Patent Grant
8183210
1. Field of the Invention
The present invention relates to reconstituted surfactants which are useful for the treatment or prophylaxis of RDS and other respiratory disorders. The present invention also relates to methods of preparing such a reconstituted surfactant and pharmaceutical compositions which contain such a reconstituted surfactant. The present invention further relates to methods for the treatment or prophylaxis of RDS and other respiratory disorders by administering such a reconstituted surfactant.
2. Discussion of the Background
The human lung is composed of a large number of small air sacs, called alveoli, in which gases are exchanged between the blood and the air spaces of the lungs. In healthy individuals, this exchange is mediated by the presence of a protein-containing surfactant complex that prevents the lungs from collapsing at the end of expiration.
Lung surfactant complex is composed primarily of lipid and contains minor amounts of various proteins. An absence of adequate levels of this complex results in malfunction of the lung. This syndrome is called Respiratory Distress Syndrome (RDS) and it commonly affects preterm infants.
Said syndrome is effectively treated with modified natural surfactant preparations extracted from animal lungs. Commercially available modified surfactant preparations are, for example, Curosurf, derived from porcine lung, Infasurf, extracted form calf lung lavage, and Survanta, a chemically modified natural bovine lung extract.
The main constituents of these surfactant preparations are phospholipids, such as 1,2-dipalmitoyl-sn-glycero-3-phosphocholine commonly known as dipalmitoyl-phosphatidylcholine (DPPC), phosphatidylglycerol (PG), and surfactant hydrophobic proteins B and C (SP-B and SP-C).
Due to the drawbacks of the surfactant preparations from animal tissues, such as the complication of the production and sterilization processes and possible induction of immune reactions, synthetic surfactants mimicking the composition of the modified natural surfactants have been developed. Said synthetic surfactants are known as reconstituted surfactants. However the development of clinically active reconstituted surfactants has turned out to be complicated since the native hydrophobic proteins are too big to synthesize, structurally complex, and unstable in pure form.
In order to replace said native hydrophobic proteins, some synthetic polypeptides partially corresponding to their sequences and analogs thereof have been proposed in the prior art and are disclosed in for example WO 89/06657, WO 92/22315, WO 98/49191, WO 95/32992, U.S. Pat. No. 6,660,833, EP 413,957, and WO 91/18015.
WO 00/47623 disclosed synthetic polypeptides which are analogs of the native protein SP-C wherein: i) cysteine residues in position 5 and 6 have been replaced by Ser residues; ii) the Val residues of the ‘center region’ of SP-C have been substituted with other neutral and hydrophobic residues selected from the group consisting of Leu, Ile, and norleucine (nL); iii) some of the neutral amino acids present in the ‘center region’ of SP-C have been replaced with bulky or polar residues selected from the group consisting of Lys, Trp, Phe, Tyr, and Ornithine. Said artificial polypeptides are characterized by the fact that they are capable of folding like the native protein SP-C and, hence, interacting properly with the surfactant lipids and, thus, do not give rise to self-oligomerization.
WO 00/76535 generically discloses pulmonary surfactant preparations comprising at least one modification of the SP-B in combination with at least one modification of the SP-C protein.
In A. J. Waring, et al., (abstract presented at the Paediatric Academy Society Annual meeting held in San Francisco on Apr. 29-May 2, 2006) a study was undertaken to examine the activity of a synthetic surfactant constituted of the SP-C-mimic, SP-Cff, that is a synthetic 34-residue SP-C with phenylalanine instead of cysteine in positions 4 and 5, and the SP-B-mimic, Mini-B.
However, according to the available literature, in animal studies, the treatment with reconstituted surfactants gives rise to poor lung gas volumes and grade of alveolar patency at the end of expiration, and a ventilation is required with a positive end expiratory pressure (PEEP) in order to achieve an in vivo activity comparable to that achieved with modified natural surfactants (see, J. Johansson, et al., J. Appl. Physiol., 2003, 95, 2055-2063; and A. J. Davis, et al., Am. J. Respir. Crit. Care Med., 1998; 157, 553-559). Thus, the available reconstituted surfactant preparations are indeed not capable of forming a stable phospholipidic film in the alveoli at the end of expiration.
