Allele-specific polymerase chain reaction diagnostic test for the functional MDR1 polymorphism in dogs

The major multidrug transporter P-glycoprotein (Pgp) contributes to the barrier function of several tissues and organs, including the brain. In a subpopulation of Collies and seven further dog breeds, a 4 base pair deletion has been described in the Pgp-encoding MDR1 gene. This deletion results in the absence of a functional form of Pgp and loss of its protective function. Severe intoxication with the Pgp substrate ivermectin has been attributed to the genetically determined lack of Pgp. An allele-specific polymerase chain reaction (PCR)-based screening method has been developed to detect the mutant allele and to determine if a dog is homozygous or heterozygous for the mutation. Based on this validation, the allele-specific PCR proved to be a robust, reproducible and specific tool, allowing rapid determination of the MDR1 genotype of dogs of at risk breeds using blood samples or buccal swabs.

Novel Use of Dual Anti-Inflammatory Therapy to Overcome Drug Resistance and Improve Functional Recovery Following Spinal Cord Injury

Over 1.2 million Americans are currently living with a traumatic spinal cord injury (SCI). Despite the need for effective therapies, there are currently no proven effective treatments that can improve recovery of function in SCI patients. Many therapeutic compounds have shown promise in preclinical models of SCI, but all of these have fallen short in clinical trials. P-glycoprotein (Pgp) is an active transporter expressed on capillary endothelial cell membranes at the blood-spinal cord barrier (BSCB). Pgp limits passive diffusion of blood-borne drugs into the CNS, by actively extruding drugs from the endothelial cell membrane. Pgp can become pathologically up-regulated, thus greatly impeding therapeutic drug delivery (‘multidrug resistance’). Importantly, many drugs that have been evaluated for the treatment of SCI are Pgp substrates. We hypothesized that Pgp-mediated drug resistance diminishes the delivery and efficacy of neuroprotective drugs following SCI. We observed a progressive, spatial spread of Pgp overexpression within the injured spinal cord. To assess Pgp function, we examined spinal cord uptake of systemically-delivered riluzole, a drug that is currently being evaluated in clinical trials as an SCI intervention. Blood-to-spinal cord riluzole penetration was reduced following SCI in wild-type but not Pgp-null rats, highlighting a critical role for Pgp in mediating spinal cord drug resistance after injury. Others have shown that pro-inflammatory signaling drives Pgp up-regulation in cancer and epilepsy. We have detected inflammation in both acutely- and chronically-injured spinal cord tissue. We therefore evaluated the ability of the dual COX-/5-LOX inhibitor licofelone to attenuate Pgp-mediated drug resistance following SCI. Licofelone treatment both reduced spinal cord Pgp levels and enhanced spinal cord riluzole bioavailability following SCI. Thus, we propose that licofelone may offer a new combinatorial treatment strategy to enhance spinal cord drug delivery following SCI. Additionally, we assessed the ability of licofelone, riluzole, or both to enhance recovery of locomotor function following SCI. We found that licofelone treatment conferred a significant improvement in hindlimb function that was sustained through the end of the study. In contrast, riluzole did not improve functional outcome. We therefore conclude that licofelone holds promise as a potential neuroprotective intervention for SCI.

