Integrin CD11b/CD18
CD11b/CD18 (Integrin αMβ2, Mac-1). The biochemical half-maximal effective concentration (EC50) for ADH-503 is 4 μM in cell-based assays. [1]

The number of CD11b+ monocytes, granulocytes, eosinophils, and macrophage subsets as well as the total number of tumor-infiltrating CD11b+ cells are decreased by ADH-503 ((Z)-Leukadherin-1 choline; 4 μM; 8 days) [1].

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ADH-503 binds CD11b and reduces myeloid cell recruitment to PDAC tissues [1]
To overcome the dosing limitation of CD11b blockade in previous studies, we developed a small molecule agonist, ADH-503, whose binding achieves a partially active CD11b conformation. ADH-503 directly alters the cytokine profile of PDAC-activated macrophages.

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ADH-503 directly alters the cytokine profile of PDAC-activated macrophages. In bone marrow-derived macrophages treated with PDAC conditioned medium, ADH-503 changed expression of >8000 RNAs within 6 hours (2-fold change). It decreased genes involved in IL-1 signaling and alternative activation markers (Arg1, YM1, Retnla), while up-regulating type I interferons (IFNα1, IFNβ) and T cell recruitment factors (CXCL9, CXCL10, CXCL11). It down-regulated TGFβ1, IL1α, and IL1β. In vivo, treatment with ADH-503 reduced tumor-infiltrating CD11b+ cells, monocytes, granulocytes, eosinophils, and macrophages. The remaining macrophages showed higher expression of MHCI, MHCII, CD80, and CD86, indicating improved antigen presentation. Gene expression profiling of TAMs from treated mice showed reduced immunosuppressive genes (IL6, TGFβ, Arginase-1, IL10) and increased CXCL10. ADH-503 increased tumor-infiltrating CD8+ cytotoxic T lymphocytes (CTLs) and CD4+ T effectors, their proliferation (Ki67+), and activation (CD44HiCD62Lneg). It also increased PD1+Eomes+High and Tim3High/PD1High CTLs, and reduced FOXP3+ regulatory T cells. It increased the number of CD8+ T cells in close proximity (<5 μm) to CK19+ PDAC cells. ADH-503 increased OVA-specific dextramer+ CD8+ T cells in tumors and draining lymph nodes. It reduced tumor-infiltrating CD11b+ cDCs2s and monocyte-derived DCs but markedly increased CD103+ cDCs1s and their MHC-I/II expression. The compound had no direct effect on PDAC cell growth in vitro. [1]

Reducing tumor burden increases overall survival. ADH-503 ((Z)-Leukadherin-1 choline; oral gavage; 30, 60, or 120 mg/kg; twice daily for 60 days) delayed tumor progression, leading to substantial differences in time-point analyses and therapy [1]. ADH-503 (oral gavage; 30, 100 mg/kg; twice daily; days 1 and 5) has an AUC0-t 30 at the mg/kg and 100 mg/kg doses, with plasma concentrations of 6950 ng.h/mL and 13962 ng.h/mL, respectively. Its highest concentration is 1716 and 2594 ng/mL. The mean half-life is 4.68 and 3.95 hours.

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ADH-503 treatment induces the accumulation of CD103+ cDCs in the tumor [1]
Due to the increase in intratumoral T cell numbers and proliferation, we explored whether these effects are driven by changes in DCs. As expected, based on their CD11b expression, we observed reduced numbers of tumor-infiltrating CD11b+ cDC2s and monocyte-derived DCs after 12 days of ADH-503 treatment (Figs. 4H and S4D). In contrast, in ADH-503-treated mice, tumor-infiltrating CD103+ cDC1s (which express CD11b at extremely low levels) were markedly increased in both number and MCH-I and MHC-II expression (Fig. 4H). These data suggest that ADH-503 reduces the numbers of potentially tolerogenic and/or CD4+ T cell-priming DCs, while enhancing cross-presenting by CD103+ cDC1s. The identity of these cDC populations was confirmed using Zbtb46gfp/+ reporter mice (Fig. S1D). To determine whether the changes in cDC1s were necessary for the increased CTL response observed in ADH-503-treated mice, we used BATF3−/− mice, which lack functional cDC1s. In contrast to wild-type controls, treatment with ADH-503 had no effect on T cell infiltration in BATF3-deficient mice (Fig. 4I). Taken together, these findings suggest that myeloid cell reprograming by ADH-503 drives cDC1 infiltration and function, leading to a reinvigorated anti-tumor T cell response.

