Discovery of Tenapanor: A First-in-Class Minimally Systemic Inhibitor of Intestinal Na+/H+ Exchanger Isoform 3.

We present herein the design, synthesis, and optimization of gut-restricted inhibitors of Na+/H+ exchanger isoform 3 (NHE3). NHE3 is predominantly expressed in the kidney and gastrointestinal tract where it acts as the major absorptive sodium transporter. We desired minimally systemic agents that would block sodium absorption in the gastrointestinal tract but avoid exposure in the kidney. Starting with a relatively low-potency highly bioavailable hit compound (1), potent and minimally absorbed NHE3 inhibitors were designed, culminating with the discovery of tenapanor (28). Tenapanor has been approved by the U.S. Food and Drug Administration (FDA) for the treatment of irritable bowel syndrome with constipation in adults.

Preclinical and Healthy Volunteer Studies of Potential Drug-Drug Interactions Between Tenapanor and Phosphate Binders.

Tenapanor (RDX5791, AZD1722), a first-in-class small molecule with minimal systemic availability, is an inhibitor of the sodium/hydrogen exchanger isoform 3. Tenapanor acts locally in the gut, where it reduces absorption of sodium and phosphate. It is being studied in patients with chronic kidney disease requiring dialysis, who are often administered phosphate binders such as sevelamer to help control hyperphosphatemia. We investigated whether coadministration of tenapanor with phosphate binders (sevelamer or calcium-based binders) impacts the pharmacodynamic effects of tenapanor. In vitro studies suggested a binding interaction between tenapanor and sevelamer, but this did not translate into altered pharmacodynamic effects in rats. An open-label, 2-way crossover study was then conducted in healthy volunteers (NCT02346890). This showed that 4 days' treatment with tenapanor hydrochloride (15 mg twice daily) with or without sevelamer carbonate (800 mg 3 times daily) resulted in comparable 24-hour stool and urinary sodium and phosphorus levels. Stool frequency, consistency, and weight were also comparable between the treatments. These results suggest that the binding between sevelamer and tenapanor observed in vitro does not translate into altered pharmacodynamic effects in humans.

Gastrointestinal Inhibition of Sodium-Hydrogen Exchanger 3 Reduces Phosphorus Absorption and Protects against Vascular Calcification in CKD.

In CKD, phosphate retention arising from diminished GFR is a key early step in a pathologic cascade leading to hyperthyroidism, metabolic bone disease, vascular calcification, and cardiovascular mortality. Tenapanor, a minimally systemically available inhibitor of the intestinal sodium-hydrogen exchanger 3, is being evaluated in clinical trials for its potential to (1) lower gastrointestinal sodium absorption, (2) improve fluid overload-related symptoms, such as hypertension and proteinuria, in patients with CKD, and (3) reduce interdialytic weight gain and intradialytic hypotension in ESRD. Here, we report the effects of tenapanor on dietary phosphorous absorption. Oral administration of tenapanor or other intestinal sodium-hydrogen exchanger 3 inhibitors increased fecal phosphorus, decreased urine phosphorus excretion, and reduced [(33)P]orthophosphate uptake in rats. In a rat model of CKD and vascular calcification, tenapanor reduced sodium and phosphorus absorption and significantly decreased ectopic calcification, serum creatinine and serum phosphorus levels, circulating phosphaturic hormone fibroblast growth factor-23 levels, and heart mass. These results indicate that tenapanor is an effective inhibitor of dietary phosphorus absorption and suggest a new approach to phosphate management in renal disease and associated mineral disorders.

Intestinal inhibition of the Na+/H+ exchanger 3 prevents cardiorenal damage in rats and inhibits Na+ uptake in humans.

The management of sodium intake is clinically important in many disease states including heart failure, kidney disease, and hypertension. Tenapanor is an inhibitor of the sodium-proton (Na(+)/H(+)) exchanger NHE3, which plays a prominent role in sodium handling in the gastrointestinal tract and kidney. When administered orally to rats, tenapanor acted exclusively in the gastrointestinal tract to inhibit sodium uptake. We showed that the systemic availability of tenapanor was negligible through plasma pharmacokinetic studies, as well as autoradiography and mass balance studies performed with (14)C-tenapanor. In humans, tenapanor reduced urinary sodium excretion by 20 to 50 mmol/day and led to an increase of similar magnitude in stool sodium. In salt-fed nephrectomized rats exhibiting hypervolemia, cardiac hypertrophy, and arterial stiffening, tenapanor reduced extracellular fluid volume, left ventricular hypertrophy, albuminuria, and blood pressure in a dose-dependent fashion. We observed these effects whether tenapanor was administered prophylactically or after disease was established. In addition, the combination of tenapanor and the blood pressure medication enalapril improved cardiac diastolic dysfunction and arterial pulse wave velocity relative to enalapril monotherapy in this animal model. Tenapanor prevented increases in glomerular area and urinary KIM-1, a marker of renal injury. The results suggest that therapeutic alteration of sodium transport in the gastrointestinal tract instead of the kidney--the target of current drugs--could lead to improved sodium management in renal disease.

