Transport of iron chelators and chelates across MDCK cell monolayers: implications for iron excretion during chelation therapy.

Iron chelators are effective at removing iron from the body in iron overload, but little is known about the handling of iron chelates by the kidney. We studied the transport of deferoxamine, deferasirox, and three hydroxypyridones, and their iron chelates, in polarized renal epithelial MDCK cells growing on Transwell inserts. Directional iron efflux was also studied in (59)Fe-loaded cells. The chelators were transported at comparable rates in the apical and basolateral directions and moved faster than their corresponding chelates, except for deferoxamine, which did not move from the basolateral to the apical side. In contrast, the chelates were transported faster in the apical-to-basolateral direction. More permeable chelators were more efficient at removing iron from iron-loaded cells compared with deferoxamine. Iron is preferentially removed from the basolateral side, and kinetic modeling suggests facilitated diffusion of chelates in some cases. Basolateral iron efflux is temperature-dependent and partially sensitive to ATP depletion. Polarized transport of chelates suggests the kidney may be involved in reabsorption of iron bound to chelators, with a temperature-sensitive facilitated removal of some iron complexes from the basolateral side. Further studies are warranted to determine if these processes may contribute to the observed nephrotoxicity of some iron chelators.

Operating characteristics of a partial-block randomized crossover bioequivalence study for dutasteride, a drug with a long half-life: investigation through simulation and comparison with final results.

Studies to establish bioequivalence (BE) of a drug are important elements in support of drug applications. A typical BE study is conducted as a single dose, randomized, 2-period crossover design. For drugs with long half lives (≥ 48 hours) and evaluation of multiple BE objectives in 1 trial, this design may not be adequate. A parallel design may then be a more appropriate choice. However, parallel designs require increased sample size, which can become substantial. One option that is a compromise between the complete randomized block design and the parallel design is a partial-block crossover design. This approach came about during the development of a combination of dutasteride and tamsulosin. Previous experience with performing single-dose dutasteride studies suggested that 28 days of washout is needed between treatments because of its half-life of 7-9 days. Simulations were performed to assess the operating characteristics of this design using a previously developed PK model. Four scenarios were developed, and each scenario was simulated 500 times. The results showed that this design demonstrated acceptable consumer and producer risk. Partial-block crossover designs should be considered for studies when the half-life of the drug is long and there are more than 2 periods.

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

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 micromol/L) maintained for at least 8 hours after oral dosing. The half-life (mean +/- SD) of the orally administered deferiprone in the IL-DFP and NIL-DFP groups was 2.91 +/- 0.78 hours and 3.61 +/- 0.90 hours, respectively, and was 2.42 +/- 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.

Pharmacokinetic disposition of the oral iron chelator deferiprone in the domestic pigeon (Columba livia).

Deferiprone is a bidentate oral iron chelator used for the treatment of iron overload in people. The purpose of this study was to determine the pharmacokinetic disposition of deferiprone in the domestic pigeon (Columba livia) and to compare the results with a previous study in the white leghorn chicken. Deferiprone (DFP) was administered orally as a suspension at a single dose of 50 mg/kg to 10 iron-loaded (IL-DFP) pigeons and 10 non--iron-loaded controls (NIL-DFP). Six NIL-DFP birds were also administered deferiprone intravenously to determine the bioavailability of the drug after a 30-day washout period. To evaluate if deferiprone induces its own metabolism, the pharmacokinetic disposition of the drug was also studied in the IL-DFP group after oral therapy with deferiprone at a dosage of 50 mg/kg q12h for 30 days. For each phase, collected blood was analyzed for deferiprone by high-performance liquid chromatography to develop a plasma concentration versus time curve. Deferiprone was rapidly absorbed from the gastrointestinal tract, with plasma concentrations effective for iron chelation maintained for at least 8 hours after administration in iron-loaded birds. The half-life (mean +/- SD) for deferiprone given orally to the IL-DFP and NIL-DFP groups was 2.98 +/- 0.85 hours and 3.26 +/- 1.25 hours, respectively, and when intravenously administered was 3.79 +/- 1.23 hours. The half-life after 30 days of treatment was 3.42 +/- 1.18 hours. Oral bioavailability was 44%. This study demonstrated that oral absorption of deferiprone is acceptable, it does not induce its own metabolism, and the drug was widely distributed in the pigeon, as it was in the chicken, with a longer half-life than that reported in mammals. Minor interspecies variations in the pharmacokinetics of deferiprone exist between chickens and pigeons.

