Millions of people worldwide suffer from chronic neuropathic pain without proper analgesic treatment options . Neuropathic pain syndromes are heterogeneous conditions that result from numerous types of nerve insult. Although the pathophysiological mechanisms underlying neuropathic pain are diverse, overactive glutamatergic transmission via N-methyl-D-aspartate receptors (NMDARs) is known to play a key role in central sensitization and consequently neuropathic pain. In addition, ketamine, an NMDAR channel-blocking antagonist, is often used for neuropathic pain [2,3]. Several nonclinical studies have shown that NMDAR antagonists such as memantine, 2-aminophosphonopentanoate, and dizocilpine (MK-801) when administered intrathecally, reduce pain associated with nerve injury, chemical damage, and inflammation [4,5,6,7,8].
Although these studies support the potential of targeting the NMDAR for developing drugs to treat neuropathic pain, several NMDAR antagonists have been reported to induce significant adverse cognitive side effects, including psychosis [9,10,11]. Antagonism of NMDARs can be achieved through blockade of the allosteric glycine B (GlyB) coagonist site . When compared with classic NMDAR antagonists, GlyB antagonists have a much better safety profile, and do not cause the adverse side effects that are associated with “classic” NMDAR antagonists [13,14].
One of the most potent and specific GlyB antagonists currently known is 7-chlorokynurenic acid (7-Cl-KYNA); however, its use in treating neuropathic pain is limited since it is very inefficient at crossing the blood-brain barrier (BBB) [15,16]. Its prodrug, L-4-chlorokynurenine (4-Cl-KYN, AV-101), is rapidly absorbed through the gut after oral administration, actively transported across the BBB, has no activity at the GlyB site , but it is converted to the active metabolite, 7-Cl-KYNA, by astrocytes [18,19]. A unique feature of the pharmacodynamics of AV-101 is the upregulation of the astrocytic enzymes responsible for this conversion during pathological conditions such as inflammation [20,21], neuronal damage , and seizures , resulting in increased levels of 7-Cl-KYNA at sites of neuronal injury or exci- totoxic insult as a result of this astrocyte activation. Peripheral nerve injury is known to result in activation of astrocytes at the site of nerve injury . Consequently, the conversion of AV- 101 to 7-Cl-KYNA in the spinal cord should be highest at sites of peripheral nerve spinal injury or inflammation . AV-101 utility in treating pain is supported by the demonstration of its potent anti-hyperalgesic actions in robust models of facilitated processing produced by peripheral tissue inflammation and nerve injury .
Human experimental pain models play an important role in bridging animal and clinical pain studies and provide the opportunity to test the potential analgesic effects of new compounds before initiation of costly clinical trials. Several human experimental pain models have been developed, and one of the most extensively studied experimentally induced pain model is the intradermal injection of capsaicin [26,27,28]. Capsaicin is the active ingredient in chili peppers that provokes a burning sensation by binding to the transient receptor potential calcium channel, subfamily V, member 1 (TRPV1). Capsaicin directly evokes pain, induces hyperalgesia and allodynia, and produces a flare response when injected intradermally . Intradermal capsaicin is a pain model that has been used to assess the analgesic effects of numerous drugs, including morphine , ketamine [32,33], pregabalin , gabapentin , lidocaine , and cannabis .
The primary objective of this study was to evaluate the safety and PK in a single dose escalation study, and of three doses of orally-administered AV-101 given once daily for 14 days in healthy volunteers. The secondary objective was to examine the antihy-peralgesic effects of 14-daily doses of orally-administered AV-101 versus placebo in healthy volunteers using the intradermal capsaicin model.
2 Material and methods
Enantiomerically pure L-4-Cl-KYN (AV-101, VistaGen Therapeutics, Inc.) was formulated as a simple dry powder-in-a-capsule as a dry blend with silicified microcrystalline cellulose (ProSolv HD90). Two AV-101 dosage strengths were prepared for clinical studies, 30 mg (Size 1 capsule) and 360 mg (Size 00 capsule). The capsules will be administered singularly, or in varying combinations, to achieve the desired dose for clinical studies. A maximum of five capsules may be dosed as a single administration for a maximum administrable dose of 1800 mg of AV-101 (five Size 00, 360-mg AV-101 capsules).
2.2 Clinical design
The Phase 1A study was a single-site (Progressive Medical Research Institute), randomized, double-blind, placebo-controlled, single ascending dose study in normal healthy male and female volunteers (IND #75,807). Seven cohorts (30, 120, 360, 720, 1080, 1440, and 1800 mg) with six subjects per cohort (1:1, AV- 101:placebo) were enrolled in this study. AV-101 was administered as a single oral dose, and subjects were dosed only once. The starting dose of 30 mg was less than 1/100 the human equivalent dose (390 mg) of the no observed adverse effect level (NOAEL) in dogs, the most sensitive animal species. The dose was to be escalated incrementally up to 1800 mg, which is slightly less than one-half the human equivalent dose of the NOAEL. More details of the study design are given in the supplemental data (Supplement 1).
