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Embryonic window in humans, when to apply the EGFR inhibitor


#1

I have been thinking about when the embryonic window actually occurs in humans. I have tried to logically reason from the patent and will try to share it with everyone in a simple way. Thoughts and comments are welcomed.
Here are some studies with mice and conclusions taken from the patent. Wound induction in these examples are most likely incisional wounding and not abrasion. No EGFR inhibition in the first four examples.

“Figure 16. No new HF are evident 11 days after wound induction in 21- day-old mice”

“Figure 4. Appearance of hair germs 12 days after wound induction”

12 days after wound induction, hair germs, with similar morphology to fetal hair germs, were observed in the wound site”

“Figure 17. 14 days after wound induction, new HF have begun to form”

“Figure 11. New hair growth 25 days

“AG1478 (150 [mu]M in 10 [mu]L volume) was administered as a single injection. 11 days after incisional wounding (1 cm<2>) to the middle of the wound near the skin surface. EGF receptor inhibitor administration led to generation of more and larger hair follicles compared with control mice that were wounded only”

"An area of the backs of 50-day old mice was subjected to depilation and removal of the epidermis using a rotating felt wheel. Fifteen days later, HF placodes, hair germs and other signs of follicle neogenesis were present "

Me again. First by day 12 signs of hair formation is seen in mice wounded by incision. By day 14 the hair follicles have begun to form. One day before any signs of hair follicles can be seen an injection with EGFR inhibitor creates more and larger hair follicles. This must be the “window of oppurtunity” for incisional wounding in mice. When the skin is abraded this seems to occur a bit later, at day 15 signs of har formation is seen, when wounding deep the hair follicles have already begun to form by now. Since the window of opportunity seem to be one day before any signs of hair germs it should be by day 14 when abrading the skin in mice.
Also it is clear, as stated by others before, that waxing the hair before will enhance the hair follicle formation:

“As depicted in Figure 20A- B, the depilated mice exhibited enhanced EDIHN relative to the non-depilated mice depicted in the previous Example by a factor of 11-fold

I will compare these findings with experiments with human skin:

"To determine whether human skin responded to EDIHN as did mouse skin, human skin was grafted onto SCID (immuno-def[iota]cient) mice and subjected to depilation by plucking and wound induction three days later. Seven days following wound induction, formation of new HF was observed in the human skin "

“adult human skin was grafted onto mice, abraded, and examined at 7 days post-abrasion. New HF were generated in the human skin, which mimicked normal hair follicle formation during fetal development”

“Figure 2 IA. EDIHN in human skin grafted to immunodeficient (scid) mice, seven days after induction of an excisional wound. Arrows indicate new HF

“Figure 21B. Dermal abrasion of human skin grafts results in EDIHN. Human adult skin (W) was grafted onto mice, abraded, and examined seven days later, by staining for S100A6 (first, second, and fourth rows) or S100A4 (third row). Hair germs (HG) and dermal papilla (DP) are indicated.”

Me again. Human skin apparently form hair follicles faster than mouse skin (yes I am disregarding the fact that the skin was grafted on mice for the moment) and incisional wounding seem to work faster than abrasion. Since the window of oppurtunity seems to be one day before any signs of hair germs I come to the conclusion that this most likely occur by day 6 in humans when abrading the skin. Since we dont know what happens when administering EGFR inhibitors before the window of oppurtunity I would like to see someone try a topical Gefitinib solution applied at day 6 post wounding. I would do it my self but these drugs are way out of my budget. Thoughts?


