Patent Application
20170103184
The present invention relates to biocompatible viscoelastic polymeric gel slurries, methods for their preparation and formulations containing them.
As a person age, facial rhytids (wrinkles) and folds develop in respond to the loss of facial fat and the decrease of the skin elasticity. Physicians have over the years tried various methods and materials to combat the facial volume loss of the soft tissue of the face. One of the most common methods is autologous fat transfer. Using this surgical method, a person's own fat is harvested from a different part of the body such as the abdomen, and then the fat is processed and prepared for injection into the dermal and soft tissue areas of the face that is requiring the volume restoration to alleviate the wrinkles and folds to achieve a more youthful appearance. Autologous fat transfer has good desirable results, however, this surgical technique is costly, painful, time consuming, has a long recovery time for the patient, and is associated with complications associated with any surgical procedure.
Systems and method are disclosed for body enhancements by modeling shape and size change in the body portion due to an implant; iteratively changing modeled body shapes or sizes until the patient is satisfied with a desired shape or size; controlling an automatic injector to deliver the implant in the patient; and monitoring injection into patient and providing feedback if needed to achieve the desired shape and size.
There are other additional aspects that will be detailed below:
In one aspect, systems and methods are disclosed for cosmetic augmentation of soft tissue using cross-linked HA that had been optimized for
The use of a particularly cross linked HA, and cross linked by forming regions of interpenetrating network (IPN) of cross linked HA by further crosslinking them. The IPN configuration gives this cross linked HA those utilities unique for this cosmetic augmentation application. The IPN core (imagine a tapioca ball) is more resistance to biodegradation in a human body than the single cross-linked material normalized for the same cross linking level. Furthermore, varying physical properties that continuously changes radiating out from the core makes the polymer tough and at the same time compliant with the local tissue for better tissue/device biocompatibility and feels more natural to the touch.
The above HA cross linking method optimized for cosmetic augmentation in certain cases may need to control delivered pharmaceutical substances to modulate local tissue response to the polymer. The pharmaceutical component makes up the multi-phase mixture with the other phase being the cross linked HA polymer.
Implementations of the above aspects may include one or more of the following. The system is biocompatible and performs controlled drug releases at strategic timing to coincide with key physiological events. For example, a fast drug release profile and no delay would be well suited for the controlled release of an anesthetic such as lidocane to relieve acute pain experienced by the patient associated with the surgical procedure. The system is also capable of a medium release profile and a medium delay of a corticosteroid or steroid such as dexamethasone or triamcinolone to co-inside with a physiological inflammatory foreign body reaction. The system can also be customized to have a medium to slow release profile and a longer delay before starting the release of an antiproliferative drug such as paclitaxel, serolimas or 5-flourouracil to stop uncontrolled healing and excessive remodeling causing unsightly scar formation orcapsular formation.
1. Molecular Weight Manipulation
Another aspect of the present invention includes methods for optimizing biodegradation profiles and control migration of the implant material through the manipulation of various types molecular weight. The system optimizes biodegradation profiles and controls migration of the implant material. The system can be formulated around various types of molecular weights such as Mn, Mw and Mz, and their polydispersity index (PDI) to optimize the biodegradation profiles to be from hypervolumic to isovolumic to hypovolumic.
2. Free Radical Scavengers Vitamins and Enzymes
HA in the body is biodegraded by two major mechanisms: oxidative and hydrolytic. Inside the cell of mammals, the mechanism is enzymatic hydrolysis by three enzymeshyaluronidase (hyase), b-d-glucuronidase, and β-N-acetyl-hexosaminidase, and outside the cell the mechanism is oxidation by oxygen derived free radical, or sometimes, they are called reactive oxygen species (ROS). These are atoms or groups of atoms with an odd (unpaired) number of electrons and can be formed when oxygen interacts with certain molecules.
ROS are produced as a normal product of cellular metabolism. In particular, one major contributor to oxidative damage is hydrogen peroxide (H2O2), which is converted from superoxide that leaks from the mitochondria. Catalase and superoxide dismutase ameliorate the damaging effects of hydrogen peroxide and superoxide by converting these compounds into oxygen and water, benign molecules. However, this conversion is not 100% efficient, and residual peroxides persist in the cell. While ROS are produced as a product of normal cellular functioning, excessive amounts can cause deleterious effects. Memory capabilities decline with age, evident in human degenerative diseases such as Alzheimer's disease, which is accompanied by an accumulation of oxidative damage. Current studies demonstrate that the accumulation of ROS can decrease an organism's fitness because oxidative damage is a contributor to senescence. In particular, the accumulation of oxidative damage may lead to cognitive dysfunction, as demonstrated in a study in which old rats were given mitochondrial metabolites and then given cognitive tests. Results showed that the rats performed better after receiving the metabolites, suggesting that the metabolites reduced oxidative damage and improved mitochondrial function. Accumulating oxidative damage can then affect the efficiency of mitochondria and further increase the rate of ROS production. The accumulation of oxidative damage and its implications for aging depends on the particular tissue type where the damage is occurring. Additional experimental results suggest that oxidative damage is responsible for age-related decline in brain functioning. Older gerbils were found to have higher levels of oxidized protein in comparison to younger gerbils. Treatment of old and young mice with a spin trapping compound caused a decrease in the level of oxidized proteins in older gerbils but did not have an effect on younger gerbils. In addition, older gerbils performed cognitive tasks better during treatment but ceased functional capacity when treatment was discontinued, causing oxidized protein levels to increase.
Furthermore, once formed these highly reactive radicals can start a chain reaction. Their chief danger comes from the damage they can do when they react with important cellular components such as DNA, or the cell membrane. Cells may function poorly or die if this occurs. To prevent free radical damage the body has a defense system of antioxidants. The free radicals and the antioxidants react with one another readily and easily.
The degradation reaction by oxygen derived free radical of HA was the results of studies using the HA present in synovial fluids. It showed that the HA was readily degraded by super oxide free radicals. This reaction is most favorable in the case of secondary free radicals. Neutrophils (polymorphonuclear leukocytes) produced the type of oxygen derived free radicals that allowed it phagocytotically consumed HA molecules. These WBC's are by far the exclusive destroyers of HA by oxygen-derived free radical mechanism. Thus, an aspect of this invention is to quench the effect of the free radical before it degrades the HA using free radical scavengers such as antioxidant vitamins.
Antioxidants are intimately involved in the prevention of cellular damage—the common pathway for cancer, aging, and a variety of diseases. Antioxidants are molecules which can safely interact with free radicals and terminate the chain reaction before vital molecules are damaged. Although there are several enzyme systems within the body that scavenge free radicals, the principle micronutrient (vitamin) antioxidants are vitamin E, beta-carotene, and in the case of HA, vitamin C is the exception. Additionally, selenium, a trace metal that is required for proper function of one of the body's antioxidant enzyme systems, is sometimes included in this category. The body cannot manufacture these micronutrients so they must be supplied in the diet.
Following are example antioxidant vitamins, their roles and recommended daily dosages:
Another aspect of this invention is the use of antioxidant enzymes to protect the longevity of HA. These enzymes can reduce the radicals and defend against ROS. They are: alpha-1-microglobulin, superoxide dismutases, catalases, lactoperoxidases, glutathione peroxidases and peroxiredoxins.
3. Anti-Hyaluronidase and Anti-Elastase
In respect to the field of cosmetic augmentation to bring back youthfulness to aging skin using cross-linked HA, an aspect of this invention uses hyaluronidase inhibitor (anti-HA) to prevent the depolymerization of HA, specifically by hyaluronidase, and to maintain the longevity of HA. Maintenance of HA longevity is important because it is directly related to the appearance of those unwanted wrinkles and the signs of aging.
