Translational modelling to predict human pharmacokinetics and pharmacodynamics of a Bruton's tyrosine kinase-targeted protein degrader BGB-16673.

BACKGROUND AND PURPOSE: Bifunctional small molecule degraders, which link the target protein with E3 ubiquitin ligase, could lead to the efficient degradation of the target protein. BGB-16673 is a Bruton's tyrosine kinase (BTK) degrader. A translational PK/PD modelling approach was used to predict the human BTK degradation of BGB-16673 from preclinical in vitro and in vivo data. EXPERIMENTAL APPROACH: A simplified mechanistic PK/PD model was used to establish the correlation between the in vitro and in vivo BTK degradation by BGB-16673 in a mouse model. Human and mouse species differences were compared using the parameters generated from in vitro human or mouse blood, and human or mouse serum spiked TMD-8 cells. Human PD was then predicted using the simplified mechanistic PK/PD model. KEY RESULTS: BGB-16673 showed potent BTK degradation in mouse whole blood, human whole blood, and TMD-8 tumour cells in vitro. Furthermore, BGB-16673 showed BTK degradation in a murine TMD-8 xenograft model in vivo. The PK/PD model predicted human PD and the observed BTK degradation in clinical studies both showed robust BTK degradation in blood and tumour at clinical dose range. CONCLUSION AND IMPLICATIONS: The presented simplified mechanistic model with reduced number of model parameters is practically easier to be applied to research projects compared with the full mechanistic model. It can be used as a tool to better understand the PK/PD behaviour for targeted protein degraders and increase the confidence when moving to the clinical stage.

A highly selective PI3Kδ inhibitor BGB-10188 shows superior preclinical anti-tumor activities and decreased on-target side effects on colon.

PI3Kδ is a key signal transduction molecule in normal and malignant B cells, as well as in T-regulatory cells, making it a promising target for treatment of hematologic malignancies through both direct killing and anti-tumor immunity regulation. BGB-10188 is a highly selective inhibitor of PI3Kδ, showing more than 3000 folds selectivity over other PI3K isoforms and no significant inhibition across tested kinases. BGB-10188 potently inhibited PI3Kδ with IC50s ranging from 1.7-16 nM through various in vitro assays and showed a long-lasting and strong target inhibition in mouse B cells in vivo. BGB-10188 showed significant antitumor effects in human B cell lymphoma xenograft models as single agent or in combination with the BTK inhibitor zanubrutinib. BGB-10188 showed significant Treg inhibition in blood but not in colon, along with less drug accumulation in colon compared with idelalisib, which is an approved PI3Kdelta inhibitor with high incidence of gastrointestinal side effects in clinic. In summary, BGB-10188 is a novel PI3Kδ inhibitor with high selectivity, potency and improved safety profile shown in preclinical studies, which is showing the potential as a best-in-class PI3Kδ inhibitor.

Discovery of the Clinical Candidate Sonrotoclax (BGB-11417), a Highly Potent and Selective Inhibitor for Both WT and G101V Mutant Bcl-2.

The approval of venetoclax, a B-cell lymphoma-2 (Bcl-2) selective inhibitor, for the treatment of chronic lymphocytic leukemia demonstrated that the antiapoptotic protein Bcl-2 is a druggable target for B-cell malignancies. However, venetoclax's limited potency cannot produce a strong, durable clinical benefit in other Bcl-2-mediated malignancies (e.g., diffuse large B-cell lymphomas) and multiple recurrent Bcl-2 mutations (e.g., G101V) have been reported to mediate resistance to venetoclax after long-term treatment. Herein, we described novel Bcl-2 inhibitors with increased potency for both wild-type (WT) and mutant Bcl-2. Comprehensive structure optimization led to the clinical candidate BGB-11417 (compound 12e, sonrotoclax), which exhibits strong in vitro and in vivo inhibitory activity against both WT Bcl-2 and the G101V mutant, as well as excellent selectivity over Bcl-xL without obvious cytochrome P450 inhibition. Currently, BGB-11417 is undergoing phase II/III clinical assessments as monotherapy and combination treatment.

