The authors would like to acknowledge Jamie Thuy for technical work. This work was supported, in part, by Research grants SC CTSI NIH/NCRR/NCATS -- UL1TR000130 (D.L.D.), AA022448 (D.L.D.), and the USC School of Pharmacy. Additionally, fast dissolving oral films containing IVM were kindly provided by Cure Pharmaceutical (Oxnard, CA).

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
<ol>
	<li>Introduction
	<ol>
		<li>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.</li>
	</ol>
	</li>
	<li>Steps
	<ol>
		<li>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.</li>
		<li>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.
		<ol>
			<li>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 .</li>
			<li>Record the weight value once the mouse has been safely placed into the weigh boat.</li>
		</ol>
		</li>
		<li>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.</li>
	</ol>
	</li>
</ol>
2. Preparation of Feeding Needle, Sucrose Solution, and Individual ODS
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
</ol>
3. Drug &#8211; Delivery
<ol>
	<li>Record the weight values as previously described in steps 1.1-1.2.</li>
	<li>After recording the weight, move the mouse to the metal grid cage top. Once there, using the dominant thumb, index finger, and 3rd 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.</li>
	<li>Grasp the scruff of the neck with the non-dominant thumb, index finger, and 3rd finger to comfortably restrain the animal with a grip that is secure. 
	Note: An experimenter with a smaller hand can also use the 4th 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.</li>
	<li>Hold the mouse in an upright position.</li>
	<li>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.
	<ol>
		<li>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.</li>
	</ol>
	</li>
	<li>Submerge the bulb tip of the gavage needle in 4.25% sucrose solution to serve as a tastant adhesive to the ODS.</li>
	<li>Press down on a pre-cut ODS with the bulb tip of the gavage needle to attach the orally dissolving strip.</li>
	<li>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.</li>
	<li>Replace the needle mid-group during the drug-dosing session to prevent the spread of infection.</li>
</ol>

