Calcipotriol toxicity in a dog

A six-montkold Labrador retriever was presented for investigation

of acute polyuria, polydipsia and haematemesis six hours following

ingestion of a tube of the topical antipsoriatic vitamin D analogue,

calcipotriol. Transient hypercalcaemia, azotaemia, protelnuria,

thrombocytopenia and ventricular arrhythmias ensued. Abdominal

ultrasonography and echocardiography revealed evidence of diffuse

soft tissue mineralisation. Despite 13 days of intensive supportive

care, the dog was euthanased due to continued haematemesis and

anorexia. Necropsy confirmed mineralisation and necrosis of multiple

organ systems consistent with vitamin D toxicity.

T. M. FAN, K. W. SIMPSON, S. TRASTI*, N. BIRNBAUM, S. A. CENTER AND A. YEAGER

Journal of Small Animal Practice (1998) 39, 581-586

Departments of Clinical Sciences and *Pathology, College of Veterinary Medicine, Cornell University, Ithaca, NY 14583, USA

Correspondence to K. W. Simpson

INTRODUCTION

Normal calcium homeostasis, critical for multiple intracellular and extracellular functions, is regulated by the activities of parathyroid hormone, vitamin D metabo- lites and calcitonin via effects in the intes- tine, kidney and bone (Chew and others 1992). In health, intestinal absorption of dietary calcium is the major regulator of serum calcium concentration, with renal tubular reabsorption playing a minor role (Zelikovic and Chesney 1989). When these mechanisms fail to conserve serum calcium concentrations adequately, skeletal resorption occurs to maintain calcium homeostasis (Brown 1991).

Vitamin D and its metabolites play a major role in serum calcium regulation. Dietary ergocalciferol and ultraviolet con- version of 7-dehydrocholesterol to chole- calciferol in the skin provide vitamin D precursors which undergo hydroxylation steps, yielding 1,25-dihydroxyergocalci- ferol and 1,25-dihydroxycholecalciferol (calcitriol), respectively. Calcitriol is the most biologically active form of vitamin D (Holick 1987, Reichel and Koeffler 1989).

Vitamin D-induced hypercalcaemia has been observed as an iatrogenic conse- quence in dogs and cats treated for hypoparathyroidism (Berger and Feldman 1987) and in young growing animals receiving excessive dietary supplementa-

tion (Chew and Capen 1980). The ingestion of plants containing calcitriol glycosides and cholecakiferol-containing rodenticides has been reported to cause vitamin D toxicity in dogs (Gunther and others 1988, Fooshee and Forrester 1990).

In addition to its effects on calcium homeostasis, calcitriol has beneficial antiproliferative effects which have been utilised for the treatment of plaque psoria- sis in humans (Binderup and Bramm 1988, Bindetup 1993). A synthetic calcitriol analogue, calcipotriol, which retains the beneficial antiproliferative properties but lacks potency in its influence on calcium homeostasis, has been developed for the treatment of human plaque psoriasis. The present report describes the distur- bance of calcium homeostasis associated with calcipotriol ingestion in a dog.

CASE HISTORY

A six-month-old, 29 kg, male castrated Labrador retriever was presented to the Cornell University emergency service for investigation of acute polyuria, polydipsia and haematemesis. Thirty-six hours before admission, the dog had ingested the contents of a 60 g tube of calcipotriol (Dovonex; Wesnvood Squibb). Within six hours, the dog had become polydipsic and polyuric, and within 12 hours had mani- fested profuse vomiting and haematemesis.

At 18 hours, the dog was examined by a veterinarian and treated with 2 litres of lactated Ringer’s solution intravenously (IV), 500 mg prednisolone sodium succi- nate (Soh-Delta Cortef; Upjohn) IV, 750 mg ampicillin trihydrate (Polyflex; Fort Dodge) subcutaneously (SC) and 60 mg frusemide (Lasix; Hoechst) W. A canine parvovirus antigen test (Idem) proved negative and a complete blood cell Lount (CBC) revealed a normal haemato- crit and leucocytosis with a mature neutrophilia. Serum biochemical abnor- malities included hypoalbuminaemia, increased alkaline phosphatase (ALP) and aspartate transferase (AST) activities,

JOURNAL OF SMALL ANIMAL PRACTICE VOL 39 DECEMBER 1998 58 1 -

Table 1. Haematological abnormalities

Compkte#oodcwnt R e f ~ m c e D.rl - 5 D.rU f lG 1. Ultrasonogram of gastric wall demonstrating increased thickness and abnormal echogenlclty. Vertical opposing arrows mark the outer and

