Summary – The possibility that the stomach, affected by general stress, might initiate a counter-response has not until recently been considered in theories of stress. We suggest that the stomach, as the most sensitive part of the gastrointestinal tract and the largest neuroendocrine organ in the body. is crucial for the initiation of a full stress response against all noxious stress pathology. The end result would be a strong protection of all organs invaded by ‘stress’. Consistent with this assumption, this coping response is best explained in terms of ‘organoprotection’. Endogenous organoprotectors (eg prostaglandins, somatostatin, dopamine) are proposed as mediators. Such an endogenous counteraction could even be afforded by their suitable application. A new gastric juice peptide, Mr 40000. named BPC, was recently isolated. Herein. a 15 amino acid fragment (BPC 157), thought to be essential for its activity, has been fully characterized and investigated. As has been demonstrated for many organoprotective agents using different models of various tissue lesions, despite the poorly understood final mechanism, practically all organ systems appear to benefit from BPC activity. These effects have been achieved in many species using very low dosages (mostly lag and ng/kg range) after ip, ig. and intramucosal (local) application. The effect was apparent already after one application. Long lasting activity was also demonstrated. BPC was highly effective when applied simultaneously with noxious agents or in already pathological, as well as chronical, conditions. Therefore, it seems that BPC treatment does not share any of the so far known limitations for ‘conventional organoprorectors’. No influence on different basal parameters and no toxicity were observed. These findings provide a breakthrough in stress theory. BPC. as a possible endogenous free radical scavenger and organoprotection mediator, could be a useful prototype of a new class of drugs, organoprotective agents.
The concept of stress (Selye, 1936; Lazarus and Folkman, 1984; Szabo and Giavin, 1990) and the concept of organoprotection (Szabo and Usadel, 1982; Pfeiffer et al, 1987) are the focus of major scientific research. However, despite these efforts, many basic and clinical aspects still remain to befully elucidated (Szabo and Glavin, 1990).
Links between the concept of stress and the concept of organoprotection: crucial role of the stomach and the stomach stress coping response
Following the tradition of Selye, hypothalamic-hypophyseal-adrenocorticai stimulation and later CRF as a key stress peptide, have been widely implicated to have a crucial role in the stress response (Axelrod and Reisine, 1984; Lazarus and Folkman, 1984; Murison and Bakke, 1990; Szabo and Giavin, 1990). On the other hand, there is growing evidence that prostaglandins (PGs), somatostatin and dopamine provide strong protection for many different organs (Szabo and Usadel, 1982; Szabo and Neumeyer, 1983; Usadei, 1988; Robert, 1979; Robert et al, 1991; Sikiric et al, 1986, 1988a,b, 1990a,b,c). Although it is accepted that virtually all of the body’s organs are affected by stressors (Szabo and Glavin, 1990), these concepts have not been developed so far. Recently, in favour of a purposeful physiologic solution, the protection of the body’s organs has been emphasized as an end result common to both concepts, and the stress coping response has been explained in terms of organoprotection (Sikirid et al, 1991a,b). In keeping with this, the commonly accepted mediators of organoprotection (Robert, 1979; Robert et al, 1991; Szabo and Usadel, 1982; Szabo and Neumeyer, 1983; Usadel, 1988; Sikiric et al, 1986; 1988a,b,c, 1990a,b,c); have been suggested to also be endogenous mediators of the stress coping response (Sikiri6 et al, 1991a, 1992a). Consequently they could be usefully applied in stress conditions.
