碳酸酐酶抑制剂乙酰唑胺的临床应用进展

碳酸酐酶(CA)能可逆性地催化CO2水合反_应,产生参与人体多种生理功能的H 和HCO3-.现在研究较多是α-CA同工酶CAⅠ,CAⅡ,CA Ⅳ,CA Ⅸ和CA Ⅻ.乙酰唑胺是一种临床应用较早的碳酸酐酶抑制剂,原用于心源性水肿和青光眼的治疗,由于不良反应较多现对上述疾病已失去实用价值.近来研究发现乙酰唑胺对众多组织细胞的CA有广谱抑制作用,使细胞内CO2水合反应正向途径受阻,从而纠正诸多病理状态的生化代谢紊乱,由此扩宽了其一系列临床新用途.文中就乙酰唑胺在急性高原病、慢性高原病、脑血管病、肿瘤、顽固性呃逆、癫痫、代谢性骨病、氧惊厥等方面临床应用的最新进展进行综述.     

Gastric mucosal protection by acetazolamide derivatives: role of carbonic anhydrase and sulfhydryls

Acetazolamide, a carbonic anhydrase inhibitor, prevents acute gastric hemorrhagic lesions induced by ethanol. We used acetazolamide and other carbonic anhydrase inhibitors to correlate their gastroprotective effects with the degree of inhibition of carbonic anhydrase. Since acetazolamide is a thiadiazole, we also investigated structurally related thiadiazoles that contain sulfhydryls to test the hypothesis that the protection against ethanol-induced gastric erosions is related to the presence of sulfhydryls. Dose-response studies with acetazolamide revealed that the protection did not correlate with the inhibition of carbonic anhydrase in the rat gastric mucosa. The carbonic anhydrase inhibitors sulfanilamide and ethoxzolamide, did not offer protection. Bismuthiol I, a thiadiazole with two sulfhydryls, was twice as protective as 2-amino-5-mercapto-1,3,4-thiadiazole with only one sulfhydryl group. We conclude that the protection by acetazolamide against ethanol-induced lesions is not related to the inhibition of carbonic anhydrase in the gastric mucosa. The gastroprotective effect of acetazolamide and its derivatives may be related to their content of sulfhydryls in an oxidized or reduced state.     

Acetazolamide binding to two carbonic anhydrase isoenzymes in human erythrocytes.

Acetazolamide binding to high activity and low activity carbonic anhydrase isoenzymes in red blood cells was studied. Inhibitory constants of 0.041 and 2.72 microM and maximum binding capacities of 17.2 and 155 microM, respectively, were found.     

Indomethacin activates carbonic anhydrase and antagonizes the effect of the specific carbonic anhydrase inhibitor acetazolamide, by a direct mechanism of action.

OBJECTIVES: In this paper we investigated the effect of indomethacin, acetazolamide and their combination in vitro and in vivo on carbonic anhydrase (CA) isozymes. METHOD: In vitro experiments followed the effect of the two substances at concentrations between 10(-8)-10(-4) M on purified human red cell CA I and II as well as on human gastric mucosa CA IV using dose-response relationships. Kinetic studies were also performed. The effects of single and combined administration of indomethacin and acetazolamide on red cell CA and on gastric acid secretion were studied in vivo. RESULTS: Indomethacin, in vitro and in vivo. induces an increase in erythorcyte CA I and CA II activity. Acetazolamide, a specific inhibitor of CA, reduces the activity of CA I and CA II from red cells. Indomethacin completely antagonizes CA activity, i.e. abolishes the inhibitory effect of acetazolamide on CA. In humans, an increase or decrease in erythrocyte CA II activity is correlated with an increase or decrease in gastric acid secretion. CONCLUSIONS: Our results show that indomethacin, a known cyclooxygenase (COX) inhibitor, is also an activator of CA. Our data also prove that indomethacin is not only an activator of CA but also antagonizes the effect of acetazolamide, a specific inhibitor of this enzyme. In view of the role of CA in acid-base balance as well as the fact that an increase or decrease in its activity is accompanied by an increase or decrease in intra- and extracellular pH, our results suggest that: firstly, CA activation induced by indomethacin might cause changes in COX activity; secondly, PGs are synthetized as a consequence of the changes in COX activity, a hypothesis that requires further study.     

