Pharmacokinetics of Inter-Alpha Inhibitor Proteins and Effects on Hemostasis After Hypoxic-Ischemic Brain Injury in Neonatal Rats

Abstract

Background

Hypoxic-ischemic (HI) brain injury is a leading cause of long-term neurodevelopmental morbidities in neonates. Human plasma-derived Inter-Alpha Inhibitor Proteins (hIAIPs) are neuroprotective after HI brain injury in neonatal rats. The light chain (bikunin) of hIAIPs inhibits proteases involved in the coagulation of blood. Newborns exposed to HI can be at risk for significant bleeding in the brain and other organs.

Objective

The objectives of the present study were to assess the pharmacokinetics (PK) and the duration of bleeding after intraperitoneal (IP) administration of hIAIPs in HI-exposed male and female neonatal rats.

Methods

HI was induced with the Rice-Vannucci method in postnatal (P) day-7 rats. After the right common carotid artery ligation, rats were exposed to 90 min of 8% oxygen. hIAIPs (30 mg/kg, IP) were given immediately after Sham or HI exposure in the PK study and serum was collected 1, 6, 12, 24, or 36 h after the injections. Serum hIAIP concentrations were measured with a competitive ELISA. ADAPT5 software was used to fit the pooled PK data considering first-order absorption and disposition. hIAIPs (60 mg/kg, IP) were given in the bleeding time studies at 0, 24 and 48 h after HI with tail bleeding times measured 72 h after HI.

Results

IP administration yielded significant systemic exposure to hIAIPs with PK being affected markedly including primarily faster absorption and reduced elimination as a result of HI and modestly because of sex-related differences. Besides, hIAIP administration did not affect bleeding times after HI.

Conclusion

These results will help to inform hIAIP dosing regimen schedules in studies of neuroprotection in neonates exposed to HI.

1. INTRODUCTION

Neonatal hypoxic-ischemic (HI) brain injury is a major cause of infant morbidity, mortality and long-term neurological impairment [1, 2]. Hypothermia is the only approved therapy to treat full-term infants exposed to HI encephalopathy. However, this therapy is only partially effective [3–5] and therapeutic strategies are not available to treat premature infants exposed to HI. Therefore, studies investigating potential adjunctive or novel neuroprotective agents for newborns exposed to HI are critically needed.

Elevations in inflammatory mediators in the systemic circulation and the brain are triggered after HI [6–8]. Therefore, anti-inflammatory agents could represent novel treatments for HI-related brain injury. Furthermore, clinical and experimental findings suggest that there are sex-related differences in response to HI. Male neonates exhibit higher incidences of injury and more long-term cognitive deficits than females exposed to similar degrees of HI-related brain injury [9–11]. Therefore, potential anti-inflammatory agents could require therapeutic tailoring based upon sex-related differences to maximize their effects, particularly because sex-related differences have been reported for other pharmacological agents [12].

Inter-Alpha Inhibitor Proteins (IAIPs) are novel immunomodulatory proteins that inhibit pro-inflammatory cytokines and have received increasing attention with respect to their contribution to many disease states including brain ischemia [13–16]. Studies suggest that a fragment of IAIPs, the light chain of IAIPs termed as bikunin, has important neuroprotective properties [13, 17, 18]. Moreover, we have recently shown that treatment with the naturally-occurring human plasma-derived form of IAIPs (hIAIPs) reduces neuronal cell death, and improves neuronal plasticity and behavioural outcomes in male neonatal rats exposed to HI [14, 19–21]. Male rats were examined in our initial studies because of their predisposition to develop HI-related brain injury and exhibit adverse neurodevelopmental outcomes [11, 14, 19, 20]. Although some aspects of the neuroprotective effects associated with hIAIPs appear to be more efficacious in male than female neonatal rats [21], we have recently shown that hIAIPs exert neuroprotective effects in both male and female neonatal rats [22]. Therefore, it is important to study the potential neuroprotective effects of hIAIPs in both male and female neonatal rats after HI and, consequently, also to determine pharmacokinetic (PK) profiles of hIAIPs after exposure to HI for both sexes.

In addition to their immunomodulatory roles, IAIPs are also serine protease inhibitors mainly found as two major forms in human plasma: Inter-alpha Inhibitor (IaI, 250 kDa) and Pre-alpha Inhibitor (PaI, 125 kDa). IAIPs are composed of light and heavy polypeptide chains [23]. The light chain, bikunin, is responsible for the serine protease inhibitory activity [24], whereas the heavy chains have an important role in stabilizing extracellular matrices of tissues and synergizing with the activity of bikunin [25, 26]. Bikunin also inhibits serine proteases of the blood coagulation system including plasmin, Factor Xa, and kallikrein [27, 28]. The proteolytic activities of serine proteases regulate the balance between clot formation and dissolution. Hence, it is also important to establish safety with regard to the effects of IAIPs on hemostasis before considering IAIPs as therapeutic agents for human neonates. The time to cessation of bleeding and spontaneous clot formation can be used as an initial approach to examine drug effects on blood coagulation.

