Fibroblast activation protein-α (FAP)

Potency and Selectivity of FAP Inhibitors for Human Recombinant FAP [1]
PNT6555, PNT6952, and PNT6522 when not chelated with metals exhibited low nanomolar IC50 values for FAP that were ∼200- to ∼1000-fold and ∼10,000- to ∼30,000-fold less, respectively, than the values for PREP and DPP4 (Table 1). The introduction of the metals reduced potency toward FAP by 2- to 48-fold, depending on the compound (Table 1). Metal chelation also reduced affinity for PREP by ≤4-fold. No inhibition of DPP4 was detectable.

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Cellular Uptake and Internalization of FAP Inhibitors [1]
The FAP dependence of the cellular uptake of natLu-PNT6555 was investigated by comparing the total uptake in HEK-mFAP versus HEK-Mock cells. Measurement of total cell-associated natLu-PNT6555 by ICP-MS demonstrated that uptake required the expression of FAP (Fig. 2A). ICP-MS measurements of the total cell bound and internalized amounts of natLu in HEK-mFAP cells after 1 h incubation with natLu-PNT6555, natLu-PNT6952 or natLu-PNT6522 indicated that natLu-PNT6555 exhibited the greatest degree of internalization (Fig. 2B).

Antitumor Activity of PNT6555, PNT6952, and PNT6522 177Lu-Radioligands [1]
177Lu-PNT6555, 177Lu-PNT6952, and 177Lu-PNT6522 were administered to HEK-mFAP tumor–bearing mice as a single dose of 15, 30, or 60 MBq. These doses were selected to enable efficacy to be directly compared with 177Lu-FAPI-46 and 177Lu-FAP-2286, previously evaluated in a HEK-FAP tumor model. 177Lu-radioligands produced dose-dependent delays in tumor growth, whereas no discernable effect was produced by unlabeled precursors (Fig. 5A). On the last day of tumor measurement before mortality in control groups, tumor growth was significantly inhibited at all doses investigated (Supplemental Fig. 4A). 177Lu-PNT6555 produced the longest tumor growth delay (Fig. 5A), with the rank order of efficacy being 177Lu-PNT6555 > 177Lu-PNT6952 > 177Lu-PNT6522. The same rank order was also reflected in animal survival (Fig. 5B). The radioligands appeared to be well tolerated, and weight loss was ≤10% and transient (Supplemental Fig. 5).

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Antitumor Activity of 225Ac-PNT6555 and 161Tb-PNT6555 [1]
To HEK-mFAP tumor–bearing mice, 225Ac-PNT6555 was administered as a single dose of 5, 25, or 50 kBq, and 161Tb-PNT6555, as a single dose of 15, 30, or 60 MBq. 225Ac-PNT6555 doses were based on single doses of 225Ac-FAPI-46 that were safe and effective in the PANC-1 xenograft model, and 161Tb-PNT6555 doses, on safe and effective single doses of radiometal targeted to L1 cell adhesion protein or folate receptor. Both 225Ac-PNT6555 and 161Tb-PNT6555 produced dose-dependent delays in tumor growth at all dose levels (Fig. 6A), and before mortality of control animals, tumor volumes were significantly reduced (Supplemental Fig. 4B). Mean tumor volume in mice that received nonradiolabeled PNT6555 appeared to be increased above that in vehicle-treated mice in the 225Ac-PNT6555 experiment. Although this effect was statistically significant, it was not pharmacologically meaningful because it was small and not seen in the 177Lu-PNT6555 (Fig. 5A) and 161Tb-PNT6555 experiments (Fig. 6A). 225Ac-PNT6555 and 161Tb-PNT6555 increased animal survival in a dose-dependent manner (Fig. 6B). Both radioligands were well tolerated, as indicated by minimal effect on body weight (Supplemental Fig. 6).

