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Biomedical subjects

C Ramachandran

Publications and source records attributed to C Ramachandran.

At least 19 recordsLinked to original sources
PubMed2006

Rapid in vivo dissolution of ketoprofen: implications on the biopharmaceutics classification system.

The aim of this paper was to investigate the in vivo dissolution behavior of ketoprofen, a Class II drug according to the Biopharmaceutics Classification System (BCS), in the upper small intestine of dogs. An intubations method was used, where no blocking balloons were used to prevent luminal drug transport along the GI tract. Our design allowed the drug to be transported freely to more distal parts of the GI tract and also, it was supported by a pharmacokinetic study. Pharmacokinetic parameters of ketoprofen were determined in dogs after administering approximately 0.27 mg kg(-1) (solution) or approximately 1.47 mg kg(-1) (suspension) in oral bolus doses. There were not statistical significant differences in plasma concentrations for both formulations, either in the maximum concentrations C(max) or AUC following oral dose administration. The rapid disappearance of ketoprofen from the intestinal lumen, reflected by low mass recovery in the supernatant and sediment of the collected intestinal fluid samples, in comparison to that recovery of the non-absorbable marker phenol red, suggests that ketoprofen is emptying into the small intestine and is rapidly dissolved and absorbed. In this study, the in vivo results clearly show that the absorption rate of ketoprofen is not dissolution limited; therefore ketoprofen would be essentially equivalent to Class I drugs and could be considered for waiver of bioavailability and bioequivalence testing.

Animals↗
PubMed2001

Structure of protein tyrosine phosphatase 1B in complex with inhibitors bearing two phosphotyrosine mimetics.

Protein tyrosine phosphatases (PTPases) are signal-transducing enzymes that dephosphorylate intracellular proteins that have phosphorylated tyrosine residues. It has been demonstrated that protein tyrosine phosphatase 1B (PTP1B) is an attractive therapeutic target because of its involvement in regulating insulin sensitivity (Elcheby et al. Science 1999, 283, 1544-1548). The identification of a second binding site in PTP1B (Puius et al., Proc. Natl. Acad. Sci. U.S.A.1997, 94, 13420-13425) suggests a new strategy for inhibitor design, where appropriate compounds may be made to simultaneously occupy both binding sites to gain much higher affinity and selectivity. To test this hypothesis and gain further insights into the structural basis of inhibitor binding, we have determined the crystal structure of PTP1B complexed with two non-peptidyl inhibitors, 4 and 5, both of which contain two aryl difluoromethylenephosphonic acid groups, a nonhydrolyzable phosphate mimetic. The structures were determined and refined to 2.35 and 2.50 A resolution, respectively. Although one of the inhibitors seems to have satisfied the perceived requirement for dual binding, it did not bind both the active site and the adjacent noncatalytic binding site as expected. The second or distal phosphonate group instead extends into the solvent and makes water-mediated interactions with Arg-47. The selectivity of the more potent of these two inhibitors, as well as four other inhibitors bearing two such phosphate mimetics for PTP1B versus seven other PTPases, was examined. In general, selectivity was modest to good when compared to PTPases Cdc25a, PTPmeg-1, PTPbeta, and CD45. However, selectivity was generally poor when compared to other PTPases such as SHP-1, SHP-2, and especially TCPTP, for which almost no selectivity was found. The implications these results have concerning the utility of dual-binding inhibitors are discussed.

Binding Sites↗
PubMed2001

Topical transfection using plasmid DNA in a water-in-oil nanoemulsion.

Expression plasmids encoding chloramphenicol acetyltransferase (CAT) or human interferon-alpha2 cDNA were formulated in water-in-oil nanoemulsions and applied to murine skin. The histological location of transfected cells was assessed by in situ DNA PCR and showed that the deposition of plasmid DNA was primarily in follicular keratinocytes. Transgene expression in the skin was monitored for 24-72 h, following topical application of either single or multiple daily doses by quantitative RT-PCR and ELISA. It was found that transgene expression was optimal at 24 h following topical application of a single dose of water-in-oil nanoemulsion containing plasmid DNA. Dose-response studies using a total dose of 3, 10 or 30 microg of plasmid DNA suggested that topical transfection using nanoemulsions is subject to both threshold and saturation effects. None of the cationic liposome formulations tested as controls mediated transgenic protein expression at levels higher than background values of the ELISAs used to assay transgenic protein. Single and multiple dose experiments using human interferon-alpha2 as a transgene indicated that the efficiency of nanoemulsion mediated transfection was most effective in the context of normal versus atrophic hair follicles. In addition, the total amount of human interferon-alpha2 present in skin appeared to accumulate as a consequence of multiple dosing. Histologic evaluation of treated skin showed no overt signs of toxicity or irritation associated with the short-term application of the nanoemulsions. The results suggest that water-in-oil nanoemulsions can be used to facilitate transfection of follicular keratinocytes in vivo.

