Linda B. Tabas

Transport mechanisms responsible for the absorption of loracarbef, cefixime, and cefuroxime axetil into human intestinal Caco-2 cells

Loracarbef, cefixime and cefuroxime axetil are beta-lactam antibiotics that are administered orally. Oral absorption of loracarbef is nearly complete, while that of cefixime and cefuroxime axetil is 30-50%. To investigate this we used the human intestinal cell line Caco-2 that possesses the proton-dependent peptide transporter that takes up cephalexin and cefaclor. Drug uptake was measured at pH 6 by high performance liquid chromatography or with radioactively labelled drug. The initial uptake rate of 1 mM cefixime was lower than that of 1 mM loracarbef. By 2 h both drugs were concentrated intracellularly against a gradient; however, the accumulation of cefixime was only 40% of that of loracarbef. The uptake rate of both drugs was sodium-independent, temperature- and energy-dependent, and was inhibited by dipeptides, cephalexin, cefaclor, but not by amino acids. Kinetic analysis of the concentration-dependence of the uptake rates for loracarbef and cefixime indicated that diffusion and a single transport system were responsible for uptake. The kinetic parameters for loracarbef and cefixime, respectively, were: Km values of 8 and 17 mM and Vmax values of 6.5 and 2 nmol/min per mg protein. Loracarbef and cefixime were competitive inhibitors of each others uptake. By contrast, cefuroxime axetil was taken up and rapidly hydrolyzed to cefuroxime by Caco-2 cells. Cefuroxime axetil uptake was not dependent on energy and was not affected by dipeptides. Thus, cefuroxime axetil apparently enters Caco-2 cells by simple diffusion. By contrast, loracarbef and cefixime share a common transport mechanism, the proton-dependent dipeptide transporter. Cefixime was taken up less well than loracarbef due to a substantial reduction in the turnover rate and decreased affinity of the transporter for cefixime.

Tricyclic isoxazoles are novel inhibitors of the multidrug resistance protein (MRP1)

Tricyclic isoxazoles were identified from a screen as a novel class of selective multidrug resistance protein (MRP1) inhibitors. From a screen lead, SAR efforts resulted in the preparation of LY 402913 (9h), which inhibits MRP1 and reverses drug resistance to MRP1 substrates, such as doxorubicin, in HeLa-T5 cells (EC(50)=0.90 microM), while showing no inherent cytotoxicity. Additionally, LY 402913 inhibits ATP-dependent, MRP1-mediated LTC(4) uptake into membrane vesicles prepared from the MRP1-overexpressing HeLa-T5 cells (EC(50)=1.8 microM). LY 402913 also shows selectivity ( approximately 22-fold) against the related transporter, P-glycoprotein, in HL60/Adr and HL60/Vinc cells. Finally, when dosed in combination with the oncolytic MRP1 substrate vincristine, LY 402913 delays the growth of MRP1-overexpressing tumors in vivo.

Cefaclor uptake by the proton-dependent dipeptide transport carrier of human intestinal Caco-2 cells and comparison to cephalexin uptake

The human Caco-2 cell line spontaneously differentiates in culture to epithelial cells possessing intestinal enterocytic-like properties. These cells possess a proton-dependent dipeptide transport carrier that mediates the uptake of the cephalosporin antibiotic cephalexin (Dantzig, A.H. and Bergin, L. (1990) Biochim. Biophys. Acta 1027, 211-217). In the present study, the uptake of cefaclor was examined and found to be sodium-independent, proton-dependent, and energy-dependent. The initial rate of D-[3-phenyl-3H]cefaclor uptake was measured over a wide concentration range; uptake was mediated by a single saturable transport carrier with a Km of 7.6 mM and a Vmax of 7.6 nmol/min per mg protein and by a non-saturable component. Uptake was inhibited by dipeptides but not amino acids. The carrier showed a preference for the L-isomer. The effect of the presence of a 5-fold excess of other beta-lactam antibiotics was examined on the initial rates of 1 mM cefaclor and 1 mM cephalexin uptake. Uptake rates were inhibited by the orally absorbed antibiotics, cefadroxil, cefaclor, loracarbef, and cephradine and less so by the parenteral agents tested. The initial uptake rates of both D-[9-14C]cephalexin and D-[3-phenyl-3H]cefaclor were competitively inhibited by cephalexin, cefaclor, and loracarbef with Ki values of 9.2-13.2, 10.7-6.2, and 7.7-6.4 mM, respectively. Taken together, these data suggest that a single proton-dependent dipeptide transport carrier mediates the uptake of these orally absorbed antibiotics into Caco-2 cells, and provide further support for the use of Caco-2 cells as a cellular model for the study of the intestinal proton-dependent dipeptide transporter.

