Varghese John

Brain and plasma pharmacokinetics and anticancer activities of cyclophosphamide and phosphoramide mustard in the rat

SummaryBy a sensitive and quantitative fluorometric assay, brain and plasma time-dependent concentration profiles were generated for phosphoramide mustard (PM) and active alkylating metabolites derived from cyclophosphamide (CPA) administration to rats. Whereas PM rapidly disappeared from plasma, with a monophasic half-life of 15.1 min, equimolar administration of CPA generated active metabolites in plasma that disappeared monoexponentially, with a composite half-life of 63 min. As a consequence, the time-dependent concentration integral of active alkylating metabolites derived from CPA administration, calculated between 5 min and infinity, was 3-fold that of PM. Pharmacokinetic parameters were calculated for each compound. The brain/plasma concentration-integral ratios of PM and active alkylating metabolites derived from CPA were 0.18 and 0.20, respectively. The cerebrovascular permeability-surface area product of PM was 7.5×10−5s−1, which is similar to that of other watersoluble anticancer agents that are restricted from entering the brain. The activities of a range of daily doses of PM and CPA were assessed against subcutaneous and intracerebral implants of Walker 256 carcinosarcoma tumor in rats. Inhibition of subcutaneous tumor growth by 50% was caused by CPA and PM doses of 6.6 and 12.0 mg/kg (daily for 5 consecutive days, starting 36 h after tumor implantation), respectively. However, administration of daily doses of up to 40 mg/kg did not significantly increase the survival of animals with intracerebral tumor implants. These studies indicate that active metabolites of CPA are restricted from entering the brain and that only subtherapeutic concentrations are achieved in brain tissue after systemic administration of CPA or PM.

Neuroprotective Sirtuin ratio reversed by ApoE4

Significance This manuscript links ApoE4-mediated signaling with Sirtuin function. Specifically, we show that ApoE4, but not ApoE3, reduces neuroprotective SirT1 levels. Our data support the hypothesis that neuronal connectivity, as reflected in the ratios of critical mediators such as sAPPα:Aβ, SirT1:SirT2, APP:p-APP, and Tau:p-Tau, is programmatically altered by ApoE4. Thus ApoE4, SirT1/2, p-Tau, and p-APP, all may be part of a signaling network that is affected in AD, providing a model for therapeutic candidate screening in AD drug discovery. These findings offer a unique insight into the mechanism by which ApoE4 confers risk for the development of Alzheimer’s disease. The canonical pathogenesis of Alzheimer’s disease links the expression of apolipoprotein E ε4 allele (ApoE) to amyloid precursor protein (APP) processing and Aβ peptide accumulation by a set of mechanisms that is incompletely defined. The development of a simple system that focuses not on a single variable but on multiple factors and pathways would be valuable both for dissecting the underlying mechanisms and for identifying candidate therapeutics. Here we show that, although both ApoE3 and ApoE4 associate with APP with nanomolar affinities, only ApoE4 significantly (i) reduces the ratio of soluble amyloid precursor protein alpha (sAPPα) to Aβ; (ii) reduces Sirtuin T1 (SirT1) expression, resulting in markedly differing ratios of neuroprotective SirT1 to neurotoxic SirT2; (iii) triggers Tau phosphorylation and APP phosphorylation; and (iv) induces programmed cell death. We describe a subset of drug candidates that interferes with the APP–ApoE interaction and returns the parameters noted above to normal. Our data support the hypothesis that neuronal connectivity, as reflected in the ratios of critical mediators such as sAPPα:Aβ, SirT1:SirT2, APP:phosphorylated (p)-APP, and Tau:p-Tau, is programmatically altered by ApoE4 and offer a simple system for the identification of program mediators and therapeutic candidates.

Posiphen as a candidate drug to lower CSF amyloid precursor protein, amyloid-β peptide and τ levels: target engagement, tolerability and pharmacokinetics in humans

Aim A first in human study to evaluate tolerability and pharmacokinetics followed by an early proof of mechanism (POM) study to determine whether the small orally, available molecule, Posiphen tartrate (Posiphen), lowers secreted (s) amyloid-β precursor protein (APP) α and -β, amyloid-β peptide (Aβ), tau (τ) and inflammatory markers in CSF of patients with mild cognitive impairment (MCI). Study design Posiphen single and multiple ascending dose phase 1 randomised, double blind, placebo-controlled safety, tolerance, pharmacokinetic studies were undertaken in a total of 120 healthy volunteers to define a dose that was then used in a small non-randomised study of five MCI subjects, used as their own controls, to define target engagement. Main outcome measures Pharmacodynamic: sAPPα, sAPPβ, Aβ42, τ (total (t) and phosphorylated (p)) and inflammatory marker levels were time-dependently measured over 12 h and compared prior to and following 10 days of oral Posiphen treatment in four MCI subjects who completed the study. Pharmacokinetic: plasma and CSF drug and primary metabolite concentrations with estimated brain levels extrapolated from steady-state drug administration in rats. Results Posiphen proved well tolerated and significantly lowered CSF levels of sAPPα, sAPPβ, t-τ, p-τ and specific inflammatory markers, and demonstrated a trend to lower CSF Aβ42. Conclusions These results confirm preclinical POM studies, demonstrate that pharmacologically relevant drug/metabolite levels reach brain and support the continued clinical optimisation and evaluation of Posiphen for MCI and Alzheimers disease.