In the current study, we utilized the well-characterized Rice-Vannucci model of neonatal HI [29] to examine effects on the PK properties of hIAIPs in order to obtain relevant PK parameters through compartmental modeling in male and female neonatal rats. Moreover, we examined the effects of a higher dose of hIAIPs in the bleeding time studies to validate that hIAIPs did not predispose to coagulation abnormalities even when given at a very high dose. Even though the current study focuses on the PK properties of a specific immunomodulatory protein, IAIPs [16, 27, 30], the study potentially has broader implications for the PK properties of other proteins that are currently administered to newborn subjects such as fresh frozen plasma, coagulation factors and antibodies.

2. MATERIALS AND METHODS

2.1. Human Inter-alpha Inhibitor Proteins (hIAIPs)

The hIAIPs were extracted from fresh frozen human plasma (Rhode Island Blood Center, RI, USA). A scalable purification process to extract hIAIPs using a monolithic anion-exchange chromatographic media (CIMmultus™, BIA Separation, Ajdovščina, Slovenia) was developed [16, 31, 32] and an additional separation step using a proprietary synthetic chemical ligand affinity chromatographic media (Prometic Bioseparations, Cambridge, UK) was applied to obtain high yield, high purity (>90%), and biologically active hIAIPs. A 50–65% yield from 1 L of plasma produces ca. 100 mg of highly pure hIAIPs. Eluted proteins were concentrated and buffer-exchanged using a tangential flow filtration device (Labscale, Millipore, Taunton, MA, USA). Analyses of the purity and biological activity of the hIAIPs were performed by using SDS-PAGE, Western immunoblot, protein assay, and competitive immunoassay (ELISA) [16, 31, 32]. The biological activity was measured based on the ability of hIAIPs to inhibit the hydrolysis of the substrate N-Benzoyl-L-arginine-p-nitroaniline HCl (L-BAPNA, Sigma St. Louis, MO) by trypsin [14, 33]. As part of the characterization of hIAIPs, endotoxin in the purified products was monitored using a limulus amebocyte lysate (LAL) endotoxin-based chromogenic test (Pierce Biotechnology, ThermoFisher Scientific, Waltham, MA, USA). The chromatographic equipment and containers/tubing were treated with 1 M NaOH to reduce or eliminate potential endotoxin contamination during the purification process. Purified hIAIPs were frozen until use.

2.2. Experimental Animals

The present study was performed after approval from the Institutional Animal Care and Use Committees of the Alpert Medical School of Brown University and Women & Infants Hospital of Rhode Island and is in accordance with the National Institutes of Health Guidelines for the Use of Experimental Animals. Wild type Wistar rats were obtained from Charles River Laboratories (Wilmington, MA, USA) on embryonic day 15 or 16 of pregnancy. The rats were housed in a temperature-controlled, 12-h light/dark-cycled facility with ad libitum access to food and water.

2.3. Hypoxic-Ischemic Procedure

Postnatal day (P) 7 Wistar rats were exposed to HI using the Rice-Vannucci method as previously described [29, 34]. On P1, litters were randomly culled to ten and balanced such that there were approximately equal numbers of males and females to reduce inter-litter variability. The P7 pups were anesthetized with isoflurane (induction: 4%; maintenance: 2%) in oxygen and underwent right common carotid artery ligation, whereas the Sham animals received a neck incision only [29, 35]. After recovery from surgery, the pups were returned to their dams for 1.5–3 h. Subjects exposed to HI were placed in a temperature-controlled oxygen/hypoxia (8% oxygen + 92% nitrogen) airtight chamber (Biospherix, Parish, NY, USA) for 90 min. One non-ligated sentinel pup per litter had a rectal temperature probe placed that had an accuracy of 0.1°C (RET-4, Physitemp, Clifton, NJ, USA) to monitor body temperature before and during HI. The sentinel pup was not included in further investigations because it has been shown that the stress of the rectal probe placement alters the outcome of the HI studies [36, 37]. Moreover, the rectal temperature has been shown to accurately reflect brain temperature in the HI rodent model [38, 39]. The rectal temperature was recorded every 10 min during HI (Fig. 1) and maintained close to 36.0°C [37, 40]. This HI model has been shown to result in approximately 60% unilateral brain injury in our laboratory [22] similar to previous reports [41, 42]. The Sham subjects were placed in a similar container but exposed to room air.

Fig. (1).

Temperature monitoring before and after carotid ligation and exposure to hypoxia. One sentinel pup per litter (n=15) was used to assess rectal temperatures before and during HI at P7. The star indicates the pups with the rectal temperature at 34.5 ± 1.1 °C (Mean ± SD) before carotid ligation and exposure to hypoxia. During the hypoxic procedure (90 min), the rectal temperature was recorded every 10 min (Mean ± SD) for each time point recorded. The open symbols are the pups before exposure to carotid ligation and hypoxia. The gray symbols indicate measurements after carotid ligation and during hypoxia.

