Study on the reduction of chemotherapy induced neutropenia in mice using glucosaminylmuramyl dipeptide.

Neutropenia is a common and often dose limiting side effect of some chemotherapy regimens. The aim of this study was to investigate the ability of an immunomodulator, glycosaminylmuramyl dipeptide (GMDP, CAS 78113-36-7, romurtide) to reduce chemotherapy induced neutropenia. BALB/c mice were treated with 200 mg kg-1 cyclophosphamide (CY) to induce a reversible neutropenia lasting approximately 6-7 days. There was no change in the duration or depth of neutropenia in mice treated with GMDP for 3 consecutive days (2.5 or 25 mg kg-1) starting the day after CY injection. In addition, at the doses used, the time of administration of GMDP relative to CY did not alter this response. However, a marked neutrophilia compared to controls was consistently observed in all cases. Neutrophil counts of up to 14 times the baseline value were measured 6-7 days after the induction of neutropenia. GMDP had no effect in the absence of CY. Less variation was seen in white cell counts of older non-SPF mice treated with CY. When the activity of GMDP (5 mg kg-1) was compared with G-CSF (granulocyte colony stimulating factor, 100 micrograms kg-1) in these animals, GMDP showed a consistent trend to reduce the length of neutropenia, however, under the conditions tested only G-CSF treatment resulted in a significant reduction in the duration of neutropenia. In the 12-week-old mice, the neutrophilia seen with both G-CSF and GMDP was much smaller than in the 8-week-old mice, and was not significantly different from that in control mice treated with CY alone.

Identification and characterisation of Xenopus moesin, a Src substrate in Xenopus laevis oocytes.

The cortical actin cytoskeleton, consisting of actin filaments and actin binding proteins, immediately underlies the inner surface of the plasma membrane and is important both structurally and in relaying signals from the surface to the interior of the cell. Signal transduction processes, initiated in the cortex, modulate numerous cellular changes ranging from modifications of the local cytoskeleton structure, the position in the cell cycle, to cell behaviour. To examine the molecular mechanisms and events associated with cortical changes. We have investigated targets of the protein tyrosine kinase, Src, which is associated with the cortical cytoskeleton, in Xenopus laevis oocytes. When a mRNA encoding an activated form of Src tyrosine kinase (d-Src) is injected into oocytes several changes are observed: proteins are phosphorylated, the rate at which progesterone matures an oocyte to an egg is accelerated, and the cortex at the site of injection appears to contract. Previous studies have implicated actin filaments in the Src-stimulated cortical rearrangements. In this study we identify two actin binding proteins-cortactin and moesin--as Src substrates in Xenopus oocytes that are Src substrates. We cloned and characterised the cDNA encoding one of those, Xenopus moesin, a member of the ezrin/radixin/moesin (ERM) family of actin binding proteins. In addition, we have determined that moesin is recruited to the cortex at the site of Src mRNA injection.

The formation of lactose plugs for hard shell capsule fills.

The purpose of this investigation was to study the effects of lubricant concentration, dosator pressure, and dosator piston height on the properties of lactose plugs. An apparatus designed to simulate Macofar 13/2 capsule filling equipment was used, and the relationship between the variables and the responses was established using a Box-Behnken three-factor, three-level experimental design followed by multiple regression. Plug porosity, both under compression and after ejection, was found to be dependent on dosator pressure. Plug weight and length were dependent on dosator piston height, and uniformity of plug weight was independent of all the factors studied. Plug ejection pressure was dependent on dosator pressure and dosator piston height, even when ejection pressure was corrected for changes in plug length. Lubricant concentration had no significant effect on any plug property, and it must be concluded that 0.5% magnesium stearate provides adequate lubrication for lactose plugs. No interaction between variables was noted, and values of the variables raised to the power 2 had no significant effect, permitting a simplified experimental design to be adopted for future work.

Studies on powder plug formation using a simulated capsule filling machine.

