Application of Cryopreserved Human Intestinal Mucosa and Cryopreserved Human Enterocytes in the Evaluation of Herb-Drug Interactions: Evaluation of CYP3A Inhibitory Potential of Grapefruit Juice and Commercial Formulations of Twenty-Nine Herbal Supplements.

Commercial formulations of 29 commonly used herbal supplements (HSs) and grapefruit juice were evaluated for drug interaction potential via quantification of their CYP3A inhibitory potential in two in vitro experimental models of human small intestine, cryopreserved human intestinal mucosa (CHIM), and cryopreserved human enterocytes (CHEs). Two CYP3A substrates were used-in the studies with CHIM, CYP3A activity was quantified via liquid chromatography tandem mass spectrometry quantification of midazolam 1'-hydroxylation, whereas in CHE, luciferin-IPA metabolism to luciferin was quantified by luminescence. Upon treatment of CHIM with the estimated lumen concentration of the HS upon each oral administration (manufacturers' recommended dosage dissolved in 200 ml of culture medium), >80% CYP3A inhibition was observed for green tea extract, St. John's wort, valerian root, horehound, and grapefruit juice. Less than 50% inhibition was observed for fenugreek, aloe vera, guarana, soy isoflavone, maca, echinacea, spirulina, evening primrose, milk thistle, cranberry, red yeast rice, rhodiola, ginkgo biloba, turmeric, curcumin, white kidney bean, garlic, cinnamon, saw palmetto berries, panax ginseng, black elderberry, wheat grass juice, flaxseed oil, black cohosh, and ginger root. The results were confirmed in a a dose-response study with HSs obtained from three suppliers for the four inhibitory HSs (green tea extract, horehound, St. John's wort, valerian root) and three representative noninhibitory HSs (black cohosh, black elderberry, echinacea). Similar results were obtained with the inhibitory HSs in CHE. The results illustrate that CHIM and CHE represent physiologically relevant in vitro experimental models for the evaluation of drug interaction potential of herbal supplements. Based on the results, green tea extract, horehound, St. John's wort, and valerian root may cause drug interactions with orally administered drugs that are CYP3A substrates, as was observed for grapefruit juice. SIGNIFICANCE STATEMENT: In vitro evaluation of 29 popular herbal supplements in cryopreserved human intestinal mucosa identified green tea extract, horehound, St. John's wort, and valerian root to have CYP3A inhibitory potential similar to that for grapefruit juice, suggesting their potential to have clinically significant pharmacokinetic interaction with orally administered drugs that are CYP3A substrates. The results suggest that cryopreserved human intestinal mucosa can be used for in vitro evaluation of drug interactions involving enteric drug metabolism.

Optimizing live-animal bioluminescence imaging prediction of tumor burden in human prostate cancer xenograft models in SCID-NSG mice.

BACKGROUND: Noninvasive live-animal longitudinal monitoring of xenograft tumor growth and metastasis by bioluminescent imaging (BLI) has been widely reported in cancer biology and preclinical therapy literature, mainly in athymic nude mice. Our own experience at calibrating BLI readout with tumor weight/volume in human prostate cancer xenograft models in haired, SCID-NSG mice through intraprostatic (orthotopic) and subcutaneous (SC) inoculations revealed either nonexistent or poor correlation (coefficient of determination, R 2 = ~0.01-0.3). The present work examined several technical and biological factors to improve BLI utility. METHODS: After ruling out promoter-luciferase (luc) specificity and luc gene loss in the cell inoculum with LNCaP-AR-luc cells expressing an androgen receptor (AR) and tagged with AR-responsive probasin promoter-luc gene, we evaluated different routes of d-luciferin administration, imaging time during the day, charge-coupled device camera image acquisition settings, and hair removal methods to improve the imaging protocol. For most imaging sessions, BLI was carried out within the same day of tumor volume measurement. After necropsy, histological and immunohistochemical (IHC) analyses were performed on the tumors to evaluate necrosis and expression of luciferase and AR, respectively. RESULTS: Injection of d-luciferin by SC route, robust image-capture setting (30 000 counts and autoexposure), imaging in the morning and thorough hair removal resulted in a substantial improvement of R2 to ~0.6. Histological analyses confirmed the lack of BLI signal in necrotic tumor masses consistent with luciferase-mediated light emission only in oxygenated adenosine triphosphate-producing viable cells. IHC staining detected heterogeneous expression of luciferase tracking generally with AR expression in nonnecrotic tumor tissues. CONCLUSIONS: Our body of work highlighted a framework to validate imaging protocols to ensure the acquisition of interpretable BLI data as an indicator of xenograft tumor burden. The vast tissue heterogeneity in prostate tumor xenografts and variable luciferase expression constrained this technology from achieving a high correlation.

