Comparative pharmacokinetics of a new oral long

Pharmaceutical Sciences 80 (2015) 9–15

Contents lists available at ScienceDirect

Pharmaceutical Sciences journal homepage: www.elsevier.com/locate/phasci

Comparative pharmacokinetics of a new oral long-acting formulation of doxycycline hyclate: A canine clinical trial Sara Melisa Arciniegas Ruiz a, Lilia Gutiérrez Olvera a, María Josefa Bernad Bernad b, Sara del Carmen Caballero Chacón a, Dinorah Vargas Estrada a,⁎ a b

Departamento de Fisiología y Farmacología, Facultad de Medicina Veterinaria y Zootecnia, Universidad Nacional Autónoma de México, México Departamento de Tecnología Farmacéutica, Facultad de Química, Universidad Nacional Autónoma de México, México

a r t i c l e

i n f o

Article history: Received 4 March 2015 Received in revised form 21 July 2015 Accepted 18 September 2015 Available online 21 September 2015 Chemical compounds studied in this article: Doxycycline hyclate (PubChem CID: 54686183) Poly(ethyl acrylate-co-methyl methacrylate-cotrimethylammonioethyl methacrylate chloride) 1:2:0.2 (EUDRAGIT RL100®, PubChem CID: 104804) Acrylic acid (PubChem CID: 6581) Keywords: Tetracyclines Long-acting Antibiotics Carbopol Eudragit Canine

a b s t r a c t Doxycicline is used in dogs as treatment of several bacterial infections, mycoplasma, chlamydia and rickettsial diseases. However, it requires long treatments and several doses to be effective. The aim of this study was to determine the pharmacokinetics of four formulations of doxycycline hyclate, administered orally, with different proportions of excipients, acrylic acid–polymethacrylate-based matrices, to obtain longer therapeutic levels than conventional formulation. Forty-eight dogs were randomly assigned in five groups to receive a single oral dose (20 mg/kg) of doxycycline hyclate without excipients (control) or a long-acting formulation containing doxycycline, acrylic acid polymer, and polymethacrylate in one of the following four proportions: DOX1(1:0.25:0.0035), DOX2(1:0.5:0.0075), DOX3 (1:1:0.015), or DOX4(1:2:0.0225). Temporal profiles of serum concentrations were obtained at several intervals after each treatment. Therapeutic concentrations were observed for 60 h for DOX1 and DOX4, 48 h for DOX2 and DOX3 and only 24 h for DOX-C. None of the pharmacokinetic parameter differed significantly between DOX1 and DOX2 or between DOX3 and DOX4; however, the findings for the control treatment were significantly different compared to all four long-acting formulations. Results indicated that DOX1 had the most adequate pharmacokinetic–pharmacodynamic relationships for a time-dependent drug and had longer release times than did doxycycline alone. However, all four formulations can be effective depend on the minimum effective serum doxycycline concentration of the microorganism being treated. These results suggest that the use of any of these formulations can reduce the frequency of administration, the patient's stress, occurrence of adverse effects and the cost of treatment. © 2015 Elsevier B.V. All rights reserved.

1. Introduction In dogs, doxycycline (DOX) is used for controlling infections caused by Staphylococcus spp, Streptococcus spp (Ross and Jones, 2004), Haemophilus spp, Bordetella bronchiseptica (Speakman et al., 2000), Mycoplasma spp, Borrelia burgdorferi, Campylobacter jejuni, and Fusobacterium spp (Cunha et al., 1982; Holmes and Charles, 2009). It is the choice for treatment of infections caused by Leptospira spp (Levett and Edwards, 2009), Brucella canis (Holmes and Charles, 2009), Haemobartonella canis and numerous tick-borne diseases, most importantly, Erhlichia canis (McClure et al., 2010). Nevertheless, these diseases have importance not only in canine health but also in human public health, because they are zoonotic diseases (McClure et al., 2010). DOX has a bacteriostatic effect by protein synthesis inhibition (Holmes and Charles, 2009); recently, anti-inflammatory and anti⁎ Corresponding author. E-mail address: [email protected] (D. Vargas Estrada).

http://dx.doi.org/10.1016/j.ejps.2015.09.012 0928-0987/© 2015 Elsevier B.V. All rights reserved.

neoplastic roles have been discovered through the inhibition of matrix metalloproteases (Lee et al., 2009; Zeng et al., 2011). Doxycycline has a better clinical efficacy at low concentrations, such as at 2 to 4 times the microorganism inhibition concentration (MIC) level for susceptible microorganisms. Therefore, the inhibition of microorganisms by the drug occurs in a time-dependent manner (Cunha et al., 2000). However, suitable treatment with DOX requires 5 mg/kg dose, twice a day, during prolonged periods ranging from 21 days to years, depending on the microorganism in treatment (Bharti et al., 2003). In spite of the benefits of treatments with DOX, its use has been limited to some extent because of adverse reactions. Oral administration can generate adverse effects such as gastric irritation, with vomiting and risk of ulcerations; tissue irritation following subcutaneous or intramuscular injection usually appears (Smith and Leyden, 2005; Xiao et al., 2013). In veterinary medicine, a long-acting injectable formulation of DOX has been evaluated for treatment in cattle (Vargas-Estrada et al., 2008), small ruminants (Vargas et al., 2008) and dogs (Gutiérrez et al.,

