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2.3 Definiciones Conceptuales
Single dose studies were both conducted using itraconazole dosages of 3 mg/kg, while multiple dose studies were both conducted using dose rates of 2.5 mg/kg. This allowed direct comparison of the effect of temperature on pharmacokinetic parameters. Results
181 show that these dosages were excessive at both temperatures, and resulted in high itraconazole plasma concentrations and significant adverse effects.
Time to maximal plasma concentration
In single dose studies at 12oC, time to maximal plasma concentration was not able to be
estimated, as most animals were not in the itraconazole terminal elimination phase at 48 hours. At 20 oC the Tmax was estimated to be between 4-8 hours. While it was not
appropriate to make statistical comparisons because of the lack of definitive data at 12
oC, it is apparent there is a marked difference in Tmax at the two temperatures using
the 3 mg/kg dose rate. The Tmax of itraconazole in reptiles has not been reported previously, but in humans was 5.7 hours (Willems et al. 2001), and in birds was 4 hours in domestic pigeons, 3.7 hours in black-footed penguins, and varied between 3.7-6.9 hours in Amazon parrots (Lumeij et al. 1995, Orosz et al. 1996, Smith et al. 2010). These studies administered itraconazole of various formulations, at different dose rates to this study, with some animals fed and others fasted, making direct comparisons inappropriate.
Absorption half-life
The absorption half-life of itraconazole in tuatara was estimated at 5.30 hours based on modelling analysis. This is slow absorption, and indicates that 50% of the administered oral dose is absorbed in 5.3 hours. Absorption is considered to be a largely passive process, and not markedly influenced by temperature, so the estimate is the same for both 12oC and 20oC.
182
Elimination half-life
In the multiple dose studies itraconazole had a mean ± SD elimination half-life of 22.0 ± 9.4 hours at 12 oC, and 7.65 ± 2.2 hours at 20 oC. This difference was statistically
significant (p=0.0083), indicating that half life was significantly lower at 20oC than 12oC.
Modelling indicated that elimination kinetics were first-order (non-saturable), as observed in other species.
Clearance
Pharmacokinetic modelling predicted clearance at 12 oC of 0.010L/h, and at 20 oC
0.0037L/h for a 1kg animal with a target itraconazole concentration of 2.4 mg/L (Tables 4.9 and 4.14). The clearance differs between temperatures by a factor of 2.86, which is very similar to the observed proportional difference in mean elimination half-lives at both temperatures (Tables 4.8 and 4.13) of 2.88.
Modelling and simulation to estimate itraconazole dosage regimens in tuatara
The observed itraconazole concentrations matched reasonably well with those predicted by modelling, indicating that itraconazole has predictable pharmacokinetics, and there can be a reasonable level of confidence in the modelling predictions.
It is apparent from these data administering the same dosage of medication at different temperatures produces markedly different plasma itraconazole concentrations. This indicates that ambient temperature control is an essential part of a hospitalisation and treatment protocol using itraconazole in tuatara. Simulations show that at 12oC,
itraconazole is not predicted to reach steady-state concentrations by the end of simulations at 70 days, as it continues to accumulate. This makes 12oC an inappropriate
183 would not be reached in an appropriate timeframe, and accumulation of the drug means adverse effects become more likely as treatment progresses. Therefore it is recommended animals are treated at 20oC, the higher end of their POTZ.
4.4.2 Adverse effects
Adverse effects were noted in both the 12oC and 20oC multiple dose studies, with
elevated bile acids being the most common problem. Bile acids are produced by the liver to aid digestion, and are considered a specific test for hepatic insufficiency (Campbell 2014). In a study in healthy green iguanas (Iguana iguana), pre-prandial bile acids were 7.5 ± 7.8 µmol/L, and post-prandial bile acids were 33.3 ± 22.0 µmol/L (McBride et al. 2006). At the time of our study there were no reference ranges for bile acids in tuatara, and healthy tuatara at Auckland Zoo had previously had bile acids ranging from <35 (the lower limit of detection of the analyser) to 41 µmol/L. Gastric emptying time in tuatara is unknown, and animals in the study were able to feed at any time of day, so no distinction between pre- and post-prandial bile acids concentrations could be made during the study. At the time of the study bile acids concentrations lower than 40 µmol/L were considered normal, between 40-60 µmol/L marginal, and above 60 µmol/L elevated, based on clinical experience and extrapolation from other reptile species.
At 12oC, elevations in bile acids (64 µmol/L) were noted in one animal on day 13 (subject
104, male), this was associated with a plasma itraconazole concentration of 15.8 mg/L, and a hydroxy-itraconazole concentration of 3.76 mg/L. On day 20 subject 105 (female), was found to have elevated bile acids (100 µmol/L); this was associated with an itraconazole plasma concentration of 13.3 mg/L, and a hydroxy-itraconazole concentration of 2.43 mg/L. On day 22 another animal (subject 112, male) was found to
184 have elevated bile acids (>200 µmol/L), the closest itraconazole measurement in this animal was 18.7 mg/L on day 20 (the last drug dose was administered on day 21), and the hydroxy-itraconazole concentration was 3.14 mg/L. Other tuatara had plasma itraconazole concentrations ranging from 10.9-22.9 mg/L and hydroxy-itraconazole concentrations of 1.86-4.76 mg/mL on day 20, and did not show elevations in bile acids.
Subject 104’s bile acids normalised to <35 µmol/L on day 22, despite itraconazole
treatment only ceasing on day 21, raising the possibility that the observed elevation on day 13 was not directly related to antifungal therapy.
