Mechanisms of Action of Biotherapeutic Agents

Jean-Paul BUTS, MD, PhD

INTRODUCTION

Although crude mixtures of microorganisms (e.g. fermented milk products or poultices of moldy bread) have been used since antiquity to treat infections, the first scientific description of a biotherapeutic effect has been made at the begining of this century by the russian Metchnikoff for which he received the Nobel Proce in 1908. Metchnikoff demonstrated that certain species of bacteries were able to enhance the proliferation of Vibrio cholerae while other species did inhibit its growth (1). A preliminary but fundamental distinction needs to be made between the concept of "PREBIOTIC" and "PROBIOTIC". A "PREBIOTIC" can be defined as a non-metabolized, non-absorbed substrate that is useful for the host by enhancing selectively the growth and/or the metabolic activity of a bacteria or of a group of bacteries (e.g. lactulose effect on the colonic flora) (2). A "probiotic" or a "biotherapeutic agent" is a living microorganism administered to promote the health of the host by treating or preventing infections due to strains of pathogens (3,4). Both terms of "probiotic" and "biotherapeutic agent" have been used in the literature to describe microorganisms that exert antagonistic activity against pathogens in vivo (4,5). Biotherapeutic agent seems to be more appropriate because it denotes a microorganism having therapeutic properties (3). Ideally, biotherapeutic agents should be innocous, act against pathogens by multiple mechanims to minimize the development of resistance and marshal host defenses to destroy the invading pathogen. An additional desirable property would be an immediate onset of action (in contrast to a vaccine that takes several weeks to stimulate antibody production).

Finally, biotherapeutic agents must be given in sufficient concentration to exert therapeutic properties, remain stable and viable before use, and survive in the intestinal ecosystem of the host to develop their therapeutic properties.

In the present chapter, we will focuse only on the mechanisms of action of biotherapeutic agents, a large class of whole microorganisms that are more and more used in clinical practise for their therapeutic properties against troubelsome pathogens.

SPECIES OF BIOTHERAPEUTIC AGENTS

Globally, biotherapeutic agents can be divided into two groups : those belonging to bacterial microorganisms and those belonging to yeast cells.

2.1. Bacterial microorganisms used as biotherapeutic agents include

-Lactobacillus acidophilus

-Lactobacillus casei GG

-Bifidobacterium longum

-Bifidobacterium bifidum used with Streptococcus thermophilus

-Enterococcus faecium SF68

2.2. The only yeast species used as biotherapeutic agent is Saccharomyces boulardii

3. MECHANISMS OF ACTION OF BACTERIAL BIOTHERAPEUTIC AGENTS (BBA)

3.1. Mode of Administration

Bacterial biotherapeutic agents (BBA) have been given to patients according to 3 modes of administration

-The rectal biotherapy inoculates into the rectal lumen a selection of BBA whose

physiological effects have not been studied. In addition to the obvious difficulty of

this practise for the patient and his doctor (6,7) it increases the risk of disseminating

undetected pathogens including HIV virus.

-The use of fermented milks or of yoghurt raises some specific problems including the

necessity of refrigeration, limited delay of conservation (6) and the need to consume

large quantities of fermented products to obtain a therapeutic effect (6).

-The use of living microorganisms dried and powdered into capsules raises no

difficulty and appears to be the more simpliest way of administration.

3.2. Pharmacokinetics of Bacterial Biotherapeutic Agents

The capacity of BBA to survive in an acidic medium has been considered as an important criteria for its efficacy as biotherapeutic agent. Many species of Lactobacilli are destroyed by gastric acidity, bile acids and pancreatic enzymes (8). For instance, the intrinsic resistance of Lactobacillus bulgarius and Streptococcus thermophilus, twos species found in yogourt, to acidity and bile is very weak (8,9) while L. acidophilus and Bifidobacteries are more resistant, with however great variations between strains (8). Recent studies (9,10) demonstrate also that Lactobacilli, Enterococcus, Bacillus cereus and Bifidobacteries do not resist to commonly prescribed antibiotics including amoxicillin, doxycyclin, cephalosporins and fluoroquinolones (10). Even when, antibiotics are administered parenterally, a rapid inactivation of these microorganisms semms to occur because of the in vivo enterohepatic cycle (10). While the ability to survive the digestive secretions in the gastrointestinal environment is thought to relate to efficacy, proliferation and persistence of BBA in the gut are apparently not of paramount importance for biotherapeutic effects provided that continuous daily administration is assumed. Lactobacillus casei GG is the only BBA shown to persist in the gastrointestinal tract following cessation of oral dosing. At day 7 after discontinuing administration, 6 of 18 subjects still harbored L. casei GG, albeit with a 100-fold decrease from state levels (11). Other BBA with demonstrated efficacies (e.g. L. acidophilus, Bifido bacterium species) can survive intestinal transit but disappear within a few days after the last dose (12,13).

