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1.
Haemophilia ; 28(4): 568-577, 2022 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-35467059

RESUMEN

INTRODUCTION: Immunogenicity causing development of anti-drug antibodies (ADAs) are major challenges in the treatment of haemophilia, as well as other diseases where proteins are used for treatment. Furthermore, it is a complication for preclinical testing of such therapies in animal models. AIM: To investigate if the immunosuppressive drug CTLA4 immunoglobulin (CTLA4-Ig) can induce tolerance in haemophilia A (HA) rats receiving recombinant human coagulation factor VIII (rhFVIII) treatment. METHODS: Two different prophylactic rhFVIII compounds were given intravenously to HA rats for 4 weeks. Both rhFVIII compounds were co-administered with commercially available CTLA4-Ig or human IgG subclass 4 (hIgG4) as control, and blood samples were collected. To functionally test if pharmacological efficacy was retained, rats were subjected to a bleeding experiment under anaesthesia at end of study. RESULTS: The mean inhibitory level after 4 weeks in rats receiving rhFVIII and hIgG4 was 85.7 BU for octocog alfa and 37.4 BU for rurioctocog alfa pegol. In contrast, co-administration with CTLA4-Ig during rhFVIII therapy prevented the formation of ADAs (both binding and inhibitory) in 14/14 rats receiving octocog alfa and in 7/7 rats receiving rurioctocog alfa pegol. Moreover, we were able to show that the pharmacological efficacy of rhFVIII was preserved. CONCLUSION: In a rat model with spontaneous bleeding, co-administration of CTLA4-Ig with rhFVIII prevented antibody formation. No FVIII antibodies were detected, demonstrating that CTLA4-Ig co-administration can be applicable as a method to prevent immunogenicity, when evaluating human proteins in preclinical systems permitting continuous pharmacokinetic and pharmacodynamic assessment.


Asunto(s)
Hemofilia A , Abatacept/farmacología , Abatacept/uso terapéutico , Animales , Anticuerpos Neutralizantes , Formación de Anticuerpos , Antígeno CTLA-4 , Factor VIII , Hemofilia A/tratamiento farmacológico , Hemofilia A/prevención & control , Hemorragia/tratamiento farmacológico , Humanos , Ratas , Proteínas Recombinantes/farmacología , Proteínas Recombinantes/uso terapéutico
2.
Eur J Pharm Sci ; 168: 106032, 2022 Jan 01.
Artículo en Inglés | MEDLINE | ID: mdl-34610450

RESUMEN

Somapacitan is a growth hormone derivative approved for once-weekly treatment of growth hormone deficiency in adults and currently in clinical development for once-weekly dosing in children. The purpose of this study was to obtain non-clinical data from rats to support the safety evaluation of the most abundant metabolites of somapacitan in humans. The aims were to identify somapacitan metabolites and their relative proportions in rat plasma, identify the structure of abundant metabolites and measure the systemic metabolite exposure at the no-observed-adverse-effect level in the rat. After a single dose of radiolabelled somapacitan and analysis by high-performance liquid chromatography with radiochemical detection, seven somapacitan-related metabolites were detected in plasma from male rats, of which six were seen in plasma from female rats. The three most abundant metabolites (M1, M2 and M3) were structurally identified from liquid chromatography and mass spectrometry data, and a fourth metabolite (P1) was characterised from its specific retention time (lacking retention to the stationary phase) in plasma analysis with reversed-phase liquid chromatography and radiochemical detection. The metabolites were products from proteolysis of the peptide backbone in somapacitan. A deamidation product of the M1 metabolite (M1B) was also identified. Following multiple, twice-weekly dosing for 4 weeks, somapacitan was the principal plasma component up to 36 h after dosing. After 36 h, metabolites M1+M1B were the most abundant plasma components. Pharmacokinetic models were developed for somapacitan and metabolite P1 and used for steady-state assessment in the rat. Comparison of our data generated from rats with data from the parallel human study demonstrated that the most abundant metabolites were present in rats at higher levels than in humans. This study has provided non-clinical safety data that contribute to an overall safety assessment of somapacitan.


