Reversing silenced AR signal with demethylating agents - A promising treatment option?

[mariobros]

I did a bit of research after reading Bradwaggoners post and came across the following. Is it possible that we have a common cytochrome P450 gene allele, which results in poor metabolism of finasteride making us especially susceptible to further inhibition CYP3A4 - and if cytochrome P450 enzymes demethylate, this could have contributed to excessive methylation and our current situation.

Influence of cytochrome P450 polymorphisms on drug therapies: Pharmacogenetic, pharmacoepigenetic and clinical aspects

The polymorphic nature of the cytochrome P450 (CYP) genes affects individual drug response and adverse reactions to a great extent. This variation includes copy number variants (CNV), missense mutations, insertions and deletions, and mutations affecting gene expression and activity of mainly CYP2A6, CYP2B6, CYP2C9, CYP2C19 and CYP2D6, which have been extensively studied and well characterized. CYP1A2 and CYP3A4 expression varies significantly, and the cause has been suggested to be mainly of genetic origin but the exact molecular basis remains unknown… We present a review of the major polymorphic CYP alleles and conclude that this variability is of greatest importance for treatment with several antidepressants, antipsychotics, antiulcer drugs, anti-HIV drugs, anticoagulants, antidiabetics and the anticancer drug tamoxifen. We also present tables illustrating the relative importance of specific common CYP alleles for the extent of enzyme functionality. The field of pharmacoepigenetics has just opened, and we present recent examples wherein gene methylation influences the expression of CYP.

tryptophan.net/chem4120/pharmacogenetics.pdf

Inhibition of Human Steroid 5β-Reductase (AKR1D1) by Finasteride and Structure of the Enzyme-Inhibitor Complex

Both AKR1D1 and 5α-reductase type 1 play important roles in the hepatic clearance of steroid hormones, suggesting that high dose finasteride may have an adverse effect on hepatic steroid metabolism. Inhibition of AKR1D1 by high dose finasteride would also deprive PXR of its natural 5β-pregnane ligands, resulting in diminished CYP3A4 induction. This could result in significant drug-drug interactions. Importantly, finasteride itself is metabolized by CYP3A4, suggesting that high dose finasteride might prevent its own metabolism (27).

ncbi.nlm.nih.gov/pmc/articles/PMC2740403/

[martinM]

Mariobros, this last post makes a lot of sense, reduction of finasteride metabolism.

[Bradwaggoner]

Well, it is certainly a possibility. This is what intrigued me the most:

“Cytochrome P450 enzymes are present in most tissues of the body, and play important roles in hormone synthesis and breakdown (including estrogen and testosterone synthesis and metabolism), cholesterol synthesis, and vitamin D metabolism”

Are those not the usual suspects in our wacky bloodwork? I haven’t found any 3a-diol-G references yet, but I assume that would actually come further down the chain and possibly related to the insufficient hormone synthesis.

[Oscar]

^There are theories about how liver issues that could be the cause of the problem (my theory thread here viewtopic.php?f=27&t=859 ) essentially the theory goes that liver enzymes have changed that are responsible for deactivating testosterone so that it can be excreted by the body. But this has nothing to do with methylation as liver enzymes do not methylate/demethylate.

[Bradwaggoner]

@Oscar

^There are theories about how liver issues that could be the cause of the problem (my theory thread here viewtopic.php?f=27&t=859 ) essentially the theory goes that liver enzymes have changed that are responsible for deactivating testosterone so that it can be excreted by the body. But this has nothing to do with methylation as liver enzymes do not methylate/demethylate.

ncbi.nlm.nih.gov/books/NBK22523/

“14. Demethylation. Methyl amines are often demethylated by cytochrome P450 enzymes.”

BTW, these enzymes are can be found anywhere in the body, not just the liver. Maybe an overlooked possibility…

[Bradwaggoner]

Just another example of demethylation related to p450 enzymes in the liver:

http://www.ncbi.nlm.nih.gov/pubmed/11353758

Involvement of CYP2B6 in n-demethylation of ketamine in human liver microsomes.
Yanagihara Y, Kariya S, Ohtani M, Uchino K, Aoyama T, Yamamura Y, Iga T.

Department of Hospital Pharmacy, Tokyo Postal Services Agency Hospital, Chiyoda-ku, Tokyo, Japan. yyanagihara@tpth.go.jp

