The antiatherosclerotic action of 1G244 - An inhibitor of dipeptidyl peptidases 8/9 - is mediated by the induction of macrophage death.

BACKGROUND: Targeting cell death to induce favorable functional and morphological changes within atherosclerotic plaques has long been postulated as a promising anti-atherosclerotic strategy. In this regard, inhibition of dipeptidyl peptidases 8/9 has received special attention in the context of chronic inflammatory diseases due to its regulatory role in macrophage death in vivo. METHODS: The present study investigates the influence of prolonged treatment with 1G244 - an inhibitor of dipeptidyl peptidases 8/9 - on the development of the advanced atherosclerosis plaque in apoE-knockout mice, using morphometric and molecular methods. RESULTS: 1G244 administration has led to a reduction in atherosclerotic plaque size in an apoE-knockout mice model. Moreover, it reduced the content of in-plaque macrophages, attributed by immunohistochemical phenotyping to the pro-inflammatory M1-like activation state of these cells. Inhibition of dipeptidyl peptidases 8/9 augmented the lytic form of death response of activated macrophages in-vitro. CONCLUSIONS: In summary, inhibition of DPP 8/9 elicited an anti-atherosclerotic effect in apoE-/- mice, which can be attributed to the lytic form of death induction in activated macrophages, as assessed by the in vitro BMDM model. This, in turn, results in a reduction of the plaque area without its transformation towards a rupture-prone morphology.

Decrease of the pro-inflammatory M1-like response by inhibition of dipeptidyl peptidases 8/9 in THP-1 macrophages - quantitative proteomics of the proteome and secretome.

BACKGROUND: Cellular peptidases are an emerging target of novel pharmacological strategies in inflammatory diseases and cancer. In this context, the dipeptidyl peptidases 8 and 9 (DPP8/9) have gained special attention due to their activities in the immune cells. However, in spite of more than hundred protein substrates identified to date by mass spectrometry-based analysis, the cellular DPP8/9 functions are still elusive. METHODS: We applied the proteomic approach (iTRAQ-2DLC-MS/MS) to comprehensively analyze the role of DPP8/9 in the regulation of macrophage activation by in-depth protein quantitation of THP-1 proteome and secretome. RESULTS: Cells pre-incubated with DPP8/9 inhibitor (1G244) prior activation (LPS or IL-4/IL-13) diminished the expression levels of M1-like response markers, but not M2-like phenotype features. This was accompanied by multiple intra- and extra-cellular protein abundance changes in THP-1 cells, related to cellular metabolism, mitochondria and endoplasmic reticulum function, as well as those engaged with inflammatory and apoptotic processes, including previously reported and novel DPP8/9 targets. CONCLUSIONS: Inhibition of DPP 8/9 had a profound effect on the THP-1 macrophage proteome and secretome, evidencing the decrease of the pro-inflammatory M1-like response. Presented results are to our best knowledge the first which, among others, highlight the metabolic effects of DPP8/9 inhibition in macrophages.

Comparative two time-point proteome analysis of the plasma from preterm infants with and without bronchopulmonary dysplasia.

BACKGROUND: In this study, we aimed to analyze differences in plasma protein abundances between infants with and without bronchopulmonary dysplasia (BPD), to add new insights into a better understanding of the pathogenesis of this disease. METHODS: Cord and peripheral blood of neonates (≤ 30 weeks gestational age) was drawn at birth and at the 36th postmenstrual week (36 PMA), respectively. Blood samples were retrospectively subdivided into BPD(+) and BPD(-) groups, according to the development of BPD. RESULTS: Children with BPD were characterized by decreased afamin, gelsolin and carboxypeptidase N subunit 2 levels in cord blood, and decreased galectin-3 binding protein and hemoglobin subunit gamma-1 levels, as well as an increased serotransferrin abundance in plasma at the 36 PMA. CONCLUSIONS: BPD development is associated with the plasma proteome changes in preterm infants, adding further evidence for the possible involvement of disturbances in vitamin E availability and impaired immunological processes in the progression of prematurity pulmonary complications. Moreover, it also points to the differences in proteins related to infection resistance and maintaining an adequate level of hematocrit in infants diagnosed with BPD.

An iTRAQ-Based Quantitative Proteomic Analysis of Plasma Proteins in Preterm Newborns With Retinopathy of Prematurity.

