Activation of free fatty acid receptors, FFAR1 and FFAR4, ameliorates ulcerative colitis by promote fatty acid metabolism and mediate macrophage polarization.

OBJECTIVE: To investigate the mechanism of action of fatty acid receptors, FFAR1 and FFAR4, on ulcerative colitis (UC) through fatty acid metabolism and macrophage polarization. METHODS: Dextran sulfate sodium (DSS)-induced mouse model of UC mice was used to evaluate the efficacy of FFAR1 (GW9508) and FFAR4 (GSK137647) agonists by analyzing body weight, colon length, disease activity index (DAI), and histological scores. Real-time PCR and immunofluorescence analysis were performed to quantify the levels of fatty acid metabolizing enzymes and macrophage makers. FFA-induced lipid accumulation in RAW264.7 cells was visualized by Oil Red O staining analysis, and cells were collected to detect macrophage polarization by flow cytometry. RESULTS: The combination of GW9508 and GSK137647 significantly improved DSS-induced UC symptoms, caused recovery in colon length, and decreased histological injury. GW9508 + GSK137647 treatment upregulated the expressions of CD206, lipid oxidation enzyme (CPT-1α) and anti-inflammatory cytokines (IL-4, IL-10, IL-13) but downregulated those of CD86, lipogenic enzymes (ACC1, FASN, SCD1), and pro-inflammatory cytokines (IL-1ß, IL-6, TNF-α). Combining the two agonists decreased FFA-induced lipid accumulation and increased CD206 expression in cell-based experiments. CONCLUSION: Activated FFAR1 and FFAR4 ameliorates DSS-induced UC by promoting fatty acid metabolism to reduce lipid accumulation and mediate M2 macrophage polarization.

TRIMETHYLAMINE OXIDE - FACTOR IN THE DEVELOPMENT OF ATHEROSCLEROSIS AND A POTENTIAL TARGET FOR DIETARY AND PHARMACOLOGICAL INTERVENTIONS.

Atherosclerotic Cardiovascular Diseases (ASCVD) are the most common cause of death worldwide. Among the well-known con¬tributors to atherosclerosis are less common ones, such as trimethylamine oxide (TMAO). This substance is formed from the oxida¬tion of trimethylamine (TMA) with the participation of flavin oxidases in the liver. TMA is produced with the involvement of the intestinal microbiota from foods rich in choline and carnitine. TMAO promotes the production of foam cells, enhances platelet aggregation, affects endothelial inflammation, and promotes atherosclerosis development. However, further research is needed to determine the effect of dietary changes on reducing TMAO levels and thus reducing incident ASCVD.

The Gut Axis Involvement in Heart Failure: Focus on Trimethylamine N-oxide.

A novel pathophysiological model of interest is the association between heart failure (HF) and the gastrointestinal system, the 'gut hypothesis'. The choline and carnitine metabolic by-product, Trimethylamine N-oxide (TMAO) is one of the more prominent molecules associated with the link between HF and the gut. Indeed, TMAO levels are increased in HF populations and higher TMAO levels are associated with poor prognosis, whereas low TMAO levels either at baseline/follow up confer better prognosis. Considering that TMAO levels seem not to be affected by guideline-HF treatment, this model could represent a novel and independent therapeutic target for HF.

Gut-restricted apical sodium-dependent bile acid transporter inhibitor attenuates alcohol-induced liver steatosis and injury in mice.

BACKGROUND: Recent studies have shown that human and experimental alcohol-related liver disease (ALD) is robustly associated with dysregulation of bile acid homeostasis, which may in turn modulate disease severity. Pharmacological agents targeting bile acid metabolism and signaling may be potential therapeutics for ALD. METHODS: The potential beneficial effects of a gut-restricted apical sodium-dependent bile acid transporter (ASBT) inhibitor were studied in a chronic-plus-binge ALD mouse model. RESULTS: Blocking intestinal bile acid reabsorption by the gut-restricted ASBT inhibitor GSK2330672 attenuated hepatic steatosis and liver injury in a chronic-plus-binge ALD mouse model. Alcohol feeding is associated with intestinal bile acid accumulation but paradoxically impaired ileal farnesoid × receptor (FXR) function, and repressed hepatic cholesterol 7α-hydrolase (CYP7A1) expression despite decreased hepatic small heterodimer partner (SHP) and ileal fibroblast growth factor 15 (FGF15) expression. ASBT inhibitor treatment decreased intestinal bile acid accumulation and increased hepatic CYP7A1 expression, but further decreased ileal FXR activity. Alcohol feeding induces serum bile acid concentration that strongly correlates with a liver injury marker. However, alcohol-induced serum bile acid elevation is not due to intrahepatic bile acid accumulation but is strongly and positively associated with hepatic multidrug resistance-associated protein 3 (MRP4) and MRP4 induction but poorly associated with sodium-taurocholate cotransporting peptide (NTCP) expression. ASBT inhibitor treatment decreases serum bile acid concentration without affecting hepatocyte basolateral bile acid uptake and efflux transporters. CONCLUSION: ASBT inhibitor treatment corrects alcohol-induced bile acid dysregulation and attenuates liver injury in experimental ALD.

