GC-NICI-MS analysis of acetazolamide and other sulfonamide (R-SO2-NH2)drugs as pentafluorobenzyl derivatives [R-SO2-N(PFB)2] and quantification of pharmacological acetazolamide in human urine

Acetazolamide(molecular mass(MM),222)belongs to the class of sulfonamides(R-SO2-NH2)and is one of the strongest pharmacological inhibitors of carbonic anhydrase activity.Acetazolamide is excreted unchanged in the urine.Here,we report on the development,validation and biomedical application of a stable-isotope dilution GC-MS method for the reliable quantitative determination of acetazolamide in human urine.The method is based on evaporation to dryness of 50 mL urine aliquots,base-catalyzed derivatization of acetazolamide(d0-AZM)and its internal standard[acetylo-2H3]acetazolamide(d3-AZM)in 30 vol%pentafluorobenzyl(PFB)bromide in acetonitrile(60 min,30C),reconstitution in toluene(200 mL)and injection of 1-mL aliquots.The negative-ion chemical ionization(NICI)mass spectra(methane)of the PFB derivatives contained several intense ions including[M]‒at m/z 581 for d0-AZM and m/z 584 for d3-AZM,suggesting derivatization of their sulfonamide groups to form N,N-dipentafluorobenzyl derivatives(R-SO2-N(PFB)2),i.e.,d0-AZM-(PFB)2 and d3-AZM-(PFB)2,respectively.Quantification was performed by selected-ion monitoring of m/z 581 and 83 for d0-AZM-(PFB)2 and m/z 584 and 86 for d3-AZM-(PFB)2.The limits of detection and quantitation of the method were determined to be 300 fmol(67 pg)and 1 mM of acetazolamide,respectively.Intra-and inter-assay precision and accuracy for acetazolamide in human urine samples in pharmacologically relevant concentration ranges were determined to be 0.3%e4.2%and 95.3%e109%,respectively.The method was applied to measure urinary acetazolamide excretion after ingestion of a 250 mg acetazolamide-containing tablet(Acemit®)by a healthy volunteer.Among other tested sulfonamide drugs,methazolamide(MM,236)was also found to form a N,N-dipentafluorobenzyl derivative,whereas dorzolamide(MM,324)was hardly detectable.No GC-MS peaks were obtained from the PFB bromide derivatization of hydrochlorothiazide(MM,298),xipamide(MM,355),indapamide and metholazone(MM,366 each)or brinzolamide(MM,384).We demonstrate for the first time that sulfonamide drugs can be derivatized with PFB bromide and quantitated by GC-MS.Sulfonamides with MM larger than 236 are likely to be derivatized by PFB bromide but to lack thermal stability.

Topical delivery of acetazolamide by encapsulating in mucoadhesive nanoparticles

The intent of this study was to provide topical delivery of acetazolamide by preparing chitosan-STPP (sodium tripolyphosphate) nanoparticles of acetazolamide and evaluate the particle size, zeta potential, drug entrapment, particle morphology;in vitro drug release and in vivo efficacy. The particles showed sustained in vitro drug release which followed the Higuchi kinetic model. The results indicate that the nanoparticles released the drug by a combination of dissolution and diffusion. The optimised formulation was having particle size 188.46 ± 8.53 nm and zeta potential + 36.86 ± 0.70 mV. The particles were spherical with a polydispersity index of 0.22 ± 0.00. Powder X-ray diffraction and differential scanning calorimetry indicated diminished crystallinity of drug in the nanoparticle formulation. In the in vitro permeation study, the nanoparticle formulation showed elevated permeation as compared to that of drug solution with negative signs of corneal damage. In vitro mucoadhesion studies showed 90.34 ± 1.12% mucoadhesion. The in vivo studies involving ocular hypotensive activity in rabbits revealed significantly higher hypotensive activity (P < 0.05) as compared with plain drug solution with no signs of ocular irritation. The stability studies revealed that formulation was quite stable.

Pharmacological potential of acetazolamide in traumatic intracranial hypertension

Traumatic brain injuries are an important cause of morbidity and mortality around the world. These types of lesions are often associated with increased intracranial pressure and cerebral edema, proper management of this can reduce tissue damage of the brain and improve brain perfusion. The use of acetazolamide is not indicated in guidelines for the management of intracranial hypertension, which is used to a great extent for the management of idiopathic intracranial hypertension. However, it is not yet known in the management of traumatic intracranial hypertension.

