Probiotic-mediated metabolism enhanced lovastatin activity in vitro
In our previous study, LCZ exhibited a substantial 54.6% degradation rate of lovastatin in an in vitro culture [11]. This observation brought attention to the potential impact of probiotic-mediated biochemical transformations on drug activity, which could even result in adverse effects due to the production of toxic compounds in some cases. Therefore, in this study, we employed non-targeted qualitative metabolomics analysis as a first step to identify LCZ-produced metabolites of lovastatin.
We monitored the metabolomic profiles of LCZ monoculture media grown with or without lovastatin at various time points (Fig. S1). The metabolomic data from all parallel samples exhibited a high level of consistency in the overlay total ion chromatograms run in both positive and negative modes (Fig. S2A, S2B), indicating the robust stability of the LC-MS system. Lovastatin was identified by its theoretical mass-to-charge ratio (405.264 m/z; Fig. S3), showing a retention time of 12.017 min, and it was only detected in the culture medium of lovastatin-containing LCZ monoculture but not in the negative culture without the drug. The concentration of lovastatin notably decreased over time.
Subsequently, we compared the metabolomic profiles of the culture media from LCZ monoculture with and without lovastatin using the MetabolitePilot™ software. MetabolitePilot™ allows for fast and efficient identification of metabolites by comparing mass spectra between samples and controls. It extracts compounds that are unique to the samples (not found in the controls) and those that share common MS/MS fragments or neutral loss with the target compound. In our analysis, a compound was considered a lovastatin metabolite when it met the following criteria: 1) it was only observed in the lovastatin-containing LCZ culture, 2) it shared common MS/MS fragments or neutral loss with lovastatin, and 3) it could be consistently identified in three independent experiments and increased as the incubation progressed.
Initially, at 0 h, lovastatin appeared as a single peak and was the only metabolite detected in lovastatin-containing LCZ culture medium (Fig. 1A). After 6 h of incubation, a new compound, referred to as M1 (m/z = 421.5240, retention time = 11.05), emerged, and its peak size continued to increase at 12 and 24 hours. This compound shared two MS/MS fragments (neutral mass: [157.1016 + 1] and [171.1344 + 1]) with lovastatin (neutral mass: [159.1161-1] and [173.1319-1]; Fig. 1B). It also exhibited a neutral loss (Δ [405.2641–303.1940]), corresponding to a fragment seen in the mass spectra of M1 (corresponding to 101.0215 m/z).
Additionally, the MetabolitePilot™ software proposed a chemical structure for M1, suggesting that it might originate from the cleavage of the ester bond within lovastatin. To confirm this, a chemical formula search was conducted using the website https://www.chemicalbook.com/StructuredSearch.htm. The search results matched M1 with lovastatin hydroxy acid, indicating that LCZ likely metabolized lovastatin into lovastatin hydroxy acid. To validate this finding, a standard solution of lovastatin hydroxy acid was purchased from Sigma-Aldrich for targeted metabolomics analysis. In three independent experiments, lovastatin hydroxy acid was only detected in the lovastatin-containing LCZ culture but not in the lovastatin-only control after 24-h incubation at 37°C (Fig. 1C), which is consistent with our hypothesis. It is worth noting that lovastatin hydroxy acid has been scientifically established as an active metabolite of lovastatin [25]. Therefore, the metabolism of lovastatin by LCZ into lovastatin hydroxy acid may enhance the drug activity and efficacy.
Probiotics, including LCZ, are not native inhabitants of the human gastrointestinal tract. Therefore, it would be necessary to monitor the changes in the viability of LCZ and its potential in lovastatin metabolism during transit through the gastrointestinal tract. Thus, a co-digestion experiment was then conducted using a DHS-IV system (Fig. S4). In the in vitro digestion system, we observed a progressive decrease in the viable count of LCZ over time (63.25 ± 9.72%, 39.60 ± 6.84%, and 24.82 ± 4.32% at 6 h, 12 h, and 24 h, respectively; Table. S3). Concurrently, the concentration of lovastatin decreased significantly when co-digested with LCZ (p < 0.05; Fig. 1D) compared to lovastatin alone. This reduction in lovastatin concentration was accompanied by an increase in the concentration of lovastatin hydroxy acid (Fig. 1E). Conversely, no such changes were observed in the negative control lacking LCZ.
