Plasma Compartment Research Articles

The pharmacokinetics of the Fab fragment of a humanized anti‐cocaine monoclonal antibody and its effects on cocaine’s distribution in mice

There is currently no FDA approved treatment for cocaine abuse. Our laboratory has created a humanized anti‐cocaine monoclonal antibody (mAb), h2E2, that has high affinity and specificity for cocaine and is cleared slowly in rodents. The mAb h2E2 sequesters cocaine in the plasma and prevents cocaine entry into the brain in rodents. The antigen binding portion, the Fab fragment, of h2E2 has the same high affinity and selectivity as its whole mAb counterpart. The Fab fragment should also sequester cocaine into the plasma compartment but is expected to have a more rapid clearance than the intact mAb. This study elucidated the pharmacokinetics of the Fab fragment and its effects on cocaine’s pharmacokinetics in mice. Catheterized mice were injected with Fab (80 mg/kg, i.v., pH=7.4, PBS) (n=8) and blood samples collected from the tip of the tail at designated time points over three days. Fab concentrations in blood samples were quantified using an ELISA assay. Two groups of catheterized mice were injected under mild restraint with Fab (82 mg/kg, i.v. pH= 7.4, PBS, generated by enzymatic digestion of whole mAb h2E2) or vehicle (PBS, pH=7.4). One hour later, mice were injected with an equimolar dose of cocaine HCl (0.56 mg/kg, i.v.). At 8 time points between 0.75 and 60 min, mice were decapitated, and trunk blood was collected. Sodium pentobarbital (50 mg/kg, i.p.) was administered 3 minutes prior to decapitation. For the 0.75 and 1.5‐minute time points, cocaine was injected into anesthetized rats. Blood (typically 0.5–2 mL) was placed into 2‐mL polypropylene tubes and then centrifuged at 5000 g for 3 minutes. Plasma was pipetted into polypropylene microcentrifuge tubes and rapidly frozen on dry ice and stored at −80°C until LC‐MS/MS analysis. This experiment was performed three times. Total cocaine concentrations were quantified using LC‐MS/MS. The pharmacokinetic profile of the Fab fragment in mice was described by a 2‐compartment model (Figure 1). The Fab fragment has a mean distribution half‐life (t1/2α) of 16.7 minutes and a terminal elimination half‐life (t1/2β) of 7.1 hours (Figure 1). In the presence of the Fab fragment, cocaine concentrations in the plasma increased by 4.5‐fold compared to vehicle animals (Figure 2). The initial volume of distribution of cocaine decreased approximately 4.5‐fold in the presence of Fab. The Fab fragment increases the concentration of cocaine in the plasma by approximately 4.5‐fold compared to vehicle controls. Although the data is not shown, it is hypothesized that the Fab fragment, like its mAb counterpart, prevents cocaine from crossing the blood‐brain barrier and entering the brain. With a 24‐fold decrease in elimination half‐life (7.1 hours) compared to the intact h2E2 (7.8 days) and predicted rapid urinary excretion, the Fab fragment may be useful for the treatment of cocaine overdoses.Support or Funding InformationThis work is supported by the National Institutes of Health, National Institute on Drug Abuse grant number U01DA039550 (to ABN).The pharmacokinetics of the Fab fragment in male miceFigure 1The pharmacokinetics of cocaine in the presence of the Fab fragmentFigure 2

Dexamethasone pharmacokinetics characteristics via sub-tenon microfluidic system in uveitis rabbits

The aim of this study was to investigate the plasma and ocular dexamethasone (DEX) pharmacokinetics characteristics of the sub-tenon microfluidic system (SMS) delivery of dexamethasone sodium phosphate (DEXP) in experimental autoimmune uveitis (EAU) rabbits. The SMS showed sustained DEX release and large amount of drug loading capacity by a miniature pump which connected with a tube that implanted into the sub-Tenon's sac of the EAU rabbit. The DEX concentrations in the plasma and different ocular compartments (cornea, aqueous humor, retina/choroid, sclera and vitreous humor) were quantified by Shimadzu LC–MS 2010 system. The SMS increased total drug delivery of DEX in ocular tissues as compared with traditional combined medication (subconjunctival injection combined with intravenous injection) in EAU rabbit. Moreover, the minimal amount of DEX was found in the blood, indicated that only a small amount of drug could enter into the bloodstream and causing minimal systematic side effect. Overall, the study provided evidence that SMS meet the large amount of drug loading capacity, continuous release drugs and moderate drug level in the EAU ocular tissue and minimal drug level in the blood, which indicated that SMS could be a feasible option for ocular application in clinical trials and maybe can replace or even discard systemic medications for the management of acute ocular inflammation disease.

