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Biomedical subjects

A Marzo

Publications and source records attributed to A Marzo.

At least 37 records · Page 2Linked to original sources
PubMed1997

L-Carnitine moiety assay: an up-to-date reappraisal covering the commonest methods for various applications.

L-Carnitine and its esters are typical endogenous substances. Their homeostatic equilibria are effectively controlled by various mechanisms which include rate-limiting enteral absorption, a multicomponent endogenous pool which is regulated according to a mammillary metabolism, an asymmetric body distribution and a saturable tubular reabsorption process leading to renal thresholds. In formal pharmacokinetic and metabolic investigations, the whole L-carnitine pool should be investigated, owing to the rapid interchange process between the various components of the pool. Free L-carnitine, as well as its acyl esters, must therefore be considered from an analytical viewpoint. L-Carnitine, acetyl-L-carnitine and total L-carnitine (the latter as an expression of the whole pool) can easily be assayed by enzyme or radioenzyme methods. Propionyl-L-carnitine and other esters containing fatty acids with more than three carbon atoms can be assayed using various HPLC approaches. Tandem mass spectrometry is another excellent approach to the assay of carnitine and its short-chain, medium-chain and long-chain esters. As L-carnitine contains a chiral carbon atom, the enantioselectivity of the assays is also considered in this review. Metabolites produced by enteral bacteria, namely tri-, di- and mono-methylamine, gamma-butyrobetaine, along with other systemic metabolites, namely trimethylamine N-oxide and N-nitroso dimethylamine, are very important in quantitative and toxicokinetic terms and require specific assay methods. This review covers the commonest methods of assaying carnitine and its esters, their impurities and pre-systemic and systemic metabolites and gives analytical details and information on their applications in pharmaceutics, biochemistry, pharmacokinetics and toxicokinetics.

Carnitine↗
PubMed1997

Gas chromatographic assay for estazolam in human plasma and results of a bioequivalence study.

This paper describes a new sensitive gas chromatographic method with electron capture detector to assay estazolam in human plasma, which has been developed and validated for pharmacokinetic purposes. The drug and the internal standard (triazolam) were extracted from plasma buffered at pH 9.0 into toluene and analysed on a widebore DB 17 column. The calibration curve covered the 1.0-200 ng ml-1 range with a mean determination coefficient of 0.9996. The quantification limit was 1.0 ng ml-1. This method was used to investigate the bioequivalence of a new formulation of estazolam in drops (test) and the formulation in tablets (reference, ESILGAN). Both formulations were administered at a single dose of 2 mg in a clinical trial carried out on 24 healthy volunteers consisting of 12 males and 12 females, following a crossover randomised design in two periods with wash-out. The test and the reference formulations proved to be fully bioequivalent according to operating guidelines, namely through 90% confidence intervals in the 0.80-1.25 range.

Adult↗
PubMed1997

Clinical pharmacokinetic registration file for NDA and ANDA procedures.

Pharmacokinetics is a relatively young branch of the life sciences, which has developed rapidly in the last 15-20 years, thanks to a series of analytical achievements that have allowed drug concentrations to be measured in biological matrices with highly selective and sensitive methods. Most old drugs and all new drugs have been investigated in depth to determine their absorption, distribution, metabolism and elimination. This has produced a huge body of data which have characterized drugs through so-called fingerprint parameters. Today, pharmacokinetics plays a vital role throughout drug development from initial non-clinical studies to clinical trials. In new drug development, simultaneous efforts in pharmacodynamic and pharmacokinetic studies have produced a high degree of synergy, the most useful expression of which is the identification of a therapeutic window, where this can be achieved. Dose linearity/proportionality, gender effect, metabolism and possible polymorphism, studies on neonates/children and on elderly and diseased patients and the population approach, relative and absolute bioavailability, possible interactions and the possible presence of a deep compartment in the distribution are other important applications of pharmacokinetics. Major new developments in pharmaceutical technology which have produced controlled-release delivery systems and the market introduction of generics would never have occurred but for the active contribution of pharmacokinetics. This review is devoted to the investigations needed to prepare appropriate pharmacokinetic registration files for new drug applications (NDAs) and abbreviated or abridged new drug applications (ANDAs) required for generic drugs. Scientific literature, operating guidelines and personal experience are the basis of this review, which describes a number of relevant examples in several specific areas.