Co-administering LCZ exacerbated lovastatin toxicity at medium and high doses
To further investigate the potential enhancement of LCZ administration on the activity and efficacy of lovastatin in vivo, a hyperlipidemic model was established in Syrian golden hamsters through the administration of an 8-week HFD. The changes in several indicators of hyperlipidemia, including body weight and serum levels of TC, TG, LDL-c, and HDL-c, were monitored before and after the 8-week HFD to evaluate the effectiveness of the hyperlipidemic model construction. At the end of the 9-week HFD, all monitored indicators showed a significant increase in the MD group compared to the Ct group (p < 0.05 in all cases; Fig. S5), confirming the successful establishment of the hyperlipidemic model. Subsequently, the Syrian golden hamsters in the MD group were randomly divided into eight groups (n = 10 per group) for various combinations of LCZ and lovastatin interventions (Fig. 2A).
Unexpectedly, some golden hamsters in the M group, H group, P + M group, and P + H group died within seven days of intervention. The intervention was immediately terminated. Blood samples were collected from the golden hamsters, and the survival rate of the remaining hamsters in these groups was monitored over the following days. The survival rate in the H + P and M + P groups was significantly lower compared to the H and M groups (p < 0.05; Fig. 2B, 2C), suggesting that LCZ administration exacerbated the toxicity of high- and medium-dose lovastatin, while low-dose lovastatin was well-tolerated. As no deaths were observed in the L group, P group, L + P group, and N group, the interventions were implemented in these groups as initially planned.
During the treatment period, the L + P group exhibited the most rapid reduction in body weight and blood lipid levels (Fig. S6), followed by the L group. By the 8th week of treatment, the body weight and the LDL-c level in the L + P group had returned to levels comparable to the Ct group (Fig. 2D, 2E), and the TG level in the L + P group was even lower than in the Ct group (Fig. 2F). Although the TC level in the L + P group remained higher than the Ct group, it was significantly lower than the other treatment groups (p < 0.05; Fig. 2G). Moreover, the golden hamsters in the L + P group had the smallest liver weight (Fig. 2I) and the fewest, smallest liver adipose droplets (Fig. 2J) compared to the Ct group. These results suggest that combined therapy of LCZ and lovastatin was more effective than administering lovastatin treatment alone. The P and N groups showed no significant differences throughout the treatment period, suggesting that LCZ alone had no therapeutic effect on hyperlipidemia. Additionally, none of the treatments were able to reduce the serum HDL-c level in the hyperlipidemic golden hamsters (Fig. 2H).
Long-term use of statins may result in some serious adverse effects, including statin-associated muscle symptoms, potential new-onset type 2 diabetes mellitus, neurological and neurocognitive effects, hepatotoxicity, renal toxicity, and side effects in the gastrointestinal, urogenital, and reproductive systems, even though it has not been proven whether these effects are legitimate [26]. Therefore, we assessed the impact of administering LCZ and low-dose lovastatin alone or in combination on biomarkers that reflect the function and health of the liver, kidney, and muscles. The results showed no significant differences in serum levels of albumin, total bilirubin, creatinine, as well as the activities of alanine aminotransferase, aspartate aminotransferase, and creatine kinase between the Ct group and the other intervention groups (L, L + P, P, and N groups; Fig. S7). These findings suggest that the administration of LCZ and low-dose lovastatin, whether alone or in combination, did not have any adverse impact on the liver, kidney, and muscle functions of golden hamsters.
Co-administering LCZ and low-dose lovastatin enhanced the drug absorption
Our in vitro experiment demonstrating the ability of LCZ to convert lovastatin into its active form, lovastatin hydroxy acid, we hypothesized that the enhanced antihyperlipidemic efficacy observed with the co-therapy of LCZ and low-dose lovastatin could be attributed to the in vivo metabolic conversion of lovastatin to lovastatin hydroxy acid by LCZ in the gut. To test this hypothesis, we measured the concentrations of lovastatin and lovastatin hydroxy acid in various gastrointestinal compartments (ileum, cecum, colon, and feces), as well as the serum, liver, and kidney of the golden hamsters. According to our hypothesis, we anticipated that extensive metabolism of lovastatin in the gut would lead to increased levels of lovastatin hydroxy acid in the intestines along with a decrease in lovastatin in the blood and the hepatic and renal circulatory systems. Intriguingly, we noted significant reductions in lovastatin concentration in the ileum, cecum, colon, and feces of the golden hamsters in the L + P group compared to the L group (Fig. 3A); however, there were no significant increases in the levels of lovastatin hydroxy acid in the samples collected from these gastrointestinal compartments (Fig. 3B). Furthermore, we observed significant increases in both lovastatin and lovastatin hydroxy acid concentrations in the serum, liver, and kidney of the hamsters in the L + P group compare to the L group (p < 0.05; Fig. 3C). These findings suggest that LCZ might not have metabolized lovastatin into lovastatin hydroxy acid in the intestine. Instead, it is more likely that LCZ enhanced the intestinal absorption of lovastatin into the bloodstream. The drug eventually reached the liver and underwent subsequent hepatic metabolism. A previous pharmacokinetic study has reported that the liver can metabolize lovastatin into lovastatin hydroxy acid [25].