Use of Optical Imaging Technology in the Validation of a New, Rapid, Cost-Effective Drug Screen as Part of a Tiered In Vivo Screening Paradigm for Development of Drugs To Treat Cutaneous Leishmaniasis.

In any drug discovery and development effort, a reduction in the time of the lead optimization cycle is critical to decrease the time to license and reduce costs. In addition, ethical guidelines call for the more ethical use of animals to minimize the number of animals used and decrease their suffering. Therefore, any effort to develop drugs to treat cutaneous leishmaniasis requires multiple tiers of in vivo testing that start with higher-throughput efficacy assessments and progress to lower-throughput models with the most clinical relevance. Here, we describe the validation of a high-throughput, first-tier, noninvasive model of lesion suppression that uses an in vivo optical imaging technology for the initial screening of compounds. A strong correlation between luciferase activity and the parasite load at up to 18 days postinfection was found. This correlation allows the direct assessment of the effects of drug treatment on parasite burden. We demonstrate that there is a strong correlation between drug efficacy measured on day 18 postinfection and the suppression of lesion size by day 60 postinfection, which allows us to reach an accurate conclusion on drug efficacy in only 18 days. Compounds demonstrating a significant reduction in the bioluminescence signal compared to that in control animals can be tested in lower-throughput, more definitive tests of lesion cure in BALB/c mice and Golden Syrian hamsters (GSH) using Old World and New World parasites.

Optimization and modeling of the remote loading of luciferin into liposomes.

We carried out a mechanistic study to characterize and optimize the remote loading of luciferin into preformed liposomes of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine/1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPC/DPPG) 7:3 mixtures. The influence of the loading agent (acetate, propionate, butyrate), the metal counterion (Na(+), K(+), Ca(+2), Mg(+2)), and the initial extra-liposomal amount of luciferin (nL(add)) on the luciferin Loading Efficiency (LE%) and luciferin-to-lipid weight ratio, i.e., Loading Capacity (LC), in the final formulation was determined. In addition, the effect of the loading process on the colloidal stability and phase behavior of the liposomes was monitored. Based on our experimental results, a theoretical model was developed to describe the course of luciferin remote loading. It was found that the highest luciferin loading was obtained with magnesium acetate. The use of longer aliphatic carboxylates or inorganic proton donors pronouncedly reduced luciferin loading, whereas the effect of the counterion was modest. The remote-loading process barely affected the colloidal stability and drug retention of the liposomes, albeit with moderate luciferin-induced membrane perturbations. The correlation between luciferin loading, expressed as LE% and LC, and nL(add) was established, and under our conditions the maximum LC was attained using an nL(add) of around 2.6µmol. Higher amounts of luciferin tend to pronouncedly perturb the liposome stability and luciferin retention. Our theoretical model furnishes a fair quantitative description of the correlation between nL(add) and luciferin loading, and a membrane permeability coefficient for uncharged luciferin of 1×10(-8)cm/s could be determined. We believe that our study will prove very useful to optimize the remote-loading strategies of moderately polar carboxylic acid drugs in general.

Novel in vivo imaging analysis of an inner ear drug delivery system: Drug availability in inner ear following different dose of systemic drug injections.