Paradoxical reduction of cerebral blood flow after acetazolamide loading:a hemodynamic and metabolic study with ^(15)O PET

Paradoxical reduction of cerebral blood flow （CBF） after administration of the vasodilator acetazolamide is the most severe stage of cerebrovascular reactivity failure and is often associated with an increased oxygen extraction fraction （OEF）. In this study, we aimed to reveal the mechanism underlying this phenomenon by focusing on the ratio of CBF to cerebral blood volume （CBV） as a marker of regional cerebral perfusion pressure （CPP）. In 37 patients with unilateral internal carotid or middle cerebral arterial （MCA） steno-occlusive disease and 8 normal controls, the baseline CBF （CBFb）, CBV, OEF, cerebral oxygen metabolic rate （CMRO2）, and CBF after acetazolamide loading in the anterior and posterior MCA territories were measured by （15）~O positron emission tomography. Paradoxical CBF reduction was found in 28 of 74 regions （18 of 37 patients） in the ipsilateral hemisphere. High CBFb （〉47.6 mL/100 mL/min, n = 7） was associated with normal CBFb/CBV, increased CBV, decreased OEF, and normal CMRO2. Low CBFb （〈31.8 mL/100 mL/min, n = 9） was associated with decreased CBFb/CBV, increased CBV, increased OEF, and decreased CMRO2. These findings demonstrated that paradoxical CBF reduction is not always associated with reduction of CPP, but partly includes high- CBFb regions with normal CPP, which has not been described in previous studies.

Synthesis and anticholinesterase activity of a novel series of acetazolamide condensed 1,4-dihydropyridines

A novel Acetazolamide condensed 1,4-dihydropyridines was set up by treating of N-(5-acetamido-1,3,4-thiadiazol-2-ylsulfonyl)-3-oxobutanamide with an aryl aldehyde and 25-30%alkali with sight amount of barium nitrate as a catalyst.Confirmation of the synthetic structure of the titled compounds(4-16)was substantiated by thin-layer chromatography(TLC),IR,^(1)H NMR,^(13)C NMR,Mass spectra(MS)and elemental analysis(C,H,and N)were finished.The titled compounds were assessed for anticholinesterase activity against acetylcholinesterase and butyl cholinesterase enzymes.The titled compound produced weak,moderate,or high anticholinesterase activity.Particularly,compound 8 demonstrated the best anticholinesterase activity of all the 1,4-dihydropyridines,with an IC_(50) estimation of 0.08μM and 2.9μM.

Aquaglyceroporins but not orthodox aquaporins are involved in the cryotolerance of pig spermatozoa

Background:Aquaporins(AQPs)are a family of transmembrane water channels that includes orthodox AQPs,aquaglyceroporins(GLPs)and super AQPs.AQP3,AQP7,AQP9 and AQP11 have been identified in boar sperm,and they are crucial for sperm maturation and osmoregulation.Water exchange is an important event in cryopreservation,which is the most efficient method for long-term storage of sperm.However,the freezethaw process leads to sperm damage and a loss of fertilizing potential.Assuming that the quality of frozenthawed sperm partially depends on the regulation of osmolality variations during this process,AQPs might play a crucial role in boar semen freezability.In this context,the aim of this study was to unravel the functional relevance of the different groups of AQPs for boar sperm cryotolerance through three different inhibitors.Results:Inhibition of different groups of AQPs was found to have different effects on boar sperm cryotolerance.Whereas the use of 1,3-propanediol(PDO),an inhibitor of orthodox AQPs and GLPs,decreased total motility(P<0.05),it increased post-thaw sperm viability,lowered membrane lipid disorder and increased mitochondrial membrane potential(MMP)(P<0.05).When acetazolamide(AC)was used as an inhibitor of orthodox AQPs,the effects on post-thaw sperm quality were restricted to a mild increase in MMP in the presence of the intermediate concentration at 30 min post-thaw and an increase in superoxide levels(P<0.05).Finally,the addition of phloretin(PHL),a GLP inhibitor,had detrimental effects on post-thaw total and progressive sperm motilities,viability and lipid membrane disorder(P<0.05).Conclusions:The effects of the different inhibitors suggest that GLPs rather than orthodox AQPs are relevant for boar sperm freezability.Moreover,the positive effect of PDO on sperm quality suggests a cryoprotective role for this molecule.

<i>Blastomyces dermatitidis</i>: Chitinase Homology Model, <i>in Silico</i>Docking, and Inhibition Assay

Blastomyces dermatitidis is a thermally dimorphic fungus that causes the disease blastomycosis. Currently there are a limited number of effective treatments, many of which have harsh side effects. Chitin, a component of the fungal cell wall is often broken down and recycled for cell wall remodeling and growth. Chitinase is the digestive enzyme capable of chitin hydrolysis. By inhibiting the chitinase we predicted that cells wouldn’t be able to divide and multiply normally, thereby leading to possible anti-fungal treatments. For this study we modeled the structure of B. dermatitidis chitinase, using homology modeling. By predicting a three-dimensional structure we were able to do additional analyses of the active site of the chitinase and predict the binding of a possible small molecule, acetazolamide, in silico. This binding allowed us to predict that this molecule might be capable of inhibiting the chitinase of B. dermatitidis. This inhibition was tested in vivo. No difference in the growth curves of the test and control organisms was observed, however there was a difference within the cell walls of the yeast cells. The cell walls appeared thicker with additional differences in cell wall orderly growth. These changes are consistent with changes that may occur as B. dermatitidis chitinases are inhibited.

