pentadecapeptide bpc 157 cream improves burn-wound healing and attenuates burn-gastric lesions in...

Burns 27 (2001) 817–827

Pentadecapeptide BPC 157 cream improves burn-wound healingand attenuates burn-gastric lesions in mice

D. Mikus a,b, P. Sikiric a,*, S. Seiwerth a,b, A. Petricevic a,b, G. Aralica a,b,N. Druzijancic a,b, R. Rucman a,b, M. Petek a,b, B. Pigac a,b, D. Perovic a,b,

M. Kolombo a,b, N. Kokic a,b, S. Mikus a,b, B. Duplancic a,b, I. Fattorini a,b,B. Turkovic a,b, I. Rotkvic a,b, S. Mise a,b, I. Prkacin a,b, P. Konjevoda b, N. Stambuk a,b,

T. Anic a,b

a Department of Pharmacology, Medical Faculty, Uni�ersity of Zagreb, Salata 11, P.O. Box 916, 10000 Zagreb, Croatiab Department of Pathology, Medical Faculty, Uni�ersity of Zagreb, Zagreb, Croatia

Accepted 23 May 2001

Abstract

The effects of the gastric pentadecapeptide BPC 157 were investigated when administered topically or systemically in burnedmice. This agent is known to have a beneficial effect in a variety of models of gastrointestinal lesions, as well as on wound orfracture healing. Deep partial skin thickness burns (1.5×1.5 cm) covering 20% of total body area, were induced under anesthesiaon the back of mice by controlled burning and gastric lesions were assessed 1, 2, 3, 7, 14 and 21 days following injury. The firstapplication of BPC 157 was immediately following burning, and thereafter, once daily, until 24 h before sacrifice. In the initialexperiments, exposure to direct flame for 5 s, the BPC 157 was applied at 10 �g or 10 ng/kg b.w. intraperitoneally (i.p.) byinjection or alternatively, topically, at the burn, as a thin layer of cream (50 �g of BPC 157 dissolved in 2 ml of distilled waterwas mixed with 50 g of commercial neutral cream (also used as local vehicle-control)), while silver sulfadiazine 1% cream was astandard agent acting locally. Others received no local medication: they were treated i.p. by injection of distilled water (distilledwater-control) or left without any medication (control). In subsequent experiments involving deeper burns (direct flame for 7 s),BPC 157 creams (50 �g, 5 �g, 500 ng, 50 ng or 5 ng of BPC 157 dissolved in 2 ml of distilled water was mixed with 50 g ofcommercial neutral cream), or vehicle as a thin layer of cream, were applied topically, at the burn. Compared with untreatedcontrols, in both experiments, in the BPC 157 cream-treated mice all parameters of burn healing were improved throughout theexperiment: less edema was observed and inflammatory cell numbers decreased. Less necrosis was seen with an increased numberof capillaries along with an advanced formation of dermal reticulin and collagen fibers. An increased number of preserved follicleswere observed. Two weeks after injury, BPC 157 cream-treated mice completely reversed the otherwise poor re-epithelization rationoted in the untreated control or mice treated with vehicle only. Tensiometry investigation showed an increased breaking strengthand relative elongation of burned skin, while water content in burned skin decreased. This was, however, not the case with thevehicle or silver sulfadiazine. Relative to the control values, in silver sulfadiazine cream-treated mice, only collagen fiber formationwas increased, in addition to a decreased inflammatory cell number. Relative to control values, BPC 157 given i.p. decreased thenumber of inflammatory cells, lowered water content in burned skin, and raised breaking strength and relative elongation ofburned skin during tensiometry. Through the experimental period, gastric lesions were continuously noted in all thermally injuredmice left without local medication and they were consistently attenuated only by BPC 157 treatments: either given i.p. (at eitherdose), or given locally (at either concentration). Other treatments (i.e. local treatment with silver sulfadiazine cream or neutralcream in mice subjected for 5 s to direct flame), led to only poor, if any attenuation. This stable gastric pentadecapeptide appearsto be active and gives a stimulation to burn healing at the defect site. The agent may act by causing an upregulation of the growthfactors, as well as influencing other local factors. © 2001 Elsevier Science Ltd and ISBI. All rights reserved.

Keywords: Pentadecapeptide BPC 157; Cream; Burn-wound healing; Burn-gastric lesions; Collagen/BMP-6; Mice

www.elsevier.com/locate/burns

* Corresponding author. Tel.: +385-1-4566-8334; fax: +385-1-492-0050.E-mail address: [email protected] (P. Sikiric).

