Patent Application
20100029973
Bone is a specialized dynamic connective tissue that serves essential mechanical, protective, and metabolic functions. To perform these functions efficiently bone must undergo a process termed bone remodelling; continuous destruction (resorption) and rebuilding (renewal) at millions of microscopic sites. During adult life bone remodelling is crucial to eliminate structurally damaged or aged bone and replace it with new healthy bone. To maintain the proper bone mass resorption and formation must be kept in equilibrium.
Various endogenous factors, such as age and diseases, as well as exogenous influences such as injuries, drug treatments and exposures may affect this equilibrium by increasing bone resorption. The result can be observed as a decrease in the bone mineral density (BMD), which measures the amount of calcium in the bones that is closely correlated to total bone mass. Known conditions that lead to a potentially pathological decrease in BMD due to increased resorption are osteopenia, osteoporosis, secondary osteoporosis, posttransplantation bone disease, Paget's disease, idiopathic juvenile bone loss, multiple myeloma, osteosarcoma, parathyroidectomy, thermal injuries, hyperparathyroidism, periodontal diseases, bone disease caused by hemodyalysis and scurvy.
With age the equilibrium between bone mass resorption and formation becomes altered, generally in favour of resorption, resulting in a reduction of bone mass termed osteopenia. This age related loss of equilibrium is generally due to both a decline in bone formation and more resorption. It is caused by the decrease in estrogen production in post-menopausal women, and the decline with age in the production of androgen in men (which is enzymatically converted to estrogen, a hormone that regulates bone metabolism directly and indirectly). Eventually this may lead to deterioration of bone architecture, decreased resistance to stress, bone fragility and susceptibility to fractures. These symptoms are collectively referred to as osteoporosis, which is a major health problem, especially in Western society, where it has been estimated that up to 85% of women and somewhat fewer men older than 45 years of age are at risk of developing osteoporosis.
In addition to age related osteoporosis, there are secondary forms of osteoporosis not caused by age-related hormonal changes, but by increased bone resorption due to exposure to glucocorticoids, hyperthyroidisem, immobilizations, heparin and immunosuppressants. The resulting conditions are generally referred to as glucocorticoid induced osteoporosis, hyperthyroidisem induced osteoporosis, immobilization induced osteoporosis, heparin induced osteoporosis, and immunosuppressant induced osteoporosis.
There is a clear direct effect of glucocorticoids on bone resorption in human cell systems, which explain the observed increase in bone resorption seen in patients treated with glucocorticoids. (Sivagurunathan S et al, J Bone Miner Res. 2005 March; 20(3):390-8. Epub 2004 Dec. 20)
Hyperthyroidism leads to secondary osteoporosis due to an increase in bone resorption, and when it is treated, a prompt decrease is found in bone resorption markers. (Inaba M, Clin Calcium. 2001; 11(7):910-4)
During long term immobilizations, disuse induces dramatic bone loss resulting from greatly elevated resorption. (Li C Y et al, J Bone Miner Res. 2005 January; 20(1):117-24. Epub 2004 Oct. 18)
Osteoporosis is considered one of the potentially serious side effects of heparin therapy. The pathogenesis is poorly understood, but it has been suggested that heparin cause an increase in bone resorption. (Gennari C et al, Aging (Milano). June; 10(3):214-24)
Immunosuppressant therapy is known to lead to secondary osteoporosis through increased bone resorption. (Kirino S et al, J Bone Miner Metab. 2004; 22(6):554-60)
In association with transplantations large dosages of immunosuppressive drugs are generally used. This leads to bone resorption, which is referred to as posttransplantation bone disease. (Cunningham J, Transplantation. 2005 Mar. 27; 79(6):629-34)
Paget's disease is not as common or as costly as osteoporosis, but it affects 3% of the population over 40, and 10% of the population over 80 years of age. Aside from causing bone fractures it can lead to severe osteoarthritis and severe neurological disorders. Paget's disease is characterized by rapid bone turnover, caused by an increased bone resorption with irregular bone formation, resulting in the formation of woven bone of a tissue type formed initially in the embryo and during growth, which is normally practically absent from the adult skeleton.
