Posibilidad de terapia génica en pacientes con enfermedades retinianas hereditarias / Possibility of genetic therapy for inherited retinal conditions

Objetivo Evaluar la posibilidad de terapia génica en pacientes con enfermedades oculares hereditarias con diagnóstico genético establecido. Los objetivos secundarios son revisar la tasa de diagnóstico genético y hacer una actualización de los genes para los cuales hay estudios clínicos o preclínicos en curso que pudieran permitir la terapia génica. Métodos Estudio observacional, retrospectivo y multicéntrico de 177 pacientes con enfermedades oculares hereditarias a quienes se realizó estudio genético.Resultados De 177 pacientes con estudio genético, se incluyeron 146. Se identificaron variantes causantes de enfermedad en 117 pacientes con lo que se obtuvo una tasa de detección de variantes del 80,1%. Se encontraron variantes patogénicas en 47 genes, siendo ABCA4 el gen más común (17,9%), seguido por CRB1 (11,9%). De los pacientes con diagnóstico genético, el 64,1% tienen una variante en un gen para el cual se ha estudiado terapia génica y solo el 40,1% presentan una variante en genes con estudios para su terapia génica en fase clínica. Conclusiones El estudio genético ha abierto nuevos horizontes en el manejo de pacientes con enfermedades oculares hereditarias. Cerca de dos tercios de los pacientes presentó variantes patogénicas en genes para los cuales se ha evaluado la posibilidad de terapia génica. Sin embargo, muchos estudios se encuentran en fase preclínica. Se debe adecuar las expectativas de los pacientes sometidos a estudio genético y sus familias (AU)

Objective To evaluate the possibility of gene therapy in patients with inherited ocular conditions and established genetic diagnosis. The secondary objectives were to determine the genetic diagnostic rate and to update the list of genes for which there are ongoing clinical trials or preclinical studies that could allow for gene therapy. Methods Observational, retrospective, multicentric study of 177 patients with inherited ocular conditions that underwent genetic testing. Results Of 177 patients with genetic testing, 146 were enrolled for this study. Disease-causing variants were identified in 117 patients (variant detection rate of 80.1%). Pathogenic variants were found in 47 genes, with ABCA4 being the most common gene (17.9%), followed by CRB1 (11.9%). 64.1% of patients with a genetic diagnosis have a variant in genes for which gene therapy has been studied and only 40.1% have a variant in genes with studies for gene therapy in clinical phase. Conclusions Genetic testing has opened new horizons in the management of patients with hereditary ocular diseases. About two-thirds of the patients had pathogenic variants in genes for which gene therapy has been evaluated. However, many studies are in the pre-clinical phase. The expectations of patients undergoing genetic study and their families should be managed accordingly (AU)

Possibility of genetic therapy for inherited retinal conditions.

OBJECTIVE: To evaluate the possibility of gene therapy in patients with inherited ocular conditions and established genetic diagnosis. The secondary objectives were to determine the genetic diagnostic rate and to update the list of genes for which there are ongoing clinical trials or preclinical studies that could allow for gene therapy. METHODS: Observational, retrospective, multicentric study of 177 patients with inherited ocular conditions that underwent genetic testing. RESULTS: Of 177 patients with genetic testing, 146 were enrolled for this study. Disease-causing variants were identified in 117 patients (variant detection rate of 80.1%). Pathogenic variants were found in 47 genes, with ABCA4 being the most common gene (17.9%), followed by CRB1 (11.9%). 64.1% of patients with a genetic diagnosis have a variant in genes for which gene therapy has been studied and only 40.1% have a variant in genes with studies for gene therapy in clinical phase. CONCLUSIONS: Genetic testing has opened new horizons in the management of patients with hereditary ocular diseases. About two-thirds of the patients had pathogenic variants in genes for which gene therapy has been evaluated. However, many studies are in the pre-clinical phase. The expectations of patients undergoing genetic study and their families should be managed accordingly.

Management of subluxated lens in young patients.

