I. Introduction

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A considerable body of experimental evidence indicates that lipid peroxidation (Komara et al., 1986; Dexter et al., 1989), the presence of iron (Riederer et al., 1989; Hirsh et al., 1991) and the depletion of natural antioxidants (Sato and Hall, 1992) seem to be a common epiphenomena of some pathologies in the central nervous system (CNS).^b

The role of iron (either free or complexed) in catalyzing oxygen-derived free radical production and, consequently, its role in the peroxidative process (Halliwell and Gutteridge, 1984; Braughler et al., 1986; Minotti and Aust, 1989) is well known, even though the involvement of this biochemical pathway with the pathogenesis of some neuropathologies remains unclear.

The radical-initiated peroxidation of neuronal, glial, vascular cell membranes and myelin is catalyzed by free iron released from hemoglobin, transferrin and ferritin by either lowered tissue pH or oxygen radicals. If unchecked, lipid peroxidation is a geometrically progressing process that will spread over the surface of the cell membrane, causing impairment to phospholipid-dependent enzymes, disruption of ionic gradients and, if severe enough, membrane lysis.

Natural or synthetic compounds with scavenger and/or chelating properties have been found in in vitro and in vivo experimental models, aimed to protect the nervous tissue from the lipid peroxidative attack (Jacobsen et al., 1990; Hara et al., 1990a; Hall et al., 1991a; Ciuffi et al., 1992), but only some of them seem to be efficient for the activity in vivo (Hall, 1987; Hall and Braughler, 1989).

Lipid peroxidation normally proceeds as a radical-driven chain reaction involving oxygen, where the lipid peroxyl radical (LOO·), formed through initiation, attacks a second unsaturated fatty acid (LH). An important endogenous inhibitor of lipid peroxidation in membranes is alpha-tocopherol (alpha-TC), that inhibits lipid peroxidation by scavenging (LOO·):

(1)

thus preventing lipid radical chain reaction from occurring (Braughler and Pregenzer, 1989). The alpha-TC· radical decomposes to tocopherolquinone and effectively terminates the chain reaction (Braughler and Pregenzer, 1989). In addition, the alpha-TC radical is much less reactive in attacking adjacent fatty acid side chains and can be converted back to alpha-TC by vitamin C.

Studies with intact membranes indicate that the 21-aminosteroids are as potent as alpha-TC as inhibitors of iron-dependent lipid peroxidation, and their reactivity is less than that for alpha-TC in scavenging (LOO·).

The chemistry of free radical formation provides several sources that may cause cell generation of superoxide free radical (·O₂) and that use (H⁺) to undergo spontaneous dismutation reaction (reaction 2). Hydrogen peroxide (H₂O₂), which results from this spontaneous dismutation, in the presence of another superoxide radical (anion), can undergo reduction to form a highly reactive hydroxyl free radical (·OH), molecular oxygen and the hydroxide ion (OH) (reaction 3). Hydroxide anion is also released when hydroxyl free radicals are formed from a series of reactions involving (Fe^3+/2+) metal ions (reactions 4 and 5):

(2)

(3)

(4)

(5)

The reaction (reaction 5) is known also as the Fenton reaction, certainly occurring in vitro.

The free radicals (·O₂) and (·OH) and the compound (H₂O₂) are generated by all aerobic cells, and their antioxidant defenses prevent these species from causing cell injury. The clamping effects occur when the rate of formation of these free radical species is increased and/or the antioxidant defenses of cells are weakened.

In these conditions, excessive production and concentrations of radical species (R·) can initiate lipid peroxidation by attacking and removing an allylic hydrogen from a (poly)unsaturated fatty acid (LH) of membrane phospholipids (reaction 6); rearrangements of the double bonds results in the formation of conjugated dienes. The resulting allylic (poly)unsaturated fatty acid radical (lipid free radical) (L·) reacts with O₂ dissolved within the membrane to form a strong oxidizing species, lipid peroxyl radical (LOO·) (reaction 7), which can extract a second allylic hydrogen (reaction 8) ion from another methylene carbon.

This autocatalytic process converts the carbons of fatty acids of membrane phospholipids to unstable and highly reactive hydroperoxide lipids radical (LOOHs), which are further fragmented to a variety of lower molecular weight products, resulting in the destruction of unsaturated fatty acids of membrane phospholipids, including malondialdehyde, ethane and pentane.

The Fe^3+/2+ can react directly with the hydroperoxide radical of lipids (reactions 9 and 10):

(6)

(7)

(8)

(9)

(10)

It was suggested that the absolute ratio of Fe³⁺ to Fe²⁺ was the primary determining factor for the initiation of lipid peroxidation reactions on the order of 1:1 to 7:1 (Braughler et al., 1986).

It should be noted that the reaction of peroxysulphenyl radical will lead to formation of the superoxide anion, thus providing a new source of H₂O₂ by dismutation reaction of the superoxide (reactions 11 and 12):

(11)

(12)

In our opinion, at first it is very important to stress that (R-SH) groups are present in many proteins with catalytic properties, i.e., enzymes; thus, their function may be altered directly. Secondly, malondialdehyde reacts with the -amino groups of lysine, causing the cross-linking of proteins and with other primary amino groups on phospholipids and nucleic acid.

As indicated, the chemistry of radicals is very complex, and it should be stressed that many of the indicated reactions have been studied in vitro. An important first concern is that the reaction (reaction 2) really happens as:

(13)

(14)

thus stressing the fact that, for each H₂O₂ formed, two electrons (e) are necessary (reaction 13). To make the reaction (reaction 3) possible, an additional (e) is required to form the third superoxide radical (that properly is an anion); therefore 3e are required for coupling (reaction 2) plus (reaction 3) reactions; reactions 4 and 5 require (3e), too.

Regarding the role of lipid peroxidation in the injury of the CNS, the mosaic lipid peroxidation is mediated by the free radical in the neurons, which forms lipid peroxides within cell membranes and organelles. These oxidized lipids alter the structure and function of membranes by tissue injury. During periods of ischemic metabolism, superoxide anion is produced by mitochondrial dysfunction, as a by-product of various enzyme-substrate reactions. Electron transport chain in mitochondria and endoplasmic reticulum are major sources of superoxide. When mitochondrial function breaks down, some of the electrons "leak" from the usual electron carriers onto oxygen, forming superoxide anion (reaction 13). This is paradoxically augmented by postischemic reperfusion, especially under hyperoxic conditions (Hall et al., 1994).

Superoxide anion is not itself particularly reactive, and it does not cross cell membranes very well. However, it can become more dangerous by either accepting a proton or by dismutating to hydrogen peroxide (reactions 2, 13 and 14). During ischemia, lactic acidosis can lead to protonation of some of the superoxide anion, and protonated superoxide anion can better penetrate the membrane, where it can initiate lipid peroxidation.

The CNS is particularly susceptible to lipid peroxidation (LeBel and Bondy, 1991) for several reasons. First, the membrane lipids of the brain are rich in polyunsaturated fatty acids, which have particularly reactive hydrogens that can participate willingly in either the initiation or the propagation phases of lipid peroxidation. Second, the brain has only modest antioxidant capacity relative to other organs; it is poor in catalase and weak in superoxide dismutase (SOD) and glutathione peroxidase (Cohen, 1988). Third, several areas of the brain are rich in intracellular iron that is released during the injury process (Youdim and Ben-Schachar, 1988). Fourth, cerebrospinal fluid (CSF) contains much less transferritin than plasma and thus does not bind excess-released iron; the transferrin that is present is essentially saturated (Halliwell and Gutteridge, 1992). Finally, the CNS is rich in monoamine neurotransmitters (dopamine, epinephrine and norepinephrine): these produce H₂O₂ when they are oxidized by monoamine oxidase.

