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Human serum albumin (HSA) is efficiently taken up by cancer cells as a source of carbon and energy. In this study, we prepared a monomodified derivative of HSA covalently linked to an EDTA derivative and investigated its efficacy to shuttle weakly anti-proliferative EDTA associating ligands such as vanadium, into a cancer cell line. HSA-S-MAL-(CH2)2-NH-CO-EDTA was found to associate both with the vanadium anion (+5) and the vanadium cation (+4) with more than thrice the associating affinity of those ligands toward EDTA. Both conjugates internalized into glioma tumor cell line via caveolae-mediated endocytosis pathway and showed potent anti-proliferative capacities. IC50 values were in the range of 0.2 to 0.3 µM, potentiating the anti-proliferative efficacies of vanadium (+4) and vanadium (+5) twenty to thirty fold, respectively. HSA-EDTA-VO++ in particular is a cancer permeable prodrug conjugate. The associated vanadium (+4) is not released, nor is it active anti-proliferatively prior to its engagement with the cancerous cells. The bound vanadium (+4) dissociates from the conjugate under acidic conditions with half maximal value at pH 5.8. In conclusion, the anti-proliferative activity feature of vanadium can be amplified and directed toward a cancer cell line. This is accomplished using a specially designed HSA-EDTA-shuttling vehicle, enabling vanadium to be anti-proliferatively active at the low micromolar range of concentration.

Tuftsin-PhosphorylCholine (TPC) is a novel bi-specific molecule which links tuftsin and phosphorylcholine. TPC has shown immunomodulatory activities in experimental mouse models of autoimmune diseases. We studied herein the effects of TPC ex vivo on both peripheral blood mononuclear cells (PBMCs) and temporal artery biopsies (TABs) obtained from patients with giant cell arteritis (GCA) and age-matched disease controls. GCA is an immune-mediated disease affecting large vessels. Levels of 18 cytokines in supernatants, PBMC viability, T helper (Th) cell differentiation of PBMCs and gene expression in TABs were analyzed. Treatment ex vivo with TPC decreased the production of IL-1β, IL-2, IL-5, IL-6, IL-9, IL-12(p70), IL-13, IL-17A, IL-18, IL-21, IL-22, IL-23, IFNγ, TNFα, GM-CSF by CD3/CD28 activated PBMCs whereas it negligibly affected cell viability. It reduced Th1 and Th17 differentiation while did not impact Th22 differentiation in PBMCs stimulated by phorbol 12-myristate 13-acetate plus ionomycin. In inflamed TABs, treatment with TPC down-regulated the production of IL-1β, IL-6, IL-13, IL-17A and CD68 gene expression. The effects of TPC were comparable to the effects of dexamethasone, included as the standard of care, with the exception of a greater reduction of IL-2, IL-18, IFNγ in CD3/CD28 activated PBMCs and CD68 gene in inflamed TABs. In conclusion our results warrant further investigations regarding TPC as an immunotherapeutic agent in GCA and potentially other autoimmune and inflammatory diseases.

Summary Treatment with helminthes and helminthes ova improved the clinical symptoms of several autoimmune diseases in patients and in animal models. Phosphorylcholine (PC) proved to be the immunomodulatory molecule. We aimed to decipher the tolerogenic potential of tuftsin–PC (TPC), a novel helminth-based compound in collagen-induced arthritis (CIA) a mouse model of rheumatoid arthritis (RA). CIA DBA/1 mice were treated with TPC subcutaneously (5 µg/0.1 ml) or orally (250 µg/0.1 ml), starting prior to disease induction. The control groups were treated with PBS. Collagen antibodies were tested by enzyme-linked immunosorbent assay (ELISA), cytokine protein levels by ELISA kits and regulatory T (Treg) and regulatory B (Breg) cell phenotypes by fluorescence-activated cell sorter (FACS). TPC-treated mice had a significantly lower arthritis score of 1.5 in comparison with control mice 11.8 (P

Recently, several papers referred to the association of different bacteria with lupus in mice and humans. This is the first report to demonstrate the effect of a compound derived from helminths on the induction of remission in mice with lupus and its association with a bacterial change. We show that several genera, including Akkermansia, are associated with clinical and serological parameters of lupus, while other genera, including butyrate-producing bacteria, are associated with amelioration of disease following tuftsin and phosphorylcholine treatment., The hygiene hypothesis claims that the lack of exposure to microorganisms in developed countries correlates with a rise in the incidence of autoimmune diseases. It was also found that helminths are able to modulate the immune response in hosts in order to survive. Consequently, several successful trials using helminths as a treatment for autoimmune patients have been reported. The helminth derivative, phosphorylcholine (PC), was discovered as an immunomodulatory molecule. We have recently shown in a murine model that when a conjugate of tuftsin and PC, termed TPC, is prophylactically administered before the onset of glomerulonephritis, it attenuates the development of systemic lupus erythematosus (SLE). The current study aimed to examine the TPC effect on the gut microbiome in a mouse model of lupus. TPC treatment altered the gut composition in the mice with active lupus, in correlation with a significant decrease in glomerulonephritis, followed by an increased level of anti-inflammatory interleukin 10 (IL-10), decreased levels of proinflammatory mediators, and expansion of the T regulatory cell population. Importantly, we found that TPC treatment altered the mouse gut microbiome composition, in correlation with a significant decrease in protein secretion and improved disease parameters. The major effects of TPC treatment on the gut microbiome included decreased abundances of Akkermansia and increased abundance of several genera, including Turicibacter, Bifidobacterium, unclassified Mogibacteriaceae, unclassified Clostridiaceae, Adlercreutzia, Allobaculum, and Anaeroplasma. Overall, our results associate microbial changes with the immunomodulation of glomerulonephritis in mice with lupus. IMPORTANCE Recently, several papers referred to the association of different bacteria with lupus in mice and humans. This is the first report to demonstrate the effect of a compound derived from helminths on the induction of remission in mice with lupus and its association with a bacterial change. We show that several genera, including Akkermansia, are associated with clinical and serological parameters of lupus, while other genera, including butyrate-producing bacteria, are associated with amelioration of disease following tuftsin and phosphorylcholine treatment.

