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Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Buccal administration of buprenorphine is commonly used to treat pain in cats. It has been argued that absorption of buprenorphine through the buccal mucosa is high, in part due to its pKa of 8.24. Morphine, methadone, hydromorphone, and oxymorphone have a pKa between 8 and 9. This study characterized the bioavailability of these drugs following buccal administration to cats. Six healthy adult female spayed cats were used. Buccal pH was measured prior to drug administration. Morphine sulfate, 0.2 mg/kg IV or 0.5 mg/kg buccal; methadone hydrochloride, 0.3 mg/kg IV or 0.75 mg/kg buccal; hydromorphone hydrochloride, 0.1 mg/kg IV or 0.25 mg/kg buccal; or oxymorphone hydrochloride, 0.1 mg/kg IV or 0.25 mg/kg buccal were administered. All cats received all treatments. Arterial blood was sampled immediately prior to drug administration and at various times up to 8 h thereafter. Bioavailability was calculated as the ratio of the area under the time–concentration curve following buccal administration to that following IV administration, each indexed to the administered dose. Mean ± SE (range) bioavailability was 36.6 ± 5.2 (12.7–49.5), 44.2 ± 7.9 (18.7–70.5), 22.4 ± 6.9 (6.4–43.4), and 18.8 ± 2.0 (12.9–23.5)% for buccal administration of morphine, methadone, hydromorphone, and oxymorphone, respectively. Bioavailability of methadone was significantly higher than that of oxymorphone.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Opioids are commonly used to treat painful conditions in cats (Robertson, 2008). Buccal administration of buprenorphine, resulting in oral transmucosal absorption, has been reported to produce thermal antinociception in cats and to be effective at reducing pain following ovariohysterectomy (Robertson et al., 2005; Catbagan et al., 2011; Steagall et al., 2013). The efficacy of buccal buprenorphine is, at least in part, thought to be due to good absorption, related to its pKa of 8.24, and cats' high buccal pH, resulting in a high unionized drug fraction (Robertson et al., 2005). Anecdotal evidence suggests that buccal buprenorphine administration is commonly used in small animal veterinary practice, to treat pain in cats, particularly after they leave the hospital (Veterinary Information Network: http://www.vin.com, consulted on 8/3/2013).

Injectable buprenorphine, as used in the studies suggesting its efficacy in the treatment of pain in cats, has been intermittently unavailable, and the buccal use of other opioids, if efficacious, would therefore be of interest. If buprenorphine owes its good efficacy following buccal administration to enough drug being available for receptor binding and therefore to good absorption related, among other factors, to its pKa, other opioids, including morphine, methadone, hydromorphone, and oxymorphone, would also be predicted to be well absorbed following buccal administration. Indeed, the pKa of morphine, methadone, hydromorphone, and oxymorphone is reported to be 8.21, 8.94, 8.59, and 8.17, respectively (DrugBank: http://www.drugbank.ca, consulted on 5/21/2013). All four drugs have been reported to produce antinociception in cats, following intramuscular (morphine, hydromorphone), intravenous (methadone, oxymorphone), subcutaneous (methadone), or buccal (methadone) administration (Robertson et al., 2003a; Lascelles & Robertson, 2004; Steagall et al., 2006; Ferreira et al., 2011; Siao et al., 2012).

The aim of this study was to characterize the bioavailability of morphine, methadone, hydromorphone, and oxymorphone following buccal administration in cats. We hypothesized that bioavailability would correlate with the unionized drug fraction, calculated on the basis of the buccal pH at the time of drug administration.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Animals

Six healthy female spayed cats, 1–2 year old, weighing 4.5 ± 0.4 kg (mean ± SD) were used. A vascular access port had been implanted under general anesthesia prior to the study, with the catheter in a carotid artery and the port subcutaneous between the shoulder blades. The port was used for blood sampling. Patency of the port was maintained by filling the port and catheter's volume with heparin (100 U/mL) 3 times per week. The study was approved by the Institutional Animal Care and Use Committee at the University of California, Davis.

