If local anesthetics do not access the binding site via the external side, how do they get to their site of action? There is much experimental evidence to show that local anesthetics must access the binding site via a hydrophobic or via a hydrophilic pathway (Fig. 22.13).64,65 The anesthetics pass through the membrane in their uncharged form (Fig. 22.13 hydrophobic pathway A). In the axoplasm, they reequili-brate with their cationic species. It is postulated that the anesthetic molecule may access the binding site via a hy-drophilic pathway by entering into the sodium channel from the interior of the pore, when the channel is open (Fig. 22.13 hydrophilic pathway C). The local anesthetic molecule is believed to bind to the binding site in its ionized form. Another possibility is that before passing all the way through the lipid membrane, the anesthetic may be able to directly access the local anesthetic binding site (Fig. 22.13 hydrophobic pathway B).

Studies using local anesthetics containing a tertiary nitrogen also confirm that the local anesthetic is accessing the binding site from either the hydrophobic or hydrophilic pathways described previously. When this type of anesthetic is applied externally, and the pH of the external media is increased, and thus the local anesthetic is predominantly in the unionized form, the onset of block is more rapid. The neutral form of the drug molecule penetrates the membrane and then accesses the binding site through pathway B or C described in Figure 22.13. The pH of the solution had no effect on sodium conduction block when the local anesthetic used was the neutral molecule benzocaine, suggesting that the change in pH affects the drug charge and not the receptor.63,66

The sodium channel has been shown to have a great deal of flexibility and can change shape when the electrical environment around the channel changes. There are at least three conformations that the sodium channel can form. (a) An open state, such as that depicted in Figure 22.12 (drawing on the left) where the sodium ion has a clear pathway from the external side of the membrane to the internal side of the membrane. (b) A "closed/inactive" state shown in Figure 22.12 (drawing on the right), where the sodium channel undergoes a conformational change to prevent sodium ion passage into the cell. The sodium channel undergoes this conformational change in response to the huge influx of sodium causing depolarization of the cell membrane. The sodium channel is now closed and inactive, it cannot open again until the membrane has reached its resting potential. (c) The third conformation of the sodium channel is formed when the membrane potential returns to the resting potential. The sodium channel is now closed but able to open when a stimulus reaches the threshold potential. At this point, the sodium channel is in a "closed/resting" state that is different from the "closed/inactive" state.

The affinity of the local anesthetics for the binding site has complex voltage and frequency dependant relationships. Affinity depends on what state the sodium channel is in as well as the specific drug being tested. In general, at resting states, when the membrane is hyperpolarized, the local anesthetics bind with low affinity. When the membrane has been depolarized and the channel is open, local anesthetics bind with high affinity. Local anesthetics also bind with high affinities when the sodium channel is in the "closed/inactive" conformation, perhaps stabilizing the inactive form of the receptor.

The ability of the local anesthetic to block conduction also depends on the targeted neuron. In general, autonomic fibers, small unmyelinated C fibers (mediating pain sensations), and small myelinated Ay fibers (mediating pain and temperature sensations) are blocked before the larger myeli-nated Ay, A3, and Aa fibers (mediating postural, touch, pressure, and motor information).67

SARs of Local Anesthetics

The structure of most local anesthetic agents consists of three parts as shown above. They contain (a) a lipophilic ring that may be substituted, (b) a linker of various lengths that usually contains either an ester or an amide, and (c) an amine group that is usually a tertiary amine with a pKa between 7.5 and 9.0.

1. The Aromatic Ring

The aromatic ring adds lipophilicity to the anesthetic and helps the molecule penetrate through biological membranes. It is also thought to have direct contact with the local anesthetic binding site on the sodium channel. The exact amino acids involved in binding depend on the sodium channel being studied as well as the state (open, closed/inactive, closed/active) of the sodium channel. The aromatic ring is believed to interact with the local anesthetic binding site in a v-v interaction or a v-cation interaction with the S6 domain of the a component of the sodium channel. Substituents on the aromatic ring may increase the lipophilic nature of the aromatic ring. An SAR study of para substituted ester type local anesthetics showed that lipophilic substituents and electron-donating substituents in the para position increased anesthetic ac-tivity.68 The lipophilic substituents are thought to both increase the ability of the molecule to penetrate the nerve membrane and increase their affinity at the receptor site. Buchi and Perlia suggested that the electron-donating groups on the aromatic ring created a resonance effect between the carbonyl group and the ring, resulting in the shift of electrons from the ring to the carbonyl oxygen. As the electronic cloud around the oxygen increased, so did the affinity of the molecule with the receptor (Fig. 22.14).69 When the aromatic ring was substituted with an electron-withdrawing group, the electron cloud around the carbonyl oxygen decreased and the anesthetic activity decreased as well.

