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]. LY2140023 has an IC50 value of 0.018 mM and is effective 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 oral dopamine metabolites dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) are dose-dependently increased by pomaglumetad methionil (LY2140023; 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, 10 and 300 mg/kg
Route of Administration: Oral; one time/day for 7 days
Experimental Results: Dose-dependent increase in dopamine metabolites DOPAC and HVA level. Preclinical methods [1]
The 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. Rat acute dosing/in vivo microdialysis study [1]
In 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.

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Rat CSF study [1]
Pomaglumetad 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.

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Study design [1]
This was an exploratory, randomized, subject-blind, placebo-controlled study including 16 healthy male subjects. The study (H8Y-FW-HBBF) was conducted. Subjects 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. The 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.

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Study drug [1]
All 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.

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Clinical Study [2]
The 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.
Eligible 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.
A 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).

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Bioanalysis. [2]
Plasma 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.
Urine 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.
For all bioanalytical methods, samples above the limit of quantification were diluted and reanalyzed to yield results within the calibrated range.

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Pharmacokinetic Analyses. [2]
Plasma 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).
Urine 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).
Although 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.
The 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.

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Safety. [2]
There 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.
Most 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.

Pharmacokinetic evaluations [1]
Mean CSF concentrations at approximately 10–12 h post-dose were 1.93 and 4.93 ng/mL for Pomaglumetad methionil/LY2140023 and LY404039, respectively, and were quantifiable for all subjects.

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Pharmacokinetics. [2]
After administration of the prodrug, the active moiety was formed rapidly and was present at the first sampling time in 18 of 21 subjects, which is consistent with previous clinical studies.
Mean PK parameters (Table 4) and profiles (Fig. 4A) were similar after dosing of Pomaglumetad methionil/LY2140023 (the prodrug) alone and when coadministered with valacyclovir. Ratios of LSM for Cmax and AUC resulted in ratios that were close to 1 with confidence intervals contained within the 0.80 to 1.25 range (Table 5).
Similarly, the plasma PK parameters (Table 4) and profiles (Fig. 4B) for LY404039 (the active moiety) were similar after dosing of the prodrug alone and when coadministered with valacyclovir. Urinary excretion of the active moiety was also similar for the prodrug alone and with valacyclovir (Table 4), as measured by the fraction excreted (fe; 0.651 and 0.595, respectively) and renal clearance (CLr; 12.8 and 12.2 l/h, respectively). Ratios of LSM for Cmax and AUC were also close to 1, and the CIs were contained within 0.80 and 1.25 (Table 5). The tmax analysis for prodrug and active moiety showed no significant differences observed for tmax (median of paired differences was 0.00 hour for prodrug and −0.07 hour for active moiety; Supplemental Table 1).
The valacyclovir plasma concentrations are limited and typically only measurable for 3 or 4 hours postdose (Phan et al., 2003), because conversion from valacyclovir (prodrug) to acyclovir (active metabolite) is rapid and efficient. Mean plasma profiles of valacyclovir were similar whether administered alone or with prodrug (Fig. 5A). As shown in Table 6, the valacyclovir plasma PK parameters and renal clearance were similar when administered alone or with the prodrug. Ratios of LSM for Cmax and AUC were near 1 with CIs contained within the 0.80 to 1.25 range (Table 5), with the exception of the lower bound of the 90% CI for AUC(0–3), which was 0.71.
After administration of the prodrug valacyclovir, the active metabolite acyclovir was formed rapidly. The acyclovir plasma PK and renal clearance were similar when administered alone or with the prodrug (Table 6). Similarly, the plasma profiles for acyclovir were similar with or without administration of the prodrug (Fig. 5B). The ratios of LSM for AUC and Cmax were close to 1, and the 90% CI were within the 0.80 to 1.25 range for acyclovir (Table 5). The tmax analysis for valacyclovir and acyclovir showed no differences observed for tmax (median of paired differences was 0.00 hour for valacyclovir and 0.00 hour for acyclovir; Supplemental Table 1).

Safety [1]
No deaths or other serious adverse events occurred during this study; however, three subjects (two placebo- and one LY-treated) were withdrawn from the study by the investigator due to post-dural puncture headache (PDPH). The most common treatment-emergent adverse events possibly related to study treatment were nausea, vomiting, dizziness, and fatigue and were generally mild to moderate in severity (Table 4). Seven subjects experienced procedural complications involving PDPH. Of these, three (two placebo- and one Pomaglumetad methionil/LY2140023-treated) were withdrawn from the study by the investigator due to PDPH. The occurrences of PDPH were resolved completely with conservative management, and no epidural patches were performed for the treatment of PDPH.

