Glutaminyl Cyclase Inhibitor PQ912 Improves Cognition in Mouse Models of Alzheimer's Disease-Studies on Relation to Effective Target Occupancy.

Numerous studies suggest that the majority of amyloid-ß (Aß) peptides deposited in Alzheimer's disease (AD) are truncated and post-translationally modified at the N terminus. Among these modified species, pyroglutamyl-Aß (pE-Aß, including N3pE-Aß40/42 and N11pE-Aß40/42) has been identified as particularly neurotoxic. The N-terminal modification renders the peptide hydrophobic, accelerates formation of oligomers, and reduces degradation by peptidases, leading ultimately to the accumulation of the peptide and progression of AD. It has been shown that the formation of pyroglutamyl residues is catalyzed by glutaminyl cyclase (QC). Here, we present data about the pharmacological in vitro and in vivo efficacy of the QC inhibitor (S)-1-(1H-benzo[d]imidazol-5-yl)-5-(4-propoxyphenyl)imidazolidin-2-one (PQ912), the first-in-class compound that is in clinical development. PQ912 inhibits human, rat, and mouse QC activity, with Ki values ranging between 20 and 65 nM. Chronic oral treatment of hAPPSLxhQC double-transgenic mice with approximately 200 mg/kg/day via chow shows a significant reduction of pE-Aß levels and concomitant improvement of spatial learning in a Morris water maze test paradigm. This dose results in a brain and cerebrospinal fluid concentration of PQ912 which relates to a QC target occupancy of about 60%. Thus, we conclude that >50% inhibition of QC activity in the brain leads to robust treatment effects. Secondary pharmacology experiments in mice indicate a fairly large potency difference for Aß cyclization compared with cyclization of physiologic substrates, suggesting a robust therapeutic window in humans. This information constitutes an important translational guidance for predicting the therapeutic dose range in clinical studies with PQ912.

Development of an UPLC-MS/MS method for quantification of Avitinib (AC0010) and its five metabolites in human cerebrospinal fluid: Application to a study of the blood-brain barrier penetration rate of non-small cell lung cancer patients.

Avitinib (AC0010) is a mutant-selective epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKI), designed to be a targeted therapeutic agent for non-small cell lung cancer (NSCLC) patients harboring EGFR active and T790M resistant mutations. A rapid and sensitive ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed and validated for the determination of Avitinib and its five metabolites (M1, M2, M4, M7, MII-6) in human cerebrospinal fluid (CSF). The samples were purified by protein precipitation and separated on a BEH C18 column (2.1×50mm, 1.7µm). Electrospray ionization (ESI) in positive ion mode and multiple reaction monitoring (MRM) were used to monitor the ion transitions at m/z 488/257, 474/403, 504/487, 434/377, 490/405, 476/391. The results indicated that the method had excellent sensitivity and specificity. The linear range covered from 0.05 to 50ng/mL for Avitinib, M1, M4, M7, and MII-6, and from 0.01 to 10ng/mL for M2. Intra-day and inter-day precisions (in terms of% RSD) were all <15% and the accuracies (in terms of% RE) were within the range of ±15%. The lower limit of quantification (LLOQ), matrix effect, extraction recovery, stability and dilution integrity were also validated and satisfied with the criteria of validation. Finally, the method was successfully applied to a blood-brain barrier (BBB) penetration rate research of NSCLC patients after an oral administration of Avitinib.

Effects of single doses of avagacestat (BMS-708163) on cerebrospinal fluid Aß levels in healthy young men.