The Phase 1B study was a single-site (University of California San Diego (UCSD)), randomized, double-blind, placebo-controlled, study involving multiple ascending doses of AV-101 in normal healthy male and female volunteers. This study was approved by the UCSD Institutional Review Board, and informed consent was obtained from each subject prior to entering the study (CTID NCT01483846). Three cohorts (AV-101 at 360, 1080, and 1440 mg/day) were enrolled in this study. Each cohort had 12 subjects on active drug and 4 subjects on placebo (oral capsules matching the AV-101 in appearance). Study drug was orally administered daily for 14 consecutive days. At a screening visit, subjects underwent laboratory assessments, physical examination, 12-lead ECG, ophthalmological examination, and neurological assessment (included tests for mental status, sensory/motor exam, coordination/gait, and ataxia), and 250 μg of capsaicin was delivered intradermally into the volar aspect of one forearm. Subjects must have reported a pain score of at least 4 out of 10 to qualify for the study. Before dosing on Day 1 and Day 14, the following data were collected: neurocognitive evaluation (Trail Making A and B Tests); and 12-lead electrocardiogram (ECG), blood pressure, heart rate, respiratory rate, and temperature . PK blood samples were collected on Days 1, 2, 14, and 15. The capsaicin-induced pain was assessed at screening, Day 1, and Day 14. On Day 1, subjects were given a paper diary to record daily self-administration of each dose, concomitant medications, and adverse events (AEs) during the 14-day treatment period. Subjects returned with any remaining study medication and the paper diary to the site on Day 7 (±1 day) and Day 14 for review.
On Day 1, subjects returned to the site in a fasted state and underwent the required assessments. Subjects were randomized to receive either AV-101 or placebo. Subjects received the first dose on Day 1, a 12-day supply of drug to take at home (Days 2–13), and a paper diary to record self-administration of study drug, concomitant medications, and adverse events (AEs). After study drug administration, subjects underwent efficacy assessments and blood draws for PK analyses. Subjects left the study site after the Day 1 assessments. On Day 2, subjects returned to the site for PK sample collection (24 h [±4h] from dose administration).
On Days 2–6 and Days 8–13, subjects took their capsule or capsules at approximately the same time each day with water and recorded it in their diary. They listed any concomitant medications taken during this timeframe. If any unexpected physical or medical condition was experienced, subjects were to call the clinic right away and report it to the study staff and to record it in their diary.
On Day 7 (±1 day), subjects returned to the site in a fasted state with their study medication and diaries (for review of the administration of doses, concomitant medications, and AEs) and to have vital signs and clinical laboratory assessments conducted.
On Day 14, subjects returned to the site in a fasted state with their diaries and any remaining study drug, and the baseline study assessments performed on Day 1 were repeated. Subjects were to receive the last dose of AV-101 or placebo at the site and undergo the capsaicin injections, efficacy assessments, and blood draws for PK analyses. They left the study site after the Day 14 assessments.
On Day 15, all subjects returned to the site for collection of a PK sample (24 h [±4h] from last dose administration) and for an eye examination. On Day 21 (±2 days), a follow-up telephone interview was conducted to monitor and record AEs.
Safety was assessed by collection of medical history at the Day 1 site visit; vital signs, clinical hematology, clinical plasma chemistry, and laboratory measures of coagulation at site visits on Days 1, 7, and 14; physical and neurocognitive examinations (Trail making A and B tests) and ataxia tests at site visits on Days 1 and 14; and an ophthalmological examination on Day 15. AEs were assessed after the first dose of study medication through Day 21 (±2 days).
2.4 PK assessments
Blood samples for PK analysis were collected from each subject on Day 1 and on Day 14 at the following time points: before study drug administration, and 0.5,1,1.5,2,4,6,8,12, and 24 h after study drug administration. The following PK parameters were derived from the plasma concentration versus time profiles to determine the single-dose and multi-dose PK profile of AV-101 and the active metabolite, 7-chlorokynurenic acid:
- Maximum concentration
- Terminal elimination half-life
- Time to maximum concentration
- Area under the plasma study drug concentration versus time curve from Time 0 to time of last measurable concentration
- Area under the plasma study drug concentration versus time curve from Time 0 extrapolated to infinity.
AV-101 quantification in human plasma. Human plasma containing AV-101, an isotopically labeled analog of AV-101, as the internal standard and lithium heparin as the anticoagulant were precipitated using a perchloric acid solution. The supernatant was recovered, neutralized, and reacted with propylchloroformate. The derivatives were isolated by liquid-liquid extraction and analyzed with HPLC using a Chiral-HSA column. The mobile phase was nebulized using heated nitrogen in a Z-spray source/interface set to electrospray positive ionization mode. The ionized compounds were detected using MS/MS.
7-Cl-KYNA quantification in human plasma. Human plasma samples containing 7-Cl-KYNA, 5,7-dichlorokynurenic acid as the internal standard and lithium heparin as the anticoagulant were precipitated with an acetonitrile:methanol solution. The supernatant was dried, reconstituted with an ammonium hydroxide solution and extracted using solid phase extraction. The extracted samples are then analyzed by reversed-phase HPLC using a Restek Allure Biphenyl column maintained at 25 °C. The mobile phase was nebulized using heated nitrogen in a Z-spray source/interface set to electrospray negative ionization mode. The ionized compounds were detected using MS/MS.