#2

Stages at which compounds of the invention may preferably be administered and/or activated include periods:

after completion of the reepithelialization process (e.g., 3-12 days, or 9- 11 days after having disrupted the skin),■ after or during the establishment of a stem cell population that will develop into a regenerated hair follicle (Ito et al, Nature 447, 316-320, May 2007),

■ prior to the expression of hair follicle differentiation markers KRT 17 and Lefl for several days after wound closure (Ito et al,

Nature 447, 316-320, May 2007),

■ after or during expression of one or more proteins including KRT 17, Lef-1, alkaline phosphatase, WntlOb, and Shh (Ito et al,

Nature 447, 316-320, May 2007),

■ characterized by the absence of KlO expression (which is expressed in normal epidermis) and/or induction of expression of Kl 6 and Kl 7 (which are not expressed in normal epidermis) (Patel et al, Journal of Investigative Dermatology, 126, 2006), ■ charactized by the elevation of one or more transcription factors including AP-I and NF-κB, primary cytokines IL- lβ and TNF-α, and matrix metalloproteases (Karimipour et al, Journal of the American Academy of Dermatology, 52, Issue 2, 2005),

■ characterized by histologic changes (Freedman et al, Dermatologic Surgery, 27 Issue 12, December 2001), including, for example: o thickening of the epidermis and dermis, o flattening of rete pegs, o vascular ectasia, o perivascular inflammation, o hyalinization of the papillary dermis with newly deposited collagen and elastic fibers, o change in orientation, density, or packing of collagen and other structures, ■ Alternatively, hair follicles may start to form before the scab falls off, in thcharacterized by detachment of the scab. Depending on the depth of the abrasion process, it may be desirable for the compounds of the invention to be administered or activated prior to or after the detachment of a scab. e case of, for example, dermabrasion.

Alternatively the compounds of the invention can be administered prior to epidermal disruption. In such embodiments, the compound may be formulated for controlled release such that the therapeutically active compound is released during reepithelialization or during a particular phase of

reepithelialization (e.g., as described above. The compound may also be formulated such that it becomes activated by an endogenous or exogenous stimulus

CONFUSING AS ALL GET IT ISN’T IT?

EVEN MORE CONFUSING:

In other experiments, continued expression of Dkkl after the 9-day period inhibited formation of new HF.
The findings of this Example show that pigmented HF can be produced by suppressing expression of Wnt 1 or by inducing expression of Dkkl during the period of re-epithelialization, then inducing expression of Wntl. In addition, the findings of this Example show that factors that inhibit neonatal hair follicle formation (e.g., Dkkl) also inhibit EDIHN, thus further supporting the notion that hair follicles formed by EDIHN are similar to normal hair follicles.

Example 11: Inhibition of EDIHN by epidermal growth factor injection.

21 day-old mice were wounded as described in previous Examples. Starting from day 11 after wounding, a time point corresponding to the point at which the wound had recently re-epithelialized, 10 mL of 1 mg/ml EGF was injected into the wound bed. EGF was injected once per day after this point for a total of 5 days. Three days later, the skin was collected, and whole-mount EDIHN assays were performed. EGF prevented HF formation as assessed by gross morphology . In addition, whole mounts of control and treated skin were analyzed with anti-K17 antibody immunostaining. All mice injected with EGF (n=4) exhibited no new HF formation (Figures 25 A-B), while control mice (n=2) had many new HF, as expected. (Figures 25 C-D).

In an additional experiment, recombinant EGF (1 microgram

(mcg)/microliter (mcl)) was injected at days 11, 13 and 15 after wounding.

Skin was collected at 18 days after wounding and stained for K17 and alkaline phosphotase. Once again, administration of EGF inhibited EDIHN.

The findings of this Example show that EGF inhibits HF formation. Thus, inhibiting EGF, EGFR, or one of the pathways in which they participate increases EDIHN- induced HF formation.


#3

Im practically certain I screwed up my own experiment, because ketoconazole is contraindicated with getfitinib-----and I used it three times----resulting in a rash all over my head…(I have thick hair though, and its well-concealed fortunately).

However, reading through the patent and reading about re-epilithialization over and over has got me thinking this:

If someone abrades their skin…they need to wait for the skin to “glaze over” and BEGIN to crust. Just as soon as the first crust starts peeling off, WHATEVER DAY THIS IS (it depends on how far down you went when you abraded, if you went too deep-----this will be longer than day three or four…it could be several days after that) THAT is the time to inhibit EGF. That is also the time to take an anti-inflammatory and anti-histamine (Cotsarialis experiments have shown in neonatal mice that hair follicles wont form in inflammed skin) also.

