Hyperlipidemia Increases Nalbuphine Brain Accumulation with Multiple Dosing without Affecting Its Analgesic Response-Its Respiratory Depression Potential Should Be Investigated in Future Studies.

Nalbuphine is associated with a significant risk of respiratory depression. Its central nervous system entry is hindered by P-glycoproteins, and lower P-glycoprotein activity is a risk factor for respiratory depression. We assessed the effect of hyperlipidemia on nalbuphine pharmacokinetics, brain and liver uptake, and analgesic response following single (2.5 mg/kg) and multiple (2.5 mg/kg/day for three days) doses in normolipidemic and hyperlipidemic rats. Trends of reduction and increase in nalbuphine Cmax and Vdss/F were observed, respectively, in hyperlipidemic rats. Negative correlations were observed between Cmax and serum lipoproteins. Serum-normalized brain and liver levels at 1 h post-dose were lower in hyperlipidemic rats, with brain and liver levels being negatively and positively correlated with TG and HDL, respectively. At steady state, marked nalbuphine accumulation was observed in hyperlipidemic rat brains (R = 1.6) compared with normolipidemic rats (R = 1.1). Nalbuphine analgesic response was not altered by hyperlipidemia at steady state. Caution should be exercised since greater brain accumulation in hyperlipidemic patients treated with nalbuphine could increase their risk of respiratory depression. Our study highlights an unexpected role of lipoproteins in drug absorption and tissue uptake. We also propose a model for reduced nalbuphine absorption based on interaction with intestinal HDL-3.

Stationary-to-migratory transition in glioblastoma stem-like cells driven by a fatty acid-binding protein 7-RXRα neurogenic pathway.

BACKGROUND: Glioblastoma (GBM) stem-like cells (GSCs) are crucial drivers of treatment resistance and tumor recurrence. While the concept of "migrating" cancer stem cells was proposed a decade ago, the roles and underlying mechanisms of the heterogeneous populations of GSCs remain poorly defined. METHODS: Cell migration using GBM cell lines and patient-derived GSCs was examined using Transwell inserts and the scratch assay. Single-cell RNA sequencing data analysis were used to map GSC drivers to specific GBM cell populations. Xenografted mice were used to model the role of brain-type fatty acid-binding protein 7 (FABP7) in GBM infiltration and expansion. The mechanism by which FABP7 and its fatty acid ligands promote GSC migration was examined by gel shift and luciferase gene reporter assays. RESULTS: A subpopulation of FABP7-expressing migratory GSCs was identified, with FABP7 upregulating SOX2, a key modulator for GBM stemness and plasticity, and ZEB1, a prominent factor in GBM epithelial-mesenchymal transition and invasiveness. Our data indicate that GSC migration is driven by nuclear FABP7 through activation of RXRα, a nuclear receptor activated by polyunsaturated fatty acids (PUFAs). CONCLUSION: Infiltrative progression in GBM is driven by migratory GSCs through activation of a PUFA-FABP7-RXRα neurogenic pathway.

TLR9: A friend or a foe.

The innate immune system is a primary protective line in our body. It confers its protection through different pattern recognition receptors (PRRs), especially toll like receptors (TLRs). Toll like receptor 9 (TLR9) is an intracellular TLR, expressed in different immunological and non-immunological cells. Release of cellular components, such as proteins, nucleotides, and DNA confers a beneficial inflammatory response and maintains homeostasis for removing cellular debris during normal physiological conditions. However, during pathological cellular damage and stress signals, engagement between mtDNA and TLR9 acts as an alarm for starting inflammatory and autoimmune disorders. The controversial role of TLR9 in different diseases baffled scientists if it has a protective or deleterious effect after activation during insults. Targeting the immune system, especially the TLR9 needs further investigation to provide a therapeutic strategy to control inflammation and autoimmune disorders.

Repression of inflammatory pathways with Boswellia for alleviation of liver injury after renal ischemia reperfusion.

