u/smurfsays

TL;DR It is not what PP405 was supposed to be as someone else has claimed.

In 2024, Amplifica announced “promising” phase I data for AMP-303, with some subjects experiencing an increase in terminal hair counts of more than 15% after one session [1]. The compound is described as a novel polysaccharide, so it appears to be based on hyaluronic acid [2]. Treatment was given on one side of the scalp and placebo on the other. The 15% increase was observed only in a subset of participants, and this was compared with baseline not placebo. This type of post hoc analysis can be misleading. They did not state that no one in the placebo group showed a more than 15% increase in hair count. What they found is that the number of subjects experiencing a greater than 15% increase in non-vellus hair count on the treatment side was significantly greater than the number subjects experiencing this level of response on the placebo side. They did not report how much hair growth increased compared with placebo. With even a small response to the drug, or random noise, it is easy to find a threshold that magnifies the significance of it. The difference between treatment and placebo could be only one percent and you could still potentially identify a threshold at which the number of subjects experiencing that extent of hair growth is significantly greater in the treatment group. Comparing responders on the treatment side to responders on the placebo side would be a more reliable comparison. We don’t know how many people responded on both sides, or from what baseline their hair counts increased. The lower the baseline reading, the less significant a 15% change becomes. Isolating responders for their comparison suggests that the treatment group as a whole did not demonstrate a significant change relative to placebo, and that even in responders there was a significant amount of hair growth on the placebo side. The press release teases a small signal that some subjects may have responded, while saying nothing of the magnitude or consistency of the response.

According to a new paper from Jiangnan University, the optimal HA molecular weight for hair growth is 268.1 kDa, demonstrating the most potent modulation for each growth factor and hair growth [3]. Medium-MW HA promotes hair growth via CD44-mediated ROS induction and phosphorylation of AKT, followed by stabilization of β-catenin. AKT stabilizes β-catenin by inactivating GSK-3, and facilitates nuclear entry via direct phosphorylation of β-catenin at ser552 [4]. However, priming of β-catenin by phosphorylation at ser45 by the destruction complex is required before GSK-3 can phosphorylate it. GSK-3-mediated regulation of β-catenin is largely AKT-independent, so the effect on Wnt signaling is more limited than canonical Wnt activation and insufficient to activate LEF1 in the absence of Wnt ligands [5]. All forms of HA stimulated VEGF in vitro and in vivo, while also inhibiting TGF-β1. Medium- and high-MW HA also markedly increased ALP and DPC migration in vitro. This suggests it has potential to extend anagen with repeated treatments, but the dermal papilla is already under oxidative stress. HA-mediated ROS generation was transient in vitro; however, the authors still caution that application of HA in such conditions may be inappropriate. Upregulation of ROS and β-catenin was more modest for High-MW HA. Pretreatment with NAC prevents ROS and β-catenin upregulation, indicating that β-catenin upregulation is dependent on ROS induction. AKT inhibition abrogated ROS generation. Taken together, the results suggest that CD44 activates AKT, while AKT-mediated ROS stabilizes its activity, thereby potentiating Wnt activation via inhibition of GSK-3 and/or direct targeting of β-catenin. Functional experiments were only performed in DP cells in vitro, while transcriptomic analysis was performed on whole mouse skin [3]. The functional location for the anagen-inducing effect of osteopontin-CD44 in vivo is in epithelial cells, but it may mirror the same regulatory network as HA, since transient ROS induction activates HFSCs [6][7].

Effects of hyaluronic acid on AKT and CD44 in human DP cells. I+II = low-MW, III= medium-MW, IV = High-MW

Effects of HA on growth factors in human DPCs in vitro

Downregulation of AKT doesn’t appear to be a contributing factor to Wnt suppression in AGA. Phosphorylated AKT and SGK1 are upregulated in bald dermal papilla cells, possibly to compensate for Wnt downregulation [8][9]. It fails to fully compensate, and it may exacerbate DPC stress.

Protein levels of TGF-β1 in bald and non-bald follicles. Activated Smad2 and Akt in bald and non-bald DPCs

In mice, medium-MW HA stimulated hair growth more robustly than other molecular weights, with earlier anagen induction, longer length, and the largest hair diameter. The amount of hair growth observed correlated with β-catenin expression in mouse skin. Measurements were taken at day 14 when treated follicles were in full anagen VI, but control follicles had only just begun entering anagen. Thus, the HF diameter increase may not meaningful. All treatments also markedly increased IGF1, and modestly inhibited BMP4 in mouse skin. Regulation of VEGF and TGF-β1 were also validated in vivo [3].

Stimulation of hair growth in mice with hyaluronic acid

Amplifica has a second CD44-based treatment in the preclinical stage [10]. AMP-203 is confirmed to be osteopontin, so AMP-203/AMP-506 might be osteopontin combined with HA and a third CD44 ligand, which could be serglycin or IGFBP4 based on their patents [11][12].

Amplifica identified this pathway by investigating transcriptional alterations in hairy moles to isolate proteins that might drive ectopic hair growth [6]. While the approach is intriguing, caution is needed when interpreting results. Body hair follicles are fundamentally different from scalp hair follicles. That which promotes body hair growth won’t necessarily promote hair growth on the scalp or in AGA. This is most obvious when observing the opposing effects of DHT and estrogen between the two regions. DHT is trichogenic in body hair, promoting vellus-to-terminal conversion, while on the scalp it promotes terminal-to-vellus conversion. DHT might even promote body hair growth via some of the same mechanisms that Amplifica has discovered. It’s possible that some trichogenic transcriptional targets of DHT in body hair have the inverse effect on scalp hair due to differential accessibility/availability of downstream effectors, or a weaker stress response [13].

The response to 17β-Estradiol is the inverse of that to DHT. It’s the most potent known stimulator of vellus-to-terminal conversion in frontal hair follicles, yet it retards body hair growth. Interestingly, osteopontin is the second most downregulated gene in male frontotemporal human follicles treated with 17β-Estradiol ex vivo. It’s possible that the observed downregulation is the result of negative feedback from upregulation of its CD44 receptor, but that would also suggest that supplementation of the ligand is insufficient.

Downregulation of osteopontin in frontotemporal human hair follicles by estrogen

Osteopontin is upregulated near the bulge in hairy moles, and knockout abrogates the precocious anagen phenotype in mice. Knockout also reduces wound-induced hair neogenesis, suggesting osteopontin might be useful for WIHN. The same phenotypes are observed in CD44 knockout, and in conditional mice treated with osteopontin, indicating that the effects of osteopontin on hair growth are mediated by epithelial CD44 [6].

Anagen progression depends on bidirectional signaling between the DP and epithelial cells. Osteopontin may enhance epithelial cell activation, which in turn enhances DP signaling, but if that DP signaling is resistant to activation in AGA, the effect would be limited. CD34+ cells were not compromised by CD44 knockout in mice [6]. This is an important epithelial cell population that’s depleted in AGA and dependent on DP signaling [14]. Osteopontin also promoted anagen entry in human occipital hair follicles grafted onto mice. It likely does the same in frontal hair follicles, but the more important question is how long it can prolong anagen in these follicles, and it’s doubtful that it can meaningfully increase hair diameter.

Follicum previously trialed an osteopontin fragment that is mutated to prevent binding of integrins while retaining its hair growth promoting ability. In preclinical testing it demonstrated a stronger effect in vivo than natural osteopontin [15]. The company published modest results from their clinical trial in 2020. After 3 months, the total hair count increased by 1.08/cm² vs placebo, while total anagen hairs increased by 8.16/cm² vs placebo [16].

AMP-601 is the last drug in Amplifica’s pipeline; it’s SCUBE3. Its function depends on whether it is secreted or membrane bound. UCI shows that it’s secreted from the DP to activate TGF-β receptors in epithelial cells, and that TGF-β inhibition abolishes SCUBE3-induced hair growth effects [17]. Presumably, it relieves the SMAD1-mediated quiescence program in HFSCs, which would trigger anagen but not reverse miniaturization [18]. Genetic studies suggest a causal role for TGF-β in AGA [19], but it appears to be overactivation in the DP driving it [8]. SCUBE3-null mice experience only a modest delay in anagen entry, with no other reported hair growth defects. Since TGF-β is a potent catagen-promoting factor upregulated by DHT, this seems less promising than HA, which inhibits TGF-β1 [3]. While not demonstrated by UCI, SCUBE proteins also facilitate Hedgehog activation by acting as chaperones for Shh, transporting it from the producing cell to the target cell [20]. SCUBE3 is likely a safer method of potentiating Hedgehog activity than activating Hedgehog with a smoothened agonist because its capacity to activate the pathway is rate-limited by Shh transcription. It’s also unlikely to significantly alter pathway activation in AGA.

In 2024, Amplifica announced “promising” phase I data for AMP-303, with some subjects experiencing an increase in terminal hair counts of more than 15% after one session [1]. The compound is described as a novel polysaccharide, so it appears to be based on hyaluronic acid [2]. Treatment was given on one side of the scalp and placebo on the other. The 15% increase was observed only in a subset of participants, and this was compared with baseline not placebo. This type of post hoc analysis can be misleading. They did not state that no one in the placebo group showed a more than 15% increase in hair count. What they found is that the number of subjects experiencing a greater than 15% increase in non-vellus hair count on the treatment side was significantly greater than the number subjects experiencing this level of response on the placebo side. They did not report how much hair growth increased compared with placebo. With even a small response to the drug, or random noise, it is easy to find a threshold that magnifies the significance of it. The difference between treatment and placebo could be only one percent and you could still potentially identify a threshold at which the number of subjects experiencing that extent of hair growth is significantly greater in the treatment group. Comparing responders on the treatment side to responders on the placebo side would be a more reliable comparison. We don’t know how many people responded on both sides, or from what baseline their hair counts increased. The lower the baseline reading, the less significant a 15% change becomes. Isolating responders for their comparison suggests that the treatment group as a whole did not demonstrate a significant change relative to placebo, and that even in responders there was a significant amount of hair growth on the placebo side. The press release teases a small signal that some subjects may have responded, while saying nothing of the magnitude or consistency of the response.

According to a new paper from Jiangnan University, the optimal HA molecular weight for hair growth is 268.1 kDa, demonstrating the most potent modulation for each growth factor and hair growth [3]. Medium-MW HA promotes hair growth via CD44-mediated ROS induction and phosphorylation of AKT, followed by stabilization of β-catenin. AKT stabilizes β-catenin by inactivating GSK-3, and facilitates nuclear entry via direct phosphorylation of β-catenin at ser552 [4]. However, priming of β-catenin by phosphorylation at ser45 by the destruction complex is required before GSK-3 can phosphorylate it. GSK-3-mediated regulation of β-catenin is largely AKT-independent, so the effect on Wnt signaling is more limited than canonical Wnt activation and insufficient to activate LEF1 in the absence of Wnt ligands [5]. All forms of HA stimulated VEGF in vitro and in vivo, while also inhibiting TGF-β1. Medium- and high-MW HA also markedly increased ALP and DPC migration in vitro. This suggests it has potential to extend anagen with repeated treatments, but the dermal papilla is already under oxidative stress. HA-mediated ROS generation was transient in vitro; however, the authors still caution that application of HA in such conditions may be inappropriate. Upregulation of ROS and β-catenin was more modest for High-MW HA. Pretreatment with NAC prevents ROS and β-catenin upregulation, indicating that β-catenin upregulation is dependent on ROS induction. AKT inhibition abrogated ROS generation. Taken together, the results suggest that CD44 activates AKT, while AKT-mediated ROS stabilizes its activity, thereby potentiating Wnt activation via inhibition of GSK-3 and/or direct targeting of β-catenin. Functional experiments were only performed in DP cells in vitro, while transcriptomic analysis was performed on whole mouse skin [3]. The functional location for the anagen-inducing effect of osteopontin-CD44 in vivo is in epithelial cells, but it may mirror the same regulatory network as HA, since transient ROS induction activates HFSCs [6][7].