Using an apparatus which simulates the action of a Macofar 13-2 dosating-type capsule-filling machine, the variation in plug weight and density with changing machine parameters has been studied. The piston ejection speed has no effect on plug properties. However increase in compression speed leads to a less consolidated powder plug and hence reduced plug weight. Application of higher pressures reduces plug weight changes, but would be expected to affect release characteristics. Comparison of axial and radial pressures generated by plugs of Starch 1500 and lubricated lactose show significant differences which can be explained by the different consolidation and elastic properties of the two solids.

Use of ATR-FTIR spectroscopy to study the diffusion of ethanol through glycerogelatin films.

The use of ATR-FTIR spectroscopy to study the permeability of a glycerogelatin film is described. Measurement of the diffusion coefficient of ethyl alcohol-d in the film showed excellent reproducibility. Comparison of results from this technique with those previously obtained using an air-flow receptor phase diffusion cell show good agreement in terms of lag time assessed diffusion coefficients. ATR-FTIR spectroscopy revealed time-dependent changes in the composition of the glycerogelatin film during the diffusional process. It was also demonstrated that the concurrent assessment of both diffusant penetration and film composition is feasible.

Construction of an intermittent-motion capsule filling machine simulator.

A pneumatically operated apparatus is described which simulates the forces generated by and the component movements encountered in a Macofar 13/2 dosator type capsule filling machine. Force transducers are fitted to the dosing piston and dosator tip, and the movement of the dosator piston and the powder bed followed by displacement transducers. Calibration of the transducers is described. The output from the transducers is collated, stored and manipulated by microcomputer. The manufacturing parameters which can be studied using this apparatus are discussed.

Pattern formation during gastrulation in the sea urchin embryo.

The sea urchin embryo follows a relatively simple cell behavioral sequence in its gastrulation movements. To form the mesoderm, primary mesenchyme cells ingress from the vegetal plate and then migrate along the basal lamina lining the blastocoel. The presumptive secondary mesenchyme and endoderm then invaginate from the vegetal pole of the embryo. The archenteron elongates and extends across the blastocoel until the tip of the archenteron touches and attaches to the opposite side of the blastocoel. Secondary mesenchyme cells, originally at the tip of the archenteron, differentiate to form a variety of structures including coelomic pouches, esophageal muscles, pigment cells and other cell types. After migration of the secondary mesenchyme cells from their original position at the tip of the archenteron, the endoderm fuses with an invagination of the ventral ectoderm (the stomodaem), to form the mouth and complete the process of gastrulation. A larval skeleton is made by primary mesenchyme cells during the time of archenteron and mouth formation. A number of experiments have established that these morphogenetic movements involve a number of cell autonomous behaviors plus a series of cell interactions that provide spatial, temporal and scalar information to cells of the mesoderm and endoderm. The cell autonomous behaviors can be demonstrated by the ability of micromeres or endoderm to perform their morphogenetic functions if either is isolated and grown in culture. The requirement for cell interactions has been demonstrated by manipulative experiments where it has been shown that axial information, temporal information, spatial information and scalar information is obtained by mesoderm and endoderm from other embryonic cells.(ABSTRACT TRUNCATED AT 250 WORDS)

The use of electron spin resonance to measure microviscosity.

The viscosities of a series of mixtures of glycerol and water were measured by electron spin resonance (ESR), photon correlation spectroscopy and Ostwald viscometry. Close agreement was obtained between the last two methods but viscosity as measured by ESR was always significantly lower. The difference, the magnitude of which depended on the spin probe used, was attributed to interaction between water and glycerol. Similar results were obtained using water-sorbitol and water-sucrose mixtures.

Formulation and in vitro evaluation of pressurized inhalation aerosols containing isotropic systems of lecithin and water.

