Introduction

Abstract Introduction Results & Discussion References

The only effective treatment of -thalassemia major, a hemoglobinopathic disorder, is to increase the hemoglobin levels by regular blood transfusion, without which the majority of the patients die within the first year of life (1, 2). Repeated blood transfusions, however, lead to an excess of iron due to the inability of humans to excrete iron produced from the breakdown of hemoglobin (2, 3). Excess iron found in thalassemic patients is distributed throughout the body, but is found in the highest concentrations within the liver and other highly perfused organs (4). The unregulated accumulation or iron causes tissue damage and failure of organs, such as the liver and heart and eventually death (5). Complications associated with the toxicity of iron after blood transfusion can, to a large extent, be alleviated by the use of specific metal scavenging agents or chelating agents to trap and allow excretion of excess and potentially toxic forms of iron from the body (1, 4-7).

DFO¹ has been available for the treatment of iron overload for >30 years. The major limiting factor of DFO is that it is inactive when administered orally and only causes sufficient iron excretion to keep pace with transfusion regimes when given either subcutaneously or intravenously over 8-12 hr for 5-7 days/week (8, 9). For this reason, many patients find it difficult to comply with the treatment, and some even stop taking the drug altogether. There is, therefore, an urgent need for the development of an orally active alternative to DFO.

The HPO class of compounds (fig. 1) are currently one of the main candidates for development of orally active iron chelating alternatives to desferral (DFO) (2, 10, 11). The 1,2-dimethyl (CP20) and 1,2-diethyl (CP94) derivatives from the HPO class of chelating agents have over the past few years been evaluated in the clinic for metabolism, pharmacokinetics, and urinary iron excretion in thalassemic patients (12-14). Both the aforementioned first-generation HPO compounds unfortunately undergo extensive phase II metabolism to form the nonchelating 3-O-glucuronide conjugate, severely restricting the clinical use of these compounds at acceptable dose levels (12, 15).

View larger version (14K): [in this window] [in a new window]   Fig. 1.   General structure of the HPOs.

The critical dependence of chelator efficacy on metabolic behavior has led the authors to design HPO compounds that do not undergo extensive conjugation reactions with glucuronic acid (16). This will permit the use of lower doses, because the bulk of the absorbed dose would retain its chelating ability. 1-Hydroxyalkyl derivatives of HPOs, such as CP102 that do not undergo such unfavorable biotransformation reactions in a variety of animal species (including humans), have since been identified (16, 17). Although the use of 1-hydroxyalkyl derivatives of HPOs may offer a significant improvement over previously evaluated HPOs, a possible disadvantage of these compounds, especially with some of the more hydrophilic analogs, is poor oral absorption and extraction by the liverthe major iron storage organ. A strategy to improve chelation efficiency further while minimizing drug-induced toxicity is to deliver the drug selectively to target iron pools such as the liver. The development of 1-hydroxyalkyl derivative esters with increased hydrophobicity is one route that has been considered to improve both drug absorption and hepatic extraction (18). To fulfill this design objective, it is essential that 1-hydroxyalkyl esters are absorbed intact from the GIT. Ideally, selective hydrolysis by hepatic carboxyesterases will allow generation of hydrophilic metabolites within hepatocytes. These metabolites will thus have the potential to chelate liver iron and, once in the systemic circulation, reticuloendothelial-derived iron without significant permeation of other tissues (fig. 2).

View larger version (9K): [in this window] [in a new window]   Fig. 2.   Absorption from the GIT and selective hydrolysis of hydrophobic esters of 1-hydroxyalkyl derivatives of HPOs by hepatic carboxyesterases.

A variety of ester types are presently being considered for use to alter the physicochemical properties of hydrophilic hydroxyalkyl hydroxypyridinones chelating agents to improve both drug absorption and allow hepatic targeting. The present study describes the metabolism and pharmacokinetics of one such compound, CP117, in the rat.

