Acetazolamide encapsulation in elastin like recombinamers using a supercritical antisolvent (SAS) process for glaucoma treatment.

Glaucoma, the second most common cause of blindness worldwide, requires the development of new and effective treatments. This study introduces a novel controlled-release system utilizing elastin-like recombinamers (ELR) and the Supercritical Antisolvent (SAS) technique with supercritical CO2. Acetazolamide (AZM), a class IV drug with limited solubility and permeability, is successfully encapsulated in an amphiphilic ELR at three different ELR:AZM ratios, yielding up to 62 %. Scanning electron microscopy (SEM) reveals spherical microparticles that disintegrate into monodisperse nanoparticles measuring approximately 42 nm under physiological conditions. The nanoparticles, as observed via Transmission Electron Microscopy (TEM) and Atomic Force Microscopy (AFM), do not exhibit aggregates, a fact confirmed by the zeta potential displaying a value of -33 mV over a period of 30 days. Transcorneal permeation tests demonstrate a 10 % higher permeation level compared to the control solution, which increases to 30 % after 2 h. Ocular irritation tests demonstrate no adverse effects or damage. Intraocular pressure (IOP) tests conducted on hypertensive rabbits indicate greater effectiveness for all three analyzed formulations, suggesting enhanced drug bioavailability during treatment. Consequently, the combination of recombinant biopolymers and high-pressure techniques represents a promising approach for advancing glaucoma therapy, emphasizing its potential clinical significance.

Development of direct compression Acetazolamide tablet with improved bioavailability in healthy human volunteers enabled by cocrystallization with p-Aminobenzoic acid.

Pharmaceutical cocrystallization has been widely used to improve physicochemical properties of APIs. However, developing cocrystal formulation with proven clinical success remains scarce. Successful translation of a cocrystal to suitable dosage forms requires simultaneously improvement of several deficient physicochemical properties over the parent API, without deteriorating other properties critical for successful product development. In the present work, we report the successful development of a direct compression tablet product of acetazolamide (ACZ), using a 1:1 cocrystal of acetazolamide with p-aminobenzoic acid (ACZ-PABA). The ACZ-PABA tablet exhibits superior biopharmaceutical performance against the commercial tablet, DIAMOX® (250 mg), in healthy human volunteers, leading to more than 50 % reduction in the required dose.

Acetazolamide and human carbonic anhydrases: retrospect, review and discussion of an intimate relationship.

Acetazolamide (AZM) is a strong pharmacological sulphonamide-type (R-SO2-NH2, pKa 7.2) inhibitor of the activity of several carbonic anhydrase (CA) isoforms, notably of renal CA II (Ki, 12 nM) and CA IV (Ki, 74 nM). AZM is clinically used for about eighty years in various diseases including epilepsy and glaucoma. Pharmacological AZM increases temporarily the urinary excretion of bicarbonate (HCO3-) and sodium ions (Na+) and sustainably the urinary pH. AZM is excreted almost unchanged over several hours at high rates in the urine. Closely parallel concentrations of circulating and excretory AZM are observed upon administration of therapeutical doses of AZM. In a proof-of-principle study, we investigated the effects of the ingestion of a 250-mg AZM-containing tablet by a healthy volunteer on the urinary excretion of organic and inorganic substances over 5 h (range, 0, 0.5, 1, 1.5, 2, 3, 4, 5 h). Measured analytes included: AZM, amino acids and their metabolites such as guanidinoacetate, i.e. the precursor of creatine, of asymmetrically (ADMA) and symmetrically (SDMA) dimethylated arginine, nitrite (O = N-O-, pKa 3.4) and nitrate (O2N-O-, pKa -1.37), the major metabolites of nitric oxide (NO), the C-H acidic malondialdehyde (MDA; (CHO)2CH2, pKa 4.5), and creatinine for correction of analytes excretion. All analytes were measured by validated isotopologues using gas chromatography-mass spectrometry (GC-MS) methods. AZM excretion in the urine reached its maximum value after 2 h and was fairly stable for the next 3 h. Time series analysis by the ARIMA method was performed. AZM ingestion increased temporarily the urinary excretion of the amino acids Leu + Ile, nitrite and nitrate, decreased temporarily the urinary excretion of other amino acids. AZM decreased sustainably the urinary excretion of MDA, a biomarker of oxidative stress (i.e. lipid peroxidation). Whether this decrease is due to inhibition of the excretion of MDA or attenuation of oxidative stress by AZM is unknown. The acute and chronic effects of AZM on the urinary excretion of electrolytes and physiological substances reported in the literature are discussed in depth in the light of its extraordinary pharmacokinetics and pharmacodynamics. Tolerance development/drug resistance to AZM in chronic use and potential mechanisms are also addressed.

