Prediction of plasma concentrations using in silico modelling and simulation approach: Case of Acebutolol.

PURPOSE: The aim of this study was to predict the plasma concentrations of acebutolol tablets with different dissolution profiles using computer modelling and evaluating whether they are bioequivalent using simulated population studies. METHODS: The dissolution behaviour of acebutolol was studied in the USP Apparatus-II using different dissolution media for pH 1.2, 4.5, and 6.8 at 37±0.5°C. The obtained dissolution data, as well as plasma concentration-time data of the reference product from the literature were used as inputs to build pharmacokinetic model of acebutolol within GastroPlus™ software (version 9.7, Simulations Plus Inc., Lancaster, CA, USA) to simulate the in vivo profiles of the drug. RESULTS: The dissolution profiles of the reference product Sectral® 400mg tablets and a locally produced generic product were>85% in 15min in three dissolution media. Simulation results demonstrated that the brand and generic products would show the same in vivo performance. Population simulation results of the ln-transformed 90% confidence interval for the ratio of Cmax, AUC0-t and AUC0-inf values for the two products were within the 80-125% interval, showing to be bioequivalent. CONCLUSION: Based on the in vitro results combined with in silico simulations using GastroPlus™, a biowaiver for immediate release acebutolol tablets is justified. Furthermore, computer modelling has shown to be a very intersting tool to prove the bioequivalence for these products.

[Quality control of mixtures for pediatric parenteral nutrition: Validation of sodium and potassium assay method]. / Contrôle qualité des mélanges pour nutrition parentérale pédiatriques : validation de la méthode de dosage du sodium et du potassium.

OBJECTIVES: Control of electrolyte concentration in mixtures for parenteral nutrition (MPN) for newborns is crucial before the release of the final product. We aimed to assess the validation of the electrolytes assay in MPN. METHODS: Electrolytes assay was performed with Ilyte Analyzer®. Validation of method was realized in accordance with ICH (International Conference on Harmonization) guideline Q2(R1) and the commission report of the French society of pharmaceutical science and technology. Linearity test solutions were prepared in triplicate using five levels of concentrations for sodium and potassium (60-140% of theoretical concentrations). Accuracy of the method was deducted from the same results of linearity. The intermediate precision was ensured by dosing the main electrolyte in six MPN, during three successive days. RESULTS: Linearity was assessed with correlation coefficients greater than 0.996 for both electrolytes. A non-significant result of comparison test of the intercept with zero (Student test) was obtained. A highly significant result of the test of existence of slopes (Fisher test) proved a linear regression for the 2 electrolytes (P<0.1%). Inter-day precision values were 2.68% and 2.65% respectively for sodium and potassium. CONCLUSION: The validation of sodium and potassium assay method was successfully performed with Ilyte Analyzer® allowing routine quality control in MPN.

[Preparation of citrulline microspheres by spray drying technique for colonic targeting]. / Préparation des microsphères de citrulline à ciblage colique par la technique de spray drying.

INTRODUCTION: Citrulline is an amino acid that becomes essential in situations of intestinal insufficiency such as short bowel syndrome. It is therefore interesting to provide the patients with dosage forms for routing citrulline to the colon. The aim of this work is to formulate microspheres of citrulline for colonic targeting by the technique of spray drying. MATERIAL AND METHODS: Eudragit(®) FS 30D was selected as polymer to encapsulate citrulline using the spray drying technique. Citrulline and Eudragit(®) FS 30D were dissolved in water and ethanol, respectively. The aqueous and the ethanolic solutions were then mixed in 1:2 (v/v) ratio. Microspheres were obtained by nebulizing the citrulline-Eudragit(®) FS 30D solution using a Mini spray dryer equipped with a 0.7mm nozzle. The microspheres have been formulated using citrulline and Eudragit(®) FS 30D. The size distribution of microspheres was determined by light diffraction. The morphology of the microspheres was studied by electron microscopy. Manufacturing yields, encapsulation rate and dissolution profiles were also studied. RESULTS AND DISCUSSION: The microspheres obtained had a spherical shape with a smooth surface and a homogeneous size except for the microspheres containing the highest concentration of polymer (90 %). The formulation showed that the size and morphology of the microspheres are influenced by the polymer concentration. Manufacturing yields were about 51 % but encapsulation rate were always very high (above 90 %). The in vitro dissolution study showed that the use of the Eudragit(®) FS 30D under these conditions is not appropriate to change the dissolution profile of the citrulline. CONCLUSION: This technique has led to the formulation of microspheres with good physical properties in terms of morphology and size. The compression of the microspheres should help to control citrulline release for colonic targeting.

