The utility of oligopeptidase in brain-targeting delivery of an enkephalin analogue by prodrug design.

In a brain-targeting prodrug approach for a metabolically stable enkephalin analogue DADLE, specific enzymes are utilized for in vivo prodrug activation. Prolyl oligopeptidase (POP) may be especially useful in this regard. In vitro metabolic stability of the putative metabolites of prodrugs having various "spacers" has shown that POP provides significantly faster release of DADLE from conjugates having dipeptidyl spacer (specifically Xaa-Pro or Xaa-Ala) than alternative peptidases utilized when single amino acids are used as spacers. In vitro half-lives measured in rat brain homogenate showed excellent correlation with CNS-mediated analgesia using the tail-flick model in rats providing, thus, an in vivo substantiation of the prodrug approach relying on POP as the peptidase to release DADLE.

A pyridinium-substituted analog of the TRH-like tripeptide pGlu-Glu-Pro-NH2 and its prodrugs as central nervous system agents.

A metabolically stable and centrally acting analog of pGlu-Glu-Pro-NH2 ([Glu2]TRH, a tripeptide structurally related to TRH (thyrotropin-releasing hormone)) was designed by replacing the amino-terminal pyroglutamyl residue with a pyridinium moiety. The analeptic action of the analog was used to optimize the efficacy of this novel CNS agent when administered intravenously in its CNS-permeable prodrug forms obtained via the reduction of the pyridinium moiety to the nonionic dihydropyridine and esterifying the central Glu with various alcohols. The maximum effect in antagonizing pentobarbital-induced narcosis in mice was achieved with the hexyl ester that was used subsequently for a comparative evaluation with a prodrug of the parent neuropeptide in the Porsolt swim test as a paradigm for antidepressant effect. The novel analog maintained its antidepressant potency but showed reduced analeptic action compared to [Glu2]TRH; thus, an increase in the selectivity of CNS-action was obtained by the incorporation of the pyridinium moiety.

Integration of mass spectrometry into early-phase discovery and development of central nervous system agents.

The early-phase discovery and development of useful central nervous system (CNS) agents present ample opportunities to exploit mass spectrometry and provide detailed compound/mixture characterization, or to make the process faster and/or more economic. Neuropeptide FF antagonists and centrally active thyrotropin-releasing hormone analogues were used as specific examples in this work. We evaluated the characterization of focused libraries of peptide derivatives by electrospray ionization, tandem mass spectrometry and liquid chromatography/tandem mass spectrometry on a quadrupole ion trap and nanoelectrospray on a Fourier transform ion cyclotron resonance mass spectrometer. Immobilized artificial-membrane chromatography was employed as a model to predict/rank new agents against lead compounds for their potential to reach the central nervous system in pharmacologically significant amounts. Measuring brain concentrations in rodents after the intravenous administration of test compounds was used as an in vivo approach, and we took advantage of microdialysis sampling that furnished samples without interfering tissue matrix and afforded the estimation of extracellular concentrations in a localized part of the brain. Overall, making atmospheric-pressure ionization mass spectrometry an integral part of the process has played a major role in increasing throughput, selectivity, specificity and detection sensitivity and thereby providing useful information about the extent or mechanism of transport and metabolic activation/inactivation in early-phase discovery and development of CNS agents.

Combinatorial lead optimization of a neuropeptide FF antagonist.

The tripeptide Pro-Gln-Arg-NH2, derivatized at the secondary amino group of the proline residue with 5-(dimethylamino)-1-naphthalenesulfonyl (dansyl-PQR-NH2), antagonizes the central anti-opioid action of neuropeptide FF in animals after systemic administration and, therefore, is a therapeutic lead to treat opiate withdrawal. For a combinatorial optimization to improve potency, libraries focused on the possible replacement of the proline and glutamine residues of this lead compound were obtained by a solid-phase split-and-mix method using coded amino acids (excluding cysteine) as building blocks. After screening for competitive binding against a radioiodinated neuropeptide FF analogue, 5-(dimethylamino)-1-naphthalenesulfonyl-Gly-Ser-Arg-NH2 (dansyl-GSR-NH2) has emerged as one of the compounds in the library with high affinity to the NPFF receptor and even with a moderate increase compared to dansyl-PQR-NH2 in its predicted ability to penetrate the central nervous system.

