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1.
Nucl Med Biol ; 94-95: 98-105, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33621898

RESUMO

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.


Assuntos
Radioisótopos de Flúor/química , Ornitina/química , Ornitina/síntese química , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada , Tomografia por Emissão de Pósitrons , Animais , Linhagem Celular Tumoral , Camundongos , Ornitina/farmacocinética , Radioquímica , Ratos , Distribuição Tecidual
2.
Hepatology ; 67(3): 1003-1013, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29080224

RESUMO

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).


Assuntos
Hiperamonemia/tratamento farmacológico , Falência Hepática Aguda/tratamento farmacológico , Ornitina/análogos & derivados , Acetatos/sangue , Adolescente , Adulto , Idoso , Amônia/sangue , Feminino , Glutamina/análogos & derivados , Glutamina/metabolismo , Humanos , Hiperamonemia/complicações , Testes de Função Renal , Fígado/patologia , Falência Hepática Aguda/complicações , Masculino , Pessoa de Meia-Idade , Ornitina/administração & dosagem , Ornitina/efeitos adversos , Ornitina/farmacocinética , Fenóis/sangue , Sistema de Registros , Resultado do Tratamento , Adulto Jovem
3.
J Clin Gastroenterol ; 47(10): 881-7, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23751856

RESUMO

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.


Assuntos
Amônia/sangue , Hemorragia Gastrointestinal/etiologia , Cirrose Hepática/fisiopatologia , Ornitina/análogos & derivados , Idoso , Estudos de Coortes , Relação Dose-Resposta a Droga , Feminino , Seguimentos , Glutamina/análogos & derivados , Glutamina/sangue , Glutamina/urina , Humanos , Infusões Intravenosas , Cirrose Hepática/complicações , Masculino , Pessoa de Meia-Idade , Ornitina/administração & dosagem , Ornitina/efeitos adversos , Ornitina/farmacocinética , Fatores de Tempo
4.
Biochem Pharmacol ; 85(1): 115-23, 2013 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-23103564

RESUMO

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.


Assuntos
Acetatos/farmacocinética , Hiperamonemia/metabolismo , Ornitina/análogos & derivados , Fenóis/farmacocinética , Acetatos/uso terapêutico , Amônia/farmacocinética , Animais , Artérias/metabolismo , Ductos Biliares , Interações Medicamentosas , Feminino , Glutamina/metabolismo , Hiperamonemia/tratamento farmacológico , Hiperamonemia/etiologia , Rim/metabolismo , Ligadura , Fígado/metabolismo , Cirrose Hepática Biliar/etiologia , Cirrose Hepática Biliar/metabolismo , Músculo Esquelético/metabolismo , Isótopos de Nitrogênio , Ornitina/farmacocinética , Ornitina/uso terapêutico , Fenóis/uso terapêutico , Ratos , Ratos Wistar , Distribuição Tecidual
5.
Metab Brain Dis ; 27(4): 479-86, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-22699997

RESUMO

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.


Assuntos
Erros Inatos do Metabolismo dos Aminoácidos/induzido quimicamente , Deficiências da Aprendizagem/induzido quimicamente , Deficiências da Aprendizagem/psicologia , Ornitina/toxicidade , Erros Inatos do Metabolismo dos Aminoácidos/psicologia , Amônia/sangue , Animais , Animais Recém-Nascidos , Aprendizagem da Esquiva/efeitos dos fármacos , Comportamento Animal/efeitos dos fármacos , Citrulina/análogos & derivados , Citrulina/sangue , Cognição/efeitos dos fármacos , Cognição/fisiologia , Deficiências do Desenvolvimento/induzido quimicamente , Modelos Animais de Doenças , Meia-Vida , Aprendizagem em Labirinto/efeitos dos fármacos , Memória/efeitos dos fármacos , Memória de Longo Prazo/efeitos dos fármacos , Ornitina/farmacocinética , Equilíbrio Postural/efeitos dos fármacos , Ratos , Ratos Wistar , Substâncias Reativas com Ácido Tiobarbitúrico/metabolismo
6.
Am J Physiol Endocrinol Metab ; 293(6): E1764-71, 2007 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-17925451

RESUMO

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.


