Novel Long-Acting Drug Combination Nanoparticles Composed of Gemcitabine and Paclitaxel Enhance Localization of Both Drugs in Metastatic Breast Cancer Nodules.

PURPOSE: To develop drug-combination nanoparticles (DcNPs) composed of hydrophilic gemcitabine (G) and hydrophobic paclitaxel (T) and deliver both drugs to metastatic cancer cells. METHODS: GT DcNPs were evaluated based on particle size and drug association efficiency (AE%). The effect of DcNP on GT plasma time-course and tissue distribution was characterized in mice and a pharmacokinetic model was developed. A GT distribution study into cancer nodules (derived from 4 T1 cells) was performed. RESULTS: An optimized GT DcNP composition (d = 59.2 nm ±9.2 nm) was found to be suitable for IV formulation. Plasma exposure of G and T were enhanced 61-fold and 3.8-fold when given in DcNP form compared to the conventional formulation, respectively. Mechanism based pharmacokinetic modeling and simulation show that both G and T remain highly associated to DcNPs in vivo (G: 98%, T:75%). GT DcNPs have minimal distribution to healthy organs with selective distribution and retention in tumor burdened tissue. Tumor bearing lungs had a 5-fold higher tissue-to-plasma ratio of gemcitabine in GT DcNPs compared to healthy lungs. CONCLUSIONS: DcNPs can deliver hydrophilic G and hydrophobic T together to cancer nodules and produce long acting exposure, likely due to stable GT association to DcNPs in vivo.

Novel drug combination nanoparticles exhibit enhanced plasma exposure and dose-responsive effects on eliminating breast cancer lung metastasis.

Early diagnosis along with new drugs targeted to cancer receptors and immunocheckpoints have improved breast cancer survival. However, full remission remains elusive for metastatic breast cancer due to dose-limiting toxicities of heavily used, highly potent drug combinations such as gemcitabine and paclitaxel. Therefore, novel strategies that lower the effective dose and improve safety margins could enhance the effect of these drug combinations. To this end, we developed and evaluated a novel drug combination of gemcitabine and paclitaxel (GT). Leveraging a simple and scalable drug-combination nanoparticle platform (DcNP), we successfully prepared an injectable GT combination in DcNP (GT DcNP). Compared to a Cremophor EL/ethanol assisted drug suspension in buffer (CrEL), GT DcNP exhibits about 56-fold and 8.6-fold increases in plasma drug exposure (area under the curve, AUC) and apparent half-life of gemcitabine respectively, and a 2.9-fold increase of AUC for paclitaxel. Using 4T1 as a syngeneic model for breast cancer metastasis, we found that a single GT (20/2 mg/kg) dose in DcNP nearly eliminated colonization in the lungs. This effect was not achievable by a CrEL drug combination at a 5-fold higher dose (i.e., 100/10 mg/kg GT). A dose-response study indicates that GT DcNP provided a therapeutic index of ~15.8. Collectively, these data suggest that GT DcNP could be effective against advancing metastatic breast cancer with a margin of safety. As the DcNP formulation is intentionally designed to be simple, scalable, and long-acting, it may be suitable for clinical development to find effective treatment against metastatic breast cancer.

Integration of Computational and Experimental Approaches to Elucidate Mechanisms of First-Pass Lymphatic Drug Sequestration and Long-Acting Pharmacokinetics of the Injectable Triple-HIV Drug Combination TLC-ART 101.

