Carbachol dimers with primary carbamate groups as homobivalent modulators of muscarinic receptors.

Although agonists and antagonists of muscarinic receptors have been known for long time, there is renewed interest in compounds (such as allosteric or bitopic ligands, or biased agonists) able to differently and selectively modulate these receptors. As a continuation of our previous research, we designed a new series of dimers of the well-known cholinergic agonist carbachol. The new compounds were tested on the five cloned human muscarinic receptors (hM1-5) expressed in CHO cells by means of equilibrium binding experiments, showing a dependence of the binding affinity on the length and position of the linker connecting the two monomers. Kinetic binding studies revealed that some of the tested compounds were able to slow the rate of NMS dissociation, suggesting allosteric behavior, also supported by docking simulations. Assessment of ERK1/2 phosphorylation on hM1, hM2 and hM3 activation showed that the new compounds are endowed with muscarinic antagonist properties. At hM2 receptors, some compounds were able to stimulate GTPγS binding but not cAMP accumulation, suggesting a biased behavior. Classification, Molecular and cellular pharmacology.

Conformational Complexity and Dynamics in a Muscarinic Receptor Revealed by NMR Spectroscopy.

The M2 muscarinic acetylcholine receptor (M2R) is a prototypical GPCR that plays important roles in regulating heart rate and CNS functions. Crystal structures provide snapshots of the M2R in inactive and active states, but the allosteric link between the ligand binding pocket and cytoplasmic surface remains poorly understood. Here we used solution NMR to examine the structure and dynamics of the M2R labeled with 13CH3-ε-methionine upon binding to various orthosteric and allosteric ligands having a range of efficacy for both G protein activation and arrestin recruitment. We observed ligand-specific changes in the NMR spectra of 13CH3-ε-methionine probes in the M2R extracellular domain, transmembrane core, and cytoplasmic surface, allowing us to correlate ligand structure with changes in receptor structure and dynamics. We show that the M2R has a complex energy landscape in which ligands with different efficacy profiles stabilize distinct receptor conformations.

Potentiation of the activation of cholinergic receptors by multivalent presentation of ligands supported on gold nanoparticles.

Gold nanoparticles (NP) with a functionalized ligand shell offer the possibility to potentiate the action of agonists at the receptor site by multivalency. In order to find out whether this can be realized for the pharmacologically important class of cholinergic receptors known to be involved in the regulation of most organ functions, carbachol-functionalized gold NPs (Au-MUDA-CCh) with an average diameter of 14 nm were synthesized. As functional read-out, cholinergic agonist-induced anion secretion was measured as increase in short-circuit current (Isc) across rat proximal colon in Ussing chambers. Similarly to the corresponding native agonist acetylcholine, Au-MUDA-CCh induced a concentration-dependent increase in Isc, which represents chloride secretion across the epithelium. This response was inhibited by atropine and hexamethonium indicating the activation of muscarinic and nicotinic receptors by the functionalized NPs. A strong potentiation of ligand-receptor interaction was a key benefit of functionalized NPs over native agonists. This was observed with physiological approaches as measurements of changes in Isc revealed a nearly equivalent response evoked by 1 pM Au-MUDA-CCh and 500 nM native CCh. To better determine this potentiation at the receptor level, pharmacological approaches based on the signaling cascade of ACh-induced activation of muscarinic receptors were used. FRET (Förster Resonance Energy Transfer) measurements performed on HEK293T cells transiently transfected with M3-R, Gαq-YFP, Gß1-wt and CFP-Gγ2, revealed that both Au-MUDA-CCh and native CCh activated G-proteins with EC50 amounting to 127 ± 0.44 fM and 224 ± 7.12 nM, respectively. Thus, the functionalization of the NPs with CCh yields a potentiation by over 106, a property that could find usage in specific targeting, activation and compensation of pathologically reduced expression of receptors of interest.

Novel long-acting antagonists of muscarinic ACh receptors.