HA is an important molecule to everything that lives on this earth. In that, it is a multifunctional high molecular weight polysaccharide found throughout the animal kingdom, especially in the extracellular matrix (ECM) of soft connective tissues. HA is thought to participate in many biological processes, and its level is markedly elevated during embryogenesis, cell migration, wound healing, malignant transformation, and tissue turnover. The enzymes that degrade HA, hyaluronidases (HAases) are expressed both in prokaryotes and eukaryotes. These enzymes are known to be involved in physiological and pathological processes ranging from fertilization to aging. Hyaluronidase-mediated degradation of HA increases the permeability of connective tissues and decreases the viscosity of body fluids and is also involved in bacterial pathogenesis, the spread of toxins and venoms, acrosomal reaction/ovum fertilization, and cancer progression. Furthermore, these enzymes may promote direct contact between pathogens and the host cell surfaces. Depolymerization of HA also adversely affects the role of ECM and impairs its activity as a reservoir of growth factors, cytokines and various enzymes involved in signal transduction. Inhibition of HA degradation therefore may be crucial in reducing disease progression and spread of venom/toxins and bacterial pathogens. Hyaluronidase inhibitors are potent, ubiquitous regulating agents that are involved in maintaining the balance between the anabolism and catabolism of HA. Hyaluronidase inhibitors could also serve as contraceptives and anti-tumor agents and possibly have antibacterial and anti-venom/toxin activities. Additionally, these molecules can be used as pharmacological tools to study the physiological and pathophysiological role of HA and hyaluronidases.
First, the preparation of the hyaluronic acid is discussed, followed by the addition of additional chemicals to enhance the use of the hyaluronic for dermal or subdermal use is discussed.
Since the hyaluronan of a recombinant Bacillus cell is expressed directly to the culture medium, a simple process may be used to isolate the hyaluronan from the culture medium. First, the Bacillus cells and cellular debris are physically removed from the culture medium. The culture medium may be diluted first, if desired, to reduce the viscosity of the medium. Many methods are known to those skilled in the art for removing cells from culture medium, such as centrifugation or microfiltration. If desired, the remaining supernatant may then be filtered, such as by ultrafiltration, to concentrate and remove small molecule contaminants from the hyaluronan. Following removal of the cells and cellular debris, a simple precipitation of the hyaluronan from the medium is performed by known mechanisms. Salt, alcohol, or combinations of salt and alcohol may be used to precipitate the hyaluronan from the filtrate. Once reduced to a precipitate, the hyaluronan can be easily isolated from the solution by physical means. The hyaluronan may be dried or concentrated from the filtrate solution by using evaporative techniques known to the art, such as lyophilization or spraydrying.
Molecular Weight
The content of hyaluronic acid may be determined according to the modified carbazole method (Bitter and Muir, 1962, Anal Biochem. 4: 330-334). Moreover, the number average molecular weight of the hyaluronic acid may be determined using standard methods in the art, such as those described by Ueno et al., 1988, Chem. Pharm. Bull 0.36, 4971-4975; Wyatt, 1993, Anal. Chim. Acta 272: 1-40; and Wyatt Technologies, 1999, “Light Scattering University DAWN Course Manual” and “DAWN EOS Manual” Wyatt Technology Corporation, Santa Barbara, Calif.
In one embodiment, the hyaluronic acid, or salt thereof, of the one embodiment has a molecular weight of about 10,000 to about 10,000,000 Da. In a more preferred embodiment it has a molecular weight of about 25,000 to about 5,000,000 Da. In a most preferred embodiment, the hyaluronic acid has a molecular weight of about 50,000 to about 3,000,000 Da.
In another embodiment, the hyaluronic acid or salt thereof has a molecular weight in the range of between 300,000 and 3,000,000; preferably in the range of between 400,000 and 2,500,000; more preferably in the range of between 500,000 and 2,000,000; and most preferably in the range of between 600,000 and 1,800,000.
In yet another embodiment, the hyaluronic acid or salt thereof has a low number average molecular weight in the range of between 10,000 and 800,000 Da; preferably in the range of between 20,000 and 600,000 Da; more preferably in the range of between 30,000 and 500,000 Da; even more preferably in the range of between 40,000 and 400,000 Da; and most preferably in the range of between 50,000 and 300,000 Da.
This example illustrates the preparation of DVS-crosslinked microparticles. Sodium hyaluronate (HA, 580 kDa, 1.90 g) was dissolved in aqueous NaOH (0.2 M, 37.5 ml) by vigorous stirring at room temperature for 3 hours until a homogenous solution was obtained. Sodium chloride (0.29 g) was added and mixed shortly. Mineral oil (10.0 g) and ABIL® EM 90 surfactant (Cetyl PEG/PPG-10/1 Dimethicone, 1.0 g) were mixed by stirring.
Divinylsulfone (DVS, 320 microliter) was added to the aqueous alkaline HA-solution and mixed for 1 min. to obtain a homogeneous distribution in the aq. phase. The water phase was then added within 2 minutes to the oil phase with mechanical stirring at low speed. An emulsion was formed immediately and stirring was continued for 30 minutes at room temperature. The emulsion was left over night at room temperature. The emulsion was neutralized to pH 7.0 by addition of aq. HCl (4 M, approx. 2.0 ml) and stirred for approx. 40 min.
This example illustrates the preparation of DVS-crosslinked microparticles with neutralization using a pH indicator. Sodium hyaluronate (HA, 580 kDa, 1.88 g) was dissolved in aqueous NaOH (0.2 M, 37.5 ml) by vigorous stirring at room temperature for 2 hours until a homogenous solution was obtained. Bromothymol blue pH indicator (equivalent range pH 6.6-6.8) was added (15 drops, blue color in solution). Sodium chloride (0.25 g) was added and mixed shortly.
Mineral oil (10.0 g) and ABIL® EM 90 surfactant (Cetyl PEG/PPG-10/1 Dimethicone, 1.0 g) were mixed by stirring.
Divinylsulfone (DVS, 320 microliter) was added to the aqueous alkaline HA-solution and mixed very vigorously for 30 to 60 seconds to obtain a homogeneous distribution in the aq. phase. The water phase was then added within 30 sec. to the oil phase with mechanical stirring at 400 RPM. An emulsion was formed immediately and stirring was continued for 30 min. at room temperature. Neutralization was performed by addition of aq. HCl (4 M, 1.6 ml) and the emulsion was left at room temperature with magnetic stirring for 4 hours. The pH indicator present in the gel particles changed color to green. pH in the emulsion was measured by pH stick to 3-4. The emulsion was left in fridge overnight. The pH indicator present in the gel particles had changed to yellow.
This example illustrates the breakage of the W/O emulsion followed by phase separation and dialysis. The crosslinked HA microparticles were separated from the W/O emulsion by organic solvent extraction. The W/O emulsion (5 g) and a mixture of n-butanol/chloroform (1/1 v %, 4.5 ml) was mixed vigorously by whirl mixing in a test tube at room temperature. Extra mQ-water (20 ml) was added to obtain phase separation. The test tube was centrifuged and three phases were obtained with the bottom phase being the organic phase, middle phase of gel particles and upper phase of clear aqueous solution. The top and bottom phases were discarded and the middle phase of gel particles was transferred into a dialysis tube (MWCO 12-14,000, Diameter 29 mm, Vol/Length 6.4 ml/cm). The sample was dialyzed overnight at room temperature in MilliQ®-water. The dialysate was changed two more times and left overnight. The resulting gel was thick and viscous and had swelled to a volume of approximately 50 ml, which correlated to 0.004 g HA/cm3.
