AVN-101: A Multi-Target Drug Candidate for the Treatment of CNS Disorders

Abstract

Lack of efficacy of many new highly selective and specific drug candidates in treating diseases with poorly understood or complex etiology, as are many of central nervous system (CNS) diseases, encouraged an idea of developing multi-modal (multi-targeted) drugs. In this manuscript, we describe molecular pharmacology, in vitro ADME, pharmacokinetics in animals and humans (part of the Phase I clinical studies), bio-distribution, bioavailability, in vivo efficacy, and safety profile of the multimodal drug candidate, AVN-101. We have carried out development of a next generation drug candidate with a multi-targeted mechanism of action, to treat CNS disorders. AVN-101 is a very potent 5-HT7 receptor antagonist (Ki = 153 pM), with slightly lesser potency toward 5-HT6, 5-HT2A, and 5HT-2C receptors (Ki = 1.2-2.0 nM). AVN-101 also exhibits a rather high affinity toward histamine H1 (Ki = 0.58 nM) and adrenergic α2A, α2B, and α2C (Ki = 0.41-3.6 nM) receptors. AVN-101 shows a good oral bioavailability and facilitated brain-blood barrier permeability, low toxicity, and reasonable efficacy in animal models of CNS diseases. The Phase I clinical study indicates the AVN-101 to be well tolerated when taken orally at doses of up to 20 mg daily. It does not dramatically influence plasma and urine biochemistry, nor does it prolong QT ECG interval, thus indicating low safety concerns. The primary therapeutic area for AVN-101 to be tested in clinical trials would be Alzheimer's disease. However, due to its anxiolytic and anti-depressive activities, there is a strong rational for it to also be studied in such diseases as general anxiety disorders, depression, schizophrenia, and multiple sclerosis.

Keywords: 5-HT7 receptor antagonists; Adrenergic alpha-2 antagonists; Alzheimer’s disease; Parkinson’s disease; anxiety; central nervous system agents; histamine H1 receptor antagonists; memory; serotonin receptor antagonists.

Fig.1

Affinities, pK_i, of AVN-101 and its major metabolites, M1 and M2, to different targets, as measured in radio-ligand binding competition assay. Assessment of the binding affinity was performed by the Contract Research Organization, Ricerka, in accordance with their protocols briefly described in [71].

Fig.2

Affinities of AVN-101 and SB742457 to 5-HT7 (A) and 5-HT2A (B) receptors in competitive radioligand binding assay. Radio-labeled 5.5 nM [³H] lysergic acid diethylamide was used for 5-HT₇ receptor and 0.5 nM [³H] ketanserin was used for 5-HT_2A receptor.

Fig.3

A) Kinetics of Ca^2 + mobilization induced by αMet5-HT (5μM) without (dashed line) and in the presence of 10μM AVN-101 (solid line) in HEK 297 cells extragenously expressing the human recombinant 5-HT_2B receptor. Arrows indicate additions of the corresponding compounds. B) Activation (squares) and inhibition of the αMet5-HT-induced rat stomach fundus contraction (circles) with AVN-101.

Fig.4

A) AVN-101-induced blockade of cell responses (SK-N-SH endogenously expressing H1 receptors and CHO-K1 exogenously expressing human recombinant H2 receptors) to corresponding agonists, 10μM histamine and 50 nM amthamine. Histamine induced Ca^2 + mobilization in SK-N-SH cells and amthamine induced cAMP accumulation in CHO-1K cells. B) AVN-101 effectively competes for the H1 and H2 receptors, expressed in CHO-K1 cells, with corresponding radio-labeled ligands, 1.2 nM [³H]Pyrilamine and 0.1 nM [¹²⁵I]Aminopotentidine.

Fig.5

4 h equilibrium concentrations of AVN-101 in dialysis apparatus, whole human blood plasma in one chamber and PBS (pH 7.2) in another. Fb –AVN-101 fraction bound to plasma proteins.

Fig.6

AVN-101-induced activation of Pgp ATPase activity.

Fig.7

Kinetics of AVN-101 (1μM) decomposition by rat and human microsomes.

Fig.8

Metabolites of AVN-101digestion identified in human (HLM) and rat (RLM) microsomes. Solid arrows indicate metabolites determined in the HLM “soup” and dashed ones indicate metabolites identified in the RLM. Grayed out areas on the structures indicate possible oxidation points.

Fig.9

Metabolism of AVN-101 in a suspension of S9 fraction isolated from rat (A and B) and human (C and D) liver in the presence of i) only phase 1 metabolism co-factor, NADPH (closed circles), ii) only phase 2 metabolism co-factor, UDPGA (closed squares), and iii) when both phase 1 and phase 2 co-factors were present (open circles). AVN-101 was tested at a concentration of 1μM (A and C) and 2μM (B and D).

Fig.10

Pharmacokinetics of AVN-101 in mice upon IP administration at a dose of 5 mg/kg.

Fig.11

AVN-101 PK in mice blood upon IP administration. A) Dose dependence of maximal concentration (C_MAX) and exposure (AUC_0 - >∞) of the AVN-101. B) Half-life of elimination (T_1/2) of the AVN-101.

Fig.12

Pharmacokinetics of AVN-101 (dose 5 mg/kg) in Wistar rats, administered through IV, IP, and PO routes.

Fig.13

A) Content of AVN-101 (μg/mL) in Wistar rat tissues 4 h after either IV (5 mg/kg) or PO (10 mg/kg) administration. B) Ratio of tissue AVN-101 concentrations, IV administration over PO administration, normalized by the corresponding dose.

Fig.14

Pharmacokinetics of AVN-101 and its metabolites, M1 and M3, in plasma (A), brain (B), and CSF (C) of Sprague Dawley rats. AVN-101 was administered as a bolus in physiologic saline solution at a dose of 2 mg/kg through the tail vein.