BACKGROUND AND PURPOSE: The aim of this study was to develop potent and long-acting antagonists of muscarinic ACh receptors. The 4-hexyloxy and 4-butyloxy derivatives of 1-[2-(4-oxidobenzoyloxy)ethyl]-1,2,3,6-tetrahydropyridin-1-ium were synthesized and tested for biological activity. Antagonists with long-residence time at receptors are therapeutic targets for the treatment of several neurological and psychiatric human diseases. Their long-acting effects allow for reduced daily doses and adverse effects. EXPERIMENTAL APPROACH: The binding and antagonism of functional responses to the agonist carbachol mediated by 4-hexyloxy compounds were investigated in CHO cells expressing individual subtypes of muscarinic receptors and compared with 4-butyloxy analogues. KEY RESULTS: The 4-hexyloxy derivatives were found to bind muscarinic receptors with micromolar affinity and antagonized the functional response to carbachol with a potency ranging from 30 nM at M1 to 4 µM at M3 receptors. Under washing conditions to reverse antagonism, the half-life of their antagonistic action ranged from 1.7 h at M2 to 5 h at M5 receptors. CONCLUSIONS AND IMPLICATIONS: The 4-hexyloxy derivatives were found to be potent long-acting M1 -preferring antagonists. In view of current literature, M1 -selective antagonists may have therapeutic potential for striatal cholinergic dystonia, delaying epileptic seizure after organophosphate intoxication or relieving depression. These compounds may also serve as a tool for research into cognitive deficits.

Intracellular Transfer of Na+ in an Active-State G-Protein-Coupled Receptor.

Playing a central role in cell signaling, G-protein-coupled receptors (GPCRs) are the largest superfamily of membrane proteins and form the majority of drug targets in humans. How extracellular agonist binding triggers the activation of GPCRs and associated intracellular effector proteins remains, however, poorly understood. Structural studies have revealed that inactive class A GPCRs harbor a conserved binding site for Na+ ions in the center of their transmembrane domain, accessible from the extracellular space. Here, we show that the opening of a conserved hydrated channel in the activated state receptors allows the Na+ ion to egress from its binding site into the cytosol. Coupled with protonation changes, this ion movement occurs without significant energy barriers, and can be driven by physiological transmembrane ion and voltage gradients. We propose that Na+ ion exchange with the cytosol is a key step in GPCR activation. Further, we hypothesize that this transition locks receptors in long-lived active-state conformations.

Integrated sudomotor axon reflex sweat stimulation for continuous sweat analyte analysis with individuals at rest.

Eccrine sweat has rapidly emerged as a non-invasive, ergonomic, and rich source of chemical analytes with numerous technological demonstrations now showing the ability for continuous electrochemical sensing. However, beyond active perspirers (athletes, workers, etc.), continuous sweat access in individuals at rest has hindered the advancement of both sweat sensing science and technology. Reported here is integration of sudomotor axon reflex sweat stimulation for continuous wearable sweat analyte analysis, including the ability for side-by-side integration of chemical stimulants & sensors without cross-contamination. This integration approach is uniquely compatible with sensors which consume the analyte (enzymatic) or sensors which equilibrate with analyte concentrations. In vivo validation is performed using iontophoretic delivery of carbachol with ion-selective and impedance sensors for sweat analysis. Carbachol has shown prolonged sweat stimulation in directly stimulated regions for five hours or longer. This work represents a significant leap forward in sweat sensing technology, and may be of broader interest to those interested in on-skin sensing integrated with drug-delivery.

Carbachol dimers as homobivalent modulators of muscarinic receptors.

A series of homodimers of the well-known cholinergic agonist carbachol have been synthesized, showing the two agonist units symmetrically connected through a methylene chain of variable length. The new compounds have been tested on the five cloned muscarinic receptors (hM1-5) expressed in CHO cells by means of equilibrium binding studies, showing an increase in affinity by rising the number of methylene units up to 7 and 9. Functional experiments on guinea-pig ileum and assessment of ERK1/2 phosphorylation on hM1, hM2 and hM3 on CHO cells have shown that the new compounds are endowed with muscarinic antagonistic properties. Kinetic binding studies have revealed that some of the tested compounds are able to slow the rate of dissociation of NMS, suggesting a bitopic behavior. Docking simulations, performed on the hM1 and hM2 receptors, give a sound rationalization of the experimental data revealing how these compounds are able to interact with both orthosteric and allosteric binding sites depending on the length of their connecting chain.

The mode of agonist binding to a G protein-coupled receptor switches the effect that voltage changes have on signaling.

