DETAILED DESCRIPTION

[0038] FIG. 1 is a flow diagram of protein glycation and lipid peroxidation and shows the involvement of reactive dicarbonyls that lead to accumulation of AGE-products and other damage on proteins, nucleic acids and lipids. FIG. 2 shows the process in more detail. Protein-AGE include protein N ^ε -(carboxymethyl)lysine residues (CML) (Ahmed, M. U., S. R. Thorpe, and J. W. Baynes. 1986. Identification of N epsilon-carboxymethyllysine as a degradation product of fructoselysine in glycated protein. J Biol Chem. 261:4889-4894) and a heterogeneous group of complex modifications such as pentosidine (Sell, DR., and V. M. Monnier. 1989. Structure elucidation of a senescence cross-link from human extracellular matrix. Implication of pentoses in the aging process. J Biol Chem. 264:21597-21602) that are characterized by their high fluorescence and ability to cause protein-protein cross-links. Accumulation of AGE-specific At fluorescence (ex. 370 rim; em. 440 mn) is a general measure of overall protein damage and it is a widely used tool of glycation research in vitro and in vivo. In some cases, reactive dicarbonyl compounds may form by auto-oxidation of the sugar itself without requiring glycation, and the presence of trace amounts of transition metal ions (Fe, Cu) has been implicated in the formation of dicarbonyl compounds and reactive oxygen species such as hydrogen peroxide as reported by Elgawish, A., M. Glomb, M. Friedlander, and V. M. Monnier. 1996. Involvement of hydrogen peroxide in collagen cross-linking by high glucose in vitro and in vivo. J Biol Chem. 271:12964-12971.

[0039] Research has demonstrated that the cell nucleus is a likely site for protein glycation in vivo by ADP-ribose (see Cervantes-Laurean, D., D. E. Minter, E. L. Jacobson, and M. K. Jacobson. 1993. Protein glycation by ADP-ribose: studies of model conjugates. Biochemistry. 32:1528-1534. Jacobson, E. L., D. Cervantes-Laurean, and M. K. Jacobson. 1994. Glycation of proteins by ADP-ribose. Mol Cell Biochem. 138:207-212. Cervantes-Laurean, D., E. L. Jacobson, and M. K. Jacobson. 1996. Glycation and glycoxidation of histones by ADP-ribose. J Biol Chem. 271:10461-10469; Jacobson, E. L., Cervantes-LAureari, D., & Jacobson, M. K. 1997. ADP-Ribose in Glycation and Glycoxidation Reactions. Jn ADP-Ribosylation in Animal Tissues. H. Koch-Nolte, editor. Plenum Press, New York. 371-379; and Jacobson, E. L., D. Cervantes-Laurean, and M. K. Jacobson. 1997. ADP-ribose in glycation and glycoxidation reactions. Adv Exp Med Biol. 419:371-379).

[0040] Oxidative stress and other conditions that cause DNA strands breaks stimulate the synthesis of nuclear polymers of ADP-ribose, which are rapidly turned over generating ADP-ribose in close proximity to the long lived histones rich in lysine and arginine residues as depicted in FIG. 3 . Based on this research a simple reaction system was established allowing the assessment of nuclear glycation damage and its supression by inhibitory substances.

[0041] As mentioned above, glycation and subsequent protein-AGE formation plays a central role in glucose toxicity. Administering the glycation inhibitor aminoguanidine effectively suppresses secondary complications in rodents with experimental diabetes (Edelstein, D., and M. Brownlee. 1992. Aminoguanidine ameliorates albuminuria in diabetic hypertensive rats. Diabetologia. 35:96-97). Aminoguanidine is thought to act as a dicarbonyl scavenger, therefore inactivating toxic reactive dicarbonyl compounds. However, aminoguanidine is a hydrazine derivative that shows systemic toxicity upon long-term administration, since it is a potent inhibitor of catalase (Ou, P., and S. P. Wolff. 1993. Aminoguanidine: a drug proposed for prophylaxis in diabetes inhibits catalase and generates hydrogen peroxide in vitro. Biochem Pharmacol. 46:1139-1144) and inducible nitric oxide synthase (Okuda, Y., S. Sakoda, H. Fujimura, and T. Yanagihara. 1998. Aminoguanidine, a selective inhibitor of the inducible nitric oxide synthase, has different effects on experimental allergic encephalomyelitis in the induction and progression phase. J Neuroimmunol. 81:201-210). The toxicity profile of aminoguanidine makes it a poor candidate for clinical use. Therefore, a need exists in the medical art for new compounds that effectively inhibit glycation and its associated pathological consequences. The following examples demonstrate the trapping reaction of the alpha-oxoaldehydes methylglyoxal and phenylglyoxal by α-amino-β, 62 -mercapto-β,β-dimethyl-ethane derivatives such as penicillamine for protection of human skin against carbonyl stress.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