Several subjects had concentration-time profiles with a last measurable sample that appeared to be an outlier or suggested multi-compartment kinetics, making it challenging to identify a terminal log-linear elimination phase, therefore a few of the last samples with measurable concentrations that were apparent outliers were not used in the analyses. Particularly for 7-Cl-KYNA, using the last two measurable samples to calculate the half-life (t½) resulted in unrealistic values for some subjects.
On Day 1 and Day 14, two intradermal injections of 250 μg of capsaicin were injected sequentially into the volar aspect of alternate forearms to produce burning pain, secondary hyperalgesia, and a flare. The capsaicin USP (United States Pharmacopeia) was prepared per the site’s standard procedure and dissolved in 20% cyclodextrin at a concentration of 10 mg/mL.
The first capsaicin injection in one forearm was given 1 h after oral administration of AV-101 or placebo, and the second capsaicin injection was given in the other forearm 2 h after the administration of AV-101 or placebo. The neurosensory testing began immediately at each capsaicin injection. The serial pain assessments using a 100mm visual analog scale (VAS) occurred at preinjection and 0, 5, 10, 15, 30, 45, and 60 min after each capsaicin injection. The examiner asked the subject to rate the intensity by using the VAS of the spontaneous pain and elicited pain from the application of a 5.18 von Frey hair, 40 °C probe, and gentle stroking with a 1-in. foam brush. The VAS consisted of a line with “no pain” written at the 0-mm end and the “worst imaginable pain” written at the 100-mm end. The distance in millimeters provided the pain measurement.
The borders of the hyperalgesic area to a 5.18 von Frey hair were determined by moving from an area of the skin that did not produce pain tangentially toward the center of the painful area at a progressively closer radius until the subject reported pain or tenderness. At least eight determinations of the hyperalgesic area borders were made using the same assessments and starting at different angles. Additionally, subjects were asked to rate the pain intensity of a 1- min 45 °C heat stimulus (brief thermal stimulation) applied to the anterior thigh at 4.5 h (±5 min) after study drug administration, then every 30 min (±5 min) through 6 h after study drug administration. A radiant heat lamp was applied to fix the skin temperature at 36°C during the pain assessments. Subjects used the VAS for all pain assessments.
All study assessment time points were anchored to Time 0, the time of study drug administration. For the capsaicin injection at 1 h after dosing, the assessment interval (i.e., time after dosing of the study drug) was 60–120 min, during which time assessments occurred at approximately 60, 65, 70, and 75 min and then every 15 min through the 120-min time point (i.e., 0, 5,10,15,30,45, and 60 min after capsaicin injection). The second injection of capsaicin was administered approximately 2 h after dosing of the clinical trial material (CTM), and the assessment interval (i.e., time after dosing of the CTM) was 120–180 min. The serial pain assessments followed the same schedule, as described after the first capsaicin injection.
2.6 Statistical analysis
For efficacy analyses, last observation carried forward (LOCF) was used to cope with missing data, with the exception that subjects who withdrew because of toxicity were considered non-responders for purposes of responder analyses. Dates and times were imputed unless needed for a calculation, in which case the least favorable date was used. In the case of determining baseline values, the last non-missing measurement before dosing was used. For analyses using the safety population, there was no imputation of missing endpoint values.
The primary efficacy endpoint was the AUPC for spontaneous pain for the time interval of 120–180 min after dosing (0–60 min after the 120-min capsaicin injection) on Day 14. For the AUPC, each dose level of AV-101 was compared to placebo. The differences between placebo and each of the active treatment groups was analyzed using an ANOVA model with treatment group as a fixed effect. The LS mean for each treatment group and for treatment group differences was provided, along with 90% CI for treatment group means, 95% CI for treatment group differences, and corresponding p-values. Since the primary objective of the study was safety and PK, a power calculation was not performed. However, based on previous studies using the intradermal capsaicin model, 3 cohorts of 16 subjects was considered adequate to fulfill the secondary objective of the study [31,32,33,34,35].
An initial Phase 1A randomized, double-blind, placebo- controlled dose escalation study evaluated the safety and PK of a single oral AV-101 dose in six cohorts (30,120, 360, 720,1080, and 1440 mg) with six subjects per cohort (1:1, AV-101:placebo) was performed. Originally the study was planned to escalate to 1800 mg, slightly less than one-half the human equivalent dose of the NOAEL in dogs (Snodgrass et al., unpublished data). In agreement with the FDA, a “PK-stopping criteria” was established to stop the dose escalation if the mean maximum concentration (Cmax) exceeded 81.6 |ag/mL. One subject in the Phase 1A Dose escalation study in the 1440 mg/day cohort met this criterion, and the dose escalation was stopped at 1440 mg (data not shown). All doses of the drug were well-tolerated, and the frequency and mild severity of the adverse events (AEs) were similar among the placebo and drug cohorts (Supplemental Table S2). Based on the excellent safety in this initial study of AV-101, even at the highest dose (1440 mg), we sought to evaluate the safety, and potential antinociception activity, of 360,1080, and 1440 mg/day of oral AV-101 in a larger number of subjects in a follow-up Phase 1B.
