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.