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Hello, everyone,

today we will study the isotope analysis.

In bioanalysis and pharmacokinetic research,

radioisotope has become indispensable

because of its high sensitivity and specificity,

wide applicability, and simple detection.

The U.S. FDA regards the pharmacokinetic data after administration

of radioisotope-labeled drugs

as an important part for the safety evaluation of new drugs.

To ensure the safety and effectiveness of radioisotope-labeled drugs,

the Chinese Pharmacopoeia has established specifications

for several radioisotope-labeled drugs.

Then, what is the radioisotope?

As you know,

the nucleus is composed of protons and neutrons.

The atom is often represented by the symbol AZXN,

where X is the element symbol,

Z is the number of protons,

A is the mass number,

and N is the number of neutrons.

Since the element symbol X determines its atomic number,

this symbol can be abbreviated as AX, such as 12C.

An atom with the same number of protons and neutrons,

at the same energy state is called a nuclide.

One element could have multiple nuclides,

such as 12C, 13C, and 14C.

These three nuclides are called isotopes.

Stable nuclides are nuclides that are not radioactive

and so do not spontaneously undergo decay,

such as 2H, 13C, 15N, and 18O.

The radionuclide is an atom that has excess nuclear energy,

making its nucleus unstable.

The radionuclide will eventually decay by emitting a particle,

transforming the nucleus into another one,

or into a lower energy state.

This process is called radioactive decay.

The most common decays

are alpha decay, beta decay, and gamma decay.

In ¦Á decay, when an unstable nucleus spontaneously emits ¦Á particles,

the mass number is reduced by 4,

and the number of protons is reduced by 2.

For example, uranium undergoes ¦Á decay.

¦Â- decay mainly occurs in light nuclides with an excess of neutrons,

such as 3H and 14C.

In ¦Â- decay,

a neutron is converted to a proton,

and this process creates an electron.

Therefore, the atomic number of the daughter nucleus increases by 1,

where the mass number remains unchanged.

¦Â+ decay mainly occurs in nuclides with insufficient neutrons,

such as 11C, 13N, 15O, and 18F.

On the contrary of ¦Â- decay,

in ¦Â+ decay a proton decays into a neutron

and then creates a positron.

Therefore, the atomic number of the daughter nucleus decreases by 1

while the mass number remains unchanged.

¦Ã decay often occurs after ¦Á or ¦Â decay.

After these decays,

the nucleus at high energy levels

dissipates excess energy by the form of ¦Ã-ray.

Please take a look at this table.

It lists the isotopes frequently used in the R&D of new drugs.

Among them,

3H and 14C are most commonly used in bioanalysis.

The half-life is approximate 12 y for 3H and 5700 y for 14C.

Thus, the correction of half-life is not necessary during experiments,

and this is convenient for the measurement and calculation.

More importantly,

low-energy ¦Â-ray particles emitted from 3H and 14C

have very low penetrating energy

and are easily to be protected.

A piece of paper or the intact human skin

can effectively block most of the radiation,

having a higher safe profile.

Radioisotope tracing technology

has been widely used in the ADME research of drugs.

This is by virtue of the following important properties.

The first is its identity with the tested substance.

The radioactive nuclide marker

is identical with the non-marker in terms of the chemical

and biological behaviors.

Second, it is distinguishable from the tested substance.

The radionuclide continuously decays

and emits detectable rays,

making it possible to quantify and locate the marker.

The decay is not interfered by impurities,

and there is no need to purify the analyte,

thus avoiding the loss caused by repeated separation and purification.

The analyte can be measured directly.

This method is highly sensitive

and can reach the level range from 10-14 to 10-18 g,

which is of special value

for the quantitative analysis of trace substances.

Due to the high sensitivity,

the dose of the tracer can be at a normal physiological level,

which does not disturb the physiological balance,

and reflect the real condition of the body.

Next, we will introduce several commonly used

radioactive detection methods.

First, let's look at the liquid scintillation counter.

Liquid scintillation counter (LSC) is the measurement of radioactivity,

which has been widely used for the detection

of radionuclides in biosamples.

LSC is generally used for the ¦Á and ¦Â rays (such as 3H and 14C)

that are low in energy, short in distance,

and could be easily absorbed by air and other substances.

In LSC,

the radiation emitted from the radioactive sample

will be placed in the scintillation fluid which transfers energy

to the solvent molecules,

and then the scintillator,

causing the scintillator molecules to be excited.

