Patent Application
20240350556
The present invention relates to compositions comprising cord blood platelet-rich plasma (CB-PRP) for the treatment of ocular diseases as well as methods of treatments of ocular diseases in which therapeutic effective amounts of blood platelet-rich plasma (CB-PRP) are administered to a subject in need thereof.
Ocular diseases or disorders are of a wide variety. Cataract, glaucoma, refractive errors are major causes of visual impairment and preventable blindness.
Notably, retinitis pigmentosa (RP) and atrophic age-related macular degeneration (d-AMD) represent a heterogeneous group of degenerations in which the genetic factor plays an important role. Together, both pathologies represent the major cause of blindness in the world. The genetic role is of considerable importance in both RP and d-AMD pathologies.
RP typically originates from genetic defects in rod photoreceptor proteins, resulting in night blindness in the early stages. Subsequently, the cone photoreceptors (responsible for day vision, visual acuity, and colour vision) also slowly die, until all effective vision is lost. An alternative scenario implies the simultaneous involvement of both rods and cones, due to a genetic defect affecting both types of photoreceptors. Numerous molecular subtypes of RP are described, which are characterized by primary degeneration of the rods, followed by secondary dysfunction/degeneration of the cones; the loss of the cones in these cases occurs as a consequence of the loss of the rods and not as a direct consequence of a genetic mutation expressed in the cones (Narayan D S, et al, 2016). Therefore, a therapeutic intervention aimed at counteracting the secondary degeneration of the cones, is not influenced, or is only minimally influenced, in its effectiveness by the presence of a genetic defect selectively expressed in the rods.
D-AMD is a multifactorial disease in which genetic and environmental factors intervene. In the d-AMD, the deterioration of the retina is often associated with the formation of small yellowish deposits (lipofuscin), known as retinal drusen, below the retina in the macular region. This phenomenon leads to a thinning and drying out of the macula, causing the retinal macular tissue (atrophic development) to lose its function and central sight loss. The amount of central vision loss is directly related to the location of the retinal atrophic development. More and more genetic alterations are reported by the literature, although environmental factors have proved to play a key role in the phenotypic manifestation.
There is currently no cure for RP and d-AMD; however, several therapeutic strategies have reached the stage of clinical trials. One of these strategies is based on the use of neurotrophic factors, which act for their anti-apoptotic activity and their putative effects on the inflammation and energy metabolism of the cones. A large number of preclinical studies (Wen R, et al 2012; Earnings V, et al 2015) and clinical studies (Sieving P A et al 2006; Pilli S, et al, 2014; Falsini B et al, 2016) suggest a potential neuroprotective effect of the neurotrophins NGF, CNTF and BDNF on the survival of the retinal cones and on the tropism of the external retina in RP from primitive rod mutations. In particular, in the 6 months following treatment with neurotrophins, there is typically an increase in the thickness of the external retina, and specifically of the external nuclear layer (Wen et al, 2012, Sieving et al., 2006) which in a significant fraction of patients is associated with an improvement in visual acuity (Sieving et al, 2006), in the electroretinogram of the cones and in the Goldmann visual field. (Falsini et al 2016).
D-AMD also appears to have numerous clinical trials under evaluation. There are several studies about the opportunity to practice conservative therapies such as oral integration using antioxidants and anti-inflammatory treatment, but also interventional therapies such as sub-retinal transplantation of pigment epithelial cells.
Many are the efforts of scientists all over the world to try to combat the states of low vision/legal blindness related to diseases such as Retinitis Pigmentosa (RP) and even more so the Senile Macular Degeneration of the Atrophic Type (d-AMD). However, within this context, a dire need still exists to develop efficient therapeutic strategies for the treatment of such severe ocular diseases.
The technical problem posed and solved by the present invention is hence that of providing an effective therapeutic approach for the treatment ocular diseases. The solution provided by the present invention is represented by a composition comprising cord blood platelet-rich plasma (CB-PRP).
As will be clearly evidenced in the experimental section of the present application, the authors of the invention have found that the administration of a composition comprising CB-PRP to a subject in need thereof, for example by way of sub-retinal injection, intravitreal injections or in the form of eye drops, depending on the disease to be treated, is particularly effective for the treatment of several ocular diseases, such as posterior segment eye diseases (PSEDs) as well as ocular surface diseases (OSDs).
Notably, sub-retinal administration of CB-PRP resulted in no serious adverse reactions such as endophthalmitis, retinal detachment, uveitis or haemorrhages, indicating the great potential of CB-PRP based compositions as safe products when placed in the contact with retinal tissue.
The authors of the invention have particularly found that the administration of CB-PRP by means of intravitreal injection to 21 patients has so far proven to be safe and effective for blocking the growth of the atrophic area. Notably, no local or systemic adverse reactions have been found during or after each single intravitreal injection.
As will be further detailed below, in one embodiment intravitreal injection of a CB-PRP composition according to any of the variants disclosed herein can be performed using first an insulin syringe to aspirate 0.1 mL of blood product and then using a 30 G needle to inject the contents of 0.05 mL into the vitreous chamber of the patient to be treated.