Statins act by competitively inhibiting hepatic HMG-CoA reductase, which reduces the synthesis of endogenous cholesterol. This triggers a negative feedback loop, causing the liver to increase the absorption and metabolism of blood cholesterol into bile acids for excretion, resulting in a lipid-lowering effect [27]. To determine whether LCZ enhances the effectiveness of lovastatin by facilitating its absorption, we measured bile acid concentrations in both the fecal and serum samples of the golden hamsters. During the intervention period, the fecal and serum levels of both total bile acids and specific bile acids, including cholic acid (CA), chenodeoxycholic acid (CDCA), deoxycholic acid (DCA), and lithocholic acid (LCA), displayed variable increases in both the L + P and L groups (Fig. S8-S9). Importantly, concurrent administration of LCZ and low-dose lovastatin (i.e., the L + P group) resulted in significantly higher elevations of the fecal and serum levels of these bile acids at the end of treatment compared to the L group (Fig. 3E-3L).
Then, we performed transcriptomics analysis on liver tissue samples taken from the golden hamsters. We used PCA to visualize the structural differences in liver transcriptomes of different groups and observed interesting group-based clustering patterns (Fig. 3M). The symbols representing the Ct and L + P groups clustered distinctly around the lower left and upper right corners of the PCA score plot, respectively. The symbols representing the L, P, and N groups clustered together in the center of the PCA score plot. These results suggested that the hepatic transcriptome structure of the Ct and L + P groups differ from those of the L, P, and N groups, which only displayed minor transcriptional divergence. The dissimilarity in the hepatic transcriptomic structure of the Ct group from the other groups probably stems from the dietary difference (between a normal diet and HFD). On the other hand, the difference in the hepatic transcriptomic structure of the L + P group from the remaining intervention groups could reflect a notable modification in hepatic gene expression as a result of the combination therapy.
A more detailed gene-level analysis identified four, nine, and 861 significant differentially expressed genes in the P, L, and L + P groups, respectively, compared to the N group (Fig. 3N; Fig. S10), which is consistent with the results of the PCA. Further gene set enrichment analysis of the 861 differentially expressed genes between the L + P and N groups revealed 70 genes of interest, which were significantly enriched in five metabolic pathways, including xenobiotic metabolism, fatty acid metabolic process, cellular lipid metabolic process, lipid metabolic process, and bile acid metabolism (adjusted p < 0.05; Fig. S11). The majority of these genes (61/70) were upregulated (Fig. 3O), indicating the activation of related metabolic processes in the liver. Notably, Cyp7a1, Slc27a2, Slc27a5, and Acsl1 were among the upregulated genes; these genes play central roles in lipid and bile acid metabolism (Fig. 3P). Additionally, members of the CYP450 family, including Cyp1a1, Cyp1a2, Cyp2e1, and Cyp3a4 (Fig. 3Q), were also upregulated. They are pivotal for drug metabolism [28]. These findings support the notion that boosting lovastatin absorption not only raises liver bile acid production, but also activates genes involved in both liver bile acid and drug metabolism. This aligns with the mechanism of action of lovastatin, implicating that LCZ co-administration may enhance the efficacy of lovastatin by promoting its absorption.
It is worth noting that the effect of LCZ co-administration appears to be two-sided. At low doses, LCZ enhanced the absorption of lovastatin, thereby augmenting its therapeutic efficacy. At higher dosages, LCZ not only enhanced lovastatin absorption but also increased drug toxicity (Fig. 2B, 2C). To test this hypothesis, we compared the serum levels of lovastatin and lovastatin hydroxy acid in golden hamsters receiving medium and high doses of the drug (10 mg/kg for M and M + P groups; 20 mg/kg for H and H + P groups) with those receiving a low drug dose (2.5 mg/kg for L and L + P groups). The results surprisingly revealed over 10-fold higher serum concentrations of lovastatin and lovastatin hydroxy acid in the higher dose groups compared to the low-dose one (Fig. S12). These notable elevations suggest that the liver of the golden hamsters may have been incapable of handling the medium and high doses of lovastatin, resulting in a considerable buildup of lovastatin and lovastatin hydroxy acid in their systemic circulatory system, ultimately leading to their death. Furthermore, co-administering LCZ with higher doses of lovastatin (M + P and H + P groups) resulted in significantly higher serum levels of lovastatin and lovastatin hydroxy acid compared to the groups that only received the drug without LCZ (p < 0.001). These observations further support the hypothesis that LCZ enhances lovastatin absorption. However, the toxicity at high doses was intolerable.
