Turning back to nerve growth factor, the main obstacle to its use in these therapies lies in the fact that concentrations of such magnitude have to be conveyed to the brain tissues that they cannot be taken via the normal administration methods. In fact, all the currently conducted studies have shown NGF effectiveness only if administered intracerebrally (intracerebroventricularly), since this molecule is unable to pass through the blood-brain barrier in therapeutic concentrations via systemic administration. The most recent therapeutic strategy for NGF use as an anti-AD drug is based on the intracerebral inoculation of genetically modified cells such that they locally release NGF (Tuszynski M.H. et al., J. Mol. Neurosci., 19(1-2):207, 2002; Tuszynski M.H. et al., Nat. Med., 11 (5):551-5,2005).
The possibility that NGF could exert a biological action on brain tissues following topical ophthalmic administration was hardly foreseeable, above all, in view of the fact that - as highlighted above - NGF is a molecule of considerable size (26,800 dalton) with a complex structure. In order for a molecule to have any action on brain tissues, then - once instilled on the eye surface - it must be able to pass through the eye tissues and reach the optic nerve and cerebrospinal fluid in order to exert its own biological activity on the brain region. In current practice, ocular administration is not used for treating brain pathologies. The foregoing is due to the fact that all the known studies concerning NGF use in brain pathologies have only employed intracerebral administration.
[0038]
In actual fact, although having a complex structure and a high molecular weight, NGF has both hydrophilic and hydrophobic structural groups which enable it to pass through the homologous (lipid and hydrophilic) anatomical barriers. Moreover, a fundamental feature of NGF is that once it reaches the target organs even at very low, but biologically active, concentrations, it can stimulate the endogenous production of NGF by the tissue itself. The presence of an endogenous fraction of NGF is clearly borne out by the results of experiments (which will be illustrated below) on the passage of NGF through tissues. These results also show that a concentration gradient is not maintained from the external surface of the eye towards the brain, as had been hypothesised for a simple diffusion mechanism through tissues.
http://www.google.co...nts/EP1948217B1
On the basis of the two quotes Metagene has provided you might be tempted to believe:
1. Only intracerebral injections of NGF are effective.
And this is because:
2. The NGF molecule is too big (28,000) daltons (daltons is basically atomic mass)
3. The NGF doesn't enter the body from the eye.
(Though the highlight section here is contradictory... the concentration gradient is NOT maintained,
this would mean that the NGF _is_ seeping into the body).
However, the patent cited by Metagene proves just the opposite:
1. Intercerebral injects are _not_ the only effective means of administration.
2 Despite the size of the protein, it is both soluble in water and soluble oil and can seemingly
travel easily through membranes of all kinds.
3. That NGF in eye drops _does_ enter the brain and travels efficiently to _all_ brain areas.
For example:
[0045]
In a first set of tests to evaluate the passage of NGF intraocularly from the external surface, on which it is administered, to the brain tissues, the aforesaid autoradiographic method was used on a group of six rabbits. Each of the rabbits received one collyrium drop (50 µl) containing 10 µg of I125 labelled NGF (concentration: 200 µg/ml) by instillation in the conjunctival fornix.
[0046]
....
Two hours following the administration of the labelled NGF, the animals were sacrificed and the brains dissected and fixed in 4% paraformaldehyde for 48 hours. Then, after incubation in 30% sucrose for 24 hours, the samples were cut with a cryostat to 15 µm thick sections, which were mounted on histology gelatinous slides, immersed in photographic emulsion (Ilford K2) and incubated for 4 weeks at 4°C. The sections were then dehydrated with ethanol, mounted with DPX after treatment with xylene and examined with a Zeiss optical microscope.
[0048]
This experiment demonstrated that, once administered on the eye surface, the labelled NGF was able to penetrate into the eye and to bond with cells of various brain tissues (including the cortex, hippocampus, basal forebrain, substantia nigra, locus coeruleus, hypothalamus and basal nucleus) expressing the specific receptor.
source: http://www.google.co...nts/EP1948217B1
Firstly, here's confirmation of my earlier post on dosage.
Here, they apply a drop of solution to the eyes of rabits. The concentration is 200mcg/mL.
The drop volume is 50 ul. It contains 10mcg of NGF (in this case, radioactively labelled NGF)
Second, they took 6 rabbits, dropped radioactively labelled NGF into their eyes and then
killed them two hours later to see where the NGF had gone. Where had the NGF gone ?
It had traversed into the entire brain. It had gone where ever the NGF receptor was present.
This is a remarkably effective delivery system.
If you now consider Metagene's first quote (above) it's clear that
.. that quote is utterly misleading.
Third, this is a remarkable finding.
NGF in eye drops finds it's way to _all_ areas of the brain.
