BPC 157 Discovery and development by DIAGEN

1.0. INTRODUCTION

2.0. GASTRIC JUICE - BPC HYPOTHESIS

Pentadecapeptide BPC 157 (abbr. for: Body Protecting Compound) has the following peptide sequence: Gly Glu Pro Pro Pro Gly Lys Pro Ala Asp Asp Ala Gly Leu Val and represents the N-terminal part of a natural BPC protein, which is actually present in gastric juice of mammals. The natural BPC protein is obtained from human or animal gastric juice by means of complicated biochemical methods (U.S. Patent No. 5,288,708). Irrespective of demanding work this way of obtaining is inappropriate also with regard to an uncertain source of raw material and a possible contamination with viruses. Therefore, by chemical synthesis (P. Sikirić, R. Ručman, M. Petek, Peptides 1998, Proceed. of 25th EPS, Budapest 1998, p. 814; Z. Pflaum and R. Ručman, Acta Chim.Slov., 2005, 52, 34–39) only the N-terminal part with the above stated sequence has been prepared. Surprisingly, it has been found that this fragment retained practically all effects of the natural BPC protein (EP 0572688 and U.S. Patent No. 6,268,346). The N-terminal part with a sequence of 15 amino acids seems to be the most responsible for the biological activity of the entire protein. The compound is very interesting from a medical point of view, as it has effect to almost all organs in extremely low concentrations (in a range from ng to mg and more per a kilogram of body weight) and without any toxic or side effects.

Pharmacological studies have shown that pentadecapeptide BPC-157 (abbr. also peptide BPC-157) introduced in an organism have the following effects:

on ulcers in any part of gastrointestinal tract (P. Sikirić et al., Life Sci., 1994; 54, PL63–68); (P. Sikirić et al., Exp. Clin. Gastroenterol., 1991, 1, 17–20);
anti-inflammatory irrespective of etiology (P. Sikirić et al., J. Physiol. /Paris/, 1997, 91, 113–122);
against an inflammatory disease of the gastrointestinal tract – Crohn’s disease (Sikirić et al., J. Physiol. /Paris/, 2001, 95, 295–301);
protective to the liver and pancreas (Prkačin et al., J. Physiol. /Paris/, 2001, 95, 315–324);
organ protective activity: protection of hepatic and pancreatic lesions, protection of endothelial cells, prevention of adhesion formation, prevention and treatment of myocardial infarction and brain stroke, protective effect in immune system; US 9,850,282
promotes healing of burns (D. Mikuš, P. Sikirić et al., Burns 2003, 29, 323–334; Burns, 2001, 127, 817–827);
promotes healing of wounds (S. Seiwerth, P. Sikirić et al., J. Physiol. /Paris/, 1997, 91, 173–178);
protective against radioactive radiation (P. Sikirić, M. Petek, R. Ručman, J. Physiol. /Paris/, 1993, 87, 313–327);
promotes healing of bone fractures (B. Sebečić, V. Miklič, P. Sikirić et al., Bone, 1999, 24, 195–202);
in interaction with adrenergic and dopaminergic systems protects mucusas in stress conditions (P. Sikirić et al., Dig. Dis. Sci., 1997, 42, 661–671);
antitumor effect in some types of tumor (ascites, melanoma) (P. Sikirić, M. Petek, R. Ručman, J. Physiol. /Paris/, 1993, 87, 313–327; S. Radeljak, S. Seiwert, P. Sikirić, Melanoma Research, 2004, 14 (4), A14–A15);
antiviral effect on herpes viruses (also zoster), poliovirus, adenovirus, Influenza virus (A and B, including the H1N1 type) and tick-borne encephalitis virus (P. Sikirić, R. Ručman, M. Petek, J. Physiol. /Paris/, 1993, 87, 313–327);
for treatment of viral infections, particularly with hepatitis A virus, ARBO viruses, West Nile, dengue types 1-4, cytomegalovirus CMV and LCM virus, feline leukemia virus; US 9,850,282;
promotes regeneration of ruptured nervous linkages (P. Sikirić et al., Dig. Dis. Sci., 41, 1604–1614; M. Gjurašin et al., Dig. Dis. Sci., 2003, 48, 1879);
treatment of neurological diseases and disorders: multiple sclerosis, myasthenia gravis, lupus erythematosus, neuropathy, dysfunction of somatosensory nerves, asthma, rhinitis, pemphigus and eczema; US 9,850,282;

