Alkylating agents are a class of cytotoxic chemotherapy drugs that work by adding alkyl groups to electron-rich nucleophilic atoms in DNA, RNA, and proteins, forming covalent bonds. This disrupts the DNA structure and prevents cell division. The two main types are monofunctional agents that bind one DNA strand, and bifunctional agents that form cross-links between both strands. Common alkylating agents include nitrogen mustards, nitrosoureas, alkyl sulfonates, and aziridines. They are used to treat many cancers but can cause toxic effects like myelosuppression, cystitis, and secondary cancers due to their DNA damaging properties. Resistance mechanisms include increased glutathione and DNA repair pathways

1. ALKYLATING AGENTS
Dr Chandragouda

7. Action sites of cytotoxic agents
S
G2
M
Alkylating agents
G1
Antibiotics
Antimetabolites
G0
Vinca alkaloids
Mitotic inhibitors
Taxoids

8. WHAT ARE ALKYLATING
AGENTS?
• Alkylation is the transfer of an alkyl
group from one molecule to another.
• They are themselves an electrophile
molecule which react with (or alkylate)
electron rich atoms in cells (nucleophile) to
form covalent bonds.

9. • These nucleophiles could be a large
variety of biomolecules
- DNA (most common)
- RNA
- Proteins

10. • Thus they react with DNA bases to disrupt
the DNA (intermediate Carbonium ion
formation)
• Most common target of such electrophiles
is N-7 atom of purine ring (Guanine)
• May also target O-6 atom of Guanine

11. • After bond formation, alkylating agents
either
- disrupt single strand of DNA
(intrastrand) - Monoalkylation
OR
- disrupt double strand of DNA
(interstarnd) – Dialkylation

12. Mechanism of action

15. Continued
Alkylating agents
Monofunctional
React with one strand
of DNA
Bifunctional
React with two strands of
DNA to produce a cross link
between them preventing
replication

16. CLASSIFICATION
1. NITROGEN MUSTARDS
Mechlorethamine, Cyclophosphamide, Ifosphamide,
Melphalan, Chlorambucil
2. ALKYL SULPHONATES
Busulphan, Treosulphan
3.NITROSOUREAS
Bischloroetylnitrosourea (BCNU,Carmustine)
Cyclohexychloroethylnitrosourea (CCNU,Lomustine)
Streptozocin
4. AZIRIDINE AND EPOXIDE
ThioTEPA, Mitomycin C,
Hexamethylmelamine(Altretamine)
5. TRIAZENES
Procarbazine, Dacarbazine
6. IMIDAZOTETRAZINES
Temozolomide

17. • Bifunctional
- Nitrogen mustards
- Nitrosoureas
- Busulphan
• Monofunctional
- Dacarbazine and Procarbazine

18. MECHLORETHAMINE
(MUSTARGEN)
• The drug is an analogue of mustard gas
• Sulpher mustard used in World War 1 – Had
devastating S/E like pulmonary toxicity, skin ulcers,
eye lesions
• In 1942, before World War II, Goodman and Gilman
were deployed to study its effects.
• They found affected people to have low lymphoid cells
and lymphoid organ aplasia
• Postulated that this could be a possible therapy for the
cancer.

19. • MOPP regimen
• Available in IV preparation, dose in MOPP
being 6 mg/m2 on Day 1 &8 of monthly
schedule
• Available as topical preparation for
cutaneous malignancies
• It has been derivatized – estramustine-
estrogen analogue, used to treat prostate
cancer.

20. MECHLORETHAMINE
contd.
• Extravasation -- painful inflammation.
• The area usually becomes indurated and
sloughing may occur.
• Rx- prompt infiltration of the area with
sterile isotonic sodium thiosulfate (1/6
molar) and application of an ice compress
for 6 to 12 hours may minimize the local
reaction

21. CYCLOPHOSPHAMIDE
• It is an oxazaphosphorine group of
nitrogen mustard compounds
• Started to be used in clinical practice in
1940s and still widely used
• Dose-1-5mg/kg (daily PO),40-50mg/kg iv
in divided doses over 2-5 days.
High dise for BMT-200mg/kg

23. Cyclophos-Contd
• CYP 2B6 and CYP 3A4 activate it.
• 4-HOCY and Aldophosphamide are degraded by
aldehyde dehydrogenase(ALDH) to
carboxyphosphamide (inactive)which is then excreted
in urine.
• Cells having high ALDH activity such as early
hematopoietic stem cells, megakaryocytes, epithelial
cells of small intestine are relatively resistant to
Cyclophosphamide.
• It is available as oral or IV preparation.