Drug administration

Buccal pH was measured by placing a strip of paper pH (Hydrion 5–9, Micro Essential Laboratory, Brooklyn, NY, USA) in the cheek pouch prior to drug administration. A 22 gauge, 2.5 cm catheter was placed in a cephalic vein, and used for intravenous drug administration. For buccal administration, drug was deposited in a cheek pouch, and the cat's mouth was held closed for 30 sec to 1 min. Morphine sulfate (15 mg/mL; Morphine Sulfate, Baxter Healthcare, Deerfield, IL, USA), 0.2 mg/kg IV1 or 0.5 mg/kg buccal (mean ± SD total volume 0.14 ± 0.01 mL); methadone hydrochloride (10 mg/mL; Dolophine Hydrochloride, aaiPharma, Wilmington, NC, USA), 0.3 mg/kg IV or 0.75 mg/kg buccal (mean ± SD total volume 0.34 ± 0.02 mL); hydromorphone hydrochloride (2 mg/mL; Hydromorphone Hydrochloride, Hospira, Lake Forest, IL, USA), 0.1 mg/kg IV or 0.25 mg/kg buccal (mean ± SD total volume 0.59 ± .05 mL); or oxymorphone hydrochloride (1 mg/mL; Opana, Endo Pharmaceuticals, Malvern, PA, USA), 0.1 mg/kg IV or 0.25 mg/kg buccal (mean ± SD total volume 1.21 ± 0.13 mL) were administered. Commercially injectable drug solutions were used and were not altered prior to administration. The order of the route was randomized according to a computer-generated list, while the order of drug was always as above. This order had been randomly selected for the entire group of cats, but the drug order was not selected randomly within cats for logistical reasons. All cats received all treatments, with at least 2 weeks between successive treatments.

Blood sampling

Blood samples (2 mL) were collected from the vascular access port prior to drug administration, and 1, 2, 4, 8, 15, 30, 60, 120, 240, and 480 min following intravenous drug administration, or 5, 10, 15, 20, 30, 45, 60, 120, 240, and 480 min following buccal drug administration. Blood was transferred to tubes containing EDTA, immediately placed on ice, and then centrifuged for 10 min at 3901 g at 4 °C within 10 min of collection. The plasma was separated and frozen at −20 °C until analysis for drug concentration.

Because the vascular access port had lost patency in one cat for both oxymorphone studies, a 20-gauge, 5-cm catheter was placed in a medial saphenous vein in that cat and used to sample blood. In addition, due to technical problems, data from 1 cat in the hydromorphone IV group were discarded and are therefore not included in the analysis.

Drug analysis

Morphine, methadone, hydromorphone, and oxymorphone were quantitated in feline plasma by LC-MS2 analysis of protein-precipitated samples. The calibration standards were prepared as follows: stock solutions were made by dissolving 10.0 mg of morphine, methadone, hydromorphone, or oxymorphone standard in 10.0 mL of methanol. Working solutions were prepared by dilution of the stock solution with methanol to drug concentrations of 1000, 100, 10, and 1 nanogram per milliliter (ng/mL). Plasma calibrators were prepared by dilution of the working solution with feline drug-free plasma following evaporation of the methanol to concentrations of 0.05, 0.1, 0.25, 0.5, 1, 5, 10, 25, 50, 100, 150, 200, 300, 400, and 500 ng/ml (morphine, methadone, hydromorphone), or 0.05, 0.1, 0.25, 0.5, 1, 5, 10, 25, 50, 100, 150, and 200 ng/mL (oxymorphone). In addition, quality control samples (plasma fortified with analytes) at concentrations of 0.3, 35, and 160 ng/mL (morphine, methadone, hydromorphone), or 0.35, 35 and 120 ng/mL (oxymorphone) were routinely included as an additional check of accuracy. The concentration of morphine, methadone, hydromorphone, or oxymorphone in each sample was determined by the internal standard (morphine-D3, methadone-D3, hydromorphone-D3, or oxymorphone-D3, respectively) method using the peak area ratio and linear regression analysis. The response was linear within the calibration range and gave correlation coefficients (R2) of 0.99 or better.

