238 replies to this topic

[JPC16]

Is it actually possible that your immuunsystem attacks the inhibitor before it can do it's job? I heard from someone who works in a pharmaceutical company that they first "humanize" molecules (antibodies) before clinical testing.

[YOLF]

huh?

[JPC16]

Never mind. Humanizing is only applied to antibodies (http://en.wikipedia....anized_antibody).

[Hebbeh]

My rat is fearless, willing and able. If we can ascertain a potential dosage range, I would consider dissolving in ethanol (vodka) or other suitable solvent so that it could be dosed by the drop with maybe 10 drops equal to target dose. Then the rat could be dosed with 1 drop (1/10 target dosage) and titrated up a drop at a time, hopefully reaching positive effects before LD100 is reached. The initial solution could be subdivided and further diluted to start with a fraction of drop to initially test toxicity and potential adverse side effects. Results and experiences would be posted to the thread as dosage increased...with posting ceasing for more than a few days being code for test terminated due to LD100 reached before intended results could be determined.

[YOLF]

My rat is fearless, willing and able. If we can ascertain a potential dosage range, I would consider dissolving in ethanol (vodka) or other suitable solvent so that it could be dosed by the drop with maybe 10 drops equal to target dose. Then the rat could be dosed with 1 drop (1/10 target dosage) and titrated up a drop at a time, hopefully reaching positive effects before LD100 is reached. The initial solution could be subdivided and further diluted to start with a fraction of drop to initially test toxicity and potential adverse side effects. Results and experiences would be posted to the thread as dosage increased...with posting ceasing for more than a few days being code for test terminated due to LD100 reached before intended results could be determined.

I don't know if I'd go past LD00... lol

[Reformed-Redan]

If the appropriate human dose can be given on the amount of C16 should be in a syringe provided I could have my rat risk it.

Edited by yadayada, 31 May 2013 - 01:02 AM.

[lostfalco]

Due to time constraints, I'm focusing on this article for now. http://www.sciencedi...014579307008952

Facts:
1) 7-day old Sprague-Dawley rats were used. Average weight for newborn Sprague-Dawley rats = 5g = .005kg. Source: http://www.bmijourna...icle/view/75/70
2) Minimum Effective Dose = 3.35mg/kg; Lethal Dose = 335 mg/kg
3) Therefore, Minimum Effect Dose = 3.35mg/1kg = x/.005kg; This equals .01675mg.
4) The average human male weighs 82kg. Source: http://www.bmijourna...icle/view/75/70
5) Using the allometric dose calculator previously provided here...http://home.fuse.net/clymer/minor/allometry.html...I entered these numbers Weight 1 = .005kg, Weight 2 = 82kg, Dose 1 = .01675, Exponent = .75
6) Therefore, Allometric Human Dose = 24.274mg = .296mg/kg
7) Therefore, Lethal Rat Dose = 1.675mg and Allometric Lethal Human Dose = 2427.435mg = 29.603mg/kg
This is obviously different than the LD50.

Please check my work and let me know if I missed anything.

Note: the article states that the BBB of young rats is more permeable than that of adult rats. Thus we should be careful to extrapolate the results to adult rats and, of course, to adult humans. Also, this says nothing about repeated dosing. Do any of the other aforementioned articles? I haven't had time to look through them.

[YOLF]

Good points lostfalco. We gotta know this stuff...

[YOLF]

So what's the price tag on this stuff going to look like? Anyone got a link to a price from a bulk supplier?

Edited by cryonicsculture, 31 May 2013 - 05:11 AM.

[lostfalco]

Googoltarian, is there another article that you want me to take a look at? If you know of any human trials or trials in which a repeated dosing schedule is followed, I'd be happy to read over them. So far, I've looked at baby rats dosed once. We are trying to calculate human adults dosed daily, right?

[JPC16]

Lostfalco


Your calculations are wrong. Minimum effective dose is 3.35µg/kg and not 3.35mg/kg.

[lostfalco]

Lostfalco


Your calculations are wrong. Minimum effective dose is 3.35µg/kg and not 3.35mg/kg.

Son of a b! I knew I shouldnt have done that in 5 minutes. Thanks for pointing that out.

[lostfalco]

So, just convert to micrograms. 1mcg = .001 milligrams

[JPC16]

I am wondering why everybody is basing their conclusion on the study with C16,while you shoud study the article about the pkr suppresion (http://www.sciencedi...092867411013754).

