Annette Dietrich

Synergistic effects of thalidomide and poly (ADP-ribose) polymerase inhibition on type II collagen-induced arthritis in mice.

The present study investigates synergistic effects of the TNF-α inhibitor thalidomide and the poly(ADP-ribose) polymerase (PARP)-inhibitor nicotinic acid amide (NAA) in male DBA/1 hybrid mice suffering from type II collagen-induced arthritis. Parameters including the arthritis index, chemiluminescence and anti-collagen antibody titers were used for the assessment of disease activity: The disease courses demonstrated clearly an inhibitory effect of thalidomide. NAA inhibited established collagen arthritis in a dose-dependent manner. The combined application of thalidomide and NAA caused a powerful synergistic inhibition of arthritis. Furthermore, thalidomide and NAA were tested ex vivo for their inhibition of the NADPH oxidase-dependent generation of reactive oxygen species by activated neutrophils and monocytes in unseparated human blood. Our data show that type II collagen-induced arthritis can be suppressed by the simultaneous inhibition of TNF-α, PARP, and NADPH oxidase.

Protection from acetaminophen-induced liver damage by the synergistic action of low doses of the poly(ADP-ribose) polymerase-inhibitor nicotinamide and the antioxidant N-acetylcysteine or the amino acid l-methionine

1. An array of therapeutically used analgetic and antirheumatic drugs cause severe liver damage. The present study investigates the hepatoprotective effects of inhibitors of NAD-dependent adenoribosylation reactions and of antioxidants in analgesic-induced hepatic injury. 2. Male NMRI mice were treated PO with 500 mg/kg of acetaminophen, and the activities of both glutamate-oxaloacetate transaminase (GOT) and glutamate-pyruvate transaminase (GPT) were determined in serum. 3. The acetaminophen-induced release of both GOT and GPT from injured liver cells could be inhibited in a dose-dependent manner, when mice were injected additionally either with increasing amounts (from (25 mg/kg to 100 mg/kg i.p.) of the PARP-inhibitor nicotinamide, with increasing amounts (from 25 mg/kg to 100 mg/kg i.p.) of the antioxidant N-acetylcysteine, or with increasing amounts (from 50 mg/kg to 300 mg/kg i.p.) of the amino acid L-methionine. 4. A combination of both nicotinamide and N-acetylcysteine (at the low dose of 12.5 mg/kg i.p. each) results in a complete protection from acetaminophen-induced release of GOT and GPT from injured liver cells. 5. A combination of both L-methionine and N-acetylcysteine or nicotinamide (at the low dose of 12.5 mg/kg IP each) resulted also in complete protection from acetaminophen-induced release of GOT and GPT.

Suppression of type II collagen-induced arthritis by N-acetyl-L-cysteine in mice.

1. The antiarthritic and anti-inflammatory efficacy of N-acetyl-L-cysteine (NAC) was tested in male DBA/1 hybrid mice suffering from type II collagen-induced arthritis. Parameters including the arthritis index and the phagocytic responses recorded by chemiluminescence in unseparated blood were used for the assessment of disease activity. 2. Mice were immunized by subdermal injection of bovine type II collagen in Freunds complete adjuvant. The treatment with NAC started at day 42 after immunization and was continued over a period of six weeks: in doses ranging up to 50 mg/kg, a dose-dependent suppression of arthritis was noted; between 50 and 200 mg/kg, the inhibition curve had a plateau [ED50 = 50 mg/(kg x day)]. 3. The arthritis index correlated positively with the generation of chemiluminescence by reactive oxygen species (ROS) produced in neutrophils and monocytes activated by 12-O-tetradecanoylphorbol 13-acetate. 4. After treatment with 100 mg/kg of NAC from day 42 after immunization over a period of six weeks, the ROS production was reduced to levels occurring in whole blood of healthy animals. 5. It is concluded that low-molecular-weight antioxidants such as NAC may be adequate for controlling oxidative stress-derived damage in rheumatic diseases by modulation of ROS-dependent signal transduction pathways.

