Showing papers by "Pharmaceutical Product Development published in 2012"

Analysis of the Lactobacillus casei supragenome and its influence in species evolution and lifestyle adaptation

Abstract: The broad ecological distribution of L. casei makes it an insightful subject for research on genome evolution and lifestyle adaptation. To explore evolutionary mechanisms that determine genomic diversity of L. casei, we performed comparative analysis of 17 L. casei genomes representing strains collected from dairy, plant, and human sources. Differences in L. casei genome inventory revealed an open pan-genome comprised of 1,715 core and 4,220 accessory genes. Extrapolation of pan-genome data indicates L. casei has a supragenome approximately 3.2 times larger than the average genome of individual strains. Evidence suggests horizontal gene transfer from other bacterial species, particularly lactobacilli, has been important in adaptation of L. casei to new habitats and lifestyles, but evolution of dairy niche specialists also appears to involve gene decay. Genome diversity in L. casei has evolved through gene acquisition and decay. Acquisition of foreign genomic islands likely confers a fitness benefit in specific habitats, notably plant-associated niches. Loss of unnecessary ancestral traits in strains collected from bacterial-ripened cheeses supports the hypothesis that gene decay contributes to enhanced fitness in that niche. This study gives the first evidence for a L. casei supragenome and provides valuable insights into mechanisms for genome evolution and lifestyle adaptation of this ecologically flexible and industrially important lactic acid bacterium. Additionally, our data confirm the Distributed Genome Hypothesis extends to non-pathogenic, ecologically flexible species like L. casei.

Apilimod inhibits the production of IL-12 and IL-23 and reduces dendritic cell infiltration in psoriasis.

Abstract: Psoriasis is characterized by hyperplasia of the epidermis and infiltration of leukocytes into both the dermis and epidermis. IL-23, a key cytokine that induces T(H)17 cells, has been found to play a critical role in the pathogenesis of psoriasis. Apilimod is a small-molecule compound that selectively suppresses synthesis of IL-12 and IL-23. An open-label clinical study of oral administration of apilimod was conducted in patients with psoriasis. Substantial improvements in histology and clinical measurements were observed in patients receiving 70 mg QD. The expression of IL-23p19 and IL-12/IL-23p40 in skin lesions was significantly reduced in this dose group, with a simultaneous increase in IL-10 observed. A decrease in the levels of T(H)1 and T(H)17 cytokines/chemokines in skin lesions followed these p19 and p40 changes. In parallel, a reduction in skin-infiltrating CD11c(+) dendritic cells and CD3(+) T cells was seen, with a greater decrease in the CD11c(+) population. This was accompanied by increases in T and B cells, and decreases in neutrophils and eosinophils in the periphery. This study demonstrates the immunomodulatory activity of apilimod and provides clinical evidence supporting the inhibition of IL-12/IL-23 synthesis for the treatment of T(H)1- and T(H)17-mediated inflammatory diseases.

Design of human granzyme B variants resistant to serpin B9

Abstract: Human granzyme B (hGB) is a serine protease involved in immune-mediated apoptosis. Its cytotoxicity makes it potentially applicable in cancer therapy. However, the effectiveness of hGB can be hampered by the cytosolic expression of a natural protein inhibitor, human Serpin B9 (hSB9). Here, we used computational approaches to identify hGB mutations that can affect its binding to hSB9 without significantly decreasing its catalytic efficiency. Alanine-scanning calculations allowed us to identify residues of hGB important for the interaction with hSB9. Some variants were selected, and molecular dynamic simulations on the mutated hGB in complex with hSB9 in aqueous solution were carried out to investigate the effect of these variants on the stability of the complex. The R28K, R201A, and R201K mutants significantly destabilized the interaction of the protein with hSB9. Consistently, all of these variants also retained their activity in the presence of the Serpin B9 inhibitor in subsequent in vitro assays of wild-type and mutated hGB. In particular, the activity of R201K hGB with and without Serpin B9 is very similar to that of the wild-type protein. Hence, R201K hGB emerges as a promising species for antitumoral therapy applications.

