RESUMO
Cisplatin, a frequently prescribed chemotherapeutic agent, serves as a clinically therapeutic strategy for a broad range of malignancies. Its primary mode of action centers around interference with DNA replication and RNA transcription, thereby inducing apoptosis in cancer cells. Nevertheless, the clinical utility of cisplatin is constrained by its severe adverse effects and the burgeoning problem of drug resistance. Ginsenosides, potent bioactive constituents derived from ginseng, possess an array of biological activities. Recent scientific investigations underscore the substantial amplification of cisplatin's anticancer potency and the mitigation of its harmful side effects when administered concomitantly with ginsenosides. This review aims to explore the underlying mechanisms at play in this combination therapy. Initially, we provide a concise introduction to the cisplatin. Then, we pivot towards illuminating how ginsenosides bolster the anticancer efficacy of cisplatin and counteract cisplatin resistance, culminating in enhanced therapeutic outcomes. Furthermore, we provide an extensive discussion on the reduction of cisplatin-induced toxicity in the kidneys, liver, gastrointestinal tract, nervous system, and ear, accompanied by immune-fortification with ginsenosides. The existing clinical combined use of cisplatin and ginsenosides is also discussed. We propose several recommendations to propel additional research into the mechanisms governing the synergistic use of ginsenosides and cisplatin, thereby furnishing invaluable insights and fostering advancement in combined modality therapy.
Assuntos
Cisplatino , Ginsenosídeos , Neoplasias , Cisplatino/uso terapêutico , Cisplatino/efeitos adversos , Cisplatino/administração & dosagem , Ginsenosídeos/uso terapêutico , Ginsenosídeos/farmacologia , Ginsenosídeos/administração & dosagem , Humanos , Animais , Neoplasias/tratamento farmacológico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Antineoplásicos/uso terapêutico , Antineoplásicos/administração & dosagemRESUMO
For the full development and utilization of Sophora tonkinensis Gagnep., this study was primarily intended to established a simple and efficient approach for the preparative purification of total flavonoids from S. tonkinensis by macroporous resin column chromatography (MRCC). The adsorption and desorption characteristics of the total flavonoids on ten macroporous resins were first studied, and AB-8 resin was chosen as the most suitable, and the adsorption data were best fitted to the pseudo-second-order kinetics model and Langmuir isotherm model. Furthermore, the technological parameters for the purification of the total flavonoids were optimized using column chromatography. After a sample one-step purification procedure, the content of the total flavonoids increased by about 4.76-fold from 12.14% to 57.82%, with a recovery yield of 84.93%. In addition, the comparative analysis of the flavonoid extracts before and after purification was performed by high-performance liquid chromatography coupled with photodiode-array detection (HPLC-PAD). The results showed that the contents of six major flavonoids in the purified product were all higher than before the purification. Therefore, the AB-8 MRCC established in this work was a promising method for the industrial-scale purification of the total flavonoids from S. tonkinensis.
Assuntos
Flavonoides/química , Extratos Vegetais/química , Resinas Vegetais/química , Sophora/química , Cromatografia Líquida de Alta PressãoRESUMO
1. TM-2 is a promising novel semi-synthetic taxane derivative with greater antitumor activity especially against resistant tumors and lower toxicity compared with docetaxel. Information on distribution and excretion of the pharmaceutical in animals, as well as biochemical information relevant to potential drug interactions should normally be evaluated prior to human clinical trials. 2. The present study investigated the tissue distribution and excretion of TM-2 in animals following intravenous administration for further advancement of the molecule. The potential inductive effect of TM-2 on cytochrome P450 iso-enzymes CYP 3A1 in rats was also evaluated. 3. The tissue distribution study in mice showed that TM-2 was rapidly dispersed in the various tissues and peak concentration levels were achieved within 0.083-1 h. The highest concentration was detected in pancreas, followed by lung, kidney, spleen, heart and liver. TM-2 was mainly excreted in the feces via the bile (0.14% of the dose) and urinary excretion was minimal (0.007%). TM-2 increased CYP3A1 enzyme activities with time and dose dependence in rat liver microsome. 4. This important data serve as a useful resource to support further research of TM-2 and allow intelligent assessment of toxicology and in vivo activity testing performed in animals.
