RPEP-00031 · 1985The patient had primary intestinal lymphangiectasia (a condition where lymph vessels in the gut leak fluid, protein, and immune cells). Biopsies confirmed the diagnosis in her intestines, lungs, and lymph nodes. Lymphocytes were literally leaking out through her gut, confirmed by finding them in her stool.
Her thymus gland was essentially absent. A biopsy showed no thymic tissue. Blood tests showed very low or absent thymulin and thymosin alpha 1. She had a selective loss of T-cells, especially CD4+ helper T-cells.
Daily thymosin fraction 5 treatment produced a brief clinical improvement. But switching to weekly injections could not sustain it. Lymphocyte numbers never increased during treatment. The authors concluded that when the body loses T-cells faster than it can replace them, even thymic hormones cannot keep up without a functioning thymus.
Sorensen, R U; Halpin, T C; Abramowsky, C R; Hornick, D L; Miller, K M; Naylor, P; Incefy, G S · Case Report
RPEP-00059 · 1987Peripheral mu-opioid receptors in the gut can independently slow gastrointestinal transit. The peptide agonist PL017 slowed gut transit at 0.37 mg/kg but did not produce pain relief until 30 mg/kg, about 80 times higher. This proves gut mu receptors work independently of brain receptors.
Delta and kappa opioid receptors in the gut also slowed transit, but less powerfully than mu receptors.
The key finding: a peptide opioid antagonist that cannot cross the blood-brain barrier blocked morphine's gut-slowing effect without reducing its painkilling activity. This pharmacological separation means constipation and pain relief work through different receptor populations (gut vs brain).
Shook, J E; Pelton, J T; Hruby, V J; Burks, T F · Animal Study
RPEP-00066 · 1988Enterochromaffin (EC) cells are the most abundant hormone-producing cells in the gut. They were known to make serotonin. Whether they also make peptides was debated for years.
Using antibodies against all three families of opioid peptide precursors, researchers tested gut tissue from dogs, guinea pigs, and humans. EC cells contained pro-dynorphin-derived peptides: dynorphin A and alpha-neo-endorphin.
They did not contain pro-opiomelanocortin derivatives (like endorphin) or pro-enkephalin derivatives. Previous reports of enkephalin in EC cells were likely due to antibody cross-reactivity.
The number and staining characteristics of opioid-positive EC cells varied considerably between species and between different segments of the gut, suggesting species-specific processing of the dynorphin precursor.
Cetin, Y · In Vitro
RPEP-00067 · 1988The myenteric plexus (the nerve network that controls gut movement) of guinea pig small intestine contained a well-defined mix of opioid peptides.
Met-enkephalin dominated at 405 pmol/g of tissue. Three other enkephalin-related peptides were present at 90 to 100 pmol/g each. Two rarer enkephalin forms (BAM-18 and Met-enkephalyl-Arg-Arg-Val-NH2) were present at 24 and 5 pmol/g.
Dynorphin-family peptides (alpha-neoendorphin, beta-neoendorphin, dynorphin A(1-8), and dynorphin B) were all present at 12 to 15 pmol/g. Full-length dynorphin A was very scarce at only 0.8 pmol/g.
No beta-endorphin was detected.
The two rare enkephalin forms (BAM-18 and MERV-NH2) had an unusual receptor profile: they preferred mu and kappa opioid receptors rather than the delta receptors that most enkephalins favor. This means they may have different functional roles in the gut.
Corbett, A D; McKnight, A T; Kosterlitz, H W · In Vitro
RPEP-00071 · 1988Morphine and other mu-opioid agonists decreased gastric acid secretion. They were more potent when injected into the brain (i.c.v.) than into the blood (i.v.), indicating a central mechanism.
The quaternary opioid antagonist naltrexone methylbromide (which cannot cross the blood-brain barrier) blocked brain-injected morphine's effect and partially blocked IV morphine's effect. This confirmed morphine reduces acid through brain receptors, even when given intravenously.
The kappa-selective agonist U-50,488H had the opposite effect: it increased gastric acid when given IV but not when given into the brain. This increase was blocked by naloxone, the muscarinic blocker atropine, and the M1-selective blocker pirenzepine.
The delta-selective agonist DPDPE did not affect acid secretion by either route.