Recently, a family of steroid compounds, 21-aminosteroids, was developed; although this family derived from glucocorticoids, it lacks glucocorticoid and mineralocorticoid activities (Jacobsen et al., 1990). The compounds in this family were shown to scavenge lipid peroxyl radicals and to inhibit iron-dependent lipid peroxidation (Braughler et al., 1988a; Braughler and Pregenzer, 1989). Moreover, they were observed to improve survival, to preserve neurons and to reduce cerebral edema in animal models of focal cerebral ischemia (Hall et al., 1988a; Young et al., 1988a).

Therefore, the 21-aminosteroids or "lazaroids," a novel series of lipid peroxidation inhibitors, were designed to be devoid of glucocorticoid receptor interactions, while simultaneously retaining a propensity for cell membrane localization and having improvements in lipid peroxidation inhibitory efficacy in comparison with methylprednisolone. In particular, one of these compounds, U-74006F (United States and United Kingdom generic name is tirilazad mesylate) was selected for clinical development, as a parenterally administered acute neuroprotective agent. The chemical structural formula of these compounds (series: F-A-E) is indicated in fig. 1. Among these compounds, U-74500A was reported to inhibit the cytotoxicity and lipid peroxidation of iron-loaded cultured endothelial cells that had been submitted to an exogenous or endogenous oxidant attack (Balla et al., 1990). Moreover, this compound reduces H₂O₂ generation by stimulated human polymorphonuclear leukocytes and decreases both chemiluminescence and H₂O₂ produced by monocytes, which are harvested from the blood of patients affected by multiple sclerosis (Fischer et al., 1990, 1991).

View larger version (20K): [in this window] [in a new window]   Fig. 1.   Chemical structures of the glucocorticoid methylprednisolone, the nonglucocorticoid steroid U-72099E, the 21-aminosteroid U-74006F (tirilazad mesylate) and the 21-aminosteroid U-74500A (lazaroids).

However, apart from the pharmacological characteristics of specific compounds, it is clear that these "lazaroids" represent very interesting molecules with important pharmacological features not yet explored. In the following chapters, these pharmacological actions will be revised with particular relevance to the brain tissue and related pathologies.

As far as the chemicophysical characteristics and bioavailability of lazaroids, these were studied using the in vivo model (Ciuffi et al., 1994) of Wistar rat treated i.p. or s.c.: (a) 12 mg/kg every 24 h, i.p.; (b) 48 mg/kg every 48 h, i.p.; (c) 48 mg/kg every 48 h, s.c.; and (d) s.c., one-fifth of the total dose dissolved in citrate vehicle. Half an hour later, the animals received the remaining part of the drug, now dissolved in PEG 4000 (1.4% aqueous solution, retard preparation).

U-74500A, following administration in the retard form, showed an inhibitory effect on lipid peroxidation in the iron-saccharate-injected brain hemicortices. On the contrary, the aminosteroid dissolved in the buffer (0.02 M citric acid monohydrate, 0.0032 M sodium citrate dihydrate, 0.077 M NaCl, pH 3) appears to be ineffective; this is probably ascribable to the short half-life of this drug (less than 10 min) once it reaches the blood.

It seems that an adequate concentration in the brain tissue that is able to inhibit the continuous iron-induced lipid peroxidation can only be achieved with a retard preparation, and it may be regarded as a possible therapeutical tool in neuropathologies that are characterized by a peroxidative attack. However, it should be noted that, in this form, U-74500A inhibits lipid peroxidation at a step before diene conjugation, and diene formation is considered to be evidence of an early or moderate alteration of the structure of polyunsaturated lipids, as a result of free radical attack (Klein, 1970); in fact, the diene measurement showed a clear decrease in iron-injected drug-treated animals (Ciuffi et al., 1994). Nevertheless, the total iron content of the brains submitted to intracortical injection was not significantly modified by the 21-aminosteroid administration. It has been reported that this lipophylic drug with chelating activity displays spectral changes in the ultraviolet (UV) range in the presence of Fe²⁺ (Braughler et al., 1988a) and inhibits in vitro iron-dependent lipid peroxidation of intact phospholipid membranes (Braughler et al., 1987a). These observations would appear to contrast with the above quoted results; however, it is to be considered that, in this model, 7 days after operation, the iron injected into the brain was almost completely re-adsorbed.

Tirilazad mesylate has been studied in animal models for the prevention of neuronal damage due to head trauma (Hall et al., 1988b), subarachnoid hemorrhage (SAH) (Kanamaru et al., 1990), spinal cord injury (Anderson et al., 1988) and stroke (Hall et al., 1988a). In these systems, tirilazad mesylate, administered intravenously, appears to reduce the moiety rate and promote neurological recovery after acute insult. Doses ranging from 0.03 to 30 mg appear to have beneficial effects in animal model free radical-induced injury after head trauma (Hall et al., 1988b).

The purpose of the trial of Fleishaker et al. (1993a), was to evaluate in humans the tolerability, pharmacological effects and pharmacokinetics of tirilazad mesylate. Doses of 0.25 mg/kg, 0.5 mg/kg, 1.0 mg/kg and 2.0 mg/kg were administered as intravenous infusions over 10 min or 30 min. The final concentration of tirilazad mesylate administered was 0.375 mg/mL, except in those subjects receiving 10 min infusions, who received the drug at a 1.5 mg/mL concentration. No significant effects of tirilazad mesylate on blood pressure, pulse or temperature were observed. No statistically, clinically significant effects of tirilazad mesylate on cardiac rhythm, as assessed by Holter recording, were observed. No effect of tirilazad mesylate on plasma cortisol or adrenocorticotrophic hormone was observed. As regards the total lymphocyte content, a statistically significant treatment-time interaction was observed: this was due to increases in lymphocyte count seen at 24 h and 48 h in the 2.0-mg/kg dose group. No significant differences among treatments in monocyte or eosinophil counts were observed. No significant treatment effects on general serum or urine chemistry or hematology assays were identified. In general, detectable tirilazad mesylate plasma concentrations were observed up to 2 h, 4 h, 8 h and 12 h for the 0.25-mg/kg, 0.5-mg/kg, 1.0-mg/kg and 2.0-mg/kg doses, respectively.

These results (Fleishaker et al., 1993a) show that tirilazad mesylate was well tolerated at the doses administered. No clinically significant effects of tirilazad mesylate on cardiovascular function or on clinical laboratory determinations were apparent. Thus, single doses of tirilazad mesylate appear to be devoid of glucocorticoid and mineralocorticoid activity in healthy male volunteers, and no safety concerns for single-dose tirilazad mesylate were identified from this study.

The pharmacokinetics of tirilazad mesylate are dose-independent for corrected values (C_inf) under single-dose conditions at doses up to 2.0 mg/kg. Tirilazad mesylate and related compounds appear to have high affinity for peripheral tissues (Cox et al., 1989). These data suggest that several tissues in humans may also have high affinity for tirilazad that was rapidly cleared from the plasma in humans. The systemic clearance of tirilazad mesylate in human approximates hepatic plasma flow and will likely be affected by those factors that affect liver blood flow. The terminal half-life observed for the two higher doses in this study was 3.7 h. In the rat, the value was of 50 h (Cox et al., 1989). A prolonged elimination phase may impact on tirilazad mesylate accumulation on multiple dosing (Fleishaker et al., 1993a). All kinetics parameters are summarized in table 1.

