RESUMEN
Activated macrophages contribute prominently to the progression and maintenance of almost all inflammatory and autoimmune diseases. Although non-specific elimination of these phagocytes has been shown to treat animal models of inflammatory disease, the same therapies have been compromised by unacceptable toxicities, because they also kill quiescent macrophages in healthy tissues. In the studies below, we exploit upregulation of folate receptor beta (FRß) on inflammatory (but not resting) macrophages to target a cytotoxic drug selectively to the inflammatory subset of macrophages. Because many of these activated macrophages are nondividing, we also employ verrucarin A as the cytotoxic payload, since it kills both mitotic and nonmitotic cells by blocking protein synthesis. By inserting a redox-sensitive self-immolative linker between the folate and verrucarin A, we further assure that release of unmodified verrucarin A is triggered primarily after internalization by an FRß-positive cell. The resulting folate-verrucarin A conjugate is shown to kill FR-expressing cells in vitro in a manner that can be inhibited by competition with 100-fold excess folic acid. The folate-verrucarin A conjugate is also shown to successfully treat a murine model of inflammatory peritonitis by eliminating inflammatory macrophages without killing other cells in the same peritonitis fluid. Based on this high specificity for inflammatory macrophages, we conclude that folate-verrucarin A warrants continued exploration as a potential therapy for inflammatory and autoimmune diseases in humans.
Asunto(s)
Modelos Animales de Enfermedad , Ácido Fólico/farmacología , Macrófagos/efectos de los fármacos , Peritonitis/tratamiento farmacológico , Tricotecenos/farmacología , Animales , Relación Dosis-Respuesta a Droga , Receptor 2 de Folato/metabolismo , Ácido Fólico/química , Macrófagos/metabolismo , Ratones , Estructura Molecular , Peritonitis/metabolismo , Relación Estructura-Actividad , Tricotecenos/químicaRESUMEN
CONTEXT: The clinical introduction of new oral anticoagulants (NOACs) has stimulated the development of tests to quantify the effects of these drugs and manage complications associated with their use. Until recently, the only treatment choices for the prevention of venous thromboembolism in orthopedic surgical patients, as well as for stroke and systemic embolism in patients with atrial fibrillation, were vitamin K antagonists, antiplatelet drugs, and unfractionated and low-molecular-weight heparins. With the approval of NOACs, treatment options and consequent diagnostic challenges have expanded. OBJECTIVE: To study the utility of thromboelastography (TEG) in monitoring and differentiating between 2 currently approved classes of NOACs, direct thrombin inhibitors (dabigatran) and factor Xa inhibitors (rivaroxaban and apixaban). DESIGN: Blood samples from healthy volunteers were spiked with each NOAC in both the presence and absence of ecarin, and the effects on TEG were evaluated. RESULTS: Both the kaolin test reaction time (R time) and the time to maximum rate of thrombus generation were prolonged versus control samples and demonstrated a dose response for apixaban (R time within the normal range) and dabigatran. The RapidTEG activated clotting time test allowed the creation of a dose-response curve for all 3 NOACs. In the presence of anti-Xa inhibitors, the ecarin test promoted significant shortening of kaolin R times to the hypercoagulable range, while in the presence of the direct thrombin inhibitor only small and dose-proportional R time shortening was observed. CONCLUSIONS: The RapidTEG activated clotting time test and the kaolin test appear to be capable of detecting and monitoring NOACs. The ecarin test may be used to differentiate between Xa inhibitors and direct thrombin inhibitors. Therefore, TEG may be a valuable tool to investigate hemostasis and the effectiveness of reversal strategies for patients receiving NOACs.