Systemic application of drugs is commonly used in clinical situations. Some of these drugs are ototoxic. Since there are few studies on in vivo monitoring of drug delivery dynamics, the time course or bioavailability of drugs in the inner ear of live animals following systemic drug application remains unknown. For instance, it is unknown whether the volume of a drug delivered systemically correlates with its inner ear pharmacokinetics. We previously established a new in vivo imaging system to monitor drug delivery in live mice. In the present study, we used this system to compare drug concentration in the inner ear over time after systemic drug injections. We used transgenic GFAP-Luc mice that harbor a firefly luciferase gene expression cassette regulated by 12 kb of murine GFAP promoter and human beta-globin intron 2. Luciferin delivered into the inner ear of these mice reacts with luciferase, and the resulting signals are detected in GFAP-expressing cells in the cochlear nerve. Thus, we assessed in the inner ear the intensity and duration of luciferin/luciferase signals after systemic injections of different volumes of luciferin. An IVIS(®) imaging system was used to observe signals, and these signals were compared to the drug dynamics of luciferin delivered through subcutaneous (sc) injections. The volume of sc-injected drug correlated significantly with photon counts measured in the inner ear. Photons were detected almost immediately after injection, peaking 20 min after injection. Drug concentration did not affect inner ear signals. Luciferin injected systemically appeared in the inner ear between highest and lowest concentration. Drug volume is an important parameter to know if the inner ear requires a higher level of the drug. We observed that it is the volume of a drug-not its concentration-that is the important factor. Indeed, the more volume of a drug injected systemically increased the concentration of that drug in the inner ear. This study provides a better understanding of in vivo drug delivery dynamics measured in the inner ear. Further studies will show whether a high dosage of drug is effective or not.

Inertial cavitation to non-invasively trigger and monitor intratumoral release of drug from intravenously delivered liposomes.

The encapsulation of cytotoxic drugs within liposomes enhances pharmacokinetics and allows passive accumulation within tumors. However, liposomes designed to achieve good stability during the delivery phase often have compromised activity at the target site. This problem of inefficient and unpredictable drug release is compounded by the present lack of low-cost, non-invasive methods to measure such release. Here we show that focused ultrasound, used at pressures similar to those applied during diagnostic ultrasound scanning, can be utilised to both trigger and monitor release of payload from liposomes. Notably, drug release was influenced by liposome composition and the presence of SonoVue® microbubbles, which provided the nuclei for the initiation of an event known as inertial cavitation. In vitro studies demonstrated that liposomes formulated with a high proportion of 1,2 distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) released up to 30% of payload following ultrasound exposure in the presence of SonoVue®, provided that the exposure created sufficient inertial cavitation events, as characterised by violent bubble collapse and the generation of broadband acoustic emissions. In contrast a 'Doxil'-like liposome formulation gave no such triggered release. In pre-clinical studies, ultrasound was used as a non-invasive, targeted stimulus to trigger a 16-fold increase in the level of payload release within tumors following intravenous delivery. The inertial cavitation events driving this release could be measured remotely in real-time and were a reliable predictor of drug release.

Neural progenitor cell survival in mouse brain can be improved by co-transplantation of helper cells expressing bFGF under doxycycline control.

Cell-based therapy of neurological disorders is hampered by poor survival of grafted neural progenitor cells (NPCs). We hypothesized that it is possible to enhance the survival of human NPCs (ReNcells) by co-transplantation of helper cells expressing basic fibroblast growth factor (bFGF) under control of doxycycline (Dox). 293 cells or C17.2 cells were transduced with a lentiviral vector encoding the fluorescent reporter mCherry and bFGF under tetracycline-regulated transgene expression (Tet-ON). The bFGF secretion level in the engineered helper cells was positively correlated with the dose of Dox (Pearson correlation test; r=0.95 and 0.99 for 293 and C17.2 cells, respectively). Using bioluminescence imaging (BLI) as readout for firefly luciferase-transduced NPC survival, the addition of both 293-bFGF and C17.2-bFGF helper cells was found to significantly improve cell survival up to 6-fold in vitro, while wild-type (WT, non-transduced) helper cells had no effect. Following co-transplantation of 293-bFGF or C17.2-bFGF cells in the striatum of Rag2(-/-) immunodeficient mice, in vivo human NPC survival could be significantly improved as compared to no helper cells or co-transplantation of WT cells for the first two days after co-transplantation. This enhancement of survival in C17.2-bFGF group was not achieved without Dox administration, indicating that the neuroprotective effect was specific for bFGF. The present results warrant further studies on the use of engineered helper cells, including those expressing other growth factors injected as mixed cell populations.

