Characterization of the methemoglobin forming metabolites of benzocaine and lidocaine.

1. Topical anesthesia with benzocaine or lidocaine occasionally causes methemoglobinemia, an uncommon but potentially fatal disorder where the blood has a reduced ability to transport oxygen. Previous in vitro studies using human whole blood have shown that benzocaine causes more methemoglobin (MetHb) formation than lidocaine, and that both compounds require metabolic transformation to form the MetHb producing species. In the current investigation, the active species of benzocaine forming the MetHb was investigated. 2. HPLC analysis of benzocaine samples incubated with human hepatic S9 showed the formation of a peak with the same UV spectrum and retention time as benzocaine hydroxylamine (BenzNOH). To confirm the activity of BenzNOH, MetHb production following exposure to the compound was determined in whole human blood using an Avoximeter 4000 CO-oximeter. 3. BenzNOH produced MetHb in a concentration dependent manner without the need for metabolic activation. Benzocaine in the presence of metabolic activation required a concentration of 500 µM to produce a similar degree of MetHb formation as 20 µM BenzNOH without activation. Previous work suggested that two metabolites of lidocaine may also form MetHb; N-hydroxyxylidine and 4-hydroxyxylidine. Of these two metabolites 4-hydroxyxylidine produced the most MetHb in whole blood in vitro in the absence of metabolic activation, however BenzNOH produced up to 14.2 times more MetHb than 4-hydroxyxylidine at a similar concentration. 4. These results suggest that the ability of benzocaine to form MetHb is likely to be mediated through its hydroxylamine metabolite and that this metabolite is inherently more active than the potentially MetHb-forming metabolites of lidocaine.

More methemoglobin is produced by benzocaine treatment than lidocaine treatment in human in vitro systems.

The clinical use of local anesthetic products to anesthetize mucous membranes has been associated with methemoglobinemia (MetHba), a serious condition in which the blood has reduced capacity to carry oxygen. An evaluation of spontaneous adverse event reporting of MetHba submitted to FDA through 2013 identified 375 reports associated with benzocaine and 16 reports associated with lidocaine. The current study was performed to determine the relative ability of benzocaine and lidocaine to produce methemoglobin (MetHb) in vitro. Incubation of 500µM benzocaine with whole human blood and pooled human liver S9 over 5h resulted in MetHb levels equaling 39.8±1.2% of the total hemoglobin. No MetHb formation was detected for 500µM lidocaine under the same conditions. Because liver S9 does not readily form lidocaine hydrolytic metabolites based on xylidine, a primary metabolic pathway, 500µM xylidine was directly incubated with whole blood and S9. Under these conditions MetHb levels of 4.4±0.4% were reached by 5h. Studies with recombinant cytochrome P450 revealed benzocaine to be extensively metabolized by CYP 1A2, with 2B6, 2C19, 2D6, and 2E1 also having activity. We conclude that benzocaine produces much more MetHb in in vitro systems than lidocaine or xylidine and that benzocaine should be more likely to cause MetHba in vivo as well.

The iron exporter ferroportin 1 is essential for development of the mouse embryo, forebrain patterning and neural tube closure.

Neural tube defects (NTDs) are some of the most common birth defects observed in humans. The incidence of NTDs can be reduced by peri-conceptional folic acid supplementation alone and reduced even further by supplementation with folic acid plus a multivitamin. Here, we present evidence that iron maybe an important nutrient necessary for normal development of the neural tube. Following implantation of the mouse embryo, ferroportin 1 (Fpn1) is essential for the transport of iron from the mother to the fetus and is expressed in the visceral endoderm, yolk sac and placenta. The flatiron (ffe) mutant mouse line harbors a hypomorphic mutation in Fpn1 and we have created an allelic series of Fpn1 mutations that result in graded developmental defects. A null mutation in the Fpn1 gene is embryonic lethal before gastrulation, hypomorphic Fpn1(ffe/ffe) mutants exhibit NTDs consisting of exencephaly, spina bifida and forebrain truncations, while Fpn1(ffe/KI) mutants exhibit even more severe NTDs. We show that Fpn1 is not required in the embryo proper but rather in the extra-embryonic visceral endoderm. Our data indicate that loss of Fpn1 results in abnormal morphogenesis of the anterior visceral endoderm (AVE). Defects in the development of the forebrain in Fpn1 mutants are compounded by defects in multiple signaling centers required for maintenance of the forebrain, including the anterior definitive endoderm (ADE), anterior mesendoderm (AME) and anterior neural ridge (ANR). Finally, we demonstrate that this loss of forebrain maintenance is due in part to the iron deficiency that results from the absence of fully functional Fpn1.

The Prevalence of Several Treatments in Preventing the Back Conversion of Acyl Glucuronide Metabolites in Abbreviated New Drug Applications.

