Pharmacokinetic Appraisal of Carprofen Delivery from Intra-Articular Nanoparticles: A Population Modeling Approach in Rabbits ^†

Abstract

Osteoarthritis is frequently treated in veterinary settings with non-steroidal anti-inflammatory drugs (NSAID) such as carprofen (CP). Its action over the articular cartilage can be enhanced by increasing drug uptake into the cartilage, alongside its site of action, and anticipating its rapid distribution towards the bloodstream. A pharmacokinetic study to evaluate carprofen nanoparticles (NP) after intraarticular administration (IA) in rabbits was performed through a modeling allometric approach. The pharmacokinetic analysis of plasma profiles showed a rapid CP distribution outwards the synovial chamber but mainly remaining in plasma (Vc = 0.14 L/5 Kg), according to its high protein-binding. The absorption data modeling showed the occurrence of two different release–absorption rate processes after nanoparticle administration in the synovial space, i.e., a fast rate process causing a burst effect and involving the 59.5% of the total CP absorbed amount and a slow rate process, involving 40.5%. Interestingly, the CP burst effect inside the joint space enhances its diffusion towards cartilage resulting in CP accumulation in about three times higher concentrations than in plasma. In line with these results, the normalized-by-dose area under the concentration vs. time curve (AUC) values after IA were 80% lower than those observed after the intravenous. Moreover, the slower slope of the concentration–time terminal phase after IA administration vs. intravenous (IV) suggested a flip-flop phenomenon (0.35 h-1 vs. 0.19 h-1). Notably, CP clearances are predictive of the pharmacokinetic (PK) profile of CP in healthy humans (0.14 L/h/5 kg vs. 2.92 L/h/70 kg) although an over-estimation has been detected for cats or dogs (10 times and 4 times, respectively). This fact could probably be attributed to inter-species metabolic differences. In summary, despite the limited number of animals used, this study shows that the rabbit model could be suitable for a predictive evaluation of the release enhancement of CP-NP towards the biophase in arthritic diseases not due to sterical retention of the formulation.

1. Introduction

Osteoarthritis is managed with COX or fosfolipase A2 inhibitors to decrease prostaglandin mediators. Carprofen (CP) is an anti-inflammatory, analgesic and antipyretic propionic acid-derivative, used in veterinary medicine [1] as an alternative to corticosteroidic management of local inflammations [2]. Intraarticular administration (IA), improves local action and reduces systemic effects. Drug delivery to synovial fluid lining with the biophase, i.e., articular cartilage surface [3] improves the quality of life of animals with inflammatory arthropaties [4]. Drug uptake to the cartilage requires high synovial fluid concentrations, anticipating its rapid distribution towards bloodstream [5] due to the thin layer of specialized cells of the synovial cavity that facilitates the drug diffusion through. Indeed, a rapid equilibrium is achieved between synovial fluid and plasma [5]. Nanoparticle formulations, using biocompatible [6] Poly lactic-co-glycolic acid (PLGA) polymers, are promising [7,8,9,10] to extend the drug residence times, bioavailability and duration of effects [4]. The aim of the current study is the in vivo evaluation of CP nanoparticles for IA administration through a modelling approach, in rabbits.

2. Experiments

2.1. Reagents and Substances and Assay Solutions

Carprofen was purchased from Sigma-Aldrich. KH2PO4, Na₂HPO₄.2H₂O, acetonitrile, methanol, ortophosphoric acid 85%, sodium acetate trihydrate and ammonium acetate were purchased from Panreac (Barcelona, Spain). Deionized water (>10 MΩ.cm) was obtained on-site. Buffers were prepared as described elsewhere [11]. Poly Lactic-co-Glycolic Acid (Resomer^® RG 753 S) was purchased from SigmaAldrich and Ethylene-Propylene Oxides Block Copolymer (Lutrol F68^®P188) was a gift sample from Basf (Barcelona, Spain). All other reagents were also of analytical grade.