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ADH-503 impairs tumor growth and improves survival in orthotopic models and KPC GEMMs [1]
To determine the impact of a CD11b agonist on tumor progression, we evaluated three syngeneic orthotopic PDAC models and KPC GEMMs (Fig. 5A–D). In all models, ADH-503 delayed tumor progression, leading to a significantly decreased tumor burden in time point analysis and improved overall survival (Fig. 5C and D). Importantly, ADH-503 had no direct effects on PDAC cell growth in vitro (Fig. S5A). To further confirm the specificity of ADH-503 for CD11b, we utilized CD11b-null (ITGAM-null) mice and found that unlike in wild-type mice, CD11b-null mice had similar tumor growth, regardless of treatment (Fig. 5E).

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ADH-503 impaired tumor growth and improved survival in orthotopic PDAC models (KP2, KI, KP2-OVA) and KPC GEMMs. It delayed tumor progression, reduced tumor burden, and improved overall survival. In KPC GEMMs, it decreased tumor grade and collagen density, and increased cleaved caspase-3 (apoptosis marker) in PDAC cells. ADH-503 enhanced the efficacy of chemotherapy (gemcitabine + paclitaxel), resulting in >90% disease control, improved survival, and reduced liver metastases (from 80% in vehicle to 12.5% in ADH-503 group and 0% in combination group). It also improved the efficacy of radiation therapy (4Gy x 5), leading to substantial tumor regression. ADH-503 rendered PDAC tumors responsive to checkpoint immunotherapy. Combination with anti-PD-1 resulted in significant tumor regression, complete regression by days 21-30, survival past 120 days, and resistance to re-challenge. Combination with anti-41BB (CD137 agonist) also induced marked tumor regression and long-term survival. In KPC GEMMs, ADH-503 plus anti-PD1/anti-CTLA4/gemcitabine (PCG) significantly increased survival and CD8+ T cell infiltration. In head-to-head comparisons, ADH-503 plus anti-PD-1 was as effective as CCR2 inhibitor (PF-04136309) plus anti-PD-1, and more effective than CSF1 neutralization or granulocyte depletion (anti-Ly6G) in combination with anti-PD-1. [1] The compound increased CD11b/CD18-dependent cell adhesion and decreased chemotaxis and transendothelial migration. It reduced leukocyte recruitment in a thioglycolate-induced peritonitis model in mice. In a rat arterial balloon injury model, it reduced neointimal thickening and macrophage infiltration. In an anti-GBM nephritis mouse model, it decreased neutrophil infiltration and proteinuria, showing better efficacy than the antagonist M1/70. In zebrafish tailfin injury, it reduced neutrophil accumulation, and its effects were reversible upon removal. [2]

Leukadherin-1, also known as LA1, is a novel and specific agonist of Complement receptor 3 (CR3) and the leukocyte surface integrin CD11b/CD18 that enhances leukocyte adhesion to ligands and vascular endothelium and thus reduces leukocyte transendothelial migration and influx to the injury sites. Complement receptor 3 (CR3, CD11b/CD18) is a multi-functional receptor expressed predominantly on myeloid and natural killer (NK) cells. Leukadherin-1 (LA1) does not modulate signal transducer and activator of transcription (STAT)-4 phosphorylation. Leukadherin-1 modulates TLR-2 and TLR-7/8-induced monocyte cytokine secretion. Targeting leukocyte trafficking using LA1, an integrin agonist, is beneficial in preventing lung inflammation and protecting alveolar and vascular structures during hyperoxia. Thus, targeting integrin-mediated leukocyte recruitment and inflammation may provide a novel strategy in preventing and treating BPD in preterm infants.

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αA domain ligand-binding assay [2]
MaxiSorp 96-well plates were coated overnight with fibrinogen (1 μg per well) in 10 mM phosphate-buffered saline (PBS, pH 7.4) and blocked with 1% BSA in PBS. Binding of purified, GST-tagged αA domain (50 μl per well of a 5 μg/ml solution) to immobilized fibrinogen was performed in TBS-based assay buffer (TBS containing 0.1% BSA, 1 mM MgCl2, 1 mM CaCl2, and 0.05% Tween 20) (TBS-Ca/Mg buffer) for 1 hour at room temperature. The αA domain was also added to uncoated wells on the plate to estimate the maximum amount of protein that could be captured and detected in each well for data normalization. Unbound αA domain was removed by washing the wells twice with TBS-Ca/Mg buffer. Subsequently, the amount of bound protein was determined by incubation with horseradish peroxidase–conjugated antibody against GST (GE, 1:2000 dilution) for 1 hour. Unbound antibody was removed by washing the wells twice with TBS-Ca/Mg buffer. Detection of bound protein was performed with 3,3′,5,5′-tetramethylbenzidine (TMB) substrate kit according to the manufacturer’s protocol. Absorbance was read with a SpectraMax M5 spectrophotometer). Absorbance values were normalized such that the mean absorbance from the input αA domain wells was set at 100%, and the results are presented as the percentage of the total input amounts of the wild-type αA domain. Assays were performed in triplicate wells, and the data shown are from one of at least three independent experiments.