This example illustrates the preparation of DVS-crosslinked HA microparticles. Sodium hyaluronate (HA, 580 kDa, 1.89 g) was dissolved in aqueous NaOH (0.2 M, 37.5 ml). Sodium chloride (0.25 g) was added and the solution was stirred by magnetic stirring for 1 hour at room temperature until a homogeneous solution was obtained. TEGOSOFT® M (10.0 g) oil and ABIL® EM 90 surfactant (Cetyl PEG/PPG-10/1 Dimethicone, 1.0 g) were mixed by stirring.
Divinylsulfone (DVS, 320 microliter) was added to the aqueous alkaline HA-solution and mixed for 1 min. to obtain a homogenoues distribution in the aq. phase. The water phase was then added within 2 min. to the oil phase with mechanical stirring (300 RPM). An emulsion was formed immediately and stirring was continued for 30 min. at room temperature.
The emulsion was neutralized by addition of stociometric amounts of HCl (4 M, 1.8 ml) and stirred for approx. 40 min. The emulsion was broken by addition of a n-butanol/chloroform mixture (1:1 v %, 90 ml) and extra MilliQ®-water (100 ml) followed by magnetic stirring. The upper phase was separated in a volume of approx. 175 ml. The organic phase was mixed with mQ-water (30 ml) for a final washing. The combined water/gel phase (205 ml) were transferred to a dialysis tube (MWCO 12-14,000, Diameter 29 mm, Vol/Length 6.4 ml/cm) and dialysed against MilliQ®-water overnight at room temperature. The conductivity were decreased to 0.67 micro-Sievert/cm after subsequent change of water (3 times) and dialysis overnight (2 nights). The microparticles were assessed by microscopy (DIC 200×), see
This example illustrates the breakage of the W/O emulsion and isolation of the gel microparticles. The gel microparticles were separated from the W/O-emulsion by organic extractions. Examples of organic solvents which were used for this extraction were mixtures of butanol/chloroform in volume ratios (v %) of 75:20 to 20.80, respectively. The weight ratio (w %) of W/O emulsion to organic solvent was approximately 1:1.
Separation in small scale: The W/O emulsion (5 g) was weighed in centrifuge tubes (50 ml). A mixture of butanol/chloroform was prepared (1:1 v %) and from this mixture 4.5 ml was added (corresponds to 5 g) to the test tube. The test tube was carefully mixed to secure that all emulsion was dissolved. The test tube was mixed by Whirl mixing and left at room temperature for phase separation. Phase separation with water phase on top and organic phase at bottom with a white emulsion phase in between was often observed. Addition of more water and organic phases improved separation. The water phase was separated by decanting and further purified or characterized.
This example illustrates a composition in which the HA microparticles were formed. A hot/cold procedure can be used with incorporation of a cold water phase B into a hot oil phase, which will shorten the time of manufacture. A non-limiting example of formulation could be as follows:
Sodium hyaluronate (HA, 580 kDa, 1.88 g) was dissolved in aqueous NaOH (0.2 M, 37.5 mL). Sodium chloride (0.25 g) was added and the solution was stirred by magnetic stirring for 1 hour at room temperature until a homogeneous solution was obtained. The oil: TEGOSOFT® M (10.0 g) and surfactant: ABIL® EM 90 (Cetyl PEG/PPG-10/1 Dimethicone, 1.0 g) was mixed by stirring. Divinylsulfone (DVS, 320 microliter) was added to the aqueous alkaline HA-solution and mixed for 1 min to obtain a homogenoues distribution in the aq. phase. The water phase was then added within 2 min to the oil phase with mechanical stirring (300 RPM). An emulsion was formed immediately and stirring was continued for 30 min at room temperature.
The emulsion was neutralized by addition of stociometric amounts of HCl (4 M, 1.8 mL) and stirred for approx. 40 min. The emulsion was transferred to a separation funnel, and broken by addition of a n-butanol/chloroform mixture (1:1 v %, 90 mL) and extra millliQ™-water (100 mL) followed by vigorous shaking. The upper phase was separated in a volume of approx. 175 mL. The organic phase was washed with millliQ™-water (100 mL). The combined water/gel phase was transferred to a dialysis tube (MWCO 12-14,000, Diameter 29 mm, Vol/Length 6.4 mL/cm) and dialysed against millliQ™-water overnight at room temperature. The conductivity was decreased to 10 micro-Sievert/cm after subsequent change of water (3 times) and dialysis overnight (2 nights).
This example illustrates the final isolation and purification of the microparticles.
100 mL particles previously isolated were re-suspended in a Na2HPO4/NaH2PO4 buffer (0.15 M, 400 mL), and stirred slowly for ½ hour. The suspension stood at 5° C. for 2 hours and solidified oil droplets were removed. The solution was then filtered through a mesh and washed further with 2×50 mL buffer. Particles were allowed to drip-dry, before characterization (
This example illustrates performance of rheological studies on particles. A particle sample is analyzed on an Anton Paarrheometer (Anton Paar GmbH, Graz, Austria, Physica MCR 301, Software: Rheoplus), by use of a 50 mm 2° cone/plate geometry. First the linear range of the visco-elastic properties G′ (Storage modulus) and G″ (Loss modulus) of the material is determined by an amplitude sweep with variable strain, γ. Secondary a Frequency sweep is made, and based on values of the visco-elastic values, G′ and G″, tan δ can be calculated as a value for week/strong gel behaviors.
This example illustrates performance of an investigation of force applied to inject at a certain speed, as a function of the homogeneity of the sample. A particle sample is transferred to a syringe applied with a needle, either 27 G×½″, 30 G×½″, and is set in a sample rig, in a texture analyzer (Stable Micro Systems, Surrey, UK, TA.XT Plus, SoftWare: Texture Component 32). The test is performed with an injection speed at 12.5 mm/min., over a given distance.
This example illustrates the preparation of DVS-cross-linked HA hydrogels with concomitant swelling and pH adjustment.
Sodium hyaluronate (HA, 770 kDa, 1 g) was dissolved into 0.2M NaOH to give a 4% (w/v) solution, which was stirred at room temperature, i.e. about 20° C., for 1 h. Three replicates were prepared. Divinylsulfone (DVS) was then added to the HA solutions in sufficient amount to give HA/DVS weight ratios of 10:1, 7:1, and 5:1, respectively. The mixtures were stirred at room temperature for 5 min and then allowed to stand at room temperature for 1 h. The gels were then swollen in 160 mL phosphate buffer (pH 4.5 or 6.5) for 24 h, as indicated in Table 1.
The pH of the gels was stabilized during the swelling step. After swelling, any excess buffer was removed by filtration and the hydrogels were briefly homogenized with an IKA® ULTRA-TURRAX® T25 homogenizer (IkaLabortechnik, DE). The volume and pH of the gels were measured (see Table 2).
The pH of the hydrogels ranged from 7.1 to 7.6 (table 2), which confirms that the swelling step can be utilized to adjust the pH in this process. All the hydrogels occupied a volume of 70 mL, which corresponds to a HA concentration of ca. 1.4% (w/v). They were transparent, coherent and homogenous. Softness increased with decreasing cross-linking degree (Table 2).
This example illustrates the preparation of highly homogenous DVS-cross-linked HA hydrogels.
