Key physicochemical property considerations in the early development of new drugs - permeability prediction

Based on the solubility and permeability factors of the drug, the Biopharmaceutical Classification System (BCS) has been formed, which has become the world's more recognized important indicator of the dosage form design and prediction of the in vitro correlation of the drug, and also pointed out that the solubility and permeability of the drug in the gastrointestinal tract is the key

The intestinal permeability of a drug refers to its ability to enter the blood circulation across the intestinal wall cells, usually measured

Drug permeability evaluation is based on the degree of absorption of human pharmacokinetic studies, such as absolute bioavailability or mass balance
.
It is generally believed that when the absolute bioavailability is not less than 85%, or not less than 85% of the drug is excreted from the urine in the form of a prototype drug or in the form of a combination of prototype drugs and metabolites (metabolites produced after absorption), the drug can be considered to have high permeability
.
Therefore, the permeability of drugs is also the focus of attention in the consistency evaluation, we need to take a variety of models to predict the permeability of drugs in vivo, to further understand the essential properties of drugs, so as to give drugs a more accurate evaluation
.

Of course, at different stages of new drug research, different research methods can be used to obtain drug permeability information to improve the efficiency of new drug development and promote new drugs forward
.
In different research stages of new drugs, the commonly used permeability prediction methods are shown in Figure 1
.

The industry usually combines parallel artificial membrane permeation technology (PAMPA) and Caco-2 cell model as permeability prediction methods in the early stage of drug development, and the current Ussing chamber rat model is also widely used
for its absorption window evaluation advantages.
The accuracy of these methods is mainly determined by the correlation between the human in vivo single-perfusion technique (Loc-I-Gut), which is the gold standard
indicating the permeability of drugs.

1 Artificial Membrane Permeability Assay (PAMPA)

PAMPA is based on an artificial, noncellular lipid membrane without porosity, active transporters, and metabolic enzymes, and is used to predict passive transcellular permeability of drug candidates
.
The PAMPA method is to divide the 96-well plate and the 96-well plate into two compartments (donor chamber and recipient chamber)
with a membrane.
Buffer solution (pH5.
0~pH7.
4) of the compound to be measured is added to the donor chamber, blank buffer (pH 7.
4 or so) is added to the recipient chamber, as shown in Figure 2, after incubation at 37 °C for a period of time, the drug concentration
on one side of the receptor chamber is determined using ultraviolet spectrophotometry.

From Figure 3, it can be seen that the permeability predicted by PAMPA of some drugs is basically the same as
that of Coca2.
The advantage of PAMPA is that there is no need for cell culture, which reduces costs and increases throughput for permeability assays
.

2Caco-2 cell model

Caco2 is a human colon cancer cell line that can divide indefinitely, and the microvilli morphology formed on the surface of the caudal membrane of Caco2 is similar to the small intestinal villi of gastrointestinal epithelial cells, and expresses numerous cell transporters, including p-glycoprotein (p-gp), breast cancer resistant protein (BCRP), multidrug-resistant protein (MPR) and other common extroverted transporters, including glucose, amino acids, oligopeptides and other absorbent types involved in the transport of substances.
It is possible to predict active transport and drug interactions with multiple transporters, a schematic diagram of which is shown in Figure 4
.

The fundamental difference between the mechanism of PAMPA and Caco-2 cell models for permeability prediction is that The Caco-2 cell model can not only predict the passive transport of compounds, but also characterize compound expulsion and active absorption.
What PAMPA predicts is simply passive transport of
compounds.
For compounds that are osmotic only by passive diffusion, the Caco-2 cell model and the PAMPA assay permeability data fall between the two correlated lines in Figure 5; The compound has an excretion effect, and the data falls in the lower right corner; The compound has active uptake or cell bypass for absorption, and the data falls in the upper left corner
.
The combination of the two allows for more accurate characterization of the transport characteristics
of the compound.

3 Ues perfusion rat model

The Uss perfusion technique is one of the most commonly used in vitro methods to study gastrointestinal permeability
.
After the rat intestine is intercepted, the serous layer tissue of the ex vivo intestinal segment is isolated, and then the intestinal slice is fixed on the hole between the incompatible mucous membrane and the serous chamber, simulating the physiological environment of the gastrointestinal tract, adding the test compound to the mucous membrane or serous side of the tissue, which can be combined with intestinal metabolism to study the transport of drugs (absorption and secretion direction), the schematic diagram of which is shown in
6.

4 Human Loc-I-Gut experiments

The jejunum is the main absorption area of most drugs in most mammals, it has the largest surface area, and is the most active carrier-mediated transport site
in the intestine.
The most commonly used and effective technique for the clinical determination of the proximal peff of the jejunum is Loc-I-Gut
.
Any in vitro model used to predict human intestinal absorption must begin with validation against the corresponding in vivo data, based on correlation
with historical Peff measurements from Loc-I-Gut.

These jejunal Peff values are converted into intestinal absorption half-life of absorption (t1/2, abs), and then compared with traditional pharmacokinetic (PK) data analysis, that is, the corresponding t1/2, abs values calculated by the blood drug concentration curve, and the effectiveness
of these jejunal Peff values is further analyzed.
The t1/2, abs values obtained from the jejunal Peff values of the selected subset of the drug are very consistent
with the "real" in vivo absorption data obtained in clinical PK studies (oral solutions or immediate-release preparations).
And the human jejunal Peff values of these drugs are location- and time-dependent, but still able to predict overall Fa
.
This all shows that Peff can reflect the "real" absorption
of drugs in the body.
Therefore, the current human Loc-I-Gut experiment is the "gold standard"
for permeability experiments.

However, the Loc-I-Gut study was conducted
in healthy subjects.
Loc-I-Gut experiments are cumbersome, expensive, and ethically involved, so they cannot be used for drug screening
on a large scale.

Permeability prediction is more about predicting the permeability of API molecules than drug products
.
Changes in pharmaceutical products generally have less effect on permeability because this is a property of the drug molecule and is less likely to change
with product and process changes.
In the drug development process of new drugs, permeability can be assessed through a variety of techniques
.
Simpler methods are preferred in the early stages of product development and when evaluating drug candidates, while more complex measurements
may be performed in later development.
Although, the efficiency of higher detections, using simpler methods, is not generally recognized in BCS classification as measured qualitatively (e.
g.
, entering high/middle/low grades) and quantitative (permeability in numerical terms), it still has predictive significance
.
If any quantitative value can be correlated with a meaningful measurement of in situ human permeability or absorption fraction, its usefulness can be further enhanced
.
For the formal BCS classification, there are specific guidance
on what is an acceptable penetration assessment method in terms of the methods used and method verification.