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.

Modulation of the Pharmacokinetics of a Radioligand Targeting Carbonic Anhydrase-IX with Albumin-Binding Moieties.

The expression of carbonic anhydrase-IX (CA-IX) in tumors can lead to a poor prognosis; thus, CA-IX has attracted much attention as a target molecule for cancer diagnosis and treatment. An 111In-labeled imidazothiadiazole sulfonamide (IS) derivative, [111In]In-DO3A-IS1, exhibited marked tumor accumulation but also marked renal accumulation, raising concerns about it producing a low signal/background ratio and a high radiation burden on the kidneys. In this study, four 111In-labeled IS derivatives, IS-[111In]In-DO2A-ALB1-4, which contained four different kinds of albumin binder (ALB) moieties, were designed and synthesized with the aim of improving the pharmacokinetics of [111In]In-DO3A-IS1. Their utility for imaging tumors that strongly express CA-IX was evaluated in mice. An in vitro binding assay of cells that strongly expressed CA-IX (HT-29 cells) was performed using acetazolamide as a competitor against CA-IX, and IS-[111In]In-DO2A-ALB1-4 did not exhibit reduced binding to HT-29 cells compared with [111In]In-DO3A-IS1. In contrast, IS-[111In]In-DO2A-ALB1-4 showed a greater ability to bind to human serum albumin than [111In]In-DO3A-IS1 in vitro. In an in vivo biodistribution study, the introduction of an ALB moiety into the 111In-labeled IS derivative markedly decreased renal accumulation and increased HT-29 tumor accumulation and blood retention. The pharmacokinetics of the IS derivatives varied depending on the substituted group within the ALB moiety. Single-photon emission computed tomography imaging with IS-[111In]In-DO2A-ALB1, which showed the highest tumor/kidney ratio in the biodistribution study, facilitated clear HT-29 tumor imaging, and no strong signals were observed in the normal organs. These results indicate that IS-[111In]In-DO2A-ALB1 may be an effective CA-IX imaging probe and that the introduction of ALB moieties may improve the pharmacokinetics of CA-IX ligands.

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.

Improved molecular recognition of Carbonic Anhydrase IX by polypeptide conjugation to acetazolamide.

The small molecule inhibitor acetazolamide (AZM) was conjugated to a set of designed polypeptides and the resulting conjugates were evaluated for their affinity to Human Carbonic Anhydrase II (HCA II) using surface plasmon resonance. The dissociation constant of the AZM-HCA II complex was 38nM and that of the AZM conjugated polypeptide (4-C10L17-AZM) to HCA II was found to be 4nM, an affinity enhancement of a factor of 10 due to polypeptide conjugation. For Human Carbonic Anhydrase IX (HCA IX) the dissociation constant of AZM was 3nM, whereas that of the 4-C10L17-AZM conjugate was 90pM, a 33-fold affinity enhancement. This dramatic affinity increase due to polypeptide conjugation was achieved for a small molecule ligand with an already high affinity to the target protein. This supports the concept that enhancements due to polypeptide conjugation are not limited to small molecule ligands that bind proteins in the mM to µM range but may be used also for nM ligands to provide recognition elements with dissociation constants in the pM range. Evaluations of two HCA IX constructs that do not carry the proteoglycan (PG) domain did not show significant affinity differences between AZM and the polypeptide conjugate, providing evidence that the improved binding of 4-C10L17-AZM to HCA IX emanated from interactions between the polypeptide segment and the PG domain found only in one carbonic anhydrase, HCA IX.

A 99mTc-Labeled Ligand of Carbonic Anhydrase IX Selectively Targets Renal Cell Carcinoma In Vivo.