Sodium hyaluronate (770 kDa, 2 g) was dissolved into 0.2M NaOH with stirring for approx. 1 hour at room temperature to give a 8% (w/v) solution. DVS was then added so that the HA/DVS weight ratio was 7:1. After stirring at room temperature for 5 min, one of the samples was heat treated at 50° C. for 2 h without stirring, and then allowed to stand at room temperature overnight. The resulting cross-linked gel was swollen into 200 ml phosphate buffer (pH 5.5) 37° C. for 42 or 55 h, and finally washed twice with 100 ml water, which was discarded. Volume and pH were measured, as well as the pressure force necessary to push the gels through a 27 G*½ injection needle (see Table 3).
The cross-linked HA hydrogel prepared according to this example exhibited a higher swelling ratio and an increased softness compared to a control hydrogel which was not heat treated (Table 3). The pressure force applied during injection through a 27 G*½ needle was more stable than that of the latter sample, indicating that the cross-linked HA hydrogel is more homogenous.
This example illustrates the in vitro biostability of DVS-cross-linked HA hydrogels using enzymatic degradation.
A bovine testes hyaluronidase (HAase) solution (100 U/mL) was prepared in 30 mM citric acid, 150 mM Na2HPO4, and 150 mM NaCl (pH 6.3). DVS-HA cross-linked hydrogel samples (ca. 1 mL) were placed into safe-lock glass vials, freeze-dried, and weighed (W0; Formula 1). The enzyme solution (4 mL, 400 U) was then added to each sample and the vials were incubated at 37° C. under gentle shaking (100-200 rpm). At predetermined time intervals, the supernatant was removed and the samples were washed thoroughly with distilled water to remove residual salts, they were then freeze-dried, and finally weighed (Wt; Formula 1).
The biodegradation is expressed as the ratio of weight loss to the initial weight of the sample (Formula 1). Weight loss was calculated from the decrease of weight of each sample before and after the enzymatic degradation test. Each biodegradation experiment was repeated three times. DVS-HA hydrogels prepared as described in example 2 (‘Heated’) were compared to DVS-HA hydrogels which had not been heat treated (‘Not heated’). For both types of gel, degradation was fast during the first four hours, and then proceeded slower until completion at 24 h. Importantly there was a significant variation of the weight loss values for the samples which had not been heated as compared to the hydrogel prepared with a heating step as described in example 2. This clearly illustrates that a highly homogenous DVS-cross-linked HA hydrogel is obtained by using the process described in example 2.
In this and in the following example, DVS-crosslinked HA hydrogels were formulated into creams and serums, that when applied to the skin increase the skin moisturization and elasticity, and provide immediate anti-aging effect, as well as film-forming effect
A typical formulation of a water-in-oil (w/o) emulsion containing 2% DVS-cross-linked HA. Each phase (A to E) was prepared separately by mixing the defined ingredients (see Table 4). Phase B was then added to phase A under stirring with a mechanical propel stirring device and at a temperature less than 40° C. Phase C was then added followed by phase D and finally phase E under stirring. Formulations were also made, wherein the HA hydrogel concentration was 4%, 6% and 8%, respectively, in Phase D, to give a range of w/o formulations.
Another typical formulation of a w/o-emulsion containing 2% DVS-crosslinked HA is shown in table 5. Each phase (A to F) in table 5 was prepared separately by mixing the defined ingredients (see Table 5). Phase B was mixed with phase A and the resulting oil phase was heated at 75° C. Phase C was also heated to 75° C. The oil phase was added to phase C at 75° C. under stirring with a mechanical propel stirring device. The emulsion was then cooled down to less than 40° C., after which phase D was added, followed by phase E and finally phase F under stirring. Formulations were also made, wherein the HA hydrogel concentration was 4%, 6% and 8%, respectively, in Phase E, to give a range of w/o formulations.
A typical formulation of a silicone serum containing 2% DVS-cross-linked HA was prepared as shown in table 6. All ingredients were mixed at the same time under very high stirring and at less than 40° C. (see table 6). Formulations were also prepared, wherein the HA hydrogel concentration was 4%, 6% and 8%, respectively, to give a range of serums.
A kinetics study showed that DVS cross-linked HA hydrogels with neutral pH are obtained after swelling in phosphate buffer (pH 7.0) for 8 to 14 hours, depending on the degree of cross-linking. A set of DVS cross-linked HA hydrogels was prepared as described in the above, using from 4 to 8% HA solution, and using various amounts of DVS cross-linker, as indicated in Table 7.
At regular intervals (every 2 hours), the hydrogels were removed during the heat-treatment and decanted, and pH was measured (see
The decrease was faster for the hydrogels that were less cross-linked, i.e., where the HA/DVS-ratio was higher. The decrease in pH is shown for the HA 6% solution and two different ratios of HA/DVS in
The rheological measurements were performed on a Physica MCR 301 rheometer (Anton Paar, Ostfildern, Germany) using a plate-plate geometry and at a controlled temperature of 25° C. The visco-elastic behavior of the samples was investigated by dynamic amplitude shear oscillatory tests, in which the material was subjected to a sinusoidal shear strain. First, strain/amplitude sweep experiments were performed to evaluate the region of deformation in which the linear viscoelasticity is valid. The strain typically ranged from 0.01 to 200% and the frequency was set to 1 Hz. Then, in the linear visco-elastic regions, the shear storage modulus (or elastic modulus G′) and the shear loss modulus (or viscous modulus, G″) values were recorded from frequency sweep experiments at a constant shear strain (10%) and at a frequency between 0.1 and 10 Hz. The geometry, the NF and the gap were PP 25, 2 and 1 mm, respectively.
G′ gives information about the elasticity or the energy stored in the material during deformation, whereas G″ describes the viscous character or the energy dissipated as heat. In particular, the elastic modulus gives information about the capability of the sample to sustain load and return in the initial configuration after an imposed stress or deformation. In all experiments, each sample was measured at least three times.
In case of the hydrogel with a higher degree of cross-linking (i.e. lower HA/DVS ratio: 10/1) G′ is one order of magnitude higher than G″, indicating that this sample behaves as a strong gel material. Briefly, the overall rheological response is due to the contributions of physical and chemical crosslinks, and to topological interactions among the HA macromolecules. The interactions among the chains bring about a reduction of their intrinsic mobility that is not able to release stress, and consequently the material behaves as a three-dimensional network, where the principal mode of accommodation of the applied stress is by network deformation. Moreover, this hydrogel was more elastic than that with a lower degree of cross-linking (i.e., higher ratio of HA/DVS:15:1). Indeed, the higher the number of permanent covalent cross-links, the larger the number of entanglements, and therefore the higher the elastic response of the hydrogel.
A DVS-cross-linked HA hydrogel was prepared using 1.5 g of sodium HA in 0.2 M NaOH to give a 6% (w/v) solution. The HA/DVS weight ratio was 10:1. The hydrogel was prepared in three replicates according to the procedure described in example 2 until the swelling step, after which it was treated as follows: After incubation in an oven at 50° C. for two hours, the hydrogel was immersed into Na2HPO4/NaH2PO4 buffer (1 L, 50 mM, pH 7.0) containing the preservative (2-phenoxyethanol/3[(2-ethylhexyl)oxy]1,2-propanediol).
The concentration of preservative was 10 mL/mL to target a final concentration of 1% (v/v) in the swollen hydrogel. It was anticipated that the preservative would diffuse into the hydrogel during the incubation, and that at the same time, microbial contamination in the buffer would be prevented.
The vessel was covered with parafilm and placed in an oven at 37° C. After 1 h, the swelling bath was removed and the hydrogel was swollen in a fresh phosphate buffer containing 10 mL/mL preservative for 6-7 h. This step was repeated until the swelling time was 12 h, whereafter the pH was measured. Swelling was continued for another 2.5 h to reach neutral pH.