EXAMPLE 1

[0042] Chemicals

[0043] All chemicals were from Sigma Chemical co. Calftissue (thymus) frozen in liquid nitrogen immediately after collection, was from Pel-Frez Biologicals.

[0044] Preparation of Glycosylated Bovine Serum Albumin (AGE-BSA)

[0045] AGE-BSA was prepared as described by Takata K H, Araki N, Shiga M, Saitoh M, Morino Y, Endocytic uptake of nonenzymatically glycosylated proteins is mediated by a scavenger receptor for aldehyde-modified proteins. Biochemistry 263: 14819-25, 1988. Briefly, 1.6 g of BSA and 3.0 g of D-glucose were dissolved in 10 mL of 0.5 M sodium phosphate buffer, pH 7.4, containing 0.05% NaN ₃ . The solution was filter sterilized through a 0.45 μm filter and incubated in the dark for 90 days at 37° C. Following dialysis against water, the sample was lyophilized.

[0046] Isolation of Histone H1 from Calf Thymus

[0047] All operations were carried out at 4° C. Chromatin was isolated from fresh calf thymus by extraction with 0.14 M NaCl, 0.05 M Na ₂ S ₂ O ₅ , as described earlier in Wondrak supra. After repeated extraction with 5% HC1) ₄ and centrifugation (1500 g), histone H1 was precipitated from the supernatant by addition of TCA (20% final concentration v/v). The histone H1 precipitate was colleted by centrifugation (12,000 g) and deionized water. After extensive dialysis (MW cut-off: 12,000-14,000) against water for 48 h, the sample was lyophilized and the protein was stored at 4° C. SDS-PAGE (12%) was used to analyze the purity of the preparation.

[0048] Glycation of histone H1 by ADP-ribose is fully inhibited by penicillamine and penicillamine derivatives. Earlier studies have reported that D-penicillamine inhibits collagen crosslinking and AGE fluorescence caused by sugars (McPherson, J. D., Shilton, B. H., and Walton, D. J. 1988. Role of Fructose in Glycation and Cross-Linking of Proteins. Biochemistry 27, 1901-1907) and reaction of D-penicillamine with aldehyde groups of proteins (Deshmukh, K., and Nimm, M. E. 1969. A Defect in the Intramolecular and Intermolecular Cross-linking of Collagen caused by Penicillamine. J.Biol. Chem. 244, 1787-1795). A number of different penicillamines and penicillamine derivatives were tested to determine the inhibition of AGE-fluorescence on histone H1 at physiological pH. The reaction conditions for the glycation of histone H1 by ADP-ribose mimic physiological conditions to the extent possible. Reaction mixtures contain 1.5 mg/ml histone H1, 1.0 mM ADP-ribose, 50 mM potassium phosphate buffer, pH 7.4, 37° C. D,L-penicillamine; L-penicillamine; D-penicillamine; D-penicillaminedisulfide and N,S-isopropylidine-D-penicillamine were tested in concentrations of 1, 5 and 10 mM. The detected reaction parameter representing the accumulation of protein damage is AGE-fluorescence ((λex=370 nm; λem = 440 nm), which is suppressed by the presence of a compound with inhibitory activity. Generation of AGE-type fluorescence (λex=355 nm; λem=405 nm) Wr;, was monitored over time at 37° C., which is in the range of the broad excitation/emission maxima of AGE compounds. To allow high throughput sample processing on 96 well microtiter plates, the reaction volumes are 300 μl. Fluorescence on the 96-well microtiter plates was measured using an automated microtiter fluorescence reader. Fluorescence of the 1 ml samples of the glycation reaction mixtures was measured using a Hitachi F-200 fluorescence spectophotometer, and prior to measurement the protein sample is dialyzed extensively against water (MW cutoff=10,00 Daltons), lyophilized and reconstituted in reaction buffer. For inhibitor screening the plate is read on a Fluoroskan II plate reader (Titertek, ICN) at the excitation/emission wavelengths set forth above at a bandwidth of 35 nm.

