Characterisation of the porcine cytokines which activate the CD131ßc common sub-unit, for potential immune-augmentation.

Early acting cytokines and growth factors such as those of the CD131 ßc subunit, may offer an alternative method to the current use of antibiotics and chemicals such as anthelmintics in maintaining Porcine (Po) health. Thus far, the recombinant Po (rPo) Granulocyte-macrophage colony-stimulating factor (GM-CSF), rPo interleukin-3 (IL-3) and rPo interleukin-5 (IL-5) proteins have been identified and cloned and the biological activity of each cytokine has been confirmed in vitro, however, in vivo immune system regulation and hematopoietic stem cell (HSC) augmentation are regulated by numerous cytokines and cellular signals within the bone marrow (BM) niche. In order to quantify the use of recombinant cytokines in augmenting the immune response, it is necessary to determine the stages of hematopoiesis induced by each cytokine and possible areas of synergy requiring further investigation. Here we used the chemotherapeutic agent 5-fluorouracil (5-FU), to chemically induce a state of myelosuppression in young pigs. This allowed for the monitoring of both the autologous BM reconstitution and recombinant cytokine induced BM repopulation, precursor cell proliferation and cellular differentiation. The recombinant cytokines PoGM-CSF, PoIL-3 and PoIL-5 were administered by intramuscular injections (i.m.) following confirmation of 5-FU induced leukocytopenia. Blood and BM samples were collected and then analysed for cell composition. Statistically significant results were observed in several blood cell populations including eosinophils for animals treated with rPoIL-5, rPoGM-CSF and basophils for animals treated with rPoIL-3. BM analysis of CD90+ and CD172a+ cells confirmed myelosuppression in week one with significant results observed between rPoIL-3 and the 5-FU control group in week two and for the rPoGM-CSF group in week three. These results have demonstrated the effects of each of these rPo cytokines within the hematopoietic processes of the pig and may demonstrate similar outcomes in other mammalian models including human.

Expression of biologically active recombinant porcine interleukin-12 from Escherichia coli.

The control of viral infections is of critical importance to livestock industries worldwide and is highlighted by costly infection outbreaks, such as that seen with foot and mouth disease virus. To ameliorate the impact of increasing problems with viral infections, new vaccine and anti-viral strategies are required and a greater understanding of the anti-viral response is essential. Furthermore, in pigs, evidence is still being gathered on the components of a defined anti-viral immune response. However, this has been greatly improved by the recent cloning and expression of critical cytokines involved in the anti-viral response. To assess the use of recombinant porcine interleukin-12 (rPoIL-12) as an immunotherapeutic and immunomodulator of swine, we have cloned and expressed rPoIL-12 as a single-chain fusion protein from Esherichia coli (E. coli). The fusion encodes the p40 and p35 subunits, linked by a glycine-serine linker and expressed as a C-terminal 6xHis tagged protein. rPoIL-12 stimulated the proliferation of human lymphoblasts and its activity on porcine cells was demonstrated by the ability of rPoIL-12 to increase the mRNA expression of porcine interleukin-18 receptor-alpha (poIL-18Ralpha) from porcine peripheral blood mononuclear cells (PoPMBCs). This data supports the inclusion of E. coli produced rPoIL-12 in immunomodulation strategies in the pig.

Quantifying amorphous content of lactose using parallel beam X-ray powder diffraction and whole pattern fitting.

The objective of this study was to demonstrate the applicability of parallel beam X-ray powder diffraction (XRPD) and a new method for whole pattern fitting to the quantification of the residual amount of amorphous content in a pharmaceutical solid using lactose as a model system. Lactose monohydrate, prepared by slurry conversion of anhydrous lactose, was mixed with different amounts of amorphous lactose produced by lyophilization. X-ray powder diffractograms of each mixture were recorded and analyzed by whole pattern fitting using Percentage Crystallinity Determination Software from Kratos Analytical Inc. The polycapillary X-ray optic, which provides a parallel beam of X-radiation, has advantages over Bragg-Brentano Optics with respect to sample height artifacts. Significant shifts in peak position with changes in sample height of lactose monohydrate were observed using Bragg-Brentano Optics while no change was detected for the polycapillary X-ray optic. A technique to normalize all diffractograms to have the same total integrated intensity was necessary to eliminate tube fluctuation effects. After normalization, the amorphous content of lactose in the range of 1-10% was reproducibly predicted (small standard deviation between samplings) using whole pattern fitting. The limit of detection was calculated to be 0.37% amorphous content. The results indicated that parallel beam XRPD and whole pattern fitting can provide accurate analysis of relatively small amounts of amorphous content in pharmaceuticals compared to typical XRPD analysis.

