10% Kcal fat, purified diet (for control low-fat group)
Non-drip water bottles
Individual clear plastic cages (28 × 28 × 12-cm) with soft bedding (e.g., corncob or sani-chips, Harlan) and nestlets (Ancare) and/or mouse igloo (Bio-Serv, VWR) for environmental enrichment
Weighing scale (range 0 to 500 g, accurate to ± 0.1 g)
1000-ml plastic beaker or bowl (receptacle used for weighing)
1. Follow Basic Protocol 1, steps 1 through 6.
2. Select mice for further study after either a fixed, pre-determined difference in mean weight gain has been achieved between mice fed the low- or high-fat diet (either 10 g or 20% to 25% is appropriate), or when weight gain begins to slow for both groups (this should occur after ~15 weeks on diet) (see Fig. 9.38.1).
Figure 9.38.1 Illustration of mean growth rate in male C57Bl6/Tac mice fed a 60% high-fat or 10% low-fat diet for 16 weeks.
SUPPORT PROTOCOL 1
ACUTE PHARMACOLOGICAL MANIPULATION IN DIO MICE AND RATS
Obese rodents are used in drug development to test experimental therapeutics designed to decrease food intake and/or to reduce body weight. Acute food intake studies are designed to assess the pharmacodynamic effects of a test drug on food intake over 24 hr. These data can then be used to guide dosing selection for chronic studies aimed at assessing the effects of the drug on body weight after repeated doses.
A single cohort of obese animals (see Basic Protocols 1 and 2) may be re-used for repeated acute studies investigating different drugs, provided a washout period of at least 1 week between studies is allowed. Prior to first testing, it is recommended that animals are habituated to dosing procedures by sham dosing daily for 2 to 3 days.
The following protocol is appropriate for both rat and mouse and is designed to provide high resolution of a compound’s effect on food intake over a 24-hr period. Efficacy is optimized by dosing compound so that maximum compound absorption is attained just prior to initiation of food intake as stimulated by the start of the animals’ dark phase. To facilitate dark phase testing, animals may be housed on a reverse light/dark cycle (see Critical Parameters).
Flexible plastic feeding tube (ranging from 5- to 8-FR/12- to 18-G; ~12-G/8-FR) for oral dosing in rats or flexible plastic feeding tube (ranging from 2- to 5-FR/18- to 22-G; ~22-G/2-FR) for oral dosing in mice
Sterile needles 25- to 27-G for i.p./s.c./i.m. dosing in rats (larger-gauged needles can be used if either larger volumes or insoluble compounds are to be administered) or sterile needles 27- to 30-G for i.p./s.c./i.m. dosing in mice (larger-gauged needles can be used if either larger volumes or insoluble compounds are to be administered)
1- to 3-ml syringes
Animal cages with steel raised grid flooring
Scale
Ultrasonic bath or probe sonicator
Prepare drug
1. Prepare compounds in an appropriate vehicle. For compounds with good solubility, use distilled water for p.o. dosing, or sterile saline for s.c., i.p., or i.m. injection.
Other vehicles may be used, including but not limited to, polyethylene glycol (PEG), a cyclodextrin, or other suspending agents. However, any intrinsic impact of a vehicle on acute food intake should be determined prior to use in drug testing, with administration route and volume taken into consideration. If necessary, disperse poorly soluble compounds with an ultrasonic bath or ultrasonic probe to obtain a full suspension or complete dissolution of compounds in vehicle.
Assess the acute effect of test substance on food intake and body weight
2. For 2 to 3 days prior to study onset, habituate subjects to injection procedures. Replicate dosing to study conditions as closely as possible, including route of administration, number of administrations per day, time of day, and vehicle solutions. Monitor body weight and food intake during this period to ensure no adverse effects of dosing. If effects are recorded, continue dosing until they are no longer apparent, or consider whether the conditions used are appropriate (e.g., volume of injection and vehicle).
Mice are particularly susceptible to the stress of injection, and therefore should be monitored accordingly.
3. Approximately 2 hr prior to lights off, weigh animals and transfer from home cages into regular test cages (one animal/cage) with raised grid flooring (no bedding), and with no access to food.
4. After ~1 to 2 hr of acclimation to this test environment, and before lights out, dose animals with test compound.
5. Immediately after lights off, provide each animal with a pre-weighed amount of food, approximately double group mean daily intake (40 to 50 g).