promotes healing of ruptured Achilles’ tendon (M. Starešinić, P. Sikirić et al., J. Orthoped. Res., 2003, 21, 976–983);
treatment of organic disorders associated with NO formation: hypertension, hypotension, anaphylaxis, circulatory and septic shock, aggregation of thrombocytes; US 9,850,282;
As original therapy in all conditions, in which rapid reorganization of blood circulation is mandatory; US 9,850,282;
Catecholaminergic dysfunction, schizophrenia, amphetamine, drug and alcohol withdrawal effects; US 9,850,282;
Prevention and elimination of disorders due to corticosteroids and NSAIDs; US 9,850,282;
Treatment of squamous degeneration of macula of the eye; US 9,850,282;
effects the immune system and disorders in cellular immune response, including AIDS and AIDS related conditions (P. Sikirić, M. Petek, R. Ručman, J. Physiol. /Paris/, 1992, 87, 313–327).

Peptide BPC-157 as a constituent part of a living organism’s own substance does not show any toxicity signs. Tests carried out on mice in order to determine LD50 as well as with the purpose of obtaining data about acute, sub chronic and chronic toxicity have been unsuccessful as this peptide applied intravenously or intraperitoneally in a broad dose range from 10 ng to 100 mg/kg of body weight, did not induce any toxic changes.

In addition, the tests of teratogenicity and genotoxicity (Salmonella microsome test) proved no signs of such action.

3.0. ISOLATION OF ENDOGENEOUS GASTRIC PROTEIN BPC

Human gastric juice is a secretion of parietal and other gland cells of gastric mucosa and contains electrolytes such as sodium chloride and hydrochloric acid, enzymes such as pepsin and other proteinases, renin, lipase, urease,lysozymes, glycoprotein-mucin, proteins and protein fragments and generally digestive principles, partially digested food residues, all this dissolved in water. Enzyme pepsin, in the presence of hydrochloric acid acts on proteins and changes them into peptones and lower peptide fragments. Pepsin alone has no digestive power and is active only in the presence of hydrochloric acid. The acidity of gastric juice can vary from pH 1 to pH 7 or even more.

There is a question, why stomach does not digest itself? The cells in the stomach wall

secreted an other enzyme, known as antipepsin, which should prevent auto digestion.

According to the last investigation is presumed that gastric juice should contain one other high-molecular compound with protein structure having organo-protective activity

which could contribute to the stomach integrity and be also of benefit for many other organs after its distribution through organism.

This compound was until now not known and not investigated. Therefore was a big challenge for researcher to isolate and investigate its structure and pharmacological activity. Due to the presumed and later found activity¹ was named Body Protecting Compound, abbrev. BPC.

First attempts in this direction have been made by pioneer work of Marijan Petek with preparation of crude, partially purified concentrate from human gastric juice²and finalized by R. Ručman in 1998 with isolation of pure compound (2 mg). The activity was assessed on animals using restraint stress model^3,4and cysteamine model for

induction of lesions⁵. The relative activity was estimated in steps from 0 (inactive) to +5 (highest activity). The beneficial effect of this concentrate has been found on gastric and duodenal lesions, induced by restraint stress or cysteamine model, respectively.

Generally this concentrate possessed very strong anti-inflammatory, anti-stress and anti-ulcerative activity and therefore was good basis for further investigation.

The composition of human gastric juice concentrate was preliminary screened with liquid chromatography, preferable for localization of active sites on chromatogram to obtain indications for further discovery.

Fig. 1.Liquid chromatogram of gastric juice concentrate; detection: UV, λ = 280 nm.

Table 1. Assessment of fragments (agents) from Fig.1., using cysteamine and restraint stress models. Based on the obtained findings the maximum activity was coined with the 248A (identified in the molecular weight region 60 kDa to 70 kDa) and 248E (identified in the region 40kDa). These substances indicated guidelines for further research.

The main problem during investigation of gastric juice components was very complex mixture of compounds present and at the same time in very low concentration of searched substance. Gastric juice was a side (waste) product of patients, who had a gastric probe inserted for reasons of their treatment. No gastric juice was taken from healthy persons.

