Radiosynthesis and evaluation of N5-(2-18F-fluoropropanyl) ornithine as a potential agent for tumor PET imaging.

OBJECTIVE: Studies have confirmed that tumorigenesis is related to an imbalance of polyamine metabolism and over-expression of oncogenes resulting in the up-regulation of ornithine decarboxylase (ODC, the first rate-limiting enzyme for regulating intracellular polyamines biosynthesis), which has become a target for anti-tumor therapy. In this study, an ornithine derivative, N5-(2-[18F]fluoropropionyl) ornithine (N5-[18F]FPO), has been prepared and its potential utility for tumor PET imaging evaluated. METHODS: N5-[18F]FPO was successfully prepared via a nucleophilic fluorination reaction and a subsequent efficient deprotection step. The in vitro and in vivo stability were determined by HPLC conducted in fetal bovine serum, saline and rat urine. Cellular uptake studies were conducted in HepG2 cells and the biodistribution and micro-PET/CT imaging performed in normal ICR mice and three tumor-bearing mice models, respectively. RESULTS: Total synthesis time of N5-[18F]FPO was about 80 min with a radiochemical yield of 15% ± 6% (uncorrected, based on 18F-, n = 6) and a high radiochemical stability can be seen in vitro and vivo. The N5-[18F]FPO exhibited fast uptake in HepG2 cells and the cellular uptake ability of N5-[18F]FPO can be inhibited by L-ornithine and DFMO, which indicated that the transport pathway of N5-[18F]FPO is similar to that of L-ornithine, interacting with ODC after being transported into the cell. The biodistribution and micro-PET/CT images demonstrate that N5-[18F]FPO was excreted by the urinary system, and excellent tumor visualization with high tumor-to-background ratios can be observed in the three tumor-bearing mice models studied. CONCLUSION: All the above results suggest that N5-[18F]FPO has the potential to be a novel radiotracer for imaging ODC expression in solid tumors.

Safety, tolerability, and pharmacokinetics of l-ornithine phenylacetate in patients with acute liver injury/failure and hyperammonemia.

Cerebral edema remains a significant cause of morbidity and mortality in patients with acute liver failure (ALF) and has been linked to elevated blood ammonia levels. l-ornithine phenylacetate (OPA) may decrease ammonia by promoting its renal excretion as phenylacetylglutamine (PAGN), decreasing the risk of cerebral edema. We evaluated the safety, tolerability, and pharmacokinetics of OPA in patients with ALF and acute liver injury (ALI), including those with renal failure. Forty-seven patients with ALI/ALF and ammonia ≥60 µM were enrolled. Patients received OPA in a dose escalation scheme from 3.3 g every 24 hours to 10 g every 24 hours; 15 patients received 20 g every 24 hours throughout the infusion for up to 120 hours. Plasma phenylacetate (PA) concentrations were uniformly below target (<75 µg/mL) in those receiving 3.3 g every 24 hours (median [interquartile range] 5.0 [5.0] µg/mL), and increased to target levels in all but one who received 20 g every 24 hours (150 [100] µg/mL). Plasma [PAGN] increased, and conversion of PA to PAGN became saturated, with increasing OPA dose. Urinary PAGN clearance and creatinine clearance were linearly related (r = 0.831, P < 0.0001). Mean ammonia concentrations based on the area under the curve decreased to a greater extent in patients who received 20 g of OPA every 24 hours compared with those who received the maximal dose of 3.3 or 6.7 g every 24 hours (P = 0.046 and 0.022, respectively). Of the reported serious adverse events (AEs), which included 11 deaths, none was attributable to study medication. The only nonserious AEs possibly related to study drug were headache and nausea/vomiting. CONCLUSION: OPA was well-tolerated in patients with ALI/ALF, and no safety signals were identified. Target [PA] was achieved at infusion rates of 20 g every 24 hours, leading to ammonia excretion in urine as PAGN in proportion to renal function. Randomized, controlled studies of high-dose OPA are needed to determine its use as an ammonia-scavenging agent in patients with ALF. (Hepatology 2018;67:1003-1013).