A Unified Management for Spontaneous CSF Leak

Background: Spontaneous CSF leak represents less frequent cause of CSF leak, but cases are more difficult to control, with the highest failure rate and recurrence despite adequate repair. The problems in these cases might be related to an underlying undiagnosed associated intracranial hyper-tension. Recognition and long-term treatment of elevated ICP is therefore critical to the successful management of these patients. Objective: To evaluate the CSF pressure in cases of spontaneous CSF rhinorrhea and to describe our same setting combined protocol to the repair of the leak, measurement and management of CSF pressure. Patients and Methods: All patients presenting to Ain Shams University Hospitals, Cairo, Egypt, with spontaneous CSF leak were included prospectively in the study. Clinical and radiologic data were collected to suspect elevated intracranial pressure. After CSF repair, CSF pressure was measured and if found to be more than 20 cmH2O, a lumboperitoneal shunt was used. Results: Twenty-seven cases, 23 women and 4 men, presented with spontaneous CSF leak. 23 patients had BMI above 30. All patients had empty sella syndrome (100%), and a meningoencephalocele was found in 13 cases (48%). CSF pressure ranged from 5 to 39 cmH2O (mean = 28.7). A pressure above 21 cmH2O was found in19 patients (70%) and subsequently had lumboperitoneal shunt in the same setting. No recurrence occurred in this subset of patients with 6 - 60 months follow-up period. Conclusion: A selective and specific same setting protocol can result in a better diagnosis and control of the accompanying elevated ICP in cases of spontaneous CSF leak. It avoids a second operative intervention, and shortens the hospital stay, with an increase in the success rate. In the same time, the smaller number of patients with normal ICP can avoid further drainage.

Inhibitory Effect of Furosemide on Carbonic Anhydrase

This study investigated the inhibitory effect of a high efficiency diuretic, furosemide, on carbonic anhydrase （CA）. First, comparing the inhibitory effect of acetazolamide, a low efficiency diuretic, on CA, shows that furosemide or acetazolamide can quickly make CA inactive when its concentration is close to the enzyme concentration, different from the usual inhibitory kinetics in which the concentration of the inhibitor is far higher than the enzyme concentration. Secondly, the reaction of the enzyme indicates that the inhibitory effect of furosemide or acetazolamide on carbonic anhydrase is quickly reversible. Finally, the degree of the inhibitory effect of furosemide and of acetazolamide on CA are compared. The results show that furosemide inhibits CA less than acetazolamide.

Anti-glaucoma potential of Heliotropium indicum Linn in experimentally-induced glaucoma

Background:Heliotropium indicum is used as a traditional remedy for hypertension in Ghana.The aim of the study was to evaluate the anti-glaucoma potential of an aqueous whole plant extract of H.indicum to manage experimentally-induced glaucoma.Methods:The percentage change in intraocular pressure(IOP),after inducing acute glaucoma(15 mLkg^(−1) of 5%dextrose,i.v.),in New Zealand White rabbits pretreated with Heliotropium indicum aqueous extract(HIE)(30–300 mgkg^(−1)),acetazolamide(5 mgkg^(−1)),and normal saline(10 mLkg^(−1))per os were measured.IOPs were also monitored in chronic glaucoma in rabbits(induced by 1%prednisolone acetate drops,12 hourly for 21 days)after treatments with the same doses of HIE,acetazolamide,and normal saline for 2 weeks.The anti-oxidant property of the extract was assessed by assaying for glutathione levels in the aqueous humour.Glutamate concentration in the vitreous humour was also determined using ELISA technique.Histopathological assessment of the ciliary bodies was made.Results:The extract significantly reduced intraocular pressure(p≤0.05–0.001)in acute and chronic glaucoma,preserved glutathione levels and glutamate concentration(p≤0.01–0.001).Histological assessment of the ciliary body showed a decrease in inflammatory infiltration in the extract and acetazolamide-treated group compared with the normal saline-treated group.Conclusion:The aqueous whole plant extract of Heliotropium indicum has ocular hypotensive,anti-oxidant and possible neuro-protective effects,which therefore underscore its plausible utility as an anti-glaucoma drug with further investigation.

Perioperative Evaluation of Cerebral Blood Flow Using ^123I-labeled N-isopropyl-p-iodoamphetamine Single-Photon Emission Computed Tomography without Blood Sampling in Patients Who Underwent Carotid Artery Stenting

The evaluation of regional cerebral vascular reserve （rCVR） with single-photon emission computed tomography （SPECT） is useful for predicting cerebral hyperperfusion following carotid artery stenting （CAS） and carotidendarterectomy （CEA）.