Effects of hyaluronic acid on AKT and CD44 in human DP cells. I+II = low-MW, III= medium-MW, IV = High-MW

Effects of HA on growth factors in human DPCs in vitro

Downregulation of AKT doesn’t appear to be a contributing factor to Wnt suppression in AGA. Phosphorylated AKT and SGK1 are upregulated in bald dermal papilla cells, possibly to compensate for Wnt downregulation [8][9]. It fails to fully compensate, and it may exacerbate DPC stress.

Protein levels of TGF-β1 in bald and non-bald follicles. Activated Smad2 and Akt in bald and non-bald DPCs

In mice, medium-MW HA stimulated hair growth more robustly than other molecular weights, with earlier anagen induction, longer length, and the largest hair diameter. The amount of hair growth observed correlated with β-catenin expression in mouse skin. Measurements were taken at day 14 when treated follicles were in full anagen VI, but control follicles had only just begun entering anagen. Thus, the HF diameter increase may not meaningful. All treatments also markedly increased IGF1, and modestly inhibited BMP4 in mouse skin. Regulation of VEGF and TGF-β1 were also validated in vivo [3].

Stimulation of hair growth in mice with hyaluronic acid

Amplifica has a second CD44-based treatment in the preclinical stage [10]. AMP-203 is confirmed to be osteopontin, so AMP-203/AMP-506 might be osteopontin combined with HA and a third CD44 ligand, which could be serglycin or IGFBP4 based on their patents [11][12].

Amplifica identified this pathway by investigating transcriptional alterations in hairy moles to isolate proteins that might drive ectopic hair growth [6]. While the approach is intriguing, caution is needed when interpreting results. Body hair follicles are fundamentally different from scalp hair follicles. That which promotes body hair growth won’t necessarily promote hair growth on the scalp or in AGA. This is most obvious when observing the opposing effects of DHT and estrogen between the two regions. DHT is trichogenic in body hair, promoting vellus-to-terminal conversion, while on the scalp it promotes terminal-to-vellus conversion. DHT might even promote body hair growth via some of the same mechanisms that Amplifica has discovered. It’s possible that some trichogenic transcriptional targets of DHT in body hair have the inverse effect on scalp hair due to differential accessibility/availability of downstream effectors, or a weaker stress response [13].

The response to 17β-Estradiol is the inverse of that to DHT. It’s the most potent known stimulator of vellus-to-terminal conversion in frontal hair follicles, yet it retards body hair growth. Interestingly, osteopontin is the second most downregulated gene in male frontotemporal human follicles treated with 17β-Estradiol ex vivo. It’s possible that the observed downregulation is the result of negative feedback from upregulation of its CD44 receptor, but that would also suggest that supplementation of the ligand is insufficient.

Downregulation of osteopontin in frontotemporal human hair follicles by estrogen

Osteopontin is upregulated near the bulge in hairy moles, and knockout abrogates the precocious anagen phenotype in mice. Knockout also reduces wound-induced hair neogenesis, suggesting osteopontin might be useful for WIHN. The same phenotypes are observed in CD44 knockout, and in conditional mice treated with osteopontin, indicating that the effects of osteopontin on hair growth are mediated by epithelial CD44 [6].

Anagen progression depends on bidirectional signaling between the DP and epithelial cells. Osteopontin may enhance epithelial cell activation, which in turn enhances DP signaling, but if that DP signaling is resistant to activation in AGA, the effect would be limited. CD34+ cells were not compromised by CD44 knockout in mice [6]. This is an important epithelial cell population that’s depleted in AGA and dependent on DP signaling [14]. Osteopontin also promoted anagen entry in human occipital hair follicles grafted onto mice. It likely does the same in frontal hair follicles, but the more important question is how long it can prolong anagen in these follicles, and it’s doubtful that it can meaningfully increase hair diameter.

Follicum previously trialed an osteopontin fragment that is mutated to prevent binding of integrins while retaining its hair growth promoting ability. In preclinical testing it demonstrated a stronger effect in vivo than natural osteopontin [15]. The company published modest results from their clinical trial in 2020. After 3 months, the total hair count increased by 1.08/cm² vs placebo, while total anagen hairs increased by 8.16/cm² vs placebo [16].

AMP-601 is the last drug in Amplifica’s pipeline; it’s SCUBE3. Its function depends on whether it is secreted or membrane bound. UCI shows that it’s secreted from the DP to activate TGF-β receptors in epithelial cells, and that TGF-β inhibition abolishes SCUBE3-induced hair growth effects [17]. Presumably, it relieves the SMAD1-mediated quiescence program in HFSCs, which would trigger anagen but not reverse miniaturization [18]. Genetic studies suggest a causal role for TGF-β in AGA [19], but it appears to be overactivation in the DP driving it [8]. SCUBE3-null mice experience only a modest delay in anagen entry, with no other reported hair growth defects. Since TGF-β is a potent catagen-promoting factor upregulated by DHT, this seems less promising than HA, which inhibits TGF-β1 [3]. While not demonstrated by UCI, SCUBE proteins also facilitate Hedgehog activation by acting as chaperones for Shh, transporting it from the producing cell to the target cell [20]. SCUBE3 is likely a safer method of potentiating Hedgehog activity than activating Hedgehog with a smoothened agonist because its capacity to activate the pathway is rate-limited by Shh transcription. It’s also unlikely to significantly alter pathway activation in AGA.

The research group has no background in dermatology research. There is only one published study testing 2dDR on hair growth, and one of the lead author’s five papers was retracted due to suspicion of fraud. They conducted no mechanistic work in hair biology.

https://preview.redd.it/c5ytisc0cygh1.png?width=843&format=png&auto=webp&s=b2473abd52690e095d752c544b7e8393bcfec75f

It was tested because it may stimulate angiogenesis. There are better angiogenesis promoters, and those are ineffective for treating human AGA. 2dDR was given to shaved mice injected with testosterone where it promoted anagen earlier than placebo. This is a poor model for human hair loss. Thousands of compounds make mouse hair grow back faster than placebo, but most of them don't do anything for human hair loss.

Since mice don't have androgen-induced hair loss, the testosterone injections only modestly delay hair growth after depilation. At best, it performed equal to 2% minoxidil in a small sample. The combination of minoxidil and 2dDR performed worse than 2% minoxidil alone, so even if it works there is no synergy and it's inferior to 5% minoxidil.

All but the most delusional people accept PP405 is cope now, so what's the next hype people are coping with?

Skip to the last paragraph for the TL;DR.

Prolactin’s effects on the hair cycle were described decades ago by Craven et al., with additional research by Foitzik and Paus demonstrating that it promotes catagen (the regression phase) [1][2]. It has since been touted as the cause of pattern hair loss by the Ray Peat community. People find this pathway compelling because hyperprolactinemia is sometimes associated with hair loss in roughly the same area affected by common pattern baldness, and because prolactin regulates shedding in mammals with seasonal coats [3][4].

https://preview.redd.it/vm7s7r9nhndh1.jpg?width=669&format=pjpg&auto=webp&s=6813e1a8e97d242a2611ad9e78b40b64b5538b57

However, it is believed that hair in this region is inherently more susceptible to all forms of hair loss, and that extremely high prolactin could induce hair loss by regulating androgens [5][30]. Still, this and other evidence suggesting a role for prolactin in androgenetic alopecia (AGA) is compelling enough to warrant consideration. That’s why I’m glad to see Absci testing their prolactin receptor (PRLR) antagonist in AGA. I will attempt to untangle the evidence and determine how much of a chance ABS-201 really has to cure common baldness.

The most compelling evidence in my view is that another monoclonal antibody (mAb) targeting the PRLR (HMI-115), worked remarkably well in stump-tailed macaques [6]. This is the best animal model available for AGA. Many primate species go bald at puberty, but the stump-tailed macaque is the best studied. Baldness in these animals begins in the frontal scalp and recedes in approximately the same pattern as humans within months of sexual maturity. As in humans, the follicles remain intact but produce finer, less pigmented hairs. Testosterone suppresses proliferation of outer root sheath (ORS) cells in coculture with dermal papilla (DP) cells from bald macaque scalp but not when the DP cells are derived from occipital scalp, which is resistant to baldness [7]. Testosterone injection in ovariectomized adult macaques is able to induce baldness, but only in frontal hair follicles [8]. This demonstrates that testosterone and genetics are sufficient and required for baldness in macaques, just as they are in humans. Furthermore, finasteride prevents baldness in these animals [9]. Taken together, this indicates that baldness in macaques is driven by DHT and shares a similar etiology with male pattern baldness in humans. Minoxidil, androgen receptor antagonists, and 5α-reductase type II inhibitors all partially reverse baldness in these animals to varying degrees, with stronger reversal observed in younger macaques having a shorter duration of baldness [10][11].

https://preview.redd.it/vhwdqgjphndh1.png?width=617&format=png&auto=webp&s=278e191fe0162da78b2a0dc30302d24c6845efa3

HMI-115 was licensed by Hopemed Inc. from Bayer; it is currently undergoing clinical trials for endometriosis and androgenetic alopecia. Preclinically, Hopemed reported substantial hair regrowth in macaques that was superior to that observed in studies using finasteride, minoxidil, RU58841, or the combination of finasteride and minoxidil [6][9][12][11][13]. It should be noted that their macaque study isn’t peer-reviewed and there was no positive control or placebo. The macaques received subcutaneous injections with the antibody once every two weeks for 28 weeks, after which some of them appear to have experienced marked regrowth on parts of their scalp. At the end of the study, they reached 216 hairs per cm² in the area where hair counts were taken, although it appears to have been substantially less effective in more frontal and proximal areas of the scalp. For comparison, rhesus macaques have 2–21x more hair follicles than humans, depending on anatomical region. Normal hair follicle density on the forehead of macaques is around 400 hairs per cm² [14]. Hair growth was maintained above baseline for at least four years after treatment was discontinued.

https://preview.redd.it/h6wan49rhndh1.png?width=827&format=png&auto=webp&s=e2b5aa6d33e9e55a6fab6b204deb05f1724f2beb

As you can see, the macaques’ hair diameter is enlarged after 28 weeks. Vellus hairs were transformed to terminal hairs, which is required to cure baldness. Nine out of the eleven animals experienced regrowth, ranging from 50–220 hairs per cm², in both males and females. The average increase in terminal hair counts was 109%, and some of the animals had been bald for decades. Five of them were more than 25 years old, equivalent to about 66 in humans. Three of these senile macaques experienced regrowth, while all 6 young animals responded to treatment. There were no apparent adverse effects.

https://preview.redd.it/90rgrsathndh1.jpg?width=828&format=pjpg&auto=webp&s=f85ae5220c0ebbdac01593e599f0ddb9ebb563fd