Administration, Topical↗
PubMed2001

Topical transport of hydrophilic compounds using water-in-oil nanoemulsions.

A variety of water-in-oil nanoemulsions were prepared using sorbitan monooleate (Span80), polyoxyethylene 20 sorbitan monooleate (Tween80), olive oil and water. The nanoemulsions were tested for their ability to facilitate transport of a model hydrophilic solute, inulin, across hairless and hairy mouse skin and hairy rat skin following topical in vitro application. The transport of inulin incorporated in water-in-oil nanoemulsions was found to be significantly higher (5- to 15-fold) than that obtained with micellar dispersions or aqueous controls. The rate and extent of inulin transport across hairy mouse skin was found to be highly dependent on the hydrophile-lipophile balance (HLB) of the surfactant mixture in the nanoemulsion. Nanoemuslions prepared using mixtures with lower HLB exhibited significantly higher rate and extent of transport. It was also found that nanoemulsion-mediated transport was independent of molecular size of the hydrophilic solute and the nature of the aqueous phase. More importantly, transport of inulin from nanoemulsions was independent of animal skin characteristics such as stratum corneum thickness and follicle-type. The combined results suggest that water-in-oil nanoemulsions that are compatible with the lipophilic sebum environment of the hair follicle facilitate efficient transport of incorporated hydrophilic solutes and imply that such transport is predominantly transfollicular in nature.

Administration, Topical↗
PubMed2001

The YRD motif is a major determinant of substrate and inhibitor specificity in T-cell protein-tyrosine phosphatase.

We have studied T-cell protein-tyrosine phosphatase (TCPTP) as a model phosphatase in an attempt to unravel amino acid residues that may influence the design of specific inhibitors. Residues 48--50, termed the YRD motif, a region that is found in protein-tyrosine phosphatases, but absent in dual-specificity phosphatases was targeted. YRD derivatives of TCPTP were characterized by steady-state kinetics and by inhibition studies with BzN-EJJ-amide, a potent inhibitor of TCPTP. Substitution of Asp(50) to alanine or Arg(49) to lysine, methionine, or alanine significantly affected substrate hydrolysis and led to a substantial decrease in affinity for BzN-EJJ-amide. The influence of residue 49 on substrate/inhibitor selectivity was further investigated by comparing subsite amino acid preferences of TCPTP and its R49K derivative by affinity selection coupled with mass spectrometry. The greatest effect on selectivity was observed on the residue that precedes the phosphorylated tyrosine. Unlike wild-type TCPTP, the R49K derivative preferred tyrosine to aspartic or glutamic acid. BzN-EJJ-amide which retains the preferred specificity requirements of TCPTP and PTP1B was equipotent on both enzymes but greater than 30-fold selective over other phosphatases. These results suggest that Arg(49) and Asp(50) may be targeted for the design of potent and selective inhibitors of TCPTP and PTP1B.

Amino Acid Substitution↗
PubMed2001

Development of a robust scintillation proximity assay for protein tyrosine phosphatase 1B using the catalytically inactive (C215S) mutant.