Materials and Methods

Chemicals. The HPOs used in the present study (CP102, CP94, and CP41) were synthesized as described previously (19). The synthesis of CP117 and CP162 is described herein. Characterization of the compounds was conducted using IR spectroscopy (Perkin-Elmer 298), proton NMR [Perkin-Elmer R32 (90 MHz)], MS [Vacuum Generators 16F (35 eV)], and elemental analysis (University of Manchester, Analytical Laboratory). Sodium dihydrogen orthophosphate, disodium hydrogen orthophosphate, sodium azide, -glucuronidase (type 1X-A from Escherichia coli), atomic absorption standard iron (1020 µg/ml in 0.1 M HCl), and iron dextran (100 mg Fe/ml) were purchased from Sigma Chemical Company (Poole, Dorset, UK). EDTA (trisodium salt Convol) was obtained from BDH (Poole, Dorset, UK). PBS was prepared and sterilized in-house. HPLC-grade acetonitrile (MeCN) was purchased from BDH. All other reagents used were of analytical grade. Sodium phosphate buffer (10 mM) was prepared using deionized water (Waters Milli-Q system). All compounds administered to animals used in the present study were prepared in PBS.

Synthesis of CP117. CP102 prepared from the aforementioned method (19) was treated with trimethylacetyl chloride (pivaloyl chloride-Aldrich) and triethylamine in dimethylformamide at 75°C for 18 hr under an inert atmosphere to afford the diester 1-(2-trimethylacetoxyethyl)-2-ethyl-3-(trimethylacetoxy)-pyridin-4-one; m.p. 133°-134°C; C₁₉H₂₉O₅N: % calculatedC, 64.9; H, 8.3; N, 3.9; % foundC, 65.2; H, 8.1; N, 3.9. The diester was subsequently hydrolyzed in water at 65°C for 4 hr to form the monoester (CP117) as a free base; m.p. 141°-144°C; IR (nujol): 3130, 1710, 1620, and 1580 cm¹; H NMR (DMSO-d6): 1.18 [s, 9H, C(CH₃)₃], 1.27 (t, 3H, CH₂CH₃), 2.85 (q, 2H, CH₂CH₃), 4.02-4.45 (m, 4H, NCH₂, and CH₂O), 5.25 (b, 1H, OH), 6.37 (d, 1H, 5-H), 7.25 (d, 1H, 6-H); MS: m/z 268 [(M+H)⁺ (100)], 267 (34); C₁₄H₂₁O₄N (CP117): % calculatedC, 62.8; H, 7.9; N, 5.2; % foundC, 62.7; H, 7.6; N, 5.3. The HCl salt for the ester was prepared by passing HCl gas through a solution of the free base in dry ethyl acetate. Subsequent addition of dry diethyl ether afforded CP117 as the hydrochloride salt; m.p. 184.5°-186°C; H NMR (DMSO-d6): 1.07 [s, 9H, C(CH₃)₃], 1.18 (t, 3H, CH₂CH₃), 3.01 (q, 2H, CH₂CH₃), 4.41 (t, 2H, CH₂O), 4.72 (t, 2H, N⁺CH₂), 7.46 (d, 1H, 5-H), 8.26 (d, 2H, 6-H), 8.5 (b, 2H, 2OH). Purity of compounds was also confirmed by HPLC.

Synthesis of CP162. Use of 1-(2-hydroxyethyl)-2-methyl-3-benzyloxy-pyridin-4-one hydrochloride, trimethylacetyl chloride, and pyridine in dimethylformamide at 60°C afforded the diester of CP40; m.p. 163°-164.5°C: C₁₈H₂₇NO₅: % calculatedC, 64.0; H, 8.0; N, 4.1; % foundC, 64.2; H, 8.3; N, 4.0.