0305-4179/01/$20.00 © 2001 Elsevier Science Ltd and ISBI. All rights reserved.PII: S0305-4179(01)00055-9

D. Mikus et al. / Burns 27 (2001) 817–827818

1. Introduction

Deep partial skin thickness burns may be used in areproducible animal model to study burn wound healingand attenuation of acute gastric mucosal lesions inresponse to antiulcer agent therapy [1–3]. An essentialfailure of healing is a common problem in both mucosalulcer and skin wound therapy (for review see Ref. [4]).This study aimed to investigate the effects on burn woundhealing and thermal injury-gastric lesions of the gastricpentadecapeptide BPC 157 (GEPPPGKPADDALVM.W. 1419 [4]). BPC 157 is known to have a beneficialeffect in a variety of gastrointestinal lesion models [5–9],including stress-ulcer [10,11], and a healing effect indifferent wound models [12–14]. BPC 157 also stimulateshealing of segmental osteoperiosteal bone defects whengiven locally either percutaneously into the bone defect,or applied intramuscularly [15]. BPC 157 solution wasshown to stimulate corneal wound healing [16].

Conditions of healing in burns are complex as a resultof vascular damage caused by burn. Dermal vascularchanges following thermal trauma result in fibrin depo-sition and vascular occlusion at the site directly destroyedby thermal energy and changes in dermal haemodynam-ics and blood vessel morphology in the areas of dermissurrounding the burn area [17]. In addition, a particularproblem for peptide application is to find a carrier thatdoes not limit their efficacy at the wound site (i.e. EGF,TGF-�) [18–20]. In previous experiments [5–16], BPC157 was effective without a special carrier. Its particularproperty of stability (i.e. not degraded in human gastricjuice for 24 h [21]) may be a relevant point. BPC 157 maybe suitably stable in a simple commercial vehicle (i.e.neutral cream) providing a suitable presence at the defectsite and able to influence the potential upregulation ofthe growth factors, as well as other local factors, actingto promote burn healing.

This study as a first step in burn wound therapy andburn injury-gastric lesion-studies also used topical appli-cation of BPC 157 at the site of the burn, administrationat a concentration 1 �g/1 g of vehicle, once daily.Pentadecapeptide BPC 157 as an intraperitoneal (i.p.)application was also used. Silver sulfadiazine cream 1%was used as a standard agent acting locally [22]. The nextstep was the analysis of the influence of this localmedication on thermal injury-induced gastric lesions andthe healing of the burn wounds itself if given dailytopically at the site of the stronger burn wounds also inlower concentrations.

2. Materials and methods

2.1. Drugs

BPC 157 (manufactured by Diagen, d.o.o., Ljubljana,

Slovenia) is a partial sequence of human gastric juiceprotein BPC, freely soluble in water at pH 7.0 and insaline. It was prepared as described before [5,6,11–16].Peptide with 99% (HPLC) purity (1-des-Gly peptide asimpurity), dissolved in saline [5,6,11–16] was used in allof the experiments. Silver sulfadiazine cream 1% (Der-mazin, Lek, Slovenia), and neutral commercial cream(Belobaza, Ljekarna Zagreb, Croatia) were commerciallypurchased.

2.2. Experimental protocol

2.2.1. AnimalsRandomly assigned male mice (NMRI-Hannover, 30

g b.w.), maintained individually in separate cages, withfree access to food and water, and no special fluidresuscitation after injury induction, were used in allexperiments, as approved by the local animal researchcommittee. In the investigations, 10 mice were used ineach of the experimental groups.

2.2.2. Lesion induction and assessmentDeep partial skin thickness burns were induced on the

back of animals by controlled burning (1.5×1.5 cm or20% of total body area), under ether anesthesia. Theanesthetized animals were exposed to direct flamethrough a 1.5×1.5 cm window in an asbestos network,for 5 or 7 s, using a modification of previously describedmethods [3–5,23,24]. This procedure resulted in nomortality. Burn healing parameter assessment includeshistological investigation, water content assessment inburn skin, and tensiometry studies (i.e. determination ofbreaking strength, and relative elongation of the burnedpart of skin) [20,25]. Gastric lesions (means�S.E.M.,mm) were assessed as described previously [5,11]. Theassessment was at 1, 2, 3, 7, 14 or 21 days followinginjury.

