Biomedical subjects

P J Chikhale

Publications and source records attributed to P J Chikhale.

10 recordsLinked to original sources

PubMed2001

Predominant functional activity of the large, neutral amino acid transporter (LAT1) isoform at the cerebrovasculature.

In this study, we identify the predominant functional expression of the large, neutral amino acid transporter (LAT1) isoform at the blood-brain barrier (BBB). An in situ rat brain perfusion technique allowed perfusion of the radiotracer [(14)C]-L-Leu (a ligand for both LAT1 and LAT2) alone or competed with excess concentration of either LAT1 or LAT2 specific amino acids. The LAT2 specific amino acid, [(14)C]-L-Asn, was perfused alone or with excess concentration of various amino acids. The brain uptake of [(14)C]-L-Leu was not significantly inhibited by LAT2 specific amino acids, but was inhibited significantly (up to 90%) by the LAT1 specific amino acid, D-Met. L-Asn did not demonstrate saturable brain uptake. These data clearly demonstrate that LAT1 is the functionally predominant isoform expressed at the BBB which is responsible for brain uptake of large, neutral amino acids. In addition, the functional activity of cerebrovascular LAT2 is insignificant, or absent.

Amino Acid Transport Systems↗

PubMed2001

Targeted drug delivery systems 6: Intracellular bioreductive activation, uptake and transport of an anticancer drug delivery system across intestinal Caco-2 cell monolayers.

We demonstrate transport across, intracellular accumulation and bioreductive activation of a conformationally constrained, anticancer drug delivery system (the CH(3)-TDDS) using Caco-2 cell monolayers (CCMs) as an in vitro model of the human intestinal mucosa. Reverse-phase High Performance Liquid Chromatography (HPLC) coupled with UV detection was used to detect CH(3)-TDDS, the bioreduction product (lactone) and the released drug (melphalan methyl ester; MME). Upon incubation of the CH(3)-TDDS with the apical (AP) surface of 21-day-old CCM, we observed rapid decrease in the AP concentration of the CH(3)-TDDS (60%/hr) as a result of cellular uptake. Rapid intracellular accumulation of the CH(3)-TDDS was followed by bioreductive activation to deplete the cellular levels of CH(3)-TDDS. The drug part (MME) and lactone, as well as CH(3)-TDDS, were detected in the basolateral (BL) chamber. Intracellular Caco-2 levels of TDDS and lactone were also detectable. Bioreductive activation of the CH(3)-TDDS was additionally confirmed by formation of lactone after incubation of the CH(3)-TDDS in the presence of freshly prepared Caco-2 cell homogenates. During transport studies of melphalan or MME alone (as control), the intact drug was not detected in the intracellular compartment or in the BL chamber. These observations demonstrate that CH(3)-TDDS has potential for improving intestinal delivery of MME. TDDS could be useful in facilitating oral absorption of MME as well as the oral delivery of other agents.

Antineoplastic Agents↗

PubMed2001

A bioreversible prodrug approach designed to shift mechanism of brain uptake for amino-acid-containing anticancer agents.

By derivatization at the N-terminus of amino acid-based anticancer agents (e.g. melphalan and acivicin) to form a drug delivery system (TDDS), we demonstrate a change in the mechanism of brain uptake from the large neutral amino acid transporter (LAT) pathway to passive. An in situ rat brain perfusion technique was used to determine the brain capillary permeability-surface area (PA) product for [(14)C]L-Leu as control (5.18 +/- 0.32 x 10(-2) mL/s/g), which was inhibited competitively (to 7-18% of control) by an excess concentration of the amino-acid-containing anticancer agents, acivicin and melphalan. However, TDDS did not compete for LAT-mediated brain uptake of the radiotracer [(14)C]L-Leu. Brain uptake of TDDS was determined after in situ brain perfusion followed by RP-HPLC along with LC-MS/MS detection of the analytes in brain samples. The PA product for CH(3)-TDDS containing melphalan (5.09 +/- 2.0 x 10(-2) mL/s/g) shows that these agents rapidly cross the blood-brain barrier. Furthermore, competition studies of CH(3)-TDDS with [(3)H]verapamil suggest that the TDDS interacts significantly with the multidrug resistant efflux system (P-glycoprotein) at the blood-brain barrier. Therefore, TDDS were shown to lack LAT-mediated brain uptake. The drug delivery systems, however, showed uptake predominantly via the passive route along with recognition by the multidrug resistant efflux protein at the cerebrovasculature.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2000

Stability of bioreductive drug delivery systems containing melphalan is influenced by conformational constraint and electronic properties of substituents.

