Journal of Alzheimer’s Disease, 2016, 53(2): 583-620.

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

Alexandre V. Ivachtchenko, Yan Lavrovsky, Ilya Okun

Alla Chem LLC, Hallandale Beach, FL, USA

Avineuro Pharmaceuticals Inc., Hallandale Beach, FL, USA

R-Pharm Overseas, Inc., San Diego, CA, USA

 

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 5-HT2C 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.

DOI: 10.3233/JAD-151146

 

Supplementary

Currently, it is well understood that many diseases have very complex etiologies with different regulatory and metabolic circuits being in a tight intercommunication to provide a stable balance of incoming and outcoming signals and metabolites that maintain an organism in a balanced state. Changes in one element of a signaling or metabolic circuit can often be compensated by the other circuits in the net. However, even small changes in several inter connected circuits, can lead to a profound amplification of the dysbalanced disease state due to such inter circuit communications. Many diseases, especially those that develop at an older age, are the result of accumulation of somatic mutations in many genes responsible for coding of key proteins maintaining the balanced signaling/metabolic net. This is true for many CNS diseases including Alzheimer’s, Parkinson’s, etc. A number of marketed drugs that have accidentally been discovered, are multi-target ligands (1,2). For such complex diseases as Alzheimer’s, which includes both the physical destruction of the neurons and changes in brain functions, memory, recognition, depression etc., often bundled with cardiovascular and metabolic disorders, the effective drugs could be those with the multi-target modality. The major challenge in the multi-modal drug design is a complexity of the target associations that represent right combination of the targets and a drug relative affinities to those targets that make a drug the drug.

In this work, we describe one of such multi-targeted drug candidates, AVN-101, with emphasis on 5-HT7 and 5-HT6 receptors exclusively located in the brain tissue, 5-HT2A and 5-HT2C receptors, adrenergic α2A, α2B, α2C, and histamine H1 receptors (Figure 1).

 

 

Figure 1. Affinities, pKi, of AVN-101 to different targets determined in radio-ligand binding competition assays.

 

Indeed, in the animal models of anxiety (elevated plus-maze, elevated platform, open field platform, and Geller-Seifer conflict), depression (Porsolt forced swim and tail suspension), Scopolamine- or MK-801-induced amnesia (passive avoidance, Morris water maze, novel object recognition), and psychosis (pre-pulse inhibition), the AVN-101 demonstrated high efficacy as anxiolytic, antidepressant, antiamnesic, and antipsychotic drug.

We speculate that while serotonin receptors are implicated in psychosis, memory, cognitive dysfunctions, depression, and anxiety, the blockade of the “accessory” receptors, which the AVN-101 also interacts with, could have positive effects in some special cases. For example, antagonism of the H1 and H2 receptor broadly present in the brain, could be efficacious in preventing CNS inflammation and/or treating multiple sclerosis (3). Blockade of the pre-synaptically located adrenergic α2A and α2C receptors could affect the CNS physiologic functions based on modulation of negative feedback circuitry controlling inhibition of the noradrenalin release (4). Due to a lack of highly selective α2B antagonists, the role of post synaptically located adrenergic α2B in mental disorders is not quite clear yet.

Permeability through a blood-brain barrier presents the major challenge in developing drugs for use in treatment of mental diseases. If it cannot permeate into the brain, a drug with even ideal multi target pattern would have no practical utility. When studying the AVN-101 permeability, we unexpectedly discovered its unusual tissue distribution behavior: The AVN-101 accumulated in rat brain very quickly, less than in 5 min, against its concentration gradient in plasma. Also, there was a clear barrier between brain and cerebrospinal fluid (CSF) (Table 1, Figure 2A).

 

Table 1. AVN-101 concentration in plasma, brain, and CSF of Sprague Dawley rats 5 min after IV administration at the dose of 2 mg/kg

Tissue Plasma Brain CSF
C, ng/mL 274 4,665 11

 

The PK data clearly showed that the AVN-101 accumulated in the brain tissue. Two mechanisms could have been responsible for the high brain concentration of the AVN-101. One mechanism – potentially greater binding affinity and capacity of the brain matter, such as lipids, proteins etc. than those in blood and CSF. Second mechanism includes participation of some active pumps on the border membranes between the brain and blood and brain and CSF.

To explore the first mechanism, we performed a dialysis experiments whereby, the brain homogenate and plasma, both diluted 1:4 with a buffer, were placed into corresponding chambers of a RED (Rapid Equilibrium Dialysis) apparatus and the AVN-101 was spiked into the plasma-containing chamber. The relative concentrations were measured in both chambers at different incubation times during the incubation at 37 °C. The rationale behind such approach was based on assumption that the dialysis membrane would serve as a model of the blood/brain barrier deprived of the active pumps and transporters. The AVN-101 was spiked into plasma to model the transition of the AVN-101 from blood to brain (Figure 2B).

