Cyclic metabolites: chemical and biological considerations.

Metabolism of xenobiotics can sometimes generate cyclic metabolites. Such metabolites are usually the result of intramolecular reactions occurring within a primary or secondary metabolite and this chemistry may lead to unexpected structures. Intramolecular chemistry is often driven by nucleophilic groups reacting with electrophilic atoms, often carbon, although radical processes also occur. Conjugation of xenobiotics or their metabolites with endogenous thiols, such as glutathione or cysteine, introduce a reactive amino group that can lead to the formation of cyclic structures. Less common than chemically driven cyclizations are enzymatically mediated ring-closures, although this may reflect our incomplete recognition of enzymatic involvement in this step of cyclic metabolite formation. While some cyclic metabolites are biologically inactive, others are biologically active. Thus, a cyclic metabolite may display desirable pharmacology, or, contribute to toxicology. When a cyclic metabolite is identified, it is important to consider the possibility that it is an artifact, i.e. metabonate, that was formed during processing of the sample, for example, through degradation or by chemical reactions with other components present in the matrix. From a medicinal chemistry perspective, a cyclic metabolite with a different chemical scaffold from the parent structure may lead to a new series of structurally novel, biologically active molecules with the same, or different, pharmacology from the parent. This review will cover a selection of cyclic metabolites from a mechanistic point of view, and when possible, discuss their biological relevance.

Molecular determinants of voltage-dependent gating and binding of pore-blocking drugs in transmembrane segment IIIS6 of the Na(+) channel alpha subunit.

Mutations of amino acid residues in the inner two-thirds of the S6 segment in domain III of the rat brain type IIA Na(+) channel (G1460A to I1473A) caused periodic positive and negative shifts in the voltage dependence of activation, consistent with an alpha-helix having one face on which mutations to alanine oppose activation. Mutations in the outer one-third of the IIIS6 segment all favored activation. Mutations in the inner half of IIIS6 had strong effects on the voltage dependence of inactivation from closed states without effect on open-state inactivation. Only three mutations had strong effects on block by local anesthetics and anticonvulsants. Mutations L1465A and I1469A decreased affinity of inactivated Na(+) channels up to 8-fold for the anticonvulsant lamotrigine and its congeners 227c89, 4030w92, and 619c89 as well as for the local anesthetic etidocaine. N1466A decreased affinity of inactivated Na(+) channels for the anticonvulsant 4030w92 and etidocaine by 3- and 8-fold, respectively, but had no effect on affinity of the other tested compounds. Leu-1465, Asn-1466, and Ile-1469 are located on one side of the IIIS6 helix, and mutation of each caused a positive shift in the voltage dependence of activation. Evidently, these amino acid residues face the lumen of the pore, contribute to formation of the high-affinity receptor site for pore-blocking drugs, and are involved in voltage-dependent activation and coupling to closed-state inactivation.

A common local anesthetic receptor for benzocaine and etidocaine in voltage-gated mu1 Na+ channels.

According to Hille's modulated receptor hypothesis, benzocaine shares a common receptor with all other local anesthetics (LAs) in the voltage-gated Na+ channel. We tested this single receptor hypothesis using mutant muscle Na+ channels of mu1-I1575A, F1579A, and N1584A transiently expressed in Hek-293t cells. Both benzocaine and etidocaine are more effective at blocking mu1-N1584A current than the wild-type current, while they are less potent at blocking mu1-F1579A current. Such concurrent changes of both benzocaine and etidocaine potency towards F1579A and N1584A mutants suggest that they share a common LA receptor. Consistent with results found in studies of native Na+ channels, permanently charged QX-314 at 1 mM is not effective at blocking wild-type, F1579A, and N1584A current via external application. In contrast, QX-314 is relatively potent at blocking I1575A current when applied externally. This increased potency of external QX-314 against the mu1-I1575A mutant has been reported previously in a study of the brain counterpart. Mutant I1575A also appears to be highly sensitive to the external divalent cation Cd2+, probably because of the presence of cysteine residues near the mu1-I1575 position in the IV-S6 segment. To our surprise, neutral benzocaine becomes more effective at blocking mu1-I1575A current than the wild-type current, whereas the opposite is found for etidocaine. We hypothesize that an increase in accessibility of external QX-314 to the mu1-I1575A mutant is accompanied by a reduction of binding towards the charged amine component.

Molecular determinants of state-dependent block of Na+ channels by local anesthetics.

