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Pharmacokinetic disposition of the oral iron chelator deferiprone in the white leghorn chicken.

Abstract: Deferiprone is a bidentate oral iron chelator used for the treatment of transfusional iron overload in people. The purpose of this study was to determine the pharmacokinetic disposition of deferiprone in the white leghorn chicken as a potential model upon which to base therapeutic regimens for the treatment of iron storage disease (hemochromatosis) in affected avian species. A suspension of deferiprone (DFP) was administered orally at a single dose of 50 mg/kg to 10 birds that were iron-loaded (IL-DFP) and 10 non iron-loaded control birds (NIL-DFP). After a 30-day washout period, 5 birds from the NIL-DFP group were used for a bioavailability study of deferiprone administered intravenously at the same dose. Blood samples were collected at varying intervals over a 24-hour period and were analyzed for deferiprone by high-performance liquid chromatography, then plasma concentration versus time curves were developed. Deferiprone was rapidly absorbed from the gastrointestinal tract of the chicken, with plasma concentrations effective for iron chelation in humans (>20 gmol/L) maintained for at least 8 hours after oral dosing. The half-life (mean [+ or -] SD) of the orally administered deferiprone in the IL-DFP and NIL-DFP groups was 2.91 [+ or -] 0.78 hours and 3.61 [+ or -] 0.90 hours, respectively, and was 2.42 [+ or -] 0.24 hours for deferiprone administered intravenously. The mean oral bioavailability was 93%. Deferiprone is well absorbed and widely distributed in the chicken, with a longer half-life than reported in mammals.

Key words: iron storage disease, iron chelator, pharmacokinetics, deferiprone, avian, white leghorn chicken, Gallus gallus f domestica

Introduction

As an essential trace element, iron is an important component of many proteins that play a key role in the metabolic pathways involved in the growth and development of organisms. However, because there is no physiologic route for the rapid excretion of excess iron, the balance of iron in the body must continually be regulated so as to not exceed physiologically appropriate amounts. (1,2) When the levels exceed the capacity of a given tissue to safely store the excess iron, serious morbidity and even mortality may ensue. (2,3)

Iron storage disease, or hemochromatosis, is characterized by excessive accumulation of iron in parenchymal organs, especially the liver and eventually the heart and the spleen, with associated functional or morphologic tissue damage. Excessive iron accumulation in tissues has been associated with predisposition to infection, neoplasia, hepatopathy, cardiomyopathy, arthropathy, and endocrine and neurodegenerative disorders. (4-9)

Various avian species are affected by iron storage disease, most notably some species of mynahs and starlings (Sturnidae), toucans (Ramphastidae), birds of paradise (Paradisaeidae), and hornbills (Bucerotidae). (10-16) Hemochromatosis has also been reported sporadically in other species of birds. (3,17-21) The majority of human patients with hereditary hemochromatosis carry a unique missense mutation (C282Y) that alters a major histocompatability--complex class l-like protein designated HFE. (2) Whether a genetic mutation occurs in avian species considered highly susceptible, such as mynahs, remains to be determined.

Once a diagnosis of hemochromatosis has been established, therapy must be instituted to prolong life. In primary human hemochromatosis, phlebotomy is performed once or twice weekly to induce iron deficiency anemia, resulting in the mobilization of stored iron. (2,22,23) This treatment has also been used in birds; however, difficulties arise because of the small size of some patients, the stress of repeated handling, and the uncertainty of the maximum safe volume of blood that can be removed at frequent intervals in a potentially compromised patient. (3,13,16)

Iron chelation therapy is used in human patients with transfusional iron overload when phlebotomy is not possible. Deferiprone (1,2-dimethyl-3-hydroxypyrid-4-one; L1; CP20), which is orally active, and deferoxamine, which is administered parenterally, are the only iron chelators clinically available for treating the disease in people. (2,24-26) Although no pharmacokinetic studies of deferoxamine have been carried out in birds, there have been 2 case reports of its successful use. (10,12) However, the stress of frequent handling for daily injections and the lack of oral efficacy would preclude the use of deferoxamine in some species affected by hemochromatosis.