A release mechanism for stored ATP in ocular ciliary epithelial cells

Purines can modify ciliary epithelial secretion of aqueous humor into the eye. The source of the purinergic agonists acting in the ciliary epithelium, as in many epithelial tissues, is unknown. We found that the fluorescent ATP marker quinacrine stained rabbit and bovine ciliary epithelia but not the nerve fibers in the ciliary bodies. Cultured bovine pigmented and nonpigmented ciliary epithelial cells also stained intensely when incubated with quinacrine. Hypotonic stimulation of cultured epithelial cells increased the extracellular ATP concentration by 3-fold; this measurement underestimates actual release as the cells also displayed ecto-ATPase activity. The hypotonically triggered increase in ATP was inhibited by the Cl−-channel blocker 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) in both cell types. In contrast, the P-glycoprotein inhibitors tamoxifen and verapamil and the cystic fibrosis transmembrane conductance regulator (CFTR) blockers glybenclamide and diphenylamine-2-carboxylate did not affect ATP release from either cell type. This pharmacological profile suggests that ATP release is not restricted to P-glycoprotein or the cystic fibrosis transmembrane conductance regulator, but can proceed through a route sensitive to NPPB. ATP release also was triggered by ionomycin through a different NPPB-insensitive mechanism, inhibitable by the calcium/calmodulin-activated kinase II inhibitor KN-62. Thus, both layers of the ciliary epithelium store and release ATP, and purines likely modulate aqueous humor flow by paracrine and/or autocrine mechanisms within the two cell layers of this epithelium.

Postgastrulation Smad2-deficient embryos show defects in embryo turning and anterior morphogenesis

SMAD2 is a member of the transforming growth factor β and activin-signaling pathway. To examine the role of Smad2 in postgastrulation development, we independently generated mice with a null mutation in this gene. Smad2-deficient embryos die around day 7.5 of gestation because of failure of gastrulation and failure to establish an anterior–posterior (A-P) axis. Expression of the homeobox gene Hex (the earliest known marker of the A-P polarity and the prospective head organizer) was found to be missing in Smad2-deficient embryos. Homozygous mutant embryos and embryonic stem cells formed mesoderm derivatives revealing that mesoderm induction is SMAD2 independent. In the presence of wild-type extraembryonic tissues, Smad2-deficient embryos developed beyond 7.5 and up to 10.5 days postcoitum, demonstrating a requirement for SMAD2 in extraembryonic tissues for the generation of an A-P axis and gastrulation. The rescued postgastrulation embryos showed malformation of head structures, abnormal embryo turning, and cyclopia. Our results show that Smad2 expression is required at several stages during embryogenesis.

Dissection of De Novo Membrane Insertion Activities of Internal Transmembrane Segments of ATP-Binding-Cassette Transporters: Toward Understanding Topological Rules for Membrane Assembly of Polytopic Membrane Proteins

The membrane assembly of polytopic membrane proteins is a complicated process. Using Chinese hamster P-glycoprotein (Pgp) as a model protein, we investigated this process previously and found that Pgp expresses more than one topology. One of the variations occurs at the transmembrane (TM) domain including TM3 and TM4: TM4 inserts into membranes in an Nin-Cout rather than the predicted Nout-Cin orientation, and TM3 is in cytoplasm rather than the predicted Nin-Cout orientation in the membrane. It is possible that TM4 has a strong activity to initiate the Nin-Cout membrane insertion, leaving TM3 out of the membrane. Here, we tested this hypothesis by expressing TM3 and TM4 in isolated conditions. Our results show that TM3 of Pgp does not have de novo Nin-Cout membrane insertion activity whereas TM4 initiates the Nin-Cout membrane insertion regardless of the presence of TM3. In contrast, TM3 and TM4 of another polytopic membrane protein, cystic fibrosis transmembrane conductance regulator (CFTR), have a similar level of de novo Nin-Cout membrane insertion activity and TM4 of CFTR functions only as a stop-transfer sequence in the presence of TM3. Based on these findings, we propose that 1) the membrane insertion of TM3 and TM4 of Pgp does not follow the sequential model, which predicts that TM3 initiates Nin-Cout membrane insertion whereas TM4 stops the insertion event; and 2) “leaving one TM segment out of the membrane” may be an important folding mechanism for polytopic membrane proteins, and it is regulated by the Nin-Cout membrane insertion activities of the TM segments.

