School of Medicine, Zhejiang University Chemotherapeutic Drugs Wei-Ping Zhang, PhD 张纬萍 Dept. of Pharmacology, School of Medicine, Zhejiang University weiping601@zju.edu.cn 2013.12.16

General Considerations for chemotherapeutic drugs Chapter 34 General Considerations for chemotherapeutic drugs

Overview I. Chemotherapy II. Chemotherapeutic agents III. Mechanisms under the action of chemotherapeutic agents IV. Bacterial Resistance V. Basic principle of clinical usage of antimicrobial agents The use of chemical substances to treat disease (??actually I think not). In its modern-day use, it refers primarily to cytotoxic drugs used to treat cancer. In its non-oncological use, the term may also refer to antibiotics (antibacterial chemotherapy).

The Birth of Modern Chemotherapy: Dreams of a “Magic Bullet” I. Chemotherapy Louis Pasteur 1822-1895 The Birth of Modern Chemotherapy: Dreams of a “Magic Bullet” Robert Koch 1843-1910 Rapid advances in the science of microbiology were made between 1857 and 1914. Louis Pasteur (pronounced: [lwi pastœʁ] December 27, 1822 – September 28, 1895) was a French chemist and microbiologist born in Dole. He is remembered for his remarkable breakthroughs in the causes and preventions of diseases. His discoveries reduced mortality from puerperal fever, and he created the first vaccine for rabies and anthrax 巴氏消毒法。 Heinrich Hermann Robert Koch ([ˈkɔx]; 11 December 1843 – 27 May 1910) was a Prussian physician. He became famous for isolating Bacillus anthracis 炭疽杆菌(1877), the Tuberculosis bacillus (1882) and the Vibrio cholerae霍乱弧菌 (1883) and for his development of Koch's postulates. [1]. Rebecca Lancefield is most famous for her serological classification 血清学分类 of beta-hemolytic streptococcal bacteria 溶血性链球菌[1], which is based on the carbohydrate composition of bacterial antigens found on their cell walls.[2] The Lancefield classification system is still used internationally today. Rebecca Lancefield 1896-1981

1928 Fleming discovers penicillin I. Chemotherapy Paul Ehrlich introduced an arsenic-containing chemical called salvarsan （阿斯凡纳明）to treat syphilis （梅毒） (1910). “Magic bullet” for treatment of syphilis 1928 Fleming discovers penicillin Rapid advances in the science of microbiology were made between 1857 and 1914.

History of Antimicrobial Therapy I. Chemotherapy History of Antimicrobial Therapy 1928 Alexander Fleming observed that the mold (fungus) Penicillium inhibited the growth of a bacterial culture. He named the active ingredient penicillin (1928).

History of Antimicrobial Therapy I. Chemotherapy History of Antimicrobial Therapy 1928 Fleming discovers penicillin (青霉素) 1932 Domagk discovers sulfonamides （磺胺类） 1940s Penicillin and streptomycin （链霉素） used widely, cephalosporins （头孢霉素） discovered 1947 Chloramphenicol （氯霉素）discovered, first broad spectrum agent 1950s Tetracycline（四环素） in use 1952 Erythromycin（红霉素） discovered (macrolides，大环内脂类) 1956 Vancomycin（万古霉素） used for penicillin-resistant S. aureus 1957 Kanamycin（卡那霉素） discovered (aminoglycosides，氨基糖苷类) 1962 Nalidixic acid（奈啶酸） discovered (quinolones，喹诺酮类) 1980s Fluoroquinolones（氟喹诺酮）, broad spectrum cephalosporins（广谱头孢类） 2000s Newer agents to combat resistant pathogens

History of Antimicrobial Therapy I. Chemotherapy History of Antimicrobial Therapy Endless way ……………… MRSA，NAM-1 Superbug……drug resistance

II. Chemotherapeutic agents

II. Chemotherapeutic agents Host Factors： patient’s age, gender, constitution, hepatic, renal function Adverse effects Resistance Pharmacokinetics Therapeutic Effects pathogenicity Interactions between drug , human body, and pathogens Immunological responses

Antineoplastic / anticancer drugs II. Chemotherapeutic agents Antimicrobial drugs Antibacterial drugs Antifungal drugs Antiviral drugs Antiparasitic durgs Antineoplastic / anticancer drugs

Ideal antimicrobial drugs II. Chemotherapeutic agents Ideal antimicrobial drugs High sensitivity Nontoxic or low-toxic (safety) Nonresistance Satisfied pharmacokinetic properties Good price

Antibacterial drugs (抗菌药) kill bacteria and arresting its growth II. Chemotherapeutic agents Antibacterial drugs (抗菌药) kill bacteria and arresting its growth antibiotics and synthetic antimicrobial agents such as sulfonamides(磺胺类) and quinolones (喹诺酮类).

