Introduction

Hypothyroidism is a common endocrine disorder in dogs with a reported prevalence from 0.2 to 0.8%.^1,2 Most dogs diagnosed with hypothyroidism will require daily replacement of orally administered thyroid hormone for life, with the therapeutic goal of achieving a euthyroid state without inducing toxicosis. From the mid-1970s through 2015, there were multiple levothyroxine sodium products on the market targeting the treatment of hypothyroidism in dogs. However, none were approved by the FDA. In an effort to decrease the number of unapproved animal drugs on the market, the FDA Center for Veterinary Medicine launched an initiative in 2010 that encouraged manufacturers of unapproved drugs to seek approval by filing a New Animal Drug Application (NADA). LLOYD, Inc. responded to the FDA request and submitted a NADA for their brand of levothyroxine sodium tablets^a for dogs. The results of the present field trial were used as substantial evidence of effectiveness in support of the NADA and registration of the first FDA-approved product for oral levothyroxine sodium supplementation in dogs.

The purpose of this study was twofold: to establish the long-term effectiveness and safety of levothyroxine sodium tablets administered to dogs diagnosed with hypothyroidism and to investigate two different dosing schedules of levothyroxine sodium, administered daily either as a single dose or divided in two and administered q 12 hr.

Materials and Methods

The study was designed as a prospective, open-label, randomized (to treatment schedule), multicenter, pivotal, clinical field effectiveness study. Dogs of varying breeds with suspected primary hypothyroidism were screened and enrolled into the study from December 2010 to June 2012. During this period, a total of 92 dogs from nine clinical study sites located in multiple geographic regions of the United States met all the enrollment criteria and were included in the study’s patient population. Prior to any screening procedure, each dog owner was informed about the risks and benefits associated with the study and documented their consent to have their dog participate in the study by signing a statement of informed consent. A set of standardized case report forms was used to collect each subject’s data.

At the initial clinic visit (Visit 1), dogs suspected of being hypothyroid were considered for the study. Screening procedures included a thorough medical history, physical examination, routine bloodwork (complete blood cell count, serum biochemistry profile), thyroid hormone panel (total thyroxine [TT4], free thyroxine [fT4], thyroid-stimulating hormone [TSH], thyroglobulin autoantibody [TgAA]), and urinalysis to determine the health and thyroid status of each dog. Clinical and historical signs frequently associated with hypothyroidism were evaluated and scored for severity (Table 1).

TABLE 1 Assessment Criteria for Clinical Signs Associated with Hypothyroidism

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Dogs meeting the following criteria for hypothyroidism based on their thyroid hormone blood levels and clinical signs were eligible for study enrollment:

• Thyroid hormone blood levels

  •  ○TT4 < 15 nmol/L or fT4 < 0.8 pmol/L; and

  •  ○TSH > 37 mU/L or TgAA > 35%

• Clinical signs: Two or more clinical signs were required to be present

  •  ○Dermatologic lesions: at least one individual lesion with an aggregate score ≥4

  •  ○Ear canal condition score ≥3

  •  ○Body condition score of 7 or greater

  •  ○Bradycardia (<70 bpm)

  •  ○Hypercholesterolemia (>343 mg/dL)

  •  ○Activity level score of 0 or 1

Dogs receiving drugs that are known or are suspected to affect thyroid function were excluded from study participation until the specified washout period for each drug had been completed. These drugs included thyroxine and thyroxine analogs, glucocorticoids, tricyclic antidepressants, diuretics, amiodarone, iodide, phenobarbital, propranolol, sulfonamides, ipodate and iopanoic acid, over-the-counter nonsteroidal anti-inflammatory drugs, heparin, and general anesthesia. With the exception of the study treatment, prohibited medications during the study included thyroxine and thyroxine analogs. To better represent field conditions, dogs with concurrent medical disorders such as other endocrinopathies (e.g., diabetes mellitus, Cushing’s disease) or chronic hepatic or renal dysfunction were allowed to enroll in the study. However, pregnant or lactating bitches or dogs intended for breeding during the study were excluded.

