Friday, February 11, 2011

Bendamustine: is it better?

Bendamustine or Treanda is being seen as the new player on the block by many oncologists and is often regarded as the place to go when the patient becomes resistant to fludarabine combinations.

As can be seen from the structural diagram, Bendamustine is an alkylating agent very similar to chlorambucil with the interpolation of a benzimidazole ring. Enthusiasts have suggested that this gives it properties similar to fludarabine, and have suggested that it will bear some resmblance to fludarabine plus cyclophosphamide when given to patients.

I have yet to see a study which demonstrates that it has purine analog-like properties, indeed Wikipedia (for what it's worth) says Bendamustine acts as an alkylating agent causing intra-strand and inter-strand cross-links between DNA bases. However, experts draw attention to this paper to suggest that it has a different action to other alkylating agents. In what follows I have attempted to precis their findings, often using their own words, but often going behind what they report.

The authors assert that the DNA single- and double-strand breaks caused by bendamustine are more extensive and significantly more durable than those caused by cyclophosphamide, cisplatinum, or carmustine. They say that the DNA damage mediated by alkylators has been associated with a regulated form of necrotic cell death whereas Bendamustine as a single agent or in combination with other anticancer agents has also shown proapoptotic activity in several in vitro tumor models.

The study used large-scale screening technologies, including the NCI In vitro Cell Line Screening Project (IVCLSP) and gene microarrays to describe potential mechanisms of action of bendamustine that might distinguish it from other alkylators. The IVCLSP program screens up to 3,000 compounds annually for potential anticancer activity. This program uses 60 human tumor cell lines representing leukemia, melanoma, and cancers of the lung, colon, brain, ovary, breast, prostate, and kidney. In a two-stage screening process, compounds are tested for growth-inhibitory activity in all 60 cell lines at a single dose of 10 ╬╝mol/L, and compounds that achieve a threshold activity are retested in all 60 cell lines in a five-dose screen. The growth-inhibitory activity of a tested compound is expressed as log (GI50, TGI, or LC50), where GI50 is the concentration required to inhibit tumor cell growth by 50%, TGI is the concentration causing total growth inhibition, and LC50 is the lethal concentration at which 50% of cells are killed. For each compound tested, 60 activity values (one for each cell line) make up the activity pattern, or fingerprint, of the compound. A Pearson correlation coefficient (PCC) of >0.8 indicates >65% agreement in the sensitivity patterns of two compounds and a high likelihood of a common mechanism of action.


The IVCLSP screen was performed for three alkylating agents: melphalan, chlorambucil, and the active metabolite of cyclophosphamide. The sensitivity patterns were shown to be similar for 25 compounds when compared to melphalan (PCC >0.839), 25 compounds compared to chlorambucil (PCC >0.839), and 23 compounds compared to the active metabolite of cyclophosphamide (PCC >0.800). The agents that matched most closely with these drugs were all alkylating agents. Direct comparisons among cyclophosphamide, chlorambucil, and melphalan showed strong correlation coefficients (0.76-0.93).

In contrast, the sensitivity pattern of bendamustine did not strongly correlate with any of the compounds tested. In fact, of the top six matches, only dacarbazine (another alkylator) showed a sensitivity agreement (r2) exceeding 50%. The remaining matches for bendamustine included a topoisomerase 1 inhibitor, an anthracycline, and an anti-metabolite, in addition to two other alkylators, including melphalan. Unfortunately they did not report any results obtained with the purine analogs, and the anti-metabolite was Fazarabine or Ara-AC. These results suggest that bendamustine has a unique mechanistic profile compared with most conventional alkylators, though the correlation with dacarbazine, the alkylator favored for the treatment of melanoma, was 0.792, which was greater than the correlation between chlorambucil and cyclophosphamide (0.762) and cyclophosphamide and melphalan (0.765)

To define the differences in molecular mechanisms of action between bendamustine and other alkylators, they conducted gene expression analyses. Affymetrix GeneChip analysis was used to compare the expression levels of >12,000 genes in SU-DHL-1 cells, a non–Hodgkin's lymphoma cell line, after 8 h of treatment with bendamustine, the active metabolite of cyclophosphamide, chlorambucil, or no drug.

