RESUMEN
Few studies have been performed to assess the risk of skin damage by cyclodextrins (CD) and they have yielded contradictory results. The present study was conducted using the corneoxenometry bioassay on human stratum corneum to compare the skin compatibility of CD currently used in pharmaceutical preparations (betaCD, gammaCD, Rameb, Dimeb, Trimeb, HP-betaCD and HP-gammaCD) and that of new amphiphilic CD derivatives, namely, the phospholipidyl-CD (DMPE-Dimeb and DMPE-Trimeb). All the tested CD were well tolerated by the stratum corneum at a concentration of 5%. However, inter-individual reactivity was larger for DMPE-Dimeb, suggesting a more aggressive trend for this compound. Cutaneous Index of Mildness values obtained confirm that Dimeb is able to extract some skin components and shows that DMPE-Dimeb performs similarly.
Asunto(s)
Ciclodextrinas/toxicidad , Epidermis/efectos de los fármacos , Piel/efectos de los fármacos , Xenobióticos/toxicidad , Ciclodextrinas/síntesis química , Células Epidérmicas , Humanos , Fosfatidiletanolaminas/síntesis química , Piel/citologíaRESUMEN
Some biological properties of new bifunctional conjugates designed for drug targeting were evaluated through in vitro experiments. Eight peptidylcyclodextrin compounds were used, which correspond to modified beta- or gamma-cyclodextrin (CD) grafted on neuropeptide substance P (SP) or a shorter derivative (SP(4-11)). Using anti-SP and anti-CD antibodies as molecular probes, we showed that the main structural features of the two moieties of these adducts were preserved. Binding experiments, using CHO cells expressing the human SP-specific NK1 receptor, demonstrated the functionality of all peptidylcyclodextrin derivatives, which exhibited IC50 values in a 10(-9)-10(-7) M range. All compounds were able to induce a pharmacological response, triggering phosphatidylinositol turnover with EC50 values in the same range as the natural ligand. Moreover, autoradiography analysis of rat spinal corn sections proved that [125I]SP binding was dose-dependently displaced by one selected compound (a gamma-CD-SP), showing a similar affinity of this adduct for the rat neurokinin 1 receptor. Our observations demonstrate that these peptidylcyclodextrins efficiently target NK1 receptor-expressing cells.
Asunto(s)
Ciclodextrinas/farmacología , Sistemas de Liberación de Medicamentos , Receptores de Neuroquinina-1/efectos de los fármacos , Sustancia P/análogos & derivados , beta-Ciclodextrinas , gamma-Ciclodextrinas , Animales , Anticuerpos/inmunología , Autorradiografía , Unión Competitiva , Células CHO , Cricetinae , Ciclodextrinas/química , Ciclodextrinas/inmunología , Diseño de Fármacos , Estructura Molecular , Receptores de Neuroquinina-1/biosíntesis , Receptores de Neuroquinina-1/genética , Proteínas Recombinantes/biosíntesis , Sustancia P/química , Sustancia P/inmunologíaRESUMEN
PURPOSE: Because of its ability to form complexes with drugs, gamma-cyclodextrin is of great potential value in pharmaceutical formulations. The biological fate of y-cyclodextrin must therefore be considered in safety evaluation, using sensitive and specific methods applicable to biological fluids. METHODS: Antibodies were raised against gamma-cyclodextrin, allowing the development of a new enzyme immunoassay. The analytical characteristics of this assay were evaluated. Rats were given a single intravenous 25 mg/kg dose of gamma-cyclodextrin. Plasma and urine samples were collected and assayed. RESULTS: This new enzyme immunoassay was sensitive (limit of detection close to 94 pg/mL) and suitable for quantification of gamma-cyclodextrin in urine and plasma after methanol extraction. The use of different linear and cyclic compounds demonstrated the high specificity of the assay. After i.v. administration, the concentration of gamma-cyclodextrin rapidly decreased in the plasma while the molecule was probably distributed into the tissues. Although urinary elimination predominates, only 50% of the injected gamma-cyclodextrin was recovered in urine, suggesting enzymatic degradation and/or tissular storage. CONCLUSIONS: This assay may provide important information on the fate of gamma-cyclodextrin inclusion complexes dedicated to drug-delivery using various modes of administration (oral, parenteral, transmucosal or dermal).
