Introduction

Hypertrophic cardiomyopathy (HCM) is the most common cardiovascular disease in domestic cats with a prevalence of 14%.^1–3 HCM is defined as asymmetric or symmetric thickening of the left ventricular posterior wall (LVPW) or interventricular septum (IVS) in the absence of any hemodynamic cause (ie, systemic hypertension, congenital disease, or fixed stenosis) or hormonal stimuli (ie, hyperthyroidism or hypersomatotropism).^1,2 The disease is typically irreversible and progressive. While some cats remain subclinical throughout life, many progress to develop 1 or a combination of severe outcomes including left-sided congestive heart failure (CHF), arterial thromboembolism (ATE), and sudden death with reported occurrence rates of 23.9%, 11.3%, and 3.4%, respectively.⁴ Despite extensive investigative efforts, current available drug therapies have failed to delay progression of disease, improve quality of life (QOL), or show survival benefit in the subclinical setting.^1,2 A contributing factor may be that many existing drug therapies target the hemodynamic consequences of HCM rather than abnormal molecular pathways or proteins involved in initiating the disease. Therefore, it is imperative to explore novel therapeutic targets to treat cats with occult HCM.

Rapamycin, also known as sirolimus, a macrolide and inhibitor of the mechanistic (previously called mammalian) target of rapamycin (mTOR), shows promise as a novel therapeutic for feline HCM.^5–10 mTOR is an atypical serine/threonine protein kinase that associates with several proteins to form 1 of 2 multiprotein complexes: mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2).^5,11,12 These 2 complexes have distinct roles in promoting cardiovascular health during both embryogenesis and postnatal life with varying up- and downstream targets. Activation of mTORC1 promotes anabolic processes that increase both protein and lipid synthesis, downregulates catabolic processes to reduce autophagy (the cell’s ability to remove damaged proteins and organelles), and plays a vital role in signaling adaptive cardiac remodeling in response to mechanical overload.⁵ Although not completely understood, mTORC2 plays a role in regulating glucose and lipid metabolism and promotes cardiomyocyte cell survival, cytoskeletal organization, and appropriate cell polarity.

Both mTORC1 and mTORC2 are essential for normal cardiovascular health; complete disruption of these complexes leads to metabolic derangements and cardiovascular disease.⁵ However, studies have demonstrated that mTORC1 activation from chronic hemodynamic stressors may be detrimental to the heart, leading to pathologic hypertrophy. While mTORC1 is acutely sensitive to rapamycin, the mTORC2 protein complex is comparatively insensitive. Prolonged chronic exposure to rapamycin is required to disrupt mTORC2 in vivo or in cell culture.¹² Inhibition of mTORC2 has been shown to result in glucose intolerance and hepatic insulin resistance.¹³ Intermittent rapamycin dosing has been shown to mitigate the deleterious effects of prolonged mTORC2 inhibition while enabling sustained inhibition of mTORC1 in many tissues.¹⁴ Numerous studies involving mouse models of HCM, as well as a human study in renal transplant patients, have demonstrated that inhibition of mTORC1 with rapamycin resulted in reversed pathologic left ventricular (LV) hypertrophy.^6–10 Additionally, a study¹⁵ of dogs demonstrated beneficial effects of intermittent dosing of rapamycin in aging hearts, including improved diastolic and systolic indices of heart function.

Once-weekly administration of a new formulation of delayed-released (DR) rapamycin has recently been developed for treatment of feline HCM. The objective of this study was to evaluate the safety and efficacy in reversing or delaying progression of myocardial hypertrophy and consequently cardiac dysfunction via once-weekly oral dosing of DR rapamycin in client-owned cats with subclinical HCM over a 6-month period. We hypothesized that once-weekly dosing of rapamycin would be well tolerated in cats and either reverse or prevent progression of LV remodeling echocardiographically.