2.2.3. Histological assessmentThe parameters investigated included edema, blood

vessel formation, and their total diameter, number ofpreserved follicles, reticulin and collagen formation,number of inflammatory cells, and number of mice withcomplete re-epithelization [3,23,24]. Standard histologi-cal slices were stained using hematoxylin–eosin (bloodvessels, edema, necrosis, inflammatory cells), Gomorisilver stain (reticulin) and Van Gieson staining (collagen).The assessed parameters according to day of sacrificewere as follows: day 1: edema, number and diameter ofblood vessels, inflammatory cells and reticulin, numberof vital follicles; days 2 and 3: as for day 1 with necrosisas well; day 14: number and diameter of blood vessels,epithelization, reticulin and collagen; day 21: number anddiameter of blood vessels, epithelization and collagen.

D. Mikus et al. / Burns 27 (2001) 817–827 819

Specifically, edema has been determined by measuringthe distance from the muscular layer to the surface ofburned area (mag. 2× , �m). Angiogenesis has beendetermined by measuring the number and sum of di-ameters of blood vessels. Two microscopic fields (mag.6.3× ) in the central part of the burned area wereanalysed to determine this parameter. Inflammatorycells have been counted in three visual fields in themiddle portion of the burned area (mag. 40× ) betweenthe deepest layer of follicles and the muscular layer ofthe skin. Necrosis has been scored using a modifiedSuzuki scale (mag. 25× ) [26]: Grade 1: necrosis withinthe epidermal layer; Grade 2: necrosis up to the deepestlayer of hair follicles. Grade 3: necrosis exceeding thedeepest layer of hair follicles; Grade 4: necrosis exceed-ing the muscular layer. The number of preserved folli-cles has been counted in three microscopic fields (mag.25× ) in the middle of the burned area and examiningthe deepest layer of hair follicles. Development of ep-ithelization has been expressed as the number of ani-mals with complete epithelization versus incompleteepithelization ratio for each group, respectively. Reti-culin and collagen have been determined as percentagesin two microscopic fields (mag. 40× ) on each marginof the burned area between the deepest follicles and themuscular layer.

Mice subjected to the same procedure (i.e. carefulshaving of the back area 24 h previously to avoid skindamage, anaesthetized at the time of burning, etc.) butnot exposed to direct flame, were used as the sham burngroup. However, no pathology was produced in shamburned mice.

All microscopic measurements were carried out bythree independent blinded observers and no significantdifferences were noted between their individual assess-ments. For morphometrical analysis, a special programSFORM (VAMS-Software Company, Zagreb, Croatia)was used.

2.2.4. Tensiometry in�estigationMeasurement of tensile breaking force and relative

elongation of the burned part of the skin was carriedout in separate experiments, immediately after sacrifice,at days 7 and 14 as described [20,25] by a special device(Technical Faculty, University of Zagreb, Croatia) us-ing formulas: breaking force (N/mm2)= force (N)/cross-section skin burned area (mm2); relativeelongation of burned part of skin=�length burnedskin immediately before breaking/initial length ofburned skin.

VSB2.2.5. Assessment of water contentIn separate experiments, a 20×20 mm specimen of

full-thickness tissue from burned sites of the back and a

20×20 mm specimen from the upper back (non-dam-aged) were taken for weight determination after sac-rifice, at day 1, 2, or 3. The tissues biopsies were placedin a drying chamber (58 °C) until stable weight wasreached for approximately 48 h [27]. The wet/dry ratioof each specimen was calculated and compared tocontrol values.

2.2.6. Medication protocolGenerally, 10 mice per experimental group were used

and medication was given once daily (first applicationimmediately after burn-injury, last application 24 hbefore sacrifice).

Initial experiments involved injury induced by directflame for 5 s, with BPC 157 cream (1 �g/1 g of vehicle,specifically 50 �g of BPC 157 dissolved in 2 ml ofdistilled water mixed with 50 g of commercial neutralcream) given locally, on the burn as a thin layer ofcream. Likewise, BPC 157 (10 �g or 10 ng/kg) givenby i.p. injection was also used. Silver sulfadiazinecream 1% (Dermazin, Lek, Slovenia) was given asa standard agent acting locally. An equal amountof neutral commercial cream (Belobaza, Belupo, Croa-tia) was given locally on the burn (local vehicle-con-trol), an equal volume of saline was given for i.p.injection (5.0 ml/kg i.p.) or no medication (zero-con-trol) was used.

To increase the level of the injury, and to ascertainthe effect of BPC 157, given at various concentrationstopically, burn was induced by direct flame for 7 s. Asbefore, BPC 157 was applied locally, at the burn, as athin layer of cream (50 �g, 5 �g, 500 ng, 50 ng or 5 ngof BPC 157 dissolved in 2 ml of distilled water wasmixed with 50 g of commercial neutral cream). Controlanimals received no medication (zero-control), or anequal amount of neutral commercial cream (Belobaza,Belupo, Croatia) locally at the burn (local vehicle-control).

