Poly(ADP-ribose)polymerase inhibition counteracts renal hypertrophy and multiple manifestations of peripheral neuropathy in diabetic Akita mice.

Poly(ADP-ribose) polymerase (PARP) activation has been implicated in the pathogenesis of diabetic complications, including nephropathy and peripheral neuropathy. This study aimed at evaluating the manifestations of both complications in diabetic Akita mice, a model of Type 1 (insulin-dependent) diabetes, and their amenability to treatment with the potent PARP inhibitor, 10-(4-methyl-piperazin-1-ylmethyl)-2H-7-oxa-1,2-diaza-benzo[de] anthracen-3-one (GPI-15427). Male non-diabetic C57Bl6/J and diabetic C57Bl/6-Ins2Akita/J (Akita) mice were maintained with or without treatment with GPI-15427, 30 mg/kg/day, for 4 weeks starting from 16 weeks of age. Sixteen week-old Akita mice displayed sensory nerve conduction velocity (SNCV) deficit, whereas the motor nerve conduction velocity (MNCV) tended to decrease, but the difference with controls did not achieve statistical significance. They also developed thermal and mechanical hypoalgesia and tactile allodynia. SNCV deficit, mechanical hypoalgesia, and tactile allodynia progressed with age whereas the severity of thermal hypoalgesia was similar in 16- and 20-week-old Akita mice. PARP inhibition alleviated, although it did not completely reverse, SNCV deficit, thermal and mechanical hypoalgesia and tactile allodynia. Sixteen-week-old Akita mice displayed MNCV deficit (41.3±2.5 vs. 51.0±1.2 m/sec in non-diabetic controls, P<0.01), axonal atrophy of myelinated fibers, kidney hypertrophy, and albuminuria. MNCV slowing, axonal atrophy, and kidney hypertrophy, but not albuminuria, were less severe in GPI-15427-treated age-matched Akita mice. Neuroprotective and nephroprotective effects of PARP inhibition were not due to alleviation of diabetic hyperglycemia, or peripheral nerve p38 mitogen-activated protein kinase activation. GPI-15427 did not affect any variables in control mice. In conclusion, the findings support an important role for PARP activation in diabetic peripheral neuropathy and kidney hypertrophy associated with Type 1 diabetes, and provide rationale for development and further studies of PARP inhibitors, for the prevention and treatment of these complications.

Dietary flavonoids as cancer prevention agents.

Dietary agents identified from fruits and vegetables contribute to keeping balanced cell proliferation and preventing cell carcinogenesis. Dietary flavonoids, combined with other components such as various vitamins, play an important role in cancer prevention. Flavonoids act on reactive oxygen species, cell signal transduction pathways related to cellular proliferation, apoptosis, and angiogenesis. Many studies demonstrate that flavonoids are responsible for chemoprevention, although mechanisms of action remain to be investigated. Overall, exciting data show that dietary flavonoids could be considered as a useful cancer preventive approach. This review summarizes recent advancements on potential cancer preventive effects and mechanic insight of dietary flavonoids.

New therapeutic and biomarker discovery for peripheral diabetic neuropathy: PARP inhibitor, nitrotyrosine, and tumor necrosis factor-{alpha}.