Measurements of vaccinia virus dissemination using whole body imaging: approaches for predicting of lethality in challenge models and testing of vaccines and antiviral treatments.

Preclinical evaluation of novel anti-smallpox vaccines and antiviral treatments often rely on mouse -challenge models using pathogenic vaccinia virus, such as Western Reserve (WR) strain or other orthopoxviruses. Traditionally, efficacy of treatment is evaluated using various readouts, such as lethality (rare), measurements of body weight loss, pox lesion scoring, and determination of viral loads in internal organs by enumerating plaques in sensitive cell lines. These methodologies provide valuable information about the contribution of the treatment to protection from infection, yet all have similar limitations: they do not evaluate dissemination of the virus within the same animal and require large numbers of animals. These two problems prompted us to turn to a recently developed whole body imaging technology, where replication of recombinant vaccinia virus expressing luciferase enzyme (WRvFire) is sensed by detecting light emitted by the enzyme in the presence of D: -luciferin substrate administered to infected animal. Bioluminescence signals from infected organs in live animals are registered by the charge-coupled device camera in IVIS instrument developed by Caliper, and are converted into numerical values. This chapter describes whole body bioimaging methodology used to determine viral loads in normal live BALB/c mice infected with recombinant WRvFire vaccinia virus. Using Dryvax vaccination as a model, we show how bioluminescence data can be used to determine efficacy of treatment. In addition, we illustrate how bioluminescence and survival outcome can be combined in Receiver Operating Characteristic curve -analysis to develop predictive models of lethality that can be applied for testing of new therapeutics and second-generation vaccines.

The efficacy of aerosol treatment with non-ionic surfactant vesicles containing amphotericin B in rodent models of leishmaniasis and pulmonary aspergillosis infection.

Amphotericin B (AMB) is used to treat both fungal and leishmanial infections, which are of major significance to human health. Clinical use of free AMB is limited by its nephrotoxicity, whereas liposomal AMB is costly and requires parenteral administration, thus development of novel formulations with enhanced efficacy, minimal toxicity and that can be applied via non-invasive routes is required. In this study we analysed the potential of non-ionic surfactant vesicles (NIV) given by nebulisation to deliver AMB to the lungs, liver and skin. Treatment with AMB-NIV resulted in significantly higher drug levels in the lungs and skin (p<0.05) compared to similar treatment with AMB solution but significantly lower plasma levels (p<0.05). Treatment with AMB-NIV resulted in a significant reduction in fungal lung burdens in a rat model of invasive pulmonary aspergillosis (p<0.05) compared to treatment with the carrier alone. Treatment with AMB-NIV but not AMB solution significantly suppressed Leishmania donovani liver parasite burdens (p<0.05) but could not inhibit the growth of cutaneous Leishmania major lesions. The results of this study indicate that aerosolised NIV enhanced pulmonary and hepatic delivery whilst minimising systemic exposure and toxicity.

Quantitation of cellular and topical uptake of luciferin-oligoarginine conjugates.

A major challenge confronting the further advancement of using molecular transporters conjugated to small molecular weight therapeutics in the clinic is the development of linkers that would allow for the controllable release of a free drug/probe only after cell entry. Development of assays that would allow for the rapid real-time quantification of transporter conjugate uptake and cargo release in cells and animals would greatly help in their development. In this chapter, we describe a imaging method that quantitatively measures transporter conjugate uptake and cargo release in real-time in both cell culture and animal models.

Bioluminescence: imaging modality for in vitro and in vivo gene expression.

Molecular imaging offers many unique opportunities to study biological processes in intact organisms. Bioluminescence is the emission of light from biochemical reactions that occur within a living organism. Luciferase has been used as a reporter gene in transgenic mice but, until bioluminescence imaging was described, the detection of luciferase activity required either sectioning of the animal or excision of tissue and homogenization to measure enzyme activities in a conventional luminometer. Bioluminescence imaging (BLI) is based on the idea that biological light sources can be incorporated into cells and animal models artificially that does not naturally express the luminescent genes. This imaging modality has proven to be a very powerful methodology to detect luciferase reporter activity in intact animal models. This form of optical imaging is low cost and noninvasive and facilitates real-time analysis of disease processes at the molecular level in living organisms. Bioluminescence provides a noninvasive method to monitor gene expression in vivo and has enormous potential to elucidate the pathobiology of lung diseases in intact mouse models, including models of inflammation/injury, infection, and cancer.