2.3. Statistical analysis

Fisher’s exact probability test two-tailed (presence/absence of re-epithelization in mice), non-parametricanalysis of variance (ANOVA) (Kruskall–Wallis one-way ANOVA by ranks) and post-hoc Wilcoxon rank-sum test (necrosis, edema, blood vessel formation, andtheir total diameter, number of preserved follicles, reti-culin and collagen formation, number of inflammatorycells, breaking tensile force, relative elongation ofburned part of skin, gastric lesions size (sum of longestlesions diameters) in mice) were used for statisticalanalysis, with a downward adjustment of the �-level tocompensate for multiple comparisons. The differenceswere considered to be significant at P�0.01 (5 s expo-


sure to direct flame) or P�0.008 (7 s exposure to directflame).

3. Results

3.1. Healing of thermal burn wounds

3.1.1. Microscope assessment of BPC 157 regimens andsil�er sulfadiazine cream in mice exposed to directflame for 5 s

A consistent damage was induced in controls whichreceived an equal volume of saline (5.0 ml/kg i.p.) ormice with no medication. Since these were not different,they are shown together (control). Mice treated withonly cream vehicle (Belobaza) showed no differencewith respect to controls, and the same poor re-epithe-lization ratio was observed in the untreated controls orin mice treated with only cream vehicle. Compared withthe controls, the BPC 157 cream-treated mice showedimprovement in all parameters of burn healingthroughout the experiment. Less edema was observedand inflammatory cell numbers were lower. Necrosiswas not so extensive and an increased number ofcapillaries along with an advanced formation of dermalreticulin and collagen fibers and an increased numberof preserved follicles were seen. Two weeks after injury,BPC 157 cream-mice completely reversed the otherwisepoor re-epithelization ratio noted in the untreated con-trol or mice treated with only cream vehicle (after a3-week period, the re-epithelization was also completedin controls (data not shown)). This was, however, notthe case with silver sulfadiazine cream-mice. Relative tothe control values, in silver sulfadiazine cream-mice,only collagen fiber formation was increased (7th, 14th,21st post-injury day), besides a decrease in inflamma-tory cell numbers at the third day. Compared with thecontrol, BPC 157 given i.p. decreases the number ofinflammatory cells seen at the third post-injury day, butother parameters were not affected (Tables 1 and 3).

3.1.2. Microscope assessment. Various concentrationsof BPC 157 cream in mice exposed to direct flame for7 s

A consistent and even more pronounced damage wasinduced in controls (Tables 2 and 4). Mice treated withvehicle only showed no difference with respect to con-trols, and the same poor re-epithelization ratio waspresent in the untreated control or mice treated withonly cream vehicle. Compared with the controls, theBPC 157 creams regularly improved wound healingparameters in mice. With respect to the control values,and the lower concentrations used (1 �g, 100 ng, 10 ng,1 ng or 100 pg/g vehicle), parameters of burn healing

were improved throughout the experiment. Less edemawas observed (1 �g–100 pg/g vehicle) and inflammatorycell numbers reduced (1 �g–100 ng/g vehicle). Lessextensive necrosis was seen (1 �g or 1 ng/g vehicle), anincreased number of capillaries (1 �g–100 pg/g vehicle),an advanced formation of dermal reticulin (1 �g or 1ng/g vehicle) and collagen fibers (1 �g or 1 ng/g vehicle)were observed, and an increased number of preservedfollicles (1 �g or 1 ng/g vehicle). At 2 weeks after injury,poor re-epithelization ratio otherwise noted in the un-treated control was completely reversed in mice treatedwith 1 �g and 100 ng BPC 157/g vehicle. Thus, it seemsthat some of the parameters of burn healing are sensi-tive to concentrations as low as 1 ng or 100 pg/gvehicle, while others need higher concentrations.

3.1.3. Measurement of water contentWater content of burned skin was assessed as de-

scribed before [26]. In the experiments using mice sub-jected to direct flame either for 5 or 7 s,cream-vehicle-treated mice showed no difference withrespect to controls, while a lower water content wasobserved in mice that received the BPC 157 (1 �g/g)cream, when they were assessed at the earliest post-in-jury interval (i.e. day 1). Interestingly, in mice subjectedto direct flame for 5 s, compared with the control, BPC157 given 10 �g/kg i.p. decreased water content at thefirst post-injury day. On the other hand, with respect tocontrols, silver sulfadiazine cream had no effect (Tables3 and 4).

