Review Bioscience Microflora Vol. 17 (1), 1-6, 1998

Pharmacokinetics of Probiotics and Biotherapeutic Agents in

Humans

Philippe MARTEAU and Tuula VESA

INSERM U290 & Gastroenterology Department Saint-Lazare Hospital, Paris, France

Received for publication, April 9, 1997

Probiotics or biotherapeutic agents can influence physiology through direct or indirect effects occurring in the

gastrointestinal tract. Knowledge on their pharmacokinetics is needed 1) to answer the questions how much probiotic should be consumed?, how often?, how long?; 2) to correlate the effects with the concentration of the probiotic at the target site; 3) to validate hypotheses such as "a probiotic should be of human origin, ... have a high survival capacity, ... adhere to the intestinal epithelium" ...; 4) to anticipate the effects of other probiotics; 5) to establish what concentrations should be present in the commercial preparations; 6) for safety. Some in vitro models have been developed to predict the survival of probiotics in vivo or their adherence to the intestinal epithelium, however, the best way to establish the pharmacokinetics of a probiotic is to measure it in vivo. Three techniques can be used: collection of feces,

pixigraphy, and intestinal intubation. The use of transit markers such as spores of Bacillus stearothermophilus is necessary to study the potential for probiotics to colonize (i.e. to persist for longer period than the inert marker). Knowledge on the pharmacokinetics of probiotics is reviewed. Some strains can adhere to cell lines such as CaCo2 or HT29. The survival of ingested probiotics differs greatly between genus and strains. Some strains are rapidly destroyed in the stomach while others such as some Bifidobacterium sp. or Lactobacillus sp. survive till feces.

Key words: probiotics; pharmacokinetics; lactobacillus; bifidobacterium; intestinal flora

INTRODUCTION

Probiotics or biotherapeutic agents are an original way of delivering active constituents to targets in the

gastrointestinal tract (Fig. 1) (12, 16, 25, 32, 35, 48, 49). Those active constituents include enzymes

(lactase or sucrase for example), immunomodulatory components, vaccinal epitopes (in the future), and com-

ponents possessing antagonistic activities against other microorganism ... (11, 12, 16, 25, 32, 35, 48, 49). Probiotics are a source for these activities, but also a vector which can protect them from acid in the stom-ach, and can deliver them at the target site where they should be active. To progress in the development of

probiotics for therapy or immunomodulation we must increase our knowledge on 1-the active constituents responsible for each effect, 2- the target sites, 3-and the pharmacokinetics of probiotics.

The biological effects of a drug or a food component depend on their concentration at the site of action. This concentration should be within the therapeutic window i.e. above the ineffective concentrations and below con-centrations with unacceptable toxicity. The same ap-

plies to probiotics and their active components. In the present review, we address the following four questions: why do we need to study the pharmacokinetics of

probiotics; how can we do this; which factors influ-

ence the survival of probiotics in the gastrointestinal tract; and what is known in this field?

WHY DO WE NEED TO STUDY THE PHARMACO- KINETICS OF PROBIOTICS?

Probiotics can influence physiology and health through direct or indirect effects in the gastrointestinal tract (GIT) (16, 25, 35, 49). Knowledge on the phar-macokinetics of a probiotic is needed for five reasons.

1-The first is to answer the questions how much of the probiotic should be consumed, how often, and for how long? the question "how much" is important, and it is surprising that so few studies were performed describing dose-effects of probiotics, or even the phar-macokinetics of increasing doses (43, 44). The answers to the questions "how often and for how long" are also depending on the pharmacokinetics. For example, if a

probiotic is able to colonize the intestinal mucosa per-manently at high concentrations, it is probably not nec-essary to administer it more than once. On the contrary, if a probiotic does not colonize (which seems to be till now the rule) it needs to be regularly administered to obtain its effects.

2- The second reason is to correlate the effects of the probiotic with its concentration (or that of its active components) at the target site. Understanding the rela-tion between the effect and the concentration at the tar-

1

2 P. MARTEAU and T. VESA

Fig. 1. Pharmacological approach of the effects of probiotics

or their active constituents in the gastrointestinal tract.

get can help to develop vectors to carry the probiotic activity to the target (for example fermented food (43)

gelatin capsules (45, 50), or microencapsulation (40)). A good example in this field is the lactase activity con-tained in yogurt bacteria. Subjects with hypolactasia do not digest sufficiently lactose. When they ingest

yogurt, the lactase activity contained in yogurt bacteria is delivered to the duodenum, but is not active there to digest lactose (20, 37). Beyond in the small bowel-

probably because the yogurt bacteria are lysed by bile salts and release lactase-the bacterial lactase becomes active and lactose is digested (23). It has been hypoth-esized that other bacteria such as Lactobacillus acido-

philus, which also contain lactase activity, were less efficient than yogurt bacteria to help in lactose diges-tion, because they were more resistant to bile and thus released less efficiently their intracellular lactase con-tent (15, 27).