BACKGROUND: The concentration of amyloid ß (Aß) peptides in cerebrospinal fluid (CSF) is a biomarker for Alzheimer's disease (AD) pathology, and has been used to evaluate the effectiveness of γ-secretase inhibition. Avagacestat is a selective γ-secretase inhibitor in development for the treatment of AD. The primary objective of this study was to assess the effects of single oral doses of avagacestat on the CSF Aß concentrations in healthy male subjects. Secondary objectives included single-dose pharmacokinetics in CSF and plasma, safety and tolerability. METHODS: This was a double-blind, placebo-controlled, randomized, single-dose study. Healthy male subjects were assigned to one of three sequential avagacestat dose panels (50, 200 and 400 mg) or placebo as single oral doses. RESULTS: 34 subjects were enrolled. Administration of a single dose of 200 or 400 mg of avagacestat resulted in a marked decrease in CSF Aß(1-38), Aß(1-40) and Aß(1-42) concentrations vs placebo; with smaller decreases observed in the 50 mg dose group. Avagacestat was quickly absorbed into the systemic circulation, with a mean time to reach maximum plasma concentration (t(max)) of approximately 1-2 h, and a CSF t(max) of approximately 3 h. Adverse events were uncommon and occurred with similar frequency in the placebo and avagacestat groups. CONCLUSION: Avagacestat was safe, well tolerated, and resulted in a notable decrease in CSF Aß concentrations, suggestive of γ-secretase inhibition. The results warrant further clinical study in patients with AD.

Endogenous ouabain regulates cell viability.

The endogenous cardiac steroid-like compounds, endogenous ouabain (EO) in particular, are present in the human circulation and are considered putative ligands of the inhibitory binding site of the plasma membrane Na(+)-K(+)-ATPase. A vast amount of data shows that, when added to cell cultures, these steroids promote the growth of cardiac, vascular, and epithelial cells. However, the involvement of the endogenous compounds in the regulation of cell viability and proliferation has never been addressed experimentally. In this study, we show that EO is present in mammalian sera and cerebral spinal fluid, as well as in commercial bovine and horse sera. The lowering of serum EO concentration by the addition of specific anti-ouabain antibodies caused a decrease in the viability of several cultured cell lines. Among these, neuronal NT2 cells were mostly affected, whereas no reduction in viability was seen in rat neuroendocrine PC12 and monkey kidney COS-7 cells. The anti-ouabain antibody-induced reduction in NT2 cell viability was significantly attenuated by the addition of ouabain and was not observed in cells growing in serum-free media. Furthermore, the addition to the medium of low concentrations (nM) of the cardenolide ouabain, but not of the bufadienolide bufalin, increased NT2 and PC12 cell viability and proliferation. In addition, at these concentrations both ouabain and bufalin caused the activation of ERK1/2 in the NT2 cells. The specific ERK1/2 inhibitor U0126 inhibited both the ouabain-induced activation of the enzyme and the increase in cell viability. Furthermore, anti-ouabain antibodies attenuated serum-stimulated ERK1/2 activity in NT2 but not in PC12 cells. Cumulatively, our results suggest that EO plays a significant role in the regulation of cell viability. In addition, our findings support the notion that activation of the ERK1/2 signaling pathway is obligatory but not sufficient for the induction of cell viability by EO.

Evaluation of miglustat treatment in patients with type III mucopolysaccharidosis: a randomized, double-blind, placebo-controlled study.

OBJECTIVE: To evaluate the efficacy and safety of oral miglustat treatment in patients with mucopolysaccharidosis type III. The primary outcome was efficacy with improvement or stabilization in at least two domains of Vineland Adaptative Behavior Scales at 6 months. The secondary outcome measured the evolution of other cognitive tests at 12 months. The safety and tolerability were assessed throughout the study. STUDY DESIGN: This was a randomized, double-blind, placebo-controlled, monocenter, institutional, phase IIb to III study. In case of efficacy at 6 months, the study would go on for another 6 months on an open design with all patients receiving miglustat. In the absence of efficacy at 6 months, the trial had to be continued for 6 more months with the initial design. RESULTS: After 6 months, efficacy was not superior in patients with miglustat. The independent review board confirmed continuing the study until 12 months. CONCLUSION: Miglustat treatment was not associated with any improvement/stabilization in behavior problems in patients with mucopolysaccharidosis type III. Miglustat has an acceptable safety profile. However, the study has confirmed that miglustat is able to pass through the blood-brain barrier without significantly decreasing ganglioside levels.

Increase of CSF tyrosine and impaired serotonin turnover in tyrosinemia type I.

