Apolipoprotein L1 (APOL1)

Researchers used tetracycline-inducible APOL1 human embryonic kidney (HEK293) cells to assess the ability of a small-molecule compound, inaxaplin, to inhibit APOL1 channel function. Inaxaplin is a functional kinase of apolipoprotein L1. It inhibits selectively inhibited APOL1 channel function in vitro [1].

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In vitro and in vivo studies showed that inaxaplin bound directly to the APOL1 protein, inhibited APOL1 channel function. Specifically, in cellular assays, inaxaplin blocked the channel function of APOL1 in a concentration-dependent manner, as shown by the reduced APOL1-induced thallium ion flux in tetracycline-inducible APOL1 HEK293 cells (Figure 1A). In this cell model, thallium flux was minimal in the absence of APOL1 expression, and APOL1-mediated thallium flux was nearly eliminated by the administration of inaxaplin (Figure 1B). Inaxaplin bound directly to APOL1 protein, as shown by a microscale thermophoresis binding assay (Figure 1C)[1].

In an APOL1 G2-homozygous transgenic mouse model, the injection of interferon gamma resulted in heavy proteinuria; in contrast, no proteinuria was seen when control Friend leukemia virus B (FVB) mice were exposed to interferon gamma. The prophylactic administration of inaxaplin (3 mg per kilogram three times daily) or vehicle for 3 days, with the first dose given on day 1 approximately 1.5 hours before the interferon gamma injection, significantly reduced the area under the curve for the mean urinary albumin-to-creatinine ratio in interferon gamma–injected G2-homozygous mice by an average of 74.1% (Figure 1D and Fig. S3).

To assess the direct target engagement of APOL1 by inaxaplin, researchers developed a microscale thermophoresis binding assay using fluorescently labeled recombinant APOL1 protein. The binding of inaxaplin to APOL1 was quantified by changes to fluorescently labeled APOL1[1].

To quantify the effect of inaxaplin on APOL1 ion flux, researchers constructed inducible, cultured, human embryonic kidney (HEK293) cell lines expressing the APOL1 reference sequence (G0) and two disease-associated variants (G1 and G2) under the control of a tetracycline promoter. Inaxaplin was assessed for its effect on thallium ion flux, a surrogate for potassium ion flux. The flow of thallium ions into the cells was quantified with the use of a thallium-sensitive dye[1].

Researchers created a transgenic APOL1 mouse model homozygous for the APOL1 G2 variant and induced kidney dysfunction by injecting G2-homozygous mice with interferon gamma; saline (vehicle) injections were used as a negative control. To evaluate the potential to reduce proteinuria, we prophylactically administered inaxaplin (at a dose of 3 mg per kilogram of body weight three times daily) or vehicle to G2-homozygous mice for 3 days before the interferon gamma injection (Fig. S1 in the Supplementary Appendix, available with the full text of this article at NEJM.org). We obtained a spot urinary albumin-to-creatinine ratio every 24 hours for 72 hours.[1]

[1]. WO2020131807.

Background: Individuals with toxic gain-of-function variants in the apolipoprotein L1 (APOL1) gene are at higher risk of developing rapidly progressive proteinuric nephropathy. Although its genetic etiology is known, there is currently a lack of effective therapies for proteinuric nephropathy in patients carrying two APOL1 variants (G1 or G2). Methods: We evaluated the ability of the small molecule compound inaxaplin to inhibit APOL1 channel function using tetracycline-induced APOL1 human embryonic kidney cells (HEK293). Furthermore, we evaluated the efficacy of inaxaplin in treating proteinuria using an APOL1 G2 homologous transgenic mouse model of proteinuric nephropathy. Subsequently, we conducted a single-arm, open-label Phase IIa clinical trial in which enoxaprilin was administered to subjects carrying two APOL1 variants, with biopsy-confirmed focal segmental glomerulosclerosis and proteinuria (urine protein/creatinine ratio ≥0.7 to <10 [protein and creatinine are in grams], and an estimated glomerular filtration rate ≥27 ml/min/1.73 m² body surface area). Subjects received enoxaprilin daily for 13 weeks (15 mg for the first 2 weeks, 45 mg for the next 11 weeks) concurrent with standard of care. The primary endpoint was the percentage change in the urine protein/creatinine ratio from baseline at week 13, and at least 80% of subjects adhered to enoxaprilin treatment. Safety was also assessed. Results: In preclinical studies, inaxaplin selectively inhibited APOL1 channel function in vitro and reduced proteinuria in mouse models. A total of 16 subjects were enrolled in the Phase IIa study. In 13 subjects who received inaxaplin and met the adherence threshold, the urine protein/creatinine ratio decreased by a mean of 47.6% from baseline at week 13 (95% confidence interval, -60.0 to -31.3). In the analysis including all subjects (regardless of inaxaplin adherence), a similar decrease as in the primary analysis was observed in all but one subject. Adverse events were mild or moderate in severity; no adverse events led to study termination. Conclusion: Targeted inhibition of APOL1 channel function with inaxaplin reduces proteinuria in patients with two APOL1 variants and focal segmental glomerulosclerosis. (Sponsored by Vertex Pharmaceuticals; ClinicalTrials.gov registration numbers: VX19-147-101, NCT04340362.) [1]