Coupled Translocation Events Generate Topological Heterogeneity at the Endoplasmic Reticulum Membrane

Topogenic determinants that direct protein topology at the endoplasmic reticulum membrane usually function with high fidelity to establish a uniform topological orientation for any given polypeptide. Here we show, however, that through the coupling of sequential translocation events, native topogenic determinants are capable of generating two alternate transmembrane structures at the endoplasmic reticulum membrane. Using defined chimeric and epitope-tagged full-length proteins, we found that topogenic activities of two C-trans (type II) signal anchor sequences, encoded within the seventh and eighth transmembrane (TM) segments of human P-glycoprotein were directly coupled by an inefficient stop transfer (ST) sequence (TM7b) contained within the C-terminus half of TM7. Remarkably, these activities enabled TM7 to achieve both a single- and a double-spanning TM topology with nearly equal efficiency. In addition, ST and C-trans signal anchor activities encoded by TM8 were tightly linked to the weak ST activity, and hence topological fate, of TM7b. This interaction enabled TM8 to span the membrane in either a type I or a type II orientation. Pleiotropic structural features contributing to this unusual topogenic behavior included 1) a short, flexible peptide loop connecting TM7a and TM7b, 2) hydrophobic residues within TM7b, and 3) hydrophilic residues between TM7b and TM8.

Use of chimeric proteins to investigate the role of transporter associated with antigen processing (TAP) structural domains in peptide binding and translocation

The transporter associated with antigen processing (TAP) comprises two subunits, TAP1 and TAP2, each containing a hydrophobic membrane-spanning region (MSR) and a nucleotide binding domain (NBD). The TAP1/TAP2 complex is required for peptide translocation across the endoplasmic reticulum membrane. To understand the role of each structural unit of the TAP1/TAP2 complex, we generated two chimeras containing TAP1 MSR and TAP2 NBD (T1MT2C) or TAP2 MSR and TAP1 NBD (T2MT1C). We show that TAP1/T2MT1C, TAP2/T1MT2C, and T1MT2C/T2MT1C complexes bind peptide with an affinity comparable to wild-type complexes. By contrast, TAP1/T1MT2C and TAP2/T2MT1C complexes, although observed, are impaired for peptide binding. Thus, the MSRs of both TAP1 and TAP2 are required for binding peptide. However, neither NBD contains unique determinants required for peptide binding. The NBD-switched complexes, T1MT2C/T2MT1C, TAP1/T2MT1C, and TAP2/T1MT2C, all translocate peptides, but with progressively reduced efficiencies relative to the TAP1/TAP2 complex. These results indicate that both nucleotide binding sites are catalytically active and support an alternating catalytic sites model for the TAP transport cycle, similar to that proposed for P-glycoprotein. The enhanced translocation efficiency of TAP1/T2MT1C relative to TAP2/T1MT2C complexes correlates with enhanced binding of the TAP1 NBD-containing constructs to ATP-agarose beads. Preferential ATP interaction with TAP1, if occurring in vivo, might polarize the transport cycle such that ATP binding to TAP1 initiates the cycle. However, our observations that TAP complexes containing two identical TAP NBDs can mediate translocation indicate that distinct properties of the nucleotide binding site per se are not essential for the TAP catalytic cycle.

Multidrug resistance mediated by a bacterial homolog of the human multidrug transporter MDR1.

Resistance of Lactococcus lactis to cytotoxic compounds shares features with the multidrug resistance phenotype of mammalian tumor cells. Here, we report the gene cloning and functional characterization in Escherichia coli of LmrA, a lactococcal structural and functional homolog of the human multidrug resistance P-glycoprotein MDR1. LmrA is a 590-aa polypeptide that has a putative topology of six alpha-helical transmembrane segments in the N-terminal hydrophobic domain, followed by a hydrophilic domain containing the ATP-binding site. LmrA is similar to each of the two halves of MDR1 and may function as a homodimer. The sequence conservation between LmrA and MDR1 includes particular regions in the transmembrane domains and connecting loops, which, in MDR1 and the MDR1 homologs in other mammalian species, have been implicated as determinants of drug recognition and binding. LmrA and MDR1 extrude a similar spectrum of amphiphilic cationic compounds, and the activity of both systems is reversed by reserpine and verapamil. As LmrA can be functionally expressed in E. coli, it offers a useful prokaryotic model for future studies on the molecular mechanism of MDR1-like multidrug transporters.

