Combined methazolamide and theophylline improves oxygen saturation but not exercise performance or altitude illness in acute hypobaric hypoxia.

NEW FINDINGS: What is the central question of this study? Does the combination of methazolamide and theophylline reduce symptoms of acute mountain sickness (AMS) and improve aerobic performance in acute hypobaric hypoxia? What is the main finding and its importance? The oral combination of methazolamide (100 BID) and theophylline (300 BID) improved arterial oxygen saturation but did not reduce symptoms of AMS and impaired aerobic performance. We do not recommend this combination of drugs for prophylaxis against the acute negative effects of hypobaric hypoxia. ABSTRACT: A limited number of small studies have suggested that methazolamide and theophylline can independently reduce symptoms of acute mountain sickness (AMS) and, if taken together, can improve aerobic exercise performance in normobaric hypoxia. We performed a randomized, double-blind, placebo-controlled, cross-over study to determine if the combination of oral methazolamide and theophylline could provide prophylaxis against AMS and improve aerobic performance in hypobaric hypoxia (∼4875 m). Volunteers with histories of AMS were screened at low altitude (1650 m) and started combined methazolamide (100 mg BID) and theophylline (300 mg BID) treatment, or placebo, 72 h prior to decompression. Baseline AMS (Lake Louise Questionnaire), blood (haemoglobin, haematocrit), cognitive function, ventilatory and pulse oximetry ( SpO2 ) measures were assessed at low altitude and repeated between 4 and 10 h of exposure to hypobaric hypoxia (PB  = 425 mmHg). Aerobic exercise performance was assessed during a 12.5 km cycling time trial (TT) after 4 h of hypobaric hypoxia. Subjects repeated all experimental procedures after a 3-week washout period. Differences between drug and placebo trials were evaluated using repeated measures ANOVA (α = 0.05). The drugs improved resting SpO2 by ∼4% (P < 0.01), but did not affect the incidence or severity of AMS or cognitive function scores relative to placebo. Subjects' performance on the 12.5 km TT was ∼3% worse when taking the drugs (P < 0.01). The combination of methazolamide and theophylline in the prescribed dosages is not recommended for use at high altitude as it appears to have no measurable effect on AMS and can impair aerobic performance.

Real-Time Quantitative In-Cell NMR: Ligand Binding and Protein Oxidation Monitored in Human Cells Using Multivariate Curve Resolution.

In-cell NMR can investigate protein conformational changes at atomic resolution, such as those changes induced by drug binding or chemical modifications, directly in living human cells, and therefore has great potential in the context of drug development as it can provide an early assessment of drug potency. NMR bioreactors can greatly improve the cell sample stability over time and, more importantly, allow for recording in-cell NMR data in real time to monitor the evolution of intracellular processes, thus providing unique insights into the kinetics of drug-target interactions. However, current implementations are limited by low cell viability at >24 h times, the reduced sensitivity compared to "static" experiments and the lack of protocols for automated and quantitative analysis of large amounts of data. Here, we report an improved bioreactor design which maintains human cells alive and metabolically active for up to 72 h, and a semiautomated workflow for quantitative analysis of real-time in-cell NMR data relying on Multivariate Curve Resolution. We apply this setup to monitor protein-ligand interactions and protein oxidation in real time. High-quality concentration profiles can be obtained from noisy 1D and 2D NMR data with high temporal resolution, allowing further analysis by fitting with kinetic models. This unique approach can therefore be applied to investigate complex kinetic behaviors of macromolecules in a cellular setting, and could be extended in principle to any real-time NMR application in live cells.

Influence of methazolamide on the human control of breathing: A comparison to acetazolamide.

