AAC Accepts, published online ahead of print on 5 December 2011 Antimicrob. Agents Chemother. doi:10.1128/AAC.

05180-11 Copyright 2011, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

1 2 3 4 5 6 7 8 9 10 11 12 13

Title: A Physiologically-Based Pharmacokinetic Model for Capreomycin Running title: A PBPK Model for Capreomycin Authors: B. Reisfeld*,1, C.P. Metzler1,, M.A. Lyons1, A.N. Mayeno1, E.J. Brooks2, M.A. DeGroote2 Key words: Mycobacterium tuberculosis, therapeutics, pharmacokinetics, computational modeling, pharmacodynamics, pbpk, mouse, human, anti-tuberculosis agents. Author affiliations:
1

Department of Chemical and Biological Engineering; Colorado State University, Fort

Collins, CO 80523
2

Department of Microbiology, Immunology, and Pathology; Colorado State University, Fort

Collins, CO 80523

Brad Reisfeld; Department of Chemical and Biological Engineering; Colorado State University; 1370 Campus

Delivery; Fort Collins, CO 80523-1370; voice: 970-491-1019, fax: 970-491-7369, email: brad.reisfeld@colostate.edu

current address Vertex Pharmaceuticals, Cambridge, MA

14 15 16 17 18 19 20 21 22 23 24 25 26 27

Abstract The emergence of multidrug-resistant tuberculosis (MDR-TB) has led to a renewed interest in the use of second-line antibiotic agents. Unfortunately, there is currently a dearth of information, data, and computational models that can be used to help design rational regimens for administration of these drugs. To help fill this knowledge gap, an exploratory physiologically-based pharmacokinetic (PBPK) model, supported by targeted experimental data, was developed to predict the absorption, distribution, metabolism, and excretion (ADME) of the second-line agent capreomycin, a cyclic peptide antibiotic often grouped with the aminoglycoside antibiotics. To account for inter-individual variability, Bayesian inference and Monte Carlo methods were used for model calibration, validation, and testing. Along with the predictive PBPK model, the first for an anti-tuberculosis agent, this study has provided estimates of various key pharmacokinetic parameter distributions and has supported a hypothesized mechanism for capreomycin transport into the kidney.

28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48

Introduction An estimated 500,000 cases of multi-drug resistant tuberculosis (MDR-TB) emerge each year with 150,000 deaths (50). MDR-TB refers to strains that are resistant to at least isoniazid and rifampicin. The World Health Organization (WHO) guidelines for treatment of MDR-TB include regimens containing Group 2 injectable agents (51). One such agent is capreomycin (CAP), a commonly used second line injectable drug with activity against many MDR-TB strains. It is generally reserved for patients who have had prior exposure to or whose isolates have documented resistance to kanamycin and streptomycin (51). Moreover, CAP has unique effectiveness against both the dormant and active forms of tuberculosis (25). However, the drug is nephrotoxic and ototoxic, especially in patients with renal impairment or in geriatric patients (26). CAP is a polypeptide antibiotic composed of four molecular analogs, IA, IIA, IB, and IIB. Its mode of action, though not fully understood, involves ribosomal inhibition of protein synthesis (22). Studies suggest that CAP binds to, and inhibits the function of, the 16S rRNA molecule of the M. tuberculosis 30S ribosomal subunit, as supported by up-regulation of a methyltransferase gene and the 16S rRNA processing protein gene (20). Due to similar nomenclature, side effects, and mode of action, CAP is often compared to, and grouped with, aminoglycoside antibiotics (AGAs) (19, 22, 24), despite its structural distinction. CAP and AGAs are nephrotoxic (26, 38), with some of the administered dose being retained in the epithelial cells of the kidney proximal tubules. The accumulation of AGAs is notable as the concentration in the kidney is much higher than that in the serum (41). In the proximal 3

49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70

tubule cells, AGAs must enter via apical membrane binding and endocytosis because the charged drug molecule cannot freely cross the cellular membrane. Megalin, a glycoprotein expressed in some specialized epithelial cells including in the renal tubule and inner ear epithelium, is the proposed endocytic receptor for such drugs (13, 41, 49). As noted earlier, CAP is known to exert both renal and ototoxicity, which supports the likelihood that megalin is responsible for uptake (38). As further evidence that megalin is an important factor in AGA uptake, a study comparing normal and genetically megalin-deficient mice was conducted by Schmitz et al. (48). In these studies, after exposure to gentamicin, wild-type mice had significant drug accumulation in the kidneys, whereas megalin-deficient mice did not. Despite its long history as an antibiotic, there is limited experimental information and few pharmacokinetic models available for the disposition of CAP. In the 1960s, Black et al. measured the pharmacokinetics of CAP in humans (3, 4) and derived peak serum concentrations and urinary excretion levels. Lee et al. (34) measured early time (up to two hours) serum levels of two different formulations of CAP in rats and looked at the impact of impurities on safety. In the present context, the most directly relevant experimental study to date is that of Le Conte et al. (33), who measured the concentrations of free and liposomal CAP in the blood, spleen, kidney, and lung of normal mice at various points (0.25, 0.5, 0.75, 1, 2, 4, and 6 hours) after administration. Notably, the concentration of CAP and the area under the curve (AUC) in the kidney were found to be much higher than that in all the other measured tissues at all time points. These investigators did not measure concentrations at later time points so that peak kidney concentrations, and decays from these concentrations, were not noted. 4

