An Vermeire

Biological containment of genetically modified Lactococcus lactis for intestinal delivery of human interleukin 10

Genetically modified Lactococcus lactis secreting interleukin 10 provides a therapeutic approach for inflammatory bowel disease. However, the release of such genetically modified organisms through clinical use raises safety concerns. In an effort to address this problem, we replaced the thymidylate synthase gene thyA of L. lactis with a synthetic human IL10 gene. This thyA− hIL10+ L. lactis strain produced human IL-10 (hIL-10), and when deprived of thymidine or thymine, its viability dropped by several orders of magnitude, essentially preventing its accumulation in the environment. The biological containment system and the bacteriums capacity to secrete hIL-10 were validated in vivo in pigs. Our approach is a promising one for transgene containment because, in the unlikely event that the engineered L. lactis strain acquired an intact thyA gene from a donor such as L. lactis subsp. cremoris, the transgene would be eliminated from the genome.

Stability and compatibility of morphine.

Morphine is a widely used analgesic for the treatment of severe cancer pain. For a large number of terminally ill patients oral administration is no longer possible and morphine is administered parenterally using portable pumps allowing comfortable treatment of the patient at home. In this situation the storage of pre-filled reservoirs and/or the administration over a longer period of time are daily practices and require data on the stability of morphine solutions. As most of these patients suffer from several other symptoms, the administration of admixtures with other drugs is common and requires information on the compatibility of morphine. Morphine degrades in aqueous solutions with the formation of mainly pseudomorphine, to a lesser extent morphine-N-oxide and probably apomorphine. From the study of the kinetics of morphine degradation it was concluded that the degradation of morphine is accelerated in the presence of oxygen and at higher pH of the solution, whereas temperature and light have only a minor influence on the degradation rate. The data reported on the stability of morphine infusion solutions kept under ambient conditions indicated that oxygen, light, the type of reservoir, the type of diluent, the salt form and the concentration of morphine do not affect the stability of morphine solutions stored for up to 3 months. Morphine solutions should preferably be stored at room temperature in order to avoid precipitation at low temperatures and water evaporation at higher temperatures causing increase in morphine concentration when stored in polymer reservoirs. Analyzing the data available on the compatibility of morphine infusion solutions revealed that differences in the formulation of the drug solutions (drug concentration, salt form, type and concentration of additives) and diluent, as well as temperature and order and ratio of mixing might affect the compatibility. Only few reports provide all necessary information, limiting the information useful for daily practice. Moreover, the majority of the compatibility studies are performed in intensive care units, where other drugs and other concentrations of morphine are required than in palliative care settings, limiting its merit for this sector.

Continuous twin screw extrusion for the wet granulation of lactose.

The suitability of continuous twin screw extrusion for the wet granulation of alpha-lactose monohydrate was studied and compared with conventional high shear granulation. The influence of process parameters (screw speed and total input rate) and formulation variables (water and polyvinylpyrrolidone (PVP) concentration) on the properties of granules (yield, particle size distribution, friability and compressibility) and tablets (tablet tensile strength, friability and disintegration time) was investigated. Variation of the formulation and process parameters had a major effect on the process feasibility. Optimization of these parameters is required to allow continuous processing and to ensure a high yield. Total input rate, screw speed and water concentration had a minor influence on the granule and the tablet properties. The addition of PVP had no major influence on the granule properties, but significantly affected the tablet characteristics. For granules formulated with and without PVP a yield above 50%, a friability below 30% and a compressibility below 15% was obtained. Tablets without PVP showed a tensile strength below 0.6 MPa, a friability above 1% and a disintegration time below 3 min, whereas tablets with PVP showed a tensile strength above 0.6 MPa, a friability below 1% and a disintegration time ranging from 8 to 15 min. High shear granulation was only possible when PVP was added and it required a higher amount of water. It was concluded that wet granulation of alpha-lactose monohydrate using continuous twin screw extrusion is a robust process and might offer a suitable alternative for high shear granulation in the pharmaceutical industry.

Evaluation of a mucoadhesive tablet for ocular use

The present study investigates the use of a polymer mixture containing Carbopol 974P and drum dried waxy maize starch to obtain prolonged drug release to the anterior eye segment. Two dosage forms with this composition are compared: a hydrated polymer dispersion and a minitablet. A model fluorescent tracer is used to study the ocular release and diffusion from the two dosage forms in humans. To evaluate the prolongation in the cornea/tearfilm compartment, the Apparent Fluorescein TurnOver (%/min) is calculated. The parameters Cmax, tmax, and C9h are used to characterize the pharmacokinetics of Na-fluorescein in the anterior chamber. Furthermore, the swelling behavior of the minitablet is evaluated macroscopically, while the degree of interaction with mucin is characterized by rheological measurements. Calculation of an acceptability score and a slug irritation potential is performed to evaluate user acceptability. In contrast to the hydrated dispersion, the minitablet significantly decreases the Apparent Fluorescein TurnOver (%/min) (P<0.05) and increases the apparent fluorescence in the anterior chamber 9 h after application of the preparation. Rheological data demonstrate the presence of elastic interactions between the polymer and mucin. The dry core of the minitablet becomes fully hydrated after approximately 2 h and is subsequently transformed into a highly concentrated gel. The acceptability of the minitablet is comparable to that of the polymer dispersion. Prolonging the release of Na-fluorescein to the anterior eye segment is only feasible with the dry preparation.