Idiopathic juvenile bone loss (osteoporosis) is a rare form of bone demineralization that occurs during childhood. The mechanism of bone loss is unclear, but some studies have found increased bone resorption. (Bertelloni S et al, Calcif Tissue Int 1992 November; 51(5):400)
Multiple myeloma is a plasma cell malignancy characterized by the high capacity to induce osteolytic bone lesions that mainly result from an increased bone resorption related to the stimulation of osteoclast recruitment and activity. (Giuliani N et al, Acta Biomed Ateneo Parmense. 2004 December; 75(3):143-52)
Osteosarcoma leads to the destruction of bone tissue, and genes for osteoclast differentiation have been found to be differentially expressed in patients with osteosarcoma, thus linking deviations in bone resorption to osteosarcoma. (Michelle B. Mintz et al, Cancer Research. 2005 March:65, 1748-1754)
Routine hemodyalysis, as performed on patients with extensive loss of retinal function, leads to an increase in bone resorption, which causes bone diseases in this patient group. (Hamano T et al, Bone. 2005 Mar. 24; Epub ahead of print)
Hyperparathyroidism is associated with skeletal changes characterized by increased resorption and fibrous replacement of bone. Parathyroidectomy in the treatment of secondary hyperparathyroidism has also been shown to lead to an increase in bone resorption, which may cause bone diseases in this patient group. (Yajima A et al, Clin Calcium. 2003; 13(3):290-4)
In thermally injured children and adults there is a dramatic decrease in bone formation which may be accompanied with an increase in bone resorption. (Shea J E et al, J Musculoskelet Neuronal Interact. 2003 September; 3(3):214-22)
Periodontal diseases are chronic infectious diseases that result in loss of alveolar bone due to bone resorption triggered through immune responses, and results from inflammatory reactions directed against periodontopathic bacteria. (Ohmori Y, Clin Calcium. 2001; 11(3):302-8)
Scurvy is caused by vitamin C depletion and leads to structural collagen alterations, defective osteoid matrix formation and increased bone resorption. Trabecular and cortical osteoporosis is common in patients with scurvy.
Bone resorption is a specific function of osteoclasts, which are multinucleated, specialized bone cells formed by the fusion of mononuclear progenitors originating from the hemopoietic compartment, more precisely from the granulocyte-macrophage colony-forming unit (GM-CFU). The osteoclast is the principal cell type to resorb bone, and together with the bone-forming cells, the osteoblasts, dictate bone mass, bone shape and bone structure. The increased activity and/or numbers of osteoclasts, relative to the activity and or numbers of bone-forming osteoblasts, dictates the development of osteoporosis and other diseases of bone loss.
For diseases in which osteoclasts presumably resorb bone at abnormally high levels and osteoblasts form bone at normal levels, the most reasonable therapeutic target for restoring the equilibrium between bone resorption and formation would be decreasing the number of osteoclasts and/or decreasing the resorption activity of the osteoclasts. The treatments now available for osteoporosis are indeed intended to suppress bone resorption, but as their effects are variable and their side effects may be substantial, there is room for improvement.
Osteoblasts are derived from bone marrow stromal cells. Upon stimulation with glucocorticoids (GR) via the GR receptor, the bone marrow stromal cells differentiate into osteoblasts. Upon stimulation with peroxisome proliferator-activated receptor (PPAR) ligands via the PPARs, the bone marrow stromal cells differentiate into adiopocytes. The PPARs are members of the steroid nuclear superfamily of receptors that are known as modulators of the expression of genes involved in lipid metabolism and fat storage, and there are three known types of PPARs; alpha, gamma and delta. Based upon this, the “lipid hyphotehsis of osteoporosis” was formed; the notion that osteoporosis may be caused by a high fat diet upregulating adipocyte differentiation at the cost of osteoblast differentiation. This hypotheses was later substantiated by findings such as that oxidized lipids inhibit differentiation of preosteoblasts, and that minimally oxidized LDL act to inhibit osteogenic differentiation through activating PPAR alpha (Parhami F et al, J Bone Miner Res, 1999 December; 14(12):2067-78), and that PPAR gamma seems to be responsible for the decrease in osteoblasts and increase in marrow fat seen in the elderly due to its inhibition of osteoblastogenesis and stimulation of adipocyte differentiation (Ali A A et al, Endocrinology. 2005 March; 146(3):1226-35. Epub 2004 Dec. 9), and that haploinnsufficency of PPAR gamma promote osteogenesis through enhanced osteoblast formation (Pei L J Clin Invest 2004 March; 113(6):805-6). Thus, most of the prior art on osteoblasts and PPARs point to a role for PPARs in inhibiting osteoblast differentiation, with the exception of one publication which reported that activating PPAR alpha, delta and gamma (with specific ligands) induced alkaline phosphatase activity (which stimulate osteoblast maturation), matrix calcification and the expression of osteoblast genes (Jackson S M et al, FEBS Lett 2000 Apr. 7; 471(1):119-24). However, the same publication also reported that at relatively high concentrations of specific PPAR gamma ligands osteoblast maturation was inhibited. Thus the prior art overall agrees with the lipid hyphotesis of osteoporosis, indicating that stimulating the PPARs inhibit osteoblast differentiation, thus decreasing bone formation.