Plasma Protein Signatures of a Murine Venous Thrombosis Model and Slc44a2 Knockout Mice Using Quantitative-Targeted Proteomics.

The plasma compartment of the blood holds important information on the risk to develop cardiovascular diseases such as venous thrombosis (VT). Mass spectrometry-based targeted proteomics with internal standards quantifies proteins in multiplex allowing generation of signatures associated with a disease or a condition. Here, to demonstrate the method, we investigate the plasma protein signatures in mice following the onset of VT, which was induced by RNA interference targeting the natural anticoagulants antithrombin and protein C. We then study mice lacking Slc44a2, which was recently characterized as a VT-susceptibility gene in human genome-wide association studies. We use a recently developed panel of 375 multiplexed mouse protein assays measured by mass spectrometry. A strong plasma protein siganture was observed when VT was induced. Discriminators included acute phase response proteins, and proteins related to erythrocyte function. In mice lacking Slc44a2, protein signature was primarily overruled by the difference between sexes and not by the absent gene. Upon separate analyses for males and females, we were able to establish a signature for Slc44a2 deficiency, in which glycosylation-dependent cell adhesion molecule-1 and thrombospondin-1 were shared by both sexes. The minimal impact of Slc44a2 deficiency on the measured plasma proteins suggests that the main effect of Slc44a2 on VT does not lay ultimately in the plasma compartment. This suggests further investigation into the role of this VT-susceptibility gene should perhaps also question the possible involvement in cellular mechanisms.

Impact of Suboptimal APOBEC3G Neutralization on the Emergence of HIV Drug Resistance in Humanized Mice.

HIV diversification facilitates immune escape and complicates antiretroviral therapy. In this study, we take advantage of a humanized-mouse model to probe the contribution of APOBEC3 mutagenesis to viral evolution. Humanized mice were infected with isogenic HIV molecular clones (HIV-WT, HIV-45G, and HIV-ΔSLQ) that differ in their abilities to counteract APOBEC3G (A3G). Infected mice remained naive or were treated with the reverse transcriptase (RT) inhibitor lamivudine (3TC). Viremia, emergence of drug-resistant variants, and quasispecies diversification in the plasma compartment were determined throughout infection. While both HIV-WT and HIV-45G achieved robust infection, over time, HIV-45G replication was significantly reduced compared to that of HIV-WT in the absence of 3TC treatment. In contrast, treatment responses differed significantly between HIV-45G- and HIV-WT-infected mice. Antiretroviral treatment failed in 91% of HIV-45G-infected mice, while only 36% of HIV-WT-infected mice displayed a similar negative outcome. Emergence of 3TC-resistant variants and nucleotide diversity were determined by analyzing 155,462 single HIV reverse transcriptase gene (RT) and 6,985 vif sequences from 33 mice. Prior to treatment, variants with genotypic 3TC resistance (RT-M184I/V) were detected at low levels in over a third of all the animals. Upon treatment, the composition of the plasma quasispecies rapidly changed, leading to a majority of circulating viral variants encoding RT-184I. Interestingly, increased viral diversity prior to treatment initiation correlated with higher plasma viremia in HIV-45G-infected animals, but not in HIV-WT-infected animals. Taken together, HIV variants with suboptimal anti-A3G activity were attenuated in the absence of selection but displayed a fitness advantage in the presence of antiretroviral treatment.IMPORTANCE Both viral (e.g., RT) and host (e.g., A3G) factors can contribute to HIV sequence diversity. This study shows that suboptimal anti-A3G activity shapes viral fitness and drives viral evolution in the plasma compartment in humanized mice.

Kinetic modeling and analysis of dynamic bioluminescence imaging of substrates administered by intraperitoneal injection.