2.2. Animals and Drug Administration

Study protocol was approved (Ref.2015_089) by the animal welfare committee (Department of Agriculture, Livestock and Fisheries, 1997). Three 7-month-old white New-Zealand healthy male rabbits (Harlan, Barcelona, Spain) weighing 3.76–3.94 kg housed under standard conditions receiving food and water ad libitum were used. After anesthesia with xylazine (Rompun^® 20 mg/mL, BayerHispania, Spain) and ketamine (Imalgene^® 100 mg/mL, Merial, Spain), a dilution of CP Rimadyl^® 50 mg/mL (Zoetis, Spain) in physiologic saline (1:1 v/v) was administered through the right ear vein at 4 mg/kg. Secondly, CP nanoparticles 4.5 mg/mL were administered intraarticularly at 0.5 mg/kg through the right knee joint according to a cross-over design with 7 days wash-out. The left ear vein was catheterized (22G × 1.00in., Henry Schein, Hong Kong, China) and Vacutainer^® tube K2E was used for sampling (Beckton&Dickinson, Madrid, Spain).

2.3. Sampling Procedures

Ethylenediaminetetraacetic blood samples (1.3 mL) were always collected at 0, 0.25, 0.5, 1, 2, 4, 6, 8, 24, 48, 72 and 96 h and cooled on ice (30 min). After centrifugation (5000 rpm/10 min), plasma was stored at −80 °C until analysis. A total of 96 h after IA administration, animals were sacrificed with pentobarbital (Dolethal^® 200 mg/mL, Vetoquinol, Alcobendas, Spain) through the auricular vein. Knees (lateral and contralateral) of one animal were excised to obtain synovial liquid, femoral articular cartilage (both condyles) and meniscus tissue. Samples were weighted in glass vials and frozen at −80 °C until analysis.

2.4. Analysis of CP in Plasma and Knee Samples

CP Solid-phase extraction in plasma samples was performed with Discovery^® DSC-18 cartridges and Visiprep DL^® vacuum manifold (Supelco) followed by HPLC-UV as described by Parton et al. [12]. Knee samples were analyzed as described by [13] and expressed in µg/g. The HPLC method was acceptably linear within the calibration range (0.51–103.50 µg/mL). The lower limit of quantification (LLOQ) was 0.51 µg/mL.

2.5. Data Analysis. Non-Compartmental/Compartmental Pharmacokinetic/Deconvolution Analysis

Individual pharmacokinetic parameters [14] were estimated using non-compartmental analysis. The in vivo CP input rate from the nanoformulation I(t) was calculated by numerical deconvolution [15] with Phoenix-WinNonlin^® 64.8.2 Certara Inc. All CP plasma concentrations (IV and IA) were simultaneously analyzed with a population approach using NONMEM ver 7.4 [16] and Xpose R package v4.2.1, as diagnostic tool. The first-order-conditional estimation method (FOCEI) with interaction was used for parameter estimation [17]. Inter-individual variability (IIV) exponentially modeled was evaluated for each pharmacokinetic parameter. Additive and combined error models were tested for residual variability. Model selection was based on: decrease in the minimum objective function (MOFV; −2xlog likelihood); parameter precision and visual inspection of goodness-of-fit plots (Gofs). A decrease in MOFV of 7.879 between nested models was statistically significant (p < 0.005). For non-hierarchical models, the model with lowest Akaike criterion (AIC) was selected [18]. One- and two-open-compartment models with linear elimination parameterized as distributional clearance (CLD), apparent distribution volumes (V), and elimination clearance (CL) were fitted to the data. Allometric weight scaling was a priori added to all disposition parameters standardized to 70 kg body weight [19,20,21]. The power parameter was 0.75 for clearances and 1.0 for distribution volumes [22].

Table 1. Tentative models for description of CP release/absorption from nanoparticles. Models are depicted in Figure 1A (models 1–2) and Figure 1B (models 3–6).

Model	Kinetics of the Release/Absorption Process	Sequential vs. Parallel	Number of Depots
1	1st order absorption (ka)	-	one
2	Zero order absorption (ka)	-	one
3	Two 1st order absorption processes (kfr, ksr)	Parallel	two
4	1st order (kfr) and zero order absorption processes (ksr)	Parallel	two
5	1st order (kfr) and zero order absorption processes (ksr)	Sequential	two
6	Two 1st order absorption processes (kfr, ksr)	Sequential	two

and Figure 1 summarize tentatives of description of CP release/absorption from nanoparticles. The descriptive/predictive capability of the final model was evaluated through Gofs and a visual predictive check (VPC) [23].

3. Results

3.1. Carprofen Concentrations

Mean ± SD plasma concentrations are displayed in Figure 2.

Table 2. Femoral cartilage (both condyles), synovial fluid, meniscus and plasma CP concentrations at 9 h after IA administration at the dose of 1.98 mg.

