(19) United States
`(12) Patent Application Publication (10) Pub. No.: US 2006/0122147 A1
`(43) Pub. Date:
`Jun. 8, 2006
`WOhrab
`
`US 2006O122147A1
`
`(54) COMBINATION PREPARATION OF
`HYALURONIC ACID AND AT LEAST OE
`LOCAL ANESTHETIC AND THE USE
`THEREOF
`
`(76) Inventor: David Wohlrab, Halle (DE)
`Correspondence Address:
`LEYDIG VOIT & MAYER, LTD
`TWO PRUDENTIAL PLAZA, SUITE 4900
`18O NORTH STETSON AVENUE
`CHICAGO, IL 60601-6780 (US)
`(21) Appl. No.:
`10/529,924
`
`(22) PCT Filed:
`Sep. 9, 2003
`(86). PCT No.:
`PCT/EPO3/10822
`(30)
`Foreign Application Priority Data
`
`Oct. 4, 2002 (DE)..................................... 102 46 340.9
`
`Publication Classification
`
`(51) Int. Cl.
`(2006.01)
`A6II 3L/728
`(2006.01)
`COSB 37/00
`(52) U.S. Cl. ................................................. 514/54; 536/53
`
`(57)
`
`ABSTRACT
`
`The invention relates to a combination preparation compris
`ing an active agent A from the group hyaluronic acid, the
`salts and fragments thereof, at least one active agent B from
`the group of local anaesthetics and derivatives thereof and
`also if necessary further additives. These combination
`preparations are used for the medical treatment of degen
`erative and traumatic diseases of all joints, for the treatment
`of articular cartilage and cartilage bone defects and also
`meniscus and intervertebral disc lesions. Such as e.g. arthro
`sis, articular rheumatism, osteochondritis dissecans, flake
`fractures, meniscus lesions and for the treatment of skin and
`mucous membrane changes, also from cosmetic aspects.
`
`ALL 2046
`PROLLENIUM V. ALLERGAN
`IPR2019-01505 et al.
`
`

`

`Patent Application Publication Jun. 8, 2006 Sheet 1 of 2
`
`US 2006/0122147 A1
`
`Figure 1.
`
`200
`
`
`
`18O
`
`160
`
`140
`
`120
`
`100
`
`5.
`
`

`

`Patent Application Publication Jun. 8, 2006 Sheet 2 of 2
`
`US 2006/O122147 A1
`
`Figure 2
`
`1200
`
`
`
`Control
`idocaine 0.1 mmol/
`
`1.
`
`* pa 0,001
`# pc 0,0001
`
`800
`
`5.
`
`800
`
`200
`
`O
`
`6
`
`12
`
`18
`
`Culture duration d .
`
`