Safety of ornithine phenylacetate in cirrhotic decompensated patients: an open-label, dose-escalating, single-cohort study.

AIMS: Confirm in patients with cirrhosis and gastrointestinal bleeding the safety of ornithine phenylacetate (OP) and assess the pharmacokinetic profile of OP and its effects on plasma ammonia. BACKGROUND: OP is a drug that has shown experimentally to decrease hyperammonemia and improve hepatic encephalopathy. OP is safe in healthy subjects and in stable patients with cirrhosis, but there are no data in decompensated cirrhosis. METHODS: We performed a study to assess safety and tolerance of OP in cirrhotic patients after an episode of upper gastrointestinal bleeding.Ten patients were included within 24 hours of an upper gastrointestinal bleeding. OP was administered as a continuous infusion up to a maximum of 10 g/24 h (0.42 g/h) for 5 days. The infusion was started at 33% of the target dose and increased at 12-hour intervals achieving target dose at 24 hours. Ammonia was also assessed in control group of 10 patients. RESULTS: No severe adverse events were observed. Mild adverse events were reported in 4 patients. Plasma ammonia (baseline: 80±43 µmol/L) showed a progressive drop between baseline and 36 hours (42±15 µmol/L), 72 hours (44±15 µmol/L), 96 hours (40±24 µmol/L), and 120 hours (33±14 µmol/L). Plasma ammonia at 24 hours was significantly higher in the control group. Plasma glutamine showed a significant decrease (-37% at day 5) and its excretion in urine as phenylacetylglutamine, a progressive rise (52±35 mmol at day 5). CONCLUSIONS: OP is a safe and well-tolerated drug in decompensated cirrhotics that may decrease plasma ammonia by inducing its appearance as phenylacetylglutamine in urine.

Interorgan metabolism of ornithine phenylacetate (OP)--a novel strategy for treatment of hyperammonemia.

Combined administration of ornithine and phenylacetate (OP) is proposed as a novel treatment of hyperammonemia and hepatic encephalopathy. Ornithine is believed to increase ammonia fixation into glutamine in muscle tissue and glutamine is subsequently thought to react with phenylacetate forming phenylacetylglutamine (PAGN) which is excreted in urine. The aim of the present study was to elucidate the interorgan metabolism of ornithine and ammonia in cirrhotic rats treated with OP in order to obtain an understanding of the underlying mechanisms of the beneficial effect of the treatment, which are largely unknown. Bile duct ligated cirrhotic rats and SHAM rats were treated with OP or saline for five days. [2,5-(15)N]Ornithine or (15)NH(4)(+) were administered intravenously and the incorporation of (15)N in amino acids as well as the content of the amino acids were subsequently determined in plasma, skeletal muscle, liver and kidney. In BDL rats, OP treatment reduced arterial ammonia concentration and increased that of glutamine 30 min after the treatment but not after 15 h. OP treatment did not increase (15)N labeling in glutamine from [2,5-(15)N]ornithine and (15)NH(4)(+) in skeletal muscle or liver. However, the extent of glutamine labeling from [2,5-(15)N]ornithine or (15)NH(4)(+) was similar in arterial blood and liver and higher than that in skeletal muscle. These findings suggest that the effect of OP was related to hepatic metabolism of ornithine. PAGN could not be detected in urine or blood in any of the rats which may explain why OP treatment only reduced arterial ammonia transiently.

Chronic postnatal ornithine administration to rats provokes learning deficit in the open field task.