Multiple dose administration, however, has been used in animal models of head injury, SAH and cerebral ischemia (Anderson et al., 1988; Silvia et al., 1987; Kanamaru et al., 1990), and multiple-dose administration is anticipated also for therapeutic intervention in humans.

The purpose of the trial of Fleishaker et al. (1993b) was to evaluate, in humans, the tolerability, pharmacological effects and pharmacokinetics of tirilazad mesylate after multiple-dose administration. The dosage of 0.5 mg/kg/day, 1 mg/kg/day, 2 mg/kg/day, 4 mg/kg/day and 6 mg/kg/day were administered in equally divided doses every 6 h as intravenous infusions over 10 min or over 30 min. The final concentration of tirilazad mesylate administered was 0.375 mg/mL. A total of 21 doses were administered, and the subjects remained in the clinic through 48 h after the last dose. As reported after single-dose administration (Fleishaker et al., 1993a), the most commonly reported medical event was local pain at the injection site. The frequency of this event increased with the increase of the dose of drug infused. The lack of differences between vehicle control and tirilazad groups suggests that the local side effect are due to the drug vehicle, rather than to the drug itself (Fleishaker et al., 1993b). The approximately even distribution of systemic medical events between active and placebo groups indicated that no systemic medical events could be attributed to tirilazad mesylate treatment. No clinically significant changes in blood pressure, pulse or cardiac rhythm were observed.

Thus, tirilazad mesylate was clinically well tolerated in these volunteers. Several subjects experienced transient increases in liver enzymes. The proportion of subjects with these transient increases was greatest in the 6 mg/kg/day-dose group (50%). Therefore, the cause of these liver function abnormalities remains unknown, but these observations suggest the need for surveillance of alanine transaminase levels in future clinical trials of tirilazad (Fleishaker et al., 1993b). Similar to results obtained previously, tirilazad mesylate exhibited no glucocorticoid, mineralocorticoid nor gonadotropic effects.

Although the analysis of concentrations of plasma tirilazad mesylate show that steady state is achieved after 5 days of dosing, longer durations of administration should not result in substantially greater accumulation of tirilazad mesylate. The pharmacokinetic parameters of tirilazad mesylate were proportional to dose, under both single-dose and multiple-dose conditions. The previous single-dose pharmacokinetic study showed linear behavior over doses of 0.25 to 2.0 mg/kg, based on evaluation of dose-corrected concentration at the end of the infusion (C_inf), systemic clearance (Cl), Cl corrected for body weight and terminal elimination rate constant (L_z) among dose groups (Fleishaker et al., 1993a). In this study (Fleishaker et al., 1993b), only data up to 6 h after the first dose were available. Thus, calculated values of L_z, plasma concentration-time curve (area under the curve), clearance and volume distribution must be viewed as suboptimal. In this case, linearity after the first dose can only really be assessed using dose-corrected C_inf. Analysis of this parameter suggested linear behavior. Because steady state was achieved by the fifth day of dosing, a more rigorous pharmacokinetics analysis could be performed using data collected after the last dose (Fleishaker et al., 1993b). The linear pharmacokinetics of tirilazad mesylate in humans contrasts with the reduced clearance exhibited at high doses in dogs.

On multiple dosing at 2 mg/kg/day or above, a terminal half-life of approximately 35 h is observed; the terminal half-life observed after a single 2-mg/kg dose is approximately 3.7 h (Fleishaker et al., 1993a). The reason for this discrepancy is that plasma tirilazad mesylate concentrations in this terminal phase fall below detectable levels after a single dose. In other words, the terminal phase is there, but it is only after accumulation of tirilazad mesylate on multiple dosing that this terminal phase may be obtained (Fleishaker et al., 1993b). Clearance of tirilazad mesylate approached liver plasma flow (Fleishaker et al., 1993a).

Tirilazad mesylate is extensively distributed in body tissues: area estimated from a single dose (V_d) was 3.33 L/kg (Fleishaker et al., 1993a). The estimate obtained using multiple-dose data ranges from 17 to 31 L/kg. The majority of tirilazad mesylate is recovered in the feces as various metabolites, and less than 12% of the dose is recovered in the urine (Stryd et al., 1992).

However, because ischemic stroke occurs primarily in an elderly (age > 65 years) population, the safety of tirilazad mesylate should be shown in this setting. Physiological changes, such as decreased cardiac output, blunted homeostatic mechanisms and diminished hepatic and renal function, occur with increasing age (Dawling and Crome, 1989). Liver size and liver blood flow also decrease with age in humans (Woodhouse and Wynne, 1988). These physiological changes in the elderly result in altered pharmacokinetic properties of several drugs. It is likely that, based on its pharmacokinetic properties in young volunteers, tirilazad mesylate will exhibit altered pharmacokinetics in the elderly (Hulst et al., 1994).

Thus, the objectives of the study of Hulst et al. (1994) were to compare the pharmacokinetics of two doses of tirilazad mesylate (1.5 and 3.0 mg/kg, as single infusions administered over 10 min) in healthy young and elderly volunteers and to assess gender-related effects on the pharmacokinetics of this drug. A secondary objective of the study was to assess the tolerability of tirilazad mesylate administration to these volunteers. Twelve healthy young volunteers and 13 healthy elderly volunteers (six men and six women in each age group) were enrolled for this study. The age range of the young volunteers was from 23 to 42 years (mean age, 33 years), and their weights ranged from 52.7 to 89.4 kg (mean weight, 67.4 kg). The age range of the elderly volunteers was from 65 to 85 years (mean age, 70 years), and their weights ranged from 49.1 to 87.5 kg (mean weight, 68.2 kg). The results of this study support the dose proportionality of tirilazad pharmacokinetics through a dose of 3.0 mg/kg.

However, as previously observed, there is a dose dependency of the observed tirilazad biological half-life (t_1/2) after single-dose administration. This has been attributed to limited assay sensitivity, which precludes the detection of the prolonged elimination phase in the concentration-time profile for tirilazad mesylate, which can be seen after higher single dose or after multiple dosing (Stryd et al., 1992). The results of this study, taken with the results of the previous single-dose study, further support this hypothesis. After a single 2.0 mg/kg dose administered to healthy young men, the mean terminal t_1/2 obtained was 3.7 h with use of an assay with a limit of quantity of 20 ng/mL (Fleishaker et al., 1993a).

However, in the study of Hulst et al. (1994), an assay with a limitation of 10 ng/mL was used, and the mean t_1/2 values of tirilazad mesylate in young male subjects were 8.1 and 14.7 h for the 1.5 mg/kg and 3.0 mg/kg doses, respectively. The longer t_1/2 values measured were attributable to the improvement in assay sensitivity, which allowed a better characterization of the terminal phase of the log concentration-time profile. The pharmacokinetics of tirilazad mesylate appear to be linear through single doses of 3.0 mg/kg, and the apparent dose dependencies of the terminal elimination rate constant (V_ss) and t_1/2 are an artifact of limited assay sensitivity (Hulst et al., 1994).

In any case, the results were consistent with decreased clearance of tirilazad in the elderly. Clearance was approximately 25% lower in the elderly volunteers than in young volunteers. Tirilazad mesylate concentrations at the end of the infusion were also higher in elderly volunteers. No significant differences were observed in t_1/2, but differences in t_1/2 may be obscured by the lack of sufficient assay sensitivity to fully characterize the terminal phase.

