Safety Assessment of PCA (2-Pyrrolidone-5-Carboxylic Acid) and Its Salts as Used in Cosmetics.

The Cosmetic Ingredient Review Expert Panel (Panel) reassessed the safety of 2-pyrrolidone-5-carboxylic acid (PCA) and sodium PCA; the Panel added 3 previously unreviewed salts (calcium, magnesium, and potassium) of PCA to this safety assessment. 2-Pyrrolidone-5-carboxylic acid and its salts are reported to function in cosmetics as skin conditioning agents-humectants. The Panel reviewed the data from the 1999 report of PCA and sodium PCA, as well as additional data included in this report, to determine the safety of these ingredients. The Panel concluded that PCA and its salts are safe in cosmetics in the present practices of use and concentration; additionally, these ingredients should not be used in cosmetic products in which N-nitroso compounds can be formed.

A Multi-Biochemical and In Silico Study on Anti-Enzymatic Actions of Pyroglutamic Acid against PDE-5, ACE, and Urease Using Various Analytical Techniques: Unexplored Pharmacological Properties and Cytotoxicity Evaluation.

In the current study, pyroglutamic acid (pGlu), a natural amino acid derivative, has efficiently inhibited the catalytic activities of three important enzymes, namely: Human recombinant phosphodiesterase-5A1 (PDE5A1), human angiotensin-converting enzyme (ACE), and urease. These enzymes were reported to be associated with several important clinical conditions in humans. Radioactivity-based assay, spectrophotometric-based assay, and an Electrospray Ionization-Mass Spectrometry-based method were employed to ascertain the inhibitory actions of pGlu against PDE5A1, ACE, and urease, respectively. The results unveiled that pGlu potently suppressed the activity of PDE5A1 (half-maximal inhibitory concentration; IC50 = 5.23 µM) compared with that of standard drug sildenafil citrate (IC50 = 7.14 µM). Moreover, pGlu at a concentration of 20 µg/mL was found to efficiently inhibit human ACE with 98.2% inhibition compared with that of standard captopril (99.6%; 20 µg/mL). The urease-catalyzed reaction was also remarkably inactivated by pGlu and standard acetohydroxamic acid with IC50 values of 1.8 and 3.9 µM, respectively. Remarkably, the outcome of in vitro cytotoxicity assay did not reveal any significant cytotoxic properties of pGlu against human cervical carcinoma cells and normal human fetal lung fibroblast cells. In addition to in vitro assays, molecular docking analyses were performed to corroborate the outcomes of in vitro results with predicted structure-activity relationships. In conclusion, pGlu could be presented as a natural and multifunctional agent with promising applications in the treatment of some ailments connected with the above-mentioned anti-enzymatic properties.

Improved in vivo antitumor effect of a daunorubicin - GnRH-III bioconjugate modified by apoptosis inducing agent butyric acid on colorectal carcinoma bearing mice.

Compared to classical chemotherapy, peptide-based drug targeting is a promising therapeutic approach for cancer, which can provide increased selectivity and decreased side effects to anticancer drugs. Among various homing devices, gonadotropin-releasing hormone-III (GnRH-III) peptide represents a suitable targeting moiety, in particular in the treatment of hormone independent tumors that highly express GnRH receptors (e.g. colon carcinoma). We have previously shown that GnRH-III[(4)Lys(Ac),(8)Lys(Dau = Aoa)] bioconjugate, in which daunorubicin was attached via oxime linkage to the (8)Lys of a GnRH-III derivative, exerted significant in vivo antitumor effect on subcutaneously developed HT-29 colon tumor. In contrast, results of the study reported here indicated that this compound was not active on an orthotopically developed tumor. However, if Lys in position 4 was acylated with butyric acid instead of acetic acid, the resulting bioconjugate GnRH-III[(4)Lys(Bu),(8)Lys(Dau = Aoa)] had significant tumor growth inhibitory effect. Furthermore, it prevented tumor neovascularization, without detectable side effects. Nevertheless, the development of metastases could not be inhibited by the bioconjugate; therefore, its application in combination with a metastasis preventive agent might be necessary in order to achieve complete tumor remission. In spite of this result, the treatment with GnRH-III[(4)Lys(Bu),(8)Lys(Dau = Aoa)] bioconjugate proved to have significant benefits over the administration of free daunorubicin, which was used at the maximum tolerated dose.

Promising toxicological biomarkers for the diagnosis of liver injury types: Bile acid metabolic profiles and oxidative stress marker as screening tools in drug development.