In the 20oC multiple dose study two animals (subjects 107 – female, and 112 – male) had
elevations in bile acids on day 13, with corresponding itraconazole concentrations of 4.893 mg/L and 6.606 mg/L, and hydroxy-itraconazole concentrations of 3.651 and 2.748 mg/L respectively. Subject 112 had persistently elevated but decreasing bile acids until day 41. One tuatara (subject 104) had marginal bile acids of 50 µmol/L on day 13, and went on to develop high bile acids of 80 µmol/L on day 20. Unaffected tuatara had itraconazole concentrations between 4.921-10.621 mg/L, and hydroxy-itraconazole concentrations of 3.53-6.129 mg/L.
Elevations in bile acids occurred earlier in the 20oC study, despite lower plasma
itraconazole concentrations at the time compared to the 12oC study (4.893-6.606 mg/L
versus 13.3-18.7 mg/L respectively). Hydroxy-itraconazole concentrations were similar in both studies when adverse effects were noted (2.748-3.651 mg/L and 2.43-3.14 mg/L). It is possible that hydroxy-itraconazole, or a different metabolite of itraconazole, is responsible for the increased bile acids (rather than the parent compound), as hydroxy-itraconazole attained higher concentrations more rapidly in the 20oC study. It
185 is also possible that, despite apparently normal bile acids concentrations on health screening prior to the 20oC study, that the 12oC study had caused subclinical liver
damage, making the liver more sensitive to itraconazole in the 20oC study.
In humans and rats, itraconazole has been noted to cause dose-dependent hepatocellular necrosis, bile duct hyperplasia, cholestasis and biliary cirrhosis, with elevation in liver enzyme activity and bilirubin (Talwalkar et al. 1999, Somchit et al. 2004, Lou et al. 2011). In one human case report, symptoms and biochemistry abnormalities including elevated bilirubin and liver enzymes continued to worsen after the cessation of itraconazole treatment, ultimately resolving four months after stopping itraconazole (Talwalkar et al. 1999). In bearded dragons administered itraconazole 5 mg/kg PO SID, five out of seven bearded dragons died, with elevations in AST reported in four of these animals (van Waeyenberghe et al. 2010); no histologic abnormalities were found in the liver on post-mortem. Bile acids were not reported in this study, and neither was CK (muscle damage can raise both CK and AST).
Uric acid concentrations were also increased in several animals in both the 12oC and
20oC studies. Uric acid is the primary product of protein catabolism in tuatara (Hill and
Dawbin 1969), and is produced by the liver and excreted via the kidneys. Uric acid can be elevated in renal disease and in dehydrated animals. Prior to this study there were no published normal concentrations of uric acid in tuatara, however our subsequent analyses showed concentrations of 42-232 µmol/L were detected in clinically healthy, well hydrated animals (Chapter 6). During the multiple dose studies, uric acid concentrations between 40-250 µmol/L were considered normal, 250-350 µmol/L
186 marginal, and above 350 µmol/L abnormal, based on clinical experience at Auckland Zoo.
In the 12oC study, one animal (subject 104) had marginally increased uric acid
concentrations on day 13, of 292 µmol/L, and progressed to more dramatic elevations on days 22 and 28, with concentrations of 436 µmol/L and 540 µmol/L, respectively. This animal also lost weight during this period, which was regained within two days of returning him to his normal enclosure, indicating that dehydration was likely the cause of both weight loss and the uric acid elevation. Tuatara obtain most of their water from dietary sources (Cree 2014), and it is possible that itraconazole therapy resulted in nausea and inappetence, as this is a commonly reported adverse effect in humans (Manire et al. 2003). It is also possible the study environment and regular handling may have contributed to altered behaviour and dietary intake, however this was not observed in voriconazole-treated animals (Chapter 5). Subject 101 also showed elevated uric acid (486 µmol/L) at the same time when marginal bile acids (52 µmol/L) were noted, on day 22. These had both resolved at the next sampling point on day 28.
In the 20oC studies subject 101 had marginal uric acid (341 µmol/L) on day 13 (the last
day of medication administration), which had normalised by day 20. Subject 104 had elevated uric acid (427 µmol/L) on day 20, which had normalised on day 27.
Animals with elevated bile acids did not always have concurrently elevated uric acid and vice versa.
White cell counts were elevated in two animals (subjects 104 and 112, both male) in the 12oC study. White blood cells are part of the immune system, and elevations commonly
187 indicate inflammation. Both tuatara had elevations in heterophils and monocytes, indicative of granulocytic inflammation. These elevations were first noted on day 22, and persisted through day 41, but had resolved by day 55. During the 20oC study white
cell numbers remained normal for all animals, however animal 105 had one instance of reactive monocytes on day 27 which were not observed again.
Overall, subjects 104 and 112 suffered the most severe adverse effects throughout both studies, as evidenced variously by elevated white cells, bile acids and uric acid concentrations. These animals had the highest plasma itraconazole concentrations and were the heaviest of the group, meaning that on an allometrically-scaled basis, these animals received more itraconazole (despite the same mg/kg dosage) than their smaller counterparts. It is possible that this higher dosage is responsible for the increased incidence of adverse effects, however sex-specific differences in itraconazole metabolism cannot be ruled out without further studies involving more animals of varying weights.
It appears that in tuatara, itraconazole toxicity manifests primarily as presumed cholestasic disease characterised by elevations in bile acids and, in more severe cases, white blood cells. Uric acid elevations, likely related to dehydration, were also present in several cases.