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Introduction: Toxicity-gaining apolipoprotein L1 (APOL1) variants are one of the factors contributing to proteinuric nephropathy (collectively referred to as APOL1-mediated nephropathy, AMKD). Despite standard treatment, patients with AMKD progress to end-stage renal disease more rapidly. Two APOL1 variants have been identified as genetic causes of AMKD, prompting the development of inaxaplin, an APOL1 channel activity inhibitor that reduces proteinuria in patients with AMKD. Methods: We conducted two Phase I clinical studies to evaluate the safety, tolerability, and pharmacokinetics of single-ascendant (SAD) and multiple-ascendant (MAD) inaxaplin in healthy subjects. In the single-dose (SAD) group, subjects were randomized to receive a single dose of enoxaprilin (dose range: 7.5 mg to 165 mg) or placebo. In the multiple-dose (MAD) group, subjects were randomized to receive multiple doses of enoxaprilin (dose range: 15 to 120 mg daily) or placebo for 14 days. We assessed safety and tolerability based on adverse events (AEs), clinical laboratory values, electrocardiogram (ECG), and vital signs. Results: A total of 178 subjects (mean age: 36.7 years; 94.9% male) were randomized in both studies' SAD/MAD groups. The proportion of subjects experiencing any adverse events was similar in the enoxaprilin group (24.6%) and the placebo group (22.7%). All adverse events were mild or moderate; no serious adverse events occurred. Headache was the most common adverse event: 10.4% in the enoxaprilin group and 2.3% in the placebo group. No drug-related treatment interruptions occurred, and no clinically significant trends were observed in laboratory values, electrocardiograms, or vital signs. Discussion/Conclusion: Enaraxaprilin at single doses up to 165 mg and at multiple daily doses up to 120 mg (for 14 days) were both safe and well-tolerated. These results are consistent with the favorable safety profile of enoxaprilin in the completed Phase IIa proof-of-concept study. In conclusion, these findings support continued evaluation of enoxaprilin as a potential precision treatment for patients with AMKD in ongoing pivotal Phase II/III trials. [2]

C21H18F3N3O3

417.381135463715

417.13

C, 60.43; H, 4.35; F, 13.66; N, 10.07; O, 11.50

2446816-88-0

147289591

White to off-white solid powder

2.1

4

6

5

30

646

2

C1[C@H]([C@@H](C(=O)N1)NC(=O)CCC2=C(NC3=C2C=C(C=C3F)F)C4=CC=C(C=C4)F)O

CTXLPYZCBOVVQK-UZLBHIALSA-N

InChI=1S/C21H18F3N3O3/c22-11-3-1-10(2-4-11)18-13(14-7-12(23)8-15(24)19(14)27-18)5-6-17(29)26-20-16(28)9-25-21(20)30/h1-4,7-8,16,20,27-28H,5-6,9H2,(H,25,30)(H,26,29)/t16-,20+/m1/s1

3-[5,7-difluoro-2-(4-fluorophenyl)-1H-indol-3-yl]-N-[(3S,4R)-4-hydroxy-2-oxopyrrolidin-3-yl]propanamide

Inaxaplin; S2SJ2RVZ6Y; UNII-S2SJ2RVZ6Y; VX-147; Inaxaplin?; CHEMBL5083170; Inaxapline; Inaxaplina;

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 : ~50 mg/mL (~119.79 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.)