Assuntos
Mutação , Doença da Deficiência de Ornitina Carbomoiltransferase/metabolismo , Ornitina Carbamoiltransferase/metabolismo , Alanina/administração & dosagem , Alanina/metabolismo , Alanina/farmacocinética , Amônia/sangue , Amônia/metabolismo , Animais , Arginina/administração & dosagem , Arginina/metabolismo , Arginina/farmacocinética , Citrulina/sangue , Citrulina/metabolismo , Modelos Animais de Doenças , Feminino , Glicina/administração & dosagem , Glicina/metabolismo , Glicina/farmacocinética , Injeções Intravenosas , Mucosa Intestinal/metabolismo , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos ICR , Camundongos Endogâmicos , Camundongos Mutantes , Modelos Biológicos , Óxido Nítrico/metabolismo , Ornitina/administração & dosagem , Ornitina/metabolismo , Ornitina/farmacocinética , Ornitina Carbamoiltransferase/genética , Doença da Deficiência de Ornitina Carbomoiltransferase/genética , Fenótipo , Fenilalanina/administração & dosagem , Fenilalanina/metabolismo , Fenilalanina/farmacocinética , Ureia/sangue , Ureia/metabolismo
7.
J Nutr ; 137(6 Suppl 2): 1646S-1649S, 2007 06.
Artigo em Inglês | MEDLINE | ID: mdl-17513441

RESUMO

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.


Assuntos
Arginina/farmacocinética , Ingestão de Alimentos/fisiologia , Jejum/metabolismo , Citrulina/farmacocinética , Humanos , Ornitina/farmacocinética
8.
Metabolism ; 54(8): 1108-14, 2005 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-16092063

RESUMO

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.


Assuntos
Absorção Intestinal , Mucosa Intestinal/metabolismo , Ornitina/análogos & derivados , Aminoácidos de Cadeia Ramificada/metabolismo , Animais , Arginina/metabolismo , Citrulina/metabolismo , Ácido Glutâmico/metabolismo , Cetoácidos/metabolismo , Ácidos Cetoglutáricos/metabolismo , Fígado/metabolismo , Masculino , Músculo Esquelético/metabolismo , Ornitina/sangue , Ornitina/metabolismo , Ornitina/farmacocinética , Prolina/metabolismo , Ratos , Ratos Wistar
9.
J Med Chem ; 48(9): 3103-6, 2005 May 05.
Artigo em Inglês | MEDLINE | ID: mdl-15857112

RESUMO

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.


Assuntos
Adjuvantes Imunológicos/síntese química , Ornitina/análogos & derivados , Ornitina/síntese química , Oxazóis/síntese química , Receptores de Prostaglandina E/antagonistas & inibidores , Adjuvantes Imunológicos/farmacocinética , Adjuvantes Imunológicos/farmacologia , Animais , Linfócitos B/efeitos dos fármacos , Linfócitos B/metabolismo , Dinoprostona/farmacologia , Cães , Humanos , Imunoglobulina E/biossíntese , Técnicas In Vitro , Ornitina/farmacocinética , Ornitina/farmacologia , Oxazóis/farmacocinética , Oxazóis/farmacologia , Ensaio Radioligante , Ratos , Receptores de Prostaglandina E Subtipo EP4 , Estereoisomerismo , Relação Estrutura-Atividade
10.
Nucl Med Biol ; 29(4): 497-503, 2002 May.
Artigo em Inglês | MEDLINE | ID: mdl-12031886

RESUMO

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.


Assuntos
Radioisótopos de Carbono/farmacocinética , Poliaminas/farmacocinética , Neoplasias da Próstata/metabolismo , Animais , Neoplasias da Mama/diagnóstico por imagem , Neoplasias da Mama/metabolismo , Humanos , Masculino , Ornitina/farmacocinética , Neoplasias da Próstata/diagnóstico por imagem , Putrescina/farmacocinética , Cintilografia , Ratos , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Espermidina/farmacocinética , Espermina/farmacocinética , Células Tumorais Cultivadas/diagnóstico por imagem , Células Tumorais Cultivadas/metabolismo , Neoplasias Urogenitais/diagnóstico por imagem , Neoplasias Urogenitais/metabolismo
11.
Am J Physiol Regul Integr Comp Physiol ; 279(3): R849-59, 2000 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-10956242

RESUMO

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].


Assuntos
Aminoácidos/sangue , Aminoácidos/farmacocinética , Troca Materno-Fetal , Cordão Umbilical/metabolismo , Útero/metabolismo , Animais , Glicemia/metabolismo , Feminino , Idade Gestacional , Técnica Clamp de Glucose , Hiperglicemia/metabolismo , Insulina/sangue , Ácido Láctico/sangue , Leucina/sangue , Leucina/farmacocinética , Lisina/sangue , Lisina/farmacocinética , Ornitina/sangue , Ornitina/farmacocinética , Oxigênio/sangue , Placenta/metabolismo , Gravidez , Ovinos
12.
Am J Physiol Regul Integr Comp Physiol ; 278(6): R1506-12, 2000 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-10848517

RESUMO

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.


