Therapeutic Applications of the Carbonic Anhydrase Inhibitors

REVIEW

Therapeutic applications of the carbonic anhydrase inhibitors

Claudiu T Supuran Inhibitors of the zinc enzyme carbonic anhydrase (CA) targeting different isozymes among Università degli Studi, Laboratorio di Chimica the 15 presently reported in humans have various therapeutic applications. These enzymes Bioinorganica, Rm 188, are efficient catalysts for the hydration of carbon dioxide to bicarbonate and protons, Via della Lastruccia 3, playing crucial physiological/pathological roles related to acid–base homeostasis, secretion I-50019 Sesto Fiorentino (Firenze), Italy of electrolytes, transport of ions, biosynthetic reactions and tumorigenesis. Many CA Tel.: +39 055 457 3005; inhibitors have been reported as antiglaucoma, anticancer and antiobesity agents, or for Fax: +39 055 457 3385; the management of a variety of neurological disorders, including epilepsy and altitude E-mail: claudiu.supuran@ unifi.it disease. Furthermore, some CA isozymes appear to be valuable markers of tumor progression in a variety of tissues/organs. Various CAs present in pathogens such as Plasmodium falciparum, Helicobacter pylori, Mycobacterium tuberculosis, Candida albicans and Cryptococcus neoformans, have started to also be investigated recently as drug targets. Recent progress in all these areas, as well as in the characterization of new such enzymes isolated in many other organisms/tissues, in addition to their detailed catalytic/inhibition mechanisms, are reviewed herein, together with the therapeutic use of these enzyme inhibitors and drug-design studies for obtaining such new-generation derivatives with improved activity.

Among the zinc enzymes extensively studied, forms (CA VA and CA VB), as well as a the carbonic anhydrases (CAs) occupy a special secreted CA isozyme (in saliva and milk; place for several reasons. These enzymes are CA VI). Among the membrane-bound CAs, ubiquitous in all kingdoms, starting with isoforms CA IV and XV are anchored to mem- archaea, bacteria, algae and green plants, and branes by means of glycosylphosphatidyl- ending with superior animals, including verte- inositol (GPI) tails, whereas isozymes IX, XII brates [1–8]. Their physiological function is and XIV are transmembrane proteins possess- essential for these organisms, as CAs catalyze a ing just one transmembrane domain [1–8]. How- very simple physiological reaction, the inter- ever, all these five isozymes have their active site conversion between CO2 and bicarbonate and outside the cell, and are thus commonly known a proton [1–8]. This reaction is critical for respi- as extracellular CAs [1–8]. ration and transport of CO2 between metabo- lizing tissues and excretion sites, secretion of Catalytic/inhibition mechanism electrolytes in a variety of tissues and organs, Isozymes pH regulation and homeostasis, CO2 fixation Over the phylogenetic tree, CAs are encoded (for algae and green plants), several metabolic by several (probably four) distinct, evolution- biosynthetic pathways (in vertebrates), tumori- arily unrelated gene families: the α-CAs genesis and signal transduction [1–8]. Inhibition (present in vertebrates, bacteria, algae and (and also activation) of these enzymes may be cytoplasm of green plants); the β-CAs (pre- exploited clinically in the treatment or preven- dominantly in bacteria, algae and chloroplasts tion of a variety of disorders [1–3]. In conse- of both mono- as well as dicotyledons); the quence, CA inhibitors (CAIs) possess a variety γ-CAs (mainly in archaea and some bacteria); Keywords: anticancer drug, of applications in therapy [1]. In mammals, 16 and the δ-CAs, evidenced so far in some antiglaucoma agent, antiobesity agent, carbonic different α-CA isozymes or CA-related pro- marine diatoms (Figure 1) [1–8]. anhydrase, enzyme inhibitor, teins (CARPs) have been described, with very CAs are metalloenzymes and most of them isozyme, sulfamate, sulfamide, different subcellular localization and tissue dis- have a Zn(II) ion within their active sites, sulfonamide, tumor-progression marker tribution [1–8]. There are several cytosolic although the δ-CAs seem to be Cd(II) enzymes forms (CA I–III and CA VII), five membrane- and the γ-CAs use Zn(II), Co(II) or Fe(II) as a part of bound isozymes (CA IV, CA IX, CA XII, metal ion cofactor [1–8]. The metal ion of CAs is CA XIV and CA XV), two mitochondrial essential for catalysis [1–8]. X-ray crystallographic

Figure 1. Metal ion coordination sphere in the four classes of carbonic anhydrases.

Thr199

A - H B O O H 2+ Asp residue O Zn N O O H N Cys residue HN N His119 Zn S Glu106 N H H S N O N N Cys residue His94 H

His96 N His residue O H

OH C 2 Cys residue D OH H2O Zn S M + Cys residue 2 S N N HN N His117 N H N His residue H His81 N H His122 M = Zn, Co, Fe or Cd

(A) α-carbonic anhydrases (CAs; hCA II numbering). (B) Porphyridium purpureum and Escherichia coli β-CAs. (C) Pisum sativum chloroplast and Methanobacterium thermoautotrophicum β-CAs. (D) γ-class CA from Methanosarcina thermophila (M = Zn(II), Co(II) or Fe(II) – which can be either tetra-, penta- or hexa- coordinated by additional water molecules; Cam numbering of the His residues) and δ-class CA from Thalassiosira weissflogii (M = Cd(II) or Zn(II) – the exact geometry of the metal ion is not known).

data carried out mainly on the α-CA family of homodimeric CA appeared like a tetramer with enzymes showed that the metal ion is situated at a pseudo 2–2–2 symmetry. The active-site the bottom of a 15-Å deep active-site cleft, Zn(II) ion was shown to be coordinated by a being coordinated by three histidine residues Cys–Asp–His–Cys tetrad that is strictly con- - (His94, 96 and 119) and a H2O/OH ion [1–8]. served among the β-CAs, and no H2O mole- The Zn-bound water is also engaged in H-bond cule was found in the Zn-liganding radius, interactions with the hydroxyl moiety of indicating that the Zn(OH)2 mechanism in α- Thr199, which in turn is bridged to the carbox- and γ-CAs is not directly applicable to the case ylate moiety of Glu106 (these amino acids are in β-CAs [9]. This first report was soon fol- conserved in all α-CAs currently known [1–5]). lowed by another, in which the structure of the These interactions enhance the nucleophilicity ‘cab’-type β-CA from the archaeon Methano- - of the Zn-bound H2O/OH ion, and orient the bacterium thermoautotrophicum (Cab) has been substrate (CO2) in a favorable location for determined to a resolution of 2.1 Å (Figure 1C) nucleophilic attack (Figure 1A) [1–5]. [10]. Cab exists as a dimer with a subunit fold The first x-ray crystallographic structure of a similar to that observed in ‘plant’-type β-CAs. member of the β-CA family has been deter- The active-site zinc ion was shown to be coor- mined by Mitsuhashi and colleagues for the dinated by the amino acid residues Cys32, enzyme isolated from the red algae Porphyrid- His87 and Cys90, with the tetrahedral coordi- ium purpureum (Figure 1B) [9]. This CA monomer nation completed by a H2O molecule [10]. The is composed of two internally repeating struc- major difference between plant- and cab-type tures, being folded as a pair of fundamentally β-CAs is in the organization of the hydropho- equivalent motifs of an α/β-domain and three bic pocket (except for the aforementioned Zn projecting α-helices. The motif is very distinct coordination). The structure also revealed a from that of either α- or γ-CAs. This HEPES buffer molecule, bound 8 Å away from

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the active-site Zn, which suggests a possible higher plants in which zinc is coordinated by two proton-transfer pathway from the active site to cysteine thiolates, one histidine and a H2O mole- the solvent [10]. The structure of the β-CA from cule [13]. The diatom CA shows no significant the dicotyledonous plant Pisum sativum at sequence similarity with other CAs and may rep- 1.93 Å resolution has also been reported resent an example of convergent evolution at the (Figure 1C) [11]. The molecule assembles as an molecular level [13]. In the same diatom, an unu- octamer with a novel dimer-of-dimers-of-dim- sual discovery has been made: the first Cd-con- ers arrangement. Two distinct patterns of containing enzyme, which is a CA-type protein [14]. servation of active-site residues were observed, The marine diatom, T. weissflogii, growing under implying two potentially mechanistically dis- conditions of low Zn, typical of the marine envi- tinct classes of β-CAs. The active site is located ronment, and in the presence of Cd, led to at the interface between two monomers, with increased levels of cellular CA activity, although Cys160, His220 and Cys223 binding the cata- the levels of TWCA1, the major intracellular lytic Zn ion and residues Asp162 (oriented by Zn-requiring isoform of CA in T. weissflogii, Arg164), Gly224, Gln151, Val184, Phe179 remained low [14]. 109Cd labelling comigrates with and Tyr205 interacting with the substrate ana- a protein band that showed this CA activity to be logue, acetic acid [11]. The substrate-binding distinct from TWCA1 on native polyacrylamide groups have a one-to-one correspondence with gel electrophoresis (PAGE) of radiolabeled the functional groups in the α-CA active site, T. weissflogii cell lysates. The levels of the Cd pro- with the corresponding residues being closely tein were modulated by CO2 in a manner that is superposable by a mirror plane. Therefore, consistent with a role for this enzyme in carbon despite differing folds, α- and β-CAs have con- acquisition. Purification of the CA-active fraction verged upon a very similar active-site design leads to the isolation of a Cd-containing protein and are likely to share a common mechanism of of 43 kDa, being clear that T. weissflogii expresses action [11]. a Cd-specific CA, which, particularly under con- The prototype of the γ-class of CA has been ditions of Zn limitation, can replace the Zn characterized from the methanogenic archaeon enzyme TWCA1 in its carbon-concentrating Methanosarcina thermophila (Figure 1D) [12]. The mechanism [14]. This enzyme is considered to crystal structures of Zn-containing and Co-sub- belong to the δ-CA class (Figure 2). stituted γ-CA from M. thermophila were The catalytic mechanism is well understood reported in the unbound form and cocrystallized for the α-CAs, and will be discussed in detail with sulfate or bicarbonate. Relative to the tetra- here. The active form of the enzyme is the basic hedral-coordination geometry seen at the active one, with hydroxide bound to Zn(II) site in the α-CAs, the active site of γ-CA contains (Figure 1A & 2A) [1–8]. This strong nucleophile additional metal-bound water ligands, so that attacks the CO2 molecule bound in a hydro- the overall coordination geometry is trigonal phobic pocket in its neighbourhood (the elusive bipyramidal for the Zn-containing enzyme and substrate-binding site comprises residues octahedral for the Co-substituted enzyme [12]. Val121, Val143 and Leu198 [1–8]) (Figure 2B), Ligands bound to the active site make contacts leading to formation of bicarbonate coordinated with the side chain of Glu62 in a manner that to Zn(II) (Figure 2C). The bicarbonate ion is then suggests this side chain to be protonated. In the displaced by a water molecule and liberated into uncomplexed Zn-containing enzyme, the side solution, leading to the acid form of the enzyme, chains of Glu62 and Glu84 appear to share a with water coordinated to Zn(II) (Figure 2D), proton; additionally, Glu 84 exhibits multiple which is catalytically inactive [1–8]. In order to conformations. This suggests that Glu84 may regenerate the basic form A, a proton-transfer act as a proton shuttle, which is an important reaction from the active site to the environment aspect of the reaction mechanism of α-CAs, for takes place, which may be assisted either by active- which a histidine active-site residue generally site residues (such as His64 – the proton shuttle in performs this function (usually His64) [1–8]. isozyme CA II) or by buffers present in the X-ray absorption spectroscopy at the Zn medium [1–8]. The process may be schematically K-edge indicates that the active site of the marine represented by Equations 1A & B. diatom, Thalassiosira weissflogii CA, is strikingly The rate-limiting step in catalysis is the sec- similar to that of mammalian α-CAs. The Zn has ond reaction, that is, the proton transfer that - three histidine ligands and a single H2O mole- regenerates the Zn-OH species of the enzyme cule, being quite different from the β-CAs of (Figure 3) [1–8]. futurefuture sciencescience groupgroup www.futuremedicine.com 357 REVIEW – Supuran

