mGluR2/3

Pomaglumetad methionil (LY2140023) is a substrate of peptide transporter 1 (PEPT1) with a strong affinity, with a Km value of about 30 µM [2]. With an IC50 of 0.018 mM, LY2140023 exhibits activity against [14C]Gly-Sar[2].

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Inhibitory Effect of the Prodrug on PEPT1 [14C]Gly-Sar Transport. [2]
The inhibitory effect of Pomaglumetad methionil/LY2140023 on the PEPT1 probe substrate [14C]Gly-Sar (25 μM) was examined at concentrations ranging from 5 to 1000 μM. The accumulation of [14C]Gly-Sar in the absence of LY2140023 was used as the positive control. The passive diffusion of [14C]Gly-Sar at each concentration was measured by conducting parallel experiments in control cells transfected with a pcDNA3.1 empty vector and the values were subtracted from the accumulation in PEPT1-transfected HeLa cells. The estimated IC50 values of the prodrug in two separate experiments were 0.023 ± 0.09 and 0.013 ± 0.07 mM, respectively, with the mean value of 0.018 mM (Table 2).

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Evaluation of Prodrug and Active Moiety Uptake by PEPT1. [2]
The uptake of amino-acid prodrug [14C]LY2140023 (30 μM; prodrug) or the active drug [14C]LY404039 (30 μM; active moiety) was conducted at pH 6.0 or 7.5 in HeLa cells transiently transfected with PEPT1 to determine if Pomaglumetad methionil/LY2140023 or LY404039 were substrates of PEPT1 (Fig. 2). The level of accumulation of the active moiety in HeLa cells transfected with PEPT1 was similar to its passive accumulation, indicating that it was not transported by PEPT1. In contrast, as illustrated in Fig. 2, prodrug uptake was both a proton- and time-dependent process, suggesting that it was a PEPT1 substrate. Although the passive permeability of the prodrug, as indicated by uptake into the pcDNA3.1 empty vector, was slightly higher than that seen for the active moiety it is still negligible compared with the pH-dependent transport mediated by PEPT1.

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Determination of IC50 of known PEPT1 Inhibitors against the Prodrug. [2]
The inhibitory potentials of known PEPT1 substrates on [14C]LY2140023/Pomaglumetad methionil (prodrug) transport were determined using the dipeptide Gly-Sar as a positive control for inhibition of [14C]LY2140023 uptake. The potential to inhibit 10 μM [14C]LY2140023 uptake was determined for several known PEPT1 substrates; the range of mean IC50 values from two separate experiments on [14C]LY2140023 uptake was between 0.46 and 25.90 mM (Table 2), with valacyclovir being the most potent and l-DOPA being the least potent inhibitor. A similar rank order of IC50 potency was observed for both [14C]Gly-Sar and the prodrug, with the exception of cephalexin being a more potent inhibitor of prodrug uptake than captopril, whereas the reverse is true on the inhibition of Gly-Sar uptake (Table 2). The inhibitory potency of LY2140023 against Gly-Sar was more potent than any tested drugs.

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Evaluation of Clinical Implication using In vitro data. [2]
To inform possible choices of inhibitors for a clinical study of PEPT1-mediated interactions, in vitro IC50 of five marketed drugs (cefadroxil, cephalexin, captopril, enalapril, and valacyclovir) were compared with their estimated concentrations in the gastrointestinal (GI) tract (I2). These concentrations (I2) were obtained by dividing the recommended clinical dose by a volume of 250 ml (volume of glass of water) (Table 3). The doses of compounds were obtained from Physicians' Desk Reference (1997). The ratios of I2/IC50 were substantially less than one (i.e., the estimated GI concentrations were lower than the mean in vitro IC50 values) for captopril (0.03 to 0.15), cephalexin (0.18 to 0.37), and enalapril (0.008 to 0.03), but were greater than one (i.e., the estimated GI concentrations were greater than the in vitro IC50 values) for cefadroxil (2.10 to 4.20) and valacyclovir (12.04 to 24.11), indicating the potential for an interaction. The I2/IC50 for Pomaglumetad methionil/LY2140023 using 80 mg dose was 48.5 greater than the ratios for valacyclovir.

The dopamine metabolites homovanillic acid (HVA) and dihydroxyphenylacetic acid (DOPAC) are increased in a dose-dependent manner by Pomaglumetad methionil (LY2140023; oral; 3-300 mg/kg; once daily for 7 days) [1].