3.3. Resistance to Colonization

Colonization resistance is the ability of normal flora to protect against the unwanted establishment of pathogens. The phenomenon is related to a complex interaction of many individual bacteria that comprise the mucosa microflora. Up to now, attempts to identify and use a single microorganism or a specific mixture of microorganisms that would have the specificity of the normal microflora to resist pathogen invasion have not been successful. An important goal of therapy with biotherapeutic agents is to stop proliferation of the pathogen until the time that the normal microflora can be reestablished (3). The ability to "gain time" to restablish resistance to colonization is probably one important mechanism of successful therapy with biotherapeutic agents.

3.4. Production of Antimicrobial Substances

Lactobacillus casei GG has been shown to produce a microcin inhibitory substance in vitro toward a broad spectrum of gram-positive and gram-negative pathogens (14). The inhibitory effect occurred between pH3 and 5 and was heat stable. The inhibitory substance was distinct from lactic and acetic acids, had a low molecular weight

3.5. Competitive Inhibition for Bacterial Adhesion Sites

This is another possible mechanism of action for BBA. A Lactobacillus strain was shown to competitively inhibit adhesion of enteropathogenic E. coli to pig ileum and interfered with bacterial attachment to the mucosal layer of ileal conducts (18). However, adhesion capacity and competitive inhibition fo L. acidophilus are not constant properties since certain L. acidophilus strains can attach in vitro to cells ressembling to enterocytes, while other strains of L. acidophilus do not (19,20). Although L. acidophilus inhibits the adhesion of several enteric pathogens to human intestinal cells in culture, when pathogen attachment preceeded L. acidophilus treatment, no inhibitory interference occurred indicating that steric hindrance of site occupation is important in the inhibition of adhesion (19,20). Thus, therapeutic use is limited to preventive application and not to a curative goal once binding of the pathogen has occurred (19). In addition, a dose-dependent inhibition against cell adhesion of several pathogens has been demonstrated only for one strain of L. acidophilus (LA1) (19).

Similar studies have been conducted with a heat killed strain of L. acidophilus which conserves in vitro binding capacities to cells, despite being heat-killed (21). In vitro, the dose of killed strain needed to inhibit the fixation of ETEC is 10 times higher than that needed to obtain the same effect with the living strain. To inhibit the attachment to cells of 50% of ETEC pathogens, very high dosis of heat-killed L. acidophilus are needed which corresponds to 2.5 x 10⁹ bacteries per ml. This dose represents an amount of 500 gelules per liter, since 1 gelule contains 5 x 10⁹ killed bacteries. Likewise, very high concentrations of L. acidophilus are needed to obtain a preventive action in vivo (21).

The suspicion that concentrations of heat-killed Lactobacillus needed to induce a physiological effect cannot be reached in vivo is confirmed by the observations that living strains of Lactobacillus have failed to prevent the development of diarrhea -induced by the administration of enterotoxigenic E. coli in adults and to reduce the duration of symptoms of the illness in a double-blind placebo controlled prospective trial, despite the efforts made to optimalize the concentration of Lactobacillus administered (22). Also, preparations of living L. acidophilus are not efficient to prevent traveller's diarrhoea whose ETEC strains are the causal agent in 40% of cases (23,24).

3.6. Effects on the Immune System

Stimulation of the immune system of the host by several Lactobacillus strains has been reported (25,26) but interpretation of the data raises many difficulties before it can be concluded that this effect significantly contributes to eradication of invasive pathogens. In a comparative study during which Lactobacillus casei GG was given in living and killed forms to infants presenting with diarrhoea due to Rotavirus, the authors found significantly more infants secreting anti-Rotavirus IgA in the treated group during the recovery period (27). However, during the period of acute diarrhoea , the level of specific antibodies was similar and very low in both groups of patients.

