ClotFoam
An Intracavitary Hemostatic Agent
for Use in Cases of Severe Hemorrhage Without Need of Compression and/or Sutures
Hemorrhage resulting from traumatic injuries is a major cause of death in accidents, and the primary cause of death
on the battlefield. The vast majority of these deaths occur in the field before the injured can be transported
to a treatment facility.
Tissue adhesives and sealants have been developed to control bleeding; but, since all existing haemostatic intracavitary
agents are designed to be used in the operating room, not in an emergency at the site of accident or in the battlefield,
hemorrhage is often lethal. Early and effective hemorrhage control can save more lives than any other measure.
While ClotFoam F10 has been designed for severe trauma, F9 is fine tuned for use in laparoscopic surgery and minimally invasive procedures.
ClotFoam can be used as an anti-adhesive hemostatic in general surgery.It is an advanced technology that bypasses the fibrinogen cleavege, producing a strong fibrin polymer, always active, always strong, always effective.
ClotFoam® is a 2nd generation fibrin sealant embedded
in a biomimetic complex polymer. Cross-linked in
situ, ClotFoam stops bleeding from severe intracavitary
trauma and achieve hemostasis without compression and/or sutures outside the operating room. It is a product in
a viscous liquid phase, composed of three parts. When delivered through a CO2
propellant, the solutions form a foamy gel that spreads
throughout the cavity to promote adhesiveness and stimulate the coagulation cascade. The foam enhances the distribution
of active clotting agents and provides a scaffold upon which the fibrin network can distribute and thereby adhere
to and bind to lacerated tissue. ClotFoam allows non-invasive application and dissemination of the agent in the
peritoneal or other body cavities; adheres and compress lacerated or wound tissue to prevent flow of blood; and
maintains the necessary components over the wound to produce a platelet plug followed by a fibrin clot that stimulates
the coagulatory cascade. Rapid formation of a strong hydrogel, effective cleavage, and rapid polymerization to
produce a functional fibrin clot over lacerated bleeding tissue ensures that the sealant remains at the site of
application without being washed away by blood: High tensile strength and adhesive strength characterize the clot
produced by the agent. ClotFoam's fibrin components can be kept in
solution for several weeks, ready-to-use.
ClotFoam was developed with the support of the Defense
Advanced Research Project Agency (DARPA), The National Heart, Blood, and Lung Institute(NHLBI) of the national
Institutes of Health (NIH); the Maryland Technology Development Corporation ( TEDCO ), and the University of Maryland.
Animal studies were conducted by Martin Bluth, MD PhD, at the animal facility of SUNY Downstate and by Grant Bochicchio,
MD, PhD at the Trauma Center, and University of Maryland at Baltimore. Chemical research was conducted at Biomedica's
laboratories located at UMBC TechCenter, in Baltimore Maryland and at the Biotech Center of the University of Maryland,
Baltimore.
Aortic and cava vein close and open cavity model Experiments Experiments were performed simultaneously at the University of Maryland School of Medicine (UMB) by Grant V. Bochicchio MD, MPH, FACS, Associate Professor of Surgery, Director of Clinical and Outcomes Research R Adams Cowley Shock Trauma Center, Study and by Michael Kilbourne, MD approved by the IACUC UMB, and at The SUNY Downstate School of Medicine, Brooklyn New York, by Martin Bluth, MD. PhD.
Methods: In this model, a midline laparotomy is made. The bowel is retracted away from the retroperitoneum and the aorta is identified. The aorta is clamped just below the renal arteries and just above the bifurcation of the iliac arteries, effectively gaining infrarenal proximal and distal aortic control. The infrarenal aorta is then pierced with a 25 gauge needle once on both the left and right sides of the vessel. After 6 seconds of uncontrolled bleeding, 500 microliters of ClotFoam is applied diffusely throughout the intraperitoneal cavity. After completion of foam application, time to hemostasis is measured. The abdomen is then closed. Immediately after injury, the rat is given Ringer's solution to maintain mean arterial pressure at about 70-80% of initial MAP (if possible) which is the current standard resuscitation technique for trauma patients. The rat is observed for 20 minutes. After 20 minutes, the animal is re-explored through the same midline incision. All of the blood is collected with pre-weighed gauze pads and total blood loss is calculated.
Results: Seventeen animals underwent aortic injury. Animals were randomized into 4 different groups. The three ClotFoam groups each had a different formulation. The final group received no treatment. Survival was 100% at 30 minutes for all animals treated with ClotFoam. No animals survived the injury in the no treatment group. All pre-injury MAP were similar. Table 1 below summarizes the outcomes measured in each group.