71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92

Especially for second-line agents, where side effects and toxicity are inherent concerns, it is critical to develop information and models that allow the examination of drug levels in specific tissue types, such as the kidney. The classical data-driven pharmacokinetic model assumes the body to be one homogenous, well-mixed vessel. To examine chemical distribution in specific tissues, a more sophisticated approach is needed. Using physiologically-based pharmacokinetic (PBPK) modeling, the body is divided into several physiologically-representative compartments organs, blood, tissues with a mass-balance for each compartment. With this approach, dose extrapolation, different routes of dosing, and animal-to-animal extrapolation may all be performed by changing relevant physiological and biochemical properties and by including appropriate allometric scaling laws (30, 31). Although traditionally used for environmental toxicants, PBPK models are increasingly used for the prediction of ADME for various drugs (45-47), including antibiotics (9, 10, 16, 32). A relatively recent advance in PBPK modeling has been the incorporation of approaches for accounting for inter-individual variability in anatomy, physiology, biochemistry, and chemical exposure (1, 6, 8, 12, 36, 39). Among other things, these approaches allow a rigorous incorporation of uncertainties, as well as predictions of chemical ADME in susceptible subpopulations. Overall, there is a knowledge gap in data and methods for predicting the ADME of secondline agents for the treatment of MDR-TB, and until totally new regimens are approved, optimized use of second-line drugs available to clinicians is a current research priority (42). The present exploratory study is meant to help fill the knowledge gap by acquiring tissue pharmacokinetic data and developing a PBPK model for CAP disposition. Although PK/PD 5

93 94 95

models have been developed (23), to our knowledge, this is the first published PBPK model for an anti-tuberculosis drug.

96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115

Materials and Methods Experimental Study The aim of the laboratory portion of this study was to determine the tissue and blood levels of CAP in mice at specified time points following subcutaneous injection. Mice. Female, six to eight week old C57BL/6 female mice were purchased from Jackson Laboratories (Bar Harbor, ME) and were housed in the Painter Center at Colorado State University. All experiments were approved by the Institutional Animal Care and Use Committee. Mice were randomly assigned to three groups: low-dose (N=24), high dose (N=24), and control (N=4). Drug. Capreomycin sulfate was purchased from Sigma Chemical Co. (St Louis, MO). Capreomycin solution was prepared by dissolving 92.5 mg (low-dose solution) and 231 mg (high-dose solution) of capreomycin sulfate in 10 ml phosphate buffer saline (1X, pH 7.4) (Fisher Scientific, Pittsburgh, PA). The vehicle solution was 1X PBS. Pharmacokinetic studies. The dosing regimen was determined through a pilot study in mice (TB Pharmacokinetic Laboratory of National Jewish Medical and Research Center, Dr. C. Peloquin), in which CAP plasma levels were measured following a single dose. Concentrations were adjusted to span doses that match human bioequivalence measures [Cmax (maximum plasma concentration), t1/2 (half life)], leading to recommended dosing levels of 100 mg/kg and 250 mg/kg for the present study. At time zero, the drug or control solution was administered subcutaneously to the mice. All mice received an injection of 0.2 ml of the

116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137

relevant solution. This corresponded to 100 mg/kg, 250 mg/kg, and 0 mg/kg capreomycin sulfate for mice in the low-dose, high-dose, and control groups, respectively. At each of the time points (0.5, 1, 2, 6, and 20 h), four mice from both the low-dose and high-dose groups were sacrificed via CO2 euthanasia followed by cervical dislocation. All of the mice in the control group were sacrificed at the 1 hr time point. Immediately following sacrifice, blood was collected by cardiac puncture, placed in a serum vial, put on ice for one hour after collection, and spun down to collect the serum; and the kidneys, lungs, spleen, and liver were harvested, weighed, and then flash frozen in cryovials at -80 C. Capreomycin analyses. Capreomycin was quantified by LC/MS/MS (vide infra). The tissues were prepared for analysis by adding water to give 100 mg tissue per ml, followed by sonication. Sonication was performed in small bursts while the tissue remained on ice in order to mitigate the effects of heat generated by the sonicator. In some cases, the larger organs were subdivided. The organs were prepared as follows in order to improve homogeneity: spleen (used in entirety due to the small size); kidneys (one kidney was used from each mouse; it was assumed that there was no preferential clearance in one kidney or the other); lung (portions of each lobe of the lung were removed and homogenized together); liver (after removal of the gallbladder, the liver was diced into small pieces, mixed, and a random sample of pieces was taken). Following sonication, 200 l of the tissue homogenate or 100 l of serum were added to a microcentrifuge tube containing 10 l of capreomycin standard or 10 l of 50% acetonitrile. The mixtures were vortexed briefly. To the tissue homogenate, 150 l of methanol + 1% formic acid was added, while 300 l of methanol + 1% formic acid was added to the serum samples to induce protein precipitation. Each sample was then vortexed 8

