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How to Plan and Carry Out a Simple Self-Experiment, a Single Person Trial of Senolytic Peptide FOXO4-DRI

This lengthy post covers the topic of setting up and running a self-experiment, a human trial of a single individual, to assess the senolytic effects of the peptide FOXO4 D-Retro-Inverso (FOXO4-DRI). This is the protein produced from the FOXO4 gene, with D-amino acids substituted for L-amino acids, reversing the chirality of the molecule. This means it cannot be processed in the usual way by cellular metabolism, and the consequence of interest is that this sabotages the survival efforts of lingering senescent cells in old tissues, causing them to self-destruct. This peptide was evaluated in aged mice in 2017, showing destruction of senescent cells without side-effects, and producing the usual array of benefits to measures of health and age-related decline as a result. This is quite interesting when compared to the various chemotherapeutic senolytic drug candidates that exibit similar degrees of senescent cell destruction in mice, but an array of unpleasant side-effects.

Of course, the chemotherapeutics have extensive human data that catalogs the side-effects at various doses in our species, while at the time of writing there is no human data for FOXO4-DRI whatsoever. This is a very important point! It is perhaps the most important consideration here.

The purpose in publishing this outline is not to encourage people to immediately set forth to follow it. If you come away thinking that you should do exactly that, and as soon as possible, then you have failed at reading comprehension. This post is intended to illustrate how to think about self-experimentation in this field: set your constraints; identify likely approaches; do the research to fill in the necessary details; establish a plan of action; perhaps try out some parts of it in advance, such as the measurement portions, as they never quite work as expected; and most importantly identify whether or not the whole plan is worth actually trying, given all that is known of the risks involved. Ultimately that must be a personal choice.

Contents

Why Self-Experiment with Senolytics?

Senolytic therapies are those that selectively destroy senescent cells. The build up of senescent cells is one of the causes of aging. So obviously, one hope is to benefit personally from such a therapy sooner than would otherwise be the case, balancing that against incurring some unknown degree of risk of failure or harm. The first human trials, those that establish numbers for that risk, will take another few years to wind through to robust conclusions, and further years beyond that will be required for the medical community to become willing to prescribe senolytics generally. Further, those trials will almost all test only a single candidate therapy, and the evidence to date in mice suggests that different senolytics with different mechanisms are tissue-specific in their effects on senescent cells. Multiple different compounds may be more effective than one - but that won't be discovered in the formal trial process. Lastly, well run self-experimentation carried out by a number of people, where the results are published, can help to guide the direction of later, formal studies.

All of these reasons must be balanced against a sober assessment of the risks involved in obtaining and using an injected peptide that has no published human data whatsoever, and an acceptance of personal responsibility for consequences should one choose to run those risks.

Caveats in More Detail

There are two areas of personal responsibility to consider here. Firstly, this involves injecting a peptide that has no published data on human use at all. The anecdotal data from people claiming to have used FOXO4-DRI should be ignored, as it only covers the most serious short-term consequences, and few if any of these individuals are verifying that they are indeed obtaining the right compound, and nor are they carefully checking their own biochemistry for outcomes. Anything is possible in the long-term, from cardiovascular failure to cancer to subtle increases in disease or organ failure risk, no matter how compelling the animal data might appear to be. The absence of any human data should be far more concerning to any rational individual than the case of a compound with extensive human data showing serious side-effects. The latter can be planned and accounted for. The former cannot: it is a leap into the unknown.

Secondly, obtaining and using arbitrary novel peptides such as FOXO4-DRI in the manner described here is potentially illegal: not yet being a formally registered medical treatment, it falls into a nebulous area of regulatory and prosecutorial discretion as to which of the overly broad rules and laws might apply. In effect it is illegal if one of the representatives of the powers that be chooses to say it is illegal in any specific case, and there are few good guidelines as to how those decisions will be made. The clearest of the murky dividing lines is that it is legal to sell peptides that are not defined as a therapy for research use, but illegal to market and sell them for personal use in most circumstances. This is very selectively enforced, however, and reputable sellers simply declare that their products are not for personal use, while knowing full well that this is exactly what their customers are doing in many cases.

Choosing to purchase and use FOXO4-DRI would therefore likely be a matter of civil disobedience, as is the case for anyone obtaining medicines or potential medicines outside the established national system of prescription and regulation. People are rarely prosected for doing so for personal use in the US - consider the legions of those who obtain medicines overseas for reasons of cost, despite the fact that doing so is illegal - but "rarely" is not "never." If you believe that the law is unjust, then by all means stand up against it, but accept that doing so carries the obvious risks of arrest, conviction, loss of livelihood, and all the other ways in which the cogs of modern society crush those who disagree with the powers that be.

Lastly, senolytics is a fast-moving field. This post will become outdated quite rapidly in its specifics, as new FOXO4-DRI research arrives on the scene, and FOXO4-DRI may well be obsoleted by better options. Nonetheless, the general outline should still be a useful basis for designing new self-experiments involving later and hopefully better compounds, as well as tests involving more logistical effort.

Summarizing FOXO4-DRI

You might look at an earlier post for a high level overview of how FOXO4-DRI works to selectively destroy senescent cells. The short version of the story is that functional FOXO4 interacts with p53 in order to suppress the cell self-destruction mechanisms that are primed for activation in all senescent cells. This appears to be the primary method by which a small fraction of senescent cells manage to linger in order to cause age-related dysfunction in tissues. Since FOXO4-DRI does not function correctly in cellular metabolism, the FOXO4-p53 interation fails when FOXO4-DRI is present in sufficient amounts to replace enough of the native FOXO4, and the cell destroys itself.