2.4. Animal and Sample Preparation for Pharmacokinetic (PK) Studies of hIAIPs

The P7 Wistar rats were randomly assigned to one of the two groups: non-ischemic sham (Sham-hIAIP; male, n=22; female, n=22), or HI (HI-hIAIP; male, n=29; female, n=29) groups. hIAIPs (30 mg/kg) were injected intraperitoneally (IP) immediately after Sham/HI treatment (Fig. 2A). IP injections were employed because intravenous injections are not readily feasible in neonatal rodents and IP injections are widely used in studies of neonatal rodents [43, 44]. The dose of 30 mg/kg of hIAIPs was selected for PK study based upon our previous studies showing significant neuroprotective efficacy in neonatal rats exposed to HI brain injury [14, 21, 22]. Four animals (male, n=2; female, n=2) without hIAIP injections were used to determine the control, non-hIAIP treated reference values for the ELISA assay for each group. The pups were weighed and sacrificed 1, 6, 12, 24, or 36 h after injection. We obtained trunk blood samples for the PK analysis of hIAIPs. Trunk blood samples (~0.4 ml per time point) were collected individually (Fig. 2A) and placed in a 2-ml polypropylene screw capped microcentrifuge tube (USA scientific, Orlando, FL, USA) and immediately centrifuged at 1200 x g at room temperature for 5 min. After removal of the fibrin clot, serum was collected by centrifugation again at 1200 x g for 5 min, and then stored at −80°C until analysis [45, 46].

Fig. (2).

(A) Study design for the pharmacokinetic study. The pups were returned to the dams for 1.5 to 3 h after the right common carotid artery ligation. Thereafter, the pups were exposed to 8% oxygen with balanced nitrogen for 90 min at a constant temperature of 36°C. Thirty mg/kg of hIAIPs were given intraperitoneally (IP) immediately (0 h, open arrow) after the termination of hypoxia. Body weight was recorded (solid circles) and the trunk blood was collected (inverted solid triangles) individually before sacrifice and at 1, 6, 12, 24, or 36 h after hIAIP injection. Serum hIAIP concentrations were measured with a specific competitive ELISA. (B) Study design for tail bleeding tests. The HI procedures were the same as for the study design shown in A except that IP hIAIPs (60 mg/kg, gray arrows) or placebo (1x PBS) were given immediately, 24 and 48 h after the termination of hypoxia. Body weights were measured before the HI (initial at −1.5 h), 0, 24, 48 and 72 h after HI injury. At 72 h after HI, the tail bleeding tests were performed for 10 min. Body weights including tail weights were measured before and after the bleeding test to estimate blood volume losses.

2.5. Competitive hIAIP ELISA

The ELISA analysis is optimal for biologics such as hIAIPs as it is based on a specific antibody reagent. A standardized quantitative competitive ELISA was developed and established for hIAIPs using a specific monoclonal antibody raised against hIAIPs (MAb 69.26) that has been developed by ProThera Biologics (Providence, RI, USA) and previously described [47]. MAb 69.26 is only reactive with hIAIPs and does not cross-react with IAIPs from other species. We used this MAb in the ELISA to specifically measure the administered hIAIPs in rat serum. Unfortunately, there is no specific antibody reagent that can only detect rat endogenous IAIP.

The ELISA analysis was performed without knowledge of the group assignment. Briefly, purified IgG of MAb 69.26 (200 ng per well) was coated on a 96-well high-binding microplate (Microlon 600, Greiner Bio-One, Monroe, NC, USA) for 1 h at room temperature. After blocking with 200 μL of 5% non-fat dried milk in phosphate-buffered saline (PBS) plus 0.05% Tween (PBS-T), 50 μL of rat serum samples or serially diluted hIAIP standards were added to the wells. Then, 50 μL of biotin-conjugated purified hIAIP (0.5 mg/mL) was added to each well. MAb 69.26 detects the light chain that contains both forms of hIAIPs (125 kDa PaI and 250 kDa IaI) by Western immunoblot analysis. The plates were incubated for 1 h at room temperature and washed with PBS-T three times. Pierce streptavidin-poly horseradish-peroxidase (Thermo Scientific, Wilmington, DE, USA) was added to each well and incubated for 1 h at room temperature to detect the bound biotinylated hIAIPs. After 5 washes, the plate was developed with 100 μL of Enhanced K-Blue TMB substrate (Neogen Corp, Lexington, KY, USA) in the dark for 15–20 min. Once the maximum color intensity was attained, the reaction was stopped with 100 μL 1N HCl solution. The absorbance at 450 nm was measured on a SpectraMAX Plus microplate reader (Molecular Devices, Sunnyvale, CA, USA). The serum hIAIP concentrations were calculated by comparing the absorbance signal to the established standard curve. Each sample was measured in triplicate and the assays were repeated at least twice on all samples. The final value was the average of the triplicate values on at least two separate assays.

2.6. PK Profiling of hIAIPs with Intraperitoneal Injections

A compartment model was used to describe hIAIP PK in both Sham- and HI-treated rats (Fig. 3). After the IP dosing of hIAIPs, absorption and mono-exponential disposition phases were observed [43]. One-compartment PK models with first-order and zero-order absorption were tested to fit the IP data. The final model selected is a one-compartment model with first-order absorption (Fig. 3) as described by:

$$\boxed{\frac{\text{dAa}}{\text{dt}}=-\text{ka}\times \text{Aa}\text{Aa}(0)=\text{Dose}\times \text{F}}$$ (1)

$$\boxed{\frac{\text{dCp}}{\text{dt}}=\text{ka}\times \frac{\text{Aa}}{\text{V}}-\frac{\text{CL}}{\text{V}}\times \text{Cp},\text{Cp}(0)=\text{Background}\text{value}(\text{ug}/\text{mL})}$$ (2)