The amount of preservative incorporated into the hydrogel was determined by UV-spectrophotometry (Thermo Electron, Nicolet, Evolution 900, equipment nr. 246-90). A 1% (v/v) solution of the preservative in phosphate buffer was first analyzed to select the wavelength. Approximately 5 mL of hydrogel were collected using a pipette. Typically, samples were collected in the center of the swollen round hydrogel, and in the north, east, south, and west “sides” of the round gel.
The samples were then transferred into a cuvette and the absorbance was read at 292 nm. Each sample was read three times and the absorbance was zeroed against a blank DVS-cross-linked HA hydrogel, containing no preservative.
The results showed that the amount of preservative incorporated in the DVS-HA hydrogel ranged between 0.91% and 1.02% (see Table 10). There was very good reproducibility between the replicates. Importantly, no significant difference between samples from the same hydrogel was observed, indicating a homogenous diffusion of the preservative into the hydrogel.
The time of degradation may be adjusted based on the polymer mixture in Table 1 below. Examples 1 and 2 below are examples of matrix incorporation of drug or drugs into a biodegradable polymer to control the releases the drugs.
Different types of biodegradable polymer may be used to control the degradation timing and/or to control the degradation by-products. Some biodegradable polymers are:
The particle sizes of the micro capsules are directly controlled by the interfacial chemistry of the organic phase and the aqueous phase. A surfactant is often used to mediate interfacial surface chemistry between an oily substance and the aqueous environment. A surfactant is a detergent that is in an aqueous solution. Surfactants are large molecules that have both polar and non-polar ends. The polar end of the molecule will attach itself to water, also a polar molecule. The non-polar end of the molecule will attract NAPL (non-aqueous phase liquid) compounds.
Examples of surfactants that are used for solubilization are:
1. Sioponic 25-9 which is a linear alcohol ethoxylate, and has a solubilization value of 2.75 g/g
2. Tergitol which is an ethylene oxide/propylene oxide with a solubilization value of 1.21 g/g
3. Tergitol XL-80N which is an ethylene oxide propylene oxide alkoxylate of primary alcohol with a solubilization value of 1.022 g/g
4. Tergitol N-10 which is an a trimethylnonalethoxylate with a solubilization value of 0.964 g/g
5. Rexophos 25/97 which is a phosphatednonylphenolethooxylate with a solubilization value of 0.951 g/g
a. Delayed 30 days
b. Controlled release over 120 days
a. Delayed 60 days
b. Controlled release over 365 days
a. Biodegradable microcapsule containing a cortical steroid delayed 30 days, controlled release over 120 days
b. Biodegradable microcapsule containing an anti-proliferative pharmaceutical delayed 60 days, controlled released over 365 days
Reconstitute in phosphate buffered saline at 0.024 g/mL concentration
The gels suitable for the use in the products according to the one embodiment can represent very many different kinds of rheological bodies varying from hard fragile gels to very soft deformable fluid-like gels. Usually, for the gels which are formed without a crosslinking reaction, for example, a conventional gelatin gel, the hardness and elasticity of the gel increases with increasing polymer concentration. The rheological properties of a crosslinked gel are usually a function of several parameters such as crosslinking density, polymer concentration in the gel, composition of the solvent in which the crosslinked polymer is swollen. Gels with different rheological properties based on hyaluronan and hylan are described in the above noted U.S. Pat. Nos. 4,605,691, 4,582,865 and 4,713,448. According to these patents, the rheological properties of the gel can be controlled, mainly, by changing the polymer concentration in the starting reaction mixture and the ratio of the polymer and the crosslinking agent, vinyl sulfone. These two parameters determine the equilibrium swelling ratio of the resulting gel and, hence, the polymer concentration in the final product and its rheological properties.
A substantial amount of solvent can be removed from a gel which had previously been allowed to swell to equilibrium, by mechanical compression of the gel. The compression can be achieved by applying pressure to the gel in a closed vessel with a screen which is permeable to the solvent and impermeable to the gel. The pressure can be applied to the gel directly by means of any suitable device or through a gas layer, conveniently through the air. The other way of compressing the gel is by applying centrifugal force to the gel in a vessel which has at its bottom the above mentioned semipermeable membrane. The compressibility of a polymeric gel slurry depends on many factors among which are the chemical nature of the gel, size of the gel particles, polymer concentration and the presence of a free solvent in the gel slurry. In general, when a gel slurry is subjected to pressure the removal of any free solvent present in the slurry proceeds fast and is followed by a much slower removal of the solvent from the gel particles. The kinetics of solvent removal from a gel slurry depends on such parameters as pressure, temperature, configuration of the apparatus, size of the gel particles, and starting polymer concentration in the gel. Usually, an increase in pressure, temperature, and filtering surface area and a decrease in the gel particle size and the initial polymer concentration results in an increase in the rate of solvent removal.
Partial removal of the solvent from a gel slurry makes the slurry more coherent and substantially changes the rheological properties of the slurry. The magnitude of the changes strongly depends on the degree of compression, hereinafter defined as the ratio of the initial volume of the slurry to the volume of the compressed material.
The achievable degree of compression, i.e. compressibility of a gel slurry, is different for different gels. For hylan gel slurries in saline, for example, it is easy to have a degree of compression of 20 and higher.
Reconstitution of the compressed gel with the same solvent to the original polymer concentration produces a gel identical to the original one. This has been proven by measuring the rheological properties and by the kinetics of solvent removal from the gel by centrifuging.
It should be understood that the polymer concentration in the gel phase of the viscoelastic mixtures according to the one embodiment may vary over broad ranges depending on the desired properties of the mixtures which, in turn, are determined by the final use of the mixture. In general, however, the polymer concentration in the gel phase can be from 0.01 to 30%, preferably, from 0.05 to 20%. In the case of hylan and hyaluronan pure or mixed gels, the polymer concentration in the gel is preferably, in the range of 0.1 to 10%, and more preferably, from 0.15 to 5% when the swelling solvent is physiological saline solution (0.15M aqueous sodium chloride).
As mentioned above the choice of a soluble polymer or polymers for the second phase of the viscoelastic gel slurries according to one embodiment is governed by many considerations determined by the final use of the product. The polymer concentration in the soluble polymer phase may vary over broad limits depending on the desired properties of the final mixture and the properties of the gel phase. If the rheological properties of the viscoelastic gel slurry are of prime concern then the concentration of the soluble polymer may be chosen accordingly with due account taken of the chemical nature of the polymer, or polymers, and its molecular weight. In general, the polymer concentration in the soluble phase may be from 0.01% to 70%, preferably from 0.02 to 40%. In the case when hylan or hyaluronan are used as the soluble polymers, their concentration may be in the range of 0.01 to 10%, preferably 0.02 to 5%. In the case where other glycosaminoglycans such as chondroitin sulfate, dermatan sulfate, etc., are used as the soluble polymers, their concentration can be substantially higher because they have a much lower molecular weight.
The two phases forming the viscoelastic gel slurries according to one embodiment can be mixed together by any conventional means such as any type of stirrer or mixer. The mixing should be long enough in order to achieve uniform distribution of the gel phase in the polymer solution. As mentioned above, the gel phase may already be a slurry obtained by disintegrating a gel by any conventional means such as pushing it through a mesh or a plate with openings under pressure, or by stirring at high speed with any suitable stirrer. Alternatively, the viscoelastic mixed gel slurries can be prepared by mixing large pieces of gel with the polymer solution and subsequently disintegrating the mixture with formation of the viscoelastic slurry by any conventional means discussed above. When the first method of preparing a mixed gel slurry according to one embodiment is used, the gel slurry phase can be made of a gel swollen to equilibrium, and in this case there is no free solvent between the gel particles, or it may have some free solvent between gel particles. In the latter case this free solvent will dilute the polymer solution used as the second phase. The third type of gel slurry used as the gel phase in the mixture is a compressed gel whose properties were discussed above. When a compressed gel slurry is mixed with a polymer solution in some cases the solvent from the solution phase will go into the gel phase and cause additional swelling of the gel phase to equilibrium when the thermodynamics of the components and their mixture allows this to occur.