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Safety of Pomaglumetad methionil/LY2140023 in the clinical study [1]
LY2140023 monohydrate was generally well tolerated, with no clinically significant safety or tolerability issues related to LY2140023 noted in this study. Adverse events reported as possibly drug-related were similar to those observed in previous clinical studies with LY2140023 monohydrate. Procedure-related complications like PDPH can occur with lumbar puncture. Overall, the safety and tolerability of the LP procedures used in this study appeared comparable to prior 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 from Lilly, which is being developed as a new treatment option for schizophrenia. LY2140023 is an oral "prodrug," meaning it is devoid of intrinsic biological activity and, once administered, is metabolized to provide the active mGlu2/3 receptor agonist called LY404039. Most currently approved antipsychotic medications work by affecting the neurotransmitters dopamine or serotonin. For LY2140023, the active substance, LY404039, is thought to work by reducing the presynaptic release of another neurotransmitter, glutamate, in brain regions where mGlu2/3 receptors are expressed. Further studies are planned or are ongoing to learn more about the safety and effectiveness, including determining an optimal therapeutic dose for LY2140023.
See also: Pomaglumetad Methionil (annotation moved to).

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Drug Indication
Investigated for use/treatment in psychosis and schizophrenia and schizoaffective disorders.

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Mechanism of Action
LY2140023 is an antipsychotic agent that is a metabotropic glutamate 2/3 receptor agonist. This agent has a new mechanism of action that is efficacious in treating schizophrenia and potentially other neuropsychiatric conditions. Once absorbed, LY2140023 is efficiently hydrolyzed to produce the active mGlu2/3 receptor agonist LY404039. LY404039 and other mGlu2/3 agonists do not directly interact with dopamine or serotonin (5-HT2A) receptors. However, 'functional' 5-HT2A receptor antagonism in the prefrontal cortex may represent a common mechanism shared by clinically effective atypical antipsychotics and mGlu2/3 receptor agonists, and may contribute to the antipsychotic actions of LY2140023.

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Effects of Pomaglumetad methionil/LY2140023 monohydrate and LY404039 on biogenic amine neurotransmitters and metabolites [1]
The present report shows that the mGlu2/3 agonist produg LY2140023 and/or LY404039 increased the CNS turnover of, in particular, the neurotransmitter DA as evidenced by the elevated levels of the metabolites DOPAC and HVA in rat brain dialysates and both rat and human CSF samples. These changes also appear to correlate with the plasma/CSF drug concentrations of LY404039. Thus, the CSF levels of these metabolites may serve as useful translational markers of LY2140023 and/or LY404039 central pharmacodynamic activity.

In the preclinical studies described herein, LY404039 and the prodrug LY2140023 both increased biogenic amine turnover in the CNS of the rat. Thus, LY2140023 monohydrate administration produced dose-dependent increases in the extracellular levels of both DOPAC and HVA (Fig. 1) in vivo in the prefrontal cortical microdialysates with no major effects on the levels of 5-HIAA. These changes were in line with recently published rat microdialysis data for LY404039 where increased levels of extracellular DA were observed in the prefrontal cortex (Rorick-Kehn et al. 2007); that study also found increased levels of DOPAC and HVA in brain tissues postmortem. In the present study, we found similar effects in the rat CSF where single doses of LY404039 produced dose-dependent increases in the levels of DOPAC and HVA, and also the NE metabolite MHPG. After dosing with the prodrug LY2140023 monohydrate for 7 days, a similar trend was observed, but the effect was less pronounced with statistically significant elevations in HVA levels observed at 10 mg/kg. Importantly, the observed effects of LY2140023 and LY404039 on biogenic amine turnover in rat microdialysate and CSF were within the range of that which demonstrated efficacy in a rat phencyclidine model of schizophrenia (Patil et al. 2007b).

In the clinical study reported herein, LY2140023 increased human CSF concentrations of DOPAC and HVA, as well as the serotonin metabolite 5-HIAA. The interpretation of lumbar concentrations of neurotransmitters and their metabolites is known to be limited by factors such as the craniocaudal gradient. However, in combination with data from the preclinical studies and the observed drug concentration effects both preclinically and clinically, these observations suggest that LY2140023 monohydrate administration also increases central dopaminergic and serotonergic turnover in humans. While significant increases in the concentrations of the NE metabolite MHPG were also observed, the lack of a statistically significant increase in DHPG concentrations in CSF precludes a clear conclusion regarding the effect of LY2140023 on central NE turnover. It is possible that the study was not sufficiently powered to adequately define the effects on noradrenergic turnover based on measurements of both NE metabolites. The observed effects of LY2140023 monohydrate on human CSF concentrations of biogenic amine metabolites occurred at a dose previously shown to be clinically efficacious in patients with schizophrenia (Patil et al. 2007b); therefore, the effects of LY2140023 monohydrate on biogenic amine metabolites in CSF were observed at a therapeutically relevant dose.

