Steady-state pharmacokinetics and pharmacodynamics of CHF3381, a novel antineuropathic pain agent, in healthy subjects

Abstract

Aims

To evaluate the safety, tolerability, pharmacokinetic and pharmacodynamic profiles of CHF3381, a dual NMDA and MAO-A inhibitor, after multiple oral doses in healthy subjects.

Methods

Forty-eight young males received CHF3381 at doses of 100 mg twice daily, 200 mg twice daily, 400 mg twice daily or placebo for 2 weeks according to a double-blind, randomized, parallel group design. Plasma and urine concentrations of the parent drug and of two major metabolites (CHF3567 and 2-aminoindane) were measured over time. MAO-A activity in plasma was estimated by measuring plasma concentrations of 3,4-dihydroxyphenylglycol. Sustained attention, memory and sedation were assessed throughout the study with standard psychometric tests.

Results

Most of the adverse events were mild in intensity, with dose regimens of 100 mg twice daily and 200 mg twice daily being indistinguishable from placebo. After 400 mg twice daily, the most frequent adverse events were mild dizziness, asthenia and insomnia. At steady-state, 400 mg twice daily slightly increased supine heart rate (+ 9 ± 2 beats min⁻¹) and diastolic blood pressure (+6 ± 2 mmHg) compared with placebo. There were no dose-dependent or consistent effects of CHF3381 on attention, motor co-ordination or memory, but 400 mg twice daily significantly decreased alertness compared with placebo. Plasma concentrations of CHF3381 peaked at around 3 h and were dose-proportional. The elimination half-life of CHF3381 was estimated to be 4–6 h. At steady-state, significant CHF3381 plasma concentrations were detected at predose with a modest accumulation (1.3–1.5 times), showing that the drug given twice daily is active over the entire 24 h period. Plasma concentrations of CHF3567 and of 2-aminoindane were also proportional to the dose of CHF3381. CHF3381 dose-dependently inhibited MAO-A activity with peak effects at steady-state of 27 ± 4%, 46 ± 2% and 65 ± 5% after 100 mg twice daily, 200 mg twice daily and 400 mg twice daily, respectively. There were no significant effects of CHF3381 on attention (rapid visual information processing), motor co-ordination (body sway) or memory (learning memory task) at any of the doses. At steady-state, there was a significant decrease in alertness (Bond & Lader visual analogue scale) in the 400 mg twice daily group compared with placebo.

Conclusions

A twice daily regimen of CHF3381 appears to be adequate from a pharmacokinetic and pharmacodynamic perspective. Plasma concentrations reached with 400 mg twice daily exceeded those observed in animals receiving pharmacologically active doses in chronic pain models.

Introduction

Neuropathic pain arises from damage to or dysfunction of neuronal tissue. There are very few drugs approved for the treatment of this condition [1]. In clinical practice, anticonvulsants, tricyclic antidepressants and membrane stabilizers are frequently used. N-methyl-d-aspartate (NMDA) receptors are believed to be involved in the mechanism of pain hypersensitivity occurring in the neurones of the dorsal horn of the spinal cord (‘central sensitization’) which leads to chronic neuropathic pain [2]. Accordingly, inhibition of NMDA receptors using the competitive antagonists like ketamine [3] or dextromethorphan [4] has been shown to lessen neuropathic pain. Unfortunately, NMDA receptors are widely distributed in the brain and this leads to unacceptable psychotomimetic effects accompanying analgesia. The development of drugs that block central sensitization without causing side-effects is being actively pursued.

The analgesic activity of tricyclic antidepressants (amitriptyline, imipramine) in neuropathic pain is believed to be linked to the activation of serotonergic and noradrenergic pathways, which are involved in the control of pain information [1]. Reversible monoamine oxidase-A (MAO-A) inhibitors (e.g. moclobemide, brofaromine) are antidepressant drugs that boost the noradrenergic system by inhibiting norepinephrine, dopamine and serotonin metabolism. Moclobemide, a reversible MAO-A inhibitor, has been shown to possess antinociceptive effects in experimental animals [5], healthy subjects [6] and patients with pain disorders [7] apparently through a central action.