Fox, D A; Burks, T F · Animal Study
RPEP-00086 · 1988Naloxone (100 to 1000 micrograms/kg) did not affect the initial stomach response to vagus nerve stimulation. But it dose-dependently enhanced the delayed response (from vagal afferent fibers). This means endogenous opioids normally restrain the delayed stomach excitation.
Met-enkephalin was markedly more potent than the mu-agonist DAGO and the kappa-agonist dynorphin A(1-13) at inhibiting both types of vagal stomach responses.
The delta-selective agonist DPDPE mimicked met-enkephalin's inhibitory effects. The delta-selective antagonist ICI 174,864 blocked DPDPE's effects. This confirmed delta-opioid receptors mediate the gastric inhibition.
The conclusion: natural opioid peptides (likely enkephalins) tonically restrain vagus nerve-driven stomach excitation through delta receptors.
Okamoto, T; Kurahashi, K; Fujiwara, M · Animal Study
RPEP-00092 · 1988Three opioid peptides caused dose-dependent contraction of isolated gallbladder muscle cells: met-enkephalin > dynorphin(1-13) > leu-enkephalin.
The contractions were blocked by the opioid antagonists naloxone and MR2266 but not by muscarinic, CCK/gastrin, or tachykinin antagonists. This confirmed the response was specifically through opioid receptors.
Differential sensitivity to preferential antagonists of mu (naloxone) and kappa (MR2266) receptors, combined with the different potencies of the three agonists, indicated the presence of mu, delta, and kappa opioid receptors on the gallbladder muscle cells.
These opioid peptides are known to be present in myenteric neurons of the gut, providing the natural source of opioid signals for gallbladder muscle.
Severi, C; Grider, J R; Makhlouf, G M · In Vitro
RPEP-00185 · 1991Met-enkephalin, leu-enkephalin, and dynorphin decreased inhibitory junction potentials in canine duodenal circular muscle via delta and mu receptors.
Bauer, A J; Szurszewski, J H · Animal Study
RPEP-00189 · 1991Pro-enkephalin fragments were released at 29-43% of tissue content loss. BAM-8 released at higher rates. The precursor BAM-18 was not released.
Corbett, A D; Gillan, M G; Kosterlitz, H W · Animal Study
RPEP-00193 · 1991VIP is diminished in intestinal smooth muscle of Crohn's patients. Substance P receptors are markedly increased at small vessels and lymph nodules in inflamed IBD intestine.
Eysselein, V E; Nast, C C · Review
RPEP-00239 · 1992VIP (20 nM-1 microM) induced neurogenic gut contractions via acetylcholine and substance P. Dynorphin (100 nM) and somatostatin (60 nM) abolished these contractions.
Katsoulis, S; Schmidt, W E; Clemens, A; Schwörer, H; Creutzfeldt, W · Animal Study
RPEP-00253 · 1992Exogenous opioids increased peristaltic threshold volume. Naloxone decreased it, indicating endogenous opioids tonically modulate peristaltic triggering.
Waterman, S A; Costa, M; Tonini, M · Animal Study
RPEP-00265 · 1993CSF beta-endorphin was significantly decreased in functional abdominal pain patients. Met-enkephalin and dynorphin were unchanged. Pain sensitivity was increased.
Jørgensen, L S; Bach, F W; Christiansen, P; Raundahl, U; Ostgaard, S; Ekman, R · Cross Sectional
RPEP-00271 · 1993Opioid peptides acting through all three receptor types (mu, delta, kappa) enhance non-adrenergic non-cholinergic inhibitory responses in the duodenum, while serotonin reduces them.
Postorino, A; Serio, R; Mulè, F; Adamo, E B; Di Giovanni, G; Marini, R · Animal Study
RPEP-00278 · 1993BPC 157 prevented liver necrosis and fatty changes in bile duct ligation, restraint stress, and CCl4 models. Effective orally and intraperitoneally. Outperformed bromocriptine, amantadine, and somatostatin.
Sikiric, P; Seiwerth, S; Grabarevic, Z; Rucman, R; Petek, M; Rotkvic, I; Turkovic, B; Jagic, V; Mildner, B; Duvnjak, M · Animal Study
RPEP-00279 · 1993BPC 157 from gastric juice shows broad organoprotection across multiple stress models. The stomach may initiate a body-wide protective response during stress via peptide mediators.