Discovery of BGB-8035, a Highly Selective Covalent Inhibitor of Bruton's Tyrosine Kinase for B-Cell Malignancies and Autoimmune Diseases.

Bruton's tyrosine kinase (BTK) plays an essential role in B-cell receptor (BCR)-mediated signaling as well as the downstream signaling pathway for Fc receptors (FcRs). Targeting BTK for B-cell malignancies by interfering with BCR signaling has been clinically validated by some covalent inhibitors, but suboptimal kinase selectivity may lead to some adverse effects, which also makes the clinical development of autoimmune disease therapy more challenging. The structure-activity relationship (SAR) starting from zanubrutinib (BGB-3111) leads to a series of highly selective BTK inhibitors, in which BGB-8035 is located in the ATP binding pocket and has similar hinge binding to ATP but exhibits high selectivity over other kinases (EGFR, Tec, etc.). With an excellent pharmacokinetic profile as well as demonstrated efficacy studies in oncology and autoimmune disease models, BGB-8035 has been declared a preclinical candidate. However, BGB-8035 showed an inferior toxicity profile compared to that of BGB-3111.

Determination of folic acid in milk, milk powder and energy drink by an indirect immunoassay.

BACKGROUND: Folic acid (FA) is essential for healthy people (reference daily intake 400 µg day⁻¹) and pregnant women (600 µg day⁻¹). Insufficient intake of FA will increase the risk of neural tube defects in newborns. In this study an indirect enzyme-linked immunosorbent assay was developed for rapid and convenient detection of FA in vitamin-fortified foods. RESULTS: A carbodiimide-modified active ester method was used to synthesise the immunogen (FA-bovine serum albumin (BSA) conjugate) to raise polyclonal antibodies for FA. The coupling ratio of FA with BSA was determined to be 14:1 (molar ratio). The detection limit of the immunoassay was 3.0 ng mL⁻¹ in buffer, 3.52 ng mL⁻¹ in energy drink, 11.91 ng mL⁻¹ in milk and 16.50 ng mL⁻¹ in milk powder. Intra- and inter-assay variability ranged from 6.6 to 15.1%. Analytical recoveries of FA-spiked samples were 88.3-108.9%. CONCLUSION: The immunoassay developed in this study can be used as a simple, rapid and accurate method for fast semi-quantitative and quantitative on-site analysis of FA in food products.

An experimental and theoretical study of pyrrole pyrolysis with tunable synchrotron VUV photoionization and molecular-beam mass spectrometry.

The pyrolysis of pyrrole (6.46% pyrrole in argon) has been performed with the tunable synchrotron vacuum ultraviolet (VUV) photoionization and molecular-beam mass spectrometry (MBMS) technique. The experiment was carried out over the temperature range of 1260-1710 K at a pressure of 267 Pa. About 30 intermediates have been identified by near-threshold measurements of photoionization mass spectra, and the corresponding mole fractions versus temperatures have been obtained. Moreover, the isomers of some pyrolysis products have been identified by the measurement of photoionization efficiency spectrum. The major products are H(2), C(2)H(2), HCN, C(3)H(4) (propyne), and C(2)H(3)N (acetonitrile). Meanwhile, some new intermediates, such as C(4)H(4)N (cyanoallyl radical) and C(2)H(2)N (cyanomethyl radical), have been determined. The major pyrolysis channels have been provided with the high-level ab initio G3B3 calculation and are well consistent with the experimental observation. The formation pathway of HCN via the cyclic carbene tautomer has been proved to be the lowest formation pathway, which is in accordance with previous theoretical work. The potential pathways of the early formed C(4)H(4)N species together with their subsequent consumption to C(2)H(2)N and C(2)H(2) have been discussed in detail. Also, the formation pathways of the major products of C(2)H(3)N and C(2)H(2) have been investigated as well.

Conformation-specific pathways of beta-alanine: a vacuum ultraviolet photoionization and theoretical study.

