Chlorambucil-Induced Myoclonus in a Cat With Lymphoma

Case Report

A 9.5-year-old, 4-kg, male castrated Persian cat was presented to the University of Illinois Veterinary Teaching Hospital for evaluation of seizures and myoclonus that began 24 hours earlier. The cat had been evaluated 9 days prior to presentation for small intestinal diarrhea of 3 months’ duration, anorexia of 1 week’s duration, and weight loss. No improvement had been observed with an empirical treatment of metronidazole, tylosin, and trimethoprim-sulfamethoxazole. The cat’s vaccination status was current.

Biopsy samples were obtained via gastroduodenoscopy by one of the authors (Gaspar) prior to referral to the University of Illinois. Histopathological examination revealed advanced, diffuse, low-grade, epitheliotropic lymphoma of the small intestine, superimposed on a chronic lymphoplasmacytic enteritis. A mild lymphoplasmacytic gastritis was also reported. The cat was treated with prednisolone acetate (2.5 mg/kg body weight intramuscularly, q 24 hours) and chlorambucil (15 mg/m² [4 mg total dose], q 24 hours for 4 days, with 17 days of rest; 21-day cycle). This is a dose-intense protocol for feline low-grade gastrointestinal lymphoma described previously by Fondacaro, et al.¹ The chlorambucil was compounded in a liquid solution (i.e., cellulose-based suspending vehicle; 6 mg/mL to be administered at a volume of 0.65 mL per os [PO], q 24 hours) by a local pharmacy and was administered orally with a syringe by the owner at home.

Treatment with prednisolone and chlorambucil began 2 days before presentation to the University of Illinois. The first dose of chlorambucil was given in the evening, followed by a second dose early the next morning, approximately 12 hours after the initial dose. It was erroneously administered 12 hours apart by the owner instead of q 24 hours as prescribed. The cat presented with neurological signs consisting of twitching and agitation, which commenced a few hours after the second dose of chlorambucil was administered. Before the onset of these signs, the cat had no prior history of neurological disease. After hospitalization for 24 hours at an emergency clinic, the cat was referred to the University of Illinois medical oncology service. The neurological signs at this point consisted of severe facial twitching and myoclonus, with muscle jerks of the head and limbs. At least two short episodes consistent with tonic-clonic seizures had been observed. No specific anticonvulsant therapy was administered before presentation. Blood work performed at the emergency clinic consisted of a serum biochemistry profile, packed cell volume (PCV), total solids (TS), and ammonia levels, and all values were found to be within reference ranges except for an elevated blood glucose (264 mg/dL; reference range, 65 to 129 mg/dL). This finding was not persistent and was attributed to stress.

On initial physical examination at the University of Illinois, the cat weighed 4 kg, was thin, and clinically, mildly dehydrated. Rectal temperature was 38.6°C, heart rate was 200 beats per minute, and respiration rate was 64 breaths per minute. Thickened intestinal loops were palpated. A linear corneal ulcer was noted in the left eye and was interpreted to be secondary to exposure keratitis, as the patient was not blinking normally. The cat displayed signs of hyperesthesia and extreme agitation. Severe facial twitching was observed. The stimuli of examination triggered spastic muscle jerks consistent with myoclonus. The owners described the cat as being normally calm and compliant. The cat was so agitated that it could barely be restrained. A complete neurological examination could not be performed because of the cat’s hyperexcitable state.

Differential diagnoses for the neurological signs included intracranial causes such as neoplasia (primary or metastatic); inflammatory diseases including nonsuppurative meningoencephalitis; vascular disease such as feline ischemic encephalopathy; hemorrhage; and infectious diseases of viral (e.g., feline infectious peritonitis [FIP], feline leukemia virus [FeLV], feline immunodeficiency virus [FIV]), parasitic (e.g., toxoplasmosis), bacterial, or fungal (e.g., cryptococcosis) origin. Extracranial causes included metabolic disturbances (e.g., hepatoencephalopathy), severe electrolyte disorders, thiamine deficiency, medication toxicity, and other toxicoses (e.g., methaldehyde). Based on the history of high-dose chlorambucil administered twice, approximately 12 hours apart, a presumptive diagnosis of chlorambucil-induced neurotoxicity was established, and supportive therapy was offered.

