HK1180968A - Dosage unit formulations of autologous dermal fibroblasts - Google Patents

Description

Dosage unit formulations of autologous dermal fibroblasts

Technical Field

The present invention relates to isolated and prepared dosage unit formulations of autologous dermal fibroblasts for injection at a site for repair and/or long term improvement of skin and soft tissue defects in a human subject.

Background

As described in U.S. patent nos. 5,591,444, 5,660,850, 5,665,372, and 5,858,390, cosmetic and aesthetic defects in an individual's skin can be corrected by injecting a suspension of autologous dermal fibroblasts into the dermal and subcutaneous tissue more adjacent to the defect. Typical defects that can be corrected by this method include wrinkles, stretch marks, depressed scars, skin depressions of non-traumatic origin, scars from acne vulgaris, and lip dysplasia. The cells are compatible with the individual tissue, preferably obtained by culturing a biopsy specimen taken from the individual, and have been expanded by subculture in a cell culture system.

The injection of materials ("injectates") into the body and particularly into the face to produce aesthetic effects began near the nineteenth century. For example, paraffin injection to correct facial contour defects enjoys a short acceptance period in years before world war I. Complications and unsatisfactory nature of long-term results, however, have led to practice abandonment. The availability of injectable silicone allows these events to begin to recur substantially early in the 1960 s. Specially manufactured "medical grade" silicone solutions, such as Dow Corning (Dow Corning) MDX 4.4011, have been used on an experimental basis at many approved testing centers in the United states. Complications such as local and systemic reactions to silicone, injectate migration, and local tissue collapse limit the use of silicone injectables. The poor results obtained by injection of non-biological materials have prompted attempts to use foreign proteins (particularly bovine collagen) as injectables. While unprocessed bovine collagen is overly immunogenic for injection into humans, removal of the C-and N-terminal peptides of bovine collagen by enzymatic degradation results in a material ("atelocollagen") that can be used in limited amounts if patients are pre-screened to exclude those that are immunoreactive. Although widely used, the material is associated with the production of anti-bovine antibodies in about 90% of individuals and significant immune complications in about 1-3% of individuals. Delustt F. (Dellustro, F.) et al, 1987, Plastic and Reconstructive Surgery 79: 581. Atelopeptide collagen in solution form has proven to be less than completely satisfactory because the material is absorbed from the injection site by the individual in a relatively short period of time and is not replaced by the host material. The retention in vivo is prolonged by glutaraldehyde cross-linking, followed by filtration through a fine screen and shearing. However, the increased and irregular viscosity makes the material difficult to use. Injectable human collagen derived entirely from an individual's own tissue sample is available, but there is no evidence that human collagen injections are more permanent than bovine collagen injections.

These problems are solved by the development of autologous fibroblast preparations. However, the problematic solutions do not represent a product that has been validated through trial and error and confirmed through clinical trials, and that has been manufactured and packaged in compliance with the requirements of the U.S. food and Drug Administration, and stored stably in prescribed doses.

It is therefore an object of the present invention to provide a prescribed dosage unit formulation of autologous dermal fibroblasts for injection into a patient for repair and long-term improvement of skin defects.

It is another object of the present invention to provide dosage unit formulations containing stem cells, precursor cells or partially differentiated cells that can be used to repair and chronically ameliorate skin defects.

Disclosure of Invention

The dosage unit consists of an autologous cell therapy product consisting of fibroblasts grown for each individual to be treated. Suspension of autologous fibroblasts grown from biopsies of each individual's own skin using Current Good Manufacturing Practice (CGMP) and standard tissue culture procedures is supplied in vials containing cryopreserved fibroblasts or precursors thereof having at least 98% fibroblast purity and at least 85% viability for supply at 1.0-2.0 x 10⁷The concentration of individual cells/mL is administered in three portions, about five weeks apart, with 1 to 6mL, preferably 2mL, of cells injected at 0.05 to 0.5mL per linear centimeter length. The subcultured dermal fibroblasts are rendered substantially free of immunogenic proteins present in the culture medium by incubating the expanded fibroblasts in a protein-free medium for a period of time. When injected into nasolabial fold wrinkles (folds on the nasal side extending to the corners of the mouth), autologous fibroblasts are thought to increase synthesis of extracellular matrix components, including collagen, thereby reducing the severity of these wrinkles. Dosage and timing of administration has proven critical to achieving clinically significant outcomes.

Although there are several approved therapies in the United States (US) for treating nasolabial fold, for exampleJuvéderm^TMAndhowever, fibroblast suspensions do not fall into the same pharmacological categories as these products.These products are structural fillers that are injected into facial tissue to smooth wrinkles and furrows by temporarily adding volume to the facial tissue. Fibroblast dosage formulations work through a very different proposed mechanism of action. The dosage formulation units described herein are suitable for treating wrinkled skin, nasolabial and bucchal folds, perioral folds, external canthus folds, periorbital folds, and interpupillary folds.

Fibroblast dosage formulations are also useful for providing pluripotent cells for tissue repair and regeneration.

Drawings

FIG. 1 is a flow chart of a standardized manufacturing process.

Fig. 2 is a schematic diagram from berne (Byrne) 2008 human molecular genetics (hum. mol.gen.) 17: R37-R41, which shows the manner in which skin-derived fibroblasts can be dedifferentiated into pluripotent stem cells, which can then differentiate into neural cells, cardiac myocytes, beta islet cells, and hematopoietic cells using epigenetic reprogramming.

FIG. 3 is a schematic representation of a method by which fibroblasts can be dedifferentiated into pluripotent cells: cell fusion (Cowan et al 2005), direct reprogramming (Takahashi et al 2007) and somatic cell nuclear transfer (berne (Byrne) et al 2007).

Figure 4 is a graphical illustration in the Evaluator active Acne Scar assessment scale (Evaluator Live ace Scar assessment scale) for physician objective assessment of Acne Scar severity using comparative assessment of relative Scar appearance under direct and tangential illumination.

Detailed Description

An autologous fibroblast product has been developed. The cell therapy product is composed of a suspension of autologous fibroblasts grown from biopsies of each individual's own skin using standard tissue culture procedures. Biopsies of skin tissue (dermal and epidermal layers) are extracted from the retroauricular region of the patient and transported via next-day delivery to the manufacturing facility at 2-8 ℃. Fibroblasts isolated from tissue via enzymatic digestion are expanded to an amount sufficient for injection into a target treatment area of a patient. The cell therapy product consists of expanded fibroblasts that are formulated to the cell concentration of the target cell therapy product and stored cryogenically in frozen vials, called frozen vial bulk drug. The final cell therapy product consists of thawed bulk cell therapy product-frozen vial cells that are thawed, washed, and prepared for patient injection.

Initial marketing approval was for the treatment of moderate to severe nasolabial folds. Current clinical development is focused on treating dermal contour deformities, vocal cord scarring, gingival retraction, restrictive burn scarring, and acne scarring. As discussed below, the dosage and dosing regimen will vary depending on the condition to be treated. While the exact mechanism is unknown, it is believed that autologous fibroblasts that constitute the active component of a fibroblast dosage formulation, when injected into a patient's skin, increase the synthesis of extracellular matrix components (such as collagen), thereby enhancing skin integrity and ultimately leading to reduction of fine lines and wrinkles.

The following definitions are used herein:

ATM analytical test method

USAN designation of AZFICEL-T autologous cultured fibroblast

BULK Collection (BULK Harveest) Material after Final Collection, before deployment in cryopreservation Medium

Current good manufacturing standard of CGMP

CS cell Stacking

DMEM Dulbecco's modified eagle's Medium

DMSO dimethyl sulfoxide

The injectable pharmaceutical PRODUCT (DRUG PRODUCT-INJECTION) is washed and reconstituted in DMEM, bottled and prepared for delivery to the clinical site

Blending frozen vial bulk DRUG (DRUG Substance-CryOVIAL) in cryopreservation media and aliquoting the material filled into frozen vials

EDTA ethylene diamine tetraacetic acid

FACS Fluorescence Activated Cell Sorting (Fluorescence Activated Cell Sorting)

FBS fetal bovine serum

GA Gentamicin and amphotericin B (Amphotericin B)

IMDM Iskoff modified Dulbecco's Medium (Iscove's modified Dulbecco's Medium)

Application of IND new drug research

PBS phosphate buffered saline

PCA personal cell analysis

QC quality control

USP United States Pharmacopeia (United States Pharmacopeia)

I. Dosage unit formulations

ACell source

1. Autologous dermal fibroblasts

The cells in the formulation exhibited typical fibroblast morphology when grown in a cultured monolayer. In particular, the cell may exhibit a long and narrow spindle-like or spindle-like appearance with an elongated extension, or the cell may appear as a larger, flat astrocyte that may have a cytoplasmic leading edge. Mixtures of these morphologies can also be observed. The cells express proteins characteristic of normal fibroblasts, including the fibroblast specific marker CD90 (Thy-1), the 35kDa cell surface glycoprotein, and extracellular matrix proteins (collagens). Fibroblast dosage formulations are autologous cell therapy products consisting of suspensions of autologous fibroblasts grown from biopsies of each individual's own skin using standard tissue culture procedures. A living sample of skin tissue (dermis and epidermis) is extracted from the retroauricular region of a patient.