Moreover, all of the aforementioned documents are silent about the problem of the alveolar patency at the end of expiration and the effect of the disclosed preparations thereof.
Thus, there remains a need for a reconstituted surfactant with improved properties in terms of lung compliance.
Accordingly, it is one object of the present invention to provide novel reconstituted surfactants which are useful for the treatment or prophylaxis of RDS and other respiratory disorders.
It is another object of the present invention to provide novel reconstituted surfactants which are capable of guaranteeing alveolar stability, and hence maintaining alveolar patency at the end of expiration without requiring ventilation with PEEP.
It is another object of the present invention to provide novel methods of preparing such a reconstituted surfactant.
It is another object of the present invention to provide novel pharmaceutical compositions which contain such a reconstituted surfactant.
It is another object of the present invention to provide novel methods of treating and/or preventing RDS and other respiratory disorders by administering such a reconstituted surfactant.
These and other objects, which will become apparent during the following detailed description, have been achieved by the inventors' discovery that particular analogues of the native SP-C protein, and preferably the polypeptides of WO 00/47623 can advantageously be combined with particular analogues of the native protein SP-B in order to provide a reconstituted surfactant preparation with improved properties in terms of lung compliance, and in particular the ability to efficaciously maintain alveolar patency at the end of expiration without requiring ventilation with PEEP.
Thus, in a first embodiment, the present invention provides a reconstituted surfactant comprising a lipid carrier, and a combination of a particular polypeptide analog of the native surfactant protein SP-C with a particular polypeptide analog of the native surfactant protein SP-B.
In particular the invention provides a reconstituted surfactant comprising :
wherein:
each X is independently an amino acid residue selected from the group consisting of I, L, and nL;
each B is independently an amino acid residue selected from the group consisting of K, R, H, W, F, Y, and Orn;
each S is independently optionally substituted with an acyl group containing 12-22 carbon atoms, preferably 16 carbon atoms, linked to the side chain via an ester bond;
Ω is an amino acid selected from the group consisting of M or M oxidized on the sulfur atom, I, L, and nL;
a is an integer having a value of 1 to 8;
b is an integer having a value of 1 to 19;
each c is independently an integer having a value of 3 to 8;
e, f, g, and p are each independently integers having a value of 0 or 1;
each n is independently an integer having a value of 0 to 3; and
with the condition that the XaBXb(BXc)n is a sequence having a maximum of 22 amino acid residues; and
wherein:
Δ is an amino acid residue independently selected from the group consisting of W and L;
each Ω is independently an amino acid residue selected from the group consisting of M, I, L, and nL;
Φ is an amino acid residue independently selected from the group consisting of R and T, preferably R; and
each f is independently an integer having a value of 0 or 1,
wherein F, G, I, P, S, V, H, L, K, R, A, C, Q, W, Y, M, and T are standard one-letter symbols for amino acid residues, nL represents an L-nor-leucine residue, and Orn represents an L-onithine residue.
The present invention also provides pharmaceutically acceptable salts of said polypeptides and their blocked N- and/or C-terminus derivatives, e.g via acetylation and amidation.
The present invention also provides pharmaceutical compositions which contain such a reconstituted surfactant.
The present invention also provides methods for improving the alveolar patency at the end of expiration by administering such a reconstituted surfactant.
The present invention also provides methods for the prophylaxis and/or treatment of respiratory distress syndrome (RDS) and other respiratory disorders, by administering to a patient in need of such treatment a therapeutically effective amount of a reconstituted surfactant comprising a lipid carrier, a polypeptide of general formula (I), and a polypeptide of general formula (II).