Protein tyrosine phosphatases are a class of enzymes that function to modulate tyrosine phosphorylation of cellular proteins and play an essential role in regulating cell function. PTP1B has been implicated in the negative regulation of the insulin signaling pathway by dephosphorylating the activated insulin receptor. Inhibiting this phosphatase and preventing the insulin-receptor downregulation has been suggested as a target for the treatment of Type II diabetes. A high-throughput screen for inhibitors of PTP1B was developed using a scintillation proximity assay (SPA) with GST-- or FLAG--PTP1B((1-320)) and a potent [(3)H]-tripeptide inhibitor. The problem of interference from extraneous oxidizing and alkylating agents which react with the cysteine active-site nucleophile was overcome by the use of the catalytically inactive C215S form of the native enzyme (GST--PTP1B(C215S)). The GST--PTP1B was linked to the protein A scintillation bead via GST antibody. The radiolabeled inhibitor when bound to the enzyme gave a radioactive signal that was competed away by the unknown competitive compounds. Further utility of PTP1B(C215S) was demonstrated by mixing in the same well both the catalytically inactive GST--PTP1B(C215S) and the catalytically active FLAG--CD45 with an inhibitor. Both a binding and kinetic assay was then performed in the same 96-well plate with the inhibition results determined for the PTP1B(C215S) (binding assay) and CD45 (activity assay). In this way inhibitors could be differentiated between the two phosphatases under identical assay conditions in one 96-well assay plate. The use of a mutant to reduce interference in a binding assay and compare with activity assays is also amenable for most cysteine active-site proteases.

Amino Acid Substitution↗
PubMed2000

Development of a method for evaluating protein tyrosine phosphatase CD45 inhibitors using Jurkat cell membrane.

A simple, high-throughput fluorescent assay was developed to measure the inhibition of membrane-bound CD45 from Jurkat cells. This assay is based on the fact that approximately 64% of PTP activity from Jurkat cell membrane is contributed by CD45. This has been proven by comparing the activity in membrane protein from wild-type Jurkat cells and CD45-negative mutant cells, and also by measuring the residual activity after depleting CD45 from Jurkat cell membrane. We have demonstrated that fluorescein diphosphate can be used as a substrate to monitor CD45 activity from Jurkat cell membrane, which allows us to easily follow CD45 activity in both fluorescent and absorbance modes in a 96-well format. Some common protein tyrosine phosphatase inhibitors have been evaluated with this assay.

Cell Membrane↗
PubMed2000

Protein tyrosine phosphatase-1B in diabetes.

A role for protein tyrosine phosphatases in the negative regulation of insulin signaling and a putative involvement in the insulin resistance associated with type 2 diabetes have been postulated since their discovery. The recent demonstration that mice lacking the protein tyrosine phosphatase-1B (PTP-1B) have enhanced insulin sensitivity validates this. Furthermore, when fed a high fat diet, these mice maintained insulin sensitivity and were resistant to obesity, suggesting that inhibition of PTP-1B activity could be a novel way of treating type 2 diabetes and obesity. This commentary reviews our current knowledge of PTP-1B in insulin signaling and its role in diabetes and discusses the development of potent and selective PTP-1B inhibitors.

Animals↗
PubMed2000

Transdermal delivery of drugs for the treatment of bone diseases.

The current status of transdermal drug delivery for the treatment of bone diseases is described in this review. The structure, physiology and function of skin and their importance in determining delivery into and across skin are discussed. Special emphasis has been devoted to a description of the major pathways of transport across the skin and the quite continuing controversy over the importance of the transfollicular route. An overview of anatomic site-dependent drug absorption is also provided and is particularly relevant to determination of transdermal patch location. Brief descriptions of the criteria for selection of transdermal drug candidate, transdermal patch designs and currently marketed transdermal products are also included. Transdermal estradiol delivery systems are examined in more detail for their clinical and biological effects. Finally, the feasibility of delivering drugs such as bisphosphonates across skin is discussed.

Administration, Cutaneous↗
PubMed2000

A general method for the rapid characterization of tyrosine-phosphorylated proteins by mini two-dimensional gel electrophoresis.

Our preliminary results are reported in the investigation of the tyrosine phosphorylation cascade triggered by the stimulation of the insulin receptor in the adipocyte cell line 3T3-L1 using a mini two-dimensional gel electrophoresis approach. The minigel format, 8 x 10 cm, was found sufficiently resolving and reproducible to study complex biological samples while considerably increasing throughput and lowering costs compared to larger gel formats. Consequently, we used the minigel format to rapidly screen a large number of samples, of which only the most relevant were then analyzed by optimized, preparative two-dimensional gels. The accurate localization and relative quantification of tyrosine-phosphorylated proteins was performed using a nonradioactive triple labeling method. After transfer onto polyvinylidene difluoride (PVDF) membranes, proteins were stained with Sypro Ruby to verify the separation quality and to localize the general region of interest for immunostaining. The membranes were subsequently blocked with polyvinylpyrrolidone-40 and probed with the relevant antibodies for visualization of the phosphorylated proteins by chemiluminescence. Finally, membranes were stained with colloidal gold to obtain a pattern reminiscent of the silver staining of a polyacrylamide gel. We believe that the presented strategy can be generalized for any gel application in which a protein has to be detected and identified based on its immunoreactivity.