Site-Specific Chemical Modification of a Cytokine Mimic for Small Molecule-Based Tumor Targeting.

The targeted delivery of bioactive proteins, such as cytokines, for cancer immunotherapy approaches mostly relies on antibodies or antibody fragments. However, fusion proteins may display low tissue penetration due to a large molecular size. Small molecule ligands with high affinity toward tumor-associated antigens provide a promising alternative for the selective delivery of cytokines to tumor lesions. We developed a one-pot procedure for the site-specific thiazolidine formation between an aldehyde bearing small molecule and the in situ generated N-terminal cysteine of a bioactive protein. Thereby, neoleukin-2/15 (Neo-2/15), a computationally engineered interleukin-2 and -15 mimic, was chemically conjugated to acetazolamide plus, a potent carbonic anhydrase IX (CAIX) ligand. The conjugate retained the biological activity of Neo-2/15 and revealed its ability to accumulate in renal cell carcinoma (SK-RC-52) xenografts upon systemic intravenous administration. The results highlight the potential of small molecule targeting moieties to drive the accumulation of a protein cargo to the respective disease site while conserving the small construct size.

Small Structural Differences Govern the Carbonic Anhydrase II Inhibition Activity of Cytotoxic Triterpene Acetazolamide Conjugates.

Acetylated triterpenoids betulin, oleanolic acid, ursolic acid, and glycyrrhetinic acid were converted into their succinyl-spacered acetazolamide conjugates. These conjugates were screened for their inhibitory activity onto carbonic anhydrase II and their cytotoxicity employing several human tumor cell lines and non-malignant fibroblasts. As a result, the best inhibitors were derived from betulin and glycyrrhetinic acid while those derived from ursolic or oleanolic acid were significantly weaker inhibitors but also of diminished cytotoxicity. A betulin-derived conjugate held a Ki = 0.129 µM and an EC50 = 8.5 µM for human A375 melanoma cells.

A Case Study on the Use of Binding Free Energies to Screen Inhibitors of Human Carbonic Anhydrase II.

We present in this article a case study on the thermodynamics of binding to human carbonic anhydrase II (HCA II) by three well-known inhibitors, viz. (a) acetazolamide (AZM) that directly binds to the catalytic Zn(II) ion at the active site, (b) non-zinc binding 6-hydroxy-2-thioxocoumarin (FC5) (c) 2-[(S)-benzylsulfinyl]benzoic acid (3G1). In each case, the crystal structure or its analogue of inhibitor-bound HCA II has been used to perform classical molecular dynamics (MD) simulation in water till 1 µ s ${1\hskip0.33em\mu s}$ . AZM and FC5 are found to undergo repeated binding and unbinding with markedly different dynamics from the partially buried, substrate-binding hydrophobic pocket near the active site. 3G1, on the other hand, is found to remain mostly at its crystallographic binding site occluded from the active site of HCA II. The associated binding free energies ( Δ G b i n d , s o l v ${{\rm \Delta }{G}_{bind,solv}}$ ) have been computed using the known MM/GBSA method and compared to the available experimental data. Our results show that Δ G b i n d , s o l v ${{\rm \Delta }{G}_{bind,solv}}$ encounters several issues including limited sampling of multiple binding sites and incorrect prediction of the affinity of the chosen ligands. Possible use of the simulation results in further construction of Markov state models is also discussed.