CHF3381 (Figure 1) is a new low-affinity, noncompetitive NMDA antagonist and reversible MAO-A inhibitor [8]. The compound has a selective affinity for the NMDA-phencyclidine site (IC₅₀ = 8.1 µm) and behaves as a MAO-A inhibitor (IC₅₀ = 7.8 µm). In primary cultures of cortical neurones, CHF3381 blocks NMDA-evoked currents with an IC₅₀ = 5 µm[9] and exhibits glycine-independent neuroprotective effects against glutamate-induced excitotoxicity [10]. The compound prevents cell damage completely in kainate seizure-induced neurodegeneration at doses unable to prevent or attenuate seizures [11]. In rats, CHF3381 penetrates the brain well giving high cerebrospinal fluid to plasma ratios [12]. The compound was shown to be active in a variety of rodent models of acute, inflammatory and neuropathic pain [13]. Unlike high affinity NMDA-receptor antagonists, CHF3381 does not display amnesic activity in mice unless doses much higher than those pharmacologically active in pain models are reached.

Figure 1.

The major metabolic pathways for CHF3381

In man, CHF3381 undergoes extensive liver metabolism with the formation of two major metabolites, CHF3567 [N-(2-indan-2-yl)glycine] catalyzed by an amide hydrolase and 2-aminoindane catalyzed by CYP2E1 (Paola Puccini, personal communication) (Figure 1). 2-Aminoindane has affinities of 91.2 µm for the nonspecific glutamate binding site of NMDA receptors and 79.1 µm for MAO-A. 2-Aminoindone was found to be active in the hot-plate test and in the formalin test in mice and in the Bennett test in rats (Villetti, personal communication). CHF3567 does not interact with NMDA receptors and MAO-A.

In healthy subjects, plasma concentrations of CHF3381 and of the two major metabolites were found to be proportional to dose [14]. The drug is excreted in urine as parent compound (2–6% of the administered dose) but mainly as metabolites (CHF3567, 50–55% and 2-aminoindane, 2–3%). Ingestion of food does not affect the extent of absorption of the drug, but the rate of absorption was reduced considerably. In healthy subjects, single oral doses of CHF3381 are well tolerated up to 600 mg [14].

In the present study we evaluated the safety, tolerability, pharmacokinetic and pharmacodynamic profiles of CHF3381 in healthy volunteers after multiple oral doses.

Methods

Study design

This was a double-blind, randomized, placebo-controlled, parallel group study of multiple oral doses of CHF3381 in 48 young healthy male subjects. Sixteen subjects were randomized in each of three panels to receive multiple oral doses of either CHF3381 (12 subjects) or placebo (four subjects) administered in capsules. The dose regimens of CHF3381 were 100, 200 and 400 mg once daily at day 1, 100, 200 and 400 mg twice daily (every 12 h) from day 2 to day 13 and 100, 200 and 400 mg once daily on day 14. Advancement to the subsequent panel was allowed after careful review of blinded safety and tolerability data of all subjects receiving the preceding dose regimen. The study was carried out at Biotrial, Rennes (France) and the study protocol was approved by the Institutional Review Board of Rennes (Comité Consultatif pour la Protection des Personnes se prêtant à la Recherche Biomédicale).

Subjects

Healthy male subjects from 18 to 35 years of age who had a body weight within 60–90 kg and a body mass index within ±20% of the ideal value were included in the study. Health was determined by medical history, complete physical examination, 12-lead electrocardiogram (ECG) and standard laboratory tests. Screening was carried out within 3 weeks from the first day of treatment administration (day 1). Subjects were asked to abstain from consuming alcoholic beverages from 48 h before the beginning of study medication and throughout the 17-day inpatient stay. Signed and dated written informed consent was obtained from all subjects.