OBJECTIVES: To evaluate visual outcomes in patients treated for lens subluxation. Secondary objectives are to report best corrected visual acuity (BCVA) in LogMAR and compare the outcomes of patients managed conservatively with those treated surgically. METHODS: Retrospective comparison of BCVA in patients under 50 years-old with lens subluxation, managed conservatively or surgically. RESULTS: A total of 49 eyes of 28 patients were included. Demographic characteristics were similar in both groups. Twenty eyes were treated surgically (40.8%) compared to 29 with medical treatment (59.2%). Marfan syndrome (79.6%) was diagnosed in 39 eyes. LogMAR BCVA post intervention was 0.35±0.31 for medical treatment and 0.39±0.32 for the surgical group, with no significant differences (P=.63). Improvements in LogMAR lines were 2.7±4.2 and 4.11±4.2 (P=.35), respectively. Two eyes in the surgery group developed ocular hypertension (0.04%), none with retinal detachment. CONCLUSIONS: The final BCVA showed no significant differences in this group of patients. BCVA depends on the visual potential of the rehabilitated eye rather than a specific type of intervention.

Manejo del cristalino subluxado en pacientes jóvenes / Management of subluxated lens in young patients

Objetivos: Evaluar los resultados visuales en pacientes tratados por subluxación del cristalino. Los objetivos secundarios son reportar la agudeza mejor corregida (AVMC) en LogMAR y comparar los resultados de los pacientes manejados de manera conservadora con aquellos manejados quirúrgicamente. Métodos: Estudio retrospectivo comparativo de AVMC en pacientes menores de 50 años con subluxación de cristalino, manejados de manera conservadora con lentes o tratados quirúrgicamente. Resultados: Se incluyeron 49 ojos de 28 pacientes. Las características demográficas fueron similares en ambos grupos; 20 ojos fueron tratados quirúrgicamente (40,8%) versus 29 con tratamiento médico (59,2%); 39 ojos con diagnóstico de síndrome de Marfán (79,6%). La AVMC LogMAR postintervención de 0,35±0,31 para el tratamiento médico y 0,39±0,32 para el grupo de manejo quirúrgico, sin encontrarse diferencias significativas (p=0,63). Las mejorías en líneas LogMAR fueron, respectivamente, 2,7±4,2 y 4,11±4,2 (p=0,35). Dos ojos del grupo cirugía evolucionaron con hipertensión ocular (0,04%), ninguno con DR. Conclusiones: La AVMC final no presentó diferencias significativas en este grupo de pacientes, dependiendo del potencial visual del ojo rehabilitado más que de un tipo específico de intervención(AU)

Objectives: To evaluate visual outcomes in patients treated for lens subluxation. Secondary objectives are to report best corrected visual acuity (BCVA) in LogMAR and compare the outcomes of patients managed conservatively with those treated surgically. Methods: Retrospective comparison of BCVA in patients under 50 years-old with lens subluxation, managed conservatively or surgically. Results: A total of 49 eyes of 28 patients were included. Demographic characteristics were similar in both groups. Twenty eyes were treated surgically (40.8%) compared to 29 with medical treatment (59.2%). Marfan syndrome (79.6%) was diagnosed in 39 eyes. LogMAR BCVA post intervention was 0.35±0.31 for medical treatment and 0.39±0.32 for the surgical group, with no significant differences (P=.63). Improvements in LogMAR lines were 2.7±4.2 and 4.11±4.2 (P=0.35), respectively. Two eyes in the surgery group developed ocular hypertension (0.04%), none with retinal detachment. Conclusions: The final BCVA showed no significant differences in this group of patients. BCVA depends on the visual potential of the rehabilitated eye rather than a specific type of intervention(AU)

Near infrared reflectance spectroscopy as a tool for the control of sheep leather defatting.