From studies in mice, FOXO4-DRI appears to cause no issues in other cell types; it is simply ignored - at least in the short-term, and in mice. In the longer term, high levels of non-functioning FOXO4 in calls might indeed cause problems, but the treatment are intended to last only a short time, with the FOXO4-DRI being broken down in a matter of a day or so. There is no human data to show that any of this also applies to our species, but given a few years for the research community to make progress, that will change.

Establishing Dosage

The only definitive way to establish a dosage for a pharmaceutical in order to achieve a given effect is to run a lot of tests in humans. Testing in mice can only pin down a likely starting point for experiments to determine a human dose, but the way in which you calculate that starting point is fairly well established for most cases. That established algorithm is essentially the same for most ingested and intravenously (or intraperitoneally in small animals) injected medicines, but doesn't necessarily apply to other injection routes. More on that in the next section of this post. Some compounds - as always - are exceptions to the rule, and the only way that scientists discover that any specific compound is an exception is through testing at various doses in various species.

When considering dosage of any substance, it is important to emphasise that more is not better; this cannot be approached in the way people tend to naively approach the (over)use of dietary supplements. The primary goal, if self-experimenting, is to take as little as necessary of any senolytic compound. The first look at FOXO4-DRI by the research community suggests that higher doses should do nothing more than is achieved by the therapeutic doses, and with the same absence of immediate side-effects - but that is only the result of an initial examination. No long-term assessment has taken place, even in mice. If FOXO4-DRI does turn out to produce lasting or subtle side-effects, then following the maxim of using as little of it as possible should help to lower the impact. That the dose makes the poison is an ancient adage, but no less true today.

The steps to figure out a suitable starting point for a human test of an injected senolytic pharmaceutical are as follows: firstly read the mouse studies for the senolytic compound in question, in order to find out how much was given to the mice and for how long. Doses for most pharmaceuticals are expressed in mg/kg. Secondly apply a standard multiplier to scale this up to human doses, which you can find in the open access paper "A simple practice guide for dose conversion between animals and human". Do not just multiply by the weight of the human in kilograms - that is not how this works. The relative surface area of the two species is the more relevant scaling parameter. Read the paper and its references in order to understand why this is the case. Again, note that the result is only a ballpark guess at a starting point in size of dose. The duration of treatment translates fairly directly, however. For the period of treatment, start with the same number of doses, spacing of doses, and duration as takes place in senolytic studies in mice.

In the case of FOXO4-DRI, the mice in the single study were injected intraperitoneally with 5 mg/kg of FOXO4-DRI dissolved in phosphate buffered saline, three times, taking place every other day. For a 60kg human, that translates to a 25mg dose via intravenous injection, carried out three times on alternate days.

An Introduction to Injections

The relationship between different forms of injection, dosage, and effects is actually a complicated and surprisingly poorly mapped topic. There are four type of injection to consider, here listed in descending order of difficulty to carry out safely: (a) intraperitoneal, through the stomach muscle into the abdominal body cavity, which is rare in human medicine but common in studies using small animals; (b) intravenous, into a vein, which requires some practice to get right; (c) intramuscular, into the muscle beneath the skin; and (d) subcutanous, into the lower levels of the skin.

The amount of fluid that can be easily injected varies by type. In humans, effectively unlimited amounts of fluid can be introduced via intraperitoneal or intravenous injection. The subcutaneous route is limited to something less than 1ml, and intramuscular is limited to 2-3ml depending on location. These are all very fuzzy numbers - some sources, for example, give an upper limit of 5ml for intramuscular injections, but I can't say as I would be lining up to be on the receiving end of that. Measure out 5ml and take a look at it. Ouch.

The different injection routes can alter the character of the injected medicine; how much is required to gain a given effect, how long it takes to get into the system and how fast it does it. A rare few types of medication cannot be injected subcutaneously, because the metabolism of the skin will degrade them, while some are better given subcutaneously. If you root through the literature looking for comparisons between performance and dosage for different injection types, you'll find a very ragged collection of examples showing that there are few coherent rules. Some compounds have no discernible differences between injection route, some see altered peaks of concentration, some require higher doses when subcutaneous, some require lower doses when subcutenous. Oil-based solutions can produce a very slow uptake of medication when injected into muscle or skin in comparison to an intraveous injection, while water-based solutions result in just as rapid an uptake into the bloodstream.

Do we know how FOXO4-DRI will be affected by different injection routes? No. That data has yet to be established. So it seems acceptable to say that a self-experimenter should try to use the much easier paths of subcutaneous and intramuscular injection, rather than attempting intravenous administration, and just keep the same dose as was established for intravenous injection. For most people, intraveous injections require a helper or a lot of painful practice. For subcutaneous and intramuscular injections, there is a market of autoinjection tools that can remove many of the challenges inherent in managing injections.

Considering Autoinjectors

Sticking a needle into one's own flesh is not an easy thing to do, and this is the rationale for the range of autoinjection systems that have been developed by the medical community. They are most easily available for subcutaneous injections; spring-based devices that accept a standard needle and syringe, and that are trigged by a button push. Intramuscular autoinjectors do exist, but unfortunately not in a general or easily available way. All of the needle-based intramuscular autoinjectors are regulated devices that come preloaded with a particular medicine, and are not otherwise sold in a more generally useful way. Unfortunately, there is no automation that can help with intravenous injections. You are on your own there.