`

`US 2006/O 122147 A1
`
`Jun. 8, 2006
`
`COMBINATION PREPARATION OF HYALURONIC
`ACID AND AT LEAST OE LOCAL ANESTHETIC
`AND THE USE THEREOF
`0001. The invention relates to a combination preparation
`comprising an active agent A from the group hyaluronic
`acid, the salts and fragments thereof, at least one active
`Substance B from the group of local anaesthetics and deriva
`tives thereof and also if necessary further additives. These
`combination preparations are used for the medical treatment
`of degenerative and traumatic diseases of all joints, for the
`treatment of articular cartilage and cartilage bone defects
`and also meniscus and intervertebral disc lesions, such as
`e.g. arthrosis, articular rheumatism, osteochondritis disse
`cans, flake fractures, meniscus lesions and for the treatment
`of skin and mucous membrane changes, also from cosmetic
`aspects.
`0002 The chemical name for hyaluronic acid is hyalu
`roman. Its chemical structure corresponds to the formula
`
`0006 Starting herefrom, it was the object of the present
`invention to provide a combination preparation which can be
`applied in various forms and in the case of which the active
`Substances can be released in a specifically delayed manner.
`0007. This object is achieved by the combination prepa
`ration with the features of claim 1. The use of the combi
`nation preparation is described in claim 15. The further
`dependent claims demonstrate advantageous developments.
`0008 According to the invention, a combination prepa
`ration consisting of an active agent A from the group
`hyaluronic acid, the physiological salts and fragments
`thereof, at least one active agent B from the group of local
`anaesthetics and derivatives thereof and also if necessary
`further additives is provided.
`0009. It was established that, due to the considerable
`molecular size of hyaluronic acid (1-6x10 Da), this must be
`split several times before it can leave the intraarticular space
`
`
`
`n = 2000-3000
`
`0003. Despite the positive clinical experiences with high
`molecular hyaluronic acid and the salts thereof (mol mass
`>1x106 Dalton), knowledge about the operating mechanism
`is incomplete. The present state of knowledge identifies
`intraarticularly applied hyaluronic acid as a lubricant (A.
`Lussier et al. (1996); J. Rheumatol. 23, 1579-1585; D. Scale
`et al. (1994); Current Therapeutic Research. 55,220-232; M.
`Wobig et al. (1998) Clinical Therapeutics. 20, 410-423).
`Furthermore, it was indicated that hyaluronic acid has
`intraarticularly anti-inflammatory properties (K. W. Mar
`shall (1997) Today's Therapeutic Trends. 15, 99-108; K. W.
`Marshall (2000) Curr. Opin. Rheumatol. 12, 468-474).
`0004 Arthrosis begins with initial damage of the carti
`lage tissue because of various causes. This results in reactive
`synovialitis which for its part causes both pathological
`changes in the synovial fluid, i.e. reduction in concentration
`and molecular weight of the hyaluronic acid, and also the
`release of inflammation mediators. This leads to secondary
`cartilage damage and hence finally to arthrosis which, in
`addition to cartilage tissue, affects all other articular struc
`tures (J. P. Pelletier et al. (1993) J. Rheumatol. 20, 19-24).
`0005. It is known that intraarticularly applied hyaluronic
`acid leads to improvement in joint mobility, to pain reduc
`tion, to inhibition of inflammation processes and, under in
`vitro conditions, to the increase of chondrocyte proliferation
`(K. Kawasaki et al. (1999) Cell Physiol. 179, 142-148; D.
`Wohlrab et al. (2000) hylan news. 2: 2-5).
`
`and be decomposed or incorporated in cartilage tissue. These
`splitting processes take hours up to several days dependent
`upon the mol mass of the hyaluronic acid.
`0010 Because of this extended intraarticular dwell time,
`in comparison to other low molecular Substances, such as
`e.g. local anaesthetics, high molecular hyaluronic acid, the
`salts or the fragments thereof are suitable as carriers for
`Substances which, without bonding of this type to a carrier
`molecule, have a significantly shortened intraarticular dwell
`time and hence a very short period of activity.
`0011 All the formulations known from the state of the art
`are possible as galenic formulation. Included herein are in
`particular intraarticularly, intradiscally, Subcutaneously,
`intracutaneously or topically applicable galenic formula
`tions.
`0012 Preferably, compounds chosen as active agent A
`are compounds from the group hyaluronic acid, the salts and
`fragments thereof and, as active agent B, compounds from
`the group of local anaesthetics and derivatives thereof,
`which compounds have together a chemical or physical
`bond, the active substance B being able to be released in a
`delayed manner. The pH value of the formulation thereby
`makes possible an optimum bond between the two active
`agents and the release of the active agent B can be controlled
`via alteration in the pH value of the Surrounding medium.
`0013 Preferably, the active agent A is contained in the
`combination preparation in a concentration between 0.001
`and 5% by weight or preferably between 0.2 and 2.0% by
`
`