Nevertheless, the present invention also advantageously provides for a pre-filled syringe suitable for intravitreal injection containing a CB-PRP composition that is ready to use for treatment of patients suffering from an ocular disease such as d-AMD.
Indeed, the inventors have particularly found that the use of such a pre-filled syringe represents a cleaner and safer approach for treatment of patients suffering from such an ocular disease by means of intravitreal injection, enabling a large-scale applicability, a reduction of the risk of possible infections, a reduction of biological and sanitary waste (e.g., needles, syringes, single-dose vials, etc. used in the conventional preparation phases prior to intravitreal administration), as well as the execution of a safer and faster procedure from a clinical point of view.
The provision of a pre-filled ready-to-use syringe for intravitreal injection can additionally make CB-PRP available to ophthalmologists who do not normally have ease of supply of this product at cord blood banks.
Hence, objects of the present invention are:
administering to said subject an effective amount of a composition comprising cord blood platelet-rich plasma (CB-PRP) by way of intravitreal injection, wherein said administering is carried out by injection using a pre-filled syringe according to any of the embodiments disclosed in the present specification and in the claims;—The use of CB-PRP for the manufacture of a medicament, such as the composition according to any of the embodiments disclosed in the present specification and in the claims, for the treatment of an ocular disease.
Additional advantages and/or embodiments of the present invention will be evident from the following detailed description.
The present invention and the following detailed description of preferred embodiments thereof may be better understood with reference to the following figures:
As used herein, the term “cord blood platelet-rich plasma” (abbreviated as CB-PRP) is referred to plasma enriched in platelets obtained from umbilical cord blood. CB-PRP is a blood component for non-transfusion use.
As used herein, the expression “ocular surface disease” (OSD) comprises a spectrum of disorders that affect the normal structure and function of the cornea, conjunctiva, eyelids, and supportive glandular network. A significant proportion of such diseases have an immune aetiology, such as in allergic and autoimmune conditions.
As used herein, the expression “posterior segment eye disease” (PSED) comprises diseases of the retina, choroid and optic nerve of the eye.
In the present specification and claims, the expression “effective amount” is referred to a “therapeutically effective amount” of the compositions of the invention and is used to denote any amount which will cause a substantial improvement in the treated disease condition (such as a subsidence of a lesion, for example) when administered to the subject in need of treatment. As further explained below, the amount will vary e.g. depending on the condition being treated, the severity of the condition, the form and concentration of composition applied.
In any part of the present description and claims the term comprising can be substituted by the term “consisting of”.
In the following, several embodiments of the invention will be described. It is intended that the features of the various embodiments can be combined, where compatible. In general, subsequent embodiments will normally be disclosed with respect to the differences with the previously described ones.
As previously mentioned, a first object of the present invention is represented by a composition comprising cord blood platelet-rich plasma (CB-PRP). According to the invention, said composition comprising CB-PRP is a hemoderivate composition.
According to the invention, CB-PRP may be obtained from umbilical cord blood by means of a method suitably selected by the skilled person among those that are already known in the art.
In one preferred embodiment of the invention, CB-PRP is prepared according to the procedure as defined by the Italian legislation on blood components as set forth in the following documents: “Decreto del Ministero della Salute, Nov. 2, 2015: Disposizioni relative ai requisiti di qualità GU n.300 del 28 dicembre 2015; Decreto del Ministero della Salute, 19 agosto 2019: Modifiche al decreto Nov. 2, 2015, recante: «Disposizioni relative ai requisiti di qualità e sicurezza del sangue e degli emocomponenti». GU n. 262 del 26-9-2019” (herein incorporated by reference).
Merely by way of example, CB-PRP may be produced from umbilical cord blood by a method including the following steps:
Preferably, the isolation step ii. of the above method is performed by centrifugation, wherein the PRP fraction is isolated as the lighter component resulting from centrifugation.
According to one embodiment of the invention, CB-PRP is prepared from different cord blood units containing an adequate amount of hematopoietic progenitors. Preferably, CB-PRP is prepared from cord blood units collected from more donors according to any of the techniques known to a person skilled in the art, which units, after collection, are subjected to one or more safety screening tests so as to check the absence of fungi, aerobic and/or anaerobic bacteria in the same.
According to one preferred embodiment of the invention, CB-PRP is prepared from at least 15 units of umbilical cord blood collected from one or more donors, in particular by means of one or more centrifugation steps so as to isolate the PRP fraction from the other blood components. Preferably the CB-PRP is prepared from units of umbilical cord blood collected from different donors, preferably at least 15 different donors.
In one preferred embodiment, the collected blood cord units are subjected to a first “soft-spin” centrifugation step so as to obtain PRP, followed by a second “hard-spin” centrifugation step and by a third step of removal of the excess platelet-poor plasma.
It is preferred that all steps necessary to produce the CB-PRP occur in sealed bags, and that sterile connections are used to transfer the CB-PRP from a bag to another, or to store aliquots of the resulting CB-PRP. Additional microbial test can be performed on the final CB-PRP product so as to check the absence of e.g. fungi, aerobic and/or anaerobic bacteria in the same.
