PD-1 signaling pathway
Programmed Cell Death 1 Receptor (PD-1) (EC50 = 0.72 nM for inhibition of PD-1/PD-L2 binding; EC50 = 0.41 nM in rat PBMC proliferation assay with hPD-L1 expressing MDA-MB231 cells) [2]
Programmed Cell Death 1 Receptor (PD-1) (equipotent antagonism toward PD-L1 and PD-L2, ) [3]

In vitro activity: AUNP-12 (also known as Aur-012, Aurigene-012, and Aurigene NP-12), a novel and potent immune checkpoint modulator developed by Aurigene Discovery Technologies, is an inhibitor of the PD-1 signalling pathway. AUNP-12 is in development for the treatment of several cancers. It is the only peptide therapeutic in this pathway and may offer more effective and safer combination opportunities compared to current approaches such as antibodies including Nivolumab (BMS), Lambrolizumab (Merck-3475), CT-011 (Curetech), MDX-1105 (BMS), MPDL3280 (GNE) and MEDI-4736 (Medimmune-AZ), or Amplimmune’s PD-L2-FC fusion protein.

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Kinase Assay: AUNP-12 displays an EC50 = 0.72 nM in the inhibition of binding PD1 to PD-L2 using hPDL2
expressing HEK293 cells, and an EC50 = 0.41 nM in a rat peripheral blood mononuclear cells (PBMC) proliferation assay using hPDL1 expressing MDA-MB231 cells. This corresponds well to the ‘sub-nanomolar potency in disruption of PD1-PDL1/2 interaction’ reported for AUNP-012.

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Cell Assay: AUNP-12 displays an EC50 = 0.72 nM in the inhibition of binding PD1 to PD-L2 using hPDL2
expressing HEK293 cells, and an EC50 = 0.41 nM in a rat peripheral blood mononuclear cells (PBMC) proliferation assay using hPDL1 expressing MDA-MB231 cells.

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1. AUNP-12 (also named Aur-012/NP-12, corresponding to compound 8 in US 2011/0318373) exhibits sub-nanomolar potency in disrupting PD-1/PD-L1/PD-L2 interaction; it has an EC50 of 0.72 nM in inhibiting PD-1 binding to PD-L2 (using hPD-L2-expressing HEK293 cells) and an EC50 of 0.41 nM in a rat PBMC proliferation assay with hPD-L1-expressing MDA-MB231 cells [2]
2. AUNP-12 is equipotent to compound 83 but less potent than compounds 34 and 45 in restoring human PBMC proliferation with recombinant hPD-L1; it is equipotent to compound 34 in the mouse splenocyte proliferation rescue assay (inhibition by MDA-MB231 cells expressing PD-L1) and more potent than compound 34 (equipotent to 83) in the IFN-γ production assay in a cytotoxic T lymphocyte assay [2]
3. AUNP-12 (NP-12) displays equipotent antagonism toward PD-L1 and PD-L2 in rescuing lymphocyte proliferation and effector functions; stimulation with anti-CD3/anti-CD28 showed complete rescue of CD4+ and CD8+ T cell proliferation, while completely abolishing the proliferation of CD4+ Foxp3+ regulatory T cells [3]
4. AUNP-12 induces sustained activation of circulatory immune cells, with their ability to secrete IFN-γ persisting up to 72 hours in vitro [2]

AUNP-12 inhibits by 44% tumor growth of B16F10 mouse melanoma cells injected subcutaneously in mice (5 mg/kg, subcutaneously once daily, 14 days); it reduces lung metastasis of B16F10 cells injected iv. in mice (5 mg/kg, subcutaneously, once daily, 11 days); it inhibits by 44% tumor growth of 4T1 cells injected orthotopically to mammary fat pad in mice (3 mg/kg, subcutaneously, once daily, 40 days). 10% of the animals treated with AUNP-12 showed complete regression and another 10% showed partial regression of tumor growth. AUNP-12 treated animals showed a mean reduction in lung metastasis, measured after euthanasia, to the extent of >60%.

