NMDA receptor;

Inwardly rectifying K+ channels (GIRK) that are activated by G proteins[1].The effects of the atypical N-methyl-D-aspartate (NMDA) receptor antagonist Ifenprodil were investigated by voltage-clamp recording of Xenopus oocytes expressing heteromeric NMDA receptors from cloned NR1 and NR2 subunit RNAs. In oocytes voltage-clamped at -70 mV, ifenprodil inhibited NMDA-induced currents at NR1A/NR2B receptors with high affinity (IC50 = 0.34 microM). The affinity of NR1A/NR2A receptors for Ifenprodil (IC50 = 146 microM) was 400-fold lower than that of NR1A/NR2B receptors. The rate of onset of inhibition by low concentrations of ifenprodil acting at NR1A/NR2B receptors was considerably slower than the onset of inhibition seen with high concentrations of ifenprodil acting at NR1A/NR2A receptors. The onset and recovery of blockade by ifenprodil at NR1A/NR2B receptors were not activity dependent. The inhibitory effects of low concentrations of ifenprodil at NR1A/NR2B receptors were not voltage dependent. In contrast, the inhibitory effects of high concentrations of ifenprodil at NR1A/NR2A receptors were partially voltage dependent, and a greater inhibition of NMDA-induced currents was seen at hyperpolarized membrane potentials than at depolarized membrane potentials. The reversal potential of NMDA currents was not altered in the presence of ifenprodil. Ifenprodil may act as a weak open-channel blocker of NR1A/NR2A receptors. The degree of inhibition seen with 100 microM ifenprodil at NR1A/NR2A receptors was not altered by changes in the concentration of extracellular glycine. However, the inhibitory effect of 1 microM ifenprodil at NR1A/NR2B receptors was reduced by increasing the concentration of glycine. Thus, part of the mechanism of action of ifenprodil at NR1A/NR2B receptors may involve noncompetitive antagonism of the effects of glycine. These results indicate that the mechanism of action of ifenprodil, as well as the potency of this antagonist, is different at NR1A/NR2B and NR1A/NR2A receptors expressed in Xenopus oocytes [1].

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G protein-activated inwardly rectifying K+ channels (GIRK, also known as Kir3) are regulated by various G-protein-coupled receptors. Activation of GIRK channels plays an important role in reducing neuronal excitability in most brain regions and the heart rate. Ifenprodil, which is a clinically used cerebral vasodilator, interacts with several receptors, such as alpha1 adrenergic, N-methyl-D-aspartate, serotonin and sigma receptors. However, the molecular mechanisms underlying the various clinically related effects of ifenprodil remain to be clarified. Here, we examined the effects of Ifenprodil on GIRK channels by using Xenopus oocyte expression assays. In oocytes injected with mRNAs for GIRK1/GIRK2, GIRK2 or GIRK1/GIRK4 subunits, ifenprodil reversibly reduced inward currents through the basal GIRK activity. The inhibition was concentration-dependent, but voltage- and time-independent, suggesting that ifenprodil may not act as an open channel blocker of the channels. In contrast, Kir1.1 and Kir2.1 channels in other Kir channel subfamilies were insensitive to ifenprodil. Furthermore, GIRK current responses activated by the cloned kappa-opioid receptor were similarly inhibited by ifenprodil. The inhibitory effects of ifenprodil were not observed when Ifenprodil was applied intracellularly, and were not affected by extracellular pH, which changed the proportion of the uncharged to protonated ifenprodil, suggesting its action from the extracellular side. The GIRK currents induced by ethanol were also attenuated in the presence of ifenprodil. Our results suggest that direct inhibition of GIRK channels by ifenprodil, at submicromolar concentrations or more, may contribute to some of its therapeutic effects and adverse side effects [2].

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In the CPE-based antiviral assay, Ifenprodil was comparable to nylidrin, with EC50 values of 6.6 and 16.9 μM for PR8 and HK, respectively (Table 1). Clenbuterol was potently active only against PR8 with an EC50 value of 9.4 μM, whereas labetalol and eliprodil were only marginally effective against HK alone with EC50 values above 44.0 μM. In contrast, ritodrine, fenoterol, and bambuterol had no antiviral activity against any viral strains. In further experiments, we tested the inhibitory compounds, even partially including nylidrin, Ifenprodil, labetalol, eliprodil, and clenbuterol, against additional influenza A and B viruses (Table 2). Nylidrin, ifenprodil, and clenbuterol were consistently effective against A/H1N1 strains, but their efficacy varied among the H3N2 viral strains, and none had antiviral activity in influenza B-infected cells. As expected, the remaining two compounds, labetalol and eliprodil, had little effect against any of these viral strains, except for A/Brisbane/59/2007 (H1N1), which was partially sensitive to eliprodil (EC50, 53.1 μM). These results indicate that nylidrin and its analogues, Ifenprodil and clenbuterol, can reliably inhibit the infection of H1N1 strains of the influenza A virus at subtoxic concentrations [4].

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Interaction of Ifenprodil with the NMDA receptor reduces the opening state of the ion channel and inhibits the influx of Ca2+ ions. With respect to this mechanism ifenprodil leads to neuroprotective, anticonvulsant and analgesic effects. The binding site of ifenprodil was first supposed at the amino terminal domain (ATD) of the GluN2B subunit. Later it could be found at the surface between the GluN1 and the GluN2B subunits. According to the first developed ligand it has been termed “ifenprodil binding site” and can cross-talk with different other binding sites of the receptor. The NMDA affinity of ifenprodil is very high (IC50 = 13.3 nM, Ki = 10 nM), but its selectivity is rather poor [5].