Safety and tolerability assessments

Safety and tolerability were evaluated using spontaneously reported adverse events, physical examination, measurements of vital functions, ECG and laboratory tests. A complete physical examination was performed at screening on day −1 and at the end of the study. Supine and standing blood pressure and pulse rate were measured at screening, on day −1 and regularly throughout the treatment period (2 and 4 h postdose). ECG was recorded at screening, on day −1, day 1, day 4, day 10 and day 14 (predose and 2 and 4 h postdose in the morning). Urinalysis was performed at screening (including drug screen), on day −1 and at the end of the study. Haematology and blood chemistry were monitored at screening, on day −1, day 7, day 14 and at the end of the study.

Assessments of CNS effects

The potential effects of CHF 3381 on attention and memory were evaluated with the rapid visual information processing (RVIP) [15] and learning memory task (LMT) [16] tests, respectively. The effects of treatments on mood and behaviour were explored with the Bond & Lader visual analogue scales (BL-VAS) [17], whereas the effects on body sway (motor co-ordination) were measured using a posturography method [18]. The rapid visual information processing (RVIP) task is a test of sustained attention, which also requires working memory for its successful execution. Single digits are presented in quick succession (100 digits min⁻¹) on a computer screen. Subjects are instructed to press a response button as quickly as possible when they detect sequences of three consecutive odds or three consecutive even digits. On average, 80 of these sequences are presented over 10 min. Every two sequences are separated by a minimum of 5 and a maximum of 30 digits. After the appearance of the third digit of an experimental target, 1500 ms are allowed for a correct response to be made. Responses obtained at any other time are counted as errors. The scores are the percentage of correctly detected targets (accuracy) and the average reaction time [15]. LMT, BL-VAS were administered as described previously [14]. The body sway (BS) test is an objective, sensitive, reliable and noninvasive method designed to assess the effects of sedative drugs and alcohol on body sway and vigilance. BS is recorded using a force-platform. Marks corresponding to the foot size are fixed to the centre of the platform so that the feet can be accurately repositioned in order to obtain reliable measurements. Subjects are asked to stand erect and motionless, looking at a plumbline placed in front of them. Body sway (1 min with eyes open and 1 min with eyes closed) is recorded as recommended by the International Society of Posturography. The length and area of the postural oscillations are then calculated [18]. RVIP and BS were performed at screening, on day −1 and day 1, day 6 and day 12 at 3 h and 3.5 h postmorning dose, respectively. LMT and BL-VAS were administered at screening, on day −1 and day 1, day 7 and day 13 at 4 h and 4.5 h postmorning dose, respectively.

Electroencephalogram (EEG) recordings were carried out in all subjects at screening and on day 13 at 2.5 h postmorning dose as described previously [14].

Blood and urine sampling

Venous blood samples (7 ml) were collected immediately prior to the dose and 0.5, 1, 2, 3, 4, 6, 8, 10, 12, 16, and 24 h on day 1, immediately before the morning dose on day 3, day 5, day 8 and day 11 and predose and 0.5, 1, 2, 3, 4, 6, 8, 10, 12, 16, 24, 36 and 48 h on day 14. Blood was collected into heparinized tubes and immediately centrifuged at 1500 g for 20 min at 4 °C. Plasma was separated and frozen at −20 °C until assayed. Two urine aliquots (10 ml) were collected just before study drug administration. On day 1, urine was collected over the 0–12 and 12–24 h periods and on day 14, over the 0–12, 12–24, 24–48 and 48–72 h intervals.

Drug and metabolite analysis

Plasma and urine concentrations of CHF3381 and of its two main metabolites, CHF3567 and 2-aminoindane, were determined using a validated HPLC method with fluorescence detection [14]. The precision of the assay for the three compounds, expressed as coefficients of variation, ranged from 0.2 to 7.6% in plasma and from 0.7 to 9.1% in urine (at 5, 100 and 400 ng ml⁻¹). The accuracy, expressed as relative percent error, ranged from −3.5 to +6.3% in plasma and from −8.9 to +3.0% in urine. Plasma and urine samples were suitably diluted with blank human matrix if the concentrations were greater than the highest standard of the corresponding calibration curves. The lower limit of quantification in plasma was 2 ng ml⁻¹ for CHF3381 and CHF3567 and 1 ng ml⁻¹ for 2-aminoindane. The corresponding limits in urine were 20 ng ml⁻¹ and 10 ng ml⁻¹, respectively.

