Effect of Different Direct Compaction Grades of Mannitol on the Storage Stability of Tablet Properties Investigated Using a Kohonen Self-Organizing Map and Elastic Net Regression Model.

This study tested 15 direct compaction grades to identify the contribution of different grades of mannitol to the storage stability of the resulting tablets. After preparing the model tablets with different values of hardness, they were stored at 25 °C, 75% relative humidity for 1 week. Then, measurement of the tablet properties was conducted on both pre- and post-storage tablets. The tablet properties were tensile strength (TS), friability, and disintegration time (DT). The experimental data were analyzed using a Kohonen self-organizing map (SOM). The SOM analysis successfully classified the test grades into three distinct clusters having different changes in the behavior of the tablet properties accompanying storage. Cluster 1 showed an obvious rise in DT induced by storage, while cluster 3 showed a substantial change in mechanical strength of the tablet including a reduction in the TS and a rise in friability. Furthermore, the data were analyzed using an Elastic net regression technique to investigate the general relationships between the powder properties of mannitol and the change behavior of the tablet properties. Consequently, we succeeded in identifying the crucial powder properties for the storage stability of the resulting tablets. This study provides advanced technical knowledge to characterize the effect of different direct compaction grades of mannitol on the storage stability of tablet properties.

A Precise Prediction Method for the Properties of API-Containing Tablets Based on Data from Placebo Tablets.

We previously reported a novel method for the precise prediction of tablet properties (e.g., tensile strength (TS)) using a small number of experimental data. The key technique of this method is to compensate for the lack of experimental data by using data of placebo tablets collected in a database. This study provides further technical knowledge to discuss the usefulness of this prediction method. Placebo tablets consisting of microcrystalline cellulose, lactose, and cornstarch were prepared using the design of an experimental method, and their TS and disintegration time (DT) were measured. The response surfaces representing the relationship between the formulation and the tablet properties were then created. This study investigated tablets containing four different active pharmaceutical ingredients (APIs) with a drug load ranging from 20-60%. Overall, the TS of API-containing tablets could be precisely predicted by this method, while the prediction accuracy of the DT was much lower than that of the TS. These results suggested that the mode of action of APIs on the DT was more complicated than that on the TS. Our prediction method could be valuable for the development of tablet formulations.

Characterization of Powder- and Tablet Properties of Different Direct Compaction Grades of Mannitol Using a Kohonen Self-organizing Map and a Lasso Regression Model.

The purpose of this study was to accumulate enhanced technical knowledge about the powder properties of direct compaction grades of mannitol that could lead to new tablet formulations. Fifteen different commercial direct compaction grades of mannitol were tested. Ten different powder properties representing flowability, particle size, specific surface area and manufacturing properties were measured. In addition, model tablets of each mannitol grade were prepared, and their disintegration time, friability, and tensile strength were measured. The data were analyzed by principle component analysis and a Kohonen self-organizing map to find correlations between powder properties. Self-organizing map clustering successfully classified the test grades into 5 distinct clusters having different powder properties. Each cluster was well characterized by statistical profiling. Subsequently, the contribution of the powder properties to the tablet properties was investigated by a least absolute shrinkage- and selection operator (Lasso) regression model. Mannitol grades with a larger particle size (D90) were prone to produce tablets with longer disintegration time, while a larger specific surface area of the particles was positively associated with tablets with higher mechanical strength. Our findings provide valuable information for the design of tablet formulations.

T2 Relaxation Study to Evaluate the Crystalline State of Indomethacin Containing Solid Dispersions Using Time-Domain NMR.

