Anyone read on any adverse effects of ghrp-6?
- Thread starter Presser
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i see where its studied for the treatment of toumors...
Treatment of tumors by administration of growth hormone releasing compounds and their antagonists
The following examples illustrate the efficacy of the most preferred compounds used in the tumor treatment of this invention.
1. Materials and Methods
a) Chemicals
Hexarelin (His-D-Mrp-Ala-Trp-D-Phe-Lys-NH<sub>2</sub>), Ala-Hexarelin (Ala-His-D-Mrp-Ala-Trp-D-Phe-Lys-NH<sub>2</sub>), Tyr-Ala-Hexarelin (Tyr-Ala-His-D-Mrp-Ala-Trp-D-Phe-Lys-NH<sub>2</sub>), MK0677 (N-[1(R) ([1,2-dihydro-1-methanesulfonylspiro-(3H-indole, 3,4'-piperidin)-1'yl]-2-(phenylmethoxy)ethyl]-2-amino-methylpropanamide-me thanesulfonate), EP80317 (HAIC-D-Mrp-D-Lys-Trp-D-Phe-Lys-NH<sub>2</sub>) and D-(Lys)<sub>3</sub> -GHRP6 (His-D-Trp-D-Lys-Trp-D-Phe-Lys-NH<sub>2</sub>) were supplied by Europeptides (Argenteuil, France). Human GHRH (GHRH 1-44) and somatostatin (somatostatin 1-14) were purchased from Bachem (Bubendorf, Switzerland). Human recombinant epidermal growth factor (EGF) and all tissue culture reagents were purchased from Sigma Chemical Co. (St. Louis, Mo., USA). <sup>3</sup> H-Thymidine was purchased from Pharmacia-Amersham Italia (Milan, Italy).
b) Human tissues
Surgical tumor specimens were collected from the Department of Biomedical Sciences and Human Oncology (Division of Pathology) of the University of Turin. A tumor fragment adjacent to that used for histopathological diagnosis was immediately frozen at -80° C. and stored for 2 to 60 months until further processed for binding studies. Samples of 13 invasive breast carcinoma (10 ductal and 3 lobular), 14 non-endocrine lung carcinomas (5 squamous cell and 9 adenocarcinomas), 11 endocrine tumors of the lung, 9 endocrine tumors of the pancreas and 12 thyroid carcinomas (7 of follicular origin and 5 of medullary origin) were used. Non-neoplastic normal tissues of the corresponding organs were also analysed in parallel with the individual tumors.
c) Tumor cell lines
Human lung carcinoma cells (CaLu1), T47D and MDA-MB231, respectively, human oestrogen dependent and oestrogen independent [color=orange ! important][font=Arial,Helvetica,sans-serif][color=orange ! important][font=Arial,Helvetica,sans-serif]breast [/font][color=orange ! important][font=Arial,Helvetica,sans-serif]cancer[/font][/color][/font][/color][/color] cell lines were purchased from the ATCC (Rockville, Md., USA). Cells were routinely cultured in 25 cm<sup>3</sup> flasks at 37° C., 5% CO<sub>2</sub> and 95% humidified atmosphere in RPMI supplemented with 10% FCS, penicillin-streptomycin and fungizone. When a subconfluent state was reached, cells were detached from the flasks with trypsin/EDTA.
d) GHRP receptor assay
GHRP receptors were measured on tumor membranes as described in G. Muccioli et al., Journal of Endocrinology, 157, 99-106, 1998, using <sup>125</sup> I-Tyr-Ala-Hexarelin as a ligand. Specific binding was calculated as the difference between binding in the absence and in the presence of excess unlabelled Tyr-Ala-Hexarelin and expressed as a percentage of the radioactivity added. Saturation and competition binding studies were analyzed with the GraphPAD Prism 2 program (GraphPAD Software, San Diego, Calif., USA).