We showed that either Ifenprodil or bupivacaine produced spinal blockades of motor function and nociception dose-dependently. On the ED50 basis, the potency of Ifenprodil (0.42(0.38-0.46) μmol; 0.40(0.36-0.44) μmol) was equal (p>0.05) to that of bupivacaine (0.38(0.36-0.40) μmol; 0.35(0.32-0.38) μmol) in motor function and nociception, respectively. At the equianesthetic doses (ED25, ED50, and ED75), duration produced by Ifenprodil was greater than that produced by bupivacaine in motor function and nociception (p<0.05 for the differences). Furthermore, both Ifenprodil and bupivacaine showed longer duration of sensory blockade than that of motor blockade (p<0.05 for the differences). Conclusions: The resulting data demonstrated that Ifenprodil produces a dose-dependent local anesthetic effect in spinal anesthesia. Ifenprodil shows a more sensory-selective duration of action over motor block, whereas the duration of anesthesia is significantly longer with Ifenprodil than with bupivacaine. [3]

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The results of the present study indicated for the first time that intrathecal Ifenprodil elicited spinal block of motor function and nociception. The spinal anesthetic effect of Ifenprodil was comparable to that of the long-acting local anesthetic bupivacaine. Ifenprodil as well as bupivacaine showed greater duration of nociceptive/sensory block than motor block. At the equianesthetic doses (ED25, ED50, and ED75), the duration of spinal anesthesia with ifenprodil was greater than that of...
Our preclinical data showed that intrathecal Ifenprodil produced a dose-dependent local anesthetic effect in spinal anesthesia. The potency of ifenprodil in spinal anesthesia was similar to that of bupivacaine, whereas duration of spinal anesthesia with ifenprodil was greater than that of bupivacaine. Furthermore, Ifenprodil as well as bupivacaine exhibited significantly sensory-specific over motor block. The neural block of ifenprodil is worth testing in further [3].

Microsomal incubations (phase I) [5]
Ifenprodil (235 μg) was dissolved in phosphate buffer (250 μL, pH 7.4, 0.1 M) containing magnesium chloride (100 μL, 45.5 mM). After adding 150 μL of the microsome preparation (7 mg protein/mL) and 5 mg NADPH/H+ the mixture was incubated in a shaker at room temperature. After 120 min, the incubation was terminated by adding an equal volume of cold acetonitrile (−20 °C). The mixture was stored in an ice bath for 10 min. Subsequently, the sample was centrifuged at 13,000 rpm for 8 min and the resulting supernatant was analyzed by HPLC–MS without further sample pre-treatment.

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Rate of metabolic degradation of Ifenprodil [5]
For calibration studies, Ifenprodil (117.5 μg) was dissolved in sodium phosphate buffer (125 μL, pH 7.4, 0.1 M). Magnesium chloride (50 μL, 45.5 mM) and rat liver microsome suspension (100 μL) were added. The calibration was performed with 100, 80, 60, 40, 20 and 0% of the parent ifenprodil concentration (117.5 μg) which was diluted with sodium phosphate buffer to obtain the desired concentration. After 120 min the reaction was stopped by addition of cold CH3CN (500 μL, −20 °C) and the internal standard eliprodil (62.75 μg, dissolved in 32.5 μL CH3CN) was added. The mixture was stored in an ice bath for 10 min and subsequently centrifuged (13,000 rpm, 8 min). The supernatant was diluted 1:40 with CH3CN/H2O (1/1) and injected into HPLC system 1. Each calibration step was carried out three times. The resulting TICs (m/z = 326.2 and m/z = 348.15) were integrated manually. Calculation of the eliprodil/Ifenprodil ratio and the resulting calibration curve were carried out using Microsoft Excel 2010. Metabolic degradation was carried out with ifenprodil (117.5 μg) dissolved in sodium phosphate buffer (125 μL, pH 7.4, 0.1 M). Magnesium chloride (50 μL, 45.5 mM), rat liver microsomes (100 μL), NADPH (2.5 mg) and UDPGA (2 mg) were added. This reaction mixture was shaken for 15, 30, 60, 90 and 120 min. Then, the reaction was stopped by addition of cold acetonitrile (500 μL, −20 °C) and eliprodil (62.75 μg, dissolved in 32.5 μL CH3CN). The mixture was stored in an ice bath for 10 min and subsequently centrifuged (13,000 rpm, 8 min). The supernatant was diluted 1:40 with acetonitrile/H2O (1/1) and injected into HPLC system 1. Each step was carried out three times. The resulting TICs (m/z 326.2 and m/z 348.15) were integrated manually. Calculation of the eliprodil/ifenprodil ratio and the resulting amount for Ifenprodil were carried out using Microsoft Excel 2010.

For examining the effect of intracellular Ifenprodil, 23 nl of 10 mM ifenprodil or 30 mM lidocaine N-ethyl bromide (QX-314) dissolved in distilled water was administered to an oocyte through an additional pipette by pressure injection using a Nanoliter injector as described previously (Kobayashi et al, 2003), and the oocyte currents were then continuously recorded for approximately 30–40 min. As the volume of a Xenopus oocyte used is ∼1 μl, the intracellular concentration of Ifenprodil or QX-314 was presumed as ∼225 or ∼674 μM, respectively. Data were fitted to a standard logistic equation by using KaleidaGraph for analysis of concentration–response relationships. The EC50 value, which is the concentration of a drug that produces 50% of the maximal current response for that drug; the IC25 and IC50 values, which are the concentrations of a drug that reduces control current responses by 25 and 50%, respectively; and the Hill coefficient (nH) were obtained from the concentration–response relationships.[2]

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Compounds and Cytopathic Effect (CPE) Reduction Assay [4]
The test compounds nylidrin hydrochloride (~95%), Ifenprodil (+)–tartrate salt (≥98%), labetalol hydrochloride (≥98%), ritodrine hydrochloride (99.6%), fenoterol hydrobromide (≥98%), eliprodil (≥98%), clenbuterol hydrochloride (≥95%), and bambuterol hydrochloride (≥98%), the M2 inhibitor amantadine hydrochloride (AMT; ≥98%) and the polymerase inhibitor ribavirin (RBV; ≥98%), oseltamivir carboxylate (OSV-C; ≥98%) were used.