[0049] The following screening scheme is used (see Table 1): AGE-fluorescence is determined at the beginning and after five days of incubation. Test compounds that are inherently fluorescent (designated false negatives) were identified by the initial fluorescence measurement (See Table 1, NADH for example). Since these are compounds of uncertain activity, they are diverted directly to the second stage of the screen. Aminoguanidine, a known glycation inhibitory agent, was used as a positive control for suppression of the increase on AGE-fluorescence. After five days incubation and plate reading, fluorescence quenchers (designated false positives) are excluded by measuring the quenching activity of the test compound by addition of AGE modified protein having known fluorescence activity to one microtiter plate well containing test compound in the complete reaction mixture. For this, AGE-BSA is used as the AGE-type fluorescence standard. This AGE-BSA test excludes false positives compounds that function by fluorescence quenching. If fluorescence quenching occurs the test compound is excluded from further screening. Potential positive compounds are further analyzed by measuring inhibition of protein-crosslinking by 12% SDS-PAGE analysis of a 3 microliter aliquot taken from the reaction well on the plate. The protein is visualized by silver staining of the gel. Untreated histone H1 and the positive control containing aminoguanidine are loaded onto the gel together with the samples of potential positive test compounds. A compound that passes the first and second stage of the screen is considered a glycation inhibitor and is further evaluated for biological activity as described below.

[0050] D-penicillamine (5 mM) and aminoguanidine (5 mM) were shown to inhibit histone H1 crosslinking measured by 12%-SDS-PAGE followed by silver staining as described ( FIG. 6 ). Crosslinking was detected with high sensitivity in a histone H1, ADP-ribose reaction system paralleling formation of high AGE-type fluorescence (C), inhibition with aminoguanidine (AG), inhibition with D-penicillamine (P).

[0051] Preparation and Structure Elucidation of the D-penicillamine-Methylglyoxal Reaction Product as 2-acetyl-5,5-dimethyl-thiazolidine-4-carboxylic Acid

[0052] To a solution of D-Penicillamine (350 mg, 2.3 mmol) in 50 ml of aqueous 0.20 M phosphate buffer (pH7.4) was added MG (40% in water/620 microiliters, 3.45 mmol). Te reaction mixture was stirred at 37° C. for 24 h. The solvent was concentrated to half volume at reduced pressure and the residue was desalted on Amberchrome CG 71 ms resin (1.5×45 cm) (TosoHaas, Philadelphia, Pa.). The column was developed with water. The UV absorbing peaks were pooled, and the water was evaporated at reduced pressure, The crude product was purified by anion exchange chromatography on a 1.5×45 column of QAE Sephadex 25 (Sigma), developed by application of a linear gradient formed between 200 ml of distilled water and 200 ml of 0.2M NH4HCO3. Fractions were collected, and absorbance at 254 nm was measured. Fractions constituting a single major peak eluting about midway in the gradient were pooled and concentrated. The 1H-NMR spectrum exhibited the following signals: (δ _H D2O in ppm): 1.03 (3H, s, CH3), 1.90 (3H, s, CO—CH3), 3.96 and 3.98 (diastereomeric, 1H, s, CH—COOH). Mass spectrometric analysis by MALD-TOF-MS revealed a (M+Na) ⁺ of 226 Daltons.

[0053] Preparation and Structure Elucidation of the D-Penicillamine-Phenylglyoxal Reaction Product as 2-benzoyl-5,5-dimethyl-thiazolidine-4-carboxylic Acid

[0054] Phenylglyoxal (10 mM) and D-penicillamine (20 mM) were reacted in 50 mM KH2PO4 buffer, pH 7.4 at room temperature. The progress of he reaction was monitored by HPLC analysis of reaction aliquots at 254 nm. After 40 minutes reaction time more than 90% conversion of the phenylglyoxal peak into a single product peak of higher retention time was observed. The reaction product was obtained by preparative HPLC, lyophilized and analyzed by 1H-NMR spectroscopy. The spectrum exhibited the following signals: (δ _H D2O in ppm): 1.38 (3H, s, CH3), 1.45 (3H, s, CH3), 4.12 (1H, s, CH—COOH), 7.42-7.82 (5H, m, ArH). UV detection was at 254 nm. As shown by the HPLC analysis of the reaction system shown in FIG. 7 , phenyglyoxal is fully reacted with D-penicillamine within 5 minutes.

