Expression of P-glycoprotein and CYP3A4 along the porcine oral-gastrointestinal tract: implications on oral mucosal drug delivery.

OBJECTIVES: To characterize the expression of Pgp and CYP3A4 along the oral-gastrointestinal (GI) tract for understanding the potential roles of CY3A4 and Pgp in oral mucosal drug delivery. DESIGN: Porcine buccal mucosa, sublingual mucosa, esophagus and jejunum, ileum and colon tissues were used for studying the mRNA and protein expression of CYP3A4 and Pgp. mRNA and protein were determined using real-time quantitative polymerase chain reaction (PCR) and western blot, respectively. The expression levels of CYP3A4 and Pgp in different segments of oral-GI tract were compared. RESULTS: Levels of Pgp mRNA were significantly lower (14-40 times lower) in buccal and sublingual mucosa than that in intestine. In contrast, higher levels of CYP3A4 mRNA were observed in the oral mucosa as compared to that in intestine, but the difference was not statistically different. The levels of Pgp protein along the oral-GI tract followed the order: sublingual ∼buccal ∼esophagus < jejunum ∼ileum ∼ colon while the expression of CYP3A4 protein in the oral mucosa was similar to that in intestine. CONCLUSION: Expression of Pgp in oral mucosa is lower than that in intestine, while the expression of CYP3A4 in oral mucosa is similar to that in intestine. Because of lower Pgp in oral mucosa, oral mucosal drug delivery can be used as an alternative strategy to avoid the coordination of Pgp and CYP3A4 metabolism in drug absorption. However, CYP3A4-dependent metabolism may play a role in oral mucosal drug delivery as in per oral-GI absorption.

Leucine-Aspartic Acid-Valine sequence as targeting ligand and drug carrier for doxorubicin delivery to melanoma cells: in vitro cellular uptake and cytotoxicity studies.

PURPOSE: To study the feasibility of Leucine-Aspartic Acid-Valine (LDV) as targeting ligand and drug carrier for targeted delivery to integrin alpha(4)beta(1) over-expressing cancer cells. METHODS: Poly(L,D,V) was randomly copolymerized using N-carboxyanhydrides of leucine, beta-benzyl-aspartic acid, and valine. Oligo(LDV), consisting of 2-6 LDV units, were synthesized by solid phase protein synthesis (SPPS) method. Binding of Leu-Asp-Val, Val-Asp-Leu, and Leu-Asn-Val, and internalization of FITC labeled LDV by wild-type and integrin alpha(4) knock-down A375 cells were studied. Cytotoxicity of poly(L,D,V)-Dox, oligo(LDV)-Dox, and doxorubicin (Dox) was also determined on wild-type, integrin alpha(4) knock-down A375 cells, and normal human epithelial keratinocytes (NHEK). RESULTS: LDV was essential for the specific binding and internalization by cells expressing integrin alpha(4)beta(1). Cytotoxicity of poly(L,D,V)-Dox and oligo(LDV)-Dox was integrin alpha(4)-dependent, while free Dox did not show this differential effect. No observable cytotoxicity trend was found when increasing LDV repeating unit. Poly(L,D,V) was relatively more effective than oligo(LDV) for the delivery of Dox to A375. CONCLUSION: LDV containing moieties bind specifically to integrin alpha(4)beta(1) expressing cancer cells. The binding, internalization, and cytotoxicity depend on the level of integrin alpha(4)beta(1) expression. Poly(L,D,V) and oligo(LDV) were both effective in the in vitro targeted delivery of Dox to integrin alpha(4)beta(1) over-expressing A375 cells.

Synthesis of folate-conjugated amphiphiles for tumor-targeted drug delivery.

