WO2006091091A1 - Biomarqueurs rmn permettant de prévoir la réactivité à la thérapie - Google Patents

Biomarqueurs rmn permettant de prévoir la réactivité à la thérapie Download PDF

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WO2006091091A1
WO2006091091A1 PCT/NL2006/000105 NL2006000105W WO2006091091A1 WO 2006091091 A1 WO2006091091 A1 WO 2006091091A1 NL 2006000105 W NL2006000105 W NL 2006000105W WO 2006091091 A1 WO2006091091 A1 WO 2006091091A1
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therapy
vitamin
osteoporotic
metabolites
calcium
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PCT/NL2006/000105
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English (en)
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Johannes Henricus Joseph Van Nesselrooij
Robert-Jan Antonius Nicolaas Lamers
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Nederlandse Centrale Organisatie Voor Toegepast Natuurwetenschappelijk Onderzoek Tno
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Publication of WO2006091091A1 publication Critical patent/WO2006091091A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N24/00—Investigating or analyzing materials by the use of nuclear magnetic resonance, electron paramagnetic resonance or other spin effects
    • G01N24/08—Investigating or analyzing materials by the use of nuclear magnetic resonance, electron paramagnetic resonance or other spin effects by using nuclear magnetic resonance
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/82—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving vitamins or their receptors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • NMR biomarkers to predict therapy responsiveness.
  • the invention relates to the treatment of osteoporosis. More specifically, it relates to biornarkers that allow to predict the response to therapy in an osteoporotic subject.
  • Post-menopausal osteoporosis is a major healthcare problem that results in significant morbidity and mortality (Genant et al. 1999). The disease affects 75 million people in the USA, Europe and Japan combined. It is characterized by low bone mass and micro-architectural deterioration of bone tissue with a consequent increase in bone fragility and susceptibility to fractures, most frequently of the vertebrae, the proximal femur and the distal forearm.
  • a study group from the WHO established a more operational definition of osteoporosis that is based on the measurement of bone mineral density (BMD): a BMD more than 2.5 SD (standard deviation) below the mean value of peak bone mass in healthy young adults.
  • BMD bone mineral density
  • Ca/vit D calcium/vitamin D
  • HRT hormone replacement therapy
  • calcitonin calcitonin
  • body fluid of a subject with osteoporosis who is responsive (i.e. a "responder") to a calcium/vitamin D therapy comprises certain metabolites which metabolites are not, or in significantly larger or smaller quantities, present in osteoporotic subjects who do not respond (i.e. "non- responders") to that therapy.
  • non- responders i.e. "non- responders”
  • 1 H NMR proton nuclear magnetic resonance
  • this difference can be used to predict the response of a subject to therapy at a pre-treatment stage. It allows to identify patients who are less likely to respond to calcium and vitamin D treatment and should therefore be considered for an alternative therapy, such as bisphosphonates, hormone replacement therapy (HRT) and/or calcitonin.
  • HRT hormone replacement therapy
  • NMR Nuclear Magnetic Resonance
  • MVDA multivariate data analysis
  • MVDA enables to visualize patterns in NMR data, which correlate with a target variable such as environment or disease by looking for significant disparities in NMR spectra (Lamers et al. (2003) Identification of disease and nutrient related metabolic fingerprints in oseoarthritic guinea pigs. J. Nutr., 133,1776-1780 ).
  • disease-specific biomarkers are used to predict the risk of contracting a disease, to set a diagnosis or to confirm a diagnosis based on clinical examination.
  • Early detection of a disease condition using a biomarker typically allows for a more effective therapeutic treatment with a correspondingly more favorable clinical outcome.
  • WO 2004/03844 describes a system for early detection of disease and development of disease -specific biomarkers using metabolic profiling. It was found that in the urine of a subject with a particular disease, such as for instance osteoarthritis, metabolites are present which are not, or in significantly larger or smaller quantities, present in healthy subjects.
  • the present invention therefore relates to a difference profile for predicting the response to a calcium/ vitamin D supplement therapy of an osteoporotic subject, comprising a plurality of spectral line positions and, optionally, corresponding signal intensities of NMR spectral lines, which express the normalized difference between one or more NMR spectra of metabolites in a body fluid of one or more responsive osteoporotic subjects prior to said therapy, and one or more corresponding NMR spectra of metabolites in a corresponding body fluid of one or more unresponsive osteoporotic subjects prior to said therapy.