Fifty subjects were enrolled in a Phase 1B Multi-dose Study, and receive oral AV-101 or placebo daily for 14 consecutive days. Subjects were healthy volunteers (23 males and 27 females) at 28-31 years of age (Supplemental Table S1). In Cohort 1,12 subjects received 360 mg AV-101, and 5 subjects received placebo. In Cohort 2, 13 subjects received 1080 mg AV-101, and 4 subjects received placebo. In Cohort 3, 12 subjects received 1440 mg AV-101, and 4 subjects received placebo. Three subjects terminated early (2 from the 1080 mg dose and 1 from placebo), due to reasons unrelated to adverse events.
Consistent with the earlier Phase 1A results, this second Phase 1B study indicated that AV-101 was well-tolerated, with no serious adverse events (SAEs). Thirty-four of 50 subjects in the study reported a total of 57 AEs (Table 1, and Supplemental Table S3). Forty-nine (85.9%) of the AEs were mild, and 8 were moderate. Nineteen subjects reported 27 AEs that were possibly related to treatment with AV-101, and 8 subjects reported 14 AEs that were possibly or probably related to placebo. There were 2 moderate intensity AEs in the 360-mg AV-101 group: one was unrelated pain in the right foot, and one was a possibly related headache. All other moderate AEs occurred in the placebo group and included nausea or vomiting (2 AEs), headache (2 AEs), and rash around the neck (1 AE) (Supplemental Table S3). The designations of AEs as treatment-related were made while the study was still blinded, and all the AEs were rapidly and completely resolved.
Overall summary of adverse events observed in the Phase 1B study.
|360 mg AV-101||1080mg AV-101||1440 mg AV-101||Pooled placebo|
|(N= 12)||(N= 13)||(N= 12)||(N= 13)|
|[n (%)]||[n (%)]||[n (%)]||[n (%)]|
|Number of AEs||16||14||10||17|
|Number of subjects with any AE||9 (75.0%)||8 (61.5%)||7 (58.3%)||10 (76.9%)|
|Number of SAEs||0 (0%)||0 (0%)||0 (0%)||0 (0%)|
|Number of AEs resulting in death||0 (0%)||0 (0%)||0 (0%)||0 (0%)|
|Number of AEs leading to discontinuation of study drug||0 (0%)||0 (0%)||0 (0%)||1 (7.7%)|
Overall, the clinical laboratory assessments indicated that there were no meaningful changes in laboratory parameters due to AV- 101 treatment, and the number of abnormal results did not present a safety concern during the study. In addition, there were no clinically significant abnormal measurements in the vital signs, physical examinations, 12-lead ECGs, ataxia tests, ophthalmological examinations, capsaicin injection site examinations, or interactions with concomitant medications during the study, and no significant changes in neurocognitive scores between Days 1 and 14. None of these measurements were reported as AEs.
Maximum plasma concentrations of AV-101 and 7-Cl-KYNA were reached between 1–2 h, depending on dose (Fig. 1 and Table 2). Mean half-life values of AV-101 were consistent across doses, ranging from 1.64 h to 1.82 h (Table 2). The highest mean Cmax and AUC0-t values for AV-101 occurred in the 1440-mg dose group and were 64.4 μg/mL (Cmax) and 196 μg h/mL (AUC0–t), respectively (Table 2), significantly lower than the stopping criteria values of 81.6 μg/mL (Cmax) and 900 μg h/mL (AUC0-t). Two individuals in the 1440-mg dose group exceeded a Cmax of 81.6 μg/mL (84.4 and 84.6 μg/mL, respectively). Whereas the highest AUC0-t values (257 μgh/mL) were observed in two other individuals in the 1440-mg dose group, which is significantly below the AUC0-t stopping criteria. For AV-101 and 7-Cl-KYNA, mean AUC0-t values were nearly dose proportional, but with an indication of a slight reduction at the highest dose (data not shown).
Phase 1B summary statistics for AV-101 pharmacokinetic parameters by dose group from treatment Day 1 (B) or Day 14 (B) for subjects receiving a daily oral dose of AV-101 for 14 days.
|Subject||Tmax (h)||Cmax (μg h/mL)||t½(h)||AUC0-t (μg h/mL)||AUC0-∞ (μg h/mL)|
|Dose = 1080mg|
|Dose = 1440 mg|
|Dose = 1080mg|
|Dose = 1440 mg|
AUC0-t, area under the plasma AV-101 concentration-time curve from Time 0 to the time of last measurable concentration; AUQ-TO, area under the plasma AV-101 concentration-time curve from Time 0 extrapolated to infinity; Cmax, maximum concentration; L-AV-101, L-4-chlorokynurenine; t½, terminal elimination half-life; Tmax, time to maximum concentration
For 7-Cl-KYNA, mean Tmax values were highest for the 1080mg dose group. Mean serum half-life values of 7-Cl-KYNA were slightly more variable across doses compared with those for AV- 101, ranging from 2.26 to 3.23 h (Supplemental Table S4). The mean Cmax values for both AV-101 and the metabolite, 7-Cl-KYNA, were approximately dose linear, but not dose proportional on both Days 1 and 14 (Supplemental Table S4). Concentration-time profiles for AV-101 were more consistently single compartment, but several had a subtle multi-compartment appearance.