A novel small molecule named tuftsin-phosphorylcholine (TPC), which is linked to the biological activity of helminths, was constructed. The current study address the effect of TPC treatment in established collagen-induced arthritis (CIA) mice and propose TPC bi-functional activity. TPC treatment was initiated when clinical score was 2 to 4. Arthritis scores in TPC treated mice were lower compared to mice treated with vehicle (P < 0.001). Joint staining showed normal joint structure in TPC-treated mice compared to control groups treated with phosphate buffered saline (PBS), phosphorylcholine, or tuftsin, which exhibited severely inflamed joints. TPC enhanced anti-inflammatory response due to increased IL-10 secretion, and reduced pro-inflammatory cytokine secretion (IL-1-β, IL-6, TNF-αP < 0.001). Furthermore, TPC therapy increased expansion of CD4+CD25+FOXP3+T regulatory cells and IL-10+CD5+CD1d+B regulatory cells. We propose that the immunomodulatory activity of TPC can be a result of a bi-specific activity of TPC: (a) The tuftsin part of the TPC shifts RAW macrophage cells from pro-inflammatory macrophages M1 to anti-inflammatory M2-secreting IL-10 (P < 0.001) through neuropilin-1 and (b) TPC significantly reduce mouse TLR4 expression via NFkB pathway by HEKTM cells (P < 0.02) via the phosphorylcholine site of the molecule. Our results indicate that TPC, significantly ameliorated established CIA by its immunomodulatory activity. These data could lead to a novel self bi-functional small molecule for treating patients with progressive RA.

In areas where helminths infections are common, autoimmune diseases are rare. Treatment with helminths and ova from helminths, improved clinical findings of inflammatory bowel disease, multiple-sclerosis and rheumatoid-arthritis. The immunomodulatory functions of some helminths were attributed to the phosphorylcholine (PC) moiety. We aimed to decipher the tolerogenic potential of Tuftsin-PC (TPC) compound in mice genetically prone to develop lupus. Lupus prone NZBXW/F1 mice received subcutaneously TPC (5 μg/1 ml), 3 times a week starting at 14 weeks age. Autoantibodies were tested by ELISA, T-regulatory-cells by FACS, cytokines profile by RT-PCR and cytokines protein levels by DuoSet ELISA. Glomerulonephritis was addressed by detection of proteinuria, and immunoglobulin complex deposition in the mesangium of the kidneys of the mice by immunofluorescence. Our results show that TPC attenuated the development of glomerulonephritis in lupus prone mice, in particular, it ameliorated proteinuria (p 0.02), and reduced immunoglobulin deposition in the kidney mesangium. TPC also enhanced the expression of TGFβ and IL-10 (p 0.001), and inhibited the production of IFNγ and IL-17 (p 0.03). TPC Significantly enhanced the expansion of CD4+CD25+FOXP3+ T-regulatory cells (Tregs) phenotype in the treated mice. These data indicate that TPC hampered lupus development in genetically lupus prone mice which was exemplified by moderate glomerulonephritis, attenuation of pro-inflammatory cytokines and enhancement of anti-inflammatory cytokines expression, as well as Tregs expansion. Our results propose harnessing novel natural therapy for lupus patients.

Despite aggressive therapy, existing treatments offer poor prognosis for glioblastoma multiforme patients, in part due to poor penetration of most drugs across the blood–brain barrier (BBB). We propose a minimal-invasive combined treatment approach consisting of local BBB disruption in the tumor in parallel to systemic drug administration. Local BBB disruption is obtained by convection-enhanced delivery of a novel BBB disruption agent, enabling efficient/targeted delivery of the systemically administered drug by the tumors own vasculature. Various human serum albumin (HSA) analogs were synthesized and screened for BBB disruption efficacy in custom in vitro systems. The candidate analogs were then delivered into naïve rat brains by convection-enhanced delivery and screened for maximal BBB disruption and minimal brain toxicity. These studies found a noncationized/neutralized analog, ethylamine (EA)–HSA, to be the optimal BBB-opening agent. Immunocytochemical studies suggested that BBB disruption by EA–HSA may be explained by alterations in occludin expression. Finally, an efficacy study in rats bearing intracranial gliomas was performed. The rats were treated by convection-enhanced delivery of EA–HSA in parallel to systemic administration of Methotrexate, showing significant antineoplastic effects of the combined approached reflected in suppressed tumor growth and significantly (~x3) prolonged survival.

To date, no truly efficacious drugs for Alzheimer's disease (AD) have been developed; moreover, all new anti-AD drugs developed since 2003 have failed. To succeed where previous ones have failed in drug development, new approaches for AD therapy are needed. Here we discuss the potential application of network medicine as a new approach to AD treatment. Unlike traditional approaches focused on a single target/pathway, network medicine targets and restores disease-disrupted networks through simultaneous modulation of numerous proteins (targets)/pathways involved in AD pathogenesis. We consider several drug candidates under development for AD therapy, including Keap1–Nrf2 regulators, endogenous neurogenic agents, and hypoxia-inducible factor 1 (HIF-1) activators. These drug candidates are multi-target ligands with the potential to further develop as network medicines, since they act as master regulators to initiate a broad range of cellular defense mechanisms/cytoprotective genes that exert their efficacy in a holistic way. We also explore their diverse mechanisms of action and potential disease-modifying effects, which may have profound implications for drug discovery.

Improved clinical findings of inflammatory bowel disease (IBD) upon treatment with helminthes and their ova were proven in animal models of IBD and in human clinical studies. The immunomodulatory properties of several helminthes were attributed to the phosphorylcholine (PC) molecule. We assessed the therapeutic potential of tuftsin-PC conjugate (TPC) to attenuate murine colitis. Colitis was induced by Dextransulfate-Sodium-Salt (DSS) in drinking water. TPC was given by daily oral ingestion (50 μg/0.1 ml/mouse or PBS) starting at day −2. Disease activity index (DAI) score was followed daily and histology of the colon was performed by H&E staining. Analysis of the cytokines profile in distal colon lysates was performed by immunoblot. Treatment of DSS induced colitis with TPC prevented the severity of colitis, including a reduction in the DAI score, less shortening of the colon and less inflammatory activity in histology. The immunoblot showed that the colitis preventive activity of TPC was associated with downregulation of colon pro-inflammatory IL-1β, TNFα and IL-17 cytokines expression, and enhancement of anti-inflammatory IL-10 cytokine expression. In the current study, we demonstrated that TPC treatment can prevent significantly experimental colitis induction in naive mice. We propose the TPC as a novel potential small synthetic molecule to treat colitis.

