Indolepropionamide

IPAM is 3-(1H-indol-3-yl)propanamide: indole-3-propionic acid (IPA) with its carboxylic acid turned into a primary amide. IPA is made from tryptophan by gut bacteria and is found in human plasma and cerebrospinal fluid. In 1999 the group that later made IPAM reported that IPA scavenged hydroxyl radicals at least twice as efficiently as melatonin, did so without the pro-oxidant side reactions they measured for vitamin C, trolox and glutathione, and at 1 µM protected cultured neurons from amyloid-β [3]. IPA was then put forward as an Alzheimer's drug candidate under the name OXIGON [4].

The inventors' objection to IPA was that its acid group is charged at body pH, so little of it enters the brain. Converting the acid to an uncharged amide was their fix [1][2].

What the 2010 paper did [1]

• Brain uptake. One-month-old male rats (6 per group) given 0.5 mg/kg into the abdomen had brain IPAM of 691, 562 and 361 pg per mg protein at 2, 4 and 8 hours. The same dose of melatonin or IPA did not raise brain levels of those indoles at these time points.
• Isolated mitochondria. At 10 nM, IPAM raised the membrane potential of brain mitochondria from 1-month-old and 20-month-old rats, by more than melatonin or IPA at the same concentration. It prevented most of the loss of potential caused by 500 nM doxorubicin, antimycin A or the uncoupler FCCP. In mouse brain mitochondria, 10 nM increased complex I and complex IV activity in young and old animals.
• Radical scavenging. In rat forebrain homogenate exposed to a hydroxyl-radical-generating mixture, the concentration that halved oxidative DNA damage (8-hydroxydeoxyguanosine) was 0.18 µM for IPAM, 1.4 µM for melatonin and 7.46 µM for IPA.
• Rotifers. In the bdelloid rotifer Philodina acuticornis, 11 animals per group, mean lifespan rose from 24.6 days to 58.5, 81.1 and 90.5 days at 10, 20 and 30 µM. At 30 µM the animals were also longer on day 15 (575 against 390 µm), produced 55 offspring against 16, and stayed fertile for 18 days against 5.

The rotifer result in context

The patent application, first filed in December 2003, more than six years before the paper, reports an earlier rotifer series by the same inventors with much smaller effects. Median lifespan was 31.8 days against 23.4 at 0.5 µM, 36.3 against 25.9 at 5 µM, and 28.8 against 25.0 at 50 µM: a gain of 40% at best, shrinking to 15% at the highest concentration. Melatonin and IPA at 5 µM did about as well (33.5 and 33.8 days) [2]. The paper reports gains of 138% to 268% at 10–30 µM and does not mention the earlier series [1].

The same group, using the same rotifer, had earlier reported a 150% lifespan extension with a different antioxidant, the nitrone LPBNAH, so very large effects are a feature of this laboratory's model and not only of IPAM [5].

No one has repeated the experiment. A different laboratory screened antioxidants in another rotifer, Brachionus manjavacas, and found no life extension from its indole compound alone. That study's methods and discussion name the compound as indole-3-propionic acid bought from a chemical supplier, at 20 µM: the parent acid, not the amide [6]. Two later papers from that laboratory describe the same test as having been of IPAM [5][7]. On the published record, then, the only attempt in a second species was negative, and it is not clear that IPAM itself was ever in the dish.

Is it really made in the body?

The paper's title calls IPAM endogenous. The evidence is a peak in rat brain extracts that leaves an HPLC column at the same time as synthetic IPAM and grows when synthetic IPAM is added to the sample. Baseline peaks were "inconsistent" and estimated at under 100 pg per mg protein; a reproducible reading (346 pg per mg protein) was obtained only one hour after loading the rats with 300 mg/kg of L-tryptophan. The paper reports no mass-spectrometric confirmation of the peak's identity and examined no human samples [1]. The patent states that significant endogenous IPAM "could not be detected" in rat brain, while asserting, without data, that the inventors had seen it in rodent bile, gut, brain and cerebrospinal fluid [2].

The authors propose two actions, on different concentration scales [1].

Electron donation to radicals. The electron-rich indole ring can give an electron to a hydroxyl radical. For the parent acid, the resulting indolyl radical was shown to end up as a stable kynuric acid instead of a reactive intermediate, which is why IPA did not drive further radical formation in the presence of iron [3]. IPAM behaved the same way in the 2010 assay: with hydrogen peroxide, iron and EDTA it lowered hydroxyl radical formation, where serotonin, 6-hydroxymelatonin and 5-methoxyindoleacetic acid raised it [1]. One oddity: in the 2010 DNA-damage assay IPA was the weakest of the three indoles, about five times less potent than melatonin, although the 1999 paper had ranked IPA above melatonin using different assays [1][3].

A direct effect on the respiratory chain. The mitochondrial effects appeared at 10 nM, well below the 0.18 µM needed to halve radical damage. From this the authors argue that IPAM is more than a scavenger: that it binds complex I at its rate-limiting step and acts as a "recyclable electron and proton carrier", reducing electron leak and so the production of reactive oxygen species at source. The support is the rise in complex I and complex IV activity, a second complex I assay based on reduction of nitroblue tetrazolium at iron-sulfur cluster N2, and displacement of radiolabelled dopamine and 2-iodomelatonin from mitochondrial binding sites. The displacement results are stated as "data not shown", as is the lack of change in complex II–III [1].

What made the rotifers live longer is not established. The paper says the mechanisms behind the lifespan, size and fertility effects "remain to be determined", and it did not measure mitochondrial function or oxidative damage in the rotifers themselves [1].

Despite the resemblance to melatonin, the paper reports no test of IPAM at melatonin receptors and no experiment on sleep or body-clock timing [1].

The patent adds one further claim: in a thioflavin T fluorescence assay, IPAM inhibited aggregation of the amyloid peptide Aβ1–40 at a 1:1 molar ratio, as melatonin and IPA did. This is the inventors' filing; the result is presented as a single figure and has not appeared in a journal [2].

There is nothing to go on. No toxicology study of IPAM has been published in any species: no lethal-dose estimate, no repeated dosing, no reproductive or genetic toxicity testing, and no human exposure.

What the two sources do record is limited. Rats received a single 0.5 mg/kg injection and were killed 30 minutes to 8 hours later for brain measurements; neither source describes how the animals behaved or any other health observation [1][2]. Rotifers kept in 10–30 µM IPAM for life lived longer, grew larger and reproduced more [1]. In the patent's earlier series the benefit at 50 µM was much smaller than at 5 µM, which the filing does not comment on, though it calls IPAM "devoid of toxicity" in these animals [2].

The parent acid showed no measurable toxicity to cultured neurons in the 1999 study [3], but that says little about the amide in a whole animal. The absence of pro-oxidant activity in a test tube [1] is a statement about one chemical reaction, not a safety finding.

No interaction study exists. The patent reports rotifer experiments combining 5 µM IPAM with 100 µM ascorbate and 100 µM trolox, in which median lifespan was 50.4 days against 28.8 days with the two vitamins alone [2]. That is a co-treatment in an invertebrate from the inventors' own filing, not evidence about combining IPAM with anything in people.