TLC-ART101 is a long-acting triple-HIV drug combination of lopinavir-ritonavir-tenofovir in one nanosuspension intended for subcutaneous injection. After a single TLC-ART 101 administration in nonhuman primates, drug concentrations in both plasma and HIV-target lymph node mononuclear cells were sustained for 2 weeks. Nevertheless, the mechanisms leading to the targeted long-acting pharmacokinetics remain elusive. Therefore, an intravenous study of TLC-ART 101 in nonhuman primates was conducted to elucidate the degree of association of drugs in vivo, estimate subcutaneous bioavailability, and refine a mechanism-based pharmacokinetic (MBPK2) model. The MBPK2 model considers TLC-ART 101 systemic drug clearances, nanoparticle-associated/dissociated species, more detailed mechanisms of lymphatic first-pass retention of associated-drugs after subcutaneous administrations, and the prediction of drug concentration time-courses in lymph node mononuclear cells. For all 3 drugs, we found a high association with the nanoparticles in plasma (>87% lopinavir-ritonavir, 97% tenofovir), and an incomplete subcutaneous bioavailability (<29% lopinavir-ritonavir, 85% tenofovir). As hypothesized by the MBPK2 model, the incomplete SC bioavailability observed is due to sequestration into a lymphatic node depot after subcutaneous absorption (unlike most intramuscular nanodrug products having near-to-injection depots), which contributes to long-acting profiles detected in plasma and target cells. This combined experimental and modeling approach may be applicable for the clinical development of other long-acting drug-combination injectables.

Three HIV Drugs, Atazanavir, Ritonavir, and Tenofovir, Coformulated in Drug-Combination Nanoparticles Exhibit Long-Acting and Lymphocyte-Targeting Properties in Nonhuman Primates.

Drug-combination nanoparticles (DcNPs) administered subcutaneously represent a potential long-acting lymphatic-targeting treatment for HIV infection. The DcNP containing lopinavir (LPV)-ritonavir (RTV)-tenofovir (TFV), Targeted-Long-Acting-Antiretroviral-Therapy product candidate 101 (TLC-ART 101), has shown to provide long-acting lymphocyte-targeting performance in nonhuman primates. To extend the TLC-ART platform, we replaced TLC-ART 101 LPV with second-generation protease inhibitor, atazanavir (ATV). Pharmacokinetics of the ATV-RTV-TFV DcNP was assessed in macaques, in comparison to the equivalent free drug formulation and to the TLC-ART 101. After single subcutaneous administration of the DcNP formulation, ATV, RTV, and TFV concentrations were sustained in plasma for up to 14 days, and in peripheral blood mononuclear cells for 8 to 14 days, compared with 1 to 2 days in those macaques treated with free drug combination. By 1 week, lymph node mononuclear cells showed significant levels for all 3 drugs from DcNPs, whereas the free controls were undetectable. Compared with TLC-ART 101, the ATV-RTV-TFV DcNP exhibited similar lymphocyte-targeted long-acting features for all 3 drugs and similar pharmacokinetics for RTV and TFV, whereas some pharmacokinetic differences were observed for ATV versus LPV. The present study demonstrated the flexibility of the TLC-ART's DcNP platform to include different antiretroviral combinations that produce targeted long-acting effects on both plasma and cells.

Extended cell and plasma drug levels after one dose of a three-in-one nanosuspension containing lopinavir, efavirenz, and tenofovir in nonhuman primates.

OBJECTIVE: To characterize a drug-combination nanoparticle (DcNP) containing water-insoluble lopinavir (LPV) and efavirenz (EFV), and water-soluble tenofovir (TFV), for its potential as a long-acting combination HIV treatment. DESIGN: Three HIV drugs (LPV, EFV, TFV) with well established efficacy and safety were coformulated into a single DcNP suspension. Two macaques were administered one subcutaneous injection and drug concentrations in plasma and mononuclear cells (in peripheral blood and lymph nodes) were analyzed over 2 weeks. Pharmacokinetic parameters and cell-to-plasma relationships of LPV, EFV, and TFV were determined. RESULTS: This three-in-one nanoformulation provided extended concentrations of all drugs in lymph node cells that were 57- to 228-fold higher than those in plasma. Levels of all three drugs in peripheral blood mononuclear cells persisted for 2 weeks at levels equal to or higher than those in plasma. CONCLUSION: With long-acting characteristics and higher drug penetration/persistence in cells, this three-in-one DcNP may enhance therapeutic efficacy of these well studied HIV drugs due to colocalization and targeting of this three-drug combination to HIV host cells.

