Closed-loop glycaemic control using an implantable artificial pancreas in diabetic domestic pig (Sus scrofa domesticus).

The performance of a completely implantable peritoneal artificial pancreas (AP) has been demonstrated in principle in a live diabetic domestic pig. The device consists of a smart glucose-sensitive gel that forms a gateway to an insulin reservoir and is designed to both sense glucose and deliver insulin in the peritoneal cavity. It can be refilled with insulin via subcutaneous ports and surgery was developed to insert the AP. Diabetes was induced with streptozotocin (STZ), the device filled with insulin (Humulin(®) R U-500) in situ and the animal observed for several weeks, during which time there was normal access to food and water and several oral glucose challenges. Blood glucose (BG) levels were brought down from >30 mmol/L (540 mg/dL) to non-fasted values between 7 and 13 mmol/L (126-234 mg/dL) about five days after filling the device. Glucose challenge responses improved ultimately so that, starting at 10 mmol/L (180 mg/dL), the BG peak was 18 mmol/L (324 mg/dL) and fell to 7 mmol/L (126 mg/dL) after 30 min, contrasting with intravenous attempts. The reservoir solution was removed after 8 days of blood glucose levels during which they had been increasingly better controlled. A rapid return to diabetic BG levels (30 mmol/L) occurred only after a further 24 days implying some insulin had remained in the device after removal of the reservoir solution. Thus, the closed loop system appeared to have particular influence on the basal and bolus needs for the 8 days in which the reservoir solution was in place and substantial impact for a further 3 weeks. No additional insulin manual adjustment was given during this period.

Target Mediated Drug Disposition Model of CPHPC in Patients with Systemic Amyloidosis.

The amyloid deposits that cause disease in systemic amyloidosis always contain the normal plasma protein, serum amyloid P (SAP) component. SAP is the target of a novel immunotherapy approach now being developed to eliminate amyloid deposits. The treatment is enabled by, and critically depends on, the use of the drug (R)-1-[6-[(R)-2-carboxy-pyrrolidin-1-yl]-6-oxo-hexanoyl]pyrrolidine-2-carboxylic acid (CPHPC, GSK2315698, Ro 63-8695), which depletes circulating SAP almost completely but leaves some SAP in amyloid deposits for specific recognition by subsequently administered therapeutic anti-SAP antibodies. Herein, we report a mechanistic model that predicts, with clinically acceptable precision, the exposure-response relationship for CPHPC, both in healthy individuals and in patients with systemic amyloidosis. The model covariates are gender, renal function, total amyloid load, and presence of hepatic amyloid, all of which are known at baseline. The model is being used to predict individualized dosing regimens in an ongoing, first-in-human study with anti-SAP antibodies.

Covalent coupling of concanavalin A to a Carbopol 934P and 941P carrier in glucose-sensitive gels for delivery of insulin.

A novel glucose-sensitive gel formulation, containing concanavalin A and specific polysaccharides, was stabilised via covalent coupling to two structurally different carbomers. The bonding was done to minimise leaching of gel components thereby preventing toxicity and preserving the working mechanism of the gel. Increased gel stability was introduced by covalently bonding amine groups present on the lysine residues of concanavalin A to carboxylic moieties on Carbopol 934P NF and 941P NF using carbodiimide chemistry. The introduction of dextran then produced a glucose-sensitive formulation that transformed from gel to sol in the presence of free glucose. Rheological examination of glucose-sensitive gels stabilised in this way and containing varying concentrations of glucose was conducted with a cone and plate viscometer used in continual rotation mode. A decrease in viscosity over the chosen glucose concentration range was exhibited by both carbomer-stabilised formulations. The subsequent testing of such formulations in in-vitro diffusion experiments revealed that the leaching of concanavalin A from the covalently coupled gels is restricted significantly with respect to non-coupled formulations. In addition, insulin delivery in response to glucose in the physiologically relevant glucose concentration range has been demonstrated using the carbomer-stabilised gels at 37 degrees C. The performance of this self-regulating drug delivery system has been improved in terms of increased gel stability with reduced component leaching.

