Factor D (Kd value = 0.54 nM)

At an IC50 value of 0.015 μM, Danicopan (ACH-4471) binds to C3b, complexes with it, and, in a dose-dependent way, inhibits the proteolytic activity of pure factor D on its natural substrate factor B. Danicopan (ACH-4471) has IC50 values between 0.0040 μM and 0.027 μM and IC90 values between 0.015 μM and 0.11 μM, which indicates that it efficiently inhibits hemolysis [1].

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Researchers report the activity of two novel small-molecule inhibitors (ACH-3856 and Danicopan (ACH4471)) of the alternative pathway component Factor D using in vitro correlates of both paroxysmal nocturnal hemoglobinuria and atypical hemolytic uremic syndrome. Both compounds bind human Factor D with high affinity and effectively inhibit its proteolytic activity against purified Factor B in complex with C3b. When tested using the traditional Ham test with cells from paroxysmal nocturnal hemoglobinuria patients, the Factor D inhibitors significantly reduced complement-mediated hemolysis at concentrations as low as 0.01 μM. Additionally the compound ACH-4471 significantly decreased C3 fragment deposition on paroxysmal nocturnal hemoglobinuria erythrocytes, indicating a reduced potential relative to eculizumab for extravascular hemolysis. Using the recently described modified Ham test with serum from patients with atypical hemolytic uremic syndrome, the compounds reduced the alternative pathway-mediated killing of PIGA-null reagent cells, thus establishing their potential utility for this disease of alternative pathway of complement dysregulation and validating the modified Ham test as a system for pre-clinical drug development for atypical hemolytic uremic syndrome [1].

Efficacy of ACH-4471 following oral delivery in non-human primates [1]
To profile the effects of continuous Factor D inhibition over a period of at least 24 hours in vivo, we administered ACH-4471 orally to 3 monkeys in two serial 200 mg/kg doses separated by 12 hours. This deliberately high dose was chosen based on an initial assessment of pharmacokinetic properties in monkeys which revealed lower oral exposure due to higher clearance than in other animal species, including rats and dogs (data not shown). The monkeys tolerated the compound well and showed no clinical abnormalities. Figure 5A shows ACH-4471 concentrations in plasma at time points from 0 hours (pre-dosing) to 30 hours (18 hours after the second dose). In parallel, serum was collected for determination of APC activity by Wieslab assay and of Factor D concentrations. APC activity was suppressed continuously by more than 95% continuously through the 30-hour time period (Figure 5B) with no significant increase in Factor D concentrations (Figure 5C). The observed stability of serum Factor D concentrations was expected because, as a small molecule, ACH-4471 should not interfere with renal Factor D clearance, in marked contrast to the effect of systemic delivery of the humanized monoclonal anti-Factor D antibody lampalizumab.29 These results demonstrate the potential utility of oral delivery of ACH-4471 for therapeutic inhibition of APC activation.

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Following oral administration of a single dose of [14C]-danicopan at 20 mg/kg, a dose that was expected to yield a good exposure to evaluate danicopan tissue distribution based on earlier studies with unlabeled danicopan, drug-derived radioactivity was rapidly absorbed and widely distributed to tissues in both pigmented (n = 10) and albino (n = 3) rats. Radioactivity was evident 1 to 8 hours postdose and became nonquantifiable at 24 hours postdose in most tissues, as demonstrated by whole-body autoradiography (Fig. 2). In pigmented rats at 24 hours postdose, radioactivity localized primarily to the uvea tract (i.e., melanin-containing ocular tissues), pigmented skin, and liver (Fig. 2A); in albino rats, radioactivity was primarily localized to the liver (Fig. 2B). Quantification of radioactivity showed that [14C]-danicopan remained quantifiable in the uveal tract of pigmented rats at 672 hours (28 days) postdose (t1/2 = 576 hours) (Fig. 2C), whereas it became undetectable in plasma and whole blood after 8 hours postdose (Fig. 2). [2]
Purpose: Complement alternative pathway (AP) dysregulation has been implicated in geographic atrophy, an advanced form of age-related macular degeneration. Danicopan is an investigational, first-in-class inhibitor of factor D, an essential AP activation enzyme. We assessed danicopan distribution to the posterior segment of the eye after oral dosing.[2]
Methods: Tissue distribution of drug-derived radioactivity was evaluated using whole-body autoradiography following oral administration of [14C]-danicopan to pigmented and albino rats. Pharmacokinetics and ocular tissue distribution were studied in pigmented and albino rabbits following single and multiple oral dosing of danicopan. The melanin binding property was characterized in vitro.[2]
Results: Radioactivity was distributed widely in rats and became nonquantifiable in most tissues 24 hours postdose except in the pigmented rat uvea (quantifiable 672 hours postdose). Danicopan binding to melanin was established in vitro. After single dosing, the maximum concentration (Cmax) and area under the curve (AUC) in neural retina and plasma were similar in both rabbit types. After multiple dosing, AUC in neural retina was 3.4-fold higher versus plasma in pigmented rabbits. Drug levels in choroid/Bruch's membrane (BrM)/retinal pigment epithelium (RPE) were similar to plasma in albino rabbits but higher in pigmented rabbits: Cmax and AUC were 2.9- and 23.8-fold higher versus plasma after single dosing and 5.8- and 62.7-fold higher after multiple dosing. In pigmented rabbits, ocular tissue exposures slowly declined over time but remained quantifiable 240 hours postdose.[2]
Conclusions: The results demonstrate that danicopan crosses the blood-retina barrier and binds melanin reversibly, leading to a higher and more sustained exposure in melanin-containing ocular tissues (choroid/BrM/RPE) and in the neural retina as compared to in plasma after repeated oral dosing in pigmented animals.[2]
Translational relevance: These findings suggest that oral danicopan possesses potential for treating geographic atrophy because AP dysregulation in the posterior segment of the eye is reported to be involved in the disease pathogenesis.