A phase I/II study of foretinib, an oral multikinase inhibitor targeting MET, RON, AXL, TIE-2, and VEGFR in advanced hepatocellular carcinoma (HCC).

Abstract: 4108 Background: Hepatocyte growth factor (HGF)/MET signalling plays a pivotal role in tumor cell proliferation, migration and invasion in HCC and circulating levels of HGF correlate with poor prog...

In vivo imaging of immunotoxin treatment using Katushka-transfected A-431 cells in a murine xenograft tumour model

Abstract: Preclinical in vivo analyses of treatment responses are an important prerequisite to evaluate new therapeutics. Molecular in vivo imaging in the far red (FR)/near infra red (NIR) is a promising method, as it enables measurements at different time points in individual animals, thereby reducing the number of animals required, while increasing statistical significance. Here, we show the establishment of a method to monitor response to treatment using fluorescent cells, expressing the epidermal growth factor receptor (EGFR), a target already used in therapy. We transfected A-431 tumour cells with the far red–emitting protein Katushka (Kat2), resulting in strong fluorescence allowing for the monitoring of tumour growth when implanted in BALB/c nu/nu mice with a CRi Maestro in vivo imager. We targeted A-431 cells with a previously reported immunotoxin (IT), consisting of the anti-EGFR antibody single-chain variable fragment (scFv) 425, fused to Pseudomonas aeruginosa Exotoxin A’ (ETA’). In addition, EGFR expression was verified using the 425(scFv) conjugated to a NIR dye BG-747 through a SNAP-tag linker. The results show the feasibility to evaluate response to treatment in vivo by FR imaging, while at the same location detecting EGFR expression. Treatment with 425(scFv)-ETA’ resulted in decelerated tumour growth, while not affecting the overall health of the animals. This is in contrast to treatment with Doxorubicin, which, although decreasing the tumour size, resulted in poor health. We developed a novel method to non-invasively determine treatment responses by in vivo imaging of multiple parameters which showed the efficacy of 425(scFv)-ETA’.

Effect of pitavastatin vs. rosuvastatin on international normalized ratio in healthy volunteers on steady-state warfarin

Abstract: Objective:Statins have been shown to impact international normalized ratio (INR) when coadministered with warfarin. The aim of this study was to assess the effect of pitavastatin compared with rosuvastatin on steady-state pharmacodynamics (PD) of warfarin by measuring INR in healthy adult subjects.Methods:Subjects received oral doses of warfarin 5 mg once daily on days 1 through 3. The dose was titrated on days 4 through 9 to reach a steady-state INR of 1.5 to 2.2. Warfarin was continued on days 10 through 21 and pitavastatin 4 mg or rosuvastatin 40 mg was administered once daily on days 14 through 22. After a 14-day washout period, the process was repeated with the alternate statin.Study number:NK-104-4.03US.Results:For pitavastatin, mean INR changed from 1.73 ± 0.18 (n = 42) on day 14 before starting statin dosing, to 1.78 ± 0.29 (n = 42) on day 22 at treatment end; the difference in INR was not significant (p = 0.219). For rosuvastatin, mean INR increased significantly from 1.74 ± 0.20 (n = 43)...

Short-chain fluorescent tryptophan tags for on-line detection of functional recombinant proteins