1

HPLC System. A Hewlett-Packard model 1090 M-II HPLC system with an autoinjector, autosampler, and diode array detector linked to a HP 900-300 data station was used to measure drug and metabolite concentrations. Treated urine samples were analyzed by reversed-phase HPLC using a Hypercarb column packed porous graphatized carbon column (10 × 0.46 cm) (Shandon Scientific Ltd., Runcorn, Cheshire, UK). A polymer PLRP-S column (15 cm × 4.6 mm, i.d. 5 µm) (Polymer Laboratories Ltd., Church Stretton, Shropshire, UK) was used to analyze treated plasma samples to allow separation of the sacrificial ester and hydrolyzed product from that of drug and its metabolite (refer to pharmacokinetic studies). The mobile phase was a mixture of aqueous NaH₂PO₄:MeCN. NaH₂PO₄ solution (10 mM) was prepared by dissolving the 1.56 g of the monobasic sodium salt of phosphoric acid in 800 ml of deionized water; 2 ml of 1 M EDTA Tris-sodium was then added and the pH adjusted to 3.0 with orthophosphoric acid before being made up to volume (1 liter). The mobile phase was pumped through the column at a flow rate of 1 ml/min. The following gradient systems were used (min/% MeCN): 0/16, 2/16, 4/55, 6/55, 8/16, 12/16, and 1/2, 5/16, 6/24, 13/27, 14/2, and 18/2 for urine and plasma samples, respectively. The eluant was monitored at 285 nm.

Urinary Recovery Studies. Urinary recovery studies were conducted in normal (intravenous and oral) and iron-overloaded rats (oral only) to compare the different routes of administration. For the oral dosing studies wherein the influence of iron overload was determined, two groups of five male Wistar rats (200-240 g) were used. One of the aforementioned groups were iron-overloaded by the administration of 4 intraperitoneal injections of iron dextran over a period of 14 days (days 1, 5, 8, and 12) such that a final total body iron loading of 500 mg Fe/kg was achieved. The other group represented an age-/weight-matched control of the iron-overloaded animals. Animals were housed in a 12-hr light/dark cycle temperature-controlled (22°C) metabolic room maintained at a humidity level of 50%. Food and water were provided ad libitum.

Analysis of Urine Samples. Iron complexes of bidentate chelating agents, such as the HPOs unlike hexadentate ligands, have a tendency to dissociate on chromatographic columns, thus preventing simple analysis of both the free ligand and that of the intact bidentate metal-chelate complex (20). To allow quantification of the drug and its metabolites, both chelating and nonchelating, three separate assays were necessary due to differences in extractability into DCM.

Assay A: Quantification of Bound and Unbound Drug {[(CP117)₃-Fe] + [CP117]} and Chelating Metabolite {[(CP102)₃-Fe] + [CP102]}. This assay measures both the free ligand and iron-bound forms of both CP117 and its chelating metabolite, CP102. Addition of a large excess of a high-affinity ligand, such as EDTA, was required to allow quantitative dissociation of the iron complexes of CP117 and CP102. Because the bidentate HPOs bind in a strict stoichiometric manner (three ligands are always associated with an iron atom), each ligand provides 2 of a total of 6 coordination positions around the metal ion (17). Data obtained from this assay can be expressed as in eq. 1, where ligand (L) is applicable to both CP117 and its metabolite, CP102.

(1)

Urine samples obtained from the studies were diluted 10-fold with 100 mM phosphate buffer (pH 7). To 100 µl of diluted urine samples was added 200 µl of the internal standard (CP94, 100 µg/ml) made up in 100 mM phosphate buffer containing 50 mM EDTA. Samples were then extracted with 10 ml of DCM. The DCM layer was then removed and evaporated to dryness in a water bath at 55°C. The residue obtained was reconstituted in 100 µl mobile phase and analyzed by HPLC.

Assay B: Quantification of Glucuronide Conjugates. Analysis of nonchelating metabolites, such as the glucuronide conjugates of CP117 and CP102 that are not extracted into DCM, was conducted by incubating urine samples with a sufficient excess of -glucuronidase to ensure complete hydrolysis to reform the parent drug. The procedure for assay B is similar to assay A, with the exception that the combined internal standard and EDTA solution also contained 5000 units/ml -glucuronidase (type IX-A from E. coli). Samples were incubated for 16 hr at 37°C to ensure complete hydrolysis of the glucuronide conjugate. This assay quantitates any species that on dissociation or hydrolysis leads to the reformation of the parent drug and measures the total drug recovered from the urine sample, including unchanged drug, its corresponding iron complex, and glucuronide conjugate (eq. 2). Differences between assays A and B, in the presence ([L]_T) and absence of -glucuronidase ([L]_t), accounts for the glucuronide conjugate (L-GLUC) present in the urine sample (eq. 3).

