Preclinical Profile of BYON3521 Predicts an Effective and Safe MET Antibody-Drug Conjugate.

MET, the cell-surface receptor for the hepatocyte growth factor/scatter factor, which is widely overexpressed in various solid cancer types, is an attractive target for the development of antibody-based therapeutics. BYON3521 is a novel site-specifically conjugated duocarmycin-based antibody-drug conjugate (ADC), comprising a humanized cysteine-engineered IgG1 monoclonal antibody with low pmol/L binding affinity towards both human and cynomolgus MET. In vitro studies showed that BYON3521 internalizes efficiently upon MET binding and induces both target- and bystander-mediated cell killing. BYON3521 showed good potency and full efficacy in MET-amplified and high MET-expressing cancer cell lines; in moderate and low MET-expressing cancer cell lines good potencies and partial efficacy were observed. In mouse xenograft models, BYON3521 showed significant antitumor activity upon single-dose administration in multiple non-MET-amplified tumor types with low, moderate, and high MET expression, including complete tumor remissions in models with moderate MET expression. In the repeat-dose Good Laboratory Practice (GLP) safety assessment in cynomolgus monkeys, BYON3521 was well tolerated and based on the observed toxicities and their reversibility, the highest non-severely toxic dose was set at 15 mg/kg. A human pharmacokinetics (PK) model was derived from the PK data from the cynomolgus safety assessments, and the minimal efficacious dose in humans is estimated to be in the range of 3 to 4 mg/kg. In all, our nonclinical data suggests that BYON3521 is a safe ADC with potential for clinical benefit in patients. A first-in-human dose-escalation study is currently ongoing to determine the maximum tolerated dose and recommended dose for expansion (NCT05323045).

Discovery of SYD5115, a novel orally active small molecule TSH-R antagonist.

The thyrotropin receptor (TSH-R) regulates the thyroid gland and is normally activated by thyrotropin. In patients with Graves' disease, TSH-R is also stimulated by stimulatory TSH-R autoantibodies leading to hyperthyroidism. In this paper, we describe the discovery of SYD5115 (67), a novel small molecule TSH-R antagonist with nanomolar potency. SYD5115 also blocks stimulating antibody induced synthesis of the thyroid hormone thyroxine (T4) in vivo, after a single oral dose. During optimization, several issues had to be addressed such as the low metabolic stability and the potential mutagenicity of our first series of compounds.

Dietary heme injures surface epithelium resulting in hyperproliferation, inhibition of apoptosis and crypt hyperplasia in rat colon.

Epidemiological and animal model studies suggest that a high intake of heme, present in red meat, is associated with an increased risk of colon cancer. The aim of this study was to elucidate the effects of dietary heme on colonic cell homeostasis in rats. Rats were fed a purified, humanized, control diet or a similar diet supplemented with 0.5 mmol heme/kg for 14 days. Fecal water cytolytic activity was determined with a bioassay, and colon epithelial cell proliferation was evaluated with (3)H-thymidine or 5-bromo-2'-deoxyuridine incorporation into DNA or by Ki-67 immunohistochemistry. Exfoliation of colonocytes was measured as the amount of rat DNA in feces, and caspase-3 expression and activity were measured to study colonic mucosal apoptosis. Dietary heme induced a >10-fold increased cytolytic activity of the fecal water and a 100-fold lower excretion of host DNA. Colons of heme-fed rats showed injured surface epithelium and an approximately 25% increase in crypt depth. Finally, dietary heme doubled colonocyte proliferation, shown by all three markers, but inhibited colonic mucosal apoptosis. In conclusion, our results demonstrate that dietary heme injures colonic surface epithelium, which is overcompensated by inhibition of apoptosis and hyperproliferation of cells in the crypts, resulting in crypt hyperplasia. This disturbed epithelial cell homeostasis might explain why a high intake of dietary heme is associated with an increased risk of colon cancer.

Breast cancer resistance protein (Bcrp1/Abcg2) limits net intestinal uptake of quercetin in rats by facilitating apical efflux of glucuronides.