Promising biomarkers were identified in adult male Crl:CD (SD) rats for the screening of new chemical entities for their potential to cause liver injury. We examined the serum biochemistry, liver histopathology, and bile acid profiles by LC-MS/MS, and the mRNA expression of transporters and CYPs by an RT-PCR after the following treatments to male Crl:CD (SD) rats: (a) bile duct ligation (BDL); (b) a single oral dose of 150 mg/kg α-naphthylisothiocyanate (ANIT); and (c) repeated oral doses of a novel pyrrolidinecarboxylic acid derivative (abbreviated as PCA) at 30, 300, and 1000 mg/kg. The serum total bile acid levels and bilirubin concentrations were found to be elevated in all of the groups. However, the bile acid component profiles of the PCA group differed significantly from BDL and ANIT models: deoxycholic acid, lithocholic acid, and sulfated bile acids were upregulated in a dose-dependent manner only in the PCA group. In addition, the PCA group demonstrated high levels of hepatic heme oxygenase-1 expression, whereas the profiles of the mRNA levels of the hepatic transporters and CYPs of all groups were found to be similar. The histopathological findings, for both the BDL and ANIT groups, were of bile duct hyperplasia, hepatocyte degeneration and necrosis. In contrast, only bile duct hyperplasia and hepatocyte degeneration were observed in the PCA group, even at a lethal dose. These results indicated that PCA induced a cholestatic condition and the increase of oxidative stress markers implies that this will also lead hepatocellular injury. In conclusion, the serum bile acid components and sulfated bile acid levels, and the expression of oxidative stress markers could provide information that aids in the diagnosis of liver injury type and helps to elucidate the mechanisms of hepatotoxicity. These findings can be extrapolated into our clinical investigation. The analysis of these crucial biomarkers is likely to be a useful screening tool in the lead optimization phase of drug discovery.

Hormonal enhancement of insecticide efficacy in Tribolium castaneum: oxidative stress and metabolic aspects.

Insect anti-stress responses, including those induced by insecticides, are controlled by adipokinetic hormones (AKHs). We examined the physiological consequences of Pyrap-AKH application on Tribolium castaneum adults (AKH-normal and AKH-deficient prepared by the RNAi technique) treated by two insecticides, pirimiphos-methyl and deltamethrin. Co-application of pirimiphos-methyl and/or deltamethrin with AKH significantly increased beetle mortality compared with application of the insecticides alone. This co-treatment was accompanied by substantial stimulation of general metabolism, as monitored by carbon dioxide production. Further, the insecticide treatment alone affected some basic markers of oxidative stress: it lowered total antioxidative capacity as well as the activity of superoxide dismutase in the beetle body; in addition, it enhanced the activity of catalase and glutathione-S-transferase. However, these discrepancies in oxidative stress markers were eliminated/reduced by co-application with Pyrap-AKH. We suggest that the elevation of metabolism, which is probably accompanied with faster turnover of toxins, might be responsible for the higher mortality that results after AKH and insecticide co-application. Changes in oxidative stress markers are probably not included in the mechanisms responsible for increased mortality.

Acute administration of 5-oxoproline induces oxidative damage to lipids and proteins and impairs antioxidant defenses in cerebral cortex and cerebellum of young rats.

5-Oxoproline accumulates in glutathione synthetase deficiency, an autossomic recessive inherited disorder clinically characterized by hemolytic anemia, metabolic acidosis, and severe neurological symptoms whose mechanisms are poorly known. In the present study we investigated the effects of acute subcutaneous administration of 5-oxoproline to verify whether oxidative stress is elicited by this metabolite in vivo in cerebral cortex and cerebellum of 14-day-old rats. Our results showed that the acute administration of 5-oxoproline is able to promote both lipid and protein oxidation, to impair brain antioxidant defenses, to alter SH/SS ratio and to enhance hydrogen peroxide content, thus promoting oxidative stress in vivo, a mechanism that may be involved in the neuropathology of gluthatione synthetase deficiency.

Synergistic immunostimulatory effect of pidotimod and red ginseng acidic polysaccharide on humoral immunity of immunosuppressed mice.

We investigated the synergistic effect of pidotimod and red ginseng acidic polysaccharide (RGAP) from Panax ginseng C.A. Meyer on humoral immune response challenged by lipopolysaccharide (LPS) and sheep red blood cells (SRBC) in immunosuppressed mice. Combined treatment with pidotimod and RGAP significantly increased the number of plaque-forming cells in the spleen in response to both LPS and SRBC, while treatment with either pidotimod or RGAP individually had no such effect. IgG levels in serum were augmented for secondary responses to SRBC in co-treated mice, but not in mice treated with either drug alone. Microscopic studies revealed that architecture of the spleen, thymus, and lymph nodes was conserved. GPT and creatinine in serum as indicators of hepatic and renal functions showed no difference compared to the control group. These results indicate that combined treatment with pidotimod and RGAP has an immunostimulatory effect in a synergistic manner on antibody response to challenge with LPS and SRBC without toxic changes.