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Rationale: Accumulating evidence suggests that the primary symptoms of schizophrenia may be associated with altered central glutamate transmission. Pomaglumetad methionil/LY2140023 monohydrate is the methionine prodrug of the selective mGlu(2/3) receptor agonist LY404039 and is currently being assessed for the treatment of schizophrenia.
Objective: The objective of this study was to evaluate the central pharmacological activity of LY2140023 monohydrate in preclinical and clinical studies.
Methods: Effects on neurotransmitter/metabolite concentrations were assessed in male rats following single oral doses of Pomaglumetad methionil/LY2140023 monohydrate (microdiasylates from the prefrontal cortex), single intraperitoneal injection of LY404039 [cerebrospinal fluid (CSF)], or LY2140023 monohydrate dosed once daily for 7 days (CSF). A clinical study in 16 healthy subjects assessed the effects of LY2140023 monohydrate 40 mg orally twice daily for 14 days in lumbar CSF.
Results: Rat studies: Acute dosing with Pomaglumetad methionil/LY2140023 monohydrate resulted in significant dose-dependent increases in extracellular concentrations of dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), but not 5-hydroxyindoleacetic acid (5-HIAA), in the prefrontal cortex. LY2140023 monohydrate dosing for 7 days elevated the concentrations of HVA in CSF, while acutely dosed LY404039 increased the concentrations of DOPAC, HVA, and methoxy-hydroxyphenylglycol (MHPG), but not 5-HIAA. Clinical study: Significant increases were seen for DOPAC, HVA, 5-HIAA, and MHPG in the CSF of subjects receiving LY2140023 monohydrate, but not placebo.
Conclusions: Pomaglumetad methionil/LY2140023 monohydrate and/or LY404039 dosing potently affected dopamine turnover and also significantly affected serotonin turnover in the human and rat central nervous systems. The measurement of biogenic amine metabolites such as DOPAC and HVA may serve as useful biomarkers of LY2140023 monohydrate and/or LY404039 central pharmacodynamic activity. [1]

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A total of 24 healthy subjects, 8 men and 16 women, with a mean age of 31.5 years (range 19 to 66 years of age) enrolled in this study. Twelve (50.0%) subjects were Caucasians, 11 ([45.8%) were African American, and 1 (4.2%) was Asian. The mean body mass index was 26.6 kg/m2. Of the 24 subjects enrolled, 20 completed the study. The four subjects discontinued for the following reasons: failure to meet eligibility criteria (1 subject was discontinued before dosing), AE (1), and protocol violation Twenty-three subjects received 1000 mg valacyclovir alone, 21 subjects received 80 mg of Pomaglumetad methionil/LY2140023 alone, and 20 subjects received valacyclovir coadministered with LY2140023. [2]

Proton- and Time-Dependent Transport of the Prodrug and its Active Moiety. [1]
The PEPT1 mediated [14C]LY2140023 (30 μM) and [14C]LY404039 (30 μM) uptake was measured in cells 24 hours post transfection. The time-dependent transport of Pomaglumetad methionil/LY2140023 and LY404039 was conducted using buffers prepared at either pH 6.0 or 7.5 as described above. The cells transfected with empty pcDNA3.1 vector were used to measure the passive diffusion of LY2140023 and LY404039. The cells were incubated for 1, 2.5, 5, 7.5, 10, and 15 minutes at room temperature and were washed, lysed, and contents quantified as described above.

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Concentration-Dependent Uptake to Measure Kinetic Parameters of the Prodrug. [1]
The concentration dependent uptake of [14C]LY2140023/Pomaglumetad methionil, ranging from 5 up to 149 μM, was determined in PEPT1 transfected HeLa cells with 2- to 3-minute incubation at room temperature. The mean passive diffusion of prodrug at each concentration was obtained in parallel experiments in HeLa cells transfected with pcDNA3.1 empty vector and subtracted from the uptake mediated by PEPT1. The corrected data were fitted by WinNonlin Professional, version 5.0.1 or 5.3 (Certara, L.P., St. Louis, MO). The kinetic parameters of the prodrug mediated by PEPT1 were estimated by utilizing the following equation:

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Inhibition. [1]
An inhibition assay was conducted using 25 μM [14C]Gly-Sar (0.278 μCi/ml) as a probe substrate to assess if the uptake of [14C]Gly-Sar was inhibited by prodrug (5 to 1000 μM). The cells were incubated for 3 minutes in uptake buffer containing 25 μM [14C]Gly-Sar and different concentrations of Pomaglumetad methionil/LY2140023 at room temperature. The uptake was corrected for background determined by parallel experiment in cells transfected with pcDNA3.1 empty vector. Cell lysing and protein quantification were performed as described above.

Seven previously reported PEPT1 substrates (Zhang et al., 2004) and Gly-Sar, a PEPT1 probe substrate, were also assessed to determine their inhibitory potential on 10 μM [14C]LY2140023 (0.193 μCi/ml) uptake mediated by PEPT1. Two separate inhibition studies were conducted as described above using ALA (0.1 to 5 mM or 0.25 to 5 mM), captopril (0.5 to 40 mM), cefadroxil (0.3 to 10 mM), cephalexin (1 to 40 mM), enalapril (1 to 15 or 1 to 20 mM), l-DOPA (2.5 to 25 mM), Gly-Sar (0.1 to 5 or 0.25 to 5 mM), and valacyclovir (0.1 to 5 mM). The solubility of compounds at these concentrations was checked by using a fiber-optic light source. For L-DOPA, the concentration range for inhibition was limited by the solubility. Less than 50% inhibition of [14C]LY2140023 uptake was reached by 25 mM L-DOPA, thus IC50 was estimated using the inhibition of Gly-Sar at 20 mM as the complete inhibition of PEPT1 (complete inhibition was assumed at 20 mM, because IC50 value of Gly-Sar was 0.99 mM).