NEW FINDINGS: What is the central question of this study? Acetazolamide and methazolamide both reduce hypoxic pulmonary vasoconstriction equally, but methazolamide does not impair skeletal muscle function. The effect of methazolamide on respiratory control in humans is not yet known. What is the main finding and its importance? Similar to acetazolamide after chronic oral administration, methazolamide causes a metabolic acidosis and shifts the ventilatory CO2 response curve leftwards without reducing O2 sensitivity. The change in ventilation over the change in log PO2 provides a more accurate measure of hypoxic sensitivity than the change in ventilation over the change in arterial oxyhaemoglobin saturation. ABSTRACT: Acetazolamide is used to prevent/treat acute mountain sickness and both central and obstructive sleep apnoea. Methazolamide, like acetazolamide, reduces hypoxic pulmonary vasoconstriction, but has fewer side-effects, including less impairment of skeletal muscle function. Given that the effects of methazolamide on respiratory control in humans are unknown, we compared the effects of oral methazolamide and acetazolamide on ventilatory control and determined the ventilation-log  PO2 relationship in humans. In a double-blind, placebo-controlled, randomized cross-over design, we studied the effects of acetazolamide (250 mg three times daily), methazolamide (100 mg twice daily) and placebo in 14 young male subjects who were exposed to 7 min of normoxic hypercapnia and to three levels of eucapnia and hypercapnic hypoxia. With placebo, methazolamide and acetazolamide, the CO2 sensitivities were 2.39 ± 1.29, 3.27 ± 1.82 and 2.62 ± 1.79 l min-1  mmHg-1 (n.s.) and estimated apnoeic thresholds 32 ± 3, 28 ± 3 and 26 ± 3 mmHg, respectively (P < 0.001, placebo versus methazolamide and acetazolamide). The relationship between ventilation ( V̇I ) and log  PO2 (using arterialized venous PO2 in hypoxia) was linear, and neither agent influenced the relationship between hypoxic sensitivity ( ΔV̇I/ΔlogPO2 ) and arterial [H+ ]. Using ΔV̇I/ΔlogPO2 rather than Δ V̇I /Δ arterial oxyhaemoglobin saturation enables a more accurate estimation of oxygenation and ventilatory control in metabolic acidosis/alkalosis when right- or leftward shifts of the oxyhaemoglobin saturation curve occur. Given that acetazolamide and methazolamide have similar effects on ventilatory control, methazolamide might be preferred for indications requiring the use of a carbonic anhydrase inhibitor, avoiding some of the negative side-effects of acetazolamide.

Sulfonamide inhibition studies of the ß-carbonic anhydrase from the newly discovered bacterium Enterobacter sp. B13.

The genome of the newly identified bacterium Enterobacter sp. B13 encodes for a ß-class carbonic anhydrases (CAs, EC 4.2.1.1), EspCA. This enzyme was recently cloned, and characterized kinetically by this group (J. Enzyme Inhib. Med. Chem. 2016, 31). Here we report an inhibition study with sulfonamides and sulfamates of this enzyme. The best EspCA inhibitors were some sulfanylated sulfonamides with elongated molecules, metanilamide, 4-aminoalkyl-benzenesulfonamides, acetazolamide, and deacetylated methazolamide (KIs in the range of 58.7-96.5nM). Clinically used agents such as methazolamide, ethoxzolamide, dorzolamide, brinzolamide, benzolamide, zonisamide, sulthiame, sulpiride, topiramate and valdecoxib were slightly less effective inhibitors (KIs in the range of 103-138nM). Saccharin, celecoxib, dichlorophenamide and many simple benzenesulfonamides were even less effective as EspCA inhibitors, with KIs in the range of 384-938nM. Identification of effective inhibitors of this bacterial enzyme may lead to pharmacological tools useful for understanding the physiological role(s) of the ß-class CAs in bacterial pathogenicity/virulence.

Chemical genetics unveils a key role of mitochondrial dynamics, cytochrome c release and IP3R activity in muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a neuromuscular disease caused by mutations in the dystrophin gene. The subcellular mechanisms of DMD remain poorly understood and there is currently no curative treatment available. Using a Caenorhabditis elegans model for DMD as a pharmacologic and genetic tool, we found that cyclosporine A (CsA) reduces muscle degeneration at low dose and acts, at least in part, through a mitochondrial cyclophilin D, CYN-1. We thus hypothesized that CsA acts on mitochondrial permeability modulation through cyclophilin D inhibition. Mitochondrial patterns and dynamics were analyzed, which revealed dramatic mitochondrial fragmentation not only in dystrophic nematodes, but also in a zebrafish model for DMD. This abnormal mitochondrial fragmentation occurs before any obvious sign of degeneration can be detected. Moreover, we demonstrate that blocking/delaying mitochondrial fragmentation by knocking down the fission-promoting gene drp-1 reduces muscle degeneration and improves locomotion abilities of dystrophic nematodes. Further experiments revealed that cytochrome c is involved in muscle degeneration in C. elegans and seems to act, at least in part, through an interaction with the inositol trisphosphate receptor calcium channel, ITR-1. Altogether, our findings reveal that mitochondria play a key role in the early process of muscle degeneration and may be a target of choice for the design of novel therapeutics for DMD. In addition, our results provide the first indication in the nematode that (i) mitochondrial permeability transition can occur and (ii) cytochrome c can act in cell death.