Bioluminescence imaging (BLI) has been found to have diverse applications in the life sciences and medical research due to its ease of use and high sensitivity. From kinetics analysis, dynamic imaging studies have significant advantages for diagnosis when compared to traditional static imaging studies. This work focuses on modeling and quantitatively analyzing the dynamic data produced from the intraperitoneal (IP) injection of D-luciferin in longitudinal BLI, aiming to provide a powerful tool for monitoring the growth of tumors. We constructed a three-compartment pharmacokinetic (PK) model and employed the standard Michaelis-Menten (M-M) kinetics to investigate the dynamic BLI data produced from the IP injection of D-luciferin. The 3 compartments were the plasma compartment, the non-specific compartment, and the specific compartment. The validity of this PK model was tested by the dynamic BLI data of MKN28M-luc xenograft mice, along with the published longitudinal dynamic BLI data of B16F10-luc xenograft mice. The R-squares between the simulated lines and the measurement were 1 and 0.99, respectively, for the mice data and the published data. In addition, the 2 kinetic macroparameters obtained reflected the rate of tumor growth in vivo. In particular, the values of macroparameters A showed a significant dependence on tumor surface area. The proposed PK model may be an effective tool for use in drug development programs and for monitoring the response of tumors to treatment.

Profiling Extracellular Long RNA Transcriptome in Human Plasma and Extracellular Vesicles for Biomarker Discovery

The recent discovery of extracellular RNAs in blood, including RNAs in extracellular vesicles (EVs), combined with low input RNA sequencing advances have enabled scientists to investigate their role in human disease. To date, most studies have been focusing on small RNAs, and methodologies to optimize long RNAs measurement are lacking. We used plasma RNA to assess the performance of six long RNA sequencing methods, at two different sites, and we report their differences in reads (%) mapped to the genome/transcriptome, number of genes detected, long RNA transcript diversity, and reproducibility. Using the best performing method, we further compare the profile of long RNAs in the EV- and no-EV-enriched RNA plasma compartments. These results provide insights on the performance and reproducibility of commercially available kits in assessing the landscape of long RNAs in human plasma and different extracellular RNA carriers that may be exploited for biomarker discovery.

A Polynomial Linear Regression Approach to Estimate Sensitive Parameters in the Novel Double Diabetes Model

Sensitivity analysis characterizes the changes in the model outputs due to the changes in the model parameters. In this article, we estimate the most sensitive parameters in the Novel Double Diabetes Model (NDDM) through the polynomial linear regression approach; this way we develop a direct relation between the sensitivity analysis and the paramter estimation. The NDDM has more than seventeen parameters, and estimating them simultanously is difficult. We select the most commonly used five parameters in the glucose-insulin dynamics for the sensitivity analysis. The model outputs-glucose concentrations in the plasma and the subcutaneous compartments are sensitive to the selected parameters whereas the insulin concentrations in the plasma and the subcutaneous compartment are sensitive only to the insulin transfer rate from the subcutaneous to the plasma compartment. System sensitivity of the model for the selected parameters is also in agreement with the individual sensitivities of the parameters. Consequently, we estimate the parameters which are more sensitive by the polynomial linear regression approach.

Blood and Gill Carbonic Anhydrase in the Context of a Chondrichthyan Model of CO2 Excretion.

Pacific spiny dogfish (Squalus suckleyi) have been widely used as a representative species for chondrichthyan CO2 excretion. Pacific spiny dogfish have a slower red blood cell (RBC) carbonic anhydrase (CA) isoform than teleost fishes, extracellular CA activity, no endogenous plasma CA inhibitor, and plasma-accessible CA IV at the gills. Thus, both the RBC and plasma compartments contribute to bicarbonate ion () dehydration at the gills for CO2 excretion in contrast to teleost fishes, in which dehydration is restricted to RBCs. We compared CA activity levels, subcellular localization, and presence of plasma CA inhibitors in the blood and gills of 13 chondrichthyans to examine the hypothesis that the dogfish model of CO2 excretion applies broadly to chondrichthyans. In general, blood samples from the 12 other chondrichthyans examined had lower RBC CA activity than teleosts, some extracellular CA activity, and no endogenous plasma CA inhibitor. While type IV-like membrane-associated CA was found in the gills in all four of the chondrichthyans examined, S. suckleyi had three times more CA activity ( μmol CO2 min-1 mg protein-1) in the microsomal (membrane) fraction of gills than the other three. In addition, unexpected variation in CA characteristics was observed between chondrichthyan species. Thus, in general, it appears that the pattern of CA distribution in fishes can be generally categorized as either chondrichthyan or teleost models. However, further studies should examine the functional significance of the within-chondrichthyan differences we observed and investigate whether CO2 excretion patterns exist along a continuum or in discrete groups.