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1. AUNP-12 (compound 8) inhibits subcutaneous B16F10 mouse melanoma tumor growth by 44% when administered at 5 mg/kg subcutaneously once daily for 14 days [2]
2. AUNP-12 reduces lung metastasis of intravenously injected B16F10 cells in mice (5 mg/kg subcutaneously once daily for 11 days), with slightly lower activity than compounds 34 and 82 [2]
3. AUNP-12 inhibits orthotopic 4T1 mammary tumor growth by 44% in mice (3 mg/kg subcutaneously once daily for 40 days); 10% of treated animals show complete tumor regression and another 10% show partial regression, with a >60% reduction in lung metastasis [2]
4. AUNP-12 (compounds 34 and 83) reduces tumor burden in orthotopically injected renal carcinoma cells in mice (5 mg/kg once daily for 21 days) with equipotent efficacy [2]
5. AUNP-12 (NP-12) shows significant efficacy comparable to commercial PD-1 antibodies in inhibiting primary tumor growth and metastasis in preclinical models of melanoma, colon cancer, and kidney cancer [3]
6. AUNP-12 exhibits additive antitumor activity in preestablished tumor models when combined with tumor vaccination or cyclophosphamide (a chemotherapeutic inducing immunologic cell death) [3]
7. In a preestablished CT26 colon cancer model, AUNP-12’s antitumor activity correlates with intratumoral recruitment of CD4+ and CD8+ T cells, and a reduction in PD-1+ T cells (CD4+ and CD8+) in tumor and blood [2][3]
8. AUNP-12 is active in vivo against E.coli sepsis in mouse models [2]
9. Dosing AUNP-12 once every three days is equally efficacious as once-daily dosing in preclinical cancer models, with no overt toxicity [2]

AUNP-12 displays an EC50 = 0.72 nM in the inhibition of binding PD1 to PD-L2 using hPDL2 expressing HEK293 cells, and an EC50 = 0.41 nM in a rat peripheral blood mononuclear cells (PBMC) proliferation assay using hPDL1 expressing MDA-MB231 cells. This corresponds well to the ‘sub-nanomolar potency in disruption of PD1-PDL1/2 interaction’ reported for AUNP-012.

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1. PD-1/PD-L2 binding inhibition assay: HEK293 cells expressing human PD-L2 were cultured and incubated with serial dilutions of AUNP-12 and recombinant PD-1 protein. The binding of PD-1 to PD-L2 on the cell surface was detected using a labeled secondary reagent (e.g., fluorescent or enzymatic label), and the EC50 value of AUNP-12 for inhibiting this interaction was calculated as 0.72 nM [2]
2. PD-1/PD-L1 functional assay (rat PBMC proliferation): Rat peripheral blood mononuclear cells (PBMCs) were isolated and co-cultured with MDA-MB231 cells expressing human PD-L1 in the presence of different concentrations of AUNP-12. PBMC proliferation was measured using a CFSE-based assay, and the EC50 of AUNP-12 was determined to be 0.41 nM [2]
3. IFN-γ production assay: Cytotoxic T lymphocytes were isolated and stimulated with tumor cells expressing PD-L1 in the presence of AUNP-12. Supernatants were collected at predetermined time points, and IFN-γ levels were quantified using an immunoassay to evaluate the effector function of T cells; AUNP-12 showed higher potency than compound 34 in this assay [2]

AUNP-12 displays an EC50 = 0.72 nM in the inhibition of binding PD1 to PD-L2 using hPDL2 expressing HEK293 cells, and an EC50 = 0.41 nM in a rat peripheral blood mononuclear cells (PBMC) proliferation assay using hPDL1 expressing MDA-MB231 cells.

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1. Mouse splenocyte proliferation rescue assay: Mouse splenocytes were isolated and cultured with MDA-MB231 cells expressing PD-L1 to induce proliferation inhibition. Serial dilutions of AUNP-12 (100 nM screening concentration) were added to the culture, and splenocyte proliferation was assessed using a CFSE-based assay. AUNP-12 (compound 8) was set as the reference with 100% activity, and its equipotency to compound 34 was confirmed in this assay [2]
2. Human PBMC proliferation assay: Human PBMCs were isolated and incubated with recombinant human PD-L1 in the presence of AUNP-12 and its derivatives (34, 45, 83). Cell proliferation was measured using a standard colorimetric or fluorescent method, and AUNP-12 was found to be less potent than 34 and 45 but equipotent to 83 in restoring proliferation [2]
3. T cell subset analysis: Immune cells were stimulated with anti-CD3/anti-CD28 antibodies in the presence of AUNP-12, then stained with specific antibodies against CD4, CD8, and Foxp3. Flow cytometry was used to analyze T cell subsets, confirming complete rescue of CD4+ and CD8+ T cell proliferation and abolition of CD4+ Foxp3+ T cell proliferation [3]