Upon confirmation of study eligibility during the second clinic visit on day 0 (Visit 2), each dog was randomized to receive 0.1 mg per 10 lb (0.022 mg per kg) body weight per day in one of two treatment groups. Group 1 dogs received the entire dose q 24 hr. Group 2 dogs received ½ dose (0.05 mg per 10 lb [0.011 mg per kg]) q 12 hr. At each study site, randomization was achieved by assigning each dog to a two-dog block based on order of enrollment. The first dog within a block was randomized to be in either Group 1 or Group 2. The second dog within a block was subsequently assigned to the other treatment group. Under this method, each site received a separate and unique randomization schedule.

The study treatment consisted of oral tablets available in multiple strengths ranging from 0.05 to 0.8 mg levothyroxine sodium per tablet. Dogs were dosed at home by their owner at approximately the same time each day for 6 mo. Owners were instructed to maintain a consistent feeding schedule in relation to dosing time(s) for the duration of the study, so that each dog was either dosed in a fed or fasted state. Owners were also instructed to observe their dogs for any adverse events and to promptly report them to their study veterinarian. Adverse events were to be reported even if they didn’t appear to be related to the levothyroxine sodium treatment.

Subsequent in-clinic evaluations occurred on days 42 ± 5 (Visit 3), 70 ± 5 (Visit 4), 126 ± 5 (Visit 5), and 182 ± 5 (Visit 6). With the exception of TgAA, the tests and procedures performed at the screening visit were repeated. During these visits, blood was collected for thyroid hormone analysis 4–6 hr following dosing and sent to a centralized laboratory^b for analysis. The centralized laboratory conducted all the thyroid hormone testing and clinical pathology testing (complete blood cell count, serum biochemistry profile, urinalysis) for the study using validated tests and methodology and quality controls, as appropriate. Reference ranges were provided by the laboratory.

Based on the results of the thyroid hormone panel and clinical response to treatment, dosage adjustments were allowed to be made following Visits 3, 4, and 5. The frequency of dosing, however, was not allowed to be altered.

The primary outcome measure and basis for clinical effectiveness was determined by the Visit 6 thyroid hormone results and whether the dog had achieved a euthyroid state. For a dog to be classified as euthyroid, the TT4, fT4, and TSH serum concentrations all had to be within the following limits: TT4 ≥ 15 nmol/L and no more than 1.4-times the upper reference range limit of 67 nmol/L (93.8 nmol/L); fT4 ≥ 8 pmol/L and no more than 1.4-times the upper reference range limit of 26 pmol/L (36.4 pmol/L); and TSH ≤ 37 mU/L.

A secondary outcome measure for effectiveness included improvement in the hypothyroid-associated clinical signs that had been present at Visit 1. During Visits 3–6, clinical signs were evaluated and rated in comparison to the Visit 1 assessment as follows: dermatological lesions and otitis externa were rated as worsened, no change, fair, good, or excellent, and body condition, heart rate, serum cholesterol, and activity level were rated as worsened, no change, or improved.

The overall safety of chronic administration of levothyroxine sodium was assessed through adverse event reporting, physical examination observations, and clinical pathology testing (hematology, serum chemistry, and urinalysis).

Results

Of the 502 dogs screened for the study, a total of 92 were enrolled. Dogs that failed screening did so because their thyroid hormone levels and/or their hypothyroid-associated clinical signs did not meet the specified entrance criteria. The study population consisted of multiple breeds, with the most common being large mixed-breed dog (n = 14), golden retriever (n = 11), Labrador retriever (n = 10), beagle (n = 7), and boxer (n = 7). Additional demographics included 44 females (43 spayed, 1 intact) and 48 males (42 castrated, 6 intact) with a median age of 6 yr (range 1–13 yr) and median weight of 35.1 kg (range 8.5–68.2 kg).

The analyses for effectiveness included 78 protocol-compliant cases. From the original 92 cases, 14 were removed in total: 3 cases for missing Visit 6 and 11 cases for noncompliance with dosing and/or the Visit 6 blood sample for thyroid hormone analysis. Cases were evenly distributed between treatment groups, with Group 1 and Group 2 each containing 39 dogs. All 92 cases were included in the analysis for safety because each dog received at least one dose of the study treatment.