Hyperornithinemia is the biochemical hallmark of hyperornithinemia-hyperammonemia-homocitrullinuria (HHH) syndrome, an inherited metabolic disease clinically characterized by mental retardation whose pathogenesis is still poorly known. In the present work, we produced a chemical animal model of hyperornithinemia induced by a subcutaneous injection of saline-buffered Orn (2-5 µmol/g body weight) to rats. High brain Orn concentrations were achieved, indicating that Orn is permeable to the blood brain barrier. We then investigated the effect of early chronic postnatal administration of Orn on physical development and on the performance of adult rats in the open field, the Morris water maze and in the step down inhibitory avoidance tasks. Chronic Orn treatment had no effect on the appearance of coat, eye opening or upper incisor eruption, nor on the free-fall righting reflex and on the adult rat performance in the Morris water maze and in the inhibitory avoidance tasks, suggesting that physical development, aversive and spatial localization were not changed by Orn. However, Orn-treated rats did not habituate to the open field apparatus, implying a deficit of learning/memory. Motor activity was the same for Orn- and saline- injected animals. We also verified that Orn subcutaneous injections provoked lipid peroxidation in the brain, as determined by a significant increase of thiobarbituric acid-reactive substances levels. Our results indicate that chronic early postnatal hyperornithinemia may impair the central nervous system, causing minor disabilities which result in specific learning deficiencies.

Interaction between murine spf-ash mutation and genetic background yields different metabolic phenotypes.

The spf-ash mutation in mice results in reduced hepatic and intestinal ornithine transcarbamylase. However, a reduction in enzyme activity only translates in reduced ureagenesis and hyperammonemia when an unbalanced nitrogen load is imposed. Six-week-old wild-type control and spf-ash mutant male mice from different genetic backgrounds (B6 and ICR) were infused intravenously with [(13)C(18)O]urea, l-[(15)N(2)]arginine, l-[5,5 D(2)]ornithine, l-[6-(13)C, 4,4,5,5, D(4)]citrulline, and l-[ring-D(5)]phenylalanine to investigate the interaction between genetic background and spf-ash mutation on ureagenesis, arginine metabolism, and nitric oxide production. ICR(spf-ash) mice maintained ureagenesis (5.5 +/- 0.3 mmol.kg(-1).h(-1)) and developed mild hyperammonemia (145 +/- 19 micromol/l) when an unbalanced nitrogen load was imposed; however, B6(spf-ash) mice became hyperammonemic (671 +/- 15 micromol/l) due to compromised ureagenesis (3.4 +/- 0.1 mmol.kg(-1).h(-1)). Ornithine supplementation restored ureagenesis and mitigated hyperammonemia. A reduction in citrulline entry rate was observed due to the mutation in both genetic backgrounds (wild-type: 128, spf-ash: 60; SE 4.0 micromol.kg(-1).h(-1)). Arginine entry rate was only reduced in B6(spf-ash) mice (B6(spf-ash): 332, ICR(spf-ash): 453; SE 20.6 micromol.kg(-1).h(-1)). Genetic background and mutation had an effect on nitric oxide production (B6: 3.4, B6(spf-ash): 2.8, ICR: 9.0, ICR(spf-ash): 4.6, SE 0.7 micromol.kg(-1).h(-1)). Protein breakdown was the main source of arginine during the postabsorptive state and was higher in ICR(spf-ash) than in B6(spf-ash) mice (phenylalanine entry rate 479 and 327, respectively; SE 18 micromol.kg(-1).h(-1)). Our results highlight the importance of the interaction between mutation and genetic background on ureagenesis, arginine metabolism, and nitric oxide production. These observations help explain the wide phenotypic variation of ornithine transcarbamylase deficiency in the human population.

Pharmacokinetics of arginine and related amino acids.

