A combination of trimethoprim and sulfamethoxazole (cotrimoxazole) is a widely used antibiotic for treating and prophylaxis infectious diseases, such as bone and joint infections, tuberculosis, and urinary tract infections [1-3]. It is considered generally safe for pediatric, adult, and pregnant patients [4,5], but there is a significant occurrence of resistance to cotrimoxazole in numerous countries [6], including Indonesia, where the uropathogenic resistance to cotrimoxazole was reported to reach 67 % [7].

Various measures are necessary to address antibiotic resistance, including co-administration with lactoferrin [8]. Lactoferrin, a peptide linked to iron ions, aids the body in defending against naturally occurring bacteria and viruses found in milk [9]. It is currently accessible as a dietary supplement in the commercial market, offering beneficial antibacterial outcomes [10,11]. Lactoferrin supplements are seldom sold as individual doses; instead, they are commonly crafted in combination with other ingredients such as colostrum, Echinacea angustifolia, vitamin C, and zinc [12]. Following the onset of the Covid-19 pandemic, there has been a rise in the utilization of supplements that incorporate lactoferrin [13], which has resulted in a growing probability of cotrimoxazole being taken together with lactoferrin-containing supplements.

At present, the interplay between cotrimoxazole and lactoferrin has not been adequately explored. What has been known is that lactoferrin is recognized for its ability to reduce the manifestation of P-glycoprotein (P-gp), a type of efflux transporter [14], while trimethoprim—one component of cotrimoxazole—can function as a substrate for P-gp, indicating that a reduction in P-gp expression can potentially enhance the absorption of trimethoprim within the gastrointestinal tract [15]. Nevertheless, there is no prior documentation of the potential interaction between lactoferrin and sulfamethoxazole, the other component of cotrimoxazole.

To address this gap, this study aimed to explore how supplements containing lactoferrin affect the pharmacokinetics of cotrimoxazole by investigating whether there is a discernible impact associated with the use of lactoferrin-containing supplements on the pharmacokinetic aspects of cotrimoxazole urinary excretion. Specifically, this study focused on assessing the elimination rate and the proportion of unchanged drug excreted in urine, which were both crucial in estimating the cotrimoxazole concentration in the urine for combating uropathogens. As this was a preliminary investigation, assessments were conducted using non-invasive urine samples.

The laboratory tests showed that the participants in the study were in good health, with the five individuals exhibiting comparable age ranges, weights, and heights (Table 1). In this study, a cross-over experimental design, rather than a parallel design, was used to control the effect of biological variation on drug pharmacokinetics more effectively.

Several pharmacokinetic models with and without covariates in the form of supplementation were compared. However, based on the goodness-of-fit parameters (i.e., BIC and shrinkage), the best model was the one without covariates based on the goodness-of-fit parameters (Table 2), which indicates that short-term administration of a supplement product containing lactoferrin does not affect the pharmacokinetic parameters of either trimethoprim or sulfamethoxazole. The final pharmacokinetic parameters are presented in Table 3.

The acceptable goodness-of-fit of the final models of trimethoprim and sulfamethoxazole was also assessed. The visual predictive check revealed that the final population of the pharmacokinetic model for trimethoprim and sulfamethoxazole demonstrated effective predictive performance, with the simulation-based prediction interval and the 95 % confidence interval appropriately encompassing the observed median, reaching the 2.5^th and 97.5^th percentiles of the data (Figure 2A-B). In the comparison between predicted and observed drug amounts in urine, a symmetrical distribution of data points was evident around the regression line (Figure 3A-B and 4A-B). The graph depicting conditional weighted residuals (CWRES) against predictions and time exhibited a random dispersion of data points around CWRES = 0, with the majority of residuals falling within the range from -2 to 2 (Figure 3C-D and Figure 4C-D).

As far as the current literature suggests, this study is the first attempt to analyse the interaction between lactoferrin-containing supplements and the antibiotic cotrimoxazole. It has been discovered that the pharmacokinetics of trimethoprim and sulfamethoxazole can effectively be modelled using a one-compartment model with first-order elimination and proportional residual error, as well as without covariates. On one hand, this aligns with the earlier assumption that the pharmacokinetic parameters of sulfamethoxazole remain unaffected by supplement administration. However, the findings contradict our hypothesis that the absorption of trimethoprim would be influenced by supplement intake.

The anticipated enhancement in trimethoprim absorption in the presence of P-gp inhibiting lactoferrin did not meet the study’s prediction. The lack of change in trimethoprim’s absorption rate may be attributed to the brief supplementation period, which was insufficient for the downregulation process of P-gp [14]. In this study, the supplementation was administered only once, concurrently with the drug, which might be the primary reason the absorption rate of trimethoprim did not change; nevertheless, further investigation is needed.

As identified in this study, the absorption constant values (k_a) for trimethoprim and sulfamethoxazole align with those reported in previous research [20,21]. Additionally, the total elimination constant (k_e) values were validated by indirectly comparing them with clearance (CL) divided by apparent distribution volume (V) within the established range [22]. Our findings indicate that the k_e values for trimethoprim and sulfamethoxazole in this study align closely to or fall within the ranges reported in prior studies [21,22]. Notably, the non-renal elimination rate constant (k_nr), which relatively lacks prior publication in pharmacokinetic studies, exhibited a minimal and potentially clinically insignificant value for trimethoprim but a relatively high value that approaches the k_e value for sulfamethoxazole. This discrepancy stems from the substantially higher urinary metabolism of sulfamethoxazole, which amounts to approximately 70 %, compared to the liver metabolism of trimethoprim, which ranges from 10-30 % [23].

To summarize, the administration of supplements containing lactoferrin demonstrated no noteworthy influence on the pharmacokinetic profiles of cotrimoxazole in a cohort of healthy volunteers. As such, no specific modifications to dosage are deemed essential. Consequently, the concomitant use of lactoferrin supplements and cotrimoxazole can be confidently encouraged without any concern for potential pharmacokinetic interactions.

Based on the pharmacokinetic modeling results using urine excretion data, no significant impact of administering supplements containing lactoferrin on the pharmacokinetic parameters of cotrimoxazole (trimethoprim and sulfamethoxazole) was observed in the single-dose administration.