[Study on the mechanism of male reproductive toxicity of metadoxine in mice and rats].

OBJECTIVE: To study the mechanism of male reproductive toxicity of metadoxine (MTDX) on mice and rats. METHODS: Mouse multiple endpoints assay and Hershberger assay were employed to evaluate the potential estrogenic and/or antiandrogenic effects of MTDX. In mouse multiple endpoints assay, MTDX (0, 640, 1500 and 4000 mg/kg, respectively) were administered once daily p.o. for 5 days in sexually matured and ovariectomied female NIH mice. Five endpoints evaluated as markers of estrogenicity included the ratio of uterine weight to body weight, incidence and extent of uterine fluid imbibition (hydrometra), vaginal epithelial cornification during estrous cycle (estrinization) and thickness of uterine epithelial cell and stroma cell. In Hershberger assay, MTDX (0, 600 and 1500 mg/kg, respectively) was administered once daily p.o. for 10 days to castrated male SD rats with or without testosterone propionate (TP, 12.5 mg/kg, i.p. for 10 days) substitution. Relative weight of androgen dependent issues was measured. RESULTS: In mouse multiple endpoints assay, ratio of uterine weight to body weight was 1.33, 1.38 and 1.31 x 10(-4) in MTDX 640, 1500 and 4000 mg/kg groups, respectively, without significant difference from that in control group (1.22 x 10(-4)). Thickness of uterine uterine epithelial cell (0.90 and 1.03 microm) and stroma cell (3.38 and 3.25 microm) in MTDX 1500 and 4000 mg/kg groups was not significantly different from the control group (0.85 microm and 2.77 microm, respectively). In Hershberger assay, relative weight of prostate plus seminal vesicle, levator ani muscle and bulbocavernous muscle was 1.13, 0.17 and 0.42, respectively, in the 1500 mg/kg group, significantly decreased as compared with those in the control group (1.46, 0.24 and 0.70, respectively) (P < 0.01). Relative weight of prostate plus seminal vesicle (1.29) in the MTDX 600 mg/kg group reduced slightly, with statistical significance (P < 0.05), as compared with that in the control group (1.46). CONCLUSIONS: In the present study, MTDX did not exhibit any estrogenic effect in mice in vivo. However, it had antiandrogenic activity in castrated male SD rats, indicating that its antiandrogenic effect may be involved in it's male reproductive toxicity.

[Reproductive toxicity of metadoxine in rats].

OBJECTIVE: To study the reproductive toxicity of metadoxine. METHODS: Male and female rats were given metadoxine before pregnancy and early gestation, i.e. to feed metadoxine to male rats for 60 days before copulation and continue feeding during copulation, and feed metadoxine to female rats for 14 days before copulation. RESULTS: No significant toxic effect was observed in the 400 mg/kg group. A few rats showed paralysis of hind leg in the 800 mg/kg group. The dosage of 1 600 mg/kg caused significant paralysis of hind legs, emaciation, and reduced weight gain. In the 1600 mg/kg group, the mating rate of male rats was significantly affected (P < 0.01). In the 800 and 1 600 mg/kg group, fertility of male rats was markedly reduced (P < 0.01). In the 800 mg/kg group, the effect on sperm counts of epididymis of male rats was markedly reduced (P < 0.05). In the 1 600 mg/kg group, testicle weight and body weight ratio and sperm counts of epididymis rate were significantly (P < 0.001) reduced. In the 1 600 mg/kg group, the fertility rate of female rats was remarkably (P < 0.001) reduced. In the 800 mg/kg group, the weight gain of pregnant rats was significantly reduced (P < 0.001). In both the 800 and 1 600 mg/kg groups, the gestation rate was greatly reduced (P < 0.001). In the 800 mg/kg group, mortality rate before nidation (P < 0.001) and average live fetus number were significantly reduced (P < 0.05). In the 400 mg/kg group, the fetal weight was significantly reduced (P < 0.001). In the 800 mg/kg group, body length, tail length, body weight and sternum development of fetal rats were significantly affected (P < 0.001). CONCLUSION: Under the presented experimental conditions, metadoxine has no teratogenic effects on SD rats and the no effect dose is 400 mg/kg. And the no effect dose for the developmental toxicity is less than 400 mg/kg.