Evaluation of lung parenchyma, blood vessels, and peripheral blood lymphocytes as a potential source of acute phase reactants in patients with COPD.

Background: Previous studies have shown that the arterial wall is a potential source of inflammatory markers in COPD. Here, we sought to compare the expression of acute phase reactants (APRs) in COPD patients and controls both at the local (pulmonary arteries and lung parenchyma) and systemic (peripheral blood leukocytes and plasma) compartments.Methods: Consecutive patients undergoing elective surgery for suspected primary lung cancer were eligible for the study. Patients were categorized either as COPD or control group based on the spirometry results. Pulmonary arteries and lung parenchyma sections, peripheral blood leukocytes, and plasma samples were obtained from all participants. Gene expression levels of C-reactive protein (CRP) and serum amyloid A (SAA1, SAA2, and SAA4) were evaluated in tissue samples and peripheral blood leukocytes by reverse transciption-PCR. Plasma CRP and SAA protein levels were measured by enzyme-linked immunosorbent assays. Proteins were evaluated in paraffin-embedded lung tissues by immunohistochemistry.Results: A total of 40 patients with COPD and 62 controls were enrolled. We did not find significant differences in the gene expression between COPD and control group. Both CRP and SAA were overexpressed in the lung parenchyma compared with pulmonary arteries and peripheral blood leukocytes. The expression of SAA was significantly higher in the lung parenchyma than in the pulmonary artery (2-fold higher for SAA1 and SAA4, P=0.015 and P<0.001, respectively; 8-fold higher for SAA2, P<0.001) and peripheral blood leukocytes (16-fold higher for SAA1, 439-fold higher for SAA2, and 5-fold higher for SAA4; P<0.001). No correlation between plasma levels of inflammatory markers and their expression in the lung and peripheral blood leukocytes was observed.Conclusions: The expression of SAA in lung parenchyma is higher than in pulmonary artery and peripheral blood leukocytes. Notably, no associations were noted between lung expression of APRs and their circulating plasma levels, making the leakage of inflammatory proteins from the lung to the bloodstream unlikely. Based on these results, other potential sources of systemic inflammation in COPD (eg, the liver) need further scrutiny.

Population pharmacokinetics of oxycodone in plasma and cerebrospinal fluid after epidural and intravenous administration

ABSTRACTObjectives: To establish the first plasma and cerebrospinal fluid (CSF) oxycodone population pharmacokinetic (PopPK) model after epidural (EPI) and intravenous (IV) oxycodone administration.Methods: The study was conducted with 30 female subjects undergoing elective gynecological surgery with epidural analgesia. A parallel single dose of EPI oxycodone with IV placebo (EPI group; n = 18) or IV oxycodone with EPI placebo (IV group; n = 12) was administered. An epidural catheter for drug administration was placed at T12/L1 and a spinal catheter for CSF sampling at L3/4. Plasma and CSF for oxycodone analysis were frequently collected. A PopPK model was built using the NONMEM software package.Results: Plasma and CSF oxycodone concentrations were evaluated using separate central plasma and CSF compartments and separate peripheral plasma and CSF compartments. Epidural space served as a depot compartment with transfer to both the plasma and CSF central compartments. The population parameters for plasma clearance and apparent distribution volumes for central and peripheral compartments for plasma and CSF were 37.4 L/h, 90.2 L, 68.9 L, 0.035 L (fixed based on literature), and 0.039 L, respectively.Conclusion: A PopPK model was developed and found to precisely and accurately describe oxycodone time-concentration data in plasma and CSF.

A preliminary investigation of circulating extracellular vesicles and biomarker discovery associated with treatment response in head and neck squamous cell carcinoma

BackgroundThere is a paucity of plasma-based biomarkers that prospectively segregate the outcome of patients with head and neck squamous-cell carcinoma (HNSCC) treated with chemoradiation therapy (CRT). Plasma extracellular vesicles (EVs) might be an alternative source for discovery of new specific markers present in patients with HNSCC, which could help to re-direct patients to appropriate curative therapies without delay.MethodsIn order to identify new markers in plasma compartments, Cholerae toxin B chain (CTB) and Annexin V (AV) were used to isolate EVs from pooled plasma samples from patients with locally advanced HNSCC who responded (CR, n = 6) or presented incomplete response (NR, n = 6) to CRT. The crude plasma and EVs cargo were screened by antibody array.ResultsOf the 370 polypeptides detected, 119 proteins were specific to NR patients while 38 were exclusive of the CR subjects. The Gene Set Enrichment Analysis (GSEA) and Search Tool for the Retrieval of Interacting Genes (STRING) database analysis indicated that the content of circulating plasma EVs might have a relevant function for the tumor intercellular communication in the HNSCC patients.ConclusionThis study provides a list of potential markers present in plasma compartments that might contribute to the development of tools for prediction and assessment of CRT response and potentially guide therapeutic decisions in this context.