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Study drug measurement [1]
Concentrations of Pomaglumetad methionil/LY2140023 and LY404039 in human plasma and CSF were measured with validated liquid chromatographic/tandem mass spectrometry (LC/MS/MS) methods. The methods were validated and implemented in accordance with the established US FDA Guidance on bioanalytical method validation and sample analysis (CDER 2001). The lower and upper limits of quantification for both compounds were 0.5 and 100 ng/mL in CSF, and 1 and 100 ng/mL in plasma. Inter-day precision values for Pomaglumetad methionil/LY2140023 and LY404039 in CSF were 3.42% and 8.53% relative standard deviation (RSD), respectively, while the inter-day accuracy ranged from −5.12% to 1.06% relative error (RE) for LY2140023 and −6.30% to −2.71% RE for LY404039. In plasma, the respective inter-day precision values for LY2140023 and LY404039 were 7.13% and 8.61% RSD, respectively, while the inter-day accuracy ranges were from −2.69% to 5.71% RE and from 0% to 11.4% RE, respectively. Samples were mixed with isotopomeric internal standards followed by solid phase extraction to isolate the analytes. LC/MS/MS analysis was conducted in positive ion mode with selected reaction monitoring settings specific for each analyte and internal standard. Analytical runs consisted of calibration standards, quality control samples, and participant samples. Samples found to be above the upper limits of quantification were diluted and reanalyzed. Analytical runs that failed to meet a priori acceptance criteria for calibration curve and quality control sample accuracy were rejected and the affected samples were reanalyzed. Concentrations of LY404039 in rat plasma and CSF were measured using a similar LC/MS/MS technique

Animal/Disease Models: Male Fischer rat (approximately 250 g) [1]
Doses: 3 mg/kg, 10 mg/kg, and 300 mg/kg
Route of Administration: Oral; one time/day for 7 days
Experimental Results: Dose-dependent increase in dopamine Levels of the metabolites DOPAC and HVA.\n\nPreclinical methods [1]
\nThe vehicle for Pomaglumetad methionil/LY2140023 was distilled water; the vehicle for LY404039 was 0.01 N NaOH. Injection volumes were 5 mL/kg. Full descriptions of laboratory methods for each of the preclinical studies are provided in the ESM. Principles of laboratory animal care were followed in all preclinical studies.\n\nRat acute dosing/in vivo microdialysis study [1]
\nIn brief, male Sprague–Dawley rats (approximately 300 g at the time of the study) were implanted with a guide cannula in the prefrontal cortex (PFC) (Paxinos and Watson 1986). Rats were allowed to acclimate overnight. Prior to the experiment, a concentric-type dialysis probe was inserted and the animals were maintained in a chamber allowing free movement. The inlet tube of the dialysis probe was perfused with artificial CSF at a final flow rate of 1.5 μL/min. Following equilibration, the fluid from the outlet was collected into a refrigerated fraction collector. Three baseline samples were collected at 30-min intervals. Pomaglumetad methionil/LY2140023 monohydrate (3, 10, or 30 mg/kg) or vehicle control was administered orally by gavage 20 min into the third sampling interval. Dialysate fractions were collected at 30-min intervals for the next 4.5 h.\n

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\n\nRat CSF study [1]
\nPomaglumetad methionil/LY2140023 monohydrate was dosed orally once daily in male Fischer rats (Harlan Labs, Indianapolis, IN, approximately 250 g at the time of the study) for 7 days at 3, 10, and 300 mg/kg with sampling 3 h after last dose. LY404039 was dosed intraperitoneally in male Sprague–Dawley rats (Harlan Labs, approximately 250 g at the time of the study) 60 min before collection of CSF from the cisterna magna.\n

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\n\nStudy design [1]
\nThis was an exploratory, randomized, subject-blind, placebo-controlled study including 16 healthy male subjects. The study (H8Y-FW-HBBF) was conducted. \n\nSubjects were randomized 3:1 to receive either 40 mg of Pomaglumetad methionil/LY2140023 monohydrate or placebo orally BID for 14 consecutive days. Dosing occurred at the investigative site, at approximately the same time each day for each subject. On day 1, a baseline lumbar puncture (LP) was performed in the morning immediately prior to the administration of the first dose of study drug. A blood sample for the determination of LY2140023 and LY404039 concentrations was collected on day 14 approximately 2 h after the last dose. The second CSF sample was collected by LP on the morning of day 15, approximately 10–12 h post-dose. A safety follow-up visit occurred 2 to 14 days after day 15.\n\nThe study was terminated before completion for reasons unrelated to the study protocol. Therefore, the number of samples available for assay was smaller than expected because of the early termination. As such, a narrower panel of metabolites was assayed than originally intended.\n