In particular it has been found that, in a model of RDS wherein the immature newborn were treated with exogenous surfactant preparations without applying PEEP, a combination of said particular analogues of the proteins SP-C and SP-B acts on the lung gas volumes which is an index of the alveolar patency at the end of expiration.
A more complete appreciation of the invention and many of the attendant advantages thereof will be readily obtained as the same become better understood by reference to the following detailed description when considered in connection with the accompanying drawings, wherein:
The respiratory function after in vivo treatment with the exogenous surfactant preparations is carried out by measuring two parameters:
i) the tidal volume which is an index of the lung compliance and
ii) the lung gas volume which is an index of the alveolar air expansion or patency at the end of expiration, and, hence, of the capability of forming a stable phospholipidic film in the alveoli at the end of expiration.
In the present application, the term “reconstituted surfactant” means a lipid carrier to which polypeptide analogues of the surfactant proteins, made through recombinant technology or synthetic methods, have been added.
The term “lipid carrier” means a mixture of phosholipids and optionally further lipid components, for example neutral lipids such as triacylglycerols, free fatty acids, and/or cholesterol.
The term “polypeptide analogues of the native surfactant protein SP-C”, includes polypeptides having an amino acid sequence in which, compared to the native proteins, one or more amino acids are missing or have been replaced by other amino acids, so long as the polypeptides, in a mixture with a lipid carrier, show pulmonary surfactant activity.
The term “polypeptide analogues of the native surfactant protein SP-B”, includes peptides having an amino acid sequence in which, compared to the native proteins, one or more amino acids are missing so long as the polypeptides, in a mixture with a lipid carrier, show pulmonary surfactant activity.
The term “mini-B” means a 34-residue polypeptide based on the N-terminal residues 8-25 and C-terminal residues 63-78 of the native SP-B protein whose structure was first generically disclosed in a presentation taken from the California NanoSystems Institute website. Its full sequence has been subsequently disclosed in the RCSB Protein Data Bank. In A. J. Waring, et al., J. Peptide Res., 2005, 66, 364-374, more information about its structure and activity has been reported.
The term “variants” means polypeptide analogues of the Mini-B peptide having an amino acid sequence in which one or more amino acids have been replaced by other amino acids, so long as the peptides, in a mixture with a lipid carrier, retain the activity of Mini-B.
The amino acid sequences are shown according to the single-letter or three-letter code with the amino acid which carries the free amino group at the left end (amino terminus) and the amino acid which carries the free carboxyl group at the right end (carboxy terminus).
The term “synergistic” means that the effectiveness of the two polypeptides is more than would be expected by summing their respective individual effectiveness in a given assay.
All the amino acid residues identified herein are in the natural L-configuration and the sequences identified herein are reported according to standard abbreviations for amino acid residues as shown in the following Table of Correspondence.
Thus, the present invention provides reconstituted surfactants comprising a lipid carrier and a combination of particular polypeptide analogues of the native surfactant protein SP-C with particular polypeptide analogues of the native surfactant protein SP-B, including the Mini-B peptide and variants thereof.
It has been found that, in a model of RDS wherein the immature newborn were treated with exogenous surfactant preparations without applying PEEP, a combination of a polypeptide of general formula (I), with a polypeptide of general formula (II) positively acts on the lung compliance. In particular, the combination of the two polypeptides was found to act in a synergistic way on the lung gas volumes which is an index of the alveolar patency at the end of expiration. Said result demonstrates that the reconstituted surfactant of the present invention is able to stabilise the phospholipid film in the alveoli at the end of expiration in a better way than a reconstituted surfactant comprising only an analog of the protein SP-C or an analog of the protein SP-B.
Moreover, the claimed reconstituted surfactant preparation also improved the respiratory function as expressed by the tidal volumes to an extent comparable or even slightly better than that achieved after administration of a modified natural surfactant.