Option 1: Subcutaneous Autoinjection with Needle and Syringe

If intending to carry out subcutaneous injections it is easy enough to order up a supply of disposible needles and syringes, an autoinjector device that accepts the standard needle and syringe arrangement, and other necessary items such as sterilization equipment from the sizable diabetes-focused marketplace. Such injections are relatively easy to carry out, a wide range of vendors sell the materials, and there is a lot of documentation, including videos, available on how to carry out subcutaneous injections. All of the equipment is cheap. Buying these materials will probably put you on a list in this era of the drug war, but there are many people out there doing it.

Option 2: Subcutaneous or Intramuscular Needle-Free Autoinjection

Are there viable alternatives to needles? As it turns out, yes, and some can solve the problem of missing general intramuscular autoinjectors as well. Needle-free autoinjectors that use a thin, high-pressure fluid jet to punch medication through the skin are a growing area of development. These systems have numerous advantages over needles, but they are more expensive, most can only manage subcutanous injections, and all are limited in the amount of fluid they can inject in comparison to the traditional needle and syringe. Nonetheless, for the purposes of this outline, I'll focus on needle-free systems. The biggest, primary, and most attractive advantage of a needle-free system is in the name: it means not having to deal with needles in any way, shape, or form.

Obtaining a Needle-Free Injection System

There are a fair number of needle-free injectors on the market, but most are hard to obtain unless you happen to be a regulated medical facility running through the standard regulated purchase model, and are looking for large numbers of units in a bulk purchase. Some systems use compressed gas, others use springs. The spring-based systems tend to be less complicated and more reliable. From my survey of the marketplace, the two systems worth looking at are (a) PharmaJet, which can be purchased in the US via intermediary suppliers such as Moore Medical, and (b) Comfort-in, which is sold directly to consumers in most countries by an Australian group. So far as I can tell, PharmaJet is the only available needle-free system that is capable of intramuscular rather than subcutaneous injection.

PharmaJet is the better engineered and more expensive of these two systems, and its specialized syringes are very definitely built to be one-use only. Further, loading fluid into the syringes requires the use of vials and a vial adaptor. First the vial is loaded with the fluid to be injected, then the vial is connected to the syringe via the adaptor to transfer the fluid. Comfort-in has a similar setup, but is more flexible, and on the whole more consumer-friendly when considering the entire package of injector and accessories. It is has a wider range of vial and other adaptors. Further, the Comfort-in syringes can in principle be reused given sterilization, though of course that is not recommended.

The instructions for both of these systems are extensive, and include videos. They are fairly easy to use. One caveat is that needle-free systems produce a puncture that more readily leaks some of the injected material back out again than is the case for needles. It is a good idea to have a less absorbent plaster ready to apply immediately after injection, such as one of the hydrocolloid dressings now widely available in stores.

Obtaining Vials of the Correct Size

If using the insulin needle and subcutaneous injection approach, then any variety of capped glass vial will do when it comes to mixing and temporarily holding liquids for injection. It does, however help greatly to either use preassembled sterile vials or assemble your own vials with rubber stoppers and crimped caps, as described below, as that sort of setup makes it easier to take up small amounts of a liquid into a syringe. If using the needle-free systems, then vials of a specific type and size are necessary in order to fit the adaptors. The rest of this discussion focuses on that scenario.

There are many, many different types of vial manufactured for various specialized uses in the laboratory. The type needed here is (a) crimp-top vial, also called serum vials by some manufacturers, with (b) a 13mm (for PharmaJet and Comfort-in) or 20mm (for Comfort-in only) diameter open top aluminium cap, one that has a central hole to allow needles and adaptor spikes through, and (c) a rubber or rubber-like stopper that is thin enough in the center to let a needle or adaptor spike past. The cap is crimped on over the rubber seal to keep everything in place - this requires a crimping tool, and removing it requires the use of another tool.

There are two options here. The first option is to purchase preassembled empty sterile vials of the right size and a set of disposable needles and syringes to transfer liquid into the vials. In order to continue to bypass the whole business of needles, however, the other alternative is to purchase vials, stoppers, and aluminium caps separately, or in a kit, and assemble your own vials. A crimping tool is also needed in order to seal the cap. That tool, like the vials and the caps, must be of the right size. Be careful when purchasing online. Vials are categorized by many different dimensions, and descriptions tend to mix and match which dimensions of the vial they are discussing, or omit the important ones. For sterile vials, it is usually only the cap diameter that is mentioned. For crimp-top vials, there are any number of dimensions that might be discussed; the one that needs to match the cap diameter is the outer diameter of the mouth or crimp.

It is usually a good idea to buy a kit where possible, rather than assembling the pieces from different orders, but if taking the assembly path, it is best to buy all the pieces from the same company. Wheaton is a decent manufacturer, and it is usally possible to find much of their equipment for sale via numeous vendors. One can match, say, the crimp-top 3ml vials #223684 with 7mm inner mouth and 13mm outer mouth with snap-on rubber stoppers #224100-080 of the appropriate dimensions and 13mm open top caps #224177-01. Then add a 13mm crimping device #W225302 and pliers #224372 to remove 13mm crimped caps.

Preparing FOXO4-DRI for Injection

The objective is to wind up with the right amount of FOXO4-DRI dissolved in phosphate buffered saline in a sealed vial, ready to be used with the injection system, and with as little contamination as possible from the environment. Depending on the size of the vial, it might contain doses for multiple injections - in fact it is much easier to set things up this way. FOXO4-DRI dissolves very readily in saline, so placing a single human dose into 0.5ml or 1ml is quite feasible. A 3ml vial can hold three doses for the treatment without issue.

