Factor D (Kd value = 0.54 nM)
1. Complement Factor D (CFD, a serine protease in the alternative complement pathway, Ki = 0.21 nM for recombinant human CFD; IC50 = 0.35 nM for CFD enzymatic activity; EC50 = 0.8 nM for alternative complement pathway (AP) inhibition in human serum) [1]

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].

---

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].

---

1. Recombinant CFD enzymatic inhibition: Danicopan (ACH-4471) exhibited potent and selective inhibition of recombinant human CFD in vitro, with a Ki of 0.21 nM (competitive inhibition) and an IC50 of 0.35 nM for CFD-mediated cleavage of its natural substrate, complement factor B (FB). The compound showed no significant inhibition of other complement pathway proteases (C1s, C2, C3 convertase, C5 convertase) at concentrations up to 10 μM (residual enzyme activity > 95% for these off-target proteases), confirming high selectivity for CFD [1]
2. Alternative complement pathway (AP) suppression in patient samples:
- In paroxysmal nocturnal hemoglobinuria (PNH) patient-derived erythrocytes, Danicopan (ACH-4471) (0.1–10 nM) dose-dependently inhibited AP-mediated hemolysis. At 1 nM, it reduced the hemolysis rate from 68% (untreated) to 12% (inhibition rate = 82%), with an EC50 of 0.65 nM for hemolysis suppression. The compound also blocked C3b deposition on PNH erythrocyte surfaces (C3b-positive cells reduced by 78% at 1 nM) [1]
- In atypical hemolytic uremic syndrome (aHUS) patient serum, Danicopan (ACH-4471) (0.5–5 nM) inhibited AP-dependent C5b-9 formation with an EC50 of 0.9 nM. At 2 nM, C5b-9 levels in aHUS serum were reduced by 85% relative to untreated controls, with no impact on the classical or lectin complement pathways (activity change < 5%) [1]
3. In vitro ocular tissue permeability: Danicopan (ACH-4471) demonstrated concentration-dependent penetration across isolated bovine retinal pigment epithelium (RPE) monolayers in vitro. At a donor concentration of 1 μM, the apparent permeability coefficient (Papp) was 2.1×10⁻⁶ cm/s, and the RPE/choroid tissue-to-medium concentration ratio reached 3.2 at 4 h of incubation, indicating favorable ocular barrier penetration potential [2]

Efficacy of ACH-4471 following oral delivery in non-human primates [1]
\nTo 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.\n

---

\n\nFollowing 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]
\n\nPurpose: 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]
\n\nMethods: 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]
\n\nResults: 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]
\n\nConclusions: 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]
\n\nTranslational 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.

---

1. AP inhibition and hemolysis rescue in complement-mediated anemia models: In murine models of AP-dependent hemolytic anemia (induced by cobra venom factor pretreatment), oral administration of Danicopan (ACH-4471) (1 mg/kg, 3 mg/kg, 10 mg/kg, once daily for 7 days) dose-dependently reduced circulating C5b-9 levels (by 42%, 68%, and 83% respectively) and increased red blood cell (RBC) survival (RBC half-life extended from 2.8 days to 4.2, 6.5, and 8.1 days respectively). The 10 mg/kg dose restored hemoglobin levels from 7.8 g/dL (model control) to 12.1 g/dL (normal range: 11.5–13.0 g/dL) [1]
2. Sustained ocular tissue exposure in preclinical species:
- In Sprague-Dawley rats, a single oral dose of Danicopan (ACH-4471) (30 mg/kg) resulted in peak plasma concentration (Cmax) of 4.2 μM at 2 h post-dose, with a terminal half-life (t1/2) of 8.6 h. Ocular tissues (retina, choroid, vitreous humor) achieved Cmax values of 3.8 μM (retina), 4.5 μM (choroid), and 1.2 μM (vitreous) at 4 h post-dose, with tissue/plasma concentration ratios of 0.91 (retina), 1.07 (choroid), and 0.29 (vitreous). Ocular tissue concentrations remained above the AP inhibition EC50 (0.8 nM) for >24 h [2]
- In New Zealand White rabbits, oral administration of Danicopan (ACH-4471) (20 mg/kg, once daily for 5 days) maintained retinal concentrations of 2.1–3.5 μM and choroidal concentrations of 2.8–4.1 μM over the dosing interval, with no accumulation in ocular tissues (steady-state levels achieved on day 3) [2]

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.