Morphology, seasonality and phylogeny of Zschokkella soleae sp. n. (Myxozoa, Myxosporea) parasite of Solea solea (L.) (Pleuronectiformes, Soleidae) from Ghar El Melh Lagoon, Tunisia.

A new Myxosporea, Zschokkella soleae sp. n., was found in the gall bladder and the bile of common sole, Solea solea (L.), from Ghar El Melh Lagoon in north-east Tunisia. This is the first record for the presence of Zschokkella species in Tunisian waters. The parasite's plasmodia are polysporic with variable size and shape. Some plasmodia appeared attached to the gall bladder epithelium while others were found free in bile. Mature spores are ellipsoidal in frontal view 13.8±0.38 µm long and 10.86±0.40 µm wide with two equal size spherical polar capsules 3.6±0.43 µm in size. The prevalence of infection seems to correlate with host size and changes over the year with maximum percentage in summer. Based on the 18S rDNA sequence data, Z. soleae sp. n. is readily distinguishable from other myxozoan DNA sequences in GenBank. Phylogenetically, the new species is placed in the freshwater Myxidium clade including several Zschokkella spp. infecting the gall bladder. Morphology, histology as well as DNA sequence analysis indicate that the examined species differs from all previously described Zschokkella species.

[Evaluation of urinary metanephrines for the diagnosis of pheochromocytoma]. / Evaluation du dosage des métanéphrines urinaires dans le diagnostic du phéochromocytome.

OBJECTIVE: evaluate the assay of urinary metanephrines in diagnosis of pheochromocytoma (PH) and determine diagnostic cut-off values. METHODS: this is a retrospective study about 87 patients suspected of pheochromocytoma,whose of 24-h urinary fractionated metanephrine was measured. These cases were collected from Internal Medecine Departments (A and B) at Charles Nicolle's Hospital. Two groups of patients were studied: a pheochromocytoma group (n=33) with a histologically-proven pheochromocytoma and a control group of 54 patients. Receiver Operating Characteristic (ROC) curves were used to determine the best sensitivities and specificities. RESULTS: The analysis of biological parameters showed that means and standard deviation of urinary fractionated metanephrines in pheochromocytoma group were significantly higher than those of control group. Sensitivity and specificity of urinary normetanephrine test (95% and 98.1% respectively) were higher than those of urinary metanephrine and 3-methoxytyramine. A correlation between urinary normetanephrine and tumor size of pheochromocytoma was found. CONCLUSION: Urinary fractionated metanephrines is an efficient biochemical test for the diagnosis of pheochromocytoma.

Aerobic training programs and glycemic control in diabetic children in relation to exercise frequency.

AIM: The aim of this study was to assess the effect of an aerobic training on glycemic control and lipid profile in diabetic children. METHODS: Thirty-three children with type 1 diabetes mellitus (T1DM) were equally divided in three groups. The control group (CG) did not perform any training intervention during the period of the investigation and the subjects were instructed to continue with their daily lifestyle patterns. The second group (G1) and the third group (G2) completed a six-month aerobic training programs. During the period of the investigation, G1 participated in one daily session (~60 min) of aerobic exercise twice weekly, while G2 performed in the form of daily session four times a week (~60 min). HbA1c and lipid profile were measured before training intervention, then after three and six months. RESULTS: The three-month periods was without significant changes of HbA1c in the two groups but significantly increased high-density lipoprotein cholesterol (HDL-C) in G1 (P<0.05). In G2, an increase of HDL-C was accompanied by a decrease of serum triglyceride (TG) (P<0.05). After six months, only HDL-C and TG levels were significantly lower in G1 (P<0.01), while in G2 HDL-C increased (68.8±5.7 mg/dL vs. 56.7 ±7.2 mg/dL; P<0.01), low-density lipoprotein cholesterol (LDL-C) (69.2±8.5 mg/dL vs. 81.6±11.8 mg/dL; P<0.01), TG concentrations decreased (60.7±6.9 mg/dL vs. 77.4±9.3 mg/dL; P<0.01) and HbA1c was significantly lower (6.8±1.1% vs. 8.2±1.5%; P<0.05), comparatively to those before training. CONCLUSION: However, the six-month periods showed that children exercising more than two times weekly significantly improved HbA1c and lipid profile. These results must encourage children with T1DM to regularly practice sporty activities for long periods.