This study evaluated poly(ADP-ribose) polymerase (PARP) inhibition as a new therapeutic approach for peripheral diabetic neuropathy using clinically relevant animal model and endpoints, and nitrotyrosine (NT), TNF-alpha, and nitrite/nitrate as potential biomarkers of the disease. Control and streptozotocin-diabetic rats were maintained with or without treatment with orally active PARP inhibitor 10-(4-methyl-piperazin-1-ylmethyl)-2H-7-oxa-1,2-diaza-benzo[de]anthracen-3-one (GPI-15,427), 30 mg kg(-1) d(-1), for 10 wk after first 2 wk without treatment. Therapeutic efficacy was evaluated by poly(ADP-ribosyl)ated protein expression (Western blot analysis), motor and sensory nerve conduction velocities, and tibial nerve morphometry. Sciatic nerve and spinal cord NT, TNF-alpha, and nitrite/nitrate concentrations were measured by ELISA. NT localization in peripheral nervous system was evaluated by double-label fluorescent immunohistochemistry. A PARP inhibitor treatment counteracted diabetes-induced motor and sensory nerve conduction slowing, axonal atrophy of large myelinated fibers, and increase in sciatic nerve and spinal cord NT and TNF-alpha concentrations. Sciatic nerve NT and TNF-alpha concentrations inversely correlated with motor and sensory nerve conduction velocities and myelin thickness, whereas nitrite/nitrate concentrations were indistinguishable between control and diabetic groups. NT accumulation was identified in endothelial and Schwann cells of the peripheral nerve, neurons, astrocytes, and oligodendrocytes of the spinal cord, and neurons and glial cells of the dorsal root ganglia. The findings identify PARP as a compelling drug target for prevention and treatment of both functional and structural manifestations of peripheral diabetic neuropathy and provide rationale for detailed evaluation of NT and TNF-alpha as potential biomarkers of its presence, severity, and progression.

Poly(ADP-ribose) polymerase (PARP) inhibition counteracts multiple manifestations of kidney disease in long-term streptozotocin-diabetic rat model.

Evidence for the important role for poly(ADP-ribose) polymerase (PARP) in the pathogenesis of diabetic nephropathy is emerging. We previously reported that PARP inhibitors counteract early Type 1 diabetic nephropathy. This study evaluated the role for PARP in kidney disease in long-term Type 1 diabetes. Control and streptozotocin-diabetic rats were maintained with or without treatment with the PARP inhibitor 10-(4-methyl-piperazin-1-ylmethyl)-2H-7-oxa-1,2-diaza-benzo[de] anthracen-3-one (GPI-15,427, Eisai Inc.), 30mgkg(-1)d(-1), for 26 weeks after first 2 weeks without treatment. PARP activity in the renal cortex was assessed by Western blot analysis of poly(ADP-ribosyl)ated proteins. Urinary albumin, isoprostane, and 8-hydroxy-2'-deoxyguanosine excretion, and renal concentrations of transforming growth factor-beta(1), vascular endothelial growth factor, soluble intercellular adhesion molecule-1, fibronectin, and nitrotyrosine were evaluated by ELISA, and urinary creatinine and renal lipid peroxidation products by colorimetric assays. PARP inhibition counteracted diabetes-associated increase in renal cortex poly(ADP-ribosyl)ated protein level. Urinary albumin, isoprostane, and 8-hydroxy-2'-deoxyguanosine excretions and urinary albumin/creatinine ratio were increased in diabetic rats, and all these changes were at least partially prevented by GPI-15,427 treatment. PARP inhibition counteracted diabetes-induced renal transforming growth factor-beta(1), vascular endothelial growth factor, and fibronectin, but not soluble intercellular adhesion molecule-1 and nitrotyrosine, accumulations. Lipid peroxidation product concentrations were indistinguishable among control and diabetic rats maintained with or without GPI-15,427 treatment. In conclusion, PARP activation plays an important role in kidney disease in long-term diabetes. These findings provide rationale for development and further studies of PARP inhibitors and PARP inhibitor-containing combination therapies, for prevention and treatment of diabetic nephropathy.

Head and neck cancer radiosensitization by the novel poly(ADP-ribose) polymerase inhibitor GPI-15427.

BACKGROUND: In this study, we tested the ability of a novel poly(adenosine diphosphate ribose) polymerase (PARP) inhibitor, 10-(4-methyl-piperazin-1-ylmethyl)-2H-7-oxa-1,2-diaza-benzo[de]-anthracen-3-one (GPI-15427), to enhance the effect of radiotherapy in a xenograft model of human head and neck squamous cell carcinoma (HNSCC). METHODS: Human xenograft HNSCC tumors were established in female nude mice: animals were treated with orally administered GPI-15427 at varied doses prior to tumor irradiation. In vitro and in vivo apoptosis analyses and neutral single-cell gel electrophoresis (comet) assay were performed, with the "tail moment" calculated to evaluate DNA double-strand break damage. RESULTS: Orally administered GPI-15427 given before radiation therapy significantly reduced tumor volume, and cells demonstrated significantly elevated mean tail moments (indicative of DNA damage) and enhanced apoptosis both in vitro and in vivo, compared with radiation-alone and control groups. CONCLUSIONS: Use of the PARP-1 inhibitor GPI-15427 induced significant sensitization to radiotherapy, representing a promising new treatment in the management of HNSCC.