Suppress the growth of other microorganisms. II. Chemotherapeutic agents Antibiotics（抗生素） Produced by various species of microorganisms (bacteria, fungi , actinomycetes) and semi-synthetic Suppress the growth of other microorganisms.

Antibacterial spectrum（抗菌谱） Narrow? Broad? II. Chemotherapeutic agents Antibacterial spectrum（抗菌谱） Narrow? Broad? Chemotherapetic index (CI)（化疗指数） CI= LD50 / ED50 CI= LD5 / ED95 Narrow spectrum drugs are only active against a relatively small number of organisms. In general, narrow spectrum antibiotics are effective against Gram-positive organisms.

II. Chemotherapeutic agents 药理作用 中毒作用 致死作用 TD50 LD5 ED95

Bacteriostatic drugs （抑菌药） inhibit the growth of microorganisms II. Chemotherapeutic agents Bacteriostatic drugs （抑菌药） inhibit the growth of microorganisms e.g. Sulfonamides, Tetracycline Bactericidal drugs （杀菌药） kill microorganisms e.g. Penicillin, Aminoglycosides

Bactericidal vs Bacterostatic II. Chemotherapeutic agents Bactericidal vs Bacterostatic

Minimum inhibitory concentration (MIC) 最低抑菌浓度 II. Chemotherapeutic agents Minimum inhibitory concentration (MIC) 最低抑菌浓度 Minimum bactericidal concentration (MBC) 最低杀菌浓度 Post antibiotic effect (PAE) 抗生素后效应 Resistance （耐药性） Cross Resistance （交叉耐药性） First expose effect （首次接触效应） 7.Minimum inhibitory concentration (MIC) The lowest concentration of an antimicrobial that will inhibit the visible growth of a microorganism after overnight incubation Used by diagnostic laboratories mainly to confirm resistance, but most often as a research tool to determine the in vitro activity of new antimicrobials, and data from such studies have been used to determine MIC breakpoints. 8. Minimum bactericidal concentration (MBC) The lowest concentration of antimicrobial that will prevent the growth of an organism after subculture on to antibiotic-free media. MBC determinations are undertaken less frequently and their major use has been reserved for isolates from the blood of patients with endocarditis 9. Post antibiotic effect (PAE) Refers to a period of time after complete removal of an antibiotic during which there is no growth of the target organism Several factors influence the presence or duration of the PAE including the type of organism, type of antimicrobial, concentration of antimicrobial, duration of antimicrobial exposure, and antimicrobial combinations.

II. Chemotherapeutic agents 最低抑菌浓度 最低杀菌浓度

最低抑菌浓度 最低杀菌浓度

II. Chemotherapeutic agents Incubate 18 to 24 hr at 37℃ Measure diameters of nongrowth zones Disk diffusion method for testing bacteria for susceptibility to specific antimicrobial drugs.

III. Mechanism of action Chloramphenicol Transacetylase

1. Inhibit synthesis of bacterial cell walls III. Mechanism of action 1. Inhibit synthesis of bacterial cell walls 2. Affecting permeability of cell membrane and leading to leakage of intracellular compounds 3. Inhibit protein synthesis 4. Affect bacterial nucleic acid metabolism 5. Block essential enzymes of folate metabolism The antimetabolites

Inhibiting synthesis of bacterial cell walls III. Mechanism of action Inhibiting synthesis of bacterial cell walls e.g. penicillins, b-lactams

III. Mechanism of action UDP-乙酰葡萄糖胺 The 1st stage precursor(UDP-acetylmuramyl-pentapeptide, 乙酰胞壁酸-5肽，又称park核苷) formation, takes place in the cytoplasm. The 2nd stage UDP- acetylmuramyl -pentapeptide and UDP-acetylglucosamin（UDP-乙酰葡萄糖胺） are linked to form a long polymer, with the release of the uridine nucleotides(尿嘧啶核苷酸). The 3rd stage the completion of the cross-link, accomplished by a transpeptidation reaction. 乙酰胞壁酸-5肽 乙酰胞壁酸-5肽