3H]ethylketocyclazocine binding to NCB-20 hybrid neurotumor cells

Ethylketocyclazocine (EKC) binds to two sites on NCB-20 neuroblastoma X Chinese hamster brain hybrid cells (KDH = 2 nM, Bmax = 21,000 sites/cell; KDL = 27 nM, Bmax = 140,000 sites/cell. The high-affinity site has been characterized as a delta opiate receptor. The low-affinity site is relatively benzomorphan-specific; opioid peptides, morphine, etorphine, and naloxone do not compete at it. Rank order of potency among benzomorphans is (+)-EKC greater than Mr 2267 greater than (+)-ketocyclazocine greater than (+)-SKF 10047 greater than bremazocine greater than cyclazocine. Among other drugs of interest that inhibit [3H]EKC binding are phencyclidine and its analogues, Ki values for which are 0.2-40 microM. Stereoselectivity is the reverse of other opioid receptors: (+)-EKC much much greater than (-)-EKC, Mr 2267 greater than Mr 2266, (+)-SKF 10047 greater than (-)-SKF 10047. The site is sensitive to trypsin, but not to N-ethylmaleimide. Binding is insensitive to nucleotides, slightly sensitive to physiological concentrations of sodium, magnesium, and manganese ions and to EDTA but not EGTA.     

3H]PN200-110 and [3H]glibenclamide binding in normal and cardiomyopathic hamsters.

1. We examined the binding of the Ca2+ channel ligand [3H]PN200-110 and the ATP-sensitive K+ channel ligand [3H]glibenclamide to brain and heart from cardiomyopathic hamsters and compared them to controls. 2. We found that [3H]PN200-110 binding site density was elevated in the heart, but not in the brain, of 30- and 180-day old cardiomyopathic hamsters when compared to controls. 3. [3H]Glibenclamide binding site density was greatly reduced in the heart of 180-day old cardiomyopathic animals compared with all other groups. 4. Quantitative autoradiography revealed that [3H]glibenclamide binding was elevated in several brain areas of 30-day old cardiomyopathic hamsters relative to controls. 5. It is concluded that alterations in both Ca2+ and K+ channels exist in the cardiomyopathic hamster.     

Metal binding functions in the design of carbonic anhydrase inhibitors

The carbonic anhydrases (CAs, EC 4.2.1.1) are zinc containing metalloenzymes which catalyse efficiently the reversible hydration of carbon dioxide to bicarbonate with discharge of a proton, playing important physiological and physiopathological functions. To date, 16 different carbonic anhydrase isoforms have been described in higher vertebrates, including humans, and some of them have been considered as important targets for inhibitors with therapeutic applications. The catalytic and structural role of zinc in these enzyme are understood in great detail, and this provided molecular basis for the design of potent inhibitors, some of which possessing important clinical applications mainly as topically acting anti-glaucoma drugs, anticancer or antiobesity agents. The metal binding function is a critically important factor in the development of isozyme-specific and organ-selective inhibitors. Discovery of compounds that possess zinc binding function different from that of the classical one (sulfonamide type) is in constant progress and can offer opportunities for developing novel pharmacological agents. In the present review we will discuss the different zinc binding function reported in the literature up to now in the design of carbonic anhydrase inhibitors.     

Design of zinc binding functions for carbonic anhydrase inhibitors

Zinc ion plays a crucial role in the protein's functions and is linked to a variety of physiological processes. It constitutes an essential component of numerous enzymes especially carbonic anhydrase (CAs, EC 4.2.1.1), a pharmaceutically-important metalloprotein which catalyses efficiently the reversible hydration of carbon dioxide to bicarbonate with discharge of a proton. The potential therapeutic applications of selective carbonic anhydrase inhibitors has become an important challenge over the last few years, as some isoforms of this enzyme on the 16 described in higher vertebrates have been found to be involved in important pathologies such as cancer, obesity and ophthalmologic diseases. Coordination of the inhibitor with the zinc ion present in the active site is an important determinant which has to be taken into consideration for the design of isozyme-specific and organ-selective inhibitors. Besides the well known sulfonamide function, others zinc binding groups have been described constituting a new platform for the development of novel pharmacological agents. In this review, recent studies on the discovery of new zinc binding function will be discussed.     