Purpose: Retinopathy of prematurity (ROP) is a vision-threatening complication of a premature birth, in which the etiology still remains unclear. Importantly, the molecular processes that govern these effects can be investigated in a perturbed plasma proteome composition. Thus, plasma proteomics may add new insights into a better understanding of the pathogenesis of this disease. Methods: The cord and peripheral blood of neonates (≤30 weeks gestational age) was drawn at birth and at the 36th postmenstrual week (PMA), respectively. Blood samples were retrospectively subdivided into ROP(+) and ROP(-) groups, according to the development of ROP. Results: The quantitative analysis of plasma proteome at both time points revealed 30 protein abundance changes between ROP(+) and ROP(-) groups. After standardization to gestational age, children who developed ROP were characterized by an increased C3 complement component and fibrinogen level at both analyzed time points. Conclusions: Higher levels of the complement C3 component and fibrinogen, present in the cord blood and persistent to 36 PMA, may indicate a chronic low-grade systemic inflammation and hypercoagulable state that may play a role in the development of ROP.

Plasma proteome changes in cord blood samples from preterm infants.

OBJECTIVE: In the presented study, we aimed to systematically analyze plasma proteomes in cord blood samples from preterm infants stratified by their gestational age to identify proteins and related malfunctioning pathways at birth, possibly contributing to the complications observed among preterm infants. STUDY DESIGN: Preterm newborns were enrolled of three subgroups with different gestation age: newborns born ≤26 (group 1), between 27 and 28 (group 2) and between 29 and 30 (group 3) weeks of gestation, respectively, and compared to the control group of healthy, full-term newborns in respect to their plasma proteome composition. RESULT: Preterm delivery is associated with multiple protein abundance changes in plasma related to a plethora of processes, including inflammation and immunomodulation, coagulation, and complement activation as some key features. CONCLUSION: Plasma proteome analysis revealed numerous gestation-age-dependent protein abundance differences between term and preterm infants, which highlight key dysregulated pathways and potential new protein treatment targets.

Prospective plasma proteome changes in preterm infants with different gestational ages.

BACKGROUND: In this study, we aimed to analyze time-resolved plasma proteome changes in preterm neonates stratified by their gestational age to detect malfunctioning pathways that derive from the systemic immaturity of the neonate and to highlight those that are differentially regulated during the early development. METHODS: Preterm newborns were enrolled in three subgroups with different gestational ages: before 26 weeks of gestation (group 1), between 27 and 28 weeks of gestation (group 2), and between 29 and 30 (group 3) weeks of gestation. Plasma protein abundances were assessed at two time points (at preterm delivery and at the 36th week of post-menstrual age) by quantitative proteomics. RESULT: The quantitative analysis of plasma proteome in preterm infants revealed a multitude of time-related differences in protein abundances between the studied groups. We report protein changes in several functional domains, including inflammatory domains, immunomodulatory factors, and coagulation regulators as key features, with important gestational age-dependent hemopexin induction. CONCLUSION: The global trend emerging from our data, which can collectively be interpreted as a progression toward recovery from the perinatal perturbations, highlights the profound impact of gestation duration on the ability to bridge the gap in systemic homeostasis after preterm labor.

The influence of AICAR - direct activator of AMP-activated protein kinase (AMPK) - on liver proteome in apoE-knockout mice.

There is a growing body of evidence that altered functioning of apoE may aggravate cellular energy homeostasis and stress response, leading to oxidative stress, mitochondrial dysfunction, endoplasmic reticulum (ER) stress and inflammation, leading to hypercholesterolemia, dyslipidemia, liver steatosis and neurodegeneration. One of the key cellular responses to mitochondria and ER-stress related processes and cellular energy imbalance is AMP-activated protein kinase (AMPK), considered as a cellular master energy sensor and critical regulator of mitochondrial homeostasis. The aim of our study was to use differential proteomics and transcriptomics approach to elucidate the effect of direct AMPK activator AICAR on liver proteome in apoE-/- mice - experimental model of atherosclerosis and moderate nonalcoholic steatosis. We applied Isobaric Tags for Relative and Absolute Quantitation (iTRAQ) labeling and two-dimensional chromatography coupled with mass spectrometry (2DLC-MS/MS) MudPIT strategy, as well as RT-PCR to investigate the changes in mitochondrial and cytosolic proteins and transcripts expression in 6-month old AICAR-treated apoE-/-. AICAR elicited induction of proteins related to mitochondrial ß-oxidation, protein degradation and energy producing pathways (i.a. tricarboxylic acid cycle members and mitochondrial adenylate kinase 2). On the other hand, AICAR repressed inflammatory and pro-apoptotic markers in the apoE-/- mice liver, alongside reduction in several peroxisomal proteins, possibly suggesting induction of anti-oxidative pexophagy.