Haematocrit (%) 39-57 52 51 47 inner margins of the wall which measures 1.5 cm in thickness. Angled White blood cell ( x 103/4) 7.5-19.9 17.2 17.0 17.2 arrows mark a thin, hyperechoic band within the mucosa. The band is the Mature neutrophil ( x i@/~l) 3.9-14.7 15.5 12.6 12.0 source of an acoustic shadow which partially obscures the margin of the Platelet count (2 x @/pi) 17S510 31 122 345 mucosa at the gastric lumen. These two sites of gastric hyperechogenlcity

- were associated with histologically apparent mineralisation

hypercalcaeniia, hypercholesterolaemia, increased blood urea nitrogen (BUN) and creatinine concentrations, hyperphosphat- aemia and hypokalaemia (Tables 1 and 2).

Thirty-six hours post-calcipotriol inges- tion, the dog was presented to the Cornell University Veterinary Medical Teaching Hospital. Physical examination revealed an irregular heart rhythm, hypersalivation, generalised abdominal pain, oropharyn- geal lesions suggestive of soft tissue mineralisation and necrosis, and firm sub- mandibular salivary glands. A CBC, serum biochemical analysis, coagulation time test, electrocardiography (ECG) and urin- alysis were performed.

Haematological abnormalities included a leucocytosis and thrombocytopenia of 31 X lo3/$ (reference range 179 to 510). A prolonged activated partial thrombo- plastin time (APTT) (26 seconds; reference range 12 to 21 seconds), and increased fibrinogen concentration

( A 2 8 g/dl; 1.05 to 5.10 g/dl) were also identified. The urinalysis revealed a specific gravity of 1.007, proteinuria of 3+ and an inactive sediment, pathological proteinuria being documented by a urine protein:creatinine ratio of 1 5.9; reference range <0.6. Serum biochemical abnormal- ities included hypokalaemia, increased creatinine and BUN concentrations, hyperphosphataernia, increased activities of.4Lc AST, alanine transferase (ALT) and creatinine kinase (CK), and hyper- cholesterolaemia (Tables 1 and 2). Serum total calcium was 2.7 mmollhtre (1.8 to 3.2 mrnol/litre) and ionised calcium was 1.13 n imol /he ( l . 18 to 1.37 mmol/litre). The ECG showed a sinus tachycardia with ventricular premature contractions.

A provisional diagnosis of vitamin D analogue toxicity was made considering the previous ingestion of calcipotriol, acute clinical manifestations of haematemesis, polyuria, polydipsia and ventricular

premature contractions, and the clinico- pathological findings suggestive of acute renal failure (elevation in creatinine, BUN and phosphorus) and disseminated intravascular coagulation (thrornbocyto- penia and prolonged APTT).

Therapeutic intervention was aimed at supporting or correcting renal, cardiac, gastrointestinal and vascular damage secondary to vitamin D analogue toxicity. The dog received intravenous fluids (0.9 per cent NaCl 120 ml/hour contain- ing 30 mmol/litre of KCl) to promote diuresis and combat acute renal failure, and a constant rate infusion of lignocaine (50 kg/kg/minute) with continuous ECG monitoring was instituted to control the activity of the ventricular premature contractions.

Famotidine (Pepcid IV; Merck) 15 mg IV every 12 hours, sucralfate (Carafate; Marion Merrell Dow) I g slurry orally every six hours and metoclopramide

FIG 2. Right parasagittal ultrasonogram of liver and kidney demonstrating markedly abnormal renal echogenicity. The kidney Is obviously hyperechoic compared to the liver which indicates nephropathy (such as diffuse nephrocalcinosis)

f lG 3. Severe minerallsation of the hard and soft palate

JOURNAL OF S U L L ANIMAL. PRACTICE VOL 39 DECEMBER 1998

Table 2. Biochemical abnormalities

FIG 4. Stomach. Severe mlneralisatlon of the pylorlc reglon (arrow). There Is also severe diffuse subserosal mineralisation as well as multifocal patches of mlneral on the duodenal mucosa

(Reglan; Robins) 6 mg SC every eight hours, or prochlorperazine edisylate (Compazine; SmithKline Beecham) 2 mg SC every eight hours, were administered to ameliorate haematemesis and to prevent gastrointestinal ulcer formation. Ampi- cillin sodium (Amp-Equine; SmithKline Beecham) at 650 mg IV every eight hours was administered to combat potential sepsis due to loss of gastrointestinal mucosal integrity. Finally, heparin sodium (Liquaemin Sodium; Organon) 2500 U SC every eight hours was administered to control disseminated intravascular coagu- lation suggested by the thrombocytopenia and prolonged APTT.

