Low-temperature micronization of a peptide drug in fluid propellant: Case study cetrorelix

Aim of this study was to elaborate an efficient method for the micronization of the decapeptide cetrorelix (a GnRH-antagonist), in order to obtain a microsuspension as basis for other pharmaceutical preparations, such as e.g. inhalation aerosols. A modified pearl-mill coupled with a cryostat was used for the micronization of cetrorelix in fluid propellant and operated under different conditions. The obtained cetrorelix suspensions were analyzed for particle size distribution, purity of cetrorelix, and for metal contamination through abrasion from parts of the mill. The method allowed an effective micronization of cetrorelix. The mean particle size of the initial cetrorelix lyophilizate bulk ware was reduced from 52.5 μ (Volume Mean Diameter, VMD) down to 14.9, 6.1 and 3.1 μm, respectively, respectively. The HPLC analysis of all cetrorelix suspensions after micronization did not show signs of decomposition as compared to the initial product. The elementary analysis of the suspensions performed by inductively coupled plasma mass spectrometry revealed a negligible amount of contaminants in the suspension (Zr=max. 0.6 ppm; Fe, Cr, Ni, Ba, below limit of quantification, i.e.<0.14 ppm). The only appreciable contaminant. Aluminum (Al=1.1 ppm), was derived from the mechanical capping of aluminum canisters prior to analysis. The Zr determination in the suspension of 0.6 ppm, is still considered to be negligible as compared to the legally tolerated limit of air contamination. By low-temperature micronization in fluid propellant, fine drug suspensions of cetrorelix for pMDIs can be directly manufactured in one-step procedure without destruction of the peptide structure and without appreciable product contamination. Published: July 12, 2001.     

Uptake of Mannose-Terminal Glucocerebrosidase in Cultured Human Cholinergic and Dopaminergic Neuron Cell Lines

Enzyme replacement therapy has been shown to be particularly effective for patients with type 1 (non-neuronopathic) Gaucher disease. However, intravenously administered glucocerebrosidase does not reverse or halt the progression of brain damage in patients with type 2 (acute neuronopathic) Gaucher disease. A previous investigation revealed that intracerebral infusion of mannose-terminal glucocerebrosidase was safe in experimental animals. The enzyme had a comparatively long half-life in the brain. It was transported by convection from the site of infusion along white matter fiber tracts to the cerebral cortex where it was endocytosed by neurons. In anticipation of intracerebral administration of mannose-terminal glucocerebrosidase to patients with type 2 Gaucher disease, it was important to learn the mechanism involved in its cellular uptake. We therefore compared the endocytosis of this enzyme by J774 macrophage cells with that in two human neuronal cell lines and a human astrocyte cell line. Mannose-terminal glucocerebrosidase was taken up by cholinergic LA-N-2 cells, but to a much lower extent than by macrophages. Considerably less of the enzyme was endocytosed by dopaminergic SH-SY5Y cells. It was not taken up by NHA astrocytes. The findings provide encouragement for an exploration of intracerebral administration of glucocerebrosidase in patients with type 2 Gaucher disease.     

From circadian rhythms to cancer chronotherapeutics

Mammalian circadian rhythms result from a complex organization involving molecular clocks within nearly all “normal” cells and a dedicated neuroanatomical system, which coordinates the so-called “peripheral oscillators.” The core of the central clock system is constituted by the suprachiasmatic nuclei that are located on the floor of the hypothalamus. Our understanding of the mechanisms of circadian rhythm generation and coordination processes has grown rapidly over the past few years. In parallel, we have learnt how to use the predictable changes in cellular metabolism or proliferation along the 24h time scale in order to improve treatment outcome for a variety of diseases, including cancer. The chronotherapeutics of malignant diseases has emerged as a result of a consistent development ranging from experimental, clinical, and technological prerequisites to multicenter clinical trials of chronomodulated delivery schedules. Indeed large dosing-time dependencies characterize the tolerability of anticancer agents in mice or rats, a better efficacy usually results from treatment administration near the least toxic circadian time in rodent tumor models. Programmable in time multichannel pumps have allowed to test the chronotherapy concepts in cancer patients and to implement chronomodulated delivery schedules in current practice. Clinical phase I and II trials have established the feasibility, the safety, and the activity of the chronotherapy schedules, so that this treatment method has undergone further evaluation in international multicenter phase III trials. Overall, more than 2000 patients with metastatic disease have been registered in chronotherapy trials. Improved tolerability and/or better antitumor activity have been demonstrated in randomized multicenter studies involving large patient cohorts. The relation between circadian rhythmicity and quality of life and even survival has also been a puzzling finding over the recent years. An essential step toward further developments of circadian-timed therapy has been the recent constitution of a Chronotherapy cooperative group within the European Organization for Research and Treatment of Cancer. This group now involves over 40 institutions in 12 countries. It is conducting currently six trials and preparing four new studies. The 19 contributions in this special issue reflect the current status and perspectives of the several components of cancer chronotherapeutics.     

