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MEDCH 561P
Immunizing and Antimicrobial Agents
http://botit.botany.wisc.edu/toms_fungi/nov2003.html
April 2, 2012Kelly Lee, Ph.D.
H-172Jkklee@u.washington.edu
Penicillium chrysogenum + Staph. Aureus
Brett Finlay, HHMI
E. coli growing under nutrient-rich conditions
Overview for Week 1
๏Impact of antimicrobials on health and health care
๏History and background
๏Mechanisms of action
๏Resistance
REFERENCES
Taylor JB, Triggle DJ (eds), Comprehensive Medicinal Chemistry II, vol 7
Lemke TL, Williams DA, Roche VF, Zito SW, Foye’s Principles of Medicinal Chemistry, 6th Edition (2008)
Murray PR, Rosenthal JS, Pfaller MA, Medical Microbiology, 6th Edition (2009)
Brunton LL, Lazo JS, Parker KL, Goodman & Gilman’s The Pharmacological Basis of Therapeutics, 11th
Edition (2005)
Drusano GL, Antimicrobial Pharmacodynamics: Critical Interactions of ‘Bug and Drug’ (2004)
Nature Reviews Microbiology 2:289-300
Wright GD, The Antibiotic Resistome: The Nexus of Chemical and Genetic Diversity (2007)
Nature Reviews Microbiology 5:175-186
Armstrong GL et al., (1999) JAMA 281:61-66
Infectious disease mortality rate 1900-1996
๏ A combination of improved sanitation, access to clean water, hygiene, vaccination, and antibiotics led to a decrease in mortality starting around the industrial revolution (late 19th century)
Armstrong GL et al., (1999) JAMA 281:61-66
Infectious disease mortality rate 1900-1996
๏ A combination of improved sanitation, access to clean water, hygiene, vaccination, and antibiotics led to a decrease in mortality starting around the industrial revolution (late 19th century)
1918 influenza pandemic~50-100 million deaths (~3% of world population)~30% of world population infected
Life in a pre-antimicrobial world
Tuberculosis (Mycobacterium tuberculosis)
Typhus (Rickettsia prowazekii)
Scarlet Fever (Streptococcus pyogenes)
Pneumonia (Streptococcus pneumonia, Haemophilus influenzae)
Diphtheria (Corynebacterium diphtheriae)
Cholera (Vibrio cholerae)
๏ Before antibiotics Streptococcus pyogenes responsible for ~50% of infant mortality
๏ S. pyogenes also frequently caused death in infected burn wounds
๏ Staphylococcus aureus fatal in nearly 80% of infected wounds
๏ Prior to antibiotics, wound infection killed more soldiers than weapons in warfare
๏ Mortaility due to infectious disease was ~20x higher in 1900 than now
Impact on health care
๏ In 2004, total global trade in antibiotics > $27 billion
๏ Beta-lactams accounted for ~45%
๏ 6 antibiotics topped $1 billion in sales:
๏ ceftriaxone (beta-lactam)
๏ amoxicillin/clavulanate (beta-lactam+beta-lactamase inhibitor)
๏ azithromycin (macrolide)
๏ clarithromycin (macrolide)
๏ ciprofloxacin (fluoroquinolone)
๏ levafloxacin (fluoroquinolone)
Taylor JB and Triggle DJ (eds), Comprehensive Medicinal Chemistry II, vol7Walsh CT, Antibiotics: Actions, origins, resistance, (2003) ASM Press, Washington DC, USA
Coates A, et al., Nature Rev. Drug Discovery (2002) 1:895-910
Impact on health care๏ Antibiotics make up a substantial fraction of prescriptions:๏ ~42% of patients admitted to hospitals receive antibiotics๏ ~50% of drug prescription costs go to antibiotics, where prescription costs amount to
10-15% of total health care costs
๏ Most likely place for an individual to acquire an antibiotic-resistant infection is the ICU
๏ U. Michigan Health System study: In 2002, 41 million antibiotic prescriptions for people suffering from colds (viral), more than one-third of patients who saw a doc about a cold (Feb 24, 2003 edition of Archives of Internal Medicine)
Selective Toxicity
Antimicrobial: Microbial secondary metabolites or synthetic compounds that in
small doses inhibit the growth and survival of microorganisms without serious
toxicity to the host (us)
Antibiotic: Natural product subset of antimicrobials
We are >90% bacterial
For our 10 trillion cells, ~100 trillion bacterial cells make their home in and on us.