The composition of the viscoelastic mixed gel slurries according to one embodiment can vary within broad limits. The polymer solution in the mixture can constitute from 0.1 to 99.5%, preferably, from 0.5 to 99%, more preferably, from 1 to 95%, the rest being the gel phase. The choice of the proper composition of the mixture depends on the properties and composition of the two components and is governed by the desirable properties of the slurry and its final use.
The viscoelastic gel mixtures according to one embodiment, in addition to the two major components, namely, the polymeric gel slurry and the polymer solution, may contain many other components such as various physiologically active substances, including drugs, fillers such as microcrystalline cellulose, metallic powders, insoluble inorganic salts, dyes, surface active substances, oils, viscosity modifiers, stabilizers, etc., all depending upon the ultimate use of the products.
The viscoelastic gel slurries according to one embodiment represent, essentially, a continuous polymer solution matrix in which discrete viscoelastic gel particles of regular or irregular shape are uniformly distributed and behave rheologically as fluids, in other words, they exhibit certain viscosity, elasticity and plasticity. By varying the compositional parameters of the slurry, namely the polymer concentration in the gel and the solution phases, and the ratio between two phases, one may conveniently control the rheological properties of the slurry such as the viscosity at a steady flow, elasticity in dynamic mode, relaxation properties, ratio between viscous and elastic behavior, etc.
The other group of properties which are strongly affected by the compositional parameters of the viscoelastic gel slurries according to one embodiment relates to diffusion of various substances into the slurry and from the slurry into the surrounding environment. The diffusion processes are of great importance for some specific applications of the viscoelastic gel slurries in the medical field such as prevention of adhesion formation between tissues and drug delivery as is discussed below in more detail.
It is well known that adhesion formation between tissues is one of the most common and extremely undesirable complications after almost any kind of surgery. The mechanism of adhesion formation normally involves the formation of a fibrin clot which eventually transforms into scar tissue connecting two different tissues which normally should be separated. The adhesion causes numerous undesirable symptoms such as discomfort or pain, and may in certain cases create a life threatening situation. Quite often the adhesion formation requires another operation just to eliminate the adhesions, though there is no guarantee against the adhesion formation after re-operation. One means of eliminating adhesion is to separate the tissues affected during surgery with some material which prevents diffusion of fibrinogen into the space between the tissues thus eliminating the formation of continuous fibrin clots in the space. A biocompatible viscoelastic gel slurry can be successfully used as an adhesion preventing material. However, the diffusion of low and high molecular weight substances in the case of plain gel slurries can easily occur between gel particles especially when the slurry mixes with body fluids and gel particles are separated from each other. On the other hand, when a viscoelastic mixed gel slurry according to one embodiment, is implanted into the body, the polymer solution phase located between gel particles continues to restrict the diffusion even after dilution with body fluids thus preventing adhesion. Moreover, this effect would be more pronounced with an increase in polymer concentration of the polymer solution phase.
The same is true when the viscoelastic mixed gel slurries according to one embodiment are used as drug delivery vehicles. Each of the phases of the slurry or both phases can be loaded with a drug or any other substance having physiological activity which will slowly diffuse from the viscoelastic slurry after its implantation into the body and the diffusion rate can be conveniently controlled by changing the compositional parameters of the slurries.
Components of the viscoelastic mixed gel slurries according to one embodiment affect the behavior of living cells by slowing down their movement through the media and preventing their adhesion to various surfaces. The degree of manifestation of these effects depends strongly on such factors as the composition of the two components of the mixture and their ratio, the nature of the surface and its interaction with the viscoelastic gel slurry, type of the cells, etc. But in any case this property of the viscoelastic gel slurries can be used for treatment of medical disorders where regulation of cell movement and attachment are of prime importance in cases such as cancer proliferation and metastasis.
In addition to the above two applications of biocompatible viscoelastic gel slurries according to one embodiment other possible applications include soft tissue augmentation, use of the material as a viscosurgical tool in opthalmology, otolaryngology and other fields, wound management, in orthopedics for the treatment of osteoarthritis, etc. In all of these applications the following basic properties of the mixed gel slurries are utilized: biocompatibility, controlled viscoelasticity and diffusion characteristics, easily controlled residence time at the site of implantation, and easy handling of the material allowing, for example its injection through a small diameter needle. The following methods were used for characterization of the products obtained according to one embodiment. The concentration of hylan or hyaluronan in solution was determined by hexuronic acid assay using the automated carbazole method (E. A. Balazs, et al, Analyt. Biochem. 12, 547-558, 1965). The concentration of hylan or hyaluronan in the gel phase was determined by a modified hexuronic acid assay as described in Example 1 of U.S. Pat. No. 4,582,865.
Rheological properties were evaluated with the Bohlin Rheometer System which is a computerized rheometer with controlled shear rate and which can operate in three modes: viscometry, oscillation and relaxation. The measurements of shear viscosity at low and high shear rates characterize viscous properties of the viscoelastic gel slurries and their pseudoplasticity (the ratio of viscosities at different shear rates) which is important for many applications of the products. Measurements of viscoelastic properties at various frequencies characterized the balance between elastic (storage modulus G′) and viscous (loss modulus G″) properties. The relaxation characteristics were evaluated as the change of the shear modulus G with time and expressed as the ratio of two modulus values at different relaxation times.
Next, various HA Crosslinking Approaches are discussed. The following reactions focus mainly on the two most reactive functional groups—the hydroxyl and the carboxyl.
1. Bisepoxide,
2. Divinylsulfone (DVS)
3. Internal Esterification
4. Photo-Cross Linking
5. Glutaraldehyde Cross Linking
6. Metal Cation Mediated Cross Linking
7. Carbodiimide Cross Linking
8. Hydrazide Cross Linking
1. Cross Linking with Residual Proteins
2. Multi-Component Reactions
3. Surface Modifications
HA Therapeutic Modification Options
HA Reactive Sites
5. Carboxyl Group Chemical Reactions
1. Esterification
2. Carbodiimide-Mediated Reactions
3. The Chemical Modification of the Carboxylic Functions of Hyaluronan by Carbodiimide Compounds is Generally Performed in Water at pH 4.75.
6. Hydroxyl Group Chemical Reactions
1. Sulfation
2. Isourea Coupling or Cyanogen Bromide Activation
3. Peroxidase Oxidation
4. Reducing End Modification
5. Amide Modifications
1. Experiment 001-12: Water in Oil Emulsion Cross-Linking Reaction
2. Experiment 001-14
3. Boundary Conditions of Components in the HA X-Linking Process
Experiment 001-16: X-Linker Mix Storage Life and Reaction Temperature
Experiment 001-17: Storage Life for 1% NaOH
2. X-Linker Storage Life—BDDE
4. X-Linker Storage Life—DVS “TBD”
5. Experiment 001-19
6. Experiment 001-21
7. Effects of X-Linking Levels
1. Experiment 001-22: BDDE (1,4-butanediol diglycidylether)
2. Experiment 001-25: DVS (Divinylsulfone)
In one embodiment, the HA can be serially cross-linked to form a system with monophasic characteristics. The forming a biocompatible cross-linked polymer as an IPN can be done by cross-linking a heteropolysaccharide to form a single cross-linked material; and performing one or more additional cross-linkings on the single cross-linked material to form a multiple cross-linked material, wherein the multiple cross-linked material has a core that lasts longer in a human body than the single cross-linked material. The result is a material with a smooth continuum from slightly cross-linked to the core which is highly cross-linked. The slightly cross-linked material enables the HA to be easily inserted into the human body with a small gauge syringe, but such slightly cross-linked material will not last long in the human body. However, the highly cross-linked material will remain longer in the human body so that the body augmentation does not need periodic touch-ups as is needed by conventional HA dermal fillers.
