The calcium‐sensing receptor: A novel target for treatment and prophylaxis of neratinib‐induced diarrhea

Abstract Diarrhea is one of the most commonly reported adverse effect of hemotherapy and targeted cancer therapies, such as tyrosine kinase inhibitors (TKI), which often significantly impact patient quality of life, morbidity, and mortality. Neratinib is an oral, irreversible pan‐HER tyrosine kinase inhibitor, which is clinically active in HER2‐positive breast cancer. Diarrhea is the most common side effect of this potent anticancer drug and the reasons for this adverse effect are still largely unclear. We have recently shown that activation of the calcium‐sensing Receptor (CaSR) can inhibit secretagogue‐induced diarrhea in the colon, therefore we hypothesized that CaSR activation may also mitigate neratinib‐induced diarrhea. Using an established ex vivo model of isolated intestinal segments, we investigated neratinib‐induced fluid secretion and the ability of CaSR activation to abate the secretion. In our study, individual segments of the rat intestine (proximal, middle, distal small intestine, and colon) were procured and perfused intraluminally with various concentrations of neratinib (10, 50, 100 nmol L−1). In a second set of comparison experiments, intraluminal calcium concentration was modulated (from 1.0 to 5.0 or 7.0 mmol L−1), both pre‐ and during neratinib exposure. In a separate series of experiments R‐568, a known calcimimetic was used CaSR activation and effect was compared to elevated Ca2+ concentration (5.0 and 7.0 mmol L−1). As a result, CaSR activation with elevated Ca2+ concentration (5.0 and 7.0 mmol L−1) or R‐568 markedly reduced neratinib‐induced fluid secretion in a dose‐dependent manner. Pre‐exposure to elevated luminal calcium solutions (5.0 and 7.0 mmol L−1) also prevented neratinib‐induced fluid secretion. In conclusion, exposure to luminal neratinib resulted in a pronounced elevation in fluid secretion in the rat intestine. Increasing luminal calcium inhibits the neratinib‐associated fluid secretion in a dose‐dependent manner. These results suggest that CaSR activation may be a potent therapeutic target to reduce chemotherapy‐associated diarrhea.

tive breast cancer. Diarrhea is the most common side effect of this potent anticancer drug and the reasons for this adverse effect are still largely unclear. We have recently shown that activation of the calcium-sensing Receptor (CaSR) can inhibit secretagogue-induced diarrhea in the colon, therefore we hypothesized that CaSR activation may also mitigate neratinib-induced diarrhea. Using an established ex vivo model of isolated intestinal segments, we investigated neratinib-induced fluid secretion and the ability of CaSR activation to abate the secretion. In our study, individual segments of the rat intestine (proximal, middle, distal small intestine, and colon) were procured and perfused intraluminally with various concentrations of neratinib (10,50, 100 nmol L −1 ). In a second set of comparison experiments, intraluminal calcium concentration was modulated (from 1.0 to 5.0 or 7.0 mmol L −1 ), both pre-and during neratinib exposure. In a separate series of experiments R-568, a known calcimimetic was used CaSR activation and effect was compared to elevated Ca 2+ concentration