The widespread presence of PGs, somatostatin and dopamine in the body as well as their crucial physiological significance (Robert, 1979; Szabo and Usadel, 1982; Szabo and Neumeyer, 1983; Usadel, 1988; Robert et al, 1991; Sikiric et al, 1986, 1988a,b, 1990a,b,c); support a hypothesis on their overall role and possible application in stress (Sikiric et al, 1991a, 1992a). In this, the stomach, previously recognized in Selye’s stress triad as a particular, but only passive target (Selye, 1936), became the focus of our interest (Sikiric et al, 1991a, 1992a). This is based on at least two lines of compelling evidence. First, among all the protective effects mentioned previously, gastroprotection was the first to be recognized. Second, the functions of putative mediators of organoprotection appear to be of the utmost importance, especially in the gastrointestinal tract (Robert, 1979; Szabo and Usadel, 1982; Szabo and Neumeyer, 1983; Usadei, 1988; Robert et al, 1991; Sikiri6 et al, 1986, 1988a,b, 1990a,b,c).
Deviating from the traditional point of view (Hernandez, 1990; Szabo and Glavin, 1990), it has been suggested that the stomach is essential for the body’s activity in stress (Sikirid et al, 1991a, 1992a) (fig 1). Confronted with general stress leading to stomach stress disturbances, the stomach itself is able to initiate, mediate and organize a feedback response against all of those noxious events by a particular organoprotective ‘stress coping response’. This endogenous counteraction (‘stomach stress coping response’) could be amplified by the suitable application of appropriate organoprotective agents and the disturbed homeostatis could be reestablished (Sikiri et al, 1991a, 1992a). Consistent with the wide range of beneficial effects of agents implicated to be organoprotective mediators (Robert, 1979; Szabo and Usadel, 1982; Szabo and Neumeyer, 1983, 1986; Usadel, 1988; Robert et al, 1991; Sikiric et al, 1986; 1988a,b, 1990a,b,c), a strong organoprotection would appear. Such stomach specificity in the organization of the stress response seems to be supported by what is known about gastroprotection (Hernandez, 1990; Szabo and Glavin, 1990), ie that stomach stress disturbances develop only when an organism cannot cope with the stressor (noxious event) at the given time. On the other hand, gastroprotection, if considered in this way, appears as a specific and essential starting point for the ability of the organism to cope with the stressor (Sikiric et al, 1991 a, 1992a).
Fig 1. Hypothesis. The stomach aspects in conventional stress terms (I) and stomach/stress/organoprotection hypothesis (IIa. lIb). I. Stomach taken as particular, but only passive target. Stomach stress response is considered only because of the local (negative) significance, lla. The stomach, specifically affected, is initiating, organizing and mediating a counteracting organoprotective response, llb. This physiological organoprotective response could be amplified by organoprotective agents application, and full organoprotection homeostasis reestablished.
Greely, 1987; Marx and Cooper, 1987; Pfeiffer et al, 1989; Grijalva and Novin, 1990; Mason and Hernandez, 1990; Henke and Ray, 1991; Bu6no and Gu& 1992). When compared to other organs, it has a specific innervation and a particular connection with the brain (Grijalva and Novin, 1990), as well as a major representation in the brain cortex (Grijalva and Novin, 1990). Interestingly, the same neural crest origin has been implicated for both brain and gut cells secreting brain-gut peptides (Oldam and Thompson, 1987). They appear very early in evolution, already in very low phyla (Krieger, 1983). Their concentration seems to be consistently higher in the gastrointestinal tract, thought to be the largest neuroendocrine organ in the body, rather than in the brain (Krieger, 1983).
The possible functional significance of these findings appears to be even more emphasized by particular dopamine system changes in the beginning of life-threatening conditions during stress. Consistent with the hypothesis that the stomach is the initiating site of the stress coping response, an increased number of dopamine receptors has been found only in the stomach mucosa, contrasting with no changes at the same time in several brain areas considered to be essential for the initiation of the stress coping response (Hernandez, 1986).
On the other hand, the stomach mucosa is completely unable to sustain anaerobic metabolism even for a short period of time and requires a continuous supply of glucose and oxygen, which seems to be quite unique in the organism (Menguy et al, 1974). Although apparently only a part of a much more complex response, these data taken together provide an insight into how the organism recognizes life-treatening conditions and initiates its coping response (Sikiri6 et al, 1991a, 1992a).
