Treatment of Ezetimibe lowers total and low-density lipoprotein cholesterol in hypercholesterolemic dogs with hyperadorenocorticism

Abstract

Ezetimibe is a cholesterol absorption inhibitor that blocks the intestinal absorption of both biliary and dietary cholesterol, thereby lowering primarily low density lipoprotein-cholesterol (LDL-chol) in human studies. This study aimed to investigate the effects of ezetimibe on dyslipidemia control in nine dogs with hypercholesterolemia. Changes in total cholesterol (T-chol) and each lipoprotein fractions were evaluated at 0, 2, and 4 months following initiation of ezetimibe treatment. A significant decrease in T-chol was observed, and a mean T-chol concentration below 400 mg/dL was achieved at 2 and 4 months. Furthermore, a significant decrease in LDL-chol was observed (−53.3% and −64.3% at 2 and 4 months, respectively). Taken together, treatment of ezetimibe could lower LDL-chol levels in dogs with hypercholesterolemia.

Hyperlipidemia is a condition that refers to an increase in serum and/or plasma levels of cholesterol, triglycerides (TG), or both. It can be primary, but is usually associated with other comorbidities. [35, 37]. The major source of cholesterol is from the diet, although it can also be synthesized by the liver and other tissues. The liver eliminates excess cholesterol through the plasma via low-density lipoprotein (LDL) and biliary secretion [17]. Primary hyperlipidemia is a hereditary disorder found in Miniature schnauzers, Shetland sheepdogs, Beagles, and other breeds. Hypertriglyceridemia predominantly occurs in Miniature schnauzers, while hypercholesterolemia occurs in Shetland sheepdogs [20, 36, 37]. Secondary hyperlipidemia can result from underlying causes such as a high-fat diet, endocrine disease (such as hyperadrenocorticism, hypothyroidism, and diabetes mellitus), obesity, protein-losing nephropathy, cholestasis, lymphoma, or exposure to certain drugs [37]. Although hyperlipidemia itself has been considered a benign condition in dogs, it is associated with other potentially life-threatening diseases such as pancreatitis, insulin resistance-induced diabetes mellitus, or seizures [37]. Furthermore, lesions of atherosclerosis in arterial vessels have been found in hyperlipidemic dogs when plasma cholesterol levels are exceedingly elevated [19].

Ezetimibe is a cholesterol absorption inhibitor that blocks the intestinal absorption of both biliary and dietary cholesterol. It works by inhibiting the intestinal sterol transporters, specifically the Niemann-Pick C1-like 1 proteins, which results in the inhibition of the absorption of cholesterol, phytosterols and certain oxysterols. Ezetimibe-based therapy is an exciting new area in the treatment of dyslipidemia [2]. Ezetimibe lowers LDL-chol levels by approximately 20% through the inhibition of cholesterol absorption by the intestines. This leads to decreased delivery of cholesterol to the liver, a decrease in hepatic cholesterol content, and an up-regulation of hepatic LDL receptors in human studies [5, 28, 34]. Ezetimibe is useful add on or alternative therapy when statin therapy is not sufficient in hypercholesterolemic patients. The efficacy of ezetimibe in lowering cholesterol levels has been reported in previous human study [18, 21]. However, a study on healthy dogs showed a lowering effect of ezetimibe on canine plasma cholesterol concentration, although its effect on dogs with hypercholesterolemia has not been investigated [6].

In the current study, the effects of ezetimibe treatment on lipoprotein profiles in dogs with hypercholesterolemia were studied.

From January 2019 to January 2021, nine dogs with hypercholesterolemia were treated with ezetimibe for 4 months at the Nippon Veterinary and Life Science University Veterinary Medical Teaching Hospital. All nine of our dogs with hypercholesterolemia also had concurrent hyperadrenocorticism (HAC). We treated these dogs, which exhibited secondary hyperlipidemia, with ezetimibe. The profiles of these nine dogs were presented in Table 1. All nine dogs were diagnosed with HAC, with two of the dogs receiving no trilostane treatment during ezetimibe therapy. Seven of the dogs had been receiving trilostane for at least 4 months before the initiation of ezetimibe administration. Additionally, there was no adjustment made to the trilostane dose both before and after the four-month period following the commencement of ezetimibe administration.

Table 1. The physical profiles of nine dogs diagnosed with hypercholesterolemia treated with Ezetimibe in this study.