| INTRODUC TI ON
Neratinib (PB-272; also called HKI-272, Nerlynx™) is an oral anticancer drug, which has clinical activity in HER2-positive breast cancer 1 and is the first anticancer drug approved by the US FDA for extended adjuvant treatment of early stage of this type of cancer following trastuzumab therapy. The mechanism of action of neratinib is well studied and described in recent literature. 2,3 The drug is a potent, small molecule, irreversible tyrosine-kinase inhibitor of human epidermal growth factor receptors (EGFRs, also known as HER-1, ErbB-1), HER2 (neu, ErbB-2), and HER4 (ErbB-4). 1,4,5 Although neratinib has a significant clinical anticancer effect, it also has the commonly reported side effect of diarrhea, 88%-91% of the patients have reported diarrheal symptoms, 6 As with all anticancer therapies, an improved treatment strategy for the agent-associated diarrhea needs to be developed to allow expanded use of these drugs while maintaining the quality of life of the patients. At present loperamide and oral rehydration have been used to improve outcomes and reduce diarrhea. 9,10 The efficacy of additional antidiarrheal interventions, such as Budesonide and Colestipol, is currently being tested clinically. While these therapies have shown promising effects in preventing neratinib-induced diarrhea, there is still an unmet outstanding need to have alternative therapies that can further abate this side effect. A better understanding of the biological mechanism(s) that causes diarrhea is also warranted.
The extracellular calcium-sensing receptor (CaSR) is a member of the G-protein-coupled receptor family and has been shown to have a variety of cellular and organ effects across a broad variety of species. [11][12][13][14][15][16] Our group has previously shown that activation of the CaSR in the colon has been very successfully in abating secretagogue-induced diarrhea. 14,15 Furthermore, CaSR has an extremely important physiological role in calcium homeostasis, 17,18 maintenance of fluid balance, 19,20 and osmotic regulation. 21 Previous studies have shown that there is high expression of CaSR along the mammalian gastrointestinal tract, 18,22,23 specifically in the intestinal epithelium, 24 enteric nerves, 13 and even on the surface of inflammatory cells. 25,26 The primary ligand for the receptor is ionized calcium (Ca 2+ o ) which binds to the surface of the two large exofacial loops of the receptor leading to activation of the receptor leading to decreases in intestinal secretion, motility, inflammation, and increased ionic and fluid absorption.
In the present study, we hypothesized that the activation of CaSR by elevating luminal intestinal calcium could reduce or abate the neratinib-induced diarrhea. Furthermore, we studied prophylactic pretreatment with oral calcium to prevent neratinib-induced fluid secretion when the drug was given post prophylactics.

| Animals
The small intestines were harvested from adult male Sprague-Dawley rats (250-365 g body weight, n = 58. All animals were obtained from Charles River Laboratories (Wilmington) and housed in climate-and humidity-controlled 12:12-hour light-dark cycled rooms at the Yale animal facility. All animals were fed standard rodent chow and then fasted for 12-15 hours prior to the study with continued free access to water.

| Perfusion solutions
For our study, we used a Ringer HEPES buffer solution (HEPES solution) with different Ca 2+ concentrations (see Table 1) with a final pH of 7.4 at 37°C.

| FITC-inulin
To quantitatively evaluate secretory-absorptive function of the various intestine segments, we have used the isothiocyanate derivative fluorescein isothiocyanate (FITC) coupled to the nonabsorbable volume marker inulin (hereinafter referred to as "FITC-Inulin"). 27,28 Inulin is a nonabsorbable sugar with a molecular weight of 3500 Da, which makes it nonpermeable through the intestinal wall. The concentration of FITC-Inulin (Sigma-Aldrich, St. Louis, MO) was 50 µmol L −1 for all arms of this study.

| Intestinal procurement and intestinal perfusion setup
Entire rat small intestine was procured, and three segments were obtained: proximal, middle, and distal each approximately 10 cm in length.  Table 1.

Calibration protocol
To calibrate the nanofluorospectrophotometer (Nanodrop 3300, Thermo Fisher Scientific Inc.) for each measurement we performed a series of three standard calibrations curves for each solution that was used as a perfusate. The mean values of these calibration curves were used as the standard curve for that series of studies. Separate calibration curves were conducted for each study and each tissue used.

Experimental sample protocol
Each sample luminal perfusate was composed of FITC-Inulin + the various solutions found in Table 1 with or without neratinib (control).

| Statistics
All studies presented are calculated for significance and plotted using the GraphPad Prism 7.01 software (GraphPad software Inc.).

| Ethical consideration
The animal handling and procurement of small intestine and colon were performed according to approved protocols of the Animal Care and Use Committee at Yale University (Protocol #2015-10253).

| Modulation in luminal Ca 2+ concentration in control conditions
In this series of studies, we measured the changes in fluid secretion with various concentrations of luminal calcium (See Table 1)

| Effects of elevated luminal Ca 2+ concentration on neratinib-induced fluid secretion in small intestine
When luminal Ca 2+ concentration was increased in the presence of

| Effects of neratinib on secretory-absorptive functions of colon
Previous studies from our group have shown that the colon also contains an active CaSR that can be stimulated by elevating the Ca 2+ concentration in the lumen, or via exposure to a calcimimetic. 13,14,18,24,30 In this series using isolated colons from rats we were able to see a rapid and sustained fluid secretion following exposure to increasing doses of neratinib ( Figure 4).

