TECHNICAL FIELD

The present invention relates to lactulose for use in treatment of Clostridioides difficile infection (CDI) and/or prevention of recurrence of CDI or a condition associated with CDI. Furthermore, the invention relates to a therapeutic combination comprising lactulose and a therapeutically effective amount of one or more antibiotic for use in treatment of Clostridioides difficile infection (CDI) and/or prevention of recurrence of CDI or a condition associated with CDI.

BACKGROUND OF INVENTION

Over the last two decades, Clostridioides difficile infection (CDI) has become one of the most common nosocomial infections, accounting for substantial morbidity and mortality as well as a significant economic burden to the healthcare system and society alike.

Clostridioides difficile (C. difficile) is a Gram-positive, spore forming, anaerobic bacillus which is widely distributed in the intestinal tract of humans and animals, and in the environment. C. difficile spores are the causative agent of CDI. The disease often results from the oral intake of acid-resistant C. difficile spores in patients with a dysbiosed intestinal microbial community.

The currently most widely used therapy of CDI is antibiotic therapy using e.g. vancomycin, metronidazole, fidaxomicin, rifaximin, or combinations thereof. The antibiotic therapy not only results in a drop of vegetative cells of C. difficile below detection limit, but also simultaneously results in collateral damage to the intestinal microbiota, by killing indigenous microbiota. Disruption of the intestinal microbiota creates a proper environment for C. difficile colonization, proliferation, and clinical disease onset. After cessation of the therapy, CDI often reoccurs. Some patients even experience continuous reoccurrences of CDI. Each subsequent relapse of CDI tends to be more severe than previous infections. High rate of CDI recurrence is a major bottleneck in the treatment of CDI with the antibiotic therapy. Some therapeutic options have been described as promising tools for prevention of CDI recurrence, like bezlotoxumab, a human monoclonal antibody blocking C. difficile toxin B, or fecal microbiota transplantation (FMT) to restore intestinal ecosystem resilience to CDI. Despite effectiveness, the routine use of these therapies is hindered by high cost or lack of approved procedures and regulatory standards.

Therefore, more cost-effective and efficacious treatment in CDI is needed. In addition, a treatment for preventing recurrence of CDI is needed.

C. difficile is ubiquitous in the environment. Food animals, horses, and household pets are frequently found positive for toxigenic C. difficile. Humans and animals share common C. difficile ribotypes suggesting potential zoonotic transmission. They can carry and shed C. difficile to the environment, and infect individuals or populations, via direct contact or via indirect transmission through raw food, or through contaminated water. This is a major public health issue. Therefore, treatment and prevention strategies against CDI in animals are necessary to control the transmission of C. difficile to humans.

The present invention solves this problem by co-administration of a therapeutically effective amount of one or more antibiotic and lactulose and prolonged administration of lactulose after the co-administation.

DRAWINGS

Figure 1. Schematic representation of the experimental design.

Figure 2. Clostridioides difficile total viable counts (TVC) and spore counts during the control (C), CDI induction (CLI), CDI stabilization (CDI), intervention (VNC), and post-intervention (PI) periods in the arm 1 (figure 2(a)), the arm 2 (figure 2(b)), and the arm 5 (figure 2(c)) of the PathoGut™QuadSHIME® setups. Numbers 1-4 and I to III in the labels on the x-axis refer to the weeks of the respective given periods and A-C the sampling points in the respective week.

Figure 3. Average acetate production during the control, CDI induction, CDI stabilization, intervention, and post-intervention periods in the distal colon (DC) reactors of the arm 1 (NCT), the arm 2 (AB NCT), the arm 5 (LAC 5 VO), the arm 6 (LAC 10 VO), the arm 7 (LAC 5 V-l), and the arm 8 (LAC 10 V-l) of the PathoGut™QuadSHIME® setups. * indicates statistically significant differences relative to the control (p < 0.05).

DETAILED DESCRIPTION

In a first aspect, the present invention provides lactulose for use in treatment of Clostrdioides difficile infection (CDI) and/or prevention of recurrence of CDI or a condition associated with CDI in a subject in need thereof, wherein the lactulose is administered with a therapeutically effective amount of one or more antibiotic, wherein lactulose is administered at a dose of 2-20 g per day, wherein the administration duration of lactulose is at least partially overlapping with that of the antibiotic and is at least 1 week longer than that of the antibiotic.

