Determination of sulfadimethoxine and 4N-acetylsulfadimethoxine in bovine plasma, urine, oral fluid, and kidney and liver biopsy samples obtained surgically from standing animals by LC/MS/MS.

A quantitative method was developed and validated to measure the concentration of sulfadimethoxine (SDM) and its major metabolite, (4)N-acetylsulfadimethoxine (AcSDM), in bovine tissues and body fluids. Liquid chromatography/tandem mass spectrometry (LC/MS/MS) gave quantitative results for these two analytes in extracts from bovine plasma, urine, oral fluid, kidney, and liver, using SDM-d(4) as internal standard (I.S.). The lower limit of quantitation (LLOQ) for both analytes in these matrices was validated at 2, 100, and 5 ng/mL in plasma, urine, and oral fluid respectively, and 10 ng/g in both kidney (cortex) and liver. The overall accuracy (average of 4 levels) is, for plasma, 104% (SDM) and 95% (AcSDM), with standard deviation of 9% (SDM) and 15% (AcSDM); for urine, 100% (SDM) and 106% (AcSDM), with standard deviation of 5% (SDM) and 6% (AcSDM); for oral fluid, 103% (SDM) and 103% (AcSDM), with standard deviation of 4% (SDM) and 4% (AcSDM); for kidney, 101% (SDM) and 111% (AcSDM), with standard deviation of 7% (SDM) and 6% (AcSDM); and for liver, 99% (SDM) and 115% (AcSDM), with standard deviation of 11% (SDM) and 9% (AcSDM). C18 SPE cartridges were used to clean-up these matrices, except for urine which was diluted directly with buffer before analysis by LC/MS/MS.

Role of a novel multifunctional excipient poly(ethylene glycol)-block-oligo(vinyl sulfadimethoxine) in controlled release of lysozyme from PLGA microspheres.

This study investigated the effect of ion-pairing of anionic polyelectrolytes: our novel poly(ethylene glycol)-block-oligo(vinyl sulfadimethoxine) (PEG-OVSDM) and poly(ethylene glycol)-block-poly(l-aspartic acid) (PEG-PAA) with cationic lysozyme on retention of protein stability during emulsification. Soluble lysozyme recovery after exposure to the deleterious interface was 42-88% (when ion-paired with PEG-OVSDM, PEG-OVSDM concentration dependent) compared to only 30% for free lysozyme. PEG-OVSDM provided a higher stabilization of lysozyme than PEG-PAA (36-60%). Lysozyme when recovered in the aqueous phase and analyzed by chromatography, enzymatic assay, fluorescence, and mass spectrometry showed no significant physicochemical change when compared with a lysozyme standard. Lysozyme was incorporated into poly(lactide-co-glycolide) (PLGA) microspheres via the typical double emulsion method. Incorporation of lysozyme complexes led to a higher encapsulation efficiency and loading amount, and a lower incidence of insoluble lysozyme aggregates compared to the control microspheres containing lysozyme only. More significantly, ion-pairing was able to dramatically reduce the initial lysozyme release to 18% compared with 50% from control microspheres and provided an overall better control of protein release. PEG-PAA was less effective than PEG-OVSDM in controlling the release probably due to weaker interactions between this polyelectrolyte and lysozyme. Manipulation of such polyelectrolyte-protein complexation may play a role in protein-controlled delivery.

Renal selective N-acetyl-L-gamma-glutamyl prodrugs: studies on the selectivity of some model prodrugs.

1. In this study, a number of structurally different N-acetyl-L-gamma-glutamyl prodrugs were investigated with respect to selective uptake by the kidney in male Wistar rats. 2. All prodrugs were tested in vitro in rat kidney slices and kidney homogenate to study their uptake and conversion. It was found that the prodrugs of para-nitroaniline (agPNA), aminophenyl acetic acid (agAFA), sulphamethoxazole (agSM), sulphadimethoxine (agSDM), propranolol (agPP) and metoprolol (agMP) were accumulated by a probenecid-sensitive carrier. The prodrug of 4'-aminoantipyrine (agAAP) was not accumulated by a probenecid- or buthionine sulphoximine-sensitive carrier. Unlike all other prodrugs, agAAP and agMP were not, or only a very limited extent converted to the parent compound in vitro. 3. agPNA, agAFA and agPP were also investigated in vivo. The tissue distribution of the prodrugs and the parent drugs was established, as was their urinary excretion and pharmacokinetic behaviour. agPNA and agAFA showed selective uptake by the kidney, in contrast to agPP which accumulated in the liver. The distribution of the parent compounds following prodrug administration was as follows: agPNA was found in kidney and plasma: agAFA in kidney only; agPP in liver only. 4. The factors which determine the selectivity of N-acetyl-L-gamma-glutamyl prodrugs are discussed. The main factors are: the transport into the kidney, the conversion rate, the residence time of the prodrug in the kidney and the presence or absence of competition for uptake and conversation by other tissues, e.g. the liver. It is concluded that this prodrug approach offers the possibility of delivering drugs selectively to the kidney, but also that it is not universally applicable.