Abstract
Ketamine is metabolized by cytochrome P450 (CYP) leading to production of pharmacologically active products and contributing to drug excretion. We identified the CYP enzymes involved in the N-demethylation of ketamine enantiomers using pooled human liver microsomes and microsomes from human B-lymphoblastoid cells that expressed CYP enzymes. The kinetic data in human liver microsomes for the ®- and (S)-ketamine N-demethylase activities could be analyzed as two-enzyme systems. The K(m) values were 31 and 496 microM for ®-ketamine, and 24 and 444 microM for (S)-ketamine. Among the 12 cDNA-expressed CYP enzymes examined, CYP2B6, CYP2C9, and CYP3A4 showed high activities for the N-demethylation of both enantiomers at the substrate concentration of 1 mM. CYP2B6 had the lowest K(m) value for the N-demethylation of ®- and (S)-ketamine (74 and 44 microM, respectively). Also, the intrinsic clearance (CL(int): V(max)/K(m)) of CYP2B6 for the N-demethylation of both enantiomers were 7 to 13 times higher than those of CYP2C9 and CYP3A4. Orphenadrine (CYP2B6 inhibitor, 500 microM) and sulfaphenazole (CYP2C9 inhibitor, 100 microM) inhibited the N-demethylase activities for both enantiomers (5 microM) in human liver microsomes by 60 to 70%, whereas cyclosporin A (CYP3A4 inhibitor, 100 microM) failed to inhibit these activities. In addition, the anti-CYP2B6 antibody inhibited these activities in human liver microsomes by 80%, whereas anti-CYP2C antibody and anti-CYP3A4 antibody failed to inhibit these activities. These results suggest that the high affinity/low capacity enzyme in human liver microsomes is mediated by CYP2B6, and the low affinity/high capacity enzyme is mediated by CYP2C9 and CYP3A4. CYP2B6 mainly mediates the N-demethylation of ®- and (S)-ketamine in human liver microsomes at therapeutic concentrations (5 microM).

PMID: 11353758 [PubMed - indexed for MEDLINE]

[Oscar]

No offence but I think this is incorrect.

Methylation/Demethylation is a chemical reaction.
en.wikipedia.org/wiki/Demethylation

Demethylation is the chemical process resulting in the removal a of methyl group (CH3) from a molecule. A common way of demethylation is the replacement of a methyl group by a hydrogen atom, resulting in a net loss of one carbon and two hydrogen atoms…
In biochemical systems, the process of demethylation is often catalyzed by an enzyme such as one of the Cytochrome P450 (CYP) family of liver enzymes

en.wikipedia.org/wiki/Methylation

In the chemical sciences, methylation denotes the addition of a methyl group to a substrate or the substitution of an atom or group by a methyl group. Methylation is a form of alkylation with, to be specific, a methyl group, rather than a larger carbon chain, replacing a hydrogen atom. These terms are commonly used in chemistry, biochemistry, soil science, and the biological sciences.
In biological systems, methylation is catalyzed by enzymes; such methylation can be involved in modification of heavy metals, regulation of gene expression, regulation of protein function, and RNA metabolism.

We are specifically talking about dna methylation (and histone methylation) which is not regulated by liver enzymes.
en.wikipedia.org/wiki/Histone_methyltransferase .

[Bradwaggoner]

I’m open to anything, and am not afraid of being wrong ,especially if it eliminates confusion and furthers our cause. Forgive me if I disagree with you though:

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC230784/

“Given the additional results that CpG/-50 of the female-specific steroid 15 alpha-hydroxylase (Cyp 2a-4) promoter is preferentially demethylated in the females, the sex-specific expressions of the P450 genes correlate very well with DNA demethylation. We also conclude that GABP is a methylation-sensitive transcription factor and is a potential transcription activator of the male-specific Cyp 2d-9 promoter.”

http://molpharm.aspetjournals.org/content/75/5/1171.abstract

Dynamic Patterns of Histone Methylation Are Associated with Ontogenic Expression of the Cyp3a Genes during Mouse Liver Maturation
Ye Li, Yue Cui, Steven N. Hart, Curtis D. Klaassen and Xiao-bo Zhong

• Author Affiliations

Department of Pharmacology, Toxicology, and Therapeutics, the University of Kansas Medical Center, Kansas City, Kansas
Address correspondence to:
Dr. Xiao-bo Zhong, Department of Pharmacology, Toxicology, and Therapeutics, The University of Kansas Medical Center, 3901 Rainbow Boulevard, Kansas City, KS 66160. E-mail: xzhong@kumc.edu
Abstract
Human cytochrome P450 3A (CYP3A) members are major drug-metabolizing enzymes in the liver. Two genes, CYP3A4 and CYP3A7, exhibit a developmental switch in gene expression during liver maturation. CYP3A4 is mainly expressed in adults, whereas CYP3A7 is dominantly expressed during the fetal and neonatal stages. Their ontogenic expression results in developmentally related changes in the capacity to metabolize endogenous and exogenous compounds. Thus, it is desirable to understand the mechanisms controlling the developmental switch. Mice also exhibit a developmental switch between Cyp3a16 (neonatal isoform) and Cyp3a11 (adult isoform) and may serve as a model to study the mechanisms controlling the developmental switch. Because the epigenetic code (e.g., DNA methylation and histone modifications) is implicated in regulating gene expression and cellular differentiation during development, the current study determined the status of DNA methylation, histone-3-lysine-4 dimethylation (H3K4me2) and histone-3-lysine-27 trimethylation (H3K27me3) around the mouse Cyp3a locus at various developmental ages from prenatal, through neonatal, to young adult. DNA was not hypermethylated in the Cyp3a locus at any age. However, increases in Cyp3a16 expression in neonatal livers and Cyp3a11 in adult livers were associated with increases of H3K4me2. Suppression of Cyp3a16 expression in adult livers coincided with decreases of H3K4me2 and increases of H3K27me3 around Cyp3a16. In conclusion, histone modifications of H3K4me2 and H3K27me3 are dynamically changed in a locus-specific manner along the Cyp3a locus. Developmental switch between Cyp3a11 and Cyp3a16 gene expression seems to be due to dynamic changes of histone modifications during postnatal liver maturation.

