Pipeline

Diagram of Cerebral brain image by cevello

Cervello Therapeutics is focusing on enhanced Fasudil treatment which has resulted in decreased prevalence of Cerebral Cavernous Malformation (CCM) lesions1, a significant decrease in mature CCM lesion burden and improved survival in murine models of CCM2, with its therapeutic benefit having been indirectly attributed to inhibition of Rho Kinase (ROCK). Cervello Therapeutics goal is to develop novel derivatives of Fasudil with markedly improved ROCK potency and kinase selectivity, as well as enhanced properties for oral dosing, to better allow for interrogation of the role of ROCK inhibition in ameliorating CCM; our efforts have resulted in compounds showing > 100-fold stronger potency for ROCK inhibition than Fasudil and hydroxy-Fasudil.

Our efforts have resulted in a novel chemical series bearing little similarity to any other ROCK inhibitor in the literature and patent landscape. Lead compounds show >100-fold improvement in potency for ROCK inhibition over fasudil and hydroxy-fasudil, along with markedly improved selectively over the human kinome. We believe that the overall profile of these molecules positions them as uniquely suitable for a potential therapeutic to help existing patients heal CCM lesions, in both familial and sporadic cases of the disease.

We have selected CVT-100069 and CVT-100077 as development candidates that have both shown clear in vivo efficacy in terms of Cerebral Cavernous Malformation (CCM) lesion load in pre-clinical studies. Based on these findings, we are currently advancing CVT-100069 and CVT-100077 into IND-enabling studies.

Structural Basis for Design of Novel Derivatives of Fasudil

Diagram for Fasudil in ROCKI

PDB 2ESM (3.20 Å) Fasudil in ROCKI

Diagram for Hydroxy-Fasudil in ROCKI

PDB 2ETK(2.96 Å) Hydroxy-Fasudil in ROCKI

Diagram of Fasudil & hydroxy-fasudil bind ROCKI

Fasudil & hydroxy-fasudil bind ROCKI with inverted binding modes.

Literature Reports of Fasudil Demonstrate Need for Improved Metabolic Stability

Mouse Liver Hepatocyte Metabolism3
CL int, in vitro 36 ± 13 μL/min/106 cells
mouse scaled CL int, in vitro 359 ± 245 ml/min/kg
human scaled CL int, in vitro 122 ± 46 ml/min/kg
In Vivo PK 3
mouse CL t 198 ± 15 mL/min/kg
mouse t1/2 0.30 ± 0.1 h
mouse CL int,in vivo 1588 mL/min/kg
human CLt 73 mL/min/kg
human t 1/2 0.26 h
human CL int,in vivo 371 mL/min/kg
Hydroxy-fasudil is the main metabolite and also bioactive.
Hydroxy-Fasudil in ROCKI

Structure-Based Design of Fasudil Analogs

Fasudil in ROCKI
PDB 2ETK (2.96 Å) Hydroxy-fasudil in ROCKI
PDB 4L6Q (2.79 Å) benzoxaborole in ROCKII
Fasudil in ROCKI
PDB 4L6Q (2.79 Å) benzoxaborole in ROCK

ROCKI & II crystal structures were used to design analogs of hydroxy-fasudil, with the primary objectives of improving potency & selectivity against the AGC kinase family.

Cervello ROCKII, ROCKI, PKA, PKBα & PKG Data Illustrate a Need for Enhanced Kinome Selectivity

IC50 Values (nM)
Compound ROCKII* ROCKI* PKA PKA/ROCKII PKBa (AKT1) PKBa/ROCKII PKG* PKG/ROCKII
Fasudil 351 476 n/a n/a n/a n/a 3,529 10
Hydroxy-fasudil 252 476 7,030 28 2605 10 4,346 17

* IC50 values were determined with 14-point dose-response curves using a fluorescence coupling enzymatic assay using purified C-terminal truncated human ROCKI enzyme kinase domain (aa 1-477) fused to GST (Carna BioSciences, Catalog # 01-109) and purified C-terminal truncated human ROCKII enzyme kinase domain (aa 1-553) fused to GST (Carna BioSciences, Catalog # 01-110). Full length cGMP-dependent protein kinase 1 (PGK or PRKG1; aa 1-686) fused to GST (Carna BioSciences, Catalog # 01-142) was used as a representative control for offtarget kinase activity within the AGC-family.

Hydroxy-fasudil is the main metabolite and also bioactive.
Hydroxy-Fasudil in ROCKI
CVT-100069 at 400 nM
CVT-100069 at 400 nM
DiscoverX Kd (nM)
Compound ROCKII ROCKI
Hydroxy-fasudil 4100 2400
CVT-100069 18 29
CVT-100077 16 16

403 non-mutant kinases in panel
(including ROCKI/II)

CVT-100077 at 400 nM
Hydroxy-Fasudil in ROCKI

  • Mutant Graph
  • S35 = 0.5% (2/401)
    S10 = 0.0% (0/401)
Percent Control
0%
0.1%
0.1 - 1%
1 - 5%
5 - 10%
10 - 35%
> 35%
  • Mutant Graph Controlled
  • S35 = 5.5% (22/401)
    S10 = 5.0% (20/403)

Literature Reported Activities of Fasudil4 & hydroxy-Fasudil5 Also Illustrate the Need for Enhanced Kinome Selectivity

IC50 Values (nM)
Compound ROCKIII PKA PKA/ROCKII PKC PKC/ROCKII PKG PKG/ROCKII
Fasudil 330 1,600 5 3,300 10 1,600 5
Hydroxy-fasudil 720 37,000 51 N/A N/A

Neither of these benchmark compounds are highly selective against the limited # of AGC kinases against which they have been profiled. Biological activity attributed to these benchmarks in cellular, tissue & in vivo assays may result in part from off-target kinase activity.

Cervello Passive Permeability & Efflux Data Demonstrates Need for Improved ADME Properties over Fasudil & hydroxy-Fasudil

MDCK Passive Permeability MDCK Efflux
Compound WT
Papp ,A-B
(10 -6 cm/s)		 OE MDR1
Papp,A-B
(10 -6 cm/s)		 OE BCRP
Papp,A-B
(10 -6 cm/s)		 WT Efflux Ratio OE MDR1 Efflux Ratio OE MDR1 EER OE BCRP Efflux Ratio OE BCRP EER
Metoprolol 35.9 29.3 34.6 0.8 0.8 1.0 0.8 1.1
Atenolol <0.2 1.0 0.008 >0.4 0.7 n/a 1.3 n/a
Terfluonomide 2.7 n/a 17.6 n/a
Hydroxy-Fasudil 0.8 2.1 0.6 1.5 3.4 2.3 8.5 5.9
  1. 1. Stroke,43:571-574 (2011)
  2. 2. Stroke,48:187-194 (2016)
  3. 3. Drug Metabolism & Disposition, 40:322-328 (2012); Asian Journal of Pharmacodynamics and Pharmacokinetics, 9:221-226 (2009)
  4. 4. Pharmacol Ther, 82:123-131 (1999)
  5. 5. Stroke, 36:2251-2257 (2005)