WO2000058733A2

WO2000058733A2 - Assay for testing the efficacity of a therapy

Info

Publication number: WO2000058733A2
Application number: PCT/GB2000/001200
Authority: WO
Inventors: Christine Booth
Original assignee: Isis Innovation Limited
Priority date: 1999-03-29
Filing date: 2000-03-29
Publication date: 2000-10-05
Also published as: WO2000058733A3; AU3447900A; GB9907234D0

Abstract

A method for assaying for a therapy of potential use in the treatment of diseases which have a component of muscle spasticity or spasm, which comprises administration of the therapy and monitoring for altered muscle morphology. Additionally, use of such therapy, which acts on the altered muscle component in treatment of such diseases.

Description

Assay for therapy

The present invention relates to an assay for a therapy which can be used to treat diseases having a component of muscle spasticity and/or muscle spasm, such as cerebral palsy.

Cerebral palsy is a disorder wherein a primary brain lesion results in the over stimulation of skeletal muscle and the development of spasticity. The disease is a neurological disorder and previous drug treatments have focused upon treating this aspect of the disease. Although the neurological component of cerebral palsy is manifested in over stimulation of muscle, the only effect known on the muscle per se is a change in the ratio of muscle fibre types seen in affected individuals when compared to normal individuals. However, this change in muscle fibre type cannot be used to assess the severity of cerebral palsy, or to follow disease progression, a limitation which also applies to other diseases having a component of muscle spasticity.

There is still a need in the art for a method which allows an assessment of the severity and progression of diseases having a component of muscle spasticity, such as cerebral palsy, and which allows analysis of the efficacy of treatment of such diseases.

The present invention sets out to overcome the problems with current diagnostic and analytical methods for such diseases.

In a first aspect the present invention relates to a method for testing the efficacy of a therapy of potential use in the treatment of diseases which have a component of muscle spasticity or spasm, which comprises administration of the therapy and monitoring for altered muscle morphology.

Suitably, the therapy is a drug, a treatment regime such as physiotherapy, or any other mechanical, electrical or physical treatment, for example. We have now. surprisingly, discovered that there are clear differences between normal and spastic muscle morphology in individuals with cerebral palsy, particularly in the levels and distribution of connective tissue components, such as collagen. It is likely that these changes contribute strongly to the bone abnormalities and contractures that characterise the disease, as increased levels of these components place an increased strain on joints and bones.

These findings indicates that there is a fibrotic aspect to the disease of cerebral palsy, and provides a new target for therapy within the affected muscle. Furthermore, the differences in collagen levels between normal and spastic muscle, and the other tissue and cellular changes provide a specific diagnostic test for severity of spasticity within cerebral palsy. This finding allows drugs or other therapies for the treatment of cerebral palsy to be tested for effectiveness by examining, for example, connective tissue components such as collagen I levels, fibronectin, total collagen levels or the other tissue specific changes such as proteoglycans.

As such, the present invention preferably relates to a method for assaying for a drug or other therapy of potential use in the treatment of cerebral palsy, which comprises administration of the drug or therapy and monitoring for altered muscle morphology.

Muscle spasticity generally occurs in conditions where there is disruption of the upper motor neuron inhibitory pathways, including cerebral palsy, stroke, brain trauma or spinal cord injury. In addition to the findings with cerebral palsy, we have determined that muscle from individuals with Perthes disease also shows similar abnormal levels of connective tissue. The cuase of Perthes disease is at present unknown, but it is a form of osteoarthritis with significant muscle involvement including atrophy and spasm. Similarities in the findings with respect to cerebral palsy and Perthes disease indicate that the present invention can be broadly applied to all diseases having a muscle spasticity or muscle spasm component.

Altered muscle morphology herein generally refers to those morphological features which are known to be different between normal muscle and spastic muscle, excluding simply a change in muscle fibre type. As such, these features are diagnostic for cerebral palsy. In the present invention, suitable morphological features have been determined by studies carried out on both human tissue samples and samples derived from an animal model of spasticity, the Spa mouse (Chai C. K. 'Hereditary spasticity in mice', 1961, J. Hereditary, 52, 241-243).