where Aa is the amount of hIAIPs at the injection site (site of absorption) (ug/kg); F indicates the bioavailability; Cp is the serum concentration of hIAIPs (ug/ml); ka is the 1^st-order absorption rate constant (1/h); V is the volume of distribution (mL/kg) and CL is the clearance from serum (mL/kg/h). Model fittings were performed with the implementation of maximum likelihood estimation in the ADAPT5 program (Biomedical Simulations Resource, University of Southern California, CA). Naive-pooled PK data for each group (Male Sham-hIAIP, Female Sham-hIAIP, Male HI-hIAIP, Female HI-hIAIP) were separately fitted for PK parameter estimation. Due to a lack of bioavailability (F) information, apparent clearance (CL/F) and apparent volume of distribution (V/F) were obtained. Due to the rapid absorption of hIAIP in the HI groups, the absorption behavior was missed, which led to difficulty in the estimation of ka. Thus, ka was fixed to 12 arbitrarily to capture the profiles. The variance model used is:

$$\boxed{V_i={({\mathrm{\sigma }}_1+{\mathrm{\sigma }}_2\cdot {\mathrm{Y}}_i)}^2}$$ (3)

where V_i represents the variance of the i^th data point, σ1 and σ2 are variance model parameters, and Y_i is the i^th model prediction. The final model was selected based on the success of minimization, modeling fittings, and reasonability and precision of parameter estimation.

Fig. (3).

Schematic of hIAIP PK model. ka, the absorption rate from the IP injection site; V, volume of distribution; Aa, amount at the site of injection; Cp, serum IAIP concentration; CL, clearance from serum.

2.7. Animal and Sample Preparation for Tail Bleeding Tests

The P7 rats were randomly assigned to one of four separate groups: sham-treated with placebo (PBS X 1, Sham-PL) or hIAIPs (Sham-hIAIP or HI treated with placebo (HI-PL) or hIAIPs (HI-hIAIP). The groups of neonatal rats used in the tail bleeding time studies were distinct from those in which the PK studies were performed. hIAIPs (60 mg/kg) or PL was given IP at 0, 24 and 48 h after sham/HI treatment (Fig. 2B). In contrast to the PK experiments, we used a dosing regimen with a higher dose of hIAIPs (60 mg/kg) given at three-time points to assure that a high dose of hIAIPs would not affect the bleeding times. The higher dose (60 mg/kg) was used so that it could potentially be used to treat more severe HI brain injury in future studies. Furthermore, the bleeding time studies were performed to ensure that the low dose of hIAIPs (30 mg/kg), which has been shown to have neuroprotective effects on neonatal HI brain injury in our previous studies [22], probably does not have side effects on coagulation pathway. Rat sex and body weight were recorded.

The tail bleeding time was measured based on previously published methods [48, 49]. Briefly, rat pups were anesthetized under 2% isoflurane at 72 h after HI and placed in the prone position. After the distal ~0.2 cm segment of the tail was removed, the tail was immediately immersed in a 50-mL Falcon tube containing prewarmed isotonic saline (0.9% NaCl, 37°C). The duration of bleeding was quantified in minutes without knowledge of the group assignment. Complete cessation of bleeding for >2 min was defined as the bleeding time. A bleeding time of >10 min was terminated by ligation of the tail [48]. The volume of blood loss was estimated from the reduction in body weight during the tail bleeding period [49]. Body weight including the tail tip was recorded immediately after the bleeding test. During the 10-min experimental period, no obvious excretion of urine or feces was observed. This method has been demonstrated to correlate significantly with the chemical assay of hemoglobin concentrations to estimate the volume of blood loss [49].

2.8. Statistics

Serum hIAIP concentrations, pup body weights, and bleeding times were expressed as mean ± standard deviation (SD). The ELISA and the bleeding time results for the male plus female group and male group were normally distributed based on the Shapiro Wilk W normality test and thus were analyzed with one-way analysis of variance (ANOVA). If significant differences were detected in the ELISA and bleeding results, multiple comparisons were made using Dunnett’s test (one-sided) for the ELISA results, or Tukey’s Honest Significant Difference (HSD) for the bleeding time results. Levene’s test was used for analyzing the homogeneity of variances for the ELISA results between male and female groups. The body weight changes for individual rat pups in the PK study were also normally distributed and analyzed with factorial ANOVA to analyze the interactive effects of multiple categorical independent variables (sex difference, sham- and HI-treatment, and time periods of HI-treatment). Body weight changes among study groups in the bleeding test study were compared across time points with two-factor ANOVA for repeated measures. Tukey’s HSD was used as a post-hoc test if a significant difference was detected. The bleeding data in the female group and the body weight changes during the bleeding test were not normally distributed. Therefore, these results were analyzed for differences between multiple independent groups using the Kruskal-Wallis ANOVA and median tests. Specific differences between treatment groups were determined with a mean rank multiple comparisons test, if a significant difference was detected with Kruskal-Wallis ANOVA. Correlations between body weight changes and serum hIAIP concentrations in the PK study, and between the bleeding time and estimated blood volume loss were determined with the Pearson product-moment correlation coefficient (r). Missing values with case-wise deletions were excluded. All statistical analyses were done using the STATISTICA package (TIBCO Software Inc., Palo Alto, CA, USA), and a P<0.05 was considered to indicate statistical significance.

