Pharmacologic synergism is not a new concept and may be defined as the combined effect of two (or more) substances that have a significantly greater effect than the effects of the individual substances used independently . Pharmacological synergism was in fact first described in ectoparasiticides [35, 36] but is also widely appreciated in antimicrobial therapy  and in cancer chemotherapy . Flumethrin and imidacloprid have never previously been used together in a single preparation for ectoparasite control in dogs and cats. Both compounds have a neuropharmacological excitatory effect but through differing molecular mechanisms and it may be speculated, based on their pharmacological mechanism of action that they would act synergistically against fleas and ticks. Neural cell membrane sodium channels are present in three different states: closed, open, and inactivated (Figure 1a). Flumethrin preferentially binds to the open state channel and then keeps the channel open resulting in a state of persistent depolarization. A low dose of imidacloprid would activate nicotinic acetylcholine receptors (nAChR) channels inducing an inward flow of current causing depolarization of the cell and activating (thus opening) sodium channels thus theoretically making them significantly more susceptible to the excitatory (and hence toxic) effects of flumethrin.
The initial investigation made use of an in vitro isolated insect nerve study. At rest, the isolated Spodoptera frugiperda ganglia usually show a low level of spontaneous spike activity of between 3 and 25 counts per second (cps). This rate increases dramatically when compounds that are known to increase the excitability of the neurons by activating of nAChR, such as imidacloprid, are applied. In contrast to this, flumethrin does not increase the activity of an almost silent nerve bundle, which could be plausibly explained by the fact that pyrethroids bind preferentially to open sodium channels (i.e. an activated neuron) and therefore will have no or little effect on a resting neuron (at least at the low concentrations used in this study). It is known that in insects the pyrethroid insecticides (such as flumethrin) bind preferentially to the open (activated) sodium channel and once bound keep the channel open . The action of pyrethroids is thus described as 'use dependant' in that they have little effect on resting nerve cells but a marked effect on stimulated cells. A low dose of imidacloprid activates nAChR channels and increases the frequency of the nerve cell electrical spiking. This translates into more sodium channels being open thus allowing flumethrin far better interaction with its target. Thus, as we have shown here, the application of both compounds together leads to a much higher level of activity than either compound alone, which demonstrates that neonicotinoid insecticides (such as imidacloprid) acting on the nAChr and pyrethroid insecticides acting on voltage dependent sodium channels cooperate at the level of the insect nervous system to produce an enhanced sensitivity to the pyrethroid component, providing a way to obtain similar levels of pest control with a reduced dose of pyrethroid. This is a classical (albeit new) example pharmacological synergism.
The next phase of the study investigated whether this principle would be visible in a well-controlled in vitro tick and flea study. The glass vial (dry surface) efficacy studies were conducted as an in vitro measure of flea and tick sensitivity to the flumethrin and imidacloprid and their combination. Using an in vitro system prior to assessment in an in vivo system provides accurate and repeatable data on the effect of the active compounds themselves without the added variable of many uncontrolled factors that a dog or cat skin and hair coat would add. What the in vitro isolated Spodoptera frugiperda ganglia studies showed in respect of the synergistic effects of imidacloprid and flumethrin was observed in this glass vial study on the whole living tick and flea organism. Although the in vitro Spodoptera nerve study demonstrated the synergism was true in an insect system, the efficacy studies conducted in the glass vial system indicated that the combination product exerted very high activities against ticks as well with Dermacentor and Ixodes being the most sensitive species followed by Rhipicephalus sanguineus.
Having demonstrated that the theory of pharmacological synergism holds true in an isolated insect nerve study and against the ticks and fleas in a glass vial system, the next phase of the study reported here evaluated the behaviour of the active ingredients in a collar matrix application device. The advantage of a collar system is the ease of application for an owner (enhancing compliance) and the potential for continuous slow release of the actives onto the dog or cats coat over an extended period of time (encouraging owner compliance further). The first way of evaluating this was through simply monitoring weight loss of the collar over time. The collars lost between 15 and 18% of their weight on the dogs and between 19 and 20% of their weight on the cats when worn over the full period for which insecticidal and acaricidal amounts of the actives are released (8 months). The fastidious grooming behaviour of cats could explain the slightly increased weight loss of the collar on the cat compared to the dog. Grooming behaviour would remove actives from the coat and result in a higher concentration gradient and hence faster collar reload of the coat in the cat than the dog.
