Patent Application
20250127810
The present invention relates to methods and apparatus for preparing plasma enriched with Interleukin Receptor 1 antagonist (“IL-1Ra”) using glass beads or polyacrylamide microbeads. Embodiments of the invention relate to preparation of platelet rich plasma (PRP) compositions enriched in IL-1Ra.
U.S. Pat. Nos. 9,962,480 and 10,617,812, by the inventor herein, include embodiments directed to systems and methods for obtaining cellular samples enriched with cells, such as a platelet rich plasma (PRP), including separations using centrifugation of a blood sample in a centrifugation tube using a density separation medium and separation of cellular components using various filter systems. The disclosure of these patents is incorporated by reference.
U.S. Pat. No. 8,734,373, by the inventor herein, describes devices and methods for preparing PRP, including using an elongated filter device adapted for tight fitting into a test tube for separation of cellular components from a plasma fraction. The disclosure of this patent is also incorporated by reference as it relates to a filter adapted to be inserted into a collection tube.
U.S. Pat. Nos. 10,167,310 and 10,519,196, also by the inventor herein, include embodiments directed to systems and methods for obtaining a plasma fraction enhanced in Interleukin-1 Receptor Antagonist (IL-1Ra). In embodiments, the disclosed methods involve subjecting a blood sample in a blood collection tube to centrifugation and incubating the plasma fraction to enrich the fraction in IL-1Ra. The disclosure of these patents is likewise incorporated by reference.
There continues to be a need in the art for methods and devices for obtaining a plasma sample enriched in IL-1Ra, with enhanced recovery of IL-1Ra, and to methods for providing PRP enriched in IL-1Ra with greater efficiency.
Some aspects of the invention pertain to simple and effective methods for obtaining a plasma sample enriched in IL-1Ra, and to methods for providing PRP enriched in IL-1Ra.
In one aspect, the invention is a method of preparing a plasma composition enriched in IL-1Ra, comprising: collecting a PRP fraction in a collection vessel or collection tube containing polymeric microbeads; mixing the polymeric microbeads with the PRP fraction to form a homogeneous mixture of microbeads and PRP; allowing the homogeneous mixture to sit for a predetermined period of time; passing the mixture through a filter (and/or centrifuging the vessel or tube containing the mixture) to separate the microbeads, along with cells and/or cellular fractions of the plasma, and to obtain the plasma composition enriched in IL-1Ra.
In embodiments, the recovery of IL-1Ra (defined as the IL-1 Ra mass in the final product after treatment divided by the IL-1Ra mass in the baseline PRP) is in a range 2.2 to 21 fold, including intermediate values, such as greater than 5 fold, and greater than 10 fold. As an increase in concentration (picograms/ml) the techniques of the invention may yield an increase in concentration of 2.8 to 25.8 fold over a baseline.
In another aspect, the invention is embodied in a method of preparing a plasma composition enriched in IL-1Ra, comprising: collecting a whole blood sample in a centrifugation tube equipped with glass beads; contacting the whole blood sample with the surface of the beads for a predetermined period of time (for example, for i) 4 to 24 hours at a temperature of 30-40° C. or ii) 5 minutes to 1 hour at room temperature); centrifuging the whole blood sample to obtain IL-1Ra enriched plasma; passing IL-1Ra enriched plasma through a filter to obtain the plasma composition enriched in IL-1Ra.
In embodiments, the recovery of IL-1 Ra (as compared to baseline) using the glass beads is greater than the recovery without the glass beads by a factor of 1.2, 1.3, 1.4 or more. In embodiments, the glass beads may be used in combination with polymeric microbeads and in combination with a separation medium (such as a gel), with or without the polymeric microbeads.
The subject matter regarded as the invention is particularly pointed out and distinctly claimed in the concluding portion of the specification. The invention, however, both as to organization and method of operation, together with objects, features, and advantages thereof, may best be understood by reference to the following detailed description when read with the accompanying drawings in which:
It will be appreciated that for simplicity and clarity of illustration, elements shown in the figures have not necessarily been drawn to scale. For example, the dimensions of some of the elements may be exaggerated relative to other elements for clarity. Further, where considered appropriate, reference numerals may be repeated among the figures to indicate corresponding or analogous elements.