The fat content is one of the variables to be controlled by the tanning industry with a view to obtaining leather for various commercial purposes. Ensuring the production of quality leather products frequently entails using some defatting treatment, particularly when the raw skin is rich in natural fat. The official method for determining fat in leather, IUC 4, is rather slow; also, it uses polluting reagents and involves powdering samples for Soxhlet extraction with low-polarity solvents. The combination of NIR diffuse reflectance spectroscopy as implemented with a fibre-optic probe and multivariate calibration is probably the best choice for the direct determination of fat in leather and the monitoring of leather defatting. In this work, a method for the determination of fat in leather and the control of the defatting process in an expeditious manner and with no sample treatment was developed. Defatting tests were conducted on leather specimens from lambs of various breeds and origins in order to span as wide as possible a range of variability in their properties and natural fat content. The NIR spectra used to construct the calibration matrices were recorded directly on the leather samples prior to and after defatting. Fat contents were determined by partial least-squares regression (PLSR), using the values obtained with the official method as references. Notwithstanding the complex nature of leather, the calibration models used provided good external predictions: the largest overall relative error, obtained by using a single calibration matrix for natural and defatted specimens, was 10%. The proposed method is therefore an advantageous alternative to the official method.

Kinetic-spectrophotometric determination of theophylline, dyphylline, and proxyphylline by use of partial least-squares regression.

A kinetic-spectrophotometric method for the determination of theophylline, dyphylline and proxyphylline, based on their azo coupling reaction with the diazonium ion of sulfanilic acid after a treatment with alkali, is proposed. The absorbance is recorded from 340 to 600 nm every second during reaction for 90 s, and calibration is performed by partial least-squares regression, using first derivative spectra values. Mixtures containing 2.5-13 micro g mL(-1) dyphylline and proxyphylline, and 2-9 micro g mL(-1) theophylline were successfully resolved with root mean squared errors of prediction (RMSEP) of 0.4, 0.3, and 0.2 for dyphylline, proxyphylline, and theophylline, respectively. The proposed method was satisfactorily applied to the determination of the three compounds in a commercially available pharmaceutical preparation and provided results similar to those obtained by HPLC.

Influence of the procedure used to prepare the calibration sample set on the performance of near infrared spectroscopy in quantitative pharmaceutical analyses.

Calibrating near infrared diffuse reflectance spectroscopy (NIRS) methods usually involves preparing a set of samples with a view to expanding the analyte concentration range spanned by production samples. In this work, the performances of the two procedures most frequently used for this purpose in near infrared pharmaceutical analysis, viz., synthetic samples obtained by weighing of the pure constituents of the pharmaceutical and doped samples made by under- or overdosing previously powdered production samples, were compared. Both procedures were found to provide similar results in the quantification of the active compound in the pharmaceutical, which was determined with a relative standard error of prediction (RSEP) of < 1.6%. However, the two types of sample preparation provide different spectra, which precludes the accurate quantification of synthetic samples from calibrations obtained with doped samples and vice versa. None of the mathematical pre-treatments tested with a view to reducing this different scattering (viz., second derivative, standard normal variate and orthogonal signal correction) could effectively solve this problem. This hinders accurate validation of the linearity of the procedure and makes it advisable to use doped samples which are markedly less different to production samples.

Multi-component kinetic-spectrophotometric analysis. Selection of wavelength and time ranges.

An empirical method for the selection of the best wavelength and time ranges which can be used in the quantification of binary mixtures, in a kinetic-spectrophotometric system, is proposed. It is based on finding those ranges which provide the least correlation between the kinetic profiles and the spectra of the products of reaction. The method was applied to the analysis of binary mixtures using simulated data with different rate constant ratios and in the presence of an interference that shows spectral overlap with the analytes. Subsequently, the proposed method was applied to the resolution of dyphylline and proxyphylline mixtures. The system studied was characterized by an elevated similarity in the kinetic behavior of the analytes under pseudo-first-order conditions and an elevated degree of spectral overlap of the products of reaction. In spite of this, satisfactory results were obtained in the quantification of the two analytes. The standard error of prediction (SEP) and the standard deviation between replicates (SDBR) did not show significant differences, being of the order of 4 and of 3% for dyphylline and proxyphylline, respectively.

Analytical control of a pharmaceutical formulation of sodium picosulfate by capillary zone electrophoresis.