The primary efficacy endpoint was the analgesic response to spontaneous pain at each dose level of AV-101 120–180 min after dosing on Day 14, with secondary endpoints for effects from 60 to 180 min. There was no significant change in the area under the pain time curve (AUPC) for the spontaneous pain assessment between the treatment and the placebo groups at 60–180 min after dosing on either Day 1 or 14 (Table 3), and likewise, there was no significant change at 120-180 min on Day 1 or 14 (Supplemental Table S5). In contrast, there were consistent reductions at 60–180 min after dosing at Day 1 between subjects that received 1080 mg AV-101 (Cohort 2) and subjects that received placebo in the least squares means of the AUPC for allodynia pain (Table 4: LS mean differences of –320.58, p-value = 0.5888 on Day 1, and –20.93, p-value = 0.9747 on Day 14), mechanical hyperalgesia pain (Table 5: LS mean differences of -400.77, p-value =0.5546 on Day 1, and –40.36, p-value = 0.9576 on Day 14), and heat hyperalgesia pain (Table 6: LS mean differences of –909.62, p-value = 0.2717 on Day 1, and -1024.72, p-value = 0.2423 on Day 14). There was also a reduction in the AUPC for heat hyperalgesia pain observed at Day 14 between subjects that received 1080 mg AV-101 (Table 6, Cohort 2: LS mean differences of –1024.72, p- value = 0.2423), but there was no meaningful difference observed at Day 14 for allodynia pain or mechanical hyperalgesia pain (Tables 4 and 5).
Phase 1B AUPC inferential analyses from spontaneous pain assessment.
|Visit||Measurement time point||Statistic||360 mg||1080 mg||1440 mg||Pooled placebo|
|Day 1||60–180 min after dosing||90% CI||1840.75, 3370.08||1819.18,3288.51||2032.00,3561.33||1890.33,3359.67|
|LS mean difference from placebo||-19.58||-71.15||171.67||NA|
|Day 14||60–180 min after dosing||90% CI||2056.43,3515.24||1588.84,3112.52||1881.64,3340.45||1472.26,2931.07|
|LS mean difference from placebo||584.17||149.02||409.38||NA|
|95% CI||-653.31,1821.65||-1116.28,1414.31||-828.10, 1646.85||NA|
Phase 1B AUPC inferential analyses for ITT population, allodynia pain.
|Visit||Measurement time point||Statistic||360 mg||1080 mg||1440 mg||Pooled placebo|
|Day 1||60–180 min after dosing||90% CI||2298.57,3753.52||1845.49,3243.36||2597.11,4052.06||2166.07,3563.93|
|LS mean difference from placebo||161.04||-320.58||459.58||NA|
|Day 14||60–180 min after dosing||90% CI||2293.23,3816.35||1738.44,3329.29||2168.23, 3691.35||1793.23,3316.35|
|LS mean difference from placebo||500.00||-20.93||375.00||NA|
|95% CI||-792.03,1792.03||-1342.00, 1300.14||-917.03,1667.03||NA|
Phase 1B AUPC inferential analyses for ITT population, mechanical hyperalgesia.
|Visit||Measurement time point||Statistic||360 mg||1080 mg||1440 mg||Pooled placebo|
|Day 1||60–180min after dosing||90% CI||2853.89,4517.36||2253.97,3852.18||3312.85, 4976.32||2654.74,4252.95|
|LS mean difference from placebo||231.78||-400.77||690.74||NA|
|Day 14||60–180 min after dosing||90% CI||2639.31,4392.77||2104.97,3963.39||2678.90,4432.35||2184.31,3937.77|
|LS mean difference from placebo||455.00||-40.36||494.58||NA|
Phase 1B AUPC inferential analyses for ITT population, heat hyperalgesia.
|Visit||Measurement time point||Statistic||360 mg||1080 mg||1440 mg||Pooled placebo|
|Day 1||60–180 min after dosing||90% CI||5241.17,7261.33||3053.20,4994.11||4258.67,6278.83||3962.81,5903.73|
|LS mean difference from placebo||1317.98||-909.62||335.48||NA|
|Day 14||60–180 min after dosing||90% CI||4258.88,6268.62||2341.35,4740.47||3952.63,5962.63||3710.75,5720.50|
|LS mean difference from placebo||548.12||-1024.72||241.87||NA|
The safety data indicate that AV-101 was safe and well tolerated in both Phase 1A and 1B studies. No serious adverse events were reported, most events being mild, and all adverse events were completely resolved. Furthermore, there was no meaningful difference in adverse events at any dose between AV-101 and placebo in both Phase 1A and 1B studies (Supplemental Tables S2 and S3). It is noteworthy that a maximum tolerated dose was not reached in this study. The dose escalation was stopped at 1440 not due to side effects, but because one subject in the Phase 1A 1440 mg dose cohort reached one of the PK-stopping criteria. In agreement with the FDA, the dose-escalation would stop if the serum maximum concentration (Cmax) of AV-101 exceeded 81.6 μg/mL. This value was determined from the Cmax at the maximum tolerated dose observed in the dog, the most sensitive species, in the preclinical toxicological studies (Snodgrass et al., unpublished data).