Specifically in humans, in normal muscle the myofibers are tightly packed and arranged in fascicles surrounded by a perimysium. However, in spastic muscles of cerebral palsy individuals this organisation is lost. The fibres are randomly arranged and sparsely distributed within a large amount of connective tissue. Furthermore, the distribution of collagen is different in the two types of muscle. In spastic muscle there is a significant increase in collagen I expression, and, therefore, an overall increase in collagen in the tissue. Moreover, in spastic muscle the endomysium is thicker and more intensely stained, as well as there being an increase in interstitial collagen I.

The increase in total collagen is likewise seen in Perthes disease. Moreover, the observed changes in human spastic muscle morphology are also found in the Spa mouse model, which indicates that this model provides a useful means for studying of drugs or other therapies for the treatment of cerebral palsy. Specifically, levels of collagens I, III, IN, N and VI are all altered in spastic muscle of 26 week old Spa mice, when compared to age-matched controls. Even in 8 week old mice, there are significant changes in levels of collagen I, III and IN, for example in spastic muscle. Given the above findings, we particularly prefer that altered morphology is studied with respect to changes in connective tissue, particularly changes in collagen.

In summary, both human and mouse data indicates that in spastic tissue the fibres are randomly arranged and sparsely distributed within a large amount of connective tissue, when compared to normal muscle tissue from age-matched controls. In spastic muscle there is a significant increase in collagen I expression, and an overall increase in total collagen in the tissue (collagen I is a standard term in the art, and relates to a specific subset of collagen fibre). Levels of the other collagen fibres V and NI also generally increase. Moreover, in spastic muscle the endomysium (basal lamina), which comprises type IN collagen, is thicker and more intensely stained, as well as there being an increase in interstitial collagen I. Any of the above morphological changes characterise the altered muscle morphology that define cerebral palsy, and may serve as markers to assess the effect of a drug or other therapy on cerebral palsy, or other diseases as previously mentioned.

The process of monitoring altered muscle morphology generally refers to any process by which a test sample is analysed to detect the specific markers of cerebral palsy as discussed above. The effect of any drug or other therapy which is being used to treat cerebral palsy may be assessed in this way. We prefer that the monitoring is carried out on an ex vivo sample, such as a tissue slice, where possible.

We prefer that altered muscle morphology is assessed by analysis of muscle fibrosis, preferably via following the changes in total collagen levels and/or collagen distribution, or the specific levels and distribution of collagen I, III, IV, N or NI, all of which are altered in individuals with diseases of muscle spasticity such as cerebral palsy. Collagens I, III and IN are particularly preferred as markers.

We prefer that collagen levels, such as collagen I levels, are identified by the use of antibodies specific for each collagen, although other suitable methods for the detection of collagen are well known in the art. In addition, the total level of collagen may be assessed by determining the amount of the collagen-specific modified amino acid hydroxyproline using, for example, high pressure liquid chromatography or a plate based assay using a suitable dye. The absolute level of collagen 1 may not need to be determined, but the ratio of different collagens to one another, for example, collagen I to collagen III may also be used diagnostically.

The present invention has identified a new muscular component in both cerebral palsy and Perthes disease, specifically that there is fibrosis in the skeletal muscle of children with the disorder. This discovery extends the scope of possible treatments for such diseases to include drugs that act upon muscle, and provides an assay for such drugs.

A drug suitable for use in the treatment of diseases with a component of muscle spasticity, such as cerebral palsy, may be any suitable compound or composition which may be administered to the human or animal body. As such, the term includes pharmaceutical compositions which include an active agent in combination with a carrier, for example. Suitable compositions and formulations will be readily apparent to the person skilled in the art.

Suitable drugs for use in the present invention are found in six main groups, although this list is not exclusive. Other suitable drugs will be readily apparent to the person skilled in the art.

i anti-inflammatory drugs such as pirfenidone; ii antifibrogenic drugs such as oxaproline-containing peptides which specifically inactivate prolyl-4 hydrolase and collagen synthesis; iii colchicine, which is known to enhance matrix metal loprotein activity and inhibits collagen secretion; iv neuronal activity blockers such as botulinum toxin, which has been shown to maintain muscle length; v anti-mitotic drugs which prevent or reduce fibroblast proliferation; and vi matrix metalloproteinases and tissue inhibitors of these enzymes.