Asunto(s)
Enanismo Hipofisario , Hormona de Crecimiento Humana , Animales , Cromatografía Líquida de Alta Presión , Femenino , Hormona del Crecimiento , Histidina , Masculino , Manitol , Fenol , Plasma , Ratas
3.
Blood ; 138(14): 1258-1268, 2021 10 07.
Artículo en Inglés | MEDLINE | ID: mdl-34077951

RESUMEN

Hemophilia A is a bleeding disorder resulting from deficient factor VIII (FVIII), which normally functions as a cofactor to activated factor IX (FIXa) that facilitates activation of factor X (FX). To mimic this property in a bispecific antibody format, a screening was conducted to identify functional pairs of anti-FIXa and anti-FX antibodies, followed by optimization of functional and biophysical properties. The resulting bispecific antibody (Mim8) assembled efficiently with FIXa and FX on membranes, and supported activation with an apparent equilibrium dissociation constant of 16 nM. Binding affinity with FIXa and FX in solution was much lower, with equilibrium dissociation constant values for FIXa and FX of 2.3 and 1.5 µM, respectively. In addition, the activity of Mim8 was dependent on stimulatory activity contributed by the anti-FIXa arm, which enhanced the proteolytic activity of FIXa by 4 orders of magnitude. In hemophilia A plasma and whole blood, Mim8 normalized thrombin generation and clot formation, with potencies 13 and 18 times higher than a sequence-identical analogue of emicizumab. A similar potency difference was observed in a tail vein transection model in hemophilia A mice, whereas reduction of bleeding in a severe tail-clip model was observed only for Mim8. Furthermore, the pharmacokinetic parameters of Mim8 were investigated and a half-life of 14 days shown in cynomolgus monkeys. In conclusion, Mim8 is an activated FVIII mimetic with a potent and efficacious hemostatic effect based on preclinical data.


Asunto(s)
Anticuerpos Biespecíficos/uso terapéutico , Hemofilia A/tratamiento farmacológico , Hemorragia/tratamiento farmacológico , Animales , Factor IXa/antagonistas & inhibidores , Factor VIIIa/uso terapéutico , Factor X/antagonistas & inhibidores , Femenino , Humanos , Masculino , Ratones Endogámicos C57BL
4.
Biochemistry ; 59(14): 1410-1419, 2020 04 14.
Artículo en Inglés | MEDLINE | ID: mdl-32208682

RESUMEN

Somapacitan, a human growth hormone derivative that binds reversibly to albumin, was investigated for human serum albumin (HSA) and HSA domain binding. Isothermal titration calorimetry (ITC) binding profiles showed high-affinity binding (∼100-1000 nM) of one somapacitan molecule and low-affinity binding (∼1000-10000 nM) of one to two somapacitan molecules to HSA. The high-affinity site was identified in HSA domain III using size exclusion chromatography (SEC) and ITC. SEC studies showed that the neonatal Fc receptor shields one binding site for somapacitan, indicating its position in domain III. A crystal structure of somapacitan in complex with HSA optimized for neonatal Fc receptor binding, having four amino acid residue replacements, identified a low-affinity site in fatty acid-binding site 6 (domain II). Surface plasmon resonance (SPR) showed these replacements affect the kinetics of the high-affinity binding site. Furthermore, small-angle X-ray scattering and SPR brace two somapacitan-binding sites on HSA.


Asunto(s)
Hormona del Crecimiento/química , Albúmina Sérica Humana/química , Sitios de Unión , Hormona del Crecimiento/análogos & derivados , Hormona del Crecimiento/metabolismo , Humanos , Cinética , Unión Proteica , Dominios Proteicos , Albúmina Sérica Humana/metabolismo , Resonancia por Plasmón de Superficie
5.
J Mol Med (Berl) ; 98(4): 585-593, 2020 04.
Artículo en Inglés | MEDLINE | ID: mdl-32108909

RESUMEN

Blocking the proteolytic capacity of urokinase-type plasminogen activator (uPA) with a monoclonal antibody (mAb) reduces arthritis progression in the collagen-induced mouse arthritis model to an extent that is on par with the effect of blocking tumor necrosis factor-alpha by etanercept. Seeking to develop a novel therapy for rheumatoid arthritis, a humanized mAb, NNC0266-0043, was selected for its dual inhibition of both the zymogen activation and the proteolytic capacity of human uPA. The antibody revealed nonlinear elimination kinetics in cynomolgus monkeys consistent with binding to and turnover of endogenous uPA. At a dose level of 20.6 mg kg-1, the antibody had a plasma half-life of 210 h. Plasma uPA activity, a pharmacodynamic marker of anti-uPA therapy, was reduced to below the detection limit during treatment, indicating that an efficacious plasma concentration was reached. Pharmacokinetic modeling predicted that sufficient antibody levels can be sustained in arthritis patients dosed subcutaneously once weekly. The anti-uPA mAb was also well tolerated in cynomolgus monkeys at weekly doses up to 200 mg kg-1 over 4 weeks. The data from cynomolgus monkeys and from human material presented here indicates that anti-uPA mAb NNC0266-0043 is suitable for clinical testing as a novel therapeutic for rheumatic diseases. KEY MESSAGES: Background: Anti-uPA therapy is on par with etanercept in a mouse arthritis model. A new humanized antibody blocks activation and proteolytic activity of human uPA. The antibody represents a radically novel mode-of-action in anti-rheumatic therapy. The antibody has PK/PD properties in primates consistent with QW clinical dosing.


