Original Article

Original Article Management of adverse side-effects after chemotherapy in macaques as exemplified by streptozotocin: case studies and recommendations Melanie L Graham1, Lucas A Mutch1, Jessica A Kittredge1, Eric F Rieke1, Nicholas A Robinson2, Elizabeth K Zolondek1, Aaron W Faig1, Theresa A DuFour1, James W Munson1 and Henk-Jan Schuurman1 1

Schulze Diabetes Institute, Department of Surgery, University of Minnesota, Minneapolis, MN 55455, USA; 2Department of Veterinary Population Medicine, College of Veterinary Medicine, University of Minnesota, St Paul, MN 55108, USA Corresponding author: M L Graham, Schulze Diabetes Institute: Animal Science/Veterinary Medicine 295, 1988 Fitch Avenue, St Paul, MN 55108, USA. Email: [email protected]

Abstract The chemotherapeutic streptozotocin is used for induction of diabetes in animal models including non-human primates. Being a cytotoxic nitrosourea compound, it can be associated with adverse events (AEs), mainly nausea and emesis, nephrotoxicity, elevated liver transaminase levels, pulmonary oedema and, most prominently, metabolic acidosis: these can be severe in some cases. The incidence and gravity are to some extent related to the characteristics of the individual animal, diagnostic tools, prompt recognition of symptoms and supportive measures. Careful animal selection, dose adaptation and supportive actions such as renal protective hydration are the main tools in managing AEs, but do not fully eliminate unavoidable and sometimes life-threatening conditions. In our centre we have built experience in a cohort of 78 cynomolgus and rhesus macaques in which six cases manifested severe AEs (8%). This experience has prompted implementation of strategies for early detection and management of adverse effects, together with an animal refinement programme. We present here specific pretreatment regimens, post-infusion laboratory evaluations, and flow charts to assess/treat metabolic acidosis and precipitating factors. Case reports of the six animals with severe AEs are presented to illustrate management of AEs, especially metabolic acidosis, and criteria for early euthanasia where appropriate. We conclude that improved monitoring and validated tools allow for optimal management of adverse effects in an early stage of their manifestation. Reduced morbidity and mortality not only improve individual animal wellbeing but also avoid model-induced confounding that diminishes the translational value of the experimental protocol.

Keywords: Adverse events, diabetes induction, non-human primate, refinement, streptozotocin Laboratory Animals 2012; 46: 178– 192. DOI: 10.1258/la.2012.011077

Animal models have proven essential in understanding the pathogenesis of type 1 (T1) diabetes and subsequently in evaluating potential therapeutic targets. Islet cell transplantation as a strategy for replacing lost b-cell mass has particularly benefited from studies in animals in translation to the clinic. Studies performed in rodents1 and non-human primates (NHPs)2 in the 1970s supported the first trials in humans in the 1980s. Animal studies continue to advance potential transplant applications and support progress in clinical transplantation. Rodents are preferentially used in basic T1 diabetes research,3 and more specifically in assessment of islet cell quality studies.4 The nude mouse bioassay is considered the gold standard for islet product testing as it Laboratory Animals 2012; 46: 178 –192

relates to quality control, islet cell potency for product release and studies assessing refinements in islet cell processing techniques or islet cell modification during manufacturing.4 NHPs, mostly macaques and baboons, are widely used in transplantation translation research5,6 because of the close phylogenetic relationship to humans and development of human-like disease and symptoms. Studies in NHPs combining efficacy and safety outcomes are often needed as part of the proof-of-concept documentation supporting the phase transition into clinical explorations. A number of characteristics make the NHP the preferred model for translational safety and efficacy studies of immunosuppressives and transplantation as opposed to rodents

Graham et al. Adverse effects of streptozotocin in macaques

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or other lower species. The outbred nature of the NHP is similar to the clinical situation, and the immune system of the NHP mimics the complexity of the human. Like humans, old-world NHPs have naturally existing antibodies (e.g. to the galactose a-1-3 galactose epitope) and show cross-reactivity with many human directed biologicals.6 On a practical level, complex multifaceted immunological, metabolic and haematological (i.e. safety) monitoring can be performed via blood sampling and ex vivo assays that parallel clinical management.7 In our islet cell transplantation programme addressing the functionality of b-cell therapy, diabetes is induced using pharmaceutical-grade streptozotocin (STZ). We prefer STZ above total surgical pancreatectomy because it spares the animal from a major survival surgery in the pretransplant period and leaves the abdomen undisturbed. Also, diabetes induction using STZ is relatively simple to perform technically, and is associated with few clinically relevant adverse events (AEs) in healthy juvenile and adult macaques.8,9 This is in contrast with total pancreatectomy where the animal undergoes major abdominal surgery in which the exocrine pancreas function is also removed, and which necessitates a substantial postoperative recovery period that requires extensive analgesic management. Hence, this approach requires surgical expertise at the highest level.5,10 After total pancreatectomy, body weight (BW) loss of 10 –15% during the first weeks after surgery is a normal finding prompted by surgical stress and associated gastrointestinal disturbances with reduced food intake, and in addition the loss of endocrine function necessitates enzyme supplementation.5,10,11 However, STZ, being a nitrosourea chemotherapeutic agent, has the disadvantages common to chemotherapeutics. STZ is an alkylating cytotoxic agent, and as such shows the toxicity profile including nephrotoxicity, hepatotoxicity, and in rare cases, mortality resulting from organ failure or severe metabolic perturbations.9,12 – 14 Most groups working with NHPs have reported complications ranging from mild laboratory abnormalities to high incidences of intercurrent death.9,10,15 – 18 We have recently published a retrospective evaluation in a large cohort of 78 cynomolgus and rhesus macaques, in which we identified risk factors for AEs.9 Based on broad multicentre experience with STZ, several improvements in prevention and reduction in both the incidence and gravity of AEs have already been realized, among others the migration to pharmaceutical-grade STZ, modifications in dosing regimens, careful animal selection and renal protective hydration protocols.9,19,20 Appreciating the complexity of managing NHPs that are at risk, we have established a programme that includes training for cooperation in frequent handling (brief or prolonged),21 reliable central vascular access22 – 24 and intensive care protocols adjusted for the unique aspects of the conscious unrestrained and socially active NHP. In our experience, cooperative training results in minimal stress that facilitates routine and productive clinical interactions;21 hence, clinical symptoms are more easily recognized and repeated frequent treatment is possible. However, even upon implementation of these concepts, unavoidable and sometimes life-threatening AEs emerge

intrinsic to cytotoxic nitrosoureas in a small percentage of at-risk animals (8% in our experience).9 Besides our recent evaluation there are only a few comprehensive reports on AEs, and there are no data regarding management and treatment of AEs in the literature. We have therefore developed flow charts and procedures for the diagnosis and treatment of certain AEs based on our experience in the large cohort mentioned above.9 Our experience has enabled us to present a number of practical recommendations for the application of STZ including the assessment of animals for suitability using eligibility criteria, establishing dosing criteria, proper handling/administration of the agent, postinfusion monitoring, and prophylaxis and treatment strategies to reduce animal discomfort. Our recommendations not only address the aspect of improved wellbeing using refinement techniques21 but also address reduction in numbers of animals. After recovery from STZ-induced AEs, these animals are still able to successfully enrol in transplantation or other studies, which avoids necessitated enrolment of additional replacement animals. In this paper we have illustrated the flow chart and procedures and recommendations in the management of severe AEs with clinical case reports describing the animals manifesting severe AEs.

Materials and methods Animals Animals were handled in accordance with the rules and regulations of the Institutional Care and Animal Use Committee of the University of Minnesota (Minneapolis, MN, USA), in compliance with the Animal Welfare Act, and adhered to principles stated in the Guide for the Care and Use of Laboratory Animals. Our institution is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC). The flow charts and procedures described in this study were developed during the period that our institution conducted diabetes induction using STZ in rhesus and cynomolgus macaques as part of an islet transplantation programme. Between December 2005 and July 2010, 78 NHPs underwent diabetes induction using STZ. The cohort comprised 54 cynomolgus macaques (Macaca fascicularis) and 24 rhesus macaques (Macaca mulatta): 59 males and 19 females. The cynomolgus macaques weighed between 2.9 and 6.5 kg (median 4.4 kg) and the rhesus macaques weighed between 3.1 and 13.0 kg (median 5.5 kg). The cynomolgus macaques’ age at induction was between 3.5 and 7.2 years and the rhesus macaques’ age was between 1.8 and 6.7 years. All animals were purpose-bred and purchased from institutionally approved commercial vendors. These commercial vendors must adhere to very specific rules regarding quality control, health and testing results, and such data need to be provided by the vendor. This can be combined with audit visits and inspection of the source facility by the Department of Research Animal Resources as deemed necessary, and the ability of the vendor to transport animals by means of an acceptable carrier (e.g. environmentally controlled vehicles using specially trained personnel).