`

`US 2006/O 122147 A1
`
`Jun. 8, 2006
`
`weight. The active agent B is preferably in a concentration
`between 0.001 and 20% by weight, preferably between
`0.001 and 5.0% by weight.
`0014 Furthermore, further additives can be contained in
`the combination preparation. There are included herein for
`example agents with radical interceptor properties, in par
`ticular tocopherol derivatives or ascorbic acid derivatives.
`Furthermore, agents of the hyaline cartilage tissue can be
`used, in particular glucosamine Sulphate derivatives or chon
`droitin sulphate derivatives. Furthermore, agents with a
`steroidal and corticoidal effect can be used, in particular
`glucocorticoids. There are possible as additives furthermore
`non-steroidal antiphlogistics which are described also as
`antirheumatics, in particular indometacin, diclofenac or sali
`cylic acid derivatives and analgesics, in particular oxicams,
`aniline or anthranilic acid derivatives. The combination
`preparation can have as additive likewise Substances with an
`inhibitory effect on prostaglandin synthesis, in particular
`lipoxygenase inhibitors, cyclo-oxygenase inhibitors and
`phospholipase A2 inhibitors. Likewise, there are possible as
`additives growth factors, in particulars retinol or bone mor
`phogenetic proteins (BMPs), vitamins, in particular vitamin
`A, C, B12 or biotin, antioxidants, in particular flavonoids or
`glutathione, and agents with water-binding properties, in
`particular urea or arginine.
`0.015 The combination preparation can be produced as
`any galenic formulation, e.g. as a solution, Suspension,
`emulsion, paste, ointment, gel, cream, lotion, Varnish, pow
`der, soap, Surfactant-containing cleaning preparation, oil,
`lipstick, lip salve, mascara, eye liner, eye shadow, rouge,
`powder, emulsion or wax makeup, Sun protection, pre-Sun
`and after-Sun preparations or as a spray.
`0016. The application of the combination preparation can
`be effected both on humans and on animals. The combina
`tion preparations according to the invention can be applied
`both in human and veterinary medicine and in cosmetics.
`0017. The application fields of the combination prepara
`tions relate to human and veterinary medical therapy, pro
`phylaxis and/or metaphylaxis of degenerative or traumatic
`articular diseases and articular function disorders, articular
`cartilage and cartilage bone defects, meniscus and interver
`tebral disc diseases. There are included herein for example
`the increase in chondrocyte proliferation, the stabilisation
`and/or regeneration of articular structures, in particular of
`the articular cartilage and menisci, the increase in joint
`mobility and the inhibition of inflammatory processes.
`0018. Likewise, the combination preparation can how
`ever by used also for treating skin and mucous membrane
`changes both from medical and cosmetic viewpoints.
`0.019 According to the invention, also the use of at least
`one active agent from the group hyaluronic acid, the salts
`and fragments thereof in combination with at least one
`active agent B from the group of local anaesthetics and
`derivatives thereof for preparing a medicament for human
`and veterinary medical therapy, prophylaxis and/or meta
`phylaxis of articular diseases and articular function disorders
