Research progress of concomitant safety pharmacology in repeated dosing toxicity test

The main purpose of repeated dosing toxicity test is to describe the toxicity characteristics of the animal after repeatedly receiving the test substance, predict the clinical adverse reactions that the test substance may cause, and determine the target organ or target tissue of the test substance toxicity. The main detection indicators include hematology, Blood biochemistry, histopathology, and drug exposure (TK), etc., to determine the dose level (NOAEL) at which no clinical adverse reactions are observed. The safety pharmacology test (Safety Pharmacology, SP) mainly studies the potential undesirable adverse effects on physiological functions when the drug is within the therapeutic range or above the therapeutic range. It mainly observes the drug’s effects on the central nervous system, cardiovascular system and respiratory system. The purpose is to find the treatment window (Margin). However, for drug preclinical safety evaluation, both types of tests have certain limitations: general toxicity tests lack functional studies of important systems or organs, and SP tests lack information on drug exposure, histopathology, and blood biochemistry.
Many high-impact factor literature reports show that short-term administration will not cause functional changes in the relevant system, but after long-term administration or even in the recovery phase of the trial, significant tissue and organ adverse reactions will occur. Torcetrapib is a cholesterol lipid transfer protein inhibitor, which has the effect of raising HDL (high density lipoprotein) and lowering LDL (low density lipoprotein). Short-term administration has no effect on the cardiovascular system. After one year of administration, it can cause an increase in aldosterone levels, increased systolic blood pressure, and increased risk of cardiotoxicity and mortality; Casopitant is a neurokinin NK1 receptor antagonist that induces Delayed effect of canine cardiotoxicity. Within one week of repeated administration, there is no significant change in canine ECG, but after 20 weeks of administration, degenerative changes in the left ventricular papillary muscle, abnormal sarcoplasmic plate structure, and troponin (CTnl) and creatine kinase (CK-MB) levels increased significantly after stopping the drug. Therefore, a single toxicity test or a single SP test cannot accurately reflect the relevant changes after the action of the drug. Only by combining the two tests reasonably can a more scientific and complete test result be obtained. The following will specifically introduce the driving factors, basic methods, relevant considerations and typical examples of the integration of SP endpoints into general toxicity tests.

SP endpoints are integrated into the drivers of general toxicity testing

Scientific factors

• (1) A single dose cannot evaluate the delayed effect of the drug

The accumulation effect of the original drug or its active metabolites in specific organs, and the results obtained from a single administration, cannot make a scientific assessment of the delayed effects of the drug. For example, amiodarone is a typical antiarrhythmic drug. The original drug and its metabolites are active. During acute exposure, the original drug has a higher concentration in serum, myocardium and ventricular cavity, but it has a significant effect on heart rate (HR) and QTc interval. No significant effect; metabolites accumulate in the ventricular cavity during repeated exposure, resulting in prolonged HR, QTc, and ventricular effective refractory period.

• (2) The specific interference mechanism of drugs on cell biochemical metabolism

The internalization of the long-term effects of receptor modulators, the inhibition of ion channel transport, and the damage or inhibition of axonal transmission can all lead to inconsistencies in the strength or mechanism of the first and last effects of drug action. For example, fluoxetine 5-HT uptake inhibitor, the original drug and its metabolites are active, acute exposure can cause hERG K+ current block, leading to prolonged QTc interval; repeated exposure interferes with hERG K+ protein membrane transport, hERG K+ current Decrease, QTc interval prolonged, catecholamine secretion imbalance, will eventually lead to torsade de pointes ventricular tachycardia, increase the risk of adverse cardiac events.

• (3) Functional change is also an important basis for drug toxicity judgment and the explanation of toxicity mechanism

Functional changes can be found before pathological and blood biochemical changes, and neurological damage often cannot be found at the tissue level. Histopathological changes can be the outcome of functional changes, not the cause, such as cardiac changes caused by tachycardia and hypertension.

Regulatory factors

Since 1997, relevant guidelines such as ICH S6/S7A/S9 and FDA have directly or indirectly pointed out that SP endpoints can be included in general toxicity tests. As pointed out in the ICH guidelines (S6), for biotech drugs with highly specific targets, SP evaluation can be used as part of the toxicology test, and SP tests can be reduced; the ICH guidelines (S9) point out that the research and evaluation of vital organs The indicators can be included in general toxicity tests.

According to the survey data of the SPS Association, most of the subjects (including SD, safety pharmacologists: 71% in North America) agreed to include the SP endpoint in the general toxicity test, and more than half of the institutions have relevant experience.