Chalcogen Bond as a Factor Stabilizing Ligand Conformation in the Binding Pocket of Carbonic Anhydrase IX Receptor Mimic.

It is postulated that the overexpression of Carbonic Anhydrase isozyme IX in some cancers contributes to the acidification of the extracellular matrix. It was proved that this promotes the growth and metastasis of the tumor. These observations have made Carbonic Anhydrase IX an attractive drug target. In the light of the findings and importance of the glycoprotein in the cancer treatment, we have employed quantum-chemical approaches to study non-covalent interactions in the binding pocket. As a ligand, the acetazolamide (AZM) molecule was chosen, being known as a potential inhibitor exhibiting anticancer properties. First-Principles Molecular Dynamics was performed to study the chalcogen and other non-covalent interactions in the AZM ligand and its complexes with amino acids forming the binding site. Based on Density Functional Theory (DFT) and post-Hartree-Fock methods, the metric and electronic structure parameters were described. The Non-Covalent Interaction (NCI) index and Atoms in Molecules (AIM) methods were applied for qualitative/quantitative analyses of the non-covalent interactions. Finally, the AZM-binding pocket interaction energy decomposition was carried out. Chalcogen bonding in the AZM molecule is an important factor stabilizing the preferred conformation. Free energy mapping via metadynamics and Path Integral molecular dynamics confirmed the significance of the chalcogen bond in structuring the conformational flexibility of the systems. The developed models are useful in the design of new inhibitors with desired pharmacological properties.

Antiproliferative effects of sulphonamide carbonic anhydrase inhibitors C18, SLC-0111 and acetazolamide on bladder, glioblastoma and pancreatic cancer cell lines.

Carbonic anhydrase IX/XII (CA IX/XII), are cell-surface enzymes typically expressed by cancer cells as a form of adaptation to hypoxia and acidosis. It has been widely reported that these proteins play pivotal roles in cancer progression fostering cell migration, aggressiveness and resistance to first line chemo- and radiotherapies. CA IX has emerged as a promising target in cancer therapy and several approaches and families of compounds were characterised in the attempt to find optimal targeting by inhibiting of the high catalytic activity of the enzyme. In the present work, different cell lines representing glioblastoma, bladder and pancreatic cancer have been exploited to compare the inhibitory and antiproliferative effect of primary sulphonamide acetazolamide (AAZ), the Phase Ib/II clinical grade sulphonamide SLC-0111, and a membrane-impermeant positively charged, pyridinium-derivative (C18). New hints regarding the possibility to exploit CA inhibitors in these cancer types are proposed.

Repurposing FDA-approved sulphonamide carbonic anhydrase inhibitors for treatment of Neisseria gonorrhoeae.

Neisseria gonorrhoeae is a high-priority pathogen of concern due to the growing prevalence of resistance development against approved antibiotics. Herein, we report the anti-gonococcal activity of ethoxzolamide, the FDA-approved human carbonic anhydrase inhibitor. Ethoxzolamide displayed an MIC50, against a panel of N. gonorrhoeae isolates, of 0.125 µg/mL, 16-fold more potent than acetazolamide, although both molecules exhibited almost similar potency against the gonococcal carbonic anhydrase enzyme (NgCA) in vitro. Acetazolamide displayed an inhibition constant (Ki) versus NgCA of 74 nM, while Ethoxzolamide's Ki was estimated to 94 nM. Therefore, the increased anti-gonococcal potency of ethoxzolamide was attributed to its increased permeability in N. gonorrhoeae as compared to that of acetazolamide. Both drugs demonstrated bacteriostatic activity against N. gonorrhoeae, exhibited post-antibiotic effects up to 10 hours, and resistance was not observed against both. Taken together, these results indicate that acetazolamide and ethoxzolamide warrant further investigation for translation into effective anti-N. gonorrhoeae agents.

Recent advances in the medicinal chemistry of carbonic anhydrase inhibitors.

