Formulating the Test Solution

Determining the Required Concentration

The primary objective in formulating the test solution is to concentrate the drug solution such that when loaded into a given pump, the animal will receive the desired dose. The information below will assist you in determining the concentration with which to load the pumps.

ALZET osmotic pumps are designed to deliver a fixed volume of test solution at a constant rate. The first step in formulating a test solution to be administered is to decide on the dose or hourly mass flow of test material you wish to deliver. To cut the mass flow rate in half, use half the concentration; to double the mass flow rate, use twice the concentration, etc. The highest mass flow of test material that the pump can deliver is set by the maximum solubility of the test agent in its vehicle (saturated solution) at the temperature of loading (usually room temperature, or about 23º C). The amount of agent (µg/hr) that a pump delivers can be adjusted by altering the concentration of agent in the test solution.

An Example:
To determine the concentration of antidiuretic hormone (ADH) for subcutaneous delivery using a Model 2001 pump in rats with diabetes insipidus, the following steps were undertaken:

Step 1. A search of the literature indicated that the normal secretion of ADH in the rat was 30 ng/day/animal, or 1.25 ng/hr/rat. Next, the concentration of ADH in the infusion medium (saline) that would be necessary to acomplish the desired infusion of 1.25 ng/hr was calculated:

Mass flow = volume flow rate x concentration, or

k_o = Q • C_d

Here k_o (µg/hr) is the mass delivery rate, C_d (µg/µl) represents the concentration of drug solution, and Q (µl/hr) the pumping rate of the pump. To calculate the concentration of agent delivered from the solution used to fill the pump, divide the total dose administered (per hour) by the pumping rate of the pump.

Step 2. The volume flow rate of the Model 2001 pump is 1 µl/hr (Q), so the required concentration (C_d) is:

C_d = 1.25 ng/hr = 1.25 ng/µl = 1.25 x 10^-3 µg/µl
1 µl/hr ccccccccccccccccccccccccccccccccccc

Step 3. The Model 2001 pump has a nominal reservoir volume of 200 µl. In order to achieve the desired concentration of 1.25 x 10^-3 µg/µl, the amount of ADH which should be dissolved in 200 µl is:

(1.25 x 10^-3 µg/µl) x 200 µl = 0.25 µg

In summary, in order to mimic the normal secretion of ADH in the rat of 30 ng/day/animal (or 1.25 ng/hr/rat), one must make a solution with a concentration of 1.25 x 10^-3 µg/µl of ADH in saline. Excess solution should be made to allow for a safety margin during pump filling.

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Considerations on formulating proteins and peptides

 

Selecting a Vehicle

When planning the administration of a given compound, it is important to select the optimum vehicle. This choice of solvent should include the following considerations:

• Solubility of the agent to be delivered
• Compatibility with the interior reservoir of the pump
• Compatibility with tissues or fluids at the site of administration
• Stability of the compound/vehicle solution for the duration of the experiment (if possible, vehicle control studies are recommended)
• Sterility

Because the volume delivery rate of ALZET pumps is fixed, the maximum administration rate of a given compound from a given pump is limited by its solubility in the chosen vehicle. Alternate vehicles may be available which better solubilize the test compound, thereby increasing its maximum administration rate. Higher doses can be administered by choosing a pump with a higher flow rate, or dividing the dose among several simultaneously-implanted pumps (assuming that the experimental animal is of sufficient size). Cyclodextrins may also be helpful to enhance solubility.

To ensure the tissue and blood compatibility of the solution administered, care should be taken during preparation and handling to maintain its sterility and isotonicity. Many media provide a hospitable environment for the growth of activity-destroying microorganisms. If the agent is supplied as a non-sterile, dry powder, it is advisable to filter-sterilize the solution during or immediately prior to filling. Microbial contamination within the pump reservoir can result in local fibrosis, inflammation, and infection.

It is wise to use standard parenteral fluids for making solutions to ensure sterility and non-pyrogenicity. Consequently, non-lactated Ringer’s solution is preferred for the subcutaneous, intraperitoneal, or intravenous infusion of water-soluble agents.