[Bradwaggoner]

BTW, I do recognize this is mice and not humans. Also, I admit I really do not have the knowledge to interpret what most of this stuff means. I just want to pass along what I find, and maybe it can be of relevance.

[Oscar]

OK just to clarify: Those studies & mariobros study are referring to the methylation status of the genes involved in liver enzymes themselves and how this alters their expression. They in no when mention that liver enzymes are involved in dna/histone methylation, which is the subject of this thread - its a dead end in other words.

{The fact that liver enzymes can deactivate testosterone through the fact they can hydroxylate hormones is an interesting idea however (see links above) . I wonder if this would be picked up on a normal blood test or would require a more in depth analysis such as mass spectrometry?}

[Bradwaggoner]

Oscar, I sort of understand what you mean, but can you please say that in layman’s terms if possible? Because when I read lines like this:

“Orphenadrine (CYP2B6 inhibitor, 500 microM) and sulfaphenazole (CYP2C9 inhibitor, 100 microM) inhibited the N-demethylase activities for both enantiomers (5 microM) in human liver microsomes by 60 to 70%, whereas cyclosporin A (CYP3A4 inhibitor, 100 microM) failed to inhibit these activities.”

I see in my little understanding (and I will paraphrase) “If you inhibit enzymes CYP2B6 and CYP2C9, you will inhibit the N-demethylase activity by 60-70% in human liver microsomes for both enantiomers (which I have no idea what this is)”. Admittedly this doesn’t even begin to explain finasteride problems, but rather only says to me that demethylation inhibition can occur if you suppress certain P450 enzymes with certain drugs.

Also, what are the implications of this? “{The fact that liver enzymes can deactivate testosterone through the fact they can hydroxylate hormones is an interesting idea however (see links above) . I wonder if this would be picked up on a normal blood test or would require a more in depth analysis such as mass spectrometry?}”

Could there be long-term effects involved with that?

[Bradwaggoner]

Ok Oscar. I have found other sources saying P450 enzymes demethylate drugs and xenobiotic compounds. But you are right, not involved in DNA or Histone demethylation. So I am with you on that.

BTW, has anyone seen this? http://biotrend.com/spec/K010-F1.pdf

[mariobros]

Yes, i think i was on a bit a far out tangent with that last post.

I’m really keen to hear some news from awor… though I’m also hoping the reason he hasn’t been around much is cause he’s feeling much improved.

[Bradwaggoner]

He probably doesn’t want to report until there is something of significance to report about. I hope it works for him.

[boston332]

@Bradwaggoner

He probably doesn’t want to report until there is something of significance to report about. I hope it works for him.

It it were me and I was having success with a treatment I’d be damn sure about it before I posted it and got 1600 people’s hopes up. Would hate to come back 3 days later and say, “oops, I was wrong.” Christ, that would probably push most of us over the edge.

Maybe thats why he hasnt posted yet.

[toadstool]

probably because its not a promising treatment.

[191]

i imagine the problem will be remethylation as in he’ll feel better for a few days and then slip back. But it the first few days are good then its overall a sign the theory is correct.

[boston332]

If he improves even temporarily thats means he’s very likely on the right track. Its a matter of tweaking the process maybe or coming up with a way to target the AR genee. He did call initial results “amazing”.

There is always the option of trying different demethylating agents.

[toadstool]

some people see amazing affects from trt in short instances… if it last 5 months, then I’ll be impressed. I’m still convinced its an thyroid/adrenal issue… at least in my case.

[Oscar]

Procaine is a monoamine oxidase inhibitor. This potentially makes it a powerful antidepressant. It must be said that its therefore not a surprise to feel good after using it. Only sustained results will be proof. en.wikipedia.org/wiki/Monoamine_oxidase_inhibitor

Procaine HCl (Gerovital H3): a weak, reversible, fully competitive inhibitor of monoamine oxidase
ncbi.nlm.nih.gov/pubmed/1109354
The effect of cocaine and other local anesthetics on central dopaminergic neurotransmission
ncbi.nlm.nih.gov/pubmed/7636732

Although the exact mechanisms behind the dopaminergic activities of procaine and dimethocaine remain unknown, it is clear that these drugs, as well as cocaine, activate dopaminergic systems in the intact animal.

Reinforcing properties of intravenous procaine in rhesus monkeys
http://dx.doi.org/10.1016/0091-3057(77)90027-2