Abstract: Conventional fluorescent proteins, such as GFP, its derivatives and flavin mononucleotide based fluorescent proteins (FbFPs) are often used as fusion tags for detecting recombinant proteins during cultivation. These reporter tags are state-of-the-art; however, they have some drawbacks, which can make on-line monitoring challenging. It is discussed in the literature that the large molecular size of proteins of the GFP family may stress the host cell metabolism during production. In addition, fluorophore formation of GFP derivatives is oxygen-dependent resulting in a lag-time between expression and fluorescence detection and the maturation of the protein is suppressed under oxygen-limited conditions. On the contrary, FbFPs are also applicable in an oxygen-limited or even anaerobic environment but are still quite large (58% of the size of GFP). As an alternative to common fluorescent tags we developed five novel tags based on clustered tryptophan residues, called W-tags. They are only 5-11% of the size of GFP. Based on the property of tryptophan to fluoresce in absence of oxygen it is reasonable to assume that the functionality of our W-tags is also given under anaerobic conditions. We fused these W-tags to a recombinant protein model, the anti-CD30 receptor single-chain fragment variable antibody (scFv) Ki-4(scFv) and the anti-MucI single-chain fragment variable M12(scFv). During cultivation in Microtiter plates, the overall tryptophan fluorescence intensity of all cultures was measured on-line for monitoring product formation via the different W-tags. After correlation of the scattered light signal representing biomass concentration and tryptophan fluorescence for the uninduced cultures, the fluorescence originating from the biomass was subtracted from the overall tryptophan signal. The resulting signal, thus, represents the product fluorescence of the tagged and untagged antibody fragments. The product fluorescence signal was increased. Antibodies with W-tags generated stronger signals than the untagged construct. Our low-molecular-weight W-tags can be used to monitor the production of antibody fragments on-line. The binding specificity of the recombinant fusion protein is not affected, even though the binding activity decreases slightly with increasing number of tryptophan residues in the W-tags. Thus, the newly designed W-tags offer a versatile and generally applicable alternative to current fluorescent fusion tags.

Evaluation of active mortality surveillance system data for monitoring hurricane-related deaths-Texas, 2008.

Abstract: Introduction The Texas Department of State Health Services (DSHS) implemented an active mortality surveillance system to enumerate and characterize hurricane-related deaths during Hurricane Ike in 2008. This surveillance system used established guidelines and case definitions to categorize deaths as directly, indirectly, and possibly related to Hurricane Ike. OBJECTIVE: The objective of this study was to evaluate Texas DSHS' active mortality surveillance system using US Centers for Disease Control and Prevention's (CDC) surveillance system evaluation guidelines. METHODS: Using CDC's Updated Guidelines for Surveillance System Evaluation, the active mortality surveillance system of the Texas DSHS was evaluated. Data from the active mortality surveillance system were compared with Texas vital statistics data for the same time period to estimate the completeness of reported disaster-related deaths. RESULTS: From September 8 through October 13, 2008, medical examiners (MEs) and Justices of the Peace (JPs) in 44 affected counties reported deaths daily by using a one-page, standardized mortality form. The active mortality surveillance system identified 74 hurricane-related deaths, whereas a review of vital statistics data revealed only four deaths that were hurricane-related. The average time of reporting a death by active mortality surveillance and vital statistics was 14 days and 16 days, respectively. CONCLUSIONS: Texas's active mortality surveillance system successfully identified hurricane-related deaths. Evaluation of the active mortality surveillance system suggested that it is necessary to collect detailed and representative mortality data during a hurricane because vital statistics do not capture sufficient information to identify whether deaths are hurricane-related. The results from this evaluation will help improve active mortality surveillance during hurricanes which, in turn, will enhance preparedness and response plans and identify public health interventions to reduce future hurricane-related mortality rates. Choudhary E, Zane DF, Beasley C, Jones R, Rey A, Noe RS, Martin C, Wolkin AF, Bayleyegn TM. Evaluation of active mortality surveillance system data for monitoring hurricane-related deaths, Texas, 2008. Prehosp Disaster Med. 2012;27(4):1-6. Language: en

Effects of multiple doses of albiglutide on the pharmacokinetics, pharmacodynamics, and safety of digoxin, warfarin, or a low-dose oral contraceptive.

Abstract: Aims: Albiglutide is a glucagon–like peptide–1 analog that is currently under investigation for the treatment of type 2 diabetes mellitus as a once–weekly injection. Three open–label phase 1 studies were conducted in healthy human participants to investigate potential pharmacokinetic (PK) and/or pharmacodynamic (PD) interactions between albiglutide and medications that may be used concomitantly. Methods: Digoxin 0.5 mg, warfarin 25 mg, or a standard, low–dose oral contraceptive containing norethindrone (NE) 0.5 mg and ethinyl estradiol (EE) 0.035 mg were administered alone and after steady–state albiglutide exposure (50 mg weekly for 4–5 weeks). The lack of a drug–drug interaction was concluded if the 90% CIs of geometric least–squares means ratio of area under the curve and maximum concentration were fully contained within 0.80 to 1.25. The effects of albiglutide on the PD of warfarin (international normalized ratio) and on the PD of a low–dose oral contraceptive (luteinizing hormone [LH], follic...

