IGF-1 LR3 1mg Peptide

IGF-1 LR3, written in the primary literature as Long [Arg3]-IGF-I or LongR3-IGF-I, is a recombinant 83-residue analogue of human insulin-like growth factor 1. It differs from the 70-residue mature hormone in exactly two ways: arginine replaces glutamate at position 3 of the IGF-1 sequence, and a 13-residue extension, Met-Phe-Pro-Ala-Met-Pro-Leu-Ser-Ser-Leu-Phe-Val-Asn, is fused to the N-terminus. Both changes were introduced by a group at the CSIRO Division of Human Nutrition and the University of Adelaide and were described in two 1992 papers in the Journal of Molecular Endocrinology.

Neither change was made to improve receptor binding. Both sit in the region of IGF-1 that contacts the six IGF binding proteins, and the analogue was built to fail that contact on purpose. In serum, native IGF-1 is almost entirely locked into a 150 kDa ternary complex with IGFBP-3 and the acid-labile subunit, a reservoir that keeps free ligand concentrations very low. Long [Arg3]-IGF-I largely escapes that reservoir, and every property the compound is known for follows from that single design decision.

The compound's documented working life is in cell culture rather than in animals or people. It is a standard insulin substitute in serum-free Chinese hamster ovary production media, it appears in stem cell differentiation media, and it is the IGF-1 receptor agonist of choice for cultures whose own cells secrete binding proteins into the medium. Long [Arg3]-IGF-I has never entered a registered human clinical trial. The clinical IGF-1 record belongs to mecasermin, which is recombinant native IGF-1, and does not transfer to this analogue.

The primary target is the type 1 IGF receptor (IGF-1R), a disulfide-linked alpha2beta2 receptor tyrosine kinase. Ligand occupancy of the extracellular alpha subunits drives trans-autophosphorylation of the beta-subunit kinase domain, which then recruits insulin receptor substrate 1 and 2 and the adaptor Shc. Two branches follow. The PI3K/Akt arm raises protein synthesis through mTORC1 and S6K1, suppresses FoxO-driven transcription of the muscle atrophy genes, and phosphorylates Bad to blunt apoptosis. The Ras/Raf/MEK/ERK arm carries the proliferative signal and cell cycle entry.

Receptor selectivity is not absolute, and the analogue does not change that. IGF-1R and the insulin receptor are close relatives that phosphorylate an overlapping substrate set, and in tissues expressing both, half-receptors assemble into IR/IGF-1R hybrids that bind IGF-1 with far higher affinity than they bind insulin. Francis and colleagues tested the Long analogues in H35 rat hepatoma cells, where the IGFs signal through the insulin receptor rather than IGF-1R, and found the analogues held roughly the same potency ratio relative to IGF-1 that they held in L6 myoblasts. Activity is therefore not confined to IGF-1R.

The enhanced potency is a binding protein phenomenon, not a receptor phenomenon, and the primary papers are unambiguous about it. King and colleagues reported that [Arg3]-IGF-I bound very poorly to bovine IGFBP-2 while binding slightly less well than native IGF-1 to the type 1 receptor on L6 myoblasts. Tomas and colleagues put a number on the receptor side: LR3IGF-I binds threefold less well than IGF-1 to the type 1 IGF receptor. What the molecule gains is free fraction, and it pays for that gain with intrinsic receptor affinity.

The experiment that settles the point is a negative control almost no product page mentions. Francis and colleagues ran the analogue series on chicken embryo fibroblasts, a line that secretes no detectable IGF binding proteins into the medium. In that system Long [Arg3]-IGF-I was less potent than native IGF-1. In cell lines that do secrete IGFBPs, the potency order ran Long [Arg3]-IGF-I and des(1-3)IGF-I ahead of Long [Gly3]-IGF-I, ahead of Long IGF-I, ahead of IGF-1. The familiar claim that the analogue is roughly three times more potent is therefore a property of the assay system rather than of the molecule, and it inverts in a binding-protein-free system. For anyone choosing a ligand, that is the operative fact.