Assessment of Collagen Type II Induced Arthritis in Mice by Whole Blood Chemiluminescence

Lucigenin-enhanced chemiluminescence can be measured in 100 microliters samples of whole, unseparated mouse blood. A procedure for doing so is here described in detail, using a standard clinical luminometer. The assay measures the TPA-induced oxidative burst from granulocytes and macrophages, which is believed to depend on the overall level of inflammation in the body. It is here applied to mice suffering from type II collagen-induced arthritis, and its relation to overt disease symptoms (the arthritis score) is characterised during the course of the disease. A correlation between the assay and the arthritis score is found at the height of the disease (r = 0.42, p = .039), but not at early or very late time points, although there is a strong hint that the results of an early assay may predict the subsequent disease course. The assay provides a rapid, convenient, quantitative and economical method of assessing disease activity, which can be carried out repeatedly on the same individual. It should be applicable in other mouse models of chronic inflammatory disease. It may find application for rapid screening of novel anti-rheumatic drugs and treatments.

Suppression of arthritis by an active center analogue of Cu2Zn2-superoxide dismutase

The anti-arthritic and anti-inflammatory efficacy of CuPu(Py)2 {[N, N′-bis(2-pyridylmethylene)-1,4-butanediamine] (N, N′, N″, N‴)}-Cu(II), a serum-stable active center analogue of Cu2Zn2-superoxide dismutase (SOD; EC 1.15.1.1), was tested in male DBA/1xB10A (4R) mice suffering from potassium-peroxochromate-induced (PIA) or collagen type II-induced arthritis (CIA). Parameters including the arthritis index, the plasma SOD activity, and the inhibition of phagocytic responses in unseparated blood were used for the assessment of disease activity. A dose-dependent suppression of arthritis was noted in both models. The ED50 was 2.5±0.4 μmol/kg/day of CuPu(Py)2 for PIA and 4.0±1.1 μmol/kg/day for CIA. The arthritis index correlated with both the levels of reactive oxygen species (ROS) generated by phorbol ester-activated neutrophils and monocytes in unseparated blood (r=0.892) and the SOD-like activity in plasma (r=0.857). CuPu(Py)2 inhibited also the lipoplysaccharide-induced release of tumor necrosis factor alpha from human monocytes and neutrophils in a dose-dependent manner. Unlike SOD, which exerts successful anti-rheumatic activity mainly upon intra-articular injection, the SOD-mimic CuPu(Py)2 can be applied systemically. Non-proteinaceous low molecular weight antioxidases may well be suited to control oxidative stress-derived damage in rheumatic diseases by modulation of ROS-dependent signal transduction pathways.

The effect of tryptophan plus methionine, 5-azacytidine, and methotrexate on adjuvant arthritis of rat.

Within the wider framework of our studies on the genesis of rheumatoid arthritis we have investigated the two signal processes in arthritis: adenoribosylation of proteins and DNA methylation. Arthritis can be induced when Freunds complete adjuvant is applied to rats. This form of arthritis can then be reduced or even totally suppressed through the application of several different substances. In the present article we have investigated if the effect of two of these substances, 5-azacytidine and methotrexate can be influenced by the application of tryptophan plus methionine. When applied singly, these latter two substances are known to reduce the formation of arthritis. This effect is intensified by a combination of tryptophan plus methionine. Application of tryptophan plus methionine without methotrexate or 5-azacytidine causes an enhanced development of an adjuvant induced arthritis.

Influence of diet free of NAD-precursors on acetaminophen hepatotoxicity in mice

Recently, we demonstrated the hepatoprotective effects of nicotinic acid amide, a selective inhibitor of poly(ADP-ribose) polymerase (PARP; EC 2.4.2.30) on mice suffering from acetaminophen (AAP)-hepatitis, suggesting that the AAP-induced liver injury involves a step which depends on adenoribosylation. The present study investigates the effects of a diet free of precursors of NAD, the substrate on which PARP acts, in female NMRI mice with AAP hepatitis and evaluates the influence of simultaneous ethanol consumption in these animals. Liver injuries were quantified as serum activities of glutamate-oxaloacetate transaminase (GOT) and glutamate-pyruvate transaminase (GPT). While AAP caused a 117-fold elevation of serum transaminase activities in mice kept on a standard laboratory diet, which was significantly exacerbated by ethanol and inhibited by nicotinic acid amide (NAA), adverse effects were noted in animals fed a diet free of precursors of NAD. In these animals, only minor increases of serum transaminase activities were measured in the presence of AAP, and unlike the exacerbation caused by ethanol in mice on a standard diet, the liver damage was inhibited by 50% by ethanol. A further 64% reduction of hepatitis was observed, when NAA was given to ethanol/AAP-mice. Our results provide evidence that the AAP-induced hepatitis and its exacerbation by ethanol can either be reduced by end-product inhibition of PARP by NAA or by dietary depletion of the enzymes substrate NAD. We see the main application of NAA as for the combinational use in pharmaceutical preparations of acetaminophen in order to avoid hepatic damage in patients treated with this widely used analgesic.