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They were housed in pairs or small groups of the same sex. They had free access to water and were fed biscuits (High-Protein Monkey Chow 5045; Purina Mills, St Louis, MO, USA) based on BW. Their diet was enriched liberally with fresh fruits, vegetables, grains, beans, nuts and a multivitamin. The NHPs participated in an environmental enrichment programme that included social play, toys, music and regularly scheduled access to large exercise and swimming areas. To facilitate venous access, a totally implantable port and catheter was placed using a previously described technique.22 – 24 NHPs were trained to cooperate in medical procedures including shifting into transport boxes for weighing, presentation of the foot for routine heelstick blood glucose (BG) measurements, presentation of the leg for access to a vascular port for blood collection or metabolic testing and drug administration, and basic physical examinations.21 Further details on these animals are presented in the publication of the detailed evaluation.9 The case studies in the present report represent six animals out of the cohort of 78 animals that experienced clinically manifest severe (grade 3) AE.9 The selection of case reports was performed on the basis of expression of AEs (renal failure, pulmonary oedema and metabolic acidosis), and irrespective of the implementation of flow charts and procedures described below. In this way, we have been able to illustrate the value of the following procedures and flow charts that were developed and implemented during the study period. Evaluation of suitability for STZ To assess eligibility for STZ administration, a physical examination and review of each individual animal’s clinical history was performed, with particular attention to factors suggesting pre-existing kidney or liver dysfunction. All animals received a central vascular access port that was used for drug or fluid administration and blood collection.22 – 24 Prior to STZ infusion animals were trained for cooperative handling without the need of chemical or physical restraint, which included acceptance of oral medications/fluids offered by hand, presenting forward facing at cage front for physical examination (e.g. palpation, auscultation and skin turgor), shifting into a transport box for weighing and stationing with presentation of a limb for vascular access.21 Cooperative handling in the home cage allowed assessment of attitude, general appearance (gait, posture, physical condition), temperament and interaction within the social group, without the influence of sedation under condition of minimal stress. The full set of eligibility and exclusion criteria is outlined in Table 1. STZ induction pretreatment Nephrotoxicity from STZ can be reduced through hydration protocols.20 We avoided full 24 h fasting (removal of all solid food and access to water only) prior to induction. Instead, highly palatable fluids were offered containing at least 55 kcal/100 mL (e.g. 50:50 mixture of fruit juice and water) cageside for oral hydration aiming for at least 100 mL/kg total consumption. Providing carbohydrates in the fluid loading protocol up to induction guards against

Table 1 Evaluation of NHPs for suitability of STZ induction Eligibility criteria

† † † † † † †

2 years of age Negative tuberculosis screen within the last 12 months Negative viral screen (macaque herpes B virus, measles, simian retrovirus D, simian immunodeficiency virus and simian T-cell leukaemia virus-1) Haemoglobin .10 g/dL Patent indwelling central vascular access port Complete laboratory analysis to include haemogram and chemistry panel at two separate timepoints, to establish individual baseline values, and absence of abnormalities Finished a training programme for cooperation with handlers for complex medical procedures, able to demonstrate ability to comply with planned study procedures

Exclusion criteria

† † † † † † †

Body condition score ,2 (thin) or weight ,2.0 kg Cytotoxic chemotherapy or nephrotoxic medication within previous six-week period Active/uncontrolled infection Active gastrointestinal disorders (e.g. diarrhoea, vomiting) Persistent elevation of kidney function tests at the time of study entry. Persistent serum elevation of CREAT or BUN, with values .1.5 times normal upper limits Persistent elevation of liver function tests at the time of study entry. Persistent serum elevation of AST, ALT, ALP or total bilirubin, with values .2.0 times normal upper limits Any medical condition that, in the opinion of the investigator or attending veterinarian, will interfere with safe STZ induction

Body condition score 4 (above normal girth-to-height ratio) is not an exclusion criterium but requires special attention in setting the dose level CREAT: creatinine; BUN: blood urea nitrogen; ALT: alanine aminotransferase; AST: aspartate aminotransferase; ALP: alkaline phosphatase NHPs: non-human primates; STZ: streptozotocin

severe hypoglycaemia after infusion, probably by sustaining a reserve of glycogen in the liver.25 A second benefit might be a reduction in nausea and vomiting, similar to reports in patients that providing carbohydrates avoids metabolic disturbance and reduces nausea and vomiting after anaesthesia.26 – 29 Ondansetron, 4 mg total dose per os, was used for antiemetic prophylaxis at least 30 min prior to induction to prevent nausea and vomiting after infusion. For comfort, animals were sedated with ketamine, 5–15 mg intramuscularly, prior to infusion. The examination immediately prior to infusion included weight, assessment for hydration by skin turgor and assessment of body condition. The body condition was scored using the girth-to-height ratio (GHtR) and Rh Obesity Index (RhOI).30,31 The RhOI is modified from the body mass index (BMI) used in humans, and is calculated as BW (kg) divided by the square of the crown-rump length (cm).31 The mid-girth circumference is divided by the height to give the GHtR as an estimate of body shape. GHtR is especially useful to give an estimate of body shape: a lower ratio indicates a leaner body shape and a higher ratio indicates a rounder body shape (i.e. obesity). We have described elsewhere the threshold values for the distinction of obesity in our colony, that is a value .0.9 for GHtR and .5.0 for RhOI.9 Warm isotonic saline (0.9% NaCl), 25–35 mL/kg intravenously, was infused over 10–30 min just prior to infusion for its renal-sparing effect.


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STZ dose calculation and administration Varying doses of STZ have been reported in the literature,20,32 – 34 ranging from 30 to 150 mg/kg in a single-dose infusion or 10 –40 mg/kg infused in multiple doses. In our experience a single injection of pharmaceutical-grade STZ at a dose of 100 mg/kg consistently induces a profound diabetic state with minimal morbidity and no mortality in normally conditioned cynomolgus or rhesus monkeys. Similar success was observed using body surface area (BSA)-based dosing to deliver a STZ dose of 1250 mg/m2, where BSA ¼ BW0.67 12/10,000.35 However, in our retrospective evaluation mentioned above we did not observe an advantage of BSA-based dosing over flat-fixed dosing. Therefore, we prefer flat-fixed dosing since fewer errors are made in calculating, preparing and administering a proper individual dose. Since there is a statistically significant association between obesity and AEs,9 a dose reduction by 20% was later implemented in animals considered to be obese, i.e. with a higher than average GHtR (.0.9) or RhOI (.5.0). Pharmaceutical-grade STZ (Zanosarw; Sicor Pharmaceuticals, Irvine, CA, USA) was supplied in 1 g vials,

Figure 1

should be stored at temperatures of 2–88C and protected from light. To prepare STZ, Zanosar was reconstituted with 9.5 mL of cold 0.9% NaCl for injection USP. The resulting pale-gold solution contained 100 mg of STZ and 22 mg of citric acid per mL. To avoid decomposition after reconstitution, immediately after preparation STZ was administered as an intravenous bolus.

Insulin therapy and evaluation of the diabetic state Insulin was initiated after three consecutive BG readings exceeding 300 mg/dL measured with a minimal time interval of 4 h in between, or anyhow 48 h after STZ administration. A sliding scale was used that combined subcutaneous glargine (Lantusw; Sanofi-Aventis, Bridgewater, NJ, USA) and lispro (Humalogw; Eli Lilly, Indianapolis, IN, USA). The starting dose of long-acting insulin was 1.0 U/kg, which was subsequently adjusted in combination with lispro at mealtime, in order to target BG levels of 100 –200 mg/dL. In animals with diabetic ketoacidosis (DKA) insulin protocols may require substantial modification based on the individual animal’s presentation

Using anion gap and clinical observations in the differential diagnosis of metabolic acidosis

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Figure 2 Fluid replacement in metabolic acidosis or renal compromise, and treatment of co-occurring clinical AE in NHPs. When acidosis has been confirmed (1) the animal should be assessed for hydration and treated (1a) for co-occurring AEs. Dehydration can be assessed by percent BW change or estimated using clinical observations. Evaluation of the laboratory values (2) determines type of fluid and supplementation required. Intensive insulin therapy can be initiated in the case of DKA (3), and corrections for electrolyte abnormalities (4) and acid/base deficit (5) can commence. Except for the most severe cases in which the animal might refuse oral fluids, administration of maintenance fluids per os is encouraged (6). This cycle is repeated until a resolution of symptoms. Organ failure that is non-responsive to treatment requires prompt euthanasia