Poly(Adenosine 5'-diphosphate-ribose) polymerase inhibition counteracts multiple manifestations of experimental type 1 diabetic nephropathy.

This study was aimed at evaluating the role for poly(ADP-ribose) polymerase (PARP) in early nephropathy associated with type 1 diabetes. Control and streptozotocin-diabetic rats were maintained with or without treatment with one of two structurally unrelated PARP inhibitors, 1,5-isoquinolinediol (ISO) and 10-(4-methyl-piperazin-1-ylmethyl)-2H-7-oxa-1,2-diaza-benzo[de] anthracen-3-one (GPI-15427), at 3 mg/kg(-1) x d(-1) ip and 30 mg/kg(-1) x d(-1), respectively, for 10 wk after the first 2 wk without treatment. PARP activity in the renal cortex was assessed by immunohistochemistry and Western blot analysis of poly(ADP-ribosyl)ated proteins. Variables of diabetic nephropathy in urine and renal cortex were evaluated by ELISA, Western blot analysis, immunohistochemistry, and colorimetry. Urinary albumin excretion was increased about 4-fold in diabetic rats, and this increase was prevented by ISO and GPI-15427. PARP inhibition counteracted diabetes-associated increase in poly(ADP-ribose) immunoreactivities in renal glomeruli and tubuli and poly(ADP-ribosyl)ated protein level. Renal concentrations of TGF-beta(1), vascular endothelial growth factor, endothelin-1, TNF-alpha, monocyte chemoattractant protein-1, lipid peroxidation products, and nitrotyrosine were increased in diabetic rats, and all these changes as well as an increase in urinary TNF-alpha excretion were completely or partially prevented by ISO and GPI-15427. PARP inhibition counteracted diabetes-induced up-regulation of endothelin (B) receptor, podocyte loss, accumulation of collagen-alpha1 (IY), periodic acid-Schiff-positive substances, fibronectin, and advanced glycation end-products in the renal cortex. In conclusion, PARP activation is implicated in multiple changes characteristic for early nephropathy associated with type 1 diabetes. These findings provide rationale for development and further studies of PARP inhibitors and PARP inhibitor-containing combination therapies.

Pharmacological inhibition of poly(ADP-ribose) polymerase activity down-regulates the expression of syndecan-4 and Id-1 in endothelial cells.

Poly(ADP-ribose) polymerase (PARP) is a family of nuclear proteins which regulate a number of cell functions, such as DNA repair, transcription, remodelling of chromatin structure, cell division and cell death. We and others have recently demonstrated that down-regulation of cellular PARP activity, using pharmacological inhibitors, impairs a number of endothelial functions and angiogenesis. In the present study, we investigated the potential mechanisms underlying the anti-angiogenic effect exerted by the potent PARP inhibitor GPI 15427, analyzing gene expression in human endothelial cells shortly after treatment with this compound. Analysis of gene and protein expression indicated that a 2-h exposure of human endothelial cells to GPI 15427 induced a rapid decrease of syndecan-4 (SDC-4), a transmembrane protein involved in modulation of cell signalling during angiogenesis that plays a role in endothelial cell migration and adhesion. Moreover, treatment with the PARP inhibitor induced a reduction of a helix-loop-helix transcription factor, the inhibitor of DNA binding-1 (Id-1), also implicated in the control of endothelial functions. We suggest that the inhibitory effect exerted by GPI 15427 on the angiogenic process is likely due to the reduced activity of specific transcription factors, such as Oct-1 and CREB that contribute to the regulation of SDC-4 and Id-1 expression, respectively. In conclusion, these results strongly suggest that PARP activity is capable of modulating molecules required for endothelial cell migration, adhesion, proliferation or differentiation during the angiogenic process.