Folic acid was derivatized specifically at its gamma-carboxyl group to retain its ligand-binding activity to the folate receptor alpha (FRalpha) present on HeLa cells. Amphiphilic molecules labeled with folic acid were prepared by conjugation of long-chain primary amines directly or via diamine linkers to the gamma-carboxyl group of folic acid. Folic acid amphiphiles labeled with fluorescent 7-amino-4-carbamoylmethylcoumarin (ACC) were also prepared to visualize the uptake of amphiphiles in folate receptor positive cells. The structures of the new compounds were verified by proton NMR and MALDI-TOF mass spectrometry. The amphiphiles form micelles in water. Critical micelle concentrations (CMCs) were determined by the pyrene fluorescence method for folic acid amphiphiles and the rise in capillary height method for the fluorescently labeled amphiphile. The CMCs of the amphiphiles were studied in buffer solution at pH 8 and ranged from 2 to 64 microM. The formation of micelle increased the solubility of paclitaxel, a model lipophilic anticancer compound, by more than 80%. A significant amount of the fluorescently tagged amphiphile was internalized into HeLa cells known to express FRsalpha when compared with Caco-2 cells that do not express FRalpha. Therefore, folate-labeled amphiphiles show promise in targeting antitumor agents to FRalpha-expressing cancer cells.

Synthesis and characterization of RGD-fatty acid amphiphilic micelles as targeted delivery carriers for anticancer agents.

Novel amphiphilic conjugates consisting of an Arg-Gly-Asp (RGD) peptide binding motif and aliphatic fatty acids of varying chain length (C10-C18) were synthesized and evaluated for their ability to form micelles and bind specifically to alphaVbeta3 integrin over-expressing tumor cells. The aphilphiles were characterized by IR, proton NMR and mass spectrometry. The size and zeta potential of the resultant micelles were ranged from 178 to 450 nm and - 13.5 to 39.6 mV, respectively. The critical micellar concentration (CMC), drug loading efficiency and tumor cell binding of these amphiphiles were determined. The CMC values, determined by pyrene fluorescent probe method, ranged from 0.02 to 0.12 mM for C14-RGD, C16-RGD and C18-RGD. The C18-RGD micelles with lowest CMC were found to increase the solubility of taxol, a model anticancer drug, by 87%. C18-RGD amphiphiles also exhibited significantly higher (12.1 +/- 1.14%, P < 0.05) binding to alphaVbeta3 integrin over-expressing human breast cancer cells (HTB-129) when compared to normal human epidermal keratinocyte (NHEK) cells (6.68 +/- 0.34). The results from this study demonstrated the feasibility of designing RGD-fatty acid amphiphiles as micellar drug delivery carriers to target to cancer cells.

Synthesis and anticancer activity of certain mononuclear Ru (II) complexes.

Bis(1,10-phenanthroline/2,2'-bipyridine) ruthenium(II)complexes containing TCP, TTZ OPBI, and BTSC ligands (where, TCP = 1-thiocarbamoyl-3,5-diphenyl-2-pyrazoline, TTZ = 2-(3,5-diphenyl-4,5-dihydropyrazol-1-yl)-4-phenylthiazole, OPBI = 2-hydroxyphenyl benzimidazole and BTSC = benzoin thiosemicarbazone) have been prepared and characterized. The spectral data suggested that the ligands were coordinated with the metal through nitrogen, sulfur and oxygen atoms. The target complexes were tested in vivo for anticancer activity against transplantable murine tumor cell line, Ehrlich's Ascitic Carcinoma (EAC). All these complexes increased the life span of the EAC-bearing mice, decreased their tumor volume and viable ascitic cell count as well as improved Hb, RBC and WBC counts. These results suggest that the Ru(II) complexes exhibit significant antitumor activity in EAC-bearing mice. It was also observed that the ruthenium complexes protected red blood cells from 2,2'-azo-bis(2-methylpropionamidine) dihydrochloride (AAPH)- induced hemolysis. The inhibitory effect was dose-dependent at a concentration of 20-120 microg/ml.

Synthesis and pharmacological activities of some mononuclear Ru(II) complexes.