3- The third reason is to validate hypotheses such as "a probiotic should be of human origin, ... have a high survival capacity, ... adhere to the intestinal epi-thelium"...

4- The fourth is to establish in what concentrations

probiotics should be present in the commercial prepa-rations.

5- And the fifth is a question of safety. Until now, very few side effects have been described in subjects consuming probiotics (1, 10, 12, 43). However, use of new probiotic microorganisms, increasing the doses or use of probiotics in fragile subjects such as subjects with acid secretion or intestinal motility disorders, in-testinal lesions, immunodeficiencies or autoimmune dis-eases could theoretically increase the risk. A few cases of fungemias have been described in patients receiving Saccharomyces boulardii (12).

HOW TO STUDY THE PHARMACOKINETICS

OF PROBIOTICS ?

The active constituents vehiculated by the probiotics are seldom known (for example lactase for lactose di-

gestion, cell wall components for immunomodulation (32, 35)...). Most pharmacokinetic studies have thus described the fate of probiotics (i.e. usually their "survial") in the gastrointestinal tract .

In vitro models. Some in vitro models have been developed to predict the survival of probiotics in vivo or their adherence to the intestinal epithelium (2, 7, 8, 13, 24, 29, 31). Static models can provide information on the sensitivity of strains to fixed pH or bile concen-trations (8). Multicompartmental dynamic models have also been developed in which a computer program al-lows to reproduce the dynamics of the gastric and bil-iary secretions, as well as the transit of the chyme in the intestine (24, 29, 31). These models should allow to predict more accurately the in vivo situation but also to study the influence of parameters such as the bile concentration or variable acid secretions (30).

In vivo techniques. The best way to establish the

pharmacokinetics of a probiotic in the GIT is to mea-sure it in vivo. Three techniques can be used to obtain samples from the gut lumen at different sites: 1- col-lection of feces or stomy effluent, 2- pyxigraphy, 3- intestinal intubation. Stomy effluent can by definition only be obtained in patients. Pyxigraphy is an elegant technique in which the subject swallows a capsule that can be opened and closed in the lumen of the GIT (39). It must then be recovered in feces before the sample is collected for analysis. This technique has not yet been used to our knowledge to study the pharmacokinetics of probiotics. Intestinal intubation is the best technique to obtain samples from any part of the GIT. The use of

perfusion techniques allows determination not only of concentrations of probiotics but also of their flow rates

(25). Whatever technique is used to obtain samples in the GIT, markers should be used if one wants to deter-mine the potential colonization property of a probiotic. We (5, 26, 38), and other (19, 33) use spores of Bacil-lus stearothermophilus as a transit marker. These do not grow and are not destroyed in the GIT, and are eas-ily enumerated on agar plates in 65°C where no other usual intestinal bacteria grow. When spores are ingested, they are eliminated in feces following an exponential curve, and become undetectable is subjects with "nor-mal" intestinal transit within 5-9 days. Using "macro-scopic" markers such as plastic pellets is a less sensitive technique since they do not allow to show that

PHARMACOKINETICS OF PROBIOTICS AND BIOTHERAPEUTIC AGENTS IN HUMANS 3

after 8 days 1% of the marker is still present in the co-lon. Colonization can be defined as the potency for a

probiotic to persist in the body for longer period than the inert marker. The sensitivity of the bacteriological techniques to detect and identify the probiotic within the endogenous flora is another critical point in the sur-vival studies. A clear identification of the strain using specific traits or probes is always the best. To increase the detection limit of the probiotic within the endog-enous ecosystem, several studies were performed using probiotic variants with antibiotic resistance, es-

pecially to streptomycin and rifampicin (5, 19). An-other method is a use of a placebo as control period. This technique is less sensitive but often sufficient, es-

pecially with probiotics exhibiting a high survival rate in the small bowel (i.e. far above the endogenous back-

ground) (26, 38). When no strain identification and non control periods are used, an increase of lactobacilli in feces after ingestion of lactobacilli can be difficult to interpret, since it may be due either to the passage of the ingested strain till feces or to an increased excre-tion of endogenous lactobacilli.