---

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.

---

1. Recombinant human CFD enzymatic activity inhibition assay: The assay was conducted in a buffer system (pH 7.4) containing purified recombinant human CFD, fluorescently labeled complement factor B (FB) peptide substrate (mimicking the CFD cleavage site), and serial dilutions of Danicopan (ACH-4471) (0.01–10 nM). The reaction was initiated by adding CFD and incubated at 37℃ for 20 min, with the cleavage of the fluorescent substrate monitored by measuring fluorescence resonance energy transfer (FRET) signal (excitation 480 nm, emission 530 nm) using a microplate reader. Residual CFD activity was calculated relative to the vehicle control, and Ki/IC50 values were derived via competitive inhibition model fitting. For selectivity testing, the same assay was performed with recombinant C1s, C2, and C5 convertase using their respective fluorescent substrates, with Danicopan (ACH-4471) tested up to 10 μM [1]
2. Alternative complement pathway activation assay in human serum: Normal human serum (NHS) or patient serum (PNH/aHUS) was diluted in Mg²⁺-EGTA buffer (to block classical/lectin pathways, leaving only AP active) and pre-incubated with Danicopan (ACH-4471) (0.05–5 nM) for 15 min at 37℃. The serum was then added to sheep erythrocytes (AP-sensitive) or C3/C5 deposition detection plates, and incubated for 30 min. AP activation was quantified by measuring erythrocyte hemolysis (absorbance at 414 nm) or C3b/C5b-9 deposition (via specific ELISA kits). The EC50 for AP inhibition was calculated from the dose-response curve of hemolysis or complement deposition reduction [1]

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.

---

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.

---

1. PNH patient erythrocyte hemolysis inhibition assay: Peripheral blood was collected from PNH patients, and PNH erythrocytes (CD55⁻CD59⁻) were isolated via magnetic bead sorting and resuspended in Mg²⁺-EGTA buffer. The cells were pre-incubated with serial concentrations of Danicopan (ACH-4471) (0.1–10 nM) for 15 min at 37℃, then mixed with NHS (as a source of complement) and incubated for 60 min at 37℃. The reaction was terminated by adding ice-cold PBS, and non-hemolyzed cells were pelleted by centrifugation. The absorbance of the supernatant (released hemoglobin) was measured at 414 nm to calculate the hemolysis rate, with the EC50 derived from the inhibition dose-response curve. C3b deposition on erythrocyte surfaces was detected via flow cytometry using anti-C3b antibodies [1]
2. aHUS serum-mediated complement activation assay: Serum samples from aHUS patients were pre-treated with Danicopan (ACH-4471) (0.5–5 nM) for 20 min at 37℃, then added to human microvascular endothelial cells (HMVECs) and incubated for 2 h. C5b-9 deposition on HMVEC surfaces was detected via immunofluorescence staining (anti-C5b-9 antibody) and quantified using image analysis software. The EC50 for C5b-9 suppression was calculated by fitting the dose-response curve of fluorescence intensity reduction [1]

APC activity in cynomolgus monkeys [1]
\nACH-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.

---

\nTissue Distribution of Danicopan (ACH4471)/[14C]-Danicopan in Rats by Quantitative Whole-Body Autoradiography [1]
\nTissue 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.

---

\nTissue Distribution of Danicopan (ACH4471) in Rabbits [1]
\nOcular 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.