3T3 Cells↗
PubMed1999

Bis(N,N-dimethylhydroxamido)hydroxooxovanadate inhibition of protein tyrosine phosphatase activity in intact cells: comparison with vanadate.

We have shown previously that bis(N,N-dimethylhydroxamido)hydroxooxovanadate (DMHV) is an excellent reversible inhibitor of protein tyrosine phosphatase (PTP) in vitro. DMHV does not carry a charge under physiological pH conditions and is anticipated to permeate cell membranes more easily than vanadate. In the present study, the efficacy of DMHV as a PTP inhibitor in intact cells was compared with that of vanadate by measuring phosphotyrosine levels in various cells treated with these compounds. DMHV was more effective in increasing both the phosphotyrosine levels of various proteins in 3T3L1 fibroblasts and the level of insulin-receptor phosphorylation in CHO cells overexpressing the human insulin receptor. DMHV was about 10- to 20-fold more effective than vanadate in increasing glucose transport and glycogen synthesis in 3T3L1 adipocytes. DMHV, unlike vanadate, also inhibited PTP in Jurkat cells. The implications of these observations are discussed.

3T3 Cells↗
PubMed1999

Phosphopeptide isomer separation using capillary zone electrophoresis for the study of protein kinases and phosphatases.

Methods for the rapid separation of phosphopeptide isomers (peptides with the same sequence but with phosphates on different residues) were developed using capillary zone electrophoresis with ultraviolet (CZE-UV) detection. Uncoated, cationic and neutral capillaries were used with both acidic and basic peptides. These methods enabled the assay of several protein kinases (mitogen activated protein kinase, protein kinase A, GST-tyrosine kinase) and phosphatases (acid, alkaline, and protein tyrosine phosphatase) and the determination of the sites of phosphorylation and dephosphorylation. Incubations of nonphosphorylated or phosphorylated peptide with kinases or phosphatases took place directly in the instrument's autosampler and were monitored over several hours using CZE-UV.

Amino Acid Sequence↗
PubMed1999

Fluorescein monophosphates as fluorogenic substrates for protein tyrosine phosphatases.

A series of novel fluorescein monophosphates aimed as substrates for protein tyrosine phosphatases (PTPs) were synthesized and evaluated against fluorescein diphosphate (FDP), the currently used fluorescent substrate for PTPs. In contrast to FDP, which is dephosphorylated to monophosphate and then to fluorescein in a sequential reaction, these monophosphates are dephosphorylated in a single step. This eliminates the complication in assaying PTPs due to the cleavage of the second phosphate group. The kinetic studies of these substrates with PTPs were performed and Michaelis-Menten parameters were obtained. These designed substrates have Km 0.03-0. 35 mM, kcat/Km of 3-100 mM-1 s-1 with CD45 and PTP1B. The results showed that the substrates with negative charge groups on the fluorescein have higher affinities for PTP1B, which are consistent with other observations. In this series, fluorescein monosulfate monophosphate (FMSP) was the best substrate observed. Since FMSP showed large increases in both absorption and fluorescence upon dephosphorylation by PTPs at pH>6.0, it is one of the most sensitive, stable and high affinity substrates reported for PTPs.

Fluoresceins↗
PubMed1999

Increased insulin sensitivity and obesity resistance in mice lacking the protein tyrosine phosphatase-1B gene.