  • osteoporotic subject refers to a person who is diagnosed with osteoporosis based on standard clinical criteria, for instance quantitative heel (calcaneal) ultrasound (QUS), blood analysis for standard biochemistry, vitamin D, parathyroid hormone (PTH) and calcium, and urine analysis for deoxypyrodinoline (DPD) and calcium.
  • the osteoporotic subject is often a female human subject of post-menopausal age, e.g. 50 years or older.
  • Ca/vit D supplementation therapy is typically prescribed only to those patients who are known to be deficient in vitamin D, as these subjects are most likely to benefit from Ca/vit D therapy. Vitamin D sufficient osteoporotic patients should be considered for an alternative therapy.
  • ⁇ . difference profile of the invention is pite&i-ably obtained from metabolite recordings of vitamin D-deficient subjects because such a metabolic profile will be most informative to predict the responsiveness of the relevant group of osteoporotic patients.
  • Vitamin-D deficiency is typically diagnosed by standard blood tests.
  • responsive indicates that a significant change in one or more relevant parameters can be observed in a subject upon exposure to calcium/vitamin D supplement therapy.
  • relevant parameters include vitamin D levels and PTH levels in the blood.
  • the PTH level in a responsive subject is increased with more than 10 pmol/1 following a 3 months therapy with oral calcium/vitamin D.
  • Calcium/vitamin D therapy refers to conventional combined calcium/vitamin D therapies prescribed for osteoporotic patients, typically a daily oral intake of calcium (e.g. 1,200 mg) and vitamin D (e.g. 400-800 units).
  • calcium e.g. 1,200 mg
  • vitamin D e.g. 400-800 units
  • Various types of commercially available drugs are suitably used in such oral supplementation therapies, for instance Adcal-D3®.
  • Fig. 1 is a representation of a factor spectrum of responders versus non- responders at 0 months. The spectrum was obtained was described below in the Experimental section. The horizontal axis shows the spectral line position in 'ppm'. The y-axis shows the signal intensity in 'regression'.
  • Fig. 2 is a representation of a score plot of NMR spectra obtained in the manner as inter alia described below in the Experimental Section.
  • component Dl is plotted on the horizontal axis.
  • component D2 is plotted on the vertical axis.
  • Outlined are clusters of NMR spectra of osteoporotic patients known to respond to a 3-months calcium/vit D supplementation therapy (responders), of NMR spectra of osteoporotic patients who did not respond to such therapy (non-reponders) and NMR spectra of non-osteoporotic controls (Controls). In all cases, the NMR spectra were recorded prior to the onset of therapy as well as after 3 months when therapy was completed.
  • Fig. 3 shows a PLS-DA score plot of responders versus non-responders.
  • sample subject number 69 was excluded from model building, resulting in a cluster of responders and a cluster of non-responders.
  • Panel B shows that, based on NMR spectra recorded prior to calcium/vit D therapy, sample subject 69 can be placed into the cluster of responders without prior knowledge about this sample. This appeared to be consistent with the clinical chemistry data showing that indeed subject 69 responded to the vit D/calcium therapy.
  • a difference profile is defined as a characteristic selection of NMR spectral lines with defined positions whose values of the signal intensities significantly differ between normalized NMR spectra of metabolites in a body fluid of responsive osteoporotic patients and normalized NMR spectra of metabolites in a body fluid of unresponsive osteoporotic patients.
  • Such a difference profile comprises the spectral line positions and optionally their corresponding signal intensities or signal intensity differences.
  • a normalized NMR spectrum is defined as an NMR spectrum in which the diversity or variation in the signal intensities of the spectral lines between samples is limited by arithmetically taking glitches into account.
  • the sum of the squares of all intensities is equated with 1. The reason for this is that it is assumed that each sample comprises an equal amount of information.
  • the absolute amount of information in each NMR spectrum is equated (equal surfaces under the NMR spectra), so that they become mutually comparable.
  • a changing signal intensity of a particular spectral line in two comparable NMR spectra indicates that the concentration, of hydrogen atoms in one of those samples has changed and that, thus, the amount of one or more chemical components containing these atoms, in this case metabolites, has changed in one of those samples.