<ol>
	<li>Brown, A. P., Dinger, N., Levine, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stress+produced+by+gavage+administration+in+the+rat.">Stress produced by gavage administration in the rat.</a> Contemp Top Lab Anim Sci. 39 (1), 17-21 (2000).</li><li>Gonzales, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alternative+method+of+oral+administration+by+peanut+butter+pellet+formulation+results+in+target+engagement+of+BACE1+and+attenuation+of+gavage-induced+stress+responses+in+mice.">Alternative method of oral administration by peanut butter pellet formulation results in target engagement of BACE1 and attenuation of gavage-induced stress responses in mice.</a> Pharmacol Biochem Behav. 126, 28-35 (2014).</li><li>Walker, M. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+less+stressful+alternative+to+oral+gavage+for+pharmacological+and+toxicological+studies+in+mice.">A less stressful alternative to oral gavage for pharmacological and toxicological studies in mice.</a> Toxicol Appl Pharmacol. 260 (1), 65-69 (2012).</li><li>Zhang, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Voluntary+oral+administration+of+drugs+in+mice.">Voluntary oral administration of drugs in mice.</a> Protocol Exchange: Nature Publishing Group. , (2011).</li><li>Froehlich, J. C., Hausauer, B. J., Federoff, D. L., Fischer, S. M., Rasmussen, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prazosin+reduces+alcohol+drinking+throughout+prolonged+treatment+and+blocks+the+initiation+of+drinking+in+rats+selectively+bred+for+high+alcohol+intake.">Prazosin reduces alcohol drinking throughout prolonged treatment and blocks the initiation of drinking in rats selectively bred for high alcohol intake.</a> Alcohol Clin Exp Res. 37 (9), 1552-1560 (2013).</li><li>Goldkuhl, R., Carlsson, H. E., Hau, J., Abelson, K. S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+subcutaneous+injection+and+oral+voluntary+ingestion+of+buprenorphine+on+post-operative+serum+corticosterone+levels+in+male+rats.">Effect of subcutaneous injection and oral voluntary ingestion of buprenorphine on post-operative serum corticosterone levels in male rats.</a> Eur Surg Res. 41 (3), 272-278 (2008).</li><li>Marx, J. O., Vudathala, D., Murphy, L., Rankin, S., Hankenson, F. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibiotic+administration+in+the+drinking+water+of+mice.">Antibiotic administration in the drinking water of mice.</a> J Am Assoc Lab Anim Sci. 53 (3), 301-306 (2014).</li><li>Ferguson, S. A., Boctor, S. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+food+wafers+for+multiple+daily+oral+treatments+in+young+rats.">Use of food wafers for multiple daily oral treatments in young rats.</a> J Am Assoc Lab Anim Sci. 48 (3), 292-295 (2009).</li><li>Turner, P. V., Brabb, T., Pekow, C., Vasbinder, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Administration+of+substances+to+laboratory+animals:+routes+of+administration+and+factors+to+consider.">Administration of substances to laboratory animals: routes of administration and factors to consider.</a> J Am Assoc Lab Anim Sci. 50 (5), 600-613 (2011).</li><li>Bala, R., Pawar, P., Khanna, S., Arora, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Orally+dissolving+strips:+A+new+approach+to+oral+drug+delivery+system.">Orally dissolving strips: A new approach to oral drug delivery system.</a> Int J Pharm Investig. 3 (2), 67-76 (2013).</li><li>Potashkin, J. A., Blume, S. R., Runkle, N. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Limitations+of+animal+models+of+Parkinson's+disease.">Limitations of animal models of Parkinson's disease.</a> Parkinsons Dis. 2011, (2010).</li><li>Machholz, E., Mulder, G., Ruiz, C., Corning, B. F., Pritchett-Corning, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Manual+restraint+and+common+compound+administration+routes+in+mice+and+rats.">Manual restraint and common compound administration routes in mice and rats.</a> Journal of Visualized Experiments JoVE. (67), e2771 (2012).</li><li>Yardley, M. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ivermectin+reduces+alcohol+intake+and+preference+in+mice.">Ivermectin reduces alcohol intake and preference in mice.</a> Neuropharmacology. 63, 190-201 (2012).</li><li>Hoggatt, A. F., Hoggatt, J., Honerlaw, M., Pelus, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+spoonful+of+sugar+helps+the+medicine+go+down:+a+novel+technique+to+improve+oral+gavage+in+mice.">A spoonful of sugar helps the medicine go down: a novel technique to improve oral gavage in mice.</a> J Am Assoc Lab Anim Sci. 49, 329-334 (2010).</li><li>Yardley, M. M., Huynh, N., Rodgers, K. E., Alkana, R. L., Davies, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oral+delivery+of+ivermectin+using+a+fast+dissolving+oral+film:+implications+for+repurposing+ivermectin+as+a+pharmacotherapy+for+alcohol+use+disorder.">Oral delivery of ivermectin using a fast dissolving oral film: implications for repurposing ivermectin as a pharmacotherapy for alcohol use disorder.</a> Alcohol. 49 (6), 553-559 (2015).</li></ol>

DD has applied for a patent as a Co-Inventor on this method and is interested in sharing this method with the scientific committee to make this technique well known and readily available for chronic animal testing (Oral Delivery of Drug Actives in Laboratory Animals Using Fast-Dissolving Oral Films (Davies, Co-Inventor, Patent Application filed 6-23-2014).

No other competing financial interests are noted.