No.	Breeds	Age (years)	Sex	Body weight (kg)	Food	Total colesterol concentration (mg/dL)		Concurrent disease before treatment	Treatment of concurrent disease
						Initial day	Treated after 4 month
1	Jack Russell Terrier	14	Spayed	5.6	Commercially available dry	843	613	Cushing’s syndrome	Trilostane (0.77 mg/kg/day)
2	Chihuahua	12	Male	4.7	N/A	882	271	Cushing’s syndrome	None
3	Chihuahua	12	Castrated	3.7	pH control dry, (RoyalCanin)	469	285	Cushing’s syndrome	Trilostane (0.41 mg/kg/day)
4	Chihuahua	12	Castrated	3.5	Commercially available dry	609	629	Cushing’s syndrome	Trilostane (1.43 mg/kg/day)
5	Toy Poodle	12	Female	4.9	Commercially available dry	427	210	Cushing’s syndrome Hypothyroidism Hypertriglyceridemia	Trilostane (1.02 mg/kg/day) Levothyroxine (20.4 µg/kg/day) Bezafibrate (5.1 mg/kg/day)
6	Toy Poodle	11	Castrated	5.8	Diabetic dry and wet (RoyalCanin)	780	536	Cushing’s syndrome Diabetes mellitus Hypertriglyceridemia	Insulin Detemir (0.86 Units/kg/day) Bezafibrate (8.6 mg/kg/day)
7	Pomeranian	10	Castrated	7.0	Commercially available dry	434	336	Cushing’s syndrome	Trilostane (1.43 mg/kg/day)
8	Welsh Corgi Pembroke	10	Spayed	8.9	Science Diet Adult 7+ dry (Hill’s)	528	329	Cushing’s syndrome Hypothyroidism	Trilostane (0.41 mg/kg/day) Levothyroxine (11.0 µg/kg/day)
9	Shetland Sheepdog	9	Castrated	12.2	Weight care dry (RoyalCanin)	404	327	Cushing’s syndrome	Trilostane (0.21 mg/kg/day)

HAC diagnosis was based on the clinical condition of the dogs (polyuria and polydipsia, abdominal distention, endocrine alopecia and muscle weakness) [7], the results of an ACTH stimulation test and the ultrasonographic appearance of the adrenal glands (2012 ACVIM consensus statement of canine hyperadrenocorticism) [3]. In addition, the patients have all maintained good nutritional status and normal hepatic function. We conducted a serum biochemistry profile, which revealed normal levels of serum bilirubin and blood proteins (albumin, total protein) and blood urea nitrogen. Informed consent was obtained from the owners following the review of the purpose, nature, potential risks, and benefits of the study.

All dogs were administered ezetimibe (Zetia, MSD Japan Co., Ltd., Tokyo, Japan) once daily. The initial dosages were determined based on body weight (0.027 ± 0.010 (mean ± SD) mg/kg/day; range: 0.012−0.042 mg/kg/day) and were further adjusted throughout the treatment period to target Total-cholesterol (T-chol) level under 400 mg/dL (reference value; 105−322 mg/dL). The pet owners administered ezetimibe to their dogs before visiting the hospital, and sample collection was performed 3 to 6 hr after administration of ezetimibe.

Blood was obtained from the peripheral vein of each dog after 0, 2 and 4 months of ezetimibe treatment. According to the product information of ezetimibe, mean maximum plasma concentrations (C_max) occur 1 to 2 hr post-administration (T_max=2.1 hr), followed by enterohepatic recycling and slow elimination. The estimated terminal half-life of ezetimibe is approximately 22 hr. In human patients, the recommended dose of ezetimibe 10 mg/day can be administered in the morning or evening without regard to food. Thus, the pet owner administered ezetimibe with meals before visiting hospital. Sample collection and processing were performed using similar methods to those described by Oda et al. [22]. Blood samples for T-chol and each lipoprotein fraction (VLDL-chol, IDL-chol, LDL-chol and HDL-chol) were collected into polypropylene tubes and allowed to clot at room temperature for 15 min. Immediately, after clotting, blood samples were centrifuged at 1,700 g for 10 min at 4°C in order to obtain serum samples. Samples were immediately stored at 4°C until further use. T-chol concentration was measured using a commercial kit (L-type WAKO CHO-M; FUJIFILM Wako Pure Chemical Co., Ltd., Osaka, Japan) and an autoanalyzer (Type 7180 Automatic Analyzer; Hitachi High-Technologies Co., Ltd., Tokyo, Japan). Furthermore, all serum samples were sent to FUJIFILM VET Systems Co., Ltd. (Tokyo, Japan) for Anion-exchange-high-performance liquid chromatography (AEX–HPLC). AEX-HPLC is a relatively new method applicable to lipoprotein analysis in humans. It can be used to determine IDL in specimens of human blood [10,11,12, 15]. In particular, IDL concentrations are considered to be a risk factor for arteriosclerotic disease in humans [16, 27]. Moreover, we reported that AEX-HPLC used to evaluate lipoprotein profiles in dogs and be a new useful indicator of hyperlipidemia in dogs [22]. Lower limit cholesterol concentrations of the measuring interval for cholesterol measurement by AEX-HPLC was 1.0 mg/dL. Intra- and inter-assay coefficient of variation (CV) values for canine cholesterol concentration in each lipoprotein fractions were <10% except for inter-assay CV values for IDL-chol and VLDL-chol (14.2% and 10.7%, respectively) [22].