碳酸酐酶抑制剂乙酰唑胺对切口痛大鼠痛行为的影响

目的观察鞘内注射碳酸酐酶抑制剂乙酰唑胺(ACT)对大鼠切口痛行为的影响。方法所有大鼠术前6天鞘内置管,随机分为5组:假手术组、假手术+ACT组、切口痛组、切口痛+ACT低剂量(2.25μg)组、切口痛+ACT高剂量(22.5μg)组,每组16只。按照Brennan法建立切口痛模型。ACT和生理盐水均在术后d 1鞘内给予。分别于术前d1(基础值)、术后d 1(给药前,给药后30、75、120、165、240min)测定大鼠的热缩足潜伏期(TWL)和机械缩足反射阈值(MWT),并予比较。结果切口痛术后d 1(给药前)与基础值相比TWL、MWT均明显降低(P<0.05);鞘内给予高剂量ACT,与给药前相比,给药后30、75、120 min TWL升高(P<0.05),但不影响大鼠的MWT;与切口痛组相比,切口痛+ACT高剂量组在给药后30、75、120 min TWL明显增高(P<0.05)。结论鞘内给予碳酸酐酶抑制剂ACT部分缓解了切口痛大鼠的热痛觉过敏,但是对机械痛觉过敏没有影响,提示碳酸酐酶可能参与了切口痛的热痛敏过程。     

Role of carbonic anhydrase in bone: plasma acetazolamide concentrations associated with inhibition of bone loss

Earlier reports from our laboratory have indicated that the carbonic anhydrase inhibitor acetazolamide blocks the hypercalcemic response to parathyroid hormone. In addition, we have reported that acetazolamide when administered by several routes partially prevents denervation-induced bone loss in a rat model of disuse osteoporosis. Continuous subcutaneous infusion required the least daily dose (8 mg/kg). The present study extends these earlier findings in several ways. It was found that in partially preventing denervation-induced bone loss: (1) incorporation of 1 M THAM [tris(hydroxymethyl)aminoethane] enhanced the potency of acetazolamide such that it was effective at daily doses of 0.6 mg/kg; (2) acetazolamide in the presence of 1 M THAM was effective at plasma concentrations as low as 50 ng/ml which are more than 500-fold less than peak plasma concentrations normally encountered in the human when acetazolamide is being used as a therapeutic agent; and (3) another inhibitor, benzolamide, was also effective by continuous subcutaneous infusion.     

The carbonic anhydrase inhibitor acetazolamide exerts antidystonic effects in the dt(sz) mutant hamster

Previous studies suggested an involvement of gamma-aminobutyric acid (GABA)-mediated excitation by an enhanced efflux of bicarbonate ions in addition to retarded development of GABAergic inhibition in the syndrome of dt(sz) mutant hamsters, a model of paroxysmal dyskinesia in which dystonic episodes occur in response to stress. Acetazolamide blocks bicarbonate regeneration in neurons and can thereby reduce GABA-mediating excitation without affecting GABA-mediated inhibition. In the present study, the effects of acetazolamide (15-60 mg/kg, i.p.) on severity of dystonia were therefore examined in dt(sz) hamsters. Acetazolamide significantly reduced the severity of dystonia at a dose of 60 mg/kg. These data are in line with several case reports from patients with paroxysmal dystonia, suggesting that acetazolamide can be useful in the treatment of this movement disorder. The mechanism of the antidystonic efficacy of acetazolamide has to be examined by further studies.     