Implication of Gut Microbiota in Cardiovascular Diseases.

Emerging evidence has identified the association between gut microbiota and various diseases, including cardiovascular diseases (CVDs). Altered intestinal flora composition has been described in detail in CVDs, such as hypertension, atherosclerosis, myocardial infarction, heart failure, and arrhythmia. In contrast, the importance of fermentation metabolites, such as trimethylamine N-oxide (TMAO), short-chain fatty acids (SCFAs), and secondary bile acid (BA), has also been implicated in CVD development, prevention, treatment, and prognosis. The potential mechanisms are conventionally thought to involve immune regulation, host energy metabolism, and oxidative stress. However, numerous types of programmed cell death, including apoptosis, autophagy, pyroptosis, ferroptosis, and clockophagy, also serve as a key link in microbiome-host cross talk. In this review, we introduced and summarized the results from recent studies dealing with the relationship between gut microbiota and cardiac disorders, highlighting the role of programmed cell death. We hope to shed light on microbiota-targeted therapeutic strategies in CVD management.

Agonism of GPR120 prevented IL-1ß-induced reduction of extracellular matrix through SOX-9.

Osteoarthritis (OA) is a whole-joint disease with extremely high prevalence. In all treatment approaches of OA, blocking the degradation of the cartilage extracellular matrix is an important treatment. In OA, overexpression of derivative enzymes leads to excessive catabolism and reduced synthesis of cartilage including type II collagen and aggrecan, which results in irreversible destruction of the joint. SOX9 is a transcription factor that regulates the synthesis of type II collagen and aggrecan and is significantly downregulated in OA. GPR120 has been reported to affect the pathophysiology of OA. In this study, we used the GPR120 agonist GW9508 and TUG891 in ATDC5 chondrocytes exposed to interleukin (IL)-1ß to investigate the involvement of GPR120 in SOX9-mediated expression of type II collagen and aggrecan. Our findings show that agonism of GPR120 can reduce inflammation by inhibiting the expression of IL-6 and IL-8 induced by IL-1ß. We also show that GW9508 and TUG891 rescue the expression of type II collagen and aggrecan by preventing the reduction of SOX9 expression. Additionally, we demonstrate that the effects of GW9508 on SOX9 expression are mediated through CREB and that GPR120 is indeed required for this effect. Thus, agonism of GPR120 by GW9508 might be a potential therapeutic strategy to halt or prevent cartilage degradation.

GW9508 ameliorates cognitive impairment via the cAMP-CREB and JNK pathways in APPswe/PS1dE9 mouse model of Alzheimer's disease.

GPR40 was utilized as the drug target to the treatment of diabetes, but the function and mechanisms ameliorating the Alzheimer's disease (AD) remain unknown. In present study, the typical APP/PS1 mouse model was applied to explore the function and mechanism of GPR40 in AD. GPR40 agonist GW9508 and antagonist GW1100 were respectively given by i.c.v. injection to activate/inhibit the GPR40 in the brain of APP/PS1 mice which illustrated the function and mechanism of GPR40 in ameliorating AD symptoms. Morris water maze test, step-through test, Y-maze spontaneous alternation test, open field test and new object recognition test were used to test the cognitive function and memory ability of mice, while molecular biology experiments such as Western blot, immunofluorescence, JC-1 were used to detect the corresponding changes of signal pathways. The results revealed that treatment with GW9508 could significantly ameliorate cognitive deficits of APP/PS1 mice, upregulate the expression levels of cAMP, p-CREB and neurotrophic factors in vivo, while GW9508 also ameliorate Aß1-42-induced neuron damage and downregulate the expression levels of pathological protein such as p-JNK, JNK and apoptosis-related proteins such as IL-6, IL-1ß, TNF-α and caspase-3 in vitro. Meanwhile, high-content screening also showed that GW9508 promoted the cellular differentiation of SH-SY5Y cells, while GW1100 reversed the effects of GW9508. These results suggested that GPR40 was an underlying therapeutic target for the treatment of AD and GPR40 agonist could be explored as the emerging AD therapeutic drug.