We report a photoionization and dissociative photoionization study of beta-alanine using IR laser desorption combined with synchrotron vacuum ultraviolet (VUV) photoionization mass spectrometry. Fragments at m/z = 45, 44, 43, and 30 yielded from photoionization are assigned to NH(3)CH(2)CH(2)(+), NH(2)CHCH(3)(+), NH(2)CHCH(2)(+), and NH(2)CH(2)(+), respectively. Some new conformation-specific dissociation channels and corresponding dissociation energies for the observed fragments are established and determined with the help of ab initio G3B3 calculations and measurements of photoionization efficiency (PIE) spectra. The theoretical values are in fair agreement with the experimental results. Three low-lying conformers of the beta-alanine cation, including two gauche conformers G1+, G2+ and one anti conformer A+ are investigated by G3B3 calculations. The conformer G1+ (intramolecular hydrogen bonding N-H...OC) is found to be another precursor in forming the NH(3)CH(2)CH(2)(+) ion, which is complementary to the previously reported formation pathway that only occurs with the conformer G2+ (intramolecular hydrogen bonding O-H...N). Species NH(2)CHCH(2)(+) may come from the contributions of G1+, G2+, and A+ via different dissociation pathways. The most abundant fragment ion, NH(2)CH(2)(+), is formed from a direct C-C bond cleavage. Intramolecular hydrogen transfer processes dominate most of the fragmentation pathways of the beta-alanine cation.

Photoionization studies on various quinones by an infrared laser desorption/tunable VUV photoionization TOF mass spectrometry.

Photoionization and dissociative photoionization characters of six quinones, including 1,2-naphthoquinone (1,2-NQ), 1,4-naphthoquinone (1,4-NQ), 9,10-phenanthroquinone (PQ), 9,10-anthraquinone (AQ), benz[a]- anthracene-7,12-dione (BAD) and 1,2-acenaphthylenedione (AND) have been studied with an infrared laser desorption/tunable synchrotron vacuum ultraviolet (VUV) photoionization mass spectrometry (IR LD/VUV PIMS) technique. Mass spectra of these compounds are obtained at different VUV photon energies. Consecutive losses of two carbon monoxide (CO) groups are found to be the main fragmentation pathways for all the quinones. Detailed dissociation processes are discussed with the help of ab initio B3LYP calculations. Ionization energies (IEs) of these quinones and appearance energies (AEs) of major fragments are obtained by measuring the photoionization efficiency (PIE) spectra. The experimental results are in good agreement with the theoretical data.

Pyrolysis of methyl tert-butyl ether (MTBE). 1. Experimental study with molecular-beam mass spectrometry and tunable synchrotron VUV photoionization.

An experimental study of methyl tert-butyl ether (MTBE) pyrolysis (3.72% MTBE in argon) has been performed at low pressure (267 Pa) within the temperature range from 700 to 1420 K. The pyrolysis process was detected with the tunable synchrotron vacuum ultraviolet (VUV) photoionization and molecular-beam mass spectrometry (MBMS). About thirty intermediates are identified from near-threshold measurements of photoionization mass spectrum and photoionization efficiency spectrum. Among them, H2, CO, CH4, CH3OH and C4H8 are the major pyrolysis products. The radicals such as methyl, methoxy, propargyl, allyl, C4H5 and C4H7 are detected. The isomers of pyrolysis products are identified as well, i.e., propyne and allene, 1,2,3-butatriene and vinylacetylene, isobutene and 1-butene, propanal and acetone. Furthermore, the mole fractions of the pyrolysis products have been evaluated under various temperatures. Meanwhile, the initial formation temperatures of different pyrolysis products can be obtained. This work is anticipated to present a new experimental method for pyrolysis study and help understand the pyrolysis and combustion chemistry of MTBE and other oxygenated fuels.

Pyrolysis of methyl tert-butyl ether (MTBE). 2. Theoretical study of decomposition pathways.