In that article there is stated that they used 0.1 and 0.2 mg/kg ip injections. Which is a lot more than the microgram-dosages. This makes me doubt if they indeed used C16 in the article which concluded that pkr suppression leads to a supermemory. But in the literature they usually refer to C16 to pkri (and not some onther inhibitor like 2-aminopurine). And the LD50 is 335µg, wright? This is 0.335mg/kg which is still lower than the used dosages seen in the pkr suppression article and therefore I think that that they indeed did use C16.
But if 3.35µg was almost just as effective as higher dosages which can be seen in this article with the young mice, then I think that dosage should be just as effective as the 0.1 mg/kg.
So according to me: 3.35µg/kg is the least (and best) effective dosage to a super memory in mice. And this is slightly less in humans.

By the way: in the article about pkr suppression they used 8 to 16 week old males (mice) which is reasonable old. And the inhibitor worked on them. The BBB was no obstacle for whatever (very probably C16) inhibtor they used.
But it could also be that you have to use more C16 in older mice because of their BBB.

[kevinseven11]

Your not suppose to take this if you are sick or stressed. Also this compound causes anxiety. So have benzos ready if you detect high energy or other signs of seizure.
This is interesting id be open to a group buy.

[JPC16]

Where does it say that?

[YOLF]

Your not suppose to take this if you are sick or stressed. Also this compound causes anxiety. So have benzos ready if you detect high energy or other signs of seizure.
This is interesting id be open to a group buy.

Can you link to a the study that mentions and describes anxiety? I'd like to know how they tested for it. How might the anxiety be minimized? Is there enough info to describe the anxiety pathology?

[megatron]

My rat is fearless, willing and able. If we can ascertain a potential dosage range, I would consider dissolving in ethanol (vodka) or other suitable solvent so that it could be dosed by the drop with maybe 10 drops equal to target dose. Then the rat could be dosed with 1 drop (1/10 target dosage) and titrated up a drop at a time, hopefully reaching positive effects before LD100 is reached. The initial solution could be subdivided and further diluted to start with a fraction of drop to initially test toxicity and potential adverse side effects. Results and experiences would be posted to the thread as dosage increased...with posting ceasing for more than a few days being code for test terminated due to LD100 reached before intended results could be determined.

Can't we just do this and start with a very low dose?

[kevinseven11]

I read somewhere that overdose would be due to seizures that would be reversible with a sedative?
I agree you can simply start in minute amounts and build up but im just saying the first testers should have life saving drugs with them.

[YOLF]

I read somewhere that overdose would be due to seizures that would be reversible with a sedative?
I agree you can simply start in minute amounts and build up but im just saying the first testers should have life saving drugs with them.

good point!

[renfr]

I read somewhere that overdose would be due to seizures that would be reversible with a sedative?
I agree you can simply start in minute amounts and build up but im just saying the first testers should have life saving drugs with them.

Seizures might be reversible but seizures cause brain damage that is irreversible and make you prone to more seizures in the future.
That's why in people with chronic seizure syndrome drugs start becoming innefective and seizures become more and more frequent.
Epileptogenicity is proportional to the number of seizures.

[Googoltarian]

There is general confusion regarding C16 safe dosing. This compound therapeutic index looks similar to caffeine: effective dose for daily user is 0.2-0.5g while 4-6g are known to cause death.

Lets say C16 will give good effects in 0,2-1mg and will kill at 13-18mg.

Not so bad

[megatron]

There is general confusion regarding C16 safe dosing. This compound therapeutic index looks similar to caffeine: effective dose for daily user is 0.2-0.5g while 4-6g are known to cause death.

Lets say C16 will give good effects in 0,2-1mg and will kill at 13-18mg.

Not so bad

That's possible to cope with. Let's say we start with 0,05mg or 0,001mg doses or something. If we dissolve 0,25mg in one ml water (i.e. 25mg/dl),
we get (0,05mg) / (0,25mg/ml) = 0,2mL which is about 3 drops (assuming 1 drop is about 0,0649 ml).

That should be manageable.

Edited by Megatrone, 02 June 2013 - 02:02 PM.

[Googoltarian]

I think JPC16 proposal for dosing is better. C16 will not dissolve in water at pH higher than 3, and in pH <3 it will decompose. DMSO is good, it could be even used to transfer this drug transdermally.