Assuntos
Antineoplásicos/farmacocinética , Indutores das Enzimas do Citocromo P-450/farmacocinética , Sistema Enzimático do Citocromo P-450/metabolismo , Taxoides/farmacocinética , Animais , Cromatografia Líquida , Humanos , Camundongos , Microssomos Hepáticos/metabolismo , Ratos , Espectrometria de Massas em Tandem , Distribuição TecidualRESUMO
A simple, specific, and sensitive ultra high performance liquid chromatography with tandem mass spectrometry method was developed and validated for the simultaneous quantification of nine compounds including a new compound, rhamnazin-3-Ο-ß-D-(6â³-ß-hydroxy-ß-methyglutaryl)-ß-D-glucoside-4'-Ο-ß-D-glucoside, in rat plasma using baicalin as an internal standard. The plasma samples were pretreated and extracted by protein precipitation with 0.2% formic acid in acetonitrile. The analytes were separated on a Thermo Syncronis C18 column by gradient elution with a mobile phase consisting of acetonitrile and 0.1% aqueous formic acid at a flow rate of 0.25 mL/min. The detection of the analytes was performed on an electrospray ionization interface operating in positive-ion and multiple reaction monitoring acquisition modes. The calibration curves of these analytes showed good linearity (r > 0.99) within the test ranges. The lower limit of quantification ranged from 0.4 to 20.1 ng/mL for the analytes. The intra- and interday precision and accuracy were all within ±15%, and the recoveries were higher than 80.0%. The validated method was successfully applied to a pharmacokinetic study of the nine flavonoids after administration of the Viscum coloratum extracts by intravenous injection.
Assuntos
Flavonoides/sangue , Flavonoides/farmacocinética , Extratos Vegetais/química , Viscum/química , Animais , Cromatografia Líquida de Alta Pressão , Flavonoides/administração & dosagem , Flavonoides/química , Injeções Intravenosas , Masculino , Estrutura Molecular , Extratos Vegetais/administração & dosagem , Extratos Vegetais/sangue , Extratos Vegetais/farmacocinética , Ratos , Ratos Wistar , Espectrometria de Massas em TandemRESUMO
A simple and sensitive method based on ultra-high-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) has been developed for the determination of TM-2, which was a novel semi-synthetic taxane derivative in beagle dog plasma. Cabazitaxel was chosen as internal standard. Following extraction by methyl tert-butyl ether, the chromatographic separation was achieved on a Thermo Syncronis C18 column (50 × 2.1 mm, 1.7 µm) by gradient elution within a runtime of 3.5 min. The mobile phase consisted of (A) acetonitrile and (B) 2 mmol/L ammonium acetate in water. The detection was accomplished using positive ion electrospray ionization in multiple reaction monitoring mode. The MS/MS ion transitions were monitored at m/z 812.39 â 551.35 for TM-2 and 836.36 â 555.26 for IS, respectively. The method was linear for TM-2 (r = 0.9924) ranging from 2.5 to 1000 ng/mL. The intra-day and inter-day precisions (relative standard deviation) were within 8.0 and 17.6%, respectively, and the accuracy (relative error) was less than 2.3%. The extraction recovery ranged from 83.1 to 97.1%. The reliable method was successfully applied to a pharmacokinetic study of TM-2 in beagle dogs after intravenous drip with different doses of 0.6, 1.2, and 2.4 mg/kg, respectively.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Espectrometria de Massas em Tandem/métodos , Taxoides/sangue , Taxoides/farmacocinética , Animais , Cães , Modelos Lineares , Masculino , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Taxoides/químicaRESUMO
RATIONALE: TM-2 (13-(N-Boc-3-i-butylisoserinoyl-4,10-ß-diacetoxy-2-α-benzoyloxy-5-ß,20-epoxy-1,13-α-dihydroxy-9-oxo-19-norcyclopropa[g]tax-11-ene) is a novel semi-synthetic taxane derivative. Our previous study demonstrated that it is a promising taxane derivative. The in vitro comparative metabolic profile of a drug between animals and humans is a key issue that should be investigated at early stages of drug development to better select drug candidates. In this study, the in vitro metabolic pathways of TM-2 in rat, dog and human liver microsomes were established and compared. METHODS: TM-2 was incubated with liver microsomes in the presence of NADPH. Two different types of mass spectrometers - a hybrid linear trap quadrupole orbitrap (LC/LTQ-Orbitrap) mass spectrometer and a triple-quadrupole tandem mass spectrometer (LC/QqQ) were employed to acquire structural information of TM-2 metabolites. Accurate mass measurement using LC/LTQ-Orbitrap was used to determine