Assuntos
Arginina/farmacocinética , Proteínas de Transporte/metabolismo , Medula Renal/enzimologia , Túbulos Renais Coletores/enzimologia , Óxido Nítrico Sintase/metabolismo , Sistemas de Transporte de Aminoácidos , Animais , Transporte Biológico/efeitos dos fármacos , Transporte Biológico/fisiologia , Proteínas de Transporte/genética , Cátions/farmacocinética , Citrulina/biossíntese , Inibidores Enzimáticos/farmacologia , Expressão Gênica/fisiologia , Homeostase/fisiologia , Medula Renal/química , Túbulos Renais Coletores/química , Lisina/farmacocinética , Masculino , NG-Nitroarginina Metil Éster/farmacologia , Ornitina/farmacocinética , RNA Mensageiro/análise , Ratos , Ratos Sprague-Dawley , Especificidade por Substrato , Trítio , Equilíbrio Hidroeletrolítico/fisiologia , beta-Alanina/análogos & derivados , beta-Alanina/farmacologia , ômega-N-Metilarginina/farmacologia
13.
Pharmacol Ther ; 85(3): 191-205, 2000 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-10739874

RESUMO

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.


Assuntos
Antineoplásicos/farmacocinética , Antagonistas do Ácido Fólico/farmacocinética , Ornitina/análogos & derivados , Pterinas/farmacocinética , Ácido 4-Aminobenzoico/química , Ácido 4-Aminobenzoico/metabolismo , Aminopterina/análogos & derivados , Aminopterina/química , Antineoplásicos/farmacologia , Sítios de Ligação , Divisão Celular , Resistência a Múltiplos Medicamentos , Ensaios de Seleção de Medicamentos Antitumorais , Ácido Fólico/metabolismo , Antagonistas do Ácido Fólico/farmacologia , Humanos , Ornitina/farmacocinética , Ornitina/farmacologia , Pterinas/farmacologia , Substâncias Redutoras , Relação Estrutura-Atividade , Tetra-Hidrofolato Desidrogenase/metabolismo , Células Tumorais Cultivadas/efeitos dos fármacos
14.
Cancer Detect Prev ; 24(6): 542-8, 2000.
Artigo em Inglês | MEDLINE | ID: mdl-11198268

RESUMO

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.


Assuntos
Adenocarcinoma/diagnóstico , Adenoma/diagnóstico , Biomarcadores Tumorais/sangue , Neoplasias do Colo/diagnóstico , Ornitina , Poliaminas/sangue , Lesões Pré-Cancerosas/diagnóstico , 1,2-Dimetilidrazina , Adenocarcinoma/sangue , Adenocarcinoma/induzido quimicamente , Adenoma/sangue , Adenoma/induzido quimicamente , Animais , Biotransformação , Peso Corporal , Divisão Celular , Cromatografia Líquida de Alta Pressão , Colo/metabolismo , Colo/patologia , Neoplasias do Colo/sangue , Neoplasias do Colo/induzido quimicamente , Pólipos do Colo/sangue , Pólipos do Colo/induzido quimicamente , Pólipos do Colo/diagnóstico , Progressão da Doença , Jejum/sangue , Hiperplasia , Masculino , Proteínas de Neoplasias/metabolismo , Ornitina/farmacocinética , Ornitina Descarboxilase/metabolismo , Lesões Pré-Cancerosas/sangue , Lesões Pré-Cancerosas/induzido quimicamente , Putrescina/sangue , Ratos , Ratos Wistar , Sensibilidade e Especificidade , Espermidina/sangue , Espermina/sangue
15.
Cancer Chemother Pharmacol ; 42(4): 300-6, 1998.
Artigo em Inglês | MEDLINE | ID: mdl-9744775

RESUMO

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.