Long-Acting Profile of 4 Drugs in 1 Anti-HIV Nanosuspension in Nonhuman Primates for 5 Weeks After a Single Subcutaneous Injection.

Daily oral antiretroviral therapy regimens produce limited drug exposure in tissues where residual HIV persists and suffer from poor patient adherence and disparate drug kinetics, which all negatively impact outcomes. To address this, we developed a tissue- and cell-targeted long-acting 4-in-1 nanosuspension composed of lopinavir (LPV), ritonavir, tenofovir (TFV), and lamivudine (3TC). In 4 macaques dosed subcutaneously, drug levels over 5 weeks in plasma, lymph node mononuclear cells (LNMCs), and peripheral blood mononuclear cells (PBMCs) were analyzed by liquid chromatography-tandem mass spectrometry. Plasma and PBMC levels of the active drugs (LPV, TFV, and 3TC) were sustained for 5 weeks; PBMC exposures to LPV, ritonavir, and 3TC were 12-, 16-, 42-fold higher than those in plasma. Apparent T1/2z of LPV, TFV, and 3TC were 219.1, 63.1, and 136.3 h in plasma; 1045.7, 105.9, and 127.7 h in PBMCs. At day 8, LPV, TFV, and 3TC levels in LNMCs were 4.1-, 5.0-, and 1.9-fold higher than in those in PBMCs and much higher than in plasma. Therefore, 1 dose of a 4-drug nanosuspension exhibited persistent drug levels in LNMCs, PBMCs, and plasma for 5 weeks. With interspecies scaling and dose adjustment, this 4-in-1 HIV drug-combination could be a long-acting treatment with the potential to target residual virus in tissues and improve patient adherence.

Mechanism-based pharmacokinetic (MBPK) models describe the complex plasma kinetics of three antiretrovirals delivered by a long-acting anti-HIV drug combination nanoparticle formulation.

Existing oral antiretroviral (ARV) agents have been shown in human studies to exhibit limited lymph node penetration and lymphatic drug insufficiency. As lymph nodes are a reservoir of HIV, it is critical to deliver and sustain effective levels of ARV combinations in these tissues. To overcome lymph node drug insufficiency of oral combination ARV therapy (cART), we developed and reported a long-acting and lymphocyte-targeting injectable that contains three ARVs-hydrophobic lopinavir (LPV) and ritonavir (RTV), and hydrophilic tenofovir (TFV)-stabilized by lipid excipients in a nanosuspension. A single subcutaneous (SC) injection of this first-generation formulation of drug combination nanoparticles (DcNPs), named TLC-ART101, provided persistent ARV levels in macaque lymph node mononuclear cells (LNMCs) for at least 1 week, and in peripheral blood mononuclear cells (PBMCs) and plasma for at least 2 weeks, demonstrating long-acting pharmacokinetics for all three drugs. In addition, the lymphocyte-targeting properties of this formulation were demonstrated by the consistently higher intracellular drug concentrations in LNMCs and PBMCs versus those in plasma. To provide insights into the complex mechanisms of absorption and disposition of TLC-ART101, we constructed novel mechanism-based pharmacokinetic (MBPK) models. Based upon plasma PK data obtained after single administration of TLC-ART101 (DcNPs) and a solution formulation of free triple-ARVs, the models feature uptake from the SC injection site that respectively routes free and nanoparticle-associated ARVs via the blood vasculature and lymphatics, and their eventual distribution into and clearance from the systemic circulation. The models provided simultaneous description of the complex long-acting plasma and lymphatic PK profiles for all three ARVs in TLC-ART101. The long-acting PK characteristics of the three drugs in TLC-ART101 were likely due to a combination of mechanisms including: (1) DcNPs undergoing preferential lymphatic uptake from the subcutaneous space, (2) retention in nodes during lymphatic first-pass, (3) subsequent slow release of ARVs into blood circulation, and (4) limited extravasation of DcNP-associated ARVs that resulted in longer persistence in the circulation.