Effects of two isometheptene enantiomers in isolated human blood vessels and rat middle meningeal artery - potential antimigraine efficacy.

BACKGROUND: Racemic isometheptene [(RS)-isometheptene] is an antimigraine drug that due to its cardiovascular side-effects was separated into its enantiomers, (R)- and (S)-isometheptene. This study set out to characterize the contribution of each enantiomer to its vasoactive profile. Moreover, rat neurogenic dural vasodilatation was used to explore their antimigraine mechanism of action. METHODS: Human blood vessel segments (middle meningeal artery, proximal and distal coronary arteries, and saphenous vein) were mounted in organ baths and concentration response curves to isometheptene were constructed. Calcitonin gene-related peptide (CGRP)-induced neurogenic dural vasodilation was elicited in the presence of the enantiomers using a rat closed cranial window model. RESULTS: The isometheptene enantiomers did not induce any significant contraction in human blood vessels, except in the middle meningeal artery, when they were administered at the highest concentration (100 µM). Interestingly in rats, (S)-isometheptene induced more pronounced vasopressor responses than (R)-isometheptene. However, none of these compounds affected the CGRP-induced vasodilator responses. CONCLUSION: The isometheptene enantiomers displayed a relatively safe peripheral vascular profile, as they failed to constrict the human coronary artery. These compounds do not appear to modulate neurogenic dural CGRP release, therefore, their antimigraine site of action remains to be determined.

Activation of GPR40 produces mechanical antiallodynia via the spinal glial interleukin-10/ß-endorphin pathway.

BACKGROUND: The G protein-coupled receptor 40 (GPR40), broadly expressed in various tissues such as the spinal cord, exerts multiple physiological functions including pain regulation. This study aimed to elucidate the mechanisms underlying GPR40 activation-induced antinociception in neuropathic pain, particularly related to the spinal glial expression of IL-10 and subsequent ß-endorphin. METHODS: Spinal nerve ligation-induced neuropathic pain model was used in this study. ß-Endorphin and IL-10 levels were measured in the spinal cord and cultured primary microglia, astrocytes, and neurons. Double immunofluorescence staining of ß-endorphin with glial and neuronal cellular biomarkers was also detected in the spinal cord and cultured primary microglia, astrocytes, and neurons. RESULTS: GPR40 was expressed on microglia, astrocytes, and neurons in the spinal cords and upregulated by spinal nerve ligation. Intrathecal injection of the GPR40 agonist GW9508 dose-dependently attenuated mechanical allodynia and thermal hyperalgesia in neuropathic rats, with Emax values of 80% and 100% MPE and ED50 values of 6.7 and 5.4 µg, respectively. Its mechanical antiallodynia was blocked by the selective GPR40 antagonist GW1100 but not GPR120 antagonist AH7614. Intrathecal GW9508 significantly enhanced IL-10 and ß-endorphin immunostaining in spinal microglia and astrocytes but not in neurons. GW9508 also markedly stimulated gene and protein expression of IL-10 and ß-endorphin in cultured primary spinal microglia and astrocytes but not in neurons, originated from 1-day-old neonatal rats. The IL-10 antibody inhibited GW9508-stimulated gene expression of the ß-endorphin precursor proopiomelanocortin (POMC) but not IL-10, whereas the ß-endorphin antibody did not affect GW9508-stimulated IL-10 or POMC gene expression. GW9508 increased phosphorylation of mitogen-activated protein kinases (MAPKs) including p38, extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinase (JNK), and its stimulatory effects on IL-10 and POMC expression were blocked by each MAPK isoform inhibitor. Spinal GW9508-induced mechanical antiallodynia was completely blocked by intrathecal minocycline, IL-10 neutralizing antibody, ß-endorphin antiserum, and µ-opioid receptor-preferred antagonist naloxone. CONCLUSIONS: Our results illustrate that GPR40 activation produces antinociception via the spinal glial IL-10/ß-endorphin antinociceptive pathway.