In addition, no difference was observed between the groups regarding the duration of symptoms, and severity of diarrhea. Thus, the rise in specific IgA appears to be late and could be theoritically important for the prevention of re-infections (28). However, this awaits further confirmation.

4. MECHANISMS OF ACTION OF SACCHAROMYCES BOULARDII

4.1. Pharmacokinetics

Physiological studies conducted in vivo in humans and in animal experimental models as well as in vitro on cultured cell lines, have conclusively demonstrated that S. boulardii act by multiple mechanisms converging to eradicate invasive pathogens to inhibit the action of toxins and to restore the absorption capacities of the small intestinal mucosa.

These mechanisms include

4.2. Competitive Inhibition for Pathogen Adhesion Sites and Microbial

Interactions

Exposure of Entamoeba histolytica trophozoites to S. boulardii, its membranes or yeast culture supernatants decreased the numbers of trophozoites able to attach to erythrocytes in vitro (37). Earlier studies showed that treatment with S. boulardii decreased mortality and morbidity in young rats infected with E. histolytica (38). CDR Sprague-Dawley young rats were infected with 5 x 10⁵ trophozoites of E. histolytica and either 1.8 x 10⁹ c.f.u./day S. boulardii or saline for 4 days. Rats were sacrified on the fifth day and autopsied. In rats treated with S. boulardii, there were significantly less lesions than in control rats and the mean healing time of lesions was significantly diminished (6 days) in S. boulardii-treated rats compared with 21 days in controls.

S. boulardii also inhibited in vivo the proliferation of C. krusei and C. pseudotropicalis but had no inhibitory action on C. tropicalis (34). Several studies have also assessed the interaction of S. boulardii with the normal gut flora. A study conducted in human volunteers which received 1 g S. boulardii per day revealed no change in the selected populations of the normal colonic flora after 4 to 5 days of exposure to the yeast (35). Concentrations of total anaerobes, Bacteroides species, and Clostridium species did not signficantly change compared with their baseline counts. The mean concentrations of the two groups of bacteria slightly increased from pre-S. boulardii to post-S. boulardii exposure. Thus in an undisturbed bowel with intact colonization resistance, S. boulardii can be introduced without apparent effect on the host microflora. When colonization resistance is disturbed or when there is an overgrowth of invasive pathogens, S. boulardii is able to reduce the concentration of several pathogenic agents responsible of diarrhea.

4.3. Anti-Secretory Effects Induced by Toxins

4.4. Inhibition of Toxin Binding to Intestinal Receptors

This effect related to S. boulardii has been extensively studied in vivo as well as in vitro on the enterotoxinogenic lesions caused by Clostridium difficile. C. difficile is a strict anaerobe which produces two well characterized toxins : toxins A and B. It is the most frequent cause of nosocomial diarrhea in adults and the pathogen causing persistent and protracted enterocolopathies (43,44) and pseudomembranous enterocolitis in children (45) as well as in adults (46), two potential lethal infections. Intestinal overgrowth of C. difficile occurs after colonization resistance has been compromised by antibiotic use, surgery or gastrointestinal pre-infections (44). The incidence of C. difficile ranges from 0.8 to 21 per cent hospitalised patients receiving antibiotics (47-50). In neonates nosocomial acquisition occurs at a very high rate (50). S. boulardii has been tested in several animal models of C. difficile-associated colitis. In each study, a significant protective effect was found whether colitis was induced by toxinogenic C. difficile itself, or by toxin A or toxin B. Corthier et al (51) found that gnotobiotic mice, who generally die rapidly after a C. difficile challenge, were protected after a single dose of S. boulardii (16 per cent survival rate), while under continuous treatment, survival rate increased up to 56 per cent. This protection rate was later found to be dependent upon the dose and viability of the yeast (52). The ability of S. boulardii to inhibit C. difficile-associated damage is lost if the yeast is administered in a non-viable form (killed by heating or by amphotericin B).

In their study, Elmer and Corthier (52) documented a dose-response effect and a good correlation between survival rate and the dose of S. boulardii given.