The question is, how does it do this?
Two choices here, either:
(a) the NGF _diffuses_ to these brain regions.
(b) the NGF causes a _cascade_of_signalling_ to (presumably millions or billions) of brain cells
for them to release (or produce) their own NGF.
You might argue, it doesn't matter which option is 'true'.
It only matters that we get the desired NGF release within the brain.
However, my view is that it does matter because:
If it's option (a) then you'll be concerned to drop a sufficiently large dose of NGF into the eye.
However, if it's option (b) then (presumably) you need a much smaller dose to kick off the signalling.
Option (a) might correspond to the 4mcg to 400mcg range (suspected of Genentech)
Option (b) might correspond to the 0.04mcg range of McMichaels
(However, here we know that they used a dosage of 10mcg from a solution of 200mcg/mL).
Fourth, The above experiment was replicated.
In this study, a total of 24 rats were used, six of which were sacrificed immediately
in order to determine the baseline values of NGF concentration in the various brain tissues.
The remaining animals were sacrificed 2 hours (6 rats), 6 hours (6 rats) and 24 hours (6 rats)
after eye-drop administration.
NGF levels in various brain tissues after NGF administration in the form of eye-drops (NGF pg/g of tissue)
HOURS (A) (B) © (D) (E) (F) (G) (H)
0 20±7 70±12 135±32 102±23 98±34 75±36 170±45 112±24
2 90±15 120±17 176±14 153±34 178±25 154±71 286±71 165±31
6 530±59 312±52 212±23 264±68 329±60 312±79 396±54 254±42
24 30±12 81±20 127±21 120±31 135±59 91±84 198±38 131±39
KEY to Columns:
(A) cerebro-spinal fluid
(B) cortex
© hippocampus
(D) basal forebrain
(E) substantia nigra
(F) locus coeru-leus
(G) hypothalamus
(H) basal nucleus
This study doesn't allow us to come to conclusions about whether option (a) or option (b) is true.
These numbers might be obtained via either route. But what this data does do is show us that
seemingly all brain areas are bathed in substantially higher levels of NGF for (at least) the
next 24 hours.
I wish they'd have sacrificed 6 more rats at 12 hours so we'd have a better idea
of how high those numbers really go.... and maybe, again at 18 hours so we get an idea of the
rate of decline from the apex.
So, in summary.
1. It's not true that _only_ inter-cerebral injections are effective.
2. It's not true that the NGF molecule is somehow 'too big' to enter the body or to cross the blood brain barrier.
3. It's not true NGF can't enter the brain from the eye.
Some pivotal points to note here are:
=> These experiments were done with 'murine' NGF.
Murine means, 'of or relating to mice'.
Rita Levi-Montalchini & Cohen decided to go the route of extracting NGF from mice (rather than cobras)
and the majority of all NGF data is based on mouse NGF. Many (perhaps most) of those papers were written by Rita.
=> There is a difference between human and mouse NGF.
Mouse NGF is denoted (mNGF)
We currently artificially synthesise human NGF. It's called recombinant human NGF (rhNGF)
To quote the second of Metagene's references (above)
There is 90 % homology in the amino acid sequence of the mature human and mNGF protein.
(source: http://www.ncbi.nlm....es/PMC4030100/)
In other words, there are 10% differences.... similar, but not the same)
=> I remember reading somewhere (but at the moment i can't back this up with a reference) that Rita
was using these eye drops for 40 years. Let's imagine that that's true.
If she started using the drops 40 years ago, that means she started around 1975. When she was in her 60's
Human recombinant NGF (rhNGF) was invented in 1995. The paper about it's creation was published in 1996.
So that suggests that Rita was using murine NGF for at least twenty years.
(She specifically chose not to use Cobra NGF, in her research, for safety reasons).
=> So, you might think: 'Great! we know the dosage, we know the source' (10mcg of mNGF, applied once per day)
But wait a moment, there are potential problems in using chemicals from other animals.
Consider the 'bio-identical hormones' movement. The estrogen given to women for Hormone Replacement Therapy
and contraception purposes is actually equine (horse) estrogen. It's extracted from the urine of pregnant horses.
This estrogen is very nearly the same, but is in fact slightly different.
It seems to do the trick. It has the expected effects.
But long term this stuff has complications... it has long term toxic effects on women.
There's a famous, and very compelling book on this, by Suzzane Summers, 'Ageless'.
Very readable, very interesting, i recommend it... you can probably get a used copy from amazon for a couple of dollars.
The 'bio-identical hormones' movement says: We want human hormones, not hormones from other animals.
Because even tiny differences between human and horse estrogen can have long term health consequences.
Playground
Edited by playground, 02 June 2015 - 10:50 AM.