The aim of this study was to demonstrate the usefulness of T2 measurements conducted with a time-domain NMR (TD-NMR) for the characterization of active pharmaceutical ingredients (APIs) containing solid dosage forms. A solid dispersion (SD) and a physical mixture (PM) consisting of indomethacin (IMC) and polyvinylpyrrolidone (PVP) were prepared at different weight ratios as test samples, and then their T2 relaxation curves were measured by TD-NMR. The T2 relaxation curve of IMC was quite different from that of PVP by nature. T2 values of the SD and PM samples became gradually shortened with increasing IMC content. No difference in T2 relaxation curves was observed between SD and PM. By analyzing the T2 relaxation curves in detail, we succeeded in precisely quantifying the IMC contents incorporated in the samples. Next, this study evaluated the T2 relaxation curves of amorphous and crystalline states of powdered IMC. T2 relaxation rate of crystalline IMC was slightly but significantly higher than that of amorphous IMC, proving that the T2 measurement was sensitive enough to detect these differences. Finally, a thermal stress was imposed on SD and PM samples at 60°C for 7 d, and then an amorphous-to-crystalline transformation occurred in IMC in the PM sample and was successfully monitored by T2 measurement. We believe that T2 measurement by TD-NMR is a promising analysis for the characterization of APIs in solid dosage forms, including SD-based pharmaceuticals.

A Novel Approach to Evaluate Amorphous-to-Crystalline Transformation of Active Pharmaceutical Ingredients in Solid Dispersion Using Time-Domain NMR.

The aim of this study was to demonstrate the usefulness of the time-domain NMR (TD-NMR) method to characterize the crystalline state of active pharmaceutical ingredients (APIs) containing a solid dispersion. In this study, indomethacin (IMC) was used as a model for poorly water-soluble API. Solid dispersions of IMC were prepared with polyvinylpyrrolidone (PVP) at different weight ratios. First, we measured the T1 relaxation behavior of solid dispersions. From the result, the T1 relaxation time (T1) changed according to the API content; the T1 tended to increase with increasing API content because the T1 value of amorphous IMC was longer than that of PVP. Next, we tried to monitor the amorphous-to-crystalline transformation of IMC in the solid dispersion during the thermal stress test. In the case of solid dispersion containing 90% IMC, a clear prolongation of the T1 could be observed during the thermal stress test. From the powder X-ray diffraction patterns, the change in T1 relaxation behavior must be caused by the IMC transformation from amorphous to crystalline. From these findings, we were successful in monitoring the IMC amorphous-to-crystalline transformation by the changes in T1 relaxation behavior. Our findings led us to conclude that TD-NMR is a novel approach for the evaluation of crystalline state of APIs in solid dispersions.

A Time-Domain NMR Study of the State of Water in Wet Granules with Different Fillers and Its Contribution to the Wet Granulation Process and to the Characteristics of Granules.

The different states of water incorporated in wet granules were studied by a low-field benchtop 1H-NMR time-domain NMR (TD-NMR) instrument. Wet granules consisting different fillers [cornstarch (CS), microcrystalline cellulose (MCC), and D-mannitol (MAN)] with different water contents were prepared using a high-speed granulator, and then their spin-spin relaxation time (T2) was measured using the NMR relaxation technique. The experimental T2 relaxation curves were analyzed by the two-component curve fitting, and then the individual T2 relaxation behaviors of solid and water in wet granules were identified. According to the observed T2 values, it was confirmed that the molecular mobility of water in CS and MCC granules was more restricted than that in the MAN granule. The state of water appeared to be associated with the drying efficiency and moisture absorption capacity of wet granules. Thus, it was confirmed that the state of water significantly affected the wet granulation process and the characteristics of the resultant granules. In the final phase of this study, the effects of binders on the molecular mobility of water in granulation fluids and wet granules were examined. The state of water in granulation fluids was substantially changed by changing the binders. The difference was still detected in wet granules prepared by addition of these fluids to the fillers. In conclusion, TD-NMR can offer valuable knowledge on wet granulation from the viewpoint of molecular mobility of water.

In silico predictions of tablet density using a quantitative structure-property relationship model.