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Not specified. However, binding studies with related compounds (leukadherins) show they bind to the ligand-binding αA domain of CD11b/CD18. [2]

Phagocytosis assay with complement iC3b-coated sheep erythrocytes (EiC3bs) [2]
Sheep erythrocytes coated with complement iC3b were prepared and used in the phagocytosis assay as described previously. Coated erythrocytes (EiC3bs) were diluted to a concentration of 1.5 × 107 to 6 × 107 cells/ml. K562 CD11b/CD18 cells were washed twice in TBS and resuspended to 1 × 106/ml, of which 40 μl (4 × 104 cells) was incubated in suspension with EiC3bs (1.2 × 106) in a total volume of 100 μl at 37°C for 25 min in the presence of 1 mM each of CaCl2 and MgCl2 (in the absence or presence of 50 to 100 μM Leukadherin-1 (LA1), LA2, or LA3), in 1 mM MnCl2, or in 10 mM EDTA. Binding was detected by visually analyzing the formation of rosettes [the binding of multiple erythrocytes (EiC3bs) to individual K562 cells] by phase-contrast microscopy, as has been described previously. For scoring, only those K562 cells that were bound to ≥3 EiC3bs were scored as positive, and >200 cells were examined in multiple fields under each condition. Binding results, showing the percentages of all cells showing rosettes in a field, are reported as histograms representing the mean ± SEM of triplicate experiments; the data shown are from one of at least three independent experiments.

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Cell viability assays [2]
Cell viability assays were performed with commercially available reagents and kits. Briefly, 1 × 104 K562 CD11b/CD18 cells or wild-type B6 neutrophils were incubated in each well of a 96-well plate with increasing amounts of the indicated compounds, and the number of viable cellswas determined with the MTS reagent, according to the manufacturers’ instructions, after 4 hours (neutrophils) or 24 hours (K562 cells) of incubation. A SpectraMaxM5 spectrophotometer was used to read the assay plates. Data are representative of at least two independent experiments.

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Western blotting analysis [2]
K562 CD11b/CD18 cells were incubated with Leukadherin-1 (LA1), LA2, or LA3 (15 μM) or fibrinogen (200 μg) in serum-free medium for 1 hour at 37°C. Cell lysates were resolved on a 10% SDS-PAGE gel and transferred to a polyvinylidene difluoride membrane by means of established protocols. Membranes were incubated with a 1:1000 dilution of an antibody against phosphorylated extracellular signal–regulated kinase 1/2 (ERK1/2) (Thr202/Tyr204), stripped with Reblot mild stripping solution, and then incubated, first with an antibody against total ERK1/2 and then with an antibody against glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and developed according to the manufacturer’s instructions. Data presented are representative of at least three independent experiments.

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Cell-based adhesion assays: K562 cells expressing CD11b/CD18 were used. Cells were incubated with ADH-503 in ligand-coated wells for 10 min at 37°C. Nonadherent cells were removed by inverting plates for 30 min. Adherent cells were quantified. The EC50 was 4 μM. [1] Bone marrow-derived macrophages were treated with PDAC conditioned medium ± ADH-503 for 7 hours, then RNA-seq and Q-PCR analysis were performed. [1] For bead labeling, blood cells were labeled with PE-conjugated microspheres. Tissues were processed for flow cytometry. [1] For chemotaxis assays (related compound LA1), neutrophils were placed in Zigmond chambers with a fMLP gradient, and migration was recorded. [2] For transendothelial migration (related compound LA1), THP-1 cells migrated across a HUVEC layer activated by TNF-α towards an MCP-1 gradient. [2]

Animal/Disease Models: KPC mouse [p48-CRE/Lox-stop-Lox(LSL)-KrasG12D/p53flox/flox][1]
Doses: 30, 60 or 120 mg/kg
Route of Administration: po (oral gavage); 60-day
Experimental Results: Delays tumor progression, results in Dramatically lower tumor burden in time point analysis, and improves overall survival.