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Cross-trial comparisons should be interpreted with caution because they are heavily confounded, but RU58841 was previously the most effective hair loss treatment evaluated in macaques. It increased hair counts by 103%, but with only 26% vellus-to-terminal conversion [6]. For finasteride, hair weight increased by 55% in the treatment group, compared with 32% for placebo [9]. Both results appear modest when compared to the HMI-115 macaque data, although the RU58841 results are also visually impressive.

https://preview.redd.it/9uplfh0whndh1.jpg?width=454&format=pjpg&auto=webp&s=925f03247b6e98f50e3225a833fd894245f682ae

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HMI-115 was developed for the treatment of endometriosis. During testing, it was incidentally discovered that it promotes hair growth in mice. Previously, another PRLR antibody (LFA102) was unsuccessfully trialed in prostate cancer [15]. Evidently, no significant hair growth was observed as a side effect in that trial. However, that drug failed to recapitulate the PRLR knockout phenotype in mice. HMI-115 is the first PRLR antibody to successfully recapitulate this phenotype, including reversible abrogation of lactation and fertility in female mice [16]. Unfortunately, this has not yet translated into much clinical success in AGA. Hopemed reported an increase of 14 non-vellus hairs per cm² for 12 men in their 6-month phase 1b trial [17]. Apparently, results were less encouraging in women since they were excluded from the phase II trial [18]. Like estrogen, prolactin appears to exert sex-dependent effects on human hair follicles [19]. In contrast to human male follicles, prolactin promotes proliferation and inhibits catagen in isolated female follicles, significantly increasing their elongation [19]. The phase II trial was completed in November 2024, and the results have yet to be reported [20]. I suspect the results were not outstanding, otherwise they would have moved to phase III by now. Efficacy was likely little to no better than in the phase 1b. It is tempting to think we can safely dismiss the prolactin pathway after this apparent failure to translate the macaque results to humans, but there is still some reason to be hopeful for ABS-201.

ABS-201 is claimed to be more potent than HMI-115. Despite being developed for human PRLR and having greater affinity for it over macaque PRLR, HMI-115 showed greater receptor occupancy in macaques in vivo. The dose in each trial was equivalent when adjusted for size and species, so species-specific differences in bioavailability and distribution may be relevant. The macaque trial showed over 90% receptor occupancy, while human trials showed only 60–80% occupancy, and it was likely less than 70%, according to the principal investigator for Hopemed and Absci. Absci expects to achieve receptor occupancies comparable to those observed in macaques [21]. Their antibody also has a longer half-life, which should allow for less frequent dosing, making it more economically feasible. They recently announced interim findings of their phase I safety study, estimating the half-life to be at least 65 days in humans. No serious adverse events were reported [22].

The antibody potency could make a difference because the PRLR has a low signaling threshold for maintaining function. Near-complete knockdown is required to block lactation and fertility. Full knockdown in mice increases hair shaft diameter, while 50% knockdown has no effect [23]. We don’t know where the threshold lies for an effect on hair growth, but something between 50% and 100% suppression is required to increase hair length or diameter in mice. If HMI-115 receptor occupancy was below 70%, there is a chance it didn’t meet the threshold required to promote hair growth. However, heterozygous mice showed no effect on hair growth, while HMI-115 apparently did reach the threshold required since hair counts increased in the trial. Additionally, the effect of PRLR knockout on hair growth is more pronounced in male mice than in females, consistent with results reported in humans thus far.

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Absci’s data also suggests that prolactin depletes CD34-positive progenitor cells in male frontotemporal scalp follicles, and this is rescued by their antibody [24]. The finding is relevant because AGA is partly driven by a failure of HFSCs to differentiate into progenitor cells. However, the ability of prolactin to deplete this pool does not prove it is the primary factor driving its depletion in human AGA. If this is an independent regulator of HFSC differentiation or progenitor proliferation, inhibiting it may increase the progenitor cell population, countering some of the suppression in AGA. That would limit its ability to fully reverse hair loss by the degree to which the strength of prolactin’s effect falls short of the primary suppressive factor in AGA. Hair loss in macaques is less severe than in humans. Their scalps don’t turn fully bald; they typically retain longer vellus hairs than humans, and their hair loss appears to be less resistant to treatment. That could indicate the primary suppressor of HFSC specification is less strongly activated in macaques than in humans, thus requiring less compensation to overcome it in macaques. Macaque immune responses differ from those in humans, and humans could have more persistent epigenetic repression. There are many reasons why this might be insufficient in humans. The fact that the PRLR is not upregulated in bald scalp suggests that knockdown may only compensate for the primary driver. The increased hair diameter observed in PRLR knockout mice is modest. The 12% increase is insignificant compared with the several-hundred-percent increase required to fully reverse miniaturization in human AGA. Unless aberrant PRLR signaling is the driver of progenitor cell depletion in AGA, its knockdown will be insufficient compensation. Still, it is one of the more promising treatments to enter clinical trials since finasteride.

At this point you may be wondering why a dopamine agonist cannot be used in lieu of a PRLR antagonist. Intrafollicular prolactin is not regulated by dopamine, and the direct, catagen-inducing effects of prolactin are independent of circulating prolactin levels except perhaps in cases of extreme hyperprolactinemia [25]. Rather, dopamine is a negative regulator of human hair growth that induces catagen ex vivo [26]. Instead, cutaneous prolactin is regulated by substance P, TNF-α, IFN-γ, TRH and estradiol [27][4][28]. Interestingly, an insertion event in the prolactin gene created an alternative promoter in primates, which may enable primate-specific tissue expression patterns and functions [29].

https://preview.redd.it/de7ls7g0indh1.png?width=555&format=png&auto=webp&s=4ec06ea87d612e5500698ddf1def9efa5965e5bd

Hair loss associated with hyperprolactinemia may be indirectly mediated by alterations in androgen metabolism because it is often accompanied by hirsutism [30][31]. In women with androgenetic hair loss, circulating prolactin levels were not elevated at baseline but only following TRH stimulation, whereas women with hirsutism showed elevated baseline prolactin levels [32]. TRH upregulates prolactin mRNA and protein in female frontotemporal and occipital follicles ex vivo, along with PRLR mRNA. However, it reduces PRLR protein expression, specifically in the ORS. At both transcriptional and protein levels, 17β-Estradiol upregulates prolactin and its receptor in the ORS and matrix of female frontotemporal and occipital hair follicles [33].

PRLR knockout appears to be well tolerated in males. All known serious effects of prolactin result from excessive production, with the exception of infertility in females and inability to lactate [34]. It is not an essential regulator of any major function in males, and knockout has no effect on male fertility in mice [35][36]. Loss-of-function mutations in the human PRLR appear to have no major consequences besides female infertility and lactation failure [37][38][39]. However, mice with PRLR knockout have markedly elevated prolactin levels due to loss of negative feedback, and can develop prolactinomas [40][41]. An open question is how much ABS-201 increases prolactin production. HMI-115 only modestly increases prolactin while PRLR-knockout mice have prolactin levels that are up to 100-fold higher than normal.

In mice, prolactin is found in the proximal inner root sheath and inner two-thirds of the ORS during early and late anagen. It shows strong expression in the ORS during catagen and weak expression during telogen. PRLR is also expressed in the ORS where it is downregulated during early anagen. While both long and short transcripts are detected, the long isoform predominates and is downregulated only from anagen I through anagen IV [1][42][43]. PRLR knockout accelerates anagen only modestly while increasing hair follicle length by just one day’s worth of growth.

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Placental lactogen I is also produced locally, with expression rising sharply during early anagen before falling rapidly. Prolactin and its receptor are localized to the epithelium of human hair follicles and are upregulated during the transition to catagen. Stimulation with supraphysiological concentrations of prolactin can induce premature catagen in human male hair follicles ex vivo from both frontal and occipital scalp [4][42]. This supports a direct role for local prolactin signaling in hair cycle regulation, but also indicates that prolactin cannot explain the pattern of hair loss [44].

STAT3/5 were identified as upregulated hub genes in an analysis of scalp skin from patients with premature AGA [45]. The PRLR recruits JAK2, which phosphorylates STAT5 and may promote apoptosis by increasing the BAX/BCL2 ratio in matrix cells during catagen [46]. Oncostatin M secreted from TREM2+ macrophages binds OSMRβ in the hair germ and CD34+ bulge cells. This maintains stem cell quiescence during early to mid-telogen by activating STAT5 independent of PRLR. Perifollicular macrophages are depleted throughout telogen, relieving STAT5-mediated quiescence [47]. Dermal macrophages could be recruited to the follicle during the anagen-to-catagen transition by increased TGF-β in late anagen. This might contribute to the onset of catagen through secreted factors such as oncostatin M and FGF5. Macrophage ablation promotes anagen, confirming they are the major source of STAT5 activation under homeostasis. However, macrophages also promote anagen by secreting Wnt ligands [48][49]. Recruitment of these macrophages may be one factor underlying the efficacy of microneedling in AGA.

Depletion of macrophages by targeting Csf1r with pexidartinib also inhibits STAT5 and induces precocious anagen. However, STAT5 inhibition after macrophage depletion is weaker than that produced by tofacitinib. This might be due to residual activation by prolactin or other cytokines such as IL-6, although this appears to be insignificant in mice under homeostatic conditions as residual activation was insufficient to delay anagen in the absence of oncostatin M signaling. Prolactin upregulates STAT5a in ORS cells from human scalp follicles in vitro, and STAT5 is strongly activated in female human skin treated with prolactin [27].

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Intriguingly, STAT5 ablation does not increase the clonogenicity of murine CD34+ bulge cells in vitro, it only increases the proliferation of P-cadherin-positive hair germ cells. JAK inhibitors act on both bulge and hair germ cells, whereas STAT5 ablation only affects hair germ cells, indicating distinct mechanisms underlying quiescence in these cell compartments. In human follicles, Absci found that prolactin increases apoptosis of bulge cells, while ABS-201 increases their proliferation ex vivo [24].

Prolactin also phosphorylates STAT3. In a study of 25 AGA patients, STAT3 expression was higher in bald follicles than in healthy follicles, but its expression did not correlate with hair loss severity [50]. STAT3 overexpression depletes K15/CD34/α6-integrin-positive HFSCs in the mouse bulge, in part, by stabilizing β-catenin and downregulating α6-integrin to facilitate migration out of the niche. This is not an effect observed in AGA, so STAT3 overexpression is unlikely to contribute to hair loss in this manner. Alternatively, it could contribute to ectopic TWIST1 expression in the ORS [51]. Prolactin also activates Akt, a pathway that is required for hair growth, but which might contribute to hair loss when overactivated. Its activation is also mediated by JAK2.

Prolactin receptor expression is regulated by NFATc1. Cyclosporine A (CsA) can suppress PRLR signaling by inhibiting the calcineurin/NFATC1 pathway. NFATC1 is normally activated by BMPs to maintain HFSC quiescence but does not directly control their differentiation [52][53][54]. CsA is well known to induce hypertrichosis, and a small trial showed marked hair regrowth in men with AGA [55][56]. However, there were only two responders in the trial, while the six non-responders and three controls also experienced strong regrowth. The trial appears flawed, but CsA is a large compound that is difficult to deliver to the follicle, and it exhibits a biphasic dose response in cultured mouse vibrissae [57]. Separately, it was found that CsA promotes hair growth by inhibiting SFRP1 in human hair follicles [58]. This might be a downstream effect of PRLR signaling through STAT5, which can activate SFRP1 [59]. CsA also prolongs anagen by preventing matrix cell apoptosis, as does the PRLR antagonist ABS-201 [60]. However, cyclosporine’s known effects on the hair cycle are PRLR-independent.