10

S.M. Arciniegas Ruiz et al. / Pharmaceutical Sciences 80 (2015) 9–15

2012). An oral formulation for horses (Zozaya et al., 2013) and a subgingival system for the localized treatment of periodontitis in beagle dogs (Polson et al., 1996; Zetner and Rothmueller, 2002) have also been evaluated. The injectable drug showed an increase in half-life in dogs (133.61 ± 6.32 h), but it caused inflammation and pain at the injection site that lasted 30 days, an aspect that can cause refusal by the dogs' owners (Gutiérrez et al., 2012). In recent decades, there has been an increased interest in the pharmaceutical industry in the development of controlled-release formulations, which include the election of the excipients according to the properties of the drug which needed modification or improvement. In this case, both acrylic acid polymer and polymethacrylate have many advantages that achieve a sustained-release. The Carbopol® is an insoluble acrylic acid polymer with gel-forming ability and mucoadhesive properties; this last property prolongs the residence time of the formulations at the site of drug absorption and reduces contact and irritation on the absorption surface (Blanco-Fuente et al., 1996; Goskonda et al., 1998; Rajesh et al., 2012). In addition, Eudragit®, a polymethacrylate polymer that has mainly been used for film-coating, also creates an inert matrix structure that allows the diffusion of the drug through pores serving as a sustained release drug and binding agent (Karthikeyini et al., 2009; Vasantha et al., 2011; EVONIK, 2013). In other studies reported that Eudragit L100 were effective stabilizers of the drug in solid dispersion and the precipitation inhibition in solution (Chauhan et al., 2013). With regard to above the goals of this study were to determine the pharmacokinetics of doxycycline hyclate administered orally in experimental formulations with different proportions of acrylic acid and polymethacrylate-based matrices. Four (4) nonirritating, long-acting formulations of doxycycline with acrylic acid polymer and polymethacrylate were developed. The hypothesis is that long-acting formulation of doxycycline would have the potential to increase the duration of therapeutic blood concentrations of the drug, reduce the frequency of administration and decrease adverse reactions compared with immediate-release products.

vaccinated and were determined to be healthy on the basis of physical examination findings. The dogs had not been medicated with any antibacterial medication for at least 30 days. During the study, all dogs received water ad libitum and were fed a commercial diet twice daily. This study was approved by the Institutional Subcommittee of Research, Care and Use of Experimental Animals according to the Mexican Official Regulation NOM-062-ZOO-1999. The study was conducted at the Facultad de Medicina Veterinaria of the Universidad Nacional Autónoma de México, Mexico City. The owners of the dogs included in this research project gave written consent for their dog's participation in the study. 2.4. Long-acting drug preparation For long-acting formulations, doxycycline hyclate, acrylic acid polymer, and polymethacrylateg were mixed in the following four different ratios: DOX1(1:0.25:0.0035), DOX2(1:0.5:0.0075), DOX3(1:1:0.015), and DOX4(1:2:0.0225). After mixing, the formulations were granulated manually by wet granulation process with ethanol (Faure et al., 2001). Granulation process includes one or more powder particles to form multiparticle entities called granules. Granules were formed by the addition of ethanol onto excipients and doxycycline until obtained homogeneous mixture. The agitation resulting in the system along with the wetting of the components within the formulation results in the aggregation of the primary powder particles to produce wet granules (Faure et al., 2001). To obtain homogenous granules, the mixture was passed through an N° 20 mesh and final size was 0.85 mm average. The granulation liquid (ethanol) was removed by drying with stove BINDER (BINDER GmbH, Germany) at 37 °C during 24 h. Excipient proportions were based on previous research (Karthikeyini et al., 2009; Kulkarni et al., 2010), manufacturing recommendations (Lubrizol Advanced Materials, 2011; EVONIK, 2013) and the handbook of pharmaceutical excipients (Rowe et al., 2012). The granules were inserted in conventional gelatin capsules according to the animals' body weight. Each dog was given one capsule administered by hand into the dog's mouth.

2. Materials and methods 2.5. Study design 2.1. Materials Doxycycline hyclate (Indukern, Mexico), polymethacrylate (EUDRAGIT RL100®; Evonik, Germany) and acrylic acid polymer (Carbopol® 971 P NF polymer; Lubrizol, Mexico) were acquired from the manufacturing companies. 2.2. Pre-formulation stage The physical and chemical characteristics of the doxycycline hyclate powder (Indukern, Mexico) were obtained by scanning electron microscopy, particle size distribution, infrared spectroscopy (Spectrometer FTIR Perkin-Elmer RX-I model, using the potassium bromide pellet method), x-ray diffraction (Siemens D5000 powder diffractometer with copper anticathode l = 1.5406 Å and Software Diffract AT 3.3 on 35 kV and 30 mA) and differential scanning calorimetry (DSC 321 METTLER TOLEDO with a heating rate of 10 °C/min with nitrogen atmosphere). Moreover, the rheological properties were evaluated, included bulk density, tapped density, true density, Carr compressibility index, Hausner ratio, porosity percentage, angle of repose, and flow velocity. The wet percentage of the powder was measured with a thermobalance (OHAUS MB 2000). All techniques were performed according to the US Pharmacopeia (USP 30, 2007). 2.3. Animals Forty-eight (48) healthy adult dogs (2 to 8 years old) of different breeds and both sexes were included in this study. The mean body weight of the dogs was 17.75 kg (range, 15 to 30 kg). The dogs were

In a crossover study, dogs were randomly assigned (in five groups, four groups of ten and control group with 8) to receive a single oral dose (20 mg/kg) of doxycycline hyclate without excipients (control) or any of the four long-acting formulations. Each dog received the treatment assigned and the control treatment with a washout period of 30 days between both treatments. The dose of 20 mg of doxycycline/kg represents the cumulative dose for 2 days of treatment, according to the recommended dosage of 5 mg of doxycycline/kg twice a day (Bharti et al., 2003; Holmes and Charles, 2009; Levett and Edwards, 2009). After administration, blood samples (3 mL) were obtained by venipuncture from each animal at 0, 1, 2, 4, 8, 12, 24, 36, 48, 60, 72 and 96 h after drug administration. The serum was immediately separated from each sample by centrifugation and was stored at −20 °C until analyzed. To evaluate acute toxicity, the animals were supervised for three days after the treatment ended. Dogs were monitored for signs of discomfort, diarrhea, or vomiting during and after each experiment. 2.6. Serum doxycycline concentration determination The serum doxycycline concentrations were determined by modified agar diffusion analysis (Okerman et al., 2004) with Bacillus cereus (ATCC 11778, American Type Culture Collection, Manassas, Va.) as a test organism on a Mueller-Hinton dehydrated growth medium (BIOXON, Becton Dickinson, Mexico City, Mexico). The drug concentrations were determined with linear regression analysis by a comparison of the diameters of the inhibition halos with the standard curve (200, 20, 10, 5, 2.5, 1.25, 0.625, 0.3125, and 0.1562 μg/mL) prepared in pooled


11

antibacterial-free canine serum. The intra-assay coefficient of variance was b4.9, and the interassay error was b4.8. The analytic assay was linear over a range of concentrations from 0.1 to 10 μg/mL, with a percentage recovery of 95 ± 1% and a coefficient of determination (r2) of 0.99 ± 0.1. The limit of detection was 0.014 μg/mL, and the limit of quantification was 0.1 μg/mL. The serum concentrations were determined using specialized software (ORIGIN PRO, version 8.6, OriginLab Corp, Northampton, Mass.), and these values were used to determine the pharmacokinetic parameters.