Looking back------I dont think someone attempting this should use anything other than water on their abraded area post wounding. If just one substance in your shampoo intereferes with the epidermal stem cells, then nothing is going to be made. Its too much trouble to go to to risk that.


#4

Cal,

The first section in this got me to thinking about your experiment:

"How does it work?

Arava tablets contain the active ingredient leflunomide, which is a type of medicine called a disease-modifiying antirheumatic drug (DMARD). It is mainly used to treat rheumatoid arthritis.

Rheumatoid arthritis is known as an autoimmune disease. The inflammation and damage to the joints in this disease result from overactivity in the immune system. Leflunomide works by suppressing the excessive activity of the immune system and modifying the process of inflammation. This actually slows progression of the underlying disease.

It is not understood exactly how leflunomide works. However, it is known that it prevents the activation and multiplication of white blood cells known as lymphocytes, which are responsible for causing inflammation.

By preventing the activation of these lymphocyte cells, blood levels of certain inflammatory chemicals that the body’s immune system produces in rheumatoid arthritis are reduced. Once these have fallen, the inflammation of the joints lessens, which helps relieve the pain.

You should not expect this medicine to start to reduce the pain in your joints for at least four to six weeks after starting treatment, because it can take this long to start working. However, your symptoms may then continue to improve for the next four to six months of treatment. After this you will then usually have to continue taking the medicine long-term to keep your arthritis under control"


#5

Benji that’s very interesting.

But does this article’s info say whether they were including a major “loading dose” or not?

I think I had that base at least somewhat covered.
I slammed 300mg of that stuff during the first 3 days, and only afterward did I back it down to 20mg/day.

That 300/3 was the original recommended way to start using Leflunomide until they discovered the lung issue. Only then did they throw out that idea and recommend starting gradually (obviously so the patient could back out sooner at signs of trouble).

So I basically just said “fcuk it” and did the whole 300/3 loading dose, lung risk or not. Whatever minor effects the stuff did have, I remember that I was feeling the stomach/crapping troubles almost immediately. Within a day or two at most.


#6

these drugs are way out of my budget…(also I have this problem)Z79. Benji which another inhibitor of egfr you used? a box with 150 compressed of gefinitib in Brazil costs 2500 USS.


#7

I think you are absolutely right about not applying before the crust falls off. benji would you be willing to sell me one of your pills? the more we are trying, the faster we will find the right path. » Im practically certain I screwed up my own experiment, because ketoconazole » is contraindicated with getfitinib-----and I used it three » times----resulting in a rash all over my head…(I have thick hair though, » and its well-concealed fortunately). » » » However, reading through the patent and reading about re-epilithialization » over and over has got me thinking this: » » » If someone abrades their skin…they need to wait for the skin » to “glaze over” and BEGIN to crust. Just as soon as the first crust starts » peeling off, WHATEVER DAY THIS IS (it depends on how far down you went when » you abraded, if you went too deep-----this will be longer than day three or » four…it could be several days after that) THAT is the time to inhibit » EGF. That is also the time to take an anti-inflammatory and anti-histamine » (Cotsarialis experiments have shown in neonatal mice that hair follicles » wont form in inflammed skin) also. » » Looking back------I dont th


#8

» Im practically certain I screwed up my own experiment, because ketoconazole
» is contraindicated with getfitinib-----and I used it three
» times----resulting in a rash all over my head…(I have thick hair though,
» and its well-concealed fortunately).
»
»
» However, reading through the patent and reading about re-epilithialization
» over and over has got me thinking this:
»
»
» If someone abrades their skin…they need to wait for the skin
» to “glaze over” and BEGIN to crust. Just as soon as the first crust starts
» peeling off, WHATEVER DAY THIS IS (it depends on how far down you went when
» you abraded, if you went too deep-----this will be longer than day three or
» four…it could be several days after that) THAT is the time to inhibit
» EGF. That is also the time to take an anti-inflammatory and anti-histamine
» (Cotsarialis experiments have shown in neonatal mice that hair follicles
» wont form in inflammed skin) also.
»
» Looking back------I dont think someone attempting this should use anything
» other than water on their abraded area post wounding. If just one substance
» in your shampoo intereferes with the epidermal stem cells, then nothing is
» going to be made. Its too much trouble to go to to risk that.