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 gavage, although somewhat promising, are associated with various limitations that preclude them from covering the full range of needs associated with drug-dosing studies such as an inability to be used in mice, or individual preference for flavor that make the drug less readily consumed9. In contrast, ODS can be formulated in a wide-variety of flavors which is important as individual preferences for flavor can change over time and flavor preferences may differ across various strains and species.
Proper animal handling is an essential component of this technique. Highlighting the importance of the habituation period, it should be noted that a given mouse may be harder to restrain if it is particularly young or unaccustomed to handling. Any difficulty grasping the mouse should subside over time as the animal is familiarized to handling and becomes less fearful of the experimenter(s). The C57 mouse strain exemplified in the representative results has been observed to habituate to handling procedures relatively quickly (within roughly 1-3 handling sessions). Although this duration may defer across various species and strains, there is no reason to assume the acclimation time-frame would escalate significantly. Novice technicians working with rodents should be cognizant that, when suspended and dangling in the air for more than a few seconds, mice are susceptible to the development of a degloving injury to the tail12. The chances of this occurring increase with the weight of the mouse as well as with the positioning of the hand, with a grasp closer to the tip of the tail more likely to cause an injury. Specific to the scope of this protocol, it is imperative that the mouse is placed on the metal grid cage top, or in an alternative apparatus, when being transferred from cage to scale, as well as in preparation for drug-delivery. Restraint during the drug-delivery process is another critical step within the protocol. ODS consumption can occur by placing the thin film near the nostrils and/or mouth of the mouse, in which case the subject will lean forward to consume the film. Alternatively the ODS can be delivered topically to the oral cavity by manually placing the film directly onto the tongue for the mouse to swallow.
Investigations that involve the assessment of stress-sensitive parameters can be confounded by the use of the standard manual restraint techniques depicted in this protocol and simple adjustments to the methodology can easily be made to accommodate studies in which restraint is prohibited. In this modified approach, the ODS could be placed directly in the cage for the animal to consume. Observations gleaned from pilot studies in our own lab indicate that this would best be performed by placing the animals in a cage without any bedding/nesting material, just for the duration of the drug delivery procedure, in order to increase the ease with which ODS consumption can be visually monitored by the experimenter(s). Moreover, it might be appropriate to initiate a training period during which time the animals are introduced to the ODS so that they can overcome the initial innate avoidance of novel food4. Of course further studies would be needed to optimize a restraint-free protocol for ODS delivery, however there is no reason to believe that such a procedure could not be done with ease.
There are some limitations to the ODS method. For instance, given that the ODS are a fast dissolving bio-adhesive polymer, the experimenter will need to consider issues of absorption and metabolism in that depending on the formulation of the strip, some of the drug could potentially bypass first pass metabolism due to a portion of the drug being absorbed via lingual or buccal mechanism. On the other hand, for CNS targeted drugs, lingual or buccal absorption could be a benefit allowing more of the compound to reach the CNS target prior to metabolism. Overall, this issue can be addressed by changing the formulation of the ODS that will result in the drug either not being readily available until it is absorbed via the gut or to take advantage of direct access to the CNS. Additionally, mice may develop conditioned taste aversion and withdraw from voluntary ODS consumption if the drug elicits aversive side effects. Although we have not observed this in our studies, one potential method to ameliorate such a situation would be to alternate the flavor of the test strip to help keep a bit of novelty for the test animal. Lastly, at high concentrations the ODS might not be palatable. However, this issue could also be addressed by changes in formulation where the drug itself is encapsulated and thus taste would no longer be an issue, or alternatively, by simply administering different flavored strips, so that there remains novelty in the flavor for the test subject.
Notably, in terms of the data reproducibility, ODS can be formulated to adhere to the tongue or cheek of the animal and designed for rapid oral disintegration to quickly release the drug, thus assuring proper dosing is achieved. And, with ODS administration, the need for gavage and other unpleasant invasive experiences for the animal is eliminated. Not only is this more humane but it also minimizes the effects of stress as a variable in the experiment. It should be noted that the ODS method can be further optimized, as in many new manufacturing processes, which may further improve the pharmacokinetics of the test compound as ODS is expanded for additional therapies. One limitation that should be pointed out regards the fact that the development of ODS is in the early stages of development. Issues concerning the loading of different physicochemical characteristic drugs over the tastant/ODS platform remains a challenge to the versatility and generalization of this technique. This is an area of ongoing investigation.
The proposed technique is a simple and novel technique that can be adopted for use in short and long term preclinical animal studies and for the delivery of medicines to animals (e.g., dogs). Drug delivery through ODS represents a new, fast, convenient and humane approach compared to the existing traditional approaches like tablets, capsules, liquids, powders, and gels and, oral gavage.

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&#39;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 molds4-5, 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.

<table><tbody><tr><td>Orally Dissolving Strip&amp;nbsp;</td><td>Cure Pharmaceutical</td><td></td><td>Available upon special request. Alternatively, ODS may also be prepared in-house.</td></tr><tr><td>Gavage Feeding Needle</td><td>VWR</td><td>7912</td><td>Our rodent studies utilized a 18 gauge feeding needle. This size may need to be adjusted for use in other mammilian species.</td></tr><tr><td>Sucrose</td><td>Sigma Aldrich</td><td>S0389-500G</td><td></td></tr></tbody></table>

utilizing an orally dissolving strip for pharmacological and toxicological studies: a simple and humane alternative to oral gavage for animals

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.