Pharmaco-economic comparative evaluation of combination chronotherapy vs. standard chemotherapy for colorectal cancer

Results of recent trials comparing combination chemotherapy consisting of 5-fluorouracil (5-FU), folinic acid (FOL), and oxaliplatin, given either as flat (A) or chronomodulated (B) infusion for metastatic colorectal cancer, were subjected to pharmaco-economic evaluation. The overall cost of treatment with the flat and chronomodulated protocols was equivalent. The expense of the delivery of medications with the chronotherapeutic arm (B) was greater than with the standard arm (A) because it was feasible to administer more courses (requiring more frequent doctor visits) and higher doses (high cost of medications) with containment of toxic reactions. Chrono-arm B was definitively more cost-effective than standard flat-arm A treatment since it made the outcome of treatment more effective; there was greater tumor response rate and longer time to progression with less treatment-associated toxicity. Finally, selection of the Melodie brand infusion pump to deliver the chronotherapy resulted in a further 18% reduction of overall costs and made it possible for patients to enjoy increased autonomy and improved quality of life.     

Plasmid delivery in the rat brain

Neurodegenerative diseases as a class do not have effective pharmacotherapies. This is due in part to a poor understanding of the pathologies of the disease processes, and the lack of effective medications. Gene delivery is an attractive possibility for treating these diseases. For the paradigm to be effective, efficient, safe and versatile vectors are required. In this study we evaluated three plasmid delivery systems for transgene expression in the rat hippocampus. Two of these systems were designed to have enhanced intracellular biodegradability. It was hypothesized that this system would be less toxic and could increase the free (non-vector) associated plasmids within the cell, leading to increased transgene activity. Polyethylenimine (PEI) and r-AAV-2 (recombinant adeno associated virus-2) were used as positive, non-viral and viral controls respectively, in the in vivo experiments. The results from the studies indicate there is a distinct difference between the various vectors in terms of total cells transfected, type of cell transfected, and toxicity. Non-viral systems were effective at transfecting both neurons and glia cells within the hippocampus, while the r-AAV-2 transfected mainly neurons. In summary, plasmid-mediated systems are effective for transgene expression within the brain and deserve further study.     

Cyotomedical therapy for insulinopenic diabetes using microencapsulated pancreatic beta cell lines

Current therapy for type 1 diabetes mellitus involves a daily regimen of multiple subcutaneous or intramuscular injections of recombinant human insulin. To achieve long-term insulin delivery in vivo, we investigated the applicability of cytomedical therapy using beta TC6 cells or MIN6 cells, both of which are murine pancreatic beta cell lines that secrete insulin in a subphysiologically or physiologically regulated manner, respectively. We examined this therapy in the insulinopenic diabetic mice intraperitoneally injected with beta TC6 cells or MIN6 cells microencapsulated within alginate-poly(L)lysine-alginate membranes (APA-beta TC6 cells or APA-MIN6 cells). The diabetic mice treated with APA-beta TC6 cells fell into hypoglycemia, whereas those injected with APA-MIN6 cells maintained normal blood glucose concentrations for over 2 months without developing hypoglycemia. In addition, we also conducted an oral glucose tolerance test using these mice. The blood glucose concentrations of normal and of diabetic mice injected with APA-MIN6 cells similarly changed over time, although the blood insulin concentration increased later in the injected diabetic mice than in the former. These results suggest that cytomedicine utilizing microencapsulated pancreatic beta cell lines with a physiological glucose sensor may be a beneficial and safe therapy with which to treat diabetes mellitus.     