The thermal decomposition pathways of MTBE have been investigated using the G3B3 method. On the basis of the experimental observation and theoretical calculation, the pyrolysis channels are provided, especially for primary pyrolysis reactions. The primary decomposition pathways include formation of methanol and isobutene, CH4 elimination, H2 elimination and C-H, C-C, C-O bond cleavage reactions. Among them, the formation channel of methanol and isobutene is the lowest energy pathway, which is in accordance with experimental observation. Furthermore, the secondary pyrolysis pathways have been calculated as well, including decomposition of tert-butyl radical, isobutene, methanol and acetone. The radicals play an important role in the formation of pyrolysis products, for example, tert-butyl radical and allyl radical are major precursors for the formation of allene and propyne. Although some isomers (isobutene and 1-butene, allene and propyne, acetone and propanal) are identified in our experiment, these isomerization reaction pathways occur merely at the high temperature due to their high activation energies. The theoretical calculation can explain the experimental results reported in part 1 and shed further light on the thermal decomposition pathways.

Identification of combustion intermediates in low-pressure premixed pyridine/oxygen/argon flames.

Combustion intermediates of two low-pressure premixed pyridine/oxygen flames with respective equivalence ratios of 0.56 (C/O/N = 1:4.83:0.20) and 2.10 (C/O/N = 1:1.29:0.20) have been identified with tunable synchrotron vacuum ultraviolet (VUV) photoionization and molecular-beam mass spectrometry techniques. About 80 intermediates in the rich flame and 60 intermediates in the lean flame, including nitrogenous, oxygenated, and hydrocarbon intermediates, have been identified by measurements of photoionization mass spectra and photoionization efficiency spectra. Some radicals and new nitrogenous intermediates are identified in the present work. The experimental results are useful for studying the conversion of volatile nitrogen compounds and understanding the formation mechanism of NO(x) in flames of nitrogenous fuels.

Colorectal cancer screening for the natural population of Beijing with sequential fecal occult blood test: a multicenter study.

OBJECTIVE: To assess the prevalence of colorectal cancer (CRC) in Beijing and the reliability of the sequential fecal occult blood test (SFOBT) for CRC screening. METHODS: Of the natural population (48 100 persons) in several Beijing communities, we screened 26 827 persons with age over 30 using the SFOBT screening program, Guaiacum Fecal Occult Blood Test (GFOBT), Immuno Fecal Occult Blood Test (IFOBT), and colonoscopies. RESULTS: The screening rate of the population was 74%. The positive rate of SFOBT was 5.6%. The prevalence of CRC in the entire population of Beijing was therefore calculated to be 36.57/10(5). Of 12 CRC detected patients, 4 cases were in stage Dukes A (33.33%), 7 cases in stage Dukes B (58.33%), only 1 case (8.34%) in stage Dukes C. CONCLUSIONS: The prevalence of CRC in Beijing is one of the highest in China. Individuals at high risk for CRC or those over 50 years of age should be considered as primary candidates for screening. SFOBT screening is a cost-effective and reliable method for early detection of CRC.

Indirect competitive immunoassay for detection of vitamin B2 in foods and pharmaceuticals.

An indirect immunoassay for the determination of vitamin B2 in food samples and vitamin tablets was developed. A carbodiimide-modified active ester method was used to synthesize the immunogen for vitamin B2. The coupling ratio of vitamin B2 to carrier protein in immunogen was 19.98:1. The titer of the polyclonal antibody was 1:64000, and the antibody showed high specificity in the presence of vitamin B2 photolytic products and other B group vitamins. The immunoassay showed detection limits (LODs) of 1.07 ng/mL in PBS, 24.6 ng/g in vitamin drink, and 0.50 mg/kg in milk powder. Recovery was 99.58-110.91% in milk powder and 70.20-100.5% in vitamin drink. Vitamin B2 samples were analyzed by high-pressure liquid chromatography (HPLC) and the immunoassay, and results showed good agreement. Finally, this method was applied to detect vitamin B2 in commercial milk powder and vitamin tablets, and the detected amount correlated well with the labeled amount.

Development of a polyclonal antibody-based enzyme-linked immunosorbent assay (ELISA) for detection of Sunset Yellow FCF in food samples.