Thyroid Hormone Blood Levels

Laboratory results indicated that by Visit 3 and continuing through Visit 6, there was a significant (P < .10) and sustained improvement in all three thyroid hormone indices (TT4, fT4, and TSH) in both treatment groups compared with the Visit 1 baseline values (Figure 1).

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The analysis of individual thyroid hormones for Visits 3–6 showed that for TT4 and fT4, the mean increase from baseline in Group 1 dogs was significantly higher than the mean increase in Group 2 dogs (TT4: P = .0019; fT4: P = .0005). The change in TSH, however, was not significantly different between groups.

The analysis for treatment success (euthyroid [TT4, fT4, and TSH serum concentration all within desired limits] at Visit 6) showed that there was not a significant difference between Group 1 and Group 2 (P = .1121). When the data from both groups were pooled, the overall success rate was 75.64%, and the lower bound of the one-sided 95% confidence interval was 66.34% (Table 2).

TABLE 2 Overall Treatment Success

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Hypothyroid-Associated Clinical Signs

The results of each evaluation were summarized according to treatment group. In both groups, all clinical variables clearly showed a trend toward improvement that increased with time (Figure 2).

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Adverse Events

Four dogs experienced serious adverse events during the course of the study. Relationship to study treatment was classified as unrelated in two cases (mast cell tumor and gastrointestinal foreign body), possibly related in one case (immune-mediated thrombocytopenia), and unknown in one case (multiple, clinically significant laboratory abnormalities). The possibly related serious adverse event involved a 5 yr old neutered male boxer who was diagnosed with immune-mediated thrombocytopenia following Visit 5 and euthanized shortly thereafter due to apparent disease progression. A necropsy was performed. Significant postmortem findings were an anemic-appearing carcass with abundant blood in the stomach and intestines. However, no ulcer, mucosal hemorrhagic lesion, or other site/source of hemorrhage was identified. The report stated that this type of lesion was considered idiopathic with few differentials, including anticoagulant rodenticides and Clostridia-associated hemorrhagic gastroenteritis. Although there is a temporal association between the diagnosis of immune-mediated thrombocytopenia and the blood loss, the dog’s platelet numbers were within the reference range just prior to death, and thrombocytopenia was not listed as a differential for the cause of death on the necropsy report.

Throughout the study, a wide variety of nonserious adverse events were documented. The most commonly reported adverse events, affecting 10% of the patient population or greater, included the following in order: abnormal laboratory results, anorexia, dermatitis, emesis, otitis externa, lethargy, polydipsia, and diarrhea (Table 3). In the majority of cases, the relationship between the adverse event and study treatment was classified by the study investigator as unlikely. Adverse events deemed possibly related to treatment were reported in eight dogs and included anorexia, tachypnea, polyuria, polydipsia, anxiety, emesis, diarrhea, borborygmus, lethargy, epistaxis, scaling, seborrhea, and skin lesion. The only adverse event to receive a classification of probably related to treatment was a single report of TT4 elevation in a dog.

TABLE 3 AEs Reported in 10% or More of the Patient Population

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Discussion

Levothyroxine sodium has been used for decades to treat hypothyroidism in humans. It was first introduced into the marketplace in the 1950s without FDA approval because it was thought not to be a new drug. By 1997, almost all levothyroxine sodium tablet manufacturers had reported product recalls to the FDA that were the result of potency and/or stability problems. These products failed to maintain potency through to the expiration date, and tablets of the same dosage strength from the same manufacturer varied from lot to lot in formulation and the amount of active ingredient present. In humans, levothyroxine sodium is a compound with a narrow therapeutic range. Small variations in product potency or bioavailability can lead to underdosing or overdosing with subsequent suboptimal responses or thyrotoxicosis. Both of these outcomes can have serious consequences.

