Research on metabolites in drug discovery and development | Hongren Pharma Share

1. Screening and optimization stage

• Identify metabolic soft spots.

• Screening for reactive metabolites.

2. Preclinical research stage

• The in vitro metabolic differences among various genera were analyzed, and the related animal species were selected for toxicological tests at the early stage of development.

• The metabolites in animal body were analyzed, and the metabolites in human body were predicted by the results of in vitro metabolic test.

3. Phase I clinical stage

• To conduct research on human metabolites and find the differences between human and animal metabolites. To evaluate specific or non-proportional metabolites in humans prior to large-scale clinical trials to predict and reduce unintended safety risks in subjects.

• Compare the metabolite profiles in animal and human plasma samples to determine initially whether the metabolites in animals are sufficient to cover the metabolites in humans.

4. End of Phase II clinical trial and/or pre-phase III clinical trial

• The material balance of rodent radiolabelling was studied. During Phase I clinical trials, researchers may also quantify human metabolites using reaction factors for radiolabelated reactions of metabolites shared by animals and humans.

• Human absorption, metabolism and excretion experiments of radioisotope tracers were carried out.

• Analysis of high proportion of metabolites/human specific metabolites; The exposure of metabolites in the animal subject to toxicology test could not cover the exposure of human metabolites; Complete corresponding non-clinical studies.

Dose group selection for clinical trials - SAD phase

• Within the range of linear PK, doses close to the clinical intended use were selected.

• If non-clinical findings suggest species differences in metabolites or a high proportion of metabolites in people of concern, early in human metabolic evaluation is recommended.

Dose group selection for clinical trials - MAD phase

• If metabolite identification is not performed at SAD stage, it can be performed at this stage. If it has been carried out in the SAD stage, it can also be chosen to continue to study the metabolites in the steady-state condition in the MAD stage.

• Human plasma samples that enter the homeostasis phase after multiple dosing with a high proportion of metabolites (relative content exceeding 10% of drug-related substances) are compared with exposure to toxicological species plasma after multiple dosing.

In vivo sample mixing protocol - Blood sample

• AUC pool (Mixed tube) : This protocol is preferred when all subjects in a dose group are combined according to AUC rules for qualitative and relatively quantitative studies.

• AUC pool (single tube) : each subject's sample is mixed separately for testing, and the blank sample is tested separately for each subject. Provide information on differences in metabolites between individual subjects.

• Near Cmax: In special cases, such as low exposure to the drug in the body, mixing can be done according to AUC rules (the amount of tail point is too much, which is equivalent to the sample being diluted); When the optimization of sample treatment and detection conditions could not satisfy the metabolite detection, the mixing scheme was chosen.

In vivo sample mixing protocol - urine and stool (bile) samples

• Mix according to equal ratio or weight ratio. Multiple subjects can be mixed or each subject can be mixed separately to obtain metabolite and metabolite profile information in urine and stool.

Dr. Cheng Zhongzhe of Hongren Biopharmaceutical combined with multiple cases of Hongren experimental platform to share further technical details. For example, research on non-clinical and clinical metabolites of innovative drug A, construction and verification of intelligent MS data processing program - midazolam, application of intelligent MS data processing program - boxyline, intelligent MS Application of data processing programs - Goserrelin, oligonucleotide drug metabolites - Formivir, radioisotopically labeled metabolites, chiral drug pharmacokinetic metabolites, etc. Interested experts and teachers are welcome to visit Hongren for exchange and discussion.

In the non-clinical research stage of innovative drug A, no high proportion of metabolites was found in vivo or in vitro studies, and FIH only tested the prototype. Human metabolites were studied in the SAD study phase, and a high proportion of metabolite M2 (>10%) and another high proportion of metabolite M1 were detected. Analysis of metabolic sites and guidance of compound synthesis.

The two metabolites were further confirmed by clinical sample detection. The prototype drug, M1 and M2 were detected simultaneously in the follow-up clinical trial dose group. At the same time, the concomitant toxicokinetics were studied in animal species for safety evaluation. The exposure levels of major metabolites in clinical and safety assessment animals were comprehensively compared to provide support for the safety evaluation of metabolites.

The fragmentation law of triterpenoid alkaloids was studied by multistage mass spectrometry, and four characteristic ions were found. The application of these characteristic ions in metabolite screening and structural analysis helps to quickly find metabolites and determine the metabolic sites occurring in the AB/CD ring.

Polypeptide drugs have a multi-charge distribution in the mass spectrum, and the response intensity is dispersed, which adds a challenge to the discovery of metabolites. At the same time, polypeptide drugs have problems such as low exposure in vivo, poor reverse-phase chromatography retention of prototype and metabolites (enhanced polarity, hydrolysis to smaller structural fragments).

Polypeptide drug B was administered subcutaneously with a peak plasma concentration of less than 30 ng/ml. The chemical derivatization method can improve the detection sensitivity and enhance the retention of reversed-phase chromatography column. At the same time, the derivatization products produce characteristic ions in the mass spectrum fragments, and the characteristic ion filtration and neutral loss can be used to quickly screen the metabolites.

Oligonucleotide drug molecules have a variety of characteristics, such as acidity, strong polarity, low ionization efficiency, multi-charge distribution, and easy to produce a large number of metal adduction ions, which bring many challenges to the study of oligonucleotide drug metabolites. Based on the rich experience in quantitative analysis of biological samples of oligonucleotides and the assistance of data processing platform BioPharma Finder, the Macro and Tenacity technical team identified 19 metabolites of Formivir (ASO).

The innovative drug C is a chiral compound, and there may be R,S- configuration transformation in vivo. The protodrug enantiomers and their metabolites were detected in human plasma samples by chiral chromatography.

After conducting radiopharmacokinetics and material balance studies on [¹⁴C] HR001, the Hongren team further studied its metabolites and radiometabolites profiles in rats. Thirty-one metabolites were identified by LC-HR-MS and detected with an online radioactive HPLC flow detector. The relative proportion of prototype drugs and their metabolites was calculated according to the peak area.