Prior to analysis, the plasma samples, controls, and calibrators were fortified with the appropriate internal standard to a final concentration of 100 ng/mL and submitted to solid phase extraction (morphine, methadone, hydromorphone: C18 200 mg/3 mL, UCT, Bristol, PA, USA; oxymorphone: Cerex Polychrom ClinII SPE 3 cc 35 mg, SPEware, Baldwin Park, CA, USA). Quantitative analyses were performed on a mass spectrometer (TSQ Quantum Ultra triple quadrupole mass spectrometer, Thermo Scientific, San Jose, CA, USA) equipped with a heated electrospray ionization probe that was kept at 355 °C. All analyses were performed in the positive ionization mode with a spray voltage set at 5000 V. The sheath and auxiliary gas used was nitrogen at 45 and 10 arbitrary units, respectively. The system was operated in the selected reaction monitoring mode with argon as the collision gas at a pressure of 1.5 mTorr. The ion transfer tube was kept at 300 °C, while the scan time and width were 0.25 s and 0.1 m/z, respectively. Data were processed using LCQuan software version 2.6 (Thermo Scientific, San Jose, CA,USA). The mass spectrometer was coupled with liquid chromatography (1100 Agilent LC system; Agilent, Santa Clara, CA, USA). Chromatographic separation employed a column (ACE C18, 100 × 2.1 mm, 3 μm, column; Mac Mod, Chadds Ford, PA) and a linear gradient of acetonitrile in water with a constant 0.2% formic acid at a flow rate of 0.35 ml/min. The acetonitrile concentration was held at 1% for 0.5 min, ramped up to 90% over 8.5 min. The injection volumes were 10.0 μl.

Detection and quantitation employed full scan LC-MS/MS transitions of initial product ions for morphine, methadone, hydromorphone, and oxymorphone (mass to charge ratio (m/z) 286.2, 310.3, 286.2, and 302.1, respectively). The response for the major product ions for morphine (m/z, 128.0), methadone (m/z, 265.1, 105.0, 77.1, 91.1, and 219.0), hydromorphone (m/z, 185.0, 157.0, 128.0, and 152.0), and oxymorphone (m/z, 284.1, 227.0, 198.0, and 181.0) was plotted and peaks at the proper retention time integrated using LCQuan. The software was also used to generate calibration curves and quantitate these analytes in all samples.

The lower limit of quantitation was 0.1 ng/mL, 0.25 ng/mL, 0.1 ng/mL, and 0.35 ng/mL, and the upper limit of quantitation was 500 ng/mL, 500 ng/mL, 500 ng/mL, and 200 ng/mL for morphine, methadone, hydromorphone, and oxymorphone, respectively. Accuracy (% nominal concentration) and precision (% relative standard deviation) at 0.3, 35 and 160 ng/mL were 104 and 9%, 110 and 6%, and 97 and 3%, respectively, for morphine; 91 and 3%, 111 and 6%, and 101 and 2%, respectively, for methadone; 92 and 12%, 95 and 10%, and 103 and 6%, respectively, for hydromorphone; and at 0.35, 35, and 120 ng/mL, they were 109 and 3%, 102 and 2%, and 93 and 8%, respectively, for oxymorphone. Accuracy and precision were considered acceptable based on FDA guidelines for bioanalytical method validation.

Bioavailability determination

Noncompartmental analysis was conducted on the time–concentration data (WinNonlin 6.2, Pharsight, Cary, NC, USA). Three to five data points were used to calculate the slope of the terminal phase and were selected by visual inspection of each individual time–concentration profile on a semi-logarithmic plot. The area under the time–concentration curve, extrapolated to infinity, was measured using the linear trapezoids method. Bioavailability was calculated for each drug as the ratio of the AUC3 following buccal administration to the AUC following intravenous administration, both indexed to their respective dose.