AUNP-12 is active in vivo in a lung metastasis model of B16F10 melanomain mice, showing a 64% reduction in metastasis at 5 mg/kg (subcutaneous, once daily, 14 days).[2]

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Pharmacokinetics of AUNP-12 in Balb/c mice[3]
All animal experimental procedures used in these studies including pharmacokinetic, pharmacodynamic, and efficacy experiments were approved by the Institutional Animal Ethical Committee based on the Committee for the Purpose of Control and Supervision on Experiments on Animals (India) guidelines. AUNP-12 was administered either intravenously or subcutaneously to the animals at a dose of 3 mg/kg to determine the pharmacokinetic parameters using 5% dextrose water as formulation. After administration, blood samples were collected at regular intervals until 24 hours and centrifuged to obtain the plasma fraction. The plasma samples were processed by SPE method and the eluent were analyzed by LC/MS-MS to determine the plasma concentration of the compound. From intravenous administration, plasma concentration after injection (C0 minutes), the area under the concentration−time curve from time zero to infinity (AUC 0−∞), the mean residence time, volume of distribution (Vdss), and clearance (CL) for each mouse were obtained. The maximum plasma concentration (Cmax), time to reach maximum plasma concentration (Tmax), and AUC 0−∞ were obtained from subcutaneous administration of AUNP-12 . On the basis of the intravenous and subcutaneous parameters, bioavailability of AUNP-12 was calculated.

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Syngeneic mouse studies[3]
In all in vivo tumor growth inhibition (TGI) studies, tumor volumes were measured two times weekly using digital calipers and the volume was expressed in mm3 using the formula V = 0.5a × b2, where a and b are the long and short diameters of the tumor, respectively. Body weights and clinical signs were monitored twice a week. AUNP-12 was dissolved in 5% dextrose water for all the in vivo studies, except for B16F10 mouse melanoma and Renca tumor models where 1 × PBS was used. Fresh formulation was prepared every day. Compound and vehicle controls were dosed subcutaneously once a day at a dosing volume of 10 mL/kg body weight.

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1. B16F10 melanoma subcutaneous tumor model: B16F10 melanoma cells were subcutaneously injected into mice. When tumors were established, AUNP-12 was administered subcutaneously at 5 mg/kg once daily for 14 days; tumor volume was measured regularly to evaluate growth inhibition [2]
2. B16F10 lung metastasis model: B16F10 cells were intravenously injected into mice, followed by subcutaneous administration of AUNP-12 at 5 mg/kg once daily for 11 days. Lungs were harvested post-euthanasia, and metastatic nodules were counted to assess anti-metastatic efficacy [2]
3. 4T1 orthotopic mammary tumor model: 4T1 cells were injected into the mammary fat pad of mice. AUNP-12 was administered subcutaneously at 3 mg/kg once daily for 40 days; tumor growth was monitored, and lung metastasis was quantified after euthanasia [2]
4. Renal carcinoma orthotopic model: Renal carcinoma cells were orthotopically injected into mouse kidneys. AUNP-12 (compounds 34 and 83) was administered at 5 mg/kg subcutaneously once daily for 21 days, and tumor burden was assessed [2]
5. CT26 colon cancer model: CT26 cells were implanted in mice to establish preclinical tumors. AUNP-12 was administered subcutaneously (dose/frequency not specified), and immune cell infiltration (CD4+/CD8+ T cells) and PD-1+ T cell levels in tumor/blood were analyzed by flow cytometry [3]
6. Combination therapy protocol: AUNP-12 was administered subcutaneously in combination with tumor vaccination or cyclophosphamide (dose/frequency not specified) in preestablished tumor models; tumor growth was monitored to evaluate additive antitumor effects [3]
7. Dosing frequency comparison: AUNP-12 was administered to tumor-bearing mice either once daily or once every three days at the same dose (5 mg/kg subcutaneous for melanoma models); tumor growth was compared to confirm equivalent efficacy [2]

1. Compared with PD-1 antibodies (half-life > 15-20 days), AUNP-12 has a shorter pharmacokinetic profile, a duration of efficacy > 24 hours, and IFN-γ secretion can continue for up to 72 hours after compound clearance [2]. 2. Specific ADME parameters (absorption, distribution, metabolism, excretion, half-life, oral bioavailability) of AUNP-12 have not yet been reported [2][3]. 3. In preclinical models, AUNP-12 showed excellent PK-PD correlation, and its efficacy was maintained despite a short systemic exposure time [2].

