Term
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Definition
alkylating agents were used in chemical warfare (mustard gas, sulfur mustards) during WWI and other conflicts
during use in war it was noted that mustard gases not only caused severe irritation and damage to eyes, skin, and mucosa, but also caused severe bone marrow suppression (marrow aplasia)
some chemotherapeutic alkylating agents are vsicants (blistering agents)
it was observed that nitrogen mustard compounds were effective in mouse models of cancer and human lymphoma in 1942
exposure to mustard gas causes severe damage (blistering) to skin, eyes, mucosa, and lungs
clinical studies using nitrogen mustard for treatment of lymphoma in humans provided the impetus for the development of the many other alkylating ageents for cancer chemotherapy
extravasation of a chemotherpeutic from a central venous catheter causes severe tissue irritation |
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Term
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Definition
[image]
two strands
3-unit repeat: phosphate-deoxyribose-base
phosphodiester backbone
base-base hydrogen bonding
A-T, G-C
shows the basic structure of DNA double helix
the backbone is composed of a phosphate linked to deoxyriboses through phosphodiester bonds
each deoxyribose is linked to a purine (adenine, guanine) or pyrimimdine (thymine, cytosine) base through a glycosidic bond
adenine pairs with thymine and guanine pairs with cytosine through hydrogen bonding
alkylating agents react with nucleophilic groups in the bases, especially N7 in guanine
other drugs covered later bind in the grooves (minor and major) formed by the double helix
groove binding and intercalation are non-covalent |
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Term
| hydrogen bonding between bases |
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Definition
A-T (2)
G-C (3)
guanine N7, O6
[image]
the 2 strands in DNA are held together by hydrogen bonding
A-T has 2 and G-C has 3 hydrogen bonds (G-C is more thermostable b/c of this fact and increases DNA melting temperature when G-C content is high)
the nitrogen atoms in the bases are susceptible to alkylation, especially the N7 in guanine
O6 in guanine is also a common target for alkylation
not all N7 and O6 sites have the same reactivity (alkylation is sequence-dependent and somewhat more reactive in S phase)
adduct formation on DNA bases can have several affects on DNA fate |
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Term
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Definition
alkylation: addition or transfer of an alkyl group (from chemotherapeutic drugs to DNA)
alkylating agents form very reactive intermediates that result in alkylation of nucleophilic groups in DNA and also proteins (thiols and amines)
BIFUNCTIONAL VS. MONOFUNCTIONAL:
an important distinction should be made between monofunctional (one reactive group) and bifunctional (2 reactive groups) alkylating agents
bifunctional agents have much greater CYTOTOXIC EFFECTS compared to monofunctional agents
this property is attributed to the ability of bifunctional agents to cross-link DNA strands
5 major types of alkylating agents are used in the chemotherapy of neoplastic diseases:
nitrogen mustards
ethyleneimines
alkyl sulfonates
nitrosoureas
triazenes |
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Term
| alkyation of guanine by mechlorethamine |
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Definition
[image]
1. reactive aziridinium intermediate
2. nucleophilic N7 guanine
3. four consequences
an example of alkylation of DNA
mechlorethamine forms an intramolecular cyclized intermediate (aziridinium ion, not shown)
the intermediate is a very reactive electrophile which can react with electron rich sites in DNA bases
a probable reaction is with N7 in guanine as illustrated above
there are 4 potential out comes of alkylation discussed next
mechlorethamine is the prototype alkylating agent |
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Term
| 4 consequences of guanine alkylation |
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Definition
[image]
1) mechlorethamine is a bifunctional alkylating agent, thus it can bind 2 DNA strands (crosslink DNA); this is a major mechanism by which alkylating agents act to cause DNA damage and induce cancer cell apoptosis
2) alkylation can open the imidazole ring in guanine; the cell will attempt to use the base excision repair mechanism to replace the guanine
3. alkylation can cause stabilization of the guanine enol tautomer resulting in base pair mismatch (hydrogen bonding of guanine with thymine instead of cytosine); O6 methylation of guanine can also result in mis-pairing with thymine
4) te cell may respond to alkylation and excise guanine in attempt to repair the DNA (base excision repair mechanism); the cell will replace the alkylated guanine with a new guanine |
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Term
| cross linking of DNA strands |
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Definition