Pharmacokinetic analysis

Model independent pharmacokinetic parameters were calculated for CHF3381, CHF3567 and 2-aminoindane. The maximum plasma concentration (C_max) and the time of its occurrence (t_max) were obtained from individual data. The area under the plasma concentration vs time curve from 0 to 12 h (AUC(0,12 h)) was calculated using the linear trapezoidal rule. The area under the plasma concentration vs time curve from 0 to infinity (AUC(0,∞)) at day 1 was calculated for CHF3381 and CHF3567 from the expression AUC(0,24 h) + C_t /λ_z, where λ_z is the slope of the log-linear regression of the terminal concentration data points. The terminal elimination half-life (t_1/2) was calculated as ln (2)/λ_z. Extent of accumulation (R) was calculated as the ratio of AUC(0,12 h) at day 14 and at day 1. The percent of dose recovered in urine (A_U) was calculated by dividing the dose by the amount excreted in the urine at day 14 in the 0–12 h period. Renal clearance (CL_R) was calculated as the amount excreted in the urine at day 14 in the 0–12-h period by AUC(0,12 h).

Pharmacodynamic assessments

Venous blood samples (6.2 ml) for the measurement of MAO-A activity were collected immediately prior to the dose and after 1, 2, 3, 4, 6, 8, 12, 16, and 24 h on day 1, immediately before the morning dose on day 3, day 5, day 8 and day 11 and predose and 1, 2, 3, 4, 6, 8, 12, 16, 24, 36 and 48 h on day 14. The free plasma concentration of 3,4-dihydroxyphenylglycol (DHPG), the deaminated metabolite of norepinephrine was used as the measure of MAO-A activity. DHPG assays were performed using a validated HPLC method with electrochemical detection [19]. Briefly, the method involved fixation of DHPG on alumina under basic conditions followed by washing with water and defixation under acid conditions. The extract was analyzed on a reversed phase column (Symmetry Shield™, RP18, 100 Å, 250 × 4.6 mm, 5 µ, Waters, Milford, Massachusetts, USA). 3,4-dihydroxybenzylamine was used as internal standard. Precision and accuracy calculated at four concentrations (100, 250, 2000 and 3500 pg ml⁻¹) were higher than or equal to 91 and 90%, respectively.

Maximum MAO-A inhibition (I_max) and the time of its occurrence (t_max) were determined from individual data. The CHF3381 plasma concentration producing a 50% MAO-A inhibition (IC₅₀) was estimated for each subject at day 14 by linear regression.

Results

Four subjects in the placebo group, four in the 100 mg twice daily group, four in the 200 mg twice daily group, and 11 in the 400 mg twice daily group complained of adverse events, of which the most frequent were mild dizziness, asthenia and insomnia. Dizziness generally lasted 90 min, asthenia 2 h and insomnia 10 days. There was one case of orthostatic hypotension in a subject receiving 400 mg twice daily and a vasovagal reaction in a subject receiving 200 mg twice daily. On day 14, there was a modest dose-dependent increase compared with placebo in heart rate measured at 2 h postdose (6 ± 2 beats min⁻¹, 8 ± 2 beats min⁻¹ and 9 ± 2 beats min⁻¹, after 100 mg twice daily, 200 mg twice daily and 400 mg twice daily, respectively). On day 14, supine and standing diastolic blood pressure were slightly but significantly increased in the 400 mg twice daily group compared with placebo (6 ± 2 mmHg, P = 0.021 and 8 ± 2 mmHg, P = 0.005, respectively). On day 14, there was also a significant increase compared with placebo in the QTc interval in the 400 mg twice daily group (10 ± 4 ms). One subject receiving 400 mg twice daily complained of mild agitation and paresthesia. Another subject in the same treatment group complained of mild bradyphrenia. No clinically significant changes in laboratory values were observed. One subject in each of the three CHF3381 dose-regimen groups showed isolated and transient elevations in serum alanine aminotransferase slightly above the upper normal range at the end of the 2-week treatment period.