Advantageously the analog of the native protein SP-C is a polypeptide of at least 20 amino acid residues and no more than 40 amino acid residues, having the sequence represented by the general formula (I) SEQ ID NO: 10:
wherein:
each X is independently an amino acid residue selected from the group consisting of I, L, and nL;
each B is independently an amino acid residue selected from the group consisting of K, R, H, W, F, Y, and Orn;
each S is independently optionally substituted with an acyl group containing 12-22 carbon atoms, preferably 16 carbon atoms, linked to the side chain via an ester bond;
Ω is an amino acid residue selected from the group consisting of M or its oxidized on the sulfur atom, I, L, and nL;
a is an integer having a value of 1 to 8;
b is an integer having a value of 1 to 19;
each c is independently an integer having a value of 3 to 8;
e, f, g and p are each independently integers having a value of 0 or 1;
n is an integer having a value comprised from 0 to 3; and
with the condition that the XaBXb(BXc)n is a sequence having a maximum of 22 amino acids, preferably comprised between 10 and 22 amino acid residues.
Preferably the polypeptide of general formula (I) is constituted of at least 30 and not more than 35 amino acid residues, more preferably not more than 33 amino acid residues.
In particular embodiments, the polypeptides of general formula (I) are constituted of 30 or 33 or 35 amino acid residues.
Preferably the polypeptide analog of the SP-C protein is represented by the general formula (Ia) (SEQ ID NO: 13) in which e and n are 0, and g is 1:
wherein
X, B, and Ω are as defined above;
a is from 1 to 8, preferably from 1 to 3; more preferably 1;
b is from 1 to 19, preferably from 5 to 15; more preferably 14; and
each f and p are independently 0 or 1.
More preferably the polypeptide analog of the SP-C protein is represented by the general formula (Ib) (SEQ ID NO: 14) in which f is 1:
wherein:
X, B, Ω, a, and b are as defined above; and
each p is independently 0 or 1.
Even more preferably, the polypeptide analog of the SP-C protein is represented by the general formula (Ic) (SEQ ID NO: 15):
wherein:
Ω is as defined above
each p is independently 0 or 1.
Examples of polypeptides of formula (Ic) are reported below:
The polypeptide (Id) has been also referred to in the prior art as SP-C33.
Most preferably the SP-C analog is a polypeptide selected from the group of polypeptides having the formulae (Ie), (If), (Ig), and (Ih).
In a preferred embodiment of the invention, the polypeptide of general formula (I) is the polypeptide (If) hereinafter referred to as SP-C33(Leu)31.
Advantageously, the analog of the native protein SP-B consists of two portions corresponding to the N-terminal residues 8-25 and to the 63-78 C-terminal part of the native SP-B protein (referred to as Mini-B peptide) or a variant thereof.
More advantageously, the analog of the native protein SP-B is a polypeptide represented by the following general formula (II) (SEQ ID NO: 11):
wherein:
Δ is an amino acid residue selected form the group consisting of W and L;
each Ω is independently an amino acid residue independently selected from the group consisting of M, I, L, and nL;
Φ is an amino acid residue selected from the group consisting of R and T, preferably R; and
each f is independently an integer having a value of 0 or 1.
In a particular embodiment of the invention, the SP-B analog is a polypeptide represented by the formula (IIa) (SEQ ID NO: 6):
In another embodiment of the invention, the SP-B analog is selected from those having the following formulae:
In a preferred embodiment, each of the polypeptides (Ia), (IIb), (IIc), and (IId) may be in the form of a disulfide-linked molecule, wherein the intramolecular disulfide linkage is between the two Cys residues in position 1 and 33 and/or between the two Cys residues in position 4 and 27.
The disulfide linked polypeptide (IIa) has been referred to in the art to as oxidized Mini-B (ox Mini-B).
The polypeptide (IIc) is hereinafter referred to as Mini-B(Leu) and its disulfide linked form as ox Mini-B(Leu).
