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Published: June 2009Print Record of Viewing
Dr. Snozek provides an overview of pharmacokinetic testing for intravenous dosing of busulfan, an alkylating agent used for bone marrow ablation prior to hematopoietic stem cell transplantation. She discusses the details of specimen collection, assay procedure, and result reporting.
Presenter: Christine Snozek, PhD, DABCC
Welcome to Mayo Medical Laboratories' Hot Topics. These presentations provide short discussion of current topics and may be helpful to you in your practice.
Our speaker for this program is Dr. Christine Snozek, Assistant Professor of Laboratory Medicine and Pathology and Associate Director of the Toxicology and Drug Monitoring Laboratory in the Division of Clinical Biochemistry and Immunology at Mayo Clinic. Dr. Snozek will provide an overview of pharmacokinetic testing for intravenous dosing of busulfan, an alkylating agent used for bone marrow ablation prior to hematopoietic stem cell transplantation. She will discuss the details of specimen collection, assay procedure, and result reporting.
Busulfan has been used for decades as an alkylating agent, which means it binds to nucleic acids during cell division, in order to kill the replicating cells. Busulfan is preferentially toxic to hematopoietic precursor cells, with comparatively little effect on mature lymphocytes, making it a useful agent to ablate bone marrow prior to hematopoietic stem cell transplant. However, the high doses necessary to successfully prepare bone marrow for stem cell engraftment carry the risk of serious toxicity, including the development of veno-occlusive disease of the liver, or VOD.
This is exactly the reason why we do pharmacokinetic testing for busulfan. Both the success of stem cell engraftment and the risk of toxicity depend on the amount of busulfan that a given patient is exposed to. Too much drug increases the likelihood of serious toxicity, but too little drug risks the entire transplant being unsuccessful. So an accurate assessment of how much busulfan each patient is exposed to is essential. To do this, we test a series of samples from patients receiving intravenous busulfan, and measure the serum drug concentration at each timepoint. We’ll look at this in more detail in a moment, but the key point here is that this series of samples allows determination of overall exposure to busulfan, which is measured as the area under the concentration-time curve. Other pharmacokinetic parameters are calculated from the AUC measurements, including busulfan clearance and an estimated dose.
Although the analysis of several timepoints can increase both costs and the difficulty of obtaining samples, it’s been shown that busulfan pharmacokinetic testing is well worth the price. Patients show improved engraftment and less-frequent toxicity when busulfan analysis is used to adjust dosing, at a cost of less than 1% of the total charges associated with stem cell transplantation.
So what do we see when pharmacokinetic testing is performed? I’ve shown here a cartoon of typical results for a patient receiving a busulfan infusion. This is not drawn to scale, but it will serve to illustrate how the AUC is determined. On this curve, the y-axis is the drug concentration in serum, and the x-axis is the time after stopping the infusion of busulfan. These are the factors that will determine the AUC, the area under the curve of concentration vs time. So to calculate the AUC, the first blood sample is taken at the time the busulfan infusion is stopped. This is called the immediate post-infusion sample, and should contain the highest concentration of busulfan. Next, to look at elimination of the drug, three more samples are drawn at timepoints of one, two, and four hours after the infusion is stopped. It is the addition of the initial two hour infusion to the four hour sampling time that makes this the 6 hour AUC.
So the patient’s ability to eliminate busulfan is assessed using all four timepoints, as seen with this elimination curve. In addition, the immediate post infusion sample defines the drug exposure calculated for the two-hour infusion. In other words, the calculation assumes that the amount of busulfan in the bloodstream throughout the infusion is the same as the concentration measured in the first post-infusion sample.
Putting it all together, then, the two-hour intravenous infusion and the four-hour series of post-infusion samples are integrated together to determine the 6-hour Area Under the Curve.