OBJECTIVE: Psychomotor impairment has been described in hypertyrosinemia types II and III (HT III). Only recently cognitive deficits have also been reported in hypertyrosinemia type I (HT I). The pathogenic mechanisms responsible are unknown. Since implementation of 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione (NTBC, Nitisinone (Swedish Orphan International)) in the treatment of HT I, plasma tyrosine elevation is a common finding as known from the other hypertyrosinemias. PATIENTS AND METHODS: With elevated tyrosine as suspected pathogenic factor in the development of cognitive deficits, we here investigated tyrosine in the cerebrospinal fluid (CSF) and serotonergic and dopaminergic neurotransmitter levels in three patients with HT I during long-term treatment with Nitisinone. In addition, Nitisinone concentrations in plasma and CSF were measured. We also assessed psychomotor and cognitive development by standardized test systems and brain morphology by magnetic resonance imaging. RESULTS: All patients presented with high tyrosine concentrations in CSF correlating with increased plasma tyrosine levels and a reduced CSF serotonin turnover. MRI revealed no structural abnormalities in the brain. All patients presented with either impaired cognitive development or behavioural abnormalities. CONCLUSIONS: We here outline the need to further study the exact pathogenic mechanisms responsible for the neurotransmitter changes observed in HT type I in order to possibly prevent cognitive dysfunction. Nitisinone has significantly improved outcome and quality of life in HT type I; however, it is also accompanied by elevated plasma and CSF tyrosine. Further studies are essential to identify the necessary dietary tyrosine restriction and the optimal Nitisinone dose.

Development and validation of sensitive and selective LC-MS/MS methods for the determination of BMS-708163, a gamma-secretase inhibitor, in plasma and cerebrospinal fluid using deprotonated or formate adduct ions as precursor ions.

BMS-708163 is a gamma-secretase inhibitor that is being developed for the treatment of Alzheimer's disease. Several LC-MS/MS methods have been developed for the determination of BMS-708163 in both plasma and cerebrospinal fluid in support of dog, rat, mouse and human studies. To support non-clinical studies, an LC-MS/MS method with a lower limit of quantitation (LLOQ) of 5 ng/mL, was developed and validated in dog, rat, and mouse plasma by using the deprotonated ion as the precursor ion. To support clinical studies, an LC-MS/MS method with LLOQ of 0.1 ng/mL, was developed and validated in human plasma by using the formate adduct as the precursor ion. Formic acid (0.01%) in water and acetonitrile was found to be the most favorable mobile phases for both deprotonated and formate adduct ions in negative electrospray ionization mode. A combination of a 3M Empore C18 plate for SPE and a Waters Atlantis dC18 analytical column for separation was used to achieve a highly selective solid phase extraction and chromatographic procedure from plasma without dry down and reconstitution steps. In the development of an assay for BMS-708163 in cerebral spinal fluid (CSF), significant non-specific binding of BMS-708163 was observed and resolved with pre- or post-spike of 0.2% Tween 20 into CSF samples. A dilute-and-shoot LC-MS/MS method with LLOQ of 0.1 ng/mL was developed and validated to assess BMS-708163 exposure in human CSF.

Safety and pharmacokinetic analysis of methotrexate administered directly into the fourth ventricle in a piglet model.

We have developed a piglet model to assess chemotherapy administration directly into the fourth ventricle as a potential treatment for medulloblastoma and other malignant posterior fossa tumors. The objective of this study was to assess safety and pharmacokinetics after methotrexate infusions into the fourth ventricle. Catheters were inserted into the fourth ventricle and lumbar cistern in five piglets. Two milligrams of Methotrexate (MTX) was infused into the fourth ventricle on five consecutive days. Safety was assessed by neurological examination, 4.7 T MRI, and post-mortem pathological analysis. MTX levels in serum and cerebrospinal fluid (CSF) were measured, and area under the concentration-time curve (AUC) was calculated for CSF samples. No neurological deficits were caused by MTX infusions. One piglet died from complications of anesthesia induction for MRI scanning. MRI scans showed accurate catheter placement without signal changes in the brainstem or cerebellum. One piglet had asymptomatic ventriculomegaly. Pathological analysis demonstrated meningitis and choroid plexitis consisting predominantly of CD-3 positive T-lymphocytes in all piglets and a small focal area of subependymal necrosis in one. In all piglets, mean peak MTX level in fourth ventricular CSF exceeded that in lumbar CSF by greater than five-fold. Serum MTX levels were undetectable or negligible. Statistically significant differences between fourth ventricle and lumbar AUC were detected at peaks (P = 0.01) and at all collection time points (P = 0.01) but not at troughs (P = 0.36). MTX can be infused into the fourth ventricle without clinical or radiographic evidence of damage. An inflammatory response without clinical correlate is observed. Significantly higher peak MTX levels are observed in the fourth ventricle than in the lumbar cistern.