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Cell-based antiviral assays were performed as previously described [19,20]. Briefly, MDCK cells were seeded on 96-well plates (3 × 104 cells per well) and grown overnight. The cells were mock-infected or infected with individual influenza strains at a multiplicity of infection (MOI) of 0.001 to 0.005 for 1 h at 33 °C or 35 °C. After the removal of unadsorbed virus, test and control compounds were serially 3-fold diluted from 100 to 0.01 µM (total ten concentration points) in serum-free MEM with 2 µg/mL tosyl phenylalanyl chloromethyl ketone (TPCK)-treated trypsin and used to treat the cells. To synchronize the reading time point, at three days post-infection (p.i.), the plates were incubated at the following temperatures: 33 °C for A/California/7/2009, A/Brisbane/59/2007, A/Perth/17/2009, A/Brisbane/10/2007, A/Victoria/361/2011-like, and B/Brisbane/60/2008); and 35 °C for PR8, HK, Lee, and A/Seoul/11/1988. The half-maximal cytotoxic concentration (CC50) and half-maximal effective concentration (EC50) were determined by measuring cell viability using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT). Immunoassays [4]
To detect viral proteins, western blot analysis was performed as described previously. MDCK cells grown to 100% confluence in 6-well plates were infected with PR8 virus at an MOI of 0.001 for 24 h at 35 °C in the presence of nylidrin, OSV-C, or RBV. Viral proteins NP, HA, and M1 were detected by immunoblotting the appropriate antibodies: mouse anti-NP, rabbit anti-HA2, and mouse anti-M1, respectively. Secondary antibodies were horseradish peroxidase (HRP)-conjugated goat anti-mouse or anti-rabbit IgG. Cellular β-actin was used as the loading control.

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Plaque Assay [4]
PR8-infected MDCK cells were mock-treated or treated with the individual compounds. After 24 h at 35 °C, culture supernatants were harvested to prepare 10-fold serial dilutions, which were used to infect naïve MDCK cells seeded in 48-well plates for 1 h. After PBS washing, they were incubated in overlay medium (MEM with 0.6% carboxymethylcellulose and 2 µg/mL TPCK-trypsin) at 33 °C for three days. Plaques were counted by crystal violet staining. For time-of-addition experiments, MDCK cells in 48-well plates were infected with PR8 (MOI, 0.001) at 4 °C for 1 h in the presence or absence of nylidrin. After washing with PBS to remove chemicals and unadsorbed virus, nylidrin was administered at 1, 2, and 4 h p.i. (−)-Epigallocatechin gallate was used as a control for blocking viral entry. Subsequently, all samples were washed with PBS at 5 h p.i., followed by incubation under the overlay medium for three days prior to crystal violet staining.

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Confocal Microscopy [4]
For fluorescence microscopy, PR8-infected MDCK cells were incubated for 5 h at 37 °C in the presence or absence of nylidrin. After fixation and permeabilization, viral NP was visualized using anti-NP antibody and Alexa Fluor 488-conjugated goat anti-mouse IgG; nuclear DNA was counterstained using 4´,6-diamidino-2-phenylindole. Images were captured on a Zeiss LSM 700 confocal microscope with analysis using the ZEN software. For investigating colocalization of viral NP with cellular Rab5 or Rab7, A549 cells were seeded in 4-well slides (4 × 104 cells per well). On the next day, cells were transfected with 0.4 μg of pEGFP-Rab5 or pEGFP-Rab7 using lipofectamine 2000, according to the manufacturer’s instructions. After 24 h, they were mock-infected or infected with PR8 at an MOI of 10 in the absence or presence of 100 μM nylidrin at 4 °C for 30 min and then at 37 °C for an additional 4 h. Viral NP protein was probed using the same primary antibody above-mentioned, but with Alexa Fluor 633-conjugated secondary antibody.

For isolating liver microsomes and for in vivo metabolism studies of Ifenprodil, Wistar rats weighing 200–320 g from a local strain were used. [5]
In vivo metabolism studies of Ifenprodil [5]
Ifenprodil was dissolved in 0.9% sterile saline solution. A single dose of 6.2 mg Ifenprodil tartrate was administered i.p. to one female rat (310 g, Wistar rat), which results in a dose of 20 mg/kg. A dose of 20 mg/kg ifenprodil was chosen, since traxoprodil, which has similar structure and pharmacological properties, was used in previous experiments in a comparable amount [12]. The rat was housed individually in a metabolism cage with water and powdered feed ad libitum. Urine was collected for 48 h in three intervals of 0–8 h, 8–24 h and 24–48 h after i.p. application of ifenprodil. The volumes of urine samples were recorded and the samples were stored at −20 °C until analysis or directly used.
Extraction was carried out with RP-18 SPE cartridges, which were pre-conditioned with 1 mL H2O and 1 mL EtOH. After application of the urine samples, the cartridge was rinsed with 10 mL of H2O. Afterwards, Ifenprodil and the corresponding metabolites were eluted with 10 mL of EtOH. The solvent was removed under reduced pressure. The resulting solid was dissolved in CH3CN:H2O 1:1 and analyzed with the different HPLC systems.