FACTORS INFLUENCING THE SURVIVAL OF

PROBIOTICS IN THE GASTROINTESTINAL TRACT

The survival of ingested microorganisms depends on the intrinsic resistance of the probiotic, host factors, and on the vehicle of the probiotic.

Intrinsic resistance of the probiotic. The intrinsic resistance of probiotics to gastric acid and bile will be discussed in the next chapter. The effect of the age of a

probiotic culture on the growth conditions or on their intrinsic resistance might be important, but remains unknown.

Host factors (14, 30, 47). The gastric acid secre-tion constitutes a major defense mechanisms against ingested microorganisms. Bile-especially bile salts-is the second important factor, but the influence of other digestive secretions such as mucus, pancreatic secre-tions, and enteric secretions seems to be limited. The

gastrointestinal motility constitutes the third major de-fense mechanism of the gut, and pathological slowing of the peristalsis results in chronic bacterial coloniza-tion of the small intestine. The equilibrium of the flora and the fate of probiotics is also dependent on micro-bial interactions including competition for substrates or for adhesion sites, and modifications of the environ-ment (through bacterial by-products or bacteriocins). Finally, the immune system is also involved in the con-trol of the flora.

Very few studies were performed to describe the

influence of host factors on the fate of probiotics. Pedrosa et al. showed that the survival of L. gasseri ADH was high in subjects with hypochlorhydria (34). Several studies demonstrated that the endogenous flora influences the pharmacokinetics of the probiotic yeast Saccharomyces boulardii. Indeed, they showed that the fecal elimination of the yeast was quicker than that of spores of Bacillus stearothermophilus, and that the sup-

pression of the endogenous flora by antibiotics enhanced the survival of Saccharomyces boulardii in the rat co-lon (3, 5, 28). A quicker fecal elimination than that of spores of Bacillus stearothermophilus has also been observed for ingested Lactococcus lactis (19).

Role of the vehicle. Few studies have assessed the influence of the vehicle on the pharmacokinetics of

probiotics. The decrease of the pH in the stomach, and the residence time of food in the stomach differ greatly with different meals. For example, the half time for

gastric emptying of water is around 15 min, that of half skimmed milk 30 min, and that of solid food more than one hour. As a consequence, the pharmacokinetics of acid sensitive probiotics can be dramatically influenced by their vehicle. Saxelin et al. showed that the survival of Lactobacillus strain GG differed when this probiotic was ingested in tablets, gelatin capsules, fermented milks, or whey drink (43-45). Some authors have pro-

posed to microencapsulate probiotics to enhance their resistance to gastric acidity (40) but the consequences on their survival in vivo and on their effects remain unknown.

PHARMACOKINETICS OF PROBIOTICS:

SOME DATA

Adhesion to the intestinal epithelium. Many stud-ies have shown that some probiotic strains can adhere to cell lines such as CaCo2 or HT29 (2, 7, 9, 13). These epithelial cell lines are of colonic or intestinal origin. The cells are polarized like in an intestinal epithelium, and many characteristics and functions of a normal epithelium are expressed on the cells. They are there-fore thought to be fair models to predict in vivo adhe-sion. Using other cell lines or colon tissues have also been proposed (42). The epithelial adhesion property differs between strains (2, 7, 9, 13, 42), and this prop-erty might be correlated with competitive exclusion

properties and immunomodulatory activities in vivo. Until now, competitive exclusion properties of adher-ing strains have only been shown in vitro. Validation of the in vitro models with in vivo data is therefore warranted, and the possibility for a probiotic to adhere to the intestinal epithelium during its intestinal transit

4 P. MARTEAU and T. VESA

has to be studied. Survival of ingested probiotics to different sites of

the gastrointestinal tract. The survival of ingested microorganisms differs among genus and strains. Some microorganisms are destroyed in the stomach while others have a high survival till feces.

Lactobacillus bulgaricus and Streptococcus ther-mophilus have a poor intrinsic resistance to acid, and are destroyed within few minutes at pH 1 and in about

1 hr at pH 3 (8). Pochart et al. observed that the con-centrations of viable yogurt bacteria reaching the duode-num after ingestion of 430 g yogurt in healthy humans were around 105 cfu/ml (37). This survival represented approximately 1% of the ingested bacteria. Using a dynamic in vitro model, we observed that 26% of "in-

gested" L. bulgaricus survived passage through the stomach when the model simulated the pH and gastric emptying of yogurt (24). Pettersson et al. reported that viable yogurt bacteria reached the ileum in 1/4 of the subjects after ingestion of 500 ml yogurt, and Lindwall & Fonden reported that after consumption of a yogurt containing 109 L. bulgaricus/g, the concentration of lac-tobacilli in the ileostomy bag was 105 to 106 cfu/ml (21, 36). Using an intestinal intubation technique, we showed that one fifth of the lactase contained in yogurt bacteria survives till the end of the small bowel and

participates to the digestion of lactose in the human gut (23). It is to note that this effect does not need the bac-teria to be of human origin, and does not require that they survive in the GIT (but even rather that they lyse there, releasing then the lactase that they contain). As the survival of yogurt bacteria is low in the upper gas-trointestinal tract, their survival and probiotic effects downstream (in the colon) are probably not relevant.