A gamma-secretase inhibitor decreases amyloid-beta production in the central nervous system.

OBJECTIVE: Accumulation of amyloid-beta (Abeta) by overproduction or underclearance in the central nervous system (CNS) is hypothesized to be a necessary event in the pathogenesis of Alzheimer's disease. However, previously, there has not been a method to determine drug effects on Abeta production or clearance in the human CNS. The objective of this study was to determine the effects of a gamma-secretase inhibitor on the production of Abeta in the human CNS. METHODS: We utilized a recently developed method of stable-isotope labeling combined with cerebrospinal fluid sampling to directly measure Abeta production during treatment of a gamma-secretase inhibitor, LY450139. We assessed whether this drug could decrease CNS Abeta production in healthy men (age range, 21-50 years) at single oral doses of 100, 140, or 280mg (n = 5 per group). RESULTS: LY450139 significantly decreased the production of CNS Abeta in a dose-dependent fashion, with inhibition of Abeta generation of 47, 52, and 84% over a 12-hour period with doses of 100, 140, and 280mg, respectively. There was no difference in Abeta clearance. INTERPRETATION: Stable isotope labeling of CNS proteins can be utilized to assess the effects of drugs on the production and clearance rates of proteins targeted as potential disease-modifying treatments for Alzheimer's disease and other CNS disorders. Results from this approach can assist in making decisions about drug dosing and frequency in the design of larger and longer clinical trials for diseases such as Alzheimer's disease, and may accelerate effective drug validation. Ann Neurol 2009.

Benzothiazinones kill Mycobacterium tuberculosis by blocking arabinan synthesis.

New drugs are required to counter the tuberculosis (TB) pandemic. Here, we describe the synthesis and characterization of 1,3-benzothiazin-4-ones (BTZs), a new class of antimycobacterial agents that kill Mycobacterium tuberculosis in vitro, ex vivo, and in mouse models of TB. Using genetics and biochemistry, we identified the enzyme decaprenylphosphoryl-beta-d-ribose 2'-epimerase as a major BTZ target. Inhibition of this enzymatic activity abolishes the formation of decaprenylphosphoryl arabinose, a key precursor that is required for the synthesis of the cell-wall arabinans, thus provoking cell lysis and bacterial death. The most advanced compound, BTZ043, is a candidate for inclusion in combination therapies for both drug-sensitive and extensively drug-resistant TB.

Rapid quantification of miglustat in human plasma and cerebrospinal fluid by liquid chromatography coupled with tandem mass spectrometry.

Miglustat (OGT 918) is an iminosugar recently introduced in therapeutic as potential alternative therapy in disorders found in several diseases such as Tay-Sachs, Gaucher or Niemann-Pick diseases. A highly sensitive liquid-chromatography-electrospray tandem mass spectrometry (LC-MS/MS) assay was developed for the quantification of miglustat in human plasma and cerebrospinal fluid (CSF). The sample preparation consists in a simple protein precipitation with a mixture of acetonitrile/methanol (75/25) which yields 100% recovery. The isocratic separation utilizes an Atlantis Hilic (3 microm, 150 mm x 2.1 mm) column, with a mobile phase of acetonitrile/water/ammonium acetate buffer (75/10/15, v/v/v) delivered at 230 microl/min. Selected reaction monitoring (SRM) mode was used with the transitions m/z 220-->158 for the miglustat and m/z 208-->m/z 146 for the miglitol (internal standard). Good linearity was observed in a range from 125 to 2500 ng/ml and from 50 to 1000 ng/ml, for plasma and CSF, respectively. The within-run precision of the assay was less than 6%, and the between-run run precision was less than 6.5%, for six replicates at each of three concentrations and evaluated on three separated days for both plasma and CSF mediums. Assay accuracy was in the range of 98-106.5%. Stability of miglustat was reported under a variety of storage conditions. The miglustat concentrations in two children are presented to demonstrate the clinical interest of this new method.