Upon emergency admission at the University of Illinois, blood glucose, serum electrolyte panel, PCV, and TS were tested. The cat was found to be anemic (PCV, 26%; reference range, 27% to 45%), but all other results were within reference ranges. Feline immunodeficiency virus antibody and FeLV antigen were tested and found to be negative by enzyme-linked immunosorbent assay (ELISA). A dose of diazepam (1 mg [0.25 mg/kg body weight] intravenously [IV] bolus) was administered. The cat was then started on crystalloids (lactated Ringer’s solution) IV at a rate of 12 mL per hour, supplemented with potassium chloride and B-complex vitamins. The corneal ulcer in the left eye was treated with topical atropine and triple antibiotic ointments. The patient also received IV injections of ranitidine (0.5 mg/kg body weight q 12 hours for gastroduodenal protection). Treatment with chlorambucil was discontinued.

On the following day (day 2), the cat displayed continuous twitching, which would progress to myoclonic activity when stimulated by noise, movement, or physical restraint. A complete blood count (CBC) and serum biochemistry profile were performed. The CBC revealed a normocytic, normochromic, nonregenerative anemia (hematocrit, 23%; reference range, 30% to 45%), attributed to chronic disease with or without gastrointestinal blood loss, and a leukocytosis (white blood cell count [WBC], 24.7 × 10³/μL; reference range, 5.5 to 19.5 × 10³/μL) characterized by a mature neutrophilia (neutrophils, 22.7 × 10³/μL; reference range, 2.5 to 12.5 × 10³/μL), mild lymphopenia (lymphocytes, 0.25 × 10³/μL; reference range, 1.7 to 7.0 × 10³/μL), and monocytosis (monocytes, 1.48 × 10³/μL; reference range, 0 to 0.9 × 10³/μL) likely caused by a combination of inflammation, stress, and exogenous corticosteroids administration. The serum biochemistry profile revealed a marginal hypocalcemia (calcium, 8.3 mg/dL; reference range, 8.4 to 10.8 mg/dL) and hypercholesterolemia (cholesterol, 146 mg/dL; reference range, 63 to 130 mg/dL). The cat was maintained on IV fluids and ranitidine, and the corneal ulcer treatment was continued. Nutritional support was initiated because of prior weight loss, gastrointestinal lymphoma, and anorexia. Anorexia may be a result of chronic nausea secondary to intestinal lymphoma, splanchnic pain, and neurological signs preventing the appropriate intake of food. Sedation with oxymorphone (0.23 mg IV) and diazepam (2 mg IV) was used to permit nasoesophageal feeding tube placement. A constant-rate infusion of liquid diet^a was initiated at 25% of the patient’s daily maintenance needs.

On day 3, the cat was resting quietly but still displayed mild facial twitching when stimulated. Treatment with oral prednisone (5 mg PO, q 24 hours) and cyproheptadine (2 mg PO, q 24 hours for appetite stimulation) was initiated. The nasoesophageal feeding was increased to 50% of the patient’s maintenance needs. On day 4, the cat was neurologically normal, and feeding was increased to 75%. Systemic antibiotic therapy was initiated (cefazolin; 22 mg/kg body weight IV, q 8 hours) for a febrile episode (40.6°C). On day 5, the temperature was in the normal range, the cat remained neurologically normal, a CBC showed improvement of all values but the nonregenerative anemia, and a percutaneous endoscopy-guided (PEG) gastric feeding tube was placed to allow continued nutritional support.

The cat continued to improve clinically and was discharged on day 7, at which time it remained neurologically normal. The corneal ulcer had also resolved. The cat was released with instructions for PEG tube feedings and prescriptions for prednisone (5 mg PO, q 24 hours), cyproheptadine (2 mg PO, q 12 hours), and famotidine (1.25 mg/kg body weight PO, q 24 hours). The antibiotics were discontinued. A modified COP (cyclophosphamide-vincristine-prednisone) protocol was to be administered by the referring veterinarian.