The resulting CB-PRP obtainable according to any of the embodiments described herein can be in liquid form, in solid form (also as a gel) or in freeze-dried form and can be stored in any device known in the art for storing platelet fractions or plasma.
The CB-PRP prepared according to any of the techniques known in the art or to any of the methods described herein can be aliquoted in sterile sealed tubes and stored at −80° C. before use.
According to one embodiment of the invention, the composition comprising CB-PRP disclosed herein is characterized by a platelet concentration of 1×106 platelets/μL.
The composition of the invention comprises one or more platelet derived growth factors such as Granulocyte colony-stimulating factor (G-CSF), Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF), Vascular-Endothelial Growth Factor (VEGF), Fibroblast Growth Factors (FGF), Platelet Derived Growth Factor (PDGF).
One aspect of the invention is referred to a composition according to any of the embodiments described herein, further comprising at least one pharmaceutically acceptable excipient and/or carrier.
Preferably, a composition comprising CB-PRP according to any of the embodiments disclosed herein is a pharmaceutical composition.
Depending on the final form of the composition the skilled person will easily select suitable carrier/s and/or excipient/s. Suitable excipients and/or carriers that can be used for the preparation of a composition according to the present invention can be selected from those commonly used in the art such as, for example, stabilizers, preservatives, solvents, pH regulators, isotonic regulators, chelating agents, cryoprotective agents, diluting agents, binding agents, antioxidants, surfactants.
The compositions of the invention may further contain other active substances such as e.g. other hemostatic agents, antioxidants agents, neurotrophic factors, vitamins or molecules for the preparation of specific ocular formulations.
The compositions of the invention can be by way of example in the form of a solution, suspension, gel, eye drops.
When in the form of eye drops the compositions of the invention may particularly comprise preservatives and/or buffers so as to control the pH of the composition. Preferably, the pH of the composition of the invention in the form of eye drops equals that of tear fluid and is about 7.4.
As previously mentioned, one further aspect of the invention is related to a composition according to any of the embodiments described herein for use in the treatment of an ocular disease.
Advantageously, the compositions of the invention can be effectively used to treat an ocular disease as well as to prevent, reduce and/or eliminate any of the symptoms caused or associated to said ocular disease.
In some embodiments, said ocular disease is an ocular surface disease (OSD) or a posterior segment eye disease (PSED).
Non-limiting examples of OSD include Dry Eye Disease (DED), blepharitis and meibomian gland dysfunction (MDG), allergic eye diseases (AED), keratitis, microbic and/or neurotrophic corneal ulcers while examples of PSED include glaucoma, age-related macular degeneration (AMD), diabetic retinopathy (DR), uveitis and retinal vassal occlusions.
Preferably, said ocular disease is selected from the group comprising retinitis pigmentosa (RP), dry age-related macular degeneration (d-AMD), glaucoma disease and neurotrophic corneal ulcers.
The compositions according to any of the embodiments described herein can be administered by way of a topic and/or intraocular administration. According to some preferred embodiments, the compositions of the invention are administered by means of sub-retinal injection or intravitreal injection. Such administration may be carried out by a surgeon or doctor by using any suitable method known in the art, e.g. in the field of ophthalmology.
In one preferred embodiment, the compositions of the present invention can be administered topically to the eye of a subject in need thereof in the form of eye-drops, e.g. according to its formulation.
The effective amount of CB-PRP in the composition of the invention for the treatment of an ocular disease may vary depending upon the exact age, as well as weight and sex of the subject being treated.
According to one preferred embodiment, the compositions of the invention can be administered in three different ways according to the type of ocular disease as indicated below:
Preferably, when administered by way of topical administration, such as in the form of eye drops, the compositions of the invention can be administered at a dose regimen of 1 drop of composition for 6 times daily for 7 days to taper down, according to the clinical ophthalmic condition.
Preferably, when administered by way of intravitreal administration, the compositions of the invention can be administered at a dose regimen of 1 injection of 0.1 or 0.05 mL of CB-PRP composition monthly for the first 6 months, wherein said injection is optionally repeated according to the clinical ophthalmic condition.
Preferably, when administered by way of sub-retinal injection, the compositions of the invention can be administered at a dose regimen of 1 injection only of 0.5 mL of CB-PRP composition (booster), which can optionally be followed by intravitreal administration according to any of the embodiments described herein, according to the clinical ophthalmic condition.
In accordance with certain embodiments, the compositions of the invention can be administered one or more times daily to a subject in need thereof. For example, the compositions according to any of the embodiments of the present invention can be administered once daily, two times daily, or about three times daily, or more. In certain embodiments, the regimen of the compositions of the invention can be administered in a subject in need thereof for a prolonged period.
All the compositions described herein may be prepared by employing standard preparation techniques known in the pharmaceutical field. The compositions can also be provided already aliquoted in single dosages or in single dosage fractions.
The compositions comprising CB-PRP according to the present invention may be employed in a method of treatment of an ocular disease in a subject in need thereof. In some embodiments said method of treatment comprises administering an effective amount of said composition to a subject in need thereof.