The term "subject" as used herein refers to a human and/or an animal, wherein the animal is particularly a sheep, a goat, a pig, cattle, a poultry, a cat, a dog, a horse, a rabbit, a rat, or a hamster.

The antibiotic herein refers to therapeutically effective antibiotics against CDI, or a condition associated with CDI. Currently, the most widely used antibiotics against CDI are vancomycin, metronidazole, fidaxomicin, rifaximin, and combinations thereof. Treatment of CDI with antibiotics is difficult, due both to antibiotic resistance and disturbance of the normal faecal microbiota. It has been found in the present invention that co-administration of a therapeutically effective amount of one or more antibiotic and lactulose with the dose 2-20 g per day and prolonged administration of lactulose surprisingly show a therapeutic effect on CDI and/or recurrence of CDI or a condition associated with CDI. Surprisingly, CDI does not reoccur even four weeks after the co-administration.

The antibiotic may be administered by any know method, e.g., intravenously, rectally, enterally, or combinations thereof, preferably enterally, more preferably orally. For severe, complicated CDI, concomitant therapy with intravenous metronidazole and oral vancomycin is often prescribed. Sometimes vancomycin per rectum is added to increase colonic drug delivery.

Lactulose belongs to non-absorbable/non-digestible oligosaccharides (NDOs) with general formula C12H22O11. NDOs further comprise, e.g., inulin, fructooligosaccharides (FOS), galactooligosaccharides (GOS), xylooligosaccharides (XOS), isomaltooligosaccharides (IMOS), glucooligosaccharides, pectin oligosaccharides (POS), mannanooligosacharides (MOS), gentiooligosaccharides (GTO), chitooligosaccharides (CHOS), soybean oligosaccharides (SOS) and polydextrose. Other NDOs may also be coadministered with a therapeutically effective amount of one or more antibiotic in treatment of CDI and/or prevention of recurrence of CDI or a condition associated with CDI, wherein a proper dose and prolonged administration of the corresponding NDOs may be applied. Optionally, combinations of the NDOs may be co-administered with the antibiotic.

The term "dose" as used herein refers to a daily quantity that is administered to a subject. The daily dose may be administered all at once or it may be spread out over several administrations throughout a day. The dose may be administered by any known method, preferably enterally, more preferably orally. The dose of 2-20 g is the optimal dose for a human. The optimal daily dose for animals is 0.03-0.3 g/kg body weight, preferably 0.05-0.25 g/kg body weight, more preferably 0.08-0.16 g/kg body weight.

During the period when the administration of lactulose is overlapped with that of the antibiotic, lactulose and the antibiotic may be administered on the same day simultaneously or separately, optionally sequentially.

In another aspect, the present invention provides lactulose for use in treatment of CDI and/or prevention of recurrence of CDI or a condition associated with CDI in a subject in need thereof, wherein the antibiotic is selected from the group consisting of vancomycin, metronidazole, fidaxomicin, rifaximin, and combinations thereof, preferably vancomycin, metronidazole, fidaxomicin, and combinations thereof, more preferably vancomycin.

In the antibiotic therapy against CDI, vancomycin, metronidazole, fidaxomicin, rifaximin or combinations thereof are currently most widely used. Preferably, vancomycin, metronidazole, fidaxomicin, or combinations thereof, more preferably, vancomycin, is used herein. For severe, complicated CDI, vancomycin and metronidazole can be used together. In another aspect, the present invention provides lactulose for use in treatment of CDI and/or prevention of recurrence of CDI or a condition associated with CDI in a subject in need thereof, wherein lactulose is administered at a dose of 3-15 g per day, preferably 5-10 g per day.

The current invention reveals that the therapeutic effect of lactulose on CDI, recurrence of CDI, and a condition associated with CDI is dose-dependent. Surprisingly, the dose of 3-15 g per day, preferably 5-10 g per day, has been found to be very effective in treatment of CDI and/or prevention of recurrence of CDI or a condition associated with CDI.

In another aspect, the present invention provides lactulose for use in treatment of CDI and/or prevention of recurrence of CDI or a condition associated with CDI in a subject in need thereof, wherein the administration duration of lactulose is 1 to 5 weeks, preferably 3 to 4 weeks, longer than that of the antibiotic.

Compared with administration of lactulose with the same duration as that of the antibiotic, the prolonged administration of lactulose has been revealed in the current invention to be effective for the therapeutic effect on CDI, recurrence of CDI and a condition associated with CDI.