e) Cell proliferation studies
[color=orange ! important][font=Arial,Helvetica,sans-serif][color=orange ! important][font=Arial,Helvetica,sans-serif]DNA [/font][color=orange ! important][font=Arial,Helvetica,sans-serif]synthesis[/font][/color][/font][/color][/color] was evaluated by <sup>3</sup> H-thymidine incorporation as described in G. Muccioli et al., Journal of Endocrinology, 153, 365-371, 1997. Starved cells were incubated with medium alone (basal) or EGF (1 ng/ml) in the absence or in the presence of different concentrations (from 10<sup>-8</sup> to 10<sup>-6</sup> mol/l) of Hexarelin, Ala-Hexarelin, Tyr-Ala-Hexarelin, MK0677, (D-Lys)<sub>3</sub> -GHRP6 or EP80317. After incubation for 20 hours, <sup>3</sup> H-thymidine was added and incubation was continued for a further 4 hours. The reaction was halted and the cells were harvested onto glass-fiber filter strips. Incorporation of <sup>3</sup> H-thymidine was measured in a scintillation counter.
Cell growth studies were carried out as described in P.Cassoni et al., Virchows Archiv, 425, 467-472, 1994. Cells were seeded in triplicate in 24-multiwell plates at a density of 5,000-10,000 cells/ml. Twenty-four hours after plating the medium was changed. Hexarelin or Ala-Hexarelin were added where requested at concentrations ranging from 10<sup>-8</sup> to 10<sup>-6</sup> mol/l. The medium was changed every 48 hours. Cells were counted at 48 and 72 or 96 hours of treatment in a double blind analysis by two independent investigators using a haemocytometer.
f) Statistical analysis
Data were expressed as means (FIGS. 1 and 2) or means±S.E.M. (FIGS. 3 to 7) unless otherwise specified. Statistical significance was determined using Mann-Whitney test (FIGS. 1 to 3) or by one-way ANOVA (FIGS. 4 to 7). All experiments were carried out at least in triplicate.
2. Results
a) Identification of receptors for GHRP and their antagonists in different human tumors
FIG. 1 shows the distribution of radiolabelled Tyr-Ala-Hexarelin binding to membranes from different endocrine and non-endocrine human tumors of various origins (*P<0.01 vs. the corresponding non-tumoral tissue). Non-endocrine tumors of the lung and breast, as well endocrine carcinomas of the pancreas and thyroid (follicular type) showed a median specific binding value which was statistically higher than that found in the corresponding non tumoral normal tissue. In contrast, no difference in the specific binding values was observed between normal tissue and endocrine tumors of the lung or thyroid (medullary type).
b) Biochemical characteristics of receptors for GHRP and their antagonists
To determine whether the binding of <sup>125</sup> I-Tyr-Ala-Hexarelin to tumor membranes shows the properties typical of ligand-receptor interaction, the binding of radiotracer was investigated in more detail in a non-endocrine carcinoma of lung origin which displayed the highest specific binding value. FIG. 2 reports the binding of <sup>125</sup> I-Tyr-Ala-Hexarelin to tumor membranes as a function of increasing concentrations of radioligand. This study revealed evidence of saturable specific binding and Scatchard analysis (upper panel) indicated the presence of a single class of high affinity sites.
The specificity of <sup>125</sup> I-Tyr-Ala-Hexarelin binding was established by determining the ability of different compounds to compete with the radioligand for the tumoral binding sites (cf. FIG. 3). The binding of radiotracer was displaced in a dose-dependent fashion by Hexarelin, Ala-Hexarelin, Tyr-Ala-Hexarelin and GHRP antagonists such as D-(Lys)<sub>3</sub> -GHRP6 and EP 80317, an (Amino-azepino-indol)<sub>1</sub> -D-(Lys)<sub>3</sub> derivative of Hexarelin which does not release GH in neonatal rats. A negligible displacement was observed in the presence of MK0677, a non-peptidyl GHRP mimetic that bind to pituitary GHRP receptors. In contrast, no competition was observed in the presence of GHRH or somatostatin.