The immune system of the freshwater zebra mussel, Dreissena polymorpha, decrypted by proteogenomics of hemocytes and plasma compartments

The immune system of bivalves is of great interest since it reflects the health status of these organisms during stressful conditions. While immune molecular responses are well documented for marine bivalves, few information is available for continental bivalves such as the zebra mussel, Dreissena polymorpha. A proteogenomic approach was conducted on both hemocytes and plasma to identified immune proteins of this non-model species. Combining transcriptomic sequences with mass spectrometry data acquired on proteins is a relevant strategy since 3020 proteins were identified, representing the largest protein inventory for this sentinel organism. Functional annotation and gene ontology (GO) analysis performed on the identified proteins described the main molecular players of hemocytes and plasma in immunity. GO analysis highlights the complementary immune functions of these two compartments in the management of micro-organisms. Functional annotation revealed new mechanisms in the immune defence of the zebra mussel. Proteins rarely observed in the hemolymph of bivalves were pinpointed such as natterin-like and thaumatin-like proteins. Furthermore, the high abundance of complement-related proteins observed in plasma suggested a strong implication of the complement system in the immune defence of D. polymorpha. This work brings a better understanding of the molecular mechanisms involved in zebra mussel immunity. SignificanceAlthough the molecular mechanisms of marine bivalves are widely investigated, little information is known for continental bivalves. Moreover, few proteomic studies described the complementarity of both hemolymphatic compartments (cellular and plasmatic) in the immune defence of invertebrates. The recent proteogenomics concept made it possible to discover proteins in non-model organisms. Here, we propose a proteogenomic strategy with the zebra mussel, a key sentinel species for biomonitoring of freshwater, to identify and describe the molecular actors involved in the immune system in both hemocytes and plasma compartments. More widely, this study provided new insight into bivalve immunity.

Adipose Specific Loss of PCPE2 Promotes Microvascular Endothelial Dysfunction and Decreased Angiogenic Potential

Procollagen C‐endopeptidase enhancer 2 (PCPE2) is an extracellular matrix protein which regulates the oligomerization of the high and low‐density lipoprotein (HDL/LDL) receptors that enable cellular uptake of HDL/LDL from the extracellular matrix under healthy conditions. To evaluate Effect of adipose PCPE2 floxed mice were crossed with mice expressing adipose specific cre recombinase. We hypothesized elevated lipid levels induced by PCPE2 KO mice vascular function and angiogenic potential.In Adipose specific PCPE2 KO we found that due to the inability of lipoprotein lipids in HDL to be taken up by the adipocytes, the concentration of HDL in the plasma compartment significantly increased. Using this model vascular function was evaluated using pressure mayo graph setup. Endothelial function was determined by dose response curves to ACH or increasing levels of shear stress. Smooth muscle function was tested by response to Papaverine. Angiogenic potential was evaluated by embedding sections of brown adipose tissue in Matrigel and vascular growth measured by sprout formation. Statistical significance was established using a student t‐test P&lt;0.05 vs. CTRL.Adipose specific PCPE2 KO resulted in elevated plasma lipid levels as elevated by insert numbers. Homozygous loss of Ad‐PCPE2 resulted in decreased endothelial function compared to control mice. Interestingly heterozygote deletion of Ad‐PCPE2 deletion trended toward increase in endothelial mediated dilation (Fig. 1A and B). Smooth muscle function was not affected by either genotype (Fig. 1C). Angiogenic Potential was significantly decreased in both heterozygote Ad‐PCPE2 KO mice compared to CTRLs (% growth Mean, SEM, N: CTRL 3.05±1.31, 6; Het 1.07±0.59*, 4)In conclusion, our data suggest that ad‐PCPE2 loss of function elevated plasma lipid levels that promote endothelial dysfunction as assessed by decreased vasodilator capacity and angiogenic potential.Support or Funding InformationFunded by MST HL127649 and HL138907; AMB R01‐HL133029‐02This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

SAT-404-Discordant NS5a but not NS3 RASs profile in liver and plasma compartments of HIV/HCV genotype 1a/4d infected patients

SAT-404-Discordant NS5a but not NS3 RASs profile in liver and plasma compartments of HIV/HCV genotype 1a/4d infected patients

Rare Detection of Antiviral Functions of Polyclonal IgA Isolated from Plasma and Breast Milk Compartments in Women Chronically Infected with HIV-1.