2. Affecting permeability of membrane Ionic- sorbent （离子吸附剂） III. Mechanism of action 2. Affecting permeability of membrane Ionic- sorbent （离子吸附剂） e.g. Aminoglycosides （氨基糖苷类） Binding to ergosterol （麦角固醇） e.g. Nystatin (制霉菌素) Amphotericin B（两性霉素） Cationic detergent e.g. polymyxins（多粘菌素） Ionic- sorbent离子吸附剂 Cationic detergent：含有多个阳离子极性基团和一个脂肪酸直链肽，其阳离子与胞浆膜中的磷脂结合，使膜功能受损。

2. Affecting permeability of membrane III. Mechanism of action 2. Affecting permeability of membrane Lipopoly -saccharide Outer membrane Peptidoglycan Cytoplasmic polymyxins

Ribosomal structure 3. Inhibiting protein synthesis III. Mechanism of action 3. Inhibiting protein synthesis Ribosomal structure Bacteria 30S + 50S 70S 30S subunit binds mRNA in initiation complex holds growing peptide chain 50S subunit accepts / translocates charged tRNAs "A" site --> Aminoacyl-tRNA (acceptor) site "P" site --> Peptidyl-tRNA (donor) site Mammals 40S + 60S 80S

3. Inhibiting protein synthesis III. Mechanism of action 3. Inhibiting protein synthesis P A ①起始阶段：抑制70S始动复合物的形成。 ②肽链延长阶段：与靶蛋白结合，使mRNA密码错译而生成异常的，无功能的蛋白质。 ③终止阶段：阻碍终止因子与核蛋白体结合，使已合成的肽链不能释放；阻止70S核蛋白体解离，阻断核糖体循环（使核糖体耗竭）。

3. Inhibiting protein synthesis III. Mechanism of action 3. Inhibiting protein synthesis P A 大环内酯类 氨基糖苷类 氯霉素 林可霉素 四环素 ①起始阶段：抑制70S始动复合物的形成。 ②肽链延长阶段：与靶蛋白结合，使mRNA密码错译而生成异常的，无功能的蛋白质。 ③终止阶段：阻碍终止因子与核蛋白体结合，使已合成的肽链不能释放；阻止70S核蛋白体解离，阻断核糖体循环（使核糖体耗竭）。 氨基糖苷类

4. Affecting bacterial nucleic acid metabolism III. Mechanism of action 4. Affecting bacterial nucleic acid metabolism (-) Break back segment (+) quinolones Gyrase:DNA回旋酶，又称拓扑异构酶II Rifampicin (利福平): inhibit DNA-dependent RNA polymerase Ridarabine (阿糖腺苷), Ganciclovir (更昔洛韦) inhibit DAN polymerase

Pteridine + PABA Dihydropteroic acid Dihydrofolic acid III. Mechanism of action 5. block essential enzymes of folate metabolism Pteridine + PABA Blocked by sulfonamides Dihydropteroic acid Dihydrofolic acid glutamate Tetrahydrofolic acid Blocked by trimethoprim NADPH Dihydropteroate synthase Dihydrofolate reductasease 蝶啶 对氨基苯甲酸 二氢蝶酸 Pteridine蝶啶 PABA 对氨基苯甲酸 Dihydropteroic acid二氢蝶酸 Dihydrofolic acid二氢叶酸 Tetrahydrofolic acid四氢叶酸 Sulfonamides磺胺类 Trimethoprim甲氧苄啶 甲氧苄啶

– Inherent features ，usually expressed by chromosomal genes IV. Bacterial Resistance Intrinsic resistance – Inherent features ，usually expressed by chromosomal genes Acquired resistance – Emerge from previously sensitive bacterial populations – Caused by mutations in chromosomal genes – Or by acquisition of plasmids or transposons Intrinsic resistance 如链球菌对氨基糖苷类抗生素耐药；G阴性杆菌对青霉素耐药 Acquired resistance 如金葡菌对青霉素产生耐药，部分可遗传；

IV. Bacterial Resistance

Bacterial Resistance- Mechanisms IV. Bacterial Resistance Bacterial Resistance- Mechanisms The drug is not active. The target is altered. The drug does not reach its target.

