Prediction of In Vivo Hepatic Clearance and Half-Life of Drug Candidates in Human Using Chimeric Mice with Humanized Liver

2011 ◽  
Vol 40 (2) ◽  
pp. 322-328 ◽  
Author(s):  
Seigo Sanoh ◽  
Aya Horiguchi ◽  
Kazumi Sugihara ◽  
Yaichiro Kotake ◽  
Yoshitaka Tayama ◽  
...  
Keyword(s):  
Half Life ◽  
Hepatic Clearance ◽  
Chimeric Mice ◽  
Drug Candidates

Immunohistochemical Staining of rFVIIIFc and rFVIII in Liver Cells Differentiates Between a VWF-Dependent and Independent Clearance Pathway in Mice

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2331-2331
Author(s):  
Arjan van der Flier ◽  
Zhan Liu ◽  
David R. Light ◽  
Haiyan Jiang
Keyword(s):  
Endothelial Cells ◽  
Half Life ◽  
Cellular Localization ◽  
Hematopoietic Cells ◽  
Chimeric Mice ◽  
Liver Sinusoid ◽  
Employment Equity ◽  
Biodistribution Studies ◽  
Equity Ownership

Abstract Introduction rFVIIIFc is a fully recombinant fusion protein consisting of a single B domain-deleted human FVIII covalently attached to the dimeric Fc domain of human IgG1. rFVIIIFc has a 1.5-fold extended half-life and decreased clearance compared to rFVIII in patients with hemophilia A (Powell, Blood 2012). The Fc region of rFVIIIFc binds to the neonatal Fc receptor (FcRn), which is part of a naturally occurring pathway that cycles IgG back into circulation, delaying lysosomal degradation. Our previous studies with FcRn-chimeric mice showed that the decreased clearance of rFVIIIFc is mediated by FcRn expressed in somatic cells and not in hematopoietic cells. Biodistribution studies with 125I-rFVIIIFc identified liver as the major organ for rFVIIIFc disposition. Furthermore, qPCR studies showed that three different cell types in liver all express FcRn: hepatocytes (HC), liver sinusoidal endothelial cells (LSEC) and Kupffer cells (KC). In primary liver cell co-cultures, rFVIIIFc and rFVIII were both internalized by somatic LSEC and HC, but not KC, in the absence of von Willebrand factor (VWF). It has been reported that in vivo, 95% of FVIII circulates as a non-covalent complex with VWF (Lenting, JTH 2007) and we found that VWF delays liver uptake of FVIII and improves the circulating half-life of rFVIIIFc in mice. Aim Compare the cellular localization of rFVIII and rFVIIIFc in the murine liver, in the presence and absence of VWF, in order to identify the cells responsible for the prolonged half-life of rFVIIIFc. Methods FVIII KO (HemA) and FVIII/VWF DKO mice were dosed with rFVIII, rFVIIIFc or the mutants rFVIIIFc-IHH and rFVIIIFc-N297A that are deficient in interacting with FcRn and FcgR, respectively. The cellular localization in the liver of rFVIII, rFVIIIFc and mutants was investigated by immunohistochemistry, using a panel of anti-human FVIII monoclonal antibodies or anti-human-IgG (Fc) for detection, along with markers for LSEC, KC, and VWF. Results In HemA mice with circulating endogenous VWF, the majority of both rFVIII and rFVIIIFc signal is found in KC, which co-stain for VWF. In contrast, neither rFVIII nor rFVIIIFc is detected in the endothelial cells of the large vessels that stained for VWF in the Weibel-Palade bodies. Closer examination shows faint vesicular staining by rFVIII in HC in contrast to a diffuse staining by rFVIIIFc in the liver sinusoid. However in FVIII/VWF DKO mice lacking VWF, neither rFVIII nor rFVIIIFc is detected in KC consistent with the notion that internalization of FVIII by KC is mediated by VWF. The majority of rFVIII is found in HC, whereas rFVIIIFc again appears as a diffused liver sinusoidal staining pattern that is more intense than that observed in the HemA mice expressing VWF. These findings suggest free rFVIII is internalized and cleared by HC, while rFVIIIFc is cycled out of the HC and localizes in the Space of Disse, hence its sinusoidal localization. Alternately, in the absence of VWF, rFVIIIFc may cycle through LSEC rather than HC. In order to distinguish these two pathways, an rFVIIIFc variant, rFVIIIFc-IHH, which is not competent to bind FcRn, was tested. In a similar manner as rFVIII, the rFVIIIFc-IHH mutant localizes into HC in DKO mice and is found predominantly in KC in HemA with endogenous VWF. In contrast, rFVIIIFc-N297A, which is not competent to bind FcgR, localizes similarly to rFVIIIFc in DKO mice, excluding a role for FcgR. Conclusions Our current immunohistochemical studies and previous biodistribution and PK studies in mice indicate that there are two parallel, linked clearance pathways for rFVIII and rFVIIIFc. rFVIII or rFVIIIFc complexed with endogenous VWF is internalized predominantly by macrophages, including KC. However, because the VWF-FVIII complex is in constant equilibrium, a fraction of free rFVIII or rFVIIIFc is available for clearance by HC. We propose that this free fraction of rFVIIIFc entering HC is then cycled by FcRn back into the liver sinusoid and into circulation, in contrast to the free rFVIII entering the HC. Staining of the rFVIIIFc-IHH mutant in HC suggests that these cells play a dominant role in vivo, however LSEC may also contribute to cycling of VWF-free rFVIIIFc. The fate of the VWF-bound rFVIIIFc fraction internalized by KC is less clear, however data using FcRn-chimeric mice suggest that FcRn expressed in hematopoietic cells, including KC contributes only marginally to the delayed clearance of rFVIIIFc. Disclosures: van der Flier: Biogen Idec: Employment, Equity Ownership. Liu:Biogen Idec: Employment, Equity Ownership. Light:Biogen Idec: Employment, Equity Ownership. Jiang:Biogen Idec: Employment, Equity Ownership.

