In order to function correctly a cell must be able to take up and release a wide range of materials; for drug therapy to be successful it must also be possible to get therapeutic substances into cells and across layers of cells such as epithelia. There are a number of possible mechanisms for transport across membranes; substances may simply diffuse across, or be carried by a range of more selective processes, depending on the substance involved.
Studies using model membranes have revealed that the phospholipid bilayer itself is remarkably impermeable to all but very small molecules such as water and ethanol, and gases such as oxygen and carbon dioxide. These compounds move across the membrane by passive diffusion, a process driven by the random motion of the molecules. Diffusion is described by Fick's law, which states that the diffusion rate R (in moles s-1) is proportional to the concentration gradient Ac/Ax:
Where Ac is the concentration difference between the outside and inside of the membrane, and Ax is the thickness of the membrane. A is the area of membrane over which diffusion is occurring, and D is a constant (for a specific molecule in a specific environment) called its diffusion coefficient. Since the area and thickness of the membrane are usually outside our control, it is evident that uptake of a molecule into a cell by passive diffusion can only be influenced by either increasing the external concentration of the drug, or by selecting our molecule so that D is large.
The diffusion coefficient of a drug is determined by a number of factors, but two are particularly important. These are the solubility of the drug, and its molecular weight. For a molecule to diffuse freely in a hydrophobic membrane it must be soluble in it, and conversely if it is to also diffuse in the extracellular fluid it must also be soluble in aqueous systems. The relative solubility of molecules in aqueous or oily environments is described by their partition coefficient, labelled P, which describes how the drug distributes itself between a pair of solvents (usually water and an oily solvent such as octanol). Hydrophobic molecules dissolve mainly in the oil and have a high partition coefficient, while hydrophilic molecules dissolve mainly in the water and have a low partition coefficient. Only for intermediate values of the partition coefficient will the drug be soluble in both the membrane and the extracellular fluid, and be free to diffuse from the extracellular fluid, across the membrane, and into the cell. Drugs which have a very low partition coefficient are poorly absorbed because they cannot dissolve in the oily membrane; conversely drugs which have a high partition coefficient cannot dissolve in the extracellular fluid and so cannot reach the membrane. Such drugs are said to be solubility-limited.
The diffusion coefficient, and hence rate of absorption, is also influenced by the molecular weight of the drug. Small molecules diffuse rapidly and so will cross the membrane more quickly than large, slowly-diffusing molecules. These concepts are illustrated in Figure 1.10, which shows how absorption depends on partition coefficient for drugs of different molecular weights. This is not based on specific drugs but is intended to illustrate the concepts. The drug with a molecular weight of 400 is rapidly absorbed for intermediate partition coefficients (P of approximately 100 or Log P=2. However, it is absorbed more slowly for larger values of P as its aqueous solubility falls, or for smaller values of P as its membrane solubility falls. The smaller drug with a molecular weight of 250 is subject to the same influence of P, but is generally absorbed more quickly due to its more rapid diffusion.
Figure 1.10 Absorption as a function of drug partition coefficient
.001 .01 .1 1 10 100 1000 10000 100000 Partition Coefficient
Figure 1.10 Absorption as a function of drug partition coefficient
Figure 1.10 shows how the diffusion of drug across a pure lipid membrane will vary depending on the properties of the drug. However, drug diffusion across real cell epithelia takes place not only through membranes, but also through small aqueous pores between cells (the paracellular route), and this enhances the absorption of hydrophilic molecules which are small enough to pass through the pores. The drug with a molecular weight of 250 can pass through these pores, but that with a higher molecular weight of 400 cannot.
Drug molecules are predominantly weakly ionizable species containing groups such as amine, carboxyl, phenyl, etc. These materials are absorbed across plasma membranes in their unionised forms, since these are non-polar; the ionised forms of the drug cannot pass through the membrane due to its hydrophobic character. Consequently, the pH of the extracellular environment is critical in determining the absorption across the membrane. Thus, for example, an acidic drug is absorbed from acidic solution if the pH is lower than the drug pK, since it will be in its unionised form. This is the basis of the pH-partition hypothesis, stressed in most classical texts of pharmacology, which discuss the absorption of drugs based on the relative degrees of ionization in the lumen and the blood.
The pH-partition hypothesis provides an indication of drug absorption, but suffers from many shortcomings. The most notable of these can be seen in the widely quoted example of the absorption of an acidic drug from the stomach, in which the drug is in its unionised state at pH 2 and so passes across the membrane. In the blood (at pH 7.4) the drug is ionized, and so cannot pass back across the membrane. This effect is referred to as ion-trapping. The conclusion is that pH and ionization are highly important in determining drug absorption. This example is logically correct but suffers from a number of errors. The gastric epithelium represents the most extreme example available of a pH gradient in vivo, but drug absorption from the stomach is minimal and most absorption takes place in the small intestine, which is normally close to pH 7. Here the ionisation of the drug in the lumen is similar to that in the blood and little ion-trapping can occur. Gastric contents delivered into the duodenum make the first few centimetres of the intestine acidic, until the chyme has been neutralised by bicarbonate. The duodenal absorptive capacity is high, but transit through this region is extremely rapid and so no significant absorption occurs.