1. In a 14-day repeated-dose toxicity study in mice, AUNP-12 was well tolerated at 100 times the effective dose, with no significant toxicity or neutralizing antibody production observed.[2]
2. No data on LD50, hepatotoxicity, nephrotoxicity, drug interactions, or plasma protein binding of AUNP-12 were provided.[2][3]
3. Preclinical studies have shown that AUNP-12 did not cause serious immune-related adverse events (irAEs), which is attributed to its shorter pharmacokinetic profile compared to PD-1 antibodies.[3]

[1]. US 2011/0318373 A1
[2]. Cancer Research, vol. 72, no. 8 Suppl. 1, Abstract 2850, 2012.
[3]. A Rationally Designed Peptide Antagonist of the PD-1 Signaling Pathway as an Immunomodulatory Agent for Cancer Therapy. Mol Cancer Ther. 2019 Jun;18(6):1081-1091.

Further in vivo studies showed that AUNP-12/AUR-012 had excellent pharmacokinetic-pharmacodynamic correlation and a duration of efficacy exceeding 24 hours. In preclinical models of melanoma, breast cancer, and renal cell carcinoma, AUR-012/AUNP-12 showed superior efficacy in inhibiting primary tumor growth and metastasis compared to currently used clinical treatments. Notably, dosing every three days was comparable to dosing once daily, and no significant toxicity or neutralizing activity was observed. [9] Analysis of immune cell proliferation after stimulation with anti-CD3/anti-CD-28 antibodies showed that the proliferation of CD4+ and CD8+ T cells was completely restored. Interestingly, AUR-012/AUNP-12 treatment completely inhibited the proliferation of CD4+ Foxp3+ T cells, indicating that the proliferation of regulatory T cells was completely suppressed. The sustained activation of circulating immune cells and their ability to secrete IFN-γ lasted for up to 72 hours, indicating that the efficacy persisted even after the compound was cleared in animal models, thus supporting a dosing interval of up to 3 days. In melanoma, breast cancer, renal cell carcinoma and colon cancer models, AUR-012/AUNP-12 showed efficacy in inhibiting primary tumor growth and metastasis. In addition, in the pre-established CT26 model, the antitumor activity of the compound was closely related to the efficacy, manifested by the recruitment of CD4+ and CD8+ T cells in the tumor and the reduction of PD1+ T cells (including CD4+ and Page7/12 positive CD8+ cells) in the tumor and blood. In a 14-day repeated-dose toxicity study, AUR-012/AUNP-12 was well tolerated at 100 times the effective dose. [2]

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AUNP-12, possibly the same compound as previously designated Aur-012, Aurigene-012 or Aurigene NP-12, is a PD-1 pathway inhibitor currently being developed for a variety of cancer indications. It is the only peptide drug in this pathway to date, and compared to existing therapies,[2-4] it may offer more effective and safer combination therapy opportunities, such as antibody drugs like Nivolumab (Bristol-Myers Squibb), Lambrulizumab (Merck-3475), CT-011 (Curetech), MDX-1105 (Bristol-Myers Squibb), MPDL3280 (GNE), and MEDI-4736 (Medimmune-AZ), or the PD-L2-FC fusion protein of Amplimmune. PD-1, programmed death receptor 1, is an immune receptor belonging to the CD28 family and plays an important role in negatively regulating immune responses. The amino acid protein structure of PD-1 includes an extracellular amino acid IgV domain, a transmembrane region, and an intracellular tail. PD-1 is expressed on the surface of activated T cells, B cells, and macrophages and has two ligands: PD-L1 and PD-L2, both of which belong to the B7 family. PD-L1 is expressed in almost all mouse tumor cell lines, while PD-L2 expression is more limited, mainly expressed by dendritic cells (DCs) and a few tumor cell lines. Blocking the PD-1 signaling pathway has been shown to restore the function of immune cells impaired in cancer and chronic infection. In recent years, significant progress has been made in using antibodies or fusion proteins to inhibit immune checkpoint proteins, including PD-1, achieving highly durable clinical efficacy and revolutionizing the prospects of cancer treatment. However, while achieving significant clinical efficacy, serious immune-related adverse events (irAEs) have become increasingly prominent due to the disruption of immune tolerance. Due to the long half-life of antibodies (>15-20 days) and the target occupancy rate of >70% that can last for months, sustained target inhibition may be one of the reasons for the serious immune-related adverse events (irAEs) observed clinically with antibodies against immune checkpoint proteins. [2]

