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We report a new spectroscopic detection scheme for molecular oxygen that achieves roughly two orders of magnitude higher sensitivity for fully rotationally resolved spectra than the current state of the art. Two-color (2 + 1') resonance-enhanced multiphoton ionization (REMPI) via the 3d Rydberg complex yields state-selective spectra with signal comparable to the intense but diffuse C 3sσ 3Πg ← X 3Σg- (2 + 1) REMPI bands without significant saturation or broadening. The resulting increase in sensitivity permitted observation of the very weak 3dπ 1Δ2 ← X 3Σg- transitions and is independent of the intermediate state. This advance in ionization efficiency and quantum state-selective sensitivity for O2 promises to aid physical and chemical studies across a wide variety of fields.

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The purpose of this study was to predict the amounts of water addition suitable for pharmaceutical formulations in wet granulation, using a high-speed mixer or a fluidized bed granulator, before granulation trials. In order to determine the suitable amount of water addition, each excipient was first subjected to kneading with water in a mortar and a refractive near-infrared moisture sensor (IR sensor) measured the amount of water at the powder surface. Further by analysis the plot (output value of the IR sensor vs. amount of added water) for each excipient, the amount of water addition for granulation was determined for it. As a second step, two model formulations were designed and suitable amounts of water for granulation were predicted by summation of the obtained excipient values. The predicted value was compared with the experimental value for high-speed mixer granulation. The predicted and experimental amounts of water addition corresponded for the two model formulations, suggesting that the above method is useful for estimating suitable amounts of addition of water for formulations before granulation trials.

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The dissolution behaviors of carbamazepine (CZP) polymorphs and pseudopolymorphs (form I, form III and dihydrate) and the bioavailabilities (BA) of each form in dogs after oral administration were investigated. Bioavailability tests were carried out at a dose of either 40 mg/body or 200 mg/body. The results of dissolution tests in JP13 first fluid (pH 1.2) at 37 degrees C indicated that the initial dissolution rate was in the order of form III>form I>dihydrate, while form III was transformed to dihydrate more rapidly than form I, resulting in decrease of the dissolution rate. The solubilities of both anhydrates (form I and form III), calculated from the initial dissolution rate of each anhydrate, were 1.5--1.6 times that of the dihydrate. At the dose of 40 mg/body, there were no significant differences in the area under the curve (AUC) between forms; their AUCs were nearly equal to that of CZP solution using polyethyleneglycol 400. These findings suggested that most crystalline powder of each form administered at the low dose was rapidly dissolved in gastrointestinal (GI) fluid. On the other hand, for the dose of 200 mg/body, significant differences in plasma concentration--time curves of CZP among polymorphic forms and dihydrate were observed. The order of AUC values was form I>form III>dihydrate. The inconsistency between the order of initial dissolution rates and that of AUC values at the high dose may have been due to rapid transformation from form III to dihydrate in GI fluids.

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The effects of operating conditions in the spray-congealing process on the release and the micromeritic properties of clarithromycin (CAM) wax matrix were evaluated. CAM wax matrix with 30% CAM, 60% glyceryl monostearate (GM) and 10% aminoalkyl methacrylate copolymer E (AMCE) was manufactured at various atomizer wheel speeds and liquid feed rates with a spray dryer. Release of CAM from the matrix exhibited a two-phase pattern, probably due to the dissolution of the fine portions broken on the surface of the matrix. The slope and the extrapolated y-intercept of the subsequent release pattern were defined as the release rate and the initial amount of release of CAM from the matrix, respectively. These release parameters, as well as the volume median diameter and the specific surface area of matrix, were selected as response variables, and multiple regression analysis was performed. For specific surface area and initial amount of release, a minimum point was observed on the contour curve when the atomizer wheel speed was constant and the liquid feed rate was varied. For the release rate, a maximum point was observed on the contour curve under the same conditions. These points were considered preferable for masking the bitter taste of CAM preparation. Microscopic observation revealed that a small spherical matrix with a smooth surface could be obtained with a high atomizer wheel speed and optimum liquid feed rate. This matrix also possessed excellent properties for taste masking, with small initial amount of release and subsequent high rate of release. In conclusion, the congealing speed of melt droplets was the dominant factor in masking the bitter taste of CAM.