Namely, because of its specific metabolic patterns and stress blood redistribution away from the stomach mucosa (‘centralization’), unlike all other organs in the body, the stomach mucosa is strongly affected in the earliest stage of lifethreatening conditions. This event could be, on the other hand, a trigger for the initiation of the stress coping response (for instance, increase of dopamine receptors in stomach mucosa) (Sikiri6 et al, 1991a, 1992a).
However, whether PGs, somatostatin and dopamine agents could indeed be applied usefully as organoprotective agents or not remains to be seen. Although dopamine is widely used in lifethreatening shock conditions and the superiority of dopamine agents over conventional therapy (eg prevention of peptic ulcer relapse unlike H2 receptors blockers) has also been suggested (Sikiri6 et al, 1991 h), their ‘organoprotective capacity’ appears to be rather limited (Sikiri~ et al, 1991a, 1992a). For instance, even from an experimental point of view, ob,ious practical limitations seem to be very likely due to the route of administration (eg somatostatin, PGs), rather high dosage (eg dopamine agonists), short time duration, repeated application (eg somatostatin, PGs), effectiveness in pretreatment only (eg somatostatin, PGs), unwanted side effects (eg PGs, dopamine) (Robert, 1979; Szabo and Usadel, 1982; Szabo and Neumeyer, 1983; Usadel, 1988; Robert et al, 1991; Sikiri~ et al, 1986, 1988a,b, 1990a,b,c).
Consequently, the search for additional mediators of the defined stress coping response, in these terms, seems to be fully justified (Sikiri~ et al, 199 la, 1992a).
BPC - a possible organoprotective mediator
A new gastric juice peptide, Mr 40000, namedBPC has recently been isolated (in preparation)and its huge range of organoprotective effects hasbeen described. In this, a 15 amino acid fragment(BPC 157) thought to be essential for its activityhas been characterized (Sikiri6 et al, 1991a,b,c,d,e,f,g, 1992a,b). Its synthesis is briefly de-scribed below.BPC 157 consists of 15 amino acids: Gly-Glu-Pro-Pro-Pro-Gly-Lys-Pro-Ala-Asp-Asp-Ala-Gly-Leu-Val. It was obtained by stepwise condensa-tion of Fmoc protected amino acids, beginningwith the first amino acid, Val, bonded to a poly-meric carrier (benzhydrilaminoresin). Diiso-propylcarodiimide was used as the couplingreagent. In each step the Fmoc protective groupwas removed with piperidine; thereafter the sec-ond and so on. All further amino acids were in-troduced using the same method until synthesiswas complete. The cleavage was done using amixture of trifluoroacetic acid/trifluoromethane-sulphonic acid/anisole (2: 17:52). The raw peptidemixture was purified by HPLC column 5 mm id,150mm length, silica RP-18, gradient elutionwith solvent system: 0,1% trifluoroacetic acid inwater/acetonitrile.All of the experiments were done using peptidewith a minimum of 95% purity.These data and the noted beneficial activityrepresenting a full range of organoprotection have been topics at many international symposia (Zagreb 1990, Berlin 1991, P6cs 1991, Balatonaliga 1992, Zagreb 1992, Athens 1992, Florence 1992, Paris 1993) and presented, albeit briefly, in many reports (Sikiri~ et al, 1991a,b,c,d,e,f,g, 1992a,b).
Because of the huge range of different organoprotective effects consistently demonstrated in our experiments, and its origin in the stomach, BPC has been proposed as a crucial organoprotective mediator of the stomach stress coping response (Sikiri6 et al, 1991a,b,c,d,e,f,g, 1992a,b). Since the 15 amino acid peptide also demonstrated the same organoprotective activity as the entire BPC, these findings will be summarized and further reviewed in the present report (tables I-VIII). Rats as well as mice, guinea pigs, rabbits and chickens were used in the experiments. The dosage range of BPC most employed has been between 10 lag and 10 ng/kg bw, ip, ig, or iv. In some studies, however, even lower dosages were effectively used.
