Safety of TCMCB07, a melanocortin-4 antagonist peptide, in dogs with naturally occurring cachexia

2.1 Animals

This study was a multi-institutional, prospective, 1-armed open-label trial in client-owned dogs with naturally occurring cachexia from the years 2016 through 2021. All procedures were approved by an Institutional Animal Care and Use Committee (University of Florida IACUC protocol #201810174, University of Missouri IACUC protocol # 8441). The following institutions participated in recruitment: University of Florida, University of Missouri, VCA Animal Diagnostic Clinic, VCA West Los Angeles, VCA Family and Oahu Veterinary Specialty Center, VCA Northwest Veterinary Specialists, and VCA Animal Specialty and Emergency Center. Client-owned dogs with naturally occurring cachexia were recruited from the institutions’ presenting pet population. Dogs of any weight were eligible for enrollment after informed client consent if, on examination, there was clinical evidence of cachexia based on body condition and muscle condition scoring and at least 5% loss of body weight within the past 3 months. On biochemistry panel, the dog's serum activity of liver-derived enzymes must have been lower than 5 times the upper limit of reference range with normal plasma or serum bilirubin. Concurrent anti-emetic therapy and therapy for an underlying disease process was allowed. Glucocorticoids were permitted if therapy had been ongoing for greater than 14 days before enrollment, there was no change in dosage upon enrollment, and no increase in body weight or body condition score had occurred. Appetite stimulants were not allowed within 2 days of the first TCMCB07 treatment. Exclusion criteria included cancer of the oral cavity, cancer of the gastrointestinal tract, or other identified disease process(es) if that process led to an inability to consume food or caused a partial or complete gastrointestinal obstruction. Additionally, dogs with active cardiogenic pulmonary edema, bradyarrhythmias, or pathologic tachyarrhythmias were not included in the trial.

After informed client consent, screening was performed to confirm eligibility for trial. This included a physical examination, complete blood count, chemistry panel, urine analysis, abdominal ultrasound, Doppler blood pressure, and 24-hour Holter monitoring. At enrollment a standardized quality of life (QOL) assessment was completed by the client¹⁹; weight, photographs, and body condition score of the dog were recorded. Three body condition scoring systems were used. The trial was initiated using the Tufts Animal Care and Condition Scale, a 5 point thin dog scale that better reflected subtle changes in thin/muscle wasted dogs than more traditional body scoring systems.²⁰ A score of 5 indicated emaciation and a score of 1 indicated an ideal body condition. As the trial progressed, 2 additional body condition scores were utilized, the 9-point Purina body condition score (score of 1-3 is too thin, 4-5 ideal, 6-9 too heavy)²¹ and the World Small Animal Veterinary Association (WSVA) muscle condition score (1 = normal 2 = mild muscle loss, 3 = moderate muscle loss 4 = severe muscle loss).^{22, 23} The screening Holter recording was reviewed and approved by a board certified veterinary cardiologist, and therapy commenced if no underlying arrhythmias occurred that would interfere with therapy.

2.2 Drug production

CPC Scientific Inc. (Sunnyvale, CA 94089) produced TCMCB07 using current Good Manufacturing Practice conditions. The active pharmaceutical component was dissolved in milliQ water at 10 mg mL⁻¹ and sterile filtered using a 0.22 μm Millex®GP PES Membrane (Merck Millipore Ltd, Tullagreen IRL). Drug was stored at −20°C and shipped on ice overnight to participating institutions. Drug was stored protected from light and stored at 4°C once received at each institution and after prescribed to pets.

2.3 Treatment

On the first day of treatment (day 1) a weight, body condition score, and physical examination were performed in addition to the first treatment of TCMCB07 and collection of blood samples via jugular or peripheral venipuncture for pharmacokinetic evaluation at time points 0, 1, 4, and 24 hours post-treatment. Dogs were dosed at 0.75 mg kg⁻¹ administered subcutaneously once every 24 hours based on results in a previous study in dogs.¹¹

Clients were taught to administer TCMCB07 by subcutaneous injection and provided with a drug administration diary. Return visits were required at day 14 and day 28, at which time the following data were collected: weight, body condition score, dog photos, quality of life form, physical examination, complete blood count, chemistry panel, and 1-hour postdrug administration pharmacokinetic (PK) sample. At day 28, Doppler blood pressure (prior to drug administration) and a 24-hour Holter monitor (applied before or after drug administration on day 28) were also collected and applied, respectively.