Translation of combination nanodrugs into nanomedicines: lessons learned and future outlook.

The concept of nanomedicine is not new. For instance, some nanocrystals and colloidal drug molecules are marketed that improve pharmacokinetic characteristics of single-agent therapeutics. For the past two decades, the number of research publications on single-agent nanoformulations has grown exponentially. However, formulations advancing to pre-clinical and clinical evaluations that lead to therapeutic products has been limited. Chronic diseases such as cancer and HIV/AIDS require drug combinations, not single agents, for durable therapeutic responses. Therefore, development and clinical translation of drug combination nanoformulations could play a significant role in improving human health. Successful translation of promising concepts into pre-clinical and clinical studies requires early considerations of the physical compatibility, pharmacological synergy, as well as pharmaceutical characteristics (e.g. stability, scalability and pharmacokinetics). With this approach and robust manufacturing processes in place, some drug-combination nanoparticles have progressed to non-human primate and human studies. In this article, we discuss the rationale and role of drug-combination nanoparticles, the pre-clinical and clinical research progress made to date and the key challenges for successful clinical translation. Finally, we offer insight to accelerate clinical translation through leveraging robust nanoplatform technologies to enable implementation of personalised and precision medicine.

Long-acting combination anti-HIV drug suspension enhances and sustains higher drug levels in lymph node cells than in blood cells and plasma.

OBJECTIVE: The aim of the present study was to determine whether a combination of anti-HIV drugs - tenofovir (TFV), lopinavir (LPV) and ritonavir (RTV) - in a lipid-stabilized nanosuspension (called TLC-ART101) could enhance and sustain intracellular drug levels and exposures in lymph node and blood cells above those in plasma. DESIGN: Four macaques were given a single dose of TLC-ART101 subcutaneously. Drug concentrations in plasma and mononuclear cells of the blood (PBMCs) and lymph nodes (LNMCs) were analysed using a validated combination LC-MS/MS assay. RESULTS: For the two active drugs (TFV, LPV), plasma and PBMC intracellular drug levels persisted for over 2 weeks; PBMC drug exposures were three- to four-fold higher than those in plasma. Apparent terminal half-lives (t1/2) of TFV and LPV were 65.3 and 476.9 h in plasma, and 169.1 and 151.2 h in PBMCs. At 24 and 192 h, TFV and LPV drug levels in LNMCs were up to 79-fold higher than those in PBMCs. Analysis of PBMC intracellular TFV and its active metabolite TFV-diphosphate (TFV-DP) indicated that intracellular exposures of total TFV and TFV-DP were markedly higher and persisted longer than in humans and macaques dosed with oral TFV prodrugs, tenofovir disoproxil fumarate (TDF) or tenofovir alafenamide (TAF). CONCLUSIONS: A simple, scalable three-drug combination, lipid-stabilized nanosuspension exhibited persistent drug levels in cells of lymph nodes and the blood (HIV host cells) and in plasma. With appropriate dose adjustment, TLC-ART101 may be a useful HIV treatment with a potential to impact residual virus in lymph nodes.

Susceptibility to quantum dot induced lung inflammation differs widely among the Collaborative Cross founder mouse strains.