Compared to the control, the genes showing the most significant modulation in the bendamustine experiments were in the following major “functional groups”: (a) response to DNA-damage stress; (b) DNA metabolism; (c) cell proliferation; and (d) cell regulation. A notable difference in the experiments with the active metabolite of cyclophosphamide was the absence of several biological processes belonging to the “DNA metabolism, DNA repair, transcription” and “cell cycle, mitotic checkpoint” groups. Chlorambucil was only highly linked to two biological processes: regulation of biological process and regulation of cellular processes. In a head to head comparison between chlorambucil and bendamustine, two main pathways were identified by this comparison as statistically different between the two compounds: nucleic acid metabolism and mitotic checkpoint-cell cycle regulation. The biological processes with the highest P value in this comparison were deoxyribonucleoside triphosphate metabolism, deoxyribonucleotide metabolism, and regulation of CDK activity.

Many pro-apoptotic genes known to possess p53-response elements in their promoter regions, and considered to be p53-dependent, were found by the microarray analysis to be induced by bendamustine. Examples of these genes are p21 (Cip1/Waf1/cyclin-dependent kinase inhibitor) and NOXA . Both genes were also induced by equitoxic concentrations of the active metabolite of cyclophosphamide and chlorambucil, but to a much lower extent. Bendamustine, but not cyclophosphamide or chlorambucil, caused an appreciable increase in the protein expression of Bax.

In addition to p53-related genes, other regulated genes related to apoptosis and identified in the top 100 modulated genes were four members of the tumor necrosis factor–receptor superfamily. Several of these genes have been shown to play a critical role in the regulation of the extrinsic apoptotic pathway.

Bendamustine induced a stronger (2.5-fold) up-regulation of the DNA repair gene exonuclease-1 (EXO1). EXO1 expression compared with that observed with cyclophosphamide (1.5-fold) or chlorambucil (1.8-fold). DNA-repair capacity has been shown to play a critical role in resistance to alkylating drugs and it may be postulated that these pathways may contribute to the different activity/resistance profiles observed for bendamustine versus cyclophosphamide and chlorambucil.

Bendamustine also caused a significantly greater increase in the proportion of cells in the S-phase of the cell cycle (∼60%) compared with chlorambucil (45%) and cyclophosphamide (37%), based on the DMSO control (37%). It is possible that a defect in mitotic checkpoints inhibits the “physiologic” arrest of the DNA alkylator–treated cells, required for efficient repair of DNA damage before cells are allowed to enter mitosis. Cells entering mitosis with significant DNA damage are reported to result in activation of the death pathway known as mitotic catastrophe. Mitotic catastrophe is a necrotic form of cell death that occurs during metaphase and is morphologically distinct from apoptosis. It can occur in the absence of functional p53 or in cells where conventional caspase-dependent apoptosis is suppressed.

To determine whether bendamustine can cause mitotic catastrophe, it was necessary to find a model in which the apoptotic effects of bendamustine could be distinguished from the potential mitotic catastrophe end point. To this end, bendamustine was tested in cell lines with deficiencies in apoptotic pathways and in the presence of an inhibitor of classic apoptotic pathways. Microscopic analysis of nuclear morphology using 4′,6-diamidino-2-phenylindole staining revealed an increased incidence of chromatin condensation and multinucleation/micronucleation, hallmarks of mitotic catastrophe, in the cell lines tested. Twenty-six percent of the bendamustine-treated cells showed micronucleation compared with only 6% in DMSO control cells. These data indicate that in addition to inducing apoptosis, bendamustine may cause mitotic catastrophe. No results are given for other alkylating agents in this test system.

What are we to make of these results? The first thing that we should note is that the results were produced in the laboratory of Salmedix, a company that has been acquired by Cephalon, who are marketing Treanda in the US. I shall comment later on Big Pharma when I get to discussing the clinical trials that have been done, but I am suspicious of a company that markets a 40-year-old drug at such an inflated price.

Second, were I refereeing this paper, I should have asked for extra data, particularly asking for similarities or not between bendamustine and fludarabine in the IVCLSP assays, and for comparisons with other alkylating agents in the mitotic catastrophe experiments.

Third, these results are all very indirect. Upregulation of genes may or may not exert a pharmacological effect. As far as I know there is no clinical study that shows that bendamustine is effective in TP53 deficient CLL as might be expected from these results. It may be that the combination of mechanism makes bendamustine a more efficient alkylating agent than chlorambucil, but with effectiveness against tumor cells comes toxicity against normal cells. Clinical studies do show more toxicty than chlorambucil.