UNLABELLED: Small organic ligands, selective for tumor-associated antigens, are increasingly being considered as alternatives to monoclonal antibodies for the targeted delivery of diagnostic and therapeutic payloads such as radionuclides and drugs into neoplastic masses. We have previously described a novel acetazolamide derivative, a carbonic anhydrase ligand with high affinity for the tumor-associated isoform IX (CAIX), which can transport highly potent cytotoxic drugs into CAIX-expressing solid tumors. The aim of the present study was to quantitatively investigate the biodistribution properties of said ligand and understand whether acetazolamide conjugates merit further development as drug carriers and radioimaging agents. METHODS: The conjugate described in this study, consisting of a derivative of acetazolamide, a spacer, and a peptidic (99m)Tc chelator, was labeled using sodium pertechnetate under reducing conditions and injected intravenously into CAIX-expressing SKRC-52 xenograft-bearing mice. Animals were sacrificed, and organ uptake as percentage injected activity of radiolabeled ligand per gram of tissues (%IA/g) was evaluated between 10 min and 24 h. Additionally, postmortem imaging by SPECT was performed. RESULTS: The acetazolamide conjugate described in this study could be labeled to high radiochemical purity (>95%, 2.2-4.5 MBq/nmol). Analysis of organ uptake at various time points revealed that the ligand displayed a maximal tumor accumulation 3 h after intravenous injection (22 %IA/g), with an excellent tumor-to-blood ratio of 70:1 at the same time point. The ligand accumulation in the tumor was more efficient than in any other organ, but a residual uptake in the kidney, lung, and stomach (9, 16, and 10 %IA/g, respectively) was also observed, in line with patterns of carbonic anhydrase isoform expression in those tissues. Interestingly, tumor-to-organ ratios improved on administration of higher doses of radiolabeled ligand, suggesting that certain binding sites in normal organs can be saturated in vivo. CONCLUSION: The (99m)Tc-labeled acetazolamide conjugate exhibits high tumor uptake and favorable tumor-to-kidney ratios of up to 3 that may allow imaging of tumors in the kidney and distant sites at earlier time points than commonly possible with antibody-based products. These data suggest that the described molecule merit further development as a radioimaging agent for CAIX-expressing renal cell carcinoma.

Targeting carbonic anhydrase IX with small organic ligands.

Carbonic anhydrase IX (CAIX) is expressed in many solid tumors in response to hypoxia and plays an important role in tumor acid-base homeostasis under these conditions. It is also constitutively expressed in the majority of renal cell carcinoma. Its functional inhibition with small molecules has recently been shown to retard tumor growth in murine models of cancer, reduce metastasis and tumor stem cell expansion. Additionally, CAIX is a promising antigen for targeted drug delivery approaches. Initially validated with anti-CAIX antibodies, the tumor-homing capacity of high-affinity small-molecule ligands of CAIX has recently been demonstrated. Indeed, conjugates formed of CAIX ligands and potent cytotoxic drugs could eradicate CAIX-expressing solid tumors in mice. These results suggest that CAIX is a promising target for the development of novel therapies for the treatment of solid tumors.

o-Benzenedisulfonimido-sulfonamides are potent inhibitors of the tumor-associated carbonic anhydrase isoforms CA IX and CA XII.

By using phthalimido-substituted aromatic sufonamides as lead molecules, a series of new sulfonamides incorporating ortho-benzenedisulfonimide moieties have been synthesized and tested against the human (h) cytosolic carbonic anhydrase (CA, EC 4.2.1.1) isozymes hCA I and hCA II and the transmembrane, tumor-associated isozymes hCA IX and hCA XII. All these compounds showed Ki values lower than 100nM and many of them showed better Kis than the reference compound acetazolamide, a clinically used sulfonamide. The tumor-associated isozymes were better inhibited than the cytosolic ones. A molecular docking within the active site of some CA isoforms, such as hCA I, explained these findings, as the benzenedisulfonimide moiety makes favorable interactions (hydrogen bonds) with amino acid residues involved in binding of inhibitors, such as Gln92, His67, and His64.

