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We have previously shown that the addition of 4-methylthio-2-oxobutanoate (MTOB) to cultures of methionine dependent neoplastic cells which lack endogenous MTOB restores their capacity to grow in the absence of exogenous methionine. Transition state inhibitors of the MTOB transaminase,responsible for the transamination of MTOB to methionine, had also been designed and selected for their capacity to inhibit the proliferation of methionine dependent neoplastic cells but not that of normal cells in culture. We now show that the transition state analogue : L-methionine ethyl esterpyridoxal(MEEP) with a structure corresponding to the oxo acid receptor covalently linked to pyridoxamine and the amine donor analogue: D-aspartate beta hydroxamate (D-AH) are efficient inhibitors of MTOB transaminase. [3H] MEEP uptake into transformed HeLa cells is similar to that in normal MRC5 cells, yet growth inhibition is seem in the transformed but not in the normal cells.MEEP irreversibly inhibits the activity of this enzyme when added to HeLa cells in culture but not that of the purified rat liver enzyme, probably due to pyridoxal phosphate already bound in the active site. On the contrary, D-AH is a noncompetitive reversible inhibitor of the purified rat liver enzyme in vitro and also inhibits intracellular HeLa MTOB transaminase. Furthermore, in HeLa cells both inhibitors induce DNA strand breaks typical of apoptotic cell death. These results provide evidence that MTOB transaminase is a potential target for antiproliferative agents which could selectively affect methionine-dependent neoplastic cells. The transition state intermediale : MEEP as an amine acceptor analogue was found to be 20 fold more effective than D-AH as the amine donor analogue in inducing apoptosis.

We examined the ability of a pyridoxal-aminoguanidine adduct with both antiglycation and antioxidant activities in vitro to protect against neuropathy and cataract in streptozotocin-diabetic rats and compared the result with that of aminoguanidine. In vivo antiglycation and antioxidant activities were also compared between the adduct and aminoguanidine. Diabetic rats were given either of the compounds in their drinking water (9 mM) for 7 weeks. Neither compound affected body weight, blood glucose level or urine volume. The adduct, but not aminoguanidine, significantly improved motor nerve conduction velocity. The time to develop cataract was longer in adduct-treated rats than in untreated and aminoguanidine-treated rats. The increase in opacification of lenses in culture medium containing high glucose levels (55.5 mM) was more efficiently attenuated by the adduct than by aminoguanidine. Adduct and aminoguanidine similarly lowered glycated hemoglobin levels. The level of urinary 8-hydroxy-2'-deoxyguanosine, a marker of oxidative DNA damage, and the level of liver malondialdehyde plus 4-hydroxy-2-alkenals, a marker of tissue lipid peroxidation, both of which were elevated by diabetes, were significantly reduced by the adduct but not by aminoguanidine. These findings indicate that the pyridoxal-aminoguanidine adduct is superior to aminoguanidine in preventing diabetic neuropathy and cataracts, and we suggest that this may be at least partly due to the higher antioxidant activity of the former.

A dimeric copper complex of the unsubstituted pyridoxal thiosemicarbazone (H(2)L), [[Cu(HL)(OH(2))](2)]Cl(2).2H(2)O, previously tested on Friend murine cell lines has been recently resynthesized to evaluate its behavior on different murine and human leukemic cell lines and has been compared, in vitro and in vivo, with its monomeric counterpart [Cu(H(2)L)(OH(2))Cl]Cl. On TS/A murine adenocarcinoma cell line in vitro, both compounds significantly inhibit cell proliferation at micromolar concentrations, although the dimeric compound is more active. Despite this cytotoxicity they lack in vivo activity on TLX5 lymphoma. The unsubstituted dimeric [[Cu(HL)(OH(2))](2)]Cl(2).2H(2)O induces apoptosis on CEM and U937 human cell lines, with IC(50) concentrations of 1.2 x 10(-5) and 6.7 x 10(-6) M, respectively, but it is inactive on K562. Moreover, it alters significantly the cell cycle of U937 and CEM lines and decreases the telomerase activity of U937. To verify if other dimeric copper complexes show relevant biological activity new complexes with N-substituted pyridoxal thiosemicarbazones have been synthesized and characterized using spectroscopic techniques. Three of them, namely [Cu(Me(2)-HL)Cl](2).6H(2)O (Me(2)-H(2)L=pyridoxal N1,N1-dimethylthiosemicarbazone) (1), [Cu(MeMe-HL)Cl](2)Cl(2).4H(2)O (MeMe-HL=pyridoxal N1,N2-dimethylthiosemicarbazone) (2), [Cu(Et-H(2)L)Cl](2)Cl(2).2H(2)O (Et-H(2)L=pyridoxal N1-ethylthiosemicarbazone) (3), were also characterized by X-ray diffractometry. These complexes are dimeric and all three present a square pyramidal coordinative geometry with the ligand showing an SNO tridentate behavior. Their biological activities have been tested in vitro on U937, CEM and K562 cell lines to ascertain their effectiveness in comparison to the corresponding unsubstituted complex [[Cu(HL)(OH(2))](2)]Cl(2).2H(2)O. Compound 1 shows weak proliferation inhibition on all three cell lines, but it does not induce apoptosis and it does not inhibit telomerase activity, compound 2 is not effective at low concentration and is toxic at higher doses; compound 3 inhibits CEM cell growth better than complex 1 but it does not exert any other biological effect.

A major obstacle to the therapeutic use of anthracyclines, highly effective anticancer agents, is the fact that their administration results in dose-dependent cardiomyopathy. According to the currently accepted hypothesis, anthracyclines injure the heart by generating oxygen free radicals. The ability of pyridoxal isonicotinoyl hydrazone (PIH) and salicylaldehyde isonicotinoyl hydrazone (SIH) -- new iron chelators -- to protect against peroxidation as well as their suitable biological, physical and chemical properties make the compounds promising candidates for pre-clinical and clinical studies. Activities of carbonyl reductase CR (1.1.1.184), dihydrodiol dehydrogenase DD2 (1.3.1.20), aldehyde reductase ALR1 (1.1.1.2) and P450 isoenzymes (CYP1A1, CYP1A2, CYP2B, CYP3A) involved in the metabolism of daunorubicin, doxorubicin and other drugs or xenobiotics were studied. Various concentrations of the chelators were used either alone or together with daunorubicin or doxorubicin for in vitro studies in isolated hepatocytes. A significant decrease of activity was observed for all enzymes only at PIH and SIH concentrations higher than those presumed to be used for therapy. The results show that PIH and SIH have no effect on the activities of the enzymes studied in vitro and allow us to believe that they will not interfere with the metabolism of co-administered drugs and other xenobiotics. Daunorubicin (Da) and doxorubicin (Dx) significantly reduce cytochrome P450 activity, but the addition of SIH and PIH chelators (50 microM) reverses the reduction and restores the activity to 70-90 % of the activity of relevant controls.

It has been known that glutamate, generated by glutamate dehydrogenase (GDH), acts as an intracellular messenger in insulin exocytosis in pancreatic beta cells. Here we demonstrate the correlation of GDH activity and insulin release in rat pancreatic islets perfused with 5'-deoxypyridoxal. Perfusion of islets with 5'-deoxypyridoxal, an effective inhibitor of GDH, reduced the islet GDH activity at concentration-dependent manner. Treatment of 5'-deoxypyridoxal up to 2 mM did not affect the cell viability. There was reduction in V(max) values on average about 60%, whereas no changes in K(m) values for substrates and coenzymes were observed. The concentration of GDH on the Western blot analysis and the level of GDH mRNA remained unchanged. The concentration of glutamate decreased by 52%, whereas the concentration of 2-oxoglutarate increased up to 2.3-fold in the presence of 5'-deoxypyridoxal. 5'-Deoxypyridoxal had no effects on inhibition by GTP and activation by ADP or L-leucine of islet GDH. In parallel with the inhibition of GDH activity, perfusion of islets with 5'-deoxypyridoxal reduced insulin release up to 2.5-fold. Although precise mechanism for correlation between GDH activity and insulin release remains to be studied further, our results suggest a possibility that the inhibitory effect of 5'-deoxypyridoxal on islet GDH activity may correlate with its effect on insulin release.