Table 1: Comparison of outcomes for aortic-injured animals with three different ClotFoam formulations and those without treatment: Resuscitation index is defined as the resuscitation MAP percentage of pre-injury MAP. P values for all outcomes and all formulations are < 0.001 compared to no agent.
| Outcome | Form-1 (N=5) | Form #2(N=5) | Form 3(N=4) | No agent(N=3) |
| Time to hemostasis (s) | 16.0 ± 6.0 | 12.2 ± 2.9 | 19.3 ± 2.5 | N/A |
| Total blood loss (ml) | 7.0 ± 0.9 | 5.2 ± 0.5 | 7.6 ± 0.6 | 16.3 ± 0.3 |
| Resuscitation index (%) | 68.2 ± 6.2 | 68.8 ± 14.0 | 73.9 ± 5.7 | 26.8 ± 2.4 |
| Resuscitation volume (ml) | 10 ± 0 | 12 ± 4.5 | 13 ± 3.6 | 20.3 ± 2.5 |
| Survival (min) | 30 ± 0 | 30 ± 0 | 30 ± 0 | 18.3 ± 2.9 |
Cava Vein Model
The second model is a liver/vena caval injury
model. In this model, a small upper midline laparotomy is made. The left lobe of the liver and the vena caval are
exposed and isolated. A small incision is created in the right lower quadrant and the ClotFoam applicator tip is
placed through that incision so that the opening is intraperitoneal but remote from where the injury will take
place. Next, the injury is created by sharply transecting the left liver lobe, and then creating a stab injury
into the vena cava. The mini-laparotomy incision is rapidly closed with staples. ClotFoam is then injected into
the closed abdominal cavity. Resuscitation, observation and blood loss measurements are collected, as mentioned
above. Given the closed cavity, time to hemostasis is not measured (Figure 1).
Eight animals underwent liver/vena caval injuries. Animals
were then randomized into 6 different groups. The 5 ClotFoam groups each had a different formulation. The final
group received no treatment. Survival was 100% at 30 minutes for all animals treated with ClotFoam. The single
animal in the no treatment group died at 18 minutes from injury. All pre-injury MAP were similar. Table 2 below
summarizes
Figure 1. The
applicator was placed into the intraperitoneal cavity. At the 20 minute mark after liver/caval injury, the animal
was opened fully to expose the injured area. As shown, ClotFoam is present (white material) in the pooled blood
of area B, demonstrating its ability to move to target tissue despite blind (and remote) intracavity application.
Table 2: Comparison of outcomes for liver/cava-injured animals conducted at UMB with five different ClotFoam formulations at various dilutions and those without treatment. Resuscitation index is defined as the resuscitation MAP percentage of pre-injury MAP.
| Outcome | Form 1 (N=4) | Form 2 (N=4) | Form 3 (=4) | No agent (N=4) |
| Total blood loss (ml) | 5.1 | 4.5 | 4.2 | 14.7 |
| Resuscitation index (%) | 73.0 | 74.7 | 92.9 | 54.3 |
| Resus citation volume (ml) | 10 | 10 | 10 | 16 |
| Survival (min) | 30 | 30 | 30 | 18 |
Aortic Vein model Experimental Procedure Spague Dawley rats were monitored for blood pressure and mean arterial pressure (MAP) via femoral catheterization and A-line placement and subjected to laparotomy as previously described in our Phase I submission. The great vessels were dissected and visualized, and the abdominal aorta was punctured with a 27 gauge needle.Blood loss occurred under high pressure. Blood pressure and (MAP) levels dropped from baseline levels of approximately 151/110 (120) to 38/31 (34) after 10 seconds. At this point ClotFoam formulation was administered. Administration took place with the mixing of proprietary components under CO2 pressure into the abdominal cavity through the device shown in Figure 3 which covered the aortic lesion ( Figure 4 ).
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| Figure 2. ClotFoam administration apparatus. Syringes of ClotFoam components. | Figure 3. Stabilization
of ClotFoam after aortic puncture. |
Figure 4 . ClotFoam administration coats the aorta in addition to other abdominal organs in a foam like fashion. |
Within 2 minutes after ClotFoam administration, the BP and (MAP) began to rise to 82/46 (57). Normal saline was administered for volume resuscitation and BP (MAP) did not drop demonstrating lack of plug leakage. Ten minutes after ClotFoam Administration the BP and (MAP) approached baseline levels (133/124 [110]) and were maintained over 2 hours when animals were sacrificed, and tissue at the site of the lesion (abdominal aorta) was obtained for histology.
