In situ regeneration of retinal pigment epithelium by gene transfer of E2F2: a potential strategy for treatment of macular degenerations.

The retinal pigment epithelium (RPE) interacts closely with photoreceptors to maintain visual function. In degenerative diseases such as Stargardt disease and age-related macular degeneration, the leading cause of blindness in the developed world, RPE cell loss is followed by photoreceptor cell death. RPE cells can proliferate under certain conditions, suggesting an intrinsic regenerative potential, but so far this has not been utilised therapeutically. Here, we used E2F2 to induce RPE cell replication and thereby regeneration. In both young and old (2 and 18 month) wildtype mice, subretinal injection of non-integrating lentiviral vector expressing E2F2 resulted in 47% of examined RPE cells becoming BrdU positive. E2F2 induced an increase in RPE cell density of 17% compared with control vector-treated and 14% compared with untreated eyes. We also tested this approach in an inducible transgenic mouse model of RPE loss, generated through activation of diphtheria toxin-A gene. E2F2 expression resulted in a 10-fold increase in BrdU uptake and a 34% increase in central RPE cell density. Although in mice this localised rescue is insufficiently large to be demonstrable by electroretinography, a measure of massed retinal function, these results provide proof-of-concept for a strategy to induce in situ regeneration of RPE for the treatment of RPE degeneration.

Phase I combination study of trabectedin and capecitabine in patients with advanced malignancies.

BACKGROUND: To determine the maximum tolerated dose (MTD), safety and pharmacokinetics of trabectedin with capecitabine in patients with advanced malignancies. DESIGN: In this Phase I, open-label, dose-finding study, patients refractory to standard therapy received trabectedin (3-h intravenous infusion, 0.4-1.3 mg/m(2), day 1) and capecitabine (2,000 or 1,600 mg/m(2)/day orally, days 2-15) every 3 weeks. Standard "3 + 3" dose escalation was used to define the MTD. Antitumor response was assessed every two cycles; adverse events (AEs) were recorded throughout. RESULTS: Forty patients received 149 cycles of treatment (median 2; range 1-11) at nine dose levels. Gastrointestinal dose-limiting toxicities in two patients at two dose levels with capecitabine at 2,000 mg/m(2)/day prompted dose reduction to 1,600 mg/m(2)/day and initiation of new trabectedin dose escalation at 0.6 mg/m(2). The MTD was capecitabine 1,600 mg/m(2)/day + trabectedin 1.1 mg/m(2). Common grade 3-4 drug-related AEs were neutropenia (20%), nausea (18%), diarrhea (15%) and palmar-plantar erythrodysesthesia (15%). One patient with cholangiocarcinoma achieved a sustained partial response, and 18 patients maintained stable disease (six for ≥6 months). CONCLUSIONS: The combination of trabectedin and capecitabine is generally well tolerated, without pharmacokinetic interactions, and shows some activity in patients with advanced cancers.

A dose-ranging study of the pharmacokinetics and pharmacodynamics of the selective apoptotic antineoplastic drug (SAAND), OSI-461, in patients with advanced cancer, in the fasted and fed state.

PURPOSE: To evaluate the safety, pharmacokinetics and determine the recommended dose of the selective apoptotic antineoplastic drug, OSI-461 administered on a twice-daily regimen to patients with advanced solid malignancies. METHODS: In this phase I trial, 33 patients were treated with OSI-461 doses ranging from 400 to 1,200 mg given twice daily in 4-week cycles. Pharmacokinetic studies were performed to characterize the plasma disposition of OSI-461 and the effect of food intake on OSI-461 absorption. Secondary biomarker studies were performed to assess the biologic activity of OSI-461 including the measurement of pGSK-3beta, a PKG substrate, and pharmacogenetic studies to identify polymorphisms of CYP3A that influence drug metabolism and of ABCG2, involved in drug resistance. RESULTS: Thirty-three patients were treated with 86 courses of OSI-461. The dose-limiting toxicities were grade 3 abdominal pain, found in one patient at the 1,000 mg BID fed dose level and all patients at the 1,200 mg BID fed dose level. There was also one episode each of grade 3 fatigue and grade 3 constipation at the 1,000 and 1,200 mg BID fed dose levels, respectively. Other common toxicities included mild to moderate fatigue, nausea, anorexia and mild elevation in bilirubin. Pharmacokinetic studies of OSI-461 revealed approximately a twofold increase in AUC(0-24) when OSI-461 was administered with food. An increase in pGSK-3beta post-dose was seen in the majority of patients and was greater at higher dose levels. No patients exhibited CYP3A4 polymorphisms, while 100% of patients were found to have the CYP3A5*3/CYP3A5*3 polymorphism. Two known polymorphisms of the ABCG2 gene, G34 --> A34 and C421 --> A421, occurred at frequencies of 11.76 and 29%, respectively. CONCLUSIONS: Toxicity and pharmacodynamic data show that the recommended oral dose of OSI-461 is 800 mg twice daily administered with food. The drug appears to be well-tolerated, and overall bioavailability appears to be markedly increased when the drug is administered with food. These results support further disease-directed evaluations of OSI-461 at a dose of 800 mg BID in combination with other chemotherapeutic agents.

Assessment of the biological and pharmacological effects of the alpha nu beta3 and alpha nu beta5 integrin receptor antagonist, cilengitide (EMD 121974), in patients with advanced solid tumors.

