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

Abstract

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 (IC 50 24.0 μM), OATP1B1 (IC 50 6.8 μM), OATP1B3 (IC 50 307 μM), OATP2B1 (IC 50 524 μM), OCT2 (IC 50 1,169 μM), MATE1 (IC 50 8.7 μM), and MATE2-K (IC 50 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.

Development of a Therapeutic Peptide for Cachexia Suggests a Platform Approach for Drug-like Peptides

“…Our in vivo evidence for the GI transport of TCMCB07 has been confirmed by additional mass balance studies from the Smith laboratory. 69 The percent GI transport for TCMCB07 from our gavage studies (∼30%) is similar to that reported by Smith et al Thus, TCMCB07 was designated as our lead anticachexia drug for further investigation.…”

“…These studies demonstrated the specific binding and transport of TCMCB07 by OATP1A2, a bile salt solute carrier transporter, potentially responsible for TCMCB07's BBB and gastrointestinal (GI) transport. 69 This solute transporter is in the right places (anatomically) and transports solutes in the right direction.…”

“…This suggested “hepatic transport” as another literature search term to uncover additional drug-like peptides. To our surprise, we rediscovered a “forgotten” class of synthetic and natural peptidessome initially designed to be potential drugsthat were ligands for a multi-specific bile salt transporter (e.g., OATP1A2) located in many tissues, including the BBB (Table , peptides 6–15). While the structural similarities between these peptides and TCMCB07 were striking, the former had half-lives too short for therapeutic utility. ,, TCMCB07 has a half-life of hours, not minutes, potentially due to relatively small differences in the peptide’s C-terminal sequence.…”

Pharmacokinetics and safety of TCMCB07, a melanocortin‐4 antagonist peptide in dogs

“…These structural features include a C‐terminus with nonpolar amino acid residues or chemical groups and cyclization or proline residue in or adjacent to the peptide's pharmacophore. Several peptides targeting MC4 were produced and TCMCB07, as the most promising candidate, was further studied 10,11 . Peripheral treatment—subcutaneous, intraperitoneal, or oral—in rodent models of LPS, renal disease, and cancer‐induced cachexia resulted in retention of both lean and fat body mass, appetite stimulation, and stable body weight.…”

“…Several peptides targeting MC4 were produced and TCMCB07, as the most promising candidate, was further studied. 10,11 Peripheral treatment-subcutaneous, intraperitoneal, or oral-in rodent models of LPS, renal disease, and cancerinduced cachexia resulted in retention of both lean and fat body mass, appetite stimulation, and stable body weight. Furthermore, TCMCB07 plasma concentrations were correlated with drug dose and the 14-day food intake and body weight gain were correlated with TCMCB07 plasma concentration.…”