IV. Bacterial Resistance 1.The drug is not active. Production of aminoglycoside-modifying enzymes and β-lactamase; B内酰胺酶 氨基糖苷类抗生素钝化酶 氯霉素乙酰转移酶 灭活大环内酯类的酯酶 核苷转移酶灭活林可霉素

IV. Bacterial Resistance 2.The target is altered Mutation of the natural target (quinolone resistance) Substitution with a resistant alternative to the native, susceptible target (methicillin（甲氧西林） resistance) A gene in the nucleoid or in a plasmid may code for production of an enzyme that is able to destroy or inactivate the antibiotic. 如肺炎链球菌对青霉素的耐药 MRSA产生青霉素结合蛋白-2a 如肠球菌对青霉素的耐药：产生酶、增加青霉素结合蛋白、减低青霉素结合蛋白与抗生素的亲和力

IV. Bacterial Resistance 2.The target is altered Target modification (ribosomal protection type of resistance to macrolides and tetracyclines)

3.The drug does not reach its target IV. Bacterial Resistance 3.The drug does not reach its target Absence, mutation or loss of the appropriate transporter or porins (膜孔蛋白） 如铜绿假单胞菌

3.The drug does not reach its target IV. Bacterial Resistance 3.The drug does not reach its target Active efflux system (主动排出系统) Efflux transporter（转运子） Accessory protein （附加蛋白） Outer membrane channel（外膜蛋白） 如大肠杆菌、金葡菌、表皮葡萄球菌等对四环素、氟喹诺酮类、大环内酯类、氯霉素、b内酰胺类耐药 外膜蛋白在外膜或者细胞壁

Active efflux system（主动排出系统 ） IV. Bacterial Resistance Active efflux system（主动排出系统 ） transporter Accessory protein Outer membrane channel

IV. Bacterial Resistance There are five families of multidrug-resistance efflux pumps: the ATP-binding cassette (ABC) superfamily, the major facilitator superfamily (MFS), the multidrug and toxic-compound extrusion (MATE) family, the small multidrug resistance (SMR) family and the resistance nodulation division (RND) family. A diagrammatic representation of the structure and membrane location of efflux pumps from each of these families is shown. Common examples of the individual proteins that form each class of efflux pump are indicated. Antibiotic substrates and examples of other substrates are also listed for each class of efflux pump. Multidrug-resistance efflux pumps expressed by Gram-negative bacteria usually have several components, and the outer-membrane protein is typically TolC. Pi, inorganic phosphate. Laura J. V. Piddock Nature Reviews Microbiology 4, 629-636 (August 2006)

The transfer of Resistance genes IV. Bacterial Resistance The transfer of Resistance genes From human  human From bacteria  bacteria Intracellular Mutations 突变 Transduction 转导 Transformation 转化 Conjugation 接合

IV. Bacterial Resistance Mutations 突变 如细菌对喹诺酮类、利福平的耐药性都是通过突变引起的；

Mutations 突变 May occur in the gene encoding The target protein IV. Bacterial Resistance Mutations 突变 May occur in the gene encoding The target protein A protein involved in drug transport A protein important for drug activation A regulatory gene or promoter affecting expression of the target, a transport protein, or an inactivating enzyme

Transduction 转导 IV. Bacterial Resistance Transduction is acquisition of bacterial DNA from a bacteriophage (噬菌体）that has incorporated DNA from a previous host bacterium within its outer protein coat. Some phages can carry plasmids that code for penicillinase, or genes encoding resistance to erythromycin, tetracycline, or chioramphenicol.

Transformation 转化 Conjugation 接合 IV. Bacterial Resistance Transformation Uptake and incorporation of DNA that is free in the environment into the host genome by homolgous recombination. Conjugation The passage of genes from cell to cell by direct contact through a sex pilus or bridge is termed comjugation. 结合需要通过性菌毛或桥接进行基因传递的过程：耐药决定基因和耐药转移基因。

IV. Bacterial Resistance

Multi-drug resistance (MDR) IV. Bacterial Resistance Multi-drug resistance (MDR) Methicillin-resistant staphylococcus aureus, MRSA 甲氧西林耐药金黄色葡萄球菌 Methicillin-resistant coagulase negative staphylococci, MRCNS 甲氧西林凝固酶阴性葡萄球菌 PBP-2a （a 78kD new PBP） MRCNS对万古霉素以外的所有抗金黄色葡萄球菌的抗菌药物耐药；

Multi-drug resistance MDR IV. Bacterial Resistance Multi-drug resistance MDR Penicillin-resistant streptococcus pneumoniae, PRSP，青霉素耐药肺炎链球菌 PBP-1a, PBP-2a, PBP-2x, PBP-2b （78-100 kD） Active efflux system （express mef(A)对大环内酯类） Vancomycin-resistant Enterococcus, VRE 万古霉素耐药肠球菌 PBP avidity ↓ van-A, van-B, van C-1, van C-2, van D, van E