Thus, the present invention also relates to use of an anti -inflammatory drug, antifibrogenic drug, colchicine. anti-mitotic drug, matrix metalloproteinases or matrix metalloproteinase tissue inhibitor in the preparation of a medicament for the treatment of a disease having a component of muscle spasticity. The use of an antifibrogenic drug, colchicine, anti-mitotic drug, matrix metalloproteinases or matrix metalloproteinase tissue inhibitor is especially preferred. We prefer that the disease to be treated is caused by a disruption of the upper motor neuron inhibitory pathways, such as cerebral palsy, stroke, brain trauma or spinal cord injury, or muscle spasm due to other causes such as Perthes disease.

In addition, the invention further provides an assay for the treatment of cerebral palsy with drugs that are aimed at the neurological component of the disorder. Diseases such as stroke, brain trauma or spinal cord injury also have disruption of the upper motor neuron inhibitory pathways, i.e. a neurological component, and are also suitable for analysis in this way. As such, the present invention relates to an assay for drugs that treat both the neurological and muscular aspects of these diseases, either separately or in combination. The present invention also extends to drugs identified by an assay as described above, which are effective in normalising the level of collagen I. III. IN N or VI. for example. Suitable drugs include both drugs aimed at the muscular component of the disease and drugs targeted at the neurological component; the present invention covers drugs in both classes.

In addition to the assay of drug efficacy, as described above, the effect of physiotherapy or other mechanical therapies which are currently used to treat diseases having a component of muscle spasticity or spasm, may be assessed by the present invention. The invention provides an alternative assay for all possible treatments of such disorders.

Administration of the drug may be in any suitable preparation and by any suitable deliver ' means. For example, the drug may be an oral preparation, nasal spray, or may be delivered by injection or inhalation. Other suitable deliver}' systems are well known to the person skilled in the art.

The discovery of muscle fibrosis in patients with cerebral palsy, for example, allows current cerebral palsy models to be tested in new ways. For example, the spastic (Spa) mouse has been currently used as a model for human cerebral palsy, as it shows similar characteristics of spasticity. However, drug tests using this mouse have currently been limited to drugs for the treatment of the neurological aspect of the disease. Botulinum toxin has been tested on the Spa mouse, for example, and is currently being tested therapeutically in cerebral palsy children. The present findings allow the Spa mouse model to be used in the testing of drugs and other therapies which act on the muscular component of the disease, in order to see if these compounds alleviate the effects of the disease. The findings may also be valuable in the treatment of related disorders. The suitability of such animal models is shown by the correspondence of human and Spa mouse fibrosis levels, as discussed above.

Therefore, the present invention also relates to the use of the Spa mouse, and other animal models of cerebral palsy, in an assay for a therapy of potential use in the treatment of cerebral palsy wherein the therapy is directed to the muscular component of the disease. In particular, we prefer that levels of collagen and total collagen are used to assess the effect of the therapy on the animal model.

The identification of a muscular component of cerebral palsy allows the identification of new markers for the disease, such as collagen I, collagen III, IV, V or VI as previously discussed. This finding also allows the identification of new biochemical markers. A marker is generally a substance whose level or activity alters directly with the parameter of interest, specifically here the severity of spasticity. Therefore, the present invention also extends to biochemical markers found in muscle which correlate with the severity of cerebral palsy or other diseases with a component of muscle spasticity or spasm and can also be used to determine the effectiveness of a trial drug or therapy.

In addition to tissue samples, the knowledge that cerebral palsy has a fibrotic element allows cerebral palsy and similar diseases to be analysed by a blood-based assay or urine-based assay. It is known that fibrotic diseases result in a greater concentration of collagen breakdown products in the blood and urine. As such, the effect of drugs or other treatments on cerebral palsy, for example, may be followed by an assessment of levels of these breakdown products. Effective drugs or treatments such as physiotherapy serve to reduce the level of collagen breakdown products in the blood or urine.