In Vitro Fluorometric IC50 Measurements [1]
Half-maximal inhibitory concentration (IC50) values of FAP ligands for recombinant human FAP, DPP4, and PREP were determined. FAP (pH 7.5), PREP (pH 7.5), and DPP4 (pH 8.0) were incubated at room temperature for 10 min with 1:10 serial dilutions of FAP ligands (PNT6555, PNT6952, and PNT6522) in 96-well plates. 7-Amino-4-methylcoumarin (AMC) fluorogenic substrates (carboxybenzyl (Z)-Gly-Pro-AMC for FAP and PREP and Gly-Pro-AMC for DPP4) were added to the reactions at a final concentration of 25 µM. After further incubation for 15 min at room temperature, enzyme activity was measured by fluorimetry (Ex380 nm:Em460 nm). A modified method with N-(4-quinolinoyl)-d-Ala-Pro (3144)–AMC as the FAP substrate was also used for IC50 assays with recombinant human and mouse FAP. The enzymes were incubated at 37°C for 10 min with 1:10 serial dilutions of FAP ligands. 3144-AMC was added at a final concentration of 25 μM, and incubation was continued for a further 30 min at 37°C before fluorimetry.
For IC50 assays with serum samples, 1:10 dilutions of human serum and Sprague–Dawley rat serum and a 1:100 dilution of mouse serum were incubated at 37°C for 10 min with 1:10 serial dilutions of FAP ligands. 3144-AMC was added at a final concentration of 25 μM (rat serum) or 50 μM (human serum and mouse serum), and incubation was continued for a further 30 min at 37°C before fluorimetry.
For IC50 assays with cell membrane FAP, HEK-mFAP cells were harvested from bulk cultures grown to approximately 80% confluency and plated in 96-well plates. After incubation overnight, 1:10 serial dilutions of FAP ligands were incubated with the cells for 1 h. 3144-AMC was added at a final concentration of 20 μM, and 37°C incubation was continued for a further 30 min before fluorimetry.

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ICP-MS Assay for In Vitro Uptake and Internalization of natLu-Chelated FAP Ligands [1]
In vitro cellular uptake of natLu-FAP ligands was investigated by inductively coupled plasma mass spectrometry (ICP-MS) analysis of total natLu associated with HEK-mFAP or HEK-Mock cells. The internalized fraction represented the natLu remaining after an acid wash to remove any cell membrane–bound inhibitor. HEK-mFAP or HEK-Mock cells were seeded in 6-well plates (Costar) at 4 × 106 cells/2 mL/well in serum-free RPMI 1640 assay medium and incubated (37°C, 5% CO2) for 18–24 h. Medium was aspirated and replaced with 1, 5, 10, or 100 nM natLu-PNT6555 for the measurement of FAP ligand uptake or with 10 nM natLu-PNT6555, natLu-PNT6952, or natLu-PNT6522 for the measurement of FAP ligand uptake and internalization. After incubation for 1 h at 37°C, the cells were washed twice with 1 mL of ice-cold phosphate-buffered saline (PBS). For the measurement of total cellular FAP ligand uptake (natLutotal), the cells were lysed by incubation at room temperature in 0.3 M NaOH for 5 min, after which the samples were passed through a 23-gauge needle to shear DNA. For the measurement of cellular internalization, 1 mL of ice-cold 50 mM glycine–100 mM NaCl (pH 2.8) buffer was added to the cells after the PBS wash step described before. After incubation at 4°C for 10 min, the cells were washed twice with 1 mL of ice-cold PBS and then lysed as described before to provide samples for the measurement of internalized FAP ligand (natLuinternal). The protein concentrations of the total uptake and internalization samples were measured by the Bradford assay (Bio-Rad). Postlysis samples (300 μL) were microwave digested with 0.5 mL of ultrapure water and 2.0 mL of ultra–high-purity nitric acid. The samples were further diluted with ultrapure water to achieve 2.8% nitric acid. An internal standard (final concentration of indium of 5 ppb) was added to the samples, and indium at 0.1 ppt was added to 500-ppt Lu standards for the creation of a standard curve. ICP-MS was conducted in low-resolution mode. Calibrant intensities were normalized to the intensities of the internal controls, and the intensities in blank samples were subtracted to create linear calibration curves. The total cellular uptake of natLu after incubation of HEK-mFAP cells with 1, 5, 10, or 100 nM natLu-PNT6555 was expressed as the absolute amount (nanograms). The percentage internalization of natLu was calculated as (natLuinternal/natLutotal) × 100.

HEK-mFAP Mouse Tumor Xenograft Model [1]
Six-week-old male Fox Chase mice with severe combined immunodeficiency were injected subcutaneously with HEK-mFAP cells. Tumor growth was determined by measurement of tumor width (W) and length (L) with calipers, and tumor volume (V) expressed in mm3 was calculated by the formula V = (W2 × 0.5L).

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Formulation and In Vivo Administration of Radioligands [1]
Radioligands diluted in PBS were administered to anesthetized mice by a single injection into the lateral tail vein.