Deferiprone is a bidentate iron ligand (ie, per molecule, it has 2 donor atoms capable of binding with iron) designed to mimic the naturally occurring chelators mimosine, tropolone, and maltol. (2,27,28) Deferiprone is rapidly absorbed from the gastrointestinal tract via the transcellular pathway and has an absorption half-life of 1-32 minutes in humans. The drug is hydrophilic; thus, deferiprone does not accumulate in lipids, such as cell membranes or the brain tissue, even though it can cross the blood-brain barrier. (28-31)

Deferiprone binds with iron in a 3:1 molecular ratio and forms a neutral complex. It binds readily chelatable non--transferrin-bound iron and likely binds loosely bound iron stored in ferritin and possibly transferrin. In people, deferiprone is metabolized in the liver, predominately (>70%) by glucuronidation, to a conjugate that lacks chelating properties. In human patients, deferiprone is also excreted unchanged in the urine; in rats, it also appears in the bile. (28)

The purpose of this study was to extend the knowledge of the pharmacokinetic properties of deferiprone to birds to establish its potential as a therapeutic agent for hemochromatosis in affected species. We investigated the pharmacokinetic disposition and bioavailability of deferiprone in the white leghorn chicken, a species that can be intravenously iron loaded, has no stainable iron in the liver of healthy birds, (32) and is large enough for the multiple blood collections required for a pharmacokinetic study. Comparisons of the pharmacokinetic disposition of deferiprone (DFP) were made between non--iron-loaded controls (NIL-DFP) and iron-loaded birds (IL-DFP) after a single oral dose. In addition, the disposition and bioavailability of deferiprone were determined in NIL-DFP birds after an intravenous dose.

Materials and Methods

As a prelude to this study, a pilot study to ensure suitable iron loading and a preliminary trial of the pharmacokinetic disposition of deferiprone were carried out in white leghorn chickens. (33) The research project described here was approved by the Animal Care Committee at the University of Guelph and the Animal Care, Research and Acquisition Committee at the Toronto Zoo, both of which operate in accordance with the guidelines of the Canadian Council on Animal Care.

Study population

Twenty adult white leghorn hens with weights of 1.31-1.72 kg were obtained from the Arkell Research Station at the University of Guelph (Guelph, Ontario, Canada) and were transported to the Toronto Zoo (Scarborough, Ontario, Canada), where this study was carried out. Birds were randomly assigned, without regard to size, to 1 of 2 treatment groups of 10 birds each (IL-DFP and NIL-DFP) then were leg banded for identification. The birds were housed in groups of 5 in cages that measured 107 X 92 X 92 cm, with plastic-mesh bottoms, and food and water were provided ad libitum in plastic bowls. All birds were fed the same breeder ration prepared at the Arkell Research Station.

After allowing a week for adjustment, the birds were given a physical examination, and 1 ml of blood was collected to establish baseline complete blood counts and serum biochemical values. The IL-DFP group was iron loaded with iron dextran (Ironol 100, P.V.U., J. Webster Laboratories Inc, Victoriaville, Quebec, Canada) at a dose of 25 mg/kg administered intravenously in the right jugular vein. This dose was based on results of the pilot study mentioned above, which demonstrated stainable hepatic iron stores 72 hours after iron administration. The 10 birds in the NIL-DFP group served as non--iron-loaded controls and did not receive intravenously administered iron dextran.

Drug administration

Oral phase: The purpose of this phase of the study was to determine the pharmacokinetic characteristics of an oral dose of deferiprone (50 mg/kg) in iron-loaded and control chickens. Seventy-two hours after iron administration, all birds were weighed, then anesthetized with isoflurane delivered by face mask. A 24-gauge intravenous catheter was placed and secured in the left or right medial metatarsal vein and was flushed with 0.5 ml of heparinized saline solution.

Birds were fasted for 12 hours before drug administration and for 2 hours after but were allowed unrestricted water intake. Deferiprone was supplied as a pure powder (Apotex Inc, Weston, Ontario, Canada). The dose for each bird (50 mg/kg) was suspended in 1 ml of propylene glycol, then gavaged into the crop, followed by 1 ml of sterile water. Birds were monitored for signs of acute toxicity for 24 hours after administration of the drug.

Intravenous phase: The purpose of this phase was to define more clearly the pharmacokinetic disposition and bioavailability of deferiprone in the control birds after intravenous administration at a dose of 50 mg/kg. After a 30-day washout period, 5 birds, which had previously received one oral dose of deferiprone, were randomly selected from the NIL-DFP control group for an intravenous study. The birds were weighed, then anesthetized with isoflurane delivered by face mask. A 24-gauge intravenous catheter was placed in the left or right medial metatarsal vein and flushed with 0.5 ml of heparinized saline solution.

Birds were fasted for 12 hours before drug administration and for 2 hours after but were allowed unrestricted water intake. The dose of deferiprone powder for each bird (50 mg/kg) was dissolved in 1 ml of warm sterile diluent (benzyl alcohol [9 mg/ml] and sterile water, pH 5.5). The catheters were flushed with 1 ml of heparinized saline solution before intravenous administration of the deferiprone solution over a 10-second period. This was followed by 2 ml of heparinized saline solution.