Asunto(s)
Ciclodextrinas/farmacocinética , Técnicas para Inmunoenzimas/métodos , alfa-Ciclodextrinas , gamma-Ciclodextrinas , Acetilcolinesterasa/metabolismo , Animales , Anticuerpos , Líquidos Corporales , Reacciones Cruzadas , Ciclodextrinas/inmunología , Ciclodextrinas/orina , Sistemas de Liberación de Medicamentos , Inyecciones Intravenosas , Masculino , Ratas , Ratas Sprague-Dawley , Sensibilidad y EspecificidadRESUMEN
We have developed a highly sensitive enzyme immunoassay for 6-monoamino-beta-CD (mono(6-amino-6-deoxy)cyclomaltoheptaose) and its parent compound (beta-CD) with a detection limit in the 100 pg/mL range. The polyclonal antibodies obtained are highly specific for the beta-cyclodextrin core and do not recognize other cyclic cyclodextrins (i.e., alpha- and gamma-CD) or linear analogues. This enzyme immunoassay can be used to quantify 6-monoamino-beta-CD in rat urine and plasma. Using this immunoassay, we have evaluated the main pharmacokinetic parameters of 6-monoamino-beta-CD after iv administration to the rat of a 25 mg/kg dose. Since this method is strictly specific to the native beta-CD form, we have demonstrated that the molecule rapidly disappeared from plasma but is probably distributed in the tissues. The urinary route appears as the predominant way of elimination since almost all the administered drug is recovered in urine. Finally, analysis of the same molecule after oral administration to the rat (25 mg/kg) demonstrates low plasma levels and that about 1% of the administered dose is excreted in urine. These experiments demonstrate the high stability of the beta-CD core irrespective of the method of administration. This immunological method could provide relevant information on the fate of beta-CD and some derivatives for drug delivery using different modes of administration (oral, parenteral, transmucosal, or dermal).
Asunto(s)
Ciclodextrinas/farmacocinética , beta-Ciclodextrinas , Administración Oral , Animales , Ciclodextrinas/administración & dosificación , Técnicas para Inmunoenzimas , Inyecciones Intravenosas , Masculino , Ratas , Ratas Sprague-Dawley , Sensibilidad y EspecificidadRESUMEN
The interactions of per (3,6 anhydro) alpha cyclodextrin (alpha 36CD) and of lead-alpha 36CD complex with biological systems were tested by NMR, ESR and electronic microscopy using erythrocytes and model membranes. It was found that the haemolytic activity of alpha 36CD alone was seven fold lower than that of natural alpha cyclodextrin (evaluated by the concentration inducing 50% haemolysis, DH50 = 35 mM). Conversely, the formation of the complex resulted in an increase of haemolytic properties, with DH50 of 1 mM. The mechanism proposed was an increased membrane diffusion by endocytosis of the complex, leading to higher amounts of intracellular lead.
Asunto(s)
Quelantes/farmacología , Ciclodextrinas/farmacología , Plomo/farmacología , Quelantes/química , Quelantes/toxicidad , Ciclodextrinas/química , Ciclodextrinas/toxicidad , Espectroscopía de Resonancia por Spin del Electrón , Eritrocitos/efectos de los fármacos , Eritrocitos/ultraestructura , Hemólisis/efectos de los fármacos , Humanos , Técnicas In Vitro , Plomo/química , Plomo/toxicidad , Espectroscopía de Resonancia Magnética , Micelas , Microscopía Electrónica , FosfolípidosRESUMEN
Different ethylated beta-cyclodextrin (Et-beta-CD) derivatives were obtained by various synthetic routes and their chemical structures and physical properties were elucidated. The aqueous solubility studies were carried out at 25 and 37 degrees C. A gas phase chromatography analysis, with head space extraction, was developed to detect the presence of residual solvents in the dry preparations. Electrospray ionization mass spectrometry allowed the determination of the average degree of substitution and the molecular mass of the Et-beta-CDs. Nuclear magnetic resonance analysis was used to elucidate the relationship between the solubility behavior and substituted positions of ethyl groups on the CD glucopyranose units. Finally, this paper deals with different physico-chemical methods used to fully characterize the different batches of Et-beta-CD to correlate the data obtained with the pharmacotechnical behavior of these cyclodextrins.