The humoral response to invading mucosal pathogens comprises multiple antibody isotypes derived from systemic and mucosal compartments. To understand the contribution of each antibody isotype/source to the mucosal humoral response, parallel investigation of the specificities and functions of antibodies within and across isotypes and compartments is required. The role of IgA against HIV-1 is complex, with studies supporting a protective role as well as a role for serum IgA in blocking effector functions. Thus, we explored the fine specificity and function of IgA in both plasma and mucosal secretions important to infant HIV-1 infection, i.e., breast milk. IgA and IgG were isolated from milk and plasma from 20 HIV-1-infected lactating Malawian women. HIV-1 binding specificities, neutralization potency, inhibition of virus-epithelial cell binding, and antibody-mediated phagocytosis were measured. Fine-specificity mapping showed IgA and IgG responses to multiple HIV-1 Env epitopes, including conformational V1/V2 and linear V2, V3, and constant region 5 (C5). Env IgA was heterogeneous between the milk and systemic compartments (Env IgA, τ = 0.00 to 0.63, P = 0.0046 to 1.00). Furthermore, IgA and IgG appeared compartmentalized as there was a lack of correlation between the specificities of Env-specific IgA and IgG (in milk, τ = -0.07 to 0.26, P = 0.35 to 0.83). IgA and IgG also differed in functions: while neutralization and phagocytosis were consistently mediated by milk and plasma IgG, they were rarely detected in IgA from both milk and plasma. Understanding the ontogeny of the divergent IgG and IgA antigen specificity repertoires and their effects on antibody function will inform vaccination approaches targeted toward mucosal pathogens.IMPORTANCE Antibodies within the mucosa are part of the first line of defense against mucosal pathogens. Evaluating mucosal antibody isotypes, specificities, and antiviral functions in relationship to the systemic antibody profile can provide insights into whether the antibody response is coordinated in response to mucosal pathogens. In a natural immunity cohort of HIV-infected lactating women, we mapped the fine specificity and function of IgA in breast milk and plasma and compared these with the autologous IgG responses. Antigen specificities and functions differed between IgG and IgA, with antiviral functions (neutralization and phagocytosis) predominantly mediated by the IgG fraction in both milk and plasma. Furthermore, the specificity of milk IgA differed from that of systemic IgA. Our data suggest that milk IgA and systemic IgA should be separately examined as potential correlates of risk. Preventive vaccines may need to employ different strategies to elicit functional antiviral immunity by both antibody isotypes in the mucosa.

Erythrocyte Partitioning Profile of Isosteviol in Human and Rat Blood

Isosteviol is a synthetic derivative of steviol glycosides with promising pharmacological properties and might find future use as a cardioprotective agent. A simple LC-MS/MS technique was developed and validated for the bioanalysis of isosteviol in plasma and erythrocytes. This method was subsequently utilized for the in vitro assessment of isosteviol's partitioning into blood compartments of humans and rats. Fresh blood samples from healthy humans and Wistar rats were equilibrated with 1, 10, and 30 µM isosteviol at 37 °C in a shaking dry-bath. The levels of isosteviol in plasma and erythrocytes partitions were determined in these samples, after separation, at intervals over a 60-minute period. The data derived was used to estimate erythrocyte-to-plasma and blood-to-plasma coefficients. Mean erythrocyte-to-plasma partition coefficients (SD) after 60 minutes of equilibration were observed to be 0.039 (0.002) and 0.040 (0.003) in humans and rats, respectively. Derived values for the blood-to-plasma ratio (SD) were 0.576 (0.001) in humans and 0.543 (0.007) in rats, whereas plasma component binding was estimated to be more than 96%. The findings suggest that isosteviol preferentially partitions into plasma compartments in humans and rats. The significance of this profile for the efficacy, tissue uptake, and retention of isosteviol will have to be further studied.

A Recombinant Humanized Anticocaine Monoclonal Antibody Alters the Urinary Clearance of Cocaine and Its Metabolites in Rats.