`is provided.
`0020. The invention is intended to be explained with
`reference to the following Examples and Figures without
`restricting it thereto.
`
`EXAMPLE 1.
`
`Physiological Compatibility of the Galenic
`Formulations According to the Invention
`Production:
`0021
`Lidocaine hydrochloride (University pharmacy of
`the Martin Luther University Halle-Wittenberg) and hyalu
`ronic acid (Aqua Biochem, Dessau) (MG 1.5x10 Da) were
`present primarily in powder form. In order to produce 2%
`parent solutions, corresponding quantities were dissolved in
`RPMI medium (Seromed, Berlin) and subsequently filtered
`in a sterile manner. In order to produce a lidocaine-hyalu
`ronic acid mixture, these parent solutions were mixed in
`equal parts. The Substance addition to the cell culture was
`effected on the 10" culture day with medium change. Cor
`responding quantities of the test Substances (parent solu
`tions) were added here so that a respective end concentration
`of 5x10 mmol/1 was achieved.
`Preparation of the Biological Material:
`0022. The tests were effected on human chondrocytes
`which were isolated from arthrotically changed knee joint
`cartilage. The cartilage tissue stemmed from femoral articu
`lar surfaces resected during implantation of total knee
`endoprostheses. Exclusively arthrotically changed cartilage
`tissue from three different donors without known relevant
`secondary diseases, in particular without rheumatoid arthri
`tis, was used.
`0023 The intraoperatively obtained bone-cartilage frag
`ments were transferred firstly into sterile L15 medium
`(Seromed, Berlin) as transport medium. Subsequently, the
`separation of the cartilage tissue from the Subchondral bone
`was effected under sterile conditions by means of a scalpel
`and also sharp severance of the tissue into pieces of approxi
`mately 1 mm. The enzymatic isolation of the chondrocytes
`from the pieces of cartilage was effected by means of
`pronase and collagenase A (Boehringer Mannheim) over a
`timespan of 16 hours.
`Test Conditions:
`0024. The isolated chondrocytes were cultivated in 24
`well plates in RPMI medium (Seromed, Berlin) with the
`addition of various antibiotics at 37° C. and 5% carbon
`dioxide in an incubator as a monolayer culture. The medium
`change was effected every 2 days. After 10 culture days,
`finally a medium change was effected and hereby the addi
`tion of the respective test substances which were dissolved
`in the culture medium. In addition, an untreated chondrocyte
`population respectively was run jointly as control.
`Implementation of the Test:
`0025) The measurement of the H thymidine incorpora
`tion as a measure of the DNA synthesis yield was effected
`24, 48 or 72 hours after addition of the substance. At the end
`of the culture time, 20 ul H-methylthymidine (specific
`activity 60.3 Ci/mmol; American Radiolabeled Chemicals
`Inc., St. Louis, USA) was added per well to the cell culture.
`Two hours after the addition of the H thymidine, the
`medium was Suctioned out of the chambers by means of a
`Cell Harvester (Berthold GmbH, Bad Wildbad). Each cul
`ture chamber was supplied with 200 ultrypsine and the cell
`suspension was suctioned off after 20 minutes via a filter.
`Subsequently, the measurement of the radioactivity of the
`
`

