Venetoclax

Materials. Two venetoclax batches were purchased from Kemprotec Ltd. (UK) (batch 1: #1705150, batch 2: #180620). One further venetoclax batch was ordered from Sigma-Aldrich (Ireland) (batch 3: #11777). Olive oil, highly refined and low acidity, capric acid, L-α-phosphatidylcholine type XI-E (PC) (768 g/mol), taurodeoxycholic acid (NaTDC) and pancreatic lipase (8 × USP) were ordered from Sigma-Aldrich (Ireland). Capmul MCM^® and Captex^® 1000 were kindly donated by Abitec corporation. A sample of Peceol^® was kindly provided by Gattefossé (France) and SIF powder version 1 was kindly donated by biorelevant.com (UK). All other chemicals and solvents were of analytical or high-performance liquid chromatography (HPLC) grade and were purchased from Sigma-Aldrich (Ireland) and used as received.
Differential scanning calorimetry (DSC). The thermal behaviour of venetoclax was studied using a TA Q1000 with a TA Refrigerated Cooling System 90 (TA Instruments, New Castle, DE, USA). The cell was purged with nitrogen at 50 mL/min. The melting temperature was measured by modulated DSC. Venetoclax (5 mg) was weighed into a T-zero pan and heated from 20 to 200 °C at 3 °C/min. The modulation amplitude was set to ± 1.0 °C and the modulation time to 60 sec.
The crystallisation behaviour of venetoclax was studied using the protocol by Baird et al. [21 (link)]. In brief, 2 mg venetoclax was weighed into a T-zero pan and heated to 165 °C at 10 °C/min, held isothermally for 3 min, cooled to −75 °C at 20 °C/min and reheated to 160 °C at 10 °C/min. As no crystallisation was observed during cooling and reheating, the heating rate was lowered to 2 °C/min. The experiments were run in triplicates. The glass-forming ability of drugs was categorised according to Baird et al. into class I (crystallisation during cooling prior to the glass transition temperature (T_g)), class II (no crystallisation during cooling, but crystallisation was observed upon reheating above T_g) and class III (no crystallisation observed during cooling nor reheating to its melting point) [21 (link)]. Additionally, the absence of crystals after heating and upon cooling was confirmed by hot-stage microscopy.
Hot-stage microscopy. Samples were placed in a glass crucible onto a Linkam THMS 600 hot stage connected to a TP94 temperature controller (Linkam Scientific Instruments, Tadworth, UK). Samples were heated to 165 °C at 10 °C/min, held isothermally for 3 min, cooled to 25 °C at 20 °C/min and continuously monitored using an Olympus BX51 with an Olympus SC100 camera operated by Olympus Stream essentials 2.3.3. Images were collected at 40× magnification and the light was polarised using the polariser U-POT and analysed with the analyser U-ANT. The absence of crystals was assumed, if no birefringence was observed. The images are presented in the Supplementary Information (Figure S2).
Cross-polarised light microscopy. The absence of crystals in the supersaturated solution was confirmed by means of cross-polarised light microscopy using an Olympus BX51 with an Olympus SC100 camera operated by Olympus Stream essentials 2.3.3. The light was polarised using the polariser U-POT and analysed with the analyser U-ANT. The absence of crystals was assumed, if no birefringence was observed.
Solubility studies. The solubility was determined in olive oil, Captex^® 1000, Peceol^® and Capmul MCM^®. Excess of venetoclax was added to 2 mL of the excipients and stirred at 200 rpm (25% power) (MIXdrive 15 HT, 2Mag AG, Germany) at 37 °C. Samples were taken after 24, 48, and 72 h. In the case of Peceol^® and Capmul MCM^®, a high amount of venetoclax dissolved instantly upon addition to the excipient (exceeding the thermodynamic solubility), which lead to precipitation after 24–48 h. Therefore, Peceol^® and Capmul MCM^® samples were left at 37 °C up to 48 h before the first sample was taken. All samples were centrifuged at 21,380× g (Mikro 200 R, Hettich GmbH, Germany) and 37 °C for 15 min. The supernatant was transferred to a new sample tube and centrifuged again under identical conditions. To solubilise the oily excipient, the supernatant was diluted in acetonitrile, ethyl acetate (1:3, v/v), followed by further 1:10 (v/v) dilution with acetonitrile:ethyl acetate (3:1, v/v). The obtained samples were diluted appropriately with mobile phase before analysis by reverse phase HPLC as described below. All samples were run in triplicates.