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\n\nStudy drug [1]
\nAll treatments were administered orally as solutions with 200 mL of room temperature water. Pomaglumetad methionil/LY2140023 monohydrate was reconstituted in a 0.42% sodium bicarbonate solution (8 mg/mL). Placebo treatment was given as 5 mL of 0.42% sodium bicarbonate solution.\n\n

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\n\nClinical Study [2]
\n \nThe study was conducted as a three-period, fixed-sequence design in which subjects received a single dose of 1000 mg valacyclovir (Period 1), a single dose of 80 mg of Pomaglumetad methionil/LY2140023 (Period 2), and then a dose of 80 mg of LY2140023 coadministered with 1000 mg valacyclovir (Period 3); all periods were separated by a 5- to 10-day washout. Serial blood samples were collected for assessment of valacyclovir, acyclovir (refer to Ganapathy et al., 1998 for structure of valacyclovir and acyclovir), LY2140023, and LY404039 pharmacokinetics (PK). Urine was collected from 0 to 6, 6 to 12, and 12 to 24 hours postdose for analysis of valacyclovir, acyclovir, and/or the active moiety in the urine. Safety was assessed by collection of adverse events, clinical laboratory evaluations, electrocardiograms, and neurologic examinations.\n
\nEligible subjects were comprised of healthy men and women between 18 and 65 years of age, inclusive, with a body mass index between 19 and 32 kg/m2, inclusive. Subjects unable to cease use of xanthines, cigarettes, or over-the-counter or prescription medication for the duration of the trial were excluded. All subjects signed written informed consent before participation in the study.\n
\nA sufficient number of subjects were enrolled to obtain 18 subjects to complete the study. This sample size was to provide at least 90% power to show the inclusion of the 90% confidence intervals (CI) of the ratio of area under the curve (AUC) geometric means between the test (LY2140023 + valacyclovir) and reference (LY2140023 alone) fall within the interval (0.80, 1.25).\n

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\n\nBioanalysis. [2]
\nPlasma samples were analyzed for valacyclovir, acyclovir, Pomaglumetad methionil/LY2140023 (prodrug), and LY404039 (active moiety) using validated turbo ion spray liquid chromatography/tandem mass spectrometric methods. For the prodrug and active moiety, the lower limit of quantification (LLQ) was 0.25 ng/ml and the upper limit of quantification (ULQ) was 100 ng/ml for both analytes (Annes et al., 2015). For valacyclovir and acyclovir, the LLQ was 100 ng/ml and the ULQ was 1000 ng/ml for both analytes.\n
\nUrine samples were analyzed for valacyclovir, acyclovir, and/or active moiety using a validated liquid chromatography/tandem mass spectrometric method. No analysis of the prodrug was performed because previous studies have shown that it is not excreted in the urine. For the active moiety, the LLQ was 50 ng/ml and the ULQ was 5000 ng/ml. For valacyclovir and acyclovir, the LLQ was 100 ng/ml and the ULQ was 20,000 ng/ml.\n
\nFor all bioanalytical methods, samples above the limit of quantification were diluted and reanalyzed to yield results within the calibrated range.\n

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\n\nPharmacokinetic Analyses. [2]
\nPlasma concentration-time data for valacyclovir, acyclovir, Pomaglumetad methionil/LY2140023, and LY404039 were analyzed by standard noncompartmental methods of analysis using WinNonlin Version 5.3. Actual sampling times were used in the analyses with the exception of predose times, which were set to 0 hour. Area under the curve (AUC) values were determined using log-linear trapezoid methods. When calculating CL/F and Vz/F for the active moiety, the dose of the prodrug was adjusted based on the molar ratio of active moiety to prodrug (0.64).\n
\nUrine concentration and volume data were measured for LY404039, valacyclovir, and acyclovir. Amounts excreted over each collection interval were summed to determine the cumulative amount excreted over the 24-hour collection interval [Ae(0–24)]. The fraction of the dose excreted (fe) was also determined. For the active moiety dose, the 0.64 correction factor was used as described previously. Similarly, for acyclovir, the valacyclovir dose was adjusted based on the molar ratio of acyclovir to valacyclovir (0.694). Apparent renal clearance was estimated using the cumulative amount excreted up to the last collection interval and plasma AUC(0–24).\n
\nAlthough PK parameters were determined for all subjects with concentration-time data, if vomiting occurred within 5 hours postdose the concentration-time data and PK parameters from that dosing period were not included in any data summaries or statistical analysis. Only one subject (in Period 2) had PK data excluded because of vomiting.\n
\nThe primary PK parameters (Cmax and AUC) for Pomaglumetad methionil/LY2140023, LY404039, valacyclovir, and acyclovir were compared when the prodrug and valacyclovir were administered alone and in combination. AUC(0–∞) was used for all analytes except valacyclovir where AUC(0–3) was assessed. Parameters were compared using linear mixed effect model where treatment (80 mg of LY2140023 administered alone, 1000 mg valacyclovir administered alone, and 80 mg of LY2140023 coadministered with 1000 mg valacyclovir) was included as a fixed factor, and subject was a random factor. The parameters were log transformed before analysis. The least squares means (LSM) for each treatment and the 90% confidence intervals (CI) for the difference in means between test and reference treatment groups were estimated from the model and back transformed from the log scale to provide estimates of the geometric means and 90% CIs for the ratio of geometric means. The analysis of tmax was based on a nonparametric method. Medians and range for treatments and the P value computed for comparison of median values using Wilcoxon signed rank test are presented.\n