Asunto(s)
Anticuerpos Monoclonales/farmacología , Antirreumáticos/farmacología , Artritis Reumatoide/etiología , Desarrollo de Medicamentos , Activador de Plasminógeno de Tipo Uroquinasa/antagonistas & inhibidores , Animales , Anticuerpos Monoclonales/uso terapéutico , Antirreumáticos/uso terapéutico , Artritis Reumatoide/tratamiento farmacológico , Artritis Reumatoide/metabolismo , Artritis Reumatoide/patología , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos , Humanos , Inmunohistoquímica , Macaca fascicularis , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Macrófagos/metabolismo , Ratones , Monocitos/metabolismo
6.
Int J Mol Sci ; 21(4)2020 Feb 11.
Artículo en Inglés | MEDLINE | ID: mdl-32053994

RESUMEN

Somapacitan is a long-acting, once-weekly, albumin-binding growth hormone (GH) derivative. The reversible albumin-binding properties leads to prolonged circulation half-life. Here, we investigated and compared somapacitan with human GH on downstream receptor signaling in primary hepatocytes and hepatocellular models and using isothermal titration calorimetry to characterize receptor binding of somapacitan in the presence or absence of human serum albumin (HSA). With non-invasive fluorescence imaging we quantitatively visualize and compare the temporal distribution and examine the tissue-specific growth hormone receptor (GHR) activation at distribution sites. We found that signaling kinetics were slightly more rapid and intense for GH compared with somapacitan. Receptor binding isotherms were characterized by a high and a low affinity interaction site with or without HSA. Using in vivo optical imaging we found prolonged systemically biodistribution of somapacitan compared with GH, which correlated with plasma pharmacokinetics. Ex vivo mouse organ analysis revealed that the temporal fluorescent intensity in livers dosed with somapacitan was significantly increased compared with GH-dosed livers and correlated with the degree of downstream GHR activation. Finally, we show that fluorescent-labeled analogs distributed to the hypertrophic zone in the epiphysis of proximal tibia of hypophysectomized rats and that somapacitan and GH activate the GHR signaling in epiphyseal tissues.


Asunto(s)
Hormona de Crecimiento Humana/análogos & derivados , Hormona de Crecimiento Humana/farmacología , Receptores de Somatotropina/metabolismo , Animales , Células Cultivadas , Femenino , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Hormona de Crecimiento Humana/farmacocinética , Humanos , Masculino , Ratones , Ratones Desnudos , Modelos Moleculares , Ratas Sprague-Dawley , Distribución Tisular
7.
Heliyon ; 5(11): e02892, 2019 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-31844758

RESUMEN

BACKGROUND: Adult onset growth hormone (GH) deficiency (AGDH) is a potentially underdiagnosed condition, caused by damage to the pituitary gland. AGHD is treated with growth hormone replacement therapy. A large variety of clinical symptoms and changes in the metabolic homeostasis can be observed and quantified. New large animal models are needed for future drug development. NEW METHOD: In this study, we evaluate methods for a new large non-primate animal model of GH deficiency in post pubertal Göttingen Minipigs (minipig). Lesions in the pituitary gland were made by stereotaxic monopolar thermo-coagulation guided by magnetic resonance imaging (MRI), and pituitary function was evaluated using insulin tolerance test (ITT) with measurements of growth hormone secretion induced by hypoglycemia. RESULTS: Lesions were successfully applied to the pituitary gland without any damage to surrounding tissue including the hypothalamus, which was confirmed by post-operative MRI and post mortem histology. Plasma levels of GH during ITT showed no decrease in secreted levels one week after surgery compared to levels obtained before surgery. COMPARISON WITH EXISTING METHODS: Compared to other GH insufficiency models, eloquent brain tissue is spared. Furthermore, alternatively to rodent models, a large animal model would allow the use of human intended equipment to evaluate disease. Using the minipig avoids social, economical and ethical issues, compared with primates. CONCLUSION: The lesions did not remove all GH production, but proof of concept is demonstrated. In addition, the ITT is presented as a safe and efficient method to diagnose GH deficiency in minipigs.