Sodium ion (Na+) channels, which initiate the action potential in electrically excitable cells, are the molecular targets of local anesthetic drugs. Site-directed mutations in transmembrane segment S6 of domain IV of the Na+ channel alpha subunit from rat brain selectively modified drug binding to resting or to open and inactivated channels when expressed in Xenopus oocytes. Mutation F1764A, near the middle of this segment, decreased the affinity of open and inactivated channels to 1 percent of the wild-type value, resulting in almost complete abolition of both the use-dependence and voltage-dependence of drug block, whereas mutation N1769A increased the affinity of the resting channel 15-fold. Mutation I1760A created an access pathway for drug molecules to reach the receptor site from the extracellular side. The results define the location of the local anesthetic receptor site in the pore of the Na+ channel and identify molecular determinants of the state-dependent binding of local anesthetics.

Circadian phase-dependent protein and tissular binding of three local anesthetics (bupivacaine, etidocaine, and mepivacaine) in mice: a possible mechanism of their chronotoxicokinetics?

The aim of this study was to investigate the possible influence of the time of administration on bupivacaine (B), etidocaine (E), and mepivacaine (M) protein and tissue (brain and heart) binding. For each anesthetic agent, a single dose of B (20 mg/kg), E (40 mg/kg), or M (60 mg/kg) was administered intraperitoneally at 10:00, 16:00, 22:00, and 04:00 h. Blood and tissue samples were collected 15 min after drug administration. This study documents significant circadian variations in protein and tissue binding of the three local anesthetic agents. We did not demonstrate a temporal relationship between the respective free and tissue levels. Thus, the temporal variations of free plasma, brain, and heart levels do not seem to be involved in the temporal changes of induced mortality.

Partitioning of local anesthetics into membranes: surface charge effects monitored by the phospholipid head-group.

The binding of the charged form of two local anesthetics, dibucaine and etidocaine, to bilayers composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) was measured simultaneously with ultraviolet spectroscopy and deuterium magnetic resonance. Because of their amphiphilic molecular structure, both drugs intercalate between the lipid molecules, increasing the surface area and imparting a positive electric charge onto the membrane. The ultraviolet (UV) binding isotherms were therefore analyzed in terms of a model which specifically took into account the bilayer expansion as well as the charge-induced concentration variations near the membrane surface. By formulating a quantitative expression for the change in surface area upon drug intercalation and combining it with the Gouy-Chapman theory, the binding of charged dibucaine and etidocaine to the lipid membrane was best described by a partition equilibrium, with surface partition coefficients of 660 +/- 80 M-1 and 11 +/- 2 M-1 for dibucaine and etidocaine, respectively (pH 5.5, 0.1 M NaCl/50 mM buffer). Deuterium magnetic resonance demonstrated further that the binding of drug changed the head-group conformation of the lipid molecules. Invoking the intercalation model, a linear variation of the deuterium quadrupole splittings of the choline segments with the surface charge density was observed, suggesting that the phosphocholine head-group may act as a 'molecular electrometer' with respect to surface charges.

Absorption of bupivacaine, etidocaine, lignocaine and ropivacaine into n-heptane, rat sciatic nerve, and human extradural and subcutaneous fat.

Absorption of four amide local anaesthetics, including a new experimental agent, ropivacaine, in n-heptane, rat sciatic nerve and human extradural and subcutaneous fat was studied in vitro. The relative n-heptane/buffer (37 degrees C) partitioning of bupivacaine:etidocaine:lignocaine:ropivacaine was 10:39:1:2.9. The absorption of bupivacaine and etidocaine into nerve tissue was identified at steady state, but in extradural and subcutaneous fat etidocaine was taken up significantly more than bupivacaine. The lowest uptake was with lignocaine, and at steady state its mean concentrations were similar in all three tissues. There was a significantly higher concentration of ropivacaine than lignocaine in all tissues. Mean tissue concentrations of bupivacaine in sciatic nerve and subcutaneous fat, on the other hand, were significantly higher than those of ropivacaine. The ratios of relative mean uptake (mg g-1), at steady state, of bupivacaine, etidocaine, lignocaine and ropivacaine were: sciatic nerve 3.3:4:1:1.8, extradural fat 4.1:8.3:1:2.3 and subcutaneous fat 3.8:10.6:1:1.9, all of which were clearly lower than the theoretical n-heptane/buffer partitioning ratios.

Clinical pharmacokinetics in pregnancy and perinatology. I. Placental transfer and fetal side effects of local anaesthetic agents.