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The aim of the study was to compare the proposed spinal anesthetic effect of Ifenprodil, an a1 adrenergic receptor antagonist, with that of the long-acting local anesthetic bupivacaine. Methods: After intrathecally injecting the rats with five different doses of each drug, the dose-response curves of Ifenprodil and bupivacaine were constructed to obtain the 50% effective dose (ED50). The spinal blockades of motor function and nociception of Ifenprodil were compared with that of bupivacaine.[3]

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Male Sprague–Dawley rats (300–350 g) were used. Intrathecal Ifenprodil caused significant motor or nociceptive blockade. Intrathecal Ifenprodil, as well as the long-lasting local anesthetic bupivacaine produced a dose-dependent local anesthetic effect in spinal anesthesia in rats (Fig. 1). The ED25s, ED50s, and ED75s of Ifenprodil and bupivacaine are presented in Table 1. On the ED50 basis, the potency of Ifenprodil in motor function and nociception was comparable to that of bupivacaine (Table 1, p > 0.05). [3]

Ifenprodil, an NMDA receptor antagonist, is an important lead compound for the development of novel GluN2B-selective NMDA receptor antagonists. While ifenprodil exhibits high affinity for the GluN2B subunit, it exhibits poor selectivity for the NMDA receptor. This characteristic, along with its rapid biotransformation, is a major drawback of ifenprodil. Identifying the major metabolic pathways of ifenprodil is crucial for optimizing the development of novel, more selective GluN2B (NMDA) receptor antagonists. This study used LC-MS(n) experiments to generate and analyze the in vitro and in vivo phase I and II metabolites of ifenprodil. In vitro experiments used rat liver microsomes and various cofactors to generate phase I and II metabolites. Ifenprodil was applied to rats, and their urine was analyzed to identify various in vivo metabolites. Phenolic compounds are the most unstable structural units in ifenprodil metabolism, as glucuronides 7 and 8 are its major metabolites. [5] It has been reported that the bioavailability of ivermoprolol is low. In humans, its peak plasma concentration occurs approximately 30 minutes after administration. [9] However, the biotransformation of ivermoprolol and the structure of its metabolites have not been elucidated. Therefore, identifying sites of metabolic instability in ivermoprolol may help in the development of more metabolically stable drugs that interact with ivermoprolol binding sites. This article reports our in vitro and in vivo phase I and II metabolism studies of ivermoprolol. [5]

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Fragmentation of ivermoprolol [5]
MSn assays of ivermoprolol revealed a fragmentation pattern that can be considered a typical feature of the ivermoprolol structure (Figure 1). The first step of fragmentation is the dehydration of the secondary alcohol (m/z 308.2003). Further fragmentation results in fragmentation at m/z 293.1752, which can be explained by the loss of the methyl group. Another important fragmentation of isifenprodil is the breaking of the CN (piperidine) single bond, resulting in the formation of a benzylpiperidine moiety at m/z 176.1424. The release of a triphenyl cation (m/z 91.0511) further confirms the presence of this fragment. This fragmentation pattern of the parent compound isifenprodil forms the basis for explaining the fragmentation studies of the generated metabolites and provides information about the structures of the formed metabolites.

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Identification and Fragmentation of Phase I Metabolites in Vitro[5]
Phase I transformation yielded a variety of metabolites, including two N-dealkylation products and several M+O metabolites (Fig. 2, Supplementary Information). The N-dealkylation metabolites were identified as 4-benzylpiperidine (1) and oxidized 4-benzylpiperidine-2-one (2). The structures of piperidine metabolites 1 and 2 were determined by fragmentation experiments. The major fragments of both metabolites were triphenyl cations with exact masses of m/z 91.0510 and m/z 91.0518, respectively (see Supplementary Information). In addition, six phase I metabolites were identified with m/z 342 [M+O+H]+, due to the introduction of an additional oxygen atom (Fig. 3a). These metabolites were analyzed by MSn experiments. The analysis revealed that oxidation occurred in the piperidine ring (3), the ortho, meta, and para positions of the phenyl moiety (4a–4c), the ortho position of the phenolic hydroxyl group (5), and the nitrogen atom forming the N-oxide (6, Fig. 3b). Three metabolites 4a–4c showed the same fragmentation pattern, indicating that their structures were very similar. The hydroxyl group of the monohydroxylated metabolite 3 was attributed to the piperidine heterocycle. The first clue was fragment m/z 192. This mass is 16 amu higher than the corresponding fragment of the parent compound. Subsequent fragmentation yielded a fragment m/z 174, representing dehydrated benzylpiperidine. These fragments confirm the position of the hydroxyl group, as fragment m/z 174 (Fig. 4) only appears when the hydroxyl group is located in the piperidine moiety. N-oxide 6 is identified by fragment m/z 192.1358 (oxidized benzylpiperidine) and m/z 174 [benzylpiperidine + O + H – H₂O]⁺. Therefore, the additional oxygen atom can only be located in the benzylpiperidine moiety of ifenprodil. The retention time is slightly increased (10.8 min) compared to ifenprodil (9.4 min), indicating the presence of N-oxide. Due to the slightly increased lipophilicity of N-oxide, its retention time is generally longer than that of the parent compound.