Carbonic anhydrase (CA, EC 4.2.1.1) is an enzyme and a very omnipresent zinc metalloenzyme which catalyzed the reversible hydration and dehydration of carbon dioxide and bicarbonate; a reaction which plays a crucial role in many physiological and pathological processes. Carbonic anhydrase is present in human (h) with sixteen different isoforms ranging from hCA I-hCA XV. All these isoforms are widely distributed in different tissues/organs and are associated with a range of pivotal physiological activities. Due to their involvement in various physiological roles, inhibitors of different human isoforms of carbonic anhydrase have found clinical applications for the treatment of various diseases including glaucoma, retinopathy, hemolytic anemia, epilepsy, obesity, and cancer. However, clinically used inhibitors of CA (acetazolamide, brinzolamide, dorzolamide, etc.) are not selective causing the undesirable side effects. One of the major hurdles in the design and development of carbonic anhydrase inhibitors is the lack of balanced isoform selectivity which thrived to new chemotypes. In this review, we have compiled the recent strategies of various researchers related to the development of carbonic anhydrase inhibitors belonging to different structural classes like pyrimidine, pyrazoline, selenourea, isatin, indole, etc. This review also summarizes the structure-activity relationships, analysis of isoform selectivity including mechanistic and in silico studies to afford ideas and to provide focused direction for the design and development of novel isoform-selective carbonic anhydrase inhibitors with therapeutic implications.

Structural Basis of Nanomolar Inhibition of Tumor-Associated Carbonic Anhydrase IX: X-Ray Crystallographic and Inhibition Study of Lipophilic Inhibitors with Acetazolamide Backbone.

This study provides a structure-activity relationship study of a series of lipophilic carbonic anhydrase (CA) inhibitors with an acetazolamide backbone. The inhibitors were tested against the tumor-expressed CA isozyme IX (CA IX), and the cytosolic CA I, CA II, and membrane-bound CA IV. The study identified several low nanomolar potent inhibitors against CA IX, with lipophilicities spanning two log units. Very potent pan-inhibitors with nanomolar potency against CA IX and sub-nanomolar potency against CA II and CA IV, and with potency against CA I one order of magnitude better than the parent acetazolamide 1 were also identified in this study, together with compounds that displayed selectivity against membrane-bound CA IV. A comprehensive X-ray crystallographic study (12 crystal structures), involving both CA II and a soluble CA IX mimetic (CA IX-mimic), revealed the structural basis of this particular inhibition profile and laid the foundation for further developments toward more potent and selective inhibitors for the tumor-expressed CA IX.

Sulphonamide inhibition profile of Staphylococcus aureus ß-carbonic anhydrase.

This paper presents the production and kinetic and inhibitory characterisation of ß-carbonic anhydrase from the opportunistic bacterium Staphylococcus aureus (SauBCA). From the eight different carbonic anhydrase (CA) families known to date, humans have only the α-form, whereas many clinically relevant pathogens have ß- and/or γ-form(s). Based on this discovery, ß- and γ-CAs have been introduced as promising new anti-infective targets. The results of this study revealed that recombinant SauBCA possesses significant CO2 hydration activity with a kcat of 1.46 × 105 s-1 and a kcat/KM of 2.56 × 107 s- 1M-1. Its enzymatic function was inhibited by various sulphonamides in the nanomolar - micromolar range, and the Ki of acetazolamide was 628 nM. The best inhibitor was the clinically used sulfamide agent famotidine (Ki of 71 nM). The least efficient inhibitors were zonisamide and dorzolamide. Our work encourages further investigations of SauBCA in an attempt to discover novel drugs against staphylococcal infections.

Optimization of Acetazolamide-Based Scaffold as Potent Inhibitors of Vancomycin-Resistant Enterococcus.