A study by Laizure et al. (1993) was conducted in Sprague-Dawley rats to determine the basic pharmacokinetics and distribution of tirilazad into the brain, heart, and liver. Rats were killed in groups of five at 0, 10, 20 and 40 min, and at 1.5, 2, 3, 4, 6 and 8 h after intravenous administration of 10 mg/kg of tirilazad mesylate. Tirilazad was assayed in plasma, heart, liver and brain tissue by high performance liquid chromatography. Tirilazad was rapidly eliminated from the plasma with a half-life of 2.4 h and clearance of 6.1 mL/min. The volume of distribution at steady state was large: 4.8 L/kg. The concentrations of tirilazad were highest in the liver and heart and lowest in the plasma and brain. Elimination from tissues paralleled elimination from plasma with half-lives of 1.9, 1.6 and 1.5 h in the brain, heart and liver, respectively. Tirilazad appears to be a highly extracted, hepatically eliminated drug, suggesting its clearance is dependent on liver blood flow, and alterations in plasma protein binding are unlikely to affect its clearance but may affect the fraction unbound (Laizure et al., 1993).

The decreased clearance in the elderly was primarily attributable to lower clearance in elderly women compared with young women, because clearance did not differ significantly between young and elderly men. Tirilazad has been characterized as a high extraction ratio compound, because clearance is dependent primarily on hepatic blood flow (Fleishaker et al., 1993a; Cox et al., 1989). Both liver weight and liver blood flow decrease with age in humans (Woodhouse and Wynne, 1988) and the apparent decrease in tirilazad clearance in elderly women may be a consequence of decreased liver blood flow in older women.

In addition to the effect of age on tirilazad pharmacokinetics, a gender-related effect was also observed (Hulst et al., 1994). Clearance of tirilazad was higher in women than in men, whereas plasma concentration-time curve and C_inf were lower in women. The gender-related effect was much more dramatic in the younger volunteers than in older volunteers. The mechanism for this gender-related effect is not known but, based on the pharmacokinetic properties of tirilazad, it may involve gender-related differences in hepatic blood flow. However, gender-related effects on blood flow have not been reported (Yonkers et al., 1992).

In fact, to elucidate these open questions, very recently, the biotransformation of tirilazad has been investigated in liver microsomal preparations from adult male and female Sprague-Dawley rats (Wienkers et al., 1995). Tirilazad metabolism in male rat liver microsomes resulted in the formation of two primary metabolites. Structural characterization by mass spectrometry demonstrated that one metabolite was formed by reduction of the delta-4-double bond in the steroid A-ring, whereas the other arose from the oxidative desaturation of one pyrrolidine ring.

Comparison of calculated intrinsic formation clearances (maximal velocity (V_max)/kinetics constant (K_m)) for both metabolites indicates that the female rat possessed a greater in vitro metabolic capacity for tirilazad biotransformation than did the male rat. Therefore, the clearance of tirilazad mesylate in the rats is mediated primarily by rat liver 5-alpha-reductase, and the capacity in the female rat is five-fold the capacity in the male. These observations correlate with documented differences in 5-alpha-reductase activity and predict a gender difference in tirilazad hepatic clearance in vivo (Wienkers et al., 1995).

Although metabolic pathways for tirilazad mesylate have not yet been completely elucidated in humans, a possible pathway may be metabolism at the steroid portion of the molecule. Lew et al. (1993) reported that a 46% higher ideal body weight normalized clearance of methylprednisolone in women compared with men. Thus, metabolic differences in metabolism at the steroid portion of tirilazad mesylate molecule may contribute to the gender-related effect on pharmacokinetics observed by Hulst et al. (1994). Further work is necessary to test this hypothesis.

Clearly, evaluation of the protein binding of tirilazad mesylate would provide an estimate of the effect of age and sex on unbound tirilazad mesylate concentrations, which would be more therapeutically relevant. Based on an estimate obtained in delipidized serum, tirilazad mesylate appears to be > 99% bound to plasma proteins in humans. Because of the lipophil properties of tirilazad and its adsorbability to surfaces, routine determinations of tirilazad protein binding in native serum from different patient populations are not currently possible. Therefore, effects of changes in tirilazad mesylate protein binding on clearance and on V_ss that were attributable to age and gender could not be assessed as part of this trial (Hulst et al., 1994).

The 21-aminosteroid mechanism of action has been studied using both in vitro and in vivo experimental models. In cell-free systems, the 21-aminosteroids are potent inhibitors of lipid peroxidation, having an IC₅₀ of 2 to 60 µM in rat brain homogenate (Braughler et al., 1987a). Lazaroids seem to inhibit lipid peroxidation by a mechanism similar to vitamin E. In addition, as a group, these drugs containing an NC=CN fragment, such as U-74500A, also possess the ability to interact with ferrous ions (Braughler and Pregenzer, 1989).

As previously reported, tirilazad mesylate is a nonglucocorticoid 21-aminosteroid that is a potent inhibitor of oxygen radical-induced, iron-catalyzed lipid peroxidation. It is a very lipophil compound that distributes preferentially to the lipid bilayer of cell membranes. It appears that the compound exerts its antilipid peroxidation action through cooperative mechanisms: (a) a radical scavenging action and (b) a physicochemical interaction with the cell membrane that serves to decrease membrane fluidity.

As regards the antioxidant effects in membrane systems, in vitro, the 21-aminosteroids are potent inhibitors of lipid peroxidation of rat brain homogenate, crude rat brain synaptosomes as the lipid source (Braughler et al., 1988a) and also rat brain synaptic plasma membranes (Braughler et al., 1987a). However, when such compounds are added in organic solution to physiological buffer, they microprecipitate. Emulsion delivery is probably a delivery technique for compounds of this class. A preliminary report suggests that U-74500A differs from U-74006F in that the former appears to interact with iron in some manner (Braughler et al., 1987a). Indeed, the concentration that inhibits 50% (IC₅₀) of U-74500A to inhibit iron-dependent lipid peroxidation in rat brain homogenates is lower in the presence of 10 µM Fe²⁺ than in presence of 200 µM Fe²⁺ (Braughler et al., 1988a). Furthermore, U-74500A has been demonstrated to display spectral changes in the ultraviolet range that are dependent upon the concentration of Fe²⁺. No iron-dependent spectral changes have been observed for U-74006F in aqueous solution. This does not rule out the possibility that U-74006F might bind iron in some manner within the membrane environment (Braughler and Pregenzer, 1989). U-74500A and tirilazad act to slow the oxidation of vitamin E during linoleic acid peroxidation and potentiate vitamin E's antioxidant efficacy (Braughler and Pregenzer, 1989). U-74500A is actually a better antioxidant than is tirilazad, especially in iron-driven peroxidation systems, possessing a lower oxidation potential than tirilazad, and it has the ability to interact with ferrous iron and to lessen its oxidation, in contrast with tirilazad, which does not (Ryan and Petry, 1993).

The effects of two 21-aminosteroids (U-74500A and U-74006F) on the oxidation and reduction of iron were investigated (Ryan and Petry, 1993). U-74500A completely prevented adenosine diphosphate (ADP):Fe(II) autoxidation, whereas U-74006F had only a slight inhibitory effect. In particular, the inhibition of Fe(II) oxidation by U-74500A was concentration-dependent, with 100% inhibition occurring at concentrations equal to or greater than 25 µM in systems containing 50 µM Fe(II). When the Fe(II)-specific chelator Ferrozine (Sigma Chemical Co. St. Louis, MO), was added to incubations containing U-74500A and ADP:Fe(II), formation of the Ferrozine:Fe(II) chromophore was slow, suggesting that U-74500A chelates Fe(II) with substantial affinity. Twenty minutes were required for complete formation of the Ferrozine:Fe(II) chromophore in the presence of U-74500A, whereas complexation in its absence was instantaneous. This phenomenon was not observed with U-74006F or ascorbate. In a system containing 25 µM ADP:Fe(II), both U-74500A (25 µM) and U-74006F (25 µM) reduce iron at rates approximately 2 and 0.1 µM/min, respectively (Ryan and Petry, 1993). U-74500A fluorescence was quenched in a concentration-dependent manner upon the addition of Fe(III), further demonstrating interactions between this compound and iron.