Arginine (ARG) and its related amino acids (AAs) ornithine (ORN) and citrulline (CIT) find a range of applications as dietary supplements in subgroups of healthy subjects (e.g., bodybuilders) and patients with acute or chronic malnutrition. These AAs appear to be well utilized in humans with, in general, a rapid return of blood concentrations to basal values (i.e., within 5-8 h) and low absolute and relative excretion in urine (<5% of administered dose). Based on published data for the maximum observed plasma concentrations (Cmax) after administration of doses in the range 5 to 10 g, CIT appeared to present relatively better absorption and systemic bioavailability than ARG and ORN. The few relevant dose-ranging studies available include 1 limited to a single subject receiving 5- to 20-g doses of ornithine alpha-ketoglutarate and another in which 8 subjects received from 5 to 15 g of CIT. Comparison of these 2 studies further indicates that CIT has higher bioavailability than ORN. The pharmacokinetics and metabolism of these AAs are modified by the coadministration of a salt such as alpha-ketoglutarate that modifies AA metabolism, as has clearly been demonstrated for ornithine alpha-ketoglutarate. Concomitant administration of a meal leads to a 15- to 30-min delay in Cmax. Finally, data from various pharmacokinetic studies together with basic physiology and biochemistry indicate that ARG is a net urea producer and ORN has a nitrogen-sparing effect, whereas CIT is neutral. However, most of the studies performed to date carry methodological weaknesses and are difficult to compare because of a number of confounding factors. To date, there have been no pharmacokinetic studies on the long-term administration of these AAs in healthy subjects despite the need to determine the safe upper limit of daily intake.

Ornithine alpha-ketoglutarate metabolism in the healthy rat in the postabsorptive state.

To gain further insight into the ability of ornithine alpha-ketoglutarate (OKG) to generate key metabolites, the aim of this work was to study the short-term metabolism, that is, 1 hour after administration, of OKG in plasma and tissues. Particular attention was paid to keto acids (alpha-ketoglutarate and branched-chain keto acids). Young (3 weeks old) male Wistar rats in the postabsorptive state received either 1.5 g/kg of monohydrated OKG (OKG group, n = 8) diluted in distilled water or an equivalent volume of saline solution at 0.9% (control group, n = 8) by gavage and were killed 1 hour later. Plasma, liver, jejunal and ileal mucosa, and the extensor digitorum longus muscle were removed to analyze amino and keto acid contents. Major metabolites detected after OKG ingestion (ornithine [ORN], alpha-ketoglutarate, proline and glutamate; OKG vs control, P < .05) and the absence of increased arginine (and even a decrease in jejunum and muscle) and citrulline levels suggested that ORN was mainly metabolized by the ORN aminotransferase pathway. In addition, significantly decreased plasma branched-chain keto acids and increased hepatic branched-chain amino acids (OKG vs control, P < .05) were observed upon OKG ingestion. Finally, glutamine accumulation restricted to the intestine, as evidenced in this short-term study, suggests that the effects of OKG on glutamine pools in other tissues in various pathological states after several days of treatment, as observed in previous studies, may be related to a long-term induction of glutamine synthetase.

Discovery of diphenyloxazole and Ndelta-Z-ornithine derivatives as highly potent and selective human prostaglandin EP(4) receptor antagonists.

Two novel classes of diphenyloxazole and Ndelta-Z-ornithine derivatives as highly potent and selective EP(4) antagonists have been discovered. The optimized diphenyloxzole 8 and Ndelta-Z-ornithine 11 effectively competed with [(3)H]PGE(2) binding to human recombinant EP(4), with K(i) values of 0.30 nM and 0.91 nM, respectively, and were selective for all members of the human prostanoid receptor family. 8 was shown to exhibit good pharmacokinetic properties in rats and dogs and potent inhibitory activity toward in vitro PGE(2)-promoted IgE synthesis.

Comparative uptake of polyamines by prostate and non-prostate cancer cell lines.

The Km and Vmax of [14C]-radiolabeled polyamines were determined for PC-3 and AT3B-1 cell lines. With PC-3 Km values are in the following order: ornithine> spermidine> spermine> putrescine, while with AT3B-1 it was spermidine> ornithine> spermine> putrescine. To determine which of these polyamines exhibit higher accumulation, the relative uptake of all the four amines was studied with prostate (PC-3, AT3B-1, LNCaP) and non-prostate (MCF-7, KLN-205, OVCAR) cell lines at 10 and 20 microM after 1 hour. Spermine and spermidine accumulated at higher levels in prostate (AT3B-1 and LNCaP) over non-prostate cell lines (p < 0.01). Putrescine accumulated more in PC-3 and LNCaP than the non-prostate cancer cells.