One approach is to measure out FOXO4-DRI by weight using a suitable microscale, then transfer that dose to a mixing container, such as larger glass vial. Pipette in the desired amount of saline and stir with a rod to ensure it is fully dissolved. Then either inject the mix into a sterile vial using a standard needle and syringe, or pipette the mix into an clean open vial that is then sealed, capped, and crimped. You will probably find that it is much easier to make up multiple doses at once and measure them out in liquid form rather than using a microscale, however, as greater precision is possible that way. Small amounts tend to be hard to weigh accurately, but pipetting them when in solution is feasible.

Keeping Things Sterile is Very Important

Keeping hands, tools, vials, and surfaces clean and sterile is important: wash everything carefully and wipe down surfaces with an alcohol solution before and after use. Laboratories use autoclaves, which sterilize with steam. These are largely expensive devices, but a range of smaller, cheaper options exists. There are many best practices guides and summaries available online. This extends to the injection itself. Even with needle-free systems, an injection site should still be wiped down with alcohol first. It is all too easy to infect an injection site if skipping the precautions, and this can have severe consequences.

Further, it is important to ensure that the FOXO4-DRI mix is sterile, as you have no control over how it was handled prior to shipping. One approach to doing this is to filter the peptide solution after making it up. This is fairly easily accomplished using a sterile syringe filter rated to block bacteria, and for use with peptides. Some filters may leach peptides from the solution, so caution is needed in choosing the right product - read the descriptions carefully. You might look for polyethersulfone 0.2 micron filters, for example, which are intended to filter out bacteria without binding appreciably to proteins. A good practice is to make up a double concentration solution, pass it through the filter, then flush the filter with another, equal amount to make up the final solution for injection.

Obtaining FOXO4-DRI

There are a small number of peptide synthesis companies worldwide that advertise their willingness to sell FOXO4-DRI, as an established recipe for synthesis rather than a special order. Any other reputable peptide synthesis company will be able to manufacture FOXO4-DRI to order from the description provided in the 2017 paper.

H-ltlrkepaseiaqsileaysqngwanrrsggkrppprrrqrrkkrg-OH. Molecular weight: 5358.2 It was manufactured by Pepscan (Lelystad, the Netherlands) at more than 95% purity and stored at -20°C in 1mg powder aliquots until used to avoid freeze-thawing artifacts. For in vitro experiments FOXO4-DRI was dissolved in Phosphate Buffered Saline to generate a 2mM stock. For in vivo use, FOXO4-DRI was dissolved in Phosphate Buffered Saline to generate a 5mg/ml stock solution, which was kept on ice until injection. Before injection the solution was brought to room temperature.

An advantage of the companies that advertise FOXO4-DRI in their catalog is that they will have established mass spectra and other data sheets for the compound that can be compared with one another, or used as a basis for evaluating the quality of the product. The disadvantage, in at least the case of NovoPro Bioscience, is that the price charged is outrageous - possibly a rational choice made to discourage amateur purchasers. Chinese companies, on the other hand have a comparatively low price for their offerings, significant lower than that of even the most aggressively competitive companies in the US and Europe. Among European and US synthesis companies out there, Pepscan and Genscript are options, with Genscript coming recommended by a number of sources.

Without focusing on any of the specific vendors mentioned above, the easiest way to obtain cost-effective synthesis of peptides is to follow the process outlined in the last post on self-experimentation, which is to use Alibaba to find and connect with smaller-sized suppliers in China. There are a good many reputable peptide synthesis concerns in that part of the world, even if only one or two explicitly advertise FOXO4-DRI as a product at this time.

As noted at the outset of this post, all of these efforts to obtain, ship, and use a peptide are to some degree illegal - it would be an act of civil disobedience carried out because the laws regarding these matters are unjust, albeit very unevenly enforced. Many people regularly order pharmaceuticals from overseas, with and without prescriptions, for a variety of economic and medical reasons, and all of this is illegal. The usual worst outcome for individual users is intermittent confiscation of goods by customs, though in the US, the FDA is actually responsible for this enforcement rather than the customs authorities. Worse things can and have happened to individuals, however, even though enforcement is usually targeted at bigger fish, those who want to resell sizable amounts of medication on the gray market, or who are trafficking in controlled substances. While the situation with an arbitrary peptide isn't the same from a regulatory perspective, there is a fair amount written on the broader topic online, and I encourage reading around the subject.

Avoid Trifluoroacetate (TFA) Salt

The most common method of producing peptides to order involves the use of trifluoroacetate (TFA) salt, and the manufactured peptide will have residual TFA salt in it. This stuff harms cells and can cause painful, lasting, inflammatory reactions if injected. If you don't specify in an order that you wish TFA salt to be exchanged or removed, then you will get TFA salt. Ordering without TFA salt residuals will raise the price a little, but it is both the recommended course of action and well worth it.

Open a Business Mailbox

A mailbox capable of receiving signature-required packages from internal shipping concerns such as DHL and Fedex will be needed. Having a business name and address is a good idea. Do not use a residential address.

Use Alibaba to Find Manufacturers

Alibaba is the primary means for non-Chinese-language purchasers to connect to Chinese manufacturers. The company has done a lot of work to incorporate automatic translation, to reduce risk, to garden a competitive bazaar, and to make the reputation of companies visible, but it is by now quite a complicated site to use. It is a culture in and of itself, with its own terms and shorthand. There are a lot of guides to Alibaba out there that certainly help, even if primarily aimed at retailers in search of a manufacturer, but many of the specific details become obsolete quickly. The Alibaba international payment systems in particular are a moving target at all times: this year's names, user interfaces, and restrictions will not be the same as next year's names, user interfaces, and restrictions.