The substructures of U-74500A consist of a steroid (U-76911) and a complex amine (U-82902E). When these compounds were assayed individually, it was found that U-82902E exhibited activities similar to those of U-74500A, whereas the free steroid had no effect (Ryan and Petry, 1993). Studies using cyclic voltametry revealed that U-74500A had relatively low oxidation potential (E = 228 mV), whereas U-74006F was much less susceptible to oxidation (E = 810 mV) (Ryan and Petry, 1993). Taken together, these data suggest that subtle effects on iron redox chemistry, which would in turn inhibit or eliminate the initiation of undesired oxidative reactions, may contribute to the potent antioxidant activities of U-74500A and U-74006F.

Tirilazad also can interact with hydroxyl radicals generated during in vitro Fenton reaction (5) (Althaus et al., 1991). In vivo studies, using the salicylate (SAL) trapping method for measurement of hydroxyl radical, have demonstrated that tirilazad administration decreases brain hydroxyl radical levels in a model of concussive head injury in mice (Hall et al., 1992, 1993a) and global cerebral ischemia/reperfusion injury in gerbils (Andrus et al., 1991). Tirilazad has also been reported to lessen the increase in hydroxyl radical concentration in rat brain produced by infusion of glutamate (Boisvert and Schreiber, 1992).

As an antioxidant in whole cells, tirilazad is effective in an in vitro model for predicting a compound's ability to prevent cell damage during periods of energy failure. Iodoacetic acid was administered (50 mM) to the cultured human astroglial cells for 4 h. This agent shuts down glycolysis and leads to subsequent irreversible breakdown of cellular membranes and, ultimately, to cell death. During the first hours, iodoacetic acid rapidly depleted cellular level of adenosine triphosphate (ATP) and decreased active uptake of tritiated aminoisobutyric acid. Subsequent irreversible cellular injuries were characterized by the release of large amounts of free arachidonic acid into extracellular medium, massive calcium influx and leakage of cytoplasmic contents. The appearance of 15-hydroxy eicosatetraenoic acid in membrane phospholipids and loss of cellular thiol groups indicated the cell constituents were being assaulted by oxidative species. These manifestations of iodoacetic acid-induced cell damage were inhibited by tirilazad, which also decreased the release of arachidonic acid (Hall et al., 1994).

The 21-aminosteroids tirilazad and U-74500A have potent stabilizing effects on cell membranes. The compounds have high affinity for the lipid bilayer because of their lipophilia and are incorporated into the lipid bilayer, where they occupy strictly defined positions and orientations (Hinzmann et al., 1992). Tirilazad is a very lipophilic compound that localizes in and protects cell membranes from peroxidative damage. Not surprisingly, this compound has been shown also to exert physicochemical effects on endothelial cell membranes. It has high affinity for vascular endothelium and protects the blood-brain barrier (BBB) against either a trauma-induced or SAH-induced permeability increase. Tirilazad appears to poorly penetrate the BBB in rats after intracarotid injection, because the penetration of tirilazad into brain parenchyma is enhanced after injury, apparently by virtue of the trauma-induced disruption of the BBB (Hall et al., 1992). The endothelial localization and protection probably is not confined to the CNS but also occurs at the hepatic level. Tirilazad does not exert any glucocorticoid receptor-mediated actions and, actually, the only demonstrated cerebroprotective mechanism of the 21-aminosteroids concerns their ability to block oxygen radical-induced lipid-peroxidation.

U-74500A is actually a more potent inhibitor of iron-catalyzed lipid peroxidation than is U-74006F, but it has not been chosen for development due to pharmaceutical instability and rapid elimination in vivo (Hall, 1992a). In addition, brief mention is made of more recently discovered antioxidants, the 2-metylaminochromans, in which the steroid moiety of U-74006F has been replaced by the more potent antioxidant chromanol structure of vitamin E (i.e., alpha-TC): U-78517F (Hall, 1992a).

In regards to effects on cerebral metabolism, tirilazad (1 mg/kg i.v. at 30 min postinjury plus 0.5 mg/kg 2 h later, in cats severely head-injured) improved the metabolic profile within the injured hemisphere measured at 4 h (Dimlich et al., 1990), particularly reducing posttraumatic lactic acid accumulation in both the cerebral cortex and the subcortical white matter.

To conclude, Hall and Braughler (1993) reviewed the current state of knowledge regarding the occurrence and possible role of oxygen radical generation and lipid peroxidation in experimental models of acute CNS injury. Although much work remains, four criteria that are logically required to establish the pathophysiological importance of oxygen radical reactions have been met, at least in part. First, oxygen radical generation and lipid peroxidation appear to be early biochemical events subsequent to CNS trauma. Second, a growing body of direct and circumstantial evidence suggests that oxygen radical formation and lipid peroxidation are linked to pathophysiological processes such as hypoperfusion, edema, axonal conduction failure, failure of energy metabolism and anterograde (Wallerian) degeneration. Third, there is a striking similarity between the pathology of blunt mechanical injury to CNS tissue and that produced by chemical induction of peroxidative injury. Fourth, compounds that inhibit lipid peroxidation or scavenge oxygen radicals can block posttraumatic pathophysiology and promote functional recovery and survival in experimental studies (Hall and Braughler, 1993).

Nevertheless, the significance of oxygen radicals and lipid peroxidation ultimately depends on whether it can be demonstrated that early application of effective anti-free radical or antiperoxidative agents can promote survival and neurological recovery after CNS injury and stroke in humans. The results of the NASCIS II clinical trial, which have shown that an antioxidant dosing regimen with methylprednisolone begun within 8 h after spinal cord injury can significantly enhance chronic neurological recovery, strongly support the significance of lipid peroxidation as a posttraumatic degenerative mechanism. However, phase III trials with the more selective and effective antioxidant U-74006F (tirilazad mesylate) will give a more clear-cut answer as to the therapeutic importance of inhibition of posttraumatic free radical reactions in the injured CNS (Hall and Braughler, 1993).

Very little is known about other biological effects of lazaroids, except that they improve endothelial cell viability at 4°C, with U-74500 being the most effective (Killinger et al., 1992). Furthermore, they inhibit growth of cultured Balb/c 3T3 clone A31 fibroblast (Singh and Bonin, 1991). U-75412E caused inhibition of cellular growth of human epithelial cell line (Wish), that was both concentration-dependent and time-dependent (Mattana et al., 1994). In particular, drug-treated cells showed a remarkable number of vacuoles and mitochondria, which were smaller, rounded and showed a widening of the intercrystal spaces in treated cells.