the accurate mass data and elemental compositions of metabolites thereby confirming the proposed structures of the metabolites. For the chemical inhibition study, selective P450 inhibitors were added to incubations to initially characterize the cytochrome P450 (CYP) enzymes involved in the metabolism of TM-2. RESULTS: A total of 12 components (M1-M12) were detected and identified as the metabolites of TM-2 in vitro. M1-M5 were formed by hydroxylation of the taxane ring or the lateral chain. Hydroxylated products can be further oxidized to the dihydroxylated metabolites M6-M10. M11 was a trihydroxylated metabolite. M12 was tentatively identified as a carboxylic acid derivative. The metabolism of TM-2 is much the same in all three species with some differences. The chemical inhibition study initially demonstrated that the formation of M2, the major metabolite of TM-2, is mainly mediated by CYP3A4. CONCLUSIONS: Hydroxylation is the major biotransformation of the TM-2 pathway in vitro. CYP3A4 may play a dominant role in the formation of M2 in liver microsomes. The knowledge of the metabolic pathways of TM-2 is important to support further research of TM-2.
Assuntos
Cromatografia Líquida/métodos , Microssomos Hepáticos/metabolismo , Espectrometria de Massas em Tandem/métodos , Taxoides/análise , Taxoides/metabolismo , Animais , Antineoplásicos , Cães , Humanos , Ratos , Taxoides/antagonistas & inibidores , Taxoides/químicaRESUMO
TM-2 (13-(N-Boc-3-i-butylisoserinoyl-4,10-ß-diacetoxy-2-α-benzoyloxy-5-ß-20-epoxy-1,13-α-dihydroxy-9-oxo-19-norcyclopropa[g]tax-11-ene) is a novel semisynthetic taxane derivative. Our previous study suggested that TM-2 is a promising antitumor analogue. In this paper, the metabolism of TM-2 was investigated in rats following intravenous administration. Two different types of mass spectrometry-hybrid linear trap quadrupole orbitrap (LTQ-Orbitrap) mass spectrometry and triple-quadrupole tandem (QQQ) mass spectrometry-were employed to acquire structural information of TM-2 metabolites. A total of 17 components were identified as the metabolites of TM-2 in bile, feces, and urine samples. Accurate mass measurement using LC-LTQ-Orbitrap-MS was used to determine the accurate mass data and elemental composition of metabolites thereby confirming the proposed structures of the metabolites. The metabolites proposed were mainly oxidates of TM-2, including methoxy, hydroxyl, dihydroxy, and trihydroxyl analogues. The major metabolic pathway of TM-2 was the hydroxylation of the taxane ring or the lateral chain. These important metabolic data serve as a useful resource to support further research of TM-2.
Assuntos
Antineoplásicos/análise , Antineoplásicos/metabolismo , Taxoides/química , Taxoides/metabolismo , Administração Intravenosa , Animais , Antineoplásicos/administração & dosagem , Cromatografia Líquida , Feminino , Masculino , Espectrometria de Massas , Conformação Molecular , Ratos , Ratos Sprague-Dawley , Taxoides/administração & dosagemRESUMO
Fufang Muji granules (FMGs) are a prominent modern prescription Chinese patent formulation derived from the Muji decoction. Utilized in clinical practice for nearly four decades, FMGs have demonstrated efficacy in treating liver diseases. However, the precise mechanism of action remains unclear. This study investigates the hepatoprotective effects of FMGs against liver fibrosis in rats based on untargeted metabolomics and elucidates their underlying mechanisms. A comprehensive model of liver fibrosis was established with 30% CCl4 (2 mL/kg) injected intraperitoneally, and a fat and sugar diet combined with high temperatures and humidity. Rats were orally administered FMGs (3.12 g/kg/d) once daily for six weeks. FMG administration resulted in improved liver fibrosis and attenuated hepatic oxidative stress and apoptosis. Furthermore, FMGs inhibited hepatic stellate cell activation and modulated transforming growth factor ß1/Smad signaling. Additionally, FMG treatment influenced the expression levels of interleukin-6, interleukin-1ß, and tumour necrosis factor alpha in the injured liver. Metabolic pathways involving taurine and hypotaurine metabolism, as well as primary bile acid biosynthesis, were identified as mechanisms of action for FMGs. Immunohistochemistry, quantitative reverse transcription polymerase chain reaction (RT-qPCR), and quantitative analysis also revealed that FMGs regulated taurine and hypotaurine metabolism and bile acid metabolism. These findings provide a valuable understanding of the role of FMGs in liver fibrosis management.