A new procedure for the analytical control of a pharmaceutical formulation by capillary zone electrophoresis (CZE) is proposed. It allows the simultaneous determination of the major compounds in the formulation: active compound (sodium picosulfate) and preservative (methylparaben), and the degradation products of the preservative, which slowly degrades by hydrolysis or by transesterification with sorbitol (sweetener in excess in the formulation) yielding p-hydroxybenzoic acid and sorbitolparaben, respectively. UV-Vis detection in the absorption maxima of the analytes and 20 mM borate solution at pH 10 as background electrolyte are used. Results are compared with those provided by the HPLC procedure. The method has also been validated using the HPLC procedure as the reference method, evaluating selectivity, accuracy, linearity and precision. The CZE procedure developed is sufficiently accurate and the precision achieved is about 1% for major and 3% for minor compounds.

Resolution of isomers of sorbitolparaben esters by chromatographic and electrophoretic techniques.

Pharmaceutical preparations usually contain preservatives and sweeteners. When parabens are used as preservatives and a polyol as a sweetener, a transesterification reaction may happen, yielding the transester polyol-paraben. The products formed in the transesterification reaction of methylparaben and sorbitol were analyzed by micellar electrokinetic chromatography and by HPLC. Up to six positional isomers of sorbitolparaben (SPB) can be produced. However, only three peaks were found by HPLC. The higher efficiency and resolution power of MEKC allowed one to resolve five peaks. Results were compared with those obtained by capillary zone electrophoresis in borate buffer, where the separation of isomers occurred in a different way, because of a complexation between SPB and borate.

Characterization of the oligosaccharides of plasma sex hormone binding globulin from noncirrhotic alcoholic patients.

In previous reports we have demonstrated high plasma levels of sex hormone-binding globulin (SHBG) in asymptomatic alcoholic men. In the present work the physicochemical properties of SHBG from plasma of noncirrhotic alcoholic patients have been further compared with SHBG of control subjects. Steroid binding to SHBG was similar for the two groups: alcoholic men, K(d) of 0.62 +/- 0.07 nM and control individuals, K(d) of 0.70 +/- 0.10 nM. The structure of oligosaccharides attached to SHBG from controls and alcoholic men were determined by using serial chromatography. Our data indicated that 7% of SHBG of control individuals was not retarded by the Con-A column, whereas approximately 30% of SHBG of alcoholic men eluted in the void volume of Con A. Approximately 46% of SHBG of alcoholics applied to Con A, possessed biantennary complex oligosaccharides, as indicated by the fact that it could be eluted with methyl-alpha-D-glucopyranoside and by its retention on wheat germ agglutinin; in contrast, when SHBG from control men was analyzed, approximately 51% was eluted with methyl-alpha-D-glucopyranoside. Approximately 9% of the biantennary complex oligosaccharides on SHBG of control men and none of those on SHBG from alcoholic men were fucosylated on the chitobiose core, as determined by chromatography on Lenn culinaris lectin. Galactosylated oligosaccharides were also present on the SHBG fraction as indicated by its interaction with Ricinus communis-I. Approximately 24% of SHBG of alcoholic men and 39% of those on SHBG from control individuals applied to Con-A were retained and could be eluted with methyl-alpha-D-mannopyranoside. Evidence based on the binding on mannoside-eluted SHBG to Con-A, wheat germ agglutinin, and R. communis-I indicated that at least the SHBG in this fraction, from alcoholics or controls, contained two glycosylation sites and that the sites were differentially glycosylated.

Development and validation of a near infrared method for the analytical control of a pharmaceutical preparation in three steps of the manufacturing process.

A near infrared diffuse reflectance spectroscopy (NIRS) procedure for the quantitative control analysis of the active compound (otilonium bromide) in a pharmaceutical preparation in three steps of the production process (blended product, cores and coated tablets) and a methodology for its validation are proposed. The analytical procedure is composed by two consecutive steps. First, the sample is identified by comparing its spectrum with a second derivative spectral library. If the sample is positively identified, the active compound is quantified by using a previously established partial least squares (PLS) calibration model. The procedure was validated by studying repeatability, intermediate precision, accuracy and linearity. To this end, an adaptation of ICH (International Conference on Harmonisation) validation methodology to an NIR multivariate calibration procedure is proposed. The relative standard error of prediction (RSEP) was < or = 1% and the suitability of the procedure for control analysis was confirmed by the results obtained analysing new production samples produced over a three-month period.

