Characterization of the cellular transport mechanisms for the anti-cachexia candidate compound TCMCB07

Background: Cachexia is a debilitating, life-threatening condition whose pathology includes reduced food intake accompanied by hypermetabolism, leading to a catabolic state. The hypothalamic melanocortin system is a critical regulator of metabolic rate with effects being mediated through the melanocortin-4 receptor (MC4R). MC4R activation is also critical to the initiation and maintenance of cachexia. A major problem in the design of anti-cachexia drugs has been the need to cross the blood-brain barrier to access the metabolic rate-controlling centres in the hypothalamus. The overwhelming majority of anti-cachexia drugs are only effective when administered intracerebroventricularly. TCMCB07 is a cyclic nonapeptide peptide MC4R Antagonist with parenteral anti-cachexia activity in both small and large animal models. This suggests it can cross the blood-brain barrier. The aim of this study was to examine potential transport mechanisms of TCMCB07 furthering its preclinical development for subsequent studies in humans.

Methods: In vitro studies were performed in transporter-transfected cells to study whether or not TCMCB07 was an inhibitor as well as substrate for OATP1A2, OATP1B1, OATP1B3, OATP2B1, OCT2, OAT1, OAT3, MATE1, MATE2-K, P-gp (MDR1), and BCRP. In vivo mass balance studies were also performed in mice to evaluate the absorption and disposition of TCMCB07 after oral and intravenous bolus administrations.

Results: TCMCB07 inhibited the uptake of prototypical substrates in cells transfected with OATP1A2 (IC₅₀ 24.0 μM), OATP1B1 (IC₅₀ 6.8 μM), OATP1B3 (IC₅₀ 307 μM), OATP2B1 (IC₅₀ 524 μM), OCT2 (IC₅₀ 1,169 μM), MATE1 (IC₅₀ 8.7 μM), and MATE2-K (IC₅₀ 20.7 μM) but not in cells transfected with OAT1 and OAT3. TCMCB07 did not affect the P-gp (MDR1)-mediated and BCRP-mediated permeability of prototypical substrates in transfected cells. Importantly, direct evidence was shown for the uptake of TCMCB07 in OATP1A2-transfected cells (i.e. V_max 236 pmol/mg, K_m 58.4 μM, and K_d 0.39 μL/mg), demonstrating that the nonapeptide was a substrate for this transporter. Mass balance studies demonstrated that 24.2% of TCMCB07 was absorbed orally in vivo (P = 0.0033) and excreted primarily in the bile after both oral and intravenous administrations.

Conclusions: OATP1A2 is the transporter responsible for the oral absorption of TCMCB07 in the intestine and for its pharmacologic response in the brain.

ABC transporters; Cachexia; Mass balance; Melanocortins; SLC transporters; TCMCB07.