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Antibodies against immune checkpoints such as PD-1 and CTLA4 have achieved breakthrough success, opening up new avenues for cancer immunotherapy. However, while demonstrating significant clinical efficacy, antibody-based therapies are increasingly characterized by severe immune-related adverse events (irAEs) due to the disruption of immune tolerance. To better manage these serious adverse reactions, we are committed to discovering a PD-1 signaling pathway antagonist with a shorter pharmacokinetic profile. In this paper, we describe a peptide antagonist, NP-12, which exhibits equivalent antagonistic effects against both PD-L1 and PD-L2 in restoring lymphocyte proliferation and effector function. In preclinical models of melanoma, colon cancer, and renal cell carcinoma, NP-12 showed significant efficacy comparable to commercially available PD-1-targeting antibodies, inhibiting primary tumor growth and metastasis. Notably, the antitumor activity of NP-12 in a pre-established CT26 model was closely related to its pharmacodynamic effects, manifested as recruitment of intratumoral CD4 and CD8 T cells, and a reduction in PD-1+ T cells (including CD4 and CD8) in both the tumor and blood. Furthermore, in pre-established tumor models, NP-12 exhibited synergistic antitumor activity when used in combination with tumor vaccines or chemotherapy drugs known to induce immune cell death (such as cyclophosphamide). In summary, NP-12 is the first rationally designed peptide therapy targeting the PD-1 signaling pathway, possessing immune-activating effects, excellent antitumor activity, and the potential for better control of immune-related adverse events (irAEs). [3] AUNP-12 is a 29-peptide therapeutic agent targeting the PD-1 immune checkpoint pathway. It is currently the only peptide-based PD-1 antagonist used to treat serious immune-related adverse events (irAEs) associated with long-acting PD-1 antibodies. [2] 2. AUNP-12 is derived from a sequence (BC loop, amino acids 24-30) in the extracellular domain of PD-1 that is crucial for ligand-receptor interaction. It was developed by nonlinearly combining human/mouse PD-1-derived peptides of 7-30 peptides. [2] 3. The structure of AUNP-12 corresponds to the 29-peptide branch described in US Patent US 2011/0318373 (component 8 is referenced). Its structure-activity relationship shows that the C-terminal chain length is crucial to the activity. C-terminal lysine acylation (long-chain fatty acids, N-maleimide) is well tolerated and can enhance activity; N-terminal serine acylation reduces activity (double N-terminal acylation can partially restore activity) [2]
4. AUNP-12 has been licensed to Pierre Fabre (rights worldwide except India) for cancer treatment development, and its combination therapy has the potential to be safer than PD-1 antibodies [2]
5. AUNP-12 (NP-12) is the first rationally designed PD-1 pathway peptide antagonist with immune-activating properties, and due to its short target occupancy time, it has a low risk of immune-related adverse events (irAEs) [3]

C142H226N40O48

3261.55

3259.65

C, 52.29; H, 6.98; N, 17.18; O, 23.55

1353563-85-5

AUNP-12 TFA

154701623

H-Ser-Asn-Thr-Ser-Glu-Ser-Phe-Lys(1)-Phe-Arg-Val-Thr-Gln-Leu-Ala-Pro-Lys-Ala-Gln-Ile-Lys-Glu-NH2.H-Ser-Asn-Thr-Ser-Glu-Ser-Phe-(1)