(Figures 1 and 2). In the month after induction the diabetic state was confirmed by measuring C-peptide and performing one or more in vivo assays for stimulation of C-peptide secretion. The induction was considered successful when the animal had demonstrated the combination of a fasting C-peptide below 0.5 ng/mL or one-third the value in the period before STZ administration, and when the animal manifested the absence of a stimulated C-peptide response upon an intravenous glucose tolerance test or arginine stimulation test (,0.3 ng/mL), and when there was persistent hyperglycaemia (BG 200 mg/dL) prior to the initiation of full exogenous insulin. These criteria are in agreement with those outlined by the International Xenotransplanation Association (IXA) consensus statement for conduct of preclinical trials.36 Diabetes induction can only be considered successful if the diabetic state is documented, and there are no

complications that prevent enrolment onto the subsequent experimental study. Follow-up Immediately after STZ infusion BG, appetite, urine and stool type/frequency, and general appearance were closely monitored. For early detection of AEs regular assessment was performed for BW, blood haematological parameters (including white blood cell counts [WBC], red blood cell counts [RBC], haemoglobin [Hb], haematocrit, platelet counts [PLT] and lymphocyte counts) and serum chemistry (creatinine [CREAT], blood urea nitrogen [BUN], alanine aminotransferase [ALT], aspartate aminotransferase [AST], alkaline phosphatase [ALP], bilirubin, albumin, bicarbonate, sodium, potassium, chloride, calcium, cholesterol and triglycerides). This was also done upon clinical indication,


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i.e. when animals presented abnormalities in general constitution or otherwise. If acidosis was suspected or there was respiratory compromise, additional blood gas analysis was performed. The observation of abnormalities prompted increased examinations and sampling for laboratory assessments. It is worth mentioning that all animals in this study were cooperatively trained, as such laboratory assessments reported were not confounded by sedation or restraint. Adverse effects assessment and monitoring In animals experiencing abnormalities the Veterinary Co-operative Oncology Group – Common Terminology Criteria for Adverse Events (VCOG-CTCAE)37 following chemotherapy was used, which we have modified for NHPs (Table 2) to score the initial severity of AEs, its progression and the response to treatment protocols. Treatment approach of metabolic acidosis Based on experience, we have developed a treatment approach of metabolic acidosis, which is presented in Figures 1 and 2. This approach is based on established practices in human and veterinary medicine,38 – 43 but modified to account for the special conditions in handling NHPs. A decision tree (Figure 1) is used to identify the likely cause of acidosis followed by a treatment regimen that aims to correct disturbances in a stepwise manner (Figure 2). The primary focus is fluid therapy and rehydration, intensive insulin therapy (in the case of DKA), and correction of electrolyte abnormalities and base deficit. Generally clinically manifest symptoms (e.g. anorexia, dehydration, hypothermia and lethargy) resolve within 48 h. However laboratory abnormalities resulting from STZ administration can take longer to resolve, in some cases up to 10 days: this is in contrast to DKA where laboratory values usually normalize in 36–48 h. This difference might be explained by the multifactorial nature of acidosis in this situation that includes lactic acidosis intrinsic to STZ administration, new onset diabetes with insulin deficit and acute renal injury that may take up to 30 days to fully recover.

Results Occurrence and monitoring of AE Following the outline presented in the Materials and methods section, the majority of animals did not experience complications following STZ infusion. However, in the few cases (6 out of 78 macaques in the cohort studied, i.e. 8%)9 that manifested clinically evident symptoms, abnormalities could quickly progress into rapid decompensation and subsequently into a life-threatening emergency situation (Table 3). Clinically relevant AEs affected in particular those animals that were considered obese, i.e. a value .0.9 for GHtR and .5.0 for RhOI, and those that did not receive STZ at an adapted dose level. This relationship between obesity and AEs became apparent in our retrospective evaluation;9 before this association was identified, obese animals received a full STZ dose, and after it was identified

a dose adaptation was implemented. In only one of six case reports below there was a dose adaptation based on obesity, which justified euthanasia in three cases. AEs present in a typical profile and in the absence of organ failure (renal or pulmonary) are generally reversible. In all cases where animals experienced a severe AE (grade 3 or higher)9 that was not responsive to treatment indicating irreversible organ failure, or any observation of unrelieved pain or distress, animals were promptly euthanized.9 The following aspects are relevant in the monitoring and treatment: Laboratory monitoring A basic haematology and chemistry panel is necessary for identifying and treating AEs. The haemogram should include a blood smear, haematocrit, Hb, PLT, RBC and WBC and differential. A minimal chemistry panel includes albumin, ALP, ALT, AST, bilirubin, CREAT, BUN, calcium, chloride, BG, HCO2 3 , potassium, sodium and total protein. Additional parameters of interest that can be added are lactate, magnesium and phosphorus. Blood gas analysis should include pH, pCO2 and pO2. Finally, measuring ketones is essential to confirm ketoacidosis.44,45 BG is not a reliable surrogate as animals might be hyperglycaemic and not ketoacidotic, and euglycaemic ketoacidosis can occur in animals with altered eating.46 Serum beta hydroxybutyrate (BHY) gives a precise measure, which is helpful when assessing treatment progress, but urine testing (Ketostixw) is an alternative in case serum testing cannot be performed. Fluid therapy, electrolyte replacement and base deficit correction We prefer to limit infusions to no longer than 1.5 h and up to four times daily, to shorten the duration necessary to cooperate with handling: consequently, oral administration during maintenance fluid is recommended. An isotonic replacement crystalloid solution of 0.9% NaCl is used until dehydration has been corrected. For intravenous fluid administration 0.9% NaCl is recommended instead of lactated Ringer’s solution (LRS). LRS is attractive because it contains lactate, a bicarbonate precursor. However, lactate is metabolized in the liver in the same way as ketone bodies, which leads to reduced hepatic metabolism: also, lactate levels are already increased after STZ infusion.47 The standard polyionic composition also does not easily allow for individualization of electrolyte (e.g. potassium) therapy. Generally, hypotonic fluids (e.g. 0.45% NaCl) should be reserved for animals with substantially elevated sodium or chloride levels and otherwise avoided because rapid shifts in osmolality can precipitate cerebral oedema. If chloride-driven acidosis is suspected, 0.45% NaCl can be substituted to diminish ongoing acidosis.48 In our unit animals move freely through the homecage and are partnered or grouped, which makes the conduct of continuous infusion of, e.g. insulin impractical. Instead, a combination of intravenous insulin lispro supplementation is used during fluid rehydration paired with subcutaneous longacting glargine. The application of subcutaneous or intramuscular insulin in the situation of DKA is not uncommon,

Renal failure (acute kidney injury)

Hypoxia

Oedema, pulmonary

.2.5 –5.0 baseline .2.5 –5.0 baseline 11.0 –15.9 mmol/L .1.5 –2.0 baseline .1.5 –2.0 baseline ,25 –20 mg/dL 6.1– 6.5 mmol/L 2.5– 2.9 mmol/L 151– 154 mmol/L 125– 129 mmol/L

pH ,baseline, but 7.3

Oral intake altered (,3 days) without significant weight loss

–

Transient laboratory abnormalities only; asymptomatic

Dyspnoea on exertion, but can Dyspnoea on exertion and tires upon ambulate without tiring ambulating Asymptomatic oedema by exam Symptomatic oedema, no respiratory only distress – ,O2 saturation with ambulation

.1.25 – 2.5baseline .1.25 – 2.5 baseline 16.0 mmol/L – ,baseline .baseline –1.5 baseline .baseline –1.5 baseline ,35 – 25 mg/dL 5.6 –6.0 mmol/L 3.0 –3.4 mmol/L 146– 150 mmol/L 130– 135 mmol/L

ALP ALT Bicarbonate, low BUN CREAT Glucose, low Potassium, high Potassium, low Sodium, high Sodium, low

Dyspnoea

–

Acidosis

Coaxing or dietary change required to maintain appetite

Transient laboratory abnormalities only; asymptomatic; fluid resuscitation required

Respiratory distress; interfering with ADL ,O2 saturation at rest; continuous O2 supplementation required

Dyspnoea with ADL

pH ,7.3 without life-threatening consequences .5.0 –10 baseline .5.0 –10 baseline 8.0– 10.9 mmol/L .2.0 –3 baseline .2.0 –3 baseline ,20 –15 mg/dL 6.6– 6.9 mmol/L 2.0– 2.4 mmol/L 155– 159 mmol/L 121– 124 mmol/L

Persistent laboratory abnormalities; symptomatic

Dyspnoea at rest; intubation/ ventilator indicated Life-threatening; intubation indicated Life-threatening; intubation or ventilation required

pH ,7.3 with life-threatening consequences .10 baseline .10 baseline ,8.0 mmol/L .3 baseline .3 baseline ,10 mg/dL .7.0 mmol/L ,2.0 mmol/L 160 mmol/L 120 mmol/L