Osteoclasts are derived from the monocyte-macrophage family. Upon stimulation of the CFU-GM with macrophage colony stimulating factor (M-CSF) form promonocytes which are immature nonadherent progenitors of mononuclear phagocytes and osteoclasts. The promonocytes may proliferate and differentiate along the macrophage pathway, eventually forming a tissue macrophage, or may differentiate along the osteoclast pathway, depending on the cytokines to which they become exposed. Unlike for osteoblasts, here is no obvious connection between osteoclast formation or activity and PPARs, and very little research has been performed to elucidate if such a connection exists. A PPAR gamma activator was found to inhibit stimulators of osteoclastogenesis in one publication, indirectly suggesting that PPAR gamma may decrease bone resorption (Mbalaviele G, et al, J Biol. Chem. 2000 May 12; 275(19):14388-93), but a more recent publication found direct evidence of the opposite; a PPAR gamma agonist enhanced bone loss, increased fat marrow volume, and increased bone resorption parameters (Sottile V et al Calcif Tissue Int. 2004 October; 75(4):329-37. Epub 2004 Jul. 13). Thus, nothing is known of PPAR alpha and delta in regards to bone resorption, and the little information there is about PPAR gamma is conflicting, although the most direct evidence point towards a role for PPAR gamma in increasing resorption.
In conclusion, the information available regarding the overall effects of the PPARs on the equilibrium between bone mass resorption and formation is very sketchy, but points toward PPAR agonists having a negative effect on the BMD, both through decreased bone formation and increased bone resorption.
Thus, when the inventor found that the PPAR alpha, gamma and delta agonist tetradecylthioacetic acid (TTA) stimulated BMD increase and inhibited bone resorption, this was quite unexpected.
The use of compounds according to the invention is thus characterized by that the compound is represented by the general formula (I),
wherein R1, R2, and R3 represent
wherein A1, A2 and A3 are chosen independently and represent an oxygen atom, a sulphur atom or an N-R4 group in which R4 is a hydrogen atom or a linear or branched alkyl group, saturated or unsaturated, optionally substituted, containing from 1 to 5 carbon atoms;
wherein R1, R2, and R3 represent
TTA is a non β-oxidizable fatty acid analogue, belonging to a group of compounds comprising
a compound represented by the general formula (I),
wherein R1, R2, and R3 represent
wherein A1, A2 and A3 are chosen independently and represent an oxygen atom, a sulphur atom or an N-R4 group in which R4 is a hydrogen atom or a linear or branched alkyl group, saturated or unsaturated, optionally substituted, containing from 1 to 5 carbon atoms;
wherein R1, R2, and R3 represent
In a preferred embodiment of a compound according to the invention at least one of R1, R2 or R3 is an alkyl.
In a preferred embodiment of a compound according to the invention at least one of R1, R2 or R3 is an alkene.
In a preferred embodiment of a compound according to the invention at least one of R1, R2 or R3 is an alkyne.
In a preferred embodiment of a compound according to the invention at least one of R1, R2 or R3 is tetradecylthioacetic acid.
In a preferred embodiment of a compound according to the invention at least one of R1, R2 or R3 is tetradecylselenoacetic acid.
Preferred embodiments of the compounds according to the invention are tetradecylthioacetic acid (TTA), tetradecylselenoacetic acid and 3-Thia-15-heptadecyne.
In a preferred embodiment of a compound according to the invention n is 0 or 1.
In a preferred embodiment of a compound according to the invention said compound is a phospholipid, wherein said phospholipid is selected from the group comprising phosphatidyl serine, phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl inositol, phosphatidyl glycerol, diphosphatidyl glycerol.
In a preferred embodiment of a compound according to the invention said compound is a triacylglycerol.
In a preferred embodiment of a compound according to the invention said compound is a diacylglycerol.
In a preferred embodiment of a compound according to the invention said compound is a monoacylglycerol.
In a preferred embodiment of a compound according to formula (II) A1 and A3 both represent an oxygen atom, while A2 represent a sulphur atom or an N-R4 group in which R4 is a hydrogen atom or a linear or branched alkyl group, saturated or unsaturated, optionally substituted, containing from 1 to 5 carbon atoms.
The compounds according to the invention are analogues of naturally occurring compounds, and as such are recognized by the same systems which process the natural compounds, including the enzymes that β- and in some cases ω-oxidize natural long chain fatty acids. The analogues differ from their naturally occurring counterparts in that they cannot be completely oxidized in this manner.