Synthesis and biological evaluation of 17beta-alkoxyestra-1,3, 5(10)-trienes as potential neuroprotectants against oxidative stress.

17beta-O-Alkyl ethers (methyl, ethyl, propyl, butyl, hexyl, and octyl) of estradiol were obtained from 3-O-benzyl-17beta-estradiol with sodium hydride/alkyl halide, followed by the removal of the O-benzyl protecting group via catalytic transfer hydrogenation. An increase compared to estradiol in the protection of neural (HT-22) cells against oxidative stress due to exposure of glutamate was furnished by higher (C-3 to C-8) alkyl ethers, while methyl and ethyl ethers decreased the neuroprotective effect significantly. Lipophilic (butyl and octyl) ethers blocking the phenolic hydroxyl (3-OH) of A-ring were inactive.

Exploratory pharmacokinetics and brain distribution study of a neuropeptide FF antagonist by liquid chromatography/atmospheric pressure ionization tandem mass spectrometry.

Dansyl-Pro-Gln-Arg-NH(2), an N-terminally modified tripeptide amide and a putative neuropeptide FF antagonist, was amenable to both positive-ion ESI and APCI. The protonated molecule yielded several fragment ions upon collision-induced dissociation in a quadrupole ion trap instrument for the development of LC/MS/MS assay methods. ESI clearly outperformed APCI in limits of detection, and was the method of choice for coupling with narrow-bore reversed-phase liquid chromatography to assess the pharmacokinetic profile and brain concentration of the neuropeptide FF antagonist in experimental animals. While plasma could be analyzed after rapid sample preparation, brain tissue required cleanup (solid phase extraction) and preconcentration before injection, and the assay was prone to matrix interference. This study indicated a rapid disappearance of dansyl-Pro-Gln-Arg-NH(2) from the plasma and the brain, and modest CNS bioavailability after intravenous administration to rats.

Targeting drugs to the brain by redox chemical delivery systems.

Chemical delivery systems (CDSs) based on the redox conversion of a lipophilic dihydropyridine to an ionic, lipid-insoluble pyridinium salt have been developed to improve the access of therapeutic agents to the central nervous system. A dihydropyridinium-type CDS or a redox analog of the drug is sufficiently lipophilic to enter the brain by passive transport, then undergoes an enzymatic oxidation to an ionic pyridinium compound, which promotes retention in the CNS. At the same time, peripheral elimination of the entity is accelerated due to facile conversion of the CDS in the body. This review discusses chemical, physicochemical, biochemical, and biological aspects in relation to the principles and practical implementation of the redox brain-targeting approach to various classes of drugs. Representative examples to the brain-enhanced delivery of neurotransmitters, steroids, anticonvulsants, antibiotics, antiviral, anticancer and antidementia agents, and neuropeptides and their analogs are presented in detail. In vivo and in vitro studies and preliminary clinical data of several novel derivatives have been promising, which could lead to a practical use of the redox CDSs after proper pharmaceutical development. The investigations accentuate the need for considering physicochemical, metabolic, and pharmacokinetic properties in designing of carrier systems that are able to target drugs into the central nervous system.

Metabolism-based brain-targeting system for a thyrotropin-releasing hormone analogue.

