Utilizing an Orally Dissolving Strip for Pharmacological and Toxicological Studies: A Simple and Humane Alternative to Oral Gavage for Animals

Summary

The following protocol describes a novel method for chronic oral drug administration using an orally dissolving strip (ODS) in lieu of the more commonly used oral gavage method. We demonstrate that preclinical, oral drug delivery using the ODS method represents a safe, convenient, and humane alternative to oral gavage.

Abstract

Prior to testing novel therapeutics in humans, short and long term preclinical (i.e., animal), repetitive pharmacological and toxicological testing is required. In most cases, the preferred route of administration is via oral delivery. At the present time, oral delivery is mostly accomplished using an oral gavage procedure, in part, because it can achieve consistent and precise dosing in the animal model. Although this method is well established it does have complications that can result in a high rate of animal attrition. To this end, the procedure introduced here describes an alternative to the oral gavage method in which the desired drug is incorporated into a tastant, orally dissolving strip (ODS) that can simply be presented to the test animal where it is then rapidly taken up with minimal manipulation of the test subject. Herein, we demonstrate that preclinical, oral drug delivery using the ODS method represents a safe, convenient, and humane alternative to oral gavage.

Introduction

In order for the successful translation of a drug from preclinical (animal testing) to clinical development, a series of well-defined, acute and chronic pharmacological and toxicological studies need to be performed in animal models using the intended clinical route of administration (which is normally the oral route). To accomplish this goal, most pre-clinical studies currently utilize an oral gavage procedure given that this method results in a consistent and precise delivery of the test compound to the animal. However, in many instances, oral gavage is not well tolerated by the animal and accumulating evidence suggests that the method is associated with a significant amount of stress induction¹.

This is especially true in the development of treatments for chronic life-long conditions that require longer-term preclinical studies, such as is the case with, but not limited to, nearly every neuropsychiatric disorder. For instance, in a transgenic mouse model of Alzheimer's disease, it was recently suggested that the gavage method itself could confound experimental results due to dosing induced stress². Similarly, in a mouse model of alcohol consumption, unpublished findings from our own laboratory have found that the repetitive insertion of the gavage tube - in and of itself - can significantly reduce the level of alcohol intake to the point of jeopardizing the integrity of the experimental paradigm (see Figure 1).

Given the aforementioned limitations associated with the oral gavage procedure, considerable effort has been put towards developing novel methods of preclinical oral drug delivery. Current alternative approaches include incorporating the test compound into peanut butter mixtures³, gelatinous molds^4-5, chocolate pellets⁶, drinking water⁷, and wafer crackers⁸, all of which are associated with varying degrees of issues including the inability to be used reliably in the animal model, lack of adoption of test compound uptake by the test subject or individual preference for flavor that makes the drug less readily consumed⁹.

The procedure introduced herein describes a method in which the desired drug is incorporated into a flavored, orally dissolving strip (ODS) that can be easily and readily be used to orally administer the test compound to the test animal. This paper focuses on demonstrating the effectiveness of the ODS method using mice. However, there is no reason to expect that the same method would not also be useful in other rodent as well as larger mammalian subjects. Clinically, the use of ODS is starting to be adopted in pediatric and geriatric patients, as a way to overcome swallowing difficulties¹⁰. We propose that ODS can also be used successfully in preclinical drug discovery programs as a safe, convenient, and humane alternative to oral gavage.

Protocol

All procedure described have been approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Southern California.

1. Animal Handling and Habituation

1. Introduction
   1. Allow mice to acclimate to the husbandry conditions and handling technique of the experiment to decrease stress during the ensuing drug administration procedures. During this time, record the body weight and food intake of each mouse; if desired, they can later be used as a non-invasive point of comparison to assess drug toxicity (optional). The former can also be used to determine weight-based drug dosing.
      Note: Anecdotally, the length of the habituation period should be determined based on the number of experimenters interacting with the animal. In our experience, mice of the C57BL/6J strain have been observed to have trouble habituating to handling by more than ~5 people within our typical week-long habituation period. If feasible, experimenter(s) are also encouraged to refrain from using scented lotions, soaps, and perfumes before handling the animals as previous reports indicate novel stimuli, such as smells, may hinder the habituation period¹¹.
2. Steps
   1. Beginning at least 5 days prior to the initiation of the study, transfer the animals to the room in which the subsequent experiments will occur.
   2. Record the weight of the mouse by firmly grasping the middle of the tail and gently lifting the animal out of the cage and into the weight boat.
      1. Place the mouse on the scale as soon as it is lifted out of the cagekeeping in mind to properly grasp the medial/proximal aspect of the tail, as to avoid degloving injuries, which can occur when grasping the distal aspect of the tail¹² .
      2. Record the weight value once the mouse has been safely placed into the weigh boat.
   3. Record the weight of the food for a recommended 2 consecutive days, in order to establish baseline food consumption levels that will help capture potential toxicities during the experiment¹³.