Studies of the Metabolism of Asialotransferrins: The Mechanism for the Hypercatabolism of Human Asialotransferrin in the Rabbit

1974 ◽  
Vol 52 (7) ◽  
pp. 645-651 ◽  
Author(s):  
E. Regoeczi ◽  
M. W. C. Hatton
Keyword(s):  
Sialic Acid ◽  
Plasma Protein ◽  
Half Life ◽  
Hepatic Clearance ◽  
The Other ◽  
Human Transferrin ◽  
Human Control ◽  
Clearance Mechanism ◽  
Catabolic Process

The behavior in vivo of human asialotransferrin in rabbits was studied in such a manner as to permit a comparison of this catabolic process with the generalized hepatic clearance mechanism for asialoglycoproteins described by Ashwell and Morell.Progressive desialylation of human transferrin in the range 50–100% yielded transferrin molecules with circulation times inversely proportional to the loss of sialic acid. The shape of plasma protein-bound radioactivity curves indicated that treatment of transferrin with neuraminidase resulted in at least two types of asialo derivatives. The biological half-life of one of them was close to 60% of that of control transferrin and the other one disappeared from the plasma with a half-life of approximately 1.5–2.5 h. Oxidation of over 50% of the terminal galactosyl groups in human asialotransferrin prolonged the circulation time of asialotransferrin.Assays of tissue radioactivities following mixed injection of human control and asialotransferrins showed that the two proteins possessed the same affinity for lung, kidney, and spleen, but asialotransferrin was preferentially taken up by the liver.On the basis of these observations, it seems likely that the mechanism which has been claimed as a generalized pathway for the clearance of asialoglycoproteins is also responsible for the rapid elimination of heterologous human asialotransferrin in the rabbit.

Kinetics of Various 99mTc-Sn-Pyrophosphate Compounds in the Rat

1977 ◽  
Vol 16 (04) ◽  
pp. 157-162 ◽  
Author(s):  
C. Schümichen ◽  
B. Mackenbrock ◽  
G. Hoffmann
Keyword(s):  
Normal Saline ◽  
Half Life ◽  
Compound A ◽  
Short Half Life ◽  
Low Concentrations ◽  
The Stability ◽  
Kinetics Of ◽  
In Vitro Stability

SummaryThe bone-seeking 99mTc-Sn-pyrophosphate compound (compound A) was diluted both in vitro and in vivo and proved to be unstable both in vitro and in vivo. However, stability was much better in vivo than in vitro and thus the in vitro stability of compound A after dilution in various mediums could be followed up by a consecutive evaluation of the in vivo distribution in the rat. After dilution in neutral normal saline compound A is metastable and after a short half-life it is transformed into the other 99mTc-Sn-pyrophosphate compound A is metastable and after a short half-life in bone but in the kidneys. After dilution in normal saline of low pH and in buffering solutions the stability of compound A is increased. In human plasma compound A is relatively stable but not in plasma water. When compound B is formed in a buffering solution, uptake in the kidneys and excretion in urine is lowered and blood concentration increased.It is assumed that the association of protons to compound A will increase its stability at low concentrations while that to compound B will lead to a strong protein bond in plasma. It is concluded that compound A will not be stable in vivo because of a lack of stability in the extravascular space, and that the protein bond in plasma will be a measure of its in vivo stability.