It will be appreciated that the present invention also provides kits for use in the assay method of the invention, and kits suitable for detecting collagen breakdown products in blood or urine. We prefer that a kit contains at least detection means for one of the collagens I, III, IV, V or VI, preferably an antibody for collagen I III, IV. V or VI, or detection means for the breakdown product of collagen, which is suitably also an antibody or other selective staining reagent.

The present invention will now be further illustrated by the following Examples and Figures, which serve to illustrate the invention but are not limiting upon it; wherein

Figure la shows a section of normal human muscle tissue stained for the extracellular matrix protein collagen I; Figures lb and lc show sections of tissue from two separate human individuals with cerebral palsy for the extracellular matrix protein collagen I:

Figure 2 shows a section of human muscle tissue in an individual with mild spasticity, stained for collagen I;

Figure 3 shows a section of human muscle tissue in two individuals with moderately severe spasticity, stained for collagen I;

Figures 4a-e shows collagen I, III, IV, V and VI expression in normal and spastic mouse muscle;

Figures 5a and 5b shows levels of collagen I and IV in normal and spastic mouse muscle;

Figure 6 shows hydroxyproline determination in normal and spastic mouse muscle;

Figure 7 shows relative immunoflorescence intensities of collagen I, III, IV, V and VI in normal and spastic mouse muscle;

Figure 8 shows relative immunoflorescence intensities of collagen I and IV in mouse muscle; and

Figures 9a and 9b shows total collagen in spastic and non spastic human skeletal muscle

Example 1 - Determination of levels of collagen I in normal muscle and spastic muscle

1.1 Method and Materials

Skeletal muscle biopsies were obtained, with full ethical approval and permission, from five non- cerebral palsy (CP) children and five children with cerebral palsy undergoing orthopaedic surgery as part of their treatment. Patients with any other neuromuscular disorder were excluded from this study. Biopsies were taken from the middle third of the quadriceps muscle and were either snap frozen or fixed in 3% paraformaldehyde/HEPES buffer. Fixed tissue was pre -treated with sheep hyaluronidase (4800 units/ml in 0.025M NaCl, 00.5 Na acetate buffer, pH 5). The sections were coded for immunohistochemistry and scored blind. The collagens were localized in 5μm thick transverse cryostat sections using specific antibodies to collagen I (the generous gift of Dr V. C. Duance, Cardiff) by incubating in the primary antibody for 1 hour followed by an incubation in a FITC-labelled secondary antibody for a further hour and mounted in Vectashield mountant. Sections were washed extensively in Tris buffered saline (0.05M Tris, 0.15M sodium chloride pH 7.4) between incubations. Controls were included where sections were treated in exactly the same way, but the primary antibody was omitted.

1.2 Results

Results of the analysis are given in Figures la - lc, Figure 2 and Figure 3.

In summary, in normal muscle, the myofibres are tightly packed and arranged in fascicles surrounded by a perimysium. This organization is lost in spastic muscles, where the fibres appear randomly arranged and sparsely distributed within a large amount of connective tissue. Severity of spasticity is correlated with levels of collagen I.

Specifically, in the non CP control (Figure 1 a) collagen I expression (indicated using arrows) is found in the endomysium around individual muscle fibres, interstitially (int) between fascicles and associated with blood vessels (bv). The muscle fibres are closely packed.

In CP muscle (Figures lb and lc). there is increased interstitial space between the fibres. Collagen I expression is higher in the endomysium, which appears brighter and thicker around each muscle fibre. There is also an increase in collagen I between the fibres in the interstitial space and the fascicles are smaller, separated by increased collagen I positive fibrotic tissue. The scale bar corresponds to 100 μm. In Figure 2, Collagen 1 expression in a mildly affected individual is shown. There is little difference in the distribution or intensity of the staining when compared to normal, non CP muscle. The scale bar corresponds to 50 μm.

Figure 3 shows muscle biopsies from children with moderately severe spasticity, stained for collagen I. Panels "a" and "b" represent biopsy samples from one individual, while panels "c" and "d" are samples from a second individual. Collagen I expression is increased in the endomysium, which appears thicker and more intensely stained. There is also an increase in the collagen I in the interstitial spaces between the fibres. The scale bar corresponds to 50 μm.