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\n\nSafety. [2]
\nThere were no serious adverse events (AEs) in this study. One subject discontinued from the study after experiencing a mild AE of urticaria that occurred approximately 4 hours after receiving 1000 mg of valacyclovir alone.\n
\nMost AEs were mild or moderate; one severe AE of headache occurred after valacyclovir alone. The most common AEs after prodrug alone were nausea, dizziness, somnolence, and headache. The AE profile for Pomaglumetad methionil/LY2140023 coadministered with valacyclovir was similar to LY2140023 alone.\n\n

Pharmacokinetic assessment [1]
Approximately 10–12 hours after administration, the mean cerebrospinal fluid concentrations of Pomaglumetad methionil/LY2140023 and LY404039 were 1.93 ng/mL and 4.93 ng/mL, respectively, and were quantifiable in all subjects.

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Pharmacokinetics. [2]
The active moiety rapidly formed after administration of the prodrug and was detectable in the first sample of 18 out of 21 subjects, consistent with previous clinical studies.
Mean pharmacokinetic parameters (Table 4) and curves (Figure 4A) were similar after administration of Pomaglumetad methionil/LY2140023 alone and in combination with valacyclovir. The least squares mean (LSM) ratios of Cmax and AUC were close to 1, with confidence intervals ranging from 0.80 to 1.25 (Table 5). Similarly, the plasma pharmacokinetic parameters (Table 4) and curves (Figure 4B) of LY404039 (active ingredient) were similar after administration of the prodrug alone and in combination with valacyclovir. Urinary excretion of the active ingredient was also similar after administration of the prodrug alone and in combination with valacyclovir (Table 4), measured as excretion fraction (fe; 0.651 and 0.595, respectively) and renal clearance (CLr; 12.8 and 12.2 L/h, respectively). The LSM ratios of Cmax and AUC were also close to 1, with confidence intervals ranging from 0.80 to 1.25 (Table 5). Analysis of the time to peak concentration (tmax) for the prodrug and active ingredient showed no significant difference (median difference for paired prodrug administration was 0.00 h, and for the active ingredient it was -0.07 h; Supplementary Table 1).
Valacyclovir plasma concentrations are limited, typically detectable only within 3 to 4 hours after administration (Phan et al., 2003), due to the rapid and efficient conversion of valacyclovir (prodrug) to acyclovir (active metabolite). The mean plasma concentration profiles of valacyclovir are similar whether administered alone or in combination with the prodrug (Figure 5A). As shown in Table 6, plasma pharmacokinetic parameters and renal clearance of valacyclovir are similar whether administered alone or in combination with the prodrug. The least squares mean (LSM) ratio of Cmax to AUC is close to 1, with confidence intervals (CI) ranging from 0.80 to 1.25 (Table 5), but the lower limit of the 90% CI for AUC(0–3) is 0.71.
The active metabolite acyclovir is rapidly generated after administration of the prodrug valacyclovir. Plasma pharmacokinetics and renal clearance of acyclovir are similar whether administered alone or in combination with the prodrug (Table 6). Similarly, the plasma concentration profiles of acyclovir were similar regardless of prodrug administration (Figure 5B). The LSM ratios of AUC and Cmax for acyclovir were close to 1, with 90% CIs ranging from 0.80 to 1.25 (Table 5). tmax analysis of valacyclovir and acyclovir showed no difference in tmax (median paired difference for valacyclovir was 0.00 h, median paired difference for acyclovir was 0.00 h; Supplementary Table 1).

Safety[1]
No deaths or other serious adverse events occurred during the study; however, three subjects (two in the placebo group and one in the LY group) were discontinued by the investigators due to post-dural headache (PDPH). The most common adverse events that occurred during treatment that were likely related to the study treatment were nausea, vomiting, dizziness, and fatigue, which were generally mild to moderate in severity (Table 4). Seven subjects experienced procedural complications related to PDPH. Three of them (two in the placebo group and one in the Pomaglumetad methionil/LY2140023 group) were discontinued by the investigators due to PDPH. All PDPHs were completely resolved with conservative treatment without epidural patch treatment.

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Safety of Pomaglumetad methionil/LY2140023 in clinical studies[1]
The LY2140023 monohydrate was generally well tolerated, and no clinically significant safety or tolerability issues related to LY2140023 were observed in this study. The reported potentially drug-related adverse events were similar to those observed in previous clinical studies of LY2140023 monohydrate. Procedure-related complications such as post-puncture headache (PDPH) may occur during lumbar puncture. Overall, the safety and tolerability of the lumbar puncture procedure used in this study were comparable to previous experience.

[1]. Effects of a novel mGlu₂/₃ receptor agonist prodrug, LY2140023 monohydrate, on central monoamine turnover as determined in human and rat cerebrospinal fluid. Psychopharmacology (Berl). 2012 Feb;219(4):959-70.

