Carbonic anhydrase inhibitors and their potential in a range of therapeutic areas

KEYWORDS:

• Carbonic anhydrase
• inhibitor
• activator
• diuretic
• sulfonamide
• epilepsy
• sleep apnea
• neuropathic pain
• cerebral ischemia
• cancer
• metastasis
• anti-infectives
• bacterial enzymes

The metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1) is ubiquitously expressed in organisms all over the phylogenetic tree, with seven genetically distinct families known to date. By equilibrating CO₂ and bicarbonate with generation of a H⁺ ion, CAs play a crucial role in pH regulation, in providing bicarbonate or H⁺ ions for electrolyte secretion, but also in several metabolic pathways such as lipogenesis, gluconeogenesis and ureagenesis. The CA inhibitors (CAIs) are clinically used as diuretics, anti-glaucoma agents and anti-epileptics, but novel applications in the management of cancer, neuropathic pain, sleep apnea, migraine, lowering intracranial pressure, and cerebral ischemia were recently reported. CA activators may be useful in the treatment of cognitive impairment and phobias. This special issue of Expert Opinion on Therapeutic Patents provides an update in these fields, with reviews dedicated to the applications in renal and central nervous system diseases, cancer and metastasis, as well as biomedical applications of prokaryotic CAs, widely abundant in pathogenic bacteria, but also in fungi and protozoa. Some patent evaluations for the use of CAIs in sleep apnea and regulation of mast cell response are also present in this special issue, demonstrating the many new fields in which modulators of the activity of these enzymes may have useful applications.

The CAs act as catalysts for the interconversion between CO₂, bicarbonate, and protons, one of the simplest chemical reactions connected with vital processes [1–6]. Indeed, CO₂, a gas already present in the primeval earth atmosphere, is generated in most oxidative metabolic processes in all organisms (except chemolithoautotrophs), whereas plants use it for photosynthesis. This is probably the reason why at least seven genetically distinct CA families are known to date, being present in organisms all over the phylogenetic tree (denominated as α-, β-, γ-, δ-, ζ-, η-, and ɵ-CAs) [7–15].

A large number of α-CA isoforms have been described in vertebrates: 15 in humans and other primates, and 16 in other mammals [1–6]. Thus, α-CAs are so widespread enzymes probably because their role is connected to tightly controlled processes such as pH regulation and metabolism [1–15]. The view of CAs as metabolic enzymes started to be considered recently, after their role in tumor metabolism has been understood in detail [5,6]. At least CA II, IX, and XII participate in pH regulation and metabolic processes within hypoxic tumors, in which the oxidative phosphorylation of glucose is impaired due to the low amount of oxygen present, and most energy is obtained by the alternative, glycolytic pathway, which leads to the formation of lactic acid [5,6,16–19]. The mitochondrial isoform CA VA was also proven to be involved in biosynthetic processes such as lipogenesis, neoglucogenesis, ureagenesis among others [20–22]. Thus, the clinical use of the CA inhibitors (CAIs) saw new developments in these areas in recent years. The sulfonamides incorporating primary SO₂NH₂ moieties [23–33] were recognized to act as potent CAIs already in the 1950s, and the first diuretic based on this class of pharmacologic agents, acetazolamide (5-acetamido-1,3,4-thiadiazole-2-sulfonamide), started to be clinically used in 1956, with this drug still being used nowadays [1]. In fact, the discovery of acetazolamide played an important role in the development of renal physiology and pharmacology and led to the discovery of many modern classes of diuretics [30–33]. Aspects related to the renal applications of CAIs, alone or in combination with other pharmacological agents, are dealt with in the paper by Supuran in this special issue [34]. Novel applications in the management of drug-induced renal injury were recently reported for classical CAIs such as acetazolamide, methazolamide and topiramate [34], whereas the involvement of renal CA isoforms in the reabsorption of nitrite (NO₂⁻), which is the most abundant reservoir of the biologically highly potent signaling molecule nitric oxide (NO), has also been investigated in some details in the last years [35,36].