Reverse micellization of nonionic surfactants in apolar media was applied to the formation of solution phase, pressurized inhalation aerosols, employing soya lecithin (SPC) and water in chlorofluorocarbon (CFC) blends. The use of a 30/70 mixture of trichlorofluoromethane (P11) and dichlorodifluoromethane (P12) resulted in the formation of stable, isotropic systems containing 0.5-2.0% (w/v) SPC and solubilized water; R (moles water/moles SPC), 0.9 to 4.28. In systems containing less than 30% P11, phase separation became apparent, particularly at higher water and surfactant concentrations. Dramatic changes in solution viscosity were noted on increasing R values and were attributed to an increase in asymmetry of SPC micelles. Dynamic fractionation of the output from pressurized aerosols using a four-stage liquid impinger showed that the respirable fraction (as measured by the percentage of emitted droplets with aerodynamic diameters less than 5.5 microns) was highly dependent on SPC concentration and R. A significant correlation between RF and actuator score, based on orifice diameter and length, was also found and confirmed that the highest RF values were achieved with the systems of lowest SPC and water concentrations sprayed through an actuator with the smallest and shortest orifice dimensions. This novel mechanism for the formulation of hydrophilic drugs as solutions within CFC-based pressurized aerosols may offer advantages over the traditional suspension approach to pulmonary drug delivery.

The effect of gelatin grade and concentration on the migration of solutes into and through glycerogelatin gels.

The diffusion of 4-hydroxybenzoic acid and phenobarbitone through glycerogelatin gels was found to be independent of the type of gelatin used. Three types of gelatin, two acid-processed and one alkali-processed were studied, and the bulk viscosities of gels prepared from them was seen to vary considerably. However, the microviscosities of the gels, as measured by ESR, showed no significant differences. Thus microviscosity was the factor governing diffusion. Gelatin concentration in aqueous solutions without glycerol influenced microviscosity and hence diffusion. This is believed to be caused by dissolution of water-soluble fractions of the gelatin. Interstices in the gelatin matrix, though reduced in size when gelatin concentration is raised, are still too large to act as physical barriers to diffusing molecules. It is suggested that hydrated gelatin forms the matrix of glycerogelatin mixtures and that the interstitial fluid, through which migration occurs, consists almost entirely of glycerol and water.

The effect of machine speed on the consolidation of four directly compressible tablet diluents.

The reduction in porosity of a powder bed on compression was found to be a function of the velocity of the punch of the press. Substances which consolidated principally by fragmentation showed relatively little velocity dependence. However, the more important deformation was in a powder's consolidation mechanism, the greater the dependence on punch velocity and hence tablet press speed.

The influence of viscosity on the migration of chloramphenicol and 4-hydroxybenzoic acid through glycerogelatin gels.

Migration of chloramphenicol and 4-hydroxybenzoic acid from solutions in 1-octanol into Glycerol Suppository Base, BP and soft gelatin capsule shells is reported. Rates of migration through the gels, quantified in terms of diffusion coefficients, are given. An electron spin resonance probing technique was used to determine the microscopic viscosity. The latter, rather than the bulk viscosity, was shown to be the major rheological influence on the rate of diffusion.

Punch velocities during the compaction process.

Velocities achieved by the upper punch of an eccentric tablet press during compaction have been compared with those predicted by equations describing the movement of the punch in an empty die. Up to the point of maximum punch penetration, actual speeds are invariably less than the predicted speed, the magnitude of the deceleration being determined by the machine speed and applied force. As the punch withdraws from the die, its speed can exceed that predicted on theoretical grounds, due to the elastic expansion of the tablet assisting punch ejection.

An in-vitro investigation of drug availability from lipophilic solutions.

An in-vitro model, previously described, has been modified and used to investigate the effects of volume and concentration on the release of 4-hydroxybenzoic acid from 1-octanol and isopropyl myristate. Solutions encapsulated in soft gelatin shells have also been examined. The quantity of solute released can be increased by reducing the volume of solvent used to prepare the solution, or by increasing the concentration of the solution. The release profile from an encapsulated solution is different from that obtained with the same, unencapsulated solution. The difference is attributed to absorption of the solute by the capsule shell.

