Eiichi Kokue

Reduced folate derivatives are endogenous substrates for cMOAT in rats

We examined the role of the canalicular multispecific organic anion transporter (cMOAT) in the biliary excretion of reduced folate derivatives in vivo and in vitro using normal [Sprague-Dawley rats (SDR)] and mutant [Eisai hyperbilirubinemic rats (EHBR)] rats whose cMOAT is hereditarily deficient. In vivo, the biliary excretion of endogenous tetrahydrofolate (H4PteGlu), 5-methyltetrahydrofolate (5-CH3-H4PteGlu), and 5,10-methylenetetrahydrofolate (5, 10-CH2-H4PteGlu) in EHBR was reduced to 8.2%, 1.9%, and 5.5% of those in SDR, respectively, whereas that of 10-formyltetrahydrofolate (10-HCO-H4PteGlu) was detected only in SDR and not in EHBR. Bile drainage caused reduction of endogenous plasma folate concentrations in SDR but not in EHBR. In vitro, significant ATP-dependent uptake of 3H-labeled 5-CH3-H4PteGlu into canalicular membrane vesicles was observed only in SDR. This ATP-dependent uptake was saturable with a Michaelis constant (Km) value of 126 microM, which was comparable with its inhibitor constant (Ki) value of 121 microM for the ATP-dependent uptake of a typical cMOAT substrate, 2,4-dinitrophenyl-S-glutathione (DNP-SG). Vice versa, DNP-SG inhibited the uptake of 5-CH3-H4PteGlu with a Ki of 35 microM, which was similar to its Km value. In addition, H4PteGlu and 5, 10-CH2-H4PteGlu also inhibited the ATP-dependent uptake of DNP-SG. These results indicate that 5-CH3-H4PteGlu and other derivatives are transported via cMOAT. Therefore, reduced folate derivatives are the first endogenous substrates for cMOAT that do not contain glutathione, glucuronide, or sulfate moieties.

Effect of split-focus approach on producing larger coagulation in swine liver.

The split-focus approach has the potential to substantially improve the throughput of coagulation high-intensity focused ultrasound (HIFU) treatment. A prototype split-focus transducer with four elements at 4.25 MHz, combined with a small imaging probe at 6.5 MHz, was constructed for transrectal treatment of a prostate. Computer simulation predicted that 1.57 times larger acoustic power than a single-spot focus was required for a split focus to heat the tissue up to the same peak temperature at 4 s from the start of insonation. When the peak temperature was 100 degrees C, the tissue volume above 70 degrees C was predicted to be 2.3 times larger for a split focus. Swine liver lobes were intraoperatively insonated with the split-focus transducer for 4 s. A lesion of coagulation necrosis 3 to 4 times larger in volume than with a single-spot focus was formed with a split focus at the same acoustic power ratio as described above. Furthermore, a lesion about 2 times larger in volume was formed when compared at the same acoustic power. These results show the great advantage of a split-focus approach over a conventional single-spot focus in coagulation treatment.

Pyrimethamine Teratogenesis by Short Term Administration in Goettingen Miniature Pig

Pyrimethamine was mixed with mashed feed and given to pregnant sows of Goettingen miniature pig through a part of the period of organogenesis. A high incidence of the major malformations such as cleft palate, club foot and micrognathia was observed in 13 out of 24 newborns from 5 pregnants which a teratogenic dose (3.6 mg/kg of body weight/day) from day 11 to day 22 of gestation, the first half of the organogenetic period. Only one cleft palate was observed among the 23 newborns from 4 pregnants administered the same dose through the second half of the period. Among the 51 newborns from 10 pregnants which the same dose through the first or second quarter, only 4 newborns showed external major malformations. However, 19 out of 38 live newborns without external major malformations in these groups died within 2 days after birth. Clinical symptoms of these neonatally dead young were similar to splayleg, a naturally occurring functional anomaly in pigs. No internal malformation was revealed by the autopsy of these dead newborns.