Poly(ADP-ribose)polymerase inhibition counteracts cataract formation and early retinal changes in streptozotocin-diabetic rats.

PURPOSE: This study evaluated the role for poly(ADP-ribose) polymerase (PARP) in diabetes-induced cataractogenesis and early retinal changes. METHODS: Control and streptozotocin (STZ)-diabetic rats were treated with or without the PARP inhibitors 1,5-isoquinolinediol (ISO; 3 mg kg(-1) d(-1) intraperitoneally) and 10-(4-methyl-piperazin-1-ylmethyl)-2H-7-oxa-1,2-diaza-benzo[de]anthracen-3-1 (GPI-15427, 30 mg kg(-1) d(-1) orally) for 10 weeks after the first 2 weeks without treatment. Lens clarity was evaluated by indirect ophthalmoscopy and slit lamp examination, and retinal changes were evaluated by immunohistochemistry and Western blot analysis. In in vitro studies, cultured human lens epithelial cells and bovine retinal pericytes and endothelial cells were exposed to high glucose or palmitate. RESULTS: PARP is expressed in lens, and poly(ADP-ribosyl)ated proteins are primarily localized in the 38- to 87-kDa range of the protein spectrum, with several minor bands at 17 to 38 kDa. The 38- to 87-kDa and the 17- to 38-kDa poly(ADP-ribosyl)ated protein expression increased by 74% and 275%, respectively, after 4 weeks of diabetes and by approximately 65% early after exposure of lens epithelial cells to 30 mM glucose. Both PARP inhibitors delayed, but did not prevent, the formation of diabetic cataract. The number of TUNEL-positive nuclei in flatmounted retinas increased approximately 4-fold in STZ diabetic rats, and this increase was prevented by ISO and GPI-15427. Both PARP inhibitors reduced diabetes-induced retinal oxidative-nitrosative and endoplasmic reticulum stress and glial activation. GPI-15427 (20 microM) prevented oxidative-nitrosative stress and cell death in palmitate-exposed pericytes and endothelial cells. CONCLUSIONS: PARP activation is implicated in the formation of diabetic cataract and in early retinal changes. These findings provide a rationale for the development of PARP inhibitors for the prevention of diabetic ocular complications.

PARP inhibition or gene deficiency counteracts intraepidermal nerve fiber loss and neuropathic pain in advanced diabetic neuropathy.

Evidence that poly(ADP-ribose) polymerase (PARP) activation plays an important role in diabetic complications is emerging. This study evaluated the role of PARP in rat and mouse models of advanced diabetic neuropathy. The orally active PARP inhibitor 10-(4-methylpiperazin-1-ylmethyl)-2H-7-oxa-1,2-diaza-benzo[de]anthracen-3-one (GPI-15427; formulated as a mesilate salt, 30 mg kg(-1) day(-1) in the drinking water for 10 weeks after the first 2 weeks without treatment) at least partially prevented PARP activation in peripheral nerve and DRG neurons, as well as thermal hypoalgesia, mechanical hyperalgesia, tactile allodynia, exaggerated response to formalin, and, most importantly, intraepidermal nerve fiber degeneration in streptozotocin-diabetic rats. These findings are consistent with the lack of small sensory nerve fiber dysfunction in diabetic PARP -/- mice. Furthermore, whereas diabetic PARP +/+ mice displayed approximately 46% intraepidermal nerve fiber loss, diabetic PARP -/- mice retained completely normal intraepidermal nerve fiber density. In conclusion, PARP activation is an important contributor to intraepidermal nerve fiber degeneration and functional changes associated with advanced Type 1 diabetic neuropathy. The results support a rationale for the development of potent and low-toxicity PARP inhibitors and PARP inhibitor-containing combination therapies.

Poly(ADP-ribose) polymerase (PARP) inhibition or PARP-1 gene deletion reduces angiogenesis.