138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158

for 10 minutes, followed by centrifugation for 10 minutes to remove cell debris and protein from the liquid portion. Supernatant was then transferred to plastic autosampler vials for analysis. Plastic vials were used as capreomycin exhibits binding to glass (37). HPLC was performed using a Waters Atlantis HILIC Silica, 5 m, 4.6 x 50 mm column with a Phenomenex C18 guard cartridge. Standard curves for CAP (from 500 ng/mL to 50 g/mL) in
matrix (control serum or tissue homogenate) were generated, by adding capreomycin sulfate

standards prepared in 50% acetonitrile: 50% H2O with 0.1% acetic acid. The PK results for CAP in the paper refer to capreomycin free base (the base form of the drug rather than the salt form; "free" does not refer to unbound drug). Lower limits of quantitation (LLOQ) for capreomycin in each tissue were determined to be as follows: kidney (50 ng/mg), serum (1 ng/mg), liver (10 ng/mg), lung (10 ng/mg), spleen (10 ng/mg). Further LC-MS/MS method details are provided in the Appendix. The use of tissue homogenates is appropriate for use in the PBPK modeling approach used here because compartments are assumed to be well mixed and homogenous with respect to drug concentration. This is in contrast to studies of drug activity and efficacy, in which results derived from these types of samples could be misleading or erroneous (40). PBPK Modeling Study The aim of this study was to develop a PBPK model, calibrated and validated with experimental data, to predict the time-dependent ADME of capreomycin in mice. Model structure and equations. The structural model for capreomycin was based on a generic whole body PBPK model (28) with subsequent modifications of the kidney 9

159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180

compartment. This model comprises compartments for the lung, skin, fat, muscle, kidney, brain, heart, bone, liver, spleen, gut, arterial blood, venous blood, and a carcass compartment that contains all tissues not accounted for in other compartments. This flexible structure allowed the prediction of drug concentrations in tissues relevant for examinations of toxicity and pharmacological efficacy relevant to TB, as well as the possibility of assessing the impact of different dosing routes. Some of the compartments are not directly relevant to the efficacy and toxicity of CAP (e.g., heart and skin); however, they are included here because they provide additional detail for scaling to other species and are often germane to studies in which drug concentrations are measured in these tissues. The connection between compartments and pathway of blood flow is shown in Figure 1. Associated with each compartment is a mass balance for the drug. In all compartments, we assumed flow-limited mass transfer, viz., the blood entering a tissue is quickly in equilibrium with the tissue. The full set of governing equations for the model is given in the Appendix (eqn A2 eqn A13). Due to similarities in certain physicochemical and pharmacodynamic properties between AGAs and capreomycin, we assumed that the mechanisms for capreomycin ADME are related to those of other AGAs (52). As noted earlier, transport of AGAs is known to be dependent on megalin endocytosis followed by lysosomal sequestration (41, 48, 49). To account for the sequestration of capreomycin in the present model, we represented the kidney by a system comprising two linked compartments: a shallow (S) and deep (D) compartment, both of which were assumed to be well mixed (Figure 2). Although the correlation is not exact, anatomically, the deep compartment would approximate the cells along the walls of the 10

181 182 183 184 185 186 187 188 189 190

kidney tubules, while the shallow compartment would represent the remainder of the kidney tissue. The absorption for the kidney compartment was modeled using an equation describing saturable kinetics, as seen in the literature for similar applications (2, 14, 18). For example, Giuliano et al. (21) demonstrated that gentamicin and netilmicin accumulation in the kidney could be described using a Michaelis-Menten (saturable kinetics) equation (eqn 1), which is traditionally used to describe an enzyme-substrate reaction. For the present model, megalin corresponds to the enzyme, v0 is the renal accumulation rate, vmax is the maximum renal accumulation rate, [S] is the capreomycin concentration (in the shallow compartment), and KM is the Michaelis constant.

191

v0 =

vmax [ S ] K M + [S ]

(eqn 1)

192 193 194 195 196 197 198 199 200

Here, although capreomycin consists of 4 distinct molecules, this mixture was treated as a lumped single chemical entity in this model.
Model parameters and accounting for variability. The following experimental inputs were

used directly in the model: body weight of the mice, and lung, liver, kidney, and spleen weights. For the remaining organs, and for approximate blood flow through each organ, values from Brown et al. (11) and Davies and Morris (17) were used. The tissue density for all of the organ systems was assumed to be equal to that of water (11). With the exception of that for the lung, all of the tissue:blood partition coefficients were set equal to one. The lung:blood partition coefficient was set equal to two based on the observation that
11

201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220

aminoglycosides are transported into the lungs by endocytosis (27), augmenting the baseline thermodynamic partitioning. A lung:blood partition coefficient greater than one is consistent with the observation that aminoglycosides are eliminated more slowly from the lung than from the serum (15, 35). The list of model parameters used in the simulations is summarized in Table A3 of the Appendix. Owing to the unique nature of the model structure, and the lack of literature values for physiological transport values for capreomycin, the model parameters listed in Table 1 were determined using calibration simulations (vide infra).
Solution Method. Simulations were performed in two steps. First, a series of calibration

simulations were conducted using a Bayesian approach (7, 8, 35) to determine the unknown parameters for the model. This approach rests on a relationship among probability distributions involving unknown parameters and available data y given by Bayes theorem
p ( | y ) p ( y | ) p ( ) . Here, the posterior distribution p ( | y ) is obtained as the product

of the prior distribution p ( ) and the likelihood p ( y | ) . The model calibration involves the identification of the parameters (Table 1) with those that are needed to complete the specification of the PBPK model (Table A3). The data y corresponds to experimentally obtained concentration-time profiles resulting from a known dose. The likelihood contains the underlying PBPK model (eqn A2 - eqn A13) calculated with the parameters . Combining the likelihood of the data with prior parameter distributions results in the posterior probability distribution for the parameters conditioned on the data. To verify the robustness of the

12

221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236

posterior estimates, several types of priors were used, including truncated-normal and uniform distributions. Following the calibration step, Monte Carlo (MC) simulations were performed, in which the PBPK model equation system was solved using values sampled from the parameter distributions found in the calibration step. In a typical MC simulation, 1000 model runs were conducted, each containing a set of parameters randomly sampled from the distributions found earlier. The result of these runs was families of time-dependent concentration profiles of capreomycin in each model compartment. All simulations were performed using GNU MCSim (5) (v. 5.2), a simulation package that is useful in solving statistical and differential equation systems, performing Monte Carlo stochastic simulations, and conducting Bayesian inference through Markov chain Monte Carlo (MCMC) simulations. For the MCMC simulations, chains of length 10,000 were used and convergence was assessed using visual inspection. All calculations were performed on a PC workstation with a 2.8 GHz dual core Intel Pentium processor and 8 GB of RAM running the Windows XP operating system.