Response surface methodology for the optimization of ubiquinone self-nanoemulsified drug delivery system

The aim of the present study was to prepare and evaluate an optimized, self-nanoemulsified drug delivery system of ubiquinone. A 3-factor, 3-level Box-Behnken design was used for the optimization procedure with the amounts of Polyoxyl 35 castor oil (X₁), medium-chain mono- and diglyceride (X₂), and lemon oil (X₃) as the independent variables. The response variable was the cumulative percentage of ubiquinone emulsified in 10 minutes. Different ubiquinone release rates were obtained. The amount released ranged from 11% to 102.3%. Turbidity profile revealed 3 regions that were used to describe the progress of emulsion formation: lag phase, pseudolinear phase, and plateau turbidity. An increase in the amount of surfactant decreased turbidity values and caused a delay in lag time. Addition of cosurfactant enhanced the release rates. Increasing the amount of the eutectic agent was necessary to overcome drug precipitation especially at higher loading of surfactants and cosurfactants. Mathematical equations and response surface plots were used to relate the dependent and independent variables. The regression equation generated for the cumulative percentage emulsified in 10 minutes was Y1=90.9–22.1X₁+5.03X₂+13.95X₃+12.13X₁X₂+15.13X₁X₃-14.40X₁ ²-6.25X₃ ². The optimization model predicted a 93.4% release with X₁, X₂, and X₃ levels of 35, 35, and 30 respectively. The observed responses were in close agreement with the predicted values of the optimized formulation. This demonstrated the reliability of the optimization procedure in predicting the dissolution behavior of a self-emulsified drug delivery system. Published: February 8, 2002.     

Preparation of dry powder dispersions for non-viral gene delivery by freeze-drying and spray-drying

Background

Dry powder dispersion devices offer potential for delivering therapeutic macromolecules to the pulmonary epithelia. Previously, freeze‐drying (lyophilisation) has been the accepted method for preparing dried formulations of proteins and non‐viral gene vectors despite the respirability of such powders being inadequate without further processing. In this study we compare the utility of freeze‐drying and spray‐drying, a one‐step process for producing dry and respirable powders, as methods for preparing non‐viral respiratory gene delivery systems.

Methods

Lipid:polycation:pDNA (LPD) vectors comprising 1,2‐dioleoyl‐3‐trimethylammoniumpropane (DOTAP), protamine sulphate and pEGFP‐N1 in 3% lactose solution were either snap‐frozen and lyophilised or spray‐dried. Lyophilised powder was used as recovered or following coarse grinding. Structural integrity of dehydrated pDNA was assessed by agarose gel electrophoresis and powder particle size determined by laser diffraction. The apparent structure of the systems was visualised by scanning and transmission electron microscopy with the biological functionality quantified in vitro (A549 human lung epithelial cell line) by Green Fluorescent Protein (GFP) associated fluorescence.

Results

Lyophilisation produced large, irregularly shaped particles prior to (mean diameter ～21 µm) and following (mean diameter ～18 µm) coarse grinding. Spray‐drying produced uniformly shaped spherical particles (mean diameter ～4 µm). All dehydrated formulations mediated reporter gene expression in A549 cells with the spray‐dried formulation generally proving superior even when compared with freshly prepared LPD complexes. Biological functionality of the LPD dry powders was not adversely affected following 3 months storage.

Conclusions

Spray‐drying has utility for producing stable, efficient and potentially respirable non‐viral dry powder systems for respiratory gene delivery. Copyright © 2002 John Wiley & Sons, Ltd.

     

Suppression of Fas-mediated hepatic apoptosis by caspase inhibitor-encapsulated nanoparticles bearing poly-(N-p-vinylbenzyl-O-β-D-galactopyranosyl-[1-4]-D-gluconamide)

To develop a drug delivery system for acute hepatic injury, we prepared Z-Asp, a general caspase inhibitor, encapsulated in poly (DL-lactic-co-glycolic acid) (50:50) (mol/mol) nanoparticles bearing poly-(N-p-vinylbenzyl-O--d-galactopyranosyl-[1-4]-d-gluconamide) (PVLA) on their surface. These nanoparticles specifically interacted with the primary cultured hepatocytes via the asialoglycoprotein receptors on surface and effectively inhibited the fulminant hepatic cell death induced by anti-mouse Fas antibody while these particles did not affect the cell death of an asialoglycoprotein receptor null cell line, A20. These nanoparticles are thus a promising therapy for acute liver injury.