3.1. Principle of isolation procedure.

Fractions (10 ng/kg) or saline (5 ml/kg) i.g. 1 h before restraint or i.p. 1 h before cysteamine	Cysteamine test 400 mg/kg s.c. means (mm)	Restraint stress for 48 hours means	Estimation of activity (0 to +5)
control- saline	5	15.8	0
S 248A	0.25	1.0	+5
S 248Ay	0.75	7.9	+3/+4
S 248Ao	2.25	1.9	+4
S 248B	3.3	4.1	+1
S 248C	1.5	6.1	+4
S 248D	1.9	4.3	+4
S 248E	0.25	0.9	+5

Step 1: Preparing of gastric juice concentrate.²

The collected gastric juice was first homogenized and centrifuged by 12.000 rpm for elimination of crude particles. The clear supernatant was dialyzed using Spectrapore dialyzing tube (M.W. cut-off: 1000) against tape water over night, following against pure water at 10 – 15 ⁰C for 20 hours. The residual liquid from tubes was cleared by filtration and lyophilized to obtain crude gastric juice concentrate.

Crude protein mixture was than purified through 5 consecutive chromatography steps with different type of chromatography. During all next steps purification steps the fractions were tested by restraint stress and cysteamine methods on experimental animals (Wistar rats). Always only fractions with the highest activity (+5) were selected for the next purification step.

Step 2: 1st Chromatography step – Capture

Fig. 2. Injected solution of Crude gastric juice and purified on Capture step of chromatography -1^st chromatography step, detection: UV λ = 280 nm.

Step 3: 2^nd Chromatography step

Fig.3. Injected solution of Fraction A from 1st chromatography step and purified on 2^nd chromatography step, detection: UV λ = 280 nm.

Step 4: 3^rd Chromatography step

Fig.4. Injected solution of Fraction B from 2^nd chromatography step and purified on 3^rd chromatography step, detection: UV λ = 280 nm.

Step 5: 4^th Chromatography step

Fig.5. Injected solution of Fraction A from 3^rd chromatography step and purified on 4^th chromatograpy step, detection: UV λ = 280 nm.

Step 6: 5^th Chromatography step – final purification

Fig.6. Injected solution of Fraction A from 4^th chromatography step and purified on 5^th chromatography step, detection: UV λ = 254 nm.

During isolation and purification procedure was observed the appearance of more active fractions, the highest in the region at about 250 kDa (S-290), then at about 70 kDa, 40 kDa and even at 25 kDa. Most probably all these fractions arise from 250 kDa protein with enzymatis or hydrolytic cleavage. The compound with molecular weight of 40 kDa was isolated according to the described procedure and considered as BPC.

The compound with highest molecular weight is very sensitive and all attempts for isolation and purification in their native form failed. It was presumed that this compound easily turns into that with M.W. 40 kDa.

In the first overview the gastric juice-derived protein BPC with molecular weight of about 40 kDa was described and beside of mucosal protective properties has very broad range of biological activities like: ulcers protective, hepatoprotective, antiviral, antiedematous, general antiinflammation activity, antimalignant activity and others

BPC 157 sequence determination

Protein BPC was investigated, analyzed and sequenced to determine amino acid sequence.

The Following N-terminal sequence has been found: GEPPPGKPADDAGLV—.

Table 2. Amino acid analysis:

Amino acid	pmoles	mole %
Alanine	1338.1	9.74
Aspartic acid	613.4	4.55
Glutamic acid	796.6	5.18
Glycine	587.5	4.30
Arginine	300.3	2.22
Histidine	186.5	1.30
Isoleucine	316.0	2.31
Leucine	379.5	2.81
Lysine	210.5	1.55
Methionine	77.6	0.56
Phenylalanine	168.4	1.25
Proline	1976.1	14.50
Serine	2816.0	20.62
Threonine	3377.1	24.72
Tyrosine	63.6	0.45
Valine	514.0	3.80

Fig. 7. IR spectrum (KBr) of BPC157

After repeated isolation and purification this sequence was confirmed, but in any case the sequence did not progress further than after 15 amino acid residues.

Isoelectric focusing indicates isoelectric point 6.7.

A protein BLAST search of this sequence performed against the human data base only matched some similar results, but far away from 100 % identity. Proteins with similar partial sequence motifs – PPPGKPQ- and – PPPGKP- are present in proline rich (PRP)

salivary protein group. The characteristically motif -PPPGKP- present in high frequency

and periodically repetitive has been found in saliva by many investigators^9,10,11.

The continuation of pharmacological investigation with purified substance BPC showed surprising effects of this compound. Above all, this native BPC is completely without any toxicity.

But, in spite of their numerous favorable effects – is unfortunately not suitable for the practical preparation of medicine for several reasons:

from gastric juice is not possible produce the required amount the compound BPC with constant quality;
gastric juice as starting substance is absolutely unsuitable due to expensive and complex isolation process and above all, it is ethically questionable;
there exists a great risk that the gastric juice is possible source of infection/biohazard.