Rheumatoid arthritis (RA) is characterized by chronic autoinflammation of the joints, with a prevalence of about 1% in Western populations. Evidence in recent years has linked RA to changes in the gut microbiota (dysbiosis). Interestingly, helminths have been shown to have therapeutic activity in RA. Specifically, a glycoprotein containing phosphorylcholine (PC) extracted from helminths was found to have immunomodulatory activity. We have previously developed a novel chimeric compound composed of tuftsin-PC (TPC) that attenuates the joint destruction in mice with collagen-induced arthritis (CIA). Here, we address the interrelationship between TPC immunomodulatory activity and the gut microbiota in CIA mice. Preventive therapy with TPC in mice with arthritis maintained a physiological arthritis score as well as a steady gut microbial environment, similar to that of healthy controls, in contrast to CIA mice with severe disease. The microbial composition differed significantly between healthy and phosphate-buffered saline-treated CIA mice, enabling classifying test samples by machine learning based on levels of a small number of bacterial species. Using these bacterial biomarkers, all TPC-treated CIA mice were classified as healthy. Thus, we describe a clear correlation between TPC treatment, healthy gut microbial communities, and prevention of arthritis. This is the first study to demonstrate the immunomodulatory effect of helminth derivatives in autoimmune diseases and the link to gut microbiota.

Background In areas where helminthes infections are common, autoimmune diseases are rare. Treatment with helminthes or their ova, improved clinical findings of several autoimmune diseases. Based on the helminthes biological activity, a novel chimeric be-specific molecule was synthetized of tuftsin-phosphoryl-choline (PC)-TPC. Objectives To study TPC treatment in established collagen induced arthritis (CIA) mice, and the mechanism of activity. Methods Arthritis was induced in DBA male mice by immunization with collagen emulsified in TB-mycobacteria at the tail base. Boost was given 3 weeks later. Treatment with TPC started when the clinical score was 2. Cytokines were measure in culture-fluid of splenocytes in-vitro. T regulatory cells and B regulatory cells were measured by FACS. TPC effect on TLR4 expression was studied using HEKTM-mTLR4 cells system and its inhibitor. M1 shift to M2 was performed in RAW macrophages differentiated to M1 by PMA followed by LPS. TPC was added and IL-6, TNFalpha, IL-10 were tested by ELISA. Results Starting TPC treatment of CIA mice after disease establishment, had a significant lower arthritis score in comparison with control vehicle subjected mice (i.e. TPC-6.8±0.8 vs vehicle-13.8±0.45; p via the phosphorylcholine end. Our data indicated that TPC significantly ameliorated established CIA by anti-inflammatory immunomodulatory activity. Conclusions Our data may lead to a novel bi-specific self small molecule for therapy of patients with advanced RA. Disclosure of Interest None declared

Bleomycin is an anticancer antibiotic effective against a range of human malignancies. Yet its usefulness is limited by serious side effects. In this study, we converted bleomycin into a prodrug by covalently linking 2-sulfo, 9 fluorenylmethoxycarbonyl (FMS) to the primary amino side chain of bleomycin. FMS—bleomycin lost its efficacy to bind transition metal ions and therefore was converted into an inactive derivative. Upon incubation in vitro under physiological conditions, the FMS-moiety undergoes spontaneous hydrolysis, generating native bleomycin possessing full anti-bacterial potency. FMS hydrolysis and reactivation takes place with a t1/2 value of 17 ± 1 h. In silico simulation predicts a narrow therapeutic window in human patients of seven hours, starting 40 min after administration. In mice, close agreement was obtained between the experimental and the simulated pharmacokinetic profiles for FMS-bleomycin. FMS-bleomycin is thus shown to be a classical prodrug: it is inactive at the time of administration and the non-modified (active) bleomycin is released with a desirable pharmacokinetic profile following administration, suggesting it may have therapeutic value in the clinic.

We found that human serum albumin (HSA) contains a single binding domain for derivatives of long-chain fatty acid (LCFA)-like molecules in which the carboxylate is replaced by sulfonate. Accordingly, we have synthesized 16-sulfo-hexadecanoic acid-N-hydroxysuccinimide ester [HO(3)S-(CH(2))(15)-CONHS], an agent that reacts selectively with the amino side chains of peptides and proteins. A macromolecule containing a single 16-sulfohexadecanoate moiety associating with albumin with a K(a) value of 0.83 ± 0.08 × 10(6) M(-1), a sufficient affinity to extend the actions in vivo of such short-lived peptides and proteins. Subcutaneous administration of insulin-NHCO-(CH(2))(15)-SO(3)(-) into mice facilitated a glucose-lowering effect 4.3 times in duration and 6.6 times in area under the curve (AUC) as compared to an in vitro equipotent amount of Zn(2+)-free insulin. Similarly, subcutaneous and intravenous administration of exendin-4-NHCO-(CH(2))(15)-SO(3)(-) to mice yielded prolonged and stable reduction in glucose level, 5-9-fold longer than that of exendin-4. Also, a single subcutaneous administration of human interferon-α2-[NH-CO-(CH(2))(15)-SO(3)(-)](3) to mice yielded circulating antiviral activity over a period of 40 h. In conclusion, a simple, hydrophilic reagent has been engineered, synthesized, and studied. Its linkage to peptides and proteins in a monomodified fashion yielded hydrophilic, prolonged acting derivatives, due to their acquired ability to associate with serum albumin after administration.

Several important pharmacological features can be integrated into injected drugs to enhance their therapeutic efficacy following administration. Short-lived peptide/protein drugs should be converted into long-lived species in vivo to avoid multiple injections. Circulating levels of anticancer agents need to be maintained within a narrow therapeutic range for prolonged period. Water-insoluble drugs must be turned into soluble species and blood–brain barrier-impermeable agents need to be modified to cross it following peripheral administrations. The derivatization requiring for achieving those desirable pharmacological features typically result in biologically/pharmacologically inactive products, unless those derivatizations can be carried out in a reversible fashion.

The finding that acetylcholinesterase (AChE) colocalizes with β-amyloid (Aβ) and promotes and accelerates Aβ aggregation has renewed an intense interest in developing new multifunctional AChE inhibitors as potential disease-modifying drugs for Alzheimer's therapy. To this end, we have developed a new class of selective AChE inhibitors with site-activated chelating activity. The identified lead, HLA20A, exhibits little affinity for metal (Fe, Cu, and Zn) ions but can be activated following inhibition of AChE to liberate an active chelator, HLA20. HLA20 has been shown to possess neuroprotective and neurorescuing activities in vitro and in vivo with the ability to lower amyloid precursor holoprotein (APP) expression and Aβ generation and inhibit Aβ aggregation induced by metal (Fe, Cu, and Zn) ion. HLA20A inhibited AChE in a time and concentration dependent manner with an HLA20A-AChE complex constant (K(i)) of 9.66 × 10(-6) M, a carbamylation rate (k(+2)) of 0.14 min(-1), and a second-order rate (k(i)) of 1.45 × 10 (4) M(-1) min(-1), comparable to those of rivastigmine. HLA20A showed little iron-binding capacity and activity against iron-induced lipid peroxidation (LPO) at concentrations of 1-50 μM, while HLA20 exhibited high potency in iron-binding and in inhibiting iron-induced LPO. At a concentration of 10 μM, HLA20A showed some activity against monoamine oxidase (MAO)-A and -B when tested in rat brain homogenates. Defined restrictively by Lipinski's rules, both HLA20A and HLA20 satisfied drug-like criteria and possible oral and brain permeability, but HLA20A was more lipophilic and considerably less toxic in human SHSY5Y neuroblastoma cells at high concentrations (25 or 50 μM). Together our data suggest that HLA20A may represent a promising lead for further development for Alzheimer's disease therapy.