BPC organoprotective pharmacodynamics
Effects on the gastrointestinal tract, inflamntation and tissqe repair In the hypothesis presented previously (Sikiri~ et al, 1991a, 1992a,b), the crucial significance of gastropr0tection has been emphasized. On the other hand, inflammation is a process thought to be essential for tissue repair and survival. These effects will be mentioned first (table I).
A plethora of different and distinctive processes are involved in the ethiopathoiogy of ulcerogenic models investigated (eg stress, cysteamine, ethanol, 2,4-dinitrophlourobenzene (DNFB)) (Menguy et al, 1974; Ishii et al, 1976; Lichtenberg et al, 1977a,b; Robert, 1979; Par6 and Glavin, 1986; Szabo and Neumeyer, 1983; Murakami et al, 1985; Sikiri6 et al, 1986, 1990c; Brki~5 et al, 1992). Bearing in mind the profound efficacy of BPC in all of these models, its protective capacity has to be very large and it could probably provide protection against all potential noxious events. Since different species were also equally protected (for instance, the gastroduodenal pathology strongly differs in chickens and in rats (Grabarevi6 et al, 1993)), this activity seems indeed to be very consistent. Interestingly, given the evidence that a strong positive effect is seen when BPC was applied as a post-treatment, a high healing capacity appears to be very likely. Also, from DNFB induced inflammatory bowel disease in mice, a model recently described by our group (Brki~ et al, 1992), it is evident that besides the mechanisms involving delayed type of hypersensitivity reactions (Sikiri~ et al, 1991e), this protection apparently involves the entire gastrointestinal tract (in preparation).
These data have to be seen in the context of a strong anti-inflammatory effect also noted in many widely used inflammation models. Consistent with our previous data (Sikiri~ et al, 1991b), these results clearly demonstrated that all of the investigated models (carrageenin, turpentine-induced paw/buccal edema (Winter et al, 1962; Willoughby et al, 1986) as well as adjuvant arthritis (Brown et al, 1971; Perper et al, 1971)) have strongly been influenced by BPC application. Likewise, an effect has also been demonstrated when BPC was applied in the presence of established arthritis lesions in rats. These data have to be considered in the light of the complexity of the different systems’ interactions implicated in the various models of inflammation.
For instance, the histamine, 5-HT, bradykinin and prostaglandins systems are implicated to be sequentially involved in the carrageenin edema formation (Gyires et al, 1985). Turpentine, known to be a highly toxic agent producing marked necrosis and intense inflammation (Kusher, 1982; Damas and Delfandre, 1984), prevented carrageenin edema formation (Damas and Delfandre, 1984). Both specific (delayed type of hypersensitivity reaction) and non-specific inflammation are suggested to be involved in the development of adjuvant arthritis (Brown et al, 1971; Perper et al, 1971). Therefore, a different and particular BPC anti-inflammatory effect is suggested. The possible meaning of these data has to be viewed in the light of the finding that unlike glucocorticoids (Wahl, 1989), no impaired wound healing could be demonstrated. In contrast, a strong positive effect was consistently noted. Using tensiometry (Mustoe et al, 1987), an increased healing was demonstrated in wounds, as well as in burns and fracture animal models (for instance, EGF and FGF were not effective in the incisional wound model and PDGF and TGF-13 were effective only in the collagen medium, but not in saline or BPC (Mustoe et al, 1987; Duel et al, 1991)).
Observation that skin wounds, and wounds in other tissues not associated with the epithelial layer such as bones, were similarly positively affected (Castor, 1981), implies a particular positive effect on connective tissue as a central component (in preparation) (table II). Of note, an apparent analgetic and antipyretic activity has also been demonstrated (table III).
Effects of BPC on other organ systems
Taking into consideration all of these activities, particularly gastroprotection, inflammation and tissue repair, the protection of other organs consistently demonstrated in our investigations is not entirely unexpected. Notably, since different models and distinctive lesion aspects have been investigated, the obtained protection in these organs appears to be consistently large as well (table IV).