[2]. In Vitro and Clinical Evaluations of the Drug-Drug Interaction Potential of a Metabotropic Glutamate 2/3 Receptor Agonist Prodrug with Intestinal Peptide Transporter 1. Drug Metab Dispos. 2017 Feb;45(2):137-144.

LY2140023 is an investigational drug developed by Eli Lilly and Company, designed to provide a new treatment option for schizophrenia. LY2140023 is an oral "prodrug," meaning it is not biologically active on its own and is metabolized after administration to produce the active mGlu2/3 receptor agonist LY404039. Most approved antipsychotics currently operate by affecting the neurotransmitters dopamine or serotonin. The active ingredient in LY2140023, LY404039, is believed to work by reducing the presynaptic release of another neurotransmitter—glutamate—in areas of the brain where mGlu2/3 receptors are expressed. Further studies are currently being planned or conducted to further understand the safety and efficacy of LY2140023, including determining its optimal therapeutic dose.

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Drug Indications
Studied for the treatment of psychosis, schizophrenia, and schizoaffective disorder.

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Mechanism of Action
LY2140023 is an antipsychotic drug belonging to the class of metabolized glutamate receptor 2/3 agonists.
This drug possesses a novel mechanism of action, effectively treating schizophrenia and potentially other neuropsychiatric disorders. Upon absorption, LY2140023 is efficiently hydrolyzed to generate the active mGlu2/3 receptor agonist LY404039. LY404039, along with other mGlu2/3 agonists, does not directly interact with dopamine or serotonin (5-HT2A) receptors. However, the "functional" 5-HT2A receptor antagonism in the prefrontal cortex may represent a common mechanism between clinically effective atypical antipsychotics and mGlu2/3 receptor agonists, and may contribute to the antipsychotic effects of LY2140023.