The intestinal absorption of the flavonoid quercetin in rats is limited by the secretion of glucuronidated metabolites back into the gut lumen. The objective of this study was to determine the role of the intestinal efflux transporters breast cancer resistance protein (Bcrp1)/Abcg2 and multidrug resistance-associated protein 2 (Mrp2)/Abcc2. To study the possible involvement of Mrp2, we compared intestinal uptake of quercetin-3-glucoside between control and Mrp2-deficient rats, using an in situ intestinal perfusion system. The contribution of Bcrp1 was determined using the specific inhibitor fumitremorgin C (FTC) in Mrp2-deficient rats. Furthermore, vectorial transport of quercetin was studied in Madin-Darby canine kidney (MDCK)II cells transfected with either human MRP2 or murine Bcrp1. In these MDCKII cells, we showed an efficient efflux-directed transport of quercetin by mouse Bcrp1, whereas in control and MRP2-transfected cells no vectorial transport of quercetin was observed. In Mrp2-deficient rats, intestinal uptake of quercetin from quercetin-3-glucoside, efflux of quercetin glucuronides to the gut lumen, and plasma concentration of quercetin were similar to that in control rats. When intestinal Bcrp1 was inhibited by FTC in Mrp2-deficient rats, total plasma concentrations of quercetin and its methylated metabolite isorhamnetin after 30 min of perfusion were more than twice that of controls (12.3 +/- 1.5 versus 5.6 +/- 1.3 muM; p < 0.01), whereas uptake of free quercetin from the intestinal lumen was not affected. Instead, inhibition of Bcrp1 lowered the efflux of quercetin glucuronides into the perfusion fluid by approximately 4-fold. In conclusion, Bcrp1 limits net intestinal absorption of quercetin by pumping quercetin glucuronides back into the lumen.

The type of sugar moiety is a major determinant of the small intestinal uptake and subsequent biliary excretion of dietary quercetin glycosides.

Quercetin is an important dietary flavonoid with putative beneficial effects in the prevention of cancer and CVD. The in vivo bioactivity of quercetin depends on its bioavailability, which varies widely between foods. We used an in situ rat intestinal perfusion model to study whether differential small intestinal hydrolysis of the sugar moiety of five naturally occurring quercetin glycosides determines the small intestinal uptake and subsequent biliary excretion of quercetin. After 30 min perfusion, a decrease of intact quercetin glycoside in perfusate was observed for quercetin-3-O-ss-glucoside (20.9 (sem 1.4) micromol/l) and quercetin-4'-O-ss-glucoside (23.5 (sem 1.6) micromol/l), but not of quercetin-3-O-ss-galactoside, quercetin-3-O-ss-rhamnoside and quercetin-3-O-alpha-arabinopyranoside. Appearance of free quercetin in perfusate and conjugated quercetin metabolites (quercetin, isorhamnetin, and tamarixetin) in portal and peripheral plasma and bile were also significantly greater after treatment with quercetin-3-O-ss-glucoside or quercetin-4'-O-ss-glucoside compared with any of the other glycosides. Thus, the type of sugar moiety is a major determinant of the small intestinal absorption of quercetin glycosides, but the position (3 or 4') of the glucose moiety does not further influence absorption. The poor bioavailability of important dietary quercetin glycosides has implications for their in vivo bioactivities.

Intestinal uptake of quercetin-3-glucoside in rats involves hydrolysis by lactase phlorizin hydrolase.

Quercetin has antioxidant, anti-inflammatory, antiproliferative and anticarcinogenic properties. In plant foods, quercetin occurs mainly bound to various sugars via a beta-glycosidic link. We hypothesized that lactase phlorizin hydrolase (LPH), an enzyme at the brush border membrane of intestinal cells, is involved in the in vivo intestinal uptake of quercetin-sugars. To study this, we measured the appearance of quercetin metabolites in plasma and perfusate after perfusing the jejunum and ileum with 50 micro mol/L quercetin-3-glucoside in an in situ rat perfusion model. LPH was inhibited by the selective LPH inhibitor N-butyldeoxygalactonojirimycin (0, 0.5, 2 or 10 mmol/L) (n = 5 rats/group). Quercetin in plasma and perfusion buffer was determined by HPLC with CoulArray detection. Results are given as means +/- SEM. In the perfusion buffer, 13.8 +/- 0.7 micro mol/L quercetin-3-glucoside was hydrolyzed during intestinal passage. Co-perfusion with 0.5, 2 and 10 mmol/L N-butyldeoxygalactonojirimycin resulted in 38% (P < 0.05), 50% (P < 0.01) and 67% (P < 0.01) less hydrolysis, respectively. Plasma concentrations of quercetin in the corresponding groups were 36% (P = 0.12), 55% (P < 0.01) and 75% (P < 0.01) lower than in controls (1.23 +/- 0.22 micro mol/L). These data suggest that LPH is a major determinant of intestinal absorption of quercetin-3-glucoside in rats.