In another aspect, the present invention provides lactulose for use in treatment of CDI and/or prevention of recurrence of CDI or a condition associated with CDI in a subject in need thereof, wherein the administration of lactulose begins on the same day or up to 1 week before that of the antibiotic.

The administration of lactulose begins either before or on the same day as that of the antibiotic. Both options have shown therapeutic effect on CDI, recurrence of CDI and a condition associated with CDI. The maximum therapeutic effect has been shown when the administration of lactulose begins on the same day as that of the antibiotic.

In another aspect, the present invention provides lactulose for use in treatment of CDI and/or prevention of recurrence of CDI or a condition associated with CDI in a subject in need thereof, wherein the condition associated with CDI is Clostridioides //77c/7e-related diarrhea, pseudomembranous colitis, toxic megacolon, perforation of the colon, or sepsis. In another aspect, the present invention provides lactulose for use in treatment of CDI and/or prevention of recurrence of CDI or a condition associated with CDI in a subject in need thereof, wherein lactulose is administered in a powder form or a liquid form, preferably a ready-to-use formulation.

In another aspect, the present invention provides a therapeutic combination comprising lactulose and a therapeutically effective amount of one or more antibiotic for use in treatment of Clostrdioides difficile infection (CDI) and/or prevention of recurrence of CDI or a condition associated with CDI in a subject in need thereof, wherein lactulose is administered at a dose of 2-20 g per day, wherein the administration duration of lactulose is at least partially overlapping with that of the antibiotic and is at least 1 week longer than that of the antibiotic.

The combination described above with the dose and prolonged administration of lactulose has been found in the present invention to be surprisingly effective against colonization of C. difficile and thus against CDI, its recurrence, or a condition associated with CDI.

In another aspect, the present invention provides a therapeutic combination comprising lactulose and the antibiotic for use in treatment of CDI and/or prevention of recurrence of CDI or a condition associated with CDI in a subject in need thereof, wherein the antibiotic is selected from the group consisting of vancomycin, metronidazole, fidaxomicin, rifaximin, and combinations thereof, preferably vancomycin, metronidazole, fidaxomicin, and combinations thereof, more preferably vancomycin.

In another aspect, the present invention provides a therapeutic combination comprising lactulose and the antibiotic for use in treatment of CDI and/or prevention of recurrence of CDI or a condition associated with CDI in a subject in need thereof, wherein lactulose is administered at a dose of 3-15 g per day, preferably 5-10 g per day.

In another aspect, the present invention provides a therapeutic combination comprising lactulose and the antibiotic for use in treatment of CDI and/or prevention of recurrence of CDI or a condition associated with CDI in a subject in need thereof, wherein the administration duration of lactulose is 1 to 5 weeks, preferably 3 to 4 weeks, longer than that of the antibiotic.

In another aspect, the present invention provides a therapeutic combination comprising lactulose and the antibiotic for use in treatment of CDI and/or prevention of recurrence of CDI or a condition associated with CDI in a subject in need thereof, wherein the administration of lactulose begins on the same day or up to 1 week before that of the antibiotic.

I n another aspect, the present invention provides a therapeutic combination comprising lactulose and the antibiotic for use in treatment of CDI and/or prevention of recurrence of CDI or a condition associated with CDI in a subject in need thereof, wherein the condition associated with CDI is Clostrdioides difficile- related diarrhea, pseudomembranous colitis, toxic megacolon, perforation of the colon, or sepsis.

In another aspect, the present invention provides a therapeutic combination comprising lactulose and the antibiotic for use in treatment of CDI and/or prevention of recurrence of CDI or a condition associated with CDI in a subject in need thereof, wherein lactulose is administered in a powder form or a liquid form, preferably in a ready-to-use formulation.

As would be clear to the skilled person, all features and aspects disclosed in combination with lactulose for use in treatment of CDI and/or prevention of recurrence of CDI or a condition associated with CDI, are equally valid in respect of the said therapeutic combination. All features of the current invention disclosed herein may freely be combined. Particularly, features described for different aspects of the current invention may be freely combined. Where known equivalents exist to specific features, such equivalents are incorporated as if specifically referred to in this specification. Further advantages and features of the current invention are apparent from the figures and non-limiting example.

