Literature

Elephant disease causes death by hemorrhage… Lyophilized platelets may help!

Authors: Jennifer C. Kishbaugh, DVM, Marc T. Valitutto, VMD, Janelle E. Over, MS, Dawn M. Zimmerman, DVM, MS, Lauren L. Howard, DVM, Dennis L. Schmitt, DCM, PhD, Carlos R. Sanchez, DVM, Suzan Murray, DVM.

The in-human care population of elephants is plagued by an endotheliotropic herpes virus that attacks young elephants and causes an acute loss of platelets with associated bleeding.  BodeVet, Inc. working with the Smithsonian Institute has begun the process of producing lyophilized platelets for zoos to have available when bleeding occurs.  A clinical trial is proposed to determine if platelet transfusion improves outcome, saving these magnificent animals from death.

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Safety Evaluation of Lyophilized Canine Platelets in a Model of CABG

Authors: Todd M. Getz, PhD, Anne S.Hale, DVM, Arthur P. Bode, PhD, Mark D. Johnson, MS, G. Michael Fitzpatrick, PhD

Project Overview: Cellphire evaluated the safety of Lyophilized Canine Platelets (LCP) in comparison to Liquid Stored Canine Platelets following intravenous administration in a model of on-pump coronary artery bypass graft (CABG) in the canine.

Wound-healing properties of trehalose-stabilized freeze-dried outdated platelets

Authors: Ruth Sum,* Sarah Hager,* Giorgio Pietramaggiori, Dennis P. Orgill, Josh Dee, Alan Rudolph, Cindy Orser, G. Michael Fitzpatrick, and David Ho

Project Overview:

Wounds treated with multiple doses of VEGF and a single dose of freeze-dried platelets reached 90% closure faster than wounds left untreated. A single administration of trehalose lyophilized and reconstituted platelet preparations enhanced diabetic wound healing, therefore representing a promising strategy for the treatment of non-healing wounds.

Loading Platelets with Biological Agents for Enhanced Local Delivery

Authors: Dr. Cindy Orser, Dr. Keith Moskowitz, Richard Cliff, Dr. Alan Rudolph, Josh Dee, Nannette Mittereder

Project Overview:

Use Platelets to engineer uptake and delivery of therapeutic compounds to specific sites

Trehalose Stabilized Freeze Dried Human Platelets, Thrombosomes®, Persist in Circulation 24 Hours After Infusion and Are Non-Immunogenic In New Zealand White Rabbits

Authors: Vibhudutta Awasthi, Josh Dee, Mike Fitzpatrick, Anna Koh, Richard Cliff, Giora Feuerstein

Project Overview:

Thrombosomes®, a human platelet derived hemostatic agent, (PDHA) is being developed to meet a critical unmet medical need, e.g. a safe PDHA that restores hemostasis in patients with a coagulopathy that does not respond to standard treatment

Trehalose Stabilized Freeze Dried Human Platelets, Thrombosomes®, Reduce Blood Loss in Thromboctopenic Rabbit Ear Bleed Model By As Much As 89.5%

Authors: Vibhudutta Awasthi, Josh Dee, Mike Fitzpatrick, Anna Koh, Richard Cliff, Giora Feuerstein

Project Overview:

Thrombosomes®, are lyophilized derivative of human platelets, is being developed for treatment of civilian or military combat related trauma and shock conditions associated with life-threatening bleeding

Trehalose Stabilized Freeze Dried Human Platelets, Thrombosomes®, Express Surface Markers, Thromboelastogram (TEG) Values and Size Distribution Similar To Two To Three Day Old Stored Platelets

Authors: Josh Dee, Mike Fitzpatrick, Anna Koh, Richard Cliff, Giora Feuerstein

Project Overview:

Thrombosomes® is lyophilized derivative of human platelets developed as a hemostatic product for treatment of civilian as well as combat related trauma and shock conditions associated with life-threatening bleeding. Thrombosomes is produced by a proprietary process that includes Trehalose, other carbohydrates, and a custom lyophilization process.