Biorelevant solubility. Fasted state-simulated intestinal fluid (FaSSIF) and fed state-simulated intestinal fluid (FeSSIF) were prepared according to the instructions by biorelevant.com. Solutions of FeSSIF were prepared and used directly, whereas FaSSIF was left at room temperature for 2 h prior to usage. Excess of venetoclax was added to 2 mL FaSSIF and FeSSIF, respectively. The suspensions were placed in a water bath shaker with 200 shakes/min at 37 °C (GLS400, Grant Instruments, UK). Samples were taken at 3, 6 and 24 h and centrifuged at 21,380× g (Mikro 200 R, Hettich GmbH, Germany) for 15 min at 37 °C. The supernatant was transferred to a new sample tube and centrifuged again under identical conditions. Samples were diluted 1:10 (v/v) with mobile phase before analysis.
The samples were analysed using an Agilent 1200 series HPLC system (Agilent Technology Inc., Santa Clara, CA, US) that comprised a binary pump, degasser, column oven, autosampler and variable wavelength detector. Data analysis was performed with EZChrom Elite^® version 3.2. Venetoclax was separated from the sample matrix with a Zorbax^® Eclipse Plus-C18 column (5 μm, 4.6 mm × 150 mm) including a Zorbax^® Eclipse Plus-C18 guard column (5 μm, 4.6 mm × 12.5 mm) at 40 °C. The mobile phase consisted of (a) acetonitrile with 0.5% (v/v) trifluoroacetic acid (TFA) and (b) water with 0.5% (v/v) TFA at a ratio of 53:47 (a:b, v/v) and was used at a flow rate of 1 mL/min. The injection volume was 20 μL and the detection wavelength was set to 316 nm. The limit of detection (LOD) was 20 ng/mL and the limit of quantification (LOQ) was 65 ng/mL, determined using the standard error of y-intercept according to the International Council for Harmonisation (ICH) Q2 guideline [22 ].
In vitro lipolysis: drug solubilisation during formulation dispersion and digestion. In vitro lipolysis was performed using a pH-stat apparatus (Metrohm AG, Herisau, Switzerland), comprising a Titrando^® 907 stirrer, 804 Ti-stand, a pH electrode (Metrohm AG, Herisau, Switzerland) and two 800 Dosino^® dosing units coupled to a 20 mL automatic burette. The system was operated by the Tiamo^® 2.2 software. The in vitro protocol was amended from Williams et al. [23 (link),24 (link)] as reported previously [25 (link)]. In brief, the buffer contained 2 mM TRIS maleate, 150 mM NaCl, and 1.4 mM CaCl₂ 2H₂O, adjusted to pH 6.5. For the digestion experiments, the buffer was supplemented with 3 mM NaTDC and 0.75 mM PC (digestion buffer) and stirred for 12 h before further usage. The pancreatin extract was prepared freshly by adding 5 mL of 5 °C digestion buffer to 1 g of porcine pancreatic enzymes (8x USP), which was vortexed thoroughly. The mixture was centrifuged for 15 min at 5 °C, 2800× g (Rotina 380 R, Hettich GmbH, Germany) and 4 mL of supernatant was recovered and stored on ice before further usage.
