Alkylating agents

Question 1

Give example of a mono-functional and bifunctional alkylating agent

Answer 1

Mono - Dacarbazine

Bi - Cyclophosphamide (CPA)

Question 2

What is the mechanism of action?

Answer 2

Highly reactive alkylate products that covalently binds onto DNA.

Example:

• Mono-functional drugs - N7 guanine is methylated - methylation causes anti tumor activity
• Bifunctional dugs, cross links (interstrand, intrastrand, DNA protein) occur to disrupt DNA replication

Question 3

How is this drug metabolised?

Answer 3

Cyclophosphamide is the prodrug and needs to be metabolised before it becomes active. This is done by CYP450 enzymes such as CYP2B6 to 4 hydroxy cyclophosphamide and DCCP (inactive). This is further metabolised (in liver but more in tumour cells) forming more inactive metabolite - carboxyphosphamide before forming phosphoramide mustard and Acrolein. Phosphoramide mustard is the anti-tumour active metabolite.

Question 4

How does drug resistance happen?

Answer 4

Resistance happens to the following 3 mechanisms:

Pre-target repair
1. Formation of DNA crosslinks is prevented by drug inactivation caused by glutathione S transferase found in tumour cells. GST also inhibits MAP kinase pathway therefore tumor cells are unable to undergo apoptosis

Post target repair

2. DNA fragmentation by repair enzymes is repaired by up-regulation of base excision repair pathway in tumour cells
3. Induction of apoptotic pathways such as p53, Bax become mutated or deficient.

Question 5

Pharmacogenetics of cyclophosphamide

Answer 5

Yule et al. 2004 - Study of cyclophosphamide in NHL in paediatrics.

The study found that variation in cyclophosphamide metabolism may influence the risk of B-NHL recurrence in children. Patients that did not relapse had low levels of inactive metabolites (DCCP and carboxyphosphamide) and high clearance of cyclophosphamide

This result suggests that the balance between the production of active and inactive metabolites by cytochrome P-450s is important in determining the treatment outcome. Studies found variants of CYP3A4 results in higher cyclophosphamide exposures and decreased survival rates.

Question 6

Tumor type treated with cyclophosphamide

Answer 6

Adjuvant breast cancer
Leukemias and lymphomas
Paediatric tumors

Question 7

Toxicity profile of cyclophosphamide

Answer 7

Haemorrhagic cystitis - caused by Acrolein (inactive metabolite of cyclophosphamide) therefore give MESNA to bind to acrolein.

Alopecia

Bone marrow suppression

Lethargy

Nausea

Question 8

How do we modify delivery of drug for more effective treatment?

Answer 8

Potential for CYP450 gene delivery.
This increases selective delivery of drug to tumour instead of host cell to enable reduction of dose and increased effectiveness of tumour cell death.

Jounaidi et al, 2004 found

• Gene therapy by coordinated delivery and expression of P450 and P450R on a single vector (retrovirus) using an internal ribosomal entry site (IRES) sequence.
• This combination of Adeno-P450 tumor cell-targeted helper adenovirus dramatically improved the low gene transfer observed with some human tumor cell lines
• It also increased tumor cell-catalyzed CPA 4-hydroxylation, CPA cytotoxicity, and in vivo antitumor activity in tumor xenograft model.