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Animal/Disease Models: Male rat[1]
Doses: 30, 100 mg/kg (pharmacokinetic/PK/PK analysis)
Route of Administration: po (oral gavage), twice (two times) daily; results on days 1 and 5: 30 mg and 100 mg, the average half-lives were 4.68 and 3.95 hrs (hrs (hours)), respectively, the maximum concentrations were 1716 and 2594 ng/mL, and the AUC0-t in plasma were 6950 and 13962 ng.h/mL/kg respectively.

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For animal experiments, ADH-503 was given at 30, 60, or 120 mg/kg and is specified in the text when not at 60mg/kg. ADH-503 was formulated for treatment in 0.5% carboxymethyl cellulose and 0.1 % Tween-80 in sterile water and administered by oral gavage twice a day (BID).

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ADH-503 was formulated in 0.5% carboxymethyl cellulose and 0.1% Tween-80 in sterile water. It was administered by oral gavage twice a day (BID) at 30, 60, or 120 mg/kg (typically 60 mg/kg). [1] For pharmacokinetic studies, ADH-503 was given orally to rats and C57/B6 mice. [1] For peritonitis studies (related compound LA1), compounds were administered intravenously or intraperitoneally 30 min before thioglycolate injection. [2] For balloon injury model (related compound LA1), compound was administered intramuscularly 30 min before injury and then every other day for 3 weeks. [2] For anti-GBM nephritis (related compound LA1), compound was injected intraperitoneally daily starting 2 hours before induction. [2] For zebrafish studies (related compound LA1), larvae were immersed in compound solution. [2]

In rats, the mean half-life of ADH503 after administration at doses of 30 and 100 mg/kg was 4.68 and 3.95 hours, respectively, with plasma Cmax and AUC0-t of 1716 and 2594 ng/mL and 6950 and 13962 ng·h/mL, respectively. These parameters were similar with repeated administration in rats at increasing doses (Fig. 2C, S2C-E). Similar pharmacokinetic characteristics were observed in C57/B6 mice (Fig. S2D). [1]

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In rats, after oral administration of ADH-503 at 30 mg/kg, the mean half-life was 4.68 hours; at 100 mg/kg, it was 3.95 hours. Cmax in plasma was 1716 ng/mL (30 mg/kg) and 2594 ng/mL (100 mg/kg). AUC0-t was 6950 ng·h/mL (30 mg/kg) and 13962 ng·h/mL (100 mg/kg). Repeat dosing showed similar parameters. In C57/B6 mice, similar PK properties were observed. [1] For related compound LA1 (leukadherin-1), no specific PK data is provided, but it is noted as a small molecule that can be delivered orally and is reversible upon removal from circulation. [2]

Studies have shown that ADH503 is well tolerated, with no adverse reactions or toxicities observed in rats after a single dose or continuous administration for 28 days (up to 1500 mg/kg/d) and in dogs after a single dose or continuous administration for 28 days (up to 1359 mg/kg/d). ADH503 administration did not cause death, clinical symptoms, or changes in body weight, and the compound is well tolerated. [1]

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[1]. Agonism of CD11b reprograms innate immunity to sensitize pancreatic cancer to immunotherapies. Sci Transl Med. 2019 Jul 3;11(499).

[2]. Small molecule-mediated activation of the integrin CD11b/CD18 reduces inflammatory disease. Sci Signal. 2011;4(189):ra57.