Theoretical approaches to physical transformations of active pharmaceutical ingredients during manufacturing processes.

Processing-induced transformations (PITs) during pharmaceutical manufacturing are well known but difficult to predict and often difficult to control. This review of the concepts of transformations is couched in terms of the issues associated with identifying rate-controlling events from the materials side and the processing side. Specifically, the approach is reconciling the characteristic time scale of the structural change(s) in the material with the time scale of the processing-induced stress. This is definitely a model (or rather a melding of a group of existing theories) in development. This overview is a 'snapshot' of the authors' attempts to identify the categories of existing theories needed to encompass all of the relevant events for each possible PIT. The ultimate goal is to establish a framework of concepts and theories for consideration, discussion, and modeling of PITs as well as to locate much of the relevant literature in the framework.

Induction of inducible nitric oxide synthase-NO* by lipoarabinomannan of Mycobacterium tuberculosis is mediated by MEK1-ERK, MKK7-JNK, and NF-kappaB signaling pathways.

Nitric oxide (NO*) expression by inducible nitric oxide synthase (iNOS) is an important host defense mechanism against Mycobacterium tuberculosis in mononuclear phagocytes. The objective of this investigation was to examine the role of mitogen-activated protein (MAP) kinase (MAPK) and nuclear factor kappaB (NF-kappaB) signaling pathways in the regulation of iNOS and NO* by a mycobacterial cell wall lipoglycan known as mannose-capped lipoarabinomannan (ManLAM). Specific pharmacologic inhibition of the extracellular-signal-regulated kinase (ERK) or NF-kappaB pathway revealed that both these signaling cascades were required in gamma interferon (IFN-gamma)-ManLAM-induced iNOS protein and NO2- expression in mouse macrophages. Transient cotransfection of dominant-negative protein mutants of the c-Jun NH2-terminal kinase (JNK) pathway revealed that the MAP kinase kinase 7 (MKK7)-JNK cascade also mediated IFN-gamma-ManLAM induction of iNOS promoter activity whereas MKK4 did not. Overexpression of null mutant IkappaBalpha, a potent inhibitor of NF-kappaB activation, confirmed that the IkappaBalpha kinase (IKK)-NF-kappaB signaling pathway enhanced IFN-gamma-ManLAM-induced iNOS promoter activity. By contrast, activated p38mapk inhibited iNOS induction. These results indicate that combined IFN-gamma and ManLAM stimulation induced iNOS and NO. expression and that MEK1-ERK, MKK7-JNK, IKK-NF-kappaB, and p38mapk signaling pathways play important regulatory roles.

Determination of average crystallite shape by X-ray diffraction and computational methods.

The objective of this work was to develop a method to estimate the average shape and habit of organic crystalline material using X-ray powder diffraction (XRPD), the single-crystal structure, and computational methods. It is proposed that the relative intensities of the peaks in an XRPD pattern from a sample exhibiting a "standard" preferred orientation correlates with the shape of the crystallites present. Models were developed to yield a quantitative "enhancement" factor for each face. The combined simple-forms morphology (CSM) of the material was then produced by indexing the observed faces and modifying the simulated Bravais-Friedel-Donnay-Harker (BFDH) morphology. The average shape of crystallites can be estimated from the CSM by multiplying each face by its enhancement factor. Acetaminophen crystals in two different habits and ibuprofen crystallized from toluene were used. The predicted shapes closely resembled the average shapes observed with microscopy. Results suggested the average shapes of the organic crystalline materials can be estimated by XRPD and the computational simulation. The current limitations are the need to "index" the faces, the size of the crystallites, and the unknown impact of a polydisperse size distribution on the calculation. The method must be used within the limits described; however, it is the only method found that may be adapted to large, more representative sample sizes. The determination of the average morphology is often a "bottle neck" in elucidating other important behaviors of large quantities of crystalline powders used in pharmaceutical development and processing.

Accelerated fluid bed drying using NIR monitoring and phenomenological modeling.

