Introduction

Hyperthyroidism is a common endocrinopathy in cats and can be seen in middle-aged and geriatric cats.^1,2 The disease process results in high serum levels of the thyroxine hormone. Increases in serum thyroxine concentrations are caused by functional adenoma or adenomatous hyperplasia of the thyroid tissue; thyroid neoplasia is uncommon.¹ Benign cases of hyperthyroidism can be treated with antithyroid medications, iodine restricted diets, radioactive iodine treatments (I-131), and thyroidectomies if palpable nodules exist.²

Medical management for the treatment of hyperthyroidism aims at limiting the excessive production of the thyroid hormone.¹ Methimazole inhibits thyroid peroxidase and interferes with iodine incorporation into tyrosyl residues of thyroglobulin, thereby inhibiting thyroid synthesis.³ Methimazole is considered an anti-thyroid medication with different formulations and routes of administration available. The dosing regimen usually includes an initial dose of 2.5 mg q 12 hr.⁴

Azathioprine is an immunosuppressive agent used primarily in dogs for immune-mediated diseases, specifically inflammatory bowel disease, immune-mediated hemolytic anemia (IMHA), and immune-mediated thrombocytopenia.⁵ Azathioprine inhibits the synthesis of purines adenine and guanine by blockage of certain enzymes resulting in the production of nonfunctional nucleic acid strands. This results in breaks in the DNA and RNA replication process which in turn inhibits the proliferation of fast-growing cells. There is also evidence of azathioprine interfering with CD28 co-stimulation of T lymphocytes resulting in apoptosis. Adverse effects of azathioprine include gastrointestinal signs, pancreatitis, hepatotoxicity, and bone marrow suppression.⁴ In dogs, it has been reported that 8% experience myelosuppression, mainly presenting as leukopenia.⁴ Azathioprine is converted from a prodrug state in the liver into the purine analogue 6-mercaptopurine (6-MP), which is then converted into 6-thioguanine nucleotides (6-TGN), which are cytotoxic.⁶ The accumulation intracellularly of 6-TGN allows for incorporation of 6-TGN into nucleic acids of hematopoietic progenitor cells, resulting in bone marrow suppression.⁶ Thiopurine methyltransferase (TPMT) is an enzyme used in the inactivation and metabolic breakdown of 6-MP to inactive metabolites.⁶ In the feline, there is low TPMT activity, resulting in higher incidence and risk for bone marrow suppression.⁶ The development of bone marrow suppression induced by azathioprine in humans has been associated with homozygosity for alleles encoding low-activity TPMT.⁶ The standardized dose for the canine starts at 2 mg/kg/day.⁴ Azathioprine is not recommended in cats because of a higher risk of myelosuppression.⁷ There is a listed dose of 1.1 to 2.2 mg/kg q 48 hr, which can cause adverse reactions.⁷ Administering azathioprine at 0.3 mg/kg q 48 hr had no major adverse reactions.⁷

Filgrastim (granulocyte colony-stimulating factor) is a cytokine hematopoietic agent used to increase the proliferation, differentiation, and activation of progenitor cells in the neutrophil–granulocyte line.⁸ Filgrastim given as a subcutaneous (SC) injection can be found at its highest concentrations in the bone marrow, thus resulting in an increase in neutrophils.⁸ Dosing in the feline and canine species is 5–10 μg/kg SC daily, although there is no consensus or evidence supporting this dosing regimen.⁴ Studies have shown some safety with pulse therapy of injections for 3–5 days in cats.⁴ Filgrastim is human in origin and therefore can be immunogenic in the veterinary field, resulting in worsening of neutropenia due to an immune response against both endogenously produced and exogenously administered granulocyte colony-stimulating factor.⁴ Most reported adverse effects in human include injection site irritation, musculoskeletal and bone pain, glomerulonephritis, splenomegaly, capillary leak syndrome, and hypotension.^4,8,9

The goal of this report is to provide information on a successful dosing regimen for filgrastim use in cats who are experiencing myelosuppression resulting from azathioprine. In this case, the cat experienced severe neutropenia, which was responsive to filgrastim injections.

Case Report

The cat was a 16 yr old spayed female domestic shorthair weighing 3.14 kg with a history of hyperthyroidism, constipation, a heart murmur, persistent tachycardia, and IRIS chronic kidney disease stage 2. The hyperthyroidism was diagnosed in March 2016 and subsequently treated and stabilized with methimazole^a (5 mg per os [PO] q 12 hr). On routine examinations, the cat had a heart murmur II-III/VI and persistent tachycardia (heart rate >180 bpm). A prescription error occurred in which methimazole was requested to be refilled, but it was incorrectly filled with azathioprine^b tablets. The prescription label read methimazole dosing. The pet was in the care of the owner’s daughter at the time, who was not familiar with the previous tablet (methimazole) description. Once the cat returned to the owner, the incorrect medication was discovered. On further investigation, it was confirmed that the cat was given azathioprine 50 mg PO q 12 hr. The patient received two doses per day for 3 days before the error was noted. The owner informed the hospital of the error, and the cat had a physical examination, and laboratory testing was performed.

