A Natural Alternative Treatment for Urinary Tract Infections: Itxasol©, the Importance of the Formulation

Genito-urinary tract infections have a high incidence in the general population, being more prevalent among women than men. These diseases are usually treated with antibiotics, but very frequently, they are recurrent and lead to the creation of resistance and are associated with increased morbidity and mortality. For this reason, it is necessary to develop new compounds for their treatment. In this work, our objective is to review the characteristics of the compounds of a new formulation called Itxasol© that is prescribed as an adjuvant for the treatment of UTIs and composed of β-arbutin, umbelliferon and n-acetyl cysteine. This formulation, based on biomimetic principles, makes Itxasol© a broad-spectrum antibiotic with bactericidal, bacteriostatic and antifungal properties that is capable of destroying the biofilm and stopping its formation. It also acts as an anti-inflammatory agent, without the adverse effects associated with the recurrent use of antibiotics that leads to renal nephrotoxicity and other side effects. All these characteristics make Itxasol© an ideal candidate for the treatment of UTIs since it behaves like an antibiotic and with better characteristics than other adjuvants, such as D-mannose and cranberry extracts.


Introduction
Urinary tract infections (UTIs) affect any part of the urinary tract and may spread through the urinary tract towards the urethra, bladder and even the kidneys [1]. It has been estimated that around 150 million people suffer from UTIs annually [2], and they are associated with an increase in morbidity and mortality [3]. UTIs are more prevalent in women than in men, which is related to the short length of the urethra favoring bacteria colonization [4][5][6], and it has been estimated that 50-70% of women will suffer at least one urinary infection in their life [6]. Of women suffering from UTIs, 20-30% might suffer from a recurrent UTI, which is defined as recurrences of uncomplicated and/or complicated UTIs, with a frequency of at least three UTIs/year or two UTIs in the last six months [7]. UTIs in women are often associated with sexual intercourse [8], poor social conditions that limit access to female hygiene products for menstruation [9] and use of contraceptive devices such as diaphragms [10,11]. In the case of postmenopausal women, recurrent UTIs are related to the low levels of estrogens that produce changes in the vaginal microbiota [12]. In the case of men, UTIs are often related to prostatitis, prostate benign hyperplasia or any kind of urinary obstructive tract pathology [13,14]. In addition, UTIs are related to smoking, which is also associated with the development of bladder cancer, and it has been reported to increase the risk of suffering from UTIs by up to 50% [15].
The main bacteria isolated from cases of UTIs (80%) is E. coli, although other pathogens have been cultured, such as Klebsiella pneumoniae and Pseudomonas aeruginosa [16]. These infections represent a major number of hospital-and community-acquired infections, acute pyelonephritis being one of the major causes of hospitalization [6,17].
Our main aim is to thoroughly review the knowledge regarding the three components of Itxasol©, a new drug compound, in relation to its efficacy to treat UTIs. Here, we analyze the mechanism of actions of the components of Itxasol©, namely, β−arbutin, UMB and NAC, and their main use related to the urinary tract.

Material and Methods
We searched PubMed, Web of Science (Clarative Analytics), and the Spanish databases Medes (https://medes.com/Public/Home.aspx, accessed on 1 June 2021) and IBECS (https://ibecs.isciii.es, accessed on 1 June 2021) for the following terms: umbelliferon/7hydroxycoumarin and urinary tract/biofilm, arbutin and urinary tract/biofilm and N-acetylcysteine and urinary tract/biofilm (for the Spanish databases, the terms were written in Spanish). Table 1 summarizes the findings of this strategy search. Although single terms were found in Spanish databases, we did not find combinations of terms. After the original search, all abstracts were read, and those that were not related to the original search were discarded.

