Comparison of Glucosamine Absorption After Administration of Oral Liquid, Chewable, and Tablet Formulations to Dogs

Introduction

Osteoarthritis is common in dogs and can be associated with aging, conformation, genetics, obesity, injury, and large body size.¹ The pathogenesis of osteoarthritis includes structural damage that leads to inflammation of the articular cartilage and synovial membrane. The inflammation can itself worsen the damage to joints, further increasing inflammation. Healthy cartilage is maintained by a balance of anabolic and catabolic processes, but this balance can shift towards degradation and cartilage resorption as osteoarthritis progresses.² Treatment modalities include the use of anti-inflammatory drugs to reduce inflammatory mediators and provide analgesia, as well as the use of nutraceutical supplements that have been advanced as chondroprotective agents.³

Glucosamine (GS) is a naturally occurring endogenous amino monosaccharide that is included in numerous supplements intended to maintain joint health. GS and chondroitin sulfate are two of the building blocks that are incorporated into the articular proteoglycans. GS, chondroitin, and similar nutraceutical agents are generally added to joint health supplements in hopes that higher concentrations of circulating cartilage precursors will encourage the net formation of hyaline cartilage.⁴

As with other veterinary nutraceutical products, there are no federal testing requirements for identity, purity, potency, or disintegration of GS formulations. Independent testing of GS supplements in people showed a potency of 41–108% of labeled amounts.⁵ However, some manufacturers of veterinary nutraceutical products do subject their products to additional, voluntary testing, which can aid in the selection of supplements that are most likely to be effective. Because GS is an ionic molecule, it is formulated as a basic salt of either hydrochloride or sulfate. Although the acid salt dissociates from the GS in the aqueous environment of the body, the identity of the salt has generated controversy.⁶ Disparate results between several large studies in people with osteoarthritis have been attributed to lower plasma and synovial fluid concentrations of GS associated with GS hydrochloride tablets, as compared to GS sulfate in liquid.⁷ However, there have been few direct comparisons of the pharmacokinetic properties or effect of GS formulations on outcome.^6,8

Although many joint supplements are available for use in dogs, few have been tested for oral bioavailability. The canine pharmacokinetic studies that have been performed examined the oral absorption of tablets only.⁹ At present, the effect of formulation on oral absorption of GS has been poorly studied in any species, and the absorption profile of orally administered liquid formulations has not been reported in veterinary medicine. The goal of the current study was to compare key pharmacokinetic parameters related to GS absorption among both liquid and tablet joint supplements in dogs.

Materials and Methods

This study was approved by the Oklahoma State University Institutional Animal Care and Use Committee. Eight adult intact male beagles, aged 2 yr, and weighing 9.9 ± 0.9 kg, were utilized in this study. Dogs were randomly assigned to one of two treatment groups, with four dogs in each group. Group 1 dogs were administered either a GS sulfate/chondroitin sulfate liquid formulation (True-Dose^a) or a chewable tablet formulation of GS hydrochloride/chondroitin sulfate (Tablet A, Cosequin^b) in a randomized, balanced cross-over design with a two week washout period between treatments. Group 2 dogs were administered either a GS sulfate/chondroitin sulfate liquid formulation (True-Dose^a) or a tablet formulation of GS hydrochloride/ chondroitin sulfate (Tablet B, Nutri-vet^c). All dogs received 1,000 mg of each GS salt formulation once daily for 8 days and were fed a meal^d immediately after. As the molecular weight of GS sulfate (277.24 g/mol) differs from that of GS hydrochloride (215.63 g/mol), the dose of glucosamine free base (GlucFB) (179.17 g/mol) in the administered GS sulfate formulation was 646.26 mg, whereas that of the GS hydrochloride formulation was 830.91 mg.

Blood samples were drawn at 0.25, 0.5, 1, 2, 4, 6, 8, and 12 hr after the first and last days of supplement administration. In addition, blood samples were obtained immediately before each daily supplement dose. Three milliliters of blood were collected into Ethylenediaminetetraacetic acid tubes from a catheter placed previously in the cephalic vein on the first and last days of dosing and by venipuncture of the jugular, cephalic, or saphenous veins on the days where a daily sample was collected. Samples were immediately placed into an ice water bath then centrifuged at 1,000 g for 10 min. Plasma was separated and stored at −80°C until assayed.