Use of the goettingen miniature pig for studying pyrimethamine teratogenesis

Although pigs are suitable animals for experimental teratology, use of miniature pigs developed as a medicobiological experimental animal is essential for this purpose. Miniature pigs are established genetically so that the background data on the naturally occurring birth defects are reliable, whereas the commercial breeds are always improved genetically to seek higher productivity. In this review the authors summarized pyrimethamine teratogenesis in Goettingen miniature pigs. Pyrimethamine administration to a pregnant sow during the early stage of the organogenetic period caused cleft palate and other birth defects, and also hind leg paralysis, a functional defect in newborns.

Disease-induced alterations in plasma drug-binding proteins and their influence on drug binding percentages in dogs.

Summary Disease‐induced variations of plasma albumin (ALB) and alph1‐acid glycoprotein (AAG) levels were investigated in dogs. Lower ALB (sometimes >50% reduction) and higher AAG (sometimes >10‐fold increase) levels were observed in dogs with various diseases. Drug binding was determined at therapeutic concentrations using normal, low‐ALB and high‐AAG dog plasma. The binding percentages of the ALB‐binding drugs decreased in low‐ALB plasma, resulting in a large increase in unbound drug, particularly for naproxen (a 13‐fold increase). The binding percentages of all AAG‐binding drugs investigated in this study increased in high‐AAG plasma, resulting in a large decrease in unbound drug, particularly for quinidine (99% decrease). The fluctuation in the unbound fraction of drugs could affect their efficacy or could cause side‐effects. Veterinary clinicians should monitor the ALB and AAG levels in the plasma of patients and correct dosage regimens according to these levels, where field conditions permit this, in order to ensure the proper usage of drugs with high affinity for ALB or AAG.

Effects of Folic Acid and Folinic Acid on Pyrimethamine Teratogenesis in Rats

Abstract The effects of folic acid (FA) and folinic acid (FNA) on the teratogenicity of the antifolate drug pyrimethamine (PYR) were examined in rats. PYR and FA were orally administered with mashed feed. FNA was intraperitoneally injected. The drugs were administered for 5 days of day 11–15 of gestation. Six groups were made; G‐I PYR 1.6 (mg/kg/day), G‐II PYR 3.6, G‐HI PYR 1.6 + FA 50, G‐IV PYR 3.6 + FNA 12, G‐V FA 50, G‐VI control. No malformations were found in G‐I, IV, V and VI. All fetuses in G‐II and III had malformations. Main malformations in G‐II and III were cleft palate and brachygnathia. Accordingly it might be demonstrated that the teratogenicity of PYR was potentiated by concurrent FA in rats.

Identification of 10-formyltetrahydrofolate, tetrahydrofolate and 5-methyltetrahydrofolate as major reduced folate derivatives in rat bile

Reduced folate derivatives in rat bile were examined using high-performance liquid chromatography with electrochemical detection (HPLC-ED). Three peaks of folate compounds were observed on the chromatograms. From the retention-time profiles and hydrodynamic voltammograms, and the profiles of ultraviolet (UV) absorbance spectra obtained by HPLC with photodiode array detection, these 3 peaks were identified as 10-formyltetrahydrofolate (10-HCO-H4PteGlu), tetrahydrofolate (H4PteGlu) and 5-methyltetrahydrofolate (5-CH3-H4PteGlu). The rates of bile secretion of 10-HCO-H4PteGlu, H4PteGlu and 5-CH3H4PteGlu were 314 +/- 181, 321 +/- 179 and 449 +/- 198 ng/h (mean +/- S.D.), respectively. 10-HCO-H4PteGlu and H4PteGlu together wtih 5-CH3-H4-PteGlu are found to be the major folate derivatives in rat bile. The nonmethylated folates, 10-HCO-H4PteGlu and H4PteGlu, may also play an important role in folate homeostasis.