24. Clinical pharmakokinetics

25. • Both Cyclophosphamide and Ifosphamide
are soluble in water, saline or alcohol as a
monohydrate and can be readily
administered orally
• Both are well absorbed and the peak
concentration appears 1-2 hours following
oral drug administration.
• Oral bioavailability is 100%

26. BUT …
Oral administered Ifosphamide results in
unacceptable incidence of neurotoxicity
which probably results from a shift in
metabolic pathways towards
dechloroethylation with increased
formation of neurotoxic compound
DI-CHLORO-ACETALDEHYDE

27. • The plasma protein binding of 4-OH-
cyclophosphamide is about 70%.
• Its active metabolites are polar
compounds, so can’t cross BBB
• This may contribute to the lack of
neurotoxicity associated with the
intravenous administration of
Cyclophosphamide

28. • The distribution of Ifosphamide is more extensive with
lower plasma protein binding compared with
Cyclophosphamide
• The active metabolite 4-hydroxy-IFO can pass the BBB
and reach cerebrospinal fluid. The concentrations of IFO
in cerebrospinal fluid are almost as high as those in
plasma .
• This may be associated with the neurotoxicity commonly
encountered in cancer patients treated with IFO.

29. • The metabolism of both CPA and IFO is an autoinducible
process. Auto-induction results in an increase in the total
clearance, increased formation of 4-OH metabolites, and
shortened t1/2/ values, following repeated administration
at 12- to 24-hour interval
• Typically observed between Day 1 and Day 5
• may reduce synthesis of proteins responsible for
CYP3A4 degradation/inactivation,

30. • Both CPA and IFO are primarily (70%)
excreted in urine in forms of metabolites and to
a less extent, in the feces [109]. However, only
10-20% is excreted unchanged in the urine
• renal clearance of IFO and its metabolites is
lower than creatinine clearance, suggesting a
substantial part of tubular reabsorption of IFO
and its metabolites when they go through the
renal tubules

31. • It is not recommended to adjust the
dosage of CPA in patients with liver
dysfunction.
• Since all active metabolites are excreted
via kidney, dose reduction is needed in
renal failure

32. Toxicity profile of
cyclophosphamide
• Metabolite Acrolein is responsible for irritation of bladder mucosa
leading to hemorrhagic cystitis.
It is advisable for patients to have enough hydration and they should be instructed to
frequently empty the bladder and not retain urine for long periods.
2-mercaptoethane sulfonate (MESNA), which dimerizes to an inactive metabolite in plasma but
hydrolyzes in urine to yield the active parent that conjugates with alkylating species and prevents
cystitis. MESNA should be administered routinely to all patients receiving ifosfamide and to any
patient who has a history of drug-induced cystitis.MESNA is usually given in divided doses every
4 hours in dosages of 60% of those of the alkylating agent.
• Antidiuretic effect produces marked fluid retention and electrolyte
abnormalities.
• Fulminant cardiac toxicity at very high doses (charactersistic
pathological picture of edema, interstitial hemorrhage and cardiac
necrosis). Cardiac failure usually begins 10 days after drug
administration.

33. Drug Interactions
• A number of drug interactions with CPA
have been reported in humans. It seems
that the underlying mechanism is inhibition
of CYP enzymes for the drug interactions
with allopurinol [163], chloramphenicol
[228], sulphaphenazole [228],
chlorpromazine [163, 229], fluconazole
[230], ranitidine [231], and thiotepa

34. • CPA reduced digoxin absorption has been
reported.
• Thiotepa inhibited the activation of CPA and
decreased the efficacy and toxicity of
CPA .Conversely, CPA induced the conversion
of thiotepa to its metabolite TEPA [244],
thioTEPA is frequently given in conjunction with
CPA in high-dose chemotherapy regimens in
preparative regimens before autologous bone-
marrow and peripheral stem-cell transplantation.

35. • Therefore, the sequence and schedule of
these two drugs should be critically
concerned and it has been recommended
that these two agents should not be
combined

36. USES
• Ifosphamide is more efficacious than
cyclophosphamide

37. IFOSPHAMIDE
• Structural isomer of cyclophosphamide, requires
activation in the liver.
• Ifosphamide only differs chemically from
Cyclophosphamide by one chloroethyl group
transpositioned from the mustard nitrogen to the ring
nitrogen
• Started to be used in clinical practice since 1970s

39. Ifos-Contd
• Intravenous mode of administration
• Available as a white powder
• Activated at a four fold slower rate than
cyclophosphamide because of lower
affinity to hepatic P 450 enzymes.
• Dose—1.2gm/m2 IV for 5 consecutive
doses

