Association of caffeine and related analytes with resistance to Parkinson disease among LRRK2 mutation carriers: A metabolomic study.

OBJECTIVE: To identify markers of resistance to developing Parkinson disease (PD) among LRRK2 mutation carriers (LRRK2+), we carried out metabolomic profiling in individuals with PD and unaffected controls (UC), with and without the LRRK2 mutation. METHODS: Plasma from 368 patients with PD and UC in the LRRK2 Cohort Consortium (LCC), comprising 118 LRRK2+/PD+, 115 LRRK2+/UC, 70 LRRK2-/PD+, and 65 LRRK2-/UC, and CSF available from 68 of them, were analyzed by liquid chromatography with mass spectrometry. For 282 analytes quantified in plasma and CSF, we assessed differences among the 4 groups and interactions between LRRK2 and PD status, using analysis of covariance models adjusted by age, study site cohort, and sex, with p value corrections for multiple comparisons. RESULTS: Plasma caffeine concentration was lower in patients with PD vs UC (p < 0.001), more so among LRRK2+ carriers (by 76%) than among LRRK2- participants (by 31%), with significant interaction between LRRK2 and PD status (p = 0.005). Similar results were found for caffeine metabolites (paraxanthine, theophylline, 1-methylxanthine) and a nonxanthine marker of coffee consumption (trigonelline) in plasma, and in the subset of corresponding CSF samples. Dietary caffeine was also lower in LRRK2+/PD+ compared to LRRK2+/UC with significant interaction effect with the LRRK2+ mutation (p < 0.001). CONCLUSIONS: Metabolomic analyses of the LCC samples identified caffeine, its demethylation metabolites, and trigonelline as prominent markers of resistance to PD linked to pathogenic LRRK2 mutations, more so than to idiopathic PD. Because these analytes are known both as correlates of coffee consumption and as neuroprotectants in animal PD models, the findings may reflect their avoidance by those predisposed to develop PD or their protective effects among LRRK2 mutation carriers.

Aminophylline, administered at usual doses for rodents in pharmacological studies, induces hippocampal neuronal cell injury under low tidal volume hypoxic conditions in guinea-pigs.

OBJECTIVES: To establish whether aminophylline, administered at usual doses for rodents in pharmacological studies, induces brain injury in systemic hypoxaemia in guinea-pigs. METHODS: A hypoxaemia (partial oxygen tension of arterial blood (PaO2) = 40-60 mmHg) model was developed by low tidal volume mechanical ventilation in guinea-pigs. KEY FINDINGS: Under hypoxic conditions, aminophylline significantly increased the concentration of brain-specific creatine kinase in the serum in a dose- and time-dependent manner. A reduced number of hippocampal neuronal cells in the CA1 region, an increase in the concentration of neuron-specific enolase (NSE) in cerebrospinal fluid (CSF), an increase in lipid hydroperoxides and a decrease in the ratio of glutathione to glutathione disulfide in the brain tissues were also observed. These effects were not observed when aminophylline at the same doses was administered under normoxic conditions (PaO2 = 80-100 mmHg). There was no difference in either serum or CSF concentrations of theophylline between normoxic and hypoxic conditions. Another methylxanthine, caffeine, did not increase the concentration of NSE in CSF. CONCLUSIONS: Aminophylline potentially induces brain damage under hypoxic conditions. We suggest that aminophylline treatment has adverse effects in patients with hypoxaemia subsequent to respiratory disorders such as asthma.

Increases in cerebrospinal fluid caffeine concentration are associated with favorable outcome after severe traumatic brain injury in humans.