Dynamic bioluminescence imaging for quantitative tumour burden assessment using IV or IP administration of D: -luciferin: effect on intensity, time kinetics and repeatability of photon emission.

INTRODUCTION: In vivo bioluminescence imaging (BLI) is a promising technique for non-invasive tumour imaging. D: -luciferin can be administrated intraperitonealy or intravenously. This will influence its availability and, therefore, the bioluminescent signal. The aim of this study is to compare the repeatability of BLI measurement after IV versus IP administration of D: -luciferin and assess the correlation between photon emission and histological cell count both in vitro and in vivo. MATERIALS AND METHODS: Fluc-positive R1M cells were subcutaneously inoculated in nu/nu mice. Dynamic BLI was performed after IV or IP administration of D: -luciferin. Maximal photon emission (PE(max)) was calculated. For repeatability assessment, every acquisition was repeated after 4 h and analysed using Bland-Altman method. A second group of animals was serially imaged, alternating IV and IP administration up to 21 days. When mice were killed, PE(max) after IV administration was correlated with histological cell number. RESULTS: The coefficients of repeatability were 80.2% (IV) versus 95.0% (IP). Time-to-peak is shorter, and its variance lower for IV (p < 0.0001). PE(max) was 5.6 times higher for IV. A trend was observed towards lower photon emission per cell in larger tumours. CONCLUSION: IV administration offers better repeatability and better sensitivity when compared to IP. In larger tumours, multiple factors may contribute to underestimation of tumour burden. It might, therefore, be beneficial to test novel therapeutics on small tumours to enable an accurate evaluation of tumour burden.

A bioluminescent HL-60 cell line to assay anti-leukaemia therapeutics under physiological conditions.

Screens for compounds and proteins with anti-cancer activity employ viability assays using relevant cancer cell lines. For leukaemia studies, the human leukaemia cell line, HL-60, is often used as a model system. To facilitate the discovery and investigation of anti-leukaemia therapeutics under physiological conditions, we have engineered HL-60 cells that stably express firefly luciferase and produce light that can be detected using an in vivo imaging system (IVIS). Bioluminescent HL-60luc cells could be rapidly detected in whole blood with a sensitivity of approximately 1000 viable cells/200 microl blood. Treatment of HL-60luc cells with the drug chlorambucil revealed that the bioluminescent viability assay is able to detect cell death earlier than the Trypan blue dye exclusion assay. HL-60luc cells administered intraperitoneally (i.p.) or intravenously (i.v.) were visualized in living mice. The rapidity and ease of detecting HL-60luc cells in biological fluid indicates that this cell line could be used in high-throughput screens for the identification of drugs with anti-leukaemia activity under physiological conditions.

Continuous delivery of D-luciferin by implanted micro-osmotic pumps enables true real-time bioluminescence imaging of luciferase activity in vivo.

Bioluminescence imaging (BLI) of luciferase reporters in small animal models offers an attractive approach to monitor regulation of gene expression, signal transduction, and protein-protein interactions, as well as following tumor progression, cell engraftment, infectious pathogens, and target-specific drug action. Conventional BLI can be repeated within the same animal after bolus reinjections of a bioluminescent substrate. However, intervals between image acquisitions are governed by substrate pharmacokinetics and excretion, therefore restricting temporal resolution of reinjection protocols to the order of hours, limiting analyses of processes in vivo with short time constants. To eliminate these constraints, we examined use of implanted micro-osmotic pumps for continuous, long-term delivery of bioluminescent substrates. Pump-assisted d-luciferin delivery enabled BLI for > or = 7 days from a variety of luciferase reporters. Pumps allowed direct repetitive imaging at < 5-minute intervals of the pharmacodynamics of proteasome- and IKK-inhibiting drugs in mice bearing tumors stably expressing ubiquitin-firefly luciferase or IkappaBalpha-firefly luciferase fusion reporters. Circadian oscillations in the olfactory bulbs of transgenic rats expressing firefly luciferase under the control of the period1 promoter also were temporally resolved over the course of several days. We conclude that implanted pumps provide reliable, prolonged substrate delivery for high temporal resolution BLI, traversing complications of repetitive substrate injections.