Methods

Results

A total of 53 cats were screened for the study. Doses administered to the 24 evaluable rapamycin-treated cats in the low-dose and high-dose groups were 0.25 to 0.38 mg/kg and 0.52 to 0.73 mg/kg, respectively. Of the cats recruited for screening, 9 were immediately excluded due to severe LVOTO (n = 4), negative or equivocal HCM status (3), body weight below inclusion criteria (1), and echocardiographic evidence of a ventricular septal defect (1). One cat was excluded from analysis after completing the study due to persistent hypercalcemia, present prior to enrollment, that was initially overlooked. A total of 43 cats were successfully enrolled. A total of 36 cats completed all follow-up visits until day 180, 38 cats completed visits up to day 120, and 41 cats completed visits up to day 60. The remaining 7 of 43 cats were excluded for the following reasons: initiation of clopidogrel during the study period (n = 1), loss of follow-up (1), owner request for withdrawal due to personal reasons unrelated to the study (1), development of left-sided CHF (1), development of left-sided CHF that resulted in humane euthanasia (1), development of seizures and death (1), and development of diabetic ketoacidosis (DKA) and death (1). The cat removed from the study due to starting clopidogrel was in the placebo group, the cat hospitalized for DKA was receiving the low-dose study drug, and all other cats withdrawn from the study were receiving the high-dose study drug.

Five cats experienced serious adverse events during the study period, 4 of which were removed from the study for reasons listed above. One serious adverse event was considered unrelated to treatment (development of a urethral obstruction), and the study subject remained in the study. Four of the cats with serious adverse events were in groups receiving rapamycin (2 cats progressed to CHF, 1 cat died suddenly, and 1 cat developed DKA) and 1 was in the placebo group; there were no difference between number of adverse events in the placebo versus treatment group (P > .99). Sixty-nine adverse events were observed during the study. Two adverse events were determined to be “probably” related to the IVP. Fourteen adverse events were determined to be “possibly” related to the IVP. The remaining 53 adverse events were determined to have an “unrelated” or “unknown” relationship to the IVP. A total of 21, 23, and 25 adverse events were observed for placebo, low-dose, and high-dose groups, respectively. No statistically significant difference between the number of adverse events between groups was noted (P = .82). A complete list of all adverse events is provided elsewhere (Supplementary Table S2).

Of the 43 cats (6 spayed females and 37 castrated males) enrolled in the study at baseline, mean age was 6 years (range, 1 to 12 years) and was similar across groups. Breed and sex distribution among treatment groups is provided elsewhere (Supplementary Table S3). Mean body weights for cats in the placebo, low-dose, and high-dose groups were 6.06 kg (range, 4.2 to 14 kg), 5.97 kg (range, 4 to 8.9 kg), and 4.94 kg (range, 3.3 to 6.3 kg), respectively. Mean body weight, respiratory rate, and heart rate were not significantly different among groups over time. No differences were observed in QOL scores, systolic BP, biochemistry or hematology values, total T4, or fructosamine between groups at baseline or at any additional time point during the study period. Proteinuria was detected in select cases in placebo, low-dose, and high-dose groups, but there was no treatment effect observed (Supplementary Tables S4–S7).

Echocardiographic values were not significantly different between the 3 study groups at baseline. Following the 180-day treatment period, significant differences in LV MWT were identified (Figure 1). Following RMANCOVA of the primary efficacy outcome variables, a significant treatment by visit effect on MWT at any location was detected. Low-dose MWT values were significantly lower than values reported in the placebo group at visit 3 (day 180; P = .01). The pooled (low- and high-dose) IVP versus placebo comparison at visit 3 (day 180) also indicated a significant difference in MWT (P = .05). Additionally, a significant treatment effect on maximum LVPWd 2-D obtained from the right parasternal imaging window was detected. A summary of the RMANCOVA statistical analysis of the efficacy outcome variables is presented (Tables 2 and 3). Baseline, day 60, and day 180 echocardiographic data are presented as arithmetic means and SDs (Supplementary Table S8). The percent of animals with a change from screening in MWT of > 0.5 mm was not significantly different between treatment groups (Supplementary Table S1).