PVA hydrogels containing beta-cyclodextrin for enhanced loading and sustained release of ocular therapeutics.

The purpose of this work is to develop poly(vinyl alcohol) (PVA) hydrogels incorporating large amounts of beta-cyclodextrin (beta-CD) in order to improve ocular drug loading and to sustain drug release. First, the mono-methacrylated-beta-CD monomer (MA-beta-CD) and the methacrylated-PVA macromer (PVAMA), with a substitution degree of 7%, are synthesized and characterized. Then, the poly(methacrylated-PVA-co-mono-methacrylated-beta-cyclodextrin) (pPVA-beta-CD) hydrogels are prepared by UV-induced polymerization of MA-beta-CD and PVAMA. The highest amount of beta-CD incorporated into the hydrogels is 30 wt%. The hydrogels are further characterized by transmittance, FT-IR, equilibrium swelling ratio (ESR), tensile tests and protein deposition. The results show that pPVA-beta-CD hydrogels possess good transmittance, while the incorporation of beta-CD in the hydrogels improves the tensile strength and decreases the ESR and protein deposition. Finally, puerarin and acetazolamide are used as models to evaluate the drug loading and in vitro release behavior of the pPVA-beta-CD hydrogels. The results indicate that the amount of drug loaded into the pPVA-beta-CD hydrogels progressively increases, while the release rate decreases with increasing beta-CD content. In particular, incorporation of beta-CD efficiently decreases the initial burst release of acetazolamide, while the release, which is almost linear, is sustained for 15 days. The pPVA-beta-CD hydrogels have potential applications as biomedical devices for sustained release of ocular drugs.

Matrix modifications modulate ophthalmic drug delivery from photo-cross-linked poly(propylene fumarate)-based networks.

In this work, different modifications of photo-cross-linked poly(propylene fumarate)/poly(N-vinyl pyrrolidone) (PPF/PNVP) matrices were studied for their effect on the release kinetics of two ophthalmic drugs. The hydrophilicity of solid PPF/PNVP matrices loaded with acetazolamide (AZ) or timolol maleate (TM) was increased by adding various amounts of poly(ethylene glycol) (PEG) or by increasing the amount of N-vinyl pyrrolidone (NVP) in the polymer mixture prior to cross-linking. The in vitro release studies that utilized high-performance liquid chromatography for quantification revealed highly accelerated drug release from the matrices with increasing contents of the hydrophilic modifier. AZ was released from matrices containing 5% PEG in 56 days, which equals approximately 25% of the release period found for the unmodified matrices. A comparable acceleration in drug release was found for TM-loaded samples modified with 5% PEG. These studies further revealed that 1% PEG is sufficient to shorten the TM release duration by one-third. A significant acceleration in drug release was also found for the samples that were fabricated from a PPF-NVP mixture with increased NVP content. Matrix water content and erosion were assessed gravimetrically. Micro-computed tomography was used to image structural changes of the release systems and shed light on the drug-release mechanism. This study showed that hydrophilic matrix modifications of PPF/PNVP matrices accelerate the drug release of two ophthalmic drugs and represent a suitable tool to adjust drug-release rates from PPF-based matrices for different therapeutic needs.

Carbonic anhydrase inhibitors: the very weak inhibitors dithiothreitol, beta-mercaptoethanol, tris(carboxyethyl)phosphine and threitol interfere with the binding of sulfonamides to isozymes II and IX.

The inhibition of the metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1) with dithiothreitol, 2-mercaptoethanol, tris(carboxyethyl)phosphine (reducing agent frequently added to enzyme assay buffers) and threitol has been investigated. The agents were very weak inhibitors of isozymes CA II and CA IX, but unexpectedly, strongly influenced the binding of the low nanomolar sulfonamide inhibitor acetazolamide (5-acetamido-1,3,4-thiadiazole-2-sulfonamide). Acetazolamide affinity for all investigated CAs diminished orders of magnitude with increasing concentrations of these agents in the assay system. DTT and similar derivatives should not be added to the assay buffers used in monitoring CA activity/inhibition, as they lead to under-estimation of the binding constants, by a mechanism probably involving the formation of ternary complexes.

