Central Nervous System Related Disorders Research Articles

In-depth Mechanism, Challenges, and Opportunities of Delivering Therapeutics in Brain Using Intranasal Route.

Central nervous system-related disorders have become a continuing threat to human life and the current statistic indicates an increasing trend of such disorders worldwide. The primary therapeutic challenge, despite the availability of therapies for these disorders, is to sustain the drug's effective concentration in the brain while limiting its accumulation in non-targeted areas. This is attributed to the presence of the blood-brain barrier and first-pass metabolism which limits the transportation of drugs to the brain irrespective of popular and conventional routes of drug administration. Therefore, there is a demand to practice alternative routes for predictable drug delivery using advanced drug delivery carriers to overcome the said obstacles. Recent research attracted attention to intranasal-to-brain drug delivery for promising targeting therapeutics in the brain. This review emphasizes the mechanisms to deliver therapeutics via different pathways for nose-to-brain drug delivery with recent advancements in delivery and formulation aspects. Concurrently, for the benefit of future studies, the difficulties in administering medications by intranasal pathway have also been highlighted.

Advances in basic research on choline and central nervous system development and related disorders

Choline is an essential nutrient that plays an integral role in all stages of the life cycle, with increasing interest in the relationship between choline and neurodevelopment. Choline is a major component in the synthesis of phospholipids, phosphatidylcholine and sphingolipids, and is an essential nutrient for methyl metabolism, acetylcholine synthesis and cell signaling. Choline plays an important role in neurogenesis and neural migration during fetal development, potentially influencing the development and prognosis of neurological disorders, but its mechanism of action is not yet clear. This article reviews the source and metabolism of choline, the effects and mechanism of choline on neurodevelopment and central nervous system related disorders.

Potential of particle size less than 15 nm via olfactory region for direct brain delivery via intranasal route

Central nervous system-related disorders are very difficult to manage with conventional therapeutics because of the selective and herculean nature of the blood-brain barrier which does not allow easy penetration of drug molecules into the brain. As a result, the efficacy of administered molecule is reduced manifold times, and difficult to ascertain the condition of the patient. An alternative route like the nose to the brain is subjectively being explored to enhance the brain's delivery. Recently various published testimonies have proved the nose to the brain can be an alternative route of administration for effective delivery to the brain. This review majorly highlights the hurdles in the delivery of the nasal route and explores the new areas available for delivery via the concept of particle size below 15 nm, which might penetrate the olfactory bulb via the paracellular spaces present in the olfactory epithelium, which starts from the brain and terminates in the nose. The success of this review might reveal new insights into drug delivery for brain targeting and the necessary delivery mechanism.

Prolonging effects of Valeriana fauriei root extract on pentobarbital-induced sleep in caffeine-induced insomnia model mice and the pharmacokinetics of its active ingredients under conditions of glycerol fatty acid ester as emulsifiers

Ethnopharmacological relevanceValeriana plant roots have traditionally been used to treat central nervous system-related disorders in European countries. Among this genus, the Japanese Pharmacopoeia registers the dried roots of V. fauriei Briq. (VF). However, insufficient pharmacological data are available for this species. Aim of the studyWe investigated the sedative effects of VF extract in a murine caffeine-induced insomnia model as well as the active ingredients and their pharmacokinetics to determine its basic pharmacological action mechanisms under conditions glycerol fatty acid ester is used as emulsifiers. Materials and methodsA murine insomnia model was created by caffeine. Samples derived from the ethanol extract of VF were administered per oral (p.o.), and caffeine was injected intraperitoneally (i.p.). Pentobarbital was injected i.p. and the sleep latency and duration were measured. To confirm the mechanism of action of VF, flumazenil, a specific γ-aminobutyric acid receptor type A (GABAA receptor) antagonist, was administered (i.p.) immediately prior to the sample administration. We examined the pharmacokinetic profiles of the active ingredients in the plasma, brain, urine, and feces of mice after the administration (p.o and intravenous (i.v.)) of VF samples. ResultsVF extract (5 g as VF/kg, p.o.) significantly shorten sleep latency and prolonged pentobarbital-induced sleep in caffeine-induced insomnia mice, partially mediated via the GABAergic nervous system, although a higher dose (10 g as VF/kg, p.o.) was required to exhibit the significant effects in normal mice. Kessyl glycol diacetate (KGD), the main constitutive compound in VF, did not shorten sleep latency but exhibited the same sleep prolonged effect at a dose related to VF extract. The concentration of kessyl glycol 8-acetate (KG8) in the plasma was higher than that of KGD in mice treated (p.o.) with VF extract. The profiles of brain concentrations of KGD and KG8 were similar to those in the plasma, and approximately 20% of those in the plasma were distributed throughout the brain. The excretions of KGD and KG8 in urine and feces was slightly detected, and an unknown large peak related to KG8 was detected in the urine of mice administered with VF extract by HPLC-MS/MS analysis. ConclusionsVF exhibits more sedative effects under stressed conditions, such as insomnia, and the major active ingredients are KGD and its metabolite KG8, which are distributed from the blood circulation into the brain by simple diffusion. KG8 is further metabolized into other metabolites that are easily excreted in the urine.