Ayurvedic medicinal plants for Alzheimer's disease: a review

Alzheimers disease is an age-associated, irreversible, progressive neurodegenerative disease that is characterized by severe memory loss, unusual behavior, personality changes, and a decline in cognitive function. No cure for Alzheimers exists, and the drugs currently available to treat the disease have limited effectiveness. It is believed that therapeutic intervention that could postpone the onset or progression of Alzheimers disease would dramatically reduce the number of cases in the next 50 years. Ayurvedic medicinal plants have been the single most productive source of leads for the development of drugs, and over a hundred new products are already in clinical development. Indeed, several scientific studies have described the use of various Ayurvedic medicinal plants and their constituents for treatment of Alzheimers disease. Although the exact mechanism of their action is still not clear, phytochemical studies of the different parts of the plants have shown the presence of many valuable compounds, such as lignans, flavonoids, tannins, polyphenols, triterpenes, sterols, and alkaloids, that show a wide spectrum of pharmacological activities, including anti-inflammatory, anti-amyloidogenic, anti-cholinesterase, hypolipidemic, and antioxidant effects. This review gathers research on various medicinal plants that have shown promise in reversing the Alzheimers disease pathology. The report summarizes information concerning the phytochemistry, biological, and cellular activities and clinical applications of these various plants in order to provide sufficient baseline information that could be used in drug discovery campaigns and development process, thereby providing new functional leads for Alzheimers disease.

Recent advances in the understanding of the processing of APP to beta amyloid peptide.

Numerous lines of evidence suggest that the 42 amino-acid long form of beta amyloid peptide (Aβ42) plays a key role in the pathogenesis of Alzheimer’s disease (AD). This evidence includes the observations that multiple missense mutations in the amyloid precursor protein (APP), presenilin-1 (PS-1) or presenilin-2 (PS-2) all result in the overproduction of Aβ42. Aβ is produced from the APP through sequential proteolytic processing events, carried out by two independent enzyme activities termed betaand gamma-secretase.3 Once the peptide is produced it can, under some conditions, go on to form amyloid deposits (senile plaques) in the brain parenchyma, which are a hallmark of AD. Efforts to block the production of Aβ have been pursued over the past several years, towards the goal of therapeutic treatment for AD. This effort has resulted in compounds that are effective in inhibiting gamma secretase-like cleavage of APP into Aβ4,5 and, very recently, have resulted in the independent identification of beta-secretase by several groups including our own.6–8 The identification of this novel, membrane bound aspartyl proteinase has increased therapeutic opportunities for Alzheimer’s disease aimed at the inhibition of Aβ peptide production.

Development of lipophilic anticancer agents for the treatment of brain tumors by the esterification of water-soluble chlorambucil.

The lipophilic derivatives of the anticancer alkylating agent chlorambucil, chlorambucil-methyl, -isopropyl and -tertiary butyl esters, were synthesized and administered i.v. to anesthetized rats. Plasma and brain concentrations of these agents and of their active metabolites, chlorambucil and phenylacetic mustard, then were determined by high-performance liquid chromatography between 5 and 60 min. Whereas large amounts of chlorambucil-tertiary butyl ester entered and were maintained in brain, lower amounts of chlorambucil-isopropyl ester and no chlorambucil-methyl ester were found in brain. The comparative brain/plasma concentration-time integral ratios of the total active agents generated from chlorambucil-tertiary butyl, -isopropyl and -methyl esters were 0.85, 0.12 and 0.06, respectively, compared to a ratio of 0.02 for chlorambucil. In vitro alkylating activity of each ester was compared to that of equimolar chlorambucil, by reaction with 4-(p-nitrobenzyl)pyridine. Each ester possessed high intrinsic alkylating activity, equal to 38.4, 57.0 and 69.9% of chlorambucil activity, for the -tertiary butyl, -isopropyl and -methyl esters, respectively. Therefore each is an active antineoplastic agent irrespective of whether or not chlorambucil is regenerated. The rates of ester hydrolysis of these derivatives to chlorambucil were measured in fresh rat blood and in liver and brain homogenates at 37°C. Chlorambucil-methyl and -isopropyl esters were hydrolysed quickly within 30 s in blood and liver, whereas chlorambucil-tertiary butyl ester was more stable with half-lives of approximately 7 h and 2 h, respectively. All proved to be relatively stable in brain homogenate. Steric hindrance around the ester linkage of chlorambucil-tertiary butyl ester reduces its affinity to and rate of hydrolysis by plasma and liver esterases, and allows it to accumulate within the brain. Chlorambucil-tertiary butyl ester maintains high levels in brain despite rapidly declining plasma concentrations and, due to these favorable pharmacokinetics and to its intrinsic anticancer activity, it possesses promising characteristics for the treatment of malignant brain tumors.