8.
Transgenic Res ; 28(Suppl 2): 151-159, 2019 08.
Artículo en Inglés | MEDLINE | ID: mdl-31321698

RESUMEN

Australia's gene technology regulatory scheme (GT Scheme) regulates activities with genetically modified organisms (GMOs, organisms modified by gene technology), including environmental releases. The scope of regulation, i.e. what organisms are and are not regulated, is set by the Gene Technology Act 2000 (GT Act) and GT Regulations 2001 (GT Regulations). The GT Act gives broad, overarching definitions of 'gene technology' and 'GMO' but also provides for exclusions and inclusions in the GT Regulations. Whether organisms developed with genome editing techniques are, or should be, regulated under countries' national GMO laws is the subject of debate globally. These issues are also under active consideration in Australia. A technical review of the GT Regulations was initiated in 2016 to clarify the regulatory status of genome editing. Proposed draft amendments are structured around whether the process involves introduction of a nucleic acid template. If agreed, amendments would exclude from regulation organisms produced using site directed nuclease (SDN) 1 techniques while organisms produced using oligonucleotide mutagenesis, SDN-2 or SDN-3 would continue to be regulated as GMOs. The review of the GT Regulations is still ongoing and no legislative changes have been made to the GT Regulations. A broader policy review of the GT Scheme was undertaken in 2017-2018 and as a result further work will be undertaken on the scope and definitions of the GT Act in light of ongoing developments.


Asunto(s)
Alimentos Modificados Genéticamente , Edición Génica/tendencias , Ingeniería Genética/tendencias , Organismos Modificados Genéticamente/genética , Unión Europea , Humanos
9.
Chem Commun (Camb) ; 55(38): 5439-5442, 2019 May 07.
Artículo en Inglés | MEDLINE | ID: mdl-31020306

RESUMEN

We report the synthesis, crystal structure, and thermally-driven phase transformation of the dicyanometallate superperovskite co-crystal [NBu4]Mn[Au(CN)2]3·[NBu4]ClO4. This phase is understandable in terms of the conventional ABX3 perovskite structure type, but with the NBu4+ A-site cation displaced onto the perovskite cage face and 1-dimensional AX' chains included within framework pores opened up by these displacements. On heating to 380 K, the co-crystal disproportionates into its two inorganic components: a bcs-structured ABX3 phase and [NBu4]ClO4. This system illustrates a new type of structural and phase complexity accessible to dicyanometallate perovskites.

10.
Bioconjug Chem ; 29(9): 3129-3143, 2018 09 19.
Artículo en Inglés | MEDLINE | ID: mdl-30168709

RESUMEN

The present work describes a series of human growth hormone (hGH) albumin binder conjugates with an extended in vivo half-life. A broad range of different conjugates were studied by varying the albumin binder structure and conjugation site. Conjugates were conveniently obtained by reductive alkylation or by alkylation to introduced cysteines using functionalized albumin-binding side chains. In vitro and in vivo profiling provided the basis for identification of position L101C in human growth hormone as the most optimal position for conjugation, where both a sufficient level of receptor binding and a suitably long half-life could yield a molecule with potential for a once-weekly dosing regimen.


Asunto(s)
Albúminas/metabolismo , Hormona de Crecimiento Humana/metabolismo , Alquilación , Animales , Área Bajo la Curva , Semivida , Oxidación-Reducción , Unión Proteica , Ratas , Ratas Sprague-Dawley
11.
J Immunol ; 200(3): 957-965, 2018 02 01.
Artículo en Inglés | MEDLINE | ID: mdl-29282305

RESUMEN

Genetic absence of the urokinase-type plasminogen activator (uPA) reduces arthritis progression in the collagen-induced arthritis (CIA) mouse model to an extent just shy of disease abrogation, but this remarkable observation has not been translated into therapeutic intervention. Our aim was to test the potential in mice of an Ab that blocks the proteolytic capacity of uPA in the CIA model and the delayed-type hypersensitivity arthritis model. A second aim was to determine the cellular origins of uPA and the uPA receptor (uPAR) in joint tissue from patients with rheumatoid arthritis. A mAb that neutralizes mouse uPA significantly reduced arthritis progression in the CIA and delayed-type hypersensitivity arthritis models. In the CIA model, the impact of anti-uPA treatment was on par with the effect of blocking TNF-α by etanercept. A pharmacokinetics evaluation of the therapeutic Ab revealed target-mediated drug disposition consistent with a high turnover of endogenous uPA. The cellular expression patterns of uPA and uPAR were characterized by double immunofluorescence in the inflamed synovium from patients with rheumatoid arthritis and compared with synovium from healthy donors. The arthritic synovium showed expression of uPA and uPAR in neutrophils, macrophages, and a fraction of endothelial cells, whereas there was little or no expression in synovium from healthy donors. The data from animal models and human material provide preclinical proof-of-principle that validates uPA as a novel therapeutic target in rheumatic diseases.


Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Artritis Experimental/patología , Artritis Reumatoide/patología , Receptores del Activador de Plasminógeno Tipo Uroquinasa/metabolismo , Membrana Sinovial/patología , Activador de Plasminógeno de Tipo Uroquinasa/antagonistas & inhibidores , Animales , Anticuerpos Monoclonales/inmunología , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Células Endoteliales/inmunología , Etanercept/farmacología , Femenino , Humanos , Hipersensibilidad Tardía/inmunología , Macrófagos/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Neutrófilos/inmunología , Membrana Sinovial/inmunología , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Activador de Plasminógeno de Tipo Uroquinasa/genética , Activador de Plasminógeno de Tipo Uroquinasa/metabolismo
12.
Int J Mol Sci ; 18(10)2017 Sep 23.
Artículo en Inglés | MEDLINE | ID: mdl-28946616

RESUMEN

Growth hormone (GH) resistance may develop as a consequence of inflammation during conditions such as inflammatory bowel disease, encompassing ulcerative colitis (UC). However, the specific role of the GH-insulin growth factor (IGF)-1-axis and/or the functional consequences of GH resistance in this condition are unclear. In situ hybridization targeting the GH receptor (GHR) and relevant transcriptional analyses were performed in patients with UC and in IL-10 knock-out mice with piroxicam accelerated colitis (PAC). Using cultured primary epithelial cells, the effects of inflammation on the molecular mechanisms governing GH resistance was verified. Also, the therapeutic potential of GH on mucosal healing was tested in the PAC model. Inflammation induced intestinal GH resistance in UC and experimental colitis by down-regulating GHR expression and up-regulating suppressor of cytokine signalling (SOCS) proteins. These effects are driven by pro-inflammatory mediators (tumor necrosis factor (TNF)-α, interleukin (IL)-1ß and IL-6) as confirmed using primary epithelial cells. Treatment of experimental colitis with GH increased IGF-1 and body weight of the mice, but had no effects on colonic inflammation or mucosal healing. The high transcriptional similarity between UC and experimental colitis accentuates the formation of intestinal GH resistance during inflammation. Inflammation-induced GH resistance not only impairs general growth but induces a state of local resistance, which potentially impairs the actions of GH on mucosal healing during colitis when using long-acting GH therapy.


Asunto(s)
Colitis Ulcerosa/metabolismo , Hormona del Crecimiento/metabolismo , Adulto , Animales , Biopsia , Estudios de Casos y Controles , Colitis Ulcerosa/diagnóstico , Colitis Ulcerosa/tratamiento farmacológico , Colitis Ulcerosa/genética , Colonoscopía , Citocinas/metabolismo , Modelos Animales de Enfermedad , Femenino , Expresión Génica , Hormona del Crecimiento/farmacología , Humanos , Inmunohistoquímica , Mediadores de Inflamación/metabolismo , Enfermedades Inflamatorias del Intestino/tratamiento farmacológico , Enfermedades Inflamatorias del Intestino/metabolismo , Enfermedades Inflamatorias del Intestino/patología , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patología , Masculino , Ratones , Ratones Noqueados , Persona de Mediana Edad , Receptores de Somatotropina/genética , Receptores de Somatotropina/metabolismo , Transducción de Señal , Adulto Joven
13.
Growth Horm IGF Res ; 35: 8-16, 2017 08.
Artículo en Inglés | MEDLINE | ID: mdl-28595133