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Phase II metabolites found in vitro [5] In the phase II reaction, ifenprodil can theoretically be converted into glucuronide, sulfate and methylated catechol derivatives, as described by traxoprodil. β-Glucuronide and sulfate can be directly generated from the hydroxyl groups present in evinprodil. Furthermore, metabolites from phase I reactions can be bound in phase II reactions. Methylated catechol derivatives can only be generated after ortho-hydroxylation of phenol to form catechol derivatives (see metabolite 5).
In vitro, glucuronidation was studied by adding UDPGA (without NADPH/H+) to a microsomal incubation mixture. Observations of glucuronide 7 [M+Glu+H]+ were recorded in both positive and negative ion modes. In positive ion mode, fragmentation of β-glucuronide metabolite 7 showed two main fragmentation pathways. Similar to evinprodil and its derivatives, dehydration was observed (m/z 484.2346 in positive ion mode). On the other hand, glucuronic acid was cleaved, producing fragment m/z 326.2134 ([evinprodil+H]+). These two fragmentation processes occur simultaneously and ultimately produce the same fragment m/z 308.2040 (see SI). Similar fragmentation was also observed in negative ion mode. Furthermore, fragmentation of glucuronic acid was identified in negative ion mode. Fragment m/z 193 represents the glucuronide anion, while m/z 175 corresponds to dehydrated glucuronic acid. Subsequent loss of water and CO2 generates fragment m/z 113, characteristic of glucuronides (Fig. 5).
Incubation of ifenprodil with NADPH/H+ and UDPGA generated additional glucuronide 8 (Fig. 2), which is formed after glucuronidation of catechol 5 produced during phase I biotransformation. The same catechol 5 can also be methylated by catechol O-methyltransferase. The addition of S-adenosylmethionine (SAM) and NADPH/H+ to the incubation mixture yielded a compound with an m/z of 356.1965, the mass of which corresponded to that of methylated catechol 9. The fragmentation of compound 9 (Fig. 6) was similar to that of isifenprodil (see Fig. 1). The formation of unsubstituted benzylpiperidine fragments with m/z of 176.1444 strongly suggests that methoxylation occurred on the phenolic hydroxyl group, rather than the benzyl group. Furthermore, fragments with m/z of 338.2129, 163, and 137 had masses 30 amu greater than the corresponding isifenprodil fragments.

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Identification of in vivo metabolites[5]
For in vivo metabolites, urine was collected from a rat over 48 hours after intraperitoneal injection of 20 mg/kg isifenprodil, divided into three time periods (0–8 hours, 8–24 hours, and 24–48 hours). The identified metabolites are shown in Figure 7. Three metabolites (11, 12, and 13) were found only in vivo. Metabolite 11 is the product of ortho-hydroxylation, methylation, and glucuronidation. The regioisomers glucuronides 12a and 12b have the same mass (m/z 518) as compound 8 but exhibit different fragmentation patterns (see Supplementary Information). They are generated by further glucuronidation of the hydroxylated metabolite 4. A fragmentation pattern similar to that of metabolite 4 allowed for the identification of the corresponding metabolites. In addition, two metabolites 13 containing an extra oxygen atom were observed, but their fragmentation patterns did not definitively determine the position of the oxygen atom. Sulfate 10, identified after the addition of PAPS to the in vitro system, was not detected in rat urine. However, whether this metabolite was not generated, or whether it was degraded in urine samples or during storage, remains to be elucidated. Ifenprodil is rapidly excreted in the initial phase (0–8 hours). During this period, ivenprodil was one of the major compounds, along with glucuronide 7 and 11, assuming that all compounds had comparable ionization factors. Glucuronization was observed to be the major metabolic pathway during the first two time periods (0–8 h, 8–24 h). ivenprodil was still detected even during the last time period of urine collection (24–48 h), indicating that ivenprodil was still excreted in its unmodified form even after 24 hours (Fig. 8).

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Metabolic stability of ivenprodil [5]
In in vitro experiments, the metabolic stability of ivenprodil in rat liver microsomes, in the presence of NADPH/H+, and UDPGA was determined. These cofactors were chosen to generate the major metabolites observed in vivo. To accurately quantify ivenprodil, calibration curves were first recorded. Different amounts of efenprodil (equivalent to 20%, 40%, 60%, 80%, and 100% of the microsomal incubation amount) were incubated with a reaction mixture without NADPH/H+. Efenprodil was also added as an internal standard (IS). A plot of the efenprodil/IS ratio against the amount of efenprodil used yielded good regression coefficients (see Supplementary Information). The concentration of efenprodil in the metabolically active mixture was determined after six incubation cycles lasting up to 120 minutes. Unexpectedly, the concentration of efenprodil increased after 60 minutes of incubation with NADPH/H+ and UDPGA. This result can be explained by the rapid degradation of glucuronide 7, leading to the regeneration of efenprodil. This theory was confirmed by incubating efenprodil with only NADPH/H+ without the addition of UDPGA, resulting in a continuous decrease in efenprodil content (Figure 9). After 60 minutes of co-incubation with two cofactors, 86 ± 2% of ifenprodil remained undetectable. After only the formation of the Phase I metabolite, the ifenprodil concentration decreased to 92.8 ± 2% after 60 minutes. Therefore, the major biotransformation of ifenprodil is induced by glucuronidation. This observation is consistent with in vivo results, which also identified glucuronide 7 and 8 as the major metabolites. ifenprodil is an important lead compound for the development of highly efficient and selective GluN2B-selective NMDA receptor antagonists, and its biotransformation was analyzed in this paper. In in vitro experiments, N-dealkylation, hydroxylation of the two aromatic rings, and hydroxylation of the piperidine moiety were identified as possible reactions. In Phase II experiments, glucuronidation of phenol was observed. Furthermore, methoxylated and sulfated metabolites were detected after incubation with the corresponding cofactors SAM and PAPS. Analysis of rat urine samples identified glucuronide, hydroxylated, and methoxylated metabolites. Glucuronide 7 was identified as the major metabolite. In summary, the phenol of ivermectin is the major structural unit that is readily biotransformed. In particular, glucuronidation of the hydroxyl group was identified as the major metabolic pathway in vitro and in vivo. These results clearly suggest that bioisosteric substitution of phenol should be performed to obtain a more metabolically stable GluN2B selective NMDA receptor antagonist. [5]

Ifenprodil interacts with other receptors in the central nervous system (CNS) (α1, 5-HT, σ1, σ2 receptors) and can cause adverse side effects such as motor dysfunction and hypotension. Nevertheless, Ifenprodil can serve as an important lead compound for the rational design of novel selective GluN2B antagonists, which are expected to become drugs for the treatment of life-threatening central nervous system diseases [5].