Cava Vein Model: Spague Dawley rats were monitored for blood pressure and mean arterial pressure (MAP) via femoral catheterization and A-line placement and subjected to laparotomy as previously described. The great vessels were dissected and visualized ( Figure 5) and in this case the vena cava was punctured with a 27 gauge needle which inflicts a low pressure injury with substantial blood loss. ClotFoam was administered as described for the aortic puncture above, and similar blood pressure and MAP readings were obtained to the aortic insult. As shown in Figure 6, 7 , ClotFaom administration to the site of the vena cava leasion provides a stable cap of foam which prohibits any further blood loss.
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| Figure 5. Visualization of great vessels (under cotton applicator). | Figure 6. Rapid oozing occuring after vena cava puncture with a 27 gauge needle |
Figure7.Stabilization of ClotFoam after vena cava puncture. |
Close Cavity Model
Sprague Dawley rats were monitored for blood
pressure and mean arterial pressure (MAP) via femoral catheterization and A-line placement. In this approach no
laparotomy was performed. Instead a #14 blade was used to slice through the upper right abdominal quadrant more
closely mimicking a knife wound. The area of wound was determined by ultrasound. Prior to the insult a small incision
(0.5-1.0 cm) was placed on the lower left abdomen ( Figure
9 ) to allow placement of the delivery apparatus
( Figure 10 )
into the abdominal cavity for ClotFoam administration.
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| Figure 8. Preparation for ClotFoam delivery in a closed abdominal wound. | Fig 9 . A small peritoneal opening (highlighted in circle) was fashioned prior to insult to allow for ClotFoam administration | Figure 10. Demostration of liver laceration by knife wound with #14 blade. As shown in highlighted circle the blade effectively traversed through the liver. |
Similar drops in blood pressure and MAP occurred after the blade was used to puncture the liver. After 10 seconds ClotFoam was administered through the lower left abdominal port under manual pressure. Once again the blood pressure stabilized and hemostatsis was maintained through 2 hours when animals were sacrificed for analysis.
Table 3: Summary of ClotFoam effects in three different hemorrhagic models: Data represent one of 3-4 experiments in each model. Details of each model are described in the text. Control represents puncture model with NS treatment alone. Numbers represent blood pressure and mean arterial pressure (MAP - in parenthesis) at baseline 10 seconds, 10 minutes and two hours post insult. NS – normal saline administration in ml. NA – not applicable (control animals died before 2 end point). TTH – total time to hemostatsis, measured as function of a sustained and maintained rise in blood pressure and MAP of 70.
| Model | N# | baseline | 10 sec | NS (ml) | 10 min | 2hrs | TTH | Survival |
| Aortic | 3 | 151/110 (120) | 38/31 (34) | 4.0 | 133/124 (110) | 131/117 (124) | 3 m | all |
| Venous | 4 | 140/105 (115) | 63/34 (41) | 3.5 | 109/69 (83) | 130/72 (97) | 2 m | all |
| Puncture | 4 | 162/102 (128) | 66/41 (50) | 4.2 | 131/69 (87) | 135/87 (102) | 2 m | all |
| Control Venous | 3 | 150/112 (128) | 50/37 (41) | 9.7 | 75/38 (48) | NA | NA | none |
| Control aortic | 3 | 153/108(119) | 42/34 (33) | 10.5 | N/A | N?A | N/A | none |
Clot Histology
Liver section samples were collected from all
animals at necropsy. Samples of liver, containing the wounded site, were preserved in 5% formalin and processed
using standard histology techniques. Fixed tissue samples were embedded in paraffin wax (melting point 56°C)
and sectioned at 2–3 µm. Glass-slide-mounted sections were then stained with hematoxylin and eosin (H&E).
Two liver sections perpendicular to the resection site were evaluated per animal.
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| Figure 11 ClotFoam adheres only to the wounded tissue | Figure 12 Bonding of ClotFoam to tissue | Figure 13 Section of lacerated inferior vena cava covered by fibrin clot following application of CloFoam |
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| Fig. 14 | Fig. 15 | Fig. 16 |
Figure 16. Samples stained with d-dimer antibody show the ClotFoam-produced fibrin network attached to pancreatic wounded tissue. The samples were collected from ClotFoam experiments in pigs
PART III Studies in Pigs
Formulation # 9 developed in the course of experiments
described in part II showed the optimal viscoelastic properties for application in military relevant animal models
(pig). This formulation served also as the “gel matter” to study interactively the quality and formation of the
fibrin clot. Formulation 9 was evaluated in the swine model. Studies were performed in liver grade III/IV wound
open and close cavity model by Grant Bochicchio, MD, FACS, at the Thomas Miller Animal facility in Baltimore .