Sikirić, P; Petek, M; Rucman, R; Seiwerth, S; Grabarević, Z; Rotkvić, I; Turković, B; Jagić, V; Mildner, B; Duvnjak, M · Review
RPEP-00286 · 1994Delta opioid receptor agonists selectively inhibited nerve-stimulated contractions in human colon tissue without affecting spontaneous contractile activity.
Chamouard, P; Rohr, S; Meyer, C; Baumann, R; Angel, F · In Vitro
RPEP-00309 · 1994BPC 157 was the only tested compound that consistently protected against gastric and duodenal ulcers in all three models, regardless of whether given before, during, or after ulcer induction.
Sikiric, P; Seiwerth, S; Grabarevic, Z; Petek, M; Rucman, R; Turkovic, B; Rotkvic, I; Jagic, V; Duvnjak, M; Mise, S · Animal Study
RPEP-00326 · 1995Intraluminal peptide factors, released in response to dietary proteins and fats, are key regulators of CCK secretion that coordinate digestion.
Liddle, R A · Review
RPEP-00348 · 1995IGF-1 had a half-life of only 2 minutes in the duodenum and ileum, but casein protein significantly protected it from degradation in the gut.
Xian, C J; Shoubridge, C A; Read, L C · Animal Study
RPEP-00356 · 1996Multiple gastrointestinal peptide hormone levels were significantly altered during acute hepatic cytonecrosis and returned toward normal during liver regeneration.
Budillon, G; Cuomo, R; Taccone, W; Panico, G; Pumpo, R; Iaquinto, G; Manzillo, G · Cross Sectional
RPEP-00377 · 1996Endogenous opioid peptides contribute to post-tetanic inhibition of gut muscle contractions, and peptidase inhibitors enhance this effect by protecting naturally released peptides from degradation.
Ozaki, M; Masuda, Y; Yamamoto, H · In Vitro
RPEP-00383 · 1996BPC-157 provided strong gastroprotection against four distinct injury types (stress, ethanol, NSAID, capsaicin neurotoxicity), suggesting a broad protective mechanism involving sensory nerve pathways.
Sikirić, P; Seiwerth, S; Grabarević, Z; Rucman, R; Petek, M; Jagić, V; Turković, B; Rotkvić, I; Mise, S; Zoricić, I; Gjurasin, M; Konjevoda, P; Separović, J; Ljubanović, D; Artuković, B; Bratulić, M; Tisljar, M; Jurina, L; Buljat, G; Miklić, P; Marović, A · Animal Study
RPEP-00384 · 1996BPC-157 demonstrated both prophylactic and therapeutic effects in bile duct ligation-induced pancreatitis, also improving concomitant gastric and duodenal lesions.
Sikirić, P; Seiwerth, S; Grabarević, Z; Rucman, R; Petek, M; Jagić, V; Turković, B; Rotkvić, I; Mise, S; Zoricić, I; Jurina, L; Konjevoda, P; Hanzevacki, M; Ljubanović, D; Separović, J; Gjurasin, M; Bratulić, M; Artuković, B; Jelovac, N; Buljat, G · Animal Study
RPEP-00428 · 1997BPC-157 both protected against NSAID-induced GI lesions and improved chronic adjuvant arthritis, demonstrating simultaneous gastroprotective and anti-inflammatory effects.
Sikiric, P; Seiwerth, S; Grabarevic, Z; Rucman, R; Petek, M; Jagic, V; Turkovic, B; Rotkvic, I; Mise, S; Zoricic, I; Konjevoda, P; Perovic, D; Simicevic, V; Separovic, J; Hanzevacki, M; Ljubanovic, D; Artukovic, B; Bratulic, M; Tisljar, M; Rekic, B; Gjurasin, M; Miklic, P; Buljat, G · Animal Study
RPEP-00429 · 1997BPC-157 demonstrated cytoprotective effects in totally gastrectomized (acid-free) rats, proving its mechanism is independent of acid reduction.
Sikirić, P; Mikus, D; Seiwerth, S; Grabarević, Z; Rucman, R; Petek, M; Jagić, V; Turković, B; Rotkvić, I; Mise, S; Zoricić, I; Perić, J; Konjevoda, P; Perović, D; Jurina, L; Hanzevacki, M; Separović, J; Gjurasin, M; Jadrijević, S; Jelovac, N; Miklić, P; Buljat, G; Marović, A · Animal Study
RPEP-00430 · 1997BPC-157's mucosal protection in stress involves interactions with both adrenergic and dopaminergic neurotransmitter systems.