Development of indole-2-carbonyl piperazine urea derivatives as selective FAAH inhibitors for efficient treatment of depression and pain

Fatty acid amide hydrolase (FAAH), aserinehydrolase with significant role in thehydrolysis of endocannabinoids, is a promising therapeutic target for peripheral and central nervous system related disorders, including pain, neuroinflammation and depression. Employing a structure-based approach, a novel series of indole-2-carbonyl piperazine urea derivatives were designed and synthesized as FAAH inhibitors for the treatment of pain-depression comorbidity. Among them, compound 4i emerged as the most potent inhibitor (IC50 = 0.12 μM) with fine selectivity versus CES2, ABHD6, MAGL and the cannabinoid receptor, which also displayed superior metabolic stability in human liver microsome and an adequate pharmacokinetic profile in rodents. Treatment of depressed rats with 4i demonstrated favorable antidepressant-like effects not only by increasing the level of BDNF in the hippocampus but also by restraining the apoptosis of hippocampal neurons. Also, 4i effectively suppressed the LPS-induced neuroinflammation in vitro. Moreover, 4i exhibited potent analgesic activity, which indicated its promising therapeutical application for pain and depression. These meaningful results shed light on FAAH inhibitors as promising pain-depression comorbidity therapeutics.

Pretreatment with Carpolobia lutea ethanol extract prevents schizophrenia-like behavior in mice models of psychosis

Ethnopharmacological relevanceCarpolobia lutea decoction is widely used as a phytotherapeutic against central nervous system-related disorders including insomnia, migraine headache, and mental illness in West and Central Tropical Africa. AimThis study was designed to investigate the antipsychotic activity of Carpolobia lutea (EECL) in mice models of psychosis. MethodsMale Swiss mice (n = 5/group) were given EECL (100, 200, 400, and 800 mg/kg), haloperidol (1 mg/kg), clozapine (5 mg/kg) and vehicle (10 mL/kg) orally before amphetamine (5 mg/kg)-induced hyperlocomotion and stereotypy, apomorphine (2 mg/kg)-induced stereotypy, or ketamine (10, 30, and 100 mg/kg)-induced hyperlocomotion, enhancement of immobility and cognitive impairment. ResultsEECL (200, 400, and 800 mg/kg) prevented amphetamine- and apomorphine-induced stereotypies, as well as reduced hyperlocomotion induced by amphetamine and ketamine, all of which are predictors of positive symptoms. Regardless of the dose administered, EECL prevented the index of negative symptoms induced by ketamine. Furthermore, higher doses of EECL (400 and 800 mg/kg) also prevented ketamine-induced cognitive impairment, a behavioral phenotype of cognitive symptoms. ConclusionPretreatment with EECL demonstrated antipsychotic activity in mice, preventing amphetamine-, apomorphine-, and ketamine-induced schizophrenia-like symptoms, with 800 mg/kg being the most effective dose.