Example 2 Identification of biochemical markers for cerebral palsy

2.1 Materials and methods

2.1.1 Human Tissue samples

Muscle biopsies can be obtained from cerebral palsy (CP) children, aged 2 to 16 years, with cerebral palsy of any neurological pattern and distribution. The age. sex. neurological involvement and ambulatory status of the patient are recorded. The range of movement of all joints in the lower limbs is recorded as an expression of the severity of their involvement. The severity of their spasticity is also assessed using the modified Ashworth Scale. Children who have had any intramuscular drug treatment for their condition are excluded. A similar approach is taken for children with other orthopaedic conditions and their muscle biopsies are used as controls. Children with any neuromuscular disorder, metabolic disease or any other muscle abnormality are excluded from this group. We prefer that a sample size of at least 20 individuals with spasticity and 20 non-spastic controls are used.

2.1.2 Spa mouse

The accumulation of connective tissue in the spastic (Spa) mouse can also be assessed. This autosomal recessive mutant is the closest animal model of human cerebral palsy available, showing many similar characteristics of spasticity including increased muscle tone and the development of contractures. It has been used by Cosgrove and Graham (Dev. Med. Child Neurology 1994. vol 36, pp379 - 385) to study the effects of intramuscular administration of botulinum toxin into spastic muscle which has been shown to prevent loss of muscle length, and this has led to its therapeutic use in CP children. The major advantage of this model is that data is obtainable across the full range of severity of spasticity and it provides essential knowledge of the condition at its early stages when therapeutic intervention is most beneficial. Such data is unobtainable from children for ethical reasons as mildly affected children do not normally undergo surgery. A scale of the severity of spasticity can be developed for this model with the same parameters as in children to enable direct comparison of results. See Examples 4-8

2.1.3 Detection of fibrosis

Stereological image analysis can be performed "blind" on coded sections to determine the percentage area in transverse muscle sections occupied by fibrotic tissue. Given the importance of fibroblasts in other fibrotic diseases and evidence for their proliferation in preliminary work, the number of fibroblasts per unit area of muscle can also be counted. An accurate index of connective tissue is the amount of total collagen, synthesised predominantly by fibroblasts. This is assayed biochemically by determining the amount of the collagen-specific modified amino acid, hvdroxyproline. and a number of different methods are available. Our preferred methods are High Pressure Liquid Chromatography (HPLC) given its accuracy with small samples, or a plate-based assay for hydroxyproline using a suitable dye.

2.2 Identification of biochemical markers

Biochemical markers are substances whose amounts or activity alter directly with the parameter of interest, in this case, clinical measures of the severity of spasticity. Potential markers can be identified which enable the progression of fibrosis to be monitored. This is a potent clinical tool as no such objective measure is currently available and is of particular importance in monitoring the outcome of drug and other therapies. Strong candidates markers are the connective tissue proteins which accumulate in fibrosis. It is possible to identify which connective tissue proteins are expressed in spastic muscle and how their expression changes with time, comparing with age matched controls. Biopsies from children are used, and a full cross-sectional study can be carried out with the Spa animal model. The connective tissue proteins expressed in normal muscle have been well characterised and include collagen types I. Ill, IV. V and IX. fibronectin and laminin. Their expression in spastic muscle has not previously been examined. Using specific antibodies, these proteins can be localised by indirect immunohistochemistry, and the cells responsible for their synthesis identified by in situ hybridization, by localization of their mRNAs as previously described. In parallel, an aliquot of tissue from the same individual can be assayed quantitatively by biochemical methods, including standard immunoprecipitation and ELISA techniques for specific proteins, and PCR and northern blotting to determine the amount of mRNA expressed, using actin and glyceraldehyde-3 -phosphate dehydrogenase as internal standards and normalising results to wet weight, total protein and total mRNA. As well as absolute concentrations of individual proteins, attention is also paid to ratios, particularly of the collagen types: it is well known from studies of other fibrotic diseases that the collagen type Ltype III ratio is an important index of fibrosis and changes in collagen fibril organization associated with fibrosis may influence the muscle's mechanical properties.