Simulataneous and independent versus antagonistic inhibition of muscle carbonic anhydrase (CA III) by acetazolamide and cyanate

The inhibition by cyanate and acetazolamide of pig muscle carbonic anhydrase III (CA III) CO₂ hydratase activity was studied in order to explore mechanistic features possibly unique to the muscle isoenzyme. The turnover number for CO₂ hydration was found to be 6000 sec^?1 with a K_m of 83 mM for CO₂. Cyanate inhibition (K_i, 3 μM) and acetazolamide inhibition (K_i, 44 μM) were both found to be noncompetitive with respect to CO₂. Significantly, acetazolamide and cyanate displayed non-exclusive binding to pig muscle carbonic anhydrase. The similarity of mode and degree of inhibition of muscle carbonic anhydrase by cyanate as compared with the inhibition of the erythrocyte isoenzymes suggests the existence of a similar metal environment. However, the observation that cyanate and acetazolamide bind simultaneously to CA III and the comparatively large K_i for acetazolamide per se appear to be more compatible with a different mode of coordination of the zinc with the sulfonamide, thus supporting a five-coordinant zinc in the catalytic mechanism of CO₂ hydration for CA III.     

New zinc binding motifs in the design of selective carbonic anhydrase inhibitors

The carbonic anhydrases (CAs, EC 4.2.1.1) are ubiquitous zinc enzymes which catalyze a very simple physiological reaction, the interconversion between carbon dioxide and the bicarbonate ion, and are involved in physiological and pathological processes. The different isozymes have been considered as important targets for inhibitors with clinical applications. Several sulfonamide carbonic anhydrase inhibitors (CAIs) were used for decades as diuretics, anti-glaucoma, anti-epileptic, anti-ulcer agents, or as drugs for treating other neurological/neuromuscular disorders, whereas presently several such agents still find wide applications in therapy, mainly as topically acting anti-glaucoma drugs, anti-cancer, or anti-obesity agents. Although sulfonamides were considered the moiety par excellence to coordinate the catalytic zinc and for designing potent CAIs, in recent years related functional groups such as sulfamate, sulfamide and others have proven to be successful in the design of selective CAIs. The present review will deal with these different zinc binding functions recently reported in literature.     

Exploiting the hydrophobic and hydrophilic binding sites for designing carbonic anhydrase inhibitors

Introduction: Carbonic anhydrases (CAs, EC 4.2.1.1) exist as five genetically distinct families (α, β, γ, δ and ζ) in organisms all over the phylogenetic tree. Due to the ubiquity of such enzymes, the selective inhibition and polypharmacology of inhibitors is an important aspect of all drug design campaigns. There are several classes of CA inhibitors (CAIs): i) metal ion binders (sulfonamides and their isosteres [sulfamates/sulfamides], dithiocarbamates, mercaptans and hydroxamates); ii) compounds anchoring to the zinc-coordinated water molecule/hydroxide ion (phenols, carboxylates, polyamines, esters and sulfocoumarins) and iii) coumarins and related compounds which apparently bind even further away from the metal ion.

Areas covered: The authors rationalize the drug design strategies of inhibitors belonging to the first two classes, based on recent X-ray crystallographic data. More precisely, this is achieved by analyzing how the hydrophobic and hydrophilic halves of the enzyme active site interact with inhibitors. This task has been eased by the recent report of β-CA-like enzymes possessing carbon disulfide and carbonyl sulfide hydrolase activities, respectively, allowing the authors to propose a general approach of structure-based drug design of CAIs.

Expert opinion: Although amazing progress has been made in the structure-based drug design of CAIs, this field is still in progress, with many constantly emerging new findings. Indeed, several new such enzymes were discovered and characterized recently and novel chemotypes were explored for finding compounds with a better inhibition profile. It is anticipated that this will continue to be one of the main frontiers in the search of pharmacologically relevant enzyme inhibitors.     