EXAMPLE

In order to investigate the efficacy of lactulose together with vancomyin in curing CDI and preventing recurrence of CDI, a model PathoGut™QuadSHIME® (SHIME: Simulator of the Human Intestinal Microbial Ecosystem) was developed. The model is highly representative of what is observed in vivo in a well-controlled colonic environment limiting the interference of external factors. The model has high stability and high reproducibility. The high stability guarantees that any effects observed truly results from the test products, while the high reproducibility allows direct comparison between the test conditions on virtually identical microbial communities.

For this study, two setups of the model were run in parallel to assess the curative and preventive efficacy of two different doses of lactulose (5g and 10g per day) together with vancomycin in CDI. Each setup consisted of a succession of three segments simulating the different parts of the human gastrointestinal tract. The first segment simulated stomach and small intestine (St+SI), while the second segment simulated proximal colon (PC) and the third segment distal colon (DC). Each segment consisted of a succession of four reactors. Thus, each setup allowed four test conditions and eight test conditions (arms 1 to 8) were conducted in parallel in this study.

The following experimental design was applied (figure 1):

Startup period :

A fecal sample was collected from a healthy volunteer who did not use antibiotics in the six months before the start of the study with a BMI < 25. Then a fecal inoculum is prepared as a 1 :5 mixture of the fecal sample and anaerobic phosphate buffer. After homogenization and removal of big particles via centrifugation, 5% of the fecal inoculum is added to the PC and DC reactors of the arms 1 to 8 and initiated a two-week startup period. This period allowed the microbial community to differentiate in the different reactors depending on the local environmental conditions (i.e., pH, carbon sources availability, retention time).

Control period :

In this period, a standard SHIME nutrient matrix, pancreatic and bile liquid were added to the St+SI reactors of the arms 1 to 8 for two weeks. The nutrient matrix consisted of the following components: 1.2 g/L arabic gum, 2 g/L pectin, 0.5 g/L xylan, 4 g/L starch, 0.4 g/L glucose, 3 g/L yeast extract, 1 g/L peptone, 3 g/L mucin and 0.5 g/L cysteine. 140 ml of the nutrient matrix and 60ml of pancreatic and bile liquid entered in the St+SI reactors every eight hours three times per day. Analysis in this period allowed to determine the baseline microbial community composition and activity in the different reactors, which was used as a control period to compare with the results from the periods with administration of one or more of clindamycin, vancomycin and lactulose.

CDI induction period:

CDI development normally originates from antibiotic treatments and subsequent exposure to C. difficile spores. Clindamycin was used as key for CDI induction in this period. The arms 1 to 8 were operated as follows in this one-week period:

Arm 1 : as a control arm with addition of C. difficile spores;

Arm 2: as an antibiotic control arm with addition of C. difficile spores and clindamycin;

Arm 3: with addition of C. difficile spores, clindamycin and lactulose (low dose 5g);

Arm 4: with addition of C. difficile spores, clindamycin and lactulose (high dose 10g);

Arms 5 to 8: with addition of C. difficile spores and clindamycin.

C. difficile spores were dosed into the PC reactors at a concentration of 10 ⁷ CFU at two time points: once at the beginning and once at the end of the induction period. Clindamycin was dosed into the PC reactors at a final concentration of 33.9 ppm, corresponding to a clinically relevant dosage. Lactulose was dosed into the St+SI reactors as a ready-to-use formulation once a day.

CDI stabilization period :

After cessation of the CDI induction period, the arms 1 to 8 were monitored for three weeks as the CDI stabilization period to check whether a stable CDI development was established. In the arms 3 and 4, the administration with lactulose was continued during the entire CDI stabilization period, i.e., the arms 3 and 4 underwent prolonged administration of lactulose.

Intervention period :

Upon obtaining a stable CDI in the arms 1 to 8, co-administration of vancomycin with lactulose were initiated for their potential to cure CDI and prevent recurrence of CDI.

The arms 1 to 8 were operated as follows in this one-week period:

Arm 1 : as a control arm without addition of vancomycin;

Arm 2: as an antibiotic control arm with addition of vancomycin;

Arm 3 and 4: stopped as CDI reoccurred;

Arm 5: with addition of vancomycin and lactulose (low dose 5g);

Arm 6: with addition of vancomycin and lactulose (high dose 10g);

Arm 7: with addition of vancomycin and lactulose (low dose 5g), wherein the administration of lactulose started one day prior to that of vancomycin and continued with that of vancomycin;

Arm 8: with addition of vancomycin and lactulose (high dose 10g), wherein the administration of lactulose started one day prior to that of vancomycin and continued with that of vancomycin.