Methods: Nine female Yorkshire crossbred swine, age 2.5 months, weighing 37 ± 2 kg, were used. All animals were maintained in an Association for Assessment and Accreditation of Laboratory Animal Care International–accredited facility, and all experimental manipulations were performed in accordance with the National Research Council's Guide for the Care and Use of Laboratory Animals.
Animals then underwent either grade 3 or 4 liver injuries via open laparotomy or by laparoscopy. For the purposes of this model, a grade 3 injury is defined as a 3 cm long full-thickness parenchymal laceration (created sharply by an 11 blade scalpel). After the liver was exposed, a spot in the middle of the liver was selected to produce the liver injury with a scalpel. The position was calculated by approximation to the suprahepatic vessels and some branches of the portal vein. The spot was marked with a marker. After the damage was induced, surgeons close the cavity,allowed for 30 seconds of massive bleeding before applying ClotFoam through a small perforation.
A grade 4 injury was a 10 cm deep parenchymal injury with a specially designed high-speed drill with a cutting drill bit creating an injury akin to a penetrating gunshot (GSW) wound (Figure 24). Injuries were highly reproducible and severity was similar between the open and laparoscopic techniques. These injuries were consistent with the American Association for the Surgery of Trauma Organ Injury Scaling system.
Figure 17: A) Schematic of high-speed liver injury drill. B) Close-up schematic of sharp rotary drill bit.
A) B)
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Animals were randomized into 4 groups to date. Group 1 (n= 2) consisted of animals who underwent grade 3 liver injuries through an open midline laparotomy and had open cavity ClotFoam application. In this group the agent was visually directed to the liver injury. Group 2 (n= 2) consisted of animals who underwent grade 3 liver injuries through and open midline laparotomy and had closed cavity ClotFoam application. In this group the agent was administered into the peritoneal cavity blindly without direct injury visualization or direction. Group 3 (n= 2) consisted of animals underwent grade 4 liver injuries through an open midline laparotomy without ClotFoam treatment (open controls). Group 4 (n= 3) underwent grade 4 liver injuries through the laparoscopic technique without ClotFoam treatment (laparoscopic controls).
In all groups, 90 cc of ClotFoam was used for treatment.
The ClotFoam was delivered via mixing syringes (Figure 24) propelled into the abdominal cavity using pressurized
carbon dioxide (approximately 50 psi). This created a homogenous hemostatic foam that actively clotted and sealed
the injuries on both the visceral and diaphragmatic aspects of the liver.
Fluid resuscitation with Lactated Ringer's (LR) was begun immediately after injury. LR was infused as necessary to re-establish a MAP within at least 80% of the preinjury MAP if possible. Resuscitation was continued for the entire observation period (1Hr.).
Results
Fourteen animals were used in the study to date.
Eight animals were grade IV liver injuries (Group 1= 4 open cavity ClotFoam applications and Group 2= 3 closed
cavity applications). End points for animals in Groups 1 and 2 are shown in Table 1. Trend of mean arterial pressures
(MAPs) are seen in Figure 1.
Table 4: Outcome measures for Grade 3 liver injuries treated with ClotFoam. Group 1 = open cavity, Group 2 = closed cavity. All values reported as mean ± SEM
| Group | Survival Time (min) | Total Blood Loss (ml) | Fluid Requirement (ml) |
| 1 (n= 4) | 60 ± 0 | 300 ± 283 | 1500 ± 283 |
| 2 (n= 4) | 60 ± 0 | 600 ± 212 | 2175 ± 742 |
Figure 19. Open Cavity Model
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Figure 20. Close Cavity Model
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Table 5: Outcome measures for Grade 4 liver injuries not treated with ClotFoam. Group 3 = open laparotomy, Group 2 = laparoscopic. All values reported as mean ± SEM
| Group | Survival Time (min) | Total Blood Loss (ml) | Fluid Requirement (ml) |
| 3 (n= 2) | 26 ± 3 | 1900 ± 424 | 3050 ± 70 |
| 4 (n= 3) | 22 ± 11 | 1700 ± 200 | 2467 ± 569 |
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| Fig. 21:
Trend of MAPs for Grade 4 liver injuries treated with ClotFoam. Group 3 = open laparotomy, Group 4 = laparoscopic. |
Fig. 22. Controls: Five
animals underwent grade 4 liver injuries (3 laparoscopic and 2 open) to validate the laparoscopic model against
the established open model. These animals were not treated with ClotFoam. Endpoints are seen in Table 2 and Figure
2. |