The extra food provides more weight, allowing easier manipulation of food and additionally accounts for individual variability in intake.
6. Coordinate the timing of initial placement into test cages, acclimation time, and dosing time so that lights out (and food presentation) occurs at a time when pharmacologically relevant concentrations of drug exposure have been obtained.
7. Measure food consumption at various time points over 24 hr (typically 1, 2, 4, 6, 8, 12, and 24 hr post-food presentation) by weighing remaining food (including food that has fallen through the grid to the cage floor) at these times. Calculate food consumption over these time intervals by subtracting food weight at each interval from the previous interval food weight. If desired, measure water consumption in the same manner.
8. After the last food and water measurement, record body weight for each animal and return to home cage.
9. Use food intake to calculate the ED50 of the test drug at various time points post administration.
These calculations can be used to estimate appropriate dose ranges and dose frequencies for chronic studies.
Detailed information on how to determine the minimum effective dose and the dose producing a half-maximal effect (ED50) can be found in Svartengren et al. (2005).
SUPPORT PROTOCOL 2
CHRONIC PHARMACOLOGICAL MANIPULATION IN DIO MICE AND RATS
In contrast to acute studies, which are designed to determine short-term drug effects primarily on food intake in DIO rodents, chronic pharmacological studies are designed to assess the effects of repeated administration of a test drug on body weight and food intake over weeks to months. Dosing may occur once or more daily, depending on information obtained from both pharmacokinetic and acute food intake studies.
A single cohort of obese animals (see Basic Protocols 1 and 2) should be used for only one chronic study, and should not be re-used. These animals should be drug naïve at the start of testing. Prior to first testing, it is recommended that animals are habituated to dosing procedures by sham dosing daily for 2 to 3 days.
The following protocol is appropriate for both rat and mouse. As with acute studies, potential for efficacy is maximized by dosing compound so that maximum compound exposure is attained just prior to initiation of food intake as stimulated by the start of the animals’ dark phase. Additional doses may be administered at other times during each day. To facilitate dark phase testing, animals may be housed on a reverse light/dark cycle (see Critical Parameters).
For materials, see Support Protocol 1.
Prepare drug
1. See Support Protocol 1, step 1.
Assess the chronic effect of test substance on food intake and body weight
2. For 2 to 3 days prior to study onset, habituate subjects to injection procedures. Replicate dosing to study conditions as closely as possible, including route of administration, number of administrations per day, time of day, and vehicle solutions. Monitor body weight and food intake during this period to ensure no adverse effects of dosing. If effects are recorded, continue dosing until they are no longer apparent, or consider whether the conditions used are appropriate (e.g., volume of injection and vehicle).
Mice are particularly susceptible to the stress of injection, and therefore should be monitored accordingly.
3. Singly house animals in home cages for the duration of chronic studies. Maintain the housing room at a constant humidity (60% to 70%) and temperature (22° to 24°C) with a fixed light/dark schedule (see Critical Parameters for temperature and light/dark cycle considerations). Change bedding every 3 to 4 days. Provide food and water ad libitum.
4. Calculate body weight and food intake at the same time each day, preferably immediately prior to dosing. Calculate 24-hr food intake by subtracting each daily food weight from the previous day’s weight. If desired, measure daily water consumption in the same manner.
5. Prior to drug treatment initiation, and after a period of sham dosing, assign DIO animals to treatment groups according to baseline body weights such that baseline group means do not differ. Obtain multiple days of baseline body weights and/or food intake prior to drug treatment initiation with sham or vehicle dosing to obtain an accurate picture of baseline food intake.
6. Dose animals with test compound daily at appropriate time(s), using most recent body weight measurement to calculate dose volume. For once-daily dosing, administer compound shortly prior to lights off as determined in acute food intake studies. Administer additional doses at various times throughout each 24-hr period, utilizing the same volume of injection.
7. Compare body weights and food intake over time between drug treatment groups.
8. Study durations can range from 10 days (subchronic) to 6 months. At the conclusion of drug treatment, effects of drug withdrawal such as rebound hyperphagia and/or weight gain may be assessed by continuing measurement of daily body and food weight in the absence of drug administration. The appearance of these effects further confirms the efficacy of the drug to reduce body weight and food intake.
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