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PURPOSE: To develop an intravenous injectable carrier composed of chitosan derivatives for taxol. METHODS: A chitosan with lauryl groups attached to amino groups to provide the hydrophobic moieties and, carboxymethyl groups attached to hydroxy groups to provide the hydrophilic moieties (N-lauryl-carboxymethyl-chitosan = LCC), was newly synthesized. The solubility of taxol in LCC micelles in aqueous solution was examined. The hemolysis test of LCC and the growth inhibition experiment of taxol-loading micelle using KB cells were also performed as in vitro assay. RESULTS: It was found that LCC solubilized taxol by forming micelles with particle sizes less than 100nm. This particle size was considered effective for passive targeting for tumors. The concentration of taxol in the micellar solution was very high, with a maximum of 2.37mg/mL. This maximum was 1000 times above that in a saturated solution of taxol at pH 7.4. Hemolysis testing as an in vitro assay indicated that LCC was safer than Polysorbate 80 (TO-10M) as intravenous surfactant in terms of induction of membrane damage. As judged by cytostatic activity against KB cells, taxol retained activity even when included in LCC micelles. LCC-entrapped taxol was more effective in cytostatic activity than free taxol in low concentrations. CONCLUSIONS: The results of solubilization capacity examination, hemolysis testing, and cytostatic activity suggest that LCC may be useful as a carrier of taxol.

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Pharmacokinetic parameters for clarithromycin (CAM) and erythromycin stearate (EMS) were obtained from a model including decomposition in the gastrointestinal tract. To confirm the accuracy of the parameters, various physicochemical properties of both drugs were examined. The ratio of the in vivo dissolution rate, the in vivo decomposition rate and the absorption rate between CAM and EMS were well correlated to the ratio of the in vitro intrinsic dissolution rate, the decomposition rate in the acidic solution, and partition coefficient, respectively. One of the reasons for the excellent absorption of CAM compared with that of EMS was the higher stability in the acidic solution and the higher partition coefficient of CAM. These findings indicate that the ratio of the partition coefficient to the decomposition rate constant in acidic solution plays an important role in determining drug bioavailability for macrolide antibiotics.

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The effect of pulverization on the dehydration behavior of 2-[(2-methylimidazol-1-yl)methyl]benzo[i]thiochromen-1-one monohydrochloride hemihydrate (GK-128), a newly developed serotonin3 receptor antagonist, was studied using powder X-ray diffraction analysis, thermogravimetry (TG), and differential scanning calorimetry (DSC). The crystalline forms of GK-128 obtained by pulverizing with a jet mill and an agale mortar were each confirmed to be the same as that of intact GK-128, since the powder X-ray diffraction patterns of the two prepared samples were identical with that of intact GK-128. However, after pulverization by jet mill, the dehydration temperature of GK-128 was markedly lowered and the DSC endothermic peak due to dehydration disappeared. The activation energy for dehydration, calculated by the Ozawa method using TG data, decreased with decreasing particle size and/or crystallinity of GK-128 crystals, e.g., the activation energies for dehydration of intact and jet-milled GK-128 were 128.1 and 75.9 kj/mol, respectively. The results of comparison of powder X-ray diffraction patterns of pulverized GK-128 and intact GK-128 and of determination of the crystal structure of GK-128 suggested that water molecules could be removed easily along the c-axis of GK-128 crystals.

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A new approach to determination of good correlations between in vivo and in vitro dissolution was studied using the optimization technique. Ibuprofen, which exhibits dissolution rate-limiting absorption, was used as a model drug. Ibuprofen capsules of two different release types were prepared, and their in vivo dissolution profiles were obtained from measurements of plasma concentration following oral administration of the capsules to beagle dogs by the mathematical deconvolution method using solution data of oral administration as a weight function. For the dissolution test to correspond to the in vivo dissolution profiles, the test was carried out at 12 levels (9 different sets of conditions) and results were analyzed with the optimization technique to deal with two factors. The first-order rate constant (kappa d) and the dissolution time at 50% (t50%) of the in vivo dissolution were selected for use as the response variables. Regression analysis was performed to describe the in vitro dissolution characteristics as functions of the pH of dissolution medium and paddle rotation speed in the paddle method. The in vivo/in vitro correlation obtained from the kappa d was better than that obtained from the t50%. The optimum conditions for dissolution testing corresponding to the in vivo kappa d were determined to be a pH 6.6 for the dissolution medium and a 56 rpm paddle rotation rate. The experimental data obtained by dissolution testing was well fit by the predicted curve derived from in vivo and in vitro dissolution profiles. This dissolution test is applicable to the formulations containing ibuprofen of particle size within the experimental range.