Characterization and in vivo evaluation of ocular bioadhesive minitablets compressed at different forces.

The influence of the compression force on the physical properties, the in vitro release and the in vivo behavior of ocular minitablets is evaluated in the present study. The bioerodible minitablets (Ø 2 mm, 6 mg) were produced at different compression forces. The crushing strength, friability, water uptake, hydration and swelling of the minitablets both in vitro as well as in vivo after application in the cul-de-sac were evaluated. The friability remained below 1% only for the minitablets made at 0.500 and 0.750 kN. The crushing strength measured was 3.53+/-0.98, 12.34+/-1.69 and 18.64+/-2.37 N for minitablets made at 0.250, 0.500 and 0.750 kN, respectively. The full hydration time equalled 20 and 30 min for minitablets compressed at 0.250 kN and 0.500-0.750 kN, respectively. Increasing the compression force resulted in a decreased swelling capacity. The in vivo release was evaluated in healthy volunteers using a non-invasive method to measure the apparent sodium fluorescein concentration in the tearfilm-cornea compartment as a function of time. The longest residence time of the fluorescent tracer at the administration site was obtained by the minitablets compressed at 0.750 kN. The in vitro release was evaluated with three different dissolution methods: the reciprocating cylinder method, vials in an oscillatory shaking bath and a static method with vials. The best correlation with the in vivo behavior of the matrix minitablets was obtained with the shaking bath method.

Cold extrusion as a continuous single-step granulation and tabletting process

The potential of cold extrusion as a continuous granulation/tabletting technique was investigated. Extrudates (X, 9 mm) were produced using twin-screw extrusion, cut manually into tablets (thickness, 4 mm) and dried at 25 degrees C for 20 h. alpha-Lactose monohydrate (200 M) was used as an excipient, PVP (Kollidon K30) and water as binders, and hydrochlorothiazide as the model drug. The influence of formulation (water content, PVP addition, drug incorporation) and process (total input rate and screw speed) parameters on the process (torque, die pressure, visual evaluation of tablets) and on the tablet properties (tensile strength, friability, disintegration time, porosity) was evaluated. Formulation, as well as process parameters, affected the process feasibility, but had only a minor effect on the tablet properties at conditions that allowed continuous tablet production. All alpha-lactose monohydrate tablets formulated without and with PVP and produced at optimum conditions showed tensile strengths above 0.7 and 1.5 MPa, friabilities below 1.0 and 0.9%, and disintegration times below 1 and 8 min, respectively. This technique allows single-step granulation/tabletting of pure alpha-lactose monohydrate, indicating that cold extrusion could be used as alternative tablet production technique for ingredients with poor compaction properties. As the tablets prepared by extrusion have a much higher porosity compared with conventional tablets, this technique could also be useful for tablet production of formulations with poor disintegrating properties.

Extrusion Granulation and High Shear Granulation of Different Grades of Lactose and Highly Dosed Drugs: A Comparative Study

Formulations containing different lactose grades, paracetamol, and cimetidine were granulated by extrusion granulation and high shear granulation. Granules were evaluated for yield, friability, and compressibility. Tablets were prepared from those granules and evaluated for tensile strength, friability, disintegration time, and dissolution. The different lactose grades had an important effect on the extrusion granulation process. Particle size and morphology affected powder feeding and power consumption, but had only a minor influence on the granule and tablet properties obtained by extrusion granulation. In contrast, the lactose grades had a major influence on the granule properties obtained by high shear granulation. Addition of polyvinylpyrrolidone (PVP) was required to process pure paracetamol and cimetidine by high shear granulation, whereas it was feasible to granulate these drugs without PVP by extrusion granulation. Granules prepared by extrusion granulation exhibited a higher yield and a lower friability than those produced by high shear granulation. Paracetamol and cimetidine tablets compressed from granules prepared by extrusion granulation showed a higher tensile strength, lower friability, and lower disintegration time than those prepared from granules produced by high shear granulation. Paracetamol tablets obtained via extrusion granulation exhibited faster dissolution than those obtained via high shear granulation. For all lactose grades studied, extrusion granulation resulted in superior granule and tablet properties in comparison with those obtained by high shear granulation. These results indicate that extrusion granulation is more efficient than high shear granulation.