41. • It is given as a treatment for a variety of
cancers, including:
1. Soft tissue sarcoma
2. Recurrent Testicular cancer
3. Head and neck cancers
4. Lymphoma (Non-Hodgkin & Hodgkin’s)
5. Osteogenic sarcoma
6. Lung cancer
7. Cervical cancer
8. Ovarian cancer

42. METABOLISM
• Ring oxidation
– by using the hepatic mixed-function
oxidase system
- leads to formation of cytotoxic
compounds
• ‘‘Side-chain’’ oxidation
- leads to formation of chloracetaldehyde
responsible for neurotoxicity and renal
toxicity

43. TOXICITY PROFILE OF
IFOSFAMIDE
• Leukopenia
• Extravasation hazard: irritant
• Alopecia
• Hemorrhagic cystitis (1-10%)
Mesna (sodium 2-sulfanylethanesulfonate) helps in ameliorating
cystitis by binding to sulph hydryl moieties and increasing the
excretion of cysteine.should be given at 60% of the ifosfamide dose)
• Neurotoxicity:- somnolence, confusion,
hallucinations, depressive psychosis.
Methylene blue used in the treatment and prophylaxis of
encephalopathy.
• Amenorrhea, oligospermia and infertility

44. • More chloroethyl side chain oxidation of
ifosphamide (upto 50%) produces
chloroacetaldehyde, which is responsible
for neurotoxicity and renal toxicity.

45. MELPHALAN
• Used in multiple myeloma as part of the
popular MP regimen.
Dosage is 0.25 mg/kg for 4 days along with prednisolone for
4 days every 4 weeks.
• Use in bone marrow myeloablative regimens
before transplantation.
• Available as oral or IV preparation.
• Most common side effect is bone marrow
suppression.

46. CHLORAMBUCIL
• Used in the treatment of CLL, lymphomas
• Used in the treatment of autoimmune
conditions.
• Common side effects include
myelosuppression , sterility, secondary
leukemia.
• Available as oral preparation.

47. ALKYL SULPHONATES
(BUSULFAN)
• Used in refractory CML as it is selectively
having suppression on myeloid series.
• Used in myeloablative regimens
Dose is 1 mg/kg every 6 hrs for 4 days to a total dose of
16 mg/kg
• Available in oral or IV preparation
• Uncommon side effect at high dose is
veno-occlusive disease of liver and
pulmonary fibrosis.

48. NITROSOUREAS
• Bischloroethylnitrosourea (BCNU,Carmustine):
used in treatment of gliomas and other
brain tumors
• Can also be used in the T/t of multiple
myeloma, lymphomas
• Available as IV preparation, can also be
used for implantation in brain tumors
• Dosage is 150-200 mg/m2 every 6 weekly
• Causes delayed myelosuppression after
4-6 weeks.

49. Nitrosoureas-Contd
• Cyclohexychloroethylnitrosourea
(CCNU,Lomustine)
• Used in brain gliomas and in relapsed
Hodgkin’s disease.
• It is a more lipid soluble alkylating
nitrosourea.
• Available as oral preparation in 10, 40, 100
mg tablets.
• Delayed myelosuppression after 4 weeks

50. STREPTOZOCIN
• In addition to the formation of DNA cross
links, it selectively targets pancreatic cells
• Used in pancreatic islet cell tumors and
carcinoid tumors
• Renal toxicity is dose limiting(manifested
by proteinuria and azotemia)
• Altered glucose metabolism either
hypoglycemia or hyperglycemia.

51. AZIRIDES AND EPOXIDES
Thiotepa:
• Myeloablative therapy at a dose of 200mg/
m2.
• Used in papillary bladder carcinoma via
intravesical route.
• It is also used in instillation into serous
cavities.

52. Mitomycin C
• Antibiotic extracted from streptomyces
spp.it is reduced under hypoxic conditions.
Mitomycin C and its reduced metabolites
produce intrastrand guanylic acid
crosslinks that produce bending of the
DNA
• Used in GIT tumors esp. anal canal
cancers
• Used in breast cancers

53. HYDRAZINE AND TRIAZENE
DERIVATIVES
• Analogous to nitrosoureas
• Metabolizes to produce alkyl carbonium
ion which alkylates DNA.

54. Procarbazine
• Phenylhydrazine derivative that was initially
developed as a MAO inhibitor.
• One of the component of MOPP regimen
• Used in primary brain tumors
• Side effects: CNS Depression or stimulation, acute
hypertension after ingestion of tyramine rich
food(wine, dark beer, bananas, cheese, yogurt).
Antabuse like action with alcohol.
Delayed myelosuppression after 4-6 weeks.
• Dose : 100mg/m2/day for 14 days.

