Carnosine Derivatives Research Articles

Salsolinol, a catechol neurotoxin, induces modification of ferritin: Protection by histidine dipeptide

1-Methyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline (salsolinol), an endogenous neurotoxin present in the mammalian brain, is known to perform a role in the pathogenesis of Parkinson's disease. In this study, we evaluated oxidative modifications of ferritin occurring after incubation with salsolinol. When ferritin was incubated with salsolinol, protein aggregation increased in a time-dependent manner. Free radical scavengers inhibited this salsolinol-mediated ferritin modification. The exposure of ferritin to salsolinol also results in the generation of protein carbonyl compounds and the formation of dityrosine. The results of this study show that free radicals may perform a pivotal role in salsolinol-mediated ferritin modification. Histidine dipeptides, such as carnosine, have been proposed to function as antioxidant agents in vivo. In this study, we also attempted to determine whether the histidine dipeptides, carnosine and N-acetyl-carnosine, could prevent salsolinol-mediated oxidative modification of ferritin. Our results showed that both carnosine and N-acetyl-carnosine significantly reduced ferritin aggregation. Both compounds effectively inhibited the formation of both carbonyl compounds and dityrosine. These results suggest that carnosine derivatives can, indeed, protect against salsolinol-mediated ferritin modification, as the consequence of free radical-scavenging activity.

Sensitive determination of carnosine in urine by high-performance liquid chromatography using 4-(5,6-dimethoxy-2-phthalimidinyl)-2-methoxyphenylsulfonyl chloride as a fluorescent labeling reagent

A simple and highly sensitive high-performance liquid chromatography procedure was developed for the determination of carnosine in urine. Carnosine was derivatized with 4-(5,6-dimethoxy-2-phthalimidinyl)-2-methoxyphenylsulfonyl chloride at 70 degrees C for 15 min in borate buffer (20 mmol l(-1), pH 9.0) to produce fluorescent sulfonamides. After hydrolysis of the reaction mixture with formic acid at 100 degrees C for 15 min, the fluorescent derivative of carnosine was separated on a reversed-phase column with a linear gradient elution using solvents of (A) acetate buffer (0.1 mmol l(-1), pH 7.0) and (B) acetonitrile at a flow-rate of 1.0 ml/min and was detected at excitation and emission wavelengths of 318 and 400 nm, respectively. The detection limit of carnosine was 4 fmol at a signal-to-noise ratio of 3. The within-day and day-to-day relative standard deviations were 2.7-4.6% and 0.4-5.2%, respectively. The concentration of carnosine in normal human urine was found to be 4.6-125 nmol (mg creatinine)(-1) (mean+/-SD: 21.6+/-26.6 nmol (mg creatinine)(-1), n=20).

Current Ocular Drug Delivery Challenges for N-acetylcarnosine: Novel Patented Routes and Modes of Delivery, Design for Enhancement of Therapeutic Activity and Drug Delivery Relationships

This review article explores the functional activity and development aspects of N-acetylcarnosine for the visual system as revealed by the use of a variety of biophysical, physiological and therapeutic ophthalmic methods. It is designed for pharmacists and more advanced ophthalmology, optometry and pharmacology researchers who wish to gain a basic understanding of the biological effects of N-acetylcarnosine for vision and to share in the excitement of the latest developments in this field. Topics under the consideration include: ophthalmic drug delivery of N-acetylcarnosine eye drops and challenging endeavors facing the pharmaceutical scientist; clinical and functional types of activity of the developed and patented N-acetylcarnosine lubricant eye drops designed as 1% N-acetylcarnosine prodrug of L-carnosine containing a mucoadhesive cellulose-based compound combined with corneal absorption promoters in a drug delivery system; management of age-related serious or disabling eye diseases in humans with N-acetylcarnosine eye drop therapeutic platform (age-related cataracts, ocular inflammation, age-related macular degeneration , macular dystrophies, ocular manifestations of diabetes , hypertonic retinopathy, primary open angle glaucoma, vitreous lesions) ; development and molecular mechanisms of ocular therapeutic activities of carnosine derivatives in the visual system. Through this article we can perceive some helpful recent patents according to the title of the issue. The biologically significant applications of carnosine mimetics including those in ophthalmology were patented by Dr. Babizhayev and the alliance Groups (WO 2004/028536 A1; WO 94/19325; WO 95/12581; WO 2004/064866 A1).