The stability of bioreductive drug delivery systems (TDDS) was monitored at various pH values and in the presence of glutathione (GSH). Results suggest that steric hindrance due to conformational constraint in TDDS led to an increase in stability of TDDS toward nucleophilic degradation under aqueous conditions. The electronic properties of substituents influenced TDDS stability at different pH values and in the presence of GSH.

Antineoplastic Agents, Alkylating↗

PubMed1995

Carrier-mediated transport of the antitumor agent acivicin across the blood-brain barrier.

The cytotoxic agent acivicin has been shown to be effective against several types of tumors. However, the clinical utilization of acivicin has been prohibited because of its dose-limiting neurotoxicity. Acivicin is believed to be transported into the brain by the large neutral amino acid (LNAA) carrier, which is expressed at the blood-brain barrier (BBB). In this study, we used an in situ rat brain perfusion technique to determine the kinetics of the LNAA carrier-mediated transport of acivicin across the BBB. We found that the Vmax of acivicin (1.05 nmol/sec/g) obtained in this study was comparable to the Vmax of L-leucine (1.07 nmol/sec/g) and other LNAAs as determined by other investigators. The Km was high compared with other LNAAs, but this could be explained by the low lipophilicity of acivicin. Acivicin transport across the BBB was inhibited by other LNAAs but not by acivicin derivatives with structural modifications at the amino or carboxyl group. The ASC (alanine, serine, cysteine) carrier system did not influence the transport of acivicin across the BBB. These results suggest that the CNS toxicity of acivicin might be reduced by coadministration of other LNAAs. Acivicin derivatives with structural modifications at the amino or carboxyl group of acivicin lack affinity for the LNAA carrier at the BBB and, therefore, will exhibit less CNS toxicity than acivicin.

Amino Acids↗

PubMed1994

Improved delivery through biological membranes. LXI: Design, synthesis, and evaluation of a lipolol-based intradermal drug targeting system for 5-fluorouracil.

This report describes initial studies with the lipolyl ester of 1-carboxypropylcarbamoyl-FU (LE-CPCFU) which was designed to enhance the dermal delivery of the antitumor agent 5-fluorouracil (FU). The design of LE-CPCFU was based upon our previous observation that sulfur-based chemical drug targeting systems were localized within the skin and improved the delivery of the parent drug to the dermal tissue (Chikhale et al., 1993; Bodor et al., 1982; Bodor and Sloan, 1982). In the in vitro test system that used freshly-excised guinea-pig skin, LE-CPCFU was found to enhance FU delivery to the skin 2- to 5-fold compared to underivatized FU. Neither LE-CPCFU nor its acid metabolite 1-carboxypropylcarbamoyl-FU (CPCFU) could be detected in the skin or receiver during the diffusion experiments even though LE-CPCFU and CPCFU were found to be reasonably stable in aqueous pH 7.4 buffer and during the analytical procedure. FU which was released from LE-CPCFU in the skin subsequently diffused into the receiver. Thus, LE-CPCFU was observed to improve FU delivery to the skin during the initial time period of the study (0-4 hr). This study indicates that LE-CPCFU in the guinea-pig skin was hydrolyzed to form FU in the skin serving as an intradermal drug delivery system for the antitumor agent. Thus, LE-CPCFU could prove to reduce the systemic toxicity of FU by enhancing the local skin concentration and minimizing the systemic concentration of the antitumor agent as compared to underivatized FU.

Absorption↗

PubMed1993

Utilization of a human intestinal epithelial cell culture system (Caco-2) for evaluating cytoprotective agents.

Human intestinal epithelial cells (Caco-2) were cultured as confluent monolayers on polycarbonate membranes in Transwells for investigating their applicability in evaluating the cytoprotective activity of sucralfate. The control experiments established a reproducible chemical method (using 0.5 mM indomethacin in Hanks' balanced salt solution) for inducing damage to the Caco-2 cell monolayers. Damage was determined by measuring changes in transepithelial electrical resistance (TEER). Twenty-day-old Caco-2 cell monolayers were significantly and reproducibly damaged (compared to buffer alone) (P < 0.001) by application of 0.5 mM indomethacin to the apical side for 1 hr. While sucralfate, at a 0.5, 2, or 5 mg/mL concentration in the buffer, was shown not to reverse (treat) the damage caused by indomethacin in this cellular model, it was able to protect (prevent) the cells from indomethacin-induced damage (P < 0.001). We observed that indomethacin-induced damage to the Caco-2 cell monolayers greatly affected the paracellular pathway since the percentage transport of [3H]methoxyinulin was significantly elevated. In contrast, protection of the Caco-2 cells with 5 mg/mL sucralfate in the presence of the damaging agent resulted in transport of the paracellular marker similar to that in the control (HBSS-treated) cell monolayers. This direct cytoprotective effect was thus independent of vascular factors at neutral pH and was observed to be dose dependent (0.5 to 5 mg/mL) when sucralfate was applied to the cells in the presence of the damaging agent.(ABSTRACT TRUNCATED AT 250 WORDS)

Cells, Cultured↗

PubMedDate not supplied

Modulating blood-brain barrier interactions of amino acid-based anticancer agents.