Ultrasonography of the abdomen revealed a hyperechoic gastric mucosal rim (Fig l) , hyperechoic renal cortices (Fig 2)

Potassium (mmol/litre) Albumin (Ulitre) BUN (nmol/litre) Creatinine (pmol/litre) Calcium (mmol/litre) Phosphate (mmol/litre) ALT (U/litre) AST (U/litre) ALP (U/litre) Cholesterol (mmol/litre) Creatinine kinase (U/litre)

44-5.7 3.8 27-31 25 36-10 26.8 44-133 389 2.2-2.9 3.0 0.85-2.0 4.85

None Not measured 21-45 238 30-102 216 3.6-5.6 8.6

None Not measured

CUrefarence Day1 w

34-5.1 3.2 30-45 24 29-10.7 37.5 44-124 265 1.8-3.2 2.7 0.75-2.1 3.2 17-85 162 16-50 1128 12-122 455 3.2-8.6 14.1 56241 51.506

Day 5

4.3 32 10.7 124 2.7 1.9 89 85 1143 13.7 337

Day ii

4.1 31 5.0 97 2.6 2.0 41 32 741

207 6.3

*rDVM Referring veterinarian. CU Cornell University

and hyperechoic splenic parenchymal foci. These findings were consistent with diffuse mineralisation involving multiple organ systems. An echocardiogram revealed pat- terns consistent with diffuse mineralisation of the endocardium and myocardium.

The dog was supported in intensive care for 13 days with intravenous fluid and total parenteral nutrition for five days. Serial CBCs and serum biochemical profiles documented gradual normalisation of the initial haematological and biochemical abnormalities (Tables 1 and 2). Add' ition- '

ally, the sinus tachycardia resolved and ventricular premature contractions became infrequent.

Despite the improvement in haemato- logical and biochemical laboratory values, and continued treatment with antiemetics and total parenteral nutrition, severe haematemesis and complete anorexia per- sisted. The dog was eventually euthanased due to the uncertainty of complete

recovery and financial considerations. Gross postmortem findings comprised

massive mineralisation of multiple organs. Most striking were changes evident in the hard palate, stomach, colon, heart, spleen, airways, and the mandibular salivary glands. The hard palate contained a large irregular area of mineralisation (Fig 3). Additionally, approximately 90 per cent of the stomach was diffusely and trans- murally mineralised (Fig 4) and a clearly delineated area, where mineralisation abruptly ended, was found to be present in the gastric fundus. Histologically, mineral deposition in the stomach was mosr severe in the smooth muscle layers, but there were patchy areas of necrosis and minerali- sation evident throughout all levels of the rnucosa (Fig 5) . Grossly, the entire colon showed diffuse serosal mineralisation, shown microscopically to be limited to the muscularis (Fig 6). The endocardium of the left atrium and ventricle and the

FIG 5. Stomach. Severe dlffuse mlnerallsatlon of muscularis (broad arrows) and patchy mineralisation throughout the mucosa (thin arrows). M Mucosa, S Submucosa, Mu = Tunlca muscularls. Haematoxylin and eosin [ H&E] x 40

FIG 6. Colon. Severe diffuse minerallsatlon of the tunlca muscularis (arrows). M Mucosa, S Submucosa, Mu Tunica muscularis. H&E x20

JOURNAL OF SMALL ANIMAL PRACTICE VOL 39 DECEMBER 1998 583 -

FIG 8. Spleen. Diffuse rnlnerallsatlon of the entire capsular surface of the spleen

FIG 7. Heart. Severe rnlnerallsatlon of the wall of the left atrium and one leaflet of the rnitral valve (arrows)

left atrioventricular (AV) valve were also severely mineralised (Fig 7). The entire sur- face of the spleen was diffusely mineralised (Fig 8) and the larynx, trachea and bronchi had diffuse rnucosal mineralisation. Many small vessels in tissues examined showed necrosis and mineralisation of the tunica intima and media. Necropsy findings collectively supported sofi tissue metastatic mineralisation consistent with hyper- calcaemia derived from vitamin D toxicity.

DISCUSSION

The strong temporal relationship between the onset of polyuria, polydipsia and haematemesis following the ingestion of calcipotriol, in conjunction with moderate hypercalcaemia (corrected calcium 3.22 mrnolllitre: reference range 2-17 to 2.94 mmolllitre), and gross and histopatholo- gical findings of soft tissue mineralisation, strongly suggest that the ingestion of calcipotriol can cause vitamin D toxicity in dogs.