Frontolimbic atrophy is associated with agitation and aggression in mild cognitive impairment and Alzheimer's disease

The neuroanatomy of agitation and aggression in Alzheimers disease is not well understood.

Structure-activity relationship of carbacephalosporins and cephalosporins: antibacterial activity and interaction with the intestinal proton-dependent dipeptide transport carrier of Caco-2 cells.

An intestinal proton-dependent peptide transporter located on the lumenal surface of the enterocyte is responsible for the uptake of many orally absorbed beta-lactam antibiotics. Both cephalexin and loracarbef are transported by this mechanism into the human intestinal Caco-2 cell line. Forty-seven analogs of the carbacephalosporin loracarbef and the cephalosporin cephalexin were prepared to evaluate the structural features necessary for uptake by this transport carrier. Compounds were evaluated for their antibacterial activities and for their ability to inhibit 1 mM cephalexin uptake and, subsequently, uptake into Caco-2 cells. Three clinically evaluated orally absorbed carbacephems were taken up by Caco-2 cells, consistent with their excellent bioavailability in humans. Although the carrier preferred the L stereoisomer, these compounds lacked antibacterial activity and were hydrolyzed intracellularly in Caco-2 cells. Compounds modified at the 3 position of cephalexin and loracarbef with a cyclopropyl or a trifluoromethyl group inhibited cephalexin uptake. Analogs with lipophilic groups on the primary amine of the side chain inhibited cephalexin uptake, retained activity against gram-positive bacteria but lost activity against gram-negative bacteria. Substitution of the phenylglycl side chain with phenylacetyl side chains gave similar results. Compounds which lacked an aromatic ring in the side chain inhibited cephalexin uptake but lost all antibacterial activity. Thus, the phenylglycl side chain is not absolutely required for uptake. Different structural features are required for antibacterial activity and for being a substrate of the transporter. Competition studies with cephalexin indicate that human intestinal Caco-2 cells may be a useful model system for initially guiding structure-activity relationships for the rational design of new oral agents.

Reversal of resistance in multidrug resistance protein (MRP1)-overexpressing cells by LY329146.

The benzothiophene LY329146 reverses the drug resistance phenotype in multidrug resistance protein (MRP1)-overexpressing cells when dosed in combination with MRP1-associated oncolytics doxorubicin and vincristine. Additionally, LY329146 inhibited MRP1-mediated uptake of the MRP1 substrate LTC4 into membrane vesicles prepared from MRP1-overexpressing cells.

Mibampator (LY451395) randomized clinical trial for agitation/aggression in Alzheimer's disease

BACKGROUND Mibampator, an amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor potentiator, was evaluated for treatment of agitation and aggression (A/A) in Alzheimers disease (AD). METHODS Outpatients (n = 132) with probable AD and A/A randomized to 12 weeks of double-blind treatment with 3-mg po mibampator or placebo were assessed using the 4-domain A/A subscale of the Neuropsychiatric Inventory (NPI-4-A/A) derived from the Neuropsychiatric Inventory. Secondary measures included the Cohen-Mansfield Agitation Inventory, Cornell Scale for Depression in Dementia, Frontal Systems Behavior Inventory (FrSBe), and Alzheimers Disease Assessment Scale-Cognitive. Efficacy was analyzed using mixed-effects model repeated measures from baseline to endpoint. Adverse events (AEs), labs, vital signs, and electrocardiograms were monitored. RESULTS Baseline characteristics were comparable between groups. Both groups improved on the NPI-4-A/A, but without group differences. Among secondaries, mibampator was significantly better (p = 0.007) than placebo only on the FrSBe. AEs were similar between groups. One death occurred in the placebo group. CONCLUSION Possible explanations for no significant group differences include caregiver, drug target engagement, and design issues. This trial is registered on ClinicalTrials.gov; ID: NCT00843518.

Clinical use of amyloid-positron emission tomography neuroimaging: Practical and bioethical considerations

Until recently, estimation of β‐amyloid plaque density as a key element for identifying Alzheimers disease (AD) pathology as the cause of cognitive impairment was only possible at autopsy. Now with amyloid‐positron emission tomography (amyloid‐PET) neuroimaging, this AD hallmark can be detected antemortem. Practitioners and patients need to better understand potential diagnostic benefits and limitations of amyloid‐PET and the complex practical, ethical, and social implications surrounding this new technology. To complement the practical considerations, Eli Lilly and Company sponsored a Bioethics Advisory Board to discuss ethical issues that might arise from clinical use of amyloid‐PET neuroimaging with patients being evaluated for causes of cognitive decline. To best address the multifaceted issues associated with amyloid‐PET neuroimaging, we recommend this technology be used only by experienced imaging and treating physicians in appropriately selected patients and only in the context of a comprehensive clinical evaluation with adequate explanations before and after the scan.