An improved assay utilizing high performance liquid chromatography with increased sensitivity and selectivity for GS in plasma was modified from a previously published method.¹⁰ Briefly, 0.1 mL of canine plasma was mixed with 0.2 mL of acetonitrile and 0.1 mL of borate buffer, followed by derivatization with 8mM FMOC-succinimide, and then subjected to solid phase extraction^e. The eluent was dried under nitrogen gas and re-dissolved in mobile phase (60% of 0.1% acetic acid, 20% acetonitrile, and 20% methanol). Samples were injected onto a 4.6 × 150 mm, 5 μM, C18 column^f. Plasma from untreated dogs was fortified with GS for the calibration curve, which ranged from 0.05–10 μg/mL. Intraday accuracy and precision of the assay were 99 and 0% (relative standard deviation) at 0.375 μg/mL, 97 and 2% at 1.75 μg/mL, and 99 and 5% at 7.5 μg/mL.

Plasma concentration versus time data after the first and last dose of each nutraceutical agent were analyzed noncompartmentally using pharmacokinetic software^g.¹¹ Peak plasma GS concentration (C_max) and the time at which peak concentrations occurred (T_max) were estimated directly from the data. The area under the plasma GS concentration-time curve to the last quantifiable sample (AUC_t) was calculated as the sum of linear trapezoids. Because doses between groups were standardized to the GS salt, rather than to the GS moiety itself, the C_max and AUC_t were normalized by dividing the relevant pharmacokinetic parameter by the dose of GlucFB that was actually administered. The mean and standard error of the mean were computed for plasma GS concentrations and for most pharmacokinetic parameters. A two-way, repeated measures analysis of variance^h was used for each study group to test whether day of administration (first dose or last dose) or nutraceutical formulation (liquid, tablet A, or tablet B) affected T_max, C_max, C_max per gram GlucFB, AUC_t, and AUC_t per gram GlucFB^h.

Results

The plasma GS concentrations were below that of the lowest calibrator (0.05 μg/mL) prior to exogenous GS administration and were uniformly <0.05 μg/mL by 24 hr after GS administration for all formulations. Plasma GS concentrations could be quantified in all dogs by 15 min after administration of the liquid formulation, whereas after administration of the tablet formulations, GS was not found in plasma until up to four hours after supplement administration (Figure 1). None of the evaluated pharmacokinetic parameters differed between the first and last dose for each of the tested formulations (P > 0.3), so the data from both sampling days 1 and 8 were combined to give mean values for each formulation (Figure 2). Formulation significantly affected the times (T_max) to peak plasma GS concentrations, which were 0.7 hr for the liquid GS, 4.2 hr for Tablet A, and 5.0 hr for Tablet B (P < 0.001; Table 1). The peak plasma GS concentrations (C_max) were highest with the liquid formulation, with a mean value of 3.5 μg/mL for the liquid formulation, 2.6 μg/mL for Tablet A, and 1.8 μg/mL for Tablet B (P = 0.01). The effect of formulation on peak concentrations was more pronounced when C_max was adjusted for the dose of GlucFB administered (P < 0.001). Formulation also affected area under the plasma concentration versus time curve (AUC), with a mean of 5.4 μg·hr/mL for the liquid formulation, 7.9 μg·hr/mL for Tablet A, and 7.1 μg·hr/mL for Tablet B (P = 0.002). However, this difference in AUC_t disappeared when AUC_t was adjusted for the dose of GS actually administered (P = 0.4).