  • a difference profile according to the invention comprises a collection of spectral line positions in a normalized NMR spectrum which enables to distinguish between osteoporotic subjects that are either responsive or unresponsive to vitamin D/ calcium supplementation therapy.
  • a difference profile comprises spectral line positions whose corresponding signal intensities are increased and/or decreased by a particular factor in the spectrum of a responsive patient in relation to a corresponding spectrum of an unresponsive patient (and vice versa).
  • This factor can be used for applying a (positive) threshold value (or reference value) for increases and a corresponding (negative) threshold value for decreases of the signal intensity.
  • Spectral line positions where the differences of the corresponding signal intensities are above or below the corresponding threshold value are included in the difference profile.
  • a threshold value which corresponds to approximately one and a half times, preferably approximately two times, more preferably approximately three times the signal to noise ratio can very suitably be used in the normalized spectrum.
  • noise in the NMR spectrum is understood to mean the signals coming from nonspecific measurement events, such as for instance machine noise, environmental fluctuations, and/or contaminations in the chemicals.
  • the value of the average signal intensity of 60-99%, preferably 70-95%, more preferably 80-90% of all spectral line positions showing a change in intensity between responsive and unresponsive individuals as a threshold value for obtaining a difference profile according to the invention.
  • a normalized spectrum of metabolites in a body fluid of an osteoporotic subject represents a set of data coming from at least two, more preferably at least three, still more preferably at least four, and even more preferably at least five subjects.
  • a difference profile can very suitably comprise 3 to 1,000 spectral line positions corresponding to possibly original spectral lines.
  • a difference profile according to the invention comprises 10 to 500, more preferably 15 to 100, and still more preferably 20 to 70 spectral Ii ne positions. Very good results have been obtained with a difference profile comprising 30 to 50 spectral line positions.
  • the number of spectral line positions from which the difference profile is built up is mainly determined by the definition of the threshold value mentioned. This threshold value, in which the value for the pitch of the noise in the normalized spectra can have been taken into account, indicates from which value differences in the height of a spectral line between responsive and unresponsive individuals are "significant".
  • a difference in height can be either positive (increase of intensity) or negative (decrease of intensity).
  • Predicting therapy responsiveness by means of a difference profile according to the iiivt ⁇ iiiun isa piefwidbl.)' ussd iu individuals '.villi properties (e.g. " ⁇ taiuiii D sled tie) which are corresponding or similar to those of the individuals from which the difference profile has been obtained.
  • any body fluid can be used, including blood(serum) and urine.
  • a body fluid is used which can be obtained in a non-invasive manner and which can be easily processed. It is most preferred that the body fluid be urine.
  • a body fluid can be used, preferably proton nuclear magnetic resonance ( 1 H NMR) spectroscopy.
  • An NMR instrument with a frequency of at least approximately 200 MHz is, in principle, suitable, but there is a preference for use of instruments with a higher frequency, such as at least approximately 300 MHz, more preferably at least approximately 400- 600 MHz.
  • samples of a body fluid can very suitably be lyophilized and the lyophilisate can then be reconstructed in a suitable buffer, for instance a sodium phosphate buffer, which is prepared on the basis of D2O.
  • a suitable acid content for such a buffer is in the range of pH 4-10, preferably of pH 4-8, and more preferably, such a buffer has a pH of approximately 6.
  • pH 4-10 preferably of pH 4-8
  • a buffer has a pH of approximately 6.
  • different samples which will be mutually compared are reconstructed in buffers of equal pH.
  • the reconstitution of the lyophilized components of a sample of a body fluid in a buffer of equal pH serves to minimize spectral differences caused by differences in pH between different samples.
  • an internal standard such as for instance TMSP (sodium trimethylsilyl-[2,2,3,3,- 2 H4J-l- propionate) or tetramethylsilane can be added.
  • an NMR spectrum can be recorded from these samples, the NMR instrument being set for 1 H NMR analysis.
  • an NMR spectrum of a sample is recorded in triplicate.
  • the measurement results are shown in chemical shift in relation to the internal standard and are expressed in "ppm” (parts per million).