[0055] The ¹ H-NMR spectrum of the reaction product formed in the reaction is shown in FIG. 8 . The product formed is a thiazolidine derivative whose proposed structure and the proposed mechanism of trapping is also shown in FIG. 8 .

[0056] Penicillamines are effective dicarbonyl scavengers and may be administered to subjects to prevent AGE formation and other types of direct damage that result from dicarbonyls in vivo. Penicillamines will be administered to the subject in sufficient doses to accomplish these therapeutic goals, and will find particular use in the treatment of diabetics.

Reaction Kinetics of Alpha-Dicarbonyl Trapping

[0057] The reaction of phenylglyoxal with test compounds were carried out in 10 mM phosphate buffer, pH 7.4 at 37° C. and were followed by HPLC analysis. The reaction kinetics were studied at a phenylglyoxal concentration of 50 micromoles and at 250 and 500 micromolar carbonyl scavenger concentration (D-penicillamine, aminoguanidine). Over the course of the reaction, aliquots were analyzed by HPLC. In the case of D-penicillamine, which required shorter sampling periods, reaction aliquots were taken every 20 seconds, and kept on dry ice until analysis. The initial reaction rates of phenylglyoxal with the test compounds were monitored by following the disappearance of phenylglyoxal over time. The reaction between phenylglyoxal and the test compounds is a second order reaction with a rate equation of −dc/dt=K _2nd {phenylglyoxal} {dicarbonyl scavenger}. The reaction was conducted in the presence of excess test compounds (ratio of 1:5 and 1:10 phenylglyoxal to test compound), to convert it to pseudo-first order reaction kinetics as demonstrated by the apparent dependency of the reaction rate constant (k _1st ) on the concentration of the test compound. A plot of Log AUC for phenylglyoxal versus time resulted in a slope equal to k _1st /2.303. The measured first order rate constant (k _st ) was then used to calculate the second order rate constant (by applying the following equation: k _2nd =k _1st {alpha-carbonyl scavenger). The calculated second order rate constants determined at the two reactant ratios were in good agreement.

[0058] Cell Culture

[0059] A continuous cell line of human epidermal keratinocytes (HaCat cells) and human dermal fibroblasts (CF-3 cells) were routinely cultured in 75 cm ² flasks and split biweekly in DMEM containing 10% fetal bovine serum and kept in a humidified atmosphere containing 5% CO ₂ at 37° C. Human keratinocytes were split using 5% trypsin and human fibroblasts employed 1% trypsin. All experiments were carried out on 6 well dishes (Falcon USA), where keratinocytes were seeded at 2×10 ⁴ cells/well and fibroblasts at 4×10 ⁴ cells/well. Cells were left overnight to attach to the plate and the appropriate carbonyl scavenger was then added 15 minutes prior to the addition of the alpha-dicarbonyl stress compounds glyoxal or methylglyoxal. Following 72 hour exposure to the stress compound, cells were counted with a Coulter counter. the protective effects of the alpha-dicarbonyl scavengers were assessed by comparing the growth of untreated cells with cells exposed to alpha-carbonyl stress+/−test compound.