Local anaesthetic agents, both of the amide type (e.g., lidocaine, bupivacaine, etidocaine) and of the ester type (e.g., 2-chloroprocaine) are widely used to relieve pain in obstetric and gynaecological practice. The pharmacokinetics of these compounds are discussed in this review, with particular emphasis on the fetal exposure and its relationship to adverse effects on the fetus. 2-Chloroprocaine is rapidly hydrolyzed by esterases, and only traces of this compound reach the fetus, even following multiple injections, suggesting safety for the fetus. The main disadvantage of this compound is the short duration of action (0.5-1 h). The amide-type agents are active for longer time periods (up to several hours). The fetal/maternal total concentration ratios were approximately 0.3 for bupivacaine and etidocaine, 0.5 for lidocaine, 0.7 for mepivacaine, and 1 for prilocaine. The low ratios of bupivacaine and etidocaine result from extensive binding (90%) of these drugs to maternal alpha1-acid glycoprotein which exceeds corresponding fetal protein binding (50%). These low fetal/maternal total concentration ratios cannot be equated with fetal safety, because fetal side effects are better related to the free drug levels. Since the amide-type anaesthetics are weak bases, fetal acidosis will increase the maternal/fetal pH gradient and will result in accumulation of free drug in the fetus and possible fetal side effects. Addition of epinephrine to the injection solution reduces the maternal blood levels of the amide-type compounds, but apparently not the fetal levels to the same extent. Because of possible unwanted effects of epinephrine (decreased uterine blood flow), the addition of this compound is not generally accepted to be of advantage. Paracervical blockade may result in elevated fetal blood levels (possibly by transarterial diffusion into uterine arteries) and possibly fetal bradycardia.

Pharmacokinetics of etidocaine in fetal and neonatal lambs and adult sheep.

Etidocaine, 2.5 mg/kg, was injected intravenously into either a nonpregnant adult sheep (N = 7), neonatal lamb (N = 7), or fetal lamb (N = 6), all with catheters in the femoral vessels and urinary bladders. Serial arterial blood and urine samples were obtained over 4 hours and analyzed for unchanged etidocaine using a gas chromatographic technique. The distribution and elimination half-lives of etidocaine in the blood of nonpregnant adults and newborn sheep were similar. The volume of distribution was significantly greater in the newborn (4.64 LK/kg) compared with the adult (1.52 L/kg) as was the total body clearance (87-4 vs 30.3 ml/min/kg). Renal clearance was also significantly greater in the newborn. The data indicate that the newborn lamb is capable of eliminating etidocaine as rapidly as the adult. Following injection of the drug into the fetus, fetal blood concentrations became undetectable after 30 minutes due to placental transfer into the maternal compartment.

[The interaction of sedativa with etidocaine in patients with brachial plexus block (author's transl)]. / Die Beeinflussung der Plasmaetidocainspiegel durch Sedativa nach supraclaviculärer Plexusanaesthesie.

The influence of diazepam and phenobarbital on plasma etidocaine concentrations is studied. It is shown that there is no statistical relevant interaction. The mean peak levels occurred within 40 min after application of local anaesthetics and amounted to 1,439 micrograms/ml. The mean half times were calculated and are 135 min.

[Epidural opiate analgesia (EOA), compatibility of opiates with tissue and CSF (author's transl)]. / Epidurale Opiatanalgesie Gewebe- und Liquorverträglichkeit der Opiate.

To estimate the compatibility of opiates with tissue we measured the pH of opiate solutions in comparison to local anaesthetics. Piritramide and opium solutions revealed very low pH values (3.96 and 3.38). Two patients, who underwent EOA for the last two and a half three weeks before their death showed, macroscopically and microscopically, no signs of effect on tissue. The examination of the compatibility of opiate solutions and local anaesthetics with CSF showed a deproteinisation of CSF after Piritramide and a rise of opacity after opium. Of the local anaesthetics tested, only Editocaine interferred by crystallizing of the substance. In conclusion, all opiates except piritramide and opium can be used in EOA. In our opinion bupivacaine but not editocaine can be used for spinal anesthesia. All opiates tested can be mixed either with bupivacaine or editocaine.

Clinical pharmacokinetics of local anaesthetics.

The introduction of the new long acting local anaesthetics, bupivacaine and etidocaine, has stimulated an expansion of interest in regional anaesthesia, particularly for obstetrical applications and pain therapy. System toxicity following injection of local anesthetics occurs albeit infrequently, and tentative correlations have been made between the onset of CNS and cardiovascular effects and circulating drug concentrations in both adults and neonates. Amongst other factors, interpretation of these relationships depends upon blood distribution and plasma binding of the agents, sampling sites and acid-base balance. The disposition kinetics and placental transfer of the amide type agents have been well characterised. In adults their clearance is almost entirely hepatic but in neonates an increase in the renal component is, in part, a reflection of the immaturity of some of the enzymes responsible for their metabolism. Ester type agents are rapidly hydrolysed by plasma pseudocholinesterase and this has led to a preference for chloroprocaine in some obstetric procedures. Major determinants of the systemic absorption of the agents after perineural administration include their physicochemical and vasoactive properties, perfusion and tissue binding at the site of injection and whether or not adrenaline has been added. In respect of blood drug concentrations achieved after various regional anaesthetic procedures, the margin of systemic safety appears to favour bupivacaine and etidocaine compared to shorter acting analogues such as lignocaine and mepivacaine. The time course of local anaesthetic remaining at the site of injection has been calculated following intravenous regional anaesthesia and peridural block. This has allowed prediction of the local and systemic accumulation of the drugs following contined dosage. Blood concentrations of local anaesthetics after perineural injection are not closely related to age, weight or pregnancy but may be influenced by diseases associated with haemodynamic changes and by other drugs given at or around the time of regional blockade.