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Biodistribution of 68Ga- and 177Lu-Radioligands In Vivo [1]
HEK-mFAP tumor–bearing mice were injected intravenously with defined doses of 68Ga-PNT6555 or 177Lu-radioligands. At designated time points, blood and tissues were collected from 3 mice per treatment and counted for radioactivity. Tissue weights were measured for determination of the percentage injected dose per gram (%ID/g).

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PET and SPECT [1]
After intravenous injection of radioligands, 68Ga imaging by small-animal PET/CT and 177Lu SPECT imaging were performed.

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Antitumor Activity of 177Lu-, 225Ac-, and 161Tb-Radioligands In Vivo [1]
Mice bearing HEK-mFAP tumors of the volumes specified in the Results section were administered 177Lu-, 225Ac-, or 161Tb-radioligands, vehicle, or precursor ligands (6 mice per group) on day 1. Health checks were performed, and body weights and tumor dimensions were measured weekly. Mortality or euthanasia was used interchangeably for plotting mouse survival curves. Tumor growth curves were plotted up to the time of the earliest incidence of a mortality/euthanasia endpoint in each of the control and test groups.

Biodistribution of Radiolabeled FAP Inhibitors in Tumor-Bearing Mice [1]
68Ga-PNT6555 and 68Ga-PNT6952 exhibited selective uptake into HEK-mFAP tumors (Fig. 3A). Small-animal PET indicated that the radioligands rapidly entered the tumors, and while they were progressively cleared from the blood and normal tissues via the kidneys and the bladder, tumor activity increased rapidly over the first 5 min after injection, and more slowly but continuously thereafter (Fig. 3C; Supplemental Fig. 2). Elimination from blood, liver, and muscle resulted in radioligand levels that were distinctly lower in normal tissues than in tumors by ∼25 min after administration (Fig. 3C), and high-contrast PET images of tumors were obtained at 60 min (Fig. 3C). All 3 177Lu-FAP radioligands exhibited selective uptake in tumors 4 h after a single dose in HEK-mFAP tumor–bearing mice (Fig. 4A; Supplemental Fig. 3). Intratumoral levels of all 3 radioligands decreased between 4 and 48 h, but high tumor-to-normal tissue ratios were maintained from 48 to 168 h. Analysis of the area under the curve (AUC) ((%ID/g)·h) for the period from 4 to 168 h indicated that the accumulation of 177Lu-PNT6555 in the tumor was significantly greater than that for 177Lu-PNT6952 or 177Lu-PNT6522 (P < 0.0001) (Table 3; Supplemental Table 1), but the increase in 177Lu-PNT6555 between 48 and 168 h was not statistically significant. Although the limited uptake in normal tissues exhibited some variation between the 3 radioligands (Table 3), these differences were not statistically significant (Supplemental Table 1). The highest levels of uptake into normal tissues occurred in kidney, liver, bone and skin for 177Lu-PNT6555, kidney for 177Lu-PNT6952, and kidney and bone for 177Lu-PNT6522 (Table 3; Supplemental Fig. 3). However, the high initial tumor uptake and kinetics of tumor retention resulted in tumor-to-normal tissue AUC ratios of 15 to 19 in these tissues (Table 3). This is illustrated by the pharmacokinetic profiles in tumor and kidney (Fig. 4A), and the retention of 177Lu-PNT6555 in the tumors was apparent in SPECT images collected from 3 to 120 h (Fig. 4B).

[1].Preclinical Development of PNT6555, a Boronic Acid–Based, Fibroblast Activation Protein-α (FAP)–Targeted Radiotheranostic for Imaging and Treatment of FAP-Positive Tumors. Journal of Nuclear Medicine, 2024 Jan 2;65(1):100-108.