The construct is an artefact of a manufacturing problem. Producing correctly folded IGF-1 in Escherichia coli is difficult because the 70-residue chain has to form three specific disulfide bonds and readily mis-pairs them. The Adelaide group solved that by expressing IGF-1 as a fusion carrying the first 46 residues of methionyl porcine growth hormone followed by the dipeptide Val-Asn. Those two residues create a unique hydroxylamine-sensitive Asn-Gly bond at the junction, so after inclusion bodies are isolated and the chain is refolded, a single chemical cleavage releases the growth factor.

Cleaving that fusion at a shorter point leaves 13 of the extension residues attached, and that truncated leader is what the word Long denotes. The extension turned out to be useful rather than merely tolerated: the hydrophobic leader improved the yield of correctly folded, biologically active product compared with refolding a normal-length IGF-1 chain. A production convenience became a design feature.

The same group made the whole matrix, which is why the structure-activity conclusion is firm rather than inferred. They produced Long IGF-I (extension only, no substitution), [Arg3]-IGF-I and [Gly3]-IGF-I (substitution only, no extension), and Long [Arg3]-IGF-I and Long [Gly3]-IGF-I with both. Comparing across that set is how the extension and the position 3 substitution were shown to contribute additively to loss of IGFBP affinity, and how it became clear that neither contributes anything to receptor affinity.

Six IGF binding proteins circulate, and IGFBP-3 dominates. IGFBP-3 binds an IGF molecule together with the acid-labile subunit to form a stable 150 kDa ternary complex that cannot leave the vascular compartment. That complex functions as an intravascular store, and it is why the free fraction of circulating IGF-1 is a small percentage of the total. Change the ternary complex and the pharmacology changes with it.

A direct demonstration comes from a rat study on IGF-1-induced hypoglycaemia. A bolus of recombinant human IGF-1 lowered blood glucose, and co-administering an equimolar amount of either glycosylated or non-glycosylated IGFBP-3 blocked that effect. A mutant IGFBP-3 with reduced acid-labile subunit affinity only partially blocked it, and the molecular mass distribution explained why: 60 percent of the wild-type binding protein was in ternary complexes against 30 percent of the mutant. Ternary complex formation, not binding protein presence, is what neutralises the ligand.

Long [Arg3]-IGF-I is the molecule built to sit outside that system. That framing also predicts where the analogue will underperform. In a culture with no binding proteins in the medium, there is no sequestration to escape and the only property that matters is intrinsic receptor affinity, where the analogue is threefold worse than the native hormone. That is precisely what was measured in chicken embryo fibroblasts.

The largest documented application is not an animal application at all. LongR3 is a standard growth factor in serum-free Chinese hamster ovary media, where it substitutes for insulin. A 2000 comparison ran two serum-free CHO lines expressing recombinant cytokine receptors on each factor. One line grew without either, the other required one of them, and under production conditions LongR3 sustained viability of both lines better than insulin did.

A 2014 study went further and asked what the supplement does to the cell. Using a purpose-built 41,304-probe CHO microarray, the group profiled CHO transcriptomes cultured with and without LongR3. The effects ran from faster growth to delayed cell death, along with a distinct signature in cytoskeletal gene expression, which the authors read as cells spending less effort building a large cytoskeleton and more on dividing.

The consequences reach product quality. In an industrial CHO line producing an Fc-fusion protein, LongR3 raised productivity and shifted the N-linked glycosylation profile of the product. Glycosylation is a critical quality attribute of a therapeutic protein, so a media growth factor that moves it is an operational variable, not a neutral additive.

Adjacent uses follow the same logic. Substituting LongR3-IGF-1 for insulin allowed embryonic stem cells to differentiate into neuroprogenitors and insulin-secreting cells, which removes an obvious confound from a protocol whose readout is insulin. In bovine in vitro maturation, 739 cumulus-oocyte complexes matured with 100 ng/mL of either IGF-1 or LongR3-IGF-1 showed no difference in meiotic progression or apoptosis, though IGFBP4 expression in oocytes and COX2 expression in cumulus cells did differ between the groups. The analogue has also been expressed as a xylanase fusion in Pichia pastoris, an alternative to the E. coli route.