Sunset Yellow FCF is widely used as food additives to make foods more attractive. Due to its abuse and potential risk to human health, Sunset Yellow FCF is precisely limited to use in food. To monitor the illegal use of Sunset Yellow FCF, a polyclonal antibody-based indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) with satisfactory sensitivity and specificity was developed. A carboxyl group was introduced to Sunset Yellow FCF, then the modified hapten was coupled with carrier proteins to synthesize the immunogen and coating antigen. The IC(50) value of 0.52 ng mL(-1) and detection limit of 25 pg mL(-1) (in buffer) were achieved by this method. The cross-reactivity values of the antibodies with six structurally related colorants were less than 1.5%, indicating the high selectivity. Three kinds of food samples (beverage, dried beancurd, braised pork) and serum were chosen to evaluate the application of the immunoassay in real systems. The limits of detection (LOD) in the above three food samples were 0.12, 0.04 and 1.11, respectively (mean+3SD). The recovery (94%-106%), intra-assay (<5%) and inter-assay (<12%) coefficients of variation in foods and serum samples were also acceptable. The results suggest that this ELISA method is a specific, sensitive and simple method for the determination of Sunset Yellow FCF additives.

Establishment of magnetic beads-based enzyme immunoassay for detection of chloramphenicol in milk.

In this research, magnetic beads-based enzyme immunoassays were investigated for rapid analysis of chloramphenicol (CAP) in milk. To improve sensitivity of CAP determination, two kinds of immunomagnetic separation methods were designed and compared. Magnetic polystyrene microspheres were conjugated with anti-CAP antibody (Method I) or goat-anti-mouse IgG (Method II). The whole determination could be finished in 1.25 h. Both methods showed high sensitivity to CAP in buffer, and obtained an IC(50) value of 0.05 ng mL(-1) for Method I and 0.4 ng mL(-1) for Method II. The methods showed high specificity, only showing a little cross-reaction towards CAP succinate. The two methods were applied to detect CAP in milk. The recovery rates were 80-106% and the coefficients of variation (CVs) were 4.7-15%. The immunomagnetic assay showed promising potential in rapid screening field for CAP analysis. Between the two methods, Method I is more sensitive, and Method II is more suitable for producing a general assay by changing a primary antibody for another analyte.

Application of an enzyme immunoassay for the quantitative determination of azo dye (Orange II) in food products.

This paper reports the preparation of polyclonal antibodies against a synthetic azo dye, Orange II, and the development of an indirect ELISA to detect Orange II in foods. The sulfonic group of Orange II was modified and linked with carrier protein to synthesise an artificial antigen. Based on the checkerboard titration, the method showed excellent sensitivity (IC50 = 0.61 ng g⁻¹) to Orange II in the linear range of 0.05-10 ng g⁻¹. The antibody had little cross-reactivity with Chromotrope FB, Gardenia Yellow, Ponceau 4R, Sunset Yellow and Sudan dyes. The ELISA had limits of detection (LOD) of 0.22, 0.97 and 0.74 ng g⁻¹ in chilli powder, chilli oil and braised pork, respectively. The limits of quantification (LOQ) of the assay were 0.91 ng g⁻¹ in chilli powder, 1.48 ng g⁻¹ in chilli oil and 1.10 ng g⁻¹ in braised pork. For food products fortified with 1-10 ng g⁻¹ Orange II, the inter- and intra-assay variations were all less than 24.0% and 18.0%, respectively. Therefore, the proposed test could be used as a rapid screening method for Orange II detection in food samples.

Preparation of monoclonal antibody for melamine and development of an indirect competitive ELISA for melamine detection in raw milk, milk powder, and animal feeds.

Melamine (MEL) has been involved in several food recalls after the discovery of severe kidney damages in children and pets poisoned by melamine-adulterated food. To detect MEL residue in foods and animal feeds, an indirect competitive ELISA (cELISA) method was developed in this study based on preparation of monoclonal antibodies (MAbs) to MEL. The immunogen was prepared by linking MEL hapten with carrier protein via carbodiimide method. The method is applicable in the range of 5.0-135.0 microg L(-1) MEL in buffer solution, with an IC(50) value of 22.6 +/- 1.9 microg L(-1). The MAbs showed high specificity with low cross-reactivity (< or =1%) toward cyanurate, ammelide, and ammeline. The method was utilized in the detection of MEL in raw milk, milk powder, and animal feeds, with detection limits of 0.1 mg L(-1) for milk, 0.2 mg kg(-1) for milk powder, and 0.5 mg L(-1) for feeds. The recovery ratio was 79-110% for all matrices. The intra-assay and interassay coefficients of variation were <12.0 and <13.0%, respectively. Finally, the application of the cELISA in quantity evaluation of MEL in various feeds from local markets was evaluated and discussed.