c) Effect of GHRP and their antagonists on <sup>3</sup> H-thymidine incorporation
Hexarelin at 10-6 mol/l was able to inhibit both basal and the EGF-stimulated <sup>3</sup> H-thymidine incorporation in human cells of lung carcinoma (cf. FIG. 4; *P<0.05, **P<0.01 vs. control). This antiproliferative effect was also observed when the cells were incubated in the presence of 10<sup>-6</sup> mol/l Ala-Hexarelin, Tyr-Ala-Hexarelin or GHRP antagonists such as (D-Lys)<sub>3</sub> -GHRP6 and EP80317. In contrast, a slight inhibition was observed in the presence of MK0677. Experiments using increasing concentrations of Hexarelin, Ala-Hexarelin, Tyr-Ala-Hexarelin, (D-Lys)<sub>3</sub> -GHRP6 and EP80317 (cf. FIG. 5) revealed that these compounds inhibited the proliferative effect of EGF on human lung carcinoma cells inhibited in a dose-dependent fashion. The EC<sub>50</sub> value was 5.6×10<sup>-8</sup> mol/l for EP80317, 6.5×10<sup>-8</sup> mol/l for Tyr-Ala-Hexarelin, 8×10<sup>-8</sup> mol/l for Hexarelin, 9×10<sup>-8</sup> mol/l for (D-Lys)<sub>3</sub> -GHRP6 and 1×10<sup>-7</sup> mol/l for Ala-Hexarelin.
d) Effect of GHRP on cell growth
In human lung carcinoma cells Hexarelin at 10<sup>-8</sup> mol/l caused a decrease in cell number compared with the control with a significant effect (-47%) only after 96 hours. This effect further increased at 10<sup>-7</sup> mol/l and 10<sup>-6</sup> mol/l and was observed at any time point tested (cf. FIG. 6; **P<0.001; ***P<0.0001 vs. control).
In human breast cancer T47D cells Hexarelin at 10<sup>-8</sup> mol/l caused a decrease in cell number compared with control with a significant effect (-54%) only after 96 hours. This effect further increased at 10<sup>-7</sup> mol/l and 10<sup>-6</sup> mol/l and was observed at any time point tested (cf. FIG. 7a; **P<0.001; ***P<0.0001 vs. control). A similar antiproliferative effect was also displayed by Ala-Hexarelin on these tumor cells (cf. FIG. 7b; **P<0.001; ***P<0.0001 vs. control).
In human breast cancer MDA-MB231 cells Hexarelin at 10<sup>-8</sup> mol/l caused a decrease in cell number compared with control with a significant effect (-33%) only after 72 hours. This effect further increased at 10<sup>-7</sup> mol/l and 10<sup>-6</sup> mol/l and was observed at any time point tested (cf. FIG. 8a; *P<0.01; **P<0.001; ***P<0.0001 vs. control). A similar antiproliferative effect was also displayed by Ala-Hexarelin on these tumor cells (cf. FIG. 8b; *P<0.01; **P<0.001; ***P<0.0001 vs. control).
These results demonstrate that synthetic growth hormone releasing peptides and their antagonists inhibit the growth of human carcinoma cells in vitro. The antiproliferative effect is mediated by a specific receptor.
Treatment of tumors by administration of growth hormone releasing compounds and their antagonists
The following examples illustrate the efficacy of the most preferred compounds used in the tumor treatment of this invention.
1. Materials and Methods
a) Chemicals
Hexarelin (His-D-Mrp-Ala-Trp-D-Phe-Lys-NH<sub>2</sub>), Ala-Hexarelin (Ala-His-D-Mrp-Ala-Trp-D-Phe-Lys-NH<sub>2</sub>), Tyr-Ala-Hexarelin (Tyr-Ala-His-D-Mrp-Ala-Trp-D-Phe-Lys-NH<sub>2</sub>), MK0677 (N-[1(R) ([1,2-dihydro-1-methanesulfonylspiro-(3H-indole, 3,4'-piperidin)-1'yl]-2-(phenylmethoxy)ethyl]-2-amino-methylpropanamide-me thanesulfonate), EP80317 (HAIC-D-Mrp-D-Lys-Trp-D-Phe-Lys-NH<sub>2</sub>) and D-(Lys)<sub>3</sub> -GHRP6 (His-D-Trp-D-Lys-Trp-D-Phe-Lys-NH<sub>2</sub>) were supplied by Europeptides (Argenteuil, France). Human GHRH (GHRH 1-44) and somatostatin (somatostatin 1-14) were purchased from Bachem (Bubendorf, Switzerland). Human recombinant epidermal growth factor (EGF) and all tissue culture reagents were purchased from Sigma Chemical Co. (St. Louis, Mo., USA). <sup>3</sup> H-Thymidine was purchased from Pharmacia-Amersham Italia (Milan, Italy).