13

237 238 239 240 241 242 243 244 245 246 247 248 249 250 251 252 253

Results

Experimental Study The measured low-dose and high-dose concentrations of capreomycin in the organs of interest are shown in Figure 3 (a-e). For all tissues except the kidney, the peak concentration of capreomycin occurred at some time prior to 0.5 hours. Within the resolution of the data, the concentrations were found to decay exponentially over time, consistent with a first-order elimination process, with both low- and high-dose data following similar elimination kinetics. The capreomycin was eliminated relatively rapidly, with concentrations falling below the LLOQ in one to two hours for all tissues, besides the kidney. For the kidney, peak concentrations for both low- and high-doses occurred around three hours after administration and then decayed relatively slowly. Concentrations of capreomycin in the kidney were still well above the LLOQ after 20 hours. The only other study in which tissue concentrations for capreomycin were measured (33) focused on time points up to six hours, and decay in concentration from the peak value was not seen. The kidney concentrations at all time points are several times those in any other compartment, consistent with the transporterenhanced uptake mechanism and supporting data described earlier.

14

254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274

Modeling Study Estimated Parameter Values Based on the calibration simulations described earlier, probability distributions of the unknown parameters were determined. Table 2 summarizes the means and standard deviations for the values in these distributions, while Figure 4 shows the distributions for each of these parameters. These parameters provide rough estimates for various transport properties for CAP that are difficult to measure directly, including the kinetics of accumulation in the kidney, the rate of renal clearance, and the rate of hepatic clearance. However, as mentioned earlier, it is difficult to compare these values to others in the literature because of the paucity of previous detailed pharmacokinetic and transport studies for capreomycin.

Comparisons Between Experimental Data and Model Predictions The MC simulations produce a family of concentration profiles in each organ compartment based on the animal and organ weights and sampling the distributions of model parameters depicted in Figure 4. The fifth and ninety-fifth percentile curves for the low-dose MC simulation results, along with the corresponding experimental data, are shown in Figure 6. Similar comparisons for the high-dose case are shown in Figure 7. For the low-dose case, agreement between simulations and experiments was generally good for all organs, with the experimental points falling within the range of simulations in most cases. The largest discrepancy appears in the peak serum concentrations for capreomycin. Part 15

275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296

of this discrepancy arises because very early time data (<0.5 hours) were not taken, and thus the specific value for the peak serum concentration, and the time at which this concentration occurs, was unknown. To better understand this phenomenon, a systematic series of simulations were conducted in which different assumptions were made about the early-time capreomycin pharmacokinetics in the serum (37). These studies showed that different posited kinetics had a significant impact on the shape and magnitude of the simulated serum concentration profiles. For example, two different assumptions regarding the drug accumulation and elimination in the serum lead to the markedly different simulated profiles depicted in Figure 5. In panel Figure 5 (a) the underlying assumption is that the actual peak concentration occurs exactly at the measured peak value, whereas in Figure 5 (b) the assumption is that the peak concentration should be determined by extrapolating the measured values using a first-order model. In general, it was found that the former model gave better overall fits to the measured data across the tissues of interest (37).

For the high-dose case, simulations for concentrations in the lung and kidney generally provided an envelope that encompassed the experimental results. Results for these compartments are particularly important because they are primary organs of interest in the treatment of tuberculosis from the perspective of efficacy (lung) and toxicity (kidney). Similar to the low-dose case, the major deficiency in the model is in the prediction of the serum concentrations, especially the peak value.

16

297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317

Discussion

Although a few studies have investigated the pharmacokinetics of capreomycin in blood (3, 4, 29, 34, 43, 44), only one study to date (33) has examined tissue concentrations of capreomycin over time, and this study focused on relatively early-time data which do not capture important late clearance events, especially for the kidney. To our knowledge no PBPK models have been published for any anti-tuberculosis drugs. Moreover, even existing PBPK models for antibiotics (9, 10, 16, 32) have not included considerations of inter-individual variability, an important consideration in the interpretation of ADME predictions for these drugs. The PBPK model for capreomycin disposition in mice developed in this study begins to close this knowledge gap. It provides predictions that are generally in good agreement with results from a corresponding experimental study and, through a Bayesian model calibration, has provided distributions for several parameters related to capreomycin transport. The approach used here has important advantages relative to classical PK or population-PK approaches. Since classical models are not based upon the true anatomy, physiology, and biochemistry of the species of interest, they cannot, in general, be used to generate reliable predictions outside the range of doses, dose routes, and species used in the studies upon which they were based. Such extrapolations, which are essential in estimating the dose-response of chemicals, can be performed more accurately using PBPK modeling approaches. Despite these advantages, there are limitations to the current study. Because of the relatively small sample size, the model and results should be viewed as exploratory in nature. In addition, very early time data were not collected in this study, making characterization of the 17

318 319 320 321 322 323 324 325 326 327 328 329 330 331

peak concentration difficult and adding uncertainty to the analysis. Future studies could make use of optimal sampling theory to design appropriate studies to capture these events in an efficient manner. Current work is focused on improving the model, principally through the inclusion of additional data; the Bayesian approach used here provides for a straightforward means of incorporating these data as they become available. Also, we are assessing the feasibility of extrapolating the model to humans using appropriate physiological parameters from Brown et al. (11) and Davies and Morris (17) and calibrating and validating using the human pharmacokinetic data from Black et al. (3, 4). In addition, because of similarities between CAP and AGAs as described earlier, the model is being extended to AGAs. Longer-term aims are to use this model, along with appropriate pharmacodynamic and toxicity data, as part of a predictive framework to help optimize drug regimens to treat MDR-TB.