Acetylcholinesterase potentiates [3H]fluorowillardiine and [3H]AMPA binding to rat cortical membranes

In addition to its action at cholinergic synapses acetylcholinesterase (AChE) has been proposed to modulate neuronal activity by mechanisms unrelated to the hydrolysis of acetylcholine. We have investigated the effects of AChE on the binding of the specific AMPA receptor agonists (S)-[3H]5-fluorowillardiine ([3H]FW) and [3H]AMPA to rat cortical membranes. Pretreatment of membranes with AChE causes a dose-dependent increase in the binding of both radiolabelled agonists with a maximal increase to approximately 60% above control. This increase is completely blocked by the specific AChE inhibitors propidium, physostigmine, DFP and BW 284C51. AChE pretreatment had no effect on [3H]kainate binding. [3H]FW binding to membranes from young (15-day-old) rats is four orders of magnitude more sensitive to AChE modulation than membranes from adult rats (EC50 values of 4x10(-5) and 0.1 unit/ml, respectively) although the total percentage increase in binding is similar. Furthermore, the AChE-induced potentiation of [3H]FW binding is Ca2+ - and temperature-dependent suggesting an enzymatic action for AChE in this system. Saturation binding experiments with [3H]FW to adult membranes reveal high and low affinity binding sites and demonstrate that the main action of AChE is to increase the Bmax of both sites. These findings suggest that modulation of AMPA receptors could provide a molecular mechanism of action for the previously reported effects of AChE in synapse formation, synaptic plasticity and neurodegeneration.     

Selective covalent binding of acrylonitrile to Cys 186 in rat liver carbonic anhydrase III in vivo

Covalent binding of reactive chemical species to tissue proteins is a common, but poorly understood, mechanism of toxicity. Identification of the proteins and the specific amino acid residues within the proteins that are chemically modified will aid our understanding of the toxification/detoxification mechanisms involved in covalent binding. Acrylonitrile (AN) is a commercial vinyl monomer that is acutely toxic and readily binds to tissue proteins. Total covalent binding of AN to tissue proteins is highly correlated with acute toxicity. Two-dimensional PAGE and autoradiography were used to locate proteins in male rat liver cytosol that are radiolabeled following administration of [2,3-(14)C]AN in vivo. Four intensely labeled spots were prominent in the autoradiogram and formed an apparent "charge-train" at approximately 30 kDa. Tryptic peptide mapping by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS was used to identify all of the spots as carbonic anhydrase III (CAIII). HPLC of the tryptic digests combined with MALDI-TOF MS was used to localize the radiolabel to tryptic fragment T22 containing amino acids 171-187. This tryptic fragment contains two Cys residues (Cys181 and Cys186) in the rat CAIII sequence. Electrospray ionization ion-trap MS was used to sequence the peptide and establish that only Cys186 was labeled. Thus, although AN is considered to be highly reactive, our data indicate that it does not react indiscriminately with rat CAIII but rather is selective for one out of five Cys residues. Rat liver CAIII has previously been shown to protect cells against oxidative stress. Our data suggest that CAIII is also capable of scavenging reactive xenobiotics and may help prevent covalent binding to more critical macromolecules.     

Characterization of [3H]brevetoxin binding to voltage-dependent sodium channels in adrenal medullary cells

We have previously reported that in bovine adrenal chromaffin cells Ptychodiscus brevis toxin-3 (PbTx-3) does not alter the veratridine-induced ²²Na influx when given alone, but increases the influx of ²²Na when co-applied with either - or -scorpion venom (Wada et al. 1992). In the present study, we characterized [³H]PbTx-3 binding in bovine adrenal chromaffin cells. [³H]PbTx-3 binding was saturable, reversible and of high-affinity with an equilibrium dissociation constant (K_d) of 32.0±4.9 nmol/1 and a maximum binding capacity B_max of 6.2 ± 1.2 pmol/4 × 10⁶ cells (4.5 ± 0.9 pmol/mg cell protein). A Hill plot revealed the lack of cooperative interaction among the binding sites. Unlabelled PbTx-3 inhibited [³H]PbTx-3 binding with an IC₅₀ of 31 nmol/l. However, tetrodotoxin, veratridine, - and -scorpion venom, or veratridine in combination with either - or -scorpion venom did not alter [³H]PbTx-3 binding. All these results suggest that PbTx-3 binds to a site (site 5) distinct from the previously known four toxin binding sites, which does not gate voltage-dependent Na channels by itself, but is specifically involved in the allosteric modulation of Na channels in adrenal medullary cells. Correspondence to: A. Wada at the above address     