Regulation of uterine and umbilical amino acid uptakes by maternal amino acid concentrations.

We tested the hypothesis that decreased fetal amino acid (AA) supply, produced by maternal hypoaminoacidemia (low AA) during hyperglycemia (HG), is reversible with maternal AA infusion and regulates fetal insulin concentration ([I]). We measured net uterine and umbilical AA uptakes during maternal HG/low AA concentration ([AA]) and after maternal intravenous infusion of a mixed AA solution. After 5 days HG, all maternal [AA] except glycine were decreased >50%, particularly essential [AA] (P < 0.00005). Most fetal [AA] also were decreased, especially branched-chain AA (P < 0.001). Maternal AA infusion increased net uterine uptakes of Val, Leu, Ile, Met, and Ser and net umbilical uptakes of Val, Leu, Ile, Met, Phe, and Arg but did not change net uteroplacental uptake of any AA. Fetal [I] increased 55 +/- 14%, P < 0.001, with correction of fetal [AA], despite the lack of change in fetal glucose concentration. Thus generalized maternal hypoaminoacidemia decreases uterine and umbilical uptakes of primarily the essential AA and decreases fetal branched-chain [AA]. These changes are reversed with correction of maternal [AA], which also increases fetal [I].

Characterization of L-arginine transporters in rat renal inner medullary collecting duct.

Previous work from our laboratory has demonstrated that the inner medullary collecting duct (IMCD) expresses a large amount of nitric oxide synthase (NOS) activity. The present study was designed to characterize the transport of NOS substrate, L-arginine, in a suspension of bulk-isolated IMCD cells from the Sprague-Dawley rat kidney. Biochemical transport studies demonstrated an L-arginine transport system in IMCD cells that was saturable and Na(+) independent (n = 6). L-Arginine uptake by IMCD cells was inhibited by the cationic amino acids L-lysine, L-homoarginine, and L-ornithine (10 mmol/l each) and unaffected by the neutral amino acids L-leucine, L-serine, and L-glutamine. Both L-ornithine (n = 6) and L-lysine (n = 6) inhibited NOS enzymatic activity in a dose-dependent manner in IMCD cells, supporting the important role of L-arginine transport for NO production by this tubular segment. Furthermore, RT-PCR of microdissected IMCD confirmed the presence of cationic amino acid transporter CAT1 mRNA, whereas CAT2A, CAT2B, and CAT3 were not detected. These results indicate that L-arginine uptake by IMCD cells occurs via system y(+), is encoded by CAT1, and may participate in the regulation of NO production in this renal segment.

The effect of side-chain, para-aminobenzoyl region, and B-ring modifications on dihydrofolate reductase binding, influx via the reduced folate carrier, and cytotoxicity of the potent nonpolyglutamatable antifolate N(alpha)-(4-amino-4-deoxypteroyl)-N(delta)-hemiphthaloyl-L- ornithine.

N(alpha)-(4-Amino-4-deoxypteroyl)-N(delta)-hemiphthaloyl-L-o rnithine (PT523) is an unusually tight-binding dihydrofolate reductase (DHFR) inhibitor and is efficiently taken up into cells via the reduced folate carrier (RFC). Unlike classical DHFR inhibitors with a glutamate side chain, such as methotrexate and aminopterin, PT523 cannot form polyglutamates. Thus, it resembles lipophilic antifolates such as trimetrexate in not requiring metabolic activation by folylpolyglutamate synthetase in order to produce its antifolate effect. However, in contrast to trimetrexate, PT523 retains growth inhibitory activity in cells with the multidrug resistance phenotype. As part of the preclinical development of this drug, we have performed systematic modification of several regions of the PT523 molecule, with the aim of defining the optimal structural features for DHFR binding, influx into cells via the RFC, and the ability to inhibit cell growth. The following structure-activity correlations have emerged from this ongoing investigation, and are discussed: (1) the hemiphthaloylornithine side chain has the optimal length; (2) the preferred location of the aromatic carboxyl group is the ortho position; and (3) replacement of the phenyl ring of the para-aminobenzoic acid moiety by naphthalene, of nitrogen at the 10-position of the bridge by carbon, and of nitrogen at the 5- and/or 8-position of the B-ring by carbon are all well tolerated. Several of the second generation analogs of PT523 are more potent DHFR inhibitors and better RFC substrates than PT523 itself, and are more potent inhibitors of tumor cell growth in culture.