A "fast-drying" method to accelerate the fluid bed drying process is presented. It relies on concepts of heat and mass transfer with real-time near-infrared (NIR) monitoring of moisture. Triplicate trials show that fast drying can reduce granulation drying time by half over single-temperature cycles. The product is equivalent in every way tested to material made using a conventional cycle even though the inlet temperature throughout the constant-rate stage was higher than the melting point of the compound. Tablets made from the fast-dried granulation exhibit equivalent physical characteristics to tablets made from granulations dried at a single, lower temperature.

Moisture sorption behavior of selected bulking agents used in lyophilized products.

To develop a rational approach for the formulation of lyophilized products, six bulking agents commonly used in freeze-dried formulations were lyophilized under identical conditions, and their moisture sorption behavior, before and after lyophilization, were determined as a function of relative humidity at 25 degrees C. The bulking agents evaluated were mannitol, anhydrous lactose, sucrose, D(+)-trehalose, dextran 40 and povidone (PVP K24). The materials were also characterized for their crystal and thermal properties by powder X-ray diffraction, DSC and TG after exposure to various relative humidity conditions. Mannitol was crystalline and non-hygroscopic both before and after lyophilization with total moisture contents of 0.1 to 0.3% w/w between 10 and 60% RH. Anhydrous lactose, sucrose and trehalose were crystalline prior to lyophilization with moisture contents of 0.86, 0.15 and 9.2%, respectively, and the crystalline materials were relatively non-hygroscopic. Upon lyophilization, they converted to the amorphous form and had moisture contents of 1.6, 2.5 and 1.2%, respectively. The amorphous materials sorbed moisture rapidly upon exposure to increasing relative humidity conditions. The amorphous lactose converted to its crystalline hydrate form at 55% RH after sorption of an additional 10% moisture. This conversion to the crystalline hydrate form was accompanied by desorption of practically all the moisture sorbed by the amorphous form. Similarly, lyophilized sucrose converted to its crystalline form after the sorption of additional 4.5% moisture at 50% RH, and the lyophilized trehalose sorbed additional 10% moisture prior to its conversion to a crystalline hydrate form at 50% RH. Dextran and povidone were amorphous and hygroscopic both before and after lyophilization and they sorbed as much as 10-20% moisture at 50% RH. It is well established that different drugs, especially proteins, need different levels of moisture for optimal stability. The results of the present study show that moisture contents of lyophilized cakes may be varied and optimized by the selection of suitable excipients.

Selection of solid dosage form composition through drug-excipient compatibility testing.

A drug-excipient compatibility screening model was developed by which potential stability problems due to interactions of drug substances with excipients in solid dosage forms can be predicted. The model involved storing drug-excipient blends with 20% added water in closed glass vials at 50 degrees C and analyzing them after 1 and 3 weeks for chemical and physical stability. The total weight of drug-excipient blend in a vial was usually kept at about 200 mg. The amount of drug substance in a blend was determined on the basis of the expected drug-to-excipient ratio in the final formulation. Potential roles of several key factors, such as the chemical nature of the excipient, drug-to-excipient ratio, moisture, microenvironmental pH of the drug-excipient mixture, temperature, and light, on dosage form stability could be identified by using the model. Certain physical changes, such as polymorphic conversion or change from crystalline to amorphous form, that could occur in drug-excipient mixtures were also studied. Selection of dosage form composition by using this model at the outset of a drug development program would lead to reduction of "surprise" problems during long-term stability testing of drug products.

Endemic blastomycosis in Mississippi: epidemiological and clinical studies.

In order to clarify the epidemiology and clinical spectrum of endemic blastomycosis, we reviewed the charts of 326 culture and/or histologically proven cases of blastomycosis in Mississippi from 1979 to 1988. Cases were dispersed throughout the state, but counties in central and south-central Mississippi reported 63% of all blastomycosis cases. The average annual incidence rate was 1.3 cases per 100,000 population. The majority of cases were in men (male to female ratio 1.7:1), and most patients were aged in their third through seventh decades (82%). Outdoor occupations were noted for only 28.9% of cases. Cases occurred throughout the year with no significant seasonal peak. Although 55% saw a physician within 7 days of onset of illness, 29% presented after 1 month. Despite early presentation, diagnosis was often delayed for more than 1 month (43.3%). Single organs were involved in 82.8% of cases. For all cases, organ systems involved included lungs (91.4%), skin (18.1%), bone (4.3%), genitourinary system (1.8%), and central nervous system (1.2%). The presence of skin or bone disease was associated with multiorgan involvement. Thirty-three patients died (11.5%), 6 of whom received no therapy. Patients who died were significantly older than those who survived. A successful outcome without relapse was noted in 86.5% of amphotericin B-treated patients and in 81.7% of ketoconazole-treated patients. The relapse rate for ketoconazole-treated patients was higher than for amphotericin B-treated patients (14% and 3.9% respectively).