BACKGROUND: Cilengitide, an antiangiogenic agent that inhibits the binding of integrins alpha(nu)beta(3) and alpha(nu)beta(5) to the extracellular matrix, was studied at two dose levels in cancer patients to determine the optimal biological dose. PATIENTS AND METHODS: The doses of cilengitide were 600 or 1200 mg/m(2) as a 1-h infusion twice weekly every 28 days. A novel dose escalation scheme was utilized that relied upon the biological activity rate. RESULTS: Twenty patients received 50 courses of cilengitide with no dose-limiting toxic effects. The pharmacokinetic (PK) profile revealed a short elimination half-life of 4 h, supporting twice weekly dosing. Of the six soluble angiogenic molecules assessed, only E-selectin increased significantly from baseline. Analysis of tumor microvessel density and gene expression was not informative due to intrapatient tumor heterogeneity. Although several patients with evaluable tumor biopsy pairs did reveal posttreatment increases in tumor and endothelial cell apoptosis, these results did not reach statistical significance due to the aforementioned heterogeneity. CONCLUSIONS: Cilengitide is a well-tolerated antiangiogenic agent. The biomarkers chosen in this study underscore the difficulty in assessing the biological activity of antiangiogenic agents in the absence of validated biological assays.

Importance of morphological findings in the progress and treatment of chest wall deformities with special reference to the value of computed tomography, echocardiography and stereophotogrammetry.

Patients with chest wall deformities have many particular anatomical, physiological, genetic, biomechanical and psychological characteristics. It is generally recognized that surgical correction of funnel chest and pigeon breast may be done for psychosocial reasons alone. Morphological and dynamic investigations are done to identify the function of the deformed chest wall and to permit objective assessment of the cosmetic and functional outcomes of the operation. There is no universally agreed standard for the diagnosis of funnel chest and pigeon breast. Non-invasive investigations, such as computed tomography (CT), echocardiography (ECG) and stereophotogrammetry (SP), improve the quality of the diagnostic assessment, including evaluation of its functional aspects, without directly influencing the decision to operate, which is still made mainly on subjective grounds. The value of these techniques in the diagnosis of chest wall deformities is assessed and the results of our personal experience are presented.

Expression, nucleotide sequence and mutational analysis of two open reading frames in the nif gene region of Anabaena sp. strain PCC7120.

A 1.8 kb transcript corresponding to a region of the Anabaena 7120 chromosome 4 kb downstream of the nifHDK operon appears 12-18 h after heterocyst induction. The DNA corresponding to this transcript was sequenced and found to contain two open reading frames, designated ORF 1 and ORF 2. Two polypeptides, of 30 kDa and 13 kDa, encoded by these ORFs were expressed in Escherichia coli. An apparent start site for the transcript, detected by S1 nuclease protection, was located 42 bp upstream of the ATG start codon of ORF 1. ORF 2 shows strong sequence similarity to ORF 6 in the nif gene region of Azotobacter vinelandii. ORF 1 was interrupted using a 1.4 kb neomycin resistance cassette and the resulting mutant grew very slowly on medium lacking combined nitrogen. The mutant had 45% of wild-type acetylene reduction activity, which could be complemented by a 2.8 kb EcoRI fragment of wild-type Anabaena DNA containing only ORF 1 and ORF 2. Thus, one or both of these ORFs is required for efficient nitrogen fixation in Anabaena.

Abnormal properties of thyroglobulin in mice with inherited congenital goiter (cog/cog).

A recessive autosomal mutation (cog) in mice that results in congenital goiter was recently described. Since the mutation has been linked to the thyroglobulin (TG) gene, we have studied the immunological and physical properties of TG in cog/cog mice. +/Cog mice, which are phenotypically normal, were used as controls. In a mouse TG RIA the displacement curve produced by cog/cog thyroid extract was not parallel to normal murine TG, and at maximum displacement 15.4% of the tracer was still bound to the antibody. Extract from +/cog thyroid tissue produced parallel and complete displacement. Sucrose density gradient velocity centrifugation followed by RIA was used to determine the sedimentation properties of cog/cog TG. An abnormal pattern was obtained; a small peak in the 3-8S area and a broad, poorly defined peak at 12S and extending to above 27S were present. By comparison, +/cog thyroids had sharp peaks at 19S and 27S. These findings suggest that normal TG contains some immunological determinants that are absent, and some that are altered, in cog/cog TG. They also indicate that the association of 12S subunits to form 19S TG in cog/cog mice is weak and abnormal. Thyroid tissue was labeled with Na125I in vivo and with [35S] methionine in vitro. In cog/cog mice iodine was incorporated predominantly into albumin and other non-TG proteins. However, by polyacrylamide gradient gel electrophoresis, distinct 125I-labeled bands comigrating with normal TG were present. The bands migrating with TG were also precipitable with anti-TG antiserum. In +/cog mice TG was the predominant iodinated molecule. With [35S]methionine labeling, cog/cog and +/cog thyroids formed TG with the same electrophoretic mobilities. These data indicate the cog/cog thyroids synthesize TG of normal, or very nearly normal, size. The immunological and sedimentation properties of this TG are abnormal, supporting the possibility that the cog mutation is in the TG gene.