Example 3 Effect of drugs on fibrosis

A small scale pilot study with factorial design can be carried out to determine if fibrosis- perturbing drugs can be identified which prevent the development of fibrosis and improve the degree of the severity of spasticity with the animal model of CP. Drugs, or appropriate controls, are administered according to published regimes which are standard in the art to batches of 5 homozygous mutant Spa mice of the same age at two clearly identifiable stages of spasticity i.e. before the development of contractures or once contractures have developed, and to homozygous normal age-matched sibling mice.

The effects of six broad groups of drugs can be investigated: i. Anti-inflammatory drugs e.g. pirfenidone which has recently been shown to reduce lung fibrosis in mice: ii. Antifibrogenic drugs such as oxaproline-containing peptides which specifically inactivate prolyl-4 hydroxylase and collagen synthesis; ii. Colchicine, which is known to enhance matrix metalloproteinase activity and inhibits collagen secretions; iv. Blockers of neuronal activity such as botulinum toxin which has been shown to maintain muscle length; v Anti-mitotic drugs which prevent or reduce fibroblast proliferation; and vi Matrix metalloproteinases and tissue inhibitors of Matrix metalloproteinases

In addition, the effect of physiotherapy can also be assessed on the level and distribution of connective tissue.

Their effect on the severity of the spasticity and on fibrosis are assayed as in section 2.1.2 at different times after the commencement of administration (range 7-28 days) with particular attention being paid to the effect of the drug on potential objective biochemical markers of severity.

Example 4 Collagen expression in normal and spastic (Spa) mouse lower leg muscles

Lower leg muscles were dissected and snap frozen on isopentane cooled on dry ice following humane killing of normal and spastic age matched mice at 8 weeks and 26 weeks of age. Collagen types I, III, IV, V and VI were localised by indirect immunohistochemistry in 5um cryostat sections which had been pretreated with hyaluronidase (4800U/rnl in acetate buffer, pH5.0 for 2 hours at room temperature) and post fixed in 10% formaldehyde for 10 minutes. Representative fields are shown from normal and spastic soleus muscles of 8 and 26 weeks as follows:

Figure 4a: Collagen I, x 40 Figure 4b: Collagen III, x 25 Figure 4c: Collagen IV, x 40 Figure 4d: Collagen V, x 25 Figure 4e: Collagen VI, x 25 Results

Morphologicall} . in the spastic muscle there is evidence of muscle fibre hypertrophλ . an increase in interstitial connective tissue (fibres are spaced further apart) and thickening of the endomysium. all of which is seen in spastic muscle of children with cerebral palsy.

The interstitial connective tissue between the myofibres contains collagens I. Ill, V and VI and appears elevated in the 26 week old spastic mouse muscle where the interstitial spaces are particularly pronounced compared to its age-matched control.

The endomysium which surrounds individual myofibres contains the basal lamina collagen, type IV, as expected. Collagen IV levels are clearly elevated in the spastic muscles at both ages examined and clearly demonstrates thickening of the endomysium.

Example 5 Thresholding of collagen I and IV immunohistochemistry

Indirect immunofluorescence was carried out on tissue sections of normal and spastic 8 week and 26 week old mice using antibodies specific for collagens I and IV. Images were collected of the soleus muscle, avoiding areas containing major blood vessels which might bias the results, of sections stained at the same time and under identical conditions at x 40 magnification using the programme "Scion Image" integrating 20 fields on-chip. No pixels were saturated. The intensity of fluorescence was measured using Scion Image using the technique of thresholding and counting total pixels up to a pre-set threshold level of 200. This level was arbitrarily set so as to visualise all immunofluorescence in the lowest intensity image of the set.

Figure 5a: Collagen I Figure 5b: Collagen IV

Results Figures 5a and 5b are matched pairs to figures 4a and 4c respectively and illustrate thresholding for collagens I and IV. They clearly show elevated levels of collagen I and IV in spastic compared to normal muscle at both 8 and 26 weeks.