Half-Life of Platelet Factor 4 (PF-4) in Plasma and Platelets from Macaca Mulatta

1977 ◽  
Vol 37 (01) ◽  
pp. 073-080 ◽  
Author(s):  
Knut Gjesdal ◽  
Duncan S. Pepper
Keyword(s):  
Macaca Mulatta ◽  
Half Life ◽  
Human Platelet ◽  
Gel Filtration ◽  
Platelet Factor 4 ◽  
Active Protein ◽  
Platelet Factor ◽  
Membrane Bound

SummaryHuman platelet factor 4 (PF-4) showed a reaction of complete identity with PF-4 from Macaca mulatta when tested against rabbit anti-human-PF-4. Such immunoglobulin was used for quantitative precipitation of in vivo labelled PF-4 in monkey serum. The results suggest that the active protein had an intra-platelet half-life of about 21 hours. In vitro 125I-labelled human PF-4 was injected intravenously into two monkeys and isolated by immuno-precipita-tion from platelet-poor plasma and from platelets disrupted after gel-filtration. Plasma PF-4 was found to have a half-life of 7 to 11 hours. Some of the labelled PF-4 was associated with platelets and this fraction had a rapid initial disappearance rate and a subsequent half-life close to that of plasma PF-4. The results are compatible with the hypothesis that granular PF-4 belongs to a separate compartment, whereas membrane-bound PF-4 and plasma PF-4 may interchange.

Turnover of Human Extrinsic (Tissue-Type) Plasminogen Activator in Rabbits

1981 ◽  
Vol 46 (03) ◽  
pp. 658-661 ◽  
Author(s):  
C Korninger ◽  
J M Stassen ◽  
D Collen
Keyword(s):  
Plasminogen Activator ◽  
Intravenous Injection ◽  
Active Site ◽  
Half Life ◽  
Degradation Products ◽  
Gel Filtration ◽  
Tissue Type ◽  
Organ Distribution ◽  
Small Rise

SummaryThe turnover of highly purified human extrinsic plasminogen activator (EPA) (one- and two-chain form) was studied in rabbits. Following intravenous injection, EPA-activity declined rapidly. The disappearance rate of EPA from the plasma could adequately be described by a single exponential term with a t ½ of approximately 2 min for both the one-chain and two-chain forms of EPA.The clearance and organ distribution of EPA was studied by using 125I-labeled preparations. Following intravenous injection of 125I-1abeled EPA the radioactivity disappeared rapidly from the plasma also with a t ½ of approximately 2 min down to a level of 15 to 20 percent, followed by a small rise of blood radioactivity. Gel filtration of serial samples revealed that the secondary increase of the radioactivity was due to the reappearance of radioactive breakdown products in the blood. Measurement of the organ distribution of 125I at different time intervals revealed that EPA was rapidly accumulated in the liver, followed by a release of degradation products in the blood.Experimental hepatectomy markedly prolonged the half-life of EPA in the blood. Blocking the active site histidine of EPA had no effect on the half-life of EPA in blood nor on the gel filtration patterns of 125I in serial plasma samples.It is concluded that human EPA is rapidly removed from the blood of rabbits by clearance and degradation in the liver. Recognition by the liver does not require a functional active site in the enzyme. Neutralization in plasma by protease inhibitors does not represent a significant pathway of EPA inactivation in vivo.

Clearance and In Vivo Release by Heparin of Human Platelet Factor 4 (PF4) in the Rabbit

1984 ◽  
Vol 52 (02) ◽  
pp. 157-159 ◽  
Author(s):  
M Prosdocimi ◽  
N Scattolo ◽  
A Zatta ◽  
F Fabris ◽  
F Stevanato ◽  
...  
Keyword(s):  
Half Life ◽  
Human Platelet ◽  
Slow Component ◽  
Platelet Factor 4 ◽  
Platelet Factor ◽  
In Vivo Release ◽  
Partial Release ◽  
Different Doses ◽  
New Zealand Rabbits

Summary13 male New Zealand rabbits were injected with two different doses (25 μg/Kg and 100 μg/Kg) of human platelet factor 4 antigen (PF4). The disappearance of the protein was extremely fast with an half-life for the fast component of 1.07 ± 0.16 and 1.76 ± 0.11 min respectively. The half-life for the slow component, detectable only with the highest dosage, was 18.8 min.The administration of 2500 I.U. of heparin 30 min after PF4 administration induced a partial release of the injected protein and its clearance from plasma was slow, with half-life of 23.3 ± 5.9 min and 30.9 ± 2.19 min respectively.