b) Human tissues
Surgical tumor specimens were collected from the Department of Biomedical Sciences and Human Oncology (Division of Pathology) of the University of Turin. A tumor fragment adjacent to that used for histopathological diagnosis was immediately frozen at -80° C. and stored for 2 to 60 months until further processed for binding studies. Samples of 13 invasive breast carcinoma (10 ductal and 3 lobular), 14 non-endocrine lung carcinomas (5 squamous cell and 9 adenocarcinomas), 11 endocrine tumors of the lung, 9 endocrine tumors of the pancreas and 12 thyroid carcinomas (7 of follicular origin and 5 of medullary origin) were used. Non-neoplastic normal tissues of the corresponding organs were also analysed in parallel with the individual tumors.
c) Tumor cell lines
Human lung carcinoma cells (CaLu1), T47D and MDA-MB231, respectively, human oestrogen dependent and oestrogen independent [color=orange ! important][font=Arial,Helvetica,sans-serif][color=orange ! important][font=Arial,Helvetica,sans-serif]breast [/font][color=orange ! important][font=Arial,Helvetica,sans-serif]cancer[/font][/color][/font][/color][/color] cell lines were purchased from the ATCC (Rockville, Md., USA). Cells were routinely cultured in 25 cm<sup>3</sup> flasks at 37° C., 5% CO<sub>2</sub> and 95% humidified atmosphere in RPMI supplemented with 10% FCS, penicillin-streptomycin and fungizone. When a subconfluent state was reached, cells were detached from the flasks with trypsin/EDTA.
d) GHRP receptor assay
GHRP receptors were measured on tumor membranes as described in G. Muccioli et al., Journal of Endocrinology, 157, 99-106, 1998, using <sup>125</sup> I-Tyr-Ala-Hexarelin as a ligand. Specific binding was calculated as the difference between binding in the absence and in the presence of excess unlabelled Tyr-Ala-Hexarelin and expressed as a percentage of the radioactivity added. Saturation and competition binding studies were analyzed with the GraphPAD Prism 2 program (GraphPAD Software, San Diego, Calif., USA).
e) Cell proliferation studies
[color=orange ! important][font=Arial,Helvetica,sans-serif][color=orange ! important][font=Arial,Helvetica,sans-serif]DNA [/font][color=orange ! important][font=Arial,Helvetica,sans-serif]synthesis[/font][/color][/font][/color][/color] was evaluated by <sup>3</sup> H-thymidine incorporation as described in G. Muccioli et al., Journal of Endocrinology, 153, 365-371, 1997. Starved cells were incubated with medium alone (basal) or EGF (1 ng/ml) in the absence or in the presence of different concentrations (from 10<sup>-8</sup> to 10<sup>-6</sup> mol/l) of Hexarelin, Ala-Hexarelin, Tyr-Ala-Hexarelin, MK0677, (D-Lys)<sub>3</sub> -GHRP6 or EP80317. After incubation for 20 hours, <sup>3</sup> H-thymidine was added and incubation was continued for a further 4 hours. The reaction was halted and the cells were harvested onto glass-fiber filter strips. Incorporation of <sup>3</sup> H-thymidine was measured in a scintillation counter.
Cell growth studies were carried out as described in P.Cassoni et al., Virchows Archiv, 425, 467-472, 1994. Cells were seeded in triplicate in 24-multiwell plates at a density of 5,000-10,000 cells/ml. Twenty-four hours after plating the medium was changed. Hexarelin or Ala-Hexarelin were added where requested at concentrations ranging from 10<sup>-8</sup> to 10<sup>-6</sup> mol/l. The medium was changed every 48 hours. Cells were counted at 48 and 72 or 96 hours of treatment in a double blind analysis by two independent investigators using a haemocytometer.
f) Statistical analysis
Data were expressed as means (FIGS. 1 and 2) or means±S.E.M. (FIGS. 3 to 7) unless otherwise specified. Statistical significance was determined using Mann-Whitney test (FIGS. 1 to 3) or by one-way ANOVA (FIGS. 4 to 7). All experiments were carried out at least in triplicate.