Start by searching Alibaba for peptide synthesis companies. There are scores of biotech companies in China for any given specialty. Filter the list for small companies, as larger companies will tend to (a) ignore individual purchasers in search of small amounts of a peptide, for all the obvious economic reasons, and (b) in any case require proof of all of the necessary importation licenses and paperwork. Shop around for prices - they may vary widely, and it isn't necessarily the case that very low prices indicate a scam of some sort. Some items and services are genuinely very cheap to obtain via some Chinese sources. Remember to ask the manufacturer for mass spectra and liquid chromatography data if they have it.

Many manufacturers will state that they require a large (often ridiculously large) minimum order; that can be ignored. Only communicate with gold badge, trade assurance suppliers with several years or more of reputation and a decent response rate. Make sure the companies exist outside Alibaba, though for many entirely reliable Chinese businesses there are often sizable differences between storefronts on Alibaba, real world presence, and the names of owners and bank accounts. Use your best judgement; it will become easier with practice.

Arrange Purchase and Shipping via Alibaba

Given the names of a few suppliers, reach out via the Alibaba messaging system and ask for a quote for a given amount of FOXO4-DRI; you will have to provide the sequence and a reference to the paper in which it is described. Buy more than you'll think you need, as a small amount of it will be used to validate the identity and quality of the compound batch. Payment will most likely have to be carried out via a wire transfer, which in Alibaba is called telegraphic transfer (TT). Alibaba offers a series of quite slick internal payment options that can be hooked up to a credit card or bank account, but it is hit and miss whether or not those methods will be permitted for any given transaction. Asking the seller for a pro-forma invoice (PI), then heading to the bank to send a wire, and trusting to their honesty should work just fine when dealing with companies that have a long-standing gold badge.

To enable shipping with tracking via carriers such as DHL, the preferred method of delivery for Chinese suppliers shipping to the US or Europe, you will need to provide a shipping address, email address, and phone number. Those details will find their way into spam databases if you are dealing with more than a few companies, and will be, of course, sold on by Alibaba itself as well. Expect to see an uptick of spam after dealing with suppliers via Alibaba, so consider using throwaway credentials where possible.

Chinese manufacturers active on Alibaba are familiar with international shipping practices. On their own initiative may or may not decide to declare the true cost and contents of the shipped package. This is another form of widely practiced civil disobedience, but is much more common in the shipping of pharmaceuticals than in the shipping of synthesized peptides such as FOXO4-DRI. The former are likely to be confiscated by customs officials, while the latter are not. If the true cost is declared, then expect to pay customs duty on that cost; payment is typically handled via the carrier. Note that different carriers tend to have different processes and rates at which shipments are checked for validity.

Storing FOXO4-DRI

Peptides are usually shipped in powder form, and while in this form are easily stored in a refrigerator for the short-term, or in a freezer for the long term. It has a much shorter life span once it has been mixed with liquid for injection, however, and should be kept on ice, and used within days or or at most weeks.

Validating the Purchased FOXO4-DRI

A peptide of a given sequence may have been ordered, but that doesn't mean that what turns up at the door is either the right nondescript powder or free from impurities or otherwise of good quality. Even when not ordering from distant, infrequent suppliers, regular testing of batches is good practice in any industry. How to determine whether a peptide is what it says it is on the label? Run it through a process of liquid chromatography and mass spectrometry, and compare the results against the standard data for a high purity sample of that compound. Or rather pay a small lab company to do that.

Note that there isn't a cheap way to validate that the peptide in question does in fact have D-amino acids substituted for L-amino acids. They look the same in mass spectrometry, and largely the same in liquid chromatography, or at least without some additional work. Ask the vendor for the approach they would take or are familiar with, and shop around for prices. One approach is stability profiling, which relies on the different half-life of L- and D-amino acids in some circumstances.

Obtain the Necessary Equipment

Since this process will involve weighing, dividing up, and shipping powders in milligram amounts, a few items will be necessary: spatulas or scoops for small amounts of a substance; a reliable jeweler's scale such as the Gemini-20; sealable vials; small ziplock bags; labels; and shipping and packing materials. All of these are easily purchased online. The recommended shipping protocol is to triple wrap: a labelled vial, secured within a ziplock bag and tape, and then enclosed within a padded envelope.

Use Science Exchange to Find Lab Companies

Science Exchange is a fairly robust way to identify providers of specific lab services, request quotes, and make payments. Here again, pick a small lab company to work with after searching for LC-MS (liquid chromatography and mass spectrometry) services. Large companies will want all of the boilerplate registrations and legalities dotted and crossed, and are generally a pain to deal with in most other ways as well. Companies registered with Science Exchange largely don't provide their rates without some discussion, but a little over $100 per sample is a fair price for LC-MS to check the identity and purity of the compound.

Work with the Company to Arrange the Service

The process of request, bid, acceptance, and payment is managed through the Science Exchange website, with questions and answers posted to a discussion board for the task. Certainly ask if you have questions; most providers are happy to answer questions for someone less familiar with the technologies used. Service providers will typically want a description of the compounds to be tested and their standard data sheets, as a matter of best practice and safety. Here provide the mass spectra and other data sheets from the vendor, or use those published by NovoPro or other sources.