Equations group of Glu106 [15–17]. One of the oxygen atoms of the SO2NH moiety also participates in a H bond with the backbone NH moiety of H2O 2+ --2+ 2+ - Thr199 [15–24]. In Figure 4, the crystal structures of A. EZn –OH + CO2 EZn –HCO3 EZn –OH2 + HCO3 the human isoform hCA II adducts with the simplest compounds incorporating a sulfamoyl moi- B. EZn2+–OH 2+ - + 2 EZn –OH + H ety (sulfamide and sulfamic acid, respectively) are shown as examples of the binding of sulfona- Two main classes of CAIs are known: the mide, sulfamate and sulfamide inhibitors [15]. 2- metal-complexing anions (inorganic inhibitors), The binegatively charged (NH)SO3 sulfamate and the unsubstituted sulfonamides and their ion and the monoanion of sulfamide - derivatives (e.g., sulfamates or sulfamides) – the NHSO2NH2 were shown to bind to the Zn(II) organic inhibitors. All inhibitors of interest for ion within the enzyme active site [15]. These two their therapeutic applications bind to the Zn(II) structures provide some close insights into why ion of the enzyme, either by substituting the this functional group (the sulfonamide) appears nonprotein Zn ligand (Figure 3A) or by adding the to have unique properties for CA inhibition: metal coordination sphere (Figure 3B), generating • It exhibits a negatively charged and most trigonal-bipyramidal species [1–8]. Sulfonamides, likely monoprotonated N coordinated to the which are the most important CAIs, bind in a Zn(II) ion tetrahedral geometry of the Zn(II) ion (Figure 3A), • Simultaneously, this group forms a H bond as in a deprotonated state, with the nitrogen atom donor to the oxygen Oγ of the adjacent of the sulfonamide group coordinated to Zn(II). Thr199 Anions may bind either in tetrahedral geometry of the metal ion or as trigonal-bipyramidal • A H bond is formed between one of the SO2 adducts, such as the thiocyanate adduct shown oxygens to the backbone NH of Thr 199 in Figure 3B [1–8]. Thus, the basic structural elements explaining X-ray crystallographic structures are available the strong affinity of the sulfonamide moiety for for many adducts of sulfonamide/sulfamate/sulfa- the Zn(II) ion of CAs were delineated in all mide inhibitors with various isozymes [1–8,15–24]. details by using these simple compounds as proto- In all these adducts, the deprotonated sulfona- typical CAIs, without the need to analyze the mide is coordinated to the Zn(II) ion of the interactions of the organic scaffold, usually enzyme, and its NH moiety participates in a H present in other inhibitors (generally belonging to bond with the Oγ of Thr199, which in turn is the aromatic/heterocyclic sulfonamide class [15]). engaged in another H bond to the carboxylate Despite important similarities of the binding of these two inhibitors to the enzyme with that of Figure 2. Schematic representation of the catalytic mechanism aromatic/heterocyclic sulfonamides of the type RSO2NH2 previously investigated, the absence for α-carbonic anhydrase-catalyzed CO2 hydration. of a C–SO2NH2 bond in sulfamide/sulfamic acid leads to a different H-bond network in the A - B O OH - neighborhood of the catalytical Zn(II) ion, + CO OH O 2 which was shown to be useful for the drug Zn2+ 2+ design of more potent CAIs, possessing differ- His119 Zn His119 ent Zn-binding functions than the classical His94 His96 His94 His96 sulfonamides [15]. A very important discovery in parasitic CAs has been reported by Krungkrai and colleagues, -BH+ B who discovered the presence of at least two different CAs in Plasmodium falciparum, the protozoa D C O causing malaria [25]. Red cells infected with OH H 2 P. f a l c i p a r u m contained CA amounts approxi- O mately twice as high as those of normal red cells. Zn2+ + H O 2 O- His119 Zn2+ The three developmental forms of the asexual His119 His94 - HCO - stages (i.e., ring, trophozoite and schizont) were His96 3 His94 His96 isolated from their host red cells and found to have stage-dependent CA activity. This enzyme

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Figure 3. Carbonic anhydrase inhibition mechanism by organic (AZ). The kinetic properties and amino-terminal (sulfonamide) and inorganic (anion) inhibitors. sequences of the purified enzymes from the parasite and host red cell were found to be different, indicating that the purified protein was a distinct EZn2+–OH + I 2+ protein (i.e., P. f a l c i p a r u m CA). In addition, the A 2 EZn –I + H2O (substitution reaction) Tetrahedral adduct aforementioned enzyme inhibitors showed anti- malarial effect against in vitro growth of P. f a l c i - parum [25]. This very important contribution B EZn2+–OH + I EZn2+–OH (I) (addition reaction) shows that CAIs may represent valuable future 2 2 drugs for the treatment of malaria. Trigonal-bipyramidal adduct CAIs & their applications

A O R B There are at least 16 clinically used drugs/orphan S drugs reported to possess significant CA inhibi- S C tory properties (Figures 5 & 6) [1,26–28], and many -HN O - N OH2 other such derivatives belonging to the sulfona- 2+ Zn 2+ mide, sulfamate or sulfamide classes are con- His119 Zn His119 stantly reported, designed and synthesized by His94 His96 His94 His96 means of rational drug-design processes [29–49]. Compounds used clinically, such as AZ Tetrahedral adduct Trigonal-bipyramidal adduct (Figure 5A) (Figure 5B) (sulfonamide) (thiocyanate) , methazolamide , ethoxzolamide (Figure 5C), the orphan drug benzolamide (Figure 5D) and dichlorophenamide (DCP; Daranide®) (Figure 5E), have been known for was then purified to homogeneity by using multi- many years and were initially developed in the ple steps of fast-liquid chromatographic tech- search for diuretics [26]. Although their diuretic niques, showing a Mr of 32, being active in use was not extensive [26–28], it was found that monomeric form (the human red cell enzyme was such enzyme inhibitors could be employed for the also purified for comparison with the parasite systemic treatment of glaucoma [26]. Thus, many enzyme in this study) [25]. The parasite–enzyme such drugs (e.g., AZ, methazolamide and DCP) activity was sensitive to well-known sulfonamide are still presently used in ophthalmology (see dis- CAIs, such as sulfanilamide and acetazolamide cussion later in the text), whereas two novel derivatives, dorzolamide (Figure 5F) and brinzolamide Figure 4. Adducts of human carbonic anhydrase II with the (Figure 5G) have been developed in the 1990s as simplest sulfonamides. topically acting antiglaucoma agents [26–28].

The antitumor sulfonamide indisulam (Figure 5H) is in Phase II clinical development for the treatment of solid tumors [50], whereas the antiepileptic sulfamate topiramate (Figure 6A) [51] and sulfonamide zonisamide (Figure 6B) [22] were recently shown to possess significant inhibitory properties against many physiologically relevant CA isozymes. The same is true for the antipsychotic sulpiride (Figure 6C) [52]. The sulfamates possessing steroidal-like scaffolds COUMATE- 667 (Figure 6D) [53] and EMATE (Figure 6E) [54] were initially discovered as steroid sulfatase (STS) inhibitors [53] and subsequently shown to be low nanomolar CAIs. These compounds are also in clinical development for the treatment of breast tumors in which both STS and some CA isozymes are overexpressed [1,53,54]. The sulfonamide cyclooxygenase (COX)-2 inhibitors celecoxib (A) Sulfamic acid hCA II-(HO)SO2(NH2) and (B) sulfamide hCA II-SO2(NH2)2, (Figure 6F) [55] and valdecoxib (Figure 6G) [23] also determined by x-ray crystallography [15]. act as potent inhibitors of many CA isozymes,

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Figure 5. Chemical compounds.

A B C O N N N O N N N N N N S O S S S N S O S O O O O O Acetazolamide Methazolamide Ethoxzolamide

D E F O O O O O S O O N N N N S S S S N C H S N 6 5 N S O O S O O O Cl N Cl Benzolamide Dichlorophenamide Dorzolamide

G H O O O O O N S S O N S S N Cl N O N O S N N O Brinzolamide Indisulam

and some of their clinical applications (e.g., the the CAIs, ranging from diuretics and antiglau- prevention of some gastrointestinal tumors) are coma agents, to anticancer, antiobesity and correlated with the strong inhibition of some antiepileptic drugs [1–8,26,27]. CAs [23,55]. The sulfimine artificial sweetener saccharin (Figure 6H) is also a very potent inhibitor of Applications of CAIs in ophthalmology several physiologically relevant mammalian CA CAIs constitute an important class of drugs in the isoforms (Table 1) [56]. armamentarium of antiglaucoma agents [1,2,26,27]. Examples of inhibitory effects of some of these In addition to the classical, systemically used sul- clinically used drugs against the mammalian iso- fonamides (AZ, methazolamide, DCP and ethox- forms CA I–XIV are shown in Table 1. Data in zolamide [1,2,26,27]), two topically acting agents Table 1 show that only isoform III is not suscepti- became available in the last years, dorzolamide and ble to inhibition by sulfonamides and sulfamates, brinzolamide [1,2,26,27]. The two drugs are effective whereas all other CA isoforms possessing catalytic in reducing intraocular pressure (IOP) by activity – CA I, II, IV, VA, VB, VI, VII, IX, XII, 20–25%, and show fewer side effects compared XIII and XIV (CA XV is not present in humans) with the systemically applied sulfonamides [1,2]. – are appreciably inhibited by many of the clini- Still, the observed side effects, including stinging, cally used drugs, with inhibition constants in burning or reddening of the eye, blurred vision, many cases in the low nanomolar range. These pruritus, and bitter taste, may lead, in many cases, very diverse inhibition profiles of the various iso- to patient noncompliance and the need to use zymes with derivatives discussed above may other drugs, or to develop topically acting sulfona- explain the very different clinical applications of mides possessing less side effects. All but the last

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Figure 6. Chemical compounds.

A O B C O O N S O O N O O O O S N N O O O N O S O O O N Topiramate Zonisamide Sulpiride

O O

D E F O S N

O N O O O O S O N

O S O N COUMATE-667 EMATE Celecoxib N O F F F O S N O G H

S O Valdecoxib O

Saccharin

side effects mentioned above are probably due to a randomized, double-blind study the efficacy of the fact that dorzolamide and brinzolamide are dorzolamide 2% and brinzolamide 1% in lowering salts of weak bases (secondary amines) with a very IOP and in evaluating side effects were recently strong acid (HCl), so that the pH of the drug solu- reported [57]. IOP decreased significantly from tion is rather acidic (generally around 5.5). Thus, baseline for both drugs, with no statistically signif- many studies were reported ultimately for the icant differences between dorzolamide 2% and development of topically acting sulfonamide CAIs brinzolamide 1% either before or after eye-drop that can be formulated in solution at near neutral administration. The most frequent side effect was pH [31,32,34,35,37,38,44,45,47]. Conversely, many clini- ocular pain in the case of dorzolamide 2% and cal studies regarding the two drugs from this class blurred vision in brinzolamide 1%. The results available up to now (dorzolamide and brinzola- suggested that dorzolamide 2% and brinzolamide mide), alone or in combination with other agents, 1% have similar IOP-lowering efficacies with dif- were reported in the last few years [57–61]. Thus, in ferent side effects [57]. The nonselective β-blocker

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Table 1. Inhibition data with the clinically used sulfonamides, the clinically used sulfamate (topiramate) and saccharin against isozymes I–XIV.

Isozyme Ki (nM) 5A* 5B* 5C* 5E* 5F* 5G* 6A* 6H* hCA I‡ 250 50 25 1200 50000 45000 250 18540 hCA II‡ 12 14 8 38 9 3 10 5950 hCA III‡ 3.105 1.105 5000 NT 8000 NT NT NT hCA IV‡ 74 6200 93 15000 8500 NT 4900 7920 hCA VA‡ 63 65 25 630 42 50 63 10060 hCA VB‡ 54 62 19 21 33 30 30 7210 hCA VI‡ 11 10 43 79 10 0.9 45 935 hCA VII‡ 2.5 2.1 0.8 26 3.5 2.8 0.9 10 hCA IX‡ 25 27 34 50 52 37 58¶ 103 hCA XII§ 5.7 3.4 22 50 3.5 3.0 3.8 633 mCA XIII‡ 17 19 NT 23 18 NT 47 12100 hCA XIV‡ 41 43 25 345 27 24 1460 773 The isoforms CA VIII, X and XI are devoid of catalytic activity and probably do not bind sulfonamides as they do not contain Zn(II) ions [1]. *Refers to the figure number from Figures 5 & 6. ‡Full-length enzyme. §Catalytic domain. ¶The data against the full-length enzyme is of 1590 nM. h: Human; m: Murine isozyme; NT: Not tested (no data available).

timolol and dorzolamide both lower IOP, but the than the combined regimen, after 90 days: IOP two agents have different mechanisms of action was lowered by 3.6 mmHg in the combined group and their effects are additive when administered versus 4.1 mmHg in the concomitant group. Dor- together [58]. Therefore, these drugs are frequently zolamide/timolol has been compared with con- used concomitantly to treat patients with open- comitant administration of timolol 0.5% and the angle glaucoma who have not adequately IOP-lowering miotic drug, pilocarpine 2.0%. This responded to first-line therapy (β-blockers or sul- non-blind, patient-preference study found that fonamide CAIs). A barrier to good compliance both regimens reduced IOP. However, the dorzola- with concomitant therapy is the need to adminis- mide/timolol combination was preferred by ter five or six drops of medication on two or four patients because of reduced frequency and severity occasions during the day. Timolol 0.5% and dor- of adverse effects and less frequent administration zolamide 2.0% have therefore been combined in a [58]. Dorzolamide/timolol was well tolerated in single formulation, reducing the number of clinical trials, the adverse effects reflected those of administrations required to two per day [58]. Clini- the individual components and no additional tol- cal trials in patients with glaucoma have demon- erability issues were identified. However, the strated that dorzolamide 2%/timolol 0.5% potential for timolol to cause cardiorespiratory (dorzolamide/timolol) is superior to monotherapy effects must be considered when prescribing this with the individual components [58]. When dor- combination, whereas dorzolamide, being a sul- zolamide/timolol administered twice daily was fonamide, can cause allergic reactions in those who compared with concomitant treatment with dor- are hypersensitive to this class of drug [58]. zolamide 2% and timolol 0.5%, each administered Similarly, the additive effect of latanoprost twice daily for 90 days, both regimens resulted in 0.005% in patients who have uncontrolled IOP marked lowering of trough IOP (measured just using timolol 0.5% and dorzolamide 2% has been before the morning dose) compared with baseline investigated [59]. Latanoprost had an additive effect (reduction in IOP = 4.2 mmHg). When the com- when used as a third drug for patients on timolol bined formulation was compared with a concomi- and dorzolamide who were in need of further IOP tant regimen that included dorzolamide 2% three reduction. These results suggested that latanoprost times a day and timolol 0.5% twice daily, the con- may be very effective in some patients with poorly comitant regimen was slightly more efficacious controlled glaucoma on multiple therapy [59].