Enzymatic synthesis of carnosine derivatives catalysed by Burkholderia cepacia lipase

A new enzymatic synthesis of α,β-dipeptides has been developed with particular focus on the preparation of carnosine (β-alanyl-α-histidine) and analogues. The lipase PS-D from Burkholderia cepacia has been used as a catalyst for the formation of the peptide bond starting from a β-lactam and a protected α-amino acid.

Protective Effects of l- and d-Carnosine on α-Crystallin Amyloid Fibril Formation: Implications for Cataract Disease

Mildly denaturing conditions induce bovine alpha-crystallin, the major structural lens protein, to self-assemble into fibrillar structures in vitro. The natural dipeptide l-carnosine has been shown to have potential protective and therapeutic significance in many diseases. Carnosine derivatives have been proposed as potent agents for ophthalmic therapies of senile cataracts and diabetic ocular complications. Here we report the inhibitory effect induced by the peptide (l- and d-enantiomeric form) on alpha-crystallin fibrillation and the almost complete restoration of the chaperone activity lost after denaturant and/or heat stress. Scanning force microscopy (SFM), thioflavin T, and a turbidimetry assay have been used to determine the morphology of alpha-crystallin aggregates in the presence and absence of carnosine. DSC and a near-UV CD assay evidenced that the structural precursors of amyloid fibrils are polypeptide chain segments that lack stable structural elements. Moreover, we have found a disassembling effect of carnosine on alpha-crystallin amyloid fibrils. Finally, we show the ability of carnosine to restore most of the lens transparency in organ-cultured rat lenses exposed to similar denaturing conditions that were used for the in vitro experiments.

Design, Synthesis, and Evaluation of Carnosine Derivatives as Selective and Efficient Sequestering Agents of Cytotoxic Reactive Carbonyl Species

Carnosine aryl derivatives as sequestering agents of RCS: Reactive carbonyl species (RCS) are cytotoxic mediators representing a novel drug target, as they are presumed to play a pathogenic role in several diseases. Carnosine is a selective RCS-sequestering agent, but is rapidly hydrolyzed by serum carnosinase. Herein we describe the in silico design, synthesis, and evaluation of a set of carnosine aryl derivatives.Reactive carbonyl species (RCS) are important cytotoxic mediators generated by lipid oxidation of polyunsaturated fatty acids (PUFAs) and represent a novel drug target, as they are presumed to play a pathogenic role in several diseases. L-Carnosine (L-CAR, beta-alanyl-L-histidine) is a specific detoxifying agent of RCS, but is rapidly hydrolyzed in human serum by carnosinase, a specific dipeptidase. Herein we describe the in silico design, synthesis, and biological evaluation of carnosine derivatives that are resistant to carnosinase and that have increased quenching efficacy. Stability against carnosinase-mediated turnover was achieved by isomerization of the histidine residue, leading to D-carnosine (D-CAR, beta-alanyl-D-histidine), which maintains the same quenching activity of L-carnosine. A molecular modeling approach was then used to design derivatives characterized by an increased quenching efficacy. The most promising candidates were synthesized, and their stability and quenching activity were evaluated. This study describes a set of aryl derivatives that are characterized by high stability in human plasma and a quenching activity toward 4-hydroxy-trans-2-nonenal (HNE), chosen as a model of RCS, up to threefold greater than D-carnosine.