2. Precursor cell

Fibroblasts may also be used to produce other cell types for tissue repair or regeneration. Derivation of Embryonic Stem (ES) cells, which are genetically identical to patients, by Somatic Cell Nuclear Transfer (SCNT), maintains the potential to cure or alleviate the symptoms of many degenerative diseases, while avoiding problems with rejection by the host immune system. Burne (Byrne) et al, Nature (Nature) 2007, 11.22, 450(7169):497-502 uses a modified SCNT method to generate rhesus monkey (rhesus macaque) blastocysts from adult skin fibroblasts, and two ES cell lines were successfully isolated from these embryos. DNA analysis confirmed that the nuclear DNA was identical to the donor somatic cells and that the mitochondrial DNA was derived from the oocyte. Both cell lines exhibit normal ES cell morphology, express key stem cell markers, are transcriptionally similar to control ES cells and differentiate into multiple cell types in vitro and in vivo. See also SeBrassican et al, Stem Cells (Stem Cells) 2009;27(6): 1255-64. Fig. 2 is a schematic diagram from berne (Byrne) 2008 human molecular genetics (hum. mol.gen.) 17: R37-R41, which shows the manner in which skin-derived fibroblasts can be dedifferentiated into pluripotent stem cells, which can then differentiate into neural cells, cardiac myocytes, beta islet cells, and hematopoietic cells using epigenetic reprogramming. See hochellinger et al, Development (Development.) 2 months 2009, 136(4):509-23, and kanawatt et al, biosystems (Bioessays.) 2 months 2009, 31(2): 134-8.

FIG. 3 is a schematic representation of a method by which fibroblasts can be dedifferentiated into pluripotent cells: cell fusion (Cowan et al, Science, 26.8.2005; 309(5739): 1369-73), direct reprogramming (Takahashi et al, Cell (Cell.) 200730;131(5): 861-72), and somatic Cell nuclear transfer (Burne (Byrne et al, 2007). The high bridge (Takahashi) et al showed that iPS cells were generated from adult dermal fibroblasts with the following four factors being the same: oct3/4, Sox2, Klf4, and c-Myc. Human iPS cells resemble human Embryonic Stem (ES) cells in morphology, proliferation, surface antigens, gene expression, epigenetic status of pluripotent cell-specific genes, and telomerase activity. In addition, these cells can be differentiated in vitro and in teratomas into cell types with three germ layers. These findings demonstrate that iPS cells can be generated from adult fibroblasts.

B.Preparation of cells

Autologous fibroblasts in the drug substance are obtained by growing from a biopsy of the recipient's own skin, followed by expansion in culture medium using standard cell culture techniques. A living sample of skin tissue (dermis and epidermis) is extracted from the retroauricular region of an individual. The starting material consisted of three 3mm punch skin biopsies collected using standard sterile specifications. The biopsy was collected by the treating physician and placed into a vial containing sterile Phosphate Buffered Saline (PBS). The samples are transported back to the manufacturing facility in transport facilities refrigerated at 2-8 ℃.

Upon arrival at the manufacturing facility, the biopsy is inspected and transferred directly to the manufacturing area after acceptance. After the process has started, the biopsy tissue is then washed, followed by enzymatic digestion. After washing, a Solution of Liberase digest enzyme (Liberase digest enzyme Solution) was added without chopping, and the tissue of the living specimen was incubated at 37.0. + -. 2 ℃ for one hour. The time of digestion of the biopsy is a critical process parameter that affects the viability and growth rate of the cells in culture. The releaser is a collagenase/neutral protease mixture obtained IN formulated form from Lonza warkersville, Inc (wolkersville, marchan), and IN unformulated form from Roche Diagnostics Corp (Indianapolis, Indiana (IN)). Alternatively, other commercially available collagenases may be used, such as Sawa Collagenase NB6 (Serva collagen NB 6) (Heidelberg, Germany (Germany)). After digestion, the enzyme was neutralized by adding initiation growth medium (IMDM, GA, 10% Fetal Bovine Serum (FBS)), and the cells were pelleted by centrifugation and resuspended in 5.0mL of initiation growth medium. Alternatively, without centrifugation, the enzyme is completely inactivated by simply adding the initial growth medium. Initial growth medium was added, and the cell suspension was subsequently seeded into T-175 cell culture flasks to initiate cell growth and expansion. Instead of a T-75 flask, a T-75, T-150, T-185 or T-225 flask may be used.

At 37 + -2.0 deg.C and 5.0 + -1.0% CO₂Cells were grown down and supplied with fresh complete growth medium every three to five days. All feeding in the process was done by removing half of the complete growth medium and replacing the same volume with fresh medium. Alternatively, complete feeding may be performed. The cells should not be kept in the T-175 flasks for more than 30 days before subculture. Confluence was monitored throughout the process to ensure adequate seeding density during culture splitting. When the confluency of cells was greater than or equal to 40% in the T-175 flask, the cells were sub-cultured by removing the spent medium, washing the cells and treating with trypsin-EDTA to release adherent cells in the flask into solution. The cells were then trypsinized and seeded into T-500 flasks for continuous cell expansion. Alternatively, one or two T-300 flasks, one cell stack (1 CS), one cell factory (1 CF) or two cell stacks (2 CS) may be used instead of the T-500 flask.

Morphology was evaluated in each sub-passage and prior to collection to monitor culture purity throughout the process. Morphology was assessed by morphological examination of the cell cultures by comparing the observed samples with visual standards. The cells exhibit a typical fibroblast morphology when grown in a cultured monolayer. The cells may exhibit a long, narrow spindle or spindle-like appearance with elongated extensions, or appear as larger, flat astrocytes that may have cytoplasmic leading edges. Mixtures of these morphologies can also be observed. Fibroblasts in the less confluent regions may have similar shapes, but are randomly oriented. The cell cultures were also evaluated for the presence of keratinocytes. Keratinocytes appear to be round as well as irregularly shaped, and at higher confluence they appear to organize into a cobblestone conformation. At lower confluency, keratinocytes can be observed in small colonies.

At 37 + -2.0 deg.C and 5.0 + -1.0% CO₂Cells were grown down and fed in T-500 flasks every three to five days and in ten-layered cell stacks (10 CS) every five to seven days. The cells should not be kept in the T-500 flask for more than 10 days before subculture. Quality Control (QC) factory tests for bulk drug substance safety include sterility and endotoxin tests. When the confluency of cells in the T-500 flask was 95% or more, the cells were subcultured into a 10CS culture vessel. Alternatively, two five-layered cell stacks (5 CS) or ten-layered cell factories (10 CF) can be used instead of 10 CS. Subculture was performed to 10CS by removing spent medium, washing cells and treating with trypsin-EDTA to release adherent cells in the flask into solution. The cells were then transferred to 10 CS. Additional complete growth medium was added to neutralize trypsin and cells from the T-500 flask were pipetted into a 2L flask containing fresh complete growth medium. The contents of the 2L bottle were transferred into 10CS and inoculated across all layers. Followed by a temperature of 37. + -. 2.0 ℃ and 5.0. + -. 1.0% CO₂Cells were grown down and supplied with fresh complete growth medium every five to seven days. The cells should not be maintained in 10CS for more than 20 days prior to subculture.

The subcultured dermal fibroblasts are rendered substantially free of immunogenic proteins present in the culture medium by incubating the expanded fibroblasts in a protein-free medium for a period of time. Primary collection: when the degree of cell confluence was 95% or more in 10CS, the cells were collected. The collection was performed by removing spent medium, washing the cells, treating with trypsin-EDTA to release adherent cells into solution, and adding additional complete growth medium to neutralize trypsin. Cells were collected by centrifugation, resuspended, and subjected to in-process QC testing to determine total viable cell count and cell viability.

For treatment of the nasolabial sulcus, the total cell count must be 3.4 × 10⁸Individual cells and viability must be 85% or higher. Alternatively, the total cell yield for other indications may be 3.4X 10⁸To 1X 10⁹Within the range of one cell. Cell count and viability at the time of collection are key parameters to ensure that there are sufficient numbers of viable cells for the drug substance to be dispensed. Cell aliquots and depleted media were tested for mycoplasma contamination if the total viable cell count was sufficient for the intended treatment. Mycoplasma testing was performed. The collected cells were conditioned and cryopreserved.

If additional cells are needed after receiving the cell count results from the primary 10CS collection, additional subcultures are performed up to a stack of cells (up to four 10 CS) (step 5a in FIG. 1). For additional subcultures, cells from the primary harvest were added to a 2L medium bottle containing fresh complete growth medium. Adding the resuspended cells to a multiple cell stack and at 37 + -2.0 deg.C and 5.0 + -1.0% CO₂And (5) cultivating. The cell stack is fed and collected as described above, except that the degree of cell confluence must be 80% or higher prior to cell collection. The collection procedure is the same as described above for the primary collection. Mycoplasma samples were collected from cells and depleted media, and cell counts and viability assays were performed as described above for primary collection.