Table 1Epithelization after injury induced by direct flame for 5 s

Number of miceBurned mice 10 mice/group; 2weeks after injury

Complete Incompleteepithelizationepithelization

Control 2 82 8Vehicle

Silver sulfadiazine cream 7 3

BPC 1579*1.0 �g/g vehicle cream 1*

10 �g/kg i.p. 4 68210 ng/kg i.p.

Burn induced by 5 s exposure to direct flame, once daily medication.Epithelization is expressed as the ratio between the number of micewith complete epithelization versus number of mice with incompleteepithelization for each group, 2 weeks after burn induction. Medica-tion (first application immediately after burn-injury, last application24 h before sacrifice) was given once daily. The effects of a variouspentadecapeptide BPC 157 regimens (topically applied in a cream, orgiven i.p.), silver sulfadiazine cream, or a vehicle (neutral cream(Belobaza)).

* P�0.01, at least versus control.


Table 2Epithelization after injury induced by direct flame for 7 s

Number of miceBurned mice 10 mice/group; 2weeks after injury

Complete Incompleteepithelization epithelization

0 10Control82Vehicle

BPC 1579*1.0 �g/g vehicle cream 1*

1*9*100 ng/g vehicle cream10 ng/g vehicle cream 3 71.0 ng/g vehicle cream 2 8

82100 pg/g vehicle cream

Burn induced by 7 s exposure to direct flame, once daily medication.Epithelization is expressed as the ratio between the number of micewith complete epithelization versus number of mice with incompleteepithelization for each group, 2 weeks after burn induction. Pentade-capeptide BPC 157 (50 �g, 5 �g, 500 ng, 50 ng or 5 ng of BPC 157dissolved in 2 ml of distilled water was mixed with 50 g of commercialneutral cream), or a vehicle (neutral cream (Belobaza)).

* P�0.008, at least versus control.

in controls (Fig. 1A).Relati�e elongation of the burned part of skin : Com-

pared with the control values at days 7 and 14, thehigher values were consistently noted in groups treatedwith BPC 157 i.p. (10 �g/kg or 10 ng/kg), as wasobserved in animals treated topically with 1 �g/g BPC157 cream. On the other hand, topical application ofthe vehicle (Belobaza) or silver sulfadiazine cream ledto values not different from control.

3.1.4.2. Mice subjected to direct flame for 7 s. Measure-ment of tensile breaking force : At days 7 and 14,assessment of tensile breaking force showed that topicalapplication of vehicle did not change the control values.On the other hand, a higher tensile breaking force thanin controls was observed in groups that received BPC157 creams 1 �g/g (days 7 and 14) or 100 ng/g (day 14)(Fig. 1B). The effectiveness of both 1 �g/g or 100 ng/gobserved at day 14 indicates an increased activity ofwound healing mechanisms to increase mechanicalstrength during the experiment. Of note, an essentiallypotentiated activity (i.e. directly, repeated topical appli-cations and/or triggering growth factors, as well asother local factors) may be associated with an addi-tional activity of the lower doses potentially postponed(i.e. seen at later period(s), if they had not been effec-tive earlier). Interestingly for longer-term therapeuticuse, this situation was also seen in some microscopicallyobserved parameters of burn healing (see also Table 4),as well as in the case of gastric lesion attenuation (seelater).

Relati�e elongation of the burned part of skin : Com-pared with the control values at days 7 and 14, highervalues were consistently noted in groups treated topi-cally with 1 �g/g or 100 ng/g pentadecapeptide BPC

3.1.4. Tensiometry in�estigation

3.1.4.1. Mice subjected to direct flame for 5 s. Measure-ment of tensile breaking force : In comparison withcontrol animals at days 7 and 14, tensile breaking forceshowed higher values in mice treated topically withBPC 157 cream (1 �g/g vehicle). Likewise, at day 14,higher values were noted in those which received i.p.pentadecapeptide BPC 157 application (10 �g or 10ng/kg). In animals treated topically with silver sulfadi-azine cream 1%, or vehicle, neutral cream, Belobaza,the observed values correspond with the values shown

Table 3Inflammation and healing parameters after injury induced by direct flame for 5 s

Therapy Days post-injury (5 s to overt flame exposure)

32 141 7 21

Bvn, Bvd, CE, W, Ic, Bvn, Ic, Bvn, Bvd, N,E, W, Ic, Bvn, Bvd, Bvn, Bvd, R, CControl tested E, W, Ic, Bvn, Bvd, N,Bvd, R, Fparameters N, R, F R, F R, F(–)Belobaza (–) (–) (–) (–) (–)