Inhibitory effect of carnosine and N-acetyl carnosine on LPS-induced microglial oxidative stress and inflammation

Chronic inflammation and oxidative stress have been implicated in the pathogenesis of neurodegenerative diseases. A growing body of research focuses on the role of microglia, the primary immune cells in the brain, in modulating brain inflammation and oxidative stress. One of the most abundant antioxidants in the brain, particularly in glia, is the dipeptide carnosine, β-alanyl- l-histidine. Carnosine is believed to be involved in cellular defense such as free radical detoxification and inhibition of protein cross-linking. The more stable N-acetyl derivative of carnosine has also been identified in the brain. The aim of the present study was to examine the role of carnosine and N-acetyl carnosine in the regulation of lipopolysaccharide (LPS)-induced microglial inflammation and oxidative damage. In this study, BV2 microglial cells were stimulated with bacterial LPS, a potent inflammatory stimulus. The data shows that both carnosine and N-acetyl carnosine significantly attenuated the LPS-induced nitric oxide synthesis and the expression of inducible nitric oxide synthase by 60% and 70%, respectively. By competitive spectrophotometric measurement and electrospray mass spectrometry analysis, we demonstrated a direct interaction of N-acetyl carnosine with nitric oxide. LPS-induced TNFα secretion and carbonyl formation were also significantly attenuated by both compounds. N-acetyl carnosine was more potent than carnosine in inhibiting the release of the inflammatory and oxidative stress mediators. These observations suggest the presence of a novel regulatory pathway through which carnosine and N-acetyl carnosine inhibit the synthesis of microglial inflammatory and oxidative stress mediators, and thus may prove to play a role in brain inflammation.

Synthesis and antioxidant activity of new homocarnosine β-cyclodextrin conjugates

Several in vitro and in vivo studies have suggested that carnosine (β-alanil- l-histidine) and homocarnosine (β-aminobutyril- l-histidine) can act as scavengers of reactive oxygen species. β-Cyclodextrin was functionalized with homocarnosine, obtaining the following new bioconjugate isomers: 6 A-[(4-{[(1 S)-1-carboxy-2-(1 H-imidazol-4-yl)ethyl]amino}-4-oxobutyl)amino]-6 A-deoxy-β-cyclodextrin and (2 A S,3 A R)-3A-[(4-{[(1 S)-1-carboxy-2-(1 H-imidazol-4-yl)ethyl]amino}-4-oxobutyl)amino]-3 A-deoxy-β-cyclodextrin. Pulse radiolysis investigations show that the β-cyclodextrin homocarnosine bioconjugates are scavengers of OH radicals because of the formation of stable imidazole-centered radicals and the scavenger ability of glucose molecules of the macrocycle. The ability of these new β-cyclodextrin derivatives to inhibit the copper(II) driven LDL oxidation was determined in comparison with that displayed by the analogous carnosine derivatives. Both the β-cyclodextrin carnosine isomers show a higher protective effect than that of free dipeptide and homocarnosine derivatives, bringing into light the role of the β-CD cavity. The ability of these new β-cyclodextrin derivatives to inhibit the copper(II) driven LDL oxidation was determined in comparison with that displayed by the analogous carnosine derivatives. Both the β-cyclodextrin carnosine isomers show a higher protective effect than that of free dipeptide and homocarnosine derivatives, bringing into light the role of the β-CD cavity.

Detoxification of cytotoxic alpha,beta-unsaturated aldehydes by carnosine: characterization of conjugated adducts by electrospray ionization tandem mass spectrometry and detection by liquid chromatography/mass spectrometry in rat skeletal muscle.