SNTSESFK(SNTSESF-NH)FRVTQLAPKAQIKE-NH2

White to off-white solid powder

-23.2

51

53

112

230

7520

33

CC[C@H](C)[C@@H](C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCC(=O)O)C(=O)N)NC(=O)[C@H](CCC(=O)N)NC(=O)[C@H](C)NC(=O)[C@H](CCCCN)NC(=O)[C@@H]1CCCN1C(=O)[C@H](C)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCC(=O)N)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](C(C)C)NC(=O)[C@H](CCCNC(=N)N)NC(=O)[C@H](CC2=CC=CC=C2)NC(=O)[C@H](CCCCNC(=O)[C@H](CC3=CC=CC=C3)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(=O)O)NC(=O)[C@H](CO)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CC(=O)N)NC(=O)[C@H](CO)N)NC(=O)[C@H](CC4=CC=CC=C4)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(=O)O)NC(=O)[C@H](CO)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CC(=O)N)NC(=O)[C@H](CO)N

ZBJUUYIGBAQYBN-QKLNNLIKSA-N

InChI=1S/C142H226N40O48/c1-12-70(6)109(137(226)165-84(37-23-26-52-144)119(208)158-81(113(151)202)43-48-105(196)197)178-125(214)87(42-47-102(148)193)159-114(203)71(7)156-118(207)82(36-22-25-51-143)164-135(224)100-40-29-55-182(100)141(230)72(8)157-126(215)90(56-68(2)3)169-121(210)86(41-46-101(147)192)166-138(227)112(75(11)191)181-136(225)108(69(4)5)177-124(213)85(39-28-54-155-142(152)153)161-127(216)92(58-77-32-18-14-19-33-77)171-120(209)83(160-128(217)93(59-78-34-20-15-21-35-78)172-134(223)97(65-186)174-123(212)89(45-50-107(200)201)163-132(221)99(67-188)176-140(229)111(74(10)190)180-130(219)95(61-104(150)195)168-116(205)80(146)63-184)38-24-27-53-154-117(206)91(57-76-30-16-13-17-31-76)170-133(222)96(64-185)173-122(211)88(44-49-106(198)199)162-131(220)98(66-187)175-139(228)110(73(9)189)179-129(218)94(60-103(149)194)167-115(204)79(145)62-183/h13-21,30-35,68-75,79-100,108-112,183-191H,12,22-29,36-67,143-146H2,1-11H3,(H2,147,192)(H2,148,193)(H2,149,194)(H2,150,195)(H2,151,202)(H,154,206)(H,156,207)(H,157,215)(H,158,208)(H,159,203)(H,160,217)(H,161,216)(H,162,220)(H,163,221)(H,164,224)(H,165,226)(H,166,227)(H,167,204)(H,168,205)(H,169,210)(H,170,222)(H,171,209)(H,172,223)(H,173,211)(H,174,212)(H,175,228)(H,176,229)(H,177,213)(H,178,214)(H,179,218)(H,180,219)(H,181,225)(H,196,197)(H,198,199)(H,200,201)(H4,152,153,155)/t70-,71-,72-,73+,74+,75+,79-,80-,81-,82-,83-,84-,85-,86-,87-,88-,89-,90-,91-,92-,93-,94-,95-,96-,97-,98-,99-,100-,108-,109-,110-,111-,112-/m0/s1

(4S)-5-amino-4-[[(2S)-6-amino-2-[[(2S,3S)-2-[[(2S)-5-amino-2-[[(2S)-2-[[(2S)-6-amino-2-[[(2S)-1-[(2S)-2-[[(2S)-2-[[(2S)-5-amino-2-[[(2S,3R)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2,6-bis[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S,3R)-2-[[(2S)-4-amino-2-[[(2S)-2-amino-3-hydroxypropanoyl]amino]-4-oxobutanoyl]amino]-3-hydroxybutanoyl]amino]-3-hydroxypropanoyl]amino]-4-carboxybutanoyl]amino]-3-hydroxypropanoyl]amino]-3-phenylpropanoyl]amino]hexanoyl]amino]-3-phenylpropanoyl]amino]-5-carbamimidamidopentanoyl]amino]-3-methylbutanoyl]amino]-3-hydroxybutanoyl]amino]-5-oxopentanoyl]amino]-4-methylpentanoyl]amino]propanoyl]pyrrolidine-2-carbonyl]amino]hexanoyl]amino]propanoyl]amino]-5-oxopentanoyl]amino]-3-methylpentanoyl]amino]hexanoyl]amino]-5-oxopentanoic acid

Aur-012; AUNP-12; 1353563-85-5; AUNP-12?; CHEMBL4635204; AUNP-12, AUR-012; EX-A7438; NONYLPHENOL POLYOXYETHYLENE ETHER; G13071; Aurigene-012

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)