RESUMEN

OBJECTIVE: Somapacitan is an albumin-binding growth hormone derivative intended for once weekly administration, currently in clinical development for treatment of adult as well as juvenile GH deficiency. Nonclinical in vivo pharmacological characterisation of somapacitan was performed to support the clinical trials. Here we present the pharmacokinetic and pharmacodynamic effects of somapacitan in rats, minipigs, and cynomolgus monkeys. METHODS: Pharmacokinetic studies investigating exposure, absorption, clearance, and bioavailability after single intravenous (i.v.) and subcutaneous (s.c.) administration were performed in all species. A dose-response study with five dose levels and a multiple dose pharmacodynamic study with four once weekly doses was performed in hypophysectomised rats to evaluate the effect of somapacitan on growth and IGF-I production. RESULTS: Pharmacokinetic profiles indicated first order absorption from the subcutaneous tissue after s.c. injections for somapacitan in all three species. Apparent terminal half-lives were 5-6h in rats, 10-12h in minipigs, and 17-20h in monkeys. Somapacitan induced a dose-dependent growth in hypophysectomised rats (p<0.001) and an increase in plasma IGF-I levels in rats (p<0.01), minipigs (p<0.01), and cynomolgus monkeys (p<0.05) after single dose administration. Multiple once weekly dosing of somapacitan in hypophysectomised rats induced a step-wise increase in body weight with an initial linear phase the first 3-4days in each dosing interval (p<0.001). CONCLUSION: The nonclinical pharmacokinetic and pharmacodynamic studies of somapacitan showed similar pharmacokinetic properties, with no absorption-limited elimination, increased clearance and increased and sustained levels of IGF-I in plasma for up to 10days after a single dose administration in all three species. Somapacitan induced a dose-dependent increase in body weight and IGF-I levels in hypophysectomised rats. Multiple dosing of somapacitan in hypophysectomised rats suggested a linear growth for the first 3-4days in each weekly dosing interval, whereas daily hGH dosing showed linear growth for approximately two weeks before reaching a plateau level.


Asunto(s)
Albúminas/metabolismo , Hormona de Crecimiento Humana/farmacocinética , Proteínas Recombinantes/farmacocinética , Albúminas/farmacocinética , Animales , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos , Enanismo Hipofisario/tratamiento farmacológico , Hormona de Crecimiento Humana/metabolismo , Macaca fascicularis , Masculino , Unión Proteica , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes/metabolismo , Porcinos , Porcinos Enanos
14.
Phys Rev Lett ; 118(6): 067201, 2017 Feb 10.
Artículo en Inglés | MEDLINE | ID: mdl-28234510

RESUMEN

The formation of a spin glass generally requires that magnetic exchange interactions are both frustrated and disordered. Consequently, the origin of spin-glass behavior in Y_{2}Mo_{2}O_{7}-in which magnetic Mo^{4+} ions occupy a frustrated pyrochlore lattice with minimal compositional disorder-has been a longstanding question. Here, we use neutron and x-ray pair-distribution function (PDF) analysis to develop a disorder model that resolves apparent incompatibilities between previously reported PDF, extended x-ray-absorption fine structure spectroscopy, and NMR studies, and provides a new and physical explanation of the exchange disorder responsible for spin-glass formation. We show that Mo^{4+} ions displace according to a local "two-in-two-out" rule on each Mo_{4} tetrahedron, driven by orbital dimerization of Jahn-Teller active Mo^{4+} ions. Long-range orbital order is prevented by the macroscopic degeneracy of dimer coverings permitted by the pyrochlore lattice. Cooperative O^{2-} displacements yield a distribution of Mo-O-Mo angles, which in turn introduces disorder into magnetic interactions. Our study demonstrates experimentally how frustration of atomic displacements can assume the role of compositional disorder in driving a spin-glass transition.

15.
J Am Chem Soc ; 138(30): 9393-6, 2016 08 03.
Artículo en Inglés | MEDLINE | ID: mdl-27414161

RESUMEN

We study the compositional dependence of molecular orientation (multipolar) and orbital (quadrupolar) order in the perovskite-like metal-organic frameworks [C(NH2)3]CuxCd1-x(HCOO)3. Upon increasing the fraction x of Jahn-Teller-active Cu(2+), we observe an orbital disorder/order transition and a multipolar reorientation transition, each occurring at distinct critical compositions xo = 0.45(5) and xm = 0.55(5). We attribute these transitions to a combination of size, charge distribution, and percolation effects. Our results establish the accessibility in formate perovskites of novel structural degrees of freedom beyond the familiar dipolar terms responsible for (anti)ferroelectric order. We discuss the implications of cooperative quadrupolar and multipolar states for the design of relaxor-like hybrid perovskites.

16.
Eur J Pharm Sci ; 86: 29-33, 2016 Apr 30.
Artículo en Inglés | MEDLINE | ID: mdl-26946443

RESUMEN

The kidneys are thought to play an important role in the clearance of recombinant human growth hormone (rhGH), but the relative importance is not clear. Obtaining knowledge of clearance pathway is an important prerequisite for the development of new long acting growth hormone analogues targeted at treatment of patients with growth hormone disorders. The purpose of this study was to investigate the relative importance of the kidneys in the clearance of rhGH. The study employed a newly validated nephrectomy rat model and a population based pharmacokinetic approach to assess renal clearance of rhGH in non-anesthetized rats, anesthetized rats and in nephrectomized anesthetized rats. Clearance in non-anesthetized rats was 290 ml/h/kg. This was reduced to 185 ml/h/kg by anesthesia and further reduced to 18 ml/h/kg by nephrectomy. As nephrectomy was able to reduce clearance with 90%, we conclude that renal clearance plays a pivotal role in the elimination of rhGH in rats.


