MULTIPLE DRUG RESISTANCE

Shubhangi GuptaM.PharmacologyIInd Sem, ISFCP

IntroductionHistoryMechanisms of Multi drug resistance

Special Cases Multi drug resistance in TB MDR with Antibotics MDR in Cancer cells MDR in HIV infection MDR in Malaria

INTRODUCTION

MDR is defined as the ability of a living cell to display resistance to a wide spectrum of drugs that are not structurally or functionally related. Organisms that display multidrug resistance can be pathologic cells, including bacterial and neoplastic (tumor) cells.

MDR occurs in many situations such as the resistance of :- • Antibiotics • Antifungal compounds• Anticancer drugs• Antiparasitic compounds

HISTORY

The concept of multidrug resistance (MDR) in tumours originated from the pioneer work of June Biedler in the early 1970s, who identified a wide profile of cross -resistance in Chinese hamster cells selected for resistance to actinomycin D.

The involvement of ABC proteins (P – glycoprotein) was first identified and named by Victor Ling in 1976, which is only the first of a wide variety of membrane transporters involved in many cellular functions and properties including MDR.

MECHANISMS INVOLVED IN MDR

Enzymatic degradation

Mutation at binding site

Down regulation of outer membrane proteins

Efflux pumps

Transduction of genes

• Methicillin-Resistant Staph aureus (MRSA)

• Vancomycin Resistant Enterococci: (VRE)

• Extended Spectrum Beta-Lactamase producing Enterobacteria. (ESBLs)

Enzymatic degradation Mechanisms of b-lactamase

N

ON

O

OH

S CH3

CH3O

RH

-lactamase

CH2

OH-lactamase

CH2

OH

N

ON

O

OH

S CH3

CH3O

RH

-lactamase

CH2

OH H2O

N

ON

O

OH

S CH3

CH3O

RH

HOH

-lactamase

CH2

OH

+Hydrolysis of Oxyimino group

Penicillin drug

Inactivated drug

Mutation at binding site

In this binding of p53 to MDR1 is blocked at site(i.e. p53 DNA-binding site) and this mutation results in enhancement of metastasis and mediate MDR

Down regulation of outer membrane proteins The outer membrane permeability is regulated by porin proteins.

Alteration in Outer membrane permeability particularly due to the decreased expression of porin proteins results in decreased influx of various drugs.

Additional resistance is also afforded by over-expressed efflux pumps that extrude a wide variety of unrelated drugs which ultimately results in a multidrug resistance (MDR).

EFFLUX PUMP

MDR is associated with increased expression of ABC drug transporter P-glycoprotein (P-gp)

Pgp, the product of MDR1 gene is a membrane protein consist of two duplicated halves each consist of hydrophobic membrane spanning segments.

Two close genes i.e. MDR1 and MDR3 are located at the long arm of chromosome 7 that encodes Pgp(30) MDR3 is not involved in drug resistance

contd…

Other proteins involved in MDR are :

•Multidrug resistance protein (MRP1)(encoded

by ABCC1)

●MRP2(encoded by ABCC2)

●MRP3(encoded by ABCC3)

●MRP4(encoded by ABCC4)

●MRP5(encoded by ABCC5)

●Breast cancer resistance protein (BCRP

encoded by ABCG2)

SPECIFIC CASES

Multiple drug resistance in TB

MDR TB: MDR-TB caused by strains of Mycobacterium tuberculosis which is resistant to both isoniazid and rifampin XDR TB: MDR + resistance to fluoroquinolone and 1 of the 3 injectable drugs (amikacin, kanamycin, capreomycin)

• Primary drug resistance:– Infected with TB which is already drug

resistant

• Secondary (acquired) drug resistance:– Drug resistance develops during treatment

Spontaneous mutations develop as bacilli proliferate to >108

Drug Mutation Rate

Rifampin 108

Isoniazid 106

Pyrazinamide 106

INHRIFPZA

INH

Drug-resistant mutants in large bacterial population

Multidrug therapy: No bacteria resistant to all 3 drugs

Monotherapy: INH-resistant bacteria proliferate

MECHANISM OF RESISTANCE IN TB:

Rifampin•Reduced binding to RNA polymerase•Clusters of mutations at “Rifampin Resistance determining Region” (RRDR)•Reduced Cell wall permeability INH•Chromosomally mediated.•Loss of catalase/peroxidase.•Mutation in mycolic acid synthesis.