18

332 333 334 335 336 337 338 339 340

Acknowledgements

The authors thank Janet Gilliland for assistance in the animal studies, Dr. Ryan Hansen for conducting the analytical chemistry analyses, and Dr. Daniel Gustafson for providing resources for pharmacokinetic analyses and for helpful comments about capreomycin pharmacokinetics. The authors also thank the anonymous reviewers for their valuable comments and suggestions to improve the quality of the paper. We gratefully acknowledge funding for this project from a Capacity Building Grant awarded by the Infectious Disease Supercluster at Colorado State University.

19

341 342 343 344 345 346 347 348

Appendix

Analysis of Capreomycin IA and IB from Mouse Tissues by LC/MS/MS Instrument: Shimadzu LC-20AD High Performance Liquid Chromatograph system (Shimadzu Corporation, Kyoto, Japan). Column: Waters Atlantis HILIC Silica 5 m, 4.6 x 50 mm (part#186002028), protected by a Phenomenex C18 Guard Cartridge (and filter frits). Vials: plastic. Injection Volume: 75 l. Loop: 100 l. Flow Rate: 800 l/min. Run Time: 4 min. Column Oven: RT. LC Gradient Conditions:
Time (min)

% Organic Phase 2% ACN 90% ACN 90% ACN 2% ACN 2% ACN

% Aqueous Phase 98% 0.1% Formic Acid in H2O 10% 0.1% Formic Acid in H2O 10% 0.1% Formic Acid in H2O 98% 0.1% Formic Acid in H2O 98% 0.1% Formic Acid in H2O

0.50 2.5 3.0 3.2 4.0 349 350 351 352 353 354 355 MS Conditions

Instrument: MDS Sciex 3200 Q-TRAP triple quadrupole mass spectrometer (Applied Biosystems, Foster City, CA) with a TurboIonSpray source. MRM Positive Ion Mode. Scan/Dwell Time: 300 msec. Transitions Monitored: Capreomycin IA (669.30 507.00 amu) Capreomycin IB (653.3 491.3 amu) DP: 71.0; CE: 45; CEP: 30; CXP: 6 DP: 55.0; CE: 48; CEP: 33.8; CXP: 4

20

356 357 358 359 360 361 362 363 364 365 366 367 368

Curtain (CUR) Gas: 20. Collision (CAD) Gas: 12 (High). Ion Spray Voltage: 2500. Source Temperature: 550C. Ion Source Gas 1: 35. Ion Source Gas 2: 30. Entrance Potential (EP): 10. Ihe: on. For analysis, both transitions were integrated as one. As capreomycin IA and IB are, by far, the major constituents, only these two components were quantified. The weight of sulfate was taken into account for quantitation, and the PK results for CAP in this paper refer to capreomycin free base (the base form of the drug rather than the salt form).

Governing Equations for PBPK Model Although the model structure is applicable for both intravenous and subcutaneous dose, in this section, we focus solely on the subcutaneous dose. Consistent with the structural model (Figure 1), for all organs except the lung, blood, kidney, and liver, a capreomycin mass balance may be written as
d ( M organ ) dt C = Qorgan CA organ Porgan ,

369 370 371 372 373 374

(eqn A2)

where Morgan is the drug mass in the organ, Qorgan is blood flow to organ, CA is the drug concentration in the arterial blood flow, Corgan is the drug concentration in the organ, and Porgan is the tissue-blood partition coefficient. Organ flows are fractions of the cardiac output, QC, which is allometrically-scaled to body weight (QC BW0.75) (11, 17). The mass of drug in the lung, MLU, is governed by

21

375 376

d ( MLU ) CLU = QLU CA . dt PLU A mass balance on the venous blood, considering the drug dose, is

(eqn A3)

377 378

C d ( MSC ) d ( MV ) = organs Qorgan organ QLU CV , dt Porgan dt where MSC is the mass of drug in the subcutaneous compartment, given by

(eqn A4)

379 380 381 382 383 384

385

d ( MSC ) = SC _ Decay MSC . dt

(eqn A5)

Here SC_Decay is the rate of drug movement from the subcutaneous compartment into the venous blood. On the arterial side, the governing equation takes the following form: d ( MA) CLU = QLU CA . dt PLU may be formulated:
d ( MKE ) = CLR CVKS dt (eqn A7)

(eqn 6)

Based on the conceptual model for the kidney (Figure 2), the following series of equations

386

d ( MKA) Vmax CVKS = dt K m + CVKS d ( MKDE ) = CLRD CVKD dt d ( MKS ) d ( MKA) d ( MKE ) d ( MKDE ) = QK (CA CVKS ) + dt dt dt dt d ( MKD) d ( MKA) d ( MKDE ) = dt dt dt

(eqn A8)

387

(eqn A9)

388

(eqn A10)

389

(eqn A11)