For instance, in liver and pancreatic lesion studies (eg bile duct with or without hepatic artery iigation, restraint stress, CC14 or ailoxan application), this complexity seems to be evident from various processes (eg blood supply impairment (ligation), all events non-specifically referred to as ‘stress’ (restraint stress-fatty liver)). Free radical formation (CC14) is implicated in the development of these lesions (Barnelli-Zazzera and Gaja, 1974; Vargas et al, 1970; Gaja et al, 1973; Schiaffonati et al, 1978; De Groot and Haas, 1980, 1981, 1982; Zawacki, 1984; Fredericks et al, 1982; Szabo, 1985; De Groot and Noll, 1985; Sikiri6 et al, 1988a, 1990c, 1991c). Likewise, a strong reduction of increased enzymes and bilirubin values was noted in BPC groups. Thus, integrating all these data, the hepatoprotective capacity of BPC (Sikiri~ et al, 1991c) is confirmed. Consistently, a strong BPC protection of both the exocrine and endocrine pancreas (Sikiri~ et al, 1991d,f) was evident in our pancreas studies (Vargas et al, 1970; Sikiri6 et al, 1988a). Namely, BPC groups consistently showed, in strong contrast with the control data, a significant lack of necrosis and inflammation in bile duct ligated rats, and preservation of cells of Langerhans islands containing insulin in the alloxan treated animals.
This view has been further supported in kidney investigations (Waugh and Pearl, 1960; Morrison and Pascoe, 1986; Sikiri~ et al, 1991g). Data obtained in the kidney assay seem also to be in line with the findings above. Both morphological and functional aspects appear to be equally involved (Sikiri6 et al, 1991g). Diuresis preservation and a dose-dependent lack of oliguria otherwise regularly noted in control animals after unilateral nephrectomy seem to be consistent with tubular epithelium protection in mercury chloride or serotonin treated rats. Interestingly, the weight of the remaining kidney in animals subjected to unilateral nephrectomy, but treated with BPC, increased less than in the corresponding controls.
Thus, in terms of the functional theory of compensatory renal hypertrophy (Johnson, 1969; Johnson and Amendola, 1969), these findings strongly emphasize a better function of the remaining kidney. This is also supported by our additional biochemical results. In addition, these data are completely in line with the effects obtained in hypertensive rats with renal artery stenosis and/or unilateral nephrectomy (Gross, 1977) (in preparation).
Mechanisms of BPC protection. Additional evidence that BPC mimics an organoprotective stomach stress response
As can be seen from the data reviewed (Sikiri6 et al, 1991a,b,c,d,e,f,g, 1992a,b) our studies were primarily focused on establishing the BPC protection in order to support the proposed hypothesis. Importantly, this protective action was also independently studied and confirmed by others (eg M6zsik et al, 1991; W Par6, A Durakovi6, K Gyires, unpublished symposium communications, Zagreb 1992). On the other hand, the mechanism(s) of such an intriguingly wide range of protection, particularly in terms of growing interest in brain-gut axis peptides (eg Hernandez, 1990), remains still a matter of strong discussion. A significant pool of BPC has been demonstated in the stomach (fig 2) as well as in the brain by our immunohistochemical studies (in preparation). A lesser increase of adrenal gland weight and reduction of thymolymphatic involution in connection with decreased gastric lesions development has also been confirmed (Par6, symposium communication). Thus, the involvement of BPC in Selye’s general adaptive syndrome seems to be very probable.