RESUMO
This study aimed to develop an integrated approach of deep eutectic solvent-based ultrasound-assisted extraction (DES-UAE) to simultaneously extract five major bioactive macamides from the roots of Lepidium meyenii Walp. Ten different DESs containing choline chloride and selected hydrogen-bond donors were prepared and evaluated based on the extracted macamide content determination using high-performance liquid chromatography (HPLC). Choline chloride/1,6-hexanediol in a 1:2 molar ratio with 20% water exhibited the most promising extraction efficiencies under the optimized parameters verified using single-factor optimization as well as Box-Behnken design. Using the optimized DES-UAE method, the extraction efficiencies of the five macamides were up to 40.3% higher compared to those using the most favorable organic solvent petroleum ether and were also superior to those of the other extraction methods, such as heating and combination of heating and stirring. Furthermore, using the macroporous resin HPD-100, the recoveries of the five target macamides from the DES extraction reached 85.62-92.25%. The 20 µg/mL group of the five macamide extracts showed superior neuroprotective activity against PC12 cell injury than that of the positive drug nimodipine. The macamide extracts also showed higher NO inhibition in LPS-stimulated RAW264.7 cells. Thus, the developed approach was a green and potential alternative that can be used to extract bioactive macamide constituents from L. meyenii in the pharmaceutical and food industries.
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In vivo, the complex process of drugs metabolism alters the change in drug composition and determines the final pharmacological properties of oral drugs. Ginsenosides are primary constituents of ginseng, whose pharmacological activities are greatly affected by liver metabolism. However, the predictive power of existing in vitro models is poor due to their inability to mimic the complexity of drug metabolism in vivo. The advance of organs-on-chip-based microfluidics system could provide a new in vitro drug screening platform by recapitulating the metabolic process and pharmacological activity of natural product. In this study, an improved microfluidic device was employed to establish an in vitro co-culture model by culturing multiple cell types in compartmentalized microchambers. Different cell lines were seeded on the device to examine the metabolites of ginsenosides from the hepatocytes in top layer and its resulting efficacy on the tumors in bottom layer. Metabolism dependent drug efficacy of Capecitabine in this system demonstrated the model is validated and controllable. High concentrations of CK, Rh2 (S), and Rg3 (S) ginsenosides showed significant inhibitory effects on two types of tumor cells. In addition, apoptosis detection showed that Rg3 (S) through liver metabolism promoted early apoptosis of tumor cells and displayed better anticancer activity than prodrug. The detected ginsenoside metabolites indicated that some protopanaxadiol saponins were converted into other anticancer aglycones in varying degrees due to orderly de-sugar and oxidation. Ginsenosides exhibited different efficacy on target cells by impacting their viabilities, indicating hepatic metabolism plays an important role in determining ginsenosides efficacy. In conclusion, this microfluidic co-culture system is simple, scalable, and possibly widely applicable in evaluating anticancer activity and metabolism of drug during the early developmental phases of natural product.
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As a steroid skeleton-based saponin, ginsenoside Rh2 (G-Rh2) is one of the major bioactive ginsenosides from the plants of genus Panax L. Many studies have reported the notable pharmacological activities of G-Rh2 such as anticancer, antiinflammatory, antiviral, antiallergic, antidiabetic, and anti-Alzheimer's activities. Numerous preclinical studies have demonstrated the great potential of G-Rh2 in the treatment of a wide range of carcinomatous diseases in vitro and in vivo. G-Rh2 is able to inhibit proliferation, induce apoptosis and cell cycle arrest, retard metastasis, promote differentiation, enhance chemotherapy and reverse multi-drug resistance against multiple tumor cells. The present review mainly summarizes the anticancer effects and related mechanisms of G-Rh2 in various models as well as the recent advances in G-Rh2 delivery systems and structural modification to ameliorate its anticancer activity and pharmacokinetics characteristics.