For the in vitro lipolysis experiment 1.583 g of lipid formulation was dispersed into 57 mL of digestion buffer for 10 min. Three 1 mL samples were taken at 2.5, 5 and 10 min from the middle of the vessel. pH of the media was adjusted and maintained at 6.5 using 0.2 M NaOH. To the remaining 54 mL (1.5 g lipid formulation) dispersion 6 mL of pancreatin extract was added to initialise digestion. After 60 min the released non-ionised free fatty acids (FFAs) were determined by a pH increase in the buffer to pH 9. An additional blank titration using the digestion buffer was performed and the released mmol of FFAs from the blank was subtracted from the mmol of FFAs released from the surfactant formulations
Samples of 4.9 mL were taken at 5, 10, 15, 30, 45 and 60 min during the digestion experiment from the middle of the vessel. In each sample and after 60 min the enzymes were inhibited by the addition of 1 M 4- Bromophenylboronic acid in methanol (5 μL per mL sample). All samples containing a lipid phase were centrifuged at 37 °C and 400,000× g for 30 min (Beckman Coulter Optima L-90K, Rotor: VTI 65.2). Samples, that did not contain a lipid phase (aqueous suspension) were centrifuged at 37 °C and 21,000× g for 30 min using a benchtop centrifuge (Micro 200 R, Hettich GmbH, Germany). The lipid phase was dissolved in a mixture of ethyl acetate and acetonitrile (3:1 v/v) and diluted with 1:10 (v/v) with a mixture of ethyl acetate and acetonitrile (1:3 v/v). The solid phase was added to 2 mL of a mixture of ethyl acetate and acetonitrile (3:7 v/v) and ultrasonicated for 10 min. The aqueous phase was diluted 1:10 (v/v) with mobile phase. The resulting lipid, solid and aqueous phase samples were subsequently centrifuged at 37 °C and 21,000× g for 30 min. The resulting supernatants were diluted further with mobile phase and analysed by HPLC as stated above.
The digestibility of Peceol^® was calculated as previously described [26 ] using the theoretical released FFAs per g of Peceol^® utilising the saponification value (166 mg) on the certificate of analysis: $\mathrm{Theoretical} \mathrm{FFAs} \left[\mathrm{mmol}\right]=\frac{\mathrm{SV} \left[\mathrm{mg}\right]}{56.1056 \left[\frac{\mathrm{g}}{\mathrm{mol}}\right]},$
where theoretical FFAs are the maximal amount of FFAs that can theoretically be released from Peceol^® in mmol per g of excipient, SV the saponification value in mg KOH per g of excipient and 56.1056 g/mol the molecular weight of KOH. The maximal absolute amount of theoretically released FFAs can be calculated by multiplying by the amount of excipient used (i.e., 1.5 g in this study). The % digested can be calculated as follows: $\% \mathrm{digested}=\frac{\mathrm{Released} \mathrm{FFAs} \left[\mathrm{mmol}\right]}{\mathrm{Theoretical} \mathrm{FFAs} \left[\mathrm{mmol}\right]}\times 100\%,$
where the released FFAs provide the total amount that is released at the end of the digestion experiment including the non-ionised FFAs determined by raising the pH to 9.0 and the theoretical FFAs were calculated using Equation (1).
Formulation development of supersaturated LBFs. The increase in venetoclax concentration above its solubility in the excipients was achieved by incorporating venetoclax at an elevated temperature, i.e., the formulations were heated, kept at elevated (isothermal) temperature and cooled to room temperature. In detail, excess venetoclax was added to Peceol^®, olive oil, Capmul MCM^®, Capex^® 1000, respectively, and suspended at 600 rpm (Stuart CD 162 heat-stir, Cole-Parmer, UK). The vial was sealed with parafilm and a continuous nitrogen stream was purged through the vial to prevent any oxidation during the heating and cooling process. While continuously stirring at 600 rpm, the suspensions were heated to 70 °C and kept at 70 °C for 10 min (Stuart CD 162 heat-stir, Cole-Parmer, UK) followed by cooling to 23 ± 2 °C by elevating the vial from the hot plate. Once the mixtures reached a temperature below 25 °C, a 0.5 mL sample was taken, and the remaining mixture was heated and cooled again using the same protocol described above. After this second cycle, a second 0.5 mL sample was taken, and the mixture was heated and cooled a third time using the same conditions as described above followed by a final 0.5 mL sample. During all heating and cooling cycles, excess venetoclax was present to investigate the maximum achievable concentration with this protocol. The samples were centrifuged at 21,380× g and 37 °C (Mikro 200 R, Hettich GmbH, Germany) for 15 min. The supernatant was transferred to a new sample tube and centrifuged again using identical conditions. The supernatant was used for HPLC analysis using the same dilutions and solvents as described for the solubility studies in the lipid excipients above. The remaining suspension after the third heating cycle was centrifuged under identical conditions as stated above and transferred to a new sample tube for stability testing and stored at room temperature. The stability of promising formulations was evaluated using polarised light (Allen Viewer Type LV28, P.W. Allen & Co Ltd., Tewkesbury, UK). Due to the high drug amount needed, these formulations and stability tests were conducted once.