Principles and clinical application of assessing alterations in renal elimination pathways

Drugs and metabolites are eliminated from the body by metabolism and excretion. The kidney makes the major contribution to excretion of unchanged drug and also to excretion of metabolites. Net renal excretion is a combination of three processes - glomerular filtration, tubular secretion and tubular reabsorption. Renal function has traditionally been determined by measuring plasma creatinine and estimating creatinine clearance. However, estimated creatinine clearance measures only glomerular filtration with a small contribution from active secretion. There is accumulating evidence of poor correlation between estimated creatinine clearance and renal drug clearance in different clinical settings, challenging the 'intact nephron hypothesis' and suggesting that renal drug handling pathways may not decline in parallel. Furthermore, it is evident that renal drug handling is altered to a clinically significant extent in a number of disease states, necessitating dosage adjustment not just based on filtration. These observations suggest that a re-evaluation of markers of renal function is required. Methods that measure all renal handling pathways would allow informed dosage individualisation using an understanding of renal excretion pathways and patient characteristics. Methodologies have been described to determine individually each of the renal elimination pathways. However, their simultaneous assessment has only recently been investigated. A cocktail of markers to measure simultaneously the individual renal handling pathways have now been developed, and evaluated in healthy volunteers. This review outlines the different renal elimination pathways and the possible markers that can be used for their measurement. Diseases and other physiological conditions causing altered renal drug elimination are presented, and the potential application of a cocktail of markers for the simultaneous measurement of drug handling is evaluated. Further investigation of the effects of disease processes on renal drug handling should include people with HIV infection, transplant recipients (renal and liver) and people with rheumatoid arthritis. Furthermore, changes in renal function in the elderly, the effect of sex on renal function, assessment of living kidney donors prior to transplantation and the investigation of renal drug interactions would also be potential applications. Once renal drug handling pathways are characterised in a patient population, the implications for accurate dosage individualisation can be assessed. The simultaneous measurement of renal function elimination pathways of drugs and metabolites has the potential to assist in understanding how renal function changes with different disease states or physiological conditions. In addition, it will further our understanding of fundamental aspects of the renal elimination of drugs.

Toward better outcomes with tacrolimus therapy: Population pharmacokinetics and individualized dosage prediction in adult liver transplantation

Patient outcomes in transplantation would improve if dosing of immunosuppressive agents was individualized. The aim of this study is to develop a population pharmacokinetic model of tacrolimus in adult liver transplant recipients and test this model in individualizing therapy. Population analysis was performed on data from 68 patients. Estimates were sought for apparent clearance (CL/F) and apparent volume of distribution (V/F) using the nonlinear mixed effects model program (NONMEM). Factors screened for influence on these parameters were weight, age, sex, transplant type, biliary reconstructive procedure, postoperative day, days of therapy, liver function test results, creatinine clearance, hematocrit, corticosteroid dose, and interacting drugs. The predictive performance of the developed model was evaluated through Bayesian forecasting in an independent cohort of 36 patients. No linear correlation existed between tacrolimus dosage and trough concentration (r(2) = 0.005). Mean individual Bayesian estimates for CL/F and V/F were 26.5 8.2 (SD) L/hr and 399 +/- 185 L, respectively. CL/F was greater in patients with normal liver function. V/F increased with patient weight. CL/F decreased with increasing hematocrit. Based on the derived model, a 70-kg patient with an aspartate aminotransferase (AST) level less than 70 U/L would require a tacrolimus dose of 4.7 mg twice daily to achieve a steady-state trough concentration of 10 ng/mL. A 50-kg patient with an AST level greater than 70 U/L would require a dose of 2.6 mg. Marked interindividual variability (43% to 93%) and residual random error (3.3 ng/mL) were observed. Predictions made using the final model were reasonably nonbiased (0.56 ng/mL), but imprecise (4.8 ng/mL). Pharmacokinetic information obtained will assist in tacrolimus dosing; however, further investigation into reasons for the pharmacokinetic variability of tacrolimus is required.