Imaging gene expression in live transgenic mice after providing luciferin in drinking water.

Mice expressing the firefly luciferase gene luc under the control of various gene promoters are used to image long-term changes in tumor growth, infection, development, and circadian rhythms. This novel approach enables ongoing regulation of gene expression to be visualized through repeated imaging of luciferase bioluminescence. Typically, luciferin, the luciferase substrate, is injected into mice before they are anaesthetized for imaging. To avoid the effects of handling and stress from injection on expression of the transgene, oral luciferin delivery methods were tested as an alternative to current methods. For unobscured imaging, a transgenic mouse line containing luc controlled by the enhancer and promoter for the major immediate-early gene of human cytomegalovirus (CMV) was crossed with a hairless albino mouse stock (HRS/J), resulting in the Hr-CMV line. Mice given food and water ad libitum readily drank 1-5 mM luciferin in water or apple juice and could be imaged repeatedly on subsequent days without any apparent adverse effects. Oral and injected luciferin produced similar patterns of luminescence in the body areas examined: abdomen, tail vertebrae, gonads, hind leg, foreleg and others, although the tail showed a slightly brighter relative luminescence after oral luciferin. These results show that luciferin is not appreciably degraded in the digestive tract and can be easily administered orally to avoid injection and any concomitant effects on behavior that could alter gene expression.

Optical imaging of luminescence for in vivo quantification of gene electrotransfer in mouse muscle and knee.

BACKGROUND: Optical imaging is an attractive non-invasive way to evaluate the expression of a transferred DNA, mainly thanks to its lower cost and ease of realization. In this study optical imaging was evaluated for monitoring and quantification of the mouse knee joint and tibial cranial muscle electrotransfer of a luciferase encoding plasmid. Optical imaging was applied to study the kinetics of luciferase expression in both tissues. RESULTS: The substrate of luciferase (luciferin) was injected either intraperitonealy (i.p.) or in situ into the muscle or the knee joint. Luminescence resulting from the luciferase-luciferin reaction was measured in vivo with a cooled CCD camera and/or in vitro on tissue lysate. Maximal luminescence of the knee joint and muscle after i.p. (2.5 mg) or local injection of luciferin (50 microg in the knee joint, 100 microg in the muscle) were highly correlated. With the local injection procedure adopted, in vivo and in vitro luminescences measured on the same muscles significantly correlated. Luminescence measurements were reproducible and the signal level was proportional to the amount of plasmid injected. In vivo luciferase activity in the electrotransfered knee joint was detected for two weeks. Intramuscular electrotransfer of 0.3 or 3 microg of plasmid led to stable luciferase expression for 62 days, whereas injecting 30 microg of plasmid resulted in a drop of luminescence three weeks after electrotransfer. These decreases were partially associated with the development of an immune response. CONCLUSION: A particular advantage of the i.p. injection of substrate is a widespread distribution at luciferase production sites. We have also highlighted advantages of local injection as a more sensitive detection method with reduced substrate consumption. Besides, this route of injection is relatively free of uncontrolled parameters, such as diffusion to the target organ, crossing of biological barriers and evidencing variations in local enzymatic kinetics, probably related to the reaction medium in the targeted organ. Optical imaging was shown to be a sensitive and relevant technique to quantify variations of luciferase activity in vivo. Further evaluation of the effective amount of luciferase in a given tissue by in vivo optical imaging relies on conditions of the enzymatic reaction and light absorption and presently requires in vitro calibration for each targeted organ.

A transgenic mouse model with a luciferase reporter for studying in vivo transcriptional regulation of the human CYP3A4 gene.