Here we describe the design and application of OSu-FMS-MAL-S-(CH(2))(15)-COOH, an agent that associates with albumin while linked to a peptide or a protein with sufficient affinity (Ka=2 to 2.6 x 10(5)M(-1)) to protract the action of short- lived peptides and proteins in vivo. Under physiological conditions this probe undergoes spontaneous hydrolysis with the concomitant reactivation of inactive conjugates. Intravenously administered (125)I-labeled-Insulin-FMS-MAL-S-(CH(2))(15)-COOH to rats shows half-life of 17+/-2h, exceeding 5.2 times that obtained with intravenously administered (125)I-labeled Insulin. In mice this derivative facilitates glucose-lowering effect over a period of 24h, yielding AUC five times greater than that obtained by a similar dose of insulin-detemir. Similarly, subcutaneous administration of Exendin-4-FMS-MAL-S-(CH(2))(15)-COOH into mice facilitated prolonged and stable reduction in glucose level, yielding a t(1/2) value of 28+/-2h, exceeding the effect of exendin-4 4.7 folds. The inactive derivative gentamicin-FMS-MAL-S-(CH(2))(15)-COOH regained its full antibacterial potency upon incubation at physiological conditions yielding a t(1/2) value of 7.1+/-0.2h. In conclusion, the albumin-binding probe we introduced enables to prolong the action of any amino containing molecule in vivo, without the drawback of inactivation that often occurs upon such derivatization.

In this review article, the structure, properties, stability and biological application of redox-active quinones are presented. A series of quinoid molecules is evaluated in terms of their ability to act as electron-transfer active compounds using cyclovoltammetric, electron paramagnetic resonance (EPR) and spin-trapping techniques. Redox potentials and electron distribution of the intermediate radical anions are shown to be decisive factors for the generation of reactive oxygen species (ROS). Mechanisms of ROS generation in dark by biological electron-transfer reaction or under photoexcitation have been proposed and experimentally verified. For site-specific damage of tumors, some quinone derivatives were covalently bound with the luteinizing hormone-releasing hormone (LH–RH or GnRH) that produces specific complexes with receptors on the surface of cancer cells. The properties of obtained conjugates to be bound with the different lines of cancer cells (αT3-1, M2R, LNCaP) were tested. EPR was used for the estimation of efficacy of ROS production by the conjugates in solution and in the complex with cancer cells. The toxicity of these conjugates as well as their stability in the stimulated oxidative stress were tested. The proposed approach could be useful in creating a new family of addressed anticancer drugs, including compounds for the treatment of tumors by photodynamic therapy.

A novel strategy to develop site-activated multifunctional chelators for targeting multiple etiologies of Alzheimer's disease is reported. The novel prochelator HLA20A with improved cytotoxicity shows little affinity for metal ions until it is activated by binding and inhibiting acetylcholinesterase (AChE), releasing an active chelator HLA20 that modulates amyloid precursor protein (APP) regulation and beta-amyloid (Abeta) reduction, suppresses oxidative stress, and passivates excess metal ions (Fe, Cu, and Zn) in the brain.

Estrogen has a key role in the regulation of skeletal growth and maintenance of bone mass. Recently, we developed peptides having estrogen-like activity as potential estrogen-based new drugs. The aim of the present study was to evaluate the influence of long-term administration of the most efficacious of these peptides, the hexapeptide EMP-1 (VSWFFE), on bone mass and development. EMP-1 was injected daily to ovariectomized (OVX) and intact young, sexually mature female mice for 10 weeks. Whole femora, including the cartilaginous growth plates were analyzed by micro-computed tomography (microCT). We found that peptide EMP-1 restrains bone growth in OVX mice: it inhibited dramatically bone longitudinal growth (40%), and decreased femoral diaphyseal diameter. Peptide EMP-1 had no effect on bone growth in normal mice, and did not influence the OVX-induced bone loss. We then developed a new microCT methodology to evaluate uncalcified and calcified growth plate parameters. In the OVX mice, peptide EMP-1 reduced volume and thickness of the uncalcified growth plate, a possible cause for the inhibition of bone longitudinal growth. Peptide EMP-1 may be used as a lead compound for the development of drugs to treat acromegalic patients.

The cyclization, under alkaline conditions, of N-benzyloxycarbonyl-L-threonine and of N-benzyloxycarbonyl-L-serine to produce 5-methyl-2-oxo-oxazolidine-4-carboxylic acid (O = C Thr1 and 2-oxo-oxazolidine-4-carboxylic acid (O = C Ser), respectively, was investigated and found to be efficient and racemization-free. Similar was the cyclization which accompanied the basic hydrolysis of N-benzyl-oxycarbonyl-L-threonyl-L-phenylalanine methyl ester and of N-benzyloxy-carbonyl-L-seryl-DL-valine ethyl ester, and which resulted in the formation of L - O = C Thr-L-Phe and L - O = C Ser-DL-Val, respectively. The reaction was applied to the synthesis of [O = C Thr1] tuftsin, an active analog of the phagocytosis stimulating peptide tuftsin. A new synthetic route to tuftsin is also described.

The facile thiolytic cleavage of the O-2,4-dinitrophenyl (Dnp) tyrosine bond was applied to the solid-phase synthesis of the 22-amino acid residue peptide H-Asp-Ala-Val-Tyr-Thr-Gly-Leu-Asn-Thr-Arg-Asn-Gln-Glu-Thr-Tyr-Glu-Thr-Leu-Lys-His-Glu-Lys-OH, corresponding to positions 62-83 in the chain of the type 1 receptor for Fce, domains expressed on the rat mucosal-type mast cells (line RBL-2H3). A method for the spectrophotometric determination of insoluble O-Dnp as well as of unprotected phenolic moieties of tyrosine was developed. It is based on monitoring S-Dnp-2-mercaptoethanol, produced upon O-Dnp thiolysis by 2-mercaptoethanol. © Munksgaard 1995. Dedicated to the memory of Dr. Susumu Funakoshi, a dear friend and a leader in peptide chemistry.