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NFATc1 directly represses transcription of cell cycle gene CDK4 in HFSCs. This maintains stem cell quiescence independent of Wnt or LDH by preventing the transition from G1 to S phase, but this mechanism is independent of PRLR [61][62]. Nuclear NFATc1 can also negatively regulate Wnt signaling downstream of the destruction complex through competitive binding with Dvl, preventing the recruitment and stabilization of β-catenin to LEF/TCF complexes [63]. CsA-associated hypertrichosis could also be related to these effects.

There is some minor evidence that prolactin could be associated with AGA. The PRL gene is within 500 kb of an AGA risk locus (rs6935891) [64]. However, the effect size is modest, and PRL is not necessarily the causal gene at this locus. The SNP is located within an intron of CASC15. Additionally, several miRNAs targeting prolactin signaling are differentially regulated in bald and non-bald plucked hair follicles [65].

Newborns have extremely high levels of serum prolactin, reaching 100–300 ng/mL, well above the common threshold of 20 ng/mL for hyperprolactinemia. Prolactin levels return to normal in the weeks to months before infants regrow their hair, suggesting a possible connection [66]. However, prenatal hormone exposure is high and there is a general postnatal hormonal surge that includes testosterone, so it is difficult to draw any link between prolactin and the shedding of lanugo [67].

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There is some animal and in vitro evidence demonstrating regulation of prolactin signaling by androgens. Castrated rats do not produce extrapituitary prolactin in the prostate. This is rescued by testosterone administration, demonstrating that prolactin production is regulated by androgens in rat prostate. In breast cancer cells, AR upregulates PRLR. [68] There is no evidence for such regulation in the hair follicle, and DHT, AR, and PRLR are not colocalized in human hair follicles [69].

In contrast to AGA, PRLR was moderately upregulated in alopecia areata lesions in one small case-control study. Any causal role in this condition would likely be mediated by immune regulation instead of direct effects on the hair follicle, but increased PRLR expression may also be a secondary effect of cell loss and subsequent reduction in the secretion of BMP inhibitors [70].

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One remaining possibility is that affinity of prolactin to its receptor is strengthened if pH is increased in bald scalp. There is a 500-fold change in the dissociation rate of prolactin from its receptor between pH 8.3 and 5.8 [71]. If hypoxia is reduced in the AGA hair follicle epithelium, this might lead to increased pH and enhanced prolactin signaling. In one breast cancer cell line, STAT5 transactivation increased approximately two-fold at pH 8 compared with pH 6 following treatment with 25 ng/mL prolactin in vitro [72]. This raises the possibility of mechanistic overlap with PP405.

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While there are some intriguing connections between prolactin and hair loss, the evidence as a whole leaves me skeptical that targeting the prolactin receptor will cure AGA. I do not expect ABS-201 to be appreciably more effective than HMI-115. Rather, it could become a useful alternative or adjuvant therapy for some individuals. Even in macaques, hair density increased to what may be only about half of normal density in the area showing the most regrowth, and some animals even lost hair during treatment. Other areas of the scalp showed more modest improvements, and there was no control group. I expect an effect similar to that of JAK inhibitors including tofacitinib or ruxolitinib, which induce modest hair growth in human AGA by inhibiting what may be the primary mediator of prolactin’s effects on the hair follicle.

There's a new verteporfin replication independent of Mascharak. In mini-pigs, they achieved better wound regeneration by following verteporfin treatment with retinoic acid after a short but unspecified time delay. RA did not appear to significantly enhance regeneration in rabbits.
Controlled microtrauma opens a regenerative window for appendage-bearing skin repair

The product contains NV-273, NV-623, and NV-624. NV-273 is a PHD2 inhibitor. It stabilizes HIF-1 alpha, which promotes glycolysis less directly than PP405. NV-623 and NV-624 were identified by screening for compounds that would stimulate DPC proliferation in vitro. The researchers used high-throughput screening of dermal papilla cells to train a computational model for a larger in silico screen, then confirmed DPC proliferation with the top hits in vitro. This isn't very interesting because DPC proliferation isn't relevant in hair loss treatment. I discussed in another thread why PP405 isn't exciting. That leaves no reason to use NOVOGRO when minoxidil is available. They do have a 5α-reductase inhibitor (NV-1065) in the pipeline, but it's not included in this product.
AI-enabled discovery of small molecules targeting complementary pathways for hair follicle rejuvenation | bioRxiv

Skip to the last paragraph for the TL;DR.

PP405 might be the most hyped hair loss treatment since Rogaine and Propecia. As always, the hair loss community is split into two camps: those who believe it will be a cure based on little more than hope, and those who believe it will fail based on nothing more than decades of disappointment. My aim here is to provide a more objective and reasoned analysis of the good, the bad, and the ugly from the scientific literature as it relates to Pelage’s supposedly breakthrough hair loss treatment.

I’ll begin with some background on what PP405 is and how it works. It’s a small molecule inhibitor of the mitochondrial pyruvate carrier (MPC) designed to artificially induce a Warburg-like state, whereby glycolytic metabolism is preferentially utilized. By preventing MPC from shuttling pyruvate into mitochondria, PP405 restricts pyruvate entry into the TCA cycle, which can limit oxidative phosphorylation (OXPHOS), while accumulation of pyruvate in the cytosol fuels glycolytic metabolism through LDH-mediated conversion of pyruvate to lactate, primarily by LDHA. The reduction of pyruvate to lactate utilizes NADH, and this reaction converts NADH back to NAD+. NAD+ is utilized in the oxidation of G3P by GAPDH, which reduces NAD+ to NADH. This is a critical step in glycolysis required for downstream ATP production. For glycolytic flux to continue, NADH must be reoxidized to NAD+. To summarize, this chain of events produces pyruvate from glucose before converting it to the end product, lactate. By inhibiting MPC, PP405 promotes the continuation of this glycolytic cycle, with the ongoing reduction of pyruvate to lactate favored over TCA entry. The accumulation of lactate is an indirect indicator of increased glycolytic activity.

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Overview of anaerobic glycolysis induced by PP405

Glycolysis provides energy rapidly when needed, but OXPHOS is much more efficient and more closely associated with differentiation. Stem cell niches rely heavily on glycolysis to support self-renewal and maintain stemness. Glycolysis typically produces less ROS than OXPHOS. This protects stem cells from ROS-associated DNA damage to support long-term maintenance. In contrast, greater ATP production from OXPHOS supports increased metabolic demands during differentiation, while ROS can act as signaling molecules that support differentiation programs.

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Typical effects of metabolism on stem cell renewal and differentiation

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This gets to the heart of the problem. As you’re probably aware, stem cell niches provide a reservoir of cells for organ regeneration, but those cells must differentiate along lineages that generate the tissue. The hair follicle stem cell (HFSC) niche is hypoxic, and glycolytic metabolism ostensibly regulates the proliferation of HFSCs and outer root sheath (ORS) progenitor cells. Glycolysis supports the activation of HFSCs, as demonstrated by Flores et al [2]. from UCLA and Pelage, and by Kim et al. at the Max Planck Institute. However, the latter group showed that the HFSC state does not depend on glycolysis, nor does glycolysis promote HFSC differentiation [3]. This is the key aspect conveniently omitted and unexplored by Pelage, limiting the translatability of their findings to male pattern baldness.

Now I’ll explain what makes this so important in the context of this treatment. Androgenetic alopecia is characterized, in part, by the progressive miniaturization of hair follicles over time. The follicles do not simply go dormant and stop growing; instead, they produce smaller hair shafts with every new cycle until the follicle is so small that it becomes invisible to the naked eye. It often takes many hair cycles for it to reach the point where the terminal-to-vellus transformation is complete.

Obviously, hair follicles don’t regenerate for a new cycle without the activation of stem cells. Therefore, their continued cycling over the course of the disease, while producing smaller hair shafts each time, shows that miniaturization isn’t driven by HFSC quiescence. Notice that everyone has vellus hairs covering most of the body. Those hairs cycle through the same process of HFSC activation. Activating HFSCs on your scalp won’t enlarge vellus follicles any more than it will enlarge the vellus follicles anywhere else on your body. Based on its MOA, expecting PP405 to make your scalp hair longer and thicker is no different from expecting it to make the hair on your arm grow as long and thick as scalp hair.

AGA does present with progressively shorter anagen phases and longer periods of dormancy, but that dormancy is a byproduct of miniaturization and less significant than the shorter anagen phase. You can activate HFSCs with something like PP405, or any other compound that promotes HFSC activation (minoxidil does this too, perhaps less directly), and some dormant hairs will start growing almost immediately, preempting their months-long dormancy phase, but this will not change the diameter or length of the hair shaft. All that will grow is a vellus hair if that’s what the follicle produced during the previous growth phase. Dormancy isn’t what makes these hairs shorter and smaller than healthy scalp hair, just as mice do not grow long hair no matter how much of a glycolysis-stimulating compound they receive. To reiterate, this is because the size of the hair shaft is programmed independently by the DP.

Mice with Mpc1 deletion in HFSCs do not produce longer or thicker hair shafts than controls, and their ear hair remains shorter than their dorsal hair. This indicates an inability to modulate organ size. When mice are depilated, MPC inhibitors trigger precocious anagen, making the hair grow back faster than it normally would, but it grows back the same size as it otherwise would have because HFSCs do not determine the size of the hair shaft. We know from other research that the size of a hair shaft is determined by the size of the dermal papilla (DP) [4], a signaling center that sits at the base of the hair follicle and drives the differentiation of HFSCs via paracrine signaling that supports Wnt activation, among other things. This is where dihydrotestosterone (DHT) is produced and causes damage. Over time, DHT produces a reduction in the number of DP cells. When the number of DP cells falls below a critical threshold, hair fails to regenerate. A progressively smaller DP secretes fewer factors supporting expansion of the progenitor cells that go on to produce the hair shaft. These are the cells that are depleted in bald scalp [5]. In contrast, HFSCs are retained in bald scalp and, as mentioned, they remain competent to activation, regularly cycling until the DP becomes too small to produce a visible hair shaft. Instead, they progressively lose the ability to differentiate into progenitor cells, presumably because reduced signaling from the DP impairs Wnt activation.

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Schematic showing requirement of Wnt activation (LEF1) for differentiation of HFSCs [6]

It has already been shown that HFSCs can activate and proliferate without Wnt, but they fail to adopt a hair follicle fate [7]. Over time, additional defects might accumulate in the hair germ that restrict its expansion. These could involve overactivation of Akt and Sgk1, forcing progenitors back into a stem cell state; apoptosis of progenitors; or EMT. Regardless of the mechanism underlying the depletion of progenitor cells, it’s inconceivable that MPC inhibition could restore the pool because its depletion is clearly independent of HFSC activation. In fact, Kim et al. showed that converting HFSCs to progenitor cells requires a metabolic switch from glycolytic metabolism to OXPHOS and glutaminolysis, while the inverse switch causes ORS progenitor cells to return to a stem cell state [3].

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Cell fate progression and reversibility of HFSCs

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The researchers even stated, three years after the founding of Pelage, that “Whether glycolytic metabolism is indeed the common denominator in HFSC activation signaling remains to be determined. [8]” Yet they had already formed a company to develop a hair loss drug based on this research.