In the Phase 1B study, 57 AEs were reported by 34 subjects, with 17 AEs (29.8%) occurring in the placebo group (Table 1, Supplemental Tables S2 and S3). There was a higher rate of AEs reported from subjects who received placebo than from subjects who received AV-101. A total of 40 AEs were reported in Phase 1B by 24 of 37 (64.9%) subjects receiving AV-101, and 17 AEs were reported by 10 of 13 (76.9%) subjects receiving placebo (Supplemental Table S3). Additionally, 49 of the 57 (85.9%) total AEs were considered mild, and the remaining 8 AEs (14.0%) were considered moderate. Of these AEs, headache (nervous system disorder) was the most commonly reported preferred term. All the AEs were completely resolved. These data support the conclusions that AV-101 is very well-tolerated, safe, and with little to no side- effects at daily doses up to 1440 mg for 14 days. In addition, there were no clinically significant abnormalities or changes in the neurocognitive examination results at Day 1 or 14 of treatment with AV-101 (data not shown). The safety of AV-101 and lack of off- target effects are consistent with the non-clinical observations that, except for binding to the GlyB site of the NMDAR, neither AV-101, nor 7-Cl-KYNA, its active metabolite, had any meaningful binding to 50 G-protein coupled receptors, ion channels and transporters important in CNS biology .
Plasma concentration-time profiles obtained for AV-101 after administration of once-daily oral doses of 360, 1080, or 1440 mg were consistent with rapid absorption of the oral dose, and first-order elimination of both AV-101 and 7-Cl-KYNA, with evidence of multi-compartment kinetics, particularly for the metabolite 7-Cl-KYNA. The mean Cmax and AUC0-t values for both AV-101 and 7-Cl-KYNA are approximately dose linear but not dose proportional on both Days 1 and 14 (data not shown).
The primary efficacy endpoint was the analgesic response to spontaneous pain at each dose level of AV-101 at 120–180 min after dosing (0-60 min after the capsaicin injection at 120-min) on Day 14. There was no significant change in the AUPC for the spontaneous pain assessment between the treatment and the placebo groups for this measurement (Supplemental Table 5). Likewise, there were no significant changes between the treatment and the placebo groups for any of the secondary efficacy endpoints (e.g., AUPC for spontaneous pain for the time interval of 120-180 min after dosing on Day 1) (Supplemental Table S5); nor for AUPC for spontaneous pain (Table 3), elicited pain from the von Frey hair (Table 5), or elicited pain from the 40°C probe (Table 6) for the time interval of 60–180 min after dosing on Day 1 or 14. However, there were consistent, but nonsignificant, decreases in the LS mean of the AUPC for allodynia pain, mechanical hyperalgesia pain, and heat hyperalgesia pain between subjects that received 1080 mg AV-101 (Cohort 2) and subjects that received placebo (Tables 4,5,6). This trend was not seen with the highest dose (1440 mg); however, this is not uncommon as therapeutic windows are seen with a variety of drugs (i.e. tricyclic antidepressants and cannabinoids) . Previous studies have demonstrated that intravenous ketamine has a dose-dependent reduction in both intradermal capsaicin and neuropathic pain; however, the administered dose was limited by side effects. If higher doses were achieved, it is possible that therapeutic window would have been observed [33,38].
Pharmacology studies conducted in rodent models have demonstrated AV-101’s antihyperalgesic activity in models of facilitated pain processing was seen at serum concentrations ranging from 150–300 μM . In addition, AV-101 has been shown to be neuroprotective activity against an intrahippocampal injection of quinolinic acid [39,40], reductions in seizures , and antidepressive activity . Although in the current studies there was a consistent trend to reduce allodynia and hyperalgesia at serum concentrations of AV-101 (145–165 μM) at the lower range of those observed in the rat studies discussed above, there was no statistically significant effect on capsaicin-induced pain. This could be due to the study being underpowered, or that a sufficient serum concentration was not achieved, but it is also true that translating preclinical animal studies to human pain states is challenging. Animals cannot communicate the intensity of pain thus limiting outcome measures to evoked pain.
In contrast, spontaneous pain measures are typically used as the primary outcome in human pain studies. Spontaneous and evoked pain dichotomy has been demonstrated in other human experimental pain studies using capsaicin in which there were reductions in secondary hyperalgesia but not spontaneous pain [35,42]. In a previous study in neuropathic pain involving another NMDA receptor glycine site antagonist (GV196771) similar to AV-101, there was also no reduction in spontaneous pain, but there was a significant reduction in mapped allodynia .