[1]. Ifenprodil discriminates subtypes of the N-methyl-D-aspartate receptor: selectivity and mechanisms at recombinant heteromeric receptors. Mol Pharmacol. 1993 Oct;44(4):851-9.

[2]. Inhibition of G protein-activated inwardly rectifying K+ channels by ifenprodil. Neuropsychopharmacology. 2006 Mar;31(3):516-24.

[3]. Ifenprodil for prolonged spinal blockades of motor function and nociception in rats. Pharmacol Rep. 2016 Apr;68(2):357-62.

[4]. In Vitro and In Vivo Antiviral Activity of Nylidrin by Targeting the Hemagglutinin 2-Mediated Membrane Fusion of Influenza A Virus. Viruses. 2020 May 25;12(5):581.

[5]. Metabolism studies of ifenprodil, a potent GluN2B receptor antagonist. J Pharm Biomed Anal . 2014 Jan:88:96-105.

4-[1-hydroxy-2-[4-(benzyl)-1-piperidinyl]propyl]phenol is a piperidine compound. N-methyl-D-aspartate (NMDA) receptors (NMDARs) are members of the ionotropic glutamate receptor family and play a crucial role in brain development and neural function. NMDARs are heterotetramers, typically composed of dimers of the GluN1 and GluN2 A-D subunits. Each subunit itself consists of an N-terminal domain (NTD), a ligand-binding domain (LBD), a transmembrane domain, and a C-terminal cytoplasmic domain. The binding of the agonist glycine (or D-serine) to the LBD of the GluN1 subunit and the binding of glutamate to the LBD of the GluN2 subunit constitute the regulatory mechanism for channel activation. Furthermore, allosteric regulators are known to bind to the N-terminal domain (NTD) of NMDARs, forming another layer of regulatory mechanisms. Ifenprodil is one such allosteric modulator, first discovered in the 1990s to bind to NMDAR, particularly those containing the GluN2B subunit. Further research showed that ifenprodil binds firmly to the interface between adjacent GluN1 and GluN2B NTD subunits, acting as a non-competitive antagonist. While ifenprodil has garnered attention for its potential neuromodulatory activity in mental illnesses, including addiction and depression, studies have also indicated its immunomodulatory effects. In an unbiased screening of compounds that reduce cell death induced by H5N1 influenza virus infection, ifenprodil was found to protect against H5N1-induced lung injury, partly by mitigating the H5N1-induced cytokine storm and reducing the infiltration of neutrophils, natural killer cells, and T cells in the lungs. Ifenprodil is currently being investigated in an ongoing Phase 2b/3 clinical trial (NCT04382924) to evaluate its potential efficacy in treating COVID-19. Ifenprodil is an orally bioavailable N-methyl-D-aspartate (NMDA) receptor antagonist with potential central nervous system (CNS) stimulatory, neuroprotective, anti-inflammatory, and anti-fibrotic activities. After administration, ifenprodil targets and binds to glutamatergic NMDA receptors (NMDARs), particularly glycine-binding NMDA receptor subunits 1 (GluN1) and 2 (glutamate-binding NMDA receptor subunit 2; NMDA-type subunit 2B; GluN2B), thereby inhibiting NMDAR signaling. This may suppress neuronal excitotoxicity and potentially enhance cognitive function. In addition, isofenprodil also inhibits G protein-coupled inward rectifier potassium (GIRK) channels and interacts with α1 adrenergic receptors, 5-hydroxytryptamine receptors, and activates σ receptors. Isofenprodil exerts its anti-inflammatory effects through its action on NMDA receptors and possibly σ-1 receptors. Although the exact mechanism has not been fully elucidated, the drug reduces the infiltration of neutrophils and T cells into the lungs and prevents the release of pro-inflammatory cytokines. This may lead to a reduction in pulmonary inflammation, inhibition of pulmonary fibrosis, and possible reduction in the severity of cough. NMDA receptors are multimeric ionotropic glutamate receptors composed of four subunits and are expressed in a variety of cells and organs, such as the brain, lungs, T cells, and neutrophils.