Gene location associated Drug-Resistant M.tuberculosis

S.NO DRUG GENE

1 Isoniazid Kat G, Inh A, KasA

2 Rifampicin rpo B

3 Ethambutol emb B

4 Streptomycin rps L

5 Pyrazinamide pnc A

6 Fluoroquinolone gyr A

Bacterial resistance to antibiotics

Bacterial Resistance to antibiotics occurs via spontaneous mutation or by DNA transfer.

Mechanisms involved in attaining Multidrug resistance:

• Enzymatic deactivation .

• Decreased cell wall permeability.

Contd…

• Alteration in target sites.

• Increased efflux mechanisms.

• Increased mutation rate.

In addition, some resistant bacteria are able to transfer copies of DNA that codes for a mechanism of resistance to other bacteria, thereby conferring resistance to their neighbours. This process is called horizontal gene transfer.

Use antibiotics only for bacterial infections Identify the causative organism if possible Use the right antibiotic; do not rely on broad-

range antibiotics Not stop antibiotics as soon as symptoms

improve; finish the full course Not use antibiotics for most colds, coughs,

bronchitis, sinus infections, and eye infections, which are caused by viruses

Neoplastic resistance

Cancer cells also have the ability to become resistant to multiple different drugs.

•Increased efflux of drug (as by P-glycoprotein, multidrug resistance-associated protein, lung resistance-related protein, breast cancer resistance protein and reproductive cancer resistance protein)

Mechanisms involved are:•Increased efflux of drug•Enzymatic deactivation (i.e, glutathione conjugation)•Decreased permeability (drugs cannot enter the cell)•Alteration in binding sites•Development of alternative metabolic pathways

The drug resistance that develops in cancer cells often results from elevated expression of particular proteins, such as cell-membrane transporters, which can result in an increased efflux of the cytotoxic drugs from the cancer cells, thus lowering their intracellular concentrations.

The cytotoxic drugs that are most frequently associated with MDR are hydrophobic, natural products, such as the taxanes (paclitaxel, docetaxel), vinca alkaloids

( vincristine, vinblastine), anthracyclines (doxorubicin, daunorubicin, epirubicin), epipodophyllotoxins (etoposide, tenipo-side), topotecan, dactinomycin, and mitomycin.

Multiple drug resistance in HIV

NDM-1 is a Metallo Beta-Lactamase essentially found in Enterobacteriaceae  (principally E. coli and K. pneumoniae).

NDM-1 stands for New Delhi Metallo-beta-lactamase-1, since it was first identified  in a Swedish patient of Indian origin, who had been admitted to hospital in New Delhi, India in 2008. The NDM-1 gene makes bacteria resistant to fluroquinolones, aminoglycosides and  almost all beta-lactams including carbapenems (imipenem, meropenem, ertapenem, doripenem).

The gene for NDM-1 is found on plasmids (DNA strands), which can easily spread from one strain of bacteria to another

MULTIPLE DRUG RESISTANCE IN MALARIAResistance developed due to the

decreased ability of the parasite to

accumulate the drug .

MDR -1 gene is associated with the

resistance in malaria.

So the modulators , for eg- verapamil :-

restored the concentration drugs which

causes resistance .

MultiDrugQuant Assay Kit

The kit provides a fast, simple and reliable ,and quantitative method for measuring the drug transport activity of the clinically most important multidrug resistance proteins : MDR1 (p-gp) and MRP1.

It was developed as a flow cytometric assay, where intracellular calcein fluorescence is measured after incubating the cells with the dye acetoxymethylester form of fluorescent calcein (calcein-AM) in the presence and absence of inhibitors of Pgp and MRP1.

Intracellular fluorescence intensities obtained with or without inhibitors are used for calculation of MDR activity factor (MAF) values, which are the quantitative measures of transport activity of Pgp and MRP1.