The compounds according to the invention may be non β-oxidizable fatty acid analogues, as represented by the formula R″CCO—(CH2)2n+1—X—R′. However, said compounds may also be more complex structures derived from one or more of said non β-oxidizable fatty acid analogues, as represented by the general formulas (I) or (II). These compounds are analogues of naturally occurring mono-, di-, and triacylglycerols, or phospholipids including phosphatidyl serine, phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl inositol, phosphatidyl glycerol, and diphosphatidyl glycerol. Said compounds may also comprise a substitution in the glycerol backbone, as shown in formula (II). Said substitution of the oxygen(s) is achieved by replacing the oxygen(s) with sulphur or a nitrogen containing group. This may block hydrolysis before uptake by the intestines, thus increasing the bioavailability of the compounds.
The above complex structures derived from one or more of said non β-oxidizable fatty acid analogues have their effect because the fatty acid analogues they comprise are not capable of being fully β-oxidized. Said complex structures may have an effect as complete structures, and as naturally resulting degradation products comprising the fatty acid analogues. Because the compounds are not able to be fully β-oxidized, they will build up, and this triggers an increase in the β-oxidation of naturally occurring fatty acids. Many of the effects of the compounds according to the invention are due to this increase in β-oxidation.
During β-oxidation, a fatty acid is enzymatically oxidized cleaved between carbons 2 and 3 (when counting from the carboxylic end of the fatty acid), resulting in the removal of the two carbon atoms on either side of the oxidation site as acetic acid. This step is then repeated on the now two carbons shorter fatty acid, and repeated again until the fatty acid is fully oxidized. β-oxidation is the usual way in which the majority of fatty acids are catabolized in vivo. The β-oxidation blocking by the compounds according to the invention is achieved by the insertion of a non-oxidizable group in the X position in the formula of the present invention. Because the mechanism for β-oxidation is well known, X is defined as S, O, SO, SO2, CH2 or Se. Anyone skilled in the art would assume, without an inventive step, that these compounds would all block β-oxidation in the same manner.
In addition, the compounds may contain more than one block, i.e. in addition to X, R′ may optionally comprise one or more heterogroups selected from the group comprising an oxygen atom, a sulphur atom, a selenium atom, an oxygen atom, a CH2 group, a SO group and a SO2 group. As an example, one may insert two or three sulphurs as X to induce a change in the degradation of the fatty acid and thus a modulated effect. Multiple sulphur atoms would also modulate the polarity and stability somewhat. From a pharmacological viewpoint it is generally desirable to be able to present a spectrum of compounds rather than just one single compound to avoid or counteract problems with resistance.
In addition to the identity of X, its position is also an issue. The distance of X from the carboxylic end of the fatty acid is defined by how many CH2 groups are positioned between X and the carboxylic end of the fatty acid, which is defined by (CH2)2n+1, where n is an integer of 0 to 11. Thus there are an odd number of CH2 groups, that is; the position of X relative to the carboxyl group is such that X eventually blocks β-oxidation. The range of n is chosen to include all variations of the fatty acid analogue which has the desired biological effect. Since β-oxidation in theory can work on infinitely long molecules, n could be infinite, but in practice this is not so. The fatty acids which normally undergo β-oxidation are usually 14 to 24 carbon atoms long, and this length is therefore most ideal for undergoing enzymatic β-oxidation. The ranges of n and R′ are thus given so that the fatty acid analogues will cover this range. (Likewise, option ii) of formulas (I) and (II) and define R to have 1 to 25 carbon groups, and option i) of formula (II) define the alkyl group to contain from 1 to 23 carbon atoms, to be analogous to naturally occurring compounds.) The total number of carbon atoms in the fatty acid backbone is preferably between 8 and 30, most preferably between 12 and 26. This size range is also desirable for the uptake and transport through cell membranes of the fatty acid analogues of the present invention.
Although all fatty acid anagoges with an odd positioning of the β-oxidation blocker X away from the carboxylic end block β-oxidation, the extent of their biological effect may be variable. This is due to the difference in biological degradation time of the various compounds. The inventors have done experiments to show the effect of moving X further from the carboxylic fatty acid end. In these experiments the activity (in nmol/min/mg/protein) of mitochondrial β-oxidation in the liver of fatty acid analogues was measured with sulphur in the 3, 5 and 7 positions relative to the carboxyl end. The activities were 0.81 for sulphur in the 3rd position, 0.61 for sulphur in the 5th position, 0.58 for sulphur in the 7th position, and 0.47 for palmitic acid, the non β-oxidation blocking control. This shows, as expected, that β-oxidation is indeed blocked by fatty acid analogues with varying positioning of the block, and that the effect thereof is lessened the further away from the carboxylic end the block is positioned at, because it takes the β-oxidation longer to reach the block so more of the fatty acid analogue is degraded by then. However, as the decline is great for going from the 3rd to 5th position, but small going from the 5th to 7th position, it is reasonable to assume that this decline will continue to be less as one moves out the chain, and thus that it will be very far out indeed before no effect (compared to the control) is seen at all.