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Characteristics of isofenprodil's inhibition of GIRK channels[2]
This study shows that isofenprodil inhibits brain-type GIRK1/2 and GIRK2 channels and cardiac-type GIRK1/4 channels in a unique manner at nanomolar or higher concentrations. The inhibitory effect of ifenprodil on GIRK channels is concentration-dependent but independent of voltage and time, primarily affecting transient currents and maintaining a stable percentage of inhibition during each voltage pulse. Our results also indicate that ifenprodil acts on the channel from the extracellular side. On the other hand, the inhibitory effects of extracellular Ba²⁺ and Cs⁺ (typical Kir channel blockers, which act by blocking the pores of open channels) are concentration-dependent, strongly voltage-dependent, and time-dependent, with relatively small effects on transient currents, but significant inhibition of steady-state currents at the end of the voltage pulse (Lesage et al., 1995). These observations suggest that ifenprodil may induce conformational changes in GIRK channels, but does not act as an open-channel blocker like Ba²⁺ and Cs⁺. The incomplete blocking of GIRK currents by ifenprodil may also be related to this mechanism of action. In this study, ivengprodil also inhibited basal free G protein βγ subunit, κ opioid receptor activation-mediated G protein, or ethanol-induced GIRK currents in oocytes. Further single-channel experiments may help elucidate the mechanism of action of ivengprodil on GIRK channels. Furthermore, ivengprodil exhibited higher inhibitory potency against GIRK1/4 channels than against GIRK1/2 and GIRK2 channels. Although at the highest concentration tested, the inhibitory potency of ivengprodil against different channels followed the order GIRK2 > GIRK1/2 ≥ GIRK1/4, the differences were not statistically significant. Additionally, Kir1.1 and Kir2.1 channels, other subfamilies of Kir channels, were insensitive to ivengprodil. Further studies using GIRK/Kir1.1 and GIRK/Kir2.1 chimeric channels, as well as GIRK mutant channels, may elucidate the key sites of action of ivengprodil on GIRK channels. In addition, high-resolution structural analysis of GIRK channels may help characterize their binding sites. Furthermore, although haloperidol is structurally related to ifenprodil (Williams, 2001), haloperidol has a weaker inhibitory effect on GIRK1/2 and GIRK1/4 channels and acts in a similar manner (Kobayashi et al., 2000). The different effects of these drugs on GIRK channels may be due to their different chemical structures or their different binding sites on GIRK channels. Studies on the relationship between GIRK channel structure and ifenprodil structure may provide a basis for designing highly effective GIRK inhibitor candidates.
Clinical and pharmacological significance[2]
It has been reported that after a single clinical dose, the concentration of ifenprodil in human plasma is approximately 0.1 μM (Aventis Pharma data). In animal studies, intramuscular injection of radiolabeled isofenprodil resulted in concentrations in the brain and heart approximately 5-8 times and 5-10 times higher than in the blood, respectively (Nakagawa et al., 1975). Therefore, our results suggest that isofenprodil may inhibit GIRK channels in the brain and heart at clinically relevant concentrations. Under physiological conditions, activation of GIRK channels induces K+ efflux, leading to membrane hyperpolarization (North, 1989); while inhibition of GIRK channels leads to membrane depolarization, thereby increasing cellular excitability (Kuzhikandathil and Oxford, 2002). Therefore, in clinical applications, isofenprodil may affect various brain and cardiac functions by inhibiting GIRK channels, which are widely expressed in the nervous system and atria (Kobayashi et al., 1995; Karschin et al., 1996).
GIRK2 knockout mice exhibit spontaneous seizures and are more susceptible to seizures induced by pentylenetetrazole (a GABAA receptor antagonist) than wild-type mice (Signorini et al., 1997). Furthermore, the resting membrane potential of neurons in GIRK knockout mice is depolarized compared to wild-type mice (Lüscher et al., 1997; Torrecilla et al., 2002). High doses of ifenprodil can enhance seizures induced by certain proconvulsants, including pentylenetetrazole (Mizusawa et al., 1976), although ifenprodil has been shown to have anticonvulsant effects (Thurgur and Church, 1998; Yourick et al., 1999), likely due to its inhibition of NMDA receptor channels (Williams, 2001) and Ca2+ channels (Church et al., 1994; Bath et al., 1996). Although ifenprodil has anticonvulsant effects, its potent blockade of neuronal GIRK channels may lead to increased susceptibility to epilepsy by increasing neuronal excitability. Interestingly, GIRK2 knockout mice showed reduced anxiety and increased motor activity in three anxiety tests (elevated cross maze, light-dark chamber, and canopy test) (Blednov et al., 2001). Ifenprodil showed an anxiolytic effect in MF1 mice in the elevated cross maze test, increasing their activity levels (Fraser et al., 1996), but no anxiolytic effect was observed in the light-dark chamber exploration test, and it had no effect on the motor activity of Wistar rats (Mikolajczak et al., 2003). This difference may be due to variations in the behavioral tests, including different ratios of light and dark compartments in the experimental setup and/or different animal species. A clinical study showed that ifenprodil can improve anxiety, reduced spontaneity, and depressive symptoms in patients with sequelae of cerebrovascular disease (Otomo et al., 1976). Therefore, the inhibitory effect of isifenprodil on neuronal GIRK channels may partially explain its clinical efficacy in treating anxiety and reduced activity, effects also observed in certain neuropsychiatric disorders. In the heart, acetylcholine opens atrial GIRK channels by activating M2 muscarinic acetylcholine receptors, ultimately leading to a bradycardia (Brown and Birnbaumer, 1990). In addition to its hypotensive effect, sinus tachycardia has been observed during isifenprodil treatment (Carron et al., 1971; Young et al., 1983; Yajima et al., 1987). Isifenprodil has no significant affinity for muscarinic acetylcholine receptors (Chenard et al., 1991). This study demonstrates that submicromolar concentrations or higher of isifenprodil inhibit cardiac GIRK1/4 channels, which are abundant in the atria (Krapivinsky et al., 1995). Therefore, in clinical practice, isofenprodil may also inhibit atrial GIRK channels. GIRK1 or GIRK4 knockout mice exhibit mild tachycardia (Bettahi et al., 2002). Furthermore, the hypotensive effect of isofenprodil may induce compensatory activation of the sympathetic nervous system, which plays a crucial role in the stimulation regulation of heart rate. In summary, our data suggest that sinus tachycardia during isofenprodil treatment may be partly related to the inhibition of atrial GIRK channels. Isofenprodil affects ethanol-related behavioral changes in animals, such as suppressing amnesia and withdrawal symptoms including seizures (Malinowska et al., 1999; Napiórkowska-Pawlak et al., 2000; Narita et al., 2000). Ethanol activates GIRK channels (Kobayashi et al., 1999; Lewohl et al., 1999). This study demonstrates that isifenprodil inhibits ethanol-induced GIRK1/2 currents. Interestingly, GIRK2 knockout mice exhibited ethanol-induced conditioned taste aversion and reduced conditioned place preference (Hill et al., 2003), and compared to wild-type mice, they were less sensitive to several acute ethanol effects, including anxiolytic effects, habitual motor excitation, and operation-induced seizures following acute ethanol administration (Blednov et al., 2001). In conclusion, isifenprodil may inhibit GIRK-related ethanol effects. Morphine is a commonly used, potent analgesic that preferentially binds to μ-opioid receptors and exerts a variety of pharmacological effects, including analgesia, euphoria, and dependence (Gutstein and Akil, 2001). μ-opioid receptors are coupled to G protein-mediated signaling pathways involving GIRK channels, adenylate cyclase, Ca2+ channels, and phospholipase C (Ikeda et al., 2002). Although morphine induces conditioned place preference in animals, suggesting a reward effect, pretreatment with ifenprodil inhibits this reward effect (Suzuki et al., 1999). However, ifenprodil has no significant affinity for opioid receptors (Chenard et al., 1991). This study indicates that ifenprodil inhibits G protein-mediated GIRK currents. Determining whether GIRK channel function is involved in the reward effect of morphine may be important. Interestingly, GIRK knockout mice exhibit reduced cocaine self-dosing (Morgan et al., 2003). In a clinical report, desipramine (a GIRK channel and norepinephrine transporter inhibitor) (Kobayashi et al., 2004b) promoted initial cocaine withdrawal (Gawin et al., 1989). Therefore, selective GIRK inhibitors may be potential treatments for cocaine abusers. Further research into the effects of ifenprodil on GIRK knockout mice may elucidate the mechanism of action of ifenprodil in morphine and cocaine addiction through GIRK-mediated action. [2]

