Leflunomide may be an effective treatment for immune-mediated disorders in dogs that do not respond well to standard therapy or have adverse effects to glucocorticoid administration (1). Though the occurence of adverse effects to leflunomide are rare they can, as reported in human medicine, include diarrhea, dermatitis, hepatotoxcity, pneumonia, neurotoxicity, and myelosuppression (2,13). In veterinary medicine, the adverse effects of leflunomide have rarely been reported (10). Therefore, further studies are needed in order to ensure the safety of this mediccation in canine patients both now and in the future. To the best of our knowledge, this is the first report of leflunomide-induced aplastic anaemia in a canine patient with granulomatous meningoencephalitis (GME).

A 10-year-old, female spayed Maltese dog was presented to veterinary hospital with a two-day history of visual impairment. After performing an ophthalmologic examination, a magnetic resonance imaging (MRI) scan, and a cerebrospinal fluid (CSF) analysis, she was tentatively diagnosed with aseptic ocular GME. She was started on prednisolone (1 mg/kg, every 12 hours) and cyclosporine (3 mg/kg, every 12 hours) therapies and two weeks after initial treatment, she regained her vision. However, four months after initial treatment, her visual impairments occurred again. A repeat MRI was performed, and a new suspected inflammatory lesion was confirmed in the right optic nerve. Due to the presence of the new lesion, we decided to change her immunosuppressive therapy from cyclosporine to cytosine arabinoside (50 mg/m² every 12 hours for 2 days repeated every 3 weeks). Even after three cycles of cytosine arabinoside treatment, her vision had not improved. Fifteen months after the first visit, a new immunosuppressant drug, leflunomide (Arava; Sanofi; 4 mg/kg/day), was added to her therapy, and cytosine arabinoside was discontinued. At this time, A complete blood count (CBC) analysis showed the following findings: white blood cell count (WBC), 19.57 × 10³/μL (reference range: 5.05-16.76 × 10³/μL); haemoglobin (Hb), 13.5 g/dL (reference range: 13.1-20.5 g/dL); haematocrit (HCT) concentration, 41.3% (reference range: 37.3-61.7%); red blood cell (RBC) count, 5,580 × 10³/μL (reference range: 5,700-8,800 × 10³/μL); mean corpuscular volume (MCV), 73.9 fL (reference range: 58.8-71.2 fL); mean corpuscular haemoglobin (MCH), 24.2 pg (reference range: 20.5-24.2 pg); mean corpuscular haemoglobin concentration (MCHC), 32.7 g/dL (reference range: 31.0-36.2 g/dL); red cell distribution width (RDW), 13.7% (reference range: 11.9-14.5%) and platelet count, 117.7 × 10³/μL (reference range: 731-400 × 10³/μL). A follow-up examination two months after starting leflunomide treatment showed that the patient’s oral mucosa was pale. On physical examination, the patient’s vital signs were as follows: heart rate, 162 beats/minute; systolic blood pressure, 130 mmHg; respiratory rate, 24 breaths/minute; and body temperature, 39.6 °C. A CBC analysis showed the following findings: WBC, 14.19 × 10³/μL; Hb, 7.8 g/dL; HCT, 24.3%; RBC, 3,250 × 10³/μL; MCV, 74.8 fL; MCH, 23.9 pg; MCHC, 32.0 g/dL; RDW, 14.7%; platelet count, 73.1 × 10³/μL; and reticulocyte count, 16.7 × 10³/μL (reference range: 0-60 × 10³/μL). Evidence of haemolysis could not be confirmed on a blood smear. Despite performing further diagnostic tests, including serum biochemistry and an abdominal ultrasound examination, the cause of the anaemia could not be identified. Additionally, general examination revealed no evidence of chronic hemorrhage such as GI hemorrhage in this patient. An iron-based supplement (Fercobsang^®, Vétaquinol SA, Lure, France; 1 ml/dose) was initially prescribed for a non-regenerative anaemia, but there was no improvement in the anaemia after 6 days of administration. A repeat complete blood count revealed the following values: packed cell volume (PCV), 21.1%; Hb, 6.8 g/dL, MCV, 74.8 fL; MCH, 32.0 pg; RDW, 15.6%; and reticulocyte count, 15.3 × 10³/μL. Although aplastic anaemia has not been reported in veterinary medicine, it has been reported following leflunomide treatment in human rheumatoid arthritis patients (10). To correct the anemia, the dose of leflunomide was reduced for 6 days and then the treatment was stopped (Fig. 1). Despite discontinuation of leflunomide treatment, the patient’s anaemia persisted, but her reticulocyte count improved (PCV, 21.4%; and reticulocyte count, 136.6 × 10³/μL) (Fig. 1). Six days after leflunomide treatment ended, the patient’s anaemia improved (with a PCV of 36.6%) and 11 days after that, her PCV was within the normal range (a PCV of 44.6%) (Fig. 1).