2. Results
a) Identification of receptors for GHRP and their antagonists in different human tumors
FIG. 1 shows the distribution of radiolabelled Tyr-Ala-Hexarelin binding to membranes from different endocrine and non-endocrine human tumors of various origins (*P<0.01 vs. the corresponding non-tumoral tissue). Non-endocrine tumors of the lung and breast, as well endocrine carcinomas of the pancreas and thyroid (follicular type) showed a median specific binding value which was statistically higher than that found in the corresponding non tumoral normal tissue. In contrast, no difference in the specific binding values was observed between normal tissue and endocrine tumors of the lung or thyroid (medullary type).
b) Biochemical characteristics of receptors for GHRP and their antagonists
To determine whether the binding of <sup>125</sup> I-Tyr-Ala-Hexarelin to tumor membranes shows the properties typical of ligand-receptor interaction, the binding of radiotracer was investigated in more detail in a non-endocrine carcinoma of lung origin which displayed the highest specific binding value. FIG. 2 reports the binding of <sup>125</sup> I-Tyr-Ala-Hexarelin to tumor membranes as a function of increasing concentrations of radioligand. This study revealed evidence of saturable specific binding and Scatchard analysis (upper panel) indicated the presence of a single class of high affinity sites.
The specificity of <sup>125</sup> I-Tyr-Ala-Hexarelin binding was established by determining the ability of different compounds to compete with the radioligand for the tumoral binding sites (cf. FIG. 3). The binding of radiotracer was displaced in a dose-dependent fashion by Hexarelin, Ala-Hexarelin, Tyr-Ala-Hexarelin and GHRP antagonists such as D-(Lys)<sub>3</sub> -GHRP6 and EP 80317, an (Amino-azepino-indol)<sub>1</sub> -D-(Lys)<sub>3</sub> derivative of Hexarelin which does not release GH in neonatal rats. A negligible displacement was observed in the presence of MK0677, a non-peptidyl GHRP mimetic that bind to pituitary GHRP receptors. In contrast, no competition was observed in the presence of GHRH or somatostatin.
c) Effect of GHRP and their antagonists on <sup>3</sup> H-thymidine incorporation
Hexarelin at 10-6 mol/l was able to inhibit both basal and the EGF-stimulated <sup>3</sup> H-thymidine incorporation in human cells of lung carcinoma (cf. FIG. 4; *P<0.05, **P<0.01 vs. control). This antiproliferative effect was also observed when the cells were incubated in the presence of 10<sup>-6</sup> mol/l Ala-Hexarelin, Tyr-Ala-Hexarelin or GHRP antagonists such as (D-Lys)<sub>3</sub> -GHRP6 and EP80317. In contrast, a slight inhibition was observed in the presence of MK0677. Experiments using increasing concentrations of Hexarelin, Ala-Hexarelin, Tyr-Ala-Hexarelin, (D-Lys)<sub>3</sub> -GHRP6 and EP80317 (cf. FIG. 5) revealed that these compounds inhibited the proliferative effect of EGF on human lung carcinoma cells inhibited in a dose-dependent fashion. The EC<sub>50</sub> value was 5.6×10<sup>-8</sup> mol/l for EP80317, 6.5×10<sup>-8</sup> mol/l for Tyr-Ala-Hexarelin, 8×10<sup>-8</sup> mol/l for Hexarelin, 9×10<sup>-8</sup> mol/l for (D-Lys)<sub>3</sub> -GHRP6 and 1×10<sup>-7</sup> mol/l for Ala-Hexarelin.
d) Effect of GHRP on cell growth
In human lung carcinoma cells Hexarelin at 10<sup>-8</sup> mol/l caused a decrease in cell number compared with the control with a significant effect (-47%) only after 96 hours. This effect further increased at 10<sup>-7</sup> mol/l and 10<sup>-6</sup> mol/l and was observed at any time point tested (cf. FIG. 6; **P<0.001; ***P<0.0001 vs. control).