Sample collection

For all phases, 700-[micro]l blood samples were collected in heparinized syringes from all birds approximately 20 minutes before and at 10, 20, 40 minutes, and 1, 1.5, 2, 4, 6, 8, 12, and 24 hours after administration of deferiprone. Catheters were removed after the final blood-sample collection at 24 hours. Collection times were established via the aforementioned pilot study carried out in chickens to evaluate the pharmacokinetic disposition of deferiprone. To account for the dead space in the catheter and to ensure that the sample was from the circulating blood volume, the first 50 [micro]l of fluid was discarded before collection of the blood sample. After sampling, approximately 500 [micro]l of heparinized saline solution was used to flush the catheter. Blood samples were transferred immediately to lithium heparin Microtainers (Becton Dickinson, Franklin Lakes, NJ, USA) and, within 20 minutes of collection, were centrifuged at 2383g for 30 minutes. Plasma was transferred to tinted 2.0-ml Sarstedt safe-seal microtubes (Sarstedt Inc, St. Leonard, Quebec, Canada) and stored at 70[degrees]C until the assay was carried out.

Assay procedure

All analyses were performed at the Faculty of Pharmacy, University of Toronto (Toronto, Ontario, Canada). Plasma samples were assayed for deferiprone by high-performance liquid chromatography (HPLC) as described by Guo et al, (34) with the modifications described below.

The HPLC system consisted of a Hamilton PRP-1 Peek column (5-[micro]m particle size, 150 X 4.6 mm; Mandel Scientific Co Ltd, Guelph, Ontario, Canada) and a Hewlett-Packard 1050 series HPLC System (Palo Alto, CA, USA) comprised a HP 1050 series pump, a HP 1050 series auto sampler, and a HP 1050 series diodearray detector that operated at ambient temperature. The detection occurred at a wavelength of 280 nm. The mobile phase consisted of 9.0 mM potassium phosphate dibasic (K[H.sub.2]P[O.sub.4]) and 8 mM ethylene diamine tetraacetic acid (adjusted to pH 7.0) mixed with acetonitrile in a 95:5 vol/ vol ratio, which was degassed, then filtered through a 0.45-[micro]m filter. The flow rate was set at 0.8 ml/min. A computer that used the HP ChemStation software (Rev.A.06.03, Hewlett-Packard, Palo Alto, CA, USA) controlled the HPLC system. Isocaffeine (Sigma, St. Louis, MO, USA) served as the internal standard. The limit of quantitation for deferiprone in plasma was 0.25 [micro]g/ml.

Plasma samples were thawed, and each sample was centrifuged to remove cryoglobulins. A mixture of 300 [micro]l plasma and 10 [micro]l isocaffeine stock solution was added to an Amicon centrifree micropartition device (Millipore Corporation, Bedford, MA, USA), with a 30 000 molecular weight cutoff. The mixture in the device was centrifuged for 25 minutes by using a fixed-angle rotor set at 1500g. The ultrafiltrate (100 [micro]l) was then analyzed by HPLC. The retention times for deferiprone and isocaffeine were approximately 7 minutes and 12 minutes, respectively.

A calibration curve from a plot of drug concentration versus area ratio of deferiprone and of internal standard was constructed. Least squared regression analysis ultimately defined the relations needed to calculate deferiprone concentration in plasma samples.

Pharmacokinetic and statistical analyses

The pharmacokinetic disposition of deferiprone in each bird was determined by noncompartmental (model-independent) methods, with nonlinear least squares regression analysis. (35) The area under the plasma concentration--time curve was calculated from time 0 to the last time point ([AUC.sub.0-t]) by using the trapezoidal method. The AUC was calculated to infinity ([AUC.sub.0-[infinity]]) by adding [AUC.sub.0-t] and the quotient of the last measured plasma concentration ([C.sub.last]) divided by the elimination rate constant (k). The elimination rate constant (k) was calculated by using the plasma concentrations of deferiprone between 240 and 720 minutes. Bioavailability was determined by using [AUC.sub.0-[infinity] for the oral dose divided by the [AUC.sub.0-[infinity]] for the intravenous dose and was expressed as a percentage. The terminal elimination half-life in the study was calculated as In 2/k. The maximum plasma concentration ([C.sub.max]) was the highest plasma concentration measured for each bird. The time to maximum concentration ([T.sub.max]) was the time at which [C.sub.max] occurred. The total area under the first moment versus time curve ([AUMC.sub.0-t]) was calculated by using the trapezoidal rule for summation of the product of [time.sup.2] and concentration at each time point. The AUMC to infinity ([AUMC.sub.0-[infinity]]) was calculated by adding the [AUMC.sub.0-t] to the quotient of [C.sub.last] x time divided by k and the quotient of Clast divided by [k.sup.2].

The mean residence time (MRT) was calculated as the quotient of [AUMC.sub.0-[infinity]] divided by [AUC.sub.0-[infinity]]. The apparent clearance of deferiprone from the plasma, in liters per hour (App.CLT), was calculated as the total dose divided by [AUC.sub.0-[infinity]]. This value was normalized (N.CLT) by dividing App.CLT by the weight of the bird; the apparent volume of distribution under steady state conditions (App.Vss) was calculated as a product of MRT and N.CLT.

