Surgical delivery of drug releasing poly(lactic-co-glycolic acid)/poly(ethylene glycol) paste with in vivo effects against glioblastoma

Introduction

The median survival of patients with glioblastoma multiforme (astrocytoma grade 4) remains less than 18 months despite radical surgery, radiotherapy and systemic chemotherapy. Surgical implantation of chemotherapy eluting wafers into the resection cavity has been shown to improve length of survival but the current licensed therapy has several drawbacks. This paper investigates in vivo efficacy of a novel drug eluting paste in glioblastoma.

Methods

Poly(lactic-co-glycolic acid)/poly(ethylene glycol) (PLGA/PEG) self-sintering paste was loaded with the chemotherapeutic agent etoposide and delivered surgically into partially resected tumours in a flank murine glioblastoma xenograft model.

Results

Surgical delivery of the paste was successful and practical, with no toxicity or surgical morbidity to the animals. The paste was retained in the tumour cavity, and preliminary results suggest a useful antitumour and antiangiogenic effect, particularly at higher doses. Bioluminescent imaging was not affected significantly by the presence of the paste in the tumour.

Conclusions

Chemotherapy loaded PLGA/PEG paste seems to be a promising technology capable of delivering active drugs into partially resected tumours. The preliminary results of this study suggest efficacy with no toxicity and will lead to larger scale efficacy studies in orthotopic glioblastoma models.     

Neuropeptide Y Attenuates Stress‐Induced Bone Loss Through Suppression of Noradrenaline Circuits

Chronic stress and depression have adverse consequences on many organ systems, including the skeleton, but the mechanisms underlying stress‐induced bone loss remain unclear. Here we demonstrate that neuropeptide Y (NPY), centrally and peripherally, plays a critical role in protecting against stress‐induced bone loss. Mice lacking the anxiolytic factor NPY exhibit more anxious behavior and elevated corticosterone levels. Additionally, following a 6‐week restraint, or cold‐stress protocol, Npy‐null mice exhibit three‐fold greater bone loss compared to wild‐type mice, owing to suppression of osteoblast activity. This stress‐protective NPY pathway acts specifically through Y2 receptors. Centrally, Y2 receptors suppress corticotropin‐releasing factor expression and inhibit activation of noradrenergic neurons in the paraventricular nucleus. In the periphery, they act to control noradrenaline release from sympathetic neurons. Specific deletion of arcuate Y2 receptors recapitulates the Npy‐null stress response, coincident with elevated serum noradrenaline. Importantly, specific reintroduction of NPY solely in noradrenergic neurons of otherwise Npy‐null mice blocks the increase in circulating noradrenaline and the stress‐induced bone loss. Thus, NPY protects against excessive stress‐induced bone loss, through Y2 receptor‐mediated modulation of central and peripheral noradrenergic neurons. © 2014 American Society for Bone and Mineral Research.     

Detection of the hemoglobin E mutation using the color complementation assay: application to complex genotyping

The color complementation assay (CCA) is a method of allele-specific DNA amplification by which competitive priming and extension of fluorescently labeled oligonucleotide primers determine the color of DNA amplification product. This diagnostic method precludes the need for radioisotopes, electrophoresis, and multiple high-stringency reaction conditions. The multiplicity of mutant globin genes present in Southeast Asians complicates clinical diagnosis and underscores the importance of DNA-based diagnostic methods. We have applied CCA to distinguish beta A and beta E alleles. Competing 15mer primers were a fluorescein-labeled complement to beta A and a rhodamine-labeled complement to beta E, identical except for their central nucleotides. A common unlabeled primer was used to amplify DNA product, the color of which was determined by the perfectly complementary primer. Color photography and spectrofluorometry, as well as a method of black-white photography that we developed to distinguish fluorescein- and rhodamine- labeled DNA, were used to record results. We applied CCA to define the complex genotype of a Thai woman with thalassemia intermedia, 96% HbE, and 4% HbF whose possible genotypes included several permutations of alpha-thalassemia, beta-thalassemia, and beta E genes. zeta-Globin gene mapping of DNA doubly digested with Bg/II and Asp 718 showed the -alpha 3.7/--SEA genotype, and CCA confirmed homozygous beta E/beta E. The CCA is useful for diagnosing the compound hemoglobin genotypes of Southeast Asians and could be applied also to prenatal diagnosis in this population.     

Cytomegalovirus infection and disease after liver transplantation

Cytomegalovirus is the single most important pathogen in clinical transplantation. Although much progress has been made in our understanding of the molecular biology and epidemiology of CMV infection and in our ability to diagnosis and treat CMV disease, it remains a major cause of morbidity but is no longer a major cause of mortality after liver transplantation. Risk factors for CMV disease after liver transplantation include donor and recipient serologic status, the use of antilymphocyte therapy, and retransplantation. CMV disease occurs early after transplantation, and the most frequent site of disease is the hepatic allograft. We have treated 79 patients with intravenous ganciclovir, with ultimate control of disease achieved in 69 patients (87.3%). Preliminary results using intravenous immunoglobulin and oral acyclovir for CMV prophylaxis in high-risk patients have been encouraging. In addition to producing clinical syndromes, CMV may have direct immunologic effects and is a marker of the net state of immunosuppression.     