Gln-Leu-Pro-Gly, a progenitor sequence for the thyrotropin-releasing hormone (TRH) analogue [Leu(2)]TRH (pGlu-Leu-Pro-NH(2)), was covalently and bioreversibly modified on its N- and C-termini (by a 1,4-dihydrotrigonellyl and a cholesteryl group, respectively) to create lipoidal brain-targeting systems for the TRH analogue. The mechanism of targeting and the recovery of the parent peptide at the target site involve several enzymatic steps, including the oxidation of the 1,4-dihydropyridine moiety. Due to the lipid insolublity of the peptide pyridinium conjugate obtained after this reaction, one of the rudimentary steps of brain targeting (i.e., trapping in the central nervous system) can be accomplished. Our design also included spacer amino acid(s) inserted between the N-terminal residue of the progenitor sequence and the dihydrotrigonellyl group to facilitate the posttargeting removal of the attached modification. The release of the TRH analogue in the brain is orchestrated by a sequential metabolism utilizing esterase/lipase, peptidyl glycine alpha-amidating monooxygenase (PAM), peptidase cleavage, and glutaminyl cyclase. In addition to in vitro experiments to prove the designed mechanism of action, the efficacy of brain targeting for [Leu(2)]TRH administered in the form of chemical-targeting systems containing the embedded progenitor sequence was monitored by the antagonistic effect of the peptide on the barbiturate-induced anesthesia (measure of the activational effect on cholinergic neurons) in mice, and considerable improvement was achieved over the efficacy of the parent peptide upon using this paradigm.

Metabolism-based drug design and drug targeting.

Even at the early stages of drug discovery and structure-based drug design, the pharmacokinetic, pharmacodynamic and toxicological consequences of drug metabolism cannot be ignored. Drug metabolism is also of interest to medicinal chemists in the design of drugs with controlled, predictable deactivation after achieving the therapeutic objective in prodrug design and in chemical-enzymatic drug targeting. In this review, the authors provide an overview of concepts that can be utilized from drug discovery to pharmaceutical development to overcome problems associated with drug metabolism, or that may be used to take advantage of 'designed-in' metabolic activation to achieve drug targeting.

Pharmacokinetics and urinary excretion of DMXBA (GTS-21), a compound enhancing cognition.

DMXBA (3-(2,4-dimethoxybenzylidene)-anabaseine, also known as GTS-21) is currently being tested as a possible pharmacological treatment of cognitive dysfunction in Alzheimer's disease. In this study, plasma and brain pharmacokinetics as well as urinary excretion of this compound have been evaluated in adult rats. DMXBA concentrations were determined by HPLC. Following a 5 mg kg-1 iv dose, DMXBA plasma concentration declined bi-exponentially with mean (+/- SE) absorption and elimination half-lives of 0.71 +/- 0.28 and 3.71 +/- 1.12 h, respectively. The apparent steady state volume of distribution was 2150 +/- 433 mL kg-1, total body clearance was 1480 +/- 273 mL h-1 kg-1, and AUC0-infinity was 3790 +/- 630 ng h mL-1. Orally administered DMXBA was rapidly absorbed. After oral administration of 10 mg kg-1, a peak plasma concentration of 1010 +/- 212 ng mL-1 was observed at 10 min after dosing. Elimination half-life was 1.740 +/- 0.34 h, and AUC0-infinity was 1440 +/- 358 ng h mL-1. DMXBA peak brain concentration after oral administration was 664 +/- 103 ng g-1 tissue, with an essentially constant brain-plasma concentration ratio of 2.61 +/- 0.34, which indicates that the drug readily passes across the blood-brain barrier. Serum protein binding was 80.3 +/- 1.1%. Apparent oral bioavailability was 19%. Renal clearance (21.8 mL h-1 kg-1) was less than 2% of the total clearance (1480 +/- 273 mL h-1 kg-1); urinary excretion of unchanged DMXBA over a 96 h period accounted for only 0.28 +/- 0.03% of the total orally administered dose. Our data indicates that DMXBA oral bioavailability is primarily limited by hepatic metabolism.

Brain-targeted delivery of a leucine-enkephalin analogue by retrometabolic design.