In Vitro Melanin-Binding Studies [2]
Solutions of two sources of melanin (natural Sepia officinalis and synthetic melanin), Danicopan (ACH4471), and chloroquine (as a positive control) were used in the assay. Danicopan (ACH4471) and chloroquine were incubated with or without melanin in 96-well plates. Melanin and bound compound were collected by centrifugation at 4000 rpm, diluted with acetonitrile containing the internal assay standard, and passed through a Waters XSelect HSS T3 2.5-µm gradient column (50 × 2.1 mm for danicopan and 30 × 2.1 mm for chloroquine). Samples and internal standards were measured using a SCIEX API-5500 triple quadrupole mass spectrometer using multiple reaction-monitoring scan modes (danicopan, 580.2/360.2; chloroquine, 320.1/247.3), and data were captured using SCIEX Analyst 1.6.2. Data curves of mean free concentration versus bound concentration were fit with a one-sided hyperbolic model using Prism 5.0 to obtain the KD as a measure for affinity and the maximum bound concentration binding (Bmax) as a measure of binding capacity.

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Assays for Factor D binding, Factor D enzymatic activity, and hemolysis mediated by APC and the classical pathway [1]
Binding kinetics and affinities of compounds to human Factor D were determined by surface plasmon resonance. Inhibition of Factor D enzymatic activity was evaluated with 80 nM purified human Factor D and the small synthetic thieoster substrate Z-Lys-SBzl, or with 0.8 nM purified human Factor D and its natural substrate C3bB. Inhibition of APC-mediated hemolytic activity was assessed with 8% normal human serum and rabbit erythrocytes; inhibition of hemolysis by the classical pathway was assessed with 0.5% normal human serum and antibody-sensitized sheep erythrocytes. Methods for these assays are described in the Online Supplementary Appendix.

Inhibition of APC-mediated cell killing using PNH and aHUS patient samples: Ham test and modified Ham test [1]
Inhibition of hemolytic assay by Factor D inhibitors was assessed using PNH erythrocytes at 1% hematocrit in GVB0/MgEGTA (pH 6.4) and 20% acidified human serum (Ham test), as previously described. Based on the same principle, the modified Ham test was performed to assess the efficacy of Factor D inhibitors in APC-mediated killing caused by aHUS patient serum.20 Both assays are described in the Online Supplementary Appendix.

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Inhibition of C3 fragment deposition [1]
C3 fragment deposition on PNH erythrocytes from PNH Patient 2 (blood group O) by 60% acidified C5-depleted human serum was assessed by flow cytometry. Erythrocytes were washed and prepared as in the hemolytic assay. Sample preparation and C3 fragment deposition measurement by flow cytometry are described in detail in the Online Supplementary Appendix.

APC activity in cynomolgus monkeys [1]
ACH-4471 was formulated in solution at 80 mg/mL in PEG400:water (70:30) (w:w). Three male cynomolgus monkeys were dosed by oral gavage with ACH-4471 at 200 mg/kg twice, 12 hours apart. Serial blood samples were collected at specified time points through 30 hours. Plasma ACH-4471 concentration was determined by LC-MS/MS with a lower limit of quantitation of 2.44 ng/mL. Pharmacokinetic parameters were calculated with non-compartmental analysis using Phoenix 6.2 WinNonlin from individual plasma concentration versus time using the linear-trapezoidal method. Serum APC activity was measured in duplicate using the APC-specific Wieslab assay. Activity at each time point was normalized to pre-dosing activity in the same animal. Serum Factor D concentrations were determined using the Human Complement Factor D Quantikine ELISA Kit.