The cat was examined on day 1 (4 days after initial azathioprine ingestion); the cat weighed 3.15 kg and was normothermic (38.4 °C), and heart rate was 160 bpm. No adverse reactions were reported by the owner at this time. A complete blood count (CBC) showed leukopenia (white blood cell count 3.3 K/µL; reference range 3.5–16 K/µL), and neutropenia (1.48 K/µL; reference range 2.50–8.50 K/µL). (It should be noted that day 1 and day 41 laboratory testing were performed at Antech laboratories, with the remainder performed in-house through IDEXX; therefore, reference ranges are not identical.) A case with poison control was opened to establish guidance from a toxicologist. The report revealed the cat received a dose of 15.88 mg/kg q 12 hr per the report; dosing for azathioprine in the feline is listed as 0.3 mg/kg q 48 hr. The poison control toxicologist advised that acute overdoses with azathioprine can result in gastrointestinal signs, including vomiting, diarrhea, and anorexia.^4,6,7 Myelosuppression can occur within 2–3 wk after ingestion. Risk of hepato- and nephrotoxicity has also been reported.^4,6,7 The toxicologist suggested checking a CBC every 3–5 days for 3 wk. The cat was discharged from the hospital for monitoring with a recheck appointment in 5 days.

On day 5, the cat reported for recheck examination and blood work. The cat was now hyporexic with episodes of vomiting and was down in weight to 2.92 kg. The physical examination vitals were within normal limits. CBC showed stable neutropenia (1.47 K/µL; reference range 2.30–10.29 K/µL). The cat was given mirtazapine (1.875 mg PO), a maropitant injection (1 mg/kg SC), and a methimazole tablet (5 mg PO) in the hospital. The cat was discharged from the hospital for monitoring by the owner at this time. Between day 5 and 9, there was no update from the owner on the cat’s condition.

On day 12, the cat returned for a recheck examination and laboratory testing. On the physical examination, the cat was hyperthermic (39.6 °C) and tachycardic (180 bpm). The owner reported that the cat had been hyporexic most days. Vomiting had occurred on two different days. The cat lost 0.32 kg of body weight. CBC showed worsening neutropenia (0.11 K/µL; reference range 2.30–10.29 K/µL) and mild anemia (28.9%; reference range 30.3%–52.3%). The cat was admitted to the hospital for supportive care. An IV catheter was placed aseptically, and lactated ringer solution was administered at 12 mL/hr (120 mL/kg/day). Supportive care included mirtazapine^c (1.875 mg PO q 24 hr), maropitant^d (1 mg/kg IV q 24 hr), pantoprazole^e (1 mg/kg IV q 12 hr), ampicillin^f (15 mg/kg IV q 8 hr), enrofloxacin^g (5 mg/kg PO q 24 hr), and methimazole (5 mg PO q 12 hr). Because of the lack of response in the cat’s bone marrow and worsening of clinical signs, filgrastim^h was ordered and shipped to the hospital. The cat was re-presented the next day. Physical examination revealed that the cat was now normothermic (101.9 °F) and had eaten well overnight. The cat was discharged with amoxicillin/clavulanate (37.5 mg PO q 12 hr), enrofloxacin (5 mg/kg PO q 24 hr), and mirtazapine (1.875 mg PO q 24 hr).

On day 14, the cat was re-presented to the hospital for the first injection of the filgrastim series. The dosage and regimen used in this case was 5 µg/kg SC daily for four injections. The cat was admitted to the hospital daily for monitoring after each injection. The cat gained 0.4 kg in body weight when compared to the previous physical examination. A CBC on day 14 revealed stable neutropenia (0.12 K/µL; reference range 2.30–10.29 K/µL) but worsening anemia (20.9%; reference range 30.3%–52.3%). Blood typing was performed to prepare for blood transfusion if necessary. Blood typing revealed type A blood group.

