By Aly W. | First published June 14, 2019 | Last modified November 29, 2020
Sex steroids like estradiol, progesterone, and testosterone can be formulated in two main different ways when it comes to intramuscular and subcutaneous injection: 1) as oil solutions; and 2) as aqueous suspensions. What we are all mostly familiar with and probably naturally default to when we think of intramuscular and subcutaneous injections is oil solutions. This is how estradiol valerate, estradiol cypionate, estradiol benzoate, estradiol enanthate, estradiol undecylate, progesterone, hydroxyprogesterone caproate, and numerous other injected sex-hormonal medications are generally formulated. However, aqueous suspensions of sex steroids also exist and are marketed. Examples include estradiol benzoate (Agofollin Depot) and progesterone (Agolutin Depot), among others. These preparations are available today, including online from sites like EU Aibolit (Reddit). Moreover, and critically, aqueous suspensions are very different from oil solutions in their properties; they are, in general, actually markedly superior to oil solutions in duration.
Yet most people are probably unaware of what sex-steroid aqueous suspensions are, let alone their existence. The purpose of this article is to shed light on them, how they differ from oil solutions, and how you can get them.
Sex steroids are typically lipophilic (“lipid-loving”) and hydrophobic (“water-avoiding”). In other words, they dissolve in and are “attracted to” lipids (e.g., fats), and they are insoluble in and are repelled by water. From chemistry, this is because sex steroids are very non-polar, whereas water is quite polar. As a result of their lipophilicity, sex steroids readily form clear homogenous solutions when mixed into oil—that is, they form oil solutions. In contrast, because sex steroids are hydrophobic, they do not easily form solutions when mixed into water—that is, they do not easily form aqueous solutions (like, e.g., salt and water). Instead of aqueous solutions, solid “clumps” or crystal particles of sex steroids can be mixed into and thereby suspended in water—that is, aqueous suspensions of crystalline sex-steroid particles can be made.
When an oil solution of a sex steroid is injected intramuscularly or subcutaneously, the injected solution is trapped within the tissue compartment it is injected into and remains there. Because the tissue fluid is a water mixture, the oil solution stays together inside the tissue compartment and does not easily separate or distribute. This is because the lipophilic fats and sex steroids within the solution are attracted to each other and are repelled by water. Instead, the fats and sex steroids at the edges of the oil solution are very slowly absorbed into the surrounding water. Once they have escaped the oil “depot” into the surrounding tissue fluid, they can be distributed into the bloodstream and then into other tissues, thereby exerting their biological effects. Eventually, the whole oil solution will be absorbed.
Oftentimes sex steroids that are used by intramuscular or subcutaneous injection are esterified with one or more lipophilic fatty acid esters. These include propanoic acid (propionate), benzoic acid (benzoate), pentanoic acid (valerate), cyclopentylpropanoic acid (cypionate), hexanoic acid (caproate), heptanoic acid (enanthate), and undecanoic acid (undecylate). Examples of such sex steroid esters include the familiar estradiol valerate, estradiol cypionate, estradiol benzoate, hydroxyprogesterone caproate, and numerous others. The attachment of a lipophilic fatty acid ester (e.g., valeric acid) to a sex steroid (e.g., estradiol) will increase the lipophilicity of the sex steroid compared to merely injecting the unesterified sex steroid in an oil solution. The longer the fatty acid ester, the more lipophilic the resulting esterified sex steroid will be. As a result, the injected sex steroid ester will escape the oil tissue depot even more slowly, lengthening the amount of time it takes for the sex steroid ester to be absorbed and therefore its duration in the body.
The tables here and here show the lengthening duration of estradiol with longer/bulkier fatty-acid esters. Whereas an intramuscular injection of estradiol or progesterone in oil solution has a duration of around a couple of days, an intramuscular injection of an oil solution of estradiol undecylate, an ester of estradiol with a long 11-carbon fatty acid chain, has a duration measured in months. And an intramuscular injection of an oil solution of hydroxyprogesterone caproate, an ester of a synthetic analogue of progesterone with a 7-carbon fatty acid chain, has a duration measured in weeks (about 1.5 to 2.5 weeks, to be exact). Once the sex steroid ester has left the oil tissue depot, it is rapidly cleaved by esterase enzymes into free unesterified steroid (e.g., estradiol) and the previously connected fatty acid (e.g., valeric acid). Hence, sex-steroid esters are prodrugs otherwise identical to their parent sex steroids in their biological properties.