The methods reduce or eliminate immunogenic proteins, thereby avoiding their introduction from animal-derived agents. To reduce process residues, cells are cryopreserved in protein-free freezing medium, then thawed and washed before being prepared for final injection to further reduce residual residues.

If additional drug substance is required after collection is complete and cells from additional subcultures are cryopreserved (step 5a in fig. 1), an aliquot of the frozen vial of drug substance is thawed and used to inoculate a 5CS or 10CS culture vessel (step 7a in fig. 1). Alternatively, a four-layered cell factory (4 CF), two 4 CFs, or two 5CS may be used instead of 5CS or 10 CS. Frozen vials of cells were thawed, washed, added to 2L medium bottles containing fresh complete growth medium, and cultured as described above, collected and cryopreserved. The cell suspension is added. The confluency of cells must be 80% or more before cell collection.

C.Preparation of cell suspensions

Upon completion of the culture expansion, the cells were collected and washed, followed by formulation to contain 1.0-2.7X 10⁷Individual cells/ml, target 2.2X 10⁷Individual cells/ml. Alternatively, the target can be adjusted within the formulation range to accommodate different indication doses. The drug substance consists of a population of viable autologous human fibroblasts suspended in a cryopreservation medium consisting of Iskoff's Modified Darber Medium (IMDM) and Profeze-CDM^TM(Longsha corporation (Lonza), Wolvel (Walkerville), Md.) plus 7.5% Dimethylsulfoxide (DMSO). Alternatively, lower DMSO concentrations can be used instead of 7.5% or CryoStor^TMCS5, or CryoStor may be used^TMCS 10 (BioLife solutions, USA), Bashal (Bothell), Washington, Inc. replaces IMDM/Profreeze/DMSO. The freezing process consists of the controlled rate freezing steps of the following ramp program:

step 1: waiting at 4.0 deg.C

Step 2: 1.0 ℃/minC/m to-4.0 ℃ (sample probe)

And step 3: 25.0 ℃/minC/m to-40 ℃ (chamber probe)

And 4, step 4: 10.0 ℃/minC/m to-12.0 ℃ (chamber probe)

And 5: 1.0 ℃/minC/m to-40 ℃ (chamber probe)

Step 6: 10.0 ℃/minC/m to-90 ℃ (chamber probe)

And 7: end up

After completion of the controlled rate freezing step, bulk drug substance vials are transferred to a cryogenic freezer for storage in the vapor phase. After low temperature freezing, the bulk drug is submitted for quality control testing. Bulk drug specifications also include cell count and cell viability tests performed prior to cryopreservation and again on frozen vial bulk drug. For product shipment, cell viability must be 85% or higher. Cell counting and viability assays were performed using an automated cell counting system (gava Technologies) that utilizes a combination of permeable and impermeable fluorescent DNA intercalating dyes to detect and distinguish between live and dead cells. Alternatively, a manual cytometric analysis using the trypan blue exclusion method may be used instead of the automated cell method described above. Alternatively, other automated cell counting systems may be used for cell counting and viability methods, including Cedex (Roche InnovatisAG), bielefeld (Bielefield), Germany (Germany)), ViaCell^TM(Beckman Coulter, Berea, Calif.), NuceloCounter^TM(New Brunswick scientific, Edison, N.J.), (New Brunswick scientific, New Jersey),(Invitrogen, division of Life Technologies, Inc., Carlsbad, plusState of rifampicin (CA)) or(Leicolo Biosciences (Nexcelom Biosciences), Lorents (Lawrence), Mass.). The frozen small bottled raw material medicine sample must meet 1.0-2.7 multiplied by 10 before leaving factory⁷Cell count specification of individual cells/ml. Sterility and endotoxin tests were also performed during factory testing.

In addition to cell count and viability, purity/identity analysis of the drug substance was performed and it was necessary to confirm that the suspension contained 98% or more fibroblasts. Common cellular contaminants include keratinocytes. Purity/identity analysis the percentage of purity of the fibroblast population was quantified using fluorescently labeled antibodies against CD90 and CD104 (cell surface markers for fibroblasts and keratinocytes, respectively). CD90 (Thy-1) is a 35kDa cell surface glycoprotein. Antibodies directed against the CD90 protein have been shown to exhibit high specificity for human fibroblasts. The CD104 integrin beta 4 chain is a 205kDa transmembrane glycoprotein that associates with the integrin alpha 6 chain (CD 49 f) to form an alpha 6/beta 4 complex. This complex has been shown to serve as a molecular marker for keratinocytes (Adams) and Watt (Watt) 1991).

Antibodies against the CD104 protein bound to 100% of human keratinocytes. Cell count and viability were determined by incubating the samples with a vitant Dye Reagent (Viacount Dye Reagent) and analyzing the samples using the Guava PCA system. The reagent consists of two dyes, a membrane permeable dye that stains all nucleated cells and a membrane impermeable dye that stains only damaged or dying cells. The use of this dye combination enables the Guava PCA system to estimate the total number of cells present in a sample and determine which cells survive, die or die. The method is specifically developed for determining the purity/identity of autologous cultured fibroblasts. The specific procedure is as follows:

Procedure

preparation of 25% (W/V) sodium azide in PBS

Preparation of FACS (fluorescence activated cell sorting) buffer：

In a 1000mL Nagen flask (Nalgene bottle), 966mL of PBS, 30mL of FBS, and 4mL of 25% sodium azide in PBS were combined.

Preparation of 1% (v/v) HuS PBS solution：

1mL of a 1% HuS PBS solution was prepared for each test sample. To a 1.5mL tube labeled as a 1% HuS aliquot, 990. mu.L PBS was added. 10 μ L of human serum was added to a tube labeled 1% HuS.

Sample receiving and preparation：

Verification results daily Guava examinations were performed on the current day. Cell count and viability analysis was performed on test vials using Guava PCA. The average cell count obtained was recorded.

For each sample set, each test vial was labeled with four 1.5mL microcentrifuge tubes as follows: "CD 90", "CD 104", "mouse" (corresponding to the mouse IgG1 isotype control) and "rat" (corresponding to the rat IgG2b isotype control).

Using the mean cell count results, the appropriate volume of cells for aliquoting was calculated so that each tube received 1X 10⁵And (4) living cells.

Example (c):

if the average viable cell count result is described as 1.5X 10⁷Individual cells/ml, then 1X 10⁵Divided by 1.5X 10⁷The appropriate volume is one cell/ml. This volume (mL) is multiplied by 1000 to convert the unit to μ L.

1×10⁵/1.5×10⁷Individual cells/mL =0.0066mL × 1000=7 μ L cell solution aliquot.

Sufficient FACS buffer was added to each tube so that the total volume would be 1 mL.

Example (c):

if 7. mu.L of cell solution is added, 7. mu.L is subtracted from 1mL or 1000. mu.L. Add 1000-7 μ L =993 μ L FACS buffer.

The test vials were mixed by continuously vortexing on the vortexing device 8 for 3 seconds.

The appropriate amount of cell solution was added to FACS buffer. Mixing was done by continuously vortexing for 3 seconds on the vortexing device 8.

The cells in each sample tube were pelleted by centrifugation at 3,000rpm for 3 minutes. If it is difficult to form an aggregate, the tube is centrifuged again at 3,000rpm for 3 minutes. If sufficient aggregate formation occurs after the second centrifugation, aspiration of the supernatant is continued. The supernatant was aspirated by means of a pipette into a waste container containing a 20% bleach solution in deionized water. Approximately 50 μ L of supernatant was left in each tube to minimize inadvertent aspiration of the cells.

200 μ L of 1% HuS in PBS was added. Mixing was done by continuously vortexing for 3 seconds on the vortexing device 8.

The tubes were incubated at 2-8 ℃ for 25 min. + -. 5 min.

mu.L of antibody or isotype control was added to each tube. Mixing was done by continuously vortexing for 3 seconds on the vortexing device 8.

The tubes were incubated at 2-8 ℃ for 25 min. + -. 5 min. And (4) avoiding light.

800 μ L of FACS buffer was added to each tube. Mixing was done by continuously vortexing for 3 seconds on the vortexing device 8.

With the aid of a pipette, aspirate the supernatant and leave about 50 μ L of supernatant in each tube to minimize inadvertent aspiration of the cells. Add 400. mu.L FACS buffer. Mixing was done by continuously vortexing for 3 seconds on the vortexing device 8.