Assuntos
Antineoplásicos , Ginsenosídeos , Neoplasias , Saponinas , Humanos , Ginsenosídeos/farmacologia , Antineoplásicos/farmacologia , Saponinas/farmacologia , Neoplasias/tratamento farmacológico , ApoptoseRESUMO
As a typical and broad-spectrum benzimidazole, mebendazole (MBZ) has long been used in human and veterinary medicine to treat parasitic infestations, and is widely employed in the aquaculture of Japanese pufferfish (Takifugu rubripes). However, there have been no studies examining the pharmacokinetic characteristics of MBZ in Japanese pufferfish. Furthermore, the presence of MBZ and its metabolites in animal-derived raw food represents a notable safety concern. Here, we investigated the metabolism of MBZ using a UPLC-Q-TOF system. Additionally, we evaluated the pharmacokinetics of MBZ and two metabolites, 2-amino-5(6)-benzoylbenzimidazole (MBZ-NH2) and 5-hydroxymebendazole (MBZ-OH), in Japanese pufferfish following intramuscular injection of 20 mg/kg MBZ. We detected three metabolites of MBZ (M1-M3), among which, 2-amino-5(6)-(a-hydroxybenzyl) benzimidazole (M3) was detected in an aquatic animal for the first time. The plasma dispositions of MBZ, MBZ-NH2, and MBZ-OH were characterized by low plasma clearance, medium distribution volume, and long terminal half-life. Moreover, these compounds were widely distributed in the muscle, from which they were rapidly cleared. The pharmacokinetics and metabolism of mebendazole in Japanese pufferfish are described for the first time in this study. Our findings provide a basis for the rational application of MBZ in Japanese pufferfish farming and contribute to our understanding of the metabolism of MBZ in cultured fish.
Assuntos
Mebendazol , Takifugu , Animais , Benzimidazóis/metabolismo , Mebendazol/metabolismo , Músculos/metabolismo , Takifugu/metabolismoRESUMO
This study was designed to investigate proteome changes in Japanese puffer fish (Takifugu rubripes) during short- and long-term frozen storage. In total, 1484 proteins were quantified, and 164 proteins were identified as differential abundance proteins (DAPs) in Japanese puffer fish from two frozen storage treatment groups (14 days and 60 days) compared with the fresh control group. Correlation analysis between the DAPs and quality traits of the puffer fish muscle showed that 106 proteins were correlated closely with colour and texture (hardness, elasticity, and chewiness). Bioinformatics analysis revealed and Western blot analysis verified that Putative prothymosin alpha species, Bridging integrator 3, NADH: the ubiquinone oxidoreductase subunit and Mx species are candidate biomarkers for puffer fish properties. This study offers valuable evidence to improve the quality control and monitoring of Japanese puffer fish during transportation and storage.
Assuntos
Biomarcadores/análise , Produtos Pesqueiros/análise , Proteínas de Peixes/análise , Takifugu , Animais , Cor , Biologia Computacional/métodos , Análise de Alimentos/métodos , Qualidade dos Alimentos , Armazenamento de Alimentos , Congelamento , Músculo Esquelético/química , Precursores de Proteínas/análise , Timosina/análogos & derivados , Timosina/análiseRESUMO
In this study, monogenean Heterobothium okamotoi was isolated and identified from the gill of diseased Tiger puffer (T. rubripes) at an industrial farm in Liaoning, North China (121.3459 E, 38.9861 N). With the completion of H. okamotoi mitochondrial genome sequencing, the full-length mitochondrial genome of H. okamotoi was assembled and analyzed. All results indicate that the complete mitochondrial genome of H. okamotoi was 14,643 bp. There were 2 rRNAs, 20 tRNAs, and 12 protein-coding genes (PCGs) all located at the heavy (H) strand. Besides, the phylogenetic tree of 19 monogeneans was constructed. The results showed that H. okamotoi and Pseudochauhanea macrorchis were clustered in a clade. To sum up, our research results would further provide essential data for systematics and evolution study of H. okamotoi.