Low penetration of oseltamivir and its carboxylate into cerebrospinal fluid in healthy Japanese and Caucasian volunteers.

Oseltamivir is a potent, well-tolerated antiviral for the treatment and prophylaxis of influenza. Although no relationship with treatment could be demonstrated, recent reports of abnormal behavior in young individuals with influenza who were receiving oseltamivir have generated renewed interest in the central nervous system (CNS) tolerability of oseltamivir. This single-center, open-label study explored the pharmacokinetics of oseltamivir and oseltamivir carboxylate (OC) in the plasma and cerebrospinal fluid (CSF) of healthy adult volunteers over a 24-hour interval to determine the CNS penetration of both these compounds. Four Japanese and four Caucasian males were enrolled in the study. Oseltamivir and OC concentrations in CSF were low (mean of observed maximum concentrations [C(max)], 2.4 ng/ml [oseltamivir] and 19.0 ng/ml [OC]) versus those in plasma (mean C(max), 115 ng/ml [oseltamivir] and 544 ng/ml [OC]), with corresponding C(max) CSF/plasma ratios of 2.1% (oseltamivir) and 3.5% (OC). Overall exposure to oseltamivir and OC in CSF was also comparatively low versus that in plasma (mean area under the concentration-time curve CSF/plasma ratio, 2.4% [oseltamivir] and 2.9% [OC]). No gross differences in the pharmacokinetics of oseltamivir or OC were observed between the Japanese and Caucasian subjects. Oseltamivir was well tolerated. This demonstrates that the CNS penetration of oseltamivir and OC is low in Japanese and Caucasian adults. Emerging data support the idea that oseltamivir and OC have limited potential to induce or exacerbate CNS adverse events in individuals with influenza. A disease- rather than drug-related effect appears likely.

Determination of dimethylarginine levels in rats using HILIC-MS/MS: an in vivo microdialysis study.

Nitric oxide (NO) is one of the most important mediators and neurotransmitters and its levels change under pathological conditions. NO production may be regulated by endogenous nitric oxide synthase (NOS) inhibitors, in particular asymmetric dimethylarginine (ADMA). Most of the interest is focused on ADMA, since this compound is present in plasma and urine and accumulation of ADMA has been described in many disease states but little is known about cerebrospinal fluid (CSF) concentrations of this compound and of its structural isomer symmetric dimethylarginine (SDMA). To determine the levels of methylarginines, we here present a new hydrophilic interaction chromatography (HILIC)-MS/MS method for the precise determination of these substances in CSF from microdialysis samples of rat prefrontal cortex (PFC). The method requires only minimal sample preparation and features isotope-labelled internal standards.

Determination of a novel gamma-secretase inhibitor in human plasma and cerebrospinal fluid using automated 96 well solid phase extraction and liquid chromatography/tandem mass spectrometry.