It is hence a further object of the present invention a method for the treatment of an ocular disease in a subject in need thereof, which method comprises:
administering to said subject an effective amount of a composition according to any of the embodiments disclosed in the present specification.
As previously mentioned, an effective amount of the composition of the invention that can be administered to said subject will vary depending on the age, weight, sex of said subject as well as depending on the severity of the ocular disease.
Preferably, the composition is administered in step i. at a dose regimen of 0.5 mL per eye.
As already mentioned above, step said administering can be carried out by way of a topic and/or intraocular administration. Preferably, the administration is carried out by means of sub-retinal injection or intravitreal injection or by means of topical administration on the ocular surface.
Merely by way of example, intravitreal injection of CB-PRP can be carried out using a 30 G syringe injecting 0.1 ml of CB-PRP directly into the vitreous chamber passing by pars plana (3.5 or 4.0 mm from the sclero-corneal limbus).
Merely by way of example, a sub-retinal injection of CB-PRP can be performed under the retina through a 41 G cannula. The injection site can be identified by the surgeon and under intraoperative conditions in the retinal region located inside the major vascular arches (posterior pole).
An ocular disease which can be treated according to any of the methods of treatment disclosed in the present specification can be an ocular disease as previously defined in the present specification or in the claims. Preferably, said ocular disease is selected from the group comprising retinitis pigmentosa (RP), dry age-related macular degeneration (d-AMD), glaucoma disease and neurotrophic corneal ulcers.
According to the present invention, “a subject in need thereof” can be a subject, preferably a human, who has been diagnosed as being affected by an ocular disease such as a disease defined in the present specification.
When sub-retinal administration is used, according to one aspect of the invention, said subject in need thereof may undergo cataract surgery and/or vitrectomy prior to administration of the composition of the invention according to any of the embodiments described herein.
In other terms, the method of sub-retinal treatment according to any of the embodiments disclosed in the present specification may further comprise one or more step prior to the administration of the composition of the invention, wherein said subject is submitted to cataract surgery and/or vitrectomy. Cataract surgery and vitrectomy procedures can be performed by means of a method suitably chosen by the skilled person among those that are already known in the art. Merely by way of example, High Speed Plana Pars Vitrectomy (10,000 cuts/minute) combined with lens phacoemulsification and simultaneously PC-IOL implantation, when required, can be performed prior to said administration. CB-PRP sub-retinal injection is carried out during the same operation. The subject undergoing said surgical operations will preferably receive peribulbar anaesthesia 30 minutes before surgery (10 ml of ropivacaine combined with hyaluronidase 300 IU). Also, the surgical field will be prepared as for standard ocular surgery with the periocular skin and the lower conjunctival fornix will be prepared with 5% povidone-iodine.
The sub-retinal method of treatment according to any of the embodiments disclosed in the present specification can further comprise at least one of the following: subjecting said subject to vitrectomy;
subjecting said subject to peripheral retinal photocoagulation;
applying to said subject scleroconjunctival closures by means of bipolar diathermy or single suture, after the sub-retinal administration of the composition of the invention, e.g. the sub-retinal administration of the CB-PRP injection of the invention.
All the additional procedures above can be performed by means of any of the standardized procedures known to a person skilled in the art.
In one aspect, the method according to any of the embodiments disclosed herein can further comprise after said sub-retinal administration:
diagnostic check may be performed immediately after said sub-retinal administration or at one or more time period after said administration, for example 1 day, 3 days, 1 week, 1 month, 3 months, 6 months and/or 12 months after step said sub-retinal administration. Subjecting the treated subject to one or more of the above-mentioned diagnostics may be useful, for example, for analysing the thickness of the outer nuclear layer (ONL) of the photoreceptors of the subject after treatment with the composition, thus for evaluating the success of the treatment and/or for monitoring the trend of the disease.
When the composition of the invention is administered topically, the term “administration” encompasses a treatment regimen in which said composition is administered in daily dosage/s for a given period of time that will depend on the pathology to be treated and on the response of the treated subject.
When the composition of the invention is administered intravitreally, the term “administration” encompasses a treatment regimen in which said formulation is administered using a 30 G syringe injecting 0.1 or 0.05 ml of CB-PRP directly into the vitreous chamber passing by pars plana (3.5 or 4.0 mm from the sclero-corneal limbus).
The composition according to any of the embodiments disclosed in the present specification and in the claims can be hence provided in a pre-filled syringe suitable for intravitreal injection as well as administered by means of a pre-filled syringe.
Therefore, it forms part of the present invention also a pre-filled syringe suitable for intravitreal injection, containing a composition comprising or consisting of CB-PRP according to any of the variants disclosed in the present specification and in the claims.
In one embodiment, the composition that is present in said pre-filled syringe is a stable ophthalmic formulation that is preferably in the form of a solution or suspension. In another embodiment, said composition is characterized by a platelet concentration of 1×106 platelets/μL and/or comprises one or more platelet derived growth factor comprising Granulocyte colony-stimulating factor (G-CSF), Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF), Vascular-Endothelial Growth Factor (VEGF), Fibroblast Growth Factors (FGF), and/or Platelet Derived Growth Factor (PDGF).