The clinical signs, clinicopathological abnormalities and pathological findings observed in the present dog are similar to those associated with vitamin D toxicity induced by cholecalciferol ingestion (Spangler and others 1979). Clinical manifestations of cholecalciferol toxicity include lethargy, weakness, anorexia, haematernesis, haematochezia, circulatory

collapse and death. Biochemical abnor- malities consist of hypercalcaemia, hyper- phosphataemia, azotaemia and increased alkaline phosphatase activity, while histo- logical lesions include locally extensive haemorrhage and necrosis of the super- ficial mucosa with mucosal mineralisation in the gastrointestinal tract, focal necrosis and mineralisation of myocytes and intimal mineralisation of medium-sized myocardial arteries in the heart. Other findings have included mineralised glomerular capillary walls, cortical tubular basement membranes and Bowman cap- sules in the kidneys, and intimal minerali- sation of blood vessels in other tissues (Gunther and others 1988).

To the authors’ knowledge, two prelim- inary reports have mentioned calcipotriol- induced vitamin D toxicity in dogs. One publication is an abstract describing a dog with hypercalcaemia and acute renal failure secondary to calcipotriol ingestion (Lesthem 1997) and the second describes four dogs accidentally ingesting calcipo- triol, with the eventual death of three of the four (Campbell 1997). Recent communi- cations with the American Society for the Prevention of Cruelty to Animals National Animal Poison Control Center in the USA have revealed that there have been over 30 reports of toxicity in dogs following acci- dental oral ingestion of calcipotriol in the USA (S. Kahn; personal communication).

Despite the apparent significance of vitamin D toxicity secondary to accidental calcipotriol ingestion in dogs, single dose oral toxicity trials determining an LD5, appear to be non-existent. Japanese studies report an LD5, of greater than 1.5 mg/kg for calcipotriol when administered percu- taneously (Imaizumi and others 1996) and results of long-term oral systemic toxicity trials with calcipotriol indicate that dogs

ingesting up to 0.9 pglkglday for 28 days do not develop hypercalcaemia. However, ingestion of 1.8 pglkglday for seven days, followed by ingestion of 3.6 pg/kg/day for seven days, induced hypercalcaemia and morphological changes in the kidneys (Leo Laboratories Canada 1995). As the serum half-life of calcipotriol is very short (approximately 100 minutes in the dog) (J. T. Mortensen; personal communica- tion), the chronic daily administration of calcipotriol to dogs may be equivalent to single oral dose toxicity as there is little, if any, systemic calcipotriol accumulation. Collectively, toxicity studies in dogs suggest that 1.8 to 3.6 pglkglday of calcipotriol may cause disturbances in calcium homeostasis. Whether the anti- proliferative effects of calcipotriol influ- ence toxicity to endothelial-lined organs has not been evaluated.

Since the dog in this present report weighed 29 kg and ingested a 60 g tube of Dovonex (0.005 per cent calcipotriol), the maximum ingested dose approximated 100 pg/kg. Based on oral systemic toxicity trials in dogs, a dosage of 1.8 to 3.6 pg/kg/day of calcipotriol would be capable of creating calcium homeostatic distur- bances. Thus, this dog ingested approxi- mately 25 to 55 times more calcipotriol than is necessary to interfere with calcium homeostasis.

Despite the definitive evidence of gross and histological mineralisation in this case, the dogs serum calcium concentration was only mildly increased (3.22 mmol/litre [2.17 to 2.94 mmol/litre], corrected for hypoalbuminaemia) for less than 24 hours. The lack of a persistent hypercalcaemia may have several causes. First, calcipotriol’s diminished influences on calcium homeo- stasis are attributedjo its short serum half- life and rapid metabolism into two

584 JOURNAL OF SMALL ANIMAL PRACTICE voi 17 L)ECEMBEK 1998

metabolites: MC 1080 and MC 1046 (Sorensen and others 1989, Kissmeyer and Binderup 1991). These metabolites are approximately 100 to 200 times less potent than calcitriol on calcium homeo- stasis (Kissmeyer and Binderup 199 1, Binderup 1993). Furthermore, the hypercalcaemia in this case may also have been attenuated by acute deposition of calcium and phosphorus into soft tissue, as the serum ca1cium:phosphorus product exceeded 60 mgldl (Chew and Meuten 1982). Lastly, the administration of 500 mg of prednisone sodium succinate and 60 mg of frusemide before referral may also have reduced the serum calcium concentration by decreasing skeletal resorption and intestinal absorption and increasing renal excretion of calcium.

