RESUMO
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.
Assuntos
Anti-Inflamatórios não Esteroides/química , Indometacina/química , Espectroscopia de Ressonância Magnética , Cristalização , Formas de Dosagem , Composição de Medicamentos , Temperatura , Difração de Raios XRESUMO
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.
Assuntos
Espectroscopia de Ressonância Magnética/métodos , Preparações Farmacêuticas/química , Transição de Fase , Cristalização , Difração de PóRESUMO
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.
Assuntos
Composição de Medicamentos/métodos , Preparações Farmacêuticas/química , Espectroscopia de Prótons por Ressonância Magnética/métodos , Água/química , Celulose/química , Umidade , Manitol/química , Tecnologia Farmacêutica/métodos , TemperaturaRESUMO
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.
Assuntos
Análise de Elementos Finitos , Lactose/química , Simulação de Dinâmica Molecular , Ácidos Esteáricos/química , Tamanho da Partícula , Propriedades de Superfície , Comprimidos/químicaRESUMO
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.
Assuntos
Adenosina Trifosfatases/antagonistas & inibidores , Lansoprazol/farmacologia , Inibidores da Bomba de Prótons/farmacologia , Adenosina Trifosfatases/metabolismo , Administração Oral , Animais , Medicamentos Genéricos , Humanos , Lansoprazol/química , Inibidores da Bomba de Prótons/química , Solubilidade , Estômago/citologia , Estômago/enzimologia , Suínos , Comprimidos , Equivalência TerapêuticaRESUMO
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.
Assuntos
Preparações Farmacêuticas/química , Comprimidos/química , Resistência à Tração/efeitos dos fármacos , Celulose/química , Composição de Medicamentos/métodos , Excipientes/química , Peso Molecular , Tamanho da Partícula , Pós/química , Pressão , Ácidos Esteáricos/químicaRESUMO
Toll-like receptors (TLRs) are innate recognition molecules for microbial products, but their direct interactions with corresponding ligands remain unclarified. LPS, a membrane constituent of gram-negative bacteria, is the best-studied TLR ligand and is recognized by TLR4 and MD-2, a molecule associated with the extracellular domain of TLR4. Although TLR4-MD-2 recognizes LPS, little is known about the physical interaction between LPS and TLR4-MD-2. Here, we demonstrate cell surface LPS-TLR4-MD-2 complexes. CD14 greatly enhances the formation of LPS-TLR4-MD-2 complexes, but is not coprecipitated with LPS-TLR4-MD-2 complexes, suggesting a role for CD14 in LPS loading onto TLR4-MD-2 but not in the interaction itself between LPS and TLR4-MD-2. A tentative dissociation constant (Kd) for LPS-TLR4-MD-2 complexes was approximately 3 nM, which is approximately 10-20 times lower than the reported Kd for LPS-MD-2 or LPS-CD14. The presence of detergent disrupts LPS interaction with CD14 but not with TLR4-MD-2. E5531, a lipid A antagonist developed for therapeutic intervention of endotoxin shock, blocks LPS interaction with TLR4-MD-2 at a concentration 100 times lower than that required for blocking LPS interaction with CD14. These results reveal direct LPS interaction with cell surface TLR4-MD-2 that is distinct from that with MD-2 or CD14.
Assuntos
Antígenos de Superfície/metabolismo , Lipídeo A/análogos & derivados , Receptores de Lipopolissacarídeos/fisiologia , Lipopolissacarídeos/metabolismo , Glicoproteínas de Membrana/metabolismo , Receptores de Superfície Celular/metabolismo , Animais , Anticorpos Monoclonais/imunologia , Lipídeo A/antagonistas & inibidores , Lipídeo A/metabolismo , Lipídeo A/farmacologia , Antígeno 96 de Linfócito , Camundongos , Receptor 4 Toll-Like , Receptores Toll-LikeRESUMO
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.
RESUMO
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.
RESUMO
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.
Assuntos
Excipientes , Manitol , Composição de Medicamentos , Tamanho da Partícula , Pós , Comprimidos , Resistência à TraçãoRESUMO
Lipid rafts are known to aggregate in response to various stimuli. By way of raft aggregation after stimulation, signaling molecules in rafts accumulate and interact so that the signal received at a given membrane receptor is amplified efficiently from the site of aggregation. To elucidate the process of lipid raft aggregation during T cell activation, we analyzed the dynamic changes of a raft-associated protein, linker for activation of T cells (LAT), on T cell receptor stimulation using LAT fused to GFP (LAT-GFP). When transfectants expressing LAT-GFP were stimulated with anti-CD3-coated beads, LAT-GFP aggregated and formed patches at the area of bead contact. Photobleaching experiments using live cells revealed that LAT-GFP in patches was markedly less mobile than that in nonpatched regions. The decreased mobility in patches was dependent on raft organization supported by membrane cholesterol and signaling molecule binding sites, especially the phospholipase C gamma 1 binding site in the cytoplasmic domain of LAT. Thus, although LAT normally moves rapidly at the plasma membrane, it loses its mobility and becomes stably associated with aggregated rafts to ensure organized and sustained signal transduction required for T cell activation.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas de Transporte/metabolismo , Ativação Linfocitária , Proteínas de Membrana , Fosfoproteínas/metabolismo , Sítios de Ligação , Colesterol/metabolismo , Citoplasma/metabolismo , Proteínas de Fluorescência Verde , Humanos , Immunoblotting , Isoenzimas/metabolismo , Células Jurkat , Cinética , Proteínas Luminescentes/metabolismo , Microscopia Confocal , Fosfolipase C gama , Fosforilação , Testes de Precipitina , Ligação Proteica , Transporte Proteico , Proteínas Recombinantes de Fusão/metabolismo , Retroviridae/metabolismo , Fatores de Tempo , Transfecção , Fosfolipases Tipo C/metabolismoRESUMO
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.