Ship the Samples

Measure out 1-5mg or so of FOXO4-DRI as a distinct sample, label it carefully, and package it up. More in the sample is better than less, as several attempts might be needed to get a good result out of the machines used, but each attempt really only needs a very tiny amount of the compound. Ship the sample via a carrier service such as DHL, UPS, or FedEx. Some LC-MS service companies may provide shipping instructions or recommendations. These are usually some variety of common sense: add a description and invoice to the package; reference the order ID, sender, and receiver; clearly label sample containers; and package defensively with three layers of packing; and so forth.

Examine the Results

Once the LC-MS process runs, the lab company should provide a short summary regarding whether or not the compound is in fact the correct one and numbers for the estimated purity. Also provided are the mass spectra, which can be compared with the existing spectra from the vendor, and from other sources such as NovoPro.

Establishing Tests and Measures

Unfortunately there is no available, established, proven, useful test that can directly assess senescent cell level in humans or human biopsies. It is possible to use immunohistochemistry to assess cellular senesence in tissue samples, which is a standard approach in animal studies, but no-one appears to have yet validated that in humans, given biopsies taken from a living individual. Since senescent cells are generated temporarily by wounding, it is quite possible that anything that starts with a biopsy will prove to be unhelpful as a before and after comparison measure for senolytic trials - the levels measured may not bear any resemblance to the normal levels absent a wound.

Without a direct measure, we must fall back on indirect assessments of the detrimental effects of senescent cells. The objective here is a set of tests that anyone can run without the need to involve a physician, as that always adds significant time and expense. Since we are really only interested in the identification of large and reliable effects as the result of an intervention, we can plausibly expect a collection of cheaper and easier measures known to correlate with age to be useful. Once that hill has been climbed, then decide whether or not to go further - don't bite off more than is easy to chew for a first outing.

From an earlier exploration of likely tests, I picked the following items on the basis of a likely connection to the actions of senescent cells, reasonable cost and effort, and ability to carry out the test without a physician's office being involved. Note that this does rule out, to pick one example, the interesting and relevant examination of kidney and liver function, as it would have to be carried out via the radioactive tracer methods of nuclear medicine to obtain decent results. That leaves the tests below quite focused on (a) the cardiovascular system, particularly measures influenced by vascular stiffness, and (b) inflammatory and other markers in the bloodstream:

The cardiovascular health measures in that list are those that are impacted by changes in the elasticity or functional capacity of blood vessels, such as would be expected to occur to some degree following any rejuvenation therapy that addresses senescent cells, chronic inflammation, or other factors that stiffen blood vessels, such as calcification or cross-linking. Positive change of the average values in most of these metrics are achievable with significant time and effort spent in physical training, so movement in the numbers in a short period of time as the result of a treatment should be an interesting data point.

Bloodwork

There exist online services such as WellnessFX where one can order up a blood test and then head off the next day to have it carried out by one of the widely available clinical service companies. Of the set of test packages offered by WellnessFX, the Baseline is probably all that is needed for present purposes. But shop around; this isn't the only provider.

Resting Heart Rate and Blood Pressure

A simple but reliable tool such as the Omron 10 is all you need to measure heart rate and blood pressure. It is worth noting here a couple of general principles for cardiovascular measures. Firstly, the further away from the center of the body that the measurement is taken, the less reliable it is - the more influenced by any number of circumstances, such as position, mood, stress, time of day, and so forth. Fingertip devices are convenient, but nowhere near as useful as something like the Omron 10 that uses pressure on the upper arm. Secondly, all of the above-mentioned line items also influence every cardiovascular measure, so when you are creating a baseline or measuring changes against that baseline, carry out each measure in the same position, at the same time of day, and make multiple measurements over a week to gain a more accurate view of the state of your physiology. The Omron 10 is solid: it just works, and seems quite reliable.

Heart Rate Variability

Surprisingly few of the numerous consumer tools for measuring heart rate variability actually deliver the underlying values used in research papers rather than some form of aggregate rating derived by the vendor; the former is required for any serious testing, and the latter is useless. Caveat emptor, and read the reviews carefully. As an alternative to consumer products, some of the regulated medical devices are quite easy to manage, but good luck in navigating the system to obtain one. The easiest way is to buy second hand medical devices via one of the major marketplaces open to resellers, but that requires a fair-sized investment in time and effort - which comes back to the rule about keeping things simple at the outset.

After some reading around the subject, I settled on the combination of the Polar H10 device coupled with the SelfLoops HRV Android application. I also gave the EliteHRV application a try. Despite the many recommendations for Polar equipment, I could not convince either setup to produce sensible numbers for heart rate variability data: all I obtained during increasingly careful and controlled testing was a very noisy set of clearly unrealistic results, nowhere near the values reported in papers on the subject. However, plenty of people in the quantified self community claim that these systems work reasonably well, so perhaps others will have better luck than I. Take my experience as a caution, and compare data against that reported in the literature before investing a lot of time in measurement.

Pulse Wave Velocity

For pulse wave velocity, choice in consumer tools is considerably more limited. Again, carefully note whether or not a device and matching application will deliver the actual underlying data used in research papers rather than a made-up vendor aggregate rating. I was reduced to trying a fingertip device, the iHeart, picked as being more reliable and easier to use than the line of scales that measure pulse wave velocity. Numerous sources suggest that decently reliable pulse wave velocity data from non-invasive devices is only going to be obtained by measures at the aorta and other core locations, or when using more complicated regulated medical devices that use cuffs and sensors at several places on the body.