In a preferred embodiment, the CB-PRP contained in a pre-filled syringe according to any of the embodiments disclosed herein is prepared from cord blood units obtained or collected from more than one donor, more preferably is prepared from units of umbilical cord blood collected from different donors and particularly from at least 15 units of umbilical cord blood collected from different donors.
The pre-filled syringe may be structurally configured according to any of the variants known to a person skilled in the art provided it is suitable for intravitreal injection. In certain embodiments, the pre-filled syringe comprises a glass or plastic body, a stopper, and a plunger for injecting the contents of the pre-filled syringe intravitreally.
20) In one preferred embodiment, the pre-filled syringe comprises a 30-gauge injection needle connected to the syringe.
Preferably, the pre-filled syringe, the injection needle or other components thereof as well as the composition comprising CB-PRP that is contained in the syringe are sterile.
The syringe according to the present invention is preferably pre-filled with a composition containing CB-PRP according to any of the variants disclosed herein under sterile conditions and can then be frozen at −4° C. When needed, it can be brought to room temperature approximately half an hour before administration to allow complete thawing.
In another embodiment, the pre-filled syringe according to any of the variants disclosed herein is resistant to temperatures ranging from −20° C. and −80° C. and particularly comprises a glass or plastic body that is suitable for storage at a temperature ranging from −20° C. and −80° C.
It also forms part of the present invention a method for the treatment of an ocular disease, preferably d-AMD, in a subject in need thereof, which method comprises:
administering to said subject an effective amount of a composition comprising CB-PRP according to any of the variants disclosed herein by way of intravitreal injection, wherein said administering is carried out by means of a pre-filled syringe according to any of the embodiments disclosed in the present specification and in the claims.
In an embodiment of the invention, the composition comprising CB-PRP is intravitreally administered from said pre-filled syringe using a 30-gauge sterile injection needle connected to it.
As previously mentioned, an effective amount of the composition of the invention that can be administered to said subject will vary depending on the age, weight, sex of said subject as well as depending on the severity of the ocular disease.
Preferably, said composition is administered using the pre-filled syringe at a dose regimen of between 0.05 to 0.1 mL per eye, in particular directly into the vitreous chamber passing by pars plana (3.5 or 4.0 mm from the sclero-corneal limbus).
It forms part of the present invention also the use of CB-PRP for the manufacture of a medicament such as the composition according to any of the embodiments disclosed in the present specification or in the claims for the treatment of an ocular disease.
In one embodiment, said manufacture may comprise one or more steps of preparation of the CB-PRP, e.g. according to any of the methods disclosed in the present specification. Said manufacture may also involve mixing CB-PRP with one or more excipient and/or carrier such as those disclosed in the present specification thereby obtaining a composition according to any of the embodiments described herein.
Examples are reported below which have the purpose of better illustrating the methodologies disclosed in the present description, such examples are in no way to be considered as a limitation of the previous description and the subsequent claims.
The primary objective of the study was the quantitative evaluation of images derived from high-resolution optical coherence tomography (structural OCT). In particular, the differences of the images obtained studied with the image subtraction software at 1, 3, 6 and 12 months for the analysis of the thickness of the outer nuclear layer (ONL) of the photoreceptors. These differences were correlated between the same eye and the untreated adelph.
Safety and efficacy study, prospective, non-randomized cohort, with blood component. Duration of the study: 12 months.
Due to the peculiarity of the study and the lack of previous similar studies on which to base hypotheses, the study was considered pilot for which we set a total sample size of (N=10 patients for RP and N=10 patients for d-AMD).
A complete general clinical and ophthalmological examination (including detailed family history, anterior segment biomicroscopy, ETDRS corrected visual acuity, direct and indirect ophthalmoscopy, intraocular pressure measurement) was performed on each patient upon enrollment.
The study was conducted in accordance with Good Clinical Practice, the ethical principles deriving from the Declaration of Helsinki and the current legislation on clinical trials. The regulatory framework to which the study referred is that relating to blood components for non-transfusion use, to which all the procedures described will be strictly adherent.
This study was approved for testing by the local ethics committee (PROT ID 3417) and was covered by a specific insurance policy (LLOYD'S A1202049503-LB), pursuant to Ministerial Decree 14/7/2009.
Sensitive patient data was collected in the data collection form or case report form (CRF), reporting only the patient's initials and an identification number consisting of a specific and unique 2-digit number.
All the documentation is available, for any checks and controls upon request to view the documentation directly to the PI of the study.
Primary outcome: absence of major ocular adverse events (bacterial or fungal septic endophthalmitis, retinal detachment, vitreous proliferative-fibrotic reaction with retinal traction, secondary glaucoma, ftisis bulbi, iris rubeosis). Absence of major general adverse events (changes in blood count and systemic inflammatory parameters: ESR, CRP)
The primary objective of the study was to identify whether the new therapy could be effective and thus guarantee personalized medicine. To estimate the efficacy, the variation of a score derived from the anatomical parameters of the structural OCT during the follow-up visits was considered.