Example 6 Hydroxyproline determination in normal and spastic mouse lower leg muscles

Total collagen was assayed in normal and spastic 8 and 26 week old mice by determining hydroxyproline based on the method of Bergman and Loxley (Analytical Chemistry, Vol 35, ppl961 - 1965. 1963). Mouse lower leg muscles (soleus and gastrocnemius) were snap frozen, freeze dried then hydrolysed in 6M hydrochloric acid at 110°C for 24 hours. Hydroxyproline was determined and calculated as μg per mg dry weight of tissue.

Results (Figure 6)

A t-test was carried out on the data, assuming unequal variances, as follows:

normal 8w spastic 8w normal 26w spastic

26w

Mean 6.196 5.922857143 5.004 7.18

Variance 2.12383 1.097157143 3.00578 1.72585

Observations 5 7 5 5

Hypothesized Mean Difference 0 0 df 7 7 t Stat 0.358190421 -2.23685462

P(T<=t) one-tail 0.365379494 0.030178493 t Critical one-tail 1.894577508 1 .894577508

P(T<=t) two-tail 0.730758989 0.060356985 t Critical two-tail 2.36462256 2.36462256

No significant difference is seen between total collagen in normal and spastic 8 week old mice, but the difference is significant at 26 weeks (P=0.05) Example 7 Relative immunofluorescence intensities of collagens types I, III, IV, V and VI in normal and spastic mouse lower leg muscles

Indirect immunofluorescence was carried out on tissue sections of normal and spastic 8 week and 26 week old mice. Images were collected of the soleus muscle, avoiding areas containing major blood vessels which might bias the results, of sections stained at the same time and under identical conditions at x40 magnification using the programme "Scion Image", integrating 20 (collagens 1, IV, V and VI) or 30 (Collagen III) exposures on-chip. No pixels were saturated. The intensity of fluorescence was measured using Scion Image using the technique of thresholding and counting total pixels up to a preset threshold level (Collagens I, IV and V: 200; collagen III: 218 and collagen VI: 190). This level was arbitrarily set so as to visualise all immunofluorescence in the lowest intensity image of the set.

Results

The graph of Figure 7 clearly demonstrates that the amount of immunofluoresence of collagens I, and IV is elevated in normal compared to spastic muscle at both 8 weeks and 26 weeks of age. The results for collagen V and VI are less clear at the 8 week time point, but these, too, are clearly elevated in the 26 week old spastic muscle.

This is consistent with an increase in fibrillar, interstitial collagens (types I, and V) associated with an increase in extracellular matrix between muscle fibres with the development of spasticity as previously described in muscle biopsies from children with cerebral palsy.

Collagen IV is a non-fibrillar, basal lamina collagen and its elevated expression in spasticity seen here is also consistent with the thickening of the endomysium previously described in the human biopsies.

Example 8 Thresholding of collagen I and IV at different threshold bands to determine the relative intensity of immunofluorescence.

Images collected as described in Example 7 were further analysed at threshold bands of 50 units (see figure 8) Highest intensity immunofluorescence is seen at the lowest threshold levels whereas low intensity immunofluorsecence is seen at high threshold levels. Results

Different profiles are seen for collagens I and IV at the different ages examined and again the collagen levels are higher in the spastic compared to normal muscle.

Example 9 Total collagen in spastic and non spastic human muscle

Total collagen was assayed by determining hydroxyproline based on the method of Bergman and Loxley (1963). Quadriceps (vastus lateralis) muscle biopsy obtained with full ethical approval and permission was snap frozen, freeze dried then hydrolysed in 6M hydrochloric acid at 110°C for 24 hours. Hydroxyproline was determined and calculated as ug per mg dry weight of tissue.

Data is presented as "controls" - normal quadriceps muscle, "Perthes" disease - a disease of the hip which is a form of osteochondritis, mild cerebral palsy (cp) and moderately severe cp, as determined on the modified Ashworth scale.

Figure 9(a) illustrates individual data, showing standard errors from 5 independent hydroxyproline assays, and Figure 9(b) illustrates means for the four groups.