A method for determination of a gamma-secretase inhibitor, cis-3-[4-[(4-chlorophenyl)sulfonyl]-4-(2,5-difluorophenyl)cyclohexyl]propanoic acid (A), in human plasma and cerebrospinal fluid (CSF) has been developed to support the clinical investigation of compound A for its potential treatment of Alzheimer's disease. The method is based on HPLC with atmospheric pressure chemical ionization tandem mass spectrometric detection (APCI-MS/MS) in the negative ionization mode using a heated nebulizer interface. The addition of phosphoric acid at the ratio of 10-30microL per milliliter of human plasma or CSF was required during clinical sample collection to stabilize an acylglucuronide metabolite (C), which was potentially present in human plasma and CSF. Tween 20 (10% solution) was added at the ratio of 20microL per milliliter of CSF during CSF sample collection to prevent the loss of compound A during the storage of clinical samples. The compound A and its analog internal standard (B) in treated plasma or CSF were isolated from human plasma or CSF using solid phase extraction (SPE) in the 96 well format. The isolated analyte and internal standard were chromatographed on a Phenomenex Synergi Polar RP analytical column (50mmx3.0mm, 4microm), using a mobile phase consisting of 60/40 (v/v, %) acetonitrile/water at a flow-rate of 0.5mL/min. Tandem mass spectrometric detection was performed using a Sciex API 3000 tandem mass spectrometer operated in the multiple reaction monitoring (MRM) mode using precursor to product ion transitions of 441-->175 for A and 469-->175 for B, respectively. The assays were validated over the concentration range of 0.5-200ng/mL for human plasma and CSF. Replicate analyses (n=5) of spiked standards for both assays yielded a linear response with coefficients of variation less than 7% and accuracy within 5% of the nominal concentrations. In addition, the assays were automated to improve sample throughput by utilizing a Packard Multi PROBEII automated liquid handling system and a Tom-Tec Quadra 96 system. Numerous clinical studies have been supported using these assays.

Plasma and cerebrospinal fluid pharmacokinetics of valproic acid after oral administration in non-human primates.

PURPOSE: Valproic acid (VPA), a widely used antiepileptic, also inhibits histone deacetylase (HDAC), and is undergoing evaluation as an anti-cancer agent. We studied the pharmacokinetics of VPA in the plasma and cerebrospinal fluid (CSF) in a non-human primate model that is highly predictive of human CSF penetration to determine if levels of VPA required to inhibit HDAC in in vivo models can be attained. METHODS: Oral VPA, 75 mg/kg, was administered to four non-human primates. Serial samples of blood (n = 4) and CSF (n = 3) were obtained for pharmacokinetic studies of total and free VPA. Plasma and CSF VPA concentrations were measured using the commercially available Abbott AxSYM VPA assay reagent system (Abbott Laboratories, Abbott Park, IL, USA). The resultant plasma and CSF data were evaluated using pharmacokinetic modeling methods. RESULTS: At a dose of 75 mg/kg, the maximum plasma concentration of VPA was 130.1 +/- 70.6 microg/ml (mean +/- standard deviation) for total drug and 53.3 +/- 44.4 microg/ml for free drug. The mean plasma area under the curve (AUC) for total drug was 680 +/- 233 microg/ml h and for free drug 146 +/- 89 microg/ml hr. The maximum CSF concentration occurred 2-3 h after administration and was 28.2 +/- 18.6 microg/ml. The CSF AUC for VPA was 108 +/- 52 microg/ml h. The CSF penetration of VPA was 12.9 +/- 5.1% for total drug and 57.0 +/- 8.7% for free drug. Disappearance from the plasma followed non-linear kinetics with a V (max) of 321.2 +/- 65.6 microg/kg/min and a K (m) of 17.2 +/- 13.7 mg/l. CONCLUSION: Valproic acid deserves further study for the treatment of CNS tumors given its high CSF penetration after oral dosing coupled with the anti-tumor activity observed in preclinical studies.

Plasma and cerebrospinal fluid pharmacokinetics of the histone deacetylase inhibitor, belinostat (PXD101), in non-human primates.

PURPOSE: Histone deacetylases (HDAC) are involved in the regulation of gene transcription. Aberrant HDAC activity has been associated with tumorigenesis, and, therefore, HDACs are potential targets for the treatment of cancers, including tumors of the central nervous system (CNS). Belinostat is a novel, potent, pan-HDAC inhibitor with antiproliferative activity on a wide variety of tumor cell lines. We studied the cerebrospinal fluid (CSF) penetration of intravenous (IV) belinostat in a non-human primate model as a surrogate for blood:brain barrier penetration. DESIGN: Five adult rhesus monkeys received increasing doses of belinostat (10-60 mg/kg) as a 30-min IV infusion. Serial blood and CSF samples were collected over 48 h. Plasma and CSF concentrations of belinostat were quantified with an LC/MS/MS assay. Pharmacokinetic parameters were calculated using non-compartmental methods, and CSF penetration is expressed as the ratio of the area under the concentration-time curve (AUC) in CSF to the AUC in plasma. RESULTS: Belinostat was cleared rapidly from plasma with a half-life of 1.0 h, a mean residence time of 0.47 h, and a clearance of 425 ml/min/m(2). CSF penetration of belinostat was limited. CSF drug exposure was <1% of plasma drug exposure and <10% of free (non-protein bound) plasma drug exposure. CONCLUSION: IV belinostat is rapidly cleared from plasma and has limited penetration into the CSF.