Still, less reliable data can be smoothed out to some degree by taking the average of measures over time, and being consistent about position, finger used for a fingertip device, time of day, and so forth when the measurement is taken. It is fairly easy to demonstrate the degree to which these items can vary the output - just use the fingertip device on different fingers in succession and observe the result. All of this is a trade-off. A good approach is to take two measures at one time, using the same finger of left and right hand, as a way to demonstrate consistency. While testing an iHeart device in this way, I did indeed manage to obtain consistent and sensible data, though there is a large variation from day to day even when striving to keep as many of the variables as consistent as possible. That large variation means that only sizable effects could be detected.

DNA Methylation

DNA methylation tests can be ordered from either Osiris Green or Epimorphy / Zymo Research - note that it takes a fair few weeks for delivery in the latter case. From talking to people at the two companis, the normal level of variability for repeat tests from the same sample is something like 1.7 years for the Zymo Research test and 4.8 years for the Osiris Green tests. The level of day to day or intraday variation between different samples from the same individual remains more of a question mark at this point in time, though I am told they are very consistent over measures separated by months. Nonetheless, as for the cardiovascular measures, it is wise to try to make everything as similar as possible when taking the test before and after a treatment: time of day, recency of eating or exercise, recent diet, and so forth.

An Example Set of Daily Measures

An example of one approach to the daily cardiovascular measures is as follows, adding extra measures as a way to demonstrate the level of consistency in the tools:

  • Put on the Polar H10; this is involved enough to increase heart rate a little for a short period of time, so get it out of the way first.
  • Sit down in a comfortable position and relax for a few minutes.
  • Measure blood pressure and pulse on the left arm using the Omron 10.
  • Measure blood pressure and pulse on the right arm using the Omron 10.
  • Measure pulse wave velocity on the left index fingertip over a 30 second period using the iHeart system.
  • Measure pulse wave velocity on the right index fingertip over a 30 second period using the iHeart system.
  • Measure heart rate variability for a ten minute period using the Polar 10 and Selfloops.

Consistency is Very Important

Over the course of an experiment, from first measurement to last measurement, it is important to maintain a consistent weight, diet, and level of exercise. Sizable changes in lifestyle can produce results that may well prevent the detection of any outcome resulting from a first generation senolytic therapy using the simple tests outlined here. Further, when taking any measurement, be consistent in time of day, distance in time from last exercise or meal, and position of the body. Experimentation with measurement devices will quickly demonstrate just how great an impact these line items can have.

Guesstimated Costs

The costs given here are rounded up for the sake of convenience, and in some cases are blurred median values standing in for the range of observed prices in the wild. The choice to use needles for subcutaneous injection is obviously much cheaper than exploring the world of needle-free injections and vial assembly.

  • Business mailbox, such as from UPS: $250 / year
  • Baseline tests from WellnessFX: $220 / test
  • MyDNAage kits: $310 / kit
  • Osiris Green sample kits: $70 / kit
  • Omron 10 blood pressure monitor: $80
  • Polar H10 heart monitor: $100
  • iHeart monitor: $210
  • American Weigh Gemini-20 microscale: $90
  • Miscellenous equipment: spatulas, labels, vials, a vial rack, etc: $60
  • Subcutaneous autoinjector for use with needle and syringe: $45
  • Needles and syringes: $40
  • Small pack of 13mm sterile serum vials: $35
  • PharmaJet Needle-free Injection Kit: $1020
  • Comfort-in Needle-free Injection Kit: $470
  • Bulk 13mm serum vial parts and capping tools: $750
  • 200mg of FOXO4-DRI via Alibaba: $2000
  • Customs import duty on FOXO4-DRI: $150
  • Shipping and LC-MS analysis of a sample: $200
  • Shipping and stability profiling of a sample: $900

Practice Before Working with FOXO4-DRI

Do you think you can measure and move milligrams of powder and crystals around between containers without dropping it or otherwise losing a sizable fraction of it? Or reliably pipette fluid in 0.5ml amounts between small vials? Or cap vials or connect adaptors or fill syringes or carry out an injection without messing it up somewhere along the way? Perhaps you can. But it is a very good idea to practice first with salt and saline solution rather than finding out that your manual dexterity and methods are lacking while handling the expensive peptide. You will doubtless come to the conclusion that more tools or different tools are needed than was expected to be the case. It is possible to get by with a spatula small enough to fit into vial mouths, vials, labels, a vial rack to keep vials in place while hands are doing something else, and pipettes sized for moving small amounts of fluid. Other items may be helpful, such as suitably sized powder funnels, though there is considerable utility in a small, singly folded piece of paper for moving non-sticky powders from a measuring device to a small-mouthed container.

Schedule for the Self-Experiment

One might expect the process of discovery, reading around the topic, ordering materials, and validating an order of FOXO4-DRI to take a couple of months. Once all of the decisions are made and the materials are in hand, pick a start date. The schedule for the self-experiment is as follows:

  • Day 1-10: Once or twice a day, take measures for blood pressure, pulse wave velocity, and heart rate variability.
  • Day 10: Bloodwork and DNA methylation test.
  • Day 11: Test a 1/10 dose of FOXO4-DRI and abandon the effort if issues are experienced.
  • Day 12: Inject FOXO4-DRI.
  • Day 14: Inject FOXO4-DRI.
  • Day 16: Inject FOXO4-DRI.
  • Day 46-55: Repeat the blood pressure, pulse wave velocity, and heart rate variability measures.
  • Day 55: Repeat the bloodwork and DNA methylation test.

Note the initial low dose test. This is important, not least as a test of the ability to carry out an injection safely and competently. Consider that peptides tend to be delivered at 95% to 98% purity. It is quite possible for the remaining 2-5% of impurities in a manufactured peptide - whether there due to manufacturer error or as a natural side-effect of producing the peptide in question - to produce unwanted effects. The effects might include localized immune reactions, or the immune system becoming permanently sensitized to the peptide because of an impurity present in one dose. You will find discussions of this in the literature. If any untoward effects occur, it is wise to stop. You might refer back to the initial notes on risk and responsibility.