  • ppm parts per million
  • a spectral line position is expressed in "ppm”
  • the signal intensity is expressed in "regression” (see also Fig. 1), as is conventional in the field.
  • the present invention also relates to a method for manufacturing a difference profile for predicting the responsiveness to vitamin D/ calcium therapy in an osteoporotic subject.
  • a difference profile according to the invention can very suitably be manufactured by means of a method comprising the step of providing a first normalized set of positions and corresponding signal intensities of spectral lines of one or more NMR spectra recorded from metabolites in a body fluid of one or more osteoporotic subjects are known to respond to vitamin D/ calcium therapy, wherein said one or more NMR spectra are recorded prior to the onset of said therapy.
  • Preferably, several osteoporotic subjects that are known to respond to vit D/Ca therapy are measured so that glitches can be arithmetically taken into account.
  • Such an arithmetic account of glitches can very suitably take place in combination with the process of normalization of the measurement data.
  • one single responsive subject can be measured, but preferably, spectra coming from a group of responsive subjects are used, more preferably a homogeneous group. Normalization of several recorded NMR spectra contributes to the reliability of a set of values obtained from a plurality of individuals. Further, normalization allows the comparison of a separately recorded spectrum with a set of previously recorded spectra.
  • a method for manufacturing a difference profile further comprises the step of providing a second set of positions and corresponding signal intensities of spectral lines in an NMR spectrum which has been recorded, prior to therapy, in a corresponding manner from metabolites in a corresponding body fluid of osteoporotic subjects that are known to be unresponsive to vitamin D/calcium supplementation therapy.
  • a method for manufacturing a difference profile comprises the step of comparing the normalized spectral line intensities corresponding to corresponding spectral line positions in the first and second set of positions of spectral lines in an NMR spectrum, and detecting the differences between them for obtaining a difference profile according to the invention.
  • Multivariate data analysis or pattern recognition can very suitably be used to visualize differences related to the different subject groups in these spectra.
  • the arithmetic method based on the Partial-Linear-Fit algorithm as described in WO 02/13228 is particularly preferred. This algorithm enables adjustment of small variations in the position of the spectral line in NMR spectra without loss of resolution.
  • the above-described Partial-Linear-Fit algorithm comprises a principal component discriminant analysis (PCDA) part.
  • PCDA principal component discriminant analysis
  • the number of variables is first reduced by means of principal component analysis (PCA).
  • PCA principal component analysis
  • the projections, so-called scores, of samples on the first principal components (PCs) are used as a starting point for linear discriminant analysis.
  • the scores of the samples are plotted in a score plot, where similar samples tend to cluster and dissimilar samples will be spaced at a larger distance from each other (see Figure 2).
  • a difference profile according to the present invention can very suitably be shown as a factor spectrum, an example of which is shown in Fig. lor as a table with spectral line positions, an example of which is shown in Table 1 below.
  • Table 1 Characteristic increasing and decreasing NMR spectral line positions in osteoporotic subjects that are responsive to Ca/ ⁇ it D therapy compared to those that are unresponsive.
  • a database comprising one or more difference profiles according to the invention. Since, in the present invention, the analytical methodology of proton nuclear magnetic resonance spectroscopy is used for obtaining numeric data concerning metabolites, the values obtained depend on the settings of the instrument and the conditions under which the measurement is carried out. Also, the absolute values depend on the reference (e.g. the int ⁇ m ⁇ l standard) used in the measuieiuteiii. A diffai-eiict ⁇ ruDla, examples of which are shown in Table 1, thus comprises values which can differ between different measurement moments and between different measurement conditions. For this reason, the values as shown in Table 1 are not absolute values.
  • the meaning of the individual values of both the spectral line positions and the possible spectral line intensities in the difference profile (see Fig. 1) for predicting therapy responsiveness thus substantially resides in their ratio and position in relation to each other and therefore in the pattern of these values. Due to deviant measurement conditions as indicated hereinabove, the ppm value of a spectral line defined in Table 1 can be located at a point with a ppm value of ⁇ 0.05 ppm as shown in Table 1.