The next way to evaluate the release kinetics of the actives from the collar was to evaluate the concentration of the actives on the surface of the collar at various time points. The results of the surface concentration of the active ingredients over time demonstrated an effective surface reload onto the collar surface that allowed for a reload of the actives down a concentration gradient from the collar surface to the animal's coat. Results of the surface analysis immediately after collar removal at various time points after collar fitting showed that approximately 2% of the starting flumethrin content in the collar is present on the collar surface for the 8 month study duration. The actives on the surface of the collar are constantly unloading onto the animal's hair coat and a dynamic equilibrium is established between actives in the collar, on the collar surface and on the animal's coat. The imidacloprid on the surface of the collars decreased slightly over the 84 days of the study from 9-12% of the starting collar content, to 6 - 8% at day 84, which suggests that removal of the active ingredient down the concentration gradient from the collar to the surface to the animal's hair coat is slightly faster than what the collar releases as actives onto its surface. These results are supported by the analysis of the concentrations of the actives in the collar over time. The change in concentration of the active ingredients in the collar matrix over the duration of the collar application in dogs and cats indicated a steady decrease due to a release of the active compounds for a time period slightly in excess of eight months. By the termination of the experiments (at 8 months) collars contained approximately 60% of their starting imidacloprid content and 80% of their flumethrin starting content. This reflects a continuous concentration gradient between the collar and the coat and a continuous coat reload of the actives from the collar for the 8 months of collar application. To substantiate a claim of efficacy of 8 months, the amount of active ingredients remaining in the collar at 8 months should still result in a sufficient concentration gradient between collar and animal surface to allow for steady release and biological efficacy for another several months beyond the claim. When evaluating efficacy against ticks and fleas in the real life situation in the numerous studies reported by Stanneck [25, 26] there was a decrease in efficacy, however, to below a 90% (for ticks) or 95% (for fleas) after 8 months. This is reflected in the product's registered label claim. This apparent contradiction (a drop in efficacy to below threshold) despite adequate collar concentrations of the active compounds) reflects the delicate balance of the physical and -chemical environments at play in such a long-term collar release matrix. These factors are influenced by numerous variables and as such are much more complex than the factors in play in the application of an active in a single-application fluid system such as with a spot on.
Finally, following evaluation of in vitro efficacy of the combination of imidacloprid and flumethrin and evaluation of the behaviour of these actives in the collar, an experiment evaluated the efficacy of hair clipped from dogs and cats wearing the collar against ticks and fleas. It has been known for many years now that the concentration of imidacloprid achieved on the coat of spot on treated animals is effective up until the end of the 4th week after application (the duration for which efficacy is claimed). Keeping the levels in the coat achieved by the spot on product in mind, we assumed that because the levels achieved by the collar device described here were never below those achieved by the spot on (for 8 months), the collar delivered imidacloprid dose would remain effective for this period. Topical flumethrin has long been known to be an effective acaricide, but the hair coat concentrations for efficacy are unknown. It has previously been established that it does not work systemically as topically applied compound is below the limit of detection in the blood of topically treated animals (Bayer Animal Health registration study ID 35642 and ID 35643, unpublished data). Taken together these unpublished results necessitated an in vitro study that evaluated the efficacy of hair harvested from dogs and cats treated with the imidacloprid: flumethrin collar device. Special attention was given to efficacy at very early time points (starting at 4 hours post collar application). Preventing tick attachment is crucial to preventing disease transmission. Including ticks that show abnormal behaviour that would prevent them from normal attachment and feeding, hair harvested from collar treated dogs and cats showed an efficacy of 99.3% after 6 hours and 100% after 12 hours (and hence for any time point thereafter for the duration of 8 months). These results strongly support a non-systemic, obviously external mode of action over the period of 8 months and provide evidence of very rapid repellent acaricidal efficacy which has also been reported from numerous other on animal studies [25–27, 40]. This is the longest time period for which a collar impregnated with an acaricide and an insecticide together has ever been shown to be capable of providing therapeutically relevant concentrations of active ingredients from its surface to the coat of a dog or cat.