Found a really good paper which is the first definitive research I've seen into the peptide composition of Cerebrolysin. The main thrust of the paper is examining the effect CRB has on FGF-2, which in elevated levels (as in AD) prevents effective progenitor differentiation into neurons.
I'll focus first on the composition information, then move on to the very interesting experiments involving neural differentiation.
Trophic factors counteract elevated FGF-2-induced inhibition of adult neurogenesis. [free full PDF]
Chen H, Tung YC, Li B, Iqbal K, Grundke-Iqbal I.
New York State Institute for Basic Research in Developmental Disabilities
The dentate gyrus of adult mammalian brain contains neural progenitor cells with self-renewal and multi-lineage potential. The lineage and maturation of the neural progenitors are determined by the composition and levels of the trophic factors in their microenvironment. In Alzheimer disease (AD) brain, especially the hippocampus, the level of basic fibroblast growth factor (FGF-2) is markedly elevated. Here we show that elevated FGF-2 enhances the division and nestin levels of cultured adult rat hippocampal progenitors but impairs neuronal lineage determination and maturation of these cells in culture. The trophic factors ciliary neurotrophic factor (CNTF), glial-derived neurotrophic factor (GDNF), and insulin-like growth factors-1 and -2 (IGF-1, IGF-2) as well as an Alzheimer peptidergic drug, Cerebrolysin(®) (CL), in which we found these neurotrophic activities, counteract the effect of FGF-2 in inducing neuronal lineage (early neurogenesis). Whereas CNTF is the most active of the neurotrophic factors studied in promoting neurogenesis, CL, probably because of a combined effect of these factors, induces similar changes but without inhibiting cell proliferation. These findings suggest that CNTF, GDNF, IGF-1, and IGF-2 are promising therapeutic targets for AD and other diseases in which neurogenesis is probably inhibited.
PMID: 16859812 [PubMed - indexed for MEDLINE]
3.3. CL contains CNTF, GDNF, IGF-2 and IGF-1 activities
Based on the above findings that CL is neurotrophic/neurogenic, it appeared likely that some of the biologically active peptides in CL might be the proteolytic products of some of the known neurotrophic factors. Therefore, we chose the ELISA to screen CL for the presence of such peptides. Because regular ELISA plates might not bind all peptides in CL equally well, we covalently linked them to Covalink plates for this assay. We found that CL contained peptides reacting with neutralizing antibodies to human CNTF, GDNF, IGF-1, and IGF-2 (Fig. 4A).
Proteolysis is enzymatic protein digestion. So this antibody reaction indicates that CRB contains active peptide fragments of these growth factors, which have some portion of the same effect.
This paper also examines the ability of CRB to encourage differentiation of adult hippocampal progenitor (AHP) cells. Nestin is a marker for immature cells that have not yet differentiated into neurons, astroglia, or oligodendroglia. Class III β-tubulin (TuJ1) is a marker for differentiated neural stem cells, and microtubule-associated protein 2 (MAP2) indicates neurons a little further along in development. Glial fibrillary acidic protein (GFAP) indicates astroglia.
They compare the effect of CRB alone to the four growth factors
As compared with the control AHPs, which were cultured in FGF-2 alone, CL decreased the percentage of nestin-positive cells by ∼20% and simultaneously increased the Tuj1- and MAP2a,b-positive cell populations by a similar percentage. CL also slightly increased the GFAP-positive cells, but had no effect on the percentage of O4-positive oligodendroglia.
...
In the presence of 5 ng/ml FGF-2, CNTF (at 1, 5, and 10 ng/ml) reduced the nestin level of these cells by almost 50%.
At the same time, at 10 ng/ml, CNTF increased MAP2a,b and, most strongly, Tuj1 levels at 5 and 10 ng/ml. Unexpectedly, 1 ng/ml, the lowest concentration of CNTF, decreased the levels of Tuj1 and MAP2a,b.
Both CL and CNTF increased the GFAP level
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The most dramatic dose response reaction, however, was obtained with CNTF, which already at 10 ng/ml induced Tuj1 expression well above the levels observed in cells treated with 5 ng/ml FGF-2. CNTF, GDNF and IGF-1 increased Tuj1 expression in a dose-dependent manner up to a concentration of 100 ng/ml studied.
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...all four factors, like CL, increased the Tuj1-positive cell population, with CNTF having the highest (~50%) and IGF-2 the lowest (20%) effect. Similarly, all factors except IGF-2 increased the percentages of BrdU-positive neurons (Tuj1-positive) from ~20% to 60% in the case of CNTF and to ∼45% in the case of GDNF, IGF-1, and CL.
...