All statistics were computed by using the SAS statistical software (PC-SAS 8.00, SAS Inc, Cary, NC, USA). All data are reported as the mean and SD. Comparisons between the means of the pharmacokinetic data were determined with the general linear model least square means procedure with a priori level of significance set at .05. In some cases, logarithmic transformations were required to normalize the data distribution to satisfy the assumptions of the model.

Results

Oral phase

All 20 birds completed the oral study. Over a postadministration period of 24 hours, there were no known adverse effects associated with oral administration of deferiprone at a single dose of 50 mg/kg. Approximately 3 hours after deferiprone administration, rust-colored urates were noted. Mean plasma concentrations over time in both the IL-DFP and NIL-DFP groups after a single oral dose of deferiprone (50 mg/kg) are shown in Figures 1 and 2. A summary of the pharmacokinetic data is presented in Table 1.

The drug was rapidly absorbed, appearing in the plasma of 9 of 10 birds in the control group and in 8 of 10 in the iron-loaded group at the first collection time of 10 minutes. Maximal plasma concentrations of 48.56 [+ or -] 13.75 [micro]g/ml and 36.18 [+ or -] 15.32 [micro]g/ml achieved by the IL-DFP and NILDFP groups, respectively, at approximately 1 hour after administration were not significantly different. The drug was still present in very low concentrations at 24 hours after administration. The terminal half-life of 2.91 [+ or -] 0.78 hours for the IL-DFP group was shorter than that of 3.61 [+ or -] 0.90 hours for the NIL-DFP group, although the difference was not statistically significant. The k value between the 2 routes of administration for the IL-DFP and NIL-DFP groups (calculated values of 0.25 [+ or -] 0.05 [hours.sup.-1] and 0.20 [+ or -] 0.05 [hours.sup.-1], respectively) was statistically significant (P = .048). Significant differences in the [AUC.sub.(0-t and 0-[infinity]]) and [AUMC.sub.(0-t and 0-[infinity])] were not observed between the 2 groups, although the means for both [AUC.sub.0-t] and [AUC.sub.0-[infinity]] were comparatively greater for the IL-DFP groups than the NIL-DFP group. There were no significant differences between treatment groups in the apparent App.CLT or the N.CLT. A significantly higher [+ or -] p.Vss was present in the IL-DFP group compared with the NIL-DFP group (2.91 [+ or -] 0.78 L/kg and 2.02 [+ or -] 1.06 L/kg, respectively; P = .030). The mean oral bioavailability was high, at 92.74 [+ or -] 16.24%, with a range of 63.68% to 100%.

[FIGURE 1 OMITTED]

[FIGURE 2 OMITTED]

Intravenous study

All 5 birds completed the intravenous study.

There were no known adverse effects associated with intravenous administration of deferiprone at a dose of 50 mg/kg. Approximately 2-3 hours after deferiprone administration, rust-colored urates were seen as in the oral administration study. Plasma concentrations over time after a single dose of deferiprone (50 mg/kg) via the oral and intravenous routes are shown in Figures 3 and 4. A summary of the pharmacokinetic data is presented in Table 2.

[FIGURE 3 OMITTED]

Mean [C.sub.max] achieved by intravenous administration was significantly greater (approximately fourfold) compared with the concentrations achieved via the oral route in the same birds (111.89 [+ or -] 74.81 [micro]g/ml and 25.90 [+ or -] 13.71 [micro]g/ml, respectively; P = .035). The intravenous dose of deferiprone was completely eliminated by 24 hours, likely owing to a short half-life of 2.42 [+ or -] 0.24 hours compared with 3.22 [+ or -] 0.81 hours after oral administration in the same birds (difference not statistically significant). However, the k value of the intravenous and oral groups (0.29 [+ or -] 0.03 [hours.sup.-1] and 0.22 [+ or -] 0.04 [hours.sup.-1], respectively) was statistically different (P = .025). The intravenous route also had a significantly shorter MRT compared with the oral route (2.70 [+ or -] 0.25 and 5.32 [+ or -] 1.06 hours, respectively; P < .001). Because, in part, of the small sample size (n = 5), no significant differences in the [AUG.sub.(0-t and 0-[infinity])] were found between the oral and intravenous routes of administration. However, the means showed trends in both the [AUC.sub.0-t] and [AUC.sub.0-[infinity]], with the oral route having greater values than the intravenous route. There were no significant differences in the App.CLT or N.CLT between the oral and intravenous routes of administration. The App.Vss was higher for the oral route compared with the intravenous route, although the difference was not statistically significant (1.62 [+ or -] 1.03 L/kg and 1.04 [+ or -] 0.27, L/ kg, respectively).