AIM: Acute kidney injury (AKI) is a sudden incident that is linked with a high lethality rate commonly due to distant organ injury. This study aims to explore the role of standardized Boswellia serrata (containing 35 % boswellic acid) in attenuating kidney and liver damage in a model of rats with renal insult. MAIN METHODS: Sprague-Dawley rats, exposed to renal injury via ischemia-reperfusion model, were administered a daily regimen of 1000 or 2000 mg/kg Boswellia for seven days then rats were sacrificed on day eight. Alanine aminotransferase, aspartate aminotransferase, serum creatinine and blood urea nitrogen, were assayed. TLR9, oxidative stress markers; namely MDA and GSH, inflammatory cytokines; namely, IL-6, IL-1ß, and TNF-α, as well as NF-κB were also measured. KEY FINDINGS: Renal ischemia-reperfusion injury (IRI) impaired renal and liver function significantly, but Boswellia attenuated this impairment in a dose-dependent fashion. Histopathological assessment of kidney and liver confirmed that Boswellia decreased damage severity. A marked increase in TLR9, NF-κB, IL-6, IL-1ß, TNF-α, and MDA besides decreased GSH levels were observed in the kidney and liver after renal IRI. Boswellia attenuated increases in TLR9, NF-κB, IL-1ß, TNF-α, and IL-6 and boosted antioxidant defences via decreasing MDA and increasing GSH in kidney and liver. Anti-inflammatory and antioxidant effects of Boswellia were mostly comparable to those of silymarin. SIGNIFICANCE: We conclude that the anti-inflammatory and antioxidant effects of Boswellia could be beneficial in ameliorating kidney and liver damage after AKI and that TLR9 might be the connection that signals liver injury in response to renal damage.

Chrysin promotes angiogenesis in rat hindlimb ischemia: Impact on PI3K/Akt/mTOR signaling pathway and autophagy.

Limb ischemia occurs due to obstruction of blood perfusion to lower limbs, a manifestation that is associated with peripheral artery disease (PAD). Angiogenesis is important for adequate oxygen delivery. The present study investigated a potential role for chrysin, a naturally occurring flavonoid, in promoting angiogenesis in hindlimb ischemia (HLI) rat model. Rats were allocated into four groups: (1) sham-operated control, (2) HLI: subjected to unilateral femoral artery ligation, (3) HLI + chrysin: received 100 mg/kg, i.p. chrysin immediately after HLI, and (4) HLI + chrysin + rapamycin: received 6 mg/kg/day rapamycin i.p. for 5 days then subjected to HLI and dosed with 100 mg/kg chrysin, i.p. Rats were killed 18 h later and gastrocnemius muscles were collected and divided into parts for (1) immunohistochemistry detection of CD31 and CD105, (2) qRT-PCR analysis of eNOS and VEGFR2, (3) colorimetric analysis of NO, (4) ELISA estimation of TGF-ß, VEGF, ATG5 and Beclin-1, and (5) Western blot analysis of p-PI3K, PI3K, p-Akt, Akt, p-mTOR, mTOR, and HIF-1α. Chrysin significantly enhanced microvessels growth in HLI muscles as indicated by increased CD31 and CD105 levels and decreased TGF-ß. Chrysin's proangiogenic effect is potentially mediated by increased VEGF, VEGFR2 and activation of PI3K/AKT/mTOR pathway, which promoted eNOS and NO levels as it was reversed by the mTOR inhibitor, rapamycin. Chrysin also inhibited autophagy as it decreased ATG5 and Beclin-1. The current study shows that chrysin possesses a proangiogenic effect in HLI rats and might be useful in patients with PAD.

Myoglobin: From physiological roles to potential implications in cancer.

Myoglobin (MB) belongs to the well-studied globin proteins superfamily. It has been extensively studied for its physiological roles in oxygen storage and transport for about a century now. However, the last two decades shed the light on unexpected aspects for MB research. Myoglobin has been suggested as a scavenger for nitric oxide and reactive oxygen species (ROS). Furthermore, MB was found to be expressed and regulated in different tissues, beyond the muscle lineage, including cancers. Current evidence suggest that MB is directly regulated by hypoxia and might be contributing to the metabolic rewiring in cancer tissues. In this article, we first discuss the MB physiological roles and then focus on the latter potential roles and regulatory networks of MB in cancer.

Experimental Evidence for Diiodohydroxyquinoline-Induced Neurotoxicity: Characterization of Age and Gender as Predisposing Factors.