Aqueous suspensions of sex steroids also have a depot effect when administered by subcutaneous or intramuscular injection, but they work in a completely different way than oil solutions. Aqueous suspensions of sex steroids consist of tiny solid crystal particles of sex steroid suspended in water. These clumpy particles of sex steroid are highly lipophilic and hydrophobic. When injected, the hydrophilic water vehicle is rapidly mixed into the fluid of the tissue compartment and absorbed by the body. But the hydrophobic sex steroid crystals are not, and instead float about in the fluid of the tissue compartment. As with oil solutions, the sex steroids at the edges of the crystals very slowly dissolve off the surface of the crystals into the surrounding water and are then distributed into the circulation and tissues. Eventually, the crystal will be fully absorbed into the body, but only after a long period of time. Since the rate of absorption is dependent on lipophilicity and hydrophobicity, fatty acid esters lengthen the durations of aqueous suspensions of sex steroids by intramuscular/subcutaneous injection similarly to the case of oil solutions of sex steroids.
In the case of aqueous suspensions, the duration of the sex steroid is additionally highly dependent on particle size. These particle sizes have ranged from nanocrystalline to microcrystalline to macrocrystalline in their range. Almost always however it is microcrystalline particle sizes that have been used. (I’ve only seen macrocrystalline preparations described a few times, specifically in research on combined injectable contraceptives, and I’m fairly sure no such preparations have ever been marketed.) Typically, there is a given particle size range for the formulation, such as 0.01 to 0.1 mm in diameter. The larger the particle sizes, the slower the absorption into the body, and the longer the duration of the preparation; the smaller the particle sizes, the faster the absorption, and the shorter the duration. When microcrystalline aqueous suspensions of sex steroids are manufactured nowadays, the particle sizes are defined and carefully controlled. Particle sizes influence duration because they result in differences in the surface area from which sex steroid ester can escape the particle. Many small particles have much greater surface area and hence dissolution rate than a few large particles.
Particle sizes are manipulated during manufacturing via micronization—the process of decreasing the diameter of larger particles, such as via milling or grinding. Whereas more micronization improves the absorption and hence bioavailability of estradiol and progesterone with oral administration by increasing surface area for absorption, less micronization decreases the speed of absorption of crystalline aqueous suspensions via intramuscular injection and thereby extends their durations by decreasing surface area for absorption.
Finally, there is a notable similarity of intramuscular/subcutaneous injections of aqueous suspensions of sex steroid to subcutaneous implantations of pellets of sex steroid. Pellet implants are basically just pure crystalline sex steroid compressed into the shape of a pellet. (Here are some examples of what they look like: Photo, Photo.) They’re inserted into subcutaneous fat in the body through a small incision using a large needle-like instrument (Diagram). Once inserted, subcutaneous pellets slowly dissolve and absorb into the body over time, eventually disappearing completely. As they’re nothing but pure crystalline hormone, they don’t need to be removed or anything of the sort. In other words, implantation of a subcutaneous pellet is essentially the same thing as a subcutaneous injection of crystalline aqueous suspensions of sex steroid; it’s just one massive crystal instead of many tiny crystals suspended in water. And, with one huge crystal, comes a very long duration—typically 6 months or more for each subcutaneous pellet of estradiol or testosterone. But, of course, injections of aqueous suspensions have the benefits of being less expensive and not requiring minor surgery.