Asunto(s)
Hormona de Crecimiento Humana/farmacocinética , Riñón/metabolismo , Animales , Hormona de Crecimiento Humana/sangre , Masculino , Nefrectomía , Ratas Sprague-Dawley
17.
J Med Chem ; 58(18): 7370-80, 2015 Sep 24.
Artículo en Inglés | MEDLINE | ID: mdl-26308095

RESUMEN

Liraglutide is an acylated glucagon-like peptide-1 (GLP-1) analogue that binds to serum albumin in vivo and is approved for once-daily treatment of diabetes as well as obesity. The aim of the present studies was to design a once weekly GLP-1 analogue by increasing albumin affinity and secure full stability against metabolic degradation. The fatty acid moiety and the linking chemistry to GLP-1 were the key features to secure high albumin affinity and GLP-1 receptor (GLP-1R) potency and in obtaining a prolonged exposure and action of the GLP-1 analogue. Semaglutide was selected as the optimal once weekly candidate. Semaglutide has two amino acid substitutions compared to human GLP-1 (Aib(8), Arg(34)) and is derivatized at lysine 26. The GLP-1R affinity of semaglutide (0.38 ± 0.06 nM) was three-fold decreased compared to liraglutide, whereas the albumin affinity was increased. The plasma half-life was 46.1 h in mini-pigs following i.v. administration, and semaglutide has an MRT of 63.6 h after s.c. dosing to mini-pigs. Semaglutide is currently in phase 3 clinical testing.


Asunto(s)
Péptido 1 Similar al Glucagón/análogos & derivados , Péptido 1 Similar al Glucagón/química , Receptor del Péptido 1 Similar al Glucagón/agonistas , Péptidos Similares al Glucagón/química , Administración Intravenosa , Animales , Línea Celular , Cricetinae , Cristalografía por Rayos X , Péptido 1 Similar al Glucagón/administración & dosificación , Péptido 1 Similar al Glucagón/farmacología , Receptor del Péptido 1 Similar al Glucagón/metabolismo , Péptidos Similares al Glucagón/administración & dosificación , Péptidos Similares al Glucagón/farmacología , Semivida , Humanos , Inyecciones Subcutáneas , Liraglutida/farmacología , Masculino , Ratones Obesos , Modelos Moleculares , Ratas Sprague-Dawley , Relación Estructura-Actividad , Porcinos , Porcinos Enanos
18.
Endocrinology ; 151(11): 5326-36, 2010 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-20826563

RESUMEN

Therapeutic use of recombinant GH typically involves daily sc injections. We examined the possibilities for prolonging the in vivo circulation of GH by introducing N-glycans. Human GH variants with a single potential N-glycosylation site (N-X-S/T) introduced by site-directed mutagenesis were expressed in HEK293 cells. In a scan of 15 different positions for N-glycosylation sites, four positions (amino acids 93, 98, 99, and 101) were efficiently utilized and did not influence GH in vitro activity. A GH variant (3N-GH) with all these sites was produced in CHOK1SV cells and contained up to three N-glycans. Two pools of 3N-GH were purified and separated according to their charge by anion-exchange chromatography. Anion-exchange HPLC revealed that the N-glycans in the two pools were very similar except for the extent of sialylation. Both 3N-GH pools circulated longer in rats than wild-type GH. The terminal half-life of 3N-GH after iv injection was 24-fold prolonged compared with wild-type GH for the pool with the most pronounced sialylation, 13-fold prolonged for the less sialylated pool, and similar to the wild-type for desialylated 3N-GH. The less sialylated 3N-GH pool exhibited a profound pharmacodynamic effect in GH-deficient rats. Over a 4-d period, a single injection of 3N-GH induced a stronger IGF-I response and a larger increase in body weight than daily injections with wild-type GH. Thus, N-glycans can prolong the in vivo circulation and enhance the pharmacodynamic effect of GH. Sialic acids seem to play a pivotal role for the properties of glycosylated GH.