Physico-chemical modification of lidocaine uptake in rat lung tissue.

The uptake of lidocaine, methyllidocaine, bupivacaine, etidocaine was studied in rat lung slices at different pH-values. The accumulation of the quaternary analogue, methyllidocaine, was not changed in the pH interval 7.0--8.0. The uptake of the three other substances was about 3--4 times lower at pH 7.0 than at pH 8.0. The rank order of distribution at a fixed cation/base ratio was bupivacaine greater than etidocaine greater than lidocaine. Interactions between lidocaine and other substances were studied in lung slices and in isolated perfused lungs. Bupivacaine and nortriptyline counteracted the accumulation of 14C-lidocaine in lung slices in a dose-dependent manner. Nortriptyline was more effective than bupivacaine. In isolated perfused lung, bolus injections of nortriptyline and lidocaine rapidly displaced 14C-lidocaine from the tissue. In this study we suggest that the base form of local anaesthetics accumulate in the lung tissue, while the cationic form binds to accessible binding sites in the cell membranes.

The identification of eight hydroxylated metabolites of etidocaine by chemical ionization mass spectrometry.

1. Eight hydroxylated metabolites of etidocaine have been identified in urine of man by g.l.c.-chemical ionization mass spectrometry, using methane and a mixture of methane and deuterium oxide as reactant gases. 2. The metabolites identified were N-(2,6-dimethyl-3- and 4-hydroxyphenyl-)-2-(N,N-ethylpropylamino)butyramides, N-(2,6-dimethyl-3- and 4-hydroxyphenyl)-2-aminobutyramides, N-(2,6-dimethyl-3- and 4-hydroxyphenyl)-2-(N-ethylamino)butyramides, and N-(2,6-dimethyl-3- and 4-hydroxyphenyl)-2-(N-propylamino)butyramides. 3. The eight metabolites represent about 10% of the oral dose of etidocaine.

Pharmacokinetics and metabolism of the anilide local anaesthetics in neonates. 11 Etidocaine.

The urinary elimination of etidocaine and several of its metabolites was investigated in neonates whose mothers had received one or more doses of etidocaine during labour. The urine collection period ranged among the neonates from 21.4 to 47.0 h post-partum. The total amounts of etidocaine and its metabolites recovered in neonatal urine represented a mean of 0.12% of the maternal dose. Some differences in the pattern of urinary metabolites were observed between neonates and adults. Mean half-life of elimination of etidocaine calculated from sigma-minus plots of the neonatal urinary data was 6.42 h. This is greater than that previously reported following intravenous administration of etidocaine to adults (2.6 h). The slower rate of elimination in neonates is probably due to an increased neonatal volume of distribution since there is evidence to show that etidocaine is extensively metabolised by the neonate.

[Diaplacentar transfer of local anaesthetics (author's transl)]. / Diaplazentarer Transfer von Lokalanästhetika.

A very rapid resorption of the amide-type local anaesthetic agents most commonly used in obstetrics results in detectable plasma levels in mother and fetus within 5 to 10 minutes, respectively, after peridural anaesthesia. The various local anaesthetics differ from each other as far as their protein-binding capacity and the resulting ratios of umbilical vein to maternal blood levels are concerned. The feto-maternal ratio is inversely correlated to the degree of protein-binding. Both, the protein-binding capacity and the feto-maternal ratio do not indicate the total amount of the anaesthetic transferred across the placenta. In contrast to measurements of the plasma concentrations only the measurement of the anaesthetic in the fetal tissue would give an exact information about the fetal uptake of local anaesthetics. Several observations hint at the possibility that there are interactions between local anaesthetics and other drugs: A competitive displacement of local anaesthetics from their protein binding sites resulting in an increase in the unbound fraction is suggested. Since the feto-maternal equilibration exists only for the unbound fraction interactions of local anaesthetics with simultaneously administered drugs should be taken into consideration.