All EEG tracings recorded at screening and on day 13 were considered normal with layouts typical of those observed in healthy subjects. At screening, the EEG parameters of the four treatments groups were well balanced with the exception of the relative power of delta waves (0–4 Hz) at both F4-T4 (P = 0.028) and T3-O1 (P = 0.002) leads, with the 400 mg group having higher percentages compared with the other groups. On day 13, the increase in the 0–4 Hz wave activities observed in the 400 mg-treated subjects moved to the 4–8 Hz band (theta waves) with a slight but significant difference compared with placebo at both F4-T4 (23.2 ± 2.3%vs 17.3 ± 1.3%, P = 0.021) and T3-O1 (23.0 ± 2.2%vs 17.7 ± 1.5%, P = 0.044) leads. Simultaneously, a general trend towards a decrease in beta 1 (12–16 Hz, P = 0.016) and beta 2 (16–20 Hz, P = 0.065) relative power was observed in the 400 mg group compared with the placebo group.

There were no dose-dependent or consistent effects of CHF3381 on attention (RVIP), motor coordination (BS) or memory (LMT). On day 13, there was a significant decrease in alertness (BL-VAS) in the 400 mg twice daily group compared with placebo (56.8 ± 4.9 mm vs 74.0 ± 4.5 mm, P = 0.025).

The time course of plasma concentrations of CHF3381 and of the two main metabolites (CHF3567 and 2-aminoindane) on day 1 and day 14 of the different dose regimens are shown in Figure 2. The corresponding pharmacokinetic parameters are summarized in Tables 1–3. Plasma concentrations of CHF3381 peaked at around 3 h. AUC(0,12 h)s were dose-proportional (r = 0.914, P < 0.001). The elimination half-life of CHF3381 was estimated to be 4–6 h. On day 14, plasma CHF3381 concentrations were determined predose with a modest accumulation (R = 1.3–1.5). The mean ratio of the AUC(0,12 h) on day 14 to the AUC(0,∞) on day 1 of CHF3381 was significantly higher than unity (1.16 ± 0.03, 1.29 ± 0.03 and 1.41 ± 0.05, after 100 mg, 200 mg and 400 mg twice daily, respectively) indicating some time-dependent nonlinearity for the parent compound. Plasma concentrations of the two metabolites, CHF3567 and of 2-aminoindane, were also proportional to dose (Figure 2). At day 14 a modest accumulation was observed for CHF3567 (R = 1.3–1.4) whereas this was much greater for 2-aminoindane (3.7–4.8 fold) due to its relatively long elimination half-life (13–14 h). The mean AUC(0,12 h)_{day 14} : AUC(0,∞)_{day 1} ratio for CHF3567 was close to unity (1.11 ± 0.03, 1.04 ± 0.03 and 1.16 ± 0.04, after 100 mg, 200 mg and 400 mg twice daily, respectively) indicating time-linearity for the major metabolite. At steady-state, 7–9% of the administered dose was found in the 0–12 h urine as unchanged drug, 57–72% as CHF3567 and 6–7% as 2-aminoindane. Renal clearances for CHF3381 ranged between 24 and 28 ml min⁻¹. The high values for the renal clearance of CHF3567 (275–311 ml min⁻¹) suggest active tubular secretion of this acid metabolite. The renal clearance (89–120 ml min⁻¹) of the other metabolite, 2-aminoindane, was similar to the glomerular filtration rate.

Figure 2.

Mean (± SEM) plasma concentrations of CHF3381 (panel a), CHF3567 (panel b) and 2-aminoindane (panel c) measured after the first (open symbols) and the last dose (closed symbols) of 100 mg twice daily (circles), 200 mg twice daily (squares) or 400 mg twice daily (triangles) of CHF3381 in young healthy males

Table 1.