The pharmacokinetic flip-flop condition was demonstrated by the following equation (Boxenbaum, 1998):

2.7. Pharmacokinetics analysis

2.8. Statistical analysis

To analyze the serum doxycycline concentration versus time curve for each individual dog after the oral administration of long-acting formulations, a computerized curve-stripping program (PKAnalyst, Micromath Scientific Software, Salt Lake City) was used. To determine the optimal pharmacokinetic model, Akaike information criterion (Yamaoka et al., 1978) and the graphical analysis of weighted residuals were used (Wagner, 1993; Gabrielsson and Weiner, 2007). For oral administration, fitted curves of doxycycline expressed the decrease in serum drug concentration as a function of time and were approximated to one compartment with first-order input and first-order output with the following equation (r2 N 0.99):

The serum doxycycline concentrations were reported as the mean ± SD (standard deviation), and the pharmacokinetics parameters of the long-acting formulations and control treatment were calculated for each dog. Data were reported as the mean ± SE (standard error). The normality and uniformity of the data were determined by Shapiro–Wilk tests; if data were not normally distributed, they were corrected by the exponential fitting method. Equality between means was evaluated by ANOVA tests and Tukey tests to obtain comparisons of the means. A value of p = 0.05 was considered significant.

CðtÞ ¼ ½ðDose Kab Þ=ðvolume Kab –Kel Þ

h i e– Kel time ‐ e‐Kabtime

C(t) is the concentration as a function of time, e is the base of the natural logarithm, Kab is the absorption rate constant, Kel is the elimination rate constant, and time refers to the time that has elapsed since the drug was administered. The pharmacokinetics parameters obtained by the curvestripping software were the following: elimination half-life (t½), calculated maximum plasma concentration (Cmax), area under the curve (AUC), area under the concentration–time curve calculated by the trapezoidal method (AUCt), area under the first moment of the concentration–time curve (AUMC), retention time (RT), and elimination rate constant (Kel). The time of peak plasma concentration (Tmax) was determined by inspecting the individual serum drug concentration–time profiles. Apparent volume of distribution at steady state was determined mathematically with the following equation: Vdss ¼ ðdose AUMCÞ=AUC2 Elimination half-life (t1/2) was calculated as follows: t½ ¼ 0:693=Kel The total body clearance (Clb) of the oral doxycycline was calculated as follows: Clb ¼ Dose=AUC The AUC0–∞ was calculated as follows: AUC0−∞ ¼ AUC þ ðClast = Kel Þ Clast is the last measurable concentration. The relative bioavailability (Frel) was calculated in the following equation as the percentage of the AUC in the experimental group relative to the control group: Frel ¼ ðAUC DOXx=AUC DOX‐CÞ 100

Rate of absorption ¼ Vz ½KC þ ðΔC=ΔtÞ Vz is the terminal exponential volume of distribution, K is the terminal disposition rate constant once drug absorption is complete, C is the serum concentration at time t, and ΔC is the change in serum concentration over the time interval Δt.

3. Results 3.1. Pre-formulation stage The granulometry data of doxycycline hyclate powder (Indukern, Mexico) were obtained by scanning electron microscopy and particle size distribution. Microscopy showed particles with prismatic shape and irregular edges. Distribution of particles indicated that 62.2% of the powder had an average size of 150 μm, 4.8% more than 150 μm and remaining percentage showed size above 250 μm. The structural properties were obtained by infrared spectroscopy, xray diffraction and differential scanning calorimetry (DSC). Infrared spectroscopy was allowed to establish the identity of active substance. X-ray diffraction showed the crystallinity of doxycycline. DSC showed degradation point or exothermic peak of 171.5 °C and fusion point or endothermic peak of 223.62 °C. Moreover, the rheological properties included: bulk density, tapped density, true density, Carr compressibility index, Hausner ratio, porosity percentage, angle of repose, and flow velocity. The tapped density was 0.6 ± 0.017 g/cm3, the bulk density was 0.5 ± 0.02 g/cm3 and the true density was 1.5 g/mL. The Carr index was 18.2 ± 2.02%, Hausner ratio was 1.22 ± 0.03, the porosity percentage was 65.5 ± 1.45%, the repose's angle was 19.42 ± 1.03° and the flow velocity was 2.62 ± 0.92 g/seg. The wet percentage of the powder was 0.26% ± 0.11. 3.2. Pharmacokinetics analysis The pharmacokinetics values of each long-acting treatment and of the control treatment are summarized in Table 1. Comparisons of Cmax, retention time, AUC, AUC0–∞, Vdss, and t½ among all treatments revealed that these parameters did not show statistically differences between DOX1, DOX2 and the control treatment, but these three formulations differed significantly with regard to DOX3 and DOX4 (p b 0.05). The relative bioavailability (Frel) of DOX3 and DOX4 were higher than DOX1 and DOX2 (p b 0.05). Significant differences were found for total body clearance (Clb) between all groups (p b 0.05). Sixty hours after administration of DOX1 and DOX4, the dogs had detectable doxycycline serum concentrations, while the dogs treated with DOX2 and DOX3 had detectable serum concentrations for fortyeight hours after administration. The four long-acting formulations lasted longer than did the control treatment, which had only 24 h of detectable serum concentrations of doxycycline (Fig. 1). However, DOX4 showed higher concentrations during the 60 h compared with DOX1,