�C(–) (–) �IcSilver sulfadiazine �C �Ccream 1%

BPC 157�E, �W, �F, �Ic, �Bvn, �F, �R,�E, �Ic, �N, �Bvn, �Bvn, �C1 �g/g cream �Ic, �N, �Bvn, �F �Bvn, �F, �R, �C�Bvn �F �C�W (–) �Ic (–) (–) (–)10 �g/kg i.p.(–) (–) �Ic10 ng/kg i.p. (–) (–) (–)

Medication (first application immediately after burn-injury, last application 24 h before sacrifice) was given once daily. The effects of various BPC157 regimens (topically applied in a cream, or given i.p.), silver sulfadiazine cream, or a vehicle (neutral cream (Belobaza)). The parameterscompared with the control, assessed as described in the text, are: E, edema; W, water content; Bvn, blood vessel number; Bvd, blood vesseldiameter; Ic, inflammatory cells; R, reticulin; C, collagen; N, necrosis; F, number of vital follicles; P�0.008 versus control: �, decrease; �, increase;(–), no influence.


Fig. 1. Tensiometry assessment. Breaking tensile force (N/mm2) (force (N)/cross-section skin burned area (mm2) (left). Relative elongation ofburned part of skin (�length of burned skin immediately before breaking/initial length of burned skin) (right). Means�S.E.M. Medication (firstapplication immediately after burn-injury, last application 24 h before sacrifice) was given once daily. (A) Injury induced by direct flame for 5 s,at day 0, days following injury. The effects of various BPC 157 regimens (topically applied in a cream, or given i.p.), silver sulfadiazine cream,or a vehicle (neutral cream (Belobaza)). P�0.01, at least versus control. (B) Injury induced by direct flame for 7 s, once daily medication.Pentadecapeptide BPC 157 (50 �g, 5 �g, 500 ng, 50 ng or 5 ng of BPC 157 dissolved in 2 ml of distilled water was mixed with 50 g of commercialneutral cream), or a vehicle (neutral cream (Belobaza)). P�0.008, at least versus control.


Table 4Inflammation and healing parameters after injury induced by direct flame for 7 s

Days post-injury (7 s to overt flame exposure)Therapy

1 2 3 7 14

E, W, Ic, Bvn, Bvn, Bvd, R, C, FE, W, Ic, Bvn, Bvd, N, Ic, Bvn, Bvd, N, R, C, FControl tested E, W, Ic, Bvn, Bvd, N,parameters Bvd, R, F R, FR, F

(–)Belobaza (–) (–) (–) (–)

BPC 157�E, �W, �F, �Ic, �Bvn, �Bvd, �Ic, �N, �F,�E, �Bvn, �Ic, �N, �R, �E, �Bvn, �Ic, �N, �R,1 �g/g cream �Bvn, �Bvd, �F,

�R, �C�F�F �R, �C�R�E, �Bvn, �Ic, �N, �R, �E, �Bvn, �Ic, �N, �R, �Bvn, �Bvd, �F,�E, �F, �Ic, �R �Bvn, �Bvd, �Ic, �N, �F,100 ng/g cream

�F�F �R, �C�R, �C�E, �Bvn, �N, �F �E, �Bvn, �N, �R, �F �Bvn, �Bvd �N, �F, �R, �Bvn, �Bvd, �F,�E, �F10 ng/g cream

�C �R, �C�E, �Bvn, �N, �F �E, �Bvn, �N, �R, �F1 ng/g cream �Bvn, �Bvd, �R, �C�E, �F �Bvn, �Bvd, �F,

�R, �C100 pg/g cream �E�E �E �Bvd �Bvn, �Bvd

Medication (first application immediately after burn-injury, last application 24 h before sacrifice) was given once daily. The effects of various BPC157 regimens (topically applied in a cream, or given i.p.), silver sulfadiazine cream, or a vehicle (neutral cream (Belobaza)). The parameterscompared with the control, assessed as described in the text, are: E, edema; W, water content; Bvn, blood vessel number; Bvd, blood vesseldiameter; Ic, inflammatory cells; R, reticulin; C, collagen; N, necrosis; F, number of vital follicles; P�0.008 versus control: �, decrease; �, increase;(–), no influence.

157 creams. On the other hand, topical application ofthe vehicle led to values not significantly differentfrom control values.

Thus, along with the already noted differencesin histology/tensiometry studies [20,25,28], thesedata show that mechanical factors, at least partly,involved in the burn-healing, may also be positivelyinfluenced by BPC 157 application, either topical orsystemic.