The biggest failing of this hypothesis is to attempt to calculate absorption from equilibrium drug distributions, when in practise the absorbed drug is swept away by the circulation. Absorption is a dynamic process involving dissolution, ionization, partition and blood flow, and consequently the correlation of pH-partition predictions with experiment is often poor.
Despite the hydrophobic nature of the cell membrane, it is necessary for a number of hydrophilic materials to enter and leave the cell. Typical examples are small amino acids and carbohydrates, which the cell requires in quantity for metabolism. Ions are also required, as the cell maintains an ion imbalance with the surroundings, with the cell having substantially more potassium and less sodium than the extracellular fluid.
Since these molecules cannot diffuse freely across the cell membrane, they are transported by a range of membrane proteins collectively called permeases. These proteins fall into two broad groups, those which allow molecules to pass into the cell down a concentration gradient, and behave like passive but selective pores, and those which actively pump molecules into cells against a concentration gradient. The former group contains transporters that allow nutrients such as glucose into the cell; this hexose transporter system is present in most mammalian cells. Since the glucose is utilized inside the cell rapidly, the internal concentration is low and the diffusion always occurs down a concentration gradient. Consequently, no input of energy is required to drive this transport system.
The second group of proteins actively accumulate materials in cells even if their concentration is higher inside the cell than outside. This requires an input of energy, usually derived from the hydrolysis of intracellular ATP, and consequently the carriers are called ATPases. The best known examples are the Na+/K+ ATPase that pumps potassium into the cell and sodium out, and the H+/K+ ATPase which pumps hydrogen ions out of the gastric parietal cells, thus acidifying the stomach contents.
An important characteristic of carrier-mediated absorption is that it is saturable. If the external concentration of the molecule being transported is extremely high, the carrier will be fully utilized and will become rate limiting. Under these conditions, increasing the external concentration of the transported molecule will have no effect on the transport rate. The maximum transport rate will be determined by the concentration of carrier molecules and the speed with which they can shuttle material across the membrane, and not on the concentration of the molecules being transported.
A number of drugs are thought to be absorbed by carrier-mediated processed rather than passive diffusion. These include amoxycillin and cyclacillin7, which show saturable kinetics, and cardioglycosides such as digitalis. The actual carrier mechanism is unclear since these materials are xenobiotics and are presumably being transported by a protein which normally serves some other purpose.
Energy must be expended in order to pump any molecule up a concentration gradient, and this is ultimately derived from ATP hydrolysis. The only transport systems that are directly coupled to ATP are those which pump ions such as Na+ and Ca2+ However, cells often have to accumulate other materials, such as amino-acids and carbohydrates, at high concentrations. This is performed by cotransport, in which the cells' ion concentration
Figure 1.11 Cotransport across membranes gradient is used as a secondary energy source (Figure 1.11). The transport proteins are systems which couple the transport of an ion to that of another molecule, so that, in allowing an ion to move out of the membrane to a lower concentration, the protein also moves a different molecule from lower to higher concentration. If the ion and molecule move across the membrane in the same direction, the process is called symport, while if they are exchanged in opposite directions it is called antiport.
An important example of this process is the absorption of glucose from the intestinal lumen by the intestinal epithelial cells. In most cells glucose is actively metabolized, so its concentration is low and it can be transported by passive transport. However, the intestinal epithelium is responsible for absorbing molecules like glucose, so it is often necessary to pump them up a concentration gradient into the epithelial cells before they can be passed into the bloodstream. This is accomplished by a sodium-glucose symport protein which couples the inward movement of a glucose molecule to that of a sodium ion. The intracellular sodium concentration is lower than in the intestinal lumen, so the inward movement of sodium is energetically favourable. A glucose molecule is simultaneously transported into the epithelial cell up a concentration gradient.
Membrane transport by diffusion or by transport proteins is only feasible for small molecules, since there is a limit to the size of pore that can be opened and closed by the conformational change of a membrane protein. Consequently, larger objects, such as macromolecules and particles, are internalized by a completely different mechanism, in which a portion of the membrane extends and envelops the object, drawing it into the cell to form a vacuole. This process is called cytosis and there are a number of variants which occur in different cells.
Endocytosis occurs when a small cavity forms on the membrane surface, which is gradually enclosed by membrane movement and finally taken within the cell (Figure 1.12). The process may be spontaneous in certain cells, and causes a small amount of extracellular
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