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Pomaglumetad methionil/The effects of LY2140023 monohydrate and LY404039 on biogenic amine neurotransmitters and their metabolites[1]
This report shows that the mGlu2/3 agonist products LY2140023 and/or LY404039 increased the turnover of the neurotransmitter DA in the central nervous system (CNS), as evidenced by elevated levels of the metabolites DOPAC and HVA in rat brain dialysis fluid and cerebrospinal fluid samples from rats and humans. These changes also appear to be associated with plasma/cerebrospinal fluid drug concentrations of LY404039. Therefore, cerebrospinal fluid levels of these metabolites may serve as effective biomarkers of the central pharmacodynamic activity of LY2140023 and/or LY404039.
In the preclinical studies described herein, both LY404039 and its prodrug LY2140023 increased the turnover of biogenic amines in the rat central nervous system. Therefore, in vivo, administration of LY2140023 monohydrate resulted in a dose-dependent increase in extracellular levels of DOPAC and HVA in the microdialysis fluid of the prefrontal cortex (Figure 1), without a significant effect on 5-HIAA levels. These changes are consistent with recently published microdialysis data from LY404039 rats, which observed elevated extracellular DA levels in the prefrontal cortex (Rorick-Kehn et al., 2007). That study also found elevated DOPAC and HVA levels in postmortem brain tissue. In this study, we observed a similar effect in rat cerebrospinal fluid, where a single injection of LY404039 led to a dose-dependent increase in DOPAC, HVA, and the NE metabolite MHPG levels. A similar trend was observed after 7 consecutive days of administration of the prodrug LY2140023 monohydrate, but the effect was weaker, with a statistically significant increase in HVA levels only observed at a dose of 10 mg/kg. Importantly, the effects of LY2140023 and LY404039 on biogenic amine turnover in rat microdialysis fluid and cerebrospinal fluid are consistent with the range of efficacy shown in a phencyclidine-induced rat model of schizophrenia (Patil et al., 2007b). In the clinical study reported in this study, LY2140023 increased the concentrations of DOPAC and HVA, as well as the serotonin metabolite 5-HIAA, in human cerebrospinal fluid. It is known that the interpretation of concentrations of lumbar neurotransmitters and their metabolites is limited by factors such as the head-tail gradient. However, combined with preclinical data and drug concentration effects observed in preclinical and clinical studies, these observations suggest that administration of the LY2140023 monohydrate also increases the turnover of central dopaminergic and serotonergic amines in the human brain. Although a significant increase in the concentration of the NE metabolite MHPG was also observed, no statistically significant increase in DHPG concentration was observed in cerebrospinal fluid; therefore, no definitive conclusions can be drawn regarding the effect of LY2140023 on central NE metabolism. The limited sample size in this study may have prevented a full determination of the effects of the two NE metabolites on norepinephrine metabolism based solely on measurements. The observed effect of LY2140023 monohydrate on the concentration of biogenic amine metabolites in human cerebrospinal fluid occurred at doses previously shown to have clinical efficacy in patients with schizophrenia (Patil et al., 2007b); therefore, the effect of LY2140023 monohydrate on biogenic amine metabolites in cerebrospinal fluid was observed at therapeutically significant doses. In this study, no significant changes were observed in the assessed concentrations of biogenic amine metabolites in the cerebrospinal fluid of the placebo group; however, due to the limited number of subjects in the placebo group, a definitive comparison between the active compound group and the placebo group was not possible. Nevertheless, prior to this clinical study, we conducted a methodological study without drug treatment to evaluate experimental techniques and identify potential sources of analytical, biological, and experimental variability in the detection of cerebrospinal fluid analytes (Patil et al., 2007a). We established analytical methods and determined the expected dynamic range and limit of quantitation for the endogenous concentration of each analyte. Cerebrospinal fluid (CSF) was obtained by lumbar puncture in healthy subjects at baseline and 2 weeks post-baseline. We analyzed the same biogenic amines and their metabolites in the CSF samples as in this study. At 2 weeks post-baseline, the ratios of DOPAC, HVA, DHPG, and MHPG in CSF compared to baseline ranged from 1.01 to 1.02, and the ratios of L-DOPA, 5-HTP, and 5-HIAA ranged from 1.11 to 1.17. Therefore, the biostability of monoamines and their metabolites in human CSF is sufficient to support studies of the effects of drug or placebo treatment.
Overall, these clinical study results are the first to demonstrate the effects of metabolized glutamate 2,3 receptor agonists on the human biogenic amine system. The exact mechanism by which LY2140023 monohydrate increases dopaminergic and serotonergic metabolism remains unclear. However, the results of clinical studies are consistent with preclinical results for this drug and other mGlu2/3 agonists, including published microdialysis and brain tissue data for LY404039 (Rorick-Kehn et al., 2007) and the mGlu2/3 agonist LY379268 (Cartmell et al., 2000). Therefore, in human and preclinical studies, doses of LY2140023 monohydrate (and/or LY404039) that have been found to induce changes in biogenic amine metabolites in cerebrospinal fluid (and/or microdialysis fluid) are comparable to doses that have shown efficacy in schizophrenia or animal models of schizophrenia.
Similar results obtained in rat and human studies using different analytical techniques (GC/MS/MS and HPLC/EC) and distinctly different biological matrices (rat interstitial fluid dialysate, rat cisterna magna cerebrospinal fluid, and human lumbar cerebrospinal fluid) further support the view that the observed changes in neurotransmitter metabolite concentrations in human lumbar cerebrospinal fluid do indeed indicate central clinical pharmacodynamic effects. Following LY2140023 monohydrate treatment, elevated levels of biogenic amines in rodents (DOPAC and HVA) and healthy subjects (DOPAC, HVA, and 5-HIAA) suggest that LY2140023 may significantly increase the metabolic turnover of dopamine and/or serotonin in the prefrontal cortex (PFC), a finding consistent with preclinical studies of atypical antipsychotics, including olanzapine (Li et al. 1998). However, unlike atypical antipsychotics, LY2140023 and its active ingredient LY404039 do not directly interact with dopamine D2 receptors (Fell et al. 2008). Therefore, it is speculated that its effect on the metabolic turnover of biogenic amine metabolites is indirectly mediated by altering glutamate release. Although LY404039 can increase the concentration of MHPG in cerebrospinal fluid, no statistically significant change in DHPG concentration in cerebrospinal fluid was observed, thus a definitive conclusion cannot be drawn regarding the effect of LY2140023 on NE metabolism. In this study, after 14 consecutive days of twice-daily administration of LY2140023 monohydrate, the concentrations of Pomaglumetad methionil/LY2140023 and LY404039 in cerebrospinal fluid and plasma were consistent with previously reported results from studies of healthy subjects taking a 40 mg dose (data not shown). Furthermore, the concentrations of the active compound LY404039 in both cerebrospinal fluid (CSF) and plasma increased linearly with increasing HVA concentrations in CSF. A similar trend was observed in rats, where both CSF and plasma LY404039 concentrations increased with increasing HVA and DOPAC concentrations. In summary, the association between biogenic amine concentrations and drug levels further supports the view that the observed pharmacodynamic changes are due to the activation of metabolized glutamate receptors 2,3 by the active ingredient LY404039 of LY2140023 monohydrate. Moreover, the relationship between central pharmacodynamic parameters and peripheral LY404039 levels suggests that plasma LY404039 concentrations may predict central pharmacokinetic and pharmacodynamic effects. If future studies confirm this, the measurement of plasma LY404039 may be sufficient to reflect central pharmacokinetic and pharmacodynamic activity, the latter based on the turnover of biogenic amine neurotransmitters. Further research is needed to assess this possibility and determine its relationship with clinical outcomes (if such a relationship exists). In summary, Pomaglumetad methionil/LY2140023 and/or LY404039 increase the metabolic turnover of dopamine (DA) in the central nervous system and may also increase the metabolic turnover of serotonin (5-HT), reflected in elevated concentrations of these neurotransmitter metabolites in cerebrospinal fluid. The effects of LY2140023 and/or LY404039 on these biomarkers are thought to be mediated by mGlu2/3 receptors; therefore, measurements of DA and 5-HT metabolites may serve as effective transconverting biomarkers of the central pharmacodynamic activity of LY2140023 and/or LY404039. The lack of significant affinity of LY2140023 or LY404039 for dopamine and serotonin receptors suggests that their effects on dopamine and serotonin metabolism are likely mediated through indirect mechanisms. [1] Although peptide transporter 1 (PEPT1) is responsible for the bioavailability of a variety of drugs, its potential role in drug interactions is poorly understood. The prodrug Pomaglumetad methionil, a metabolite glutamate receptor 2/3 agonist, utilizes PEPT1 to enhance absorption and bioavailability. In vitro studies have been conducted to guide decisions on conducting clinical drug interaction studies and to inform clinical study design. In vitro studies identified the prodrug (Pomaglumetad methionil/LY2140023 monohydrate) as a substrate of PEPT1 with a Km value of approximately 30 µM, while the active moiety (LY404039) was not a substrate of PEPT1. Furthermore, among eight known PEPT1 substrates evaluated in vitro, valacyclovir was the most potent inhibitor of PEPT1-mediated prodrug uptake (IC50 = 0.46 mM). Therefore, a clinical drug interaction study was conducted to assess the potential interaction between the prodrug and valacyclovir in healthy subjects. The results showed that the combination therapy had no effect on the pharmacokinetics of the prodrug, valacyclovir, and their active fraction. Although in vitro studies have shown that the prodrug and valacyclovir may interact through PEPT1, no interaction between the two drugs was found in in vivo studies. Because PEPT1 has a high capacity and is abundantly expressed in the gut, it appears to be difficult to saturate. Therefore, even compounds with high affinity for this transporter are unlikely to have clinical interactions with PEPT1. [2]