Investigation of film curing stages by dielectric analysis and physical characterization.

A dielectric analysis (DEA) method was developed to monitor the curing of Eudragit RS30D plasticized with 20% acetyltributyl citrate and to investigate the effects of over curing on the barrier properties of the film. Permittivity (epsilon') obtained from DEA is related to the degree of mobility of permanent dipoles present in the sample. Changes in the epsilon' values were observed in various film sample types cured at 60 degrees C. Four distinct regions of curing were identified from the epsilon' versus time curves; namely, under-cured, optimally-cured, over-cured, and super-cured. The epsilon' values were high in under-cured films, and decreased to a minimum when the films became optimally-cured. Continued curing beyond the optimum stage caused an increase in the epsilon' values, corresponding to an over-cured stage, whereas in the super-cured region, the values decreased again. The changes in dielectric properties manifested the changes in the structure of the film, which correlated with the dissolution rates of the active drug from the film-coated beads as well as with the changes on their surface morphology at the same stage of cure. A mechanism of film curing was proposed based not only on the dielectric properties and dissolution data but also on the amount of plasticizer remaining in the film-coated beads, the changes in film thickness, and the surface electron micrographs of the cured film-coated beads.

Aggregation behavior of fosinopril sodium--a new angiotensin-converting enzyme inhibitor.

Fosinopril sodium is an effective new angiotensin-converting enzyme (ACE) inhibitor that is very useful for the clinical treatment of hypertension. After oral administration, fosinopril sodium is only partially absorbed (about one third of the drug). We studied the solution behavior of fosinopril sodium in aqueous media by a combination of high-resolution nuclear magnetic resonance (NMR) spectroscopy and laser light scattering (LLS). LLS characterizes the self-association properties of fosinopril sodium in solution, and NMR chemical shifts provide information on molecular conformation and interactions. The results revealed that fosinopril sodium has a micelle-like self-association behavior with a critical micelle concentration (cmc) approximately 1.5 mg/mL. Hydrophobic interactions could induce formation of micellar aggregates, which had a narrow hydrodynamic size distribution, with an average diameter of approximately 150 nm at concentrations above the cmc. The surface activity and self-association of fosinopril sodium may be responsible for its early observed concentration-dependent stability in aqueous solution, unexpected decrease in solubility in the presence of metal ions, as well as the limited absorption in clinical studies.

Dielectric characterization of thermodynamic first order events in model frozen systems intended for lyophilization.

The degree and ease with which permanent dipoles in a frozen sample orient in an applied electric field is affected during thermal transitions. This motion can be monitored with dielectric analysis (DEA) at low AC frequencies (< approximately 300,000 Hz). The systems characterized with respect to their behavior in the frozen state consisted of common lyophile excipients in aqueous solutions known to exhibit typical thermodynamic first order transitions. Prior to freezing, solution samples were placed on an interdigitated electrodes and served as the dielectric component of the resulting capacitor. Plots of derivative of dielectric permittivity with respect to time (or temperature) showed the presence of frequency independent peaks (signifying a first order event) at temperatures that coincided with eutectic temperatures (ca. -22 degrees C for sodium chloride-water and ca. -5 degrees C for mannitol-water), amorphous to crystalline transition temperatures (ca. -25 degrees C for mannitol-water), and/or freezing point depression values in water. The values obtained by DEA correlated well with differential scanning calorimetry (DSC) and literature values. DEA appears to offer added insight over established techniques by not only determining the temperature at which these events occur, but also by defining the thermodynamic order of the event.

Prediction of lyophile collapse temperature by dielectric analysis.

A new method for predicting lyophile collapse temperatures based upon dielectric analysis (DEA) of frozen two component systems is presented. The method, called the take off frequency model (TOF), relies both on the inherent ability of DEA to detect molecular motion and on the abrupt change in viscosity experienced by a frozen sample undergoing a glass-liquid transition. Collapse temperatures for binary glass forming systems (an antibiotic, sucrose, trehalose, or sorbitol, with water) were in good agreement with the values reported in the literature. DEA was easily able to detect glass transitions poorly defined by differential scanning calorimetry (DSC). Conservative lyophilization cycles for simple systems can be quickly determined on the basis of the TOF model.