Association of an endogenous inhibitor of nitric oxide synthase with cerebral vasospasm in patients with aneurysmal subarachnoid hemorrhage.

OBJECT: Delayed cerebral vasospasm after subarachnoid hemorrhage (SAH) may be evoked by the decreased availability of nitric oxide (NO). Increased cerebrospinal fluid (CSF) levels of asymmetric dimethyl-L-arginine (ADMA), an endogenous inhibitor of NO synthase (NOS), have been associated with the course and degree of cerebral vasospasm in a primate model of SAH. In this study, the authors sought to determine if similar changes in CSF ADMA levels are observed in patients with SAH, and whether these changes are associated with NO and NOS metabolite levels in the CSF and the presence of cerebral vasospasm. METHODS: Asymmetric dimethyl-L-arginine, L-arginine, L-citrulline, and nitrite levels were measured in CSF and serum samples collected during the 21-day period after a single aneurysmal SAH in 18 consecutive patients. Samples were also obtained in a control group consisting of seven patients with Chiari malformation Type I and five patients with spontaneous intracerebral hemorrhage without SAH. Vasospasm, defined as a greater than 11% reduction in the anterior circulation vessel diameter ratio compared with the ratio calculated from the initial arteriogram, was assessed on cerebral arteriography performed around Day 7. RESULTS: In 13 patients with SAH, arteriographic cerebral vasospasm developed. Cerebrospinal fluid ADMA levels in patients with SAH were higher than in those in the control group (p < 0.001). The CSF ADMA level remained unchanged in the five patients with SAH without vasospasm, but was significantly increased in patients with vasospasm after Day 3 (6.2 +/- 1.7 microM) peaking during Days 7 through 9 (13.3 +/- 6.7 microM; p < 0.001) and then gradually decreasing between Days 12 and 21 (8.8 +/- 3.2 microM; p < 0.05). Nitrite levels in the CSF were lower in patients with vasospasm compared to patients without vasospasm (p < 0.03). Cerebrospinal fluid ADMA levels positively correlated with the degree of vasospasm (correlation coefficient [CC] = 0.88, p = 0.0001; 95% confidence interval [CI] 0.74-0.95) and negatively correlated with CSF nitrite levels (CC = -0.55; p = 0.017; 95% CI -0.81 to -0.12). CONCLUSIONS: These results support the hypothesis that ADMA is involved in the progression of cerebral vasospasm. Asymmetric dimethyl-L-arginine and its metabolizing enzymes may be a future target for treatment of cerebral vasospasm after SAH.

Association between cerebrospinal fluid levels of asymmetric dimethyl-L-arginine, an endogenous inhibitor of endothelial nitric oxide synthase, and cerebral vasospasm in a primate model of subarachnoid hemorrhage.