Ldha is ostensibly required for hair growth, since deletion of Ldha in murine HFSCs prevents their activation [2]. However, targeting this downstream mechanism may fail to activate other programs that are also important or required for cell cycle entry. Even if the drug is sufficient to trigger anagen entry, it will not rescue dysfunctional lineage progression in AGA, as OXPHOS and Wnt are required for this.

The assertion that Wnt is downstream of glycolytic flux in HFSCs is based on the observation of low Wnt expression in Ldha-null HFSCs [2], but acting downstream of a process does not establish direct regulation by that process. Wnt activation requires paracrine signaling between dermal papilla and epithelial cells. In a healthy hair follicle, signals from the DP activate the hair germ before the HFSC bulge. The hair germ then activates bulge stem cells while crosstalk enhances Wnt activation to promote lineage progression [9]. In bald follicles, a dysfunctional DP may be unable to participate in that crosstalk, impairing Wnt activation and lineage progression [10].

Additional pathways are involved in AGA, but Wnt is the most relevant one for this discussion. Lactate can activate HFSCs and progenitors prematurely, but without Wnt-activating signals from the DP, it can’t push HFSCs to differentiate into progenitors and replenish the depleted progenitor pool in bald scalp. Without that, there are not enough of these cells with which to build the new hair.

Furthermore, reduced glycolytic activity hasn’t been observed in mpAGA, and there is no genetic association with this pathway in the disease. LDHA deficiency is not associated with hair loss despite individuals with LDHA loss-of-function mutations expressing very low LDH activity in scalp hair follicles described as “virtually devoid of LDH activity [11][12][13].”

Further support for the continued activation of HFSCs in bald scalp comes from the fact that sebocytes are derived from the same HFSC pool that produces the hair follicle [14]. AGA is characterized by progressive miniaturization of the follicle with concomitant sebaceous gland hyperplasia. Rather than failing to activate, these stem cells preferentially differentiate along another trajectory in the absence of Wnt, becoming sebocytes and epidermal cells instead of hair follicle progenitors, and the hair cycle is shortened from years to weeks [15][4]. Activating the cell cycle with glycolysis can’t correct this defect. To reiterate, it only accelerates production of the same tissue that the cells would otherwise build, which in late-stage baldness is a sebaceous gland, interfollicular epidermis, and a tiny vellus hair [7][16].

Many drugs activate HFSCs, and several do so through the same pathway or mechanism as PP405. Hypoxia stabilizes HIF-1α, a master regulator that promotes glycolysis over OXPHOS. Stemoxydine recapitulates hypoxic signaling in human hair follicles, including upregulation of glycolytic genes. Although MPC inhibition is the most direct way to stimulate glycolysis, Stemoxydine efficiently enhances it based on L’Oreal’s testing. Unfortunately, it only increased hair density by 4% compared to placebo [17].

UCLA’s research demonstrates that activation of the beta-2 adrenergic receptor (ADRB2) is what stimulates glycolysis in HFSCs in vivo to support their activation. The researchers showed that procaterol induces glycolysis and precocious anagen in mice, comparable to PP405 [18]. Yet procaterol does not appear to have any noticeable effect on AGA. Perhaps it would be more effective at higher local concentrations, but if stimulating glycolysis were the cure for hair loss, we would expect to see some improvement in hair growth among men taking beta-2 agonists. NFATC1 inhibitors, such as cyclosporine, also activate HFSCs. Minoxidil does this quite well, among other effects, albeit most likely indirectly. The indirect nature of minoxidil’s ability to induce anagen in AGA suggests there is typically no defect in the intrinsic capacity of HFSCs to produce lactate, and that there would be no additive effect when combining these compounds. Few agents induce precocious anagen in mice faster than minoxidil. A multitude of agents that increase IGF-1 or KGF can activate HFSCs; the literature is replete with examples of such failed treatments.

Adrb2 is activated in HFSCs by norepinephrine released from the sympathetic nerve [19]. This nerve wraps around the arrector pili muscle (APM), a small muscle attached to the hair follicle bulge. Without the APM, innervation of HFSCs is lost. Adrb2 activation promotes lactate production through Creb signaling. Beta-2 activation stimulates adenylyl cyclase, leading to the accumulation of cAMP. cAMP activates Pka, which phosphorylates Creb to facilitate its nuclear translocation. Production of glycolytic intermediates is increased following cAMP stimulation [18].

Beta-2 signaling also represses FGF18 [19], which regulates histone H2A ubiquitination in HFSCs and thereby controls the expression of multiple cell cycle genes. This controls stem cell quiescence independently of other known HFSC quiescence regulators Nfatc1, TCF3/4, BMP6 and Foxc1 [20].

In mice, Creb signaling promotes glycolytic flux in HFSCs and triggers anagen, similarly to Mpc inhibition [21]. cAMP accumulation promoted by forskolin is sufficient for HFSC activation. cAMP accumulation and anagen induction were also observed following topical PDE inhibition. However, only some Creb+ cells are proliferative, and only some proliferative cells are Creb+, suggesting Creb may only correlate with HFSC activation. Nevertheless, anagen induction upon cAMP stimulation indicates this is one mechanism by which Adrb2 activates HFSCs and triggers anagen [18]. In addition to glycolysis, pathways upregulated by Creb activation in mouse epidermis include OXPHOS, cholesterol biosynthesis, TCA and FAO [21].

Sympathectomy (removal of the sympathetic nerve) and Adrb2 knockout produce the same delayed-hair-cycle phenotype, with the former being rescued by the Adrb2 agonist procaterol. This confirms that Adrb2 mediates the effect of the APM-sympathetic nerve unit on the hair cycle [19].

Because Adrb2 knockout only delays anagen rather than inducing miniaturization, and FGF18 is significantly downregulated in bald scalp (GSE36169), it appears that loss of ADRB2 does not drive follicular miniaturization, but it could contribute to prolonged kenogen in vellus hairs. The authors acknowledge the speculative nature of targeting this pathway in AGA and the fact that Adrb2 loss merely delays activation rather than preventing it. They suggest ADRB2 agonists might be useful in AGA, but refrain from the sort of definitive claims that Pelage has made regarding this pathway [19].

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There is speculation that because the APM becomes detached from follicles in bald scalp, its detachment and downstream effects drive hair loss. On the contrary, these Adrb2 and sympathetic nerve knockout experiments provide support for APM detachment being a consequence of hair loss rather than its cause. The APM makes hair stand erect for thermoregulation and connects the sympathetic nerve to HFSCs in the bulge. In response to cold, the muscle contracts, causing hair to stand erect and trap warm air, while the nerve simultaneously secretes norepinephrine to signal dormant hair follicles to regenerate. The APM is not required for mammalian hair growth, as some mammals don’t have an APM, including sea otters and pinnipeds [22]. Despite this, sea otters have the densest hair of any animal.

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sea otter

Rhesus monkeys lack an APM in both thick vibrissae and vellus hairs of the lips and eyelids [23]. Rat and mouse vibrissae have an APM, but it doesn’t connect to the bulge compartment [24]. During morphogenesis, the hair follicle is generated before the APM forms and innervation occurs because Shh produced by the hair follicle is required for APM development [19]. Absence of innervation doesn’t prevent initial follicle downgrowth.

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APM formation with establishment of sympathetic nerve innervation

Likewise, the APM connection is severed during hair transplantation, yet this doesn’t prevent the follicle from growing normally before the connection is reestablished [25]. Therefore, APM detachment doesn’t appear to prevent regeneration. Nor does it prevent lab-grown hair follicle organoids from developing before establishing attachment to the APM [26].

Estrogen has a remarkable ability to reverse AGA, indicating that, with the right signaling, either the APM is not required for reversal or its attachment can be reestablished. Rather than driving hair loss, APM detachm0ent in bald scalp may be another epiphenomenon of the Wnt downregulation that drives hair loss. APM attachment is governed by the Wnt target gene nephronectin, which is required to establish the anchor to which the APM attaches downstream of Wnt activation in the bulge [27]. Wnt signaling in the bulge depends on factors secreted by the DP, which becomes dysfunctional as a consequence of DHT [28].

Nephronectin binds α8β1 integrin on mesenchymal cells, anchoring them to the bulge, after which the APM attaches to these cells. As AGA progresses, Wnt signaling may become sufficiently reduced that nephronectin is no longer expressed, causing the APM to lose its anchor. In nephronectin-knockout mice, the APM can instead localize above the bulge due to compensatory upregulation of EGFL6. Such compensation may not occur following detachment in wild-type mice or humans.

Further evidence against APM detachment as the cause of AGA is that deletion of nephronectin doesn’t affect DP size. Hair follicle development is normal, but anagen entry is accelerated and telogen is shortened because altered localization of α8β1+ cells reduces the physical separation between the DP and hair germ [27]. As previously mentioned, severing the sympathetic nerve or deleting Adrb2 delays anagen but does not alter hair follicle morphology [19]. Since the APM-sympathetic nerve unit activates HFSCs through Adrb2 activation, deletion of Adrb2 should recapitulate the AGA phenotype if APM detachment were the driver of AGA. All of this evidence indicates that the APM and sympathetic nerve regulate the timing of the hair cycle rather than the size of the hair shaft, and there’s no evidence that glycolytic flux regulates anything beyond HFSC activation and proliferation.

Flores et al. also observed robust lactate induction and precocious anagen within six to nine days after application of the GP130 modulator RCGD423 to activate STAT3 [2].

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Lactate induction by STAT3 activation using RCGD423

STAT3 and STAT5 were identified as upregulated hub genes in an analysis of scalp skin from patients with premature AGA [29]. In another study of 25 patients, RT-qPCR showed stronger STAT3 expression in bald follicles than in healthy follicles, though it did not correlate with hair loss severity [30]. STAT3 overexpression in AGA may therefore already promote lactate accumulation. STAT5, by contrast, acting downstream of oncostatin M and NFATC1/prolactin, could repress HFSC activation in AGA through as-yet-unresolved mechanisms, potentially including the activation of SFRP Wnt-ligand antagonists in the bulge [31][32][33].

Commonly prescribed TZDs are also known to inhibit MPC, yet they show little to no effect on AGA [34]. PKM2 catalyzes the conversion of PEP to pyruvate during glycolysis. Its activation also accelerates the hair cycle in mice and enhances WIHN [35]. β-catenin and nuclear β-catenin were markedly elevated in KY19382-treated wounds compared with control wounds, but not in wounds treated with the PKM2 activator. Nuclear β-catenin was slightly reduced in K15+ HFSCs of mice treated with PKM2 activator. PKM2 expression was decreased in alopecia areata patients (GSE45512), but not in AGA.

In young, but not aged, mice, rapamycin, metformin and αKG activate HFSCs and accelerate anagen through autophagy, as does the AMPK activator AICAR [36]. This activation is mediated by increased Ldh activity that fuels glycolysis [37]. Metformin has been reported to stimulate hair growth in cicatricial alopecia [38][39][40], but the effect may be mediated by PPAR regulation rather than LDH. It may also be beneficial in alopecia areata [41]. If it has any impact on AGA, the effect has gone unnoticed clinically. Likewise, there are only anecdotal reports of modest hair growth with rapamycin. These drugs are so widely used that a meaningful effect on a disease as common and visible as AGA would likely have been noticed by now.