PSC 833, an inhibitor of P-glycoprotein inhibits 1,2-dimethylhydrazine-induced colorectal carcinogenesis in male Fischer F344 rats.

BACKGROUND: The expression of P-glycoprotein (Pgp) is intimately associated with cancer development. In order to explore the therapeutic value of Pgp as a target for chemotherapy, we studied the effect of PSC 833 (PSC), a potent inhibitor of Pgp, on 1,2-dimethylhydrazine (1,2-DMH)-initiated colorectal carcinogenesis in rats. MATERIALS AND METHODS: Male Fischer 344 rats, initiated with 1,2-DMH coupled with partial hepatectomy, were exposed to dietary 1% orotic acid for 22 weeks. They were then fed either the AIN93G basal diet (BD) or BD containing PSC (a daily dose of 15 mg/kg body weight) for 35 weeks. RESULTS: PSC significantly inhibited colorectal tumor multiplicity by 53% and tumor burden by 74%. PSC-mediated inhibition was evident in tumors as small as 2 mm in diameter and remained effective throughout the course of tumor growth. Histological assessment showed that PSC significantly inhibited tumor progression to colorectal adenocarcinoma by 63%. CONCLUSION: Collectively, this study indicates that PSC inhibited experimental colorectal carcinogenesis initiated with 1,2-DMH in rats.

Validated HPLC assay for iron determination in biological matrices based on ferrioxamine formation.

A simple, robust and reproducible HPLC method has been developed and validated for iron determination in biological matrices. It is based on chelation with desferrioxamine (DFO) and the measurement of the chelate ferrioxamine (FO). The method was developed to permit monitoring of iron bio-kinetics and estimation of iron status in experimental animals. The chromatography was performed on a stainless steel XTerra MS C18 column (Waters; 250 mm x 4.6 mm i.d., 5 microm) using a gradient of Tris-HCl buffer (10mM, pH 5) and acetonitrile. The method was validated in terms of selectivity, linearity (0.3-80 nmol on-column), limit of detection (0.2 nmol on-column), low limit of quantification (0.3 nmol on-column), recovery (91-102%), intra- and inter-day reproducibility, stability, and robustness. The method's universal applicability was illustrated by monitoring plasma and heart iron kinetic profiles in rats after a single intraperitoneal (i.p.) injection of 200mg/kg iron dextran.

Pharmacokinetic studies of a novel 1,2,4-thiadiazole derivative, inhibitor of Factor XIIIa, in the rabbit by a validated HPLC method.

Activated Factor XIII (FXIIIa) stabilizes fibrin clot by covalent cross-linking of fibrin strands in the fibrin, making it resistant to physiological and pharmacologically induced fibrinolysis. Inhibition of Factor XIIIa offers a novel approach to treatment of thrombosis. Selected derivatives of 1,2,4-thiadiazoles, presently in discovery and development, may offer new treatment strategies as inhibitors of Factor XIIIa. In order to evaluate its pharmacokinetic (PK) profile and to facilitate the selection of drug candidates for drug discovery and development process, we developed and validated a simple and selective reversed-phase high-performance liquid chromatographic method (RP-HPLC) with UV detection for the determination of N-[6-(imidazo[1,2-d][1,2,4]thiadiazol-3-ylamino)hexyl]-2-nitrobenzensulfonamide (5624) in rabbit plasma. The plasma protein precipitation and sample preparation was achieved by using acetonitrile, followed by organic phase evaporation to dryness and the residue reconstitution in the mobile phase. The 5624 recovery from the plasma was about 90%. Chromatography was performed on a C18 column using a gradient of acetonitrile in water as a mobile phase. A chemically related compound, N-[6-(imidazo[1,2-d][1,2,4]thiadiazol-3-ylamino)hexyl]naphthalene-1-sulfonamide (5422), was used as an internal standard. Limit of detection (LOD), based on signal to noise ratio>3, was 0.2 microM (on-column amount of about 7 ng), while limit of quantification (LOQ), based on signal to noise ratio>10, was 0.5 microM (on-column amount of about 20 ng). The plasma samples for the PK study were collected at defined time points during and after 5624 slow intravenous infusion (25 mg/kg) to male White New Zealand rabbits and analyzed by RP-HPLC method. The PK parameters, such as half-life, volume of distribution, total clearance, elimination rate constant etc., were determined. The PK profile of 5624 offered insights in the design and development of additional new compounds, derivatives of 1,2,4-thiadiazole, with desired PK properties.