Introduction of antimicrobials impacts the pathogens as well as our flora.
Implications for side effects and the emergence/harboring of resistance.
Ways commensal bacteria impact our health
๏ In many cases the flora-host interaction is mutualistic. Commensal bacteria provide for us:๏ Aid in digestion of food and production of vitamins๏ Processing of nutrients and drugs in our gut๏ Prevent establishment of pathogenic competitors๏ Immunity๏ Imbalance can impact asthma
Organs and internal tissues are normally sterile. Commensal bacteria do
colonize “exterior” including skin, gut, respiratory tract, mouth, eyes,
urogenital tract, etc.
Normal flora
Something to keep in mind for the future:Native flora is specific to an individual, impact of treatment may differ significantly
๏ Appears human microbiome of the gut may be categorizable into 3 main “enterotypes”, each dominated by a main genus
๏ Not related to nation, ethnicity, gender, or age๏ There may be a link between the enterotype found in an individual and
susceptibility to disorders/diseases “Enterotypes of the human gut microbiome”Arumugam et al., Nature (2011)
472:7343
Impact on our microbial flora
๏ The antimicrobials are not so specifically targeted that they knock out only the primary pathogen, they can act potently against other species of the flora.
๏ Can compromise the balanced bacterial ecology, especially of the gut๏ e.g. leading to diarrhea, Clostridium difficile overgrowth
๏ The flora can also be reservoirs for transferable resistance factors (R-factors).๏ After antibiotic treatment, R-factors can be detected even during the course of
the therapy, and persist for years
Sources of pathogenic bacterial infections
๏ Some of the commensal bacteria may become pathogens if they gain access to normally sterile internal sites through wounds, medical device insertion, etc. OPPORTUNISTIC PATHOGENS.
๏ Compromised immune systems also can create opportunities for pathogenesis.
๏ Some pathogens are extrinsic and not related to our commensal flora.
Organs and internal tissues are normally sterile. Commensal bacteria do
colonize “exterior” including skin, gut, respiratory tract, mouth, eyes,
urogenital tract, etc.
Some non-commensal pathogens๏ Mycobacterium tuberculosis (“acid-fast”; waxy mycolic acid cell surface)
๏ Acinetobacter baumannii (gram-, highly resistant to most antimicrobials; significant
source of hospital-acquired infections)
๏ Klebsiella pneumoniae (gram-, significant source of hospital-aquired infections)
๏ Stenotrophomonas maltophilia (gram-, naturally resistant to broad spectrum
antimicrobials; medical devices)
๏ Burkholderia cepacia (gram-; naturally resistant to many antimicrobials including
polymyxin; transmissible, pathogenic; cystic fibrosis)
๏ Neisseria gonorrhoeae
๏ Treponema pallidum
๏ Chlamydia trachomatis
๏ Bacillus anthracis (gram+; environmental exposure, bioweapon)
๏ Salmonella typhi
History๏ 1877 concept of “antibiosis” first documented when Louis Pasteur observed that a “common bacterium”
could inhibit the growth of Bacillus anthracis
๏ 1908 Paul Erlich (w/ Sahachiro Hata) developed Salvarsan a synthetic organoarsenical treatment for
syphilis and trypanosomiasis (sleeping sickness). Erlich developed the “magic bullet concept”, proposing
that one could screen chemical compounds for selective anti-microbial activity.
๏ 1928 Alexander Fleming observed Penicillium notatum (now classified P. chrysogenum) killing staphylococci
๏ 1931-1936 Gerhard Domagk with others at Bayer Lab discovered and tested Prontosil, the first
sulfonamide drug, effective against gram+ bacteria
๏ 1939 Rene Dubos isolated gramicidin (Bacillus brevis), one of the first commercially produced antibiotics to
prove highly effective as a topical treatment for wounds and ulcers; used in World War II
๏ 1940 Flory, Chain, Heatley led industrialization of penicillin, increased yields 100-fold
๏ 1945 Benjamin Duggar discovered the first of the tetracyclines, the first broad-spectrum antibiotics from
Streptomyces, a soil bacterium
๏ 1948 Guiseppe Brotzu isolated first cephalosporins from Cephalosporium acremonium active against
Salmonella typhi that expresses beta-lactamases (knock out penicillins).
๏ 1952 Selman Waksman (and Albert Schatz) streptomycin from Streptomyces griseus against TB;
aminoglycosides from soil bacteria; coined the term “antibiotics”
๏ In late 1999 the development of a drug called Zyvox was finally announced. It’s in a new class of antibiotics
called the Oxazolidinones. It’s claimed to be effective against multi-resistant strains of bacteria.