This work was approved by the Nippon Veterinary and Life Science University Animal Research Committee (Acceptance Number: 28S-20).

Results are presented as individual plot and line at median. Statistical significance was determined using one-way or two-way repeated measures (RM) ANOVA and Bonferroni’s post-hoc test using GraphPad Prism 8 analysis software (GraphPad Software, Inc., San Diego, CA, USA). Differences were considered significant at values of P<0.05.

During the treatment with ezetimibe, a significant decrease in T-chol was observed (P<0.05, one-way RM ANOVA). Lower levels were recorded at 2 and 4 months compared to the pretreatment level (adjusted P<0.05, Bonferroni’s post-hoc test) (Fig. 1A). The mean T-chol levels changed from 597.3 (before ezetimibe treatment) to 386.8, and 392.9 mg/dL at 0, 2, and 4 months, respectively. Moreover, a significant decreases in LDL-chol was observed following ezetimibe treatment (−53.3% and −64.3% at 2 and 4 months, respectively) (P<0.05, one-way RM ANOVA). Moreover, the Bonferroni’s post-hoc test showed significant differences between 2 and 4 months relative to the pretreatment (adjusted P<0.05, Bonferroni’s post-hoc test) (Fig. 1B). A significant decrease was also found in HDL-chol (P<0.05, one-way RM ANOVA, −13.9% at 4 month), with lower levels being recorded at 4 month than at 0 month (adjusted P<0.05, Bonferroni’s post-hoc test) (Fig. 1E). Additionally, no significant difference were observed in VLDL-chol and IDL-chol over the treatment period (Fig. 1C and 1D) (one-way RM ANOVA). The mean dose of ezetimibe was 0.027 ± 0.010 and 0.040 ± 0.023 mg/kg/day at 2 and 4 months, respectively (Fig. 1F).

Fig. 1.

Change in total cholesterol (A), each lipoprotein concentrations (low-density lipoprotein (LDL) cholesterol (B), very-low-density lipoprotein (VLDL) cholesterol (C), intermediate-density lipoprotein (IDL) cholesterol (D) and high-density lipoprotein (HDL) cholesterol (E)) and the dose of ezetimibe (F) in nine Cushing’s syndrome dogs throughout 4 months of ezetimibe treatment. Individual “raw” values are expressed as dots. Line are expressed as median. Asterisks indicate significant differences (P<0.05) as compared with the 0-month value.

In this study, ezetimibe administration significantly decreased T-chol and LDL-chol in dogs with hypercholesterolemia from pretreatment to at two months treatment. Furthermore, we used nine dogs with hypercholesterolemia, all of which were diagnosed with hyperadrenocorticism. Therefore, ezetimibe was effective for treating secondarily induced hypercholesterolemia. As the reason, dietary and biliary cholesterol absorption was inhibited by ezetimibe administration. These hypercholesterolemia dogs were treated with a mean dose of ezetimibe from 0.027 ± 0.010 mg/kg/day [min-max: 0.012–0.042] at 2 month to 0.040 ± 0.023 mg/kg/day [min-max: 0.012–0.082] at 4 month. However, T-chol and each lipoprotein fractions did not changed from 2 to 4 months of treatment, indicating that the dose increase did not correspond with the efficacy of decreasing cholesterol over the short-term period. The reason for the dose-independent action of ezetimibe is as follows. Cholesterol is mainly acquired from two sources: dietary cholesterol, which is absorbed in the intestine, and intracellularly synthesized cholesterol, which is mainly synthesized in the liver. Ezetimibe selectively inhibits only the intestinal absorption of cholesterol, which selectively blocks the uptake of biliary and dietary cholesterol in the small intestine [23, 24]. Thus, ezetimibe does not have a lowering effect on hepatic cholesterol production. An increase in hepatic sterol excretion might occur to compensate for the loss of dietary and biliary cholesterol absorption at 4 months in the current study [6].