Poly(ADP-ribose) polymerase (PARP)-1 has recently been shown to promote tumour progression. Since angiogenesis is an essential requirement for tumour growth, we examined whether PARP inhibition/deletion might affect endothelial cell functions. To this end, the influence of PARP inhibitors on endothelial cell proliferation, migration, tube formation and angiogenesis in PARP-1 knock-out mice, using an in vivo matrigel plug assay, was investigated. The results indicated that the PARP inhibitor GPI 15427 (IC50 on endothelial PARP: 237 +/- 27 nM), at concentrations devoid of cytotoxic effects (0.5-1 microM), abrogated migration in response to vascular endothelial growth factor or placenta growth factor, hampered formation of tubule-like networks and impaired angiogenesis in vivo. The anti-angiogenic effect of the PARP inhibitor was confirmed in PARP-1 knock-out mice that displayed a defect of angiogenesis induced by growth factors. These results provide evidence for targeting PARP for anti-angiogenesis, adding novel therapeutic implications to the use of PARP inhibitors in cancer treatment.

Sirtuin inhibition increases the rate of non-homologous end-joining of DNA double strand breaks.

Sirtuins (type III histone deacetylases) are an important member of a group of enzymes that modify chromatin conformation. We investigated the role of sirtuin inhibitor, GPI 19015, in double strand break (DSB) repair in CHO-K1 wt and xrs-6 mutant cells. The latter is defective in DNA-dependent protein kinase (DNA-PK)-mediated non-homologous end-joining (D-NHEJ). DSB were estimated by the neutral comet assay and histone gammaH2AX foci formation. We observed a weaker effect of GPI 19015 treatment on the repair kinetics in CHO wt cells than in xrs6. In the latter cells the increase in DNA repair rate was most pronounced in G1 phase and practically absent in S and G2 cell cycle phases. The decrease in the number of histone gammaH2AX foci was faster in xrs6 than in CHO-K1 cells. The altered repair rate did not affect survival of X-irradiated cells. Since in G1 xrs6 cells DNA-PK-dependent non-homologous end-joining, D-NHEJ, does not operate, these results indicate that inhibition of sirtuins modulates DNA-PK-independent (backup) non-homologous end-joining, B-NHEJ, to a greater extent than the other DSB repair system, D-NHEJ.

Inhibition of poly(ADP-ribose) polymerase prevents irinotecan-induced intestinal damage and enhances irinotecan/temozolomide efficacy against colon carcinoma.

Poly(ADP-ribose) polymerase (PARP) inhibitors enhance the antitumor activity of the topoisomerase I inhibitor irinotecan (CPT-11), which is used to treat advanced colorectal carcinoma. Since PARP inhibitors sensitize tumor cells also to the methylating agent temozolomide (TMZ) and clinical trials are evaluating CPT-11 in combination with TMZ, we tested whether the PARP inhibitor GPI 15427 (10-(4-methyl-piperazin-1-ylmethyl)-2H-7-oxa-1,2-diaza-benzo[de]anthracen-3-one) increases the efficacy of CPT-11 + TMZ against colon cancer. Moreover, due to the ability of PARP inhibitors to avoid cell death consequent to PARP-1 overactivation, we evaluated whether oral administration of GPI 15427 provides protection from the dose-limiting intestinal toxicity of CPT-11. The results of colony formation assay indicated that GPI 15427 increased the antiproliferative effects (combination index <1) of TMZ + SN-38 (the active metabolite of CPT-11) against colon cancer cells. Accordingly, GPI 15427 (40 mg/kg/dayx5 days per os) in combination with TMZ (10 mg/kg/dayx5 days) + CPT-11 (4 mg/kg/dayx5 days) significantly reduced the growth of tumor xenografts. Oral administration of GPI 15427 (40 mg/kg/q2x3 days) prevented intestinal injury and diarrhea induced by CPT-11 (30 mg/kg/day x 3 days) reducing inflammation and PARP-1 overactivation, as evidenced by immunohistochemical staining of intestinal tissue with antipoly(ADP-ribose) antibody (Ab). In conclusion, the PARP inhibitor represents a novel strategy to enhance the antitumor efficacy and reduce toxicity of chemotherapy in colon cancer.

Poly(ADP-ribose) glycohydrolase activity mediates post-traumatic inflammatory reaction after experimental spinal cord trauma.

