4-aminobiphenyl (0.125-0.5 mM), a bladder carcinogen in humans but not in rats, gave a weak positive response in rats cells and a more marked response in humans cells. In terms of DNA-damaging potency, 4-aminobiphenyl, cyclophosphamide, phenacetin and 4 nitrilotriacetic acid were more active in human than in rat cells, whereas the converse occurred with 2-naphthylamine. Consistently with the results observed in vitro statistically significant dose-dependent increases in the average frequency of DNA breaks were detected in the urinary bladder mucosa of rats given p.o. single doses corresponding to 14 and 12 LD50 of six of the seven test compounds; the only one which gave a substantially negative response was 4-aminobiphenyl. With the exception of N-nitrosodi-n-butylamine which caused DNA damage in liver and of phenacetin and nitrilotriacetic acid which caused damage in kidney in agreement with their tumorigenic activity, any substantial evidence of DNA lesions in these two organs was absent in rats treated with 12 LD50 of the other 4 test compounds. These findings give evidence that urinary bladder genotoxic carcinogens may be identified by the DNA damage/Comet assay using as targets cells of urinary bladder mucosa, and show that the effect may be quantitatively different in cells from rats and from human donors.

DNA damage and micronuclei induced in rat and human kidney cells by six chemicals carcinogenic to the rat kidney.

Six chemicals, known to induce kidney tumors in rats, were examined for their ability to induce DNA fragmentation and formation of micronuclei in primary cultures of rat and human kidney cells, and in the kidney of intact rats. Significant dose-dependent increases in the frequency of DNA single-strand breaks and alkali-labile sites, as measured by the Comet assay, and in micronuclei frequency, were obtained in primary kidney cells from both male rats and humans of both genders with the following subtoxic concentrations of five of the six test compounds: bromodichlorometane (BDCM) from 0.5 to 4 mM, captafol (CF) from 0.5 to 2 microM, nitrobenzene (NB) from 0.062 to 0.5 mM, ochratoxin A (OTA) from 0.015 to 1.215 microM, and trichloroethylene (TCE) from 1 to 4 mM. Benzofuran (BF), consistent with its carcinogenic activity for the kidney of female, but not of male rats, at concentrations from 0.125 to 0.5 mM gave positive responses in cells from females but did not induce DNA damage and increased the frequency of micronuclei in cells from males to a lower extent; in contrast, it was active in cells from humans of both genders. DNA-damaging and micronuclei-inducing potencies were similar in the two species. In agreement with these findings, statistically significant increases in the average frequency of both DNA breaks and micronucleated cells were obtained in the kidney of rats, given p.o. a single dose (1/2 LD50) of the six compounds, BF in this assay being more genotoxic in female than in male rats. Taken as a whole, these findings give further evidence that kidney carcinogens may be identified by short-term genotoxicity assays, using as target kidney cells, and show that the six chemicals tested produce, in primary cultures of kidney cells from human donors, effects similar to those observed in rats.

Genotoxicity and carcinogenicity studies of bronchodilators and antiasthma drugs.

This survey is a compendium of genotoxicity and carcinogenicity information of bronchodilators and antiasthma drugs. Data from 46 marketed drugs were collected. Of these 46 drugs, 25 (54.3%) did not have retrievable genotoxicity or carcinogenicity data. The remaining 21 (45.7%) had at least one genotoxicity or carcinogenicity test result. Of these 21 drugs, 10 had at least one positive finding: three tested positive in at least one genotoxicity assay, eight in at least one carcinogenicity assay, and one of them gave positive results in both genotoxicity assay and carcinogenicity assay. Concerning the predictivity of genetic toxicology findings for the result(s) of long-term carcinogenesis assays, 15 drugs had both genotoxicity and carcinogenicity data: seven of them (46.6%) were neither genotoxic nor carcinogenic, 6 (40.0%) were carcinogenic in at least one sex of mice or rats but tested negative in genotoxicity assays, 1 (6.7%) tested positive in genotoxicity assay but was non-carcinogenic, and 1 (6.7%) gave positive responses in both genotoxicity and carcinogenicity assay. Only 11 (23.9%) of the 46 drugs considered had all data required by current guidelines for testing of pharmaceuticals, but a large fraction of them were developed and marketed prior to the present regulatory climate.

Involvement of the mu-opioid receptor gene polymorphism A118G in the efficacy of detoxification of alcohol dependent patients.

INTRODUCTION: The A118G (rs 1799971) polymorphism in the mu-opioid receptor gene (OPRM1) has been reported to be associated with alcohol addiction. METHODS: In this study 109 patients diagnosed with alcohol dependence in accordance with DMS-IV criteria and 95 healthy subjects were enrolled and everyone has been genotyped. RESULTS: The percentage of alcoholic patients with higher than normal gamma-glutamyl transferase (GGT) levels significantly decreased after six months of standard detoxification treatment, both in patients with A/A genotype and in the other ones with A/G genotype. However, the percentage of alcohol dependent patients with the A/A genotype recorded a slight decrease of the GGT and the mean corpuscolar volume of erythrocytes (MCV) combination marker after six months of therapy (30% vs 12%), while subjects with the A/G genotype showed no variation. CONCLUSION: This finding suggests that alcohol dependent patients with the A/A genotype could have a faster restoration of their liver function than those ones with the A/G genotype: it might be possible that the presence of G allele confers on these patients a reduced ability in abstaining from drinking alcohol.

Correlation between induction of DNA fragmentation in lung cells from rats and humans and carcinogenic activity.

Six chemicals, known to induce lung tumors in rats, were examined for their ability to induce DNA fragmentation in primary cultures of rat and human lung cells, and in the lung of intact rats. Significant dose-dependent increases in the frequency of DNA single-strand breaks and alkali-labile sites, as measured by the single-cell gel electrophoresis (Comet) assay, were obtained in primary lung cells from male rats with the following, minimally toxic, concentrations of the six test compounds: N-nitrosodimethylamine (NDMA; 2.5-10 mM), hydrazine (HZ; 0.5-4 mM), cadmium sulfate (CD; 31.2 and 62.5 µM), 4,4'-methylene bis (2-chloroaniline) (MOCA; 31.2-125 µM), isobutyl nitrite (IBN; 7.8-31.2 µM) and tetranitromethane (TNM; 1.9-15.6 µM). Similar degrees of DNA fragmentation were obtained in primary human lung cells; however, due to inter-donor differences, the minimum effective concentrations were in some donors lower and in others higher than in rats, and IBN induced DNA damage only in one of three donors. The DNA-damaging potency of HZ was higher in rats than in humans, and the opposite was true for MOCA. In agreement with these findings, statistically significant increases in the average frequency of DNA breaks were obtained in the lung of rats given a single oral dose (1/2 LD50) of the six test compounds. These findings give evidence that genotoxic lung carcinogens may be identified by use of the DNA fragmentation/Comet assay on rat lung cells as targets cells, and show that the six compounds tested produce in primary cultures of lung cells from human donors DNA-damaging effects substantially similar to those observed in rats.