The CD11b agonist GB1275 is a small molecule CD11b (integrin α-M; ITGAM; integrin α-M chain) agonist with high oral bioavailability and potential immunomodulatory activity. Upon administration, GB1275 targets and binds to CD11b, thereby activating it. This leads to CD11b-mediated signaling, promoting pro-inflammatory macrophage polarization while inhibiting immunosuppressive macrophage polarization. This reduces the infiltration of tumor-associated macrophages (TAMs) and myeloid-derived suppressor cells (MDSCs) in the tumor microenvironment (TME), promotes anti-tumor immune responses, induces the production of cytotoxic T lymphocytes (CTLs), and inhibits tumor growth. CD11b, a member of the integrin family of cell adhesion receptors, is highly expressed on immune system cells and acts as a negative regulator of immunosuppression, capable of activating innate anti-tumor immunity. While immune checkpoint therapy has revolutionized cancer treatment, not all types of tumors benefit from it. Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal malignancy with very limited response to immunotherapy. Extensive immunosuppressive myeloid cell infiltration in PDAC tissues is considered a major mechanism of immunotherapy resistance. In preclinical studies, strategies simultaneously targeting monocyte or granulocyte migration or macrophage survival, in combination with immune checkpoint therapy, have shown promising promise, and these studies have translated into ongoing clinical trials for the treatment of pancreatic cancer and other cancer types. However, compensatory mechanisms by untargeted monocytes, granulocytes, and/or tissue-resident macrophages may limit the efficacy of such strategies. CD11b/CD18 are integrin molecules highly expressed on the cell surface of these myeloid cell subsets and play important roles in cell migration and function in inflamed tissues. This paper demonstrates that partial activation of CD11b by the small molecule agonist (ADH-503) leads to repolarization of tumor-associated macrophages, a reduction in the number of tumor-infiltrating immunosuppressive myeloid cells, and enhanced dendritic cell responses. These effects, in turn, enhance anti-tumor T-cell immunity and enable immune checkpoint inhibitors to exert their therapeutic effects in previously unresponsive pancreatic ductal adenocarcinoma (PDAC) models. These data suggest that the molecular agonistic effects of CD11b can reprogram immunosuppressive myeloid cell responses and potentially circumvent the limitations of current clinical strategies in overcoming immunotherapy resistance. [1]

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ADH-503 is a small molecule agonist of CD11b that reprograms innate immunity to sensitize pancreatic cancer to immunotherapies. It targets multiple myeloid cell lineages (monocytes, granulocytes, macrophages) to overcome resistance to checkpoint blockade. It is currently on track for Phase I single agent clinical testing. The combination of ADH-503 with anti-PD-1 or anti-41BB led to complete tumor regression and long-term immunologic memory in PDAC models. [1] The related compound LA1 (leukadherin-1) is a small molecule agonist that binds to the αA domain of CD11b/CD18, increasing cell adhesion and reducing migration, and has shown efficacy in inflammatory disease models. [2]

C27H28N2O5S2

524.651624679565

524.143

C, 61.81; H, 5.38; N, 5.34; O, 15.25; S, 12.22

2055362-74-6

Leukadherin-1;344897-95-6;(Z)-Leukadherin-1;2055362-72-4; 2055362-74-6 (choline)

145711124

Brown to reddish brown solid powder

1

7

6

36

721

0

S1C(N(C(/C/1=C/C1=CC=C(C2C=CC(C(=O)[O-])=CC=2)O1)=O)CC1C=CC=CC=1)=S.OCC[N+](C)(C)C

GOWDQYRMBCOOJR-JHMJKTBASA-M

InChI=1S/C22H15NO4S2.C5H14NO/c24-20-19(29-22(28)23(20)13-14-4-2-1-3-5-14)12-17-10-11-18(27-17)15-6-8-16(9-7-15)21(25)261-6(2,3)4-5-7/h1-12H,13H2,(H,25,26)7H,4-5H2,1-3H3/q+1/p-1/b19-12-

2-hydroxy-N,N,N-trimethylethan-1-aminium (Z)-4-(5-((3-benzyl-4-oxo-2-thioxothiazolidin-5-ylidene)methyl)furan-2-yl)benzoate

ADH-503 choline; ADH-503; ADH 503; ADH503; Leukadherin-1 choline; 2-Hydroxy-N,N,N-trimethylethan-1-aminium (Z)-4-(5-((3-benzyl-4-oxo-2-thioxothiazolidin-5-ylidene)methyl)furan-2-yl)benzoate; THN3VQ67CA; UNII-THN3VQ67CA; 4-[5-[(Z)-(3-benzyl-4-oxo-2-sulfanylidene-1,3-thiazolidin-5-ylidene)methyl]furan-2-yl]benzoate;2-hydroxyethyl(trimethyl)azanium; LA1

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)

MEthanol : ~100 mg/mL (~190.60 mM)
DMSO : ~21.43 mg/mL (~40.85 mM)
Ethanol : ~3.33 mg/mL (~6.35 mM)

Solubility in Formulation 1: ≥ 2.14 mg/mL (4.08 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 21.4 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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.

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Solubility in Formulation 2: ≥ 2.14 mg/mL (4.08 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 21.4 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 3: 2.08 mg/mL (3.96 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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