22

390 391 392 393 394 395 396 397 398 399 400 401 402

d ( MK ) d ( MKS ) d ( MKD) = + dt dt dt

(eqn A12)

Here, MKE is the mass of drug excreted from the kidney, MKDE is the mass of drug excreted from the deep compartment and returned to blood flow, MKA is the mass of drug accumulating in the deep compartment of the kidney, MKS is the mass of drug in the shallow compartment, CLR is the rate of capreomycin clearance from kidney blood flow, CLRD is the rate of capreomycin clearance from the deep kidney compartment, CVKS is the capreomycin concentration in the well-mixed blood in the kidney that is then mixed with the venous blood, CVKD is the capreomycin concentration in the well-mixed deep kidney compartment, and Vmax and Km are Michaelis-Menten parameters for accumulation. Finally, for the liver compartment, we have d ( ML) = QLA CA + QS CVS + QG CVG QL CVL CLH CVL , dt (eqn A13)

where CVL is the concentration of drug in the liver, and CLH is the hepatic clearance rate (CLH = CLHCBW).

23

403 404

24

405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436

References

1.

Allen, B. C., C. E. Hack, and H. J. Clewell. 2007. Use of Markov chain Monte Carlo analysis with a physiologically-based pharmacokinetic model of methylmercury to estimate exposures in US women of childbearing age. Risk Analysis 27:947-959. Alonso, I. G., J. M. Lanao, M. C. Saez, A. A. Dominguez-Gil, and A. DominguezGil. 1987. Non-linear tissue binding of amikacin in rats: the effect of renal impairment. Eur J Drug Metab Pharmacokinet 12:193-201. Black, H. R., R. S. Griffith, and J. F. Brickler. 1963. Preliminary Laboratory Studies with Capreomycin. Antimicrob Agents Chemother (Bethesda) 161:522-9. Black, H. R., R. S. Griffith, and A. M. Peabody. 1966. Absorption, excretion and metabolism of capreomycin in normal and diseased states. Ann N Y Acad Sci 135:974-82. Bois, F. February 24, 2009 2009, posting date. MCSim manual, version 5.3.0. Free Software Foundation, Inc. [Online.] Bois, F. Y. 1999. Analysis of PBPK models for risk characterization. Uncertainty in the Risk Assessment of Environmental and Occupational Hazards 895:317-337. Bois, F. Y., A. Gelman, J. Jiang, D. R. Maszle, L. Zeise, and G. Alexeef. 1996. Population toxicokinetics of tetrachloroethylene. Arch Toxicol 70:347-55. Bois, F. Y., M. Jamei, and H. J. Clewell. 2010. PBPK modelling of inter-individual variability in the pharmacokinetics of environmental chemicals. Toxicology 278:256267. Brightman, F. A., D. E. Leahy, G. E. Searle, and S. Thomas. 2006. Application of a generic physiologically based pharmacokinetic model to the estimation of xenobiotic levels in rat plasma. Drug Metab Dispos 34:84-93. Brocklebank, J. R., R. Namdari, and F. C. Law. 1997. An oxytetracycline residue depletion study to assess the physiologically based pharmokinetic (PBPK) model in farmed Atlantic salmon. Can Vet J 38:645-6. Brown, R. P., M. D. Delp, S. L. Lindstedt, L. R. Rhomberg, and R. P. Beliles. 1997. Physiological parameter values for physiologically based pharmacokinetic models. Toxicol Ind Health 13:407-84. Chiu, W. A., M. S. Okino, and M. V. Evans. 2009. Characterizing uncertainty and population variability in the toxicokinetics of trichloroethylene and metabolites in

2.

3. 4.

5. 6. 7. 8.

9.

10.

11.

12.

25

437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 13. 14.

mice, rats, and humans using an updated database, physiologically based pharmacokinetic (PBPK) model, and Bayesian approach. Toxicology and Applied Pharmacology 241:36-60.
Christensen, E. I., and H. Birn. 2002. Megalin and cubilin: multifunctional endocytic receptors. Nat Rev Mol Cell Biol 3:256-66. Clewell, R. A., and H. J. Clewell, 3rd. 2008. Development and specification of physiologically based pharmacokinetic models for use in risk assessment. Regul Toxicol Pharmacol 50:129-43. Craig, W. A., J. Redington, and S. C. Ebert. 1991. Pharmacodynamics of amikacin in vitro and in mouse thigh and lung infections. J Antimicrob Chemother 27 Suppl C:29-40. Craigmill, A. L. 2003. A physiologically based pharmacokinetic model for oxytetracycline residues in sheep. J Vet Pharmacol Ther 26:55-63. Davies, B., and T. Morris. 1993. Physiological parameters in laboratory animals and humans. Pharm Res 10:1093-5. DeWoskin, R. S., and C. M. Thompson. 2008. Renal clearance parameters for PBPK model analysis of early lifestage differences in the disposition of environmental toxicants. Regul Toxicol Pharmacol 51:66-86. Fiegel, J., L. Garcia-Contreras, M. Thomas, J. VerBerkmoes, K. Elbert, A. Hickey, and D. Edwards. 2008. Preparation and in vivo evaluation of a dry powder for inhalation of capreomycin. Pharm Res 25:805-11. Fu, L. M., and T. M. Shinnick. 2007. Genome-wide exploration of the drug action of capreomycin on Mycobacterium tuberculosis using Affymetrix oligonucleotide GeneChips. J Infect 54:277-84. Giuliano, R. A., G. A. Verpooten, L. Verbist, R. P. Wedeen, and M. E. De Broe. 1986. In vivo uptake kinetics of aminoglycosides in the kidney cortex of rats. J Pharmacol Exp Ther 236:470-5. Global Alliance for TB Drug Development 2008. Capreomycin. Tuberculosis 88:89-91. Goutelle, S., L. Bourguignon, R. W. Jelliffe, J. E. Conte, Jr., and P. Maire. Mathematical modeling of pulmonary tuberculosis therapy: Insights from a prototype model with rifampin. J Theor Biol 282:80-92.