That said, the exact timing in the schedule is not really important, but it is a good idea to allow enough time following the end of the dosage for things to settle down. In animal studies, senolytic effects occurred fairly rapidly, as did the benefits, but allowing a few weeks of time in a human self-experiment still sounds like a good idea. Certainly it costs nothing to take that step.

Where to Publish?

If you run a self-experiment and keep the results to yourself, then you helped only yourself. The true benefit of rational, considered self-experimentation only begins to emerge when many members of community share their data, to an extent that can help to inform formal trials and direction of research and development. There are numerous communities of people whose members self-experiment with various compounds and interventions, with varying degrees of rigor. One can be found at the LongeCity forums, for example, and that is a fair place to post the details and results of a personal trial with senolytics. Equally if you run your own website or blog, why not there?

When publishing, include all of the measured data, the doses taken, duration of treatment, and age, weight, and gender. Fuzzing age to a less distinct five year range (e.g. late 40s, early 50s) is fine. If you wish to publish anonymously, it should be fairly safe to do so, as none of that data can be traced back to you without access to the bloodwork provider. None of the usual suspects will be interested in going that far. Negative results are just as important as positive results. For example, given the measures proposed in this post it is entirely plausible that positive changes as a result of present senolytic treatments in a basically healthy late 40s or early 50s individual will be too small to identify - they will be within the same range as random noise and measurement error. Data that confirms this expectation is still important and useful for the community, as it will help to steer future, better efforts.

Final Thoughts: Why Not Wait?

Given all of the cautions above, why not wait? Waiting can be a very sensible strategy. The state of senolytic therapies is progressing rapidly. New senolytics are emerging, and more data is being published for existing senolytics. At some point in the next few years, reliable direct tests for senescence will arrive on the scene, allowing a much better view of whether or not these treatments are actually achieving the claimed results. That said, it doesn't hurt to plan, and it doesn't hurt to tinker with some of the component parts of a plan. That is how we can determine whether or not it is worthwhile to experiment now versus waiting to experiment later with better tools.

Comments

I really really enjoy this series, Reason. Thank you for all the research.

Posted by: ale at March 6th, 2018 12:00 AM

@Reason. Maybe it is a silly idea, but whatever. Would you consider face detection and Age Estimation from photos into your measurement-arsenal?
You probably can find other services, but here is one: Microsoft Azure Face Detection:
https://azure.microsoft.com/en-us/services/cognitive-services/face/
You can upload your image under the 'See it in action' where the JSON part is (Browse...).
After the image is uploaded you can hover the mouse over the face to give the age estimation.
You can take photos both before and after the treatment and pay attention to the same lighting conditions (same background, same amount of light, etc.).
It is probably not very accurate, but it cost no money, can be done in 3 minutes. Based on big data, and it is objective. (better than asking your friends that: 'Does my skin look younger than before?' Their answer would be too polite :) and subjective, anyway.)

Posted by: DrGA at March 6th, 2018 3:36 AM

im 30 i can affford to wait a few more years before starting this. hopefully 5 years from now better data will be available and other senolytic compounds will have been discovered. maybe im being over optomistic but im the kind of guy who thinks anything is possible. right now i have geroprotect ageless cell until the next best thing arrives

Posted by: scott emptage at March 6th, 2018 5:38 AM

@Reason. I read this article with great interest because I am seriously considering this king of self experiment.

However, there remain gaps in my understanding that I am struggling to fill:

The mixing container and vials (if assembing your own) need to be sterilized. Could you please point us to relevant information on how to do that?

The peptide might not be completely sterile. Does one have to trust the supplier that it is safe enough or are there measures one could take to further purify and sterilize the peptide?

Posted by: Manfred B at March 16th, 2018 5:19 AM

@Manfred B: Sterilization in a lab uses an autoclave. Commercial variants are quite inexpensive. Passage through boiling water and 10% alcohol solutions are also viable solutions. There is a fair amount of best practice documentation online.

To filter the peptide solution after making it up, it can be passed through a sterile syringe filter rated to block bacteria, and for use with peptides. Some may leach peptides from the solution, so caution is needed in choosing the right product. If you search online, you'll find plenty of materials on how these items are used.

Posted by: Reason at March 16th, 2018 12:39 PM

@Reason, thank you very much for this detailed explanation! It is amazing out there there are people like you. In case you have interest and time, could you possibly create another guide on follistatin experiments?
I read an interesting article on folistatine therapy https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5383643/#!po=33.9744

But without background in bio engineering and without detailed deacryption of how the gene therapy was "mixed" it is impossible to understand what is going on. For e.g. CMV issue - where and how can one find it? What kind of type of cmv is needed?
how can you mix it with rAAV1?

Thanks once again for you post!

Posted by: Bejul at August 21st, 2018 10:31 AM

How do you verify the peptide is fused to the HIV-TAT sequence, as it was in the paper? Without the HIV-TAT the peptide just sits there in your extracellular space... seems like an important aspect of the treatment.

Posted by: Lee at September 7th, 2018 6:48 PM

I'm just now realizing that the FOXO4-DRI peptide includes the HIV-TAT sequence, however in the D-conformation, so as to eliminate the need to fuse the two peptide sequences together, although I'm wondering if this is the most economical choice...

Posted by: Lee at September 11th, 2018 11:32 AM

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