DIAGNOSTIC ROLE OF EXOSOMES IN VARIOUS DISEASES

Exosomes are lipid bilayer- enclosed membrane bound extracellular vesicles (EVs) that secreted by cells under physiological conditions called extracellular vesicles (EV). EVs participate in are natural carrier which can transfer DNAs, RNAs, proteins, and lipids in autocrine, paracrine and endocrine manner. Exosome cargo having cell cycle related mRNA from cancer promotes proliferation of vascular endothelial cells and exosome play a significant role in angiogenesis by vascular endothelial cells. Exosome cargos showed significant role in pathogenesis of central nervous system related disease and exosomal content can act as biomarkers for specific central nervous system related disorders. It has been observed that viral infected cell release exosome more frequently as compared to non- infected cells; which might be act as indicator of viral infection. It was found that exosomes act as extracellular communicator in ischemic signaling and myocardial repair and several studies evidently prove that in adult heart, exosome secreted by myocardial tissue involved in hetero-cellular communication. It has been suggested that exosomes could play an important role in prognosis of various disease like cancer, disease related to nervous system, infections disease and cardiovascular disease. Exosomes secretion plays important role in disease pathogenesis and may be serve as an important biomedical tool as a biomarker in early detection of various disease.

A Mechanistic approach of Peroxisome Proliferator-Activated Receptors and its subtypes on Clinical and preclinical model of Neurodegenerative disorders

Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors, belonging to the nuclear receptor family, which has high expression of three structurally homologous PPARs isotypes (PPARα, PPARβ/δ, and PPARγ) in brain. Several studies have discovered role of PPARs in oxidative stress, mitochondrial dysfunction, neuroinflammation and production of the toxic proteins in various neurodegenerative disorders such as Parkinson disease, Alzheimer’s disease, Huntington disease, Amyotrophic Lateral Sclerosis, Multiple sclerosis etc. Currently available drugs provide symptomatic relief, but disease progression cannot be stopped, because of their unclear molecular approach. The ability of PPAR to modulate the pathways involved in these conditions paved a path for future studies. Due to increasing challenges to treat central nervous system related disorders, hence PPARs have attracted much attention nowadays. In this review, we discussed various mechanisms of PPARs subtypes in neurodegenerative disorders. We congregate the molecular evidences which support PPARs as a therapeutic target to treat neurodegenerative disorders from preclinical and clinical studies and provide a basis for the potential therapeutic use of PPAR ligands in human diseases.

Recent Advances in the Endogenous Brain Renin-Angiotensin System and Drugs Acting on It.

The RAS (renin-angiotensin system) is the part of the endocrine system that plays a prime role in the control of essential hypertension. Since the discovery of brain RAS in the seventies, continuous efforts have been put by the scientific committee to explore it more. The brain has shown the presence of various components of brain RAS such as angiotensinogen (AGT), converting enzymes, angiotensin (Ang), and specific receptors (ATR). AGT acts as the precursor molecule for Ang peptides—I, II, III, and IV—while the enzymes such as prorenin, ACE, and aminopeptidases A and N synthesize it. AT1, AT2, AT4, and mitochondrial assembly receptor (MasR) are found to be plentiful in the brain. The brain RAS system exhibits pleiotropic properties such as neuroprotection and cognition along with regulation of blood pressure, CVS homeostasis, thirst and salt appetite, stress, depression, alcohol addiction, and pain modulation. The molecules acting through RAS predominantly ARBs and ACEI are found to be effective in various ongoing and completed clinical trials related to cognition, memory, Alzheimer's disease (AD), and pain. The review summarizes the recent advances in the brain RAS system highlighting its significance in pathophysiology and treatment of the central nervous system-related disorders.

English

Neuropathic pain is the consequence of abnormal processing in the peripheral or central nervous system (CNS) elicited by neuronal injury. Due to its heterogeneous nature and important unwanted adverse effects of the commonly prescribed psychoactive drugs like benzodiazepines (BDZ), the treatment of neuropathic pain has remained a challenge for the scientific community. Flavonoids initially isolated from plants and used as tranquilizers in Folkloric medicine, have been reported to possess selective affinity for BDZ binding site. These positive ionotropic modulators of γ-amino butyric acid-A (GABAA) receptors enhance the chloride ion flux and provide a strong inhibitory effect. Therefore, for the treatment of central nervous system-related disorders such as neuropathic pain, these selective GABAA receptor modulators stand amongst the strongest candidates. This review provides an update on research development that has confirmed the activity of different flavonoids on GABAA receptors. Key words: Neuropathic pain, flavonoid, γ-amino butyric acid-A (GABAA) receptors, animal models neuropathy.

Feasibility and Utility of Tele-Neurorehabilitation Service in India: Experience from a Quaternary Center.