Differential contribution of isoaspartate post-translational modifications to the fibrillization and toxic properties of amyloid ß and the Asn23 Iowa mutation.

Mutations within the Aß (amyloid ß) peptide, especially those clustered at residues 21-23, are linked to early-onset AD (Alzheimer's disease) and primarily associated with cerebral amyloid angiopathy. The Iowa variant, a substitution of an aspartic acid residue for asparagine at position 23 (D23N), associates with widespread vascular amyloid and abundant diffuse pre-amyloid lesions significantly exceeding the incidence of mature plaques. Brain Iowa deposits consist primarily of a mixture of mutated and non-mutated Aß species exhibiting partial aspartate isomerization at positions 1, 7 and 23. The present study analysed the contribution of the post-translational modification and the D23N mutation to the aggregation/fibrillization and cell toxicity properties of Aß providing insight into the elicited cell death mechanisms. The induction of apoptosis by the different Aß species correlated with their oligomerization/fibrillization propensity and ß-sheet content. Although cell toxicity was primarily driven by the D23N mutation, all Aß isoforms tested were capable, albeit at different time frames, of eliciting comparable apoptotic pathways with mitochondrial engagement and cytochrome c release to the cytoplasm in both neuronal and microvascular endothelial cells. Methazolamide, a cytochrome c release inhibitor, exerted a protective effect in both cell types, suggesting that pharmacological targeting of mitochondria may constitute a viable therapeutic avenue.

Optimization of methazolamide-loaded solid lipid nanoparticles for ophthalmic delivery using Box-Behnken design.

The purpose of the present study was to optimize methazolamide (MTZ)-loaded solid lipid nanoparticles (SLNs) which were used as topical eye drops by evaluating the relationship between design factors and experimental data. A three factor, three-level Box-Behnken design (BBD) was used for the optimization procedure, choosing the amount of GMS, the amount of phospholipid, the concentration of surfactant as the independent variables. The chosen dependent variables were entrapment efficiency, dosage loading, and particle size. The generated polynomial equations and response surface plots were used to relate the dependent and independent variables. The optimal nanoparticles were formulated with 100 mg GMS, 150 mg phospholipid, and 1% Tween80 and PEG 400 (1:1, w/v). A new formulation was prepared according to these levels. The observed responses were close to the predicted values of the optimized formulation. The particle size was 197.8 ± 4.9 nm. The polydispersity index of particle size was 0.239 ± 0.01 and the zeta potential was 32.7 ± 2.6 mV. The entrapment efficiency and dosage loading were about 68.39% and 2.49%, respectively. Fourier transform infrared spectroscopy (FT-IR) study indicated that the drug was entrapped in nanoparticles. The optimized formulation showed a sustained release followed the Peppas model. MTZ-SLNs showed significant prolonged decreasing intraocular pressure effect comparing with MTZ solution in vivo pharmacodynamics studies. The results of acute eye irritation study indicated that MTZ-SLNs and AZOPT both had no eye irritation. Furthermore, the MTZ-SLNs were suitable to be stored at low temperature (4 °C).

The Impact of Acetazolamide and Methazolamide on Exercise Performance in Normoxia and Hypoxia.

Doherty, Connor J., Jou-Chung Chang, Benjamin P. Thompson, Erik R. Swenson, Glen E. Foster, and Paolo B. Dominelli. The impact of acetazolamide and methazolamide on exercise performance in normoxia and hypoxia. High Alt Med Biol. 24:7-18, 2023.-Carbonic anhydrase (CA) inhibitors are commonly prescribed for acute mountain sickness (AMS). In this review, we sought to examine how two CA inhibitors, acetazolamide (AZ) and methazolamide (MZ), affect exercise performance in normoxia and hypoxia. First, we briefly describe the role of CA inhibition in facilitating the increase in ventilation and arterial oxygenation in preventing and treating AMS. Next, we detail how AZ affects exercise performance in normoxia and hypoxia and this is followed by a discussion on MZ. We emphasize that the overarching focus of the review is how the two drugs potentially affect exercise performance, rather than their ability to prevent/treat AMS per se, their interrelationship will be discussed. Overall, we suggest that AZ hinders exercise performance in normoxia, but may be beneficial in hypoxia. Based upon head-to-head studies of AZ and MZ in humans on diaphragmatic and locomotor strength in normoxia, MZ may be a better CA inhibitor when exercise performance is crucial at high altitude.