3.5. CNTF neutralizing antibody inhibits the neurogenic effects of CNTF and CL
We postulated that the neurotrophic/neurogenic activity of CL was at least partly due to the presence of an active fragment of CNTF in this preparation. To test this hypothesis, we investigated the effect of a CNTF neutralizing antibody on CL-, and as a positive control, on CNTF-induced changes on the levels of nestin and Tuj1 in AHPs...Western blot analysis of the treated cells revealed that addition of the antibody to CNTF indeed inhibited the neurotrophic/neurogenic activities of both CL and CNTF, as determined by an increase in the level of nestin, a marker for immature cells, and a decrease in the level of the neuronal marker Tuj1.
4. Discussion
...
In CL, we found CNTF, GDNF, IGF-1, and IGF-2 peptides presumed to be biologically active. Of these trophic factors, CNTF was by far the most active towards neurogenesis, followed by GDNF and IGF-1, which had approximately the same activity as CL. In contrast, IGF-2 had lesser neurogenic activity at high FGF-2 but had the strongest ability of all factors to drive the AHPs towards more mature neuronal stages at low FGF-2 concentration.
...
By far the most important amongst the four trophic factors found in CL is CNTF because of its strong neurotrophic and neurogenic characteristics and, importantly, because it was the only factor studied that not only fully counteracted the strong mitogenic activity of elevated FGF-2 but also reduced nestin to the same level as was found at low-level FGF-2.
...
CNTF is known to be a strong inducer of the glial lineage in cultured fetal precursors, which almost totally differentiate into astroglia within 2 days. In contrast, in the present study, the effect of CNTF on adult hippocampal progenitors was different. Only a few cells presumed to be previously uncommitted differentiated into astroglia, whereas the large majority of the cells strongly expressed the early neuronal marker Tuj1.
This was obviously a limited model, primarily relevant to increased FGF-2 in AD. Presumably, the 5ng/mL amount of FGF-2 used as a control in some instances is closer to normal amounts in the healthy brain. So to some extent, these actions can probably be expected in healthy individuals, and likely constitute a large portion of CRB's actions.
I don't know if we've arrived yet at a comprehensive idea of active components. They didn't test for presence of NGF or BDNF fragments; perhaps their presence is ruled out somehow. The study demonstrates that CNTF fragments are responsible for a large portion of the effects in this model. Whether this would be true for other measures remains to be seen. But as CNTF Peptide 6 was selected for development because of its strong performance in a largely AD-specific model, I think a strong case can be made that full-spectrum Cerebrolysin might still be a better choice for general nootropic usage. At least until a wide range of studies are available.
Speaking of which, a later research summary by these same authors links this work to the new Ebewe patent, and Peptide 6:
Development of neurotrophic peptides for enhancement of neurogenesis and associated behavioral improvement
Inge Grundke-Iqbal, Muhammad O. Chohan, Bin Li, Julie Blanchard, Lukas Wanka, Khalid Iqbal
Background: Levels of neurotrophic factors and the dentate gyrus neurogenesis are dysregulated in AD brain. The anti-dementia drug Cerebrolysin, which contains several neurotrophic activities, enhances the dentate gyrus neurogenesis and improves cognition in adult rats. To assess the neurogenic/neurotrophic (ngc/ntc) activities and corresponding neurobehavioral effect in mice of 4-11 mer synthetic peptides corresponding to the active regions of CNTF and the possible molecular mechanisms of action of these peptides. Methods: An 11-mer peptide, Peptide 6, and several 4-mer peptides of Peptide 6 were designed based on a biologically active region of CNTF. These peptides were administered to female retired breeders (C57 BL6 mice) either subcutaneously as a slow release bolus or daily IP injections at a dose of 5 to 50 nmoles peptide/day for 4-5 weeks. On days 2-6 of the treatment with the test peptide, the animals received IP 100 mg BrdU/kg/day. Results: Administration of Peptide 6 subcutaneously as a slow release bolus at a dose of 5 nmoles/day for 4 weeks enhanced the dentate gyrus neurogenesis and hippocampal-dependent spatial memory in normal adult mice. This peptide increased proliferation/survival of newborn neurons, along with a general neurotrophic effect. Of the four 4-mer peptides (Peptides 6A, 6B, 6C, and 6D), Peptide 6C was found to enhance most the dentate gyrus neurogenesis in mice. Both Peptide 6 and Peptide 6C increased the expression levels of MAP2 and synaptophysin in the dentate gyrus, and improved cognition in Morris water maze. These peptides had plasma half-lives of over 6 hours and were blood brain permeable. Peptide 6 was found to inhibit LIF-induced haptoglobin secretion from HepG2 cells. Thus, Peptide 6 appears to enhance neurogenesis in adult mice as a competitive inhibitor to LIF which is known to decrease overall neurogenesis in the subventricular zone. Conclusions: Peptide 6 and its neurogenic/neurotrophic fragments are promising candidate drugs for enhancing cognition through increase in ntg/ntc activity in AD and other learning and memory disorders as well as for promoting general cognitive health.
Edited by chrono, 16 July 2010 - 01:23 AM.