Caffeine, the most widely consumed psychoactive drug and a weak adenosine receptor antagonist, can be neuroprotective or neurotoxic depending on the experimental model or neurologic disorder. However, its contribution to pathophysiology and outcome in traumatic brain injury (TBI) in humans is undefined. We assessed serial cerebrospinal fluid (CSF) concentrations of caffeine and its metabolites (theobromine, paraxanthine, and theophylline) by high-pressure liquid chromatography/ultraviolet in 97 ventricular CSF samples from an established bank, from 30 adults with severe TBI. We prospectively selected a threshold caffeine level of > or = 1 micromol/L (194 ng/mL) as clinically significant. Demographics, Glasgow Coma Scale (GCS) score, admission blood alcohol level, and 6-month dichotomized Glasgow Outcome Scale (GOS) score were assessed. Mean time from injury to initial CSF sampling was 10.77+/-3.13 h. On initial sampling, caffeine was detected in 24 of 30 patients, and the threshold was achieved in 9 patients. Favorable GOS was seen more often in patients with CSF caffeine concentration > or = versus < the threshold (55.6 versus 11.8%, P=0.028). Gender, age, admission CGS score, admission blood alcohol level, and admission systolic arterial blood pressure did not differ between patients with CSF caffeine concentration > or = versus < the threshold. Increases in CSF concentrations of the caffeine metabolites theobromine and paraxanthine were also associated with favorable outcome (P=0.018 and 0.056, respectively). Caffeine and its metabolites are commonly detected in CSF in patients with severe TBI and in an exploratory assessment are associated with favorable outcome. We speculate that caffeine may be neuroprotective by long-term upregulation of adenosine A1 receptors or acute inhibition of A2a receptors.

Pharmacokinetics of theophylline in Guinea pig tears.

It is well recognized that the theophylline (TP) concentration in human tears correlates well with the free TP concentration in human plasma. However this correlation was found only in a very narrow range of concentrations of TP, and pharmacokinetic analysis of TP in tears has not been carried out for a wide range of concentrations of TP. The aims of this investigation were to develop a simple kinetic model for TP in guinea pig plasma (total [Cf+b] and free [Cf]), cerebrospinal fluid (CSF) [C](CSF) and tears [C](T), and to examine whether [Cf], [Cf+b] and [C](CSF) can be predicted from [C](T) using the resulting kinetic parameters. [Cf+b], [Cf], [C](CSF) and [C](T) were determined by GC/EI-SIM following bolus i.v. injection of TP in doses of 10, 50 and 100 mg/kg into guinea pigs. The wide range of concentrations of [Cf+b] could be quantitatively described by a two-compartment model with non-linear elimination kinetics and individual volume distribution of TP at each dose. [C](T) and [C](CSF) were analyzed using passive diffusion models with and without the pH-partition theory, respectively. The value of [Cf] could be predicted from the value of [C](T). Thus, the measurement of [C](T) which can be collected non-invasively would be a useful method for the therapeutic drug monitoring of TP.

Evaluation of cerebrospinal fluid concentration and plasma free concentration as a surrogate measurement for brain free concentration.

This study was designed to evaluate the use of cerebrospinal fluid (CSF) drug concentration and plasma unbound concentration (C(u,plasma)) to predict brain unbound concentration (C(u,brain)). The concentration-time profiles in CSF, plasma, and brain of seven model compounds were determined after subcutaneous administration in rats. The C(u,brain) was estimated from the product of total brain concentrations and unbound fractions, which were determined using brain tissue slice and brain homogenate methods. For theobromine, theophylline, caffeine, fluoxetine, and propranolol, which represent rapid brain penetration compounds with a simple diffusion mechanism, the ratios of the area under the curve of C(u,brain)/C(CSF) and C(u,brain)/C(u,plasma) were 0.27 to 1.5 and 0.29 to 2.1, respectively, using the brain slice method, and were 0.27 to 2.9 and 0.36 to 3.9, respectively, using the brain homogenate method. A P-glycoprotein substrate, CP-141938 (methoxy-3-[(2-phenyl-piperadinyl-3-amino)-methyl]-phenyl-N-methyl-methane-sulfonamide), had C(u,brain)/C(CSF) and C(u,brain)/C(u,plasma) ratios of 0.57 and 0.066, using the brain slice method, and 1.1 and 0.13, using the brain homogenate method, respectively. The slow brain-penetrating compound, N[3-(4'-fluorophenyl)-3-(4'-phenylphenoxy)propyl-]sarcosine, had C(u,brain)/C(CSF) and C(u,brain)/C(u,plasma) ratios of 0.94 and 0.12 using the brain slice method and 0.15 and 0.018 using the brain homogenate method, respectively. Therefore, for quick brain penetration with simple diffusion mechanism compounds, C(CSF) and C(u,plasma) represent C(u,brain) equally well; for efflux substrates or slow brain penetration compounds, C(CSF) appears to be equivalent to or more accurate than C(u,plasma) to represent C(u,brain). Thus, we hypothesize that C(CSF) is equivalent to or better than C(u,plasma) to predict C(u,brain). This hypothesis is supported by the literature data.

