Small nucleic acid drug non-clinical DMPK research concerns and examples | Suzhou T20 Macro and tough theme report sharing

Non-clinical evaluation Forum

Dr. Jiang Hongliang, founder and Chief Scientist of Hongren Biopharmaceutical

In the keynote speech, Dr. Jiang, combined with the rich project experience accumulated by Hongren Pharmaceutical, introduced in detail the difficulties and concerns of non-clinical DMPK research on small nucleic acid drugs, gave detailed professional insights and looked forward to the future research development direction.

The main biological analysis methods of small nucleic acid drugs include chromatograph-based detection methods, PCR and hybrid ELISA.

1. Biological analysis of nucleic acid drugs based on chromatography

HPLC based separation technology analysis methods (HPLC and HPLC-MS) and hybridization-based high performance liquid chromatography fluorescence method are carried out with reference to the requirements of chromatographic analysis method validation.

1. Hybridization-based HPLC-fluorescence

1) Main features:

• Sensitivity (up to 1ng/mL)

• Wide detection range (1000x)

• Good specificity

• No sample processing required (hybridization with fluorescent probes of specific nucleic acid sequences)

• High accuracy + good repeatability (±15%)

• Not susceptible to nucleic acid modification

• Long injection time (20-30 min)

• Chromatographic separation of nucleic acid drug and its metabolites, internal standard

2) Difficulties/Key points of nucleic acid drug analysis:

• Analysis of single strand nucleic acid and design of single strand probe directly

• To analyze one strand of double-stranded nucleic acid, in addition to designing a fluorescent probe for the target analysis strand, it is also necessary to design a termination probe for the other strand.

• Both single-stranded and double-stranded nucleic acids need to be designed to quenched the excess probe anti-probe

2, HPLC-HRMS (Liquid Chromatography-High resolution mass spectrometry)

1) Main features:

• High resolution, accurate determination of molecular mass (deviation less than 5ppm)

• When the resolution is greater than 35,000, the isotopic overlap effect of each charge state ion can be eliminated

• Good selectivity

• Good repeatability and reproducibility

• The original drug and its metabolites were detected

• m- signal of ions [M-MH] in different charge states of analyte

2) Difficulties/Key points of nucleic acid drug analysis:

• Device resolution is limited by the duty cycle, and nucleic acid drug analysis may be slightly inadequate to distinguish between isotopic peaks, multicharged ions, metabolites, and other matrix interfering substances

3, HPLC/MS-MS (liquid chromatography and tandem mass spectrometry)

1) Main features:

• Good selectivity

• The original drug and its metabolites were detected

• High throughput (~5 minutes per sample)

• Wide detection linear range (1000x)

• Enzymes and other specific reagents are not required

• High accuracy and reproducibility (±15%)

• Method development is challenging

2) Analysis of nucleic acid drugs difficult/key points:

• ESI-MS parameter optimization: Confirm the charge distribution of analytes and select suitable MRM ion pairs, optimize the SNR of MRM ion pairs, and reduce the formation of cationic adducts. Ion pair reagent balance; Take sensitivity into account.

• Chromatographic separation column and mobile phase condition optimization: ESI-MS compatibility; Ion pair reagent -RPLC (HFIP/TEA, etc.); HILIC (small nucleic acid retention on column) mode; The column temperature was increased by 45~70℃, and the AS/SS chain status was maintained.

• Non-specific adsorption and residue solutions: provisioning of reserve and working fluids (0.5% plasma, chelating agent EDTA); Low adsorption material consumables (bioinert column); Bioinert liquid LC system.

Second, qPCR/ddPCR analysis of small nucleic acid drugs

qPCR/ddPCR is a powerful means of gene analysis, through the specificity of primer design, nucleic acid drugs can be analyzed. The advantages include: high sensitivity, good accuracy, high throughput, low cost, simple sample processing (plasma generally only needs to be diluted), but the disadvantages are: the method is affected by the sequence composition of nucleic acid drugs, relatively weak specificity, and can not obtain the information of degradation products.

PCR requires at least two pairs of primers or probes, usually 20nt length per primer; In this way, the detection of DNA or RNA above 45-60nt can be directly designed by the traditional PCR method qPCR primers for detection. Many nucleic acid drugs, such as siRNA and microRNA, are around 20nt, which is too short for traditional PCR methods. For using PCR to solve the detection of short chain nucleic acid, you can first extend the analyte and then detect!

Take qPCR for the detection of siRNA drugs as an example. Before the PCR reaction, the traditional method needs to extract nucleic acid, which requires the use of nucleic acid extraction kit. Attention should be paid to evaluating the recovery rate of the kit, and the recovery efficiency of internal reference gene correction can be added. The PCR extraction process not only increases the operation and makes the process complicated, but also brings errors due to the difference in recovery rate. An alternative to nucleic acid extraction is to dilute plasma with triton diluent, heat it up, and then direct PCR. This method is easy to operate and has good accuracy, but it is not suitable for all cases and needs to be evaluated during method development.

3. Hybridization ELISA analysis of small nucleic acid drugs

The principle of hybrid ELISA is similar to ELISA. ELISA is a commonly used assay method to detect protein drugs by detecting antibodies and capturing the specific affinity of the antibody to the analyte to identify the grasping analyte in the matrix. The hybridization ELISA is a method of detecting nucleic acid drugs, using a detection reagent (nucleic acid paired with the analyte) and a capture reagent (nucleic acid also paired with another part of the analyte) can also specifically identify the analyte, so as to analyze nucleic acid drugs.

Features of hybrid ELISA:

1, advantages: high sensitivity, good specificity (does not cross with genomic DNA or miRNA precursors), high throughput, improved and not strictly purified RNA (can be crude extraction detection), purified nucleic acid can make the method anti-interference.

2, disadvantages: primer design to ensure accuracy specificity, high requirements for parallel operation (avoid contamination, negative positive control), accuracy and precision is relatively low, can not get the information of degradation products.

Summary: The appropriate bioassay method was selected according to the length of small nucleic acid drug chain.

Selection of research system for small nucleic acid drug metabolism. Hepatocytes, liver microsomes and liver S9 system are often used in the study of metabolism of small molecule compounds. Small nucleic acid drugs are metabolized by nuclease, which is widely present in various tissues and organs. The metabolic system suitable for conventional small molecule compounds may not be suitable for small nucleic acid drugs. The selection of the appropriate metabolic study system (nuclease, serum or plasma, liver S9, liver microsome, liver homogenate, liver cell, lysosome, target tissue) can best predict the situation in vivo.

The identification of small nucleic acid metabolites was studied based on high resolution mass spectrometry (LC-HRMS). The main metabolites of small nucleic acid drugs are usually predictable, and depending on their sequence and chemical modifications, the multicharged states can likewise be treated by deconvolution.

There are 4 methods for the determination of small nucleic acid drug plasma protein binding (PPB) :

1, balanced dialysis method: lack of commercial balanced dialysis membrane (interception molecular weight more than 20K), non-specific binding

2, ultra-fast centrifugal method: low recovery of siRNA (molecular settlement difference), limited by flux

3, ultrafiltration method: non-specific binding, low recovery (interception molecular weight less than 20K), protein leakage (interception molecular weight more than 50K)

4. Electrophoretic migration (EMSA) : limited by flux