(2)

(3)

Assay C: Quantification of the Iron Complex. To determine the relative contributions of the free ligand and the corresponding iron complexes in assay A, a method that directly measures one of the aforementioned species is required. Iron content in the urine samples from both normal and iron-overloaded animals was measured using an atomic absorption spectrometer. Values obtained are corrected for background excretion for both normal and iron-overloaded animals. Data obtained from the direct measurement of iron in assay C can, therefore, be used to solve eq. 1 and provide information on the free ligand concentrations in urine (eq. 4) (L₃-Fe = iron complex):

(4)

When more than one iron binding species is present (e.g. CP102 and CP117), the proportion of the individual chelating moieties associated with the metal is directly related to the ratio of the total ligands present (i.e. [CP102]_T and [CP117]_T). This is due to the ability of bidentate chelating agents to undergo rapid ligand exchange reactions that can lead to the formation of mixed iron complexes due to the fact that the affinity constants for iron (III) are virtually identical.

Pharmacokinetic Studies. Male Wistar rats weighing ~350-400 g were housed under conditions described for urinary recovery studies. Animals were surgically prepared before administration of CP117. The jugular vein and carotid artery cannulae (0.58 mm i.d., polythene; Portex, Hyte, Kent, UK) were implanted under light anesthesia using a mixture of fentanyl and fluanisone (Hypnorm, Janssen Pharmaceuticals Ltd., Grove, Oxford, UK) and midazolam (Hypnovel, Roche Products Ltd., Welwyn Garden City, Herts, UK) at a dose of 2.5, 0.08, and 5 mg/kg, respectively. The inserted cannulae were secured with sutures before being exteriorized in the dorsal intracapsular area. The surgical incision was closed using biodegradable sutures and the wound swabbed with heparinized saline-dipped cotton wool. The patency of the cannulae was maintained by flushing with heparinized saline (200 µl, 100 units/ml). Rats (N = 6) were allowed a recovery period of 24 hr before the administration of 450 µmol/kg of CP117 prepared in normal saline (450 µmol/ml) via the jugular cannulae as a single bolus injection. For oral pharmacokinetic studies, only the carotid artery was cannulated and the drug administered by gavage.

Pharmacokinetic Analysis. Data obtained was analyzed using noncompartmental analysis. AUC₀ after intravenous bolus and oral doses were determined by using the trapezoidal rule from time 0 (determined by extrapolation) to the last sampling time point t*, and the AUC from the last sampling time point t* to infinity was determined by dividing the last plasma concentration C* by the terminal elimination rate constant (_z). Terminal elimination rate (_z) was determined from the slope of the regression line fitted to the log plasma concentration-time data of the terminal phase by the method of least squares. t_1/2 was calculated by dividing ln 2 by the elimination rate constant. The total plasma CL was calculated using eq. 5. Contribution of CL_R to CL was determined by dividing the amount of drug recovered unchanged in the urine (oral) in time t* divided by the AUC in the same time, obtained via the same route of administration. AUMC is the area under the curve of the product of concentration and time vs. time. The AUMC from time 0 to t* was calculated by means of the trapezoidal rule and from t* to infinity being estimated using eq. 6, where C* is the concentration at last sampling time t*. Vd_ss was calculated from eq. 7, the MRT was determined by eq. 8 and MAT was calculated by eq. 9. t_max and C_max after oral administration were assessed by visual inspection of the blood concentration-time curve. The oral bioavailability (F) was calculated by the AUC ratio method [F = (AUC_po)/(AUC_iv)] after the administration of 450 µmol/kg of appropriate compound.