Asunto(s)
Ciclodextrinas/química , Alquilación , Fenómenos Químicos , Química Física , Cromatografía de Gases y Espectrometría de Masas , Espectroscopía de Resonancia Magnética , Espectrometría de Masas , Peso Molecular , Solubilidad , Solventes , AguaRESUMEN
Crucial conditions for the pharmacological use of active compounds are their ability to cross the biological barriers and reach their intracellular target. In the case of two antiviral pyridopurine derivatives, 1 and 2, this included essentially the membranes and the nucleic acids. Thus the interactions of 1 and 2 with model membranes and oligonucleotides were studied using NMR spectroscopy. It was found that these hydrophobic molecules can be incorporated into the model membranes at the terminal methyl group level, inducing dynamic perturbations in the bilayer. In the presence of the synthetic oligonucleotide ACATGT, both molecules can intercalate aspecifically in AT and GC systems. Inclusion complexes of 1 and 2 beta-cyclodextrins with a 1:1 stoichiometry, were also prepared. This led to to propose two galenic forms 1 and 2, i.e. included in phospholipid vesicles in the form of a beta-cyclodextrin complex
Asunto(s)
Aminas/química , Ciclodextrinas/química , Preparaciones Farmacéuticas/química , Dimiristoilfosfatidilcolina/química , Alimentos , Espectroscopía de Resonancia MagnéticaRESUMEN
The thermodynamic ionization constants (pK(a)(1), pK(a)(2), and pK(a)(3)) of ginkgolide B (9H-1,7a-(epoxymethano)-1H,6aH-cyclopenta[c]furo[2,3-b]furo-[3',2':3,4]cyclopenta[1,2-d]furan-5,9,12-(4H)-trione, 3-tert-butylhexahydro-4,7b,11-trihydroxy-8-methyl-) in aqueous solution have been settled by pH-metric and NMR studies. The three macroscopic pK(a) values as well as the water solubility and the water/n-octanol partition coefficient have been extracted from pH-metric data by means of a nonlinear regression methodology. NMR spectroscopy provided confirmation of the values of the macroscopic constants, information about the effective ionization pathways, and an estimation of the proportions of the various forms under physiologically relevant conditions.
RESUMEN
Spiralin is the major protein of the plasma membrane of several spiroplasmas. Neither the function of this protein nor the crystallographic structure is known. Analysis of the primary structure of spiralin from Spiroplasma melliferum BC3 suggests the presence of an amphipathic peptide in the 143-162 region (Chevalier, C., Saillard, C. and Bové, J.M. (1990) J. Bacteriol. 172, 6090-6097). The structure of a synthetic peptide, H2N-L-N-A-V-N-T-Y-A-T-L-A-K-A-V-L-D-A-I-Q-N-amide, corresponding to this fragment has been examined by 1H-NMR spectroscopy. This 20 amino acid peptide adopts a random coil structure in solution, but the addition of trifluoroethanol stabilizes a structure exhibiting alpha-helical character. The 1H-NMR spectrum has been fully assigned in CF3CD2OD/H2O (30:70, v/v) and the three-dimensional structure has been elucidated using NMR-derived distance information. The calculated structures have been obtained by dynamical simulated annealing or distance geometry followed by simulated annealing. Both sets of structures have been energy-minimized using CHARMm potential. The resulting structures are very similar in terms of constraint violations and energies. It is demonstrated that whereas the first three residues exhibit a large flexibility, the remaining sequence is helical.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/química , Espectroscopía de Resonancia Magnética , Fragmentos de Péptidos/química , Estructura Secundaria de Proteína , Secuencia de Aminoácidos , Modelos Moleculares , Datos de Secuencia Molecular , Soluciones , Spiroplasma/químicaRESUMEN
Antibodies were raised against cyclomaltoheptaose (beta-cyclodextrin), providing a highly sensitive enzyme immunoassay for beta-cyclodextrin and some derivatives with a detection limit of 120 pg/mL. Investigations of cross-reactivities with a wide variety of linear and cyclic maltooligosaccharides demonstrate that the antibodies are highly specific for cyclomaltoheptaose and a number of derivatives. The epitope is probably located on the secondary hydroxyl groups of the rim side. This enzyme immunoassay is shown to be suitable to detect cyclomaltoheptaose in urine and in plasma.