History
๏ 1943 Mary Hunt, a lab worker in Peoria, Illinois, found a moldy cantaloupe at a market sporting a strain
of P. chrysogenum that combined with improvements in fermentation recipes, increased yields so
dramatically that 2.3 million doses were available by D-Day, 1944.
๏ By 1942, the first patient was treated for septicemia, using up half of the entire world’s supply of
penicillin
History
Wright GD (2007) Nature Reviews Microbiology 5:175
History๏ 2000 linezolid (Zyvox; Pfizer) approved by FDA. Represents a new class of synthetic antimicrobials
called the oxazolidinones (discovered in the 1980s). Effective against multi-resistant strains of
gram+ bacteria.
๏ 2003 daptomycin (Cubicin; Cubist and Novartis) from Streptomyces roseosporus approved by FDA
(discovered in 1980s). A lipopeptide that acts by depolarizing the membrane of gram+ bacteria.
๏ 2005 tigecycline approved (Tygacil; Wyeth). Glycylcycline class anti-microbial with broad spectrum
activity including against MRSA. Structurally similar to tetracycline.
Future?๏ Many fewer antibiotics are in development and being approved now than in previous decades due
to economic and regulatory factors
๏ 1983-1987: 16 approved๏ 1988-1992: 14๏ 1993-1997: 10๏ 1998-2002: 7๏ 2003-2007: 4๏ 2007-present: 2
๏ But bacterial resistance to existing therapeutics is increasing
What Fleming saw
A Kirby-Bauer disc test
Don Stanlons; phil.cdc.gov
disc saturated with antibioticplated with
Staph. Aureus
Where do antimicrobials come from?
www.flickr.com/photos/ ajc1/2902232380/
ANTIBIOTICS:Natural products ofmicrobial warfare
ANTIBACTERIALS:Synthetic products from
chemical screens
Arsphenamine (Salvarsan, compound 606)
1910 used to treat syphilis
the first chemotherapeutic drug
Penicillium chrysogenum
Streptomyces
Where do antimicrobials come from?ANTIBIOTICS:
Natural products ofmicrobial warfare
ANTIBACTERIALS:Synthetic products from
chemical screens
๏ Beta-lactams:๏ Penicillins๏ Cephalosporins๏ Carbapenems๏ Monobactams
๏ Aminoglycosides๏ Macrolides๏ Tetracyclines๏ Daptomycin (Cubicin)๏ Vancomycin๏ Chloramphenicol๏ Bacitracin๏ Phosphomycin๏ Polymyxin
๏ Sulfa drugs (sulfonamides)๏ Quinolones๏ Linezolid (Zyvox)
Production of pigmented secondary metabolites by Streptomyces colonies. (a) Typical colonial morphologies of Streptomyces isolated from the soil. Colonies often excrete colored pigments, providing a visual recording of secondary metabolite biosynthesis. The chemically diverse compounds represent a vast array of bioactive compounds that often have pharmaceutical applications. (b) A panoramic view of Streptomyces coelicolor colonial morphology. Both
Actinobacteria: especially genus Streptomyces
๏Erythromycin (Saccharopolyspora erythrea)
๏Neomycin (S. fradiae)
๏Streptomycin (S. griseus)
๏Tetracycline (S. rimosus)
๏Vancomycin (S. orientalis)
๏Daptomycin (S. roseosporus)
๏Rifamycin (Amycolatopsis rifamycinica/S. mediterranei)
๏Chloramphenicol (S. venezuelae)
๏Puromycin (S. alboniger)
๏Lincomycin (S. lincolnensis)
๏Cefoxitin (S. lactamdurans)
๏ Approximately two-thirds of the antibiotics (natural products) in use are from Streptomyces, including:
Thompson CJ et al. (2002) Genome Biology 3:reviews1020
๏Medicinal chemistry starting from the natural products to produce
derivatives with greater potency, broader antimicrobial spectrum, lower
toxicity, and improved pharmacokinetic profile, and to overcome
resistance
๏ 1950s and ‘60s much work along these lines, especially for the beta-
lactams
Semi-synthetic derivatives
General concepts
Bioavailability
๏ Before the anti-microbials such as penicillin, arsenicals, and sulfa drugs,
topical antiseptics were the only tools available for treating infection.
๏ Penicillin in particular provided low host toxicity, high potency that
could get to the site of infection and permeate it.