[image]
blue represents the deoxyribose-phosphate backbone
bifunctional agents can bind 2 guanines (itnra-strand and inter-strand reactions) as illustrated in the figure
cross-linking interferes with DNA replication (and transcription)
cross-linking between adenine and guanine may also occur
nucleotide sequence is an important determinant of cross-linking reactivity
the dashed lines in the figure denote 2 or 3 hydrogen bonds |
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Term
| alkylating agents interfere with DNA replication |
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Definition
[image]
strand separation (helicase) inhibited by INTER-strand crosslinking
duplication (polymerase) inhibited by INTRA-strand crosslinking
a major mechanism of alkylating agents is interference with DNA replication and transcription through inter-strand and intra-strand crosslinking
the 2 strands that make up the double helix must be separated (requires helicase) before the template strand can be duplicated or transcribed
interstrand crosslinking can stop strand separation (interferes with helicase) and intra-strand crosslinking can stop DNA duplication and transcription (interferes with polymerase or may generate a mutation |
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Term
| cell fate after DNA damage |
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Definition
[image]
bifunctional agents are more cytotoxic (active apoptosis) compared to monofunctional agents
monofunctional agents are more carcinogenic (base alkylation can lead to permanent base change)
the following may occur in a cancer cell or normal cell:
1) DNA is damaged by an alkylating agent, but is repaired (likely base excision repair mechanism) before apoptosis is triggered
2) the DNA is damaged and not repaired
3) if DNA is repaired (before apoptosis become irreversible) then the cancer cell can progress through the cell cycle and produce progeny
4) damage to DNA that is not repaired triggers cell cycle arrest in G1 phase and activation of the apoptosis pathway; this pathway is programmed cell death and requires certain intracellular signaling molecules (p53 tumor suppressor gene)
5) if apoptosis is not triggered (p53 is not activated), cycle may continue and produce cells with permanent mutation |
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Term
| important features of apoptosis (programmed cell death) |
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Definition
TRIGGER (INDUCER):
a trigger or initiator must be present (intracellular or extracellular)
PROTEIN CLEAVAGE
caspases breakdown nucleus membrane and cytoskeleton proteins, and activate other factors in apoptosis pathway
DNA BREAKDOWN
DNA is fragmented by endonucleases
PHAGOCYTIC RECOGNITION
cells that have undergone apoptosis are removed by phagocytes |
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Term
| triggers and signaling of apoptosis |
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Definition
[image]
1. major inducers (trigger) of apoptosis; these include injury by radiation, toxins (chemotherapeutic drugs) and free radicals (may be produced by chemotherapeutic drugs); DNA damage is a major route of alkylating agent action
2. pro-apoptotic Bcl-2 members become activated and cause increased permeability of mitochondria and release of cytochrome c; next, initiator (cleave other caspases) caspases are activated
3. executioner caspases cause breakdown of the cell cytoskelton and activation of endonucleases which cause DNA fragmentation; apoptotic bodies are formed (membrane-bound cell fragments)
4. certain ligands expressed on apoptotic bodies are recognized by phagocytic cells
note: withdrawal from growth factors and immune cytotoxicity (T cells release granzyme B) are other important apoptosis triggers and mechanisms of anti-cancer drug action (discussed later in the course) |
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Term
| cancer cell sensitivity to alkylating agentsq |
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Definition
the traditional thought is cancer cells are rapidly proliferating, making them more sensitive to alkylating agents and other anti-cancer drugs
recent studies suggest proliferation rate is only part of the answer explaining drug sensitivity
cancer cells may be closer to the threshold of apoptosis making them more drug sensitive
the mitochondria in cancer cells may be "primed" for apoptosis |
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Term
| general toxicity of alkylating agents |
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Definition
BONE MARROW SUPPRESSION
often dose limiting
all alkylating agents cause bone marrow suppression
however, there are differences in timing of lowest point (nadir), duration and recovery of blood counts
marrow suppression is the most often dose-limiting adverse effect of alkylating agents
immune (humoral and cellular systems) suppression is a consequence dramatically increasing probability of infection
MUCOSAL