Signaling by many heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors (GPCRs) is either enhanced or attenuated by changes in plasma membrane potential. To identify structural correlates of the voltage sensitivity of GPCR signaling, we chose muscarinic acetylcholine receptors (the M1, M3, and M5 isoforms) as a model system. We combined molecular docking analysis with Förster resonance energy transfer (FRET)-based assays that monitored receptor activity under voltage clamp conditions. When human embryonic kidney (HEK) 293 cells expressing the individual receptors were stimulated with the agonist carbachol, membrane depolarization enhanced signaling by the M1 receptor but attenuated signaling by the M3 and M5 receptors. Furthermore, whether membrane depolarization enhanced or inhibited receptor signaling depended on the type of agonist. Membrane depolarization attenuated M3 receptor signaling when the receptor was bound to carbachol or acetylcholine, whereas depolarization enhanced signaling when the receptor was bound to either choline or pilocarpine. Docking calculations predicted that there were two distinct binding modes for these ligands, which were associated with the effect of depolarization on receptor function. From these calculations, we identified a residue in the M3 receptor that, when mutated, would alter the binding mode of carbachol to resemble that of pilocarpine in silico. Introduction of this mutated M3 receptor into cells confirmed that the membrane depolarization enhanced, rather than attenuated, signaling by the carbachol-bound receptor. Together, these data suggest that the directionality of the voltage sensitivity of GPCR signaling is defined by the specific binding mode of each ligand to the receptor.

Conformationally restrained carbamoylcholine homologues. Synthesis, pharmacology at neuronal nicotinic acetylcholine receptors and biostructural considerations.

Exploration of small selective ligands for the nicotinic acetylcholine receptors (nAChRs) based on acetylcholine (ACh) has led to the development of potent agonists with clear preference for the α4ß2 nAChR, the most prevalent nAChR subtype in the central nervous system. In this work we present the continuation of these efforts aimed at increasing this subtype selectivity by introduction of conformational restriction in the carbamoylcholine homologue, 3-(dimethylaminobutyl) dimethylcarbamate (DMABC). Our results highlight the importance of the N-carbamoyl substitution in α4ß2-subtype selectivity. Moreover, we have confirmed the non-linear conformation of DMABC bound to nAChRs suggested by recent crystal structures of the compound in complex with the Lymnaea stagnalis ACh binding protein.

Role of Telokin in Regulating Murine Gastric Fundus Smooth Muscle Tension.

Telokin phosphorylation by cyclic GMP-dependent protein kinase facilitates smooth muscle relaxation. In this study we examined the relaxation of gastric fundus smooth muscles from basal tone, or pre-contracted with KCl or carbachol (CCh), and the phosphorylation of telokin S13, myosin light chain (MLC) S19, MYPT1 T853, T696, and CPI-17 T38 in response to 8-Bromo-cGMP, the NO donor sodium nitroprusside (SNP), or nitrergic neurotransmission. We compared MLC phosphorylation and the contraction and relaxation responses of gastric fundus smooth muscles from telokin-/- mice and their wild-type littermates to KCl or CCh, and 8-Bromo-cGMP, SNP, or nitrergic neurotransmission, respectively. We compared the relaxation responses and telokin phosphorylation of gastric fundus smooth muscles from wild-type mice and W/WV mice which lack ICC-IM, to 8-Bromo-cGMP, SNP, or nitrergic neurotransmission. We found that telokin S13 is basally phosphorylated and that 8-Bromo-cGMP and SNP increased basal telokin phosphorylation. In muscles pre-contracted with KCl or CCh, 8-Bromo-cGMP and SNP had no effect on CPI-17 or MYPT1 phosphorylation, but increased telokin phosphorylation and reduced MLC phosphorylation. In telokin-/- gastric fundus smooth muscles, basal tone and constitutive MLC S19 phosphorylation were increased. Pre-contracted telokin-/- gastric fundus smooth muscles have increased contractile responses to KCl, CCh, or cholinergic neurotransmission and reduced relaxation to 8-Bromo-cGMP, SNP, and nitrergic neurotransmission. However, basal telokin phosphorylation was not increased when muscles were stimulated with lower concentrations of SNP or when the muscles were stimulated by nitrergic neurotransmission. SNP, but not nitrergic neurotransmission, increased telokin Ser13 phosphorylation in both wild-type and W/WV gastric fundus smooth muscles. Our findings indicate that telokin may play a role in attenuating constitutive MLC phosphorylation and provide an additional mechanism to augment gastric fundus mechanical responses to inhibitory neurotransmission.