The purpose of this study was to explore the potential of a quantitative structure-property relationship (QSPR) model to predict tablet density. First, we calculated 3381 molecular descriptors for 81 active pharmaceutical ingredients (API). Second, we obtained data that were merged with a dataset including powder properties that we had constructed previously. Next, we prepared 81 types of tablet, each containing API, microcrystalline cellulose, and magnesium stearate using direct compression at 120, 160, and 200 MPa, and measured the tablet density. Finally, we applied the boosted-tree machine learning approach to construct a QSPR model and to identify crucial factors from the large complex dataset. The QSPR model achieved statistically good performance. A sensitivity analysis of the QSPR model revealed that molecular descriptors related to the average molecular weight and electronegativity of the API were crucial factors in tablet density, whereas the effects of powder properties were relatively insignificant. Moreover, we found that these descriptors had a positive linear relationship with tablet density. This study indicates that a QSPR approach is possibly useful for in silico prediction of tablet density for tablets prepared using more than a threshold compression pressure, and to allow a deeper understanding of tablet density.

1H NMR Relaxation Study to Evaluate the Crystalline State of Active Pharmaceutical Ingredients Containing Solid Dosage Forms Using Time Domain NMR.

The aim of this study was to demonstrate the usefulness of the time domain nuclear magnetic resonance (TD-NMR) method to characterize the crystalline state of active pharmaceutical ingredients (APIs) containing solid dosage forms. In this study, carbamazepine and indomethacin are used as models for poorly water-soluble APIs. First, we measured the T1 and T2 relaxation behavior of crystalline and amorphous APIs. From the results, we were able to confirm that the T1 relaxation time measured by TD-NMR is an effective parameter for distinguishing between crystalline and amorphous states in powdered APIs. We then examined physical mixtures of APIs with polyvinylpyrrolidone and their solid dispersion. The results indicated that TD-NMR allows the evaluation of not only the crystalline form of APIs but also the miscibility of APIs and polymers. In the final phase of the study, we conducted continuous monitoring of the crystalline state of APIs incorporated into physical mixtures during the thermal stress test. Conversion to crystalline forms of the APIs was successfully monitored based on the T1 relaxation behavior. Our findings led us to conclude that TD-NMR is useful as a new approach to evaluate the crystalline state of APIs.

Inhibition of Gastric H+,K+-ATPase Activity in Vitro by Dissolution Media of Original Brand-Name and Generic Tablets of Lansoprazole, a Proton Pump Inhibitor.

To investigate the inhibitory effect of a commercial proton pump inhibitor (lansoprazole) on the gastric proton pump H+,K+-ATPase in vitro, we used orally disintegrating (OD) tablets including original brand-name and generic tablets. In the course of the development of generic products, dissolution and clinical tests are necessary to ensure their bioequivalence to the original brand-name products; by contrast, there is almost no opportunity to demonstrate their activity in vitro. This study initially compared the similarity of the dissolution of test generic tablets with that of the original brand-name tablets. The dissolution tests for 15 and 30-mg lansoprazole tablets found their dissolution properties were similar. Subsequently, the dissolution media were sampled and then their effects on the H+,K+-ATPase activity were measured using tubulovesicles prepared from the gastric mucosa of hogs. We confirmed that the inhibitory effects of the generic tablets on H+,K+-ATPase activity were consistent with those of the original brand-name tablets. Furthermore, lansoprazole contents in each tablet estimated from their inhibitory effects were in good agreement with their active pharmaceutical ingredient content. To our knowledge, this is the first technical report to compare the in vitro biochemical activity of lansoprazole OD tablets between the original brand-name and generic commercial products.

Determining the Influence of Granule Size on Simulation Parameters and Residual Shear Stress Distribution in Tablets by Combining the Finite Element Method into the Design of Experiments.

