Increased lipophilicity and subsequent cell partitioning decrease passive transcellular diffusion of novel, highly lipophilic antioxidants.

Oxidative stress is considered a cause or propagator of acute and chronic disorders of the central nervous system. Novel 2, 4-diamino-pyrrolo[2,3-d]pyrimidines are potent inhibitors of iron-dependent lipid peroxidation, are cytoprotective in cell culture models of oxidative injury, and are neuroprotective in brain injury and ischemia models. The selection of lead candidates from this series required that they reach target cells deep within brain tissue in efficacious amounts after oral dosing. A homologous series of 26 highly lipophilic pyrrolopyrimidines was examined using cultured cell monolayers to understand the structure-permeability relationship and to use this information to predict brain penetration and residence time. Pyrrolopyrimidines were shown to be a more permeable structural class of membrane-interactive antioxidants where transepithelial permeability was inversely related to lipophilicity or to cell partitioning. Pyrrole substitutions influence cell partitioning where bulky hydrophobic groups increased partitioning and decreased permeability and smaller hydrophobic groups and more hydrophilic groups, especially those capable of weak hydrogen bonding, decreased partitioning, and increased permeability. Transmonolayer diffusion for these membrane-interactive antioxidants was limited mostly by desorption from the receiver-side membrane into the buffer. Thus, in this case, these in vitro cell monolayer models do not adequately mimic the in vivo situation by underestimating in vivo bioavailability of highly lipophilic compounds unless acceptors, such as serum proteins, are added to the receiving buffer.

Novel, highly lipophilic antioxidants readily diffuse across the blood-brain barrier and access intracellular sites.

In an accompanying article, an in vitro assay for permeability predicts that membrane-protective, antioxidant 2,4-diamino-pyrrolo[2, 3-d]pyrimidines should have improved blood-brain barrier (BBB) permeation over previously described lipophilic antioxidants. Using a first-pass extraction method and brain/plasma quantification, we show here that two of the pyrrolopyrimidines, one of which is markedly less permeable, readily partition into rat brain. The efficiency of extraction was dependent on serum protein binding, and in situ efflux confirms the in vitro data showing that PNU-87663 is retained in brain longer than PNU-89843. By exploiting inherent fluorescence properties of PNU-87663, its distribution within brain and within cells in culture was demonstrated using confocal scanning laser microscopy. PNU-87663 rapidly partitioned into the cell membrane and equilibrates with cytoplasmic compartments via passive diffusion. Although partitioning of PNU-87663 favors intracytoplasmic lipid storage droplets, the compound was readily exchangeable as shown by efflux of compound from cells to buffer when protein was present. The results demonstrated that pyrrolopyrimidines were well suited for quickly accessing target cells within the central nervous system as well as in other target tissues.

Pyrrolopyrimidines: novel brain-penetrating antioxidants with neuroprotective activity in brain injury and ischemia models.

A novel group of antioxidant compounds, the pyrrolopyrimidines, has been discovered recently. Many of these possess significantly improved oral bioavailability (56-70% in rats), increased efficacy and potency in protecting cultured neurons against iron-induced lipid peroxidative injury and as much as a 5-fold increase in brain uptake compared with the 21-aminosteroid antioxidant compound, tirilazad mesylate (U-74006F), described earlier. They appear to quench lipid peroxidation reactions by electron-donating and/or radical-trapping mechanisms. Several compounds in the series, such as U-101033E and U-104067F, demonstrate greater ability than tirilazad to protect the hippocampal CA1 region in the gerbil transient (5-min) forebrain ischemia model. Delaying treatment until 4 hr after the ischemic insult still results in significant CA1 neuronal protection. U-101033E is still effective in salvaging a portion of the CA1 neuronal population when the ischemic duration is extended to 10 min. In addition, U-101033E has been found to be protective in the context of focal cerebral ischemia, reducing infarct size in the mouse permanent middle cerebral artery occlusion model, in contrast to tirilazad which is minimally effective. These results suggest that antioxidant compounds with improved brain parenchymal penetration are better able to limit certain types of ischemic brain damage than those which are localized in the cerebral microvasculature. However, the activity of U-101033E in improving early post-traumatic recovery in mice subjected to severe concussive head injury is similar to that of tirilazad. Last, the oral bioavailability of many pyrrolopyrimidines suggests that they may be useful for certain chronic neurodegenerative disorders in which lipid peroxidation plays a role.

Pharmacological characterization of U-101387, a dopamine D4 receptor selective antagonist.