The in vivo rodent work is unusually well controlled because it came from the same laboratory that built the molecule. The core model is a 150 g male rat given 20 micrograms of dexamethasone daily to induce catabolism. IGF-1 by implanted osmotic minipump produced dose-dependent effects on body weight and nitrogen retention, with the highest dose tested, 695 micrograms delivered daily, giving a 6 g gain over seven days against a 19 g loss in the dexamethasone-only group and an 18 g gain in pair-fed controls. Both low-IGFBP-affinity analogues, des(1-3)IGF-I and LR3IGF-I, were roughly 2.5-fold more potent.

The mechanism was measured on both sides of protein turnover rather than inferred. Muscle protein synthesis was assessed at the end of treatment, and breakdown was tracked throughout by 3-methylhistidine excretion, which fell from 83.5 to 65.1 micromol per kg over seven days at the top dose against 54.9 in pair-fed controls. The dexamethasone-induced changes in liver, spleen and heart weight moved back toward normal, and the gain in total carcass weight was nitrogen rather than fat.

A companion paper examined the gut specifically and found it to be the most IGF-responsive tissue in the model, with total gut weight rising by up to 60 percent and gut weight as a fraction of body weight by up to 32 percent. Stomach, small intestine and colon were all affected, and histology showed cross-sectional mass increasing rather than length, with proportional gains in wet weight, protein and DNA per unit length in both the mucosa and the muscularis. That gut trophic effect is a consistent finding across the low-IGFBP-affinity analogue literature and is rarely mentioned on product pages.

Timing mattered as much as amount. A later study infused LR3IGF-I continuously at 280 micrograms daily starting either two days before, concurrent with, or two days after a six-day dexamethasone infusion. In young rats, early treatment beat delayed treatment on nitrogen balance (90 against 31 mg of nitrogen daily), on carcass nitrogen gain, and on protein fractional synthesis rate. Older weight-stable female rats were markedly less responsive, and only early and concurrent treatment reduced the catabolic response at all.

The most recent controlled in vivo data on this specific analogue come from a University of Colorado group studying fetal growth restriction in sheep, and the arc of three studies is more informative than any single result. In the first, catheterised late-gestation fetuses received a one-week intravenous infusion of LR3 IGF-1 (n = 8) or saline (n = 8), followed by a metabolic study measuring uterine and umbilical blood flow, nutrient uptake and fetal protein kinetics. Heart, adrenal and spleen weights rose and skeletal myoblast proliferation increased, but total fetal weight did not differ significantly and blood flow and umbilical glucose, lactate and oxygen uptake were unchanged. Umbilical amino acid uptake and fetal amino acid concentrations fell. The authors read this as more efficient use of available nutrients rather than increased delivery.

The second study followed an unwelcome observation from the first: fetal insulin secretion had fallen. A 90-minute infusion reduced fetal plasma insulin and cut insulin concentrations during a hyperglycaemic clamp by 66 percent against controls. Islets isolated immediately after that infusion secreted normally in vitro, so the acute suppression looked functional and reversible rather than a structural defect in the beta cell.

The third study tested the analogue in the population it had been proposed for and it did not work. Growth-restricted fetal sheep received either LR3 IGF-1 at 1.17 micrograms per kg per hour (n = 7) or vehicle (n = 7) for one week. Body weight, insulin, glucose, oxygen and glucose-stimulated insulin secretion were all unchanged between groups. Circulating amino acids fell in the treated group, consistent with increased utilisation, and the authors concluded that amino acid, insulin, glucose or oxygen availability would likely have to be raised alongside the analogue for growth to follow. This is the clearest published example of the compound being tested against a real endpoint and missing it.

Product pages generally describe the analogue as uniformly anabolic. The livestock literature does not agree, and the disagreement is instructive rather than marginal. A four-day infusion of LR3IGF-I at 180 micrograms per kg per day in finisher pigs decreased average daily gain and food intake, and lowered plasma IGFBP-3, IGF-1 and insulin concentrations.

The likely reason is feedback. Mean plasma growth hormone fell by 23 percent and the area under the growth hormone pulse curve fell by 60 percent. Co-administering porcine growth hormone alongside the analogue raised mean growth hormone concentrations but did not restore the pulse area and did not rescue growth performance, with insulin, IGFBP-3 and endogenous IGF-1 remaining suppressed. Native IGF-1 at the same infusion rate also suppressed growth hormone and insulin but left average daily gain unchanged.