Electrospray/VUV single-photon ionization mass spectrometry for the analysis of organic compounds.

For the comprehensive analysis of organic compounds, especially thermal labile and nonpolar compounds, an electrospray/vacuum ultraviolet (VUV) single-photon ionization (ES-SPI) method was developed. The fine droplets of the sample solution from the electrospray process were directed through a quartz capillary and two skimmers to form a molecular beam into a high vacuum ionization chamber. The neutral sample molecules were softly ionized with tunable VUV light and analyzed with a reflection time-of-flight mass spectrometer (RTOF-MS). The ionization energy (IE) and appearance onsets of fragments were obtained based on the photoionization efficiency (PIE) spectrum. The isomers can also be distinguished. With this new method, clean (fragment-free) mass spectra of nonpolar compounds, such as benzene, cyclohexane, and some thermal labile solid compounds (triphenylamine, thioacetamide, and urea) have been obtained without any tedious pretreatment. The components of complex mixtures (gasoline and kerosene) can be identified. Furthermore, quantitative analysis of the components can be obtained based on photoionization cross section data. This method may be used for quantitative analysis of small biomolecules and natural products.

Intramolecular hydrogen transfer in the ionization process of alpha-alanine.

Photoionization and dissociative photoionization of alpha-alanine have been studied with infrared laser desorption/tunable synchrotron vacuum ultraviolet (VUV) photoionization mass spectrometry (IR LD/VUV PIMS) and theoretical calculations. By scanning photoionization efficiency (PIE) spectra, appearance energies (AEs) of the fragments CH(3)CHNH(3)(+) (m/z = 45, doublet state) and CH(3)CH = NH(2)(+) (m/z = 44, singlet state) are measured to be 9.53 and 9.21 +/- 0.05 eV, respectively, which are consistent with the theoretical values of 9.61 and 9.37 eV with ab initio G3B3 calculations. Formation of the CH(3)CHNH(3)(+) ion produced via intramolecular hydrogen transfer and subsequent decarboxylation processes is competitive to that of the CH(3)CH[double bond, length as m-dash]NH(2)(+) ion derived from a direct C-C(O) bond cleavage. The photoionization mass spectrum obtained at the photon energy of 10.0 eV shows that the intensity of CH(3)CH[double bond, length as m-dash]NH(2)(+) is much stronger than that of the CH(3)CHNH(3)(+) fragment. The formation of the CH(3)CHNH(3)(+) and CH(3)CH[double bond, length as m-dash]NH(2)(+) ions is thought to be thermodynamically and kinetically favorable, respectively. Detailed formation pathways for the two cations have been proposed by using ab initio calculations. The calculated results provide a clear picture of the photoionization and dissociative photoionization processes of the alpha-alanine cation.

The characterization of selected drugs with infrared laser desorption/tunable synchrotron vacuum ultraviolet photoionization mass spectrometry.

Some selected drugs including captopril, fudosteine and racecadotril have been analyzed by infrared (IR) laser desorption/tunable synchrotron vacuum ultraviolet (VUV) photoionization mass spectrometry (PIMS). The molecular ions of captopril and racecadotril are exclusively observed without any fragments at near threshold single-photon ionization (SPI). However, fudosteine easily forms fragments even at a photon energy near the ionization threshold, indicating the instability of its molecular ion. For these drugs, a number of fragments are yielded with the increase of photon energy. The structures of such fragments proposed by IR LD/VUV PIMS are supported by electron ionization time-of-flight mass spectrometry (EI-TOFMS) results. Fragmentation pathways are discussed in detail.