Drug Pathways and Chemical Concepts

Prof. Sally Boudinot

17. Penicillin

n 1929, Sir Alexander Fleming made a discovery that was to change the course of medical practice and perhaps humanity: penicillin. Prior to penicillin, bacterial infections were widespread, and deaths from pneumonia, scarlet fever, osteomyelitis, and syphillis were common. The discovery of penicillin indeed changed the course of medicine, but it was the synergism of chemistry and pharmaceutics that made the dosing and use of penicillin effective. Fleming was awarded the Nobel Prize in Physioogy or Medicine in 1945.

pen2.gif (5027 bytes)
Molecular model of penicillin

The discovery of penicillin by Fleming is widely known, but it was years after his discovery that the use of penicillin became widespread. The purification process to isolate the drug from the biological cultures in which it was made took years to develop, and it was terribly inefficient.   To obtain the penicillin needed to treat one patient for one day, it took 100 liters of the fermentation broth! In the early days, penicillin was extremely expensive, with prices in the twenty dollar per dose range! The antibiotics that we routinely complain about today don't approach that price.

Perhaps the most efficient purification process was by the soldiers that were treated in those early days; penicillin was recovered from the urine of the patients who had been treated and then used to treat other patients! This is recycling at its best!

The first penicillin that was widely used is benzylpenicillin, or Penicillin G. It was, for years, widely used for more bacterial infections than any other antibiotic. It was the prototype for the
development of the entire class of drugs now known as the "penicillins". Penicillin G possesses many of the qualities of an "ideal" antibiotic. It is relatively non-toxic to the host (or patient) and it is effective against a wide variety of organisms including fungi.

pen.gif (1337 bytes)
Structure of Penicillin G
Nother method of representation: angles represent the location of carbon atoms, hydrogens on carbons are not shown.  Pay particular attention to the four-membered ring called a -lactam.

Penicillin G is a highly acidic drug, with a pKa of 2.74. (Acidity in this case is a relative term.
Let's not forget that most drugs are weakly ionizable, defined as <5% ionized in aqueous solution. As drugs go, however, Penicillin G has a pKa that indicates it is more likely to ionize
than many other drugs.)  The free acid form is only sparingly soluble in water, so sodium, potassium, and calcium salts were made available. Let's try to predict their absorption from oral administration!

gitract.jpg (19869 bytes)

First, in the stomach where the pH =2:
pcn1.gif (2890 bytes)
pcn2.gif (915 bytes)
pcn3.gif (1049 bytes)
In  the duodenum, where the pH = 6:
pcn4.gif (2822 bytes)
or 99.95% ionized and only 0.05% unionized

And in the small intestines where the pH is 7.5:
pcn5.gif (2853 bytes)
This shows that there are 57,544 ionized molecules for every one unionized molecule. At the small intestines, penicillin G is 99.9983% ionized and only 0.0017% unionized.

So it would appear that conditions are very favorable for complete absorption to take place in
the stomach since the unionized form is overwhelmingly dominant. Is that what we find? Well,
no. You see, there are other chemical processes taking place! As it turns out, penicillin is quite unstable under acid conditions. The penicillin molecule is subject to several hydrolytic reactions, all yielding therapeutically inactive compounds. One of the three reactions that takes place in the presence of water is that the amide side chain is attacked in the presence of hydrogen ions. So in the acidic environment of the stomach, penicillin is broken down immediately and cannot get to the duodenum in active form!

[The term "penicillin resistance" is a result of another chemical reaction. Many microorganisms have enzymes that break down the "beta-lactam" ring portion of the penicillin molecule. These enzymes, called penicillinases, differ from one strain of bacteria to another, and different enzymes may have different degrees of specificity toward different "penicillins". The broken "beta-lactam ring" of the resulting molecule yields a compound that has no therapeutic activity. ]

Early attempts at improving the oral absorption of penicillin G exemplify the close relationship to pharmaceutics and chemistry. One of the first solutions tried was to administer each dose of penicillin G with a buffer (antacid) in an attempt to modify the acidity of the stomach. Drugs
such as magnesium trisilicate, sodium citrate, calcium carbonate, and aluminum hydroxide were co-administered with the oral penicillin, and the result was an increased absorption of the penicillin. But the doses required were still quite large, and production and purification of
penicillin was still an issue.