`

`US 2006/O 122147 A1
`
`Jun. 8, 2006
`
`cells in the filter paper was effected by means of a liquid
`scintillation meter (WINSPECTRAL 1414, Wallace-ADL
`GmbH, Freiburg, Deutschland).
`0026. The results for the physiological compatibility of
`the galenic formulations according to the invention are
`represented in FIG. 1.
`0027) FIG. 1 shows the effect of hyaluronic acid (hya)
`(1.5x10 Da, 5x10 mmol/l), lidocaine (lido) (5x10
`mmol/l) and hyaluronic acid-lidocaine mixture (hya +lido)
`(respectively 5x10 mmol/l) on the incorporation of H
`thymidine by human chondrocytes (N=3) cultivated in vitro
`after 48 h. Each measurement value is the mean of 8
`individual measurements.
`
`EXAMPLE 3
`The Optimal Bonding of the Local Anaesthetic or
`Local Anaesthetics or the Derivatives of These
`Compounds to Hyaluronic Acid and/or the
`Physiologically Compatible Salts of Hyaluronic
`Acid and also the Fragments of these Compounds
`in the Example of Lidocaine
`
`0033)
`
`TABLE 1.
`
`shows subsequently the proportion of free lidocaine with
`different lidocaine concentrations (conc. hyaluronic acid = 0.05%).
`
`Conc. lidocaine%
`
`EXAMPLE 2
`
`Influence of Lidocaine Upon the Proliferation of
`Human Chondrocytes
`
`Production:
`0028 Lidocaine hydrochloride (University pharmacy of
`the Martin Luther University Halle-Wittenberg) was present
`primarily in powder form. This was dissolved in a corre
`sponding quantity in RPMI medium (Seromed, Berlin) so
`that an end concentration of 0.1 mmol/l lidocaine was
`present. Sterile filtration was effected subsequently. The
`addition of substance to the cell culture was effected after the
`2" culture day during each medium change. Corresponding
`quantities of the test Substances (parent solutions) were
`added here so that a respective end concentration of 5x10
`mmol/l was achieved.
`Preparation of the Biological Material:
`0029. The preparation of the cartilage tissue and the
`chondrocytes isolated therefrom was effected analogously to
`the methods represented in Example 1.
`Test Conditions:
`0030 The isolated chondrocytes were cultivated in 24
`well plates in RPMI medium (Seromed, Berlin) with the
`addition of various antibiotics at 37° C. and 5% carbon
`dioxide in an incubator as a monolayer culture. The medium
`change was effected every two days. After the first medium
`change, the addition of lidocaine in the cell culture medium
`was effected in a concentration of 0.1 mmol/l. In addition, an
`untreated chondrocyte population respectively was run
`jointly as control. The culture duration was 6, 12 or 18 days.
`Implementation of the Test:
`0031) The measurement of the H thymidine incorpora
`tion as a measure of the DNA synthesis yield was effected
`at the respective end of the culture duration analogously to
`the method represented in Example 1. The results of the tests
`are represented in FIG. 2.
`0032 FIG. 2 shows the effect of lidocaine (0.1 mmol/l)
`on the H thymidine incorporation by human chondrocytes
`(N=6) cultivated in vitro. Substance addition on the second
`culture day. Each measurement value is the mean of 8
`individual measurements.
`
`Free
`lidocaine%
`
`pH 6.9
`pH 7.9
`
`S4
`34
`
`55
`81
`
`70
`79
`
`71. 87 89
`82 89 91
`
`93
`105
`
`Experimental Conditions:
`0034 3D CE system of the company Hewlett Packard
`with fused silica capillary 40.0 (48.5) cm with internal
`diameter 50 lum, temp.: 25° C., pressure injection: 50
`mbarxsec, voltage: +30 kV. UV detection: cathodeside at
`w=195 nm and 200 nm, injection time: 200 sec. Throughout
`the injection time, 7.5 cm of the capillary was filled with the
`sample in order to achieve optimal separation of the peaks.
`0035. By means of electrophoretic frontal analysis, it was
`able to be shown that an interaction took place between
`hyaluronic acid (hya) and lidocaine. If the same percentage
`proportions of hya and lidocaine or if percentage-wise less
`lidocaine than hya is present, the greatest proportion of
`lidocaine is bonded to hya. The mechanism of the interaction
`is based on incorporation of the lidocaine in the helix-like
`coil of the hya, there also at pH value of 7.9, when lidocaine
`is present semi-undissociated (pKS value=7.9 in the pres
`ence of hya). In addition, ionic bonds are involved in the
`interaction, since at pH 6.9 when lidocaine is present com
`pletely dissociated, less free lidocaine was able to be
`detected (except with a lidocaine concentration=0.025%)
`EXAMPLE 4
`Delayed Release of the Local Anaesthetic or Local
`Anaesthetics or the Derivatives of this Compound
`From Formulations Which Contain Hyaluronic
`Acid and/or the Physiologically Compatible Salts
`of Hyaluronic Acid and of Fragments of These
`Compounds, in the Example of Lidocaine
`
`0036)
`
`TABLE 2
`
`shows Subsequently the flux of lidocaine through a dialysis membrane
`with and without hyaluronic acid (hya) in the donor compartment.
`
`pH value
`
`3.1
`
`6.O
`
`6.5
`
`6.9
`
`7.7
`
`9.0
`
`O.32
`O.27
`
`O.3SS
`O.256
`
`O.315 O42 (0.35
`O.234
`0.27 0.23
`
`0.09
`O.04
`
`lidocaine
`Flux
`mg hi lidocaine +
`cm2.
`hya
`
`