β-arbutin
Arbutin is a glycoside derived from extracts of leaves of Arctostaphylos uva-ursi, plants from genus Bergenia or other plants that belong to genus Ainsliaea and Calluna. These plants have been traditionally used for the treatment of urinary tract infections in Europe, America and Asiatic countries [52]. After its administration, arbutin is transformed into hydroquinone and glucose ( Figure 1). The mechanism of action of hydroquinone (HQ) is related to the destruction of the bacteria wall, which leads to leakage of intracellular content and bacteria death [53]. One of the major advantages of β-arbutin treatment is that the amount of derived HQ that might reach the urinary tract after HQ excretion in urine is around 65% of the β-arbutin administrated [54]. The antibacterial activity of β-arbutin has been tested in several studies that demon  The antibacterial activity of β-arbutin has been tested in several studies that demonstrated that it can destroy both Gram-negative and positive bacteria as well as fungi. In this list, we can find Pseudomonas aeruginosa, Staphylococcus aureus, S. aureus MRSA K31 (clinical, antibiotic-resistant strain), Enterococcus faecalis, Escherichia coli, Escherichia coli ESBL R194 (clinical, antibiotic-resistant strain), Enterococcus faecalis HLAR (clinical, antibiotic-resistant strain), Bacillus subtilis and Candida albicans [53,55,56]. In Table 2, we show the minimum inhibitory concentration of HQ against different pathogens. In fact, in vivo, it has been reported in a randomized controlled trial that β-arbutin in combination with other plant extracts, such as berberine and birch, significantly reduced the incidence of recurrent cystitis [61]. The use of β-arbutin as a substitute for antibiotic treatment for UTIs is under study in a double-blind, randomized and controlled clinical trial on women aged 15 to 75 years old, but the results are yet to be published [62].
One concern regarding the safety of arbutin is that hydroquinone might be nephrotoxic [63,64]. Extract of strawberry tree Arbutus unedo, which contains arbutin, has been proven to be safe for the kidney in rats [65]. These extracts did not show any side effects in kidney function or affect the integrity of the DNA in the kidney cells. It has also been demonstrated that the administration of arbutin to humans was not toxic for lymphocytes, and it did not induce damage to the DNA of these cells [66].
Inflammation is an important consequence of UTIs. Regarding this effect, it has been demonstrated that β-arbutin reduces the kidney inflammation produced by the administration of lipopolysaccharide to rats. It was concluded that the anti-inflammatory effects of β-arbutin were mediated by inhibition of the Akt signaling pathway [67]. These results were dependent on the activity of arbutin, because when a specific arbutin activity inhibitor was administered, the beneficial effects of arbutin were abolished. Of note, arbutin, in a polyherbal mixture administered to diabetic rats, resulted in a decrease in blood sugar and level of cholesterol as well as cardiovascular risk, while it restored several histopathological changes produced by diabetes in the liver, pancreas and kidney [68]. The main actions of β-arbutin are summarized in Table 3. Table 3. Main actions of β-arbutin.

Action Main Findings/Use Reference
Antibacterial action Inhibits Pseudomonas aeruginosa growth at 128 mg/mL [56] Demonstration of antibacterial activity [55] Arbutin destroys bacteria through wall cellular disruption (Gram + and Gram −) [53] Reduction in bacterial load in prevention of UTI recurrence [61] Clinical trial to reduce the use of antibiotics administering b-arbutin (results not yet published) [62] Anti-inflammatory Attenuated damage induced by lipopolysaccharide in rat [67] Anti-diabetic Ameliorates hyperglycemia, hyperlipidemia and histopathological changes in pancreas, kidney and liver in a diabetes rat model [68]

lipopolysaccharide in rat
Anti-diabetic Ameliorates hyperglycemia, hyperlipidemia and histopathological changes in pancreas, kidney and liver in a diabetes rat model [68]