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Temporal comparison of maximum wall thickness (MWT; mm) at any location between placebo (gray) and low- (pink) and high-dose (teal) delayed-release rapamycin. The * and ** denote a P value of .05 for pooled dose groups compared to placebo and .01 for low dose compared to placebo, respectively. Values are reported as mean ± SEM.

Citation: Journal of the American Veterinary Medical Association 261, 11; 10.2460/javma.23.04.0187

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The relationship between baseline MWT and NTproBNP values to progression/response was evaluated to determine whether baseline parameters were useful predictors of treatment outcome. A statistically significant relationship between NTproBNP values at baseline and change in MWT from baseline to day 180 was observed. Higher baseline NTproBNP values were positively correlated (r² = 0.26; P = .001) with a change in MWT from baseline (Figure 2). When placebo- and rapamycin-treated groups were compared, a more pronounced MWT progression from baseline to day 180 in cats receiving placebo with baseline NTproBNP > 100 pmol/L was noted, although this did not reach statistical significance (P = .11; Figure 3). Neither baseline MWT nor baseline cTNI predicted change in MWT at day 180 (r² = 0.09, P > .23; and r² = 0.11, P > .31, respectively).

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Pearson correlation of baseline N-terminal pro-B-type natriuretic peptide (NTproBNP) with change in MWT (mm) from baseline to day 180. The best-fit line is shown in bright pink. The 95% CI is shaded in light pink.

Citation: Journal of the American Veterinary Medical Association 261, 11; 10.2460/javma.23.04.0187

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Baseline NTproBNP values and MWT changes (%) from baseline to day 180 in placebo (gray) and low- (pink) and high-dose (teal) delayed-release rapamycin groups. Color-matched curved best-fit lines are shown for each dose group by use of a nonlinear regression model and least-squares method in Prism (version 9.0; GraphPad Software). A dashed vertical line at 100 pmol/L denotes the cut point used for an abnormal NTproBNP in cats.

Citation: Journal of the American Veterinary Medical Association 261, 11; 10.2460/javma.23.04.0187

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Midway into the study period, previously used US CTnI kits were discontinued by the manufacturer, Siemens Healthineers, and no longer available for use by the reference laboratory (Texas A&M Gastrointestinal Laboratory); for this reason, subsequent analyses were conducted using an HS kit. Mean baseline results for US kits were 0.63 ng/mL (range, 0.05 to 1.84 ng/mL), 0.57 ng/mL (range, 0 to 1.9 ng/mL), and 0.60 ng/mL (range, 0 to 1.98 ng/mL) for low-dose, high-dose, and placebo groups, respectively. At day 180, mean values using HS kits were numerically higher in the placebo group at 491.45 pg/mL (range, 14.63 to 1,996.57 pg/mL) than in the low-dose group at 254.39 pg/mL (range, 9.01 to 808.6 pg/mL) and in the high-dose group at 244.03 pg/mL (range, 15.02 to 445.9 pg/mL), but these differences did not reach statistical significance (P = .25).

Discussion

This was the first study evaluating the effects of rapamycin in cats with subclinical HCM. Furthermore, this study evaluated these effects in the absence of observed pressure overload. The data presented here demonstrate that once-weekly low-dose (0.3 mg/kg) DR rapamycin significantly alters the primary outcome variable, MWT, over a 6-month period. Furthermore, oral low- and high-dose DR rapamycin were well tolerated with no significant differences in adverse effects compared to placebo. Therefore, results of this study suggest that this new formulation of rapamycin may effectively prevent or delay progression of LV hypertrophy in cats with subclinical HCM, establishing DR rapamycin or inhibition of the mTOR pathway as the first promising new approach for treatment of subclinical feline HCM.