Duodenal acidity "sensing" but not epithelial HCO3- supply is critically dependent on carbonic anhydrase II expression.

Carbonic anhydrase (CA) is strongly expressed in the duodenum and has been implicated in a variety of physiological functions including enterocyte HCO(3)(-) supply for secretion and the "sensing" of luminal acid and CO(2). Here, we report the physiological role of the intracellular CAII isoform involvement in acid-, PGE(2,) and forskolin-induced murine duodenal bicarbonate secretion (DBS) in vivo. CAII-deficient and WT littermates were studied in vivo during isoflurane anesthesia. An approximate 10-mm segment of the proximal duodenum with intact blood supply was perfused under different experimental conditions and DBS was titrated by pH immediately. Two-photon confocal microscopy using the pH-sensitive dye SNARF-1F was used to assess duodenocyte pH(i) in vivo. After correction of systemic acidosis by infusion of isotonic Na(2)CO(3), basal DBS was not significantly different in CAII-deficient mice and WT littermates. The duodenal bicarbonate secretory response to acid was almost abolished in CAII-deficient mice, but normal to forskolin- or 16,16-dimethyl PGE(2) stimulation. The complete inhibition of tissue CAs by luminal methazolamide and i.v. acetazolamide completely blocked the response to acid, but did not significantly alter the response to forskolin. While duodenocytes acidified upon luminal perfusion with acid, no significant pH(i) change occurred in CAII-deficient duodenum in vivo. The results suggest that CA II is important for duodenocyte acidification by low luminal pH and for eliciting the acid-mediated HCO(3)(-) secretory response, but is not important in the generation of the secreted HCO(3)(-) ions.

Cardioprotective effects of N-methylacetazolamide mediated by inhibition of L-type Ca2+ channel current.

Our objective was to examine the effects of N-methylacetazolamide (NMA), a non­carbonic anhydrase inhibitor, on ischemia-reperfusion injury. Isolated rat hearts were assigned to the following groups: 1) Non-ischemic control (NIC):110 min of perfusion and 2) Ischemic control (IC): 30 min of global ischemia and 60 min of reperfusion (R). Both groups were repeated in presence of NMA (5 µM), administered during the first 10 min of R. Infarct size (IS) was measured by TTC staining. Developed pressure (LVDP) and end-diastolic pressure (LVEDP) of the left ventricle were used to assess systolic and diastolic function, respectively. The content of P-Akt, P-PKCε, P-Drp1 and calcineurin Aß were measured. In cardiomyocytes the L-type Ca2+ current (ICaL) was recorded with the whole-cell configuration of patch-clamp technique. The addition of NMA to non-ischemic hearts decreased 15% the contractility. In ischemic hearts (IC group), NMA decreased IS (22 ± 2% vs 32 ± 2%, p < 0.05) and improved the post-ischemic recovery of myocardial function. At the end of R, LVDP was 54 ± 7% vs 18 ± 3% and LVEDP was 23 ± 8 vs. 55 ± 7 mmHg ¨p < 0.05¨. The level of P-Akt, P-PKCε and P-Drp1 increased and the expression of calcineurin Aß decreased in NMA treated hearts. Peak ICaL density recorded at 0 mV was smaller in myocytes treated with NMA than in non-treated cells (-1.91 ± 0.15 pA/pF vs -2.32 ± 0.17 pA/pF, p < 0.05). These data suggest that NMA protects the myocardium against ischemia-reperfusion injury through an attenuation of mitochondrial fission by calcineurin/Akt/PKCε-dependent pathways associated to the decrease of ICaL current.

Methazolamide Attenuates the Development of Diabetic Cardiomyopathy by Promoting ß-Catenin Degradation in Type 1 Diabetic Mice.