12


Table 1 Pharmacokinetics values expressed as mean ± standard error (SE) after a single oral administration of doxycycline hiclate (20 mg/kg) to dogs treated with doxycycline hyclate without excipients (control) and one of the four long-acting formulations of doxycycline varying in their proportions of acrylic acid polymer and polymethacrylate; Control, DOX1 (1:0.25:0.0035), DOX2 (1:0.5:0.0075), DOX3 (1:1:0.015) and DOX4 (1:2:0.0225). a b c Values within a row with no common superscript differ significantly (p b 0.05). Mean ± SE

Control (without excipients)

DOX1 (1:0.25:0.0037)

DOX 2 (1:0.5:0.0075)

DOX 3 (1:1:0.015)

DOX 4 (1:2:0.0225)

t½ (h) Cmax (μg/mL) T Cmax (h) AUC (μg∗h/mL) AUC ∞ (μg∗h/mL) AUCt (μg∗h/mL) AUMC (μg∗h2/mL) RT (h) Kel (h−1) Vdss (L/Kg) Clb (mL/min/Kg) Frel (%)

7.54 ± 0.17ᵃ 2.03 ± 0.28ᵃ 2 22.1 ± 2.52ᵃ 24.18 ± 2.47ᵃ 18.65 ± 2.06ᵃ 239.92 ± 21.793ᵃ 10.82 ± 0.25ᵃ 0.09 ± 0.002ᵃ 10.003 ± 1.37ᵃ 0.91 ± 0.1ᵃ –

8.5 ± 0.46ᵃ 2.63 ± 0.106ᵃ 2 32.46 ± 0.66ᵃ 34.54 ± 0.75ᵃ 37.91 ± 1.15ᵃ 403 ± 29.01ᵃ 12.37 ± 0.66ᵃ 0.08 ± 0.004ᵃ 7.61 ± 0.29ᵃ 0.61 ± 0.12ᵇ 146.89 ± 2.98ᵃ

12.02 ± 0.92ᵃ 2.41 ± 0.88ᵃ 4 41.57 ± 3.08ᵃ 45.11 ± 3.42ᵃ 36.73 ± 2.47ᵃ 747.42 ± 100.97ᵃ 17.34 ± 1.33ᵃ 0.06 ± 0.006ᵇ 8.34 ± 0.29ᵃ 0.501 ± 0.04ᵇ 178.91 ± 15.32ᵃ

17.36 ± 0.42ᵇ 4.11 ± 0.208ᵇ 4.5 ± 1.02 106.35 ± 4.49ᵇ 112.68 ± 4.41ᵇ 95.54 ± 3.85ᵇ 2710.18 ± 156.63ᵇ 25.26 ± 0.62ᵇ 0.04 ± 0.001ᵇ 4.84 ± 0.16ᵇ 0.19 ± 0.008ᶜ 481.69 ± 19.16ᵇ

15.21 ± 0.99ᵇ 4.11 ± 0.21ᵇ 3.5 ± 1.1 88.6 ± 5.05ᵇ 94.04 ± 5.43ᵇ 84.08 ± 3.84ᵇ 2018.95 ± 29.01ᵇ 22.01 ± 1.42ᵇ 0.05 ± 0.003ᵇ 5.02 ± 0.27ᵇ 0.23 ± 0.013ᶜ 422.93 ± 26.04ᵇ

t½ = elimination half rate; Cmax = calculated maximum plasma concentration; Tmax = time of maximum plasma concentration; AUC = area under the curve; AUC∞ = area under the concentration–time curve from zero up to ∞ with extrapolation of the terminal phase; AUCt = area under the concentration–time curve calculated by the trapezoidal method; AUMC = area under the first moment of the concentration–time curve; RT = retention time; Kel = elimination rate; Vdss = apparent volume of distribution at steady state; Clb = Total body clearance; Frel = relative bioavailability.

and DOX3 had higher concentrations during the forty-eight hours treatment period, compared to DOX2. A pharmacokinetic flip-flop condition was demonstrated by use of the following equation for the rate of absorption: Vz (KC + [ΔC/Δt]). For DOX1 (doxycycline hyclate, polymethacrylate and acrylic acid polymer [1:0.25:0.0035]) the demonstrated plasma concentration–time (ΔC/Δt) data at 36 and 60 h was 0.008 μg/mL/h. At the midpoint of this time period (48 h), KC was 0.4 μg/mL/h. Because KC was much greater than ΔC/Δt, the rate of absorption was approximately equal to the rate of elimination. For DOX2 (doxycycline hyclate, polymethacrylate and acrylic acid polymer [1:0.5:0.0075]), the dogs' plasma concentration–time (ΔC/Δt) data at 24 and 48 h was 0.02 μg/mL/h. At the midpoint of this time period (36 h), KC was 0.3 μg/mL/h. For DOX3 (doxycycline hyclate, polymethacrylate and acrylic acid polymer [1:1:0.015]), ΔC/Δt was 0.06 μg/mL/h, and KC was 0.7 μg/mL/h. Finally, for DOX4 (doxycycline hyclate, polymethacrylate and acrylic acid polymer [1:2:0.0225]), ΔC/ Δt was 0.029 μg/mL/h, and KC was 0.5 μg/mL/h. For all cases, because KC was much greater than ΔC/Δt, the rate of absorption was approximately equal to the rate of elimination. Therefore, a flip-flop condition

existed, and the four formulations can be regarded as true long-acting formulations. The animals did not show any unusual sign of discomfort after treatments. The dogs did not vomit or have diarrhea during the study or afterwards. Monitoring was performed one month after each dog participated in each experiment. No adverse effects were found. 4. Discussion 4.1. Pre-formulation stage According to the shape of particles, equidimensional shapes with sharp edges had less flowablility than spherical shapes or smooth edges. The size of particles indicated very thin powder; this could generate segregation of particles. X-ray diffraction, DSC and infrared spectroscopy had adequate match with previous descriptions for doxycycline (Leypold et al., 2003; Heinemann et al., 2013). However, the compressibility properties were adequate, Hausner ratio, Carr index and angle of repose demonstrated suitable flow capacity with low velocity. Wet granulation was used to avoid segregation and improve flow velocity, Segregation could occur due to differences in the size or density of the component of the mix (Faure et al., 2001). 4.2. Pharmacokinetics study