Formulations for in vivo and in vitro studies. For the in vivo used powder venetoclax formulation, 100 mg venetoclax were weighed into hard gelatine capsules of size 00. For the in vivo used Peceol^® suspension, 50 mg venetoclax was weight into hard gelatine capsules of size 000 and 1 mL of Peceol^® (melted at 50 °C and cooled to 37 °C) was added. Venetoclax was dispersed by vigorous shaking.
In the in vitro experiments, the powder formulation was resembled by an aqueous suspension. The aqueous suspension was prepared by adding 50 mg venetoclax to 1 mL water followed by ultrasonication. The suspensions were stirred constantly to prevent sedimentation before usage. The Peceol^® suspension was prepared by melting Peceol^® at 50 °C and cooling it to 37 °C before adding 50 mg venetoclax to 1 mL Peceol^® followed by an over-night stir.
The supersaturated lipid solution was prepared by adding 300 mg venetoclax to 6 mL Peceol^® (50 mg/mL). The mixture was stirred at 600 rpm (Stuart CD 162 heat-stir, Cole-Parmer, UK) and sealed with parafilm. A continuous nitrogen stream into the vial removed oxygen throughout the manufacturing process. After suspending the drug particles, the obtained suspension was slowly heated to 70 °C (Stuart CD 162 heat-stir, Cole-Parmer, UK). The mixture was kept at 70 °C for 10 min and cooled to 25 °C while continuously stirring at 600 rpm. Subsequently, the mixture was heated a second time under the same conditions as stated above and cooled to room temperature. The absence of crystals was confirmed using cross-polarised light microscopy. For the in vivo study, sLBF was administered in hard gelatine capsules of size 000 (1 mL per capsule).
In vivo study. All experiments were approved and conducted with licences issued by the Health Product Regulatory Authority, Ireland (project licence AE19130/P058) as directed by the EU Statutory instruments of the EU directive 2010/63/EU (Protection of Animals used for Scientific Purposes). Local ethical approval was granted by University College Cork Animal Experimentation Ethics Committee (AEEC). A randomised, three-way cross-over study in 3 male landrace pigs (15–17 kg) was conducted and each pig received a single dose of 100 mg venetoclax. Pigs were fed approximately 175 g of standard weanling pig pellet feed twice daily. In the fasted study legs, the final feed of 175 g was given 24 h prior to dosing. As part of the study design, any remaining food was removed 16 h before dosing. However, no food remained at this point in any of the groups. On day 1 of the experiment, an intravenous catheter was inserted from the ear vein into the jugular vein under general anaesthesia, which was used for blood sampling throughout the study. On day 3, following an overnight fast of 16 h, pigs were administered either a reference capsule with venetoclax powder or two capsules of either venetoclax Peceol^® suspension or a venetoclax supersaturated lipid solution (sLBF) with the aid of a dosing gun followed by 50 mL of water via a syringe. In order to control the water intake with the dosage forms, the water availability was restricted for 3 h post-dosing. At all other times, water was available ad libitum. To facilitate handling during the oral administration, an intramuscular dose of ketamine (5 mg/kg) and xylazine (1 mg/kg) was administered in both studies. Blood samples were collected after 0.5, 1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, and 24 h in heparinised tubes. Upon collection, blood samples were immediately centrifuged at 3000× g, 4 °C for 5.5 min (Eppendorf 5702 R, Rotor A-4-38, Eppendorf Ltd., Stevenage, UK). The supernatant plasma was harvested and stored at −20 °C until further analysis. A 6 day washout period was maintained between the study legs. Due to a rupture and spillage of one sLBF capsule during oral dosing to one of the pigs, this experimental unit was excluded, resulting in n = 2 for the sLBF study group.