Studies that have compared sex steroids administered by intramuscular injection in oil solutions versus sex steroids administered by intramuscular injection in microcrystalline aqueous suspensions have generally found that the duration is considerably longer with aqueous suspensions than with oil solutions. For example, whereas an intramuscular injection of estradiol benzoate in oil solution has a duration of 4 to 6 days, an intramuscular injection of estradiol benzoate in aqueous suspension has a duration of 21 days (Wiki-Table). That is longer than the duration of estradiol cypionate in oil solution, and approaches the duration of estradiol enanthate in oil solution. In addition, whereas an intramuscular injection of progesterone in oil solution typically has a duration of about 2 to 3 days, an intramuscular injection of progesterone in aqueous suspension has a duration of 7 to 14 days (Wiki-Table). This duration is comparable to that of hydroxyprogesterone caproate in oil solution. Some aqueous suspensions can even have rather extreme durations; for example, medroxyprogesterone acetate (Depo-Provera, Depo-SubQ Provera) is always formulated as a microcrystalline aqueous suspension and has a duration of at least 3 months and as long as 6 to 9 months (Wiki). Even longer durations have been accomplished in studies.
Here is a full list of aqueous suspensions of sex steroids for use by intramuscular/subcutaneous injection that are known to have been marketed. Most of them were introduced in the 1950s and have been discontinued, but several remain marketed: estradiol benzoate (Agofollin Depot), progesterone (Agolutin Depot), testosterone isobutyrate (Agovirin Depot), and medroxyprogesterone acetate (Depo-Provera, Depo-SubQ Provera 104), as well as the combinations estradiol benzoate/testosterone isobutyrate (Folivirin) and estradiol cypionate/medroxyprogesterone acetate (Cyclofem). One of the more unfortunate casualties that is no longer marketed is estradiol benzoate/progesterone (Sistocyclin). In any case, both Agofollin Depot and Agolutin Depot remain available, and could be used together to allow for a similar preparation.
Agofollin Depot (estradiol benzoate), Agolutin Depot (progesterone), and Folivirin (estradiol benzoate/testosterone isobutyrate) all are marketed today only in the Czech Republic and Slovakia. However, they all seem to be available from EU Aibolit, a common site that DIY transgender people appear to buy their medications from (Reddit). I have not tried ordering them and am not aware of anyone who has, but it may be possible. If someone is curious enough and decides to try ordering, please leave a comment and let us know how things go!
Here are the medication leaflets for the above products (in Czech and translated into English):
- Agofollin Depot (estradiol benzoate): PDF; Google Translate
- Agolutin Depot (progesterone): PDF; Google Translate
- Agovirin Depot (testosterone isobutyrate): PDF; Google Translate
- Folivirin (estradiol benzoate/testosterone isobutyrate): PDF; Google Translate
The company that manufactures these products, BB Pharma, is notably also the company that produces the well-known Neofollin (estradiol valerate in oil solution for intramuscular injection) in Europe.
One last thing to note is that crystalline aqueous suspensions require larger needle gauges than normal to allow the crystal particles through the lumen of the needle. I am not sure exactly what size needles are needed. Another thing to note is that aqueous suspensions might be more irritating and prone to local site reactions than oil solutions, but I am not totally certain on that. One actual promoted benefit of aqueous suspensions is that, in contrast to oil solutions, there is no oil, and hence there is no risk of allergic reactions to the oil.
Taken together, aqueous suspensions of sex steroids generally have considerably longer durations when administered by intramuscular or subcutaneous injection than oil solutions of the same sex steroids. Formulations of estradiol benzoate (Agofollin Depot) and progesterone (Agolutin Depot), among others, are available on the market for use by intramuscular/subcutaneous injection and can possibly be purchased online. Estradiol benzoate and progesterone in aqueous suspension in particular have substantially longer and more useful durations than their shorter-acting oil-based counterparts; up to 3 weeks in the case of estradiol benzoate and up to 2 weeks in the case of progesterone. Considering the apparent injectable estradiol “shortage” as well as the limitations and inconveniences of all other routes of progesterone (e.g., poor efficacy of oral, short duration with conventional injections, inconvenience of rectal administration, etc.), aqueous suspensions of these sex-steroid medications may be a highly valuable alternative.
It would appear that Agofollin Depot (estradiol benzoate), Agolutin Depot (progesterone), and similar products have recently been discontinued by their manufacturer. See the comments here for more information.
Agolutin Depot is back in stock on, though not the others.