The polypeptides of general formulae (I) and (II) may be prepared according to synthetic methods or recombinant techniques well known to the person skilled in the art. An excellent summary of the many techniques available may be found in J. M. Steward and J. D. Young, “Solid Phase Peptide Synthesis,” W.H. Freeman Co., San Francisco, 1969; and J. Meienhofer, “Hormonal Proteins and Peptides,” Vol. 2, p. 46, Academic Press (New York), 1983, for solid phase peptide synthesis; and in E. Schroder and K. Kubke, “The Peptides,” Vol. 1, Academic Press (New York), 1965, for classical solution synthesis.
The polypeptides of the present invention can also be prepared using the solid-phase synthetic technique initially described by Merrifield, in J. Am. Chem. Soc., 85: 2149-2154 (1963). Other polypeptide synthesis techniques may be found, for example, in M. Bodanszky et al., “Peptide Synthesis”,: John Wiley & Sons, 2d Ed., (1976) as well as in other reference works known to those skilled in the art.
Appropriate protective groups for use in such syntheses will be found in the above texts as well as in J. F. W. McOmie, “Protective Groups in Organic Chemistry,” Plenum Press, New York, N.Y. (1973).
For example, the polypeptides of general formula (I) may be prepared according to the method disclosed in WO 00/47623, while the polypeptides of general formula (II) may be prepared according to the method reported in A. J. Waring, et al., J. Peptide Res., 2005, 66, 364-374.
The present invention also provides the pharmaceutically acceptable salts of the polypeptides of general formulae (I) and (II) and their blocked N- and/or C-terminus derivatives, e.g., via acetylation and amidation. Pharmaceutically acceptable salts include for example, salts of hydrochloric acid, acetic acid, and trifluoroacetic acid.
The reconstituted surfactant of the present invention may be prepared by mixing a solution or a suspension of the polypeptides of general formula (I) and (II) and lipids and by subsequently drying the mixture, otherwise they may be prepared by lyophilisation or spray-drying.
Preferably, the polypeptides of general formula (I) and the polypeptides of general formula (II) are present in the reconstituted surfactants of the invention in a fixed amount and quantitative ratio as a fixed combination.
The proportion of the polypeptides of general formulae (I) and (II) relative to the reconstituted surfactant can vary. Advantageously each polypeptide may be present in an amount of 0.5 to 10 wt. % based on the total weight of the surfactant (w/w), preferably 1 to 5 wt. %, most preferably 1 to 3 wt. %.
In a preferred embodiment, the reconstituted surfactant comprises 1 to 3% by weight of the polypeptide (If), and 1 to 3% by weight of the polypeptide (IIa), preferably in the oxidized form.
In another preferred embodiment, the reconstituted surfactant comprises 1 to 3% by weight of the polypeptide (If), and 1 to 3% by weight of the polypeptide (IIc), preferably in the oxidized form.
Advantageously, the lipid carrier comprises the phospholipids that are contained in natural pulmonary surfactant preparations, for example phosphatidylcholines (PC) such as dipalmitoylphosphatidylcholine (DPPC) and palmitoyloleoylphosphatidylcholine (POPC), and phosphatidylglycerols (PG), such as palmitoyloleoylphosphatidylglycerol (POPG) and dipalmitoylposphatidylglycerol (DPPG).
Other phospholipids which can be advantageously used are phosphatidylinositols (PI), phosphatidylethanolamines (PE), phosphatidylserines, and sphingomyelins (SM).
In a particular embodiment, the lipid carrier may comprise further components, for example neutral lipids such as triacylglycerols, free fatty acids, and/or cholesterol.
Advantageously the reconstituted surfactant according to the present invention comprises 90 to 99% by weight of a lipid carrier, preferably 92 to 98% by weight, more preferably 94 to 96% by weight, and 1 to 10% by weight of the sum of both peptides, preferably 2 to 8% by weight, more preferably 4 to 6% weight, based on the total weight of the lipid and the polypeptides of formulae (I) and (II).