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.
Figure 1 illustrates the effects of ivermectin (IVM) on preference drinking when orally administered using a gavage vehicle. This pilot study had 3 groups: N/A, placebo, and IVM representing no gavage, corn oil (vehicle) gavage, and IVM (10 mg/kg, diluted in corn oil) gavage respectively. Mice were treated for 10 days, 5 consecutive days per week for 2 weeks. As can be see below, IVM was able to significantly reduce 10E intake across the study when compared to the placebo group. Compared to N/A, the ethanol intake values of the placebo group (~5 g/kg), are much too low (48% lower than the non-gavaged N/A group), and thus, may not be an accurate representation, or "model", of social drinking behavior (~10-15 g/kg). Meaning that an unknown factor associated with the gavage procedure managed to jeopardize the integrity of the drinking paradigm.
Figure 2 illustrates the same experiment subsequently performed utilizing the ODS delivery system15. The study had 3 groups: 1) no oral film, 2) key lime flavored oral film with no IVM and 3) key lime flavored oral film with IVM (0.21 mg, which is equivalent to the 10 mg/kg administered in the aforementioned study) referred to as control, placebo and IVM, respectively. Mice were treated for 20 days, 5 consecutive days per week for 4 weeks. There was no significant difference in ethanol intake between the N/A and placebo treated mice, indicating that the ODS placebo group represented a valid control with which a comparison to the IVM group could then be made. In agreement with previous work13,15, IVM significantly and consistently reduced 10E intake.
A pictorial representation of the technique is shown in Figure 3.
<img alt="Graph of 10E intake over 10 days; comparison of N/A, placebo, IVM treatments shows intake trends." src="/files/ftp_upload/53770/53770fig1.jpg" data-altupdatedat="1747911167000" data-alt="Figure 1"> 
Figure 1. Gavaged Control Mice are Unable to Maintain Ethanol Intake Levels Representative of the 24 hr TBC Paradigm. Male C57BL/6J mice were treated for 10 days (5 consecutive days per week for 2 weeks). Vehicle gavage treatment of the placebo group significantly altered ethanol intake values by 48% when compared to the non-gavage N/A group. Although IVM significantly reduced 10E intake compared to placebo, the low 10E intake values of the placebo group added a second confound to the study, which could have the effect of compromising the usefulness of the group as an adequate drinking control for the study. Inset graphs illustrate the average 10E intake over the 10 day treatment. Values represent the mean ± SEM for n = 6-8 mice/group. +++P &lt;0.001 versus Placebo, and ***P &lt;0.001 versus N/A, Turkey Post-Hoc Test. <a href="https://www.jove.com/files/ftp_upload/53770/53770fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="10E intake comparison chart showing placebo, N/A, IVM treatment impact over 20-day ODS treatment." src="/files/ftp_upload/53770/53770fig2.jpg" data-altupdatedat="1747911167000" data-alt="Figure 2"> 
Figure 2. ODS Treatment of IVM. Female C57BL/6J mice were treated for 20 days (5 consecutive days per week for 4 weeks). Placebo ODS treatment had no significant effect on 10E intake, relative to the non-treated N/A group, maintaining the validity of placebo ODS as an adequate social drinking control. IVM significantly reduced 10E intake compared to placebo control. Inset graphs illustrate the average 10E intake over the 20 day treatment. Values represent the mean ± SEM for n = 6-8 mice/group. ***P &lt;0.001 versus Placebo, Turkey Post-Hoc Test.. This figure has been modified from Yardley et al.15 <a href="https://www.jove.com/files/ftp_upload/53770/53770fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Lab mouse oral gavage procedure, gloved hands, syringe; animal testing research method." src="/files/ftp_upload/53770/53770fig3.jpg" data-altupdatedat="1747911167000" data-alt="Figure 3"> 
Figure 3. Pictorial Representation of ODS Delivery. Photographic depiction of the procedure. <a href="https://www.jove.com/files/ftp_upload/53770/53770fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Utilizing an Orally Dissolving Strip for Pharmacological and Toxicological Studies: A Simple and Humane Alternative to Oral Gavage for Animals at JoVE.com

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

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.