Fourth, nothing in this study suggests that bendamustine behaves like fludarabine, for all it possesses a benzimidazole ring.

Fifth, I feel I must reiterate my criticisms of the pivotal trial that allowed the FDA to grant approval for bendamustine.

319 patients were randomly assigned to receive either Bendamustine or Chlorambucil (162 Bendamustine, 157 Chlorambucil). The overall response rate was 68% for Bendamustine and 31% for Chlorambucil. CRs were 31% for Bendamustine and 2% for Chlorambucil. Median progression-free survival was 21.6 months for Bendamustine and 8.3 months for Chlorambucil. Bendamustine seems astonishingly better than Chlorambucil, though this does seem a very poor result with Chlorambucil, compared with, say, the LRF CLL4 trial, where the median progression free survival was 22 months.

Toxicity was worse with Bendamustine. Grade 3 or 4 adverse events occurred in 40% for Bendamustine and 19% for Chlorambucil.

This paper is published in JCO which probably means it was turned down by Blood. The criticism which any Blood reviewer would have made (and I have reason to believe was made) would have been that the dose of chlorambucil was too low. They have answered this criticism by declaring that the dose of chlorambucil was equivalent to 60 mg/meter squared which compares well with comparisons with fludarabine (Rai = 40), alemtuzumab (Hillmen = 40) and FC (Catovsky = 70).

That seems to be alright, but then I read the paper more carefully. The two drugs were given according to quite different formulae. Bendamustine was given in a dose of 100mg/sq meter for two days every 4 weeks, while Chlorambucil was given in a dose of 0.8 mg/kg for two days every two weeks. It seems strange that two different calculations were used and even stranger when I see that rather than weighing the patients they used something called Broca's normal weight.

I didn't have a clue what Broca's normal weight was and I am pretty sure that most people reading the paper won't have either. So I Googled it. It turns out to be the height in centimetres minus 100. Does this give the same answer as weighing? By no means. I am 176cm high so my Broca weight is 76kg. Alas my scales make me 90 kg.

If I calculate the dose of chlorambucil I would have got under the LRF4 formula it would have been 148 mg, but under the Bendamustine paper formula it would have been 123 mg. I'm afraid my ideal weight is a little less than my actual weight. For my mother the discrepancy would have been even more. She is only 5ft 3, but weighs about the same as me. The Bendamustine paper would have given her 96 mg of Chlorambucil while the LRF4 calculation would have given her 139 mg. Since most people today weigh considerably more than their ideal weight, it looks to me that the chlorambucil dose given in this trial was too low for a fair comparison.

Since I wrote about this, it has emerged that to get the full benefit from chlorambucil it needs to be given for longer than 6 months- preferably up to twelve.

My conclusion then is that Bendamustine is just a way of getting adequate doses of an alkylating agent into a patient, but I am prepared to be convinced that is more than that if soeone would only show me the data.

7 comments:

~ chris said...

Perhaps the CLL10 trial will answer some of these questions?

Anonymous said...

interesting article!

Debbie

Anonymous said...

"Broca's normal weight"

You don't have to be a scientist(which I am not)to know something is wrong when two different methods are used to determine dosing....something very wrong....

please, continue to poke holes in "questionable" data

Brian Koffman said...

Do you have any data on the efficacy of bendamustine for 11q del? It seems that 11q del do better with alkylating agents and certainly with cytoxan, but is that a general effect? I am also wondering if bendamustine + R might be a less immune suppressive than FCR?

Terry Hamblin said...

The German CLL8 trial demonstrated that FCR was better than FC especially for Del 11q. I don't know of any evidence that alkylating agents are better for del 11q despite Rick Furman's recent post. It may be that B+R will have the same benefit but I don't know of any evidence that this is true.

Wayne said...

Terry,

Does the finding that Bendamustine increases BAX have clinical importance given that BAX is one of several pro-apoptotic proteins?

You state: "Bendamustine was tested in cell lines with deficiencies in apoptotic pathways...." Are tests of this nature done only in-vitro and if so how reliable are the findings when applied to the microenvironment of lymphnodes and bone marrow?

Are results from this level of testing detailed enough to match Bendamustine to a subtype of patient if enough were known about a patient's genetic & epigenetic dysfunction?

WWW

Terry Hamblin said...

As you surmise these are in vitro results on cell lines so they can only suggest clinical trials. I don't think they are detailed enough or comprehensive enough to draw any other conclusions.