Adult↗
PubMed1997

Absorption and distribution of naproxen in rats orally treated with naproxen betainate sodium salt monohydrate. Comparison with naproxen.

The S-naproxen betainate sodium salt monohydrate (naproxen-betaNa, CAS 104124-26-7, Aprenin) was synthesized to improve bioavailability and tolerability of naproxen. 24 albino rats were treated with naproxen-betaNa (84 mg/kg) and 24 with S-naproxen (naproxen) (50 mg/kg) by the oral route, the doses being equimolar. The animals were sacrificed and naproxen was assayed in timed plasma samples drawn off over a 24-h period and in tissues excised 1 h after administration. Peak concentrations of naproxen proved to be higher with naproxen-betaNa than with naproxen as such. The area under the curve of naproxen concentrations observed with the two administrations overlapped as did concentrations of the drug in the lungs, myocardium and liver. Naproxen concentrations in the gastric wall after naproxen-betaNa proved to be lower than after administration of naproxen as such, which allowed the authors to assume that naproxen-betaNa has a better gastric tolerability.

Animals↗
PubMed1997

Comparative bioavailability study on naproxen betainate sodium salt monohydrate and naproxen sodium salt in healthy volunteers.

The S-naproxen betainate sodium salt monohydrate (naproxen-betaNa, CAS 104124-26-7, Aprenin, test drug), and the sodium salt of S-naproxen (reference), were administered to twelve healthy volunteers of both sexes according to a crossover design, in a single dose of one 575 mg capsule of test, containing 342 mg of S-naproxen and two 275 mg tablets of reference, containing 502 mg of S-naproxen. Blood samples were drawn off over a 24-h period before (time 0) and after administration at foreseen time intervals. Naproxen was measured in plasma by a validated HPLC assay with UV detection which was able to detect 1 microgram/ml and proved to be linear in the range 1-100 micrograms/ml. The non-compartmental pharmacokinetic parameters obtained were statistically processed according to the EU guidance note on bioavailability and bioequivalence Cmax, AUC0-24h and AUC0-infinity were normalized to the dose of 502 mg of naproxen and log-transformed before statistical analysis to assess bioequivalence. Dose-normalized values of plasma concentrations encountered with the two formulations proved to overlap, with the exception of the first sampling time which showed naproxen concentrations that were higher with test drug than with reference. The specific test for bioequivalence led to 90% confidence intervals within the 80-125% range with target pharmacokinetic parameters, whereas the time to peak (tmax) observed with the test and reference drugs did not differ to any statistically significant degree when analysed with Wilcoxon's non-parametric test. It is concluded that the test drug should be declared bioequivalent with the reference drug in terms of dose-normalized concentrations, despite the more rapid increase in plasma concentrations of naproxen observed at the first sampling time with test drug.

Adult↗
PubMed1997

Myocardial fatty acid pattern in rats fed on an erucic acid enriched diet.

Individual lipid classes and their fatty acid pattern in myocardium of rats fed on a diet containing 10% erucic acid ethyl ester (cis-13-docosenoic acid ethyl ester) were investigated and compared to rats fed on a normal diet. Two groups of rats were treated for 10 consecutive days with the erucic acid ethyl ester diet and the standard diet, respectively. After extracting total lipids from the myocardium of the rats, the individual lipid classes and the percentage of fatty acids in phospholipids, free fatty acids, diglycerides and triglycerides were measured. The data obtained demonstrate that the erucic acid ethyl ester diet induces a marked increase in free fatty acids and in triglycerides and marked differences in fatty acid pattern in triglycerides, free fatty acids and diglycerides, but only marginal differences in phospholipids, which seem to be carefully preserved as fundamental components of cell and mitochondria membranes.

Animals↗
PubMed1996

Investigation of xenobiotic metabolism by CYP2D6 and CYP2C19: importance of enantioselective analytical methods.