To achieve this goal, it was important to identify structural changes in precise retinal anatomical components that play a key role in visual recovery. Among these we distinguish: 1) Ellipsoid zone (Ellipsoid zone), Outer Nuclear layer (outer nuclear layer) and Ganglion complex layer (layer of the ganglion cell complex).
The occurrence of a sudden worsening of the disease and/or bacterial infections and/or inflammatory reactions reasonably connected to the treatment with CB-PRP in three consecutive patients constituted a fundamental rule of stopping the trial.
Preparation of Cord Blood Enriched with Platelet Plasma.
CB-PRP is a blood component for non-transfusion use produced according to procedures defined by the Italian legislation on blood components (Decree of the Ministry of Health, 2 Nov. 2015: Provisions relating to the quality requirements GU No. 300 of 28 Dec. 2015; Decree of the Ministry of Health, 19 Aug. 2019: Amendments to the decree of 2 Nov. 2015, containing: “Provisions relating to the quality and safety requirements of blood and blood components”. OJ no. 262 of 26-9-2019). The basic materials for the production of CB-PRP are cord blood units collected at the UNICATT cord blood bank of the Policlinico Gemelli IRCCS Foundation. These units are allogeneic solidarity donations for haematological patients who are candidates for hematopoietic stem cell transplantation: in this field, the use of cord blood represents a consolidated therapeutic practice for decades of experience. Cord blood units that can be used for transplantation must contain an adequate amount of hematopoietic progenitors. Units that did not meet the cell thresholds established for transplantation were not frozen and, with the consent of the donors, were intended for further clinical and research use. According to the aforementioned Italian legislation, couples who want to donate cord blood obtain eligibility after a thorough medical consultation. Furthermore, immediately after collection, infectious screening tests are performed on the maternal blood sample (serology and genome for HIV, HBV, HCV, and serological test for syphilis) or on the unit (blood cultures for fungi, aerobic and anaerobic bacteria). Only units with negative tests were used for subretinal injection.
The CB-PRP was made up of a pool of 15 units. Shortly after collection, each unit was subjected to a “soft-spin” centrifugation to obtain CB-PRP: the platelet concentration was then determined which will be normalized to 1×10{circumflex over ( )}9/L, through a “hard-spin” and subsequent removal of excess platelet-poor plasma. CB-PRP was then recovered and stored at −80° C. pending microbial testing. Once 15 CB-PRPs with negative tests were obtained, in order to avoid any disparity in the concentration of growth factors between the different units, they were thawed and collected in a single bag, suitable for the preparation of blood components for non-transfusional use. The pool was then fractionated into 1 ml aliquots in sterile sealed vials, which were stored again at −80° C. until use. This process made the intervention homogeneous for all patients who participated in the study. All steps required to prepare the CB-PRP pool were performed in sterile cryogenic blood component bags, and sterile connections were used to transfer CB-PRP from one bag to another and to fractionate the CB-PRP aliquots.
The surgical approach was a 23 Gauge (G) (10,000 cuts/minute) High Speed Plana Pars Vitrectomy combined with lens phacoemulsification and PC-IOL implantation simultaneously when required. Constellation Vision System (Alcon Laboratories, Inc., Fort Worth, TX) was the tool used for all procedures. All patients received peribulbar anesthesia 30 minutes before surgery (10 ml of ropivacaine combined with hyaluronidase 300 IU). The periocular skin and the lower conjunctival fornix were cleaned with repeated passages of 5% iodo-povidone. As previously described, prior to introducing 23 G trocar cannulae, the conjunctiva is partially displaced to misalign the conjunctiva from the sclera and an angled incision was made. Three valved cannulas were positioned at 2 and 11 o'clock for service sclerotomies and one in the lower temporal sector for infusion. They were positioned 3.5 mm from the limbus. Phacoemulsification was performed through 2 corneal incisions: a main (tunnel) incision of 2.2 mm and a service incision of 0.9 mm. During the vitrectomy, all eyes received the detachment and removal of the posterior hyaloid membrane if it had not already been separated from the underlying retinal layers. A “core vitrectomy” was then performed. Diluted triamcinolone acetonide will always be used to highlight the residual vitreous. The procedures described up to now are standardized procedures for cataract removal and vitrectomy. [28]
A sub-retinal injection of 0.5 ml of CB-PRP was performed under the retina through a 41 G cannula.
The injection site was identified by the surgeon and under intraoperative conditions, in the retinal region located inside or outside the major vascular arches.
A complete vitrectomy and eventual peripheral retinal photocoagulation, where necessary, for suspected rhematogenous areas was then performed. The filtered air was used as an internal buffer. After removal of the cannula, sclerotomies were checked and, if necessary, scleroconjunctival closures were applied using bipolar diathermy or a single suture (Vicryl 7-0; Ethicon Inc.). After surgery, patients were recommended to maintain a prone position for several hours for the 3 days following surgery.