Given that rapamycin induces a more than threefold increase in lactate in murine hair follicles, comparable to that induced by MPC inhibitors [42] [43], and accelerates the hair cycle within the same timeframe, it is unlikely that PP405 would fare much better than rapamycin in the absence of evidence for a specific defect requiring direct MPC targeting. Most evidence, including the effects of minoxidil, argues against such a defect. Rapamycin did modestly prolong hair growth in one of two murine studies (no p-value provided). Rapamycin and α-KG were the fastest-acting of the tested compounds. Dietary α-KG also increased hair density in Rex rabbits and promoted the nuclear localization of β-catenin in DPCs in vitro [44].

 Another experimental molecule capable of facilitating glycolytic reprogramming is IM176OUT05, which acts by inhibiting OXPHOS. In preclinical models, it produced similar effects to Pelage’s molecules [45]. The UCLA team behind Pelage also demonstrated HFSC activation and accelerated telogen-anagen transition in mice following transient inhibition of the electron transport chain (ETC), the machinery that drives OXPHOS. The compounds they used to stimulate glycolysis were phenformin, rotenone, and antimycin [46]. Phenformin was withdrawn from the market but was never reported to cause hair growth. In contrast, genetic ablation of the ETC in mice causes hair follicle dystrophy with severe impairment of K15+ HFSC differentiation, a possibility discussed earlier in this article [47].

DP cells rely on OXPHOS, which is impaired in bald scalp and may dampen Wnt activation through endoplasmic reticulum (ER) stress [48][49]. Restoring OXPHOS is more likely to show efficacy in AGA than forced glycolysis. Researchers tested one of the same MPC inhibitors that promotes hair growth in mice in human hair follicles ex vivo [50]. They found that it caused ER stress and inhibited Wnt signaling, leading to cell cycle arrest in epithelial progenitor and matrix cells.

The results contradict those observed by Flores et al. in mice [2]. One of the researchers behind Pelage suggested that the dose may have been too high in this study. However, the effect was seen at 10 μM, while Flores used the same drug at 20 μM on mice with positive results. Off-target effects on mitochondrial membrane potential are only observed at concentrations exceeding 10 μM, specifically at 100 μM [2][51].

Cell proliferation of human hair follicles exposed to MPC inhibitor ex vivo

In light of the way PP405 has been presented by Pelage, the media, and the hair loss community, it is striking that one of the researchers at Pelage told a reporter they were concerned the drug would kill all the hair follicles in the clinical trial and they were glad to be wrong about that [52]. It’s difficult to reconcile that concern with prior statements implying it’s a breakthrough treatment that will reverse miniaturization. How could they be so sure about the latter when they weren’t even sure that it would not kill follicles?

It’s possible that the drug stimulates initial hair growth in dormant follicles while worsening the underlying condition over the long term through activation of the integrated stress response (ISR). Deletion of Mpc1 in mouse HFSCs produced no apparent adverse effects, but this does not guarantee it will be harmless in other cell compartments. The data on HFSCs from Flores et al. are reassuring. However, broad-based and chronic induction of glycolysis is very different from HFSC-specific Mpc1 knockout or the transient drug-induced glycolysis in mouse experiments. Forced glycolysis in other follicular compartments could compromise their normal function. UK-5099 has also been found to cause mitochondrial dysfunction in LNCaP cells, producing a twofold increase in ROS production together with reduced ATP production and mitochondrial membrane potential as a consequence of forced glycolysis even under normoxia [53].

Further evidence of increased glycolysis in bald DPCs comes from a recent analysis of mitochondrial function. It was found that IRG1 (ACOD1) is downregulated both in DPCs exposed to DHT and in DPCs from bald scalp. This change increases methylation of DDX1, an enzyme that regulates alternative splicing of genes involved in Ca²⁺ flux. EZH2-mediated methylation of DDX1 inhibits its enzymatic activity and impairs calcium influx, which might be prevented by IRG1. In vitro, IRG1 knockdown upregulates the senescence markers p16INK4a, p21, and p53 and alters DPC metabolism, leading to severe impairment of the TCA cycle and enhanced glycolysis [54].

Around the same time Flores et al. (2017) was published, it was independently reported that HIF-1α is the active paralog in the bulge and, although inactive in DPCs, can be stabilized in non-balding DP cells to force a shift toward glycolysis. As discussed, hypoxia stabilizes HIF-1α, which then promotes anaerobic glycolysis. The authors hypothesized that this could reduce oxidative stress in bald DPCs [55].

This compensatory strategy for impaired OXPHOS has been explored in conditions such as neurodegeneration. Acute OXPHOS dysfunction is compensated for by glycolysis, but that’s insufficient to prevent long-term progression as the underlying dysfunction worsens. Chronic glycolysis may even exacerbate mitochondrial dysfunction. It’s believed that restoring metabolic efficiency is required to reverse disease rather than compensating for it through metabolic reprogramming [56][57][58]. In some cases, inhibiting glycolysis might even alleviate dysfunction by limiting the production of pro-inflammatory metabolites that accelerate mitochondrial damage [59].

A proteomic analysis does show downregulation of LDHA and a reduced glycolytic signature in hair follicles from women with female pattern hair loss (FPHL) compared with healthy controls, suggesting that the treatment might be more effective in women. By contrast, men with AGA showed a signature of increased glycolysis. It should be noted that there was a large age gap between the men with AGA and the two healthy controls in this study [66].

There’s an interesting case report tangentially related to this subject involving a man with an IDH-mutation-driven tumor [67]. Within one month of beginning treatment with an IDH1/2 inhibitor, he developed hypertrichosis, trichomegaly, and increased hair growth on his bald scalp. The growth continued to increase for at least five months after treatment began.

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The work by Flores, Lowry, et al. is welcome. Unfortunately, their research does not establish a connection to AGA, and PP405 is unlikely to be a breakthrough treatment. To show how little effort was put into establishing a connection to AGA pathogenesis before Pelage was founded, a 2020 dissertation claims that AGA is caused by DHT acting as an allosteric inhibitor of the androgen receptor, thereby blocking testosterone from activating HFSCs [84]. You only need the most basic understanding of the disease to know that is completely wrong. They did not seem to care about demonstrating any role for this mechanism in the disease, or even learning basic facts about AGA, before proceeding to human trials. Those trials have yet to produce any meaningful data. Based on the evidence presented here, I would expect PP405 to prompt hair growth earlier than would otherwise occur, which might modestly increase the number of visible hairs at any given time. This is not guaranteed, because there may be additional brakes on the hair cycle that can’t be circumvented with lactate. The wild card is whether the DP responds to MPC inhibition and what effect that might have. It could produce modest compensatory increases in DP mitochondrial respiration that support hair growth. Alternatively, downregulation of HIF1A might reduce AR activity and TWIST1 activation if HIF-1α is ectopically activated in the AGA DP [85], but that is speculative. It could also cause mitochondrial dysfunction over time, as it did in human hair follicles ex vivo. What is difficult to imagine is a mechanism by which MPC inhibition would significantly reverse hair follicle miniaturization.

The hype behind this drug needs to die. Mouthbreathers keep arguing that it has to be a breakthrough because researchers at UCLA and investors at Google Ventures must be geniuses who couldn't possibly be wrong. As of 2020, the UCLA researchers thought androgenetic alopecia was caused by DHT acting as an allosteric inhibitor of the AR that prevents testosterone from activating HFSCs. No wonder they think their drug is a cure. They can't even get basic facts about AGA right, yet they and you people think they know how to cure it. This is why credentialism and appeals to authority are stupid.

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Dissertation

Skip to the last paragraph for the TL;DR. The article exceeds Reddit's character limit so I had to cut a lot of it out.

PP405 might be the most hyped hair loss treatment since Rogaine and Propecia. As always, the hair loss community is split into two camps: those who believe it will be a cure based on little more than hope, and those who believe it will fail based on nothing more than decades of disappointment. My aim here is to provide a more objective and reasoned analysis of the good, the bad, and the ugly from the scientific literature as it relates to Pelage’s supposedly breakthrough hair loss treatment.

I’ll begin with some background on what PP405 is and how it works. It’s a small molecule inhibitor of the mitochondrial pyruvate carrier (MPC) designed to artificially induce a Warburg-like state, whereby glycolytic metabolism is preferentially utilized. By preventing MPC from shuttling pyruvate into mitochondria, PP405 restricts pyruvate entry into the TCA cycle, which can limit oxidative phosphorylation (OXPHOS), while accumulation of pyruvate in the cytosol fuels glycolytic metabolism through LDH-mediated conversion of pyruvate to lactate, primarily by LDHA. The reduction of pyruvate to lactate utilizes NADH, and this reaction converts NADH back to NAD+. NAD+ is utilized in the oxidation of G3P by GAPDH, which reduces NAD+ to NADH. This is a critical step in glycolysis required for downstream ATP production. For glycolytic flux to continue, NADH must be reoxidized to NAD+. To summarize, this chain of events produces pyruvate from glucose before converting it to the end product, lactate. By inhibiting MPC, PP405 promotes the continuation of this glycolytic cycle, with the ongoing reduction of pyruvate to lactate favored over TCA entry. The accumulation of lactate is an indirect indicator of increased glycolytic activity.

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Overview of anaerobic glycolysis induced by PP405

Glycolysis provides energy rapidly when needed, but OXPHOS is much more efficient and more closely associated with differentiation. Stem cell niches rely heavily on glycolysis to support self-renewal and maintain stemness. Glycolysis typically produces less ROS than OXPHOS. This protects stem cells from ROS-associated DNA damage to support long-term maintenance. In contrast, greater ATP production from OXPHOS supports increased metabolic demands during differentiation, while ROS can act as signaling molecules that support differentiation programs.

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Typical effects of metabolism on stem cell renewal and differentiation

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This gets to the heart of the problem. As you’re probably aware, stem cell niches provide a reservoir of cells for organ regeneration, but those cells must differentiate along lineages that generate the tissue. The hair follicle stem cell (HFSC) niche is hypoxic, and glycolytic metabolism ostensibly regulates the proliferation of HFSCs and outer root sheath (ORS) progenitor cells. Glycolysis supports the activation of HFSCs, as demonstrated by Flores et al [2]. from UCLA and Pelage, and by Kim et al. at the Max Planck Institute. However, the latter group showed that the HFSC state does not depend on glycolysis, nor does glycolysis promote HFSC differentiation [3]. This is the key aspect conveniently omitted and unexplored by Pelage, limiting the translatability of their findings to male pattern baldness.

Now I’ll explain what makes this so important in the context of this treatment. Androgenetic alopecia is characterized, in part, by the progressive miniaturization of hair follicles over time. The follicles do not simply go dormant and stop growing; instead, they produce smaller hair shafts with every new cycle until the follicle is so small that it becomes invisible to the naked eye. It often takes many hair cycles for it to reach the point where the terminal-to-vellus transformation is complete.

Obviously, hair follicles don’t regenerate for a new cycle without the activation of stem cells. Therefore, their continued cycling over the course of the disease, while producing smaller hair shafts each time, shows that miniaturization isn’t driven by HFSC quiescence. Notice that everyone has vellus hairs covering most of the body. Those hairs cycle through the same process of HFSC activation. Activating HFSCs on your scalp won’t enlarge vellus follicles any more than it will enlarge the vellus follicles anywhere else on your body. Based on its MOA, expecting PP405 to make your scalp hair longer and thicker is no different from expecting it to make the hair on your arm grow as long and thick as scalp hair.