The cross-link time resulting from the use of a stable, non-aqueous suspension of a delayed cross-linker according to the preferred embodiment may be controlled by varying any one or all of the following:
1) the cross linking compound used,
2) the particle size of the HA in suspension,
3) the pH of the fluid containing the HA,
4) the concentration (i.e., loading) of the HA suspension,
5) the temperature of the solution.
Illustratively, when used under similar conditions, the type of molecular weight of the HA compound may be employed effectively to control the exact cross-linking time of the water-soluble solution. More particularly, suspensions of larger molecular weight HA cross-link more slowly than suspensions of low molecular weight acid.
With respect to the particle size of the suspended halyuronic acid, as particle size increases, the time required for the cross-linking of a water-soluble polymer solution increases. Conversely, as the particle size decreases, the time required for the cross-linking of a water soluble decreases.
The pH of the water soluble polymer solution prior to its cross-linking may be used to control cross-link time. The pH of the water soluble polymer solution affects the solubility rate of the stable, non-aqueous suspension of a delayed cross-linker. Specifically, as the pH of the water soluble polymer solution increases, the solubility rate of the cross-linker suspension increases if the suspension contains a majority of HA particles, whereas the solubility rate of the cross-linker suspension decreases if the suspension contains a majority of borax particles. Conversely, as the pH of the water soluble polymer solution decreases, the solubility rate of the cross-linker suspension decreases if the suspension contains a majority of boric acid particles, whereas the solubility rate of the cross-linker suspension increases if the suspension contains a majority of HA particles.
Both the concentration (i.e., loading) of the stable, non-aqueous suspension of a delayed HA cross-linker in the water soluble polymer solution and the content of the cross-linker suspension affect the cross-link time of a water soluble polymer solution similarly. As either the concentration of the suspension of delayed HA cross-linker in the water-soluble polymer solution or the content of the cross-linker suspension increase, the cross-link time of the water soluble polymer solution decreases. Conversely, as either the concentration of the suspension of the delayed boron cross-linker in the water soluble polymer solution and the content of the cross-linker suspension decrease, the cross-link time of the water soluble polymer solution increases.
Temperature may be used to alter the cross-link time of a water soluble polymer solution. As the temperature of the water soluble polymer solution increases, its cross-link time decreases. Conversely, as the temperature of the water soluble polymer solution decreases, its cross-link time increases. Furthermore, the cross-link time of a water-soluble polymer may be increased or decreased depending upon the clay type utilized in the formulation of the stable, non-aqueous suspension of a delayed HA cross-linker.
In addition, materials such as polymeric microspheres, polymer micelles, soluble polymers and hydrogel-type materials can be used for providing protection for pharmaceuticals against biochemical degradation, and thus have shown great potential for use in biomedical applications, particularly as components of drug delivery devices. The design and engineering of biomedical polymers (e.g., polymers for use under physiological conditions) are generally subject to specific and stringent requirements. In particular, such polymeric materials must be compatible with the biological milieu in which they will be used, which often means that they show certain characteristics of hydrophilicity. They also have to demonstrate adequate biodegradability (i.e., they degrade to low molecular weight species. The polymer fragments are in turn metabolized in the body or excreted, leaving no trace). Biodegradability is typically accomplished by synthesizing or using polymers that have hydrolytically unstable linkages in the backbone. The most common chemical functional groups with this characteristic are esters, anhydrides, orthoesters, and amides. Chemical hydrolysis of the hydrolytically unstable backbone is the prevailing mechanism for the degradation of the polymer. Biodegradable polymers can be either natural or synthetic. Synthetic polymers commonly used in medical applications and biomedical research include polyethyleneglycol (pharmacokinetics and immune response modifier), polyvinyl alcohol (drug carrier), and poly(hydroxypropylmetacrylamide) (drug carrier). In addition, natural polymers are also used in biomedical applications. For instance, dextran, hydroxyethylstarch, albumin and partially hydrolyzed proteins find use in applications ranging from plasma substitute, to radiopharmaceutical to parenteral nutrition. In general, synthetic polymers may offer greater advantages than natural materials in that they can be tailored to give a wider range of properties and more predictable lot-to-lot uniformity than can materials from natural sources.
In one embodiment, the linker is a dicarboxylic acid with at least three atoms between the carbonyls and contains a heteroatom alpha to the carbonyl forming the ester, the release half-life is less than about 10 hours; when Linker is a dicarboxylic acid with at least three atoms between the carbonyls with no heteroatom alpha to the carbonyl forming the ester, the release half-life is more than about 100 hours; wherein when Linker is a dicarboxylic acid with two atoms between the carbonyls and Tether contains a nitrogen with a reactive hydrogen, the release half-life of the HA is from about 0.1 hours to about 20 hours; wherein the release half-life being measured in 0.05M phosphate buffer, 0.9% saline, pH 7.4, at 37° C.; with the proviso that the conjugate is not PHF-SA-Gly-CPT, PHF-(methyl)SA-Gly-CPT, PHF-(2,2-dimethyl)SA-Gly-CPT, PHF-(2-nonen-2-yl)SA-Gly-CPT, PHF-SA-Gly-Taxol, or PHF-SA-Gly-Illudin.
In some embodiments, the polyal is an acetal. In other embodiments, the polyal is a ketal. In some embodiments, the acetal is PHF. In some embodiments, Ri is H. In other embodiments, Ri is CH3. In some embodiments, R2 is —CH(Y)—C(O)—, wherein Y is one of the side chains of the naturally occurring amino acids. In some embodiments, R2 is an aryl group. In some embodiments, R2 is anheteroaryl group. In other embodiments, R2 is an aliphatic ring. In some embodiments, R2 is an aliphatic chain. In some embodiments, R2 is a heterocyclic aliphatic ring. In some embodiments, Ri and R2 when taken together with nitrogen to which they are attached form a ring. Other embodiments are known to those skilled in the art. For example, some embodiments are discussed in US2010/036413, the content of which is incorporated by reference.
In another embodiment, a biocompatible cross-linked IPN polymer can be done by cross-linking a heteropolysaccharide to form a first cross-linked material; and by performing one or more additional cross-linking of the first cross-linked material to form a multiple cross-linked material. The result monophasic HA can be used for augmenting soft tissue with the biocompatible cross-linked polymer.
Besides the foregoing methods of obtaining IPN and semi-IPN by crosslinking both of the components of the blend, semi-IPN can also be obtained by the polymerization of a monomer in the presence of a crosslinking agent and in the presence of the natural acidic polysaccharide or a semisynthetic ester-type derivative thereof.
In the following examples, the HA composition percentage is varied from 75% to 99% of the total composition while the cross linker percentage is varied between 1 and 25% as follows:
As the percentage of HA increases, the material is soft, but less resistant to biodegration. As more cross-linker is introduced, the material becomes more hardened and lasts longer. The multiple serially cross-linking processes provide advantages of being soft to the touch, yet long lasting. The varying mechanical/physical properties that constantly becomes softer while remaining tough radiating out from the IPN makes the polymer tough and at the same time compliant with its surrounding for better biocompatibility and feels more natural to the touch. The IPN is an intimate combination of two or more polymer systems, both in network form, at least one of which is synthesized or cross-linked in the immediate presence of the other. If one of the two polymers is in network form (cross-linked) and the other is a linear polymer (not cross-linked), a semi-IPN results. The term IPN currently covers new materials where the at least two polymers in the mixture are not necessarily bound together, but the components are physically associated.