Insoluble 2-mercaptopyridine and 2-mercapto-nitrobenzene derivatives were prepared by modification of commercially available polystyrene. Applicability of these polymers as reagents for the thiolytic removal of the 2-nitrophenyl-sulphenyl amino-protecting group and as supports for preparation of polymeric active esters was evaluated. Polymeric 2-mercaptopyridine (PMP) was found efficient for both purposes. It was used in the stepwise synthesis of Leu-enkephalin via the polymeric reagent approach, serving as an Nps-cleaver. Polymeric esters derived from PMP and Boc-amino acids proved to be excellent acylators. Their usefulness is exemplified in the preparation of two dipeptides, which were produced rapidly and in high yields and purity.

The thiolysis of Nim-2, 4-dinitrophenyl-histidine peptides with 2-mercaptoethanol demonstrates an optimal rate between pH 8.5 and 9.0. Base lability of both reactants and product was investigated as a possible cause for the pH optimum. N-2, 4-Dinitrophenylimidazole, a model for Nim-2, 4-dinitrophenyl-histidine peptides, 2-mercaptoethanol and the product of thiolysis, 2, 4-dinitrophenyl-S-mercaptoethan-2-ol, were subjected to aqueous basic conditions (pH 8.5 to 12.0). The reactions were followed spectrophotometrically, and products were identified by comparison of their ultra-violet spectra with commercially available or synthetic compounds. Mechanisms and rate constants for base hydrolysis of 2, 4-dinitrophenyl-thio ethers are presented. Although N-2, 4-dinitrophenylimidazole and 2, 4-dinitrophenyl-S-mercaptoethan-2-ol dohydrolyze, it is the oxidation of 2-mercaptoethanol to 2-mercaptoethanol disulfide which results in the decreasing rate of thiolysis above pH 9.0.

Serum amyloid A (SAA) is an acute-phase serum protein which exists in the body in a complex with high-density lipoprotein (HDL3). It is involved in chronic inflammation and neoplastic diseases in an as yet unknown manner. Toward an understanding of the possible physiological role of SAA we initiated a study of its association with blood proinflammatory cells with which it may interact functionally in vivo. In the following we describe the binding characteristics of recombinant human SAA to human neutrophils (polymorphonuclear leukocytes; PMNLs) and their plasma membranes. Scatchard analysis of rSAA binding and displacement curves revealed Kd in the nanomolar range. The C-terminal domain of the protein, i.e. amino acid residues 77-104, which might reside in serum following SAA degradation and amyloid A formation, was found to inhibit efficiently the binding of the whole protein to neutrophils. The interaction of SAA, and of its related peptides while complexed in HDL3, with human PMNs was also studied. The results suggest that SAA may be involved, in an as yet unknown manner, in the neutrophil-associated inflammatory mechanism.

2-Mercaptopyridine was used to effect the selective, mild and efficient cleavage of the o-nitrophenylsulphenyl amino-protecting group from several amino acids and peptides. By utilization of this reagent a stepwise synthesis of the tetrapeptide Thr-Lys-Leu-Arg([Leu3]tuftsin) was successfully achieved. The potential use of 2-mercaptopyridine in mechanized peptide synthesis via the polymeric reagents approach is discussed.

We attempted to engineer a novel long-acting insulin based on the following properties: (i) action as a prodrug to preclude supraphysiological concentrations shortly after injection; (ii) maintenance of low-circulating level of biologically active insulin for prolonged period; and (iii) high solubility in aqueous solution. A spontaneously hydrolyzable prodrug was thus designed and prepared by conjugating insulin through its amino side chains to a 40kDa polyethylene glycol containing sulfhydryl moiety (PEG(40)-SH), employing recently developed hetero-bifunctional spacer 9-hydroxymethyl-7(amino-3-maleimidopropionate)-fluorene-N-hydroxysucinimide (MAL-Fmoc-0Su). A conjugate trapped in the circulatory system and capable of releasing insulin by spontaneous chemical hydrolysis has been created. PEG(40)-Fmoc-insulin is a water-soluble, reactivatable prodrug with low biological activity. Upon incubation at physiological conditions, the covalently linked insulin undergoes spontaneous hydrolysis at a slow rate and in a linear fashion, releasing the nonmodified immunologically and biologically active insulin with a t(1/2) value of 30h. A single subcutaneous administration of PEG(40)-Fmoc-insulin to healthy and diabetic rodents facilitates prolonged glucose-lowering effects 4- to 7-fold greater than similar doses of the native hormone. The beneficial pharmacological features endowed by PEGylation are thus preserved. In contrast, nonreversible, "conventional" pegylation of insulin led to inactivation of the hormone.

We synthesized two carminic acid (7-alpha-d-glucopyranosyl-9,10-dihydro-3,5,6,8-tetrahydroxy-1-methyl-9,10-dioxo-2-anthracene carboxlic acid, CA)-GnRH conjugates to be used as a model for potential photoactive targeted compounds. CA was conjugated to the epsilon-amino group of [d-Lys(6)]GnRH through its carboxylic moiety or via a beta-alanine spacer (beta-ala). Redox potentials of CA and its conjugates were determined. We used electron spin resonance (ESR) and spin trapping techniques to study the light-stimulated redox properties of CA and its CA-GnRH conjugates. Upon irradiation, the compounds stimulated the formation of reactive oxygen species (ROS), that is, singlet oxygen ((1)O(2)) and oxygen radicals (O(2)(-*) and OH(*)). Both conjugates exhibited higher ROS production than the non-conjugated CA. The bioactivity properties of the CA conjugates and the parent peptide, [d-Lys(6)]GnRH, were tested on primary rat pituitary cells. We found that the conjugates preserved the bioactivity of GnRH as illustrated by their capability to induce ERK phosphorylation and LH release.