The influence of granule size on simulation parameters and residual shear stress in tablets was determined by combining the finite element method (FEM) into the design of experiments (DoE). Lactose granules were prepared using a wet granulation method with a high-shear mixer and sorted into small and large granules using sieves. To simulate the tableting process using the FEM, parameters simulating each granule were optimized using a DoE and a response surface method (RSM). The compaction behavior of each granule simulated by FEM was in reasonable agreement with the experimental findings. Higher coefficients of friction between powder and die/punch (µ) and lower by internal friction angle (αy) were generated in the case of small granules, respectively. RSM revealed that die wall force was affected by αy. On the other hand, the pressure transmissibility rate of punches value was affected not only by the αy value, but also by µ. The FEM revealed that the residual shear stress was greater for small granules than for large granules. These results suggest that the inner structure of a tablet comprising small granules was less homogeneous than that comprising large granules. To evaluate the contribution of the simulation parameters to residual stress, these parameters were assigned to the fractional factorial design and an ANOVA was applied. The result indicated that µ was the critical factor influencing residual shear stress. This study demonstrates the importance of combining simulation and statistical analysis to gain a deeper understanding of the tableting process.

Modeling of quantitative relationships between physicochemical properties of active pharmaceutical ingredients and tensile strength of tablets using a boosted tree.

OBJECTIVES: The aim of this study was to explore the potential of boosted tree (BT) to develop a correlation model between active pharmaceutical ingredient (API) characteristics and a tensile strength (TS) of tablets as critical quality attributes. METHODS: First, we evaluated 81 kinds of API characteristics, such as particle size distribution, bulk density, tapped density, Hausner ratio, moisture content, elastic recovery, molecular weight, and partition coefficient. Next, we prepared tablets containing 50% API, 49% microcrystalline cellulose, and 1% magnesium stearate using direct compression at 6, 8, and 10 kN, and measured TS. Then, we applied BT to our dataset to develop a correlation model. Finally, the constructed BT model was validated using k-fold cross-validation. RESULTS: Results showed that the BT model achieved high-performance statistics, whereas multiple regression analysis resulted in poor estimations. Sensitivity analysis of the BT model revealed that diameter of powder particles at the 10th percentile of the cumulative percentage size distribution was the most crucial factor for TS. In addition, the influences of moisture content, partition coefficients, and modal diameter were appreciably meaningful factors. CONCLUSIONS: This study demonstrates that BT model could provide comprehensive understanding of the latent structure underlying APIs and TS of tablets.

Relationships between response surfaces for tablet characteristics of placebo and API-containing tablets manufactured by direct compression method.

In this study, we evaluated the correlation between the response surfaces for the tablet characteristics of placebo and active pharmaceutical ingredient (API)-containing tablets. The quantities of lactose, cornstarch, and microcrystalline cellulose were chosen as the formulation factors. Ten tablet formulations were prepared. The tensile strength (TS) and disintegration time (DT) of tablets were measured as tablet characteristics. The response surfaces for TS and DT were estimated using a nonlinear response surface method incorporating multivariate spline interpolation, and were then compared with those of placebo tablets. A correlation was clearly observed for TS and DT of all APIs, although the value of the response surfaces for TS and DT was highly dependent on the type of API used. Based on this knowledge, the response surfaces for TS and DT of API-containing tablets were predicted from only two and four formulations using regression expression and placebo tablet data, respectively. The results from the evaluation of prediction accuracy showed that this method accurately predicted TS and DT, suggesting that it could construct a reliable response surface for TS and DT with a small number of samples. This technique assists in the effective estimation of the relationships between design variables and pharmaceutical responses during pharmaceutical development.

Palmitoylation of LAT contributes to its subcellular localization and stability.

Palmitoylation is a protein modification for trafficking to lipid raft. Without palmitoylation, linker for activation of T cells (LAT), an adaptor molecule mediating T cell receptor signaling, is unable to localize in lipid rafts and to mediate T cell activation. We here show a novel role for palmitoylation in LAT trafficking to the plasma membrane and in the stability of the LAT protein. The human LAT mutant lacking palmitoylation was unable to traffic to the plasma membrane despite the presence of transmembrane portion. The mouse LAT mutant lacking palmitoylation was unstable and susceptible to degradation via the proteasome pathway. The human LAT mutant became unstable when the extracellular portion was swapped for that from mouse, indicating that both palmitoylation and the extracellular portion regulate the stability of LAT. These results suggest that palmitoylation has an important role in trafficking to the plasma membrane and the stability of LAT.