Though quinoline anti-infective agents-associated neurotoxicity has been reported in the early 1970s, it only recently received regulatory recognition. In 2019, the European Medicines Agency enforced strict use for quinoline antibiotics. Thus, the current study evaluates the relation between subacute exposure to diiodohydroxyquinoline (DHQ), a commonly misused amebicide, with the development of motor and sensory abnormalities, highlighting age and gender as possible predisposing factors. Eighty rats were randomly assigned to eight groups according to their gender, age, and drug exposure; namely, four control groups received saline (adult male, adult female, young male, and young female), and the other four groups received DHQ. Young and adult rats received DHQ in doses of 176.7 and 247.4 mg/kg/day, respectively. After 4 weeks, rats were tested for sensory abnormality using analgesiometer, hot plate, and hind paw cold allodynia tests, and for motor function using open field and rotarod tests. Herein, the complex behavioral data were analyzed by principal component analysis to reduce the high number of variables to a lower number of representative factors that extracted components related to sensory, motor, and anxiety-like behavior. Behavioral outcomes were reflected in a histopathological examination of the cerebral cortex, striatum, spinal cord, and sciatic nerve, which revealed degenerative changes as well demyelination. Noteworthy, young female rats were more susceptible to DHQ's toxicity than their counterparts. Taken together, these findings confirm previous safety concerns regarding quinoline-associated neurotoxicity and provide an impetus to review risk/benefit balance for their use.

Expression of Myoglobin in Normal and Cancer Brain Tissues: Correlation With Hypoxia Markers.

BACKGROUND: Myoglobin (MB) is increasingly recognized as a key player in cancer growth and metastasis. Low oxygen tensions, commonly associated with highly aggressive and recurrent cancers, have been shown to regulate its expression in several cancers such as lung, neck, prostate and breast cancer. However, it is not yet known whether it contributes to the growth and spread of brain cancers especially Glioblastoma multiforme (GBM). METHODS: Here we investigate the expression of MB, and its correlation with the hypoxia markers carbonic anhydrase IX (CAIX) and lactate dehydrogenase A (LDHA), in human tissue microarrays of multiple organ tumors, brain tumors, and GBM tumors, and their respective cancer-adjacent normal tissues. Correlation between MB protein expression and tumor grade was also assessed. RESULTS: We show that MB protein is expressed in a wide variety of cancers, benign tumors, cancer-adjacent normal tissues, hyperplastic tissue samples and normal brain tissue, and low oxygen tensions modulate MB protein expression in different brain cancers, including GBM. Enhanced nuclear LDHA immune-reactivity in GBM was also observed. Finally, we report for the first time a positive correlation between MB expression and brain tumor grade. CONCLUSION: Our data suggest that hypoxia regulate MB expression in different brain cancers (including GBM) and that its expression is associated with a more aggressive phenotype as indicated by the positive correlation with the brain tumor grade. Additionally, a role for nuclear LDHA in promoting aggressive tumor phenotype is also suggested based on enhanced nuclear expression which was observed only in GBM.

Myoglobin variants are expressed in human glioblastoma cells­hypoxia effect?

Glioblastoma multiforme (GBM) is the most aggressive human brain cancer. Little is known regarding how these cells adapt to the harsh tumor microenvironment, and consequently survive and resist various treatments. Myoglobin (MB), the oxygen­binding hemoprotein, has been shown to be ectopically expressed in different human cancers and cell lines, and its expression is hypothesized to be an adaptation mechanism to hypoxia. The aim of the present study was to determine whether cancer­related and hypoxia­responsive MB mRNA splice variants are expressed in human GBM cells and glioblastoma tumor xenografts, and whether their expression is induced by hypoxia and correlated with hypoxia markers [lactate dehydrogenase A (LDHA), glucose transporter 1 (GLUT1), vascular endothelial growth factor (VEGF) and carbonic anhydrase IX (CAIX)]. Conventional reverse transcription (RT)­PCR, DNA sequencing, RT­quantitative PCR and immunohistochemistry were conducted to investigate MB expression in hypoxia­sensitive (M010b, M059J) and ­tolerant (M059K, M006xLo) GBM cell lines that also exhibit differential response towards radiation, rendering them a valuable translational GBM model. It was revealed that cancer­related MB variants 9, 10, 11 and 13 were expressed in GBM cells under normoxia, and following hypoxia, their expression exhibited modest­to­significant upregulation that correlated with hypoxia markers. It was also demonstrated that MB was upregulated in hypoxic microregions of glioblastoma tumor xenografts that were stained in matched tumor regions of serial tumor sections with the hypoxia markers, pimonidazole, CAIX, VEGF and LDHA. The present study identified myoglobin as a potential contributor to the hypoxia adaptation and survival strategies of glioblastoma, and may explain the aggressiveness and frequent recurrence rates associated with GBM.

Updates on Aptamer Research.