Quantum dots (QDs) are engineered semiconductor nanoparticles with unique physicochemical properties that make them potentially useful in clinical, research and industrial settings. However, a growing body of evidence indicates that like other engineered nanomaterials, QDs have the potential to be respiratory hazards, especially in the context of the manufacture of QDs and products containing them, as well as exposures to consumers using these products. The overall goal of this study was to investigate the role of mouse strain in determining susceptibility to QD-induced pulmonary inflammation and toxicity. Male mice from 8 genetically diverse inbred strains (the Collaborative Cross founder strains) were exposed to CdSe-ZnS core-shell QDs stabilized with an amphiphilic polymer. QD treatment resulted in significant increases in the percentage of neutrophils and levels of cytokines present in bronchoalveolar lavage fluid (BALF) obtained from NOD/ShiLtJ and NZO/HlLtJ mice relative to their saline (Sal) treated controls. Cadmium measurements in lung tissue indicated strain-dependent differences in disposition of QDs in the lung. Total glutathione levels in lung tissue were significantly correlated with percent neutrophils in BALF as well as with lung tissue Cd levels. Our findings indicate that QD-induced acute lung inflammation is mouse strain dependent, that it is heritable, and that the choice of mouse strain is an important consideration in planning QD toxicity studies. These data also suggest that formal genetic analyses using additional strains or recombinant inbred strains from these mice could be useful for discovering potential QD-induced inflammation susceptibility loci.

The glutathione synthesis gene Gclm modulates amphiphilic polymer-coated CdSe/ZnS quantum dot-induced lung inflammation in mice.

Quantum dots (QDs) are unique semi-conductor fluorescent nanoparticles with potential uses in a variety of biomedical applications. However, concerns exist regarding their potential toxicity, specifically their capacity to induce oxidative stress and inflammation. In this study we synthesized CdSe/ZnS core/shell QDs with a tri-n-octylphosphine oxide, poly(maleic anhydride-alt-1-tetradecene) (TOPO-PMAT) coating and assessed their effects on lung inflammation in mice. Previously published in vitro data demonstrated these TOPO-PMAT QDs cause oxidative stress resulting in increased expression of antioxidant proteins, including heme oxygenase, and the glutathione (GSH) synthesis enzyme glutamate cysteine ligase (GCL). We therefore investigated the effects of these QDs in vivo in mice deficient in GSH synthesis (Gclm +/- and Gclm -/- mice). When mice were exposed via nasal instillation to a TOPO-PMAT QD dose of 6 µg cadmium (Cd) equivalents/kg body weight, neutrophil counts in bronchoalveolar lavage fluid (BALF) increased in both Gclm wild-type (+/+) and Gclm heterozygous (+/-) mice, whereas Gclm null (-/-) mice exhibited no such increase. Levels of the pro-inflammatory cytokines KC and TNFα increased in BALF from Gclm +/+ and +/- mice, but not from Gclm -/- mice. Analysis of lung Cd levels suggested that QDs were cleared more readily from the lungs of Gclm -/- mice. There was no change in matrix metalloproteinase (MMP) activity in any of the mice. However, there was a decrease in whole lung myeloperoxidase (MPO) content in Gclm -/- mice, regardless of treatment, relative to untreated Gclm +/+ mice. We conclude that in mice TOPO-PMAT QDs have in vivo pro-inflammatory properties, and the inflammatory response is dependent on GSH synthesis status. Because there is a common polymorphism in humans that influences GCLM expression, these findings imply that humans with reduced GSH synthesis capabilities may be more susceptible to the pro-inflammatory effects of QDs.

Heme oxygenase expression as a biomarker of exposure to amphiphilic polymer-coated CdSe/ZnS quantum dots.

Because of their unique optical properties, quantum dots (QDs) have become a preferred system for ultrasensitive detection and imaging. However, since QDs commonly contain Cd and other heavy metals, concerns have been raised regarding their toxicity. QDs are thus commonly synthesised with a ZnS cap structure and/or coated with polymeric stabilisers. We recently synthesised amphiphilic polymer-coated tri-n-octylphosphine oxide - poly(maleic anhydride-alt-1-tetradecene (TOPO-PMAT) QDs, which are highly stable in aqueous environments. The effects of these QDs on viability and stress response in five cell lines of mouse and human origins are reported here. Human and mouse macrophages and human kidney cells readily internalised these QDs, resulting in modest toxicity. TOPO-PMAT QD exposure was highly correlated with the induction of the stress response protein heme oxygenase-1 (HMOX1). Other stress biomarkers (glutamate cysteine ligase modifier subunit, NAD(P)H, necrosis) were only moderately affected. HMOX1 may thus be a useful biomarker of TOPO-QDOT QD exposure across cell types and species.