Microfluidic Chip for Site-Specific Neuropharmacological Treatment and Activity Probing of 3D Neuronal "Optonet" Cultures.

The study introduces a "brain-on-a-chip" microfluidic platform that hosts brain-like 3D cultures ("optonets") whose activity and responses to flowing drugs are recorded optically. Optonets are viable, optically accessible 3D neural networks whose characteristics approximate cortical networks. The results demonstrate the ability to monitor complex 3D activity patterns during extended site-specific, reversible neuropharmacogical exposure, suggesting an interesting potential in drug screening.

ß-Adrenoceptor-Mediated Relaxation of Carbachol-Pre-Contracted Mouse Detrusor.

AIMS: To study the ß-adrenoceptor subtypes involved in the relaxation responses to (-)-isoprenaline in carbachol-pre-contracted (CCh) mouse detrusor muscle with intact and denuded mucosa. METHODS: Isolated muscle strips from the urinary bladder of male C57BL6 mice or ß2-adrenoceptor knockout mice were pre-contracted with CCh, 1 µM and relaxed with increasing concentrations of the ß-adrenoceptor (ß-AR) agonist (-)-isoprenaline and forskolin. For estimating the ß-AR subtypes involved, subtype-selective receptor blockers were used, that is, CGP 20712A (ß1-ARs), ICI 118,551 (ß2-ARs), and L748,337 (ß3-ARs). RESULTS: Unlike in KCl-pre-contracted muscle, the mucosa did not affect the sensitivity of the relaxation response to (-)-isoprenaline in CCh-pre-contracted murine detrusor strips. Increasing concentrations of (-)-isoprenaline produced a biphasic concentration-relaxation response without any difference both during the presence and absence of mucosa. The relaxation fraction produced by low (-)-isoprenaline concentrations was mediated by ß2-AR as evidenced by a shift of the concentration-response curve to higher concentrations with ICI 118,551, but not with CGP 20712A and L748,337, and by the absence of this fraction in ß2-AR-KO mice. The relaxation response with low sensitivity to (-)-isoprenaline was not affected by any of the ß-AR subtype-selective blockers and was the only response detected in detrusor strips from ß2-AR-KO mice. CONCLUSIONS: In CCh-pre-contracted mouse detrusor, ß2-ARs are responsible for the relaxation component with high sensitivity to (-)-isoprenaline as indicated by the conversion of a biphasic into a monophasic CRC with ICI 118,551 or by its absence in ß2-AR KO mice. The mucosa does not impair relaxation under these conditions.

Molecular determinants of allosteric modulation at the M1 muscarinic acetylcholine receptor.

Benzylquinolone carboxylic acid (BQCA) is an unprecedented example of a selective positive allosteric modulator of acetylcholine at the M1 muscarinic acetylcholine receptor (mAChR). To probe the structural basis underlying its selectivity, we utilized site-directed mutagenesis, analytical modeling, and molecular dynamics to delineate regions of the M1 mAChR that govern modulator binding and transmission of cooperativity. We identified Tyr-85(2.64) in transmembrane domain 2 (TMII), Tyr-179 and Phe-182 in the second extracellular loop (ECL2), and Glu-397(7.32) and Trp-400(7.35) in TMVII as residues that contribute to the BQCA binding pocket at the M1 mAChR, as well as to the transmission of cooperativity with the orthosteric agonist carbachol. As such, the BQCA binding pocket partially overlaps with the previously described "common" allosteric site in the extracellular vestibule of the M1 mAChR, suggesting that its high subtype selectivity derives from either additional contacts outside this region or through a subtype-specific cooperativity mechanism. Mutation of amino acid residues that form the orthosteric binding pocket caused a loss of carbachol response that could be rescued by BQCA. Two of these residues (Leu-102(3.29) and Asp-105(3.32)) were also identified as indirect contributors to the binding affinity of the modulator. This new insight into the structural basis of binding and function of BQCA can guide the design of new allosteric ligands with tailored pharmacological properties.

Monitoring intracellular calcium in response to GPCR activation using thin-film silicon photodiodes with integrated fluorescence filters.