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Multinucleus formation experiments confirmed that nilidil targets HA2-mediated membrane fusion by blocking pH-dependent conformational changes in HA. As mentioned above, the dependence of antiviral efficacy on viral subtype also provides further evidence for this hypothesis: nilidil, ifenprodil, and clenbuterol were effective against all tested A/H1N1 strains; in contrast, among the tested H3N2 strains, nilidil and ifenprodil had limited efficacy against only A/Hong Kong/8/1968 and A/Seoul/11/1988 strains (Tables 1 and 2). This observation is noteworthy because the antiviral efficacy of most HA2 fusion inhibitors, whether therapeutic antibodies or small molecule drugs, tends to depend on the viral subtype or HA group; group I includes H1, H2 and H5, while group II includes H3 and H7. The strain-specific activity of these compounds against the H3N2 subtype virus suggests that optimizing nylidrin through chemical modification may be a viable approach to identifying broad-spectrum fusion inhibitors. [4] Influenza A virus is one of the major human respiratory pathogens, causing seasonal epidemics and unpredictable periodic pandemics every year. Despite the clinical use of vaccines and antiviral drugs, the antigenic diversity and drug resistance of the virus make it a persistent threat to public health, highlighting the need to develop novel antiviral drugs. In a high-throughput screening based on cell culture, the β2-adrenergic receptor agonist nylidrin was identified as a compound against influenza A virus. The molecule is effective against a variety of H1N1 subtype isolates, but has limited activity against the H3N2 subtype, and the activity depends on the strain. By studying the antiviral activity of its chemical analogs, we found that ifenprodil and clenbuterol also had reliable inhibitory effects on influenza A H1N1 virus strains. Field-based pharmacophore models combined with comparisons of active and inactive compounds revealed the importance of the positive and negative electrostatic modes of phenylaminoethanol derivatives. Addition time experiments and visualization of nucleoprotein NP intracellular localization results showed that nilidil inhibited early steps in the viral life cycle. Finally, we found that nilidil targets hemagglutinin 2 (HA2)-mediated membrane fusion by blocking conformational changes of HA under acidic pH conditions. In mouse models, pre-incubation of mouse-adapted influenza A H1N1 virus with nilidil completely blocked intranasal viral infection. This study shows that nilidil can provide a core chemical framework for the development of inhibitors that act directly on influenza A virus invasion. [4]

C21H27NO2.1/2C4H6O6

800.98

325.204

C, 68.98; H, 7.55; N, 3.50; O, 19.97

23210-58-4

Ifenprodil glucuronide;66516-92-5

3689

White to off-white solid powder

493.5ºC at 760mmHg

178-180ºC

248.7ºC

0mmHg at 25°C

1.584

2

3

5

24

353

0

CC(C(C1=CC=C(C=C1)O)O)N2CCC(CC2)CC3=CC=CC=C3.CC(C(C1=CC=C(C=C1)O)O)N2CCC(CC2)CC3=CC=CC=C3.[C@@H]([C@H](C(=O)O)O)(C(=O)O)O

DMPRDSPPYMZQBT-CEAXSRTFSA-N

InChI=1S/2C21H27NO2.C4H6O6/c2*1-16(21(24)19-7-9-20(23)10-8-19)22-13-11-18(12-14-22)15-17-5-3-2-4-6-17;5-1(3(7)8)2(6)4(9)10/h2*2-10,16,18,21,23-24H,11-15H2,1H3;1-2,5-6H,(H,7,8)(H,9,10)/t;;1-,2-/m..1/s1

4-(2-(4-benzylpiperidin-1-yl)-1-hydroxypropyl)phenol; (2R,3R)-2,3-dihydroxysuccinate (2:1)

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 1 mg/mL (2.50 mM) (saturation unknown) in 1% DMSO + 99% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: 12.22 mg/mL (30.51 mM) in 0.5% CMC-Na/saline water (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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 (Please use freshly prepared in vivo formulations for optimal results.)