Thus, it is reasonable to include as compounds of the present invention, fatty acid analogues and other compounds represented by the general formulas (I) and (II), (which comprise said fatty acid analogue(s),) which block β-oxidation at different distances from the carboxylic end of the analogues, as the compounds of the present invention all do indeed block β-oxidation, even if the effect thereof can be modulated. This modulation will after all differ under wearying conditions; in different tissues, with wearying dosages, and by changing the fatty acid analogue so that it is not so easily broken down, as will be described next. Thus it is reasonable to include in the formula all distances of the β-oxidation blocker from the carboxylic end of the fatty acid analogue which are biologically relevant.
Although fatty acid analogues as described with a block in the X position cannot undergo β-oxidation, they may still undergo ω-oxidation. This is a much less common and slower biological process, which oxidizes the fatty acid not from the carboxylic end, but rather from the methyl/hydrophobic head group, here termed R′. In this pathway the carbon atom at the ω-end of the fatty acid is hydroxylated by a member of the cytochrome P450 enzyme family. This hydroxylated fatty acid is then converted into an aldehyde by an alcohol dehydrogenase, and subsequently this aldehyde is converted into a carboxyl group by an aldehyde dehydrogenase. As a consequence, the final product of the pathway is a dicarboxylic fatty acid, which can be degraded further by ω-oxidation from the ω-end.
ω-oxidation is believed to be the main pathway for degradation of the fatty acid analogues as described with a block in the X position. Experiments were thus performed where R′ was changed to block w-oxidation, by introducing a triple bond at the methyl end of the fatty acid analogue. This resulted in the fatty acid analogue 3-thia-15-heptadecyn, which when tested showed the expected result: a substantially increased degradation time in vivo. This is important for the use of the fatty acid analogues in pharmaceutical preparation, as it may potentiate the effects of the β-oxidizable fatty acid analogues by further slowing down their breakdown.
Again, as with the blocking of β-oxidation, it is routine to find other fatty acid analogues witch would block ω-oxidation in exactly the same manner, based upon knowledge of how ω-oxidation occurs. A double bond will for instance have the exact same effect as the triple bond did, and it is therefore included in the definition of the methyl/hydrophobic head group end of the molecule, here termed R′, that it may be saturated or unsaturated. A branch may also block oxidation, so R′ is defined as linear or branched.
In order to block ω-oxidation by the insertion of a substitute in R′, said R′ may be substituted in one or several positions with heterogroups selected from the group comprising an oxygen atom, a sulphur atom, a selenium atom, an oxygen atom, a CH2 group, a SO group and a SO2 group. R′ may also be substituted with one or more compounds selected from the group comprising fluoride, chloride, hydroxy, C1-C4 alkoxy, C1-C4 alkylthio, C2-C5 acyloxy or C1-C4 alkyl.
Thus the compounds according to the present invention are either fatty acids analogous to naturally occurring fatty acids, which are not capable of being β-oxidized, or naturally occurring lipids comprising said fatty acid analogues. In vivo, the fatty acid analogues show a strong preference for being incorporated into phospholipids. In some cases it is indeed advantageous to mimic nature and incorporate the fatty acid analogues in naturally occurring lipids, such as mono-, di-, and triglycerides and phospholipids. This changes the absorption of the compounds (when comparing fatty acids to fatty acids incorporated in larger lipid structures) and may increase the bioavailability or stability.
As an example, one could make a complex by including a fatty acid(s) which are not capable of being β-oxidized into a triacylglycerol. Such compounds are encompassed by formulas (I) and (II). If such a triacylglycerol was taken orally, for instance in an animal feed product, it would probably be transported like any triacylglycerol, from the small intestine in chylomicrons and from the liver in the blood in lipoproteins to be stored in the adipose tissue or used by muscles, heart or the liver, by hydrolyzes of the triacylglycerol into glycerol and 3 free fatty acids. The free fatty acids would at this point be the parent compound of the present invention, and not a complex anymore.
Yet other possible glycerophospholipid derivatives of the fatty acids of the present invention includes, but are not limited to, phosphatidyl cholines, phosphatidyl ethanolamines, phosphatidyl inositols, phosphatidyl serines and phosphatidyl glycerols.
Another esterification of fatty acids found in vivo which could be easily used to make a complex for a compound of the present invention would be to make the alcohol or polyalcohol corresponding to the fatty acid, for example one could make a sphingolipid derivative such as ceramide or sphingomyelin by making the corresponding amino alcohol. Like the glycerophospholipid complexes, such complexes would be very water insoluble and less hydrophilic. These kinds of hydrophobic complexes of the present invention would pass easier through biological membranes.
Other possibilities of polar complexes of the present invention may be, but are not limited to, lysophospholipids, phosphatidic acis, alkoxy compounds, glycerocarbohydrates, gangliosiedes, and cerebrosides.