Influence of metformin on mitochondrial subproteome in the brain of apoE knockout mice.

Neurodegenerative diseases are the set of progressive, age-related brain disorders, characterized by an excessive accumulation of mutant proteins in the certain regions of the brain. Such changes, collectively identified as causal factors of neurodegeneration, all impact mitochondria, imminently leading to their dysfunction. These observations predestine mitochondria as an attractive drug target for counteracting degenerative brain damage. The aim of this study was to use a differential proteomic approach to comprehensively assess the changes in mitochondrial protein expression in the brain of apoE-knockout mice (apoE(-/-)) and to investigate the influence of prolonged treatment with metformin - an indirect activator of AMP-activated protein kinase (AMPK) on the brain mitoproteome in apoE(-/-) mice. The quantitative assessment of the brain mitoproteome in apoE(-/-) revealed the changes in 10 proteins expression as compared to healthy C57BL/6J mice and 25 proteins expression in metformin-treated apoE(-/-) mice. Identified proteins mainly included apoptosis regulators, metabolic enzymes and structural proteins. In summary, our study provided proteomic characteristics suggesting the decrease of antioxidant defense and structural disturbances in the brain mitochondria of apoE(-/-) mice as compared to healthy controls. In this setting, the use of metformin changed the expression of several proteins primarily involved in metabolic processes, the regulation of apoptosis and the structural maintenance of mitochondria, what could potentially restore their native functionalities.

Mitochondrial aldehyde dehydrogenase activation by Alda-1 inhibits atherosclerosis and attenuates hepatic steatosis in apolipoprotein E-knockout mice.

BACKGROUND: Mitochondrial dysfunction has been shown to play an important role in the development of atherosclerosis and nonalcoholic fatty liver disease (NAFLD). Mitochondrial aldehyde dehydrogenase (ALDH2), an enzyme responsible for the detoxification of reactive aldehydes, is considered to exert protective function in mitochondria. We investigated the influence of Alda-1, an activator of ALDH2, on atherogenesis and on the liver steatosis in apolipoprotein E knockout (apoE(-/-)) mice. METHODS AND RESULTS: Alda-1 caused decrease of atherosclerotic lesions approximately 25% as estimated by "en face" and "cross-section" methods without influence on plasma lipid profile, atherosclerosis-related markers of inflammation, and macrophage and smooth muscle content in the plaques. Plaque nitrotyrosine was not changed upon Alda-1 treatment, and there were no changes in aortic mRNA levels of factors involved in antioxidative defense, regulation of apoptosis, mitogenesis, and autophagy. Hematoxylin/eosin staining showed decrease of steatotic changes in liver of Alda-1-treated apoE(-/-) mice. Alda-1 attenuated formation of 4-hydroxy-2-nonenal (4-HNE) protein adducts and decreased triglyceride content in liver tissue. Two-dimensional electrophoresis coupled with mass spectrometry identified 20 differentially expressed mitochondrial proteins upon Alda-1 treatment in liver of apoE(-/-) mice, mostly proteins related to metabolism and oxidative stress. The most up-regulated were the proteins that participated in beta oxidation of fatty acids. CONCLUSIONS: Collectively, Alda-1 inhibited atherosclerosis and attenuated NAFLD in apoE(-/-) mice. The pattern of changes suggests a beneficial effect of Alda-1 in NAFLD; however, the exact liver functional consequences of the revealed alterations as well as the mechanism(s) of antiatherosclerotic Alda-1 action require further investigation.

Influence of atorvastatin on angiotensin I metabolism in resting and TNF-α -activated rat vascular smooth muscle cells.