Increased sensitivity to testicular toxicity of transplacental benzo[a]pyrene exposure in male glutamate cysteine ligase modifier subunit knockout (Gclm-/-) mice.

Polycyclic aromatic hydrocarbons (PAHs), like benzo[a]pyrene (BaP), are ubiquitous environmental pollutants formed by the incomplete combustion of organic materials. The tripeptide glutathione (GSH) is a major antioxidant and is important in detoxification of PAH metabolites. Mice null for the modifier subunit of glutamate cysteine ligase (Gclm), the rate-limiting enzyme in GSH synthesis, have decreased GSH concentrations. We investigated the effects of Gclm deletion alone on male fertility and spermatogenesis and its effect on the sensitivity of male embryos to the transplacental testicular toxicity of BaP. Gclm-/- males had dramatically decreased testicular and epididymal GCL enzymatic activity and total GSH concentrations compared with Gclm+/+ littermates. Ratios of reduced to oxidized GSH were significantly increased in Gclm-/- testes. GSH reductase enzymatic activity was increased in Gclm-/- epididymides. We observed no changes in fertility, testicular weights, testicular sperm head counts, or testicular histology and subtle changes in cauda epididymal sperm counts, motility, and morphology in Gclm-/- compared with Gclm+/+ males. Prenatal exposure to BaP from gestational day 7 to 16 was dose dependently associated with significantly decreased testicular and epididymal weights, testicular and epididymal sperm counts, and with vacuolated seminiferous tubules at 10 weeks of age. Gclm-/- males exposed prenatally to BaP had greater decreases in testicular weights, testicular sperm head counts, epididymal sperm counts, and epididymal sperm motility than Gclm+/+ littermates. These results show no effects of Gclm deletion alone on male fertility and testicular spermatogenesis and subtle epididymal effects but support increased sensitivity of Gclm-/- males to the transplacental testicular toxicity of BaP.

Lack of maternal glutamate cysteine ligase modifier subunit (Gclm) decreases oocyte glutathione concentrations and disrupts preimplantation development in mice.

Glutathione (GSH) is the most abundant intracellular thiol and an important regulator of cellular redox status. Mice that lack the modifier subunit of glutamate cysteine ligase (Gclm), the rate-limiting enzyme in GSH synthesis, have decreased GSH synthesis. Nicotinamide nucleotide transhydrogenase, an inner mitochondrial membrane protein, catalyzes the interconversion of reduced nicotinamide adenine dinucleotide and reduced nicotinamide adenine dinucleotide phosphate; reduced nicotinamide adenine dinucleotide phosphate is required for reduction of GSH disulfide. Previous work supports roles for GSH in preimplantation development. We hypothesized that Gclm-/- mice have increased preimplantation embryonic mortality and that this effect is enhanced by absence of a functioning Nnt gene. Gclm-/- females produced significantly fewer pups per litter than Gclm+/+ littermates. Numbers of oocytes ovulated in a natural estrous cycle or upon superovulation did not differ by genotype. Fewer uterine implantation sites were observed in the Gclm-/- females. Prepubertal Gclm-/- and Gclm+/+ females were superovulated, then mated overnight with a Gclm+/+ male. At 0.5 d postcoitum, Gclm-/- females had significantly lower percentages of zygotes with two pronuclei and higher percentages of zygotes with one pronucleus than Gclm+/+ or Gclm+/- females. At 3.5 d postcoitum, a significantly lower percentage of blastocyst stage embryos was recovered from uteri of Gclm-/- females than Gclm+/+ females. Embryonic development to the blastocyst stage, but not the two-cell stage, was significantly decreased after in vitro fertilization of oocytes from Gclm-/- females compared with Gclm+/+ females. The Nnt mutation did not enhance the effects of Gclm genotype on female fertility. These results demonstrate critical roles for maternal GSH in supporting normal preimplantation development.