[FIGURE 4 OMITTED]

Discussion

Knowledge of the pharmacokinetic behavior and bioavailability of a drug used for treatment is of great importance in understanding the relation between dose and efficacy. (36,37) This study was undertaken to determine the pharmacokinetic disposition of deferiprone in an avian model, because the drug may have potential in treating hemochromatosis in valuable and often rare or endangered species. Our findings indicate that deferiprone can be administered orally or intravenously to white leghorn chickens at a single dose of 50 mg/kg without signs of acute toxicity. The drug can be measured in the plasma by using HPLC methodology designed for human patient studies.

Deferiprone is rapidly absorbed from the gastrointestinal tract in the chicken, with measurable plasma concentrations evident in most birds within 10 minutes. The [C.sub.max] of deferiprone in the control and iron-loaded birds (36 [micro]g/ml [260 [micro]mol/L] and 48 [micro]g/ml [345 [micro]mol/L], respectively) were similar to those reported by Matsui et al, (30) when corrected for dose (17.49 [+ or -] 2.08 [micro]g/ml, with a 25 mg/kg oral dose), but higher than [C.sub.max] reported in people administered deferiprone orally at a dose of 50 mg/kg (158.8 [+ or -] 82.9 [micro]mol/L). (27,28) The [C.sub.max] was also higher than that reported in dogs dosed at 100 mg/kg (24.3-52.4 [micro]g/ml). (38) Maximal concentrations were achieved at approximately 1 hour in the chicken, which was similar to findings in people. (27,28,30)

The mean oral bioavailability of deferiprone in the non-iron-loaded chicken (93%) is higher than that reported in the rabbit (72%), (39) rat (60%), (40) and dog (61%-76%). (38) This observed high bioavailability indicated that the drug was well absorbed from the intestinal tract and that the first pass effect or hepatic extraction of deferiprone in the chicken was low. To be orally active, a drug must overcome the anatomical and physiologic barriers of the gastrointestinal tract, and the enzymatic barriers of the intestines and liver. (37) Deferiprone is a small molecule (molecular weight = 139), with a partition coefficient of 0.2. It is absorbed primarily by diffusion into the enterocytes, possibly via the transcellular pathway, thereby using 95% of the intestinal surface area. (28) However, one study that investigated the effect of amino acids on absorption of deferiprone suggested that in addition to transcellular diffusion, carrier-facilitated pathway might be involved. (38) The details of this proposed pathway remain to be elucidated.

The unique anatomy of the avian upper-digestive tract may influence absorption of deferiprone, which in people occurs in the stomach and the intestine. (2,28) As with other drugs, it is unlikely that absorption occurs in the avian crop because of the squamous nature of the epithelium. However, the acidic pH of the crop may aid in increasing the solubility of deferiprone. (36) The formulation of the drug may also influence absorption in birds, because the storage function and emptying pattern of the crop may decrease the availability of tablet formulations compared with capsules, solutions, or suspensions. A stable solution progresses rapidly through the crop, esophagus, proventriculus, and ventriculus, and is available for intestinal absorption within minutes after administration, especially in the absence of food. (36) A suspension was used in this study, which likely maximized drug absorption. The peristaltic and retroperistaltic activity of the avian intestinal tract may also serve to enhance absorption by increasing surface contact time in the small intestine. (41) Further studies are needed to establish the site of deferiprone absorption in birds.

In human patients, administration of deferiprone with food delays the drug's rate of absorption but does not affect the extent of absorption. (28,30) In this study, the birds were fasted to decrease potential variability in absorption associated with food administration. Although the avian species that most commonly develop iron storage disease lack a crop, fasting before drug administration deserves further study in affected species with a well-developed crop, because emptying time of the crop in the domestic chicken is 3.25-19 hours, depending on the amount of grain fed. (36,41) Plasma deferiprone concentrations were low but still measurable at 24 hours in the oral study compared with concentrations below the detection limit of the assay in the intravenous study. The significantly greater elimination rate constant in the intravenous group suggested continued absorption of the drug during the elimination phase when given orally.