Reduction of activated macrophages after ischaemia–reperfusion injury diminishes oxidative stress and ameliorates renal damage

Abstract: Background. Macrophages are major effectors of the local inflammatory response syndrome (LIRS) and influence the extent of ischaemia/reperfusion injury, thereby impacting organ function. Several subgroups of macrophages exist, representing distinct modes of action. The specific role of the subset expressing Fc gamma receptor (FcγR) 1 in the activated state of macrophages is poorly defined. Methods. We induced a LIRS via 30 min of ischaemia in uninephrectomized rats, transgenic for the human FcγR1. Six hours after reperfusion, the treatment group was injected with a recombinant immunotoxin (IT) H22(scFv)ETA’ targeted against human FcγR1, which induced apoptosis of target cells. The placebo group received normal saline (NS). Contralateral kidneys served as healthy controls (Ctr). After 24 h of reperfusion, the animals were analysed. Results. Targeted treatment with IT resulted in preserved renal function [NS versus IT treatment and baseline (creatinine: 69.2 ± 2.6, 54.7 ± 3.4 and 27.3 ± 1.0 μmol/L; P < 0.001)]. The number of all infiltrating monocytes were significantly reduced (CD68-positive cells per view field: NS 3.8± 0.4, IT 2.5± 0.2 and Ctr 1.2± 0.4; P < 0.05), renal histology improved and there was a reduced expression of renal fibronectin (NS 4.0 ± 0.4, IT 2.3± 0.2 and Ctr 1.1 ± 0.1; P < 0.001). Following IT administration, we also observed less expression of renal monocyte chemoattractant protein-1-positive cells per view field (NS 19.0 ± 1, IT 10.1 ± 0.8 and Ctr 2.0± 0.3; P < 0.001) as well as reduced systemic and local oxidative stress [serum malondialdehyde (MDA): NS 340 ± 30, IT 224± 36 versus baseline 140± 5 nmol/mL; P < 0.01]; renal MDA arbitrary units of fluorescence intensity: NS 3.7± 0.2, IT 1.8± 0.3 and Ctr 0.4± 0.2; P < 0.001. Conclusions. Reduction of FcγR1-up-regulated monocytic cells leads to preserved renal function and morphology in a rat model of ischaemia-triggered LIRS. Our results show that targeting activated macrophages is a valuable approach for ameliorating ischaemia-induced tissue injury.

Summary of Pharmacokinetics and Tissue Distribution of a Broad-Spectrum Fluoroquinolone, JNJ-Q2.

Abstract: This article summarizes key pharmacokinetic properties of JNJ-Q2, a broad-spectrum fluoroquinolone, being developed for acute bacterial skin and skin structure infections and community-acquired bacterial pneumonia. Two randomized placebo-controlled studies and one open-label study are presented: single and multiple ascending-dose studies evaluating intravenous (IV) and oral pharmacokinetics, absolute bioavailability accumulation, and lung penetration. Fifty-seven participants received JNJ-Q2, which was safe and well tolerated. The half-life was 13 to 20 hours, clearance (CLtot ) was 4.41 to 6.22 L/h, volume of distribution (Vd ) was 86 to 148 L, renal clearance (CLren ) was 0.58 L/h, and the fraction excreted in urine (%Fe) was 12%. Accumulation ratio was 1.63, and absolute bioavailability was 0.65. The lung penetration study demonstrated substantial concentration of JNJ-Q2 in epithelial lining fluid and alveolar macrophages with ratios to free plasma of 50.6 and 156.9, respectively, at 6 hours postdose. JNJ-Q2 doses under consideration for future clinical trials are 150 mg for IV and 250-mg tablets for oral administration.