Dopamine D2-like receptors play an important role in the pharmacotherapy of psychotic disorders. Molecular and cellular techniques have identified distinct gene products (D2-long, D2-short, D3 and D4) displaying the D2 receptor pharmacology. However, the contribution of each subtype in antipsychotic effects of or their physiological role remain unclear. Here we describe the pharmacological effects of a selective D4 antagonist, U-101387. U-101387 displayed moderately high affinity (Ki = 10 nM) and selectivity for the dopamine D4.2 receptor expressed in clonal cell lines. It lacked measurable affinity for not only other dopamine receptors but also noradrenalin, serotonin and histamine receptor families (Ki > 2000 nM). It fully and dose-dependently antagonized quinpirole-induced cAMP inhibition (without producing any effect by itself) in stably transfected cells. U-101387 also displayed excellent oral bioavailability, brain penetration and other pharmacokinetic characteristics. Unlike classical neuroleptics (e.g., haloperidol), U-101387 neither blocked acute behavioral effects of amphetamine or apomorphine nor did it alter spontaneous locomotion by itself. Additionally, U-101387 was without effect in behavioral and biochemical tests predictive of extrapyramidal and neuroendocrine side effects. Consistent with the lack of autoreceptor function of D4, acute administration of U-101387 failed to alter dopamine neuronal firing by itself or reverse the inhibition produced by dopamine agonists and to affect monoamine turnover in areas innervated by the mesencephalic or hypothalamic dopamine neurons. However, U-101387 potently induced c-fos mRNA expression in the infralimbic/ventral prelimbic cortex to a level similar to that produced by the atypical antipsychotic, clozapine. This is consistent with the predominantly cortical distribution of the D4 receptor. Taken together, these results demonstrate that the D4-selective antagonist, U-101387, produces effects that are distinct from those of the nonselective D2 antagonists as well as D3-preferring agents. U-101387 offers a unique tool to understand the role of dopamine D4 receptors in diseases involving central dopamine systems.

Neuroprotective efficacy of microvascularly-localized versus brain-penetrating antioxidants.

The 21-aminosteroid (lazaroid) tirilazad mesylate has been demonstrated to be a potent inhibitor of lipid peroxidation and to reduce traumatic and ischemic damage in a number of experimental models. Currently, tirilazad is being actively investigated in phase III clinical trials in head and spinal cord injury, ischemic stroke and subarachnoid hemorrhage. This compound acts in large part to protect the microvascular endothelium and consequently to maintain normal blood-brain barrier (BBB) permeability and cerebral blood flow autoregulatory mechanisms. However, due to its limited penetration into brain parenchyma, tirilazad has generally failed to affect delayed neuronal damage to the selectively vulnerable hippocampal CA1 and striatal regions. Recently, we have discovered a new group of antioxidant compounds, the pyrrolopyrimidines, which possess significantly improved ability to penetrate the BBB and gain direct access to neural tissue. Several compounds in the series, such as U-101033E, have demonstrated greater ability to protect the CA1 region in the gerbil transient forebrain ischemia model with a post-ischemic therapeutic window of at least four hours. In addition, U-101033E has been found to reduce infarct size in the mouse permanent middle cerebral artery occlusion model in contrast to tirilazad which is minimally effective. These results suggest that antioxidant compounds with improved brain parenchymal penetration are better able to limit certain types of ischemic brain damage compared to those which are localized in the cerebral microvasculature. On the other hand, microvascularly-localized agents like tirilazad appear to have better ability to limit BBB damage.

HPLC-chemiluminescence and thermospray LC/MS study of hydroperoxides generated from phosphatidylcholine.

Lipid hydroperoxides generated from phosphatidylcholine (PC) by two commonly employed phosphatidylcholine hydroperoxide (PCOOH) generation methods were examined by HPLC-chemiluminescence (CL) and thermospray LC/MS assay. This HPLC-CL assay is specific for hydroperoxides. In the HPLC-CL chromatograms, a major peak eluted at 4.7 min for the samples generated by the photooxidation of PC in the presence of methylene blue. The direct LC/MS analysis of the hydroperoxides contained in this peak determined that the hydroperoxides are mono- and di-PCOOH. Quantitation showed that over 90% of the hydroperoxides generated by photooxidation are PCOOH. In contrast, a different major peak appeared at 3.7 min for the hydroperoxides generated by the incubation of PC with the azo compound AMVN. We determined by LC/MS analysis that the hydroperoxides contained in this peak were not equivalent to either mono- or di-PCOOH. Indeed, 70%-95% of the hydroperoxides generated by AMVN incubation were not PCOOH, but rather a large portion were AMVN-derived hydroperoxides. The hydroperoxides contained in the 4.7-min peak (i.e., PCOOH) were preferentially responsive to cytochrome c-luminol CL cocktail (about 100-fold more responsive than the hydroperoxides in the 3.7-min peak), whereas the hydroperoxides in the 3.7-min peak (including AMVN-derived hydroperoxide) were preferentially responsive to microperoxidase-isoluminol CL cocktail (about 20-fold more responsive than the PCOOH), suggesting a substrate specificity for the CL cocktail.