Exacerbation of acetaminophen hepatotoxicity by thalidomide and protection by nicotinic acid amide

1. The effects of racemic thalidomide (D[+]/L[-] alpha-phthalimido-glutarimide) on acetaminophen (AAP)-induced hepatitis were tested in male NMRI mice (n = 133) and quantified as serum activities of glutamate-oxaloacetate transaminase (GOT) and glutamate-pyruvate transaminase (GPT). 2. A 2.1-fold increase of GOT and a 1.9-fold increase of GPT activities (P < 0.001) were observed in mice treated perorally with 500 mg/kg of AAP plus 150 mg/kg of thalidomide (Thal). In the absence of AAP, Thal did not display any detectable hepatotoxic effects. 3. The Thal-induced exacerbation of AAP hepatotoxicity was completely inhibited by nicotinic acid amide, a selective inhibitor of poly(ADP-ribose) polymerase (PARP) (P < 0.0001), suggesting a possible influence of Thal on the hepatic metabolism of NAD-adenoribosylation. 4. We see the main application of nicotinic acid amide as for the combinational use in pharmaceutical preparations of AAP in order to avoid hepatic damage in patients treated with AAP and Thal.

The therapeutic effect of a combination of cofpropamine, a caffeine derivative, and cyclophosphamide on the development of adjuvant arthritis of rats and collagen arthritis of mice.

1. Cofpropamine (Cofa), a caffeine derivative that inhibits polyadenoribosylation, enhances the therapeutic effect of cyclophosphamide (CPA) in two animal models of arthritis. 2. The development of adjuvant arthritis of rats is reduced by treatment with 2 x 50 mg/kg IP CPA and 2 x 50 mg/kg IP Cofa. 3. The development of collagen arthritis in mice is prevented by treatment with 12.5 mg/kg IP CPA and 150 mg/kg IP Cofa three times per week.

The Influence of Antagonists of Poly(Adp-Ribose)Polymerase on the Activity Of Antirheumatic Drugs on the Development of Adjuvant Arthritis in Rats and on the Induction of Tyrosine Aminotransferase in the Liver of Rats

Poly-adenoribosylation is a posttranslational modification of chromatin-bound proteins. Recently, there has been increasing evidence that poly-adenoribosylation is involved in the genesis of cancer but also in the genesis of autoimmune diseases such as rheumatoid arthritis. The synthesis of poly(ADP-ribose) by the enzyme poly(ADP-ribose)polymerase depends on the intracellular levels of NAD and this depends on the supply with the precursers tryptophan, nicotinamide, and nicotinic acid. Poly-adenoribosylation is a protective cellular response to DNA damage which leads to a decrease in the NAD content of cells (Kroger et al., 1960). The consequence is a depletion of NAD with a reduced poly(ADP-ribose) accumulation and altered biological responses such as the expression of disease specific genes (Jacobson et al., 1992, Ehrlich, et. al., 1995). The question has been raised whether agents or conditions that influence the NAD-poly(ADP-ribose)metabolism do have an effect on the pathogenesis of diseases such as rheumatoid arthritis. Previously, we reported that a combination of benzamide or a diet without tryptophan and nicotinamide in combination with D-penicillamine leads to an improvement of adjuvant arthritis in rats (Kroger and Ehrlich, 1994). We continued these studies and investigated the effects of the poly(ADP-ri- bose)polymerase antagonists nicotinamide, benzamide, caffeine-derivatives and a diet lacking tryptophan and nicotinamide alone and in combination with the antirheumatic drugs endoxan (cyclophosphamide), D-penicillamine, auranofin, voltaren, and methotrexate on the development of adjuvant arthritis of rats. In another set of experiments we studied the influence of D-penicillamine, benzamide, and cofpropamine (a caffeine-derivative) on the induction of tyrosine aminotransferase in the liver of rats with adjuvant arthritis set on a diet without tryptophan and nicotinamide.