Vancomycin-resistant enterococci (VRE) are the second leading cause of hospital-acquired infections (HAIs) attributed to a drug-resistant bacterium in the United States, and resistance to the frontline treatments is well documented. To combat VRE, we have repurposed the FDA-approved carbonic anhydrase drug acetazolamide to design potent antienterococcal agents. Through structure-activity relationship optimization we have arrived at two leads possessing improved potency against clinical VRE strains from MIC = 2 µg/mL (acetazolamide) to MIC = 0.007 µg/mL (22) and 1 µg/mL (26). Physicochemical properties were modified to design leads that have either high oral bioavailability to treat systemic infections or low intestinal permeability to treat VRE infections in the gastrointestinal tract. Our data suggest the intracellular targets for the molecules are putative α-carbonic and γ-carbonic anhydrases, and homology modeling and molecular dynamics simulations were performed. Together, this study presents potential anti-VRE therapeutic options to provide alternatives for problematic VRE infections.

Visualizing drug binding interactions using microcrystal electron diffraction.

Visualizing ligand binding interactions is important for structure-based drug design and fragment-based screening methods. Rapid and uniform soaking with potentially reduced lattice defects make small macromolecular crystals attractive targets for studying drug binding using microcrystal electron diffraction (MicroED). However, so far no drug binding interactions could unambiguously be resolved by electron diffraction alone. Here, we use MicroED to study the binding of a sulfonamide inhibitor to human carbonic anhydrase isoform II (HCA II). We show that MicroED data can efficiently be collected on a conventional transmission electron microscope from thin hydrated microcrystals soaked with the clinical drug acetazolamide (AZM). The data are of high enough quality to unequivocally fit and resolve the bound inhibitor. We anticipate MicroED can play an important role in facilitating in-house fragment screening for drug discovery, complementing existing methods in structural biology such as X-ray and neutron diffraction.

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

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

Synthesis, characterization and carbonic anhydrase I and II inhibitory evaluation of new sulfonamide derivatives bearing dithiocarbamate.

In this study, novel dithiocarbamate-sulfonamide derivatives (3a-3k) were synthesized to investigate their inhibitory activity on purified human carbonic anhydrase (hCA) I and II. The IC50 and Ki values of the compounds were calculated to compare their inhibition profiles on hCA I and II isoenzymes. Acetazolamide was used as the standard inhibitor in the enzyme inhibition assay. Compounds 3a, 3e, 3g, 3h, 3j and 3k showed notable inhibitory effects against hCA I and II. Among these compounds, compound 3h was found to be the most active derivate against both the hCA I and II enzymes with Ki values of 0.032 ± 0.001 µM and 0.013 ± 0.0005 µM, respectively. The cytotoxicity of compounds 3a, 3e, 3g, 3h, 3j and 3k toward NIH/3T3 (mouse embryonic fibroblast cell line) was observed and the compounds were found to be non-cytotoxic. Furthermore, molecular docking studies were performed to investigate the interaction types between compound 3h and the hCA I and II enzymes. As a result of this study a novel and potent class of CA inhibitors with good activity potential were identified.

Bacterial ι-carbonic anhydrase: a new active class of carbonic anhydrase identified in the genome of the Gram-negative bacterium Burkholderia territorii.

The carbonic anhydrases (CAs, EC 4.2.1.1) catalyse a simple but physiologically crucial reversible reaction, the carbon dioxide hydration with the production of bicarbonate and protons. In the last years, and especially, to the rapid emergence of the bacterial antibiotic resistance that is occurring worldwide, the understanding of the function of bacterial CAs has increased significantly. Recently, a new CA-class (ι-CA) was discovered in the marine diatom T. pseudonana. It has been reported that bacterial genomes may contain genes with relevant homology to the diatom ι-class CA. Still, the catalytic activity of the enzyme encoded by the gene was not investigated. Thus, herein, for the first time, we cloned, expressed, and purified the recombinant bacterial ι-CA (acronym BteCAι) identified in the genome of Burkholderia territorii. The recombinant BteCAι resulted in a good catalyst for the hydration of CO2 to bicarbonate and protons, with a kcat of 3.0 × 105 s -1 and kcat/KM of 3.9 × 107 M -1 s -1, and is also sensitive to inhibition by the sulphonamide acetazolamide. Furthermore, with the aid of the protonography, it has been demonstrated that BteCAι can be present as a dimer. This result is corroborated by the construction of a molecular model of BteCAι, which showed that the enzyme is formed by two equivalent monomers having a structure similar to a butterfly.