For infusions into the brain (either via the cerebral ventricles or into solid tissue), the preferred vehicle is artificial cerebrospinal fluid (CSF). (Recipe)

A solution that generates gases within the pump makes the pumping rate highly unpredictable. Also, solutions with precipitated solute particles must be filtered before use. Solutions should be at room temperature when filling the pump.

ALZET pumps are capable of delivering homogeneous viscous solutions with a viscosity of less than 100,000 centipoise, including vehicles such as PEG 300, PEG 400, propylene glycol, and glycerol. Suspensions may be delivered from the pump if they do not precipitate. To ensure uniform delivery, suspensions must remain homogenous throughout the duration of delivery.

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Vehicle Compatibility with the Pump Reservoir

The pumps are compatible with aqueous solutions, dilute acids and bases, dilute or low concentrations of DMSO, and ethanol. Suitable vehicles are listed below. In general, natural oils and most organic solvents are not compatible with ALZET pumps. Contact with these substances can result in pump malfunction.

If your preferred solvent does not appear on the list below:

• Contact ALZET Technical Support to check on the compatibility of your solvent with the pumps
• Use a Lynch coil with the ALZET pumps to deliver your solution (What is this?)
• Use an ALZAID® Chemical Compatibility Test Kit to test the solvent or solvent/drug mixtures. (Cannot be used for solvents including ethanol.)

 

Commonly used solvents known to be compatible with ALZET pumps:

• Acids, with pH greater than 1.8
• Bases, with pH less than 14
• Cremophor EL, up to 25% in water
• Culture media (1% benzyl alcohol as bacteriostatic)
• Cyclodextrins
• Dextrose, up to 5%, in water or NaCl
• N,N-Dimethyl formamide (DMF), up to 25% in water
• DMSO, up to 50% in water or polyethylene glycol
• DMSO, up to 50% in ethanol (< or =15%) and water
• Ethanol, up to 15% in water
• Glycerol
• 1-Methyl-2-Pyrrolidone, up to 12.5% in water
• Phosphate buffer
• Polyethylene glycol 300 or 400, neat or in water
• Propylene glycol, neat or in water
• Ringer’s solution (with or without lactate)
• Saline, 0.9% (or other aqueous salt solution)
• Serum (rat, mouse, etc.)
• Solutol, up to 30% in water
• Triacetin, up to 5% in water
• Tween 80, up to 2%
• Water, distilled

Note: An agent or solvent that is incompatible with the pump’s reservoir material still can be pumped using an ALZET pump. This is done by attaching an “external reservoir” to the pump. The contents of the external reservoir are displaced by the action of the pump. The external reservoir can be created easily, such as from coiled PE-60 catheter tubing, in a technique known as the Lynch coil. More about this simple technique

Stability

Long-term, constant delivery of test agents can be obtained only if the test agents are stable in solution at body temperature for the duration of infusion. While it is important to use sterile solutions, especially if test materials are potential substrates for microorganisms, it is equally important to choose a vehicle of the right characteristics. Some considerations are pH, ionic strength, redox potential, etc. Some antioxidants, buffers, and similar compounds may suffice to stabilize, but the choice may have to be made empirically, rather than on theoretical grounds. It may be helpful to consult with an industrial or hospital pharmacist, or with the manufacturer of the test agent, to obtain assistance with regard to optimum formulations.

As a preliminary method for assessing the stability of a test material at 37°C, first fill a small sterile test tube with the sterile material and cap tightly. Incubate at 37°C for the expected duration of the planned experiment and analyze for specific activity. Compare the activity to that of a freshly made sample. If the respective activities are equivalent, the test material is stable in the above conditions. Providing the vehicle is compatible with the pump reservoir ( refer to list of compatible solvents ), then positive results for an agent's stability using the above test will probably be indicative of stability within the pump. However, it is recommended that stability testing include an in vitro pumping rate test (Method) to ultimately ascertain compatibility of the test material inside the pump.

 

 

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