Unionized drug fraction calculation

The fraction of unionized drug (%) was calculated as inline image, where pH is the buccal pH at the time of drug administration.

Statistical analysis

Normal distribution of the parameters was verified using the Shapiro–Wilk test. Bioavailability of the 4 drugs was compared using a repeated measures anova, followed by the Tukey's test for pairwise comparisons. Correlation between bioavailability and unionized drug fraction, and bioavailability and volume of drug used for buccal administration was calculated. Significance was set at P < 0.05. Because of violations of the normality assumption for many pharmacokinetic parameters, parameters are presented as median (range). Bioavailability data are reported as mean ± SE (range).

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Changes in plasma drug concentration over time are shown in Fig. 1. Parameters obtained from noncompartmental analysis of time–concentration data following administration of morphine, methadone, hydromorphone, and oxymorphone by intravenous and buccal route are summarized in Tables 1 and 2, respectively. The bioavailability of methadone was significantly higher than that of oxymorphone. Bioavailability was negatively correlated to the unionized drug fraction (R = −0.43; P = 0.04) and to the volume of drug administered by buccal route (R = −0.53; P = 0.0092) (Table 2).

Table 1. Median (range) parameters obtained by noncompartmental analysis of time arterial concentration data following intravenous administration of 0.1 mg/kg morphine (n = 6), 0.3 mg/kg methadone (n = 6), 0.1 mg/kg hydromorphone (n = 5), or 0.1 mg/kg oxymorphone (n = 6) in cats
ParameterMorphineMethadoneHydromorphoneOxymorphone
  1. AUC, area under the time–concentration curve extrapolated to infinity; AUC extrapolation, fraction of AUC extrapolated; AUMC, area under the first moment curve extrapolated to infinity; C0, concentration at time 0; Cl, clearance; λz, slope of the terminal phase; t1/2 λz, terminal half-life; MRT, mean residence time; Vz, volume of distribution of the terminal phase; Vss, volume of distribution at steady-state.

AUC (ng.min/mL)4882 (3538–5353)41938 (34664–58174)1468 (1364–1986)2767 (2279–4906)
AUC extrapolation (%)0.4 (0.2–0.7)29.5 (23.4–43.6)2.4 (1.7–3.6)2.4 (1.7–9.2)
AUMC (ng.min2/mL)293754 (196813–413327)14293717 (9332289–23977501)76224 (64558–111105)210064 (175550–653198)
C0 (ng/mL)376 (259–550)492 (371–904)173 (88–312)243 (95–1425)
Cl (mL/min/kg)42 (37.4–58.5)7.2 (5.3–8.7)68.1 (50.3–73.3)36.2 (20.4–43.9)
λz (/min)0.011 (0.01–0.012)0.003 (0.002–0.004)0.014 (0.013–0.016)0.007 (0.004–0.007)
t1/2 λz (min)63.6 (57.6–68.7)237.2 (186.6–302.3)48.7 (44.3–54.9)106.7 (94.6–180.6)
MRT (min)63.8 (54.7–77.8)320.1 (245.5–412.2)53 (46.9–55.9)79 (74.6–133.2)
Vz (mL/kg)3818 (3264–5626)2422 (1635–2869)4929 (3535–5144)5482 (4148–9031)
Vss (mL/kg)2832 (2302–3341)2260 (1617–2724)3410 (2816–4097)2817 (2486–3379)
Table 2. Mean ± SD buccal pH and calculated unionized drug fraction prior to drug administration, and drug volume administered, mean ± SE (range) bioavailability, and median (range) parameters obtained by noncompartmental analysis of time arterial concentration data following buccal administration of 0.5 mg/kg morphine, 0.75 mg/kg methadone, 0.25 mg/kg hydromorphone, or 0.25 mg/kg oxymorphone 6 in cats
ParameterMorphineMethadoneHydromorphoneOxymorphone
  1. Cmax, maximum observed concentration; Tmax, time at which Cmax was observed; F, bioavailability. See Table 1 for remainder of key.