Sulfonamide Inhibition Studies of the ß-Class Carbonic Anhydrase CAS3 from the Filamentous Ascomycete Sordaria macrospora.

A new ß-class carbonic anhydrase was cloned and purified from the filamentous ascomycete Sordaria macrospora, CAS3. This enzyme has a higher catalytic activity compared to the other two such enzymes from this fungus, CAS1 and CAS2, which were reported earlier, with the following kinetic parameters: kcat of (7.9 ± 0.2) × 105 s-1, and kcat/Km of (9.5 ± 0.12) × 107 M-1∙s-1. An inhibition study with a panel of sulfonamides and one sulfamate was also performed. The most effective CAS3 inhibitors were benzolamide, brinzolamide, dichlorophnamide, methazolamide, acetazolamide, ethoxzolamide, sulfanilamide, methanilamide, and benzene-1,3-disulfonamide, with KIs in the range of 54-95 nM. CAS3 generally shows a higher affinity for this class of inhibitors compared to CAS1 and CAS2. As S. macrospora is a model organism for the study of fruiting body development in fungi, these data may be useful for developing antifungal compounds based on CA inhibition.

Automated and enhanced extraction of a small molecule-drug conjugate using an enzyme-inhibitor interaction based SPME tool followed by direct analysis by ESI-MS.

We report a novel, fast, and automatic SPME-based method capable of extracting a small molecule-drug conjugate (SMDC) from biological matrices. Our method relies on the extraction of the drug conjugate followed by direct elution into an electrospray mass spectrometer (ESI-MS) source for qualitative and quantitative analysis. We designed a tool for extracting the targeting head of a recently synthesized SMDC, which includes acetazolamide (AAZ) as high-affinity ligand specific to carbonic anhydrase IX. Specificity of the extraction was achieved through systematic optimization. The design of the extraction tool is based on noncovalent and reversible interaction between AAZ and CAII that is immobilized on the SPME extraction phase. Using this approach, we showed a 330% rise in extracted AAZ signal intensity compared to a control, which was performed in the absence of CAII. A linear dynamic range from 1.2 to 25 µg/ml was found. The limits of detection (LOD) of extracted AAZ from phosphate-buffered saline (PBS) and human plasma were 0.4 and 1.2 µg/ml, respectively. This with a relative standard deviation of less than 14% (n = 40) covers the therapeutic range. Graphical abstract.

Potentially repurposable drugs for schizophrenia identified from its interactome.

We previously presented the protein-protein interaction network of schizophrenia associated genes, and from it, the drug-protein interactome which showed the drugs that target any of the proteins in the interactome. Here, we studied these drugs further to identify whether any of them may potentially be repurposable for schizophrenia. In schizophrenia, gene expression has been described as a measurable aspect of the disease reflecting the action of risk genes. We studied each of the drugs from the interactome using the BaseSpace Correlation Engine, and shortlisted those that had a negative correlation with differential gene expression of schizophrenia. This analysis resulted in 12 drugs whose differential gene expression (drug versus normal) had an anti-correlation with differential expression for schizophrenia (disorder versus normal). Some of these drugs were already being tested for their clinical activity in schizophrenia and other neuropsychiatric disorders. Several proteins in the protein interactome of the targets of several of these drugs were associated with various neuropsychiatric disorders. The network of genes with opposite drug-induced versus schizophrenia-associated expression profiles were significantly enriched in pathways relevant to schizophrenia etiology and GWAS genes associated with traits or diseases that had a pathophysiological overlap with schizophrenia. Drugs that targeted the same genes as the shortlisted drugs, have also demonstrated clinical activity in schizophrenia and other related disorders. This integrated computational analysis will help translate insights from the schizophrenia drug-protein interactome to clinical research - an important step, especially in the field of psychiatric drug development which faces a high failure rate.