Increased endothelin-1 production in patients with chronic heart failure.

Endothelin-1 (ET-1) concentrations are elevated in patients with congestive heart failure (CHF), although the cause of this increase remains uncertain. We hypothesized that abnormalities in ET-1 production, clearance, or a combination of these may be the cause of elevated ET-1 concentrations in chronic CHF. The kinetics of clearance of ET-1 were measured with (125)I-labeled ET-1 in eight patients with CHF and five age-matched normal individuals. In both normal subjects and the CHF group, the kinetics of ET-1 clearance were best described by a three-compartment model. The steady-state volume of distribution of ET-1 was significantly greater in the CHF group compared with normal subjects (25.2 +/- 3.9 vs. 13.8 +/- 2.1 l/kg; P < 0.05). The total clearance rate from plasma was greater in the CHF group (0.119 +/- 0.018 vs. 0.047 +/- 0.013 l.kg(-1).min(-1); P = 0.05). The total body production rate of ET-1 was also significantly higher in patients with CHF (0.21 +/- 0.03. vs. 0.06 +/- 0.02 ng.kg(-1).min(-1); P < 0.05). It appears that increased ET-1 production rather than decreased clearance is the cause of elevated ET-1 concentrations in patients with chronic CHF.

Phase I and pharmacokinetic study of fostriecin given as an intravenous bolus daily for five consecutive days.

Fostriecin (CI-920) is a potent inhibitor of protein phosphatase 2A (PP2A) and protein phosphatase 4(PP4) found to have anticancer activity in preclinical testing. A phase I study was conducted to evaluate the maximum-tolerated dose (MTD), toxicity profile, and pharmacokinetics (PK) of this drug. Forty-six patients were treated with escalating doses of fostriecin (2-47 mg/m2) administered as a daily bolus infusion for five consecutive days. PK studies were performed at different time points following administration of fostriecin. Dose-limiting toxicities included: elevation of creatinine, bilirubin, and hepatic transaminases; nausea, anorexia, lethargy, and hypotension. PK studies were compatible with a two-compartment model. Regression analysis revealed a significant relationship between dose and clearance; however, the r2 value was only 0.168 indicating a low predictive value for the model. No significant difference was seen in PK parameters with repeated dosing during the same cycle. Although no tumor responses were seen, 16 patients had stable disease with a median duration response of 2.6 months. The study was closed before reaching MTD due to problems with the supply of fostriecin from the National Cancer Institute of the United States (NCI US). New methods for synthesizing fostriecin have recently been described and therefore further development of this unique anticancer agent may be warranted.

Metabolic and pharmacokinetic evaluation of a novel 3-hydroxypyridinone iron chelator, CP502, in the rat.

A recently synthesized 3-hydroxypyridinone derivative with an amido function at the 2-position, CP502 (1,6-dimethyl-3-hydroxy-4-(1H)-pyridinone-2-carboxy-(N-methyl)-amide hydrochloride), exhibited high in vitro iron chelating potency (pFe3+ =21.7). It was targeted as a new iron-chelating candidate for further development in early pre-clinical testing. To evaluate its pharmacokinetics, including oral bioavailability, metabolic and disappearance profiles, studies were conducted in Sprague Dawley male rats. A single 150 mg/kg intravenous and oral dose was given to male Sprague Dawley rats (N=6, B.Wt. 250g). The rats were placed in metabolic cages and fasted overnight before the dosing. Venous blood samples (200 microL per withdrawal) were collected at defined time points before (blank) and up to 28 h post administration. Urine and feces were collected before dosing (blank) and in 24 h intervals up to 72 h post administration. Plasma CP502 concentration versus time profiles were consistent with two-compartment distribution, and the oral bioavailability approached 100%. Total clearance and mean residence time (i.v.) were 1.02 L/kg/h and 1.10 h, respectively. Simultaneous computer fitting yielded V1 and Vss estimates of 0.96 L/kg and 1.74 L/kg, respectively. CP502 was mainly excreted unchanged via urine (45.29+/-9.40 % of total dose) or as glucuronide (6.46+/-1.22% of total dose). High iron chelation potential and favorable pharmacokinetic and metabolic profiles indicate that CP502 is a promising candidate for further development.

