Endari treatment ameliorates sickle cell-related disruption in intestinal barrier functions and is associated with prolonged survival in sickle cell mice

ABSTRACT

Background

Endari (L-glutamine) is a conditional amino acid that reduces the frequency of vaso-occlusive crisis (VOC) in sickle cell disease (SCD).

Aim

To investigate whether Endari could ameliorate intestinal barrier function and improve survival outcomes in SCD.

Methods

We treated female Townes SCD mice with Endari and evaluated their intestinal barrier functions by measuring the recovery of orally administered fluorescein isothiocyanate (FITC)-conjugated dextran 4 kDa in serum, and serum intestinal fatty acid binding proteins (iFABP) and lipopolysaccharide (LPS) concentrations by ELISA. We also explored the impact the Endari has on the survival of the SCD mice that underwent repeated experimentally-induced VOC.

Results

Compared to SCD mice treated with water only, Endari-treated mice showed improved intestinal barrier functions, with decrease in the barrier permeability and reduction in the translocation of lipopolysaccharides from the intestinal lumen into the circulation. These changes occurred after only 4 weeks of Endari treatment. Improved intestinal barrier function was also associated with prolonged survival in Endari-treated SCD mice after repeated experimentally-induced VOC.

Conclusion

Our findings provide the evidence supporting the beneficial effects of Enadri in improving intestinal barrier function and associated survival outcomes in SCD.

KEYWORDS:

• Endari
• L-glutaime
• intestinal barrier
• survival outcome
• sickle cell disease
• Townes mice
• intestinal permeability‌
• vaso-occlusive crisis

Introduction

Despite improved supportive care and increased availability of treatment options for sickle cell disease (SCD), the life expectancy in these patients continue to fall behind by more than two decades when compared to those without the disease. In the last six years, three new treatments for SCD have been approved by the United States Food and Drug Administration (FDA). They include Endari (L-glutamine) [1], Oxbryta (voxelotor) [2], and Adakveo (crizanlizumab) [3]. These agents either reduce the frequency of painful vaso-occlusive crisis (VOC) or increase the hemoglobin concentrations. However, hydroxyurea remains the first-line therapy in SCD. In addition to reducing the frequency of VOC, hydroxyurea also improves survival in patients [4,5]. Among these three newer agents approved by the FDA, there has not been any preclinical or clinical data on how they might affect survival.

Sickle erythrocytes exhibit an imbalance of the nicotinamide adenine dinucleotide (NAD) redox reactions so that there is a lower ([NADH]:[NAD⁺ + NADH]) ratio compared to normal erythrocytes [6]. L-glutamine restores the redox imbalance within the erythrocytes [7], thereby reducing the oxidative stress, and the risks for HbS polymerization and hemolysis. It has also been found to reduce adhesion of sickle red cells to endothelial cells [8]. However, the Phase 3 pivotal study that led to the marketing authorization by the FDA, treated SCD patients did not show any changes in the hemoglobin or reticulocyte counts compared to the placebo group [1], although the frequency of painful VOC was lower. Therefore, the exact mechanisms of action of Endari leading to the clinical benefits of reduction in the frequency of painful VOC remain to be elucidated.

Among the three newer agents approved by the FDA, Endari is the least expensive. Data on its efficacy in reducing the frequency VOC are also more robust than Oxbryta; and being an oral agent, it is more acceptable to patients than Adakveo that requires intravenous infusion every four weeks. In this current study, we hypothesized that Endari treatment ameliorates intestinal barrier function associated with SCD. We also explored whether improvement in intestinal barrier function in Endari-treated SCD mice was associated with improved survival.

Materials and methods

Mice

Five-week-old female Townes SCD mice (homozygous for Hba^tm1(HBA)Tow and homozygous for Hbb^{tm2(HBG1,HBB*)Tow}), created on a B6:129 genetic background that express SCD phenotype, were purchased from Jackson Laboratory, ME, USA, and housed in the SUNY Upstate Medical University vivarium. Experiments were started after one week of acclimatization. All procedures were conducted in accordance with PHS policy and were approved by the IACUC at SUNY Upstate Medical University, New York.