Wound Healing Properties Of Reconstituted Freeze-Dried Platelets

Authors: D. Ho, G. Pietramaggiori, K. Moskowitz, R. Sum, J. Dee, A. Rudolph, C. Burch, W. Pebley, D. Orgill, C. Orser

Project Overview:

The use of fresh platelets has gained value in medicine as an essential part of wound treatments. This is not surprising since platelets contain a number of bioactive factors that contribute to the process of wound healing such as: platelet derived platelet-derived growth factor (PDGF) and transforming growth factor (TGF). Fresh platelets’ short shelf life limits platelet based therapies. If platelets can be stabilized in a freeze-dried form (FDP) then long-term storage and pathogen inactivation methods become possible.

Dried platelets in a Swine Model of Liver Injury

Authors: Kenji Inaba,* Galinos Barmparas,* Peter Rhee,† Bernardino C. Branco,‡ Michael Fitzpatrick,§ Obi T. Okoye,* and Demetrios Demetriades*

Project Overview: Lyophilization may facilitate production of a safe, portable, easily storable, and transportable source of platelets for bleeding patients. The objective of this study was to examine the impact of lyophilized human and porcine platelets in a swine liver injury model of nonsurgical hemorrhage.

Supporting Scientific Research:

Assessment of platelet growth factors in supernatants from rehydrated freeze-dried equine platelets and their effects on fibroblasts in vitro

Authors: Fern Tablin, VMD, PhD; Naomi J. Walker, BA; Sara E. Hogle, DVM; Suzanne M. Pratt, DVM; Jeffrey W. Norris, PhD

Objective:

To determine whether platelet growth factors are preserved in supernatants obtained from rehydrated trehalose-stabilized, freeze-dried (lyophilized) equine latelets and whether those growth factors stimulate fibroblast proliferation and migration and enhance fibroblast-associated contraction in a collagen gel assay.

Temporal Growth Factor Release from Platelet-Rich Plasma, Trehalose Lyophilized Platelets, and Bone Marrow Aspirate and Their Effect on Tendon and Ligament Gene Expression

Authors: Taralyn McCarrel and Lisa Fortier

Abstract:

Platelet-rich plasma (PRP) has generated substantial interest for tendon and ligament regeneration because of the high concentrations of growth factors in platelet a-granules. This study compared the temporal release of growth factors from bone marrowaspirate (BMA), PRP, and lyophilized platelet product (PP), and measured their effects on tendon and ligament gene expression. Blood and BMA were collected and processed to yield PRP and plasma. Flexor digitorum superficialis tendon (FDS) and suspensory ligament (SL) explants were cultured in 10%plasma inDMEM(control), BMA,PRP, or PP. TGF-b1 and PDGF-BBconcentrations were determined at 0, 24, and 96 h of culture using ELISA. Quantitative RT-PCR for collagen types I and III (COL1A1, COL3A1), cartilage oligomeric matrix protein (COMP), decorin, and matrix metalloproteinases-3 and 13 (MMP-3, MMP-13) was performed. TGF-b1 and PDGF-BB concentrations were highest in PRP and PP. Growth factor quantity was unchanged in BMA, increased in PRP, and decreased in PP over 4 days. TGF-b1 and platelet concentrations were positively correlated. Lyophilized PP and PRP resulted in increased COL1A1:COL3A1 ratio, increased COMP, and decreased MMP-13 expression. BMA resulted in decreased COMP and increased MMP-3 and MMP-13 gene expression. Platelet concentration was positively correlated with COL1A1, ratio of COL1A1:COL3A1, and COMP, and negatively correlated with COL3A1, MMP-13,and MMP-3.White blood cell concentration was positively correlated withCOL3A1,MMP3,andMMP13,and negatively correlated with a ratio of COL1A1:COL3A1, COMP, and decorin. These findings support further in vivo investigation of PRP and PP for treatment of tendonitis and desmitis.