Although iron chelation therapy with deferoxamine (DFO) results in improved life expectancy of patients with thalassemia, compliance with parenteral DFO treatment is unsatisfactory, underlining the need for alternative drugs and innovative ways of drug administration. We examined the chelating potential of pyridoxal isonicotinoyl hydrazone (PIH) analogs, alone or in combination with DFO, using hypertransfused rats with labeled hepatocellular iron stores and cultured iron-loaded rat heart cells. Our in vivo studies using 2 representative PIH analogs, 108-o and 109-o, have shown that PIH analogs given orally are 2.6 to 2.8 times more effective in mobilizing hepatocellular iron in rats, on a weight-per-weight basis, than parenteral DFO administered intraperitoneally. The combined effect of DFO and 108-o on hepatocellular iron excretion was additive, and response at a dose range of 25 to 200 mg/kg was linear. In vitro studies in heart cells showed that DFO was more effective in heart cell iron mobilization than all PIH analogs studied. Response to joint chelation with DFO and PIH analogs was similar to an increase in the equivalent molar dose of DFO alone, rather than the sum of the separate effects of the PIH analog and DFO. This finding was most likely the result of iron transfer from PIH analogs to DFO, a conclusion supported directly by iron-shuttle experiments using fluorescent DFO. These findings provide a rationale for the combined, simultaneous use of iron-chelating drugs and may have useful, practical implications for designing novel strategies of iron chelation therapy.

The vitamin B(6) compounds pyridoxine (PN), pyridoxamine (PM), pyridoxal (PL), and pyridoxamine 5'-phosphate (PMP) inhibited the diphenolase activity of mushroom tyrosinase. PM showed the highest inhibition; the control activity was inhibited by 38% at 1.5 mM. Each PL, PN, and PMP showed about 30% inhibition at the same concentration. Lineweaver-Burk plots showed that PM and PN were mixed-type inhibitors with K(I) values of 4.3 and 5.2 mM, respectively. Because PM and PN cannot form a Schiff base with a primary amino group of the enzyme, their inhibition is not attributable to the formation of the Schiff base. Alternatively, their quenching function of reactive oxygen species (ROS) was postulated to be responsible for the inhibition. Thus, the inhibitory effect of ROS was examined. The representative singlet oxygen quenchers l-histidine, sodium azide, Trolox, and anthracene-9,10-dipropionic acid (AAP) inhibited the activity. The specific scavenger of superoxide, proxyl fluorescamine, also inhibited the activity. The scavengers of hydroxyl radical, d-mannitol and dimethyl sulfoxide, showed no inhibition. The fluorescence of AAP was decayed during the diphenolase reaction, and PM inhibited the decay. AAP was also a mixed-type inhibitor. The results showed that the vitamin B(6) compounds inhibited the diphenolase activity by quenching ROS (probably singlet oxygen) generated during some reaction step of the diphenolase reaction.

Previous work from our laboratory demonstrated that pyridoxal isonicotinoyl hydrazone (PIH) has in vitro antioxidant activity against iron plus ascorbate-induced 2-deoxyribose degradation due to its ability to chelate iron; the resulting Fe(III)-PIH(2) complex is supposedly unable to catalyze oxyradical formation. A putative step in the antioxidant action of PIH is the inhibition of Fe(III)-mediated ascorbate oxidation, which yields the Fenton reagent Fe(II) [Biochim. Biophys. Acta 1523 (2000) 154]. In this work, we demonstrate that PIH inhibits Fe(III)-EDTA-mediated ascorbate oxidation (measured at 265 nm) and the formation of ascorbyl radical (in electron paramagnetic resonance (EPR) studies). The efficiency of PIH against ascorbate oxidation, ascorbyl radical formation and 2-deoxyribose degradation was dose dependent and directly proportional to the period of preincubation of PIH with Fe(III)-EDTA. The efficiency of PIH in inhibiting ascorbate oxidation and ascorbyl radical formation was also inversely proportional to the Fe(III)-EDTA concentration in the media. When EDTA was replaced by the weaker iron ligand nitrilotriacetic acid (NTA), PIH was much more effective in preventing ascorbate oxidation, ascorbyl radical formation and 2-deoxyribose degradation. Moreover, the replacement of EDTA with citrate, a physiological chelator with a low affinity for iron, also resulted in PIH having a higher efficiency in inhibiting iron-mediated ascorbate oxidation and 2-deoxyribose degradation. These results demonstrate that PIH removes iron from EDTA (or from either NTA or citrate), forming an iron-PIH complex that cannot induce ascorbate oxidation effectively, thus inhibiting iron-mediated oxyradical formation. These results are of pharmacological relevance because PIH has been considered for experimental chelating therapy in iron-overload diseases.

Iron overload secondary to beta-thalassemia and other iron-loading anemias is the most serious obstacle to be overcome in the treatment of these diseases, since there is no physiological mechanism for excretion of the excess iron acquired by chronic blood transfusion. Due to the inconvenience and cost of the current iron chelation therapy, the search for an orally available iron chelator is ongoing. Pyridoxal isonicotinoyl hydrazone (PIH) and many of its analogs are effective at mobilizing iron in vivo and in vitro at doses that are not toxic. PIH analogs were approximately equally effective at binding 59Fe within K562 cells; their efficacy depended upon the kinetics of release of the iron-chelator complex from the cell, which was correlated inversely with the lipophilicity of the chelators. Addition of BSA, which has a well-characterized affinity for lipophilic species, to the extracellular medium enhanced iron-chelator efflux, such that all analogs caused 59Fe release from the cells as quickly as it was chelated; this suggests that BSA acts as an extracellular sink for the iron-chelator complexes, many of which are highly lipophilic. The toxicity of the free chelators varied over two orders of magnitude, and was correlated with the amount of intracellular 59Fe-chelator complexes, implicating the complexes in the mechanism of toxicity of the chelators. Understanding the structural features that determine the efficacy and toxicity of iron chelators in biological systems is of value in the selection of PIH analogs for in vivo examination.

Analogs of pyridoxal isonicotinoyl hydrazone (PIH) are of interest as iron chelators for the treatment of secondary iron overload and cancer. PIH, salicylaldehyde isonicotinoyl hydrazone (SIH), and 2-hydroxy-1-naphthylaldehyde isonicotinoyl hydrazone (NIH), the toxicity of which vary over two orders of magnitude, were selected for a study of their mechanisms of toxicity. PIH analogs and their iron complexes caused concentration- and time-dependent apoptosis in Jurkat T lymphocytes and K562 cells. Bcl-2 overexpression was partially anti-apoptotic, suggesting mitochondrial mediation of apoptosis. Since the pan-caspase inhibitor zVAD-fmk did not reduce lysosomal and mitochondrial destabilization, these events occur upstream of caspase activation. In contrast, phosphatidylserine externalization and the development of apoptotic morphology were inhibited significantly, indicating the role of caspases in mediating these later events. Since overexpression of CrmA had no effect on apoptosis, caspase-8 is not likely involved. Fe(3+) complexes of SIH and NIH, which accumulated in 59Fe-labeled mouse reticulocytes during incubation with the chelators, also caused apoptosis. BSA, which promotes release of the complexes from cells, reduced the toxicity of SIH and NIH, suggesting that the induction of apoptosis by PIH analogs involves toxic effects mediated by their Fe(3+) complexes. Moreover, analogs of these agents lacking the iron-chelating moiety were non-toxic.

Aminoguanidine improved the erythrocyte filterability by 4%, pyridoxyliden-aminoguanidine by 11% and pyridoxal by 13% in healthy subjects. In diabetic patients the aminoguanidine effect on erythrocyte filterability was improved by 7%, PAG effect by 9% and pyridoxal effect by 15% in comparison to the control group. The other investigated haematological variables in both groups were within the range of the physiological standard. All of the tested substances demonstrated a mild protective influence on erythrocyte elasticity both in healthy subjects and diabetic patients. Significant elasticity improvement was obtained only by pyridoxal (p<0.01) in patients with diabetes mellitus. (Fig. 4, Ref. 18.).

In the ongoing search for an effective, orally active iron-chelator, the capacity of a series of halogenated analogs of pyridoxal isonicotinoyl hydrazone (PIH) to bind intracellular 59Fe and cause its release from cells was investigated. Reticulocytes labeled with 59Fe(2)-transferrin in which heme synthesis was inhibited by succinylacetone were used as a model of 59Fe mobilization. The kinetics of iron binding were similar for all the chelators tested (half-time of approximately 1 hr), and all bound more than twice as much 59Fe as PIH. The rate of release of the 59Fe-chelator complexes from cells depended upon the structure of the chelators. Ortho-substituted analogs were more effective at mobilizing cellular iron than meta and para isomers, due to a more efficient release of the iron complexes from the cell. The iron-chelator complexes which were released slowly from cells had a high affinity for erythrocyte ghost membranes, indicating the role of membrane permeability in the release mechanism of the complexes. The addition of BSA to the extracellular medium increased the extent of iron release by lipophilic analogs in a concentration-dependent manner, presumably by acting as a sink for the lipophilic complexes. The affinity of BSA for the chelators and their Fe(3+) complexes, determined spectrophotometrically, demonstrated that all chelators and their iron complexes bound BSA with dissociation constants ranging from 7,000 to >500,000 M(-1). Understanding the importance of the rate of release of the iron-chelator complex will direct the search for iron-chelators with improved efficacy., (Copyright 2002 Elsevier Science Inc.)