Effective chelation therapy in people is related to both dose and the level of iron stores. Initial concentrations of deferiprone exceeding 100-200 [micro]mol/L (14-28 [micro]g/ml) in plasma are extremely effective because of the potential of such concentrations to remove iron from saturated transferrin. Plasma concentrations of 20-50 [micro]mol/L (3-7 [micro]g/ml) can maintain nontransferrin-bound iron levels below 2 [micro]mol/L. (2,42) Plasma deferiprone concentrations remained > 100 [micro]mol/ L for 6 hours after oral administration in both the iron-loaded and the control chickens, and remained above 20 [micro]mol/L for more than 8 hours but less than 12 hours for both groups. Therefore, chelation therapy with deferiprone appears to have effective potential. Although this has been proven in experimental trials, clinical cases are needed for further substantiation. (43)

The mean oral elimination half-life for deferiprone was shorter but not significantly different in the iron-loaded group compared with the control group (2.91 [+ or -] 0.78 hours and 3.61 [+ or -] 0.90 hours, respectively). This half-life is longer than reported in patients with thalassemia (1.5-2.7 hours) and considerably longer than the half-life of 1.2-1.3 hours in healthy human volunteers. (27,29-31) In people, the difference in half-life between iron-loaded patients and normal subjects has been attributed to a larger App.Vss in patients with thalassemia associated with substantially more circulating iron. (31) Our findings are partially consistent with this observation, because the App.Vss was significantly larger in the iron-loaded group compared with the control group (2.91 L/kg and 2.02 L/kg, respectively); however, the trend in half-life was reversed in our study because of the greater N.CLT in the control group compared with the iron-loaded group.

An important difference between the chicken and the human data relates to the method of iron loading. Whereas, in patients with thalassemia, iron is accumulated over a long period of time, resulting in greater storage in deep tissue compartments, the chickens were given a single intravenous dose, and it is likely that much of the iron would not have accumulated in deep tissue compartments. This would minimize the "sink" condition, which may explain the large App.Vss in patients with thalassemia compared with the chickens in this study. (30,31) In this study, the App.Vss was approximately 30 times greater than the plasma volume and 3 times greater than the total body water of healthy white leghorn chickens. (41) This extensive volume of distribution offers great potential for removal of excessive iron stores in parenchymal tissues.

Based on similar [T.sub.max] in chickens and people, the longer half-life of deferiprone in the chicken could be based on different eliminating properties of the drug. This was supported by the N.CLT of 0.25 [+ or -] 0.05 L/h per kilogram and 0.33 [+ or -] 0.12 L/h per kilogram in iron-loaded and control chickens, respectively, which is approximately half the clearance of 0.6 [+ or -] 0.18 L/h per kilogram in human patients with [beta]-thalassemia. Deferiprone is converted to an inactive metabolite by glucuronidation in the liver. (28) Although the enzymes involved in phase I (oxidation and reduction) and phase II (conjugation) reactions are known to exist in the avian liver, the enzymes involved in the process of glucuronidation have been poorly described in birds. (36) Nevertheless, one of the enzymes involved in glucuronidation, UDP-glucuronyl transferase, was shown to have low activity in the chicken (44) and the ostrich (Struthio camelus) (45) compared with the rat. Thus, a decreased rate of metabolism of deferiprone, because of lower glucuronidation activity in the avian liver, could cause the reduced clearance and lengthen the half-life in the white leghorn chicken compared with humans.

In people, the kidney is an important excretory organ for unchanged deferiprone, its chelate, and the glucuronide conjugate. (2,28) This route of excretion also occurs in the chicken, as rust-colored urine was noted within 3 hours after administration of the drug. The excretion of the conjugate is slower than the parent compound, and accumulation of the glucuronide derivative was noted in a patient with impaired renal function. (27) The glomerular filtration rate in the chicken is approximately 2.90 ml/kg per minute, half that of mammals at 4.8 ml/kg per minute. (36) This difference in the glomerular filtration rate may also contribute to the longer half-life observed in both the iron-loaded and control birds compared with human patients.

The elimination rate constant of deferiprone administered intravenously was greater than that of the oral route of administration. This suggests that the oral absorption rate imparted an inexplicable influence on the elimination profile but not to the extent that the absorption half-life would be longer than the elimination half-life. (37) In addition, owing to the fact that the kidneys excrete deferiprone, the half-life also may be shorter in the intravenous study because of first pass excretion via the renal portal system, because the drug was administered in the medial metatarsal vein. Drugs administered in the metatarsal veins may be excreted by the renal tubules of the kidney on the injection side before entering the general circulation. (41) However, this would be the case only if deferiprone was excreted by the tubular epithelium, a point that we were unable to ascertain. Renal extraction of the drug would elevate the calculated bioavailability of deferiprone and may contribute to the significantly higher value seen in the chicken compared with mammalian species. Further studies are needed to evaluate the influence of the renal portal system on intravenous deferiprone kinetics in birds.