(5)

(6)

(7)

(8)

(9)

Absorption Studies. Absorption studies were conducted using the in situ isolated rat gut loop model. Male Wistar rats were anesthetized as described previously in the pharmacokinetic studies. Although still under anesthesia, a 10-cm section of the jejunum was isolated and the two ends ligated with nylon sutures. Mesenteric vein branching from the isolated vein was cannulated (0.58 mm i.d., polythene; Portex). CP117 at a dose of 450 µmol/kg (prepared in 1 ml PBS) was administered via a ligated cannulae into the isolated gut loop. Blood samples were collected both before and after drug administration via the mesenteric vein for 60 min at 5-min intervals into heparinized microcentrifuge tubes containing 10 µl of sacrificial ester (CP162, 100 mg/ml) and processed as described for pharmacokinetic studies.

Statistical Analysis. Statistical analysis was determined by Student's paired and unpaired t test (Statworks). p  0.05 was considered statistically significant.

	Results and Discussion

Abstract Introduction Results & Discussion References

Metabolism and pharmacokinetic studies are crucially important for the design of safe and effective chelating agents due to their narrow therapeutic safety margin. One such requirement is to ensure that chelating agents are not metabolically degraded to metabolites that lack the ability to bind iron. This would allow the use of lower doses and correspondingly decreasing the risk of inducing toxic side effects. The use of 1-hydroxyalkyl derivatives of HPOs identified by the authors that are either nonmetabolized or selectively metabolized will overcome the aforementioned limitations. Further therapeutic benefit can be gained by ensuring that the chelating moiety, either as the drug or chelating metabolite, is delivered to target site(s) at an appropriate concentration, rate, and duration to ensure interception of iron from targeted body stores of the metal.

Hepatic iron is one of the two major pools of this metal in the body and is therefore an obvious target for the design of new therapeutic agents. Data generated by us (18) and others (19, 21) suggest that iron bound within hepatocytes and from the extracellular space is strictly compartmentalized and behave independently of each other due to their inability to permeate biological membranes. Chelation of hepatic iron leads to exclusive excretion via the bile and that of extracellular iron via the urine (22). Recovery data obtained from iron-overloaded animals in comparison to normal animals are potentially useful, because they provide information on the ability of chelating agents to access the hepatic iron pool. The difference in urinary recovery of the drug/metabolite between the two groups arises, because chelation of hepatic iron will lead to a decrease in the recovery of the drug/metabolite in the urine. Such studies also provide an indication on the likely in vivo efficacy of a chelating agent.

Urinary Recovery Study. Urinary recovery studies of CP117 were conducted in normal (intravenous and oral, N = 5) and iron-overloaded animals (oral only, N = 5) to assess the influence of iron. Iron loading was achieved by using iron dextran, a polymeric form of iron that is selectively taken up by reticuloendothelial cells and subsequently redistributed via plasma transferrin and when transferrin saturation occurs, as nontransferrin bound iron to parenchymal iron stores such as hepatocytes. CP117 was also injected as an intravenous bolus to normal animals to compare routes of drug administration.

View larger version (45K): [in this window] [in a new window]   Fig. 3.   Seventy-two hour urinary recovery of CP117 and its metabolites in normal (intravenous and oral, N = 5) and iron-overloaded (500 mg Fe/kg; oral only, N = 5) rats postadministration at 450 µmol/kg (oral only, N = 5). GLUC, glucuronide.

Pharmacokinetic Study. Urinary recovery studies, although useful, do not provide information to account for the quantitative behavior of the drug with respect to time. Information relating to these aspects can be obtained from pharmacokinetic investigations. The pharmacokinetics of CP117 was studied at a fixed dose of (450 µmol/kg, N = 6) via both the intravenous and oral routes. Plasma concentration-time plots of CP117 and its hydrolytic product, CP102, postintravenously and orally are depicted in fig. 4, a and b, respectively. The pharmacokinetic parameters of CP117 postintravenous and oral administrations are tabulated in tables 1 and 2.

View larger version (19K): [in this window] [in a new window]   Fig. 4.   Plasma drug concentration-time profile of CP117 and its metabolite (CP102) after bolus (a) intravenous (N = 6) and (b) oral (N = 6) administrations of 450 µmol/kg CP117.

View larger version (17K): [in this window] [in a new window]   Fig. 5.   Mesenteric vein concentration-time profile of CP117 and CP102 after bolus administration of CP117 (N = 4) into a 10-cm isolated gut loop section of a rat via a ligated cannula.