Postprandial lysophospholipid suppresses hepatic fatty acid oxidation: the molecular link between group 1B phospholipase A2 and diet-induced obesity.

Decrease in fat catabolic rate on consuming a high-fat diet contributes to diet-induced obesity. This study used group 1B phospholipase A(2) (Pla2g1b)-deficient mice, which are resistant to hyperglycemia, to test the hypothesis that Pla2g1b and its lipolytic product lysophospholipid suppress hepatic fat utilization and energy metabolism in promoting diet-induced obesity. The metabolic consequences of hypercaloric diet, including body weight gain, energy expenditure, and fatty acid oxidation, were compared between Pla2g1b(+/+) and Pla2g1b(-/-) mice. The Pla2g1b(-/-) mice displayed normal energy balance when fed chow, but were resistant to obesity when challenged with a hypercaloric diet. Obesity resistance in Pla2g1b(-/-) mice is due to their ability to maintain elevated energy expenditure and core body temperature when subjected to hypercaloric diet, which was not observed in Pla2g1b(+/+) mice. The Pla2g1b(-/-) mice also displayed increased postprandial hepatic fat utilization due to increased expression of peroxisome proliferator-activated receptor (PPAR)-alpha, PPAR-delta, PPAR-gamma, cd36/Fat, and Ucp2, which coincided with reduced postprandial plasma lysophospholipid levels. Lysophospholipids produced by Pla2g1b hydrolysis suppress hepatic fat utilization and down-regulate energy expenditure, thereby preventing metabolically beneficial adaptation to a high-fat diet exposure in promoting diet-induced obesity and type 2 diabetes.

Reduced absorption of saturated fatty acids and resistance to diet-induced obesity and diabetes by ezetimibe-treated and Npc1l1-/- mice.

The impact of NPC1L1 and ezetimibe on cholesterol absorption are well documented. However, their potential consequences relative to absorption and metabolism of other nutrients have been only minimally investigated. Thus studies were undertaken to investigate the possible effects of this protein and drug on fat absorption, weight gain, and glucose metabolism by using Npc1l1(-/-) and ezetimibe-treated mice fed control and high-fat, high-sucrose diets. Results show that lack of NPC1L1 or treatment with ezetimibe reduces weight gain when animals are fed a diabetogenic diet. This resistance to diet-induced obesity results, at least in part, from significantly reduced absorption of dietary saturated fatty acids, particularly stearate and palmitate, since food intake did not differ between groups. Expression analysis showed less fatty acid transport protein 4 (FATP4) in intestinal scrapings of Npc1l1(-/-) and ezetimibe-treated mice, suggesting an important role for FATP4 in intestinal absorption of long-chain fatty acids. Concomitant with resistance to weight gain, lack of NPC1L1 or treatment with ezetimibe also conferred protection against diet-induced hyperglycemia and insulin resistance. These unexpected beneficial results may be clinically important, given the focus on NPC1L1 as a target for the treatment of hypercholesterolemia.

Development and physiological regulation of intestinal lipid absorption. III. Intestinal transporters and cholesterol absorption.

Intestinal cholesterol absorption is modulated by transport proteins in enterocytes. Cholesterol uptake from intestinal lumen requires several proteins on apical brush-border membranes, including Niemann-Pick C1-like 1 (NPC1L1), scavenger receptor B-I, and CD36, whereas two ATP-binding cassette half transporters, ABCG5 and ABCG8, on apical membranes work together for cholesterol efflux back to the intestinal lumen to limit cholesterol absorption. NPC1L1 is essential for cholesterol absorption, but its function as a cell surface transporter or an intracellular cholesterol transport protein needs clarification. Another ATP transporter, ABCA1, is present in the basolateral membrane to mediate HDL secretion from enterocytes.

Carboxyl ester lipase deficiency exacerbates dietary lipid absorption abnormalities and resistance to diet-induced obesity in pancreatic triglyceride lipase knockout mice.