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The reduction in IOP by topical dorzolamide brinzolamide plus timolol (14.7%). In addition, 2% and oral methazolamide (5 mg/kg) in dogs more patients experienced ocular discomfort has also been investigated in order to determine (stinging and burning) after dorzolamide plus if the combination of both drugs would reduce timolol (13.1%) than after brinzolamide plus IOP more than either drug administered alone. timolol (1.7%) in this study [61]. Topical dorzolamide 2%, administered twice Medical treatment of cystoid macular edema and three times a day, significantly decreased (CME) is another major indication for CAIs [1,62]. IOP in glaucomatous dogs on the first day Initial observations were based on experimental (twice daily 7.6 mmHg and three times a day data that suggested that AZ can increase fluid 16.4 mmHg), which was greater by day 5 (twice absorption across the retinal pigment epithelium daily 10.4 mmHg and three times daily [1,62]. CAIs have also been shown to possess other 13.9 mmHg). Oral methazolamide also signifi- direct effects, both on retinal and retinal pig- cantly lowered IOP in both eyes. Oral meth- ment epithelial cell function, by inducing an azolamide combined with topical dorzolamide acidification of the subretinal space, a decrease of 2% (instilled in the eye from day 3 to 5) also sig- the standing potential as well as an increase in nificantly lowered IOP of both eyes for all days retinal adhesiveness [1,62]. It is thought that acid- and, for day 5, the mean IOP was decreased by ification of the subretinal space is responsible for 7.9 mmHg (methazolamide plus dorzolamide) the increase in fluid resorption from the retina. and 7.5 mmHg (methazolamide only). Topical Several clinical studies have suggested that dorzolamide (instilled in the drug eye for day patients with CME due to retinitis pigmentosa 1–5) combined with oral methazolamide and uveitis may react more favorably to treat- (administered from day 3 through to day 5) sig- ment with CAIs than other etiologies, such as nificantly lowered IOP in the eye on day 1 diabetic maculopathy or macular edema after (5 pm, 9.6 mmHg), for day 3 (11 am and 5 pm) retinal vein occlusion [1,62]. A normal clinical and for all of day 5 for both eyes (5 pm: control starting dose of systemic CAI is 500 mg/day, eye 9.5 mmHg; drug eye 9.0 mmHg). Topical which should be continued for at least 1 month dorzolamide (2%) instilled three times a day to see an effect on CME [62]. produced similar IOP declines compared with Recently, the involvement of CA I (an abun- the combination of oral methazolamide and dant isoform with a rather unknown physiologic dorzolamide 2% administered twice daily. Thus, function [1]) in eye pathology has emerged [63]. dorzolamide (2%) instilled twice or three times Thus, it is known that excessive retinal vascular a day caused significant decreases in IOP in occlusion/permeability contributes to the glaucomatous dogs. Twice-daily instillations pathogenesis of proliferative diabetic retinopa- caused progressive declines in IOP from day 1 to thy and diabetic macular edema, leading causes day 5. Dorzolamide (2%) combined with oral of vision loss in adults. Using mass spectros- methazolamide (5 mg/kg twice daily) produced copy-based proteomics, Gao and colleagues similar but not additional declines in IOP [60]. detected 117 proteins in human vitreous and A comparison study of topical brinzolamide elevated levels of extracellular CA I in vitreous 1% twice daily with dorzolamide 2% twice daily, from individuals with diabetic retinopathy, sug- each given with timolol 0.5% twice daily, has gesting that retinal hemorrhage and erythrocyte also been reported [61]. Both treatment regimens lysis contribute to the diabetic vitreous pro- significantly reduced IOP at all time points: teome [63]. Intravitreous injection of CA I in rats brinzolamide plus timolol by -3.6 to -5.3 mmHg was then shown to increase retinal vessel leakage (-14.2 to -21.9%) and dorzolamide plus timolol and to cause intraretinal edema. CA I-induced by -3.6 to -5.1 mmHg (-14.1 to -21.2%). Clini- alkalinization of vitreous increased kallikrein cally relevant IOP reductions (decreases activity and its generation of factor XIIa revealed 5 mmHg or greater or absolute IOP values of a new pathway for contact system activation. 21 mmHg or less) were manifested by CA I-induced retinal edema was decreased by 50–89.3% of patients under brinzolamide plus complement 1 inhibitor, a neutralizing antibody timolol and by 43.9–85.4% under dorzolamide to prekallikrein- and bradykinin-receptor antag- plus timolol. The treatments were equivalent in onism. Subdural infusion of CAI in rats mean IOP lowering. In general, both regimens induced cerebralvascular permeability, suggest- were well tolerated. However, more patients ing that extracellular CA I could have broad rel- experienced at least one adverse event with dor- evance to neurovascular edema [63]. Inhibition of zolamide plus timolol (32.8%) compared with the enzyme activity of the extracellular CA I by futurefuture sciencescience groupgroup www.futuremedicine.com 363 REVIEW – Supuran

sulfonamide/sulfamate/sulfamide inhibitors such tumors. Indeed, both fluorescent inhibitors could thus provide new therapeutic opportuni- [67] or positively charged, membrane-imper- ties for the treatment of hemorrhage-induced meant sulfonamides [68] that were shown to retinal and cerebral edema, a highly relevant inhibit only these extracellular CAs have recently medical problem. been developed as CA IX inhibitors and used as proof-of-concept tools for demonstrating that CAIs & cancer CA IX constitutes a novel and interesting target There are many connections between CA and for anticancer drug development, as well as for cancer [1,2,64,65]. Some CA isozymes are predomi- imaging purposes of hypoxic tumors [67–69]. nantly found in cancer cells, lacking from their The design of fluorescent sulfonamides that normal counterparts [1,2,64,65]. CA IX was shown preferentially inhibit the activity of CA IX, to be highly overexpressed in many cancer types showing reduced penetration through the and was present in few normal tissues in a quite plasma membranes and binding to hypoxic limited amount [64,65]. CA IX expression is cells expressing CA IX, was reported by Cecchi strongly induced by hypoxia present in many and colleagues [67]. These inhibitors represent tumors, being regulated by the hypoxia-induci- promising candidates for developing anticancer ble factor (HIF) transcription factor and corre- therapies based on tumor-associated CA isolated with a poor response to classical chemo- zyme inhibition and offer very interesting tools and radio-therapies [64,65]. CA IX was recently for imaging and further investigation of shown to contribute to acidification of the hypoxic tumors, due to the fact that they only tumor environment by efficiently catalyzing the accumulate in such tissues [66,67]. - hydration of CO2 to HCO3 and protons with A series of positively charged, membrane- its extracellularly situated active site [66], leading impermeant sulfonamides were obtained by both to the acquisition of metastasic phenotypes reaction of aminobenzolamide [5-(4-aminoben- and chemoresistance with weakly basic anti- zenesulfonylamino)-1,3,4-thiadiazole-2-sulfon- cancer drugs [66,67]. Thus, Svastova and col- amide] with tri-/tetra-substituted pyrilium salts leagues proved that CO2 is a significant source of possessing alkyl, aryl or combinations of alkyl acidity in tumors (in addition to lactic acid) [66], and aryl groups at the pyridinium ring [68]. The thereby indicating the relevant contribution of compounds were assayed for the inhibition of CA IX in the tumor acidification processes. This four physiologically relevant isozymes: the seminal study provided the first evidence for the cytosolic hCA I and II, the membrane-anchored role of CA IX in the control of the extracellular bCA IV and the membrane-bound, tumor-asso- pH (pHe) in tumors [66]. It was shown that CA ciated isozyme hCA IX. They showed potent IX can acidify the pH of the culture medium in inhibitory activity against all investigated iso- hypoxia but not in normoxia. This acidification zymes, although with different profiles. For could be perturbed by deletion of the enzyme CA I, the derivatives showed inhibition con- active site or by inhibiting the CA IX catalytic stants in the range of 3–12 nM, for CA II in the domain with selective sulfonamide CAIs, which range of 0.20–5.96 nM, against CA IV in the were shown to bind only to hypoxic cells express- range of 2.0–10.3 nM and against CA IX in the ing CA IX [66]. These findings suggested that range of 3–45 nM. These compounds were hypoxia regulates both expression and activity of demonstrated to be membrane impermeant due CA IX in order to enhance the extracellular acid- to the permanent positive charge present in ification processes, which may have important their molecules. Some of these derivatives were implications for tumor progression [66]. Con- also tested for their inhibitory activity against - - versely, inhibition of this enzymatic activity by the Cl /HCO3 anion exchanger (AE)1. Two specific and potent inhibitors was shown to derivatives showed inhibitory activity in the low revert these processes, establishing a clear-cut micromolar range, whereas one compound was role for CA IX in tumorigenesis and proving this inactive at these concentrations. The high affin- enzyme to be a druggable target. The develop- ity of these derivatives for the tumor-associated ment of a wide range of potent and selective isozyme CA IX and their membrane impermea- CA IX inhibitors might thus provide useful tools bility make this type of CAI an interesting can- for highlighting the exact role of CA IX in didate for the selective inhibition of only the hypoxic cancers, to control the pH (im)balance tumor-associated isozyme and not the cytosolic of tumor cells and to develop novel diagnostic or types, for which they also show high potency. therapeutic applications for the management of Furthermore, it was proved for the first time in

364 Therapy (2007) 4(3) futurefuture sciencescience ggrouproup Therapeutic applications of the carbonic anhydrase inhibitors – REVIEW

this study that the CA–AE metabolon can be Parkkila’s group investigated the effect of AZ, inhibited by the same type of sulfonamide a potent CAI (Table 1), on the invasive capacity of derivatives [68]. four renal carcinoma cell lines (Caki-1, Caki-2, In a recent report, the drug design, synthesis, ACHN and A-498) [70]. It was found that AZ and transepithelial transport of a group of 10 µM inhibited the relative invasion rate of thioureido sulfonamide CAIs, which have been these cell lines by between 18 and 74%. The obtained by reaction of isothiocyanate-substi- Caki-2 and ACHN cell lines displayed the high- tuted aromatic sulfonamides with amines, was est responsiveness, and their responses clearly also investigated [69]. These compounds have depended on the AZ concentration in the cul- potent inhibitory properties against CA IX with ture medium. Immunocytochemical and west- Ki values in the range of 10–37 nM and Papp val- ern blotting results identified the presence of ues greater than 0.34 × 10-6 cm/s for the absorp- CA isozyme II in the cytoplasm of all four cell tive transepithelial transport in Caco-2 cells. In lines and CA XII on the plasma membrane in these cells, one of these compounds was shown three of four cell lines. Because AZ alone to be a substrate for efflux transporters such as reduced invasiveness of these cancer cells p-glycoprotein (p-gp). p-gp activity is not likely in vitro, it was concluded that the CAs over- to be rate limiting for intestinal absorption, but expressed in renal cancer cells contribute to might be useful when targeting hypoxic tumors invasiveness [70]. This valuable study constituted expressing both p-gp and CA IX [69]. another demonstration that CAIs may be used Indisulam is a sulfonamide anticancer drug in the management of tumors that overexpress discovered at Eisai Co. and is in Phase II clinical one or more CA isozymes. development for the treatment of solid tumors In a series of important studies [71–78], [5–7]. The combination of indisulam with various Parkilla’s, Pastorek’s and Harris’ groups investi- antitumor drugs, such as carboplatin, oxalipla- gated the expression of various CA isozymes tin, capecitabine or their salts, for the prevention (mainly CA IX and XII) and hypoxia as markers or treatment of cancer was recently reported [27]. of tumor progression in different organs/tissues. Indisulam is a cell-cycle inhibitor and a potent CA IX was shown to be expressed in the baso- CAI. The combinations have been found to have lateral plasma membrane of normal biliary epi- strong synergistic activity. The human breast thelial cells, but not in hepatocytes. Pastorek’s cancer cell line HBC4 was implanted subcutane- group recently showed that, in the biliary epi- ously in nude mice and, when tumor volume thelial tumors, immunostaining for CA IX was reached 114 mm3, the mice were treated with mainly localized at the basolateral surface of the indisulam at dosages of: epithelial cells, as in normal mucosa [71]. All non- • (I) 30.625 mg/kg/day for 5 days invasive dysplastic lesions and 57% of invasive lesions of the gall bladder expressed this isozyme. • (II) 1.3125 mmol/kg/day for 14 days or In the liver, 78% of cholangiocellular malignant sequentially with (I) on day 1–5 lesions showed a positive reaction for CA IX, • (II) on day 6–19 whereas only 33% of hepatocellular carcinomas The minimum relative tumor volume (mRTV) showed a weak immunoreaction [71]. The con- between the treatment groups was determined and clusion was that abnormal expression of CA IX it was found that (II) alone elicited no reduction may be linked to malignant transformation of in tumor growth, while the mRTV for (I) and the hepatobiliary cells, this enzyme being a promis- combination groups were 24 and 18% of original ing marker for biliary differentiation in hepato- tumor size, respectively. Out of 18 mice divided biliary neoplasms [71]. In another study [72], the equally among the groups, the only tumor-free same group examined the expression of CA IX mouse was in the combination group [27]. Novel in non-small-cell lung cancers. Of 107 cases sulfonamide-containing indole compounds struc- analyzed, 39 (36.4%) had strong mem- turally related to indisulam, their pharmacologi- brane/cytoplasmic expression of CA IX and cally acceptable salts and their hydrates were were grouped as positive. The staining was con- claimed by Eisai scientists [27]. In addition, neovas- fined around areas of necrosis, and a significant cularization inhibitors, anticancer agents, metasta- association of CA IX expression with the extent sis inhibitors and anti-inflammatory agents for the of necrosis was noted. Nevertheless, 38 out of 74 treatment of rheumatoid arthritis comprising these cases with focal or extensive necrosis did not compounds as the active component were also express this enzyme [72]. A direct association of claimed [27]. CA IX expression with epidermal growth factor futurefuture sciencescience groupgroup www.futuremedicine.com 365 REVIEW – Supuran