Fig.15

The AVN-101 distribution in blood, plasma, and cells as well as in CSF and brain of Sprague Dawley male rats administered IV with 2 mg/kg dose of the drug. The tissue samples were collected 5 min after the drug injection.

Fig.16

Kinetics of AVN-101 diffusion through semi-permeable membrane of a 48-well dialysis plate. A) Donor chamber contained diluted with PBS 20% plasma with 1μM AVN-101 and acceptor chamber contained rat brain homogenate (A), both chambers contained 20% rat plasma with 1μM AVN-101 in the donor chamber (B), and both chambers contained PBS with 1μM AVN-101 in the donor chamber (C).

Fig.17

Ratio of time spent in open arm over the closed arm (A), ratio of number of visits into open arm over closed arm (B), number of defecations (C), and total number of visits (D) in the elevated plus-maze test. Significance (Student t-test) of difference between each of the experimental groups and placebo (control) group: ^*p < 0.05; ^**p < 0.01; ^***p < 0.005; and ^****p < 0.001.

Fig.18

Ratio of time spent in open arm over the closed arm (A), ratio of number of visits into open arm over closed arm (B), number of defecations (C), and total number of visits (D) in the elevated plus-maze test. Significance (Student t-test) of differences between each of the experimental groups and placebo (control) group: ^*p < 0.05; ^**p < 0.01; ^***p < 0.005.

Fig.19

Cumulative time in frozen state of male BALB/c mice in the elevated platform test. Group 1 –control (saline vehicle), Group 2 - Lorazepam (0.05 mg/kg) and Group 3 - AVN-101 (0.2 mg/kg). Each group included at least 8 animals. Mean values ± SE. Difference from placebo group: *p < 0.05 (ANOVA’s Fisher LS test).

Fig.20

Time duration spent by male BALB/c mice in the central zone of open-field platform. Significance of the difference of test groups, Lorazepam and AVN-101 from control (placebo) group: ^*p < 0.05; ^**p < 0.01 (ANOVAs Fisher LS test).

Fig.21

Relative changes of rat responses during unpunished (open bars) and punished (closed bars) sessions in the Geller-Seifter conflict test.

Fig.22

Effect of Fluoxetine and Desipramine (both at 15 mg/kg) and AVN-101 (0.05 mg/kg) on mice immobility in (A) the Porsolt forced swim test and (B) tail suspension test. ^*p < 0.05, ^**p < 0.01, ^***p < 0.001 (Student t-test).

Fig.23

Latency time before the first entry of BALB/c mice into the dark box (A), duration of staying in the white compartment (B) and number of entries into the dark compartment (C) measured 24 h after the electric shock was given (mean ± SEM). Difference from scopolamine-treated placebo group: ^*p < 0.05; ^**p < 0.01; ^***p < 0.001 (Student t-test).

Fig.24

Latency time before the first entry of Spraque Dawley rats into the dark box (A), duration of staying in the white compartment (B), and number of entries into the dark compartment (C) measured 24 h after the electric shock was given (mean ± SEM). Difference from scopolamine-treated placebo group: ^*p < 0.05; ^**p < 0.01; (Student t-test).

Fig.25

Latency time for the first entry into the dark box (A), duration of staying in the white compartment (B) and number of entries into the dark compartment (C) measured 24 h after the electric shock was given (mean ± SEM). Difference from MK-801-treated placebo group (Placebo 2): ^*p < 0.05 (Student t-test).

Fig.26

The Morris water maze test for reversal of scopolamine-induced amnesia in male BALB/c mice. Ordinate axis: the time (mean ± SE) spent in the quadrant where the “safe” platform was located during two previous training days. Difference from placebo 2 group: ^*p < 0.05; ^**p < 0.01 (Student t-test).

Fig.27

Effect of the AVN-101 and positive control drugs, memantine and SB-742457, on novel object recognition index in male BALB/c mice (mean ± SE) upon scopolamine (A) or NK-801-induced amnesia (B). Difference from group administered with scopolamine (Placebo 2): ^*p < 0.05; ^**p < 0.01 (Student test).

Fig.28

Effect of AVN-101 and Haloperidol on apomorphine-induced disruption of pre-pulse inhibition in SHK male mice. Difference from group administered with Apomorphine (Placebo 2): ^*p < 0.05; ^**p < 0.01 (Student test).

Fig.29

Q-T prolongation in guinea pigs by AVN-101 and positive control amiodaron.

Fig.30

QT interval in Rhesus Macaca mulatta upon IV administration of positive control drug amiodaron (15 mg/kg) or AVN-101 (1 mg/kg).

Fig.31

Effect of AVN-101 and control substances on reverse mutation rate (Ames test) of Salmonella typhimurium strains TA98 (light gray), TA1535 (dark gray), and TA1537 (black). Results for the highest AVN-101 concentration tested, 50μM, are shown. Positive controls for TA98 (4-NOPD), TA1535 (Na-azide), and TA1537 (9-AA), were tested at respective concentrations of 15μg/mL, 2μg/mL, and 50μg/mL. Effects of the compounds are presented as a fold increase over negative control, non-treated bacteria cultures (mean ± SE). Dotted line shows negative control level and dashed line represents the 3-fold threshold that is considered as a significant level for mutagenic effect.

Fig.32

Kinetics of AVN-101 and its metabolites, M1 and M2, in plasma of human healthy volunteers after single AVN-101 PO administration at different doses.

Fig.33

Relation between an AVN-101 dose upon PO administration and blood concentration of AVN-101 and its two metabolites, M1 and M2, in humans. A) Peak concentration, C_max (mean ± SD). B) Exposure, AUC (mean ± SD).