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ANESTHETIC PHARMACOLOGY

The Effects of Fentanyl-Like Opioids and Hydromorphone on Human 5-HT_3A Receptors

From the Klinik und Poliklinik für Anästhesiologie und Operative Intensivmedizin, Universitätskliniken Bonn, Bonn, Germany.

Address correspondence and reprint requests to Dr. Martin Barann, Department of Anesthesiology and Intensive Care Medicine, University of Bonn, Sigmund-Freud Str. 25, 53127 Bonn, Germany. Address e-mail to martin.barann{at}ukb.uni-bonn.de.

	Abstract

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BACKGROUND: 5-HT₃ receptors are involved in various physiologic functions, including the modulation of emesis. 5-HT₃ antagonists are clinically widely used as potent antiemetics. Emesis is also a side effect of opioid analgesics. Intriguingly, the natural opioid morphine shows specific interactions with human 5-HT₃ receptors at clinically relevant concentrations. In the present study, we investigated whether this is a general effect of opioids, even when they are structurally diverse. Therefore, another morphine (phenanthrene-type) derivative, hydromorphone, and fentanyl including its (4-anilinopiperidine-type) derivatives were tested.

METHODS: Whole-cell patches from human embryonic kidney-293 cells, stably transfected with the human 5-HT_3A receptor cDNA, were used to determine the opioid effects on the 5-HT (3 µM)-induced currents using the patch clamp technique (voltage-clamp).

RESULTS: None of the fentanyl derivatives affected currents through the 5-HT_3A receptor (3 µM 5-HT) significantly in the clinically relevant nanomolar concentration range (IC₅₀ values >30 µM). In contrast, hydromorphone was considerably more potent (IC₅₀ = 5.3 µM), slowing the current activation- and desensitization-kinetics significantly (at 3 µM by a factor of 1.9 and 2.4, respectively), similar to morphine. At concentrations much higher than clinically relevant, but within the range predicted from Meyer-Overton correlations for nonspecific interactions, the fentanyl derivatives all showed at least a tendency to suppress current amplitudes, but they had diverse effects on the activation- and desensitization-kinetics of 5-HT_3A receptors.

CONCLUSIONS: Only morphine and hydromorphone, but not the fentanyl derivatives, reduced 5-HT-induced current amplitudes and slowed current kinetics near clinically relevant concentrations. The high potencies of morphine and hydromorphone, when compared to their lipophilicities, suggest a specific interaction with 5-HT_3A receptors. In contrast, the effects of fentanyl-type opioids appear to be of unspecific nature. Because the rank order of opioid potencies for human 5-HT_3A receptors is opposite of that for opioid receptors, the site involved is structurally different from opioid receptor binding sites. In agreement with recent data on different phenols, a phenolic OH-group (which morphine and hydromorphone possess) may contribute to specific interactions of morphine and hydromorphone with the 5-HT_3A receptor. Future clinical studies could test whether corresponding differences in emetogenicity between different classes of opioids will be found.

	Introduction

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Within the central nervous system, 5-HT₃ receptors are found primarily in the limbic system, brainstem and spinal cord. The 5-HT₃ receptor is a cation-permeable ligand-gated ion channel. It is directly and indirectly involved in several physiological and pathological processes, including emesis and nausea, gastrointestinal motility and visceral pain.1

5-HT₃ receptors in the area postrema and at vagal afferencies mediate emesis.2,3 Antagonists of this receptor, such as ondansetron, are clinically used to prevent nausea and emesis associated with chemotherapy and general anesthesia.3,4 Ondansetron, for example, reduces the relative risk for the occurrence of postoperative nausea and vomiting by 26%.5 On the other hand, emesis is a well-known side effect after opioid administration.

The interactions between the serotonergic and the opioid system have been known for a long time. For example, it has been demonstrated that 5-HT_4A receptor activation prevents the respiratory depression of opioid respiratory depression.6 The direct effects of morphine on 5-HT₃ receptors have also been described.7,8 The 5HT₃ receptor has several subunit variants, i.e., A, B, C, D, and E. The 5-HT_3A receptor is the only functional homopentameric type. Because of its simple stoichiometry, the homomeric 5-HT_3A receptor is a useful model for initial studies of molecular drugs actions. Morphine potently inhibits at low micromolar concentrations (Table 1), not only 5-HT_3A receptor-mediated peak currents,8,9 but as we reported in our previous study also slows down current activation and desensitization and that part of its action appears to be competitive with 5-HT.9 In contrast to the suppression of the peak-current and the current activation, the effect on the desensitization amounts to an enhancement (i.e., an increase of charge flow over time). The aim of the present study was, therefore, to investigate whether these effects are typical for other morphine derivatives and structurally different classes of opioids and what the underlying structural requirements are.

