A novel 22bp deletion in a Tunisian phenylketonuria family.

Phenylketonuria (PKU) is an autosomal recessive metabolic disorder caused by a deficiency of phenylalanine hydroxylase. To date, more than 530 mutations in the PAH gene have been reported. In Tunisia, this disease seems to be the result of point mutations, few studies have been published about molecular defects of PKU in our country. In this study, we report a novel deletion in exon 6 of two brothers in a Tunisian family after DHPLC analysis and sequencing of the exon 6 of the PAH gene.

Continuous direct compression: Development of an empirical predictive model and challenges regarding PAT implementation.

In this study, an empirical predictive model was developed based on the quantitative relationships between blend properties, critical quality attributes (CQA) and critical process parameters (CPP) related to blending and tableting. The blend uniformity and API concentration in the tablets were used to elucidate challenges related to the processability as well as the implementation of PAT tools. Thirty divergent ternary blends were evaluated on a continuous direct compression line (ConsiGma™ CDC-50). The trials showed a significant impact of the impeller configuration and impeller speed on the blending performance, whereas a limited impact of blend properties was observed. In contrast, blend properties played a significant role during compression, where changes in blend composition significantly altered the tablet quality. The observed correlations allowed to develop an empirical predictive model for the selection of process configurations based on the blend properties, reducing the number of trial runs needed to optimize a process and thus reducing development time and costs of new drug products. Furthermore, the trials elucidated several challenges related to blend properties that had a significant impact on PAT implementation and performance of the CDC-platform, highlighting the importance of further process development and optimization in order to solve the remaining challenges.

In-depth analysis of the long-term processability of materials during continuous feeding.

This study determined the feasibility of long-term continuous powder feeding and its effect on the overall process performance. Additionally, quantitative relationships were established between material properties, process settings and screw feeding responses during gravimetric feeding. Twelve divergent raw materials were processed over longer periods using a GEA Compact Feeder integrated in a continuous direct compression line (ConsiGma™ CDC-50). The resulting gravimetric feeding responses were combined with the material properties and process settings into an overall PLS model. The model elucidated the impact of the material descriptors for density; powder flow; particle size; compressibility; permeability and wall friction angle on the feeding process. Furthermore, long-term processing of the materials exhibited challenges related to the processability and refill consistency where a significant impact of the compressibility and cohesive/adhesive properties of the materials was observed. Overall, this approach provided insights into the feasibility of long-term continuous feeding which is not possible through 'short-term' feeding trials. Additionally, throughout this study, the need for material-specific adjustments of the feeding and refill equipment was highlighted.

Determination of a quantitative relationship between material properties, process settings and screw feeding behavior via multivariate data-analysis.

In this study, a quantitative relationship between material properties, process settings and screw feeding responses of a high-throughput feeder was established via multivariate models (PLS). Thirteen divergent powders were selected and characterized for 44 material property descriptors. During volumetric feeder trials, the maximum feed capacity (FCCmax), the relative standard deviation on the maximum feed capacity (RSDFCmax), the short term variability (STRSD) and feed capacity decay (FCdecay) were determined. The gravimetric feeder trials generated values for the mass flow rate variability (RSDLC), short term variability (STRSD) and refill responses (Vrefill and RSDrefill). The developed PLS models elucidated that the material properties and process settings were clearly correlated to the feeding behavior. The extended volumetric feeder trials pointed out that there was a significant influence of the chosen screw type and screw speed on the feeding process. Furthermore, the process could be optimized by reducing the feeding variability through the application of optimized mass flow filters, high frequency vibrations, independent agitator control and optimized top-up systems. Overall, the models could allow the prediction of the feeding performance for a wide range of materials based on the characterization of a subset of material properties greatly reducing the number of required feeding experiments.

TPLS as predictive platform for twin-screw wet granulation process and formulation development.