Samples were collected using guava pca:

cytosoft 2.1.5 is turned on and "Guava Express" in the Main Menu is clicked. Click on "New Data Set" and follow the screen command to generate a New file. It is ensured that 2000 is input in the event (Events To Acquisition of the Guava Express Acquisition screen) To acquire part of the Guava Express Acquisition screen. Settings for mouse IgG1 isotype controls were adjusted. Sample fractions, batches, and isotype controls or antibodies (e.g., "DR 01200502001 mouse") are entered within the sample information control panel to identify each sample in the sample ID field.

Mouse IgG1 isotype control was vortexed continuously for 3 seconds on vortex setting 8, loaded into the Guava PCA and clicked on "Settings" (Settings). The message window will appear and "Adjust Settings (Adjust Settings)" will be selected. Another message window will appear prompting the loading of the control sample, selecting "OK".

An "adjust settings" screen should appear and the system will automatically set the threshold to exclude background fluorescence. One FSC Gain intensity (Gain intensity) was chosen to center the defined cell clusters above 10e3 in the lower right quadrant on the X-axis of the FSC versus viability (PM 1) plot (e.g., "X2"). FSC threshold bars on the FSC versus viability (PM 1) graph were located to exclude debris.

PM2 was adjusted to 300V. PM1 was adjusted to between 300-400V so that the negative control population was located between FSC versus PM1 and PM1 versus 10e0 and 10e1 on the PM2 dot plot.

Collection of mouse IgG1 isotype control：

Mouse IgG1 isotype control was vortexed continuously for 3 seconds on vortex setting 8, loaded into Guava PCA and clicked "take Next Sample (Acquire Next Sample)". It is ensured that all default gating options are set to ungated. On the bar graph of PM1 fluorescence versus count, a marker was set at the default position. Click markers were set such that the percentage of histogram events for PM1 gated samples was about 1.0%.

Collection of CD90 samples：

Sample fractions, batches, and isotype controls or antibodies (e.g., "DR 01200502001CD 90") are entered within the sample information control panel to identify each sample in the sample ID field. Check to set the PM1 fluorescent label to the same setting as the mouse IgG1 isotype control sample.

Adjusting settings for rat IgG2b isotype controls：

Rat IgG1 isotype control was vortexed continuously for 3 seconds on vortex setting 8, loaded into the Guava PCA and clicked "set". An "adjust settings" screen should appear and the system will automatically set the threshold to exclude background fluorescence. One FSC gain intensity was selected to center the defined cell clusters in the lower right quadrant on the X-axis of the FSC versus viability (PM 1) plot (e.g., "X2") at 10³Above. FSC threshold bars on the FSC versus viability (PM 1) graph were located to exclude debris. PM2 was adjusted to 300V. PM1 was adjusted to between 300-400V so that the negative control population was located between FSC versus PM1 and PM1 versus 10e0 and 10e1 on the PM2 dot plot.

Control samples of rat IgG1 isotype：

Rat IgG1 isotype control was vortexed continuously for 3 seconds on vortex setting 8, loaded into the Guava PCA and clicked "take next sample". All gating options are set to non-gating. On the bar graph of PM1 fluorescence versus count, a marker was set at the default position. Click markers were set such that the percentage of histogram events for PM1 gated samples was about 1.0%. The position of the marker is displayed in a numerical value in a "marker position" box.

Collection of CD104 samples：

The CD104 tube was vortexed continuously for 3 seconds on vortex setting 8, loaded into the guava pca and clicked "take next sample". Check to set the PM1 fluorescent label to the same setting as the rat IgG1 isotype control sample.

Analysis of samples Using Guava PCA：

Click "execute (Go)" is analyzed from the capture screen. Click on "Print (Print)", and then click on "OK (OK)". Each printed report is signed with a name and date.

Purity calculation：

PM1 event count [ total events labeled with CD90 (CD 90) ] -PM1 event count [ total events labeled with anti-CD 90 (mouse IgG 1) ] = total events labeled with CD90 (normalized for background/non-specific fluorescence) (e.g. 1918 events-20 events =1898 events).

PM1 event counts [ total events labeled with CD104 (CD 104) ] -PM1 event counts [ total events labeled with anti-CD 104 (rat IgG2 b) ] = total events labeled with CD104 (normalized for background/non-specific fluorescence) (e.g. 31 events-23 events =8 events).

Total events labeled with CD90 (normalized for background/non-specific fluorescence) + total events labeled with CD104 (normalized for background/non-specific fluorescence) = total normalized labeled events (e.g. 1898 events +8 events =1906 events).

Purity% was calculated as follows: 1898 total CD90 events/1906 total normalized marker events × 100=99.58, or 100%.

System suitability and specification：

A successful Bead Check (Bead Check) must be performed before starting the analysis.

The total particle count must be collected 2000.

The purity percentage of all samples must be > 98%.

Alternative manufacturing method

Alternatively, cells can be sub-cultured from T-175 flasks (or alternatives) or T-500 flasks (or alternatives) into spinner flasks containing microcarriers as cell growth surfaces. Microcarriers are small bead-like structures that serve as growth surfaces for anchorage-dependent cells in suspension culture. The microcarriers are designed to produce large cell yields in small volumes.

In this apparatus, a volume of complete growth medium in the range of 50mL to 300mL is added to a 500mL, 1L or 2L sterile disposable spinner flask. Sterile microcarriers were added to the spinner flask. At 37 + -2.0 deg.C and 5.0 + -1.0% CO₂The cultures were either kept static in an incubator or placed on a stir plate at low RPM (15-30 RRM) for a short period of time (1-24 hours) to allow the cells to adhere to the carriers. After the attachment period, the speed of the rotating plate is increased (30-120 RPM). Cells are supplied with fresh complete growth medium every one to five days or when the medium appears to be depleted according to a color change.

At regular intervals cells were collected by sampling microcarriers, isolating cells and performing cell counting and viability analysis. The cell concentration of each vector was used to determine when to scale up the culture. When enough cells were produced, the cells were washed with PBS and harvested from microcarriers using trypsin-EDTA, and the spinner flasks were seeded in larger amounts of microcarriers and larger volumes of complete growth medium (300 mL-2L). Alternatively, additional microcarriers as well as complete growth medium can be added directly to the spinner flask containing the existing microcarrier culture, allowing for direct inter-bead transfer of cells without trypsin treatment and re-seeding. Alternatively, if enough cells are produced from the initial T-175 or T-500 flask, the cells can be seeded directly into a scaled-up amount of microcarriers.

After the attachment period, the speed of the rotating plate is increased (30-120 RPM). Cells are supplied with fresh complete growth medium every one to five days or when the medium appears to be depleted according to a color change. When the concentration reached the cell count required for the desired indication, the cells were washed with PBS and harvested using trypsin-EDTA. All factory testing, cryopreservation and preparation of injectable pharmaceutical products will follow the procedures described in sections C and D.

The microcarriers used in the disposable spinner flasks can be made of the following materials: polymer blends (poly blends), such as BioNOC(Secey bioengineering, Inc., distributed by Bellco Biotechnology, Inc., Vanilla, Vineland, N.J.). and(New Brunswick Scientific, Edison, N.J.); gelatin, such as Cultispher-G (Percell Biolytica), Astrop (Astrop), Sweden (Sweden)); cellulose, e.g. Cytopore^TM(general electric health group (GE Healthcare), piscatavir (Piscataway), New Jersey (NJ)); or coated/uncoated polystyrene, e.g. 2D MicroHex^TM(Neken (Nunc), Wisbardon (Weisbaden), Germany (Germany));(GE Healthcare), Piscataway, N.J.. or Hy-Q Sphere^TM(Seimer Feishale science & technology (ThermoScientific Hyclone), Rougen (Logan), Utah (UT)).

Alternatively, the polymer blend may be supported on a 2D microcarrier (e.g., BioNOC)And) Using an automated corrugation system (e.g. FibraStage)^TM(New Brunswick Scientific, Edison, N.J.) or(Seyu Bioengineering, Inc. (Cesco Biotechnology), distributed by Bellco Biotechnology, Van. Van.Lane (Vineland), N.J. (NJ))) instead of a rotary flask apparatus. Cells were sub-cultured from T-175 (or surrogate) or T-500 flasks (or surrogate) into corrugated flasks containing microcarriers and the appropriate amount of complete growth medium and placed into the system. The system pumps media onto the microcarriers to feed the cells and aspirates the media away to allow oxidation in repeated fixed cycles. Cells were monitored, fed, washed and collected in the same order as described above.

Alternatively, the cells may be processed using an automated system. After digestion of the biopsy tissue or after completion of the first subculture (T-175 flask or surrogate), the cells can be seeded into an automated device. One approach is an Automated Cell Expansion (ACE) system, which is a series of commercially available or specially manufactured components linked together to form a cell growth platform in which cells can be expanded without human intervention. Cells were expanded in a cell tower consisting of a stack of discs capable of supporting anchorage-dependent cell attachment. The system automatically circulates the medium and trypsinizes for collection after the cell expansion phase is complete.