Therefore a good idea rises itself that it would be much more reasonable to synthesize the peptide fragment, which was determined by sequencing as N-terminal pentadecapeptide

having amino acid sequence: GEPPPGKPADDAGLV, following with pharmacological assessment and comparison of activity. In any case, synthetic peptide, having the same activity as parent compound BPC is more suitable and acceptable for medicine development.

Although the investigation of BPC full structure is continuated, hitherto obtained results are very valuable and guidance for development of new medicine.

4. 0. SYNTHESIS OF PENTADECAPEPTIDE BPC 157.

Pentadecapeptide BPC 157 was first time synthesized by R. Ručman (Diagen) in 1993 using a solid phase synthesis¹², a method which is based on the classic Merrifield synthesis¹³, where peptide is synthesized stepwise on a modified solid polystyrene support (resin). The resin is functionalized with an acid labile linker that allows for acidic cleavage usually trifluoroacetic acid, hydrofluoric acid (HF) or trifluoromethanesulfonic acid.

In the contrary, for the synthesis of protected, partially protected or very sensitive peptides the HYCRAM^™polymeric resin is more suitable^14.15 This method has the environmental

advantage that there is no longer any need for strong acid as HF or trifluoroacetic acid to release any peptide acid, even fully protected from polymeric support. The release of the peptide from polymeric resin occurs with Pd₀(triphenylphosphine)₄ noble metal catalyst under neutral conditions in the presence of an allyl receptor as dimedone or morpholine and under strict esclusion of oxygen.

4. 1. Pentadecapeptide BPC 157

Chemical name:

Glycyl-L-glutamyl-L-prolyl-L-prolyl-L-prolyl-glycyl-L-lysyl-L-prolyl-L-alanyl-L-aspartyl-L-asparty-L-alanyl-glycyl-l-leucyl-L-valine.

Recommended name: Bepecin

Code names: BPC 157

Chemical Abstract Services Reg. No,: 137525-51-0 (free peptide)

The structural formula is GEPPPGKPADDAGLV or with three letter codes:

H₂N Gly Glu Pro Pro Pro Gly Lys Pro Ala Asp Asp Ala Gly Leu Val OH

Fig. 8. Structural formula of pentadecapeptide BPC 157

Molecular Formula

The Molecular formula of free peptide is C₆₂H₉₆N₁₆O₂₂.

Molecular Mass

The Molecular weight of is 1419,5 Da as free peptide

Specifical opt- rotation: [α]²⁰_D = -155⁰ (c = 1, water)

IR spectrum (KBr): 3450, 3300, 2950, 2880, 1650, 1550, 1460, 1400, 1340, 1250,

1180, 1070 cm^-1

¹H NMR spectrum (20% D₂O/H₂O: δ = 0.9 (14 H,m), 1.4 (10H,t,J,1,7 Hz), 1,6 (8H,m),

2 (19H,m), 2,36 (7H,m), 2,7 (5H,m), 3 (2H,t,J 7,1 Hz), 3.9 (15H,m),

4,15 (2H,m), 4,25 (2H,m), 4,4 (2H,m), 4,6 (2H,m), 7,64 (1H,d J 8,6

Hz), 8 (1H,d,J 7,2Hz), 8,15 (1H,d,J 6,8Hz), 8,25 (2H,t,J 5,2 Hz),

8,33 (1H,d,J 7,2 Hz), 8,4 (2H,m), 8,45 (1H,d,J 5,1 Hz).

Isoelectrical point: 4.03.

4.6.1. Pentadecapeptide BPC 157 acetate

Generally, usual salt forms of many peptides are acetate, mostly on account of suitable solubility of salts in water. Pentadecapeptide acetate is common commercial form of this compound. Usually contains less than 1 % of acetate, what is about 0.25 mol to one mole of

pentadecapeptide. This low concentration of acetate is attributed to the intermolecular neutralization of the N-terminal amino group and the ε-amino group of lysine by carboxylate groups on the C-terminal or on the side chain of the aspartate and glutamate residues.

Molecular formula of acetate: C₆₂H₉₈N₁₆O₂₇ x 0.25 CH₃COOH

Specifical opt. rotation: [α]²⁰_D= -172,6 ⁰ (DI, H₂O, c = 5.2)

Mass analysis: (M+H)⁺ = 1420; (M+2H)²⁺/2 = 710.9

Fig. 9.IR (KBr) spectrum of pentadecapeptide BPC 157 acetate

Fig. 10. ¹H NMR spectrum of pentadecapeptide BPC 157 acetate

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