Assuntos
Carbamazepina/química , Indometacina/química , Química Farmacêutica/métodos , Cristalização/métodos , Formas de Dosagem , Imageamento por Ressonância Magnética/métodos , Polímeros/química , Povidona/química , Pós/química , Espectroscopia de Prótons por Ressonância Magnética/métodosRESUMO
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 200â¯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.
Assuntos
Modelos Químicos , Relação Quantitativa Estrutura-Atividade , Comprimidos/química , Celulose/química , Simulação por Computador , Excipientes/química , Ácidos Esteáricos/químicaRESUMO
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.
Assuntos
Desenho de Fármacos , Comprimidos/química , Acetaminofen/química , Celulose/química , Composição de Medicamentos , Excipientes/química , Lactose/química , Niacina/química , Placebos/química , Pressão , Piridoxina/química , Salicilamidas/química , Amido/química , Ácidos Esteáricos/químicaRESUMO
The earliest biochemical events at the plasma membrane that lead to gene activation appear to depend not only on the local concentration of signaling molecules, but also on the mobility of these molecules at the site of signaling. To elucidate the process of signal transduction after receptor engagement in the immune system, it is important to analyze the mobility of signaling molecules in living lymphocytes. Current knowledge of the changes in intracellular localization and dynamic movements of signaling molecules during lymphocyte activation is limited. Here, we describe a method for known as fluorescence recovery after photobleaching, used to measure the diffusion mobility of a signaling molecule in a T cell line after T cell receptor stimulation. This method is a useful tool in studies of spatiotemporal regulation in immunoreceptor signaling.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Recuperação de Fluorescência Após Fotodegradação/métodos , Linfócitos/química , Proteínas de Membrana , Transdução de Sinais/fisiologia , Animais , Apresentação de Antígeno , Proteínas de Transporte/biossíntese , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Linhagem Celular , Cricetinae , Recuperação de Fluorescência Após Fotodegradação/instrumentação , Imunofluorescência , Proteínas de Fluorescência Verde , Humanos , Células Jurkat , Proteínas Luminescentes/biossíntese , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Ativação Linfocitária/imunologia , Ativação Linfocitária/fisiologia , Linfócitos/metabolismo , Linfócitos/fisiologia , Microdomínios da Membrana/química , Microdomínios da Membrana/metabolismo , Microdomínios da Membrana/fisiologia , Microscopia Confocal , Microesferas , Muromonab-CD3/metabolismo , Fosfoproteínas/biossíntese , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Complexo Receptor-CD3 de Antígeno de Linfócitos T/imunologia , Complexo Receptor-CD3 de Antígeno de Linfócitos T/fisiologia , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais/imunologia , Software , Coloração e Rotulagem , TransfecçãoRESUMO
Toll-like receptor 4 (TLR4) mediates lipopolysaccharide (LPS) signaling in a variety of cell types. MD-2 is associated with the extracellular domain of TLR4 and augments TLR4-dependent LPS responses in vitro. Moreover, mice lacking MD-2 (MD-2(-/-)) do not respond to LPS, survive endotoxin shock, and are susceptible to Salmonella typhimurium infection. Here, we further show that B cells lacking MD-2 do not up-regulate CD23 in response to LPS. TLR4 predominantly resides in the Golgi apparatus without MD-2. MD-2 is essential for LPS responses in vivo.
Assuntos
Antígenos de Superfície/farmacologia , Linfócitos B/imunologia , Lipopolissacarídeos/farmacologia , Glicoproteínas de Membrana/farmacologia , Animais , Técnicas de Cultura de Células , Fibroblastos , Complexo de Golgi , Humanos , Antígeno 96 de Linfócito , Glicoproteínas de Membrana/genética , Camundongos , Receptores de Superfície Celular/genética , Receptores de IgE/biossíntese , Salmonelose Animal , Salmonella typhimurium/patogenicidade , Transdução de Sinais , Receptor 4 Toll-Like , Receptores Toll-Like , Regulação para CimaRESUMO
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.
Assuntos
Antígenos de Superfície/metabolismo , Antígenos de Superfície/farmacologia , Proteínas de Transporte/metabolismo , Proteínas de Transporte/farmacologia , Lipopolissacarídeos/toxicidade , Glicoproteínas de Membrana/metabolismo , Glicoproteínas de Membrana/farmacologia , Receptores de Superfície Celular/metabolismo , Animais , Escherichia coli/patogenicidade , Ligantes , Antígeno 96 de Linfócito , Camundongos , Transdução de Sinais , Receptor 4 Toll-Like , Receptores Toll-Like , TransfecçãoRESUMO
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.