INTRODUCTION: Vascular smooth muscle cells (VSMCs) are essential for maintaining vasculature homeostasis and function. By influence on its growth and activation both proinflammatory cytokines and peptides of the renin-angiotensin system (RAS) are potent regulators of VSMCs. Interestingly, angiotensin (Ang) II and Ang-(1-7) elicit opposite effects on VSMC activation, differentiation and proliferation. It has been suggested that statins, besides anti-inflammatory effects, may also modulate VSMC activation by their influence on the RAS. METHODS: The effect of atorvastatin on Ang I metabolism in a culture of explanted rat VSMCs was examined by liquid chromatography-mass spectrometry (LC-MS); expression of mRNA of the main RAS enzymes in VSMC was assessed by real-time polymerase chain reaction (PCR). RESULTS: In VSMC culture Ang-(1-7) was identified as a major product of Ang I metabolism. In this setting, TNF-α (1 ng/ml) caused a decrease in the conversion of Ang I to Ang-(1-7). This effect was accompanied by a decrease of mRNA expression of neutral endopeptidase (NEP) and angiotensin converting enzyme 2 (ACE2) and increase of mRNA of ACE. Interestingly, atorvastatin (3 µM) attenuated the effects of TNF-α on Ang-(1-7) production as well as reversed the influence of TNF-α on ACE and ACE2 expression. CONCLUSIONS: Enhancement by atorvastatin of the ACE2/Ang-(1-7) axis in VSMCs could represent a new and beneficial mechanism on cardiovascular action of this widely used drug.

The influence of angiotensin-(1-7) Mas receptor agonist (AVE 0991) on mitochondrial proteome in kidneys of apoE knockout mice.

Excessive action of angiotensin II on mitochondria has been shown to play an important role in mitochondrial dysfunction, a common feature of atherogenesis and kidney injury. Angiotensin-(1-7)/Mas receptor axis constitutes a countermeasure to the detrimental effects of angiotensin II on AT1 receptors. The aim of the study was to assess the effects of angiotensin-(1-7) peptidomimetic AVE0991 on the kidney mitochondrial proteome in widely used animal model of atherosclerosis (apoE(-/-) mice). Proteins changed in apoE(-/-) mice belonged to the groups of antioxidant enzymes, apoptosis regulators, inflammatory factors and metabolic enzymes. Importantly, AVE0991 partially reversed atherosclerosis-related changes in apoE(-/-) mice.

Elevated interleukin-1ß serum level after chronic peripheral salsolinol administration.

The catechol isoquinoline derivatives are endogenous compounds present in the mammalian brain and the representative one is referred to as salsolinol. It may be formed from aromatic amines leading to neurotoxic N-methyltetrahydroquinolinium ions that may play a role in the etiology of Parkinson's disease (PD). Neuroinflammation and apoptosis is thought to be a major contributor to the neuronal degeneration in PD. The alteration of inflammatory cytokines in the brain, cerebral spinal fluid and plasma of PD patients supports the existence of functional interconnections between the immune and nervous systems. In animal studies, chronic administration of salsolinol induced parkinsonian-like symptoms, both peripherally and centrally. However, still little has been known about the effects of salsolinol on the pro-inflammatory cytokine production or mast cells activation in the gastrointestinal tract. Male Wistar rats were subjected to continuous intraperitoneal dosing of salsolinol (200 mg/kg in total) with osmotic mini-pumps for two or four weeks and fed with either standard or high fat diet. An equivalent group of rats served as the appropriate controls. At the end of the experiment animals were decapitated and blood samples as well as tissue fragments were collected. Serum samples were assayed immunoenzymatically for IL-11ß and by liquid chromatography-mass spectrometry for histamine. Tissue fragments from gastric antrum, duodenum and proximal colon were formalin fixed, paraffin-embedded and stained with either hematoxylin and eosin or toluidine blue. Once activated, mast cells might secrete a range of neurosensitizing and pro-inflammatory molecules, increasing gut-blood and blood-brain barrier permeability. Cytokines mediate the activity of immune cells and may affect brain neurochemistry. The results of the present work serve as an additional support for the existence of an interrelationship between the nervous and immune system.

Proteomic analysis of liver mitochondria of apolipoprotein E knockout mice treated with metformin.