Alternatively, the ACE system may be scaled down to a single batch unit form that contains disposable components consisting of: cell growth surfaces, delivery conduits, media and reagents, and permanent mounts housing mechanisms for heating/cooling, media transfer, and performing automated programming cycles, as well as computer processing capabilities. After acceptance, each sterile irradiated ACE disposable unit is opened from its packaging and loaded with media and reagents by hanging a pre-filled bag and connecting the bag to existing tubing through a sterile connector. The process continues as follows:

in the biosafety cabinet (BSC), a suspension of cells from biopsies that have been subjected to enzymatic digestion is introduced into a "pre-growth chamber" (small cell at the top of the cell tower) that has been filled with an initial growth medium containing antibiotics. The disposable is transferred from the BSC to the permanent ACE unit already in place.

After about three days, the cells within the pre-growth chamber were trypsinized and introduced into the cell tower itself, which was pre-filled with complete growth medium. Here, by CO₂The "bubbling action" caused by the injection forces the medium to circulate at a rate that causes the cells to spiral down and settle in an evenly distributed manner on the surface of the disk.

Cells were multiplied for about seven days. At this point, confluency (the method was unknown at the time of writing) was checked to verify that the culture was growing. At this point, the complete growth medium was also replaced with fresh complete growth medium. CGM was replaced every seven days for three to four weeks. At the end of the incubation period, the confluency is checked again to verify that there is sufficient growth to potentially obtain the desired amount of cells for the intended treatment.

If the culture is sufficiently confluent, it is collected. The depleted medium (supernatant) is drained from the vessel. PBS was then pumped into the vessel (to wash the media, FBS from the cells) and drained almost immediately. trypsin-EDTA was pumped into the container to detach the cells from the growth surface. The trypsin/cell mixture was drained from the container and into a rotating separator. The cryopreservative was pumped into a container to flush any residual cells from the disc surface and likewise to a rotating separator. The cells were collected by rotating the separator and then resuspended uniformly in transport/injection medium. From the rotating separator, the cells will be sent or collected by an automated cell counting device for cell counting and viability testing by laboratory analysis. Once a certain number of cells have been counted and the appropriate cell concentration has been reached, the collected cells are delivered into a collection vial, which can be removed to aliquot the sample for cryo-freezing.

Alternatively, automated robotic systems may be used to feed, sub-culture, and collect cells throughout the length or in a portion of the process. Cells can be introduced directly into the robotic apparatus after digestion and seeding into T-175 flasks (or alternatives). The device may have the ability to cultivate cells, perform cell counting and viability analysis, and feed and transfer into larger culture vessels. The system may also have computerized cataloging functionality to track individual batches. Existing technology or custom systems may be used for the robot options.

D.Dosage unit

The frozen vial bulk drug used to prepare the final dosage unit consists of fibroblasts that are collected from the final culture vessel, formulated to the desired cell concentration and stored cryogenically in the frozen vial. Storing frozen vials of the drug substance in a container made of IMDM and Profreeze^TMAdding 7.5% DMSO to obtain a concentration of 2.2 × 10⁷Individual cells per ml of target. After undergoing a controlled rate freezing cycle, the frozen vial of bulk drug is stored frozen in the gas phase of a liquid nitrogen freezer.

The collected cells were pooled, formulated in cryopreservation media including Profreeze, DMSO, and IMDM media, aliquoted into frozen vials, and stored frozen in liquid nitrogen as frozen vial bulk drug material by controlled rate freezing.

The caps and vials were radiation sterilized and received aseptically from the manufacturer. The required volume of bulk material required for treatment is removed from frozen storage, thawed and pooled. Cells were washed with 4 × bulk volume of PBS and centrifuged at 150 × g for 10 min (5 ± 3 ℃). After this time, the cells were washed with 4 × bulk volume of DMEM by resuspension and centrifuged at 150 × g for 10 min (5. + -. 3 ℃). The washed cells were resuspended in phenol red-free DMEM to 1.0-2.0X 10⁷Target concentration of individual cells/ml. Alternatively, it can useFRS (BioLife Solutions, Bothell, Wash.) for a second 4 × wash and final resuspension. The final sterile frozen vial container was then manually filled to a volume of 1.2 ml/container in a biosafety cabinet. Storing the injectable pharmaceutical product at 2-8 deg.C until it is transported to the administration site in a transport container refrigerated at 2-8 deg.C.

Alternatively, the drug substance vial may be removed from cryogenic storage and transported directly to the drug administration site for dilution and administration. In the direct injection concept, cells were harvested and prepared for use at higher cell concentrations (3.0-4.0X 10)⁷Individual cells/ml, and 2.2X 10⁷Individual cells/ml of current target). While awaiting injection, the frozen vials were transported to the study site on dry ice or in a liquid nitrogen dewar (dewar). The vials are thawed at the administration site by hand or with a heat block and the frozen cells are diluted 1:1 at the study site using typical injection diluents such as bacteriostatic water, sterile water, sodium chloride or phosphate buffered saline. Alternatively, DMEM may be used as the diluent. This concept eliminates the need to wash and prepare fresh injection suspensions for overnight transport to the research site.

Alternatively, cells freshly collected from flasks or cell stacks can be placed inDMEM adjusted to 1.0-2.0X 10⁷Target concentration of individual cells/ml, subjected to all bulk collection and frozen vial bulk drug testing as described above, and delivered freshly as a final injection product overnight to the administration site in a refrigerated delivery vehicle at 2-8 ℃. In such a case, sterility and mycoplasma testing may be performed upstream of collection to allow time for results prior to shipment.

Method of administration

A.Preparation of dosage units

The Azficel-T injectable drug consisted of a suspension of autologous fibroblasts, which survived themselves in each patient, in Darber Modified Eagle's Medium (DMEM) without phenol red. Azficel-T fibroblast dosage formulations were supplied in two 2mL vials, each containing 1.2mL of 1.0-2.0X 10⁷Individual cells/ml drug. The sterile cell suspension is intended for intradermal injection. Initially, prior to the specification of Azficel-T fibroblast dosage formulation by the FDA, the cell dosage of Azficel-fibroblast dosage formulation was based on the number of cells administered by the clinician. 1.5-7.0X 10 per 1.2-1.4mL of injection was used successfully⁷Dose range for individual cells. Viscosity becomes a problem for fibroblast dosage formulation injections at concentrations above this range. This dose range translates to 1.1-5.8X 10⁷Individual cells/ml.

In the first clinical study conducted under IND, 0.5-3.0X 10 was formulated⁷Individual cells/ml. In the second clinical study, 1.0-3.0X 10 was formulated⁷Individual cells/ml. The standard factory specifications of all subsequent clinical studies are 1.0-2.0 × 10⁷Individual cells/ml.

In later clinical studies IT-R-005 and IT-R-006, a formulation provided in each of two 2.0mL cryovials was 1.0-2.0X 10⁷cells/mL of a 1.2mL solution of fibroblasts. Of the 2.4mL provided, 2.0mL is intended for injection at a dose of 0.1mL per linear centimeter length for a total of 20cm maximum on both sides of the faceThe nasolabial sulcus length is highly predicted. An excess of 0.2mL is contemplated to ensure that 1.0mL can be obtained from the vial to treat nasolabial folds up to 10 linear centimeters in length.

B.Administration dosage unit

The vial was warmed to room temperature and gently inverted to resuspend the settled cells. The cell suspension is withdrawn from the vessel using a small unit syringe equipped with a removable or fixed needle. Short, sharp needles and small unit syringes (e.g., 0.5mL insulin syringes) are recommended to achieve better injection control and reduce the risk of inflammation. Intradermal injection of the product requires 29 gauge or 30 gauge needles. However, a 21 gauge removable needle syringe with a larger bore may be used to assist in withdrawing product from the container. Once withdrawn, the 21 gauge needle can be switched to 30 gauge and the product administered.

One batch consisted of three injections of treatment. For treatment of nasolabial folds, a single injection treatment (batch) requires two 2mL vials containing 1.2 mL/vial of azficel-T fibroblast dosage formulation.

C.The condition to be treated

The aging process of the skin is initiated by both intrinsic and extrinsic factors. Factors contributing to intrinsic or natural aging are structural and functional factors. Structurally, the epidermis becomes thinner, the keratinocytes adhere less and the dermal-epidermal junction flattens. Functionally, the number and biosynthetic capacity of fibroblasts is reduced and the dermis becomes atrophic and relatively acellular and avascular. Exposure to ultraviolet radiation is the primary cause of extrinsic aging or photoaging. Extrinsic aging is characterized by loss of elasticity, increased roughness and dryness, irregular pigmentation, deep wrinkle formation, leathery appearance, blister formation, and impaired wound healing. The visible appearance of aging, particularly facial wrinkles and furrows, is a common outcome that patients seek to reduce. Options for treating facial lines, wrinkles and furrows include surgery, neurotoxins, fillers, lasers, non-exfoliative therapy, microdermabrasion (microdermabrasion), and chemical peeling. Many of these treatments vary in safety, efficacy, and duration of action in treating signs of aging. It is believed that the formulations described herein increase the number of collagen-producing cells in the dermis, which may have a multifactorial effect on improving the skin.