15.

16. 17. 18.

19.

20.

21.

22. 23.

26

469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502

24.

Hall, R. G., R. D. Leff, and T. Gumbo. 2009. Treatment of active pulmonary tuberculosis in adults: current standards and recent advances. Insights from the Society of Infectious Diseases Pharmacists. Pharmacotherapy 29:1468-81. Heifets, L., J. Simon, and V. Pham. 2005. Capreomycin is active against nonreplicating M. tuberculosis. Ann Clin Microbiol Antimicrob 4:6. Holdiness, M. R. 1984. Clinical pharmacokinetics of the antituberculosis drugs. Clin Pharmacokinet 9:511-44. Honeybourne, D. 1994. Antibiotic Penetration into Lung Tissues. Thorax 49:104106. Jones, H. M., N. Parrott, K. Jorga, and T. Lave. 2006. A novel strategy for physiologically based predictions of human pharmacokinetics. Clin Pharmacokinet 45:511-42. Kanazawa, Y., and T. Kuramata. 1964. [Measurement of Capreomycin in Body Fluids]. J Antibiot B 17:190-2. Kirman, C. R., L. M. Sweeney, M. E. Meek, and M. L. Gargas. 2003. Assessing the dose-dependency of allometric scaling performance using physiologically based pharmacokinetic modeling. Regul Toxicol Pharmacol 38:345-67. Knaak, J. B., M. A. al-Bayati, and O. G. Raabe. 1995. Development of partition coefficients, Vmax and Km values, and allometric relationships. Toxicol Lett 79:8798. Laplanche, R., G. M. Meno-Tetang, and R. Kawai. 2007. Physiologically based pharmacokinetic (PBPK) modeling of everolimus (RAD001) in rats involving nonlinear tissue uptake. J Pharmacokinet Pharmacodyn 34:373-400. Le Conte, P., F. Le Gallou, G. Potel, L. Struillou, D. Baron, and H. B. Drugeon. 1994. Pharmacokinetics, toxicity, and efficacy of liposomal capreomycin in disseminated Mycobacterium avium beige mouse model. Antimicrob Agents Chemother 38:2695-701. Lee, S. H., J. Shin, J. M. Choi, E. Y. Lee, D. H. Kim, J. W. Suh, and J. H. Chang. 2003. The impurities of capreomycin make a difference in the safety and pharmacokinetic profiles. Int J Antimicrob Agents 22:81-3. Leggett, J. E., B. Fantin, S. Ebert, K. Totsuka, B. Vogelman, W. Calame, H. Mattie, and W. A. Craig. 1989. Comparative antibiotic dose-effect relations at several dosing intervals in murine pneumonitis and thigh-infection models. J Infect Dis 159:281-92.

25. 26. 27. 28.

29. 30.

31.

32.

33.

34.

35.

27

503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536

36.

Lyons, M. A., R. S. H. Yang, A. N. Mayeno, and B. Reisfeld. 2008. Computational toxicology of chloroform: Reverse dosimetry using Bayesian inference, Markov chain Monte Carlo simulation, and human biomonitoring data. Environmental Health Perspectives 116:1040-1046. Metzler, C. 2010. Masters Thesis: Physiologically based pharmacokinetic modeling for prediction of pharmacokinetic parameters of capreomycin Colorado State University Fort Collins Mingeot-Leclercq, M. P., and P. M. Tulkens. 1999. Aminoglycosides: nephrotoxicity. Antimicrob Agents Chemother 43:1003-12. Mork, A. K., and G. Johanson. 2010. Chemical-Specific Adjustment Factors for Intraspecies Variability of Acetone Toxicokinetics Using a Probabilistic Approach. Toxicological Sciences 116:336-348. Mouton, J. W., U. Theuretzbacher, W. A. Craig, P. M. Tulkens, H. Derendorf, and O. Cars. 2008. Tissue concentrations: do we ever learn? J Antimicrob Chemother 61:235-7. Nagai, J., and M. Takano. 2004. Molecular aspects of renal handling of aminoglycosides and strategies for preventing the nephrotoxicity. Drug Metab Pharmacokinet 19:159-70. NIAID Tuberculosis Working Group. 2007. NIAID Research Agenda MultidrugResistant and Extensively Drug Resistant Tuberculosis. Organick, A. B., and E. M. Wilson. 1968. Multiple drugs in retreatment of chronic pulmonary tuberculosis. Results with capreomycin and ethambutol. Dis Chest 53:56070. Peloquin, C. A. 1993. Pharmacology of the antimycobacterial drugs. Med Clin North Am 77:1253-62. Peters, S. A. 2012. Physiologically based pharmacokinetic (PBPK) modeling and simulations : principles, methods, and applications in the pharmaceutical industry, Wiley, Hoboken, N.J. Reddy, M. B. 2005. Physiologically based pharmacokinetic modeling: science and applications, Wiley-Interscience, Hoboken, N.J. Reisfeld, B., A. N. Mayeno, M. A. Lyons, and R. S. H. Yang. 2007. Physiologicallybased pharmacokinetic and pharmacodynamic modeling p. xxii, 814 p. In S. Ekins (ed.), Computational toxicology : risk assessment for pharmaceutical and environmental chemicals. Wiley-Interscience, Hoboken, N.J.