The different clinical entities of osteochondromas, hereditary multiple exostoses (HME) and non-familial solitary exostosis, are known to express localized exostoses in their joint metaphyseal cartilage. In the current study biopsies of osteochondromas patients were screened with respect to a number of cellular and molecular parameters. Specifically, cartilaginous biopsy samples of nine HME patients, 10 solitary exostosis patients and 10 articular cartilages of control subjects were collected and cell cultures were established. Results obtained showed that one of the two HME samples that underwent DNA sequencing analysis (HME-1) had a novel mutation for an early stop codon, which led to an aberrant protein, migrating at a lower molecular weight position. The EXT-1 mRNA and protein levels in chondrocyte cultures derived from all nine HME patients were elevated, compared with solitary exostosis patients or control subjects. Furthermore, cell cultures of HME patients had significantly decreased pericellular heparan sulphate (HS) in comparison with cultures of solitary exostosis patients or control subjects. Immunohistochemical staining of tissue sections and Western blotting of cell cultures derived from HME patients revealed higher levels of heparanase compared with solitary exostosis patients and of control subjects. Further investigations are needed to determine whether the low pericellular HS levels in HME patients stem from decreased biosynthesis of HS, increased degradation or a combination of both. In conclusion, it appears that due to a mutated glycosyltransferase, the low content of pericellular HS in HME patients leads to the anatomical deformations with exostoses formation. Hence, elevation of HS content in the pericellular regions should be a potential molecular target for correction.

Affecting an estimated 5% of adults over 65 years of age, Parkinson's disease and Alzheimer's disease are the most common neurodegenerative disorders. Accumulating evidence suggests that oxidative stress induced by the breakdown of iron homeostasis is a major contributor to the neuronal loss observed in neurodegeneration. Thus, brain-permeable iron chelators may present potential therapeutic benefits. In the present study, iron-chelating hydroxamate groups were introduced into the NAP (NAPVSIPQ) peptide, whose neuroprotective qualities have been widely demonstrated. Our experiments revealed that the novel dihydroxamate peptide 3 is capable of inhibiting iron-catalyzed hydroxyl radical formation and lipid peroxidation, abilities that are not part of the repertoire of its parent peptide. In addition, peptide 3 was superior to native NAP in protecting human neuroblastoma cell cultures against the toxicity of hydrogen peroxide. These results suggest that NAP-based iron chelators deserve further investigation in the search for drug candidates for neurodegeneration.

Traditionally, drug design programs are focused on optimizing the specificity of lead compounds against a carefully selected drug target. Disappointingly, this approach to discover a "magic bullet" drug has not met with the expected success for CNS disorders. Transcriptomics and proteomic profiling of neurodegenerative diseases have indicated that they are poly-etiological in origin and that the processes leading to neuronal death are multifactorial. An emerging concept is the design of drug ligands that modulate multiple drug targets identified for a particular disease. In this review we explore some examples of multifunctional drugs which may be useful in the treatment of neurodegenerative diseases, such as Alzheimer's and Parkinson's disease.

Accumulation of iron at sites where neurons degenerate in Parkinson's disease (PD) and Alzheimer's disease (AD) is thought to have a major role in oxidative stress induced process of neurodegeneration. The novel non-toxic lipophilic brain- permeable iron chelators, VK-28 (5- [4- (2- hydroxyethyl) piperazine-1-ylmethyl]- quinoline- 8- ol) and its multi-functional derivative, M-30 (5-[N-methyl-N-propargylaminomethyl]-8-hydroxyquinoline), as well as the main polyphenol constituent of green tea (-)-epigallocatechin-3-gallate (EGCG), which possesses iron metal chelating, radical scavenging and neuroprotective properties, offer potential therapeutic benefits for these diseases. M-30 and EGCG decreased apoptosis of human SH-SY5Y neuroblastoma cells in a neurorescue, serum deprivation model, via multiple protection mechanisms including: reduction of the pro-apoptotic proteins, Bad and Bax, reduction of apoptosis-associated Ser139 phosphorylated H2A.X and inhibition of the cleavage and activation of caspase-3. M-30 and EGCG also promoted morphological changes, resulting in axonal growth-associated protein-43 (GAP-43) implicating neuronal differentiation. Both compounds significantly reduced the levels of cellular holo-amyloid precursor protein (APP) in SH-SY5Y cells. The ability of theses novel iron chelators and EGCG to regulate APP are in line with the presence of an iron-responsive element (IRE) in the 5'-untranslated region (5'UTR) of APP. Also, EGCG reduced the levels of toxic amyloid-beta peptides in CHO cells over-expressing the APP "Swedish" mutation. The diverse molecular mechanisms and cell signaling pathways participating in the neuroprotective/neurorescue and APP regulation/processing actions of M-30 and EGCG, make these multifunctional compounds potential neuroprotective drugs for the treatment of neurodegenerative diseases, such as PD, AD, Huntington's disease and amyotrophic lateral sclerosis.

In an attempt to develop an efficient chemotherapeutic agent targeted at malignant cells that express receptors to gonadotropin releasing hormone (GnRH) we coupled [D-Lys6]GnRH covalently to an emodin derivative, i.e. emodic acid (Emo) to yield [D-Lys6(Emo)]GnRH. Emodin is a naturally occurring anthraquinone which is widely used as a laxative and has other versatile biological activities. Physico-chemical studies employing electron paramagnetic resonance and electrochemistry of the conjugate as well as the (Emo) moiety showed that these compounds could be easily reduced either chemically, photochemically or enzymatically to their corresponding semiquinones. In the presence of oxygen the semiquinones generated reactive oxygen species (ROS), mainly superoxide and hydroxyl radicals, which were detected by the spin trapping method. Moreover, upon irradiation with visible light these compounds produced ROS and a highly reactive excited triplet state of Emo, which by itself may cause the oxidation of certain electron acceptors such as amino acids and bases of nucleic acids. Thus, [D-Lys6]GnRH-photosensitizer conjugates may be potentially used for targeted photodynamic chemotherapy aimed at treating cancer cells that carry GnRH receptors. These conjugates may also induce cytotoxicity in the dark similar to common conventional chemotherapeutic agents. The peptidic moiety, [D-Lys6]GnRH, was found to be stable toward highly reactive ROS generated either from enzymatic reduction or upon photoirradiation. The physico-chemical properties of Emo were only marginally influenced by the peptidic [D-Lys6]GnRH carrier.