[megatron]

Oh, sorry. I thought it was water soluble. Yea, his method seems OK.

[JPC16]

I just wrote a pretty decent report about how I am going to execute my experiments once I get the C16. I am really serious about this. I pretty much wanted to try the compound out from the first minute I heard about it (after doing some serious research about this), which was only a week later when the actual article about it got published. So that is almost 2 years already of which I spended most of my time of it getting my hands on C16. But every time I contacted a chemical company that sold C16, they always got on to me, or they asked me to deliver a shitload of paperwork I coudn't provide.
But eventually Yadayada recomended me to contact Googoltarian. Which I did and he orderd the compound for me against an arranged payment. And now here we are.

I don't know about you guys but now when it is finaly here, I am ready to test this out for myself!
I think you also don't have to worry about seizures. What the drug actually does is induce seizures somewhere on the CA1 region of the hippocampus, but it is harmless. It is this seizure type activity that is the perfect kind of network activity that has to be maintainded to induce LTP and therefore store an event in the long term memory. These seizures are therefore (I think) something like spindle waves that occure during sleep.
The PKR knockout mice had this going on al the time in their brain and there were no side effects reporte about this (I think).

Below you can find my report. I know it isn't perfect, but I have been really busy with exams and shit.
I already apologize for my crappy English and possible inaccuracies or mistakes.



Introduction

The main goal of this experiment is to assess the effectivity of the PKR inhibitor (pkri) C16 in terms of memory enhancement in humans. So far, scientific literature only contains one study wherein research has been done about the nootropic values of pkri (http://ac.els-cdn.co...69bb02573dd29bb ). To further investigate its nootropic properties, two in vivo studies will be done.

The first one will investigate the activity of pkri in an other mammal besides mice. The goals is to reproduce the same results that were seen in the original studies. The second in vivo test is to investigate if it is successfully active in humans whitout downsides (side effects).


Method and materials

Because the dosages that are used are very small, it will be easier to dissolve an exact as possible measured quantity of C16 in DMSO (max. solubility: 12mg/ml).
For example: If 5mg were dissolved in 50 ml if DMSO, one has 0,1mg C16 for every ml (which is the dosage used in the original study).

The reason why this method of measurement will be applied is because it is easier and safer in usage. It is safer because one only has to weigh the substance once (which reduces human errors), and it only has to be weighed in the mg range and not the µg range which as well makes it easier.
So if a standard solution is premade it will be easier just to measure one ml of DMSO-solution (with the C16 in it) when the experiments are executed. The standard solution with pkri dissolved in it will be further kept below freezing temperature to prevent degradation of the compound.

One ml is very small in size and can be easily ejected in the back of the mouth of the test animal with a (sterilized) pipette.

The capsules for human trial will be of the 000 type which are large enough to contain one ml (volume of capsule= 1,37ml and density of DMSO is about the same as water).
http://www.erowid.or...p.sizechart.pdf


Actual dosing

The dosage used in the study that reported nootropic potential used minimaly 0,1mg/kg. How the researchers arrived at that dosage is not explaind.

If we base ourselfs on previous research, http://ac.els-cdn.co...4716c21217ed01c, we know that the minimal effective dosage for 7 day old mice is 3,35µg/kg. Higher doses only lead to marginally better PKR inhibition. So you can conclude that 3,35µg/kg is pretty much “just as effective” as higher dosages. This makes it in my opinion the best starting dosage.

Administration will happen from under limit to upper limit in small incremental dosages.

If we impliment the following formula to translate the mice dosage to human (and canine) dosage:


(http://www.fasebj.or...2/3/659.full#T1)

• 
  Human:

We get:

(3,35µg/kg) * (3/37) = 0,2716 µg/kg

A 70kg weighing individual would need the following dosage:

(0,2716 µg/kg) * (70kg) = 19,012 µg = 0,019012 mg

To get this dosage:

dissolve 5mg in 1000ml DMSO = 0,005mg/ml

4ml of this solution (= 4 type 000 capsules) gives us: 0,020 mg which is slightly above the effective dosage but definitely still between the under- and upper limit.

• 
  Dog:

(3,35µg/kg) * (3/20) = 0,5025 µg/kg

A 12 kg weighing dog would need the following dosage:

0,5025 µg/kg * 12kg = 6,03 µg = 0,00603 mg

Upper-and underlimit:
(Upper limit does not mean the maximum safe dosage, but it does mean the maximum dosage used in this experiment!)