Fig. 1. Serum doxycycline concentrations (Mean ± SD) in 48 healthy adult dogs randomly assigned (in groups of 12) to receive a single oral dose (20 mg/kg) of doxycycline hyclate without excipients (control; squares) and one of the four long-acting formulations containing doxycycline, acrylic acid polymer and polymethacrylate in proportions as follows: DOX1 (1:0.25:0.0035; circles), DOX2 (1:0.5:0.0075; up triangles), DOX3 (1:1:0.015; down triangles), DOX4 (1:2:0.0225; diamonds). Each dog received the treatment assigned and control treatment. The time of treatment administration was zero hours.

Few pharmacokinetics studies of oral doxycycline in dogs have been published (Michel et al., 1979; Van Gool et al., 1988), and none of those studies included controlled-release oral formulations for dogs. Prior investigations have reported on a perioral long-acting formulation in horses (Zozaya et al., 2013), a perioral gel for periodontitis treatment in humans (Mundargi et al., 2007; Chadha and Bhat, 2012) and a subgingival system for localized periodontitis treatment in beagle dogs (Polson et al., 1996; Zetner and Rothmueller, 2002) and in humans (Polson et al., 1997; Mundargi et al., 2007). This study reports the pharmacokinetics values of this antibiotic administered orally in dogs and includes the pharmacokinetics of four long-acting formulations that were designed for this species. The main purpose of designing long-acting formulations of doxycycline hyclate, acrylic acid polymer, and polymethacrylate was to obtain, with a single administration, serum doxycycline concentrations equivalent in practical terms to many administrations of a conventional doxycycline product. Being a time-dependent antibacterial drug, it could be expected that the clinical efficacy of the long-acting formulations of doxycycline should be at least equivalent to the clinical efficacy of immediate-release preparations administered orally once or twice daily, with a considerable reduction in workload, cost, and animal stress.


In the present study, DOX1 and DOX4 were clinically efficacious for sixty hours but showed differences in pharmacokinetics values; DOX1 had a Cmax of 2.63 ± 0.28 μg/mL, while DOX2 reached 4.11 ± 0.21 μg/mL. Similar data were obtained with DOX2 and DOX3; DOX2 had a Cmax of 2.41 ± 0.88 μg/ml, and DOX3 was 4.11 ± 0.208 μg/ml. Nevertheless, doxycycline is a time-dependent drug, meaning is not necessary to have huge peaks when the concentration is over the minimal inhibitory concentration specific for the microorganism treated (Cunha et al., 2000). Doxycycline shows the best clinical efficacy with low concentrations, 2 to 4 times the MIC, in this case the inhibition of the microorganisms occurs in a timedependent way. At higher concentrations, 8 to 16 times the MICs of the microorganism treated, doxycycline exhibits concentrationdependent killing (Cunha et al., 2000). After Cmax, the serum concentrations of DOX4 and DOX3 declined slowly but with a significantly greater decline than that observed for DOX1 and DOX2. Predictably for a highly lipid soluble drug, a high apparent volume of distribution at steady state was achieved after oral administration of the drug for the four long-acting formulations and the control treatment, and it would be expected to have had widespread tissue distribution (Riond and Riviere, 1988; Abd El-Aty et al., 2004). Total body clearance (Clb) is a measure of drug elimination from the body; it indicates the volume of plasma from which the drug is completely removed, or cleared, in a given time period. The obtained clearance values of the four long-acting formulations and control treatment were very low, indicating that the organism was very efficient in removing the drug (Jambhekar and Breen, 2009). The high volume of distribution and the low total body clearance indicated that doxycycline was quickly absorbed, widely distributed and slowly eliminated in the body. The four long-acting formulations showed better values in these parameters than did the control treatment, although the control treatment exhibited a similar pattern. The AUC values were expected. As AUC is inversely proportional to Clb, patients with low clearance have high AUC (Jambhekar and Breen, 2009). For long-acting formulations, it is predicted that the absorption rate would be lower than the elimination rate (Yáñez et al., 2011); in all four long-acting formulations the elimination rates were very slow but were greater than the absorption rates. This finding is not unusual for longacting preparations that exhibit flip-flop kinetics and may also explain the relative bioavailability, which reaches unusual values (Table 1). In turn, to demonstrate flip-flop pharmacokinetics, the overall appearance of the serum concentration versus time profile of the drug must be taken into account. If a much longer elimination half-life following extravascular dosing had been observed, compared with the IV route, it would suggest that flip-flop pharmacokinetics was occurring (Yáñez et al., 2011). However, this study design is not possible with doxycycline because IV administration of this drug is not recommended (Riond and Riviere, 1988; Riond et al., 1992). The absolute bioavailability of doxycycline for long-acting formulations was not determined, because data on the IV kinetics of the drug were needed, and the risk of cardiovascular toxicity was avoided (Riond and Riviere, 1988). Cardiac toxicity of this drug has been reported in calves (Yeruham et al., 2002) and rats (El-Neweshy, 2013). Higher blood concentrations were obtained with DOX4 (1:2:0.0225), it had the higher content of excipients. That means than the release improved with higher proportions of excipients. However, doxycycline is a time-dependent drug, and is not necessary to have huge peaks while the concentration maintained over the minimal inhibitory concentration specific for the microorganism treated, and that effect was obtained with DOX1. The pharmaceutical form is administered to remain in the upper portion of the gastrointestinal tract, such as the stomach or the duodenum, in order to increase systemic bioavailability of drug included in the dosage form. In the case of doxycycline, it is maintained in the upper portion of the gastrointestinal tract so that the bioavailability of

