Controlled release process to recover heterologous glycosylphosphatidylinositol membrane anchored proteins from CHO cells

Kennard, Malcolm L.; Food, M. R.; Jefferies, Wilfred A.; Piret, James M.

doi:10.1002/bit.260420411

Cited by 15 publications

(10 citation statements)

References 17 publications

(1 reference statement)

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“…The expression then fell steadily to less than half of the maximum expression by 10 days. This observation was similar to a previous study based on p97-transfected wild-type CHO (CHO-WTB) cells (Kennard et al, 1993). Based on the measurement of the concentration of p97 in the phosphatidylinositolphospholipase C (PI-PLC) solution used to treat 108 cells, it was possible to determine the number of molecules of p97 per cell.…”

Section: Resultssupporting

confidence: 86%

A novel iron uptake mechanism mediated by GPI-anchored human p97.

et al. 1995

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The established process for iron uptake into mammalian cells involves transferrin and its receptor. Here, the role of the glycosyl‐phosphatidylinositol (GPI)‐linked transferrin homologue, melanotransferrin or p97, was studied using CHO cell lines defective in the transferrin receptor (TR) and transfected with human TR and/or human p97. The presence of p97 doubled the iron uptake, which could be explained by the binding of one atom of iron to one molecule of p97. The internalization of iron was shown to be temperature sensitive and saturated at a media iron concentration of 2.5 micrograms/ml with a Vmax of 0.1 pmol Fe/10(6) cell/min and a Km of 2.58 microM for p97. Treatment of the cells with either phosphatidylinositol‐phospholipase C or monoclonal antibodies against p97 resulted in over a 50% reduction and a 47% increase in the iron uptake respectively. These data identify p97 as a unique cell surface GPI‐anchored, iron binding protein involved in the transferrin‐independent uptake of iron in mammals.

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Section: Resultssupporting

confidence: 86%

A novel iron uptake mechanism mediated by GPI-anchored human p97.

et al. 1995

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“…Conjugates (p97-ADR) were purified on 5 mL D-Salt Excellulose (Pierce Chemical Co.) desalting columns equilibrated with PBS. Purified p97-ADR was assessed by SDS-PAGE, anion exchange chromatography and Western blot analysis using the primary anti-human p97 monoclonal mouse antibody L235 [63]. Individual conjugates were designated as SYN002, SYN018, SYN019, and SYN020.…”

Section: Methodsmentioning

confidence: 99%

A Unique Carrier for Delivery of Therapeutic Compounds beyond the Blood-Brain Barrier

Karkan¹,

Pfeifer

Vitalis

et al. 2008

PLoS ONE

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BackgroundTherapeutic intervention in many neurological diseases is thwarted by the physical obstacle formed by the blood-brain barrier (BBB) that excludes most drugs from entering the brain from the blood. Thus, identifying efficacious modes of drug delivery to the brain remains a “holy grail” in molecular medicine and nanobiotechnology. Brain capillaries, that comprise the BBB, possess an endogenous receptor that ferries an iron-transport protein, termed p97 (melanotransferrin), across the BBB. Here, we explored the hypothesis that therapeutic drugs “piggybacked” as conjugates of p97 can be shuttled across the BBB for treatment of otherwise inoperable brain tumors.ApproachHuman p97 was covalently linked with the chemotherapeutic agents paclitaxel (PTAX) or adriamycin (ADR) and following intravenous injection, measured their penetration into brain tissue and other organs using radiolabeled and fluorescent derivatives of the drugs. In order to establish efficacy of the conjugates, we used nude mouse models to assess p97-drug conjugate activity towards glioma and mammary tumors growing subcutaneously compared to those growing intracranially.Principal FindingsBolus-injected p97-drug conjugates and unconjugated p97 traversed brain capillary endothelium within a few minutes and accumulated to 1–2% of the injected by 24 hours. Brain delivery with p97-drug conjugates was quantitatively 10 fold higher than with free drug controls. Furthermore, both free-ADR and p97-ADR conjugates equally inhibited the subcutaneous growth of gliomas growing outside the brain. Evocatively, only p97-ADR conjugates significantly prolonged the survival of animals bearing intracranial gliomas or mammary tumors when compared to similar cumulated doses of free-ADR.SignificanceThis study provides the initial proof of concept for p97 as a carrier capable of shuttling therapeutic levels of drugs from the blood to the brain for the treatment of neurological disorders, including classes of resident and metastatic brain tumors. It may be prudent, therefore, to consider implementation of this novel delivery platform in various clinical settings for therapeutic intervention in acute and chronic neurological diseases.

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“…The conformation of the p97 after conjugation was assumed to be retained as demonstrated by Western blot analysis (data not shown). In addition, the ability to measure p97 concentration using the immunofluorescence assay [13], which involved the use of two anti-p97 monoclonal antibodies, also confirmed that the conformation and hence activity of p97 had been retained.…”

Section: Preparation Of the P97-adr Conjugatesmentioning

confidence: 60%

“…Three separate batches of conjugate were prepared from ADR-SMCC produced by Method 2 and were called SYN018, SYN019 and SYN020. Western blot analysis on the conjugates was carried out according to standard procedures using the primary anti p97 monoclonal mouse antibody L235 [13].…”

Section: Conjugation Of P97 To Adrmentioning

confidence: 99%