Lecture 10 [Notes]

Chemotherapeutics are defined as certain specific chemicals that can be put 
into the human body: chemical agents, antibiotics, antibodies

They must fulfill the following criteria:

	1. they must destroy or prevent the activity of a parasite
	   WITHOUT injury to the host cell or cause ONLY LIMITED
	   damage to the host cell (SELECTIVE TOXICITY)
	2. they must be able to come into contact with the parasite
	   mainly by penetration through the tissues or cells
	3. they must NOT interfere with the host's natural defenses

Historical Overview

Mercury compounds were used to treat syphilis during the Renaissance. In the 
1880's an extract of the chinchona bark (quinine) was discovered as a treatment for 

malaria. The establishment of most modern day work began with Joubert, Tyndall and Pasteur.

One of the first chemotherapeutic agent came from the Gram - bacterium Pseudomonas. 
It produces a water soluble green dye, known as Pyocyanous, that inhibits 
Gram + rods - unfortunately this compound also hemolyzes red blood cells.

In 1899, Metchnikoff, a Russian microbiologist (who also noted phagocytosis of white blood 
cells), worked with the Gram + Lactobacillus acidophilus. The growth of 
this organism in the intestine will modify the intestinal pH. It changed the normally 
alkaline small intestine to acid. This in turn killed off large numbers of Gram - rods, 
such as the typhoid and Shigella organisms. To accomplish this the patients had 
to drink sour milk. Unfortunately there were a number of serious side effects.

During the time period 1906-1910, the chemist Paul Ehrlich sought a cure for syphilis. 
After several years and many trials he discovered Salvarsan (the 606th compound tested). 
This arsenic compound (arspenamide) served as the definitive treatment of syphilis for 
over 40 years - until the availability of penicillin

In 1924, Drs. Dath and Gratin from the University of Pennsylvania introduced Actinomycetin. 
They developed a technique which involved the filtrate of a lysed culture of a strain 
of actinomyces. It was active against Gram + cocci and spore forming rods but not effective 
against Gram - rods. Their technique became the basis for future work in extraction of 

In 1929, Alexander Fleming made his initial observations on the fungus Penicillium. 
This ultimately led to the isolation and commercial development of penicillin.

During 1936-1937 Dr. Damagh discovered sulfanilamide. This represented a big breakthrough
because it could be prepared commercially. The sulfa drugs provided most of the microbial
control of wounds during World War II. In 1942, penicillin became more available and 
practical but its use was not widespread.

After 1938, American microbiologists began to appreciate the importance of penicillin. 
Florey (1940) harvested experimental penicillin through the urine of treated patients 
and recrystalized it because there was so little available for use.

In 1939, Rene' Dubois discovered Gramicidin - unfortunately it was too toxic for 
protracted human use.
In 1944 two graduate students working for Selman Waksman of Rutgers University isolated 
and purified Streptomycin. This was the first of the broad spectrum antibiotics 
(works against both Gram + and Gram - organisms). Waksman received the Nobel Prize, 
Rutgers got the patent, and the graduate students got their degrees. 
The discovery of streptomycin, in conjunction with the avail-
ability of penicillin, ushered in what is known as The Age of Antibiotics.

	Modes of Action (How and Where Chemotherapeutic Agents Work)
	see also - Major Spectrum of Chemotherapeutic Agents

	1. Inhibition of cell wall synthesis - attacks an enzyme
	   which is involved in cross linkage of NAM-NAG; penicillin
	   is an analog of D-alanine and substitutes for it.
	2. Combines with the cell membrane and destroys it perme-
	   ability. Polymyxin-B works against some strains of
	   Pseudomonas. Poly-B has a + charged region which binds to
	   the - charged phospholipid in the cell membrane. The 	          
           lipid-soluble portion of Poly-B reacts with the rest of
	   the membrane making it porous.
	3. Inhibitors of Protein Synthesis - most of the broad spec-
	   trum antibiotics work this way. While most work only on 	          
           the smaller bacterial ribosomes, some can also interfere
	   with normal host cell protein synthesis. Long term use 
	   must be avoided for this reason.
	4. Alteration of Nucleic Acids - reacts with guanine residue
	   of DNA and links two strands together so that replication
	   is impossible. This prevents the formation of mRNA since 
	   it acts on the site of synthesis of m-RNA.

	Major Spectrum of Some Chemotherapeutic Agents

Compete with PABA (Para-AminoBenzoic Acid)
	Para-aminosalicylic acid (PAS)
Compete with Pyridoxine (Vitamin B6)
	Isonictotinic acid hydrazide (INH)
Inhibit cell wall peptidoglycan synthesis
Inhibit protein synthesis by binding to 50S subunit of ribosome
	Macrolide Antibiotics
Inhibit protein synthesis by binding to 30S subunit of ribosome
	Other aminoglycoside antibiotics
Disruption of cell membranes
	Polyene antibiotics
		Amphotericin B
Inhibit DNA synthesis (prevent replication and transcription)
	Nalidixic acid
Inhibit RNA synthesis
Inhibit purine synthesis (Adenine and Guanine)

	Complications of Chemotherapy

     A serious problem encountered with many chemotherapeutic agents is an allergic 
reaction developed by many patients. This type of sensitivity, called HYPERSENSITIVITY, 
elicits various reactions characteristic of allergic conditions. Skin rashes and fever 
are the most common manifestations, but a number of deaths have been attributed directly 
to antibiotic hypersensitivity.
     Penicillin is the antibiotic most frequently administered. Therefore, it is not 
surprising that it is responsible for more side reactions than any other drug. 
Paradoxically, penicillin is one of the least toxic of the antibiotics. It can be taken 
by most people in enormous quantities with no undesirable results.