epithelial cells, infection risk
the rapidly dividing epithelial cells of the intestinal mucosa are very susceptible to the cytotoxic effect of alkylating agents
mucosal damage increases risk of bacterial sepsis
CNS
emesis (rapid and delayed)
emesis can occur acutely after IV administration (activation of CTZ in CNS)
the cytotoxic effect on GI epithelial can also cause emesis days after adminstration
emesis is very common with chemotherapeutic agents
incidence is >60% for many alkylating agents
CARCINOGENIC
leukemia (years)
some alkylating agents can increase risk of developing leukemia and other types of cancer
this effect is observed years after therapy
OTHERS
pulmonary fibrosis, hepatic veno-occulsion, renal
effects are less predictable (compared to bone marrow and GI effects) |
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Term
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Definition
[image]
of the nitrogen mustards only mechlorethamine is a strong vesicant |
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Term
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Definition
[image]
used for leukemia and lymphomas
may cause AML
good oral bioavailability, 99% albumin bound; hepatic metabolism
more gradual myelosuppressive onset
especially toxic for lymphocytes
side effects are similar to other alkylating agents
myelosuppression limits dose
t1/2 is 1.5 hours
drug undergoes complete hepatic metabolism
associated with development of acute myelogenous leukemia (AML) |
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Term
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Definition
[image]
multiple myeloma
incomplete and variable oral absorption
20-50% in stool
phenylalanine moiety (AA transporter)
used for multiple myeloma (MM) and epithelial ovarian cancer
MM is a differentiated B-cell malignancy
this disease is characterized (diagnostics features) by increased numbers of plasma cells (mature B cells) in bone marrow, bone lesions (caused by inflammation response in bone) and high serum levels of antibody (usually IgG)
similar structure as chlorambucil, but less oral bioavailability
much less hepatic metabolism (found in stool and urine unchanged
the rationale was to design a drug that could be taken up by the aromatic amino acid transporter which may increase intracellular concentration in cancer cells (some cancers may have high transporter expression) |
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Term
| multiple myeloma (MM) diagnostic features |
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Definition
MM is a malignancy of the B cell lineage
the malignant B cells are differentiated and most of them are contained in the bone marrow
bone marrow aspiration reveals high number of plasma cells
lesions in the skull visible on x-ray
bone lesions are believed to be caused from interaction of plasma cells with bone stromal cells and subsequent activation of the stromal cells
the stromal cells in turn activate the osteoclasts which destroy bone
the malignant B cells produce over abundance of imuunoglobulin which shows up on plasma electrophoresis |
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Term
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Definition
[image]
broad spectrum use (leukemia, lymphoma, multiple myeloma, ovarian, breast, neuroblastoma)
prodrug, hepatic activation (variable)
phosphoramide metabolite therapeutic
acrolein metabolite causes HEMORRHAGIC CYSTITIS
HYDRATION important
rare, syndrome of inappropriate ADH SECRETION
in addition, used for organ rejection and autoimmune disease treatment
not a vesicant
requires hepatic activation to hydroxycyclophosphamide
prodrug activation is interpatient variable and is saturable
accumulation of acrolein in the bladder causes hemorrhagic cystitis (inflammation of the bladder causing bleeding and presence of RBCs in urine)
another metabolite (phosphoramide mustard) is thought to be responsible for anti-tumor effects
coadministration with mesna (a sulfhydryl) reduces hemorrhagic cystitis
hydration also helpful for reducing concentration of acrolein in bladder
bladder fibrosis may occur with or without cystitis
some reports found link between elevated ADH (insertion of water channels in renal tubule and conservation of water resulting in serum sodium dilution, hyponatremia) and cyclophsophamide use
[image]
the prodrug cyclophosphamide is activated by hydroxylation to 4-hydroxycyclophosphamide which can further be oxidized to 4-ketocyclophosphamide or undergo reversible ring cleavage to aldophosphamide
acrolein and phosphoramide are potent cytotoxic metabolites of aldophsophamide
accumulation of acrolein in the bladder causes hemorrhagic cystitis (inflammation of the bladder causing bleeding)
coadministration with mesna (sulfhydryl) reduces hemorrhagic cystitis
[image] |
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Term
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Definition
[image]
cyclphosphamide analog
testicular cancer, sarcomas
hepatic activation
toxicity concerns same as cyclophosphamide