Buccal pH8.3 ± 0.38.4 ± 0.28.6 ± 0.28.7 ± 0.4
Unionized drug fraction (%)56.7 ± 14.423.9 ± 6.743.4 ± 11.173.0 ± 18.5
Drug volume (mL)0.14 ± 0.010.34 ± 0.020.59 ± 0.051.21 ± 0.13
AUC (ng.min/mL)4357 (1683–5369)45481 (24643–76247)506 (131–1546)1405 (835–2264)
AUC extrapolation (%)2.3 (0.9–3.6)36.9 (26.8–49.9)24.5 (0.9–62.3)7.2 (3.9–34.8)
AUMC (ng.min2/mL)547337 (288047–621927)17322989 (8373955–37221875)100862 (37859–856881)226280 (158480–1039093)
Cmax (ng/mL)29.7 (7–73.3)109.1 (48.7–140.7)3.1 (0.4–13.4)6.7 (4–12.8)
Tmax (min)40.5 (9–123.2)52.5 (5–119.9)28 (15–120.9)32.2 (21–60)
λz (/min)0.008 (0.007–0.011)0.002 (0.002–0.003)0.005 (0.002–0.01)0.005 (0.002–0.007)
t1/2 λz (min)85.7 (65.6–94.6)293 (230.4–330.6)153.9 (71.2–397.9)129.6 (101.3–306.9)
MRT (min)126.8 (98–171.1)430.2 (339.8–488.2)229.3 (101.4–568.1)172.8 (134.1–458.9)
F (%)36.6 ± 5.2 (12.7–49.5)44.2 ± 7.9 (18.7–70.5)22.4 ± 6.9 (6.4–43.4)18.8 ± 2.0 (12.9–23.5)
image

Figure 1. Mean ± SD arterial plasma drug concentration over time following IV administration (closed circles) of 0.1 mg/kg morphine (a; n = 6), 0.3 mg/kg methadone (b; n = 6), 0.1 mg/kg hydromorphone (C; n = 5), or 0.1 mg/kg oxymorphone (d; n = 6), or buccal administration (open circles) of 0.5 mg/kg morphine (a; n = 6), 0.75 mg/kg methadone (b; n = 6), 0.25 mg/kg hydromorphone (c; n = 6), or 0.25 mg/kg oxymorphone (d; n = 6) in cats.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

This study characterized the bioavailability of morphine, methadone, hydromorphone, and oxymorphone following buccal administration in cats. A large variability in bioavailability was seen between individuals, limiting the statistical power of the drug comparison, and resulting in statistical significance for the difference between oxymorphone and methadone only, although the bioavailability of morphine and methadone tended to be larger than that of hydromorphone and oxymorphone.