Measurements of retinal thicknesses, electrophysiological results of the electroretinogram (ERG), visual evoked potentials (PEV) and visual acuity were analyzed by multivariate analysis of variance for repeated measures (ANOVA). A test of normality was performed preliminarily to confirm that the distribution of the data approximates the Gaussian one. The changes recorded in the various times of the trial (1-12 months) were compared both with the baseline measures with the changes recorded in the untreated contralateral eyes. In the main analyzes, a P value<0.05 was considered statistically significant. Multiple comparisons were evaluated by Tukey's post-hoc test.
Of the 20 eyes hypothesized for the study, we have currently enrolled 13 eyes from 13 patients and treated 7 eyes (4 patients with RP and 3 with d-AMD) selected at our retinal disease and maculopathy clinics currently followed in our outpatient clinic (age range: 18-68 years).
No serious adverse reactions were found in all eyes of treated patients and who received subretinal CB-PRP. Specifically, uveitis, retinal detachment, vitreous haemorrhage and endophthalmitis were not observed in any eye. In all eyes we observed a regular postoperative course. As for the eyes of patients treated by RP, the results of visual acuity show an improvement at 3 months of patient n. 2 (DO) moving from a baseline of 19 letters read ETDRS to 33 letters ETDRS at 1 month and then 25 letters at 3 months. Patient no. 1 (GP), on the other hand, showed a decrease in visual acuity going from 3+30 letters ETDRS at baseline to 0+0 letters at 1 month and remaining at 0+0 letters ETDRS at 3 months. However, the patient felt satisfied as she reported seeing the colors of the brighter objects and better defining the contour of the objects at a distance (unfortunately these parameters are not well quantifiable with specific ophthalmic tests). The other 2 patients treated while maintaining a stable visual acuity at 1 month compared to the baseline (0+0 and 0+8 patient n.3 and n.4 respectively), were satisfied as they claimed to see better the colors of the objects. Regarding the analysis of the electrophysiological results, we did not record significant differences in ERG and VEP for the 2 patients who have a 3-month follow-up. Central macular thickness analysis showed no significant changes at either 1 or 3 months from baseline.
For the 3 eyes of patients suffering from d-AMD at 1 month after the injection we did not observe a significant increase in the number of letters read by the patients but also in this case the patients were not considered dissatisfied as they reported having, in some way, improved the quality of vision. Also in this case the analysis of the electrophysiological results of ERG, PEV and of the central macular thickness did not show significant changes at 1 month compared to the baseline.
In the 7 eyes we treated with subretinal CB-PRP, there were no serious adverse reactions such as endophthalmitis, retinal detachment, uveitis or haemorrhages. Regarding the parameters evaluated such as the electroretinogram and the visual evoked potentials, even if with a short follow-up, we did not observe significant changes compared to the baseline data, making us understand that CB-PRP is a safe product when placed on contact with the retinal tissue. The central retinal thickness (CMT) measured with modern high definition optical coherence tomographs (OCT), although showing a significant increase at 1 week, then approaches the baseline values already at 1 month in all patients. A careful analysis of the OCT images, both at 1 and 3 months (only 2 patients), shows a clearer stratification of the retina, especially of the outer layers. This finding could be compatible with the hypothesis that the growth factors contained in the CB-PRP could stimulate the retinal cells to realign themselves correctly in a retinal stratification that could be functional again. Instead, we reported conflicting results for what concerns visual acuity, that is, although all patients we injected report seeing better, we were not able to quantify this improvement. In one case (GP) we had a significant worsening of visual acuity after the injection but also in this case the patient reported seeing better the outlines of things and colors. Recently we are evaluating the importance of the different CB-PRP injection sites as we believe that the saving of the macular region from the retinal detachment bubble (which is obtained when the CB-PRP is injected below the retina) can bring the same number of neurotrophic factors, avoiding the trauma of the separation of the neuroepithelium of the retinal pigment epithelium in the macular region. Since CB-PRP had never been evaluated until now for what concerns the injection inside the eye and in the subretinal spaces, this treatment could probably find the maximum of its rationale for use in the early stages of the disease. The cases we treated, for precautionary reasons, were patients with very advanced pathology in both the RP and d-AMD groups. In the near future, the surgical approach could be different, preferring, for example, the intravitreal rather than the subretinal injection route given the great surgical experience and high-cost instrumentation required linked to the latter. It could also be important to anticipate the timing of the CB-PRP injection precisely in order to prevent the development of a disabling disease.
In conclusion, the subretinal injection of CB-PRP has been shown to be safe and without serious adverse reactions in the cases followed so far. Its real efficacy remains to be clarified by performing this injection in a greater number of patients and with longer follow-ups with regard to RP and d-AMD. The intravitreal route of administration remains an area still to be explored and which would entail greater practicality and ease of use of the CB-PRP for intra-ocular use.
It forms part of the present description also the following:
The objective of the present study is to evaluate the efficacy of intravitreal injections of Umbilical Cord Blood Platelet-rich Plasma (CB-PRP) in order to reduce or stabilize the atrophic progression in dry Age-related Macular Degeneration (AMD).