Sikirić, P; Mazul, B; Seiwerth, S; Grabarević, Z; Rucman, R; Petek, M; Jagić, V; Turković, B; Rotkvić, I; Mise, S; Zoricić, I; Jurina, L; Konjevoda, P; Hanzevacki, M; Gjurasin, M; Separović, J; Ljubanović, D; Artuković, B; Bratulić, M; Tisljar, M; Miklić, P; Sumajstorcić, J · Animal Study
RPEP-00431 · 1997BPC-157 counteracted both L-NAME (NO blocker) and L-arginine (NO donor) effects on gastric mucosa and blood pressure, confirming its role as an NO system modulator.
Sikirić, P; Seiwerth, S; Grabarević, Z; Rucman, R; Petek, M; Jagić, V; Turković, B; Rotkvić, I; Mise, S; Zoricić, I; Konjevoda, P; Perović, D; Jurina, L; Separović, J; Hanzevacki, M; Artuković, B; Bratulić, M; Tisljar, M; Gjurasin, M; Miklić, P; Stancić-Rokotov, D; Slobodnjak, Z; Jelovac, N; Marović, A · Animal Study
RPEP-00517 · 1999Endogenous opioid peptides regulate visceral pain sensitivity, gastrointestinal motility, and secretion through mu, delta, and kappa receptors in the brain, spinal cord, and enteric nervous system, with dysfunction potentially underlying IBS symptoms.
Corazziari, E · Review
RPEP-00528 · 1999BPC-157 attenuated catalepsy and gastric ulcers induced by haloperidol, fluphenazine, and reserpine at both 10 μg/kg and 10 ng/kg doses, demonstrating broad protective effects against neuroleptic side effects.
Jelovac, N; Sikiric, P; Rucman, R; Petek, M; Marovic, A; Perovic, D; Seiwerth, S; Mise, S; Turkovic, B; Dodig, G; Miklic, P; Buljat, G; Prkacin, I · Animal Study
RPEP-00551 · 1999BPC-157 significantly attenuated alloxan-induced gastric lesions in rats (200 mg/kg SC) and mice (400 mg/kg IP), reducing lesion severity and accelerating healing over the observation period.
Petek, M; Sikiric, P; Anic, T; Buljat, G; Separovic, J; Stancic-Rokotov, D; Seiwerth, S; Grabarevic, Z; Rucman, R; Mikus, D; Zoricic, I; Prkacin, I; Sebecic, B; Ziger, T; Coric, V; Turkovic, B; Aralica, G; Rotkvic, I; Mise, S; Hahn, V · Animal Study
RPEP-00559 · 1999BPC-157 provided long-lasting cytoprotection lasting days after administration in gastrectomized rats with duodenal ulcers and reflux esophagitis, significantly outlasting ranitidine and sucralfate.
Sikiric, P; Jadrijevic, S; Seiwerth, S; Sosa, T; Deskovic, S; Perovic, D; Aralica, G; Grabarevic, Z; Rucman, R; Petek, M; Jagic, V; Turkovic, B; Ziger, T; Rotkvic, I; Mise, S; Zoricic, I; Sebecic, B; Patrlj, L; Kocman, B; Sarlija, M; Mikus, D; Separovic, J; Hanzevacki, M; Gjurasin, M; Miklic, P · Animal Study
RPEP-00560 · 1999BPC-157 promoted significantly more angiogenesis and granulation tissue formation than H2-blockers, omeprazole, or sucralfate, identifying its primary healing mechanism as enhanced new tissue growth rather than acid suppression.
Sikiric, P; Separovic, J; Anic, T; Buljat, G; Mikus, D; Seiwerth, S; Grabarevic, Z; Stancic-Rokotov, D; Pigac, B; Hanzevacki, M; Marovic, A; Rucman, R; Petek, M; Zoricic, I; Ziger, T; Aralica, G; Konjevoda, P; Prkacin, I; Gjurasin, M; Miklic, P; Artukovic, B; Tisljar, M; Bratulic, M; Mise, S; Rotkvic, I · Animal Study
RPEP-00561 · 1999BPC-157 demonstrates tissue-protective and regenerative effects across gastrointestinal, musculoskeletal, neurological, and cardiovascular systems, consistently accelerating healing through angiogenesis, anti-inflammation, and NO modulation.