`

`US 2006/O 122147 A1
`
`Jun. 8, 2006
`
`TABLE 2-continued
`
`shows Subsequently the flux of lidocaine through a dialysis membrane
`with and without hyaluronic acid (hya) in the donor compartment.
`
`pH value
`
`3.1
`
`6.0
`
`6.5
`
`6.9
`
`7.7
`
`9.0
`
`Difference %
`
`Flux lidocaine -
`Flux lidocaine + hya
`
`15.6
`
`27.8
`
`25.8
`
`35.7 34.3
`
`55.6
`
`Experimental Conditions:
`0037 diffusion cell with diffusion surface (A)=15.9 cm
`and a sodium cellulose Xanthate (nephrophan) dialysis mem
`brane, volume (V) of the donor (DK) and of the acceptor
`compartment (AK)=20 ml, diffusion time=4 h, temp.: 37°
`C., concentration of the hya in the donor compartment=
`0.25% and the initial concentration of the lidocaine in the
`donor compartment=0.05%.
`Calculation of the Flux:
`
`CAKVAK
`Fitty = At
`
`Thereby:
`0038 CA=Concentration of the lidocaine in the AK and
`0039) t=diffusion time.
`0040. With reference to the results which were obtained
`in the dialysis cell, it was shown that the flux of the lidocaine
`through this pore membrane was reduced considerably in the
`presence of the hya in the donor compartment. The most
`pronounced is the effect at pH=9.0, there lidocaine is present
`extensively undissociated. This confirms the results which
`were described in Example 3 that the mechanism of the
`interaction between lidocaine and hya is based on incorpo
`ration of the lidocaine in the helix-like coil of the hya. But
`also at pH values between 6.9 and 7.7, a strong reduction in
`the lidocaine flux can be observed. This confirms that also
`ionic bonds and the interaction between lidocaine and hya
`are involved.
`0041) If the pH value is moved into the acidic range, e.g.
`after pH=3.1, the hya there is present extensively undisso
`ciated, the lidocaine flux is reduced less strongly. This shows
`clearly that also ionic bonds are involved in the interaction.
`0042. It can be established in total that a strong delay
`effect with respect to the release of the lidocaine from the
`lidocaine-hya complex can be achieved. As a result, the
`effect of the lidocaine in biological systems (e.g. in the knee
`joint) can be considerably extended.
`
`1. Combination preparation consisting of at least one
`active agent A from the group hyaluronic acid, the salts and
`fragments thereof, an active agent B from the group of local
`anaesthetics and derivatives thereof and if necessary further
`additives for human and Veterinary medical therapy, pro
`phylaxis and/or metaphylaxis of degenerative or traumatic
`
`articular diseases and articular function disorders and also
`articular cartilage and cartilage bone defects.
`2. Combination preparation according to claim 1, wherein
`the active agents A and B are bonded to each other chemi
`cally or physically, and in that the active agent B can be
`released in a delayed manner.
`3. Combination preparation according to claim 1, wherein
`the active agent A is contained in a concentration between
`0.001 and 5% by weight, preferably between 0.2 and 2% by
`weight.
`4. Combination preparation according to claim 1, wherein
`the active agent B is contained in a concentration between
`0.001 and 20% by weight, preferably between 0.001 and 5%
`by weight.
`5. Combination preparation according to claim 1, wherein
`agents with radical interceptor properties, in particular toco
`pherol derivatives and/or ascorbic acid derivatives, are con
`tained as additive.
`6. Combination preparation according to claim 1, wherein
`agents of the hyaline cartilage tissue, in particular glu
`cosamine Sulphate derivatives and/or chondroitin Sulphate
`derivatives, are contained as additive.
`7. Combination preparation according to claim 1, wherein
`agents with a steroidal or corticoidal effect, in particular
`glucocorticoids, are contained as additive.
`8. Combination preparation according to claim 1, wherein
`non-steroidal antiphlogistics, in particular indometacin,
`diclofenac or salicylic acid derivatives, are contained as
`additive.
`9. Combination preparation according to claim 1, wherein
`analgesics, in particular oxicams, aniline or anthranilic acid
`derivatives, are contained as additive.
`10. Combination preparation according to claim 1,
`wherein agents with an inhibitory effect on prostaglandin
`synthesis, in particular lipoxygenase inhibitors, cyclo-oxy
`genase inhibitors and phospholipase A2 inhibitors, are con
`tained as additive.
`11. Combination preparation according to claim 1,
`wherein growth factors, in particular retinol or bone mor
`phogenetic proteins (BMPs), are contained as additive.
`12. Combination preparation according to claim 1,
`wherein vitamins, in particular vitamin A, C, B12 or biotin,
`are contained as additive.
`13. Combination preparation according to claim 1,
`wherein antioxidants, in particular flavonoids or glutathione,
`are contained as additive.
`14. Combination preparation according to claim 1,
`wherein agents with water-binding properties, in particular
`urea or arginine, are contained as additive.
`15. Use of hyaluronic acid, the salts and fragments thereof
`in combination with at least one active agent from the group
`of local anaesthetics and derivatives thereof for preparing a
`medicament for human and Veterinary medical therapy,
`prophylaxis and/or metaphylaxis of degenerative or trau
`matic articular diseases and articular function disorders.
`16. Use according to claim 15 for human and veterinary
`medical therapy, prophylaxis and/or metaphylaxis of articu
`lar cartilage and cartilage bone defects.
`17. Use according to claim 15 for human and veterinary
`medical therapy, prophylaxis and/or metaphylaxis of menis
`cus and intervertebral disc diseases.
`18. Use according to claim 15 in order to increase the
`proliferation of chondrocytes.
`
`

`

`US 2006/O 122147 A1
`
`Jun. 8, 2006
`
`5
`
`19. Use according to claim 15 in order to stabilise and/or
`regenerate articular structures, in particular of the articular
`cartilage and of the meniscus.
`20. Use according to claim 15 in order to increase
`articular mobility.
`
`21. Use according to claim 15 in order to inhibit inflam
`matory processes.
`22. Use according to claim 15 in order to treat skin or
`mucous membrane changes.
`9.
`
`k
`
`.
`
`.
`
`.
`
`.
`
`