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Tissue Distribution of Danicopan (ACH4471)/[14C]-Danicopan in Rats by Quantitative Whole-Body Autoradiography [1]
Tissue distribution of Danicopan (ACH4471) was measured by quantitative whole-body autoradiography following a single oral dose of 20 mg/kg [14C]-danicopan to pigmented Long–Evans and albino Wistar Han rats. This dose was chosen based on the results from an earlier plasma pharmacokinetic study with unlabeled danicopan; thus, it was expected to yield a good exposure signal (i.e., radioactivity) to evaluate danicopan tissue distribution. Rats were euthanized at 1, 2, 4, 8, 24, 72, 168, 336, 504, and 672 hours postdose, and carcasses were frozen in hexane/dry ice and stored at or below −20°C before processing and sectioning. Frozen samples were embedded in 1% carboxymethylcellulose matrix, mounted on a microtome stage maintained at −20°C, and sectioned in approximately 40-µm–thick sagittal sections. Sections were labeled with calibration standards of [14C]-glucose mixed with blood at various concentrations, visualized using a [14C]-sensitive phosphor-imaging plate, and scanned using a GE Amersham Molecular Dynamics Typhoon 9410 image acquisition system, with a scanning resolution of 50 µm.

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Tissue Distribution of Danicopan (ACH4471) in Rabbits [1]
Ocular tissue and plasma distributions of danicopan were evaluated in pigmented DB and albino NZW rabbits. Based on efficacious regimens in human studies and pharmacokinetic/pharmacodynamic modeling, danicopan was administered orally at 7.5, 15, or 50 mg/kg with single or multiple dosing to cover the projected range of therapeutic exposures in the posterior segment of the eye; for multiple dosing, rabbits received danicopan approximately every 12 hours for 14 days and a single dose on the morning of day 15. Rabbits were euthanized, and plasma and ocular tissues were collected at various time points either after a single dose or after the morning dose on day 15 (i.e., the last dose of the multiple-dosing regimen) with one exception: Samples were only collected at the approximate time to maximum concentration (tmax; hour 1) from NZW rabbits in the multiple-dosing study at 50 mg/kg. Danicopan concentrations were determined by liquid chromatography/mass spectrometry.

Factor D inhibitor inhibits alternative pathway of complement (APC) activity following oral dosing in cynomolgus monkeys. (A) ACH-4471 concentrations at the indicated time points in plasma samples collected from 3 cynomolgus monkeys following oral dosing with ACH-4471 at 200 mg/kg at 0 and 12 hours (arrows). Key pharmacokinetic parameters were calculated as follows: maximum concentration (Cmax), 4020±1480 ng/mL; time at Cmax (Tmax), 15.3±1.2 hours (3.3±1.2 hours following the second dose), and 30-hour exposure level (AUC0-30), 48,300±19,100 ng/mL/h. Parameters were calculated as mean ± standard deviation (SD) from the 3 animals. (B) APC activity assessed by Wieslab assay in serum samples collected at the indicated time points. Activity in each sample was normalized to the activity in the same animal at 0 h (pre-dosing). Mean±SD are shown from duplicate assay values. (C) Serum FD concentrations at the indicated time points. [1]

[1]. Small-molecule factor D inhibitors selectively block the alternative pathway of complement in paroxysmal nocturnal hemoglobinuria and atypical hemolytic uremic syndrome. Haematologica. 2017 Mar;102(3):466-475.

[2]. Danicopan, an Oral Complement Factor D Inhibitor, Exhibits High and Sustained Exposure in Ocular Tissues in Preclinical Studies. Transl Vis Sci Technol. 2022;11(10):37.

Danicopan is an organic molecular entity.
Danicopan is under investigation in clinical trial NCT03459443 (A Proof of Concept Study for a 12 Month Treatment in Patients With C3 Glomerulopathy (C3G) or Immune-Complex Membranoproliferative Glomerulonephritis (IC-MPGN)).
Danicopan is an orally bioavailable inhibitor of complement factor D (FD; CFD), a serine protease that cleaves complement factor B, with potential complement system inhibiting activity. Upon administration, danicopan targets, binds to and blocks the activity of FD, and thereby inhibits cleavage of complement factor B into Ba and Bb in the alternative pathway of the complement cascade. This inhibits FD-mediated signaling and activation of the alternative complement pathway (ACP), blocks complement-mediated hemolysis in paroxysmal nocturnal hemoglobinuria (PNH) and prevents ACP-induced tissue damage. FD plays a key role in the activation of the ACP.