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TM-2 known as a potential antitumor drug is a novel semi-synthetic taxane derivative. As drug-protein interactions contribute to insights into pharmacokinetic and pharmacodynamic properties, we elucidated the binding of TM-2 to plasma protein. In this study, a simple, rapid and reliable method was developed and validated employing equilibrium dialysis for the separation of bound and unbound drugs and ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) for the quantitation. Protein binding reached equilibrium within 24 h of incubation at 37 °C. After liquid-liquid extraction with methyl tert-butyl ether, the samples were separated on Thermo Syncronis UPLC® C18 (2.1 mm×50 mm, 1.7 µm), and acquisition of mass spectrometric data was performed in multiple reaction monitoring (MRM) mode via positive electrospray ionization. The assay was linear over the concentration rang of 5-2000 ng/mL. The intra- and inter-day precisions were 0.1%-14.8%, and the accuracy was from -6.4% to 7.0%. This assay has been successfully applied to a protein binding study of TM-2 in rat, human and beagle dog plasma. TM-2 showed high protein binding of 81.4%±6.5% (rat), 87.9%±3.6% (human) and 79.4%±4.0% (beagle dog). The results revealed that there was an insignificant difference among the three species.
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TM-2 is a novel semi-synthetic taxane derivative, selected for preclinical development based on its greater anticancer activity and lower toxicity compared with docetaxel. In this study, a rapid and sensitive analytical method based on ultra performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been developed for the determination of TM-2 in rat plasma. The biological samples were extracted with methyl tert-butyl ether and separated on a C18 column (50 mm × 2.1 mm, 1.7 µm) using a mobile phase consisting of acetonitrile and 2 mM ammonium acetate. The standard curves were linear over the range 5-1000 ng/mL in rat plasma. The precision (relative standard deviation, RSD, %) were within 14.5%, and the accuracy (relative error, RE, %) ranged from -1.56 to 2.36%. Recovery and matrix effect were satisfactory in rat plasma. The validated method was successfully applied to pharmacokinetic studies after intravenous administration of TM-2 to rats. The pharmacokinetics of TM-2 in rats were characterized by a large volume of distribution and a long half-life of elimination after single dose (4, 8, and 16 mg/kg), and a good correlation was observed between AUC and dose. The preclinical data will be useful for the design of subsequent trials of TM-2.
Assuntos
Antineoplásicos/farmacocinética , Taxoides/farmacocinética , Animais , Antineoplásicos/análise , Antineoplásicos/sangue , Cromatografia Líquida , Relação Dose-Resposta a Droga , Feminino , Masculino , Ratos , Espectrometria de Massas em Tandem , Taxoides/análise , Taxoides/sangueRESUMO
In order to investigate the pharmacokinetics of triamcinolone acetonide palmitate (TAP) which is a lipid-soluble prodrug of triamcinolone acetonide (TA), a rapid, simple, sensitive and reproducible UPLC-MS/MS method has been developed and validated for the simultaneous determination of TAP and TA in beagle dog plasma. After simple liquid-liquid extraction, the analytes and internal standard (dexamethasone, DEX) were separated on Phenomenex Luna C18 column (50 mm × 2.1mm, 1.7 µm) using a mobile phase consisting of solvent A (acetonitrile) and solvent B (0.1% ammonia solution) at a flow rate of 0.2 ml/min with gradient elution. Acquisition of mass spectrometric data was performed in multiple reaction monitoring (MRM) mode via positive electrospray ionization using the ion transitions of m/z 673.5â397.3, 435.3â415.3 and 393.3â355.3 for TAP, TA and IS, respectively. The method was of satisfactory specificity, sensitivity, precision and accuracy over the concentration range of 1-1,000 ng/ml for TAP and 0.5-500 ng/ml for TA. The intra- and inter-day precisions for both TAP and TA were 3.2% to 18.7% and the accuracy was in the range of -8.4% to 6.8%. The mean recoveries of TAP, TA and IS were 86.7-104.7%. The method was successfully applied to a long-term pharmacokinetic study of TAP and TA after 28-day repeated intravenous administration of TAP lipid emulsion injection to beagle dogs.