G-protein coupled receptor (GPCRs) drug discovery is a thriving strategy in the pharmaceutical industry. The standard approach uses living cells to test millions of compounds in a high-throughput format. Typically, changes in the intracellular levels of key elements in the signaling cascade are monitored using fluorescence or luminescence read-out systems, which require external equipment for signal acquisition. In this work, thin-film amorphous silicon photodiodes with an integrated fluorescence filter were developed to capture the intracellular calcium dynamics in response to the activation of the endogenous muscarinic M1 GPCR of HEK 293T cells. Using the new device it was possible to characterize the potency of carbachol (EC50=10.5 µM) and pirenzepine (IC50=4.2 µM), with the same accuracy as standard microscopy optical systems. The smaller foot-print provided by the detection system makes it an ideal candidate for the future integration in microfluidic devices for drug discovery.

Design, synthesis and binding affinity of acetylcholine carbamoyl analogues.

A series of acetylcholine carbamoyl analogues, cyclised at the carbamate moiety or at the cationic head or at both, were tested for binding affinity at muscarinic and neuronal nicotinic receptors (nAChRs). While no muscarinic affinity was found, submicromolar Ki values, similar to that of carbachol, were measured at α4ß2 nAChRs for the enantiomers of 5-dimethylaminomethyl- and 5-trimethylammoniomethyl-2-oxazolidinone, 2 and 2a, and for (S)-N-methylprolinol carbamate (S)-3. Methylation of oxazolidinone nitrogen of 2 and 2a and of N-methylprolinol nitrogen of (S)-3 and, even more, hybridization of cyclic carbamate substructure (oxazolidinone) with cyclic cationic head (N-methylpyrrolidine) markedly lower the nicotinic affinity. Docking results were consistent with SAR analysis highlighting the interaction capabilities of (R)-2a and (S)-3 and the negative effect of intracyclic nitrogen methylation and of double cyclisation.

A simple method for quantifying functional selectivity and agonist bias.

Activation of seven-transmembrane (7TM) receptors by agonists does not always lead to uniform activation of all signaling pathways mediated by a given receptor. Relative to other ligands, many agonists are "biased" toward producing subsets of receptor behaviors. A hallmark of such "functional selectivity" is cell type dependence; this poses a particular problem for the profiling of agonists in whole cell test systems removed from the therapeutic one(s). Such response-specific cell-based variability makes it difficult to guide medicinal chemistry efforts aimed at identifying and optimizing therapeutically meaningful agonist bias. For this reason, we present a scale, based on the Black and Leff operational model, that contains the key elements required to describe 7TM agonism, namely, affinity (K(A) (-1)) for the receptor and efficacy (τ) in activating a particular signaling pathway. Utilizing a "transduction coefficient" term, log(τ/K(A)), this scale can statistically evaluate selective agonist effects in a manner that can theoretically inform structure-activity studies and/or drug candidate selection matrices. The bias of four chemokines for CCR5-mediated inositol phosphate production versus internalization is quantified to illustrate the practical application of this method. The independence of this method with respect to receptor density and the calculation of statistical estimates of confidence of differences are specifically discussed.

Dissecting the conserved NPxxY motif of the M3 muscarinic acetylcholine receptor: critical role of Asp-7.49 for receptor signaling and multiprotein complex formation.

Acetylcholine challenge produces M(3) muscarinic acetylcholine receptor activation and accessory/scaffold proteins recruitment into a signalsome complex. The dynamics of such a complex is not well understood but a conserved NPxxY motif located within transmembrane 7 and juxtamembrane helix 8 of the receptor was found to modulate G protein activation. Here by means of receptor mutagenesis we unravel the role of the conserved M(3) muscarinic acetylcholine receptor NPxxY motif on ligand binding, signaling and multiprotein complex formation. Interestingly, while a N7.49D receptor mutant showed normal ligand binding properties a N7.49A mutant had reduced antagonist binding and increased affinity for carbachol. Also, besides this last mutant was able to physically couple to Gα(q/11) after carbachol challenge it was neither capable to activate phospholipase C nor phospholipase D. On the other hand, we demonstrated that the Asn-7.49 is important for the interaction between M(3)R and ARF1 and also for the formation of the ARF/Rho/ß Î³ signaling complex, a complex that might determine the rapid activation and desensitization of PLD. Overall, these results indicate that the NPxxY motif of the M(3) muscarinic acetylcholine receptor acts as key conformational switch for receptor signaling and multiprotein complex formation.