Transducin (T), a guanine nucleotide binding regulatory protein composed of alpha-, beta-, and gamma-subunits, serves as an intermediary between rhodopsin and cGMP phosphodiesterase during signaling in the visual process. Pyridoxal 5'-phosphate (PLP), a reagent that has been used to modify enzymes that bind phosphorylated substrates, was probed here as an affinity label for T. PLP inhibited the guanine nucleotide binding activity of T in a concentration dependent manner, and was covalently incorporated into the protein in the presence of [3H]NaBH4. Approximately 1 mol of 3H was bound per mol of T. GTP and GTP analogs appreciably hindered the incorporation of 3H to T, suggesting that PLP specifically modified the protein active site. Interestingly, PLP modified both the alpha- and beta-subunits of T. Moreover, PLP in the presence of GDP behaved as a GTP analog, since this mixture was capable of dissociating T from T:photoactivated rhodopsin complexes.

Vitamin B6 derivatives protect the retinal neurons from excitotoxic injury in vitro. However, their in vivo role in a process involving excitotoxicity, such as ischemia, remains unknown. We studied potential protective effects of pyridoxal 5'-phosphate (PLP) and pyridoxal hydrochloride (pyridoxal) on the retinal neurons in a monkey model of transient global ischemia. Daily intravenous injections (15 mg/kg) of pyridoxal and PLP were performed for consecutive 10 days. On the sixth day, whole brain complete ischemia was produced by clipping the innominate and the left subclavian arteries for 20 min. The monkeys were sacrificed 5 days after ischemia and their retinas were processed for histological analysis. The ischemia induced a marked cellular injury in the retina as shown by the loss of ganglion cells and the reduction of thickness of the ganglion cell, inner plexiform, and inner nuclear layers. PLP significantly prevented the ganglion cell loss and the reduction of thickness of the ganglion cell layer. Pyridoxal significantly prevented the ganglion cell loss as well as the reduction of thickness of ganglion cell, inner plexiform and inner nuclear layers. These results suggest that PLP and pyridoxal counteract the postischemic neuronal death in the adult primate retina, offering a potential for a novel pharmacotherapy of retinal ischemic injury.

Pyridoxal isonicotinoyl hydrazone (PIH) is an iron chelator with antioxidant activity, low toxicity and is useful in the experimental treatment of iron-overload diseases. Previous studies on x-ray diffraction have revealed that PIH also forms a complex with Cu(II). Since the main drug of choice for the treatment of Wilson's disease, d-penicillamine, causes a series of side effects, there is an urgent need for the development of alternative copper chelating agents for clinical use. These chelators must also have antioxidant activity because oxidative stress is associated with brain and liver copper-overload. In this work we tested the ability of PIH to prevent in vitro free radical formation mediated by Cu(II), ascorbate and dissolved O2. Degradation of 2-deoxyribose mediated by 10 microM Cu(II) and 3 mM ascorbate was fully inhibited by 10 microM PIH (I50 = 6 microM) or 20 microM d-penicillamine (I50 = 10 microM). The antioxidant efficiency of PIH remained unchanged with increasing concentrations (from 1 to 15 mM) of the hydroxyl radical detector molecule, 2-deoxyribose, indicating that PIH does not act as a hydroxyl scavenger. On the other hand, the efficiency of PIH (against copper-mediated 2-deoxyribose degradation and ascorbate oxidation) was inversely proportional to the Cu(II) concentration, suggesting a competition between PIH and ascorbate for complexation with Cu(lI). An almost full inhibitory effect by PIH was observed when the ratio PIH:copper was 1:1. A similar result was obtained with the measurement of copper plus ascorbate-mediated O2 uptake. Moreover, spectral studies of the copper and PIH interaction showed a peak at 455 nm and also indicated the formation of a stable Cu(II) complex with PIH with a 1:1 ratio. These data demonstrated that PIH prevents hydroxyl radical formation and oxidative damage to 2-deoxyribose by forming a complex with Cu(II) that is not reactive with ascorbate (first step of the reactions leading to hydroxyl radical formation from Cu(II), ascorbate and O2) and does not participate in Haber-Weiss reactions.

Supraphysiological doses of vitamin B6 has been reported to suppress tumor growth and metastasis in rodents. To examine if its anticancer effect is due to suppression in angiogenesis, this study was conducted to investigate the antiangiogenic effect of pyridoxal 5'-phosphate (PLP), pyridoxine, pyridoxal and pyridoxamine in an ex vivo serum-free matrix culture model using rat aortic ring. Rat aortic rings were incubated with PLP or pyridoxine (25 micromol/l to 5 mmol/l). Higher concentrations of PLP (2.5 and 5 mmol/l) and pyridoxine (5 mmol/l) caused complete inhibition of microvessel outgrowth. However, the addition of pyridoxine at 2.5 mmol/l did not show complete inhibition of angiogenesis. PLP inhibited microvessel outgrowth almost completely at a concentration of 500 micromol/l and showed antiangiogenic effect in a dose-dependent manner within the range of 25-500 micromol/l. At 250 micromol/l, pyridoxal was as effective as PLP, but pyridoxamine was inactive, implying that the aldehyde group relates to the antiangiogenic effect. These results indicated the antiangiogenic effect of PLP and pyridoxal, and suggested that the antitumor effect of high levels of vitamin B6 might be mediated through suppression of angiogenesis.

Pyridoxal isonicotinoyl hydrazone (PIH) is able to prevent iron-mediated hydroxyl radical formation by means of iron chelation and inhibition of redox cycling of the metal. In this study, we investigated the effect of PIH on Fe(II)-citrate-mediated lipid peroxidation and damage to isolated rat liver mitochondria. Lipid peroxidation was quantified by the production of thiobarbituric acid-reactive substances (TBARS) and by antimycin A-insensitive oxygen consumption. PIH at 300 microM induced full protection against 50 microM Fe(II)-citrate-induced loss of mitochondrial transmembrane potential (deltapsi) and mitochondrial swelling. In addition, PIH prevented the Fe(II)-citrate-dependent formation of TBARS and antimycin A-insensitive oxygen consumption. The antioxidant effectiveness of 100 microM PIH (on TBARS formation and mitochondrial swelling) was greater in the presence of 20 or 50 microM Fe(II)-citrate than in the presence of 100 microM Fe(II)-citrate, suggesting that the mechanism of PIH antioxidant action is linked with its Fe(II) chelating property. Finally, PIH increased the rate of Fe(II) autoxidation by sequestering iron from the Fe(II)-citrate complex, forming a Fe(III)-PIH, complex that does not participate in Fenton-type reactions and lipid peroxidation. These results are of pharmacological relevance since PIH is a potential candidate for chelation therapy in diseases related to abnormal intracellular iron distribution and/or iron overload.

Some chelators of the pyridoxal isonicotinoyl hydrazone class have antiproliferative activity that is far greater than desferrioxamine (DFO). In this study, DFO was compared with one of the most active chelators (311) on the expression of molecules that play key roles in cell-cycle control. This was vital for understanding the role of iron (Fe) in cell-cycle progression and for designing chelators to treat cancer. Incubating cells with DFO, and especially 311, resulted in a decrease in the hyperphosphorylated form of the retinoblastoma susceptibility gene product (pRb). Chelators also decreased cyclins D1, D2, and D3, which bind with cyclin-dependent kinase 4 (cdk4) to phosphorylate pRb. The levels of cdk2 also decreased after incubation with DFO, and especially 311, which may be important for explaining the decrease in hyperphosphorylated pRb. Cyclins A and B1 were also decreased after incubation with 311 and, to a lesser extent, DFO. In contrast, cyclin E levels increased. These effects were prevented by presaturating the chelators with Fe. In contrast to DFO and 311, the ribonucleotide reductase inhibitor hydroxyurea increased the expression of all cyclins. Hence, the effect of chelators on cyclin expression was not due to their ability to inhibit ribonucleotide reductase. Although chelators induced a marked increase in WAF1 and GADD45 mRNA transcripts, there was no appreciable increase in their protein levels. Failure to translate these cell-cycle inhibitors may contribute to dysregulation of the cell cycle after exposure to chelators. (Blood. 2001;98:842-850)

Friedreich's ataxia (FA) is a crippling neurodegenerative disease that is due to iron (Fe) overload within the mitochondrion. One therapeutic intervention may be the development of a chelator that could remove mitochondrial Fe. We have implemented the only well characterized model of mammalian mitochondrial Fe overload to examine the Fe chelation efficacy of novel chelators of the 2-pyridylcarboxaldehyde isonicotinoyl hydrazone (PCIH) class. In this model we utilize reticulocytes treated with the haem synthesis inhibitor succinylacetone which results in mitochondrial Fe-loading. Our experiments demonstrate that in contrast to desferrioxamine, several of the PCIH analogues show very high activity at mobilizing (59)Fe from (59)Fe-loaded reticulocytes. Further studies on these ligands in animals are clearly warranted considering their potential to treat FA.