Cytochrome p450 3A4 (CYP3A4) plays an important role in drug metabolism, and the enzymatic activity of CYP3A4 contributes to many adverse drug-drug interactions. Here we describe a transgenic mouse model that is useful in monitoring the in vivo transcriptional regulation of the human CYP3A4 gene. A reporter construct consisting of 13 kilobases of the human CYP3A4 promoter controlling the firefly luciferase gene was used to generate a transgenic mouse line [FVB/N-Tg(CYP3A4-luc)Xen]. Reporter gene expression was assessed using an in vivo imaging system (IVIS) in anesthetized mice. Basal expression of the reporter was highest in liver and kidney, and moderate in the duodenum in male transgenic mice, whereas the basal luciferase activity was highest in the duodenum and lower in kidney and liver in females. Injections of pregnenolone, phenobarbital, rifampicin, nifedipine, dexamethasone, 5-pregnen-3beta-ol-20-one-16alpha-carbonitrile (PCN), and clotrimazole resulted in a time-dependent induction of luciferase expression, primarily in liver, that peaked at 6 h post injection. The greatest induction was found with clotrimazole, dexamethasone, and PCN, whereas the lowest induction followed pregnenolone, phenobarbital, and rifampicin injection. In general, male mice responded to these drugs more strongly than did females. Our results suggest that the human CYP3A4 promoter functions in transgenic mice and that this in vivo model can be used to study transcriptional regulation of the CYP3A4 gene.

Optical imaging of cardiac reporter gene expression in living rats.

BACKGROUND: Studies of cardiac gene transfer rely on postmortem analysis using histologic staining or enzyme assays. Noninvasive imaging of the temporal and spatial characteristics of cardiac gene expression in the same subject offers significant advantages. METHODS AND RESULTS: Rats underwent direct myocardial injection via left thoracotomy with adenovirus-expressing firefly luciferase (Ad-CMV-Fluc; n=30). The reporter substrate D-luciferin was injected intraperitoneally. Serial images were acquired by use of a cooled charged couple detector (CCD) camera. Results are expressed as relative light unit per minute (RLU/min). Rats transduced with 1x10(9) plaque-forming units show decremental cardiac luciferase activity over time: 152 070+/-21 170 (day 2), 195 806+/-62 630 (day 5), 7250+/-2941 (day 8), and 2040+/-971 RLU/min (day 14). To assess the detection sensitivity, serially diluted titers of Ad-CMV-Fluc were injected: 1x10(9) (195 393+/-14 896), 1x10(8) (33 777+/-18 179), 1x10(7) (417+/-91), 1x10(6) (185+/-64), 1x10(5) (53+/-1), and control (54+/-1) (P<0.05 for 1x10(9), 1x10(8), and 1x10(7) plaque-forming units versus control adenovirus-expressing mutant thymidine kinase [Ad-CMV-HSV1-sr39tk]; n=3). Finally, rats were euthanized, and in vitro luciferase activity correlated with in vivo CCD signals (r2=0.92). CONCLUSIONS: This study demonstrates for the first time the feasibility of imaging the location, magnitude, and time course of cardiac reporter gene expression in living rats. Cardiac gene therapy studies could be aided with wider application of this approach.

In vivo imaging of NF-kappa B activity.

A wide range of human disorders involves inappropriate regulation of NF-kappaB, including cancers and numerous inflammatory conditions. Toward our goal to define mechanisms through which NF-kappaB leads to the development of disease, we have developed transgenic mice that express luciferase under the control of NF-kappaB, enabling real-time in vivo imaging of NF-kappaB activity in intact animals. We show that in the absence of extrinsic stimulation, strong luminescence is evident in lymph nodes in the neck region, thymus, and Peyer's patches. Treating mice with TNF-alpha, IL-1alpha, or LPS increased the luminescence in a tissue-specific manner, with the strongest activity observed in skin, lungs, spleen, Peyer's patches, and the wall of the small intestine. Liver, kidney, heart, muscle, and adipose tissue displayed less intense activities. Also, exposure of skin to a low dose of UV radiation increased luminescence in the exposed areas. Furthermore, induction of chronic inflammation resembling rheumatoid arthritis produced strong NF-kappaB activity in the affected joints, as revealed by in vivo imaging. Thus, we have developed a versatile model for monitoring NF-kappaB activation in vivo.