Death

Death

Death

Death

– – Death Death Death Death Death Death – –

Death

Death

–

Death

Death

Death

–

Death

Grade 5

Veterinary Co-operative Oncology Group – Common Terminology Criteria for AEs (VCOG-CTCAE) following chemotherapy.37 The VCOG-CTCAE have been specifically developed for dogs and cats with modifications from the standard National Cancer Institute (NCI) common terminology for patients ( particularly in constitutional signs and recognition of pain): this is easier to translate to NHPs than the NCI criteria that are applied to human patients Modifications from canine/feline VCOG-CTCAE guidelines based on NHP characteristics; The VCOG-CTCAE criteria for AE in laboratory values include the fold change in relation to normal. However, for NHPs wide ranges of ‘normal’ values have been reported in the literature, and therefore a more conservative approach should be used, i.e. the animals baseline ( pre-STZ) values ADL: activities of daily living (eating, sleeping, defaecating and urinating); PPN/TPN: peripheral parenteral nutrition/total parenteral nutrition; IV: intravenous; SC: subcutaneous; BUN: blood urea nitrogen; ALT: alanine aminotransferase; NHPs: non-human primates; STZ: streptozotocin

Renal

Respiratory

Laboratory and metabolic

Vomiting

Nausea

Moderate pain or distress; pain or analgesics interfering with function but not ADL .10 –20% from baseline; nutritional support indicated

Grade 4

Compromised, severely restricted in Disabled, must be force fed ADL, ambulatory only to the point and helped to perform ADL of performing ADL Severe pain or distress; pain or Disabling analgesics severely interfering with ADL .20% baseline –

Grade 3

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Grade 2 Moderate lethargy causing some difficulty with performing ADL

Life-threatening Of 3– 5 days duration; associated consequences; with significant weight loss or .5 days duration malnutrition; IV fluids, tube feeding or TPN indicated Increased oral fluids indicated; Parenteral (IV or SC) fluids indicated IV fluids indicated .24 h Life-threatening (e.g. dry mucous membranes ,24 h haemodynamic collapse) Loss of appetite (e.g. decreased Salivation or food avoidance (i.e. grimacing Salivation or food avoidance (i.e. Salivation or food avoidance food intake from baseline) at food or turning away) on offering grimacing at food or turning away) (i.e. grimacing at food or ,12 h on offering .12 –24 h turning away) on offering .24 h ,3 episodes in 24 h 3–5 episodes in 24 h; ,3 episodes/day .5 episodes in 24 h; vomiting .4 Life-threatening (e.g. for .2 days but ,5 days days; IV fluids or PPN/TPN haemodymnamic collapse) indicated .24 h

5– 10% from baseline; intervention not indicated

Weight loss

Anorexia

Mild pain or distress not interfering with function

Pain (distress)

Grade 1

Mild lethargy

Adverse event

Lethargy/fatigue

................................................................................................................................................

Laboratory Animals

Gastrointestinal

Constitutional

Table 2 Grading of main clinical and laboratory adverse events

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Table 3 Demographic and clinical data in cases with severe adverse events

Gender

Body condition (GHtR)

STZ dose (mg/kg) [mg/m2]

5.0

F

NS

100 [1,394]

7.6

M

1.10

100 [1,596]

Pulmonary oedema 3 Rhesus 5.2

13.0

M

1.11

100 [1,900]

4

11.3

M

1.15

100 [1,815]

Primary metabolic acidosis 5 Cyno 6.6 4.4

F

0.94

100 [1,328]

6

F

1.27

80 [1,285]

Age (years)

Weight (kg)

Renal failure 1 Cyno

7.1

2

5.7

Case

Species

Cyno

Rhesus

Cyno

6.0

7.2

7.3

Clinical symptoms

Outcome

Post-STZ emesis (2/48 h). Intermittent anorexia, dehydration, hypothermia, lethargy, ataxia Post-STZ emesis (2/48 h), then no clinical symptoms present until day 82 post-STZ, seizure

Acute renal failure, euthanasia

Severe respiratory distress manifest ,4 h post-infusion Laboured breathing and decreased O2 saturation

Acute pulmonary oedema, euthanasia Favourable

Transient anorexia, emesis (1), abdominal pain Transient anorexia, dehydration, hypothermia, lethargy, ataxia, and anaemia

Favourable

Progressive renal failure uraemia, euthanasia

Favourable

In a large series of 78 animals, 8% experienced grade 3 or higher clinical adverse events.9 These six cases are presented GHtR: girth-to-height ratio; STZ: streptozotocin; Cyno: cynomolgus

because similar protocols are used in human and veterinary patients.40,49 – 51 When electrolyte disturbances are present, electrolytes can be added to saline used for resuscitation, adapting to a proper correction of both volume depletion and electrolyte abnormalities. The most common electrolyte imbalance in animals receiving STZ is hypokalaemia which is expected as potassium moves from the extracellular space to the intracellular space in response to insulin administration. Potassium supplementation should be done (Figure 2) with special precaution in order not to exceed the recommended infusion rate. We generally allow 6–8 h to elapse between supplementation and re-evaluation to avoid oversupplementation. Hypophosphataemia occurs similar to hypokalaemia and is also a known AE after STZ infusion. STZ-induced renal damage has been described as insidious with hypophosphataemia as the first presenting laboratory abnormality followed by glycosuria, proteinuria, and increases in serum CREAT and BUN.52 We have not developed a supplement protocol for hypophosphataemia, but rely on surrogate markers for detection and focus on correction of primary deficit. In the future, monitoring of serum phosphate is desired, as phosphate supplementation in severe hypophosphataemia has shown benefit.53 The occurrence of severe hypophosphataemia is evident as haemolytic anaemia and rhabdomyolysis do occur. The ATP depletion that occurs in severe hypophosphataemia leads to a loss in membrane integrity in both erythrocytes and muscle cells.54 Blood transfusion In animals with anaemia, ABO-typed whole-blood transfusions are performed at 10– 20 mL/kg: we reduce individual infusions by 50% in animals with respiratory compromise. Whole blood can be leukocyte-depleted to reduce risk of antibody development or transmission of infectious agents. Evidently, blood donors should be extensively

screened prior to donation. We have not seen complications associated with a blood transfusion, and are impressed by the way in which a blood transfusion can dramatically improve the clinical condition (see the reports on case 4 with acute pulmonary oedema, and case 6 with metabolic acidosis below). Aside from improving haemodynamic stability, the contribution of the anticoagulant should be mentioned. We use ACD-Aw as the anticoagulant in blood collection, which contains citrate that is converted to bicarbonate by the liver. In normal individuals this results in mild alkalosis, but in animals with STZ-induced AE it serves as an additional base deficit correction.55 Adjunct supportive care Perhaps most important during treatment for the primary condition is to use adjunct care strategies to reduce and eliminate discomfort. Ondansetron (Figure 2) is most effective in prevention or treatment of nausea and vomiting, and superior to H1 receptor agonists (e.g. promethazine). Generalized abdominal pain may occur in the situation of acidosis, and evidently the cause of pain should be treated: pain relievers like ketoprofen (1 mg/kg intramuscularly), buprenorphine (0.01 –0.03 mg/kg intramuscularly) or equivalents can be used in addition to the primary treatment course (Figure 2). Renal failure The kidney is the first target of STZ toxicity.56 Two cases of renal injury in animals with documented obesity are presented (Tables 3 and 4, Figure 3). In both cases renal injury manifested itself within five days after injection by a substantial elevation of CREAT accompanied by uraemia and absent DKA. One animal had to be promptly euthanized because of severe metabolic acidosis secondary to renal injury, and one animal remained in excellent clinical condition despite

Pre þ5 þ6 þ7 þ8 þ9 þ10 þ11 þ14 þ21 þ34

Pre þ4 þ6 þ7 þ8 þ9 þ10 þ14 þ21 þ34

0 (þ5 h) 0 (þ10 h)

0.6 2.5 1.7 1.7 2.1 1.9 2.0 1.3 1.0 0.7 0.9

0.94 1.36 1.42 1.57 1.87 1.48 1.66 1.36 1.34 1.29

0.8 1.13

–

14 46 30 23 27 17 18 16 23 10 7

14 10 15 17 8 6 7 9 12 23

18 24

–

22 33 71 66 117 148 236

15 61 79

148 120 133 136 135 139 137 139 138 141 142

145 140 139 141 139 149 139 144 145 142

153 139

–

147 132 142 139 138 134 133

149 136 133

3.9 3.7 3.1 2.6 2.5 2.9 2.6 4.9 4.5 4.5 4.0

3.3 4.1 3.4 3.4 2.9 3.2 4.0 4.0 3.6 3.5

3.5 3.8

–

4.1 3.7 4.6 3.8 4.9 6.1 6.9

3.9 4.5 3.1

†

Day in relation to streptozotocin infusion; BHY: beta-hydroxybutyrate; BUN: blood urea nitrogen Measured as part of the venous blood gas panel ‡ Measured via urinalysis

Case 6

Metabolic acidosis Case 5

Case 4

0 (þ4 h)