• 
  Humans:

Under limit: 0,2716 µg/kg

Upper limit: 10 times the dosage (as seen in C16 study in 7 day old mice) = 2,716µg/kg

• 
  Dogs

Under limit: 0,00603 mg

Upper limit: 0,0603 mg

Cognitive testing

Human

Testing of memory ability in humans will be done through memory tests on: http://www.cambridgebrainsciences.com/
All these tests (memory categorie) are indeed short term memory tests and working memory tests. But test animals from the original study had an “one-trial-memory” induced, which means that only one trial was enough for the information to be transferred into their long term memory. This means that an improvement should be seen as well in short term memory tests.

Dog

As for testing the long term memory in dogs, I invented the following simple memory test which more or less taxes the spatial memory in the same way the morris water maze test does in mice.

To commence: A treat will be shown and given to at first excite the dog.
Next, a second treat will be placed somewhere in the room where the dog is situated in, but he is at the same time prevented from taking the treat prematurely.
The dog will then be placed outside the room where the treat was placed in. At that moment, the dog will be distracted for exactly 5 minutes. This time will be held consistent when the entire test will be done over again (multiple trials).
The dog is after 5 minutes of diversion, reëxposed to the initial room. The dogs time of trying to find the treat back will be timed.
During every trial a different spot for the treat to be placed will be chosen.

It goes without saying, I think, that every test (human and canine) will be done more than once (an average of 5 trials). What also goes futher without saying is that of each type of test, control tests (baseline tests) will be done done!
The scent of the treat shoud probably be masked with a certain spray to rule out that the dog finds the treat by smelling it and not by relying on its memory.


Toxicity reports

To investigate the possible toxicity of the compound, 2 bloodtests will be done. A controle bloodtest before experimentation and one after usage of the substance.
The bloodtests will monitor the following aspects:

• 
  gamma-glutamyl transferase
• 
  alanine aminotransferase
• 
  aspartaat aminotransferase
• 
  C-reactive protein (CRP)

These are all liver enzymes (and one protein synthesized by the liver), which pariculary interst me since I will orally take the compound. That means it will pass my digestive tract and therefore possibly stress my liver.
CRP will check for inflammation because PKR is involved in the stress pathway

[Googoltarian]

Few remarks:

-did you check if DMSO liquefies gelation capsules?

-you know that DMSO solidifies at 19°C and it takes few hours for 1L bottle to melt at RT after storage at lower temperatures?

-DMSO should be degassed, and dissolving should be done under protective gas

-DMSO solution should be divided (for example 1L to 20x50ml) and stored in freezer

-you can measure 1ml with syringes with accuracy comparable to pipette (10cm long, 1ml syringe)

-while experimenting on yourself could be in worst case scenario considered foolish by bystanders, doing same thing on animal without license could be prosecuted as animal abuse - this idea should officially remain theoretical

[JPC16]

I know that DMSO solidifies at 19°C. You dissolve the C16 in the "warm" DMSO, it hardens and then you freeze it. So if you know the DMSO is for instance exactly 50 ml, which you then weigh(the frozen dmso) which should be something around 51g. So 1/50 of that clumb of DMSO should be 1ml and contain x amount of C16.

The beauty of it is that you are operating at a mg scale and not microgram scale. So if you want to dose the x amount of C16 dissolved in one ml DMSO, you cut of 1/50 of the lumb of hard frozen dmso. The piece(s) can then be used in a capsule.
But I couldn't find anything about gel caps being liquefied by dmso. I know dmso dissolves a lot of things, but if you use hard starch capsules it wil take a while. What doen't matter since the cap will be made on the spot.

I also can drop the animal experiment, and only try it out on myself. I am pretty confident in the inhibitor's safety.
But what is the plan exactly? Are we first trying to replicate the results we have seen in other animals first, and investigate its safety in the proces?

[YOLF]

I think JPC16 proposal for dosing is better. C16 will not dissolve in water at pH higher than 3, and in pH <3 it will decompose. DMSO is good, it could be even used to transfer this drug transdermally.

DMSO is controversial, try something else.

[Googoltarian]

DMSO crystallizes differently than water. Believe me, you won't cut it easily

I just made quick experiment - I did put empty gelatin capsule in 10ml DMSO - after 3 min I touched it with a spatula and it broke to pieces.