Similar to our findings, previous in vitro and in vivo studies in mouse and rat models, as well as humans, have also reported either regression or reduction of LV remodeling in response to rapamycin administration, although the vast majority of these studies evaluated LV hypertrophy in the context of pressure overload.^6–9,21 Nonetheless, investigations that evaluated the effects of rapamycin on LV hypertrophy following aortic banding in mice consistently demonstrated regression of LV cardiac remodeling assessed both echocardiographically and histologically compared to mice receiving a vehicle control.^6–8,21 Mice receiving rapamycin had an attenuated increase in cardiomyocyte size and heart weight and a reduction in interstitial fibrosis. These studies also demonstrated a normalization of gene expression altered by pressure overload–induced LV hypertrophy.^7,8 Most notably, while aortic banding led to increased expression of MYH7 and reduced expression of SERCA2a, rapamycin-treated mice reverted to normal SERCA2a expression and enhanced expression of MYH7 and MYH6 compared to their vehicle-treated controls.⁷ These studies showed that rapamycin therapy was associated with a reduction in several downstream targets of the mTOR pathway important in translation and protein synthesis, including p70 ribosomal S6 kinase (S6K1) activity in the acute and chronic setting^6–8 and eukaryotic translation initiation factor-4E in the chronic setting.⁸ Interestingly, when S6K1 activity was evaluated in rapamycin-treated rats that received aortic constriction or sham procedure, both groups had a reduction in S6K1 activity compared to vehicle-treated controls, suggesting that rapamycin appears to target pathways involved in LV hypertrophy by mechanisms independent of the effects of pressure overload alone.²¹

Finally, in humans, the effects of rapamycin on LV hypertrophy in kidney transplant recipients have been explored.¹⁰ Although calcineurin inhibitors (cyclosporine or tacrolimus) are most commonly used, some patients do not tolerate these medications due to calcineurin-inhibitor–induced nephrotoxicity. A 2008 study¹⁰ demonstrated significantly reduced LV mass index and IVSd wall thickness in renal transplant recipients receiving rapamycin over the course of 1 year compared to those receiving cyclosporine or tacrolimus. Although these patients were also receiving antihypertensive therapy with similar BPs achieved, significant regression in LV mass index and IVSd measures were only observed in patients on rapamycin therapy. This suggests that rapamycin acted through mechanisms independent of hemodynamic load.

While rapamycin-specific in vivo effects on cardiac health in cats have not been previously evaluated to the authors’ knowledge, a feline model of induced right ventricular pressure overload demonstrated that many of the downstream pathways of mTOR activation are involved in feline right ventricular hypertrophy, further emphasizing that rapamycin may be a beneficial target for feline LV hypertrophy, whether it be due to pressure overload or primary HCM.²² Furthermore, feline cardiomyocytes have been evaluated in vitro, in which rapamycin effectively downregulates or augments downstream targets of mTOR.^23,24

It is noteworthy that a significant effect of rapamycin on LV hypertrophy was seen in the low-dose rapamycin group as opposed to the high-dose treatment group. This is postulated to be due to the dosing strategy utilized. Intermittent and low-dose administration of rapamycin has been shown to partially inhibit mTORC1 without impacting mTORC2, therefore maximizing its effects on LV remodeling while minimizing adverse side effects secondary to mTORC2 inhibition.^5,12,13 Inhibition of mTORC2 has been shown to result in glucose intolerance and hepatic insulin resistance.¹³ In the current study, a single cat with risk factors for DM (obese body condition and prescribed a satiety diet) at baseline went on to develop overt DM and DKA during the study period, suggesting that mTORC2 susceptibility to inhibition may be dependent on individual patient factors. This cat had changes on day 120 bloodwork and urinalysis consistent with a diagnosis of DM that were not initially recognized by the clinician. Per the study protocol, discontinuation of therapy and removal of the cat from the study should have occurred at this point, as glucose intolerance is a known potential effect of rapamycin in other species. The occurrence of DKA in this cat on the current study suggests that caution is warranted in administering rapamycin to feline patients with risk factors for DM and that patients should be carefully monitored during dosing for development of DM. Overall, more cats in the treatment groups experienced serious adverse events compared to the placebo group; however, this did not reach statistical significance. Aside from the aforementioned patient with DKA, all other serious adverse events represented reported disease progression (CHF and sudden death) in patients with advanced, subclinical HCM. Importantly, activity of mTORC1 and mTORC2, as well as their downstream targets, were not directly measured, and as such, this hypothesis cannot be entirely confirmed.