๏ The drug must get to its target
๏ Attain adequate concentrations to produce desired effect w/o toxicity
๏ Tissue penetration
๏ Penetrate biofilms
๏ Bacterial cell penetration to bind to the target
Therapeutic Control of Infection
๏ Bacteriostatic: some antimicrobials do not necessarily kill the bacteria
๏ Break the logarithmic growth phase, allowing the immune system to deal
with the infection
๏ Bactericidal: kill the bacterium
๏ Weaken the cell wall, leading to lysis (e.g. penicillins)
๏ Disrupt DNA replication (quinolones: DNA gyrase and topoisomerase IV)
๏ Disrupt RNA synthesis (rifampin: RNA polymerase)
๏ Disrupt protein synthesis (some however are bacteriostatic)
๏ Some drugs that are bacteriostatic at lower concentrations can be -cidal at
higher concentrations
MIC: Minimum Inhibitory ConcentrationMBC: Minimum Bactericidal Concentration
medium drug+
medium drug-
MBC
MIC
[drug]
KILLED partially inhibited
notinhibited
Minimum Inhibitory Concentration: lowest concentration of drug that gives no visible growth after 24h incubation
Minimum Bactericidal Concentration: concentration of drug that gives no visible growth even in the absence of drug
Concentration-time curve
conc
entr
atio
n
MIC
CMAX
time post-administration
AUC (Area Under the Curve)
๏ MIC: minimum inhibitory concentration
๏ AUC: a measure of the total exposure to the drug
๏ Cmax: maximum concentration attained
Time-dependent killing
๏ Beta-lactams
๏ Length of time where concentration>MIC is most important
๏ So long as concentration>MIC, absolute concentration does not matter too much
๏ Related to number of target enzymes for the drug and their saturation
๏ Frequent, lower doses
conc
entr
atio
n
MIC
t>MIC
CMAX
time post-administration
Concentration-dependent killing
๏ Aminoglycosides and fluoroquinolones
๏ AUC/MIC is the critical index for effective control
๏ Concentration-dependent mechanism of killing
๏ Relates to barriers in drug getting to target site.
๏ Less frequent, higher doses
conc
entr
atio
n
MIC
CMAX
time post-administration
Time versus concentration-dependent activity
Craig WA, “Pharmacokinetic/pharmacodynamic parameters: rationale for antibacterial dosing of mice and men,” (1998) Clin. Infect. Dis. 26:1-12
beta-lactamaminoglycoside fluoroquinolone
Post-antibiotic effect (PAE)
๏ Post-antibiotic effect: after initial dosing, even when concentration in the
blood drops below the MIC, microbial growth may be inhibited
๏ Believed to be due to either drugs that remain bound or are
concentrated in/near infection or due to damage to the bacteria that
they must repair before they can recommence their growth
AUC/MIC but not concentration-dependent killing
๏ Example: Vancomycin, macrolides (e.g. azithromycin)๏ AUC/MIC is the critical index for effective control, but higher concentrations not
necessarily more effective above a certain point๏ Strong post-antibiotic effect (PAE), likely reflects antibiotic-induced cell damage๏ The duration of the PAE is increased by increasing the AUC/MIC๏ When MIC is relatively high and AUC/MIC is low, these drugs may act similarly to
time-dependent agents
conc
entr
atio
n
MIC
CMAX
time post-administration
Time versus concentration-dependent activity
Antimicrobialclass Goal of therapy index
type Iconc-dependent
killing long PAE
aminoglycosidesdaptomycin
fluoroquinolonesketolides (telithromycin)
maximize concentrationAUC/MICCmax/MIC
type IItime-dependent
killing minimal PAE
beta-lactamslinezolid
erythromycin, clarithromycinmaximize duration of
exposure
type IIItime-dependent
killingmoderate to
long PAE
tetracyclinesglycopeptide (vancomycin)
clindamycinmaximize total exposure AUC/MIC
t>MIC
Example AUC/MIC, T>MIC values
๏ Fluoroquinolones vs gm+ bacteria AUC/MIC>40
๏ Fluoroquinolones vs gm- bacteria AUC/MIC>125
๏ Aminoglycosides, Cmax/MIC~8-10
๏ Beta-lactams, T>MIC comparable to dosing interval
๏ Vancomycin, AUC/MIC >125-400
Varability in bioavailability across populations
๏ The same dosage to a population of patients will result in a broad range of effective drug concentrations (exposures) in those patients.
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