A brain-targeted chemical delivery system (CDS) based on retrometabolic drug design was applied to a Leu-enkephalin analogue, Try-D-Ala-Gly-Phe-D-Leu (DADLE). The molecular architecture of the peptide CDS disguises its peptide nature from neuropeptide-degrading enzymes and provides lipophilic, bioreversible functions for the penetration through the blood-brain barrier. These functions were provided by a targetor, a 1,4-dihydrotrigonellyl group, on the N-terminus and a bulky, lipophilic ester group on the C-terminus. A spacer amino acid residue was also inserted between the targetor and the parent peptide to assure the release of DADLE by specific enzymes. Four CDSs were synthesized by segment-coupling method that proved to be superior to sequential elongation in obtaining this type of peptide conjugates. Intravenous injection of the compounds produced a significant and long-lasting response in rats monitored by the tail-flick latency measurements. CDSs having the bulkier cholesteryl group showed a better efficacy than those having the smaller 1-adamantaneethyl ester. The spacer was the most important factor to manipulate the rate of DADLE release and, thus, the pharmacological activity; proline as a spacer produced more potent analgesia than alanine. The antinociceptive effect of the CDSs was naloxone-reversible and methylnaloxonium-irreversible, confirming that central opiate receptors were solely responsible for mediating analgesia induced by the peptide CDS that delivered, retained, and then released the peptide in the brain.

Redox derivatives of tranylcypromine: syntheses, properties, and monoamine oxidase inhibitor activity of some chemical delivery systems.

Several brain-targeting chemical delivery systems (CDS) based on a dihydropyridine----pyridinium salt type redox system were synthesized for the monoamine oxidase (MAO) inhibitor tranylcypromine (TCP). The dihydronicotinate moiety was chemically attached to the amino group of TCP by either an amide or substituted carbamate linkages. Physicochemical studies of the new derivatives, including chromatographic Rm determinations, were performed. Only the substituted carbamate-type derivatives manifested an increased lipophilicity relative to the parent compound. In vitro oxidation stability studies were also performed on selected derivatives using a ferricyanide-mediated method. Results of this assay showed that the dihydropyridine-type derivatives oxidized to the respective quaternary salt forms with stabilities which empirically correlated with other effective CDSs. Preliminary in vivo studies performed in rats indicated that some of the new derivatives exerted significant biological activity.

Antihypertensive activity of redox derivatives of tryptophan.

The essential amino acid, tryptophan, has been shown to lower blood pressure in rats when administered orally or intravenously. In order to potentially enhance this action, a brain-targeting chemical delivery system (CDS) approach was applied to this compound. The CDS is based on a dihydropyridine----pyridinium ion redox system, chemically analogous to the naturally occurring NADH----NAD+ system. The dihydropyridine moiety containing carrier is chemically attached to the amino group by an amide-type bonding while the carboxylic acid functionality is esterified to various alcohols. Physicochemical studies of the new derivatives were performed. The determined chromatographic Rm values indicate an increased lipophilicity for the CDSs compared to the parent compound. Oxidation stability studies performed on selected compounds using a ferricyanide-mediated method showed that the CDSs are oxidized to the respective quaternary salt forms. Activity studies performed in deoxycorticosterone acetate induced hypertensive rats, demonstrated that the delivery system for tryptophan reduced blood pressure more efficiently for a longer time than did the parent compound.

Application of a brain-targeting chemical delivery system to 9-amino-1,2,3,4-tetrahydroacridine.

Several chemical delivery systems (CDS) were synthesized for the cholinesterase inhibitor 9-amino-1,2,3,4-tetrahydroacridine (THA). The derivatives prepared were substituted with a 1,4-dihydropyridine in equilibrium pyridinium salt redox system at the amino functionality. These compounds were synthesized by acylation of the 9 amino group of THA with nicotinic anhydride under forced conditions, followed by a selective N-alkylation of the pyridine ring and regioselective reduction of the resulting quaternary salts. Lipophilicity parameters indicated increased lipophilic indices for various CDS's compared to the THA. Oxidation studies showed that dihydronicotinamides readily converted to the quaternary salt, both chemically and enzymatically. The transport forms of THA were also shown not to interact with acetylcholinesterase in vivo. In vivo distribution studies in the rat indicated that high and sustained levels of the pyridinium quaternary ion derivative were present in the central nervous system (CNS). In addition, THA was produced in the CNS from the quaternary salt precursor in low concentrations, indicating a slow but sustained release. The CDS for THA were found to be less acutely toxic than THA.