Oxidation of polyunsaturated fatty acids containing phospholipids in tissue generates lipid hydroperoxides, which are further degraded to several products, among which unsaturated aldehydes such as 4-hydroxy-trans-2-nonenal (HNE) play an important role in mediating the pathological effects of oxidative stress. While the reaction of HNE with glutathione (GSH) is a well recognized pathway of detoxification in biological systems, no data are available on HNE interactions with carnosine, a dipeptide (beta-alanyl-L-histidine) present in high concentration in skeletal muscle. The aim of this work was to study the quenching ability of carnosine towards HNE and to characterize the reaction products by electrospray ionization tandem mass spectrometry (ESI-MS/MS), using GSH as a model peptide. GSH incubation with HNE in 1 mM phosphate buffer (pH 7.4) results in the complete disappearance of HNE within 1 h owing to the formation of a Michael adduct, S-(4-hydroxynonanal-3-yl)glutathione. The reaction of HNE with carnosine was studied in different molar ratios and monitored up to 24 h by high-performance liquid chromatography (HPLC) (HNE consumption), MS/MS (infusion) and liquid chromatography mass spectrometry (LC/MS) experiments. Carnosine, although less reactive than GSH, significantly quenched HNE (48.2 +/- 0.9% HNE consumption after 1 h; carnosine:HNE molar ratio 10 : 1). Two reaction products were identified: the Michael adduct, N-(4-hydroxynonanal-3-yl)carnosine involving the imidazolic nitrogen of histidine, and the imine adduct, involving the amino group of the beta-alanine residue. Definitive structure assignment was achieved by chemical reduction with NaBH(4) and multinuclear magnetic resonance experiments. To understand whether carnosine acts as a quencher of unsaturated aldehydes in biological matrices, rat skeletal muscle homogenate was incubated with HNE and the formation of conjugated adducts was determined by LC/MS analysis. Three main products were detected and identified as Michael adducts of HNE with GSH, carnosine and anserine (the N-methylated derivative of carnosine, present in high concentrations in rat muscle). The results indicate that beside GSH, histidine-containing dipeptides could be involved in the detoxification pathway of reactive aldehydes from lipid peroxidation generated in skeletal muscle during physical endurance.

Synthesis of New Carnosine Derivatives ofβ-Cyclodextrin and Their Hydroxyl Radical Scavenger Ability

Several in vitro and in vivo studies have suggested that carnosine can act as a scavenger of reactive oxygen species and intracellular proton buffer. On the other hand, carnosinase is a specific peptidase able to destroy the biological active dipeptide. To overcome this constraint, β-cyclodextrin (β-CD) was functionalized with carnosine to give the following new compounds: 6A-[(3-{[(1S)-1-carboxy-2-(1H-imidazol-4-yl)ethyl]amino}-3-oxopropyl)amino]-6A-deoxy-β-cyclodextrin (1), 6A-[(β-alanyl-L-histidyl)amino]-β-cyclodextrin (2), and (2AS,3AR)-3A-[(3-{[(1S)-1-carboxy-2-(1H-imidazol-4-yl)ethyl]amino}-3-oxopropyl)amino]-3A-deoxy-β-cyclodextrin (3). Pulse-radiolysis investigation showed that the β-CD derivatives 1–3 are excellent scavengers of OH. radicals. Their activity is not only due to the formation of the stable imidazole-centered radical, but also to the scavenger ability of the glucose moieties of the macrocycle (Scheme). This effect is independent of the disposition of the imidazole ring. In fact, the quenching constant values are similar for the three compounds.

Carbonic anhydrase activators: high affinity isozymes I, II, and IV activators, incorporating a beta-alanyl-histidine scaffold.