A covalently stabilised glucose responsive gel formulation with a Carbopol carrier.

A novel glucose-responsive gel formulation was stabilised via covalent coupling to a carbomer resin. The gel formed between the plant lectin, concanavalin A and specific polysaccharides was stabilised to minimise leaching of gel components into the surroundings. This was required to prevent toxicity and to preserve the working mechanism of the formulation. Increased gel stability was introduced by covalently bonding amine groups present on the lysine residues of concanavalin A to carboxylic moieties on Carbopol 974P NF using carbodiimide chemistry. The introduction of dextran then produced a glucose-responsive formulation that transformed from gel to sol in the presence of free glucose. The rheological properties and in vitro component and insulin release of the carbomer-stabilised gel were evaluated. A decrease in viscosity over a chosen glucose concentration range was exhibited by the carbomer-based gel. The testing of such a formulation in in vitro diffusion experiments revealed that the leaching of concanavalin A from the covalently coupled gels was restricted significantly with respect to a non-coupled gel. Insulin delivery in response to glucose in the physiologically relevant glucose concentration range was demonstrated using the carbomer-stabilised gel at 37 degrees C. The performance of this novel self-regulating drug delivery system has been improved in terms of increased gel stability with reduced component leaching.

In vitro iontophoretic release of lithium chloride and lidocaine hydrochloride from polymer electrolytes.

Ionically conducting polymers, frequently known as polymer electrolytes, are potential candidates as hosts for drugs to be delivered iontophoretically. The iontophoretic delivery of lithium or lidocaine from polymer electrolyte films through a cellophane membrane was examined using different delivery current regimes. Thin, mechanically strong, polymer electrolyte films were fabricated from poly(ethylene oxide) (PEO) with lithium chloride or lidocaine hydrochloride. Experiments showed that iontophoretic transport of both lithium chloride and lidocaine hydrochloride might be achieved from these PEO-based films. Cation transport number determinations give values for PEO-based films of about 0.4 for lithium chloride systems and 0.12 for lidocaine hydrochloride systems. The mechanism of transport from these PEO-based polymer electrolyte films allows the delivery of ionic salts such as lithium chloride and lidocaine hydrochloride to be controlled solely by current, thus providing a system that can deliver precise amounts of drug.

Physical characterization of polymer electrolytes as novel iontophoretic drug delivery devices.

Polymer electrolytes are solid-like materials formed by dispersing a salt at the molecular level in a high molecular weight polymer such as poly(ethylene oxide) (PEO). They have been extensively studied for use in electrochemical applications such as batteries and display devices. This paper considers a novel application of polymer electrolytes as the basis of iontophoretic drug delivery systems. Polymer electrolyte films were cast from solutions of PEO and various drug salts using either water or an acetonitrile/ethanol mixture as the solvent. These films were characterized by variable-temperature polarizing microscopy (VTPM), differential scanning calorimetry (DSC), and alternating current (AC) impedance analysis. The films were around 100-micron thick and mechanically strong; the optical and thermal methods provided evidence that the polymer electrolytes had crystalline and amorphous phases, although some drugs may exist in films as nanodispersions. The amorphous phase is important as ions have greater mobility in this phase and therefore allow a current to be passed when the material is incorporated into a device such as one suitable for drug delivery by iontophoresis. The AC impedance analysis showed that the conductivity of the films varied between 10(-6) and 10(-3) S cm-1, depending on the salt, casting solvent, and temperature. Two drugs in particular were shown to be promising candidates in these systems: lidocaine hydrochloride and lithium chloride.

Differential expression of the vitellogenin gene in the spruce terminal weevil feeding on resistant versus susceptible host trees.