In addition to the study’s primary outcome variables, evaluation of baseline characteristics that may predict response to therapy were explored. This is of particular interest in feline HCM, as previous studies have documented some cats with little progression over the lifetime of this disease.⁴ In this dataset, NTproBNP was moderately correlated with response to therapy by change in MWT over time, while baseline cTNI and baseline MWT were not significant. Unfortunately, due to a change in methodology with the reference laboratory for measurement of cTNI during the study period, exploratory evaluation of cTNI was limited to baseline assessment. Future pivotal studies should aim to further explore these relationships to predict the most robust drug responders.

It is important to evaluate our findings considering certain limitations. Our primary end point of altered MWT was chosen on the basis of the known pathophysiology of HCM and prior studies demonstrating favorable efficacy in the management of maladaptive LV hypertrophy. Future pivotal studies investigating the impact of rapamycin on previously observed prognostic markers of feline HCM are warranted. Additionally, this study only evaluated dosing of DR rapamycin in subclinical cats with HCM over a 6-month period without evidence of significant LVOTO. It is unclear how rapamycin will impact this population of cats over a longer period of time or in cats with significant LVOTO, although animal models of pressure overload would suggest that cats with LVOTO would likely benefit from this drug as well.^6–8 Further studies are required to determine whether the benefits observed in this study ultimately result in a prolonged life-span, reduced risk of complications associated with HCM (ie, CHF, ATE, arrhythmia generation, and sudden death), and improved QOL. Furthermore, whether rapamycin will have a similar impact if started in patients after development of one of these severe outcomes remains unclear. A study⁷ of mice with LV hypertrophy due to aortic banding showed improved LV remodeling in mice with both compensated and decompensated LV hypertrophy, suggesting that rapamycin may be beneficial in both the subclinical and symptomatic phases of disease. The precise mechanisms of action of the drug on the hypertrophied LV myocardium remain unknown; future studies interrogating drug impact on gene and protein expression at the level of the feline heart are warranted. Finally, while we utilized statistical testing to minimize differences between groups at baseline and a P value threshold that reflected the exploratory nature of this initial nonpivotal study, the possibility of a type I error must be considered.

It is important to address the remarkable heterogeneity of genotypic and phenotypic expression of feline HCM and how this may impact the effect of rapamycin in a larger population of affected cats. While this may have impacted our results to some degree, there still appears to be a clear treatment effect on LV hypertrophy in cats receiving low-dose rapamycin. Future studies could aim to evaluate the impact of pharmacogenetics on the individual patient’s response to rapamycin. Although this nonpivotal study reached an adequate sample size to resolve statistical significance for MWT, the possibility of both type I and type II error remains.

In conclusion, this study showed that DR rapamycin is a promising novel drug for continued study of the treatment of subclinical feline HCM that may prevent or delay progression of LV hypertrophy. Results of our study demonstrated that once-weekly dosing of DR rapamycin was well-tolerated at both doses studied. The significant treatment by visit effect on MWT at any location detected in the low-dose group on day 180 supports the effectiveness of 0.3 mg/kg once-weekly DR rapamycin in reducing LV hypertrophy in feline subclinical HCM. The greater treatment effect observed in the low-dose group compared to the high-dose group may represent a type II error given the small cohort size (n = 10) in the high-dose group at day 180, or may reflect a more favorable balance of mTORC1 suppression and activity across the weekly dosing interval while minimizing long-term adverse effects via mTORC2 inhibition. Future studies are needed to elucidate the impact of this drug on long-term survival and determine whether rapamycin delays the onset of other HCM disease outcomes.

Supplementary Materials

Supplementary materials are posted online at the journal website: avmajournals.avma.org