A versatile polypeptide platform for integrated recognition and reporting: affinity arrays for protein-ligand interaction analysis.

A molecular platform for protein detection and quantification is reported in which recognition has been integrated with direct monitoring of target-protein binding. The platform is based on a versatile 42-residue helix-loop-helix polypeptide that dimerizes to form four-helix bundles and allows site-selective modification with recognition and reporter elements on the side chains of individually addressable lysine residues. The well-characterized interaction between the model target-protein carbonic anhydrase and its inhibitor benzenesulfonamide was used for a proof-of-concept demonstration. An affinity array was designed where benzenesulfonamide derivatives with aliphatic or oligoglycine spacers and a fluorescent dansyl reporter group were introduced into the scaffold. The affinities of the array members for human carbonic anhydrase II (HCAII) were determined by titration with the target protein and were found to be highly affected by the properties of the spacers (dissociation constant Kd=0.02-3 microM). The affinity of HCAII for acetazolamide (Kd=4 nM) was determined in a competition experiment with one of the benzenesulfonamide array members to address the possibility of screening substance libraries for new target-protein binders. Also, successful affinity discrimination between different carbonic anhydrase isozymes highlighted the possibility of performing future isoform-expression profiling. Our platform is predicted to become a flexible tool for a variety of biosensor and protein-microarray applications within biochemistry, diagnostics and pharmaceutical chemistry.

Dissolution of poorly crystalline apatite crystals by osteoclasts determined on artificial thin-film apatite.

Poorly crystalline apatite (PCA) crystals introduced into bone tissue should be stable for a definite period before they are dissolved as a result of a host response. In this report, the dissolution of PCA crystals by the action of osteoclasts was studied on artificial thin films. These consisted of PCA crystals having similar crystallographic properties to bone crystals which were developed for assaying the osteoclast activity in vitro. The dissolution of minerals by osteoclasts decreased along with the decreased amount of labile phosphate and hydrogen phosphate domains of apatite crystals, which were caused by the crystal maturation temperature. A profound effect on mineral dissolution by pH in the culture medium was also shown. Low acidity considerably increased mineral dissolution, whereas a slight alkalinity totally blocked mineral dissolution. There was little difference in the mineral dissolution behavior of osteoclasts near the physiologic pH. In addition, it was determined whether mineral dissolution by osteoclasts was dependent on the destruction of the organic matrix. Nocodazole was introduced to inhibit the secretion of hydrolytic enzymes, and acetazolamide was added to inhibit acid production by the osteoclasts. There was no significant change as a result of nocodazole addition on mineral dissolution or by the addition of acetazolamide on degradation of collagen. These results indicate that small changes in the physicochemical properties of apatite crystals can decrease resorption by osteoclasts, which can be highly activated at low pH. These results also suggest that mineral dissolution and organic degradation by osteoclasts are self-regulating.

[Drug-induced urinary calculi in 1999]. / Les lithiases urinaires médicamenteuses en 1999.

Drug-induced urolithiasis are observed in 1.6% of the urinary calculi in France. Drugs crystals are identified in two thirds of these stones. Other drugs are responsible for stones which have an apparent metabolic origin (one third of the cases). Stone analysis using physical methods such as infrared spectroscopy is needed to unambiguously identify stones containing drugs. The inquiry is an important step to identify lithogenetic drugs which do not crystallize in the stones. The main substances which were identified in stones over the past decade were indinavir monohydrate (31.4%), triamterene (11.1%), sulphonamides (10.5%) and amorphous silica (4.5%). The main drugs involved in the nucleation and growth of metabolic stones were calcium and vitamin D supplementation (15%) and long-term treatment with carbonic anhydrase inhibitors (8%). Stone prevention is based on drug withdrawal or change in dosage with additional measures including an increase of diuresis and, if necessary, changes in the urine pH.