2. The Linker

The linker is usually an ester or an amide group along with a hydrophobic chain of various lengths. In general, when the number of carbon atoms in the linker is increased, the lipid solubility, protein binding, duration of action, and toxicity increases.70 Esters and amides are bioisosteres having similar sizes, shapes, and electronic structures. The similarity in their structures means that esters and amides have similar binding properties and usually differ only in their stability in vivo and in vitro. For molecules that only differ at the linker functional groups, amides are more stable than esters and thus have longer half-lives than esters. Plasma protein binding may

-dipole attraction

Figure 22.14 • Schematic representation of the binding of an ester-type local anesthetic agent to a receptor site. (Reprinted with permission from Buchi, J., Perlia, X.: Design of local anesthetics. In Ariens, E. J. [ed.] Drug Design. New York, Academic Press, 1972, Vol. 3, p. 243.)

-dipole attraction

Figure 22.14 • Schematic representation of the binding of an ester-type local anesthetic agent to a receptor site. (Reprinted with permission from Buchi, J., Perlia, X.: Design of local anesthetics. In Ariens, E. J. [ed.] Drug Design. New York, Academic Press, 1972, Vol. 3, p. 243.)

be more prevalent for the amide anesthetics as well, contributing to the increased half-life.71 Individual drugs are discussed in the drug monograph section.

As described previously, the nature of the substituents on the aromatic ring can affect the electronic nature of the linker and can contribute to the drug's potency and stability. Substituents on the aromatic ring may also confer a steric block to protect the linker from metabolism. Thus, the binding affinity and stability of the anesthetic molecule is affected by the linker as well as the functional groups on the aromatic ring. In general, ester groups are more susceptible to hydrolysis than amide functional groups because of the prevalence of esterases in the blood and the liver. The first ester type local anesthetic synthesized was procaine (Novocain) in 1905. Procaine metabolism can be seen in Figure 22.15. The para-aminobenzoic acid (PABA) metabolite, common to the ester class of drugs, is believed to be responsible for the allergic reactions some patients have experienced with local anesthetics.72 3. The Nitrogen

Most local anesthetics contain a tertiary nitrogen with a pKa between 7.5 and 9.5. Therefore, at physiological pH, both the cationic and neutral form of the molecule exists. At physiological pH, the ionized to unionized form of the anesthetic can be calculated using the Henderson-Hasselbalch equation:

Extensive work using both internally and externally applied compounds, changing the pH of the solution, and using permanently charged anesthetic analogs has led to the present theory of anesthetic SARs. Namely, that the anesthetic compounds bind to the anesthetic receptor site on the sodium channel in the ionized form. From Figure 22.13, it can be seen that the molecule can penetrate the nerve membrane in its neutral form and then reequilibrate with its cationic form on the internal side of the membrane. Permanently charged, quaternary anesthetics applied to the external side of the nerve membrane do not penetrate and cannot access the local anesthetic binding site.

To keep the anesthetic soluble in commercial solutions, most preparations are acidified. In an attempt to decrease pain on injection and to increase the onset of action, some practitioners advocate adding sodium bicarbonate to the commercial preparation. By adding sodium bicarbonate, the solution will become less acidic and more of the drug will be found in the neutral form. The neutral form will thus cross the nerve membrane quicker and have a quicker onset of action. Although this theoretically makes sense, many studies have found no difference in the onset of action between alkalinized and nonadjusted anesthetics. Manufacturers formulate solutions at a pH that gives them adequate shelf life. When the pH is increased, the stability of the preparation decreases and outright precipitation can occur if too much of the water-soluble cationic form is converted to the anesthetic base. If this practice is followed, very careful titration of the added base is required. The solution should be observed for precipitation and the solution must be used immediately.

Vasoconstrictors Used in Combination with Local Anesthetics

Many anesthetic preparations are commercially available combined with the vasoconstrictor epinephrine. Some anesthetics are also combined with other agents such as norepi-nephrine, phenylephrine, oxymetolazone, or clonidine to achieve a desired formulation. The epinephrine in the anesthetic solution has multiple purposes. As a vasoconstrictor, the injected epinephrine will constrict capillaries at the injection site and thus limit blood flow to the area. The local anesthetic will thus stay in the immediate area of injection longer and not be carried away to the general circulation. This will help keep the drug where it is needed and allow minimal drug to be absorbed systemically. Thus, anesthetics with epinephrine used for infiltration anesthesia consistently result in lower plasma levels of the anesthetic. This will reduce the systemic toxicity from the anesthetic and increase f/ \ O ,CH2"CH3 cholinesterase i/ O ,CH2-CH3

H2N^ VC-O-CH2-CH2-N cholmesterase , H2N^ VC-OH + HO-CH2-CH2-N

Procaine para-aminobenzoic acid (PABA)

Figure 22.15 • Metabolism of procaine.

the duration of anesthetic activity at the site of injection. The lack of blood flow in the immediate area will also decrease the presence of metabolizing enzymes and this also increase the duration of action of the anesthetic locally. The characteristic blanching that follows epinephrine infiltration anesthesia also makes suturing or manipulating the area easier because of the lack of blood flow in the area. It is not recommended that anesthetics with a vasoconstrictor be used in tissue served by end-arterial blood supply (fingers, toes, ear-lobes, etc.). This is to prevent ischemic injury or necrosis of the tissue. Epinephrine has also been shown to counteract the myocardial depressant effects of bupivacaine when added to a bupivacaine epidural solution.73

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