I’m having a very hard time following all this. Here is my only question – would applying the topical egf-r too early (say, first day of wounding) be counter productive to the experiment? It is all timing. We all agree that applying the stuff too late would certainly produce nothing but how about too early? It is better to be too early than too late if you don’t get penalized for it.


#9

Z79,
You can go to HLT and email me there. My addy there is Michael Barry.

I dont know how I could send a pill in the mail though. Wouldn’t they just confiscate it. I know mail is screened for powders now. That problematic.

You’d only need one pill to make a cream. One pill contains 250mgs. Thats like 250 doses of propecia. The patents highest composition of a cream was only something like 5% or some such. In other words, one pill would go a long way if your topical was right. You’d simply have to read the patent and see what exact topical preperations they were talking of using. I dont think it was going to be alcohol of any kind, but creams. Thats why I think Dermovan from the drug store or Cetaphil would probably be acceptable.

Cal,
That goes for you too. If you want one tablet of getfitinib, I’ll give you both one if you can think of a way for me to send it to you where I dont get arrested or something (LOL). Generic getfitinib isn’t supposed to be sold, etc. Im Michael Barry at HLT (I think you already know that).


#10

» I have been thinking about when the embryonic window actually occurs in
» humans. I have tried to logically reason from the patent and will try to
» share it with everyone in a simple way. Thoughts and comments are
» welcomed.
» Here are some studies with mice and conclusions taken from the patent.
» Wound induction in these examples are most likely incisional wounding and
» not abrasion. No EGFR inhibition in the first four examples.
»
» “Figure 16. No new HF are evident 11 days after
» wound induction in 21- day-old mice”
»
» “Figure 4. Appearance of hair germs 12 days after wound
» induction”
»
» “12 days after wound induction, hair germs, with similar
» morphology to fetal hair germs, were observed in the wound site”
»
» “Figure 17. 14 days after wound induction, new HF have begun
» to form”
»
» “Figure 11. New hair growth 25 days
»
» “AG1478 (150 [mu]M in 10 [mu]L volume) was administered as a single
» injection. 11 days after incisional wounding (1 cm<2>) to the middle
» of the wound near the skin surface. EGF receptor inhibitor administration
» led to generation of more and larger hair follicles compared with control
» mice that were wounded only”
»
» "An area of the backs of 50-day old mice was subjected to depilation and
» removal of the epidermis using a rotating felt wheel. Fifteen
» days later
, HF placodes, hair germs and other signs of
» follicle neogenesis
were present "
»
» Me again. First by day 12 signs of hair formation is seen in mice wounded
» by incision. By day 14 the hair follicles have begun to form. One day
» before any signs of hair follicles can be seen an injection with EGFR
» inhibitor creates more and larger hair follicles. This must be the “window
» of oppurtunity” for incisional wounding in mice. When the skin is abraded
» this seems to occur a bit later, at day 15 signs of har formation is seen,
» when wounding deep the hair follicles have already begun to form by now.
» Since the window of opportunity seem to be one day before any signs of hair
» germs it should be by day 14 when abrading the skin in mice.
» Also it is clear, as stated by others before, that waxing the hair before
» will enhance the hair follicle formation:
»
» “As depicted in Figure 20A- B, the depilated mice exhibited
» enhanced EDIHN relative to the non-depilated mice depicted in the previous
» Example by a factor of 11-fold
»
» I will compare these findings with experiments with human skin:
»
» "To determine whether human skin responded to EDIHN as did mouse
» skin, human skin was grafted onto SCID (immuno-def[iota]cient) mice and
» subjected to depilation by plucking and wound induction three
» days later
. Seven days following wound induction, formation of
» new HF was observed in the human skin "
»
» “adult human skin was grafted onto mice, abraded, and
» examined at 7 days post-abrasion. New HF were generated in
» the human skin, which mimicked normal hair follicle formation during fetal
» development”
»
» “Figure 2 IA. EDIHN in human skin grafted to immunodeficient (scid)
» mice, seven days after induction of an excisional wound. Arrows
» indicate new HF
»
» “Figure 21B. Dermal abrasion of human skin grafts results in
» EDIHN. Human adult skin (W) was grafted onto mice, abraded, and examined
» seven days later, by staining for S100A6 (first, second, and fourth
» rows) or S100A4 (third row). Hair germs (HG) and dermal papilla (DP)
» are indicated.”
»
» Me again. Human skin apparently form hair follicles faster than mouse skin
» (yes I am disregarding the fact that the skin was grafted on mice for the
» moment) and incisional wounding seem to work faster than abrasion. Since
» the window of oppurtunity seems to be one day before any signs of hair
» germs I come to the conclusion that this most likely occur by day
» 6
in humans when abrading the skin. Since we dont know what happens
» when administering EGFR inhibitors before the window of oppurtunity I would
» like to see someone try a topical Gefitinib solution applied at day 6 post
» wounding. I would do it my self but these drugs are way out of my budget.
» Thoughts?
I think the formation of hair germs is more gradual than in a one day window.
And I keep coming back to the fact that EGF only comes into play during epithelization. Also, the act of keeping the wound open and thus delaying epithelization gives the cells more time to differentiate into HF cells WHEN EGFR INHIBITOR IS APPLIED. It’s just my take on it but I believe the window is primarily at the epithelization stage and there is no effect both before and after this stage. In other words, it won’t harm the process to inhibit EGF beforehand and get EGF activity down real low before wounding and it also won’t hurt if EGF remains low after differentiation is complete. I think maybe we’re bestowing this procedure with some kind of ‘Harry Potter-like mysticism’ as to the window. This belief is reinforced deliberately by wording of the patent.
Just my opinion and not based on any medical knowledge.