Structure and function of lipid rafts in human activated T cells.

Lipid rafts, specialized membrane microdomains enriched in sphingolipids and cholesterol, have been shown to function as signaling platforms in T cells. Surface raft expression is known to be increased in human T cells upon activation, and this increased raft expression may account for efficient signaling capability and decreased dependency for co-stimulation in effector and/or activated T cells. However, raft-mediated signaling ability in activated T cells remains to be clarified. In this study, we analyzed the structure and function of lipid rafts in human activated T cells. We demonstrated that raft protein constituents are dramatically changed after activation along with an increase in lipid contents. T cells stimulated with anti-CD3 plus anti-CD28 antibodies showed an increase not only in surface monosialoganglioside GM1 expression but also in total amounts of raft-associated lipids such as sphingomyelin, cholesterol and glycosphingolipids. Raft proteins increased after activation include Csk, Csk-binding protein and Fyn, the molecules known to be involved in negative regulation of T cell activation. Consistent with the increase in expression of these proteins, TCR-mediated Ca(2+) response, a response dependent on raft integrity, was clearly inhibited in activated T cells. Thus, the structure and function of lipid rafts in human activated T cells seem to be quite distinct from those in naive T cells. Further, human activated T cells are relatively resistant to signaling, at least transiently, by TCR re-stimulation even though their raft expression is increased.

Ligand-dependent Toll-like receptor 4 (TLR4)-oligomerization is directly linked with TLR4-signaling.

Toll-like receptor 4 (TLR4) and MD-2 recognize lipid A, the active moiety of microbial lipopolysaccharide (LPS). Little is known about mechanisms for LPS recognition by TLR4/MD-2. We here showed, by using in vitro transfectants, ligand-induced TLR4-oligomerization, which required both membrane CD14 and MD-2. We previously reported that lipid IVa, a lipid A precursor, is agonistic on mouse TLR4/MD-2 but antagonistic on human TLR4/MD-2 and chimeric mouse TLR4/human MD-2. Lipid IVa triggered oligomerization of mouse TLR4/MD-2 but not human TLR4/MD-2 or chimeric mouse TLR4/human MD-2. Further, lipid IVa inhibited lipid A-dependent oligomerization of chimeric mouse TLR4/human MD-2. These results demonstrate that ligand-induced TLR4-oligomerization is directly linked with TLR4-signaling and suggest that MD-2 has an important role in regulating TLR4-oligomerization.

Skeletal muscle targeting in vivo electroporation-mediated HGF gene therapy of bleomycin-induced pulmonary fibrosis in mice.

Lung fibrosis is a common feature of interstitial lung diseases, and apoptosis and fibrinogenesis play critical roles in its formation and progression. Hepatocyte growth factor (HGF) is one of the ideal therapeutic agents for prevention of lung fibrosis because of its antiapoptotic and fibrinolytic effects. The aim of this study is to establish nonviral HGF gene therapy of bleomycin-induced lung fibrosis avoiding the viral vector-related side effects. C57BL/6 mice were injected with 3.0 mg/kg body weight of bleomycin intratracheally. Following bleomycin injection, 50 microl of pUC-HGF (1 mg/ml) was injected into each of the quadriceps muscle. Immediately after plasmid injection, in vivo electroporation was performed with pulse generator. Skeletal muscle-targeting electroporation induced transgene expression on day 1 and persisted for 4 weeks, and human HGF was also detected in the lung. In mice transferred with HGF, pathological score (1.0+/-0.3 vs 3.2+/-0.6), TUNEL-positive cell index (4.5+/-1.1 vs 14.2+/-3.1), and hydroxyproline content (9.0+/-1.3 vs 14.4+/-5.1 micromol/g) were significantly reduced compared with the control. Furthermore, survival rate of HGF mice was significantly improved compared with the control. Our data indicate that HGF gene therapy with a single skeletal muscle-targeting electroporation has a therapeutic potential for bleomycin-induced lung fibrosis and this strategy can be applied as a practical gene therapy protocol for various organs.