For many years, different probing techniques have mainly relied on antibodies for molecular recognition. However, with the discovery of aptamers, this has changed. The science community is currently considering using aptamers in molecular targeting studies because of the many potential advantages they have over traditional antibodies. Some of these possible advantages are their specificity, higher binding affinity, better target discrimination, minimized batch-to-batch variation, and reduced side effects. Overall, these characteristics of aptamers have attracted scholars to use them as molecular probes in place of antibodies, with some aptamer-based targeting products being now available in the market. The present review is aimed at discussing the potential of aptamers as probes in molecular biology and in super-resolution microscopy.

ω-3 and ω-6 Fatty Acids Modulate Conventional and Atypical Protein Kinase C Activities in a Brain Fatty Acid Binding Protein Dependent Manner in Glioblastoma Multiforme.

Glioblastoma multiforme (GBM) is a highly infiltrative brain cancer with a dismal prognosis. High levels of brain fatty acid binding protein (B-FABP) are associated with increased migration/infiltration in GBM cells, with a high ratio of arachidonic acid (AA) to docosahexaenoic acid (DHA) driving B-FABP-mediated migration. Since several protein kinase Cs (PKCs) are overexpressed in GBM and linked to migration, we explored a possible relationship between B-FABP and levels/activity of different PKCs, as a function of AA and DHA supplementation. We report that ectopic expression of B-FABP in U87 cells alters the levels of several PKCs, particularly PKCζ. Upon analysis of PKCζ RNA levels in a panel of GBM cell lines and patient-derived GBM neurospheres, we observed a trend towards moderate positive correlation (r = 0.624, p = 0.054) between B-FABP and PKCζ RNA levels. Analysis of PKC activity in U87 GBM cells revealed decreased typical PKC activity (23.4%) in B-FABP-expressing cells compared with nonexpressing cells, with no difference in novel and atypical PKC activities. AA and DHA modulated both conventional and atypical PKC activities in a B-FABP-dependent manner, but had no effect on novel PKC activity. These results suggest that conventional and atypical PKCs are potential downstream effectors of B-FABP/fatty acid-mediated alterations in GBM growth properties.

Long-Term Effect of Docosahexaenoic Acid Feeding on Lipid Composition and Brain Fatty Acid-Binding Protein Expression in Rats.

Arachidonic (AA) and docosahexaenoic acid (DHA) brain accretion is essential for brain development. The impact of DHA-rich maternal diets on offspring brain fatty acid composition has previously been studied up to the weanling stage; however, there has been no follow-up at later stages. Here, we examine the impact of DHA-rich maternal and weaning diets on brain fatty acid composition at weaning and three weeks post-weaning. We report that DHA supplementation during lactation maintains high DHA levels in the brains of pups even when they are fed a DHA-deficient diet for three weeks after weaning. We show that boosting dietary DHA levels for three weeks after weaning compensates for a maternal DHA-deficient diet during lactation. Finally, our data indicate that brain fatty acid binding protein (FABP7), a marker of neural stem cells, is down-regulated in the brains of six-week pups with a high DHA:AA ratio. We propose that elevated levels of DHA in developing brain accelerate brain maturation relative to DHA-deficient brains.

Effects of serum lipoproteins on cyclosporine A cellular uptake and renal toxicity in vitro.

In-vitro studies were performed to shed light on previous findings that showed increased uptake of cyclosporine A in the kidneys and liver of hyperlipidemic rats, and increased signs of kidney toxicity. Hepatocytes were obtained from rats, cultured, and exposed to a diluted serum from hyperlipidemic rats. Some cells were also exposed to lipid-lowering drugs. After washing out the rat serum or lipid-lowering drugs, cells were exposed to cyclosporine A embedded in serum lipoproteins. Pretreatment with hyperlipidemic serum and lipid-lowering drugs was associated with an increased uptake of cyclosporine A. As expected, atorvastatin caused an increase in low density lipoprotein receptor and a decrease in MDR1A mRNA in the hepatocytes. A decrease in NRK-52E rat renal tubular cellular viability caused by cyclosporine A was noted when cells were preincubated with diluted hyperlipidemic serum. This was matched with evidence of hyperlipidemic-serum-associated increases in the NRK-52E cellular uptake of cyclosporine A and rhodamine-123. The findings of these experiments suggested that in hyperlipidemia the expression and (or) the functional activity of P-glycoprotein was diminished, leading to greater hepatic and renal uptake of cyclosporine A, and renal cellular toxicity.

Interaction of brain fatty acid-binding protein with the polyunsaturated fatty acid environment as a potential determinant of poor prognosis in malignant glioma.