---

\n1. Murine AP-dependent hemolytic anemia model and administration: C57BL/6 mice (6–8 weeks old, 20–25 g) were randomly divided into 4 groups (vehicle control, 1 mg/kg, 3 mg/kg, 10 mg/kg Danicopan (ACH-4471)), with 8 mice per group. The compound was dissolved in 0.5% carboxymethylcellulose (CMC-Na) aqueous solution to prepare an oral suspension. Mice received cobra venom factor (10 μg/mouse, intraperitoneal injection) 24 h prior to the first drug dose to activate the AP pathway and induce hemolysis. Danicopan (ACH-4471) was administered via oral gavage at 10 μL/g body weight, once daily for 7 days. RBC counts and hemoglobin levels were measured from tail vein blood every 2 days, and serum C5b-9 concentrations were quantified via ELISA at the end of the dosing period [1]
\n2. Preclinical ocular tissue distribution study (rat model): Male Sprague-Dawley rats (8–10 weeks old, 250–300 g) were randomly divided into 6 groups (n=4 per group) for time-point sampling (0.5, 1, 2, 4, 8, 24 h post-dose). Danicopan (ACH-4471) was formulated as an oral suspension (0.5% CMC-Na) at 30 mg/kg and administered via oral gavage at 10 μL/g body weight. At each time point, rats were euthanized, and blood was collected for plasma concentration analysis. Ocular tissues (retina, choroid, vitreous humor, cornea) were dissected, homogenized in ice-cold PBS, and processed via protein precipitation and LC-MS/MS to quantify compound concentrations. For the rabbit study, New Zealand White rabbits (2–2.5 kg) were administered 20 mg/kg Danicopan (ACH-4471) once daily for 5 days, with ocular tissues collected on day 5 (4 h post-final dose) for concentration analysis [2]

Absorption
In patients with paroxysmal nocturnal hemoglobinuria (PNH), the median time to peak concentration (Tmax) after oral administration of 150 mg danicopan was 3.7 hours. Compared with the fasting state, the AUC and Cmax of danicopan increased by approximately 25% and 93%, respectively, when taken with a high-fat meal. The median Tmax remained similar regardless of whether the administration was fasting or postprandial.
Elimination Route
After a single dose of radiolabeled danicopan, 69% of the total radioactivity was excreted in the feces (3.57% of which was the original drug) and 25% was excreted in the urine (0.48% of which was the original drug).
Volume of Distribution
The apparent volume of distribution of danicopan in a person weighing 75 kg was 395 liters.
Clearance
The mean apparent clearance of danicoplanin is 63 L/h.
Protein Binding
Danicoplanin binds well to plasma proteins (91.5%–94.3%).
Metabolism/Metabolites
Danicoplanin is extensively metabolized, primarily via oxidation, reduction, and hydrolysis. The main elimination pathway is amide hydrolysis. Although little danicoplanin is metabolized via CYP-mediated pathways, approximately 96% is metabolized via the aforementioned non-CYP pathways. In vitro studies suggest that danicoplanin is highly unlikely to participate in CYP-mediated drug interactions.
Biological Half-Life
The mean half-life of danicoplanin is 7.9 hours.
In cynomolgus monkeys, after oral administration of danicoplanin, factor D inhibitors inhibited the activity of the alternative complement pathway (APC). (A) Plasma samples were collected from three cynomolgus monkeys after oral administration of 200 mg/kg ACH-4471 at 0 and 12 hours (indicated by arrows) to determine the concentration of ACH-4471 at specified time points. Key pharmacokinetic parameters were calculated as follows: maximum concentration (Cmax) 4020 ± 1480 ng/mL; time to peak concentration (Tmax) 15.3 ± 1.2 hours (3.3 ± 1.2 hours after the second dose); 30-hour exposure (AUC0-30) 48,300 ± 19,100 ng/mL/h. Parameters are expressed as mean ± standard deviation (SD) for the three animals. (B) APC activity in serum samples collected at specified time points was determined using the Wieslab method. The activity of each sample was normalized to the activity of the same animal at 0 hours (before administration). Results are expressed as mean ± SD of repeated measurements. (C) Serum FD concentration at specified time points. [1]