Main pharmacokinetic parameters (mean ± SEM) for CHF3381 in young healthy males (n = 12 per group) after different multiple oral doses given twice daily

	100 mg twice daily		200 mg twice daily		400 mg twice daily
Pharmacokinetic parameter	Day 1	Day 14	Day 1	Day 14	Day 1	Day 14*
Cmax (ng ml−1)	652 ± 36	760 ± 46	1209 ± 80	1522 ± 102	2232 ± 121	3216 ± 179
tmax (h)#	2.0 (0.5–4.0)	3.0 (1.0–4.0)	3.0 (0.5–4.0)	3.0 (1.0–4.0)	3.0 (0.5–3.0)	3.0 (1.0–3.0)
AUC(0,12 h) (ng ml−1 h)	3036 ± 139	3811 ± 242	6016 ± 365	8513 ± 613	11 624 ± 536	17 593 ± 1213
AUC(0,∞) (ng ml−1 h)	3262 ± 167		6629 ± 476		12 671 ± 644
t1/2 (h)		4.2 ± 0.3		5.7 ± 0.5		4.4 ± 0.3
R		1.25 ± 0.04		1.41 ± 0.04		1.54 ± 0.07
AU (%)		7.1 ± 1.0		7.2 ± 1.2		8.5 ± 0.9
CLR (ml min−1)		25.7 ± 2.3		23.7 ± 3.8		28.2 ± 3.6

R = accumulation ratio. A_U = percent of dose recovered in urine in the 0–12 h period.

^#Median and range.

^*n = 11.

Table 3.

Main pharmacokinetic parameters (mean ± SEM) for 2-aminoindane in young healthy males (n = 12 per group) after different multiple oral doses of CHF3381 given twice daily

	100 mg twice daily		200 mg twice daily		400 mg twice daily
Pharmacokinetic parameter	Day 1	Day 14	Day 1	Day 14	Day 1	Day 14*
Cmax (ng ml−1)	17.7 ± 0.7	56.0 ± 3.3	41.91 ± 4.0	134.4 ± 10.5	77.7 ± 4.2	304.3 ± 21.0
tmax (h)#	7.0 (4.0–10.0)	3.0 (2.0–8.0)	8.0 (6.0–12.0)	4.0 (2.0–8.0)	8.0 (4.0–12.0)	4.0 (2.0–8.0)
AUC(0.12 h) (ng ml−1 h)	152 ± 4	565 ± 31	326 ± 16	1361 ± 07	665 ± 30	3136 ± 10
t½ (h)		14.2 ± 0.6		12.7 ± 0.7		13.9 ± 0.8
R		3.72 ± 0.18		4.18 ± 0.27		4.82 ± 0.27
AU (%)		6.8 ± 0.4		5.9 ± 0.8		6.9 ± 0.8
CLR (ml min−1)		120.0 ± 7.5		88.3 ± 12.3		88.9 ± 10.5

R = accumulation ratio. A_U = percent of dose recovered in urine in the 0–12 h period.

^#Median and range.

^*n = 11.

Table 2.

Main pharmacokinetic parameters (mean ± SEM) for CHF3567 in young healthy males (n = 12 per group) after different multiple oral doses of CHF3381 given twice daily

	100 mg twice daily		200 mg twice daily		400 mg twice daily
Pharmacokinetic parameter	Day 1	Day 14	Day 1	Day 14	Day 1	Day 14*
Cmax (ng ml−1)	384 ± 16	449 ± 17	719 ± 52	797 ± 51	1292 ± 02	1663 ± 188
tmax (h)#	4.0 (1.0–4.0)	4.0 (3.0–4.0)	4.0 (3.0–6.0)	4.0 (3.0–4.0)	4.0 (3.0–6.0)	4.0 (3.0–6.0)
AUC(0,12 h) (ng ml−1 h)	2515 ± 94	3272 ± 119	4807 ± 306	5959 ± 350	8869 ± 711	12 280 ± 1066
AUC(0,∞) (ng ml−1 h)	2971 ± 129		5773 ± 345		10 537 ± 815
t1/2 (h)		8.71 ± 0.74		7.4 ± 0.5		7.2 ± 0.4
R		1.31 ± 0.03		1.25 ± 0.04		1.38 ± 0.07
AU (%)		71.5 ± 3.3		58.5 ± 4.3		56.8 ± 4.6
CLR (ml min−1)		310.8 ± 16.7		288.4 ± 28.5		274.5 ± 28.4

R = accumulation ratio. A_U = percent of dose recovered in urine in the 0–12 h period.