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Drug Indication
Treatment of paroxysmal nocturnal haemoglobinuria.

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Paroxysmal nocturnal hemoglobinuria and atypical hemolytic uremic syndrome are diseases of excess activation of the alternative pathway of complement that are treated with eculizumab, a humanized monoclonal antibody against the terminal complement component C5. Eculizumab must be administered intravenously, and moreover some patients with paroxysmal nocturnal hemoglobinuria on eculizumab have symptomatic extravascular hemolysis, indicating an unmet need for additional therapeutic approaches. We report the activity of two novel small-molecule inhibitors of the alternative pathway component Factor D using in vitro correlates of both paroxysmal nocturnal hemoglobinuria and atypical hemolytic uremic syndrome. Both compounds bind human Factor D with high affinity and effectively inhibit its proteolytic activity against purified Factor B in complex with C3b. When tested using the traditional Ham test with cells from paroxysmal nocturnal hemoglobinuria patients, the Factor D inhibitors significantly reduced complement-mediated hemolysis at concentrations as low as 0.01 μM. Additionally the compound ACH-4471 significantly decreased C3 fragment deposition on paroxysmal nocturnal hemoglobinuria erythrocytes, indicating a reduced potential relative to eculizumab for extravascular hemolysis. Using the recently described modified Ham test with serum from patients with atypical hemolytic uremic syndrome, the compounds reduced the alternative pathway-mediated killing of PIGA-null reagent cells, thus establishing their potential utility for this disease of alternative pathway of complement dysregulation and validating the modified Ham test as a system for pre-clinical drug development for atypical hemolytic uremic syndrome. Finally, ACH-4471 blocked alternative pathway activity when administered orally to cynomolgus monkeys. In conclusion, the small-molecule Factor D inhibitors show potential as oral therapeutics for human diseases driven by the alternative pathway of complement, including paroxysmal nocturnal hemoglobinuria and atypical hemolytic uremic syndrome.[1]

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In conclusion, Factor D is a promising target for oral therapy of diseases driven by APC dysregulation. Based on results presented here, and guided by additional assessments of its pharmacology, pharmacokinetic properties, and safety and toxicology, ACH-4471 has been selected for clinical development in PNH and is currently in phase I clinical study.[1]

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In summary, we showed that danicopan, a complement factor D inhibitor, crosses the BRB and binds melanin reversibly, leading to a higher and more sustained exposure not only in melanin-containing ocular tissues such as choroid/BrM/RPE but also in the neural retina as compared to plasma after repeated oral dosing in pigmented animals. Additionally, no ocular safety signals were observed with oral dosing of danicopan in the multiple animal studies across several species and strains. Danicopan is currently being investigated in a phase 2 clinical study in patients with GA (NCT05019521).[2]

C26H23BRFN7O3

580.40832734108

579.102

C, 53.80; H, 3.99; Br, 13.77; F, 3.27; N, 16.89; O, 8.27

1903768-17-1

1903768-17-1;Danicopan HCl;

118323590

White to off-white solid powder

3.3

1

8

6

38

891

2

BrC1=CC=CC(=N1)NC([C@@H]1C[C@H](CN1C(CN1C2C=CC(C3=CN=C(C)N=C3)=CC=2C(C(C)=O)=N1)=O)F)=O

PIBARDGJJAGJAJ-NQIIRXRSSA-N

InChI=1S/C26H23BrFN7O3/c1-14(36)25-19-8-16(17-10-29-15(2)30-11-17)6-7-20(19)35(33-25)13-24(37)34-12-18(28)9-21(34)26(38)32-23-5-3-4-22(27)31-23/h3-8,10-11,18,21H,9,12-13H2,1-2H3,(H,31,32,38)/t18-,21+/m1/s1

(2S,4R)-1-{[3-acetyl-5-(2-methylpyrimidin-5-yl)-1H-indazol1-yl]acetyl}-N-(6-bromopyridin-2-yl)-4-fluoropyrrolidine2-carboxamide

ACH-4471; ACH4471; ACH 4471; 1903768-17-1; ACH-4471; Danicopan free base; Danicopan [USAN]; VOYDEYA; JM8C1SFX0U; ACH-0144471; ACH 0144471; ACH0144471; Danicopan free base

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 : ~125 mg/mL (~215.37 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (3.58 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% 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 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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Solubility in Formulation 2: ≥ 2.08 mg/mL (3.58 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 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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