As the dose of S. boulardii was increased from 3 x 10⁸/ml to 3.3 x 10¹⁰ ml in drinking water given to C. difficile infected mice, the survival rate increased linearly from 0 to 85% (52).

In hamsters, S. boulardii was found to reduce significantly from 100% to 28% mortality induced by clindamycin, an experimental model of C. difficile infection (53, 54). Although several studies have documented that S. boulardii reduced the levels of C. difficile in hamster fecal pellets (53,54), the most prominent effect of S. boulardii treatment is the decrease in concentrations of C. difficile toxins A and B (52,53,55). In gnotobiotic mice and in normal mice and hamsters, there is a decrease in toxin A and/or cytotoxin B levels after exposure to S. boulardii (53,56,57). Czerucka et al (5) documented a decrease in the percentage rounding of intestinal cells due to C. difficile if S. boulardii was added to the cell culture, but another study failed to confirm this protective effect (59). Likewise, several studies have shown that S. boulardii inhibited the formation of histological lesions in the cecum due to the toxins of C. difficile in mice and hamsters (54,56,57).

The toxin A intestinal receptor for C. difficile is a protease-sensitive high molecular weight glycoprotein (60). Pothoulakis et al (59) have demonstrated that S. boulardii produces a protease retained by a 100-kDa molecular weight filter which decreases fluid secretion in a rat loop model but appears to have no effect on cell tissue layer damage caused by C. difficile such as the human lung fibroblast (IMR-90) or the rat basophilic leukemia (RBL) cell lines. The S. boulardii protease was able to inhibit binding of purified [³H]-labeled enterotoxin to rabbit ileal brush border membranes by 37 per cent, to reduce enterotoxin-induced fluid secretion by 55 per cent in rat ileal loops and to decrease mannitol permeability by 93% in rat ileal loops. In addition, when S. boulardii was given orally for 3 days to rats, a challenge by pure enterotoxin failed to increase fluid secretion or permeability. In contrast, when S. boulardii and toxin A were co-administered, no protective effect of S. boulardii was observed. Thus, S. boulardii has apparently no action on the toxins of C. difficile but could alter or degrade their receptor sites on the apical membrane of enterocytes.

4.5. Immunological Effects

In vitro, S. boulardii activates the complement directly and fixes the C3b fraction. Phagocytosis of S. boulardii by mononuclear cells is complement-dependent (61).

Thus, by oral administration of biotherapeutic agents, both local (intestinal) and systemic effects appear to be involved in their activity.

4.6. Trophic Effects on Intestinal Mucosa

In 1986, we have examined the interaction of S. boulardii with the intestinal mucosa of the host. Oral administration of S. boulardii (1 g per day during 8 days) to seven human volunteers produced no effect on intestinal morphology (conventional histology of mucosal biopsies), villus height and crypt depth (64). Likewise, electron microscopic examination of duodeno-jejunal mucosa in rats given S. boulardii and mice showed no invasion of S. boulardii into sub-epithelial mucosal layers without morphological changes of the villi or changes in crypt depth (57,64). Using a three-dimensional microdissection technique of human intestinal biopsies, Jahn et al (65) confirmed recently that after S. boulardii treatment there was no statistical difference in villi surface area nor in crypt depth.

Thus in humans as in rats, S. boulardii enhances the expression of disaccharidases and alkaline phosphatase which may improve the absorption of carbohydrates, usually defective in acute and chronic diarrheal disorders.

In a recent study (66), we demonstrated in rats who underwent a 60% proximal enterectomy that treatment with S. boulardii during 8 days post-surgery not only enhanced markedly disaccharidase activities but also significantly stimulated the Na⁺-dependent uptake of D-glucose, measured in brush border membrane vesicles as a function of incubation time and as a function of D-glucose concentration in the incubation media, compared to matched resected and transected controls (66). This demonstrates unequivocally that S. boulardii can optimalize the quality of the adaptive response of the small intestine, implying a potential therapeutic benefit in situations where upregulation of intestinal nutrient absorption in advantageous.