The aim of the present study was to examine the role of poly-(ADP-ribose) glycohydrolase (PARG) on the modulation of the inflammatory response and tissue injury associated with neurotrauma. Spinal cord trauma was induced in wild-type (WT) mice by the application of vascular clips (force of 24 g) to the dura via a two-level T(6) to T(7) laminectomy. Spinal cord injury in WT mice resulted in severe trauma characterized by edema, neutrophil infiltration, and cytokine production followed by recruitment of other inflammatory cells, production of a range of inflammation mediators, tissue damage, apoptosis, and disease. The genetic disruption of the PARG gene in mice or the pharmacological inhibition of PARG with GPI 16552 [N-bis-(3-phenyl-propyl)9-oxo-fluorene-2,7-diamide] (40 mg/kg i.p. bolus), a novel and potent PARG inhibitor, significantly reduced the degree of spinal cord inflammation and tissue injury (histological score), neutrophil infiltration, cytokine production (tumor necrosis factor-alpha and interleukin-1beta), and apoptosis. In a separate experiment, we have clearly demonstrated that PARG inhibition significantly ameliorated the recovery of limb function. Taken together, our results indicate that PARG activity modulates the inflammatory response and tissue injury events associated with spinal cord trauma and participate in target organ damage under these conditions.

Structural optimization of thiol-based inhibitors of glutamate carboxypeptidase II by modification of the P1' side chain.

A series of thiol-based inhibitors containing a benzyl moiety at the P1' position have been synthesized and tested for their abilities to inhibit glutamate carboxypeptidase II (GCP II). 3-(2-Carboxy-5-mercaptopentyl)benzoic acid 6c was found to be the most potent inhibitor with an IC(50) value of 15 nM, 6-fold more potent than 2-(3-mercaptopropyl)pentanedioic acid (2-MPPA), a previously discovered, orally active GCP II inhibitor. Subsequent SAR studies have revealed that the phenoxy and phenylsulfanyl analogues of 6c, 3-(1-carboxy-4-mercaptobutoxy)benzoic acid 26a and 3-[(1-carboxy-4-mercaptobutyl)thio]benzoic acid 26b, also possess potent inhibitory activities toward GCP II. In the rat chronic constriction injury (CCI) model of neuropathic pain, compounds 6c and 26a significantly reduced hyperalgesia following oral administration (1.0 mg/kg/day).

Treatment with PARP-1 inhibitors, GPI 15427 or GPI 16539, ameliorates intestinal damage in rat models of colitis and shock.

Poly (ADP-ribose) polymerase-1 (PARP-1), a nuclear enzyme activated by DNA strand breaks, plays a detrimental role during inflammation. As inflammation is important in the development of colitis and ischemia/reperfusion (I/R) injury of the intestine, we investigated the effects of 10-(4-methyl-piperazin-1-ylmethyl)-2H-7-oxa-1,2-diaza-benzo[de]anthracen-3-one (GPI 15427) and 2-(4-methyl-piperazin-1-yl)-5H-benzo[c][1,5]naphthyridin-6-one (GPI 16539), two novel and potent inhibitors of PARP-1, in a rat model of gut injury and inflammation, splanchnic artery occlusion (SAO)shock and dinitrobenzene sulfonic acid (DNBS)-induced colitis. We report here for the first time that post-injury administration of GPI 15427 and GPI 16539 exerts potent anti-inflammatory effects by reducing inflammatory cell infiltration and histological injury, and delaying the development of clinical signs in both in vivo models. Furthermore, GPI 15427 and GPI 16539 treatment diminished the accumulation of poly(ADP-ribose) in the ileum of splanchnic artery occlusion-shocked rats and in the colons of dinitrobenzene sulfonic acid-treated rats. Thus, GPI 15427 and GPI 16539 exhibited anti-inflammation activity against damage caused by intestinal ischemia/reperfusion and colitis. GPI 15427 and GPI 16539 may be useful for treating gut ischemia and inflammation.

PARG activity mediates intestinal injury induced by splanchnic artery occlusion and reperfusion.