Investigations into the genetic polymorphism of drug metabolism have involved specific models to screen poor and extensive metabolisers of xenobiotics. Debrisoquine, sparteine, S-mephenytoin and dextromethorphan are particularly well known. They have been extensively described in the literature and are used to phenotype human subjects before performing investigations with new drugs which are believed to be under the control of a genetic polymorphism. Dextromethorphan, debrisoquine and sparteine are good substrates for CYP2D6, whereas the S-enantiomer of mephenytoin is a good substrate for CYP2C19, both being two isozymes of cytochrome P-450. In many drugs, the hepatic microsomal oxidative metabolism involving stereogenic centres congregates either with CYP2D6 or with CYP2C19 or, in certain cases, with both of them. The availability of both CYP2D6 from poor and extensive metabolisers and an enantioselective assay would allow genetic polymorphism in drug biotransformation to be investigated in vitro ex vivo at an early stage of drug development before the IND (investigational new drug). Single-dose investigations in vivo can also be performed when only minimal pre-clinical toxicological data are available and produce more reliable results than in vitro studies. This paper focuses on the problem of genetic polymorphism in drug development and specifically discusses some relevant knowledge gained in the last two decades on enantioselective bioassays. Specific examples are given.

Aryl Hydrocarbon Hydroxylases↗
PubMed1996

Toxicokinetics of endogenous substances: a neglected issue.

Some specific problems related to the pharmacokinetics and toxicokinetics of endogenous substances are reviewed in this paper with specific reference to baseline and its variation, reversible dynamic pools, saturable tubular reabsorption, the stability of the parent compound, poorly absorbed substances, analytical procedures and statistical analysis. Compared to xenobiotics, endogenous substances require special care in planning pharmacokinetic and toxicokinetic investigations as they possess homeostatic equilibria. The presence of a baseline, its variation according to specific rhythms, age, sex or simple fluctuation, active absorption and passive diffusion, saturable enzyme processes, renal threshold, the distribution of the exogenous drug in relation to that already present in the body and a possible balance between endogenous synthesis and dietary intake are the main mechanisms which allow homeostatic equilibria to be preserved and restored when impaired. The presence of multicomponent dynamic pools requires not only the substance administered but also the entire pool to be assayed, to determine the components that can be originated from the drug administered. Various analytical approaches are discussed and some specific examples given. The above mechanisms frequently involve saturable processes which lead to non-linear kinetics. Clearance and, for substances excreted via urine not interacting with plasma proteins, renal clearance, are important parameters to check linearity/non-linearity, and give more accurate information than the half-life of the elimination phase, which with this kind of substance varies in non-linearity and baseline fluctuation or rhythms. Appropriate statistical approaches are suggested to analyse proof of absorption, length of treatment, gender difference and the linearity/proportionality assessment of absorption. Possible discrepancies in the dose-absorption relationship when dose-linearity and dose non-proportionality are simultaneously encountered are carefully discussed and attributed to the presence of the baseline. This review is aimed at stimulating the attention of scientists and regulatory authorities to several unsolved issues in the toxicokinetics and pharmacokinetics of endogenous substances, which appear to have been overlooked in current operating guidelines.

Animals↗
PubMed1995

Determination of ticlopidine in human plasma by high-performance liquid chromatography and ultraviolet absorbance detection.

A simple HPLC method has been developed for the determination of ticlopidine in human plasma. Plasma samples were buffered at pH 9 and extracted with n-heptane-isoamyl alcohol (98.5:1.5, v/v). Imipramine was used as internal standard. Chromatography was performed isocratically with acetonitrile-methanol-0.05 M KH2PO4 (20:25:55, v/v) at pH 3.0 containing 3% triethylamine at a flow-rate of 1 ml/min. A reversed-phase column, Supelcosil LC-8-DB, 15 cm x 4.6 mm I.D., 5 microns particle size, was used. The effluent was monitored by UV absorbance detection at 235 nm. The method showed good accuracy, precision and linearity in the concentration range 5-1200 ng/ml. The limit of quantitation was 5 ng/ml, with a precision (C.V.) of 8.91%, which is the same as that achieved by other authors with a previously published GC-MS method. The procedure described in this paper is simple and allows the routine assessment of ticlopidine plasma concentration in pharmacokinetic studies following therapeutic doses in human subjects.