Although there can be large structural differences between different compounds of the invention, the biological functions of all compounds are expected to be similar because they all block β-oxidation in the same manner. This inability of the lipid analogues to be β-oxidized (and in some cases, ω-oxidized,) causes the analogues to build up in the mitochondria, which triggers the β-oxidation of the in vivo naturally occurring fatty acids, which in turn leads to many of the biological effects of the fatty acid analogues of the present invention. (Berge R K et al. (2002) Curr Opin Lipidol 13(3):295-304)
The peroxisome proliferator-activated receptor (PPAR) family are pleiotropic regulators of cellular functions such as cellular proliferation, differentiation and lipid homeostasis (Ye J M et al. (2001) Diabetes 50:411-417). The PPAR family is comprised of three subtypes; PPARα, PPARβ, and PPARγ. A fatty acid analogue according to formula (I); TTA, has been used previously by the present inventors to test the various biological effects of the fatty acid analogues. TTA is a potent ligand of PPARα (Forman B M, Chen J, Evans R M (1997) Proc Natl Acad Sci 94:4312-4317; Gottlicher M et al. (1993) Biochem Pharmacol 46:2177-2184; Berge R K et al. (1999) Biochem J 343(1):191-197). As a PPARα activator TTA stimulate the catabolism of fatty acids by increasing their cellular uptake. Lowering the plasma triglyceride levels with TTA caused a shift in liver cellular metabolism, towards PPARα regulated fatty acid catabolism in mitochondria. (Grav H J et al. (2003) J Biol Chem 278(33):30525-33). TTA also activate PPARβ and PPARγ as well as PPARα (Raspe E et al. (1999) J Lipid Res 40:2099-2110). It is believed that the fatty acid analogues according to formula (I) will have the same effects as PPAR activators as exemplified by TTA, since their biological effects on fatty acid oxidation is the same, and they are believed to be PPAR activators due to their ability to block fatty acid oxidation.
In the current invention, TTAs effect on bone resorption in vivo and bone mineral density (BMD) in rats was investigated. In addition, the effects on these parameters of the PPARα agonists Wyeth 14,643 and fenofibrate, and the PPARγ agonist pioglitazone was also investigated.
The in vivo experiments on rats showed an increase in femoral BMD for rats treated with PPARα agonists Wyeth 14,643 and fenofibrate, of 7% and 5.7% respectively. As discussed, there was little prior art available to suggest what one should have expected for PPARα agonists. The PPARγ agonist pioglitazone induced a decrease in femoral BMD of −3%, which is as expected from the prior art. TTA is both a PPARα and PPARγ agonist, so one would most probably assume that the result of it being a PPARα agonist was unclear, while the result of it being a PPARγ agonist would be a reduction in BMD, and the overall effect of TTA was thus not very predictable, but probably leaning towards a reduction in BMD. TTA did however show the unexpected result of increasing the femoral BMD by 5%.
The in vitro experiments on preosteoblasts showed similar results for resorption. PPARα agonists Wyeth 14,643 and fenofibrate, as well as PPARα and PPARγ agonist TTA, stimulated OPG and interlukin 6 (IL-6) release from preosteoblasts, while the PPARγ agonist pioglitazone did the opposite. OPG is an important inhibitor of bone resorption, while IL-6 is an important stimulator of bone resorption. Thus, the overall effect is that of bone resorption inhibition by Wyeth 14,643, fenofibrate and TTA, and bone resorption stimulation by pioglitazone.
Seeing as an increase in BMD may be caused by an increase in bone formation or a decrease in bone resorption, the increase in BMD for Wyeth 14,643, fenofibrate and TTA may be due to the decrease in bone resorption, and the decrease in BMD for pioglitazone may be due to the increase in bone resorption. However, the changes in bone resorption may also only be partially responsible for the changes in BMD; there may also be a concurrent change in bone formation.
The BMD is closely correlated to total bone mass, and loss thereof is associated with a host of diseases. Loss of bone mass due to an increase in resorption compared to that seen in healthy individuals is also associated with diseases, as discussed earlier. By increasing the BMD and decreasing the resorption TTA and the other fatty acid analogues according to the present invention are therefore potentially beneficial in treating or preventing these diseases associated with a loss in BMD and/or increase in bone resorption.
As a pharmaceutical medicament the compounds of the present invention may be administered directly to the animal by any suitable technique, including parenterally, intranasally, orally, or by absorption through the skin. They can be administered locally or systemically. The specific route of administration of each agent will depend, e.g., on the medical history of the recipient human or animal.