In recent years, the interest in continuous manufacturing techniques, such as twin-screw wet granulation, has increased. However, the understanding of the influence of the combination of raw material properties and process settings upon the granule quality attributes is still limited. In this study, a T-shaped partial least squares (TPLS) model was developed to link raw material properties, the ratios in which these raw materials were combined and the applied process parameters for the twin-screw wet granulation process with the granule quality attributes. In addition, the predictive ability of the TPLS model was used to find a suitable combination of formulation composition and twin-screw granulation process settings for a new API leading to desired granule quality attributes. Overall, this study helped to better understand the link between raw material properties, formulation composition and process settings on granule quality attributes. Moreover, as TPLS can provide a reasonable starting point for formulation and process development for new APIs, it can reduce the experimental development efforts and consequently the consumption of expensive (and often limited available) new API.

[Reliability of immunoturbidimetric urine albumin determination for normal and moderately high values]. / Fiabilité du dosage immunoturbidimétrique de l'albumine urinaire pour les valeurs normales ou modérément élevées.

The value of albuminuria determination is commonly accepted in the monitoring of renal function, particularly in diabetic patients. This may be achieved in different ways and using different assay kits. The aim of this study was to assess the reliability of immunoturbidimetric measurements by comparing the results obtained by two different methods in our laboratory. Therefore we measured albuminuria of 54 patients monitored for their diabetes, with normal to moderately high albuminuria, according to two methods: Microalbumin reagent (Beckman Coulter) on a Unicel DxC 600 (Beckman Coulter) and Albumin Turbiquant reagent (Dade Behring, Siemens) on a Turbitimer system (Dade Behring, Siemens). It was then possible to study the correlation between the two techniques. Our results suggest that although this correlation is excellent, the values differ significantly: the results observed with the Siemens method are higher by about 10%. It seems legitimate to think that a correction should be applied, depending on the used method. In addition, it appears that standardization is necessary, concerning analytical stage but although urine collection, pre-analytical stage or reporting and interpretation of results.

A multivariate approach to predict the volumetric and gravimetric feeding behavior of a low feed rate feeder based on raw material properties.

In this study, the volumetric and gravimetric feeding behavior of 15 pharmaceutical powders on a low feed rate feeder was correlated with their material properties through a multivariate approach. The powders under investigation differ substantially in terms of material properties, making the selected powders representative for powders typically used in pharmaceutical manufacturing. The material properties were described by 25 material property descriptors, obtained from a rational selection of critical characterization techniques that provided maximal information with minimal characterization effort. From volumetric feeding experiments (i.e., powder feed rate not controlled), the maximum feeding capacity (maximum feed factor (FFmax)) and optimal hopper fill level at which the feeder should be refilled during gravimetric feeding (feed factor decay (FFdecay)) were obtained. During gravimetric feeding experiments (i.e., powder feed rate controlled), the variability on the feed rate (relative standard deviation (RSD)) and the difference between the setpoint and mean feed rate (relative error (RE)) were determined. Partial least squares (PLS) regression was applied to correlate the volumetric and gravimetric feeding responses (Y) with the material property descriptors (X). The predictive ability of the developed PLS models was assessed by predicting the feeding responses of two new powders (i.e., validation set). Overall, the volumetric feeding responses (FFmax and FFdecay) were predicted better than the gravimetric feeding responses (RSD and RE), since in gravimetric mode the impact of material properties on the feeding behavior is reduced due to the control system of the feeder. Especially RE was weakly correlated with material properties as RE of most powders varied around zero with only a small numerical variation. Interestingly, this confirms that the control system is working properly and that the feeder is capable of feeding different powders accurately at low feed rates. The developed models allowed to predict the feeding behavior of new powders based on their material properties. Consequently the number of feeding experiments during process development can be greatly reduced, thereby leading to a more efficient and faster development of new drug products.

Impact of material properties and process variables on the residence time distribution in twin screw feeding equipment.

Screw feeders are integrated as dispensing units in most continuous manufacturing platforms. Hence, characterizing and modelling the residence time distribution (RTD) of materials in feeders is indispensable to understand the traceability of raw materials from the drum till tablet, enabling the separation of non-confirming material. The proposed methodology addressed this leap in knowledge by characterizing materials, performing RTD trials according to an experimental design, applying RTD models and establishing a partial least square (PLS) regression model that links the material properties and process variables with the RTD responses as outputs. Results showed that RTD in screw feeders can be represented by a combination of plug-flow and mixed-flow. Three variables were found to impact the residence time distribution in feeders: flow rate, hopper level and conditioned bulk density. Interestingly, the plug-flow fraction was not affected by variation in flow rate or material properties. Consequently, simple PLS models could be developed that use density and flow rate to predict RTD at a given hopper level. This approach is powerful for RTD prediction based on bulk density in the early phases of development when control strategies for clinical manufacturing need to be established and material availability is still limited.