The tissue non-specific alkaline phosphatase (TNAP) knock-out mouse is a model of infantile hypophosphatasia displaying impaired bone mineralization, epileptic seizures, apnoea, abnormal apoptosis in the thymus, abnormal lumbar nerve roots, and postnatal death. Administration of vitamin B6 suppresses the epileptic seizures in TNAP-/- mice. This paper examines to what extent the diverse abnormalities seen in these mice are due to impaired utilization of vitamin B6, using two complementary approaches: administration of vitamin B6 to TNAP null mice and deprivation of vitamin B6 in wild-type and TNAP heterozygous mice. Administration of exogenous pyridoxal HCl delayed the onset of epileptic attacks and increased the life span of TNAP-/- mice. The episodes of apnoea ceased and the appearance of lumbar nerve roots improved, but hypomineralization and accumulation of osteoid continued to worsen with age. Control mice fed a vitamin B6-depleted diet developed epileptic seizures indistinguishable from those observed in TNAP-/- mice, abnormal apoptosis in the thymus, and thinning of the nerve roots, but showed no evidence of bone mineralization abnormalities. Depletion of vitamin B6 did not affect the ability of primary cultures of osteoblasts to deposit bone mineral in vitro. While abnormal metabolism of vitamin B6 explains many of the abnormalities in this mouse model of infantile hypophosphatasia, it is not the basis of the abnormal mineralization that characterizes this disease.

We have previously reported that pyridoxine shows UVA-induced cytotoxicity. Four other vitamin B6 compounds (pyridoxal, pyridoxamine, pyridoxal phosphate, and pyridoxamine phosphate) are metabolically more important in vivo than pyridoxine. These compounds were examined for UVA phototoxicity to cultured human fibroblasts. The cytotoxicity was measured by post-UVA irradiation colony-forming ability. All the B6 compounds except pyridoxal phosphate showed cytotoxicity. Pyridoxamine phosphate, which is the most important form of vitamin B6 in vivo, had the strongest cytotoxic effect. To examine the involvement of reactive oxygen species in the phototoxicity, we performed an electron spin resonance study using the spin trapping agent, 5,5-dimethyl-1-pyrroline N-oxide, and diethylenetriaminepentaacetic acid. We failed to detect radicals derived from vitamin B6. The cytotoxic effect remained in UVA-irradiated solutions for at least 30 min after the end of UVA irradiation. Hydrogen peroxide was produced in the solution, but the amount was not enough to cause cytotoxicity. In addition, the cells from xeroderma pigmentosum patients who belong to group A or C showed survival curves similar to those of normal fibroblasts. This suggests that cyclobutane pyrimidine dimers or 6-4 photoproducts of DNA were not involved in this damage. These findings suggest that UVA-induced vitamin B6 cytotoxicity is caused by toxic photoproducts resulting from irradiated vitamin B6.

Iron is released in a free desferrioxamine-chelatable form when erythrocytes are challenged by an oxidative stress. The release of iron is believed to play an important role in inducing destructive damage (lipid peroxidation and hemolysis) or in producing membrane protein oxidation and generation of senescent cell antigens (SCA). In this report, we further tested the hypothesis that intracellular chelation of iron released under conditions of oxidative stress prevents erythrocyte damage or SCA formation. Fluor-benzoil-pyridoxal hydrazone (FBPH), an iron-chelating molecule of the family of aromatic hydrazones, was prepared by synthesis and used for the above purpose after the capacity of the product to enter cells had been ascertained. GSH-depleted mouse erythrocytes were incubated with the oxidant drug phenylhydrazine in order to produce iron release, lipid peroxidation, and hemolysis. FBPH at a concentration of 200 microM prevented lipid peroxidation and hemolysis in spite of equal values of iron release. FBPH was active even at a lower concentration (100 microM) when the erythrocytes were preincubated with it for 15 min. No preventive effect was seen when FBPH saturated with iron was used. Prolonged aerobic incubation (60 hr) of erythrocytes produced iron release and formation of SCA as determined by autologous immunoglobulin G (IgG) binding. The IgG binding was detected by using an anti-IgG antibody labeled with fluorescein and by examining the cells for fluorescence by confocal microscopy. FBPH prevented SCA formation in a dose-related manner. These results lend further support to the hypothesis that iron release is a key factor in erythrocyte ageing.

We reported that pyridoxal phosphate (PAP), a coenzyme form of vitamin B6, strongly inhibits activities of cathepsin B and weakly inhibits those of cathepsins S, K, and C in vitro. Either intraperitoneal injection or peroral administration of medication doses of vitamin B6 in the diet caused dose-dependent inhibition of hepatic cathepsins B, L, S, and C, and the inhibition was exhibited much more significantly in the case of a high protein diet than in a low protein diet. Administration of vitamin B6 induced the suppression of immune responses against ovalbumin (OVA) mediated by helper T lymphocyte type-2, based on the suppression of antigen processing by cathepsin B inhibition, as in the case of CA-074 administration, a cathepsin B specific inhibitor. Ovalbumin-dependent production of immunoglobulins IgE, IgG1 and interleukin IL-4 was suppressed by administration of medication doses of pyridoxal (PA) or pyridoxine (PI), while the production of IgG2alpha and interferon (INF)-gamma mediated by helper T lymphocyte type 1 was not changed. Administration of medication doses of vitamin B6 caused the inhibition of intracellular cathepsin B activity due to suppression of the functions of helper T lymphocyte type-2.

We found that pyridoxal phosphate shows considerable inhibition of cathepsins. CLIK-071, in which the phosphate ester of position 3 of pyridoxal phosphate was replaced by propionate, strongly inhibited cathepsin B. Three new types of synthetic pyridoxal propionate derivatives showing specific inhibition of cathepsin K were developed. New synthetic pyridoxal propionate derivatives, -162, -163, and -164, in which the methyl arm of position 6 of CLIK-071 was additionally modified, strongly inhibited cathepsin K and cathepsin S weakly, but other cathepsins were not inhibited. CLIK-166, in which the position 4 aldehyde of CLIK-071 is replaced by a vinyl radical and position 5 is additionally modified, showed cathepsin K-specific inhibition at 10(-5) M. Pit formation due to bone collagen degradation by cathepsin K of rat osteoclasts was specifically suppressed by administration of CLIK-164, but not by inhibitors of cathepsin L or B., (Copyright 2000 Academic Press.)

In previous studies, decreased growth of tumor cells by vitamin B-6 treatment has been attributed to modulation of steroid hormone action. Therefore, the growth-inhibiting properties of pyridoxal (PL) supplementation were studied in estrogen receptor-positive, MCF-7 and T-47D, and estrogen receptor-negative, BT-20, breast cancer cell lines. Cell counting and [3H]thymidine incorporation into DNA were used to assess growth, and analysis of pS2 expression was used to determine whether PL supplementation affected estrogen action. Treatment with 100 or 300 mM PL resulted in dose-dependent decreases in total cell numbers in the absence (26-85% and 72-98%, respectively) and presence (38-42% and 88-98%, respectively) of estradiol in all cell lines studied compared with control cells cultured without PL supplementation. Similar decreases in DNA synthesis were observed in response to PL supplementation. Incorporation of [3H]thymidine into DNA of cells cultured with 100 or 300 microM PL was decreased by 30-90% and 96-99%, respectively, in the absence and by 32-40% and 82-99%, respectively, in the presence of estradiol. Northern analysis showed that expression of the estrogen-sensitive gene pS2 was not affected by either concentration of PL. These results indicate that PL supplementation regulates breast cancer cell growth in vitro via a mechanism that appears to be steroid independent.