#11

» I dont know how I could send a pill in the mail though. Wouldn’t they just
» confiscate it. I know mail is screened for powders now. That problematic.
»

Go to the store and buy the cheapest vitamins. put the pill in the with the vitamins and send the whole thing of vitamins.


#12

» » I dont know how I could send a pill in the mail though. Wouldn’t they
» just
» » confiscate it. I know mail is screened for powders now. That
» problematic.
» »
»
» Go to the store and buy the cheapest vitamins. put the pill in the with
» the vitamins and send the whole thing of vitamins.

Ooohh . . . check out the big brain on CMS!


#13

» » I have been thinking about when the embryonic window actually occurs in
» » » I think the formation of hair germs is more gradual than in a one day
» window.
» And I keep coming back to the fact that EGF only comes into play during
» epithelization. Also, the act of keeping the wound open and thus delaying
» epithelization gives the cells more time to differentiate into HF cells
» WHEN EGFR INHIBITOR IS APPLIED. It’s just my take on it but I believe the
» window is primarily at the epithelization stage and there is no effect both
» before and after this stage. In other words, it won’t harm the process to
» inhibit EGF beforehand and get EGF activity down real low before wounding
» and it also won’t hurt if EGF remains low after differentiation is
» complete. I think maybe we’re bestowing this procedure with some kind of
» ‘Harry Potter-like mysticism’ as to the window. This belief is reinforced
» deliberately by wording of the patent.
» Just my opinion and not based on any medical knowledge.