Let's take a look at the calculations for the duodenum again, and see how absorption would take place there. If the penicillin were to travel to the duodenum for absorption, there would be 99.95% of the amount as ionized drug. We see from our calculations that for every 1820 molecules of ionized drug there would be 1 molecule of unionized. Remember too that these
physiologic processes like absorption are an equilibrium phenomenon. If that one molecule of unionized penicillin crosses the lipid membrane to the blood where the concentration of penicillin is zero, then immediately an ionized molecule grabs an available hydrogen ion, becomes unionized,and immediately crosses the membrane. This process happens so quickly that it would appear almost instantaneous. The fluid at the surface of the duodenum is very concentrated with penicillin, ready to be converted to the unionized form as long as there are available hydrogen ions. As soon as unionized drug crosses the membrane, more drug is converted.

Remember,too, that the VERY large surface are of the duodenum provides plenty of opportunities for this absorption to take place. So even though mathematically it appears that absorption in the duodenum is unlikely, that is not exactly the case. It would be LESS likely to occur there than in the stomach, all else being equal. But with drug absorption, there are so many processes taking place simultaneously that to look at one process by itself would be misleading.

Now, back to our journey to discover how to get the most out of our penicillin dose. Another method aimed at improving the concentration of penicillin in the blood was to incorporate the salt form into an oily or waxy matrix, creating a depot effect. The salt dissolved slowly through the oily substances, and was released more slowly. This, too, produced higher levels of penicillin in the blood. But the quest continued. Finally an aqueous injectible form of penicillin was developed that would go directly into the body , and higher concentrations of penicillin were achieved by using smaller doses.

The injectible form of pencillin was an aqueous suspension, not a solution. The rather
water-insoluble procaine salt of penicillin G was used in this formulation, and it dissolved slowly enough in water to produce therapeutic levels of the drug.

Further modifications to the penicillin molecule have produced dozens of compounds. Changes in the amide side change have reduced the acid catalysis, resulting in improved absorption from the stomach. Members of the penicillin class of drugs have pKas within the
range of 2.5-3, so all would have the unionized form dominate in the stomach, as we saw in the example calculations for penicillin G. The newer penicillin derivatives are much more stable in an acidic environment, and are thus rapidly and readily absorbed from the stomach.


hospital.jpg (16939 bytes) Another problem with penicillin is that is excreted almost exclusively in the urine, and very rapidly.  In the early penicillin G days, 70% of an injectible dose  was recovered in the urine within two hours. That is why British field hospitals in 1943 were able to  recycle their insufficient supply of   penicillin from the urine of  their soldier patients.  And that is why it was so difficult to maintain therapeutic levels of penicillin in the blood long enough for the drug to be effective.

As soon as the drug was in the blood, it was filtered out through the kidneys. More importantly, the kidney has specialized transport mechanisms to remove ionized weak acids and bases from the blood. This process is called active tubular secretion. Remember that at the higher pH of the blood (7.4), these acidic compounds would be predominantly in the ionized state, and susceptible to removal from the blood by the active tubular secretion process. Chemists and pharmacologists then got together to try to determine a method to block the filtration of penicillin in the renal tubules. They were looking for an organic acid that would decrease the renal tubular secretion of penicillin, but that would not be excreted itself. (This is not a pH effect, but a modification of the renal filtration.)

A benzoic acid derivative was developed that fit the bill. N-dipropyl benzoic acid, or probenecid, was discovered, to inhibit  the active tubular renal secretion of penicillin and prolong its circulation in the blood.  Probenecid is coadministered with parenteral and oral penicillin in the treatment of gonorrhea and syphillis, and is still widely used today to maintain therapeutic levels of antibiotics for a longer period.


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Prof. Sally Boudinot
College of Pharmacy
University of Georgia
Athens, GA