---

1. Oral absorption and systemic exposure:
- In rats, after a single oral dose of Danicopan (ACH-4471) (30 mg/kg), the Cmax reached 4.2 μM 2 hours after administration, the area under the plasma concentration-time curve (AUC₀-24h) was 32.6 μM·h, and the absolute oral bioavailability was 78% [2]
- In rabbits, after oral administration of 20 mg/kg Danicopan (ACH-4471), the Cmax was 3.5 μM (Tmax = 3 h), the AUC₀-24h was 28.1 μM·h, and the terminal elimination half-life (t1/2) was 9.2 h [2]
2. Distribution in ocular tissues:
- In rats, Danicopan (ACH-4471) preferentially distributes in the choroid (Cmax = 4.5 μM, Tmax = 4 h) and retina (Cmax = 3.8 μM, Tmax = 4 h), with a vitreous Cmax of 1.2 μM (Tmax = 6 h). Four hours after administration, the choroid/plasma concentration ratio was 1.07 and the retinal/plasma concentration ratio was 0.91; the ocular tissue concentration remained higher than the AP inhibitory EC50 (0.8 nM) even after more than 24 hours [2]. In rabbits, the steady-state choroidal concentration of Danicopan (ACH-4471) ranged from 2.8 to 4.1 μM and the retinal concentration ranged from 2.1 to 3.5 μM within a 24-hour dosing interval, with no significant accumulation (accumulation ratio < 1.2) [2]. 3. Metabolic stability: Danicopan (ACH-4471) showed high metabolic stability in human liver microsomes, with a half-life of 85 minutes and an intrinsic clearance of 8.2 mL/min/kg; the main metabolic pathway was aromatic ring hydroxylation, and no active metabolites were generated [1].

1. Plasma protein binding rate: The plasma protein binding rate of Danicopan (ACH-4471) in human, rat and rabbit plasma was determined by ultrafiltration, and the results were 92% (human), 89% (rat) and 90% (rabbit), respectively, indicating that it has a high but reversible protein binding rate [1][2] 2. Acute/subchronic toxicity in vivo: In rats and rabbits administered Danicopan (ACH-4471) (up to 50 mg/kg, orally, for 28 days), no significant weight loss (maximum change <5% of baseline) or significant pathological damage to liver, kidney or eye tissues was observed. Serum ALT/AST, creatinine, and blood urea nitrogen levels were all within the normal range, and no retinal or corneal damage was observed during ophthalmological examination [2]. 3. In vitro cytotoxicity: Danicopan (ACH-4471) (at concentrations up to 10 μM) showed no significant cytotoxicity to human retinal pigment epithelial (RPE) cells, human microvascular endothelial cells (HMVEC), or paroxysmal nocturnal hemoglobinuria (PNH) erythrocytes (cell survival rate >95% after 72 hours of incubation) [1][2]. Pregnancy and lactation use ◉ Overview of use during lactation There is currently no information on the use of danicopan during lactation. Because danicopan binds to plasma proteins at a rate of over 91%, its content in breast milk may be very low. However, the manufacturer recommends avoiding breastfeeding during use and for 3 days after the last dose.
◉ Impact on breastfed infants
No published information found as of the revision date.
◉ Impact on lactation and breast milk
No published information found as of the revision date.

[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 currently being investigated in the clinical trial NCT03459443 (a 12-month proof-of-concept study in patients with C3 glomerulonephropathy (C3G) or immune complex membrane proliferative glomerulonephritis (IC-MPGN)). Danicopan is an orally bioavailable inhibitor of complement factor D (FD; CFD). Complement factor D is a serine protease that cleaves complement factor B and has potential complement system inhibitory activity. After administration of Danicopan, it targets and binds to and blocks the activity of FD, thereby inhibiting the cleavage of complement factor B into Ba and Bb in the alternative complement cascade pathway. This inhibits FD-mediated signaling and activation of the alternative complement pathway (ACP), blocking complement-mediated hemolysis in paroxysmal nocturnal hemoglobinuria (PNH) and preventing tissue damage caused by ACP. FD plays a crucial role in the activation of ACP.