Blood polyamine levels after oral ornithine load, a diagnostic marker of hyperproliferative premalignant and malignant stages in a model of colon carcinogenesis.

This study was performed to determine whether a single oral dose of ornithine (Orn), the substrate of ornithine decarboxylase (ODC), increases blood concentrations of polyamines premalignant stage, and whether blood polyamine levels could be used as predictive markers of cancer development. Male Wistar rats were divided into two groups, control and 1,2-dimethylhydrazine (DMH)-treated rats. DMH (20 mg/kg body weight) was injected intraperitoneally once weekly for 10 weeks. Five, 7, and 10 weeks after the last injection when premalignant aberrant crypt foci have developed in the colon, blood levels of putrescine (PUT), spermidine (SPD), and spermine (SPM) were estimated before and after an oral load of ORN. The results showed that after a single oral load of Orn, blood PUT, but not SPD and SPM, concentrations were significantly higher in DMH-treated rats compared with control rats, indicating enhancement of ODC activity. These results support the view that the increased blood concentration of PUT after administration of Orn may be a useful marker to detect hyperproliferative premalignant and malignant stages of cancer development.

Pharmacokinetics, antifolate activity and tissue distribution of PT523 in SCC VII tumor-bearing mice.

PURPOSE: To monitor the pharmacokinetics of PT523 and methotrexate in C3H mice with transplanted SCC VII tumors; to compare the impact of PT523 and methotrexate on tumor and normal host 5,10-methylenetetrahydrofolate levels; and to synthesize [14C]PT523 and determine its time-dependent tissue distribution in tumor and host tissues. METHODS: C3H mice bearing SCC VII tumors were given i.p. PT523 or methotrexate. Plasma drug levels and tumor, gut and marrow 5,10-methylenetetrahydrofolate were assayed. [14C]PT523 was synthesized and administered i.v. to tumor-bearing mice for tissue distribution analysis. RESULTS: Areas under the curve, mean residence times, whole body clearances, apparent distribution volumes, and plasma protein binding of PT523 vs methotrexate were, respectively, 4311 vs 6472 microM x min(-1); 20 vs 16 min; 0.56 vs 0.36 ml min(-1); 532 vs 325 ml x kg(-1); and 70% vs 30%. Both PT523 and methotrexate caused time-dependent declines in 5,10-methylenetetrahydrofolate in tumor and marrow, but not in gut mucosa [corrected]. Gut levels began to recover within 4 h in the PT523-treated group only. [14C]PT523 distributed mainly into the liver, duodenum, kidneys, lungs, tumor, pancreas and muscle; less into the spleen, blood cells, heart, brain and testicles; and very little into gut [corrected. Only 35% of the dose was excreted, and 2.9-fold more in feces than urine. CONCLUSIONS: Despite its more rapid clearance, accumulation of PT523 in extravascular tissues was greater than that of methotrexate. Consequently, less PT523 was recovered in feces and urine and its apparent volume of distribution was greater. PT523 selectively depleted 5,10-methylenetetrahydrofolate pools in tumor and, less persistently, in marrow, but spared the gut mucosa [corrected]. [14C]PT523 tissue distribution correlated with organ mass and blood supply.

Macula densa nitric oxide synthase: expression, regulation, and function.