At least 9 of the various CA isoforms are present in the human central nervous system (CNS) and choroid plexus, but their functions are poorly understood, although it is clear that they are involved in crucial functions within these organs [34]. CA inhibition in the CNS was exploited therapeutically for obtaining anticonvulsant agents already in the 1970s, with acetazolamide and methazolamide the most used such agents, in some forms of epilepsy [37–39]. The mechanisms by which CAIs exert antiepileptic action are rather complex and there is not a definitive consensus among researchers on many aspects related to this pharmacological effect of drugs such as topiramate, zonisamide, or sulthiame [37–40]. Inhibition of brain CAs leads to a diminished formation of bicarbonate and also changes the brain pH, contributing thus to an antiepileptic effect by several different pathways [37–40]. Among the new applications recently claimed in the fields, were those of combination therapies of a CAI, preferentially topiramate, with other pharmacological agents, such as an aldosterone derivative, or benzodiazepines as a therapeutic option for obstructive sleep apnea, with several patents which are discussed in the Patent evaluation by Angeli and Supuran [41]. There are several other pharmacologic applications of the CAIs related to the inhibition of CNS isoforms, among which idiopathic intracranial hypertension (IIH) [42,43], cerebral ischemia [44], neuropathic pain [45–47], and migraine [48]. Drug design campaigns and the proof of concept study regarding the druggability of various CA isoforms for the management of these conditions were recently published [43–47] and are discussed in detail in one of the reviews present in this special issue [34].

CA activation was reported in an early stage of CA research, but the field remained highly controversial till recently, when work with highly purified enzymes and very precise techniques, such as the stopped-flow assay, undoubtedly demonstrated that the CA activators (CAAs) exist and that they take part to the catalytic cycle [49–53]. However, up until recently there seem to be no pharmacological applications for the CAAs [54]. Recently it has been unraveled that CAAs may be of great relevance in pathologies related to the pharmacological enhancement of synaptic efficacy, spatial learning, and memory, with the mechanistic details responsible for such phenomena reported by Blandina’s group [54]. Administration of CAAs of the amino acid type was shown to lead to an enhanced spatial learning, which was antagonized by the simultaneous administration of a sulfonamide inhibitor, such as acetazolamide [54]. Impairments of the fear memory consolidation were also observed in the course of such experiments, which might pave the way for pharmacologic applications in novel therapies for phobia and post-traumatic shock [54]. The administration of the CAA rapidly activated the extracellular signal-regulated kinase (ERK) pathways, which is involved in a critical step of memory formation, both in the cortex and the hippocampus, the two brain areas involved in memory processing [54]. Such interesting results might also lead to the use of CAAs for memory therapy in aging or in neurodegenerative conditions such as Alzheimer’s disease [54].

A second review article from the Special issue deals with the inhibition of the tumor associated isoforms CA IX and XII, which may lead to novel therapies for the treatment of hypoxic, metastatic solid tumors [55]. As mentioned above [6,16–19], several CA isoforms are predominantly present in tumors, in which they are involved in pH regulation and metabolic processes. It has been demonstrated that their specific inhibition with sulfonamides, sulfamates, or coumarins [56–59] has profound effects on the inhibition of the primary tumor growth, metastases formation and reduction in the cancer stem cells number, three mechanisms unique only to this class of pharmacological agents [60–62]. Thus, a large number of drug design studies were performed in the last 10 years for the design of CA IX/XII – selective inhibitors [1,3–6,55], with one such compound, SLC-0111 presently in Phase Ib/II clinical trials as an antitumor/antimetastatic agent [63]. All these aspects are dealt with in detail in the review by Nocentini and Supuran in this Special issue [55].

A new aspect of CAI research is analyzed in the Patent evaluation by Winum [64] published in the Special issue. It has recently been claimed that CA activity modulators (inhibitors or activators) may be also useful for the treatment of allergic diseases, viral, bacterial, or fungal infections, or mastocytosis, based on their effects on mast cell-mediated inflammation [64].

The last review in the Special issue, by Supuran and Capasso [65] deals with the biomedical applications of CAs of prokaryotic origin. As mentioned above [1,2,4,7], CAs from at least three classes (the α-, β- and γ-CA) are present in eukaryotes, in which they play crucial functions related to pH regulation, photosynthesis, acclimation to environmental conditions (e.g. extreme pH conditions, extreme temperatures, high concentrations of salts, etc. [1,2,4,7]), and metabolism [2,7]. Many representatives of such enzymes were cloned and characterized in detail from many pathogenic and nonpathogenic bacteria among which Helicobacter pylori, Vibrio cholerae, Brucella suis, Mycobacterium tuberculosis, etc. (to mention only the most relevant ones) [66–72]. Successful drug design campaigns [1,2,4,7,73] were also reported for many such enzymes from pathogenic bacteria in the search of agents able to overcome the extensive drug resistance observed with the classical antibiotics [74]. However, there seems to be a lack of interest from drug companies for developing anti-infectives with an alternative mechanism of action based on CA inhibition. This review article brings the attention to these underexplored anti-infective drug targets, which, if properly considered, may represent a breakthrough to the field.

Overall, the very interesting and highly diverse topics treated in these review articles and Patent evaluations prove again, if necessary, the many aspects in which the CA research may bring highly innovative applications, in resolving challenging problems with which our evolving society is confronted nowadays.

Declaration of interest

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

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