receptor, c-erbB-2 and MUC1 expression was studies provided clinical evidence that CA IX also noted. Survival analysis showed that CA IX expression is upregulated in hypoxic human cer- expression is related to poor prognosis. Multi- vical tumors and is associated with a poor prog- variate analysis also revealed that CA IX expres- nosis. CA IX may act as an intrinsic marker of sion was a significant prognostic factor tumor hypoxia and poor outcome after radiation independent of angiogenesis. It was concluded therapy. The level of CA IX expression may be that CAIX is an important molecule in non- used to aid in the selection of patients who small-cell lung cancer, the upregulation of which would benefit most from hypoxia-modification occurs in highly hypoxic/necrotic regions of the therapies or bioreductive drugs [74]. CA12, the tumors. The expression of CA IX was linked to gene encoding isozyme XII, has also been identi- the expression of a constellation of proteins fied as a hypoxia-inducible gene [75]. The expres- involved in angiogenesis, apoptosis inhibition, sion of CA IX and CA XII in relation to necrosis and cell–cell adhesion disruption, which and early breast tumor progression in 68 cases of explained the strong association of this enzyme ductal carcinoma has been recently examined with poor clinical outcome [72]. [75]. CA IX expression was rare in normal epithe- A similar study was then reported in a cohort lium and benign lesions, but was present focally of patients with invasive breast cancer [73]. The in ductal carcinomas (50% of cases) and in asso- majority of patients were treated with adjuvant ciated invasive carcinomas (29%). In compari- hormonal or chemotherapy. The frequency of son, CA XII was frequently expressed in normal CA IX expression, its association with recog- breast tissues (89%), in ductal carcinomas (84%) nized prognostic factors and the relationship and in invasive breast lesions (71%). Neither with outcome was evaluated by univariate and CA IX nor CA XII expression was associated multivariate statistical analyses. CA IX expres- with regional or overall proliferation. Assessment sion was present in 49 (48%) out of 103 cases. of mammographic calcification showed that The level of CA IX expression was found to be CA XII expression was associated with the significantly associated with tumor necrosis absence of calcification. These results demon- (p < 0.001), higher grade and negative estrogen strate that induction of CAIX and CAXII receptor status [73]. CA IX expression was associ- occurs in regions adjacent to necrosis in these ated with worse relapse-free survival and overall tumors [75]. Furthermore, these data suggested survival in an unselected cohort of patients with that proliferation status does not influence invasive breast carcinoma. The potential role of expression of either CA isozyme in breast tissues, CA IX as a marker of hypoxia within breast car- that hypoxia may be the dominant factor in the cinomas was also indicated by a significant regulation of CA IX and that factors related to association with necrosis [73]. differentiation, as determined by tumor grade, There is increasing evidence that hypoxia-reg- dominated the regulation of CA XII. The exist- ulated gene expression influences tumor aggres- ence of differential regulation and associations siveness, contributing to the poorer outcome of with an aggressive phenotype may be important patients with hypoxic tumors [74]. The role of the in the development of selective CAIs useful to transcriptional complex HIF-1 as an important prevent/treat tumor invasion [1,2,67]. In another mediator of hypoxia-regulated gene expression is study, the expression and localization of CA IX one of the best documented pathways. Recently, in head and neck squamous cell carcinoma it has emerged that certain tumor-associated CAs (HNSCC) was examined and related to the loca- can be added to the list of known HIF-respon- tion of tumor microvessels, angiogenesis, necro- sive genes [72–74]. In such a study, it was proved sis and stage [76]. CA IX was induced by hypoxia that the tumor-associated CA IX is correlated in three HNSCC cell lines and overexpressed in with the level of hypoxia in human cervical HNSCC tumor tissue. Overexpression was tumors [74]. There was a significant positive corre- localized to the perinecrotic area of the tumor lation between the level of tumor hypoxia and the on immunostaining, and the percentage area of extent of CA IX expression. A retrospective study the tumor expressing CA IX was significantly of 130 squamous cell cervical carcinomas demon- higher with more tumor necrosis and advanced strated that a semiquantitative immunohisto- stage. CA IX was overexpressed in HNSCC chemical analysis of CA IX expression in tumor because of hypoxia and may be a potential biopsies is a significant and independent prog- biomarker for hypoxia in this tumor. Overex- nostic indicator of overall survival and metastasis- pression may help to maintain the intracellular free survival after radiation therapy [74]. These pH, giving tumor cells a survival advantage and

366 Therapy (2007) 4(3) futurefuture sciencescience ggrouproup Therapeutic applications of the carbonic anhydrase inhibitors – REVIEW

enhancing resistance to radiotherapy and chem- fluid removal from the retina to the choroid. In a otherapy. Thus, CA IX is considered a potential 40-year-old female patient on adjuvant chemo- target for future therapy in HNSCC [76]. In a therapy for breast cancer with an isolated recent study, the localization of isozymes CA I, choroid metastasis, clinical and radiological II, IX and XII in normal large intestine and remission was achieved after orbital radio- colorectal tumors has been investigated [77]. therapy, chemotherapy and AZ treatment [75]. While the normal mucosa of the large intestine Thus, AZ may possess another, only slightly showed high expression for CA I and II, the explored up to now, beneficial clinical use in intensity of the immunostaining for both iso- patients with choroid metastasis [75]. zymes decreased in benign lesions and was very weak in malignant tumors. The reciprocal pat- CAIs in obesity tern of expression observed for these cytoplasmic Obesity, a multifactorial disorder characterized isozymes and transmembrane CA IX and XII in by an excess of adipose tissue, represents a chal- intestinal tissue specimens supported the sugges- lenging medical problem in Western countries, tion that CA IX and XII may be functionally with, for example, 65% of the US population involved in tumor progression to malignancy affected [8]. Developing countries, such as China, and/or in invasion [77]. While CA I and II were are also not immune to this epidemic, since it shown to be prominent in normal colorectal was reported that obesity doubled in women and mucosa, playing a role in regulation of pH almost tripled in men from 1989 to 1997 in this homeostasis and water and ion transport, loss of country [8]. Obesity is caused by an excessively expression of these cytoplasmic isozymes was positive energy balance, with the energy intake shown to consistently accompany progression to exceeding the expenditure, but the exact etiology malignant transformation [77]. of the disease is largely unknown [8]. Although In another study, the presence of CA XII diet, physical activity and behavioral modifica- along the human nephron and collecting duct, tions should theoretically help in controlling this together with its cellular and subcellular local- condition, very rarely, patients undergoing these ization, have been investigated [78]. CA XII has strategies can lose more than 5–10% of their been revealed to be present in the basolateral body weight and maintain this loss for a suffi- plasma membrane of the epithelial cells in the ciently long period in order to experience thick ascending limb of Henle and distal convo- improvements in blood pressure, plasma lipid luted tubules, and in the principal cells of the levels, blood glucose and diabetic control [8]. collecting ducts. A weak basolateral signal was Thus, phamacological interventions for the also detected in the epithelium of the proximal treatment of obesity are essential. Paradoxically, convoluted tubules. In addition to the normal the drugs available for the treatment of obesity kidney specimens, this immunohistochemical are very few, their mechanism of action hardly study included 31 renal tumors [78]. CA XII understood and their side effects generally quite showed moderate or strong plasma membrane- serious [8]. associated expression in most oncocytomas and Among the many CA isozymes evidenced so clear-cell carcinomas. The segmental, cellular far in diverse organisms all over the phyloge- and subcellular distribution of CA XII along the netic tree, CA VA and CA VB are present in human nephron and collecting duct suggested mitochondria and are shown to be involved in that it may be one of the key enzymes involved several biosynthetic processes, such as ureagene- in normal renal physiology, particularly in the sis [1–8], gluconeogenesis [1–8] and lipogenesis regulation of water homeostasis [78]. High (both in vertebrates, such as rodents, as well as expression of CA XII in some renal carcinomas invertebrates, such as the locust [8,80–83]). may contribute to its role in von Hippel–Lindau Indeed, in several important biosynthetic proc- carcinogenesis [78,79]. esses involving pyruvate carboxylase (PC), Breast carcinoma is the most frequent cancer acetyl CoA carboxylase (ACC) and carbamoyl - in women and is the second leading cause of phosphate synthetases I and II, HCO3 and not death [75]. Choroid metastasis of breast carci- CO2, is the real substrate of these carboxylating - noma can be found either at presentation or in enzyme. The provision of enough HCO3 is remission, being also frequently encountered in assured mainly by catalysis involving the mito- disseminated breast cancer with multiple-organ chondrial isozymes CA VA and CA VB (proba- metastasis. It has recently been proposed that the bly assisted by the high-activity cytosolic edema-reducing effect of AZ might be used for isozyme CA II) (Figure 7) [8,84–89]. futurefuture sciencescience groupgroup www.futuremedicine.com 367 REVIEW – Supuran

Thus, mitochondrial PC is needed for the several CA isozymes are critical to the entire efflux of acetyl groups from the mitochondria to process of fatty acid biosynthesis: CA VA and/or the cytosol where the fatty acid biosynthesis CA VB within the mitochondria (to provide takes place [82]. Practically, pyruvate is carboxy- enough substrate to PC) and CA II within the lated to oxaloacetate in the presence of bicarbo- cytosol (for providing sufficient substrate to nate and PC. The bicarbonate needed for this ACC). It was, in fact, demonstrated that inhibi- process is generated under the catalytic influence tion of CAs by sulfonamides (e.g., trifluoro- of the mitochondrial isozymes (CA VA and/or methanesulfonamide [TFM], a very potent but CA VB). The mitochondrial membrane is unstable CAI [8]) can decrease lipogenesis in adi- impermeant to acetyl-CoA, which reacts with pocytes in cell culture [82,83]. In such experi- oxaloacetate, leading to citrate, which is there- ments, an indiscriminate inhibition of all CA after translocated to the cytoplasm by means of isozymes present in these tissues is achieved by the tricarboxylic acid transporter. In the cytosol, the used inhibitor, such as, for example, TFM or the citrate is cleaved and regenerates acetyl-CoA AZ [82–84]. and oxaloacetate. As oxaloacetate is unable to Topiramate is an antiepileptic drug possessing cross the mitochondrial membrane, its decarbox- potent anticonvulsant effects due to a multi- ylation regenerates pyruvate, which can be then factorial mechanism of action: CA inhibition, transported into the mitochondria by means of blockade of sodium channels and kain- the pyruvate transporter (Figure 7) [8]. The acetyl- ate/α-amino-3-hydroxy-5-methylisoxazole-4-pro- CoA generated in the cytosol is in fact used for pionic acid (AMPA) receptors, CO2 retention de novo lipogenesis, by carboxylation in the pres- secondary to inhibition of the red cell and brain ence of ACC and bicarbonate, with formation of CA isozymes, as well as enhancement of γ-ami- - malonyl-CoA. The HCO3 needed in this proc- nobutyric acid (GABA)ergic transmission [51]. A ess is furnished by the CA II-catalyzed conver- side effect of this drug observed in obese patients - sion of CO2 to HCO3 . Subsequent steps was the loss of body weight [90], although no involving the sequential transfer of acetyl groups pharmacological explanation of this phenome- lead to longer-chain fatty acids [8]. As a whole, non has been provided. Furthermore, topiramate

Figure 7. The transfer of acetyl groups from the mitochondrion to the cytosol (as citrate) for the provision of substrate for de novo lipogenesis.

Mitochondrion Cytosol

[Pyruvate]c HCO - 3 [Pyruvate]m -CO2 PC Oxaloacetate Oxaloacetate -CO2 Ac-CoA

[Citrate]m [Citrate]c CoA Krebs cycle Ac-CoA - ACC HCO3 Malonyl-CoA

-CO2 Fatty acids Pyruvate transporter

Tricarboxylate transporter Therapy

All steps involving bicarbonate also need the presence of carbonic anhydrase (CA) isozymes: CA VA and CA VB in the mitochondrion and CA II in the cytosol (see discussion in the text). Adapted from [13].

368 Therapy (2007) 4(3) futurefuture sciencescience ggrouproup Therapeutic applications of the carbonic anhydrase inhibitors – REVIEW