Asunto(s)
Hormona de Crecimiento Humana/metabolismo , Polisacáridos/metabolismo , Animales , Línea Celular , Ensayo de Inmunoadsorción Enzimática , Glicosilación , Semivida , Humanos , Distribución Aleatoria , Ratas , Ratas Sprague-Dawley
19.
Pharm Res ; 26(12): 2543-50, 2009 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-19847627

RESUMEN

Therapeutic proteins are a diverse class of drugs consisting of naturally occurring or modified proteins, and due to their size and physico-chemical properties, they can pose challenges for the pharmacokinetic and pharmacodynamic studies. Physiologically-based pharmacokinetics (PBPK) modelling has been effective for early in silico prediction of pharmacokinetic properties of new drugs. The aim of the present workshop was to discuss the feasibility of PBPK modelling of macromolecules. The classical PBPK approach was discussed with a presentation of the successful example of PBPK modelling of cyclosporine A. PBPK model was performed with transport of the cyclosporine across cell membranes, affinity to plasma proteins and active membrane transporters included to describe drug transport between physiological compartments. For macromolecules, complex PBPK modelling or permeability-limited and/or target-mediated distribution was discussed. It was generally agreed that PBPK modelling was feasible and desirable. The role of the lymphatic system should be considered when absorption after extravascular administration is modelled. Target-mediated drug disposition was regarded as an important feature for generation of PK models. Complex PK-models may not be necessary when a limited number of organs are affected. More mechanistic PK/PD models will be relevant when adverse events/toxicity are included in the PK/PD modelling.


Asunto(s)
Química Farmacéutica , Modelos Biológicos , Farmacocinética , Sustancias Macromoleculares/farmacocinética , Péptidos/química , Péptidos/farmacocinética , Péptidos/fisiología
20.
Clin Cancer Res ; 13(12): 3630-6, 2007 Jun 15.
Artículo en Inglés | MEDLINE | ID: mdl-17575227

RESUMEN

PURPOSE: Human interleukin-21 (IL-21) is a pleiotropic class I cytokine that activates CD8(+) T cells and natural killer cells. We report a phase 1 study of recombinant human IL-21 in patients with surgically incurable metastatic melanoma. The primary objective was to investigate safety and tolerability by determining dose-limiting toxicity (DLT). The secondary objectives were to identify a dose response for various biomarkers in the peripheral blood, estimate the minimum biologically effective dose, determine the pharmacokinetics of IL-21, determine if anti-IL-21 antibodies were induced during therapy, and measure effects on tumor size according to Response Evaluation Criteria in Solid Tumors. EXPERIMENTAL DESIGN: Open-label, two-arm, dose escalation trial of IL-21 administered by i.v. bolus injection at dose levels from 1 to 100 microg/kg using two parallel treatment regimens: thrice weekly for 6 weeks (3/wk) or three cycles of daily dosing for 5 days followed by 9 days of rest (5+9). RESULTS: Twenty-nine patients entered the study. IL-21 was generally well tolerated and no DLTs were observed at the 1, 3, and 10 microg/kg dose levels. In the 3/wk regimen, DLTs were increased in alanine aminotransferase, neutropenia, and lightheadedness with fever and rigors. DLTs in the 5+9 regimen were increased in aspartate aminotransferase and alanine aminotransferase, neutropenia, fatigue, and thrombocytopenia. The maximum tolerated dose was declared to be 30 microg/kg for both regimens. Effects on biomarkers were observed at all dose levels, including increased levels of soluble CD25 and up-regulation of perforin and granzyme B mRNA in CD8(+) cells. One partial tumor response observed after treatment with IL-21 for 2 x 6 weeks (3/wk) became complete 3 months later. CONCLUSIONS: IL-21 is biologically active at all dose levels administered and is generally well tolerated, and phase 2 studies have commenced using 30 microg/kg in the 5+9 regimen.


Asunto(s)
Antineoplásicos/administración & dosificación , Interleucinas/administración & dosificación , Melanoma/tratamiento farmacológico , Adulto , Anciano , Antineoplásicos/efectos adversos , Antineoplásicos/farmacocinética , Relación Dosis-Respuesta a Droga , Ensayo de Inmunoadsorción Enzimática , Femenino , Granzimas/efectos de los fármacos , Humanos , Subunidad alfa del Receptor de Interleucina-2/sangre , Subunidad alfa del Receptor de Interleucina-2/efectos de los fármacos , Interleucinas/efectos adversos , Interleucinas/farmacocinética , Masculino , Dosis Máxima Tolerada , Persona de Mediana Edad , Perforina , Proteínas Citotóxicas Formadoras de Poros/efectos de los fármacos , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/efectos adversos , Proteínas Recombinantes/farmacocinética
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