3H]cytisine binding to nicotinic cholinergic receptors in brain

Cytisine, a ganglionic agonist, competes with high affinity for brain nicotinic cholinergic receptors labeled by any of several nicotinic 3H-agonist ligands. Here we have examined the binding of [3H]cytisine in rat brain homogenates. [3H]Cytisine binds with high affinity (Kd less than 1 nM), and specific binding represented 60-90% of total binding at all concentrations examined up to 15 nM. The nicotinic cholinergic agonists nicotine, acetylcholine, and carbachol compete with high affinity for [3H]cytisine binding sites, whereas among nicotinic receptor antagonists only dihydro-beta-erythroidine competes with high affinity (in the nanomolar range). Comparison of binding in several brain regions showed that [3H]cytisine binding is higher in the thalamus, striatum, and cortex than in the hippocampus, cerebellum, or hypothalamus. The pharmacology and brain regional distribution of [3H]cytisine binding sites are those predicted for neuronal nicotinic receptor agonist recognition sites. The high affinity and low nonspecific binding of [3H]cytisine should make it a very useful ligand for studying neuronal nicotinic receptors.     

High affinity binding of [3H]paroxetine and [3H]imipramine to human platelet membranes

Paroxetine, one of the most potent and specific serotonin uptake inhibitors, was tritiated and used for binding studies with human platelet membranes. Specific, high affinity binding was demonstrated. The binding was compared with [3H]imipramine binding; it was found that the maximal binding (Bmax) was the same for [3H]paroxetine and [3H]imipramine, whereas the affinity was much higher for [3H]paroxetine (KD 0.08 nM and 0.56 nM for paroxetine and imipramine binding, respectively). IC50 was calculated for the inhibition of [3H]paroxetine and [3H]imipramine binding by a number of antidepressants; the corresponding Hill coefficients were also calculated.     

Specific binding of [3H]prazosin to myocardial cells isolated from adult rats

The characteristics of alpha-adrenoceptors in rat myocardium were investigated by specific binding of [3H]prazosin to cells isolated from adult rat heart by perfusion with collagenase and hyaluronidase. The cells were incubated in Krebs-Ringer bicarbonate buffer gassed with 95% O2 and 5% CO2 at 31 degrees with the appropriate concentrations of the different ligands. Non-specific binding was defined by the addition of 10(-5) mole/l. phentolamine. The binding of [3H]prazosin was saturable and reached equilibrium within 15 min. Scatchard analysis showed a straight line giving an apparent dissociation constant, Kd, equal to 155.9 +/- 8.0 pmole/l. and a maximal number of binding sites equal to 76.7 +/- 11.1 fmole/mg protein. Inhibition of specific [3H]prazosin binding by different adrenergic blockers showed the order of potency characteristic of alpha 1-adrenoceptors: prazosin much greater than phentolamine greater than yohimbine much greater than propranolol. Inhibition by adrenergic agonists showed the order of potency: adrenaline greater than noradrenaline = phenylephrine greater than isoprenaline. The same orders of potency were observed in the presence of propranolol. However, propranolol slightly decreased the affinity for noradrenaline and phenylephrine. Hofstee analyses of the inhibition curves showed two binding components for all ordinary alpha-adrenoceptor blockers and agonists including unlabelled prazosin. In contrast, [3H]prazosin showed only one binding component. Both binding components were of the alpha 1-adrenoceptor subtype according to the order of potency of blockers. The different ligands had different affinity ratios for the two binding components giving them different profiles. Trifluoperazine, a phenothiazine compound, also had high affinity for the [3H]prazosin binding sites. This drug, however, apparently detected one class of binding sites only, as interpreted from the Hofstee analysis. Hill analyses of the inhibition data consistently yielded Hill constants, nH, in the range 0.75-0.85 except for [3H]prazosin, where nH = 1.02 and for trifluoperazine, where nH = 1.07. Although the two binding components may serve different functions, it seems impossible at present to relate the negative and the positive inotropic components, respectively, of the alpha-adrenergic inotropic response observed in functional studies only to one or the other binding component.