Given the extent of soft tissue minerali- sation and necrosis identified upon necropsy, it remains intriguing that only mild hypercalcaemia was identified within the first 24 hours following ingestion of calcipotriol. It can be speculated that calcipotriol may exert the majority of its calcaemic actions intracellularly, resulting in massive cellular death yet minimal elevations in extracellular calcium concentrations.

An unexpected biochemical finding in this dog was the increased creatinine kinase activity of 51,506 U/litre (58 to 241 U/litre). Since this enzyme is predom- inantly located in skeletal muscle, myocardium and brain, damage to these tissues is implicated. Increased cytosolic ionised calcium is an important mecha- nism initiating cell death following a vari- ety of cell injuries. High concentrations of cytosolic calcium can result from enhanced extracellular entry and/or redistribution from intracellular compartments (Trump and others 1984, 1997, Trump and Berezesky 1996). It is likely that a massive transient hypercalcaemia followed ingestion of calcipotriol, resulting in high cytosolic calcium concentration derived from increased absorption of extracellular calcium. Tissues known to have metabolic functions tightly regulated by calcium ion

JOURNAL OF SMALL ANIMAL PRACTICE VOL 39 C

transport could be predisposed to cell injury in such cases.

Other clinicopathological abnormali- ties observed in the present case, and not usually associated with vitamin D toxicity, include hypoalbuminaemia, hypercholes- terolaemia and pathological proteinuria. The severe protein-losing nephropathy observed in this dog contrasts with the mild renal tubular and glomerular degen- eration observed in dogs with vitamin D nephropathy (Spangler and others 1979). Renal lesions associated with hyper- calcaemia and acute renal failure are attributed to afferent renal arteriole vaso- constriction, activation of phospholipases and disruption of mitochondrial oxidative phosphorylation (Grauer and Lane 1995). Although the protein-losing nephropathy observed in the present case may have been caused by renal tubular and glomerular cell death secondary to renal ischaemia and increased cytosolic calcium, the lack of an active urine sediment following calcipo- trio1 ingestion would not be consistent with this conclusion.

The dog in this report presented ini- tially with severe thrombocytopenia and a prolonged APTT. Activation of human platelets depends predominantly on the influx of extracellular calcium via specific calcium receptor channels. In addition, calcium channel blockers have been shown to inhibit normal platelet aggregation, illustrating the important role of calcium (MacIntyre and Shaw 1983). Induction of platelet aggregation by exposure to high extracellular calcium concentrations has also been shown to occur in guinea-pig and rabbit platelets (Miyamae and others 1995). The thrombocytopenia observed in this dog may have been induced initially by calcium-mediated platelet aggregation and perpetuated by disseminated intravas- cular coagulation related to vascular and soft tissue necrosis.

Necropsy findings confirmed patho- logical mineralisation of multiple organ systems. Pathological mineralisation may be categorised as dystrophic or metastatic. Dystrophic mineralisation is the deposi-

EMBER 1998

tion of calcium salts in non-viable or dying tissue in the absence of calcium metabolic derangements. In contrast, metastatic mineralisation occurs in normal tissue, is associated with disturbances in calcium homeostasis, and affects primarily the interstitial tissues of the blood vessels, kidneys, lungs and gastric mucosa (Cotran and others 1994). In the present case, pathological mineralisation was considered to be primarily metastatic due to the hypercalcaemia documented. However, necrosis observed in multiple organ sys- tems argues for a component of dystrophic mineralisation. The present authors pre- sume that the massive soft tissue minerali- sation observed was initially metastatic, with dystrophic mineralisation occurring as a secondary mechanism.

Conclusions 0 The oral ingestion of calcipotriol, a syn- thetic calcitriol analogue, was associated with findings consistent with vitamin D toxicity in a dog. 0 Oral systemic toxicity studies of calcipotriol in dogs indicate that 1.8 to 3.6 pg/kg/day may cause hypercalcaemia and morphological changes in the kidney. 0 Transient hypercalcaemia in this dog following oral ingestion of calcipotriol may have been due to calcipotriol's rapid conversion to less active metabolites (MC 1046 and M C 1080) and its short plasma half-life (100 minutes in the dog). 0 Biochemical findings associated with calcipotriol ingestion may include hyper- phosphataemia, azotaemia, increased ALP and creatinine kinase activity, transient hypercalcaemia, hypercholesterolaemia and hypoalbuminaemia. 0 Urinalysis and haematological findings associated with calcipotriol ingestion may include pathological proteinuria, thrombo- cytopenia and prolonged coagulation times. 0 Following calcipotriol ingestion in this dog, normalisation of biochemical and haematological abnormalities could not be used as prognostic indicators as permanent organ mineralisation and dysfunction pre- cluded resolution of toxic effects.

585 -

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