Glutamate cysteine ligase (GCL) transgenic and gene-targeted mice for controlling glutathione synthesis.

The tripeptide glutathione (GSH) has important antioxidant properties, scavenges free radicals, and serves as a cofactor for glutathione S-transferase conjugation of many xenobiotics. GSH is synthesized in two steps. The first and, often, rate-limiting step is the formation of γ-glutamylcysteine, which is catalyzed by the inducible heterodimeric enzyme glutamate cysteine ligase (GCL). The two subunits of GCL are the catalytic subunit (GCLC) and the modifier subunit (GCLM). In this unit, the generation and basic characterization methodologies of transgenic mouse models that have been developed to (1) conditionally over express both GCL subunits; (2) lack GCLM (Gclm null); and (3) create a hybrid between Gclm conditional over-expressing mice on a Gclm null genetic background are discussed. These models can be used to explore the fundamental role of GCLC and GCLM in GSH synthesis, as well as the toxicological role of GSH and its synthesis in xenobiotic metabolism and response to oxidative stress.

Modulating GSH synthesis using glutamate cysteine ligase transgenic and gene-targeted mice.

Glutathione (GSH) is an important antioxidant and cofactor for glutathione S-transferase conjugation. GSH synthesis is catalyzed by glutamate cysteine ligase (GCL), composed of catalytic (GCLC) and modifier (GCLM) subunits. Transgenic mice that conditionally over express GCL subunits are protected from acetaminophen induced liver injury. Gclm null mice exhibit low GSH levels and enhanced sensitivity to acetaminophen. When Gclm expression and GCL activity are restored in Gclm conditional transgenic X Gclm null mice, they become resistant to APAP-induced liver damage. These animal models are a valuable resource for investigating the role of GSH synthesis in modulating oxidative damage and drug-induced hepatotoxicity.

Glutamate cysteine ligase modifier subunit deficiency and gender as determinants of acetaminophen-induced hepatotoxicity in mice.

The analgesic and antipyretic drug acetaminophen (APAP) is bioactivated to the reactive intermediate N-acetyl-p-benzoquinoneimine, which is scavenged by glutathione (GSH). APAP overdose can deplete GSH leading to the accumulation of APAP-protein adducts and centrilobular necrosis in the liver. N-acetylcysteine (NAC), a cysteine prodrug and GSH precursor, is often given as a treatment for APAP overdose. The rate-limiting step in GSH biosynthesis is catalyzed by glutamate cysteine ligase (GCL) a heterodimer composed of catalytic and modifier (GCLM) subunits. Previous studies have indicated that GCL activity is likely to be an important determinant of APAP toxicity. In this study, we investigated APAP toxicity, and NAC or GSH ethyl ester (GSHee)-mediated rescue in mice with normal or compromised GCLM expression. Gclm wild-type, heterozygous, and null mice were administered APAP (500 mg/kg) alone, or immediately following NAC (800 mg/kg) or GSHee (168 mg/kg), and assessed for hepatotoxicity 6 h later. APAP caused GSH depletion in all mice. Gclm null and heterozygous mice exhibited more extensive hepatic damage compared to wild-type mice as assessed by serum alanine aminotransferase activity and histopathology. Additionally, male Gclm wild-type mice demonstrated greater APAP-induced hepatotoxicity than female wild-type mice. Cotreatment with either NAC or GSHee mitigated the effects of APAP in Gclm wild-type and heterozygous mice, but not in Gclm null mice. Collectively, these data reassert the importance of GSH in protection against APAP-induced hepatotoxicity, and indicate critical roles for GCL activity and gender in APAP-induced liver damage in mice.

Multicenter study of acetaminophen hepatotoxicity reveals the importance of biological endpoints in genomic analyses.