3.2. Gastric lesions in burned mice

3.2.1. BPC 157 regimens (topically applied in a cream,or gi�en i.p.) and sil�er sulfadiazine cream in miceexposed to o�ert flame for 5 s

Deep partial skin thickness burns consistently in-duced reproducible acute gastric mucosal lesions[15,28]. Accordingly, in the experiments using 5-s ex-posure to flame, gastric lesions are present through-out the entire experimental period in all thermallyinjured mice left without local medication. Interest-ingly, when compared to controls, BPC 157 givenonce daily i.p., attenuates the lesion at both early (i.e.at post-burn day 3) and later stages (i.e. at post-burndays 14 and 21). Likewise, this antiulcer effect is evenmore prominent with BPC 157 cream 1 �g/g vehicle:it is evident from post-burn day 3 to the end of theexperiment. Silver sulfadiazine cream decreased stom-ach damage at two points, at post-burn days 7 or 21,but not before, whereas the vehicle treatment tempo-

rarily attenuated gastric lesions at post-burn day 3(Fig. 2A).

3.2.2. Various concentrations of BPC 157 cream inmice exposed to direct flame for 7 s

With the deep partial skin thickness burns-model[15,29] a longer exposure to direct flame (i.e. 7 s)produced even larger thermal-injury-related gastric le-sions. As may be expected, in comparison with thecontrol, vehicle treatment was completely ineffectiveagainst this exaggerated gastric lesion (Fig. 2B). Onthe other hand, a prominent antiulcer effect wasdemonstrated following topical application of BPC157 creams. Interestingly, against the background ofthe inevitable larger gastric lesion seen regularly inthe controls, all concentrations of BPC 157 creamscounteract gastric lesion development. Shown throughthe whole experimental period, this antiulcer effect isconsistent with the attenuation of stress and othergastrointestinal lesion, noted in the previous BPC 157studies using systemic application [5,10,11].

4. Discussion

The benefit of creams containing BPC 157 on burnwound and burn-induced stress gastric ulcers may beconsidered from both local (i.e. healing of the burn-wound) [3,23,24] and systemic (i.e. interruption of thenegative chain of events that otherwise inevitably leadto stress stomach lesions) [1,2] points of view. The


gastric pentadecapeptide BPC 157 appears to have aspecial relationship to bone morphogenetic protein-6precursor (BMP-6), a molecule known to be involvedin keratinocyte differentiation and wound healing[29,30]. This molecule is also found in the intestine[31] and has been consistently implicated in recentmolecular investigation of BPC-157 patterns [32]. Inits structure it shows analogy to several regions ofbone morphogenetic protein-6 precursor (BMP-6),and numerous fragments of BMP-6 precursormolecule are contained within the consensus sequenceof BPC 157 molecule. In addition to the molecularsimilarity to BMP-6, the fragments of BPC 157 alsocorrespond to various collagen fragments, importantin the wound healing [33]. Consequently, the similar-ity to collagen fragments may explain the fact thatBPC 157 has strong osteogenic effects [15,32], butdoes not require the addition of collagen matrix forits effects [15,32] unlike other peptides [20,29,30].

Re-epithelization, a desired final result, likely ap-pears to be enhanced due to advanced reticulin andcollagen formation, less edema and necrosis, im-proved blood vessel formation and more preservedfollicles noted in BPC 157 cream-treated mice. Thisfinding is also supported in the experiments usingvarious concentrations of BPC 157 creams, and sub-stantiated by the demonstration of increased tensilebreaking force, or increased relative elongation of theburned part of skin during tensiometry. Dermal vas-cular changes following thermal trauma include fibrindeposition and vascular occlusion at the site directlydestroyed by thermal energy and changes in dermalhaemodynamics and blood vessel morphology in theareas of dermis surrounding the burn area [17]. Sinceit is effective when given topically, BPC 157 may bepresent directly at the site of burn injury in suffi-ciently high concentration permitting an effectivestimulation of all parameters of burn healing, andpossibly preventing vascular occlusion. To supportthis, an increase in the number of capillaries (1st to14th post-injury day) was noted in the BPC 157cream-treated mice, along with its effect on angiogen-esis [14] and NO-system modulation [34]. Conse-quently, besides a local attenuation of the burninjuries, it may promptly accelerate healing of gastriclesions following local absorption. This shows, com-pared with control values, different patterns of heal-ing in BPC 157 and silver sulfadiazine creamregimens. A marked attenuation of burn-producedgastric lesions was seen along with accelerated burnwound healing involving all parameters when BPC157 creams were used for topical treatment. Alterna-tively, with the silver sulfadiazine cream, only a fewparameters of burn healing were improved (i.e. colla-

gen) in parallel with a relatively weak and delayedattenuation of thermal injury-induced gastric lesions.As observed in other wound healing models [12,13,15]systemic application of BPC 157 in burn healingmodels increased breaking strength and relative elon-gation of the burned part of skin during tensiometry,and reduced water content in burned skin.