A novel class of tight binding carbonic anhydrase (CA) activators was designed by using histamine and histidine as lead molecules. Carnosine (beta-Ala-His) derivatives were synthesized by reaction of appropriately derivatized beta-alanines with imidazole/carboxy-protected histidine in the presence of carbodiimides, followed by removal of the various protecting groups. The derivatized beta-alanines mentioned above were in turn obtained by coupling of 4-fluorophenylsulfonylureido amino acids (fpu-AA) or 2-toluenesulfonylureido amino acids (ots-AA) with beta-Ala. Some structurally related dipeptides with the general formula fpu/ots-AA1-AA2 (AA, AA1, and AA2 represent amino acyl moieties) were also prepared by a similar strategy and used thereafter for obtaining CA activators incorporating a modified tetrapeptide scaffold. Many of the new tri-/tetrapeptide derivatives reported here proved to be efficient in vitro activators of three CA isozymes. Very good activity was detected against hCA I and bCA IV, for which some of the new compounds showed affinities in the 1-20 nM range (h = human; b = bovine isozymes), whereas against hCA II, their affinities were in the range of 10-40 nM. Ex vivo experiments showed some of the new activators to strongly enhance cytosolic red cell CA activity after incubation with human erythrocytes. This new class of CA activators might lead to the development of drugs/diagnostic tools for the management of CA deficiency syndromes, as well as for the pharmacological enhancement of synaptic efficacy, spatial learning, and memory. This may constitute a new approach for the treatment of Alzheimer's disease and other conditions in need of achieving memory therapy.

Histidine-containing dipeptides as endogenous regulators of the activity of sarcoplasmic reticulum Ca-release channels

It is shown that histidine-containing dipeptide carnosine ( β-alanyl- l-histidine), which is present in skeletal muscles in millimolar concentrations, decreases the rate of Ca 2+ accumulation by the heavy fraction of sarcoplasmic reticulum from rabbit skeletal muscles. This effect results from the ability of carnosine to induce a rapid Ca 2+ release from the heavy sarcoplasmic reticulum vesicles via activation of the ruthenium red-sensitive Ca-channels. The effect of carnosine is dose-dependent that indicates the presence of saturable site(s) for carnosine in the molecules of Ca-channels. The C 0.5 value for carnosine (the concentration that induces the half-maximal Ca 2+ release) is 8.7 mM. The 1 N-methylated derivative of carnosine, i.e., anserine, also induces a rapid Ca 2+ release with the half-maximal effect at 2.7 mM. Conversely, neither histidine nor β-alanine (both separately and in the mixture) cause Ca 2+ release. In addition, carnosine increases the sensitivity of Ca-channels to their well-known activators (caffeine, AMP, and Ca 2+) and decreases inhibitory effect of low concentrations of Mg 2+. It is concluded that carnosine as a component of skeletal muscles can be an endogenous regulator of the sarcoplasmic reticulum Ca-channel activity.

Detection of pork in processed meal: experimental comparison of methodology: W.N. Sawaya, T. Saeed, M. Mameesh, E. El-Rayes, A. Husain, S. Ali and H. Abdul Rahman, Food Chem. 37, 201–219

It is shown that histidine-containing dipeptide carnosine (β-alanyl-l-histidine), which is present in skeletal muscles in millimolar concentrations, decreases the rate of Ca2+ accumulation by the heavy fraction of sarcoplasmic reticulum from rabbit skeletal muscles. This effect results from the ability of carnosine to induce a rapid Ca2+ release from the heavy sarcoplasmic reticulum vesicles via activation of the ruthenium red-sensitive Ca-channels. The effect of carnosine is dose-dependent that indicates the presence of saturable site(s) for carnosine in the molecules of Ca-channels. The C0.5 value for carnosine (the concentration that induces the half-maximal Ca2+ release) is 8.7 mM. The 1N-methylated derivative of carnosine, i.e., anserine, also induces a rapid Ca2+ release with the half-maximal effect at 2.7 mM. Conversely, neither histidine nor β-alanine (both separately and in the mixture) cause Ca2+ release. In addition, carnosine increases the sensitivity of Ca-channels to their well-known activators (caffeine, AMP, and Ca2+) and decreases inhibitory effect of low concentrations of Mg2+. It is concluded that carnosine as a component of skeletal muscles can be an endogenous regulator of the sarcoplasmic reticulum Ca-channel activity.