^#Median and range.

^*n = 11.

CHF3381 dose-dependently inhibited MAO-A activity. On day 1, maximum inhibition occurred after 2–3 h from the first dose and was 14 ± 5%, 35 ± 2% and 50 ± 7% after 100 mg, 200 mg and 400 mg, respectively. On day 14, peak inhibition increased to 27 ± 4%, 46 ± 2% and 65 ± 5% after 100 mg twice daily, 200 mg twice daily and 400 mg twice daily, respectively (Figure 3). A significant residual inhibition was measured at predose (6 ± 6%, 16 ± 4% and 39 ± 6% after 100 mg twice daily, 200 mg twice daily and 400 mg twice daily regimens, respectively). Inspection of plots of CHF3381 plasma concentration vs changes from baseline of MAO-A activity at both day 1 and day 14 indicate the absence of hysteresis. The mean (SD) CHF3381 plasma concentration producing a 50% MAO-A inhibition (IC₅₀) was estimated to be 1478 ± 119 ng ml⁻¹ based on linear regression (r = 0.861 ± 0.016).

Figure 3.

Inhibition of MAO-A after placebo and different dose regimens of CHF3381 in young healthy males at steady state. Placebo bid (n = 12) (○), 100 mg bid (n = 12) (•), 200 mg bid (n = 12) (▪), 400 mg bid (n = 11) (▾)

Discussion

This study showed that CHF3381 is well tolerated in young healthy subjects when given in multiple oral doses up to 400 mg twice a day for 2 weeks. The maximum tolerated dose was not achieved in this study, since serious or severe adverse events were not observed. The main adverse events likely to be associated to CHF3381 administration were dizziness, asthenia and insomnia. With the 400 mg twice daily dose regimen their frequency was higher than in the placebo group, but their severity was judged by the subjects to be mild except in one case of moderate orthostatic hypotension. Dizziness and asthenia were also observed after single doses of 200 mg or higher [14]. Dizziness is frequently reported for other NMDA antagonists such as ketamine [20], dextromethorphan [21] and memantine [22]. Asthenia has also been reported for ketamine [3]. Insomnia has been described in demented patients during treatment with memantine [23]. Insomnia has been also associated with MAO-A selective inhibitors [24, 25].

Heart rate and blood pressure were statistically significantly affected by CHF3381, but the effects were not judged to be clinically significant. The isolated cases of palpitations and orthostatic hypotension (400 mg twice daily) and vasovagal attack (200 mg twice daily) did not lead to discontinuation of treatment. QTc interval in the 400 mg twice daily group was slightly higher compared with placebo (+ 10 ± 4 ms, P = 0.021). However, none of subjects receiving 400 mg twice daily had abnormal QTc intervals (> 440 ms), the maximum value being 427 ms for subject 36 on day 4 (2 h post dose). A higher prolongation of the QTc interval compared with baseline (+ 42 ms) was observed in subject 41 on day 1 (4 h post dose), but the actual value was still normal (416 ms). QTc interval is defined by Bazett's formula ((mean QT interval)/SQR(mean RR interval)). Thus, it is evident that if heart rate increases (RR interval decreases), the QTc ratio increases. Thus, the modest higher QTc intervals we observed with 400 mg twice daily compared with placebo could simply reflect the modest increase in heart rate found at this dose. In vitro studies have shown that CHF3381 (10 µm) does not bind to receptor site 2 of sodium channels, adenosine triphosphate-sensitive potassium channels, small-conductance calcium-activated potassium channels SKca, N-type or L-type calcium channels (Villetti, personal communication).

Several NMDA antagonists have shown important haemodynamic and cardiovascular effects in humans that may restrict their clinical use [26]. Dose-dependent blood pressure elevation has been described for ketamine in geriatric patients [27]. Dextrorphan causes transient but clinically significant hypotension and later hypertension in patients with acute stroke [28]. The good cardiovascular tolerability of CHF3381 shown in this study after administration of single doses supports the suggestion that low-affinity, noncompetitive antagonists may be better tolerated than high-affinity competitive antagonists [29].