Poly (ADP-ribosyl)ation, an early post-translational modification in response to DNA damage, is catalyzed by poly (ADP-ribose) polymerase (PARP-1) and catabolized by poly(ADP-ribose) glycohydrolase (PARG). The aim of this study was to investigate the role of PARG on the modulation of the inflammatory response caused by splanchnic ischemia and reperfusion. SAO shock in rats and wild-type (WT) mice was associated with a significant neutrophil infiltration in the ileum and production of tumor necrosis factor-alpha (TNF-alpha). Reperfused ileum tissue sections from SAO-shocked WT mice and rats showed positive staining for P-selectin and ICAM-1 localized mainly in the vascular endothelial cells. Genetic disruption of the PARG gene in mice or pharmacological inhibition of PARG by PARG inhibitors significantly improved the histological status of the reperfused tissues associated with reduced expression of P-selectin and ICAM-1, neutrophil infiltration into the reperfused intestine, and TNF-alpha production. These results suggest that PARG activity modulates the inflammatory response in ischemia/reperfusion and participates in end (target) organ damage under these conditions.

Brain distribution and efficacy as chemosensitizer of an oral formulation of PARP-1 inhibitor GPI 15427 in experimental models of CNS tumors.

We previously demonstrated that intravenous or intra-cerebral administration of poly (ADP-ribose) polymerase-1 (PARP-1) inhibitors increases the antitumor activity of temozolomide (TMZ), an oral anticancer drug used for the treatment of malignant melanoma and primary or secondary brain tumors. Since the oral route has a number of advantages in terms of safety and convenience with respect to intravenous injection, in this study we tested whether administration per os of the novel PARP-1 inhibitor GPI 15427 allows sufficient absorption of the compound and achievement of brain concentrations capable of enhancing the efficacy of TMZ against tumors growing at the CNS. Pharmacokinetics analysis of GPI 15427 levels in plasma and brain was assessed in Sprague-Dawley rats after oral dosing, by liquid chromatography and tandem mass spectrometry. Antitumor activity of oral GPI 15427 in association with TMZ was evaluated in BD2F1 mice injected intracranially with B16 melanoma or L5178Y lymphoma. Pharmacokinetics studies revealed that GPI 15427 possesses a substantial oral bioavailability (plasma Cmax after a single dose of 40 mg/kg: 1041+/-516 ng/ml). Moreover, the brain levels and brain/plasma ratios of GPI 15427 (3.37 at 0.5 h and 3.19 at 1 h) indicated that the compound readily penetrates the blood-brain barrier. GPI 15427 (10 or 40 mg/kg/per os) was then administered for five days, 1 h before TMZ (100 mg/kg/i.p.), to tumor-bearing mice. The results indicated that GPI 15427+TMZ was well tolerated and significantly increased life-span of the animals with respect to TMZ. In conclusion, PARP-1 inhibitor GPI 15427 is efficacious as chemosensitizer for the treatment of tumors located at the CNS site when it is administered by oral route.

Treatment with a novel poly(ADP-ribose) glycohydrolase inhibitor reduces development of septic shock-like syndrome induced by zymosan in mice.