Supporting both attenuation of burn-induced gas-tric lesions and promotion of burn-healing with verylow concentrations of the BPC 157 creams, all thebeneficial effects of the BPC 157 had been previouslydescribed in different experimental models [12–16].These included an angiogenesis model (with syntheticsponge implantation [14]), accelerated collagen, reti-culin and blood vessel formation, increased tensilebreaking force (skin incision wounds [12]), raisedbursting pressure (colon–colon anastomoses [13]), andan accelerated healing in special and complex condi-tions (healing of chronic segmental osteoperiostealbone [15]). Burn injury induces a pronounced inflam-matory reaction, involving various vasoactive media-tors such as histamine [35], serotonin [36],leukotrienes [37] and prostaglandins [38] with the re-sulting effects on vascular permeability and edema[39]. In relation to the events in burn patients [40],BPC 157 strongly inhibited the effect of calciumionophore A23187 [9,41,42], known to induce his-tamine release from mast cells and tissues (for review,see Ref. [43]), and it reduced the A23187 promoted-release of leukotriene and other inflammatory media-tors (i.e. MPO, TXB2) as noted in inflamed animaltissues and human blood [9,41,42]. Thus, along withthe verified effects of BPC 157, a parallel inhibitionof their damaging overactivity in burned tissue is verylikely.

Finally, taking the present findings together withprevious reports [4–16,21,34], this suitably stable gas-tric pentadecapeptide can achieve an adequate concen-tration to give a significant stimulus to burn woundhealing at the defect site. Due to its structural charac-teristics, it may potentially provide an upregulation ofgrowth factors, as well as other local factors. Thistriggering is not unexpected since the pentadecapeptideis stable and not degraded in human gastric juice evenfor 24 h, unlike other peptides, i.e. human-EGF, hu-man-TGF [21]. The observed effectiveness of this pen-tadecapeptide applied without a special carrier,dissolved in distilled water, in a simple commercialvehicle, is a major advantage over its relationship withthe added carrier [18,19]. For instance, besides thepreviously mentioned limitations for BMP-6 and TGF-� [20,29,30]), only topical application of EGF ointmentcontaining a protease inhibitor has ameliorative sys-temic effects [19]. Possibly, the lack of otherwise presentserum proline peptidases required for the systemic


Fig. 2. Gastric lesion assessment. Severity of gastric lesions was described by the sum of longest lesion diameters (means�S.E.M., mm).Medication (first application immediately after burn-injury, last application 24 h before sacrifice) was given once daily. (A) Injury induced bydirect flame for 5 s, at day 0, days following injury. The effects of various pentadecapeptide BPC 157 regimens (topically applied in a cream, orgiven i.p.), silver sulfadiazine cream, or a vehicle (neutral cream (Belobaza)). P�0.01, at least versus control. (B) Injury induced by direct flamefor 7 s, once daily medication. Pentadecapeptide BPC 157 (50 �g, 5 �g, 500 ng, 50 ng or 5 ng of BPC 157 dissolved in 2 ml of distilled water wasmixed with 50 g of commercial neutral cream), or a vehicle (neutral cream (Belobaza)). P�0.008, at least versus control.

cleaving of proline containing peptide bonds [44] mayfurther promote this local BPC 157 stability and itsprolonged therapeutic potential after topicalapplication.

5. Conclusion

In summary, when compared with the control val-ues, the activity of the pentadecapeptide BPC 157 oninflammatory cells, edema, reticulin, collagen, necro-sis, blood vessel formation, number of preserved folli-cles, re-epithelization and stress-gastric lesion atten-

uation, tensile breaking strength, and water content inburned skin, suggests that this substance may act togive a more accelerated healing in burns of treatedmice. In addition, BPC 157 appears to attenuatestress-induced gastric lesions. This stable gastric pen-tadecapeptide may reach an adequate concentration togive a significant stimulation to burn healing at thedefect site, without special carrier. The mechanisms bywhich it acts are unclear but may be linked with itsparticular structural characteristics and may involve itin exerting an upregulation of growth factor synthesis,as well as influences on other local inflammatory fac-tors.


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