Reproductive maturation and oviposition of the spruce terminal weevil (Pissodes strobi, Peck) are inhibited by resistant Sitka spruce (Picea sitchensis (Bong) Carr.). Vitellogenin is an egg yolk protein precursor which accumulates during the maturation of eggs. We describe the molecular cloning and characterization of a fragment of the vitellogenin gene from the spruce terminal weevil. The DNA sequence of this fragment has high identity to vitellogenin sequences from other insects and less to nematode and vertebrate vitellogenins. It hybridizes on Northern blots to a single 6.0 kb mRNA that is expressed only in females, and only after they have commenced reproductive development. Vitellogenin gene expression is induced by treatment with juvenile hormone, and is differentially regulated in insects feeding on resistant or susceptible trees. The effects of resistance on vitellogenin gene expression were studied to confirm our earlier findings and to provide a sensitive bioassay for identifying resistance factors.

A digital image analysis system for comparing groups of small nematodes.

A digital imaging system was developed for measuring various physical characteristics of individual nematodes and for comparing groups of nematodes. The equipment consists of a microscope, a video camera, a video digitizer, interactive displays, and a computer. Various physical and mathematical methods were incorporated, algorithms devised, and computer software written for image acquisition, editing, and analysis. To test the system, four populations of an isolate of the pinewood nematode, Bursaphelenchus xylophilus, subjected to 100% relative humidity at 22 C for 0, 12, 24, or 48 hours were compared. The results showed that the system can be used to measure physical parameters of individual nematodes and to differentiate groups of nematodes.

Image texture features for cell nuclei based on absorbance surface curvature.

The absorbance values of a cell nucleus, as measured at each pixel location, can be considered as forming a surface in a three-dimensional space. The principal curvatures of differential geometry can then be evaluated for this surface. From these curvatures, two new variables are computed that, when averaged over all pixels in the nucleus, are viewed as image texture measures. These two measures were found to be effective for differentiating insect populations and compared well with other features used in the TICAS system. This paper presents the results of comparing the performance of these two features with the performance of other features for several populations of cells.

Surface curvature as a measure of image texture.

Methods from the theory of surfaces in differential geometry are applied to the gray tone intensity and absorbance surfaces defined by a digital image. In particular, the first fundamental form, the second fundamental form, the two principal curvatures, the Gaussian curvature, and mean curvature from classical differential geometry are presented. New curvatures are introduced which appear to be more appropriate for the purposes of image texture. For each curvature, a texture feature is generated. These features are applied to biological cell nuclei and it is found that they are useful for purposes of discrimination.

A computer-assisted cell identification system.

A computer and optical microscope image processing system for the acquisition, editing and analysis of quantitative, multivariate, cell nuclear data is described. The experimental equipment and methods are discussed, the mathematics used are presented, and the additions, modifications and deletions to the TICAS 11/45 set of computer programs for the analysis of the data are outlined.

A nonparametric approach to the discrimination of two cell populations.

An approach to the statistical testing of differences between two cell populations the elements of which are characterized by multivariate data is described. The approach is based on the Fisher discriminant and the Kruskal-Wallis test. The method, which is sufficient but not necessary, makes no assumptions about the normality of the data or about the equality of the covariance matrices for each population.

Use of pectinase to dissociate plant nuclei for squash preparations: effect of hydration procedures.

Scale tissues taken from cones of pendent Douglas-fir Pseudotsuga menziesii (Mirb.) Franco were treated with pectinase to dissociate nuclei for Feulgen staining. Hydration of fixed tissues with water or a graded series of ethyl alcohol before pectinase treatment resulted in distention and faint staining of nuclei and inadequate disintegration of cell walls. These problems were overcome by adopting citrate buffer hydration. Analysis of digitized scans of nuclei provided information about the distribution of the stained material in density classes and nuclear size which is usually unavailable through the conventional double wavelength DNA measuring method.

Image enhancement for light microscopy.

Digitized light microscope images of low contrast histological samples were processed through image enhancement computer techniques. Original digital data and their square root transformations were linearly expanded to produce a wider range of brightness levels in the enhanced images. These treatments resulted in significantly improved images compared to those obtained by direct photomicrography.