Effect of TMAO, a Gut-Bacteria Metabolite, on Dry Eye in a Rat Model.

PURPOSE Trimethylamine oxide (TMAO) is an osmolyte used by saltwater animals to protect their cells from hyperosmotic environment. Here, we studied if TMAO may exert beneficial effect in dry eye disease (DED) which results from hyperosmotic tear film. MATERIALS AND METHODS Female, 12-week-old Sprague-Dawley rats underwent either removal of extra- and infraorbital lacrimal glands (dry eye group) or sham surgery (sham group). A 1-week topical treatment with either 0.9% NaCl (control) or 1% TMAO in 0.9% NaCl solution was started 4 weeks after the surgery. Fluorescein score (FS), blink rate (BR), and histological picture of cornea were assessed. RESULTS At baseline, corneas did not stain with fluorescein and there was no difference between the groups in BR. There was a significant increase in FS and BR after the removal of lacrimal glands (p < 0.0001 and p = 0.0003, respectively). Accordingly, the dry eye group showed significantly higher FS and BR than the sham group (p = 0.0003 and p = 0.0005, respectively). Treatment with TMAO but not with 0.9% NaCl significantly reduced FS and BR (p = 0.01 and p = 0.005, respectively); however, FS and BR did not return to baseline (p = 0.0045 and p = 0.0065, respectively). In comparison to the control group, treatment with TMAO did not affect epithelial thickness or the number of layers of epithelium layers. CONCLUSIONS We have found that in a rat model of DED, the topical treatment with TMAO improves clinical picture, however does not lead to the evident histopathological recovery.

Gut Microbiota in Human Immunodeficiency Virus-Infected Individuals Linked to Coronary Heart Disease.

Background: Human immunodeficiency virus (HIV) infection is an independent risk factor for coronary heart disease (CHD) and is associated with perturbation of the gut microbiota. Methods: We analyzed gut microbiota in 30 HIV-infected individuals with CHD (CHD+) and 30 without CHD (CHD-) of the HIV-HEART study group. Results: Gut microbiota linked to CHD was associated with lower α-diversity. Despite insignificant differences in ß-diversity, co-occurrence networks of bacterial genera clearly diverged between CHD+ and CHD- individuals. Multidimensional scaling separated HIV-infected individuals into 2 microbiome clusters, dominated by the genus Prevotella or Bacteroides. The relative abundance of 49 other genera was significantly different between both clusters. The Prevotella-rich cluster was largely composed of men who have sex with men (MSM) (97%), whereas the Bacteroides-rich cluster comprised both MSM (45%) and heterosexual individuals (55%). MSM of the Bacteroides-rich cluster were characterized by reduced α-diversity, advanced immunological HIV stage, longer antiretroviral therapy with more ART regimens, and longer use of protease inhibitors, compared with Prevotella-rich MSM. Conclusions: Community structures of gut microbiota rather than individual species might facilitate risk assessment of CHD in HIV-infected individuals. Sexual behavior appears to be an important factor affecting gut microbiota ß-diversity and should be considered in future studies.

Validation of Matrix Metalloproteinase-9 (MMP-9) as a Novel Target for Treatment of Diabetic Foot Ulcers in Humans and Discovery of a Potent and Selective Small-Molecule MMP-9 Inhibitor That Accelerates Healing.

Diabetic foot ulcers (DFUs) are a significant health problem. A single existing FDA-approved drug for this ailment, becaplermin, is not standard-of-care. We previously demonstrated that upregulation of active matrix metalloproteinase (MMP)-9 is the reason that the diabetic wound in mice is recalcitrant to healing and that MMP-8 participates in wound repair. In the present study, we validate the target MMP-9 by identifying and quantifying active MMP-8 and MMP-9 in human diabetic wounds using an affinity resin that binds exclusively to the active forms of MMPs coupled with proteomics. Furthermore, we synthesize and evaluate enantiomerically pure ( R)- and ( S)-ND-336, as inhibitors of the detrimental MMP-9, and show that the ( R)-enantiomer has superior efficacy in wound healing over becaplermin. Our results reveal that the mechanisms of pathology and repair are similar in diabetic mice and diabetic humans and that ( R)-ND-336 holds promise for the treatment of DFUs as a first-in-class therapeutic.

Chronic, low-dose TMAO treatment reduces diastolic dysfunction and heart fibrosis in hypertensive rats.