The MRT, the average time that introduced molecules reside in the body, was approximately half as long for intravenous administration compared with oral administration of deferiprone (2.70 hours and 5.32 hours, respectively). This difference is expected with an intravenous bolus, because all molecules begin their residence at the same time. Therefore, the MRT for the oral route represented the sum of the MRT for the intravenous route and the mean absorption time. Thus, in the white leghorn chicken, the apparent mean absorption time for the total dose of deferiprone is approximately 2.62 hours. (35,37)

In summary, oral administration of deferiprone in the white leghorn chicken at a dose of 50 mg/kg achieves suitable plasma concentration levels for effective iron chelation therapy for at least 8 hours, even in non-iron-loaded controls. Based on its half-life, a twice-daily dosage regimen appeared suitable and was experimentally effective over 30 days, (43) although a single dosage of 100 mg/kg may be as effective. (2) Further clinical studies in affected bird species are needed to ensure that the drug is efficacious at this dosage. Deferiprone is rapidly absorbed from the gastrointestinal tract and has a large apparent volume of distribution. Further pharmacokinetic studies of the effect of varied doses and the cumulative effects of multiple dose administration are needed in the chicken, as well as studies in other birds to evaluate interspecies differences. Studies are also needed to determine whether deferiprone is excreted entirely by the kidneys or whether fecal excretion occurs in the chicken. Whether deferiprone is eliminated by glomerular filtration or tubular excretion in birds also needs to be determined. As well, the disposition and elimination of the glucuronide metabolite deserves further study in birds.

Acknowledgments: The Toronto Zoo Foundation and the Ontario Veterinary College's Pet Trust Fund supported this research. Gratitude is extended to Dr. Graham Crawshaw, the keepers, and the veterinary technicians (Animal Health Center, Toronto Zoo) for their assistance. We thank Ann Bell (Arkell Research Station), Dr. Bruce Hunter (Ontario Veterinary College), and William Sears (statistician, Ontario Veterinary College) for their contributions to the project; Dr. Gerry Dorrestein for his editorial insight; and the staff of the Animal Health Laboratory, University of Guelph for their assistance. Apotex Inc (Weston, Ontario, Canada) generously supplied deferiprone as a pure powder. This work was performed as part of a thesis for the Doctor of Veterinary Science degree (DVSc) for Douglas Whiteside.

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Douglas P. Whiteside, DVM, DVSc, Ian K. Barker, DVM, PhD, Peter D. Conlon, DVM, PhD, Angelo Tesoro, Jake J. Thiessen, PhD, Kay G. Mehren, DVM, Dipl ACZM, Robert M. Jacobs, DVM, PhD, Dipl ACVP, and Michael Spino, Pharm D

From the Departments of Pathobiology (Whiteside, Barker, Jacobs) and Biomedical Sciences (Conlon), Ontario Veterinary College, University of Guelph, Guelph, Ontario N1G 2Wl, Canada; the Toronto Zoo, 361 A Old Finch Avenue, Scarborough, Ontario M1B 5K7, Canada (Whiteside, Mehren); the Faculty of Pharmacy, University of Toronto, 19 Russell Street, Toronto, Ontario M5S 2S2, Canada (Tesoro, Thiessen, Spino); and Apotex Inc, 150 Signet Drive, Toronto, Ontario M9L 1T9, Canada (Spino). Present address (Whiteside): Calgary Zoo Animal Health Centre, 1625 Centre Avenue East, Calgary, Alberta T2E 8K2, Canada.
Table 1. Comparison of pharmacokinetic parameters after oral
administration of a single dose of deferiprone (50 mg/ kg) to
10 iron-loaded and 10 control white leghorn chickens. (a)

 Parameter Iron loaded (b) Control (b)

[AUC.sub.(0-t)],
 [micro]g x h/ml 195.96 [+ or -] 35.38 165.56 [+ or -] 63.85
[AUC.sub.([0-
 [infinity]),
 [micro]g x h/ml 202.05 [+ or -] 32.29 169.50 [+ or -] 63.22
k, [h.sup.-1] 0.25 [+ or -] 0.05 0.20 [+ or -] 0.05
Elimination
 half-life, h 2.91 [+ or -] 0.78 3.61 [+ or -] 0.90
[C.sub.max],
 [micro]g/ml 48.56 [+ or -] 13.75 36.18 [+ or -] 15.32
[T.sub.max], min 53 [+ or -] 36.22 55 [+ or -] 25.93
[AUMC.sub.(0-t)],
 [micro]g x 894.36 [+ or -] 189.61 849.51 [+ or -] 272.77
 [h.sup.2]/ml
[AUMC.sub.
 (0-[infinity]), 947.08 [+ or -] 207.42 927.89 [+ or -] 277.59
 [micro]g x
 [h.sup.2]/ml
MRT, h 4.70 [+ or -] 0.84 5.76 [+ or -] 1.50
App.CLT, L/h 0.39 [+ or -] 0.08 0.53 [+ or -] 0.20
N.CLT, L/kg x h 0.25 [+ or -] 0.05 0.33 [+ or -] 0.12
App.Vss, L/kg 2.91 [+ or -] 0.78 2.02 [+ or -] 1.06

