Table 3.

Pharmacological effects of AOAA in various in vivo experimental models and the proposed underlying pharmacological mechanism(s) action. The listed studiess [88,175,178,296,299,320,394,396,408,409,410,411,412,413,414,415,416,417,418,419,420,421,422,423,424,425,426,427,428,429] provides a selection of the in vivo physiology and efficacy studies obtained with AOAA in various animal studies over the last six decades.

Animal Model	Dose of AOAA	Effects of AOAA; Proposed Mechanism of Action	Reference
Methionine sulfoximine or thiosemicarbazide induced seizures in mice, Sprague-Dawley rats, and cats	23–50 mg/kg i.p. single dose	AOAA dose-dependently decreased the incidence of convulsions and improved survival. The mechanism of action was proposed to be inhibition by AOAA of GABA-T activity in the CNS and subsequent elevation of brain GABA content; in support of this hypothesis, brain GABA levels were measured and were found to be increased at the same doses of AOAA where functional benefits were also noted.	[408]
Endocochlear potentials in response to 6 kHz tone bursts in anesthetized guinea pigs	10–80 mg/kg i.v. single dose	AOAA dose-dependently attenuated the generation of endocochlear potentials. The mechanism of action was not identified, but observations of this type have subsequently led to clinical trials with AOAA in patients with tinnitus.	[409]
Isonicotinic acid hydrazide-induced seizures in male Swiss albino mice	23 mg/kg i.p. single dose	AOAA dose-dependently decreased the incidence of convulsions. The mechanism of action was proposed to be due to a combined inhibition by AOAA of GABA-T activity (which inhibits GABA degradation) and of glutamate decarboxylase activity (which catalyzes GABA production from glutamate), and the resulting changes in the brain GABA content are the function of these two combined enzymatic effects.	[410,411]
Pentobarbital metabolism in mice	30 mg/kg i.v. single dose	AOAA increased pentobarbital plasma levels and decreased the plasma levels of pentobarbital metabolites. The mechanism of action was not identified, but it was suggested to relate to an AOAA-induced broad suppression of cellular bioenergetics.	[412]
Cobalt-induced epilepsy in male piebald rats	2.5–10 mg/kg i.p. single dose	AOAA reduced the frequency of epileptic spikes in the secondary foci of cobalt epileptic rats. The mechanism of action was proposed to be inhibition of GABA-T activity in the brain; however, the protective effect of AOAA was more pronounced at the lower dose (5 mg/kg) while the enhancement of CNS GABA-T levels was more pronounced at higher doses, where the functional benefit of AOAA was less pronounced.	[413]
Memory consolidation in male Sprague-Dawley rats	25 mg/kg/day i.p. for 8 days	In the shuttlebox shock avoidance used, controls animals showed learning both within and across sessions, while AOAA-treated only showed learning within sessions but exhibited a lack of consolidation across sessions. Because GABA plays a role in memory consolidation, the mechanism was hypothesized to relate to the inhibitory effect of AOAA on GABA-T, but no pharmacological mechanism was investigated in the study.	[414]
Hyperbaric oxygen induced seizures in chicken	2.5 mg/kg s.q. single dose	AOAA decreased the onset and duration of the convulsions. The mechanism of action was proposed to be inhibition by AOAA of the GABA-T activity in the CNS and an elevation of central GABA levels, but no biochemical markers were measured.	[415]
Dichlorovinylcysteine induced nephrotoxicity model in male NMRI mice	40 mg/kg i.p. single dose	AOAA attenuated the generation of various lipid peroxidation markers. The mechanism of action was not directly explored but was presumed to be related to an antioxidant effect of AOAA.	[416]
Circulating glucose and insulin and glucagon levels in control and streptozotocin-diabetic female Wistar rats	30 mg/kg i.p. single dose	In control animals, AOAA significantly increased circulating insulin levels (but not glucose or glucagon levels). In the diabetic animals, AOAA protected against the development of streptozotocin-induced hyperglycemia. Streptozotocin caused a 50% drop in plasma insulin levels in the rats; this effect was largely absent in the AOAA-treated streptozotocin animals. The proposed mechanism relates to AOAA’s effect on some peripheral GABA-T system and subsequent increases in peripheral GABA levels, but no direct measurements were provided.	[417]
Male Wistar rats subjected to stroke (transient middle cerebral artery occlusion)	2.5, 5, 10 or 50 mg/kg i.p. single dose	AOAA at 10 and 50 mg/kg significantly reduced stroke volume and brain edema and improved neurological scores, without affecting post-ischemic cerebral blood flow, brain malondialdehyde content, SOD, or glutathione peroxidase activity. The mechanism of action was proposed to be inhibition of CBS activity by AOAA in the brain, but no biochemical markers were measured.	[418]