The practical reading is that the analogue's effect depends on how tightly the species regulates its own somatotropic axis and on whether the animal is catabolic to begin with. Rats made catabolic with a glucocorticoid respond well. Normal finisher pigs respond by shutting down endogenous growth factor production. Extrapolating a rat result to another species is exactly the step this literature shows to be unsafe.

A figure of 20 to 30 hours is repeated on essentially every vendor page, usually alongside a claim that native IGF-1 lasts ten minutes. The 20 to 30 hour number does not appear in the primary literature on this analogue, and no citation is offered for it anywhere it is repeated. What can be said with a source is that free native IGF-1 is cleared in minutes while IGF-1 held in the IGFBP-3 and acid-labile subunit ternary complex persists for hours, and that Long [Arg3]-IGF-I is largely excluded from that complex.

The one published measurement points the other way. An antidoping laboratory validated an immunopurification and high-resolution mass spectrometry method for IGF-1 analogues and then tracked them in rats after a single 100 micrograms per kg dose delivered into skeletal muscle. Unchanged Des(1-3)-IGF-I and R3-IGF-I remained detectable to 24 hours. Unchanged LongR3-IGF-I disappeared rapidly after 4 hours. What persisted were N-terminally clipped degradation products: a newly described Des(1)-LongR3-IGF-I, plus Des(1-10)- and Des(1-11)-LongR3-IGF-I, the last of which was detectable to 16 hours. The same products formed when the analogues were incubated in human whole blood in vitro.

The two observations are not in conflict once separated. Escaping the binding proteins raises the free fraction of ligand available to the receptor; it does not make the intact molecule resistant to proteolysis, and the exposed hydrophobic N-terminal extension is a natural substrate for aminopeptidase trimming. An unbound protein is also cleared faster than one parked in a 150 kDa complex. Anyone designing an in vivo experiment around this compound should treat the 20 to 30 hour figure as unsourced and design against the measured data.

Two well-documented findings show why identity confirmation on this compound needs more than a purity percentage. In 2010 a Cologne doping control laboratory analysed a confiscated injection vial and identified its contents by nano-UPLC and high-resolution mass spectrometry of both the intact and trypsinised protein. It was Long-R3-IGF-I carrying a hexahistidine tag on the C-terminus, joined by a Leu-Glu linker. His tags are added during recombinant production for affinity purification and are enzymatically removed when placed at the N-terminus; a C-terminal tag left in place marks the material as a by-product of a biochemical research production run rather than something made to be administered.

The second finding is oxidation. The antidoping method development study reported abundant oxidised forms in black market IGF-1 analogue products, to the point that the authors concluded a detection method has to monitor the mono-oxidised species alongside the native one. The chemistry is predictable: this molecule carries three methionine residues, two of them in the N-terminal extension, and methionine sulfoxide formation is one of the commonest degradation routes for a lyophilised protein exposed to air or peroxide-contaminated excipients.

The molecular weight in circulation is also worth checking. Almost every published specification gives 9,117.5 or 9,117.6 g/mol. That is the calculated mass of the reduced 83-residue chain. The correctly folded protein carries three disulfide bonds and therefore weighs about 6 daltons less, roughly 9,111.5 g/mol, and an intact-mass measurement on properly folded material should land there. A quoted mass of 9,117 alongside a claim of correct folding is internally inconsistent. One widely copied formula, C423H684N122O128S7, is simply wrong: a protein with six cysteines and three methionines must contain nine sulfur atoms, not seven.

The practical consequence is that a single HPLC purity number is not sufficient characterisation for this compound. Reversed-phase HPLC separates by hydrophobicity and will resolve an oxidised variant poorly at best. SDS-PAGE cannot resolve a 6 dalton disulfide difference or an 840 dalton tag with confidence. Intact-mass spectrometry answers all three questions at once: correct sequence mass, correct disulfide state, and the presence or absence of an unremoved purification tag.