Asunto(s)
Ciclodextrinas/análisis , Técnicas para Inmunoenzimas , beta-Ciclodextrinas , Animales , Anticuerpos , Conformación de Carbohidratos , Secuencia de Carbohidratos , Reacciones Cruzadas , Ciclodextrinas/inmunología , Datos de Secuencia Molecular , Estructura Molecular , Conejos/inmunología , Sensibilidad y Especificidad , Relación Estructura-ActividadRESUMEN
The geometry and structural features of the inclusion complexes of beta-cyclodextrin (beta-CD) with the chiral antiamnesic drugs (+/-)-1-benzyl-4-hydroxymethylpyrrolidin-2-one (WEB-1868). (+/-)-1-benzenesulfonyl-5-ethoxypyrrolidin-2-one (RU-35929), and (+/-)-1-(3-pyridinlysulfonyl)-5-ethoxypyrrolidin-2-one (RU-47010) were studied by the molecular modeling method (MacroModel interactive computer program). Docking procedures yielded the most stable complexes, which showed the aromatic ring of the guests inside the cavity and the pyrrolidinone ring out from the side of the beta-CD secondary hydroxyl groups. The binding energies were essentially due to hydrogen-bonded structures involving the C=O group of the guests. Selective interactions allowed chiral discrimination, and accordingly, separate beta-CD complexes of the R and S enantiomers of each guest compound were studied. The almost round beta-CD structure, in all the cases, assumed an elliptic shape on passing from the isolated molecule to the docked complex. The optimized structures and conformations of beta-CD and its inclusion compounds showed acceptable general agreement with information from proton nuclear magnetic resonance studies.
Asunto(s)
Simulación por Computador , Ciclodextrinas/química , Modelos Químicos , Psicotrópicos/química , beta-Ciclodextrinas , Espectroscopía de Resonancia Magnética/métodos , Modelos Moleculares , Conformación Molecular , Sensibilidad y Especificidad , EstereoisomerismoRESUMEN
Nuclear magnetic resonance at high field has been used to investigate the stoichiometries of pharmacologically important beta-cyclodextrin:steroid complexes. This technique provides evidence for the existence of true inclusion complexes and allows unequivocal determination of the stoichiometry for complexes obtained in the solid state, as well as in solution. The present data clarify previously published determinations of the stoichiometry of these complexes and show the influence of the nature and position of functional groups on the molecular ratio in the complex. The observed behavior can be rationalized using representative steroids and allows prediction of the strength of inclusion and the most probable stoichiometry.
Asunto(s)
Ciclodextrinas/química , Esteroides/química , beta-Ciclodextrinas , Espectroscopía de Resonancia Magnética , Estructura Molecular , SolubilidadRESUMEN
[Nle7]-endothelin was synthesized and studied by 1H NMR and distance geometry calculations. The NMR study was performed first in DMSO-d6 and then in 50% acetonitrile/water since this peptide aggregates in pure water. In both cases, all spin systems were identified and assigned with the aid of two-dimensional spectroscopy (2D): COSY (for scalar couplings) and NOESY (for dipolar couplings). On the basis of the acetonitrile/water NMR parameters, and using the DISGEO program, a three-dimensional structure of [Nle7]-endothelin is proposed and discussed.
Asunto(s)
Endotelinas/química , Espectroscopía de Resonancia Magnética , Acetonitrilos , Secuencia de Aminoácidos , Dimetilsulfóxido , Endotelinas/síntesis química , Datos de Secuencia Molecular , Conformación Proteica , Soluciones , AguaRESUMEN
Quasielastic incoherent neutron scattering has been used to investigate the rate of local translational diffusion of lipid molecules in phospholipid bilayers of dipalmitoyl-phosphatidylcholine. The measured translational diffusion constants (4 x 10(-10) m2 s-1 at 63 degrees C and 1.4 x 10(-11) m2 s-1 at 30 degrees C) are considerably faster than those deduced using other less direct methods, but are in agreement with those measured in soap-water lyotropic liquid crystals, and with calculated values. This disagreement is attributed to differences in the time and distance scales characterising the various measurements. Quasielastic neutron scattering experiments observe fast motions over molecular distances, whereas other methods tend to measure a rate of diffusion which is averaged over macroscopic distances, and may thus contain contributions from long distance slow diffusive motions such as diffusion between the bilayers.
Asunto(s)
1,2-Dipalmitoilfosfatidilcolina/química , Fluidez de la Membrana , Lípidos de la Membrana/química , Difusión , Técnicas In Vitro , Membrana Dobles de Lípidos , Neutrones , Dispersión de RadiaciónRESUMEN
Sarafotoxin-S6b has been synthesized and studied by (1)H NMR in 50 50 acetonitrile/water mixture. All spin systems were identified and assigned with the aid of 2D experiments. On the basis of these data, a 3D structure of sarafotoxin is proposed and compared to that of [Nle(7)]endothelin obtained in the same conditions. From this study, it appeared that sarafotoxin-S6b and [Nle(7)]endothelin roughly share the same 3D structure, the main differences being located in the 4-7 loop bearing the sequence variation.