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Subsequently, a clinical study was conducted to evaluate the role of Pomaglumetad methionil/LY2140023 as a substrate and inhibitor of PEPT1. The combination administration of LY2140023 with valacyclovir did not affect the pharmacokinetics of either drug or their respective active fractions (LY404039 or acyclovir), indicating that there was no clinical drug interaction between the prodrug and valacyclovir. The data also showed that the presence of either the prodrug or valacyclovir did not affect the conversion of LY2140023 or the prodrug of valacyclovir to its active moiety. The lack of interaction between the prodrug and valacyclovir during their conversion to their respective active moieties is expected, as different enzymes are responsible for their activation. Dehydropeptidase 1 has been shown to cleave the prodrug into its active moiety (Moulton et al., 2015), while valacyclovirase (a biphenyl hydrolase-like protein) cleaves valacyclovir into acyclovir (Marsillach et al., 2014). Neither the CL/F nor CLr of the two active moieties changed, indicating that co-administration of the drugs does not affect their renal clearance. Furthermore, a change in Tmax value might be observed if a weak interaction with PEPT1 were present. However, no change in Tmax value was observed in any of the entities studied. Additionally, the AUC (0–3 h) of valacyclovir did not change significantly, again indicating the absence of interaction at PEPT1.
In this study, we illustrate how in vitro studies can guide the design of clinical drug interaction studies based on transporter-based drug interactions. Screening multiple drugs competing for the transporter in vitro can determine the order of inhibitory efficacy and analyze potential drug interactions in relation to the oral dose of the compound. Thus, unnecessary in vivo studies can be avoided while focusing on the most relevant potential drug interactions. Although in vitro studies suggest that there may be drug interactions between the prodrug and valacyclovir, based on other intestinal transporter guidelines, in vivo drug interaction studies have shown that the two drugs do not interact through PEPT1. Therefore, our results clearly demonstrate that even novel molecular entities with high affinity for the transporter are unlikely to have clinical drug interactions at PEPT1. [2]

C12H20N2O8S2

384.425801277161

384.066

956385-05-0

Pomaglumetad methionil hydrochloride;635318-26-2;Pomaglumetad methionil anhydrous;635318-55-7

56842185

Typically exists as solid at room temperature

0.691

5

10

7

24

644

5

S1(C[C@@](C(=O)O)([C@@H]2[C@@H](C(=O)O)[C@H]12)NC([C@H](CCSC)N)=O)(=O)=O.O

HQMAHCKDJKNYEK-LBMFEJOUSA-N

InChI=1S/C12H18N2O7S2.H2O/c1-22-3-2-5(13)9(15)14-12(11(18)19)4-23(20,21)8-6(7(8)12)10(16)17;/h5-8H,2-4,13H2,1H3,(H,14,15)(H,16,17)(H,18,19);1H2/t5-,6+,7+,8-,12-;/m0./s1

(1R,4S,5S,6S)-4-[[(2S)-2-amino-4-methylsulfanylbutanoyl]amino]-2,2-dioxo-2λ6-thiabicyclo[3.1.0]hexane-4,6-dicarboxylic acid;hydrate

Pomaglumetad methionil; LY2140023 Monohydrate; Pomaglumetad methionil [USAN]; UNII-WE665JB15R; pomaglumetad metionilo; WE665JB15R; LY-2140023 Monohydrate; ...; 956385-05-0;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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