37.

38. 39.

40.

41.

42. 43.

44. 45.

46. 47.

28

537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553

48.

Schmitz, C., J. Hilpert, C. Jacobsen, C. Boensch, E. I. Christensen, F. C. Luft, and T. E. Willnow. 2002. Megalin deficiency offers protection from renal aminoglycoside accumulation. J Biol Chem 277:618-22. Ward, D. T., S. J. McLarnon, and D. Riccardi. 2002. Aminoglycosides increase intracellular calcium levels and ERK activity in proximal tubular OK cells expressing the extracellular calcium-sensing receptor. J Am Soc Nephrol 13:1481-9. World Health Organization. 2010 Multidrug and extensively drug-resistant TB (M/XDR-TB): 2010 global report on surveillance and response. WHO/HTM/TB/2010.3. World Health Organization., and Stop TB Initiative (World Health Organization). 2010. Treatment of Tuberculosis: guidelines for national programmes, 4th ed, World Health Organization, Geneva. Zietse, R., R. Zoutendijk, and E. J. Hoorn. 2009. Fluid, electrolyte and acidbase disorders associated with antibiotic therapy. Nature Reviews Nephrology 5:193-202.

49.

50.

51.

52.

29

554 555 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 571 572 573 574 Figure 1. PBPK model structure

Figure Captions

Figure 2. Conceptual model of the kidney Figure 3. Low dose and high dose pharmacokinetic data in various tissues (N=4): (a) serum, (b) kidney, (c) lung, (d) liver, and (e) spleen. Mean SD are shown. Scales for the abscissa and ordinate differ for each plot. Data points below the LLOQ are not shown. LLOQ (ng/mg): kidney (50.0), serum (1.0), liver (10.0), lung (10.0), spleen (10.0). Figure 4. Parameter distributions found in the calibration simulations for (a) Km, (b) Vmax, (c) CLRD, (d) CLR, (e) CLHC, (f) SC_Decay. Figure 5. Measured and predicted serum concentration for the low-dose group based on (a) using the measured peak concentration as the actual peak value, and (b) extrapolating to determine the peak concentration Figure 6. Low dose pharmacokinetic data and model predictions in various tissues: (a) serum, (b) kidney, (c) lung, (d) liver, and (e) spleen. The experimental data are shown with symbols, and the fifth and ninety-fifth percentile curves for the simulation data are shown with dashed lines. Data points below the LLOQ are not shown. Figure 7. High dose pharmacokinetic data and model predictions in various tissues: (a) serum, (b) kidney, (c) lung, (d) liver, and (e) spleen. The experimental data are shown with symbols, and the fifth and ninety-fifth percentile curves for the simulation results are shown with dashed lines. Data points below the LLOQ are not shown.

30

575 576

Tables

31

577 578
Symbol KM Vmax CLR CLRD CLHC SC_Decay

Table 1. Parameters to be determined through model calibration

Units ng/kg ng/h l/h l/h l h-1 kg-1 h-1 Description (relevant compartment) Michaelis constant in accumulation kinetics (kidney) Michaelis-Menten maximum accumulation rate (kidney) Overall renal clearance (kidney) Deep renal compartment clearance (kidney) Hepatic clearance rate (liver) Subcutaneous dose decay rate (subcutaneous); release into venous blood.

579

32

580 581
Table 2. Summary statistics for the parameters estimated in the calibration simulations
Parameter Km (ng/kg) Vmax (ng/h) CLR (kg/h) CLRD (kg/h) CLHC (l hr-1 kg-1) SC_Decay (h-1) Mean SD 5.55 x 108 0.46 x 108 1.04 x 106 0.08 x 106 0.012 0.001 3.47 x 10-6 0.25 x 10-6 3.97 0.27 0.902 0.164

582

33

583
BW

Table A3. Parameters for use in PBPK modeling

from individual mouse data partition coefficients BP (blood:plasma) PLU (lung:blood) PBR (brain:blood) PF (fat:blood) PH (heart:blood) PM (muscle:blood) PB (bone:blood) Fractional tissue weights (17) VLUC (lung) VBRC (brain) VFC (fat) VHC (heart) VMC (muscle) VBC (bone) VSKC (skin) VKC (kidney) VKSC (shallow kidney) VKDC (deep kidney) VSpC (spleen) VGC (GI) VLC (liver) VVC (venous blood) VAC (arterial blood) VRCR (carcass) 0.0044 (exp) 0.018 0.070 0.004 0.384 0.107 0.165 0.014 (exp) 0.010 0.004 0.0037 (exp) 0.042 0.05 (exp) 0.0327 0.0163 1 - sum of all others 1 2 1 1 1 1 1 Fractional tissue flows (fraction of cardiac output) (17) QLUC (lung) QBRC (brain) QFC (fat) QHC (heart) QMC (muscle) QBC (bone) QSKC (skin) QKSC (shallow kidney) QSC (spleen) QGC (GI tract) QLAC (hepatic artery) QCRC carcass 1.0 0.033 0.043 0.066 0.159 0.110 0.058 0.091 0.01 0.13 0.02 0.28 PSK (skin:blood) PKS (shallow kidney:blood) PS (spleen:blood) PG (gut:blood) PL (liver:blood) PCR (carcass:blood) 1 1 1 1 1 1

584

34