AGA does present with progressively shorter anagen phases and longer periods of dormancy, but that dormancy is a byproduct of miniaturization and less significant than the shorter anagen phase. You can activate HFSCs with something like PP405, or any other compound that promotes HFSC activation (minoxidil does this too, perhaps less directly), and some dormant hairs will start growing almost immediately, preempting their months-long dormancy phase, but this will not change the diameter or length of the hair shaft. All that will grow is a vellus hair if that’s what the follicle produced during the previous growth phase. Dormancy isn’t what makes these hairs shorter and smaller than healthy scalp hair, just as mice do not grow long hair no matter how much of a glycolysis-stimulating compound they receive. To reiterate, this is because the size of the hair shaft is programmed independently by the DP.

Mice with Mpc1 deletion in HFSCs do not produce longer or thicker hair shafts than controls, and their ear hair remains shorter than their dorsal hair. This indicates an inability to modulate organ size. When mice are depilated, MPC inhibitors trigger precocious anagen, making the hair grow back faster than it normally would, but it grows back the same size as it otherwise would have because HFSCs do not determine the size of the hair shaft. We know from other research that the size of a hair shaft is determined by the size of the dermal papilla (DP) [4], a signaling center that sits at the base of the hair follicle and drives the differentiation of HFSCs via paracrine signaling that supports Wnt activation, among other things. This is where dihydrotestosterone (DHT) is produced and causes damage. Over time, DHT produces a reduction in the number of DP cells. When the number of DP cells falls below a critical threshold, hair fails to regenerate. A progressively smaller DP secretes fewer factors supporting expansion of the progenitor cells that go on to produce the hair shaft. These are the cells that are depleted in bald scalp [5]. In contrast, HFSCs are retained in bald scalp and, as mentioned, they remain competent to activation, regularly cycling until the DP becomes too small to produce a visible hair shaft. Instead, they progressively lose the ability to differentiate into progenitor cells, presumably because reduced signaling from the DP impairs Wnt activation.

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Schematic showing requirement of Wnt activation (LEF1) for differentiation of HFSCs [6]

It has already been shown that HFSCs can activate and proliferate without Wnt, but they fail to adopt a hair follicle fate [7]. Over time, additional defects might accumulate in the hair germ that restrict its expansion. These could involve overactivation of Akt and Sgk1, forcing progenitors back into a stem cell state; apoptosis of progenitors; or EMT. Regardless of the mechanism underlying the depletion of progenitor cells, it’s inconceivable that MPC inhibition could restore the pool because its depletion is clearly independent of HFSC activation. In fact, Kim et al. showed that converting HFSCs to progenitor cells requires a metabolic switch from glycolytic metabolism to OXPHOS and glutaminolysis, while the inverse switch causes ORS progenitor cells to return to a stem cell state [3].

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Cell fate progression and reversibility of HFSCs

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The researchers even stated, three years after the founding of Pelage, that “Whether glycolytic metabolism is indeed the common denominator in HFSC activation signaling remains to be determined. [8]” Yet they had already formed a company to develop a hair loss drug based on this research.

Ldha is ostensibly required for hair growth, since deletion of Ldha in murine HFSCs prevents their activation [2]. However, targeting this downstream mechanism may fail to activate other programs that are also important or required for cell cycle entry. Even if the drug is sufficient to trigger anagen entry, it will not rescue dysfunctional lineage progression in AGA, as OXPHOS and Wnt are required for this.

The assertion that Wnt is downstream of glycolytic flux in HFSCs is based on the observation of low Wnt expression in Ldha-null HFSCs [2], but acting downstream of a process does not establish direct regulation by that process. Wnt activation requires paracrine signaling between dermal papilla and epithelial cells. In a healthy hair follicle, signals from the DP activate the hair germ before the HFSC bulge. The hair germ then activates bulge stem cells while crosstalk enhances Wnt activation to promote lineage progression [9]. In bald follicles, a dysfunctional DP may be unable to participate in that crosstalk, impairing Wnt activation and lineage progression [10].

Additional pathways are involved in AGA, but Wnt is the most relevant one for this discussion. Lactate can activate HFSCs and progenitors prematurely, but without Wnt-activating signals from the DP, it can’t push HFSCs to differentiate into progenitors and replenish the depleted progenitor pool in bald scalp. Without that, there are not enough of these cells with which to build the new hair.

Furthermore, reduced glycolytic activity hasn’t been observed in mpAGA, and there is no genetic association with this pathway in the disease. LDHA deficiency is not associated with hair loss despite individuals with LDHA loss-of-function mutations expressing very low LDH activity in scalp hair follicles described as “virtually devoid of LDH activity [11][12][13].”

Further support for the continued activation of HFSCs in bald scalp comes from the fact that sebocytes are derived from the same HFSC pool that produces the hair follicle [14]. AGA is characterized by progressive miniaturization of the follicle with concomitant sebaceous gland hyperplasia. Rather than failing to activate, these stem cells preferentially differentiate along another trajectory in the absence of Wnt, becoming sebocytes and epidermal cells instead of hair follicle progenitors, and the hair cycle is shortened from years to weeks [15][4]. Activating the cell cycle with glycolysis can’t correct this defect. To reiterate, it only accelerates production of the same tissue that the cells would otherwise build, which in late-stage baldness is a sebaceous gland, interfollicular epidermis, and a tiny vellus hair [7][16].

Many drugs activate HFSCs, and several do so through the same pathway or mechanism as PP405. Hypoxia stabilizes HIF-1α, a master regulator that promotes glycolysis over OXPHOS. Stemoxydine recapitulates hypoxic signaling in human hair follicles, including upregulation of glycolytic genes. Although MPC inhibition is the most direct way to stimulate glycolysis, Stemoxydine efficiently enhances it based on L’Oreal’s testing. Unfortunately, it only increased hair density by 4% compared to placebo [17].

UCLA’s research demonstrates that activation of the beta-2 adrenergic receptor (ADRB2) is what stimulates glycolysis in HFSCs in vivo to support their activation. The researchers showed that procaterol induces glycolysis and precocious anagen in mice, comparable to PP405 [18]. Yet procaterol does not appear to have any noticeable effect on AGA. Perhaps it would be more effective at higher local concentrations, but if stimulating glycolysis were the cure for hair loss, we would expect to see some improvement in hair growth among men taking beta-2 agonists. NFATC1 inhibitors, such as cyclosporine, also activate HFSCs. Minoxidil does this quite well, among other effects, albeit most likely indirectly. The indirect nature of minoxidil’s ability to induce anagen in AGA suggests there is typically no defect in the intrinsic capacity of HFSCs to produce lactate, and that there would be no additive effect when combining these compounds. Few agents induce precocious anagen in mice faster than minoxidil. A multitude of agents that increase IGF-1 or KGF can activate HFSCs; the literature is replete with examples of such failed treatments.

Adrb2 is activated in HFSCs by norepinephrine released from the sympathetic nerve [19]. This nerve wraps around the arrector pili muscle (APM), a small muscle attached to the hair follicle bulge. Without the APM, innervation of HFSCs is lost. Adrb2 activation promotes lactate production through Creb signaling. Beta-2 activation stimulates adenylyl cyclase, leading to the accumulation of cAMP. cAMP activates Pka, which phosphorylates Creb to facilitate its nuclear translocation. Production of glycolytic intermediates is increased following cAMP stimulation [18].

Beta-2 signaling also represses FGF18 [19], which regulates histone H2A ubiquitination in HFSCs and thereby controls the expression of multiple cell cycle genes. This controls stem cell quiescence independently of other known HFSC quiescence regulators Nfatc1, TCF3/4, BMP6 and Foxc1 [20].

In mice, Creb signaling promotes glycolytic flux in HFSCs and triggers anagen, similarly to Mpc inhibition [21]. cAMP accumulation promoted by forskolin is sufficient for HFSC activation. cAMP accumulation and anagen induction were also observed following topical PDE inhibition. However, only some Creb+ cells are proliferative, and only some proliferative cells are Creb+, suggesting Creb may only correlate with HFSC activation. Nevertheless, anagen induction upon cAMP stimulation indicates this is one mechanism by which Adrb2 activates HFSCs and triggers anagen [18]. In addition to glycolysis, pathways upregulated by Creb activation in mouse epidermis include OXPHOS, cholesterol biosynthesis, TCA and FAO [21].

Sympathectomy (removal of the sympathetic nerve) and Adrb2 knockout produce the same delayed-hair-cycle phenotype, with the former being rescued by the Adrb2 agonist procaterol. This confirms that Adrb2 mediates the effect of the APM-sympathetic nerve unit on the hair cycle [19].

Because Adrb2 knockout only delays anagen rather than inducing miniaturization, and FGF18 is significantly downregulated in bald scalp (GSE36169), it appears that loss of ADRB2 does not drive follicular miniaturization, but it could contribute to prolonged kenogen in vellus hairs. The authors acknowledge the speculative nature of targeting this pathway in AGA and the fact that Adrb2 loss merely delays activation rather than preventing it. They suggest ADRB2 agonists might be useful in AGA, but refrain from the sort of definitive claims that Pelage has made regarding this pathway [19].

There is speculation that because the APM becomes detached from follicles in bald scalp, its detachment and downstream effects drive hair loss. On the contrary, these Adrb2 and sympathetic nerve knockout experiments provide support for APM detachment being a consequence of hair loss rather than its cause. The APM makes hair stand erect for thermoregulation and connects the sympathetic nerve to HFSCs in the bulge. In response to cold, the muscle contracts, causing hair to stand erect and trap warm air, while the nerve simultaneously secretes norepinephrine to signal dormant hair follicles to regenerate. The APM is not required for mammalian hair growth, as some mammals don’t have an APM, including sea otters and pinnipeds [22]. Despite this, sea otters have the densest hair of any animal.

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sea otter

Rhesus monkeys lack an APM in both thick vibrissae and vellus hairs of the lips and eyelids [23]. Rat and mouse vibrissae have an APM, but it doesn’t connect to the bulge compartment [24]. During morphogenesis, the hair follicle is generated before the APM forms and innervation occurs because Shh produced by the hair follicle is required for APM development [19]. Absence of innervation doesn’t prevent initial follicle downgrowth.

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APM formation with establishment of sympathetic nerve innervation

Likewise, the APM connection is severed during hair transplantation, yet this doesn’t prevent the follicle from growing normally before the connection is reestablished [25]. Therefore, APM detachment doesn’t appear to prevent regeneration. Nor does it prevent lab-grown hair follicle organoids from developing before establishing attachment to the APM [26].

Estrogen has a remarkable ability to reverse AGA, indicating that, with the right signaling, either the APM is not required for reversal or its attachment can be reestablished. Rather than driving hair loss, APM detachment in bald scalp may be another epiphenomenon of the Wnt downregulation that drives hair loss. APM attachment is governed by the Wnt target gene nephronectin, which is required to establish the anchor to which the APM attaches downstream of Wnt activation in the bulge [27]. Wnt signaling in the bulge depends on factors secreted by the DP, which becomes dysfunctional as a consequence of DHT [28].

Nephronectin binds α8β1 integrin on mesenchymal cells, anchoring them to the bulge, after which the APM attaches to these cells. As AGA progresses, Wnt signaling may become sufficiently reduced that nephronectin is no longer expressed, causing the APM to lose its anchor. In nephronectin-knockout mice, the APM can instead localize above the bulge due to compensatory upregulation of EGFL6. Such compensation may not occur following detachment in wild-type mice or humans.