The multiply cross linking process is akin to a discrete or digital process where the HA is first cross-linked, then the result is cross-linked a second time, then third cross-linked is done, thus forming serial cross-linking additions. This discrete or digital process is in contrast to the conventional continuous process. In one embodiment, the IPN center can be where ever relative aqueous front exists.
It should be mentioned that for the purpose of HA longevity, the more hydrophobic a cross linker is the better because hydrolysis is not favored. Sterically hindered cross linker is also preferred for the same reason mentioned. However, hydrophobicity in this case will make the HA polymer less biocompatible and will likely illicit unwanted foreign body reactions. The type of cross linker used for any part of the process will also make a difference in longevity, biocompatibility and physical properties. Application requirement will dictate the ideal polymer composition that gives the balance of properties.
Through the serial cross-linking steps, the cross-linked HA (hyaluronic acid) molecular macro structures are interpenetrated cross-linked highest at the surface that is interfacing the basic aqueous media and lowest toward the center core. After the initial cross-linking reaction step, the cross-linked HA chains lost significant mobility. Thus, an IPN (interpenetrating network) polymer is formed readily with subsequent sequential cross-linking reactions.
The rheology of cross-linked HA may be characterized as having non-Newtonian fluid behaviors. According to theory in regards mixing in a two-dimensional cavity flow, the key to effective mixing lies in producing repetitive stretching and folding, an operation referred to as a “horse-shoe-map”. The mixing can be done as a mixing of viscous Newtonian and non-Newtonian fluids, as described by Chavan et al, “Mixing of Viscous Newtonian and Non-Newtonian Fluids, pp 211-252, the content of which is incorporated by reference. Alternatively, the mixing can be done according to “Stretching and mixing of non-Newtonian fluids in time-periodic flows”, by Paulo E. Arratia, the content of which is incorporated by reference. The scaling of the mixing processes can be done as described in Wilkens et al. “How to Scale Up Mixing Processes in Non-Newtonian Fluids”, the content of which is incorporated by reference.
In the end product, the cross-linking level is non-uniform throughout the HA polymer matrix, the polymer chains become bi-axially oriented. The orientation is the result of the polar medium the HA polymer resides in.
Various implementations of mixing the reactants are described below:
Since all of the components used in the reaction are water soluble, and the reaction product is not water soluble, the cross-linked HA polymer can be purified using DI water. Furthermore, water will swell the reaction product many folds which allows the impurities to easily diffuse out of the polymer and be eliminated. Continuous flushing with DI water will speed up the purification process, and effectively rid the reaction product of unwanted impurities.
The pH of the water before mixing with the cross-linked HA and after mixing with the cross-linked HA is a good indirect indicator of the cleansing effectiveness along the process. The of the water pH before and after should not significantly changed.
The other methods, used for characterization of the products according to one embodiment are described in the following examples which illustrate preferred embodiments of one embodiment without, however, being a limitation thereof. Variations and modifications can, of course, be made without departing from the spirit and scope of the invention. For example, the HA can be used as facial fillers, dermal fillers, butt fillers, breast fillers, and other body part fillers. The implants of the present invention further can be instilled, before or after implantation, with indicated medicines and other chemical or diagnostic agents. Examples of such agents include, but are not limited to, antibiotics, chemotherapies, other cancer therapies, brachy-therapeutic material for local radiation effect, x-ray opaque or metallic material for identification of the area, hemostatic material for control of bleeding, growth factor hormones, immune system factors, gene therapies, biochemical indicators or vectors, and other types of therapeutic or diagnostic materials which may enhance the treatment of the patient.
Advantages of one IPN embodiment can include one or more of the following. A natural feel is achieved through viscoelastic harmony of properties between the existing tissue and the implant. This can be done by manipulating the viscous component of the implant through flow properties by way of the particle size and particle size distribution ratios. The elastic component is intrinsic within the material tertiary structure (molecular weight and steric hindrance) and cross linking densities. The interpenetrating polymer network hydrogels have a number of desirable properties. These properties include high tensile strength with high water content, making the interpenetrating polymer network hydrogels excellent for use in dermal filling applications. Other advantages and features include: longevity without touch up, hyper-volumic degradation, anatomic compliant and iso-osmotic controlled, among others.
In one embodiment, the system photographs a patient's body in 3D before her breast or butt procedure, captures linear and volumetric measurements, and creates an exact three dimensional replica of her body on screen. The doctor examines this model with the patient during the consultation, and performs a virtual breast or butt augmentation, breast or butt lift, or breast or butt reconstruction on the 3D model to visualize the expected result in advance of an actual surgical procedure. The photo-realistic result can be viewed from all angles, and implant size adjusted to most closely meet the patient's needs. This allows women for the first time to select implant size, shape, and position based on the expected outcome on their own body.
In another embodiment, a 3D webcam is used with two cameras spaced roughly the same distance apart as human eyes, for the stereoscopic effect. 3D data acquisition and object reconstruction can be performed using stereo image pairs. Stereo photogrammetry or photogrammetry based on a block of overlapped images is the primary approach for 3D mapping and object reconstruction using 2D images. Close-range photogrammetry where cameras or digital cameras can be used to capture the close-look images of objects, e.g., breast or butts, and reconstruct them using the very same theory as the aerial photogrammetry.
Once the 3D model of the implant is finalized, the patient may wish to view the “try on” implants in combination with various articles of clothing to more fully determine how the implants will affect the patient's appearance. A library of wardrobe can be placed over the patient, so the patient can preview her implants with various items of clothing. Photorealistic images of the patient can be generated for the patient to consult family or friends as to which size implants gives the most favorable appearance. Thus, the system provides patients with the ability to realistically determine how a range of implant sizes will change their appearance.
A relatively large amount of hyaluronic acid, for example an entire syringe, is emptied into one area creating a large volume of the hyaluronic acid material in the deep tissue that does not break down readily. The deep volume or bolus can be sculpted by the doctor to enlarge or change the shape of the buttock or breast. The system injectable material comes in packages of 25, 50, 100, and 200 cc in volume. The delivery system is completely sterile and can be used in an outpatient setting or doctor's office. Since the volume of the system can be adjusted accordingly by the physician, the amount of soft tissue augmentation is limited only by the site. The system can also be additive just like MIBA Medical's Restor, Restylane or Allergan's Juvederm for augmenting facial wrinkles.
The present invention has been described particularly in connection with a breast, butt, or body implant, but it will be obvious to those of skill in the art that the invention can have application to other parts of the body, such as the face, and generally to other soft tissue or bone.
Accordingly, the invention is applicable to replacing missing or damaged soft tissue, structural tissue or bone, or for cosmetic tissue or bone replacement.
Although the present invention has been described in relation to particular embodiments thereof, many other variations and modifications and other uses will become apparent to those skilled in the art. It is preferred, therefore, that the present invention be limited not by the specific disclosure herein, but only by the appended claims. The other methods, used for characterization of the products according to one embodiment are described in the following examples which illustrate preferred embodiments of one embodiment without, however, being a limitation thereof. Variations and modifications can, of course, be made without departing from the spirit and scope of the invention.
| Number | Date | Country | |
|---|---|---|---|
| Parent | 14881028 | Oct 2015 | US |
| Child | 15331861 | US |