OBJECT: Decreased availability of nitric oxide (NO) has been proposed to evoke delayed cerebral vasospasm after sub-arachnoid hemorrhage (SAH). Asymmetric dimethyl-L-arginine (ADMA) inhibits endothelial NO synthase (eNOS) and, therefore, may be responsible for decreased NO availability in cases of cerebral vasospasm. The goal of this study was to determine whether ADMA levels are associated with cerebral vasospasm in a primate model of SAH. METHODS: Twenty-two cynomolgus monkeys (six control animals and 16 with SAH) were used in this study. The levels of ADMA, L-arginine, L-citrulline, nitrites, and nitrates in cerebrospinal fluid (CSF) and serum were determined on Days 0, 7, 14, and 21 following onset of SAH. Cerebral arteriography was performed to assess the degree of vasospasm. Western blot analyses of the right and left middle cerebral arteries (MCAs) were performed to assess the expression of eNOS, type I protein-arginine methyl transferase (PRMT1) and dimethylarginine dimethylaminohydrolase (DDAH2). Cerebrospinal fluid levels of ADMA remained unchanged in the control group (six animals) and in animals with SAH that did not have vasospasm (five animals; p = 0.17), but the levels increased in animals with vasospasm (11 animals) on Day 7 post-SAH (p < 0.01) and decreased on Days 14 through 21 (p < 0.05). Cerebrospinal fluid levels of ADMA correlated directly with the degree of vasospasm (correlation coefficient = 0.7, p = 0.0001; 95% confidence interval: 0.43-0.83). Levels of nitrite and nitrate as well as those of L-citrulline in CSF were decreased in animals with vasospasm. Furthermore, DDAH2 expression was attenuated in the right spastic MCA on Day 7 post-SAH, whereas eNOS and PRMT1 expression remained unchanged. CONCLUSIONS: Changes in the CSF levels of ADMA are associated with the development and resolution of vasospasm found on arteriograms after SAH. The results indicate that endogenous inhibition of eNOS by ADMA may be involved in the development of delayed cerebral vasospasm. Inhibition of ADMA production may provide a new therapeutic approach for cerebral vasospasm after SAH.

[Development of drug delivery system for intrathecal administration and its therapeutic effect on cerebral vasospasm and ischemia].

To date, the pharmacologic approach to cerebral vasospasm and ischemia has been hampered in part by an inability to attain sufficiently high concentrations of drugs in the cerebrospinal fluid (CSF). To overcome this limitation of current drug therapy, we have developed a sustained-release preparation of the protein kinase inhibitor fasudil. Experimental cerebral vasospasm in rats and dogs was induced by double injection of autologous arterial blood into the cisterna magna. Focal cerebral ischemia in rats was induced by middle cerebral artery occlusion using an intraluminal suture technique. A single intrathecal injection of liposomal fasudil can maintain a therapeutic the drug concentration in the CSF due to the sustained-release property of liposomes, significantly decreasing intact size of acute ischemia and decreasing vasoconstriction of the basilar artery in cerebral vasospasm. This novel approach for the treatment of cerebral vasospasm and ischemia may have significant potential for use in the clinical setting.

Plasma and cerebrospinal fluid pharmacokinetics of SU5416 after intravenous administration in nonhuman primates.

PURPOSE: SU5416 is a small, lipophilic synthetic molecule that selectively inhibits the tyrosine kinase activity of the VEGF receptor Flk-1/KDR. The role of this agent in brain tumors is currently being investigated. Pharmacokinetic studies of SU5416 have been performed in humans; however, there have been no studies of its penetration in the cerebrospinal fluid (CSF). We studied the pharmacokinetics of SU5416 in plasma and CSF after intravenous (i.v.) administration using a nonhuman primate model that is highly predictive of the CSF penetration in humans. EXPERIMENTAL DESIGN: SU5416 (85 mg/m(2), about 3.8 mg/kg) was administered i.v. over 20 min to four nonhuman primates. Serial plasma and CSF samples were obtained prior to, during, and after completion of the infusion for determination of SU5416 concentrations. SU5416 was measured in plasma and CSF using high-performance liquid chromatography (HPLC). Concentration-versus-time data were modeled using model-independent and model-dependent methods. RESULTS: Peak plasma concentrations ranged from 6.3 to 14.5 microM and the mean plasma AUC was 620+/-180 microM.min. Disappearance of SU5416 from the plasma was best described by a one-compartment model with a half-life of 39+/-2.9 min. The volume of distribution was 36+/-11 l/m(2) and the clearance was 0.62+/-0.2 l/min per m(2). SU4516 was not quantifiable in the CSF. CONCLUSIONS: There is minimal penetration of SU5416 into the CSF after i.v. administration. The very low CNS exposure to SU5416 after i.v. dosing suggests that this agent is not optimal for the treatment of leptomeningeal tumors.