The capacities of survival of L. acidophilus, L. reuteri, L. rhamnosus strain GG in acid conditions are higher than that of L. bulgaricus (25). About 1-10% of L. acidophilus ingested in fermented products were found to survive until the ileum in several human stud-ies using intestinal intubation techniques (6, 25, 26, 41). In one of our studies, the concentrations of lactobacilli flowing through the ileum after ingestion of a cup of milk product containing 108 cfu/ml of Yoplait-Al strain were 100 times higher than the concentrations after in-

gestion of a control meal (36); the passage lasted for more than 5 hr, and no permanent colonization of the small bowel was observed. Studies in healthy volun-teers ingesting different probiotic preparations showed that fecal concentrations of L. acidophilus, L. reuteri, L. rhamnosus strain GG reached around 106 cfu/g (6, 17, 22, 25, 26, 41, 43-46, 50). Some Bifidobacterium

sp. from fermented dairy products exhibited a high sur-vival in the GIT i.e. 30% of the ingested bacteria could be recovered in the ileum and in feces (5, 26, 38). Concentrations above 106 cfu/g in the small bowel and around 108 cfu/g in the colon were reached (5, 26, 38). After cessation of the oral administration of these

probiotic strains, the kinetics of their fecal elimination paralleled that of an inert marker indicating the absence of colonization (5).

Several human studies were performed to describe the pharmacokinetics of Saccharomyces boulardii in man, a yeast which has proven efficient in decreasing the risk of antibiotic associated diarrhea and the risk of recurrence of Clostridium difficile colitis (3, 4, 13, 34). About 60% of the ingested yeasts were recovered as dead cells in feces; the survival in feces was about 0.1%-which is about the same as what was measured with S. cerevisiae (19)-; concentrations of living yeasts were above 105 cfu/g in the colon (3, 4, 12, 28).

Colonization of the gastrointestinal tract by

probiotics? Some authors observed the persistence of some probiotic strains in the feces of a few subjects for longer periods than what can be expected from a "nor-mal persistence" of a "normal person" with a "normal intestinal transit" (17, 25). This might indicate coloni-zation, but this remains unproved. Probiotics are usu-ally excreted within a few days after their ingestion in feces at the same rate or even quicker than a transit marker (3, 4, 19, 25). However, Johansson et al. pro-vided interesting results which suggest a colonization of the jejunal and rectal mucosa with alimentary lacto-bacilli (18). These authors administered a soup con-taining 19 different lactobacilli to healthy volunteers, and searched for colonization in the jejunum and rec-tum using probes on biopsies which were taken on days one and eleven after ingestion of the lactobacillus soup. Two strains of Lactobacillus plantarum were detected 11 days after their ingestion. One (Lp299) was of hu-man origin; the other (Lp299v) had been initially iso-lated from a sour dough. Confirmation of this result and testing of other strains with the same methodology seems an important issue both for fundamental research in intestinal ecology and for safety aspects of probiotics.

CONCLUSIONS

Knowledge on the pharmacokinetics of probiotics is increasing owing to progresses of in vitro models, sampling techniques in the human gut, and tools for reliable identification of probiotics within complex eco-systems. Strains differ in their survival capacity at the different levels of the gastrointestinal tract and in their

PHARMACOKINETICS OF PROBIOTICS AND BIOTHERAPEUTIC AGENTS IN HUMANS 5

adhesion properties to epithelial cells in vitro. Coloni-zation has not been proven but has been suggested at least in one study with two strains (18). Studies are needed to establish the adhesion of probiotics to the epithelium in vivo, potential colonization, and the influence of the vehicle in which the probiotic is in-

gested. Dose response studies should be done. All these efforts, which are also needed for safety reasons, should help to confirm or refute hypotheses such as "a probiotic should be of human origin, ... have a high survival ca-

pacity,... adhere to the intestinal epithelium"... Last but not least, they should give an answer to the ques-tions: how much probiotic should be consumed; how often; for how long; and what concentrations of

probiotics should be present in the commercial prepa-rations?

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