Hypoxia-induced central apneas in ventilated C57BL/6J mice	30 mg/kg i.p. single dose	AOAA reduced the percentage of animals expressing one or more apneas during reoxygenation. AOAA-treated mice also exhibited a smaller coefficient of variation for frequency during reoxygenation, suggesting improved respiratory stability. The mechanism of action was proposed to be inhibition of CBS activity in the CNS, but no biochemical markers were measured.	[419]
Cisplatin nephrotoxicity in male C57BL/6 mice or F344 rats	100 mg/kg p.o., single dose	AOAA protected against the biochemical (plasma BUN) and histological (renal tubular alterations) damage induced by cisplatin. The mechanism of action was proposed to be inhibition of cysteine S-conjugate b-lyase activity by AOAA (and/or an inhibitory effect of AOAA on some other PLP-dependent enzyme, most likely a transaminase). However, no experiments were conducted to delineate the molecular mechanism of AOAA’s action.	[420,421]
Tumor growth in female BALB/c nude mice bearing MDA-MB-231 human breast cancer subcutaneous xenografts	10 mg/kg/day i.p. for 14 days	AOAA significantly inhibited tumor growth. Based on complementary in vitro studies, the mechanism of AOAA’s action was proposed to relate to the suppression of tumor cell bioenergetics, in particular due to AOAA-mediated inhibition of tumor cell aspartate aminotransferase activity (an enzyme which functions in tandem with malate dehydrogenase to regulate mitochondrial electron transport).	[394]
Complete Freund adjuvant (CFA)-induced mechanical hyperalgesia model in adult Sprague-Dawley rats	5, 15 or 45 mg/kg/day i.p. single dose	AOAA dose-dependently attenuated mechanical hyperalgesia due to an inhibition of the hyperexcitability of dorsal root ganglion neurons. In these neurons, CFA up-regulated CBS mRNA transcription and subsequent translation of CBS protein. The mode of AOAA’s action was proposed to be related to inhibition of CBS activity, and the consequent prevention of the H2S-mediated opening of tetrodotoxin-resistant voltage-gated sodium channels.	[422]
Tumor growth in female athymic nude mice bearing subcutaneous xenografts of HCT116 colon cancer cells or human patient-derived colon cancer xenografts (PDTX). Liver metastasis model (nude mice, intracecal HCT116 implantation)	1, 3 or 9 mg/kg/day i.p. for 2 weeks	AOAA (at 9 mg/kg/day, but not at the lower doses) suppressed tumor growth. The underlying mechanisms was proposed to relate to the AOAA-induced inhibition of intratumor CBS, inhibition of intratumor H2S production, which, in turn, inhibits cellular bioenergetics and reduces tumor angiogenesis. The effect of AOAA was independent of the tumor’s K-ras status. The effects of AOAA were reproduced by the AOAA prodrug YD0171, which, however, was more potent (effective at 0.5 and 1 mg/kg/day). YD0171 (at 3 mg/kg/day for 3 weeks), caused the regression of established HCT116 subcutaneous xenografts. YD0171 also inhibited liver metastasis formation in an intracecal HCT116 implantation model.	[88,296,320]
Athymic Balb/c mice bearing SUM149, SUM159, or HCC1954 MDA-MB-231 xenografts; MMTV-rTtA-TetO-myc mouse mammary tumor model	5 mg/kg/day i.p. or 0.5 mg/kg/day i.p. in the TetO-myc model	AOAA suppressed the growth of the UM149, SUM159 xenografts, but did not affect the growth of HCC1954 xenografts. AOAA was also effective in the TetO-myc model. In the MDA-MB-231 xenografts, AOAA did not inhibit tumor growth alone, but potentiated the growth-suppressant effect of paclitaxel. The underlying mechanisms was proposed to relate to the inhibition of intratumor GOT activity, as it is associated with increased C-MYC expression in the tumors and the subsequent increased reliance of the tumor cells on glutaminolysis.	[369]
Male BALB/c mice subjected to burn injury	10 mg/kg/day i.p. for 6 days	AOAA attenuated the degree of burn-induced oxidative stress in various tissues. It also reduced plasma levels of various circulating mediators (IL-6, IL-10). It improved various plasma markers of multiorgan failure. The effects were attributed to AOAA’s effect as an inhibitor of CBS.	[175]
Female athymic nude mice bearing subcutaneous xenografts of various human colon cancer tumor lines	5 or 10 mg/kg/day i.p. for 2–4 weeks (depending on the growth of the particular cell line graft)	AOAA dose-dependently reduced tumor growth of the HCT116, DLD1, RKO, and HT29 xenografts, but did not affect the growth of SW40 or LoVo xenografts). The underlying mechanisms was proposed to relate to the inhibition by AOAA of glutamate pyruvate transaminase 2 (GPT2) in the tumor cells. This hypothesis was supported by the findings that the growth of PIK3CA mutant xenograft tumors (which express GPT2) were inhibited by AOAA, but GPT2 knockdown tumors were not. (It should be noted, however that the latter tumors showed a significantly slower baseline proliferation rate in the absence of AOAA).	[423]