In one of the embodiments of the present invention, the reconstituted surfactant comprises about 96% by weight of a lipid carrier, about 2% by weight of a polypeptide of general formula (I) and about 2% by weight of a polypeptide of general formula (II).
In a particular embodiment, the lipid carried only consists of phospholipids more preferably of a mixture of: (1) DPPC; and (2) a palmitoyloleylphospholipid selected from POPG or a mixture thereof with POPC in weight ratios ranging from 95:5 to 50:50, preferably from 80:20 to 60:40.
The weight ratio between DPPC and POPG ranges preferably from 75:25 to 65:35, and is more preferably about 68:31. In the case of DPPC:POPG:POPC mixtures, the phospholipids are preferably used in weight ratios of about 60:20:20 or about 68:15:16.
In another embodiment, the lipid carrier may consist of DPPC, DPPG, and cholesterol.
In a preferred embodiment of the invention, the reconstituted surfactant comprises from 1 to 5% by weight of one the polypeptides of general formula (Ia), from 1 to 5% by weight of one of the polypeptides of general formula (II) and a mixture of DPPC and POPG in a weight ratio of about 68:31.
The administration of the reconstituted surfactant of the invention is carried out in a manner known to the person skilled in the art, preferably by intratracheal installation (infusion or bolus) or by nebulisation.
The present invention also provides pharmaceutical compositions comprising the reconstituted surfactant of the invention. Said compositions are advantageously administered in the form of a solution, dispersion, suspension, or dry powder. Preferably said compositions comprise the reconstituted surfactant dissolved or suspended in a suitable solvent or resuspension medium.
Preferably said pharmaceutical compositions are supplied as suspension in a buffered physiological saline aqueous solution in single-use glass vials. Advantageously, the reconstituted surfactant concentration (expressed as phospholipid content) is in the range of from about 2 to about 160 mg of surfactant per ml, preferably between 10 and 100 mg/ml, more preferably between 20 and 80 mg/ml.
Said compositions may further comprise electrolytes, such as calcium, magnesium and/or sodium salts, preferably calcium chloride.
The pharmaceutical compositions according to the present invention are suitable for the prophylaxis and/or treatment of respiratory distress syndrome (RDS) in prematurely born babies or other diseases related to a surfactant-deficiency or dysfunction including RDS in adults (ARDS), meconium aspiration syndrome (MAS), and bronchopulmonary dysplasia (BPD).
The present reconstituted surfactants may also be useful for the prophylaxis and/or treatment of other respiratory disorders such as pneumonia, bronchitis, COPD (chronic obstructive pulmonary disease), asthma, and cystic fibrosis as well as for the treatment of serous otitis media (glue ear).
Other features of the invention will become apparent in the course of the following descriptions of exemplary embodiments which are given for illustration of the invention and are not intended to be limiting thereof.
The polypeptide SP-C33(Leu)31 was prepared by standard SPPS (Solid Phase Peptide Synthesis) methods based on Fmoc chemistry and consecutive TFA-cleavages. In total 186.0 g of crude SP-C33(Leu)31 were obtained. The polypeptide was purified by subjecting the material to counter current distribution (CCD) using H2O/n-BuOH/AcOEt/AcOH 4:1:4:1 as a biphasic solvent system. This purification yielded 78.9 g of SP-C33(Leu)31 with a purity of >60%. The final purification was carried out by preparative HPLC using PLRP-S as the stationary phase in a steel column of 50×300 mm applying a linear gradient of 25% B to 100% B in 75 minutes. The mobile phase consisted of buffer A=0.1% TFA in ACN/H2O 1:4 and buffer B=0.1% TFA in IPA. The purified polypeptide was dissolved in 90% AcOH and passed through a column packed with Dowex ion exchange resin (acetate form) to furnish, after lyophilisation, 5.8 g (=5.4%) of the final product as the acetate.