Concerned about the health and welfare of the public, the FDA announced in 1997 that orally administered drug products containing levothyroxine sodium are new drugs and mandated that by 2001, all such products must undergo the New Drug Application or Abbreviated New Drug Application approval process before they could be marketed.^5,6 Further tightening of levothyroxine sodium tablet standards occurred in October 2007, when the FDA petitioned the United States Pharmacopeia to change the potency standard from 90–110% of label claim to 95–105% of label claim.⁷ By October 2009, all levothyroxine sodium tablet manufacturers producing product for humans were required to meet this new specification.

As in humans, levothyroxine sodium is used to treat hypothyroidism in dogs. Similar to the pre-2001 products for humans, none of the approximately 10 different levothyroxine sodium products marketed for dogs were approved for use by the FDA. In 2010, the FDA announced an initiative to address unapproved animal drugs and actively sought out and encouraged manufacturers of unapproved products, such as levothyroxine sodium tablets, to undergo the NADA approval process.

Unlike the experience in the human sector, the motivation to regulate levothyroxine sodium tablets for dogs stemmed primarily from a compliance factor and not from a safety perspective, as there is in fact a much wider margin of safety in the dog compared with humans. The benefits of an FDA-approved drug include more predictability in manufacturing, including stability of the product; labeling consistent with FDA regulations; and government review of promotion and advertising materials to ensure they're not false or misleading. Additionally, with approved drugs, manufacturers are required to maintain constant safety surveillance and submit pharmacovigilance reports on a routine basis to the Agency.

There is a lot of information in the literature about the bioavailability differences between levothyroxine sodium tablets developed for humans and the problems associated with interchanging brands. However, not much exists on the same subject for dogs. Recommended dosing guidelines for the dog include using a brand name or proven generic equivalent of levothyroxine sodium tablets when initiating therapy and, once a maintenance dose is established, discouraging the substitution of one brand for another. If a substitution is made, the dog’s response to treatment should be monitored closely and the dose adjusted if necessary. Whether these dosing guidelines exist from a historical perspective and the use of nonbioequivalent products or from veterinary experience in the field, or both, there is a strong need to regulate the manufacture of levothyroxine sodium tablets and provide a reliable and consistent formulation manufactured to Good Manufacturing Practice standards for veterinarians to use in dogs.

The present study was conducted to provide substantial evidence of effectiveness in support of the NADA and registration of levothyroxine sodium tablets for the treatment of canine hypothyroidism. Careful consideration was given to the design of the study by reviewing the literature and the many studies that have been conducted in hypothyroid dogs. Two publications in particular, Le Traon et al. (2009) and Dixon et al. (2002), were key in forming the basic design elements of the study, including selection of the patient population, clinical evaluations, and therapeutic monitoring.^3,8

Response to treatment was rapid and sustained, and by Visit 3, at least one of the thyroid panel hormones (TT4, fT4, and TSH) had returned to desired therapeutic ranges in greater than 80% of dogs in both treatment groups and remained that way for the duration of the study. This is in line with the recognition that TSH and peak TT4 levels are usually within their reference ranges within approximately 2 wk after the start of therapy and that there is limited change unless an actual dose adjustment is made.⁸ The significantly higher increases in TT4 and fT4 levels at Visits 3–6 (from baseline) for Group 1 dogs compared with Group 2 can be explained by a combination of the treatment schedule, the pharmacokinetic parameters of levothyroxine sodium, and the timing of blood sampling. Dogs in Group 1 received their entire 24-hr dose 4–6 hr before sampling and therefore exhibited higher peak hormone levels compared with dogs in Group 2, who only received half of their 24-hr dose within the same time frame.