Umbelliferon (UMB)
UMB ( Figure 2) is a coumarin (7-hydroxycoumarin) that can be extracted from fruits [2] and plants and has demonstrated multiple activities, such as antitumoral, antioxidant (with the ability to quench free radicals), antihyperglycemic, anti-arthritic and anti-inflammatory activities, as well as hepatic and cerebral protective functions [17,[69][70][71][72][73][74][75].  UMB activity as an antibiotic was described as earlier as 1978. However, it has not been used due to a lack of knowledge about its mechanism of action and its actions in the kidney [46]. Although knowledge of the antibiotic action of UMB is scarce, it has recently been demonstrated that derivates of UMB from plants of genus Ferula are useful against periodontal bacteria, inhibiting bacteria growth and biofilm formation [76]. In agreement with these data, it has been described that UMB can inhibit the formation of the biofilm created by Staphylococcus epidermidis, impairing intracellular adhesion by downregulation in the expressions of genes related to adherence function [77]. The MIC of UMB against different pathogens is shown in Table 4. Coumarins are useful against fungus such as C. albicans [80,81]. Thus, this efficacy might lie in the fact that UMB can produce internal changes, such as an increase in ROS, DNA fragmentation and externalization of phosphatidyl serine, all of them being related to an increase in apoptosis [82]. The use of UMB to stop biofilm production in a model of C. elegans showed downregulation of the expression of genes related to the formation of filaments and adhesion as well as reducing the formation of biofilm in this nematode in general [83]. Besides fungi, it has also been reported that coumarins reduce the virulence and biofilm formation of E. coli O157:H7. Coumarins downregulate the expression of curli and motility genes, which results in fewer fimbriae and the production of biofilm [84]. In the same sense, UMB prevents the formation of biofilm by the methicillin-resistant Staphylococcus epidermidis strain at a dose of 500 mg/mL but without affecting the growth of this bacteria [77].
UMB has also been reported as a good nephron protector when it is used in combination with cisplatin, which is one of the chemotherapeutic agents used against different cancers but with limited use given its nephrotoxicity [85]. In a mouse model of cisplatin-induced acute kidney injury (AKI), it was demonstrated that UMB reduced the nephrotoxicity associated with cisplatin use. These effects were achieved by increased cell tubular proliferation upon enhanced expression of sox9 transcription factor. Another kidney-protective effect of UMB was the reduction of necrosis in kidney cells produced by the inhibition of the RIPK1/RIPK3/MLKL pathway. In a model of methotrexate-induced kidney injury, the administration of UMB reduced the expression of inflammation mediators P 38 MAPK and NF-κB and the pro-apoptotic molecules BAS and caspase3 while increasing anti-apoptotic levels of the BLC-2 molecule [86].
On the other hand, UMB has proven useful in treating the nephropathy produced in diabetic rat models induced with streptozotocin. In this model, UMB reduced the creatinine level in plasma, renal oxidative stress levels and levels of tissue and circulating TGF-β1 [1]. In another similar rat model study, in addition to the reduction of glucose blood levels, the beneficial effects of UMB were related to a decrease in inflammatory mediators, such as TNF-α, IL-6 and IL-1β; a reduction in the levels of mesenchymal-epithelial markers, such as podocin and CD2AP; and the reversal of some of the histopathological changes mediated by a diabetic status in the kidney [87]. In fact, this inhibition of the TGF-β1 pathway has also been reported in carbon tetrachloride-induced liver fibrosis in rats. After treatment with UMB, liver cells reduced the activation of the Smad/TGF-β1 pathway; downregulated the expression of NF-κB, collagen I and III and α-Smad; increased the levels of glutathione; and upregulated the expression of PPARγ. The histology result was a decrease in liver fibrosis [88]. The principal actions of UMB are summarized in Table 5.

N-Acetyl L-Cysteine (NAC)
The NAC molecule ( Figure 3) is well known for its antioxidant, anti-inflammatory and mucolytic actions [90,91]. Its mechanism of action is related to its ability to reduce the levels of reactive oxygen species (ROS), such as • OH, NO 2 and CO 3 − , given that it is a precursor of glutathione, a renowned natural ROS scavenger [92]. Moreover, there is compelling evidence regarding NAC as a molecule that destroys biofilms and also reduces their formation via bacteria and fungus [93,94]. Although the mechanism by which NAC reduces biofilm formation is still not clear, a recent report by Li and co-workers demonstrated that NAC can penetrate the bacteria wall, stopping protein synthesis and leading to bacteria death [95]. Table 6 summarizes the recent reports demonstrating the ability of NAC to both inhibit biofilm formation and destroy already formed ones. Besides being useful against UTIs, several pieces of evidence point out NAC as a kidney protector [96]. Moreover, a recent study demonstrated that the administration of NAC protected Wistar rats with a kidney injury against long warm renal ischemia. In particular, NAC administration resulted in an improvement in biochemical parameters and renal function when compared to the placebo group [97].

Discussion
UTIs have a high economic and social burden and affect large segments of the population, such as children, pregnant women, healthy pre-and post-menopausal women and patients undergoing catheterization and diabetics. In addition, there is a very high risk of relapse in UTIs, and it is common for these episodes to be caused by the same bacteria or something different [6]. Unfortunately, many of the current treatments fail to resolve UTIs and therefore lead to complications and relapses, and in many cases, patients also experience the adverse effects of medications [110,111].