 

Figure 2. A. PK of the AVN-101 in plasma, brain, and CSF in Sprague Dawley rats intravenously administered with 2 mg/kg AVN-101. B. Dialysis of the AVN-101 spiked into rat plasma against rat brain homogenate (both the plasma and homogenate are diluted 1:4 with a buffer. As a control, dialysis of the AVN-101 dissolved in the buffer against the buffer.

 

First, while accumulation into brain is achieved at a very high speed, CMAX is attained at 5 minutes or less (Figure 2A), free diffusion of the AVN-101 through the semipermeable membrane in the dialysis apparatus is quite slow: diffusion T1/2 = 237 min in plasma vs. brain homogenate dialysis system and even in the buffer vs. buffer dialysis system the T1/2 = 75 min (Figure 2B). Besides, in both dialysis systems, AVN-101 in plasma against brain homogenate and AVN-101 in the buffer against buffer, the AVN-101 concentration tends to equilibrate at the brain/plasma ratio of 1. If the brain matter would have binding capacity higher than that of plasma, one could expect the brain/plasma ratio to be higher than 1. So, the relatively high brain concentration of the AVN-101 and its very fast accumulation in the brain are not caused by the simple dissolution of the compound in the brain matter and rather indicates participation of pumps facilitating transport of the AVN-101 into brain against its concentration gradient. The PK data (Figure 2A), while surprisingly, also indicates that the CSF circulation has a tight barrier against AVN-101 on the blood/CSF and brain/CSF borders.

We have attempted to assess if serotonin transporter that has been shown to exist in the blood brain border (5) could be responsible for the AVN-101 transport into the brain. The conception of the experiment was construed to see if a serotonin transporter blocker, fluoxetine (10 mg/kg, IV) could prevent the counter gradient accumulation of the AVN-101 (2 mg/kg, IV) in brain. The male Spraque Dawley rats were administered first with the fluoxetine (or PBS as a control) and 40 min later, with the AVN-101. Three animals per group were sacrificed in a CO2 chamber at the indicated time points. The CSF and blood were drawn and then brains were removed. The brains were homogenized in 1:4 PBS. The blood and CSF sample were also diluted 1:4 with the PBS and the AVN-101 was extracted with acetonitrile for further LC/MS-MS analysis.

The data presented in Figure 3 demonstrate absence of any significant participation of the serotonin transporter into the facilitated entry and accumulation of the AVN-101 in the brain as well as substantially reduced concentration of the drug in the CSF circulation system.

 

 

Figure 3. Pharmacokinetics of the AVN-101 in blood, brain, and CSF in male Sprague Dawley rats administered IV (2 mg/kg) through a tail vein. 40 minutes prior to the AVN-101 treatment, the rats were administered with either 10 mg/kg Fluoxetine or vehicle (0.9% NaCl salt solution).

 

Importance of the study: our data suggests that multi-modal ligands targeting different receptor systems could be beneficial for treatment diseases of complex etiology. Further work to identify relative roles of the receptor systems in maintaining a homeostatic balance of different signalling and metabolic circuitries needs to be facilitated to enable rational design of drugs with right multi receptor target combination. The AVN-101 of the present work may represent the relevant compound to mediate this effort. Another discovery made with the AVN-101 shows that there some active pumps might be present in the blood/brain and CSF/brain border cells that could facilitate permeation and even accumulation of a drug in the brain.

 

References:

  1. Lipinski CA. Phenotypic and In Vivo Screening: Lead Discovery and Drug Repurposing. In: Morphy JR, Harris CJ, editors. Designing Multi-Target Drugs. Royal Society of Chemistry; 2012. p. 86–93.
  2. Hornberg JJ. Simple Drugs Do Not Cure Complex Diseases: The Need for Multi-Targeted Drugs. In: Morphy JR, Harris CJ, editors. Designing Multi-Target Drugs. Royal Society of Chemistry; 2012. p. 1–13.
  3. Saligrama N, Noubade R, Case LK, del Rio R, Teuscher C. Combinatorial roles for histamine H1-H2 and H3-H4 receptors in autoimmune inflammatory disease of the central nervous system. Eur J Immunol. 2012 Jun;42(6):1536–46.
  4. Philipp M, Brede M, Hein L. Physiological significance of alpha(2)-adrenergic receptor subtype diversity: one receptor is not enough. Am J Physiol Regul Integr Comp Physiol. 2002 Aug 1;283(2):R287-295.
  5. Nakatani Y, Sato-Suzuki I, Tsujino N, Nakasato A, Seki Y, Fumoto M, et al. Augmented brain 5-HT crosses the blood-brain barrier through the 5-HT transporter in rat. Eur J Neurosci. 2008 May;27(9):2466–72.

 

Contact:

Ilya Okun, Ph.D.

Vice President (retired)

AllaChem LLC, 1835 E. Hallandale Beach Blvd., #442

Hallandale Beach, FL 33009

e-mail: ilyaokun@sbcglobal.net

Phone: 858-775-6705

Fax: 858-630-3330

 

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