55. Dacarbazine
• Metabolized in liver to release a methyl diazonium that
methylates DNA,RNA.
• Used in ABVD regimen for Hodgkin’s
lymphoma.
• Used in malignant melanoma.
• Side effects: myelosuppression, highly emetogenic,
vesicant
• Dose: 375mg/m2 in ABVD regimen
200-250mg/m2/day x 5 days in melanoma

56. Temozolomide:
• Triazene analogue that acts
as a prodrug and spontaneously decomposes
to form methyl diazonium ion (MTIC).
• It is lipophilic and crosses the blood brain barrier with
concentrations in the CNS approximating 30% of
plasma concentrations.
• Used in the treatment of relapsed gliomas and
melanomas.
• Dose is 150mg/m2 for 5 days every 28 days.
• Patient is advised to avoid sun exposure for several
days after drug intake

57. MECHANISMS OF RESISTANCE
1. Alkylating agents are potent electrophiles that react
with electron rich molecules like glutathione (GSH).
GSH- sulphydryl transferase enzyme catalyses the
conjugation of GSH to electrophiles thereby resulting
in inactivation. Hence cells containing high levels of
GSH or GSH-SH show resistance to alkylating
agents.
2. Membrane transporter MDR protein can efflux GSH
conjugates from the cell leading to resistance
3. Metallothionein binds melphalan and phosphoramide
mustard. Exposure of cells to zinc increases
metallothionein in cells and increases resistance to
melphalan, doxorubicin and cisplatin.

58. MOR--Contd
4. Repair of DNA damage produced by alkylating
agents with enzyme O6alkylguanine alkyl
transferase.(responsible for resistance to
temozolomide)
5. Enhanced repair of cross links produced by
the alkylating agents with nucleotide excision
repair enzymes.

59. TOXICITY PROFILE
HEMATOPOIETIC TOXICITY
1. Cyclophosphamide causes granulocyte
depletion with relative platelet sparing.Bone
marrow recovery occurs by 21 days.
2. Nitrosoureas produce unusually delayed bone
marrow suppression with nadirs of both
granulocytes and platelets at 5-6 weeks.
3. Severe granulocytopenia and
thrombocytopenia occurs with busulfan with
relative sparing of lymphocytes.

60. GASTROINTESTINAL TOXICITY
1. Nausea and vomiting are significant side
effects.
2. Cyclophosphamide causes delayed emesis as
late as 8 hrs after drug administration.
3. Significant mucositis, stomatitis and diarrhea
can occur after high dose myeloablative
therapy with melphalan and thioTEPA.

61. RENAL AND BLADDER TOXICITY
1. Hemorrhagic cystitis seen with
cyclophosphamide and ifosfamide (can be
lessened by adequate hydration and continuous irrigation of the
bladder with MESNA and frequent bladder emptying)
2. At high cumulative doses nitrosoureas
produce a dose related renal toxicity
(increases in serum creatinine appear after completion of therapy,
renal biopsy shows prominent glomerulosclerosis, basement
membrane thickening and interstitial fibrosis)

62. PULMONARY TOXICITY
1. Long term busulfan leads to tachypnea, cyanosis
and severe pulmonary insufficiency due to interstitial
fibrosis.
2. Cyclophosfamide, BCNU produce pulmonary
insuffiency.
3. Melphalan leads to pulmonary fibrosis after therapy.
( direct cytotoxicity to alveolar epithelium leading to alveolitis and fibrosis)

63. GONADAL TOXICITY
1. In men a depletion of testicular germ cells
leads to aspermia. However testicular
damage is reversible.
2. In women, a high incidence of amenorrhea
and ovarian atrophy is associated with
cyclophosphamide or melphalan.
3. Teratogenicity: maximum in the first trimester.

64. CARCINOGENESIS
1. A fulminant AML preceded by a phase of
myelodysplasia is found in some patients treated
with melphalan, cyclophosphamide, chlorambucil,
nitrosoureas.
2. Rate of occurrence of acute leukemia in patients of
ovarian cancer who survive for 10 yrs after t/t with
alkylating agents is as high as 5-10%
3. Incidence more with high dose myeoablative
regimens.
4. AML develops 4-6yrs after drug exposure.
( chromosome 5 or 7 deletion)
5. Bladder cancers in patients treated with ifosfamide
or cyclophosfamide.

65. IMMUNOSUPPRESSION
1. Humoral and cellular immunity
suppressed
2. Maximum effect seen with
cyclophosphamide

66. FUTURE PERSPECTIVES
1. Drug combinations with enzymes
(ADEPT)
2. Drug combinations with viral targeting
agents.
3. Canfosfamide

67. THANK YOU