0.8 1.9 4.8 5.2 6.9 9.7 19.8

0.7 2.9 7.98

Potassium (mEq/L) 3.1 – 5.5

112 83 102 101 95 105 105 109 102 99 105

108 111 109 118 119 129 117 118 113 112

116 102

–

111 101 105 103 98 90 79

119 95 94

Chloride (mEq/L) 94 –110

23.0 3.7 5.0 5.3 6.3 12.0 13.1 14.1 12.7 24.3 20.5

20.7 11.4 7.3 3.2 5.3 7.3 9.9 14.2 17.9 16.5

– 16.9/18.0†

17.7†

22.2 15.3 21.9 21.0 22.6 24.4 23.6

– 3.7† 3.0†

Bicarbonate (mEq/L) 17 –32

17 37 29 32 36 25 22 21 27 21 22

19.6 18.7 26.1 23.2 17.6 15.9 16.1 15.8 17.7 17.0

18 24

–

18 19 20 20 22 26 37

– 37 36

Anion Gap 6 –17

– 15.67 9.34 7.2 11.05 5.47 2.89 1.87 3.54 3.06 0.46

– – – 0‡ – – – 0‡ – –

– 0.19

–

– 0.28 0.18/0‡ 0.11 0 0.28 –

– – 0‡

Ketones BHY (mmol/L) 0– 3

– 7.02 7.13 7.17 7.21 7.29 7.37 – 7.33 – –

– – – 7.15 7.16 7.18 7.23 7.32 – –

– 7.34

7.04

– – – – – – –

– 6.94 6.9

pH 7.32 –7.43

Blood Gas

– 42 35 48 43 24 27 – 35 – –

– – – 21 21 26 25 49 – –

– 33

10

– – – – – – –

– 134 61

pO2 (mmHg) 25– 47

– 25.5 23.7 18.4 21.1 30.6 26.7 – 29.7 – –

– – – 44 48 38 47 22 – –

– 33.5

65.1

– – – – – – –

– 17 13

pCO2 (mmHg) 40– 50

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Pulmonary oedema Case 3

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Pre þ5 þ7

Sodium (mEq/L) 135 –145

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Case 2

Renal failure Case 1

Normal Values

Electrolytes

Creatinine (mg/dL) 0.4– 1.0

BUN (mg/dL) 7–28

Kidney tests

Table 4 Serum abnormalities observed in cases with severe adverse events

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Figure 3 Histopathology. (a) Case 2, kidney: diffuse severe renal tubular dropout with replacement by loose fine connective tissue with oedema. Glomeruli are relatively unaffected. Haematoxylin and eosin (HE), bar ¼ 500 mm. (b) Case 2, kidney: severe renal tubular dropout. The few remaining renal tubules are degenerative or necrotic (asterisk). The featured glomerulus is relatively unaffected. HE, bar ¼ 50 mm. (c) Control animal after STZ infusion, kidney: centrally there is a small area of mature fibrous connective tissue surrounding and embedding a low number of renal tubules (asterisk). HE, bar ¼ 50 mm. (d) Control animal after STZ infusion, kidney: segmental areas of renal tubules have vacuolated cytoplasm with reduced eosinophilic tinctorial properties (tubular degeneration). There are some small areas of tubular regeneration (asterisk). HE, bar ¼ 100 mm. (e) Normal macaque kidney: all renal tubules have a moderate amount of strongly eosinophilic cytoplasm with little or no vacuolation. HE, bar ¼ 100 mm. (f ) Case 3, lung: the medium calibre vessel has a marked perivascular oedematous expansion (asterisk). HE, bar ¼ 100 mm. (g) Case 3, lung: oedema fluid radiates from the medium calibre vessel expanding the perivascular space and infiltrating the alveolar spaces (asterisk). HE, bar ¼ 20 mm. (h) Normal macaque lung: alveolar septa are thin with adequate covering by type I pneumocytes. Note that the alveolar spaces are free of any eosinophilic homogenous material (oedema fluid). HE, bar ¼ 20 mm

chronic renal failure, until symptomatic uraemia on day 82 post-infusion prompted immediate euthanasia. Case 1 A 7.1-year-old obese cynomolgus female experienced acute renal failure and secondary metabolic acidosis. This animal was induced prior to the initiation of anthropometric measures. It is noteworthy that this case occurred prior to initiation of complete chemistry panels in 2009 (absent

HCO2 3 ). In this case we also used LRS instead of 0.9% NaCl, because LRS is considered a beneficial isotonic alternative to saline in cases of acute kidney injury due to drug toxicity.57 We have discontinued the use of LRS due to the intrinsic risk of lactic acidosis following STZ, because in this condition LRS is contraindicated. In this case it could be argued that 0.9% NaCl should have been used nonetheless, to correct the hyponatraemia and hypochloraemia. Historically, fluid management was less

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aggressive due to reports of post-infusion pulmonary oedema. However, underestimating fluids during renal decompensation, as is apparent in this case, can lead to rapid loss of kidney function. In the first 24 h after STZ infusion two episodes of vomiting were observed. On day 1 appetite was decreased, BG was normal (57 mg/dL) in the morning and slightly elevated in the afternoon, urine and stool were normal. On day 2 appetite and condition were scored normal; the animal developed hyperglycaemia (325 mg/dL) and sliding scale combination glargine and lispro insulin therapy was initiated in the afternoon and continued for the duration of the study (median 0.81 U/ kg/day). On the morning of day 5, the animal exhibited behavioural changes including lethargy and ataxia, was uncooperative, and had a core body temperature of 33.38C. Kidney parameters were abnormal, and venous blood gas showed severe metabolic acidosis (Table 4): WBC was 36.4109/L, Hb 12.6 g/dL and PLT 476109/L. The animal was immediately treated with 20 mL/kg warm LRS given intravenously and supplemented with 1.5 U regular insulin. Core body temperature improved to 378C within 2 h of rehydration, and the animal began taking oral fluid itself (approximately 60 mL) and selective eating. Supportive intravenous fluid, 20 mL/kg LRS, was repeated in the afternoon and a warming lamp was applied cageside. The animal’s temperature and BG stabilized (36.38C and 48– 112 mg/dL, respectively). The following day, BG continued to be stable, but appetite declined. On the early morning of day 7, Kussmaul breathing was observed along with mild ataxia, and core body temperature was 33.78C. The animal was cooperative for supportive fluid administration (20 mL/kg LRS solution intravenously) and blood collection. Kidney parameters had worsened, and venous blood gas showed a worsening metabolic acidosis. Urinalysis revealed protein 3þ, glucose 3þ, negative ketones, blood 3þ, WBC 21 –50 and RBC .100. The combined laboratory assessments suggested progressive and severe renal injury and the animal was euthanized. No necropsy was performed. Case 2 A 5.7-year-old obese cynomolgus male experienced progressive renal failure. Within 6 h after STZ infusion the animal became hyperglycaemic: two episodes of vomiting were observed during the first 48 h after infusion. On day 1, BG was normal in the early morning (71 mg/dL) and the animal’s appetite was excellent; the BG increased to 320 mg/dL in the afternoon. Combination lispro and glargine insulin therapy was initiated on day 2 and continued for the duration of the study (median 1.1 U/kg/day). The animal was in excellent clinical condition; however, laboratory abnormalities included raised CREAT and BUN, and low sodium (Table 3). Blood ketones and BHY were negative (0.28 mmol/L). Haematology values were in the normal range. The animal was treated with intravenous administration of 0.9% NaCl per protocol. The rise in CREAT and BUN was consistent with a score 2 injury to the kidney, and subsequently the animal was scheduled for additional per os fluids (500 mL 50:50 fruit juice:water daily) to supplement hydration and for follow-up

laboratory assessment at day 14 or as clinically indicated. Raised values of CREAT and BUN persisted throughout the follow-up indicative of chronic renal failure (Table 4). Haematology values were in the normal range. Urinalysis (day þ15) revealed protein 1þ, glucose 3þ, negative ketones, blood 1þ, WBC 0 and RBC 0– 5. The animal remained in excellent clinical condition despite the evident decline in renal function. It was decided to follow the course of the animal under conventional medical management (intravenous/per os fluid therapy along with continued exogenous insulin) as long as the clinical condition did not deteriorate, since it is possible to recover renal function (i.e. tubular regeneration) after a nephrotoxic drug insult given a sufficient recovery period although this is variable between individuals. Since STZ infusion the animal lost only 4% BW and scored on daily assessments normal with respect to attitude, appetite, urine, faeces and social interactions: this condition continued till day 82. During cooperative handling on the evening of that day, a 20 –30 s seizure occurred followed by 10 s in which the animal was ataxic. Three episodes were observed during a 30 min observation period. In between these episodes the animal was bright, alert, responsive, cooperative with handlers and was eating normally. Considering the history of chronic renal failure, the animal was euthanized. Necropsy examination revealed tubular dropout within both kidneys, with a diffusely pale reddish to brown renal cortex and medulla. Histology showed replacement of .80% of the cortex by a loose fine myxoid to fibrous material which contained low numbers of lymphocytes and plasma cells (Figures 3a and b). A few regions in the cortex showed minimal evidence of tubular regeneration. Glomeruli were relatively unaffected. The severe loss of both proximal and distal convoluted tubules was consistent with the clinical chemistry findings of renal failure. This case is representative of severe diffuse tubular dropout which was most likely the consequence of the sudden exposure to STZ: there was insufficient recovery due to the lack of functional renal mass remaining, hence the presence of oedema and what appeared to be a few remaining basement membranes (Figure 3b). However the glomeruli were not part of the process and were either not susceptible or much less susceptible to STZ, they were relatively unaffected. For comparison, the ‘typical’ post-STZ kidney features featured by other animals are presented in Figures 3c and d and a normal kidney in Figure 3e. Figure 3c demonstrates the extent to which the kidney can be affected. The small focus of fibrous connective tissue is indicative of an insult that occurred a fair time earlier, hence had the time to heal (with the generation of fibrous connective tissue). This is in contrast to the present animal in Figures 3a and b regarding the remaining functional renal mass. This example in Figure 3c has abundant functional renal mass (i.e. greater than 25% of total renal mass that is minimally required to sustain physiological normality). With respect to the tubules, the reparative response of the kidney is relatively limited and can essentially be one of two things, either replacement by fibrous connective tissue or regeneration of tubules. The pattern of connective tissue is often segmental (i.e. running from cortex to medulla) because