Development and validation of a method for the analysis of a pharmaceutical preparation by near-infrared diffuse reflectance spectroscopy.

A near-infrared (NIR) spectroscopic method based on the use of a fiber optical probe for the analysis of a commercially available pharmaceutical preparation is proposed. The analyte is identified by comparison with a second-derivative spectral library, using the correlation coefficient as the discriminating parameter. Once a sample has been positively identified, the active principle is quantified with partial least-squares (PLS) calibration. The proposed method was validated for use as a control method; to this end, the selectivity of the identification process, and the repeatability, intermediate precision, accuracy, linearity, and robustness of the active principle quantitation, were assessed.

Sex hormone-binding globulin in non-cirrhotic alcoholic patients during early withdrawal and after longer abstinence.

In recently intoxicated non-cirrhotic male alcohol-misusing and -dependent patients, we studied, during early withdrawal and more prolonged abstinence, the rate of changes of sex hormones and their binding globulin (SHBG), the prevalence of hypo-androgenism and possible determinant factors of SHBG increase. Twenty-one alcoholics and 21 controls were studied. SHBG plasma levels, sex hormones (SH), cortisol, insulin and thyroid hormones were measured at admission and discharge. SHBG and SH were also determined on days 2, 4 and 7 after admission and on weeks 2, 6 and 12 after discharge. SHBG showed a 3-fold increase, decreasing slowly during the first 10 days, but remaining above control values. Luteinizing hormone was also increased. Free testosterone (Tf) was low at admission and correlated negatively with SHBG during the first 10 days. By day 10, Tf reached normal values, despite SHBG remaining elevated. The other sex hormones were normal. Neither insulin nor thyroid hormones correlated with SHBG. Cortisol was high at admission and then normalized. Clinical hypo-androgenism was found in 33-50% of patients, but did not correlate with SHBG or SH. During follow-up, nine patients relapsed. In those remaining abstinent, SHBG continued decreasing, reaching normal levels in the 12th week. In those who relapsed, SHBG remained high or even increased further. Gamma-glutamyltransferase showed similar but faster changes. We conclude that excessive alcohol ingestion is associated with marked increases of SHBG which slowly revert during abstinence. High SHBG does not fully explain the low Tf values or the presence of clinical hypo-androgenism in alcoholics. This SHBG response to ethanol makes it a potential marker of excessive alcohol intake.

Kinetic spectrophotometric determination of hydrocortisone acetate in a pharmaceutical preparation by use of partial least-squares regression.

A kinetic spectrophotometric method for the determination of hydrocortisone acetate based on its condensation with isonicotinic acid hydrazide is proposed. The method is applied to the determination of hydrocortisone acetate in a commercially available pharmaceutical preparation, presented as a pomade, that also contains another corticosteroid and additional active compounds. The operating procedure involves dissolving the pomade in chloroform and the addition of the reagent solution directly to the cuvette, in this way avoiding the previous extraction of analytes from the insoluble pomade matrix required by the alternative HPLC procedure. Calibration is performed by partial least-squares regression, using absorbance or first derivative spectra values recorded each minute during the first 30 min of reaction. Use of first derivative spectra overcomes possible scattered light problems produced by excipients precipitating, and produced slightly better results than absorbance data. The relative standard deviation obtained for 11 replicates analysed on different days was approx. 1.5%. The proposed method improves both accuracy and precision of the classical initial rate method and the precision of the HPLC procedure.