3. RESULTS

3.1. Serum Concentration-time Curves of hIAIPs After 30 mg/kg IP Administration in Male and Female Neonatal Rats with or without Exposure to HI Brain Injury

Fig. (4) contains the time-course changes in serum hIAIP concentrations over 36 h after IP administration of hIAIPs (30 mg/kg, open arrows). The serum concentrations of hIAIPs significantly increased 1 h after the hIAIP injection (ANOVA, P = 0.001) in the Sham male pups (Fig. 4, left upper panel) compared to the reference values, (solid circle at time zero) and reached its maximum concentration at 6 h (Cmax = 313.2 ± 25.6 μg/mL, ANOVA, P < 0.001) before returning to reference values by 36 h after injection (ANOVA, P = 0.31). Similarly, serum hIAIP concentrations in Sham female pups increased 1 h after hIAIP administration, and reached maximum concentrations 6 h (Cmax = 326.4 ± 101.0 μg/mL, ANOVA, P < 0.001) after injection (Fig. 4, left lower panel).

Fig. (4).

hIAIP serum concentrations in postnatal rats with and without exposure to HI. Serum hIAIP concentration (mg/L) versus time (1, 6, 12, 24, 36 h after hIAIP IP injections) for the males and females in the Sham-hIAIP and HI-hIAIP groups after IP hIAIP doses (30 mg/kg). Arrows indicate the time of hIAIP injection. Data are presented as Mean ± SD, one-way ANOVA with Dunnett (one sided) post hoc analysis, *p < 0.05 vs. the reference values (male: closed circle, n = 2; female: closed diamond, n = 2). Sham-hIAIP: Male n = 22: 1 h after hIAIP IP n = 4, 6 h after hIAIP IP n = 5, 12 h after hIAIP IP n = 5, 24 h after hIAIP IP n = 5, 36 h after hIAIP IP n = 3; Female n = 22: 1 h after hIAIP IP n = 4, 6 h after hIAIP IP n = 8, 12 h after hIAIP IP n = 4, 24 h after hIAIP IP n = 3, 36 h after hIAIP IP n = 3, respectively; HI-hIAIP: Male n = 29: 1 h after hIAIP IP n = 8, 6 h after hIAIP IP n = 5, 12 h after hIAIP IP n = 6, 24 h after hIAIP IP n = 4, 36 h after hIAIP IP n = 6, Female n = 29: 1 h after hIAIP IP n = 8, 6 h after hIAIP IP n = 5, 12 h after hIAIP IP n = 6, 24 h after hIAIP IP n = 6, 36 h after hIAIP IP n = 4. Variability in liter numbers in each group due to liter size.

In contrast, the highest serum hIAIP concentrations were already observed by 1 h after injection compared with the other sampling times (Fig. 4, right upper panel, Male: Cmax = 255.4 ± 122.4 μg/mL, ANOVA; P = 0.006; Fig. 4, right lower panel: Female: Cmax = 445.7 ± 274.6 μg/mL, ANOVA, P = 0.005) in the HI males and females. The concentrations of hIAIPs decreased to reference values in 24 h after injection (ANOVA, Male: P = 0.36; Female: P = 0.56). Furthermore, the serum hIAIP concentrations in HI female pups (Fig. 4, lower panels) exhibited larger standard deviations compared with the HI male pups (Fig. 4, upper panel, Levene’s test, P = 0.017) particularly in the HI females 1 h after injection (Fig. 4, right lower panel, Levene’s test, P = 0.008).

3.2. PK Profiles of IP-Administered hIAIPs in Neonatal Rats

The model fittings for the Sham and HI groups are shown in Fig. (5) for both sexes. The goodness of fitting indicates that our current model captures the currently available data reasonably well. The PK parameters (ka, AUC, V/F, CL/F, T-half) for IP administration of hIAIP for the four groups are summarized in Table 1. The 1st-order absorption rate constant (ka, 1/h) in Sham groups was slightly smaller in females than in males (0.21 versus 0.50 1/h). However, the HI groups unexpectedly exhibited much faster absorption rates, which resulted in incomplete capture of the absorption phase based on the sampling schedule. Therefore, the ka was fixed to a reasonably large value. The area under the curve to infinity (AUC_inf, μg·h/ml) was greater in the HI than in the Sham groups in both males (9260 versus 6290 μg·h/ml) and females (9610 versus 7620 μg·h/ml). In addition, the females have greater hIAIP exposure (AUC_inf) than the males in both the Sham and HI groups. The Sham groups generally had smaller volumes of distribution (V/F, mL/kg) than the HI groups for each sex (91.0 versus 122.2 mL/kg in males, and 58.1 versus 77.2 mL/kg in females). The males had greater V/F than the females in both Sham and HI groups. Males had a similar hIAIP clearances [CL/F, mL/(kg·h)] to the females in both the Sham and HI groups. However, the HI pups, in general, had lower CL/F values compared with the Sham pups in the males [3.66 versus 5.08 mL/(kg·h)] and in the females [3.30 versus 4.21 mL/(kg·h)]. The half-life (T-half, h) of hIAIPs was much longer in the HI than in the Sham groups. Females in both the Sham and HI groups exhibited shorter hIAIP half-lives than males.