| Elevated luminal Ca 2+ concentration effects on luminal neratinib exposure in colon
These studies were designed to determine if CaSR activation via elevated levels in luminal Ca 2+ (5 and 7 mmol L −1 ) could block

| Memory effects of CaSR activation prior to neratinib exposure
Following the positive effects of elevating luminal Ca 2+ to abate neratinib-induced fluid secretion we examined pre-exposure to elevated Ca 2+ prevented neratinib-sensitive fluid secretion ( Figure 6D).

| Effects of R-568 (calcimimetic) on neratinib-induced fluid secretion in the small intestine and colon
We next compared the effects of R-568 (400 nmol L −1 ) with a normal

| D ISCUSS I ON
Targeted therapies such as TKIs are showing promising clinical efficacy in cancer patients, however, the side effects of the therapy can in some cases adverse symptoms that may lead to the cessation of treatment. 31-34 A very common adverse effect from chemotherapy or certain TKIs, such as neratinib, is diarrhea which can lead to dramatic loss of fluids and electrolytes, and under severe conditions also lead to malabsorption of nutrients due to the accelerated transit time along the intestine. 35,36 In the present study, we investigated if activation of the CaSR either concurrently or prophylactically prior to exposure to neratinib could lead to a reduction in the loss of fluid and electrolytes, which would allow patients an improved quality of life while taking the drug.

| Effects of neratinib on fluid secretion in the small intestine
In these studies, we examined and confirmed that exposure to neratinib in the lumen led to a dose-dependent increase in fluid secretion in proximal, middle, and distal segments of the small intestine (See

| Modulation of neratinib-induced fluid secretion in the colon by CaSR activation
The effects of CaSR activation in the colon are well defined by our laboratory regarding secretagogue-induced diarrhea. 13,15,23,39 In this

| Memory effects of CaSR activation prior to neratinib exposure
One additional question was whether the beneficial effects of Ca 2+ could be given prior to the neratinib and still maintain the protective antisecretory effect in the gut. We were in the studies shown in Figure 6 able to demonstrate that exposure for 15 minutes to an elevated Ca 2+ solution would be sufficient to maintain the antisecretory effects when Ca 2+ concentration returned to the reduced level and neratinib was added. This observation suggests that a short stimulation of the CaSR can provide a "memory effect" whereby the intestine becomes temporarily resistant to neratinib-induced diarrhea. This is an important finding in that it suggests that a prophylactic dose of Ca 2+ followed by neratinib and Ca 2+ may provide an even more complete prevention.

| Effects of R-568 (calcimimetic) on neratinibinduced fluid secretion in small intestine and colon
To provide additional evidence of the effect(s) of activation of CaSR on the secretory-absorptive function rat intestine and colon we used R-568, a small-molecule calcimimetic. This data shows that intraluminal exposure to elevated Ca 2+ concentration or R-568 with normal Ca 2+ concentration activate CaSR and have similar effects on reduction of the neratinib-induced fluid secretion.
In closing, these findings demonstrate that activation of the CaSR by calcium can abate the pharmaceutical induced diarrhea associated with exposure to neratinib and abate the side effects of rapid loss of fluid and electrolytes. Both the prophylactic and acute administration of oral Ca 2+ with neratinib therapy provides new important opportunities for improved patient outcomes.

ACK N OWLED G EM ENTS
The study was supported in part by a grant from PUMA Biotechnology, and the Charles Ohse fund from the department of surgery. We thank PUMA for the generous gift of neratinib for this study.

D I SCLOS U R E S
TL and JG have no conflict of interest. AL, EO, and ID are employees of Puma Biotechnology.

AUTH O R CO NTR I B UTI O N S
TL was involved in experimental design, conducting the experiments, and writing the manuscript. AL, EO, and ID were involved in data evaluation and editing of the manuscript. JG was involved in all aspects of the experimental design, manuscript writing, and editing.