was shown to reduce energy and fat gain in lean Zonisamide, is another antiepileptic drug (Fa/?) and obese (fa/fa) Zucker rats [91]. Con- used as adjunctive therapy for refractory partial versely, our group recently demonstrated that seizures [96]. Owing to its multiple mechanisms topiramate is a very potent (low nanomolar) of action, it shows a broad spectrum of anti- inhibitor of several CA isozymes, such as CA I, convulsant activity and has been shown to be II, IV, VA and VAB, and the x-ray crystal struc- effective in several types of seizures, including par- ture of its complex with hCA II has also been tial and generalized seizures, tonic–clonic seizures determined, revealing the basical molecular and absence seizures in patients unresponsive to interactions that explain the high affinity of other anticonvulsants [96]. Recent clinical studies topiramate for the CA active site [51]. have demonstrated additional potential for its Considering these in vitro findings and the therapeutic use in neuropathic pain, bipolar disor- reported clinical side effects of the drug that der, migraine, obesity, eating disorders and Par- caused weight loss in humans and animals [90,91], kinson’s disease, similarly to topiramate [96,97]. we also investigated in detail the inhibition of Being an aliphatic sulfonamide, we have investi- the mitochondrial isozymes hCA VA [92] and gated whether this compound will also interact hCA VB [93] with this drug, showing the com- with the CAs [22]. pound to be a good inhibitor of both (Ki in the Unill recently, zonisamide was considered to range of 30–63 nM). Thus, we hypothesized act as a weak CAI, with a Ki of 4.3 µM against that the inhibition of mitochondrial and/or the cytosolic isozyme hCA II [22]. We proved that cytosolic CA isozymes leading to impaired lipid this is not true. Indeed, testing zonisamide in biosynthesis may represent a means of control- classical assay conditions of CO2 hydrase activity ling weight loss with pharmacological agents of hCA II, with incubation times for the enzyme such as topiramate or any other sulfamate/sul- and inhibitor solution of 15 min, a Ki of fonamide CAIs that show effective CA VA/CA 10.3 µM has been obtained. However, when the VB/CA II inhibition profiles. As a consequence, incubation time between enzyme and inhibitor selective inhibition of mitochondrial CA iso- was increased to 1 h, the obtained Ki was zymes (CA VA and CA VB) may lead to dimin- 35.2 nM, of the same order of magnitude as that ished lipid biosynthesis and a novel of the clinically used sulfonamides/sulfamates pharmacological approach for the treatment of AZ, methazolamide, ethoxzolamide and topiram- obesity [8]. ate (Ki values in the range of 5.4–15.4 nM). Inhi- Recently, Lynch’s group showed that the bition of the human mitochondrial isozyme hCA antipsychotic drug olanzapine (1–8 mg/kg), VA with these compounds has also been tested by but not clozapine, increased body weight in means of a dansylamide competition-binding female rats only [94]. Weight changes were assay, which showed zonisamide and topiramate detectable within 2–3 days and were associated to be effective inhibitors, with Ki values in the with hyperphagia starting approximately 24 h range of 20.6–25.4 nM [22]. The x-ray crystallo- after the first dose. Chronic administration of graphic structure of the adduct of hCA II with the drug (12–29 days) led to adiposity, hyper- zonisamide has also been examined at a resolu- leptinemia and mild insulin resistance, but no tion of 1.70 Å, showing that the sulfonamide lipid abnormalities or changes in D2-receptor moiety participates in the classical interactions density were observed. Topiramate, which has with the Zn2+ ion, through coordination to the reversed weight gain from atypical antipsy- deprotonated sulfonamide moiety, and also inter- chotics in humans [95], also attenuated weight acting with the Thr199 and Glu106 residues. gain in rats. Olanzapine, but not clozapine, The benzisoxazole ring of zonisamide has been acutely lowered plasma glucose and leptin [94]. shown to be oriented toward the hydrophobic Indeed, patients treated with atypical antipsy- area of the active site, establishing a large number chotic drugs commonly gain excess weight. of strong van der Waals interactions (<4.5 Å) Because obesity is associated with considerable with residues Gln92, Val121, Phe131, Leu198, morbidity and decreased life expectancy, treat- Thr200 and Pro202 [22]. ment of weight gain in these patients is criti- Zonisamide, similarly to topiramate, in con- cal. Topiramate was shown to promote weight junction with a reduced-calorie diet (deficit of loss in healthy obese subjects, patients with 500 kcal/day), resulted in an additional mean bipolar disorder, eating disorder and schizo- 5-kg weight loss compared with diet alone [98]. phrenia, without aggravation of their This regimen was well-tolerated in obese female psychotic symptoms [95]. patients. In a randomized, controlled trial, futurefuture sciencescience groupgroup www.futuremedicine.com 369 REVIEW – Supuran

zonisamide therapy was started at 100 mg/day β-adrenergic blockers, alcohol, benzodiazepines, orally, with a gradual increase to 400 mg/day and gabapentin and CAIs, but few detailed studies a further increase to 600 mg/day for patients los- in this field are available [101]. In this context, it ing less than 5% of body weight at the end of must be mentioned that, although long sus- 12 weeks [99]. The zonisamide group (n = 19) pected from histochemical evidence for CA had a mean weight loss of 9.2 kg (1.7 kg; 9.4% activity on neurons and observations that CAIs loss) at week 32 compared with 1.5 kg (0.7 kg; enhance the extracellular alkaline shifts associ- 1.8% loss) for the placebo group (n = 17). ated with synaptic transmission, an extracellular Zonisamide was well tolerated, with few adverse CA located in the brain had not been identified effects [53]. In this preliminary trial, zonisamide until recently [102]. A candidate for this CA was and hypocaloric diet resulted in greater weight suggested by the recent discovery of membrane loss than placebo and hypocaloric diet in the CA XIV, whose mRNA is expressed in mouse treatment of obesity [99]. and human brain as well as several other tissues. CA XIV was found by immunostaining on neu- Miscellaneous applications of CAIs ronal membranes and axons in both mouse and Since CAs are ubiquitous enzymes, their inhibi- human brain. The highest expression was seen tion may have other types of applications in on large neuronal bodies and axons in the anter- addition to those already mentioned above. olateral part of pons and medulla oblongata. Thus, CAIs have been used in the treatment of Other CA XIV-positive sites included the the primary periodic paralyses (PPs), but their hippocampus, corpus callosum, cerebellar white efficacy has not been demonstrated in double- matter and peduncles, pyramidal tract and blind, placebo-controlled trials [100]. Therefore, choroid plexus. These observations made the efficacy of DCP, a potent CAI in the treat- CA XIV a likely candidate for the extracellular ment of episodic weakness in primary PPs, has CA postulated to have an important role in recently been tested [69]. Two multicenter, rand- modulating excitatory synaptic transmission in omized, double-blind, placebo-controlled cross- brain [102]. over trials, one involving 42 subjects with By inhibition of CA activity, the regional hypokalemic periodic paralysis (HypoPP) and blood flow (rBF) in organs increases as hyper- the other involving 31 subjects with potassium- capnia develops [1,103]. However, the effects of sensitive periodic paralysis (PSPP) have been per- AZ-induced vasodilation have not been esti- formed. In each trial, two 8-week treatment peri- mated with respect to vessel size and organs ods were separated by an active washout period until recently [103]. The aim of a recent study of at least 9 weeks. The primary outcome varia- was solely to determine the diameter of the cap- ble in the HypoPP trial was the occurrence of an illaries in various organs that respond to CA intolerable increase in attack severity or fre- inhibition by AZ. While the systolic blood pres- quency; the primary outcome variable in the sure (SBP), pH, hemoglobin concentration and PSPP trial was the number of attacks per week. base excess did not change, the partial pressure The HypoPP trial had 13 subjects who exhibited of arterial O2 (PaO2) increased significantly and a preference for either DCP or placebo, and 11 the partial pressure of arterial CO2 (PaCO2) of these preferred DCP. In the PSPP trial, DCP decreased significantly with AZ. The rBF was significantly reduced attack rates relative to pla- calculated by using three different sizes (15, 25, cebo [69,100]. DCP also significantly reduced and 50 µM) of colored microspheres (CMs). attack rates relative to placebo in the HypoPP The rBF measured with 15-µM CMs in the subjects. It was concluded that the drug is effec- brain, kidneys and liver increased in response to tive in the prevention of episodic weakness in AZ, and the rBF in these organs differed both HypoPP and PSPP [100]. between size of CM. However, the rBF, calcu- Essential tremor (ET) is another common lated by using the different sizes of CM in the movement disorder that often causes functional stomach and abdominal muscle, did not change disability, potentially leading to physical and after the administration of AZ. AZ-induced emotional difficulties [101]. The paucity of data vasodilation occurred in all sizes of vessels in the available regarding the underlying pathophysio- liver, the small- and medium-sized vessels in logic mechanism of ET hinders the develop- kidneys and in the larger capillaries in the brain ment of innovative approaches to [103]. Thus, CAIs may be successfully used as pharmacotherapeutic treatments. Options for diagnostic tools in magnetic resonance imaging drug therapy include the use of primidone, or positron-emission tomography [1,103].

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The study by Komai and colleagues was with Ki values in the range of 1090–6680 nM. designed in order to clarify the effect of Zn defi- Better inhibitors were detected among derivatives ciency on NaCl preference, the lingual trigeminal bearing 2- or 4-amino-, 4-aminomethyl or and taste nerves transduction and CA activity of 4-hydroxymethyl moieties or among halogenated the tongue surface and salivary gland [104]. Male sulfanilamides (Ki values of 608–955 nM). Some Sprague–Dawley rats, 4 weeks old, were divided clinically used compounds, such as AZ, meth- into four groups and fed Zn-deficient, low-Zn azolamide, ethoxzolamide, DCP, dorzolamide, and Zn-sufficient diets with free access and pair- brinzolamide, topiramate, sulpiride and indisu- feeding. After taking part in the preference tests lam, or the orphan drug benzolamide, showed for 42 days, the rats took part in the chorda tym- effective hCA VI inhibitory activity, with Ki val- pani and lingual trigeminal nerve recordings, ues of 0.8–79 nM. The best inhibitors were brin- then finally they were sacrificed and the tongue zolamide and sulpiride (Ki values of and submandibular gland were excised to meas- 0.8–0.9 nM), the latter compound also being a ure CA activity. NaCl preference increased only CA VI-selective inhibitor. The metallic taste after 4 days of the feeding of Zn-deficient and reported as a side effect after the treatment with low-Zn diets, which means that the taste abnor- systemic sulfonamides may be due to the inhibi- mality appeared abruptly in Zn deficieny, even in tion of the salivary CA VI. Some of the com- marginal deficiency. Reduced CA activities of the pounds investigated in this study might be used taste-related tissues in the Zn-deficient group as additives in toothpastes for reducing the acidi- paralleled well with the decreased taste and fication produced by the relevant CO2 hydrase lingual trigeminal nerve sensitivities [104]. activity of enamel CA VI, which leads to the for- - The secretory isozyme CA VI was previously mation of protons and HCO3 and may play a identified as an essential component of mamma- role in carcinogenesis [106]. lian saliva [1], and recently it has been shown to be An immune-mediated reaction to pancreatic an elementary component of human milk [105]. structures has been postulated for the pathogen- The 42-kDa glycopolypeptide purified from esis of chronic pancreatitis (CP) [107]. Several human milk in CAI affinity chromatography reports demonstrate the presence of antibodies shared 100% homology with salivary CA VI in to the pancreatic ductal epithelium in some the protein sequence analysis (40% coverage), patients suffering from CP. Serum antibodies to and its digestion with PNGase F (N-glycosidase) CA I (anti-CA I) and II (anti-CA II) were shown resulted in a polypeptide backbone similar in size to be present in patients affected by idiopathic to salivary CA VI. Quantification of CA VI in CP. A significant correlation between anti-CA I milk using a time-resolved immunofluorometric and anti-CA II serum levels in control subjects assay revealed an approximately eight-times- and in CP patients has been observed. No corre- higher concentration in human colostrum than lation was found between serum antibody levels in mature milk, the latter corresponding to the and any of the following variables: length of dis- levels previously detected in human saliva. The ease, alcohol consumption, smoking habits, high concentration in the colostrum, in particu- pancreatic surgery, pancreatic calcifications, dia- lar its functional and structural stability in an betes and steatorrhea. Serum levels of anti-CA I acidic milieu and its growth-supporting role in and anti-CA II are thus quite elevated in the taste buds, suggested that milk CA VI is an patients suffering from CP [107]. essential factor in normal growth and develop- Seizures are one of the most common neuro- ment of the infant alimentary tract [105]. This logical disorders. The triggering mechanisms by secretory isozyme of human origin, hCA VI, has which seizures occur remain unclear, but are been cloned, expressed and purified in a bacterial related to a rapid change in ionic composition, expression system recently [106]. The kinetic including an increase in intracellular potassium parameters for the CO2 hydration reaction concentration and pH shifts within the brain [108]. proved that hCA VI possessed a significant cata- pH buffering of extra- and intra-cellular spaces is - lytic activity for the physiological reaction in the mainly carried out by the CO2/HCO3 buffer, the same order of magnitude as the ubiquitous iso- equilibration of the two species being assured by form CA I or the transmembrane, tumor-associ- the many CA isoforms present in the brain (e.g., ated isozyme CA IX. A series of sulfonamides and CA I, II, IV, VB, VII, XII and XIV) [108–110]. one sulfamate have been tested for their interac- Some CAIs, such as AZ and methazolamide, tion with this isozyme. Simple benzenesulfona- have been used as anticonvulsants in the treat- mides were rather ineffective hCA VI inhibitors, ment of epilepsy, with rather scarce success [108]. futurefuture sciencescience groupgroup www.futuremedicine.com 371 REVIEW – Supuran