Lipid A antagonist, lipid IVa, is distinct from lipid A in interaction with Toll-like receptor 4 (TLR4)-MD-2 and ligand-induced TLR4 oligomerization.

Toll-like receptor 4 (TLR4) and MD-2 recognizes lipid A, the active moiety of microbial lipopolysaccharide (LPS). Little is known about mechanisms for LPS recognition by TLR4-MD-2. Here we show ligand-induced TLR4 oligomerization, homotypic interaction of TLR4, which directly leads to TLR4 signaling. Since TLR4 oligomerization normally occurred in the absence of the cytoplasmic portion of TLR4, TLR4 oligomerization works upstream of TLR4 signaling. Lipid IVa, a lipid A precursor, is agonistic on mouse TLR4-MD-2 but turns antagonistic on chimeric mouse TLR4-human MD-2, demonstrating that the antagonistic activity of lipid IVa is determined by human MD-2. Binding studies with radioactive lipid A and lipid IVa revealed that lipid IVa is similar to lipid A in dose-dependent and saturable binding to mouse TLR4-human MD-2. Lipid IVa, however, did not induce TLR4 oligomerization, and inhibited lipid A-dependent oligomerization of mouse TLR4-human MD-2. Thus, lipid IVa binds mouse TLR4-human MD-2 but does not trigger TLR4 oligomerization. Binding study further revealed that the antagonistic activity of lipid IVa correlates with augmented maximal binding to mouse TLR4-human MD-2, which was approximately 2-fold higher than lipid A. Taken together, lipid A antagonist lipid IVa is distinct from lipid A in binding to TLR4-MD-2 and in subsequent triggering of TLR4 oligomerization. Given that the antagonistic activity of lipid IVa is determined by MD-2, MD-2 has an important role in a link between ligand interaction and TLR4 oligomerization.

Nonviral gene gun mediated transfer into the beating heart.

Several techniques for gene transfer into the heart have been developed, including direct injection of naked plasmid DNA into the myocardium and coronary infusion of various viral vectors. However, complications and side effects with those methods have been reported. In this study, to resolve these problems, the authors investigated the feasibility of nonviral gene transfer into the beating heart with the hand held gene gun. The genes pCAGGS/CTLA4-EGFP were coated around the surface of gold particles. Three sizes of gold particles (0.6, 1.0, and 1.6 microm in diameter) and three settings of helium gas pressure (200, 250, and 300 psi) were examined. Gene transfer into the rat beating heart was performed using the hand held gene gun. EGFP expressions were detected by fluorescence microscopy from day 1 to 3 weeks after bombardment. The most prominent expressions were detected with the combination of 1.0 microm gold particles and 300 psi helium gas pressure. In this study, the present authors showed that non-viral gene transfer into the beating heart was feasible with the hand held gene gun. This technique is effective for gene transfer into the heart and may be one of the most useful methods for gene therapy for many cardiovascular diseases in the future.

Essential role for ERK2 mitogen-activated protein kinase in placental development.

BACKGROUND: Extracellular signal-regulated kinase 2 (ERK2) has been implicated in cell proliferation, differentiation, and survival. However, its role in vivo remains to be determined. RESULTS: Here we show that the targeted disruption of the mouse ERK2 gene results in embryonic lethality by E11.5 and severe abnormality of the placenta. In these animals, the labyrinthine layer of the placenta is very thin and few foetal blood vessels are observed. ERK2 mutants can be rescued by the transgenic expression of ERK2, demonstrating that these abnormalities are caused by ERK2-deficiency. Although ERK2-deficient fetuses are much smaller than wild-type littermates, this seems to be secondary to malfunction of the placenta. When the placental defect is rescued by tetraploid-aggregation, ERK2-deficient foetuses grow as well as littermate controls. CONCLUSION: These observations indicate that ERK2 is essential for placental development and suggest that ERK2 in the trophoblast compartment may be indispensable for the vascularization of the labyrinth.