The mice were randomly divided into two groups (n = 18 in each group): Group 1 mice received water supplemented with Endari (3 g/L) daily, and Group 2 mice only water. A water deprivation protocol that we previously described, and validated on microscopy showing induction of sickle erythrocytes, was used to induce VOC [9]. Briefly, the mice were kept under strict water deprivation from 7:00 pm to 7:00 am for seven consecutive nights every two weeks starting Week 2. To mirror the frequent worsening of VOC as patients with SCD age [10], we subjected the mice to two 15-minute periods of cold exposure, one week apart towards the end of the study, by transferring the mice to cages cooled from outside by ice-water. The schema of VOC induction and cold exposure is shown in Figure 1. Endari treatment was continued in Group 1 SCD mice throughout the study period.

Figure 1. Treatment schema. Thirty-six 6-week old Townes model SCD female mice were randomly divided into two groups and treated either with or without Endari (3 g/L) mixed in drinking water. Normal drinking water and Endari-water were replaced daily. Mice were kept under strict water deprivation for 12 h seven consecutive nights every two weeks starting Week 2. After 100 days, mice were subjected to two 15-minute periods of cold exposure, one week apart.

In vivo intestinal permeability assay

Intestinal permeability was assessed by quantifying fluorescein isothiocyanate (FITC) conjugated dextran 4 kDa in the serum after oral gavage. Briefly, mice were placed in a fresh cage and food and water were withdrawn for 4 hours. After weighing each mouse, 60 mg/100 g body weight of freshly prepared FITC-Dextran 4k (MW 4000 Da; Sigma-Aldrich) solution, prepared in sterile PBS, was administered by oral gavage. Peripheral blood was collected 3 h post-gavage to evaluate the permeability of the small intestine [9,11]. 20-fold diluted serum was transferred into a black flat-bottom 96-well plate (Corning). FITC-Dextran 4k concentrations were analyzed with a fluorescence 96-well plate reader (Molecular Devices, SpectraMax i3x Multi-Mode Microplate Reader) with excitation and emission wavelengths of 488 and 520 nm, respectively. Serum samples were always protected from light.

ELISA

Commercial enzyme-linked immunosorbent assays (ELISA) kits of intestinal barrier integrity markers were used to measure intestinal fatty acid binding proteins (iFABP) (MyBiosource) and lipopolysaccharide (LPS) (Cloud-Clone Corp.) from serum according to the manufacturer's instructions.

Statistics

All experiments were carried out in triplicates. Differences in the measured parameters between the two groups were analyzed using Student t-tests. A two-sided p value of ≤0.05 was considered statistically significant. The survival comparison between the two groups was performed using Kaplan-Meier method.

Results

As expected, there was no difference in spleen weights between the two groups of SCD mice (Figure 2(A)). Examination of the intestinal barrier function revealed significant improvements after only 4 weeks of Endari treatment. There were no differences in the levels of FITC-Dextran 4k recovered in serum and concentration of serum LPS between the two groups of mice at baseline. Both groups of mice had increased concentration of serum LPS (Figure 2(B)) after one-month Endari treatment. However, there was a significant reduction in serum LPS concentration (p = 0.0001) in SCD mice after four weeks of Endari treatment compared to control mice. Similarly, the concentration of FITC-Dextran 4k recovered in serum of Endari-treated mice was significantly low (p = 0.03) compared to control mice (Figure 2(C)), confirming improved intestinal barrier functions in the Endari-treated mice. FITC-Dextran 4k permeability across the intestine after four weeks was also improved (p = 0.02) compared to the baseline levels in the same set of Endari-treated SCD mice. However, there was no difference in serum iFABP levels in control versus Endari-treated mice (Figure 2(D)).

Figure 2. (A) There was no difference in spleen weights between control and Endari-treated SCD mice; Endari improved intestinal permeability as evident form (B) significant reduction in the serum LPS in SCD; and (C) FITC-Dextran 4 kDa recovery in serum; (D) No difference was observed in serum iFABP between the two treatment groups; (E) Probability of survival using the Kaplan-Meier estimator, showing improved survival in Endari-treated mice (77.8%) compared to non-Endari-treated mice (38.9%).

Endari treatment significantly improved the survival of the SCD mice (Figure 2(E)). Four mice treated with Endari and 11 in the control group died. The probability of survival at 114 days was 38.9% in the control group and doubled at 77.8% in the Endari-treated group (p = 0.0353). Endari treatment was associated with a Hazard Ratio of 0.3246 (95% CI: 0.1138–0.9253).