The use of autologous human fibroblasts for the treatment of contour deformities was pioneered by the William K.Boss, MD, Vice Chairman of the Department of Medical orthopedics, university of Hakensaka (Vise Charrman of the Department of Plastic Surgery at Hakens university Medical Center). His work in the area of dermal repair began in 1992. He removed a small piece of skin from the patient's wrist, cultured autologous fibroblasts from the biopsy, injected the autologous cells into the fold in the patient's wrist skin, and observed that the fold disappeared over time. He observed the area for years and noticed that the wrinkles at the test site remained corrected. No adverse reaction to the single treatment.

A small clinical trial was performed at 3 months in 1995. Initially, autologous cultured fibroblasts were injected in two to three injections into selected wrinkles and scars in about 12 individuals. After 12 months of follow-up, progressive improvement was noted in each individual. Bos (dr. boss) and mad doctor (dr. marko) reported that treatment of more than 100 patients, with no signs of allergic or adverse reactions observed over an observation period of more than 2 and a half years. Autologous fibroblasts were commercially available in the united states at 12 months 1995. The U.S. commercial experience includes about 100 clinicians in the fields of dermatology, facial plastic surgery, and reconstructive plastic surgery, who treat patients with facial wrinkles, scars, lip dysplasia, burns, and other problems. Patient experiences of nearly 1,000 patients were reported, of which 354 were included in the efficacy population reported in the review of information.

A pilot study using intradermal injection of fibroblast cell suspension for treatment of significant facial wrinkles and depressed facial scars was conducted on 10 healthy adults at the University of California Los Angeles University (UCLA). Nine out of ten patients noted 60-100% improvement achieved by treatment after two (2) injection sessions at 3 week intervals; similar observations were observed by the clinician. The size reduction of all treated deformities was confirmed by optical measurement (laser) to be 10-85%. Under a microscope, there is evidence of increased thickness and density of the dermal layer.

Studies have shown that injection of fibroblast suspension into the contour defect of the dermis can correct damaged dermis and subcutaneous tissue. The dermis of the skin contains fibroblasts, which are primarily responsible for secreting extracellular matrix components, such as collagen and elastin, which provide mechanical strength and integrity to the skin. Although the exact mechanism is unknown, fibroblast cell suspensions contain autologous fibroblasts that, when injected into a patient's skin, increase the synthesis of extracellular matrix components, thereby enhancing skin integrity, ultimately resulting in the reduction of fine lines and wrinkles. The effect of the therapy is not immediate, but provides a gradual improvement in the appearance of lines and wrinkles over time.

Fibroblast suspensions were initially evaluated in a clinical study conducted by doctor williams (dr. williamboss) in 1995 and subsequently marketed in the united states as a cosmetic treatment for facial contour deformities from 12 months in 1995 to 2 months in 1999. After 2 months 1999, the FDA was required to regulate all somatic therapies under the PHS act.

Upon FDA announcement that fibroblast cell suspensions will fall under new cell and tissue regulations, commercial circulation has ceased and clinical development has begun under new drug research application No. 8641 (IND). Studies on the treatment of facial wrinkles and folds were conducted in two phase II studies (study IT-R-001 and IT-R-007) and five phase III studies (study IT-R-002, IT-R-003A, IT-R-003B, IT-R-005, and IT-R-006). Study IT-R-003A, IT-R-003B, IT-R-005 and IT-R-006 provided robust, well-controlled data that demonstrated the safety and efficacy profile of fibroblast cell suspensions. Fibroblast cell suspensions were studied in the treatment of acne scars (study IT-A-008; IND 13455). 99 individuals received fibroblast suspension treatment during the study IT-A-008. Fibroblast suspensions were also developed for other indications, including treatment of restrictive burn scar (IND No. 13308), vocal cord scar (IND No. 9892), and gingival repair (IND No. 10805).

The earlier 001 and 002 tests provided supportive exploratory data that guided the design of the 003A/B and 005/006 tests. Study IT-R-001 was a phase II double-blind, randomized and placebo-controlled study of fibroblast cell suspensions for the treatment of cockles (treatment of nasolabial and labial-buccal sulci, perioral striatum, glabellar striatum, acne scar, and forehead). During the acute phase of the study, each individual received a fibroblast suspension (0.5X 10)⁷、1.0×10⁷Or 2.0X 10⁷One cell/ml) or placebo, with administration occurring about two weeks apart. Individuals (40) were treated with fibroblast suspension (N = 30) or placebo (N = 10) during the acute phase of the study.

Study IT-R-002 was a phase III double-blind, randomized and placebo-controlled study of fibroblast cell suspensions for the treatment of facial contour deformities and scars. During the acute phase of the study, each individual received a composition containing 2.0X 10⁷Three treatments of single cell/ml fibroblast suspension or placebo, administered every 14 ± 7 days. Individuals (151) were treated with fibroblast suspension (N = 112) or placebo (N = 39) during the acute phase of the study. In both studies, individuals (213) were treated with fibroblast suspension (N = 100) or placebo (N = 113) during the acute phase of the study.

While study IT-R-003B showed efficacy at both combined primary endpoints, one of the combined primary endpoints in study IT-R-003A failed to meet the statistical significance criteria (for investigator evaluation), thus leading to two new protocols as key phase III trials of fibroblast suspensions (study IT-R-005 and IT-R-006). It is believed that the reason for the missed endpoint in 003A is that suboptimal administration is included. The cell concentration remained the same, but the delivered volume increased. Other causes were identified, including training techniques and the time between injections.

Study IT-R-005 and IT-R-006 are phase III multicenter, double-blind, randomized, placebo-controlled studies of the efficacy and safety of fibroblast suspensions in treating nasolabial folds. These studies were performed according to the same protocol that is compliant with the FDA SPA protocol. During the acute phase of these studies, each individual received a composition containing 1.0-2.0X 10⁷Three treatments of single cell/ml fibroblast suspension or placebo, administered every 5 weeks ± 1 week. In study IT-R-005, 83 individuals were treated with fibroblast suspension and 92 individuals were treated with placebo. In study IT-R-006, 98 individuals were treated with fibroblast suspension and 99 individuals were treated with placebo.

Study IT-R-007 is a phase II multicenter, open label study of the safety and efficacy of fibroblast cell suspensions in treating facial wrinkles and folds, designed to obtain safety and efficacy data on the use of fibroblast cell suspensions for uses other than nasolabial folds. During the acute phase of the study, each individual received up to 6mL of a composition containing 1.0-2.0X 10⁷Two treatments of single cell/ml fibroblast suspension, administered every 5 weeks ± 10 days. During the acute phase of the study, 45 subjects were treated with fibroblast suspension. This study exposed individuals to a fibroblast suspension at a total dose of three times the total dose used in the 005/006 study.

The population of individuals enrolled in the phase III critical efficacy test of fibroblasts, study IT-R-005 and IT-R-006 (005/006), was predominantly caucasian women between the ages of 50 and 60. The individual ages ranged from 23-82 years with an average age of 56.1 years. The severity of the individual's nasolabial fold when enrolled in the study ranged from 3-5 on the evaluators' wrinkle severity assessment scale, with the right side wrinkles having an average score of 3.7 and the left side wrinkles having an average score of 3.6.

The populations selected into phase III IT-R-003A and IT-R-003B (003A/B) studies were similar to those in 005/006. Women accounted for 94% of the population in the 003A/B study and 95% of the population were caucasian. The mean age of the individuals in both studies was 54.1 years. While the 003A/B regimen was very similar in design to the 005/006 regimen, the 003A/003B allowed for the inclusion of individuals with a broader range of wrinkle severity, with scores for primary nasolabial sulcus deformity on the assessor scale ranging from 2-5 at the time of inclusion, but the average severity score was similarly 3.9.

All key efficacy studies use a combined primary endpoint consisting of an individual self-assessment means as well as assessments by clinical researchers.

In the 005/006 study, the combined primary efficacy endpoints were: evaluation of individual wrinkles: the individual's active period was assessed comprehensively for wrinkles in the lower portion of the face at visit 6 using a 5-point wrinkle assessment scale, where response was defined as two points or better improvement on the scale when compared to baseline.

Evaluator wrinkle severity assessment: blinded evaluator active period assessments were performed on resting bilateral nasolabial fold wrinkles at visit 6 using a 6-point sequential wrinkle severity scale using light guidance, where response was defined as two points or better improvement on the scale compared to baseline. These endpoints were selected to provide a fair assessment (rated by an uninformed physician) and clinical relevance (individual's opinion of their own appearance). Evaluators the scale used for wrinkle severity assessment was the 6-point sequential wrinkle severity scale for assessment of nasolabial folds (NLF) developed and validated by blue bob et al, plastic surgery and reconstructive surgery (plat Reconstr Surg.) 2001108(6): 1735-50. The blue Bole Scale (Lemperle scale) has been validated to detect a point of improvement in the severity of nasolabial fold wrinkles. However, because the assessment of appearance can be subjective and tends to vary, the success of this endpoint is defined as the improvement of two points per NLF, i.e., for a given patient, both the left and right NLFs must have two point improvements in the assessor's assessment in order to be considered a responder. The blue bobble scale is a recognized metric in the field of dermatology and has been successfully used in critical clinical trials of FDA approved products in similar indications.