Previous studies have demonstrated that aroylhydrazone iron (Fe) chelators of the pyridoxal isonicotinoyl hydrazone (PIH) class have high Fe chelation efficacy both in vitro and in vivo. Depending on their design, these drugs may have potential as agents for the treatment of Fe overload disease or cancer. Considering the high potential of this class of ligands, we have synthesized seven novel aroylhydrazones in an attempt to identify Fe chelators more efficient than desferrioxamine (DFO) and more soluble than those of the PIH class. These compounds belong to a new series of tridentate chelators known as the 2-pyridylcarboxaldehyde isonicotinoyl hydrazones (PCIH). In this study we have examined the Fe chelation efficacy and antiproliferative activity of these chelators including their effects on the expression of genes (WAF1 and GADD45) known to be important in mediating cell cycle arrest at G1/S. From seven chelators synthesized, three ligands, namely 2-pyridylcarbox-aldehyde benzoyl hydrazone (PCBH), 2-pyridylcarboxaldehyde m-bromobenzoyl hydrazone (PCBBH), and 2-pyridylcarboxaldehyde 2-thiophenecarboxyl hydrazone (PCTH), showed greater Fe chelation activity than DFO and comparable or greater efficiency than PIH. These ligands were highly effective at both mobilizing 59Fe from cells and preventing 59Fe uptake from 59Fe-transferrin and caused a marked increase in the RNA-binding activity of the iron-regulatory proteins (IRP). Our studies have also demonstrated that compared with the cytotoxic Fe chelator, 2-hydroxy-1-naphthylaldehyde isonicotinoyl hydrazone (311), these ligands have far less effect on cellular growth and 3H-thymidine, 3H-leucine, or 3H-uridine incorporation. In addition, in contrast to 311, which markedly increased WAF1 and GADD45 mRNA expression, PCBH and PCTH did not have any effect, whereas PCBBH increased the expression of GADD45 mRNA. Collectively, these results demonstrate the potential of several of these ligands as agents for the management of Fe overload disease.

Glycoprotein IIb (GPIIb) is the alpha-subunit of the platelet membrane receptor GPIIb/IIIa, which plays a major role in platelet aggregation. Vitamin B6 has been reported to be an antiaggregative agent, although its mechanism is not well known. To understand the molecular mechanism of vitamin B6 on antiaggregation, we analyzed the effects of various forms of vitamin B6 on the expression of human GPIIb promoter using chloramphenicol acetyl transferase (CAT) as the reporter gene. The GPIIb promoter region was amplified by polymerase chain reaction (PCR), cloned into pBLCAT3 to drive the CAT reporter gene and transfected into human erythroleukemia (HEL) cells. Transient expression of the GPIIb promoter was determined after transfected cells were treated with 1 microM pyridoxine (PN), pyridoxal (PL), pyridoxal-5-phosphate (PLP), or 4-deoxypyridoxine (4-dex) for 48 h. Our results show that the GPIIb promoter activity was down-regulated to 54, 35 and 63% in the presence of PN, PL and PLP, respectively, as compared to an untreated control whose promoter activity was 100%. However, no adverse effect on GPIIb promoter was detected by 4-dex, which is an antagonist of vitamin B6. The down-regulation effect of vitamin B6 on GPIIb promoter activity may lead to a reduction of GPIIb protein expression and thus be detrimental to platelet aggregation.

Previous studies have demonstrated that 2-hydroxy-1-naphthaldehyde isonicotinoyl hydrazone (NIH) and several other aroylhydrazone chelators possess anti-neoplastic activity due to their ability to bind intracellular iron. In this study we have examined the structure and properties of NIH and its FeIII complex in order to obtain further insight into its anti-tumour activity. Two tridentate NIH ligands deprotonate upon coordination to FeIII in a meridional fashion to form a distorted octahedral, high-spin complex. Solution electrochemistry of [Fe(NIH-H)2]+ shows that the trivalent oxidation state is dominant over a wide potential range and that the FeII analogue is not a stable form of this complex. The fact that [Fe(NIH-H)2]+ cannot cycle between the FeII and FeIII states suggests that the production of toxic free-radical species, e.g. OH. or O2.-, is not part of this ligand's cytotoxic action. This suggestion is supported by cell culture experiments demonstrating that the addition of FeIII to NIH prevents its anti-proliferative effect. The chemistry of this chelator and its FeIII complex are discussed in the context of understanding its anti-tumour activity.

To analyze the functional share of individual cathepsins, we developed powerful and specific inhibitors for individual cathepsins using computer graphics of substrate binding pockets based on X-ray crystallography. These new inhibitors were named CLIK group. Epoxy succinate peptide derivatives, CLIK-066, 088, 112, 121, 148, 181, 185 and 187, are typical specific inhibitors for cathepsin L. Aldehyde derivatives CLIK-060 and CLIK-164 showed specific inhibition against cathepsin S and cathepsin K, respectively. We found that pyridoxal phosphate (PLP), a coenzyme form of vitamin B6, inhibits all cathepsins and also new artificially synthesized pyridoxal derivatives, CLIK-071 and -072, in which the phosphate esters of PLP were replaced by propionic acid, exhibited strong inhibition for cathepsins. Furthermore, CLIK-071 was easy to incorporate into cells and showed powerful inhibition for intracellular cathepsins. Using these selective inhibitors, the allotment of individual cathepsin functions in cells has been studied as follows. Cathepsin L and/or K participate in bone resorption based on bone type-1 collagen degradation and the L-type protease inhibitors suppressed the bone resorption. Cathepsins B and S participate in antigen presentations based on antigen processing and invariant chain degradation, respectively. Also cathepsin L participates in cell apoptosis mediated by caspase III activation.

Pyridoxal isonicotinoyl hydrazone (PIH) has previously been studied for use in iron chelation therapy in iron-overload diseases. It is an efficient in vitro antioxidant due to its Fe(III) complexing activity (Schulman, H. M., et al. Redox Report 1:373-378; 1995). Pathologies associated with iron-overload include hepatic and other cancers. Since oxidative alterations of DNA can be linked to the development of cancer, we decided to study whether PIH protects DNA against in vitro oxidative stress. We report here that pUC-18 plasmid DNA is damaged by *OH radicals generated from Fe(II) plus H2O2 or from Fe(II) plus hypoxanthine/xanthine oxidase. The DNA damage was quantified by determining the diminution of supercoiled DNA forms after oxidative attack using agar gel electrophoresis. Micromolar amounts of PIH (20-30 microM) were able to half-protect DNA from iron (1-7.5 microM)-mediated *OH formation. The antioxidant capacity of PIH was significantly higher than that of some of its analogs and desferrioxamine. PIH and some of its analogues could also inhibit the oxidative degradation of 2-deoxyribose caused by Fenton reagents. Since we observed that PIH enhances the Fe(II) autoxidation rate, measured by the ferrozine technique, PIH may limit *OH formation and consequently DNA damage by decreasing the amount of Fe(II) available to catalyze Fenton reactions.

Iron overload is a major life-threatening complication of thalassemia major and other iron-loading anemias treated by regular blood transfusions. Although the clinical manifestations of iron overload may be prevented by desferrioxamine, the only iron-chelating drug in routine clinical use, this treatment requires subcutaneous infusion of desferrioxamine for 12 hours each day. New orally effective iron chelators are urgently needed, and pyridoxal isonicotinoyl hydrazone (PIH), which was first recognized as an effective iron chelator in vitro and subsequently in vivo, shows promise for the treatment of iron overload. More recently, over 40 analogs of PIH were synthesized, and some of them proved to be very potent in mobilizing 59Fe in vitro from 59Fe-labeled cells. In this study, we show that PIH analogs such as pyridoxal benzoyl hydrazone, pyridoxal p-methoxybenzoyl hydrazone (PMBH), pyridoxal m-fluorobenzoyl hydrazone (PFBH), and pyridoxal-2-thiophenecarboxyl hydrazone, compounds previously shown to mobilize iron from cells in vitro, are also effective in vivo. All of these chelators significantly enhanced biliary excretion of iron (measured by atomic absorption spectrophotometry) following their intraperitoneal (IP) and/or oral administration to rats. The most effective was PFBH, which increased iron concentration in the bile about 150-fold, as compared with basal biliary iron concentration, within 1 hour following a single IP dose of 0.2 mmol/kg body weight. In contrast, desferrioxamine increased the biliary iron concentration only 20-fold to 30-fold under the same conditions. Moreover, while control rats excreted approximately 0.8 microg Fe in 2 hours, treatment with PFBH, PMBH, and desferrioxamine resulted in cumulative excretions of 87, 59, and 22 microg Fe, respectively, in the same period of time. Interestingly, PMBH was also quite effective following gastric administration, resulting in a 6-hour cumulative value of 34 microg Fe. These compounds are nontoxic and are inexpensive and easy to make. Their further evaluation as candidate drugs for the treatment of iron overload is warranted.