The bioanalysis of drugs that undergo acyl glucuronidation presents an analytical challenge due to poor stability of acyl glucuronide metabolites in biological matrices. The objective of this study was to investigate the impact of back conversion of acyl glucuronide metabolites on drug concentration measurement in bioequivalence (BE) studies submitted to Abbreviated New Drug Applications (ANDAs). The prevalence of several treatments for preventing the back conversion of acyl glucuronide metabolites and the results of incurred sample reanalysis (ISR) were analyzed. In total, 322 ANDAs for 26 drugs known to generate acyl glucuronide metabolites were surveyed. Many studies have applied multiple preventive treatments during the clinical and bioanalytical phases. More than two-thirds (67.2%) of the studies utilized procedures of lowering the temperature for sample collection during clinical phase. Fewer studies have utilized procedures for lowering the pH of plasma samples (12.3%) or adding enzyme inhibitors (4.4%) in the clinical phase. A small fraction (16.9%) validated the pre-study method in the presence of the acyl glucuronide metabolites. The majority (62.2%) of the studies employed the procedure of lowering the pH during the sample extraction process in the bioanalytical phase. Among the studies that had significantly higher (p-value < 0.01 by sign test) ISR results than the corresponding original concentration values, 41 BE studies did not carry out any preventive treatments during the bioanalysis phase, suggesting that back conversion of acyl glucuronide metabolites to parent drugs may be present in these studies. The awareness of appropriate treatments of study samples for possible back-conversions of acyl glucuronide metabolites is expected to assist generic drug applicants in improving the quality of their future applications.

Remodeling the exocrine pancreas at metamorphosis in Xenopus laevis.

At metamorphosis the Xenopus laevis tadpole exocrine pancreas remodels in two stages. At the climax of metamorphosis thyroid hormone (TH) induces dedifferentiation of the entire exocrine pancreas to a progenitor state. The organ shrinks to 20% of its size, and approximately 40% of its cells die. The acinar cells lose their zymogen granules and approximately 75% of their RNA. The mRNAs that encode exocrine-specific proteins (including the transcription factor Ptf1a) undergo almost complete extinction at climax, whereas PDX-1, Notch-1, and Hes-1, genes implicated in differentiation of the progenitor cells, are activated. At the end of spontaneous metamorphosis when the endogenous TH has reached a low level, the pancreas begins to redifferentiate. Exogenous TH induces the dedifferentiation phase but not the redifferentation phase. The tadpole pancreas lacks the mature ductal system that is found in adult vertebrate pancreases, including the frog. Exocrine pancreases of transgenic tadpoles expressing a dominant negative form of the TH receptor controlled by the elastase promoter are resistant to TH. They do not shrink when subjected to TH. Their acinar cells do not dedifferentiate at climax, nor do they down-regulate exocrine-specific genes or activate Notch-1 and Hes-1. Even 2 months after metamorphosis these frogs have not developed a mature ductal system and the acinar cells are abnormally arranged. The TH-dependent dedifferentiation of the tadpole acinar cells at climax is a necessary step in the formation of a mature frog pancreas.

Chimeric Ply187 endolysin kills Staphylococcus aureus more effectively than the parental enzyme.

Peptidoglycan hydrolases are an effective new source of antimicrobials. A chimeric fusion protein of the Ply187 endopeptidase domain and LysK SH3b cell wall-binding domain is a potent agent against Staphylococcus aureus in four functional assays.

Inhibition of G-protein-coupled inward rectifying K+ channels by intracellular acidosis.

G-protein-coupled inward rectification K(+) (GIRK) channels play an important role in modulation of synaptic transmission and cellular excitability. The GIRK channels are regulated by diverse intra- and extracellular signaling molecules. Previously, we have shown that GIRK1/GIRK4 channels are activated by extracellular protons. The channel activation depends on a histidine residue in the M1-H5 linker and may play a role in neurotransmission. Here, we show evidence that the heteromeric GIRK1/GIRK4 channels are inhibited by intracellular acidification. This inhibition was produced by selective decrease in the channel open probability with a modest drop in the single-channel conductance. The inhibition does not seem to require G-proteins as it was seen in two G-protein coupling-defective GIRK mutants and in excised patches in the absence of exogenous G-proteins. Three histidine residues in intracellular domains were critical for the inhibition. Individual mutation of His-64, His-228, or His-352 in GIRK4 abolished or greatly diminished the inhibition in homomeric GIRK4. Mutations of any of these histidine residues in GIRK4 or their counterparts in GIRK1 were sufficient to eliminate the pH(i) sensitivity of the heteromeric GIRK1/GIRK4 channels. Thus, the molecular and biophysical bases for the inhibition of GIRK channels by intracellular protons are illustrated. Because of the inequality of the pH(i) and pH(o) in most cells and their relatively independent controls by cellular versus systemic mechanisms, such pH(i) sensitivity may allow these channels to regulate cellular excitability in certain physiological and pathophysiological conditions when intracellular acidosis occurs.