Discussion

Chlorambucil is a nitrogen mustard derivative that acts as a bifunctional alkylating agent.²³ Chlorambucil forms biadducts that are mainly interstrand deoxyribonucleic acid (DNA) cross-links, as well as ribonucleic acid (RNA) cross-links. By the latter mechanism, chlorambucil interferes with protein synthesis. The drug also has immunosuppressive properties. Different dosing regimens have been described in the veterinary literature, ranging from 2 to 8 mg/m² PO, q 2 days, to 20 mg/m² PO, q 21 days.³ High doses can be associated with an increased potential for myelosuppression and gastrointestinal toxicity, which are the most commonly recognized adverse effects of the drug. These adverse effects are rare at lower doses. The dose (15 mg/m² PO, q 24 hours for 4 days, on a 21-day cycle) used in the case reported here had been reported based on a series of 29 cats treated for low-grade intestinal lymphomas.¹ In this report, toxicity was minimal and did not include neurotoxicity.

Chlorambucil is prescribed for a variety of conditions in veterinary oncology, including chronic lymphocytic leukemia (CLL), low-grade lymphoma, as part of maintenance protocols for intermediate to high-grade lymphoma, mast cell tumors, multiple myeloma, and to replace cyclophosphamide when sterile hemorrhagic cystitis is diagnosed in a given patient.^13–10 Another use of chlorambucil in veterinary medicine is for immunosuppression in conditions such as dermatological (e.g., pemphigus foliaceus, eosinophilic granuloma complex), hematological (e.g., hemolytic anemia), or renal (e.g., glomerulonephritis) immune-mediated diseases.^11–14

Chlorambucil was introduced in 1953 as a more stable derivative of nitrogen mustard.¹⁵ Neurotoxic adverse effects in human patients were recognized as early as 1957 in cases of inadvertent overdose, and they were confirmed through toxicity studies with high-dose chlorambucil in rats.¹⁶¹⁷ High-dose chlorambucil has been used in laboratory animals (i.e., rabbits, cats, monkeys) as models for experimental epilepsy.^18–22 Although chlorambucil-induced neurotoxicity remains uncommon in humans (<40 reported cases in the human literature as of 2001), it has been well recognized.¹⁵ Neurotoxicity is associated with treatment for nephrotic syndrome in children, in rare cases of overdose or accidental ingestion, in high-dose pulse anticancer chemotherapy regimens, and in rare cases at standard doses in adults.^1623–36 In addition, at least two reports (four patients) of prednimustine-induced myoclonus have been published as well.³⁷³⁸ Prednimustine is an ester of prednisolone and chlorambucil, specifically developed for treating tumors containing gluco-corticoid receptors. In the reported cases in humans and in studies of laboratory animals (i.e., rats, cats, rabbits), the symptoms were mainly myoclonus, uncontrollable tremors, muscular twitching, hallucinations (in humans), agitation, and focal or generalized tonic-clonic seizures or both.^16–38

The exact mechanism of chlorambucil-induced neurotoxicity has not been clearly established. A chemical or functional, rather than structural, effect appears obvious, since signs resolved after discontinuation of the drug in all reported nonlethal cases.^{1516182023–38} After oral administration, chlorambucil is metabolized to phenylacetic mustard. The monohydroxy and dihydroxy hydrolysis products of chlorambucil and its alkylating metabolites may yield to chloroacetaldehyde. This substance is thought to be neurotoxic because of its structural similarity to metabolites of ethanol (i.e., acetaldehyde) and chloral hydrate (i.e., trichloroacetaldehyde).³⁶ It has been suggested that the concomitant use of corticosteroids in many of the reported cases in humans could play a role in the neurotoxic effects observed. Because of the action on cortical neuron excitability level, corticosteroids decrease the dose of neurotoxic agents (including chlorambucil metabolites) required to initiate seizures.³⁶ In children with nephrotic syndrome, an additional postulated factor that could predispose to chlorambucil neurological side effects is hypoproteinemia. Since chlorambucil is extensively bound to proteins, decreased plasma protein concentration could alter pharmacokinetics of the drug and thus increase central nervous system (CNS) side effects in this group of patients.³³³⁵ This situation could also potentially be seen in animals with gastrointestinal lymphoma and protein-losing enteropathy. Hypoproteinemia was not a complication in the case reported here.