Recent Advances in the Development of Antimicrobial Peptides (AMPs): Attempts for Sustainable Medicine?

2018 ◽  
Vol 25 (21) ◽  
pp. 2503-2519 ◽  
Author(s):  
Anne Kokel ◽  
Marianna Torok
Keyword(s):  
Side Effects ◽  
Antimicrobial Peptides ◽  
Potential Drug ◽  
Green Technologies ◽  
Specific Antibodies ◽  
Structural Modifications ◽  
Drug Candidates ◽  
Induce Resistance ◽  
Conventional Antibiotics

Background: Since the first isolation of antimicrobial peptides (AMPs) they have attracted extensive interest in medicinal chemistry. However, only a few AMP-based drugs are currently available on the market. Despite their effectiveness, biodegradability, and versatile mode of action that is less likely to induce resistance compared to conventional antibiotics, AMPs suffer from major issues that need to be addressed to broaden their use. Notably, AMPs can lack selectivity leading to side effects and cytotoxicity, and also exhibit in vivo instability. Several strategies are being actively considered to overcome the limitations that restrain the success of AMPs. Methods: In the current work, recent strategies reported for improving AMPs in the context of drug design and delivery were surveyed, and also their possible impact on patients and the environment was assessed. Results: As a major advantage AMPs possess an easily tunable skeleton offering opportunities to improve their properties. Strategic structural modifications and the beneficial properties of cyclic or branched AMPs in term of stability have been reported. The conjugation of AMPs with nanoparticles has also been explored to increase their in vivo stability. Other techniques such as the coupling of AMPs with specific antibodies aim to increase the selectivity of the potential drug towards the target. These strategies were evaluated for their effect on the environment highlighting green technologies. Conclusion: Although further research is needed taking into account both environmental and human health consequences of novel AMPs, several of these compounds are promising drug candidates for use in sustainable medicine.

Flavonoids as P-gp Inhibitors: A Systematic Review of SARs

2019 ◽  
Vol 26 (25) ◽  
pp. 4799-4831 ◽  
Author(s):  
Jiahua Cui ◽  
Xiaoyang Liu ◽  
Larry M.C. Chow
Keyword(s):  
Molecular Mechanisms ◽  
Metastatic Cancer ◽  
Drug Candidates ◽  
Chemical Structures ◽  
Transporter Family ◽  
Promising Area ◽  
New Generation ◽  
Nontoxic Inhibitors

P-glycoprotein, also known as ABCB1 in the ABC transporter family, confers the simultaneous resistance of metastatic cancer cells towards various anticancer drugs with different targets and diverse chemical structures. The exploration of safe and specific inhibitors of this pump has always been the pursuit of scientists for the past four decades. Naturally occurring flavonoids as benzopyrone derivatives were recognized as a class of nontoxic inhibitors of P-gp. The recent advent of synthetic flavonoid dimer FD18, as a potent P-gp modulator in reversing multidrug resistance both in vitro and in vivo, specifically targeted the pseudodimeric structure of the drug transporter and represented a new generation of inhibitors with high transporter binding affinity and low toxicity. This review concerned the recent updates on the structure-activity relationships of flavonoids as P-gp inhibitors, the molecular mechanisms of their action and their ability to overcome P-gp-mediated MDR in preclinical studies. It had crucial implications on the discovery of new drug candidates that modulated the efflux of ABC transporters and also provided some clues for the future development in this promising area.

Current Approaches to Drug Discovery for Chagas Disease: Methodological Advances

2019 ◽  
Vol 22 (8) ◽  
pp. 509-520
Author(s):  
Cauê B. Scarim ◽  
Chung M. Chin
Keyword(s):  
Drug Discovery ◽  
Chagas Disease ◽  
Drug Candidates ◽  
Lead Compounds ◽  
New Drug ◽  
Compound Libraries ◽  
Screening Assays ◽  
Cruzi Infection

Background: In recent years, there has been an improvement in the in vitro and in vivo methodology for the screening of anti-chagasic compounds. Millions of compounds can now have their activity evaluated (in large compound libraries) by means of high throughput in vitro screening assays. Objective: Current approaches to drug discovery for Chagas disease. Method: This review article examines the contribution of these methodological advances in medicinal chemistry in the last four years, focusing on Trypanosoma cruzi infection, obtained from the PubMed, Web of Science, and Scopus databases. Results: Here, we have shown that the promise is increasing each year for more lead compounds for the development of a new drug against Chagas disease. Conclusion: There is increased optimism among those working with the objective to find new drug candidates for optimal treatments against Chagas disease.

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