This study evaluated the contributions of carboxyl ester lipase (CEL) and pancreatic triglyceride lipase (PTL) in lipid nutrient absorption. Results showed PTL deficiency has minimal effect on triacylglycerol (TAG) absorption under low fat dietary conditions. Interestingly, PTL(-)(/)(-) mice displayed significantly reduced TAG absorption compared with wild type mice under high fat/high cholesterol dietary conditions (80.1 +/- 3.7 versus 91.5 +/- 0.7%, p < 0.05). Net TAG absorption was reduced further to 61.1 +/- 3.8% in mice lacking both PTL and CEL. Cholesterol absorption was 41% lower in PTL(-/-) mice compared with control mice (p < 0.05), but this difference was not exaggerated in PTL(-/-), CEL(-/-) mice. Retinyl palmitate absorption was reduced by 45 and 60% in PTL(-/-) mice (p < 0.05) and PTL(-/-), CEL(-/-) mice (p < 0.01), respectively. After 15 weeks of feeding, the high fat/high cholesterol diet, wild type, and CEL(-/-) mice gained approximately 24 g of body weight. However, body weight gain was 6.2 and 8.6 g less (p < 0.01) in PTL(-/-) and PTL(-/-), CEL(-/-) mice, respectively, despite their consumption of comparable amounts of the high fat/high cholesterol diet. The decrease body weight gain in PTL(-/-) and PTL(-/-), CEL(-/-) mice was attributed to their absorption of fewer calories from the high fat/high cholesterol diet, thereby resulting in less fat mass accumulation than that observed in wild type and CEL(-/-) mice. Thus, this study documents that PTL and CEL serve complementary functions, working together to mediate the absorption of a major portion of dietary fat and fat-soluble vitamin esters. The reduced lipid absorption efficiency due to PTL and CEL inactivation also resulted in protection against diet-induced obesity.

Class B type I scavenger receptor is responsible for the high affinity cholesterol binding activity of intestinal brush border membrane vesicles.

Recent studies have documented the importance of Niemann-Pick C1-like 1 protein (NPC1L1), a putative physiological target of the drug ezetimibe, in mediating intestinal cholesterol absorption. However, whether NPC1L1 is the high affinity cholesterol binding protein on intestinal brush border membranes is still controversial. In this study, brush border membrane vesicles (BBMV) from wild type and NPC1L1-/- mice were isolated and assayed for micellar cholesterol binding in the presence or absence of ezetimibe. Results confirmed the loss of the high affinity component of cholesterol binding when wild type BBMV preparations were incubated with antiserum against the class B type 1 scavenger receptor (SR-BI) in the reaction mixture similar to previous studies. Subsequently, second order binding of cholesterol was observed with BBMV from wild type and NPC1L1-/- mice. The inclusion of ezetimibe in these in vitro reaction assays resulted in the loss of the high affinity component of cholesterol interaction. Surprisingly, BBMVs from NPC1L1-/- mice maintained active binding of cholesterol. These results documented that SR-BI, not NPC1L1, is the major protein responsible for the initial high affinity cholesterol ligand binding process in the cholesterol absorption pathway. Additionally, ezetimibe may inhibit BBM cholesterol binding through targets such as SR-BI in addition to its inhibition of NPC1L1.

Group 1B phospholipase A2-mediated lysophospholipid absorption directly contributes to postprandial hyperglycemia.

Postprandial hyperglycemia is an early indicator of abnormality in glucose metabolism leading to type 2 diabetes. However, mechanisms that contribute to postprandial hyperglycemia have not been identified. This study showed that mice with targeted inactivation of the group 1B phospholipase A2 (Pla2g1b) gene displayed lower postprandial glycemia than that observed in wild-type mice after being fed a glucose-rich meal. The difference was caused by enhanced postprandial glucose uptake by the liver, heart, and muscle tissues as well as altered postprandial hepatic glucose metabolism in the Pla2g1b-/- mice. These differences were attributed to a fivefold decrease in the amount of dietary phospholipids absorbed as lysophospholipids in Pla2g1b-/- mice compared with that observed in Pla2g1b+/+ mice. Elevating plasma lysophospholipid levels in Pla2g1b-/- mice via intraperitoneal injection resulted in glucose intolerance similar to that exhibited by Pla2g1b+/+ mice. Studies with cultured hepatoma cells revealed that lysophospholipids dose-dependently suppressed insulin-stimulated glycogen synthesis. These results demonstrated that reduction of lysophospholipid absorption enhances insulin-mediated glucose metabolism and is protective against postprandial hyperglycemia.

The relative ligand binding preference of the murine ileal lipid binding protein.

The ileal lipid binding protein (ILBP), a member of the intracellular lipid binding protein family, is a 14-kDa protein that has bile and fatty acids as possible physiological ligands. The ligand binding specificity of this protein is not well characterized. Therefore, we studied the lipid binding activity of purified recombinant murine ILBP (mILBP) in vitro. These studies demonstrated by direct analysis the interaction of mILBP with naturally occurring bile and fatty acids. The rank order of binding preference for fatty acids, or unconjugated and conjugated bile acids, was assessed. Among fatty acids, mILBP preferred species that had longer chain length and increased saturation, similar to other members of the intracellular lipid binding protein family. Among the bile acids, mILBP showed the greatest preference for conjugated species that contained a doubly hydroxylated steroid moiety. The results demonstrate that mILBP exhibits a preference for certain species of bile and fatty acids.