Fig. (5).

PK model fitting of hIAIP serum profiles in male (upper panel) and female (lower panel) Sham and HI rats that received 30 mg/kg doses. Solid lines: model predictions; dots: observed data.

Table 1.

PK parameter estimates of hIAIP after IP injection in neonatal rats.

Parameters	Sham (Male)			Sham (Female)			HI (Male)			HI (Female)
	Mean	95% CI	CV%	Mean	95% CI	CV%	Mean	95% CI	CV%	Mean	95% CI	CV%
Ka (1/h)	0.50	0.25 – 0.74	23.9	0.21	0.044 – 0.38	37.6	12 (Fixed)			12 (Fixed)
AUCinf (ug·h/ml)	6290			7620			9260			9610
V/F (mL/kg)	91.0	68.6 – 113.4	11.7	58.1	27.9 – 88.3	24.8	122.2	99.2 – 145.2	9.4	77.2	51.3 – 103.2	16.8
CL/F (mL/(kg·h))	5.08	4.33 – 5.84	7.0	4.21	3.54 – 4.88	7.5	3.66	2.54 – 4.77	15.3	3.30	2.1 – 4.5	18.1
T-half (h)	12.4			9.56			23.1			16.2
Background values (μg/mL)	21.5	5.1 – 38.0	36.3	35.3	20.2 – 50.4	19.4	31.4	18.1 – 44.7	21.2	22.6	0 – 148.0	277.5

Symbols: ka, absorption rate constant; AUC, area under the curve; V/F, volume of distribution; CL/F, clearance; T-half, half-life; %CV, coefficient of variation × 100, and the 95% confidence intervals (CI).

3.3. Body Weight Changes in Neonatal Rats with HI Injury After IP hIAIPs Administration

Changes in body weight can possibly influence the outcomes of pharmacokinetic studies [50]. In the present study, the baseline body weight of the individual pups was recorded before exposure to HI at −3/−4.5 h (Fig. 2 A). The body weight changes of the individual pups were calculated from the difference in body weight measured before and after hIAIP administration. Fig. (6) shows body weight plotted as the body weight changes in grams for the males and females with or without exposure to HI that received hIAIPs IP 0 h after exposure to Sham or HI (Fig. 2A, open arrow). Values are plotted for the four groups 1, 6, 12, 24, or 36 h after hIAIPs was given (Fig. 2A). Body weight changes were lower in the HI-hIAIP group compared to the Sham-hIAIP group in both males (Factorial ANOVA, P < 0.001) and females (Factorial ANOVA, P < 0.001) over the study period. Moreover, compared to the 1 h study point, reductions in body weight changes (g) were observed at 12 h (Factorial ANOVA, P) in the HI-hIAIP-treated males, whereas increases were observed at 12 h, 24 h and 36 h (Factorial ANOVA, P < 0.01) in the Sham-hIAIP-treated males and females. Increases in body weight changes (g) were observed at 36 h (Factorial ANOVA, P < 0.001) in the HI males, and at 24 h and 36 h (Factorial ANOVA, P < 0.01) in the HI females. Significant (P < 0.05) differences were observed between the Sham and HI male and female groups. Statistical differences were also observed between Sham males and females (Factorial ANOVA, P < 0.001), but not between the HI males and females (Factorial ANOVA, P = 0.198).

Fig. (6).

Body weight changes (g) in neonatal male and female rats plotted against time in hours after hIAIP IP administration with and without HI exposure. Compared to the Sham-hIAIP group, percent body weight changes decreased in the HI-hIAIP group in both males and females over the study periods. Compared to the body weight changes (g) at 1 h, the reduction was observed at 12 h in the HI-hIAIP-treated males, whereas increases occurred at 12, 24, and 36 h in the Sham-hIAIP-treated males and females, at 36 h in the HI-hIAIP-treated males, and at 24 and 36 h in the HI-hIAIP-treated females. Animal numbers same as Fig. 4, except for females in HI-hIAIP group (Female n = 30: 1 h after hIAIP IP n = 8, 6 h after hIAIP IP n = 6, 12 h after hIAIP IP n = 6, 24 h after hIAIP IP n = 6, 36 h after hIAIP IP n = 4). Values are mean ± SD. Factorial ANOVA with Tukey HSD post hoc analysis. *P < 0.05, each time point vs. 1 h. # P < 0.05, Sham-hIAIP-treated vs. HI-hIAIP-treated pups over the study periods.

3.4. hIAIP IP Administration does not change Bleeding Times or Blood Volume Losses in Neonatal Rats with and or without HI Brain Injury

The effects of hIAIPs on the coagulation system were determined by the duration of tail bleeding in the Sham- and HI-treated neonatal rats after three doses of hIAIP (Fig. 2B, gray arrows, 60 mg/kg). Fig. (7A) contains the tail bleeding time (min) plotted for the male and female groups that were treated with hIAIPs or placebo at 0, 24 and 48 h after exposure to Sham or HI. The bleeding times did not differ among the study groups in the cohort (male + female, ANOVA, P = 0.63), male (Fig. 7A, ANOVA, P = 0.89), and female (Fig. 7A, Kruskal-Wallis, P = 0.24) groups.