more narrow indication compared to cyclophosphamide
sarcoma is malignancy that originates from connective tissue cells (bone, muscle, cartilage, fat cells)
sarcomas are further divided into origin of specific connective tissue type
drug activated by hydroxylation
given with mesna to prevent hemorrhagic cystitis |
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Term
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Definition
[image]
leukemia, lymphomas
first clinically used nitrogen mustard
VERY REACTIVE, VESICANT
EXTRAVASATION concern (local thiosulfate treatment)
very reactive, very short half-life in blood stream due to spontaneous degradation and reaction with tissue proteins and hydrolysis
IV use only
extravasation a concern with this drug (local thiosulfate is treatment for extravasation)
extravasation causes local tissue sloughing and severe inflammation
replaced by other (more stable) alkylating agents |
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Term
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Definition
[image]
administered as phosphate
rational design for prostate cancer
combined estradiol with nitrogen mustard analog
interferes with MICROTUBULE POLYMERIZATION
inhibits mitosis
side effects from estrogen activity
dephosphorylated during absorption
it was designed as a treatment for prostate cancer
the rationale was the estrogen part of the molecule would facilitate nuclear uptake into prostate cancer cells and alkylating group would bind DNA and cause apoptosis
it was later found that the drug does not alkylate in vivo and unexpectedly binds to proteins that regulate microtubule function
[image]
b/c of the microtubule interference activity this drug may also interfere with mitosis
side effects are attributed to the estrogenic activity (impotence, gynecomastia, fluid retention)
estrogens are released |
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Term
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Definition
chronic lymphocytic leukemia (CLL) and non-Hodgkin lymphoma (NHL)
effective against resistance due to p53 inactivation
resistance does arise from base excision repair
in vitro research shows effective against resistant cells due to p53 mutation (causing low expression or low activity)
tumor cells use base excision repair as resistance mechanism |
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Term
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Definition
[image]
potential tri-functional
breast, BLADDER, ovarian cancers
1. free radical generation
2. breaks bond between base and deoxyribose
3. abasic site generation
after IV administration parent drug is desulfurated to TEPA (triethylenephosphoramide)
parent drug directly infused into bladder for bladder cancer treatment
also used to ablate bone marrow before stem cell transplantation
crosses BBB (more neurotoxic effects)
though to react with guanine N7 and release free radicals which break the bond (glycosidic) between base and deoxyribose
alkylated guanine is released |
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Term
altretamine, hexamethylmelamine
[image] |
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Definition
[image]
ovarian cancer
parent compound without alkylating activity
cytotoxicity attributed to metabolites
generation of FORMALDEHYDE
NEUROTOXICITY (central and peripheral)
use of pyridoxine?
mechanism uncertain
thought that metabolites may have alkylating activity
formaldehyde generation may account for some antitumor activity
side effects include ataxia, mood change, confusion, dizziness
neurotoxic effects are reversible and may be decreased by co-administration of pyridoxine, a vitamin B6 derivative (this has not been thoroughly investigated, some reports conclude that pyridoxine lowers anti-tumor response) |
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Term
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Definition
chronic myelogenous leukemia, bone marrow transplantation
in vitro INTRA-STRAND crosslinking and "sulfur stripping"
pulmonary fibrosis and liver VENO-OCCLUSION (high dose)
an alkyl sulfonate
selective for myeloid lineage cells
intra-strand, but not inter-strand DNA crosslinking observed using virus DNA
high reactivity with thiols (sulfur stripping) may have therapeutic effect
at high doses is associated with pulmonary fibrosis and liver venous occlusion (endothelial cell damage in liver sinusoids)
after endothelial cell damage, hepatocyte necrosis and liver fibrotic deposition follows
standard dose causes only myelosuppression
[image]
endothelial cells (not shown) line sinusoids
the structure of the liver's functional units, or lobules
blood enters the lobules through branches of the portal vein and hepatic artery, then flows through small channels called sinusoids that are lined with endothelial cells primary liver cells (hepatocytes)
the hepatocytes remove toxic substances, including alcohol, from the blood, which then exits the lobule through the central vein (the hepatic venule)