Malignant gliomas are the most common adult brain cancers. In spite of aggressive treatment, recurrence occurs in the great majority of patients and is invariably fatal. Polyunsaturated fatty acids are abundant in brain, particularly ω-6 arachidonic acid (AA) and ω-3 docosahexaenoic acid (DHA). Although the levels of ω-6 and ω-3 polyunsaturated fatty acids are tightly regulated in brain, the ω-6:ω-3 ratio is dramatically increased in malignant glioma, suggesting deregulation of fundamental lipid homeostasis in brain tumor tissue. The migratory properties of malignant glioma cells can be modified by altering the ratio of AA:DHA in growth medium, with increased migration observed in AA-rich medium. This fatty acid-dependent effect on cell migration is dependent on expression of the brain fatty acid binding protein (FABP7) previously shown to bind DHA and AA. Increased levels of enzymes involved in eicosanoid production in FABP7-positive malignant glioma cells suggest that FABP7 is an important modulator of AA metabolism. We provide evidence that increased production of eicosanoids in FABP7-positive malignant glioma growing in an AA-rich environment contributes to tumor infiltration in the brain. We discuss pathways and molecules that may underlie FABP7/AA-mediated promotion of cell migration and FABP7/DHA-mediated inhibition of cell migration in malignant glioma.

The ability of polycyclic aromatic hydrocarbons to alter physiological factors underlying drug disposition.

As part of everyday life, people are exposed to polycyclic aromatic hydrocarbons (PAHs). Sources of PAHs include cigarette smoke, ingestion of contaminated food and water or specifically charcoal-grilled meat, and occupational exposure (e.g., the coal industry). PAH compounds are well known to have enzyme-inducing effects, especially on the cytochrome P450 (CYP) family of enzymes, including CYP1A. Enhanced clearance of CYP1A-metabolized drugs as a result of PAH exposure is well established. However, there are examples where PAH-containing sources enhanced the clearance or altered the disposition of some non-CYP1A-metabolized drugs. It has been shown that not only do these compounds induce CYP1A isoforms, but they also can alter the expression of other CYPs, such as 1B1/2 and 2E1, certain phase II enzymes, some transport proteins (in animal models and cell lines), levels of plasma proteins (e.g., α1-acid glycoprotein and lipoproteins), and liver mass. Changes in any of these parameters can lead to changes in the biological disposition of a wide variety of drugs by altering either their concentrations in blood or tissues. Identification of patients with elevated enzyme activities or otherwise altered physiological parameters as a consequence of exposure to PAH could serve to lessen the risks and optimize therapeutic benefits of drug therapy. In this article, the pharmacokinetic properties of PAH, the possible mechanisms by which they can alter drug disposition, and specific examples are discussed.

A liquid chromatography-mass spectrometry method for nicotine and cotinine; utility in screening tobacco exposure in patients taking amiodarone.

A liquid chromatographic mass spectrometric (LC-MS) assay for the quantification of nicotine and cotinine in human specimens was developed. Human serum and urine (100 µL) were subjected to liquid-liquid extraction. For glucuronidated cotinine, serum was alkalinized and hydrolyzed before extraction. The dried samples were reconstituted and run using gradient flow reverse-phase liquid chromatography with MS detection. The ions utilized for quantification of nicotine, cotinine and milrinone (internal standard) were 162.8, 176.9 and 211.9 m/z, respectively. The mean recoveries were over 80% for cotinine and nicotine with excellent linearity between nominal concentrations and peak area ratios, over a wide concentration range. The percentage coefficient of variation and mean error of the inter- and intra-day validations were <15% for nicotine and cotinine. Analysis of serum from cardiac patients receiving amiodarone suggested that a number of patients were either active smokers or exposed to second-hand smoke. Significant concentrations of nicotine and cotinine were measured in the urine of a known smoking volunteer. The method was highly specific, sensitive and applicable as a tool in detecting and monitoring the passive exposure to tobacco smoke using small specimen volumes (0.1 mL).

The effect of beta-naphthoflavone on the metabolism of amiodarone by hepatic and extra-hepatic microsomes.