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PURPOSE: We applied non-invasive and real-time method with in vivo ESR spectroscopy to determining pharmacokinetics and metabolism of lipid emulsion as a drug carrier in living mice. METHODS: A spin-labeled triglyceride (SL-TG) was newly synthesized and lipid emulsion containing SL-TG was prepared. In vivo ESR spectra in mice were observed after intravenous administration of the lipid emulsion. RESULTS: In vivo ESR spectra consisted of three components, coinciding with the in vitro spectra of SL-TG particles, free and immobilized fatty acids. The amount of the components depended on both the observing domain and the period after administration. In the chest, all three components were observed, while SL-TG particle was lacking in the abdomen. The half-life of the lipid particles in the chest was 2 hr. CONCLUSIONS: Non-invasive and real-time analysis of drug carriers in living animal is successfully accomplished using an in vivo ESR method.

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The correlation between in vivo and in vitro dissolution of clarithromycin (CAM) tablets was examined. In vivo dissolution rate constants in the stomach and the intestine were obtained from analysis of the urinary excretion data of CAM following oral administration to humans in the fasting or postprandial state using a pharmacokinetic model including gastrointestinal transit. In the present study, the flow-through cell method with moderate agitation was used, as the in vitro dissolution test related to the in vivo dissolution rate constants. Both the effects of pH of the dissolution medium and the volumetric solvent flow rate on the dissolution rate in the flow-through cell method were examined. The pH of the dissolution medium and the flow rate were related to the in vitro dissolution rate. Therefore, the conditions of the flow-through cell method in correlation with the in vivo dissolution rates in the stomach and intestine were determined by controlling the flow rate at pH 3.0 and 6.8 dissolution medium. The urinary excretion of CAM, simulated by substituting the in vitro dissolution rate constants into the equation, were consistent with the in vivo data. The in vitro tests corresponding to the in vivo dissolution in the stomach and intestine following a single oral administration in the fasting or postprandial state for a CAM tablet were established.

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PURPOSE: The stability of lipid emulsions (LE) containing various cosurfactants (oleic acid, cholesterol, Tween 80, or HCO-60) was evaluated using the maximum total interaction energy, Vtmax and the energy barrier for coalescence, W. METHODS: The Vtmax and W were calculated from the zeta potential and the rate of increase in LE particle size, respectively. RESULTS: The Vtmax and W of LE containing the oleic acid were 0.598 x 10(-19) J and 3.03 x 10(-19) J, respectively, while those of LE without the cosurfactant were 0.141 x 10(-19) J and 1.36 x 10(-19) J. CONCLUSIONS: These findings suggest that oleic acid prevents the flocculation and coalescence of LE. The Vtmax and W of LE containing the cholesterol were 0.435 x 10(-19) J and 0.63 x 10(-19) J, respectively, suggesting that the cholesterol prevents the flocculation of LE but does not affect the coalescence. Analysis of the stability of LE was performed by the separate considerations of the flocculation and coalescence.

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When dissolution is the rate-limiting step in absorption of a drug, both it and gastrointestinal transit can play dominant roles in determining absorption. Therefore, a pharmacokinetic model including the process of dissolution, gastrointestinal transit rate and gastrointestinal pH profile was proposed to explain the behavior of drug formulations in vivo. Clarithromycin (CAM), which exhibits pH-dependent solubility and has high partition coefficients, was used as a drug to test the model, and the effect of a meal on serum concentrations of CAM after a single oral administration to healthy volunteers was evaluated. The results of pharmacokinetic analyses of serum concentration-time data for a single oral administration of CAM to humans in the fasting and postprandial states were well fitted by the new pharmacokinetic model. By substituting the pharmacokinetic parameters obtained into the model equations, the in vivo rate of dissolution could be determined. Pharmacokinetic parameters affecting drug bioavailability were also examined using the model. Factors influencing the behavior of a drug formulation in the gastrointestinal tract were determined. These findings indicate that the model we have proposed can be used to evaluate the behavior of drug formulations in vivo.

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The rates of release of prostaglandin E1 (PGE1) from lipid particles into aqueous solution were obtained by the dialysis method, for parenteral lipid emulsion (Lipo-PGE1) diluted 10-times with buffered solutions of various pH. The findings, which were the rates of release of PGE1, were used to calculate the amount of PGE1 distributed in lipid particles when Lipo-PGE1, diluted 100-times with various pH levels of the buffered solution, was administered by intravenous drip infusion. More than 90% of PGE1 was retained in the lipid particles and intravenously infused when transfusion fluid pH was less than 5.5 and 2 ml of Lipo-PGE1 and 198 ml of transfusion fluid had been mixed and were administered over 2 h. Results from simulation showed that half of the PGE1 was retained in lipid particles and was infused, if Lipo-PGE1 was diluted 100-times with pH 8 transfusion fluid. Though PGE1 was sparingly soluble in an aqueous solution, these findings demonstrated that a significant amount of PGE1 was retained in lipid particles. Thus, this dosage form is expected to be highly effective for a drug delivery of PGE1 in clinical treatment.