The type 1 brain nitric oxide synthase (bNOS) isoform occurs in macula densa (MD) cells where it functions to vasodilate the afferent arteriole and blunt expression of tubuloglomerular feedback (TGF). Dietary salt restriction enhances bNOS expression, yet microperfusion studies with NOS inhibitors imply that it is functionally inactive. We thus assessed the hypothesis that reduced L-arginine (L-Arg) availability during low salt (LS) intake limits MD NO generation. Maximal TGF responses were recorded during Henle's loop perfusion with artificial tubular fluid (ATF). Microperfusion of L-Arg into the MD of LS, but not normal or high-salt (HS) rats blunted maximal TGF responses (8.0 +/- 0.4 to 6.0 +/- 0.5 mm Hg; N = 23; P < 0.01). Response to L-Arg was stereospecific, inhibited by coperfusion with monomethyL-L-arginine (L-NMA), and dependent on system y+ transport, because it was blocked by coperfusion with the competitors L-lysine or L-homoarginine. Absorption of [3H]-L-Arg from the perfused loop, via an L-Arg- or L-homoarginine-inhibitable process, was enhanced during HS. Salt restriction thus diminishes TGF attenuation by NO in the MD despite enhanced bNOS expression because of limited delivery and/or uptake of L-Arg via system y+. This defines a novel mechanism of renal microcirculatory adaptation to salt restriction via L-Arg-dependent changes in TGF.

Transport of arginine and ornithine into isolated mitochondria of Saccharomyces cerevisiae.

In this work we have characterised the transport of L-arginine and L-ornithine into mitochondria isolated from a wild-type Saccharomyces cerevisiae strain and an isogenic arg11 knock-out mutant. The Arg11 protein (Arg11p) is a mitochondrial carrier required for arginine biosynthesis [Crabeel, M., Soetens, O., De Rijcke, M., Pratiwi, R. & Pankiewicz, R. (1996) J. Biol. Chem. 271, 25011-25019]. Reconstitution experiments have confirmed that it is an L-ornithine carrier also transporting L-arginine and L-lysine by order of decreasing affinity, but not L-histidine [Palmieri, L., De Marco, V., Iacobazzi, V., Palmieri, F., Runswick, M. & Walker, J. (1997) FEBS Lett. 410, 447-451]. Evidence is presented here that the mitochondrial inner membrane contains an L-arginine and L-ornithine transporting system distinct from Arg11p, in keeping with the arginine leaky phenotype of arg11 knock-out mutants. The newly characterised carrier, which we propose to name Bac1p (basic amino acid carrier), behaves as an antiporter catalysing the electroneutral exchange of the basic amino acids L-arginine, L-lysine, L-ornithine and L-histidine and displays the highest affinity for L-arginine (Km of 30 microM). L-Arginine uptake has a pH optimum in the range of 7.5-9 and is inhibited by several sulphydryl reagents, by pyridoxal 5'-phosphate and by cations.

Ornithine alpha-ketoglutarate metabolism after enteral administration in burn patients: bolus compared with continuous infusion.

Ornithine alpha-ketoglutarate (OKG) has been successfully used as an enteral supplement in the treatment of catabolic states, including burn injury. However, specific questions remain unanswered concerning burn patients, including OKG metabolism and metabolite production, appropriate mode of administration, and dose. We thus performed a kinetic study and followed plasma ornithine and OKG metabolite concentrations on day 7 postburn in 42 (35 men, 7 women) consecutive burn patients aged 33 +/- 2 y with a mean (+/-SEM) total burn surface area (TBSA) of 31 +/- 1%. Patients were randomly assigned to receive OKG as a single bolus (10 g; n = 13) or in the form of a continuous gastric infusion (10, 20, or 30 g/d over 21 h; n = 13) or an isonitrogenous control (n = 16). Plasma pharmacokinetics of ornithine followed a one-compartment model with first-order input (r = 0.993, P < 0.005). OKG was extensively metabolized in these patients (absorption constant = 0.028 min-1, elimination half-life = 89 min), with the production of glutamine, arginine, and proline; proline was quantitatively the main metabolite [in OKG bolus, area under the curve (AUC)0-7h: proline, 41.4 +/- 5.6 mmol.min/L; glutamine, 20.4 +/- 5.7 mmol.min/L; and arginine, 7.3 +/- 1.9 mmol.min/L]. Proline production was dose-dependent and quantitatively similar between modes of OKG administration. Glutamine and arginine production were not dose-dependent and were higher in the bolus group than in the infusion group. Overall, the bolus mode of OKG administration appeared to be associated with higher metabolite production compared with continuous infusion in burn patients, especially for glutamine and arginine.

