Investigation of utilization of nanosuspension formulation to enhance exposure of 1,3-dicyclohexylurea in rats: Preparation for PK/PD study via subcutaneous route of nanosuspension drug delivery
© Chiang et al; licensee Springer. 2011
Received: 10 March 2011
Accepted: 7 June 2011
Published: 7 June 2011
1,3-Dicyclohexylurea (DCU), a potent soluble epoxide hydrolase (sEH) inhibitor has been reported to lower systemic blood pressure in spontaneously hypertensive rats. One limitation of continual administration of DCU for in vivo studies is the compound's poor oral bioavailability. This phenomenon is mainly attributed to its poor dissolution rate and low aqueous solubility. Previously, wet-milled DCU nanosuspension has been reported to enhance the bioavailability of DCU. However, the prosperities and limitations of wet-milled nanosuspension have not been fully evaluated. Furthermore, the oral pharmacokinetics of DCU in rodent are such that the use of DCU to understand PK/PD relationships of sEH inhibitors in preclinical efficacy model is less than ideal. In this study, the limitation of orally delivered DCU nanosuspension was assessed by a surface area sensitive absorption model and pharmacokinetic modeling. It was found that dosing DCU nanosuspension did not provide the desired plasma profile needed for PK/PD investigation. Based on the model and in vivo data, a subcutaneous route of delivery of nanosuspension of DCU was evaluated and demonstrated to be appropriate for future PK/PD studies.
In recent years, researchers have demonstrated that various epoxyeicosatrienoic acid (EETs) regioisomers cause either vasodilatation or vasoconstriction in a number of vascular beds [1–3] and that they hold anti-inflammatory properties . There is compelling evidence from the literature that increasing the levels of EETs demonstrates anti-inflammatory, cardio-protective [5–8] antihypertensive, and renal vascular protective effects during disease states. These properties make this pathway an extremely attractive target for intervention. Based on these findings, soluble epoxide hydrolase (sEH) inhibition is a potentially attractive pharmacological approach to treat human hypertension. It has been reported that 1,3-dicyclohexyl urea (DCU) is a potent sEH inhibitor and inhibits human vascular smooth muscle (VSM) cell proliferation in a dose-dependent manner [9, 10]. Because of the anti-inflammatory and antihypertensive properties of sEH inhibition, DCU can be used as a model sEH inhibitor to further investigate decreased VSM cell proliferation, a crucial pathologic mechanism in the progression from systemic hypertension to the atherosclerotic state [4, 11, 12]. However, despite having high in vitro potency, the utility of DCU to investigate sEH is limited based both on its short t1/2 in rats [13–15] and its low aqueous solubility, which makes oral delivery of DCU to maintain prolonged and constant exposure difficult. Such an issue is not DCU specific. It is well acknowledged in the pharmaceutical industry today that an increasing number of lipophilic drug candidates are providing scientists with the growing challenge of reaching desired exposures in vivo. Approaches to deliver poorly soluble molecules have been developed for both clinical and preclinical activities [14–17]. However, in the early phase of drug discovery where large numbers of potential candidates are screened, development of suitable formulations in time for a drug candidate's in vivo evaluation remains a big challenge. In general, formulations made at this early stage need to be prepared on a small scale using common excipients with little lead development time and the assurance of reliable delivery of target concentration levels.
Recently, nano- and microparticle drug delivery has been widely used in the pharmaceutical industry as a tool to overcome exposure issues [17–23]. Previously, much improved exposures were reported when nanosuspension formulations were used to deliver DCU [13–15]. Improvements in oral exposure by a DCU nanosuspension formulation enabled a dose-dependent efficacy study in a diseased animal model . Despite the success of demonstrating preclincal efficacy, further utilization of DCU as a tool to evaluate target PK/PD relationships in chronic animal models  remains challenge. A short t1/2 coupled with a high drug plasma peak to trough (P/T) ratio was observed when DCU nanosuspension was dosed orally in rats .
In order to have full confidence of chemistry strategy for drug research, a full understanding of PK/PD relationships is essential when new targets are explored. The short apparent oral t1/2 (2.6 h)  and the high plasma P/T ratio limits the ability of dosing DCU nanosuspension orally to characterize PK/PD relationships in detail. In this case, the short t1/2 of DCU required twice daily (b.i.d.) to three times daily (t.i.d.) dosing to cover the target plasma IC50 and multiples. In addition, the high plasma P/T ratio confounds the researcher's ability to understand IC50 coverage requirements needed for in vivo efficacy. For example, it is very difficult to determine if the observed efficacy is driven by maximum plasma concentration (C max) or minimum plasma concentration (C min) when such a steep drop of DCU plasma exposure is encountered . Unless full PK/PD relationships can be determined, the drug target candidate profile for first in class targets cannot be established with confidence; consequently, chemistry strategy cannot be implemented without risks.