The Drosophila bifocal gene encodes a novel protein which colocalizes with actin and is necessary for photoreceptor morphogenesis.

Photoreceptor cells of the Drosophila compound eye begin to develop specialized membrane foldings at the apical surface in midpupation. The microvillar structure ultimately forms the rhabdomere, an actin-rich light-gathering organelle with a characteristic shape and morphology. In a P-element transposition screen, we isolated mutations in a gene, bifocal (bif), which is required for the development of normal rhabdomeres. The morphological defects seen in bif mutant animals, in which the distinct contact domains established by the newly formed rhabdomeres are abnormal, first become apparent during midpupal development. The later defects seen in the mutant adult R cells are more dramatic, with the rhabdomeres enlarged, elongated, and frequently split. bif encodes a novel putative protein of 1063 amino acids which is expressed in the embryo and the larval eye imaginal disc in a pattern identical to that of F actin. During pupal development, Bif localizes to the base of the filamentous actin associated with the forming rhabdomeres along one side of the differentiating R cells. On the basis of its subcellular localization and loss-of-function phenotype, we discuss possible roles of Bif in photoreceptor morphogenesis.

Interaction with protein phosphatase 1 Is essential for bifocal function during the morphogenesis of the Drosophila compound eye.

The gene bifocal (bif), required for photoreceptor morphogenesis in the Drosophila compound eye, encodes a protein that is shown to interact with protein phosphatase 1 (PP1) using the yeast two-hybrid system. Complex formation between Bif and PP1 is supported by coprecipitation of the two proteins. Residues 992 to 995 (RVQF) in the carboxy-terminal region of Bif, which conform to the consensus PP1-binding motif, are shown to be essential for the interaction of Bif with PP1. The interaction of PP1 with bacterially expressed and endogenous Bif can be disrupted by a synthetic peptide known to block interaction of other regulatory subunits with PP1. Null bif mutants exhibit a rough eye phenotype, disorganized rhabdomeres (light-gathering rhodopsin-rich microvillar membrane structures in the photoreceptor cells) and alterations in the actin cytoskeleton. Expression of wild-type bif transgenes resulted in significant rescue of these abnormalities. In contrast, expression of transgenes encoding the Bif F995A mutant, which disrupts binding to PP1, was unable to rescue any aspect of the bif phenotype. The results indicate that the PP1-Bif interaction is critical for the rescue and that a major function of Bif is to target PP1c to a specific subcellular location. The role of the PP1-Bif complex in modulating the organization of the actin cytoskeleton underlying the rhabdomeres is discussed.

Antiproliferative and antitumor effects of alpha-interferon in renal cell carcinomas: correlation with the expression of a kidney-associated differentiation glycoprotein.