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The particle size of lipid emulsion (LE) is changed by flocculation and coalescence. This change in particle size was studied using values obtained for maximum total interaction energy (Vtmax) for flocculation and activation energy for coalescence (E). Vtmax was calculated using DLVO theory, and E was calculated from the rate of increase in particle size in LE. Two LEs (PC99LE and PC70LE) were prepared from lecithins containing 99% and 70% phosphatidylcholine, respectively. The Hamaker constants for PC99LE and PC70LE were found to be 1.4 x 10(-22) J and 3.1 x 10(-21) J, respectively. Vtmax for PC99LE was 4.7 kT at 121 degrees C, and E was 1.5 x 10(-19) J, while Vtmax for PC70LE at 121 degrees C was 151 kT and E was 3.2 x 10(-19) J. These findings suggest that PC99LE readily underwent flocculation and coalescence with increase in particle size, but that the particle size of PC70LE changed little. The degrees of flocculation and coalescence of LE were determined separately using values of Vtmax and E. These parameters are thus quite useful in predicting the stability of LE.

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Chemical mediators released from human hepatic macrophages (HHM phi) in primary cultures were analyzed for their secretory function and probable contribution to the modulation of the host defense system and metabolism in liver cirrhosis. In our basic studies, HHM phi increased dose dependently the release of superoxide (O2-) and interleukin-1 (IL-1) when stimulated by opsonized zymosan, up to 1000 micrograms/dish. PGE2 production showed a relatively narrow range of dose dependency, and larger doses led to a reduction of PGE2 yield in some samples. Next, we compared the mediator release from the HHM phi of patients with liver cirrhosis with that from HHM phi in normal liver. O2- released from HHM phi of 8 patients with liver cirrhosis was significantly decreased (controls, n = 20) (P < 0.01). IL-1 released from the HHM phi of 6 cirrhotic patients tended to be higher than that from the HHM phi of 10 control patients, but the difference was not statistically significant (P < 0.10). PGE2 production, however, was about the same in the two groups. These results suggest that cultured HHM phi have certain basic characteristics in releasing mediators with highly potent specific activities and also that these secretory abilities may change in liver cirrhosis. In conclusion, the analysis of cultured HHM phi may be a very practical way to clarify their inherent abilities and participation in the complicated clinical features of liver cirrhosis.

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Expression of type 1 and type 2 chain carbohydrate antigens during the course of morphogenesis of human embryonic pancreas was investigated using specific monoclonal antibodies and compared with the carbohydrate antigen profiles of human pancreatic cancers. The type 2 chain antigens, such as stage-specific embryonic antigen 1 (Le(x)) and I-antigens, appeared much earlier than the type 1 chain antigens; the epithelial cells of primitive foregut were Le(x)+I-antigen- in the embryos at Carnegie stages 16-23, while the pancreatic primordial cells, which had differentiated from the Le(x)+ gut epithelial cells, were Le(x)-I-antigen+ at Carnegie stages 22-23. The type 1 chain antigens, such as Le(a), Le(b), Le(c), and their sialylated derivatives, were not expressed in any cells at these stages and appeared much later in the pancreas of the 10-12-week embryos, when the primitive pancreatic ductal cells in the primordia exhibited an extensive budding of the daughter cells. At this stage, Le(a) appeared and was expressed strongly in the epithelial cells of primitive pancreatic ducts as well as in the daughter cells that were destined to differentiate into future centroacinar cells; Le(b) was localized in the daughter cells which were to become future acinar cells; and Le(c) was specifically expressed in the daughter cells which were to form future Langerhans islets. With regard to the sialylated derivatives of Le(a), expression of the 2-3 sialyl Le(a) antigen was limited to the epithelial cells of the primitive pancreatic ducts, while the 2-6 sialyl Le(a) antigen was strongly expressed in the future centroacinar cells, which had differentiated from the corresponding daughter cells. Among these antigens, the Le(a) and 2-3 sialyl Le(a) antigens showed the highest incidence in human pancreatic cancer tissues. These results indicate that the expression of these carbohydrate antigens in embryonic pancreas is differentiation dependent and cell lineage specific and that most human pancreatic cancer cells mimic the carbohydrate antigen profile of the epithelial cells of the primitive pancreatic ducts in human embryos.