Examples of parenteral administration include subcutaneous, intramuscular, intravenous, intra-arterial, and intra-peritoneal administration
As a general proposition, the total pharmaceutically effective amount of each of the non β-oxidizable fatty acid analogues administered parenterally per dose will preferably be in the range of about 1 mg/kg/day to 200 mg/kg/day of patient body weight for humans, although, as noted above, this will be subject to a great deal of therapeutic discretion. A dose of 5-50 mg/kg/day is most preferable.
If given continuously, the compounds of the present invention are each typically administered by 1-4 injections per day or by continuous subcutaneous infusions, for example, using a mini-pump. An intravenous bag solution may also be employed.
For parenteral administration, in one embodiment, the compounds of the present invention are formulated generally by mixing each at the desired degree of purity, in a unit dosage injectable form (solution, suspension, or emulsion), with a pharmaceutically acceptable carrier, i.e., one that is non-toxic to recipients at the dosages and concentrations employed and is compatible with other ingredients of the formulation.
Generally, the formulations are prepared by contacting the compounds of the present invention each uniformly and intimately with liquid carriers or finely divided solid carriers or both. Then, if necessary, the product is shaped into the desired formulation. Preferably the carrier is a parenteral carrier, more preferably a solution that is isotonic with the blood of the recipient. Examples of such carrier vehicles include water, saline, Ringer's solution, and dextrose solution. Non-aqueous vehicles such as fixed oils and ethyl oleate are also useful herein, as well as liposomes.
The carrier may suitably contain minor amounts of additives such as substances that enhance isotonicity and chemical stability. Such materials are non-toxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, succinate, acetic acid, and other organic acids or their salts; antioxidants such as ascorbic acid; immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids, such as glycine, glutamic acid, aspartic acid, or arginine; monosaccharides, disaccharides, and other carbohydrates including cellulose or its derivatives, glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; counterions such as sodium; and/or non-ionic surfactants such as polysorbates, poloxamers, or PEG.
For oral pharmacological compositions such carrier material as, for example, water, gelatine, gums, lactose, starches, magnesium-stearate, talc, oils, polyalkene glycol, petroleum jelly and the like may be used. Such pharmaceutical preparation may be in unit dosage form and may additionally contain other therapeutically valuable substances or conventional pharmaceutical adjuvants such as preservatives, stabilising agents, emulsifiers, buffers and the like. The pharmaceutical preparations may be in conventional liquid forms such as tablets, capsules, dragees, ampoules and the like, in conventional dosage forms, such as dry ampoules, and as suppositories and the like.
In addition the compounds of the present invention, i.e. the fatty acid analogue according to formula (I), may be used in nutritional preparations, as defined earlier, in which case the dosage of the fatty acid analogue preferable is as described pharmaceuticals or less. In animal fodder, the amount of non β-oxidizable fatty acid analogue can be up to 10 times that in products for human consumption, that is, up to 2 g/kg/day of animal body weight.
In many cases it is obvious whether the use of fatty acid analogues in accordance with the present invention is appropriate or not, and this choice can be made by the skilled medical professional without undue experimentation. If a person is for instance about to undergo aggressive immunosuppressant therapy, it would be routine for the skilled medical professional to determine if bone loss could be prevented by the use of compounds according to formula (I). However, there may be instances where it is necessary to first determine if bone metabolism impairment has occurred, or to determine the bone density, or to determine the boner resorption.
There are several techniques currently used by medical professionals to measure bone metabolism impairment. One can perform a bone cell density measurement, or an ultrasound densitometer test, or measure bone metabolism parameters directly. Bone metabolism parameters comprise markers present in the blood such as BAP (a marker of osteogenesis), DPD (a bone resorption marker), NTX, corrected Ca, and the SOS value (an index of bone metabolism impairment).
Several methods are available to measure bone density, but currently the most widely used technique is DEXA (Dual Energy Xray Absorptiometry). This is the method used to determine efficacy in the recent large clinical trials, and to characterize fracture risk in large epidemiological studies. Older methods such as single photon absorptiometry do not predict hip fractures as well as DEXA. Three companies manufacture these densitometers: Hologic, Norland, and Lunar.
Newer techniques such as ultrasound appear to offer a more cost-effective method of screening bone mass. Ultrasound measurements are usually performed at the calcaneous and it is not possible to measure sites of osteoporotic fracture such as the hip or spine. Adding an ultrasound measurement to a DEXA does not improve the prediction of fractures.
Quantitative computed tomography (QCT) is another method for measuring bone density. QCT of the spine must be done following strict protocols in laboratories that do these tests frequently; in community settings the reproducibility is poor. The QCT measurements decrease more rapidly with aging, so the “T scores” in older individuals will be much lower than DEXA measurements.
Several techniques can measure bone density at the hand, radius or ankle. These include single energy absorptiometry, metacarpal width or density from hand xrays. Magnetic resonance imaging is a new method of measuring bone density.