The role of morphine-6-glucuronide (M6G) in pain control

Morphine-6beta-D-glucuronide (M6G) is an analgesically active metabolite of morphine, accounting for approximate to10% of the morphine dose when administered by systemic routes to humans. Although M6G is more hydrophilic than morphine, it crosses the blood-brain barrier, albeit relatively slowly. For this reason, it is generally thought that, after chronic dosing, M6G contributes significantly to the analgesic effects of systemically administered morphine. Owing to its polar nature, M6G is cleared from the systemic circulation primarily via renal elimination. As M6G accumulates in patients with renal impairment, there is an increased risk of M6G-induced respiratory depression in renal failure patients who are being dosed chronically with systemic morphine. Consistent with its analgesic and respiratory depressant properties, M6G binds to the p-opioid receptor in a naloxone-reversible manner. Although the affinity of M6G for the mu-opioid receptor is similar to or slightly less than that of morphine, preclinical studies in rodents show that M6G is one to two orders of magnitude more potent than morphine when administered by central routes. This major discrepancy between the markedly higher intrinsic antinociceptive potency of M6G relative to morphine, despite their similar p-opioid receptor binding affinities, is difficult to reconcile. It has been proposed that M6G mediates its pain-relieving effects through a novel 'M6G opioid receptor', while others have argued that M6G may have higher efficacy than morphine for transduction of intracellular events. When administered by parenteral routes to rodents, M6G's antinociceptive potency is no more than twofold higher than morphine. In humans, the analgesic efficacy and respiratory depressant potency of M6G relative to morphine have been assessed in a number of short-term studies involving the intrathecal or intravenous routes of administration. For example, in hip replacement patients, intrathecal M6G provided excellent postoperative analgesia but the occurrence of late respiratory depression in 10% of these patients raised serious concern about safety. In postoperative patients, intravenous M6G administered by means of patient-controlled analgesia (PCA), or bolus plus PCA, produced no analgesia in one study and limited analgesia in another. Similarly, there was a lack of significant analgesia in healthy volunteers who received intravenous M6G for the alleviation of experimental pain (carbon dioxide applied to the nasal mucosa). In contrast, satisfactory analgesia was produced by bolus doses of intravenous M6G administered to patients with cancer pain, and to healthy volunteers with experimentally-induced ischaemic, electrical or thermal (ice water) pain. Studies to date in healthy volunteers suggest that intravenous M6G may be a less potent respiratory depressant and have a lower propensity for producing nausea and vomiting than morphine. However, it is unclear whether equi-analgesic doses of M6G and morphine were compared. Clearly, more extensive short-term trials, together with studies involving chronic M6G administration, are necessary before the potential clinical utility of M6G as an analgesic drug in its own right can be determined.

Area-under-the-curve monitoring of prednisolone for dose optimization in a stable renal transplant population