Theoritically, such amounts of polyamines are able to influence intestinal enzyme expression. Indeed, it has been shown that marked changes in intestinal disaccharidase and aminopeptidase activities and in the production of endoluminal s-IgA occur in infant rat small intestine in response to oral supplies in spermine and spermidine equivalent to 1000 nmol per day of polyamines (68). When suckling rats were treated with an amount of spermine (500 nanomoles per day) equivalent to the polyamine content of the yeast (679 nanomoles/100 mg) similar patterns of enzymatic responses were observed consisting in significant increases in sucrase (2.5 fold increase) and maltase (+ 24%) specific and total activities.

In response to 1000 nanomoles of spermine, the stimulation of the enzyme activities was proportionally greater, including a 4.6 fold increase in sucrase and a 70% increase in maltase. Similarly, weanling rats treated with S. boulardii or with the equivalent amount of spermine (500 nanomoles) exibited significant and similar increases in the specific activity of sucrase (157%) and maltase (+47.5%). Thus, the comparison presented here between 100 mg lyophilized S. boulardii containing 679 nanomoles of polyamines and 500 nanomoles of spermine given orally to suckling and weanling rats revealed to produce similar patterns of enzymatic responses. As previously noted (69), the stimulation of sucrase and maltase by oral spermine is dose-dependent, is more sensitive than for other microvillous enzymes (lactase, aminopeptidase) and becomes detectable for doses of spermine exceeding 250 nmol/day. Both S. boulardii (62) and oral spermine (69) have been shown to enhance significantly the intestinal production of the polymeric immunoglobulin receptor in weanling rats treated from d. 20 to d. 30 post-partum.

Besides changes in enzyme activities, oral treatment with S. boulardii (68) resulted in parallel changes in polyamine concentrations in both intestinal mucosa (+21.4%) and endoluminal fluid (+48 to 316% in the jejunum and + 60.8 to 150% in the ileum). The variations in the three polyamine levels measured in the intestinal mucosal (putrescine: +7%, spermidine : +21.9%, spermine : + 21.4%) were proportional to their concentration measured in the yeast lyophilized preparation. Spermine and spermidine that represented 44 and 55 % respectively, of the total amount of polyamines supplied by S. boulardii increased in the same proportions in the intestinal mucosa (+ 21.4 and + 21.9%). In concordance, with the negligible amount of putrescine provided by the yeast (1.4 %), mucosal putrescine levels varied very little and were unaffected by the oral treatment. Also, the ration of spermidine to spermine was equivalent in treated rats (1.79) and controls (1.80).

Experimental evidence indicates that the uptake of endoluminal polyamines by brush border membrane vesicles is a selective and saturable absorptive process dependent to a large extent on their endoluminal concentration (70-73). In samples of jejunal and ileal fluid collected by intestinal flushing and filtered to discard yeast cells, from S. boulardii-treated rats, spermidine and spermine were increased by 48 to 316% over controls, whereas changes in putrescine levels were not significant. Because endoluminal polyamines (especially putrescine) originate from several sources including food (74,76), intestinal secretions and microbial flora (77), variations in concentration were much greater in the gut lumen than in mucosa.

Taken together these data indicate that at the dose used, lyophilized S. boulardii exerts trophic effects on the small intestinal mucosa that are likely mediated by the release of spermidine and spermine in the endoluminal compartment. These substances could be released by the yeast intestinal catabolism rather than secreted by viable cells during their transit. Indeed, only traces of putrescine were detected in the yeast culture media after 96 hours without evidence of spermine or of spermidine secretion in the media.

Further support for our conclusion is provided by two observations

First, less than 3% of the oral dose of yeast cells is recovered in a viable form in stools, indicating that progressive endoluminal catabolism occurs during intestinal transit (33) and second, microflora-derived polyamines can modulate mucosal tickness, causing even intestinal obstruction by excessive bowel hypertrophy (77).

In view of the well-known physiologic effects of exogenous polyamines on cell maturation, enzyme expression, membrane transport mechanisms, and epithelial cell renewal, the quantification of substantial amounts of polyamines in S. boulardii could have important clinical implications. Because S. boulardii use is indicated in acute enteropathies and gastro-intestinal microflora disturbances, rational use of the yeast preparation requires that potential trophic effects for the prevention of chronic persistent and protracted enterocolopathies be further investigated in infants and children.

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