Studies with electroencephalographic (EEG) monitoring, as well as studies with ¹⁴C-labeled chlorambucil, suggest subcortical structures as being the primary site of neurological insult.¹⁹²⁰ Marked EEG abnormalities have been described in laboratory animals and in some cases of toxicity in humans.^{18–2224–3133–36} One report of children with nephrotic syndrome treated with chlorambucil revealed EEG abnormalities in two of the nine patients, despite the absence of overt neurological signs. These EEG changes disappeared after discontinuation of chlorambucil therapy.²⁹ Early studies with very high-dose chlorambucil in cats (i.e., up to 40 mg/kg body weight per dose) suggested age-related differences in the CNS reaction to the drug. Kittens 25 to 30 days old had massive myoclonic flexor spasms, whereas older kittens (i.e., 3 months old) had typical grand mal seizures or purely tonic seizures.²⁰ Furthermore, the threshold to chlorambucil-induced neurotoxicity appeared to be higher in more mature animals.¹⁹²⁰ Electroencephalography was not available and was not performed in this case. In addition, this was not performed in many of the reported cases in the human literature.

A number of other antineoplastic agents are known to be neurotoxic to some degree. Vinca alkaloids (i.e., vincristine, vinblastine, and vinorelbine), taxanes (i.e., paclitaxel and docetaxel), platinum compounds (i.e., cisplatin and oxali-platin), cytarabine, fluorouracil, ifosfamide, methotrexate when given intrathecally, procarbazine, and fludarabine have all been recognized as potentially neurotoxic.^39–41 For some of these drugs (such as vincristine, the platinum compounds, and the taxanes), neurotoxicity may be the dose-limiting toxicity in humans and may be observed in up to 70% of the patients treated.³⁹ Neurotoxicity as a side effect of chemotherapy has been rarely observed in small animal patients. Drugs associated with neurotoxicity in veterinary medicine include vincristine, cisplatin, and fluorouracil.⁴¹ Fluorouracil causes lethal neurotoxicity in cats and is contraindicated in that species.

Although other differential diagnoses were not all completely ruled out by the use of cerebrospinal fluid analysis, magnetic resonance imaging, and EEG, the authors believe that this represents the first reported case of chlorambucil-induced neurotoxicity in the clinical veterinary literature. A challenge with chlorambucil could have been done to confirm the causal relationship but was not performed. Measurement of chlorambucil blood concentrations is not widely available and has not been performed in any case reported in the human literature. The causal association between the treatment with chlorambucil and the observed myoclonus and seizures is inferred from the following facts, based on a previously published algorithm:⁴² no previous history of neurological signs before this episode; appropriate time interval between drug intake and event based on laboratory animal studies; resolution of signs after discontinuation of chlorambucil; neuromuscular events not explained by the clinical state of the patient or concurrent therapy; no subsequent neurological signs seen in the cat as of this writing (3 months); and signs observed in the patient of this study that are entirely consistent with those reported in the human literature and with those seen in the early experimental toxicity studies of cats.

Another case, not treated by the authors but brought to their attention,^b was treated for multicentric nodal lymphoma with a different high-dose chlorambucil protocol. This 16.5-year-old spayed female cat weighing 4 kg received multiple treatments of chlorambucil (8 mg total dose q 14 days). Mild twitching of the head and limbs was noted, starting 24 hours following the administration of each dose of chlorambucil. These neurological signs resolved 96 to 120 hours after each treatment. In addition, generalized motor seizure activity was observed following the 12th dose of chlorambucil. This cat was not myelosuppressed following chlorambucil administration, which indicates that neurological signs, such as myoclonus and seizures, could be the dose-limiting toxicity in some chlorambucil-treated patients. This case illustrates a challenge with chlorambucil and, therefore, the causal relationship between chlorambucil administration and neurological signs observed (i.e., twitching, seizure) according to the algorithm previously described. Recommendation to discontinue chlorambucil administration was made.

Conclusion

Although the patient reported in this study and the other described in the Discussion section represent the first published cases of chlorambucil-induced neurotoxicity in a veterinary patient, one of the authors (Gaspar) has seen another feline patient treated with the same protocol that developed tonic-clonic seizures that abated after discontinuation of therapy. Anecdotally,^c at least two other cats had seizures while on the same protocol.

Neurotoxicity, in the form of myoclonus or tonic-clonic seizures, must be considered a potential adverse effect of chlorambucil therapy in veterinary patients, especially when used in higher intensity dosing regimens. Although this adverse effect remains a rare entity, careful observation is warranted in such patients, and discontinuing the drug is advised if signs such as muscle twitching, myoclonus, or seizures develop. Patients with a previous history of seizures or other CNS diseases, hepatic dysfunction, or hypoproteinemia, could be at higher risk for chlorambucil neurotoxicity.