[Prematurity and neonatal screening]. / Prématurité et dépistage néonatal.

Neonatal screening for metabolic and endocrine diseases has clear benefits for full-term infants by providing early diagnosis and treatment to prevent or limit the severe adverse outcomes which occur in untreated infants. The situation of preterm infants is more complicated, with an increased risk of false-positive as well as false-negative screening results. The aim of this work was to evaluate the impact of prematurity in screening strategies in order to answer practical questions: is there a need or not to adjust cut-off values in accord to gestational parameters? Is there a need or not to repeat screening tests at a later age?

Impact of blend properties on die filling during tableting.

Based on characterization of a wide range of fillers and APIs, thirty divergent blends were composed and subsequently compressed on a rotary tablet press, varying paddle speed and turret speed. The tablet weight variability was determined of 20 grab samples consisting of each 20 tablets. Additionally, the bulk residence time, ejection force, pre-compression displacement, main compression force, die fill fraction and feed frame fill fraction were determined during each run. Multivariate data analysis was applied to investigate the relation between the process parameters, blend characteristics, product and process responses. Blends with metoprolol tartrate as API showed high ejection forces. This behavior could be linked to the high wall friction value of metoprolol tartrate. The main responses related to the die filling could be predicted via a PLS model based on blend characteristics. Tablet weight variability was highly correlated with the variability on pre-compression displacement and main compression force. A good predictive model for tablet weight variability was obtained taking the porosity, wall friction angle, flowability, density, compressibility and permeability into account. Additionally, turret speed and paddle speed were included in the calibration of the model. The applied approach can save resources (material, time) during early drug product development.

First ISNS Reference Preparation for Neonatal Screening for thyrotropin, phenylalanine and 17alpha-hydroxyprogesterone in blood spots.

BACKGROUND: Neonatal screening for congenital disorders like phenylketonuria (PKU), congenital hypothyroidism (CH) and congenital adrenal hyperplasia (CAH) is generally performed in dried blood spots on filter paper. The analytes of interest for testing for PKU, CH and CAH are phenylalanine, thyrotropin (TSH) and 17alpha-hydroxyprogesterone (17OHP), respectively. The International Society for Neonatal Screening (ISNS) decided to prepare a combined reference preparation for the three analytes on filter paper Schleicher & Schuell #903, Whatman BFC180 and Toyo Roshi 545. This 'First ISNS Reference Preparation for Neonatal Screening for TSH, phenylalanine and 17OHP in blood spots' (1st ISNS-RPNS) has been prepared by the RIVM (Bilthoven). METHOD: The number of filter paper cards prepared, each with two sets of six blood spot calibrators, was 480, 42 and 69 for Schleicher & Schuell #903, Whatman BFC180 and Toyo Roshi 545, respectively. The volume of blood dispensed was 50 microl. The range of concentrations for TSH was 1-121 mIU/L blood, for phenylalanine 65-865 micromol/L blood and for 17OHP 2.2-302 nmol/L blood. RESULTS: The linearity of the blood spot calibrators and the homogeneity of the batch (only tested for Schleicher & Schuell) were good. The differences between the three filter papers were small: i.e. the potency of the ISNS-RPNS on Whatman and Toyo Roshi in terms of Schleicher & Schuell was between 0.98 and 1.09 for the three analytes. CONCLUSION: The 1st ISNS-RPNS for TSH, phenylalanine and 17OHP can be said to be suitable as formal reference preparation and as a source for (re)calibrating kit calibrators.

[Interest of Prom-Test in the follow-up of premature rupture of membranes]. / Place du Prom-Test dans la prise en charge de la rupture prématurée des membranes.