The complexes [SnR2(L)] (R = Me, Et, Bu, Ph; H2L = pyridoxal thiosemicarbazone) have been prepared and characterized. In the light of the spectral properties of the complexes in the solid state (IR, mass, Mössbauer) the bideprotonated thiosemicarbazonato anion is O(phenolic)-, N(3)-, S-bonded to the tin atom which probably has trigonal bipyramidal coordination with N(3) atom and R groups occupying equatorial positions. NMR ( 1H, 13C and 119Sn) data in CDCl3 or DMSO-d6 suggest that this coordinative picture remains in these solutions. The ethyl, butyl and phenyl derivatives suppress proliferation of Friend erithroleukaemia cells (FLC). Of the pyridoxal thiosemicarbazone complexes so far evaluated. [SnBu2(L)] and [SnPh2(L)] showed the lowest thresholds for inhibition of FLC proliferation. The effects of these compounds on DMSO-induced differentiation of FLC, DNA synthesis and reverse transcriptase were also assayed.

Cell proliferation during the early phase of growth in regenerating amphibian limbs requires a permissive influence of nerves. Based on analyses of proliferative activity in denervated blastemas, it was proposed that nerves provide factors important for cells to complete the proliferative cycle rather than for mitogenesis itself. One such factor, the iron-transport protein transferrin (Tf), is abundant in regenerating peripheral nerves where it is axonally transported and released at growth cones. Using blastemas in organ culture, which have been widely used in previous investigations of the neural effect on growth, it was shown here that the growth-promoting activity of neural extract was completely removed by immuno-absorption with antiserum against Tf and restored by addition of Tf. Purified Tf or a low molecular weight ferric ionophore were as active as the neural extract in this assay, indicating that the trophic effect of Tf involves its capacity for iron delivery. Both Tf and ferric ionophore also maintained DNA synthesis in denervated blastemas in vivo. A dose-response assay indicated that purified axolotl Tf stimulates growth of cultured blastemal cells at concentrations as low as 100 ng/mL. The Tf mRNA in axolotl nervous tissue was shown by northern analysis to be similar in size to that of liver. These results are discussed together with those from previous in vitro studies of blastemal growth and support the hypothesis that cell division in the blastema depends on axonally released Tf during the early, nerve-dependent phase of limb regeneration.

The partition coefficients P between n-octanol and water of pyridoxal isonicotinoyl hydrazone and 34 analogues have been determined experimentally; the values indicate that the partition coefficients calculated for these compounds, and previously reported (P. Ponka, D.R. Richardson, J.T. Edward, and F.L. Chubb. Can. J. Physiol. Pharmacol. 72: 659-666. 1994; D.R. Richardson, E.H. Tran, and P. Ponka. Blood, 86: 4294-4306. 1994), are too low by 2-3 orders of magnitude. The calculations, using Rekker's additive method, failed because the molecules have two or more hydrophilic sites close together. More recent additive schemes (CLOGP, KOWWIN, ACD/LogP) also failed. The only reliable method was the semi-empirical method of Hansch. This requires the experimental determination of the partition coefficient of at least one representative in each series of compounds of related structure. In the present paper, determination of log P of three representatives enabled us to calculate the partition coefficients of the other 32 compounds with acceptable accuracy. The new results show that apochelators have maximum activity in releasing 59Fe from reticulocytes when they have log P = 2.8 (P = 630), and not log P = 0 (P = 1), as reported by Ponka et al. (P. Ponka, D.R Richardson, J.T. Edward, and F.L Chubb. Can. J. Physiol. Pharmacol. 72: 659-666 1994).

This study examined if complexation with metals increased the antiproliferative activities of chelators of the pyridoxal isonicotinoyl hydrazone (PIH) class. Addition of iron(III) to some PIH analogs markedly depressed their activities, whereas it had little effect on others. The gallium(III) complex of PIH, but not its copper(II) complex, was more efficient than the apochelator at inhibiting [3H]thymidine incorporation.

The subthalamic nucleus (SThN) provides a glutamate mediated excitatory drive to several other component nuclei of the basal ganglia, thereby significantly influencing locomotion and control of voluntary movement. We have characterised functionally the metabotropic glutamate (mGlu) receptors in the SThN using extracellular single unit recording from rat midbrain slices. SThN neurones fired action potentials spontaneously at a rate of 10 Hz which was increased by the group I/II mGlu receptor agonist (1S,3R)-1-aminocyclopentane-1,3-dicarboxylate (1S,3 R-ACPD; 1-30 microM) and the group I selective agonist (S, R)-dihydroxyphenylglycine (DHPG; 1-30 microM). However, both the group II selective agonist (1S,1'R,2'R,3'R)-2-(2,3-dicarboxycyclopropyl)glycine (DCG-IV; 1 microM) and the group III selective agonist (S)-2-amino-4-phosphonobutanoic acid (L-AP4; 10 microM) were without effect, indicating that the excitation was mediated by a group I mGlu receptor. The excitation caused by DHPG (3 microM) was reversed by co-application of the mGlu receptor antagonist (+)-alpha-methyl-4-carboxyphenylglycine (MCPG; 500 microM). Thus a group I mGlu receptor mediates excitation of SThN neurones, and suggests a use for group I mGlu receptor ligands for treatment of both hypo- and hyperkinetic disorders of basal ganglia origin, such as Parkinson's disease and Huntington's disease.

Iron (Fe) chelators of the pyridoxal isonicotinoyl hydrazone (PIH) class may be useful agents to treat Fe overload disease and also cancer. These ligands possess high activity at mobilizing 59Fe from neoplastic cells, and the present study has been designed to examine whether their marked activity may be related to an energy-dependent transport process across the cell membrane. Initial experiments examined the release of 59Fe from SK-N-MC neuroblastoma (NB) cells prelabelled for 3 h at 37 degrees C with 59Fe-transferrin (1.25 microM) and then reincubated in the presence and absence of the chelators for 3 h at 4 degrees C or 37 degrees C. Prelabelled cells released 4-5% of total cellular 59Fe when reincubated in minimum essential medium at 4 degrees C or 37 degrees C. When the chelators desferrioxamine (DFO; 0.1 mM) or PIH (0.1 mM) were reincubated with labelled cells at 4 degrees C, they mobilized only 4-5% of cellular 59Fe, whereas as 37 degrees C, these ligands mobilized 21% and 48% of cell 59Fe, respectively. The lipophilic PIH analogue, 311 (2-hydroxy-1-naphthylaldehyde isonicotinoyl hydrazone; 0.1 mM), which exhibits high anti-proliferative activity, released 10% and 53% of cellular 59Fe when reincubated with prelabelled cells at 4 degrees C and 37 degrees C, respectively. Almost identical results were obtained using the SK-Mel-28 melanoma cell line. These data suggest that perhaps temperature-dependent mechanisms are essential for 59Fe mobilization from these cells. Interestingly, the metabolic inhibitors, 2,4-dinitrophenol, oligomycin, rotenone, and sodium azide, markedly decreased 59Fe mobilization mediated by PIH, but had either no effect or much less effect on 59Fe release by 311. Considering that an ATP-dependent process was involved in 59Fe release by PIH, further studies examined 4 widely used inhibitors of the multi-drug efflux pump P-glycoprotein (P-gp). All of these inhibitors, namely, verapamil (Ver), cyclosporin A (CsA), reserpine (Res) and quinine (Qui), decreased 59Fe mobilization by PIH but had little or no effect on 59Fe release mediated by analogue 311. Further, both CsA and Ver increased the proportion of ethanol-soluble 59Fe within cells in the presence of PIH, suggesting inhibited transport of the 59Fe complex from the cell. However, when PIH-mediated 59Fe release was compared between a well characterized Chinese hamster ovary cell line (CHRB30) expressing high levels of P-gp and the relevant control cell line (AuxB1), no appreciable difference in the kinetics of 59Fe release were found. In contrast, it was intriguing that the CHRB30 cells released more 59Fe into control medium (i.e., without PIH) than the AuxB1 control line (16.7% compared to 5.9%, respectively). In summary, the results suggest that a temperature- and energy-dependent process was involved in the efflux of the PIH-59Fe complex from the cells. In contrast, 59Fe release mediated by 311 was temperature-dependent but not energy-dependent, and could occur by simple diffusion or passive transport. Further studies investigating the membrane transport of Fe chelators may be useful in designing regimes that potentiate their anti-neoplastic effects.