Few CNS adverse events were observed in this study, with only single and mild cases of bradyphrenia, agitation and paresthesia with the 400 mg twice daily doses. No psychotomimetic episodes were observed. None of the dose regimens impaired memory or attention. However, a decrease in alertness was observed at steady-state in the 400 mg twice daily group, 4.5 h after the morning dose. This effect has also been observed after single doses of 300 mg or 600 mg in healthy subjects [14]. The EEG data in the present study indicated a possible moderate relaxant effect of CHF3381 at 400 mg twice daily, the effect being consistent with the decrease in alertness. Overall, the central effects of CHF3381 appear modest compared with other potent NMDA antagonists like ketamine or selfotel, again suggesting that low-affinity noncompetitive antagonists are well tolerated in humans [30].

The AUCs and C_max of CHF3381 and its two major metabolites were found to be proportional to the dose. However, some departure from linearity was observed on day 14, AUC(0,12 h)s being significantly higher than AUC(0,∞)s measured on day 1. The reason for this time-dependent nonlinearity is unclear but a decrease in plasma clearance has been hypothesized (Davide Verotta, personal communication). The main pharmacokinetic parameters showed relatively low variability with coefficients of variation ranging from 15% to 27% for total exposure (AUC(0,∞)) and from 20% to 31% for terminal half-life (t_1/2). At steady-state CHF3381 plasma concentrations were detected predose and had an accumulation ratio of 1.3–1.5, suggesting that a twice a day regimen is appropriate from a pharmacokinetic point of view. The accumulation of the major metabolite, CHF3567, was also modest (1.3–1.4). Conversely, significant accumulation (4–5 fold) was observed at steady-state for 2-aminoindane due to its relatively long elimination half-life (13–14 h). This may explain the minor haemodynamic effects observed at day 14, since 2-aminoindane has been shown to possess amphetamine-like activity [31]. However, peak plasma 2-aminoindane concentrations at steady state were still about 10 times lower than those of parent compound.

This study has demonstrated that CHF3381 produces a dose-dependent inhibition of DHPG plasma concentration, which is a good marker of the magnitude and duration of MAO-A inhibition in humans [19, 32–34]. Values measured after administration of placebo in the present and in previous studies [19] are reasonably stable, confirming the lack of significant diurnal variation [35]. A dose-dependent decrease in plasma concentrations of DHPG has been shown with other reversible and selective MAO-A inhibitors like moclobemide [36], befloxatone [19], brofaromine [37] and toloxatone [38]. The extent of the effect of CHF3381 on DHPG concentrations increased at steady-state with significant trough inhibition measured at predose, thus suggesting that a twice a day regimen is more than adequate. The data did not support the existence of hysteresis, thus suggesting a direct relationship between plasma CHF3381 concentration and MAO-A inhibition.

In vitro, CHF3381 interacts with the MAO-A enzyme and the NMDA ion channels with similar affinity (IC₅₀s of 7.8 and 8.1 µm, respectively) [10]. The affinity for MAO-A enzyme corresponds to a concentration of 1768 ng ml⁻¹, quite close to the IC₅₀ for MAO-A inhibition we found in the present study (1478 ng ml⁻¹). Thus, there is good agreement between the in vitro and in vivo activity of CHF3881 with respect to MAO-A inhibition. Because the affinity of CHF3381 for MAO-A and NMDA receptors is similar, one can speculate that when CHF3381 plasma concentrations reach 1,500–2000 ng ml⁻¹, significant NMDA occupancy should be achieved in humans. These plasma concentrations were approached and exceeded in the present study with the 200 mg twice daily and the 400 mg twice daily dose regimens, respectively. Plasma concentrations of about 2000 ng ml⁻¹ are also achieved by effective oral doses of CHF3381 (100 mg kg⁻¹) in rat neuropathic pain models [13]. Thus, CHF3381 plasma concentrations achieved after the 400 mg twice daily dose regimen should be high enough to produce a significant block of NMDA receptors, and exceed those observed in animals receiving pharmacologically active doses.