Several studies have suggested negative effects of trimethylamine oxide (TMAO) on the circulatory system. However, a number of studies have shown protective functions of TMAO, a piezolyte and osmolyte, in animals exposed to high hydrostatic and/or osmotic stress. We evaluated the effects of TMAO treatment on the development of hypertension and its complications in male spontaneously hypertensive rats (SHRs) maintained on water (SHR-Water) and SHRs drinking TMAO water solution from weaning (SHR-TMAO). Wistar-Kyoto (WKY) rats were used as normotensive controls to discriminate between age-dependent and hypertension-dependent changes. Telemetry measurements of blood pressure were performed in rats between the 7th and 16th weeks of life. Anesthetized rats underwent echocardiographic, electrocardiographic, and direct left ventricular end-diastolic pressure (LVEDP) measurements. Hematoxylin and eosin as well as van Gieson staining for histopathological evaluation were performed. Plasma TMAO measured by chromatography coupled with mass spectrometry was significantly higher in the SHR-Water group compared with the WKY group (~20%). TMAO treatment increased plasma TMAO by four- to fivefold and did not affect the development of hypertension in SHRs. Sixteen-week-old rats in the SHR-Water and SHR-TMAO groups (12-wk TMAO treatment) showed similar blood pressures, angiopathy, and cardiac hypertrophy. However, the SHR-TMAO group had lower plasma NH2-terminal pro-B-type natriuretic peptide, LVEDP, and cardiac fibrosis. In contrast to age-matched WKY rats, 60-wk-old SHRs showed hypertensive angiopathy and heart failure with preserved ejection fraction. Compared with the SHR-Water group, the SHR-TMAO group (56-wk TMAO treatment) showed significantly lower plasma NH2-terminal pro-B-type natriuretic peptide and vasopressin, significantly lower LVEDP, and cardiac fibrosis. In conclusion, a four- to fivefold increase in plasma TMAO does not exert negative effects on the circulatory system. In contrast, increased dietary TMAO seems to reduce diastolic dysfunction in pressure-overloaded hearts in rats. NEW & NOTEWORTHY Chronic, low-dose trimethylamine oxide (TMAO) treatment that increases plasma TMAO by four- to fivefold reduces plasma NH2-terminal pro-B-type natriuretic peptide and vasopressin, left ventricular end-diastolic pressure, and cardiac fibrosis in pressure-overloaded hearts in hypertensive rats. Our study provides evidence that a moderate increase in plasma TMAO does not have a negative effect on the circulatory system. In contrast, increased dietary TMAO seems to reduce diastolic dysfunction in the pressure-overloaded heart.

Work in Progress: Drugs in Development.

Primary biliary cholangitis is an archetypal autoimmune disease that causes cholestasis, fibrosis, and liver failure. Ursodeoxycholic acid and obeticholic acid are approved for its treatment. Not all patients respond, some are intolerant, many have ongoing symptoms, and new therapies are required. Herein we describe drugs in development and potential future biological targets. We consider compounds acting on the farnesoid X receptor/fibroblast growth factor 19 pathway, fibrates and other agonists of the peroxisome proliferator-activated receptor family, transmembrane-G-protein-receptor-5 agonists, and several immunological agents. We also consider the roles of bile acid reuptake inhibitors, nalfurafine, and fibrates in pruritus management.

The effect of NAD-299 and TCB-2 on learning and memory, hippocampal BDNF levels and amyloid plaques in Streptozotocin-induced memory deficits in male rats.