Male Wistar rats subjected to experimental subarachnoid hemorrhage induced by double blood injection; effect of L-cysteine	5 mg/kg i.p. single dose	AOAA suppressed the neuroprotective effect of L-cysteine. Its mechanism of action was proposed to be inhibition of CBS-induced H2S production. The authors’ working hypothesis is that L-cysteine increases CBS-derived H2S production, and this produces neuroprotective effects. Unfortunately, the effect of AOAA (in the absence of L-cysteine) was not tested in the study.	[424]
Female athymic nude mice bearing subcutaneous xenografts of NCM356 colon epithelial cells overexpressing CBS	9 mg/kg/day i.p. for 2 weeks	AOAA significantly decreased the size of established tumors. The underlying mechanisms was proposed to relate to the inhibition of intratumor CBS activity by AOAA and the consequent inhibition of intratumor H2S production. Metabolomic and pharmacological studies also implicated a role for the pentose phosphate pathway in the CBS-mediated enhancement of tumor growth.	[303]
Experimental allergic encephalomyelitis model in C57BL/6 mice induced by a myelin oligodendrocyte glycoprotein peptide fragment	35 mg/kg/day i.p. for 7 days	Disease severity was suppressed by AOAA. The effect of AOAA was associated with significant changes in immune cell populations. The percentage of IL-17-producing T cells was reduced while the percentage of FOXP3+ T cells increased, while the percentage of IFNγ + cells was unaffected in the central nervous system. The ratio of FOXP3+ cells to IL-17+ cells increased by AOAA. AOAA markedly reduced the total number of mononuclear cells infiltrating into the central nervous system. Based on complementary in vitro and in vivo studies, the mechanism proposed to underlie AOAA’s action was proposed to relate to the suppression of immune cell bioenergetics, in particular due to AOAA-mediated inhibition of GOT1 activity, which produces an increase in 2-hydroxyglutarate levels in differentiating TH17 cells, which in turn results in the hypermethylation of the Foxp3 gene locus and inhibited Foxp3 transcription, which ultimately regulates the differentiation towards TH17.	[178]
Male Sprague-Dawley rats subjected to an experimental model of chronic alcoholism (chronic ethanol consumption)	5 mg/kg/day i.p. for 2 weeks	Alcoholism produced learning and memory deficits (assessed by the Morris water maze test). AOAA improved latency and swimming distance parameters and improved the animals’ performance in the spatial probe test. AOAA also prevented the down-regulation of myelin basic protein expression and protected against the deterioration of mitochondrial ultrastructure. The mechanism of action was proposed to be inhibition of CBS activity by AOAA in the brain; the AOAA-induced normalization of hippocampal H2S levels provided some experimental support for this theory. AOAA also induced complex changes in gene expression and antioxidant levels in the brain of the animals.	[425,426]
Male Swiss albino mice subjected to stroke (transient middle cerebral artery occlusion) in combination with remote ischemic preconditioning	50 mg/kg i.p. single dose	AOAA suppressed the neuroprotective effect of remote ischemic preconditioning. Its mechanism of action was proposed to be inhibition of CBS-induced H2S production. The authors’ working hypothesis is that stroke down-regulates CBS expression in the CNS, and this down-regulation is prevented by preconditioning. Unfortunately, the effect of AOAA on stroke (in the absence of preconditioning) was not tested in the study.	[427]
Male and female SOD1G93A mice, a model of familial ALS	8.75 mg/kg/day i.p. for 100 days	AOAA significantly improved motor performance (Rotarod test) in the female (but not male) animals and tended to extend survival. The underlying mechanisms was proposed to relate to an up-regulation of CBS in ALS, which, in turn, elevates H2S to cytotoxic concentrations. Thus, it was hypothesized that inhibition of CBS activity with AOAA reduces neuronal and glial H2S levels to physiological (cytoprotective) levels. The gender difference was proposed to relate to higher levels of CNS H2S levels in females with ALS than males with ALS.	[428]
Male athymic nude mice bearing subcutaneous human colon cancer cell line xenografts	9 mg/kg/day i.p. 5 days per week for 4 weeks	AOAA potentiated the inhibitory effect of oxaliplatin on tumor growth, but on its own, did not exert a significant inhibitory effect. The underlying mechanisms was proposed to relate to the AOAA-induced inhibition of intratumor CBS and the subsequent inhibition of intratumor H2S production, with a consequent suppression of cellular bioenergetics and of tumor angiogenesis. The potentiation of oxaliplatin’s antitumor effect was hypothesized to be related to an enhancement by AOAA of oxaliplatin-induced tumor cell apoptosis.	[318]

i.p. = intraperitoneal administration; i.v. = intravenous administration; p.o. = per os (oral administration); s.q. = subcutaneous administration.