RESUMEN
The inclusion complex of indomethacin sodium salt in beta-cyclodextrin has been studied by proton NMR at high magnetic field. The continuous variation technique was used to evidence the formation of a soluble 1:1 complex in aqueous solution at physiological pH. The effective association constant was determined by the Benesi-Hildebrand procedure to be 760 M-1 at 303 K. This technique requires NMR measurements in the presence of a very large excess of one of the complex components and, since both beta-cyclodextrin and the sodium salt of indomethacin are sparingly soluble in water, NMR spectrometers operating at very high magnetic fields were used. Besides the effective association constant, the Benesi-Hildebrand approach allows a precise determination of all NMR parameters of the pure inclusion complex which may be used for a complete analysis of the geometry of this complex in solution.
Asunto(s)
Ciclodextrinas/análisis , Dextrinas/análisis , Indometacina/análisis , Almidón/análisis , beta-Ciclodextrinas , Concentración de Iones de Hidrógeno , Espectroscopía de Resonancia MagnéticaRESUMEN
The 1H-n.m.r. spectra of various dermatan sulphate preparations present, besides the major signals of the basic disaccharide unit, several other minor signals. We have assigned most of them by n.m.r., using two-dimensional proton-proton double-quantum-correlation and nuclear-Overhauser-effect spectroscopy experiments. This allowed us to identify 2-O-sulphated L-iduronic acid and D-glucuronic acid residues as well as 6-sulphated N-acetylgalactosamine (presumably 4-O-sulphated as well). 2-O-Sulphated iduronic acid was present to similar extents (6-10% of total uronic acids) in pig skin dermatan sulphate and pig intestine dermatan sulphate, whereas glucuronic acid represented 17% of the uronic acid of pig skin dermatan sulphate and was virtually absent (1%) from the other preparation. 6-O-Sulphated N-acetylgalactosamine was present in minor amounts in pig intestine dermatan sulphate only. The influence of sulphation of iduronic acid units on their conformation was assessed by using chemically oversulphated pig intestine dermatan sulphate. Introduction of sulphate groups in this unit in dermatan sulphate tends to shift the conformational equilibrium towards the 1C4 conformer.
Asunto(s)
Condroitín/análogos & derivados , Dermatán Sulfato/análisis , Galactosamina/análisis , Glucuronatos/análisis , Ácido Idurónico/análisis , Ácidos Urónicos/análisis , Animales , Ácido Glucurónico , Espectroscopía de Resonancia Magnética , Conformación Molecular , PorcinosRESUMEN
The 1H-n.m.r. 3J values for the L-iduronic acid (IdoA) residues for solutions in D2O of natural and synthetic oligosaccharides that represent the biologically important sequences of dermatan sulfate, heparan sulfate, and heparin have been rationalized by force-field calculations. The relative proportions of the low-energy conformers 1C4, 2S0, and 4C1 vary widely as a function of sequence and of pattern of sulfation. When IdoA or IdoA-2-sulfate units are present inside saccharide sequences, only 1C4 and 2S0 conformations contribute significantly to the equilibrium. This equilibrium is displaced towards the 2S0 form when IdoA-2-sulfate is preceded by a 3-O-sulfated amino sugar residue, and towards the 1C4 form when it is a non-reducing terminal. For terminal non-sulfated IdoA, the 4C1 form also contributes to the equilibrium. N.O.e. data confirm these conclusions. Possible biological implications of the conformational flexibility and the counter-ion induced changes in conformer populations are discussed.
Asunto(s)
Glicosaminoglicanos , Ácido Idurónico , Ácidos Urónicos , Conformación de Carbohidratos , Secuencia de Carbohidratos , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Datos de Secuencia Molecular , SulfatosRESUMEN
The conformation in solution of the pentasaccharide methyl glycoside (As-G-A*-Is-AM; 1), which represents the binding site of heparin for Antithrombin III, has been investigated using molecular mechanics and 1H-n.m.r. spectroscopy. The pentasaccharide has a rather rigid (As-G-A*) and a more flexible (Is-AM) region. A simplified model of 1, comprising two conformations, corresponding to the 1C4 and the 2S0 forms of the iduronate residue, and modified at the G-A* glycosidic linkage with respect to the energy minimum, reproduces most of the observed 3J values and n.O.e. enhancements. The possible role in the binding to Antithrombin III of a low-energy conformer, not observed in solution, is discussed.