When the scintillator molecule returns to the ground state,

it emits photons.

The number of photons is proportional to the energy of the ray.

The photomultiplier tube converts light energy into electrical pulses.

These steps, thereby,

completes the conversion from radiation to light,

and then to electrical energy.

Thus, the rays could be quantitatively measured.

The determination of LSC mostly uses homogeneous measurement.

The sample exists in the scintillation fluid in the form of a true solution,

and the radioactive material is surrounded by the scintillation fluid.

The homogeneous solution should be clear and preferably colorless.

Further, it should not undergo phase separation and precipitation.

For insoluble biological tissues, excreta,

and macromolecules, acid-base reagents

can be used to dissolve molecules in scintillation fluid for measurement.

Next, we will introduce the autoradiography technique.

In autoradiography (ARG for short),

photosensitive materials are exposed to radiation

to detect the tissue distribution of radionuclides

or their labeled compounds.

Autoradiography could obtain the image of the distribution of markers,

which can accurately locate the markers

and are highly sensitive.

This method can measure drugs labeled with 3H, 14C,

and other nuclides.

According to the observation range and resolution,

autoradiography can be classified into macroscopic ARG,

optical microscopic ARG,

and electron microscopic ARG.

The quantitative whole-body autoradiography in macroscopic ARG

provides data of the tissue distribution

of the drug in animals,

and has become an important method

in the pharmacokinetic study.

Here is an example.

When studying the distribution of bisphenol A (BPA)

in pregnant and newborn mice,

14C-BPA was injected into the pregnant mice.

The mice were sacrificed at different times,

and sections of frozen mice were prepared.

The whole-body autoradiography

was used to observe the distribution of BPA in the mice.

This is the HE staining and autoradiography of mice

obtained one hour after i.p. injection of 14C-BPA.

HE staining can effectively locate tissues.

In autoradiography, the redder the color,

the higher the drug concentration.

As you can see,

14C-BPA was distributed throughout the body

with higher drug concentrations in the kidney, liver,

and local stomach.

Finally, let's study the radionuclide imaging.

Real-time quantitative determination of the drug concentration

in different tissues of human body

is extremely important and challenging for new drug development.

The traditional studies of tissue distribution need large numbers

of animal sacrifice at different time points.

Moreover, the data extrapolation to humans

may have great error due to the specie differences.

Radionuclide imaging technology effectively addresses this problem.

It is non-invasive,

and could introduce radiopharmaceuticals into the body

and then detect them with the nuclear medicine imaging devices.

The three-dimensional in-vivo images of the radiopharmaceutical

are obtained by the computer,

thus realizing the dynamic, continuous,

and non-invasive observation of drug distribution

and change in the body.

At present, there are two main imaging techniques.

One is single-photon emission computed tomography (SPECT),

and the other is positron emission tomography (PET).

Today I will introduce PET.

After injecting short-lived nuclides with positron radioactivity,

such as 11C, 13N, 15O, and 18F into the human body,

the emitted positrons will travel a short distance in tissues

until they meet and annihilate with the electrons.

As a result, two photons of 551 keV are emitted in opposite directions,

and are received by a PET scanner outside the body.

The resulting image could measure the real-time tissue drug

concentrations in living body.

At present,

many countries have carried out clinical trial projects using PET

and other molecular imaging methods,

including human pharmacokinetic tests,

pharmacodynamic studies, safety evaluations,

and dose optimizations.

FDA has approved multiple projects using PET results

as the primary endpoint.

This is the result that we found out using PET

as a keyword on the ClinicalTrials website.

Let's take a look at one example.

Multidrug resistance-related protein (MRP1)

is a drug efflux transporter,

and its function is closely related

to a variety of central nervous system diseases.

Therefore, it is necessary to develop PET probes

to evaluate brain MRP1 function in vivo.

The researchers labeled a candidate compound with 18F

and used PET to profile its fate in the brain.

This probe drug underwent metabolism after entering the brain,

and the metabolite could be excreted into the blood by MRP1.

Then the in-vivo study was performed in wild-type mice

and MRP1 knockout mice for comparison.

The brain concentration of the radionuclides of knockout mice

was much larger than that of wild-type mice,

thus confirming that the probe

could detect the function of MRP1 in the brain.

That's all, thank you for watching.