The potential mechanism of LCZ in promoting lovastatin absorption
To elucidate how LCZ enhances lovastatin absorption, we analyzed the fecal metagenomes and metabolomes of the golden hamsters across multiple time points. At week 0 (baseline), a total of 429 bacterial species (including 11 major species with a relative abundance > 1%) were identified across all hamster groups (Fig. S13); no significant intergroup differences were detected in any of these species (Fig. S14A). Considering the impact of age and diet on gut microbiota, we first examined and ruled out age- and diet-related natural variations in the gut microbiota, which were likely unrelated to the intervention of LCZ and/or lovastatin. This was accomplished by evaluating differences in the fecal microbiota of two groups: Ct and N groups. In the Ct group, significant changes were noted in the relative abundance of 101 species (FDR < 0.05; Fig. S14B) through the experiment, which were associated with the process of aging. These species were excluded from further analysis to eliminate the effects of confounding variables. 87 species in the N group showed significant changes in abundance related to diet and were also excluded (Fig. S14C). After excluding species that varied with age and diet, 241 bacterial species remained for further analysis.
Subsequently, we identified specific changes in the fecal microbiota related to the interventions (LCZ or lovastatin) based on two criteria: 1) species that displayed a significant change following LCZ and/or lovastatin treatment, and 2) species that consistently maintained the same change pattern at both 12 and 16 weeks. A total of 23 species in the L + P group, 16 species in the L group, and four species in the P group met both criteria (Fig. 4A, S15). These species are regarded as intervention-responsive species. A notable finding from the comparative abundance analysis was that, at the end of the treatment, nine bacterial species in the L + P group showed a significant increase in relative abundance compared to the L group (Fig. S16). This result highlights the unique impact of combination therapy on the gut microbiota composition.
To eliminate the potential confounding effects of age and diet-related natural variations, we implemented a similar exclusion process in the fecal metabolomics dataset. Initially, a total of 2,428 metabolites were identified across all groups. To accurately evaluate the effects of LCZ on gut metabolism, we excluded 584 metabolites that showed significant intergroup differences at baseline (week 0; Fig. S17A). Additionally, throughout the experiment, 264 metabolites in the Ct group and 671 metabolites in the N group exhibited significant changes, which could be attributed to the effects of aging and a high-fat diet (Fig. S17B, S17C). These metabolites were also excluded from subsequent analyses. Following that, we identified 27, 29, and 10 metabolites that exhibited intervention-associated differential abundance in the L + P, L, and P groups, respectively (Fig. 4B, S18). Comparative abundance analysis revealed that 18 metabolites in the L + P group showed a significant increase in relative signal intensity compared to the L group (which received a low-dose drug without LCZ) by the end of the treatment (Fig. S19). This finding provides further evidence of the distinct impact of combination therapy on gut metabolism.
To identify the potential interactions between gut microbiota and metabolic changes, we performed Spearman’s correlation analysis on the nine bacteria exhibiting increased abundance and the 18 metabolites with elevated signal intensity in the L + P group. The analysis revealed significant positive correlations (adjusted p < 0.05, r > 0.6) among 15 metabolites and all nine bacterial species (Fig. 4C), suggesting close potential biosynthetic interconnection among the identified differential abundant features.
We then explored the potential interactions between gut metabolites and lovastatin. To assess their compatibility, we followed the solubility principle, which states that polar compounds dissolve polar solvents (“like dissolves like”) and vice versa. To compare the polarity of the 15 gut microbial interactive metabolites with that of lovastatin, we computed their octanol-water partition coefficient (LogP). Notably, 12 of the 15 metabolites exhibited LogP values ranging from 3.26 to 5.26 (Fig. 4D), which is close to the LogP value of lovastatin (i.e., 4.26). This suggests a similar solubility profile between these metabolites and lovastatin, indicating that they may have a compatible solubility that allows their dissolution and potentially enhances the intestinal absorption of lovastatin. Conversely, the LogP of only two of the 21 significantly increased metabolites in the L group (compared to the L + P group) fell within the range of 3.26 to 5.26, and the remaining ones had very low or even negative LogP values (Fig. 4E), indicating a discordant solubility profile between these metabolites and lovastatin. Therefore, it is highly probable that the significantly increased metabolites in the intestine of golden hamsters in the L + P group contribute to the solubilization of lovastatin, facilitating drug absorption through improved compatibility.