In the present study, there were no significant changes in the concentrations of the biogenic amine metabolites assessed in the CSF of the placebo-treated subjects; however, due to the limited number of placebo-treated subjects in the present study, a definitive comparison between the active compound and placebo groups cannot be made. Nevertheless, prior to conducting the present clinical study, a methodological study absent of drug treatment had been performed to evaluate experimental techniques and identify possible sources of analytical, biological, and experimental variability that might occur during the assessment of analytes in the CSF (Patil et al. 2007a). Analytical methods were established with quantitative dynamic ranges and limits of quantification for the anticipated endogenous concentrations of each analyte. Cerebrospinal fluid was obtained by lumbar puncture at baseline and 2 weeks after baseline in healthy subjects. Aliquots of CSF were analyzed for the same biogenic amines and metabolites evaluated in the current study. The ratios of CSF monoamine and metabolite concentrations determined 2 weeks after baseline compared with baseline ranged from 1.01 to 1.02 for DOPAC, HVA, DHPG, and MHPG and from 1.11 to 1.17 for L-DOPA, 5-HTP, and 5-HIAA. Therefore, the temporal biological stability of monoamines and monoamine metabolites in human CSF was considered sufficient to support the investigation of drug or placebo treatment effect.

Overall, the results of the clinical study provide the first demonstration of an effect of an agonist at metabotropic glutamate 2,3 receptors on biogenic amine systems in humans. The exact mechanism whereby LY2140023 monohydrate increases dopaminergic and serotonergic turnover is unknown. However, the results from the clinical study are consistent with preclinical studies with this 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 both humans and in the preclinical setting, the doses of LY2140023 monohydrate (and/or LY404039) that were found to induce biogenic amine metabolite changes in the CSF (and/or microdialysate) were comparable to doses that were associated with evidence of efficacy in either schizophrenia or an animal model of schizophrenia.

The similarity of result from the studies in rats and humans using varied analytical techniques (GC/MS/MS versus HPLC/EC) and distinctly different biological matrices (dialysates from brain parenchymal interstitial fluid in rats, cistern magna CSF in rats, and lumbar CSF in humans) further supports the notion that the observed changes in human lumbar CSF neurotransmitter metabolite concentrations are indeed indicative of a central clinical pharmacodynamic effect. The LY2140023 monohydrate treatment-associated increases in biogenic amines in rodents (DOPAC and HVA) and healthy subjects (DOPAC, HVA, and 5-HIAA) suggest that LY2140023 may particularly increase dopamine and possibly serotonin turnover in the PFC, a finding observed in preclinical studies with atypical antipsychotics including olanzapine (Li et al. 1998). Unlike atypical antipsychotics, however, LY2140023 and the active agent LY404039 do not directly interact with the dopamine D2 receptor (Fell et al. 2008). The observed effects on biogenic amine metabolite turnover are therefore presumed to be mediated indirectly via changes in glutamate release. While there was an increase in CSF MHPG with LY404039, no clear conclusions can be made regarding the effect of LY2140023 on NE turnover as no statistical significant change in CSF DHPG concentrations was observed.

CSF and plasma Pomaglumetad methionil/LY2140023 and LY404039 concentrations following 14 days of LY2140023 monohydrate BID administration in the present study were within the same range of concentrations previously reported in studies of healthy subjects administered 40-mg doses (data not shown). Furthermore, individual concentrations of the active compound LY404039 in both CSF and plasma increased linearly with increasing CSF concentrations of HVA. A similar trend for increasing concentrations of HVA and DOPAC was seen in the rat, both for CSF and plasma concentrations of LY404039. Taken together, the association of biogenic amine concentrations with drug levels further supports the notion that the observed pharmacodynamic changes are in response to metabotropic glutamate 2,3 activation by LY404039, the active component of LY2140023 monohydrate. Furthermore, the relationship between central pharmacodynamic markers and peripheral LY404039 levels suggest that plasma LY404039 concentrations may predict central pharmacokinetic and pharmacodynamic effects. If confirmed in future studies, plasma LY404039 determinations may suffice to reflect central pharmacokinetic and central pharmacodynamic activity, with the latter based on biogenic amine neurotransmitter turnover. Further studies are required to evaluate this possibility and define the relationship to clinical outcome, if such a relationship exists.