Bioanalysis. The plasma concentrations of venetoclax were determined by reversed phase HPLC. The Agilent 1260 series HPLC system (Agilent Technology Inc., Santa Clara, CA, US) comprised a binary pump, degasser, temperature controlled autosampler, column oven and diode array detector. The system was operated, and the data analysed with EZChrom Elite^® version 3.3.2. A Zorbax^® Eclipse Plus-C18 column (5 μm, 4.6 mm × 150 mm) with a Zorbax^® Eclipse Plus-C18 guard column (5 μm, 4.6 mm × 12.5 mm) was used for the separation of venetoclax. The mobile phase consisted of water and acetonitrile with 0.5% (v/v) TFA at a ratio of 47:53 (v/v) and was used at a flow rate of 1.0 mL/min. The sample and column temperature were set at 5 °C and 40 °C, respectively, and the detection wavelength was set to 250, 290 and 316 nm. Venetoclax was extracted from the plasma samples by liquid-liquid extraction. To 500 μL of pig plasma, 50 μL vemurafenib (internal standard dissolved in acetonitrile) and 450 μL acetonitrile was added. The mixture was mixed thoroughly, and 1 mL of ethyl acetate was added followed by mixing for 15 s. The mixture was centrifuged at 25 °C, 11,500× g for 5 min (Mikro 200 R, Andreas Hettich GmbH & Co. KG, Germany). The supernatant (1.5 mL) was recovered and transferred to a new sample tube. Subsequently, the supernatant was dried at 60 °C under a nitrogen stream. To the remaining plasma sample, 1 mL of ethyl acetate was added and the mixture was thoroughly mixed and centrifuged using the same condition as above. The supernatant was recovered and transferred to the same sample tube that contained the supernatant of the plasma sample after the first centrifugation (which was dried as described above). After drying of the supernatant, the residues were reconstituted in 100 μL mobile phase (excluding TFA), followed by centrifugation at 25 °C, 11,500× g for 5 min (Mikro 200 R, Andreas Hettich GmbH & Co. KG, Germany). The injection volume used for HPLC analysis of the supernatant was 50 μL. The LOD and LOQ in plasma by this method were 6 and 20 ng/mL, respectively, determined using the standard error of y-intercept according to ICH Q2 guideline [22 ]. Linearity was confirmed between 25 and 2.5 μg/mL.
Data analysis. A one-way analysis of variance (one-way ANOVA) was performed for the lipolysis data using Tukey’s post-hoc test to compare the different formulation performances and Bartlett’s test to check for equal variance. The pharmacokinetic parameters were calculated using Gastro Plus^® version 9.5 (Simulations Plus Inc., Lancaster, CA, US) and in-house intravenous data. The plasma concentration profiles were analysed by non-compartmental analysis.
The statistical analysis for all in vivo parameters was performed using a one-way ANOVA after using Bartlett’s test to check for equal variance. The pairwise comparison of the groups was based on Tukey’s multiple range test. All statistical analyses were calculated using GraphPad Prism^® 5 (GraphPad Software Inc., San Diego, CA, US).

CD34⁺ blasts cultured in R20 media were stimulated with 10 ng/ml of human recombinant IFN-γ. Unstimulated (PBS) blasts were included for comparison. After 12 h of culture, blasts with or without IFN-γ stimulation were counted and plated in a U-bottom 96-well plate (5 × 10⁴ per well), with or without 10 ng/ml of human recombinant IFN-γ, along with titrated concentrations of venetoclax [Cat# S8048, Selleck Chemicals]. After 24 h of incubation, cell viability was assessed using the CellTiter-Glo luminescent cell viability assay [Cat# G7572, Promega] following the manufacturer’s protocol. IC50 values were calculated using GraphPad Prism version 10.1.0.