The fentanyl-like opioids belong to a class of drugs chemically distinct from morphine, possessing a higher affinity to µ-opioid receptors (Table 1). Here we present the effects of fentanyl and the structurally related opioids alfentanil, sufentanil and remifentanil on human 5-HT₃ receptors, and compare them with those of morphine-type opioids, i.e., hydromorphone and morphine. We were interested in whether the two groups of opioids share common effects on 5-HT_3A receptors. In order to decide whether the effects on 5-HT_3A receptors were nonspecific, specific or both [Figure 12b in Ref. 10], we compared the experimental drug potencies at 5-HT_3A receptors with those exclusively predicted by drug lipophilicities. To decide whether a possible specific site on 5-HT_3A receptors might be similar to a recognition site on opioid receptors, we compared the rank order of opioid potencies at 5-HT_3A receptors with that at opioid receptors (Table 1).

	METHODS

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Cell Culture
Human embryonic kidney-293 cells stably transfected with human 5-HT_3A receptors9 were grown as mono-layers on culture plates (NUNC, Wiesbaden, Germany) in DMEM Nutrient Mix F12 medium containing 10% heat-inactivated fetal calf serum, penicillin (100 I.U./mL), streptomycin (100 µg/mL), geneticine (0.75 µg/mL) and glutamine (292 µg/mL). The cells were cultured at 37°C in a humidified atmosphere (5% CO₂). Cells were transferred to 35-mm dishes (NUNC). They were used 2–6 days after transfer, before the cell layer became confluent.

Electrophysiology
Currents through the 5-HT₃ receptor were measured with the patch-clamp technique. A patch-clamp amplifier (EPC-7; List Electronic, Darmstadt, Germany) was used with the output filter set between 65 and 500 Hz (sampling rate 125–1000 Hz) and pClamp software (Axon Instruments, Foster City, CA). 5-HT (3 µM) was applied for 60 s at –30 or –60 mV in a standard voltage-clamp experiment in whole cell configuration. Capacitative transients and series resistance were measured and compensated using the internal compensation circuitry of the amplifier. A series resistance compensation of up to 70% was used. The currents were digitized with an interface (Digidata 1200, Axon Instruments, Molecular Devices Corporation, CA) and stored on an IBM 586-compatible PC.

The baseline control response to 3 µM 5-HT was measured before and after recovery from drug application. Three successive current measurements under identical experimental conditions were averaged to reduce noise effects. A washout time of at least 90 s was allowed for recovery of the receptors from desensitization, and only measurements with a recovery of a minimum of 75% were used. To correct for rundown-effects, the mean value of control and recovery were taken as the average control current.

The solutions were applied via a perfusion pipette positioned close to the cell. The solution (extracellular solution) applied to the patch had the following composition: NaCl 150 mM, KCl 5.6 mM, CaCl₂ 1.8 mM, MgCl₂ 1.0 mM, HEPES 10 mM, pH 7.4. Patch pipettes with resistances of 1.5–3 M were filled with "intracellular" solution containing: KCl 140 mM, EGTA 10 mM, MgCl₂ 5 mM, HEPES 10 mM, adjusted to a pH of 7.4. To minimize loss of drug due to interaction with plastic material, drug solutions were stored in glass reservoirs and teflon tubing was used. The recordings were performed at room temperature (21 ± 1°C).

Data Analysis
Data analysis was performed with pClamp 6/8 software (Axon Instruments). Graph Pad Prism 3.03 software (Graph Pad, San Diego, CA) was used to create graphics.

The concentration-response curves for opioids were fitted by the Hill equation, when possible:

i = 1 – cⁿ/(cⁿ + ICⁿ₅₀)

i: remaining peak current as fraction of the maximal (control) current,

c: opioid concentration,

n: Hill coefficient.

IC₅₀: opioid concentration causing half-maximal inhibition.

The time courses of agonist-induced activation and desensitization, f(t), were fitted (pClamp 6/8, Axon Instruments) either separately using single exponential functions:

f(t) = a₀ + a₁e^{– t/1}

or simultaneously with a bi-exponential function:

f(t) = a₀ + a₁e^–t/1+ a₂e^–t/2

Drugs and Solutions
5-hydroxytryptamine (creatinine sulfate) was obtained from Sigma, München, Germany; hydromorphone from Abbott, Wiesbaden, Germany; alfentanil, fentanyl, sufentanil from Janssen-Cilag, Neuss, Germany; and remifentanil from GlaxoSmithKline, München, Germany. Drug solutions were prepared daily from aqueous stock solutions (10–50 mM).