Synthesis, physical properties, toxicological studies and bioavailability of L-pyroglutamic and L-glutamic acid esters of paracetamol as potentially useful prodrugs.

Paracetamol ester prodrugs with L-pyroglutamic and L-glutamic acid, biosynthetic precursors of glutathione, have been synthesized to reduce paracetamol hepatotoxicity and improve bioavailability. The toxicological studies of paracetamol esters show that only L-5-oxo-pyrrolidine-2-paracetamol carboxylate reduces toxicity after administration of an overdose. The glutathione hepatic values in mice obtained by intraperitoneal injection of the ester are superimposable on controls and the oral LD50 was found to be greater than 2000 mg kg-1 and the intraperitoneal LD50 was 1900 mg kg-1. These results taken together with hydrolysis and bioavailability data show that ester is a potential candidate as a prodrug of paracetamol.

Neurochemical effects of L-pyroglutamic acid.

The effect of L-pyroglutamic acid, a metabolite that accumulates in pyroglutamic aciduria, on different neurochemical parameters was investigated in adult male Wistar rats. Glutamate binding, adenylate cyclase activity and G protein coupling to adenylate cyclase were assayed in the presence of the acid. L-pyroglutamic acid decreased Na(+)-dependent and Na(+)-independent glutamate binding. Basal and GMP-PNP stimulated adenylate cyclase activity were not affected by the acid. Furthermore, rats received unilateral intrastriatal injections of 10-300 nmol of buffered L-pyroglutamic acid. Vehicle (0.25 M Tris-Cl, pH 7.35-7.4) was injected into the contralateral striatum. Neurotoxic damage was assessed seven days after the injection by histological examination and by weighing both cerebral hemispheres. No difference in histology or weight could be identified between hemispheres. These results suggest that, although capable of interfering with glutamate binding, pyroglutamate did not cause a major lesion in the present model of neurotoxicity.

General pharmacology of pidotimod and testing for drug interactions.

Pidotimod ((R)-3-[(S)-(5-oxo-2-pyrrolidinyl) carbonyl]-thiazolidine-4-carboxylic acid, PGT/1A, CAS 121808-62-6) is a new biological response modifier. General pharmacology and interactions with some drugs were tested. The drug, at doses of 200 mg/kg i.p. and 400 mg/kg p.o., did not affect the normal behaviour, did not modify the responses to stimulation of autonomic nervous system or central nervous system. Pidotimod did not display any cardiovascular or respiratory effect up to 125 mg/kg i.v. in 3 animal species. The drug did not show antimicrobial or antifungal activities nor interact with some of the most common therapeutics (antibiotics, tolbutamide, pentobarbital, antihypertensives, chlorothiazide, warfarin, non-steroidal antiinflammatory agents). On the basis of these results pidotimod shows a safe profile; moreover it does not interact with many therapeutic agents.

Toxicological evaluation of pidotimod.

This paper reports the toxicological evaluation of pidotimod ((R)-3-[(S)-(5-oxo-2-pyrrolidinyl) carbonyl]-thiazolidine-4-carboxylic acid, PGT/1A, CAS 121808-62-6). Its acute toxicity in mice, rats and dogs was very low after oral, i.v., i.m. and i.p. administration. The repeated administration studies in rats were performed for 4 months via the i.p. route and for 12 months via the oral route. Pidotimod did not show toxic effects at dosages up to 200 mg/kg i.p. and 800 mg/kg p.o. These dosages correspond to 32.5 times the maximum dosage intended for clinical use. The repeated administration studies in dogs were performed for 26 weeks via the i.m. route and for 52 weeks via the oral route. Pidotimod did not show toxic effects at dosages up to 300 mg/kg i.m. and 600 mg/kg p.o.. It did not affect male or female rat fertility at dosages up to 600 mg/kg by oral and 500 mg/kg by i.v. route. The compound was not teratogenic in rats (600 mg/kg p.o. and 1000 mg/kg i.v.), with no effects on subsequent embryofoetal development at dosages up to 1000 mg/kg/day, and in rabbits (300 mg/kg p.o. and 500 mg/kg. i.v.). There were no peri- and postnatal toxic effects in rats (600 mg/kg p.o. and 500 mg/kg i.v.). Local tolerability of pidotimod after i.m. administration was very good. In conclusion pidotimod is characterized by a high safety margin in all animal species.

Genotoxicity testing of pidotimod in vitro and in vivo.