Prior to testing novel therapeutics in humans, short and long term preclinical (i.e., animal), repetitive pharmacological and toxicological testing is ...

utilizing-an-orally-dissolving-strip-for-pharmacological

Universidad San Sebastian

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JoVE Journal

Medicine

12.6K Views.  University of Southern California School of Pharmacy. 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.

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

A Novel Procedure for Evaluating the Reinforcing Properties of Tastants in Laboratory Rats: Operant Intraoral Self-administration

This paper describes a novel method for studying the bio-behavioral basis of addiction to food. This method combines the surgical component of taste reactivity with the behavioral aspects of operant self-administration of drugs. Under very brief general anaesthesia, rats are implanted with an intraoral (IO) cannula that allows delivery of test solutions directly in the oral cavity. Animals are then tested in operant self-administration chambers whereby they can press a lever to receive IO infusions of test solutions. IO self-administration has several advantages over experimental procedures that involve drinking a solution from a spout&#160;or operant responding for solid pellets or solutions delivered in a receptacle. Here, we show that IO self-administration can be employed to study self-administration of high fructose corn syrup (HFCS). Rats were first tested for self-administration on a progressive ratio (PR) schedule, which assesses the maximum amount of operant behavior that will be emitted for different concentrations of HFCS (i.e.&#160;8%, 25%, and 50%). Following this test, rats self-administered these concentrations on a continuous schedule of reinforcement (i.e.&#160;one infusion for each lever press) for 10 consecutive days (1 session/day; each lasting 3 hr), and then they were retested on the PR schedule. On the continuous reinforcement schedule, rats took fewer infusions of higher concentrations, although the lowest concentration of HFCS (8%) maintained more variable self-administration. Furthermore, the PR tests revealed that 8% had lower reinforcing value than 25% and 50%. These results indicate that IO self-administration can be employed to study acquisition and maintenance of responding for sweet solutions. The sensitivity of the operant response to differences in concentration and schedule of reinforcement makes IO self-administration an ideal procedure to investigate the neurobiology of voluntary intake of sweets.

The current study evaluates a novel procedure for assessing the reinforcing effects of palatable solutions in laboratory rats: intraoral self-administration. To this end, operant responding (i.e.&#160;lever pressing) for intraoral infusions of sweet solutions at different concentrations was measured on continuous and progressive ratios schedules of reinforcement.

This paper describes a novel method for studying the bio-behavioral basis of addiction to food. This method combines the surgical component of taste ...

Behavior

An Intestine/Liver Microphysiological System for Drug Pharmacokinetic and Toxicological Assessment

The recently introduced microphysiological systems (MPS) cultivating human organoids are expected to perform better than animals in the preclinical tests phase of drug developing process because they are genetically human and recapitulate the interplay among tissues. In this study, the human intestinal barrier (emulated by a co-culture of Caco-2 and HT-29 cells) and the liver equivalent (emulated by spheroids made of differentiated HepaRG cells and human hepatic stellate cells) were integrated into a two-organ chip (2-OC) microfluidic device to assess some acetaminophen (APAP) pharmacokinetic (PK) and toxicological properties. The MPS had three assemblies: Intestine only 2-OC, Liver only 2-OC, and Intestine/Liver 2-OC with the same media perfusing both organoids. For PK assessments, we dosed the APAP in the media at preset timepoints after administering it either over the intestinal barrier (emulating the oral route) or in the media (emulating the intravenous route), at 12 &#181;M and 2 &#181;M respectively. The media samples were analyzed by reversed-phase high-pressure liquid chromatography (HPLC). Organoids were analyzed for gene expression, for TEER values, for protein expression and activity, and then collected, fixed, and submitted to a set of morphological evaluations. The MTT technique performed well in assessing the organoid viability, but the high content analyses (HCA) were able to detect very early toxic events in response to APAP treatment. We verified that the media flow does not significantly affect the APAP absorption whereas it significantly improves the liver equivalent functionality. The APAP human intestinal absorption and hepatic metabolism could be emulated in the MPS. The association between MPS data and in silico modeling has great potential to improve the predictability of the in vitro methods and provide better accuracy than animal models in pharmacokinetic and toxicological studies.