Synthesis and biological evaluation of N alpha-(4-amino-4-deoxy-10-methylpteroyl)-DL-4,4-difluoroornithine.

N alpha-(4-Amino-4-deoxy-10-methylpteroyl)-DL-4,4-difluoroornithi ne (AMPte-DL-4,4-F2Orn, 4) was synthesized and evaluated as an inhibitor of human folypoly-gamma-glutamate synthetase (FPGS), dihydrofolate reductase (DHFR), and cell growth. Synthesis of 4 involved the use of a protected form of DL-4,4-difluoroornithine 9 which was derived from DL-4,4-difluoroglutamic acid. Biological activities of 4 were compared directly to those of the corresponding nonfluorinated compound N alpha-(4-amino-4-deoxy-10-methylpteroyl)-L-ornithine (AMPte-L-Orn, 3). Although the fluorinated analogue is a potent inhibitor of DHFR, it is a poor inhibitor of FPGS. However, the compound is transported across the cell membrane and inhibits cell growth, presumably due to the inhibition of DHFR. The data obtained with the fluorinated analogue are in contrast to those of the corresponding nonfluorinated compound 3, which is a potent inhibitor of both FPGS and DHFR but shows very low cytotoxicity due to poor transport.

Dihydrofolate reductase binding and cellular uptake of nonpolyglutamatable antifolates: correlates of cytotoxicity toward methotrexate-sensitive and -resistant human head and neck squamous carcinoma cells.

Several mechanisms have been demonstrated to be independently involved in methotrexate (MTX) resistance, including increased dihydrofolate reductase (DHFR) activity, decreased membrane transport, and decreased conversion to noneffluxing polyglutamates by folylpolyglutamate synthetase. We conducted the present study to test the hypothesis that nonpolyglutamatable antifolates with an N delta-hemiphthaloyl-L-ornithine side chain could be more potent than MTX against MTX-sensitive and -resistant human carcinoma cells via tighter DHFR binding, more efficient cellular uptake, the ability to bypass defective polyglutamation, or a combination. Two nonpolyglutamatable antifolates, N alpha-(4-amino-4-deoxypteroyl)-N delta-hemiphthaloyl-L-ornithine (PT523) and the new B-ring analogue N alpha-[4-[N-(2,4-diamino-5-chloroquinazolin-6-yl)methyl]amino] benzoyl-N delta-hemiphthaloyl-L-ornithine (PT619), were tested as inhibitors of purified recombinant human DHFR and were found to bind somewhat better to the enzyme than MTX as determined by competitive radioligand binding assay. PT523 and PT619 were 9- and 14-fold, respectively, more active than MTX as inhibitors of parental SCC25 human and neck squamous carcinoma cell growth in 72-hr cultures. Moreover, there was an even greater increase in relative potency against two previously described MTX-resistant cell lines with an increased DHFR content and a decreased ability to convert MTX to polyglutamates: SCC25/R1 (selected with MTX) and SCC25/CP (selected with cisplatin but collaterally resistant to MTX). Both PT523 and PT619 very efficiently inhibited [3H]MTX uptake by SCC25 cells in a 1-hr assay, with PT523 being 11-fold more potent and PT619 being 17-fold more potent than MTX. Greater inhibition of [3H]MTX uptake with PT523 and PT619 than with MTX was also observed in SCC25/R1 and SCC/CP cells. However, the increase in activity of PT523 and PT619 relative to MTX in uptake experiments was less than that in growth-inhibition assays, especially for SCC25/CP cells. This suggested that additional cytotoxicity determinants may exist in these resistant cells.