Anidulafungin—challenges in development and validation of an LC-MS/MS bioanalytical method validated for regulated clinical studies

Abstract: Anidulafungin is a semi-synthetic echinocandin with antifungal activity, usually administered as an intravenous infusion. In order to determine the pharmacokinetics (PK) of anidulafungin in pediatric patients, a sensitive high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) bioanalytical method (M1) was developed and validated for quantification of anidulafungin in plasma. During analysis of incurred samples (samples collected from patients enrolled in a clinical study) an isobaric chromatographic interference was observed. The source of interference was identified as an anidulafungin open-ring form (D1) and its impact on the quantification of anidulafungin was investigated. It was found that accurately quantifying anidulafungin in incurred samples required chromatographic separation of the open-ring form from anidulafungin. The method was redeveloped to achieve the appropriate baseline separation and to avoid experimental conditions that favored opening the anidulafungin ring. The extraction of anidulafungin from plasma by protein precipitation remained unchanged, but the changes in chromatography warranted validation of a new method, M2, 2 years after M1 was validated. Incurred samples from three studies that were previously analyzed by M1 and were within confirmed long-term frozen stability were then reanalyzed by M2. Although the incurred sample reproducibility tests on those samples passed for each of the two methods, comparison of concentrations from the same samples obtained by M1 and M2 revealed that an overestimation of anidulafungin following the M1 method exceeded acceptance criteria. The new HPLC-MS/MS method (M2) is applicable for quantification of anidulafungin within a nominal range 50–20,000 ng/mL and requires a 50 μL human plasma aliquot. A linear, 1/concentration squared weighted, least-squares regression algorithm was used to generate the calibration curve and its parameters were used to quantitate the incurred samples. The inter-assay accuracy in heparin human plasma validation ranged from −4.33 to 0.0386 % and precision was ≤7.32 %. The method M2 was validated for use in regulated bioanalysis and is presently used to quantitate anidulafungin in plasma samples from clinical studies.

Plasma proteomic analysis in patients with obstructive sleep apnea syndrome

Abstract: Proteomics is one of the strategies to evaluate molecular mechanisms underlying obstructive sleep apnea syndrome (OSAS). To examine the pathophysiological significance of plasma proteomics in OSAS, the plasma samples from severe OSAS patients (n = 6) with obese (BMI > 30) and non-OSAS patients (n = 6) with non-obese (BMI < 30) were subjected to proteomic profiling. Many proteins regarding inflammation and immune response, including complement proteins, two acute-phase reactants ceruloplasmin and serum amyloid P-component, were found to be highly expressed in severe OSAS patients. Protein changes responsible for immune modulation and inflammation may be a feature of OSAS patients.

Desarrollo de nuevos medicamentos: oportunidades y beneficios para el Perú

Abstract: El desarrollo de drogas innovadoras permite la obtencion de nuevos medicamentos para asi prevenir y tratar mejor las enfermedades, ello mejora la calidad de vida y la hace mas productiva; por tanto, la mision de la investigacion farmaceutica es desarrollar drogas seguras y eficaces. Los ensayos clinicos permiten evaluar los perfiles de seguridad y eficacia de nuevos medicamentos, dispositivos medicos y pruebas diagnosticas. La investigacion y el desarrollo de nuevas drogas es un proceso largo y costoso en donde por cada 5000 a 10 000 nuevos compuestos que ingresan a las pruebas preclinicas, solo uno es aprobado. En la actualidad, el desarrollo de drogas muestra un crecimiento de 7,6% con respecto al 2011. Segun ClinicalTrials.gov, el 5% de los ensayos se implementan en Latinoamerica, en donde, Peru ocupa el quinto lugar, con un descenso de estudios aprobados desde el ano 2009. De otro lado, segun el Reporte Global de Competitividad del Foro Economico Mundial, Peru ocupa el puesto 61 con retos principalmente en el funcionamiento de sus instituciones publicas, inversion en I&D y capacidad tecnologica. La complejidad de la I&D de medicamentos explica la busqueda de locaciones competitivas para el desarrollo de estudios clinicos. La Investigacion Clinica es una industria humanizada por su plataforma etica enunciada en las guias de buenas practicas clinicas, y que exige de nuestro pais desarrollar un valor diferenciador que contribuya con el desarrollo de conocimiento y su competitividad.