It's not possible. You need a more concentrated stock solution. Your 5 uM solution is 4 ug/ml:

             5 x 10-6 mol/L x 808 g/mol = 4 x 10-3 g/L = 4 mg/L = 4 ug/ml

A typical 20 gram (0.02 kg) mouse will require 0.2 mg of the drug:

            10 mg/kg x 0.02 kg = 0.2 mg

To get 0.2 mg of the drug will require 50 ml of the stock solution:

        0.2 mg/(4 mg/L) = 5 x 10-2 L = 50 ml

You need a stock solution that is 500 x more concentrated, or 2.5 mM (2 mg/ml), to use 100 ul.

Thanks Adam.  I thought that could have been an issue.

Hamed, Could you explain your question a bit more?

The acute toxicity can be described by the parameter Maximum Tolerated Dose (MTD), the largest dose that can be given before acute toxicity becomes apparent. The MTD sets an upper limit on the amount of drug you can give.

It's simple enough if the plan is to give a single dose. The MTD is determined by doing a single ascending dose (SAD) trial  But if you want to do multiple doses, you need to find out the MTD for the multiple dose regimen, requiring a multiple ascending dose (MAD) trial.

The timing and required size of the (multiple) dose(s) will be determined by the pharmacokinetics and pharmacodynamics (PK/PD) of the compound.The MTD just puts an upper limit on the amount of drug that can be given.

Hi Noorulain, I don't know of a specific reference. I have seen this type of study done in drug discovery projects.

hope this will help

Dr. Adam can you please help me with one question?

I know the MIC of a particular drug X, but I don't know what dosage of drug should be administered to animal model for toxicity studies. Is there a formula or correlation that can be used to calculate dosage with given MIC?

The goal of initial toxicity studies is to identify the maximal tolerated dose (MTD), so the MIC is not relevant. In a single ascending dose (SAD) study, the dose is increased until acute toxicity is observed.

The MIC becomes relevant when working out the dose for in vivo efficacy experiments.

Dr. Adam,

I need your guidance .

I have just completed my antimicrobial studies and established synergism between drug X and drug Y  (commercially available drugs). how do I proceed with dose selection in Animal model? 

This is a very complex area, particularly when two drugs are involved. However, you can get some help from the fact that these are commercial drugs, so there is probably already a considerable amount of information available about the pharmacokinetics (PK) and pharmacodynamics (PD) of each of the drugs. From this information, you should be able to work out how much of each drug to dose, and on what schedule, to achieve the necessary blood level. First, find out what the PD driver for efficacy is. For antibiotics, it may be AUC/MIC or time above MIC, for example.

Here is a reference on antibiotic PD:

http://www.antimicrobe.org/history/PK-PD%20Quint.asp

Combine this information with the PK of each drug to figure out how much to dose to cover the PD magnitude. Remember that the relevant blood concentration is not the total concentration, but the free concentration, i.e. not plasma protein bound. The plasma protein binding free fraction of the drugs should also be available in the literature.

Since the two drugs are synergistic, the amount of each member of the combination needed for efficacy should be lower than the amount of compounds when used alone. Therefore, the efficacious dose regime for each compound separately can be used as an upper bound for the dose of each one in the combination.

If you can decide on a fixed ratio of the two drugs, or on a fixed concentration of one of them, based on the MICs and/or PK/PD, then you can reduce the size of the experiment.

I strongly recommend you consult an expert on antibiotic PK/PD before beginning. Otherwise, you could use more animals than necessary, which would be unethical.

hi adam,

i have a mice with the weight of 26g and i need to administer a dose of 500mg/kg treatment at a maximum amount of 200ul only according to guidelines. i have prepared the stock solution of 12mg/ml. how do i calculate this? please help

thanks

The dose is 0.026 kg x 500 mg/kg = 13 mg.

If the dose is administered in a volume of 200 µl, the concentration must be 13 mg/0.2 ml = 65 mg/ml.

You either need to make a more concentrated solution, or increase the volume of the dose, or spread the dose out over time using an infusion.

thanks alot adam! appreciate the tip.

Dear dr i have extract not antibiotic and i have tested mic 6mg/ml how much dose i should give to rat for example weight 100gm how to calculate it

There is not a calculation for this. First, consider using mice instead of rats. They are much smaller, so you need less of your substance. If you are testing for efficacy against an infection, you should dose as much as possible at first, even multiple doses per day. Later, you can measure pharmacokinetics and pharmacodynamics if the first experiment gives a positive result.

Removing background and non-specific binding when using BODIPY 493/503, a fluorescent dye often used for staining lipids (like lipid droplets in cells), is crucial for obtaining clear and specific results. Here are some strategies to minimize background and non-specific binding:

1. Optimize Staining Conditions:

• Concentration: Adjust the concentration of BODIPY 493/503. Start with a recommended concentration (often around 1-10 µM) and optimize based on your specific application.
• Incubation Time: Shorten or lengthen the incubation time as needed. Longer incubation can sometimes increase non-specific binding.
• Temperature: Perform the staining at room temperature or 4°C, as recommended by your specific protocol.

2. Washing Steps:

• After staining, wash the cells thoroughly with a suitable buffer (like PBS) to remove unbound dye.
• Multiple washes might be necessary to reduce background.

3. Use of Blocking Agents:

• Use blocking agents in your staining buffer. For example, BSA (Bovine Serum Albumin) or gelatin can be used to block non-specific sites.

4. Control Experiments:

• Perform control experiments to differentiate between specific and non-specific staining. For instance, stain cells known not to have the target lipid droplets.

5. Optimize Fixation:

• If you are fixing cells, optimize your fixation protocol. Over-fixation can increase background, while under-fixation can lead to poor morphology and staining.

6. Mounting Medium:

• Use an appropriate mounting medium that reduces autofluorescence and enhances the fluorescence signal.

7. Imaging Parameters:

• Adjust the microscope settings. Increasing the signal-to-noise ratio can be achieved by adjusting parameters such as gain, exposure time, and pinhole size (in confocal microscopy).
• Use appropriate filters that match the excitation and emission spectra of BODIPY 493/503 to ensure specific detection.

8. Sample Preparation:

• Ensure that your samples are prepared consistently and cleanly to reduce artifacts and background.

9. Dilution Buffers:

• The choice of dilution buffer for the dye can also impact non-specific binding. PBS or other physiological buffers are commonly used.

10. Environmental Factors:

• Be aware of environmental factors that might affect staining, such as pH and ionic strength of the buffers.

11. Data Analysis:

• Use image analysis software to subtract background fluorescence and enhance signal specificity in post-processing.

Remember, the key is to optimize each step of your protocol and perform adequate controls to distinguish specific signal from background noise. If problems persist, it may be helpful to consult the technical support of the dye supplier or seek advice from experienced colleagues.

l Perhaps this protocol list can give us more information to help solve the problem.