Methazolamide (MTZ), a carbonic anhydrase inhibitor, has been shown to inhibit cardiomyocyte hypertrophy and exert a hypoglycemic effect in patients with type 2 diabetes and diabetic db/db mice. However, whether MTZ has a cardioprotective effect in the setting of diabetic cardiomyopathy is not clear. We investigated the effects of MTZ in a mouse model of streptozotocin-induced type 1 diabetes mellitus (T1DM). Diabetic mice received MTZ by intragastric gavage (10, 25, or 50 mg/kg, daily for 16 weeks). In the diabetic group, MTZ significantly reduced both random and fasting blood glucose levels and improved glucose tolerance in a dose-dependent manner. MTZ ameliorated T1DM-induced changes in cardiac morphology and dysfunction. Mechanistic analysis revealed that MTZ blunted T1DM-induced enhanced expression of ß-catenin. Similar results were observed in neonatal rat cardiomyocytes (NRCMs) and adult mouse cardiomyocytes treated with high glucose or Wnt3a (a ß-catenin activator). There was no significant change in ß-catenin mRNA levels in cardiac tissues or NRCMs. MTZ-mediated ß-catenin downregulation was recovered by MG132, a proteasome inhibitor. Immunoprecipitation and immunofluorescence analyses showed augmentation of AXIN1-ß-catenin interaction by MTZ in T1DM hearts and in NRCMs treated with Wnt3a; thus, MTZ may potentiate AXIN1-ß-catenin linkage to increase ß-catenin degradation. Overall, MTZ may alleviate cardiac hypertrophy by mediating AXIN1-ß-catenin interaction to promote degradation and inhibition of ß-catenin activity. These findings may help inform novel therapeutic strategy to prevent heart failure in patients with diabetes.

Imatinib and methazolamide ameliorate COVID-19-induced metabolic complications via elevating ACE2 enzymatic activity and inhibiting viral entry.

Coronavirus disease 2019 (COVID-19) represents a systemic disease that may cause severe metabolic complications in multiple tissues including liver, kidney, and cardiovascular system. However, the underlying mechanisms and optimal treatment remain elusive. Our study shows that impairment of ACE2 pathway is a key factor linking virus infection to its secondary metabolic sequelae. By using structure-based high-throughput virtual screening and connectivity map database, followed with experimental validations, we identify imatinib, methazolamide, and harpagoside as direct enzymatic activators of ACE2. Imatinib and methazolamide remarkably improve metabolic perturbations in vivo in an ACE2-dependent manner under the insulin-resistant state and SARS-CoV-2-infected state. Moreover, viral entry is directly inhibited by these three compounds due to allosteric inhibition of ACE2 binding to spike protein on SARS-CoV-2. Taken together, our study shows that enzymatic activation of ACE2 via imatinib, methazolamide, or harpagoside may be a conceptually new strategy to treat metabolic sequelae of COVID-19.

Evaluation of the therapeutic potential of carbonic anhydrase inhibitors in two animal models of dystrophin deficient muscular dystrophy.

Duchenne Muscular Dystrophy is an inherited muscle degeneration disease for which there is still no efficient treatment. However, compounds active on the disease may already exist among approved drugs but are difficult to identify in the absence of cellular models. We used the Caenorhabditis elegans animal model to screen a collection of 1000 already approved compounds. Two of the most active hits obtained were methazolamide and dichlorphenamide, carbonic anhydrase inhibitors widely used in human therapy. In C. elegans, these drugs were shown to interact with CAH-4, a putative carbonic anhydrase. The therapeutic efficacy of these compounds was further validated in long-term experiments on mdx mice, the mouse model of Duchenne Muscular Dystrophy. Mice were treated for 120 days with food containing methazolamide or dichlorphenamide at two doses each. Musculus tibialis anterior and diaphragm muscles were histologically analyzed and isometric muscle force was measured in M. extensor digitorum longus. Both substances increased the tetanic muscle force in the treated M. extensor digitorum longus muscle group, dichlorphenamide increased the force significantly by 30%, but both drugs failed to increase resistance of muscle fibres to eccentric contractions. Histological analysis revealed a reduction of centrally nucleated fibers in M. tibialis anterior and diaphragm in the treated groups. These studies further demonstrated that a C. elegans-based screen coupled with a mouse model validation strategy can lead to the identification of potential pharmacological agents for rare diseases.

Pharmacological characterisation of apical Na+ and Cl- transport mechanisms of the anal papillae in the larval mosquito Aedes aegypti.