13

the drug is increased (Holmes and Charles, 2009). Retention of the pharmaceutical dosage form in the desired portion of the gastrointestinal tract can be achieved by delaying the expulsion of the dosage form from the gastrointestinal tract using techniques such as bioadhesion, floatation, swelling, or a combination of any of the foregoing (Shen et al., 2003). Bioadhesion is the ability of any material to adhere to a biological tissue, such as a mucous membrane. Bioadhesion improves residence time, which normally is a problem caused by stomach emptying and intestinal peristalsis, and from displacement by ciliary movement in oral dosage administration (Ponchel et al., 1987; Cleary et al., 2004). Bioadhesive properties of polymers are affected by both the nature of the polymer and by the nature of the surrounding media. Bioadhesives include natural and synthetic materials, which can stick to a mucous membrane (Bromberg et al., 2004; Cleary et al., 2004). In our pharmaceutical form, acrylic acid polymer was used to give this adhesive property and with this increase the residence time in digestive tract before oral administration, allowing the release for prolonged time. Additionally, to provide a solution to the problem of potential drug resistance and side effects, we can generate control in the release of drug. Release rate controlling agents for controlled release pharmaceutical dosage forms include, hydrophilic release controlling agents, hydrophobic release controlling agents, and mixtures thereof (Shen et al., 2003; EVONIK, 2013). In our formulation we used Eudragit RL100, it is hydrophobic release controlled agent, which allows release the drug more slowly, increasing the duration of the therapeutic blood concentrations. In other studies reported that in solid state, Eudragit L100 were effective stabilizers of the drug in solid dispersion. Drug– polymer interaction is believed to play a significant role in the precipitation inhibition of drug in solution and amorphous stabilization in solid state. (Chauhan et al., 2013). Controlled release and increased residence time in the upper portion of the gastrointestinal tract, increasing the bioavailability of doxycycline, while simultaneously allowing the quantity of doxycycline dosed to be reduced relative to the amount of immediate release doxycycline typically required to achieve a similar result in a patient. Benefits of the controlled delivery of drugs include the maintenance of serum drug concentrations at optimal therapeutic levels for a more prolonged time interval, reduction in handling and consequently, a possible improvement in drug-administration compliance (Brayden, 2003). In this context, the four long-acting preparations described here provided, with a single oral administration, useful serum concentrations of this antibacterial drug for a longer time in comparison with a conventional doxycycline product. The quantitative/qualitative microbiological agar diffusion technique used in this trial to determine the serum concentrations of doxycycline has been regarded as sufficiently reliable to substitute for high performance liquid chromatography analysis (Bocker, 1983). Furthermore, because it determines the active fractions of the drug, the technique offers more clinically meaningful data than do concentrations derived from purely chemical methods. In turn, this allows speculations on the relationships between serum concentrations, clinical efficacy and dosing intervals for specific pathogens. In that context, PK/PD compliance can be achieved when the serum concentrations of the drug are barely above or at the MIC level of the involved pathogen, but for as long as possible within the dosing interval (Bousquet et al., 1998). Therefore, the MIC level is still the most important measure of drug potency. Considering the above and the fact that serum concentrations of doxycycline are ideally never below the MIC at any time during the dosing interval (Skúlason et al., 2003), it is safe to regard all four long-acting formulations as drug preparations that possess good PK/PD ratios to control bacterial diseases in dogs, compared to conventional doxycycline. However, it should be noted that these values have not been validated for doxycycline.