A recombinant humanized anticocaine monoclonal antibody, h2E2, has shown potential in the preclinical phases for the treatment of cocaine abuse. The standard tests for cocaine usage are the detection of benzoylecgonine (BE) and cocaine in the urine. This includes workplace drug screens as well as in clinical trials for potential treatments of cocaine abuse. By sequestering cocaine into the plasma compartment, h2E2 prevents cocaine from entering the brain. Due to the altered disposition of cocaine in the presence of h2E2, we investigated the effects of h2E2 on cocaine and metabolite levels in the urine of rats to clarify the use of BE as an endpoint measurement for effectiveness in future clinical trials. The urine concentrations of cocaine and metabolites were considerably altered in the presence of h2E2. After a single injection of h2E2 (120 mg/kg) and cocaine hydrochloride (0.56 mg/kg), the concentration of cocaine and BE excreted into the urine of rats decreased by 92% and 91%, respectively, from vehicle controls. Due to the significant decrease in urinary excretion, BE is not an appropriate indicator of cocaine usage in the presence of h2E2. Another endpoint measurement must be selected for the measurement of cocaine usage in the upcoming clinical trials of h2E2. In contrast to the effects on cocaine and BE urinary excretion, there was a 3-fold increase in ecgonine methyl ester (EME) in the presence of h2E2. Therefore, we conclude that EME is a more appropriate measurement of cocaine intake in the presence of h2E2.

A Three-Compartment Pharmacokinetic Model to Predict the Interstitial Concentration of Talaporfin Sodium in the Myocardium for Photodynamic Therapy: A Method Combining Measured Fluorescence and Analysis of the Compartmental Origin of the Fluorescence.

To evaluate the effectiveness of photodynamic therapy occurring in the interstitial space of the myocardium, we estimated the interstitial concentration of talaporfin sodium in the canine myocardium by constructing a three-compartment pharmacokinetic model based on measured changes in talaporfin sodium plasma concentration and myocardial fluorescence. Differential rate equations of talaporfin sodium concentration in the plasma, interstitial space, and cell compartment were developed with individual compartment volume, concentration, and rate constants. Using measured volume ratios based on histological examinations, we defined that the myocardial fluorescence consisted of the linear addition of fluorescence generated from these three compartments. The rate constants were obtained by fitting to minimize the sum of the squared errors between the measured talaporfin sodium concentrations and the calculated concentrations divided by the number of data points using the conjugate gradient method in MATLAB. We confirmed that this fitting operation may be appropriate, because a coefficient of determination between the measured talaporfin sodium changes and the calculated concentrations using our equations was 0.99. Consequently, to estimate the interstitial concentration in the canine myocardium, we propose a three-compartment pharmacokinetic model construction methodology using measured changes in talaporfin sodium plasma concentration and changes in myocardial fluorescence.

Hydroxyurea Exposure in Lactation—a Pharmacokinetics Study (HELPS)