Both these sulfonamides are used either in com- activity-dependent pH changes, as demonstrated bination therapy with other antiepileptic medi- for other CAIs [116]. Such pH shifts might have a cations in both children and adults or in deep impact on neuronal excitability because refractory epilepsy [108–110]. AZ might be useful many ion channels, gap junctions and neuro- in partial, myoclonic, absence and primary gen- transmitter receptors are highly sensitive to eralized tonic–clonic seizures uncontrolled by change in pH [112]. Local administration of AZ other classical antiepileptic drugs [110]. However, has a similar effect to systemic administration of such sulfonamides did not provide effective topiramate or AZ, suggesting that the effect of long-term therapy for epileptic patients due to these drugs on the initial alkalinization is reasons currently not well understood [108–110]. through inhibition of central CA [117]. Both Zonisamide is a synthetic 1,2-benzisoxazole- drugs decreased initial alkalinization, which 3-methanesulfonamide with potent anticonvul- would be consistent with an antiepileptic effect, sant properties [111]. The sulfamoyl group on and may actually contribute to their antiepileptic zonisamide was expected to suppress seizures in actions in vivo [117]. However, the change in pH a similar way to AZ through inhibition of CA, regulation is not a fundamental anticonvulsant but it was claimed that CA inhibition does not mechanism since carbamazepine and phenytoin, appear to be the primary mechanism of action two antiepileptic drugs, have no effect on the of this drug [111]. Zonisamide prevents repetitive alkalinization [117]. Thus, the effect of CA inhibi- neuronal firing by blockade of voltage-sensitive tion on the antiepileptic action of topiramate is Na+ channels. It also reduces voltage-dependent not well understood. T-type Ca2+ channels, facilitating dopaminergic CAIs were also shown to be effective in the and serotoninergic neurotransmission [111]. prevention and treatment of mountain sickness However, it has recently been reported that and high-altitude cerebral edema [118–120]. These zonisamide is also quite an effective CAI against conditions affect, to varying degrees, all travellers many of the physiologically relevant isoforms to high altitudes, being characterized by a com- [22]. It is unknown at this point to what extent bination of symptoms such as headache, insom- the CA inhibitory properties of the drug have a nia, anorexia, nausea, dizziness, vomiting, contribution to its antiepileptic action. dyspnea, muscle weakness, oliguria, peripheral Topiramate is a sulfamate fructopyranose edema and retinal hemorrhage [118–120]. In more derivative currently available for the treatment of serious cases, pulmonary or cerebral edema were partial-onset epilepsy [112]. As an antiepileptic also observed [118–120]. The primary cause of drug, it is known to be clinically effective against mountain sickness is related to reduced oxygen simple or complex partial seizures and also against content in the air at high altitudes, which leads generalized tonic–clonic seizures [112]. Besides its to hypoxemia and all the symptoms mentioned ability to block the voltage-gated Na+ channel, to above. AZ, alone or in combination with dexam- potentiate GABAergic transmission and to block ethasone, is useful in markedly reducing these the kainate/AMPA receptor, topiramate occupies symptoms, due to the increased arterial oxygen a particular place among the new anticonvulsants concentrations after red blood cell/brain enzyme due to its ability to inhibit CA [113]. In this inhibition by the sulfonamide drug [118–120]. It is respect, topiramate-induced changes of GABA- not clear whether methazolamide or other clini- ergic depolarizations were supposed to be based cally used CAIs may have a better mode of action - on a decreased intracellular HCO3 concentra- compared with AZ in preventing acute moun- tion, which may be caused by an inhibition of tain sickness, since few comparative studies have neuronal CA [112,114]. It has been reported by been reported [120]. our group that topiramate strongly inhibits several CA isoforms, among which are the cytosolic Conclusion CA II and CA VII present in the brain [51,115]. CAs continue to be surprising enzymes, as many Moreover, its x-ray crystallographic structure in exciting new discoveries are constantly emerging, complex with hCA II revealed a tight associa- even though these are quite ‘old’ enzymes, being tion, with a network of seven strong H bonds first discovered in 1933, and thoroughly investi- fixing the inhibitor within the active site, in gated since then. In the last few years, a host of addition to Zn(II) coordination through the interesting such reports have been made, first of all ionized sulfamate moiety [51]. The inhibitory regarding the catalytic/inhibition mechanism as effect of topiramate on interstitial and intracel- well as isolation/characterization of new isozymes, lular CAs could modify extra- and intra-cellular in addition to CAs of nonvertebrate origin.

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The x-ray crystallographic structure adducts of membrane-impermeant compounds that inhibit hCA II with the simplest sulfonamides (sulfamide only the membrane-associated isoforms (e.g., and sulfamate) have been reported. These com- CA IX) and not cytosolic (e.g., CA I and II). By pounds are considered to be the mother of all sul- using fluorescent CAIs, a proof-of-concept study fonamides, since they incorporate in their showing the role of CA IX in the tumor acidifica- structure the essential Zn-binding function tion processes was reported, and the fact that this present in all potent CAIs currently known, with- phenomenon may be reversed by inhibiting the out any other aromatic/heterocyclic scaffold gen- catalytic activity of the enzyme with CA IX-selec- erally present in other inhibitors investigated in tive and potent inhibitor. Furthermore, B-con- detail. The x-ray crystallographic structures of taining CA IX inhibitors have been reported, many hCA II and I adducts with sulfonamides which may be useful in B neutron capture ther- and sulfamates currently reported ultimately apy. In another important study, hypoxia-activat- helped in a better understanding of the molecular able CA IX prodrugs containing disulfide bonds interactions between the inhibitor and the were reported. Such bulky, dimeric sulfonamides enzyme, which may lead to a rational drug design are very weak CA I, II and IX inhibitors, but by of inhibitors with reduced side effects and selectiv- reduction in hypoxic conditions present in ity for the target isoform. Among these structures, tumors, they lead to thiols that act as low there are also those with some widely used clinical nanomolar inhibitors of these isozymes. agents, some of which have originally been Various ophthalmologic and antiobesity appli- designed for other applications, such as the anti- cations of the CAIs were also reported in the pat- epileptics topiramate and zonisamide, the antitu- ent and scientific literature, together with novel mor sulfonamide indisulam, the antipsychotic derivatives that act as anticonvulsants. sulpiride, the steroid sulfatase inhibitors COU- There are currently very few pharmacological MATE and EMATE, the COX-2 inhibitors approaches for the treatment of obesity, and most celecoxib and valdecoxib and the artificial sweet- are unsatisfactory. Furthermore, this disease is ener saccaharin. All these compounds show appre- widespread both in the developed and developing ciable inhibition (in the low nanomolar range) of world, and the number of affected individuals is some physiologically relevant human CAs. constantly increasing. Thus, new and effective In the last few years, most of the catalytically approaches are needed for the development of active isozymes have been investigated in detail antiobesity agents possessing different mecha- for their inhibition profile against the most nisms of action. A new approach for the treat- important classes of CAIs, the sulfonamides, sul- ment and prophylaxis of obesity may be based on famates and sulfamides. This is highly important the inhibition of CAs, enzymes involved in several in the search of isozyme-selective compounds steps of de novo lipogenesis, both in the mito- and for reducing side effects of the presently chondria and the cytosol of cells. Topiramate and available drugs. Indeed, physiologically quite rel- zonisamide, clinically used antiepileptic drugs evant isoforms, such as CA IV, VA, VB, VII, IX, also showing strong CA inhibitory properties, XII, XIII and XIV, have been investigated for the possess a highly desired side effect in obese first time in detail for their inhibition with many patients – induction of weight loss. It has been types of such compounds, and several important recently proved by our groups that both topiram- drug-design studies have also been reported in ate and zonisamide act as very potent in vitro which compounds with selectivity for one or inhibitors of several CA isozymes (including other such isozymes were obtained. However, to CA II, CA VA and CA VB), and that this might date, few compounds show a good selectivity for explain their antiobesity effects. A program of any CA isoform with clinical relevance, although designing such antiobesity sulfonamide CAIs also important advances have been reported in the could lead to the detection of several interesting design of compounds with a good selectivity lead molecules. CAIs targeting isozymes involved ratio for CA VA over CA II, CA IX over CA II or in lipogenesis may represent a new approach for CA XIII over CA II (CA II is the ubiquitous, cat- the treatment and prevention of obesity. alytically very effective isoform, and this is the Several important findings relate to the full reason why many-times its inhibition is regarded structural characterization of CAs belonging to as detrimental). the β- and γ-families (characteristic of bacteria, In targeting the tumor-associated isoform plants and Archaea, respectively). Rather shock- CA IX, important advances have been reported ing was the finding that, in some β-CAs, there is - in the design of fluorescence labels and of no H2O molecule or OH coordinated to the futurefuture sciencescience groupgroup www.futuremedicine.com 373 REVIEW – Supuran

catalytically essential Zn(II) ion, making these families in most of them, although the non-mam- enzymes quite different to those belonging to malian CAs are highly uninvestigated at the the α- and γ-CA families. Thus, the catalytic present time. Thus, in my opinion, there will be mechanism of β-CAs is not fully understood at major breakthroughs in this field with the discov- this time. ery of many new such enzymes and/or novel gene Many novel representatives of CAs belonging families encoding for CAs. to the three families mentioned above were iso- It is less probable that novel CAs will be discov- lated and characterized in some other organisms ered in mammalians/humans, since the entire less investigated up to now, such as bacteria (e.g., genome of such species has already been analyzed Helicobacter pylori, Neisseria gonorrheae, in detail, and 15 isozymes was detected in pri- Escherichia coli and Mycobacterium tuberculosis) mates, and 16 in nonprimates. However, the fungi (Candida albicans and Cryptococcus neofor- physiological function of many of these isoforms mans) or in the protozoa causing malaria is currently largely not understood, and we esti- (P. f a l c i p a r u m ). However, few inhibition studies mate that important progress will occur in this of these enzymes are currently available. These field, especially considering the fact that all these discoveries may soon have important therapeutic isozymes have now been characterized, from the consequences in the treatment of infections biochemical point of view, and their inhibition caused by pathogenic agents, in cases where profiles have also been investigated with many potent and selective inhibitors for the pathogenic classes of organic and inorganic inhibitors. As a over the host enzymes could be detected. consequence, it is possible that new CAIs will be The data presented herein clearly show that developed, some of them with selectivity for vari- CAs and their inhibitors may play an essential ous isoforms. Indeed, the presently available drugs role in the development of new therapeutic belonging to this class indiscriminately inhibit approaches against a multitude of disorders, in most isozymes. It is probable that the future will addition to the classical ones for which such see the development of isozyme-selective inhibi- agents were and are still in use. tors for isozymes I, II, VA, VB, VII, IX, XII, XIII and XIV, which will lead to drugs with less side Future perspective effects. It is also probable that many new such tar- As CAs are widespread enzymes in most organisms gets belonging to the bacterial/fungal/protozoan all over the phylogenetic tree, and as they deal with CAs will be validated and lead to novel types of some of the very simple substrates (such as CO2 agents fighting diseases provoked by such patho- - and HCO3 ), it is rather probable that the future gens. The first results already obtained in this field will see the discovery of many new representatives are very promising. of these catalysts in many different species. In fact, the recent reports of genomes of many bacterial Acknowledgements (pathogenic) species revealed a high number of Research from the author’s laboratory was financed in part by an CAs belonging to the α-, β-, γ- and/or δ-CA gene EU grant of the 6th framework programme (EUROXY project).

Executive summary

• Carbonic anhydrases (CAs) are widespread enzymes in bacteria, archaea and eukaryotes that catalyze a critically important - physiologic reaction – hydration of CO2 to HCO3 and protons. • These enzymes are inhibited by several classes of compounds, such as sulfonamides, sulfamates and sulfamides, among others.

• A number of such derivatives are clinically used inhibitors, such as acetazolamide, methazolamide, ethoxzolamide, dichlorophenamide, dorzolamide, brinzolamide, topiramate, zonisamide, sulpiride, celecoxib and valdecoxib. Several compounds are in clinical development, such as indisulam and COUMATE-667.

• CA inhibitors (CAIs) are used in therapy as antiglaucoma, diuretic, antiobesity and antitumor drugs/diagnostic tools. Some compounds are also used as anticonvulsants or for the treatment of other neurological disorders.

• Most presently available CAIs show undesired side effects due to indiscriminate inhibition of CA isoforms other than the target.

• Many new CAIs are being developed in the search of isozyme-selective compounds as potential drugs with less side effects.

• Bacterial, fungal and protozoan CAs present in many pathogens have recently started to be considered as potential targets for the development of inhibitors with therapeutic applications.

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Bibliography 13. Cox EH, McLendon GL, Morel FM et al.: 23. Di Fiore A, Pedone C, D’Ambrosio K, 1. Supuran CT, Scozzafava A, Conway J: The active site structure of Thalassiosira Scozzafava A, De Simone G, Supuran CT: Carbonic Anhydrase: its Inhibitors and weissflogii carbonic anhydrase 1. Carbonic anhydrase inhibitors: valdecoxib Activators. CRC Press, Boca Raton, FL, Biochemistry 39, 12128–12130 (2000). binds to a different active site region of the USA 1–363 (2004). 14. Lane TW, Morel FM: A biological function human isoform II as compared to the 2. Supuran CT, Scozzafava A: Carbonic for cadmium in marine diatoms. Proc. Natl structurally related cyclooxygenase II anhydrase inhibitors and their therapeutic Acad. Sci. USA 97, 4627–4631 (2000). “selective” inhibitor celecoxib. Bioorg. Med. potential. Expert Opin. Ther. Pat. 10, 15. Abbate F, Supuran CT, Scozzafava A, Chem. Lett. 16, 437–442 (2006). 575–600 (2000). Orioli P, Stubbs MT, Klebe G: The 24. Menchise V, De Simone G, Alterio V et al.: 3. Supuran CT, Scozzafava A: Applications of sulfonamide group as an ideal anchor for the Carbonic anhydrase inhibitors: stacking carbonic anhydrase inhibitors and design of potent human carbonic anhydrase with Phe131 determines active site binding activators in therapy. Expert Opin. Ther. inhibitors: role of hydrogen-bonding region of inhibitors as exemplified by the Pat. 12, 217–242 (2002). networks in ligand binding and drug design. x-ray crystal structure of a membrane- 4. Scozzafava A, Mastrolorenzo A, J. Med. Chem. 45, 3583–3587 (2002). impermeant antitumor sulfonamide Supuran CT: Modulation of carbonic 16. Ferraroni M, Briganti F, Chegwidden WR complexed with isozyme II. J. Med. Chem. anhydrase activity and its applications in et al.: Structure of the human carbonic 48, 5721–5727 (2005). therapy. Expert Opin. Ther. Pat. 14, anhydrase (hCA) isoenzyme I Michigan 1 25. Krungkrai SR, Suraveratum N, 667–702 (2004). variant in the absence and in the presence of Rochanakij S, Krungkrai J: Characterisation 5. Supuran CT, Scozzafava A, Casini A: exogenous zinc ions: the first CA binding of carbonic anhydrase in Plasmodium Carbonic anhydrase inhibitors. Med. Res. two zinc ions. Biochemistry 41, 6237–6244 falciparum. Int. J. Parasitol. 31, 661–668 Rev. 23, 146–189 (2003). (2001). (2001). 6. Supuran CT, Vullo D, Manole G, Casini A, 17. Kim CY, Chandra PP, Jain A, 26. Supuran CT, Scozzafava A, Casini A: Scozzafava A: Designing of novel carbonic Christianson DW: Fluoroaromatic- Development of sulfonamide carbonic anhydrase inhibitors and activators. Curr. fluoroaromatic interactions between anhydrase inhibitors. In: Carbonic Med. Chem. Cardiovasc. Hematol. Agents 2, inhibitors bound in the crystal lattice of Anhydrase: its Inhibitors and Activators. 49–68 (2004). human carbonic anhydrase II. J. Am. Chem. Supuran CT; Scozzafava A, Conway J (Eds). 7. Pastorekova S, Parkkila S, Pastorek J, Soc. 123, 9620–9627 (2001). CRC Press, Boca Raton, FL, USA 67–147 Supuran CT: Carbonic anhydrases: current 18. Whittington DA, Waheed A, Ulmasov B (2004). state of the art, therapeutic applications et al.: Crystal structure of the dimeric 27. Scozzafava A, Mastrolorenzo A, and future prospects. J. Enzyme Inhib. Med. extracellular domain of human carbonic Supuran CT: Carbonic anhydrase inhibitors Chem. 19, 199–229 (2004). anhydrase XII, a bitopic membrane protein and activators and their use in therapy. 8. Supuran CT: Carbonic anhydrase overexpressed in certain cancer tumor cells. Expert Opin. Ther. Pat. 16, 1627–1644 inhibitors in the treatment and prophylaxis Proc. Natl Acad. Sci. USA 98, 9545–9550 (2006). of obesity. Expert Opin. Ther. Pat. 13, (2001). 28. Supuran CT: Carbonic anhydrases: catalytic 1545–1550 (2003). 19. Stams T, Nair SK, Okuyama T, Waheed A, and inhibition mechanisms, distribution and 9. Mitsuhashi S, Mizushima T, Yamashita E Sly WS, Christianson DW. Crystal structure physiological roles. In: Carbonic Anhydrase: et al.: X-ray structure of β-carbonic of the secretory form of membran-associated its Inhibitors and Activators. Supuran CT; anhydrase from the red alga, Porphyridium human carbonic anhydrase IV at 2.8 Å Scozzafava A, Conway J (Eds). CRC Press, purpureum, reveals a novel catalytic site for resolution. Proc. Natl Acad. Sci. USA 93, Boca Raton, FL, USA 1–23 (2004).