Human leukocyte alpha-interferon (IFN-alpha) has significant antitumor activity in advanced renal cell carcinoma (RC), with approximately 15% (range, 5 to 29%) of patients subjected to IFN-alpha therapy exhibiting a major objective response. We assayed 16 RC cell lines for intrinsic sensitivity to the growth-inhibitory effects of recombinant IFN-alpha. Similar to results observed in patients, cultured RCs could be divided into those that are inhibited by IFN-alpha and those that are not. In addition, the IFN-alpha-sensitive or -resistant phenotype of cultured RCs was correlated with surface expression of six unrelated kidney-associated differentiation antigens. The expression of one antigen, a Mr 160,000 glycoprotein (gp160), was found to correlate with resistance to IFN-alpha. Proliferation of seven RC cell lines expressing gp160 (gp160+) was not significantly inhibited by IFN-alpha at concentrations as high as 3000 units/ml. In contrast, proliferation of eight of nine RC cell lines lacking expression of gp160 (gp160-) was markedly inhibited by IFN-alpha. The effect of IFN-alpha on gp160+ and gp160- RC xenografts in nu/nu mice was examined. In separate experiments, two gp160+ RC cell lines and five gp160- RC cell lines were injected s.c. into nu/nu mice; one half of the mice were subsequently treated with 10(6) units of IFN-alpha i.p. 3 times a wk, and one half received no IFN-alpha. Tumors appeared at the sites of inoculation in all mice given injections of gp160+ RC cell lines within 10 to 25 days regardless of INF-alpha therapy. Mice given injections of gp160- RC cell lines, but not receiving IFN-alpha, also formed tumors. In contrast, gp160- RC cell lines injected into mice that were treated with IFN-alpha exhibited a marked sensitivity, as demonstrated by either no tumor formation or delayed tumor formation. We conclude that the absence of gp160 expression by RCs may be predictive of sensitivity to the antitumor effects of IFN-alpha and, thus, provide a basis for identifying IFN-responsive patients.

The Drosophila homolog of human AF10/AF17 leukemia fusion genes (Dalf) encodes a zinc finger/leucine zipper nuclear protein required in the nervous system for maintaining EVE expression and normal growth.

Sibling neurons in the embryonic central nervous system (CNS) of Drosophila can adopt distinct states as judged by gene expression and axon projection. In the NB4-2 lineage, two even-skipped (eve)-expressing sibling neuronal cells, RP2 and RP2sib, are formed in each hemineuromere. Throughout embryogenesis, only RP2, but not RP2sib, maintains eve expression. In this report, we describe a P-element induced mutation that alters the expression pattern of EVE in RP2 motoneurons in the Drosophila embryonic CNS. The mutation was mapped to a Drosophila homolog of human AF10/AF17 leukemia fusion genes (alf), and therefore named Dalf. Like its human counterparts, Dalf encodes a zinc finger/leucine zipper nuclear protein that is widely expressed in embryonic and larval tissues including neurons and glia. In Dalf mutant embryos, the RP2 motoneuron no longer maintains EVE expression. The effect of the Dalf mutation on EVE expression is RP2-specific and does not affect other characteristics of the RP2 motoneuron. In addition to the embryonic phenotype, Dalf mutant larvae are retarded in their growth and this defect can be rescued by the ectopic expression of a Dalf transgene under the control of a neuronal GAL4 driver. This indicates a requirement for Dalf function in the nervous system for maintaining gene expression and the facilitation of normal growth.

Sequence of the Streptomyces thermoviolaceus CUB74 alpha-amylase-encoding gene and its transcription analysis in Streptomyces lividans.

The alpha-amylase (Amy)-encoding gene (amy) of Streptomyces thermoviolaceus CUB74, previously cloned in Escherichia coli and S. lividans and localised on a 1.7-kb BamHI-SphI genomic DNA fragment, has been sequenced. A single open reading frame of 1380 bp, which could encode an Amy protein of 460 amino acids (aa), was identified. The deduced aa sequence of the thermophilic Amy is similar (up to 69.5%) to the mesophilic Amy of S. griseus, S. limosus, S. venezuelae and S. hygroscopicus. A 40% sequence similarity was found between the extracellular forms of the S. thermoviolaceus and the pig pancreatic Amy. In addition, the activity of the S. thermoviolaceus Amy is strongly inhibited by tendamistat, a potent inhibitor of mammalian Amy. The nucleotide sequence at the 5' end of amy was able to initiate transcription in S. lividans and contains a promoter whose sequence is identical to the promoters of the S. limosus, S. venezuelae and S. griseus amy.

Drosophila DPP2C1, a novel member of the protein phosphatase 2C (PP2C) family.