blood flow through the sinusoids moves from hepatic artery and portal (connecting GI to liver) vein to the hepatic venule as indicated by the direction of the arrow
flow of blood through the sinusoids is necessary for metabolism of drugs, alcohol, and some molecule ingested in diet
endothelial cells line the inside of the sinusoids (endothelial cells not shown in the above figure)
some alkylating agents carry risk of veno-occlusive disease in which the endothelial cells are damaged
damage to other cell types may follow including hepatocytes
other alkylating agents may cause haptic veno-occlusion (more recently it has been proposed that this disease be called sinusoidal obstruction syndrome) |
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Term
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Definition
metastatic MELANOMA, Hodgkin's disease
adds methyl group to O6 guanine (AGT, alkyltransferase confers resistance)
cell-cycle nonspecific (like others)
other mechanisms considered
high rate of nausea and emesis (>90%)
meylosuppression common
a triazene
thought to add methyl group to O6 of guanine which can result in mis-pairing (G-T)
resistance to this drug is attributed to production of a tumor cell enzyme that removes the O6 methylation (AGT, O6-alkylguanine-DNA alkyltransferase)
AGT is viewed as a repair mechanism in normal cells
other anti-cancer mechanisms are possible (may act as purine analog, an antimetabolite)
like other alkylating agents, the drug is cell cycle non-specific
onset of emesis can occur within hours after administration or be delayed (days) due to GI epithelial damage |
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Term
| possible outcomes of O6 methylation |
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Definition
[image]
a. the O6 alkylguanine DNA alkyltransferase (AGT) protein scans double stranded DNA for alkylation at the O6 position of guanine; covalent transfer of the alkyl group to the conserved active site cysteine inactivates the AGT protein and restores the guanine
b. if repair of the CH3-G modification does not occur, a permanent mutation (A-T) or a strand break can result |
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Term
carmustine and lomustine
[image] |
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Definition
[image]
lipophilic, ENTERS CNS
effective for brain tumors (GLIOMA)
GLIADEL WAFER impolant (carmustine) in brain
alkylation of DNA and carbamoylation of PROTEINS
resistance attrituted to TUMOR PRODUCTION OF AGT
carmustine wafer indicated for high grade glioma
thse drugs crosslink DNA
it is unclear the role of carbamoylation in therpeutic mechanism (potential for inactivation of enzymes involved in cell cycle)
tumors can become resistant to DNA alkylation through production of AGT which removes alkyl group
guanine alkylation in the promoter region of AGT is associated with more successful treatment outcome
[image]
1. O6 guanine alkylation
2. AGT PROMOTER alkylation important
3. carbamoylation of cell cycle proteins?
carmustine can spontaneously decompose (hydrolysis of anhydride link) to both alkylating and carbamoylating (isocyanate) products
alkylating product attacks guanine O6 and may crosslink DNA
alkylation of the AGT gene promoter region correlates with increased effectiveness of this drug (blocks transcription)
proteins can be carbamoylated on epsilon amino of lysine residues
therapeutic effect of protein carbamoylation is uncertain (may inactivate cyclin proteins required for progression of cell cycle) |
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Term
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Definition
[image]
metastatic islet cell carcinoma
TRANSPORTED BY GLUT
bone marrow suppression
streptozocin consists of methylnitrosourea linked to carbon 2 of glucose
the glucose moiety enhances uptake of the drug into islet cells of the pancreas by the GLUT transporter
the methylnitrosourea moiety alkylates DNA
high dose of this drug can induce diabetes mellitus (animal models) |
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Term
| mechanism of resistance to alkylating agents |
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Definition
increased intracellular concentration of nucleophiles
enhanced DNA repair:
1. BASE EXCISION
2. AGT
3. nucleotide excision less likely
enzymatic drug inactivation
the tumor cells may increase production of nucleophiles (i.e. thiols such as glutathione) that can react with and neutralize alkylating agents
a potential strategy is to deplete cells of glutathione in attempt to increase anti-tumomr activity, but this is likely to increase toxic side effects
tumor cells may increase production of DNA repair mechanisms such as base excision repair or AGT (O6-alkylguanine-DNA alkyltransferase)
generally, adducts larger than ones produced by alkylating agents are remomved by nucleotide excision (i.e. platinum comoplexes)
inside tumor cells drugs may be enzymatically inactivated (i.e. cyclophosphamide by action of aldehyde dehydrogenase) |
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