Supportive of the thyroid hormone results, the secondary outcome measure of hypothyroidism-associated clinical signs clearly showed a trend toward improvement that increased with time. Activity level and serum cholesterol showed the greatest initial improvement by Visit 3, whereas dermatological signs and body condition improved more slowly over a period of months. Similarly, Dixon et al. in 2002 reported that clinical resolution of metabolic signs such as lethargy and mental dullness can be expected within 2 wk of starting therapy, whereas other abnormalities, including dermatological signs, may take up to 3 mo to resolve.⁸

By the end of the study, 80% of dogs had daily maintenance doses of levothyroxine sodium ranging from 0.08 to 0.12 mg per 10 lb (0.0018–0.026 mg per kg) body weight (median 0.11 mg per 10 lb [0.024 mg per kg] body weight), thus confirming the selection of 0.1 mg per 10 lb (0.022 mg per kg) body weight per day as an appropriate starting dose. Dosing guidelines for levothyroxine sodium supplementation, however, recommend clinicians use a starting dose of 0.2 mg per 10 lb (0.04 mg per kg) body weight per day, or twice the amount used in this present study.⁹ Multiple other reports within the literature also support a lower starting dose and seem to indicate that with current levothyroxine formulations, a more appropriate starting dose should be half the recommended value.^3,4,8

The overall adverse event profile reflected an aging population of dogs, many of whom had concurrent and chronic medical conditions. The majority of commonly reported nonserious adverse events were attributed to the disease state itself or were a result of the transition from a hypothyroid state to a euthyroid state. Nonserious adverse events classified with a probable or possible relationship to treatment were attributed to suboptimal thyroid hormone levels (necessitating a dosage adjustment) or a transient worsening of the dermatologic condition before improving after initiation of levothyroxine supplementation.⁸

Of the four reported serious adverse events, only one describing immune-mediated thrombocytopenia in a boxer was classified as possibly related to treatment with levothyroxine sodium. In the companion Target Animal Safety Study, dogs who received 0.2 or 0.6 mg levothyroxine sodium per 10 lb (0.044 or 0.132 mg per kg) body weight per day for 6 mo had mild decreases in mean platelet counts compared with dogs who received no levothyroxine sodium.¹⁰ The boxer in this efficacy study did not have evidence of oversupplementation. The TT4 and fT4 serum concentrations were within the endogenous reference ranges, and the TSH was not suppressed. Although the dog did experience weight loss between the autoimmune thrombocytopenia diagnosis and when it was euthanized, it did not have other clinical signs (tachycardia, hyperexcitability) associated with hyperthyroidism. Overall, the type and frequency of reported adverse events were expected considering the length of the study and targeted patient population; no abnormal trends or unusual findings were observed.

The statistically significant differences in lymphocyte percentage and segmented neutrophil count between the two treatment groups were not clinically relevant, as the mean values for Group 1 and Group 2 dogs fell within or close to the normal expected ranges, the absolute differences were not great, and there was not a consistent unifying pathophysiological explanation for the findings.

The findings that seven individual dogs had red blood cell indices above the reference range and three individual dogs had increases in liver enzymes were not unexpected. Thyroid hormone status is well known to affect erythropoiesis and the resultant red blood cell indices.^2,11,12 Increases in hematocrit are associated with increased blood viscosity, with the rate of viscosity increase becoming more pronounced at a hematocrit of 60%.¹³ At a hematocrit of 70%, the viscosity is twice that of dogs with a normal hematocrit. Among the seven dogs with red blood cell indices above the reference range at Visit 6, one had a hematocrit of 61%; this was the only one with a value >60%. None of these dogs had clinical signs (injected conjunctival blood vessels, brick-red mucous membranes, ataxia, seizures, blindness, behavior changes) associated with hyperviscosity.

Thyroid hormone status is also associated with alterations in liver enzymes, with increased values reported for both hypothyroid and hyperthyroid individuals.^2,10,14,15 Increases in liver enzyme activity in hypothyroid dogs and humans at the initial follow-up after the start of levothyroxine sodium treatment have been previously reported.^7,16

Conclusion

This field study successfully demonstrated the long-term safety and effectiveness of levothyroxine supplementation using LLOYD, Inc.’s brand of levothyroxine sodium tablets^a for the treatment of dogs with hypothyroidism and hypothyroid-associated clinical signs. The results of this study also supported a daily starting dose of 0.1 mg per 10 lb (0.022 mg per kg) body weight and showed that the treatment regimen has no effect on clinical outcome.