1H NMR and UV-Vis spectroscopic characterization of sulfonamide complexes of nickel(II)-carbonic anhydrase. Resonance assignments based on NOE effects.

The binding of acetazolamide, p-fluorobenzensulfonamide, p-toluenesulfonamide, and sulfanilamide to nickel(II)-substituted carbonic anhydrase II has been studied by 1H NMR and electronic absorption spectroscopies. These inhibitors bind to the metal ion forming 1:1 complexes and their affinity constants were determined. The 1H NMR spectra of the formed complexes show a number of isotropically shifted signals corresponding to the histidine ligands. The complexes with benzene-sulfonamides gave rise to very similar 1H NMR spectra. The NMR data suggest that these aromatic sulfonamides bind to the metal ion altering its coordination sphere. In addition, from the temperature dependence of 1H NMR spectra of the p-fluorobenzenesulfonamide adduct, a conformational change is suggested. The T1 values of the meta-like protons of the coordinated histidines have been measured and resonance assignments based on NOE experiments were performed.

[3H]acetazolamide binding to carbonic anhydrase in normal and transformed cells.

The binding of [3H]acetazolamide (AZ), a carbonic anhydrase (CA) inhibitor, to soluble and particulate forms of CA was investigated. Sources for the assays were purified CA II, adult rat cortical, oligodendrocyte and neuronal enriched preparations; cultured murine glial cells, rat C-6 glioma, rat hepatoma and human glioblastoma cells. CA enzyme activity in the same preparations was also assayed by following change in pH during incubation. A gel permeation chromatographic method was developed to assess [3H]AZ binding to soluble CA, while glass fiber filter vacuum filtration was used for particulate CA binding. Saturable specific binding of [3H]AZ to rat cortical soluble and particulate CA preparations was demonstrated. Computer-assisted data analysis estimated the binding parameters of [3H]AZ to soluble rat cortical CA to be Bmax = 0.38 +/- 0.13 pmol/mg protein and Kd = 34.7 +/- 17.5 nM. The rat cortical particulate fraction Bmax was 2.05 +/- 0.28 pmol/mg protein with a Kd of 107.1 +/- 24.2 nM. Purified bovine CA-II bound 1.15 +/- 0.19 pmol [3H]AZ/mg protein with a Kd of 54.0 +/- 3.4 nM. The pH optima for [3H]AZ binding to soluble and particulate CA was between 6.5 and 7.5. Binding was linear with respect to protein up to 1.0 mg/mL. The particulate fraction bound 3-4 times more [3H]ligand per unit protein than the soluble fraction. Interestingly, no detectable CA enzyme activity or [3H]AZ binding was observed in the soluble or particulate fractions of human glioblastoma, rat C-6 glioma or rat hepatoma cells. Binding of [3H]AZ to other soluble enzymes or proteins was negligible. In competition binding experiments, a rank order of inhibition of [3H]AZ binding to rat cortical CA by established CA inhibitors was: dichlorphenamide greater than acetazolamide greater than or equal to benzolamide greater than methazolamide greater than hydrochlorothiazide greater than or equal to sulfanilamide. [3H]AZ binding was not affected by other classes of pharmacologic characterizing agents. The binding of [3H]AZ to the CA enzyme molecule is highly specific and sensitive and may prove useful in vitro or in situ as a probe for this enzyme.

Cyst function in polycystic kidney disease: nongradient cysts.