Fig. (7).

(A) The effects of hIAIP IP administration on bleeding times in neonatal rats with and without HI-related brain injury. Tail bleeding times (min) plotted for the study groups. The hIAIPs (60 mg/kg) were given to the P7 rats at 0, 24, and 48 h after HI insult. Bleeding time was recorded 72 h after the HI insult over 10 min. Scatter dot plots show that statistical differences were not observed among study groups in male (Sham: open circle; HI: closed circles) and female (Sham: open diamond; HI: closed diamond) pups. Values are mean ± SD. One-way ANOVA or Kruskal-Wallis test with Tukey HSD post hoc analysis. Sham: male n = 10, female n = 5; Sham-hIAIP: male n = 9, female n = 10; HI-PL: male n = 10, female n = 5; HI-hIAIP: male n = 8, female n = 11. (B) Body weight changes before and after bleeding time tests in neonatal rats with and without HI brain injury. Body weight changes (mg) are plotted for the study groups. Body weight changes were used to estimate blood volume losses for bleeding time experiments. Scatter plots show that differences were not observed in body weight changes among the study groups. Data are presented as Mean ± SD, and circles for males and diamonds for females. Kruskal-Wallis test with Tukey HSD post hoc analysis. Sham: male n = 10, female n = 5; Sham-hIAIP: male n = 8, female n = 8; HI-PL: male n = 9, female n = 5; HI-hIAIP: male n = 8, female n = 10.

Volumes of blood loss during the bleeding tests were also estimated from the reductions in body weight. Fig. (7B) plots the differences in body weight of pups before and after tail bleeding for the male and female groups. Differences were not detected in the cohort (male + female, Kruskal-Wallis, P = 0.31), male (Fig. 7B, Kruskal-Wallis, P = 0.38), and female (Fig. 7B, Kruskal-Wallis, P = 0.89) rats, indicating that blood volume losses were similar among the study groups. Correlations were not observed between bleeding times and body weight changes in any of the study groups (data not shown). These results suggest that tail bleeding time may not be correlated with the volume of blood loss after IP hIAIP doses in pups with or without HI brain injury.

4. DISCUSSION

The objective of the present study was to determine PK profiles of hIAIPs in male and female neonates with and without exposure to HI brain injury. Although we examined prepubertal neonatal rats, sex-related differences have previously been reported in HI brain injury in similarly aged rats [10, 11]. In addition, we determined whether hIAIP administration affected bleeding times because bikunin inhibits components of the coagulation system [27, 28] and exposure to HI can result in bleeding in newborns. The major findings are: 1) HI is associated with rapid absorption, reduced CL/F, and increased AUC, V/F, and T-half values of hIAIPs in males and females; 2) IP administration results in good systemic hIAIP levels in all groups; 3) sex has modest effects on AUC, V/F, CL/F, and T-half of hIAIPs in neonatal rats exposed to HI; and 4) even a high dose of hIAIPs does not affect bleeding times in neonatal rats exposed to HI.

Absorption kinetics that describe drug movement from sites of administration into the systemic circulation are complicated and consist of pre-systemic degradation, direct capillary absorption, and indirect lymph-mediated absorption for therapeutic proteins such as hIAIPs and monoclonal antibodies. Knowledge about key determinants of absorption of biologics is incomplete particularly in neonates that have been exposed to HI. A semi-mechanistic model has been proposed to explore recombinant human tumour necrosis factor-α in rats following different routes of injection and results suggested that after IP injections both lymphatic and non-lymphatic routes contribute to systemic absorption [51]. In the current study, hIAIPs reached the highest serum concentrations at the initial sampling time, 1 h after IP administration, in the HI-hIAIP- and 6 h in the Sham-hIAIP-treated neonatal rats. The sex-related differences in the ka (1st-order absorption rate constant) were small but the differences between Sham-hIAIP- and HI-hIAIP-treated groups were significant based upon the observed rapid absorption in the hIAIP in HI groups. The AUCs showed greater (>25%) exposure to hIAIPs in the HI-hIAIP- than Sham-hIAIP-treated neonatal rats. These findings suggest that exposure to HI potentially increases the rate of absorption of IP administered hIAIPs in neonatal rats. One potential mechanism could relate gastrointestinal tract membrane injury after acute inflammation caused by 90 min systemic hypoxia [52], which could potentially facilitate the non-lymphatic mediated transfer of proteins such as hIAIPs into the bloodstream. The HI exposed groups appear to exhibit larger volumes of distribution compared with the Sham groups. There were several reports demonstrating that HI has the potential to damage the cerebral microvasculature, which could render the brain more ‘leaky’ for hIAIP distribution [53–56]. Besides, the neonatal rats exposed to carotid ligation were also exposed to systemic hypoxia for 90 min, which could also render other systemic vasculatures ‘leaky’ to proteins. The volumes of distribution (V/F) of hIAIPs were greater than the plasma volume in rats, which is 4.68 ± 0.57 mL/100 g (Mean ± SD, body weight < 120 g) [57]. This suggests that hIAIPs were not necessarily confined to the plasma vascular space but could distribute into various body tissues. Similar to the Sham-hIAIP-treated groups, the males had a larger V/F of hIAIPs (>55%) compared to females in the HI-hIAIP-treated groups. The HI-hIAIP-treated groups showed reduced CL/F and prolonged elimination T-half of hIAIPs for each sex compared with the Sham-hIAIP-treated groups. Conversely, previous findings suggest that inflammation resulting from sepsis and necrotizing enterocolitis in premature infants resulted in rapid reductions in endogenous IAIPs from the system circulation [58, 59]. These findings potentially indicate that inflammation mediated rapid clearances, metabolism or utilization of IAIPs from the systemic circulation more likely result from infection-inflammation related events than from HI-related inflammation. Although detailed mechanisms of hIAIP elimination from the systemic circulation were not examined, one potential mechanism could be proteolysis. The reason systemic hIAIPs were eliminated slower in the HI-exposed rats requires further investigation along with the effects of inflammation on exogenously administered IAIPs.