Background:Neurological rehabilitation service in developing countries like India is a great challenge in view of limited resources and manpower. Currently, neurological rehabilitation with a multidisciplinary team is limited to a few major cities in the country. Tele-neurorehabilitation (TNR) is considered as an alternative and innovative approach in health care. It connects the needy patients with the health-care providers with minimum inconvenience and yields cost-effective health care.Aim:The aim of this study was to study the socioclinical parameters, feasibility, and utility of TNR services in India.Methodology:A retrospective file review of TNR consultations provided through Telemedicine Center at a quaternary hospital-based research center in south India between August 2012 and January 2016.Results:A total of 37 consultations were provided to the patients belonging to four districts of Karnataka. The mean age of the patients was 34.7 (±19.5) years, 23 (62.1%) were aged between 19 and 60 years, and 31 (83.8%) were male. Thirty-one patients (83.8%) had central nervous system-related disorders such as stroke, cerebral palsy, and tubercular meningitis with sequelae or neuromuscular disorders such as Guillain–Barre Syndrome and Duchenne muscular dystrophy. Twelve patients (32.4%) were advised to consult higher centers in the vicinity, and the rest was referred to the district hospital.Conclusion:The findings suggest that TNR services are feasible, effective, and less resource intensive in delivering quality telemedicine care in India. More clinical studies are required to elucidate its full utility at different levels and in different parts of the country.

Symptoms of menopause - global prevalence, physiology and implications.

The symptoms of menopause can be distressing, particularly as they occur at a time when women have important roles in society, within the family and at the workplace. Hormonal changes that begin during the menopausal transition affect many biological systems. Accordingly, the signs and symptoms of menopause include central nervous system-related disorders; metabolic, weight, cardiovascular and musculoskeletal changes; urogenital and skin atrophy; and sexual dysfunction. The physiological basis of these manifestations is emerging as complex and related, but not limited to, oestrogen deprivation. Findings generated mainly from longitudinal population studies have shown that ethnic, geographical and individual factors affect symptom prevalence and severity. Moreover, and of great importance to clinical practice, the latest research has highlighted how certain menopausal symptoms can be associated with the onset of other disorders and might therefore serve as predictors of future health risks in postmenopausal women. The goal of this Review is to describe in a timely manner new research findings on the global prevalence and physiology of menopausal symptoms and their impact on future health.

Perinatal Outcomes After Treatment With ADHD Medication During Pregnancy.

To analyze perinatal outcomes after maternal use of attention-deficit/hyperactivity disorder (ADHD) medication during pregnancy. The study included singletons born between 2006 and 2014 in Sweden. Data on prescription drug use, pregnancies, deliveries, and the newborn infants' health were obtained from the Swedish Medical Birth Register, the Prescribed Drug Register, and the Swedish Neonatal Quality Register. We compared infants exposed to ADHD medication during pregnancy with infants whose mothers never used these drugs and infants whose mothers used ADHD medication before or after pregnancy. Analyses were performed with logistic regression. Among 964 734 infants, 1591 (0.2%) were exposed to ADHD medication during pregnancy and 9475 (1.0%) had mothers treated before or after pregnancy. Exposure during pregnancy increased the risk for admission to a NICU compared with both no use and use before or after pregnancy (adjusted odds ratio [aOR], 1.5; 95% confidence interval [CI], 1.3-1.7; and aOR, 1.2; 95% CI, 1.1-1.4, respectively). Infants exposed during pregnancy had more often central nervous system-related disorders (aOR, 1.9; 95% CI, 1.1-3.1) and were more often moderately preterm (aOR, 1.3; 95% CI, 1.1-1.6) than nonexposed infants. There was no increased risk for congenital malformations or perinatal death. Treatment with ADHD medication during pregnancy was associated with a higher risk for neonatal morbidity, especially central nervous system-related disorders such as seizures. Because of large differences in background characteristics between treated women and controls, it is uncertain to what extent this can be explained by the ADHD medication per se.

Sex and Gender Differences in Central Nervous System-Related Disorders.