The flow cytometry analysis confirmed the antiproliferative effect, a large number of cells were blocked in the G2/M phase, without apparently degenerative phenomena (Mattana et al., 1994). Different phases of nuclear fragmentation (apoptosis) were also evident when the cells were incubated with 6 µM U-75412E for 48 h. Reduced deoxyribonucleic acid (DNA) stainability observed in apoptotic cells was a consequence of a partial loss of DNA due to activation of endogenous endonuclease (Darzynkiewicz et al., 1992; Hotz et al., 1992). Cell growth inhibition by lazaroids was probably due to a cytotoxic action of the compounds in these experimental conditions. The release of the intracellular enzyme lactate dehydrogenase, used as an indicator of cytotoxicity, confirmed these data (Mattana et al., 1994).

Furthermore, scanning electron microscopy experiments showed that treated cells exhibited damage to the cell surface that could be ascribed to the high lipophilia of the molecule. In addition, U-75412E caused ultrastructural damage, as shown by transmission electron microscopy, indicating that tubulin could be quite a specific target for the lazaroid toxicity. In conclusion, these data suggest that lazaroid U-75412E has a cytotoxic effect at concentrations above 1 µM in Wish cells (Mattana et al., 1994).

	II. Central Nervous System Trauma

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Lipid hydrolysis with subsequent eicosanoid production is an early pathochemical event in the injured spinal cord (Anderson et al., 1985; Demediuk et al., 1985; Hsu et al., 1985; Jonsson and Daniell, 1976). However, lipid hydrolysis with the release of arachidonate may be closely tied to peroxidation-induced changes in membrane calcium permeability. In addition, a synergistic interaction between calcium and lipid peroxidation during cell damage has been demonstrated (Braughler et al., 1985; Malis and Bonventre, 1986).

Many factors are involved in the pathogenesis of traumatic brain edema. The initial mechanical disruption of capillary endothelial cells (Long, 1982) allows excess movement of fluid into the brain, but vascular thrombosis quickly prevents further edema formation from this source (Tornheim, 1985). A more important cause of traumatic brain edema appears to be the release or activation of chemical mediators, such as bradykinin, serotonin, histamine, arachidonic acid, leukotrienes, excitatory amino acids and free radicals, and failure of the BBB (Black and Hoff, 1985; Chan et al., 1984; Wahl et al., 1988). Although it is accepted that chemical mediators play a role in brain edema development, the importance of each mediator has yet to be determined. In CNS trauma, tissue hemorrhage initiates free radical formation, and iron compounds catalyze the generation of the highly reactive hydroxyl radical and stimulate membrane lipid peroxidation (Halliwell and Gutteridge, 1985).

Lipid peroxides and oxygen reactive species are thought to be involved in major physiological or pathological events, such as inflammation, radiation damage, mutagenesis, cellular aging and reperfusion damage (Halliwell, 1987). Evidence of the potential role of oxidants in the pathogenesis of many diseases suggests that antioxidants may be used in the therapy or prevention of these diseases.

The 21-aminosteroids, specifically designed to localize within cell membranes and to inhibit lipid peroxidation reactions (Braughler et al., 1987a,b, 1988a,b; Braughler and Pregenzer, 1989), have shown activity in in vivo models of experimental CNS trauma (Anderson et al., 1988; Hall et al., 1988b; Braughler et al., 1989). In contrast to metylprednisolone, U-74006F has no steroidal side effects or peripheral vasodilator activity, is more potent (Hall et al., 1988a) and has no deleterious effect on blood pressure. These characteristics make this compound a candidate for possible treatment of CNS injury (Sanada et al., 1993).

Regarding the effects on BBB permeability, free radicals are known to increase BBB permeability. It is possible that the effect of tirilazad to protect the BBB is due to either a reduced formation of hydroxyl radicals or perhaps a protection of the microvascular endothelium from hydroxyl radical-induced lipid peroxidation.

Preventing and reducing secondary brain injury have been foci of recent research on CNS trauma (Sanada et al., 1993). Although the precise mechanism of delayed injury after mechanical trauma is unclear, several metabolic derangements have been implicated. These include influx of calcium ions (Hubschmann and Nathanson, 1985; Siesjo and Wieloch, 1985), tissue lactic acidosis, free radical generation and tissue peroxidation (Kontos and Wei, 1986), production of prostaglandins and leukotrienes (Kiwak et al., 1985) and membrane depolarization by release of excitatory amino acid neurotransmitters (Choi and Tecoma, 1988). A variety of antioxidants, or free radical scavengers, have been proposed to treat CNS injury (Faden, 1985). Among the agents tested are vitamins C and E, selenium, coenzyme Q₁₀, megadose corticosteroids and high-dose opiate antagonists. None of these agents, however, has led to major improvement in neurological function after CNS trauma (Sanada et al., 1993).

Mechanical trauma of the spinal cord causes destruction of gray and white matter with consequent loss of function (Anderson et al., 1988). Nerve cells and axons can be damaged directly by the physical deformation of the spinal cord and/or by a cascade of pathochemical events that are initiated by the original mechanical trauma. It is this biochemical injury that would be susceptible to pharmacological treatment if the mechanisms were understood (Anderson et al., 1988). Lipid peroxidation (Anderson et al., 1985; Demediuk et al., 1985; Hall and Braughler, 1982; Malis and Bonventre, 1986), phospholipid hydrolysis with production of eicosanoids (Anderson et al., 1985; Demediuk et al., 1985; Hsu et al., 1985) and depletion of energy stores with increased lactic acid formation (Anderson et al., 1976, 1985; Braughler and Hall, 1983) are the earliest biochemical events detected thus far in injured spinal cord tissue. Disruption of cell membranes by peroxidative and hydrolytic process may be intimately involved in the initiation and/or propagation of the posttraumatic autodestruction of spinal cord tissue.

Thus, agents that protect cell membranes by quenching these peroxidative reactions and/or by limiting lipolysis should be effective in improving neurological recovery (Anderson et al., 1988). However, it appears that a major portion of posttraumatic neuronal necrosis after spinal cord (or brain) injury does not result from differences in primary injury, but rather occurs as a secondary pathophysiological process. The injury is due to a series of molecular events that lead to gradual derangements, e.g., vascular and neuronal degeneration, thus destroying the anatomic substrate necessary for the neurological recovery. Thus, the functional recovery can be facilitated by appropriate therapies targeted to interrupt the molecular processes involved in the secondary degeneration phenomena.

High doses of methylprednisolone sodium succinate (MP) promote functional recovery in animals with spinal cord injury (Braughler et al., 1987b). A primary action of MP in the injured or ischemic CNS is believed to be its ability to inhibit lipid peroxidation and to preserve the structural and functional integrity of biological membranes (Anderson and Means, 1985; Anderson et al., 1985; Braughler, 1985). Treatment of human CNS injuries with MP has been complicated by its biphasic dose-response characteristics (Braughler and Hall, 1982, 1983; Hall, 1985; Hall et al., 1984) and its glucocorticoid receptor-mediated activity (Braughler and Hall, 1985; Hall and Braughler, 1982). The realization that the membrane-protective capabilities of MP were separate from its hormonal activity stimulated an intensive drug-development effort to identify and prepare unique compounds specifically targeted for the treatment of human CNS trauma and ischemia. U-74006F has proved to be a substantially more potent and effective treatment than MP in several different models of acute CNS trauma and ischemia (Hall, 1988; Hall et al., 1988a,b). The slow CNS tissue uptake of vitamin E requires chronic dosing, making it an impractical agent for treatment of acute neuronal injury.

There are many data about the effects of lazaroids in experimental spinal cord and head injury, evaluating a variety of functional or biochemical parameters. Spinal cord white matter blood flow was measured by hydrogen clearance in the injured segment before and at various times up to 4 h after injury. After 4 h postinjury, spinal cord white matter blood flow was decreased by 63.5%, whereas the spinal cord white matter blood flow measured 4 h postinjury in cats treated with a single 10-mg/kg dose of U-74006F was of about normal value. The mechanism of action of U-74006F in antagonizing posttraumatic development is believed to involve the ability of the compound to inhibit iron-dependent lipid peroxidation in CNS (Hall, 1988).