We exposed a microphysiological system (MPS) with intestine and liver organoids to acetaminophen (APAP). This article describes the methods for organoid production and APAP pharmacokinetic and toxicological property assessments in the MPS. It also describes the tissue functionality analyses necessary to validate the results.

The recently introduced microphysiological systems (MPS) cultivating human organoids are expected to perform better than animals in the preclinical tests ...

Chemistry

Reducing Research Animal Stress with Micropipette-Guided Drug Administration

Use of Micropipette-Guided Drug Administration as an Alternative Method to Oral Gavage in Rodent Models

Oral gavage (OG) with the use of a cannula attached to a syringe is one of the most common methods used to deliver precise dosing of compounds to the stomach of research animals. Unfortunately, this method comes with difficulties for both the operator and the research animal. Studies have shown that OG may lead to complications, including esophagitis, perforation of the esophagus, and inadvertent tracheal drug administration. In addition, OG is associated with increased plasma and fecal corticosterone levels (due to stress), altered blood pressure, and increased heart rate, which could negatively influence or bias study results. A previously developed alternative method termed micropipette-guided drug administration (MDA) incentivizes the animal to consume treatments readily in a minimally invasive manner. Herein, we present examples of the use of the MDA technique with treatments reconstituted in different vehicles and demonstrate effective delivery of the varied treatments to multiple different mouse strains. We further demonstrate that MDA is a technique that decreases the timing and invasiveness of drug administration and does not affect the gut microbiome composition as assessed by quantitative analysis of core gut microbial species. Overall, MDA may offer a less stressful and effective alternative to OG.

Here, we describe micropipette-guided drug administration (MDA) as an alternative method to oral gavage that incentivizes the research animal to ingest treatments readily with minimal stress and discomfort.

Oral gavage (OG) with the use of a cannula attached to a syringe is one of the most common methods used to deliver precise dosing of compounds to the ...

Cancer Research

Manual Restraint and Common Compound Administration Routes in Mice and Rats

Being able to safely and effectively restrain mice and rats is an important part of conducting research. Working confidently and humanely with mice and rats requires a basic competency in handling and restraint methods. This article will present the basic principles required to safely handle animals. One-handed, two-handed, and restraint with specially designed restraint objects will be illustrated. Often, another part of the research or testing use of animals is the effective administration of compounds to mice and rats. Although there are a large number of possible administration routes (limited only by the size and organs of the animal), most are not used regularly in research. This video will illustrate several of the more common routes, including intravenous, intramuscular, subcutaneous, and oral gavage. The goal of this article is to expose a viewer unfamiliar with these techniques to basic restraint and substance administration routes. This video does not replace required hands-on training at your facility, but is meant to augment and supplement that training.

Working safely and humanely with research rodents requires a core competency in handling and restraint methods. This article will present the basic principles required to safely handle and effectively administer compounds to mice and rats.

Being able to safely and effectively restrain mice and rats is an important part of conducting research.  Working confidently and humanely with mice and ...

Biology

Oral Intubation of Adult Zebrafish: A Model for Evaluating Intestinal Uptake of Bioactive Compounds

Most pathogens invade organisms through their mucosa. This is particularly true in fish as they are continuously exposed to a microbial-rich water environment. Developing effective methods for oral delivery of immunostimulants or vaccines, which activate the immune system against infectious diseases, is highly desirable. In devising prophylactic tools, good experimental models are needed to test their performance. Here, we show a method for oral intubation of adult zebrafish and a set of procedures to dissect and prepare the intestine for cytometry, confocal microscopy and quantitative polymerase chain reaction (qPCR) analysis. With this protocol, we can precisely administer volumes up to 50 &#181;L to fish weighing approximately 1 g simply and quickly, without harming the animals. This method allows us to explore the direct in vivo uptake of fluorescently labelled compounds by the intestinal mucosa and the immunomodulatory capacity of such biologics at the local site after intubation. By combining downstream methods such as flow cytometry, histology, qPCR and confocal microscopy of the intestinal tissue, we can understand how immunostimulants or vaccines are able to cross the intestinal mucosal barriers, pass through the lamina propria, and reach the muscle, exerting an effect on the intestinal mucosal immune system. The model could be used to test candidate oral prophylactics and delivery systems or the local effect of any orally administered bioactive compound.