Background: Renal transplant recipients were noted to appear cushingoid while on low doses of steroid as part of a triple therapy immunosuppression of cyclosporin A (CsA), prednisolone, and azathioprine. Methods: The study group comprised adult renal transplant recipients with stable graft function who had received their renal allograft a minimum of 1 year previously (43 studies undertaken in 22 men and 20 women) with median daily prednisone dose of 7 mg (range 3-10). The control group was healthy nontransplant subjects [median dose 10 mg (10-30)]. Prednisolone bioavailability was measured using a limited 6-hour area under the curve (AUC), with prednisolone measured using specific HPLC assay. Results: The median prednisolone AUC/mg dose for all transplant recipients was significantly greater than the control group by approximately 50% (316 nmol(.)h/L/mg prednisolone versus 218). AUC was significantly higher in female recipients (median 415 versus 297 for men) and in recipients receiving cyclospotin (348 versus 285). The highest AUC was in women on estrogen supplements who were receiving cyclosporin (median 595). A significantly higher proportion of patients on triple therapy had steroid side effects compared with those on steroid and azathioprine (17/27 versus 4/15), more women than men had side effects (14/16 versus 7/22), and the AUC/mg prednisone was greater in those with side effects than without (median 377 versus 288 nmol-h/L/mg). Discussion: The results are consistent with the hypothesis that CsA increases the bioavailability of prednisolone, most likely through inhibition of beta-glycoprotein. The increased exposure to steroid increased the side-effect profile of steroids in the majority of patients. Because the major contributor to AUC is the maximum postdose concentration, it may be possible to use single-point monitoring (2 hours postdose) for routine clinical studies.

Milk thistle and indinavir: a randomized controlled pharmacokinetics study and meta-analysis

Objectives: To determine whether ingestion of milk thistle affects the pharmacokinetics of indinavir. Methods: We conducted a three-period, randomized controlled trial with 16 healthy participants. We randomized participants to milk thistle or control. All participants received initial dosing of indinavir, and baseline indinavir levels were obtained (AUC(0-8)) (phase I). The active group were then given 450 mg milk-thistle extract capsules to be taken t.i.d. from day 2 to day 30. The control group received no plant extract. On day 29 and day 30, indinavir dosing and sampling was repeated in both groups as before (phase II). After a wash-out period of 7 days, indinavir dosing and sampling were repeated as before (phase III). Results: All participants completed the trial, but two were excluded from analysis due to protocol violation. There were no significant between-group differences. Active group mean AUC(0-8) indinavir decreased by 4.4% (90% CI, -27.5% to -26%, P=0.78) from phase I to phase II in the active group, and by 17.3% (90% CI, -37.3% to +9%, P=0.25) in phase III. Control group mean AUC(0-8) decreased by 21.5% (90% CI, -43% to +8%, P=0.2) from phase I to phase II and by 38.5% (90% CI, -55.3% to -15.3%, P=0.01) of baseline at phase III. To place our findings in context, milk thistle-oindinavir trials were identified through systematic searches of the literature. A meta-analysis of three milk thistle-indinavir trials revealed a non-significant pooled mean difference of 1% in AUC(0-8) (95% CI, -53% to 55%, P=0.97). Conclusions: Indinavir levels were not reduced significantly in the presence of milk thistle.

Uptake and metabolism of ginsenoside Rh2 and its aglycon protopanaxadiol by Caco-2 cells

The uptake and metabolism profiles of ginsenoside Rh2 and its aglycon protopanaxadiol (ppd) were studied in the human epithelial Caco-2 cell line. High-performance liquid chromatography-mass spectrometry was applied to determine Rh2 and its aglycon ppd concentration in the cells at different pH, temperature, concentration levels and in the presence or absence of inhibitors. Rh2 uptake was time and concentration dependent, and its uptake rates were reduced by metabolic inhibitors and influenced by low temperature, thus indicating that the absorption process was energy-dependent. Drug uptake was maximal when the extracellular pH was 7.0 for Rh2 and 8.0 for ppd. Rh2 kinetic analysis showed that a non-saturable component (K-d 0.17 nmol (.) h(-1) (.) mg(-1) protein) and an active transport system with a K-m of 3.95 mumol (.) l(-1) and a V-max of 4.78 nmol(.)h(-1) (.)mg(-1) protein were responsible for the drug uptake. Kinetic analysis of ppd showed a non-saturable component (K-d 0.78 nmol (.) h(-1) (.) mg(-1) protein). It was suggested that active extrusion of P-glycoprotein and drug degradation in the intestine may influence Rh2 bioavailability.