14


5. Conclusion Considering doxycycline's MICs and that it is a time-dependent antibacterial drug, the best pharmacokinetic–pharmacodynamic profile would be achieved when serum concentrations of the drug are never less than the MIC for the infecting microorganism at any time during the dose interval (Craig, 1998; Del Castillo, 2013). On that basis, for susceptible bacteria, doxycycline alone (without excipients) should be administered every 24 h, whereas with the dosing interval for the four long-acting formulations evaluated in the present study could be increased to every 48 and 60 h. Less frequent dosing should improve prescription compliance among owners of dogs requiring treatment and should decrease patient stress levels. The decision to use one formulation or another would depend on the microorganism targeted for treatment. Undoubtedly, clinical trials and toxicological studies are needed to assess if these preparations can be regarded as potentially useful in this species. However, based on the manufacturing reports (Lubrizol Advanced Materials, 2011; Rowe et al., 2012; EVONIK, 2013) the levels of excipients (acrylic acid polymer and polymethacrylate) used in this study are less than known toxic doses. Assessment of the safety of these preparations in the species of interest is warranted. References Abd El-Aty, A.M., Goudah, A., Zhou, H.H., 2004. Pharmacokinetics of doxycycline after administration as a single intravenous bolus and intramuscular doses to non-lactating Egyptian goats. Pharmacol. Res. 49, 487–491. Bharti, A.R., Nally, J.E., Ricaldi, J.N., Matthias, M., Diaz, M., Lovett, M., Levett, P., Gilman, R., Willig, M., Gotuzzo, E., Vinetz, J., 2003. Leptospirosis: a zoonotic disease of global importance. Lancet Infect. Dis. 3, 757–771. Blanco-Fuente, H., Anguiano-Igea, S., Otero-Espinar, F., 1996. In-vitro bioadhesion of carbopol hydrogels. Int. J. Pharm. 142 (169), 174. Bocker, R., 1983. Analysis and quantitation of a metabolite of doxycycline in mice, rats, and humans by high-performance liquid chromatography. J. Chromatogr. 274, 255–262. Bousquet, E., Pommier, P., Wessel-Robert, S., Morvan, H., Benoit-Valiergue, H., Laval, A., 1998. Efficacy of doxycycline in feed for the control of pneumonia caused by Pasteurella multocida and Mycoplasma hyopneumoniae in fattening pigs. Vet. Rec. 143, 269 (29). Boxenbaum, H., 1998. Pharmacokinetics tricks and traps: flip-flop models. J. Pharm. Pharm. Sci. 1 (3), 90–91. Brayden, D., 2003. Novel drug delivery strategies in veterinary medicine. Ir. Vet. J. 56, 310–316. Bromberg, L., Temchenko, M., Alakhov, V., Hatton, T.A., 2004. Bioadhesive properties and rheology of polyether–modified poly(acrylic acid) hydrogels. Int. J. Pharm. 282 (1–2), 45–60. Chadha, V., Bhat, K., 2012. The evaluation of doxycycline controlled release gel versus doxycycline controlled release implant in the management of periodontitis. J. Indian Soc. Periodontol. 16 (2), 200–206. Chauhan, H., Hui-Gu, C., Atef, E., 2013. Correlating the behavior of polymers in solution as precipitation inhibitor to its amorphous stabilization ability in solid dispersions. J. Pharm. Sci. 102 (6), 1924–1935. Cleary, J., Bromberg, L., Magner, E., 2004. Adhesion of polyether–modified poly(acrylic acid) to mucin. Langmuir 20 (22), 9755–9762. Craig, W.A., 1998. Pharmacokinetic/pharmacodynamic parameters: rationale for antibacterial dosing of mice and men. Clin. Infect. Dis. 26, 1–10. Cunha, B., Sibley, C., Ristuccia, A., 1982. Doxycycline. Ther. Drug Monit. 4 (2), 115–135. Cunha, B., Domenico, P., Cunha, C., 2000. Pharmacodynamics of doxycycline. Clin. Microbiol. Infect. 6 (5), 270–273. Del Castillo, J.R., 2013. Tetracyclines. In: Giguere, S., Prescott, J., Dowling, P.M. (Eds.), Antimicrobial Therapy in Veterinary Medicine, 3th ed. Ames, Iowa, Wiley Blackwell, pp. 257–268. El-Neweshy, M.S., 2013. Experimental doxycycline overdose in rats causes cardiomyopathy. Int. J. Exp. Pathol. 94, 109–114. Evonik Industries AG, 2013. Polymethacrylate application guidelines. 12th ed. Evonik Industries AG, Darmstadt, Germany, pp. 1–8 (Chapter 6). Faure, A., York, P., Rowe, R., 2001. Process control and scale-up of pharmaceutical wet granulation processes: a review. Eur. J. Pharm. Biopharm. 52, 269–277. Gabrielsson, J., Weiner, D., 2007. Parameter estimation. In: Gabrielsson, J., Weiner, D. (Eds.), Pharmacokinetic and Pharmacodynamic Data Analysis, Concepts and Applications, 4th ed. Swedish Pharmaceutical Press, Stockholm, pp. 21–44. Goskonda, V., Reddy, I., Durrani, N., Wilber, W., Khan, M., 1998. Solid-state stability assessment of controlled release tablets containing Carbopol 971P. J. Control. Release 54, 87–93. Gutiérrez, L., Velasco, Z., Vázquez, C., Vargas, D., Sumano, H., 2012. Pharmacokinetics of an injectable long-acting formulation of doxycycline hyclate in dogs. Acta Vet. Scand. 54, 35–43. Heinemann, F., Leypold, C., Roman, C., Schmitt, M., Schneider, S., 2013. X-ray crystallography of tetracycline, doxycycline and sancycline. J. Chem. Crystallogr. 43, 213–222.