  • the invention further relates to a method for predicting the responsiveness to vitamin D/ calcium therapy in an osteoporotic subject, comprising the steps of providing an NMR spectrum of metabolites in a body fluid of an osteoporotic subject which is considered for vitamin D/ calcium therapy and determining whether said NMR spectrum is characteristic of a responsive or an unresponsive subject, using a difference profile according to the invention, which difference profile has been determined for a corresponding body fluid.
  • a determination step can be carried out visually, but also arithmetically.
  • the invention also provides a method for identifying a biomarker for predicting the responsiveness to vitamin D/ calcium therapy in an osteoporotic subject, comprising manufacturing a difference profile according to the invention and identifying one or more metabolites which are characterized by one or more defined spectral lines in said difference profile, which one or more metabolites, alone or in combination, characterize said biomarker.
  • biomarkers are understood to mean one or more organic compounds or their metabolites, or specific patterns or specific amounts of several organic compounds or their metabolites, which can be found in the body fluid of an osteoporotic subject and which are correlated with the responsiveness to Ca/vit D supplementation therapy of that subject.
  • the present invention furthermore provides a method for the identification of biomarkers.
  • a biomarker according to the invention can be one single substance or metabolite, but also a specific combination of substances or metabolites. In the latter case, it can also be considered a set of biomarkers.
  • a biomarker is a specific combination of metabolites which can be found in a specific pattern of concentrations or amounts in a body fluid, preferably urine, and which can be derived from a difference profile.
  • a biomarker is also understood to mean moieties of organic compounds or of metabolites.
  • the identification of a metabolite which is characterized by one or more defined spectral lines in a difference profile can, for instance, be done by the coupling of a mass spectrometer to an NMR instrument and the subsequent analysis of the metabolite corresponding to one or more defined spectral lines by means of mass spectrometry (MS).
  • MS mass spectrometry
  • a skilled person is familiar with mass spectrometry for the identification of organic compounds and metabolites.
  • determining the identity of a metabolite corresponding to one or more defined spectral lines can also be done by recording the NMR spectrum from known metabolites and comparing it to the NMR spectral lines in a difference profile according to the invention.
  • Table 2 lists characteristic metabolites and their spectral line position that can be used as biomarker to predict whether an osteoporotic subject will benefit from Ca/vitD therapy. Table 2:
  • Unidentified Region Most likely to be metabolites 6.86-7.98 (hetero)aromates
  • the 1 H chemical shifts of particular characteristic metabolites for responsive osteoporotic patients are, for instance (values ⁇ 0.05 ppm): malonic acid appears as a singlet at 3.11 ppm (assignment CH2); valine appears as a doublet at 1.07 ppm (assignment CH3) and as a doublet at 3.69 ppm (assignment CH).
  • the level of these metabolites appears to be increased in laspuiideis arid are thus attrauiiva fur List; &s predictive biomarker to distinguish between responders and non-responders before onset of calcium/vit D therapy.
  • Biomarkers that are decreased in responders relative to non-responders are also given in Table 2. These include alanine, lactic acid, histidine, 1-methylhistidine, formic acid, ⁇ -glucose and ⁇ -glucose. Furthermore, some regions of spectral line positions were found that probably represent (cyclo)alkanes, alkynes or (hetero)aromates.
  • the metabolites of Table 2 can suitably be used as biomarkers according to the present invention for predicting therapy responsiveness in a patient, where increases in the concentration of the biomarkers indicate, for instance, the (increased) degradation or conversion of the base material from which these metabolites originate.
  • metabolites are secreted in a body fluid (e.g. in the blood or urine) in free condition or in a derived form, for instance conjugated or bound in another manner.
  • a body fluid e.g. in the blood or urine
  • the metabolites described can be used as a biomarker in any condition in which they may be found in the body fluid.
  • the present invention further relates to a method for predicting the responsiveness to calcium/vit D therapy in an osteoporotic subject, comprising measuring a biomarker according to the invention in a body fluid, preferably urine.
  • a biomarker according to the invention is preferably non-invasive, and preferably comprises the detection, in a body fluid of an osteoporotic subject in which therapy responsiveness can be expected e.g. a vitamin D- deficient subject, of a quantitative change in the occurrence of a biomarker in relation to a value for that biomarker which is found in a body fluid of an unresponsive subject and which quantitative change corresponds to the regression of that biomarker in the difference profile for the responders compared to non-responders.