Long [Arg3]-IGF-I is a folded 9 kDa protein produced by bacterial expression and in vitro refolding, not a solid-phase synthetic peptide, and its failure modes are those of a protein. The three disulfide bonds are the fragile part. Thiol contamination, alkaline pH or elevated temperature can promote disulfide scrambling, and a scrambled molecule can retain its exact mass and its apparent HPLC purity while having lost receptor binding entirely. Activity loss with no change in the certificate of analysis is the characteristic signature.

Chemical degradation runs on two clocks. Methionine oxidation affects the three methionines and is driven by dissolved oxygen, light and trace metals. Asparagine deamidation and aspartate isomerisation proceed faster in solution than in the lyophilised state and faster still at neutral to alkaline pH. Both are why the compound ships lyophilised and why the shelf life of a solution is measured in weeks rather than the two years quoted for the dry powder.

Two handling problems are specific to working at the concentrations this molecule is active at. Below roughly 1 microgram per mL, a 9 kDa protein adsorbs measurably onto glass and polypropylene surfaces, which is why cell culture protocols routinely include a carrier protein or a non-ionic surfactant in the dilution buffer. And each freeze-thaw cycle drives aggregation at the ice-water interface, so single-use aliquots preserve activity that repeated cycling on one vial does not.

The literature offers a useful check on all of this. In the fetal sheep and rodent work, biological activity was confirmed against a functional endpoint rather than assumed from a purity figure. For a compound whose potency depends on an intact fold and an intact N-terminal extension, a proliferation or receptor phosphorylation assay is the only characterisation that reports on the property actually being purchased.

Des(1-3)IGF-I is the other classic low-IGFBP-affinity analogue and the closest comparison. It is a 67-residue truncation missing the N-terminal Gly-Pro-Glu tripeptide, and it occurs naturally in fetal brain rather than being wholly engineered. In the original Adelaide comparisons it matched Long [Arg3]-IGF-I for potency in IGFBP-secreting cultures, and the two were treated as an equivalent pair throughout the rat catabolism work. The antidoping pharmacokinetic study separates them clearly: unchanged Des(1-3)-IGF-I was detectable in rats to 24 hours after a single administration, where unchanged LongR3-IGF-I was gone within 4.

Mechano growth factor is a different molecule despite the shared IGF-1 origin. It is a splice variant of the IGF-1 gene induced in skeletal muscle by mechanical loading, and its distinguishing activity sits in its unique C-terminal E domain peptide, which acts on satellite cell proliferation through a receptor that is not IGF-1R. A study of Long [Arg3]-IGF-I says nothing about mechano growth factor and the reverse is equally true.

The growth hormone secretagogues in the same catalogue category operate one level upstream and are not substitutes. Compounds such as ipamorelin, CJC-1295 and tesamorelin act on the pituitary to raise endogenous growth hormone, which raises hepatic IGF-1 production, which is then captured into the normal IGFBP system. Long [Arg3]-IGF-I bypasses the entire axis and, as the pig data show, actively suppresses it. An experiment intended to probe pituitary regulation and an experiment intended to probe IGF-1R signalling call for different compounds.

There is no registered human clinical trial of Long [Arg3]-IGF-I. A ClinicalTrials.gov search returns no interventional study of this analogue at any phase, and the compound was never developed for human use in the first place: it was built as a laboratory reagent and reached commercial scale through cell culture media supply.

The human IGF-1 record that does exist belongs to a different molecule. Mecasermin is recombinant native IGF-1, licensed for severe primary IGF-1 deficiency, and its safety and efficacy data describe a molecule that circulates in the normal ternary complex. Long [Arg3]-IGF-I was engineered specifically to escape that complex, which is the parameter that governs free ligand exposure. No part of the mecasermin record transfers.

The analogue does appear in one human-facing context: antidoping. Long [Arg3]-IGF-I, Des(1-3)-IGF-I and R3-IGF-I are prohibited substances in sport under the World Anti-Doping Agency's growth factor category, and validated immunopurification and mass spectrometry detection methods for all three have been published. The confiscated-vial case report and the survey of black market product quality both come out of that programme, which is also why the best available data on what is actually in an uncontrolled vial comes from doping control laboratories rather than from manufacturers.