Further evidence against APM detachment as the cause of AGA is that deletion of nephronectin doesn’t affect DP size. Hair follicle development is normal, but anagen entry is accelerated and telogen is shortened because altered localization of α8β1+ cells reduces the physical separation between the DP and hair germ [27]. As previously mentioned, severing the sympathetic nerve or deleting Adrb2 delays anagen but does not alter hair follicle morphology [19]. Since the APM-sympathetic nerve unit activates HFSCs through Adrb2 activation, deletion of Adrb2 should recapitulate the AGA phenotype if APM detachment were the driver of AGA. All of this evidence indicates that the APM and sympathetic nerve regulate the timing of the hair cycle rather than the size of the hair shaft, and there’s no evidence that glycolytic flux regulates anything beyond HFSC activation and proliferation.

Flores et al. also observed robust lactate induction and precocious anagen within six to nine days after application of the GP130 modulator RCGD423 to activate STAT3 [2].

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Lactate induction by STAT3 activation using RCGD423

STAT3 and STAT5 were identified as upregulated hub genes in an analysis of scalp skin from patients with premature AGA [29]. In another study of 25 patients, RT-qPCR showed stronger STAT3 expression in bald follicles than in healthy follicles, though it did not correlate with hair loss severity [30]. STAT3 overexpression in AGA may therefore already promote lactate accumulation. STAT5, by contrast, acting downstream of oncostatin M and NFATC1/prolactin, could repress HFSC activation in AGA through as-yet-unresolved mechanisms, potentially including the activation of SFRP Wnt-ligand antagonists in the bulge [31][32][33].

Commonly prescribed TZDs are also known to inhibit MPC, yet they show little to no effect on AGA [34]. PKM2 catalyzes the conversion of PEP to pyruvate during glycolysis. Its activation also accelerates the hair cycle in mice and enhances WIHN [35]. β-catenin and nuclear β-catenin were markedly elevated in KY19382-treated wounds compared with control wounds, but not in wounds treated with the PKM2 activator. Nuclear β-catenin was slightly reduced in K15+ HFSCs of mice treated with PKM2 activator. PKM2 expression was decreased in alopecia areata patients (GSE45512), but not in AGA.

In young, but not aged, mice, rapamycin, metformin and αKG activate HFSCs and accelerate anagen through autophagy, as does the AMPK activator AICAR [36]. This activation is mediated by increased Ldh activity that fuels glycolysis [37]. Metformin has been reported to stimulate hair growth in cicatricial alopecia [38][39][40], but the effect may be mediated by PPAR regulation rather than LDH. It may also be beneficial in alopecia areata [41]. If it has any impact on AGA, the effect has gone unnoticed clinically. Likewise, there are only anecdotal reports of modest hair growth with rapamycin. These drugs are so widely used that a meaningful effect on a disease as common and visible as AGA would likely have been noticed by now.

Given that rapamycin induces a more than threefold increase in lactate in murine hair follicles, comparable to that induced by MPC inhibitors [42] [43], and accelerates the hair cycle within the same timeframe, it is unlikely that PP405 would fare much better than rapamycin in the absence of evidence for a specific defect requiring direct MPC targeting. Most evidence, including the effects of minoxidil, argues against such a defect. Rapamycin did modestly prolong hair growth in one of two murine studies (no p-value provided). Rapamycin and α-KG were the fastest-acting of the tested compounds. Dietary α-KG also increased hair density in Rex rabbits and promoted the nuclear localization of β-catenin in DPCs in vitro [44].

 Another experimental molecule capable of facilitating glycolytic reprogramming is IM176OUT05, which acts by inhibiting OXPHOS. In preclinical models, it produced similar effects to Pelage’s molecules [45]. The UCLA team behind Pelage also demonstrated HFSC activation and accelerated telogen-anagen transition in mice following transient inhibition of the electron transport chain (ETC), the machinery that drives OXPHOS. The compounds they used to stimulate glycolysis were phenformin, rotenone, and antimycin [46]. Phenformin was withdrawn from the market but was never reported to cause hair growth. In contrast, genetic ablation of the ETC in mice causes hair follicle dystrophy with severe impairment of K15+ HFSC differentiation, a possibility discussed earlier in this article [47].

DP cells rely on OXPHOS, which is impaired in bald scalp and may dampen Wnt activation through endoplasmic reticulum (ER) stress [48][49]. Restoring OXPHOS is more likely to show efficacy in AGA than forced glycolysis. Researchers tested one of the same MPC inhibitors that promotes hair growth in mice in human hair follicles ex vivo [50]. They found that it caused ER stress and inhibited Wnt signaling, leading to cell cycle arrest in epithelial progenitor and matrix cells.

The results contradict those observed by Flores et al. in mice [2]. One of the researchers behind Pelage suggested that the dose may have been too high in this study. However, the effect was seen at 10 μM, while Flores used the same drug at 20 μM on mice with positive results. Off-target effects on mitochondrial membrane potential are only observed at concentrations exceeding 10 μM, specifically at 100 μM [2][51].

Cell proliferation in human hair follicles ex vivo after exposure to MPC inhibitor

In light of the way PP405 has been presented by Pelage, the media, and the hair loss community, it is striking that one of the researchers at Pelage told a reporter they were concerned the drug would kill all the hair follicles in the clinical trial and they were glad to be wrong about that [52]. It’s difficult to reconcile that concern with prior statements implying it’s a breakthrough treatment that will reverse miniaturization. How could they be so sure about the latter when they weren’t even sure that it would not kill follicles?

It’s possible that the drug stimulates initial hair growth in dormant follicles while worsening the underlying condition over the long term through activation of the integrated stress response (ISR). Deletion of Mpc1 in mouse HFSCs produced no apparent adverse effects, but this does not guarantee it will be harmless in other cell compartments. The data on HFSCs from Flores et al. are reassuring. However, broad-based and chronic induction of glycolysis is very different from HFSC-specific Mpc1 knockout or the transient drug-induced glycolysis in mouse experiments. Forced glycolysis in other follicular compartments could compromise their normal function. UK-5099 has also been found to cause mitochondrial dysfunction in LNCaP cells, producing a twofold increase in ROS production together with reduced ATP production and mitochondrial membrane potential as a consequence of forced glycolysis even under normoxia [53].

Further evidence of increased glycolysis in bald DPCs comes from a recent analysis of mitochondrial function. It was found that IRG1 (ACOD1) is downregulated both in DPCs exposed to DHT and in DPCs from bald scalp. This change increases methylation of DDX1, an enzyme that regulates alternative splicing of genes involved in Ca²⁺ flux. EZH2-mediated methylation of DDX1 inhibits its enzymatic activity and impairs calcium influx, which might be prevented by IRG1. In vitro, IRG1 knockdown upregulates the senescence markers p16INK4a, p21, and p53 and alters DPC metabolism, leading to severe impairment of the TCA cycle and enhanced glycolysis [54].

Around the same time Flores et al. (2017) was published, it was independently reported that HIF-1α is the active paralog in the bulge and, although inactive in DPCs, can be stabilized in non-balding DP cells to force a shift toward glycolysis. As discussed, hypoxia stabilizes HIF-1α, which then promotes anaerobic glycolysis. The authors hypothesized that this could reduce oxidative stress in bald DPCs [55].

This compensatory strategy for impaired OXPHOS has been explored in conditions such as neurodegeneration. Acute OXPHOS dysfunction is compensated for by glycolysis, but that’s insufficient to prevent long-term progression as the underlying dysfunction worsens. Chronic glycolysis may even exacerbate mitochondrial dysfunction. It’s believed that restoring metabolic efficiency is required to reverse disease rather than compensating for it through metabolic reprogramming [56][57][58]. In some cases, inhibiting glycolysis might even alleviate dysfunction by limiting the production of pro-inflammatory metabolites that accelerate mitochondrial damage [59].

A proteomic analysis does show downregulation of LDHA and a reduced glycolytic signature in hair follicles from women with female pattern hair loss (FPHL) compared with healthy controls, suggesting that the treatment might be more effective in women. By contrast, men with AGA showed a signature of increased glycolysis. It should be noted that there was a large age gap between the men with AGA and the two healthy controls in this study [66].

There’s an interesting case report tangentially related to this subject involving a man with an IDH-mutation-driven tumor [67]. Within one month of beginning treatment with an IDH1/2 inhibitor, he developed hypertrichosis, trichomegaly, and increased hair growth on his bald scalp. The growth continued to increase for at least five months after treatment began.

https://preview.redd.it/279ru4ip3u7h1.jpg?width=687&format=pjpg&auto=webp&s=2a2eae0dac38980a4dbbdb7389dcd75e84003dbe

The work by Flores, Lowry, et al. is welcome. Unfortunately, their research does not establish a connection to AGA, and PP405 is unlikely to be a breakthrough treatment. To show how little effort was put into establishing a connection to AGA pathogenesis before Pelage was founded, a 2020 dissertation claims that AGA is caused by DHT acting as an allosteric inhibitor of the androgen receptor, thereby blocking testosterone from activating HFSCs [84]. You only need the most basic understanding of the disease to know that is completely wrong. They did not seem to care about demonstrating any role for this mechanism in the disease, or even learning basic facts about AGA, before proceeding to human trials. Those trials have yet to produce any meaningful data. Based on the evidence presented here, I would expect PP405 to prompt hair growth earlier than would otherwise occur, which might modestly increase the number of visible hairs at any given time. This is not guaranteed, because there may be additional brakes on the hair cycle that can’t be circumvented with lactate. The wild card is whether the DP responds to MPC inhibition and what effect that might have. It could produce modest compensatory increases in DP mitochondrial respiration that support hair growth. Alternatively, downregulation of HIF1A might reduce AR activity and TWIST1 activation if HIF-1α is ectopically activated in the AGA DP [85], but that is speculative. It could also exacerbate mitochondrial dysfunction over time, as it did in human hair follicles ex vivo. What is difficult to imagine is a mechanism by which MPC inhibition would significantly reverse hair follicle miniaturization.

Excellent results as expected. There's no chance that PP405 can come close to this. They even showed a statistically significant increase in hair counts after only two months, which was the earliest time point evaluated. It was well-tolerated with an adverse event rate similar to the placebo group. They report no clinically significant differences in heart rate, blood pressure, or ECG changes compared to placebo, and no observed shedding or cardiac-associated adverse events of special interest.

>Rapid and robust hair growth was achieved with VDPHL01 treatment as demonstrated by a mean increase in non-vellus target area hair count (TAHC) of 30.3 hairs/cm² (once daily dosing; p<0.0001) and 33.0 hairs/cm² (twice daily dosing; p<0.0001) versus 7.3 hairs/cm² for placebo at Month 6

>Approximately 79.3% (QD) and 86.0% (BID) of patients reported any improvement in patient-reported outcomes (PRO) versus 35.6% of placebo patients; 48.4% (QD; p<0.0001) and 62.9% (BID; p<0.0001) of patients reported ‘improved’ or ‘much improved’ outcomes at Month 6 versus 13.4% of placebo patients

>The consistency of clinically meaningful hair growth reported by patients was further supported by investigator perception of hair growth, with investigators grading 72.0% (QD; p<0.0001) and 84.4% (BID; p<0.0001) of male patients as having improved hair coverage at Month 6 (p<0.0001). Rapid onset of hair growth was also observed at the earliest measured time point measured in the trial, with statistically significant separation from placebo on TAHC and IGA as early as Month 2.

https://ir.veradermics.com/news-releases/news-release-details/veradermics-oral-vdphl01-achieved-early-consistent-and-robust