The overexpression of fibroblast activation protein-α (FAP) in solid cancers relative to levels in normal tissues has led to its recognition as a target for delivering agents directly to tumors. Radiolabeled quinoline-based FAP ligands have established clinical feasibility for tumor imaging, but their therapeutic potential is limited due to suboptimal tumor retention, which has prompted the search for alternative pharmacophores. One such pharmacophore is the boronic acid derivative N-(pyridine-4-carbonyl)-d-Ala-boroPro, a potent and selective FAP inhibitor (FAPI). In this study, the diagnostic and therapeutic (theranostic) potential of N-(pyridine-4-carbonyl)-d-Ala-boroPro-based metal-chelating DOTA-FAPIs was evaluated. Methods: Three DOTA-FAPIs, PNT6555, PNT6952, and PNT6522, were synthesized and characterized with respect to potency and selectivity toward soluble and cell membrane FAP; cellular uptake of the Lu-chelated analogs; biodistribution and pharmacokinetics in mice xenografted with human embryonic kidney cell-derived tumors expressing mouse FAP; the diagnostic potential of 68Ga-chelated DOTA-FAPIs by direct organ assay and small-animal PET; the antitumor activity of 177Lu-, 225Ac-, or 161Tb-chelated analogs using human embryonic kidney cell-derived tumors expressing mouse FAP; and the tumor-selective delivery of 177Lu-chelated DOTA-FAPIs via direct organ assay and SPECT. Results: DOTA-FAPIs and their natGa and natLu chelates exhibited potent inhibition of human and mouse sources of FAP and greatly reduced activity toward closely related prolyl endopeptidase and dipeptidyl peptidase 4. 68Ga-PNT6555 and 68Ga-PNT6952 showed rapid renal clearance and continuous accumulation in tumors, resulting in tumor-selective exposure at 60 min after administration. 177Lu-PNT6555 was distinguished from 177Lu-PNT6952 and 177Lu-PNT6522 by significantly higher tumor accumulation over 168 h. In therapeutic studies, all 3 177Lu-DOTA-FAPIs exhibited significant antitumor activity at well-tolerated doses, with 177Lu-PNT6555 producing the greatest tumor growth delay and animal survival. 225Ac-PNT6555 and 161Tb-PNT6555 were similarly efficacious, producing 80% and 100% survival at optimal doses, respectively. Conclusion: PNT6555 has potential for clinical translation as a theranostic agent in FAP-positive cancer. [1]

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The selective inhibition of FAP over the dipeptidyl peptidases and PREP by 3099, which was achieved by the pyridin-4-carbonyl blocking group at the N terminus, and d-alanine at P2, respectively (25), was maintained in PNT6555, PNT6952, and PNT6522. When expressed as a cell membrane protein in HEK-mFAP cells, the catalytic site of FAP was found to be pharmacologically accessible to the d-Ala-boroPro–based ligands and to be essential for the cellular uptake of natLu-PNT6555 by HEK-mFAP cells. In vivo, 68Ga-PNT6555 and 68Ga-PNT6952 were selectively retained in HEK-mFAP tumors, resulting in PET images with high tumor-to-background contrast. The biodistribution of 177Lu-PNT6555, 177Lu-PNT6952, and 177Lu-PNT6522 in HEK-mFAP tumor–bearing mice confirmed the selective targeting of the HEK-mFAP tumors and revealed that 177Lu-PNT6555 exhibited the greatest tumor accumulation, consistent with its greater cellular internalization compared with natLu-PNT6952 and natLu-PNT6522 in vitro.[1]

C31H48BN7O11

C31H48BN7O11

705.35

C, 52.77; H, 6.86; B, 1.53; N, 13.90; O, 24.94

2715113-34-9

168489413

Typically exists as White to off-white solid at room temperature

7

15

14

50

1140

2

B([C@@H]1CCCN1C(=O)[C@@H](C)NC(=O)C2=CC=C(C=C2)CNC(=O)CN3CCN(CCN(CCN(CC3)CC(=O)O)CC(=O)O)CC(=O)O)(O)O

PTVMRWQEZPTMFA-RDGATRHJSA-N

InChI=1S/C31H48BN7O11/c1-22(31(48)39-8-2-3-25(39)32(49)50)34-30(47)24-6-4-23(5-7-24)17-33-26(40)18-35-9-11-36(19-27(41)42)13-15-38(21-29(45)46)16-14-37(12-10-35)20-28(43)44/h4-7,22,25,49-50H,2-3,8-21H2,1H3,(H,33,40)(H,34,47)(H,41,42)(H,43,44)(H,45,46)/t22-,25+/m1/s1

2-[4-[2-[[4-[[(2R)-1-[(2R)-2-boronopyrrolidin-1-yl]-1-oxopropan-2-yl]carbamoyl]phenyl]methylamino]-2-oxoethyl]-7,10-bis(carboxymethyl)-1,4,7,10-tetrazacyclododec-1-yl]acetic acid

BDBM609722; DOTA- aminomethyl-Bz- D-Ala-boroPro; US11707539, Compound 6555; 2715113-34-9

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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

DMSO (~250 mg/mL (354 mM)

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.)