Vancomycin was dosed into the PC reactors at a final concentration of 125 ppm, corresponding to a clinically relevant dosage. Lactulose was dosed into the St+SI reactors as a ready-to-use formulation once a day.

Post-intervention period :

Considering that recurrence of CDI is a major bottleneck in treatment of CDI, a post-intervention period was particularly taken into account to assess recurrence of CDI. During this three-week period, the administration with the low and high dose of lactulose was continued in the arms 5 to 8, i.e., the arms 5 to 8 underwent prolonged administration of lactulose. As recurrence of CDI was not observed after three weeks of post-intervention, an additional week of postintervention was implemented with the sole focus of monitoring recurrence of CDI (figure 2(c)).

Considering that the C. difficile spores play an important role in recurrence of CDI, a distinction was made between total viable counts TVC and spore counts, as shown in figure 2. It can be deduced whether the test products are active against vegetative cells only, or whether they also lead to elimination of C. difficile spores.

It was observed that the C. difficile spores were gradually washed out in the control arm 1 during the CDI stabilization period. Neither C. difficile vegetative cells nor spores were detected (figure 2(a)), indicating that a healthy intestinal microbiota was able to prevent colonization of C. difficile and thereby prevent CDI and its recurrence.

The addition of clindamycin during the CDI induction period on the other hand resulted in a dysbiosed intestinal microbial composition, rendering the intestinal microbiota susceptible to pathogenic colonization. CDI did reoccur during the CDI stabilization period in the antibiotic control arm 2 and in the arm 5 (starting from the sampling point CDI III (B) in figures 2(b) and 2(c)). CDI was maintained during at least four consecutive sampling points, indicating a stable CDI development (starting from the sampling point CDI III (B) to VNC (A) in figures 2(b) and 2(c)).

Upon the administration of vancomycin, C. difficile could no longer be detected in the antibiotic control arm 2 during the latter part of the intervention period and the first part of the post-intervention period (starting from the sampling point VNC (C) to PI 2 (A) in figure 2(b)), illustrating susceptibility of C. difficile vegetative cells to vancomycin. Approximately one and a half week after cessation of the administration of vancomycin, CDI reoccurred in the antibiotic control arm 2, which was apparent from the outgrowth of C. difficile vegetative cells (starting from the sampling point PI 2 (C) in figure 2(b)). CDI was maintained until the end of the post-intervention period, indicating a stable recurrence of CDI (figure 2(b)). Upon the co-administration of lactulose (low dose 5g) with vancomycin in the arm 5, CDI did not reoccur, even not after four weeks of post-intervention (figure 2(c)). This indicated that dosing 5g lactulose per day exerted inhibitory effects on the proliferation of C. difficile.

Short-chain fatty acid (SCFA) production results from carbohydrate metabolism by a wide range of intestinal microbiota and is related with various health effects. Acetate is one of the most abundant SCFAs. As an important regulator in the pH of the intestine, acetate helps to keep the environment stable. In this way, it helps to keep the intestine acidic enough for the beneficial microbiota to thrive and survive but deter the opportunistic ones from entering and sticking around. Acetate also helps other species to thrive and survive and supports the diversity of the beneficial intestinal microbiota. For example, the acetate produced by bacteria, such as Bifidobacteria, helps to nourish the butyrate-producing intestinal microbiota. Beneficial effects of the co-administration of lactulose with vancomycin on SCFA production in the intestine therefore include, for example, an increase of acetate production.

As C. difficile specifically colonizes distal colon regions, samples were taken from the DC reactors to analyse the concentration of acetate three times a week.

The administration of vancomycin in the antibiotic control arm 2 resulted in a decrease in the acetate level during the intervention period (figure 3), indicating creation of a dysbiosed microbial community. In comparison, no decrease in the acetate level was observed with the co-administration of vancomycin and lactulose in the arms 5 to 8 during the intervention period. This indicated that lactulose exerted a protective effect on at least part of the intestinal microbial community from being affected by vancomycin.

The prolonged administration of lactulose in the arms 5 to 8 strongly stimulated acetate production in the distal colon, reaching significance (p<0.05) in the postintervention period as in the control period. The reason for this is that acetate is one of the major end products of fermentation of lactulose. The prolonged administration aided thereby supporting the healthy indigenous intestinal microbiota.