In order to overcome this issue, the delivery of DCU via intravenous (IV) infusion route was explored. Similar to oral delivery, IV delivery of DCU was limited by the poor aqueous solubility of DCU. The poor aqueous solubility of DCU is such that it cannot be formulated for IV delivery without a high percentage of organic cosolvents which is incompatible with animal models in terms of efficacy. An alternative IV formulation using nanosuspension has also been evaluated in rats and demonstrated as a valuable option . However, due to the complexity of the setup, such technique is only suitable for short term study (i.e., 2-4 h). The tool of delivering DCU to a chronic model for preclinical PK/PD still remains unanswered.
In this research, a drug surface area-based in vivo absorption model was established to evaluate the limitation of oral dosing DCU nanosuspension with respect to in vivo coverage. Due to the limitation of an inadequate t1/2 and a high plasma P/T ratio associated with oral dosing of DCU nanosuspension, it was concluded that an adequate and sustained coverage without a high plasma P/T ratio was not easily achievable by the oral route. In this investigation, a subcutaneous (SC) route of delivery of the nanosuspension of DCU was tested and was found to be suitable for future PK/PD studies. The findings confirmed our previous hypothesis and strongly support the use of SC dosing of DCU nanosuspension in the disease model (rat) to evaluate PK/PD relationships.
Materials and methods
HPLC grade acetonitrile was obtained from Burdick & Jackson (Honeywell Burdick & Jackson, Muskegon, MI, USA), the reagent grade formic acid was obtained from EM Science (Omnisolve, EM Science, Gibbstown, NJ, USA), and 1,3-dicyclohexyl urea, Tween 80 were purchased from Sigma-Aldrich (Sigma-Aldrich Corp., St. Louis, MO, USA).
Lead-free glass beads (0.5-0.75 mm) were purchased from Glen Mill (Glen Mill's, Inc., Clifton, NJ, USA) and were preconditioned in-house. The water purification system used was a Millipore Milli-Q system (Millipore, Billerica, MA, USA). The XRPD pattern was recorded at room temperature with a Rigaku (Rigaku Americas Corp., The Woodlands, TX, USA) MiniFlex II desktop X-ray powder diffractometer. Radiation of Cu Kα at 30 kV-15 mA was used with a 2θ increment rate of 3°/min. The scans ran over a range of 2-40° 2θ with a step size of 0.02° and a step time of 2 s. The powder samples were placed on a flat silicon zero background sample holder. The particle size distribution of a regular suspension and nanosuspension was measured by using a Mictrotrac® S3500 (Mictrotrac, Inc., Montgomeryville, PA, USA) instrument. Triplicates were measured for each sample, and the average was used for the final particle size distribution. The particle size distribution was calculated based on the general purpose (normal sensitivity) analysis model and the following refractive indices (RI): particle RI, 1.58; absorption, 1.0; and dispersant RI, 1.38.
For the particle size reduction, a bench scale wet milling devise was developed as described by Chiang et al. . To prepare a nanosuspension stock formulation (50 mg/mL), bulk DCU, an appropriate amount of glass beads (1.5 times weight by weight of the final formulation), and a vehicle containing 0.5% (w/w) Tween 80 in phosphate saline (pH 7.4) were added in a scintillation vial to the desired volume. The mixture was then stirred on at 1,200 rpm for a period of 24 h with occasional shaking to prevent a buildup of the drug around the vial. The stock formulation was harvested by filtration to remove the glass beads. The same vehicle (0.5% (w/w) Tween 80 in phosphate saline pH 7.4) was used to prepare the regular suspension. For the regular suspension, a formulation was made by directly suspending bulk DCU in the vehicle. Formulation concentrations were verified by liquid chromatographic tandem mass spectrometric (LC/MS/MS).
Male Sprague-Dawley rats weighing between 290 and 350 g, obtained from Charles Rivers Laboratories (Charles Rivers Laboratories, Inc., Wilmington, MA, USA), were housed in a room with an ambient temperature of 22°C ± 1°C on a 12-h light/dark cycle. The animals were allowed 7 days to acclimate and were given ad libitum access to standard rat chow (0.5% NaCl) (Baxter Healthcare Corporation, Deerfield, IL, USA) and tap water until the initiation of the experiment . The current study was conducted in accordance with the institutional guidelines for humane treatment of animals and was approved by the IACUC of Genentech. For dosing, each group of three male Sprague-Dawley rats was given either a 30-mg/kg subcutaneous dose of DCU formulated as regular suspension or nanosuspension. Oral dosing followed the same guidelines . At the initiation of the study, the rats weighed from 297 to 329 g. Blood samples (approximately 0.2 mL per sample) were collected from each animal via jugular vein cannulae at the following time points: predose; 5, 15, and 30 min post dose; and 1, 2, 4, 8, and 24 h post dose. All samples were collected into tubes containing potassium ethylenediaminetetraacetic acid as an anticoagulant. Blood samples were centrifuged within 30 min of the collection, and plasma was harvested. Plasma samples were stored at approximately 70°C until analysis for DCU concentrations by a LC/MS/MS assay method.