Hydroxyurea is a potent and safe disease-modifying therapy for both children and adults with sickle cell anemia (SCA). Several decades of research data have documented clinical efficacy characterized by fewer acute complications, improved organ function, and reduced mortality. Relatively sparse data exist, however, regarding the risks and benefits of hydroxyurea for mothers with SCA during pregnancy and lactation. Mothers with SCA who wish to breastfeed their babies are discouraged from taking hydroxyurea, due to mostly theoretical concerns about transfer of the drug into breast milk. Determination of the pharmacokinetics (PK) of hydroxyurea in breast milk, along with accurate measurement of the amounts transferred to infants during lactation, are warranted since hydroxyurea is now commonly prescribed to women of child-bearing years.We designed the prospective HELPS trial (Hydroxyurea Exposure in Lactation: a Pharmacokinetics Study, NCT02990598) with the following objectives: (1) determine the PK profile of hydroxyurea in blood and breast milk; (2) calculate the distribution of hydroxyurea between plasma and milk compartments; (3) measure the amount of hydroxyurea excreted into the milk and urine; and (4) create population PK profiles to estimate infant drug exposure. Lactating women were recruited to receive a single oral dose of hydroxyurea (two 500mg capsules). Breast milk, blood, and urine were collected before the dose and then at scheduled timepoints, including 15, 30, 60, 120, 180, 240, 360, 480, 720, and 1440 minutes. Hydroxyurea concentrations were measured using a validated HPLC assay.A total of 16 women (14 healthy volunteers and 2 with SCA; 11 white and 5 black) were recruited into HELPS; the median age was 31 years (range 26-38 years) and the median weight was 76.3 kg (range 48.8-115.9 kg). All women were currently lactating and breastfeeding their infants for a median duration of 6 months (range 2-22 months). After the 1000mg dose, peak plasma hydroxyurea concentrations ranged from 10-40 µg/mL, typically at 60-120 minutes after dosing although absorption varied among participants. Hydroxyurea was measured in breast milk at ~80% of the plasma concentration, with both compartments having a similar PK profile for each participant, from absorption through distribution and clearance. No hydroxyurea was detected in either plasma or breast milk 24 hours after the dose. With this frequent collection schedule, an average of 2.2 ± 1.0 mg of hydroxyurea transferred into breast milk over the 24-hour timeframe, with the majority transferred in the first 3-6 hours. Most of the drug was excreted into the urine, with an average of 542 ± 178 mg (range 295-876 mg) measured over the 24-hour timeframe; over 90% of urinary excretion occurred in the first 9 hours after the dose.The collection schedule was then modified to assess drug accumulation and equilibrium in more physiologically relevant 3-hour intervals, which reduced the average amount of hydroxyurea transfer to 1.1 mg; this 50% reduction in drug transfer indicated a rapid equilibrium between milk and plasma compartments. Two lactating women with SCA had similar hydroxyurea PK curves for plasma and milk to the 14 healthy volunteers. PK analysis based on the frequent milk collection schedule and a standard 150 mL/kg intake per 24 hours concluded that 0.41 ± 0.14 mg hydroxyurea will transfer per kg of infant weight per day. Using the World Health Organization definition of Relative Infant Dosage (RID), defined as the infant dose (mg/kg/day) compared to the maternal dose (mg/kg/day), this infant hydroxyurea exposure is 3.1%, where <5% is generally considered safe and acceptable. However, since 48% of the drug transfers in the first 3 hours, the RID would be 1.6% if the mother fed her baby before the dose and waited 3 hours before the next feeding, and <1% when using a single “pump and dump” process.The HELPS trial provides definitive measurements and PK data regarding hydroxyurea in human breast milk, confirming that drug does transfer but has a rapid equilibrium between plasma and milk. Breastfeeding mothers will transfer only a small amount of hydroxyurea to their infants, so lactation during hydroxyurea therapy should not be contraindicated. These data are relevant for expanded access to hydroxyurea as a disease-modifying therapy to lactating women around the world. DisclosuresWare:Novartis: Membership on an entity's Board of Directors or advisory committees; Global Blood Therapeutics: Other: advisory board; Bristol Myers Squibb: Research Funding; Addmedica: Research Funding; Agios: Other: advisory board; Nova Laboratories: Consultancy; Biomedomics: Research Funding.

Minimal physiologically-based pharmacokinetic model to investigate the effect of pH dependent FcRn affinity and the endothelial endocytosis on the pharmacokinetics of anti-VEGF humanized IgG1 antibody in cynomolgus monkey

In this study, we developed a first minimal physiologically-based pharmacokinetic (mPBPK) model to investigate the complex interaction effects of endocytosis rate/FcRn binding affinity at both acidic/physiological pH on the pharmacokinetics (PK) of the anti-VEGF IgG1 antibodies. The data used in this study were the PK of the native IgG and humanized anti-VEGF IgG1 antibodies with a wide range FcRn-binding at both acidic and physiological pH in the cynomolgus monkey. The basic structure of the developed mPBPK models consisted of plasma, tissue and lymph compartments. The tissue compartment was subdivided into vascular, endothelial and interstitial spaces. Non-equilibrium binding mechanism was used to describe the FcRn-IgG interaction in the endosome. The fittings in the final model with three pH systems in the endosome compartment showed a good fit based on the visualization of the fitted graphs and the coefficient of variations of the estimated parameters (CV < 50%). The quantitative endocytosis/FcRn binding affinity PK relationships was constructed using the final model to provide better understanding of complex interaction effects of endocytosis rate and FcRn binding on PK of anti-VEGF IgG1 antibodies. This result may serve as an important model-based drug discovery platform to guide the design and development of the future generation of anti-VEGF IgG1 or other therapeutic IgG1 antibodies. In addition, the mPBPK model developed in cynomolgus monkey was successfully used to predict the PK of the anti-VEGF IgG1 antibody (bevacizumab) in human subjects.