On the basis of organ ablation studies and in the generally suggested actions of many agents, some additional conclusions about the mode of action could be proposed (table V), although this has not been fully solved. It is obviously complex and probably mediated by, at least, the hormones of the adrenals, parathyroids, thyroid, and ovarian glands (Sikiri~ et al, 1991a, 1992a). The evidence that the same effect was not counteracted by many other procedures (among them orchidectomy) would suggest a specific and sex-related mechanism (Sikiri~ et al, 1992a). A control of ACTH secretion seems to be likely from the studies of the BPC effect on aniline induced adrenal gland lesions (Kovacs et al, 1970). Notably, the interactions with dopamine (nigrostriatum), PGs, somatostatin, serotonin, glycine and GABA systems (Waugh and Pearl, 1960; Robert, 1979; Szabo and Neumeyer, 1983; Hernandez, 1986; Usadel, 1988; Adams and Odunze, 1991; MOhler, 1992; Hughes and Woodruff, 1992) could be speculated upon (tables I, V, VI): Likewise, the protective action of BPC could be attributed, partially, to a free radical scavenging effect (Slater et al, 1985) (table IV). The reduction of mitochondrial respiration and the oxidative metabolism of l-methyl~ 4-phenyl-l,2,3,6-tetra-hydropyridine (MPTP) to l-methyl-4-phenyl pyrimidine, a positively charged species, were suggested as the critical features in the MPTP neurotoxic process (Heikkila et al, 1984; Adams and Odunze, 1991). As indicated before for CC14 liver lesions, a strong BPC effect has been noted in MPTP induced Parkinson’s disease in mice. That BPC 157 indeed penetrates the brain very effectively (an essential factor for antioxidant-treatment (Adams and Odunze, 1991)) was demonstrated in our pharmacokinetic studies (in preparation) (table VI).
Whether BPC could be an endogenous free radical scavenger as seems very likely, particularly in terms of the definition of Slater et al (1985), or not, remains to be further established. Another point would also be endothelium protection, suggested to be an essential substrate for organoprotection (Szabo et al, 1985). Consistently, a strong endothelium protection was demonstrated in our Monastral blue studies in ethanol gastric lesions in rats (in preparation) (see table I).
Nevertheless, it is generally accepted that true cytoprotective events are not limited to a particular organ morphology, but also occur within a variety of interacting and poorly understood biochemical events (Pfeiffer et al, 1989). Thus, the preservation of cell biochemical functions may finally be a common denominator for BPC beneficial activity.
The possibility that BPC, when applied, indeed mimics the organoprotective stomach stress response seems to be further supported by clear evidence for additional intriguing protective effects (eg in tissue repair, radioprotection, antiviral activity, bleeding time reduction, heparin bleeding reduction (in preparation)). In agreement with this, there seems to be no obvious toxicity (LD could not be achieved, despite application of dosages as high as 100 mg/kg bw) and some particular effects such as vitamin A induced malformation reduction, testis protection (germinative epithelium/spermatogenic cells) (noted also in high dosage testosterone toxicity (Je~.ek et al, 1993) studies) (in preparation). For instance, in favour of the suggested stress/organoprotection hypothesis and noted BPC anti-viral activity (in preparation), PGs as well as the dopamine agent amantadine demonstrated anti-viral activity (Parkes, 1974; Santoro et al, 1980). Particularly in terms of the possible role of gut hormones in cancer (Townsend et al, 1987), of some interest would also be the recently reported inhibitory effect on tumor growth in vivo and cyclophosphamide induced hematopoiesis disturbances in mice (Bura et al, 1993; Lang et al, 1993) (table VII). An additional interesting point would be a neuroprotection based on the evidence obtained in the widely used models of Parkinson’s disease (Adams and Odunze, 1991), depression (Willner, 1991), and stroke (Uemura et al, 1990) as well (in preparation).
A particular physiological role in the maintenance of homeostasis seems to be likely from the evidence that BPC has no effect on many variables (such as behaviour, diuresis, blood pressure, temperature, gastrointestinal motility and secretion) in normal circumstances (Frank, 1976) or in vitro conditions (Lefebvre, gastrointestinal motility studies (personal communication); coagulation factors in human blood), otherwise noted to be strongly influenced by BPC treatment if they are disturbed. Further support comes from the evidence that the synthesized 15 amino acid BPC peptide implicated to be of crucial significance for the organoprotective actions of the entire BPC (Sikiri6 et al, 1991a, 1992a), indeed shares its organoprotection as well (in preparation).