Permeability of the developing and mature blood-brain barriers to theophylline in rats.

1. In the present study, the uptake of theophylline and L-glucose into the adult and neonatal rat brain has been investigated. Steady state cerebrospinal fluid (CSF) and brain concentrations of theophylline were reached within 1 h following a single intraperitoneal (i.p.) injection, whereas steady state CSF and brain concentrations of L-glucose were not approached until after 5 h. 2. Steady state brain:plasma and CSF:plasma concentration ratios for theophylline and L-glucose in neonatal rats were significantly higher than ratios in adult rats. Erythrocyte:plasma ratios for theophylline in neonatal rats were also significantly higher than ratios in adult rats. Steady state ratios for theophylline were significantly higher than those for L-glucose in both neonatal and adult rats. 3. Respiratory acidosis (pH 6.9-7.0) did not affect steady state CSF:plasma or brain:plasma ratios for theophylline in neonatal or adult rats. In contrast, steady state CSF:plasma and brain:plasma ratios for L-glucose were increased by respiratory acidosis. 4. The lower steady state CSF:plasma, brain:plasma and erythrocyte:plasma ratios for theophylline in adult rats are likely to be due to a higher concentration of plasma proteins in adult blood compared with neonates, with a greater retention of protein-bound (non-exchangeable) theophylline in adult blood, and are unlikely to be due to p-glycoprotein-mediated efflux of theophylline at the adult blood-brain barrier.

Theophylline-induced seizures in a 6-month-old girl. Serum and cerebrospinal fluid levels.

The case is presented of a 6-month-old girl with theophylline-induced focal seizures associated with tachycardia, tremor and irritability. The serum level of theophylline was 20.0 micrograms/ml 30 min after the onset of seizures, and declined to half this level at 19 h. The ratio of the concentration in cerebrospinal fluid (CSF; 8.9 micrograms/ml) to serum (16.4 micrograms/ml) was 0.54 at 4.5 h after the onset. Despite the cessation of seizures, the EEG showed periodic high voltage delta waves over the right hemisphere and left-sided flatness. Since theophylline diffuses easily through the blood-brain barrier in infancy, serum levels should be maintained below 15 micrograms/ml unless severe bronchial asthma occurs.

Effect of pretreatment with anticonvulsants on theophylline-induced seizures in the rat.

Seizures, often with fatal outcome, are a manifestation of pronounced theophylline toxicity. Prodromal symptoms are not always apparent, and the seizures are reported to be, in certain cases, refractory to treatment with anticonvulsant drugs. The purpose of this investigation was to examine, by an established animal model, which of the commonly used anticonvulsants can reduce the central nervous system sensitivity to theophylline neurotoxicity and what should be the preferred treatment in cases in which theophylline toxicity is anticipated. The anticonvulsant agents in doses that are found to be effective in other types of experimentally induced seizures in rats, clonazepam 5 mg/kg, diazepam 5 mg/kg, phenytoin 8 mg/kg, phenobarbital 100 mg/kg, valproic acid 150 mg/kg, and magnesium sulphate 300 mg/kg, or the vehicle (controls) were administered intravenously to Lewis female rats. Thirty minutes later, theophylline was infused at a constant rate of 1.3 mg/min until onset of maximal seizures. Theophylline concentrations in the cerebrospinal fluid, brain, and serum were assayed by a high-performance liquid chromatography method. It was found that pretreatment with either clonazepam, diazepam, phenobarbital, or valproic acid increased the central nervous system thresholds to the theophylline-induced seizures, whereas phenytoin and magnesium sulphate did not attenuate the sensitivity of the brain to the stimulatory action of this widely used bronchodilator. Therefore, whenever theophylline toxicity is suspected, treatment with either diazepam, clonazepam, phenobarbital, or valproic acid can reduce the hazard associated with theophylline-induced seizures.

Theophylline concentrations in serum, saliva, and cerebrospinal fluid in patients with essential tremor.

Theophylline concentrations in saliva and serum were compared in 20 patients with essential tremor. There was a strong correlation between the concentrations in saliva and serum (r = 0.9, slope of 0.48). The ratio between unbound and bound theophylline as assessed by ultrafiltration was 0.30. This ratio was similar to that between theophylline concentrations in CSF and serum. The theophylline concentration in serum was not predictable from that obtained in saliva in individual cases. Moreover, the levels in saliva were about 20% higher than the unbound concentrations of theophylline in serum.