Effect of PSC 833, an inhibitor of P-glycoprotein on N-methyl-N-nitrosourea induced mammary carcinogenesis in rats.

Studies in our laboratory on the role of P-glycoprotein (Pgp, coded by mdr1 gene) have led to the hypothesis that over-expression of Pgp is closely associated with the development of cancer. It was conceived therefore that inhibitors of Pgp should inhibit the development of cancer. We have reported that PSC833 (PSC), a potent inhibitor of Pgp, inhibits the development of liver cancer in rats. Similarly, based on the intrinsic over-expression of Pgp in experimental mammary carcinogenesis, we studied the effect of PSC on N-methyl-N-nitrosourea induced mammary cancer in female Sprague-Dawley rats. The study indicates that PSC at daily dietary doses of 15 (PSC15) and 30 mg/kg (PSC30) body wt resulted in dose-dependent inhibition of the incidence as well as the growth of mammary tumors. Compared with controls, PSC15 and PSC30 inhibited: (i) mean tumor multiplicity by 32 and 67%, (ii) median tumor burden by 46 and 93% and (iii) incidence of ulcerated tumors by 40 and 82%, respectively. Most remarkably, PSC delayed median tumor incidence by 8 weeks, and exerted a 100% inhibitory effect on the incidence of large tumors, 4 cm(3) and greater. In all the cases, although the inhibitory effect of PSC was evident at both doses, only PSC30 exhibited statistical significance. A possible compounding effect that was also observed in PSC30-treated rats was a decrease in body weight gain not attributed to diminished food consumption. All in all, consistent with recent reports, which have demonstrated inhibition of cancer development by compromising Pgp function, this study introduces a novel role for Pgp in breast cancer and potentially an unexplored therapeutic approach in treating the disease.

Improved high-performance liquid chromatographic method for the pharmacokinetic studies of a novel iron chelator, CP502, in rats.

An improved reverse-phase high-performance liquid chromatographic method (RP-HPLC) for the determination of a novel iron chelator CP502 (1,6-dimethyl-3-hydroxy-4-(1H)-pyridinone-2-carboxy-(N-methyl)-amide hydrochloride) in rat plasma, urine and feces was developed and validated. The separation was performed on a polymeric column using a mobile phase composed of 1mM ethylenediaminetetra-acetic acid disodium salt (EDTA), acetonitrile, methanol and methylene chloride. Separation of CP502 from plasma, urine or feces endogenous compounds was achieved by gradient elution. Retention times of CP502 and its major metabolite (glucuronide) were about 13 and 4 min, respectively. The method was validated in terms of limit of detection (LOD), limit of quantification (LOQ), selectivity (endogenous from plasma, urine or feces), linearity, extraction recovery, robustness (column selection, mobile phase composition, detection mode, internal standard (IS) selection, analyte stability), day-to-day reproducibility and system suitability (repeatability, peak symmetry and resolution). The method is applicable to bioavailability and pharmacokinetic studies of CP502 in rats.

Source of a micro-nutrient in a semi-synthetic basal diet as a causative factor in inducing urinary calculi in rats and its inhibition by PSC 833, a potent inhibitor of P-glycoprotein.