RATIONALE: Alzheimer's disease (AD) is the most common form of dementia characterized by a progressive decline in cognitive function. The serotonergic system via the 5-HT1A receptor and 5-HT2A receptor is proposed to affect the cognitive process. OBJECTIVE: In the present study, the effects of NAD-299 (5-HT1AR antagonist) and TCB-2 (5-HT2AR agonist) on learning and memory processes, hippocampal brain-derived neurotrophic factor (BDNF) levels, neuronal necrosis, and Aß plaque production have been investigated on the intracerebroventricular (icv) injection of streptozotocin (STZ)-induced memory deficits in rats. METHODS: Fifty-four adult male Wistar rats (250-300 g) were divided into six groups (n = 9 in each group): control, sham-operated, AD (icv-STZ (3 mg/kg, 10 µl)), AD+NAD-299 (5 µg/1 µl icv for 30 days), AD+TCB-2 (5 µg/1 µl icv for 30 days), and AD+NAD-299 + TCB-2 (NAD-299 (5 µg/0.5 µl icv) and TCB-2 (5 µg/0.5 µl icv) for 30 days). Following the treatment period, rats were subjected to behavioral tests of learning and memory. Then, hippocampal BDNF, amyloid-beta (Aß) plaque, and neuronal loss were determined by ELISA Kit, Congo red staining, and Nissl staining, respectively. RESULTS: The results of behavioral tests showed that icv-STZ injection decreased the discrimination index in the novel object recognition (NOR) test. In the passive avoidance learning (PAL) task, icv-STZ injection significantly decreased step-through latency (STLr) and increased time spent in dark compartment (TDC). Treatment with NAD-299, TCB-2, and NAD-299 + TCB-2 attenuated the STZ-induced memory impairment in both NOR and PAL tasks. icv-STZ induced a decrease in hippocampal BDNF levels and increased Aß plaques production in the brain, whereas treatment with NAD-299, TCB-2, and NAD-299 + TCB-2 reduced Aß plaques in the brain and increased the hippocampal BDNF level. Results of Nissl staining showed that icv-STZ injection increased neuronal loss in the hippocampus, while treatment with NAD-299, TCB-2, and NAD-299 + TCB-2 reduced hippocampal neurodegeneration. CONCLUSION: These findings suggest that 5-HT1AR blockade by NAD-299 and 5-HT2AR activation by TCB-2 improve cognitive dysfunction in icv-STZ-treated rats, and these drugs may potentially prevent the progression of AD.

Structure-based design of free fatty acid receptor 1 agonists bearing non-biphenyl scaffold.

The free fatty acid receptor 1 (FFA1) enhances the glucose-stimulated insulin secretion without the risk of hypoglycemia. However, most of FFA1 agonists have a common biphenyl moiety, leading to a relative deprivation in structure types. Herein, we describe the exploration of non-biphenyl scaffold based on the co-crystal structure of FFA1 to increase additional interactions with the lateral residues, which led to the identification of lead compounds 3 and 9. In induced-fit docking study, compound 3 forms an edge-on interaction with Trp150 by slightly rotating the indole ring of Trp150, and compound 9 has additional hydrogen bond and δ-π interactions with Leu135, which demonstrated the feasibility of our design strategy. Moreover, lead compounds 3 and 9 revealed improved polar surface area compared to GW9508, and have considerable hypoglycemic effects in mice. This structure-based study might inspire us to design more promising FFA1 agonists by increasing additional interactions with the residues outside of binding pocket.

Management of Chronic Hepatic Itch.

Hepatic itch remains among the most agonizing symptoms for affected patients and a major clinical challenge for physicians. Pruritus may occur in almost all liver diseases, particularly those with cholestatic features. Hepatic itch arises irrespective of the severity of the underlying liver disease or extent of cholestasis. Antihistamines are ineffective in hepatic itch. Therapeutic recommendations consist of a guideline-based stepwise approach, starting with the anion exchange resin cholestyramine, followed by rifampicin, naltrexone, and sertraline. Bezafibrate and ileal bile acid transporter inhibitors are promising future treatment options. Experimental and invasive procedures should be reserved for refractory pruritus.

Trimethylamine N-oxide (TMAO) is a counteracting solute of benzyl alcohol for multi-dose formulation of immunoglobulin.

Multi-dose formulation of biopharmaceuticals contains an antimicrobial preservative, which may facilitate aggregation of the proteins due to their hydrophobic protein binding. Here, we have investigated the effects of co-solvents on heat-induced protein aggregation in the presence of an antimicrobial preservative. Human immunoglobulin G (IgG) and benzyl alcohol were chosen because of their wide usage as a pharmaceutical protein and a preservative. Trimethylamine N-oxide (TMAO) was found to be the most effective additive in suppressing benzyl alcohol-induced IgG aggregation among the additives tested. Interestingly, TMAO was less effective in increasing the thermal-unfolding temperature of IgG than trehalose, indicating that the observed suppression of IgG aggregation by TMAO in the presence of benzyl alcohol is not simply due to its stabilization effect of the protein structure. The solubility of benzyl alcohol increased with increasing concentrations of TMAO, suggesting that the hydrophobic interaction of TMAO with benzyl alcohol is responsible for the observed aggregation suppression. Thus, TMAO may be a potential aggregation suppressor in multi-dose formulation of biopharmaceuticals.