The polypeptide SP-C33 was prepared in an analogous manner.
The polypeptide ox-Mini-B(Leu) was prepared by standard SPPS (Solid Phase Peptide Synthesis) methods based on Fmoc chemistry and consecutive TFA-cleavages. The crude polypeptide was purified with preparative HPLC using a TFA system and isolated by lyophilisation. Air oxidation of the purified peptide yielded the monocyclic sequence with the disulfide bond between Cys1 and Cys33. The monocyclic peptide was purified with preparative HPLC using a TFA system and isolated by lyophilisation. The second disulfide bridge between Cys4 and Cys27 was formed using iodine. After oxidation, the product was purified with preparative HPLC using a TFA system and isolated by lyophilisation. 1.12 g (=1.7%) of the final compound were isolated with a purity of >89%.
The polypeptide ox Mini-B was prepared in an analogous manner.
The surfactant preparations were assayed in premature newborn rabbits, obtained by hysterectomy at the gestational age of 27 days. The experiments were performed without applying a positive end expiratory pressure (PEEP). As the SP-C analog, the polypeptide referred to as SP-C33 was used which was prepared according to Example 1. As the analog of the protein SP-B, oxidized Mini-B (ox Mini-B) was used which was prepared according to Example 2.
The animals were treated at birth with reconstituted surfactant preparations containing, as lipid carrier, the phospholipid mixture DPPC:POPG in the ratio 68:31 w/w. The phospholipids were mixed with 2 or 4% w/w SPC-33, 2% w/w ox Mini-B, or 2% w/w SPC-33 plus 2% w/w ox Mini-B. Animals receiving the same dose of Curosurf® served as positive controls, and non-treated littermates served as negative controls. All surfactant preparations were administered at a concentration of 80 mg/ml and at a standard dose of 2.5 ml/kg.
The immature newborn rabbits were ventilated in parallel with a standardized sequence of peak insufflation pressures. To open up the lungs, pressure was first set at 35 cmH2O for 1 minute. After this recruitment manoeuvre, pressure was lowered to 25 cmH2O for 15 minutes and further on to 20 and 15 cm H2O. Finally, the pressure was raised again to 25 cmH2O for 5 minutes, after which the lungs were ventilated for additional 5 minutes with nitrogen and then excised for gas volume measurements. Both lung gas volumes and tidal volumes expressed as ml/kg were measured, and the results, given as median values, are reported in
From
As shown in
Where a numerical limit or range is stated herein, the endpoints are included. Also, all values and subranges within a numerical limit or range are specifically included as if explicitly written out.
Obviously, numerous modifications and variations of the present invention are possible in light of the above teachings. It is therefore to be understood that, within the scope of the appended claims, the invention may be practiced otherwise than as specifically described herein.
All patents and other references mentioned above are incorporated in full herein by this reference, the same as if set forth at length.
| Number | Date | Country | Kind |
|---|---|---|---|
| 06021521 | Oct 2006 | EP | regional |
This application is a continuation of International Patent Application No. PCT/IB2007/002841, filed on Sep. 28, 2007, and claims priority to European Patent Application No. 06021521.7, filed on Oct. 13, 2006, both of which are incorporated herein by reference in their entireties.
| Number | Name | Date | Kind |
|---|---|---|---|
| 20080242589 | Curstedt et al. | Oct 2008 | A1 |
| 20090075892 | Johansson et al. | Mar 2009 | A1 |
| 20090088379 | Johansson et al. | Apr 2009 | A1 |
| Number | Date | Country |
|---|---|---|
| 1 481 665 | Dec 2004 | EP |
| 00047623 | Aug 2000 | WO |
| WO 0047623 | Aug 2000 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 20100004173 A1 | Jan 2010 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/IB2007/002841 | Sep 2007 | US |
| Child | 12422581 | US |