The follow-up of premature rupture of membranes requires an hospitalization. The aim of this work was to evaluate the interest of the Prom-Test in the action to be taken in the case of a suspicion of premature rupture of membranes, a negative result allowing to not hospitalize the patient. 105 tests have been carried out producing 73 negative results, 23 positive and 9 doubtful. The childbirth, started or not, occurred much more quickly when the test is positive. Only 13 of the 73 negative tests resulted in a hospitalization including 11 for beginning of delivery. A correct use of the test can prevent unnecessary days of hospitalization.

[Hyperhomocysteinaemia and type 2 diabetes]. / Hyperhomocystéinémie et diabète de type 2.

OBJECTIVES: Hyperhomocysteinaemia is associated with cardiovascular events in nondiabetic individuals. The present study was aimed to explore the implication of hyperhomocysteinemia in development of cardiovascular events in patients with type 2 diabetes. DESIGN: A total of 185 patients with type 2 diabetes (115 women and 70 men, 30 to 93 years of age) have been included consecutively in the ambulatory unit at the Saint-Philibert Hospital. For each patient the concentration of homocysteine, cholesterol and triglyceride levels and HbA1c have been measured. In the studied population, 121 patients presented cardiovascular events (myocardial infarctus, peripheric arteriopathy, cerebrovascular accident). RESULTS: The patients with cardiovascular events were older, the concentration of homocysteine and creatinine were higher. The plasma homocysteine levels adjusted for age and creatinine levels were higher in patients with cardiovascular events than in patients without cardiovascular events (15.4 +/- 3.52 micromol/L and 13.13 +/- 2.26 micromol/L respectively; p = 2. 10(-5)). CONCLUSIONS: Hyperhomocysteinemia is an independant risk factor for cardiovascular events in type 2 diabetes, independent of age and renal function.

Developmental aspect of phenylalanine hydroxylase in the rat -- hormonal influences.

Interpretations of the development of phenylalanine hydroxylase (PAH) in rat liver have been controversial, and the mechanism of ontogenic changes have not yet been elucidated. Fetal PAH activity at a gestational age of 21 days appeared to reach 32% that of adult male level at birth. The in vivo effectiveness of fetal PAH activity was correlated with enhancement of blood tyrosine, while amino-transferase activity appeared only after birth. No sex difference was noted in weaning rats, whereas, in adult females, PAH activity was only 42% that of males. Investigating hormonal influences on liver PAH activity we noted no change of enzyme activity following hydrocortisone, ACTH and estradiol treatment. However, 4 days of testosterone treatment in weaning female rats increased PAH activity (X1.7). Therefore, testosterone could explain increased PAH activity in adult males.

Phenylalanine analogues as inhibitors of phenylalanine-hydroxylase from rat liver. New conclusions concerning kinetic behaviors of the enzyme.

The conversion of phenylalanine to tyrosine is catalysed by phenylalanine-hydroxylase. The substrate phenylalanine shows two effects: (1) allosteric transition at low phenylalanine concentrations, (2) excess substration inhibition. The molecular structure of phenylalanine-hydroxylase has not yet been elucidated. However, a tetrameric structure has been proposed. The Kinetic analysis with respect to substrate analogues suggest the existence of three types of sites on each protomer: (1) a catalytic site, (2) a non-competitive inhibitory site, (3) a positive cooperative site. Use of the enzyme's natural cofactor, tetrahydrobiopterin, has been emphasized to ensure good interpretation of the kinetic results of the phenylalanine-hydroxylase effectors.

A new experimental model of hyperphenylalaninemia in rat. Effect of p-chlorophenylalanine and cotrimoxazole.

A new experimental model of hyperphenylalaninemia was proposed. Combination of p.chlorophenylalanine, strongly inhibitor of phenylalanine hydroxylase, and cotrimoxazole, presumably inhibitor of dihydropteridine reductase, produced a good inhibition of phenylalanine hydroxylation in vivo. Thus phenylalaninemia reached values similar to those found in PKU patients, without administration of excess phenylalanine. Tyrosine concentrations remained near the control values and a phenylketonuria occurred.