Amiodarone is a potent antiarrhythmic drug with several limiting side effects, some of which have been correlated with increased levels of its more toxic metabolite, desethylamiodarone. Elevated serum desethylamiodarone to amiodarone ratios are associated with a risk of amiodarone-induced pulmonary toxicity. Polycyclic aromatic hydrocarbons such as beta-naphthoflavone are known to increase desethylamiodarone levels in rat in vivo. In this article we investigated if this increase was solely due to increased formation as a result of cytochrome P450 (CYP) 1A1 and 1A2 induction in different rat hepatic and extra-hepatic tissues. Additionally, the effect of amiodarone treatment on CYP1A1 and 1A2 gene expression and activity was investigated. In rats, beta-naphthoflavone was found to increase desethylamiodarone forming activity in lung and kidney microsomes. Amiodarone increased beta-naphthoflavone mediated induction of CYP1A1 gene expression in liver, lung and kidney. However, there was no significant change in CYP1A activity. As expected, the data indicated that the increase in desethylamiodarone levels in vivo was partly due to increased formation through CYP1A1 induction, although increased formation was only evident in some extra-hepatic tissues. Amiodarone treatment did not affect basal or induced CYP1A activity.

Assessment of the inactivation potential of desethylamiodarone on human CYP1A1.

Desethylamiodarone was reported to inactivate human CYP1A1. To assess this, two protocols were implemented employing dilution and non-dilution of the preincubation mixture. Inactivation studies performed with diluted preincubation mixtures showed no inactivation of CYP1A1 by desethylamiodarone. However there was evidence for a mixed competitive inhibition (competitive and the uncompetitive inhibition constants of 2.1 microM and 9.6 microM, respectively) of CYP1A1 by desethylamiodarone. NADPH addition and/or replenishment were found to be important factors in determining the control activity in inactivation studies.

The effect of CYP1A induction on amiodarone disposition in the rat.

In the treatment of cardiac arrhythmias, amiodarone (AM) has emerged as a primary therapeutic agent. In addition to other cytochrome P450 (CYP), 1A1 and 1A2 facilitate the biotransformation of AM to the pharmacologically and toxicologically active metabolite, desethylamiodarone (DEA). The exposure to polycyclic aromatic hydrocarbons can induce these isoforms. This study was aimed at investigating the effect of CYP1A induction on the disposition of AM, after single and multiple intravenous doses, using beta-naphthoflavone (BNF) treated rats to model induction of CYP1A. After a single dose (25 mg/kg), the plasma AUC of DEA were significantly induced ( approximately 3-fold). With multiple doses, AM AUC(0-24 h) was significantly reduced in the BNF plasma (30%), lung (35%), liver (48%), kidney (52%), heart (34%), and intestine (43%). In contrast the DEA AUC(0-24 h) was increased significantly in the BNF plasma (36%), lung (56%), liver (101%), kidney (65%), and heart (73%). The DEA/AM ratios of the AUC(0-24 h) were increased in the BNF plasma, lung, liver, kidney, and heart by 1.9-, 2.5-, 3.8-, 3.4-, and 2.7-fold, respectively. In both groups of rats, the highest concentrations of AM and DEA were in the lung. Exposure to BNF was shown to increase DEA concentrations in the rat.

The metabolism of amiodarone by various CYP isoenzymes of human and rat, and the inhibitory influence of ketoconazole.

PURPOSE: To evaluate the metabolism of amiodarone (AM) to desethylamiodarone (DEA) by selected human and rat cytochrome P450, and the inhibitory effect of ketoconazole (KTZ). METHODS: Some important CYP isoenzymes (rat CYP1A1, 1A2, 2C6, 2C11, 2D1, 2D2, and 3A1 and human CYP1A1, 1A2, 2D6 and 3A4) were spiked with various concentrations of AM to determine the relative kinetic parameters for formation of DEA in the presence and absence of various concentrations of KTZ. RESULTS: The formation of DEA was observed when AM was exposed to each of the CYP tested, although the rates were varied. Human CYP1A1 followed by 3A4 had the highest intrinsic clearance (CLint) for DEA formation whereas in rat, CYP2D1 followed by CYP2C11 had the highest CLint. Human and rat CYP1A2 seemed to have the lowest CLint. At high concentrations of AM and KTZ, near those expected in vivo, significant inhibition of all isoforms except for rat CYP1A2 was observed. At lower concentration ranges of both drugs, the inhibitory constant was determined. At these levels, KTZ was found to potently inhibit human CYP1A1 and 3A4 and rat 2D2 and 1A1. CONCLUSION: Human CYP1A1 and 3A4 and rat CYP2D1 and 2C11 were most efficient in converting AM to DEA. For DEA formation, the in vivo administration of KTZ could inhibit other CYP isoforms besides CYP3A in human and rat.