Buccal administration of opioids is thought to be preferable to oral administration, because of the expectation that the drug gets absorbed through the buccal mucosa, avoiding the first-pass effect seen with absorption through the gastrointestinal tract (Stanley & Ashburn, 1992; Guindon et al., 2007). Diffusion of drugs across membranes is governed by the concentration gradient across the membrane, the ratio of the surface area available for diffusion to the thickness of the membrane, and a permeability constant (Chiou, 1996; Kramer et al., 2009). Factors known to influence uptake through the buccal mucosa include the size of the molecule, the relative solubility in polar and nonpolar solvents, the type of solvent or vehicle in which the drug is applied, and its degree of ionization at buccal pH (Squier & Johnson, 1975; Stanley & Ashburn, 1992). Because cats have been reported to have a high buccal pH, between 8 and 9 (Robertson et al., 2003b) and because many opioids are weak bases with a pKa between 8 and 9, buccal absorption of opioids is expected to be good in that species. The negative correlation between bioavailability and calculated unionized drug fraction is surprising. It should be noted that, although significant, the correlation is weak (approximately 18% of the variation in bioavailability related to the variation in calculated unionized drug fraction). It is generally assumed that a larger unionized drug fraction is desirable for absorption through mucous membranes, because it correlates with lipid solubility. The negative correlation suggests that, in this case, the opposite was observed, that is, that a larger ionized drug fraction improved bioavailability. The significance could be a type I error. Alternatively, either ionization may actually be important for initial absorption, for example, to favor distribution in the saliva and allow contact with a larger area of buccal mucosa, or factors related to ionization or drug pKa in relation to buccal pH, but different from ionization itself, influence drug absorption; while correlation indicates some relation, it does not determine that the variables are causally related. Finally, and most importantly, the buccal pH was measured prior to drug administration. Because these drugs are formulated in aqueous, acidic solutions, it is likely that the administration itself resulted in some local change in buccal pH. The pH of interest would be the pH following drug administration, in the area of contact with the buccal mucosa. This would, however, be difficult to measure, and the measurement itself may interfere with drug absorption.

The correlation between volume administered (Table 2) and bioavailability is also relatively weak, albeit significant (approximately 28% of the variation in bioavailability related to the variation in volume administered). Several factors may explain this relationship. The larger the volume administered, the more likely that some of the drug would be swallowed or spitted out before it could be absorbed. The mean time–concentration plot for buccal administration of hydromorphone, and possibly morphine (Fig. 1), shows 2 concentration peaks, possibly related to absorption through the oral mucosa, and the gastrointestinal tract. Also, because the commercial drug solutions have a similar pH, the larger the volume administered, the larger the likely decrease in buccal pH, reducing the unionized drug fraction.

Time–concentration profiles in this study were derived from arterial drug concentrations. Arterial blood sampling was preferred to venous blood sampling for several reasons: (i) the arterial blood concentration is the input to the site of effect and is more likely to be directly associated with the magnitude of effect than venous concentrations; (ii) venous drug concentrations depend on the actual vein being sampled (contrary to arterial drug concentrations), implying that the only venous concentration relevant to the effect would be obtained from sampling the vein draining the site of effect (e.g., jugular vein for opioids, which act in the central nervous system); and (iii) jugular venous concentrations overestimate arterial concentrations significantly following buccal drug administration, because blood perfusing the buccal mucosa drains in the external jugular vein (Chiou, 1989a,b; Hedges et al., 2013). Comparisons with studies using venous sampling and extrapolations to venous concentrations should be made with caution, as venous concentrations (other than jugular venous) would be expected to be lower than arterial concentrations at the early time points and higher at the late time points.

The order of drug administration was not randomized within cats, for logistical reasons. Because of this, a time effect (i.e., an effect related to the number of studies) on time–concentration profiles cannot be excluded. It is, however, deemed unlikely based on the fact that less than 10% of blood volume was removed during each study and that 2 weeks would be expected to be sufficient for restoration of full blood volume.

Comparison of time–concentration profiles following buccal and oral administrations may provide additional information on whether some of the drug is likely swallowed and undergoes absorption through the gastrointestinal tract and may help understand some of the reasons for the high variability between individuals. In any case, the results presented here are likely representative of the absorption and disposition of the drugs studied, when administered at the doses used, using unaltered commercial injectable solutions containing the drug concentrations used in this study.

In conclusion, in this study, following buccal administration to cats, the bioavailability of methadone was significantly larger than that of oxymorphone. Further studies on the analgesic effect of these drugs following buccal administration are needed to establish the optimal dose and dosing interval in cats.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

This study was funded by the Winn Feline Foundation, the George Sydney and Phyllis Redmond Miller Trust, and the Center for Companion Animal Health, School of Veterinary Medicine, University of California, Davis.

Notes
  1. 1

    Intravenous

  2. 2

    Liquid chromatography-mass spectrometry

  3. 3

    Area under the time–concentration curve

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References