The scale used for individual wrinkle assessment is based on published scales used by Cohen and Holmes (Holmes), orthopedic and reconstructive surgery (plant Reconstr Surg.) 200415;114(4): 964-76. As with the evaluator assessments, two points of improvement were established as criteria for successful response to treatment.

The 003A/B study was designed to have a similar combined primary endpoint, but using an individual assessment tool different from the 005/006 study. As specified in the 003A/B protocol. The combined primary efficacy endpoint was the efficacy of the fibroblast dose formulation injection in the primary nasolabial sulcus at 6 months follow-up assessed using the investigator's 6-point sequential scale and the individual's VAS.

The 6-point order scale referred to in the protocol was the same blue Bob scale used in the 005/006 study, although the 005/006 study provided more descriptive text for each point on the scale than used in the 003A/B study. Visual analog scales are used for individual assessment. This scale requires individuals to rank each contour abnormality from 0 (no defect) to 100 (very severe defect) by placing a marker on the 10cm line. While both studies met with improved statistical significance using this assessment tool, the VAS scale was replaced with the sequential individual wrinkle assessment scale in the 005/006 study to improve clinical relevance as well as interpretability of the individual assessment data.

005/006 the results of the two studies showed a very significant difference in response between IT-treated individuals and placebo-treated individuals, as measured by the two combined primary endpoints.

In the 003A/B study, statistically significant differences in response between IT-treated and placebo-treated individuals were observed for three of the four endpoints. In study IT-R-003A, IT-treated individuals were rated by evaluator evaluation as a higher percentage of responders than placebo-treated individuals, but the differences did not meet statistical significance.

The studies of IT-R-005 and IT-R-006 were performed simultaneously using the same study protocol, and thus there was no difference in study design. Studies of IT-R-003A and IT-R-003B were also performed simultaneously under independent protocols. Nevertheless, the results were still different within each study group. All primary endpoints were successfully met in the 005/006 study, but of these, the 005 study reported responder rates in the fibroblast suspension treatment group of 57% and 33% for individual and evaluators evaluations, respectively, and the 006 study reported responder rates of 46% and 19% for the same measure. In the 003A study, 21% of the individuals in the IT treatment group were scored as responders in the evaluator evaluation, while 48% of the individuals in the fibroblast dose formulation group responded to this measure in study IT-R-003B.

In the 005/006 study, there was no significant difference in patient demographics (gender, race, age, or baseline severity) between the two trials. However, variability in the rate of reaction was observed between sites within each study. Subsequently, all the investigators involved were trained to use the same technique for nasolabial sulcus injection, and it was expected that they could produce "blisters" as expected for injection into the papillary dermis. This was not done prior to the IT-R-003 test. Differences in the evaluation methods were also observed. Site differences when combined with these observations lead to the conclusion that: many inconsistencies between sites may arise due to lack of general training in both injection techniques and evaluation.

005/006 studies were designed after reviewing and analyzing the data from the 003A/B study. As a result of this review, many modifications were made to design 005/006 solutions compared to the 003A/B study. A summary of modifications that may contribute to the differences in primary endpoint results observed is provided in table 6.

Effect of dose and dosing regimen

Facial wrinkled skin and nasolabial folds

Selected doses (1.0-2.0X 10 up to 2 mL) for treatment of nasolabial folds⁷0.1 mL/mL administered per linear centimeter length) based not only on data generated in clinical trials conducted at IND 8641, but also on information obtained in the united states and abroad through commercial use of fibroblast dosage formulations. Study IT-R-001 is a placebo-controlled phase II dose range study evaluating 0.5, 1.0 and 2.0X 10⁷Safety and primary efficacy of individual cells/mL fibroblast suspension (0.1 mL per linear centimeter length) versus placebo in treating facial wrinkles. In the highest dose group (2.0X 10)⁷Individual cells/ml), best results were obtained from baseline to four months after the initial injection (primary efficacy time point for study IT-R-001). Thus, this is the cell density used in all subsequent studies.

In the 003A/B study, the dose per linear centimeter length was the same as used in the other azficel-T fibroblast dose formulation studies for this indication (0.1 mL per linear centimeter length). However, the total dose per treatment was limited to 1mL over a total treatment area of 10cm in total. This difference was because study 003A/B was not useful in treating a wrinkle sometimes referred to as the mesio-labial sulcus, which is a furrow or wrinkle extending downward from the corner of the mouth toward the chin. The total dose was increased to 2mL in 005/006 to allow treatment of the mesio-labial folds as it is often associated with nasolabial folds and is therefore a component contributing to the overall aesthetic effect. While the total dose allowed was not the only difference between the 003A/B regimen and the 005/006 regimen, this increase in the volume of product administered was considered to be a contributing factor to the successful results obtained from the 005/006 study.

The recommended dosing interval of 5 weeks ± 1 week was determined based on the feedback provided by the investigator from the 003A/B study and the results of the 005/006 trial. The 003A/B investigator suggested the use of esalic (isologen), which was insufficient to allow the inflammation induced by one injection to subside before the next injection was administered between treatments in those studies (7 to 14 days). This duration of inflammation was only noted during injection and was not reported as an adverse event. When successive injections were administered at longer time intervals allowed by the protocol, researchers reported that the injection site was more likely to appear closer to normal skin, which resulted in greater control over injection depth and volume. This allowed the time interval between injections to be extended in the 005/006 study, first to 4 weeks and then to 5 weeks ± 1 week. This variation is also considered to be a contributing factor to the results of a successful 005/006 study.

Primary efficacy analysis of the Intent-to-Treat Population (Intent to Treat) in the 005/006 study evaluated the treatment response between the fibroblast suspension and the placebo treated group as measured in combination with the primary efficacy endpoint. For the assessor wrinkle severity assessment at visit 6 (in combination with the primary efficacy endpoint), 26% of IT randomized individuals showed two-point improvement in bilateral nasolabial folds relative to 7% of placebo randomized individuals. When only those individuals actually receiving at least one treatment of either the fibroblast dose formulation or placebo were included in the analysis (modified intent to treat (MITT) population), the percentage of two-point responses on the assessor wrinkle severity assessment was 30% in IT-treated individuals compared to 8% for placebo-treated individuals. One-point response rate in the MITT population on evaluator wrinkle severity evaluation was 64% in fibroblast dose formulation treated individuals and 36% in placebo treated individuals. Finally, in the demonstration of the onset of action observed in microseconds with IT, photographic assessment of efficacy by assessors and individuals showed a highly statistically significant difference in treatment response between IT-treated individuals and placebo-treated individuals. When the assessor compared the individual photographs taken at baseline side-by-side with photographs taken at the last efficacy follow-up, 57% of the IT-treated individuals in the MITT population and only 20% of the placebo-treated individuals in this population were scored improved. On photographic evaluation (individual improvement evaluation) of the individuals, 67% of the IT-treated individuals and 26% of the placebo-treated individuals showed improvement when the individuals compared their own baseline photographs with photographs taken at the last efficacy follow-up. The response rate obtained using this assessment approach is increased compared to the active-period assessment results, showing a microsecond, gradual onset of action that may not be apparent to the individual or assessor prior to comparing appearance to baseline.

In summary, for the reasons discussed above, the preferred dose for treating nasolabial fold lines is to inject 1 to 2mL of the dose formulation into the superficial papillary dermis of the fold at a dose distribution of 0.1mL per linear centimeter length per treatment period, preferably for three treatment periods, separated by five weeks plus or minus seven to ten days.

The preferred dose for treating wrinkled skin in multiple facial regions (i.e., "full-face") is by injecting 5 to 6mL of the dose formulation into the superficial papillary dermis per treatment session, preferably for one or two treatment sessions, separated by five weeks plus or minus seven to ten days, according to the treatment map in fig. 4, with a dose distribution of 0.05mL per linear centimeter length.

Acne scar

The appropriate dose for treating acne scars can be determined using a validated method and an active grading scale of acne scar severity objectively assessed by the physician, i.e., the "assessor active acne scar assessment scale" which employs a comparative assessment of relative scar appearance under direct and tangential illumination, see fig. 2.