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the insult was either blood-borne or was due to a lack of blood and due to the vascular supply of the kidney. Figure 3d presents a typical post-STZ kidney featuring regeneration of tubules. In this case there is some effect on the tubules, but this is not sufficient to give a clinical response. The tubules show a mild segmental tubular degeneration (see figure legend for description); however, in this case there has been a sufficient period for some isolated pockets of tubular regeneration. It is noteworthy that there was sufficient renal mass remaining to sustain normal physiological function to allow an essentially normal clinical picture with concurrent renal degeneration/regeneration. The insult was not that severe (i.e. retaining the basement membrane scaffold) that regeneration is not possible, otherwise there would have been a fibrous connective tissue replacement (rather than regeneration). Figure 3e presents a normal kidney which has all the regular features: the tubular epithelial cells have sufficient eosinophilic cytoplasm and basally located nuclei, and are arranged in a regular manner with no debris in the lumina. The glomeruli are regularly arranged and separated (it is noteworthy that in the case of tubular dropout they come much closer and more irregularly together). In necropsy investigation of the present case, the brain did not reveal any abnormalities. Hence, the central nervous system signs were attributed to accumulation of toxic metabolites during renal decompensation – uraemic encephalopathy (BUN, CREAT and other toxins normally eliminated via the renal route). Acute pulmonary oedema An acute pulmonary reaction to chemotherapy, termed ‘chemotherapy lung’, is a known complication for agents in the nitrosourea class like STZ.58 The histological appearances include interstitial oedema or fibrinous organizing pneumonia in early cases, and bland oedema with proteinaceous fluid filling most alveolar spaces in severe cases. On the other hand, renal impairment as a causative factor should also be considered, as disturbances in the fluid electrolyte balance can promote oedema. Two cases of pulmonary oedema in animals with documented obesity are presented (Tables 3 and 4, Figure 3). In both cases pulmonary oedema was apparent within 6 h after STZ injection, and this was associated with a varying degree of respiratory distress. The first animal developed severe respiratory distress that was not responsive to loop diuretic treatment, and necessitated prompt euthanasia. The second animal responded to conventional medical treatment that included loop diuretics and supplemental oxygen, and recovered fully. Case 3 A 5.2-year-old obese rhesus male developed severe pulmonary oedema. Within 2 h after STZ infusion the animal became hyperglycaemic (BG 338 mg/dL): no vomiting was observed and the animal was eating well. Approximately 4 h after infusion the animal exhibited severe respiratory distress and was cyanotic (Table 4); BG was 509 mg/dL. A life-threatening condition developed in less than 15 min. Over the course of 30 min three doses of 2 mg/kg

intravenous furosemide were administered. The clinical condition continued to deteriorate and the animal was promptly euthanized. More than 40 mL of blood was found in the thorax; all lung lobes were mottled pink to red, wet and heavy, and cut surface oozed with a whitish foam. Histologically, the perivascular adventitia of most medium and large calibre vessels ( predominantly arteries) were markedly expanded by a pale eosinophilic homogenous to fine fibrillar, occasionally beaded, material representing fibrin and serum proteins (Figures 3f and g; for comparison a normal lung is shown in Figure 3h). Alveolar spaces frequently contained homogenous eosinophilic material ( protein-rich oedema fluid). The microscopic appearance of the lungs confirmed the clinical sign of hypoxia due to severe perivascular pulmonary oedema which most likely reflects an idiosyncratic drug reaction. Interestingly, Figures 3f and g show essentially a selective failure of the vascular endothelium allowing protein and fluid leakage from the vessels. This is in contrast to the observations in the kidney where the glomeruli which are comprised of vascular endothelium are relatively unaffected: this difference points to a selective mechanism of the pulmonary vasculature. In this animal acute severe perivascular oedema was also observed in the brain. The only other abnormality observed was centrilobular hepatocellular vacuolar degeneration in the liver, which could be attributed to the obese state of the animal. Case 4 A 6.0-year-old obese rhesus male developed pulmonary oedema. Within 6 h after STZ infusion the animal became hyperglycaemic (BG 319 mg/dL): no vomiting was observed and the animal was eating well. The animal was dehydrated as assessed by skin turgor. Laboratory investigations revealed hypernatraemia and hyperchloraemia early after infusion confirming dehydration. The animal was rehydrated intravenously with 30 mL/kg NaCl 0.9%. Approximately one hour after rehydration the animal manifested laboured breathing: furosemide was administered, first 2 mg/kg intramuscularly, and one hour later 6 mg/kg intravenously. Laboured breathing worsened in the next hour and two additional intravenous doses of 2 mg/kg furosemide were given at 30 min intervals. The animal’s condition continued to deteriorate: the restless behaviour, inconsistency in working with handlers, in combination with dyspnoea and poor oxygen saturation (dropping to 88%) were most consistent with pulmonary oedema. An ABO-matched blood transfusion (65 mL) was given to correct the oxygen-carrying capacity. The condition then improved, the animal started selective eating, and cooperation with handlers was improved. Follow-up laboratory investigations revealed a recovery in electrolytes, HCO2 3 16.9 mEq/L and BG 537 mg/dL. Blood ketones and BHY were negative (0.19 mmol/L). The reduction in HCO2 3 in combination with venous blood gas values confirmed mild acidosis. One day later, day 1, the animal’s attitude and activity had returned to normal; however laboured breathing was still observed and oxygen saturation had dropped to 80%. The animal was placed in a modified transport box

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that was equipped with high flow supplemental oxygen for 45 min. One hour later the oxygen saturation was 86%, so the animal was returned to this box for another 30 min. The laboured breathing resolved during this session and the oxygen saturation improved to 98%. At day 7 the animal’s clinical pathology had normalized, and the animal was successfully enrolled in an experimental study protocol approximately two weeks later. Metabolic acidosis STZ is strongly linked to metabolic acidosis through a number of mechanisms.59 We have developed procedures to aid in differential diagnosis (Figure 1) and also support measures for treating metabolic acidosis and fluid therapy in acute renal injury (Figure 2). STZ is known to cause type B lactic acidosis60 which can occur in the presence of liver or kidney disease, or DKA. Two case reports of metabolic acidosis in animals with documented obesity are presented (Tables 3 and 4). One animal was the only one in this series that received an adjusted STZ dose. In both cases severe metabolic acidosis was apparent within seven days after STZ injection, and this was characterized by clinical symptoms ( poor appetite, decreased activity) and a significant base deficit. The first animal developed severe metabolic acidosis while maintaining acceptable kidney function. The second animal developed severe DKA followed by acute renal failure that was reversible. Case 5 A 6.6-year-old obese cynomolgus female developed metabolic acidosis. After STZ infusion appetite was decreased in the evening, BG was slightly elevated (130 mg/dL), and urine and stool were normal. On day 1, appetite and condition were scored normal and the BG remained normal (40 mg/dL morning and 42 mg/dL afternoon). On day 2, appetite and condition were scored normal, the animal’s BG was elevated in the morning and afternoon (222 mg/ dL and 224 mg/dL, respectively) but failed to reach the standard .300 mg/dL threshold used to initiate insulin therapy. On day 3, appetite and condition were scored normal: a sliding scale glargine and lispro insulin combination therapy was initiated (median 1.3 U/kg/day). On day 4, routine laboratory values revealed a slight increase in CREAT and an already abnormal bicarbonate level (Table 4); haematology values were in the normal range. On day 6, the animal’s appetite in the morning was poor and vomiting was observed in the afternoon. The animal was otherwise normal and cooperative: laboratory assessment revealed metabolic acidosis peaking in severity on day 7 and recovering at around day 14 after intensive medical management (Table 4). This included fluids, bicarbonate and insulin infusion per protocol immediately after the diagnosis of metabolic acidosis was evident. Clinical observations manifested abnormalities starting day 6 in the morning: the animal was active, alert and cooperative but had poor appetite followed by vomiting in the afternoon. While no additional vomiting occurred, the clinical status was stable with poor appetite until day 9 in the afternoon when huddling was observed, at which point the