Most peptide and protein drugs are short-lived species in vivo with a circulatory half-life of several minutes. This is particularly valid for non-glycosylated proteins with a molecular mass of less than 50 kDa. Since peptide/protein drugs are not absorbed orally, prolonged maintenance of therapeutically active drugs in the circulatory system is of primary clinical importance. Another major obstacle of injected polypeptide drugs is the elevated concentration of 100–1000 times above the therapeutical level that may be present in the circulatory system shortly after administration. Such overdosing may lead to undesirable side effects such as over-stimulation or down-regulation of receptor sites. In this review we describe two new strategies that overcome these two problems of systemically injected peptide/protein drugs. The first strategy includes Fmoc and FMS derivatization of peptides, proteins and low molecular-weight drugs, converting them to inactive prodrugs that undergo reactivation with desirable pharmacokinetic patterns in body fluids. Based on this Fmoc/FMS-technology, we have developed a second strategy, reversible pegylation. Inactive pegylated peptide/protein drugs release the native active parental molecule at slow rates, and in homogeneous fashion under physiological conditions, thus facilitating prolonged therapeutic effects, following a single administration.

Luliberin analogues modified at the N-terminus were synthesized to search for drugs exerting a cytotoxic effect on cells of hormone-dependent tumors. A synthetic scheme effective in the preparation of analogues containing fatty acid residues was proposed. The cytotoxic effect of the peptides was studied on a number of cell lines of human tumors in vitro. The dependence of the antitumor effect on the length of peptide chain, amino acid sequence, and structure of the N-terminal group was demonstrated. Modification with palmitic acid was found to result in highly active compounds in the case of analogues containing more than ten aa, whereas modifications with lauric, caproic, or trimethylacetic acid led to compounds with significantly lower activities. Analogues of luliberin containing a palmitic acid residue and effectively inhibiting the growth of tumor cells in vitro were synthesized.

The steroidogenic enzyme 21-hydroxylase (21OH) is the main autoantigen in autoimmune primary adrenal failure (Addison’s disease). Autoantibodies against 21OH are immunological markers of an ongoing autoimmune process but are not directly involved in the tissue destruction. Autoreactive T cells are thought to mediate tissue damage, but the T cell antigen(s) has not been identified. To find out whether 21OH contains important immunodominant epitopes for T cells, we first immunized BALB/c and SJL inbred mouse strains with recombinant 21OH and showed that lymph node cells proliferated effectively following in vitro stimulation with recombinant 21OH (stimulation indices (SI) 20–40). We further synthesized a series of peptides based on 21OH with amino acid sequences with propensity to bind to major histocompatibility complex class II molecules. Only a few peptides could trigger lymphocytes of 21OH-primed mice to proliferate. One of these, 21OH (342–361), stimulated effectively 21OH-primed lymph node cells of SJL mice (SI = 4–8) and also, although to a lesser extent, of BALB/c mice (SI = 2.5). When SJL mice were immunized with 21OH (342–361), the immunizing peptide as well as peptide 21OH (346–361) triggered a significant proliferative response (SI = 24). A peptide from another part of 21OH, namely 21OH (191–202), did not stimulate the 21OH (342–361)-primed cells. Moreover, stimulation of lymph node cells of mice immunized with 21OH (342–361) with 21OH resulted in a significant proliferative response. We conclude that 21OH (342–361) is an immunodominant determinant for T cells in SJL and probably BALB/c mice. 21OH (342–361) corresponds to the substrate binding site of the enzyme. The p342–361 region may be involved in the pathogenesis of autoimmune adrenal failure in humans.

Polymyxin B (PMXB) blocks the action of insulin on glucose uptake in vitro. In vivo ,i t reverses hypoglycemia induced by exogenous insulin. Here we have treated mature male rats daily with PMXB over a period of two weeks. This therapy has decreased body weight by 11%, adipose fat mass by 46% and triglyceride levels by 39%, with no indication of liver or kidney toxicity. Two suboptimal parameters, however, were a decrease in food intake in the first week of treatment and some increase in fasting glucose levels. We have screened for PMXB-analogs having less associating affinity with rat-muscle phospholipids, and revealed that the same therapy using PMXB-derived peptide (nona-PMXB) is most optimal. This PMXB-analog is devoid of antibacterial activity and is four times less toxic than PMXB. Nona-PMXB therapy lower by 10, 32, 35 and 6% body weight gain, fat mass, circulating triglycerides and fasting glucose levels, respectively, in spite of normal or even elevated food intake in nona-PMXB treated rats. In summary, we found that nona-PMXB therapy is capable if inducing leanness in mature rats, particularly at the expense of decreasing fat-mass in adipose tissue. By and large, we suggest that lowering the action of insulin, on fat build-up solely, may be a therapeutically feasible task to fight with human adiposity in the future.

Vasoactive intestinal peptide (VIP), calcitonin gene-related peptide (CGRP) and angiotensin 2 are key neuropeptides that innervate the sexual organs. For further understanding of neuropeptide involvement in female sexual function, we investigated peptide receptor mRNA expression using reverse transcription-polymerase chain reaction (RT-PCR) in the rat vagina and clitoris, and alteration during the shift from the proestrus to the estrus phase. VIP, angiotensin 2 and CGRP receptor subtypes transcripts were found to be expressed in the vagina and the clitoris. Significantly increased levels of angiotensin 2 and CGRP receptor subtypes transcripts were observed in the vagina as compared to the clitoris. Significant increases in the expression of the VIP receptor type 2 (VPAC2) mRNA and parallel increases in a novel VIP responsive gene, activity-dependent neuroprotective protein (ADNP) mRNA were detected in the rat vagina during the estrus phase. The expression pattern of neuropeptide receptors in the female sexual organs suggest an intimate involvement of the corresponding neuropeptides in female sexual function.

Antioxidants and iron chelating molecules are known as neuroprotective agents in animal models of neurodegenerative disorders such as Alzheimer's disease (AD) and Parkinson's disease (PD). In this study, we designed and synthesized a novel bifunctional molecule (M10) with radical scavenging and iron chelating ability on an amino acid carrier likely to be a substrate for system L, thus targeting the compound to the central nervous system (CNS). M10 had a moderate iron affinity in HEPES buffer (pH 7.4) with logK(3)=12.25+/-0.55 but exhibited highly inhibitory action against iron-induced lipid peroxidation, with an IC(50) value (12microM) comparable to that of desferal (DFO). EPR studies indicated that M10 was a highly potent *OH scavenger with an IC(50) of about 0.3 molar ratio of M10 to H(2)O(2). In PC12 cell culture, M10 was at least as potent as the anti-Parkinson drug rasagiline in protecting against cell death induced by serum-deprivation and by 6-hydroxydopamine (6-OHDA). These results suggest that M10 deserves further investigation as a potential agent for the treatment of neurodegenerative disorders such as AD and PD.