The basic method of SP endpoint included in general toxicity test

The SP endpoint is used in repeated dosing toxicity tests to detect the acute and chronic effects of important organ functions and the relationship between the toxicity of important organ functions and TK (drug exposure); finally, the correlation between related organ functions can be analyzed It also satisfies the animal 3R principle to the greatest extent, and most of the SP indicators can be integrated into the toxicity test. See the table for details.

Cardiovascular system (CV)

It mainly includes methods such as anesthesia (almost not used now), non-anaesthetic restriction, vest telemetry and implantable telemetry. Each method is specifically introduced in the following table:
image
Some scholars have conducted a comparative study on the three methods, and the results show that the data of the telemetry system fluctuates little, and restraining the animal will cause the heart rate to rise and interfere with the judgment of the HR\QT interval.

Nervous System (CNS)

Most neurotoxic drugs do not cause obvious neurohistological changes. Various related neurobehavioral evaluations are important indicators for detecting whether drugs have an effect on the nervous system. Neuropsychiatric drugs, tumor drugs (small molecule targeting) and other drugs that can break through the blood-brain barrier should all be tested for their effects on the nervous system.

The main detection methods are:
(1) FOB or Irwin test, mainly applicable to dogs, monkeys, rats and mice. Specific evaluation indicators can be specifically designed based on experiments.

(2) Spontaneous animal activities, mainly including observation in cages and field trials. Video tracking methods are commonly used to observe large animals in cages, and a few use radio frequency identification (RFID) technology to freely monitor animal activity changes and behavior habits. Cage observation is simple, but limited, and sedative drugs are not applicable. The mine field test is suitable for rodents, but the new environment is easy to cause animal stress, and the animals are affected by noise and time, and usually require training or the establishment of a satellite group.

Respiratory system

Generally use vest telemetry technology (applicable to dogs and monkeys) and cavity method (applicable to large and small animals). Both methods require the detection environment to be quiet and free of interference, and large animals adapt to wearing vests in advance. The cavity method also needs to adapt to the environment in the cavity in advance, strictly control the temperature and humidity of the room, and it needs to be tested in batches according to the number of chambers. However, the vest telemetry technology generally only measures respiratory frequency and tidal volume. The cavity method can measure expiratory time, inspiratory time, respiratory frequency and tidal volume and other related indicators.

Urinary system

Urinary system testing is divided into general excretion function testing and glomerular filtration rate (GFR) or renal blood flow (clearance) testing. The general excretion function testing methods are mainly hematology testing (such as urea, creatinine, blood sugar, etc.) and urine testing (urine protein, urine glucose, electrolytes, pH, etc.), but there are many interference factors, and the kidneys have strong compensatory functions, which are not easy to detect. change. Glomerular filtration rate (GFR) and renal blood flow (clearance) detection generally use probe drug method (small animals can set up satellite group), such as inulin and hippuric acid. Some scholars used Guanmutong water decoction to act on rats in a 4-week toxicity test, and compared the two methods. The results showed that the sensitivity of the probe drug method was significantly higher than that of general functional testing methods such as hematology.

Urinary system testing is divided into general excretion function testing and glomerular filtration rate (GFR) or renal blood flow (clearance) testing. The general excretion function testing methods are mainly hematology testing (such as urea, creatinine, blood sugar, etc.) and urine testing (urine protein, urine glucose, electrolytes, pH, etc.), but there are many interference factors, and the kidneys have strong compensatory functions, which are not easy to detect. change. Glomerular filtration rate (GFR) and renal blood flow (clearance) detection generally use probe drug method (small animals can set up satellite group), such as inulin and hippuric acid. Some scholars used Guanmutong water decoction to act on rats in a 4-week toxicity test, and compared the two methods. The results showed that the sensitivity of the probe drug method was significantly higher than that of general functional testing methods such as hematology.

Digestive system (GI)

Commonly used detection methods for the digestive system are: (1) Fecal properties (number of particles), suitable for rats, mice and large animals, and is the simplest and most effective method for monitoring gastrointestinal changes; (2) Nutritional carbon paste method, suitable for large and small animals This method is widely used, but the data fluctuates greatly, and charcoal has an adsorption effect, which may affect some drugs; (3) The magnetic dipole oral filling method is suitable for large animals and can be continuously tracked and recorded; (4) ) Sensing telemetry capsule is a new type of technology that is commonly used in dogs. It can continuously monitor changes in temperature, pressure, pH and other indicators. However, the capsule is larger and takes a long time to discharge into the duodenum.