 Parameter P value

[AUC.sub.(0-t)],
 [micro]g x h/ml .204
[AUC.sub.([0-
 [infinity]),
 [micro]g x h/ml .164
k, [h.sup.-1] .048 *
Elimination
 half-life, h .081
[C.sub.max],
 [micro]g/ml .073
[T.sub.max], min .889
[AUMC.sub.(0-t)],
 [micro]g x .674
 [h.sup.2]/ml
[AUMC.sub.
 (0-[infinity]), .863
 [micro]g x
 [h.sup.2]/ml
MRT, h .065
App.CLT, L/h .056
N.CLT, L/kg x h .061
App.Vss, L/kg .030 *

(a) [AUC.sub.(0-t)] indicates area under the plasma concentration-time
curve from time 0 to the last time point; [AUC.sub.([0-[infinity]),
area under the plasma concentration time curve calculated to infinity;
k, elimination rate constant; [C.sub.max], maximum plasma
concentration; [T.sub.max], time to maximum concentration;
[AUMC.sub.(0-t)], area under the first moment versus time
curve; [AUMC.sub.(0-[infinity]), area under the first moment
versus time curve calculated to infinity; MRT, mean residence
time; App.CLT, apparent rate of clearance from the plasma;
N.CLT, normalized apparent rate of clearance from the plasma; App.Vss,
apparent volume of distribution.

(b) Mean [+ or -] SD for 10 birds in each group.

* Significant difference between groups (P [less than or equal to] .05).

Table 2. Comparison of pharmacokinetic parameters after oral or
intravenous administration of a single dose of deferiprone
(50 mg/kg) to control white leghorn chickens. (a)

Parameter Oral (b) Intravenous (b)

[AUC.sub.(0-t)],
 [micro]g x h/ml 191.82 [+ or -] 69.91 135.44 [+ or -] 33.72
AUC.sub.
 (0-[infinity]),
 [micro]g x h/ml 195.90 [+ or -] 69.46 135.44 [+ or -] 33.72
k, [h.sup.-1] 0.22 [+ or -] 0.04 0.29 [+ or -] 0.03
Elimination
 half-life, h 3.22 [+ or -] 0.81 2.42 [+ or -] 0.24
[C.sub.max],
 [micro]g/ml 25.90 [+ or -] 13.71 111.89 [+ or -] 74.81
[T.sub.max], min 70 [+ or -] 27.38 10 [+ or -] 0.00
[AUMC.sub.(0-t)],
 [micro]g
 x [h.sup.2]/ml 932.73 [+ or -] 298.80 366.74 [+ or -] 104.05
[AUMC.sub.(0-
 [infinity])],
 [micro]g x
 [h.sup.2]/ml 1002.01 [+ or -] 296.94 366.74 [+ or -] 104.05
MRT, h 5.32 [+ or -] 1.06 2.70 [+ or -] 0.25
App.CLT, L/h 0.44 [+ or -] 0.19 0.55 [+ or -] 0.12
N.CLT, L/kg x h 0.29 [+ or -] 0.12 0.39 [+ or -] 0.10
App.Vss, L/kg 1.62 [+ or -] 1.03 1.04 [+ or -] 0.27

Parameter P value

[AUC.sub.(0-t)],
 [micro]g x h/ml .143
AUC.sub.
 (0-[infinity]),
 [micro]g x h/ml .118
k, [h.sup.-1] .025 *
Elimination
 half-life, h .066
[C.sub.max],
 [micro]g/ml .035 *
[T.sub.max], min .001
[AUMC.sub.(0-t)],
 [micro]g
 x [h.sup.2]/ml .004 *
[AUMC.sub.(0-
 [infinity])],
 [micro]g x
 [h.sup.2]/ml .002 *
MRT, h <.001 *
App.CLT, L/h .314
N.CLT, L/kg x h .193
App.Vss, L/kg .263

(a) [AUC.sub.(0-t)] indicates area under the plasma
concentration-time curve from time 0 to the last time point;
[AUC.sub.(0-[infinity])], area under the plasma concentration-time
curve calculated to infinity; k, elimination rate constant;
[C.sub.max], maximum plasma concentration; [T.sub.max], time to
maximum concentration; [AUMC.sub(0-t)], area under the first moment
versus time curve; [AUMC.sub(0-[infininty)], area under the first
moment versus time curve calculated to infinity; MRT, mean
residence time; App.CLT, apparent rate of clearance from the
plasma; N.CLT, normalized apparent rate of clearance from the
plasma, App. Vss, apparent volume of distribution.

(b) Mean [+ or -] SD for 5 birds for each route of administration.

* Significant difference between route of administration (P [less
than or equal to] 0.05).
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Author:Whiteside, Douglas P.; Barker, Ian K.; Conlon, Peter D.; Tesoro, Angelo; Thiessen, Jake J.; Mehren,
Publication:Journal of Avian Medicine and Surgery
Date:Jun 1, 2007
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