animal was additionally treated with analgesic (ketoprofen 1 mg/kg intramuscularly). On day 10, both the animal’s clinical condition and appetite had improved to normal and continued to improve to baseline on day 14. The animal was successfully enrolled in an experimental transplantation protocol approximately two months later. Case 6 A 7.2-year-old obese cynomolgus female developed severe DKA. Within 4 h after STZ infusion the animal became hyperglycaemic (BG 301 mg/dL); no vomiting was observed and the animal was eating well. Sliding scale combination glargine and lispro insulin therapy was initiated in the afternoon on day 1 and continued for the duration of the study (median 2.76 U/kg/day). On the morning of day 5, the animal exhibited behavioural changes, lethargy, coordination problems and dehydration as assessed by skin turgor. Appetite was decreased, BG was moderately elevated: also kidney markers were increased and there was hyponatraemia (Table 4). Urine and stool were normal, and core body temperature was 35.68C. WBC was 24.08 109/L, Hb 10.4 g/dL and PLT 586109/L. Blood ketones measured by BHY were critically high (15.7 mmol/L). The laboratory values revealed severe DKA with peak severity on day 5 (Table 4). Medical management starting that day included treatment per protocol with fluids, bicarbonate, potassium and insulin infusion that continued to day 12. In addition, on day 7 the animal received an ABO-matched blood transfusion (100 mL) to correct for anaemia (Hb 7.8 g/dL). Clinical observations consistent with DKA manifested from day 5 in the morning, and included poor appetite and decreased activity. The animal’s status was stable from day 5 to day 7, but did not sufficiently improve. On day þ8, after the blood transfusion on day 7, the condition markedly improved with respect to attitude, appearance and activity level, but appetite remained selective. On day 12 slight swelling of the abdomen was observed indicative of ascites; this suspicion was also based on relative poor nutrition during the prior seven days, resulting in low albumin (1.9 g/dL) in combination with fluid loading for DKA treatment. Day 12 marked the first day in which appetite returned to normal, and except for an unkempt haircoat the animal was clinically normal. On day 16, the animal’s clinical condition had improved to baseline. Laboratory values returned to an acceptable range on day 21; however, evidence of ketones persisted till day 30. The animal was successfully enrolled in a transplant protocol in the same year.

Discussion Considering the widespread use of STZ in NHPs, it makes sense to outline strategies for prevention and reduction of STZ (chemotherapy)-induced AEs, in accordance with the concern for the wellbeing of animals enrolled in such trials. The strategies presented are based on our long and broad experience in a large cohort of cynomolgus and rhesus macaques. Dose adaptation to compensate for individual animal’s attributes (e.g. species, age and body condition) and supportive measures in case of clinical manifestations are the main tools for toxicity control to


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avoid morbidity and mortality. Since a toxic AE can present in an abrupt way requiring quick intervention, we found it useful to implement protocols for detection, scoring and treatment of the primary AE: this regards primarily nausea and emesis, nephrotoxicity, metabolic acidosis, elevated liver transaminase levels and pulmonary oedema. Such protocols have to be taken with some flexibility because the incidence and gravity of AEs are to some extent related to the individual’s characteristics (e.g. age and obesity), diagnostic tools, prompt recognition of symptoms and supportive measures. Taking into consideration that intensive clinical care relies extensively on reliable vascular access and cooperation of the animal with complicated medical procedures, we recommend that NHPs undergo appropriate training and instrumentation prior to STZ administration.21 In our experience this is a very relevant aspect in prevention and treatment, in particular because it enables early detection of clinical AE in the absence of confounding characteristics of restraint stress, and also because it avoids the need for sedation in an already compromised animal. Severe metabolic alterations have been reported in animals subjected to physical restraint, even if applied for a time period of less than 5 min.61 Healthy monkeys demonstrated the ability to recover rapidly. Metabolic stress resulting from restraint negatively affects recovery efficiency in clinically compromised animals, adds burden and can be lethal in debilitated animals especially in case of a hepatic or renal disorder. The case studies presented serve to illustrate this phenomenon: both early detection and treatment under conditions of low stress not only add to the general wellbeing but also avoid animals having to be taken off the study and subjected to euthanasia, and can enrol in studies after full recovery. Furthermore, it is essential to identify animals with risk factors for STZ induction so that they might be excluded or appropriate adjustments made. Without dose adjustment, at-risk animals are more likely to experience high-grade AE that are unlikely to recover under even the best intense medical management (i.e. irreversible organ failure). We conclude that a combination of approaches enables prevention, early detection and subsequent management of AE caused by STZ in NHPs, and recommend these for experimental studies in NHP diabetes models. This not only improves the wellbeing of an individual animal by a reduction in morbidity and mortality but also has a broader perspective, namely avoidance of model induced confounding that diminishes the translational value of experimental studies in diabetic animals. REFERENCES 1 Kemp CB, Knight MJ, Scharp DW, Lacy PE, Ballinger WF. Transplantation of isolated pancreatic islets into the portal vein of diabetic rats. Nature 1973;244:447 2 Scharp DW, Murphy JJ, Newton WT, Ballinger WF, Lacy PE. Transplantation of islets of Langerhans in diabetic rhesus monkeys. Surgery 1975;77:100 –5 3 Von Herrath M, Nepom GT. Animal models of human type 1 diabetes. Nat Immunol 2009;10:129 –32 4 DAIT, NIAID, NIH. Purified human pancreatic islets, in vivo islets function. Document No. 3104, A04, effective date 7 July 2008. See http://

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www.isletstudy.org/CITDocs/3104,%20A04%20In%20Vivo% 20Islets%20Function.pdf (last checked 12 June 2011) Howard CF Jr. Nonhuman primates as models for the study of human diabetes mellitus. Diabetes 1982;31(Suppl. 1, Part 2):37 –42 Schuurman HJ. Xenotransplantation. Drug Discov Today Dis Models 2008;5:81 –7 Hering BJ, Wijkstrom M, Graham ML, et al. Prolonged diabetes reversal after intraportal xenotransplantation of wild-type porcine islets in immunosuppressed nonhuman primates. Nat Med 2006;12:301 –3 Rood PP, Buhler LH, Bottino R, Trucco M, Cooper DK. Pig-to-nonhuman primate islet xenotransplantation: a review of current problems. Cell Transplant 2006;15:89– 104 Graham ML, Mutch LA, Rieke EF, et al. Refining the high-dose streptozotocin-induced diabetic nonhuman primate model: an evaluation of risk factors and outcomes. Exp Biol Med 2011;236:1218 – 30 Ericzon BG, Wijnen RM, Kubota K, vd Bogaard A, Kootstra G. Diabetes induction and pancreatic transplantation in the cynomolgus monkey: methodological considerations. Transpl Int 1991;4:103 –9 Yeo CJ, Cameron JL, Sohn TA, et al. Six hundred fifty consecutive pancreaticoduodenectomies in the 1990s: pathology, complications, and outcomes. Ann Surg 1997;226:248 – 57; Discussion 257– 60 Schacht RG, Feiner HD, Gallo GR, Lieberman A, Baldwin DS. Nephrotoxicity of nitrosoureas. Cancer 1981;48:1328 –34 Schein PS, O’Connell MJ, Blom J, et al. Clinical antitumor activity and toxicity of streptozotocin (NSC-85998). Cancer 1974;34:993 –1000 King PD, Perry MC. Hepatotoxicity of chemotherapy. Oncologist 2001;6:162 –76 Theriault BR, Thistlethwaite JR Jr, Levisetti MG, et al. Induction, maintenance, and reversal of streptozotocin-induced insulin-dependent diabetes mellitus in the juvenile cynomolgus monkey (Macaca fascicularis). Transplantation 1999;68:331 – 7 Litwak KN, Cefalu WT, Wagner JD. Streptozotocin-induced diabetes mellitus in cynomolgus monkeys: changes in carbohydrate metabolism, skin glycation, and pancreatic islets. Lab Anim Sci 1998;48:172– 8 Jones CW, Reynolds WA, Hoganson GE. Streptozotocin diabetes in the monkey: plasma levels of glucose, insulin, glucagon, and somatostatin, with corresponding morphometric analysis of islet endocrine cells. Diabetes 1980;29:536– 46 Pitkin RM, Reynolds WA. Diabetogenic effects of streptozotocin in rhesus monkeys. Diabetes 1970;19:85 – 90 Skinner R. Strategies to prevent nephrotoxicity of anticancer drugs. Curr Opin Oncol 1995;7:310 –15 Rood PP, Bottino R, Balamurugan AN, et al. Induction of diabetes in cynomolgus monkeys with high-dose streptozotocin: adverse effects and early responses. Pancreas 2006;33:287– 92 Graham ML, Rieke EF, Mutch LA, et al. Successful implementation of cooperative handling eliminates the need for restraint in a complex nonhuman primate disease model. J Med Primatol 2011 [Epub ahead of print] Graham ML, Rieke EF, Wijkstrom M, et al. Risk factors associated with surgical site infection and the development of short-term complications in macaques undergoing indwelling vascular access port placement. J Med Primatol 2008;37:202 –9 Graham ML, Rieke EF, Dunning M, et al. A novel alternative placement site and technique for totally implantable vascular access ports in non-human primates. J Med Primatol 2009;38:204 – 12 Graham ML, Mutch LA, Rieke EF, et al. Refinement of vascular access port placement in nonhuman primates: complication rates and outcomes. Comp Med 2010;60:479 – 85 Henriksen MG, Hessov I, Dela F, Hansen HV, Haraldsted V, Rodt SA. Effects of preoperative oral carbohydrates and peptides on postoperative endocrine response, mobilization, nutrition and muscle function in abdominal surgery. Acta Anaesthesiol Scand 2003;47:191 – 9 Smith AF, Vallance H, Slater RM. Shorter preoperative fluid fasts reduce postoperative emesis. BMJ 1997;314:1486 Hausel J, Nygren J, Thorell A, Lagerkranser M, Ljungqvist O. Randomized clinical trial of the effects of oral preoperative carbohydrates on postoperative nausea and vomiting after laparoscopic cholecystectomy. Br J Surg 2005;92:415 –21 Nygren J, Thorell A, Ljungqvist O. Preoperative oral carbohydrate nutrition: an update. Curr Opin Clin Nutr Metab Care 2001;4:255 – 9