A vessel or tube for use with the invention may be vacuum or non-vacuum, made of glass or plastic. In embodiments, a collection tube used with the invention is made of durable plastic such as polyacrylate (PA) or polyethylene terephthalate (PET), polyethylene terephthalate glycol (PETG) or polyethylene naphthalate (PEN). In other embodiments, the tube or vessel may be glass or coated glass.
In embodiments, the vessel or tube is provided with a separation medium, such as a gel, with a density in a range of 1.04-1.08 g/ml, and in embodiments between 1.073-1.078 g/ml. In embodiments, an anticoagulant is provided in the tube or vessel, such as heparin salts, citrate/citric (e.g., sodium citrate/citric acid), citrate/citric-dextrose (acid-citrate-dextrose ACD), citrate-dextrose-phosphate, oxalate, and oxalate-fluoride salt. In embodiments the vessel or tube is provided without gel. In embodiments, the vessel or tube is provided without anticoagulant.
The vessel or tube may be preinserted with glass beads and/or polymer microbeads with high rate of water absorption, such as polyacrylamide (PAA) microbeads. Alternatively, glass beads may be used, of a type conventionally used for incubation of cellular samples. Examples of both systems are provided below with sometimes surprisingly different result depending on the combination of process steps.
The above vessel or tube may be filled with the 10-100 ml of whole blood, for example 10-50 ml of whole blood. Alternatively, the glass beads or the polymer microbeads may be preinserted in a second vessel or tube which is then used to collect the plasma or the PRP fraction produced in a separate vessel or tube.
Where anticoagulant and glass beads are preinserted into a collection tube, the tube may be either incubated for 4-24 hours or placed in an ambient temperature for zero to 1 hour, followed by centrifugation at 1000 g to 3000 g, for example at 1500 g where a fraction of plasma enriched in IL-1Ra may be separated by sleeve filter as described in the aforesaid U.S. Pat. No. 10,519,196 or directly transferred to syringe or to a syringe via disc filter of 0.22-0.45 micron to separate cells and cells residue and microbeads and to enable the plasma with the enriched IL-1 Ra to be stored at appropriate temperature, for example −5 to −20° C. which allows dosage forms of the IL-1Ra to be stored and prepared for use. The above filtration procedure may be processed after a second centrifugation, for example at 2000-4000 g for 5-10 minutes to deposit cells, cells fractions and microbeads.
Where the first vessel or collection tube is provided without glass beads or polymer microbeads, a blood sample may be centrifuged for several minutes, for example 5-10 minutes at 1000-3000 g, for example at 1500 g to obtain separated plasma with an upper fraction of platelet poor plasma (PPP) and lower fraction (still above gel) of PRP enriched in monocytes and lymphocytes but depleted in erythrocytes and granulocytes due to the specific properties of the gel that act as a separator. PPP is removed, for example 50%-75% of the whole plasma volume is removed, and the cells are dispersed in the remaining plasma to prepare the IL-1Ra enriched composition.
The PRP fraction may be inserted into the second collection tube with glass beads and/or plastic microbeads. When the second tube is preinserted with glass beads only, incubation may proceed at, for example, 37 centigrade with or without 5% CO2 for 1-24 hours, for example 8-16 hours and the plasma enriched with IL1Ra is collected via disc filter, for example having a pore size of 0.22-0.45 micron to eliminate cells and to provide concentrated plasma with IL-1 Ra at the time of processing, or after 3-12 months from the process time when stored at the appropriate temperature, such as −5 to −20 centigrade.
When a collection tube is preinserted only with polymer microbeads or with polymer micro beads and glass beads, the collecting of the enriched IL-1Ra plasma can be done immediately or up to 1 hour, for example product may be withdrawn after 15-30 minutes via sleeve filter with pore size of 0.22-40 micron and/or via disc filter with preferred pore size of 0.22 micron to 0.45 micron or directly to the syringe.