Compatibility and stability of ternary admixtures of morphine with haloperidol or midazolam and dexamethasone or methylprednisolone

The concentration range over which compatible admixtures of morphine hydrochloride with haloperidol lactate (Haldol) or midazolam hydrochloride (Dormicum) and dexamethasone-21-sodium phosphate (Decadron and Decadron Pack) or methylprednisolone-21-sodium succinate (Solu-Medrol) can be prepared was determined by visual evaluation of the solutions at 22 degrees C. The compatibility was evaluated for admixtures prepared in a ratio morphine hydrochloride (D1)/drug 2 (D2)/drug 2 (D3) in a ratio 10/1/1 to 10/1/10 (v/v/v). The solutions of morphine hydrochloride used were 10, 20, 30, 40 and 50 mg/ml prepared in water and isotonized with sodium chloride or dextrose. The drug solutions were used undiluted and diluted 1/5 (v/v) in water. All admixtures were prepared by adding the corticosteroid as D2 and as D3 in order to evaluate the influence of the order of mixing on the compatibility. The stability of the drugs in the compatible admixtures was evaluated during storage for 28 days at 22 degrees C and protected from light. Visual inspection, high performance liquid chromatography (HPLC) analysis, pH and osmolality determinations were performed. For each drug combination incompatibility was observed with increasing ratio and/or concentration of the drug solutions. Within the range of compatibility the concentrations of the three drugs could be increased so to allow adequate symptom control with all drug combinations. For a similar admixture a higher concentrations of corticosteroid could be obtained using dexamethasone-21-sodium phosphate versus methylprednisolone-21-sodium succinate and a higher concentration of dexamethasone-21-sodium phosphate could be obtained without incompatibility using Decadron Pack versus Decadron. The admixtures for which the stability was evaluated were stable for 28 days (> 95% of the initial concentration). None of these admixtures showed any visual changes during storage, except for some of the admixtures prepared using undiluted Decadron, in which small crystals were seen after 1-28 days. The initial pH of the admixture ranged from 3.99 to 6.06 and varied less than 0.10 during storage. The initial osmolality of the admixtures ranged from 170 to 323 mOsm/kg and remained almost constant during storage.

The solubility of morphine and the stability of concentrated morphine solutions in glass, polypropylene syringes and PVC containers.

Abstract Morphine solutions are frequently used in palliative settings for the treatment of severe cancer pain. There is, however, no complete information concerning the solubility, isotonisation and shelf-life of these solutions. The solubility limits of morphine hydrochloride (M) were determined as 50 mg/ml in water and 5% dextrose, and 30 mg/ml in 0.9% NaCl at 22°C, figures which decreased to 30 and 20 mg/ml, respectively at 4°C. Isotonisation of the M solutions with NaCl or dextrose did not cause any solubility problems at room temperature. The stability of isotonic M solutions and M solutions in water was investigated over a concentration range of 10–50 mg/ml. All solutions were stored in borosilicate glass, polypropylene syringes and PVC containers at 4, 22 and 40°C in the absence of light. Samples were taken immediately after preparation and after 1, 3, 7 and 14 days, 1, 2 and 3 months of storage. All samples were evaluated visually (colour and precipitation) and pH and osmolality were measured. Determination of morphine, morphine- N -oxide, pseudomorphine and apomorphine was done with a reversed-phase ion-pair HPLC assay. During storage at 4°C of M solutions at a concentration above 20 mg/ml, a white precipitate was formed that was difficult to redissolve. In all samples the pH and the osmolality remained nearly unchanged over the study period, except when stored in PVC containers at 22 and 40°C where there was a gradual increase of the osmolality during storage. In the solutions stored in PVC containers at 22 and 40°C an increase in M concentration of up to 105% of the theoretical concentration was detected after 1 month and 1 week, respectively. In all samples only two degradation products were found: morphine- N -oxide and pseudomorphine. During storage the concentration of both degradation products gradually increased, but remained below 0.4% for morphine- N -oxide and below 2% for pseudomorphine. The type of reservoir and the composition of the solution had only a minor influence on the degradation of M. This study indicates that concentrated M solutions are stable for 3 months under all conditions tested, but should be stored at 22°C to avoid precipitation.

Optimisation of electrical conductance measurements for the quantification and prediction of phase separation in o/w-emulsions, containing hydroxypropylmethylcelluloses as emulsifying agents

Abstract Different hydroxypropylmethylcelluloses, Methocel E and K, and one methylcellulose, Methocel A (Colorcon, Kent), were evaluated for their emulsifying properties. Eight o/w emulsions were prepared with 20%, (w/w) arachidic oil and 80% (w/w) aqueous phase containing the emulsifying agent in different concentrations. Methocel K100-LV, Methocel E50-LV and Methocel A4C were used in a 2% (w/w) concentration, while Methocel A15-LV was used in a 1, 2, 3 and 4% (w/w) concentration. The stability of the emulsions was assessed with four different techniques: visual inspection, electrical conductance, droplet size (Coulter counter) and viscosity measurements. The Methocel A15-LV 1% emulsion showed a visual instability after only 3 days, followed by the 2% emulsion (6 days) and the E50-LV (2%) emulsion (55 days). The Methocel A15-LV 3 and 4%, the Methocel K100-LV (2%) emulsion and the A4C (2%) emulsion showed no visual instability after 90 days. For all emulsions, the difference in electrical conductance between the upper and lower part of the conductance cell, especially designed for the stability study, gradually increased with ageing. Droplet size measurements showed no major differences in droplet size as a function of time, probably due to the fact that before measurements the emulsions had to be gently shaken before sample taking. The conclusion of this study is that the electrical conductance measurements in the specially designed cells can be used to quantify and predict the phase separation in o/w-emulsions.