In human breast cancer T47D cells Hexarelin at 10<sup>-8</sup> mol/l caused a decrease in cell number compared with control with a significant effect (-54%) only after 96 hours. This effect further increased at 10<sup>-7</sup> mol/l and 10<sup>-6</sup> mol/l and was observed at any time point tested (cf. FIG. 7a; **P<0.001; ***P<0.0001 vs. control). A similar antiproliferative effect was also displayed by Ala-Hexarelin on these tumor cells (cf. FIG. 7b; **P<0.001; ***P<0.0001 vs. control).
In human breast cancer MDA-MB231 cells Hexarelin at 10<sup>-8</sup> mol/l caused a decrease in cell number compared with control with a significant effect (-33%) only after 72 hours. This effect further increased at 10<sup>-7</sup> mol/l and 10<sup>-6</sup> mol/l and was observed at any time point tested (cf. FIG. 8a; *P<0.01; **P<0.001; ***P<0.0001 vs. control). A similar antiproliferative effect was also displayed by Ala-Hexarelin on these tumor cells (cf. FIG. 8b; *P<0.01; **P<0.001; ***P<0.0001 vs. control).
These results demonstrate that synthetic growth hormone releasing peptides and their antagonists inhibit the growth of human carcinoma cells in vitro. The antiproliferative effect is mediated by a specific receptor.
S
saudades
Guest
That's very interesting, good to know. Thanks for the informative post.
Southern Comfor
Banana
Soooo I read that and have NO clue what it meant... either it will make my dick grow (good) or shrivel up and fall off (bad)...... ummmm so which one of those should I be looking out for?
irishpride
MuscleChemistry Registered Member
shouldnt make a difference if mcgaret really loves you he would love you with or without a dick and thats what really counts
S
saudades
Guest
normalsucks said:
Well, my wife wakes up to me poking her in the back every morning while I'm on it so I'd have to say it makes me big, lol!
S
saudades
Guest
I've been taking approximately 500mcg/day. I will get extremely hungry about an hour and a half later. I've been staying pretty lean even though I'm eating more. It is like taking 3-4 iu of HGH/day for me. As long as I keep up the calories, I can see the weight on the scale increase.
S
saudades
Guest
Okay, I'm wondering if anyone else has experienced this. Granted I'm 39 years old, but I have just verified that I am actually taller than I used to be. I seem to be inching towards 6' 1". I think using HGH started something. Considering I always had low test, my growth plates may not have closed when they were supposed to. Using GHRP-6 could be continuing where the HGH left off since it makes me produce my own HGH. Damn, where was this stuff when I was 25, lol?
irishpride
MuscleChemistry Registered Member
really? damn thats some crazy stuff right there... i have noticed a slight increase in foot size since i starting using peptides years ago but luckily my head is still the same shape and size LOLsaudades said:
S
saudades
Guest
I'm quite serious. Even after using Immense growth factor for the first time, I went down to Brazil for the second time to visit family there, and everyone commented that I looked taller. Even a small fraction of an increase will make a difference.
I'd normally agree that it would be impossible, but there have been cases of adults whose growth plates have not closed all the way or others that have had sudden strange growth spurts. They weren't much--usually 1/2" to 1" at the most. There is always going to be exceptions to the rule.
My older brother doesn't seem too happy about it. We've been the same height for a long time, and now I'm looking down at him, and I'm enjoying every moment of it.
I'd normally agree that it would be impossible, but there have been cases of adults whose growth plates have not closed all the way or others that have had sudden strange growth spurts. They weren't much--usually 1/2" to 1" at the most. There is always going to be exceptions to the rule.
My older brother doesn't seem too happy about it. We've been the same height for a long time, and now I'm looking down at him, and I'm enjoying every moment of it.
irishpride
MuscleChemistry Registered Member
i am not doubting you bud, hell you would no better than anyone else how tall you were and how tall you are now, try and find some studies if you can about how this can/does occur...
or you could just take off your high heals you slut you
or you could just take off your high heals you slut you
S
saudades
Guest
Lol, that was in response to John44. It has been interesting to say the least. I hope it continues.
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