In one embodiment, whole blood is collected in a glass collection vessel containing density separation gel and glass microbeads, in the absence of anticoagulant, followed by incubating the PRP fraction for 30 minutes to 24 hours. Following incubation, the mixture may be centrifuged at 900 to 2500 g to separate the fractions. To collect IL-1Ra concentrate, the fraction enriched in IL-1Ra may be passed through a 0.2 to 1.0 micron filter separating the microbeads, cells and cellular fractions. Alternatively, the mixture may be centrifuged at 3000-5000 g to remove these components. The resulting plasma composition enriched in IL-1Ra may be used for injection to a patient.
The injection of IL-1Ra enriched plasma can be performed immediately after the above procedures or even after 1-12 months, if the IL-1Ra enriched plasma is stored at an appropriate temperature, such as between −5 to −20° C.
In embodiments, IL-1Ra enriched plasma products prepared according to the methods described herein may be effective as a treatment, such as by injection, for both early and late stage orthopedic pathological conditions and/or to decelerate natural progression of chronic orthopedic diseases. The IL-1Ra enriched plasma products may be effective in patients with early symptoms and those with family history looking for preventive therapies. In embodiments, the products may be used as prophylactic therapy in orthopedic surgery or as a symptom modifier option after injury. Injection intervals can be performed according to severity and reappearance of symptoms.
The autologous IL-1Ra therapy is free of immunological response and provides a non-surgical, minimally invasive therapy without pharmacologic treatment. The IL-1Ra obtained is quite concentrated and substantially free of contamination by erythrocytes and pro-inflammatory white blood cells.
Referring to
Referring to
To induce the production of IL-1Ra that is derived from a blood separation platform, e.g., TropoCells® and fused in Tropokine™ P with polyacrylamide (PAA) microbeads.
As shown in Table 1 below, ELISA assessment of one sample showed a nearly 10-fold increase in the IL-1Ra concentration obtained according to the procedure above, using PAA beads. The “fold” calculation is obtained by dividing the IL-1Ra mass in the final product after treatment by the IL-1Ra mass in the baseline PRP.
Tropokine™ IL-1Ra enriched plasma using glass beads (Suggested Protocol)
To induce the production of IL-1ra by incubating whole blood in Tropokine™ G tube with glass beads.
ELISA assessment verified that using the glass beads The “fold” was calculated by dividing the IL-1Ra mass of the baseline after treatment with beads by the IL-1Ra mass in the baseline. Whole blood with glass beads-blood in a glass tube with glass beads Whole blood without glass beads-blood in glass tube without glass beads.
Whole blood samples were obtained from four donors and IL-1Ra was measured, reported as concentration (pg/ml) and total mass (pg), at baseline and after processing according the techniques described above, using glass tubes with microbeads only, plastic syringes with microbeads only, glass collection tubes only; glass tube with glass beads and microbeads; and glass tubes with glass beads plus separation gel plus microbeads, with the following results:
The volume of the samples is reported in TABLE 5
The fold increase (total mass basis) is reported in TABLE 6
In this example, whole blood was collected in a glass tube containing density separation gel and glass microbeads in the absence of anticoagulant. The vessel was incubated for 30 minutes to 24 hours (to prepare different amounts of IL-1Ra in the final product), followed by centrifuging the tube at 1500 g for 10 minutes. The plasma composition enriched in IL-1Ra was obtained directly from the plasma fraction via syringe, passing the mixture through a 0.2 to 1.0 micron disc filter. Table 7 provides shows the average mass in picograms of the IL-1Ra obtained in the samples after different periods of incubation.
While the invention has been described with respect to a limited number of embodiments, these should not be construed as limitations on the scope of the invention, but rather as exemplifications of some of the preferred embodiments. Other possible variations, modifications, and applications are also within the scope of the invention. Accordingly, the scope of the invention should not be limited by what has thus far been described, but by the appended claims and their legal equivalents.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/IL2022/050924 | 8/23/2022 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63236130 | Aug 2021 | US |