We have recently screened 36 analogues of the lipophilic iron (Fe) chelator, pyridoxal isonicotinoyl hydrazone (PIH), for their antiproliferative effect (Richardson et al, Blood 86:4295, 1995). Of these compounds, 1 chelator derived from salicylaldehyde benzoyl hydrazone (206) and 4 ligands derived from 2-hydroxy-1-naphthylaldehyde benzoyl hydrazone (308, 309, 311, and 315) showed pronounced antiproliferative activity, being far more effective than desferrioxamine (DFO). The present study was designed to investigate in detail the mechanism of action of these PIH analogues in a variety of neoplastic cell lines. This investigation showed that the analogues were far more active than DFO at inhibiting cellular proliferation and 3H-thymidine, 3H-leucine, and 3H-uridine incorporation. Additional experiments showed that, in contrast to DFO, the 5 analogues were potent at preventing 59Fe uptake from transferrin (Tf) and increasing 59Fe release from cells at concentrations as low as 10 micromol/L. Examination of the distribution of 59Fe in neoplastic cells using native polyacrylamide gel electrophoresis (PAGE)/59Fe-autoradiography showed that most of the 59Fe taken up from Tf was incorporated into ferritin, although 3 other previously unrecognized components (bands A, B, and C) were also identified. Band C comigrated with 59Fe-citrate and was chelated on incubation of neuroblastoma cells with DFO, PIH, or the PIH analogues, with this compartment being the main intracellular target of these ligands. Further work showed that the effects of the chelators at inducing characteristics consistent with apoptosis or necrosis were cell line-specific, and while DFO increased the percentage of cells in the G0/G1 phases in all cell types, the effect of analogue 311 on the cell cycle was variable depending on the cell line. This study provides further evidence for the potential use of these Fe chelators as anticancer agents.

Gallium nitrate is active against lymphoma and bladder cancer; however, little is understood about tumor resistance to this drug. Transferrin, the iron transport protein, increases gallium uptake by cells, whereas pyridoxal isonicotinoyl hydrazone (PIH), an iron chelator, transports iron into cells. Therefore, we examined whether these metal transporters would increase the cytotoxicity of gallium in gallium nitrate-resistant CCRF-CEM cells. Transferrin, in increasing concentrations, enhanced the cytotoxicity of gallium nitrate. One mg/ml transferrin decreased the 50% inhibitory concentration of gallium nitrate from 1650 to 75 micrometer in gallium-resistant cells and from 190 to 150 micrometer in gallium-sensitive cells. Transferrin also enhanced the cytotoxicity of gallium even at drug concentrations that were not growth inhibitory. The gallium chelate Ga-PIH inhibited the growth of both gallium nitrate-resistant and -sensitive cells. Fifty micrometer Ga-PIH inhibited cellular proliferation by 50%, whereas similar concentrations of PIH or gallium nitrate were not growth inhibitory. However, because higher concentrations of PIH also inhibited cell growth, the cytotoxicity of Ga-PIH was greater than PIH only at concentrations of <100 micrometer. Cross-titration experiments demonstrated that the cytotoxicity of PIH was partially reversed by gallium nitrate, whereas the cytotoxicity of gallium nitrate was enhanced by PIH. Our studies suggest that Ga-PIH warrants further evaluation as a potential antineoplastic agent. Because transferrin increases the cytotoxicity of gallium nitrate in transferrin receptor-bearing, gallium nitrate-resistant cells, future clinical trials of this drug should incorporate the development of strategies to increase plasma transferrin levels.

Numerous studies have suggested that iron (Fe) chelators such as desferrioxamine (DFO) may be useful antitumor agents (Blatt and Stitely, Cancer Res 47:1749, 1987; Becton and Bryles, Cancer Res 48:7189, 1988). Recent work with several analogues of the lipophilic Fe chelator, pyridoxal isonicotinoyl hydrazone (PIH), indicate that some of these ligands are considerably more efficient than DFO both in terms of their Fe chelation efficacy and at preventing 3H-thymidine incorporation by neuroblastoma (NB) cells (Richardson and Ponka, J Lab Clin Med 124:660, 1994). Considering this fact, the present study was designed to test the antiproliferative effect of a wide range of PIH analogues to identify the most active compounds. A total of 36 ligands have been examined that were synthesized by condensation of three types of aromatic aldehydes (pyridoxal, salicylaldehyde, and 2-hydroxy-1-naphthyladehyde) with a range of acid hydrazides. The effects of these chelators were assessed using the human NB cell line, SK-N-MC. Although PIH was far more effective than DFO at preventing Fe uptake from transferrin, it was less effective than DFO at preventing cellular proliferation (DFO ID50 = 22 mumol/L; PIH ID50 = 75 mumol/L). In contrast, 14 PIH analogues were far more efficient than DFO at preventing proliferation (ID50 = 1 to 7 mumol/L) and may have potential as antitumor agents. The most effective compounds were those hydrazones derived from 2-hydroxy-1-naphthylaldehyde. Most of the PIH analogues were considerably more effective than DFO at both preventing 59Fe uptake from 59Fe-transferrin and in mobilizing 59Fe from prelabeled NB cells. In addition, a linear relationship between Fe chelation efficacy and antiproliferative activity was found only for hydrazones derived from salicylaldehyde. Apart from gallium (Ga) nitrate having an antiproliferative effect by itself, this metal potentiated the antiproliferative effect of PIH but not that of DFO. Spectrophotometric studies showed that PIH could chelate Ga, and it can be suggested that, like the PIH-Fe complex that donates Fe to reticulocytes (Ponka et al, Biochim Biophys Acta 718:151, 1982), the PIH-Ga complex may efficiently bestow Ga to NB cells. The results suggest that analogues of PIH deserve further vigorous investigation because they may be useful therapeutic agents for the treatment of cancer.

Histidine, arginine, tyrosine, lysine and cysteine residues of protein S1 were modified with diethyl pyrocarbonate & rose bengal, 2,3-butanedione (diacetyl), tetranitromethane, pyridoxal 5-phosphate, and N-ethyl-maleimide, respectively. Modification of the residues and the number of modified residues were determined by either fluorescence or UV spectroscopy. The effect of chemical modification on the function of protein S1 was studied with respect to nucleic acid (poly U and M13 ssDNA) binding and ribosome binding properties of the protein. We tested S1 binding to these two types of polynucleotides because of their reported (Draper et al. (1977) PNAS 74, 4786-4790) binding to two different sites on S1. The results indicate that histidine and lysine residues of S1 play an important role in the binding of S1 to both types of nucleic acids and that histidine and to some extent tyrosine residues are involved in the binding of S1 to ribosomes. The Data indicate the need for a re-evaluation of the two nucleic acid binding-site model.

The mechanisms of iron uptake from transferrin and the effects of iron chelators on these processes were investigated in human neuroblastoma cells. This study was performed because numerous reports have indicated that neuroblastoma cells contain iron-rich ferritin and are also especially sensitive to iron chelation by deferoxamine. The mechanisms of iron and transferrin uptake were examined in the human neuroblastoma cell line SK-N-MC by using human transferrin labeled with iodine 125 and iron 59. Internalized and membrane-bound 59Fe and 125I-transferrin were separated with the protease pronase. Total internalized and membrane 125I-transferrin uptake was biphasic with time, whereas total and internalized 59Fe uptake was linear. Iron uptake from transferrin was prevented by incubation at 4 degrees C and also by lysosomotrophic agents. In addition, 59Fe uptake occurred by two processes. The first process was consistent with receptor-mediated endocytosis involving internalization of transferrin bound to specific binding sites. Iron uptake also occurred by a second process, which was not saturable up to a transferrin concentration of 0.06 mg/ml. In terms of quantitative iron uptake, however, the second process was far less important than receptor-mediated endocytosis. Deferoxamine (0.25 mmol/L) only slightly increased 59Fe release from prelabeled cells; the orally effective iron chelator pyridoxal isonicotinoyl hydrazone (0.25 mmol/L) was six times more effective. Moreover, when pyridoxal isonicotinoyl hydrazone (0.2 mmol/L) was added together with labeled transferrin over a 2-hour incubation, 59Fe uptake from transferrin decreased to 18% of the control value, whereas deferoxamine (0.2 mmol/L) had no appreciable effect. Even though deferoxamine (0.1 mmol/L) had little effect on 59Fe uptake or release, it reduced uptake of tritiated thymidine to 33% of the control value after a 24-hour incubation. Three analogs of pyridoxal isonicotinoyl hydrazone, pyridoxal benzoyl hydrazone (#101), pyridoxal p-methoxybenzoyl hydrazone (#107), and pyridoxal m-fluorobenzoyl hydrazone (#109), had chelation activities comparable to that of pyridoxal isonicotinoyl hydrazone and were more effective than either deferoxamine or pyridoxal isonicotinoyl hydrazone at preventing tritiated thymidine uptake. These results suggest that the pyridoxal isonicotinoyl hydrazone analogs have potential as effective antiproliferative agents and deserve further investigation.