The protocol describes intubating adult zebrafish with a biologic; then dissecting and preparing the intestine for cytometry, confocal microscopy and qPCR. This method allows administration of bioactive compounds to monitor intestinal uptake and the local immune stimulus evoked. It&#160;is relevant for testing the intestinal dynamics of oral prophylactics.

Most pathogens invade organisms through their mucosa. This is particularly true in fish as they are continuously exposed to a microbial-rich water ...

Immunology and Infection

Disposable Dosators for Pulmonary Insufflation of Therapeutic Agents to Small Animals

Development of new therapeutic products requires efficacy testing in an animal model. The pulmonary route of administration can be utilized to deliver drugs locally and systemically. Evaluation of dry powder aerosols necessitates an efficient dispersion mechanism to maintain high concentrations in an exposure chamber or for direct endotracheal administration. While solutions exist to expose animals by passive inhalation to dry powder aerosols, most require masses of powder in large excess of the dose delivered. This precludes conducting early feasibility studies as insufficient drug is available at the research or early development stage to support the dose delivery requirements for conventional aerosol delivery systems. When designing an aerosol drug product, aerodynamic performance can relate directly to delivery efficiency and efficacy. Dispersion of powder into an aerosol requires energy input sufficient to overcome interparticulate forces, and particle engineering approaches can substantially improve aerosol performance. We have developed a dispersion system (dosator) which can aerosolize engineered dry powder aerosols efficiently for the purpose of direct pulmonary insufflation, dispersion into an exposure system or generation for analytical purposes.

During development of drugs for pulmonary delivery, it is necessary to evaluate pharmacokinetics and efficacy in an animal model. We present a method to build a disposable aerosol dispersion system from of-the-shelf components that can be used to administer intrapulmonary dry powder aerosol to rodents.

Development of new therapeutic products requires efficacy testing in an animal model. The pulmonary route of administration can be utilized to deliver ...

Dry Powder and Nebulized Aerosol Inhalation of Pharmaceuticals Delivered to Mice Using a Nose-only Exposure System

Obstructive respiratory diseases like asthma and chronic obstructive pulmonary disease (COPD) are currently treated by inhaled anti-inflammatory and bronchodilator drugs. Despite the availability of multiple treatments, both diseases are growing public health concerns. The majority of asthma patients are well controlled on current inhaled therapies but a substantial number of patients with severe asthma are not. Asthma affects an estimated 300 million people worldwide and approximately 20 percent have a severe form of the disease. In contrast to asthma, there are few effective therapies for COPD. An estimated 10% of the population has COPD and the trend in death rates is increasing for COPD while decreasing for other major diseases. Although developing drugs for inhaled delivery is challenging, the nose-only inhalation unit enables direct delivery of novel drugs to the lung of rodents for pre-clinical efficacy and safety/toxicology studies. Inhaled drug delivery has multiple advantages for respiratory diseases, where high concentration in the lung improves efficacy and minimizes systemic side effects. Inhaled corticosteroids and bronchodilators benefit from these advantages and inhaled delivery may also hold potential for future biologic therapies. The inhalation unit described herein can generate, sample for characterization, and uniformly deposit a drug aerosol in the lungs of rodents. This enables the pre-clinical determination of the efficacy and safety of drug doses deposited in the lungs of rodents, key data required before initiating clinical development.

The inhalation unit described herein can generate, sample for characterization, and uniformly deposit a drug aerosol in the lungs of rodents. This enables the pre-clinical determination of the efficacy and safety of drug doses deposited in the lungs; key data enabling clinical inhaled drug development.

Obstructive respiratory diseases like asthma and chronic obstructive pulmonary disease (COPD) are currently treated by inhaled anti-inflammatory and ...