Ethanol enhances carbachol-induced protease activation and accelerates Ca2+ waves in isolated rat pancreatic acini.

Alcohol abuse is a leading cause of pancreatitis, accounting for 30% of acute cases and 70-90% of chronic cases, yet the mechanisms leading to alcohol-associated pancreatic injury are unclear. An early and critical feature of pancreatitis is the aberrant signaling of Ca(2+) within the pancreatic acinar cell. An important conductor of this Ca(2+) is the basolaterally localized, intracellular Ca(2+) channel ryanodine receptor (RYR). In this study, we examined the effect of ethanol on mediating both pathologic intra-acinar protease activation, a precursor to pancreatitis, as well as RYR Ca(2+) signals. We hypothesized that ethanol sensitizes the acinar cell to protease activation by modulating RYR Ca(2+). Acinar cells were freshly isolated from rat, pretreated with ethanol, and stimulated with the muscarinic agonist carbachol (1 µM). Ethanol caused a doubling in the carbachol-induced activation of the proteases trypsin and chymotrypsin (p < 0.02). The RYR inhibitor dantrolene abrogated the enhancement of trypsin and chymotrypsin activity by ethanol (p < 0.005 for both proteases). Further, ethanol accelerated the speed of the apical to basolateral Ca(2+) wave from 9 to 18 µm/s (p < 0.0005; n = 18-22 cells/group); an increase in Ca(2+) wave speed was also observed with a change from physiologic concentrations of carbachol (1 µM) to a supraphysiologic concentration (1 mM) that leads to protease activation. Dantrolene abrogated the ethanol-induced acceleration of wave speed (p < 0.05; n = 10-16 cells/group). Our results suggest that the enhancement of pathologic protease activation by ethanol is dependent on the RYR and that a novel mechanism for this enhancement may involve RYR-mediated acceleration of Ca(2+) waves.

Electrochemical detection of interaction between Thioflavin T and acetylcholinesterase.

The electrochemical oxidation of the benzothiazole dye Thioflavin T (ThT) was found to be modulated by its interaction with electric eel acetylcholinesterase (AChE). Modifications of AChE by trace amounts of small molecule inhibitors such as carbachol and paraoxon were detectable electrochemically using minimal reagents and with greater sensitivity than attainable through conventional fluorescence approaches. This property appears to be unique to ThT, since its closely related neutral derivative BTA-1 only interacts with AChE, but is not significantly affected by the presence of small molecule inhibitors.

In vitro compatibility study of cephalosporin with intraocular irrigating solutions and intracameral medications.

BACKGROUND: To study the compatibility of cephalosporins with intraocular irrigating solutions and intracameral medications commonly used in cataract surgery. DESIGN: The was an in vitro experiment conducted in the Research Laboratory of the Department of Microbiology, the Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, Hong Kong. SAMPLES: Three cephalosporins--cefazolin, cefuroxime and ceftazidime--were separately diluted and mixed with irrigating solutions and intracameral medications to form 192 samples and 12 control solutions. METHODS: The cephalosporins were dissolved in normal saline and further diluted to the concentration of 1 mg in 0.1 mL with normal saline, Ringer's solution, balanced salt solution and fortified balanced salt solutions. These were mixed with balanced salt solutions or fortified balanced salt solutions, with adrenaline, acetylcholine or carbachol and kept at 37°C for 2 h. The concentrations of free cephalosporins were measured with rapid high-performance liquid chromatography at baseline (0 h) and at 2 h. MAIN OUTCOME MEASURES: Free concentrations of cephalosporins at 2 h were compared with mean baseline (0 h) value. A difference of 3 standard deviations or more was considered statistically significant. RESULTS: At 2 h there was a significant drop in the cefuroxime concentration in preparations in which cefuroxime was diluted with normal saline (P < 0.01). In all preparations, the final concentrations of cephalosporins were higher than the minimal inhibitory concentrations (MIC(90)) for microbials commonly isolated from the external eye. CONCLUSION: Cefazolin, cefuroxime and ceftazidime were compatible with irrigating solutions and intracameral medications commonly used in cataract surgery.