The anal papillae of freshwater mosquito larvae are important sites of NaCl uptake, thereby acting to offset the dilution of the hemolymph by the dilute habitat. The ion-transport mechanisms in the anal papillae are not well understood. In this study, the scanning ion-selective electrode technique (SIET) was utilized to measure ion fluxes at the anal papillae, and pharmacological inhibitors of ion transport were utilized to identify ion-transport mechanisms. Na(+) uptake by the anal papillae was inhibited by bafilomycin and phenamil but not by HMA. Cl(-) uptake was inhibited by methazolamide, SITS and DIDS but not by bafilomycin. H(+) secretion was inhibited by bafilomycin and methazolamide. Ouabain and bumetanide had no effect on NaCl uptake or H(+) secretion. Together, the results suggest that Na(+) uptake at the apical membrane occurs through a Na(+) channel that is driven by a V-type H(+)-ATPase and that Cl(-) uptake occurs through a Cl(-)/HCO(3)(-) exchanger, with carbonic anhydrase providing H(+) and HCO(3)(-) to the V-type H(+)-ATPase and exchanger, respectively.

A potential new therapeutic system for glaucoma: solid lipid nanoparticles containing methazolamide.

Methazolamide (MTA) is an antiglaucoma drug; however, there are many side effects of its systemic administration with insufficient ocular therapeutic concentrations. The aim of this study was to formulate MTA-loaded solid lipid nanoparticles (SLNs) and evaluate the potential of SLNs as a new therapeutic system for glaucoma. SLNs were prepared by a modified emulsion-solvent evaporation method and their physicochemical characteristics were evaluated. The pharmacodynamics was investigated by determining the percentage decrease in intraocular pressure. The ocular irritation was studied by Draize test. Despite a burst release of SLNs, the pharmacodynamic experiment indicated that MTA-SLNs had higher therapeutic efficacy, later occurrence of maximum action, and more prolonged effect than drug solution and commercial product. Formulation of MTA-SLNs would be a potential delivery carrier for ocular delivery, with the advantages of a more intensive treatment for glaucoma, lower in doses and better patient compliance compared to the conventional eye drops.

Larvae of the midge Chironomus riparius possess two distinct mechanisms for ionoregulation in response to ion-poor conditions.

This study examined the role of the anal papillae of the freshwater (FW) chironomid larva Chironomus riparius in ionoregulation under ion-poor conditions. The scanning ion-selective electrode technique (SIET) was utilized to characterize the species, direction, and rates of inorganic ion transport by the anal papillae following acute and long-term exposure to ion-poor water (IPW). The major inorganic ions in the hemolymph of larvae treated as above were measured using standard ion-selective microelectrodes. The anal papillae of C. riparius are sites of net NaCl uptake and H(+) secretion under FW and IPW conditions and are not likely to be a major contributor of K(+) exchange. Acute and long-term exposure to IPW increased total net transport of Na(+), Cl(-), and H(+) by the anal papillae, but the mechanisms underlying the increase under the two conditions were different. Acute IPW exposure increased the magnitude of net ion fluxes at sites along the anal papillae, while long-term IPW exposure resulted in increased size of the anal papillae with no change in the magnitude of net ion fluxes. The contribution of the anal papillae to observed alterations of hemolymph ion activities upon exposure to IPW is discussed. Inhibitors of the Na(+)/H(+) exchangers (EIPA) and carbonic anhydrase (methazolamide) provide evidence for Na(+)/H(+) and Cl(-)/HCO(3)(-) exchange mechanisms in the anal papillae. This study demonstrates that C. riparius larvae employ two different mechanisms to upregulate the total net transport of ions by the anal papillae, and these mechanisms are at least partially responsible for regulating hemolymph ion activity.

Preparation and evaluation of in situ gelling ophthalmic drug delivery system for methazolamide.

PURPOSE: In this study, a thermosensitive in situ gelling vehicle was prepared to increase the precorneal resident time and the bioavailability of methazolamide (MTA). METHOD: Poloxamer analogs were used as the gelling agents, and the in situ gel was obtained by using a cold method. The gelation temperature, rheological properties, in vitro release as well as in vivo evaluation (the elimination of MTA in aqueous humor and intraocular-lowering effect) of the optimized formulations were investigated. RESULTS: The optimum concentrations of poloxamer analogs for the in situ gel-forming delivery system were 21% (w/w) poloxamer 407 and 10% (w/w) poloxamer P188. This formulation was able to flow freely under nonphysiological conditions and underwent sol-gel transition in the cul-de-sac upon placement into the eye. In vitro release studies demonstrated a diffusion-controlled release of MTA from the poloxamer solutions over a period of 10 hours. In vivo evaluation indicated that the poloxamer solutions had a better ability to retain drug than MTA eyedrops did. CONCLUSION: These results suggested that in situ gelling ophthalmic drug delivery system may hold some promise in ocular MTA delivery.

