We recommend that users take steps to verify delivery from ALZET pumps. There are two techniques which can provide information about how a pump has functioned during the infusion period:
- Measurement of plasma levels during infusion
- Measurement of the residual volume in the pump reservoir after explantation
To verify pump delivery in vitro, refer to the recommended protocol.
Note: the weight of a partially empty or discharged pump cannot be used to determine the quantity of drug delivered because the pump imbibes water during operation. Likewise, cutting open a spent pump is not a reliable means of verifying pump performance.
Monitoring blood or plasma levels of the drug administered at several
points during the course of administration provides the most reliable
and quantitative information for verifying pump functionality. This
allows one to detect when such levels reach steady state and whether
variables unrelated to pump functionality (e.g. metabolic tolerance)
influence experimental results.
In the event that determination of circulating blood levels is not possible or is technically undesirable, another method of verifying pump functionality is to measure the residual drug solution which remains in the pump reservoir at the time the pump is explanted.
What is it?
Residual volume is the solution remaining inside the pump reservoir at the end of the infusion period. At the completion of its stated duration of infusion (from one day to four weeks depending upon the model), the pump reservoir will still contain some of the solution originally loaded into it.
How is it measured?
Use a filling tube attached to a syringe to aspirate the remaining solution from the reservoir of the pump. (Use of a needle risks puncturing the reservoir and drawing solution from the osmotic chamber instead, which could result in an erroneously high measurement.) Subtract the volume removed from the initial loading volume. This net amount of solution, divided by the elapsed time, provides a measure of the average release rate. This method is not as quantitative as measuring plasma levels of the compound. On occasion, the pump's reservoir collapses, making it difficult to extract solution from its interior.
How much residual volume should there be?
The residual volume can be predicted from the initial volume loaded in the pumps (fill volume), pumping rate, and duration of infusion. If the pumps were used in an environment other than 37º C, please use our ambient temperature calculator first, to obtain the adjusted flow rate for your pump model.
Example: A Model 2002 pump with a mean pumping rate of 0.45 µl/hr is filled with 225 µl and implanted for 14 days. Upon removal, 70 µl are aspirated from the reservoir. Based on this amount, can one assume that the pump functioned properly during infusion? Yes, this is an appropriate amount to recover from this pump. It would have infused approximately 151 µl during the 14-day infusion period:
Mean Pumping Rate x Infusion Duration (in hours) = Volume Infused
0.45 µl/hr x (24 hrs x 14 days) = 151 µl
This would leave a residual volume of 74 µl:
Actual Fill Volume – Volume Infused = Residual Volume
225 µl – 151 µl = 74 µl
Why is there a residual volume?
The residual volume represents a safety margin in the design of these
devices. The reservoirs are deliberately made slightly larger than is
necessary for the specified duration of pumping so that on the last
day of the nominal period of pumping, there is enough fluid left in
the reservoir to support constant delivery at the specified rate. This
safety margin also allows for time to prime the pumps, and provides
some flexibility in case the experiment cannot be terminated immediately.
ALZET pumps are supplied with a specified volume pumping rate determined in isotonic saline at 37°C (± 0.5°C). In the event that the user finds it necessary to test the pumping rate, DURECT recommends that the following steps be taken:
- Indicator dye (e.g., FD&C Blue No. 1)
- Filling tubes (order)
- ALZET pumps & flow moderators (order)
- 20 ml test tubes with caps (or other water tight closure)
- Incubator or water bath
Step 1. Prepare a solution of known concentration of an indicator dissolved in isotonic saline. The amount of indicator pumped out of the pump will be the basis for the pumping rate calculation. Therefore, the indicator solution should be compatible with the pump and be easy to analyze in isotonic saline. One such indicator is FD&C blue #1 dye at 10 mg/ml for all 100 µl and 200 µl sized pumps (except Model 2001D), and 1.0 mg/ml for Model 2001D and all 2 ml sized pumps.*
Step 2. Fill the ALZET pump with the indicator solution. ( Filling procedure )
Step 3. Place the ALZET pump into a 20 ml test tube pre-filled with 15 ml of isotonic saline and pre-incubated at 37°C (± 0.5°C). Cap the tube to prevent evaporation.
Step 4. Record the starting time of the incubation.
Step 5. Transfer the ALZET pump to a new test tube filled with 15 ml of isotonic saline approximately every 12 hours. Record the precise time of the transfer and/or the time interval between transfers.
Step 6. Repeat Step 5 daily for the duration of the pump. (For example, 7 days for Model 2001)
Step 7. Analyze for the concentration of indicator in each test tube against a standard of known concentration to determine the amount of indicator pumped during each time interval. From this amount and the concentration of the indicator in the solution initially loaded into the ALZET pump, calculate the volume pumped during each interval. Divide by the time interval in hours to obtain the average hourly volume pumping rate.
*Note: When FD&C blue #1 dye is used, the recommended wavelength for spectrophotometric analysis is 630 nm.
- Ensure around-the-clock exposure to test agents at predictable levels
- Permit continuous administration of short half-life proteins and peptides
- Provide a convenient method for the chronic dosing of laboratory animals
- Minimize unwanted experimental variables and ensure reproducible, consistent results
- Eliminate the need for nighttime or weekend dosing
- Reduce handling and stress to laboratory animals
- Small enough for use in mice or very young rats
- Allow for targeted delivery of agents to virtually any tissue
- Cost-effective research tool
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Researchers are saying...
"Utilizing continuous minipump administration enhances the knockdown to 63% of control." (p. 619) This compared with 40% by twice daily administration. "The results of the present study confirm the utility of osmotic minipump administration of antisense oligonucleotides." (p. 621) Falk et al., Pharmacol Biochem Behav 2002;72:617-622.