Results

Statisitical data is as follows:

control perthes control mild cp control moderately severe cp

Mean 4.596 8.987 4.596 5.301 4.596 9.503

Variance 2.012 2.992 2.012 6.438 2.012 8.512

Observations 4.000 3.000 4.000 4.000 4.000 4.000

Hypothesized Mean 0.000 0.000 0.000 df 4.000 5.000 4.000 t Stat -3.585 -0.485 -3.025

P(T<=t) one-tail 0.012 0.324 0.019 t Critical one-tail 2.132 2.015 2.132

P(T<=t) two-tail 0.023 0.648 0.039 t Critical two-tail 2.776 2.571 2.776 [control + mild cp [control -+- moderately perthes] perthes] severe cp

Mean 6.477 5.301 6.477 9.50275

Variance 7.513 6.438 7.513 8.51 197292

Observations 7.000 4.000 7.000 4

Hypothesized Mean 0.000 0.000 df 7.000 6.000 t Stat 0.719 -1.691

P(T<=t) one-tail 0.248 0.071 t Critical one-tail 1.895 1.943

P(T<=t) two-tail 0.496 0.142 t Critical two-tail 2.365 2.447

Total collagen is significantly higher (P=0.05) in children with moderately severe cp but not with mild cp compared to the control group. Children with Perthes disease also have significantly higher total collagen.

Claims

1 A method for testing the efficacy of a therapy of potential use in the treatment of diseases which have a component of muscle spasticity or muscle spasm, which comprises administration of the therapy and monitoring for altered muscle morphology.

2 A method according to claim 1 , wherein the therapy comprises a drug treatment.

3 A method according to claim 1 or 2, wherein the therapy comprises a mechanical or electrical treatment regime

4 A method according to claim 3, wherein the mechanical regime is physiotherapy.

5 A method according to any preceding claim, wherein altered morphology is a change in connective tissue.

6 A method according to claim 5, wherein the change in connective tissue is a change in the total collagen level and/or collagen distribution.

7 A method according to claim 6, wherein the total level of collagen is assessed by determining the amount of hydroxyproline in a muscle sample.

8 A method according to claim 5, wherein the change in connective tissue is a change in the specific levels and/or distribution of collagen I, III, IV, V or VI.

9 A method according to claim 8, wherein the change in connective tissue is a change in the level of collagen I, III or IV.

10 A method according to claim 6 wherein the change in collagen levels and/or distribution is a change in the ratio of different collagens to one another. 1 1 A method according to any preceding claim, wherein the monitoring step is carried out on an ex vivo sample.

12 A method according to any preceding claim, wherein the disease causes disruption of the upper motor neuron inhibitory pathways.

13 A method according to claim 12. wherein the disease is cerebral palsy, stroke, brain trauma or spinal cord injury.

14 A method according to claim 13, wherein the disease is cerebral palsy.

15 A method according to any of claims 1 to 1 1. wherein the disease is Perthes disease.

16 Use of an animal model of a disease having a component of muscle spasticity or muscle spasm in an assay for a therapy of potential use in the treatment of such a disease, wherein the therapy is directed to the muscular component of the disease.

17 Use according to claim 16, wherein the disease is cerebral palsy.

18 Use according to claim 17, wherein the animal model is the Spa animal model

19 A kit for use in the assay method of claims 1 -15. comprising detection means for at least one of the collagens I, III, IV, V or VI.

20 A kit for use in the assay method of claim 1 -15. comprising detection means for detecting collagen breakdown products in blood or urine.

21 Use of an agent which is an anti-inflammatory drug, antifibrogenic drug, colchicine, an anti-mitotic drug, matrix metalloproteinase or tissue inhibitor of matrix metalloproteinases in the preparation of a medicament for the treatment of a disease having a component of muscle spasticity or muscle spasm. 22 Use according to claim 21 , wherein more than one agent is present in the medicament.

23 Use according to claim 21 or 22, wherein the disease causes disruption of the upper motor neuron inhibitory pathways.

24 Use according to claim 23, wherein the disease is cerebral palsy, stroke, brain trauma or spinal cord injury.

25 Use according to claim 21 or 22, wherein the disease is Perthes disease.