Discussion

Despite reducing the frequency of painful VOC [12] but prolonging survival [4,5], hydroxyurea is not widely accepted by patients with SCD [13]. Reasons contributing to the low clinical uptake of the medication include skin and nail discoloration and development of leg ulcers in some patients. However, hydroxyurea has remained the first-line therapy for treating SCD. Among the rest of the FDA approved medication including Oxbryta and Adakveo, Endari remains the most cost-effective option, especially for low-income patients.

In this study, we set out to determine the direct effect of Endari in ameliorating the intestinal barrier disruption without the complication due to repeated VOC. We chose to study this after just four weeks of Endari treatment in an attempt to exclude the detrimental effects of repeated VOC on the intestinal barrier because VOC-induced ischemia incur injury to the enterocytes and may induce breach of the intestinal barrier. Our results suggest that we were successful in this objective since, by studying the barrier functions at only 4 weeks, we did not observe any difference between the two groups in the iFABP, a serum biomarker of enterocyte damage.

Our findings represent the first evidence that Endari not only help in reducing the frequency of painful VOC but may also have a positive impact on life-expectancy in SCD. These results are consistent with clinical improvements. SCD patients on Endari for 72 weeks had significantly lower number of annual VOCs, reduced rate of annual hospitalizations, fewer number of days spent in hospital, and decreased events of acute chest syndrome (ACS) compared to baseline [14].

The beneficial effects of Endari could be due to the restoration of the redox imbalance within the sickled erythrocytes. This would reduce the oxidative stress and lower the risks for HbS polymerization and hemolysis [6–8,15]. Endari could also help to improve SCD pathophysiology and survival outcome by ameliorating intestinal barrier function. We and others have previously demonstrated high intestinal dysbiosis and permeability in SCD [9,16]. Expression of tight junction genes such as occludin, Zonula occludens-1 (Zo-1), junctional adhesion molecule (Jam), and epithelial cadherin (E-cad) are disrupted in the small and large intestines in SCD [11,17]. Both dysbiosis and permeability of intestine increases during VOC. Increased intestinal permeability causes luminal pathogen-associated molecular patterns (PAMPs) such as LPS to translocate across the gut barrier. Circulating LPS trigger inflammation by activating and ageing neutrophils [18]. The increase in circulating aged neutrophils (CANs) is a hallmark of VOC-induced pathogenesis and worsening of SCD pathophysiology. Reduction of intestinal dysbiosis and improvement in barrier integrity lowers CANs and reduces VOC in SCD [19–21]. Since Endari (L-glutamine) has an important function in intestinal health, it may improve the barrier function and help reduce the translocation of LPS, as evident from our study.

Both small and large intestines are major sites of glutamine consumption because of high glutaminase activity. Glutamine is used by the intestinal cells to upregulate intestinal stem cell proliferation, fortify tight junctions, inhibit apoptosis, and help to maintain intestinal immunity. Glutamine is also a substrate for intestinal cells for the synthesis nucleotides (purines, pyrimidines, and amino sugars) [22], and is used for aminogenesis, the citric acid cycle, formation of the antioxidants NADPH, and maintenance of the intestinal surface integrity by mediating the synthesis of N-acetylglucosamine and N-acetylgalactosamine [23,24]. Thus, Endari may improve the intestinal barrier function, thereby reducing the circulating PAMP-induced inflammation and lower the severity of VOCs. As a consequence, the life-expectancy in SCD increased significantly with Endari treatment.

Limitations of this study include the use of SCD mouse model to test laboratory-induced VOC. The use of a mouse model of SCD is warranted to test our hypothesis since Endari was approved only six years ago. Therefore, clinical data available for such an analysis is very limited, especially many patients taking Endari are also taking hydroxyurea, thereby compounding result analysis since hydroxyurea is associated with prolonged survival in patients with SCD. While outside the scope of this current study, it would be interesting to compare in future studies, the survival of Endari-treated SCD mice was treated with hydroxyurea-treated mice. The results of such a study may provide vital information on whether Endari could be as an upfront therapy, instead of hydroxyurea, in SCD.

Acknowledgement

Endari was kindly provided by Emmaus Medicals, Inc.

Disclosure statement

Seah H. Lim is on the Board of Directors of Emmaus Medicals, Inc.

Data availability statement

All data generated during this study are available from the corresponding author on reasonable request.

Additional information

Funding

The author(s) reported there is no funding associated with the work featured in this article.