Multi-drug resistance MDR IV. Bacterial Resistance Multi-drug resistance MDR 4. The 3rd generation-cephalosporins -resistant Extended spectrumβ-lactamases, ESBL 超广谱β- 内酰胺酶 Class I chromosone mediated β-lactamases I类染色体介导的β- 内酰胺酶 E.g. 大肠埃希菌、克雷伯肺炎杆菌、阴沟肠杆菌 广谱酶均为质粒介导，对第三代敏感 超广谱β- 内酰胺酶大部分由质粒介导，少部分由染色体介导

Multi-drug resistance MDR IV. Bacterial Resistance Multi-drug resistance MDR Carbapenem (碳青霉烯) –resistant：对亚胺培南的铜绿假单胞菌敏感 OprD porin Metalβ-lactamases （金属β- 内酰胺酶 ） 6. Quinolone-resistant escherichia coli（大肠埃希菌）, AREC Active efflux system Cross-resistance 亚胺培南通过 Opr D porin通道进入铜绿假单胞菌，而细菌会发生突变，使Opr D porin通道表达减少 superbug or super bacterium

Antimicrobial resistance among ICU patients

Ceftriaxone头孢曲松 Levofloxacin左氧氟沙星 Moxifloxacin莫西沙星

Antimicrobial drugs -Characteristics Basic principle of clinical usage of antimicrobial agents Antimicrobial drugs -Characteristics Some laboratory techniques that are useful in the diagnosis of microbial diseases

Antimicrobial drugs -Characteristics Basic principle of clinical usage of antimicrobial agents Antimicrobial drugs -Characteristics According to bio-activity Anti G+ antibiotic Anti G- antibiotic Broad-spectrum antibiotic Anti mycobacterium antibiotic Anti anaerobe antibiotic - lactamase inhibitor Mycobacterium 分支杆菌 Anaerobe 厌氧菌

Antimicrobial drugs -Characteristics Basic principle of clinical usage of antimicrobial agents Antimicrobial drugs -Characteristics According to the chemical structure： -lactams (-内酰胺类）；Penicillins（青霉素类）；Cephalosporins（头孢菌素类）; Aminoglycosides(氨基糖苷类); Macrolides（大环内酯类）; Lincosamides（林可胺类）;Vancomycins（万古霉素类） Tetracyclines（四环素类）； Chloramphenicol (氯霉素)

8. Antimycobacterial agents (抗结核分支杆菌类 ) 9. others: Basic principle of clinical usage of antimicrobial agents 5. Quinolones (喹诺酮类 ) 6. Sulphonamides (磺胺类 ) 7. Nitrofurans (硝基呋喃类) 8. Antimycobacterial agents (抗结核分支杆菌类 ) 9. others: Oxazolidinones（恶唑烷酮类） Streptogramins（链阳菌素类）

Some clinical situation in which prophylactic antibiotics Basic principle of clinical usage of antimicrobial agents Some clinical situation in which prophylactic antibiotics

Some clinical situation in which prophylactic antibiotics Basic principle of clinical usage of antimicrobial agents Some clinical situation in which prophylactic antibiotics

1）抗菌药物对病毒无治疗作用，除非伴有细菌感染或继发感染，一般病毒感染不应该使用抗菌药物； Basic principle of clinical usage of antimicrobial agents 防止抗菌药物的不合理应用的注意点： 1）抗菌药物对病毒无治疗作用，除非伴有细菌感染或继发感染，一般病毒感染不应该使用抗菌药物； 2）原因未明的发热者，除非伴有感染，一般不使用抗菌药物治疗； 3）应尽量避免抗菌药物的局部应用，否则可引起细菌耐药和变态反应； 4）使用抗菌药物剂量要适宜，疗程要足够。

Summary I. Chemotherapy II. Chemotherapeutic agents terminology III. Mechanisms under the action of chemotherapeutic agents Inhibiting synthesis of bacterial cell walls Affecting permeability of membrane Inhibiting protein synthesis Affecting bacterial nucleic acid metabolism Block essential enzymes of folate metabolism

IV. Bacterial Resistance Intrinsic resistance – Inherent features ，usually expressed by chromosomal genes Acquired resistance – Emerge from previously sensitive bacterial populations – Caused by mutations in chromosomal genes – Or by acquisition of plasmids or transposons V. Basic principle of clinical usage of antimicrobial agents