Nonalcoholic fatty liver disease (NAFLD) is strongly associated with insulin resistance. Metformin, a widely known anti-diabetic drug, used for patients with type 2 diabetes mellitus, is also claimed to be useful in treatment of NAFLD. However, both the clinical efficacy and the putative mechanisms underlying the clinical effects of metformin in treating NAFLD are unclear. Adenosine monophosphate-activated protein kinase (AMPK), the primary molecular target for metformin, is a known regulator of mitochondrial function. Thus, we used a proteomic approach to investigate the effect of metformin on liver mitochondria of apolipoprotein E knockout (apoE(-/-)) mice, an animal model of NAFLD. Two-dimensional electrophoresis coupled with mass spectrometry was applied to study the changes in liver mitochondrial protein expression in 6-month old metformin-treated apoE(-/-) mice as compared to non-treated animals. Collectively, 25 differentially expressed proteins were indentified upon metformin treatment including proteins related to metabolism, oxidative stress and cellular respiration. The most up-regulated protein was glycine N-methyltransferase (GNMT) - an enzyme, whose deficiency was shown to be directly related to the development of NAFLD. Our results clearly point to the strong mitochondrial action of metformin in NAFLD. Up-regulation of GNMT may represent an important mechanism of beneficial action of metformin in NAFLD treatment.

Beneficial effect of amantadine on postoperative pain reduction and consumption of morphine in patients subjected to elective spine surgery.

OBJECTIVE: To analyze the effect of coadministration of morphine and amantadine on postoperative pain reduction and morphine consumption in patients after elective spine surgery. METHODS: In double-blinded study, 60 patients (ASA physical status I-II) were randomized into two groups. Group A was given oral amantadine 50 or 100 mg 1 hour before surgery and 8, 20, 32 hours after operation. Group P received placebo at identical times. Pain was assessed using numerical rating scale before first administration of morphine and in 2, 3, 4, 6, 24, and 48 hours after operation. The amounts of morphine consumed were recorded up to 48 hours after surgery. Blood samples were taken twice in 2 hours after surgery and plasma levels of morphine and its main metabolites were measured. RESULTS: As compared with placebo, amantadine significantly reduced intra-operative Fentanyl use and sensation of postoperative pain. Up to 48 hours after operation, the cumulative consumption of morphine was 25% lower in the amantadine group. Moreover, intensity of nausea and vomiting tended to be lower in A group. Starting from 12th hour after surgery, the level of postoperative sedation was lower in patients who received amantadine, as compared with placebo group. No significant differences in plasma levels of morphine ant its metabolites were observed between A and P groups. CONCLUSIONS: Pre- and postoperative administration of amantadine significantly reduced fentanyl use during operation, as well as reduced the postoperative pain and decreased morphine consumption in young patients undergoing orthopedic surgery.

The influence of salsolinol on basic rat metabolism.

Parkinson's disease (PD) is associated with a broad spectrum of non-motor symptoms, which are poorly understood and foremost, may precede motor impairment. These symptoms include weight changes and gastrointestinal dysregulation. In our experiment, we applied salsolinol given peripherally and continuously in rats to induce changes in the enteric nervous system, which might be similar to those observed in PD patients. Surprisingly, we noted decrease in body weight and alteration in body fat contents of the animals during salsolinol exposure. The blood glucose levels, lipid profile and hepatic enzymes levels were assessed as well. While lipid profile, postprandial blood glucose and hepatic enzymes levels remained indifferent, postprandial triglyceridemia was significantly lower in all salsolinol-treated rats in comparison with the control, which might be related to disturbed absorption. We also suggest that diminished body weight gain and lower adipose tissue accumulation in salsolinol-treated animals were due to delayed gastric emptying together with disturbed gut function resulting in absorptive dysfunction.

Metabolism of bradykinin in aorta of hypertensive rats.

Alterations in the formation and metabolism of bradykinin (Bk) are hypothesized to play a role in the pathophysiology of hypertension, atherosclerosis and vascular complications of diabetes. However, despite its prominent role in cardiovascular regulation, studies on bradykinin have been limited by various difficulties in accurate measurements of this peptide in biological samples. In this study, using the LC-ESI-MS method we estimated the conversion of exogenous Bk to its main metabolites - Bk-(1-5) and Bk-(1-7) - in endothelial cell culture and in fragments of aorta of normotensive (WKY) and hypertensive rats (SHR). The effects of angiotensin converting enzyme (ACE) and neutral endopeptidase (NEP) inhibitors were more pronounced in SHR: perindoprilat inhibited Bk-(1-5) formation by 49 % and 76 % in WKY and SHR rats, respectively, and tiorphan tended to decrease formation of Bk-(1-5) in both groups of animals. The degradation of bradykinin and generation of both metabolites were significantly higher in the aorta of SHR rats than in WKY controls. Our results show that even in relatively early hypertension (in 4-month old SHR rats) inactivation of Bk by aorta wall is enhanced.