Following the initial 28-day study period, if a good response was noted (such as increased appetite, activity level, weight gain, or any of these), clients were permitted to have their dog continue TCMCB07 treatment daily. These dogs were required to follow up monthly for 2 months and then once every 3 months for the following: physical examination, QOL form, weight, body condition score, dog photos, complete blood count, chemistry panel, urine analysis, and drug plasma concentration sample 1-hour post TCMCB07 administration. Dogs that showed improvement but had not returned to a normal body condition were allowed to continue at an increased dosage of 0.75 mg kg⁻¹ every 12 hours if this was considered to be in the best interest of the dog in the opinion of the owner and treating clinician.

2.4 Plasma concentration determination

Plasma concentrations of TCMCB07 were assessed on days 1 (timepoints 0, 1, 4, and 24 hours post TCMCB07 administration), and 1-hour postadministration on days 14, 28, and any additional follow up visits for dogs on long-term treatment based on the T_max in Beagle dogs of 1 hour.¹¹ Whole blood (1–3 mL) was collected using jugular or peripheral venipuncture into lithium heparin tubes and kept on ice until centrifugation. Blood was centrifuged at 13 000 rpm at 4°C for 2.5 minutes, and plasma was extracted and stored at −80°C for batch analysis. Plasma concentrations of the active pharmaceutical ingredient was measured as previously described.¹¹ The maximum, or peak concentration (C_max) is described.

2.5 Statistics

Continuous data sets (body weight, blood chemistry, CBC, blood pressure, heart rate, etc) were analyzed utilizing parametric tests, either Paired t test for before and after comparisons or repeated measures analysis of variance (ANOVA) followed by post hoc analysis when appropriate. Ordinal data sets (body condition score, quality of life, etc) were analyzed using either Wilcoxon sign ranked (before vs after) or Friedman analysis as appropriate. A probability level of ≤.05 was used for significance.

3.1 Clinical findings

Twenty-five dogs were deemed eligible for enrollment and screening based on history, examination, and initial bloodwork. After informed client consent and the initial testing to confirm eligibility, 23 dogs were deemed eligible to start TCMCB07 therapy; 2 dogs were withdrawn by investigators due to underlying arrhythmias detected on the 24-hour Holter monitor. Five dogs did not start TCMCB07 therapy due to client withdrawal (rapid disease progression of neurologic disease—suspected forebrain lesion n = 1 or declined to start after screening n = 4; Table S1).

Eighteen dogs with cachexia started treatment in the 4-week open label trial. Sixteen were neutered males and 2 were spayed females. Dog breeds included: mixed breed (n = 8), Labrador retriever (n = 3), and 1 each of the following: American cocker spaniel, border collie, Bernese mountain dog, Chihuahua, Maltese, American pit bull terrier, and wolf hybrid. The reason for presentation and evaluation included cancer in 8 dogs: intermediate-to-large cell lymphoma (n = 3) and 1 each of the following: osteosarcoma, intestinal carcinoid with liver metastasis, thyroid and salivary carcinoma (in the same dog), metastatic prostate adenocarcinoma, and hepatobiliary neoplasm with intrahepatic metastasis. Other underlying diagnoses were as follows: suspected age-related cachexia without detectable underlying pain (n = 2) and 1 each of the following: chronic pain, congestive heart failure due to dilated cardiomyopathy, congestive heart failure due to degenerative valve disease (ACVIM stage C), inflammatory effusive disease of unknown origin manifesting primarily as pleural effusion, lymphoplasmacytic gastritis, intervertebral disc disease, generalized neurologic disorder of unknown etiology, and hepatopathy with chronic intervertebral disc disease.