We report the molecular cloning, chromosome mapping and developmental transcription pattern of a putative serine/threonine protein phosphatase 2C (PP2C), DPP2C1, from Drosophila melanogaster. The 6-kb transcript of this first Drosophila PP2C gene encodes a 1428-aa deduced protein. The DPP2C1 protein contains a approximately 330-aa PP2C-like catalytic domain flanked by extensive N- and C-terminal sequences showing no similarities to other PP2Cs. The dpp2c1 gene maps to 4E1-2 on the X chromosome, 1.5 kb upstream of the ddlc1 gene. Northern blot analyses showed that dpp2c1 transcription is developmentally regulated, accumulating maximally during early (0-6 h) and late (12-24 h) embryogensis. The presented molecular characterisation provides the basis for a genetic dissection of DPP2C1 function.

Regulation of a thermostable alpha-amylase of Streptomyces thermoviolaceus CUB74: maltotriose is the smallest inducer.

We have examined induction and repression by various sugars and carbon sources of the synthesis of a thermostable alpha-amylase in its natural host, S thermoviolaceus CUB74. The smallest molecule capable of inducing synthesis of the enzyme was maltotriose whereas maltose had no effect which might suggest a different control system from that found in other streptomycete amylases. Addition of mannitol to the growth medium impeded the alpha-amylase induction whereas glucose had no effect. After cloning of its gene into a new streptomycete host, S lividans TK24, the S thermoviolaceus alpha-amylase could not be induced by any of the sugars tested.

Chemoradiotherapy in non-small cell lung cancer: paclitaxel/carboplatin/radiotherapy in regionally advanced disease.

Based on superior results observed with combined-modality therapy over radiotherapy alone and on the authors' previous work with carboplatin and standard daily thoracic radiotherapy in patients with advanced, unresectable non-small cell lung cancer, a phase II study was designed to incorporate radiosensitizing doses of paclitaxel (Taxol; Bristol-Myers Squibb Company, Princeton, NJ) into the carboplatin/radiotherapy regimen, to improve the therapeutic index and define the toxicities. Thirty-two patients have been entered. Paclitaxel 45 mg/m2/wk was administered over 3 hours prior to carboplatin (100 mg/m2/wk) and thoracic radiotherapy (1.8 Gy/d 5 days a week; total dose, 60 to 65 Gy). No grade 4 toxicities occurred. Seven patients had chemotherapy doses delayed because of grade 3 neutropenia, and one patient had grade 3 mucositis/esophagitis that required hospitalization. Median survival has not yet been reached, and all patients are being followed. These preliminary data demonstrate the feasibility of combined concurrent chemoradiotherapy, with acceptable toxicities. Further investigation is needed to optimize carboplatin dosage with adaptive control using formulas based on pharmacokinetics and pharmacodynamics. Full-dose induction chemotherapy regimens to maximize the systemic effects of chemotherapy should precede concurrent chemoradiotherapy in future studies.

Protection against herpes simplex virus-induced eye disease after vaccination with seven individually expressed herpes simplex virus 1 glycoproteins.

PURPOSE: To compare the efficacy of each of seven expressed herpes simplex virus 1 (HSV-1) glycoproteins as vaccines to protect against ocular disease after primary ocular HSV-1 infection. METHODS: Mice were vaccinated three times with equal amounts of each of seven individually expressed HSV-1 glycoproteins (gB, gC, gD, gE, gG, gH, and gI) and then ocularly challenged with McKrae, a corneal disease-producing strain of HSV-1. Viral clearance from the eye, blepharitis, keratitis, and neovascularization were determined at various times after infection. RESULTS: Mice vaccinated with gD or gB had the best protection against eye disease. Vaccination with gI, gC, or gE produced moderate protection against eye disease. Vaccination with gG produced less protection, and vaccination with gH produced no apparent protection against eye disease. CONCLUSIONS: These results suggest that when used as vaccines, different HSV-1 glycoproteins provide different levels of protection against HSV-1-induced eye disease. Based on comparison with the authors' previously published results, the ability of each glycoprotein to protect against eye disease correlated with the ability of the glycoprotein to induce high serum neutralizing antibody titers and killer cell activity. Results suggest that the effectiveness of these seven glycoproteins in protecting against eye disease can be ranked as follows: gD > gB > gI > (gC = gE) > gG > gH.

Considerations in optimizing radiation therapy for non-small cell lung cancer.