OBJECTIVE: Poly(ADP-ribose) is synthesized from nicotinamide adenine dinucleotide by poly(ADP-ribose) polymerase (PARP) and degraded by poly(ADP-ribose) glycohydrolase (PARG). The activation of the PARP/PARG pathway has been found in a variety of animal models of diseases, including septic shock-like syndrome. We have previously demonstrated that PARP inhibition by 3-ami-nobenzamide or GPI 6150 ameliorates multiple organ dysfunctions induced by zymosan. In the present study, we investigated whether similar effect could be achieved through PARG inhibition to break the cycle of poly(ADP-ribose) turnaround. DESIGN: Experimental study. SETTING: University laboratory. SUBJECTS: Male CD mice (20-22 g). INTERVENTIONS: We tested the effects of GPI 18214 (40 mg/kg intraperitoneally bolus), a novel and potent PARG inhibitor, at 1 and 6 hr after zymosan (500 mg/kg, administered intraperitoneally as a suspension in saline) on the development of septic shock-like syndrome in mice. Organ failure and systemic inflammation in mice were assessed 18 hrs after administration of zymosan and/or GPI 18214 and monitored for 12 days (for loss of body weight and mortality). MEASUREMENTS AND MAIN RESULTS: At 18 hrs after zymosan administration, we found a significant increase of peritoneal exudates, leukocyte infiltration in peritoneal cavity as well as an infiltration of neutrophils in lung and ileum tissues and subsequent lipid peroxidation, and increased production of plasma tumor necrosis factor-alpha and interleukin-1 beta. Furthermore, zymosan administration induced significant liver, lung, pancreas, intestine, and kidney dysfunction as well as a systemic toxicity and significant loss of body weight. At the end of observation period (12 days), 90% of zymosan-treated mice were dead. GPI 18214 (40 mg/kg intraperitoneally, 1 and 6 hrs after zymosan) treatment significantly reduced peritoneal exudates, inflammatory cell infiltration, and organ injury and mortality rate in zymosan-treated mice. CONCLUSIONS: This study supports early studies that show efficacy from blocking the poly(ADP-ribose) pathway in septic shock-like syndrome model. It provides evidence that GPI 18214, a PARG inhibitor, attenuates the degree of zymosan-induced nonseptic shock in mice, suggesting that PARG may be an alternative therapeutic target for shock treatment.

Systemic administration of GPI 15427, a novel poly(ADP-ribose) polymerase-1 inhibitor, increases the antitumor activity of temozolomide against intracranial melanoma, glioma, lymphoma.

PURPOSE: Temozolomide (TMZ) is a DNA methylating agent that has shown promising antitumor activity in recent clinical trials against high grade gliomas, metastatic melanoma, and brain lymphoma. In this study, we tested whether systemic administration of GPI 15427, a novel poly(ADP-ribose) polymerase (PARP-1) inhibitor capable of crossing the blood-brain barrier, could enhance the efficacy of TMZ against metastatic melanoma, glioblastoma multiforme, and lymphoma growing in the brain. EXPERIMENTAL DESIGN: Murine B16 melanoma or L5178Y lymphoma cells were injected intracranially in syngeneic mice. An orthotopic xenograft of the human SJGBM2 glioblastoma multiforme was implanted in nude mice. Animals were treated with TMZ + GPI 15427 using a schedule of 40 mg/kg/i.v. GPI 15427 + 100 mg/kg/i.p. TMZ for 3 days. The efficacy of drug treatment was assessed by: (a) the increase of mouse survival and life span; and (b) the suppression of melanoma metastases to lung after i.v. injection of B16 cells. RESULTS: In all models, systemic administration of GPI 15427 shortly before TMZ significantly increased life span of tumor-bearing mice with respect to untreated controls or to groups treated with either GPI 15427 or TMZ only. Moreover, GPI 15427 increased the antimetastatic effect of TMZ. CONCLUSIONS: These data indicate that systemic administration of the poly(ADP-ribose) polymerase-1 inhibitor GPI 15427 significantly enhances TMZ antitumor efficacy against solid or hematological neoplasias even when located at the central nervous system site.

Reaction of Resorcinol with alpha,beta-Unsaturated Ketones.

Products of the acid-catalyzed reaction of resorcinol with alpha,beta-unsaturated ketones have been found to fall into two classes. The first type of product is illustrated by 4-(3,4-dihydro-7-hydroxy-2,4,4-trimethyl-2H-1-benzopyran-2-yl)-1,3-benzenediol, 1, formed in 91% yield by the reaction of resorcinol with 4-methyl-3-penten-2-one (mesityl oxide). The second type of product is illustrated by (C(2)-symmetric) 2,2'-spirobi(7-hydroxy-4,4-dimethylchroman),4, formed in 85% yield by the reaction of resorcinol with 2,6-dimethyl-2,5-heptadien-4-one (phorone). A number of examples of reactions leading, in quite good yields, to products analogous to 1are presented, as well as some reactions that fail. Flavan 1 is identical to the compound formed by the acid-catalyzed reaction of acetone with excess resorcinol. Compound 1 and a steroidal analogue of 1 have been found to be fluorescent.