Theophylline neurotoxicity is unaffected by glycerol-induced renal failure.

Glycerol acute renal failure (ARF) was examined to see if it alters theophylline (Th) neurotoxicity in rats. Concentrations of Th in serum, cerebrospinal fluid and in brain at seizure onset were similar in control and ARF rats infused with Th. Thus, glycerol ARF fails to alter Th neurotoxicity, an effect similar to that noted previously with uranyl nitrate but not with ureter ligation.

Influence of adenosine on cerebral blood flow during hypoxic hypoxia in the newborn piglet.

This study investigated the role of adenosine in the regulation of neonatal cerebral blood flow (CBF) during moderate (arterial PO2 = 47 +/- 9 Torr) and severe (arterial PO2 = 25 +/- 4 Torr) hypoxia. Twenty-eight anesthetized and ventilated newborn piglets were assigned to four groups: 8 were injected intravenously with the vehicle (controls, group 1); 13 received an intravenous injection of 8-phenyltheophylline (8-PT), a potent adenosine receptor blocker, either 4 mg/kg (group 2, n = 6, mean cerebrospinal fluid (CSF) levels less than 1 mg/l) or 8 mg/kg (group 3, n = 7, mean CSF levels less than 3.5 mg/l); and 7 received an intracerebroventricular injection of 10 micrograms 8-PT (group 4). During normoxia, CBF was not altered by vehicle or 8-PT injections. In group 1, 10 min of moderate and severe hypoxia increased total CBF by 112 +/- 36 and 176 +/- 28% (SE), respectively. Compared with controls, the cerebral hyperemia during moderate hypoxia was not altered in group 2, attenuated in group 3 (to 53 +/- 13%, P = NS), and completely blocked in group 4 (P less than 0.01). CBF increase secondary to severe hypoxia was attenuated only in group 4 (74 +/- 29%, P less than 0.05). CSF concentrations of adenosine and adenosine metabolites measured by high-performance liquid chromatography increased during hypoxia. Arterial O2 content was inversely correlated (P less than 0.005) to maximal CSF levels of adenosine (r = 0.73), inosine (r = 0.87), and hypoxanthine (r = 0.80).(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of chronic caffeine administration on theophylline concentrations required to produce seizures in rats.

Caffeine as well as the antiasthmatic drug theophylline can cause seizures when administered to humans or animals in excessive doses. Studies on rats have shown rapid development of functional tolerance to caffeine-induced seizures whereas repeated pretreatment with theophylline had no significant effect on the theophylline concentrations required to produce seizures. The purpose of this investigation was to determine whether chronic exposure to caffeine can affect susceptibility to the convulsant effect of theophylline. Rats received caffeine, 40 mg/kg, or solvent twice a day for 7 days as an intravenous injection. On the eighth day, theophylline was infused intravenously until the onset of maximal seizures. At this pharmacologic end point, rats pretreated with caffeine had significantly higher theophylline concentrations in the brain and cerebrospinal fluid than did control (solvent-pretreated) animals. Although the concentration differences were relatively small (approximately 11%), they demonstrate in principle the development of caffeine-induced tolerance to the neurotoxic effect of theophylline. Additional experiments showed that the caffeine effect on theophylline neurotoxicity is not acutely mediated by paraxanthine, a major metabolite of caffeine.

Distribution of enprofylline and theophylline between plasma and cerebrospinal fluid.

Six patients undergoing diagnostic lumbar myelography were studied with respect to plasma and cerebrospinal fluid (CSF) concentrations of two xanthine drugs--enprofylline and theophylline. Three patients received enprofylline and three patients received theophylline, 200 mg t.i.d., and plasma and CSF were sampled on the morning of the third day of treatment. CSF plasma ratios averaged 0.095 with enprofylline (range of 0.094-0.097) and 0.36 with theophylline (range of 0.35-0.37). The different ratios of the two drugs contrast to their similar plasma protein binding, about 50%. Different lipophilicity or differences with regard to transport out of the CSF may explain the discrepancy.

Caffeine as a potential risk factor for theophylline neurotoxicity.