So why go to all this effort? It’s important to remember that the goal of measuring the AUC is to ascertain how much busulfan a patient is being exposed to. As with any drug, there can be a large amount of variability between patients receiving busulfan. Variation in factors such as metabolic rate or elimination can affect how long the drug remains in a patient’s system, potentially leading to ineffective therapy or toxicity. The AUC is used as a means of comparing patients and ensuring that each one receives the proper amount of busulfan. It’s important to note that the literature regarding busulfan ablation uses several different measures of overall drug exposure; these can include the 6 hour AUC, an AUC that is extrapolated to infinity, the steady-state concentration, or the trough concentration. Here at Mayo, we’ve chosen the 6 hour AUC because of the balance it provides. The number of samples provides detailed information about each patient’s busulfan exposure, without being prohibitively difficult. More importantly, the AUC calculation is based only on measured values, rather than relying on a computed algorithm to extrapolate predicted serum levels.
The busulfan pharmacokinetic test was established based on a protocol of 16 total doses of 0.8 mg/kg body weight, spread over four days. As I mentioned, there are four total samples required for the analysis. These are drawn after the conclusion of a 2-hour infusion, and are taken immediately after the infusion is stopped, then one, two, and four hours after the infusion ends. The report provided will include the 6 hour area under the curve, a calculated elimination rate, and the estimated dose adjustment needed to reach an optimal level of busulfan exposure. For this protocol, optimal busulfan exposure is seen at an AUC of 1100 umol/min. Toxicity is more likely with AUCs above 1500 umol/min, while AUCs below 900 umol/min are typically less effective. Please note that we can perform busulfan analysis for other administration protocols, and we can also provide concentration data for use with other dose optimization algorithms. Please contact the lab if you’re interested in discussing these options.
Each set of samples should be submitted to the lab with a patient information sheet. In addition to the patient’s name and busulfan dose, it is essential that the lab receives accurate information about the patient’s age, and the weight used to calculate the appropriate dose. If an adjusted weight is used in calculating busulfan dose, for example, with young children or in overweight patients, the adjusted weight should be provided in the patient information. Finally, please provide accurate times for the infusion start and stop, and for each of the samples drawn.
Completing the patient information sheet properly is the first step in ensuring accurate results for busulfan. However, I’d like to take this opportunity to highlight the most common problem we see with busulfan testing, and to provide some tips on how to prevent it from happening. The problem we see most often is referred to as an atypical curve. What this means is that the first post-infusion sample does not show the highest concentration of busulfan. I’ll show an example of this visually, then discuss some reasons why it happens.
Going back to the demonstration cartoon with the normal curve in blue, I’ve added an example of an atypical curve, that’s shown in pink. As you can see, the first sample does not show the highest concentration of busulfan, which indicates that there is a problem with one or more of the samples. Because of this, we are unable to determine an accurate AUC. One question we’re commonly asked is whether we can correct for an atypical curve. The problem with this sort of correction is that it’s usually not possible to determine which sample or samples are the source of the atypical curve. For example, what if the first two samples were accidentally switched? If this were the case, the “corrected” results would look like this… (refer to slide)
With an elimination curve showing relatively low exposure to busulfan.
However, it’s possible that only the first sample is problematic. We see this commonly with samples drawn from the same intravenous line as was used to administer the busulfan. What happens is that the saline used to flush the remaining drug from the line gets mixed with the blood drawn in the first sample. In other words, the post-infusion sample is falsely diluted, and the first concentration should lookmore like this (refer to slide) giving a curve showing relatively high exposure to busulfan.
As you can see, the effect of having the first two samples switched is considerably different from the effect of diluting the first sample. The busulfan AUCs are very different for the two scenarios. Unfortunately, it is generally not possible to discern which of these types of error is the source of a given atypical curve, meaning that the results are uninterpretable.
So to summarize, busulfan pharmacokinetic testing is used to optimize and individualize dosing of this drug for stem cell transplant. Both toxicity and outcomes are improved when this sort of analysis is performed, leading to better treatment and quality of life for the patient. The most common problem we see is an atypical curve, where the immediate post-infusion sample does not have the highest concentration of drug. There are two major reasons why this occurs, and therefore two ways to prevent it: first, careful attention to labeling should eliminate accidentally switched samples. And second, following established line draw protocols for flushing the intravenous line and discarding waste blood before drawing the sample should prevent accidental dilution of the post-infusion timepoint. These easy fixes should ensure that your busulfan results are accurate and meaningful.