Drug migration into soft gelatin capsule shells and its effect on in-vitro availability.

Analysis of the shells and contents of soft gelatin capsules containing acetomenaphthone, ephedrine, 4-hydroxyenzoic acid or phenobarbitone, dissolved in isopropyl myristate, revealed that the percentage of solute taken up by the shells increased with increasing aqueous solubility of the substrate. Thus no acetomenaphthone, which has a negligible aqueous solubility, was found in the shells, in comparison with 92% of the 4-hydroxybenzoic acid, which has a significant solubility in water. Uptake was not influenced by the solubility in isopropyl myristate. The effect of the oily solvent was studied using blends of 1-octanol and isopropyl myristate in which either 4-hydroxybenzoic acid or phenobarbitone were dissolved. Solute release shows that both release and migration can be predicted from a knowledge of the aqueous solubility of the solute and its partition coefficient between water and the non-polar solvent. Samples of capsules containing 4-hydroxybenzoic acid in isopropyl myristate were withdrawn at various stages of the manufacturing process, and the distributions between shell and contents noted. Most of the transfer took place while the capsules were being dried in rotating basket driers, and at this point 67% of the acid had migrated. This increased to 92% during tray drying, and remained so for at least 6 months after manufacture.

Multiple compression of powders in a tablet press.

Lactose, magnesium carbonate, phenacetin, microcrystalline cellulose and directly compressible starch compacts were repeatedly compressed in a tablet press, the tablets not being ejected from the die between compressions. When the interval between compressions was 1.3 s, all substances showed a decrease in the maximum force exerted by the tablet press, this being attributed to the formation of a structure of lower porosity. As the intervals between compressions is increased, the decrease in force is reduced, and in the case of microcrystalline cellulose and directly compressible starch, the force exerted at the second compression is greater than that exerted by the first.

An in vitro model for assessing drug availability from lipophilic vehicles.

An apparatus for studying the rate of release of a solute from a water-immiscible solvent into an acidic, aqueous liquid, followed by permeation through a simulated lipid membrane, is described. The object was to imitate the absorption of a drug after oral administration in a soft gelatin capsule. Rate constants of transfer were determined for a series of substituted benzoic acids, using octanol and isopropyl myristate as solvents. The quantity of solute in the acidic phase did not correlate with the solvent-water distribution coefficient, but was linearly related to the rate constant for transfer from solvent to water. A dynamic system was therefore postulated, rather than one in which the two phases are in equilibrium. In vivo studies on two of the solutes confirmed the in vitro observations. No simple relationship could be derived between blood concentrations and any in vitro parameter, but the rank order of magnitude of the blood concentrations fitted the postulated dynamic mechanism.

Inter-relationships between solubilities, distribution coefficients and melting points of some substituted benzoic and phenylacetic acids.

Ten 4-hydroxy and 4-alkoxy benzoic and phenylalkanoic acids have been investigated. Solubilities in aqueous buffer at pH 1.2 were determined, together with distribution coefficients between the buffer and either octanol or isopropyl myristate. When plotted against the total number of carbon atoms in the side chains, log octanol/water distribution coefficients gave two parallel straight lines, one for the substituted benzoic acids, and the other for the substituted phenylalkanoic acids. The slopes approximated to 0.5, the generally accepted value for methylene. Similar plots could be obtained with isopropyl myristate, provided the hydroxy acid results were ignored, and also when log aqueous solubilities were plotted against carbon number, although there was considerable scatter. The differences between the distribution coefficient results were explained in terms of solute-solvent interactions, and the scatter attributed to variations in the heats of fusion of the solutes. Yalkowsky's equation (1977), linking aqueous solubilities and melting points with distribution coefficients, was applied to the results, and found to be of limited predictive value.