Theophylline can cause life-threatening seizures when administered in excessive doses. The plasma concentrations associated with this neurotoxic effect vary widely among patients. To determine the reasons for the wide variation, an animal model of theophylline-induced seizures was developed and has now been used to determine the effect of pre-exposure to caffeine on theophylline-induced neurotoxicity. Male adult rats received an iv infusion of either caffeine citrate or sodium citrate solution for 15 min. Theophylline was then infused at a relatively rapid rate until onset of maximum seizures. A third group of rats received a rapid infusion of caffeine only until onset of seizures. Samples of blood, brain, and cerebrospinal fluid were obtained at that time for determination of caffeine and theophylline concentrations by HPLC. Prior exposure to caffeine was associated with a statistically significant reduction in the total amount of theophylline required to produce seizures and caused theophylline concentrations at all sampling sites to be significantly lower than in controls. Caffeine alone required a larger total dose and higher concentrations than theophylline alone to produce seizures. It is concluded that acute exposure to caffeine can increase the risk of theophylline-induced neurotoxicity.

Kinetics of drug action in disease states. XXVI: Effect of fever on the pharmacodynamics of theophylline-induced seizures in rats.

This investigation was designed to determine the effect of fever on the neurotoxicity of theophylline as reflected by the concentrations of this drug that cause convulsions in experimental animals. Fever was produced in male, inbred, adult Lewis rats (approximately 180 g) by sc injection of brewer's yeast; an elevation of body temperature of 1.2 +/- 0.4 degrees C (mean +/- SD) was achieved at the time of the pharmacodynamic measurements. Theophylline was infused iv at a rate of 1.03 mg/min until the onset of maximal seizures. Drug concentrations in serum, serum water, brain, and cerebrospinal fluid (CSF) at that time were determined by high-performance liquid chromatography. Compared with the control group, the group of febrile rats had statically significantly lower serum protein concentrations, decreased serum protein binding of theophylline, and slightly increased theophylline concentrations in the CSF at the onset of seizures. Inasmuch as theophylline concentrations in the CSF reflect the concentrations of this drug in the biophase, the results of this study show that fever does not increase the sensitivity of the central nervous system to the neurotoxic effects of theophylline in rats. In fact, a statistically significant positive correlation between theophylline concentrations in the CSF and body temperature was found in this investigation, suggesting a decreased sensitivity of the animals to the neurotoxic effects of theophylline at higher body temperature.

Theophylline neurotoxicity in pregnant rats.

The purpose of this investigation was to determine whether the neurotoxicity of theophylline is altered in advanced pregnancy. Sprague-Dawley rats that were 20 days pregnant and nonpregnant rats of the same age and strain received infusions of aminophylline until onset of maximal seizures which occurred after 28 and 30 minutes respectively. Theophylline concentrations at this endpoint in serum (total) and CSF were similar but serum (free) and brain concentrations were slightly different in pregnant rats. Theophylline serum protein binding determined by equilibrium dialysis was lower in pregnant rats. Fetal serum concentrations at onset of seizures in the mother were similar to maternal brain and CSF concentrations and correlated significantly with the former. It is concluded that advanced pregnancy has a negligible effect on the neurotoxic response to theophylline in rats.

Caffeine disposition in the pregnant rabbit. II. Fetal distribution of caffeine and paraxanthine during chronic maternal caffeine administration.

Twenty-three New Zealand White rabbits received a continuous intravenous infusion of caffeine during gestation. The amniotic fluid/maternal plasma concentration ratio was higher for caffeine than for its major metabolite, paraxanthine, throughout gestation, and increased near term for both compounds. Both compounds distributed nearly homogeneously to fluids and tissues of the 29-day fetus, with mean fetal/maternal concentration ratios of 0.7 for paraxanthine and 0.9 for caffeine. The free fraction of caffeine was constant during gestation (about 0.8), while that of paraxanthine increased from 0.25 to 0.4. Similar results were observed in 3 Dutch Belted rabbits given caffeine in their drinking water and sacrificed at 29 days of gestation.

Role of adenosine in regulation of cerebral blood flow: effects of theophylline during normoxia and hypoxia.