We report a serendipitous finding of urinary calculi in rats fed a semi-synthetic basal diet. This observation was made during ongoing studies to evaluate the inhibitory effect of PSC 833, a potent inhibitor of P-glycoprotein, on development of tumors in rodent tumor model systems. A large number of specific-pathogen-free (SPF) female Sprague-Dawley and SPF male Fischer 344 rats being fed the diet were euthanized when it became evident clinically that they were uremic. At necropsy, the renal pelvis, ureters, and urinary bladder contained numerous calculi. The presence of urinary calculi was determined to be related to the source of a Food Chemical Codex grade of choline bitartrate. Rats being fed the same basal diet containing the United States Pharmacopia grade of choline bitartrate failed to develop urinary calculi. Interestingly, rats treated with the P-glycoprotein inhibitor were at significantly reduced risk of developing urinary calculi. This finding highlights how something seemingly innocuous as a minor dietary constituent can have a profound impact and, thereby, affect experimental outcome.

Effect of PSC 833, an inhibitor of P-glycoprotein, on 1,2-dimethylhydrazine-induced liver carcinogenesis in rats.

The present study explores the hypothesis that over-expression of P-glycoprotein (Pgp, product of mdr1) is intimately associated with liver cancer development and therefore inhibitors of Pgp should inhibit the development of liver cancer. Accordingly, we determined the effect of PSC833 (PSC), a potent inhibitor of Pgp, on experimental liver carcinogenesis in rats. To study the effects of PSC on liver cancer development, a daily dose of 30 mg PSC/kg body wt (PSC30) was chosen based on an initial dose-response experiment. Accordingly in experiment 1, PSC30 was fed to rats initiated by 1,2-dimethylhydrazine coupled with two-thirds partial hepatectomy and promoted for 22 weeks with 1% dietary orotic acid. Surprisingly, in contrast to our earlier observations in rats without hepatic nodules, in rats bearing hepatic nodules, PSC30 was found to be toxic. Because of this, PSC30 diet was discontinued after 5 weeks and the rats were transferred to basal diet (BD). The rats were killed 10 and 25 weeks thereafter. Cumulative results indicate that PSC30 exhibited a 40% decrease in the incidence of hepatocellular carcinoma (HCC; 15 of 18 in the BD group compared with eight of 17 in the PSC30 group; P = 0.08) coupled with significant reduction of tumor multiplicity (54%; P < 0.05) and tumor burden (61%; P < 0.005) compared with controls. In experiment 2, 15 mg PSC/kg body wt (PSC15) was fed for 20 weeks to rats similarly initiated and promoted for 35 weeks. PSC15 inhibited the incidence of HCC by 75% (four of four in the BD group compared to one of four in the PSC30 group; P = 0.15) and significantly reduced tumor burden by 55% (P < 0.05). The lack of statistical significance of inhibition on tumor incidence reflects the small sample size. Taken together the results indicate a possible intrinsic role for Pgp in liver cancer development and introduce another promising unexplored therapeutic approach in liver cancer treatment.

Tiaprofenic acid: an uncommon cause of cystitis often misdiagnosed as interstitial cystitis.

OBJECTIVE: To bring to the attention of Urologists that the medication tiaprofenic acid (Surgam, Albert-tiafen), a non-steroidal anti-inflammatory drug has been found to cause chemical cystitis. METHODS: Patients presenting between November 1994 and November 1995 with a confirmed or presumed diagnosis of interstitial cystitis and who were taking tiaprofenic acid are reported. RESULTS: Thirty-six patients presented with a confirmed or presumed diagnosis of interstitial cystitis. One woman without irritative symptoms presented with an abnormal ultrasound finding of a thickened bladder. Eight of these 37 patients were diagnosed with chemical cystitis from tiaprofenic acid. These eight patients (one man and seven women) with ages ranging between 46 and 79 (mean 67 years), were taking tiaprofenic acid from 22 to 84 months (mean 41.8 months), for the treatment of osteoarthritis. All eight patients had findings of inflammation at cystoscopy. Symptoms resolved from two to eight weeks (mean 5.3 weeks) in six out of the eight patients (one patient was asymptomatic and one is awaiting resolution two weeks after discontinuation), and persistence of bladder inflammation (as seen on cystoscopy) lingered greater than three months in two patients and five months one patient, after the medication was stopped. CONCLUSION: Symptoms of frequency, nocturia and suprapubic pain are associated with long term ingestion of tiaprofenic acid. Simple discontinuation of the drug will relieve symptoms usually within six weeks. The mechanism of inflammation is unknown.