A protein toxin, termed peditoxin, containing an active prosthetic group was isolated and purified from the globiferous pedicellariae of the sea urchin Toxopneustes pileolus (Lamarck). The prosthetic group, called pedoxin, is a small molecular mass substance (206 daltons) with an empirical formula of C10H10N2O3 and is comprised of a heterocyclic lactone structure formed from pyridoxal and glycine. Administered subcutaneously or intramuscularly to mice in sublethal doses, pedoxin markedly reduced basal body temperature and led to sedation and anesthetic coma accompanied by muscular relaxation. At higher doses, it leads to convulsion and death (LD50 200 mg/kg). The apoprotein, a cytochrome b-like heme protein called pedin (molecular mass, 10,000 daltons) is itself not toxic, but the purified holoprotein is extremely toxic, causing anaphylaxis-like shock and death in experimental animals at low doses (LD50 70 micrograms/kg). Small amounts of the prosthetic group added to holoprotein preparations caused the toxicity to be greatly enhanced, suggesting that holoprotein preparations also contain apoprotein capable of being activated by the low molecular weight toxin. The structure of pedoxin was determined to be 3-hydroxy-2-methyl-5-methoxy-4-pyridineformyl-glycyliden ester and was confirmed by total synthesis.

Analogues of the iron (III) chelator, pyridoxal isonicotinoyl hydrazone (PIH) show high potential as orally active agents for the treatment of iron-overload diseases, such as thalassemia. In the present study, the n-octanol-water partition coefficients of 30 analogues of PIH were measured by thin-layer chromatography and also calculated using the additive schemes of Rekker. The purpose was to examine the relationship between lipophilicity of the apochelator and its ability to promote the release of 59Fe from reticulocytes loaded with nonheme 59Fe. Interestingly, maximum activity occurred when the partition coefficient of the apochelator was approximately 1 (log P = 0). Considering the results in the context of the design and synthesis of more active analogues of PIH, it can be suggested that before initiating synthesis, a useful indication of biological activity can be determined by examining the lipophilicity of the molecule, using the schemes of Rekker to calculate the partition coefficient. By using this strategy, analogues of PIH with inappropriate lipophilicity can be excluded before initiating the expensive process of screening in biological models.

The iron chelators desferrioxamine (DFO), pyridoxal isonicotinoyl hydrazone (PIH), 2,2'-bipyridine, diethylenetriamine penta-acetic acid (DTPA) and 1,2 dimethyl-3-hydroxy pyrid-4-one (CP20) were analysed for their ability to change 59Fe uptake and release from the brain of 15- and 63-day rats either during or after intravenous injection of 59Fe-125I-transferrin. DTPA was the only chelator unable to significantly reduce iron uptake into the brain of 15-day rats. This indicates that iron is not released from transferrin at the luminal surface of brain capillary endothelial cells. CP20 was able to reduce iron uptake in the brain by 85% compared to 28% with DFO. Only CP20 was able to significantly reduce brain iron uptake in 63 day rats. Once 59Fe had entered the brain no chelator used was able to mediate its release. All of the chelators except CP20 had similar effects on femur iron uptake as they did on brain uptake, suggesting similar iron uptake mechanisms. It is concluded that during the passage of transferrin-bound iron into the brain the iron is released from transferrin within endothelial cells after endocytosis of transferrin.

In the vitamin B6 deficiency-induced hypertensive (B6DHT) rat there is an increased influx of calcium into the vascular smooth muscle. Vitamers which possess vitamin B6 activity blocked the in vitro calcium influx into the caudal artery of B6DHT rats and the BAY K 8644-induced influx into artery segments from vitamin B6-supplemented (control) rats. BAY K 8644 did not increase further the calcium influx into caudal artery segments from B6DHT rats. However, when this influx was partly decreased by Nifedipine or by pyridoxal phosphate, BAY K 8644 antagonized their effects indicating that calcium influx mediated by the dihydropyridine-sensitive calcium channel is a site of the calcium influx defect in the B6DHT rat.

Pyridoxal and cis-diamminedichloroplatinum (II) (cis-DDP) have a different mode of interaction with DNA. Cis-DDP caused extensive transforming inactivation of pBR322 DNA but did not form strand breaks in DNA molecules. On the other hand, pyridoxal formed frank strand breaks in the DNA chains and decreased DNA transforming activity. A higher level of DNA transforming inactivation was obtained during simultaneous application of both compounds than would be an additive effect of these compounds applied to DNA independently. This synergistic effect can be ascribed to the introduction of thermolabile sites by the combined action of cis-DDP and pyridoxal. These lesions were converted by heat-treatment to DNA strand breaks and detected electrophoretically. Using two different methods, cis-DDP and pyridoxal, during simultaneous application, interacted with DNA with higher efficiency.

Lysyl oxidase, which cross-links collagen and elastin, was obtained from chick embryo bone and cartilage and its substrate, elastin, from aorta. The enzyme was studied using an improved assay which enabled the stability of the substrate to be monitored. The enzyme was fully inhibited in vivo by beta-aminopropionitrile, semicarbazide, thiosemicarbazide and isoniazid and in vitro by beta-aminopropionitrile and semicarbazide but only partially by thiosemicarbazide and isoniazid. Penicillamine, which solubilizes collagen by labilizing Schiff base cross-links in vivo and which prevents stable cross-link formation in vitro indirectly by binding to aldehyde groups on collagen, was shown to have no direct inhibitory effect on lysyl oxidase in vivo or in vitro. Homocysteine, which also solubilizes collagen by a mechanism similar to penicillamine does not inhibit lysyl oxidase either in vivo or in vitro. Pyridoxal reversed the inhibition of lysyl oxidase by semicarbazide and isoniazid in vivo but was unable to reverse that produced by either beta-aminopropionitrile or thiosemicarbazide. These results can be explained by the presence of a sulphydryl group near the active site of lysyl oxidase, which can form a complex with the nitrile group on beta-aminopropionitrile or with the thiol group on thiosemicarbazide leading to irreversible inhibition.

Human erythrocytes rapidly convert vitamin B6 to pyridoxal-P and contain soluble phosphatase activity which dephosphorylates pyridoxal-P at a pH optimum of 6-6.5. This phosphatase was purified 51,000-fold with a yield of 39% by ammonium sulfate precipitation and chromatography on DEAE-Sepharose, Sephacryl S-200, hydroxylapatite, and reactive yellow 86-agarose. Sephacryl S-200 chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the enzyme was a dimer with a molecular mass of approximately 64 kDa. The phosphatase required Mg2+ for activity. It specifically catalyzed the removal of phosphate from pyridoxal-P, pyridoxine-P, pyridoxamine-P, 4-pyridoxic acid-P, and 4-deoxypyridoxine-P at pH 7.4. Nucleotide phosphates, phosphoamino acids, and other phosphorylated compounds were not hydrolyzed significantly nor were they effective inhibitors of the enzyme. The phosphatase showed Michaelis-Menten kinetics with its substrates. It had a Km of 1.5 microM and a Vmax of 3.2 mumol/min/mg with pyridoxal-P. The Vmax/Km was greatest with pyridoxal-P greater than 4-pyridoxic acid-P greater than pyridoxine-P greater than pyridoxamine-P. The phosphatase was competitively inhibited by the product, inorganic phosphate, with a Ki of 0.8 mM, and weakly inhibited by pyridoxal. It was also inhibited by Zn2+, fluoride, molybdate, and EDTA, but was not inhibited by levamisole, L-phenylalanine, or L(+)-tartrate. These properties of the purified enzyme suggest that it is a unique acid phosphatase that specifically dephosphorylates vitamin B6-phosphates.