PD-1 signaling pathway[1]

NP-12 TFA exhibits equipotent antagonistic effects on PD-L1 and PD-L2, hence promoting the proliferation of lymphocytes and their ability to perform effector tasks [1]. With average EC50 values against rmPD-L1 and rmPD-L2 of 17 nM and 16.6 nM, respectively, NP-12 TFA restores the proliferation in the mouse splenocyte assay system [1]. Additionally, NP-12 TFA has been shown to dramatically mitigate the inhibition of in vitro human PBMC proliferation mediated by recombinant human PD-L1 and PD-L2, with average EC50 values against PD-L1 and PD-L2 of 63.3 nM and 44.1 nM, respectively [1].

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%.

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.

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.

AUNP-12 is active in vivo in a lung metastasis model of B16F10 melanoma in 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]. 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 aimed to discover 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 demonstrated 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 the number of PD-1+ T cells (including CD4 and CD8) in the tumor and blood. In addition, in pre-established tumor models, NP-12 showed enhanced 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, with immune-activating effects, excellent antitumor activity, and the potential to better control immune-related adverse events (irAEs). [3]

C142H226N40O48.C2HF3O2

3375.57

AUNP-12;1353563-85-5

White to off-white solid powder

(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 TFA

AUNP-12 TFA; 1353563-85-5; AUNP12 TFA; NP-12 TFA; CHEMBL4635204; AUNP-12, AUR-012 TFA; NONYLPHENOL POLYOXYETHYLENE ETHER; G13071 TFA

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

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.)