CO2 hydration. J. Biol. Chem. 275, 13589–13594 (1996). 29. Scozzafava A, Briganti F, Ilies MA, 5521–5526 (2000). 20. Lindahl M, Vidgren J, Eriksson E et al.: Supuran CT: Carbonic anhydrase inhibitors: 10. Strop P, Smith KS, Iverson TM, Ferry JG, Crystallographic studies of carbonic synthesis of membrane-impermeant low Rees DC: Crystal structure of the “cab”- anhydrase inhibition. In: Carbonic molecular weight sulfonamides possessing type β class carbonic anhydrase from the Anhydrase. Botrè F, Gros G, Storey BT in vivo selectivity for the membrane-bound archaeon Methanobacterium (Eds). VCH, Weinheim, Germany 111–118 versus cytosolic isozymes. J. Med. Chem. 43, thermoautotrophicum. J. Biol. Chem. 276, (1991). 292–300 (2000). 10299–10305 (2001). 21. Alterio V, Vitale RM, Monti SM et al.: 30. Scozzafava A, Supuran CT: Carbonic 11. Kimber MS, Pai EF: The active site Carbonic anhydrase inhibitors: x-ray and anhydrase inhibitors: synthesis of architecture of Pisum sativum β-carbonic molecular modeling study for the N-morpholylthiocarbonylsulfenylamino anhydrase is a mirror image of that of interaction of a fluorescent antitumor aromatic/heterocyclic sulfonamides and α-carbonic anhydrases. EMBO J. 19, sulfonamide with isozyme II and IX. J. Am. their interaction with isozymes I, II and IV. 1407–1418 (2000). Chem. Soc. 128, 8329–8335 (2006). Bioorg. Med. Chem. Lett. 10, 1117–1120 12. Iverson TM, Alber BE, Kisker C, Ferry JG, 22. De Simone G, Di Fiore A, Menchise V (2000). Rees DC: A closer look at the active site of et al.: Carbonic anhydrase inhibitors. 31. Barboiu M, Supuran CT, Menabuoni L γ-class carbonic anhydrases: high-resolution Zonisamide is an effective inhibitor of the et al.: Carbonic anhydrase inhibitors. crystallographic studies of the carbonic cytosolic isozyme II and mitochondrial Synthesis of topically effective intraocular anhydrase from Methanosarcina isozyme V: solution and x-ray pressure lowering agents derived from 5-(O- thermophila. Biochemistry 39, 9222–9231 crystallographic studies. Bioorg. Med. Chem. aminoalkylcarboxamido)-1,3,4-thiadiazole- (2000). Lett. 15, 2315–2320 (2005). 2-sulfonamide. J. Enzyme Inhib. 15, 23–46 (2000).

futurefuture sciencescience groupgroup www.futuremedicine.com 375 REVIEW – Supuran

32. Briganti F, Tilli S, Mincione G, Mincione F, 40. Scozzafava A, Banciu MD, Popescu A, hydroxamates with MMP inhibitory Menabuoni L, Supuran CT: Carbonic Supuran CT: Carbonic anhydrase properties act as efficient inhibitors of CA anhydrase inhibitors. Metal complexes of 5- inhibitors: synthesis of Schiff bases of isozymes I, II, and IV, and (2-chlorophenyl)-1,3,4-thiadiazole-2- hydroxybenzaldehydes with aromatic N-hydroxysulfonamides inhibit both these sulfonamide with topical intraocular sulfonamides and their reactions with zinc enzymes. J. Med. Chem. 43, 3677–3687 pressure lowering properties: the influence arylsulfonyl isocyanates. J. Enzyme Inhib. (2000). of metal ions upon the pharmacological 15, 533–546 (2000). 50. Abbate F, Casini A, Owa T, Scozzafava A, activity. J. Enzyme Inhib. 15, 185–200 41. Supuran CT, Scozzafava A: Carbonic Supuran CT: Carbonic anhydrase (2000). anhydrase inhibitors: aromatic sulfonamides inhibitors: E7070, a sulfonamide anticancer 33. Supuran CT, Scozzafava A, Ilies MA, and disulfonamides act as efficient tumor agent, potently inhibits cytosolic isozymes I Briganti F: Carbonic anhydrase inhibitors: growth inhibitors. J. Enzyme Inhib. 15, and II, and transmembrane, tumor- synthesis of sulfonamides incorporating 597–610 (2000). associated isozyme IX. Bioorg. Med. Chem. 2,4,6-trisubstituted-pyridinium- 42. Supuran CT, Scozzafava A: Carbonic Lett. 14, 217–223 (2004). ethylcarboxamido moieties possessing anhydrase inhibitors – Part 94. 1,3,4- 51. Casini A, Antel J, Abbate F et al.: Carbonic membrane-impermeability and in vivo thiadiazole-2-sulfonamidederivatives as anhydrase inhibitors: SAR and x-ray selectivity for the membrane-bound antitumor agents? Eur. J. Med. Chem. 35, crystallographic study for the interaction of (CA IV) versus the cytosolic (CA I and 867–874 (2000). sugar sulfamates/sulfamides with isozymes CA II) isozymes. J. Enzyme Inhib. 15, 43. Supuran CT, Briganti F, Tilli S, I, II and IV. Bioorg. Med. Chem. Lett. 13, 381–401 (2000). Chegwidden WR, Scozzafava A: Carbonic 841–845 (2003). 34. Ilies M, Supuran CT, Scozzafava A et al.: anhydrase inhibitors: sulfonamides as 52. Abbate F, Coetzee A, Casini A, Ciattini S, Carbonic anhydrase inhibitors: antitumor agents? Bioorg. Med. Chem. 9, Scozzafava A, Supuran CT: Carbonic sulfonamides incorporating furan-, 703–714 (2001). anhydrase inhibitors: x-ray crystallographic thiophene- and pyrrole-carboxamido 44. Scozzafava A, Menabuoni L, Mincione F, structure of the adduct of human isozyme groups possess strong topical intraocular Mincione G, Supuran CT: Carbonic II with the antipsychotic drug sulpiride. pressure lowering properties as aqueous anhydrase inhibitors: synthesis of Bioorg. Med. Chem. Lett. 14, 337–341 suspensions. Bioorg. Med. Chem. 8, sulfonamides incorporating DTPA tails and (2004). 2145–2455 (2000). of their zinc complexes with powerful 53. Lloyd MD, Pederick RL, Natesh R et al.: 35. Clare BW, Supuran CT: Carbonic topical antiglaucoma properties. Bioorg. Crystal structure of human carbonic anhydrase inhibitors. Part 86. A QSAR Med. Chem. Lett. 11, 575–582 (2001). anhydrase II at 1.95 Å resolution in study on some sulfonamide drugs which 45. Casini A, Mincione F, Ilies MA, complex with 667-coumate, a novel anti- lower intra-ocular pressure, using the ACE Menabuoni L, Scozzafava A, Supuran CT: cancer agent. Biochem. J. 385, 715–720 non-linear statistical method. Eur. J. Med. Carbonic anhydrase inhibitors: synthesis (2005). Chem. 35, 859–865 (2000). and inhibition against isozymes I, II and IV 54. Abbate F, Winum JY, Potter BV et al.: 36. Scozzafava A, Banciu MD, Popescu A, of topically acting antiglaucoma Carbonic anhydrase inhibitors: x-ray Supuran CT: Carbonic anhydrase sulfonamides incorporating cis-5- crystallographic structure of the adduct of inhibitors: inhibition of isozymes I, II and norbornene-endo-3-carboxy-2-carboxamido human isozyme II with EMATE, a dual IV by sulfamide and sulfamic acid moieties. J. Enzyme Inhib. 16, 113–123 inhibitor of carbonic anhydrases and derivatives. J. Enzyme Inhib. 15, 443–453 (2001). steroid sulfatase. Bioorg. Med. Chem. Lett. (2000). 46. Chazalette C, Riviere-Baudet M, 14, 231–234 (2004). 37. Scozzafava A, Menabuoni L, Mincione F, Scozzafava A, Abbate F, Ben Maarouf Z, 55. Weber A, Casini A, Heine A et al.: Briganti F, Mincione G, Supuran CT: Supuran CT: Carbonic anhydrase inhibitors, Unexpected nanomolar inhibition of Carbonic anhydrase inhibitors: interaction of boron derivatives with carbonic anhydrase by COX-2-selective perfluoroalkyl/aryl-substituted derivatives isozymes I and II: a new binding site for celecoxib: new pharmacological of aromatic/heterocyclic sulfonamides as hydrophobic inhibitors at the entrance of opportunities due to related binding site topical intraocular pressure-lowering agents the active site as shown by docking studies. recognition. J. Med. Chem. 47, 550–557 with prolonged duration of action. J. Med. J. Enzyme Inhib. 16, 125–133 (2001). (2004). Chem. 43, 4542–4551 (2000). 47. Mincione F, Starnotti M, Menabuoni L, 56. Köhler K, Hillebrecht A, Innocenti A, 38. Casini A, Scozzafava A, Mincione F, Scozzafava A, Casini A, Supuran CT: Heine A, Supuran CT, Klebe G: Saccharin, Menabuoni L, Ilies MA, Supuran CT: Carbonic anhydrase inhibitors: 4-sulfamoyl- a potent inhibitor of carbonic anhydrases. Carbonic anhydrase inhibitors: water- benzenecarboxamides and 4-chloro-3- Science (2007) (In Press). soluble 4-sulfamoylphenylthioureas as sulfamoyl-benzenecarboxamides with strong 57. Seong GJ, Lee SC, Lee JH, Chu YK, topical intraocular pressure-lowering agents topical antiglaucoma properties. Bioorg. Hong YJ: Comparisons of intraocular- with long-lasting effects. J. Med. Chem. 43, Med. Chem. Lett. 11, 1787–1791 (2001). pressure- lowering efficacy and side effects 4884–4892 (2000). 48. Supuran CT, Clare BW: Orbital symmetry of 2% dorzolamide and 1% brinzolamide. 39. Mastrolorenzo A, Scozzafava A, in QSAR: some Schiff’s base inhibitors of Ophthalmologica 215, 188–191 (2001). Supuran CT: Antifungal activity of Ag(I) carbonic anhydrase. SAR QSAR Environ. 58. Ormrod D, McClellan K: Topical and Zn(II) complexes of aminobenzolamide Res. 12, 17–29 (2001). dorzolamide 2%/timolol 0.5%: a review of (5-sulfanilylamido-1,3,4-thiadiazole-2- 49. Scozzafava A, Supuran CT: Carbonic its use in the treatment of open-angle sulfonamide) derivatives. J. Enzyme Inhib. anhydrase and matrix metalloproteinase glaucoma. Drugs Aging 17, 477–496 15, 517–531 (2000). inhibitors: sulfonylated amino acid (2000).