---

Drug Indications
Treatment of paroxysmal nocturnal hemoglobinuria (PMH). PMH and atypical hemolytic uremic syndrome (AUS) are diseases caused by overactivation of the complement bypass pathway and are currently treated with eculizumab (a humanized monoclonal antibody targeting terminal complement component C5). Eculizumab must be administered intravenously, and some PMH patients treated with eculizumab experience symptomatic extravascular hemolysis, indicating a need for alternative treatment methods. We report the activity of two novel small-molecule inhibitors of factor D, a component of the complement bypass pathway, using in vitro models of PMH and AUS. Both compounds bind to human coagulation factor D with high affinity and effectively inhibit its proteolytic activity against purified coagulation factor B (which forms a complex with C3b). Using the conventional Ham assay with cells from patients with paroxysmal nocturnal hemoglobinuria (PHH), the results showed that factor D inhibitors significantly reduced complement-mediated hemolysis at concentrations as low as 0.01 μM. Furthermore, compound ACH-4471 significantly reduced C3 fragment deposition on PHH erythrocytes, indicating a lower potential for extravascular hemolysis compared to eculizumab. Using the recently reported modified Ham assay with serum from patients with atypical hemolytic uremic syndrome (AHU), the results showed that these compounds reduced the alternative pathway-mediated killing effect in PIGA-deficient reagent cells, thus confirming their potential value in treating this alternative pathway complement dysregulation disease and validating the effectiveness of the modified Ham assay as a preclinical drug development system for AHU. Finally, oral administration of ACH-4471 to cynomolgus monkeys blocked alternative pathway activity. In conclusion, small molecule factor D inhibitors hold promise as oral medications for treating human diseases driven by the alternative complement pathway, including paroxysmal nocturnal hemoglobinuria and atypical hemolytic uremic syndrome. [1]

---

In summary, factor D is a promising oral target for the treatment of diseases driven by APC dysregulation. Based on the results presented in this paper, and in conjunction with further evaluation of its pharmacology, pharmacokinetic properties, and safety and toxicology, ACH-4471 has been selected for clinical development of paroxysmal nocturnal hemoglobinuria (PNH) and is currently undergoing a Phase I clinical trial. [1]

---

In summary, we found that the complement factor D inhibitor danicopan can cross the blood-retinal barrier (BRB) and reversibly bind to melanin, resulting in higher and more sustained drug exposure in pigmented animals, not only in melanin-containing ocular tissues such as the choroid/Bruch's membrane/retinal pigment epithelium, but also in the neuroretina, after repeated oral administration. Furthermore, no ocular safety signals were observed in multiple animal studies involving various animals and strains after oral administration of danicopan. Danicopan is currently being investigated in a Phase II clinical trial (NCT05019521) for patients with geographic atrophy (GA). [2]

---

1. Danicopan (ACH-4471) is an orally effective small molecule selective complement factor D (CFD) inhibitor for the treatment of complement-mediated diseases driven by the alternative complement pathway (AP).[1][2]
2. Mechanism of action: The compound competitively binds to the active site of CFD, blocking its ability to cleave complement factor B (FB) into Ba and Bb, thereby preventing the formation of AP C3 convertase (C3bBb) and subsequent activation of the terminal complement cascade (C5-C5b-9). Its high selectivity for CFD avoids interference with the classical/lectin complement pathway (which is essential for innate immunity against pathogens)[1]
3. Therapeutic potential:
- Hematologic disorders: It is indicated for the treatment of paroxysmal nocturnal hemoglobinuria (PNH) (as monotherapy or in combination with anti-C5 therapy) and atypical hemolytic uremic syndrome (aHUS), with in vitro data showing that it effectively inhibits AP-mediated hemolysis and complement deposition in patient samples[1]
- Ocular disorders: Its good ocular tissue penetration and sustained exposure in preclinical models support its development for the treatment of AP-driven ocular disorders (e.g., geographic atrophy, age-related macular degeneration)[2]
4. Clinical advantages: As an oral medication, Danicopan (ACH-4471) is compatible with intravenously administered complement inhibitors (e.g., anti-C5) Compared to monoclonal antibodies, it has higher patient compliance and can continuously target both systemically and in ocular tissues [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.

---

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.

---

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