Proteomic analysis of changes in protein expression in liver mitochondria in apoE knockout mice.

The involvement of both apolipoprotein E (apoE) and mitochondria in lipid metabolism is widely recognized, however there is surprisingly scarce data about the putative mitochondrial action(s) of this protein. The aim of the study was to screen the alterations in liver mitochondrial proteome caused by apoE deficiency. We applied 2DE-LC-MS/MS methodology to investigate the changes in liver mitochondrial protein expression in 6-months old apoE(-/-) mice as compared to C57BL/6J controls. ApoE(-/-), but not C57BL/6J mice developed visible atherosclerotic changes in aorta and mild, diffuse steatosis of the liver. Collectively, 18 differentially expressed proteins were identified in mitochondria, related to apoptosis, antioxidant and detoxifying mechanisms of mitochondria, as well as lipid metabolism and transport. In conclusion, differential proteomic approach revealed several lines of proteomic evidence that mitochondrial function in the liver of apoE(-/-) mice could be altered as a result of overlapping of pathological and compensatory changes in expression of proteins.

Co-administration of dextromethorphan and morphine: reduction of post-operative pain and lack of influence on morphine metabolism.

We investigated co-analgesic effect of dextromethorphan in adolescent post-operative patients with idiopathic scoliosis. In a double-blind study, 60 patients with ASA physical status I-II were randomised into two groups. Group dextromethorphan (n = 30; age: 15.9 +/- 2.4 years) was given oral dextromethorphan 30 or 45 mg 1 hr before surgery and 8, 20 and 32 hr after operation. Group placebo (n = 30; age: 16.5 +/- 2.7 years) received placebo at identical times. Post-operative analgesic requirements were assessed using nurse-controlled analgesia system. Pain was assessed using numeric rating scale before first administration of morphine and at 2, 3, 4, 6, 24 and 48 hr after operation. Blood samples were taken 20 min. after the first use of morphine (within 1 hr after operation). The total use of analgesics during surgery was lower in the dextromethorphan group. The dose of morphine providing relief immediately after surgery, as well as total analgesic requirements in the first and second day after surgery did not differ between groups. Subjectively evaluated pain intensity score (numeric rating scale) was lower for the dextromethorphan patients in the first 4 hr, but not later after surgery. Plasma levels of morphine, morphine-6-glucuronide and morphine-3-glucuronide did not differ between groups. Dextromethorphan did not influence morphine glucuronidation, in terms of promotion of formation of any morphine glucuronides. In conclusion, in young patients subjected to spine surgery, addition of dextromethorphan to morphine reduced pain only in early post-operative period. In such patients, co-analgesic action of dextromethorphan was not associated with significant changes in plasma levels of morphine metabolites.

[Metformin--an inhibitor of early stages of protein glycation]. / Metformina--inhibitor wczesnych etapów glikacji bialek.

Nonenzymatic glycation of proteins is associated with the long-term diabetes complication. The aim of this work was to examine in vitro the infuence of metformin on glycated proteins formation by mass spectrometry (ESI/MS, LC/MS/MS) and spectrofluorimetric method. Obtained results suggest that metformin dose-dependently inhibits early stage of Maillard reaction, although with the weaker potency than known glycation inhibitor aminoguanidine.

[Evaluation of morphine metabolism in presence of amantadine and dextromethorphan in human serum by high performance liquid chromatography coupled with mass spectrometry (LC/MS) method]. / Metabolizm morfiny w obecnosci amantadyny i dekstrometorfanu u ludzi--ocena metoda wysokosprawnej chromatografii cieczowej sprzezonej ze spektrometria mas (LC/MS).

In the present study we assessed by LC/MS method the influence of amantadine and dextromethorphan on morphine metabolism in humans in order to explain clinically observed phenomenon of amplification of analgesic action of morphine by these drugs. Neither dextromethorphan nor amantadine influenced the rate of morphine degradation and concentration of morphine metabolites. Thus, our results suggest pharmacodynamical, not pharmacokinetic mechanism of interaction.