Initial studies of the efficacy of U-74006F in experimental acute head injury have been carried out to determine the ability of the compound to improve early neurological recovery and survival of head-injured mice (Hall et al., 1988b). Unanesthetized male CF-1 mice were subjected to a 900-gcm head injury produced by a 50-g weight that was dropped 18 cm (Hall, 1985). Administration of a single dose of i.v. U-74006F significantly improved the 1 h postinjury neurological status (grip test score) over a broad range of dosages (0.003 to 30 mg/kg). A 1 mg/kg i.v. dose given within 5 min and again at 1.5 h after a severe injury, in addition to improving early recovery, increased the 1-week survival to 78.6% compared with 27.3% in vehicle-treated mice. The compound was also effective in enhancing early recovery after a more moderate injury.

The study of Anderson et al. (1988) demonstrates the remarkable effectiveness of a nonglucocorticoid 21-aminosteroid, U-74006F, administered through the venous cannula, in enhancing neurological recovery in female adult mongrel cats traumatized by compression of the spinal cord with a 180-gm weight for 5 min. Beginning at 30 min after injury, cats were given an intravenous bolus of either vehicle or U-74006F. Two hours later, the treated cats received a second intravenous bolus of one-half the original loading dose; at 6 h postinjury, the cats received a third intravenous bolus, again one-half of the original loading dose. Immediately after this last injection, a continuous intravenous infusion was started and continued for 42 h. Thus, the animals were treated for the first 48 h postinjury. The cats were divided into nine groups: one vehicle-treated group and eight U-74006F-treated groups. The dose levels tested (the initial loading dose and total dose) were: 0.01 mg/kg (0.048 mg/kg/48 h); 0.03 mg/kg (0.16 mg/kg/48 h); 0.1 mg/kg (0.48 mg/kg/48 h); 1.0 mg/kg (4.8 mg/kg/48 h); 3.0 mg/kg (16.0 mg/kg/48 h); 10 mg/kg (48 mg/kg/48 h) and 30 mg/kg (160 mg/kg/48 h). All cats were allowed to recover for 4 weeks, and their functional recovery was evaluated on a weekly basis. The neurological evaluation procedure used is based on observing and rating the mobility of a freely moving animal in various controlled situations. Over the initial 2 weeks following injury, there was no statistically detectable recovery in any of the U-74006F-treated groups as compared with vehicle-treated controls. However, at 2 weeks, the mean recovery scores for all drug-treated groups, with the exception of the lowest dose, tended to be higher than the vehicle-treated groups, with the exception of the lowest dose. By 3 weeks postinjury, all treatment groups receiving total U-74006F doses of 1.6 m/kg/48 h and higher (with exception of the group receiving 16.0 mg/kg/48 h) showed statistically better recovery than the vehicle-treated cats. This pattern of recovery was sustained through the fourth and final week of evaluation.

The molecular mechanism(s) by which U-74006F promoted neurological recovery in this model of spinal cord injury is not known (Anderson et al., 1988). U-74006F completely lacks any glucocorticoid, mineralocorticoid or other hormonal activity (Braughler et al., 1988b; Hayes and Murad, 1980). Hence, it is unlikely that any CNS protective functions of U-74006F are mediated through glucocorticoid receptors. Physiologically, U-74006F has been shown to prevent the development of white matter ischemia following a severe contusion of the spinal cord (Hall, 1988). Moreover, U-74006F has the ability to partially restore posttraumatic spinal cord blood flow, even after it has declined significantly (Hall, 1988).

Some data are consistent with the dose-response findings for U-74006F in less complex models of CNS trauma (Hall et al., 1984; McCall et al., 1987). The broad range of effective doses for U-74006F, its remarkable potency, its lack of glucocorticoid receptor-mediated activity and the lack of any adverse side effects should make the clinical utility for this 21-aminosteroid significantly greater than that of MP for the treatment of human CNS trauma. The beneficial effect of antioxidant doses of methylprednisolone, administered within 8 h after spinal cord injury, can improve 3-month, 6-month and 12-month neurological recovery in humans (Bracken et al., 1990, 1992). This observation supports the view that posttraumatic lipid peroxidation is a critical degenerative mechanism that can be effectively interrupted with an antioxidant agent.

The study of Dimlich et al. (1990) was designed to evaluate further the effect of U-74006F on the acute pathophysiology of experimental head injury, involving severe unilateral cerebral contusion in cats. The parameters tested included magnitude and territory of vasogenic edema, brain swelling and cerebral metabolic function. Conditioned mongrel female cats were anesthetized with ketamine hydrochloride and, at 30 min after head or sham injury, each cat was intravenously injected with either 1 mg/kg of U-74006F or a comparable volume of its vehicle (0.02 M citric acid; 0.003 M sodium citrate; 0.08 M sodium chloride). A second treatment (0.5 mg/kg) was administered 2.5 h after injury. Four hours after injury, a styrofoam cup was fixed to the calvaria, and liquid nitrogen was poured over the skull for 20 min for in situ fixation of brain tissue. The frozen heads were coronally sliced at 5-mm intervals in a 20°C cold room. Brain samples (5 to 10 mg) were weighed and extracted (Wagner et al., 1985). Lactate, ATP and phosphocreatine were assayed in perchloric acid extracts using enzymatic fluorometric techniques (Lowry and Passonneau, 1972). Glucose and glycogen were determined in non-perchlorate-treated samples of homogenate, using the fluorometric procedure of Passonneau and Lauderdale (1974). Metabolites and edema (specific gravity) were measured bilaterally in the cerebral cortex and white matter. The magnitude of edema and metabolites in tissue with vasogenic edema was similar in vehicle-treated and drug-treated cats. By contrast, the cortex and nonedematous white matter neighboring contusion in drug-treated cats had lactate, glucose and glycogen levels that suggested an improved metabolic state over vehicle treatment. Most metabolites were not affected by trauma or treatment in the uncontused hemisphere. These results suggest that postinjury treatment with the nonglucocorticoid steroid U-74006F may benefit the metabolism of nonedematous tissue adjacent to contusion (Dimlich et al., 1990).

Regarding the effect on cerebral metabolism, tirilazad (1 mg/kg i.v. at 30 min postinjury, plus 0.5 mg/kg 2 h later, in cats severely head-injured) improved metabolic profile within the injured hemisphere measured at 4 h (Dimlich et al., 1990). In particular, the drug reduced posttraumatic lactic acid accumulation in both the cerebral cortex and the subcortical white matter.