Simultaneous high-performance liquid chromatographic determination of residual sulphamonomethoxine, sulphadimethoxine and their N4-acetyl metabolites in foods of animal origin.

A rapid and sensitive method for the determination of residual sulphamonomethoxine, sulphadimethoxine and their N4-acetyl metabolites in beef, pork, chicken and eggs by high-performance liquid chromatography (HPLC) was developed. The extraction of these compounds was performed using a mixture of 90% (v/v) acetonitrile solution and hexane (5:4, v/v) to minimize the fat content followed by purification by alumina column chromatography. These extracts contained sulphonamide analytes which were free from interfering compounds when examined by HPLC using a LiChrosorb RP-18 column. The average recoveries from spiked meat and egg were in excess of 80% with relative standard deviations between 0.4 and 5.0%. The practical limits of detection were 0.01 ppm for all samples.

Renal tubular excretion of the N4-acetyl metabolites of sulphasomidine and sulphadimethoxine in the dog.

To investigate whether dogs are able to excrete acetylated drugs by active transport, the plasma kinetics and renal excretion of the N4-acetyl metabolites of sulphasomidine and sulphadimethoxine were studied in the beagle dog after a rapid intravenous bolus injection. Two doses of N4-acetylsulphasomidine (1050 and 105 mg) and one dose of N4-acetylsulphadimethoxine (472 mg) were administered on separate occasions. The renal clearance (CLR) was as follows: N4-acetylsulphasomidine (1050 mg) 34 mL min-1; N4-acetylsulphasomidine (105 mg) 28 mL min-1; and N4-acetylsulphadimethoxine (472 mg) 24 mL min-1. CLR was higher than expected on the basis of the measured glomerular filtration rate, indicating that the N4-acetyl metabolites may be excreted by the renal tubules by active tubular transport. Saturation of the excretion process of N4-acetylsulphasomidine occurred with a transport maximum of 930 +/- 190 micrograms min-1 and a Michaelis-Menten constant of 37 +/- 10 micrograms mL-1. It may be concluded that the dog renal organic anion transport system is able to secrete acetylated sulphonamides.

Distribution and elimination of sulphadimethoxine and its metabolites in treated chicken.

Sulphadimethoxine (SDM), and its metabolites, N4-acetyl SDM, N1-(2-methyl-6-hydroxy-4-pyrimidinyl) sulphanilamide (6-OH-SDM), N1-(6-methyl-2-hydroxy-4-pyrimidinyl) sulphanilamide (2-OH-SDM), N1-(2,6-dihydroxy-4-pyrimidinyl) sulphanilamide (2,6-diOH-SDM) and SDM N1-glucuronide in chicken tissues were extracted, partially purified by Bond Elute SCX cartridges, and assayed and identified by HPLC/LC-MS after administration of SDM to chickens. During the administration and 24 h after withdrawal, SDM and 6-OH-SDM were observed in almost all tissues and excreta. N4-Acetyl SDM and 2,6-diOH-SDM were observed in some tissues, but 2-OH-SDM and SDM N1-glucuronide were observed in a few limited tissues. Twenty four hours after withdrawal, SDM and its metabolites, except 6-OH-SDM, decreased. SDM and its metabolites were eliminated from all tissues within 48 h of withdrawal.

Extraction and liquid chromatographic analysis of sulfadimethoxine and 4-N-acetylsulfadimethoxine residues in channel catfish (Ictalurus punctatus) muscle and plasma.

A simple and rapid method was developed for the simultaneous extraction and liquid chromatographic (LC) determination of sulfadimethoxine (SDM) and 4-N-acetylsulfadimethoxine (N-acetyl SDM) in channel catfish muscle and plasma. Tissues fortified at 0, 50, 100, 200, 400, and 1000 ppb were examined. Matrix solid phase dispersion (MSPD) was used for muscle extraction. Plasma was extracted with a modified MSPD procedure in which 100 microL plasma and 400 mg C18 were blended by Vortex mixing in a disposable chromatographic column. Recovery of SDM based on radioactivity was 79% for muscle and 67% for plasma. Standard curves based on extracted fortified samples were used for quantitation of N-acetyl SDM. LC run times of 12 min were obtained using a microbore analytical column and an isocratic mobile phase of aqueous 0.017M phosphoric acid-acetonitrile at ratios of 71:29 for muscle and 73:27 for plasma extracts. Method detection limits were 26 ng SDM and 26 ng N-acetyl SDM/g muscle, and 33 ng SDM and 11 ng N-acetyl SDM/mL plasma. Intra-assay variation was < 10% for both compounds at all concentrations examined. Inter-assay variation for SDM was 13% for muscle and 14% for plasma, and for N-acetyl SDM was 11% for muscle and 10% for plasma.