Furthermore, the ability of the intradermal capsaicin model to accurately predict the antinociceptive effects of a drug in the human clinical pain state can vary depending on the mechanism of action. For example, the antidepressants duloxetine (public unpublished data, AbbVie, Inc.), amitriptyline  and desipramine  failed to reduce pain in this model in spite of proven clinical effectiveness in a variety of neuropathic pain states. However, the opioids and N-type calcium channel modulators have been shown to reduce capsaicin induced pain [31,33]. These findings suggest that drugs that have a direct effect on spinal nociceptive pathways (e.g. opioids and N-type calcium channel modulators) reduce capsaicin induced pain, whereas drugs that uncouple the sensory perceptions from memory and emotional responses (e.g., antidepressants) may have no effect on capsaicin induced pain, but can have significant therapeutic benefit in reducing clinical pain states.
The lack of response to capsaicin induced pain in this study is supported by a recent study evaluating a T-type calcium channel modulator failed to show an effect in capsaicin induced pain . T-type calcium channels have been shown to mediate the transition between the tonic and phasic firing of thalamic neurons suggesting a dissociative effect on pain . NMDA antagonists have been shown to treat refractory depression [47,48,49]; therefore, since it has been recently demonstrated that AV-101 has rapid antidepressant activities  through its potential antidepressive properties, it could still effectively treat neuropathic pain similar to other antidepressants. Furthermore, the consistent reduction in the heat pain from 60–120 min using the 1080 mg dose suggest that there is also some intrinsic analgesic effect similar to the study of a glycine antagonist for neuropathic pain .
The safety data indicate that AV-101 was extremely safe and well tolerated in both Phase 1A and 1B studies. There was no meaningful difference in adverse events at any dose between AV-101 and placebo in either Phase study. Although, AV-101 did not reach statistical significance in reducing pain in the capsaicin induced- pain model, there were consistent reductions, in allodynia pain and mechanical and heat hyperalgesia. There are several limitations to this study. First, dose selection may have been a factor as there were few side effects and average plasma levels were below the PK stopping criteria. However, results suggest that the mid dose (1080 mg) was the optimum dose and higher doses may have been ineffective despite tolerability. Other studies have demonstrated a therapeutic window of analgesia with the cannabinoids in both the intradermal capsaicin model and clinical pain state [34,50]. Second, the study may have been underpowered to evaluate efficacy as we used a randomization ratio of 4:1, active:placebo. However, the study was designed with the primary purpose of assessing safety and PK in which this ratio is typically used. The efficacy assessment using the capsaicin model was a secondary purpose. Third, as discussed above, the relationship of human experimental pain models to the clinical pain state is limited by the lack of studies correlating the two.
The safety profile, PK data, and an indication of an intrinsic analgesic effect, suggest that future clinical trials of AV-101 in neuropathic pain are justified. Finally, it is noteworthy that among the mild AEs reported from both the Phase 1A dose-escalation and the Phase 1B study reported herein, there were voluntary reports of positive and agreeable “feelings of well-being” within 2 h of dosing. Although these were coded as “euphoria”, these reports were not related to the hallucinogenic nor dissociative out-of-body experiences typically reported with the use of ketamine, and other NMDAR antagonists. Combined Phase 1A and 1B data suggest that approximately 10% (5 of 54) of the subjects given AV-101, but none (n = 30) of the subjects given placebo, expressed positive feelings of well-being. These were reported most often in the highest dose (1440 mg) of AV-101 (Supplemental Table S6). This is notable and may suggest, similar to other modulators of the NMDAR [47,49], that AV-101 may have antidepressive properties in humans as it does in preclinical animal models . This is currently being evaluated in an ongoing Phase 2a study in major depressive disorder (Clinicaltrials.gov identifier: NCT02484456).
- AV-101 is an oral prodrug producing a potent brain NMDAR GlyB-site antagonism.
- AV-101 has excellent safety profile, similar to placebo.
- AV-101 exhibited nonsignificant decreases in allodynia and hyperalgesia.
- Reports of feelings of well-being with AV-101 suggest anti-depressant activity.
Funding: This work was funded by the following NIH grants: NIDA 1R43DA018515 and 2R44DA018515; and NINDS R43NS047808.
Conflict of interest: R. Snodgrass and A. Cato have equity positions in VistaGen Therapeutics, which has commercial rights to AV-101 (L-4- chlorokynurenine). K. Grako, R. Lane and A. Cato are employees of Cato Research, which has drug development contracts with Vista-Gen. M. Wallace and A. White have no conflicts or competing interests.
Dougherty PM, Palecek J, Paleckova V, Sorkin LS, Willis WD. The role of NMDA and non-NMDA excitatory amino acid receptors in the excitation of primate spinothalamic tract neurons by mechanical, chemical, thermal, and electrical stimuli. J Neurosci 1992;12:3025–41 https://www.ncbi.nlm.nih.gov/pubmed/1353793.