On day 15 and 16, the cat returned to the hospital for the second and third injections of filgrastim SC. No obvious immediate adverse reactions were noted at this time. General physical examinations were similar to previous examinations, and the cat had regained a good appetite. Weight continued to stay stable. CBC on day 15 showed worsening neutropenia (0.1 K/µL; reference range 2.30–10.29 K/µL) but improving anemia (24.5%; reference range 30.3%–52.3%). CBC on day 16 showed an increase in neutrophils (0.15 K/µL; reference range 2.30–10.29 K/µL) and worsening anemia (22.2%; reference range 30.3%–52.3%). A blood agglutination test was performed on day 16 to rule out IMHA. No agglutination on the slide was seen, making IMHA less likely. The cat was presented for the fourth and final injection of filgrastim on day 18. The neutropenia improved (0.154 K/µL; reference range 2.30–10.29 K/µL) as did the anemia (22.6%; reference range 30.3%–52.3%). Slide agglutination test was repeated and remained negative. A CBC was rechecked on day 19, 27, and 34, and neutropenia and anemia continued to improve, as can be seen in Table 1.

TABLE 1 Neutrophil Concentrations and PCV

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On day 41, the cat was presented for difficulty walking. Vitals were within normal limits. A whole-body radiograph revealed mild spinal arthritis. A CBC showed neutrophilia (11.59 K/µL; reference range 2.30–10.29 K/µL). Gabapentin was recommended for pain but was declined at this time. Antibiotics were discontinued. The last CBC was rechecked on day 49, and neutrophil counts were within reference range (Table 1). Informed consent for treatment was obtained from the cat’s owners, and contemporary standard of care was provided to the cat for the duration of the treatment period.

Discussion

In this single feline case report, the use of filgrastim was perceived to have a successful time response in normalizing the neutrophil cell count after severe neutropenia secondary to azathioprine toxicity. The regimen that was used in this case was 5 µg/kg SC once a day for four doses. It should be noted that consecutive dosing occurred for the first three injections. The fourth injection was separated by two days due to a scheduling conflict. The cat did experience mild signs of pain days after the course of injections, which has been reported as a side effect after filgrastim injections in humans.^4,8,9 The owner reported an increase in stiff gait and an increase in vocalization when being picked up or when limbs were touched on day 41. Bone pain is the most common adverse event with filgrastim.^8,9 The indications for filgrastim use in human medicine include a decrease in time to neutrophil count recovery and to reduce the duration of neutropenia and neutropenia-related complications.⁹ In this case, the indications were the same as in human medicine.

The use of azathioprine in cats is not recommended because of a high potential for bone marrow toxicity.¹⁰ The importance of TPMT in the breakdown of 6-MP has serious repercussions in the TPMT-deficient patient. In the TPMT enzyme deficient patient, 6-MP is not catabolized into inactive metabolites.⁶ This in turn increases the cytotoxic thioguanine nucleotides, resulting in continued suppression of immune cell replication leading to increased risk of myelosuppression.⁶ In Foster et al., erythrocyte TPMT enzyme activity in 41 cats was measured. Results were compared to humans and canines. The feline exhibited erythrocyte TPMT activity below the normal range of humans.⁶ The mean erythrocyte TPMT activity in the cats was 2.4 ± 0.4 nmol (range 1.2–3.9 nmol) per hour per milliliter of red blood cells.⁶ Human TPMT enzyme activity ranged from 8 to 15 U/mL red blood cells. A small group of dogs also had their blood samples analyzed and revealed similar TPMT activity to the human range (5.5–13.1 U/mL).⁶ In humans, genetic variants in this gene are associated with low enzyme activity, which can lead to increased toxicoses including bone marrow suppression.^6,11 This concludes that cats on average exhibit a lower TPMT enzyme concentration and in turn results in higher incidence of myelosuppression.⁶ In this case, because the cat experienced severe neutropenia, it is possible that the cat had low TPMT activity at baseline.

On analysis of CBCs over the course of examination of the cat, it can be seen that the neutrophil cell line was the predominant cell that was impacted by the toxicity of azathioprine. Neutropenia rebounded after the course of filgrastim injections in the case reported here, which was also reported in case reports by Paul et al. in 2008¹⁰ and Vijayalakshmi et al. in 2017.¹² Filgrastim regulates the production and release of functional neutrophils from the bone marrow within 24 hr of administration.⁴ The neutrophil count in the case reported here increased within 48 hr after the first filgrastim injection (Table 1). Anemia has been reported to occur secondary to azathioprine toxicity as seen in this case and Rinkardt et al. in 1996.¹³

Prescription drug errors occur throughout the medical field, including veterinary medicine and have been identified as one of the most prominent issues in patient safety.^10,14 These errors, also known as medication errors, can be defined as any preventable event that may cause or lead to inappropriate medication use or patient harm while the medication is in the control of the healthcare professional, patient, or consumer.^10,15

Conclusion

Filgrastim can successfully be used in cats after bone marrow toxicity resulting in neutropenia following an azathioprine overdose. Prescription errors can occur in veterinary medicine potentially putting our patients at risk. Care should be taken to eliminate prescription errors.

The author acknowledges the assistance of Dr. Rebecca Senter for editorial assistance and the support of Dr. Mimi Noonan.