Asunto(s)
Anticoagulantes/sangre , Antitrombinas/sangre , Inhibidores del Factor Xa/sangre , Tromboelastografía/métodos , Tromboembolia Venosa/tratamiento farmacológico , Adolescente , Anticoagulantes/administración & dosificación , Antitrombinas/administración & dosificación , Bencimidazoles/administración & dosificación , Bencimidazoles/sangre , Dabigatrán , Inhibidores del Factor Xa/administración & dosificación , Humanos , Morfolinas/administración & dosificación , Morfolinas/sangre , Pirazoles/administración & dosificación , Pirazoles/sangre , Piridonas/administración & dosificación , Piridonas/sangre , Rivaroxabán , Sensibilidad y Especificidad , Tiofenos/administración & dosificación , Tiofenos/sangre , Tromboembolia Venosa/prevención & control , Adulto Joven , beta-Alanina/administración & dosificación , beta-Alanina/análogos & derivados , beta-Alanina/sangreRESUMEN
A folate receptor targeted didemnin B conjugate was synthesized using a hydrophilic peptide spacer linked to folate via a releasable disulfide carbonate linker. Cell cytotoxicity and TNF-α inhibition in RAW264.7 macrophage-like cells exhibited IC(50)s of 13 and 5 nM, respectively. Folate didemnin B was found to be â¼50-100 fold more potent than didemnin B itself. More importantly, activity of the prodrug was blocked by excess folic acid, demonstrating receptor-mediated cellular uptake of the conjugate.
Asunto(s)
Depsipéptidos/síntesis química , Depsipéptidos/farmacología , Ácido Fólico/química , Inflamación/tratamiento farmacológico , Animales , Carbono/química , Relación Dosis-Respuesta a Droga , Interacciones Hidrofóbicas e Hidrofílicas , Concentración 50 Inhibidora , Macrófagos/citología , Ratones , Modelos Biológicos , Modelos Químicos , Péptidos/química , Profármacos/farmacología , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Rapid identification of infectious pathogens constitutes an important step toward limiting the spread of contagious diseases. Whereas antibody-based detection strategies are often selected because of their speed, mutation of the pathogen can render such tests obsolete. In an effort to develop a rapid yet mutation-proof method for pathogen identification, we have explored the use of "immutable ligands" to capture the desired microbe on a detection device. In this "proof-of-principle" study, we immobilize pyoverdine, a siderophore that Pseudomonas aeruginosa must bind to obtain iron, onto gold-plated glass chips and then examine the siderophore's ability to capture P. aeruginosa for its subsequent identification. We demonstrate that exposure of pyoverdine-coated chips to increasing dilutions of P. aeruginosa allows detection of the bacterium down to concentrations as low as 10(2)/mL. We further demonstrate that printing of the siderophore in a periodic pattern on the detection chip enables a sensitive method of detecting the bound pathogen by a Fourier transform analysis of light scattered by the patterned chip. Because unrelated bacteria are not captured on the pyoverdine chip, we conclude that pyoverdine can be exploited for the specific binding and identification of P. aeruginosa. It follows that the utilization of other microbe-specific "immutable ligands" may allow the specific identification of their cognate pathogens.