Discussion
UTIs have a high economic and social burden and affect large segments of the population, such as children, pregnant women, healthy pre-and post-menopausal women and patients undergoing catheterization and diabetics. In addition, there is a very high risk of relapse in UTIs, and it is common for these episodes to be caused by the same bacteria or something different [6]. Unfortunately, many of the current treatments fail to resolve UTIs and therefore lead to complications and relapses, and in many cases, patients also experience the adverse effects of medications [110,111].
Taken together, a new compound that combines antimicrobial and anti-inflammatory action and that does not have side effects is required to manage these diseases. Thus, in this study, we reviewed the main uses of arbutin, UMB and NAC, and the major actions of these three molecules are summarized in Figure 4.
There are new natural alternatives that, through an adequate formulation of different compounds, could contribute to solving this unmet need that constitutes the management of urinary tract infections. Itxasol© is a formulation that produces a synergistic effect thanks to its three components that act against urinary tract infections and improve diuresis. Itxasol© combines antibiotic properties of a natural bactericide and bacteriostat of the broad spectrum with antibiofilm (both prevention and elimination), nephroprotective, anti-inflammatory and antioxidant properties.
Taken together, a new compound that combines antimicrobial and anti-inflammatory action and that does not have side effects is required to manage these diseases. Thus, in this study, we reviewed the main uses of arbutin, UMB and NAC, and the major actions of these three molecules are summarized in Figure 4. There are new natural alternatives that, through an adequate formulation of different compounds, could contribute to solving this unmet need that constitutes the management of urinary tract infections. Itxasol© is a formulation that produces a synergistic effect thanks to its three components that act against urinary tract infections and improve diuresis. Itxasol© combines antibiotic properties of a natural bactericide and bacteriostat of the broad spectrum with antibiofilm (both prevention and elimination), nephroprotective, anti-inflammatory and antioxidant properties.
There are other coadjutants that have been used against UTIs, such as cranberry extracts and D-mannose or combinations of them or with extracts from other plants [111]. D-Mannose has been suggested as a coadjutant in the treatment of UTIs. However, this monosaccharide that acts by inhibiting the adherence of bacteria (E.coli) to urothelium [112] lacks antibacterial activity [113] and causes a considerable number of cases of diarrhea (around 8%) as a side effect [114]; moreover, its efficacy in regard to preventing or reducing UTI incidence remains clear [115]. UMB's high bioavailability in the urinary tract is the characteristic that differentiates it from other coadjutants, such as curcumin (anthocyanosises), which can be found in red cranberry anthocyanosises. This feature makes it a suitable candidate for treating UTIs [116]. Of note, neither D-mannose nor cranberry extracts have an established dosage, and both are limited to the action of preventing the adherence of bacteria to the urothelium.
One of the limits of our review is that the combined effect of the molecules that form Itxasol© is not completely described. However, the antimicrobial activity of the three molecules has been tested in urinary catheters colonized with Enterococcus faecalis. In this study, it was demonstrated that these three molecules were able to reduce the formation of biofilms. In fact, after 72 h of treatment, the catheters treated with UMB (300 mg), arbutin (60 mg) and N-acetylcysteine (150 mg) showed a significant reduction in loaded biofilms and loaded bacteria [117]. It is important to highlight that the concentrations of the different compounds in the formulation are important since, in this study, UMB seems to work better at high doses (300 mg) accompanied by low doses of NAC (150 mg). There are other coadjutants that have been used against UTIs, such as cranberry extracts and D-mannose or combinations of them or with extracts from other plants [111]. D-Mannose has been suggested as a coadjutant in the treatment of UTIs. However, this monosaccharide that acts by inhibiting the adherence of bacteria (E. coli) to urothelium [112] lacks antibacterial activity [113] and causes a considerable number of cases of diarrhea (around 8%) as a side effect [114]; moreover, its efficacy in regard to preventing or reducing UTI incidence remains clear [115]. UMB's high bioavailability in the urinary tract is the characteristic that differentiates it from other coadjutants, such as curcumin (anthocyanosises), which can be found in red cranberry anthocyanosises. This feature makes it a suitable candidate for treating UTIs [116]. Of note, neither D-mannose nor cranberry extracts have an established dosage, and both are limited to the action of preventing the adherence of bacteria to the urothelium.
One of the limits of our review is that the combined effect of the molecules that form Itxasol© is not completely described. However, the antimicrobial activity of the three molecules has been tested in urinary catheters colonized with Enterococcus faecalis. In this study, it was demonstrated that these three molecules were able to reduce the formation of biofilms. In fact, after 72 h of treatment, the catheters treated with UMB (300 mg), arbutin (60 mg) and N-acetylcysteine (150 mg) showed a significant reduction in loaded biofilms and loaded bacteria [117]. It is important to highlight that the concentrations of the different compounds in the formulation are important since, in this study, UMB seems to work better at high doses (300 mg) accompanied by low doses of NAC (150 mg).

Conclusions
Itxasol© represents a promising tool in the treatment and prevention of ITUs, and it acts mainly as a broad-spectrum antibiotic protecting the kidney. It may be used for an extended time without generating bacterial resistance, which makes it an excellent alternative treatment to actual antibiotics and other coadjutants. The design of new controlled and randomized trials is necessary to confirm the potential of Itxasol© in comparison with current treatments.

List of Abbreviations
UTIs urinary tract infections UMB umbelliferon NAC N-acetyl L-cysteine TMP-SMX trimethoprim/sulfamethoxazole MIC minimum inhibitory concentration