Tissue	Concentration (µg/g) *
Cartilage	0.997
Meniscus	0.099
Synovial fluid	0.049
Plasma	0.3

* Once the distribution equilibrium was reached, the highest concentrations occurred in cartilage followed by meniscus, plasma and then synovial fluid.

summarizes the comparative CP concentrations in knee tissues at 9 h post-IA-administration.

3.2. Pharmacokinetic Analysis

Table 3. Mean ± SD values of the main pharmacokinetic parameters estimated by the non-compartmental approach, after IV and IA administration of CP to rabbits at 4 mg/kg and 1.98 mg, respectively.

Parameter	Intravenous Administration	Intra-Articular Administration
λz (h−1)	0.3565 ± 0.1546	0.1892 ± 0.0436
t1/2λz (h)	2.16 ± 0.76	3.78 ± 0.78
AUC (mg/L)·h	65.03 ± 20.90	6.73 ± 0.38
AUC/D	4.24 ± 1.36	3.40 ± 0.19
AUCextrap (%)	4.18 ± 4.06	28.29 ± 2.92
CL (L/h)	0.2533 ± 0.0831	-
Vi (L)	0.2058 ± 0.0273	-
Vss (L)	0.4403 ± 0.0758	-
Vdarea (L)	0.7963 ± 0.414	-
Cmax (mg/L)	75.67 ± 12.40	1.84 ± 0.19
Cmax/D	4.93 ± 0.81	0.93 ± 0.96
Tmax (h)	-	0.25 (0.08–0.5)
F (%)	-	94.48 ± 27.83

λz: apparent elimination rate constant; t1/2_λz: elimination half life; AUC: area under the concentration vs. time curve; D: dose in mg/kg.; AUC_extrap: percentage of extrapolated area; CL: plasma clearance; V_i: initial distribution volume; Vd_area: distribution volume associated to the terminal phase; V_ss: distribution volume at steady-state; C_max; peak concentrations; T_max; time to peak concentration after IA administration (median and range); F: bioavailability after IA administration, estimated as the ratio of dose normalized AUC values after IA administration to dose normalized AUC values after IV administration. Peak concentrations after the intraarticular administration were rapidly achieved (median T_max = 0.25 h). The higher apparent half-life after IA compared to IV, suggested a flip-flop phenomenon from nanoparticles, providing a slower release/absorption rate than elimination process.

summarizes the main non-compartmental disposition/absorption parameters. Caution should be taken because the high extrapolation percentage after IA administration (>20%) suggests that release/absorption process and bioavailability (51.4%) could not be accurately characterized.

A total of 47 CP plasma concentrations were analyzed by the population approach. Concentrations below the LLOQ (23.4%) were replaced by LOQ/2. CP disposition was best described by a two-compartment model. IIV was only associated with CL. The CP release/absorption from nanoparticles consisted of two first-order processes (K_fR: fast and K_sR: slow). Thus, the IA dose was partitioned into fast (F_fR·F1·Dose_IA) and slow (F_sR·F1·Dose_IA) absorption compartments, where F_fR refers to the fraction of the administered dose for the fast absorption and F_sR to the slow absorption (F_sR = 1 − F_fR). F1 and Dose_IA the absolute bioavailabilty and the input from the IA CP dose at time of administration, respectively. The VPC (Figure 3) confirmed the descriptive/predictive model capability. The circles represent the observed data. Dashed lines depict the 2.5th and 97.5th percentiles of the simulated concentrations. The solid line corresponds to the 50th percentiles of the simulated concentrations. VPC showed that most of the data fell within the 90% prediction interval and were symmetrically distributed around the median both after iv and IA administrations. The final population pharmacokinetic parameters are shown in

Table 4. Mean (relative standard error (RSE%)) values of the disposition and absorption pharmacokinetic parameters estimated by the final model.