Figure 1. Medication dosage, PCV and reticulocyte before and after leflunomide treatment. Graph showing dosage of leflunomide, PDS and Iron based supplement, PCV, reticulocyte value from the time of admission with leflunomide to the period before and after treatment.

Leflunomide is an isoxazole derivative that inhibits dihydro-orotate dehydrogenase (DHODH), which is the rate-limiting enzyme in the de novo synthesis of pyrimidines, and inhibits effector T cell proliferation (12). Due to its anti-inflammatory properties, leflunomide has been used as a therapeutic agent for various immune-mediated diseases including rheumatoid arthritis (3,10).

In human medicine, the most commonly reported adverse effects of leflunomide include diarrhoea, nausea, hepatotoxicity, hypertension, and hair loss (7). According to one report, leflunomide was discontinued within one year in 67% of cases due to adverse drug reactions. Additionally, several cases of leflunomide induced anaemia have been reported in the human medical literature although the underlying mechanism is still unknown. Wüsthof et al reported that a 32-year-old woman diagnosed with rheumatoid arthritis developed severe aplastic anaemia after taking leflunomide for 60 days, although her arthritis improved (14). Toyokawa et al reported that patients with arthritis developed thrombocytopenia, lymphopenia, and macrocytic anaemia after taking leflunomide (11). Although further studies are needed to determine the mechanism, it is known that leflunomide-induced anaemia is caused by bone marrow failure due to impaired pyrimidine biosynthesis, and in most cases, the anaemia may be accompanied by pancytopenia (1,9). Adverse events that may be associated with leflunomide administration in veterinary medicine include diarrhoea, lethargy, unexplained bleeding, thrombocytopenia, and increased liver enzyme activity (10). However, currently, there are no case reports of non-regenerative anaemia after leflunomide administration.

In this case, reticulocyte numbers were determined using a cationic dye (Oxazine 750) and a light-scatter technique on the ADVIA 2120i (Siemens Healthineers, Erlangen, Germany) that quality assessment was performed weekly previous described (6). The presence of anemia and regeneration was determined based on previous study for dogs (red blood cell count <5.7 × 10⁶/μL, hematocrit <37%, hemoglobin <12.9 g/dL, reticulocyte >80 × 10³/μL) (6). A general examination revealed no evidence of chronic bleeding, and the patient did not have non-regenerative, microcytic, and hypochromic anemia, which is typical of iron deficiency anemia. Instead, two months after leflunomide administration, the patient presented with non-regenerative, macrocytic, and normochromic anemia. Bone marrow examination was not performed because the owner of the patient did not provide consent for the procedure. However, inhibitors of pyrimidine DNA synthesis often cause drug-induced aplastic macrocytic anaemia, similar to that caused by anticancer drugs (5,8). In addition, no other cause of anaemia was observed; moreover, since the anaemia improved after discontinuation of leflunomide, it is highly likely that the patient in this report had anaemia secondary to leflunomide. According to a previous case report, leflunomide-induced anaemia improved after discontinuation of leflunomide without the need for additional drug therapies (4). Based on these reports, we discontinued leflunomide treatment, and the patient’s PCV returned to the normal reference interval 15 days later.

Unfortunately, our case was unable to determine whether the anemia that occurred was a dose-dependent side effect of leflunomide. In this patient, leflunomide was prescribed at a dose of 4 mg/kg/day (3). According to a previous study, the dose causing leflunomide adverse effects was also investigated. Significant dose differences were found between dogs with adverse events (median: 2.9 mg/kg/day; range: 1.8-3.6 mg/kg/day) and dogs without adverse events (median: 1.6 mg/kg/day; range: 0.8-4.3 mg/kg/day) (10).

To the best of our knowledge, this is the first case report of reversible aplastic anaemia following treatment with leflunomide in dog patient with meningoencephalitis. Based this case, the rare incidence of non-regenerative, macrocytic, normochromic, non-haemolytic anaemia following leflunomide treatment should be considered. Our case highlights the importance of recognizing leflunomide as a drug that can induce aplastic (non-regenerative) anaemia. In addition, if an adverse reaction to leflunomide is suspected, therapy should be discontinued immediately and, if necessary, symptomatic treatment should be started.

We report transient aplastic anemia in a dog with granulomatous meningoencephalitis following leflunomide treatment. This well-documented case of aplastic anaemia associated with leflunomide may help inform the veterinary and medical communities about the potential complications linked to this medication.

This research was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (RS-2023-00240708).

GHL and JHA drafted the initial manuscript. GHL, JHA, SMP, JHL, YIO, KWS acquired, analyzed and interpreted the data; GHL, JHA, HYY revised the article; and HYY approved the final version of the article prior to submission. All authors have read and approved the manuscript.

The authors have no conflicting interests.