A series of mononuclear Ru(II) complexes of the type [Ru(M)2(U)]2+, where M = 2,2'-bipyridine/1,10-phenanthroline and U = tpl (Ru1), 4-Cl-tpl (Ru2), 4-CH3-tpl (Ru3), 4-CH3O-tpl (Ru4), and 4-NO2-tpl (Ru5), -pai (Ru6), where tpl = thiopicolinanilide and pai = 2-phenyl-azo-imidazole, have been prepared and characterized by IR, UV-Vis, 1H NMR, 13C-NMR, FAB-Mass spectrophotometer, and elemental analysis. The complexes display metal-ligand charge transfer (MLCT) transitions in the visible region. The title complexes were subjected to in vivo anticancer activity tests against a transplantable murine tumor cell line, Ehrlich's ascitic carcinoma (EAC) and in vitro antibacterial activity against Gram positive and Gram negative microorganisms. Ru1-Ru6 were found to increase the life span of the tumor hosts by 19-52%, and decreased tumor volume and viable ascitic cell count. The results of the present study clearly demonstrated the tumor inhibitory activity of the ruthenium chelates against transplantable murine tumor cell line. The treatment with ruthenium complexes could be secondary to tumor regression or due to the action of the compounds itself. The significant antibacterial activity was observed for Ru1-Ru4 against microorganisms like Vibrio cholera 865, Staphylococcus aureus 6571, and Shigella flexneri as compared to that of standard drug chloramphenical. Ru5 showed moderate activity against S. aureus 8530. However, all the complexes fail to show significant antibacterial activity against V. cholera 14033 and Shigella sonnai.

Antineoplastic and antibacterial activity of some mononuclear Ru(II) complexes.

These ligands (L) show a bidentate behavior, forming octahedral ruthenium complexes. The title complexes were subjected to in-vivo anticancer activity tests against a transplantable murine tumor cell line, Ehrlich's Ascitic Carcinoma (EAC) and in-vitro antibacterial activity against several Gram positive and Gram negative bacterial strains. [Ru(bpy)2(ihqs)]Cl2 and [Ru(bpy)2 (hc)]Cl2 (where bpy = 2,2'-bipyridine, ihqs = 7-iodo-8hydroxy quinoline-5-sulphonic acid and hc = 3-hydroxy coumarin) showed promising antitumor activity. Treatment with these complexes prolonged the life span of EAC bearing mice as well as decreased their tumor volume and viable ascitic cell count. All the tested complexes exhibited mild to moderate antibacterial activity.

Synthesis, anticancer and antibacterial activity of some novel mononuclear Ru(II) complexes.

In search of potential anticancer drug candidates in ruthenium complexes, a series of mononuclear ruthenium complexes of the type [Ru(phen)(2)(nmit)]Cl(2) (Ru1), [Ru(bpy)(2)(nmit)]Cl(2) (Ru2), [Ru(phen)(2)(icpl)]Cl(2) (Ru3), Ru(bpy)(2)(icpl)]Cl(2) (Ru4) (phen=1,10-phenanthroline; bpy=2,2'-bipyridine; nmit=N-methyl-isatin-3-thiosemicarbazone, icpl=isatin-3-(4-Cl-phenyl)thiosemicarbazone) and [Ru(phen)(2)(aze)]Cl(2) (Ru5), [Ru(bpy)(2)(aze)]Cl(2) (Ru6) (aze=acetazolamide) and [Ru(phen)(2)(R-tsc)](ClO(4))(2) (R=methyl (Ru7), ethyl (Ru8), cyclohexyl (Ru9), 4-Cl-phenyl (10), 4-Br-phenyl (Ru11), and 4-EtO-phenyl (Ru12), tsc=thiosemicarbazone) were prepared and characterized by elemental analysis, FTIR, (1)H-NMR and FAB-MS. Effect of these complexes on the growth of a transplantable murine tumor cell line (Ehrlich Ascites Carcinoma) and their antibacterial activity were studied. In cancer study the effect of hematological profile of the tumor hosts have also been studied. In the cancer study, the complexes Ru1-Ru4, Ru10 and Ru11 have remarkably decreased the tumor volume and viable ascitic cell count as indicated by trypan blue dye exclusion test (p<0.05). Treatment with the ruthenium complexes prolonged the lifespan of Ehrlich Ascites Carcinoma (EAC) bearing mice. Tumor inhibition by the ruthenium chelates was followed by improvements in hemoglobin, RBC and WBC values. All the complexes showed antibacterial activity, except Ru5 and Ru6. Thus, the results suggest that these ruthenium complexes have significant antitumor property and antibacterial activity. The results also reflect that the drug does not adversely affect the hematological profiles as compared to that of cisplatin on the host.