There are important sex differences in the brain that seem to arise from biology as well as psychosocial influences. Sex differences in several aspects of human behavior and cognition have been reported. Gonadal sex steroids or genes found on sex chromosomes influence sex differences in neuroanatomy, neurochemistry and neuronal structure, and connectivity. There has been some resistance to accept that sex differences in the human brain exist and have biological relevance; however, a few years ago, it has been recommended by the USA National Institute of Mental Health to incorporate sex as a variable in experimental and clinical neurological and psychiatric studies. We here review the clinical literature on sex differences in pain and neurological and psychiatric diseases, with the aim to further stimulate interest in sexual dimorphisms in the brain and brain diseases, possibly encouraging more research in the field of the implications of sex differences for treating these conditions.

Quorum Sensing Peptides Selectively Penetrate the Blood-Brain Barrier.

Bacteria communicate with each other by the use of signaling molecules, a process called ‘quorum sensing’. One group of quorum sensing molecules includes the oligopeptides, which are mainly produced by Gram-positive bacteria. Recently, these quorum sensing peptides were found to biologically influence mammalian cells, promoting i.a. metastasis of cancer cells. Moreover, it was found that bacteria can influence different central nervous system related disorders as well, e.g. anxiety, depression and autism. Research currently focuses on the role of bacterial metabolites in this bacteria-brain interaction, with the role of the quorum sensing peptides not yet known. Here, three chemically diverse quorum sensing peptides were investigated for their brain influx (multiple time regression technique) and efflux properties in an in vivo mouse model (ICR-CD-1) to determine blood-brain transfer properties: PhrCACET1 demonstrated comparatively a very high initial influx into the mouse brain (Kin = 20.87 μl/(g×min)), while brain penetrabilities of BIP-2 and PhrANTH2 were found to be low (Kin = 2.68 μl/(g×min)) and very low (Kin = 0.18 μl/(g×min)), respectively. All three quorum sensing peptides were metabolically stable in plasma (in vitro) during the experimental time frame and no significant brain efflux was observed. Initial tissue distribution data showed remarkably high liver accumulation of BIP-2 as well. Our results thus support the potential role of some quorum sensing peptides in different neurological disorders, thereby enlarging our knowledge about the microbiome-brain axis.

Studies on Pharmacokinetic Drug Interaction Potential of Vinpocetine.

BackgroundVinpocetine, a semi-synthetic derivative of vincamine, is a popular dietary supplement used for the treatment of several central nervous system related disorders. Despite its wide use, no pharmacokinetic drug interaction studies are reported in the literature. Due to increasing use of dietary supplements in combination with conventional drugs, the risk of adverse effects is on the rise. As a preliminary step to predict a possibility of drug interaction during concomitant use of vinpocetine and conventional drugs, this study was carried out to evaluate the effects of vinpocetine on three main regulators of pharmacokinetic drug interactions namely, cytochromes P450 (CYPs), P-glycoprotein (P-gp), and Pregnane X receptor (PXR).MethodsInhibition of CYPs was evaluated by employing recombinant enzymes. The inhibition of P-gp was determined by calcein-AM uptake method in transfected and wild type MDCKII cells. Modulation of PXR activity was monitored through a reporter gene assay in HepG2 cells.ResultsVinpocetine showed a strong inhibition of P-gp (EC50 8 μM) and a moderate inhibition of recombinant CYP3A4 and CYP2D6 (IC50 2.8 and 6.5 μM) with no activity towards CYP2C9, CYP2C19 and CYP1A2 enzymes. In HLM, competitive inhibition of CYP3A4 (IC50 54 and Ki 19 μM) and non-competitive inhibition of CYP2D6 (IC50 19 and Ki 26 μM) was observed. Activation of PXR was observed only at the highest tested concentration of vinpocetine (30 μM) while lower doses were ineffective.ConclusionStrong inhibition of P-gp by vinpocetine is indicative of a possibility of drug interactions by altering the pharmacokinetics of drugs, which are the substrates of P-gp. However, the effects on CYPs and PXR indicate that vinpocetine may not affect CYP-mediated metabolism of drugs, as the inhibitory concentrations are much greater than the expected plasma concentrations in humans.

Structure-Based Design of a Potent, Selective, and Brain Penetrating PDE2 Inhibitor with Demonstrated Target Engagement.

Structure-guided design led to the identification of the novel, potent, and selective phosphodiesterase 2 (PDE2) inhibitor 12. Compound 12 demonstrated a >210-fold selectivity versus PDE10 and PDE11 and was inactive against all other PDE family members up to 10 μM. In vivo evaluation of 12 provided evidence that it is able to engage the target and to increase cGMP levels in relevant brain regions. Hence, 12 is a valuable tool compound for the better understanding of the role of PDE2 in cognitive impairment and other central nervous system related disorders.