Sikiric, P · Review
RPEP-00562 · 1999BPC-157 maintained cytoprotective activity even when prostaglandin synthesis was blocked by indomethacin, unlike standard anti-ulcer agents, indicating prostaglandin-independent protective mechanisms for adaptive cytoprotection against endogenous irritants.
Sikirić, P; Seiwerth, S; Desković, S; Grabarević, Z; Marović, A; Rucman, R; Petek, M; Konjevoda, P; Jadrijević, S; Sosa, T; Perović, D; Aralica, G; Turković, B · Animal Study
RPEP-00620 · 2000Complete dopamine system failure (combined haloperidol + reserpine) produced severe gastric lesions. BPC-157 provided superior healing compared to ranitidine, atropine, and other agents while also reducing associated catalepsy.
Sikiric, P; Separovic, J; Buljat, G; Anic, T; Stancic-Rokotov, D; Mikus, D; Duplancic, B; Marovic, A; Zoricic, I; Prkacin, I; Lovric-Bencic, M; Aralica, G; Ziger, T; Perovic, D; Jelovac, N; Dodig, G; Rotkvic, I; Mise, S; Seiwerth, S; Turkovic, B; Grabarevic, Z; Petek, M; Rucman, R · Animal Study
RPEP-00651 · 2001BPC-157, omeprazole, and bromocriptine healed haloperidol-induced gastric lesions, while atropine, lansoprazole, and cimetidine failed, demonstrating the lesions are dopamine-mediated and BPC-157 acts through dopaminergic mechanisms.
Bilic, I; Zoricic, I; Anic, T; Separovic, J; Stancic-Rokotov, D; Mikus, D; Buljat, G; Ivankovic, D; Aralica, G; Prkacin, I; Perovic, D; Mise, S; Rotkvic, I; Petek, M; Rucman, R; Seiwerth, S; Sikiric, P · Animal Study
RPEP-00679 · 2001Topical BPC-157 cream accelerated burn wound healing (faster re-epithelialization and contraction) while simultaneously attenuating distant burn-associated gastric lesions in mice, demonstrating both local and systemic effects from topical application.
Mikus, D; Sikiric, P; Seiwerth, S; Petricevic, A; Aralica, G; Druzijancic, N; Rucman, R; Petek, M; Pigac, B; Perovic, D; Kolombo, M; Kokic, N; Mikus, S; Duplancic, B; Fattorini, I; Turkovic, B; Rotkvic, I; Mise, S; Prkacin, I; Konjevoda, P; Stambuk, N; Anic, T · Animal Study
RPEP-00687 · 2001Gut peptides (ghrelin, CCK, GLP-1, PYY) regulate appetite through neural and hormonal pathways that are significantly disrupted by gastrointestinal surgery, requiring surgical awareness for optimal patient management.
Näslund, E; Hellström, P M; Kral, J G · Review
RPEP-00691 · 2001BPC-157 provided sustained chronic cytoprotection against alcohol-induced gastric lesions, with effectiveness maintained throughout prolonged alcohol exposure alongside liver protection documented in companion study.
Prkacin, I; Aralica, G; Perovic, D; Separovic, J; Gjurasin, M; Lovric-Bencic, M; Stancic-Rokotov, D; Ziger, T; Anic, T; Sikiric, P; Seiwerth, S; Staresinic, M; Mise, S; Rotkvic, I; Jagic, V; Rucman, R; Petek, M; Turkovic, B; Marovic, A; Sjekavica, I; Sebecic, B; Boban-Blagaic, A; Ivasovic, Z · Animal Study
RPEP-00692 · 2001BPC-157 both prevented (when co-administered) and reversed (when given after damage) chronic alcohol-induced portal hypertension and liver lesions in rats, measured by direct portal vein pressure and histological assessment.