  • therapy responsiveness e.g. a vitamin D- deficient subject
  • a biomarker according to the invention can be measured in a body fluid in different manners. For instance, NMR and/or Mass Spectrometry (MS) can be applied to a sample of a body fluid. But other analytical methods can also be used for this purpose, such as ELISA or a related methodology.
  • MS Mass Spectrometry
  • microsystem technologies for instance by using a "microfluidics” instrument or a microelectromoctianic ⁇ yctr.tn (MEMS) in combination with, fur iiisLtixwa, specific; fluorescent enzymes or other manners of detection by means of which the biomarkers found in a body fluid can be quantitatively and/or qualitatively measured.
  • MEMS microelectromoctianic ⁇ yctr.tn
  • the present invention also relates to an apparatus for using a method for the prediction of therapy responsiveness in a subject by measuring a biomarker according to the invention.
  • Such an apparatus preferably comprises a solid carrier with immobilized binding partners for this biomarker thereon.
  • the nature of such binding partners depends on the biomarker which will be measured, but can, for instance, comprise an antibody or a peptide as a specific binding partner which is able to specifically bind the biomarker.
  • An apparatus according to the invention further preferably comprises a system for quantitative detection of binding between the biomarker and the immobilized binding partners.
  • Such a system can comprise either direct detection (for instance by applying fluorescent labels on the biomarker) or indirect detection (for instance by applying a secondary binding partner to the biomarker, which secondary binding partner comprises a detectable label).
  • direct detection for instance by applying fluorescent labels on the biomarker
  • indirect detection for instance by applying a secondary binding partner to the biomarker, which secondary binding partner comprises a detectable label.
  • Phase 1 included screening, routine diagnostic tests (biochemistry for renal function, hepatic function and calcium/vitamin D homeostasis) and treatment with calcium and vitamin D when necessary.
  • Phase 2 consisted of further biochemical assessments, including metabolite analysis by NMR.
  • the population under study consisted of osteoporosis patients and non-osteoporotic subjects.
  • the subjects were female and they were aged 50 years or older.
  • the subjects completed a witnessed written informed consent form for phase 2 of the study.
  • Subjects had a satisfactorily documented medical history, and blood biochemistry (for renal and liver function) and urinalysis with no clinically significant abnormalities for their age.
  • the following subjects were excluded from the study: subjects with any evidence or history of clinically significant renal or hepatic dysfunction, subjects with any history of metastatic disease, multiple myeloma, or any other condition resulting in hypercalcaemia, subjects who had participated in a clinical trial in the three months prior to study commencement and subjects who were on therapy known to affect calcium or bone metabolism.
  • Dosing Subjects who were osteoporotic were prescribed 1200 mg oral calcium and ROOTT vitamin T) which they took for the duration of the. trial Calcium/vitamin D was not prescribed to those who acted as controls (i.e. non-osteoporotic subjects).
  • Sampling Approximately 15 ml of venous blood was drawn from a forearm vein at screening. This was used for biochemical analyses, NMR and pattern recognition. A further 15 ml sample was drawn at 3 months. Urine was analysed at screening and 3 months.
  • Osteoporotic subjects were recruited from the King's College Hospital (KCH) osteoporosis clinic.
  • a standard NHS (National Health Services) osteoporosis work-up was performed at screening. This included: quantitative heel (calcaneal) ultrasound (QUS), blood analysis for standard biochemistry, vitamin D, PTH and calcium, and urine analysis for deoxypyrodinoline (DPD) and calcium.
  • Subjects with osteoporosis were started on oral calcium and vitamin D supplements. Those with normal QUS did not receive any Ca/vitamin D supplementation, and acted as controls. After 3 months of treatment, urine and blood analyses were repeated. The difference in vitamin D levels and PTH levels were assessed to distinguish between subjects who responded well to Ca/vitamin D treatment and those who did not respond well to the treatment.
  • Subjects were identified by a unique code. Therefore when blood samples and results were analysed, the analysts were blinded to the identity of the subject, and whether they were on oral calcium and vitamin D treatment or not.