Of the 18 dogs enrolled, 2 dogs suffered progressive lymphoma at day 7 (n = 1) and day 21 (n = 1) of trial, these dogs were removed from trial and humanely euthanized (neither dog received follow up therapy for lymphoma or palliative care). Two other dogs missed their week 4 visit by 6 and 19 days, and were removed from study evaluation for a total of 14 evaluable dogs (Table S2). Twelve of 14 dogs had concurrent medication lists completed; concurrent medications included metoclopramide (n = 1), maropitant (n = 3), ondansetron (n = 1), cyclosporine (n = 1), tramadol (n = 2), gabapentin (n = 4), nonsteroidal anti-inflammatory drugs (n = 3), diazepam (n = 1), intermittent capromorelin (n = 1), omeprazole (n = 1), phenylpropanolamine (n = 1), prazosin (n = 1), trazodone (n = 1), metronidazole (n = 1), prednisone (started prior to trial entry according to enrollment criteria, n = 2), furosemide (n = 1), spironolactone (n = 1), and pimobendan (n = 2). Eight dogs continued therapy beyond the 4-week trial period. Four dogs were lost to follow up after 56 days, 1 lost to follow up after 180 days, and the other dogs remained on drug for 150, 300, and 390 days (Figure 1).

During treatment with TCMCB07, cachectic dogs had a significant increase in body weight, Tufts 5-point thin dog body condition score, and WSAVA 4-point muscle condition score. The mean body weight was significantly increased on days 14 and 28 compared to day 1: on day 1 of trial the mean body weight was 18.6 kg which increased to 19.2 kg on day 14 and 19.5 kg on day 28 (P < .02; Figure 2). The median Tufts 5-point thin dog scale score (n = 14 dogs) was 3 (thin) on all time points measured days 1 (score range 2–4), 14 (score range 2–4), and 28 (score range 1–4), however, there was a significant improvement on day 28 compared to day 1 (P < .004) as dogs' scores improved over time (Figure 3). Similar results were found with the WSAVA muscle condition score (n = 12), in which the median score was 3 (moderate muscle wasting) on days 1 (range 2–4), 14 (range 2–4), and 28 (range 1–4), but a significant improvement was noted on day 28 of trial (P < .02; Figure 4). No change was noted when using the Purina 9-point body condition scoring system (n = 9). The median body condition score using the Purina 9-point system was 3 (too thin) with a range of 2–4 on days 1, 14, and 28 of trial (P = .36). A representative dog in trial is provided in Figure 5 and Figures S1–S3. In addition, 4 of 14 dogs experienced a change of pigmentation to their coat at day 28 or later, when therapy was extended past the 28-day trial period (Figure 6). Tables S3 and S4 summarize weight and Tufts body condition score data in all 14 evaluable dogs.

Fourteen clients completed the quality of life questionnaires at all timepoints; however, not all clients answered all questions. Quality of life questionnaires were evaluated and quality of life ratings were compared between days 1, 14, and 28 (Table 1). Two variables were statistically significant over the course of the study period. Pet owners perceived their dog appeared to be panting less over the course of the trial (P < .002) and that the general health of their dog had improved (P < .03; Figure 7).

3.2 Clinical pathology data

Hematology on all 14 dogs revealed no significant differences between days 1, 14, and 28 (Table 2). There were also no significant differences between any of the chemistry panel values between days 1, 14, and 28 with the exception of blood urea nitrogen (Table 3). Blood urea nitrogen was increased at days 14 and 28 when compared to day 1 (day 1 mean 21.79 mg dL⁻¹, day 14 mean 31.79 mg dL⁻¹, and day 28 mean 30.43 mg dL⁻¹, P < .004; Figure 8). The increases were not correlated with increases in creatinine, decreases in red blood cell count, or other clinical variables.

3.3 Cardiovascular data

There were no significant changes in systolic blood pressure between screening and day 28 of trial (Figure 9). When comparing the screening and day 28 Holter monitor, there was no difference in the maximum heart rate. There was an increase in the mean and minimum heart rate between day 0 and day 28 of the study (P ≤ .03). There were no increases in the occurrence of ventricular ectopy or maximum pauses in heartbeat between days 0 and 28 (Figure 10).

3.4 Plasma concentration data

On day 1 of drug therapy, the plasma C_max of TCMCB07 was a mean of 1.52 ± 0.2 μg mL⁻¹ standard error of the mean (SEM) 1 hour after drug administration. At 4 hours, plasma concentrations were 1.23 ± 0.3 μg mL⁻¹ (mean ± SEM) and in most subjects, by 24 hours after injection, TCMCB07 was below the level of detection. On day 28, plasma levels again peaked at 1 hour after injection at 1.21 ± 0.2 μg mL⁻¹ (mean ± SEM).