2. Preparation of Feeding Needle, Sucrose Solution, and Individual ODS

1. Use an autoclave to sterilize the stainless steel gavage needles in preparation for the experiment. Prepare 2 needles/group and 1 additional needle to be used as a back-up if need be.
   Note: This study utilized a 1.5 inches, curved, 18 G, stainless steel feeding needle with a 2.25 mm ball at the end of the needle.
2. Add 20 ml of water to 0.85 g of sucrose to create a 4.25% (w/v) sucrose solution.
   Note: Depending on the parameters under assessment other low doses of sucrose could potentially also be used¹⁴.
3. Prepare the oral dissolving strip (ODS) for each animal by cutting the standard rectangular test strip into a piece that is 0.5 cm inches in diameter using a commercially available single quarter inch hole-puncher. The rectangular test strip used in this demonstration was formulated to contain 6 mg of ivermectin (IVM), and when cut to this size, allocates a 0.21 mg dosage to each individual circular piece approximating a 10 mg/kg dose for each animal (our desired dose/animal). This dose can be adjusted by concentrating/diluting the overall formulation on the rectangular strip and/or cutting the strip into different size.

3. Drug – Delivery

1. Record the weight values as previously described in steps 1.1-1.2.
2. After recording the weight, move the mouse to the metal grid cage top. Once there, using the dominant thumb, index finger, and 3^rd finger, gently pull back on the tail. This will cause the mouse to inadvertently grip the wire cage top and stretch out its back, stretching its body away from the experimenter.
3. Grasp the scruff of the neck with the non-dominant thumb, index finger, and 3^rd finger to comfortably restrain the animal with a grip that is secure.
   Note: An experimenter with a smaller hand can also use the 4^th finger, of the same hand, to pull back the scruff of the back and obtain a more secure hold of the mouse. Additionally, depending on individual preference, ring finger, and/or pinky finger may also be used to secure the tail however this is not necessary¹².
4. Hold the mouse in an upright position.
5. Caution: Observe the color of the mucosal membranes of the mouse. A purple/blue appearance is an indication that the experimenter is cinching the scruff too tightly and the mouse cannot breathe.
   1. If this occurs, immediately put the mouse back down on the metal cage top and allow it to rest for ~2-3 min before reattempting to restrain the animal.
6. Submerge the bulb tip of the gavage needle in 4.25% sucrose solution to serve as a tastant adhesive to the ODS.
7. Press down on a pre-cut ODS with the bulb tip of the gavage needle to attach the orally dissolving strip.
8. Present the ODS to the mouse by placing it near the nostrils and/or mouth, and allow the animal to consume the film. Alternatively the ODS can be delivered topically to the oral cavity by placing the thin strip directly onto the tongue for the mice to swallow.
9. Replace the needle mid-group during the drug-dosing session to prevent the spread of infection.

Results

In the following representative investigations, social drinking was modeled using a 24 hr two-bottle choice (TBC) paradigm as previously described¹⁵. Briefly, mice had access to two bottles of solution, one of which contained water, and the other a 10% (v/v) ethanol solution. Subjects were subsequently assigned to drug treatment groups so that the average 10E (10% ethanol/90% water v/v) intake was similar across groups.

Discussion

Oral gavage is associated with numerous complications that result in potentially compromised data collection and high rates of animal attrition¹. Here a simple method of oral drug delivery is introduced through which mice (and potentially other animals) can easily and reliably consume the drug of choice through an orally dissolving strip (ODS). Notably, the use of this method represents a safe, convenient, and humane alternative to oral gavage.

Other current alternatives to oral gav...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Orally Dissolving Strip	Cure Pharmaceutical		Available upon special request. Alternatively, ODS may also be prepared in-house.
Gavage Feeding Needle	VWR	7912	Our rodent studies utilized a 18 gauge feeding needle. This size may need to be adjusted for use in other mammilian species.
Sucrose	Sigma Aldrich	S0389-500G