We studied the effects of the methylxanthine theophylline, an adenosine receptor blocker, on cerebral circulation. Cerebral blood flow (CBF) was measured by the retroglenoid outflow and microsphere techniques, and pial circulation changes were observed through a closed cranial window. Intraperitoneal administration of theophylline in normoxic animals resulted in a biphasic response of pial vessels and CBF. At low concentrations (0.05 mumol/g) of theophylline, pial vessel diameter and CBF decreased, whereas vasodilatation and hyperemia were observed at higher levels. After intraperitoneal administration of either 0.05 or 0.2 mumol/g, hypoxic hyperemia was attenuated both during short (c. 30 s) and sustained (c. 2-3 min) hypoxia, as was hypoxic pial arteriolar vasodilatation. These actions of theophylline appear to be due to adenosine receptor blockade, since micromolar concentrations were achieved in cerebrospinal fluid (CSF), and no increases in adenosine 3',5'-cyclic monophosphate concentrations in brain were noted. Moreover, theophylline (either intraperitoneal or topical) blocked pial vasodilatation caused by topically applied adenosine, but had little effect on hypercarbic hyperemia or pial vasodilatation induced by topically applied acetylcholine. The results of these studies suggest that adenosine is involved in the maintenance of resting cerebral vascular tone and has a paramount role in the regulation of CBF during hypoxia.

The effect of theophylline on regional cerebral blood flow responses to hypoxia in newborn piglets.

Theophylline attenuates cerebral hypoxic hyperemia in several adult models and this is thought to be due to receptor-mediated antagonism of adenosine, a proposed mediator of hypoxic hyperemia. This attenuation of hypoxic hyperemia reduces cerebral oxygen delivery and may thus jeopardize cerebral oxidative metabolism. With these considerations in mind, and because theophylline is widely used in neonatal medicine, the present study was designed to investigate the effect of theophylline on regional cerebral blood flow, cerebral oxygen delivery, and cerebral metabolic rate for oxygen during normoxia and hypoxia in the newborn piglet model. In 16 newborn piglets, regional cerebral blood flow (microspheres) increased 250-350% during hypoxia (PaO2 20-30 torr), while cerebral oxygen delivery and cerebral metabolic rate for oxygen were maintained at normoxic levels. Eight of these piglets were then given 10 mg/kg theophylline ethylenediamine intravenously and studies during normoxia and hypoxia were repeated; the remaining eight piglets served as time controls. Regional cerebral blood flow, cerebral oxygen delivery, and cerebral metabolic rate for oxygen during normoxia and hypoxia were not influenced by theophylline, despite plasma theophylline levels of 55-65 mumol/liter, and cerebrospinal fluid theophylline levels of 30-40 mumol/liter. These negative results are reassuring with respect to hypoxic cerebral blood flow control in theophylline-medicated infants. However, they do not support a role for adenosine as a mediator of cerebral hypoxic hyperemia in this model.

Kinetics of drug action in disease states. XVIII. Effect of experimental renal failure on the pharmacodynamics of theophylline-induced seizures in rats.

Theophylline, the bronchodilating agent, can cause life-threatening, generalized seizures when plasma concentrations exceed the usual therapeutic concentration range. However, the plasma concentrations of theophylline associated with this neurotoxic effect vary widely between patients. To determine the reasons for the wide variation, and thereby to facilitate prevention or early treatment of theophylline-induced neurotoxicity, an animal model of theophylline-induced seizures was developed and has now been used to determine the effect of experimental renal failure on the concentrations of theophylline that cause convulsions. Adult female rats were subjected to bilateral ureteral ligation or injected with uranyl nitrate to produce renal failure or dysfunction. Sham-operated and saline-injected rats, respectively, served as controls. Theophylline was infused i.v. at either 1.03 or 2.06 mg/min until the onset of maximal seizures. Renal failure due to ureter ligation was associated with a substantial reduction of the dose of drug required to produce seizures, the concentrations of total and free (unbound) theophylline in serum and the concentrations of theophylline in the brain and cerebrospinal fluid at onset of seizures. The concentrations of theophylline metabolites were very low and did not account for the enhanced neurotoxicity. No apparent change in the neurotoxicity of theophylline was observed in rats with uranyl nitrate-induced renal dysfunction. The results of the investigation on ureter-ligated rats are consistent with recent clinical findings of a higher incidence of theophylline-induced neurotoxicity in azotemic patients. The experimental methodology may therefore be suitable for the prospective identification of other potential clinical risk factors for theophylline neurotoxicity.