Preparation Of Gushukang (GSK) Granules for In Vivo and In Vitro Experiments

Traditional Chinese herbal medicine plays a role as an alternative method in treating many diseases, such as postmenopausal osteoporosis (POP). Gushukang (GSK) granules, a marketed prescription in China, have bone-protective effects in treating POP. Before administration to the body, one standard preparation procedure is commonly required, which aims to promote the release of active constituents from raw herbs and enhance the pharmacological effects as well as therapeutic outcomes. This study proposes a detailed protocol for using GSK granules in in vivo and in vitro experimental assays. The authors first provide a detailed protocol to calculate the animal-appropriate dosages of granules for in vivo investigation: weighing, dissolving, storage, and administration. Second, this article describes protocols for micro-CT scanning and the measurement of bone parameters. Sample preparation, protocols for running the micro-CT machine and quantification of bone parameters were evaluated. Third, serum-containing GSK granules are prepared, and drug-containing serum is extracted for in vitro osteoclastogenesis and osteoblastogenesis. GSK granules were intragastrically administered twice per day to rats for three consecutive days. Blood was then collected, centrifuged, inactivated, and filtered. Finally, serum was diluted and used for performing osteoclastogenesis and osteoblastogenesis. The protocol described here can be considered a reference for pharmacological investigations of herbal prescription medicines, such as granules.

Preparation Of Gushukang GSK Granules for In Vivo and In Vitro Experiments

This article provides a detailed protocol for preparing a working solution of Gushukang granules for animal studies and GSK granule containing serum for in vitro experiments. This protocol can be applied to pharmacological investigations of herbal medicines as well as prescriptions for both in vivo and in vitro experiments.

Traditional Chinese herbal medicine plays a role as an alternative method in treating many diseases, such as postmenopausal osteoporosis (POP). Gushukang ...

Intraventricular Drug Delivery and Sampling for Pharmacokinetics and Pharmacodynamics Study

Although the blood-brain barrier (BBB) protects the brain from foreign entities, it also prevents some therapeutics from crossing into the central nervous system (CNS) to ameliorate diseases or infections. Drugs are administered directly into the CNS in animals and humans to circumvent the BBB. The present protocol describes a unique way of treating brain infections through intraventricular delivery of antibiotics, i.e., polymyxins, the last-line antibiotics to treat multi-drug resistant Gram-negative bacteria. A straightforward stereotaxic surgery protocol was developed to implant a guide cannula reaching into the lateral ventricle in rats. After a recovery period of 24 h, rats can be injected consciously and repeatedly through a cannula that is fitted to the guide. Injections can be delivered manually as a bolus or infusion using a microinjection pump to obtain a slow and controlled flow rate. The intraventricular injection was successfully confirmed with Evans Blue dye. Cerebrospinal fluid (CSF) can be drained, and the brain and other organs can be collected. This approach is highly amenable for studies involving drug delivery to the CNS and subsequent assessment of pharmacokinetic and pharmacodynamic activity.

Delivery of therapeutics directly into the central nervous system is one way of circumventing the blood-brain barrier. The present protocol demonstrates intracerebroventricular injection for subsequent collection of cerebrospinal fluid and bodily organs. This facilitates the investigation of drug pharmacokinetics and pharmacodynamics in animal models for developing new treatments.

Although the blood-brain barrier (BBB) protects the brain from foreign entities, it also prevents some therapeutics from crossing into the central nervous ...

Neuroscience

Self-Administration of Drugs in Mouse Models of Feeding and Obesity

Preclinical studies in mice often rely on invasive protocols, such as injections or oral gavage, to deliver drugs. These stressful routes of administration have significant effects on important metabolic parameters including food intake and body weight. Although an attractive option to circumvent this is to compound the drug in rodent food or dissolve it in water, these approaches also have limitations as they are affected by drug stability at room temperature for extended periods of time, the drug's solubility in water, and that the dosing is highly dependent on timing of food or water intake. The constant availability of the drug also limits translational relevance on how drugs are administered to patients. To overcome these limitations, drugs can be mixed with highly palatable food, such as peanut butter, allowing mice to self-administer compounds. Mice reliably and reproducibly consume the drug/peanut butter pellet in a short time frame. This approach facilitates a delivery approach with minimal stress compared with an injection or gavage. This protocol demonstrates the approach of drug preparation, animal acclimatization to placebo delivery, and drug delivery. The implications of this approach are discussed in studies related to timing of drug administration and the circadian rhythm.

The overall goal of this procedure is to describe a method for self-administration of drugs that can be used in mouse models of feeding and obesity.