  • Blood samples were collected in a vacutainer tube. Immediately after collection, blood was mixed well and put in a cooled box. Within 15-30 minutes after collection samples were centrifuged for 10 minutes at ca. 2000 x g at ca. 4°C. After centrifugation, 1 aliquot of ca. 5 ml plasma/serum (minimum 2 ml) was stored directly at ⁇ -20°C until analysis.
  • Free induction decays were collected as 64K data points with a spectral width of 8.000 Hz; 45 degree pulses were used with an acquisition time of 4.10 s and a relaxation delay of 2 s.
  • the spectra were acquired by accumulation of 128 FIDs.
  • the signal of the residual water was removed by a pre- saturation technique in which the water peak was irradiated with a constant frequency during 2 s prior to the acquisition pulse.
  • the spectra were processed using the standard Varian software. An exponential window function with a line broadening of 0 R HV and.
  • the filters with a molecular-mass cutoff of 10 kDa (Microcon YM-IO, Millipore) were spin-rinsed with 0.5 mL of 0.05 M NaOH followed by 2 x 0.5 mL de-ionized water to avoid contamination of the ultrafiltrate with glycerin.
  • Deproteinisation of the plasma samples by filtration was performed by centrifugation (Ih at 10000 rpm) of 0.5 mL plasma over a filter followed by the centrifugation (Ih at 10000 rpm) of 0.5 mL de-ionised water in order to rinse the filtration membrane.
  • filtrates were freeze-dried and reconstructed in 750 ⁇ L of sodium phosphate buffer (pH 6.0, made up with D2O) containing 1 mM sodium trimethylsilyl-[2,2,3,3,- 2 H4]-l-propionate (TMSP) as internal standard.
  • sodium phosphate buffer pH 6.0, made up with D2O
  • TMSP trimethylsilyl-[2,2,3,3,- 2 H4]-l-propionate
  • Plasma samples were deproteinised by extraction of 100 ⁇ L plasma with 600 ⁇ L isopropanol. After centrifugation (5 min at 10000 rpm) the supernatant was isolated and concentrated to dryness. The residue was dissolved in 750 ⁇ L MeOD prior to NMR analysis.
  • NMR Spectroscopy - NMR spectra were recorded in triplicate in a fully automated uiaimei uxx a Vaxiaii UNITY 400 MHz BpwLnmieLer using a proton NME ssL-up operating at a temperature of 293 K.
  • Free induction decays (FIDs) were collected as 64K data points with a spectral width of 8.000 Hz; 45 degree pulses were used with an acquisition time of 4.10 s and a relaxation delay of 2 s.
  • the spectra were acquired by accumulation of 512 FIDs.
  • the signal of the residual water was removed by a pre-saturation technique in which the water peak was irradiated with a constant frequency during 2 s prior to the acquisition pulse.

Abstract

L’invention concerne le traitement de l’ostéoporose. Plus spécifiquement, elle concerne des biomarqueurs permettant de prévoir la réaction à une thérapie chez un sujet souffrant d’ostéoporose. Elle concerne également un profil de différence permettant de prévoir la réaction à une thérapie à base de calcium / de vitamine D d’un sujet souffrant d’ostéoporose, de préférence un sujet humain, comprenant une pluralité de positions de ligne spectrale RMN et, en option, d’intensités de signaux correspondants de lignes spectrales RMN, qui expriment la différence normalisée entre un ou plusieurs spectres RMN de métabolites dans un fluide corporel d’un ou de plusieurs sujets souffrant d’ostéoporose connus pour réagir à une thérapie de calcium / de vitamine D, et un ou plusieurs spectres RMN correspondants de métabolites dans un fluide corporel correspondant d’un ou de plusieurs sujets souffrant d’ostéoporose connus pour ne pas réagir à ladite thérapie, les spectres RMN étant enregistrés avant ladite thérapie. Elle concerne de plus un procédé permettant de prévoir la réactivité à la thérapie de vitamine D/ calcium chez un sujet souffrant d’ostéoporose utilisant un profil de différence de l’invention.
PCT/NL2006/000105 2005-02-28 2006-02-28 Biomarqueurs rmn permettant de prévoir la réactivité à la thérapie WO2006091091A1 (fr)

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US10478117B2 (en) 2015-04-08 2019-11-19 Ramot At Tel-Aviv University Ltd. Early detection of reduced bone formation with an NMR scanner

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