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Clarithromycin (6-O-methylerythromycin), a new 14-membered macrolide antibiotic, has been studied to clarify its physicochemical properties and stability in acidic solution, as compared with erythromycin (EM). The solubility of clarithromycin (CAM) in distilled water was lower than that of EM and decreased with increasing temperature. The solubilities of CAM and EM in the phosphate buffer solution at 37 degrees C decreased with an increasing pH and kept constant above pH 9. From pH-solubility profiles, the dissociation constants of CAM and EM were determined to be 8.76 and 8.36, respectively. The partition coefficient of CAM took a higher value than that of EM and increased with an increasing pH. In the acidic solution, the decomposition of CAM and EM obeyed the pseudo-first order kinetics. From the decomposition rate constants, the half life (T1/2) of CAM and EM were determined. In pH 1.39, CAM degraded with a T1/2 of 17 min while EM kinetics corresponded to a T1/2 of 3 s. Therefore, CAM was 340-fold more stable in pH 1.39 and markedly more stable in the acidic solution than EM.

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The effect of biological response modifiers, such as interleukin-2 (IL-2) and streptococcal preparation OK432, on the functions of hepatic macrophages was investigated. The macrophages, even with no exogenous stimulation, produced superoxide anion (O2-) and tumor necrosis factor (TNF), displayed cytotoxicity against K562 cells and cytostasis against P815 cells and expressed immune-region-associated antigen (Ia). IL-2 administered in vitro or in vivo enhanced O2- production by hepatic macrophages and the intravenous injection of OK432 also enhanced O2 production. Furthermore, IL-2 added to the culture medium of hepatic macrophages isolated from OK432-injected rats augmented O2- production even more. The TNF production and Ia expression of the macrophages were also increased by the intravenous injection of OK432. As with O2- production, the cytotoxicity of the cells was enhanced by OK432 injection or by IL-2 added to the culture medium and the combination of OK432 and IL-2 augmented their cytotoxicity even more. Thus, the present study suggested that IL-2 and OK432 induce the augmentation of the antitumor activity of hepatic macrophages, partly as a result of the increase in production of O2- and TNF and Ia expression.

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The authors investigated the tissue distribution of two kinds of sialylated derivatives of Lewis A (Le(a)) antigen in patients with cancers of the digestive system using specific monoclonal antibodies, and evaluated the significance of determining the 2-3 and 2-6 sialylated Le(a) antigen levels for the diagnosis of cancer. In most specimens from patients with cancers of the pancreas, biliary tract, stomach, and colon, the 2-3 sialylated Le(a) antigen was strongly expressed in cancer cells. However, 2-6 sialylated Le(a) antigen was less frequently expressed in cancer cells. The former is therefore more specific to cancer than the latter. Also, the serum level of the 2-3 sialylated Le(a) antigen was significantly higher than that of the 2-6 counterpart in patients with cancers of pancreas, biliary tract, stomach, and colon. The resulting ratio of serum 2-3/2-6 sialylated Le(a) antigens was frequently high in patients with malignancy and was low in patients with benign disorders of these digestive organs. Therefore, the 2-3/2-6 sialylated Le(a) antigen ratio is a useful for the differential diagnosis of malignant disorders in these organs. However, liver disorders were found to be exceptional in that both antigens were mostly absent in hepatocellular carcinoma (HCC) cells in immunohistologic examination, as well as in nonmalignant parenchymal liver cells. Only the epithelial cells of the intrahepatic bile ducts expressed the 2-6 sialylated Le(a) antigen strongly, and expressed the 2-3 sialylated Le(a) antigen moderately. The levels of both antigens were sometimes high in patients with liver disorders, but the ratio always remained low in patients with HCC as well as benign liver disorders such as cirrhosis or chronic hepatitis. The sialylated Le(a) antigens, which sometimes accumulate in the sera of patients with HCC, were concluded to originate from the epithelial cells of the proliferating small bile ducts, and those serum antigens cannot be considered as evidence for the presence of liver cancer cells.

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In the present study, the function of hepatic macrophages and the modulation of hepatocytes by sepsis-elicited hepatic macrophages were investigated in rats with induced sepsis. The functional state of hepatic macrophages was determined by the following indicators: phagocytic index, protein-synthesizing capacity, and superoxide (O2-) producing capacity. These indices of changes in hepatic macrophages were much higher in rats with sepsis than in healthy controls. Moreover, the activated hepatic macrophages had some biological properties which were different from those of the resident Kupffer cells. It was found that protein synthesis by cultured hepatocytes was inhibited in the co-culture system of hepatocytes and sepsis-elicited hepatic macrophages, and that the supernatant of hepatic macrophages from rats with sepsis also reduced the protein-synthesizing capacity of cultured hepatocytes. Thus, activated hepatic macrophages may play a role in inducing hepatic dysfunction in sepsis.

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