Assuntos
Antineoplásicos/farmacocinética , Carcinoma de Células Escamosas/metabolismo , Antagonistas do Ácido Fólico/farmacocinética , Neoplasias Experimentais/metabolismo , Ornitina/análogos & derivados , Pterinas/farmacocinética , Animais , Antineoplásicos/farmacologia , Área Sob a Curva , Radioisótopos de Carbono , Antagonistas do Ácido Fólico/farmacologia , Injeções Intravenosas , Marcação por Isótopo , Masculino , Metotrexato/farmacocinética , Metotrexato/farmacologia , Camundongos , Camundongos Endogâmicos C3H , Ornitina/síntese química , Ornitina/farmacocinética , Ornitina/farmacologia , Pterinas/síntese química , Pterinas/farmacologia , Tetra-Hidrofolatos/metabolismo , Distribuição Tecidual
16.
Kidney Int Suppl ; 67: S53-7, 1998 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-9736254

RESUMO

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.


Assuntos
Sistema Justaglomerular/enzimologia , Óxido Nítrico Sintase/biossíntese , Óxido Nítrico Sintase/metabolismo , Animais , Arginina/farmacocinética , Ligação Competitiva/fisiologia , Transporte Biológico/fisiologia , Pressão Sanguínea , Proteínas de Transporte/fisiologia , Homeostase/fisiologia , Sistema Justaglomerular/irrigação sanguínea , Sistema Justaglomerular/química , Lisina/farmacocinética , Masculino , Microcirculação/fisiologia , Óxido Nítrico Sintase Tipo I , Ornitina/farmacocinética , Ratos , Ratos Sprague-Dawley , Circulação Renal/fisiologia
17.
Eur J Biochem ; 258(2): 702-9, 1998 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-9874237

RESUMO

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.


Assuntos
Arginina/farmacocinética , Proteínas de Transporte/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Membrana Transportadoras , Mitocôndrias/metabolismo , Ornitina/farmacocinética , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Aminoácidos/farmacocinética , Transporte Biológico , Proteínas de Transporte/genética , Deleção de Genes , Concentração de Íons de Hidrogênio , Cinética , Proteínas de Membrana/genética , Proteínas de Transporte da Membrana Mitocondrial , Saccharomyces cerevisiae/genética
18.
Am J Clin Nutr ; 65(2): 512-8, 1997 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-9022538

RESUMO

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.


Assuntos
Queimaduras/metabolismo , Ornitina/análogos & derivados , Ornitina/sangue , Ornitina/farmacocinética , Adulto , Arginina/sangue , Nutrição Enteral , Feminino , Meia-Vida , Humanos , Infusões Parenterais , Masculino , Ornitina/administração & dosagem , Ornitina/metabolismo , Distribuição Aleatória
19.
J Med Chem ; 39(13): 2536-40, 1996 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-8691451

RESUMO

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.


Assuntos
Inibidores Enzimáticos/síntese química , Antagonistas do Ácido Fólico/síntese química , Ornitina/análogos & derivados , Peptídeo Sintases/antagonistas & inibidores , Tetra-Hidrofolato Desidrogenase/metabolismo , Transporte Biológico , Divisão Celular/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Antagonistas do Ácido Fólico/química , Antagonistas do Ácido Fólico/farmacocinética , Antagonistas do Ácido Fólico/farmacologia , Humanos , Espectroscopia de Ressonância Magnética , Modelos Químicos , Estrutura Molecular , Ornitina/síntese química , Ornitina/química , Ornitina/farmacocinética , Ornitina/farmacologia , Células Tumorais Cultivadas
20.
Mol Pharmacol ; 48(4): 758-65, 1995 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-7476904

RESUMO

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.


Assuntos
Antimetabólitos Antineoplásicos/farmacocinética , Antineoplásicos/farmacocinética , Carcinoma de Células Escamosas/metabolismo , Antagonistas do Ácido Fólico/farmacocinética , Neoplasias de Cabeça e Pescoço/metabolismo , Metotrexato/farmacologia , Ornitina/análogos & derivados , Pterinas/farmacocinética , Quinazolinas/farmacologia , Tetra-Hidrofolato Desidrogenase/metabolismo , Antimetabólitos Antineoplásicos/farmacologia , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Carcinoma de Células Escamosas/tratamento farmacológico , Divisão Celular/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos , Ensaios de Seleção de Medicamentos Antitumorais , Antagonistas do Ácido Fólico/metabolismo , Antagonistas do Ácido Fólico/farmacologia , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Humanos , Cinética , Metotrexato/farmacocinética , Ornitina/metabolismo , Ornitina/farmacocinética , Ornitina/farmacologia , Pterinas/metabolismo , Pterinas/farmacologia , Ácidos Pteroilpoliglutâmicos/metabolismo , Tetra-Hidrofolato Desidrogenase/efeitos dos fármacos , Trítio , Células Tumorais Cultivadas
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