A lamellar liquid crystal with fosinopril sodium.

The location and conformation of fosinopril sodium (FS) in a lamellar liquid crystal of water, sodium dodecyl sulfate, and decanol was studied by low-angle X-ray diffraction. The result showed the FS molecule to be located within the amphiphilic part of the liquid crystalline structure with the polar parts anchored at the water/polar part interface. An area per molecule in the range of 95-100 A2 showed not only the polar groups but also the benzene ring to be located at this interface.

Solid-state NMR and IR for the analysis of pharmaceutical solids: polymorphs of fosinopril sodium.

The two polymorphic modifications of fosinopril sodium have been characterized as to their differences in melting behaviour, powder X-ray diffraction patterns, Fourier transform infrared spectra (FTIR), and solid-state 31P- and 13C-NMR spectra. The polymorphs were found to be enantiotropically related based upon melting point, heat of fusion, and solution mediated transformation data. Analysis of the solid-state FTIR and 13C-NMR data indicated that the environment of the acetal side chain of fosinopril sodium differed in two polymorphs, and that there might be cis-trans isomerization about the C6-N peptide bond. These conformational differences are postulated as the origin of the observed polymorphism.

Structural properties of polyethylene glycol-polysorbate 80 mixture, a solid dispersion vehicle.

The structural properties of the mixtures of polysorbate 80 with various polyethylene glycols (PEG), viz., PEG 1000, PEG 1450, PEG 3350, and PEG 8000, have been investigated by powder X-ray diffraction (XRD) and differential scanning calorimetric studies. These mixtures may be used as solid dispersion vehicles to insure complete dissolution of poorly water-soluble drugs. Although polysorbate 80 is a liquid at room temperature, the PEG-polysorbate 80 mixtures with up to 75% (w/w) polysorbate 80 were solid. The XRD studies revealed that the crystal structures (d-spacings) of the PEGs (M(r) 1000, 1450, 3350, and 8000) did not change with increasing amounts of polysorbate 80 in the mixture. The intensities of the XRD peaks, however, varied approximately in proportion to the concentration of PEG present. Similarly, the differential scanning calorimetric studies showed that the melting behavior of a PEG-polysorbate 80 mixture was similar to that of the PEG used. The lowering of the mp of a particular PEG due to the presence of 50% (w/w) polysorbate 80 in the mixture was < 6 degrees C, and the decrease in mp was < 12 degrees C in the presence of 75% (w/w) polysorbate 80. When enthalpies of fusion of the mixtures were normalized for the amounts of PEGs present, they were similar to those of pure PEGs. These results indicate that the crystalline structure of PEG in a PEG-polysorbate 80 mixture is substantially the same as that of the pure PEG, and that polysorbate 80 is incorporated into the amorphous region of PEG solid structure.

Stimulation of 22Na+ efflux from rat forebrain membrane vesicles by L-glutamic acid, L-aspartic acid and kainic acid.

A glass fiber filter assay method is described for measuring 22Na+ efflux stimulated by L-glutamic acid, L-aspartic acid and kainic acid from osmotically sensitive membrane vesicles prepared from rat brain. L-Glutamic acid and L-aspartic acid showed the greatest efficacy for the stimulation of 22Na+ efflux with EC50 values of 3 microM. Kainic acid produced 28% of the maximal efflux seen with L-glutamic acid or L-aspartic acid with an EC50 value of 1.5 microM. Quisqualic acid never showed statistically significant increases in 22Na+ efflux over control experiments. N-Methyl-D-aspartic acid showed no detectable efflux activity in this preparation. DL-2-Amino-4-phosphonobutyric acid (APB) inhibited up to 40% of the 50 microM L-glutamic acid-stimulated or 50 microM L-aspartic acid-stimulated 22Na+ efflux with an IC50 value of 1.5 nM. Calcium was required for the inhibitory action of APB, but not for the stimulatory actions of L-glutamic, L-aspartic, or kainic acids. L-Glutamic, L-aspartic, and kainic acids at concentrations above 100 microM were found to inhibit rather than to stimulate 22Na+ efflux. Veratridine (1 microM) had no influence on the 22Na+ efflux component which was produced by L-glutamic or kainic acids. We are unable to firmly establish the mechanism for the stimulated 22Na+ efflux.