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Table 1 Mean (± Standard Error of the Mean) Pharmacokinetic Parameters for GS Following the Daily Administration of 1,000 mg of Three Formulations of GS Salt to Eight Beagle Dogs

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Discussion

The administration of all three nutraceutical products increased plasma GS concentrations, indicating that all three formulations were bioavailable in dogs. However, the formulations differed from one another with respect to their pharmacokinetic profile. The oral pharmacokinetics of Tablet A, Cosequin^b, have been previously reported in dogs that received 1500 mg of GS hydrochloride.⁹ In that prior study, the mean C_max per gram GlucFB for Tablet A was 6.4 μg/mL, as compared to 3.1 μg/mL in the present study. The T_max for Tablet A of 4.2 hr was also much longer in the present study as compared to the previous report of approximately 1.3 hr.⁹ However, this discrepancy may be explained by the use of fasting before and after tablet administration in the previous study, whereas dogs were fed a meal with the supplement in the present study. As dogs are likely to receive nutraceutical supplements in conjunction with a meal, dogs in the present study were fed in order to more closely match typical home environments.

Both the rate and the extent of xenobiotic absorption can affect the pharmacokinetic parameters that were examined in the present study. However, the AUC is the parameter that best reflects the extent of absorption since AUC is a measure of total body exposure. Therefore, it was not surprising that AUC_t was lower for the liquid formulation as compared to the tablet formulations since the presence of the sulfate salt decreased the dose of the GS moiety (GlucFB) in the liquid formulation. When the dose of GlucFB that was actually administered was accounted for, AUC_t did not differ between formulations. This similarity in dose adjusted AUC_t among the three formulations indicated that the extent of GS absorption into systemic circulation, or relative bioavailability, was similar among formulations. Therefore, the profound effect of formulation on the time of maximal absorption and peak GS concentrations, T_max and C_max, demonstrated that the formulation affected the rate, rather than the extent, of GS absorption. In general, if the extent of absorption is constant, then T_max and C_max will be inversely related. As a consequence, a xenobiotic that is slowly absorbed will have a longer T_max and a lower C_max as compared to a rapidly absorbed xenobiotic. Indeed, the high peak concentrations occurring soon after the administration of the liquid GS formulation were consistent with rapid absorption. Unlike tablets, liquids do not require a dissolution process in order to partition into the aqueous environment of the gastrointestinal tract for absorption, thus speeding their movement across the mucosa.¹² The much longer T_max, and consequently lower C_max, associated with both tablet formulations was also consistent with slow dissolution and absorption of the tablet GS formulations as compared to the liquid formulation.

The potential consequences of the differences in absorption profiles between the liquid and tablet formulations are unclear. The administration of GS sulfate to horses has previously been shown to result in higher plasma and synovial concentrations as compared to GS hydrochloride.⁸ Similarly, higher plasma concentrations of GS have been associated with higher GS concentrations in synovial fluid.^8,13 In human studies, it has been postulated that the GS sulfate is more efficacious for attenuation of osteoarthritis than is GS hydrochloride because the sulfate salt produces higher plasma GS concentrations.⁷ Since the anti-inflammatory effects of GS on equine cartilage cell culture are dose dependent and higher plasma concentrations of GS result in higher concentrations in synovial fluid, it may be that higher plasma concentrations of GS are more important than are lower concentrations present over a longer time period. However, there is currently poor evidence in general for the efficacy of most nutraceutical agents to modify joint health in both people and veterinary species.^7,14 The importance of nutraceutical formulation on promoting joint health is even more poorly understood. The formulation of joint health supplements clearly affects the oral absorption kinetics of GS. Although the current study found that a liquid formulation of GS was more rapidly absorbed than two tablet formulations, these results cannot be generalized to other nutraceutical formulations since differences between similarly labeled GS formulations can be profound.⁵ Therefore, well-controlled, prospective clinical trials comparing the effects of different GS formulations are needed to explore the effect of plasma GS concentrations on prevention or amelioration of osteoarthritis in dogs.

Conclusion

Given the general lack of regulation and testing of nutraceutical products, pharmacokinetic studies can aid the practitioner in selecting formulations that are bioavailable and capable of entering the systemic circulation at potentially therapeutic concentrations. GS formulations differ with respect to their absorption characteristics, with the liquid supplement tested here producing higher peak concentrations as compared to tablet formulations. However, the importance of such pharmacokinetic differences to patient response remains to be investigated.