DCU plasma concentrations were quantified by using LC/MS/MS. Briefly, an internal standard (in-house compound) was added to samples followed by protein precipitation involving the addition of acetonitrile. Chromatography of DCU was achieved using a HALO Phenyl Hexyl column (2 × 50 mm, 2.7 μM particle size) (Advanced Materials Technology, Wilmington, DE, USA). The mobile phase used was 0.1% formic acid (A) and acetonitrile with 0.1% formic acid (B). A gradient was used and is described as follows: 10% B at 0 min and hold for 0.2 min, linear gradient to 95% B at 0.8 min and hold until 1.2 min, back to 10% B at 1.25 min and hold until 2.0 min. The total run time was 2.0 min, and the flow rate was 0.75 mL/min. An AB Sciex QTRAP 5500 mass spectrometer was used for detection. The MRM transition monitored for DCU was m/z 225.4 to m/z 100.2. The lower limit of quantitation was 0.013 μM (S/N = 6) in plasma.
where A is the drug absorbed, V is the volume of distribution, Cp is the plasma concentration, K is the elimination rate constant, and t is time.
where M is the mass of the drug remaining in the stomach, D is the drug dosed, Ke is the stomach empting rate, and t is the time.
Result and discussion
The use of nanoparticles and particle size reduction in general to increase in vivo exposure for poorly soluble drugs is well practiced [17–23]. Reducing the particle size increases the surface area available to the dissolution media and thus increases the overall apparent drug dissolution. This can be estimated by the equation developed by Noyes and Whitney. Despite the understanding of surface area impact on the drug dissolution, the degree of impact on absorption by dosing nanoparticles remains unclear . In theory, the best usage of utilizing nanoparticles to improve in vivo exposure (dissolution) is to dose it within the dissolution control range. In which the higher surface area of the nanoparticles is translated into a higher in vivo exposure.
An oral dose of DCU nanosuspension has been reported to greatly improve the in vivo exposure . However, the overall limit of improvement that a nanosuspension formulation can provide for orally dosed DCU is not well understood . In order to understand the degree of improvement provided by an oral nanosuspension formulation, simulations were performed using a Wagner-Nelson equation-based model that was established in-house in order to assess the amount of drug absorbed (dA) as a function of time [26, 27]. In this model, the stomach empting time was taking into consideration. A log linear gastro transit model  was used to estimate the amount of drug available (W) in the small intestine for absorption. The surface area of the DCU was estimated by assuming a sphere shape particle and a true density (d) of 1.3 cm3/gm. The total surface area (A) was estimated by first obtaining the particle volume (V) using the equation of V = 3/4 πr 3, and then total particle number (n) using the equation of n = ((drug weight)/V/d). The total surface area of the dose was estimated by the equation A = (4 πr 2) × n. The unit surface area by weight (A/W) was calculated to estimate the surface area reduction after the absorption took place, and the total residual surface area (RA) was calculated for each time point. The absorption efficiency (AE) was calculated by taking the ratio of the amount of drug available and was divided by the RA (AE = W/RA), and the absorption constant (K) was calculated as AE/δT.
Predicted dug absorption (at C max) versus in vivo data (Wagner-Nelson equation) based on the surface area model
Dose/Drug absorbed in mg (impact by surface area only)
In vivo(Wagner-Nelson equation) mg
Total surface area of the drug dose (cm2)
Absorption efficiency (AE) mg/cm2
3 mg/kg (regular suspension)
10 mg/kg (nano suspension)
30 mg/kg (nano suspension)
SC dose exposure comparison (nanosuspension versus regular suspension)
C max(μM) ± STDEV
C min24hr(μM) ± STDEV
AUC0-t(h*μM) ± STDEV
t1/2(h) ± STDEV
30 mg/kg (nanosuspension)
0.82 ± 0.03
0.20 ± 0.02
11.0 ± 0.5
10.2 ± 0.8
30 mg/kg (regular suspension)
0.13 ± 0.02
0.06 ± 0.02
2.0 ± 0.8
34.4 ± 21.4
It is well-known that safety and efficacy are the two major concerns of any new therapeutic target. Failure to fully understand either target safety or efficacy in the early development process often results in a more costly failure later in the clinic or even postmarketing. For this reason, the pharmaceutical industry spends significant resources on early target evaluation in order to minimize the risk in moving forward. However, such a process often relies on finding a suitable compound to interrogate the target which may take a considerable time and is not cost effective. Here, we describe an effort with a less than ideal model compound, DCU, utilizing nanosuspension formulation and careful evaluation using PK modeling and simulation. This approach helped us identify clear advantages of using nanosuspension. In addition, we were able to evaluate SC delivery of DCU which has distinct advantages when compared to what has been previously described in literature. We firmly believed that using the systematic approach will enable earlier "preclinical proof of concept studies" and ultimately save both time and resources when investigating new and novel targets. Further research is needed to continue the development in this area.
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