The role of plasma protein binding on the metabolism and renal excretion of sulphadimethoxine and its metabolite N4-acetylsulphadimethoxine in pigs.

The effects of plasma protein binding on the elimination of sulphadimethoxine (SDM) were examined after intravenous administration of 6.25, 12.5, 25, 50, 100 and 150 mg/kg to pigs. At an early stage of the experiment, the animals were anaesthetised by inhalation of enflurane to obtain a more exact relationship between plasma concentration and the renal excretion. SDM and its acetylated conjugate, N4-acetylsulphadimethoxine (N4-SDM) were detected in plasma and urine of all animals, and the recovery of the doses was almost complete in two animals with negligible renal excretion of SDM. The percentages of plasma protein binding of SDM and N4-SDM were almost similar, and ranged from 30 to 95%, depending on the plasma concentration. The metabolic clearance of SDM by acetylation increased when the plasma protein binding decreased. These results suggested that the main elimination route of SDM in pigs is acetylation, and that the plasma protein binding can have a large effect on the elimination of SDM in pigs. The effect of plasma protein binding on the renal clearance of SDM was not so evident, because urine pH had a much greater effect on it. The deacetylation of N4-SDM was detected after 25 mg/kg intravenous administration of N4-SDM, which suggests that the metabolic clearance of SDM is part of an acetylation-deacetylation equilibrium. Saturation of the active tubular reabsorption of SDM and of the active tubular secretion of N4-SDM was also suggested after higher doses of SDM.

Sulfadimethoxine-bucolome interaction in rabbits: role of N4-acetylsulfadimethoxine, a major metabolite of sulfadimethoxine.

The role of N4-acetylsulfadimethoxine (N4-AcSDM), a major metabolite of sulfadimethoxine (SDM), in protein binding and pharmacokinetic interactions between SDM and bucolome (BCP) was investigated in rabbits. When SDM and BCP were intravenously co-administered, BCP indirectly reduced the serum protein binding of SDM by causing a marked increase of N4-AcSDM concentration in serum, and significantly increased the steady-state volume of distribution (Vss) and total body clearance (C1tot) of SDM. In addition, the co-administration of N4-AcSDM was found to increase Vss and C1tot of SDM. These results lead us to conclude that N4-AcSDM plays an important role in protein binding and pharmacokinetic interactions between SDM and BCP in rabbits. Several investigations have demonstrated that a metabolite plays an important role in drug-drug interaction. For example, Sellers et al. (1) reported that when warfarin and chloral hydrate are co-administered, a major metabolite of chloral hydrate, trichloroacetic acid, reduces the plasma protein binding of warfarin and enhances its anti-coagulant activity. Our previous paper (2) showed that probenecid indirectly reduces the plasma protein binding of sulfadimethoxine (SDM) by causing a marked increase in the plasma concentration of N4-acetylsulfadimethoxine (N4-AcSDM), which is a major metabolite of SDM (3) and strongly displaces SDM from its plasma protein binding sites (2). However, as yet the role of this metabolite in drug-drug interaction has not been fully examined.(ABSTRACT TRUNCATED AT 250 WORDS)

[Displacing effect of serum protein binding on intestinal absorption of sulfadimethoxine].

The displacing effect of serum protein binding on the intestinal absorption of sulfadimethoxine (SDM) in rabbits was examined by using N4-acetylsulfadimethoxine (N4-AcSDM), a major metabolite of SDM, as a displacing drug. N4-AcSDM markedly decreased the in vitro serum protein binding of SDM, while many drugs including phenylbutazone and salicylic acid did not display such a marked decreasing-effect. The intravenous administration of N4-AcSDM clearly decreased the in situ intestinal absorption of SDM. As expected, the intravenously administered N4-AcSDM enhanced the serum concentration of unbound SDM in the common jejunal vein. However, the intravenously administered N4-AcSDM caused no change in the transfer of SDM across the intestinal membrane. These results indicate that the displacement of serum protein binding can become one of factors decreasing the intestinal absorption of SDM.

Characterization of binding sites for sulfadimethoxine and its major metabolite, N4-acetylsulfadimethoxine, on human and rabbit serum albumin.