Allosteric modulation of the mouse Kir6.2 channel by intracellular H+ and ATP.

The ATP-sensitive K+ (K(ATP)) channels are regulated by intracellular H+ in addition to ATP, ADP, and phospholipids. Here we show evidence for the interaction of H+ with ATP in regulating a cloned K(ATP) channel, i.e. Kir6.2 expressed with and without the SUR1 subunit. Channel sensitivity to ATP decreases at acidic pH, while the pH sensitivity also drops in the presence of ATP. These effects are more evident in the presence of the SUR1 subunit. In the Kir6.2 + SUR1, the pH sensitivity is reduced by about 0.4 pH units with 100 microM ATP and 0.6 pH units with 1 mM ATP, while a decrease in pH from 7.4 to 6.8 lowers the ATP sensitivity by about fourfold. The Kir6.2 + SUR1 currents are strongly activated at pH 5.9-6.5 even in the presence of 1 mM ATP. The modulations appear to take place at His175 and Lys185 that are involved in proton and ATP sensing, respectively. Mutation of His175 completely eliminates the pH effect on the ATP sensitivity. Similarly, the K185E mutant-channel loses the ATP-dependent modulation of the pH sensitivity. Thus, allosteric modulations of the cloned K(ATP) channel by ATP and H+ are demonstrated. Such a regulation allows protons to activate directly the K(ATP) channels and release channel inhibition by intracellular ATP; the pH effect is further enhanced with a decrease in ATP concentration as seen in several pathophysiological conditions.

Molecular determinants for activation of G-protein-coupled inward rectifier K+ (GIRK) channels by extracellular acidosis.

Synaptic cleft acidification occurs following vesicle release. Such a pH change may affect synaptic transmissions in which G-protein-coupled inward rectifier K(+) (GIRK) channels play a role. To elucidate the effect of extracellular pH (pH(o)) on GIRK channels, we performed experiments on heteromeric GIRK1/GIRK4 channels expressed in Xenopus oocytes. A decrease in pH(o) to 6.2 augmented GIRK1/GIRK4 currents by approximately 30%. The channel activation was reversible and dependent on pH(o) levels. This effect was produced by selective augmentation of single channel conductance without change in the open-state probability. To determine which subunit was involved, we took advantage of homomeric expression of GIRK1 and GIRK4 by introducing a single mutation. We found that homomeric GIRK1-F137S and GIRK4-S143T channels were activated at pH(o) 6.2 by approximately 20 and approximately 70%, respectively. Such activation was eliminated when a histidine residue in the M1-H5 linker was mutated to a non-titratable glutamine, i.e. H116Q in GIRK1 and H120Q in GIRK4. Both of these histidines were required for pH sensing of the heteromeric channels, because the mutation of one of them diminished but not abolished the pH(o) sensitivity. The pH(o) sensitivity of the heteromeric channels was completely lost when both were mutated. Thus, these results suggest that the GIRK-mediated synaptic transmission is determined by both neurotransmitter and protons with the transmitter accounting for only 70% of the effect on postsynaptic cell and protons released together with the transmitter contributing to the other 30%.

Molecular basis for the inhibition of G protein-coupled inward rectifier K(+) channels by protein kinase C.

G protein-coupled inward rectifier K(+) (GIRK) channels regulate cellular excitability and neurotransmission. The GIRK channels are activated by a number of inhibitory neurotransmitters through the G protein betagamma subunit (G(betagamma)) after activation of G protein-coupled receptors and inhibited by several excitatory neurotransmitters through activation of phospholipase C. If the inhibition is produced by PKC, there should be PKC phosphorylation sites in GIRK channel proteins. To identify the PKC phosphorylation sites, we performed systematic mutagenesis analysis on GIRK4 and GIRK1 subunits expressed in Xenopus oocytes. Our data showed that the heteromeric GIRK1/GIRK4 channels were inhibited by a PKC activator phorbol 12-myristate 13-acetate (PMA) through reduction of single channel open-state probability. Direct application of the catalytic subunit of PKC to excised patches had a similar inhibitory effect. This inhibition was greatly eliminated by mutation of Ser-185 in GIRK1 and Ser-191 in GIRK4 that remained G protein sensitive. The PKC-dependent phosphorylation seems to mediate the channel inhibition by the excitatory neurotransmitter substance P (SP) as specific PKC inhibitors and mutation of these PKC phosphorylation sites abolished the SP-induced inhibition of GIRK1/GIRK4 channels. Thus, these results indicate that the PKC-dependent phosphorylation underscores the inhibition of GIRK channels by SP, and Ser-185 in GIRK1 and Ser-191 in GIRK4 are the PKC phosphorylation sites.