Prkacin, I; Separovic, J; Aralicia, G; Perovic, D; Gjurasin, M; Lovric-Bencic, M; Stancic-Rokotov, D; Staresinic, M; Anic, T; Mikus, D; Sikiric, P; Seiwerth, S; Mise, S; Rotkvic, I; Jagic, V; Rucman, R; Petek, M; Turkovic, B; Marovic, A; Sebecic, B; Boban-Blagaic, A; Kokic, N · Animal Study
RPEP-00697 · 2001BPC-157 attenuated cysteamine-induced colon and duodenal lesions, including in gastrectomized rats, demonstrating acid-independent healing and extending its protective effects to the entire gut.
Sikiric, P; Seiwerth, S; Grabarevic, Z; Balen, I; Aralica, G; Gjurasin, M; Komericki, L; Perovic, D; Ziger, T; Anic, T; Prkacin, I; Separovic, J; Stancic-Rokotov, D; Lovric-Bencic, M; Mikus, D; Staresinic, M; Aralica, J; DiBiaggio, N; Simec, Z; Turkovic, B; Rotkvic, I; Mise, S; Rucman, R; Petek, M; Sebecic, B; Ivasovic, Z; Boban-Blagaic, A; Sjekavica, I · Animal Study
RPEP-00700 · 2001BPC-157 attenuated both intratracheal HCl-induced lung lesions and associated gastric damage in rats, demonstrating cytoprotective effects extending beyond the GI tract to the respiratory system.
Stancic-Rokotov, D; Slobodnjak, Z; Aralica, J; Aralica, G; Perovic, D; Staresinic, M; Gjurasin, M; Anic, T; Zoricic, I; Buljat, G; Prkacin, I; Sikiric, P; Seiwerth, S; Rucman, R; Petek, M; Turkovic, B; Kokic, N; Jagic, V; Boban-Blagaic, A · Animal Study
RPEP-00707 · 2001Small intestinal ADM mRNA and protein were significantly upregulated during polymicrobial sepsis, with the gut releasing ADM into portal circulation, establishing the intestine as a major source of circulating ADM during sepsis.
Zhou, M; Chaudry, I H; Wang, P · Animal Study
RPEP-00736 · 2002CCK/CCK-A receptor dysfunction is implicated in gallbladder and pancreatic diseases, while gastrin/CCK-B receptor abnormalities may contribute to GI cancer development, positioning these peptide systems as therapeutic targets.
Jensen, Robert T · Review
RPEP-00792 · 2003BPC-157 maintained duodenal ulcer healing after sialoadenectomy while cimetidine and ranitidine lost effectiveness, demonstrating BPC-157's independence from salivary gland-derived protective factors — a unique pharmacological property.
Bedekovic, Vlado; Mise, Stjepan; Anic, Tomislav; Staresinic, Mario; Gjurasin, Miroslav; Kopljar, Mario; Kalogjera, Livije; Drvis, Petar; Boban Blagaic, Alenka; Batelja, Lovorka; Seiwerth, Sven; Sikiric, Predrag · Animal Study
RPEP-00797 · 2003Ghrelin displays integrated central (GH, appetite, neuroprotection) and peripheral (cardiac, adipose, gastric) activities through widespread GHS-R distribution, functioning as a systemic metabolic coordinator rather than a single-function hormone.
Bona, G; Bellone, S · Review
RPEP-00810 · 2003Bidirectional cross-reactivity between ghrelin/GHRP-6 and the motilin receptor was demonstrated: ghrelin activated the motilin receptor and motilin activated GHS-R, linking GH regulation and gut motility at the receptor level.
Depoortere, Inge; Thijs, Theo; Thielemans, Leen; Robberecht, Patrick; Peeters, Theo L · In Vitro
RPEP-00843 · 2003TNF-alpha inhibited TFF3 transcription through NF-κB activation in intestinal cells, creating a pathogenic feedback loop where inflammation simultaneously damages the gut AND suppresses its natural repair peptide.
Loncar, M B; Al-azzeh, E-d; Sommer, P S M; Marinovic, M; Schmehl, K; Kruschewski, M; Blin, N; Stohwasser, R; Gött, P; Kayademir, T · In Vitro
RPEP-00865 · 2003Beta-casomorphin-7 and casein hydrolysate stimulated mucus release in rat jejunum through mu-opioid receptor activation, providing a direct mechanism for dairy-derived gut protection through opioid peptide signaling.
Trompette, Aurélien; Claustre, Jean; Caillon, Fabienne; Jourdan, Gérard; Chayvialle, Jean Alain; Plaisancié, Pascale · Animal Study