In order to gain an understanding of protein binding of sulfadimethoxine (SDM) and its major metabolite, N4-acetylsulfadimethoxine (N4-AcSDM), the binding of SDM and N4-AcSDM to human and rabbit serum albumin (HSA and RSA) was investigated using circular dichroism (CD), fluorescence and dialysis techniques. The CD spectral characteristics of the compounds bound to the albumins suggested that the drug-binding sites on the HSA and RSA had somewhat different asymmetries. The binding constants for SDM-HSA and -RSA interaction were smaller than those for N4-AcSDM. Two specific drug-binding sites were found on RSA, similarly to HSA, from the results of competitive displacement using fluorescence probes. Moreover, SDM and N4-AcSDM were found to share the same first binding site on the albumins. It can be presumed from the displacement data with a series of p-aminobenzoates that the characteristics of the binding sites (such as depth and width of the hydrophobic cleft) for SDM and N4-AcSDM on RSA may be almost the same, but the characteristics of these drug-binding sites on HSA may be somewhat different.

Plasma/muscle ratios of sulfadimethoxine residues in channel catfish (Ictalurus punctatus).

Channel catfish (n = 84) maintained at a water temperature of 27 degrees C were used in a feeding study to determine the plasma to muscle concentration ratios of sulfadimethoxine (SDM) and 4-N-acetylsulfadimethoxine residues. Sulfadimethoxine medicated feed was provided free choice at 42 mg SDM/kg body weight once daily for 5 days and the plasma and muscle concentrations of SDM were determined at selected withdrawal times (6, 12, 24, 48, 72, and 96 hours) following the last dose. Considerable variation in total SDM tissue concentration among fish within a sampling period was observed. For fish (n = 12) at six hours post-dose, total SDM concentrations ranged from 1.4-24.8 micrograms/mL and 0.6-12.6 micrograms/g, with mean total SDM concentrations of 9.1 micrograms/mL and 5.3 micrograms/g for plasma and muscle, respectively. However, a mean plasma:muscle concentration ratio of 1.8:1 +/- 0.3:1 was obtained over all concentrations and sampling periods. The plasma:muscle 95% t distribution interval for individual fish was 1.2:1 to 2.4:1. A correlation coefficient of 0.967 was obtained for the relationship between plasma and muscle total SDM concentration among individual fish (n = 25). Results of this study indicate that plasma total SDM concentration may be used to identify samples containing violative SDM muscle residue. No fish contained total SDM muscle residues greater than the FDA tolerance (0.1 microgram/g) by 48 hours following the final dose.

Elimination half-lives of sulphadimethoxine and its N4-acetyl metabolite in tissues of laying hens.

The depletion rates of sulphadimethoxine (SDM) and its metabolite N4-acetylsulphadimethoxine (N4-AcSDM) were estimated in blood and various tissues of laying hens. The tissue contents (ppm) of SDM and N4-AcSDM after the withdrawal of SDM, which was fed to hens at 400 ppm diet for 5 successive days, were determined by HPLC. The elimination half-life (t1/2) of N4-AcSDM in the liver, ovary and muscle was estimated to be 4.3 h with a 95% confidence interval from 3.6 to 5.3 h. No significant difference between t1/2 of N4-AcSDM in the tissues and that of SDM (4.4 h) in the blood, kidney, muscle, ovary and adipose tissue was observed. On the other hand, the t1/2 of N4-AcSDM in the kidney (8.1 h) was significantly longer than that in the above 3 tissues.

Effect of phenylbutazone on serum protein binding and pharmacokinetic behavior of sulfadimethoxine in rabbits, dogs and rats.

The effect of phenylbutazone (PBZ) on the in vitro binding of sulfadimethoxine (SDM) to serum or albumin was compared among rabbits, dogs and rats. In rabbits, a major metabolite of SDM, N4-acetylsulfadimethoxine (N4-AcSDM), markedly reduced the in vitro binding of SDM, and PBZ significantly increased the serum concentration of N4-AcSDM after SDM administration. PBZ did not affect the in vitro binding of SDM. These findings indicate that in rabbits, PBZ indirectly reduces the in vivo binding of SDM through the interaction of PBZ with N4-AcSDM. In dogs, both PBZ and N4-AcSDM caused the reduction in the in vitro binding of SDM. However, unlike rabbits, the contribution of N4-AcSDM to the in vivo binding of SDM appeared to be negligible in dogs. In rats, PBZ or N4-AcSDM had little effect on the in vitro binding of SDM. The co-administration of PBZ significantly increased the total body clearance and steady-state volume of distribution of SDM in rabbits. Such changes in pharmacokinetic behavior were not observed in dogs and rats.