The large neutral amino acid (LNAA) transporter at the blood-brain barrier (BBB) mediates brain uptake of amino acid-based anticancer agents (e.g., melphalan and acivicin). In this study, we blocked the amino acid terminus of the anticancer agents using a bioreductive drug delivery system (TDDS). This molecular modification of the anticancer agents is expected to prevent LNAA carrier-mediated transport across the BBB. In this study, we demonstrate that the parent amino acid containing anticancer agents are substrates for the LNAA transporter at the BBB, whereas the TDDS is not recognized by the LNAA transporter. An in situ rat brain perfusion technique was used to determine competition for LNAA carrier-mediated transport at the BBB using [14C]L-leucine. The BBB capillary permeability-surface area (PA) product for the radiotracer [14C]L-leucine (control) was determined to be 5.18 +/- 0.32 x 10(-2) ml/s/g (100%). The control PA value for [14C]L-leucine was competitively inhibited (down to 7-18% of control) by excess L-phenylalanine as well as by excess concentration of the anticancer amino acids, melphalan and acivicin, showing competition for the LNAA transporter at the BBB. In contrast, brain perfusion of [14C]L-leucine in presence of excess TDDS resulted in no competition for brain uptake of [14C]L-leucine via the LNAA transporter. Thus, bioreversible derivatization of the parent anticancer amino acids resulted in blocking the amino acid functional group, thereby leading to loss of recognition for the cerebrovascular LNAA transporter at the BBB.

Algorithms↗

PubMedDate not supplied

Carboplatin hypersensitivity.

Carboplatin has established an important role in many different cancers. As its use increased, the documented cases of hypersensitivity also picked up. Although the mechanism of these reactions remains unknown, the immediate type of hypersensitivity reaction mediated by IgE may be involved. It takes a while for the reaction to develop, but cases are reported even after 1st cycle. The incidence of hypersensitivity is highest at about 8th cycle of therapy with decline after that. These reactions themselves ranged from facial flushing or itching to seizures, dyspnea, and anaphylaxis. Many physicians currently do not use skin testing prior to 8th cycle of carboplatin therapy and retreat their patients with carboplatin after the first hypersensitivity reaction. Therefore, it is suggested that skin test should be conducted prior to the 8th cycle, preferably before the 6th cycle, as hypersensitivity tends to increase on the 6th cycle-treatment. Methods published so far involve: desensitization, skin testing, switching therapy to another platinum analogue, and premedication. Despite all the process, the most effective drug toxicity prevention method remains skin testing prior to 8th cycle. It can accurately predict patients who will develop hypersensitivity reactions. Other methods so far have not shown consistent results.

Adult↗

PubMedDate not supplied

Metabolism of L-alpha-methyldopa in cultured human intestinal epithelial (Caco-2) cell monolayers. Comparison with metabolism in vivo.

Our objective was to evaluate the utility of an in vitro cell culture system (Caco-2) to study the pathways for the metabolism of L-alpha-methyldopa (MD) in the intestinal mucosa during its transepithelial transport. Caco-2 cell monolayers grown onto polycarbonate membranes in Transwells have been reported to exhibit morphological and biochemical properties similar to those of the human intestinal mucosa. After oral administration, MD has been reported to undergo extensive first-pass metabolism during its transport across the intestinal mucosa. In earlier studies, our laboratory showed that MD was rapidly transported across Caco-2 cell monolayers consistent with a carrier-mediated mechanism involving the large neutral amino acid transporter (Hu and Borchardt, 1990). Reversed-phase, ion-pair HPLC coupled to an electrochemical (amperometric) detector operating in the oxidative mode was used in this study to detect MD and its metabolites in the transport and cellular media. After rapid cellular uptake from the apical surface of Caco-2 cell monolayers, MD was predominantly metabolized to form MD-sulfate and MD-glucuronide and 3-O-methyl-methyldopa and its sulfated conjugate. Although methyldopamine was detected in minor quantities, it was present mainly as the sulfate and glucuronide conjugate. Although the cellular levels of MD and its metabolites rapidly reached steady-state, the corresponding basolateral levels continuously increased after an initial small lag-time. We observed that the 25-day-old Caco-2 cell monolayers used in this study were able to metabolize MD to a greater extent than those cultured for 11 days (p < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Biological Transport↗