Acetonitriles↗
PubMed1995

Open questions in bioequivalence.

Bioequivalence studies are mainly required to demonstrate the interchangeability of multisource pharmaceutical products, usually called generics. A pivotal investigation projected and conducted in compliance with specific guidelines allows a replicative company to register a generic drug via an ANDA (abbreviated new drug application) procedure. Serious problems are often encountered in planning and conducting these studies, which are not covered by operating guidelines, and call for protocols to be set up on a case-by-case basis. The main problems involve the high variability of some active ingredients, how to manage baseline with endogenous substances, drugs with a long terminal half-life, drugs under genetic polymorphic metabolism, drugs possessing stereogenic centre(s), and drugs which cannot ethically be given to healthy volunteers. These problems are extensively covered in this paper and, where appropriate, possible solutions are suggested.

Half-Life↗
PubMed1995

Bioequivalence. An updated reappraisal addressed to applications of interchangeable multi-source pharmaceutical products.

This paper reviews study procedures for bioequivalence trials, mainly addressed to the New Drug Application (NDA) of generic drugs, strictly referring to EU and USA guidelines on this matter. Specific attention is devoted to the most appropriate experimental designs, the size of the volunteer sample, the ethical issues involved, statistics to assess bioequivalence and the accepted standard format for final research reports. Some aspects which create serious problems in bioequivalence trials, most of which not fully covered by the EU and USA specific guidelines, are comprehensively discussed. These include a) drugs with elevated variability; b) endogeneous substances and the management of baseline value; c) modified release formulations; d) prodrugs; e) restrictions to be contained in forthcoming guidelines on chiral medicinal products; f) superbioavailability; g) drugs with elevated half-life and h) cases in which bioequivalence trials should not be needed. As generic drugs cost less than the innovator product, agencies have facilitated their NDA procedures by requiring a dossier on chemistry and pharmacy and a pivotal bioequivalence study to demonstrate that the generic formulation is fully interchangeable with the innovator product. Bioequivalence is thus the key requirement for an NDA of a generic drug, and trials should be planned, conducted and reported in the most appropriate way. With this in mind, this review is an up-to-date reappraisal that should stimulate the attention of scientists and regulatory authorities on some open questions on bioequivalence.

Dosage Forms↗
PubMed1995

Interference of heparin in plasma levocarnitine determination with radioenzyme assay.

This paper describes the interference of heparin (CAS 9005-49-6) in whole plasma used for free and total levocarnitine (L-carnitine, CAS 541-15-1) analysis. Alkaline hydrolysis required for total L-carnitine measurement does not overcome the problem. The same interference is also present in long-chain acyl-L-carnitine assay, because heparin precipitates together with proteins and long-chain esters of L-carnitine in the acid insoluble fraction of perchloric acid treated plasma sample. In this case an appropriate cleaning of the sample is recommended before the assay.

Acetylcarnitine↗
PubMed1994

High-performance liquid chromatography and capillary electrophoresis of L- and D-carnitine by precolumn diastereomeric derivatization.

A new method for the simultaneous assay of D- and L-enantiomers of carnitine is described. The method is based on precolumn derivatization with (+)-1-(9-fluorenyl)ethyl chloroformate [(+)FLEC] producing a diastereomeric derivative which can be detected both by UV absorbance and fluorescence detection. Also acyl esters of carnitine can be processed with this method, after alkaline hydrolysis. The D-enantiomer of carnitine and acylcarnitine can be detected at a concentration as low as 0.2% in the raw material and in pharmaceuticals. Assays can be carried out using an autoinjector either by HPLC or capillary electrophoresis (CE) because the derivative proved to be very stable. Its application is proposed for the routine assay of the enantiomeric excess of L-carnitine and their acyl esters in pharmaceutical products.