Secuelas oculares debidas a infección herpética en un grupo de niños chilenos / Ocular sequels caused by herpetic infection in chilean children

Estudio prospectivo, multicéntrico, en el cual investigadores entrenados seleccionaron y siguieron clínica y virológicamente (aislamiento viral, anticuerpos monoclonales y PCR) pacientes de 0 a 18 años de edad con diagnóstico clínico de infección ocular por HSV. Se efectuó determinación de la agudeza visual (AV) por el Test de Mirada Preferencial, HOTV, E Snellen u optotipos de Snellen, con la mejor corrección óptica, examen refractivo bajo cicloplegia, estudio completo de estrabismo, topografía, paquimetría y fotografía. Se enrolaron entre 1993 a 1997 16 niños, dos de ellos con compromiso bilateral (18 ojos). En todos salvo un niño, se identificó HSV tipo I. Mediana de edad 5 años. El diagnóstico de ingreso fue el edema disciforme en 45 por ciento y en 33 por ciento de los casos una queratitis epitelial dendrítica. El 87,5 por ciento de los pacientes con edema disciforme presentó al menos una recurrencia por año. Los pacientes con compromiso epiteliar presentaron en todos los casos AV > 20/40, a diferencia de los pacientes con edema disciforme en que 71,4 por ciento presentaron AV final < de 20/50. El leucoma corneal fue severo en 62,5 por ciento de los ojos con edema disciforme y desarrollaron estrabismo 50 por ciento de los casos. En tres casos se requiere trasplante corneal. La enfermedad ocular herpética afecta principalmente a población joven y sana. El diagnóstico más frecuente fue el edema disciforme que presentó evolución crónica, requirió un gran número de consultas médicas y de tratamiento prolongado (entre 3 y 6 meses) y presentaron la mayor frecuencia de secualas. En general, el 78 por ciento de los pacientes estudiados desarrolló algún tipo de secuela visual

Chiral and nonchiral determination of ketoprofen in pharmaceuticals by capillary zone electrophoresis.

The new method for the enantiomeric resolution of various 2-arylpropionic acids by capillary zone electrophoresis (CZE) using heptakis-tri-O-methyl-beta-cyclodextrin as chiral selector was applied to the determination of ketoprofen in different commercially-available pharmaceutical preparations. The analyte was determined under chiral and nonchiral conditions (viz. in the presence and absence of 50 mM heptakis-tri-O-methyl-beta-cyclodextrin in the background electrolyte), with significantly similar results and relative standard deviations from 1.2 to 6.5% in both cases. The limits of detection and determination for the inactive enantiomer, R-(-)-ketoprofen, were calculated to be 7.0 x 10(-7) and 1.6 x 10(-6) M, respectively. The proposed method was successfully used to determine enantiomeric purity in the drugs studied, with results comparable to those provided by the chiral HPLC method.

Separation of profen enantiomers by capillary electrophoresis using cyclodextrins as chiral selectors.

A method for resolving the enantiomers of various 2-arylpropionic acids (viz. ketoprofen, ibuprofen and fenoprofen) by capillary zone electrophoresis (CZE) using a background electrolyte (BGE) containing a cyclodextrin as chiral selector is proposed. The effects of the type of cyclodextrin used and its concentration on resolution were studied and heptakis-2,3,6-tri- O-methyl-beta-cyclodextrin was found to be the sole effective choice for the quantitative enantiomeric resolution of all the compounds tested. The influence of pH, BGE concentration, capillary temperature and addition of methanol to the BGE on resolution and other separation-related parameters was also studied. The three compounds studied can be enantiomerically resolved with a high efficiency in a short time (less than 20 min) with no capillary treatment. This makes the proposed method suitable for assessing the enantiomeric purity of commercially available pharmaceuticals.

Near-infrared analytical control of pharmaceuticals. A single calibration model from mixed phase to coated tablets.

A new method for analysis of the active compound in a commercial pharmaceutical formulation in different steps of the production cycle, based on near-infrared diffuse reflectance spectroscopy, is proposed. The analysis includes three different steps of the production cycle: granules ready for compression (mixed phase), cores (intermediate) and coated tablets (final product). Satisfactory predictive results for production samples, independently of its origin in the production cycle, require calibration with laboratory-made samples covering the concentration range involved in the manufacturing process, and also production samples from various production batches and different steps, which introduce the variation sources inherent in such a process. A global and sole model was found to determine the active compound during the production cycle, with errors of prediction less than 1.8% in all cases. Tablets can be individually analysed with high accuracy and precision, so a content uniformity analysis may be performed.