The chart below gives some indication of the range of reactions associated with various 

	Adverse Reactions and Major Contraindications for the Use of
	Various Antibiotics
Antibiotic               Most Common Reaction or Contraindication 
Penicillins			Hypersensitivity shown by about 5 % of
Cephalosporins			similar to penicillin
Chloramphenicol		        Irreversible aplastic anemia
Erythromycin			Relatively nontoxic; jaundice in about
					0.4% of cases when used for over 10 days
Lincomycin & 			Diarrhea, severe colitis
Tetracyclines			Permanent staining of teeth and bones if
					given during last half of pregnancy up
					to 8 years of age; increased photosensi-
					tivity in some adults; gastrointestinal
Streptomycin &			8th nerve damage (may be irreversible),
Aminoglycosides		        skin eruptions; dizziness
Polymyxins			Toxic to kidneys
Nalidixic Acid			Gastrointestinal upset; rash; headache;
Trimethoprim			Rash; fever; kidney and liver damage;
					very rarely - cases of aplastic anemia
Sulfonamides			Similar to Trimethoprim

	Antibiotic Resistance (Why Some Organisms Become Resistant)
   1. the sensitive target structure may be missing in the 	 
	 resistant form (cell wall, enzyme, ribosome)
   2. the cellular structure that is the target of the antibiotic
	 may undergo an alteration so that it no longer binds the
	 antibiotic but can still carry out its normal function
   3. the resultant organisms may be impermeable to the anti-
      biotic (e.g. it may have developed a capsule)
   4. the organism may be able to modify the antibiotic to an
	 inactive form; e.g. certain organisms produce the enzyme
	 penicillinase which inactivates penicillin - 
	 PPNG (Penicillinase Producing Neisseria gonorrhea)

(see also: "Antibiotics that Resist Resistance," Science, Vol.270, pp.724-727, November 3, 1995)

No one drug is effective against all pathogens, so that care must be taken to select 
the appropriate drug.

	Additional Reasons for Ineffectiveness of Certain Chemicals

1. Many chemicals are inhibitory to pathogens in culture (IN          
       VITRO) BUT are ineffective against the same pathogen in an
       infected host (IN VIVO)
	a. the chemical may be inactivated or destroyed by the host
	b. the chemical may be poorly absorbed or rapidly excreted
	c. a high concentration must be maintained at the site of
	   the infection
	d. the pathogen may be alive in the host at some site in the
	   body where the drug cannot penetrate (e.g. dead tissue)
2. In some cases, treatment of the infectious disease requires
   more than the use of drugs.
3. Drugs may have toxic side effects or cause allergic reactions.
	a. the pathogen may become drug resistant
	b. the drug may reduce or destroy the body's normal flora
	c. drugs may permit SUPERINFECTION - a natural pathogen,
	   normally held in check by the normal flora, is able to
	   flourish once the normal flora is gone' e.g. Candida
	d. the ability of the body to develop immunity to the patho-
	   gen may be reduced if the pathogen is rapidly eliminated
	   by drug treatment
	e. drug interaction - two or more drugs taken together may
	   create a dangerous condition or inactivate one of the
	   drugs - consult your pharmacist or AccuFays computer

In very few infections is the drug alone responsible for a cure. Most of the defense and 
immunity systems of the body are essential to bring about a cure, even when a highly 
effective drug is used.

Drugs may also be used to prevent future infections in people who are unusually 
susceptible to them - chemoprophylaxis - e.g. surgical patients; penicillin to 
prevent streptococcal sore throats in rheumatic fever patients.

Antibiotic - defined as a chemical substance produced by a living organism that is 
capable of killing or inhibiting the growth of microorganisms.

Three groups of microorganisms are responsible for the production of most of the 
antibiotics used in medicine.
   1. fungi - especially the genus Penicillium (penicillin and
	 griseofulvin); provides molecular backbone for semisynthetic penicillins
   2. bacteria of the genus Bacillus (bacitracin, polymyxin)
   3. actinomycetes (filamentous bacteria) of the genus
	 Streptomyces (streptomycin, chloramphenicol, erythromycin,
	 tetracycline); most antibiotics come from this group

Antibiotics capable of effective action on both Gram + AND Gram -
bacteria are known as BROAD SPECTRUM antibiotics.

Testing of Antibiotics
	activity is measured by determining the smallest amount of 	
the agent needed to inhibit the growth of a test organism

1. (MIC) Minimum Inhibitory Concentration - often called the tube
   dilution technique. It does not provide an absolute constant 
   for a given agent since it is affected by: the kind of test 
   organism used, inoculum size, composition of culture medium,
   and incubation conditions such as temperature, pH, aeration.
   With rigorous standardization, comparisons are possible. This
   procedure does not distinguish between CIDAL or STATIC agents
   since the chemical agent is present throughout the growth period.

2. Agar Diffusion or Disk Diffusion Method - most widely used 
   method. The standardized version is known as the KIRBY-BAUER
   test. A zone of no growth called a ZONE OF INHIBITION occurs
   if the organisms are killed by the concentration of antibiotic
   in the paper disk. The size of the zone is affected by: the
   sensitivity of the test organism, type of culture medium, 
   incubation conditions, rate of diffusion of the agent within
   the medium, and concentration of the antibiotic. In order to
   interpret the results, comparison with standardized tables is 
   required. These tables provide information on the size of the
   zone (in vitro) required to be effective within the body (in vivo).
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