Novel collagen-based wafers as a drug delivery method for local analgesia in deer antlers : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Veterinary Sciences at Massey University, Manawatu, New Zealand
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Date
2021
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Massey University
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Abstract
Introduction
This study provided a practical and novel solution for post-operative pain mitigation and wound management after velvet antler removal in red deer (Cervus elaphus). Currently, there are no topical methods to mitigate pain for an extended period of time in deer following surgical removal of antlers. The current methods licensed in New Zealand provide only peri-operative analgesia with short-term effects and raise animal welfare concerns about whether animals are still in pain when the effect has worn off, especially in the deer industry in which a large number of animals are being managed.
Materials and methods
In vitro study: In vitro drug release test (IVDRT) was conducted using the Franz diffusion cell to assess the drug release rates of lidocaine and bupivacaine in two different phases of the pilot and main studies. The pilot in vitro study contained 9 treatment groups and 3 control groups (n=3), which were classified based on collagen extraction technique, whether modified with zinc oxide-polyvinylpyrrolidone (ZnO-PVP) nanoparticles and the difference in the order of adding local anaesthetics and ZnO-PVP nanoparticles. The main in vitro study was comprised of 4 treatment groups of 5%, 10%, and 25% ZnO-PVP nanoparticles (n=6) proportional to dry collagen weight and a control group. In both pilot and main in vitro studies, the samples were taken every 15 minutes in the first hour and every 2 hours up to 12 hours. LC-MS and HPLC were used for the quantification of the samples in the pilot and main in vitro studies, respectively.
MNT validation study: Forty male deer (stags) were assigned for the MNT validation study on three alternative days. A handheld algometer (Wagner FPX50) was used for mechanical nociceptive threshold (MNT) assessment of four antler sites (cranial, medial, caudal, lateral) in both right and left antlers. Animal body weight (kg) and antler length (cm) were recorded to investigate the correlation with MNT. The MNT readings from three days were compared with each other. In addition, the MNT reading from all four antler sites and the right and left antlers were compared with each other.
In vivo study: Eighteen stags sorted into three groups of 6 animals in each (2 treatment groups and 1 control group) for the pilot in vivo study, and forty yearling age stags assorted into four groups of 10 animals (three treatments and one control), were used in the main in vivo study. All animals had both antlers removed after administration of local anaesthesia. The control group in both pilot and main in vivo studies received a ring block of 4% articaine hydrochloride only, whilst the treatment groups received modified (with ZnO-PVP) or non-modified collagen composite wafers to the wound sites. The modified collagen composite wafers had 50% ZnO-PVP for the pilot in vivo study and had 0%, 5% or 25% ZnO-PVP proportional to dry collagen weight for the main in vivo study. A handheld algometer (Wagner FPX50), was used for mechanical nociceptive threshold (MNT) assessment at different time points (0, 4, 24, 72 hrs, 7 days and 14 days). Thermal imaging with a forward-looking infrared (FLIR) camera was performed for the detection of temperature differences between the groups. Digital photography of the wounds was performed for further quantitative wound healing analysis.
Pharmacokinetic study: Blood samples were drawn from deer after the application of collagen composite wafers at time points t0, t1, t2, t4, t6, t8, t12, and t24 hours for the pilot study and at time points t0, t1, t2, t4, t6, t8, and t24 hours for the main in vivo study. The plasma was
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analysed with LC-MS to calculate pharmacokinetic parameters with the non-compartmental method such as Cmax, Tmax, AUC, AUMC, half-life, the volume of distribution and clearance.
Statistical analysis: Higuchi model was mainly incorporated to calculate drug release rates for the in vitro studies. For in vivo studies, the statistical analyses were performed with a linear model for repeated measurements that accounted for the fixed effects of day, antler, location within antler or antler sites, antler length and weight of deer as covariates, and the random effect of animals.
Results
IVDRT did not show any statistically significant difference between the treatment groups; however, the treatment groups had significantly slower release compared to the control group in the pilot in vitro study. IVDRT in the main in vitro study showed the slowest release rate in the treatment group with 25% ZnO-PVP compared to the other groups for both lidocaine and bupivacaine. The control group had the most rapid drug release rates compared to the treatment groups, particularly for lidocaine. Furthermore, lidocaine showed a considerably slower release compared to bupivacaine when zinc oxide nanoparticles were incorporated, and the results significantly differed. MNT validation results showed that antler length (cm) and animal body weight (kg) are directly and positively correlated with the baseline MNT readings. The MNT readings from four sites of antlers, including cranial, medial, caudal and lateral aspects, did not have any significant difference from each other. In addition, the MNT readings from the right and left antlers did not show any significant difference from each other.
In vivo results in the pilot study showed a lack of collagen composite wafer adherence for the non-modified wafers (PT2) and 50% adherence for the modified wafers (PT1) in the pilot study. As a result of the main in vivo study, 90%, 70%, and 45% were in group 25%NP (T1), 5%NP (T2), and 0%NP (T3) to the wounds, respectively. A significant difference was observed in the recovery rates of PT1 compared to the control group (P<0.0001) for the pilot study. For the main in vivo study, all three treatment groups also showed a significant difference compared to each other: T1 vs. T2 (P<0.01), T1 vs. T3 (P<0.05), and T2 vs. T3 (P<0.0001). In addition, the treatment groups showed a significantly slower recovery rate from analgesia compared to the control group (P<0.0001 for all). All the treatment groups in the main study demonstrated analgesia beyond 6 hrs and up to 10 hrs.
The pharmacokinetics study showed significantly smaller Cmax for T1 and T2 compared to T3 only for bupivacaine. Tmax showed significantly smaller values for T1 compared to T2 for only bupivacaine. Both AUC (0-24), AUC (0-∞), and AUMC (0-∞) showed smaller values for T1 and T2 compared to T3.
Conclusion
The physically modified collagen composite wafer with zinc oxide-PVP nanoparticles, containing a short-acting (lidocaine) and a long-acting (bupivacaine) local anaesthetic, is a novel method to sustain drug delivery of local anaesthetics after the surgical removal of velvet antlers. Our suggested treatment can deliver analgesia to the wounded antler for up to 10 hours and is a safe and convenient method to use by farmers in the deer industry. Furthermore, the collagen wafer is very adhesive to the wound and can help facilitate wound healing of deer antlers.
Description
Figures 1.6 (=Shoulders & Raines, 2009 Fig 1) and 1.7 (=Li & Mooney, 2016 Fig 3) were removed for copyright reasons.
Keywords
Animal anesthesia, Local anesthetics, Drug delivery systems, Collagen, Antlers, sustained release, pain, Lidocaine, Bupivacaine, Articaine, collagen, composite, analgesia, algometry, Mechanical Nociceptive Threshold, local anaesthesia, Red Deer