In the current study, we started at a relatively low dose of ezetimibe (0.01–0.03 mg/kg) referencing a previous study that used healthy dogs [6]. The recommended dosage of ezetimibe in human patients is 10 mg daily (approximately 0.1–0.2 mg/kg body weight) when administered as monotherapy or in combination with satin [18, 21]. Therefore, if there are no clinical side effects associated with the use of ezetimibe after the first 1–2 months, a gradual increase in the dose of ezetimibe may be necessary. Furthermore, there is a potential for cholesterol levels to decrease with long-term ezetimibe treatment. Additionally, it is worthwhile to consider the option of increasing the dosage.

To achieve further reduction in cholesterol levels, it should investigate the combination therapy with other medications such as statins and fibrates. Statin is the first-choice of cholesterol-lowering drug and is a 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitor in human studies. HMG-CoA reductase is an enzyme that limits the conversion of HMG-CoA to mevalonic acid, a precursor of sterols, including cholesterol. Inhibition of this enzyme initially leads to a reduction of cholesterol synthesized in the liver [24]. In human patients, combination therapy of ezetimibe and statin is recommended if target reduction in LDL levels is not achieved by ezetimibe or statin monotherapy [8]. Additionally, in this study, we also used two cases of combination therapy with bezafibrate (Table 1, case 5 and 6). Bezafibrate, dual and pan-peroxisome proliferator-activated receptors (PPAR) co-agonist, inhibits the cholesterol biosynthesis pathway, which starts from acetyl-CoA to mevalonic acid. Bezafibrate leads to considerable raising of HDL cholesterol and reduces triglycerides, improves insulin sensitivity and reduces blood glucose level, significantly lowering the incidence of cardiovascular events and new diabetes in patients with features of metabolic syndrome [13, 14, 26, 29, 31, 32]. Bezafibrate is primarily used to treat patients with hypertriglyceridemia; however, it was also report describing the efficacy of bezafibrate in combination with ezetimibe for treating dyslipidemia including hypercholesterolemia [30, 33]. In this study, the dosage of trilostane and other medication remained unchanged both before and after the four-month period following the commencement of ezetimibe administration. Therefore, ezetimibe treatment would be effective in lowering total cholesterol and LDL-chol. Future studies should investigate the combination therapy of ezetimibe and statins or fibrates in dogs with hyperlipidemia.

In previous studies, cholesterol and lipoprotein concentrations have been investigated in dogs with hypothyroidism, diabetes mellitus, and Cushing’s syndrome. Dogs with hypothyroidism have been shown to have increased levels of VLDL, LDL, and HDL-chol; dogs with diabetes mellitus have been shown to have increased VLDL and HDL-chol; dogs with Cushing’s syndrome have been shown to have increased LDL-chol [1, 9, 22, 25]. The nine dogs used in this study had Cushing’s syndrome dog and were concurrent with hypothyroidism (n=2) or diabetes mellitus (n=1). If these endocrine diseases are poorly controlled, it might be associated with increasing cholesterol during study period. However, no increase in trilostane doses was required during the study period. Moreover, other treatment for concurrent disease such as dose of insulin injection for diabetes mellitus and levothyroxine sodium tablets medication for hypothyroidism remained during study period. Therefore, the reduction in total cholesterol, especially LDL-chol, in the current study was likely due to the effect of ezetimibe through blocking the uptake of biliary and dietary cholesterol in the small intestine.

Side effects of ezetimibe are generally mild and gradually disappeared as the medication is adjusting. Common side effects include stomach (abdominal) pain and diarrhea in human patients [4]. In this study, no clinical side effect was associated with the use of ezetimibe.

In conclusion, ezetimibe treatments reduced the concentration of T-chol and LDL-chol concentration in hypercholesterolemic dogs with HAC, and were effective in the amelioration of dyslipidemia. Moreover, this study firstly used ezetimibe in dogs with hypercholesterolemia.

CONFLICT OF INTEREST

No potential conflict of interest was reported by the author.