In conclusion, Pomaglumetad methionil/LY2140023 and/or LY404039 increased CNS DA turnover and possibly also increased 5-HT turnover, as reflected by the increased CSF concentrations of the metabolites derived from these neurotransmitters. The effects of LY2140023 and/or LY404039 on these markers are presumed to be mGlu2/3 receptor-mediated, so the measurement of DA and 5-HT metabolites may serve as useful translational markers of LY2140023 and/or LY404039 central pharmacodynamic activity. The lack of any significant affinity of LY2140023 or LY404039 for dopamine and serotonin receptors suggests that the impact on DA and 5-HT turnover is likely mediated through an indirect mechanism. [1]

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Despite peptide transporter 1 (PEPT1) being responsible for the bioavailability for a variety of drugs, there has been little study of its potential involvement in drug-drug interactions. Pomaglumetad methionil, a metabotropic glutamate 2/3 receptor agonist prodrug, utilizes PEPT1 to enhance absorption and bioavailability. In vitro studies were conducted to guide the decision to conduct a clinical drug interaction study and to inform the clinical study design. In vitro investigations determined the prodrug (Pomaglumetad methionil/LY2140023 monohydrate) is a substrate of PEPT1 with Km value of approximately 30 µM, whereas the active moiety (LY404039) is not a PEPT1 substrate. In addition, among the eight known PEPT1 substrates evaluated in vitro, valacyclovir was the most potent inhibitor (IC50 = 0.46 mM) of PEPT1-mediated uptake of the prodrug. Therefore, a clinical drug interaction study was conducted to evaluate the potential interaction between the prodrug and valacyclovir in healthy subjects. No effect of coadministration was observed on the pharmacokinetics of the prodrug, valacyclovir, or either of their active moieties. Although in vitro studies showed potential for the prodrug and valacyclovir interaction via PEPT1, an in vivo study showed no interaction between these two drugs. PEPT1 does not appear to easily saturate because of its high capacity and expression in the intestine. Thus, a clinical interaction at PEPT1 is unlikely even with a compound with high affinity for the transporter. [2]

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Subsequently, a clinical study was designed to evaluate Pomaglumetad methionil/LY2140023 as both a substrate and an inhibitor of PEPT1. The coadministration of LY2140023 and valacyclovir did not affect the PK of each other or their respective active moieties (LY404039 or acyclovir), indicating no clinical DDI between the prodrug and valacyclovir. The data also showed that the presence of the prodrug or valacyclovir did not affect the conversion of prodrug to its active moiety for either LY2140023 or for valacyclovir. The lack of interaction on the conversion of the prodrug and valacyclovir to its corresponding active moieties was expected, because different enzymes are responsible for their activation. Dehydropeptidase 1 has been shown to cleave the prodrug to its active moiety (Moulton et al., 2015) and valacyclovirase (biphenyl hydrolase-like protein) to cleave valacyclovir to acyclovir (Marsillach et al., 2014). For both active moieties there was no change in the CL/F or the CLr, indicating that coadministration of the drugs did not affect the renal clearance of each other. Furthermore, if there is a weak interaction at PEPT1, a shift in Tmax values may be observed. However, no shift in Tmax was observed for any of the entities studied. Also, for valacyclovir there was no significant change in the AUC(0–3 hours), again indicating no interaction at PEPT1.

In this study, we illustrated how in vitro studies can guide the design of clinical DDI studies for transporter-based interactions. In vitro screening of inhibitory potencies of multiple drugs that compete at the transporter could give the rank order of inhibitory potencies and an analysis for potential for DDI in relation to oral dose of the compound. Therefore, unnecessary in vivo studies could be avoided, while focusing on the most relevant potential for DDI. Although the in vitro study indicated the potential for a DDI between the prodrug and valacyclovir according to guideline for other intestinal transporters, an in vivo DDI study showed no interaction of these two drugs via PEPT1. Therefore, our results clearly illustrated that a clinical DDI at PEPT1 is highly unlikely even with a NME with high affinity for the transporter.[2]

C12H18N2O7S2

366.41052

366.055543

C, 39.34; H, 4.95; N, 7.65; O, 30.57; S, 17.50

635318-55-7

Pomaglumetad methionil hydrochloride;635318-26-2;Pomaglumetad methionil;956385-05-0

25195354

Typically exists as solid at room temperature

1.9±0.1 g/cm3

600.3±55.0 °C at 760 mmHg

316.8±31.5 °C

0.0±3.7 mmHg at 25°C

1.661

-2.02

4

9

7

23

644

5

CSCC[C@H](N)C(N[C@@]1(C(O)=O)CS(=O)([C@H]2[C@H](C(O)=O)[C@H]21)=O)=O

VOYCNOJFAJAILW-CAMHOICYSA-N

InChI=1S/C12H18N2O7S2/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)/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

635318-55-7; LY 2140023; UNII-3V85EZ3KFQ; Pomaglumetad methionil free base; 3V85EZ3KFQ; Pomaglumetad methionil anhydrous; Pomaglumetad methionil [USAN:INN]; LY-2140023;

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)

DMSO : ~13.33 mg/mL (~36.38 mM)

Solubility in Formulation 1: ≥ 1.33 mg/mL (3.63 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 13.3 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
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: 1.33 mg/mL (3.63 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 13.3 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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.

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Solubility in Formulation 3: ≥ 1.33 mg/mL (3.63 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 13.3 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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