Hunter-killer peptide (HKP) for targeted therapy.

Hunter-killer peptides (HKPs) are short chimeric molecules (∼20 amino acids) consisting of two functional domains: a “targeting” domain (5-10 amino acids) that facilitates receptormediated binding and internalization into the cytosol of targeted cells and a proapoptotic domain (∼14 amino acids) designed to be nontoxic outside cells but toxic when internalized into targeted cells by the disruption of mitochondrial membranes. HKPs have shown promise as therapeutic agents in animal models of several human diseases. HKPs such as HKP-1, with the amino acid sequence NH2-c(CNGRC)-GG-D(KLAKLAK)2COOH (1) (which includes a cyclic disulfide bridge between the cysteine residues), are targeted to the angiogenic vasculature of tumors and have strong anticancer activity in models of breast and prostate cancer, reducing tumor volume and metastasis and prolonging survival. HKPs targeted to normal prostate vasculature reduce the size of the prostate gland and have a strong anticancer effect in a transgenic adenocarcinoma of the mouse prostate model (TRAMP). HKPs targeted to the synovial vasculature have strong anti-inflammatory effects in a mouse model of collagen-induced arthritis. Finally, HKPs targeted to the normal blood vessels that feed fat deposits decrease obesity in a transgenic mouse model of obesity. A new generation of HKPs currently under development holds the promise of bringing this class of therapeutic agents from the bench to the clinic.

Importance of the caspase cleavage site in amyloid-β protein precursor.

Reports from multiple laboratories have now been published analyzing the critical nature of the caspase cleavage site of amyloid-β protein precursor (AβPP) for cell death induction, synaptic loss, hippocampal atrophy, long-term potentiation, memory loss, neophobia, and other aspects of the Alzheimers phenotype. Here we review the results and implications of these studies for the understanding of Alzheimers disease pathophysiology and the potential development of therapeutics that target this site in AβPP.

Chapter 21. Alzheimer's Disease: Current Therapeutic Approaches

Publisher Summary This chapter discusses the status of the active Alzheimers disease (AD) therapeutic approaches as well as some areas that are only beginning to see medicinal chemistry activity. Though the exact pathogenic mechanisms of AD remain elusive, it is known that many neurotransmitter systems are dysfunctional, neuronal calcium homeostasis is upset, processes are in motion that produce senile plaque and neurofibrillary tangles, and there is an inflammatory response associated with the disease. The lack of any proven biochemical diagnostic method for AD makes selection of the therapeutic targets difficult. The cholinergic hypothesis of AD is based on the reported reduction of cholinergic markers, such as acetylcholinesterase (AChE) and choline acetyltransferase (ChAT). According to this hypothesis medial forebrain, cholinergic neurons undergo progressive retrograde degeneration accounting for some of the cognitive impairments in patients with AD. Clinical studies on the AChE inhibitor tacrine (THA) in a small group of patients, with moderate to severe AD that showed improvements in cognitive function, resulted in intensified efforts to develop novel acetylcholine esterase inhibitors. Though subsequent clinical studies have produced mixed results, THA has been recommended by the FDA advisory panel for the approval for AD treatment. Cholinerrgic agonists, acting directly on muscarinic receptors, may improve the cholinergic dysfunction seen in AD in which the basal forebrain muscarinic neurons that predominantly express the presynaptic M2 receptors have been found to atrophy. The formation and the biological properties of amyloid plaque is an area of major interest (80) though considerable controversy surrounds the role of amyloid plaque in AD associated neuro-degeneration. If amyloid derived senile plaque is a primary cause of neuronal degeneration, then the inhibition of amyloid formation could provide agents with the potential to slow down or even stop the progression of AD. Senile plaque consists largely of a peptide fragment (An) derived from Alzheimer amyloid precursor protein (APP). Amyloid may be indirectly neurotoxic via the induction of an inflammatory response that could result in toxicity to neurons surrounding the inflamed plaque.

Design and synthesis of hydroxyethylamine (HEA) BACE-1 inhibitors: structure-activity relationship of the aryl region.

The structure-activity relationship of the prime region of hydroxyethylamine BACE inhibitors is described. Variation in the aryl linker region with 5- and 6-membered heterocycles provided compounds such as 33 with improved permeability and reduced P-gp liability compared to benzyl amine analog 1.