Holmes, N., Charles, P., 2009. Safety and efficacy review of doxycycline. Clin. Med. Ther. 1, 471–482. Jambhekar, S.S., Breen, P., 2009. Basic phamacokinetics. Second ed. Pharmaceutical Press, USA, pp. 53–56. Karthikeyini, C., Jayaprakash, S., Abirami, A., Halith, M., 2009. Formulation and evaluation of aceclofenac sodium bilayer sustained release tablets. Int. J. ChemTech Res. 1 (4), 1381–1385. Kulkarni, S.V., Kumar, R., Shankar, M.S., Rao, S., Kumar, A., Ramesh, B., 2010. Formulation and in vitro evaluation of sustained release matrix tablet of stavudine. Asian J. Pharm. Clin. Res. 3 (3), 215–217. Lee, H., Park, J.W., Kim, S.P., Lo, E., Lee, S., 2009. Doxycycline inhibits matrix metalloproteinase-9 and laminin degradation after transient global cerebral ischemia. Neurobiol. Dis. 34, 189–198. Levett, P., Edward, C., 2009. Leptospirosis. In: Evans, A.S., Brachman, P.S. (Eds.), Bacterial infections of human: epidemiology and control. Springer, New York, pp. 439–456. Leypold, C., Reiher, M., Brehm, G., Schmitt, C., Schneider, S., Matousekb, P., Towrieb, M., 2003. Tetracycline and derivatives-assignment of IR and Raman spectra via DFT calculations. Phys. Chem. Chem. Phys. 5, 1149–1157. Lubrizol Advanced Materials Inc, 2011. Toxicity of Carbopol polymers as a class. Technical Data Sheet 93. Lubrizol Advanced Materials Inc, Countryside, Ill, pp. 1–3. McClure, J., Crothers, M., Schaefer, J., Stanley, P., Needham, G., Ewing, S., Stich, R., 2010. Efficacy of a doxycycline treatment regimen initiated during three different phases of experimental ehrlichiosis. Antimicrob. Agents Chemother. 54, 5012–5020. Michel, G., Mosser, J., Fauran, F., 1979. Serum kinetics of doxycycline polyphosphate in dogs. Eur. J. Drug Metab. Pharmacokinet. 4 (1), 43–48. Mundargi, R., Srirangarajan, S., Agnihotri, S., Patil, S., Ravindra, S., Setty, S., Aminabhavi, T., 2007. Development and evaluation of novel biodegradable microspheres based on poly(d, l-lactide-co-glycolide) and poly(epsilon–caprolactone) for controlled delivery of doxycycline in the treatment of human periodontal pocket: in vitro and in vivo studies. J. Control. Release 119 (1), 59–68. Okerman, L., Croubels, S., Cherlet, M., De Wasch, K., De Backer, P., Van Hoof, J., 2004. Evaluation and establishing the performance of different screening tests for tetracycline residues in animal tissues. Food Addit. Contam. 21, 145–153. Polson, A.M., Southard, G., Dunn, R., Yewey, G., Godowski, K., Poison, A.P., Fulfs, J., Laster, J., 1996. Periodontal pocket treatment in beagle dogs using subgingival doxycycline from a biodegradable system. I. Initial clinical Responses. J. Periodontol. 67, 1176–1184. Polson, A.M., Garrett, S., Stoller, N., Bandt, C., Hanes, P., Killoy, W., Southard, G., Duke, S., Bogle, G., Drisko, C., Friesen, L., 1997. Multi-center comparative evaluation of subgingivally delivered sanguinarine and doxycycline in the treatment of periodontitis. II. Clinical results. J. Periodontol. 68 (2), 119–126. Ponchel, G., Touchard, F., DuchQne, D., Peppas, N.A., 1987. Bioadhesive analysis of controlled-release systems. i. fracture and interpenetration analysis in poly(acrylic acid)-containing systems. J. Control. Release 5, 129–141. Rajesh, M., Narayanan, N., Chacko, A., 2012. Formulation and evaluation of mucoadhesive microcapsules of aceclofenac using methyl cellulose and carbopol as mucoadhesive polymers. Int. J. Pharm. Pharm. Sci. 4 (4), 362–366. Riond, J., Riviere, J., 1988. Pharmacology and toxicology of doxycycline binding to plasma albumin of several species. J. Vet. Pharmacol. Ther. 12, 253–260. Riond, J., Riviere, J., Duckett, W., Atkins, C., Jernigan, A., Rikihisa, Y., Spurlock, S., 1992. Cardiovascular effects and fatalities associated with intravenous administration of doxycycline to horses and ponies. Equine Vet. J. 24 (1), 41–45. Ross, J.E., Jones, R.N., 2004. MIC quality control guidelines for doxycycline when testing gram positive control strains by the reference methods. Diagn. Microbiol. Infect. Dis. 50, 295–297. Rowe, R.C., Sheskey, P.J., Cook, W.G., Fenton, M.E., 2012. Handbook of Pharmaceutical Excipients. Seventh ed. Pharmaceutical Press, London (pp 118-123; 373-376). Shen, S., Jasti, B., Li, X., 2003. Design of controlled-release drug delivery systems. In: Kutz, M. (Ed.), Standard Handbook of biomedical Engineering & Design. McGraw-Hill, New York, pp. 161–179. Skúlason, S., Ingólfsson, E., Kristmundsdóttir, T., 2003. Development of a simple HPLC method for separation of doxycycline and its degradation products. J. Pharm. Biomed. Anal. 33, 667–672. Smith, K., Leyden, J., 2005. Safety of doxycycline and minocycline: a systematic review. Clin. Ther. 27 (9), 1329–1342. Speakman, A.J., Dawson, S., Corkill, J.E., Binns, S., Hart, S., Gaskell, R., 2000. Antibiotic susceptibility of canine Bordetella bronchiseptica isolates. Vet. Microbiol. 71, 193–200. US pharmacopeia and national formulary, 2007. USP 30–NF 25 vol. 2. US Pharmacopeia Convention, Rockville, Md, pp. 1553–1554. Van Gool, F., Santoul, C., Giuseppin-Huet, A., 1988. Doxycycline: pharmacokinetics and suggested dosage in dogs and cats. Tijdschr. Diergeneeskd. 13, 1189–1193. Vargas, D., Gutiérrez, L., Juárez, I., Sumano, H., 2008. Pharmacokinetics of an injectable long action parenteral formulation of doxycycline hyclate in goats. Am. J. Vet. Res. 69 (8), 1–6. Vargas-Estrada, D., Gracia-Mora, J., Sumano, H., 2008. Pharmacokinetic study of an injectable long-acting parenteral formulation of doxycycline hyclate in calves. Res. Vet. Sci. 84, 477–482. Vasantha, P., Puratchikody, A., Mathew, S., Balaraman, A., 2011. Development and characterization of Eudragit based mucoadhesive buccal patches of salbutamol sulfate. Saudi Pharm. J. 19 (4), 207–214. Wagner, J.G., 1993. Simple linear models. In: Wagner, J.G. (Ed.), Pharmacokinetics for the Pharmaceutical Scientist. Technomic Publishing Co, Lancaster, Pa, pp. 1–14. Xiao, S., Zhao, L., Hart, J., Semrad, C., 2013. Gastric mucosal necrosis with vascular degeneration induced by doxycycline. Am. J. Surg. Pathol. 37, 259–263. Yamaoka, K., Nakagawa, T., Uno, T., 1978. Application of Akaike's information criterion (AIC) in the evaluation of linear pharmacokinetic equations. J. Pharmacokinet. Biopharm. 6 (2), 165–175.

S.M. Arciniegas Ruiz et al. / Pharmaceutical Sciences 80 (2015) 9–15 Yáñez, J., Remsberg, C., Sayre, C., Forrest, M., Davis, N., 2011. Flip-flop pharmacokinetics — delivering a reversal of disposition: challenges and opportunities during drug development. Ther. Deliv. 2 (5), 643–672. Yeruham, I., Perl, S., Sharony, D., Vishinisky, Y., 2002. Doxycycline toxicity in calves in two feedlots. J. Vet. Med. B Infect. Dis Vet. Public Health 49, 406–408. Zeng, S., Zhou, X., Tu, Y., Yao, M., Han, Z., Gao, F., Li, Y., 2011. Long-Term MMP inhibition by doxycycline exerts divergent effect on ventricular extracellular matrix deposition and

15

systolic performance in stroke-prone spontaneously hypertensive rats. Clin. Exp. Hypertens. 33 (5), 316–324. Zetner, K., Rothmueller, G., 2002. Treatment of periodontal pockets with doxycycline in beagles. Vet. Ther. 3 (4), 441–452. Zozaya, H., Gutierrez, L., Bernad, M., Sumano, H., 2013. Pharmacokinetics of a peroral single dose of two long-acting formulations and an aqueous formulation of doxycycline hyclate in horses. Acta Vet. Scand. 55, 21–28.