Acylation↗
PubMed1994

Plasma concentration, urinary excretion and renal clearance of L-carnitine during pregnancy: a reversible secondary L-carnitine deficiency.

Plasma concentration, urinary excretion and renal clearance of free, total and esterified L-carnitine were monitored monthly in 14 women during the last 6 months of pregnancy and 1 month after delivery. Plasma concentration and renal clearance measured 1 month after delivery overlapped with normal values for females of comparable age, and were considered the reference values for further comparisons. Plasma concentration of free, total and esterified L-carnitine decreased during pregnancy, reaching values as low as half of those measured 1 month after delivery, whereas urinary excretion and renal clearance, mainly of L-carnitine esters, increased, with renal clearance reaching a peak at the 16th week of pregnancy. Pregnancy thus leads to a reversible secondary deficiency of L-carnitine. The involvement of L-carnitine in the excretion of an excess of acyl-S-coenzyme A groups to prevent a possible systemic acidosis, as well as hormonal changes and a reduction of L-carnitine biosynthesis, could play a significant role in the variations in L-carnitine metabolism encountered in pregnancy. As physiological components of L-carnitine are excreted via a saturable tubular reabsorption, their threshold seems to follow plasma concentration, even when they decrease markedly, as in pregnancy.

Adolescent↗
PubMed1994

On definition and use of the term bioavailability.

In common usage, the rate of absorption of an active ingredient or its therapeutic moiety is generally not mentioned in the context of bioavailability. In this communication it is shown that exclusion of the rate of absorption may have serious consequence on the interpretation of bioavailability depending on the underlying model for the system under study. In the case of endogenous substances, the term "bioavailability" is ambiguous unless one specifies whether it refers to availability of the exogenous substance only or the sum total of the exogenous and endogenous substances.

Absorption↗
PubMed1994

The body of a trial master file for a pivotal phase I study.

This paper describes in detail the documentation which would form the body of a Trial Master File for Phase I pivotal investigations filed to render easily and totally auditable all the documentation. The body of a Trial Master File is formed by formal documents (study protocol, research report), by documents to be attached to the research report (clinical report, analytical validation report, statistical report, blank case report form, blank consent form, approval from Ethics Committee), by documents to be kept in file (investigator's brochure, clinical source documentation, clinical trial supplies, filled case report forms, monitoring reports, auditing reports, letters, contracts, faxes, phone notes) and by confidential documents that must be filed in the Clinical Unit only (signed consent forms which contain the subjects' identification).

Clinical Trials, Phase I as Topic↗
PubMed1994

Serum and urine levels of levocarnitine family components in genetically diabetic rats.

Serum concentration and urinary excretion of levocarnitine (L-carnitine, CAS 541-15-1) family components were evaluated in a Wistar derived strain of genetically diabetic rats BB/BB, in comparison with normal Wistar rats, and their control rats BB/WB of both sexes. BB/BB diabetic animals have lower serum concentration of total-L-carnitine (TC), L-carnitine (LC), acetyl-L-carnitine (ALC), and short chain L-carnitine esters (SCLCE) than both the strains of non-diabetic rats, as previously observed in streptozotocin diabetic rats. No or marginal variations between control and diabetic rats were detected in cumulative urinary excretion of L-carnitine family components. A strain difference was observed between Wistar and BB/WB non-diabetic rats, BB/WB showing higher serum concentration and lower cumulative urinary excretion of LC and TC than Wistar animals. Renal clearance of L-carnitine components proved to be markedly higher in BB/BB diabetic rats, as previously shown in streptozotocin rats. The reduction of serum concentration of the carnitines endogenous pool may explain this finding. The lack of an increased urinary excretion of L-carnitine components in diabetic animals despite the high increase of diuresis suggests that the saturable tubular reabsorption of L-carnitine family components also in diabetes is the primary mechanism to preserve the homeostatic equilibria of the L-carnitine family, the variation in serum concentration being attributable to the complex systemic metabolic alterations typical of diabetes. In agreement with previous investigations, male animals of all the strains showed higher serum concentration and urinary excretion of L-carnitine components as compared to females.

Acetylcarnitine↗