376 Therapy (2007) 4(3) futurefuture sciencescience ggrouproup Therapeutic applications of the carbonic anhydrase inhibitors – REVIEW

59. Susanna R Jr, Nicolela MT, Oga E: inhibitors of the cancer-associated isozyme patient with choroid metastasis of breast Additive effect of latanoprost to the IX is dependent on efflux transporters. carcinoma. Int. J. Clin. Pract. 55, 488–490 combination of timolol and dorzolamide. Bioorg. Med. Chem. 14, 2418–2427 (2001). J. Glaucoma 9, 183–186 (2000). (2006). 80. Spencer IM, Hargreaves I, 60. Gelatt KN, MacKay EO: Changes in 70. Parkkila S, Rajaniemi H, Parkkila AK et al.: Chegwidden WR. Effect of the carbonic intraocular pressure associated with topical Carbonic anhydrase inhibitor suppresses anhydrase inhibitor acetazolamide on lipid dorzolamide and oral methazolamide in invasion of renal cancer cells in vitro. Proc. synthesis in the locust. Biochem. Soc. Trans. glaucomatous dogs. Vet. Ophthalmol. 4, Natl Acad. Sci. USA 97, 2220–2224 16, 973–974 (1988). 61–67 (2001). (2000). 81. Chegwidden WR, Spencer IM. Carbonic 61. Michaud JE, Friren B; The International 71. Saarnio J, Parkkila S, Parkkila AK et al.: anhydrase provides bicarbonate for de novo Brinzolamide Adjunctive Study Group: Transmembrane carbonic anhydrase, lipogenesis in the locus. Comp. Biochem. Comparison of topical brinzolamide 1% MN/CA IX, is a potential biomarker for Physiol. 115B, 247–254 (1996). and dorzolamide 2% eye drops given twice biliary tumours. J. Hepatol. 35, 643–649 82. Chegwidden WR, Dodgson SJ, Spencer IM: daily in addition to timolol 0.5% in (2001). The roles of carbonic anhydrase in patients with primary open-angle 72. Giatromanolaki A, Koukourakis MI, metabolism, cell growth and cancer in glaucoma or ocular hypertension. Am. J. Sivridis E et al.: Expression of hypoxia- animals. In: The Carbonic Anhydrases – New Ophthalmol. 132, 235–243 (2001). inducible carbonic anhydrase-9 relates to Horizons. Chegwidden WR, Edwards Y, 62. Wolfensberger TJ: The role of carbonic angiogenic pathways and independently to Carter N (Eds). Birkhäuser Verlag, Basel, anhydrase inhibitors in the management of poor outcome in non-small cell lung Switzerland 343–363 (2000). macular edema. Doc. Ophthalmol. 97, cancer. Cancer Res. 61, 7992–7998 (2001). 83. Lynch CJ, Fox H, Hazen SA, Stanley BA, 387–397 (1999). 73. Chia SK, Wykoff CC, Watson PH et al.: Dodgson SJ, Lanoue KF: Role of hepatic 63. Gao BB, Clermont A, Rook S et al.: Prognostic significance of a novel hypoxia- carbonic anhydrase in de novo lipogenesis. Extracellular carbonic anhydrase mediates regulated marker, carbonic anhydrase IX, in Biochem. J. 310, 197–202 (1995). hemorrhagic retinal and cerebral vascular invasive breast carcinoma. J. Clin. Oncol. 84. Hazen SA, Waheed A, Sly WS, Lanoue KF, permeability through prekallikrein 19, 3660–3668 (2001). Lynch CJ: Differentiation-dependent activation. Nat. Med. 13, 181–188 (2007). 74. Loncaster JA, Harris AL, Davidson SE expression of CA V and the role of carbonic 64. Pastorekova S, Pastorek J: Cancer-related et al.: Carbonic anhydrase (CA IX) anhydrase isozymes in pyruvate carbonic anhydrase isozymes and their expression, a potential new intrinsic marker carboxylation in adipocytes. FASEB J. 10, inhibition. In: Carbonic Anhydrase: its of hypoxia: correlations with tumor oxygen 481–490 (1996). Inhibitors and Activators. Supuran CT, measurements and prognosis in locally 85. Atwood PV: The structure and mechanism Scozzafava A, Conway J (Eds). CRC Press, advanced carcinoma of the cervix. Cancer of action of pyruvate carboxylase. Int. J. Boca Raton, FL, USA 253–280 (2004). Res. 61, 6394–6399 (2001). Biochem. Cell Biol. 27, 231–249 (1995). 65. Thiry A, Dogné JM, Masereel B, 75. Wykoff CC, Beasley N, Watson PH et al.: 86. Alldred JB, Reilly KE: Short-term regulation Supuran CT: Targeting tumor-associated Expression of the hypoxia-inducible and of acetyl CoA carboxylase in tissues of carbonic anhydrase IX in cancer therapy. tumor-associated carbonic anhydrases in higher animals. Prog. Lipid Res. 35, 371–385 Trends Pharmacol. Sci. 27, 566–573 ductal carcinoma in situ of the breast. Am. (1997). (2006). J. Pathol. 158, 1011–1019 (2001). 87. Forster RE, Dodgson SJ: Membrane 66. Svastova E, Hulikova A, Rafajova M et al.: 76. Beasley NJ, Wykoff CC, Watson PH et al.: transport and provision of substrates for Hypoxia activates the capacity of tumor- Carbonic anhydrase IX, an endogenous carbonic anhydrase in vertebrates. In: The associated carbonic anhydrase IX to acidify hypoxia marker, expression in head and Carbonic Anhydrases – New Horizons. extracellular pH. FEBS Lett. 577, 439–445 neck squamous cell carcinoma and its Chegwidden WR, Edwards Y, Carter N (2004). relationship to hypoxia, necrosis, and (Eds). Birkhäuser Verlag, Basel, Switzerland 67. Cecchi A, Hulikova A, Pastorek J et al.: microvessel density. Cancer Res. 61, 263–280 (2000). Carbonic anhydrase inhibitors. Design of 5262–5267 (2001). 88. Parkkila S: An overview of the distribution fluorescent sulfonamides as probes of 77. Kivela AJ, Saarnio J, Karttunen TJ et al.: and function of carbonic anhydrase tumor-associated carbonic anhydrase IX Differential expression of cytoplasmic isozymes in mammals. In: The Carbonic that inhibit isozyme IX-mediated carbonic anhydrases, CA I and II, and Anhydrases – New Horizons. Chegwidden acidification of hypoxic tumors. J. Med. membrane-associated isozymes, CA IX and WR, Edwards Y, Carter N (Eds). Birkhäuser Chem. 48, 4834–4841 (2005). XII, in normal mucosa of large intestine Verlag, Basel, Switzerland 79–94 (2000). 68. Casey JR, Morgan PE, Vullo D, and in colorectal tumors. Dig. Dis. Sci. 46, 89. Parkkila S, Parkkila AK, Kivelä J: Role of Scozzafava A, Mastrolorenzo A, 2179–2186 (2001). carbonic anhydrase and its inhibitors in Supuran CT: Carbonic anhydrase 78. Parkkila S, Parkkila AK, Saarnio J et al.: biological science related to inhibitors. Design of selective, membrane- Expression of the membrane-associated gastroenterology, neurology and nephrology. impermeant inhibitors targeting the carbonic anhydrase isozyme XII in the In: Carbonic Anhydrase: its Inhibitors and human tumor-associated isozyme IX. J. human kidney and renal tumors. Activators. Supuran CT, Scozzafava A, Med. Chem. 47, 2337–2347 (2004). J. Histochem. Cytochem. 48, 1601–1608 Conway J (Eds). CRC Press, Boca Raton, 69. Vullo D, Steffansen B, Brodin B, Supuran (2000). FL, USA 283–301 (2004). CT, Scozzafava A, Nielsen CU: Carbonic 79. Sari R, Camci C, Kutlu R, Totan Y, 90. Gordon A, Price LH: Mood stabilization anhydrase inhibitors: transepithelial Sevinc A, Buyukberber S: The efficacy of and weight loss with topiramate. Am. J. transport of thioureido sulfonamide acetazolamide on visual functions in a Psychiatry 156, 968–969 (1999).

futurefuture sciencescience groupgroup www.futuremedicine.com 377 REVIEW – Supuran

91. Picard F, Deshaies Y, Lalonde J, Samson P, 102. Parkkila S, Parkkila AK, Rajaniemi H et al.: 111. Leppik IE: Zonisamide: chemistry, Richard D: Topiramate reduces energy and Expression of membrane-associated mechanism of action, and fat gains in lean (Fa/?) and obese (fa/fa) carbonic anhydrase XIV on neurons and pharmacokinetics. Seizure 13(Suppl. 1), Zucker rats. Obesity Res. 8, 656–663 (2000). axons in mouse and human brain. Proc. Natl S5–9 (2004). 92. Vullo D, Franchi M, Gallori E, Antel J, Acad. Sci. USA 98, 1918–1923 (2001). 112. Herrero AI, Del Olmo N, Gonzalez- Scozzafava A, Supuran CT: Carbonic 103. Taki K, Oogushi K, Hirahara K, Gai X, Escalada JR, Solis JM: Two new actions of anhydrase inhibitors. Inhibition of Nagashima F, Tozuka K: Preferential topiramate: inhibition of depolarizing mitochondrial isozyme V with aromatic and acetazolamide-induced vasodilation based GABA(A)-mediated responses and heterocyclic sulfonamides. J. Med. Chem. on vessel size and organ: confirmation of activation of a potassium conductance. 47, 1272–1279 (2004). peripheral vasodilation with use of colored Neuropharmacology 42, 210–220 (2002). 93. Nishimori I, Vullo D, Innocenti A, microspheres. Angiology 52, 483–488 113. Ilies MA, Masereel B, Rolin S et al.: Scozzafava A, Mastrolorenzo A, (2001). Carbonic anhydrase inhibitors: aromatic Supuran CT: Carbonic anhydrase 104. Komai M, Goto T, Suzuki H, Takeda T, and heterocyclic sulfonamides incorporating inhibitors. The mitochondrial isozyme VB Furukawa Y: Zinc deficiency and taste adamantyl moieties with strong as a new target for sulfonamide and dysfunction; contribution of carbonic anticonvulsant activity. Bioorg. Med. Chem. sulfamate inhibitors. J. Med. Chem. 48, anhydrase, a zinc-metalloenzyme, to normal Lett, 12, 2717–2726 (2004). 7860–7866 (2005). taste sensation. Biofactors 12, 65–70 (2000). 114. Leniger T, Thone J, Wiemann M: 94. Albaugh VL, Henry CR, Bello NT et al.: 105. Karhumaa P, Leinonen J, Parkkila S, Topiramate modulates pH of hippocampal Hormonal and metabolic effects of Kaunisto K, Tapanainen J, Rajaniemi H: CA3 neurons by combined effects on - - olanzapine and clozapine related to body The identification of secreted carbonic carbonic anhydrase and Cl /HCO3 weight in rodents. Obesity 14, 36–51 anhydrase VI as a constitutive glycoprotein exchange. Br. J. Pharmacol. 142, 831–842 (2006). of human and rat milk. Proc. Natl Acad. Sci. (2004). 95. Lin YH, Liu CY, Hsiao MC: Management USA 98, 11604–11608 (2001). 115. Vullo D, Voipio J, Innocenti A et al.: of atypical antipsychotic-induced weight 106. Nishimori I, Minakuchi T, Onishi S, Carbonic anhydrase inhibitors. Inhibition of gain in schizophrenic patients with Vullo D, Scozzafava A, Supuran CT: the human cytosolic isozyme VII with topiramate. Psychiatry Clin. Neurosci. 59, Carbonic anhydrase inhibitors. DNA aromatic and heterocyclic sulfonamides. 613–615 (2005). cloning, characterization, and inhibition Bioorg. Med. Chem. Lett. 15, 971–976 96. Zareba G: Zonisamide: review of its use in studies of the human secretory isoform VI, a (2005). epilepsy therapy. Drug Today (Barc.) 41, new target for sulfonamide and sulfamate 116. Pasternack M, Voipio J, Kaila K: 589–597 (2005). inhibitors. J. Med. Chem. 50, 381–388 Intracellular carbonic anhydrase activity and 97. Canitano R: Clinical experience with (2007). its role in GABA-induced acidosis in Topiramate to counteract neuroleptic 107. Frulloni L, Bovo P, Brunelli S et al.: Elevated isolated rat hippocampal pyramidal induced weight gain in 10 individuals with serum levels of antibodies to carbonic neurones. Acta Physiol. Scand. 148, 229–231 autistic spectrum disorders. Brain Dev. 27, anhydrase I and II in patients with chronic (1993). 228–232 (2005). pancreatitis. Pancreas 20, 382–388 (2000). 117. Aribi AM, Stringer JL: Effects of 98. Kim CS: Zonisamide effective for weight 108. Reiss WG, Oles KS: Acetazolamide in the antiepileptic drugs on extracellular pH loss in women. J. Fam. Pract. 52, 600–601 treatment of seizures. Ann. Pharmacother. regulation in the hippocampal CA1 region (2003). 30, 514–519 (1996). in vivo. Epilepsy Res. 49, 143–151 (2002). 99. Gadde KM, Franciscy DM, 109. Katayama F, Miura H, Takanashi S: Long- 118. Bernhard WN, Schalik LM, Delaney PA, Wagner HR 2nd, Krishnan KR: Zonisamide term effectiveness and side effects of Bernhard TM, Barnas GM: Acetazolamide for weight loss in obese adults: a randomized acetazolamide as an adjunct to other plus low-dose dexamethasone is better than controlled trial. JAMA 289, 1820–1825 anticonvulsants in the treatment of acetazolamide alone to ameliorate symptoms (2003). refractory epilepsies. Brain Dev. 24, of acute mountain sickness. Aviat. Space 100. Tawil R, McDermott MP, Brown R Jr et al.: 150–154 (2002). Environ. Med. 69, 883–886 (1998). Randomized trials of dichlorphenamide in 110. Parkkila S, Parkkila AK, Kivela J: Role of 119. Bradwell AR, Wright AD, Winterborn M, the periodic paralyses. Working Group on carbonic anhydrase and its inhibitors in Imray C: Acetazolamide and high altitude Periodic Paralysis. Ann. Neurol. 47, 46–53 gastroenterology, neurology and nephrology. disease. Int. J. Sports Med. 13, 63–64 (2000). In: Carbonic Anhydrase: its Inhibitors and (1992). 101. Koller WC, Hristova A, Brin M: Activators. Supuran CT, Scozzafava A, 120. Porcelli MJ, Gugelchuk GM: A trek to the Pharmacologic treatment of essential tremor. Conway J (Eds). CRC Press, Boca Raton, top: a review of acute mountain sickness. Neurology 54(Suppl 4), S30–S38 (2000). FL, USA 283–301 (2004). J. Am. Osteopath. Assoc. 95, 718–720 (1995).

378 Therapy (2007) 4(3) futurefuture sciencescience ggrouproup