Asunto(s)
Pseudomonas aeruginosa/aislamiento & purificación , Sideróforos/química , Microscopía Fluorescente , Oligopéptidos/química , Pseudomonas aeruginosa/patogenicidad , Albúmina Sérica Bovina/química , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Espectrofotometría UltravioletaRESUMEN
In order to avoid the toxicities associated with prescription drug use today, we have explored novel methods for delivering drugs selectively to pathologic cells, thereby avoiding the collateral damage that accompanies their uptake by healthy cells. In this Account, we describe our quest for the ideal targeted therapeutic agent. This effort began with a search for ligands that would bind selectively to pathologic cells, displaying no affinity for healthy cells. After identification of an optimal targeting ligand, effort was focused on construction of linkers that would carry the attached drug to pathologic cells with receptors for the selected ligand. In the case of cancer, we exploited the well-characterized up-regulation of folate receptors on malignant cells to target folate-linked pharmaceuticals to cancer tissues in vivo. Drugs that have been linked to folic acid for tumor-selective drug delivery to date include (i) protein toxins, (ii) chemotherapeutic agents, (iii) gene therapy vectors, (iv) oligonucleotides (including small interfering RNA (siRNA)), (v) radioimaging agents, (vi) magnetic resonance imaging (MRI) contrast agents, (vii) liposomes with entrapped drugs, (viii) radiotherapeutic agents, (ix) immunotherapeutic agents, and (x) enzyme constructs for prodrug therapy. Current clinical trials of four folate-linked drugs demonstrate that folate receptor-targeting holds great promise for increasing the potency while reducing toxicity of many cancer therapies. In the course of developing folate-conjugated drugs for cancer, we discovered that folate receptors are also overexpressed on activated (but not resting or quiescent) macrophages. Recognizing that activated macrophages either cause or contribute to such diseases as rheumatoid arthritis, Crohn's disease, atherosclerosis, lupus, inflammatory osteoarthritis, diabetes, ischemia reperfusion injury, glomerulonephritis, sarcoidosis, psoriasis, Sjogren's disease, and vasculitis, we initiated studies aimed at developing folate-conjugated imaging and therapeutic agents for the diagnosis and treatment of such diseases. In very brief time, significant progress has been made towards identification of clinical candidates for targeted treatment of several inflammatory and autoimmune diseases. This Account summarizes the discovery and development of a variety of folate-targeted drugs for the diagnosis and therapy of cancers and inflammatory/autoimmune diseases.
Asunto(s)
Antiinflamatorios/química , Antineoplásicos/química , Enfermedades Autoinmunes/tratamiento farmacológico , Proteínas Portadoras/efectos de los fármacos , Diseño de Fármacos , Neoplasias/tratamiento farmacológico , Receptores de Superficie Celular/efectos de los fármacos , Antiinflamatorios/uso terapéutico , Antineoplásicos/uso terapéutico , Enfermedades Autoinmunes/diagnóstico , Proteínas Portadoras/química , Receptores de Folato Anclados a GPI , Ácido Fólico/química , Humanos , Neoplasias/diagnóstico , Receptores de Superficie Celular/químicaRESUMEN
Bacillus subtilis spores (a simulant of Bacillus anthracis) have been imaged by two-photon luminescence (TPL) microscopy, using gold nanorods (GNRs) functionalized with a cysteine-terminated homing peptide. Control experiments using a peptide with a scrambled amino acid sequence confirmed that the GNR targeting was highly selective for the spore surfaces. The high sensitivity of TPL combined with the high affinity of the peptide labels enables spores to be detected with high fidelity using GNRs at femtomolar concentrations. It was also determined that GNRs are capable of significant TPL output even when irradiated at near infrared (NIR) wavelengths far from their longitudinal plasmon resonance (LPR), permitting considerable flexibility in the choice of GNR aspect ratio or excitation wavelength for TPL imaging.
RESUMEN
We describe an integrated approach for detection of diagnostic markers using in situ assembled optical diffraction gratings in combination with immunomagnetic capture. Folate receptor (FR), a serum protein indicative of various cancers, was chosen as a model system to demonstrate the potential of the method. Magnetic beads coupled to FR antibody were used to capture FR from serum. The FR-bound magnetic beads self-assembled onto microcontact-printed folate-coupled BSA (F-BSA) patterns to form diffraction gratings which served to detect FR by measuring the diffraction intensities caused by laser illumination. The FR-containing beads, upon binding to the F-BSA surface, served as intrinsic signal enhancement agents, circumventing the need for additional enzymatic signal amplification or fluorescent labeling steps. With this approach, a detection sensitivity of 700 fM (20 pg/mL) was achieved. The potential use of this approach in clinical diagnostics was demonstrated by measuring FR concentration in blood samples obtained from cancer patients.