Protein tyrosine phosphatase-1B (PTP-1B) has been implicated in the negative regulation of insulin signaling. Disruption of the mouse homolog of the gene encoding PTP-1B yielded healthy mice that, in the fed state, had blood glucose concentrations that were slightly lower and concentrations of circulating insulin that were one-half those of their PTP-1B+/+ littermates. The enhanced insulin sensitivity of the PTP-1B-/- mice was also evident in glucose and insulin tolerance tests. The PTP-1B-/- mice showed increased phosphorylation of the insulin receptor in liver and muscle tissue after insulin injection in comparison to PTP-1B+/+ mice. On a high-fat diet, the PTP-1B-/- and PTP-1B+/- mice were resistant to weight gain and remained insulin sensitive, whereas the PTP-1B+/+ mice rapidly gained weight and became insulin resistant. These results demonstrate that PTP-1B has a major role in modulating both insulin sensitivity and fuel metabolism, thereby establishing it as a potential therapeutic target in the treatment of type 2 diabetes and obesity.

Animals↗
PubMed1999

[Difluro(phosphono)methyl]phenylalanine-containing peptide inhibitors of protein tyrosine phosphatases.

Peptides containing the non-hydrolysable phosphotyrosine analogue 4-[difluro(phosphono)methyl]phenylalanine [Phe(CF2P)] were synthesized and tested as inhibitors of the protein tyrosine phosphatases (PTPs) PTP1B, CD45, PTPbeta, LAR and SHP-1. We have identified peptides containing two adjacent Phe(CF2P) residues as potent inhibitors of PTPs. The tripeptide having the sequence Glu-Phe(CF2P)-Phe(CF2P) is a potent and selective inhibitor of PTP1B. This peptide inhibits PTP1B with an IC50 of 40 nM, which is at least 100-fold lower than with other PTPs. A second tripeptide, Pro-Phe(CF2P)-Phe(CF2P), is most potent against PTPbeta, with an IC50 of 200 nM, and inhibits PTP1B with an IC50 of 300 nM. These data suggest that it is possible to develop selective, active-site-directed, reversible, potent inhibitors of PTPs.

Catalytic Domain↗
PubMed1999

Vanadate inhibition of protein tyrosine phosphatases in Jurkat cells: modulation by redox state.

Vanadate is a potent reversible inhibitor of protein tyrosine phosphatases (PTP) in vitro. Vanadate has been shown to increase the phosphotyrosine levels in some cell types whereas in others, like the Jurkat T-lymphoma, vanadate has no effect. The reason for the apparent lack of effect of vanadate in Jurkat cells was investigated in this study. Alteration of the redox state of these cells by reducing the glutathione level with 1-chloro-2,4-dinitrobenzene (DnpCl) had no effect on phosphotyrosine levels. However, the cells became sensitive to vanadate, as measured by an increase in phosphotyrosine levels on a wide range of proteins including the MAP kinases. The increase in phosphotyrosine levels most likely results from inhibition of cellular PTP and suggests that protein tyrosine kinases are constitutively active in cells, resulting in a dynamic phosphorylation-dephosphorylation cycle. The mode of inhibition of PTP by vanadate was investigated by measuring the PTP activity of Jurkat membranes isolated after treatment of cells with vanadate and DnpCl. In contrast to the reversible inhibition of PTP in vitro, the effect of vanadate in the presence of DnpCl was irreversible, raising the possibility that it is peroxovanadate formed in situ that is responsible for the inhibition of PTP in intact cells.

Calcium-Calmodulin-Dependent Protein Kinases↗
PubMed1999

Multidrug resistance in human tumors--molecular diagnosis and clinical significance.

BACKGROUND: Multidrug resistance (MDR) of human tumors is one of the major reasons for the failure of chemotherapy in refractory cancer patients. MDR can be intrinsic or acquired, depending on the time of its occurrence, either at diagnosis or during chemotherapy. Molecular investigations in MDR during the last two decades have resulted in the isolation and characterization of genes coding for P-glycoprotein, multidrug resistance-associated protein, lung resistance-related protein, drug resistance-associated protein, breast cancer resistance protein, and adenosine triphosphate-binding cassette protein. Several molecular probes, primer pairs, and monoclonal antibodies have been developed over these years to quantify the regulation and expression of these drug resistance markers in tumor cells. Methodologies have also been standardized to estimate the gene amplification, mRNA and protein expression, and functionality of drug resistance proteins in clinical specimens from cancer patients. METHODS AND RESULTS: This review describes these drug resistance genes and techniques for detection and quantification of their expression and function. CONCLUSIONS: Because these markers have clinical significance and usefulness, currently available technology warrants the application of these markers in clinical oncology.

Antibodies, Monoclonal↗