[Chemistry of urinary mannosides excreted in mannosidosis]. / Etude chimique des 'mannosides urinaires excrétés au cours de la mannosidose

Mannose-rich oligosaccharides have been isolated from urines of 5 patients with mannosidosis. Their compositon and structure were determined. Three of them have been previously described by Norden et al: alpha p-Manp-(1 leads to 3) beta-d-Manp-(1 leads to 4) d-GlcNAcp; alpha-p-Manp-(1 leads to 2), alpha-d-Manp-(1 leads to 3) beta-d-Manp-(1 leads to 4) d-GlcNAc and alpha-d-Manp-(1 leads to 2) alpha-d-Manp-(1 leads to 2) alpha-d-Manp-(1 leads to 3) beta-d-Manp-(1 leads to 4) d-GlcNAcp, but the four others are new entities: alpha-d-Manp-(1 leads to 3) (alpha-d-Manp-(1 leads to 2) alpha-d-Manp-(1 leads to 2) alpha-d-Manp-(1 leads to 6) beta-d-Manp-(1 leads to 4) GlcNAcp; alpha-d-Manp-(1 leads to 2) alpha-d-Manp-(1 leads to 3) (alpha-d-Manp-(1 leads to 2) alpha-d-Manp-(1 leads to 6) beta-d-Manp-(1 leads to 4) GlcNAcp; alpha-d-Manp-(1 leads to 2), alpha-d-Man-(1 leads to 3) (alpha-d-Manp-(1 leads to 6) beta-d-Manp-(1 leads to 4) GlcNAp and alpha-d-Manp-(1 leads to 2) alpha-d-Manp-(1 leads to 3) (alpha-d-Manp-(1 leads to 6) beta-d-Manp-(1 leads to 4) GlcNAcp. These structures are related to the glycans of "oligomannosidic type" present in numerous glycoproteins. All possess a N-acetylglucosamine residue in terminal reducing position and reinforce the hypothesis of Kobata et al. and Montreuil et al. that catabolism of glycans N-glucosidically linked to the protein moiety begins by the aciton of a beta-endo-N-acetylglucosaminidase.

Unconjugated pteridines in bronchoalveolar lavage as indicators of alveolar macrophage activation.

Bronchoalveolar lavage (BAL) has shown great efficacy in clarifying the role of immune processes in many disorders of the lower respiratory tract. Following the in vitro demonstration that neopterin is an indicator of the activation of macrophages, neopterin was measured in the BAL fluid and cells from patients with various pulmonary diseases. In most of the patients, high levels of neopterin were found in the serum, BAL fluid and BAL cells. Because neopterin in BAL fluid results from local production as well as from plasma transudation, neopterin in BAL cells seems to reflect the macrophage stimulation more directly. In addition, the correlation between cellular neopterin and lymphocyte count was found to be more significant than the correlation between cellular neopterin and macrophage count. Neopterin in BAL fluid and cells may be a useful measurement in the investigation and elucidation of pulmonary pathologies involving the cellular immune system.

Cerebrospinal fluid neopterin levels in children with central nervous system leukemia.

Cerebrospinal fluid (CSF) neopterin levels were determined by high-pressure liquid chromatography in 48 normal children and in 15 children with meningeal relapse of hematologic malignancies (13 acute lymphoblastic leukemia and 2 high-grade lymphomas). When meningeal relapse was diagnosed, all patients had CSF neopterin levels higher than mean normal value +2 standard deviations. No significant correlation between the blast count in the CSF and neopterin levels was observed. CSF data before relapse were available in 10 children: the neopterin values at relapse were significantly higher than values observed at diagnosis. In 3 patients, elevated neopterin levels preceded the occurrence of neurologic signs and the detection of blast cells in CSF by 15 to 30 days. In the absence of infection, the rise of CSF neopterin levels in patients with hematologic malignancies indicates an active phase of the disease. This could reflect a cell-mediated immunologic process induced by malignant cells. The measurement of CSF neopterin should be helpful in the monitoring of patients to detect early meningeal relapse.

Unconjugated pteridines in human milk.

Defective de novo synthesis of tetrahydrobiopterin (BH4) has been reported as being responsible for neonatal hyperphenylalaninemia. The presence of BH4 and its derivatives in human milk is confirmed but the vitamin-like effectiveness of the BH4 supplied by human milk appeared doubtful.