The study of McIntosh et al. (1992), evaluated the effect of the nonglucocorticoid 21-aminosteroid U-74006F, an inhibitor of iron-dependent lipid peroxidation, on the development of regional cerebral edema after lateral fluid-percussion brain injury. Male Sprague-Dawley rats were anesthetized and subjected to fluid-percussion brain injury of moderate severity centered over the left parietal cortex (2.5 to 2.6 atms). Fifteen minutes after brain injury, animals randomly received an i.v. bolus of either U-74006F (3 mg/kg) followed by a second bolus (3 mg/kg) at 3 h or buffered sodium citrate vehicle. An additional group of 12 surgically prepared but uninjured animals served as preinjury controls. At 48 h after injury, animals were sacrificed, and brain tissue was assayed for water content and regional cation concentrations. With the use of specific gravimetric techniques, no significant differences were observed in posttraumatic cerebral edema between drug-treated and control-treated animals. However, using wet weight/dry weight methodology, McIntosh et al. (1992) found that administration of U-74006F significantly reduced water content in the right hippocampus (controlateral to the site of injury) compared with saline-treated animals. U-74006F also significantly prevented the postinjury increase in sodium concentrations in the ipsilateral hippocampus and thalamus. Regional concentrations of potassium were unaltered after drug treatment. Administration of U-74006F significantly reduced postinjury mortality, from 28% in control animals to zero in treated animals. These results suggest that lipid peroxidation may be involved in the pathophysiological sequelae of brain injury and that 21-aminosteroids may be beneficial in the treatment of brain injury (McIntosh et al., 1992).

The purpose of the study of Sanada et al. (1993) was to further evaluate the effect of U-74006F on neurological outcome and cerebral edema after head injury in rats. The rats were anesthetized with chloral hydrate (0.35 g/kg, i.p.). Through a 4.0-mm diameter craniectomy in the right temporal region just above the zygoma, a flanged polyethylene tube filled with isotonic saline was placed over the dura, securely fixed to the skull and connected to the fluid percussion device (Sullivan et al., 1976). The rats were subjected to an impact pressure of 4.5 atm for 15 msec and immediately transferred into a chamber supplied continuously with 7% oxygen. The PaO₂ was maintained at a hypoxic level for 45 min and then normoxia was restored. The animals were treated with U-74006F at 1, 3, 10 or 30 mg/kg intravenously at 3 min and at 3 h after impact injury. Neurological function was evaluated 24 h after injury, and three categories were scored: motor function, rotaroad walking and activity. The measurement of water content was determined by microgravimetry in the coronal slices obtained from the impact site, from the frontal, temporal and parietal cortex, and from caudate putamen and thalamus from both ipsilateral and controlateral hemispheres. In this study, only the 10-mg/kg dose (20 mg/kg total) of U-74006F significantly improved motor function 24 h after fluid percussion-hypoxic brain injury, but no improvement was evident when the dose was increased to 30 mg/kg (60 mg/kg total), which may indicate a relatively narrow effective dose range for rats.

Whether U-74006F has any adverse effects at doses higher than 30 mg/kg in rats has yet to be demonstrated. Steroids show biphasic actions on cell membranes, stabilizing them at relatively low concentrations and lysing them at higher concentrations (Lewis et al., 1970).

U-74006F had a differential effect on the three categories of the neurological evaluation. A statistically significant improvement was detected in motor score, but not in rotaroad walking nor activity, and the brain water content was not reduced by U-74006F at any dose (Sanada et al., 1993). This compound has reduced arachidonic acid-induced vasogenic edema and ischemic edema in rats subjected to middle cerebral artery (MCA) occlusions (Hall et al., 1988a; Wahl et al., 1988). Despite its ability to scavenge or inhibit the formation of free radicals, U-74006F did not reduce brain water content in this study.

Lipid peroxidation, however, is only one of the events in the complex process that results in traumatic cerebral edema. Oxygen free radicals have been implicated as a causal factor in neuronal cell loss following both cerebral ischemia and head injury. In this research, Althaus et al. (1993) studied simultaneously the effect of lazaroid U-74006F both in incomplete ischemia and in head injury. The conversion of SAL to dihydroxybenzoic acid (DHBA) in vivo was used to study the formation of hydroxyl radical (OH·) following CNS injury. Bilateral carotid occlusion (BCO) in gerbils and concussive head trauma in mice were selected as models of brain injury. The lipid peroxidation inhibitor, tirilazad mesylate (U-74006F), was tested for its ability to attenuate (OH·) formation in these models. In addition, U-74006F was studied as a scavenger of (OH·) in an in vitro assay based on the Fenton reaction. For in vivo experimentation, (OH·) formation was expressed as the ratio of DHBA to SAL (DHBA/SAL) measured in brain. In the BCO model, (OH·) formation increased in whole brain with 10 min of occlusion followed by 1 min of reperfusion. DHBA/SAL was also found to increase in the mouse head injury model at 1 h postinjury. In both models, U-74006F (1 or 10 mg/kg) blocked the increase in DHBA/SAL following injury (Althaus et al., 1993). In vitro, reaction of U-74006F with (OH·) gave a product with a molecular weight that was 16-fold greater than U-74006F, indicative of (OH·) scavenging. The authors speculate that U-74006F may function by blocking oxyradical-dependent cell damage, thereby maintaining free iron (which catalyzes hydroxyl radical formation) concentrations at normal levels (Althaus et al., 1993). It is believed that U-74006F acts at the cell membrane level during reperfusion by inhibiting lipid peroxidation and significantly reduces the incidence of postischemic spinal cord injury following temporary aortic occlusion (Fowl et al., 1990), as well as locomotor function in cats (Anderson et al., 1991).

The aim of a recent study (Schneider et al., 1994) was to determine whether brain edema induced by a cryogenic injury can be influenced by the 21-aminosteroid U-74389F. A cortical freezing lesion was applied to the right parietal region of Sprague-Dawley rats under ketamine-xylazine anesthesia. Systemic blood pressure was monitored in the peritraumatic period. Four different doses (A to D) of U-74389F were studied for their effect on posttraumatic brain swelling and edema. Respective control groups received only the solvent, citric acid buffer. The doses were as follows: (A) 3 mg/kg b.w., i.p. (total dose) 30 min before, 1 and 12 h posttrauma; (B) 9 mg/kg b.w., i.v. 30 min before, 1 and 12 h posttrauma; (C) 25 mg/kg b.w., i.v. 30 min before, 1, 6 and 12 h posttrauma; and (D) 50 mg/kg b.w., i.v. 15 min before, 15 and 30 min as well as 1, 2, 6 and 12 h posttrauma. Twenty-four h after trauma, brains were removed, and hemispheric swelling and water content were determined from the difference between wet weight and dry weight. Application of the 21-aminosteroid U-74389F (Schneider et al., 1994) moderately reduced posttraumatic brain swelling in all treatment groups: (A) 5%, (B) 9%, (C) 12% and (D) 14%. In parallel with this, the increase in water content of traumatized hemisphere was marginally lowered by U-74389F in all groups: in (C) e.g., from 1.9 ± 0.1% to 1.7 ± 0.1%, P = 0.07. These two findings taken together indicate that the 21-aminosteroid U-74389F moderately reduces posttraumatic swelling and edema (Schneider et al., 1994).

The neurochemical sequelae of traumatic brain injury and their therapeutic implications have been reviewed recently and extensively by McIntosh (1994). A general comment at the end of this paragraph is that in head injury, major foci regarding the supported therapeutic efficiency of lazaroids have been the membrane damage resulting from the free radical cascade and the disruption in cellular ionic homeostasis, with less attention to other factors related to the pathological mechanisms of this disease: for example, the excitotoxic effects of pathological release of amino acid neurotransmitters.

At present, the final analyses of phase III trials of the antagonism of the initiation and propagation of the free radical cascade by tirilazad and polyethylene glycol-bound superoxide dismutase are nearing completion, as reported by Marshall and Marshall (1995).

	III. Subarachnoid Hemorrhage

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Sustained cerebral arterial narrowing, occurring days after SAH, is commonly referred to as cerebral vasospasm (Findlay et al., 1991) and is defined as a reduction in vessel caliber of 10% or greater as compared with the baseline value (Kanamaru et al., 1991). This effect is widely accepted as an important complicati