Tirilazad mesylate protects stored erythrocytes against osmotic fragility.

The hypoosmotic lysis curve of freshly collected human erythrocytes is consistent with a single Gaussian error function with a mean of 46.5 +/- 0.25 mM NaCl and a standard deviation of 5.0 +/- 0.4 mM NaCl. After extended storage of RBCs under standard blood bank conditions the lysis curve conforms to the sum of two error functions instead of a possible shift in the mean and a broadening of a single error function. Thus, two distinct sub-populations with different fragilities are present instead of a single, broadly distributed population. One population is identical to the freshly collected erythrocytes, whereas the other population consists of osmotically fragile cells. The rate of generation of the new, osmotically fragile, population of cells was used to probe the hypothesis that lipid peroxidation is responsible for the induction of membrane fragility. If it is so, then the antioxidant, tirilazad mesylate (U-74,006f), should protect against this degradation of stored erythrocytes. We found that tirilazad mesylate, at 17 microM (1.5 mol% with respect to membrane lecithin), retards significantly the formation of the osmotically fragile RBCs. Concomitantly, the concentration of free hemoglobin which accumulates during storage is markedly reduced by the drug. Since the presence of the drug also decreases the amount of F2-isoprostanes formed during the storage period, an antioxidant mechanism must be operative. These results demonstrate that tirilazad mesylate significantly decreases the number of fragile erythrocytes formed during storage in the blood bank.

Kinetic evaluation of lipophilic inhibitors of lipid peroxidation in DLPC liposomes.

The authors have developed a kinetic method that allows one to obtain relative reactivity constants for lipophilic antioxidants in free radical systems. Two experimental model systems were developed: (a) a methanolic solution using AMVN as the free radical initiator and linoleic acid as the substrate, and (b) a multilamellar vesicle system composed of dilinoleoylphosphatidylcholine and AAPH as the substrate and the initiator, respectively. The use of these two systems allows researchers not only to determine the intrinsic reactivity of a potential antioxidant, but also to evaluate its potency in a membranous system where the contribution of the physical properties of the antioxidant to the inhibition of lipid peroxidation is important. These results show that all antioxidants tested acted in these systems as free radical scavengers, and they validate the synergism between intrinsic scavenging ability and membrane affinity and/or membrane-modifying physical properties in the inhibition of lipid peroxidation.

Purification of an endogenous digitalislike factor from human plasma for structural analysis.

In previous reports, we described the isolation and characterization of an endogenous digitalislike factor (EDLF). In this report, we describe a unique combination of bioassay and large-scale purification methodology that made possible the purification of sufficient quantities of this inhibitor of Na+,K(+)-ATPase for structural analysis. Using an initial XAD-2 extraction and preparative high-performance liquid chromatography followed by a batch enzyme affinity extraction and two subsequent semipreparative chromatographic steps, 300 l of human plasma was processed, yielding 31 micrograms (53 nmol) of pure EDLF and representing purification on a dry weight basis in excess of 0.6 billionfold. Four divergent pieces of evidence, including chromatographic, mass spectrometric, immunoreactive, and binding characteristics, suggested that the EDLF purified in the present study was either ouabain or an isomer of ouabain. This material may represent a plasma-borne, naturally occurring, selective, high-affinity ligand for the digitalis binding site that may play a significant role in the modulation of the sodium pump and thereby cellular electrolyte homeostasis in humans.

An improved method for the identification and quantitation of biological lipids by HPLC using laser light-scattering detection.

We have developed a simplified and improved high performance liquid chromatography (HPLC) method for the detection and quantitation of tissue lipid using a new laser light-scattering detector (Varex model ELSD II). This detector has a limit of sensitivity of 50 ng for neutral lipid and 200 ng for most phospholipids with excellent reproducibility. By coupling the ELSD II with a ternary gradient normal phase HPLC system, we were able to separate and quantify the major lipid constituents of extracted tissue. This system was used to profile and quantitate the major lipids from rat brain, liver, and heart with greater sensitivity than other available techniques, with the exception of high performance thin-layer chromatography (HPTLC). However, the convenience of HPLC allows for a significant improvement in analysis time with only a threefold reduction in sensitivity when compared to HPTLC.