Investigation of key interactions between the second extracellular loop of dopamine D2 receptor and several hydroxy-N-{[2-(4-phenyl-piperaziny-1-yl)ethyl]phenyl}-nicotinamides

Dopaminergic receptor system has been in focus for the development of new pharmacotherapeutic agents, targeting number of central nervous system related disorders, like drug addiction, schizophrenia, depression, and Parkinson's disease, just to name a few. So far crystal structure for human D2 receptor is not known, despite it?s vital function and importance as therapeutic target. Here we present, recent progress in determination of the key receptor-ligand interactions for available arylpiperazine like ligands, using D2 receptor model, based upon crystal D3 receptor structure. To determine key interactions responsible for high dopaminergic activity, we used computer docking analysis together with experimental data. Total of 4 dopaminergic ligands showing moderate to high affinity, were tested and obtained results rationalized using ligand structures docked into proposed D2 receptor model.

Effects of Telfairia occidentalis (Fluted Pumpkin; Cucurbitaceae) in Mouse Models of Convulsion, Muscle Relaxation, and Depression

Telfairia occidentalis (Cucurbitaceae) is a leafy vegetable used in soup and folk medicine in southern Nigeria. Ethnobotanical survey revealed that preparations of the plant are used in the treatment of central nervous system-related disorders including convulsion. This study was conducted to investigate the effect of the hydroethanolic leaf extract of T. occidentalis in mouse models of convulsion, muscle relaxation, and depression. The strychnine and isoniazid convulsion, traction and climbing muscle relaxation, and forced swim and tail suspension depression tests were used in this study. The extract was administered orally (p.o.) at dose range of 25-800 mg/kg while distilled water (10 mL/kg p.o.) served as negative control. Diazepam (5 mg/kg p.o.) was used as positive control in the convulsion and muscle relaxation models while imipramine (64 mg/kg p.o.) served the same purpose in the depression tests. T. occidentalis significantly increased the onset (P<.001) and reduced the duration of convulsion (P<.05, .01) in the strychnine test and increased the time to death (P<.05, .01, .001) in the isoniazid model. The extract insignificantly increased the reaction time in the traction test while it significantly increased the time in the climbing test (P<.001). In the forced swim and tail suspension models, T. occidentalis significantly (P<.001) and dose-dependently increased the duration of immobility. The results obtained in this study suggest that the hydroethanolic leaf extract of T. occidentalis possesses anticonvulsant and muscle relaxant properties, thus justifying its folkloric use.

High prevalence of multidrug-resistant Pneumococcal molecular epidemiology network clones among Streptococcus pneumoniae isolates from adult patients with community-acquired pneumonia in Japan

A total of 141 Streptococcus pneumoniae isolates from patients with community-acquired pneumonia were collected from May 2003 through October 2004. The strains were tested for antimicrobial agent susceptibility, serotype and genotype by multilocus sequence typing (MLST) and the presence of the pilus rlrA islet. MLST analysis identified 49 sequence types (STs), of which 19 were novel. eBURST analysis using the MLST database (3773 STs) grouped the isolates into 27 clonal complexes and three singletons. A total of 92 (65.2%) isolates were related to ten of the 43 international Pneumococcal Molecular Epidemiology Network (PMEN) clones; major clones found were multidrug-resistant Netherlands3-31 [clonal complex (CC) 180], Taiwan19F-14 (CC271), Taiwan23F-15 (CC242), and Colombia23F-26 (CC138) (the latter new to Asia). We adopted univariate and multiple logistic regression models to identify factors associated with PMEN CCs. Multivariate analysis showed that multidrug resistance (OR 6.3; 95% CI 2.0–22.9), carriage serogroups (OR 7.2; 95% CI 2.5–23.7), prevalence of rlrA (OR 12.6; 95% CI 3.6–59.7) and central nervous system-related disorders (OR 7.7; 95% CI 1.8–48.4) were independently associated with PMEN CCs. Our data indicate that multidrug-resistant PMEN clones are highly prevalent, contributing to the high frequency of resistance to antimicrobial agents in Japan, and suggest that certain predisposing factors in patients contribute to the high frequency of these clones.