Irradiation therapy for lung cancer is mostly restricted to conventional methods. To improve therapeutic ratio, we have combined a treatment planning and a gene therapy approach. Three-dimensional conformal radiotherapy is described as carried out by methods of gene therapy for radiation protection using the manganese-superoxide-dismutase transgene delivered by inhalation gene transfer. These methods may improve therapeutic outcomes in lung cancer.

Keratoconus detected by videokeratography in candidates for photorefractive keratectomy.

BACKGROUND: Patients with corneal shape abnormalities should be identified prior to photorefractive keratectomy (PRK). We used videokeratography screening to detect subclinical corneal abnormalities, including keratoconus, which might have been missed by conventional clinical evaluation. METHODS: One hundred forty-six apparently normal myopic eyes (-1.00 to -7.00 diopters [D] with less than 1.50 D of cylinder) of 91 consecutive patients who were candidates for PRK were screened by videokeratography. RESULTS: In 6 of 91 patients (7 of 146 eyes), unsuspected corneal shape abnormalities were detected by videokeratography. Two patients had definite keratoconus and three were classified as keratoconus suspects by inferior corneal steepening (Rabinowitz I-S index ranging from 1.62 to 6.20 D). One patient had early pellucid marginal degeneration. CONCLUSIONS: Keratoconus suspects and contact lens-induced changes resembling keratoconus are present in the "normal" myopic population that presents for refractive surgery. Videokeratographic screening is the only effective means of identifying these and other corneal shape abnormalities.

Inhibition of peridural fibrosis after laminectomy using low-dose external beam radiation in a rat model.

OBJECTIVE: Clinical studies have revealed a significant association between the presence of extensive postlumbar discectomy peridural scar formation and the reoccurrence of low back and radicular pain. Low-dose perioperative radiation therapy has been shown to inhibit scar formation. Its effect on peridural fibrosis, however, has not been studied. METHODS: Thirty male Sprague-Dawley rats underwent L5 laminectomies. Ten rats each received a single fraction of 700-cGy external beam radiation to the lumbar spine 24 hours before surgery; 10 rats each received 700 cGy 24 hours after surgery. The remaining 10 rats served as a control group. All of the rats were killed 30 days after surgery. The spines were harvested, and axial histological sections through the laminectomy defect were evaluated. Each specimen was scored for extent, density, and arachnoidal involvement by fibrosis. RESULTS: There was a statistically significant difference between the treatment and control groups regarding the extent of fibrosis along the dura (P < 0.001), the density of fibroblasts (P < 0.005), and the arachnoid involvement (P < 0.01). There was no difference in fibrosis reduction between the groups receiving pre- and postlaminectomy radiation. CONCLUSION: Low-dose external beam radiation therapy administered before or after laminectomy in a rat model significantly decreases the extent, density, and arachnoidal involvement of peridural fibrosis. This technique may improve the outcome of patients who undergo reoperations for recurrent radicular and/or low back pain after successful lumbar discectomy in whom there is a significant amount of peridural fibrosis.

Australian-Indonesian collaboration in veterinary arbovirology--a review.

Australian-Indonesian collaboration in veterinary development programs has led to significant advances in the study of arboviruses. This paper reviews the resulting knowledge of arboviral infections of livestock in Indonesia. The first recognized arboviral disease of animals in Indonesia was bovine ephemeral fever. Serology indicates that the virus is widespread, as are related rhabdoviruses. Local sheep appear resistant to bluetongue disease, but imported sheep have suffered mortalities. Bluetongue viral serotypes 1, 7, 9, 12, 21 and 23 have been isolated from sentinel cattle; 1, 21 and 23 at widely separate locations. Bluetongue serotype 21 has been isolated from Culicoides spp. Serological reactors to Akabane virus are widespread, as are reactors to the flavivirus group. Japanese encephalitis, isolated from sentinel pigs, is the flavivirus of most veterinary importance but the limit of its easterly distribution is unknown. Many of the arboviruses present in Indonesia are also present in Australia and elsewhere in Asia. Their patterns of mobility among countries in the region are largely undescribed, but there are opportunities for further regional collaboration.