Measuring the bone density gives a good indication of how healthy the bones are. However, if it is not as one would desire, a bone density measurement in itself does not provide information regarding on whether there this is due to too much resorption or too little new bone formation, and to find out one must measure bone resorption and/or formation directly.
Bone resorption markers are released in circulation as byproducts of osteoclast action on bone and include cross-links for collagen type I. Bone formation markers are released during osteoblast synthesis of new bone protein matrix, which can be assessed by measuring circulating osteocalcin.
Bone resorption can be measured by the Crosslaps method. Crosslaps is degradation products of C-terminal telopeptides of type I collagen in human serum and plasma, which can be measured in urine with specific RIA and ELISAs. Since more than 90% of the organic matrix of bone consists of type 1 collagen, measuring its degradation products in urine makes crosslaps a potential specific marker of bone resorption. A pronounced and significant increase (47-142%) in Crosslaps at menopause indicate that it is a very sensitive marker of metabolic bone changes taking place at menopause. The correlation between Crosslaps and the rate of loss measured by single photon absorptiometry has been shown to be much higher than with Pyridinoline and Deoxypyridinoline. Crosslaps has also been used to predict the rate of bone loss considering a bone loss of more than 3% per year. Crosslapse has a specificity of 80% and a sensitivity of more than 70%, it can thus be used as a potentially useful screening parameter in the risk assessment of diseases like postmenopausal osteoporosis and Paget's disease. Crosslaps values decreases substantially in response to replacement therapies thus suggesting its usefulness in monitoring treatment efficacy. Crosslaps have been cleared by the FDA, and is currently used for research only, but is expected to be available for general testing soon. It is developed by Nordic bioscience diagnostics.
Another method for measuring bone resorption which is currently used clinically is measuring pyridinoline and deoxypyridinoline (Pyr and D-Pyr). Pyridinoline cross links are released into the circulation during bone resorption and are excreted as both free and bound to C and N terminal ends of type 1 collagen. These are measured by different methods, the most sensitive being HPLC. More recently sensitive chemiluminescence based assays have also been made available.
Fasting urinary calcium measured in a morning sample and corrected for creatinine excretion is the cheapest marker of bone resorption. It is useful to detect marked changes in bone resorption but lacks sensitivity especially in conditions characterized by subtle alteration of bone turnover such as osteoporosis. Hydroxyproline is found mainly in collagen and represents about 13% of the amino acid content of the molecule. Hydroxyproline is highly metabolized before being excreted and is poorly correlated with bone resorption as assessed by calcium kinetic and bone histomorphometry.
The preparation of non β-oxidizable fatty acid analogues according to the present invention is disclosed in detail in the applicant's earlier Norwegian patent applications no. 20005461, 20005462, 20005463 and 20024114. These documents also describe toxicity studies of TTA. Preparation of mono-, di-, and triglycerides and nitrogen comprising lipids according to the invention is disclosed in detail in U.S. patent application Ser. No. 10/484,350. The preparation of phospholipids including serine, ethanolamine, choline, glycerol, and inositol according to the invention is disclosed in detail in the applicant's earlier Norwegian patent application no. 20045562.
The effect of the PPAR agonists on release of osteoprotegerin (OPG), RANKL and IL6 from the preosteoblast cell line MC3T3-E1 was investigated. Wyeth 14,643 was found to inhibit the differentiation of human monocytes into osteoclasts in a dose-dependent manner, and also inhibited the proliferation of the preosteoclast cell line RAW264.7. Wyeth 14643, fenofibrate and TTA were also shown to stimulate OPG release and inhibit IL6 release from preosteoblasts, while no effects on RANKL could be detected. Pioglitazone, on the other hand, tended to inhibit the OPG release and stimulated the release of IL6.
Fifty female Fischer rats were divided into 5 groups and were given methocel (control group), Wyeth 14,643, fenofibrate, tetradecylthioacetic acid (TTA) and pioglitazone (50 mg/kg body weight) by intragastric gavage for 4 months. Body weight was registered throughout the study. BMD was measured by double x-ray absorptiometry (DXA). Histomorhometry of femur was performed, and mechanical strength in the femoral shaft and collum femoris was measured.
There was no difference in body weight between control rats and the treatment groups at the end of the study. After 4 months femoral BMD was significantly higher in rats treated with Wyeth 14,643 (7%), fenofibrate (5.7%) and TTA (5%) than in control rats. In rats treated with pioglitazone, femoral BMD tended to be lower than in control rats (−3%).
| Number | Date | Country | Kind |
|---|---|---|---|
| 20053519 | Jul 2005 | NO | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/NO2006/000262 | 7/10/2006 | WO | 00 | 5/6/2009 |