Trehalose lyophilized platelets for wound healing

Authors: Giorgio Pietramaggiori, MD; Arja Kaipainen, MD, PhD; David Ho, PhD; Cindy Orser, PhD; Walter Pebley, PE; Alan Rudolph, PhD, MBA; Dennis P. Orgill, MD, PhD

Abstract:

Fresh platelet preparations are utilized to treat a wide variety of wounds, although storage limitations and mixed results have hampered their clinical use. We hypothesized that concentrated lyophilized and reconstituted platelet preparations, preserved with trehalose, maintain and possibly enhance fresh platelets’ ability to improve wound healing. We studied the ability of a single dose of trehalose lyophilized and reconstituted platelets to enhance wound healing when topically applied on full-thickness wounds in the genetically diabetic mouse. We compared these results with the application of multiple doses of fresh platelet preparations and trehalose lyophilized and reconstituted platelets as well as multiple doses of vascular endothelial growth factor (VEGF) and wounds left untreated. Trehalose lyophilized and reconstituted platelets, in single and multiple applications, multiple applications of fresh platelets and multiple applications of VEGF increased granulation tissue deposition, vascularity, and proliferation when compared with untreated wounds, as assessed by histology and immunohistochemistry. Wounds treated with multiple doses of VEGF and a single dose of freeze-dried platelets reached 90% closure faster than wounds left untreated. A single administration of trehalose lyophilized and reconstituted platelet preparations enhanced diabetic wound healing, therefore representing a promising strategy for the treatment of nonhealing wounds.

Single Dose Toxicity Study Of Canine Thrombosomes™ In Dogs Followed By A 14-Day Observation Period

Study Director: Jonathan C. White, M.S.A.

Abstract:

This study was conducted for Cellphire, Inc., to evaluate the safety of the test article, Canine Thrombosomes™, following a single infusion dose of 10x the native platelet protein per animal (10x) or 3 doses of 1x the native platelet protein per animal (3x). Two treatment groups of six male and six female beagle dogs were to be administered the test article at respective doses of 1x or 10x, with half the animals necropsied on each Day 2 and 15. Animals receiving the 1x dose were to receive doses three times on Day 1, approximately 2 hours between doses, followed by one administration of Fresh Canine Platelets (100 mL). Due to a shortage of test article, three animals/sex were administered the 1x or 10x dose then scheduled for necropsy on Day 15 and two animals/sex were dosed for the scheduled Day 2 necropsy. Due to a dosing error, the two animals/sex scheduled for the Day 2 necropsy, were actually administered a single dose of approximately 2.4x followed by the fresh canine platelets. Animals in the 10x dose group received a single administration of the test article followed by a single dose of fresh canine platelets. One additional group of six animals/sex served as the control and received a single dose of the vehicle, Thrombosomes™ Buffer at a fixed volume of 105 mL, the mean dose volume administered to the 10x dose group animals. The test article or vehicle was administered to all groups via intravenous infusion.

Based on the results of this study, canine thrombosomes administered as a single dose of 10x, a single dose of 2.4x (misdosed group) or 3 doses of 1x administered approximately 2 hours apart were considered non-adverse.

Clinical Development of Stabilized Platelets for In-fusible Clinical Applications

Investigator: William T. Phillips, MD

Co-Investigators: Vibhudutta Awasthi, PhD and Beth Goins, PhD

Abstract:

To study the circulation kinetics of freeze-dried platelets (FDP) in normal rabbits and to investigate the effect of re-injection of human freeze-dried platelets (thrombosomes).

There were no observed differences in accumulation of endogenous platelets and FDPs in other tissues. It is clear that accumulation in blood, liver and spleen were substantial among endogenous fresh platelets and various FDPs. The pattern was identical when the data was registered in per organ basis. It is important to note that the regardless of the source of freeze-dried platelets, biodistribution of various FDPs in rabbits was remarkably similar.