that certainly could be true Baccy…however we have seen that DKK-1 expession before re-epilithialization is complete can indeed thwart Hair Follice Formation in the mice via their experiemnts.
'The desire not to be on an internal egf antagonist any longer than possible is due to the side effects. It really is awful stuff. It causes hyperkeratinization of hair follicles, which means your hair follicles have keratinocytes die within the hair shaft very quickly and do not slough off properly and get stuck in the opening in the dermis where the hair exits the body. This leads to severe inflammatory acne in a matter of days. I have very good skin----Im just now clearing up. You would not believe how bad (painful too) my face and shoulders and upper back “broke out”. I looked like an ugly teenager. This was from seven straight days of getfitinib at 250 mgs a pill. Needless to say, it would be better if a topical could be compounded, but that topical would have to be able to penetrate dying upper skin layers—a unique task. If you could get the one to two day window “down”, you’d probably have it. When the epidermal stem cells “decide” to make hairs and begin to go about it, I imagine the rest of the process follows rote pretty much. Its not as if they will change their minds. The patent covered days (6-9) in one embodiment. The fact Follica felt it necessary to cover such a short period of time does say something. Since getfitinib stays in the body longer than a day…One could just take two pills if they had the right two days lined up and caught it just right.

I sincerely hope that YOU, Baccy, hit a homerun with diluted tannic acid.

I also “think” that some people might only need anti-inflammatories and anti-histamines to get a result. The mice with human skin on their backs didn’t have a egf-antagonist applied, but did get more hair when one was applied. If somebody depilated, waited three days, abraded, and did the anti-inflammatories when the wound glazed over with keratinocytes…its hard for me to believe they’d get nothing. Im sadly confident that no anti-infective should be used on this skin during this time though.


#14

Needless to say, it would be better if a topical could be compounded, but that topical would have to be able to penetrate dying upper skin layers—a unique task.

I don’t see how Folica could expect to ultimately sell this whole gig if they can’t get Gentifilb into the skin topically somehow. I just don’t see a very widespread market for something that involves making yourself systemically sick like what happened to you, Benji.

However –

Oral Leflunomide hardly did sh*t to me in comparison. Even with the serious “loading dose” at the beginning of 300 mg in the first 3 days. And I suspect my experiment failed for lack of immune suppressants, not because of a failure to inhibit EGF-R.

Maybe I will reconsider another run of oral Leflunomide after all. If we can’t get Genfilitib into the skin and we can’t withstand it orally either, this is the next thing on the table.

It will probably pose a real trick to time it right with the wounding & loading doses. But I see no other obvious failure points if that was indeed handled right.


#15

Cal,
They INJECTED a synthetic EGF antagonist in some of the experiments. If one of the EGF inhibitors was alchohol or water soluble…


#16

Alcohol/water-soluable would be great. I think Gen & alcohol or water is the next thing we need to investigate.

(Wasn’t there some idea that Genfilitib might have a decent shot at getting through the skin? Like from some other research data somewhere? I thought this was partly why we all zeroed in on Genfilitib as the probable EGF-R inhibitor for Folica’s deal in the first place.)

Here’s another crazy thought. It’s certainly not the next thing I would try, but I’ll start thinking about it if we can’t make a topical work:

I wonder about taking a smaller amount of both Gen & Lefl at the same time. Like maybe take 200 mg (rather than the 300mg loading dose) of Leflunomide a day or two before you want to get the effect, and then take a single Genfilitib pill on the perfect day to make absolutely sure you got the EGF-R inhibited at that time. Then take just a little more Leflunomide for a few days to finish the job.

This seems like a decent way to get the lion’s share of the effect from Leflunomide (read: no vomiting & acne), but still use Genfilitib’s immediate-action properties to help you bullseye the exact day to begin.

Of course, ALL OF THAT is predicated on the idea that having both Leflunomide & Genfilitib in your system at once is safe. It may not be even remotely safe, I don’t know. I don’t know who to even ask about this idea either.


#17

» I have been thinking about when the embryonic window actually occurs in
» humans.

It is clear as a bell that the embryonic window opens after re-epithelialization. The original Nature paper says as much. Furthermore, most of the original experiments (published in Nature) as well as the patent experiments - for both creating and suppressing hair follicles - utilize a waiting period to demonstrate when the crucial interval is for manipulating follicle generation.


#18

» I think you are absolutely right about not applying before the crust falls
» off.

That’s why I’ve decided to extend the window on the meds. At Day 13, my scabs are all beginning to fall off so from I shall continue with the topicals for another 8 to 10 days. Some of the existing hair, althouigh not all of it has begun to grow back.