Methazolamide in high-altitude illnesses.

As a carbonic anhydrase inhibitor and a methylated lipophilic analogue of acetazolamide, Methazolamide has higher lipid solubility, less plasma protein binding and renal excretion, and fewer side effects, compared to acetazolamide. Methazolamide can increase systemic metabolic acidosis and sequentially improve ventilation and oxygenation level. The increased oxygenation level leads to reduced reactive oxygen species (ROS) production, relived cerebral edema, mitigated hypoxic pulmonary vasoconstriction, abrogated hypoxic fatigue, and decreased excessive erythrocytosis. In addition to the effect as a carbonic anhydrase inhibitor, methazolamide directly activates the transcription factor anti-oxidative nuclear factor-related factor 2 (Nrf2) and inhibits interleukin-1ß (IL-1ß) release. These pharmacological functions of methazolamide are beneficial for the prevention and treatment of high-altitude illnesses. Besides, methazolamide causes less fatigue side effects than acetazolamide does. It is also worth noting that several studies suggested that a lower dose of methazolamide has similar prophylaxis and treatment efficacy in acute mountain sickness (AMS) to a higher dose of acetazolamide. Given methazolamide's advantages over acetazolamide, methazolamide may thus represent an alternative for acetazolamide when taken for high-altitude illnesses prophylaxis and treatment. However, more in-depth clinical trials are needed to fully evaluate this efficacy of methazolamide.

Inhibitors of cytochrome c release with therapeutic potential for Huntington's disease.

Release of mitochondrial cytochrome c resulting in downstream activation of cell death pathways has been suggested to play a role in neurologic diseases featuring cell death. However, the specific biologic importance of cytochrome c release has not been demonstrated in Huntington's disease (HD). To evaluate the role of cytochrome c release, we screened a drug library to identify new inhibitors of cytochrome c release from mitochondria. Drugs effective at the level of purified mitochondria were evaluated in a cellular model of HD. As proof of principle, one drug was chosen for in depth evaluation in vitro and a transgenic mouse model of HD. Our findings demonstrate the utility of mitochondrial screening to identify inhibitors of cell death and provide further support for the important functional role of cytochrome c release in HD. Given that many of these compounds have been approved by the Food and Drug Administration for clinical usage and cross the blood-brain barrier, these drugs may lead to trials in patients.

Methazolamide and melatonin inhibit mitochondrial cytochrome C release and are neuroprotective in experimental models of ischemic injury.

BACKGROUND AND PURPOSE: The identification of a neuroprotective drug for stroke remains elusive. Given that mitochondria play a key role both in maintaining cellular energetic homeostasis and in triggering the activation of cell death pathways, we evaluated the efficacy of newly identified inhibitors of cytochrome c release in hypoxia/ischemia induced cell death. We demonstrate that methazolamide and melatonin are protective in cellular and in vivo models of neuronal hypoxia. METHODS: The effects of methazolamide and melatonin were tested in oxygen/glucose deprivation-induced death of primary cerebrocortical neurons. Mitochondrial membrane potential, release of apoptogenic mitochondrial factors, pro-IL-1beta processing, and activation of caspase -1 and -3 were evaluated. Methazolamide and melatonin were also studied in a middle cerebral artery occlusion mouse model. Infarct volume, neurological function, and biochemical events were examined in the absence or presence of the 2 drugs. RESULTS: Methazolamide and melatonin inhibit oxygen/glucose deprivation-induced cell death, loss of mitochondrial membrane potential, release of mitochondrial factors, pro-IL-1beta processing, and activation of caspase-1 and -3 in primary cerebrocortical neurons. Furthermore, they decrease infarct size and improve neurological scores after middle cerebral artery occlusion in mice. CONCLUSIONS: We demonstrate that methazolamide and melatonin are neuroprotective against cerebral ischemia and provide evidence of the effectiveness of a mitochondrial-based drug screen in identifying neuroprotective drugs. Given the proven human safety of melatonin and methazolamide, and their ability to cross the blood-brain-barrier, these drugs are attractive as potential novel therapies for ischemic injury.