A series of novel derivatives of neuropeptides with a metal-chelating moiety was synthesized and examined for various properties related to iron (Fe) chelation and neuroprotective action. All derivatives chelated Fe to form stable Fe complexes in water. Some strongly inhibited Fe-induced lipid peroxidation with an IC(50) value of about 12 microm. In PC12 cell culture, several compounds, at concentrations as low as 1 microm, attenuated serum-free stimulated cell death and improved cell survival by 20-35%. At this concentration, these analogs also protected against 6-hydroxydopamine (6-OHDA)-induced cell death, increasing cell viability by 20-30%. Electron paramagnetic resonance (EPR) studies indicated that besides being good Fe chelators, these analogs act as radical scavengers to directly scavenge hydroxyl radicals. Together, the data indicate that some of the analogs could be further developed as possible neuroprotective agents for treatment of neurodegenerative diseases such as Parkinson's, Alzheimer's, and Huntington's diseases, Friedreich's atxia, amyotrophic, and lateral sclerosis where Fe misregulation has been reported.

Blood infections caused by gram-negative bacteria are one of the major challenges facing modern medicine, despite treatment with the available conventional antibiotics (22). Mortality rates in the range of 20 to 80% for septicemia caused by gram-negative bacteria have been reported. Antibiotic treatment is often administered when the disease reaches an advanced stage, usually when symptoms appear, by which point insufficient time remains for the antibiotic to kill the pathogen before the onset of irreversible tissue damage. Moreover, in many cases the antibiotic is given before sensitivity tests have been performed to identify an effective treatment. The emergence of bacterial strains resistant to conventional antibiotics, the lack of a rapid means of diagnosing the infection, and the generally unknown antibiotic sensitivity pattern of the infecting bacteria are probably among the major causes of inefficient therapy and high mortality rates (26). Most of these blood infections are caused by opportunistic pathogens that are not usually capable of initiating bacteremia in otherwise healthy individuals. Host defense mechanisms of the innate, nonclonal immune system serve as the principal pathway for effective elimination of pathogens (11). Potent components of innate immunity are the macrophages and polymorphonuclear leukocytes (PMNs) that mediate the early clearance of bacteria by the phagocytic process (1, 23). Agents that enhance phagocytosis may promote clearance of the pathogen and termination of the infectious process. In some cases the phagocytotic process is mediated by adhesins expressed on the bacterial surface, in a process termed nonopsonic phagocytosis (18). However, the interaction of many bacteria with phagocytic cells is greatly facilitated by opsonins, which act as a bridge between the surfaces of these two types of cells (5, 8). The development of agents that can function as opsonins may provide a useful new approach to terminating the infectious process by enhancing bacterial attachment to phagocytic cells, followed by ingestion and digestion of the pathogen. For an agent to function as an opsonin, it must contain a moiety that recognizes a specific target molecule on the bacterial surface and another that recognizes specific receptors on phagocytic cells. Moreover, its toxicity must be relatively low, and most importantly, there should be little likelihood for the emergence of strains resistant to its action. In the present study, we describe the synthesis of antimicrobial peptide conjugates that act both as opsonins to enhance destruction by phagocytic cells and as agents that permeabilize the bacterial membrane to enhance eradication by hydrophobic antibiotics and other antimicrobial agents. The peptides are derived from polymyxin B (PMB) or polymyxin E (PME) covalently linked to a short chemotactic peptide. Early studies have established that although polymyxin-based antibiotics are relatively toxic, they can be rendered 10 to 15 times less toxic by cleaving the lipid moiety from the molecule (7, 10). These polymyxin-based peptides lack direct bactericidal activity but retain their ability to bind to lipopolysaccharides (LPSs) on the bacterial surface and to permeabilize the outer membrane (OM) to hydrophobic antibiotics that cannot otherwise penetrate the bacteria, as well as to other bactericidal agents, such as the complement (33). Since resistance to the parent polymyxins is rare (25), it is expected that resistance to these polymyxin-derived peptides will be rare as well. Indeed, we did not find strains resistant to the permeabilizing activity of a polymyxin B (PMB) nonapeptide (PMBN) among 59 PMB-sensitive strains tested (21). Moreover, administration of the nonapeptide in combination with erythromycin protected mice against a lethal dose of an erythromycin-resistant strain of Klebsiella pneumoniae (19). More recently, we showed that the polymyxin B nonapeptide binds to LPS, which is consistent with previous studies showing that the parent PMB and PME molecules bind to lipid A of LPS on bacterial surfaces (16, 25, 31). Encouraged by these results, we reasoned that conjugation of PMBN or PMEN to a moiety that recognizes specific receptors on phagocytic cells may confer opsonic activity on the molecule. We chose to use formyl methionine-leucine-phenylalanine (fMLF), a chemotactic peptide, as our conjugant because it has been studied extensively for its property of binding to specific receptors on phagocytic cells (17). It will be shown that conjugates consisting of polymyxin peptides covalently linked to fMLF enhance the bactericidal activities of conventional antibiotics, act as opsonins in promoting the phagocytic destruction of bacteria in vitro, and protect mice from lethal infections and bacteremia in vivo.

Several multifunctional iron chelators have been synthesized from hydroxyquinoline pharmacophore of the iron chelator, VK-28, possessing the monoamine oxidase (MAO) and neuroprotective N-propargylamine moiety. They have iron chelating potency similar to desferal. M30 is a potent irreversible rat brain mitochondrial MAO-A and -B inhibitor in vitro (IC50, MAO-A, 0.037 +/- 0.02; MAO-B, 0.057 +/- 0.01). Acute (1-5 mg/kg) and chronic [5-10 mg/kg intraperitoneally (i.p.) or orally (p.o.) once daily for 14 days]in vivo studies have shown M30 to be a potent brain selective (striatum, hippocampus and cerebellum) MAO-A and -B inhibitor. It has little effects on the enzyme activities of the liver and small intestine. Its N-desmethylated derivative, M30A is significantly less active. Acute and chronic treatment with M30 results in increased levels of dopamine (DA), serotonin(5-HT), noradrenaline (NA) and decreases in DOPAC (dihydroxyphenylacetic acid), HVA (homovanillic acid) and 5-HIAA (5-hydroxyindole acetic acid) as determined in striatum and hypothalamus. In the mouse MPTP (N-methy-4-phenyl-1,2,3,6-tetrahydropyridine) model of Parkinson's disease (PD) it attenuates the DA depleting action of the neurotoxin and increases striatal levels of DA, 5-HT and NA, while decreasing their metabolites. As DA is equally well metabolized by MAO-A and -B, it is expected that M30 would have a greater DA neurotransmission potentiation in PD than selective MAO-B inhibitors, for which it is being developed, as MAO-B inhibitors do not alter brain dopamine.