EXAMPLE 3

[0060] Glycated Proteins as Photosensitizers of DNA Damage in Skin Photoaging. D-Penicillamine inhibits genotoxic consequences of AGE-photosensitization. Accumulation of AGEs on dermal elastin and collagen occurs during normal skin aging in humans. The hypothesis was tested that the intra- and extracellular accumulation of the complex yellow-brown AGE-chromophores contributes to skin aging and carcinogenesis induced by chronic exposure to sunlight. As a possible molecular mechanisms for a detrimental synergism of AGE-formation and exposure to sunlight, photosensitized DNA damage by AGEs was assessed in a simple in vitro system. Irradiation of covalently closed circular ΦX-174 DNA with increasing doses of solar simulated light (SSL) in the presence of AGE-BSA was used to detect photosensitized DNA nicking as a measure of DNA photodamage (panel A). Upon exposure to SSL (0.8-16 J/cm ² ), the damage was concentration dependent with respect to AGE-BSA as the photosensitizer (panel B). Unmodified BSA displayed no such photosensitizing activity. Addition of several antioxidants modulated the photosensitization effect (panel C). Mannitol (a hydroxyl radical quencher) and NaN ₃ (a sinlet oxygen quencher) blocked the photosensitized DNA cleavage, whereas catalase and SOD were not effective, indicating the involvement of photoactivated oxygen and hydroxyl radicals. The thiol-antioxidant and RCS-scavenger D-penicillamine inhibited the photosensitization effect of AGE-BSA in a dose dependent relationship (panel D). Results are shown in FIG. 10 .

[0061] Photosensitized DNA damage in skin is thought to be an important mechanism of UVA phototoxicity. Taken together with a recently published report on reduced viability of human dermal fibroblasts exposed to UVA irradiation in the presence of protein modified by AGEs, these preliminary results suggest that glycated skin proteins can function as photosensitizers of DNA damage. Future research efforts should clarify the genotoxic consequences of skin glycation implicated in this in vitro study.

EXAMPLE 4

[0062] Inhibition of AGE-Photosensitization of Human Skin Cells by d-Penicillamine

[0063] Human HaCat keratinocytes growing on 35 mm dishes were exposed to solar simulated light (1.2 kJ/m ² UVB and 23 kJ/m ² UVA) in the presence or absence of AGE-BSA as a model of a protein with advanced glycation endproducts. Control populations were treated in exactly the same way as described above, but were not exposed to solar simulated light (SSL). Exposure of HaCat cells to SSL effected a 20% growth inhibition compared to cells which were not exposed (see below). However, cells exposed to SSL in the presence of 10 mg/mL AGE-BSA were growth inhibited by 80%, showing that AGE-BSA is a photosensitizer, effecting a 4 fold increase in growth inhibition as compared to SSL alone. Exposure to 10 mg/mL AGE-BSA alone in the absence of SSL had no effect on cell growth. Results are shown in FIG. 16 .

[0064] Hence, AGE-BSA is completely non-toxic in the absence of SSL, but can exert significant toxicity when photoactivated by SSL. Furthermore, 10 mM D-penicillamine, previously shown to exert no toxic effects on HaCat cells, completely reversed the photosensitization effect. Therefore, D-penicillamine is an effective inhibitor of photosensitization exerted by glycated proteins and related age-pigments in human skin.

EXAMPLE 5

[0065] Human skin consists of keratinocytes and fibroblasts growing in a collagen matrix. Skin aging, as well as certain pathological conditions, e.g. diabetes, leads to the collagen matrix becoming glycated.

[0066] The experiment described above was repeated in exactly the same way, but CF-3 fibroblasts and glycated collagen (AGE-collagen) was used (see above). Exposure to 2 mg/mL AGE-collagen with SSL also showed a photosensitization effect. It is therefore feasible that D-penicillamine can be used topically to inhibit photosensitization effects in human skin relevant for the prevention of skin photoaging and skin photocarcinogenesis. Result are shown in FIG. 17 .

[0067] The results from the above experiments show that α-amino-β,β-mercapto-β,β-dimethyl-ethane derivatives (pharmacophore), especially penicillamines, are useful in prevention of AGE-related damage to the skin of a subject, particularly mammals such as humans. The results also show that these compounds provide effective protection of skin cells and genetic toxicity induced by photoaging. This can be accomplished, e.g., by systemic delivery through oral, parenteral, e.g., intravenous, topical or other suitable delivery means. A sufficient dose of the agent will be given to produce the desired effect in the subject, which can be any animal, mammal, reptile, etc. The dose will vary upon a variety of factors known to those skilled in the art, e.g., weight, desired therapeutic endpoint, weight of the subject, etc. 1

[0068] Other embodiments of the invention will be readily apparent to those skilled in the art and are meant to be within the scope of the claims appended hereto.

[0069] All cited references are hereby incorporated by reference.