Change during follow ups
Other outcome measures: Evaluation of Therapy Safety
Evaluation of major ocular adverse events (bacterial or fungal septic endophthalmitis, retinal detachment, vitreous proliferative-fibrotic reaction with retinal traction, secondary glaucoma, phthisis bulbs, iris rubeosis), studied at slit-lamp evaluation in the anterior and posterior chambers.
A sample size of 36 patients affected by d-AMD has been enrolled.
Inclusion Criteria:
CB-PRP is a blood component for non-transfusion use produced according to procedures defined by the Italian legislation on blood components (Decree of the Ministry of Health, 2 Nov. 2015: Provisions relating to the quality requirements GU No. 300 of 28 Dec. 2015; Decree of the Ministry of Health, 19 Aug. 2019: Amendments to the decree of 2 Nov. 2015, containing: “Provisions relating to the quality and safety requirements of blood and blood components”. OJ no. 262 of 26-9-2019).
The basic materials for the production of CB-PRP are cord blood units collected at the UNICATT cord blood bank of the Policlinico Gemelli IRCCS Foundation. These units are allogeneic solidarity donations for haematological patients who are candidates for hematopoietic stem cell transplantation: in this field, the use of cord blood represents a consolidated therapeutic practice for decades of experience. Cord blood units that can be used for transplantation must contain an adequate amount of hematopoietic progenitors. Units that did not meet the cell thresholds established for transplantation were not frozen and, with the consent of the donors, were intended for further clinical and research use. According to the aforementioned Italian legislation, couples who want to donate cord blood obtain eligibility after a thorough medical consultation. Furthermore, immediately after collection, infectious screening tests are performed on the maternal blood sample (serology and genome for HIV, HBV, HCV, and serological test for syphilis) or on the unit (blood cultures for fungi, aerobic and anaerobic bacteria). Only units with negative tests were used for subretinal injection.
The CB-PRP was made up of a pool of 15 units. Shortly after collection, each unit was subjected to a “soft-spin” centrifugation to obtain CB-PRP: the platelet concentration was then determined which will be normalized to 1×10{circumflex over ( )}9/L, through a “hard-spin” and subsequent removal of excess platelet-poor plasma. CB-PRP was then recovered and stored at −80° C. pending microbial testing. Once 15 CB-PRPs with negative tests were obtained, to avoid any disparity in the concentration of growth factors between the different units, they were thawed and collected in a single bag, suitable for the preparation of blood components for non-transfusion use. The pool was then fractionated into 1 ml aliquots in sterile sealed vials, which were stored again at −80° C. until use. This process made the intervention homogeneous for all patients who participated in the study. All steps required to prepare the CB-PRP pool were performed in sterile cryogenic blood component bags, and sterile connections were used to transfer CB-PRP from one bag to another and to fractionate the CB-PRP aliquots.
The photographs reported in
The procedure consists in a trans-scleral puncture to access the vitreous cavity, with subsequent injection of CB-PRP, in particular by means of a pre-filled syringe according to the invention. Preferably, a 30-gauge injection needle connected to the syringe is used to inject the contents of 0.05 mL of the CB-PRP composition into the vitreous chamber of the patient.
The study respected the guiding principles of the World Medical Association Declaration of Helsinki and was approved by the Ethics Committee. Informed consent was collected after an extensive description of study characteristics to patients. According to the study design, patients were randomized in 3 different schedules of treatment: monthly, bimonthly and every 3 months. The same amount of 0.05 mL of intravitreal CB-PRP per course of treatment was administered. Twenty-one eyes of 21 patients received CB-PRP in one eye and sham injection in the fellow eye. 30 The administration of CB-PRP by means of intravitreal injection to 21 patients has so far proven to be safe and effective for blocking the growth of the atrophic area. Notably, no local or systemic adverse reactions have been found during or after each single intravitreal injection.
In particular, no type of intraocular inflammatory or discomfort were referred from the patients or observed by ophthalmologist at follow-up after intravitreal injections. Indeed, the maintenance of the transparency of the dioptric, allowed an accurate collection of multimodal imaging methods to document macular status (
The applicability of intravitreal administration finds ample space in daily clinical practice. In fact, an ever-increasing number is expected for this method of administration for new emerging initial therapies also in dry-AMD.
The use of a pre-filled ready-to-use syringe containing CB-PRP according to the invention represents a cleaner and safer approach for treatment of patients suffering from d-AMD, enabling a large-scale applicability. Notably, the use of a pre-filled syringe loaded with CB-PRP for patients undergoing injection of this blood product allows to reduce the number of possible infections, reduce the quantity of CB-PRP thrown away for non-use, and to enable a safer and faster procedure from a clinical point of view. In conclusion, CB-PRP can be considered a safe regenerative therapy that could offer new hope for patients with dry-AMD. CB-PRP could be considered a valid alternative for patients with maculopathy waiting for more targeted therapies able to maintain or restore the central vision.
| Number | Date | Country | |
|---|---|---|---|
| 63264185 | Nov 2021 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 18056624 | Nov 2022 | US |
| Child | 18762199 | US |