Studies on brain biogenic amines in methanolic extract of Cuscuta reflexa Roxb. and Corchorus olitorius Linn. seed treated mice.

The methanolic extract of both Cuscuta reflexa stem and Corchorus olitorius seed showed marked protection against convulsion induced by chemoconvulsive agents in mice. The catecholamines contained were significantly increased in the processed extract treated mice. The amount of GABA, which is most likely to be involved in seizure activity, was increased significantly in mice brain after a six week treatment. Results of the present study revealed that both the processed extracts showed a significant anticonvulsive property by altering the level of catecholamines and brain amino acids in mice.

Evaluation of psychopharmacological effects of petroleum ether extract of Cuscuta reflexa Roxb. stem in mice.

The petroleum ether extract of Cuscuta reflexa Roxb. stem (PECR) was evaluated for its psychopharmacological activities in several experimental models using Swiss albino mice. The PECR was found to cause significant reduction in spontaneous activity and exploratory behavioral profiles. It also showed reduction in muscle relaxant activity by rotarod, 30 degrees inclined screen tests and showed significant analgesic properties as well as potentiated remarkably the pentobarbitone sodium, diazepam and meprobamate--induced sleeping time. All these results were compared with respective controls for the evaluation of significance. The presence of steroids in the PECR might he responsible for psychopharmacological activities.

Chemical and toxicological evaluation of methanol extract of Cuscuta reflexa Roxb. stem and Corchorus olitorius Linn. seed on hematological parameters and hepatorenal functions in mice.

Methanol extract of Cuscuta reflexa Roxb. stem (MECR) contain flavonoids (0.2%) and Corchorus olitorius Linn. seed (MECO) was found to contain steroids and cardenolide glycosides. Effects of multiple weekly dose of MECR (25, 50, 75 mg/kg, i.p.) and MECO (15, 20, 25 mg/kg, i.p.) on liver and kidney functions and hematological parameters in mice were studied. No significant alteration of RBC count and hemoglobin content was observed in all dose level of treatment in MECR and MECO treated mice whereas significant increase of clotting time was seen in moderate and high doses in both case. MECR and MECO both caused significant increase in WBC count only in high dose level of treatment. Both the extracts in medium and high dose level increased SGOT, SGPT, NPN and plasma cholesterol significantly. Serum alkaline phosphatase and total bilirubin were also increased by both moderate and high dose level of treatments in MECR and MECO treated mice respectively. Low dose of both the extract did not exhibit any significant change of creatinine and serum protein level. But high dose level of MECR and MECO significantly increased creatinine level. Increase in plasma cholesterol may be due to decrease in cholesterol catabolism owing to liver dysfunction of due to the intake of MECO itself as it was found to be steroid in nature. Elevated level of SGOT, SGPT and serum alkaline phosphatase activity in moderate and high dose level of weekly treated mice may be due to improper liver function following the treatment. Increased urea, non protein nitrogen and creatinine content in blood have been observed with impaired renal function. The slightly higher toxicity in case of MECO treated mice may be due to the presence of cardenolide glycosides in the ME of C. olitorius seed. However, low doses of MECR and MECO (25 and 15 mg/kg, i.p. respectively) did not exhibit any remarkable change on liver and kidney functions and hematological parameters.