TABLE 1 evaluation scale for acne scar in active period of evaluators

The severity of acne scars was assessed by scar counts of atrophic and hypertrophic/keloid scars in a study conducted by Linton (Layton) et al at the Leeds General Infirmary. Atrophic scars-morphologically defined as atrophic cones of the macula or follicular macula-converting to a score in the range of 1 to 6, representing 1-5, 6-10, 11-25, 26-50, 51-100, and more than 100 scars, respectively. A icicle-like scar is described as a scar with irregular edges, jagged edges, and sharp boundaries, with steep sides leading to the fibrous substrate. Atrophic scars of the macula are soft and extensive, with the base often easily crumpling. Follicular macular atrophic scars are described as small white perifollicular papules or maculopapules. The authors quantified keloid and hypertrophic scars separately because of their greater extent of disfigurement. 2. The scoring configurations of 4 and 6 represent 1 to 3, 4 to 7 or more than 7 scars of this type, respectively. Keloid scars are described as scars which are hard and extend beyond the boundaries of the starting inflammatory acne scar, whereas hypertrophic scars are defined as areas which are less raised and accommodate the primary acne scar. Total scar scores were then obtained by adding scores from both atrophic and hypertrophic varieties. Such total scores can be calculated separately for the face, chest, and back to provide a comprehensive system for scar assessment, and also to provide a means for assessing effective dosages and treatment regimens.

In a preferred embodiment, acne scars are treated by injecting into the superficial papillary dermis at a dose distribution of 0.1mL per square centimeter scar area, 2 to 12mL of a fibroblast dose formulation for each treatment period, for one to three treatment periods, separated by fourteen days plus or minus three days.

Burn injury

A potentially serious long-term consequence of a burn reaching the deep dermis is a post-injury scar. The formation of restrictive scars from severe burns can impede the normal activity of the affected area and limit the range of motion (flexion, adduction, and/or extension). In fact, "scar and contracture of burn affecting function are still the most depressed late complications of burn" (Wujiawu, plasty and reconstructive surgery (last Reconstr Surg.) 4 months 1999; 103(4): 1198-. These scars can have a significant negative impact on quality of life by causing pain and reduced functionality. Depending on the location, restrictive burn scars can cause significant damage to the upper limbs and loss of function. Fibroblast dosage formulations are particularly useful for treating restrictive burn scars on the upper extremities, particularly to reduce the extent of injury and related disability experienced by an individual.

The preferred dose for treating the restrictive burn scar is to inject 1 to 10mL of the dose formulation per treatment period into the palpated restrictive band at a dose distribution of 0.1-0.5mL per square centimeter of scar area, preferably for one to five treatments, two to six weeks apart.

Case reports from the uk, in which previously available fibroblast formulations could be used, showed successful application to burn scars and wounds without adverse effects.

The kriss engelfeld, london (dr. chris Inglefield) reported cases of men with multiple burns in the neck, lower face, eyes and ear regions. The subject has previously received a fibroblast formulation for the facial wrinkles and thus stores the cells at the time he is burned. This enables him to receive facial burn treatment within six weeks of his initial injury. He successfully received three additional treatments three months after injury. Each treatment was performed under local anesthesia and contained 3-4ml of cells administered by 30-40 injections. The subject has previously been treated with a skin graft. The individual reported improved activity in his face and neck and improved texture and flexibility of his skin within three weeks of first treatment.

Graggori chernoff, indiana university w.w.gregory chernoff has also used fibroblast formulations in ten difficult-to-heal laser burn wounds. Although these wounds did not heal for three to nine months prior to treatment with fibroblasts, he noted complete healing six weeks after the final injection of cells (Boss et al, 2000 Clinical Plastic Surgery 27:613- & 626).

In the united states, two burn victims have benefited from using fibroblast formulations in a complimentary fashion, with a substantial improvement in their refractory burn wounds. the victim of the Oklahoma City Bombing explosive case (Oklahoma City Bombing) of Karah horse tragically left a scar in the explosion. After several months of surgery, laser resurfacing, and other treatments to save her life and improve her appearance, she was treated with fibroblasts to aid in failed skin grafts and other difficult to heal wounds that remained on her face and neck. After treatment, the refractory wound closes and heals. In addition, she noted an improvement in the appearance of her scar.

Another individual with 12 refractory wounds on the forehead and zygomatic areas due to laser burns was treated with a fibroblast formulation based on a gift-style use. The only replacement she was treated as a cow's skin graft. After treatment, most wounds healed within six weeks of injection.

The following case report indicates that fibroblast formulations may not only improve subacute or healing burn wounds, but also improve long-standing, well-healed but debilitating restrictive burn scars.

Clise engelfeld, london, uk (dr. chris Inglefield) reported cases of a woman with multiple burns lasting two years before presentation, resulting in large facial scars, limiting the expression of her left side face. Injury to the individual has serious negative psychological consequences and she feels that she has "lost her own". The face of the individual received three treatments, 3mL each. Within six to eight weeks, the individual, her family and treating physician noted significant improvement, making her mother feel that her large daughter "returned". Her facial ability to move about is improved as is her skin texture and appearance. The same individual then receives therapy for the contracting scar and the refractory wound in her hands. Prior to therapy, it was difficult for an individual to manipulate small objects during her jewelry making process. Within four weeks after treatment, the wound had healed and she had improved her ability to perform jewelry-making tasks.

Another individual who was treated for long-term wounds lasting 10 years on the neck, shoulders and upper arm was reported by mark Palmer, litz, uk. Prior to treatment, the range of motion of the neck and shoulders of the individual was severely limited and her long-term pain required daily opiate analgesia. The individual received a total of two 4mL treatments in her neck region. Even after the first treatment, the individual reported an improvement in range of motion and skin texture. With the second treatment, the range of motion of the individual was close to normal and striking changes in scar appearance and texture were noted.

The same individual then receives treatment for upper arm scarring. These scars were severely disabling before treatment, so that she could not lift her arms more than 90 ° due to pain. By three months after a single treatment of two contracting scar bands (1.5 mL per band), the scar had almost disappeared and the individual had recovered 180 ° of range of motion without pain.

Claims (15)

1. A dosage formulation comprising 1.0 x 10⁷To 2.7X 10⁷(ii) cells/ml, wherein at least 98% of the cells are autologous human fibroblasts or precursors thereof, of which at least 85% are viable; and optionally a cryopreservation medium.

2. The dosage formulation of claim 1, comprising a cryopreservation Medium consisting of Iscove's Modified Dulbecco's Medium (IMDM) and ProfreezeTM plus 7.5% dimethyl sulfoxide (DMSO).

3. The dosage formulation of claim 1, wherein 98% or more of said cells are fibroblasts.

4. The dosage formulation of claim 1, comprising 1, 2, 3, or 6 mL.

5. A method for treating skin defects comprising injecting an effective amount of a composition comprising 1.0 x 10 to the site to be treated⁷To 2.7X 10⁷A dosage formulation of individual cells/ml, wherein at least 98% of said cells are autologous human fibroblasts or precursors thereof, wherein at least 85% are viable, said method being performed in two or three treatments, said treatments being separated by four or five weeks, plus or minus seven to ten days.

6. The method of claim 5, for treating wrinkles, nasolabial and bucchal folds, perioral folds, external canthus folds, periorbital folds, and interphalangeal folds.

7. The method of claim 5, comprising injecting 0.05 to 0.5mL of the dose formulation per linear centimeter length.

8. The method of claim 5, comprising injecting 2 to 6mL per treatment.

9. The method of claim 5 for treating nasolabial fold comprising administering 1 to 2mL of the dose formulation per treatment period, injected into the superficial papillary dermis of the fold at a dose distribution of 0.1mL per linear centimeter length, for three treatment periods, separated by five weeks plus or minus seven to ten days.

10. The method of claim 5 for treating wrinkled skin in multiple facial regions, the method comprising injecting 5 to 6mL into the superficial papillary dermis per treatment session at a dose distribution of 0.05mL per linear centimeter length, the method continuing for one or two treatment sessions, the treatment sessions being separated by five weeks plus or minus seven to ten days.

11. A method according to claim 5 for treating acne scars comprising injecting 2 to 12mL into the superficial papillary dermis per treatment period at a dose distribution of 0.1mL per square centimeter scar area, said method lasting for one to three treatment periods, said treatment periods being separated by fourteen days plus or minus three days.

12. The method of claim 5 for treating a restrictive burn scar comprising injecting 1 to 10mL per treatment period into a palpated restriction band of the burn scar at a dose distribution of 0.1-0.5mL per square centimeter scar area, the method lasting for one to five treatments, the treatments separated by two to six weeks.

13. A method of providing pluripotent cells for tissue repair or regeneration, comprising:

providing a composition comprising 1.0 x 10⁷To 2.7X 10⁷A dosage formulation of individual cells per milliliter, wherein at least 98% of the cells are autologous human fibroblasts or precursors thereof, of which at least 85% are viable; and

means are provided for dedifferentiating the cells.

14. The method of claim 13, wherein the cell is dedifferentiated by cell programming, cell fusion, or somatic cell transfer.

15. A pluripotent cell formulation comprising cells of 1.0X 10⁷To 2.7X 10⁷Dedifferentiation of individual cell/ml dosage formulationsIn the dosage formulation, at least 98% of the cells are autologous human fibroblasts or precursors thereof, of which at least 85% are viable.