Parameter	Units	Final Model Parameter Estimate (RSE%)
Disposition parameters
CL	L/h·70 kg	2.00 (8.25)
VC	L/70 kg	3.60 (9.36)
CLD	L/h·70 kg	3.08 (4.90)
VP	L/70 kg	4.28 (14.21)
Release/Absorption parameters
F	%	51.4 (9.82)
KaFR	h−1	7.38 (9.17)
KaSR	h−1	0.0667 (15.59)
FFR	%	0.595 (1.20)
FSR	%	0.405
TlagSR	h	0.5 (0.11)
Inter-individual variability
IIVCL	%	19.21 (62.6)
Residual variability
Proportional	%	25.51 (29.34)

CL = plasma clearance; V_C and V_P = volumes of distribution for central and peripheral compartments; CL_D = intercompartmental clearance between central and peripheral compartments; IIV and residual variability given as coefficient of variation (%). Ka_FR: initial rapid release rate constant; Ka_SR: slow release rate constant. F: Bioavailability; F_FR: fraction of drug released during the initial faster phase; F_SR: fraction of drug released during the slow phase; T_lagSR: lag-time of the slow phase release. All final parameter estimates are shown with the relative standard error (RSE) indicated by italic numbers in parentheses, demonstrating an acceptable precision.

.

Individual in vivo cumulate release/absorption and input rate I(t) profiles from the assayed IA nanoformulation are shown in Figure 4. An initial first-order kinetics burst effect was followed by a more sustained release.

4. Discussion

Anatomical similarities of rabbits with other species (dog, cat) suggest the extrapolation of in vivo CP release/absorption to these species. As previously reported [12,24,25], CP disposition was best described by a two-compartment model. The steady-state distribution volume (V_ss = 0.1126 L/kg mean live bodyweight) was in line with other results in rabbits [26], dogs 0.1192 L/kg [24] or cats (0.1506 L/kg) [27]. Considering a total body-water of 0.61 L/kg in live rabbits [28], the low V_ss values suggest that carprofen is mainly confined to plasma according to its high protein-binding [29] as other NSAIDs [2].

Predicted CP clearance (1.99 L/h/70 kg) was in agreement with results in healthy volunteers [29] (100 mg IV: 2.916 L/h). However, somewhat higher CL was predicted for 7.1–15.8 kg dogs (0.0447 vs. 0.01487 L/h·kg) and for 1.9–6.0 kg cats (0.058 vs. 0.006 L/h/kg). Prediction failure of the allometric CP model is probably due to large interspecies differences in metabolic patterns [22]. The metabolic pathway is only known in dogs, cat and humans [30] but not in rabbits.

The CP bioavailability from the nanoparticles (51.4%) was lower than expected, since the IA route obviates first-pass effects. Analytical limitations preventing the complete characterization of the release/absorption profile (extrapolated areas >20%), or a higher iv variability than anticipated probably due to the low number of animals can be the cause. The flip-flop phenomenon of IA nanoparticles support a longer residence time than conventional formulations. The CP release/absorption profile was best described by an initial burst effect (fast release component) due to the rapid release/absorption of the unencapsulated fraction into central compartment with a rate constant of 7.38 h⁻¹ (K_fR), resulting in a high concentration gradient between synovial fluid and plasma at initial times. Afterwards, a sequential second process with a slower first order release rate constant (K_sR = 0.0667 h⁻¹) of microencapsulated CP occurred. The fast process contributed to the 60% of total CP reaching the systemic circulation while the slow release prevented CP from leaving the joint space rapidly. Poorly-optimized inclusion of the non-ionic surfactant beneath the PLGA matrix [31] could have contributed to such an undesirable initial burst effect, so improvement of the formulation would be required to extend the CP residence time in the synovial cavity. Additionally, the reduced intraarticular volume in rabbits has demanded the injection of a minimal volume (0.4 mL) that, although being highly concentrated to migrate towards the cartilage, does not allow the build-up of a relevant drug reservoir for a significant modified release. In any case, CP plasma levels resulted to be higher than the half maximal inhibitory concentration (IC50) for Cox2 [32] both after IV and IA administrations. The lower albumin concentrations in synovial fluid (60%) in rabbits compared to plasma resulted in higher total plasma CP concentrations than in synovial cavity once the distribution equilibrium reached, as also reported in sheep [25] and in horse [2].

The cumulative input profile predicted by deconvolution was useful to optimize previous in vitro evaluation tests. Indeed, inadequate in vitro release profiles [33] have been due to, probably, an inappropriate membrane type.

5. Conclusions

In vivo characterization of a new CP nanoformulation for IA administration has been performed in rabbits. The pharmacokinetic profile was scalable to other species. The CP burst effect inside the joint space enhances its diffusion towards cartilage and plasma. Although a limited number of animals, the rabbit model seems suitable for a predictive evaluation of the release enhancement of CP (not due to sterical retention of the formulation) towards the biophase of arthritic diseases.