It is important to point out that hIAIPs have a much longer T-half (Table 1) than its light chain (bikunin). The T-half of bikunin is 3–10 min [60], which requires large amounts of protein and frequent intravenous infusions for use in therapeutic strategies [13, 18]. The heavy chains of hIAIPs stabilize and construct the extracellular matrix and synergize with the activity of the bikunin [25, 26]. Consequently, hIAIPs may have advantages over bikunin as therapeutic agents requiring less frequent and smaller doses. Nonetheless, more than one dose of hIAIPs per day could be required to achieve optimal neuroprotective effects.

Body weight and maturation can affect drug PK [61]. Therefore, it is important to monitor body weight changes particularly after adverse events such as exposure to HI. Although the body weight also increased in the HI-hIAIP-treated group after exposure to HI, the increases remained below those of the Sham-hIAIP group. These findings are consistent with work showing that HI-related insults impair somatic growth as indicated by lower daily body weight changes [62, 63]. Therefore, the HI-related changes in body weight also could have affected the PK of the hIAIPs in neonatal rats.

Neonates with HI-related brain injury are at risk for hemorrhage [64, 65]. Although we have previously shown that the administration of hIAIPs has important neuroprotective effects on HI-related brain injury [14, 19–22], bikunin can inhibit several components of the coagulation system including plasmin, factor Xa, and kallikrein [27, 28]. Therefore, we examined the effects of hIAIPs on bleeding times in the neonatal rats with and without exposure to HI as an initial effort to examine their effects on hemostasis in neonates. We did not find differences in the bleeding times between placebo- and hIAIP-treated male and female rats with and without exposure to HI brain injury. Therefore, systemic treatment with hIAIPs most likely does not exert adverse effects on the coagulation system at least as measured by bleeding time. The light chain bikunin is responsible for the serine protease inhibitory activity of hIAIPs [24]. Bikunin has been shown to inhibit serine proteases including plasmin, Factor Xa, Factor XIIa, and kallikrein in vitro [27, 28, 66], and to prolong the APTT [66]. However, none of the serine proteases appear to be physiological targets of bikunin in vivo because plasma contains other inhibitors that bind to these proteases more avidly than bikunin [24]. Treatment with hIAIPs also does not affect platelet counts in neonatal rats exposed to HI [21], which when combined with our present findings suggests that even a very high dose of hIAIPs most likely does not affect hemostasis in neonates. Although we did not examine the enzyme-inhibiting capacity of hIAIP on Factor XIIa or kallikrein and PT/APTT, it would be of interest to investigate the effects of hIAIPs on these coagulation factors in future work.

There are several limitations to our study along with opportunities for future study. The bioavailability of IP hIAIP was not examined as this requires intravenous administration, which is not readily feasible in neonatal rats. Thus, some PK parameters were combined with bioavailability. The present studies have a narrow age range and duration of evaluation after hIAIPs. PK behavior of hIAIPs also needs to be evaluated at later time points because of the long terminal half-life of IAIPs after HI. Sampling at very early time points in the HI groups is required to characterize the fast absorption kinetics. It would also be important to perform a dose-response study including higher hIAIP doses to assess dose linearity, provide improved exposure-response evaluations, and allow a better basis to translate our findings to studies in human subjects. The faster hIAIP absorption in the animals exposed to HI could have resulted in damage to the intraperitoneal membrane resulting from systemic hypoxia [52]. Nonetheless, these are the first studies to begin to examine the PK properties of hIAIPs, which we have shown to have important neuroprotective effects after HI in the neonate [14, 19–22].

CONCLUSION

IP administration of hIAIPs resulted in good systemic hIAIPs exposure and did not affect bleeding times in neonatal rats with and without exposure to HI. HI was associated with increased and rapid absorption, increased AUC, reduced CL, and prolonged T-half values of hIAIPs. These findings combined with our previous studies [14, 19–22] suggest that hIAIPs represent reasonable neuroprotective agents for HI-related brain injury. Sex has modest effects on the hIAIP PK, suggesting the need for additional PK and efficacy assessments in males and females. These studies provide important preliminary insights into determinants of hIAIP exposure in this neonatal model of HI brain injury. Furthermore, we speculate that our findings also have broader implications for other proteins that could be used as therapeutic agents in neonates exposed to HI.