The mutagenic potential of pidotimod ((R)-3-[(S)-(5-oxo-2- pyrrolidinyl)carbonyl]-thiazolidine-4-carboxylic acid, PGT/1A, CAS 121808-62-6) was assessed in a series of five assays designed to measure gene mutation, chromosomal damage and primary DNA damage. All tests were carried out in accordance with appropriate EEC and OECD Guidelines. No indications of mutagenic potential were observed in any of the assays.

Chronic intrastriatal L-pyroglutamate: neuropathology and neuron sparing like Huntington's disease.

Continuous injection of L-pyroglutamate (L-PGA) into the rat striatum induces a lesion with three regions: a necrotic core, a rim of pyknotic cells, and a peripheral spongiose region. The L-PGA was administered through an implanted intrastriatal cannula coupled to an Alzet osmotic pump loaded with one of three doses of L-PGA (3, 5, or 13 times the normal amount of L-PGA/g wet wt rat forebrain (23 nmol/g). The magnitude of the lesion was dependent upon the concentration of buffered L-PGA and the duration of continuous pumping. The necrotic region contained macrophages and neutrophils, while condensed neurons and oligodendroglial cells were present in the pyknotic region. The spongiose region contained vacuolated neuropil and degenerating nerve cells and oligodendroglia. The spongiose region blends with the normal neuropil. A population of aspiny neurons were identified throughout the spongiose region. These neurons stained positive for NADPH diaphorase and demonstrated a somatostatin-like immunoreactivity similar to that of the aspiny neurons spared in Huntington's disease and in the neurotoxin-induced striatal-lesioned rat models of Huntington's disease.

Does monosodium glutamate cause flushing (or merely "glutamania")?

Monosodium glutamate is widely regarded as the provocative agent in the "Chinese restaurant syndrome," of which flushing is regarded as part of the reaction. Six subjects were monitored by laser Doppler velocimetry for changes in facial cutaneous blood flow during challenge with monosodium glutamate and its cyclization product, pyroglutamate. Additionally, records of patients challenged with monosodium glutamate in the laboratory were reviewed. No flushing was provoked among the twenty-four people tested, eighteen of whom gave a positive history of Chinese restaurant syndrome flushing. These results indicate that monosodium glutamate-provoked flushing, if it exists at all, must be rare. Monosodium glutamate and its cyclization product, pyroglutamate, may provoke edema and associated symptoms.

Neurotoxic effects of endogenous materials: quinolinic acid, L-pyroglutamic acid, and thyroid releasing hormone (TRH).

The previously reported, dose-related, and selective neurotoxic action of 100 to 200 nmol quinolinic acid on intrastriatal injection was confirmed. A slight neurotoxicity was obtained with 250 nmol thyroid releasing hormone but not with a similar dose of L-pyroglutamic acid.

L-pyroglutamate: an alternate neurotoxin for a rodent model of Huntington's disease.

Intrastriatal injections of L-Pyroglutamate (L-PGA) in mice produced behavioral and neuropathological effects that resemble in part the kainate-injected rat striatal model of Huntington's Disease (HD). The behavioral responses induced after unilateral injections of L-PGA included circling, postural asymmetry of head and trunk and possible dyskinesias. The neuropil in the injected striatum contained dilated profiles, degenerating neurons and oligodendroglia, and numerous phagocytic microglial-like cells. A dose response relation existed. The size of the lesion (expressed as a percent volume of the striatum destroyed) ranged from 1 +/- 0.18% at 0.02 mumoles to 20.2 +/- 3.97% at 200 mumoles L-PGA (pH = 7.3). L-PGA is a weak neurotoxin when compared to kainic acid. Several factors raise interest in the possible role of L-PGA in HD, including the recently reported elevated plasma levels of L-PGA in some HD patients, and these considered in the discussion.

Pyroglutamate kinetics and neurotoxicity studies in mice.

Plasma and brain kinetics of L-glutamic (GA) and L-pyroglutamic (PY) acids were studied after oral administration of monosodium glutamate (MSG) or pyroglutamate to adult mice. Oral MSG (0.5 g/kg) increased plasma GA and PY levels 4.5 and 1.8 times, respectively. A small increase in brain PY (1.3 times the basal level) but not in brain GA, was observed. Oral administration of pyroglutamate (0.5 g/kg) increased plasma PY levels 56 times in adult mice and 69 times in infant mice. No lesions in the arcuate nucleus of the hypothalamus were observed when pyroglutamate was administered orally to infant mice at doses of 2 and 4 g/kg.