Implication of altered levels of plasma α1‐acid glycoprotein and its ‐derived sialic acid on plasma protein binding of trimethoprim in pigs in physiological and pathological states

In growing pigs (0 to 158 days after birth), pregnant sows (0 to 90 days), and in pigs suffering from respiratory disease (Actinobacillus pleuropneumoniae), the plasma levels of alpha(1)-acid glycoprotein (AGP) and its derived sialic acid were determined, as was the meaning of their altered levels on the plasma protein binding of trimethoprim. The AGP level was very high immediately after birth (more than 10,000 mu g/ml), decreased markedly during 2 weeks after birth (about 700 mu g/ml) and thereafter stayed at a constant level (about 400 mu g/ml). Pregnant sows had a low AGP level with a narrow variation throughout pregnancy (about 190 to 260 mu g/ml). Pigs infected with A. pleuropneumoniae showed an increased AGP level (mean value; 732 mu g/ml) with a wide variation (range: 170-1,840 mu g/ml). N-acetylneuraminic acid (NANA) and N-glycolylneuraminic acid (NGNA) were sialic acid subtypes detected in porcine plasma. In growing pigs, the time course of changes in NANA concentrations was consistent with that of AGP, whereas that of NGNA was different, implying that NANA is a sialic acid subtype derived from porcine AGP, in contrast to NGNA. The relationship between AGP and NANA levels in growing pigs could be expressed by the following equation: NANA=0.14 x AGP + 159 mu g/ml, whereas that in pigs with respiratory disease could be expressed by NANA=0.O67 x AGP + 357 mu g/ml, indicating a low fraction of NANA in AGP in diseased pigs. The regression lines between the AGP level and the plasma protein binding of trimethoprim < or = 2,000 mu g of AGP/ml were similar as follows: binding(%)=0.O23 x GP + 34 in growing pigs and binding(%)=0.O22 x GP+29 in diseased pigs, implying a minor role of sialic acid residues in the binding of basic drugs to AGP. In conclusion, the wide change in plasma AGP levels in diseased pigs as well as during the initial growth phase can alter the plasma protein binding of basic drugs such as trimethoprim, probably leading to a change in drug disposition. The low sialylation of AGP in diseased pigs may not have a great influence on the binding of basic drugs to AGP, implying the quantitative importance of AGP.

α1‐Acid glycoprotein‐binding as a factor in age‐related changes in the pharmacokinetics of trimethoprim in piglets

This study examined the effects of plasma alpha 1-acid glycoprotein (AGP) on the protein binding and pharmacokinetics of trimethoprim (TMP) in piglets. The piglets were given 5 mg/kg of TMP intravenously at 1, 14 and 28 days after birth. The plasma AGP level was highest at day 1. Fourteen days after birth, the level decreased by about 90% of that at day 1. The level at 28 days was almost the same as that at 14 days. Plasma protein bindings of TMP depended on the AGP level but not on the albumin level. The percentage of plasma protein binding decreased from 85 to 45%, and the AGP level also decreased from 6,000 to 700 micrograms/ml. The altered protein binding of TMP affected pharmacokinetic parameters such as total body clearance (CLtot), distribution volume and therefore the elimination rate constant. These parameters correlated well with the percentage binding to plasma proteins. Maturational development in the capacity to eliminate TMP was also indicated by the increase in total body clearance of unbound drug (CLtotub), which directly reflects the elimination capacity of the body. However, its contribution to the increase in CLtot was considered not to be large. CLtotub increased twofold 14 days after birth, whereas CLtot increased about ninefold. The increase in CLtot therefore, may result from both the maturational development in elimination capacity and the AGP-dependent decrease in plasma protein binding. It is concluded that the decrease in plasma AGP level observed in piglets is one of major factors affecting the pharmacokinetics of TMP.

Effects of Pyrimethamine and Folic Acid on Plasma Level of 5-Methyltetrahydrofolic Acid in Rats

Abstract: The relationship between pyrimethamine (PYR) teratogenesis and the plasma level of 5‐methyltetrahydrofolic acid (5MF), known as an active form of folate in plasma, was studied in rats using an HPLC‐ECD method for 5MF determination. The rat received, for 5 days, in‐feed PYR with or without folic acid (FA), which was previously reported as a potentiator of PYR teratogenesis. PYR alone caused a dose‐dependent decrease in the plasma 5MF level after the 5 day dosings. A potentiated decrease in the 5MF level was observed after the concomitant dosing of PYR with FA. The concomitant dosing of PYR 1.6 mg/kgBW/day (subteratogenic dose of PYR alone) with FA 50 mg/kg/day, which was 100% teratogenic dose in our previous report, decreased the 5MF level more than that after the dosing of PYR 3.6 mg/kg/day alone (100% teratogenic dose of PYR alone).