Gene expression profiling is a widely used technique with data from the majority of published microarray studies being publicly available. These data are being used for meta-analyses and in silico discovery; however, the comparability of toxicogenomic data generated in multiple laboratories has not been critically evaluated. Using the power of prospective multilaboratory investigations, seven centers individually conducted a common toxicogenomics experiment designed to advance understanding of molecular pathways perturbed in liver by an acute toxic dose of N-acetyl-p-aminophenol (APAP) and to uncover reproducible genomic signatures of APAP-induced toxicity. The nonhepatotoxic APAP isomer N-acetyl-m-aminophenol was used to identify gene expression changes unique to APAP. Our data show that c-Myc is induced by APAP and that c-Myc-centered interactomes are the most significant networks of proteins associated with liver injury. Furthermore, sources of error and data variability among Centers and methods to accommodate this variability were identified by coupling gene expression with extensive toxicological evaluation of the toxic responses. We show that phenotypic anchoring of gene expression data is required for biologically meaningful analysis of toxicogenomic experiments.

An RT-PCR-based strategy to estimate full-length CYP2D6 mRNA copy number.

The goal of this study is to develop an analytical tool to assess cytochrome P450 2D6 (CYP2D6) levels in the form of full-length transcripts. CYP2D6 RNA in test samples was evaluated by co-amplification with an internal RNA control in a reverse-transcribed polymerase chain reaction (RT-PCR). The internal CYP2D6 RNA control was constructed by internally deleting 474 bp of CYP2D6 RNA, allowing simultaneous amplification of the test RNA together with the internal control RNA in a single RT-PCR reaction. With sequential dilution of test RNA, the CYP2D6 mRNA transcript levels in test samples were estimated. The full-length RT-PCR strategy allowed semiquantitative assessments of CYP2D6 RNA transcripts with a sensitivity limit of 500 copies for CYP2D6 RNA transcripts, 2500 copies/microg total human liver RNA, and 10% intraday coefficient of variation (CV). In a method validation study, the CYP2D6 activity appeared to relate more closely to full-length CYP2D6 mRNA concentration than a short-sequence of CYP2D6 RNA estimated with a real-time quantitative RT-PCR assay. We have developed an efficient semiquantitative assay and demonstrated its suitability for estimating full-length CYP2D6 mRNA transcripts in cells and tissues.

Only truncated, not complete cytochrome p450 2D6 RNA transcript and no detectable enzyme activity are expressed in human lymphocytes.

Genotyping of the highly polymorphic cytochrome p450 2D6 (CYP2D6) permits a gross classification of individual phenotype (viz. ultra-rapid, extensive, and poor metabolizers). It does not, however, provide a precise prediction of CYP2D6 activity, particularly in an individual possessing at least one functional CYP2D6 allele. It has been suggested that the level of mRNA expression or enzyme activity in lymphocytes, isolated from blood, could potentially provide a better quantitative estimate of in vivo hepatic enzymatic activity in human subjects. Although short sequences of CYP2D6 mRNA have been detected in human lymphocytes by reverse transcriptase-polymerase chain reaction (RT-PCR) that suggests the potential use of lymphocyte RNA as a readily accessible biomarker, it is not known whether a functional enzyme is expressed in human lymphocytes. In this study, human lymphocyte activity was assessed with a CYP2D6-specific, high-turnover probe substrate that is severalfold more sensitive than traditional markers of CYP2D6 (e.g., dextromethorphan). CYP2D6 catalytic activity could not be detected in homogenates of human lymphocytes, even at high protein concentrations and with supplementation of enzyme cofactors. Further RT-PCR analysis of lymphocytes collected from eight human donors revealed the presence of only a fragment, but not the complete transcript, of CYP2D6 mRNA. Northern blot RNA transcript analysis also failed to indicate the presence of the full-length transcript in lymphocytes. Collectively, these data indicate that human lymphocytes express neither the full-length CYP2D6 mRNA transcript nor functional enzyme activity. Therefore, the utility of lymphocytes as a functional biomarker for CYP2D6 enzyme activity is not clear at present.