Calculation of Lipophilicity and Predicted IC₅₀ Values
The Meyer-Overton correlation from Figure 12B [published in Ref. 10], in which several ligand-gated ion channels and a wide range of anesthetics have been pooled, was used to predict anesthetic IC₅₀ values (in mol/l): log (IC₅₀^{uncharged drug}) = –1.568 – 0.9898 x log (P_{octanol/water}).

Total IC₅₀ (ionized plus uncharged drug) was calculated from IC₅₀^{uncharged drug}, using respective pKa values of drugs, assuming a pH of 7.4. Octanol/water partition coefficients, (P_{octanol/water}), were calculated with the program aLogPs.11

Limitations of Data Aquisition
As the recording of entire concentration-response curves was not possible on identical patches, mean IC₅₀ values and their standard deviations could not be calculated. Instead, IC₅₀ values and estimates of their standard errors were calculated from fitting a single concentration-response curve to the entire data set (Graph Pad Prism 3.02). As this was an exploratory study, the number of experiments needed to reach statistical significance could not be estimated in advance with a power analysis. In addition, the further characterization of the molecular mechanism(s) of morphine and its derivatives requires a higher time resolution than that of whole cell measurements, e.g., the use of excised patches combined with rapid solution exchange (<2 ms).

	RESULTS

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All drugs were applied in equilibrium, i.e., 90 s before and during the 5-HT (3 µM) pulse. The original current traces in the absence and in the presence of opioids, as well as the molecular drug structures, are shown in Figure 1. The different opioids caused various degrees of reduction of current-amplitudes (Fig. 2) and showed a variable pattern of effects on the kinetics of activation and desensitization of control currents (_act = 272 ± 119 ms and _des = 10.03 ± 9.7 s, means ± sd of n = 107 patches). These effects were analyzed as percentage of the respective control values from the same patches. Significance was tested using the paired t-test, comparing absolute values for time constants in the presence and in the absence of drugs (Fig. 3).

View larger version (12K): [in this window] [in a new window]   Figure 1. Left column: Molecular structures of morphine, hydromorphone, fentanyl, sufentanil, remifentanil and alfentanil, right column: traces of the 5-HT-induced currents and the effect of the opioids. The drugs were applied 90 s before plus during the 5-HT pulse.

View larger version (10K): [in this window] [in a new window]   Figure 2. Concentration-dependent effects of four fentanyl-like opioids, hydromorphone, and morphine on 5-HT (3 µM)-induced currents through human 5-HT3A receptors. The data for morphine are taken from.9 Shown are means ± sd of n = 4–7 different experiments. Only the data for morphine, hydromorphone, and remifentanil could be fitted according to the Hill-equation, the remaining data are connected by a line.

View larger version (11K): [in this window] [in a new window]   Figure 3. Comparison of the effects on the activation-(black) and desensitization-(gray) kinetics of the 5-HT (3 µM)-induced current by the different opioids. Note that only morphine and hydromorphone caused strong retardation of onsets and offsets of 5-HT-induced currents [morphine data taken from Ref. 9]. Means ± sd of n = 4–7, *P < 0.05, paired Student’s t-test.

The concentration-response curves for the reduction in current amplitudes caused by the fentanyl derivatives are incomplete because of the high concentration of drug needed to demonstrate an effect (Fig. 2). In comparison, hydromorphone (this study) and morphine (taken from Ref. 9) were much more potent. Values and estimates for IC₅₀ values are given in Table 1. In contrast to morphine and hydromorphone, at concentrations 1 µM (vertical line), no effects of fentanyl-like opioids on human 5-HT_3A were detectable (Fig. 2). At concentrations above 1 µM, increasing with concentration, various effects were observed:

Fentanyl, at high concentrations (3 µM), produced a slight peak inhibition (e.g., a suppression of 23% for 10 µM fentanyl, Fig. 2), but did not cause any significant changes in the desensitization kinetics; the activation kinetics were slightly accelerated by 10 µM fentanyl (Fig. 3).

Remifentanil, at low concentrations (0.01 and 0.1 µM), had no effects on current amplitudes, whereas at concentrations above 3 µM (up to 100 µM) a reduction of the current could be seen (Fig. 2). There were no significant changes with respect to the current kinetics (Fig. 3).

