Summary /

ML154 is found to have > 100 fold selective against the NPSR with nanomolar potency (Figure 1) vs a vasopressin receptor V1B that has 27% identity with NPSR. In addition the probe is profiled against 8 other targets (Table 1).

Figure 1: Concentration-response of the probe molecule, CID 46930969, in functional assays of NPSR antagonism: cAMP signaling IC50 = 45nM (black) and calcium signaling IC50 = 1nM (blue), 4 replicates each.

Table 1:  The probe, CID 46930969, was profiled against 8 other targets at @ 10 μM at Cerep®. 90% activity was discovered in the mu agonist displacement assay.

Summary /

IC50 determination in a radiolabeled displacement assay determined that the ML154’s activity against the mu-receptor was >500-fold less potent compared to the IC50 in the 125I-NPS displacement assay (Table 2 and Figure 2).

Table 2. ML154 activity: NPSR vs. mu-opioid receptor (MOP). An IC50 of 57 nM in the mu-agonist displacement assay at Cerep® is observed. Although there is a 10-fold shift in potency between the MOP activities between Cerep and our assay provider, we have to be cognizant of this activity during in vivo studies.

Figure 2: CID 46930969 mu receptor activity (assay provider).

Summary /

ML154  (CID 46930969) is profiled at 10 uM against a GRCR panel of 55 targets at Cerep® in binding assays.  IC50s against targets which recorded > 90% inhibition at 10 uM (Patnaik 2010).

Summary /

The stability in mouse liver microsomes spurred an evaluation of the pharmacokinetics of ML154 (CID 46930969) at a 10 mpk doses in mice (Tables 3 and 4). There were no signs of adverse effects with this dose. The mice appeared healthy with compound concentrations ranging from 0.05 to 1.5 uM in the plasma and 30 to 93 nM in the brain. The compound concentration in the brain was still above the in vitro IC50 at 24h. By comparison, the reported in vivo rat PK of a prior art compound from Merck at 30 mg/kg IP dose reveals the following tissue levels at 0.5 h: Plasma/Brain/CSF (nM) = 4344, 1690, 101 and at 2.0 h: Plasma/Brain/CSF (nM) =2297, 665, 45. SHA68 (1b) reaches the following concentrations in mice after a 50 mpk IP dose at 15 min: Plasma/Brain, 88, 6.3 uM and at 2.0 h: 1.1, 2.1 uM. Thus, these two compounds appear to be rapidly cleared from the plasma and the brain, and this is consistent with the in vitro microsomal stability data generated by NCGC. ML154 seems to maintain steady concentrations well above the IC50 for 24 h when it is administrated IP at a dose of 10 mg/kg. This may be critical for in vivo activity, as SHA68 (prior art compound) only shows partial efficacy in a hyperlocomotion mouse model (Patnaik 2010).

Table 3. Comparison of the microsomal stability of ML154 and 2 other known NPSR antagonists. 

Table 4. ML154 Mouse PK Paramaters and Curve for 10 mpk IP dose.

Summary /

Activity of ML154 in animal (rodent) model, where hyperactivation of the NPS signaling causes a suppression of food intake, is assessed. Rats with intracranial implants were habituated to a sweetened diet. The food intake was measured at 15, 30, and 60 min in a control group and in animals where NPS was administered icv (Figure 3). Animals were balanced between groups so that the pre-test baseline intake was matched. The NPS induced reduction in food intake (orange line) compared to controls (green) was completely reversed by intracerebroventricular (icv) administering of ML154 (10 ug) prior to NPS administration (6-8 animals/group).

Figure 3. ML154 Food Intake icv Experiment showed reversal of the food intake suppression.