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29 Klemetti S, Kinnunen I, Suominen T, et al. The effect of preoperative fasting on postoperative pain, nausea and vomiting in pediatric ambulatory tonsillectomy. Int J Pediatr Otorhinolaryngol 2009;73:263 –73 30 Clingerman KJ, Summers L. Development of a body condition scoring system for nonhuman primates using Macaca mulatta as a model. Lab Anim 2005;34:31 –6 31 Jen KL, Hansen BC, Metzger BL. Adiposity, anthropometric measures, and plasma insulin levels of rhesus monkeys. Int J Obes 1985;9:213 – 24 32 Shibata S, Kirchhof N, Matsumoto S, et al. High-dose streptozotocin for diabetes induction in adult rhesus monkeys. Transplant Proc 2002;34:1341 – 4 33 Koulmanda M, Qipo A, Chebrolu S, O’Neil J, Auchincloss H, Smith RN. The effect of low versus high dose of streptozotocin in cynomolgus monkeys (Macaca fascicularis). Am J Transplant 2003;3:267 – 72 34 Junod A, Lambert AE, Stauffacher W, Renold AE. Diabetogenic action of streptozotocin: relationship of dose to metabolic response. J Clin Invest 1969;48:2129 – 39 35 Wijkstrom M, Kirchhof N, Reynolds N, Gruessner AC, Hering BJ. Dosing streptozotocin based on body surface area is superior to dosing based on body weight. Cell Transplant 2003;12:195 36 Cooper DK, Casu A. The International Xenotransplantation Association consensus statement on conditions for undertaking clinical trials of porcine islet products in type 1 diabetes – chapter 4: pre-clinical efficacy and complication data required to justify a clinical trial. Xenotransplantation 2009;16:229 – 38 37 Veterinary Co-operative Oncology Group (VCOG). Veterinary Co-operative Oncology Group – Common Terminology Criteria for Adverse Events (VCOG-CTCAE) following chemotherapy or biological antineoplastic therapy in dogs and cats v1.0. Vet Comp Oncol 2004;2:195 –213 38 Shankar V, Haque A, Churchwell KB, Russell W. Insulin glargine supplementation during early management phase of diabetic ketoacidosis in children. Intensive Care Med 2007;33:1173 – 8 39 Kerl ME. Diabetic ketoacidosis: treatment recommendations. Compend Contin Educ Pract Vet 2001;23:330 –9 40 Chastain CB, Nichols CE. Low-dose intramuscular insulin therapy for diabetic ketoacidosis in dogs. J Am Vet Med Assoc 1981;178:561 – 4 41 Feldman EC, Nelson RW. Diabetic ketoacidosis. In: Feldman EC, Nelson RW, eds. Canine and Feline Endocrinology and Reproduction. 2nd edn. Philadelphia: WB Saunders, 1996:392 –421 42 Greco DS. Pancreatic disorders: diabetic ketoacidosis and insulinoma – treatment of DKA. In: Wingfield WE, Raffe MC, eds. The Veterinary ICU Book. Jackson, WY, USA: Teton NewMedia, 2002:846– 8 43 Kitabchi AE, Umpierrez GE, Murphy MB, et al. Management of hyperglycemic crises in patients with diabetes. Diabetes Care 2001;24:131 –53 44 Rewers A, McFann K, Chase HP. Bedside monitoring of blood beta-hydroxybutyrate levels in the management of diabetic ketoacidosis in children. Diabetes Technol Ther 2006;8:671 –6 45 Laffel L. Ketone bodies: a review of physiology, pathophysiology and application of monitoring to diabetes. Diabetes Metab Res Rev 1999;15:412 –26

46 Burge MR, Hardy KJ, Schade DS. Short-term fasting is a mechanism for the development of euglycemic ketoacidosis during periods of insulin deficiency. J Clin Endocrinol Metab 1993;76:1192 – 8 47 Macintire DK. Emergency therapy of diabetic crises: insulin overdose, diabetic ketoacidosis, and hyperosmolar coma. Vet Clin North Am Small Anim Pract 1995;25:639 – 50 48 Skellett S, Mayer A, Durward A, Tibby SM, Murdoch IA. Chasing the base deficit: hyperchloraemic acidosis following 0.9% saline fluid resuscitation. Arch Dis Child 2000;83:514 –16 49 Umpierrez GE, Latif K, Stoever J, et al. Efficacy of subcutaneous insulin lispro versus continuous intravenous regular insulin for the treatment of patients with diabetic ketoacidosis. Am J Med 2004;117:291 –6 50 Della Manna T, Steinmetz L, Campos PR, et al. Subcutaneous use of a fast-acting insulin analog: an alternative treatment for pediatric patients with diabetic ketoacidosis. Diabetes Care 2005;28:1856 – 61 51 O’Brien MA. Diabetic emergencies in small animals. Vet Clin North Am Small Anim Pract 2010;40:317 – 33 52 Isnard-Bagnais C, Deray G. Anticancer Drugs. Clinical Nephrotoxins: Renal Injury from Drugs and Chemicals. 2nd edn. Dordrecht: Kluwer Academic Press, 2003:360 53 Hruska KA, Levi M, Slatopolsky E. Disorders of phosphorus, calcium, and magnesium metabolism. In: Schrier RW, ed. Diseases of the Kidney and Urinary Tract. 8th edn. Philadelphia: Wolters Kluwer Health/ Lippincott Williams & Wilkins, 2007:2311 54 Adams LG, Hardy RM, Weiss DJ, Bartges JW. Hypophosphatemia and hemolytic anemia associated with diabetes mellitus and hepatic lipidosis in cats. J Vet Intern Med 1993;7:266 –71 55 McBryde KD, Bunchman TE. Continuous renal replacement therapy. In: Wheeler DS, Wong HR, Shanley TP, eds. Pediatric Critical Care Medicine: Basic Science and Clinical Evidence. London: Springer, 2007:1235 56 Safirstein RL. Renal diseases induced by antineoplastic agents. In: Schrier RW, ed. Diseases of the Kidney and Urinary Tract. 8th edn. Philadelphia: Wolters Kluwer Health/Lippincott Williams & Wilkins, 2007:1069 –73 57 Merck Sharp & Dohme Corp. Acute Kidney Disease. The Merck Veterinary Manual. Whitehouse Station, NJ, USA: Merck Sharp & Dohme Corp, 2011 58 Carnus P. Drug-induced and iatrogenic infiltrative lung disease. In: Costabel U, duBois RM, Egan JJ, eds. Diffuse Parenchymal Lung Disease. Basel: Karger, 2007:222 59 Richardson RMA, Halperin ML. Metabolic acidosis. In: Suki WN, Massry SG, eds. Therapy of Renal Diseases and Related Disorders. 2nd edn. Norwell: Kluwer Academic Publishers, 1991:177 60 Shahab I, Patterson WP. Renal and electrolyte abnormalities due to chemotherapy. In: Perry MC, ed. The Chemotherapy Source Book. 4th edn. Philadelphia: Wolters Kluwer Health/Lippincott Williams & Wilkins, 2008:223– 33 61 Bush M, Custer R, Smeller J, Bush LM. Physiological measures of nonhuman primates during physical restraint and chemical immobilization. J Am Vet Med Assoc 1977;171:866 – 9

(Accepted 10 January 2012)