Interleukin 2 induction of lymphokine-activated killer (LAK) activity in the peripheral blood and bone marrow of acute leukemia patients. I. Feasibility of LAK generation in adult patients with active disease and in remission

The feasibility of in vitro interleukin 2 (IL-2) activation and expansion of mononuclear cells (MNCs) derived from adult patients with acute myelogenous leukemia (ANLL) was studied. Patients' natural killer (NK) and lymphokine-activated killer (LAK) cell activity was compared with that of normal donors in terms of: (a) cytolytic activity (four- hour 51Cr release assay) against an NK-sensitive target (K562), NK- resistant targets (Raji/Daudi), and fresh/cryopreserved autologous and allogeneic leukemic blasts; (b) proliferation and expansion in culture with 1,000 U/mL recombinant IL 2 (rIL 2); and (c) the cell surface phenotype of the cultured cells. In 21 of 24 patients with active disease (AP) MNCs derived from the peripheral blood (PBL) or bone marrow (BM) could be cultured and expanded in the presence of rIL 2. These cultures initially contained between 30% and 50% blasts, and during 2 to 4 weeks of culture destruction of blasts and enrichment of up to 60% in cells with the morphology of large granular lymphocytes (LGLs) was observed. Expansion in culture varied between two- and 100- fold. MNCs from all patients in remission (RP) could be activated by rIL 2 and expanded up to 30-fold after 1 to 3 weeks in culture. NK activity of fresh PBLs from AP was significantly lower than in normal controls, whereas NK activity of RP was within the normal range. High levels of postactivation NK and LAK activity on K562/Raji/Daudi and on fresh/cryopreserved leukemic blasts was generated in approximately 50% of cases of AP and in most RP. Cell surface phenotype studies showed that cultured cells derived from ANLL patients were significantly enriched (up to 40%) in NKH-1 (Leu 19) positive cells, with RP LAK cells also expressing a high proportion of CD16 positive cells (up to 40%). This study has shown that it is feasible to activate and significantly expand killer cells derived from active disease and remission ANLL patients during 1 to 3 weeks culture with IL 2 with good maintenance of cytolytic activity. Both initial NK activity and LAK generation was optimal in remission patients. Based on data from this study, a clinical protocol has been developed for treatment of early relapse ANLL patients with LAK cells cultured for 1 to 3 weeks and systemic IL 2.     

Effects of monoclonal antibody therapy in patients with chronic lymphocytic leukemia

A phase I clinical trial was initiated to treat patients with stage IV B-derived chronic lymphocytic leukemia (CLL) with the IgG2a murine monoclonal antibody T101. This antibody binds to a 65,000-mol wt (T65) antigen found on normal T lymphocytes, malignant T lymphocytes, and B- derived CLL cells. All of the patients had a histologically confirmed diagnosis of advanced B-derived CLL and were refractory to standard therapy, and more than 50% of their leukemia cells reacted with the T101 antibody in vitro. The patients received T101 antibody two times per week, over two to 50 hours by intravenous administration in 100 mL of normal saline containing 5% human albumin. Twelve patients were treated with a fixed dosage of 1, 10, 50, or 100 mg, and one patient was treated with 140 mg of antibody. It was demonstrated that patients given two-hour infusions of 50 mg developed pulmonary toxicity, with shortness of breath and chest tightness. This toxicity was eliminated when infusions of 50 or 100 mg of T101 were prolonged to 50 hours. All dose levels caused a rapid but transient decrease in circulating leukemia cell counts. In vivo binding to circulating and bone marrow leukemia cells was demonstrated at all dose levels with increased binding at higher dosages. Antimurine antibody responses were not demonstrated in any patients at any time during treatment. Circulating free murine antibody was demonstrated in the serum of only the two patients treated with 100 mg of antibody as a 50-hour infusion and the patient treated with 140 mg of antibody over 30 hours. Antigenic modulation was demonstrated in patients treated at all dose levels but was particularly apparent in patients treated with prolonged infusions of 50 and 100 mg of antibody. We were also able to demonstrate antigenic modulation in lymph node cells, which strongly suggests in vivo labeling of these cells. Overall, T101 antibody alone appears to have a very limited therapeutic value for patients with CLL. The observations of in vivo labeling of tumor cells, antigenic modulation, antibody pharmacokinetics, toxicity, and antimurine antibody formation may be used in the future for more effective therapy when drugs or toxins are conjugated to the antibody.