Alfentanil (0.03–30 µM) reduced the peak current at highest concentrations (30 µM) only by 12% (Fig. 2). Within this high concentration range (10–30 µM), an acceleration of current desensitization was detectable (by 49%–77%, Fig. 3).

Sufentanil, the most potent µ-opioid receptor agonist, exhibited nearly no effect on the peak of 5-HT-evoked peak currents, even at concentrations up to 10 µM (Fig. 2). In contrast, an acceleration of the current desensitization was observed, whereas the current activation was not affected in the concentration range from 0.01 µM up to 10 µM (Fig. 3). As can be seen from the original traces (Fig. 1), the acceleration of the current desensitization is visible as a reduction of the area over the current curve.

Hydromorphone, a morphine derivative, was, in contrast to the four fentanyl-type opioids much more potent in inhibiting the peak-currents (Fig. 2, IC₅₀ = 5.3 µM) and caused a considerable slowing of the current activation and the current desensitization (Fig. 3). As can be seen in Figure 3, such significant attenuation of current activation—and desensitization—kinetics is typical only for morphine and hydromorphone.

	DISCUSSION

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Distinct Effects by Fentanyl Derivatives and Morphine Derivatives
In the present study, we found that the effects of fentanyl-type opioids on human 5-HT_3A receptors clearly differ from those of hydromorphone and morphine: the fentanyl derivatives were at least a factor of 30–100 less potent in inhibiting 5-HT-induced currents than the morphine derivatives (Table 1), and fentanyl derivatives did not slow down the current kinetics as did hydromorphone and morphine (Fig. 3).

Specific and Nonspecific Effects
Comparing the experimental IC₅₀ values for 5-HT_3A receptor inhibition with calculated IC₅₀ values (Table 1), which are predicted from a linear regression between lipophilicity and anesthetic potency (Methods and Fig. 12b in Ref. 10), there is another clear distinction between morphine derivatives and fentanyl derivatives. The measured potencies of morphine and hydromorphone are high, and three to four orders of magnitude stronger than the linear regression would predict. This suggests specific interactions between morphine and hydromorphone with 5-HT_3A receptors. In contrast, the potencies of fentanyl derivatives are low and seem to follow the predictions from the regression line, suggesting that their effects could be due to nonspecific interactions with 5-HT_3A receptors based on lipophilicity.

Specific Morphine Interaction Site
Comparing the chemical structure of the drugs studied here (Fig. 1), it is obvious that all fentanyl derivatives differ substantially from hydromorphone and morphine, which belong to the so called "phenanthren-class." One characteristic of the two morphine derivatives studied here is the phenolic OH-group. The agonist 5-HT itself contains a phenolic OH-group. It was found that the 5-HT derivative 5-OH-indole,12 similar to morphine,9 prolongs 5-HT₃ receptor desensitization. Furthermore, even phenol itself, but not benzene (which lacks this group), causes a slowing of desensitization kinetics.13 Thus, it is tempting to speculate that the phenolic OH-group is important for the specific interaction of morphine and hydromorphone with 5-HT₃ receptors, consistent with the low potencies and the absence of slowed desensitization kinetics of the fentanyl derivatives lacking such OH-group.

Clinical Potencies and 5-HT_3A Receptor Potencies Differ
Table 1 also compares the clinical (analgesic) potencies with experimental (5-HT_3A receptor-inhibition) potencies of opioids. It is evident that the rank-order found at human 5-HT_3A receptors is opposite to the analgesic rank order of potency: the most potent 5-HT_3A-inhibitors, morphine and hydromorphone, are the least potent analgesics (Table 1). Thus, although a specific action of morphine with 5-HT₃ receptors is suggested by the findings in this paper, it also seems clear that a putative morphine-sensitive site of action on 5-HT₃ receptors is structurally dissimilar from typical opioid receptor binding sites.

Clinical Implications
It is shown in the present study that human 5-HT₃ receptors are more sensitive and more specific targets for morphine and hydromorphone than for fentanyl-type opioids. This result corresponds to the first clinical observations suggesting that opioids differ in emetogenic risk14 as well as affect the antiemetic potency of 5-HT₃ receptor antagonists.15 However, more and improved clinical data are needed before this consensus could be confirmed.

	ACKNOWLEDGMENTS

 
We thank Ms C. v.d. Bussche for carefully maintaining cell culture. This study was supported by the DFG (BA 1454).

	Footnotes

 
Accepted for publication March 5, 2008.

	REFERENCES

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