Parsons CG, Danysz W, Quack G, Hartmann S, Lorenz B, Wollenburg C, Baran L, Przegalinski E, Kostowski W, Krzascik P, Chizh B, Headley PM. Novel systemically active antagonists of the glycine site of the N-methyl-D-aspartate receptor: electrophysiological, biochemical and behavioral characterization. J Pharmacol Exp Ther 1997;283:1264–75 https://www.ncbi.nlm.nih.gov/pubmed/9400002.
Rao TS, Gray NM, Dappen MS, Cler JA, Mick SJ, Emmett MR, Iyengar S, Monahan JB, Cordi AA, Wood PL. Indole-2-carboxylates, novel antagonists of the N-methyl-D-aspartate (NMDA)-associated glycine recognition sites: in vivo characterization. Neuropharmacology 1993;32:139–47 https://www.ncbi.nlm.nih.gov/pubmed/8383813.
Heyes MP, Saito K, Crowley JS, Davis LE, Demitrack MA, Der M, Dilling LA, Elia J, Kruesi MJ, Lackner A, Larsen SA, Lee K, Leonard HL, Markey SP, Martin A, Milstein S, Mouradian MM, Pranzatelli MR, Quearry BJ, Salazar A, Smith M, Strauss SE, Sunderland T, Swedo SW, Tourtellotte WW. Quinolinic acid and kynurenine pathway metabolism in inflammatory and non-inflammatory neurological disease. Brain 1992;115 (Pt 5):1249-73 https://www.ncbi.nlm.nih.gov/pubmed/1422788.
Chiarugi A, Dello Sbarba P, Paccagnini A, Donnini S, Filippi S, Moroni F. Combined inhibition of indoleamine 2, 3-dioxygenase and nitric oxide synthase modulates neurotoxin release by interferon-gamma-activated macrophages. J Leukoc Biol 2000;68:260–6 https://www.ncbi.nlm.nih.gov/pubmed/10947071.
Wang H, Bolognese J, Calder N, Baxendale J, Kehler A, Cummings C, Connell J, Herman G. Effect of morphine and pregabalin compared with diphenhydramine hydrochloride and placebo on hyperalgesia and allodynia induced by intradermal capsaicin in healthy male subjects. J Pain 2008;9:1088–95, http://dx.doi.org/10.1016/jjpain.2008.05.013.
Wallace MS, Ridgeway 3rd B, Leung A, Schulteis G, Yaksh TL. Concentration-effect relationships for intravenous alfentanil and ketamine infusions in human volunteers: effects on acute thresholds and capsaicin-evoked hyperpathia. J Clin Pharmacol 2002;42:70–80 https://www.ncbi.nlm.nih.gov/pubmed/11808827.
Wallace M, Schulteis G, Atkinson JH, Wolfson T, Lazzaretto D, Bentley H, Gouaux B, Abramson I. Dose-dependent effects of smoked cannabis on capsaicin-induced painand hyperalgesiain healthy volunteers. Anesthesiology 2007;107:785–96, http://dx.doi.org/10.1097/01.anes.0000286986.92475.b7.
Gottrup H, Juhl G, Kristensen AD, Lai R, Chizh BA, Brown J, Bach FW, Jensen TS. Chronic oral gabapentin reduces elements of central sensitization in human experimental hyperalgesia. Anesthesiology 2004;101:1400–8, http://dx.doi.org/10.1097/00000542-200412000-00021.
Reitan RM, Davison LA. Clinical neuropsychology: current status and applications. Washington, DC: V.H. Winston & Sons; 1974.
Zanos P, Piantadosi SC, Wu HQ, Pribut HJ, Dell MJ, Can A, Snodgrass HR, Zarate Jr CA, Schwarcz R, Gould TD. The prodrug 4-chlorokynurenine causes ketamine-like antidepressant effects, but not side effects, by NMDA/glycineB-site inhibition. J Pharmacol Exp Ther 2015;355:76–85, http://dx.doi.org/10.1124/jpet.115.225664.
Murrough JW, Iosifescu DV, Chang LC, Al Jurdi RK, Green CE, Perez AM, Iqbal S, Pillemer S, Foulkes A, Shah A, Charney DS, Mathew SJ. Antidepressant efficacy of ketamine in treatment-resistant major depression: a two-site randomized controlled trial. Am J Psychiatry 2013;170:1134–42, http://dx.doi.org/10.1176/appi.ajp.2013.13030392.
Preskorn S, Macaluso M, Mehra DO, Zammit G, Moskal JR, Burch RM, Group G-CS. Randomized proof of concept trial of GLYX-13, an N-methyl-D-aspartate receptor glycine site partial agonist, in major depressive disorder nonresponsive to a previous antidepressant agent. J Psychiatr Pract 2015;21:140–9, http://dx.doi.org/10.1097/01.pra.0000462606.17725.93.
Portenoy RK, Ganae-Motan ED, Allende S, Yanagihara R, Shaiova L, Weinstein S, McQuade R, Wright S, Fallon MT. Nabiximols for opioidtreated cancer patients with poorly-controlled chronic pain: a randomized, placebo-controlled, graded-dose trial. J Pain 2012;13:438–49, http://dx.doi.org/10.1016/jjpain.2012.01.003.
Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.sjpain.2017.05.004.