Previous studies from this laboratory have demonstrated active sodium transport by cyst epithelia obtained from human polycystic kidneys. Cysts which maintained a steep sodium gradient between cyst fluid and plasma (gradient cysts) exhibited conductive amiloride sensitive sodium transport. Cysts which failed to maintain a sodium gradient between cyst fluid and plasma (nongradient cysts) were insufficiently characterized. In the present study, we report flux and electrical parameters of 23 nongradient cysts studied in vitro. Nongradient cysts exhibit low PD, low Isc, and high conductance. Unidirectional fluxes of sodium and chloride varied from approximately 14.4 to greater than 250 microEq.h-1.cm-2 and net flux was not significantly different from zero. There was no apparent effect on flux or electrical parameters of amiloride, ouabain, acetazolamide, or bumetanide. There was a very high correlation between unidirectional flux of sodium and chloride in individual cysts which was similar to the predicted relationship for diffusional fluxes. This correlation suggested that movement of sodium and chloride across cyst membranes was passive via an aqueous channel. Estimates of ionic permeability exceeded those determined for proximal nephron by almost one order of magnitude. We conclude that nongradient cysts are nonfunctional and, regardless of their origin, do not function analogously to the proximal nephron.

Maleic acid-induced impaired conversion of 25(OH)D3 to 1,25(OH)2D3: implications for Fanconi's syndrome.

Conversion of 25-hydroxyvitamin D3 [25(OH)D3] to 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] occurs exclusively in the renal cortex. To determine whether a disorder of the renal cortical tubule capable of causing Fanconi's syndrome can also impair the synthesis of 1,25(OH)2D3, we investigated whether conversion of 25(OH)D3 to 1,25(OH)2D3 was reduced by maleic acid. In vitamin D-deficient rats, maleic acid was administered i.v. over two hours. Thirty minutes after its initiation, when the complex renal tubule dysfunction had occurred, 3H-25(OH)D3 was administered i.v. as a bolus. Five hours afterwards, the amount of 3H-1,25(OH)2D3 recovered in the kidney, small intestine mucosa, and blood was one-third to one-half that in tissues of control rats that received acetazolamide or only saline or were subjected only to the surgical procedure. The glomerular filtration rate, as measured by inulin clearance, did not decrease significantly with maleic acid. In intact vitamin D-deficient chicks, 24 and 22 hr after i.p. administration of maleic acid and 14C-vitamin D3, respectively, the amount of 14C-1,25(OH)2D3 recovered in small intestine mucosa was reduced by one-half when compared to saline-treated controls. In kidney homogenates and isolated renal tubules of vitamin D-deficient chicks, activity of 25-hydroxyvitamin D3-1-hydroxylase was diminished immediately after maleic acid was administered in vivo or added in vitro to the incubation medium, respectively. These data provide the first demonstration that the renal capacity to convert 25(OH)D3 to 1,25(OH)2D3 can be substantially impaired in vivo by a renal disorder in which the glomerular filtration rate is not reduced.

Effect of edetate disodium and reduced glutathione on absorption of acetazolamide from GI tract of rats.

The absorption of acetazolamide suspensions from in situ rat gastric and intestinal loop segments was studied. In 1 hr, 66.2 and 64.3% remained unabsorbed from the rat stomach and intestine, respectively. Although 1% (w/v) reduced glutathione and 1% (w/v) (24 mM) edetate disodium had no effect on gastric absorption, drug absorption from the rat intestine (1 hr) was increased 1.5 and 2 times, respectively. It was hypothesized that the relatively poor intestinal absorption was due primarily to the formation of a pH-dependent (pH 4.5-10), nonabsorbable complex between acetazolamide and carbonic anhydrase present in the gut and that reduced glutathione acted as an inhibitor to promote intestinal absorption. Equilibrium dialysis studies showed that reduced glutathion could reduce the fraction of drug bound to human carbonic anhydrase B by one-half when present in a molar ratio 10 times that of acetazolamide; edetate disodium had no effect on the in vitro binding. It was, therefore, assumed that edetate disodium promoted an increase in intestinal absorption by altering the permeability of intestinal epithelium. Based upon present experimentation, however, the alteration of intestinal epithelium by reduced glutathione cannot be ruled out.