Asunto(s)
Biomarcadores/sangre , Proteínas Portadoras/sangre , Separación Inmunomagnética , Receptores de Superficie Celular/sangre , Proteínas Portadoras/química , Ensayo de Inmunoadsorción Enzimática , Receptores de Folato Anclados a GPI , Humanos , Reacción en Cadena de la Polimerasa , Receptores de Superficie Celular/química , Albúmina Sérica Bovina/químicaAsunto(s)
Carbunco/prevención & control , Bacillus anthracis/aislamiento & purificación , Técnicas Biosensibles/instrumentación , Bioterrorismo/prevención & control , Microscopía Confocal/instrumentación , Secuencia de Aminoácidos , Datos de Secuencia Molecular , Óptica y Fotónica/instrumentación , Péptidos/química , Esporas Bacterianas/aislamiento & purificaciónRESUMEN
A folate receptor targeted camptothecin prodrug was synthesized using a hydrophilic peptide spacer linked to folate via a releasable disulfide carbonate linker. The conjugate was found to possess high affinity for folate receptor-expressing cells and inhibited cell proliferation in human KB cells with an IC(50) of 10nM. Activity of the prodrug was completely blocked by excess folic acid, demonstrating receptor-mediated uptake.
Asunto(s)
Camptotecina/síntesis química , Camptotecina/farmacología , Proteínas Portadoras/química , Profármacos/síntesis química , Profármacos/farmacología , Receptores de Superficie Celular/química , Unión Competitiva/efectos de los fármacos , Camptotecina/antagonistas & inhibidores , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Receptores de Folato Anclados a GPI , Ácido Fólico/farmacología , Humanos , Técnicas In Vitro , Estructura Molecular , Profármacos/química , Estereoisomerismo , Factores de TiempoRESUMEN
In this report, we describe the development of a quartz crystal microbalance biosensor for detection of folate binding protein (FBP). Using a simple folate-BSA conjugate adsorbed onto a Au-coated quartz sensor, a detection limit of 30 nM was achieved. Binding of FBP to the sensor surface could be blocked at concentrations as high as 1 microM with a 100-fold excess of folic acid, indicating the specificity of the folate-FBP interaction and the absence of nonspecific binding to the functionalized surface. Moreover, capture could be achieved in the presence of blood serum, making the assay amenable to the analysis of bodily fluids. Further signal enhancement based on an anti-FBP antibody and protein-A-coated gold nanosphere sandwich assay extended the detection limit to 50 pM (approximately 3 orders-of-magnitude improvement). Given the overexpression of FBP in certain malignancies and inflammatory disorders, we expect the methodology described here to be useful to detect FBP as a possible biomarker for disease diagnosis.
Asunto(s)
Técnicas Biosensibles/métodos , Ácido Fólico/química , Proteínas/análisis , Proteínas/química , Cuarzo/química , Anticuerpos/inmunología , Cristalización , Oro/química , Estructura Molecular , Nanotubos/química , Unión Proteica , Sensibilidad y Especificidad , SueroRESUMEN
We move beyond antibody-antigen binding systems and demonstrate that short peptide ligands can be used to efficiently capture Bacillus subtilis (a simulant of Bacillus anthracis) spores in liquids. On an eight-cantilever array chip, four cantilevers were coated with binding peptide (NHFLPKV-GGGC) and the other four were coated with control peptide (LFNKHVP-GGGC) for reagentless detection of whole B. subtilis spores in liquids. The peptide-ligand-functionalized microcantilever chip was mounted onto a fluid cell filled with a B. subtilis spore suspension for approximately 40 min; a 40 nm net differential deflection was observed. Fifth-mode resonant frequency measurements were also performed before and after dipping microcantilever arrays into a static B. subtilis solution showing a substantial decrease in frequency for binding-peptide-coated microcantilevers as compared to that for control peptide cantilevers. Further confirmation was obtained by subsequent examination of the microcantilever arrays under a dark-field microscope. Applications of this technology will serve as a platform for the detection of pathogenic organisms including biowarfare agents.